Proinsulin folding

Processing of insulin. Insulin is synthesized by membrane-bound polysomes in the /3 cells of the pancreas. The primary translation product is preproinsulin, which contains a 24-residue signal peptide preceding the 81-residue proinsulin molecule. The signal peptide is removed by signal peptidase, cutting between Ala (—1) and Phe (+1), as the nascent chain is transported into the lumen of the endoplasmic reticulum. Proinsulin folds and two disulfide bonds crosslink the ends of the molecule as shown. Before secretion, a trypsinlike enzyme cleaves after a pair of basic residues 31, 32 and 59, 60 then a carboxypeptidase B-like enzyme removes these basic residues to generate the mature form of insulin. 

Figure 7 Maturation of insulin. Insulin is synthesized as preproinsulin that contains an N-terminal signal sequence. After translocating into the ER, the signal sequence is cleaved off by the signal peptidase and the resulting proinsulin folds into a stable conformation. Three disulfide bonds are formed between cysteine side chains. The connecting sequence (Chain C) is cleaved off in the Golgi by proprotein convertases to form the mature and active insulin molecule, which is then secreted.

Proinsulin folds into specific 3D structure and disulfide bonds form... 

Proinsulin folds to adopt the correct orientation of the prevailing disulphide bonds plus other relevant conformational constraints whatsoever on account of its primary structure exclusively. 

A small number of proteins, and again insulin is an example, are synthesized as pro-proteins with an additional amino acid sequence which dictates the final three-dimensional structure. In the case of proinsulin, proteolytic attack cleaves out a stretch of 35 amino acids in the middle of the molecule to generate insulin. The peptide that is removed is known as the C chain. The other chains, A and B, remain crosslinked and thus locked in a stable tertiary stiucture by the disulphide bridges formed when the molecule originally folded as proinsulin. Bacteria have no mechanism for specifically cutting out the folding sequences from pro-hormones and the way of solving this problem is described in a later section. 

This concept of reversible chemical crosslinking of the chains to drastically decrease the enthalpic penalty of the folding process and to exploit the highly favored disulfide loop formation in the A-chain with m = 4, has been further developed into artificial peptide linker chains that can be excised selectively by enzymatic processing, to allow for bioexpression of the artificial proinsulins on an industrial scale. 95,96 However, to apply this approach rationally to other double-stranded cystine peptides, knowledge about their three-dimensional structure is essential. 

Insulin is synthesized as proinsulin. After synthesis and folding, a section of the molecule (the C peptide) is excised, leaving the A and B peptides connected via disulfide bridges. Thus, native insulin, lacking the C peptide, lacks some of the information necessary to direct the folding process. 

Osterbye, T., Jorgensen, K.H., Fredman, P., Tranum-Jensen, J., Kaas, A., Brange, J., Whittingham, J.L. and Buschard, K., Sulfatide promotes the folding of proinsulin, preserves insuhn crystals, and mediates its monomerization, Glycobiology 11 (2001) 473-479. 

The neutral mutation rate differs for each nucleotide in a gene and usually serves as a good indicator for the functional importance of encoded amino acid residue. For example, the C-peptide of proinsulin evolves at a rate (0.526PAM/10 years) much more rapid than that of the A- and B-chains of insulin (0.071 PAM(point accepted mutation)/10 years) because the C-peptide appears to promote the protein folding and is removed pro-teolytically after the insulin has folded to its correct conformation. The greater divergent rate of the C-peptide than that of the A and B-chains reflects the fewer constraints on its... 

The polypeptide chains of all proteins are synthesized by the process described above. This mechanism gives rise to primary polypeptide chains, which are often further modified—for example, by cleavage into smaller peptides, by stmctural modification of selected amino acid residues, by splicing of the polypeptide chain, or by the formation of covalent bonds between polypeptide chains. Some of these secondary modifications are related to the correct folding of polypeptide chains and to the production of active enzymes or peptide hormones from inactive precursors (e.g., insulin from proinsulin). Also, the transport of proteins within the cell or the secretion of extracellular proteins is often linked to structural changes in polypeptide chains either during or after completion of synthesis. 

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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