Exam 3 Flashcards
(257 cards)
1
Q
The nucleus
A
- stores and maintains DNA in eukaryotic cells
- where DNA replication and transcriptions occurs
2
Q
Genes
A
- protein coding segments of DNA
- nestled within ~3.2 billion base pairs of DNA
- all cells within an organisms have roughly the same DNA content
3
Q
Do all cells express the exact same 20,00 genes or do they differentiate by expressing different subsets of genes?
A
they differentiate
4
Q
Transcription
A
- Turns DNA into RNA
5
Q
3 major types of RNA
A
- tRNA
- mRNA
- rRNA
6
Q
Central dogma
A
- dna transcribed into mRNA translated into protien
7
Q
Which enyme transcribes DNA into RNA?
A
RNA polymerase
8
Q
4 general stages of transcription
A
1) promoter binds and orients RNA pol with help of transcription factors
2) transient local unwinding and RNA pol progression 5’-3’
3) elongation of RNA (5’ (upstream) to 3’ (downstream)) until termination signal is reached (AAUAAAA)
4) RNA and pol release DNA re-duplexes
- after transcription significant RNA processing occurs (ex: capping, splicing)
9
Q
termination signal for eukaryotic RNA pol2
A
AAUAAA
10
Q
template strand
A
- the one DNA strand that is copied into RNA
- the opposite strand is the coding strand
- occasionally genes overlap in which case 1 strand codes for 1 gene and the other strand codes for the other gene
11
Q
NTP
A
nucleotide triphosphate
12
Q
Pol 1
A
- in the nucleolus
- transcribe 3/4 of the rRNA
13
Q
Pol 2
A
- in the nucleoplasm
- transcribes mRNA, snRNA’s, microRNA’s
14
Q
Pol 3
A
- in the nucleoplasm
- transcribes tRNA, and 1/4 of the rRNA’s
15
Q
RNA polymerases transcribe…
A
- different types of RNA
16
Q
Promoters
A
- DNA where RNA pol binds to begin transcription
- vary somewhat in DNA sequence
- basal machinery (ex:RNA pol) and multiple transcription factors are needed at the promoter to amplify/inhibit transcritption
17
Q
Core eukaryotic promoter “cis” elements
A
- is ~200bp up or downstream from the start site
- recruit RNA pol 2 to allow basal levels of transcription
- built into the DNA
- Initiator (Inr sequence) near the start
- TATA box (w/ or w/o BRE) ~25 bases upstream of the start OR downstream promoter element (DPE)
- specific and robust transcription typically requires other proximal control elements (ex:CAAT, GC box) and/or distant enhancer elements
18
Q
RNA pol is recruited to core promoters via…
A
- basal accessory actors such as TATA Binding Protein (TBP)
- TFIID (containing TBP) binds TATA box first (or DPE) then recruits Other basal accessory factors (Ex. TFII: A,B,F,H) to help recruit/stabilize RNA Pol 2
- TFIIH is helicase (mostly) and kinase: phosphorylates Pol 2 to release from Initiation Complex
19
Q
Manner of Regulation of Gene Transcription
A
- Most gene expression is regulated in a time/cell type specific fashion
- is induced via intracellular/ extracellular “signaling” via transcription factors.
- Constitutive “housekeeping” genes are always expressed in all cells, all the time.
- Ex. Actin, Tubulin, GAPDH
- Other regions of genome are always quiet (heterochromatin).
- Ex. X-inactivation
20
Q
Gene Expression is Often Regulated By…
A
- Transcription Factor Proteins (some act as repressors)
- Ex. LacI (Inhibitor/Repressor): typically binds the Operator/Promoter region and blocks RNA pol
21
Q
operon
A
- Group of Genes with Related Function Under Control of Same Promoter.
- Common in bacteria- not in eukaryotes
- Produce polycistronic mRNA
22
Q
Lactose Triggers Induction of Lac Operon
by…
A
- by Binding LacI Repressor Allowing Transcription Initiation
- Lactose in environment leaks in across the few constitutively open permease channels.
- Lactose binds LacI, changes LacI shape
- LacI repressor can no longer bind operon and inhibit RNA Polymerase
- lacZ, lacY, and lacA expressed (in presence of lactose in environment)
23
Q
Example of transcription factors acting as positive regulators of transcription
A
- Positive Regulation of Lac Operon via CRP (cAMP Receptor Protein)
- cAMP binds to CRP changing its shape
- CRP can now bind near Lac promoter and interact with/stabilize RNA polymerase.
24
Q
Cis control elements function
A
- recruit and bind regulatory transcription factors
- typical eukaryotic gene promoter have multiple
25
core promoter
- Required for Transcription
- General Transcription Factors (TFIID, etc..) and RNA Polymerase binds
- itself may produce “Basal” transcription
26
proximal control elements
- Variable and gene specific
- Close to promoter
- Recruit Regulatory Transcription Factors Such as: “GC box” binds SP1, “CAAT box” binds CAAT box binding protein.
27
Transcription factors are proteins that...
- Bind Basal Transcription Machinery or Alter chromatin status
- Both of which enhance (or inhibit) transcription efficiency.
28
Transcription Factors Bind to ...
- Specific DNA Consensus Sequences
- Many different Transcription factor families: Helix-turn-Helix motif, Leucine Zipper, Zinc Finger, etc...
29
Transcription Factors Typically Have a _____
and a_____
-DNA Binding Domain
- Trans-Activation Domain.
30
Transcription Factor Activity is Often Modulated via...
- Phosphorylation
- PO4 Adds (-) Charge to Protein, Changes its Shape and Function.
- Ex. CREB = cAMP Response Element Binding Protein
31
CBP
- CREB Binding Protein
- catalyzes histone acetylation, and RNA polymerase stabilization.
