human recombinant proinsulin  (Eli Lilly)

Journal: Protein Science : A Publication of the Protein Society

Article Title: Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis

doi: 10.1002/pro.4949

Figure Lengend Snippet: After nonreducing SDS‐PAGE, post‐gel disulfide reduction improves detection of proinsulin in distinct folded states. (a) Schematic of different immunoblotting methods to detect proinsulin misfolding. At left: Conventional immunoblotting after nonreducing SDS‐PAGE is super‐sensitive to misfolded disulfide‐linked complexes of proinsulin. Red arrow pathway: Treatment of samples with DTT + heating after nonreducing SDS‐PAGE converts all proinsulin contained within the gel to reduced monomers. Using a fixed percentage of acrylamide ensures even transfer efficiency across the gel. This method enhances detection of proinsulin monomers (particularly native monomers) after nonreducing SDS‐PAGE and provides a more quantitative estimate of the distribution of differently‐folded proinsulin species. (b) Cartoon highlighting the impact of the blotting method described in panel A for pancreatic β‐cells after nonreducing SDS‐PAGE. (c) Immunblotting with anti‐insulin or with monoclonal antibodies (generated by Hytest) targeting different regions of rodent proinsulin C‐peptide (A cartoon above shows the location of antibody binding on the proinsulin C‐peptide). Identical samples of INS1E cell lysates were resolved by SDS‐PAGE under nonreducing (lanes 1, 3, 5, 7, 9, 11, 13, and 15) or reducing conditions (lanes 2, 4, 6, 8, 10, 12, 14, and 16). After running, the gels were divided, and lanes 3,4; 7,8; 11,12; and 15,16 were treated with 100 mM DTT + heating for post‐gel disulfide reduction (indicated below as – or +) before electrotransfer and immunoblotting with the indicated antibodies. Immunoblotting with anti‐insulin that cross‐reacts with proinsulin show dramatic signal loss (for both proinsulin and insulin, dotted red lines ) upon post‐gel disulfide reduction. The curved blue arrows connecting lanes 5–7, 9–11, or 13–15 bearing samples that were run under nonreduced conditions show dramatically improved detection of proinsulin monomers by post‐gel disulfide reduction. (d) 293 T cells were transfected with proinsulin “keep one bond” constructs—keep‐B19/A20, keep‐B7/A7, or keep‐A6/A11. After 24 h, the cells were lysed and identical aliquots of cell lysate were resolved by SDS‐PAGE (12% NuPAGE) under nonreduced (first 6 lanes) or reduced conditions (last three lanes). After electrophoresis, a portion of the nonreduced gel underwent post‐gel disulfide reduction with 100 mM DTT + heating to 60°C for 15 min (middle three lanes) prior to electrotransfer and immunoblotting with anti‐human proinsulin. The detection of proinsulin monomers by nonreducing SDS‐PAGE increased after post‐gel disulfide reduction with DTT + heating ( curved blue arrows ). (e) Immunoblots were quantified by densitometry; the relative recovery of proinsulin monomers before and after post‐gel disulfide reduction is shown ( n = 4 independent experiments; mean ± SD; * p < 0.05; ns = non‐significant). (f) 293 T cells were either untransfected (“U”) or transfected to express WT hPro‐CpepMyc or that bearing the L(A16)P mutation (here simply labeled as A16P), and culture media was collected overnight. The samples were resolved by 12% NuPAGE under nonreduced (first 5 lanes) or reduced conditions (last 5 lanes) and the nonreduced gel underwent post‐gel disulfide reduction (indicated at bottom) before electrotransfer and immunoblotting with anti‐human proinsulin. Cyclophilin B (CypB) is a loading control. (g) Quantitation of native proinsulin monomers recovered as a fraction of total proinsulin ( n = 4 independent experiments; mean ± SD; ** p = 0.0022).

Article Snippet: After nonreducing SDS‐PAGE of purified recombinant human proinsulin expressed in Escherichia coli (N‐terminally 6xHis‐tagged, purchased from Creative Biomart, Shirley, NY; with no attempt for in vitro refolding to the native state) increased detection of monomers, with decreased detection of intermolecular disulfide‐linked forms, was observed upon post‐gel disulfide reduction (Supplemental Figure , quantified in ).

