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      Uncoupling Proteostasis and Development in Vitro with a Small Molecule Inhibitor of the Pancreatic Endoplasmic Reticulum Kinase, PERK*

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          Abstract

          Background: PERK controls unfolded protein load in the ER and promotes a latent gene expression program whose relative contributions to cell physiology are incompletely understood.

          Results: Acute PERK inhibition deregulates protein synthesis and promotes accumulation of misfolded pro-insulin.

          Conclusion: PERK contributes to proteostasis acutely.

          Significance: The proteostatic activity of PERK can be uncoupled from its latent role in gene expression.

          Abstract

          Loss-of-function mutations in EIF2AK3, encoding the pancreatic endoplasmic reticulum (ER) kinase, PERK, are associated with dysfunction of the endocrine pancreas and diabetes. However, to date it has not been possible to uncouple the long term developmental effects of PERK deficiency from sensitization to physiological levels of ER unfolded protein stress upon interruption of PERK modulation of protein synthesis rates. Here, we report that a selective PERK inhibitor acutely deregulates protein synthesis in freshly isolated islets of Langerhans, across a range of glucose concentrations. Acute loss of the PERK-mediated strand of the unfolded protein response leads to rapid accumulation of misfolded pro-insulin in cultured beta cells and is associated with a kinetic defect in pro-insulin processing. These in vitro observations uncouple the latent role of PERK in beta cell development from the regulation of unfolded protein flux through the ER and attest to the importance of the latter in beta cell proteostasis.

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          Diabetes mellitus and exocrine pancreatic dysfunction in perk-/- mice reveals a role for translational control in secretory cell survival.

          The protein kinase PERK couples protein folding in the endoplasmic reticulum (ER) to polypeptide biosynthesis by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha), attenuating translation initiation in response to ER stress. PERK is highly expressed in mouse pancreas, an organ active in protein secretion. Under physiological conditions, PERK was partially activated, accounting for much of the phosphorylated eIF2alpha in the pancreas. The exocrine and endocrine pancreas developed normally in Perk-/- mice. Postnatally, ER distention and activation of the ER stress transducer IRE1alpha accompanied increased cell death and led to progressive diabetes mellitus and exocrine pancreatic insufficiency. These findings suggest a special role for translational control in protecting secretory cells from ER stress.
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            Endoplasmic reticulum stress signaling in disease.

            The extracellular space is an environment hostile to unmodified polypeptides. For this reason, many eukaryotic proteins destined for exposure to this environment through secretion or display at the cell surface require maturation steps within a specialized organelle, the endoplasmic reticulum (ER). A complex homeostatic mechanism, known as the unfolded protein response (UPR), has evolved to link the load of newly synthesized proteins with the capacity of the ER to mature them. It has become apparent that dysfunction of the UPR plays an important role in some human diseases, especially those involving tissues dedicated to extracellular protein synthesis. Diabetes mellitus is an example of such a disease, since the demands for constantly varying levels of insulin synthesis make pancreatic beta-cells dependent on efficient UPR signaling. Furthermore, recent discoveries in this field indicate that the importance of the UPR in diabetes is not restricted to the beta-cell but is also involved in peripheral insulin resistance. This review addresses aspects of the UPR currently understood to be involved in human disease, including their role in diabetes mellitus, atherosclerosis, and neoplasia.
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              The PERK Eukaryotic Initiation Factor 2α Kinase Is Required for the Development of the Skeletal System, Postnatal Growth, and the Function and Viability of the Pancreas

              Phosphorylation of eukaryotic initiation factor 2α (eIF-2α) is typically associated with stress responses and causes a reduction in protein synthesis. However, we found high phosphorylated eIF-2α (eIF-2α[P]) levels in nonstressed pancreata of mice. Administration of glucose stimulated a rapid dephosphorylation of eIF-2α. Among the four eIF-2α kinases present in mammals, PERK is most highly expressed in the pancreas, suggesting that it may be responsible for the high eIF-2α[P] levels found therein. We describe a Perk knockout mutation in mice. Pancreata of Perk −/− mice are morphologically and functionally normal at birth, but the islets of Langerhans progressively degenerate, resulting in loss of insulin-secreting beta cells and development of diabetes mellitus, followed later by loss of glucagon-secreting alpha cells. The exocrine pancreas exhibits a reduction in the synthesis of several major digestive enzymes and succumbs to massive apoptosis after the fourth postnatal week. Perk −/− mice also exhibit skeletal dysplasias at birth and postnatal growth retardation. Skeletal defects include deficient mineralization, osteoporosis, and abnormal compact bone development. The skeletal and pancreatic defects are associated with defects in the rough endoplasmic reticulum of the major secretory cells that comprise the skeletal system and pancreas. The skeletal, pancreatic, and growth defects are similar to those seen in human Wolcott-Rallison syndrome.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                28 December 2012
                12 November 2012
                12 November 2012
                : 287
                : 53
                : 44338-44344
                Affiliations
                [1]From the Metabolic Research Laboratories, University of Cambridge, and National Institute for Health Research Cambridge Biomedical Research Centre, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 0QQ, United Kingdom
                Author notes
                [1 ] To whom correspondence may be addressed. Tel.: 44-1223-769-092; Fax: 44-1223-768-639; E-mail: hph23@ 123456medschl.cam.ac.uk .
                [2 ] To whom correspondence may be addressed. Tel.: 44-1223-768-940; Fax: 44-1223-768-639; E-mail: dr360@ 123456medschl.cam.ac.uk .
                Article
                M112.428987
                10.1074/jbc.M112.428987
                3531748
                23148209
                4aa91e3b-903f-467f-ad41-91a0a2b39ebe
                © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Non-Commercial License applies to Author Choice Articles

                History
                : 18 October 2012
                : 6 November 2012
                Categories
                Cell Biology

                Biochemistry
                endoplasmic reticulum stress,insulin synthesis,pancreatic islets,protein misfolding,protein synthesis,unfolded protein response,insulin metabolism,translation initiation

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