Final - New Material Flashcards
(243 cards)
1
Q
What maintains human blood glucose levels at about 5.5 mM?
A
The opposing actions of insulin and glucagon
2
Q
How does eating affect insulin levels?
A
-Causes a rise in glucose levels, which in turn causes increased secretion of insulin from the beta cells of the endocrine pancreas
3
Q
True or False: Insulin acts to increase blood glucose levels
A
FALSE: Insulin acts to DECREASE blood glucose levels
–> Does this by promoting glucose uptake and storage in muscle (fat) and adipose tissue (fat) and glucose storage in liver (glycogen)
4
Q
What six things do insulin levels affect in humans (that we need to know)?
A
i) Glucose uptake
ii) Amino acid uptake
iii) Acetyl-Coa –> Fatty acids
iv) Pyruvate –> Glycolysis (gluconeogenesis)
v) Protein synthesis
vi) Glucose –> Glycogen
5
Q
Is insulin anabolic or catabolic?
A
Insulin is anabolic
–> Signals building the of macromolecules, cells, and tissues
6
Q
What kind of receptor is the insulin receptor?
A
Receptor Tyrosine Kinase (RTK)
7
Q
Describe the configuration of the insulin receptor
A
- The Insulin Receptor Is a Monomer of Two Different Proteins Linked by Disulfide Bonds
- Insulin receptor (IR) monomer is comprised of an external α-subunit linked by disulfide bonds to the internal β-subunit
- -> Two of these monomers are linked together by disulfide bonds so that IR is already functionally ‘dimeric’ without insulin
8
Q
What happens to the insulin receptor when insulin binds?
A
- When insulin binds, it causes a conformational change in the β– subunits that activates their kinase activity and brings the cytoplasmic
domains closer together so they can phosphorylate each other. - Thus, the two β–subunits transphosphorylate each other, just as occurs with other RTKs
9
Q
What is Insulin Receptor Substrate (IRS)?
A
- IRS (insulin receptor substrate) is an adaptor protein, although it is more commonly called a docking protein that is recruited by its PTB domain to the
phosphotyrosines on IR. IRS itself becomes phosphorylated on many tyrosines by the actions of IR. - - These P-Tyr serve as recognition sites for other
signaling proteins as described for other RTKs
10
Q
What does the stimulation of the MAPK pathway via insulin receptor do? What part of the insulin receptor stimulates this pathway?
A
- Grb2 on the insulin receptor stimulates MAPK
- Leads to cell proliferation and Gene transcription
11
Q
What part of the insulin receptor causes DIRECT stimulation of cell proliferation and gene transcription when it binds to a phosphate group on IRS?
A
PLCgamma
12
Q
What is The Phoshatidylinositide 3-Kinase (PI3K, PI 3-K)?
A
• Major kinase in cell growth and survival
• Interacts with many signaling networks,
leading to different biological outcomes
• Pathway altered frequently in cancers
• Promotes cell proliferation and chemo
resistance
13
Q
True or False: PI 3-kinases Are a Family of Protein Kinases That Phosphorylate the 3 position of Phosphoinositols
A
FALSE: PI 3-kinases Are a Family of LIPID Kinases
That Phosphorylate the 3 position of Phosphoinositols
14
Q
What is the difference between IP3 and PIP3?
A
IP3: Stimulates the release of Ca2+ from the ER
PIP3: Recruits proteins to the membrane
15
Q
Describe the structure of PI 3-K
A
- PI 3-K has two subunits, p85 and p110
- PI 3-kinase converts the membrane phospholipid ‘PIP2’ into ‘PIP3’
16
Q
What recognizes PIP3?
A
PIP3 Is Recognized by PH Domain-Containing
Proteins (ex: Akt / PKB)
17
Q
What does Akt / PKB do?
A
- Akt (Protein Kinase B, PKB) recognizes and binds PIP3, which brings it in proximity to PDK1 (another PH domain-containing protein). PDK1 is a serine threonine protein kinase
- PDK1 phosphorylates Akt, activating and releasing it from the membrane; Akt can now phosphorylate its downstream target proteins on ser/thr.
- These targets tend to promote cell growth, cell proliferation, and cell survival
18
Q
What are the three targets of Akt?
A
- mTORC
- MDM2
- BAD
19
Q
What does mTORC do when phosphorylated by Akt?
A
Increases cell growth and proliferation
20
Q
What does MDM2 do when phosphorylated by Akt?
A
- This is an E3 ubiquitin ligase that targets p53 –> This leads to inappropriate cell survival
- –> No cell cycle arrest
- –> No DNA repair
- –> No apoptosis
21
Q
What does BAD do when phosphorylated by Akt?
A
Inhibits Bad to PREVENT apoptosis
22
Q
What is PTEN?
A
PTEN is a lipid phosphatase and it dephosphorylates PIP3 on the 3’ position to yield PIP2. PH domain-containing proteins will no longer be recruited to the membrane. PTEN thus is a tumor suppressor of this pathway, and it is mutated in many cancers.
23
Q
What does Binding of a growth factor or insulin to an RTK activate?
A
Binding of a growth factor or insulin to an RTK activates PI 3-kinase, a lipid kinase.
24
Q
What does PIP3 serve as recognition site for?
A
PIP3 serves as recognition site for PH domain containing kinases such as PDK1 and Akt (PKB).
25
When does PDK1 phosphorylates and activates Akt?
PDK1 phosphorylates and activates Akt when it is docked at the membrane
26
What does activated Akt do? What is the ultimate consequence of this?
Activated Akt phosphorylates many proteins, including TOR, MDM2, and BAD. The ultimate consequence is an increase glucose uptake and utilization, increase cell growth, increase cell division, overriding of cell cycle checkpoints, and inhibition apoptosis.
27
What is PI 3-K signaling negatively regulated by?
PI 3-K signaling is negatively regulated by the lipid phosphatase PTEN, which dephosphorylates PIP3 back to PIP2.
28
List the pathway from Akt --> mTORC
Akt --> Tsc2 (phosphorylated by Akt)--> Rheb-GTP --> mTORC
--> Tsc2 is a GAP for Rheb (which inactivates Rheb). Akt phosphorylates Tsc2, which inactivates it, allowing Rheb to become activated via the addition of a GTP, which in turn can activate mTORC
29
What does Rapamycin do in mammals?
