Block 1 Flashcards

Question

The Nucleosome:

Answer 1

1. The Nucleosome: the fundamental and smallest unit of chromatin packaging a. There are millions of nucleosomes in each nucleus, each shaped like a rugby ball i. Using electron microscopy, these can be seen as beads along a string (string being linear DNA) ii. As in bulk chromosome, each nucleosome is half protein, half DNA iii. The protein component of each beat consists of an octomer (8) of histone molecules, plus a few non-histone molecules

Answer 2

i. Grouped within 5 related protein “families” a. H1 family: termed “linker” histones because they are located between the nucleosomal beads b. H2A, H2B, H3, H4: termed “core” histones i. Two molecules of each core histone make-up each nucleosome; hence each nucleosome contains an octamer (8) of histone molecules ii. All the histones are highly basic due to the enrichment of lysine+ and arginine+ residues, and are evolutionarily highly conserved, indicating their vital cellular function

Answer 3

histones function to inhibit transcription, globally repressing the genome

Answer 4

i. In order for transcription to occur, the histones must be modified via acetylation of the lysine+ and arginine+ residues to neutralize the positive charges 1. This results in repulsion of histones from DNA gene sites causing “chromatin opening,” a process that allows entry of transcriptional machinery

Answer 5

i. In contrast, methylation of histone H3 causes formation of the non-transcribable ‘hetero’-chromatin by serving as a binding surface for the non-histone protein HP1 (heterochromatin protein 1) 1. HP1 is bound, other proteins (i.e., transcription factors) can’t

Answer 6

i. Phosphorylation of serine and threonine is usually associated with the repression of transcription that occurs during condensation of chromatin into recognizable chromosomes as the cell enters M-phase (mitosis) of the cell cycle

Answer 7

Methylation and phosphorylation INHIBIT transcription Acetylation ALLOW transcription

Answer 8

a. The posttranslational modifications of histones occur primarily at specific residues located within the histone amino-terminal tails protruding from the surface of the nucleosome i. Histone modifications impact chromosome function though at least two distinct mechanisms: 1. Histone modifications alter the electrostatic charge of the histone resulting in a structural change in histones or their binding to DNA a. This is exemplified by histone acetylation/deacetylation 2. Histone modifications provide binding sites for regulatory proteins containing protein domains that recognize specific histone modifications a. HP1 uses its chromodomain to bind methylated lysines residues on histone H3 during heterochromatin formation ii. Histone Modifications are carried out by specific histone modifying enzymes (histone acetyltransferases, methyltransferases, etc)

Answer 9

i. Epigenetics modifications produce heritable changes in gene function that occur without a change in the sequence of DNA

Answer 10

1. Another type of epigenetics modification is methylation of DNA, which is when a methyl group is added to the 5’ position of the pyrimidine ring in cytosine residues in CpG dinucleotides a. DNA methylation is carried out by DNA methyltransferases (DNMTs) b. This represses gene expression by blocking binding of TFs that normally activate genes c. Can synergize with repressive histone modifications to promote heterochromatin formation i. Methylated CpG in DNA is bound by methyl CpG binding proteins (MBPs) ii. MBPs recruit histone deacetylases (HDACs) iii. MBPs recruit histone methyltransferases (HMTs) 1. Methylated histones are recognized and bound by HP1, completely silencing gene

Answer 11

a. CLINICAL RELEVANCE: Importance of epigenetic changes in diagnosis and treatment of cancer i. Epigenetic modifications of DNA and histones can combine to suppress expression of tumor suppressing genes, whose products control cell growth by regulating the processes of mitosis and cell division ii. DNMT inhibitors and HDAC inhibitors have been tested for use as anti-cancer agents 1. HDAC inhibitors directly inactivate HDAC enzymatic activity 2. DNMT inhibitors incorporate into DNA in the place of Cytosine, rendering CpG dinucleotides incapable of methylation by DNMT

Answer 12

a. Nucleosomes are approx. 50% (histone) protein, 50% DNA i. Each nucleosome is considered to contain approx. 200 pb DNA ii. Each nucleosome forms by combining two histone dimers (H2A/H2B) with one histone tetramer (2x H3/H4) to form the histone octamer iii. Non-Histone: any chromatin protein that is not a histone (DNA repair enzymes, DNA and RNA polymerases, and TFs) b. Transcribing chromatin does have nucleosomes! i. Modifications during gene activation 1. The dissolution of one or two nucleosomes from the gene promoter and of histone H1 from linker DNA 2. Histone acetylation by histone acetyl transferases (HATs)

Answer 13

1. The 30-Nanometer Fiber: a. Six 200 bp nucleosomes are packed into a solenoid arrangement as one 1200 pb fiber with the help of Histone H1 i. Solenoid: 6 nucleosomes per turn

Answer 14

1. Loops: a. An average of 50 1,200 bp 30 nm fibers are coiled into a loop containing 60,000 pb of DNA b. The bases of each loop are attached to a proteinaceous structure termed the nuclear matrix, which contains important structural and gene regulatory machinery c. Looping circularizes the DNA, enabling localized genes to become supercoiled, which is required for transcription d. Genes located in one loop can interact with up- and downstream regulatory sequences in adjacent loops, such as enhancers

Answer 15

1. Minibands: a. Tandems of loops that encircle the chromosome axis b. Proximity of loops on adjacent minibands would permit regulatory sequences in distal loops to interact

Answer 16

a. The formation of individual chromosomes at the onset of mitosis reflects hyper-condensation of chromatin, which is mediated by hyper-phosphorylation of chromatin proteins, especially histone H1 i. Each chromosome contains a single linear DNA molecule of an average 75 million bp DNA ii. Centromere: locus of repetitive DNA in the middle of the chromosome iii. Two telomeres: loci of repetitive DNA that cap the ends of the chromosome

Answer 17

i. Two telomeres: loci of repetitive DNA that cap the ends of the chromosome 1. Contain a repeating sequence of GGGTTA up to 15,000 bps 2. At each cell division, several repeats are lost in somatic cells causing shortening of telomeres and cellular senescence 3. Telomeres in germ-line cells are maintained at full-length via an enzyme termed telomerase, which adds on GGTTA segments after cell division

Answer 18

CLINICAL SIGNIFICANCE: telomerase is normally quiescent in somatic cells, but can be mutated and activated, causing abnormal maintenance of telomere length and cellular immortality that result in tumor formation

Answer 19

The nuclear matrix maintains the dynamic structural organization of the interphase nucleus by organizing the genome into domains that regulate gene expression and cell replication Three Components of nuclear Matrix: 1. Nuclear Envelope/Pore Complex/Nuclear Lamina 2. Nucleolus 3. Internal Nuclear Matrix

Answer 20

1. Nuclear envelope/Pore Complex/Nuclear Lamina

Answer 21

a. The nuclear envelope i. A double membrane that encloses the perinuclear space, which is continuous with the lumen of the endoplasmic reticulum (ER) ii. It is divided into segments, limited by nuclear pores iii. The outer membrane (faces cytoplasm) contains ribosomes for protein translation iv. The inner nuclear membrane contains integral proteins that bind the nuclear lamina, which in turn attaches to marginal heterochromatin

Answer 22

i. Nuclear pores between segments of the envelope are ~10 nm diameter ii. The pore complex regulates passage of proteins into, and proteins/RNAs out of, the nucleus 1. Proteins smaller than ~40kD diffuse thru, larger proteins are under complex regulation

Answer 23

i. Each pore has three strata of proteins arranged as an octamer: (1) cytoplasmic stratum (2) nuclear stratum and (3) middle stratum 1. Strata contain ~100 different proteins termed nucleoporins 2. Nucleoporins in the cytoplasmic stratum serve as “docking sites” for proteins called nuclear cargo proteins that contain nuclear localization signals (NLSs) a. NLSs are short amino acid sequences that contain positive charged basic amino acids (lysine [K] and arginine [R])

Answer 24

1. Proteins larger than 45 kD must have a NLS to get in 2. NLS-Receptor Proteins are in the cytoplasm a. Example: importin, a heterodimer consisting of subunits importin- and importin- i. Importin- binds the NLS of a nuclear cargo protein ii. Then the complex migrates to the pore iii. Importin- binds a nucleoporin in the pore complex iv. Then, energy-dependent transport thru the pore v. Once in the nucleus, the nuclear cargo protein is released from importin, which is cycled back thru the nuclear pore complex

Answer 25

a. Proteins may have to be de-phosphorylated | b. Proteins may have to be released from a cytoplasmic masking protein to be recognized by importin

Answer 26

1. Exportins mediate nuclear protein export a. Exportins contain leucine-rich NESs (Nuclear Export Sequences) in their amino acid sequence b. Many if not must nuclear proteins have both an NLS and an NES 2. RNA export does not depend on type of sequence of RNA a. RNA export is mediated by RNA-BPs which may be bound to an exportin protein

Answer 27

1. The same pore can accommodate protein import as well as export of protein and RNA

Answer 28

i. Energy for nuclear import and export is provided by a GTP-BP termed Ran-GTP (“ran” in and out of the nucleus) 1. Within nucleus, Ran-GTP binds to both empty nuclear import receptors and cargo-bound export receptors, which migrate to the cytoplasm where Ran-GTP is hydrolyzed to Ran-GDP, releasing all bound moieties a. Empty nuclear import receptors migrate back into nucleus b. Nuclear import receptors bind proteins with NLSs before migrating back to nucleus

Answer 29

i. CLINICAL SIGNIFICANCE: Drugs cannot readily penetrate the nucleus so nuclear structures are poor targets for therapeutic intervention 1. Knowing how substances are targeted to the nucleus, approaches may be designed that enable the development of drugs that can target specific nuclear structures and genes

Answer 30

i. The nuclear lamina is subjacent to the inner nuclear envelope, to which it is bound 1. In interphase nuclei, the nuclear lamina maintains the nucleus as a sphere 2. During mitosis, the lamina breaks down due to hyperphosphorylation of lamina proteins

Answer 31

i. The nuclear lamina’s proteins are termed lamins A, B, and C ii. Lamin A (LMNA) gene: Expresses Lamins A and C, which interact with the marginal heterochromatin iii. Lamin B binds to the lamin-B receptor (LBR) on the inner nuclear membrane 1. When the nuclear envelope falls apart during mitosis, lamin B remains bound to the LBR, mediating re-formation of the nuclear envelope segments at the start of the next interphase

Answer 32

i. CLINICAL SIGNIFICANCE: Laminopathies – mutations of the LMNA gene disrupt the lamina 1. Diseases range from several rare cardiac and skeletal muscular dystrophies to a severe form of premature aging (progeria), whch is caused by deletion of the part of the gene that generates the mRNA encoding lamina A

Answer 33

a. The site of ribosomal RNA (rRNA) production, comprised of portions of ten chromosomes (each contributing ~40 rRNA genes) i. Nucleoli do not have a limiting membrane b. Cells need lots of ribosomes to live i. To satisfy the cell’s demand, transcription of rRNA genes occurs constantly via RNA Polymerase I 1. Physical gene assumes the shape of a “Christmas tree”

Answer 34

1. The Internal Nuclear Matrix (98% of the nuclear matrix) a. Consists mostly of chromatin: DNA, Histones (H1, H2A, H2B, H3, H4) and non-histones (TFs, RNA and DNA polymerases, etc.) b. Matrix proteins are disease-specific and tissue-specific i. Some of the nuclear matrix proteins in normal cells and cancer cells is different – of diagnostic value (used in diagnostic tests for bladder cancer and cervical carcinoma)

Answer 35

a. Chromosomes occupying specific locations in the interphase nucleus i. Telomeres stick to the nuclear envelope while centromeres stuck to the opposite side of the envelope ii. Chromosomes occupy the same site of the interphase nucleus, generation after generation iii. Chromosome specific territories in the nucleus have been shown using fluorescent in situ hybridization (FISH) of DNA – also called chromosome painting

Answer 36

a. DNA replication and transcription occur in the nuclear matrix i. Although most nuclear material is removed during preparation of the nuclear matrix, sites of transcription are retained as indicated by in situ hybridization

Answer 37

i. In S-phase, DNA is replicated in groups of short sequences termed “replicons” – after matrix preparation, these replicons are retained in the matrix

Answer 38

• Duration of each cell-cycle revolution is termed the generation time (Tg) o During each Tg the cell goes through four cell-cycle phases: ♣ (1) G1 Phase (Gap 1) – duration is highly variable • Cells in G1 have four choices: o Senescence (i.e., G0) o Differentiation (also G0) o Apoptosis cell death o Proliferation entry into cell cycle ♣ (2) S-Phase (Synthesis [of DNA] Phase) • Chromosomes are duplicated, 2N 4N DNA • Histones are synthesized to make-up the new chromosome ♣ (3) G2 Phase (Gap 2) • Preparation for mitosis occurs; late in G2 the centrosome is duplicated • Hyper-phosphorylation of histone and non-histone proteins occurs in late G2 ♣ (4) M Phase (Mitosis): • Cells become spherical • Nuclear membrane disintegrates • Chromatin condenses into chromatids (2/chromosome) • Chromosomes align on equatorial plan, then chromatids segregate into “daughter” cells

Answer 39

• External Factors “1o messengers” o Secreted molecules and peptides – GFs, cytokines, hormones – which interact with a cognate receptor that is in the cell membrane (or, for steroid/retinoid hormones, in the cytoplasm) ♣ Factors that ACTIVATE the cell-cycle (examples): FGFs, IGFs, Wnts ♣ Factors that INHIBIT the cell-cycle: TGF

Answer 40

• Internal Factors “2o messengers” o “Early Response” genes: c-myc, fos, and jun ♣ Rapidly (15 min) respond to growth signals o “Delayed Response” genes: CDKs and cyclins ♣ the CDKs and cyclins comprise heterodimers termed cyclin-dependent protein kinases (Kinases are enzymes that phosphorylate target proteins) • The CDK subunit is the catalytic subunit, which phosphorylates the target (substrate) o There are several different CDKs (CDKs 4, 2, and 1) o During the cell-cycle, the content of each CDK remains constant • The cyclic subunit is the regulatory subunit, which regulates the activity of the kinase heterodimer o There are several cyclins (Cyclins D, E, A, and B) o During the cell-cycle, the content of each cyclin increases

Answer 41

o CLINICAL CORRELATION: Each of the proteins described to this point – Growth Factors, Growth Factor Receptors, early response genes, and CDKs/cyclins – are termed proto-oncogenes ♣ When a proto-oncogene is mutated, the protein it encodes becomes pathologically over-active stimulating the cell-cycle to induce cancer

Answer 42

• Regulation by extracellular growth factors is prominent in early G1, diminishing in late G1 before the restriction point (R) o In response to growth factors, intracellular cyclins regulate discrete steps during interphase ♣ The content of cyclins D, E, A, and B increases during the cell-cycle in that sequence to activate the cell-cycle phases • These increases occur concomitant with declining content of p27, a cell-cycle inhibitor Cyclins DEAB

Answer 43

1. G1 Phase is activated by cyclinD/CDK4 heterodimer: a. During early G1, growth factor activity induces sustained increase in cyclin D i. A classical example is initiated by the Wnt growth factors, which induce increased content of the cytoplasmic protein -catenin, which migrates to the nucleus to activate transcription of the c-myc gene, which in turn activates the cyclin D gene Wnt GF cytoplasmic -catenin –migrates to the nucleus c-myc gene cyclin D genei. Other factors also activate cyclin D transcription 1. GFs induce intracellular ras, a small GTPase-binding protein, which activates the MAP kinase pathway to effect cyclin D production

Answer 44

a. Cyclin D binds CDK4 to form cyclinD/CDK4 heterodimer

Answer 45

a. CyclinD/CDK4 phosphorylates retinoblastoma (Rb) protein, forming P~Rb i. In the phosphorylated state, Rb releases its hold on a transcription factor protein termed E2F ii. Rb inhibits the cell-cycle; P~Rb releases this inhibition

Answer 46

a. E2F activates genes encoding cyclin E and cyclin A, increasing their content

Answer 47

CyclinD + CDK4 CyclinD/CDK4 phosphorylates Rb P~Rb releases E2F activates gene for cyclin E and cyclin A

