8/16/17

Question 1

Two types of protein degradation pathways

Answer 1

Ubiquitin-proteasome

Lysosomal

Question 2

Ubiquitin-proteasome pathway

Answer 2

Selective process for short lived and damaged/misfolded proteins

ATP to add ubiquitin to Lys and get chain of them, ATP to make proteasome function

Question 3

Ubiquitination of proteins

Answer 3

Use ATP to add ubiquitin to E1

E1 transfers ubiquitin to E2

E2 works with E3 to add ubiquitin to the target

E3 is a ubiquitin ligase, many different kinds since they are selective for each protein

Question 4

Disorders of ubiquitination

Answer 4

Cancer: change in stability of proteins for cell division can create uncontrolled growth

Neurodegenerative: proteins aggregate if decreased degradation

Immune disorders, muscle wasting, diabetes

Question 5

Types of autophagy

Answer 5

Macroautophagy

Microautophagy

Chaperone-mediated autophagy

Question 6

Macroautophagy

Answer 6

Isolation membrane (phagophore) is a double membrane that begins to expand to engulf organelles, ribosomes, and protein aggregates

Autophagosome after isolation membrane fully engulf

Autophagosomefuses with lysosome to degrade cargo and inner membrane of the Autophagosome

Question 7

Microautophagy

Answer 7

Lysosomes invaginate and eat cytosolic components and small organelles

The invagination becomes an autophagic tube

Vesicles bud off the autophagic tube into lysosome lumen

Question 8

Chaperone-mediated autophagy

Answer 8

Highly selective unlike other 2 autophagies

Proteins have KEFRQ amino acid sequence or similar that bind to Hsc70 or Hsc73, targeted to go to lysosomal membrane

Protein binds to LAMP-2 at the membrane, oligimerize into a translocator to allow it to enter for degradation in the lysosome

Question 9

Autophagy-related genes

Answer 9

Stressors that induce expression of genes for autophagy

Nutrient depravation, growth factor withdrawal, infection, hypoxia, DNA damage

Question 10

mTOR

Mammalian target of rapamycin

Answer 10

Master regulator protein that promotes cell growth and proliferation by promoting anabolic processes and by limiting autophagy

mTOR inhibits expression of autophagy-related genes when healthy, upregulate when under cellular stress

Question 11

Pharmacodynamics vs pharmacokinetics

Answer 11

Kinetics: what body does to drug
ADME- adsorption, distribution, metabolism, excretion

Dynamics: what drug does to body

Question 12

Inert binding site

Answer 12

Endogenous molecule that binds to a drug but doesn’t lead to a response

Drugs bind to albumin but no effect

Question 13

Drug targets beside receptors

Answer 13

Enzymes: statins inhibit HMGCoA Reductase, lower intra-hepatic cholesterol synthesis

Transport proteins: digitalis inhibits Na-K ATPase

Structural proteins: colchicine binds tubulin

Question 14

Agonist

Answer 14

Affinity for a receptor AND activates intrinsic activity

Question 15

Orthosteric binding site

Answer 15

The main binding site

Allosteric is off to the side

Question 16

Inverse agonist vs antagonist

Answer 16

Inverse agonists generates a response below baseline

Antagonist: produces no effect, neutral

Question 17

Competitive antagonist

Answer 17

Binds to the same site of the agonist

Can be overcome by high conc. of agonists

Reduce agonist potency

Question 18

Noncompetitive antagonist

Answer 18

Bind to a site different than the agonist

Prevent binding of the agonist or prevent agonist from activating the receptor

Reduce agonist potency

Question 19

Drug selectivity

Answer 19

Drug has high affinity for a receptor

Produces intended effect with little side effects

Question 20

Therapeutic index and margin of safety

Answer 20

TD 50 / ED 50

LD 1 / ED 99

Question 21

Drug Receptor types

Answer 21

Ligand-gated ion channels: change in membrane potential or ion concentration

G protein-coupled receptors: intracellular protein phosphorylation

Enzyme-linked receptors: intracellular protein and receptor phosphorylation

Intracellular receptors: intracellular protein phosphorylation and altered gene expression

Question 22

Clinical case of defect in transport of molecules across the plasma membrane

CF

Answer 22

8 month old with poor growth and chronic cough

Bloating, diarrhea, and failure to thrive whether breast fed or formula

Developed daily cough and respiratory difficulty

Got asthma at 5 months

Large, greasy, foul-smelling stool and failure to thrive

Question 23

CF Symptoms

Answer 23

Salty sweat

Progressive respiratory damage

Chronic digestive problems

Obstruction of airways and bacterial infections from abnormally thick mucous

Pancreatic ducts are blocked, digestive enzymes can’t reach intestines

Die from pulmonary disease

Question 24

CF mutation

Answer 24

Most due to single AA mutation, many different mutations known

CFTR gene on q arm of chromosome 7

Protein misfolded in ER so never get to plasma membrane even though partly functional

Question 25

Predicted CF protein structure

Answer 25

2 membrane spanning domains that form chloride ion channel 2 nucleotide binding domains that bind and hydrolyze ATP Regulatory domain, not in other ABC transporters C terminal anchored to cytoskeleton by PDZ interacting domain

