The generation of insulin-containing secretory granules from proinsulin in the lumen of the endoplasmic reticulum (ER) can be described in 4 steps: formation of intramolecular disulfide bonds, formation of proinsulin-zinc-calcium complexes, proteolytic cleavage of proinsulin to yield insulin, translocation of the granules across the cytosol to the plasma membrane. Transcription of the human insulin gene INS is activated by 4 important transcription factors: Pdx-1, MafA, Beta2/NeuroD1, and E47. The transcription factors interact with each other at the promoters of the insulin gene and act synergistically to promote transcription. Expression of the transcription factors is upregulated in response to glucose. The preproinsulin mRNA is translated by ribosomes at the rough endoplasmic reticulum (ER) and the preproinsulin enters the secretion pathway by virtue of its signal peptide, which is cleaved during translation to yield proinsulin. Evidence indicates that the preproinsulin mRNA is stabilized by glucose. In the process annotated in detail here, within the ER, three intramolecular disulfide bonds form between cysteine residues in the proinsulin. Formation of the bonds is the spontaneous result of the conformation of proinsulin and the oxidizing environment of the ER, which is maintained by Ero1-like alpha The cystine bonded proinsulin then moves via vesicles from the ER to the Golgi Complex. High concentrations of zinc are maintained in the Golgi by zinc transporters ZnT5, ZnT6, and ZnT7 and the proinsulin forms complexes with zinc and calcium. Proinsulin-zinc-calcium complexes bud in vesicles from the trans-Golgi to form immature secretory vesicles (secretory granules) in the cytosol. Within the immature granules the endoproteases Prohormone Convertase 1/3 and Prohormone Convertase 2 cleave at two sites of the proinsulin and Carboxypeptidase E removes a further 4 amino acid residues to yield the cystine-bonded A and B chains of mature insulin and the C peptide, which will also be secreted with the insulin. The insulin-zinc-calcium complexes form insoluble crystals within the granule The insulin-containing secretory granules are then translocated across the cytosol to the inner surface of the plasma membrane. Translocation occurs initially by attachment of the granules to Kinesin-1, which motors along microtubules, and then by attachment to Myosin Va, which motors along the microfilaments of the cortical actin network. A pancreatic beta cell contains about 10000 insulin granules of which about 1000 are docked at the plasma membrane and 50 are readily releasable in immediate response to stimulation by glucose or other secretogogues. Docking is due to interaction between the Exocyst proteins EXOC3 on the granule membrane and EXOC4 on the plasma membrane. Exocytosis is accomplished by interaction between SNARE-type proteins Syntaxin 1A and Syntaxin 4 on the plasma membrane and Synaptobrevin-2/VAMP2 on the granule membrane. Exocytosis is a calcium-dependent process due to interaction of the calcium-binding membrane protein Synaptotagmin V/IX with the SNARE-type proteins.
