Unit III - Cellular Physiology Flashcards
Properties & Phenotype of Transformed Cells
Altered morphology Loss of contact inhibition Anchorage independence Immortalization Reduced requirement for mitogenic growth factors High saturation density Increased transport of glucose Sustained angiogenesis De-differentiated Invasive Metastatic Clonal in origin
Familial Retinoblastoma
Autosomal dominant cancer susceptibility resulting from inherited heterozygosity in the RB tumor suppressor gene on 13, followed by LOH of RB in a single cell; presents as bilateral retinal tumors
Loss of heterozygosity
Results from duplication of an inherited mutant tumor suppressor gene during S phase, followed by a rare mitotic cross-over event between homologs during M phase; in some cases, one daughter cell will end up with both functional tumor suppressor genes and one daughter cell will end up with NO functional tumor suppressor genes, becoming tumorigenic
Familial Adenomatous Polyposis (FAP) & Wnt Signalling
Results from inheritance of one defective copy of the APC tumor suppressor gene followed by LOH; 90% of affected will develop colon cancer by age 50
Normally, WNT growth factor stimulates the Frizzled receptor, which signals APC to release B-catenin in the cytoplasm; B-catenin then moves to the nucleus and binds TCF which signals transcription of c-myc, a TF that promotes cellular proliferation (oncogene)
Familial Breast Cancer
Caused by inherited heterozygosity in BRCA1 or BRCA2 genes followed by LOH; normally, BRCA1 and BRCA2 function in the ATM/CHK2 DNA-damage check point & repair system
p53
P53 is a tumor suppressor gene that is up-regulated in response to DNA damage; ATM/CHK2 phosphorylate p53 in response to DNA damage, activating p53 to bind DNA and promote transcription of p21, which inactivates S-CDK complexes, thereby inhibiting progression through the cell cycle
Missense mutations in p53 code for a mutant protein that disrupts the normal tetramer structure of p53, “poisoning” it
erB2
ErB2 encodes an integral membrane protein kinase that is amplified in 20% of breast cancers; Herceptin is a monoclonal antibody specific for the ErB2 receptor
Von Hippel Lindau Syndrome - Mechanism, Phenotype, & Treatment
Caused by mutation in the VHL gene; normally, VHL binds hypoxia-inducible factor (HIF) subunit alpha and targets it for ubiquitination; in the absence of VHL protein, HIF-a binds HIF-B and translocates into the nucleus to promote transcription of VEGF
Phenotypic tumors: CNS & retina hemangioblastoma, renal cell carcinoma, pheochromocytoma
Target therapies: VEGFR TKIs Sunitinib & Sorafenib
Li-Fraumeni Syndrome
Caused by an inherited mutation in the p53 tumor suppressor gene, which predisposes the affected to many forms of cancer
Diagnostic criteria:
- Sarcoma diagnosed before 45 years old
- 1st degree relative with any cancer under 45 years old
- 1st or 2nd degree relative with any cancer under 45 years old, or sarcoma at any age
SNARE Complex Formation & Disassembly
Formed by association between helical domains of Syntaxin, SNAP-25, and VAMP proteins; hydrophobic surfaces of each helix orient toward each other and form a stable complex; an “ionic bubble” is formed by 1 charged residue on each helix
SNARE complexes are dissassembled by ATPases NSF and alpha-SNAP and N-sec-1 acts as a molecular chaperone to help re-fold SNARE proteins
Viral Fusion (Ex: HIV)
Viral fusion proteins have a transmembrane domain embedded in the viral envelope and a fusion peptide motif - a stretch of hydrophobic AAs that become “activated” to insert into the host cell membrane
Ex: HIV FP gp120 recognizes and binds CD4 expressed on the host cell membrane; this activates FP gp41 to insert into the host cell membrane
3 classes of lipids in a membrane
- Phospholipids
- Sphingolipids
- Cholesterol
Cholesterol Regulation Pathway
SREBP contains a bHLH TF domain that regulates LDLR gene; SREBP is held in the ER and bound by Insig/SCAP when cholesterol is high; when cholesterol is low, Insig releases SCAP and SCAP signalling recruits COPII, which transports the SCAP/SREBP complex into the Golgi where the TF is cleaved from SREBP by S1P and S2P
Voltage-gated channel structure