32
Enhancers Likely “Loop back” to ...
- Join Core/Proximal Promoter Elements
- “Trans” Activators help Bind/Stabilize Basal Transcription Machinery.
- Coactivators: Histone Acetyltransferase (HAT), SWI/SNF modify/re-position nucleosomes
33
Combinatorial Model of Gene Expression
- Helps explain why only certain genes are expressed in specific cell types/times.
- Different cells express different suites of transcription factors driving gene specific expression in different cell types.
- chromatin status also affects expression levels.
34
Nucelosomes Form an Impediment for...
- RNA Polymerase, and can negatively affect transcription efficancy.
- Chemical Modifications Affect Nucleosome Structure/ DNA Packing State
- (Heterochromatin –vs- Euchromatin)
35
Chromatin Must be Remodeled During ...
- transcription
36
Common Histone Chemical Modifications Affect...
- DNA packaging
- Precise “histone code” Dictates transcriptional availability
37
Histone Acetylation Enhances Transcription- by...
- by removing positive charge on histone lysines
38
Histone Methylation can repress or activate transcription- depending on...
- which lysine
- DNA Methylation also inhibits transcription- particularly at “CG islands”)
39
Methylation of DNA Cytosines Represses Transcription By...
Inhibiting Transcription Factor/ Polymerase Binding
40
Significant Eukaryotic RNA Processing Occurs...
- post-transcription
- 5'cap
- poly(A) tail
- splicing
- chemical modification (methylation)
- additions/deletions
41
All mRNA Receive
- a 5'Cap and a PolyA tail
42
5'cap
- Stabilizes mRNA
- Positions Ribosome
- is 7-methylguanosine
43
PolyA tail
- 50-250 nucleotide AAAA tail
- placed by a Poly(A) Polymerase
- AAUAAA upstream, GU rich downstream of tail
- Contributes to Nuclear Export into Cytoplasm
- Confers mRNA stability, and Ribosome recognition,
- Modulates mRNA Stability via Length (LongerAAA = more stable
44
How does RNA pol know where to go?
promoters
45
TATA box
- recruits TATA binding proteins
- most common promoter
- if a core promoter element doesn't have a TATA box it has to have a downstream promoter element (DPE)
46
x-inactivation
- random
- extra chromosome in females is tightly coiled and muted (mostly) so there isn't double the expression of proteins
47
Substrate Induction of Lactose Catabolic Pathway in Bacteria
- if e.coli is in an environment with lactose then galactoside permease will create a few pores in the plasma membrane to allow lactose in (used for energy)
- if not in the environment LacY, LacA, and LacZ are turned off and Lacl gene is turned on
48
LacY, LacA, LacZ, and LacL
- LacY: galactoside permease
- LacA: acetylase (acetylates lactose as it enters cell)
- LacZ: beta-galactosidase
- LacL: represses gene transcription
49
CREB
- part of cyclic AMP
- very common
- binds to CRE and if phosphorylated it also binds to CBP (puts acetyl group of chromatin to make it more like euchromatin and more transcriptionally active)
- enhances transcription
50
"Distal" enhancer sequence
- affect transcription rates from afar
- core promoter required
- variable distance/ orientation (up/downstream)
- contributes by looping back
- believed to possibly loosen up and stabilize chromatin
51
protein/gene expressed is determined by...
- transcription factors available
- chromatin status
52
Histones
- specific histones have specific amino acids in them
- if we change the histone's a.a code it changes the gene being expressed
- some can be post transcriptionally modified (changes how they fold and how tightly they package)
- when acetylation occurs histone becomes more heterochromatin like and more transcriptionally active
53
DNA methylation
- inhibits transcription particularly at CG islands (tend to be tightly bound and transcriptionally quite)
54
Rett's syndrome
protein expression level are either over or under expressed
55
How is mRNA degradation rate controlled?
- Via 5’ Cap and PolyA Tail Length.
- Longer polyA tail = longer mRNA half-life.
- PolyA Tail Length Dictates Stability: 20min-24hrs
56
Silencing
- making the opposite oligo of mRNA we want to silence
- influences mRNA stability
- quality control methods that also helps with differentiation
- silencing w/ microRNA's can be used to (KO) genes in lab to study the gene's function
57
Short (~20nucleotide) expressed MicroRNA’s (miRNA) control...
- stability and translation rates of 1000’s of different mRNA’s by binding to mRNA 3’UTR.
58
Splicing
- the large introns (noncoding) are spliced out (excised)
- exons that code for protein are spliced together
- introns and exons are roughly equal in number within organisms
59
Spliceosome Complex
- ~50 proteins that Precisely Splices Introns Out of mRNA.
- Composed of snRNP’s (small nuclear ribonucleoproteins)
- Upstream GU interacts with downstream AG (spliced out)
- Can be modulated (alternative splicing of exons)
- can dramatically effect protein variability
60
Alternative mRNA Splicing
- Allows for Amplification of Possible Protein Products from Single Gene.
- Ex: Drosophila DSCAM Gene (Down’s Syndrome Cell Adhesion Molecule)
- 38,016 possible "spliceforms" of the same gene product
- gets rid on introns and splices different version of exons together (each exon have multiple versions of it)
61
individual exons often code for...
individual protein domains
62
What RNA demonstrates significant processing?
- tRNAs
- once transcribed...
- leader sequence is removed
- replacement of nucleotides w/ CCA
- chemical modification of bases
- excision of introns
- processing
63
Export of mRNA Through Nuclear Pores
- requires appropriate protein complement to be present on mature mRNA.
- If Introns, uncapped, or no PolyA tail are present then mRNA can’t leave
64
Examples of appropriate protein complement for export of mRNA through nuclear pores
- snRNP’s: should be gone from mature mRNA- prior to export.