Techniques: SDS Page, Western Blot, Bioprocessing, Generated, Binding Assay, Electrotransfer, Transfection, Construct, Electrophoresis, Mutagenesis, Labeling, Control, Quantitation Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis

doi: 10.1002/pro.4949

Figure Lengend Snippet: Folding status of endogenous (intracellular and secreted) proinsulin from Min6 cells. (a) Identical aliquots of Min6 cell lysate and 6 h conditioned media were resolved by nonreducing 12% NuPAGE. After electrophoresis, the gel was cut in fourths ( dotted lines ) and each quarter treated as indicated (below the image) prior to electrotransfer to nitrocellulose for immunoblotting with anti‐rodent proinsulin. Improved detection of secreted proinsulin ( blue arrow , as well as intracellular proinsulin monomer bearing native disulfide bonds) is noted after post‐gel disulfide reduction (optimized with DTT + heating). (b) Min6 cells in complete medium were incubated for 4 h at 37 or 29°C, as indicated. Secretion of proinsulin (Proins) from cells to media is shown at both temperatures ( curved blue arrows; solid or dashed, respectively ). The non‐native proinsulin monomer band is identified with a red arrow . Cyclophilin B (Cyp B) is a loading control. (c) Quantitation of native proinsulin intracellularly from replicate experiments like those shown in panel B as a fraction of total (cells + media) proinsulin ( n = 3 independent experiments; mean ± SD; * p = 0.045). Each point represents an independent experiment (mean ± SD). (d) Min6 cells were either untreated or treated with Monensin (15 μM for 90 min) as indicated. Secretion of proinsulin (Proins) from cells to media under both conditions is indicated ( curved blue arrows; solid or dashed , respectively ). The non‐native proinsulin monomer band is identified with a red arrow . Cyclophilin B (Cyp B) is a loading control. In panels 2a and 2d, nonspecific bands in the media migrating at >50 kDa are largely derived from serum albumin, observed in similar blotting exposures of culture medium that was never incubated with cells. (e) Quantitation of native proinsulin intracellularly from replicate experiments like those shown in panel D as a fraction of total (cells + media) proinsulin ( n = 4 independent experiments; mean ± SD; * p = 0.012).

Article Snippet: After nonreducing SDS‐PAGE of purified recombinant human proinsulin expressed in Escherichia coli (N‐terminally 6xHis‐tagged, purchased from Creative Biomart, Shirley, NY; with no attempt for in vitro refolding to the native state) increased detection of monomers, with decreased detection of intermolecular disulfide‐linked forms, was observed upon post‐gel disulfide reduction (Supplemental Figure , quantified in ).

Techniques: Electrophoresis, Electrotransfer, Western Blot, Incubation, Control, Quantitation Assay, Derivative Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis

doi: 10.1002/pro.4949

Figure Lengend Snippet: Proinsulin folding response to pharmacological perturbation of ER homeostasis. (a) Identical wells of Min6 cells were treated ± Antimycin A (6 μM) for 1.5 h, and equal aliquots of cell lysate or media were resolved by 12% NuPAGE under nonreducing (lanes 1–8) or reducing conditions (lanes 9–12). Identical nonreduced samples were either not exposed (lanes 1–4) or exposed (lanes 5–8) to post‐gel disulfide reductions (as indicated below) prior to electrotransfer and immunoblotting with anti‐rodent proinsulin (Proins). Non‐native proinsulin monomers and disulfide‐linked proinsulin dimers are indicated ( red arrows ); native proinsulin monomers migrate faster ( blue arrow ). (b) Quantitation of native proinsulin monomers recovered as a fraction of total proinsulin ( n = 3 independent experiments; mean ± SD; * p = 0.0045). (c) Identical wells of Min6 cells were untreated or treated with PERK inhibitor (2 μM GSK2656157, overnight, as indicated above), and equal aliquots of cells (C) or media (M) were resolved by 12% NuPAGE under nonreducing (lanes 1–8) or reducing (lanes 9–16) conditions. Identical nonreduced samples were either exposed (lanes 1–4) or not exposed (lanes 5–8) to post‐gel disulfide reduction (as indicated below) prior to electrotransfer and immunoblotting with anti‐rodent proinsulin (Proins). Proinsulin monomers bearing native ( blue arrow ) or non‐native ( red arrow ) disulfide bonds are indicated. (d) Quantitation of native proinsulin monomers recovered as a fraction of total proinsulin ( n = 5 independent experiments; mean ± SD; * p = 0.0062). (e) Human islets (nondiabetic donor) were untreated (DMSO) or treated with PERK inhibitor (2 μM GSK2656157, 24 h) and equal aliquots of cells or media were resolved by 12% NuPAGE under nonreducing (lanes 1–8) or reducing (lanes 9–12) conditions. nonreducing or reducing conditions as indicated at bottom. Identical nonreduced samples were either not exposed (lanes 1–4) or exposed (lanes 5–8) to post‐gel disulfide reduction (as indicated below) prior to electrotransfer and immunoblotting with anti‐human proinsulin (Proins). Proinsulin monomers bearing native ( blue arrow ) or non‐native ( red arrow ) disulfide bonds are indicated.