4 membrane-spanning domains, each containing 6 alpha helices (S1-S6). S4 helices have positively charged residues (Lys or Arg) which form the voltage sensor. S5 and S6 helices are connected by the P loop and assemble to form the ion-conducting pathway
Kv = 4 separate polypeptides Nav = 1 polypeptide
NaV Mechanism
Activation: Depolarization causes repulsive electrostatic interactions with the positive charge on S4, causing the activation gate to swing open
Inactivation: The inactivation gate is formed by the cytoplasmic loop which connects repeats III and IV; it swings up into a binding site on the inner portion of the channel causing current to decay to 0
Removal of inactivation: The inactivation gate leaves it’s binding site, allowing deactivation to occur in which the activation gate swings shut and the channel is re-set
2 Mechanisms of Glucose Transport
- Glucose is pumped across the apical membrane via secondary active transport driven by the Na gradient
- Facilitated diffusion into muscle cells followed by immediate phosphorylation to G-6-P; these GLUT receptors are exocytosed to the membrane in response to insulin signalling from pancreatic beta cells
Uses of Na powered secondary active transport
- Na/Ca exchanger uses the inward leak of Na+ to pump Ca2+ out of the cell
- Na/H exchanger uses the inward leak of Na+ to pump H+ out of the cell
Microtubule structure
The basic subunit of a microtubule is a heterodimer of alpha and beta tubulin, which bind GTP and align in tandem to form a protofilament with a free beta subunit at the (+) end and a free alpha subunit at the (-) end; 13 protofilaments assemble to form a microtubule ~25 nm in diameter
Centrosome
AKA microtubule organizing center (MTOC); most cells contain a single MTOC with a pair of centrioles located near the nucleus; microtubules grow from the pericentriolar material with their (-) ends anchored in the complex and their (+) ends growing into the cell
Kinesin Structure & Function
Exists as a homodimer; each subunit consists of a head group which binds microtubules, a coil motif, and a tail group which binds an adapter molecule for a specific cargo
Kinesin is an ATPase that moves cargo along microtubules toward the (+) end
Dynein
Dynein transports cargo toward the (-) end of microtubules; ex: it is responsible for retrograde transport of materials from axonal terminals to neuronal cell bodies
Role of centrosome during mitosis
During mitosis, the centrosome is duplicated and one moves to each pole of the dividing cell; astral microtubules radiate out from the centrosomes; (+) ends of kinetochore microtubules attach to centromeres of chromosomes; (+) ends of overlap microtubules slide against each other, creating a force that pushes the poles apart while (-) directed motors separate daughter chromosomes and move them toward the centrosomes
Intermediate filament structure & function
Basic subunit is composed of two globular protein domains linked by an alpha helical region; the subunit forms tetramers and 8 tetramers twist into a rope-like filament 10 nm in diameter
Actin microfilament structure
In the presence of ATP, globular actin (G-actin) monomers assemble to form two-stranded, helical filaments (F-actin) 7 nm in diameter with (+) and (-) ends
FH2/Formin nucleates linear networks of actin filaments; Arp2/3 nucleates branched networks
4 main roles of actin in cell function
Epithelial cell polarity - anchors TJ/AJ proteins
Contraction - via interactions with Myosin
Motility - via polymerization in the filopodia and lamellipodia
Division - via actomyosin ring formation
Nernst Equation
E = (60/Z) x log (Co/Ci)
Osmolarity vs. Tonicity
Osmolarity describes the total concentration of solutes in a solution, whereas tonicity describes the effect of a solution on a cell (hypertonic or hypotonic)
Ionic driving force
Vm - Eion
Donnan Rule
[K]o[Cl]o = [K]i[Cl]i
Na/K Pump - Mechanism
While ATP-bound, the pump binds 3 intracellular Na+ ions; ATP hydrolysis phosphorylates the pump and triggers the closing of the inner gate and the opening of the outer gate; 3Na+ molecules are released extracellularly and 2K+ molecule are bound from the ECF; dephosphorylation of the pump triggers closing of the outer gate and opening of the inner gate; 2K+ are released intracellularly, and ATP binds again.
