The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Leena Haataja - One of the best experts on this subject based on the ideXlab platform.
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distinct states of Proinsulin misfolding in midy
Cellular and Molecular Life Sciences, 2021Co-Authors: Leena Haataja, Ming Liu, Billy Tsai, Balamurugan Dhayalan, Anoop Arunagiri, Michael A Weiss, Anis Hassan, Kaitlin Regan, Peter ArvanAbstract:A precondition for efficient Proinsulin export from the endoplasmic reticulum (ER) is that Proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) "interchain" disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third Proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a "lose-A6/A11" mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of Proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed Proinsulin disulfide bond formation. Here, we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep + 2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic β-cells. Three of these mutants, however, must disrupt the Cys(A6)-Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of Proinsulin folding and ER export, because when introduced into the Proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type Proinsulin. A growing molecular understanding of Proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.
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predisposition to Proinsulin misfolding as a genetic risk to diet induced diabetes
bioRxiv, 2021Co-Authors: M Alam, Leena Haataja, Anoop Arunagiri, M Torres, Daniel J Larkin, John W Kappler, N Jin, Peter ArvanAbstract:Throughout evolution, Proinsulin has exhibited significant sequence variation in both C-peptide and insulin moieties. As the Proinsulin coding sequence evolves, the gene product continues to be under selection pressure both for ultimate insulin bioactivity and for the ability of Proinsulin to be folded for export through the secretory pathway of pancreatic {beta}-cells. The substitution Proinsulin-R(B22)E is known to yield a bioactive insulin, although R(B22)Q has been reported as a mutation that falls within the spectrum of Mutant INS-gene induced Diabetes of Youth (MIDY). Here we have studied mice expressing heterozygous (or homozygous) Proinsulin-R(B22)E knocked into the Ins2 locus. Neither females nor males bearing the heterozygous mutation develop diabetes at any age examined, but subtle evidence of increased Proinsulin misfolding in the endoplasmic reticulum is demonstrable in isolated islets from the heterozygotes. Moreover, males have indications of glucose intolerance and within a few week exposure to a high-fat diet, they develop frank diabetes. Diabetes is more severe in homozygotes, and the development of disease parallels a progressive heterogeneity of {beta}-cells with increasing fractions of Proinsulin-rich/insulin-poor cells, as well as glucagon-positive cells. Evidently, sub-threshold predisposition to Proinsulin misfolding can go undetected, but provides genetic susceptibility to diet-induced {beta}-cell failure.
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distinct states of Proinsulin misfolding in midy
bioRxiv, 2021Co-Authors: Leena Haataja, Ming Liu, Billy Tsai, Balamurugan Dhayalan, Anoop Arunagiri, Michael A Weiss, Anis Hassan, Kaitlin Regan, Peter ArvanAbstract:A precondition for efficient Proinsulin export from the endoplasmic reticulum (ER) is that Proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) "interchain" disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third Proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a "lose-A6/A11" mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of Proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed Proinsulin disulfide bond formation. Here we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep+2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic {beta}-cells. Three of these mutants, however, must disrupt the Cys(A6)-Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of Proinsulin folding and ER export, because when introduced into the Proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type Proinsulin. A growing molecular understanding of Proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.
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biological behaviors of mutant Proinsulin contribute to the phenotypic spectrum of diabetes associated with insulin gene mutations
Molecular and Cellular Endocrinology, 2020Co-Authors: Heting Wang, Ming Liu, Leena Haataja, Cecile Saintmartin, Li Ding, Ruodan Wang, Wenli Feng, Hua Shu, Zhenqian Fan, Peter ArvanAbstract:Insulin gene mutation is the second most common cause of neonatal diabetes (NDM). It is also one of the genes involved in maturity-onset diabetes of the young (MODY). We aim to investigate molecular behaviors of different INS gene variants that may correlate with the clinical spectrum of diabetes phenotypes. In this study, we concentrated on two previously uncharacterized MODY-causing mutants, Proinsulin-p.Gly44Arg [G(B20)R] and p.Pro52Leu [P(B28)L] (a novel mutant identified in one French family), and an NDM causing Proinsulin-p.(Cys96Tyr) [C(A7)Y]. We find that these Proinsulin mutants exhibit impaired oxidative folding in the endoplasmic reticulum (ER) with blocked ER export, ER stress, and apoptosis. Importantly, the Proinsulin mutants formed abnormal intermolecular disulfide bonds that not only involved the mutant Proinsulin, but also the co-expressed WT-Proinsulin, forming misfolded disulfide-linked Proinsulin complexes. This impaired the intracellular trafficking of WT-Proinsulin and limited the production of bioactive mature insulin. Notably, although all three mutants presented with similar defects in folding, trafficking, and dominant negative behavior, the degrees of these defects appeared to be different. Specifically, compared to MODY mutants G(B20)R and P(B28)L that partially affected folding and trafficking of co-expressed WT-Proinsulin, the NDM mutant C(A7)Y resulted in an almost complete blockade of the ER export of WT-Proinsulin, decreasing insulin production, inducing more severe ER stress and apoptosis. We thus demonstrate that differences in cell biological behaviors among different Proinsulin mutants correlate with the spectrum of diabetes phenotypes caused by the different INS gene mutations.
