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. 2010 Oct 11;5(10):e13333.
doi: 10.1371/journal.pone.0013333.

Mutant INS-gene induced diabetes of youth: proinsulin cysteine residues impose dominant-negative inhibition on wild-type proinsulin transport

Ming Liu  1 , Leena Haataja, Jordan Wright, Nalinda P Wickramasinghe, Qing-Xin Hua, Nelson F Phillips, Fabrizio Barbetti, Michael A Weiss, Peter Arvan
Affiliations

Mutant INS-gene induced diabetes of youth: proinsulin cysteine residues impose dominant-negative inhibition on wild-type proinsulin transport

Ming Liu et al. PLoS One. .

Erratum in

  • PLoS One. 2010;5(10) doi: 10.1371/annotation/6d5e12f2-defc-48b5-84f6-43253f593a2a

Abstract

Recently, a syndrome of Mutant INS-gene-induced Diabetes of Youth (MIDY, derived from one of 26 distinct mutations) has been identified as a cause of insulin-deficient diabetes, resulting from expression of a misfolded mutant proinsulin protein in the endoplasmic reticulum (ER) of insulin-producing pancreatic beta cells. Genetic deletion of one, two, or even three alleles encoding insulin in mice does not necessarily lead to diabetes. Yet MIDY patients are INS-gene heterozygotes; inheritance of even one MIDY allele, causes diabetes. Although a favored explanation for the onset of diabetes is that insurmountable ER stress and ER stress response from the mutant proinsulin causes a net loss of beta cells, in this report we present three surprising and interlinked discoveries. First, in the presence of MIDY mutants, an increased fraction of wild-type proinsulin becomes recruited into nonnative disulfide-linked protein complexes. Second, regardless of whether MIDY mutations result in the loss, or creation, of an extra unpaired cysteine within proinsulin, Cys residues in the mutant protein are nevertheless essential in causing intracellular entrapment of co-expressed wild-type proinsulin, blocking insulin production. Third, while each of the MIDY mutants induces ER stress and ER stress response; ER stress and ER stress response alone appear insufficient to account for blockade of wild-type proinsulin. While there is general agreement that ultimately, as diabetes progresses, a significant loss of beta cell mass occurs, the early events described herein precede cell death and loss of beta cell mass. We conclude that the molecular pathogenesis of MIDY is initiated by perturbation of the disulfide-coupled folding pathway of wild-type proinsulin.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. MIDY proinsulins form abnormally increased amounts of disulfide-linked protein complexes.
293T cells were transfected with vector expressing preproinsulin wild type (‘WT’) or preproinsulin missense mutants in which the described mutation is within the B-chain, C-peptide, or A-chain. At 40 h post-transfection, cells were pulse-labeled with 35S-amino acids for 1 h and then chased for the times indicated. For completeness, chase media and cell lysates were mixed, but none of the MIDY proinsulins are appreciably secreted (see Figure S1). A. At both time points, samples were immunoprecipitated with anti-insulin followed by Tris-tricine-urea-SDS-PAGE under both nonreducing (gels on left) and reducing conditions (gels on right), followed by fluorography. B. The fractional recovery of the native isoform of newly-synthesized proinsulin (fastest migrating band under nonreduced conditions) at 4 h of chase was compared against the recovery from the same sample under reducing conditions (considered to represent total at that chase time). The relative recovery for proinsulin-WT served as a positive control (ie, set to 100%). Results are expressed as mean ± s.d. from two independent experiments.
Figure 2
Figure 2. Misfolded proinsulin with or without free cysteine thiols.
A. 293T cells transiently expressing mouse proinsulin-WT (‘WT’) or proinsulin-DelCys (‘DelCys’) were pulse-labeled with 35S-amino acids for 1 h without chase. Cells were lysed and immunoprecipitated with anti-insulin, followed by Tris-tricine-urea-SDS-PAGE under both nonreducing and reducing conditions as indicated, followed by fluorography. “N” = proinsulin with native disulfide pairs; “I” = proinsulin disulfide isomer(s). Note that proinsulin-DelCys has identical gel mobility under nonreducing and reducing conditions. B. 293T cells transiently expressing empty vector, proinsulin-WT, proinsulin-C(A7)Y, or proinsulin-DelCys were pulse-labeled with 35S-amino acids for 1 h and chased for 1 h. Cell lysates (‘C’) and chase media (‘M’) were immunoprecipitated with