L5 - Protein degradation

Question 1

Protein degradation: what type of control is it, how speedy is it, and what forms can it take?

Answer 1

Amount of enzyme - “Coarse” control

Not very fast

Can induce synthesis/inhibit degradation
Can repress activation/activate degradation

Question 2

Protein turnover: is the constant among all proteins, what is the half-life, what does a long half-life mean, and what are some examples of short/long-living proteins?

Answer 2

Cellular proteins are degraded at different rates - varying from minutes to infinity

Half-life = time taken for 50% of protein to be degraded

long t₁/₂ = stable

Short-lived proteins:
* enzymes that catalyze committed steps
* regulatory proteins
* transcription factors

Long-lived proteins
* Constant catalytic proteins
* Structural proteins

Short - HMG-CoA reductase (11mins)
Long - histones, haemoglobin (RBC lifespan), crystallin

Question 3

How may protein degradation vary?

Answer 3

Tissue distribution - lactic acid dehydrogenase:
* Heart t₁/₂ - 1.6 days
* Muscle t₁/₂ - 31 days
* Liver t₁/₂ - 16 days

Regulated processes - Acetyl CoA carboxylase:
Fed t₁/₂ - 48 hours
Fasted t₁/₂ - 18 hours

Question 4

Pathways of protein degradation, what are protease examples, and what do they break down?

Answer 4

Degradation mediated by proteases:
* Intestinal - exogenous dietary proteins (GI tract, stomach, intestine)
* Lysosomal - long-lived cellular proteins, extracellular proteins, cell organelles
* Ubiquitin - most intracellular proteins
* Proteasome - Defective proteins

Question 5

Lysosomal protein degradation: what is it, how selective is it, what can influence its activity, and what gets broken down?

Answer 5

Membrane encapsulated organelle resulting in bulk degradation of extracellular/long-lived cellular proteins

Most non-selective protein degradation

Nutrients and growth factors

• Ingested materials in cells
• Obsolete cell components

Question 6

Lysosomal enzymes: how many are there, what pH do they work at, what happens if they enter the cytosol, and what maintains lysosome pH?

Answer 6

50 different degradative enzymes

Acid hydrolases - active at pH 5 (inside lysosome)

Inactive if released into cytosol (pH 7.2)

pH maintained by a proton pump in the lysosomal membrane - requires ATP (mitochondria)

Question 7

Acid hydrolases in lysosomes: what types are there?

Answer 7

• Nucleases
• Proteases
• Glycosidases
• Lipases
• Phosphatases
• Sulfatases
• Phospholipases

Question 8

Lysosomal protein degradation: what methods to it are there, what is the rate, how is it regulated, when does autophagy, and how selective is it?

Answer 8

• Endocytosis for extracellular proteins
• Autophagy for cellular proteins/organelles

Rate varies 1-10% total cell protein / hr ??

Regulated by delivery

Autophagy increases when cells are starved

In well-nourished cells, lysosomal protein degradation is non-selective (non-regulated)

LECCY

Question 9

UPS: what is it, what benefits are there for it using selective degradation,

Answer 9

Ub-proteasome system, tags molecules for degradation in proteasomes

• Regulation (short-lived proteins)
• To remove unwanted proteins
• To remove mutant or damaged proteins:
• Synthesis of proteins is not very accurate (defective proteins)
• Susceptible to damage by environmental factors – oxidation
• Genetic mutation results in synthesis of mutant protein

Question 10

Ubiquitin: what is it, what does it have on its C-terminal, how does it attach to molecules, and is anything used for attachment?

Answer 10

76 amino acid small peptide “TAG”

C-terminal Gly - isopeptide bond with e-amino group of Lys residues on the substrate

Attached as either monoubiquitin or polyubiquitin chains

Attaching Ub requires ATP

Question 11

Ubiquitination process

Answer 11

1 - Ubiquitin is activated by forming a link (thioester bond) to “enzyme 1” (E1)

2 - Ubiquitin is transferred to one of several types of “enzyme 2” (E2).

