- Please check and comment entries here.
Identifying Insulin Granule Proteins
Only four studies have attempted to investigate ISG proteins by proteomic analysis to date. These studies employ various combinations of density gradient centrifugations, in silico analyses, and immunoprecipitation techniques . As a result, Li and colleagues identified 81 total ISG proteins from the INS-1 rat beta-cell line, while Schvartz et al. identified 140 ISG proteins, Hickey et al. identified 51 ISG proteins, and Brunner et al. identified 130 ISG proteins from the INS-1E rat beta-cell line. Proteomic data obtained from these four studies on ISG proteins from INS-1 or INS1-E cells produced a total of 5 proteins that were consistently identified. These were: Insulin-1 (Ins1), Insulin-2 (Ins2), Carboxypeptidase E (CPE), Chromogranin-A (CgA) and Prohormone convertase 2 (PC2). Rat beta-cells synthesize two different forms of insulin encoded by the Ins1 and Ins2 gene that share 90% homology [66,67], hence two insulin forms found in these proteomes. Though different isolation techniques would influence the proteins identified, one would expect that using similar cell lines would result in more than a handful of proteins consistently identified across all four studies.
1. Intravesicular Proteins
The most consistently identified intravesicular proteins in the proteomic studies were the previously well-characterised ISG proteins insulin (Ins1 and Ins2), CPE, PC2 and CgA . Discovery of proinsulin processing of labelled insulin  and CgA  have allowed subsequent studies to identify localization of PC1/3 , PC2  and CPE  as ISG localized enzymes. While all proteomes identified PC2 and CPE, PC1/3 was discovered only in two studies . Other intravesicular proteins identified were from the chromogranin-secretogranin protein family. CgA in particular was identified in all four studies, with full-length CgA believed to be important for the biogenesis of granules in beta-cells . Interestingly, CgA knockout mice display a reduced islet number, beta-cell to alpha-cell ratio and plasma insulin levels ; however, they exhibit normal blood glucose levels, as a result of compensation from other granin proteins . CgB has been suggested to not be specifically involved in granule formation but instead is essential in the secretion of insulin and other islet hormones such as somatostatin and glucagon . However, through pulse-chase labelling of CgB, Bearrows et al. show that in the absence of CgB, there is a delay in proinsulin trafficking from the TGN followed by a reduction in nascent ISGs at the plasma membrane . CgB was identified in three of the four ISG proteomes (all but Li et al.). Significantly, aside from the full-length granins, PC1/3 and PC2 also cleave granins to form active peptides . Beta-granin is an example of a CgA derived peptide identified by Li et al. and is proposed to inhibit insulin secretion through unknown mechanisms . This emphasises technical challenges in peptide identification in proteomics analysis, to differentiate the presence and eventual function of both granins and their derived peptides in future studies.
Hydrolases were found in two of the proteomics analyses . Cathepsins B and L were identified by Brunner et al. and are most intriguing as these proteins have been previously shown by electron microscopy to localise in immature ISGs, while cathepsin L alone remains in mature ISGs . While some hydrolases have previously been described within ISGs , other hydrolases present in proteomic analysis may be appearing due to crinophagy processes of ISGs with lysosomes . As such, further validation of hydrolase proteins will be essential to help elucidate their role in ISG biogenesis and processing. Particularly, the validation of cathepsins present in immature and mature ISGs demonstrates that these enzymes may follow sorting mechanisms out of immature ISGs via the mannose 6-phosphate receptor . This adds weight to the ‘sorting by retention’ and ‘sorting by exit’ hypotheses in ISGs, in which immature ISGs may target proteins either for retention in maturing granules or exit towards the lysosome .
2. Membrane Proteins
A substantial proportion of ISG proteins identified by the proteomic analyses were membrane-bound or membrane-associated proteins. Of this group, the most commonly identified were synaptobrevin proteins (VAMPs), including Vamp3 , Vamp7 and Vamp8 . VAMPs interact with their cognate t-SNAREs and other proteins that mediate the fusion of vesicles to the target membrane , which in turn interact with a variety of presynaptic proteins and q-SNAREs to form the complete SNARE complex . Vamp2 was first described as an ISG localised v-SNARE protein  by cDNA cloning and confocal microscopy. Brunner et al. then identified Vamp2 in their proteomics analysis and following this, Hickey et al. used Vamp2 antibodies to immuno-purify ISGs. Surprisingly, Hickey et al. and Li et al. do not identify Vamp2 in their proteomes, with Hickey et al. suggesting that it and many other docking proteins potentially remained on the immunoaffinity beads . If these membranal proteins were left unidentified, this may explain why fewer proteins (51) were identified in comparison to other proteomes.