Rapamycin inhibits the ser/thr kinase activity of TOR.
--> TOR coordinates cell growth with the environmental conditions. When inactivated by rapamycin, it can no longer do this.
30
Describe Rapamycin
• Rapamycin is a bacterial peptide discovered in a bacterial sample from the island of Rapa Nui (Easter
Island) in the South Pacific.
• It was originally developed as an antifungal drug, but this was abandoned once it became clear that it had strong immunosuppressant and anti - proliferative actions.
• Mostly used now to prevent rejection in organ transplants, esp. of kidneys
31
What is the central role of TOR in cell growth?
TOR is a ser/thr kinase and it phosphorylates proteins in a number of key pathways
--> Ex: glucose uptake and utilization by TOR, which ultimately leads to an increase in lipid synthesis
32
What Is the Basis for Detection of Metastases by PET Scans?
The Excessive Production of Glucose in Response to Signaling Through PI 3-K and TOR
33
Describe how TOR stimulates protein synthesis
- Signaling of growth factors through RTKs and nutrients both activate TOR. As TOR is a ser/thr
kinase, it phosphorylates many proteins to stimulate translation.
- 4E-BP is an inhibitor of eIF4E. When TOR phosphorylates 4EBP, it no longer inhibits eIF4E, and translation is stimulated.
- S6K (S6 kinase) is an kinase that phosphorylates the ribosomal protein S6, which increases the ability of ribosomes to translate mRNAs encoding ribosomal components.
34
Define apoptosis
- Apoptosis is ‘programmed cell death’ - a specific
biochemical pathway to remove cells that are not
needed or that are damaged, especially by
extensive DNA damage
• Apoptosis is controlled by a family of proteins that
promote (pro-apoptotic) or inhibit (anti-apoptotic) it.
35
Describe BAD and how phosphorylation of it by Akt affects signaling
• BAD is a pro-apoptotic protein that promotes
apoptosis by forming a complex with Bcl2, one of
the key anti-apoptotic proteins, and inhibits it.
• Akt phosphorylates BAD; when it does so, it
inactivates it and hence frees up Bcl2, which inhibits
apoptosis. Akt thus promotes cell survival indirectly
by inhibiting apoptosis.
36
How does Signaling Through RTKs Promote Cell
| Survival?
- By inhibition of Bad
- Phosphorylation of BAD by Akt is inhibitory for apoptosis.
- -> Phosphorylated Bad releases Bcl2, which is anti-apoptotic.
- -> When Bad is inactive, apoptosis is blocked and cells survive even when they shouldn’t.
37
What Five Major Signaling Pathways Are
| Activated by GPCRs, RTKs, or Both?
- PKA
- CaM-Kinase
- PKC
- MAP Kinase
- Akt Kinase
38
What are cytokines?
– Cytokines are small proteins, peptides, or glycoproteins that can be made by virtually any cell. Thus, they differ from the true ‘hormones,’ which by definition come only from endocrine glands.
– Cytokines are very analogous to hormones in their affinities for receptors.
– Cytokines act locally and hormones act systemically, but both can enter the blood stream
39
True or False: Cytokine receptors have intrinsic enzyme activity
FALSE: Cytokine receptors do NOT have intrinsic enzyme activity
– Cytokine receptors associate tightly with cytoplasmic tyrosine kinases such as Src or members of the JAK tyrosine kinase family
40
Describe the shape of cytokine receptors
Cytokine Receptors Transverse the Membrane
Once, Function as Dimers, and Are Closely
Linked to Intracellular Tyrosine Kinases
41
What are transcription factors for cytokine receptors?
STATs
42
What are the ligands of cytokine receptors (other than cytokines)?
```
– Prolactin
– Erythropoietin
– Leptin
– Interleukins
– Interferons
```
43
What do Activated Cytokine Receptors do?
- Activated Cytokine Receptors Activates the Jak-STAT Pathway
- Stats activate many genes, including those
involved in the immune response, hematopoiesis, Myc, Fos, mammary gland development, milk production, etc
44
Where are STATs found in their inactive state?
In their inactive state, STATs are found in the
| cytosolic compartment as monomers
45
What are STAT proteins recruited by? What happens once the STATs are recruited?
- The STAT proteins are recruited via their SH2
domains to the phosphorylated cytokine receptor
- Once there, a JAK will phosphorylate the STAT,
causing it to come off the receptor. The SH2
domain of one STAT will recognize the
phosphotyrosine on another STAT, and they will
dimerize.
- Dimerization of STAT leads to its nuclear
localization, DNA binding, and regulation of
transcription.
46
How is specificity in the JAK/STAT pathway achieved when there are over 30 hormones that utilize this pathway?
• In part, specificity comes from multiple genes
encoding both JAK kinases (4) and STATs (7)
• These JAK kinases interact with different cytokine
receptors and phosphorylate distinct STATs that
recognize different genes
• Not all cells have all of the isoforms of the kinases
or the STATs
47
What does erythropoietin (Epo) induce?
Red blood cell formation
--> Used by runners to enhance performance (ethically wrong)
48
Describe Receptor Serine/Threonine Protein Kinases (TGFβ Receptor Family)
• These are the receptors for a related family of about
33 ligands in humans including TGFbs (transforming
growth factor βs), BMPs (bone morphogenetic
proteins), anti-Mullerian hormone (AMH, MIS),
inhibins, and the activins.
• During development, these proteins regulate pattern
formation, influence proliferation, differentiation,
tissue remodeling, and promote cell death
• In adults, they are involved in cell homeostasis,
tissue repair and remodeling, inhibition of cell
proliferation, and immune regulation
49
What do Transforming Growth Factor-betas (TGFβ) do?
Inhibition of cell proliferation
50
What are all of the members of TGFβs?
Transforming growth factor β, activins, inhibitins, and nodals
51
What do Members of the BMP Subgroup of
| the TGFβ Superfamily contribute to?
Osteogenesis and Chondrogenesis
52
What are Two inflammatory cytokines that activate NF-kB?
TNF-a and interleukin-1 (IL-1)
53
What is NF-kB activity is regulated by?
inflammation, infection, and other stressful situations such as ionizing radiation
54
What are NF-kB proteins?
NF-kB proteins are latent transcription factors that are
present in most animal cells and are central to stressful,
inflammatory, and innate immune responses.