- Cap Binding Complex (CBC): contributes to mRNA stability, and helps initiate translation in cytosol should be present- prior to export
- Nuclear Export Receptor: protein complex deposited onto mRNA after appropriate splicing and polyadenylation should be present- prior to export
65
How to tell if 2'cap is present on mRNA
- the presence of CBC which is a cap protein
66
Differential Gene Expression Underlies ...
-Cell Phenotype Differences.
- All cells within an organism typically contain the same DNA sequence.
- different cells (Ex. kidney versus neuron) express different genes/proteins.
- individual cells can “change over time” based on signaled protein expression changes.
67
How DNA MicroArray displays differential gene expression between normal and cancer cells. (steps)
1) isolate mRNA
2) use reverse transcription to make cDNAs labeled with fluorescent dyes
3) combine cDNAs and hybridize to DNA microarray
68
Mechanisms cells use to Regulate Gene Expression.
- Genomic: Chemical modification of DNA and Histones (Methylation, Acetylation)
- Transcriptional: Combinatorial model of gene expression (Express Different Transcription Factors)
- Post-Transcriptional: Differential mRNA Splicing, RNA Degradation (ex:length / stability of the tail)
- Protein Level: Translation Rate, Protein Degradation, Post-Translational Modifications (PO4)
69
Ribosomes “Translate” ____ Into _____.
mRNA, proteins
70
Proteins are typically ___ functional directly after translation, why?
- not
- Significant post-translational processing occurs to make a protein fold/function “just right.”
71
_______ mRNA is Released Out Via Nuclear Pores.
- Processed/Mature
- Ribosomes associate with mRNA in Cytoplasm.
72
What makes cells different from each other?
- What genes they express and when
- How they make proteins and certain levels of proteins
73
Ribosomes associate with _____ in cytoplasm and “____” it to make proteins
- mRNA, read
- Ribosomes clamp down on mRNA to read the code and translate them into protein
74
Ribosomes are _____ _______ complexes assembled in the ________.
- large cytosolic (Protein/RNA), nucleolus
- some are free in the cytoplasm others are bound to the E.R
- subunits are made by several RNA and require rRNA in order to make the ribosome complex
75
mRNA Polypeptide “Code”
- untranslated: 5' end, 5' cap
- coding sequence: start codon, coding genes, stop codon (UAG, UAA, UGA)
- untranslated: 3' end, Poly(A) tail
76
Ribosome “Reads” mRNA in a _ - _ direction.
- 5'-3'
77
start and stop codons
- start: AUG / methionine (where ribosomes usually sit)
- stop: UAG, UAA, UGA
78
that mRNA typically contains 5’ and 3’ __________ regions.
- untranslated
- the entire mRNA is not translated
79
Untranslated regions may
may have to do with the stability of mRNA or recruiting the ribosomes to the right space
80
genetic triplet code is...
- 3 nucleotides coding for a specific amino acid
- universal: code works in every organism known to date
- unambiguous: the sequence only codes for 1 specific amino acid
- degenerate: amino acids can be coded for by multiple codons (ex: Lys is AAA and AAG)
- non-overlapping: triplet codons are discrete
- 61/64 code for a.a the other 3 are stop codons
81
Translation Overview
(3 steps)
- Initiation: Ribosomes scan for 5’ AUG (Kozak consensus sequence: ACCAUGG )
- Elongation: AA shuttled in by tRNA’s and linked via peptide bond (N-terminus to C-Terminus)
- Termination: UAG stop codon reached and protein factor releases polypeptide.
82
How do ribosomes know where to go?
the 5' cap may direct them
83
AUG sequence requires _____ ______ to be a start codon
kozak sequence
84
Ribosomes Function
- Reads mRNA Code and Appropriately Positions Two Incoming tRNA’s to Catalyze Peptide Bond Formation Between Amino Acids.
85
Ribosome sites
- A (aminoacyl): “Arrival” new tRNA
- P (peptidyl): tRNA/aa “Peptide bonds” (within nascent polypeptide)
- E (exit): empty (no a.a) tRNA “Exit” site
86
Peptide bonds are...
- Strong Covalent Chemical Bonds that keep amino acids together
- ribosomes catalyze the formation of peptide bonds
87
nascent polypeptide chain grows...
- NH2 – COO- (N-terminus to c-terminus)
- C-terminus is where the next amino acid attaches to
- the first amino acid is on the n-terminus and the last is on the c-terminus
88
How do we get rid of proteins?
proteases
89
tRNA
- Carry Individual Amino Acids to Ribosome
- several types
90
tRNA structure
- stem/loop
- Amino acids are attached to CCA of 3’ end via ester bond (right amino acids need to go the right tRNA)
- “3” base-pair anticodon hybridizes transiently with mRNA codon. (critical)
91
wobble hypothesis
- mRNA and tRNA line up in ribosome in such a way that allows some flexibility in pairing 3rd anti-codon base.Thus, last nucleotide can vary yet still code for same aa.
- (the 1st and 2nd a.a are whats important for coding for main acids (allows flexibility with the 3rd)
- is how we can have multiple codons for the same amino acid
92
Aminoacyl-tRNA Synthetase
- Links Appropriate Amino Acid to tRNA.
- 20 different synthetases for 20 different tRNA’s.
- Specificity results from unique tRNA sequences near the 3’ end and the anticodon.
- ATP required
93
steps of Aminoacyl-tRNA Synthetase
1) Amino acid and ATP bind to Synthetase
2) ATP attaches to the amino acid causing it to lose pyrophosphate
3) tRNA binds to Synthetase and AMP unattaches
4) aminoacyl tRNA (activated amino acid) detach from Synthetase
94
Translation Initiates at...
- First AUG in Appropriate Consensus Sequence.