Chen H, Jawahar S, Qian Y, Duong Q, Chan G, Parker A, Meyer JM, Moore KJ, Chayen S, Gross DJ, Glaser B, Permutt MA, Fricker LD.; ''Missense polymorphism in the human carboxypeptidase E gene alters enzymatic activity.''; PubMedEurope PMCScholia
Rutter GA, Hill EV.; ''Insulin vesicle release: walk, kiss, pause ... then run.''; PubMedEurope PMCScholia
Kambe T, Yamaguchi-Iwai Y, Sasaki R, Nagao M.; ''Overview of mammalian zinc transporters.''; PubMedEurope PMCScholia
Jackson RS, Creemers JW, Farooqi IS, Raffin-Sanson ML, Varro A, Dockray GJ, Holst JJ, Brubaker PL, Corvol P, Polonsky KS, Ostrega D, Becker KL, Bertagna X, Hutton JC, White A, Dattani MT, Hussain K, Middleton SJ, Nicole TM, Milla PJ, Lindley KJ, O'Rahilly S.; ''Small-intestinal dysfunction accompanies the complex endocrinopathy of human proprotein convertase 1 deficiency.''; PubMedEurope PMCScholia
Kaufmann JE, Irminger JC, Mungall J, Halban PA.; ''Proinsulin conversion in GH3 cells after coexpression of human proinsulin with the endoproteases PC2 and/or PC3.''; PubMedEurope PMCScholia
Bratanova-Tochkova TK, Cheng H, Daniel S, Gunawardana S, Liu YJ, Mulvaney-Musa J, Schermerhorn T, Straub SG, Yajima H, Sharp GW.; ''Triggering and augmentation mechanisms, granule pools, and biphasic insulin secretion.''; PubMedEurope PMCScholia
Itoh Y, Tanaka S, Takekoshi S, Itoh J, Osamura RY.; ''Prohormone convertases (PC1/3 and PC2) in rat and human pancreas and islet cell tumors: subcellular immunohistochemical analysis.''; PubMedEurope PMCScholia
Kaarsholm NC, Ko HC, Dunn MF.; ''Comparison of solution structural flexibility and zinc binding domains for insulin, proinsulin, and miniproinsulin.''; PubMedEurope PMCScholia
Matern HT, Yeaman C, Nelson WJ, Scheller RH.; ''The Sec6/8 complex in mammalian cells: characterization of mammalian Sec3, subunit interactions, and expression of subunits in polarized cells.''; PubMedEurope PMCScholia
Qiao ZS, Min CY, Hua QX, Weiss MA, Feng YM.; ''In vitro refolding of human proinsulin. Kinetic intermediates, putative disulfide-forming pathway folding initiation site, and potential role of C-peptide in folding process.''; PubMedEurope PMCScholia
Huang L, Kirschke CP, Gitschier J.; ''Functional characterization of a novel mammalian zinc transporter, ZnT6.''; PubMedEurope PMCScholia
Tsuboi T, Ravier MA, Xie H, Ewart MA, Gould GW, Baldwin SA, Rutter GA.; ''Mammalian exocyst complex is required for the docking step of insulin vesicle exocytosis.''; PubMedEurope PMCScholia
Min CY, Qiao ZS, Feng YM.; ''Unfolding of human proinsulin. Intermediates and possible role of its C-peptide in folding/unfolding.''; PubMedEurope PMCScholia
Liu M, Ramos-Castañeda J, Arvan P.; ''Role of the connecting peptide in insulin biosynthesis.''; PubMedEurope PMCScholia
Chang SG, Choi KD, Jang SH, Shin HC.; ''Role of disulfide bonds in the structure and activity of human insulin.''; PubMedEurope PMCScholia
Kadima W.; ''Role of metal ions in the T- to R-allosteric transition in the insulin hexamer.''; PubMedEurope PMCScholia
Jackson RS, Creemers JW, Ohagi S, Raffin-Sanson ML, Sanders L, Montague CT, Hutton JC, O'Rahilly S.; ''Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene.''; PubMedEurope PMCScholia
Liu M, Li Y, Cavener D, Arvan P.; ''Proinsulin disulfide maturation and misfolding in the endoplasmic reticulum.''; PubMedEurope PMCScholia
Bailyes EM, Shennan KI, Usac EF, Arden SD, Guest PC, Docherty K, Hutton JC.; ''Differences between the catalytic properties of recombinant human PC2 and endogenous rat PC2.''; PubMedEurope PMCScholia
Chimienti F, Devergnas S, Pattou F, Schuit F, Garcia-Cuenca R, Vandewalle B, Kerr-Conte J, Van Lommel L, Grunwald D, Favier A, Seve M.; ''In vivo expression and functional characterization of the zinc transporter ZnT8 in glucose-induced insulin secretion.''; PubMedEurope PMCScholia