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role of Proinsulin self association in mutant ins gene induced diabetes of youth
bioRxiv, 2019Co-Authors: Jinhong Sun, Leena Haataja, Dennis Larkin, Yi Xiong, Wei Chen, Saiful A Mir, Rachel Madley, Arfah Anjum, Balamurugan Dhayalan, Nischay RegeAbstract:Abstract Abnormal interactions between misfolded mutant and wild-type (WT) Proinsulin in the endoplasmic reticulum (ER) drive the molecular pathogenesis of Mutant-INS-gene induced Diabetes of Youth (MIDY). How these abnormal interactions are initiated remains unknown. Normally, Proinsulin-WT dimerizes in the ER. Here, we suggest that the normal Proinsulin-Proinsulin contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that Proinsulin Tyr-B16, which is a key residue in normal Proinsulin dimerization, helps confer dominant-negative behavior of MIDY mutant Proinsulin-C(A7)Y. Substitutions of Tyr-B16 with ether Ala, Asp, or Pro in Proinsulin-C(A7)Y each decrease the abnormal interactions between the MIDY mutant and Proinsulin-WT, rescuing Proinsulin-WT export, limiting ER stress, and increasing insulin production in β-cells and human islets. This study reveals the first evidence indicating that noncovalent Proinsulin-Proinsulin contact initiates dominant-negative behavior of misfolded Proinsulin, pointing to a novel therapeutic target to enhance bystander Proinsulin export for increased insulin production.
Peter Arvan - One of the best experts on this subject based on the ideXlab platform.
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distinct states of Proinsulin misfolding in midy
Cellular and Molecular Life Sciences, 2021Co-Authors: Leena Haataja, Ming Liu, Billy Tsai, Balamurugan Dhayalan, Anoop Arunagiri, Michael A Weiss, Anis Hassan, Kaitlin Regan, Peter ArvanAbstract:A precondition for efficient Proinsulin export from the endoplasmic reticulum (ER) is that Proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) "interchain" disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third Proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a "lose-A6/A11" mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of Proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed Proinsulin disulfide bond formation. Here, we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep + 2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic β-cells. Three of these mutants, however, must disrupt the Cys(A6)-Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of Proinsulin folding and ER export, because when introduced into the Proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type Proinsulin. A growing molecular understanding of Proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.
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predisposition to Proinsulin misfolding as a genetic risk to diet induced diabetes
bioRxiv, 2021Co-Authors: M Alam, Leena Haataja, Anoop Arunagiri, M Torres, Daniel J Larkin, John W Kappler, N Jin, Peter ArvanAbstract:Throughout evolution, Proinsulin has exhibited significant sequence variation in both C-peptide and insulin moieties. As the Proinsulin coding sequence evolves, the gene product continues to be under selection pressure both for ultimate insulin bioactivity and for the ability of Proinsulin to be folded for export through the secretory pathway of pancreatic {beta}-cells. The substitution Proinsulin-R(B22)E is known to yield a bioactive insulin, although R(B22)Q has been reported as a mutation that falls within the spectrum of Mutant INS-gene induced Diabetes of Youth (MIDY). Here we have studied mice expressing heterozygous (or homozygous) Proinsulin-R(B22)E knocked into the Ins2 locus. Neither females nor males bearing the heterozygous mutation develop diabetes at any age examined, but subtle evidence of increased Proinsulin misfolding in the endoplasmic reticulum is demonstrable in isolated islets from the heterozygotes. Moreover, males have indications of glucose intolerance and within a few week exposure to a high-fat diet, they develop frank diabetes. Diabetes is more severe in homozygotes, and the development of disease parallels a progressive heterogeneity of {beta}-cells with increasing fractions of Proinsulin-rich/insulin-poor cells, as well as glucagon-positive cells. Evidently, sub-threshold predisposition to Proinsulin misfolding can go undetected, but provides genetic susceptibility to diet-induced {beta}-cell failure.