- Generally, initial Methionine is removed post translationally- so mature proteins have different NH2 termini
- Eukaryote 40S ribosome attaches to mRNA 5’cap and “scans” for first AUG in a Kozak consensus sequence (ACCAUGG)
95
Shine-Dalgarno sequence
- sequence 3-9 purines (AGGA)
- upstream of AUG
- binds/orients 30S ribosome subunit (There is a complementary pyrimidine stretch in 16S rRNA)
- in prokaryotes
96
Translation steps require ___
- GTP
97
Initiation factors
- help ribosomes hop on mRNA
- requires GTP
- 3 of them
98
Polypeptide elongation
- EF-Tu(GTP) nonselectively shuttle incoming tRNA’s to “A” site.
- If codon/anticodon complementary GTP is hydrolyzed, and EF-Tu leaves (Error rate ~1:10,000) (EF-Tu(GTP) regenerated via EF-Ts).
- Two tRNA/AA’s closely juxtaposed allow peptide bond formation. (No direct energy required) rRNA catalyzes peptide bond formation- acts as a “ribozyme” (peptidyl transferase)
- EF-G(GTP) translocates mRNA 3 bases relative to 30S subunit “Empty” tRNA exits at “E” site
- Growing peptide exits through tunnel ~40aa/sec
- Next tRNA arrives and Nascent polypeptide immediately starts folding with help of chaperones
99
polypeptide elongation summary
- Works by ribosome shifting down a triplet codon
- Random process where multiple tRNAs try to sit in the tRNA until the right one comes around and can sit
- EF-Tu: pop in the tRNAs
- Ribosome: catalyzes peptide bond transformation
- Work reasonably fast
- Enery required in multiple parts of the process (ATP and GTP)
- Nascent tries to fold into the most stable form possible
100
Translation Termination
- requires energy (GTP)
- No tRNA’s exist that base pair with Stop codons
- Release factor protein mimics tRNA structure, and binds to “A” sites: UAG,UAA,UGA triggering release of polypeptide from the “P”site tRNA.
- Ribosome components disassemble and are recycled
101
Antibiotics
- Many Common Antibiotics Inhibit Prokaryotic Ribosomes by fitting into the ribosome of bacteria to block them from making proteins (causing the bacteria to die)
- Small Differences in Ribosome Structure Between Eukaryotes and Prokaryotes
- Can be Taken Advantage of to Make Selective Drugs.
102
Translation Rates Can be Controlled
via
- Translation Factor” proteins on UTR’s. (Note: similarity to transcription control)
- Ex. Control of Ferritin Translation Rate via IRE Translational Repressor. (controlling translation of the protein)
- is less common
- can repress translation
103
Ferritin
- globular protein complex
- stores intracellular iron keeping iron soluble and non-toxic
104
microRNA's (miRNA)
- control stability and translation rates of many different mRNA’s especially by binding to 3’UTR.
- may be expressed in cells that are NOT supposed to express target protein (another form of quality control)
- as long as the miRNA is completely opposite of the target RNA it will bind and either block out ribosomes or degrade the rna
105
miRNA example
- Let-7 miRNA
- Lin-41 miRNA
106
why does miRNA control translation?
to allow for more control of production of proteins and better response to the environment
107
Translation Efficiency (Protein Product Formation) Correlates with...
- mRNA Stability
- mRNA degradation rate is affected by 5’ Cap, PolyA Tail Length, and homologous microRNA expression.
108
Post-Translational Protein “Processing”
- Initial Methionine is typically removed (by methionine aminopeptidase)
- Folding (Chaperones) (w/o the protein isn't functional)
- Protein targeting
- Protein-Protein Interactions
- Protein Splicing
- Cleavage (Zymogens)
109
Post-Translational Chemical Modifications (often reversible)
- Phosphorylation
- Methylation
- Acetylation
- Glycosylation
- Lipid anchors
- Ubiquitylation
110
Protein Folding is Critical To...
- Function
- Occurs immediately after (even during) translation.
- Dictated primarily via chemical bonding/ interactions between amino acids-coded for within primary acid structure.
- Ex. Positive charged amino acids attract negatively charged amino acids.
111
Protein-Protein Interactions
- Transcription Factor Complexes Stabilize Basal Transcription Machinery (RNA pol.)
- Proteins often come together to work on the central dogma (protein-protein interactions is absolutely needed)
112
Chemical Modifications
- Affect Protein Structure/Function (Ex. Histones)
- Acetylation, Methylation, Phosphorylation
- kinases add phosphates for phosphorylation
113
what amino acids can be phosphorylated
serine, threonine and tyrosine
114
Phosphorylation Changes Rb Structure so
- that it can no longer bind/sequester E2F.
115
How does phosphorylation affect protein structure? How do PO4 groups get put on target proteins? How do they get taken back off?
- it alters the protien's charge which alters it's shape
- kinase
- protein phosphatase hydrolyze the phosphoric acid
116
Protein “Splicing”
- Can Occur After Translation.
- Introns spliced out (sometimes self-catalyzed “intramolecular”).
- less common than mRNA splicing
117
Protein cleaving
- Certain Proteins (Ex. Caspases) Need to be Cleaved to Become Active.
- Inactive Zymogens
- Can “self” activate if at high enough local concentration (clusters).
- NH2 (prodomains) are cleaved of inactive procaspases at the cleaving site
- after caspase is active and has a 2 large and 2 small subunits
118
Protein targeting
- Proteins Need to Be Targeted to the Appropriate Cellular Location.
- to get to the right location the targets must be built in
- Ex. Nuclear Localization Signal (NLS) Targets Proteins to Nucleus.
119
Nuclear Localization Signal:
- Short stretches of (+) Lys and Arg (typical target signal).