Article Snippet: After nonreducing SDS‐PAGE of purified recombinant human proinsulin expressed in Escherichia coli (N‐terminally 6xHis‐tagged, purchased from Creative Biomart, Shirley, NY; with no attempt for in vitro refolding to the native state) increased detection of monomers, with decreased detection of intermolecular disulfide‐linked forms, was observed upon post‐gel disulfide reduction (Supplemental Figure , quantified in ).

Techniques: Electrotransfer, Western Blot, Quantitation Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis

doi: 10.1002/pro.4949

Figure Lengend Snippet: Proinsulin misfolding in the ER of YIPF5‐I98S iPSCs. (a) Control or YIPF5‐I98S (YIPF5*) iPSCs were differentiated to stage 7 β‐like cells prior to analysis. Media containing secretion from human islets was collected as a control to identify the position of native human proinsulin as detected by immunoblotting under nonreduced conditions ( lanes 1 and 7 ). Lysates of β‐like cells were analyzed under nonreduced ( lanes 2,3 and 8,9 ) or reduced conditions ( lanes 5,6 and 11,12 ). Identical nonreduced samples were either exposed ( lanes 1–3 ) or not exposed ( lanes 7–9 ) to post‐gel disulfide reduction (as indicated above) prior to electrotransfer and immunoblotting with anti‐human proinsulin (Proins). Intracellularly, the mutant β‐like cells exhibited a decreased abundance of native proinsulin ( blue arrow ). Non‐native proinsulin monomers are shown with red arrow . The same samples were analyzed by immunoblotting with guinea pig anti‐insulin, which primarily recognizes disulfide‐linked two‐chain mature insulin ( lanes 8,9 ). Cyclophilin B (CypB) is a loading control. (b) Quantitation of native proinsulin ( blue arrow ) as a fraction of total cellular proinsulin (recovered under reduced conditions) from at least three independent differentiation experiments like that shown in panel A (mean ± SD; * p = 0.044). (c) Quantitation of mature insulin (normalized to the loading control) from the same samples as those quantified in panel B (mean ± SD; * p = 0.032). In panels D and E, Stage 7 islet‐like clusters differentiated from embryonic stem cells from WT or YIPF5‐I98S were implanted under the kidney capsule of immunocompromised NOD/SCID‐γ mice and then retrieved for processing 3 months thereafter (the WT cell images are above and YIPF5‐I98S cell images are below). (d) Co‐immunostaining for insulin (INS, panel 2), proinsulin (PROINS, panel 3), or merged image (panel 4); counter‐stained with Hoechst (panel 1). (e) Co‐immunostaining for proinsulin (PROINS, panel 2), the ER protein calreticulin (CALR, panel 3) or merged image (panel 4); counter‐stained with Hoechst (panel 1). Note that in YIPF5‐I98S β‐like cells, the accumulated proinsulin colocalized with calreticulin.

Article Snippet: After nonreducing SDS‐PAGE of purified recombinant human proinsulin expressed in Escherichia coli (N‐terminally 6xHis‐tagged, purchased from Creative Biomart, Shirley, NY; with no attempt for in vitro refolding to the native state) increased detection of monomers, with decreased detection of intermolecular disulfide‐linked forms, was observed upon post‐gel disulfide reduction (Supplemental Figure , quantified in ).

