Cell Physiology Flashcards
1
Q
What is the loss of heterozygosity?
A
Heterozygosity = two different copies of one gene. Loss thereof can lead to oncogenic factors getting turned on
2
Q
What is Knudson’s “two hit” hypothesis?
A
Start with 2 chromosomes, 2 normal copies of the gene
Get a mutation in one copy of gene –> pre-malignant state
Get a mutation in other copy of gene –> carcinoma
Idea has now evolved, and hits can encompass multiple loci
3
Q
What are some cancers that are inherited in an autosomal dominant fashion?
A
Familial Adenomatous Polyposis (FAP-APC gene), Familial Retinoblastoma (RB gene), familial Breast and Ovarian Cancer (BRCA1 and BRCA2 genes) and Wilms tumor syndromes
4
Q
What are some cancers that are inherited in an autosomal recessive fashion?
A
Xeroderma pigmentosa (XP genes), Ataxia-telangiectasia (AT gene), Bloom’s syndrome and Fanconi’s congenital aplastic anemia (FA genes)
5
Q
What is the significance of the Rb gene?
A
It was the first elucidated tumor suppressor gene.
It’s a tumor suppressor –> it inhibits growth
6
Q
What are some biochemical properties of the Rb protein?
A
When it’s phosphorylated, it’s inactive
Phosphorylated during S or G2 phase of the cell cycle in rapidly dividing cells
Dephosphorylated in G1 or G0 of non-dividing cells –> it’s active –> preventing cell from dividing
It’s targeted by some viral cancers (HPV) that produce a protein that binds to Rb protein and inactivate it
7
Q
How does the loss of Rb lead to malignancy?
A
Rb must be inactivated (via phosphorylation) for the cell to proceed with division/proliferation. Gets inactivated by CDKs, growth factors
If there is no Rb, the cell doesn’t know when to divide or not –> it divides all the time without stopping –> CANCER
8
Q
What is the genetic mutation in familial retinoblastoma?
A
They’re heterozygous for Rb mutation –> only need one additional knockout to get cancer.
Likely to get cancer in both eyes or elsewhere (small cell lung cancer)
9
Q
What is APC?
A
Adenomatous polyposis coli gene
= tumor suppressor
Mutation in this leads to familial adenomatous polyposis (FAP)
10
Q
What are the biochemical events that occur in FAP?
A
Cancer is caused by a mutation in the APC gene
APC binds to β-catenin and keeps it in the cytoplasm (inactive)
Normally, Wnt binds to cytoplasmic receptors and causes the release of β-catenin
β-catenin then goes into nucleus & binds to a family of TFs called TCF
TCF cause expression of the c-myc oncogene –> cell growth
If there is a mutation in APC, β-catenin is always in the nucleus –> always causing cell growth
11
Q
What do BRCA1 & BRCA2 do?
A
They’re both tumor suppressors
They regulate checkpoints, or the response of the cell to DNA damage
If they’re mutated, then cell will proliferate despite DNA damage
12
Q
What happens in mutations of p53? Why are they so bad?
A
p53 has four subunits. If even one is mutated, that “spoils” the whole thing –> dominant negative mutation
Mutant type is more stable ☹
Thus, 75% of mutations in p53 are missense, not frameshift
Mutated p53 proteins can bind to normal p53 proteins and inactive them!
13
Q
Why is p53 “the guardian of the genome”?
A
p53 is a transcription factor that regulates the transcription of ~300 genes that prevent cells from replicating with damaged or foreign DNA
p53 is also important in regulating apoptosis when DNA is damaged
14
Q
How does HPV act as an oncogenic virus in cells?
A
Acts by creating proteins that inactivate both Rb (through E7) and p53 (through E8), which are both tumor suppressor proteins –> double kill shot!
15
Q
How do viral oncogenes work?
A
c-onc = normal copy of the oncogene. When it gets picked up by the virus –> v-onc
Viruses will incorporated their ds-DNA into our genomes, and then in a mistranscription event, a nearby oncogene will also be transcribed –> both the viral mRNA and oncogenic mRNA are incorporated into the viral capsid. Thus, the virion becomes cancerous. This is how ALV –> RSV (Rouse’s Sarcoma Virus)
16
Q
What are some examples of viral oncogenes?
A
v-src = creates membrane-bound protein kinase
v-erb-B = protein that is similar to the receptor for epithelial growth factor
v-abl = creates a kinase that phosphorylates tyrosine residues. Similar to c-abl that’s translocated to BCR-ABL in CML
17
Q
What the relevance of n-myc in cancer?
A
It’s amplified in neuroblastoma
It’s a member of the c-myc family of oncogenes (promoters of cell growth)
18
Q
What is the relevance of the HER2/neu/Erb2 gene in cancer?
A
It’s amplified in ~20% of breast cancers
19
Q
What genes can be mutated in human bladder cancer cells?
A
c-ras = point mutations causes a protein product that is always on
20
Q
What is Herceptin?
A
A monoclonal antibody specific for the protein product of the HER/neu/Erb2 oncogenes in breast cancer cells
They can reverse the transformed phenotype of the cell
21
Q
What does Gleevac do specifically?
A
It acts as an ATP mimic and prevents the kinase from phosphorylating
22
Q
What are the parts of a phospholipid?
A
Polar head group
Phosphate connecting polar head group and glycerol backbone
Glycerol 3-phosphate backbone
Fatty acyl chains (16C or 18C), saturated or unsaturated
23
Q
What are the parts of a sphingolipid?
A
Sphingosine backbone + amide group + fatty acid = ceramide
Phosphate linker
Polar head group
*If you add a sugar instead of a polar head group = glucosylceramide
24
Q
What is the structure of cholesterol?
A
Hydrophilic head: -OH on top
Rigid steroid rings; fatty acid tail
25
What is the function of cholesterol?
