Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
A prominent role of PDIA6 in processing of 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
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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.
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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.