Hyperkalemia
Acute increase in [K]o often caused by potassium release from cells following crush injury, or secondary to acidosis (i.e. in DKA); may cause depolarization of cardiac cells resulting in cardiac arrhythmia
Treatment: CBIGK
Blood pH, venous vs. arterial
Arterial: 7.34 - 7.44
Venous: 7.28 - 7.42
Venous blood is more acidic because it carries more CO2 in equilibrium with carbonic acid
Henderson Hasselbach equation (general and bicarbonate-specific)
General: pH = pKa + log ([base]/[acid])
Bicarbonate-specific: pH = 6.1 + log ([HCO3-]/(.03)(PCO2)
Clinical Presentations related to acid/base chemistry
Normal bicarbonate = 24mM
Normal pCO2 = 40 mmHg
Normal CO2 concentration = 1.2mM
Hyperventilation is a compensatory response to acidosis
What molecular phenomena are responsible for the refractory period of an AP?
The absolute refractory period is caused by closing of the NaV inactivation (h) gate; although the activation (m) gate may be open in response to depolarization, no Na+ current will flow
The relative refractory period is caused by K+ channels that remain open; it will take a larger depolarizing stimulus to reach threshold if outward K+ conductance is still high
Cardiac Arrhythmia in Hyperkalemia - Mechanism & Treatment
Increasing [K]o reduces the driving force on K+ to leave the cell, causing depolarization which move Vm closer to threshold; this can either induce APs or inhibit APs by chronically inactivating Na+ channels - either way, it leads to Maverick pacemakers
Ca2+ interacts with the outer cellular membrane, ‘screening’ fixed negative charges via electrostatic interactions and effectively hyperpolarizing the membrane; this increases the threshold for the cell to fire an AP and ‘quiets’ the arrhythmia
6 functions of ER
Synthesis of lipids
Control of cholesterol homeostasis
Storage of Ca2+
Synthesis of proteins on membrane-bound ribosomes
Co-translationa folding of proteins & early post-translational modification
Quality control
4 Functions of the Golgi
Synthesis of complex sphingolipids
Additional post-translational modification of proteins and lipids
Proteolytic processing
Sorting of proteins & lipids for post-Golgi compartments
Necrosis
Mitochondria undergo Ca2+ induced high amplitude swelling and become non-functional; without ATP, the Na/K pump fails, Na+ accumulates within the cell, water follows, and the cell swells and bursts releasing pro-inflammatory cellular contents into the extracellular space
Intrinsic pathway of apoptosis
Normally, anti-apoptotic members of the BCL-2 protein family, BCL-2 and BCL-XL, guard the mitochondrial membrane. As a result of some suicide signal, “pro-apoptotic” members such as Bim and PUMA are made; they move to the mitochondrion and replace BCL-2 and BCL-XL. There, they associate with Bax, which acts on the membrane, making it permeable to cyt-c. Relase of cyt-c into the cytoplasm activates Apaf-1, Apaf-1 activates Casepase-9, and Casepase-9 activates Caspase-3 (the executioner), which cleaves over 700 substrate proteins, leading to apoptosis
Extrinsic Pathway of apoptosis
Cytotoxic T cells express a ligand FasL (CD95L) which recognizes the surface molecule Fas (CD95) on an abnormal cell; binding of FasL to Fas recruits an intracellular adaptor molecule FADD, which activates Casepase 8; Casepase 8 activates Caspase 3, which carries out apoptosis.