Serve to increase membrane stiffness and thickness
26
Describe the asymmetry of membrane bilayers
Exoplasmic face = PC (phosphatidylcholine), sphingomyelin, & glycolipids
Cytoplasmic face = PE (phosphatidylethanolamine), PS (--serine), & PI (--inositol)
Cholesterol is distributed evenly
Phospholipids do not switch sides! If they do, it's a sign of cell death
27
Explain cholesterol synthesis
Cholesterol is synthesized from glycerol (3C)
First enzyme: HMGCoA reductase. (Statins block this!)
Cholesterol is both brought in & synthesized in the cell. Statin regulatory element binding protein (SREBP) has a TF that can upregulate transcription of BOTH the LDL receptor (intake) and 30 synthesis proteins (synthesis)
28
How does the sensor to detect cellular levels of cholesterol work?
In the ER membrane (5% cholesterol)
TF is a bHLH attached to the transmembrane SREBP
SREBP is held in ER until cholesterol is low and then it's moved to Golgi, where TF is cleaved & can move into the nucleus
29
What are the proteins that bind SREBP and regulate cholesterol levels?
SCAP (SREBP cleavage activation protein) and Insig
Insig binds SCAP only when cholesterol is high, & binding blocks a signaling part of SCAP. This signaling domain is recognized by a COPII coat protein, which delivers the protein complex to the Golgi for cleavage
1. When cholesterol levels are low SCAP-SREBP complex dissociates from Insig.
2. SCAP escorts SREBP to the Golgi by vesicular transport.
3. The bHLH transcription factor is released from SREBP by RIP
4. S1P is luminal, S2P is within the membrane – cleavage by both is required for activation
5. Nuclear bHLH SREBP moves to the nucleus, binds to DNA promoters, and activates many genes to produce more LDLR to bring cholesterol into the cell and to increase all the enzymes involved in cellular synthesis of cholesterol.
30
What is unusual about how the SREBP protein is cleaved in times of low cholesterol?
It's cleaved in the transmembrane domain (not aqueous environment) via RIP, regulated intramembrane proteolysis.
Also seen in Alzheimer's
31
What is Von Hippel-Lindau?
An autosomal dominant condition that's caused by a mutation in the VHL tumor suppressor gene. Highly penetrant!
Leads to cystic & highly vascularized tumors in spinal cord, eyes, ears, kidneys, pancreas, & genitourinary tract
40% of people will eventually develop clear cell renal cell carcinoma
VHL is most common cause of inherited ccRCC
32
What are the different types of VHL?
Type 1: total/partial VHL loss, improper folding = hemangioblastoma (benign, highly vascular tumor in brain & spine; originates from vascular system), RCC, low risk of pheochromocytoma (tumor of adrenal gland)
Type 2: missense mutation = hemangioblastoma, low/high risk of RCC, high risk of pheochromocytoma
33
How does the VHL protein work?
Under normoxic conditions, HIF is hydroxylated and ubiquitinated by VHL --> gets degraded
Under hypoxic conditions, HIF is not hydroxylated, cannot get marked by VHL --> goes into nucleus as a TF
HIF upregulates growth factors (VEGF, PDGF, TGF) --> angiogenesis --> survival of cancer cells!
^One reason why these tumors are so highly vascularized)
34
What are the treatment options for RCC?
Immunotherapy: high doses of IL2 (RCC suppresses immune function) - high toxicity, must be administered in ICU
VEGF inhibitors: suppress VEGF or downstream pathway, try to prevent angiogenesis
mTOR inhibitors: mTOR is upregulated in ~20% of RCC. Important AE: pneumonitis (14%)
35
What do SNAREs do?
SNARE = soluble SNF attachment protein receptor
They regulate membrane fusion
36
What are the three main classes of SNAREs and where are they located?
VAMP = vesicle associated membrane protein. Located on vesicle
SNAPs = synaptosome associated protein. Bound to cytosolic side of target membrane
Syntaxin = also in target membrane
37
How does intracellular membrane fusion occur?
Alpha helix coiled-coiled structures form between VAMPs, SNAPs, and syntaxin --> very stable structure! Overcome the resistance forces between membranes and bring them together
38
How are the alpha helix coiled-coil structures created during membrane fusion broken up and recycled?
NSF & αSNAP use ATP hydrolysis to disassemble the SNARE complex
Sec1 protein binds to & refolds syntaxin to active conformation
39
How is specificity of fusion achieved?
Cells have over 18 SNAREs, 9 SNAPs, etc. --> this allows specificity of fusion. Only the place where this specific complex will form will allow fusion.
40
Explain viral membrane fusion
2 alpha helices
One is transmembrane domain, the other is hydrophobic domain initially buried in the peptide
Once it binds to host membrane, hydrophobic portions are exposed, form a coiled-coil, and bring the two membranes together
41
How is the influenza protein activated?
By a low pH
Influenza is phagocytized and taken to lysosome, where the low pH activates the fusogenic protein --> virus invades the cell
42
How is the HIV virus activated?
By receptor binding activation
Fusogenic protein is a dimer of 2 proteins, gp120 and gp41. gp120 sits on top of gp41 and hides it. gp120 binds to receptors on T-cells --> conformational change --> exposure of gp41 --> membrane fusion
43
What are typical values for the volumes of plasma, extracellular fluid, and intracellular fluid?
Plasma = 3 L
ECF = 13 L
"Extra space" = 5 L (eyes, lumen of gut, sweat glands, kidneys, etc.)
ICF = 27 L
Total adult volume: 45 L of fluids
44
What is the ionic composition of ICF and ECF?
Na inside = 14 mM -- Na outside = 140 mM
K inside = 145 mM -- K outside = 5 mM
Cl inside = 5 mM -- Cl outside = 145 mM
Ca inside = 0.0001 mM -- Ca outside = 1 mM
H inside = 0.0001 mM -- H outside = 0.00004 mM = 40 nm
H2O = 55,000 mM
HCO3- = 25 mM
Max urine mosM = 1200
Plasma mosM = 300 mM
45
What are the 3 mechanisms that cells have evolved to prevent from swelling & bursting?
