Proinsulin, proteolytic processing

Proinsulin is proteolytically processed in the coated secretory granules, yielding mature insulin and a 34-amino acid connecting peptide (C peptide, Figure 11.1). The C peptide is further proteolytically modified by removal of a dipeptide from each of its ends. The secretory granules thus contain low levels of proinsulin, C peptide and proteases, in addition to insulin itself. The insulin is stored in the form of a characteristic zinc-insulin hexamer, consisting of six molecules of insulin stabilized by two zinc atoms. 

Figure 11.1 Proteolytic processing of proinsulin, yielding mature insulin, as occurs within the coated secretory granules...

FIGURE 23-5 Insulin. Mature insulin is formed from its larger precursor preproinsulin by proteolytic processing. Removal of a 23 amino acid segment (the signal sequence) at the amino terminus of preproinsulin and formation of three disulfide bonds produces proinsulin. Further proteolytic cuts remove the C peptide from proinsulin to produce mature insulin, composed of A and B chains. The amino acid sequence of bovine insulin is shown in Figure 3-24. 

Proteolytic Processing Many proteins are initially synthesized as large, inactive precursor polypeptides that are proteolytically trimmed to form their smaller, active forms. Examples include proinsulin, some viral proteins, and proteases such as chymotrypsinogen and trypsino-gen (see Fig. 6-33). 

Conversion of proinsulin to insulin and C-peptide in secretory granules involves site-specific cleavages at the Arg-Arg and Lys-Arg sequences (Figure 22-6) these serve as signals for proteolytic processing of many other proteins. Cleavage occurs at the C-terminal end of each pair by trypsin-like enzymes and is followed by... 

Moore, H.P.H., Walker, M.D., Ue, F. and Kelly, R.B. (1983) Expressing a human proinsulin DNA in a mouse ACTH-secreting cell. Intracellular storage, proteolytic processing and secretion on stimulation. Cell 35 531-538. 

Insulin is synthesized as preproinsulin that is proteolytically processed in the P cells of the islets of Lang-erhans in the pancreas, to give proinsulin. After synthesis and folding, a section of the molecule (the C peptide) is excised, leaving the A and B peptides connected via disulfide bonds. Thus, native insulin, lacking the C peptide, lacks some of the information necessary to direct the folding process. 

Nearly all bacterial proteins are synthesized with a formyl-methionine as the first amino acid, as described above. Yet most proteins isolated from cell culture do not contain an N-terminal fMet or Met residue, indicating that the maturation process involves proteolytic removal of this amino acid. Some proteolytic events release a functional protein from a synthesized precursor, or proprotein. An example is insulin, which is released from its proinsulin precursor by excision of an internal 33-residue peptide. Another type of proteolytic processing is the removal of N-terminal signal peptides, which target some proteins for insertion into membranes or for secretion from the cell. 

These processes are shown for insulin in Figure 3.27. After the polypeptide synthesis (primary structure), the signaling protein is removed and the proinsulin is folded into its secondary structure using thiol-disulfide oxidation and transported into a vesicle. Here, acknowledgement proteolytic enzymes, known as prohormone convertases (PCI and PC2), remove the C peptide segment and the exoprotease carboxypep-tidase E produces the insulin molecule that has a molecular weight of 5,808 g/mol (Dalton). Six of these units will then make the quaternary structure shown in Figure 3.27. 

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