Mechanism of CFTR-mediated secretion
Gut epithelia cells draw Cl- into the cell through a Na/K/Cl co-transporter in the basolateral membrane which pumps 3 Na and 3K into the cell along with 6 Cl; Cl- then leaks across the apical membrane through the CFTR channel which is opened by parasympathetic stimulation during digestion; Na+ and H20 follow Cl- through the pericellular shunt, secreting an isotonic solution of NaCl
Composition of NPCs
Nuclear Pore Complexes (NPCs) are comprised of ~30 distinct nucleoporins (Nups) repetitively arranged in distinct subcomplexes
NPCs contain FG domains which are disordered repeat sequences rich in phenylalanine and glycine which act as “tethers” to dock cargo
Ran Cycle
In the cytoplasm, the Importin/NLS complex is translocated through the NPC and into the nucleus; in the nucleus, Ran-GEF exchanges GDP for GTP, which triggers the release of cargo; Ran-GTP can then associate with the exportin/NES complex and escort it through the NPC to the cytoplasm, where Ran-GAP hydrolyzes GTP to GDP, releasing the exported cargo; Ran-GDP is then escorted back into the nucleus by NFT2 for re-use
Nuclear export of mRNA
ALY protein recognizes binding sites in mRNA and recruits NXF1/NXT1 proteins which associate with mRNA via substrate-binding domains; ALY adapter protein then disassembles and the mRNA/NXF1/NXT1 complex moves through the nucleoporin via NPC-binding domains
Translocation of ER-bound proteins
Signal recognition particle (SRP) recognizes and binds the ER signal sequence on a newly formed polypeptide, inducing a pause in translation; the SRP then delivers the nascent polypeptide and ribosome to the SRP receptor on the ER membrane, next to the translocon. Membrane-bound signal peptidase then cleaves the ER signal sequence from secreted proteins, allowing the translocon to release the hydrophobic signal into the membrane where it is degraded
Types of Vesicular Coat Proteins
COPI - Facilitates budding of vesicles from Golgi to ER
COPII - Facilitates budding of vesicles from ER to Golgi
Clathrin - Facilitates budding of vesicles from Golgi to plasma membrane (with AP2 and GTPase Dyamin)
N-linked glycosylation
Targaret asparagine AAs on protein domains within the ER lumen are glycosylated; precursor oligosaccharides are transferred to the Asn as an intact unit in a reaction catalyzed by membrane-bound oligosaccharyl transferase enzyme, associated with each translocon.
Mechanism of ER quality control
Calnexin (an ER TM protein) and Calreticulin (a soluble ER protein) bind to glucose within oligosaccharide chains on misfolded proteins; glucosidase removes the glucose and correctly folded proteins are able to exit the ER; if protein is not correctly folded, it is recognized by a glucosyltransferase (GT) which puts glucose back onto the sugar chain, enabling Calnexin/Calreticulin to bind the protein again
Ubiquitination & Proteolysis
Ligase enzymes E1, E2, and E3 attach 4+ ubiquitin molecules to proteins; ubiquitin is recognized by the proteosome caps, which use ATP to unfold the protein and feed it into a central cylinder where proteolytic cleavage takes place by Beta subunits that cut the polypeptide into strings of 7-9 AAs
Chaperone-mediated autophagy
HSC70 recognizes a specific 5 AA motif in misfolded proteins and associates with a large protein complex to bring these misfolded proteins to a lysosomal membrane receptor LAMP-2A
Steps of macroautophagy
- Induction
- Vesicle nucleation
- Vesicle expansion
- Cargo targeting
- Vesicle closure
- Vesicle fusion with endosome
- Vesicle fusion with lysosome
Mechanism of Ras activation
Growth factor ligand binding to RTK triggers dimerization and autophosphorylation of the RTK; SH2 domain in Grb2 adaptor protein binds to phosphotyrosine residues on the RTK; Grb2 SH3 domains bind proline-containing peptide Sos, a Ras GEF; Sos activates Ras through proximity
Mechanism of TKI vs. antibodies
Antibodies (i.e. Cetuximab) block the extracellular ligand binding site on EGFR, preventing dimerization
Targeted kinase inhibitors (TKIs) i.e. Gefinitib bind in the phosphorylation site of EGFR, blocking ATP from binding and preventing phosphorylation & downstream signaling
Tumors may be resistant to TKIs (acquired or primary resistance)
5 mechanisms of signaling termination