1. Membrane impermeable to water
2. Cell wall
3. Balance cell contents osmotically with outside environment
46
What are reflection coefficients?
Reflection coefficient of 1 = non-permeable
| Reflection coefficient of 0 = same permeability as water
47
What is osmolarity?
Concentration of solute particles - a 1 M solution of CaCl2 gives a 3 osM solution (3 ion/mole)
48
What is tonicity?
Measure of solution in a cell --> hypotonic means lacking in solution, cell will swell. Hypertonic means too much solution, cell will shrink.
49
What are equivalents?
Calculated by converting to mosM & multiplying mosM by valence of the ion
50
What is the Donnan Rule?
[K]i[Cl]i = [K]o[Cl]o
51
What is the Driving Force on an ion?
Vm - E of the ion
52
Define pKa
pKa = -log [H+ ][A-] / [HA]
The lower the pKa, the stronger the acid. Higher = weaker acid.
53
What is the Henderson-Hasselbalch Equation?
pH = pKa + log [A-]/[HA]
54
What is the H-H equation for the bicarbonate buffer system in ECF?
pH = 6.1 + log [HCO3-]/ .03Pco2
55
What are normal blood pH, [HCO3-], and pCO2?
Blood pH = 7.4
[HCO3-] = 24 mM
pCO2 = 40 mmHg
56
What is log(2)?
0.3
57
What is the pH range of maximal buffering capacity?
+/- 1 pH away from where [HA] = [A-]
58
What does straightforward DKA look like?
Tachypnea, nausea, vomiting, diffuse belly pain, dehydrated, ill appearing
Polyuria, polydipsia (drinking a lot), and weight loss = very suspicious for diabetes
Breathing deeply & rapidly, nausea, and vomiting = very suspicious for ketoacidosis
59
What are the major metabolic disturbances in DKA?
Hyperglycemia: plasma glucose >200 mg/dL
Acidosis: blood pH < 7.3
or [HCO3-] < 15 mmol/L
(comes from ketoacids in your blood)
Potassium derangements: normal levels are between 3.5-4.5 mEq/L
Kidneys keep Na, so lose K to urine. Elevated H+ in blood, so H/K transporter puts more K into plasma to get rid of H. Elevated plasma K, overall lower body levels of K.
Dehydration: lots of glucose lost in urine, so lots of water lost through osmosis
60
What is the mechanism for the release of insulin?
1. Glucose enters beta cell in pancreas
2. Glucose undergoes glycolysis, produces ATP
3. ATP inhibits a K channel that allows K to exit cell; K builds up in cell
4. K buildup causes depolarization of cell, Vm increases
5. Voltage-gated Ca channels open, allowing Ca to rush inside
6. Increase in intra-cellular Ca causes insulin-containing vesicles to fuse with plasma membrane and release contents extracullularly
61
What does insulin do?
Insulin makes you STORE ENERGY
```
Liver
+ glucose uptake, + glycogen synthesis
+ lipogenesis
- ketogenesis
- gluconeogenesis
```
Muscle
+ glucose uptake, + glycogen synthesis
+ protein synthesis
Adipose
+ glucose uptake
+ lipid synthesis
+ triglyceride synthesis
62
What is Cushing's Triad?
Irregular/agonal breathing, hypertension, and bradycardia
= a sign of increased intracranial pressure
63
What are some of the warning signs for cerebral edema in DKA?
Dilated/fixed pupils, headache, altered mental status, irregular/agonal breathing, bradycardia, hypertension
Treatment is to raise the osmolality of the blood with mannitol, a sugar alcohol
64
What happens with Vm and Ek when you have hypokalemia?
Lower extracellular levels of K --> Ek decreases (you need more of a charge difference when there's more of a concentration difference)
Low extracellular K --> cell wants to release more K --> membranes react against that and close
Decreased permeability to K --> Vm moves away from Ek
End result: cell depolarizes
65
What are treatments for hyperkalemia?
CBIGK
Calcium, bicarbonate, insulin + glucose, Kayexalate
Calcium relieves cardiac arrhythmias
Bicarbonate alkalinizes the blood --> K is brought into cells
Insulin + glucose = more ATP = more Na/K pump action
Kayexalate = exchanger bound to Na that then selectively binds to K ions in the blood
Extreme: dialysis
66
What is Li-Fraumeni Syndrome?
A hereditary autosomal dominant cancer syndrome associate with a mutation in the p53 gene
67
What are the diagnostic criteria for Li-Fraumeni?
A proband with a sarcoma under 45 years of age
AND a first-degree relative with any cancer under 45 years of age
AND a first- or second-degree relative with a cancer before 45 or a sarcoma at any age
68
Describe the structure of the voltage-gated K channel
4 membrane-spanning separate polypeptide domains
Each domain contains 6 alpha helices (S1-S6)
***must memorize!!***
S4 domains have positive Lys or Arg residues every 3 positions - these "sense" voltage
S5 and S6 helices & the connecting "P loop" assemble to form the ion conducting pathway and "selectivity filter"
69
Describe the structure of the voltage-gated Na and Ca channels
Have four polypeptide domains that are linked together as 4 repeats (I-IV)
Each domain contains 6 alpha helices
S4 has pos Lys & Arg every 3 residues; are the voltage-sensors
S5 & S6 & P loop form conducting pathway and selectivity filter
70
What is the role of dehydration of the ions in ion channels?
Ions are bound to water in order to stabilize them - in order to compensate for getting rid of water molecule interactions, which are thermodynamically unstable, amino acids within ion channel stabilize ions
71
How does the K channel activation gate work?
There is an activation gate that swings on a hinge
When cell is negatively charged intracellularly, the activation gate is horizontal, and the positive end is close to cytosolic side
When inside of cell is positive (depolarization), positive end of activation gate swings toward extracellular side, channel opens, and K ions flow out
When inside of cell is negative again, gate swings closed = *deactivation*
72
How do the Na channel activation gate and inactivation gate work?
