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Isolation and Proteomics of the Insulin Secretory Granule

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Isolation and Proteomics of the Insulin Secretory Granule H OH metabolites OH Review Isolation and Proteomics of the Insulin Secretory Granule Nicholas Norris , Belinda Yau * and Melkam Alamerew Kebede Charles Perkins Centre, School of Medical Sciences, University of Sydney, Camperdown, NSW 2006, Australia; [email protected] (N.N.); [email protected] (M.A.K.) * Correspondence: [email protected] Abstract: Insulin, a vital hormone for glucose homeostasis is produced by pancreatic beta-cells and when secreted, stimulates the uptake and storage of glucose from the blood. In the pancreas, insulin is stored in vesicles termed insulin secretory granules (ISGs). In Type 2 diabetes (T2D), defects in insulin action results in peripheral insulin resistance and beta-cell compensation, ultimately leading to dysfunctional ISG production and secretion. ISGs are functionally dynamic and many proteins present either on the membrane or in the lumen of the ISG may modulate and affect different stages of ISG trafficking and secretion. Previously, studies have identified few ISG proteins and more recently, proteomics analyses of purified ISGs have uncovered potential novel ISG proteins. This review summarizes the proteins identified in the current ISG proteomes from rat insulinoma INS-1 and INS-1E cell lines. Here, we also discuss techniques of ISG isolation and purification, its challenges and potential future directions. Keywords: insulin secretory granule; beta-cells; granule protein purification 1. Insulin Granule Biogenesis and Function Citation: Norris, N.; Yau, B.; Kebede, The insulin secretory granule (ISG) is the storage vesicle for insulin in pancreatic M.A. Isolation and Proteomics of the beta-cells. It was long treated as an inert carrier for insulin but is now appreciated as a Insulin Secretory Granule. Metabolites regulatory structure all on its own. There is a continuous turnover of insulin granules in 2021, 11, 288. https://doi.org/ the beta-cell, which is highly specialised in its capacity for ISG biogenesis, and insulin 10.3390/metabo11050288 represents the most abundant protein within the beta-cell at 5–10% of total cell protein mass [1]. Production of insulin first begins in the rough endoplasmic reticulum with the Academic Editor: Amedeo Lonardo synthesis of preproinsulin [2]. The signal peptide of preproinsulin is cleaved to form proinsulin, which is folded and trafficked to the Golgi complex [3]. Here, proinsulin is Received: 12 April 2021 packaged with other proteins destined for secretion into a budding immature ISG at the Accepted: 28 April 2021 trans-Golgi network via a mechanism termed ‘sorting by entry’[4]. Following the release Published: 30 April 2021 of these granules from the trans-Golgi network, maturation of the immature ISG includes acidification of the granule lumen by ATP-dependent proton pumps and promotion of Publisher’s Note: MDPI stays neutral endoprotease convertases (PC1/3 and PC2) activity that cleave proinsulin to form free with regard to jurisdictional claims in C-peptide and mature insulin, comprised of the A and B chains bound together by two published maps and institutional affil- inter-chain disulfide bonds [5–7]. Through a secondary mechanism called ‘sorting by iations. retention’, proinsulin and other proteins are retained in the immature ISG (Davidson et al., 1988), while in parallel, proteins such as clathrin are removed from the immature ISG via ‘sorting by exit’[8,9]. Finally, insulin crystallises with zinc cations (Zn2+), assembling an ~300 nm dense-core mature ISG [10]. From its point of synthesis, proinsulin enters an ISG Copyright: © 2021 by the authors. within 4 hours [5] and is processed into insulin in a mature ISG within 40 minutes [9]. Licensee MDPI, Basel, Switzerland. The ISG has a half-life of 3–5 days within the beta-cell cytoplasm [11] (Figure1), and This article is an open access article are ultimately destined for secretion or degradation. Upon glucose stimulation, ISG are distributed under the terms and motivated to undergo exocytosis, which requires the coordination of cellular machinery conditions of the Creative Commons Attribution (CC BY) license (https:// present both on ISGs and at the plasma membrane. It is therefore likely that ISG com- creativecommons.org/licenses/by/ position contributes to exocytosis, though the variables that determine whether an ISG 4.0/). eventually undergoes secretion are still unclear. Only 1–2% of total ISG content is released Metabolites 2021, 11, 288. https://doi.org/10.3390/metabo11050288 https://www.mdpi.com/journal/metabolites Metabolites 2021, 11, x FOR PEER REVIEW 2 of 16 sition contributes to exocytosis, though the variables that determine whether an ISG even- tually undergoes secretion