Sept 5 - Cellular Quality Controll

Question 1

What fraction of newly synthesized proteins are rapidly degraded?

Answer 1

1/3

Question 2

2 major sites of protein degradation?

Answer 2

1. Late endosomes/lysosomes - degradation of proteins, organelles (autophagy), membrane proteins, cytosolic components (microautophagy)
2. Cytosol

Question 3

Autophagy

Answer 3

Degradation of a cell’s own components

Question 4

What are some things contained by lysosomes?

Answer 4

1. Acid hydrolases - require pH 4.5-5.0, which are largely inactive at cytosolic/ER pH and are inactivated by weak bases like NH4Cl or chloroquine.
2. Proteases, glycosidases, reductases, esterases

Question 5

Chloroquine

Answer 5

Weak base that can inactivate lysosome

Question 6

Leupeptin

Answer 6

Chemical inhibitor that can inactivate lysosomes (inactivates sulfahydryl and serine proteases)

Question 7

Pepstatin A

Answer 7

Inactivates acid proteases in lysosomes

Question 8

Bfilomycin, concanamycin

Answer 8

Blocks acidification, V-type ATPase in lysosomes

Question 9

How do you know if your protein is degraded in the lysosome?

Answer 9

1. Degradation is inhibited by weak bases or inhibitors of the vacuolar proton pump
2. Degradation is inhibited in cells with mutations in lyosomal hydrolases
3. Degradation is inhibited by lysosomal protease inhibitors.

Question 10

Lactacystin

Answer 10

Proteasomal inhibitor

Question 11

Concanamycin

Answer 11

Lysosomal inhibitor

Question 12

MG132

Answer 12

Proteasomal inhibitor

Question 13

What was the experiment called where they fractionated the rabbit reticulocyte lysate + that was breaking down the radioctive protein?

Answer 13

Anion exchange chromatography. Put it through a column.
Fraction I - Ubiquitin
Fraction IIA - the proteasome
Fraction IIB - E1-3

Question 14

Ubiquitin

Answer 14

76 aa polypeptide, heat stable, highly conserved from prokaryotes to eukaryotes.

Question 15

proteasome

Answer 15

heat labile, stabilized by ATP, large (450 kda by gel filtration chromatography)

Question 16

How did they further distinguish the components of Fraction IIB?

Answer 16

Using affinity chromatography on Ub-sepharose. Basically this involved seeing what binds Ub and then eluting and fractionating that.

Question 17

E1

Answer 17

There is only 1 of these in the entire cell. Ubiquitin activating enzyme - uses energy of ATP hydrolysis to form a covalent thioester bond with Ub - activates Ub for transfer to E2
Cytosolic protein.
Single function - activate ubiquitin.
Interacts with E2 (ubiquitin conjugating enzymes)

Question 18

E2

Answer 18

Ubiquitin conjugating enzyme (Ubc). Forms covalent thioester bond with Ub that is accepted from E1, often does the actual transfer of Ub to substrate or to E3 (ATP independent reaction) - specificity for interaction with E3.
(Many of these - tens. Cytosolic, can be membrane bound. Involved in diverse metabolic functions. Conserved E2 domain involved in conjugation. Specific E2s have different N- or C-terminal extensions involved in conjugation.

Question 19

E3

Answer 19

Ubiquitin protein ligase. Facilitates transfer of Ub from E2 to substrate - each E3 has specificity for certain substrates, in most cases it links the E2 to the substrate. Catalytic and non-catalytic versions.

E3 confers substrate specificity.

Hundreds of these. Some participate in covalent linkage, others bridge interactions between target/substrate and E2. Most not conserved (structurally diverse). Subfamilies - Ring and Hect.

Question 20

RING finger proteins

Answer 20

An E3 subfamily - no catalytic activity - rely on E2. These comprise the vast majority of E3s

Question 21

HECT domain proteins

Answer 21

Catalytically active - there are 2 dozen of these.

Question 22

Where does ubiquitin attach on E2 and HECT?

