Lecture 12: Protein Misfolding and Disease I

Question 1

What parts of proteins does Hsp70 bind to? In what conformation are the proteins in at this time?

Answer 1

hydrophobic patches

intermediate conformation - not native

Question 2

What are the domains of Hsp60?

Answer 2

GroEL/GroES

Question 3

When do chaperones come into play? About what percent of proteins need this?

Answer 3

• cell is overwhelmed with misfolded proteins

- small percent of proteins require chaps

Question 4

By what three mechanisms does GroEL/GroES seem to work?

Answer 4

1. isolation (forms Anfinsen box)
2. forced unfolding
3. confinement (in box)

Question 5

What is the function of GroEL/GroES?

Answer 5

help proteins fold/unfold

Question 6

What is the function of E1?

Answer 6

activating enzyme
activates Ub
ATP driven
forms E1-Ub bond

Question 7

What are the roles of E2 and E3?

Answer 7

E2 = conjugating enzyme
E3 = ligating enzyme
target protein is bound to E3
E2 transfers activated Ub to target protein +E3
E3 catalyzes final transfer step of Ub to protein

Question 8

Where are Ub-tagged proteins degraded?

Answer 8

Proteasome

Question 9

How many concatenated Ub molecules are needed for protein-proteasome binding?

Answer 9

4

Question 10

What are the subunits of the proteasome?

Answer 10

20S core particle

19S regulatory particle caps

Question 11

What immune response can proteins degraded by the proteasome activate? Where do the peptides pass through to do so?

Answer 11

MHC Class 1

ER

Question 12

What are 4 diseases associaited with the Ub-proteasome pathway?

Answer 12

Cancer
Neurodegenerative diseases
Cystic Fibrosis
Autoimmune disease

Question 13

How is the Ub-proteasome pathway associated with cancer?

Answer 13

faster growth makes cells more dependent on proteasome

ca have increased degradation of tumor supressors

Question 14

How is the Ub-proteasome pathway associated with neurodegenerative diseases (Alzheimer’s, Parkinson, Huntington)?

Answer 14

accumulation of Ub-proteins has been seen in plaques

have found some disease-causing mutations in pathway

Question 15

How is the Ub-proteasome pathway associated with Cystic fibrosis?

Answer 15

Ub-proteasome is supposed to clear delta-F508 CFTR

Question 16

How is the Ub-proteasome pathway associated with autoimmune diseases?

Answer 16

improper processing of peptide antigens

Question 17

What is the role of p53? How is it activated? What does it trigger? What does it prevent?

Answer 17

Txn factor
Activated by DNA damage
Triggers cell cycle arrest or apoptosis
Prevents accumulation of xs mutations

Question 18

What are three aspects which can be altered by p53 mutation?

Answer 18

1. DNA contact
2. Stability
3. Zinc binding

Question 19

What motifs and secondary structures are important in p53?

Answer 19

beta sheets –> beta clam
alpha helices and loops = DNA binding site
Zinc ion

Question 20

What is the most common outcome of missense mutations?

Answer 20

loss of thermodynamic stability

Question 21

How does loss of stability often affect the Ub-proteasome pathway?

Answer 21

leads to increased degredation

Question 22

How does loss of stability of p53 affect the Ub-proteasome pathway?

Answer 22

exhibits neg feedback with pathway

mutant p53 accumulates

Question 23

What is the molecular logic behind using crevice binders to stabilize stability mutants?

Answer 23

design small molecule
fits into solvent-accessible area
binds that protein only
take into account size/shape and chem complementarity

Question 24

What is the molecular logic behind using MDM2 blockers when p53 is mutated?

Answer 24

MDM2 blockers prevent p53 from binding E3
–> p53 accumulates
even if mutate, some might be functional
can reactivate p53 by other means

Question 25

What protein is missing from target tissues in Cystic fibrosis?

Answer 25

Delta-F508 CFTR

Question 26

What mutation characterizes Cystic fibrosis?

Answer 26

ABSENCE of F508 (not change in structure)

Question 27

Where is F508 located on the CFTR?

Answer 27

nucleotide binding domain 1 (NBD1)

Question 28

Of what family is CFTR? What does it do?

Answer 28

ABC membrane protein (ATP binding casette) | pumps solutes in and out

Question 29

What are treatment options for delta-F508 CFTR? Are any of them great?

Answer 29

Not great | 25 degrees C, small organic molecules, overexpressing chaps, inhibiting Ub-proteasome, stimulating CFTR funcion

Question 30

What are the Z-type and S-type mutations in alpha1-antitrypsin deficiency?

Answer 30

``` Z-type = Glu342 --> Lys S-type = Glu342 --> Val ```

Question 31

What is the role of Alpha1-AT?

Answer 31

Serine protease inhibitor (serpin) | in plasma - wound healing

Question 32

Where is Alpha1-AT released?

Answer 32

sites of inflammation

Question 33

What diseases are characterized by Alpha1-AT deficiency?

Answer 33

lung diseases - emphysema | liver disease - cirrhosis, cancer

Question 34

What secondary structure forms the active, inhibitory reactive center of Alpha1-AT? Is this cleaved or uncleaved?

Answer 34

Beta-sheet (RCL inserted into Beta-sheet) | cleaved

Question 35

What secondary structure forms the inactive reactive center of Alpha1-AT? Is this cleaved or uncleaved?

Answer 35

alpha-helix far away from Beta-sheet | uncleaved

Question 36

What structure of Alpha1-AT acts as a molecular mousetrap?

Answer 36

Reactive center loop (RCL)

Question 37

Is the cleaved or uncleaved form of Alpha1-AT more stable why?

Answer 37

cleaved | RCL inserted into beta sheet

Question 38

When antithrombin is in the locked form, how does its function change?

Answer 38

Goes from protease inhibitor --> angiogenesis inhibitor

Question 39

What family is antithrombin a member of?

Answer 39

Serpin (serine protease inhibitor)

Question 40

What can go wrong with the serpin activation cycle of alpha1-AT involving structural weakness?

Answer 40

Beta sheet needs to be flexible enough to accept RCL, but at the right times to have inhibitory function If beta sheet is structurally weakened, it might accept RCL too often

Question 41

What can go wrong with the serpin activation cycle of alpha1-AT at the shutter region?

Answer 41

- Glu264-Tyr38 Hydrogen bond forms right under beta sheet | - removing H bond can weaken beta sheet

Question 42

In alpha1-AT, is insertion of RCL into beta sheet reversible?

Answer 42

No - permanent insertion, permanent inhibitory function

Question 43

What happens to alpha1-AT in liver disease?

Answer 43

beta sheet opens at abnormal time RCL from second protein enters irreversibly forms beads on strings which accumulate and cause failure