anti-insulin and analyzed as in A. C. 293T cells transiently expressing proinsulin-C(A7)Y or proinsulin-DelCys were pulse-labeled with 35S-amino acids for 1 h and chased in complete medium for the times indicated. For one sample, 10 µM lactacystin (‘Lac’) was added to the chase medium bathing transfected cells. At each chase time, cells were lysed, immunoprecipitated with anti-insulin, and the immunoprecipitates incubated with or without AMS as described in Methods, and analyzed by reducing Tris-Tricine-urea-SDS-PAGE and fluorography. As a fraction of all proinsulin bands recovered per lane, thiol-consumed (nonreactive) and thiol-available (AMS reactive) subfractions of newly-synthesized proinsulin-C(A7)Y were quantified by scanning densitometry; the average and range from two independent such experiments is shown at right. Note that proinsulin-DelCys has no reactivity with AMS.
Figure 3
Figure 3. Effect of mutant proinsulins on insulin storage derived from co-expressed nonmutant proinsulin in pancreatic beta cells (and effect of mutant proinsulins on proinsulin export in 293T cells).
A. INS832/13 cells, which co-store human insulin in secretory granules, were transiently transfected with pCMS-GFP to co-express mouse preproinsulins (as indicated) and cytosolic GFP. Transfected cells were recovered after 48 h by fluorescence-activated cell sorting and were extracted with acid-ethanol. A human insulin-specific radioimmunoassay was used to measure hormone storage in mature secretory granules, normalized to cell number. When mouse proinsulin-WT was expressed, the amount of human insulin stored served as a positive control (ie, set to 100%). Results shown are the mean ± s.d. in at least three independent experiments. *p<0.05 compared with mouse proinsulin-WT. B. INS-1 cells were co-transfected with wild-type human preproinsulin and wild-type or mutant mouse preproinsulin. Transfected cells were incubated for 28 h with fresh medium before collection for measurement of secreted human insulin using human insulin specific radioimmunoassay. C. 293T cells were co-transfected to express 1) human proinsulin-WT and co-express 2) mouse proinsulin-WT or missense mutants in which the described mutation is within the B-chain, C-peptide, A-chain, or proinsulin-DelCys at a plasmid ratio of 1 : 2. Beginning at 24 h post-transfection, cells were incubated for 16 h with high-glucose DMEM plus 10% FBS. Media were collected and a human proinsulin-specific radioimmunoassay was used to measure secretion of co-expressed human proinsulin-WT. When mouse proinsulin was replaced by empty vector, the amount of human proinsulin-WT secretion served as a positive control (ie, set to 100%). Medium collected from 293T cells expressing only mouse proinsulin-WT served as a negative control for the specificity of the human proinsulin radioimmunoassay (while independent measurements not shown proved ample secretion of rodent proinsulin-WT in these samples). Results are expressed as mean ± s.d. from at least three independent experiments. *p<0.05 compared with mouse proinsulin-WT.
Figure 4
Figure 4. Co-expression of mutant proinsulins with proinsulin-WT and an unrelated secretory protein demonstrates protein-specific dominant-negative inhibition of proinsulin export.
A. A Myc-epitope tag was inserted into the C-peptide of human proinsulin-WT to form the construct known as hProCpepMyc, whose peptide sequence is shown. B. 293T cells were transfected to express either human proinsulin or hProCpepMyc. At 48 h post-transfection, cells were pulse-labeled with 35S-amino acids for 1 h and chased for 1 h. Cell lysates (‘C’) and chase media (‘M’) were immunoprecipitated with anti-insulin or anti-myc antibodies. There was no recovery of untagged proinsulin-WT with anti-myc antibodies in either cells or medium (not shown). Immunoprecipitates were analyzed by nonreducing Tris-tricine-urea-SDS-PAGE and fluorography. Note that hProCpepMyc is secreted efficiently and is recovered equally with anti-insulin and anti-myc immunoprecipitation, but the protein has a higher apparent molecular mass than proinsulin-WT. C. 293T cells were triply co-transfected to express hProCpepMyc, mouse proinsulins (as indicated) and α1-antitrypsin at a plasmid ratio of 2 : 4 : 1, respectively. At 48 h post-transfection, cells were pulse-labeled with 35S-amino acids for 1 h and either lysed at the zero chase time (‘0’) or chased for 3 h. Cell lysates (‘C’) and chase media (‘M’) were immunoprecipitated with anti-insulin (upper set of gels) and anti-α1-antitrypsin (lower set of gels). Proinsulin immunoprecipitates were analyzed by Tris-tricine-urea-SDS-PAGE while α1-antitrypsin was analyzed by conventional SDS-PAGE, both under reducing conditions. Note that while the secretion efficiency of untagged mutant proinsulin and tagged proinsulin-WT varied, a1-antitrypsin was efficiently secreted in every case.