Answer 48

1. S-Phase is activated by cyclinE/CDK2 and cyclinA/CDK2 heterodimers a. This depends on E2F-induced activation of genes encoding cyclins E and A i. When content of cyclin E increases, the restriction checkpoint (R) regulated by cyclinE/CDK2 is breeched 1. Once breeched, the cell is committed to complete the cycle, all the way through G2 phase 2. This commitment ensures that the whole genome is replicated during S-phase 3. Failure to replicated all of the DNA results in cancer b. During S phase, ‘DNA replication complexes’ which are ‘poised’ at multiple origins of DNA replication, are activated via phosphorylation of cyclinA/CDK2 i. This mechanism prevents formation of new, inappropriate DNA replication complexes ii. This prevents the DNA from being replicated more than once c. The end result of the cell-cycle is the precise apportionment of 1.0 genome to each daughter cell Cyclin E restriction checkpoint is breeched cell is committed to complete cycle CyclinA/CDK2 phosphorylates (and activates) “DNA replication complexes”

Answer 49

1. M-Phase is activated at the G2/M boundary upon de-phosphorylation of the cyclinB/CDK1 heterodimer a. This de-phosphorylation is mediated by a phosphatase termed cdc25 i. After it is de-phosphorylated, the cyclinB/CDK1 dimer enters the nucleus to phosphorlate many target proteins, causing: 1. Nuclear envelope breakdown (NEB) 2. Mitotic spindle assembly 3. Metaphase arrest

Answer 50

Activated at mid-metaphase via the Anaphase Promoter Complex (APC)

Answer 51

• Activity of cyclin/CDK heterodimers is antagonized by proteins suppressing the cell-cycle o Under the influence of such inhibitory proteins, cells are maintained in a prolonged G1-phase termed G0 ♣ The genes that encode for these inhibitory suppressor proteins are termed tumor suppressor genes ♣ Mutation of a gene encoding a tumor suppressor protein can cause deactivation of that protein, resulting in cancer

Answer 52

♣ Binds E2F-1, preventing E2F-1 from transcribing genes encoding cyclins E and A ♣ Rb is a “classic” tumor suppressor gene because when it is mutated, tumors of the retina termed “retinoblastomas” occur ♣ Rb, as well as proteins of related function such as p107 and p130, are termed “pocket proteins”

Answer 53

o p21: Inhibits the cell-cycle by directly inhibiting CDK-2 and CDK-4

Answer 54

o p27: similar to p21

Answer 55

o p53 – the most prominent tumor suppressor ♣ Inhibits the cell-cycle via two functions • (1) p53 induces transcription of the tumor suppressor p21 • (2) p53 promotes apoptosis (cell death) ♣ Is mutated in over ½ of human cancers ♣ Is referred to as the “sentinel of the genome,” since it induces killing of cells that contain inappropriately replicated DNA resultant from mistakes made during S-phase

Answer 56

Cancer: What is it? • Cancer is a genetic disease displaying: o Loss of cellular differentiation o Increased proliferation and invasiveness of cells o Changes in chromosomes: re-arrangement, loss, gain Cancer: What causes it? • Cancer is caused by inherited mutations and/or environmental insults to DNA during aging o As we age, our cells evolve toward malignancy o Cancer is definitely a genetic disease

Answer 57

1. Proto-oncogenes: about 70 genes that encode proteins that activate the cell cycle a. Genes encoding cell surface receptors, factors, cyclins, and CDKs b. When mutated, these are termed oncogenes i. Mutated to be over-expressed, or the proteins the they encode have intensified activity ii. Result is up-regulation

Answer 58

1. Tumor Suppressing Genes: inhibit the cell-cycle a. When mutated, products of these genes no longer “work” b. About 20 tumor suppressors have been discovered, including:

Answer 59

a. Diseased cells must be removed before they can colonize, so apoptosis is a normal and necessary mechanism b. Stimulation of apoptosis causes tumors to regress c. Apoptosis also regulates embryonic development, as shape (morphology) is attained – example: digit formation of fingers

Answer 60

i. Macrophages release TNF (tumor necrosis factor) ii. TNF binds TNFR cell membrane receptor iii. A balance between pro-apoptotic factors + anti-apoptotic factors is established iv. Pro-apoptotic signals induce “leakiness” of the outer cell membrane of mitochondria v. Cytochrome-C escapes to the cytoplasm vi. Cytochrome-C activates Caspase, a protease that destroys chromatin, which fragments into 200 bp (i.e. nucleosomal) pieces vii. Blebs appear on the cell surface and the cell defoliates (explodes)

Answer 61

a. Relationship to cancer: In B cell lymphoma, a gene that normally inhibits apoptosis (bcl-2) is mutated i. bcl-2 is an anti-apoptotic factor, but when mutated it is abnormally active ii. Too much inhibition of apoptosis occurs, resulting in tumor formation

Answer 62

1. Genes that induce cellular immortality: Telomerase a. “Telomere hypothesis” – upon attaining a given length, the shortened telomere signals the cell to become senescent i. This is a major regulatory mechanism in the aging process b. In embryonic stem cells, and germ cells (sperm/egg), the telomere is restored to its original length after each cell division via an enzyme: telomerase i. Telomerase is normally inhibited during aging of somatic cells so they normally become senescent after a critical number of cell divisions ii. If the gene encoding telomerase is mutated, telomerase becomes active in somatic cells, rendering them immortal – this can cause carcinogenesis

Answer 63

a. Normally DNA damage causes the cell-cycle to stop until DNA repair enzymes fix the damaged DNA b. If genes that encode DNA repair proteins are mutated, the proteins are inactivated, allowing clones of abnormal cells to arise i. Recently documented in the instance of colo-rectal cancer

Answer 64

• DNA synthesis is semiconservative o Each daughter DNA molecule ends up with one of the original strands and one newly synthesized strand • DNA is synthesized in the 5’ 3’ direction • A 3’-OH primer and a template are required o DNA polymerase I activity requires a single unpaired strand to act as a template and a primer strand to provide a free hydroxyl group at the 3’ end, to which a new nucleotide unit is added o The catalytic mechanism of elongation likely involves two Mg2+ ions, coordinated to the phosphate groups of the incoming nucleotide triphosphate and to three Asp residues, two of which are highly conserved in all DNA polymerases • DNA replication is semi-discontinuous o There is a leading strand and a lagging strand ♣ The leading strand is transcribed in the direction of movement of the replication fork ♣ The leading strand is transcribed in the opposite direction, away from the movement of the replication fork • Still transcribed 5’ 3’, the lagging strand uses an RNA primer to provide the free 3’ OH • It is transcribed in segments called Okazaki fragments

Answer 65

1. Separation of the two complementary strands at an origin of replication (a specific place(s) in the genome) 2. Formation of the replication fork (primers and Okazaki fragments) 3. Chain Elongation 4. Removal of the primers

Answer 66

``` • One protein, 3 catalytic activities o 5’ 3’ polymerase o 5’ 3’ exonuclease o 3’ 5’ exonuclease Exonuclease: Proofreading activity: removes incorrectly base paired nucleotides ```

Answer 67

* Polymerase mispairs dC with dT, which impedes translocation of DNA polymerase I to the next site * Polymerase repositions the mispaired 3’ terminus into the 3’ 5’ exonuclease site which is behind the polymerase activity when the enzyme is oriented in its movement along the DNA * Exonuclease hydrolyzes the mispaired dC * The 3’ terminus repositions back to the polymerase site * Polymerase incorporates the correct nucleotide, dA

Answer 68

• Process of DNA replication is functionally conserved between pro and euk organisms o Many of the proteins are different but they function in an analogous manner o A major difference in the replication process is that in prokaryotic replication this is one origin of replication and in eukaryotic replication there are multiple

Answer 69

* SSB – binding to single-stranded DNA * DnaB protein (helicase) – DNA unwinding; primosome constituent * Primase (DnaG protein) – RNA primer synthesis; primosome constituent * DNA polymerase III – New strand elongation * DNA polymerase I – Filling of gaps; excision of primers * DNA ligase – Ligation * DNA gyrase (DNA topoisomerase II) – Supercoiling

Answer 70

• Structures at the ends of eukaryotic chromosomes • Have tandem repeats usually of T1-4G1-4 (with A-C on opposing strand) • Can be tens of thousands of bp long in mammals • TG strand is longer than its complement, leaves a 3’-overhang of several hundred bases • Telomerase is an RNA-dependent DNA polymerase (reverse transcriptase) that carries its own RNA template o The internal template of RNA of telomerase binds t and basepairs with the TG primer of DNA o Telomerase adds more T and G residues to the TG primer, then repositions the internal template RNA to allow the addition of more T and G residues that generate the TG strand of the telomere o The complementary strand is synthesized by cellular DNA polymerases after priming by RNA primase

Answer 71

• Mis-Incorporation of nucleotides during DNA replication o Adenine tautomer base pairs like Guanine • Inherent chemical instability of bases (NONEZYMATIC) o Deamination of CU; 100 bp/day o Hydrolysis of N-glycosidic bond – depurination; 5000 bp/day • Environmental Mutagens o Deaminating agents (CU; GX) ♣ Nitrous acid precursors • Sodium Nitrite (NaNO2), Sodium Nitrate (NaNO3), Nitrosamine • Ionizing Radiation o UV light can cause pyrimidine dimers

Answer 72

o Deaminating agents (CU; GX) ♣ Nitrous acid precursors • Sodium Nitrite (NaNO2), Sodium Nitrate (NaNO3), Nitrosamine o Alkylating agents (adding a methyl group) – methylated G binds with T ♣ Nitrogen mustards ♣ Aflatoxin ♣ Benzo(a)pyrene ♣ AAF (N-2-acetyl-2-aminofluorene) o Intercalating agents – lead to insertion or deletion of one or more base pairs, alter reading frames ♣ Frameshift mutations caused by flat aromatic molecules: • Ethidium bromide, acridines

Answer 73

1. Mismatch Repair: Mismatches 2. Base-Excision Repair: Remove abnormal/damaged bases 3. Nucleotide-Excision Repair: Remove large structural damages (thymine dimers) 4. Direct Repair: Systems that recognize and repair with specific damages Pyrimidine dimers (photolyase) 5. Recombinational Repair: Uses sister chromatid to repair damaged region

Answer 74

• Methylation of DNA strands can serve to distinguish parent (template) strands from newly synthesized strains o The methylation occurs at the N6 of adenines in (5’)GATC sequences ♣ This sequence is a palindrome because present in opposite orientations on the two strands o After a few minutes, the new strand is methylated and the two strands can no longer be distinguished

Answer 75

o MutS recognizes the mismatch o MutH recognizes the (5’)GATC o MutS and MutL form a complex at the mismatch o DNA is threaded through the complex such that the complex moves simultaneously in both directions along the DNA until it encounters a MutH protein bound at the hemimethylated GATC sequence o MutH cleaves the unmethylated strand on the 5’ side of the G in this sequences o A complex consisting of DNA helicase II, SSB, and one of several exonucleases then degrades the unmethylated DNA strand from that point toward the mismatch ♣ The exonuclease that is used depends on the location of the cleavage site relative to the mismatch o The resulting gap is filled in by DNA polymerase III, and the nick is sealed by DNA ligase (Not shown)

Answer 76

Nucleotide (BASE) Excision Repair on human DNA • Some proteins involved in humans excision repair identified in patients with xeroderma pigmentosum (XP) • Pathway: o An excinuclease binds to DNA at the site of a bulky lesion and cleaves the damaged DNA strand on either side of the lesion o The DNA segment –of 13 nucleotides or 29 nucleotides- is removed with the aid of a helicase o The gap is filled in by DNA polymerase o The remaining nick is filled in with DNA ligase

Answer 77

• Some forms of DNA damage can be enzymatically reversed – for example, the removal of alkyl groups at the O6 position of Guanine o Alkylation of guanine occurs from environmental mutagens and by cancer chemotherapeutic drugs o MGMT (O-6-methylguanine-DNA methyltransferase) is the enzyme that repairs O6 methyl guanine ♣ It works by direct removal of the alkyl group o This enzyme can interfere with some forms of chemotherapy that rely on DNA damage by alkylation Therefore, inhibition of MGMT can facilitate cancer chemotherapy

Answer 78

• Some lesions of DNA, such as double-stranded breaks, double-stranded cross-links, and lesions in single stranded regions, cannot be repaired using information from the complementary strand – Information must come from a separate homologous strand • These types of lesions result from ionizing radiation and oxidative reactions o Repair of these types of lesions is mediated by homologous genetic recombination called recombinational repair

Answer 79

• Xeroderma Pigmentosum (XP) – a rare skin disease o Individuals are extremely sensitive to UV light and develop skin cancer which metastasize - Patients often die before age 30 o XP results from a defect in the nucleotide excision repair mechanism for thymine dimers ♣ Recessive disease – fewer than 1,000 cases known worldwide ♣ Wide range of symptoms • Blindness and deafness • Blistering or freckling on minimum sun exposure • Developmental disabilities • Dwarfism and hypergonadism • Increased skin and eye cancers • Mental Retardation

Answer 80

• Werner’s Syndrome: premature aging (adult progeria) – due to defects in a DNA helicase • Bloom Syndrome: small body size, sun sensitivity, hypo and hyper-pigmented lesions, immunodeficiency, cancer, diabetes, and lung diseases – due to abolition of Ligase I activity needed to complete DNA repair • Fanconi’s anemia, Ataxia telangiectasia, and Gardner’s Syndrome o All believed to be due to defects in ligase activity • Breast Cancer: Some forms of breast cancer are due to defects in breast cancer susceptibility genes (BRCA Genes) o BRCA genes participate in DNA repair

Answer 81

• Proteins spontaneously fold based on their sequence of amino acids (primary structure) o most hydrophobic (apolar) amino acids within the core o most hydrophilic (polar) amino acids on the surface

Answer 82

• Secondary structures involve H-bonds between peptide bond carbonyl (C=O) groups and peptide bond amide hydrogens (>N-H) form first o -helices, parallel and anti-parallel -pleated sheets

Answer 83

• There are 20 different genetically coded amino acids found in proteins o Each aa (except proline) has a carboxyl group (-COOH), an amino group (-NH3), and a distinctive side change (R group) bonded to the -carbon atom

Answer 84

• If the -carbon of an amino acid is attached to four different chemical groups, then that carbon is chiral and the amino acid is optically active o Glycine is the only amino acid without optical activity because its -carbon is attached to two Hydrogen atoms o AAs (except glycine) exist in either of two mirror image forms (enantiomers) termed the D and L, which have identical chemical properties in all respects except their solutions cause polarized light to rotate in opposite directions ♣ All amino acids found in mammalian proteins are of the L-configuration

Answer 85

``` o Aliphatic amino acids possess side chains comprised of saturated carbon backbones – the subtle differences in the side chains allow for tight packing within the protein interior (7 –> GAVLIMP) – Grab All Vaginas Like Its My Pussy ♣ Glycine ♣ Alanine ♣ Valine ♣ Leucine ♣ Isoleucine ♣ Methionine ♣ Proline ```

Answer 86

o Aromatic amino acids possess side chains derived from either benzene (Phe and Tyr) or indole (Trp); each contains a methylene spacer (-CH2-) between the aromatic ring and the -carbon, this minimizes steric repulsion between the ring and the polypeptide chain backbone (3 –> PTT) ♣ Phenylalanine ♣ Tyrosine ♣ Tryptophan

Answer 87

``` o Uncharged polar amino acids have side chains that do not ionize at physiological pH (Met is sometimes classified in this group because of the slightly polar nature of its sulfur-containing side chain) (5 –> STAGC) Stop Touching All Girls C*nts ♣ Serine ♣ Threonine ♣ Asparagine ♣ Glutamine ♣ Cysteine ```

Answer 88

``` o Charged polar amino acids have side chains that can carry either a positive charge or a negative charge; although formally classified in other groups, the side chains of Tyr and Cys can ionize with pKa values close to physiological pH (5 –> LAHAG) Like All Hoes, Ashley Gags ♣ Lysine + ♣ Arginine + ♣ Histidine + ♣ Apartate + ♣ Glutamate + ```

Answer 89

• The pKa of a group is the pH at which it is 50% protonated and 50% unprotonated o At pH values lower than the pKa, there is a higher concentration of H+ in solution and >50% of the side chain group is protonated o At pH values higher than the pKa, the solution has fewers H+ and <50% of the group is protonated

Answer 90

• At some intermediate pH, the amino acid, peptide, or protein will have a net charge of zero; this pH is termed the isoelectric point, or pI

