After proinsulin is translocated in to the lumen of the ER, it is after that delivered in transport “COP-coated vesicles” to the cis-Golgi apparatus [93] (Figure 6.4). Up until relatively recently, it was believed that newly synthesized proinsulin was passed from the cis-Golgi network “stack” via the medial– to the trans-stack of the Golgi apparatus stacks in “COP”-coated vesicles, yet a landmark study conducted in β cells using electron microscope tomography showed that the Golgi apparatus is actually one continuous organellar compartment, and not a collection of stacks [94]. As such, newly synthesized proinsulin traverses through the lumen of the β cell’s Golgi apparatus to the trans-Golgi network (TGN) [94], where it accumulates in clathrin-coated regions [93]. This is the site of secretory granule biogenesis (Figure 6.4). The means by which newly synthesized proinsulin (and others choose proteins destined to the β granule), is specifically targeted to sites of β-granule biogenesis in the TGN remains a matter of debate [95]. However, it is known to be a highly efficient process, along with >99% efficient of newly synthesized proinsulin sorted to the β granule and regulated secretory pathway under typical conditions [91,95].
Proteolytic enzymes of proinsulin conversion
The severe site for processing of proinsulin to biologically energetic insulin is the immature secretory granule compartment of the β cell [91,93] (Figure 6.4). Production of insulin (and C-peptide) occurs via limited proteolysis of the proinsulin precursor molecule, which is catalyzed by two Ca2+-dependent endopeptidases, PC2 and PC1/3 and a Ni2+-dependent exopeptidase, CP-H [88,91]. There are two dibasic sites on the human proinsulin molecule: Arg3, Arg32 and Lys64, Arg65, that signal limited endoproteolytic cleavage of proinsulin to excise the C-peptide moiety and to provide insulin along with its disulphide-linked A- and B-chains correctly aligned (Figure 6.3). Endoproteolytic peptide bond cleavage of proinsulin occurs on the carboxylic edge of the Arg31, Arg32 or Lys64, Arg65, followed by rapid and individual exopeptidic removal of the newly exposed straightforward amino acids by CP-H [88,91]. Both distinct β-granule proinsulin-processing endopeptidase tasks were originally found as Ca2+-dependent along with an acidic pH max and were later identified as the PC1/3 and PC2 endopeptidase genes [88,91].
A scheme of proinsulin conversion is illustrated in Figure 6.5. Proinsulin conversion could occur by two feasible routes. Either PC2 initial cleaves on the carboxylic edge of Lys64-Arg65 to generate a split 65,66 proinsulin intermediate, followed by CP-H trimming of the newly exposed lysine and arginine residues to generate des 64,65 proinsulin. after that PC1/3 can easily after that cleave des 64,65 proinsulin at Arg31, Arg32, which with each other along with CP-H trimming of the exposed arginine residues, yields insulin and C-peptide (Figure 6.5). Alternatively, PC1/3 initial could cleave at the carboxylic edge of Arg31, Arg32 to generate a split 32,33 proinsulin intermediate, followed by CP-H trimming of the revealed arginine residues to generate des 31,32 proinsulin. PC2 can easily after that cleave des 32,33 proinsulin at Lys64-Arg65, which with each other along with CP-H trimming of the lysine and arginine residues, yields insulin and C-peptide (Figure 6.5). However, PC2 has actually a considerably more powerful preference for the des 31,32 proinsulin substrate compared to proinsulin, whereas PC1/3 has actually an equivalent preference for proinsulin or des 64,65 proinsulin substrates [88,91]. As such, in humans, the sequential processing of proinsulin via des 31,32 proinsulin is the predominant route, where PC1/3 cleaves intact proinsulin first, followed by PC2 cleavage of des 31,32 proinsulin (Figure 6.5). This is consistent along with the presence of the des 31,32 proinsulin conversion intermediates in the human flow yet negligible levels of des 64,65 proinsulin [91].