Answer 22

On the catalytic cysteine

Question 23

K48 versus K63

Answer 23

Polyubiquitylation with K48 targets substrates for degradation by the proteasome.
K63 chains have other outcomes - such as forming signaling platforms

Question 24

Mono-ubiquitylation

Answer 24

These have other functions, they are not used for degradation.

Question 25

What’s the best control for ubiquitylation?

Answer 25

Use a mutant E3 that does not facilitate ubiquitylation.

Question 26

Other ways to test for ubiquitylation

Answer 26

Immunoprecipitate protein under right conditions (+MG132 blocks proteasome)
Immunoblot with antibodies to ubiquitin - can use antibodies specific for K63 and K48 chains.
In vitro ubiquitylation assays -add protein E1, E2, E3, ATP, Ub

Question 27

What is Itch?

Answer 27

Hect type E3 ubiquitin ligase

Question 28

What are some tags you can use?

Answer 28

Flag tag

HA tag

Question 29

How do E3 ubiquitin ligases recognize their substrates?

Answer 29

1. Nonspecific recognition - unfolded protein response - need to recognize a broad array of proteins - relies on chaperones.
2. Substrate specific recognition - post-translational modifications and domain specific recognition. Phosphorylation of a given site may be recognized by E3 ligase for example.

Question 30

How is E3 activity regulated?

Answer 30

1. Increased expression
2. Phosphorylation or de-phosphorylation of E3 or substrate
3. Association with adaptors

Question 31

UBPs

Answer 31

Ubiquitin specific processing protease - takes ubiquitin off things

Question 32

DUbs

Answer 32

De ubiquitinating enzyme - DUB - takes ubiquitin off things

Question 33

What might a protein with a Dub end that removes K63 chains, and with a ubiquitin end that adds K48 chains be doing?

Answer 33

With the dub end, might be removing signaling Ubs. With the ubiquitylating end it might be driving it toward the proteosome

Question 34

Ikappa beta

Answer 34

inhibitor of nfkb. it is constantly being ubiquitylated in cells where nfkb is on

Question 35

What size is the proteasome?

Answer 35

Core 20S complex

Associates in vivo with 19S to form 26S proteasomes

Question 36

20S proteasome

Answer 36

Core proteinase - 28 subunits arranged in a cylindar. Comprises 1% of total cell protein in a typical cell.
7 subunits per donut. - 7 different alpha 7 different beta subunits.
3 of the beta subunits are catalyitcally active.
2 inner donuts composed of catalytically active beta subunits.
2 outer donuts composed of alpha subunits which regulate assembly and activity of beta subunits and bind 19s and PA28 regulatory complexes.

By itself, the 20s proteasome can degrade denatured proteins in the absence of ubiquitin - the substrate must be denatured.

Question 37

19S complex

Answer 37

20 additional protein subunits. Forms a cap on one or both ends. ATP dependent association with 20s proteasome. Recognizes and binds ubiquitin - confers UBIQUITIN DEPENDENCE of degradation. Confers ATP DEPENDENCE of degradation through ATP dependent protein unfolding activity - necessary to thread peptide into central cavity of 20s proteasome.
Confers processivity to degradation by ATP motor activity.
Contains isopeptidase activity - releases ubiquitin from substrate.

Question 38

Chaperones

Answer 38

Mediate assembly of ATPase ring to the core particle

Question 39

Monoubiquitination

Answer 39

No targeting to proteasome

Question 40

SUMO-1/Smt3p

Answer 40

101 aa peptide with homology to ubiquitin - single conjugates attached to substrates. Substrates include proteins involved in nuclear import, transcriptional regulation, chromatin remodeling, DNA replication and repair, and viral proteins.

Question 41

FAT10

Answer 41

A small ubiquitin like modifier that can degrade proteins in a ubiquitin independent manner, encoded within the human MHC locus and upregulated by cytokine signaling

Question 42

Nedd8

Answer 42

Another ubiquitin like polypeptide with E1 and E2-like enzymes, couples to cell cycle dependent ubiquitin ligase in a manner dependent on tumor suppressor VHL

Question 43

ISG-15

Answer 43

has 2 ub-like domains

Question 44

Pup

Answer 44

prokaryotic ubiquitin-like protein, foundin TB