Sept 11 Protein Folding, Misfolding and Degradation

Question 1

what is protein folding?

Answer 1

in vitro conversion between native and denatured conformations

Question 2

what is the native conformation?

Answer 2

the “right” conformation

Question 3

what is misfolding of a protein?

Answer 3

it goes into the wrong conformation
for example, an exposed hydrophobic patch
this makes the protein insoluble

Question 4

what is being done through a “folding pathway”

Answer 4

the spontaneous refolding of a denatured protein
same thing happens on new proteins being made on ribosomes
N-terminal region starts to fold before the C-terminal region is synthesised

Question 5

what are chaperones, what do they do and how do they do it? (7 points)

Answer 5

chaperones facilitate the folding of proteins
they help guide the folding protein along productive pathways, by permitting partially misfolded proteins to return to the proper folding pathway
they work by recognising exposed hydrophobic patches
can also disassemble potentially toxic protein aggregates that form due to protein misfolding
can assemble and dismantle large multiprotein complexes
mediate transformations between inactive an active forms of some proteins

Question 6

where are chaperones upregulated?

Answer 6

where misfolded proteins accumulate

Question 7

how do chaperones work? (what mechanism specifically)

Answer 7

work through ATP dependent cycles of binding to and releasing from misfolded “client” molecules at exposed hydrophobic patches
by blocking the exposed hydrophobic patch, the chaperone keeps the folding/refolding protein out of trouble while productive folding occurs
(ATP binding and hydrolysis)

Question 8

what are the two major classes of chaperones?

Answer 8

molecular chaperones
operate as single molecules
chaperonins
form a multisubunit “refolding” chamber

Question 9

how do chaperonins work?

Answer 9

form an enclosed chamber made up of inward facing protein-binding subunits that undergo ATP binding/hydrolysis and conformation change
give the protein time and environment to refold properly

Question 10

what is the significance of chaperones/chaperonins?

Answer 10

some proteins can fold and refold properly without help, but majority of cellular proteins require help to adopt proper 3D structures or correct mistakes
they are essential for life
very ancient (in both pro and eukaryotes)
highly conserved in AA sequence throughout evolution
if they were not there, cells would have a crippling burden of nonfunctional misfolded proteins

Question 11

what happens to proteins that are irretrievably misfolded?

Answer 11

they are destroyed by proteolytic cleavage into small fragments

Question 12

what are the steps in the ubiquitin system for protein degradation?

Answer 12

step 1:
poly-ubiquitin “tags” damaged or misfolded proteins for degradation
step 2:
ubiquitin tagged proteins are fed into a multisubunit chamber where the subunits form inward facing proteases

Question 13

what is ubiquitin?

Answer 13

a 76 residue long protein that can be covalently linked to lysine residues on target proteins

Question 14

what do E3 ubiquitin ligases do?

Answer 14

recognise misfolded or damaged proteins
thought to recognise hydrophobic patches or oxidised amino acids
they can also recognise and target for degradation particular “normal” proteins that the cells needs to degrade for regulation purposes
for examples: cyclins during the cell cycle, need to be degraded

Question 15

what happens to proteins that cannot be properly refolded with chaperones?

Answer 15

degraded by the multiubiquitination/proteasome system
BUT that system is imperfect and leads to accumulation of aggregates of insoluble proteins

Question 16

in what form do those misfolded proteins accumulate?

Answer 16

accumulate in the form of amyloids
important aspect of several neurodegenerative diseases
show as plaques and tangles when seen with a microscope

Question 17

what can cause denaturation?

Answer 17

extremes of heat or pH
or being exposed to denaturants, such as urea or guanidine hydrochloride at high concentrations

Question 18

what are the three ways to regulate protein activity?

Answer 18

1. cells can increase or decrease the level of protein by altering the rate of synthesis, rate of degradation or both
2. cells can change the intrinsic activity of the protein (through covalent and non covalent interactions)
3. change in the location of the concentration of proteins within the cell itself

Question 19

what are the especially important roles that protein degradation plays in a cell?

Answer 19

removes proteins that are potentially toxic/harmful
provides proteostasis= maintaining proteins and activity at appropriate levels and making rapid adjustments to those levels if conditions change

Question 20

what are proteasomes?