Dodson G, Steiner D.; ''The role of assembly in insulin's biosynthesis.''; PubMedEurope PMCScholia
Poitout V, Hagman D, Stein R, Artner I, Robertson RP, Harmon JS.; ''Regulation of the insulin gene by glucose and fatty acids.''; PubMedEurope PMCScholia
Kambe T, Narita H, Yamaguchi-Iwai Y, Hirose J, Amano T, Sugiura N, Sasaki R, Mori K, Iwanaga T, Nagao M.; ''Cloning and characterization of a novel mammalian zinc transporter, zinc transporter 5, abundantly expressed in pancreatic beta cells.''; PubMedEurope PMCScholia
Dunn MF.; ''Zinc-ligand interactions modulate assembly and stability of the insulin hexamer -- a review.''; PubMedEurope PMCScholia
Orci L, Ravazzola M, Amherdt M, Madsen O, Vassalli JD, Perrelet A.; ''Direct identification of prohormone conversion site in insulin-secreting cells.''; PubMedEurope PMCScholia
Smeekens SP, Montag AG, Thomas G, Albiges-Rizo C, Carroll R, Benig M, Phillips LA, Martin S, Ohagi S, Gardner P.; ''Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3.''; PubMedEurope PMCScholia
Kirschke CP, Huang L.; ''ZnT7, a novel mammalian zinc transporter, accumulates zinc in the Golgi apparatus.''; PubMedEurope PMCScholia
Chimienti F, Favier A, Seve M.; ''ZnT-8, a pancreatic beta-cell-specific zinc transporter.''; PubMedEurope PMCScholia
This CandidateSet contains sequences identified by William Pearson's analysis of Reactome catalyst entities. Catalyst entity sequences were used to identify analagous sequences that shared overall homology and active site homology. Sequences in this Candidate set were identified in an April 24, 2012 analysis.
Proinsulin in proinsulin-zinc-calcium complexes is cleaved by endopeptidases Convertase 1/3 and Convertase 2. The exopeptidase Carboxypeptidase E then removes 2 amino acids from the carboxyl termini. Unlike the proinsulin-zinc calcium complex, the insulin-zinc-calcium complex is not soluble and forms crystals inside the secretory granules.
A beta cell contains about 10 000 secretory granules. Of these, about 1000 are docked at the inner surface of the plasma membrane and a subset of about 100 docked granules form the "readily releasable" pool (granules which are released within about 5 minutes of glucose stimulation). As inferred from rat MIN6 cells, docking occurs by interaction between EXOC3/Sec6 located on the membrane of the secretory granule and EXOC4/Sec8 located at the plasma membrane (Tsuboi et al. 2005). Additional components (EXOC1, EXOC2, EXOC5, EXOC6, EXOC7, EXOC8) form the Exocyst Complex. EXOC7 binds the plasma membrane (Matern et al. 2001).
Cystine bonds are formed in Proinsulin-1 between cysteine residues 31 and 96, cysteine residues 43 and 109, and cysteine residues 95 and 100. Ero1-like alpha does not directly catalyze the oxidation of cysteines to cystine. Instead it maintains a suitably oxidizing environment for the reactions to occur . Though Ero1-like alpha can act via specific isomerases such as P4HB/PDI, there is currently no evidence that formation of cystine bonds in insulin requires a specific isomerase. Interestingly, even in beta cells of wild type animals, trace amounts of incorrectly bonded proinsulin can be detected. Thus, the formation of correct cystine bonds may involve a period of bond shuffling.
Insulin-containing secretory vesicles are translocated along microtubules (polymerized tubulin) from the trans-golgi to the cellular cortex. Motor activity is provided by Dynamin-1 but the complex that connects the secretory granule to the Kinesin-1 is not yet fully known. The process is stimulated by intracellular calcium ions (Ca2+).