Answer 91

• Methionine and Cysteine, the only amino acids containing sulfur (S), have unique roles and sensitivities

Answer 92

o In newly synthesized (nascent) proteins, Met is always the first or N-terminal amino acid, but it also occurs at various positions throughout a polypeptide o Under some conditions, the S of Met is oxidized, impairing protein function

Answer 93

• The thiol or sulfhydroyl group of cysteine can spontaneously oxidize to form a disulfide linking two cysteine residues within a protein o Disulfide bonds form between specific cysteine residues because chain folding places the –SH groups in close proximity ♣ Disulfides rarely occur in intracellular proteins, but are common in extracellular proteins where their presence increases conformational stability of the structure ♣ Disulfide bonds are important in stabilizing the circulating peptide hormone insulin

Answer 94

• Metamorphic proteins exist in an ensemble of distinct structures of approximately equal energy that are in equilibrium (envision a thermodynamic folding funnel with two wells of similar depth)

Answer 95

• -Keratin – a primary component of hair and nails o Consists of two right-handed -helices that are intertwined in a left-handed supercoil called an -coiled coil ♣ Bonding between coils is both non-covalent (hydrophobic interactions, ionic bonds, H-bonds) and covalent (disulfide bonds between adjacent cysteine residues) ♣ Moderate number of disulfide bonds allow hair to stretch but then return to its original shape • A much higher number of disulfide cross-links makes nails (and horns, claws, and hooves) rigid ♣ Belongs to a large family of coiled coil proteins that also includes the intermediate filaments that provide internal scaffolding for cells and myosin and tropomysin, which are involved in muscle action

Answer 96

• Collagen – the most abundant protein in the body (20-25% of total protein) and exists as a superfamily of molecules whose type and organization are dictated by the structural roles each plays in different organs o Typical collagen molecule is a long, rigid structure in which three polypeptides are wound around each other in a rope-like triple helix o The individual collagen chains ( chains) are left-handed helices (NOT left-handed helices) ♣ 3 of them are wound around each other in a right-handed triple helix ♣ Multiple triple helices interdigitate to make collagen microfibrils

Answer 97

o Hydroxyproline and hydroxylysine are formed through post-translational hydroxylation reactions of proline and lysine and serve to stabilize the triple-helical structure o The enzymes that catalyze the hydroxylation reactions require ascorbate (Vit C), a deficiency of which results in scurvy ♣ The symptoms of scurvy (e.g., bleeding gums) are due in part to the decreased tensile strength of collagen

Answer 98

o Synthesis, post-translational modification, and triple helix assembly occur inside the cell, then the resulting procollagen molecule is secreted into the extracellular matrix where specific peptidases cleave N- and C- terminal propeptides ♣ Once removed, the mature collagen triple helix (tropocollagen) self-assembles into fibrils, with subsequent cross-linking to form mature collagen fibers

Answer 99

• Elastin – A connective tissue protein with rubber-like properties that is found in the lings, large arterial walls, and elastic ligaments o Comprised predominately of small, non-polar amino acids (glycine, alanine, valine) and is also rich in proline and lysine – contains few few hydroxyl derivatives o Elastin is synthesized from a precursor, tropoelastin, which is secreted into the extracellular space where it interacts with specific glycoprotein microfibrils ♣ Some lysyl side chains are oxidized to form allysine residues which cross-link with lysine amino groups of neighboring polypeptides to produce elastin

Answer 100

o Role of -antitrypsin (1-AT) in elastin degredation ♣ 1-AT is a small protein that inhibits a number of proteolytic enymes in bodily fluids ♣ Crucial function of 1-AT is to inhibit elastase, which is released by neutrophils and functions to degrade elastin • Elastase destroys the alveolar epithelium if unregulated by 1-AT • There are both genetic and environmental causes of insufficient functional 1-AT and both can lead to emphysema o Genetic: 1-AT deficiency is predominantly due to the inheritance of two prominent mutant alleles, Z and S ♣ The Z allele causes a more severe deficiency and is due to an E342K substitution that causes 1-AT to be retained inside the cell o Environmental: 1-AT contains a methionine residue that is essential for binding to target proteins (i.e., elastase) ♣ Elements in cigarette smoke cause the oxidation of the Met, which renders 1-AT unable to bind and inactivate elastase ♣ Heavy smokers can develop emphysema even if they have no mutant 1-AT alleles

Answer 101

• Adult hemoglobin (Hb) is a tetramer of two and two subunits o Each subunit contains a heme cofactor to which O2 can be reversibly bound on the Fe2+ atom o Hemoglobins of other subunit compositions occur developmentally ♣ Most important: Fetal Hb (HbF): comprised of 22 • O2 binding to Hb is cooperative such that binding of each O2 molecule to the tetramer increases the affinity with which the next O2 binds o Similarly, dissociation of each O2 decreases affinity for those remaining so they dissociate more easily o This cooperativity results in a sigmoidal O2 binding curve and that greatly increases the ability of Hb to release O2 in tissues

Answer 102

o (1) High concentrations of H+ (lower pH) o (2) CO2 o (3) 2,3-diphosphoglycerate (DPG) o (4) Increasing Temperature (Fever)

Answer 103

• Sickle Cell Disease is an inherited mutation of the - globin gene causing a change from Glu to Val at position 6 o Presence of Val at position 6 allows deoxyHbS to polymerize into microfibrils that distort RBC shape and cause both hemolysis and vaso-occlusive disease

Answer 104

• On both chromosome 16 (alpha-globin family) and chromosome 11 (beta-globin family), the genes are expressed developmentally from 5’ 3’ • Expression of embryonic alpha-globin (zeta) occurs only briefly, and then the adult form (alpha) dominates through fetal growth and into adulthood • Each chromosome 16 has 2 alpha-globin genes (a person has 4 total); each is active and codes about ¼ of expressed alpha-globin o Consequently, a defect in 1 or 2 alpha-gene clusters is not too serious • Expression of embryonic beta-globin (epsilon) is also brief but followed by a fetal form (gamma) that does not disappear until after birth o HbF is clinically important • There are two forms of adult Hb: HbA and HbA2 o HbA2 usually accounts for only a few present of adult Hb • Each chromosome 11 has only one functional beta-globin gene o Beta-globin gene defects are more likely to have clinical consequences

Answer 105

• Hemoglobin is a specialized protein designed to transport oxygen from the lungs to the peripheral tissues where oxygen tension is low o Metabolism in the peripheral tissues generates CO2 and H+ that are transported back to the lungs, in part by hemoglobin

Answer 106

• O2 has very low solubility in plasma o >98% of the O2 that reaches tissues is carried in RBCs bound to Hb o Situation is different for CO2 ♣ RBCs contain carbonic anhydrase, which catalyzes the rapid reversible hydration of CO2 to carbonic acid (H2CO3) • H2CO3 then rapidly and spontaneously dissociates to bicarbonate (HCO3-) and a H+ • CO2 and HCO3- are soluble in plasma and RBC cytosol and most of the CO2 made in tissues returns to the lungs as those species o About 14% of the CO2 made is carried bound to Hb

Answer 107

• Hemoglobin is a heterotetrameric protein having the subunit composition ()2 o The alpha and beta subunits have similar sequences and tertiary structures o Both subunits are evolutionarily related to myoglobin, a monomeric protein abundant in muscle that is designed to store O2 ♣ Both proteins contain a Fe2+-protoporphryin IX prosthetic group that is responsible for binding O2 • Note: Fe2+ is the ferrous form of iron that is capable of binding O2 • Fe3+ is the ferric form that cannot bind O2, and is present in an inactive form of hemoglobin called methemoglobin

Answer 108

• The partial pressure of dissolved oxygen in aqueous solution is proportional to the partial pressure in the gas phase o Myoglobin gives a normal binding curve, which is hyperbolic in shape o Hemoglobin shows sigmoidal cooperative binding of the oxygen that is a direct result of its more complex subunit structure

Answer 109

• Without O2 bound, the heme Fe2+is pulled away from the plane of the porphyrin ring by a His residue of the polypeptide chain o When O2 binds, it pulls the Fe2+ back into the plane of the ring and that moves the His residue and its whole section of the polypeptide chain o That in turn causes the Hb subunits to shift relative to one another to an arrangement that favors the R-conformation

Answer 110

o The reciprocal relationship between O2 and H+ is termed the Bohr effect, or isohydric shift o Changes in H+ binding result from a shift in the pKa of specific residues (mostly histidines) due to charges microenvironment effects trigged by conformational changes in the hemoglobin molecule

Answer 111

• DPG is a negative allosteric effector of O2 binding o DPG binds to a specific site in a cleft between the beta subunits ♣ Binding stabilized mostly by ionic interactions o Special regulatory mechanisms exist in RBCs to control the concentration of 2,3 DPG in order to fine tune the affinity of hemoglobin for O2 in response to changes in metabolism and environment ♣ This obviates the need to induce the synthesis of different hemoglobin isoforms of altered O2 affinities (which is not possible for human RBCs since they lose the capacity to synthesize protein during their terminal differentiation)

Answer 112

1. Messenger RNAs: encode amino acid sequences of the polypeptides found in the cell (5% of total RNA, most complex) 2. Transfer RNAs: match specific amino acids to triplet codons in mRNA during protein syn (~15% of total RNA (and snRNA in eukaryotes) 3. Ribosomal RNAs: the constituents and catalytic appropriate amino acids (~80% of total RNA, least complex)

Answer 113

o MicroRNA: appears to regulate the expression of genes, possibly via binding to specific nucleotide sequences o Other functions: Ribonucleic acids act as genomic material in viruses

Answer 114

• Ribonucleic acids are synthesized in cells using DNA as a template in transcription o Transcription is tightly regulated in order to control the concentration of each protein • Being mainly single-stranded, many RNA molecules can fold into compact structures with specific functions o Some RNA molecules can act as catalysts (ribozymes), often using metal ions as cofactors • Most Eukaryotic ribonucleic acids are processed after synthesis o Elimination of introns; joining of exons o Poly-adenylation of the 3’ end o Capping the 5’ end

Answer 115

• Basic Reaction: o Nucleoside triphosphates added to the 3’ end of the growing RNA strand o Chemically the RNA polymerization reaction is very similar to DNA synthesis, occurring in the 5’ to 3’ direction o The 3’ hydroxyl group of the existing RNA chain undergoes a nucleophilic attack on the alpha-phosphate of the incoming nucleotide o The growing chain is complementary to the template strand in DNA o The synthesis is catalyzed by an enzyme (RNA polymerase) ♣ Two Mg2+ ions are used as cofactors among many Asp residues

Answer 116

• In prokaryotes, a single RNA polymerase synthesizes mRNA, rRNA, and tRNA • Prokaryotic RNA polymerase is a multisubunit enzyme o RNA polymerase holoenzyme has five core subunits of 2’ plus a sixth, o The core enzyme is responsible for polymerization but it lacks specificity and cannot recognize the promoter ♣ The “sigma” factor allows the holoenzyme to recognize promoter regions on the DNA o RNA pol lacks 3’ 5’ exonuclease, so it has a high error rate of 1/104-1/105 ♣ You don’t have to worry about inheriting them because they are largely unstable, so it doesn’t matter o RNA binds to promoter regions to initiate transcription

Answer 117

* Promoter: site for binding RNA polymerase * Operator: binding sites for repressor of activator * Structural gene: often times many related genes transcribed as a single unit * Together they are called an operon

Answer 118

o (1) Binding phase – the initial interaction of the RNA polymerase with the promoter leads to formation of a closed complex, in which the promoter DNA is stable bound but not unwound ♣ The sigma subunit helps provide specificity ♣ A 12 to 15 bp region of DNA is then unwound to form an open complex o (2) Initiation phase – encompasses transcription initiation and promoter clearance

Answer 119

• Elongation – once commenced, the sigma subunit is released and it replaced by the protein NusA o The polymerase leaves the promoter and becomes committed to elongation of the RNA

Answer 120

• Termination – once transcription is complete, the RNA is released, the NusA protein dissociates, and the RNA polymerase dissociates from the DNA

Answer 121

• DNA Template Strand: serves as template for RNA polymerase

Answer 122

• DNA Coding Strand: the non-template strand; has the same sequence as the RNA transcript

Answer 123

The Sigma Subunit of the Holoenzyme Enables RNA Polymerase to Recognize Promoters – Not All Promoters Are Created Equal • Different sigma factors recognize different promoters • Sigma increases specificity of RNAP, but decreases affinity • Strong promoters cause frequent initiation and tend to conform closely to the consensus • Different sigma factors lead to differential gene expression: Change in sigma utilization can be used to regulate developmental changes in gene expression

Answer 124

• Elongation proceeds in the 5’ to 3’ direction partially through the energy released by cleavage of the phosphate bond in the incoming ribonucleotide, and by subsequent hydrolysis of pyrophosphate to inorganic phosphate o The sigma factor dissociates from the holoenzyme complex immediately after elongation begins • Multiple RNA polymerase complexes load onto the promoter region in sequential fashion, allowing the gene to be transcribed continuously o The rate of elongation by E. coli polymerase is about 30 nt/second o Elongation is extremely processive ♣ Once sigma dissociates, a single RNAP can synthesize thousands of nucleotides before it dissociates

Answer 125

• A transcription bubble forms during the process of elongation o Negative superhelicity in the double-stranded DNA facilitates the melting of the two strands of DNA and the formation of this bubble o The RNA transcript is only base paired with the DNA template in this region, leaving a growing chain behind as the DNA helix reforms behind the elongation complex

Answer 126

• Bacteria termination of transcription occurs by protein-dependent or protein-independent mechanisms, both of which rely on a hairpin loop structure

Answer 127

♣ Active signals for termination lie in nascent RNA chain – information is in the structure • Palindromic GC-rich region followed by AT o The hairpin is formed at the palindromic sequence, reducing the length of the RNA-DNA hybrid • RNAP pauses at GC • dA-rU form weak hybrid A-T, therefore DNA duplex reforms • Core RNAP has less affinity for dsDNA than ssRNA therefore complex dissociates

Answer 128

♣ Due to the interaction of a terminator protein (Rho) with the RNA polymerase elongation complex as it pauses at a hairpin loop in the nascent transcript • The Rho protein is an ATP-dependent RNA-DNA helicase that disrupts the RNA-DNA hybrid, leading to disassembly of the elongation process o Rho binds to rut site, a common CA-rich sequence (Rho utilization element) o Rho processes in an ATP dependent manner until termination site is reached

Answer 129

o (1) RNA polymerase I – Synthesizes pre-ribosomal RNA ♣ Precursors for 28S, 18S, and 5.8 rRNAs o (2) RNA polymerase II – responsible for synthesis of mRNA ♣ Very fast (500-1000 nucleotides/sec) ♣ Specifically inhibited by mushroom toxin -amanitin ♣ Can recognize thousands of promoters o (3) RNA polymerase III – makes tRNAs and some small RNA products o Mitochondria have their own RNA polymerase

Answer 130

* Rifampicin: Inhibits bacterial RNAPS, important in treatment of tuberculosis * -amanitin: Mushroom toxin, potent inhibitor of RNA polymerase II * Actinomycin D: Intercalates between two G-C base pairs in DNA * Daunorubicin: Intercalates between base pairs * Cordycepin: Chain terminator that lacks 3’ OH

Answer 131

o Enhances stability: protection from nucleases o Enhances translation efficiency o Modifications: ♣ 7-methyl-guanosine added by guanylyltransferase ♣ methyl groups sometimes added to riboses by methyl transferases using S-Adenosyl Methionine (SAM) as a donor o Note: only mRNA is modified with a cap, not tRNA or rRNA

Answer 132

o Important for mRNA stability o Helps in translation o Added downstream of polyadenylation signal in 3’ UTR in a context-dependent manner ♣ 3’ UTR contains cleavage and polyadenylation signals o Nascent mRNA is considerably longer than length of RNA preceding poly A site

Answer 133

o Genes of nucleated cells are fragmented o Some genes contain more than 90% intronic sequences o Splicing usually occurs in order from the 5’ to 3’ end of the mRNA precursor, but not always

Answer 134

o CFTR encodes the chloride channel which when mutated causes cystic fibrosis (CF) ♣ CFTR gene is composed of 27 exons covering over 189,000 base pairs of DNA ♣ The processed mRNA (introns removed) is approximately 6,000 nucleotides • only 3% of the gene is finally seen in the mRNA that is translated into protein

