AC Lecture 32: Protein quality control

Question 1

What kinds of damage leads to misfolding and aggregation of proteins?

Answer 1

physical forces (e.g. heat)
chemical forces (e.g. oxidation

Question 2

What is proteostasis?

Answer 2

cellular quality control processes that continually monitor the proteome for misfolded or aggregated proteins and target them for degradation and disposal

Question 3

What are the stages for polypeptide folding?

Answer 3

burst phase (0-5ms)
intermediate phase (5-100ms)
rate-limiting

Question 4

Why did molecular chaperone proteins evolve?

Answer 4

to promote folding and prevent protein aggregation in physiological conditions (which are make it difficult to refold denatured proteins due to molecular crowding and relatively high temperatures)

Question 5

What happens in the burst phase of polypeptide chain folding?

Answer 5

formation of secondary structure and collapse of the hydrophobic core

Question 6

What happens in the intermediate phase of polypeptide chain folding?

Answer 6

formation of molten globule intermediate which has characteristics of both folded and unfolded proteins

Question 7

What happens in the rate-limiting phase of polypeptide chain folding?

Answer 7

attainment of native structure
marked by conversion of molten globule via global repacking of hydrophobic side chains and the association of domains that were folded independently in the intermediate stages

Question 8

List some families of molecular chaperones.

Answer 8

Heat Shock Proteins:

Hsp70, Hsp60 (chaperonin), Hsp90 families

Question 9

Describe the Hsp chaperones.

Answer 9

chaperone proteins that bind and release polypeptides in a manner that is dependent on ATP binding, hydrolysis, and nucleotide exchange

Question 10

Describe the process of protein folding in a chaperonin family molecular chaperone.

Answer 10

1. unfolded protein binds the rim of a chaperonin (which looks like a barrel with 2 stacks of rings, each 7 subunits)
2. Lid structure displaces protein into the cavity of the barrel (which is lined with hydrophilic amino acids to drive folding)
3. polypeptide can then fold in a sequestered and protected environment
4. lid dissociates due to changes in conformation of the large subunit as ATP is hydrolyzed

Question 11

How is gene transcription for molecular chaperones controled?

Answer 11

Heat Shock Transcription factor (Hsf)- which responds to the presence of unfolded proteins or heat shock or other types of proteotoxic stress

Question 12

Describe the structure of the proteasome (26S).

Answer 12

central catalytic core (20S)
regulatory cap (19S)- recognizes ubiquitinylated substrates and deubiquitinylated substrates as well as prepares them for proteolysis (via protein unfolding if necessary)

Question 13

How are proteins degraded in cells?

Answer 13

in both the cytosol and nucleus it is largely done by proteasomes (large gated proteases)
target substrates via covalent linkages to multiple copies of ubiquitin

Question 14

List the activities of the eukaryotic proteasome.

Answer 14

chymotrypsin-like (cleaves after hydrophobic amino acids)
trypsin-like (cleaves after basic amino acids)
peptidyl-glutamyl peptide hydrolyzing activity (cleaves after acidic amino acids)

Question 15

List the additional activities of mammalian proteasomes.

Answer 15

cleavage after branched chain amino acids

cleavage between small, neutral amino acids

Question 16

What enzymes are necessary for ubiquitinylation?

Answer 16

E1 (ubiquitin-activating enzyme; small amount)
E2 (ubiquitin-conjugating enzyme)
E3 (ubiquitin ligase; specifies substrate selection)

Question 17

Describe the process of ubiquitinlyation.

Answer 17

1. ubiquitin-adenylate is formed
2. transfer of activated ubiquitin to thiol site in E1
3. E2 accepts ubiquitin from E1
4. E2 transfers ubiquitin to the protein substrate with the help of E3

Question 18

How is ubiquitination linked to molecular chaperones?

Answer 18

CHIP (C-terminal Hsp interacting protein)

binds to Hsp70 and catalyzes ubiquitinylation of misfolded proteins

Question 19

What causes protein aggregation?

Answer 19

when misfolded proteins overwhelm the ubiquitin/proteasome pathway

Question 20

What are the types of aggregates?

Answer 20

amorphous assemblies of misfolded proteins (bound together by hydrophobic interactions)
ordered assemblies of amyloid fibers (formed by stacked beta-sheet structures)

Question 21

How are aggreagates cleared?

Answer 21

largely inaccessible to proteosome

cleared by the autophagic system

Question 22

Which human disorders are characterized by the presence of organized protein aggregates?

Answer 22

amyloid diseases (e.g. Alzheimer's disease)
Lewy Bodies (e.g. Parkinson disease)
polyglutamine repeat diseases (e.g. Huntington, SMA, DRPLA, SCA)
prion diseases (e.g. Creutzfeld-Jakob, fatal familial insomnia)

Question 23

What are the characteristics of Alzheimer’s disease?

Answer 23

extracellular amyloids of APP
intracellular deposits (or neurofibrillary tangles) of phosphorylated Tau (microtubule binding protein)

Question 24

What causes PD?

Answer 24

loss of dopaminergic neurons in the substantia nigra

aggregates of Leqy Bodies enriched in alpha-synuclein