In the presence of high concentrations of zinc and calcium, proinsulin spontaneously forms soluble complexes containing 6 molecules of proinsulin, 2 zinc ions, and 1 calcium ion. Zinc Transporters ZnT5, ZnT6, and ZnT7 are found in the membrane of the Golgi in pancreatic cells (and also in many other cell types). They play a role in maintaining the high zinc concentration in the Golgi lumen and thus catalyze the formation of the proinsulin-zinc-calcium complex. Other transporters, such as the newly identified ZnT9 and ZnT10, may also be involved but this is presently unknown.
Insulin-containing secretory granules are bound to Myosin Va via Rab27a in a complex of uncertain composition. Myosin Va moves along the cortical actin network (actin at the periphery of the cytoplasm), carrying the granules to the inner surface of the plasma membrane. A beta cell contains about 10 000 secretory granules. Of these, about 1000 are docked at the inner surface of the plasma membrane and a subset of about 100 docked granules form the "readily releasable" pool (granules which are released within about 5 minutes of glucose stimulation). Docking occurs by interaction between EXOC3/Sec6 located on the membrane of the secretory granule and EXOC4/Sec8 located at the plasma membrane. Additional components (EXOC1, EXOC2, EXOC5, EXOC6, EXOC7, EXOC8) form the Exocyst Complex.
Transcription of the human insulin gene INS is activated by 4 important transcription factors: Pdx-1, MafA, Beta2/NeuroD1, and E47. The transcription factors interact with each other at the promoters of the insulin gene and act synergistically to promote transcription. Expression of the transcription factors is upregulated in response to glucose.
The preproinsulin mRNA is translated by ribosomes at the rough endoplasmic reticulum (ER) and the preproinsulin enters the secretion pathway by virtue of its signal peptide, which is cleaved during translation to yield proinsulin. Evidence indicates that the preproinsulin mRNA is stabilized by glucose.
In the process annotated in detail here, within the ER, three intramolecular disulfide bonds form between cysteine residues in the proinsulin. Formation of the bonds is the spontaneous result of the conformation of proinsulin and the oxidizing environment of the ER, which is maintained by Ero1-like alpha
The cystine bonded proinsulin then moves via vesicles from the ER to the Golgi Complex. High concentrations of zinc are maintained in the Golgi by zinc transporters ZnT5, ZnT6, and ZnT7 and the proinsulin forms complexes with zinc and calcium.
Proinsulin-zinc-calcium complexes bud in vesicles from the trans-Golgi to form immature secretory vesicles (secretory granules) in the cytosol. Within the immature granules the endoproteases Prohormone Convertase 1/3 and Prohormone Convertase 2 cleave at two sites of the proinsulin and Carboxypeptidase E removes a further 4 amino acid residues to yield the cystine-bonded A and B chains of mature insulin and the C peptide, which will also be secreted with the insulin. The insulin-zinc-calcium complexes form insoluble crystals within the granule
The insulin-containing secretory granules are then translocated across the cytosol to the inner surface of the plasma membrane. Translocation occurs initially by attachment of the granules to Kinesin-1, which motors along microtubules, and then by attachment to Myosin Va, which motors along the microfilaments of the cortical actin network.
A pancreatic beta cell contains about 10000 insulin granules of which about 1000 are docked at the plasma membrane and 50 are readily releasable in immediate response to stimulation by glucose or other secretogogues. Docking is due to interaction between the Exocyst proteins EXOC3 on the granule membrane and EXOC4 on the plasma membrane. Exocytosis is accomplished by interaction between SNARE-type proteins Syntaxin 1A and Syntaxin 4 on the plasma membrane and Synaptobrevin-2/VAMP2 on the granule membrane. Exocytosis is a calcium-dependent process due to interaction of the calcium-binding membrane protein Synaptotagmin V/IX with the SNARE-type proteins.
Original Pathway at Reactome: http://www.reactome.org/PathwayBrowser/#DB=gk_current&FOCUS_SPECIES_ID=48887&FOCUS_PATHWAY_ID=264876
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Zinc
Calcium Complex in docked granuleAnnotated Interactions