Figure 5
Figure 5. MIDY proinsulins cause co-expressed proinsulin-WT to become abnormally engaged in disulfide-linked protein complexes.
A. 293T cells were co-transfected to express human proinsulin-WT and co-express untagged or Myc-tagged proinsulin-C(A7)Y or proinsulin-DelCys, using a plasmid ratio of 1 : 2. At 24 h post-transfection, the cells were incubated in fresh high glucose DMEM containing 10% FBS for 16 h and media were analyzed using a human proinsulin-specific radioimmunoassay. Medium collected from cells transfected with empty vectors served as a negative control; medium collected from cells transfected human proinsulin-WT and mouse proinsulin-WT served as a positive control (ie, set to 100%). Results are expressed as mean ± s.d. from at least three independent measurements. Note that presence of the Myc-tag neither caused nor prevented dominant-negative inhibition of co-expressed human proinsulin-WT. B. 293T cells were co-transfected to express untagged human proinsulin-WT and co-express Myc-tagged proinsulin-WT or missense mutants, using a plasmid ratio of 1 : 1. At 48 h post-transfection, cells were pulse-labeled with 35S-amino acids for 1 h, lysed, and immunoprecipitated with anti-insulin followed by analysis under nonreducing (‘NR’) and reducing (‘R’) conditions as indicated (note that NR and R lanes were run at opposite ends of the gel and upon fluorography were spliced after imaging to juxtapose the two conditions for each sample). The fractional recovery of co-expressed untagged human proinsulin-WT under nonreducing conditions was compared against the recovery of the same untagged proinsulin-WT in these samples under reducing conditions (in the Figure, the bands in question connected by a blue line). With the relative recovery of untagged human proinsulin-WT co-expressed in the presence of tagged proinsulin-WT serving as a positive control (ie, set to 100%), the bar graph below quantifies the bands (and the dominant-negative effect) from three replicate experiments. C. Islets isolated from wild-type control mice were pulse-labeled with 35S-amino acids for 20 min without chase. The lysates were immunoprecipitated with anti-insulin, followed by analysis under nonreducing (‘NR’) and reducing (‘R’) conditions as indicated. Islets isolated from male Akita mice were similarly labeled, lysed, and the lysates divided for immunoprecipitation with anti-insulin or co-precipitation with anti-BiP. Under reducing conditions, the wild-type and mutant gene products of Akita islets could be separated and their full recovery quantified. Under nonreducing conditions, the recovery of the native disulfide isomer of wild-type proinsulin was calculated relative to total recovery of the wild-type gene product under reducing conditions. Note that the wild-type translation product is the fastest band on the nonreducing gel and the slowest band on the reducing gel (also note that NR and R lanes were run at opposite ends of the gel and upon fluorography were spliced after imaging to juxtapose the two conditions for each sample). The bar graph below quantifies this ratio for wild-type proinsulin in Akita islets relative to that obtained in wild-type (‘WT’) islets ± s.d., from three independent experiments (p<0.05 compared to wild-type islets). At right, recombinant human proinsulin-WT or -C(A7)Y were expressed and labeled in 293T cells to serve as molecular mass markers for the positions of these proteins under reducing conditions.
Figure 6
Figure 6. 1H-NMR analysis of mutant insulins.
A. Expanded ball-and-stick model of the β-turn from residues B20-B23, highlighting positive φ angle at B23 (circular arrow at left). The Cα atom of GlyB23 is shown in red; amide nitrogen atoms are shown in blue. B. 1H-NMR spectra of DKP-insulin analogs in D2O (pD 7.0): top to bottom, the MIDY mutant ValB23 (protein concentration 70 µM), ValB23 (protein concentration 500 µM); the remaining spectra were obtained at a protein concentration of 500-600 µM: LeuB25-DKP-insulin, LeuA3-DKP-insulin, and DKP-insulin (parent spectrum). The aliphatic region is shown at right, and aromatic region at left; aromatic spin systems of PheB24 and TyrB26 are indicated. (C. and D.) Histograms of NMR