Re-uptake, degradation, or diffusion of an extracellular signaling molecule
Phosphatases - de-phosphorylate kinase cascades
Phosphodiesterases - hydrolyze cAMP and cGMP; activity increased by allosteric binding of substrate and phosphorylation by c-NMP dependent kinases
Intrinsic GTPase activity
Constitutively active terminators, i.e. Ca2+ pumps
4 types of cellular signaling
Paracrine - from signaling cell to target cell over a short distance
Contact-dependent - requires physical contact between a membrane-bound mediator on the signaling cell and a receptor on the target cell
Endocrine - from signaling cell to target cell over a long distance
Synaptic - mediated by release of neurotransmitter at a synaptic cleft
Mechanism of G-protein coupled signaling
Inactivated GCPRs are bound intracellularly to a heterotrimeric G-protein, composed of an alpha subunit(-GDP) and a beta/gamma subunit; agonist binding triggers a conformational change that favors the disassociation of GDP from the alpha subunit; GTP quickly binds to and activates G-alpha; G-alpha dissociates from beta-gamma (via SwitchII) and both subunits diffuse through the membrane to reach their effector proteins
G-alpha is a GTPase, which hydrolyzes GTP to GDP; hydrolysis triggers G-alpha-GDP to re-bind the beta-gamma subunit as well as the receptor;
GAPs accelerate the process of GTP hydrolysis, shortening the lifespan of the signaling pathway
Beta-1 adrenergic receptor
Binds norepinephrine to activate Gs protein; G-alpha subunit activates adenylyl cyclase, converting ATP to cAMP; cAMP activates PKA; PKA phosphorylates voltage-gated Ca2+ channels and Ryanodine Receptors in the SR, increasing intracellular concentration of calcium, leading to increased heart rate and contractility
Antagonists: propranolol, metoprolol (HTN)
Alpha-1 adrenergic receptor
Alpha-1 adrenergic receptor binds norepinephrine; activation of Gq protein activates PLC, which cleaves membrane lipid PIP2 into IP3 (cytosolic) and DAG (membrane-bound);
IP3 binds to IP3-receptor in ER and releases Ca2+
DAG-PKC stimulates Ca2+ through L-type, voltage gated Ca2+ channels
Increased intracellular Ca2+ stimulates smooth muscle contraction in peripheral vasculature, shifting blood flow from the skin to the viscera, increasing blood pressure
Antagonist: Prazosin (HTN)
m2 muscarinic cholinergic receptor signaling via alpha in the heart
M2 AchR binds agonist Ach; activated G-i protein antagonizes the effect of G-s on adenylyl cyclase; G-i can dominantly inhibit AC and the production of c-AMP; cAMP is degraded by PDE and so the sympathetic response is shut down, leading to decreased heart contraction
m2 muscarinic cholinergic receptor signaling via beta-gamma in the heart
Ach binds m2 AChR, activating G-i protein; activated beta-gamma subunit activates the GIRK membrane-bound K+ channel, which allows K+ efflux from the cell, causing cellular hyperpolarization and decreased excitability, leading to decreased heart rate contraction
Antagonist: Atropine, increases heart rate
Beta-2 adrenergic receptor signaling in the lungs
Epinephrine binds B2AR, activating Gs protein; Gs alpha subunit activates AC, which produces cAMP; cAMP activates PKA; PKA phosphorylation inhibits smooth muscle contraction, leading to bronchodilation
Agonist: Albuterol
m3 muscarinic cholinergic receptor signaling in the lungs
ACh binds m3AChr, activating Gq protein; Gq-alpha activates PLC, which cleaves PIP2 into IP3 and DAG leading to Ca2+ release and increased bronchoconstriction
Antagonist: Ipratropium, asthma inhaler
GCPR Desensitization
Activated GCPR conformation is a substrate for G-protein receptor kinase (GRK), which phosphorylates the receptor; phosphorylation signals binding of B-arrestin to the receptor; B-arrestin serves as an adaptor molecule for receptor endocytosis machinery; endocytosed receptors can be degraded or re-sensitized by phosphatases and returned to the membrane
Ca2+ movement from sources into cytoplasm
Ligand-gated Ca2+ channels in the plasma membrane (nACh, glutamate)
Voltage-gated Ca2+ channels in the plasma membrane
Store-operated Ca2+ channels in the plasma membrane - activated in response to depletion of Ca2+
Ryanodine Receptor in the ER/SR
IP3 Receptor in the ER/SR
Movement of Ca2+ from sources to sink is electrochemically passive