Question 26

Model of CFTR channel gating

Answer 26

1. R domain phosphorylated by PKA 2. Allows binding of ATP to NBD1 3. Hydrolysis of ATP by NBD1 opens channel (transiently) 4. PKA phosphorylates more sites on the R domain 5. NBD2 binds ATP to stabilize open channel 6. ATP hydrolysis at NBD2 releases ADP, channel closes 7. R domain dephosphorylation closes channel CFTR is cAMP activated and ATP gated ion channel

Question 27

Difference between CFTR and other ABC proteins

Answer 27

Has an R domain Allows flow of ions down their electrochemical gradient, ABCs use ATP to go against gradient

Question 28

Location of CFTR

Answer 28

Epithelial cells in the lung, liver, pancreas, digestive tract, respiratory tract, and skin

Question 29

6 types of CFTR mutations

Answer 29

1. Lack CTFR synthesis (premature stop codon) 2. Defective protein processing (misfolded CTFR) 3. Defective channel regulation/gating (gating defect) 4. Defective chloride conductance (restricted Cl- movement through channel) 5. Reduced amount of CFTR protein (alternative splicing) 6. Accelerated turnover of surface CFTR 1-3 more severe, 4-6 are milder disease forms

Question 30

Class II mutation of CFTR

Answer 30

Immature protein synthesized, most common mutation, F508del occurs on the surface of NBD1 Immature protein cuz partial glycosylation, not released from ER for transport to Golgi Degraded by ubiquitin-proteasomal pathway

Question 31

Salt reabsorption from sweat ducts for CF

Answer 31

Secretory cells operate normally In the reabsorptive duct epithelial Na+ channels in the apical membrane bring sodium ions in the cells to eventually be transported out of the basolateral membrane to the blood Na+ enters but Cl- doesn't since CFTR is the only anionic transporter, disrupts electrochemical gradient, little NaCl reabsorbed Sweat duct has low permeability for water so only ions move Sweat chloride conc. is the best physiological marker of CF

Question 32

Why salty sweat in CF

Answer 32

Failure of NaCl reabsorption and low water permeability in sweat duct Normal water permeability in secretory coil

Question 33

CFTR in airway epithelium

Answer 33

Normally CFTR open to allow Cl- to flow into lumen of lung (and pancreas) Cl- in lumen slows Na+ from lumen into the cells due to charge High NaCl conc. causes osmosis so there is normal airway surface liquid to clear pathogens In CF Na+ go into the cells since Cl- stuck there, little ASL to help fight pathogens

Question 34

Symptoms of CF in the lungs

Answer 34

Severe cough- to remove excess mucus Breathlessness- shortage of oxygen leads to increased tiredness and lack of energy Infections- bacteria trapped in mucus

Question 35

Pancreatic juice flow with CFTR

Answer 35

CFTR pumps out Cl-, so Na+ and H2O follow Increased volume of pancreatic juice, HCO3- also comes out to modulate pH In CF the Cl- stays in pancreatic epithelial cells so Na+ enter, ducts filled with viscous pancreatic juice (mucus) Sticky mucus blocks duct so digestive enzymes not reach duodenum

Question 36

CF pancreas problems

Answer 36

Exocrine pancreatic secretions have lower bicarbonate and volume, blocks ducts and obstructs flow of digestive enzymes Degradation of acinar cells and pancreatic fibrosis Pancreatic sufficient (mild) or pancreatic insufficient (severe) disease Pancreatic insufficiency has maldigestiin of fats/proteins, increased fecal loss, steatorrhea (pale loose smelly shits, associated with poor weight gain) Leads to chronic pancreatitis, possible pancreatic atrophy of endocrine cells that causes Cystic fibrosis-related diabetes food is not digested and absorbed so patients don't put on mass and suffer from malnutrition Trapped digestive enzymes damage beta cells so get diabetes

Question 37

Genotype and phenotype correlations for CF

Answer 37

Good correlation: Class I-III mutations lead to pancreatic insufficiency, IV and V lead to PS Moderate correlation: sweat chloride conc. and pancreatic status Weak correlation: lung function No clinical features unless less than 10% of normal CTFR function

Question 38

CF Treatments

Answer 38

Gene therapies are unsuccessful Pharmacotherapy that rescue CFTR function Gastrointestinal treatment: vitamin supplements, high caloric intake that is low in fat and high in protein Lung transplant if real bad

Question 39

Reason for persistence of CTFR gene mutations

Answer 39

Heterozygotes express 50% of normal CFTR levels, less fluid loss from cholera Europeans have the mutations mostly

Question 40

Cholera and CF

Answer 40

Toxin enters cell, passes through ER where A1 subunit detach and then resold in cytoplasm, A1 ADP-ribosylates the alpha subunit of a G protein, activated G protein activates adenylyl cyclase to make cAMP cAMP activates PKA that phosphorylates R domain of CFTR Cl- flows out too much cuz alpha subunit of G protein can't turn off the signal due to the ADP-ribosylation High salt conc. outside creates osmotic pressure Less CFTR means less water movement out and less diarrhea