parameters, highlighting MIDY-associated perturbation. For each of DKP-insulin (“wt”), LeuA3-DKP-insulin, LeuB25-DKP-insulin, and ValB23-DKP-insulin, black and gray bars correspond to A-chain probes and B-chain probes, respectively. A-chain probes [A2(γ',δ), A10(γ',δ), and A19(δ,ε)] reflect its helix-turn-helix conformation (see Table S1) whereas B-chain probes [B15(δ), B24(δ,ε,ξ), and B26(δ,ε)] monitor conformation of the C-terminal β-strand and its packing against the central B-chain α-helix (LeuB15). C. Normalized secondary chemical shifts changes (scs; defined as differences between observed chemical shifts and those tabulated from random-coil values). Significant attenuation of chemical shifts is observed only for the MIDY mutant ValB23-DKP-insulin. A- and B-chain (black and gray bars, respectively) represent mean scs values of A- and B-chain probe resonances (ND, not determined). D. Line widths of the above A-chain or B-chain resonances in corresponding 2D-TOCSY spectra (Figure S2) were normalized by the line widths of corresponding resonances in the wild-type spectrum. Significant peak broadening was observed only in the spectrum of ValB23-DKP-insulin, which became more severe as the protein concentration was increased to 500 µM. At 500 µM ValB23-DKP-insulin exhibits generalized broadening indicative of protein aggregation; only resonances from A19(δ,ε) and B26(δ,ε) could be used to quantify line widths because the other probe resonances were either too broad or unresolved. B23 bars thus under-estimate extent of resonance broadening.
Figure 7
Figure 7. ER stress and ER stress reponse alone do not block wild-type proinsulin secretion.
A. Wild-type proinsulin secretion from cells co-expressing wild-type α1-antitrypsin (“wt-AAT”) or mutant Hong Kong-null α1-antitrypsin (“HKN AAT-myc”). 293T cells co-transfected with proinsulin and wt-AAT (left panels) or HKN AAT-myc (right panels) were pulse-labeled with 35S-amino acids and chased for the times indicated. Cell lysates and media were immunoprecipitated with anti-AAT (upper panels) or anti-insulin (lower panels) and analyzed by SDS-PAGE and fluorography. During the chase, wt-AAT shifted upwards to a Golgi-glycosylated form and was secreted from cells (“C”) to medium (“M”); proinsulin was secreted in parallel. B. Quantification at 2.5 h of chase of the percent of proinsulin recovered in the medium, from three independent experiments. C. 293T cells co-transfected with proinsulin, BiP-luciferase, and CMV-Renilla luciferase were treated with tunicamycin (‘TUN’, 0.1 µg/mL) for 16 h. At this time, the cells were pulse-labeled with with 35S-amino acids for 30 min and chased for 2.5 h. The media were collected and cells were lysed and analyzed by immunoprecipitation with anti-insulin (the proinsulin band is shown). D. The ratio of simultaneous BiP-firefly to CMV-renilla luciferase activities are quantified from the experiment shown in panel C. E. At 2.5 h of chase from two independent experiments like that shown in panel C, the percent of total proinsulin recovered in the medium is expressed as mean ± s.d.
Figure 8
Figure 8. MIDY mutants induce ER stress in pancreatic beta cells.
A. Min6 cells were transiently transfected with the BiP promoter-firefly luciferase plasmid, the pRL-CMV-driven Renilla luciferase reference plasmid, and empty vector or proinsulin expression plasmids at ratio 1 : 2 : 5 (this ratio helps ensure that BiP-luciferase serves as a reporter from cells synthesizing exogenously expressed proinsulins). At 48 h post-transfection, the cells were lysed and a ratio of firefly/renilla luciferase was measured. The relative activity of the BiP promoter in cells expressing mutant proinsulins was compared to that in cells expressing wild-type (“WT”) proinsulin, which served as a negative control. In a separate co-transfection Min 6 cells were treated overnight with tunicamycin (‘Tun’, 1 µg/mL), which served as a positive control for ER stress induction. Results are expressed as mean ± s.d. from at least four independent experiments. *<0.05 compared with proinsulin-WT B. Min6 cells were co-transfected to express BiP-luciferase and either hProCpepMyc-C(A7)Y or hProCpepMyc-DelCys. At 48 h after transfection, the cells were lysed and BiP-luciferase activity was measured along with the steady-state level of proinsulin mutants by Western blotting with anti-myc. Neither mutant is secreted by from the cells to the medium; note that DelCys is not deficient for induction of ER stress response.
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