- Recognized by Importin.
120
Peroxisomal Targeting Signal
- = SKL at Protein Carboxy Terminus.
- Pex5p recognizes protein-SKL in cytosol, transports, and helps import into peroxisome.
- Add/subtract SKL signal shows it is necessary and sufficient.
121
Lipid-Anchors
- Directs Proteins to Cellular Membranes (target proteins for where they need to go)
- Sometimes Reversible
- is a complicated lipid groups added post translationally to proteins
- Is how rab5a sinks into endosome membranes
- Ex. Prenylation targets Rab5a to Endosome membranes
122
Precise Folding is _______ for Protein Function.
- critical (for function)
- proteins that mis-fold tend to aggregate, and perhaps even cause cell death (seen in Alzheimer patients especially)
- Information required for proper folding resides in the primary amino acid sequence.
123
chymotrypsinogen
cuts up proteins
124
Chaperones
- Help Nascent (or Damaged) Proteins Fold Properly.
- Detect improperly folded proteins- and “help” them to fold.
- are upregulated in response to stress- (aka. heat shock proteins).
- require ATP.
- found in the e.r (because the e.r has lots of ribosomes)
125
Cytosolic Chaperones:
- Hsp60 (GroEL)
- Hsp70 (Works in concert with Hsp40)
- Hsp90
126
ER Resident Chaperones
BIP, Calnexin, Hsp90B
127
Hsp60 (GroEL)
- A Large Multi-protein Barrel Shaped Chaperone that helps “refold” mis-folded proteins.
- 1mDa
- Double ring 14mer barrel structure.
127
How might a chaperone recognize an improperly folded protein?
Molecular cues (think opposites attract)
128
How does Hsp60 (GroEL) work?
- Hydrophobic entrance recruits (only) improperly folded peptides that have “exposed” hydrophobic patches of amino acids
- ATP hydrolysis stimulates conformational changes that cause substrate entry into barrel.
- Hydrophilic interior barrel amino acids somehow promote proper substrate folding.
- is an example of protein-protein interaction
129
Heat Shock Protein 70 (Hsp70)
- Promotes Proper Protein Folding by binding to “exposed” hydrophobic patches in newly synthesized proteins.
- ATP required
130
How Heat Shock Protein 70 (Hsp70) works
- Hsp70 binds hydrophobic patches (~7 amino acids) in nascent protein substrate, and (with help from Hsp40) “coats” polypeptide as it leaves the ribosome (ensuring new protein doesn’t inappropriately fold/aggregate).
- Once entire protein is translated, other proteins (Ex. BAG-1) induce Hsp70 to release full-length nascent protein so it can properly fold
131
Is protein folding always successful? What should a cell do if it can’t fold a protein? (ex: mutant protein X)
- no
- get rid of it
132
2 main cellular protein degradation pathways (quality control pathway)
- lysosomal
- proteasomal
133
lysosomal pathway
- Extracellular proteins (endocytosis)
- Bacteria (phagocytosis)
- Autophagy (Aggregates and organelles)
- “Large scale”
- less specific than proteasomal
134
proteasomal
- Mostly unwanted intracellular proteins
- Ex. Mutant Proteins
- “Small scale” (requires Ub tag)
- more specific than lysosomal
135
lysosome
designed to get rid of things coming from a variety of places
136
endocytosis
- creating a vacuole to bring into the cell that can then be targeted for the lysosome to be degraded
- early endosome -> endocytosis -> late endosome /MVB -> lysosomal degradation
- rab11 (AD), BIN1(AD), VPS34(AD), LRRK2 (PD)
137
autophagy
- Old organelles and unwanted cytoplasmic entities (Ex. protein aggregates) can also be encapsulated and recycled to lysosome
- normal process but unknown how the membrane knows where to go
- upregulate in certain pathologic states (ex: severe starvation, aggregation in disease)
138
autophagy steps
- vesicle nucleation: isolation membrane forms
- vesicle elongation: membrane start surrounding unwanted materials
- autophagosome forms: membrane completely surrounds unwanted material
- docking and fusion: lysosome with lysosomal hydrolase fuses with the autophagosome
- vesicle breakdown and degradation
139
what happens to Individual Proteins That Can’t Be Folded Correctly (even with help of chaperones)
they are typically sent to 26S proteasome for degradation
140
CHIP
- is an E3-Ub-Ligase
- interacts with Chaperones (Hsp70)
- seems to help triage cellular proteins using ub-ligase for either folding and/or proteasomal degradation.
141
TAU
- Microtubule Associated Protein that is known to form aggregates in Alzheimer’s Disease.
- commonly misfolds
- leads to tauopathy (neurodegenerative disorders due to misfolding of tau)
- meant to stabilize proteins
142
proposed role of Hsp70/CHIP in tau protein triage
- is tau binds with a productive co-factor is forms a function protein
- if tau has ubiquitin put on it by CHIP it goes down an alternative ubiquitin-indpependent degradation pathway
- Tau get ub -> ub chain forms -> proteins are aggregated (excess tau ub) and form tau aggregates or the it's degraded by the proteasome
143
Proteasomes
- “Barrel Shaped” Protein Recycling Factories that degrade mutant or unwanted proteins.
- multimeric protein complex
- opposite of ribosomes
- is the degradative organelle (degrades into short peptides)
- very active in the immune system
- potentially multiple version of them that are slightly different
144
ubiquitin/ proteasome pathway
- ub conjugation: Ubiquitin tree is ligated onto target protein (Ex. cyclin or mutated Tau) (trade E2 for the tree)
- Ub-tagged proteins are sent to 26S Proteasome for degradation.
- with ATP peptides and ub-specific proteases are formed
- deubiquitination occurs resulting in ub
- with ATP ub binds to E1
- ub activation: ub trades it's E1 for E2
145
Proteins Destined For Proteasomal Degradation Are Tagged with?
