A prominent role of PDIA6 in processing of misfolded proinsulin

Dhana G Gorasia, Nadine L Dudek, Helena Safavi-Hemami, Rochelle Ayala-Perez, Ralf B Schittenhelm, Philippa M Saunders, Sheena Wee, Jon E Mangum, Michael J Hubbard, Anthony W Purcell

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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 (betaTC-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.
Original languageEnglish
Pages (from-to)715 - 723
Number of pages9
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1864
Issue number6
DOIs
Publication statusPublished - 2016

Cite this

Gorasia, Dhana G ; Dudek, Nadine L ; Safavi-Hemami, Helena ; Ayala-Perez, Rochelle ; Schittenhelm, Ralf B ; Saunders, Philippa M ; Wee, Sheena ; Mangum, Jon E ; Hubbard, Michael J ; Purcell, Anthony W. / A prominent role of PDIA6 in processing of misfolded proinsulin. In: Biochimica et Biophysica Acta - Proteins and Proteomics. 2016 ; Vol. 1864, No. 6. pp. 715 - 723.
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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 (betaTC-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.",
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Gorasia, DG, Dudek, NL, Safavi-Hemami, H, Ayala-Perez, R, Schittenhelm, RB, Saunders, PM, Wee, S, Mangum, JE, Hubbard, MJ & Purcell, AW 2016, 'A prominent role of PDIA6 in processing of misfolded proinsulin', Biochimica et Biophysica Acta - Proteins and Proteomics, vol. 1864, no. 6, pp. 715 - 723. https://doi.org/10.1016/j.bbapap.2016.03.002

A prominent role of PDIA6 in processing of misfolded proinsulin. / Gorasia, Dhana G; Dudek, Nadine L; Safavi-Hemami, Helena; Ayala-Perez, Rochelle; Schittenhelm, Ralf B; Saunders, Philippa M; Wee, Sheena; Mangum, Jon E; Hubbard, Michael J; Purcell, Anthony W.

In: Biochimica et Biophysica Acta - Proteins and Proteomics, Vol. 1864, No. 6, 2016, p. 715 - 723.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - A prominent role of PDIA6 in processing of misfolded proinsulin

AU - Gorasia, Dhana G

AU - Dudek, Nadine L

AU - Safavi-Hemami, Helena

AU - Ayala-Perez, Rochelle

AU - Schittenhelm, Ralf B

AU - Saunders, Philippa M

AU - Wee, Sheena

AU - Mangum, Jon E

AU - Hubbard, Michael J

AU - Purcell, Anthony W

PY - 2016

Y1 - 2016

N2 - 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 (betaTC-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.

AB - 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 (betaTC-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.

UR - http://www.ncbi.nlm.nih.gov/pubmed/26947243

U2 - 10.1016/j.bbapap.2016.03.002

DO - 10.1016/j.bbapap.2016.03.002

M3 - Article

VL - 1864

SP - 715

EP - 723

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 6

ER -