Rab proteins were also found to be enriched with ISG fractions. Rab proteins are a family of GTPases from the Ras superfamily  that modulate several stages of vesicle trafficking and fusion of ISGs with the plasma membrane . Through proteomic analysis and colocalisation imaging, Brunner’s study illustrated that both VAMP8 and Rab37 are novel ISG associated proteins that colocalise with ISGs of INS1-E cells . Previous to this, only 30 proteins were described as ISG associated proteins in beta-cells  and information surrounding the trafficking of ISGs was limited. Their proteomic analyses and validation of novel proteins suggested a more complex trafficking process than previously established in beta-cells. Other SNARE complex proteins present in the proteomes include syntaxin5 and 12, (Stx5, Stx12)  and granuphilin . However, these proteins are believed to be localised to the plasma membrane  and not on ISG membranes, suggesting that they were present in contaminant co-purification with ISG fractions.
Many ATPase subunits were commonly identified in the four proteomic analyses, most notably the vacuolar-H+ ATPases (V-type). These V-type ATPases have been previously shown to be localized to ISGs in beta-cells , and are important in producing and maintaining a proton gradient by acidifying the granule . This facilitates the maturation of ISGs  as well as maintaining a suitable pH for intravesicular enzymes . Many other subunits of ATPases identified are lysosomal isoforms and should be validated as to whether they are genuine ISG proteins or proteins co-purified with ISGs.
3. Other Proteins
The remaining proteins identified with non-specific or unknown localization in ISGs are often grouped in these studies. These include cytoskeletal, cytoplasmic and organelle localized proteins. The cytoplasmic proteins identified range from mis-folding chaperones  and isomerases (PDIA3)  to N-ethylmaleimide sensitive fusion protein . Whether these proteins are genuinely ISG-associated, or technical contaminants, requires further validation. Different cytoskeleton-associated proteins are found across all four proteomes. Alpha-centractin , alpha and beta-actin  and kinesin subunits  are some examples of cytoskeletal associated proteins identified. ISGs are transported along microtubules by kinesins  and cytoskeleton remodelling is critical for ISG trafficking during glucose-stimulated insulin secretion . The presence of these proteins is therefore unsurprising, though are likely present due to co-purification of these proteins through the isolation of ISGs. Indeed, the presence of proteins localized to the ER, Golgi, mitochondria and lysosomes are also commonly observed across all four studies. Examples include Erp44 (ER), Glg1 (Golgi), SHMT (mitochondria) and Lamp1 (lysosomes) . It is difficult to prevent the copurification of these proteins using present isolation techniques and their co-localisations with ISGs need further validation.
The presence of isomerases and proteins involved in protein folding is quite surprising. Hickey et al. in particular find a striking number of chaperone proteins (~20% of proteins identified) . Recent studies have shown that ER chaperone proteins are vital in proinsulin handling and insulin-like growth factor folding ; however, none of these ER-resident proteins have been shown to be localized in ISGs. Interestingly, Stanniocalcin-1 (STC1) or its precursors were found in three of the four proteomes (Li, Schvartz, Brunner). STC1 is found in many tissue types such as muscle, kidney, adrenal and lung . Human STC1 protein is described as an uncoupler of oxidative phosphorylation in mitochondria , and has been implicated in apoptotic mechanisms and carcinogenesis . Its function in beta-cells is not well understood, however; immunocytochemistry, and in situ ligand binding and hybridization  show that STC1 colocalizes with insulin in mouse pancreatic beta-cells. The abundance of these chaperones, alongside identification of proteins such as STC1, illustrates the importance of ISG proteomics as a rich source of data to potentially identify novel ISG proteins that may modulate different processes of ISG biogenesis, trafficking, and secretion. Altogether, these studies highlight the importance of developing improved purification techniques that restrict isolation of ISGs to granules post-sorting and packaging from the TGN, and before degradation.
This entry is adapted from 10.3390/metabo11050288
- Orci, L.; Ravazzola, M.; Amherdt, M.; Madsen, O.; Vassalli, J.-D.; Perrelet, A. Direct identification of prohormone conversion site in insulin-secreting cells. Cell 1985, 42, 671–681.