55
T or F: Many signaling systems rely on the activation of latent gene regulatory proteins (transcription factors) to transmit the signal to the DNA that gene transcription must be altered.
TRUE
56
What are Stats (cytokine receptors) and Smads (receptor ser/thr kinases) activated by?
Phosphorylation
57
What are β-catenin, NF-κB, Notch, and Hedgehog/sonic hedgehog activated by?
Regulated degradation of a component in the signaling pathway
58
What does regulated proteolysis involve?
Regulated proteolysis’\ involves polyubiquitination followed by proteasomal degradation. It differs
from the proteolysis triggered by misfolded proteins in that the targets of the E3 ubiquitin ligases are very specific – not just an exposed hydrophobic region
59
What causes Wnt signaling?
Lack of phosphorylation of the protein to be degraded
--> If the protein IS phosphorylated, it would go through the NF-kB pathway
60
What is a degron?
A degradation signal
61
List the steps of the Wnt pathway
Wnt (the transmembrane protein) --> Dsh --> APC + GSK3 --> Beta-Catenin --> Gene Regulation and Cell Proliferation
62
List the steps of the Jak/Stat pathway
Cytokine (transmembrane receptor) --> Jak --> Stat --> Gene Regulation
63
List the steps of the TGFBeta pathway
TGFBeta (membrane protein) --> R-Smad --> Smad4 --> Gene Regulation
64
List the steps of the ANP Pathway
ANP (membrane protein) --> Guanlylyl cyclase --> PKG
65
List the steps of the NO pathway
NO --> Guanylyl cyclase --> PKG
66
T or F: Wnts are a family of secreted ligands that act as contact-dependent mediators to control many aspects of development. They also contribute to cancer formation
FALSE: Wnts are a family of secreted ligands that act as PARACRINE mediators to control many aspects of development
67
What pathway uses the Frizzled family of cell surface receptors?
Wnt
68
True or False: GPCRs are the only cell surface receptors that transverse the membrane 7 times
FALSE: Frizzled does this too, but G proteins don't activate Frizzled, so it's considered a different thing
69
What is the co-receptor necessary for activation of Frizzled?
LRP
70
What does the Wnt pathway accomplish?
The Wnt pathway acts by regulating the proteolysis of β- catenin, which functions both in cell adhesion and as a transcription factor
71
What keeps Wnt responsive genes inactive?
Wnt responsive genes are kept inactive by the Groucho co-repressor bound to the LEF1/TCF activators
72
Where is β- catenin in its inactive state?
In its inactive state, the b-catenin that is not bound to cell-cell adherens junctions is sequestered in the cytoplasm in a “degradation” complex with 4 other proteins
73
What four proteins are part of the β- catenin degradation complex? (actually need to know this card)
```
• Axin – scaffold protein
• APC – (Adenomatous polyposis coli),
scaffold protein
• GSK3 – (glycogen synthase kinase 3),
ser/thr protein kinase
• CK1 – (casein kinase 1), ser/thr kinase
```
74
How is β- catenin marked for degradation?
Phosphorylation of b-catenin by CK1 and then
by GSK3 marks it for ubiquitination and rapid
degradation.
75
What do Wnt ligands do to Frizzled and LRP?
Wnt ligands cause the Frizzled and LRP receptors to cluster together, which activates the Dishevelled (Dsh) scaffold protein
Somehow, Dsh recruits GSK1 and CK1 to the receptor complex, and LRP gets phosphorylated.
- -> Axin is also brought to the receptor.
- -> All of this disrupts the ‘degradation complex’, so b-catenin is no longer phosphorylated and therefore is no longer recognized by an E3 ligase.
76
What does b-catenin do when it is no longer marked for degradation (via phosphorylation)? (Wnt pathway)
- b-catenin goes to the nucleus, kicks off the Groucho repressor and binds to LEF1/TCF, activating gene transcription.
•Target genes for b-catenin include Myc, which is a transcription factor that promotes cell growth and proliferation.
77
What is Apc?
Apc is a tumor suppressor gene. Tumor suppressor genes are recessive so both alleles typically need to be inactivated to cause cancer
78
What 2 receptor pathways activate NF-kB (that we care about)?
- Cytokine
| - Infection
79
What 3 things does NF-kB do (that we care about)?
- Anti-apoptosis
- Proliferation
- Inflammation
80
Where is NF-kB in its inactive state?
In its inactive state, NF-kB is stored in the cytoplasm with an inhibitory protein, I-kB, which hides its NLS
81
What triggers the NF-kB pathway?
Binding of an inflammatory cytokine to its receptor triggers a protein kinase cascade that leads to the
phosphorylation of I-kB by I-kB kinase.
82
What is TAK1? What pathway is it involved in?
TAK1 is a serine/threonine protein kinase. Involved in NF-kB
83
What does phosphorylated I-kB do?
The phosphorylated I-kB is recognized by a specific E3 ligase, which ubiquitinates I-kB (now it is polyubiquinated I-kB)
84
What does Polyubiquitinated I-kB do?
Polyubiquitinated I-kB is recognized and degraded by the proteasome
85
T/F: NF-kB binds to a specific DNA regulatory sequence in the enhancers/promoters of target genes to activate gene transcription
TRUE
86
What does Removal of I-kB do?
Removal of I-kB exposes NLS on NF-kB so that it can enter the nucleus
87
Compare Wnt signaling to NF-kB signaling
Wnt signaling > no phosphorylation of β-catenin and thus its stabilization
NF-κB signaling > phosphorylation and destabilization of I-κB > free NF-κB
88
What are the two other names for nuclear receptors (NR)?
intracellular receptors or steroid receptors
89
What are NRs?
They are ligand-activated transcription factors, and the
family of receptors is activated by a wide variety of lipophilic (hydrophobic) molecules
- Once ligand is bound, the NRs undergo a conformational change and regulate the transcriptional activity of target genes by binding to specific DNA sequences.
• The NRs direct diverse relatively long term physiological processes such as reproduction, metabolism, development, immune responses, wound repair
90
What are the Normal Physiological Effects of Estrogen
| in Both Males and Females?