Na activation gate works just like K gate (on a hinge, pos end swings toward EC side when cell depolarizes, Na floods in) = fast step
After activation gate opens, inactivation gate closes (*inactivation*). Removal of inactivation = slow step
73
What is the inactivation gate on the Na channel formed by?
The cytoplasmic III-IV linker between the domains
Located near the cytoplasmic side
74
What does tetrodotoxin (TTX) do?
It's a neurotoxin that binds to Na channels and prevents action potentials --> DEATH :(
Structure is a charged molecule that binds extracellularly to the entrance of the Na channel, independent of activation/inactivation gates. Has no effect intracellularly.
75
How does lidocaine work?
Lidocaine is a tertiary amine & alternates between protonated (charged) and deprotonated (uncharged)
Protonated lidocaine can only cross the membrane when the activation gate is open and the inactivation gate is not blocking the channel. Thus, it is state-dependent
Protonated lidocaine will act on receptor from intracellular side & close it, producing numbing effects
76
What is the threshold for an action potential?
Where the inward flow of Na exactly matches the outward flow of K
Voltage-gated Na channels respond to depolarization (increased positivity)
If influx of Na > efflux of K, channels will open --> further influx of Na --> positive-feedback loop
77
During an action potential, why is there a refractory period?
After inactivation gate has closed, it takes some time for it to reopen
There is an "absolute refractory period," where nothing will depolarize the membrane, and a "relatively refractory period," where an excessively large depolarization is required to initiate an action potential
78
Describe how changes in membrane resistance, membrane capacitance, and internal resistance affect the passive spread of voltage along an axon
Higher internal resistance = slower current internally
Higher membrane resistance = faster current internally, as it will take longer to cross membrane and so will just spread down axon
Higher membrane capacitance = slower spread of current, as time will be spent building up the charge on the membrane
79
Why are axons poor passive conductors over long distances?
They have high internal resistance (lots of molecules in the way), low membrane resistance (lots of leakage), and high membrane capacitance (negative charge inside, pos charge outside = capacitance)
80
What is the length constant of an axon?
λ=0.5 √(Rm/Ri )
=distance by which voltage has dropped to 37% of its initial value
81
Why is myelination and larger diameter of an axon good?
Myelination provides increased membrane resistance, decreased membrane capacitance, and allows for saltatory conduction (faster)
Larger diameter = more charge can flow through at any time
82
What are the consequences of demyelination in axons?
More K channels will be "naked" and exposed
Demyelination causes proliferation of Na channels along the axon --> increased Na entry --> slowing of nerve conduction
Clinical symptoms: fatigue, spasticity, sexual dysfunction, bladder dysfunction, walking impairment, pain, mood instability
83
What is the architecture of the nuclear pore complex (NPC)?
Made up of 30 distinct proteins called nucleoporins (Nups) repetitively arranged
They have FG repeats (phenylalanine, glycine) --> form patches that are separated by hydrophilic regions
FG repeats can rapidly interact, associate, & dissociate – critical to trafficking process
84
What are karyopherins?
They transport cargo into/out of nucleus! =importins/exportins
Interact directly with cargo (beta), or use an adaptor protein (alpha)
Contain a Ran-GTP binding domain = Ran-mediated
85
How does nuclear importation work?
1. In cytoplasm, the cargo protein with a nuclear localization signal (NLS) binds to a nuclear import receptor (NIR) and enters nucleus via NPC
2. In nucleus, Ran-GTP is required for release of the cargo and binds to the NIR --> sends back to cytoplasm
3. In cytoplasm, Ran-GTP is hydrolyzed by GTPase activating protein (GAP) and Ran-GDP then dissociates from NIR
4. NIR is then free to bind to cargo again
86
How does nuclear export work?
1. In nucleus, cargo with a nuclear export signal (NES) binds to a nuclear export receptor (NER), which then binds to Ran-GTP
2. The whole complex is delivered to the cytoplasm via NPC
3. In cytoplasm, GAP hydrolyzes Ran-GTP, causing release of NER and cargo
4. NER returns to nucleus *by itself*
87
What is the driving force of directionality for nuclear import/export?
High Ran-GTP in nucleus
Low Ran-GTP in cytosol
88
What is one change in a nuclear import complex that can cause disease?
Normal cell: BRCA1-RAD51 gets imported into nucleus & is involved in DNA repair. Retained in cell because NESs on the two are occluded by being bound to other things.
Diseased cell: exposure of NES continuously. You get tumors because DNA repair mechanisms can't do their job
89
What are the major functions of the ER?
1. Synthesis of lipids
2. Control of cholesterol homeostasis
3. Ca2+ storage (rapid uptake & release)
4. Synthesis of proteins on membrane-bound ribosomes
5. Co-translational folding of proteins, post-translational mods, and post-translational insertion into membrane
6. QC
90
Describe co-translational translocation
1. Protein gets translated by ribosome
2. Nascent strand has ER signal sequence
3. ***Signal recognition particle (SRP)***, which is composed of proteins & RNA, recognizes this signal sequence on the nascent protein and binds
4. Binding of SRP causes a pause in translation
5. SRP-bound ribosome attaches to SRP receptor, which is bound to translocon, in ER membrane
6. Translocon opens, allowing polypeptide chain through; translation starts again
7. Signal peptidase cleaves signal sequence from protein
8. Completed protein folds within the ER lumen
91
What are the major functions of the Golgi?
1. N-linked GLYCOSYLATION happens on asparagine residues near the amino terminal of the protein
2. Synthesis of complex SPHINGOLIPIDS from the ceramide backbone
3. Additional POST-TRANSLATIONAL MODIFICATIONS of proteins and lipids. For example, sulfation takes place in the trans Golgi and TGN (trans-Golgi network; misshapen membrane at very end of Golgi), near end of Golgi processing
4. PROTEOLYTIC processing
5. SORTING of proteins and lipids for post-Golgi compartments
92
What are 3 vesicle coats and how they function in transport?
