Cell Physiology Flashcards
What is the loss of heterozygosity?
Heterozygosity = two different copies of one gene. Loss thereof can lead to oncogenic factors getting turned on
What is Knudson’s “two hit” hypothesis?
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
What are some cancers that are inherited in an autosomal dominant fashion?
Familial Adenomatous Polyposis (FAP-APC gene), Familial Retinoblastoma (RB gene), familial Breast and Ovarian Cancer (BRCA1 and BRCA2 genes) and Wilms tumor syndromes
What are some cancers that are inherited in an autosomal recessive fashion?
Xeroderma pigmentosa (XP genes), Ataxia-telangiectasia (AT gene), Bloom’s syndrome and Fanconi’s congenital aplastic anemia (FA genes)
What is the significance of the Rb gene?
It was the first elucidated tumor suppressor gene.
It’s a tumor suppressor –> it inhibits growth
What are some biochemical properties of the Rb protein?
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
How does the loss of Rb lead to malignancy?
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
What is the genetic mutation in familial retinoblastoma?
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)
What is APC?
Adenomatous polyposis coli gene
= tumor suppressor
Mutation in this leads to familial adenomatous polyposis (FAP)
What are the biochemical events that occur in FAP?
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
What do BRCA1 & BRCA2 do?
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
What happens in mutations of p53? Why are they so bad?
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!
Why is p53 “the guardian of the genome”?
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
How does HPV act as an oncogenic virus in cells?
Acts by creating proteins that inactivate both Rb (through E7) and p53 (through E8), which are both tumor suppressor proteins –> double kill shot!
How do viral oncogenes work?
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)
What are some examples of viral oncogenes?
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
What the relevance of n-myc in cancer?
It’s amplified in neuroblastoma
It’s a member of the c-myc family of oncogenes (promoters of cell growth)
What is the relevance of the HER2/neu/Erb2 gene in cancer?
It’s amplified in ~20% of breast cancers
What genes can be mutated in human bladder cancer cells?
c-ras = point mutations causes a protein product that is always on
What is Herceptin?
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
What does Gleevac do specifically?
It acts as an ATP mimic and prevents the kinase from phosphorylating
What are the parts of a phospholipid?
Polar head group
Phosphate connecting polar head group and glycerol backbone
Glycerol 3-phosphate backbone
Fatty acyl chains (16C or 18C), saturated or unsaturated
What are the parts of a sphingolipid?
Sphingosine backbone + amide group + fatty acid = ceramide
Phosphate linker
Polar head group
*If you add a sugar instead of a polar head group = glucosylceramide
What is the structure of cholesterol?
Hydrophilic head: -OH on top
Rigid steroid rings; fatty acid tail
What is the function of cholesterol?
Serve to increase membrane stiffness and thickness
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
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)
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
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
- When cholesterol levels are low SCAP-SREBP complex dissociates from Insig.
- SCAP escorts SREBP to the Golgi by vesicular transport.
- The bHLH transcription factor is released from SREBP by RIP
- S1P is luminal, S2P is within the membrane – cleavage by both is required for activation
- 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.
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
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
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
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)
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%)
What do SNAREs do?
SNARE = soluble SNF attachment protein receptor
They regulate membrane fusion
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
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
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
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.
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
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
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
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
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
What are the 3 mechanisms that cells have evolved to prevent from swelling & bursting?
- Membrane impermeable to water
- Cell wall
- Balance cell contents osmotically with outside environment
What are reflection coefficients?
Reflection coefficient of 1 = non-permeable
Reflection coefficient of 0 = same permeability as water
What is osmolarity?
Concentration of solute particles - a 1 M solution of CaCl2 gives a 3 osM solution (3 ion/mole)
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.
What are equivalents?
Calculated by converting to mosM & multiplying mosM by valence of the ion
What is the Donnan Rule?
[K]i[Cl]i = [K]o[Cl]o
What is the Driving Force on an ion?
Vm - E of the ion
Define pKa
pKa = -log [H+ ][A-] / [HA]
The lower the pKa, the stronger the acid. Higher = weaker acid.
What is the Henderson-Hasselbalch Equation?
pH = pKa + log [A-]/[HA]
What is the H-H equation for the bicarbonate buffer system in ECF?
pH = 6.1 + log [HCO3-]/ .03Pco2
What are normal blood pH, [HCO3-], and pCO2?
Blood pH = 7.4
[HCO3-] = 24 mM
pCO2 = 40 mmHg
What is log(2)?
0.3
What is the pH range of maximal buffering capacity?
+/- 1 pH away from where [HA] = [A-]
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
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
What is the mechanism for the release of insulin?
- Glucose enters beta cell in pancreas
- Glucose undergoes glycolysis, produces ATP
- ATP inhibits a K channel that allows K to exit cell; K builds up in cell
- K buildup causes depolarization of cell, Vm increases
- Voltage-gated Ca channels open, allowing Ca to rush inside
- Increase in intra-cellular Ca causes insulin-containing vesicles to fuse with plasma membrane and release contents extracullularly
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
What is Cushing’s Triad?
Irregular/agonal breathing, hypertension, and bradycardia
= a sign of increased intracranial pressure
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
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
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
What is Li-Fraumeni Syndrome?
A hereditary autosomal dominant cancer syndrome associate with a mutation in the p53 gene
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
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”
