PDI reductase acts on Akita mutant proinsulin to initiate retrotranslocation along the Hrd1/Sel1L-p97 axis

Abstract

In mutant INS gene-induced diabetes of youth (MIDY), characterized by insulin deficiency, MIDY proinsulin mutants misfold and fail to exit the endoplasmic reticulum (ER). Moreover, these mutants bind and block ER exit of wild-type (WT) proinsulin, inhibiting insulin production. The ultimate fate of ER-entrapped MIDY mutants is unclear, but previous studies implicated ER-associated degradation (ERAD), a pathway that retrotranslocates misfolded ER proteins to the cytosol for proteasomal degradation. Here we establish key ERAD machinery components used to triage the Akita proinsulin mutant, including the Hrd1-Sel1L membrane complex, which conducts Akita proinsulin from the ER lumen to the cytosol, and the p97 ATPase, which couples the cytosolic arrival of proinsulin with its proteasomal degradation. Surprisingly, we find that protein disulfide isomerase (PDI), the major protein oxidase of the ER lumen, engages Akita proinsulin in a novel way, reducing proinsulin disulfide bonds and priming the Akita protein for ERAD. Efficient PDI engagement of Akita proinsulin appears linked to the availability of Hrd1, suggesting that retrotranslocation is coordinated on the lumenal side of the ER membrane. We believe that, in principle, this form of diabetes could be alleviated by enhancing the targeting of MIDY mutants for ERAD to restore WT insulin production.

FIGURE 1:

The Hrd1-Sel1L complex promotes ERAD of Akita. (A) 293T cells expressing Akita-Myc were transfected with a scrambled or Hrd1-specific siRNA. Cells were then treated with cycloheximide for the indicated time and harvested, and the resulting WCE was analyzed by using the indicated antibodies. (B) The Akita band intensity in A and D was quantified with ImageJ (National Institutes of Health, Bethesda, MD). Data represent mean ± SD of at least three independent experiments. (C) As in A, except that cells were transfected with GFP-FLAG, WT Hrd1-Myc, or C291A Hrd1-Myc construct. (D) As in A, except cells were transfected with a siRNA against Sel1L. The black vertical line indicates that an intervening lane from the same immunoblot has been spliced out.

FIGURE 2:

p97 facilitates Akita degradation. (A) As in Figure 1C, except that 293T cells were transfected with WT p97-His or QQ p97-His. (B) As in Figure 1B, except that the Akita band intensity in A is quantified. (C) 293T cells expressing Akita-Myc or not were transfected with an empty vector or QQ p97-His as indicated. Cells were lysed in a RIPA buffer and Akita immunoprecipitated from the resulting WCE using a Myc antibody. The precipitated material was subjected to SDS–PAGE and immunoblotted with the indicated antibodies. The WCE was also immunoblotted.

FIGURE 3:

PDI exerts a key role in regulating Akita degradation. (A) As in Figure 1A, except that 293T cells were transfected with a PDI-specific siRNA for 3 d. (B) As in Figure 1C, except that the Akita band intensity in A and C–E are quantified. (C) As in A, except where indicated cells transfected with a PDI-specific siRNA for 3 d were cotransfected with a PDI construct resistant to the PDI-specific siRNA. (D) As in A, except hat cells were transfected with an ERp57-specific siRNA. (E) As in A, except that cells were transfected with an ERdj5-specific siRNA. (F) 293T cells expressing either a control or PDI-specific siRNA were cotransfected with ΔCys-Myc and treated or not with MG132 as indicated. (G) Cells expressing WT proinsulin-Myc or ΔCys-Myc were treated with DTT (10 μM) in the presence of MG132 and the WCE analyzed as in F. (H) 293T cells expressing the indicated Myc-tagged mutant proinsulins were cotransfected with PDI C56A-FLAG. The cells were lysed and immunoprecipitated with M2 FLAG antibody-conjugated beads, analyzed by reducing SDS–PAGE, and immunoblotted using the appropriate antibodies. The WCE was also directly immunoblotted with anti-Myc or anti-FLAG antibodies. pTarget was the plasmid used as a control for the mutant proinsulin constructs.

FIGURE 4:

PDI acts as a reductase against Akita. (A) 293T cells expressing Akita were transfected with or without PDI C56A-FLAG or PDI C400A-FLAG. Tagged PDI proteins were immunoprecipitated from the resulting WCE using a FLAG antibody. The precipitated material was subjected to nonreducing or reducing SDS–PAGE as indicated and immunoblotted using the appropriate antibodies. The WCE was also immunoblotted with a Myc antibody. (B) 293T cells expressing Akita and transfected with either a scrambled or PDI-specific siRNA were treated with or without MG132. The resulting WCE was subjected to nonreducing or reducing SDS–PAGE as indicated and immunoblotted using the indicated antibodies. The black vertical line indicates that an intervening lane from the same immunoblot has been spliced out. (C) Lanes 1–4, WCE in B was treated with or without DTT, layered over a 20% sucrose cushion, and centrifuged. The top and bottom fractions were separated and subjected to SDS–PAGE, followed by immunoblotting using a Myc antibody. Lanes 5 and 6, WCE was subjected to SDS–PAGE, followed by immunoblotting using the indicated antibodies. (D) His-tagged PDI purified from bacteria is shown in the Coomassie gel. Immunoprecipitated Akita-Myc from PDI-knockdown cells was incubated with PDI or IAA-modified PDI in the presence of 1 mM GSH and 1 mM GSSG, and the samples were subjected to nonreducing SDS–PAGE, followed by immunoblotting with a Myc antibody. Bottom graph, the intensity level of Akita dimer and trimer was quantified by ImageJ. Data represent the mean ± SD of three independent experiments. The black vertical line indicates that an intervening lane from the same immunoblot has been spliced out. (E) Cells expressing Akita transfected with a scrambled or Hrd1-specific siRNA were cotransfected with either PDI C56A-FLAG or PDI C400A-FLAG. The ability of tagged PDI protein to interact with Akita was analyzed as in A.

FIGURE 5:

PDI, Hrd1, and p97 promote ERAD of Akita in pancreatic β-cells. (A) As in Supplemental Figure S2C, except that rat pancreatic INS-1 832/13 β-cells were used and GFP-FLAG was expressed instead of ERp57 C60A-FLAG. The Akita band intensity is quantified as in Figure 1B. (B) As in Figure 1C, except that the experiment was performed in INS-1 832/13 β-cells and GFP-FLAG was transfected instead of vector. The Akita band intensity is quantified as in Figure 1B. (C) As in Figure 2A, except that the experiment was performed in INS-1 832/13 β-cells and GFP-FLAG was transfected instead of vector. The Akita band intensity is quantified as in Figure 1B.

FIGURE 6:

Model depicting ERAD of Akita. To dispose of Akita via the ERAD pathway, PDI reduces the disulfide bonds in this mutant proinsulin to initiate its retrotranslocation into the cytosol (step 1). The precise pairing of disulfide bonds in Akita proinsulin is not known. In the second step, Akita retrotranslocates across the ER membrane by using the Hrd1/Sel1L membrane complex (step 2). Once it is presented to the cytosol, Akita becomes polyubiquitinated using Hrd1’s catalytic activity (step 3). In the final step, p97 propels polyubiquitinated Akita into the cytosol, targeting it to the proteasome for degradation (step 4).