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distinct states of Proinsulin misfolding in midy
bioRxiv, 2021Co-Authors: Leena Haataja, Ming Liu, Billy Tsai, Balamurugan Dhayalan, Anoop Arunagiri, Michael A Weiss, Anis Hassan, Kaitlin Regan, Peter ArvanAbstract:A precondition for efficient Proinsulin export from the endoplasmic reticulum (ER) is that Proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) "interchain" disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third Proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a "lose-A6/A11" mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of Proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed Proinsulin disulfide bond formation. Here we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep+2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic {beta}-cells. Three of these mutants, however, must disrupt the Cys(A6)-Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of Proinsulin folding and ER export, because when introduced into the Proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type Proinsulin. A growing molecular understanding of Proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.
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biological behaviors of mutant Proinsulin contribute to the phenotypic spectrum of diabetes associated with insulin gene mutations
Molecular and Cellular Endocrinology, 2020Co-Authors: Heting Wang, Ming Liu, Leena Haataja, Cecile Saintmartin, Li Ding, Ruodan Wang, Wenli Feng, Hua Shu, Zhenqian Fan, Peter ArvanAbstract:Insulin gene mutation is the second most common cause of neonatal diabetes (NDM). It is also one of the genes involved in maturity-onset diabetes of the young (MODY). We aim to investigate molecular behaviors of different INS gene variants that may correlate with the clinical spectrum of diabetes phenotypes. In this study, we concentrated on two previously uncharacterized MODY-causing mutants, Proinsulin-p.Gly44Arg [G(B20)R] and p.Pro52Leu [P(B28)L] (a novel mutant identified in one French family), and an NDM causing Proinsulin-p.(Cys96Tyr) [C(A7)Y]. We find that these Proinsulin mutants exhibit impaired oxidative folding in the endoplasmic reticulum (ER) with blocked ER export, ER stress, and apoptosis. Importantly, the Proinsulin mutants formed abnormal intermolecular disulfide bonds that not only involved the mutant Proinsulin, but also the co-expressed WT-Proinsulin, forming misfolded disulfide-linked Proinsulin complexes. This impaired the intracellular trafficking of WT-Proinsulin and limited the production of bioactive mature insulin. Notably, although all three mutants presented with similar defects in folding, trafficking, and dominant negative behavior, the degrees of these defects appeared to be different. Specifically, compared to MODY mutants G(B20)R and P(B28)L that partially affected folding and trafficking of co-expressed WT-Proinsulin, the NDM mutant C(A7)Y resulted in an almost complete blockade of the ER export of WT-Proinsulin, decreasing insulin production, inducing more severe ER stress and apoptosis. We thus demonstrate that differences in cell biological behaviors among different Proinsulin mutants correlate with the spectrum of diabetes phenotypes caused by the different INS gene mutations.
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cells deploy a two pronged strategy to rectify misfolded Proinsulin aggregates
Molecular Cell, 2019Co-Authors: Corey N Cunningham, Peter Arvan, Anoop Arunagiri, Jeffrey M Williams, Jeffrey Knupp, Billy TsaiAbstract:Insulin gene coding sequence mutations are known to cause mutant INS-gene-induced diabetes of youth (MIDY), yet the cellular pathways needed to prevent misfolded Proinsulin accumulation remain incompletely understood. Here, we report that Akita mutant Proinsulin forms detergent-insoluble aggregates that entrap wild-type (WT) Proinsulin in the endoplasmic reticulum (ER), thereby blocking insulin production. Two distinct quality-control mechanisms operate together to combat this insult: the ER luminal chaperone Grp170 prevents Proinsulin aggregation, while the ER membrane morphogenic protein reticulon-3 (RTN3) disposes of aggregates via ER-coupled autophagy (ER-phagy). We show that enhanced RTN-dependent clearance of aggregated Akita Proinsulin helps to restore ER export of WT Proinsulin, which can promote WT insulin production, potentially alleviating MIDY. We also find that RTN3 participates in the clearance of other mutant prohormone aggregates. Together, these results identify a series of substrates of RTN3-mediated ER-phagy, highlighting RTN3 in the disposal of pathogenic prohormone aggregates.