Clathrin
Located at trans-Golgi network
Involved in endocytosis (plasma membrane, on intracellular side)
COPI (coat protein I)
MOVE BACKWARDS
Backwards from one part of Golgi to another
Backwards from Golgi to ER
COPII
Forms vesicles on ER membrane, which go and fuse with the Golgi
93
How does vesicle coat assembly work?
Coat proteins bind to proteins that recognize target membrane protein & cargo protein
When the coat forms a vesicle, has the right cargo
Dynamin wraps itself around neck of vesicle & strangulates it – vesicle gets released
Almost as soon as this release, the coat proteins dissociate – then it can get targeted to another organelle, or for exocytosis
94
What are the key clinical features of cholera infection?
Dehydration - sunken eyes, dry/chapped lips, less urination
Severe dehydration - skin turgor!
Profuse, water diarrhea (like "rice porridge")
95
Describe the actions of cholera toxin’s A and B subunits
A subunit = active site
B subunit = transport molecule
B subunit binds to the GM1 glanglioside receptor on the surface of the cell.
A subunit cleaves off and binds to G protein; stimulates adenylate cyclase to produce cAMP.
cAMP activates CFTR.
Massive efflux of Cl ions --> extreme loss of water
96
Describe the role of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in cholera
cAMP activates CFTR, which opens and causes a massive efflux of chloride ions. This causes massive amounts of water loss – secretory diarrhea
97
Describe the physiology behind oral rehydration solutions
Relies on solute-coupled sodium cotransporters. Despite the fact that you’re losing chloride ions (and thus water), the thinking is that if you bring sodium, glucose, and other solute back across apical membrane, you can draw water back in
98
What is pinocytosis?
A mode of endocytosis in which small particles are brought into the cell, forming an invagination, and then suspended within small vesicles
**Uses clathrin!!!**
99
What are caveolae?
They are a special type of lipid raft -- are small invaginations of the plasma membrane
Utilized by cholera toxin, folic acid, & albumin
100
Describe two types of molecular chaperones
Hsp70
Help fold a protein by binding to exposed hydrophobic patches in incompletely folded proteins
Hsp60
Form large barrel-shaped structures to act as an “isolation chamber” into which misfolded proteins are fed to prevent aggregation & help them to refold
***have GroES cap!!***
101
How does a proteasome work, and how is ubiquitination involved?
Proteasome degrades proteins!
Proteins get marked for degradation by attachment of at least 4 ubiquitin proteins
Alpha subunits (on rim of proteasome) guide misfolded protein in
Beta subunits cleave --> end up with polypeptides 7-9 aa in length --> get recycled
102
What is the importance of mannose-6-phosphate?
It's a sorting signal for lysosomal proteins.
Binds to a receptor --> receptor is targeted to vesicles that fuse with the endosome --> in endosome, receptor/M6P-tagged protein dissociate from vesicle --> endosome delivers the protein to the lysosome; receptor is recycled
103
What are characteristic plasma membrane events of apoptosis?
In a normal cell, all the phosphatidylserine head groups are on the inner leaflet of the plasma membrane
Early in process of apoptosis, phosphatidylserine flips from inner leaflet to outer leaflet of plasma membrane
Becomes equalized in inner/outer distribution via scramblase
Phagocytes recognize the phosphatidylserines and use them to get the cell inside
Membrane also undergoes "boiling" action = zeiosis
104
What are characteristic cytoplasm events of apoptosis?
Early in apoptosis, cell shrinks to about 1/3 of its size
Eventually tears itself apart into apoptotic bodies
105
What are characteristic events in the nucleus of apoptosis?
Defining morphological feature of apoptosis = COLLAPSE OF THE NUCLEUS
Chromatin becomes supercondensed and forms beads
106
What are some of the differences between necrosis and apoptosis?
Necrosis = occurs during ischemia, mitochondria swell, Na/K pump eventually fails, cell bursts & releases contents --> INFLAMMATION
Apoptosis = no inflammation, cell shrinks, nucleases, macrophages "eat it alive"
107
What are the role of caspases in apoptosis?
"Executioner" proteins
Caspase 9 is the main initiator caspase for the intrinsic pathway
Caspase 8 and 9 both activate the effector caspase 3. Caspase 3 causes all the changes – flipping across membranes, etc.
108
What happens during the intrinsic pathway in apoptosis?
A cell gets signaled (via radiation, etc.), and it decides to die
Normally, mitochondrial membrane is “guarded” by Bcl family genes that associate with the mitochondrial membrane & are anti-apoptotic
Signal for apoptosis: pro-apoptotic factors move to mitochondria & replace Bcl --> --> --> Caspase 9
109
What happens during the extrinsic pathway in apoptosis?
Killer T cells activate apoptosis in other cells
Fas ligand on the Killer cells interacts with a death receptor (Fas) that’s on most cells in your body
Fas arranges for the activation of Caspase 8 – the initiator caspase for the extrinsic pathway
Caspase 8 activates Caspase 3
110
What is morphogenetic death?
Apoptosis that occurs during development to determine final shapes of body parts and organs (limbs, brain, thymus, etc.)
111
What does FLIP do in apoptosis?
FLIP = a protein related to caspase-8, but proteolytically-inactive
It competes with caspase-8 for binding to FADD, and thus inhibits apoptosis signaling
There are viral FLIPs! (herpes, KSV) ►Clever pathogens will develop (or, in the case of viruses, steal) anti-apoptotic genes to keep the cell alive until they can finish their replicative cycle
CELL = ZOMBIE
112
What is macroautophagy?
Signaling leads to formation of double-membrane vesicle that encapsulates a bunch of proteins, organelles, etc. (autophagosome)
This then fuses with the lysosome & those acidic enzymes then degrade everything inside
113
What is autophagy?
The basic mechanism by which a cell breaks down and recycles various parts via lysosomes. Autophagy is how that stuff gets delivered to the lysosomes.
114
What is chaperone-mediated autophagy?