Answer 135

• The intron is spliced out in the form of a lariat o Steps: 2 transesterifications ♣ (1) 2’ OH attacks 5’ splice site ♣ (2) Newly formed 3’ OH attacks 3’ splice and exons ♣ (3) Lariat form of intron with 2’-5’ phosphodiester bond formed

Answer 136

Disease of splicing • Aortic aneurysm – weakened and bulging area in the aorta • Arachnodactyly – fingers are abnormally long and slender in comparson to the palm of the hand • Dural ectasia – the brain and the spinal cord are surrounded by fluid contained by a embrane called the dura, which is primarily connective tissue o The enlargement of this membrane is reffered to as “dural ectasia” • Proposed Mechanism: FBN1 mutations resulted in the production of abnormal fibrillin protein that, when incorporated into microfibrils along with normal fibrillin, resulted in structurally inferior connective tissue o This is consistent with aortic aneurism, a dural ectasia, but not arachnodactyly • Revised Mechanism: Fibrillin was homologous with the family of latent transforming growth factor (TGF-) binding proteins (LTBPs), which serve to hold TGF- in an inactive complex in various tissues, including the extracellular matrix o Researchers showed that fibrillin can bind TFG- o Hypothesis: abnormal fibrillin, or reduced levels of fibrillin, in connective tissue might result in an excess of active TGF- • New therapy? o It has been found that blocking TGF- with neutralizing antibodies leads to the normalization of lung development in affected mice

Answer 137

• Systemic lupus erythematosus: an autoimmune disease in which serum antibodies to U1 are formed, blocking proper splicing from occurring o Other connective diseases also exhibit serum antibodies to U1 • Globinopathies: many diseases of globin are caused by splicing defects • Spinal Muscular Atrophy (SMA): a mutation in exon 7 of the SMN-1 gene leads to decreased survival of motor neurons • Becker Muscular Dystrophy (BMD): a mutation in exon 27 of the dystrophin gene affects the dystrophin protein and muscle fiber formation • Frontotemporal Dementia with Parkinsonism (FTDP-17): a mutation in exon 10 of the Tau microtubule associated protein gene which promotes a more stable protein o Mutant Tau proteins form abnormal filamentous structures in the brains of FTDP-17 patients

Answer 138

* Functionally related genes in prokaryotes are organized into operons * The genes of an operon are coordinately controlled: an operon is a coordinated unit of gene expression

Answer 139

o A regulatory gene: example, LacI gene codes for repressor protein o A promoter: nucleotide sequence that serves as a recognition motif for RNAP binding o An operator: nucleotide sequence that binds repressor protein

Answer 140

• Inducers bind to the repressor to weaken affinity for operator o In the lac operon, the inducers are allolactose (side product of B-Galactosidase) and IPTG (gratuitous inducers)

Answer 141

• Lac Repressor binds tightly to the operator as a tetramer in a tethered dimer configuration: searches DNA in one-dimension to find operator o It is constitutively expressed from its own promoter

Answer 142

• Common patterns of regulation of transcription initiation o (1) Repressor binds to the operator in the absence of the molecular signal ♣ The external signal causes dissociation of the repressor to permit transcription o (2) Repressor binds in the presence of the signal ♣ The repressor dissociates and transcription continues when the signal is removed

Answer 143

• Common patterns of regulation of transcription initiation o (1) Activator binds in the absence of the molecular signal and transcription proceeds ♣ When the signal is added, the activator dissociates and transcription is inhibited o (2) Activator binds in the presence of the signal ♣ It dissociates only when the signal is removed

Answer 144

• Glucose is used up first in E. coli, then other carbon sources are utilized o When glucose goes down, cAMP goes up o CAMP binds to CAP (Catabolite Activator Proteins aka CRP) o cAMP-CAP binds to promoter, binding facilitates RNAP binding to promoter ♣ DNA binding properties of CAP • CAP binding site shows two-fold rotational symmetry • CAP binding creates additional sites for RNAP interaction • So glucose is absent, the lac operon transcription is stimulated by CRP-cAMP • When lactose is absent ~no transcription o Whether [glucose] is high or low, if lactose is absent, the repressor stays bound ♣ No transcription even when CPR-cAMP binds o When [glucose] is high, cAMP is low, and lactose is present, there will be LOW levels of lac operon expression o When [glucose] is low, cAMP is high, and lactose is present, there will be HIGH levels of lac operon expression

Answer 145

1. Lactose must be present to form allolactose to bind to repressor and cause it to dissociate from operator (reducing repression) 2. [Glucose] must be low so that cAMP can increase, bind to CRP, and the complex can bind near the promoter • When lactose is low, repressor is bound inhibition • When lactose is high, repressor dissociates permitting transcriptionally • When glucose is high, CRP is not bound transcription is dampened • When glucose is low, cAMP is high and CRP is bound activation

Answer 146

``` • Differences in the strategies for gene regulation in prokaryotes and eukaryotes: o Prokaryotes ♣ Polycistronic mRNAs ♣ Coupled Transcription/Translation o Eukaryotes ♣ Monocistronic mRNAs ♣ Uncouple Transciption/Translation ```

Answer 147

• Transcription factors may be useful drug targets: o Human cancers are dependent on inappropriate activity of oncogenic transcription factors o New strategies of targeting transcription factors include disrupting protein-protein interactions and blocking binding at the epigenetic level by modulating chromatin accessibility

Answer 148

o (1) Transcription activators (enhancer binding proteins) ♣ Proteins that bind to upstream activator sequences (UAS) that are often palindromic o (2) Coactivators ♣ Act indirectly (with other proteins, not with DNA) o (3) Basal Transcription Factors o (4) Architectural regulators to facilitate DNA looping o (5) Chromatin modification/remodeling proteins

Answer 149

o (1) Pol II is recruited to the DNA by TFs o (2) The transcription bubble forms o (3) The CTD is phosphorylated during initiation ♣ The polymerase escapes the promoter o (4) Transcription elongation is aided by elongation factors after TFIIE and TFIIH dissociate o (5) Elongation factors dissociate ♣ The Carboxyl Terminal Domain (CTD) is dephosphorylated as transcription terminates, a process facilitated by transcription termination factors

Answer 150

• RNA polymerase II and its associated basal transcription factors form a preinitation complex (PIC) at the TATA box, a process that is facilitated by transcriptional activators acting through co-activators (Mediator and/or TFIID) o Composite promoter with typical sequence elements and protein complexes found in eukaryotes ♣ The carboxyl terminal domain (CTD) of pol II is an important point of interaction with Mediator and other protein complexes ♣ Transcriptional activators bind to enhancer sequences (UAS) and coactivators ♣ HMG proteins are architectural regulators that facilitate DNA looping to allow machinery at a distance access to transcriptional start site o Transcriptional repressors function by many mechanisms, including binding to DNA to displace activator proteins, or interacting with other protein complexes to disrupt initiation

Answer 151

• A pentose sugar may be attached in an N-glycosidic linkage to the purine or pyrimidine base, in which case the compound is called a nucleoside

Answer 152

o A phosphate may be esterified to one of the OH groups, typically attached to the C-5’ of the nucleoside pentose, in which case the compound is called a nucleotide

Answer 153

• Uric acid is the least soluble purine base and exists at or near saturation levels in most individuals • (1) Primary gout is characterized by hyperuricemia due to a variety of inherited metabolic abnormalities • (2) Secondary gout is characterized by excessive uric acid production due to a coexisting acquired condition ♣ Long term treatment of gout involves administration of allopurinol, which inhibits the rate of uric acid production

Answer 154

Disease of nucleotide metabolism ♣ Xanthine is the second most insoluble purine base and can also precipitate when overproduced, a condition known as xanthinuria ♣ Xanthine can results from a variety of abnormalities, the most common of which is a deficiency of xanthine dehydrogenase, estimated to occur in 1 in 6,000-60,000 individuals

Answer 155

♣ Adenosine deaminase deficiency • A deficiency of adenosine deaminase (ADA) is associated with a severe combined immunodeficiency involving T-cell and B-cell dysfunction • The mechanism is not completely understood, but it is likely due to accumulation of dATP, particularly in lymphocytes o dATP is an effective inhibitor of ribonucleotide reductase and, consequently, of DNA synthesis and cell division ♣ Purine nucleoside phosphorylase (PNP) deficiency • A deficiency of purine nucleoside phosphorylase (PNP) is associated with impairment of T cell function o PNP-deficient individuals under excrete uric acid and over excrete PN subtrates o Accumulation of dGTP in T-cells with resulting inhibition of ribonucleotide reductasae and DNA replication has been proposed as an explanation for immune dysfunction in this disorder

Answer 156

• Lesch-Nyhan Syndrome – an inherited disorder of salvage synthesis o HPRT is lacking or is present at very low levels (<1% of normal) in these individuals ♣ The HPRT locus is located on the X chromosome, but because this disorder is very debilitating, affected individuals do not reproduce – therefore, males are almost exclusively affected o This syndrome is associated with aggressive behavior, self-mutilation, and mental retardation o Because of the disruption in salvage synthesis, these individuals also display high levels of uric acid and gout o High levels of PRPP and increased rates of de novo synthesis are observed because reduced synthesis of purine nucleotides by the salvage pathway results in inadequate feedback inhibition of PRPP synthetase and ATase ♣ Increased levels of PRPP lead to an increase in de novo synthesis and a resulting overproduction of uric acid o Identified mutations in the HPRT gene have variable effects on enzyme activity (0.1 to ~10% residual) ♣ Severity of enzyme deficiency is correlated with the degree of neurological complications o Although administration of allopurinol to Lesch-Nyhan patients relieves the complication of gout, there is no known treatment for the neurological problems ♣ The brain has 10-20 times the HPRT enzymatic activity found in other tissues, thus it is possible that high levels of purine catabolites are toxic to the developing brain, or perhaps the lack of HPRT leads to an imbalance in purine nucleotides at critical times during development

Answer 157

• Orotic aciduria-UMP synthase deficiency o Characterized by an excessive amount of orotic acid in the urine and accompanying metaloblastic anemia ♣ An apparent defect in the UMP synthase gene renders both OPRT and OMP decarboxylase dysfunctional, although it is the deficiency of the former which would result in the accumulation of orotic acid ♣ Lethal, unless treated by pyrimidine replacement, i.e., uridine • Uracil is ineffective as therapy • Pyrimidine 5’-nucleotidase deficiency o Characterized by vastly increased levels of erythrocyte pyrimidine ribonucleotides (particularly cytidine nucleotides) and an accompanying hereditary anemia • Dihydropyrimidine dehydrogenase (also called dihydrouracil dehydrogenase) deficiency o Characterized by elevated levels of uracil and thymine in body fluids o Convulsive disorder characterized by microencephaly • Dihydropyrimidinase deficiency o Characterized by elevated levels of dihydropyrimidines in body fluids o Variable neurological symptoms

Answer 158

Amino Acid, tRNA, mRNA, Amino Acid tRNA synthetases, Ribosomes, Initiation, Elongation, and Termination Factors, ATP and GTP

Answer 159

• Amino acids: All amino acids in a protein o Must be present at the time of protein synthesis o Essential amino acids

Answer 160

``` • tRNAs: a.k.a. adaptor molecules with highly ordered structure o At least 1 per amino acid o Actually >50 in humans o Contain two important sites ♣ (1) amino acid attachment site ♣ (2) anticodon sequence ```

Answer 161

• mRNAs: Translated in the 5’ 3’ direction o In E. coli, transcription and translation are closely coupled regulation of protein expression is largely controlled at transcriptional level

Answer 162

Amino Acid tRNA synthetases: Family of enzymes that attach amino acids to corresponding tRNAs

Answer 163

• Ribosomes: Ribonucleoprotein complexes; consist of two subunits: o Prok – 30S + 50S = 70S o Euk – 40S + 60S = 80S

Answer 164

• ATP and GTP: energy sources o Cleavage of 4 high energy bonds (ATP and GTP) are required for the addition of one amino acid o Initiation and termination require additional ATP and GTP

Answer 165

Ribosomes: • Overall, Euks and Proks are very similar • Both have two subunits with mRNA running between • In eukaryotes, larger (80S), more complex, contain > 80 proteins • Chloroplasts and mitochondria have ribosomes simpler than those in bacteria • Ribosomal subunits are identified by their S (Svedberg unit) values, sedimentation coefficients that refer to their rate of sedimentation in a centrifuge

Answer 166

The genetic code: • There are 20 common, genetically encoded amino acids • The genetic code for proteins consists of triplets of nucleotides o A four-letter code in groups of two is insufficient (16)

Answer 167

o There are 64 different combinations of 3 base codons (43) o 61 of these code for the 20 amino acids (then there are 3 stop codons) 5’ … CCGUAGU AUG CGA GGU GGA CUA UAA GGAUAAC … 3’

Answer 168

o It is specific – a specific codon always codes for the same amino acid o It is essentially universal (mito is slightly different) o It is redundant (or degenerate) – a given amino acid may have more than one codon (synonyms, mostly differ in 3rd position) o It is non-overlapping and comma-less ♣ The code is read from a fixed starting point as a continuous sequence of bases, three at a time ♣ Frameshift mutations occur if the reading frame is altered

Answer 169

• The code is written in the 5’ 3’ direction • Third base is less important in binding to tRNA • First codon establishes the reading frame o If the reading frame is thrown off by a base or two, all subsequent codons are out of order • 61/64 codons code for amino acids • Three are termination codons: UAA, UGA, UAG • AUG = initiation codon (as well as Met codon)

Answer 170

UAG, UGA, UAA do not code for amino acids (no tRNAs)

Answer 171

o Silent mutations – codon base is changed to codon that codes for same a.a. UCA = Serine UCU = Serine

Answer 172

o Missense mutations – codon is changed to codon that codes for a different a.a. UCA = Serine CCA = Proline

Answer 173

o Nonsense mutations – codon is changed to become a termination factor UCA = Serine UAA = Terminate

Answer 174

* Most amino acids have more than one codon (Arg, Leu, and Ser have 6) * Only Met and Trp have a single codon * Some codons are less subjective to causing a mutation in an amino acid sequence because of degeneracy or because of the abundance of such tRNAs

Answer 175

• tRNA anticodons recognize codons o Binding follows the rules of complementary base pairing o mRNA is read 5’ 3’ by a flipped anticodon

Answer 176

Molecular Recognition of Codons in mRNA by tRNA • tRNA anticodons recognize codons o Binding follows the rules of complementary base pairing, mRNA is read 5’ 3’ by a flipped anticodon • Wobble Hypothesis o tRNAs can recognize more than one codon for a specific amino acid due to “wobble” at the 5’ end of the tRNA anticodon, forming non-traditional base pairs o The result is that there need not be 61 tRNAs to read 51 codons • Suppressor tRNAs o Nonsense suppressors are tRNAs whose anticodons have been mutated such that they incorporate an amino acid at termination codons (i.e., they suppress the normal termination of a protein)

Answer 177

complementary

Answer 178

anticodon; | hydrogen bonding

Answer 179

Antiparallel

Answer 180

• Wobble Hypothesis helps to explain degeneracy of the code: o Degenerate code allows certain mutations to still code for the same amino acid ♣ “Silent” mutations – different nucleotide in DNA but same amino acid in protein • Mutation in first base of a codon usually produces a conservative substitution o Example: GUU Val but AUU Leu

Answer 181

1. Activation of amino acids a. tRNA is aminoacylated 2. Initation of translation a. mRNA and aminoacylated tRNA bind to ribosome 3. Elongation a. Cycles of aminoacyl-tRNA binding and peptide bond formation until a STOP codon is reached 4. Termination and ribosome cycling a. mRNA and protein dissociate, ribosome recycled 5. Folding and post-translational rocessing a. Catalyzed by a variety of enzymes

Answer 182

* Charging tRNA with amino acids * Requires Aminoacyl-tRNA Synthetase, ATP, Mg2+ Amino Acid + tRNA + ATP aminoacyl-tRNA + AMP + PPi

Answer 183

o Each enzyme binds a specific amino acid and the matching tRNA o Most cells contain 20 different aminoacyl-tRNA synthetases, one for each amino acid o Some cells contain less than 20 synthetases; in this case, one amino acid is converted to another after charging the tRNA o Aminoacyl-tRNA synthetases must be specific for both amino acid and tRNA ♣ Matching each amino acid with correct tRNA can be viewed as the “second genetic code” ♣ The “code” is in molecular recognition of a specific tRNA molecule by a specific synthetase o Only a few nucleotides in tRNA confer the binding specificity: Anticodon region

