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CBB > Proteolysis > Flashcards

Proteolysis Flashcards

1
Q

What is proteolysis?

A

The breakdown of proteins into polypeptides or amino acids, through enzymic cleavage of peptide bonds.

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2
Q

What is cellular proteolysis for?

A

Required for quality control and removal of misfolded or mutant proteins.
Full proteolytic cleaveage is the biological end point for protein activity.

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3
Q

What are examples of needing cellular proteolysis?

A

Cellular receptors
Transcription Factors
Cell cycle proteins.

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4
Q

What is the major site of proteolysis?

A

Lysosomes - contain hydrolytic enzymes involved in protein degradation into small polypeptides and proteins.
Degrade lipids, DNA, cells, organells and pathogens.
Involved in nutrient sensing and recycling of biomolecules.

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5
Q

What is the 26S proteasome?

A

A macromolecular protein complex involved in the degradation of proteins into small polypeptides, through ubiquitin-mediated proteolysis.

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6
Q

What is the function of the 26S proteasome?

A

Quality control of protein synthesis - targets misfolded and mutant protein.
Regulates protein activities.
Recycling of peptides and amino acids.
Generation of peptides for antigen presentation.

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7
Q

What is ubiquitin?

A

A polypeptide which is covalently conjugated via its C-terminal Glycine to a lysine in the target protein or another ubiquitin to form polyubiquitin chains - Ubiquitylation.

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8
Q

Which poly-ubiquitylated chains are important?

A

Polyubiquitylated proteins with Lysine 11 or 48 ubiquitin linkages are important in being recognised by the 26S proteasome and degraded, and targeting substrates for degradation.

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9
Q

What are the steps of the ubiquitin proteasome system?

A

Activation
Conjugation
Ligation
Elongation

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10
Q

What is the activation of Ubiquitin?

A

Ubiquitin is activated through ATP.
It is then conjugated by high energy thioester linkages and activated by the E1 enzyme.
The ubiquitin is then transferred to the conjugated enzyme by the thioester linkages.

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11
Q

How is ubiquitin transferred?

A

Ubiquitin is transferred from E2 to the substrate in the presence of E3.
Or for different types of E3, ubiquitin forms an intermediate with E3, and is then transferred to the substrate.
Allows for ubiquitilation on a lysine residue.

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12
Q

What is elongation of ubiquitin ligase?

A

The ubiquitin ligase undergoes repeated poly-ubiquitylation to form a poly-ubiquitin chain.
This is then recognised by the proteasome and degraded in an ATP dependent matter into polypeptides and amino acids.

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13
Q

What is the reversal of ubiquitylation?

A

Enzymes - deubiquitylases (DUBs) and USPs can cleave off the ubiquitin from the substrate.
Rpn 11 cleaves the chain, removes ubiquitin, and released the chain from the substrate.

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14
Q

What are the sub-types of ubiquitin chains?

A

Mono chains - for protein interactions and localisation
K48 and 11 - for proteasomal degradation.
K63 - activation, DNA repair, Lysosomal targeting
Linear chain - NF-kB activation.

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15
Q

How is the specificity of protein ubiquitylation controlled?

A

Through the massive diversity of E3 ubiquitin ligases.
There is only 1 or 2 E1.
40 E2, and these can couple with multiple E3 ligases.
There is 800 E3 ligases, which can have many susbtrates.

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16
Q

What is APC/C?

A

The Anaphase-Promoting Complex/Cyclosome, a multimeric E3 ubiquitin ligase.
APC11 possesses the ligase activity.
Regulates progression through mitosis and G1.
Ubiquitylates cell cycle proteins - cyclins A and B, and securin.

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17
Q

What are the activators of APC?

A

Cdc20 and Cdh1
Used in recruitment of substrates to APC.
Cdc20 is a proto-oncogene.

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18
Q

What are the E2 ubiquitin conjugating enzymes in APC?

A

UbcH10, also a proto-oncogene.
Ube2S
Promotes ubiquitylation of the substrate in complex with ABC11

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19
Q

How does the APC control sister chromatin segregation in mitosis?

A

Once the sister chromatins are aligned on the metaphase plate, the APC is activated by Cdc20, which promotes ubiquitylation of Securin.
This allows activation of Separase, which cleaves the cohesing ring which keeps sister chromatin together, so they are pulled into separate daughter cells.
The Cdc20 is degraded by the APC, and CbH1 allows for mitotic exit by promoting degradation of Cyclin B1.

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20
Q

How does ubiquitin-mediated proteolysis drive the cell cycle?