- Rindler, M.J. Carboxypeptidase E, a peripheral membrane protein implicated in the targeting of hormones to secretory granules, co-aggregates with granule content proteins at acidic pH. J. Biol. Chem. 1998, 273, 31180–31185.
- Hendy, G.N.; Li, T.; Girard, M.; Feldstein, R.C.; Mulay, S.; Desjardins, R.; Day, R.; Karaplis, A.C.; Tremblay, M.L.; Canaff, L. Targeted ablation of the chromogranin a (Chga) gene: Normal neuroendocrine dense-core secretory granules and increased expression of other granins. Mol. Endocrinol. 2006, 20, 1935–1947.
- Hoflehner, J.; Eder, U.; Laslop, A.; Seidah, N.G.; Fischer-Colbrie, R.; Winkler, H. Processing of secretogranin II by prohormone convertases: Importance ofPC1 in generation of secretoneurin. FEBS Lett. 1995, 360, 294–298.
- Laslop, A.; Weiss, C.; Savaria, D.; Eiter, C.; Tooze, S.A.; Seidah, N.G.; Winkler, H. Proteolytic processing of chromogranin B and secretogranin II by prohormone convertases. J. Neurochem. 1998, 70, 374–383.
- Davidson, H.W.; Peshavaria, M.; Hutton, J.C. Proteolytic conversion of proinsulin into insulin. Identification of a Ca2+-dependent acidic endopeptidase in isolated insulin-secretory granules. Biochem. J. 1987, 246, 279–286.
- Hutton, J.C.; Davidson, H.W.; Peshavaria, M. Proteolytic processing of chromogranin A in purified insulin granules. Formation of a 20 kDa N-terminal fragment (betagranin) by the concerted action of a Ca2+-dependent endopeptidase and carboxypeptidase H (EC 3.4. 17.10). Biochem. J. 1987, 244, 457–464.
- Muller, L.; Lindberg, I. The cell biology of the prohormone convertases PCI and PC2. Prog. Nucleic Acid Res. Mol. Biol. 1999, 63, 69–108.
- Bennett, D.L.; Bailyes, E.; Nielsen, E.; Guest, P.; Rutherford, N.; Arden, S.; Hutton, J. Identification of the type 2 proinsulin processing endopeptidase as PC2, a member of the eukaryote subtilisin family. J. Biol. Chem. 1992, 267, 15229–15236.
- Guest, P.C.; Ravazzola, M.; Davidson, H.W.; Orci, L.; Hutton, J.C. Molecular heterogeneity and cellular localization of carboxypeptidase H in the islets of Langerhans. Endocrinology 1991, 129, 734–740.
- Li, M.; Du, W.; Zhou, M.; Zheng, L.; Song, E.; Hou, J. Proteomic analysis of insulin secretory granules in INS-1 cells by protein correlation profiling. Biophys. Rep. 2018, 4, 329–338.
- Schvartz, D.; Brunner, Y.; Coute, Y.; Foti, M.; Wollheim, C.B.; Sanchez, J.C. Improved characterization of the insulin secretory granule proteomes. J. Proteom. 2012, 75, 4620–4631.
- Kim, T.; Tao-Cheng, J.-H.; Eiden, L.E.; Loh, Y.P. Chromogranin A, an “on/off” switch controlling dense-core secretory granule biogenesis. Cell 2001, 106, 499–509.
- Portela-Gomes, G.; Gayen, J.; Grimelius, L.; Stridsberg, M.; Mahata, S. The importance of chromogranin A in the development and function of endocrine pancreas. Regul. Pept. 2008, 151, 19–25.
- Obermüller, S.; Calegari, F.; King, A.; Lindqvist, A.; Lundquist, I.; Salehi, A.; Francolini, M.; Rosa, P.; Rorsman, P.; Huttner, W.B. Defective secretion of islet hormones in chromogranin-B deficient mice. PLoS ONE 2010, 5, e8936.
- Bearrows, S.C.; Bauchle, C.J.; Becker, M.; Haldeman, J.M.; Swaminathan, S.; Stephens, S.B. Chromogranin B regulates early-stage insulin granule trafficking from the Golgi in pancreatic islet beta-cells. J. Cell Sci. 2019, 132, jcs231373.
- Udupi, V.; Lee, H.-M.; Kurosky, A.; Greeley, G.H., Jr. Prohormone convertase-1 is essential for conversion of chromogranin A to pancreastatin. Regul. Pept. 1999, 83, 123–127.