* Cardiovascular and neuronal activity; protects against dementia
* Liver, fat, and bone cell metabolism
* Reproduction – development and function
* Immune responses
91
True or False: Estrogen is a mitogen
TRUE: Estrogen receptor targets genes involved in cellular proliferation, cellular differentiation, cell survival. Thus, it is a mitogen
92
T/F: Testosterone is a mitogen
TRUE
93
The receptors for many lipophilic hormones,
metabolites, and xenobiotic endocrine disruptors
are intracellular proteins that function as ___________ - ____________ transcription factors
Ligand-activated
94
T/F: NRs are found in all kingdoms of life
FALSE: They're only found in animals
95
What are Orphan NRs?
Ligands and functions are unknown
96
List the 4 main functional domains of an NR
- DNA binding domain
- Ligand binding domain
- Transcriptional activation 1
- Transcriptional activation 2
97
What is a Type I NR?
Bind to inverted repeats as homodimers. May be
| cytoplasmic and/or nuclear (the steroid receptors)
98
What is a Type II NR?
Bind to direct repeats as heterodimers with RXR. Are
| nuclear. Usually called the orphan receptors
99
What are steroid hormones derived from?
All steroid hormones are derived from cholesterol.
– They are lipophilic and enter cells by passive diffusion.
– Vitamin D is also a steroid hormone, although it binds to a Type II receptor
100
T/F: Ligands for the Type II receptors are varied in
| structure and are all hydrophilic
FALSE: Ligands for the Type II receptors are varied in
| structure but are also LIPOPHILIC (hydroPHOBIC)
101
Describe ligands for type I NRs
1. Are all derived from cholesterol
2. Are present in serum in pM to nM amounts
3. Bind NR with high specificity
4. Bind NR with high affinity
102
What is PPARα?
- Binds to Type II NRs
| - the fibrate receptor. Lowers serum triglycerides
103
What is PPARγ?
- Binds to Type II NRs
| - the glitazone receptor. Lowers serum glucose
104
What is LXR?
- Binds to Type II NRs
| - the oxysterol receptor. Lowers serum cholesterol
105
What is FXR?
- Binds to Type II NRs
| - the bile acid receptor. Raises serum cholesterol
106
T/F: Type II NRs have high ligand-binding specificity
F: Low binding specificity, are somewhat promiscuous
107
What are Selective Receptor Modulators (SRMs)?
Ligands for specific nuclear receptors. The goal is to develop tissue-specific regulators.
108
What are SERMs?
Selective ER modulators (birth control -agonists, chemotherapy – tissue dependent)
-A type of SRM
109
What are SARMs?
Selective AR modulators (steroid abuse-agonists,
chemotherapy - antagonists)
- A type of SRM
110
What is agonist or antagonist activity determined by in SRMs?
Agonist or Antagonist Activity Is Determined by Tissue -Specific Co-Regulatory Proteins
* The conformation of ER changes depending on which SERM binds.
* The proteins that recognize the different ER forms sometimes vary with different cell/tissue types.
* The differences in the intercellular proteins provides the basis for tissue-selective agonism/antagonism.
111
What motif to NRs use to bind DNA?
Zinc finger
112
What are Response Elements for Steroid Receptors (Type I NRs)??
Inverted repeats (palindromes)
• Response elements are called HREs (hormone response elements), SREs (steroid response elements), or by the receptor name such as GRE for glucocorticoid response element. These HREs are usually enhancers
113
What are response elements for Type II NRs?
Direct repeats
114
Give a Summary of the Mechanism of Action of Type I NRs
• The NR resides in the cytoplasm and/or the nucleus
in a heat shock protein inhibitory complex.
• The binding of steroid hormone to the NR releases
the chaperone complex, and two receptors dimerize.
• The dimerized NRs travel to the correct site on
chromosomes and binding to their HREs. In the
process, chromatin structure is often opened up.
• Coactivators associate with the NRs and gene
transcription is increased.
115
How do NRs Directly and Indirectly Regulate Gene Transcription?
Although NRs have major effects on the transcription of genes, each receptor type binds directly to relatively few genes. However, those genes may encode
transcription factors, which then go on to bind to other genes. Thus, NRs can indirectly regulate a large number of genes through transcriptional cascades. Bottom line: they have many indirect effects and their effects can be quite delayed because of this.
116
Give a Summary of the Mechanism of Action of Type II NRs
* Type II NRs bind to DNA as heterodimers with a common heterodimerization partner, RXR (retinoid X receptor)
* Type II nuclear receptors are typically bound to DNA in a corepressor complex in the absence of ligand (rather than in a hsp complex), which silences transcription.
* Binding of ligand to the non-RXR receptor partner releases the corepressor molecules, which are replaced by coactivators.
* Coactivators associate with the NRs and gene transcription is increased.
117
List Characteristics of Apoptosis
• Cytoskeleton collapses; cells becomes more compact
• Nuclear envelope disassembles and chromatin
condenses
• Nuclear DNA breaks up into nucleosomal-sized
fragments
• Mitochondrial function is lost
• Phosphatidylserine flips from the inside to the outside
of the plasma membrane bilayer
• Plasma membrane is altered (blebbing), allowing
dying cells to be engulfed by macrophages via
phagocytosis
118
What is a TUNEL assay? What is it used for?
TUNEL assays add a fluorescent nucleotide to DNA ends. The DNA ends are extended with the template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT). TdT adds fluorescently labeled dUTPs to the blunt ends of
fragmented DNA. Large numbers of DNA fragments leads to bright fluorescent dots in apoptotic cells.
--> Used to detect apoptotic nuclei
119
What does having a phosphatidylserine on the outside of a cell do?
Having phosphatidylserine on the outside of the cell serves as an ‘eat me’ signal, which is why phagocytes engulf it.
120
What do caspases use as an active site for hydrolysis?
Caspases utilize an active site CYSTIENE residue as the
| mechanism of hydrolysis.
121
T/F: Caspases are synthesized directly into their active form
FALSE: Caspases are synthesized as inactive proenzymes that need to be activated by cleavage to remove an inhibitory pro-peptide segment.
122
Where do caspases cleave?
Caspases cleave the peptide bond at a specific four amino acid sequence on the C-terminal side of an ASPARTATE residue
123
List characteristics of initiator caspases
– Initiator Caspases (start the ‘caspase cascade’)
• Caspases 8 and 9
• Exist as inactive, soluble monomers in the cytosol
before the apoptotic signal
• Function is to activate the executioner caspases
124
List characteristics of executioner caspases
Executioner (Effector) Caspases (cut target proteins)
• Caspase 3
• Exist as inactive, soluble dimers in the cytosol before
the apoptotic signal
• Function is to degrade cellular proteins
125
Give an Overview of Caspase Activation
• Caspases are originally made in an inactive,
procaspase form.