Ming Liu - One of the best experts on this subject based on the ideXlab platform.
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distinct states of Proinsulin misfolding in midy
Cellular and Molecular Life Sciences, 2021Co-Authors: Leena Haataja, Ming Liu, Billy Tsai, Balamurugan Dhayalan, Anoop Arunagiri, Michael A Weiss, Anis Hassan, Kaitlin Regan, Peter ArvanAbstract:A precondition for efficient Proinsulin export from the endoplasmic reticulum (ER) is that Proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) "interchain" disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third Proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a "lose-A6/A11" mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of Proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed Proinsulin disulfide bond formation. Here, we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep + 2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic β-cells. Three of these mutants, however, must disrupt the Cys(A6)-Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of Proinsulin folding and ER export, because when introduced into the Proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type Proinsulin. A growing molecular understanding of Proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.
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distinct states of Proinsulin misfolding in midy
bioRxiv, 2021Co-Authors: Leena Haataja, Ming Liu, Billy Tsai, Balamurugan Dhayalan, Anoop Arunagiri, Michael A Weiss, Anis Hassan, Kaitlin Regan, Peter ArvanAbstract:A precondition for efficient Proinsulin export from the endoplasmic reticulum (ER) is that Proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) "interchain" disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third Proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a "lose-A6/A11" mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of Proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed Proinsulin disulfide bond formation. Here we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep+2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic {beta}-cells. Three of these mutants, however, must disrupt the Cys(A6)-Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of Proinsulin folding and ER export, because when introduced into the Proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type Proinsulin. A growing molecular understanding of Proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.
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biological behaviors of mutant Proinsulin contribute to the phenotypic spectrum of diabetes associated with insulin gene mutations
Molecular and Cellular Endocrinology, 2020Co-Authors: Heting Wang, Ming Liu, Leena Haataja, Cecile Saintmartin, Li Ding, Ruodan Wang, Wenli Feng, Hua Shu, Zhenqian Fan, Peter ArvanAbstract:Insulin gene mutation is the second most common cause of neonatal diabetes (NDM). It is also one of the genes involved in maturity-onset diabetes of the young (MODY). We aim to investigate molecular behaviors of different INS gene variants that may correlate with the clinical spectrum of diabetes phenotypes. In this study, we concentrated on two previously uncharacterized MODY-causing mutants, Proinsulin-p.Gly44Arg [G(B20)R] and p.Pro52Leu [P(B28)L] (a novel mutant identified in one French family), and an NDM causing Proinsulin-p.(Cys96Tyr) [C(A7)Y]. We find that these Proinsulin mutants exhibit impaired oxidative folding in the endoplasmic reticulum (ER) with blocked ER export, ER stress, and apoptosis. Importantly, the Proinsulin mutants formed abnormal intermolecular disulfide bonds that not only involved the mutant Proinsulin, but also the co-expressed WT-Proinsulin, forming misfolded disulfide-linked Proinsulin complexes. This impaired the intracellular trafficking of WT-Proinsulin and limited the production of bioactive mature insulin. Notably, although all three mutants presented with similar defects in folding, trafficking, and dominant negative behavior, the degrees of these defects appeared to be different. Specifically, compared to MODY mutants G(B20)R and P(B28)L that partially affected folding and trafficking of co-expressed WT-Proinsulin, the NDM mutant C(A7)Y resulted in an almost complete blockade of the ER export of WT-Proinsulin, decreasing insulin production, inducing more severe ER stress and apoptosis. We thus demonstrate that differences in cell biological behaviors among different Proinsulin mutants correlate with the spectrum of diabetes phenotypes caused by the different INS gene mutations.