Techniques: Control, Western Blot, Electrotransfer, Mutagenesis, Quantitation Assay, Immunostaining, Staining

Journal: Protein Science : A Publication of the Protein Society

Article Title: Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis

doi: 10.1002/pro.4949

Figure Lengend Snippet: Misfolded proinsulin accumulates upon disruption of ER‐to‐Golgi trafficking. (a) Min6 cells were treated ± Brefeldin A (BFA, 10 μg/mL) for 10 min in the absence or presence of 5 mM DTT (as indicated) before analysis by 12% NuPAGE under nonreducing conditions or reducing (last lane used as a MW marker of fully reduced proinsulin) followed by immunoblotting with anti‐rodent proinsulin (CCI‐17). Secreted proinsulin from untreated Min6 cells ( far left ) is a marker of endogenous native proinsulin. Native intracellular proinsulin is shown with blue arrow; proinsulin disulfide‐linked dimers and misfolded proinsulin monomers are shown with red arrows. (b) Isolated pancreatic islets from Ins2‐KO mice were treated as outline at the top of panel: first a 4 h treatment with cycloheximide (first lane: CHX) followed by two washes with PBS and a further 45 min culture in complete medium in the absence (Vehicle) or presence of BFA (10 μg/mL). The islets were then lysed and resolved by straight 12% NuPAGE under nonreducing (left panel) or reducing (right panel) conditions. The last lane in each panel shows the position of proinsulin (Proins) secreted overnight from Ins2‐KO islets as an internal control. The nonreducing gel was treated with DTT + heating prior to transfer for immunoblotting with anti‐rodent proinsulin. Native proinsulin is shown ( blue arrow ); non‐native monomers and disulfide‐linked proinsulin dimers are also observed ( red arrows ). Cyclophilin B (Cyp B) is a loading control. (c) Quantitation of native proinsulin monomers recovered as a fraction of total proinsulin from experiments performed as in panel B ( n = 3 independent experiments; mean ± SD; ** p = 0.0099). (d) Isolated human islets were treated as described for rodent islets in panel B; BFA treatment decreased the recovery of native proinsulin ( blue arrow ) and increased misfolded proinsulin ( red arrows ).

Article Snippet: After nonreducing SDS‐PAGE of purified recombinant human proinsulin expressed in Escherichia coli (N‐terminally 6xHis‐tagged, purchased from Creative Biomart, Shirley, NY; with no attempt for in vitro refolding to the native state) increased detection of monomers, with decreased detection of intermolecular disulfide‐linked forms, was observed upon post‐gel disulfide reduction (Supplemental Figure , quantified in ).

Techniques: Disruption, Marker, Western Blot, Isolation, Control, Quantitation Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis

doi: 10.1002/pro.4949

Figure Lengend Snippet: Amelioration of proinsulin misfolding in β‐cells. (a) INS1E cells were pre‐treated with brefeldin A for 2 h (“BFA pre‐treat”) followed either by an additional 90 min in the presence of cycloheximide (“BFA con't + CHX”) or BFA washout involving a quick PBS wash followed by addition of complete RPMI medium containing CHX plus Monensin (“BFA → CHX + Mon”). Cell lysates from each group were either resolved by 12% NuPAGE under reduced conditions, or under nonreduced conditions with post‐gel disulfide reduction before electrotransfer and immunoblotting with anti‐rodent proinsulin. Native proinsulin monomers ( blue arrow ) and disulfide‐linked dimers and non‐native proinsulin monomers ( red arrows ) are indicated. β‐actin (β‐act, shown above) is a loading control. (b) Quantitation (from 4 independent experiments; mean ± SD) of disulfide‐linked proinsulin dimers; nonnative proinsulin monomers; and native proinsulin monomers are shown with p ‐values or nonsignificant changes (ns) indicated on the figure. (c) INS1E cells were pre‐treated for 60 min in phosphate buffered saline plus 20 mM 2‐deoxyglucose (“2DG”), followed either by an additional 30 min in the presence of CHX (“→2DG, 30 min”) or by washout involving replacement with complete RPMI medium containing CHX plus Monensin (“→RPMI, 30 min”). Cell lysates from each group were either resolved by 12% NuPAGE under reduced conditions, or under nonreduced conditions with post‐gel disulfide reduction before electrotransfer and immunoblotting with anti‐rodent proinsulin. Native proinsulin monomers ( blue arrow ) and disulfide‐linked dimers and non‐native proinsulin monomers ( red arrows ) are indicated. Hsp90 (shown below) is a loading control. (d) Quantitation (from at least four independent experiments; mean ± SD) of disulfide‐linked proinsulin dimers; nonnative proinsulin monomers; and native proinsulin monomers are shown with p ‐values or nonsignificant changes (ns) indicated directly on the figure.