A

Waves of cyclin synthesis and degradation drives the temporal progression of the cell cycle.
Increases in cyclin levels are controlled transcriptionally.
Reduction in cyclin leves are controlled by ubiquitin-mediated protolysis.

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21
Q

How is dysregulating of E3 ubiquitin ligases linked to cancer?

A

Mdm2 proto-oncogenes product is overexpressed in many cancers.
BRCA1 tumour suppressor is inactivated in breast and ovarian cancer.

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22
Q

How is dysregulation of E3 ubiquitin ligases linked to neurodegeneration?

A

Parkin loss of function by mutations is linked to Parkinson’s disease.
Parkin is also a disease modifier and promotes motor neuron loss in amyotrophic lateral sclerosis (ALS).

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23
Q

How is dysregulation of E3 ubiquitin ligases linked to cardiovascular disease?

A

MuRF1 and Atrogin 1 expression is misregulated in atrophy and hypertrophy.
Parkin mediates mitrophagy in cardiac injury.

24
Q

What is the structure of the 26S proteasome?

A

20S proteolytic core, capped by alpha chains with Beta chains in the centre.
1 or both sides of the proteasome is a 19S regulatory particle, composed of a base and a lid.

25
Q

What is the function of the 26S protealytic core?

A

There is a mechanism for recognising the ubiquitylated substrate by the 19S recognising particle.
The substrate is then unfolded and translocated into the 20S protealytic core, where peptidases chop the the substrate into peptides, and release it from the proteasome.

26
Q

What is the structure of the 20S proteasome core?

A

Barrel-shaped symmetrical structure
4 heteromeric rings
2 copies of each of 14 subunits - 7 a-subunits on each end, 7 B-subunits in the centre.
Proteolysis occurs in the central channel.

27
Q

What are the proteolytic enzyme activities associated with the 20S proteasome?

A

B1 - caspase like - cleaves after acidic residuss e.g. aspartic acid and glutamic acid.
B2 - Trypsin like - cleaves after basic residues e.g. lysine and arginine.
B5 - chymotrypsin - cleaves hydrophobic residues - valine and isoleucine.

28
Q

What are proteasome inhibitors?

A

Useful in cancer therapeutics:
Induce cell growth and cell death.
Classes - peptide aldehydes, boronates, epoxyketone, B-lactone.

29
Q

What is the role of the 19S lid?

A

The lid recognises the poly-ubiquitin chain.
3 receptors on the 26S proteasome - Rpn13 and Rpn10 recognise the chain.

30
Q

What are chaperone proteins?

A

Sometimes substrates are recruited through Chaperone proteins.
This has ubiquitylated associated domains, and ubiquitin like domains, to associate with the receptors.

31
Q

What is the role of the 19S base in substrate unfolding?

A

Ubiquitin is released and the chain is unfolded in an ATP dependent manner.
The chain is then fed thorugh the proteolytic core of the 20S proteasome.
The ATP ring forms a closed gate, and on binding of the substrate in the presence of ATP, the gate opens.
This allows feeding in of the subtrate and entry into the proteolytic core.

32
Q

What are the characteristics of lysosomes?

A

Membrane-bound organelles.
Acidic pH - for hydrolytic enzyme functioning.
The marker is acid phosphatase.
Primary lysosomes are formed in the Golgi.
Secondary lysosomes are formed by fusion.

33
Q

How are hydrolases targeted to the lysosomes?

A

Mannose-6-phosphate is added to N-linked oligosaccharides of lysosomal hydrolases in the Golgi.
This then targets it to the lysosome.

34
Q

What cellular pathways couple with lysosomes?

A

Endocytosis - receptors and ligands can be internalised to form endosomes. These fuse with compononents of damaged organelles to form lysosomes.
Phagocytosis - bacteria form a phagosome in the cell, fuses with the endosome to form the lysosome.

35
Q

What are the endocytic routes for receptor degradation?

A

Unbound receptors are recycled slowly.
Ligand binding accelerates endocytosis.
The receptor complex uncouples in the endosome, and products target to the lysosome.
Hydrolases the degrade the ligand and receptor.
Alternatively are recycled to the plasma membrane.

36
Q

What is the role of Parkin in Parkinson’s disease?

A

Parkinson’s is associated with protein aggregate formation of damaged proteins.
Parkin is E3, and has a role in lysine-linked polyubiquitin chains in targeting to lysosomes.
PINK1 phosphorylates ubiquitin at S65 to recruit and activate Parkin to damaged mitochondria.

37
Q

What are the roles of lysosomes in cellular processes?