• Extrinsic or intrinsic signals trigger the assembly of adaptor proteins with the procaspases.
• Upon binding of the adaptor proteins (FADD or Apaf), the procaspases dimerize and are activated, trans-cleaving the procaspase into a large and small subunit.
• The initiator caspases then clip the executioner caspase dimer, and the execution dimer undergoes a
conformational change that makes it active.
126
What is a nuclear lamin? What cleaves these?
Executioner caspases cleave nuclear lamins, the
| structural protein responsible for the nuclear envelope and nuclear integrity
127
What do executioner caspases do to Mdm2?
Executioner caspases degrade Mdm2, the E3 ubiquitin
ligase that targets p53 for ubiquitination and thus
proteasomal degradation. Thus, without Mdm2, p53
remains high and can signal cells to die.
128
What is CAD (caspase-activated DNase)?
• CAD (caspase-activated DNase) is the enzyme
responsible for DNA cleavage during apoptosis
– CAD is normally inhibited by an inhibitor (iCAD)
– Executioner caspases inactivate iCAD by cleaving it
– This releases an active CAD, which fragments DNA between nucleosomes
129
T/F: DNA cut by CAD forms a ladder of fragments based on the frequency of cutting between nucleosomes.
TRUE
130
What is the difference between the extrinsitc and intrinsic apoptotic pathways?
Extrinsic: External cell signals
Intrinsic: Internal cell signals
--> In either case, protein:protein interactions mediated by specific modular interaction domains are particularly
important to these signaling pathways
131
What is DD in apoptosis?
Death Domain
132
What is DED in apoptosis?
Death Effector Domain
133
What is CARD in apoptosis?
caspase-associated recruitment domains
134
What are death receptors?
• Death receptors are transmembrane proteins that have an extracellular ligand binding domain, a single transmembrane domain, and an internal death domain
(DD). TNF and Fas are the major examples.
•The receptors (and ligands) are homotrimers.
• Liganded death receptors cluster and interact with the FADD (Fas-associated death domain) adaptor proteins
• FADD recruits an initiator caspase (caspase 8) with a death effector domain (DED)
135
Describe Death by FasL (extrinsic apoptotic pathway)
* The extrinsic pathway starts with a ligand trimer binding to a death receptor trimer such as FasR.
* The receptor has a DD that attracts an adaptor protein such as FAAD that has both a DD and a DED.
* The adaptor protein attracts a complex that contains several initiator caspases (typically caspase 8)
136
Give examples of external apoptotic signals
- TNF-alpha
- Fas
- TRAIL
--> All are members of the TNF (tumor necrosis factor) family, also known as death receptors
137
What is DISC?
```
- The aggregate of receptor, adaptor, and procaspase is
called DISC (death-inducing signaling complex).
• The aggregation (dimerization) of the procaspases ‘activates’ them so they can trans-cleave (clip the caspase into 2 fragments) each other
```
138
What happens once the procaspases dimerize in extrinsitc apoptosis?
• Once the procaspases dimerize, the procaspase
dimer becomes active and can trans-cleave itself.
• This is followed by a conformational change that
enables the initiator caspase to move around the cytosol and activate executioner caspases by proteolytic cleavage.
139
List the steps of the intrinsic apoptotic pathway
p53 --> Mitochondria --> Cytochrome C --> Caspase --> Caspase 3 --> Apoptosis
140
List the steps of the extrinsic apoptotic pathway
Death Receptors (FasR) receive death signal --> FADD --> Caspase 8 --> Caspase 3 --> Apoptosis
141
What is the signal for intrinstic apoptosis?
Cytochrome c from inside the mitochondria
142
What does released cytochrome C do in apoptosis?
- Released cytochrome c binds to the procaspase activating adaptor protein Apaf-1 (an adaptor protein), causing its aggregation into an apoptosome.
• Apaf-1 contains the CARD protein interaction domain,
which binds the initiator procaspase 9.
• Procaspase 9 is activated while in the apoptosome
143
What triggers release of cytochrome C from the mitochondria?
• The Bcl2 family of proteins (15 members) control the
release of cytochrome c.
• Bcl-2 family members come in two functional classes:
– Anti-apoptotic (survival): e.g., Bcl2
– Pro-apoptotic (death): e.g., Bad, Bax
• Pro- and anti-apoptotic Bcl2 proteins bind to each other to form hetero-oligomers.
• The balance between the activities of the pro- and antiapoptotic proteins determines whether a cell lives or dies by the intrinsic pathway
144
What does Bcl2 do? (intrinstic apoptotis)
Inhibit pore formation by the BH123 proteins -- prevents clustering and cytochrome c release
--> reside in the outer mitochondrial membrane
-Act mainly by binding to and inhibiting the action of
pro-apoptotic family members, i.e. Bcl2 inhibits
Bax
• Act to prevent inappropriate apoptosis
145
What does Bax do? (intrinstic apoptosis)
Form the pores that let out cytochrome C
--> found on mitochondrial membrane (Bak) or free in the cytoplasm (Bax)
- Apoptotic signals make these form oligomers on the outside of the mitochomdrial membrane. Formation of the oligomers forms a pore that allows release of cytochrome c
146
What does Bad do? (intrinstic apoptosis)
Inhibit the anti-apoptotic proteins
--> The BH3 domain mediates interactions between pro- and anti-apoptotic family members
--> BAD Is a Target of the PI 3-Kinase Pathway
147
T/F: p53 is pro-apoptotic, and it is the most commonly
| mutated tumor suppressor gene in humans
TRUE
--> p53 is a transcription factor
148
What is the 'cell-cycle control system' or 'cell cycle clock'?
- cell-cycle control system or cell cycle clock governs
progression through the cell cycle.
• The term cell cycle refers to the ordered series of events that lead to cell division and the production of two daughter cells.
149
What are The two most basic functions of the cell cycle?
* Chromosomal replication: to faithfully produce two copies of the genetic information.
* Chromosomal segregation: to ensure each daughter cell receives a copy of the entire genome.
150
What is cytokinesis?
Cell division
151
What are the phases of the cell cycle?