Answer 184

• Prokaryotes often produce polycistronic mRNAs o The RNA component of the ribosome (16S rRNA) is involved in the correct positioning of the ribosome at the translation start site o The Shine-Dalgarno sequence pairs with the 16S rRNA – positions ribosome in the right place to start translation • Eukaryotes produce monocistronic mRNAs • Initiation Codon: recognized by special initiator tRNA o Facilitated by: ♣ IF-2 in E. coli ♣ eIF-2 in humans o In bacteria and mitochondria, initiator tRNA carries N-formyl methionine o In humans there is no formylation • fMet or Met is the first amino acid in a peptide o First codon is AUG o All organisms have two tRNAs for Met ♣ Even eukaryotes, whose proteins begin with Met, not fMet, still use a special tRNA for initiaton

Answer 185

* Peptidyl transferase catalyzes peptide bond formation | * Ribosome moves one codon using energy from hydrolysis of GTP bound to EF-G

Answer 186

• General idea: because of this coupling, regulation is largely controlled at the level of transcription

Answer 187

• Generally conserved, but some steps are more intricately regulated o Ribosomes ♣ Eukaryotic: 80S, can be dissociated into: • 60S large subunit (5S, 28S RNAs, and unique 5.8S) • 40S small subunit (18S RNA) ♣ Prokaryotic: 70S, can be dissociated into • 50S large subunit (5S, 28S RNAs) • 30S small subunit (16S RNAs)

Answer 188

♣ In eukaryotes: the initiator amino acid is methionine and it is encoded by MET-tRNAi ♣ In prokaryotes: proteins start with N-formylmethionine

Answer 189

♣ The selection of AUG (Met codon) to initiate translation differs in prokaryotes and eukaryotes • In prokaryotes, initiation occurs at an AUG adjacent to a purine rich sequence called the Shine-Dalgarno sequence, which base pairs with the 16S RNA • In eukaryotes, the AUG nearest the 5’ end of the mRNA is generally utilized as the initiator in MET o The 40S ribosome loads at the 5’ end of the message and ‘scans’ o This stepwise movement requires energy (ATP) and helicase

Answer 190

1. eIF1A and eIF3 help keep 60S and 40S dissociated, eIF1 helps to bind ternary complex 2. Ternary complex (eIF2-GTP:met-RNA) binds to 40S along with eIF5B-GTP to create the 43S preinitiation complex 3. 43S complex binds eIF4F bound CAP of mRNA to form 48S complex 4. 48S complex unwinds any helix structure and sans to find first AUG (eIF4 has helicase activity) 5. Once AUG found, 60S ribosomal subunit binds and releases any of the initiation factors, and the 80S initiation complex is formed

Answer 191

• Stimulate: o eIF4E is a subunit of eIF4, and it recognizes and binds to the 5’ cap of the mRNA and promotes binding of mRNA to the 40S subunit o eIF4E needs to be phosphorylated to be active o Factors that stimulate translation through eIF-4E proteins: ♣ Tumor Necrosis Facter alpha ♣ Insulin ♣ Epidermal Growth Factor ♣ Platelet Derived Growth Factor ♣ Nerve Growth Factor ♣ Interleukin 1 • Inhibit: o Phosphorylations of eIF2 blocks formation of 43S preinitiation complex o Polio virus causes the proteolysis of cap binding protein (eIF4E) allowing its own uncapped mRNA to be translated

Answer 192

• Diptheria Toxin: blocks eukaryotic translation by inhibiting translocation; ADP ribosylation of EF2-translocase (eukaryotic version of EF-G) o Diptheria toxin blocks eukaryotic translation by inhibiting translocation o ADP ribosylation of EF2-translocase inactivation of all of the host cell EF-2 molecules causes death of the cell o Attachment of the ADP ribosyl group occurs at an unusual derivative of histidine called diphthamide

Answer 193

• Proteolytic Processing: occurs in the ER, Golgi apparatus, secretory cesicles; i.e., zymogens, inactive precursors of secreted enzymes (preforms), include many digestive proteases o Familial Hyperproinsulinemia: abnormally processed insulin due to deficiency in enzymes that process insulin ♣ Afflicted individuals are normal in glucose metabolism

Answer 194

• Phosphorylation: most often hydroxyl groups of serines or threonines, sometimes tyrosine, by protein kinases; can often increase or decrease the function of a protein or allow protein:protein interaction

Answer 195

• Glycosylation: carbohydrate chains attached to hydroxyl groups of serines or threonines (O-linked) or asparagine (N-linked); occurs in stepwise manner in ER and Golgi; often found in proteins destined for secretion or part of the plasma membrane o I-Cell Disease: Lack glycosyl transferase therefore cannot properly target lysosomal enzymes: patients have abnormally high levels of lysosomal enzymes in their sera and other bodily fluids ♣ Disease is characterized by psychomotor retardation, coarse facial features, and restricted joint movement ♣ Usually death occurs by age 8

Answer 196

• Hydroxylation: i.e., proline and lysine residues of collagen o Scurvy causes decrease in hydroxyproline synthesis o Type IV Ehlers-Danlos syndrome is deficient in lysyl hydroxylase

Answer 197

• Ubiquitination: protein trafficking and protein turnover, i.e., degradation o In ekaryotes, proteins are linked to the protein ubiquitin via activating enzyme E1, conjugating enzyme E2, and ligating enzyme E3 o Ubiquinated proteins are cleaved by the 26 protein complex o Ubiquitin is very highly conserved

Answer 198

• Protein Targeting: Proteins move from the site of synthesis to: o Exit cell o Become part of the membrane o Enter a subcellular compartment, etc ♣ Most have a signal sequence at or near N-terminus

Answer 199

• Lipids, along with amino acids, nucleotides, and carbohydrates, are the building blocks from which most cellular macromolecules are constructed • “Lipids” – a chemically diverse group of compounds defined by their insolubility in water (i.e., are hydrophobic molecules) o Their functions are also diverse and include storage form of energy, structural elements of membranes, hydrophobic anchors for proteins, cellular signaling hormones, lipid-soluble vitamins, and emulsifying agents in the digestive tract

Answer 200

* Each organelle has a unique lipid and protein composition * “Fluid Mosaic” lipids and proteins move laterally in the plane of the membrane * Membranes are not uniform (micro domains or “rafts”)

Answer 201

o Cardiolipin: important component of the inner mitochondrial membrane ♣ Required for function of enzymes involved in mitochondrial energy metabolism

Answer 202

o Barth Syndrome: Deficient cardiolipin synthesis ♣ Infantile death ♣ Cardiomyopathy ♣ Decreased mitochondrial ATP production

Answer 203

• Fatty acids are carboxylic acids composed of a hydrocarbon chain with a carboxyl group at one end and a methyl group at the other end (called the -carbon) o Most lipids contain fatty acids or are derived from fatty acids ♣ Exception is cholesterol which is synthesized from acetate • Sterate 18:0 --- Saturated/unbranched • Oleate 18:19 --- Unsaturated/branched

Answer 204

• Glycerophospholipids contain a glycerol backbone linked to two fatty acid-derived tails by ester linkages • The fatty acid components attached to C1 and C2 are hydrophobic • Saturated fatty acid is attached to the C1 carbon • Unsaturated fatty acid, such as arachidonic acid, is attached to the C2 carbon • The molecule attached to C3 of glycerol is hydrophilic (phosphate) • Phosphatidic acid (PA) is the precursor in the synthesis of glycerophospholipids and triacylglycerols o Just an –H at the head-group substituent

Answer 205

• PA is formed by adding two fatty acids (from the respective acyl-CoA derivatives) onto glycerol-3-phosphate by acyl transferases • Glycerol-3-phophate is primarily derived from reduction of the glycolytic intermediate dihydroxyacetone phosphate • Phosphatidic acid can also be formed by: o (1) Phosphorylation of diacylglycerol (DAG) by DAG Kinase or o (2) Phospholipase D-mediated hydrolysis of glycerophospholipid

Answer 206

• Phosphatidates are derivatives of the basic parent compound, phosphatide acid (PA) • Polar groups esterified to the phosphate moiety • Cytidine triphosphate (CTP) is involved in the synthesis o The second most important role for CTP (after nucleic acid synthesis) is phospholipid synthesis

Answer 207

o Alveolar structures ♣ Basic units for gas exchange ♣ Have large surface areas with high surface tension at the air-water interface that opposes lung function o Pulmonary surfactant is synthesized/secreted by alveolar type II epithelial cells ♣ Secreted surfactant forms a film covering the epithelial cells ♣ Pulmonary surfactant reduces surface tension in lung alveoli, thereby decreasing their tendency to collapse during expiration ♣ Surfactant deficiency is the primary cause of neonatal respiratory distress syndrome (RDS), and surfactant supplementation in premature infants with RDS significantly reduces mortality in these patients o Pulmonary surfactant is a complex mixture of lipids and proteins with dipalmitoylphosphatidylcholine (DPPC) being the most abundant phospholipid in the mixture o Lipids constant ~90% of the composition of pulmonary surfactant

Answer 208

• Phospholipases o Cleave phospholipids o Phospholipid remodeling o Release of arachidonic acid o Intracellular signaling • Some remove fatty acids from the C1 (phospholipase A1) or C2 (phospholipase A2) position of the glycerol backbone, which permits re-esterification reactions with other fatty acyl CoAs or release of arachidonic acid (from C2) to be used to synthesize eicosanoids (paracrine hormones) • Other phospholipases cleave the C3 phosphodiester to produce either diacylglycerol, DAG (phospholipase C) or phosphatidic acid (Phospholipase D)

Answer 209

• Phospholipase C cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two products that are active in cell signaling: o (1) Diacylglycerol (DAG) o (2) Inositol 1,4,5-triphosphate (IP3) ♣ IP3 triggers release of Ca2+ from the ER, which activates protein kinase C (PKC) involved in cell signaling events

Answer 210

• Some extracellular proteins can be associated with the outer surface of the plasma membrane by a glycosyl phosphatidylinositol (GPI) anchor o These are covalently bound to the C-terminal residue • Some intracellular proteins can be associated with the cytosolic side of membranes through specific interactions with: o Long-chain fatty acids (i.e., palmitic acid 16:0, and myristic acid 14:0) o Isoprenoids such as the polyisoprenes farnesyl (C15) and geranylgeranyl (C20) • In contrast, integral membrane proteins span a lipid bilayer o The region of the protein in the lipid bilayer is enriched in hydrophobic amino acids that interact with the nonpolar fatty acid side chains of the phospholipids

Answer 211

* Sphingolipids are found in most membranes, but are particularly abundant in cells of the central nervous system and brain * Like glycerophospholipids, sphingolipids contain a polar head group and two nonpolar tails * Sphingolipids do not contain glycerol, but one molecule of the long-chain amino alcohol, sphingosine * Ceramide is the base compound of the sphingolipids (phosphatidic acid serves this role for the glycerophospholipds)

Answer 212

♣ Phosphocholine or phosphoethanolamine as their polar group, and because of the presence of phosphate, are also classified as phospholipids ♣ Abundant in myelin, the membranous sheath that surrounds and insulates the axons of some neurons

Answer 213

o Have polar head groups with one (cerebrosides) or more (globosides) carbohydrates o Found primarily on the outer surface of the plasma membrane o Also called neutral glycolipids because they are uncharged at pH 7

Answer 214

o Have oligosaccharides with one or more N-acetylneuraminic acid (Neu5Ac) ♣ Neu5Ac is a sialic acid o Are negatively charged glycolipids due to the presence of sialic acid o Found at their highest concentration in the nervous system and brain

Answer 215

• Defects in the recycling center (lysosome) • Family of progressive, degenerative diseases with multi-organ involvement o Skeletal deformities, mental retardation, heart disease, decreased life expectancies are common features) • ~50 different diseases (e.g., Tay-Sachs, Niemann-Pick) o Only FDA approved treatments for about 6 of them • Genetic disorders: individually rare, altogether incidence is ~1 in 6,000 live births (as a group they are among the most common genetic disorders in children) • Caused by deficiency of an enzyme necessary to breakdown macromolecules, resulting in accumulation of undegraded material in the lysosome Sphingolipidoses continued… • Degradation occurs in lysosomes by the action of hydrolytic enzymes, including phospholipases and glycosidases • When activity of one of the hydrolytic enzymes is decreased or absent due to a genetic error, then the substrate accumulates in the lysosomes of the tissue(s) responsible for its catabolism • Tay-Sachs and Niemann-Pick are two examples of the lysosomal storage diseases caused by the abnormal accumulation of sphingolipids o Patients have progressive neurodegeneration and exhibit severe neurological defects because molecules are highly enriched in the brain and CNS tissues

Answer 216

• Fatty acid derivatives that are short-lived and have a variety of effects on human tissues and cells: o Inflammation o Fever and pain associated with injury o Reproductive function o Formation of blood clots o Regulation of blood pressure • Family of paracrine hormones that act as short-range signaling molecules, affecting tissues near the cells that produce them o They mediate their biological effects by interacting with cell surface G-protein coupled receptors (GPCRs)

Answer 217

• Three classes: Prostaglandins (5 member ring), Thromboxanes (6 member ring) and Leukotrienes (linear) o All eiconsanoids are synthesized from arachidonic acid, a 20-carbon polyunsaturated fatty acid ♣ The precursor to arachidonic acid is the essential fatty acid linoleic acid o Arachidonic acid is a component of membrane phospholipids, which makes it readily available for eicosanoid synthesis following phospholipase A2 cleavage in response to hormonal or other stimuli

Answer 218

Dietary Linoleic Acid is required for synthesis of arachidonic acid and eicosanoids • Mammals cannot convert oleate to linoleate or -linolenate, which are therefore required in the diet as essential fatty acids

Answer 219

• Arachidonic acid is cyclized and oxidized by the bifunctional enzyme cyclooxygenase (COX), also called prostaglandin H2 synthase • Mammals have two isoforms of COX that differ in their expression: o COX1: constitutively expressed in many cells o COX2: exhibits inducible expression in selected cell types • The resulting 5-membered ring (e.g., PGH2) distinguishes prostaglandins from other eicosanoids • Thromboxanes are then synthesized from PGH2 by thromboxane synthase in platelets that converts the cyclopentane ring into a six-membered oxygen containing (oxane) ring

Answer 220

• Unlike the ring-containing eicosanoids, leukotrienes are linear molecules with 4 double bonds (3 of the double bonds are in series, forming a “triene”) • Lipoxygenase converts arachidonic acid to leukotrienes and catalyze the incorporation of 2 atoms of oxygen, forming a hydroperoxy (-OOH) group on the molecule o Leukotrienes differ from each other due to differences in the position of this peroxide group • 5-Lipoxygenase produces the major leukotriene members, generating 5-hydroperoxyeicosatetraenoate (5-HPETE) o 5-HPETE is converted to leukotriene A4 (LTA4), which can be further modified to generate other leukotrienes

Answer 221

1. Corticosteroids (prednisone) – potent anti-inflamatory drugs a. Surpress COX2 synthesis b. Inhibit phospholipase A2 mediated release of arachidonic acid from membrane phospholipids c. Decreased production of pro-inflammatory eicosanoids 2. Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) – ibuprofen/aspirin a. Inhibit prostaglandin and thromboxane synthesis, but not leukotriene synthesis b. Inhibit both isoforms of COX c. Aspirin, but not ibuprofen, irreversibly inactivates the cyclooxygenase activity of both COX1 and COX2 by acetylating the enzyme and blocking the enzyme’s active site i. Side-effect of COX1 inhibition: stomach irritation 1. Prostaglandins regulate gastric mucins production, which are highly glycosylated proteins of the gastric mucosa that protect the stomach 3. Low dose of aspirin decreases risk of heart attack and stroke by reducing thromboxane synthesis a. Thromboxane induces platelet aggregation, an early step in blood clotting 4. COX2 specific inhibitors (Celebrex, Vioxx) a. Side effect: increased risk of heart attack and stroke, suggests an imbalance in the fine tuning expression levels of the different eicosanoids b. Only Celebrex remains available in the U.S.

