Protein Misfolding And Disease 1

Question 0

Hsp70 and Hsp60

Answer 0

Chaperone classes

Hsp70 binds to proteins to cover up sticky hydrophobic patches when they are in intermediate but not native conformations (during synthesis)

Hsp60 forms large and hollow double donut, misfolded protein enter cavity, groES cap seals in polar environment, ATP hydrolysis used to unfold, if doesn’t refold by time cap dissociates, is expelled back into cell to refold, may take several rounds

Question 1

Chaperones

Answer 1

Proteins to prevent aggregation during folding, especially during high temperatures
Usually used to fold large, multi-domain proteins

Question 2

GroEL/GroES

Answer 2

Mechanisms include:
Isolation (anfinsen box)
Forced unfolding (iterative annealing)
Confinement (same as anfinsen but active and protective container)

Question 3

Protein degradation

Answer 3

Regulated by ubiquitin/proteasome pathway

Proteins are targeted for degradation by covalent ligation with ubiquitin which requires ATP

Question 4

Degradation by proteasome

Answer 4

High molecular mass multifunctional protease, 26S
Substrates are not only old or damaged proteins but also short lived proteins functioning as regulatory factors in cell cycle progression, cell growth, gene expression, inflammatory response, immune surveillance.
Polyubiquinated proteins bind, ATP used to unfold protein
Deubiquinating enzymes cleave away ubiquitin to be reused
Protein cleaved into peptides, transported through ER for antigen presentation or recycled by further degradation by free endo- and exopeptidases.

Question 5

Diseases of ubiquitin-proteasome pathway

Answer 5

Cancer (inc growth rate makes cancer cells more dependent on proteasome, inc degradation of tumor suppressors)
Neurodegenerative diseases (accumulation of ubiquinated proteins in plaques etc)
Cystic fibrosis
Autoimmune disease (improper processing of peptide antigens)

Question 6

Molecular basis of disease

Answer 6

Loss of protein function
Formation of alternate conformations that are toxic
Gain of function

Question 7

Function can be perturbed by

Answer 7

Direct knockout-mutation of a residue essential for function (substrate binding, catalysis
Destabilization-mutation pushes equilibrium toward the unfolded state (side chain in tightly packed hydrophobic core changed to one of different size, shape or charge)
Toxic conformation-mutation shifts equilibrium towards incorrectly folded state (surface charged residue changed to hydrophobic one, causes aggregation)

Question 8

P53 related cancers

Answer 8

Half of all tumors have point mutations in p53, in DNA binding domain
Transcription factor activated by DNA damage or other and triggers cell cycle arrest/apoptosis
Has a single zinc ion necessary for site-specific DNA binding

Question 9

P53 mutations

Answer 9

DNA contact-reducing DNA binding without changing overall protein structure or stability

Destabilizing-mutations don’t change DNA binding residues, they decrease thermodynamic stability (native and intermediate forms) by disrupting hydrophobic, electrostatic, H bonding, van der waals; Most common outcome; leads to increased degradation by ubiquitin/proteasome pathway in others but mutant p53 actually accumulates because of negative feedback loop with own ubiquitin ligase MDM2.

Zinc binding

Question 10

Treat stability mutants by

Answer 10

Designing small molecule to bind protein and stabilize the folded state
Can treat many diseases
Or block interaction between p53 and MDM2 to allow mutated p53 to accumulate in cell so that even a small percentage may be functional

Question 11

Cystic fibrosis

Answer 11

Thick, sticky mucus in lungs, pancreas, intestine
Inability to absorb nutrients
Buildup of fluids in lungs causes infection and lung degeneration
Most caused by deletion of Phe508 in cystic fibrosis transmembrane conductance regulator (CFTR)
Mutation alters the folding pathway of CFTR, gets degraded
Treatments?:over expressing chaperones, inhibiting degradation, stimulating CFTR function

Question 12

Alpha1-anitrypsin (a1-AT) deficiency

Answer 12

Lung disease (emphysema) and liver disease (cirrhosis)

a1-AT is a serine protease inhibitor (serpin) that inhibits neutrophil elastine (serine protease that is from activated neutrophils at inflammation sites, digests connective tissue by cleaving proteins)
If activity of neutrophil elastine is unchecked by a1-AT, then connective tissue damage
Mechanism: target protease binds reactive center loop (RCL) of a1-AT and cleaves it, RCL inserts into beta sheets and drags protease into middle of a1-AT and unfold (mousetrap)

Mutation in a1-AT (S type) may cause aggregation in liver cells because beta strand is weakened, gets into center even without serine protease cleaving