* G1 = Gap #1 (growth phase 1)
* S = DNA synthesis (DNA replication)
* G2 = Gap #2 (growth phase 2)
* M = when the nucleus and cytoplasm divide
152
What is Go phase?
The variability in cell cycle times is due to the variability in the length of Go. Go is a resting phase where the cell is neither dividing nor preparing to divide.
153
Describe prophase
The Duplicated Chromosomes Are Condensed and the Mitotic Spindle Forms Between the Two Centrosomes
The nomenclature for the genomic state during prophase is 2N,4C. There are 2 copies of every chromosome, but 4 chromatids. (Interphase is 2N, 4C after DNA replication)
154
Definition: Centrosomes
Centrosomes: organelles that are the microtubule organizing center and act as the spindle pole during mitosis.
155
Definition: Mitotic spindles
Mitotic spindles: the microtubules and associated proteins that pull the chromatids apart during mitosis.
156
Definition: Kinetochore
Kinetochore is a protein structure that connects the centromere of a chromosome to microtubules of the mitotic spindle
157
Describe prometaphase
The Nuclear Envelope Disintegrates and the Chromosomes Attach to the Mitotic Spindle via Their Kinetochores
2N,4C
158
Describe metaphase
the Chromosomes Are Lined Up on the Equator with Each Chromatid of a Chromosome Attached to Opposite Poles
2N,4C
159
Describe anaphase
The Sister Chromatids Are Pulled to Opposite Sides of the Cell
4N,4C
160
Definition: Ploidy
The number of sets of chromosomes in a cell
161
What is chromosome number?
the number of sets of centromeres (N)
162
What is number of chromatids?
the number of copies of each DNA molecule (C)
163
Describe telophase
4N, 4C
The Segregated Chromosomes Decondense and a New Nuclear Envelope Forms Around Each Set
164
Describe cytokinesis
The Cytoplasm Divides and Two Daughter Cells Are Formed
2N, 2C
165
```
T/F:
46 chromosomes = 2N
92 chromosomes = 4N
46 chromatids = 2C
92 chromatids = 4C
```
TRUE
166
What are checkpoint controls in cell replication?
Negative controls, known as checkpoint controls
operate to prevent cells from continuing division if
the cell is not ready, most notably if the DNA is
damaged or if mitotic spindle attachment is not
complete.
167
At what three points in the cell cycle does it pause to determine if everything is good to go forward?
- -> G2 into mitosis
- -> Metaphase into Anaphase
- -> G1 phase into S phase
168
What do Cyclin-dependent protein kinase complexes (cyclin-Cdks) do?
They regulate the activities of multiple proteins involved in cell replication such at those involved in entry into the cell cycle, DNA replication, and mitosis. The cyclin-Cdks do that by phosphorylating these proteins at specific regulatory serines or threonines.
169
What are Cyclin-dependent kinases (Cdks)?
• are serine/threonine protein kinases that phosphorylate proteins involved in
the cell cycle
• are inactive unless bound to cyclins
• Cdk specificity is determined by the cyclin bound to it
170
What are Cyclins?
- are specialized Cdk-activating proteins but have no enzyme activity
• help direct Cdks to the target proteins
• are only present during the cell cycle stage that they trigger, i.e. the amount
of these proteins changes in a cyclical fashion due to regulated proteolysis.
171
There are 4 Cell Cyclins: G1/S, S, M, and G1. Three of these are required by all cells, and one is not required by all cells. Which are which?
All cells require the G1/S cyclins, the S-cyclins, and the
| M-cyclins. The G1 cyclin is not required by stem cells
172
Describe Activation of Cdk by Cyclin and CAK
(A) Without cyclin bound, the Cdk is in an inactive state because the active site is
blocked by a region of the Cdk called the T-loop.
(B) The binding of cyclin causes the T-loop to move out of the way, which partially
activates the Cdk
(C) Phosphorylation of Cdk by CAK changes the shape of the T-loop even more,
allowing the Cdk access to substrates and full activity
173
What interferes with kinase activity in the cell cycle?
Cdk-inhibiting proteins (CKI’s, CIPs, INKs)
174
What is responsible for the regulated degradation of cyclins in the cell cycle?
The E3 ubiquitin ligases SCF and APC/C are responsible for the regulated degradation of the cyclins.
175
Describe cyclins in G1 and S phase
• During G1 and S phases, the SCF E3 ubiquitin ligase complex ubiquitinates cyclins D and E and thus targets them for degradation
– Cyclins D and E and other targets get phosphorylated
(GSK3b) and thus are recognized as substrates for the
SCF E3 ligase
176
Describe cyclins in anaphase
• The anaphase-promoting E3 ubiquitin ligase complex
(APC/C) ubiquitinates the S- and M-cyclins
– APC/C gets activated by binding of another protein (cdc20) at the metaphase-anaphase transition and targets the M cyclin and other proteins
177
What is SCF activity is dependent upon ?
SCF activity is dependent upon association with an F-box protein
-> The activity of SCF seems to be constant, but the
ability to bind to the target protein is regulated.
178
What does Cdc20 do (cell cycle)?
APC/C target selection is controlled by the activating
| subunit, Cdc20. Cdc20 is inactive unless all the kinetochores are attached to microtubules.
179
Describe how Regulated Proteolysis of the Cyclins During the Cycle Is Triggered by Two Major Ubiquitin Ligase Complexes
- SCF typically recognizes target proteins when they are phosphorylated (activation of the target)
- APC/C is activated by the binding of the Cdc20 protein to it (activation of the ubiquitin ligase)
180
What are CAK, Wee1, and Cdc25? (cell cycle)
CAK: Cdk-activating kinase
Wee1: tyrosine kinase
Cdc25: tyrosine phosphatase
181
What is EF2? (cell cycle)
E2F is a transcription factor that is required to move the
cell from G1 to S.
AP1 = Fos + Jun
182
What four major mechanisms regulate the restriction checkpoint in the cell cycle?
1. Induction of the D cyclins (D1, D2, D3) by
mitogens
2. Induction of the SCF E3 ubiquitin ligase and
degradation of the CKIs (cyclin inhibitory
proteins)
3. Inhibition of Rb, an inhibitor of E2F
4. Induction and activation of E2F, a transcriptional
activator required to induce the synthesis of
proteins needed in S phase, including Cyclin E
183
How Does the Cell Get to G1?
No active cyclin-Cdk so the cells go into G1 (or G0).
184
How are cells kept in G1?