- Schmid, G.M.; Meda, P.; Caille, D.; Wargent, E.; O’Dowd, J.; Hochstrasser, D.F.; Cawthorne, M.A.; Sanchez, J.-C. Inhibition of insulin secretion by betagranin, an N-terminal chromogranin A fragment. J. Biol. Chem. 2007, 282, 12717–12724.
- Hickey, A.J.; Bradley, J.W.; Skea, G.L.; Middleditch, M.J.; Buchanan, C.M.; Phillips, A.R.; Cooper, G.J. Proteins associated with immunopurified granules from a model pancreatic islet beta-cell system: Proteomic snapshot of an endocrine secretory granule. J. Proteome Res. 2009, 8, 178–186.
- Brunner, Y.; Coute, Y.; Iezzi, M.; Foti, M.; Fukuda, M.; Hochstrasser, D.F.; Wollheim, C.B.; Sanchez, J.C. Proteomics analysis of insulin secretory granules. Mol. Cell. Proteom. 2007, 6, 1007–1017.
- Kuliawat, R.; Klumperman, J.; Ludwig, T.; Arvan, P. Differential sorting of lysosomal enzymes out of the regulated secretory pathway in pancreatic beta-cells. J. Cell Biol. 1997, 137, 595–608.
- Hutton, J.C. Insulin secretory granule biogenesis and the proinsulin-processing endopeptidases. Diabetologia 1994, 37, S48–S56.
- Davidson, H.W.; Hutton, J.C. The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing. Biochem. J. 1987, 245, 575–582.
- Sandberg, M.; Borg, L.H. Intracellular degradation of insulin and crinophagy are maintained by nitric oxide and cyclo-oxygenase 2 activity in isolated pancreatic islets. Biol. Cell 2006, 98, 307–315.
- Klumperman, J.; Kuliawat, R.; Griffith, J.M.; Geuze, H.J.; Arvan, P. Mannose 6–phosphate receptors are sorted from immature secretory granules via adaptor protein AP-1, clathrin, and syntaxin 6–positive vesicles. J. Cell Biol. 1998, 141, 359–371.
- Arvan, P.; Castle, D. Sorting and storage during secretory granule biogenesis: Looking backward and looking forward. Biochem. J. 1998, 332, 593–610.
- Lin, R.C.; Scheller, R.H. Mechanisms of synaptic vesicle exocytosis. Annu. Rev. Cell Dev. Biol. 2000, 16, 19–49.
- Lam, P.P.; Ohno, M.; Dolai, S.; He, Y.; Qin, T.; Liang, T.; Zhu, D.; Kang, Y.; Liu, Y.; Kauppi, M. Munc18b is a major mediator of insulin exocytosis in rat pancreatic β-cells. Diabetes 2013, 62, 2416–2428.
- Iorio, V.; Festa, M.; Rosati, A.; Hahne, M.; Tiberti, C.; Capunzo, M.; De Laurenzi, V.; Turco, M. BAG3 regulates formation of the SNARE complex and insulin secretion. Cell Death Dis. 2015, 6, e1684.
- Gaisano, H.Y. Recent new insights into the role of SNARE and associated proteins in insulin granule exocytosis. Diabetes Obes. Metab. 2017, 19 (Suppl. 1), 115–123.
- Qin, T.; Liang, T.; Zhu, D.; Kang, Y.; Xie, L.; Dolai, S.; Sugita, S.; Takahashi, N.; Ostenson, C.-G.; Banks, K. Munc18b increases insulin granule fusion, restoring deficient insulin secretion in type-2 diabetes human and Goto-Kakizaki rat islets with improvement in glucose homeostasis. EBioMedicine 2017, 16, 262–274.
- Regazzi, R.; Wollheim, C.; Lang, J.; Theler, J.; Rossetto, O.; Montecucco, C.; Sadoul, K.; Weller, U.; Palmer, M.; Thorens, B. VAMP-2 and cellubrevin are expressed in pancreatic beta-cells and are essential for Ca(2+)-but not for GTP gamma S-induced insulin secretion. EMBO J. 1995, 14, 2723–2730.
- Martinez, O.; Goud, B. Rab proteins. Biochim. Biophys. Acta (BBA) Mol. Cell Res. 1998, 1404, 101–112.
