Oxygen Sensing Part II

Question 1

Common functions of proteases

Answer 1

Protein digestion: typsin, chymotrypsin, pepsin

Clotting cascade: thrombin

Blood pressure control: angiotensin converting enzyme

Regulation of cell death: caspases

Viral life cycle: HIV protease, Hepatitis C protease

Protein quality control and turnover: proteasome

Lysosomal pathway: cathepsin

Question 2

Primary chemical functions of proteases

Answer 2

1. Activate water to perform a nucleophilic attack on a peptide bond
2. Twist and thereby destabilize the peptide bond
3. Directly attack the peptide bond in order to form a less stable intermediate for water to attack

In summary, to fix the problem that water is a bad nucleophile and amide carbonyls are bad electrophiles

Question 3

Serine proteases

Answer 3

Trypsin

Chymotrypsin

Thrombin

Question 4

Aspartyl proteases

Answer 4

HIV protease

pepsin

presenilin

Question 5

Cysteine protease

Answer 5

Caspases

Cathepsins

Deubiquitinases

Question 6

Metallo proteases

Answer 6

Angiotensin converting enzyme

Question 7

Threonine protease

Answer 7

Proteasome

Question 8

HIV protease contortion of peptide bond

Answer 8

Question 9

Autophagy of cytoplasm outline

Answer 9

Question 10

Critical Parameters of Proteolytic Control

Answer 10

Access - How does the protease access an unfolded peptide sequence?

Specificity - Where does the protease cleave?

Regulation - What regulates the activity of this protease?

Question 11

A highly dynamic protein is. . .

Answer 11

synthesized and degraded at a high rate

Question 12

One example of a highly dynamic protein would be. . .

Answer 12

a regulatory transcription factor

Question 13

One example of a non-dynamic protein would be. . .

Answer 13

cytoskeletal elements

Question 14

Phagophore

Answer 14

Membrane which expands to pinch off cyotplasm for autophagy

Question 15

Mitophagy

Answer 15

Autophagy of mitochondria

Question 16

Modes of autophagy

Answer 16

Question 17

Lysosomal storage disorders

Answer 17

Disorders resulting in a deficiency of a lysosomal enzyme, leading to a buildup of the associated substrate in lysosomes.

Question 18

Non-degradation roles of ubiquitination

Answer 18

Epigenetic marker for readers

Protein trafficking

Question 19

Ubiquitination occurs at. . .

Answer 19

The epsilon amino group of lysine sidechains

which reacts with the C-terminal glycine of ubiquitin

Question 20

K48 Ubiquitination

Answer 20

Question 21

K63 Ubiquitination

Answer 21

Question 22

K11 Ubiquitination

Answer 22

Question 23

Linear Ubiquitination

Answer 23

Question 24

Where does the energy for ubiquitination come from?

Answer 24

E1 enzymes utilize energy from ATP hydrolysis to form a thioester bond with ubiquitin, transfering the ATP’s hydrolysis energy to this thioester.

This thioester transfered to E2 and its energy is preserved, and finally it is utilized in an irreversible attachment of the ubiquitin to its target protein via the formation of an amide bond.

Some specialized E3 ligases also form a thioester with ubiquitin and then transfer this ubiquitin to the target protein.

Question 25

Types of E3 ligase

Answer 25

Question 26

Structure of an E3 Ligase

Answer 26

Where RING = Really Interesting New Gene

Receptors recognize targets and scaffolds position the E2 to transfer its ubiquitin. Once the ubiquitination has occurred, the E2 must dissociate in order to be reloaded by an E1 to continue polyubiquitination.

Ubiquitination continues until the target diffuses or the ubiquitin chain becomes too long to fit into the active site.

Question 27

How is ubiquitination regulated?

Answer 27

By expression of E3 ligases and by having E3 receptors that only recognize phosphorylated proteins that have been “targeted” for ubiquitination and degradation.

Question 28

Proteasome structure

Answer 28

Alpha chains on the core particle make up the proteasome gate

Beta chains on the core particle make up the catalytic threonine protease core

The 19S regulatory particle contains docking sites for ubiquitin that position the protein to be threaded through the regulatory particle’s cannal with some aid from ATP hydrolysis for unfolding

Question 29

“Coding genes”, by definition, encode

Answer 29

mRNA/protein

NOT tRNA, rRNA, lncRNA, miRNA, etc etc etc

Only mRNA counts because only it is translated

Question 30

The function of the mediator can best be described as a multi-protein complex that. . .

Answer 30

Helps recruit and stimulate the activity of RNA polymerase II

Question 31

Levels of HIF1b vs HIF1a

Answer 31

HIF1b is constitutively expressed,

HIF1a varies in concentration in response to oxygen levels.

Question 32

VHL

Answer 32

Receptor for the E3 complex that ubiquitinates HIF1a.

Requires HIF1a to be hydroxylated by prolyl hydroxylase in order to recognize. Thus, when oxygen levels are low and prolyl hydroxylase cannot bind oxygen to catalyze the reaction, HIF1a is not degraded, enabling its transcriptional activity.

Question 33

K_d rate definition

Answer 33

k_off/k_on

Question 34

What protein functions as the human body’s oxygen sensor and initiates the respose to hypoxia?

Answer 34

Prolyl hydroxylase