Ca2+ movement from cytoplasm into sources
Ca2+ is moved from sinks to sources by transporters, which pump against an electrochemical gradient
Ca2+ pumps use ATP active transport to pump Ca2+ out of the cytoplasm into the extracellular space or ER/SR lumen; PMCa ATPase and SERCa ATPase
Na+/Ca2+ exchangers extrude 2 Ca2+ across the plasma membrane using the energy of leak of 3 Na+ into the cell down its electrochemical gradient
Stem Cells
Totipotent - Can differentiate into any cell of the body; derived from zygotes
Pluripotent - can differentiate into cells from any of the 3 germ layers; adult somatic cells can be induced to exhibit pluripotency by expression of reprogramming factors followed by DNA editing/repair and re-differentiation
Multipotent - can differentiate into multiple cell types within the same tissue
Protein Kinase structural features
Glycine-rich loop: Binds ATP in the closed conformation and positions gamma-phosphate for phosphorylation reaction; allows ADP to escape in the open conformation; this nucleotide exchange is the rate-limiting step of the phosphorylation reaction
Many (not all) kinases require phosphorylation of an activation loop in order to form the right conformation for activity
Diagnosis and Presentation of DKA
In the setting of diabetes, cells may become starved for glucose and undergo beta oxidation of fatty acids to produce ATP; this pocess generates hydrogen ions & ketone bodies
Presentation: Hyperglycemia, ketonemia/ketonuria, metabolic acidosis, polyuria, dehydration, K+ imbalances, rapid breathing
Pathophysiology of cholera
Vibrio cholerae secretes cholera toxin (CT); CT-Beta binds to the GM1 ganglioside receptor on the lumenal surface of cells in the small intestine, at which point the alpha subunit is cleaved off and endocytosed; it binds to intracellular Gs-alpha, stimulating adenylyl cyclase to produce cAMP, which activates CFTR to efflux chloride ions, followed by water, into the intestinal lumen.
Clinical features & treatment of cholera
Watery feces with presence of mucus (up to 1L/hr)
Vomiting
Severe and rapid dehydration (tachycardic, skin tenting)
ORT solutions take advantage of sodium co-transporters in the apical membrane; giving Na along with glucose or AAs drives Na co-transport into the cell through the apical membrane; as Na is transported from the lumen into the cell, Cl- and water follow
Multiple Sclerosis
MS is an auto-immune disease characterized by inappropriate reaction of T-cells and B-cells to myelin in the brain, causing inflammatory demyelination and decreased speed of conduction in nerves
Clinical presentation: fatigue, walking impairment, muscle weakness, loss of balance, spasticity, pain
Mechanism of insulin release
Glucose entering the pancreatic beta cell through the GLUT2 transporter undergoes glycolysis, leading to an increase in ATP; this signals the ATP-sensitive K-channel to close, preventing outward leak of K; build-up of intracellular K depolarizes the membrane, activating a voltage-gated Ca channel and leading to Ca influx, which signals exocytosis of insulin-containing secretory granules.
Potassium derangements in DKA
- In response to dehydration, the body conserves sodium in order to create an osmotic force that helps conserve water; the body activates aldosterone, which stimulates an antiport mechanism that takes in sodium at the expense of excreting potassium into the urine
- Acidosis leads to the uptake of H+ ions in exchange for the release of K+ into the ECF (hyperkalemia)
Therefore, patients may present with hyperkalemia despite overall potassium depletion
Cerebral edema in DKA
Total body osmolarity increases in DKA due to hyperglycemia and sodium retention secondary to dehydration; therefore, giving fluids that are normally isotonic may actually be hypotonic to this patient, causing movement of water across the BBB via osmosis and swelling of the brain
Giving insulin improves ketoacidosis; decreased H+ in the ECF drives K+ back into cells, which can create an osmotic gradient pulling water into the cells in the brain
Fixed, dilated pupils (CN III palsy)
Cushing’s triad (hypertension, bradycardia, irregular respirations)
Mental status changes
Treatment: IV mannitol, which raises the effective osmolarity of the blood and pulls water out of the brain in order to decrease swelling