- Cheviet, S.V.; Coppola, T.; Haynes, L.P.; Burgoyne, R.D.; Regazzi, R. The Rab-binding protein Noc2 is associated with insulin-containing secretory granules and is essential for pancreatic β-cell exocytosis. Mol. Endocrinol. 2004, 18, 117–126.
- Wang, Z.; Thurmond, D.C. Mechanisms of biphasic insulin-granule exocytosis–roles of the cytoskeleton, small GTPases and SNARE proteins. J. Cell Sci. 2009, 122, 893–903.
- Mizuno, K.; Fujita, T.; Gomi, H.; Izumi, T. Granuphilin exclusively mediates functional granule docking to the plasma membrane. Sci. Rep. 2016, 6, 1–12.
- Hatanaka, M.; Tanabe, K.; Yanai, A.; Ohta, Y.; Kondo, M.; Akiyama, M.; Shinoda, K.; Oka, Y.; Tanizawa, Y. Wolfram syndrome 1 gene (WFS1) product localizes to secretory granules and determines granule acidification in pancreatic β-cells. Hum. Mol. Genet. 2011, 20, 1274–1284.
- Barg, S.; Huang, P.; Eliasson, L.; Nelson, D.J.; Obermüller, S.; Rorsman, P.; Thévenod, F.; Renström, E. Priming of insulin granules for exocytosis by granular Cl− uptake and acidification. J. Cell Sci. 2001, 114, 2145–2154.
- Gharanei, S.; Zatyka, M.; Astuti, D.; Fenton, J.; Sik, A.; Nagy, Z.; Barrett, T.G. Vacuolar-type H+-ATPase V1A subunit is a molecular partner of Wolfram syndrome 1 (WFS1) protein, which regulates its expression and stability. Hum. Mol. Genet. 2013, 22, 203–217.
- Orci, L.; Ravazzola, M.; Storch, M.-J.; Anderson, R.; Vassalli, J.-D.; Perrelet, A. Proteolytic maturation of insulin is a post-Golgi event which occurs in acidifying clathrin-coated secretory vesicles. Cell 1987, 49, 865–868.
- Orci, L.; Ravazzola, M.; Amherdt, M.; Madsen, O.; Perrelet, A.; Vassalli, J.D.; Anderson, R.G. Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles. J. Cell Biol. 1986, 103, 2273–2281.
- Heaslip, A.T.; Nelson, S.R.; Lombardo, A.T.; Previs, S.B.; Armstrong, J.; Warshaw, D.M. Cytoskeletal dependence of insulin granule movement dynamics in INS-1 beta-cells in response to glucose. PLoS ONE 2014, 9, e109082.
- Wang, B.; Lin, H.; Li, X.; Lu, W.; Kim, J.B.; Xu, A.; Cheng, K.K. The adaptor protein APPL2 controls glucose-stimulated insulin secretion via F-actin remodeling in pancreatic β-cells. Proc. Natl. Acad. Sci. USA 2020, 117, 28307–28315.
- Ghiasi, S.M.; Dahlby, T.; Andersen, C.H.; Haataja, L.; Petersen, S.; Omar-Hmeadi, M.; Yang, M.; Pihl, C.; Bresson, S.E.; Khilji, M.S. Endoplasmic reticulum chaperone glucose-regulated protein 94 is essential for proinsulin handling. Diabetes 2019, 68, 747–760.
- Varghese, R.; Wong, C.K.; Deol, H.; Wagner, G.F.; DiMattia, G.E. Comparative analysis of mammalian stanniocalcin genes. Endocrinology 1998, 139, 4714–4725.
- Ellard, J.P.; McCudden, C.R.; Tanega, C.; James, K.A.; Ratkovic, S.; Staples, J.F.; Wagner, G.F. The respiratory effects of stanniocalcin-1 (STC-1) on intact mitochondria and cells: STC-1 uncouples oxidative phosphorylation and its actions are modulated by nucleotide triphosphates. Mol. Cell. Endocrinol. 2007, 264, 90–101.
- Iversen, P.; Sorensen, D.; Benestad, H. Inhibitors of angiogenesis selectively reduce the malignant cell load in rodent models of human myeloid leukemias. Leukemia 2002, 16, 376–381.
- Zaidi, D.; Turner, J.K.; Durst, M.A.; Wagner, G.F. Stanniocalcin-1 co-localizes with insulin in the pancreatic islets. Int. Sch. Res. Not. 2012, 2012, 834359.