3 - “Enzyme 3” (E3) catalyses the transfer of ubiquitin from E2 to a Lys e-amino group of the target protein

Question 12

What is the prevalence of UPS enzymes in the genome?

Answer 12

The genes of the UPS constitute ~5% of the genome

E1 (ubiquitin-activating enzyme) - 1/2 activating enzymes

E2 (Ubiquitin-conjugating enzymes) - 10/20 conjugating enzymes

E3 (Ubiquitin-protein ligases) - 500-800 ubiquitin ligase for driving specificity

Question 13

Proteasome: what is it, what is its structure, what regulates entry into it, and what occurs within the proteasome?

Answer 13

Organelle that degrades proteins

• Hollow cylindrical supramolecule
• Four cyclic heptamer
• 2 central β-subunits, 1 α-subunit on each side of the β-subunits
• 28 polypeptides (4 heptamers, 7 structures per heptamer)

Caps on the ends of the organelle

Inside, different proteins degrade the molecules into short 8 amino acid chains. Ubiquitin is not degraded, though, it is released intact

Question 14

Hydrolysis peptide bonds after:

Answer 14

hydrophobic a.a. = CHYMOTRYPSIN-LIKE - 5

acidic a.a. = (-) CASPASE-LIKE -1

basic a.a. = (+) TRYPSIN-LIKE -2

LECCY

Question 15

Regulated protein degradation is an exergonic process, yet it is dependent on ATP, explain:

Answer 15

? it releases more energy than is used

• Specificity requires ATP
• ATP required for protein unfolding in proteasome

Question 16

Ubiquitination specificity: what determines whether a protein is ubiquitinated?

Answer 16

E3 ubiquitin ligases:
* Hundreds of different E3 ligases exist
* They Provide substrate specificity for ubiquitination
* Each ubiquitinates different proteins
* Can undergo activation themselves or interact with specific motifs in target proteins

Question 17

Recognition of proteins needing to be ubiquitinated: what process makes proteins get recognised?

Answer 17

• Phosphorylation using ATP
• Protein dissociation to unmask ubiquitinate sites
• Creation of destabilising N-terminus

Question 18

The N-end rule: what is it, what affects it, and what protein sis responsible for detecting those residues?

Answer 18

The rule stating that the half-life of cytosolic protein is determined by N-terminal amino acid residue

The identity of N-terminal amino acid residues

E3 ligase which recognises these proteins – E3α/Ubr1p

Question 19

Highly stabilising residues

Answer 19

• Alanine
• Cysteine
• Glycine
• Methionine
• Proline
• Valine
• Serine
• Threonine

Question 20

Intrinsically destabilising residues

Answer 20

• Arginine
• Histidine
• Isoleucine
• Leucine
• Lysine
• Tryptophan
• Tyrosine
• Phenylalanine

Question 21

PEST Sequences: what are they, what sites do they contain, and what do they do (?)?

Answer 21

Regions, 12 to 60 residues long, in proteins with a predominance of proline (P), glutamate (E), serine (S), and threonine (T)

Often contain phosphorylation sites necessary for degradation eg G1 cyclins, STAT1 transcription factor, IkBa

Initial regulatory event performed by protein kinase followed by recognition of the protein substrate by an E3 ubiquitin ligase, resulting in protein degradation

Question 22

Regulated ubiquitination of HMG-CoA reductase: what is it used for, why is it not always needed, how regulated is it, what protein ????, what enzyme is involved in the process, and what is the process behind it?

Answer 22

Cholesterol synthesis

Cholesterol can be obtained from the diet or synthesised - accumulation of sterols induces rapid degradation of HMG-CoA reductase -> cholesterol synthesis decreases

Highly regulated process

Protein levels of the first committed step in cholesterol synthesis

Bipartite:
* cytoplasmic domain carries out catalysis
* Membrane domain senses signals that lead to degradation

PROCESS:
1 - Sterol binds the sterol sensing domain within HMG-CoA reductase
2 - Induces binding to ubiquitin ligase (gp78)
3 - gp78 ubiquitinates HMG-CoA reductase
4 - Ubiquitinated HMG-CoA reductase pulled out of the membrane and degraded