By Rb (a tumor suppressor)
Mitogens stimulate the inactivation of Rb by inducing Cyclin D and activating cyclin D-Cdk.
185
What do mitogens do?
Mitogens Stimulate Synthesis of the D-Cyclins
The mitogen-activated transcription factors have short half lives, ex. Myc has a half life of 25 min. This means
they have to be synthesized de novo before each round of cell division. Thus, the mitogens must be there to stimulate each round.
186
Give a Summary of Entry into G1 and Passage Through R
• Degradation of Cyclin B (M cyclin) initiates G1
• Mitogenic stimulation begins the cell cycle by
inducing transcription factors such as Myc, AP-1
(Fos & Jun), β-catenin, STATs, or by activating
nuclear receptors
– Induction of cyclin D
– Induction of SCF and degradation of CKIs
– Inactivation of Rb and activation of E2F
– Synthesis of G1/S cyclin and other proteins
needed for DNA synthesis
187
What does TGFβ do in the cell cycle?
TGFβ functions as a tumor suppressor and opposes cell
proliferation by preempting the mitogenic pathway.
• TGFβ prevents progression into G1 by
– Inhibiting the induction of Myc
– Inducing the transcription of CKIs
– Keeping Rb unphosphorylated
– Rb inhibits E2F so the G1-S cyclin is not synthesized
– Rb and TGFβ are thus major tumor suppressors of
cell cycle progression through the Restriction point
188
What detects damage to DNA?
A cascade of DNA-dependent kinases
--> These include the ATM and ATR ser/thr protein
kinases
189
What do ATM/ATR do?
ATM/ATR Activation Blocks the Cell Cycle in Two Major Ways:
1. ATM and ATR activate the Chk1 and Chk2 ser/thr
protein kinases. Chk1/Chk2 phosphorylate the Cdc25 tyrosine phosphatase, which leads to its degradation.
(Cdc25 removes the inactivating phosphoryl from
Cdks. Without Cdc25, Cdks cannot be activated.)
2. Both ATM/ATR and the Chk kinases increase the
levels of the p53 transcription factor. p53 regulates genes involved in cell cycle checkpoint controls, among other functions
190
Describe how p53 Induces Target Genes Involved in
| Inhibiting the Cycle
• In undamaged cells, p53 is highly unstable because it
interacts with Mdm2, which is an E3 ubiquitin ligase that
targets p53 to the proteasome.
• Phosphorylation of p53 by Chk1/Chk2 reduces its
binding to Mdm2, so p53 becomes stable.
• p53 is a transcription factor and it induces tumor
suppressor genes such as the CKIs (p21) and proapoptotic genes such as Bax.
191
Describe The Metaphase-to-Anaphase Transition
| Spindle-Assembly Checkpoint
- Separase must be activated by the destruction of
securin to get chromatid separation.
- Only when all microtubules are attached does APC/C get activated by Cdc20. D
192
What are the two heritable properties of cancer cells?
1. They reproduce in defiance of normal
restraints on cell growth and division
2. They invade and colonize space normally
reserved for other cells
– Any abnormal growth is called a tumor
• Benign tumors can not invade other tissues
• Malignant tumors can invade.
– A tumor is a true cancer if it is malignant.
193
True or False: Cancers are not inherited. However, we can inherit predisposition (susceptibility) to cancer.
TRUE
194
Describe how X mosaicism in females affects cancer
In females, because of X mosaicism, one would expect tumors to be a mosaic of cells with one or the other X inactivated, but typically the entire tumor has the same X inactivated, suggesting there was a single initiating cancer cell.
195
True or False: A Single Mutation Is Typically Not
| Enough to Cause Cancer
TRUE
196
What is a carcinoma?
Epithelial cell cancer
197
What is a sarcomas?
Connective or muscle tissue cancer
198
T/F: Dominant mutations are usually gain-of-function, whereas recessive mutations are usually loss-of-function
TRUE
199
What can Conversion of a Proto-Oncogene to an Oncogene Can Cause?
1. increased levels of the proto-oncoprotein, which
will inappropriately increase signaling.
Ex: Increased amounts of RTKs will make the cells
hypersensitive to otherwise-limiting amounts of GF
2. alterations in signaling proteins such as in RTKs
that make the signaling ligand-independent.
Ex: Mutations that delete the external domain of RTKs or change their dimerization properties so that activation
of the RTK is ligand-independent signaling
3. new fusion proteins are generated that have
hyperactive signaling properties.
200
T/F: In the hereditary form of cancer, all cells in the body lack function of one of the two genes that encode Rb.
TRUE
201
In most cases, a single mutation in a tumor suppressor gene is recessive. What is the exception?
p53 is the exception.
202
T/F: CpG is a palindrome when double-stranded.
TRUE
203
What are the 5 Hallmarks of Cancer?
```
– Self-sufficiency in growth signals
– Insensitivity to antigrowth signals
– Reprogramming energy metabolism
– Limitless replicative potential
– Sustained angiogenesis
```
204
What can Mutations in genes encoding GF receptors do?
Mutations in genes encoding GF receptors can cause changes in the receptors such that they dimerize and are active without mitogen. These include amino acid substitutions (red dots) or truncation of the external domain. Alternatively, too much receptor can be made.
205
What is a Philadelphia chromosome?
• A reciprocal translocation between the 9 and 22 chromosomes creates the Philadelphia
chromosome. Transcription generates the BCR-ABL fusion protein from the BCR and Able
tyrosine kinase genes.
• The function of BCR is unknown but Abl is a membrane-associated tyrosine kinase (like Src).
• The tyrosine kinase activity of the BCR-ABL fusion protein is always active, inappropriately
signaling cells to proliferate.
• When this fusion protein occurs in bone marrow, depending on the actual chromosomal
breakpoint, it leads to chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), or
chronic neutrophilic leukemia.
206
How do leukemias occur?
the JAK/STAT pathway gets activated even without cytokines
207
What are the two major tumor suppressors?
The p53 and retinoblastoma (RB) protein
208
What are the functions of p53?
* Promotes cell cycle arrest when DNA is damaged
* Triggers DNA repair mechanisms
* Initiates apoptosis when DNA damage is too severe
* Blocks angiogenesis
* Blocks excessive mitogen signaling
209
Where are mutations most commonly found in p53?
