A prominent role of PDIA6 in processing of misfolded proinsulin

https://doi.org/10.1016/j.bbapap.2016.03.002Get rights and content

Highlights

  • Several chaperones were identified to be associated with both wild type and Akita mutant proinsulin.

  • Significantly more PDIA6, but not PDI, associated with Akita mutant proinsulin

  • P58IPK was found to associate only with the Akita mutant proinsulin.

  • For the first time a substrate (misfolded proinsulin) is identified for PDIA6.

Abstract

Despite its critical role in maintaining glucose homeostasis, surprisingly little is known about proinsulin folding in the endoplasmic reticulum. In this study we aimed to understand the chaperones involved in the maturation and degradation of proinsulin. We generated pancreatic beta cell lines expressing FLAG-tagged proinsulin. Several chaperones (including BiP, PDIA6, calnexin, calreticulin, GRP170, Erdj3 and ribophorin II) co-immunoprecipitated with proinsulin suggesting a role for these proteins in folding. To investigate the chaperones responsible for targeting misfolded proinsulin for degradation, we also created a beta cell line expressing FLAG-tagged proinsulin carrying the Akita mutation (Cys96Tyr). All chaperones found to be associated with wild type proinsulin also co-immunoprecipitated with Akita proinsulin. However, one additional protein, namely P58IPK, specifically precipitated with Akita proinsulin and approximately ten fold more PDIA6, but not other PDI family members, was bound to Akita proinsulin. The latter suggests that PDIA6 may act as a key reductase and target misfolded proinsulin to the ER-degradation pathway. The preferential association of PDIA6 to Akita proinsulin was also confirmed in another beta cell line (βTC-6). Furthermore, for the first time, a physiologically relevant substrate for PDIA6 has been evidenced. Thus, this study has identified several chaperones/foldases that associated with wild type proinsulin and has also provided a comprehensive interactome for Akita misfolded proinsulin.

Introduction

The insulin molecule is synthesised as preproinsulin and translocated to the endoplasmic reticulum (ER), where immediately after translocation the signal peptide is cleaved by a signal peptidase to generate proinsulin [1]. Proinsulin contains three disulphide bonds; two disulphides linking the A and B chains of insulin and an additional intrachain disulphide bond within the A chain [2]. A number of ER-resident chaperones/folding enzymes, such as protein disulphide isomerase (PDI), are thought to assist in the folding of proinsulin to ensure the correct formation of disulphide bonds prior to its translocation from the ER to the Golgi apparatus [3], [4]. In the trans-Golgi, proinsulin is assembled into proinsulin hexamers in the presence of zinc ions, and packaged into vesicles that bud from the trans-Golgi network [1], [2]. Within the secretory granules proteolytic cleavage of the proinsulin hexamers by proconvertases and carboxypeptidases at the B chain/C-peptide junction and the A chain/C-peptide junction leads to the formation of insulin hexamers and C-peptides that are released into the circulation upon glucose stimulation [1], [5].

In addition to its important role in controlling glucose homeostasis, insulin is a significant autoantigen in type 1 diabetes (T1D), however it is unclear what triggers the autoreactive immune response to proinsulin [6], [7], [8]. Aberrant forms of proinsulin may be more immunogenic than the native hormone as it has been shown that CD4 + T cells isolated from T1D patients specifically recognize misfolded proinsulin [9], [10]. In addition, transgenic mice expressing mutant proinsulin (Akita mutation) where the cysteine at position 7 in the A chain, which normally engages in a crucial disulphide bond, is mutated to tyrosine develop diabetes [11], [12]. In addition to this mutant form of proinsulin, Akita mice express wild type proinsulin from three alleles (two Ins1 and one Ins2), which should be more than sufficient to control glucose homeostasis and avoid diabetes. However, these mice experience beta cell loss a few weeks after birth and subsequently develop diabetes in the absence of inflammation [12], [13].

Proteomics studies have shown that proteins thought to play a role in insulin biosynthesis (e.g. PDI, ERO1L-β) are altered in beta cells when exposed to proinflammatory cytokines [14], [15]. We hypothesised that changes in the abundance of these molecules may lead to accumulation of misfolded proinsulin that in turn leads to ER stress. Very little is known about chaperone-assisted proinsulin folding and misfolding. A recent study by Pottekat et al. characterised 230 proteins that interacted with proinsulin/insulin during its biosynthesis [16]. Of these 230 proteins, 21 were found to interact with proinsulin at early stages of synthesis and folding [16]. However, an interactome for misfolded proinsulin has yet to be determined. Of note, Hartley et al. [17] identified genes that were affected by the inducible expression of mutant proinsulin in a beta cell line, but did not focus on investigating the proteins that interact with mutant proinsulin.

Here we were interested in identifying the foldases and chaperones involved in proinsulin production (folding and misfolding). FLAG-tagged proinsulin and mutant Akita proinsulin were expressed in the NIT-1 pancreatic beta cell line. Importantly, the tags provided unique immunoreactivity with anti-FLAG-monoclonal antibodies, and so enabled specific immunoprecipitation of proinsulin and associated molecules, irrespective of the conformation or processing of the introduced insulin molecule. Moreover, FLAG-tagged Akita proinsulin was specifically immunoprecipitated using this approach avoiding precipitation of endogenous wild type insulin associated with the use of insulin-specific antibodies. We identified several chaperones and foldases that associated with both wild type and Akita proinsulin and also a few that specifically associated with the mutant proinsulin. We also found evidence of a stable association between protein disulphide isomerase A6 (PDIA6), a PDI family member, and proinsulin which has not been directly reported in the past.