Answer 222

• Several leukotrienes have pro-inflammatory effects • Elevated levels of certain leukotrienes are associated with asthma and anaphylactic shock (induce contraction of the smooth muscle lining the airways of the lungs, thereby increasing bronchoconstriction) • Some asthma medications target the leukotriene pathway by inhibiting: o 5-lipoxygenase activity o the interaction of leukotrienes with their cell surface receptors

Answer 223

``` • Precursor for the synthesis of: o Steroid Hormones o Bile Acids o Vitamin D • Structural component of membranes ```

Answer 224

• Found in all plasma and intracellular membranes • Regulates the fluidity of the lipid bilayer • Most cholesterol in membranes occurs in an unesterified form • Amphiphathic lipid o Extensive nonpolar region – 4 fused rings o Small polar region – hydroxyl group

Answer 225

* Cells tightly regulate the amount of unesterified cholesterol, with excess cholesterol being esterified and packaged as lipid droplets inside cells * Unesterified membranes Esterified lipid droplets + inside lipoprotein particles

Answer 226

• Cholesterol exhibits low solubility in water but its concentration in human serum is high • Cholesterol’s high solubility in SERUM is due to it being packaged into spherical particles called lipoprotein particles (e.g., LDL, HDL) o LDL carries cholesterol to tissues/cells that need it • Composed of outer shell of phospholipids, unesterified cholesterol, and plasma lipoproteins (e.g., Apo B 100) o Inside: cholesterol esters and triacylglycerol

Answer 227

• All 27 carbons are derived from acetate • De novo synthesis of cholesterol occurs in virtually all cells, with the greatest capacity in liver, intestine, adrenal cortex, and reproductive tissues • The biosynthesis of cholesterol can be broken into 4 stages involving 37 steps o The rate-limiting step is carried out by HMG-CoA Reductase ♣ Inhibitors have been designed to target this enzyme o Blocking de novo synthesis of cholesterol is used as a means to lower serum cholesterol levels (High serum LDL correlate w increased risk of CVD and stroke) o Statins, such as Lipitor, are competitive inhibitors of HMG-CoA Reductase

Answer 228

• Alter protein activity/function without having to synthesize a whole new protein • Modifications are a mechanism to increase the functional diversity of a limited gene pool o >200 different modified amino acids have been found in proteins o Post-translational modifications (“PTMs”) increase the number of letters in amino acid alphabet and lead to a combinatorial explosion of diversity ♣ Multiple PTMs provides cells with a means to integrate different pathways and coordinate responses to different physiological conditions

Answer 229

o Carbohydrates (~1/2 of all proteins undergo glycosylation) ♣ Linkages: N-linked (Asn-X-Ser/Thr) O-linked (Ser) o Phosphate: (Ser, Thr, Tyr; ~1/3 of all proteins undergo phosphorylation) o Hydroxyl: Pro; Acetyl: Lys; ADP-Ribose (Arg) o Ubiquitin (76 aa protein; Lys) o Fatty acids (Cys, Gly); Lipids (Cys), Glycosylphosphatidyl-inositol (GPI)

Answer 230

• The most common protein modification – 50% of all proteins • Names “glycan” and “oligosaccharide” are used interchangeably o Polymer containing 2 or more monosaccharides joined together by O-glycosidic linkages (Glycogen) o Carbohydrates are the building blocks to form oligosaccharides on proteins o Carbohydrates can be joined by different linkages

Answer 231

• Nucleotide sugars: o Monosaccharides must be “activated” to high-energy compounds before they can function as immediate precursors in glycoprotein/glycolipid synthesis • Synthesis occurs in the cytoplasm (exception is CMP-Neu5Ac) Nucleoside triphosphate + sugar 1-phosphate nucleoside diphosphate sugar + PPi • Examples: UDP-Gal, UDP-Glc, UDP-GlcNAc, and GDP-Man • Key Points: o Glucose (Glc) is the major in vivo source for the synthesis of nucleotide sugars o ATP is required for the synthesis of the early intermediates in the pathway (i.e., sugar 1-phosphate or sugar 6-phosphate) ♣ UTP, GTP, or CTP are used at late stages in the pathway o Glutamine is required for the synthesis of UDP-GlcNAc

Answer 232

• Cause by a deficiency of galactose-1-phosphate uridyltransferase (GALT) o Infants deficient in GALT fail to thrive, and have hepatomegaly, jaundice, and cataracts o Accumulation of galactose and galactose-1-phosphate, and the metabolites galactitol and galactonate contribute to symptoms o Treatment: eliminate lactose and galactose from the diet o Newborn screening in many states, including WI, for GALT deficiency

Answer 233

• UDP-glucose (UDP-Glc) is used by glycogen synthase to make glycogen • UD-glucuronic acid (UDP-GlcA) is the substrate for several families of glucouronyltransferases involved in o Glycoprotein/Proteoglycan biosynthesis o Drug detoxification of xenobiotics (e.g. clinical drugs, environmental toxins) o Excretion of steroid hormones o Heme metabolism and excretion of bilirubin (red blood cell turnover) • Resident integral membrane proteins of the endoplasmic reticulum • >300 different glycosyltransferases • Specific o (1) sugar that is transferred o (2) acceptor substrate o (3) type of linkage generated • Anomeric/C1 carbon of the sugar becomes glycosidically bonded to the hydroxyl group of the acceptor

Answer 234

• The majority of glycosylation reactions occur in the lumen (interior) of the ER and Golgi (secretory pathway) • Proteins move through the secretory pathway (ER, Golgi, plasma membrane) in transport vesicles o (1) Secreted and transmembrane proteins enter the ER as they are being synthesized by ribosomes o (2) Proteins exit the ER in transport vesicles o (3) Proteins travel through the cisternae of the Golgi apparatus o (4) Proteins are sorted at the trans Golgi Network (TGN) o (5) Soluble proteins are secreted from the cell ♣ A transmembrane protein will be present in the plasma membrane with its glycans exposed to the extracellular space

Answer 235

1. N-Glycosidic Linkage a. Requires the triplet sequence Asn-X-Ser(Thr) b. Found in membrane and secretory glycoproteins 2. O-Glycosidic Linkage

Answer 236

• Preassembly of a glycan (oligosaccharide) chain occurs on a lipid called dolichol phosphate (dolichol-P), a membrane-associated isoprenoid derivative o Mevalonic acid serves as the precursor of dolichol-P, as well as cholesterol o The regulation of dolichol-P is directly related to cholesterol metabolism • Translation of protein in the rough endoplasmic reticulum • Oligosaccharyltransferase (OST) catalyzes the co-translational transfer of the 14 residue oligosaccharide chain (Glc3Man9GlcNAc2) from dolichol-P to the Asn residue in certain Asn-X-Ser(Thr) sequences of growing polypeptide chains o This reaction occurs exclusively in the lumen of the ER • SUMMARY: o 14 different glycosyltransferases: ♣ First 7 steps occur on sytosolic face of RER ♣ Last 7 steps occur in the lumen of RER

Answer 237

• The specific location of the various processing enzymes in the ER or the cis, medial, or trans Golgi cisternae provide a mechanism for controlling their sequential action on newly synthesized glycoproteins in both space and time o The final oligosaccharide structure assembled on a glycoprotein is dictated to a large extent by the order in which that glycoprotein encounters specific processing glycosyltransferases and glycosidases • In contrast to glycosyltransferases that function to add carbohydrates to proteins or lipids, glycosidases are hydrolytic enzymes that remove carbohydrates o Glycosidases are grouped into families based on the type of carbohydrate they remove; (e.g., glucosidase, mannosidase) • The initial N linked oligosaccharide structure (Glc3Man9GlcNAc2) is recognized by chaperones in the ER called calnexin and calreticulin, which are lectins (proteins that bind to a specific glycan structure) that assist newly synthesized proteins to fold into their native conformation o These chaperones are part of the “quality control mechanism” in the ER that facilitates protein folding and recognizes mis-folded proteins that are subsequently targeted for degradation in the cytosol by the proteasome

Answer 238

• Found in proteoglycans – a glycoprotein that contains a specific type of oligosaccharide/glycan called a glycosaminoglycan o Proteoglycans often contain large numbers of glycosaminoglycan chains

Answer 239

• Proteoglycans consist of a protein backbone to which glycosaminoglycan chain(s) are covalently attached o Glycosaminoglycans are negatively charged polysaccharides composed of repeating disaccharide units o The disaccharide unit consists of an amino sugar and an uronic acid, both of which may be sulfated, thus contributing to the polyanionic properties of the molecule • The addition of carbohydrates occurs in a stepwise fashion in the Golgi o The glycosoaminoglycans chondroitin sulfate, heparin, heparin sulfate, and dermatan sulfate are attached to proteins via a Xyl-Ser O-linkage • Hyaluronic acid, which is not covalently attached to protein and is not sulfated, serves as a scaffolding platform for large numbers of proteoglycans in the extracellular matrix • Proteoglycans are found in almost all mammalian tissues as ubiquitous components of the cell surface and extracellular matrix o They are especially prominent in connective tissues o In the cornea, proteoglycans serve as “spacers for collagen,” facilitating collagen’s arrangement which is critical for the transparency of the cornea

Answer 240

• Glycans linked to serine (Ser) or threonine (Thr) • Found in mucous secretions or in the cell surface • Synthesized by many epithelial cells and by goblet cells of the tracheobronchial, gastrointestinal, and genitourinary tracts • Hydrate and protect epithelial cells, but have other functions as well (e.g., fertilization) • Example of a clinically relevant mucin: o The Cancer Antigen 125 (CA125) is found on the mucin Muc16 o CA125/Muc16 is used as a marker for the diagnosis of ovarian cancer, with the majority of ovarian cancer patients having elevated CA125 levels o A decline in serum CA125 levels is associated with a response to therapy

Answer 241

• Glycosylation is critical for normal development: o Numerous genetic defects of glycan synthesis involving defective synthesis of: ♣ N-Glycans: large family of diseases called "congenital disorders of glycosylation” (CDGs) ♣ Glycosaminoglycans (e.g., Chondrodysplasias) ♣ O-linked Glycans: -dystroglycan (at least 5 different muscular dystrophies) • Protein folding/conformation o Quality control mechanism in the ER

Answer 242

• Erythropoietin (EPO) is a growth factor secreted by the kidney: stimulates the production of red blood cells (induces the proliferation and differentiation of erythroid progenitors) • Used in the treatment of anemia caused by bone marrow suppression (e.g., after cancer chemotherapy) o EPO is also used as a performance-enhancing drug (Lance Armstrong)

Answer 243

* Erythropoietin is a 165 amino acid protein that contains N-linked oligosaccharides, carbohydrate constitutes ~40% of the mass of this glycoprotein * Glycans are critical for the function of erythropoietin because the nonglycosylated protein exhibits only 10% activity

Answer 244

• Heparin – single most widely used drug in the world today, acts as an anticoagulant • Forms a high affinity complex with antithrombin o Upon binding heparin, antithrombin undergoes a conformational change which increases its activity 1,000 to 10,000 fold o Antithombin inhibits two principle procoagulant proteases, Factor Xa and thrombin, thereby decreasing the production of fibrin clots

Answer 245

1. Cellular recognition (leukocyte adhesion selectins bind immune system cells to the sites of injury in the inflammatory response potential target for new therapeutic agents to control inflammation 2. Intracellular targeting (lysosomal enzymes, lysosomal storage disorders) 3. Binding sites for bacterial toxins/parasites (cholera toxin, Helicobacter pylori, flu virus)

Answer 246

• Humans can be divided into different groups according to the presence or absence of serum antibodies (against a carbohydrate structure on glycoproteins and glycolipids) that would agglutinate red blood cells isolated from other humans • The antigens are oligosaccharides determined by allelic glycosyltransferases • Example: Type A person receives blood from Type B person o Type A person views type B RBCs as foreign and produces anti-B antibodies which agglutinate the transfused RBCs o Note: Non-primate mammals express glycans not present in humans; limits using non-primate mammalian organs for transplantation into humans

Answer 247

♣ Glycoprotein “cancer antigens” have been detected in melanomas, gliomas, neuroblastomas, and breast, pancreatic, lung, prostate, and kidney cancers ♣ Clinical diagnostic procedures are being developed to detect abnormal glycosylation on the surfaces of malignant cells

Answer 248

o Glycation: a non-enzymatic process that effects mostly long-lived proteins of the lens, plasma and RBCs ♣ Glucose becomes covalently attached to -amino groups of lysine residues in hemoglobin forming “glycated” hemoglobin called HbA1c • Patients with diabetes mellitus have high concentrations of blood glucose and therefore high amounts of HbA1c • The changes in the concentration of HbA1c in diabetic patients can be used to follow the effectiveness of insulin treatment • Glycated proteins undergo further structural changes to generate heterogeneous compounds termed Advanced Glycation End products (AGEs) that can damage other proteins by cross-linking them, causing pathological changes

Answer 249

o Influenza virus uses host glycoproteins and glycolipids which contain sialic acid as cell surface receptors, allowing the virus to infect cells ♣ Human-adapted viruses bind sialic acid 2,6 galactose found on epithelial cells of the human upper respiratory tract (URT) ♣ Avian-adapted viruses bind sialic acid 2,6 glactose found on epithelial cells of the avian upper respiratory tract [in humans, present in the lower respiratory tract (LTR)] o Glycosylation Inhibitors as antiviral agents: ♣ Virus encodes a surface sialidase that cleaves terminal sialic acid residues from glycoproteins that helps the virus through the mucosa (rich in sialic-acid containing mucins) of the respiratory tract and in the release of the virus from infected cells ♣ Sialidase (Neuraminidase) inhibitors: • FDA approved drugs are analogs of sialic acid o 1) Oseltamivir (Tamiflu, tablet) o 2) Zanamivir (Relenza, inhaler) • Drugs do not kill the virus but slow the virus replication down to a level where the immune system can more easily destroy it

Answer 250

o Incomplete degradation ♣ Catabolism of glycolipids, glycoproteins, and proteoglycans occurs in the lysosome • Their carbohydrate units are degraded by the sequential action of hydrolytic enzymes (glycosidases) solely from the non-reducing end of the oligosaccharide • Exoglycosidases

Answer 251

• 4-hydroxyl proline: Collagen is constructed of the repeating tripeptide unit Gly-X-Y, where X is proline and Y is 4-hydroxy proline o Prolyl 4-hydroxylase requires Vitamin C (ascorbate) for its activity • Scurvy is caused by a lack of vitamin C: decreased levels of 4 hydroxy proline destabilizes collagen resulting in degeneration of connective tissue

Answer 252

• Lysine modification: Acetylation of lysine -amino groups in histones inhibits their interactions with DNA • Acetylation removes the positive charge on the lysine side chain, thus inhibiting the ability of histones, which are rich in lysine and arginine residues, to interact with negatively charged DNA o HAT – Histone Acetyltransferases o HDAC – Histone Deacetylases • Common modification with ~2,000 proteins identified that undergo acetylation o Nearly all enzymes involved in glycolysis, gluconeogenesis, TCA cycle, fatty acid oxidation, urea cycle, and glycogen metabolism are acetylated • REVERSIBLE (deacetylases remove acetyl group)

Answer 253

• The hydroxyl side chain of Serine, Threonine, and Tyrosine can undergo phosphorylation o ~1/3 of all proteins undergo phosphorylation o ~500 kinases and ~500 phosphatases in the human genome • Protein kinases use ATP as their substrate to attach phosphate to proteins • Protein phosphatases remove the phosphate (reversible modification) • Introduces a negatively charged group into a protein • Can have multiple effects: o Cause a dramatic change in protein conformation (e.g., insulin receptor) to convert an inactive protein into an active protein (“on-off switch”) o Serve as a docking/binding site for other proteins in signaling cascades (e.g., erythropoietin signaling pathway that stimulates the formation of RBCs) o Serve as a “rheostat” to modulate the activity of enzymes (e.g., glycogen synthase has at least 9 different phosphorylation site, and the pattern of phosphorylation modulates the activity of the enzyme)

Answer 254

• Bacterial Toxins, such as diphtheria, cholera, and pertussis toxins, act by modifying crucial host cell proteins by the addition of an ADP-ribose molecule o The reaction is catalyzed by enzymes that use NAD+ as the substrate • Cholera toxin modifies a trimeric G protein (GS) that renders it permanently active in signaling adenylyl cyclase, resulting in high cAMP levels o In intestinal epithelial cells, the increased cAM levels trigger massive water loss o Severe dehydration and electrolyte loss are the major pathologies in cholera

Answer 255

• Protein degradation serves two important roles in the cell: o (1) Removes improperly folded and damaged proteins to prevent the accumulation of abnormal proteins o (2) Maintains the appropriate levels of normal proteins and for permitting rapid changes in these levels, which allows cells to respond to changing conditions • Two major degradation pathways in mammalian cells: o (1) Proteosome (2) Lysosome

Answer 256

• Ubiquitin (Ub) tags proteins for degradation and is one of the most highly conserved proteins known (76 amino acids) • ATP-dependent pathway involving 3 enzymes: o E1 Ub-activating enzyme o E2 Ub-conjugating enzyme o E3 Ub ligase ♣ ~700 different E3s help choose which protein should be covalently modified by ubiquitin ♣ carboxyl-terminal glycine residue of ubiquitin is linked to lysine residues of the target protein • Proteins with a polyubiquitin chain are degraded by a large complex called the proteasome that is located in the cytosol o The proteasome hydrolyzes ATP to unfold proteins prior to their proteolysis by the inner chamber (i.e., 20S core particle has proteolytic activity) of the proteasome o *Proteasome is not membrane bound like the lysosome*

Answer 257

* Mutations in BRCA1 (the breast cancer susceptibility locus) are seen in ~10% of brease and ovarian cancer * BRCA1 protein is a tumor suppressor gene (and its mutation affects DNA repair) * BRCA1 is an E3 ubiquitin ligase whose activity is abolished by mutations found in familiar breast and ovarian cancers

Answer 258

o Common function: promotes the interaction of proteins with the lipid bilayer of a membrane ♣ Palmitoylation – Palmitic Acid (C16) is linked to a protein ♣ Myristoylation – Myrisric Acid (C14) is linked to a protein

Answer 259

o Intermediate of cholesterol biosynthetic pathway, farnesyl pyrophosphate (C15), is covalently attached to C-terminal cysteine (Cys) residue o Ras must be farnesylated to associate with the plasma membrane and is essential for the transforming activity of oncogenic variants of Ras ♣ Clinical trials have used farnesyltransferase inhibitor drugs o Farnesylation and Disease ♣ Hutchinson-Gilford Progeria Syndrome • Rapid aging disease • Children die at ~13 years, generally from MIs or strokes • Disease is caused by the accumulation of a mutant form of lamin A (retains a farnesyl group rather than being converted to mature lamin A which lacks a farnesyl group) o Lamin A is a structural protein of the nuclear lamina • Clinical trials are ongoing using farnesyltransferase inhibitor drugs

Answer 260

o GPI is attached to the C-terminus of a target protein through the phosphoethanolamine portion of the molecule ♣ The two fatty acid moieties of GPI are imbedded in the bilayer

Answer 261

‘Peripheral’ = all blood that’s not in bone marrow

Answer 262

to convey: – nutrients & waste – endocrine functions (hormones) – gas exchange: O2, CO2

Answer 263

Plasma: fluid phase after centrifugation with heparin (prevents clotting) – 90% water, 10% protein – Contrast with Serum, which is the fluid phase after clotting & centrifugation to remove clotting factors & cells.

Answer 264

A. Granulocytes: have specific granules a. neutrophils: b. eosinophils: c. basophils: 34-71% 0-7% 0-1% Differential Count B. Agranulocytes: a. lymphocytes: b. monocytes: 19-53% 5-12%

Answer 265

RBC (varies w altitude): male: 4.6 - 6.1 million cells/mcL female: 4.2 - 5.4 million cells/mcL complete WBC: 4,000 - 10,000 cells/mcL

Answer 266

7.5 microm diameter = the internal standard

Answer 267