- PolyUbiquitin “Trees.”
146
Ubiquitin (Ub)
- 76aa protein
- Tags other “substrate” proteins for destruction
- has internal Lys’s
147
PolyUb trees are put onto ___ by ____
- lys residues of substrate (Ub itself has internal Lys’s)
- a series of enzymes (E1,E2,E3,E4)
148
E1
- “Activates” Ub and transfers it to E2
- only 1 known type
149
E2
- Carries activated Ub to E3’s
- (Several, Ex. Ubc6,Ubc7)
150
E3
- Ub Ligase- attaches Ub to specific protein substrates (E3 does the ligating)
- Many: Ex. Mdm2, APC, CHIP
151
E4
- Elongates “Ub tree” on certain substrates
152
Degron
- located on substrates
- signals for destruction (PEST, cyclin destruction box, N-end rule)
153
What are the signals for ub?
- Pest sequence
- cyclin destruction box
- n-end rule (that depend what specific amino acid is on that terminus determines degration rate)
154
26S Proteasome
- is a protein recycling factory
- Ubiquitous- Located in every cell, throughout the cell (nucleus and cytoplasm)
- made up of 19s cap + 20s core
- Other “caps” may help localize 26S or modulate peptidase activity
- Proteins enter and are digested inside chamber, 4-20aa peptides are released.
155
20S Proteasome
- core polypeptide degradation
- Houses interior catalytic sites
- Sequesters Proteolytic Active Sites
156
19S Regulatory Particle
- cap polypeptide bonding
- Recognizes ub tagged protein and Unfolds protein substrates (they're too big otherwise)
157
20s proteasome core structure
- Large barrel-shaped Multi-Protein Complex 700kD
- 7 fold symmetry
- Alpha subunits form “Pore” (1 on each side of the beta subunit)
- Beta subunits sequester 3 proteolytic active sites (is in the middle)
- (Trypsin-like, Chymotrypsin-like, Caspase-like)
- both subunits have different proteins
158
Regulators of 20S Proteasome
- 19S Regulatory Particle (ATPase) is required for in vivo degradation and it recognizes Ub, opens 20S pore, unfolds, and pumps in substrates
- Others: 11S REG’s (α, β, γ) and PA200 Increase rates of degradation…
- Chimera’s = 19S - 20Proteasome - 11SREG
159
Proteasome Summary
- Ubiquitin tag is ligated onto unwanted target protein via a series of enzymes (E1, E2, E3).
- Ub-tagged proteins are sent to 26S Proteasome.
- 19S recognizes and unfolds/pumps substrate protein into 20S catalytic core.
- Ubiquitin tree is recycled.
- 20S core digests/recycles unwanted substrate into short peptides.
160
Rough ER is Studded with
- Ribosomes that Translate “Secretory Proteins” into the ER Lumen
- ER also contains many proteasomes to degrade misfolded proteins
161
ERAD (ER- associated degradation)
- ER Translated Proteins That Fail to Fold Properly are subject to a special proteolytic quality control pathway,
- Unwanted ER substrates are basically pumped out of ER lumen Through SEC61 translocon channel Into awaiting proteasomes.
- the proteosomes wait to degradate recently synthesized misfolded proteins right outside the ER
162
Proteasomes are one more form of
gene regulation
163
Increase a protein product
- Up Regulate Transcription
- Stabilize mRNA
- Up Regulate Translation
- Stabilize Protein
164
Decrease a protein product:
- Down Regulate
- Sequester
- Degrade Protein
165
main levels of gene expression
- genome (nucleus)
- transcription (nucleus)
- RNA processing and nuclear export (nucleus)
- translation (cytoplasm)
- post-translation (cytoplasm)
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genome level of gene expression
- chromatin
- possible gene amplification or deletion (rare)
- possible DNA rearrangements (rare)
- chromatin condensation
- DNA methylation
- Histone acetylation, changes in HMG proteins, nuclear matrix
167
transcription level of gene expression
- gene available for expression
- transcriptions controlled by transcriptional factors
168
RNA processing and nuclear export level of gene expression
- primary RNA transcript (pre-mRNA)
- RNA splicing and processining
- mRNA in nucleus: transport of mRNA to cytoplasm
169
translation level of gene expression
- mRNA in cytosol
- mRNA degradation (turnover) -or-
- translation (controlled by initiation factors and translational repressors
170
post-translational level of gene expression
- polypeptide product in cytosol or ER
- protein folding and assembly
- possible polypeptide cleavage
- possible modification
- possible important into organelles
- functional protein: protein degradation (turnover)
171
Modulation of p53 Levels Is partially regulated by
- its targeted destruction via the proteasome.
- Upon detection of DNA damage, ATM adds a PO4 to p53 so that Mdm2 can no longer target it for destruction (p53 stabilized) a quick way to “upregulate” it.
- p53 can then act as a transcription factor-
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Mdm2
a Ub-Ligase that targets p53 for 26S degradation.
173
How might PO4 affect p53 so that Mdm2
can no longer recognize it?
- po4 causes p53 to stabilize and changes p53’s structure.
- If p53 is po4 is folds a different way and the degron is no longer exposed so MDM2 doesn’t see it
174
Mutant CFTR is Destroyed via
- ERAD Thus, CFTR is not trafficked to plasma membrane (loss of function)
175
Cells Expend Significant Energy to...
- Maintain Proper Protein Structure.
- Either chaperones fold correctly or target “unfoldable” proteins to proteasome.
- Mutant/Misfolded Proteins Tend to Aggregate (which lead to neuronal cell death) and Likely causes disease
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diseases caused by protein aggregation
- Alzheimers
- Parkinsons
- Huntingtons
- Prion Disease (Mad Cow, Creutzfeld Jacob, etal.)