Article Snippet: After nonreducing SDS‐PAGE of purified recombinant human proinsulin expressed in Escherichia coli (N‐terminally 6xHis‐tagged, purchased from Creative Biomart, Shirley, NY; with no attempt for in vitro refolding to the native state) increased detection of monomers, with decreased detection of intermolecular disulfide‐linked forms, was observed upon post‐gel disulfide reduction (Supplemental Figure , quantified in ).

Techniques: Electrotransfer, Western Blot, Control, Quantitation Assay, Saline

Journal: Protein Science : A Publication of the Protein Society

Article Title: Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis

doi: 10.1002/pro.4949

Figure Lengend Snippet: After nonreducing SDS‐PAGE, post‐gel disulfide reduction improves detection of proinsulin in distinct folded states. (a) Schematic of different immunoblotting methods to detect proinsulin misfolding. At left: Conventional immunoblotting after nonreducing SDS‐PAGE is super‐sensitive to misfolded disulfide‐linked complexes of proinsulin. Red arrow pathway: Treatment of samples with DTT + heating after nonreducing SDS‐PAGE converts all proinsulin contained within the gel to reduced monomers. Using a fixed percentage of acrylamide ensures even transfer efficiency across the gel. This method enhances detection of proinsulin monomers (particularly native monomers) after nonreducing SDS‐PAGE and provides a more quantitative estimate of the distribution of differently‐folded proinsulin species. (b) Cartoon highlighting the impact of the blotting method described in panel A for pancreatic β‐cells after nonreducing SDS‐PAGE. (c) Immunblotting with anti‐insulin or with monoclonal antibodies (generated by Hytest) targeting different regions of rodent proinsulin C‐peptide (A cartoon above shows the location of antibody binding on the proinsulin C‐peptide). Identical samples of INS1E cell lysates were resolved by SDS‐PAGE under nonreducing (lanes 1, 3, 5, 7, 9, 11, 13, and 15) or reducing conditions (lanes 2, 4, 6, 8, 10, 12, 14, and 16). After running, the gels were divided, and lanes 3,4; 7,8; 11,12; and 15,16 were treated with 100 mM DTT + heating for post‐gel disulfide reduction (indicated below as – or +) before electrotransfer and immunoblotting with the indicated antibodies. Immunoblotting with anti‐insulin that cross‐reacts with proinsulin show dramatic signal loss (for both proinsulin and insulin, dotted red lines ) upon post‐gel disulfide reduction. The curved blue arrows connecting lanes 5–7, 9–11, or 13–15 bearing samples that were run under nonreduced conditions show dramatically improved detection of proinsulin monomers by post‐gel disulfide reduction. (d) 293 T cells were transfected with proinsulin “keep one bond” constructs—keep‐B19/A20, keep‐B7/A7, or keep‐A6/A11. After 24 h, the cells were lysed and identical aliquots of cell lysate were resolved by SDS‐PAGE (12% NuPAGE) under nonreduced (first 6 lanes) or reduced conditions (last three lanes). After electrophoresis, a portion of the nonreduced gel underwent post‐gel disulfide reduction with 100 mM DTT + heating to 60°C for 15 min (middle three lanes) prior to electrotransfer and immunoblotting with anti‐human proinsulin. The detection of proinsulin monomers by nonreducing SDS‐PAGE increased after post‐gel disulfide reduction with DTT + heating ( curved blue arrows ). (e) Immunoblots were quantified by densitometry; the relative recovery of proinsulin monomers before and after post‐gel disulfide reduction is shown ( n = 4 independent experiments; mean ± SD; * p < 0.05; ns = non‐significant). (f) 293 T cells were either untransfected (“U”) or transfected to express WT hPro‐CpepMyc or that bearing the L(A16)P mutation (here simply labeled as A16P), and culture media was collected overnight. The samples were resolved by 12% NuPAGE under nonreduced (first 5 lanes) or reduced conditions (last 5 lanes) and the nonreduced gel underwent post‐gel disulfide reduction (indicated at bottom) before electrotransfer and immunoblotting with anti‐human proinsulin. Cyclophilin B (CypB) is a loading control. (g) Quantitation of native proinsulin monomers recovered as a fraction of total proinsulin ( n = 4 independent experiments; mean ± SD; ** p = 0.0022).