``` Carbonic Anhydrase (CA): CO2 --> carbonic acid --> HCO3- (bicarbonate) • Then,“band-3”pumpsHCO3- outoftheRBC. ```

Answer 268

Band 3: an integral cell membrane protein with 2 functions • exports HCO3- • binds ankyrin, which in turn binds to spectrins, which are structural proteins that function to maintain biconcave shape!

Answer 269

Because the cytoskeleton is defective, spherocytes form. • Spherocytes can respire, they are functional. • But, they are fragile, degraded (hemolysis) in the spleen, causing anemia.

Answer 270

Leukocytes • ...are transient in blood; ‘work’ in the tissues. • ...function in defense. • ...include “granulocytes” & “agranulocytes”.

Answer 271

34-71% (most common WBC); ~15 μm • nucleus  highly lobulated –  polymorphonuclear (“polys”) • Neutrophils have 2 types of granules: 1. lysosomes 2. “specific” granules that do not stain  are ‘neutro’-philic. • Why are neutrophils ‘polymorphonuclear’?  to facilitate passage between cells, to enter tissue spaces  The ‘poly’ nucleus is caused by activation of caspase, which degrades nuclear lamins ( a very limited apoptosis).

Answer 272

Neutrophil function: anti-bacterial • • mutation of NADPH oxidase causes persistent bacterial infection neutrophil lifespan: days – – Bacteria emit signals that attract neutrophils. 3-step attack: 1. granulereleasetodegradebacteria 2. phagocytosiseatsbacteria(microphage) 3. NADPHoxidase • produces superoxide anions (O2-) that kill bacteria via a “respiratory burst”

Answer 273

0-7%; ~15 μm • bi-lobed nucleus • Specific granules are red (i.e. eosinophilic) & large, with a crystalloid center. – contains major basic protein, which kills parasitesEosinophils Have 3 Functions: 1. kill parasites (major basic protein) 2. phagocytize antigen-antibody complexes 3. secrete leukotrienes – Eosinophils increase during parasitic infections & allergic reactions. – Eosinophils in the lungs secrete leukotrienes, causing asthma. 1. BV leakiness  edema 2. bronchioles  constrict asthma 3. mucous glands  mucous Short-term treatment: target smooth muscle cells to dilate bronchioles. – β2-adrenergic agonists (SMCs relax) – ACh antagonists Long-term treatment: inhaled corticosteroids, & and drugs that block – leukotriene production – leukotriene cell receptors – eosinophil lifespan: weeks

Answer 274

nucleus obscured by blue specific granules • specific granules  heparin & histamine • similar to “mast” cellsAntigen (Ag) invades. In response to Ag, plasma cellsIgE. IgE binds receptors on basophil/mast cell. Later in life, when the same Ag enters: – basophil lifespan: years (basophils have “memory”) • epinephrine – β-adrenergic agonist • • anti-histamine steroids • Ag binds IgE on the basophil/mast cell surface. • In response, the basophil/mast cell... i.  granules  histamine ii.  leukotrienes

Answer 275

Monocytes: 5-12%; 15-20 μm • largest WBC • nucleus indented, up to horseshoe-shape In tissues, monocytes differentiate into: • Atigen-Presenting Cells (APCs) • Macrophages Lifespan is months.

Answer 276

Lymphocytes: 19-53%; ~12 μm • B (bone marrow) & T (thymus) lymphocytes – histologically indistinguishable – T cells predominate in peripheral blood (several subtypes) – B cells predominate in tissues  plasma cells  antibodies

