L18: Protein Breakdown

Question 1

Why degrade proteins?

Answer 1

Regulation of protein levels

Defective proteins (incorrectly inserted AA, misfolded partially or unfolded, incorrectly modified, damaged by oxidation or proteolytic cleavage -> carbonyl group)

Question 2

Lysosomes

Answer 2

Vesicles 0.2-0.5 um in diameter

Bounded by single membrane

Contain hydrolytic enzyme

Formed from Golgi apparatus

Digest molecules from outside of cell and inside cell

Question 3

Protein degradation

Answer 3

Proteins- extracellular, membrane and intracellular

Acidic environment: helps unfold protein to make more susceptible proteases

Acidic proteases

Question 4

Macroautophagy (lysosome autophagy)

Answer 4

Regulated catabolic process

Degrade macromolecular & organelle constituents

Question 5

Steps in macroautophagy

Answer 5

Phagophore: cup-shaped structure formed in cytoplasm (formed from ATG proteins and associated lipids)

Autophagosome- closure of phagophore around sequestered material. Double membrane

Fusion with lysosome

Question 6

Microautophagy

Answer 6

Lysosomal membrane undergoes invagination of protrusion to enclose cargo for degradation

Question 7

Chaperone mediated autophagy

Answer 7

Hsc70-containing chaperone complex recognises protein -> bind to receptor

Delivery to lysosome-associated membrane protein type-2a (LAMP 2A)

Question 8

Proteasomes

Answer 8

Eukaryotic cells- 20S proteosomes and 26S proteosomes

26S proteosome- 2 x 19S regulators plus 20S proteosome

19S regulators- cap structures & selected ubiquinated proteins for degradation

Eukaryotic proteins are targeted for proteasome destruction by ubiquitination

Question 9

Ubiquination

Answer 9

Covalent addition of ubiquitin. Ubiquitin highly conserved, 76 residue (8.5kD) protein found widely in eukaryotes

Widely utilised form of posttranslational modification in eukaryotes (but not prokaryotes)

Marks proteins for regulated proteolytic destruction by proteosome

Ub bunds onto lysine residue

Attach ub group through c terminal to AA residues -> repeated -> ub bound to lysine through c terminus. Lysine binds to c terminus of other ub and so on

Question 10

Ways which proteins can be ubiquinated

Answer 10

Mono-ubiquitylation -> histone regulation, regulate DNA repair and gene expression

Multi-ubiquitylation -> endocytosis

Polyubiquitylation -> proteasomal degradation. Protein degradation

Polyubiquitylation -> DNA repair

Question 11

Proteins involved in ubiquination process

Answer 11

E1: Ubiquitin-activating enzyme

E2: ubiquitin-carrier protein

E3: ubiquitin-protein ligase. Different ligases with differing substrate specificity

Question 12

Ubiquination steps

Answer 12

1. Terminal carboxyl group of ubiquitin is joined to E1. ATP dependent step
2. Ubiquitin transferred to E2
3. E3 transfers activated to the epsilon-amino group of Lys residue of target protein

Question 13

Muscle weakness in ageing

Answer 13

Loss of myofibres -> cell death

Decreased myofibre size

Protein synthesis < protein catabolism

Question 14

Protein catabolism

Answer 14

Proteins have finite lifespan

Different proteins in same tissue have different half-lives. <1hr to a week for liver enzymes. Stability correlates with sequence at N-terminus

Proteins in different tissues have different half-lives

Question 15

Cathepsins: example of protein targeting and post-translational modifications

Answer 15

Major lysosomal proteases

Signal peptide: directs insertion of nascent polypeptide chain into ER

ER: signal peptide is cleaved with assistance of proregion. Disulfide bond formation. N-linked glycosylation with high-mannose glycans

Golgi: mannose residues phosphorylated -> mannose 6-phosphate. M6p routes protein into endosomal/lysosomal compartment via m6p receptor. Portion not converted to m6p form and is shunted into exocytosis pathway

Endosome: acidification. Activation: cleavage of proregion and proteolytic processing in lysosome into heavy and light chains

Question 16

Polyubiquitinylation

Answer 16

Free carboxyl group of ubiquitin’s carboxyl-terminal is to

Question 17

How proteins are degraded in proteosomes

Answer 17

Polyubiquitinylated target recognised by proteosome cap

Deubiquitinase enzymes remove ubiquitin groups

ATP hydrolysis in proteosome cap drives unfolding and transfer to central cavity

Central cavity possesses proteolytic activity

Cleave proteins into short peptides of about 7-9 AA -> released into cytoplasm for degradation by cytoplasmic peptidases