Proteins have specific sequence (KFERQ) that allows Hsc70 to bind, other host of proteins bind, deliver it to the lysosome
In the lysosome, it’s degraded
115
Describe the process of macroautophagy
1. Induction
a. Nutrient starvation, growth factor-mediated starvation, exposure to chemo drugs, rapamycin, etc.
2. Vesicle Nucleation
a. “Phagophore”
3. Vesicle Expansion
a. “Omegasome”
4. Cargo targeting
5. Vesicle Closure
a. =autophagosome
b. Recruits proteins & organelles
6. Double membrane fuses with something else to make a vesicle
a. Can fuse with endosomes from outside = “amphisome”
b. Can directly fuse with lysosomes = “autolysosome”
7. Enzymes degrade things that were in autophagosome
8. Macromolecule precursors are then released back into cytoplasm
9. Recycling! ☺
**Atg genes regulate steps of this process**
116
Describe microtubules
Building blocks are heterodimers of the protein tubulin (α and β)
Each tubulin (α and β) has a binding site for GTP. The GTP bound to a tubulin is trapped and is not hydrolyzed
The GTP bound to β tubulin can be hydrolyzed and is exchangeable
Once β tubulin hydrolyzes GTP, it forms a little kink --> filament bends backwards, breaks apart
The only reason this doesn’t happen & destroy the MTs is because of the GTP cap on new tubulin heterodimers – keep them from curving back. Cap = stability
--> Regulated by MT capping proteins
Bind to the ends of the MTs and stabilize them. Bind to the GTP cap & part of stabilizing complex
Also regulated by MT severing proteins: cut an MT in the middle – now do not have GTP cap; MTs will fall apart
117
Describe intermediate filaments
Rope-like, fibrous structures of about 10nm diameter
PROTEINS: keratins, vimentins, and neurofilament proteins --> far more heterogeneous
Fall into two categories: cytoplasmic IFs and nuclear lamins
Nuclear lamins = filamentous proteins that form a stabilizing meshwork lining the inner membrane of the nuclear envelope to provide anchorage for chromosomes and nuclear pores.
All IF proteins = long molecules w/ α-helical domain --> forms coiled-coil with another monomer --> dimers associate anti-parallel --> form tetramers --> IF polymerization
**They are not polarized**
118
What is the MTOC?
Microtubule-organizing center
Two main functions: organization of flagella/cilia and growth of mitotic spindle apparatus
Structure: a pair of centrioles making up a centrosome. On the centrosome are of rings of γ-tubulin which initiate MT growth --> MTs anchored at - end, grow at + end
119
What do spastin and katanin do?
They're microtubule-severing proteins --> increase microtubule instability by cutting & exposing GDP-rich parts of microtubules
120
What do Colchicine, Vinblastin, and Vincristine do?
They inhibit MT polymerization
Block mitosis & thus are of interest in cancer treatments
121
What are some examples of molecular motors for microtubules?
Kinesins -- go to (+) end
Dyneins -- go to (-) end
Kinesins:
- they’re a coiled-coil; there are many different kinds
- they bind to adaptor molecules (>100 different kinds), which provide specificity for binding of cargo vesicle
- head binds to MT, tail binds to adaptor protein/cargo
Important for axonal transport
122
Describe the kinesin cycle
Kinesin Cycle
- When ADP is bound --> kinesin is released
- When ATP is bound --> kinesin is bound to MT
- Hydrolysis of ATP --> ADP will change the conformation of the head domain and make a little kink --> moving forward
123
How is actin formed?
G-actin + ATP --> two-stranded, helical filaments (F-actin)
A trimer of actin monomers is necessary to initiate nucleation (formation)
Actin-ATP units polymerize at positive end
Actin-ADP units de-polymerize at negative end (treadmilling)
124
What are the two major proteins involved in actin formation?
Arp2/3 and Formin
Arp2/3 (actin-related protein)
- looks like an actin dimer
- attaches to an actin monomer --> now you have the actin trimer necessary for nucleation & creation of an actin strand
- creates new filaments AT ANGLES --> branched network
- key for cell motility
Formin (FH2)
- creates long actin cable filaments that are PARALLEL
- key for cell division
125
Which drugs interact with actin formation?
Phalloidin
Extracted from the highly toxic fungus Amanita phalloides ("death cap" mushroom), which binds to and stabilizes F-actin (causing a net increase in actin polymerization)
126
What happens in microvilli inclusion disease?
Broadly: microvilli are lost
Myosin V helps deliver cargo vesicles along actin --> this is mutated in microvilli inclusion disease
Myosin V deliver vesicles that have a regulator that induces formation of a terminal web
Mutant = no terminal web, no anchor for microvilli actin fibers to bind to
127
What is the molecule motor used for actin filaments, and how does it work?
Myosin --> binds to actin
Myosin forms coiled-coil structures with tails & heads – binds together with other myosin bundles to form large bipolar assemblies
ATP-driven myosin heads “walk” along actin filaments = sliding mechanism of muscle contraction
10% of the time will be attached to myosin (ATP bound)
90% of the time will not
Muscle contraction works because of multiple heads, so overall binding - you don't want too much binding b/c muscles will be stiff
128
How does a cell move?
At growing end, Arp 2/3 actin will polymerize at head & grow. Protrusion of fillopodia and lamellipodia is driven by polymerization of actin meshworks at the leading edge.
At retracting end, Formin filaments will cause retraction together with myosin II
RhoGTPases respond to signals (chemotaxis, etc.)
129
Describe the actomyosin ring in cell division
Actin plays a key role during cytokinesis
The formation & contraction of the actomyosin ring drives the formation of the cleavage furrow and separation of the daughter cells
--> also determine symmetry of cell separation
Regulated by RhoGTPase
When it’s bound to GTP, it’s active. When it’s activated, it’ll activate Formin --> forms contractile ring
Activation is dependent on astral MTs – some of these have Rho attached to tips. As they go to midzone region & overlap with MTs from other side, they activate RhoA, etc.
130
What are some examples of 2nd messengers?
