8/16/17 Flashcards
Two types of protein degradation pathways
Ubiquitin-proteasome
Lysosomal
Ubiquitin-proteasome pathway
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
Ubiquitination of proteins
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
Disorders of ubiquitination
Cancer: change in stability of proteins for cell division can create uncontrolled growth
Neurodegenerative: proteins aggregate if decreased degradation
Immune disorders, muscle wasting, diabetes
Types of autophagy
Macroautophagy
Microautophagy
Chaperone-mediated autophagy
Macroautophagy
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
Microautophagy
Lysosomes invaginate and eat cytosolic components and small organelles
The invagination becomes an autophagic tube
Vesicles bud off the autophagic tube into lysosome lumen
Chaperone-mediated autophagy
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
Autophagy-related genes
Stressors that induce expression of genes for autophagy
Nutrient depravation, growth factor withdrawal, infection, hypoxia, DNA damage
mTOR
Mammalian target of rapamycin
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
Pharmacodynamics vs pharmacokinetics
Kinetics: what body does to drug
ADME- adsorption, distribution, metabolism, excretion
Dynamics: what drug does to body
Inert binding site
Endogenous molecule that binds to a drug but doesn’t lead to a response
Drugs bind to albumin but no effect
Drug targets beside receptors
Enzymes: statins inhibit HMGCoA Reductase, lower intra-hepatic cholesterol synthesis
Transport proteins: digitalis inhibits Na-K ATPase
Structural proteins: colchicine binds tubulin
Agonist
Affinity for a receptor AND activates intrinsic activity
Orthosteric binding site
The main binding site
Allosteric is off to the side
Inverse agonist vs antagonist
Inverse agonists generates a response below baseline
Antagonist: produces no effect, neutral
Competitive antagonist
Binds to the same site of the agonist
Can be overcome by high conc. of agonists
Reduce agonist potency
Noncompetitive antagonist
Bind to a site different than the agonist
Prevent binding of the agonist or prevent agonist from activating the receptor
Reduce agonist potency
Drug selectivity
Drug has high affinity for a receptor
Produces intended effect with little side effects
Therapeutic index and margin of safety
TD 50 / ED 50
LD 1 / ED 99
Drug Receptor types
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
Clinical case of defect in transport of molecules across the plasma membrane
CF
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
CF Symptoms
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
CF mutation
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
Predicted CF protein structure
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
Model of CFTR channel gating
- R domain phosphorylated by PKA
- Allows binding of ATP to NBD1
- Hydrolysis of ATP by NBD1 opens channel (transiently)
- PKA phosphorylates more sites on the R domain
- NBD2 binds ATP to stabilize open channel
- ATP hydrolysis at NBD2 releases ADP, channel closes
- R domain dephosphorylation closes channel
CFTR is cAMP activated and ATP gated ion channel
Difference between CFTR and other ABC proteins
Has an R domain
Allows flow of ions down their electrochemical gradient, ABCs use ATP to go against gradient
Location of CFTR
Epithelial cells in the lung, liver, pancreas, digestive tract, respiratory tract, and skin
6 types of CFTR mutations
- Lack CTFR synthesis (premature stop codon)
- Defective protein processing (misfolded CTFR)
- Defective channel regulation/gating (gating defect)
- Defective chloride conductance (restricted Cl- movement through channel)
- Reduced amount of CFTR protein (alternative splicing)
- Accelerated turnover of surface CFTR
1-3 more severe, 4-6 are milder disease forms
Class II mutation of CFTR
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
Salt reabsorption from sweat ducts for CF
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
Why salty sweat in CF
Failure of NaCl reabsorption and low water permeability in sweat duct
Normal water permeability in secretory coil
CFTR in airway epithelium
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
Symptoms of CF in the lungs
Severe cough- to remove excess mucus
Breathlessness- shortage of oxygen leads to increased tiredness and lack of energy
Infections- bacteria trapped in mucus
Pancreatic juice flow with CFTR
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
CF pancreas problems
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
Genotype and phenotype correlations for CF
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
CF Treatments
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
Reason for persistence of CTFR gene mutations
Heterozygotes express 50% of normal CFTR levels, less fluid loss from cholera
Europeans have the mutations mostly
Cholera and CF
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