Answer 277

``` • fragments of megakaryocyte cytoplasm • 300,000/l; ~2 m • granulomere – clotting & growth factors (PDGF) • hyalomere – microtubules • clinical: thromboembolism ```

Answer 278

• The cytosol contains many of the enzymes involved in intermediary metabolism and is packed with ribosomes • All proteins start their synthesis on ribosomes free in the cytosol o ~50% of the proteins made on these ribosomes remain in the cytosol as permanent residents o Actively synthesizing ribosomes that attach to the endoplasmic reticulum (ER) membrane function to remove selected proteins from the cytosol

Answer 279

• (1) Transmembrane proteins, which are only partly translocated across the ER membrane and become inbedded in it • (2) Water soluble proteins, which are fully translocated across the ER membrane and are released into the lumen of the ER o These include secretory proteins and proteins destined for the lysosome

Answer 280

• ~50% of proteins in the cell complete their synthesis on ER-bound ribosomes SRP: Ribonucleoprotein composed of a single 7SL RNA molecule of 300 nucleotides plus 6 different polypeptide chains • Cycles between the ER membrane and the cytosol • Binds to the signal sequence and to the ribosome to function in “signal recognition” and “elongation arrest” of the polypeptide chain

Answer 281

• No consensus sequence (primary sequence differs between signal sequences) • Adopt a similar three-dimensional structure that is recognized by SRP • Usually occurs at the amino terminus of a protein o Those that occur at the amino terminus are cleaved co-translationally by signal peptidase

Answer 282

o a hydrophilic amino-terminal domain which typically contains a net positive charge o A hydrophobic core domain with a minimum length of 7 residues that typically forms an -helix o A polar carboxyl-terminal domain of 4-6 residues

Answer 283

* Expressed exclusively in the ER * Integral membrane protein with its SRP binding site exposed to the cytosol * Functions to bind SRP in the SRP-nascent polypeptide chain-ribosome complex which targets the complex to the ER membrane (SRP receptor does NOT bind free SRP) * GTP regulates this process

Answer 284

* Cleavage of amino-terminal signal sequence occurs co-translationally in lumen of ER * Water soluble protein, fully translocated across the ER membrane, folded into its native conformation

Answer 285

* Requires ATP hydrolysis and occurs through an aqueous pore or channel called a “translocon” * Translocation and protein synthesis are usually coupled since unfolded polypeptide chains are the preferred substrate for translocation * Translocated polypeptide chains fold into their native conformation in the lumen of the ER with the assistance of other proteins called chaperones

Answer 286

• “Stop-Transfer sequences” are hydrophobic, -helical sequences which function to anchor the protein in the membrane o Proteins which span the membrane many times contain numerous stop-transfer sequences • Hydrophobic interactions between the non-polar amino acids and the fatty acyl groups of the membrane lipids firmly anchor the protein in the membrane o This is in contrast to lipid-linked membrane proteins in which the palmitoyl, myristoyl farnesyl, or acyl chains of GPI imbed only within the inner or outer leaflet of a bilayer

Answer 287

• The golgi apparatus contains a collection of membrane-bound cisternae resembling a stack of plates that has two distinct sides: o The cis face is juxtaposed to the ER o The trans face is distended into a tubular reticulum called the trans Golgi network (TGN) • Each cisterna is a distinct compartment with its own set of processing enzymes, and proteins are modified in successive stages as they move from compartment to compartment across the stack in a cis to trans direction o The TGN is the site in the cell where proteins that have been synthesized on ER-bound ribosomes are sorted for transport to lysosomes, secretory granules, or the plasma membrane

Answer 288

• Proteins (soluble or membrane-bound) are transported to the cell surface by a nonselective “default pathway” unless they carry signals that direct them elsewhere o Constitutive Pathway: common to all cells – soluble proteins are continuously secreted without intracellular storage o Regulated Secretory Pathway: present (in addition to the constitutive pathway) only in certain cells such as exocrine cells, endocrine cells, and neurons – a subset of secretory proteins is sorted to storage organelles (called secretory granules) from which they are released only upon stimulation of the cell

Answer 289

* Lysosome has ~60 different hydrolytic enzymes used for the controlled intracellular digestion of macromolecules * Degraded macromolecules are recycled and used in pathways for energy production (TCA Cycle) or in biosynthetic pathways (protein synthesis) * All hydrolytic enzymes are acid hydrolases that are optimally active near the pH 5 maintained within lysosomes * V-type ATPases are responsible for acidifying intracellular compartments; lysosomes are the most acidified compartment in mammalian cells with a pH of 5.0 or less

Answer 290

• Numerous functions are carried out by lysosomes including: o (1) disposal of abnormal proteins o (2) downregulation of cell surface signaling receptors (e.g., EGF receptor) o (3) Release of endocytosed nutrients (e.g., cholesterol from LDL) o (4) Degradation of pathogenic organisms (phagocytosis) o (5) Cellular survival (autophagy)

Answer 291

o (1) phagocytosis o (2) endocytosis o (3) autophagy ♣ “Self-eating” – a major catabolic, energy-producing pathway that involves the lysosomal degradation of cytoplasmic proteins and organelles ♣ Housekeeping function (Quality Control mechanism) to eliminate damaged organelles and protein aggregates ♣ During starvation, autophagy provides an internal source of nutrients for energy generation and thus survival

Answer 292

• Protein aggregates and damaged organelles accumulate within specific neurons of patients with: o Alzheimer’s disease o Parkinson’s disease o Huntington’s disease o Amyotrophic Lateral Sclerosis (ALS/Lou Gehrig’s Disease) • Regulation of autophagic pathway is potential therapeutic target in these neurodegenerative diseases

Answer 293

* Lysosomal enzymes (i.e., acid hydrolases) are synthesized in the ER and transported through the Golgi apparatus * Mannose 6-phosphate receptors are the delivery trucks that recognize the “zip code” and carry the ~60 different newly synthesized mannose 6-phosphate tagged hydrolytic lysosomal enzymes (packages) from the Golgi to pre-lysosomal compartments

Answer 294

o (1) GlcNAc phosphotransferase ♣ Recognizes a protein signal on lysosomal enzymes ♣ Transfers GlcNAc-1-PO4 from the sugar nucleotide uDP-GlcNAc to specific mannose residues o (2) -N-acetylglucosaminidase (“uncovering enzyme”) ♣ Cleaves off the GlcNAc residue, generating the mannose 6-phosphate recognition marker o (3) Mannose 6 phosphate receptors ♣ Recognizes mannose 6-phosphate residues on lysosomal enzymes ♣ Transports the lysosomal enzymes from the Golgi to a prelysosomal compartment (i.e., endosomes)

Answer 295

• Loss of any one lysosomal enzyme causes disease o ~50 different lysosomal storage diseases (Tay-Sachs, Niemann-Pick) • Results in accumulation of undegraded material • FDA approved therapies exist only for a few of these diseases • Enzyme replacement therapy (ERT) involves intravenous injection of the missing enzyme every 2 weeks o Treatment depends upon receptor-mediated endocytosis of mannose 6-phosphate tagged recombinant acid hydrolase enzyme via cell surface mannose 6-phosphate receptors o Annual cost to patient: $200,000-$400,000

Answer 296

• Arise from defects in lysosomal enzyme targeting because of a deficiency in the enzyme (GlcNAc phosphotransferase) that generates the mannose 6-phosphate tag on proteins destined for the lysosome o The patient’s cells lack multiple lysosomal enzymes in their lysosomes and abnormally high levels of lysosomal enzymes are present in plasma and other body fluids o The disease is characterized by severe psychomotor retardation, hepatosplenomegaly, many skeletal abnormalities, coarse facial features, and restricted joint movement o Symptoms are present at birth and progress until death, usually by age 10

Answer 297

* Patients accumulate undegraded glycogen | * Pompe disease is also classified as a glycogen storage disease

Answer 298

o (1) a coat protein-covered vesicle buds from a donor compartment o (2) coat proteins are released from the vesicle o (3) the uncoated vesicle binds to a specific target compartment o (4) fusion of the uncoated vesicle to the target membrane

Answer 299

o SNAREs are a family of integral membrane proteins ♣ Each vesicle has its own v-SNARE which pairs in a unique match with a cognate t-SNARE found only at the intended target membrane ♣ This matching ensures specificity of the fusion event • Example: vesicles budding from the ER contain an ER v-SNARE that will only bind to a t-SNARE found on the cis-Golgi

Answer 300

• SNARE proteins provide the specificity in the fusion of vesicles with target membranes • Many transport vesicles move through the cell along “tracks” of microtubules o Vesicles are NOT just freely floating

Answer 301

• Tetanus and botulism are caused by extremely potent neurotoxin proteins that act by inhibiting the release of neurotransmitter from presynaptic nerve endings o Tetanus and botulinal toxins are produced by certain anaerobic bacteria (Clostridium) whose spores are widely distributed in the soil o Tetanus and botulism toxins are peptidases that cleave SNARES essential for synaptic vesicle fusion to the plasma membrane • Botulinum blocks the release of acetylcholine and results in a flaccid paralysis • Tetanus blocks the release of the inhibitory neurotransmitter -aminobutyric acid (GABA) and results in a spastic paralysis (“lockjaw”)

Answer 302

* Botox – diluted botulinal toxin that is injected into the skin/muscle (paralysis occurs only at the site of the injection) to lessen the appearance of wrinkles * Botox is administered for numerous conditions related to overactive muscle contraction: brow wrinkling, back pain, bladder spasms, migraine headaches, writer’s cramp, incontinence, patients with cerebral palsy

Answer 303

• An efficient pathway for taking up specific macromolecules from the extracellular fluid • This process provides a selective concentrating mechanism that increases the efficiency of internalization of particular ligands more than 1000-fold so that even minor components for the EC fluid can be specifically taken up in large amounts without taking up a correspondingly large volume of EC fluid o Nutrients (LDL, transferrin, vit B12) o Growth Factors (EGF) o Polypeptide hormones (Insulin) o Viruses o Toxins Fate of receptor And Ligand ----------> Clathrin-Coated Pits and Vesicles

Answer 304

• Clathrin: major protein component of coated pits/vesicles o Triskelion-shaped (“3-legged”) protein complex – 3 heavy and 3 light chains o Forms distinctive polyhedral protein baskets or cages o Lines coated pits that occupy ~2% of the plasma membrane surface o Recycles: dissociates from the vesicle membrane and returns to the cell surface to form a new coated pit ♣ This removal of clathrin coat is required before the vesicle can fuse to the endosome ♣ V-SNARE and T-SNARE are involved

Answer 305

• The internal pH of endosomes is lowered by the presence of vacuolar ATPases (V-Type ATPases) in its membrane o V-type ATPases are a distinct family of ATP-dependent proton pumps that function exclusively in the hydrolytic direction to generate pH and electrical gradients across membranes o The acidic pH of the lumen of the endosome facilitates the dissociation of receptors from their ligands

Answer 306

• Transferrin cycle transports iron into cells via the transferrin receptor o This cycle takes 16 minutes to occur and a typical liver cell can internalize 20,000 iron atoms/min o pH regulated process o Vesicle fusion events involve SNARES o Apo = iron-free (Apotransferrin)

Answer 307

o 1) Elevated concentration of LDL in the plasma o (2) Deposition of LDL-derived cholesterol in tendons and skin (xanthomas) and in arteries (atheromas) o (3) Inheritance as an autosomal dominant trait with a gene dosage effect ♣ Homozygotes are more severely affected than heterozygotes

Answer 308

• Results from mutations that affect the structure and function of the LDL receptor

Answer 309

• Heterozygotes number 1 in 500 persons, placing FH among the most common inborn errors of metabolism o Heterozygotes have two-fold elevations in plasma cholesterol from birth • Homozygotes number 1 in 1 million persons, exhibit severe hypercholesterolemia (650 to 1000 mg/dl) and coronary heart disease begins in childhood • Treatment is directed at lowering the plasma level of LDL (direct correlation between high LDL levels and atherosclerosis) • LDL receptor mutations (over 400 known mutations) can be divided into 5 classes based on their phenotypic effects on the receptor o Three of these classes result in abnormal targeting

Answer 310

• Signal transduction is fundamental to life o Enables cells to communicate with each other and to respond to an alteration in their environment (changes in pH, osmotic strength, oxygen, nutrients, etc) o Signal transduction alters cell behavior o Coordinates homeostasis in differentiation, development, and metabolism • Aberrant signal transduction can cause disease o Example: mutations in Ras, such as Gly12 to Val mutation, can initiate carcinogenesis by promoting uncontrolled cell proliferation

Answer 311

* Extracellular signal molecules mediate cell-cell communication * Reception of the signals depends on receptor proteins * The receptor activates one or more intracellular pathways * Intracellular signaling pathways distribute the signals to appropriate effector proteins

Answer 312

• In most cases, signaling receptors are transmembrane proteins on the cell surface o When bound by a ligand, the receptor becomes activated and generates various intracellular signals that alter the behavior of the cell In other cases, the receptor proteins are inside the target cells and are stimulated by small/hydrophobic signal molecules that diffuse across the target cell’s plasma membrane

Answer 313

• Contact-Dependent: especially important during development and in immune responses • Paracrine: occurs between different cell types; the signaling between cells of the same type is referred to as autocrine signaling o Cancel cells often rely on autocrine signaling for their survival and proliferation • Synaptic: coordinates behavior of cells in remote parts of the body • Endocrine: relies on diffusion and blood flow, and it is relatively slow

Answer 314

* Slow responses: increased cell growth and division require changes in gene expression and protein synthesis * Fast responses: responses such as movement, secretion, or metabolism often involve rapid phosphorylation of the effector proteins in the cytoplasm

Answer 315

• Different receptors respond differently because the downstream activating factors are different; even if it is the same receptor, there can be different signaling cascades

Answer 316

• Nitric Oxide (NO) stimulates guanylyl cyclase o For smooth muscle relaxation, autonomic nerves in the vessel wall release acetylcholine, which acts on the nearby endothelial cells by stimulating NO synthesis ♣ NO diffuses across the membrane and binds to guanylyl cyclase in the smooth muscle cells ♣ Activated guanylyl cyclase produces cyclic GMP, which induces rapid relaxation of smooth muscle cells

Answer 317

o Steroid hormone receptors translocate into the nucleus open ligand binding o Ligand binding induces conformational changes in the nuclear receptors and affect their interaction with either inhibitory proteins or coactivator proteins ♣ This determines the function of nuclear receptors to regulate transcription either negatively or positively

Answer 318

♣ The gene products then induce expression of other genes (delayed response, also called “secondary response”) ♣ Some of the early response gene products suppress the expression of primary response genes (an example of negative feedback regulation)

Answer 319

♣ Only certain types of cells have the receptors ♣ Each of these cell types contains a different combination of other cell-type-specific gene regulatory proteins that collaborate with the activated receptor to influence the transcription of specific sets of genes

Answer 320

o (1) Ion-Channel coupled receptors: ♣ Also known as transmitted-gated channels or ionotropic receptors) involved in rapid synaptic signaling o (2) G-Protein coupled receptors: ♣ Indirectly regulate plasma membrane-bound enzymes or ion channels, which is mediated by a trimeric GTP-binding protein (G-Protein) o (3) Enzyme-coupled receptors ♣ Either function as enzymes or associate directly with enzymes that they activate

Answer 321

1. Molecular switches relay the signal to the next signaling component in the chain 2. Scaffold proteins bring two or more signaling proteins together so that they can interact more quickly and efficiently 3. Primary signals are transduced to produce large amounts of small intracellular mediators or many copies of a downstream signaling protein, which amplify the original signal a. A small number of EC signal molecules can evoke a large intracellular response b. When there are multiple amplification steps in a relay chain, the chain is often referred to as a signaling cascade 4. A coincidence detector receives signals from two or more signaling pathways and integrates them before relaying a signal onward 5. Signals can spread from one signaling pathway to another, creating branches in the signaling stream, thereby increasing the complexity of the response 6. Some proteins anchor one or more signaling proteins in a pathway to a particular structure in the cell where the signaling proteins are needed Some proteins modulate the activity of other signaling proteins and thereby regulate the strength of signaling along a pathway

Answer 322

o Many intracellular signaling proteins behave like molecular switches ♣ Two important mechanisms that control “active” and “inactive” status of proteins are phosphorylation and GTP-Binding o Other mechanisms are available, including cAMP-binding, Ca2+-binding, and protein modifications such as ubiquitlyation o About 30% of human proteins contain covalently attached phosphate on Ser/Thr or Tyr ♣ The human genome encodes 518 protein kinases and 137 protein phosphatases

Answer 323

o GTP binding proteins switch between an “on” state when GTP is bound and an “off” state when GDP is bound ♣ In the “on” state, they have intrinsic GTPase activity and shut themselves off by hydrolyzing their bound GT to GDP

Answer 324

♣ (1) Monomeric GTP-binding proteins (Small GTPases) | ♣ (2) Trimeric GTP-binding proteins (G-Proteins)

Answer 325

(GEFs) promote the release of bound GDP in exchange of GTP to activate small GTPases

Answer 326

(GAPs) increase the rate of GTP hydrolysis of small GTPases

Answer 327

• Scaffold proteins: keep signaling proteins in close proximity and regulate the specificity of signaling • Many receptor tyrosine kinsases generate specific docking sites on themselves to interact with signaling proteins o This occurs transiently in response to extracellular signal • Phospholipid molecules (Phosphoinositides) can also generate docking sites in the plasma membrane to recruit intracellular signaling proteins

Answer 328

• The assembly of signaling complexes depends on various highly conserved, small interaction domains, which are found in many intracellular signaling proteins o Src homology 2 (SH2) domains and phosphotyrosine-binding (PTB) domains bind to phosphorylated tyrosine o Src homology 3 (SH3) domains bind to short proline-rich amino acid sequences o Pleckstrin homology (PH) domains bind to the charged head groups of specific phosphoinositides

Answer 329

• Positive feedback loop sustains signaling even after the signal strength drops o This can contribute to prolonging the effects of a stimulus • Negative feedback loop counteracts the effects of a stimulus and thereby abbreviates and limits the level of the response

Answer 330

• Prolonged exposure to a stimulus decreases the cell’s response to the level of stimulus • Desensitization can occur via diverse mechanisms that regulate either receptors or intracellular signaling proteins, or the production of inhibitory proteins o Receptor Sequestration (use of endosome) o Receptor Down-Regulation (use of lysosome) o Receptor Inactivation o Inactivation of Signaling Protein o Production of Inhibitory Protein

Answer 331

o A single peptide chain that threads back and forth across the lipid bilayer seven times

Answer 332

• GPCR activates a trimeric GTP binding protein (made of , , and subunits) by acting as a guanine nucleotide exchange factor (GEF) for the G-protein • The subunit has GTPase activity which is enhanced by a specific regulator of G-protein signaling (RGS) o RGSs are the counterparts of GAPs that regulate small GTPases o There are about 25 RGS proteins encoded in the human genome

Answer 333

o Normal [cAMP] in the cytosol is about 10-7 o A nonomolar signal that activates adenylate cyclase can increase [cAMP] twenty fold in seconds • Levels of cAMP are also regulated by cAMP phosphodiesterases that hydrolyze cAMP to adenine 5’ Monophophate • In most animal cells, cAMP exerts its effects mainly by activating the Ser/Thr kinase, cAMP-dependent protein kinase (PKA)

Answer 334

o PKA consists of two regulatory and two catalytic subunits ♣ cAMP binds to the regulatory subunits and induces their dissociation from the catalytic subunits o PKA mediates short term as well as long term responses ♣ Long term responses require gene transcription, which is mainly regulated through cAMP Response Element (CRE)

Answer 335

o Cholera toxin transfers ADP ribose from NAD+ to the subunit of GS, inhibiting GTP hydrolysis ♣ This sustains active conformation of GS and stimulation of adenylyl cyclase ♣ The resulting prolonged cAMP production in the intestinal epithelial cells causes a large efflux of Cl- and water into the gut, thereby causing diarrhea that characterizes cholera

Answer 336

o Pertussis toxin (whooping cough) catalyzes ADP ribosylation of the subunit of GI, preventing the protein from interacting with GPCRs ♣ As a result, the G protein remains in GDP-bound form and is unable to regulate its target proteins ♣ Because GI can’t inhibit AC, there is an increase in cAMP

Answer 337

• Phospholipase C generates IP3 and diacylglycerol (DAG) from PIP2. IP3 is a water soluble molecule that diffuses into the cytosol and acts as a small intracellular mediator

Answer 338

• There are there general modes of desensitization of GPCR: o (1) Receptor inactivation: GPCR is altered and cannot interact with G proteins o (2) Receptor sequestration: GPCR is temporarily internalized o (3) Receptor downregulation: GPCR is destroyed in lysosomes after internalization • In each case, GPCRs are phosphorylated by PKA, PKC, or GPCR Kinases (GRKs), indicating the presence of feed back regulation • Arrestin prevents phosphorylated GPCR from interacting with G proteins o It also couples GPCR to the clathrin-dependent endocytosis machinery

Answer 339

• Many extracellular signal proteins act through receptor tyrosine kinases (RTKs) o RTK has only one transmembrane segment • About 60 human genes encode RTKs, which can be classified into more than 16 structural subfamilies, each dedicated to its complementary family of protein ligands • ****RTK activations requires the receptor chains to dimerize, bringing the kinase domains of two receptor chains together so that they can become activated and cross-phosphorylate each other on multiple tyrosines (transautophosphorylation) **** • Exception: the receptors for insulin and IGF1 are ()2 dimers, and ligand binding rearranged their transmembrane receptor chains, moving the two kinase domains in the subunits close together

Answer 340

• Transautophosphorylation of RTK contributes to the receptor activation process in two ways: o (1) Tyr phosphorylation within the kinase domain increases kinase activity o (2) Tyr phosphorylation outside the kinase domain creates high-affinity docking sites for binding of specific intracellular signaling proteins that contain SH2 domains or PTB domains • There are about 115 SH2 domains and about 295 SH3 domains encoded in the human genome • Exception: The receptors for insulin and IGF1 do not generate docking sites on the receptor but use a specialized docking protein called insulin receptor substrate 1 (IRS1)

Answer 341

• The Ras superfamily consists of various families of monomeric GTPases o Only the Ras and Rho families relay signals from cell-surface receptors o A Ras or Rho family member can coordinately spread the signal along several distinct downstream signaling pathways, thereby acting as a signaling hub • H-, K-, and N-Ras are the three major Ras proteins in humans and they work in almost the same way

Answer 342

o Raf/MEK/ERK pathway is a three layered signaling cascade o Plays a pivotal role in controlling cell growth and differentiation o Its dysregulated signaling can lead to disease

Answer 343

o 30% of human tumors have hyperactive mutant forms of Ras (mainly Gly12 to Val mutation), which contribute to the uncontrolled proliferation of cancer cells

Answer 344

* Many small molecule inhibitors or therapeutic antibodies that target RTK/Ras/MAPK signaling have been developed and tested for their efficacy * Kinases are a major therapeutic target because they can be effectively inhibited by a specific ATP analog or an allosteric inhibitor

Answer 345

• Phosphotydylinositol (PI) undergoes reversible phosphorylation at multiple sites on its inositol head group to generate a variety of phosphorylated PI lipids called phosphoinositides • Phosphoinositide 3-Kinase (PI3K) phosphorylates inositol phospholipids o PI3K class 1a – activated by RTKs o PI3K class 1 b – activated by GPCRs • PI3,4,5-triphoshate (PI(3,4,5)P3) serves as a lipid docking site for a small subset of proteins that contain pleckstrin homology (PH) domain • The PI3K-AKT pathway is the major pathway activated by insulin, and plays a central role in promoting cell survival and growth in response to many other signals

Answer 346

• Cytokine receptors do not have kinase activity and, thus, mediate their signaling in association with the cytoplasmic tyrosine kinase Janus kinase (JAK) o More than 30 cytokines and hormones, including interferons, erythropoietin, growth hormones, etc. bind to the cytokine receptors to activate the JAK-STAT pathway o There are four known JAKs and at least six STATs in humans

Answer 347

• Transforming Growth Factor- (TGF) superfamily consists of a large number of structurally related secreted dimeric proteins o TGF/activin family and the larger bone morphogenetic (BMP) family • TGF family members mediate development, immune responses, and cell proliferation and death

Answer 348

• Human genome contains 518 putative protein kinase genes that are either membrane associated or intracellular o 90 Tyr Kinases o 428 Ser/Thr Kinases • Phospho-Ser, Phospho-Thr, and Phospho-Tyr account for 86.4%, 11.8%, and 1.8%, respectively, of the phosphorylated amino acids o Tyr phosphorylation is used for signaling of high specificity • Protein phosphorylation used in signal transduction is reversed by dephosphorylation, which is mediated by either Protein Tyr Phosphatases (PTPs) or Ser/Thr Phosphatases o 107 protein Tyr phosphatases are encoded in the human genome, including some dual-specificity phosphatases that also dephosphorylate Ser and Thr o Only 30 Ser/Thr phosphatases are encoded, further supporting that Tyr phosphorylation is utilized more specifically than Ser/Thr phosphorylation for cell signaling

Answer 349

• Notch cleavage (3 cleavage sites) is mediated by -secretase o One of its essential subunits is Presenilin o Mutations in Presenilin are a frequent cause of early onset of familial Alzheimer’s disease, a form of presenile dementia o -secretase containing mutant Presenilin contributes to Alzhemier’s disease by generating amyloid plaques, which may injure nerve less (-secretase loses its specificity)

Block 1 Flashcards

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