- Ca2+ enters through ion channels
- cAMP is generated by adenylate cyclase
- IP3 (inositol triphosphate) and DAG (diacylglycerol) are generated by PLC (phospholipase C)
- NO (nitric oxide) generated by NOS (nitric oxide synthase)
131
What does phosphodiesterase (PDE) do?
It breaks down cAMP and cGMP into AMP and GMP
132
How does Viagra work?
Acts on catalytic site of PDE --> breaks down cGMP to GMP --> reduces intracellular Ca levels --> smooth muscle relaxation --> vasodilation --> penile erection
133
How do receptor tyrosine kinases get activated?
Ligand binding to receptor on extracellular side --> dimerization of receptors --> activates catalytic activity of the kinase --> autophosphorylation of tyrosine on cytoplasmic side
134
Explain the molecular mechanism of stimulation of Ras GTPase by receptor tyrosine kinases
Phosphorylation of tyrosines on receptor --> binding by Grb2 --> binds Sos (a Ras GEF = GTP exchange factor)
This brings Sos to the plasma membrane, where it interacts with Ras --> activation by removing GDP, attaching GTP (GEF action)
**more detail**
Grb2 binds to receptor TK via SH2 domain, which recognizes 3 aa on the receptor
Sos binds to Grb2 via the SH3 domain, which binds to proline domains
135
What are two receptor tyrosine kinase agents that act as anti-cancer drugs?
Antibodies
- block ligand binding to the receptor (extracellularly)
- prevents receptor DIMERIZATION, activation of growth factors
TKI (Tyrosine Kinase Inhibitors)
- block TKR kinase activity – bind in substrate-binding (usually ATP) site of the kinase
- inhibit CATALYTIC activity
136
What is the membrane topology of a G protein-coupled receptor?
- 7 helical transmembrane domains
- N-terminus outside; C-terminus inside
- 1st and 7th domain fold back to each other to form a barrel structure
- ligand binding occurs in pocket on extracellular side
Ligand binds --> conformational changes --> down to intracellular side --> changes the way it interacts with G protein
137
How do G protein-coupled receptors activate G proteins?
Resting state: trimeric G protein bound to GDP
```
Activate = NUCLEOTIDE EXCHANGE
Upon ligand (agonist) binding, receptor catalyzes GDP dissociation (=rate-limiting step)
```
GTP then binds very quickly to nucleotide-free G α-subunit --> additional conformational changes --> active state of G-protein complex
Active state = G-α-subunit-GTP dissociates from receptor & βγ-subunit --> effectors
138
How do G protein-coupled receptors turn off G proteins?
= NUCLEOTIDE HYDROLYSIS
| α-subunit is a GTPase --> hydrolyzes bound GTP to GDP --> subunits reassociate & recouple to receptor
139
β1-adrenergic receptor signaling – in the heart
G-protein coupled receptor in the heart
- via Gs
- stimulates AC (adenylyl cyclase)
- cAMP production from ATP (by AC)
- cAMP activates PKA (protein kinase A)
- PKA phosphorylates proteins --> Ca2+ influx increases --> increased heart rate and contraction
- agonists: norepinephrine, epinephrine, isoproterenol
- antagonists: propranolol, metropolol
o These drugs are β-blockers – they block activation of β-receptor and reduce blood pressure (and heart rate)
140
α1-adrenergic receptor signaling
G-coupled protein receptor in the peripheral vasculature
- via Gq
- stimulates PLC activation
- PLC cleaves PIP2
- PIP2 releases IP3 and DAG
- IP3 & DAG cause influx of Ca2+
- Ca2+ triggers smooth muscle contraction --> peripheral vasoconstriction decreases blood flow to skin --> increases blood pressure & shifts blood flow to heart, lungs, and skeletal muscle
- Agonists: norepinephrine, epinephrine, or phenylephrine
- Antagonists: prazosin
o These drugs are α-blockers – they also reduce bp
141
What is mTOR?
= mammalian target of rapamycin
MTOR is a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription
Acts on, among other things, CDK2
142
What does prazosin do?
It's an α-blockers --> causes decrease in bp
143
m2-muscarinic cholinergic receptor signaling
G-protein coupled receptor in the heart
- via Gi
- counters the actions of Gs --> suppresses AC activity --> Ca2+ influx decreases --> decreased heart contraction
- also targets K channels
o βγ-subunit acts on K channel called GIRK & activates it (opens it)
o loss of K hyperpolarizes the cell & makes it less excitable
o Makes it harder to open the Ca2+ channel --> decreased Ca2+ influx --> decreased heart rate & contraction
144
What do atropine and epinephrine do?
Atropine is a muscarinic antagonist --> would increase the heart rate via blocking the parasympathetic system
Epinephrine is a β-agonist --> would also increase the heart rate via the sympathetic system
145
What do caffeine and theophylline act upon?
They inhibit PDEs, which convert cAMP into AMP and thereby reduce Ca influx
Thus, they have a stimulatory effect
146
β2-adrenergic receptor signaling – in the LUNGS
- via Gs
- stimulates AC (adenylyl cyclase)
- cAMP production from ATP (by AC)
- cAMP activates PKA (protein kinase A)
- PKA inhibits smooth muscle contraction (different!!!)
- Smooth muscle relaxation --> bronchodilation (and dilation of vasculature of blood to lungs/heart/muscle)
- Albuterol is a β2-selective agonist
o Causes bronchodilation
147
m3-muscarinic cholinergic receptor signaling – in the LUNGS
- via Gq
- triggers PLC --> PIP2 --> IP3 + DAG --> Ca2+ influx --> stimulates smooth muscle contraction --> bronchoconstriction
- Ipratropium is a muscarinic antagonist
o Prevents bronchoconstriction; relieves acute asthma symptoms
148
What does GRK do?
Involved in receptor desensitization:
If you turn on a G-protein coupled receptor receptor for a long time, it favors activation of a kinase called GRK --> phosphorylates the receptor --> β-arrestin binds to phosphorylated receptor --> inhibits re-binding of G-proteins; now they’re uncoupled :(
β-arrestin also favors endocytosis of these receptors – they get removed from the plasma membrane
149
How does cAMP activate PKA?