Article Snippet: Recombinant human proinsulin (expressed in E. coli ) was purchased from Creative Biomart (Catalog # INS‐315H).

Techniques: SDS Page, Western Blot, Bioprocessing, Generated, Binding Assay, Electrotransfer, Transfection, Construct, Electrophoresis, Mutagenesis, Labeling, Control, Quantitation Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis

doi: 10.1002/pro.4949

Figure Lengend Snippet: Folding status of endogenous (intracellular and secreted) proinsulin from Min6 cells. (a) Identical aliquots of Min6 cell lysate and 6 h conditioned media were resolved by nonreducing 12% NuPAGE. After electrophoresis, the gel was cut in fourths ( dotted lines ) and each quarter treated as indicated (below the image) prior to electrotransfer to nitrocellulose for immunoblotting with anti‐rodent proinsulin. Improved detection of secreted proinsulin ( blue arrow , as well as intracellular proinsulin monomer bearing native disulfide bonds) is noted after post‐gel disulfide reduction (optimized with DTT + heating). (b) Min6 cells in complete medium were incubated for 4 h at 37 or 29°C, as indicated. Secretion of proinsulin (Proins) from cells to media is shown at both temperatures ( curved blue arrows; solid or dashed, respectively ). The non‐native proinsulin monomer band is identified with a red arrow . Cyclophilin B (Cyp B) is a loading control. (c) Quantitation of native proinsulin intracellularly from replicate experiments like those shown in panel B as a fraction of total (cells + media) proinsulin ( n = 3 independent experiments; mean ± SD; * p = 0.045). Each point represents an independent experiment (mean ± SD). (d) Min6 cells were either untreated or treated with Monensin (15 μM for 90 min) as indicated. Secretion of proinsulin (Proins) from cells to media under both conditions is indicated ( curved blue arrows; solid or dashed , respectively ). The non‐native proinsulin monomer band is identified with a red arrow . Cyclophilin B (Cyp B) is a loading control. In panels 2a and 2d, nonspecific bands in the media migrating at >50 kDa are largely derived from serum albumin, observed in similar blotting exposures of culture medium that was never incubated with cells. (e) Quantitation of native proinsulin intracellularly from replicate experiments like those shown in panel D as a fraction of total (cells + media) proinsulin ( n = 4 independent experiments; mean ± SD; * p = 0.012).

Article Snippet: Recombinant human proinsulin (expressed in E. coli ) was purchased from Creative Biomart (Catalog # INS‐315H).

Techniques: Electrophoresis, Electrotransfer, Western Blot, Incubation, Control, Quantitation Assay, Derivative Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis

doi: 10.1002/pro.4949

Figure Lengend Snippet: Proinsulin folding response to pharmacological perturbation of ER homeostasis. (a) Identical wells of Min6 cells were treated ± Antimycin A (6 μM) for 1.5 h, and equal aliquots of cell lysate or media were resolved by 12% NuPAGE under nonreducing (lanes 1–8) or reducing conditions (lanes 9–12). Identical nonreduced samples were either not exposed (lanes 1–4) or exposed (lanes 5–8) to post‐gel disulfide reductions (as indicated below) prior to electrotransfer and immunoblotting with anti‐rodent proinsulin (Proins). Non‐native proinsulin monomers and disulfide‐linked proinsulin dimers are indicated ( red arrows ); native proinsulin monomers migrate faster ( blue arrow ). (b) Quantitation of native proinsulin monomers recovered as a fraction of total proinsulin ( n = 3 independent experiments; mean ± SD; * p = 0.0045). (c) Identical wells of Min6 cells were untreated or treated with PERK inhibitor (2 μM GSK2656157, overnight, as indicated above), and equal aliquots of cells (C) or media (M) were resolved by 12% NuPAGE under nonreducing (lanes 1–8) or reducing (lanes 9–16) conditions. Identical nonreduced samples were either exposed (lanes 1–4) or not exposed (lanes 5–8) to post‐gel disulfide reduction (as indicated below) prior to electrotransfer and immunoblotting with anti‐rodent proinsulin (Proins). Proinsulin monomers bearing native ( blue arrow ) or non‐native ( red arrow ) disulfide bonds are indicated. (d) Quantitation of native proinsulin monomers recovered as a fraction of total proinsulin ( n = 5 independent experiments; mean ± SD; * p = 0.0062). (e) Human islets (nondiabetic donor) were untreated (DMSO) or treated with PERK inhibitor (2 μM GSK2656157, 24 h) and equal aliquots of cells or media were resolved by 12% NuPAGE under nonreducing (lanes 1–8) or reducing (lanes 9–12) conditions. nonreducing or reducing conditions as indicated at bottom. Identical nonreduced samples were either not exposed (lanes 1–4) or exposed (lanes 5–8) to post‐gel disulfide reduction (as indicated below) prior to electrotransfer and immunoblotting with anti‐human proinsulin (Proins). Proinsulin monomers bearing native ( blue arrow ) or non‐native ( red arrow ) disulfide bonds are indicated.

