Protein Degradation

Question 1

Protein turnover

Answer 1

-Proteins turn over continually
-Adult humans turn over ~2% of their protein per day
-Cells continuously synthesize proteins from and degrade proteins to amino acids
—Recycling
-A protein’s level is determined by synthesis and degradation rate
—To study one rate, the other process must be inhibited
-Half-lives range from 10 min for Cyclin D1 to 4 months for hemoglobin

Question 2

Why are cells wasting so much energy turning proteins over?

Answer 2

Continual turnover serves to:
-Respond to environmental changes
—Ramp up/down metabolic processes
-Control cell cycle
-Eliminate damaged proteins, arising from chemical modifications in cell’s reactive environment

Question 3

What are lysosomes?

Answer 3

-Membrane bound organelles involved in protein degradation
-Maintain an internal pH of 5
-Contain ~50 hydrolytic enzymes, including proteins called cathepsins
-Lysosomal degradation is largely non-selective

Question 4

What is a characteristic of lysosomal enzymes?

Answer 4

-They have acidic pH optima
-They are non-functional at cytosolic pH
-Occurs because if they get out of lysosome, you don’t want them to harm anything

Question 5

How is intercellular material degraded in the lysosome?

Answer 5

-Material is degraded in lysosome by fusing with other vacuoles
-Intercellular “garbage” can be packaged into vacuoles through a process called “autophagy”

Question 6

How is extracellular material degraded in the lysosome?

Answer 6

Material is degraded in lysosome when taken up by endocytosis

Question 7

What is chloroquine

Answer 7

-Antimalarial drug that inhibits the lysosome
-A weak base that freely penetrates the lysosome where it becomes pronated, this charged form accumulates and raises lysosomal pH
-Inhibits the lysosome

Question 8

What maintains low lysosomal pH

Answer 8

-Vacuolar ATPAses

Question 9

Ubiquitin-based degradation basic characteristics

Answer 9

-Selective
-ATP-dependent
-Occurs in nucleus and cytosol
-Ubiquitin is ubiquitous and abundant 76 amino acid protein
-Highly conserved protein

Question 10

How are proteins selected for degradation in ubiquitin-based degradation?

Answer 10

-Proteins are marked by covalently linking ubiquitin to them

Question 11

How is ubiquitin expressed? What are DUBs?

Answer 11

-Expressed as a polyprotein containing several ubiquitin units (no gene that encodes a single ubiquitin)
-These polyproteins are processed by proteases, called deubiquitinases (DUBs) to yield single ubiquitin
-DUBs also remove ubiquitin from targets in the cell

Question 12

How does ubiquitin attach and where?

Answer 12

-Attaches to substrates/targets by its carboxyl-terminal Gly residue to the ε-amino group of Lys residues on target proteins
-Bond to protein is similar to an amino bond

Question 13

Steps to attachment/ligation of ubiquitin to target proteins

Answer 13

-C-terminus of ubiquitin is activated with ATP by ubiquitin activating enzyme (E1)
—aka AMPylation
-Terminal carboxyl group of ubiquitin is conjugated via a thioester bond to a Cys on E1
—Yeast and humans only have one E1
-Ubiquitin is then transferred to Cys on a ubiquitin conjugating enzyme (E2), forming a second thioester bond
—11 in yeast; 20+ in mammals
-A ubiquitin-protein ligase (E3) transfers the activated Ub from the E2 to a lysine group on the target protein, forming an isopeptide bond
—~644 E3 ligases (more than the number of protein kinases)
-SEE DIAGRAM (L21 pg 17)

Question 14

Why are there more E2’s than E1’s?

