Protein Degradation

Question 1

Overview of proteins?

Answer 1

Macronutrients - protein, carbs and fats
Sources of protein - meat/fish, dairy, nuts/seeds and legumes
Proteins contains: C,O, H and N (nitrogen comes entirely from proteins)

Question 2

How are proteins broken down?

Answer 2

Proteases:
Stomach - pepsin
Duodenum - chymotrypsin, trypsin, elastase
Small intestine - endo- & exo-peptidases

Question 3

How can we store proteins?

Answer 3

There is no effective storage for proteins
Amino acids can be polymerised into proteins but this is functional not storage
We can degrade these proteins to acquire amino acids is needed

Question 4

What are the half lives of proteins?

Answer 4

Proteins have different half lives e.g. From a few minutes to several weeks
Half life is related to function
Control-point enzymes have short half lives
Collagen has a longer half life (60-70 days)

Question 5

What is nitrogen balance?

Answer 5

If you are healthy:
nitrogen intake = nitrogen excretion

Negative nitrogen balance: intake < excretion (internal illness)
Occurs during fasting/illness – body is breaking down proteins for energy

Positive nitrogen balance: intake > excretion
Occurs during growth, pregnancy – body is building new tissue, after major surgery

Question 6

What can result in muscle mass change?

Answer 6

Increasing muscle mass during excercise - exemplifies protein turnover as protein synthesis must exceed protein breakdown
Opposite is muscle atrophy - during starvation/fasting, in the absence of exercise, after breaking a bone and it was in plaster and older immobile people

Question 7

Why do we degrade proteins?

Answer 7

1. to store amino acids in the form of proteins and release them when needed
2. to eliminate abnormal proteins, where accummulation would be harmfull
3. to regulate cellular metabolism by eliminating unnecessary enzymes and regulatory proteins

Question 8

What does rate of protein degradation depend on?

Answer 8

Half life of the protein

Nutritional and hormonal state

Question 9

What can be involved in protein degradation?

Answer 9

Lysosomes

Proteosomes (ubiquitin)

Question 10

Describe lysosomes?

Answer 10

Lysosomes are acidic (pH<5) organelles
They contain 50 degradative enzymes including cathepsin proteases (aspartyl, serine, but most are cysteine proteases)
Low pH is maintained by proton pumps (to protect the cell against lysosomal leakage)

Question 11

What do lysosomes do?

Answer 11

Lysosomes degrade extracellular substances taken up by endocytosis
They also degrade intracellular material, portions of cytoplasm (microautophagy) or entire organelles (macroautophagy), by fusion with vacuoles

Products of digestion (e.g. amino acids) are released back into the cell for recycling

Question 12

What happens with lysosomes under normal conditions?

Answer 12

Non-selective pathways

Therefore any/all proteins can be degraded

Question 13

What are stressed conditions for a lysosome?

Answer 13

Oxidative stress
Exposure to toxic/denaturing cells
Starving

Question 14

What happen with lysosomes under stressed conditions? why? example?

Answer 14

Selective pathways are induced only degrading proteins that contain KFERQ (lys, Phe, Glu, Arg, Gln)

Allows starving effects to be minimised in the brain

Example - Heat shock protein binds to KFERQ motif and allows control of the removal of damaged/oxidised proteins

Question 15

What diseases are related to lysosomes?

Answer 15

Normal autophagy is affected in diseases such as Alzheimer’s, Parkinson’s and Huntingdon’s disease
Lysosomal storage diseases (e.g. Niemann-Pick type C, Gaucher’s disease) cause neurodegeneration

Question 16

Describe the proteosome?

Answer 16

Large multi-protein complex - 2100 kDa with a central 20S proteasome (core particle) and one or two 19S caps (regulatory particles)
Barrel structure has a hollow core comprising three chambers

28 protein subunits = 4 rings
Outer rings comprise 7 α-type subunits
Inner rings comprise 7 β-type subunits.

Three of the β -type subunits have catalytic activity - threonine proteases
Three active sites have different specificities: β1 cleaves after acid residues, β2 after basic residues, β5 after hydrophobic residues Proteins are cleaved into 8-9 amino acid peptides - further degraded in the cytosol

Question 17

Describe the proteosome cap?

Answer 17

When the cap is associated it is active
The cap is responsible for: recognition of polyubiquitinated protein substrates, unfolding them and feeding them into the 20S core (ATP dependent)

It is not very well understood

Question 18

What does the proteosome do?

Answer 18

This is highly selective in the nucleus and cytoplasm
They tag proteins with ubiquitin - the proteins become unfolded and hydrolysed in the proteasome

It helps control of the amount of proteins in cells, and so the rate of many cellular processes e.g. cell cycle/division, apoptosis, DNA transcription & repair, immune function

Occurs only in eukaryotes, as prokaryotes lack ubiquitin ATP dependent process

Question 19

Describe ubiquitin?

Answer 19

Small, 76 amino acid protein
Found in eukaryotes only
Highly conserved

It marks the protein for degredation - so the proteosome knows to degrade it

Question 20

How does ubiquitin bind to the protein?

Answer 20

1. The COO end of Ub attaches via a thioester bond to E1 of the ubiquitin-activating enzyme
2. Ub is tranfered to a cys sulfhydryl group on E2
3. Ub transferred to the e-amino group of a lysine residue on the target protein, by E3 (forming an iso peptide bond)

Further Ub molecules are added the same way (needs at least 4)

Question 21

What is the iso peptide bond?

Answer 21

A peptide bond which is not part of the peptide backbone of a protein, but involves side-chain amino or carboxyl groups

Question 22

What is the variation of ubiquitination?

Answer 22

Not all proteins are ubiquitinated equally

N-end rule:
N-terminal Asp, Arg, Leu, Lys & Phe are destabilising – half-lives of just a few minutes
N-terminal Ala, Gly, Met, Ser, Thr & Val are stabilising – half-lives of many hours

PEST proteins:
Proteins with segments rich in Pro, Glu, Ser and Thr are degraded rapidly

Question 23

What is another form of ubiquitination?

Answer 23

Monoubiquitination can be reversible and has a regulatory role in modifying protein activity
Can also be used in cells as a form of metabolic control - like de/phosphorylation

Question 24

What do bacteria have to degrade proteins?

Answer 24

They have similar assemblies of self-compartmentalised proteases
E. coli has 2 proteases Lon & Clp which operate similarly to eukaryotic proteasomes
Not very selective, and the active sites are hidden to be protected so a substrate will have to go into the barrel in order to hydrolysed

Question 25

What diseases are linked to proteosomes?

Answer 25

Regulating muscle wasting diseases
Decreased proteasome activity has been observed in Parkinson’s disease, Alzheimer’s disease, cardiovascular disease

Bortezomib (Velcade) is a UPS inhibitor, available for the treatment of cancers (e.g. multiple myeloma)