Article Snippet: Recombinant human proinsulin (expressed in E. coli ) was purchased from Creative Biomart (Catalog # INS‐315H).

Techniques: Electrotransfer, Western Blot, Quantitation Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis

doi: 10.1002/pro.4949

Figure Lengend Snippet: Proinsulin misfolding in the ER of YIPF5‐I98S iPSCs. (a) Control or YIPF5‐I98S (YIPF5*) iPSCs were differentiated to stage 7 β‐like cells prior to analysis. Media containing secretion from human islets was collected as a control to identify the position of native human proinsulin as detected by immunoblotting under nonreduced conditions ( lanes 1 and 7 ). Lysates of β‐like cells were analyzed under nonreduced ( lanes 2,3 and 8,9 ) or reduced conditions ( lanes 5,6 and 11,12 ). Identical nonreduced samples were either exposed ( lanes 1–3 ) or not exposed ( lanes 7–9 ) to post‐gel disulfide reduction (as indicated above) prior to electrotransfer and immunoblotting with anti‐human proinsulin (Proins). Intracellularly, the mutant β‐like cells exhibited a decreased abundance of native proinsulin ( blue arrow ). Non‐native proinsulin monomers are shown with red arrow . The same samples were analyzed by immunoblotting with guinea pig anti‐insulin, which primarily recognizes disulfide‐linked two‐chain mature insulin ( lanes 8,9 ). Cyclophilin B (CypB) is a loading control. (b) Quantitation of native proinsulin ( blue arrow ) as a fraction of total cellular proinsulin (recovered under reduced conditions) from at least three independent differentiation experiments like that shown in panel A (mean ± SD; * p = 0.044). (c) Quantitation of mature insulin (normalized to the loading control) from the same samples as those quantified in panel B (mean ± SD; * p = 0.032). In panels D and E, Stage 7 islet‐like clusters differentiated from embryonic stem cells from WT or YIPF5‐I98S were implanted under the kidney capsule of immunocompromised NOD/SCID‐γ mice and then retrieved for processing 3 months thereafter (the WT cell images are above and YIPF5‐I98S cell images are below). (d) Co‐immunostaining for insulin (INS, panel 2), proinsulin (PROINS, panel 3), or merged image (panel 4); counter‐stained with Hoechst (panel 1). (e) Co‐immunostaining for proinsulin (PROINS, panel 2), the ER protein calreticulin (CALR, panel 3) or merged image (panel 4); counter‐stained with Hoechst (panel 1). Note that in YIPF5‐I98S β‐like cells, the accumulated proinsulin colocalized with calreticulin.

Article Snippet: Recombinant human proinsulin (expressed in E. coli ) was purchased from Creative Biomart (Catalog # INS‐315H).