Section snippets

Tissue culture

The SV40 transformed NIT-1 and βTC-6 insulinoma cell lines [18], [19] were grown in DMEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat inactivated foetal calf serum (FCS) (Sigma-Aldrich, St Louis, MO, USA), 2 mM l-glutamine (MP Biomedicals, Santa Ana, CA, USA), 100 units/ml benzyl-penicillin (CSL, Melbourne, Victoria, Australia), 0.1 mg/ml streptomycin sulphate (Sigma-Aldrich), 0.05 mM β-mercaptoethanol (Sigma-Aldrich), 5 mM HEPES buffer (MP Biomedicals) and 0.1 mM non-essential

Generation of cell lines expressing FLAG-tagged proinsulin

The constructs containing murine preproinsulin with a FLAG-tag engineered at either the C or N terminus of the proinsulin molecule were transfected into NIT-1 cells. The transfectants were screened for the expression of FLAG-tagged proinsulin by western blot and stably transfected clonal cell lines were established (Fig. 1A and B). Clone 6 of FLAG-C-terminal proinsulin and clone 1 of FLAG-N-terminal proinsulin were chosen for further experiments as they expressed high levels of tagged

Discussion

Protein misfolding is increasingly being recognized as central to the progression of many diseases, including diabetes. The misfolding of proinsulin both in vitro and in vivo involves predisposition to disulphide mispairing [32], [33]. Chaperones and folding enzymes play a crucial role both in ensuring the proper folding of proinsulin and the degradation of misfolded proinsulin. The goal of the present study was to examine the chaperones/folding enzymes involved in these processes. To identify

Transparency Document

Transparency document.

Acknowledgements

This work was supported by a grant from the Juvenile Diabetes Research Foundation International (17-2012-134). AWP and SW acknowledge fellowship support from the Australian National Health and Medical Research Council (1085017). HSH is supported by a Marie Curie Fellowship from the European Union (CONBIOS 330486). MJH acknowledges fellowship support from Melbourne Research Unit for Facial Disorders. RBS acknowledges fellowship support from the Swiss National Science Foundation.

References (58)

  • G. Rajpal

    Action of protein disulfide isomerase on proinsulin exit from endoplasmic reticulum of pancreatic beta-cells

    J. Biol. Chem.

    (2012)
  • L. Zhang

    GRP78, but not protein-disulfide isomerase, partially reverses hyperglycemia-induced inhibition of insulin synthesis and secretion in pancreatic {beta}-cells

    J. Biol. Chem.

    (2009)
  • D. Eletto

    Protein disulfide isomerase A6 controls the decay of IRE1alpha signaling via disulfide-dependent association

    Mol. Cell

    (2014)
  • B. Tsai

    Protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin

    Cell

    (2001)
  • R. van Huizen

    P58IPK, a novel endoplasmic reticulum stress-inducible protein and potential negative regulator of eIF2alpha signaling

    J. Biol. Chem.

    (2003)
  • J. Tao

    Crystal structure of P58(IPK) TPR fragment reveals the mechanism for its molecular chaperone activity in UPR

    J. Mol. Biol.

    (2010)
  • S. Oyadomari

    Cotranslocational degradation protects the stressed endoplasmic reticulum from protein overload

    Cell

    (2006)
  • Y. Ihara

    Calnexin discriminates between protein conformational states and functions as a molecular chaperone in vitro

    Mol. Cell

    (1999)
  • R.G. Marques et al.

    C-peptide: much more than a byproduct of insulin biosynthesis

    Pancreas

    (2004)
  • J.G. Tang et al.

    Formation of native insulin from the scrambled molecule by protein disulphide-isomerase

    Biochem. J.

    (1988)
  • M.F. Dunn

    Zinc–ligand interactions modulate assembly and stability of the insulin hexamer — a review

    Biometals

    (2005)
  • D. Dubois-LaForgue

    T-cell response to proinsulin and insulin in type 1 and pretype 1 diabetes

    J. Clin. Immunol.

    (1999)
  • Y. Hassainya

    Identification of naturally processed HLA-A2-restricted proinsulin epitopes by reverse immunology

    Diabetes

    (2005)
  • A. Toma

    Recognition of a subregion of human proinsulin by class I-restricted T cells in type 1 diabetic patients

    Proc. Natl. Acad. Sci. U. S. A.

    (2005)
  • S.I. Mannering

    The insulin A-chain epitope recognized by human T cells is posttranslationally modified

    J. Exp. Med.

    (2005)
  • S.I. Mannering

    The A-chain of insulin is a hot-spot for CD4 + T cell epitopes in human type 1 diabetes

    Clin. Exp. Immunol.

    (2009)
  • J. Wang

    A mutation in the insulin 2 gene induces diabetes with severe pancreatic beta-cell dysfunction in the Mody mouse

    J. Clin. Invest.

    (1999)
  • T. Kayo et al.

    Mapping of murine diabetogenic gene mody on chromosome 7 at D7Mit258 and its involvement in pancreatic islet and beta cell development during the perinatal period

    J. Clin. Invest.

    (1998)
  • L. Leroux

    Compensatory responses in mice carrying a null mutation for Ins1 or Ins2

    Diabetes

    (2001)
  • Cited by (0)

    1

    Current address: Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.

    2

    Current address: Institute of Molecular and Cell Biology, Agency for Science Technology and Research, 61 Biopolis Drive, Singapore 138673, Singapore.

    View full text