- Tauopathies
- Picks Disease
- Spinocerebellar Ataxia’s
177
Aggregation of Misfolded Proteins
Is a Common Pathologic Hallmark of Neurodegenerative Disease.
178
Alzheimer's
- Arrowhead: Intracellular Tau tangles;
- Arrow: Extracellular Amyloid plaque
179
Pick's disease
Tau inclusions
180
Prion disease
PrPSc amyloid deposition
181
Parkinson's disease
alpha synuclein Lewy bodies
182
Machado-Joseph's disease
(SCA3) Intranuclear polyQ inclusions of mutant Ataxin-3
183
Higher power micrograph of mutant ataxin
3 polyQ inclusion.
184
The 20S Proteasome Redistributes to
- polyQ Nuclear Aggregates
- Suggesting that the Proteasome is Attempting to Degrade Them.
185
How Might Protein Aggregation Affect Proteasomal Degradation?
- Proteasome may have a harder time isolating proteins
- Might not be able to unfold proteins that are aggregated or it may clog up the proteasome
- unknow for sure still
186
Let7 miRNA
- inhibits translation of Lin-41(involved in epidermal cell development).
- targets/inhibits Hbl-1 in neurons.
- acts as a “master regulator” by binding to and inhibiting translation of multiple mRNA’s in different tissues/times during life of the organism.
187
Lin41 miRNA
is constitutively expressed, but translation is inhibited after initial developmental stages by Let-7 miRNA.
188
polypeptide elongation requires
GTP
189
Mutant CFTR
- ~ 600 different mutations cause CF, Most common is a deletion of 3 nucleotides = delta 508 (Phe)
- Delta 508 mutants are not trafficked to plasma membrane correctly (degraded in ER)
- Cl- (and Na+) do not leave, H20 doesn’t follow (thick mucus)
190
transcription coactivators
- Ex. SWI/SNF Complex, CBP
- transiently displace nucleosomes allowing efficient transcription.
191
Cells Communicate With Each Other Using
Diffusible Extracellular Signals
192
Ligand binds receptor- causing
“changes” inside receiving cell.
193
short distance cell communication
- paracrines
- autocrines
194
long distance cell communication
- neurotransmitters
- hormones
195
Most Signaling Molecules are Either...
protein or lipid derivatives
196
lipid derivatives
- steroids
- eicosanoids
197
Most Receptors are
- Plasma Membrane Proteins
- Although lipophilic signalling molecules (steroids) have intracellular receptors
198
Receptors
- Specific for ligand
- Subject to inhibition
- Note: same ligand can bind multiple receptors (receptor dictates affect)
199
Many Steroid Receptors Are
- Intracellular (Cytoplasmic or Nuclear).
- Hydrophobic
200
Ligand bound receptor translocates to? Why?
nucleus to affect transcription of specific genes.
201
signal molecules
- dopamine
- norepinephrine
- epinephrine
202
Lipophilic messenger steps
- lipophilic messenger diffuses across the cell membrane
- messenger bind with cytoplasmic or nuclear receptor to make a hormone-receptor complex (in nucleus)
- hormone receptor complex bings to hormone response element (HRE)
- mRNA forms and exits the nucleus to form protein
203
Genes Containing _______ _________ ______Consensus Sequences are “_______” in response to steroid
- appropriate regulatory region
- signaled
204
Response element for Glucocorticoids
5' ag AA cannntg TT ct 3'
205
Response element for Estrogen
5' ag GT cannntg AC ct 3'
206
Response element for Thyroid hormone
5' ag GT catg AC t 3'
207
Steroid response elements
- similar to each other
- are scattered throughout genome in various promoters.
- each steroid typically controls multiple downstream genes.
208
Ligand binds ___________ aspect of receptor- changing receptor ________.
- extracellular
- conformation
209
Intracellular part of receptor does?
transduces signal
210
Production of second messengers (Ex. cAMP), Phosphorylation cascades (Kinases) May...
alter gene expression or have more localized effects (Ex. reorganize cytoskeleton)
211
receptor- ligand binding steps
- receptor- ligand binding
- signal transduction via second messengers
- cellular responses
- changes in gene expression
212
Three major Plasma Membrane receptor subclasses
- Ion channels
- G-protein coupled
- Tyrosine Kinase
213
Ligand Gated Ion Channel Example
Nicotinic Acetylcholine Receptor: Na+ influx and cell depolarization
214
Ligand Gated Ion Channel Steps
- closed channel is opened with messenger bind to receptor
- ions (Na+, K+, Cl-) move through open channel
- change in electrical properties of cell
215
G-Protein Coupled Receptors
- Major Class (super-family) of Signal Transduction Receptors That Signal Via Associated G-proteins.
- All of which display Magnificent 7 (Transmembrane domains)
- Major target for pharmaceutical intervention (~50% of all drugs
- Ex. Muscarinic Acetylcholine receptor.
216
Many G-Protein Coupled Receptors Have
various receptor subtypes that all act via associated heterotrimeric G-proteins
217
Heterotrimeric G-proteins Transduce
Signal From Receptor
218
Heterotrimeric G-proteins Transduce Signal From Receptor Steps
- Binding of ligand changes receptor conformation so it can now interact with G-protein.
- G1 conformation change (to active form), so it now prefers GTP over GDP.
- G alpha separates from beta y and modulates downstream target protein function (Ex. Adenyly cyclase).
- Beta y - help localize/anchor to plasma membrane, sometimes “affects” own target (Ex. K+ channel).