Techniques: Control, Western Blot, Electrotransfer, Mutagenesis, Quantitation Assay, Immunostaining, Staining

Journal: Protein Science : A Publication of the Protein Society

Article Title: Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis

doi: 10.1002/pro.4949

Figure Lengend Snippet: Misfolded proinsulin accumulates upon disruption of ER‐to‐Golgi trafficking. (a) Min6 cells were treated ± Brefeldin A (BFA, 10 μg/mL) for 10 min in the absence or presence of 5 mM DTT (as indicated) before analysis by 12% NuPAGE under nonreducing conditions or reducing (last lane used as a MW marker of fully reduced proinsulin) followed by immunoblotting with anti‐rodent proinsulin (CCI‐17). Secreted proinsulin from untreated Min6 cells ( far left ) is a marker of endogenous native proinsulin. Native intracellular proinsulin is shown with blue arrow; proinsulin disulfide‐linked dimers and misfolded proinsulin monomers are shown with red arrows. (b) Isolated pancreatic islets from Ins2‐KO mice were treated as outline at the top of panel: first a 4 h treatment with cycloheximide (first lane: CHX) followed by two washes with PBS and a further 45 min culture in complete medium in the absence (Vehicle) or presence of BFA (10 μg/mL). The islets were then lysed and resolved by straight 12% NuPAGE under nonreducing (left panel) or reducing (right panel) conditions. The last lane in each panel shows the position of proinsulin (Proins) secreted overnight from Ins2‐KO islets as an internal control. The nonreducing gel was treated with DTT + heating prior to transfer for immunoblotting with anti‐rodent proinsulin. Native proinsulin is shown ( blue arrow ); non‐native monomers and disulfide‐linked proinsulin dimers are also observed ( red arrows ). Cyclophilin B (Cyp B) is a loading control. (c) Quantitation of native proinsulin monomers recovered as a fraction of total proinsulin from experiments performed as in panel B ( n = 3 independent experiments; mean ± SD; ** p = 0.0099). (d) Isolated human islets were treated as described for rodent islets in panel B; BFA treatment decreased the recovery of native proinsulin ( blue arrow ) and increased misfolded proinsulin ( red arrows ).

Article Snippet: Recombinant human proinsulin (expressed in E. coli ) was purchased from Creative Biomart (Catalog # INS‐315H).

Techniques: Disruption, Marker, Western Blot, Isolation, Control, Quantitation Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis

doi: 10.1002/pro.4949

Figure Lengend Snippet: Amelioration of proinsulin misfolding in β‐cells. (a) INS1E cells were pre‐treated with brefeldin A for 2 h (“BFA pre‐treat”) followed either by an additional 90 min in the presence of cycloheximide (“BFA con't + CHX”) or BFA washout involving a quick PBS wash followed by addition of complete RPMI medium containing CHX plus Monensin (“BFA → CHX + Mon”). Cell lysates from each group were either resolved by 12% NuPAGE under reduced conditions, or under nonreduced conditions with post‐gel disulfide reduction before electrotransfer and immunoblotting with anti‐rodent proinsulin. Native proinsulin monomers ( blue arrow ) and disulfide‐linked dimers and non‐native proinsulin monomers ( red arrows ) are indicated. β‐actin (β‐act, shown above) is a loading control. (b) Quantitation (from 4 independent experiments; mean ± SD) of disulfide‐linked proinsulin dimers; nonnative proinsulin monomers; and native proinsulin monomers are shown with p ‐values or nonsignificant changes (ns) indicated on the figure. (c) INS1E cells were pre‐treated for 60 min in phosphate buffered saline plus 20 mM 2‐deoxyglucose (“2DG”), followed either by an additional 30 min in the presence of CHX (“→2DG, 30 min”) or by washout involving replacement with complete RPMI medium containing CHX plus Monensin (“→RPMI, 30 min”). Cell lysates from each group were either resolved by 12% NuPAGE under reduced conditions, or under nonreduced conditions with post‐gel disulfide reduction before electrotransfer and immunoblotting with anti‐rodent proinsulin. Native proinsulin monomers ( blue arrow ) and disulfide‐linked dimers and non‐native proinsulin monomers ( red arrows ) are indicated. Hsp90 (shown below) is a loading control. (d) Quantitation (from at least four independent experiments; mean ± SD) of disulfide‐linked proinsulin dimers; nonnative proinsulin monomers; and native proinsulin monomers are shown with p ‐values or nonsignificant changes (ns) indicated directly on the figure.

Article Snippet: Recombinant human proinsulin (expressed in E. coli ) was purchased from Creative Biomart (Catalog # INS‐315H).

Techniques: Electrotransfer, Western Blot, Control, Quantitation Assay, Saline