Lysosomes

Question 1

What are the characteristics of lysosomes?

Answer 1

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.

Question 2

How are hydrolases targeted to the lysosomes?

Answer 2

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

Question 3

What cellular pathways couple with lysosomes?

Answer 3

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.

Question 4

What are the endocytic routes for receptor degradation?

Answer 4

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

Question 5

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

Answer 5

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.

Question 6

What are the roles of lysosomes in cellular processes?

Answer 6

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

Question 7

What are signal peptides?

Answer 7

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

Question 8

What are pro-peptides for?

Answer 8

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.

Question 9

What are the secretory vesicles?

Answer 9

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

Question 10

What is pro-peptide cleavage?

Answer 10

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

Question 11

What are PCSKs?

Answer 11

Proprotein Convertases Subtilisin/Kexin
e.g. Furin

Question 12

What is the specificity of PCSK?

Answer 12

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.

Question 13

Which PCSKs prefer which amino acids?

Answer 13

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

Question 14

What is the localisation of PCSKs?

Answer 14

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.

Question 15

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

Answer 15

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

Question 16

How can distinct hormones be made from one protein?

Answer 16

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

Question 17

How is PCSK used in insulin maturation?

Answer 17

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.

Question 18

What is the role of PCSK9 in LDL receptor recycling?

Answer 18

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.

Question 19

How can PCSK9 be used to treat hypercholesterolemia?

Answer 19

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.

Question 20

What is the role of PCSK in viral glycoprotein cleavage?

Answer 20

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

Question 21

What are the outcomes of Matrix metalloproteinases?

Answer 21

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

Question 22

What are secretases?

Answer 22

Liberate active domains from membrane-bound proteins.

Question 23

What is the structure of MMPs?

Answer 23

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.

Question 24

What do dysregualted MMPs cause?

Answer 24

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

Question 25

What is alpha-secretase?

Answer 25

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.