Answer 20

very large, multi-subunit, protein-degrading molecular machines that influence many different cellular functions

Question 21

what are the different cellular functions affected by proteasomes? (5)

Answer 21

the cell cycle
transcription and DNA repair
programmed cell death (apoptosis)
recognition of and response to infection by foreign organisms
removal of misfolded proteins

Question 22

what are the three key steps in the degradation of a protein by proteasomes?

Answer 22

1. the protein is tagged to target it for proteasomal degradation
   (cells can control the tagging and therefore the rate)
2. proteasomes bind to the protein via the tag and unfold the protein as it is transferred to an internal chamber
3. protein cutting subunits of the proteasome within the chamber degrade the target protein into small peptides (fragments) which are released into the cytosol (cytoplasm) for further processing

Question 23

what happens to the short peptides after the exit the proteasome?

Answer 23

further degraded rapidly by peptidase enzymes in the cytoplasm, and are eventually converted to “free” amino acids

Question 24

how do cells “tag” proteins that should be degradated?

Answer 24

covalently attach to the protein a linear chain of multiple copies of a 76-residue polypeptide called ubiquitin
(ubiquitinylation)
after the protein enters the proteasome, the ubiquitin unbinds and can be recycled for further cycles

Question 25

how can allosteric interactions affect the activity of a protein?

Answer 25

allostery is the change in a protein’s tertiary or quaternary structure or both, induced by the noncovalent binding of a ligand
when a ligand binds to a side A (allosteric binding site), it affects the activity of site B (allosteric effector)
allosteric can be positive or negative (increase or decrease activity)
negative allostery is involved in end product inhibition (negative feedback) –> restricting excessive buildup of the product

Question 26

explain the structure and mechanism of the Hsp70 chaperone?

Answer 26

structure: the chaperon has three parts. the nucleotide binding domain, with ATP present
the substrate binding site and an unnamed third part
Hsp70 is an ATPase –> hydrolyses ATP

1. if ATP is present, the substrate binding site is open
2. the unfolded protein binds on the substrate binding site
3. the chaperone will hydrolyse and release a Pi, ADP left
4. the release of Pi causes a conformational change which closes the substrate binding site, therefore the aggregation/misfolding is blocked
5. the protein is released or it can bind again for another round

Question 27

explain the structure and the mechanism of group 1 chaperonins (found in bacteria)

Answer 27

form an enclosed chamber made up of 7 inward facing subunits that undergo ATP binding/hydrolysis and conformation change
huge cylindrical supramolecular assemblies
–> hollow chambers in which the protein to be fixed enters
there is also a lid

mechanism:
1. the partially folded/misfolded protein enters the chamber, with the release of Pi from ATP opening the lid
2. inside, the environment is different than the environment of the cytoplasm
–> hydrophobic patches are isolated on the inside
3. after the conformational changes, the properly folded protein is released or an improperly folded is recycled back into the mechanism

Question 28

what is the difference between group 1 and group 2 chaperonins?

Answer 28

group 1 are found in bacteria and mitochondria
group 2 are found in eukaryotes, and there are also structural differences
made of 8 protein subunits, and there is also no lid, but a conformational change/orientation that opens and closes the protein

Question 29

explain the process of the ubiquitin/proteasome system for protein degradation (until the protein enters the proteasome)

Answer 29

1. E1 (ubiquitin activating enzyme) activates and links ubiquitin to itself
2. ubiquitin is transferred from E1 to E2 (ubiquitin conjugating enzyme)
3. ubiquitin binds to the side chain of the target protein (not the backbone) and forms an isopeptide bond with the protein
4. E3 (ubiquitin ligase) recognises proteins that need to be destroyed (exposed hydrophobic patches) and assists E2 in transferring ubiquitin from E2 to the protein
5. ubiquitin can attach to itself on the protein, making a chain of ubiquitin proteins attached on the protein to be destroyed
6. ubiquitin tagged proteins are fed into a multisubunit chamber in which the subunits form the inward facing proteases (proteasome)

Question 30

what is the structure of a proteasome and how does it work?

Answer 30

proteins in the cap of the proteasome recognise and bind to polyubiquitin (the chain of Ub on the protein)
the ubiquitins are removed by hydrolysis and the target protein is unfolded (using energy from ATP)
the target protein is then fed into the central chamber in the core
inside the chamber, the hydrophobic patches are isolated and there are inward facing proteases that degrade proteins to short amino acids or peptides
this isolates the active proteases from the cytoplasm