are still unclear. Only 1-2% of total ISG content is released upon a single glucose stimulation [12]. Plasma membrane proximity [12,13] and docking [14,15] have long been suggested to contribute to an ISG’s secretory capacity. More recently, ISG motility [16] and age [16–18] have also been shown to significantly contribute to an ISG’s propensity for translocation to the plasma membrane and its necessity for docking [16,18]. Finally, an ISG’s fusion capacity–whether the ISG collapses or is recycled–may also be intrinsically regulated [19]. ISGs that are not secreted are targeted to the lysosome for degradation, either through autophagosome-dependent or independent pathways [20]. As insulin accounts for a large proportion of protein synthesis in pancreatic beta-cells [21], ISG homeostasis is essential to maintaining beta-cell function [22]. In autophagosome-dependent degrada- tion, ISGs are engulfed by autophagosomes and subsequently fuse with the lysosomes, degrading ISG contents [22,23]. Autophagosome-independent degradation involves the fusion of ISGs with the lysosomes directly (crinophagy) [24]. Apart from whole ISG deg- Metabolites 2021, 11, 288 2 of 16 radation, many proteases involved may also directly influence insulin turnover. For ex- ample, insulin has been shown to be degraded by insulin-degrading enzyme (IDE) in beta- cells and deletion or inhibition of this enzyme perturbs insulin secretion in beta-cells upon[25,26]. a single glucose stimulation [12]. Plasma membrane proximity [12,13] and dock- ing [14It, 15is ]now have appreciated long been that suggested all these to processe contributes are to not an ISG’sonly externally secretory regulated capacity. Moreby the recently,ISG environment, ISG motility and [16 proteins] and age both [16 –in18 and] have on alsoISG beencan modulate shown to both significantly the processing contribute and totrafficking an ISG’s propensityof ISGs, ultimately for translocation controlling to the granule plasma mobility, membrane secretion and its capacity, necessity and for degra- dock- ingdation. [16,18 Our]. Finally, current an review ISG’s focuses fusion capacity–whether on the continuing the pursuit ISG collapses to characterise or is recycled–may ISG-localised alsoproteins be intrinsically from pancreatic regulated beta-cells. [19]. Figure 1. The insulin secretory pathway. (A) Proinsulin is synthesised and folded at the endoplasmic reticulum, trafficked to the trans-Golgi network and sorted into budding immature insulin secretory granules. (B) Immature insulin secretory granules undergo maturation where proinsulin is cleaved into mature insulin and condenses with zinc to form the dense core within the mature insulin secretory granule. (C) Aged mature insulin secretory granules that do not undergo secretion are trafficked for degradation within lysosomes or autophagosomes. ISGs that are not secreted are targeted to the lysosome for degradation, either through autophagosome-dependent or independent pathways [20]. As insulin accounts for a large proportion of protein synthesis in pancreatic beta-cells [21], ISG homeostasis is essential to maintaining beta-cell function [22]. In autophagosome-dependent degradation, ISGs are engulfed by autophagosomes and subsequently fuse with the lysosomes, degrading ISG contents [22,23]. Autophagosome-independent degradation involves the fusion of ISGs with the lysosomes directly (crinophagy) [24]. Apart from whole ISG degradation, many proteases involved may also directly influence insulin turnover. For example, insulin has been shown to be degraded by insulin-degrading enzyme (IDE) in beta-cells and deletion or inhibition of this enzyme perturbs insulin secretion in beta-cells [25,26]. It is now appreciated that all these processes are not only externally regulated by the ISG environment, and proteins both in and on ISG can modulate both the processing and trafficking of ISGs, ultimately controlling granule mobility, secretion capacity, and Metabolites 2021, 11, x FOR PEER REVIEW 3 of 16 Figure 1. The insulin secretory pathway. (A) Proinsulin is synthesised and folded at the endoplasmic reticulum, trafficked Metabolites 2021, 11, 288 3 of 16 to the trans-Golgi network and sorted into budding immature insulin secretory granules. (B) Immature insulin secretory granules undergo maturation where proinsulin is cleaved into mature insulin and condenses with zinc to form the dense core within the mature insulin secretory granule. (C) Aged mature insulin secretory granules that do not undergo secretion are trafficked for degradationdegradation. within lysosomes Our currentor autophagosomes. review focuses on the continuing pursuit to characterise ISG- localised proteins from pancreatic beta-cells. The ISG is key to beta-cell identity. Pathological dysfunction related to insulin occurs The ISG is key to beta-cell
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