Question

1. Insulin is a protein that is secreted into the blood to regulate blood sugar levels.  Describe the process of insulin production and secretion, including where it is translated, how it is modified and processed, and how it is ultimately secreted from the cell.  Be sure to point out any organelles that are involved in insulin production, as well as any important features of the insulin protein structure, along with all modifications and processing steps that occur from its initial translation to its secretion to the outside of the cell.  You can do this in a list format or flow chart format if preferred, but be sure to cover all of the aspects that are asked for in the question.

2.   Experiments have shown that the addition of the drug colchicine to cultured fibroblasts inhibits the movement of transport vesicles. Based on protein sorting/transport, the cytoskeleton, and the action of colchicine, explain why this result is observed.

3.   For each of the following, briefly describe the effect on the cytoskeleton that would most likely result. Be sure to indicate which cytoskeletal component would be affected. (8 pts)
a.   A mutation in cofilin that inhibits its activity
b.   A mutation in profilin that causes it to be over-active
c.   A mutation in the Arp2/3 complex that causes it to be over-active
d.   A mutation in the CLASP protein that inhibits its activity

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Answer 1

Insulin is produced by cells in the pancreas, called the islets of Langerhans. The insulin production involves the following steps:

TRANSLATION:

The insulin mRNA is translated as a single chain precursor called preproinsulin, and removal of its signal peptide during insertion into the endoplasmic reticulum generates proinsulin.

MODIFICATION:
Proinsulin consists of three domains: an amino-terminal B chain, a carboxy-terminal A chain and a connecting peptide in the middle known as the C peptide.

Within the endoplasmic reticulum, proinsulin is exposed to several specific endopeptidases which excise the C peptide, thereby generating the mature form of insulin.

Primary Structure:

The monomeric insulin consists of the 21 amino acid residue “A” chain and 30 amino acid residue “B” chain bound by disulfide linkages. The monomer consists of three disulfide linkages, including two between the A and B chains (A7-B7, A20-B19) and one within the A chain (A7-A11)

Secondary Structure:

The secondary structure of the A chain contains two antiparallel α-helices, formed between the residues A2-A8 and A13 to A19. These two helices are connected by residues A9 to A12. This conformation brings the two ends of the A chain into close proximity, allowing them to exist side by side.

The secondary structure of the B chain contains both alpha helices and β-sheets

Tertiary Structure:

The overall tertiary structure of the insulin monomer (A and B chain joined by disulfide bonds) is highly organized and stabilized by specific amino acid side chain interactions. These interactions influence ligand-receptor binding kinetics.

PACKAGING:

Insulin is packaged in the Golgi into secretory granules which accumulate in the cytoplasm.

SECRETION:

Insulin secretion involves a sequence of events in β-cells that lead to fusion of secretory granules with the plasma membrane.

On appropriate stimulation, insulin is secreted from the cell by exocytosis and diffuses into islet capillary blood.