Most mutations in p53 are in the DNA binding domain,
| meaning it can still form a tetramer.
210
What is the Wraburg effect?
aerobic glycolysis: most cancer cells have a high rate of glycolysis and lactate production even when oxygen is present.
Cancer cells thus have
anaerobic respiration! This is similar to what happens in rapidly proliferating cells in embryos and during tissue repair. This generates large amounts of building blocks
AND allows cancer cells to live in hypoxic conditions.
211
What does the p53 Pathway do?
regulates responses to stress and to DNA damage
212
What does the Rb pathway do?
Initiation of the cell division cycle
213
What does The RTK/Ras/PI3K Pathway do?
Transmits signals for cell growth from the exterior of the cell to the nucelus
214
What are driver mutations?
Driver mutations cause or contribute to oncogenesis. They contribute growth advantage to the cells.
215
What are passenger mutations?
Passenger mutations do not contribute to oncogenesis and occur because of the instability of the genome of cancer cells. They are ‘carried along’ but have no significant role in the cancer.
216
T/F: CSCs can evolve from either the cell-of-origin (which could be a stem cell) or from more mature progenitors that have sustained genetic and epigenetic mutations.
TRUE
217
T/F: Cancer stem cells (CSCs) are more sensitive to chemotherapy
FALSE: They're less sensitive
* CSCs have higher levels of free-radical scavengers, which makes them more resistant to DNA damage induced by ionizing radiation therapy
* CSCs have increased DNA damage checkpoint characteristics and DNA repair capacities
* CSCs are more resistant to chemotherapy, in part because they have increased numbers of multidrug resistance transporters (ABC transporters) that export the drugs out of the cell
218
What is the current prevalent hypothesis about recurring cancer?
The prevalent hypothesis is that recurrent cancers (cancers that come back) are largely the consequence of CSCs slowly repopulating the area.
219
Definition: Cancer stem cells
CSCs are functionally defined as cells within a tumor that can sell-renew and that
can give rise to the heterogeneous lineages of cancer cells that comprise the
tumor. They are thought to be responsible for tumor growth, therapy resistance,
and metastasis of some cancers. (Sometimes called tumor initiating cells.)
220
T/F: Signaling is typically paracrine. It can be autocrine, particularly in cancer.
TRUE
221
What do Type I TGFβ receptors do?
Phosphorylate R-Smads
Both receptor subunits possess serine/threonine protein kinase activity.
222
What do type II TGFβ receptors do?
Bind the ligand, phosphorylate Type I receptors
Both receptor subunits possess serine/threonine protein kinase activity.
223
T/F: TGFβ receptors and ligands are very specific
FALSE: They're promiscuous
224
What are TGFβ target genes?
Myc - repression
p21, p15 - induction
Cdc25 - repression
Cyclin D - repression
225
What happens after an R-Smad is dephosphoylated?
Once an R-Smad is dephosphorylated, it cycles out of the nucleus.
226
What are the consequences of TGF-b Signaling in Addition to Transcriptional Regulation?
- Chromatin remodeling
- Transcriptional regulation
- miRNA processing / regulation
227
How does activation of Type II receptors in TGFb signalling affect tyrosine phosphorylation?
Activation of Type II receptors can induce tyrosine phosphorylation of both the Type I and Type II receptors. This allows them to be recognized by other signaling molecules such ShcA and Grb2, which are both adaptor proteins.
228
T/F: Smads can regulate miRNAs
TRUE
229
what happens when TGFβ changes from a tumor suppressor protein to a tumor promoter protein ?
Downstream alterations that usurp the normal functions of the pathway to cause growth promotion
230
Definition: Natriuresis
the excretion of sodium in the urine via action of the kidneys, and usually refers to the excess excretion of sodium (diuretic, anti-diuretic)
231
What is an ANP?
- ANPs are hormones secreted primarily by the heart in response to high blood pressure but they are also
• ANPs primarily act by relaxing smooth muscle cells in blood vessels and stimulating excretion of Na+ and H2O. They regulate salt and water balance and thus blood pressure
- The ANP receptors have guanylyl cyclase activity in their cytosolic domains
232
What do ANPs do?
Regulate blood pressure
233
What is a trget of PKG in the ER?
The IP3-gated Ca2+ channel, which is inhibited by phosphorylation
234
What does NOs use as a nitrogen donor to oxidize N to NO?
NOS uses arginine as the nitrogen donor and O2 to oxidize N to NO
235
List the family of Nitric Oxide Synthases that Make NO
* Neuronal constitutive NOS (nNOS) –signaling in the nervous system. Enzyme activity is regulated.
* Endothelial constitutive NOS (eNOS) – signaling in the vascular system. Enzyme activity is regulated.
* Induced NOS (iNOS) – killing of microbes, viruses (and surrounding cells). Gene transcription is regulated (induced).
236
How are nNOS and eNOS activated?
nNOS and eNOS are activated by the binding of Ca2+-CaM. Ca2+ is released by neurotransmitter action, sheer stress, or pressure and binds to CaM (calmodulin). Both nNOs and eNOS release small pulses of NO (pM).
237
What is iNOS transcription induced by?
iNOS transcription is induced in phagocytes (macrophages, monocytes, and neutrophils) primarily by invading microbes or inflammation, which elicit the release of interferon-γ or TNFα. As a result, the phagocytes make large amounts of NO (nM).
238
Where is NO produced? What is the target of NO?
Produced: Endothelial cells
Target: Smooth muscle cells (binds to cytosolic (soluble) guanylyl cyclase, by covalently binding to the Fe of the heme group in the guanylyl cyclase )
239
How does NO activate gyanylyl cyclase?
Binding of NO to intracellular (soluble) guanylyl cyclase activates the guanylyl cyclase, which then makes cGMP. cGMP activates protein kinase G (PKG).
240
T/F: Unlike other signaling systems, the ligand (NO) binding to the ‘receptor’ is covalent and not readily reversible. Often, the guanylyl cyclase will remain active until it is degraded.
TRUE
241
T/F: High NO levels oxidize proteins and DNA in microbes and viruses, which ‘kills’ them.
TRUE
242
What do Ligand-gated extracellular ligand channels typically bind?
Ligand-gated extracellular ligand channels typically bind neurotransmitters.
243
What do Ligand-gated intracellular ligand channels typically bind?
Ligand-gated intracellular ligand channels typically bind ions or nucleotides