Answer 14

To enable substrate specificity and diverse regulation patterns

Question 15

What is necessary for a target protein to be degraded? (ubiquitin-based degradation)

Answer 15

-For a target protein to be degraded, it must be linked to a chain of at least 4 tandemly linked ubiquitins
-Ubiquitin chains are linked via internal Lys residues
-Lys 48 of Ub forms an isopeptide bond with the C-terminal carboxyl of the next ubiquitin
-The ε-amino group of a Lys residue of one ubiquitin is linked to the terminal carboxylate of another

Question 16

Polyubiquitin chains (what are they, what generates them, linkage)

Answer 16

-Can be 50+ ubiquitins long
-Generated by E3s, how E3s switch from transferring a Ub to a target protein to synthesize a polyUb chain is unknown
-Lys/amino group linkage determines chain geometry
-Different types of linkages have different signaling outcomes beyond degradation

Question 17

Degrons (what are they, where are they)

Answer 17

-Specific sequences of amino acids that indicates protein should be degraded
-For many proteins, the amino-terminal amino acid residue (N-degron) is an important degradation signal E3
-Residue may only be exposed afterproteolytic cleavage, may be added after protein synthesis, and may be impacted by modification (like N-terminal acetylation)
-Also called the “N-end rule”
-Other degrons include cyclin destruction boxes

Question 18

N-end rule (what is it, what does it result from)

Answer 18

-Half-lives of many proteins vary with the identity of N-term AAs (the N-end rule)
-Rule results from actions of a RING E3 called E3a, which recognizes the destabilizing residues for ubiquitination

Question 19

What do K48 Polyubiquitin chains do?

Answer 19

-Target a protein to the 26S proteasome for degradation

Question 20

26S proteasome structure

Answer 20

-~2100 kDa
-Complex of three components:
-One 20S catalytic unit arranged as a barrel
-Two 19S regulatory units that control access to the interior of the 20S catalytic subunit

Question 21

26S proteasome basic characteristics

Answer 21

-Exists in the nucleus and cytoplasm
-Rivals the ribosome in size and complexity
-Exists in all eukaryotes and some archaea

Question 22

19S subunits of 26S proteasome

Answer 22

-Regulatory units, control access to interior of catalytic subunit (20S)
-Contains ubiquitin receptors that bind specifically to polyubiquitin chains
-Use ATP to unfold polyubiquitinated chains and direct them into the 20S core
-Contain an isopeptidase (a DUB) that cleaves off intact ubiquitin molecules (so it can be reused)

Question 23

20S subunits of 26S proteasome

Answer 23

-Catalytic unit
-Has three types of active stirs in the β subunits, each with a different specificity
-Substrates are degraded in a processive manner without intermediate release
-Substrates are reduced to peptides ranging from 7 to 9 amino acids before release

Question 24

What degrades peptides to amino acids?

Answer 24

Cytosolic peptidases

Question 25

Bortezomib (Velcade) (basics/therapeutic use)

Answer 25

-Inhibits the proteasome
-Used as a therapy for multiple myeloma
-Mechanism of why in anticancer is unclear, proteasome inhibition may prevent degradation of pro-apoptotic factors

Question 26

How does Bortzomib work

Answer 26

-Substrate analog (resemble the “normal” substrate, but act as competitive inhibitors)
-Resembles a tripeptide
-Active boron atom binds the catalytic site of the 26S proteasome

Question 27

Bacterial proteases

Answer 27

-Lack 20S proteasome or homologs
-Have ATP-dependent proteolytic assemblies that share barrel architecture
-Compartmentalized proteases
-Two E. coli proteins, Lon and Clp, mediate 80% od E. coli protein degradation in inner cavity of barrel

Question 28

Monoubiquitination

Answer 28

-Proteasome-indepdent
-Proteins can be monoubiquitinated on more than one Lys
-Ubiquitin itself has 7 Lys residues than can be ubiquitinated
-Ubiquinated proteins are recognized by ubiquitin-binding domains (UBD)

Question 29

polyUb that are proteasome independent

Answer 29

-Can result in DNA repair, protein kinases, and other results
-K48 and K63

Question 30

Monoubiquitination example

Answer 30

Mono-ubiquitination of PCNA at Lys164 recruits an error-prone translesion DNA polymerase, whereas polyubiquitination at same Lys164 with Lys63-linked chains recruits error-free DNA polymerase

Question 31

Ubiquitin-like proteins

Answer 31

-Ubiquitin-like modifiers also have E1, E2, E3 enzymes
—These are diffeent proteins that have diverged from ubiquitin
-SUMO (small ubiquitin-related modifier, 18% identical to Ub)
—Sumoylation of pCNA at Lys164 promotes normal DNA replication
-NEDD8 (50% identical to Ub)