A

Cellular clearance - endocytosis.
Lysosomal exocytosis
Plasma membrane repair.
Reservoir for amino acids and ions.
Transcriptional regulation
Lipid catabolism

38
Q

What are signal peptides?

A

Most secretory proteins are synthesised with signal peptides (pre-peptides) at the N-terminus and removed after sorting by proteolysis.

39
Q

What are pro-peptides for?

A

Folding - intramolecular chaperones
Assembly of multimers
Latency - most bioactive polypeptides must be inactive within the secretory pathway.
Must be removed for activity.
Allows for rapid response to cellular need.

40
Q

What are the secretory vesicles?

A

The constitutive pathway delivers contents directly to the plasma membrane.
The regulated pathway vesicles form a pool of contents, awaiting a secretagogue for release.

41
Q

What is pro-peptide cleavage?

A

Essential for activation
Cleavage sites contain at least 2 basic residues, removed after cleavage by carboxypeptidase.
Sequence homologous to bacterial subtilisins.

42
Q

What are PCSKs?

A

Proprotein Convertases Subtilisin/Kexin
e.g. Furin

43
Q

What is the specificity of PCSK?

A

Cleavage mostly C-terminal to Arg.
Specificity is dictated by sequence 2-5 amino acids N-terminal to the bond cleaved.
Amino acids recognised are basic.
Site selection is modified by pH.
Substrate range dictated by physical proximity.

44
Q

Which PCSKs prefer which amino acids?

A

PCSK1 - arg arg
PCSK2 - Lys arg
PCSK3 - Arg, any, Lys, Arg

45
Q

What is the localisation of PCSKs?

A

PCSK 1 and 2 are in the regulated secretory pathway.
PCSK 4,5,6,9 are in the constitutive pathway.
PCSK 3,5,7 are transmembrane species.
PCSK 3 (Furin) recycles through the plasma membrane and endosomal compartments.

46
Q

What is the role of PCSKs in pro-peptide cleavage?

A

Provides control for mature protein production.
Allows tissue-specific expression of peptide hormones form common precursors - e.g. POMC.

47
Q

How can distinct hormones be made from one protein?

A

POMC is a protein which through PCSK1 can generate 2 hormones, then through further modifications by PCSK2 can generate 4 more hormones.

48
Q

How is PCSK used in insulin maturation?

A

Insulin is synthesised as pre-pro-insulin
The signal pre-peptide is cleaved.
This generates pro-insulin inactive precursor, which facilitates correct protein folding.
Processing by PCSK2 and 1 yields mature insulin within the regulated secretory vesicle at pH5.5.

49
Q

What is the role of PCSK9 in LDL receptor recycling?

A

PCSK9 promotes LDL receptor degradation in the lysosome.
This prevents LDLR recycling to the plasma membrane, and reduces its number at the membrane.
This reduces the clearance of LDL from circulation.
This is non-proteolytic, but uses PCSKs.

50
Q

How can PCSK9 be used to treat hypercholesterolemia?

A

Favours LDL recycling.
LDLR targeted by PCSK9, limits number of receptor.
LDL is broken down.
Antibody favours LDLR binding.
More efficient clearing of LDL in circulation.

51
Q

What is the role of PCSK in viral glycoprotein cleavage?

A

Viruses exploit PCSKs for the maturation of envelope glycoproteins.
HIV1 gp160 requires PCSK7 for activation - allows HIV entry into cells.

52
Q

What are the outcomes of Matrix metalloproteinases?

A

Act on the ECM:
Wound healing
Tissue remodelling
Immunity and inflammation
Ovulation
Pregnancy

53
Q

What are secretases?

A

Liberate active domains from membrane-bound proteins.

54
Q

What is the structure of MMPs?

A

Have a cysteine residue in pro-domain, in latent form coordinates with zinc ion in catalytic domain.
Inhibits the function of MMP.
On pro-domain removal, it is active because of no-coordination with zinc - cysteine-switch mechanism.

55
Q

What do dysregualted MMPs cause?

A

Cardiovascular disease - hypertension, heart failure.
Cancer - leukemia, lung, breast, colorectal.
Neurodegenerative - alzheimers, parkinsons.
Lung disease - COPD, bronchial asthma.
Diabetes
Rheumatoid arthritis.

56
Q

What is alpha-secretase?

A

Member of ADAMs family.
Possess MMP and transmembrane domains.
Active at the cell surface.
Responsible for sheddase activities - inserted into plasma membrane, secretase cleaves protein and released from cell surface.

CBB (17 decks)

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