PKA is bound to regulatory subunits. Binding of cAMP causes release from these regulatory subunits; phosphorylation and activation of catalytic subunits of PKA
150
How does CDK2 get activated?
CDK2 kinase activity only turns on if one phosphate has been added, one phosphate has been removed, and cyclin is present to activate it
151
How do kinases work?
A kinase has a small & large lobe. ATP binds in the cleft between the lobes (but basically to the small lobe). Protein substrate usually binds to large lobe.
A ***glycine-rich loop*** in small lobe clamps down on ATP; positions γ-phosphate correctly.
“Closed conformation” of the glycine loop in the small lobe forces the γ-phosphate into the right position for phosphorylation (=fast reaction).
“Open conformation” of the glycine loop allows exchange of ADP molecule --> new ATP (=slow reaction). Kinase activity requires alternating open and closed conformations.
Some (but not all) kinases have ***activation loop*** on large lobe that needs to be phosphorylated in order to work.
152
What does parvalbumin do?
It's a cytoplasmic buffer of Ca2+ - restricts the spatial and temporal spread of the ions
153
What do buffers of Ca2+ do?
Restrict the spread of ions
Buffers also serve as a temporary storage site for Ca2+ -- allow muscle contraction & relaxation to take place rapidly, despite the fact that Ca2+ extrusion transport processes are operating slowly
154
What does calsequestrin do?
In the ER/SR lumen, high-capacity low affinity buffers (like calsequestrin) allow large quantities of Ca2+ to be stored without the generation of a large gradient – the molecules stick a lot of Ca2+ onto themselves, which is vital in order to prevent a large gradient, but release it rapidly (important for IP3 and ryanodine receptors – there needs to be a higher gradient of Ca2+ in the ER/SR)
155
How does Ca2+ enter the cell?
- ion channels
- voltage- and ligand-gated Ca2+ channels
- store-operate Ca2+ channels
156
How does Ca2+ move from ER/SR into the cytoplasm?
- IP3 receptors
| - ryanodine receptors
157
How does Ca2+ get extruded from the cytoplasm into the extracellular space?
- PMCA pumps use ATP to pump Ca2+ extracellularly
| - Na+/Ca2+ exchanges move 3 Na+ ions in and 1 Ca2+ ion out – derive energy from Na+ gradient
158
How does Ca2+ get extruded from the cytoplasm into the lumen of the ER/SR?
SERCA pumps use ATP to move Ca2+ into lumen of ER/SR
159
What are C2 domains?
The C2 domain of PKC is activated by Ca2+ --> PKC sticks to membrane --> phosphorylates various substrates
C2 domains are also important in synaptotagmin – has 2, C2A and C2B. Synaptotagmin is stuck into synaptic vesicle (ball full of neurotransmitter). C2A and C2B are sticking out of the vesicle. When Ca2+ binds to them, the vesicle fuses to the presynaptic membrane
160
What are EF hands?
Calmodulin has 4 “EF hand” Ca2+-binding domains, 2 at either end. When Ca2+ binds, calmodulin can go bind to other things (ion channels, protein kinases/phosphatases, cyclic nucleotide phosphodiesterases)
EF hand motif is found on many other Ca2+ effectors, including parvalbumin (cellular Ca2+ buffer), calpain (Ca2+-activated protease), and troponin
161
What are the four major components of the ECM?
1. Glycosylaminoglycans (GAGs), which usually form proteoglycans
2. Fibrous proteins, like collagen and elastin
3. Multidomain adaptor proteins, like fibronectin and laminin
4. Water and many solutes
162
What are GAGs?
Polysaccharide chains with disaccharide repeats
1 sugar = amino sugar
1 sugar = uronic acid
High negative charge --> becomes very hydrated --> forms gels
163
What are proteoglycans?
Covalently linked complexes of GAGs and proteins
Core protein has serine, which attaches a special tetrasaccharide, which then allows polymerization of disaccharide repeats
Polymerized sugars may be modified further (sulfation)
164
What are two types of fibrous proteins?
Collagens: ~20 different kinds. Collagen I is most abundant. Collagen IV is in basal lamina.
Elastin: network of elastic fibers in the ECM that provide elasticity
165
What are some multidomain adaptor proteins?
They help to organize the matrix and attach cells to it
Fibronectin:
- large, dimeric glycoprotein
- linked by disulfide bonds
- "type III fibronectin repeat" binds to integrins
Laminin:
- three subunits (α,β,γ)
- form an asymmetric, disulfide-linked cross
- found in the basal lamina only
166
What do matrix metalloproteases do?
Remodel the ECM by eating up the matrix
= allow cell migration, facilitate cell signaling
Important in development in tissues and used by pathogens to invade tissues.
Sometimes they unmask cryptic cell binding sites to promote cell binding or migration.
Promote cell detachment. Activate growth factors. Release ECM bound extracellular signals. Highly regulated and can be specific.
167
What are some cellular adhesion molecules?
Cadherins:
- homodimer transmembrane protein
- requires calcium
- homophilic binding to other cells via other cadherins
Ig-CAMs:
- also homophilic binding mechanism
- do NOT form dimers or require Ca
Integrins:
- heterodimers with α and β subunits
- heterophilic binding = large variety
- ligands include ECM proteins laminin, fibronectin, collagen
168
What are the sources of androgen in the body relevant to prostate cancer?
1. Testes – 90-95% of systemic testosterone
2. Adrenal glands – 5-10% of systemic testosterone
3. Intracrine androgen production in the prostate cancer cells themselves
169
What do enzalutamide and abiraterone do in terms of treating resistance in prostate cancer?
Abiraterone is a specific blocker of CYP 17 --> eliminates testosterone completely
Enzalutamide binds to AR --> inhibits nuclear translocation
170
How are voltage-gated Na and K channels activated?
K only has activation gate
Na has activation & inactivation gate