- Galpha eventually hydrolyzes GTP (to GDP) becoming inactive- and re-able to rebind beta y
219
Different G-protein Subclasses
- alpha a
- alpha olf
- alpha I
- alpha q
220
alpha s
- “Stimulate” Adenylyl cyclase
- Ex. beta-adrenergic, Dopamine-1
221
alpha olf
- Stimulate Adenylyl cyclase
- odorant receptors
222
alpha I
- “Inhibit” Adenylyl cyclase
- Ex. 2-adrenergic, opiate, Dopamine-2
223
alpha q
- Phospholipase C
- Acetylcholine muscarinic type 5
224
Adenylate Cyclase
- is a Major Target for Many G-protein Linked Receptors.
- Produces cAMP (second messenger)- which stimulates Protein Kinase A (PKA)
- Gs/olf = stimulate adenylate cyclase
- Gi = inhibits adenylate cyclase
225
PKA phosphorylates
- many downstream targets- changing their shape/function.
- (Ex. CREB, K+ channels)
226
Protein Kinases
- Add PO4 to Substrate.
- Serine, Threonine Kinases (Ex. PKA) add PO4 to Ser, Thr, of substrate.
- Tyrosine Kinases (Ex. NGF Receptor) auto-phosphorylate own Tyr.
227
Phosphorylation of a protein substrate causes
a strong negative charge to be added which modulates protein folding/shape and consequent function.
228
Phosphorylation is a __________ post-translational modification? allowing?
- reversible
- Phosphotases take PO4 group back off
- Thus, some proteins can “toggle” back in forth between different shapes/functions, based on their phosphorylation status.
229
Two cAMP (Produced by Adenylyl Cyclase) Activate
Protein Kinase A (PKA)
230
PKA phosphorylates a number of
downstream targets on their Ser and Thr residues Such as: K+ channels, Tau, and the transcription factor CREB.
231
Phosphorylation of CREB by PKA Allows it
- to Bind CBP
- Ultimately Up-regulating Expression of Genes Containing CRE’s in their promoter.
232
CBP has ______ _____________ activity. Which?
- histone acetyltransferase
- activity- which promotes euchromatin formation, and helps to stabilize the transcription machinery complex.
233
Downstream targets of CREB include
genes involved in learning and memory
234
why so many steps in cell signaling?
to help amplify and allow for more tweaking at each step (more diversification of cells)
235
Transduction Cascades Allows
Amplification/Diversity of Signal
236
Ligand activated receptor activates
multiple G-proteins, which in turn activate multiple adenylyl cyclases, etc...
237
transduction cascade
- transmitter
- transmitter activates receptor
- receptor activates G-proteins
- G-protein stimulates adenylyl cyclase to convert ATP to cAMP
- cAMP activates protein kinase A (multiple downstream targets)
238
PKA phosphorylation inhibits
K+ channels
239
Receptor Sub-Type Dictates
- Ultimate Physiologic Effect
- Same ligand (Ex. Norepi) can have dramatically different effect on downstream cells depending on which receptor the cell is expressing.
240
NE beta Receptor
- Gs
- Stimulates Adenylyl cyclase
- Increases PKA Activity
- Increases PO4 of downstream Targets
- Ex. Vasodilate
241
NE alpha 2 Receptor
- Gi
- Inhibits Adenylyl cyclase
- Decreases PKA Activity
- Decreases PO4 of downstream Targets
- Ex. Vasoconstrict
242
G alpha q Activates
The Phospholipase C/ PKC Signaling Pathway
243
G alpha q Activating The Phospholipase C/ PKC Signaling Pathway Steps
- G alpha q interacts with and “turns on” Phospholipase C and changes shape
- Phospholipase C cleaves PIP2 into bioactive IP3 and DAG molecules.
- DAG activates Protein Kinase C (PO4 many targets Ex. Na+ channels)
- IP3 travels to ER and binds the IP3 receptor to allow Ca++ release from ER lumen
244
Ca++ Exerts Many of its Effects Via
Protein Calmodulin
245
Protein Calmodulin
- Small protein with four Ca++ binding domains.
- Binding of Ca++ ions changes calmodulin’s conformation allowing interaction with other proteins such as Kinases
- (CaM Kinase I, II, IV etc...).
246
G alpha q Signaling Via IP3 Leads to
Ca++ Release from ER Storage
247
Calcium is a major signaling molecule
And therefore has _____ sequestration/release pathways
many
248
Concentration of Ca++ is ~______ times lower in Cytosol (than extracellular)
10,000
249
Growth Factor Tyrosine Kinase Receptor signaling can also lead to....
- intracellular Ca++ release.
- Ex. of Overlapping signaling Pathways.
250
Growth Factor Associated Receptors
- are proteins kinases
- Act as Dimers
- Dimerization causes them to auto-phosphorylate each other Ser/Thr, Tyr
- Can affect multiple downstream pathways (Ras, PKC, etc..)
251
Growth Factors Are Secreted Molecules That...
Act At Short Range to Affect cell growth, division, development, and response to injury
252
Cells not only need “nutrients” to survive, develop, and differentiate but also require
- growth factor- signals
- different growth factor receptors are expressed in different cell types.
253
Trophic Factors
- Are Required to Keep Cells Alive.
- by inhibiting Cytochrome C release from mitochondria- and consequent Apoptosis
- Growth Factors also may increase cyclin expression
254
Epidermal Growth Factor (EGF)
- Tyrosine Kinase Receptor
- Expressed in many tissue types
- Mutations in EGFR are linked to lung cancer.
255
How EGF receptor works
- Ligand binding causes EGF Receptor dimerization, and autophosphorylation of Tyr residues.
- Phosphorylated Tyr are recognized by SH2 binding domains of proteins such as GRB2 and Phospholipase C.
256
Downstream Signaling from EGF Receptor Via GRB2
triggers
- gene expression changes allowing epithelial cell survival and growth