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a reference map of the human Proinsulin biosynthetic interaction network
bioRxiv, 2019Co-Authors: Duc T Tran, Ming Liu, Saiful A Mir, Anita Pottekat, Salvatore Loguercio, Insook Jang, Alexandre Rosa Campos, Reyhaneh LahmyAbstract:The beta-cell secretory protein synthetic machinery is dedicated to insulin production, and recent reports suggest that both Proinsulin misfolding and accompanying beta-cell oxidative stress could be common features in type 2 diabetes (T2D). Despite the critical role of insulin in organismal homeostasis, the precise network of interactions from early Proinsulin synthesis and folding in the ER, to its subsequent trafficking through the secretory pathway, remain poorly defined. In the present study we utilized a human Proinsulin-specific monoclonal antibody for affinity purification mass spectrometry, to yield unbiased profiling of the Proinsulin interactome in human islets. The data reveal that human Proinsulin interacts with a network of ER folding factors (including chaperones: e.g. ERDJ5, ERDJ3, GRP94, and BiP; and oxidoreductases: e.g. QSOX1, DUOX2, and PRDX4) that are remarkably conserved across both genders and 3 ethnicities. Knockdown of one of the most prominent hits, peroxiredoxin-4 (PRDX4) in MIN6 beta-cells, rendered Proinsulin more susceptible to misfolding. Additionally, oxidant exposure in human islets enhanced Proinsulin:BiP interactions with augmented Proinsulin misfolding. Finally, oxidant exposure in human islets also led to sulfonylation of PRDX4, a modification known to inactivate peroxiredoxins. Interestingly, we observed significantly higher levels of sulfonylated (inactive) PRDX4 in islets from patients with T2D compared to that of normal islets. Taken together, these data provide a detailed reference map of the human Proinsulin interaction network and suggest critical unrecognized areas for study in insulin biosynthesis, beta cell function, and T2D.
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pdia1 p4hb is required for efficient Proinsulin maturation and s cell health in response to diet induced obesity
eLife, 2019Co-Authors: Insook Jang, Ming Liu, Anita Pottekat, Jing Yong, Juthakorn Poothong, Jacqueline Lagunasacosta, Adriana Charbono, Zhouji Chen, Donalyn Scheuner, Pamela ItkinansariAbstract:Regulated Proinsulin biosynthesis, disulfide bond formation and ER redox homeostasis are essential to prevent Type two diabetes. In s cells, protein disulfide isomerase A1 (PDIA1/P4HB), the most abundant ER oxidoreductase of over 17 members, can interact with Proinsulin to influence disulfide maturation. Here we find Pdia1 is required for optimal insulin production under metabolic stress in vivo. s cell-specific Pdia1 deletion in young high-fat diet fed mice or aged mice exacerbated glucose intolerance with inadequate insulinemia and increased the Proinsulin/insulin ratio in both serum and islets compared to wildtype mice. Ultrastructural abnormalities in Pdia1-null s cells include diminished insulin granule content, ER vesiculation and distention, mitochondrial swelling and nuclear condensation. Furthermore, Pdia1 deletion increased accumulation of disulfide-linked high molecular weight Proinsulin complexes and islet vulnerability to oxidative stress. These findings demonstrate that PDIA1 contributes to oxidative maturation of Proinsulin in the ER to support insulin production and s cell health.
Christopher J. Rhodes - One of the best experts on this subject based on the ideXlab platform.
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persistence of pancreatic insulin mrna expression and Proinsulin protein in type 1 diabetes pancreata
Cell Metabolism, 2017Co-Authors: Clive Wasserfall, Leena Haataja, Christopher J. Rhodes, Amanda L Posgai, Harry S Nick, Martha Campbellthompson, Dawn E Beachy, Irina Kusmartseva, Maria Beery, Ezio BonifacioAbstract:Summary The canonical notion that type 1 diabetes (T1D) results following a complete destruction of β cells has recently been questioned as small amounts of C-peptide are detectable in patients with long-standing disease. We analyzed protein and gene expression levels for Proinsulin, insulin, C-peptide, and islet amyloid polypeptide within pancreatic tissues from T1D, autoantibody positive (Ab+), and control organs. Insulin and C-peptide levels were low to undetectable in extracts from the T1D cohort; however, Proinsulin and INS mRNA were detected in the majority of T1D pancreata. Interestingly, heterogeneous nuclear RNA (hnRNA) for insulin and INS-IGF2, both originating from the INS promoter, were essentially undetectable in T1D pancreata, arguing for a silent INS promoter. Expression of PCSK1, a convertase responsible for Proinsulin processing, was reduced in T1D pancreata, supportive of persistent Proinsulin. These data implicate the existence of β cells enriched for inefficient insulin/C-peptide production in T1D patients, potentially less susceptible to autoimmune destruction.
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Increased Secretory Demand Rather than a Defect in the Proinsulin Conversion Mechanism Causes HyperProinsulinemia in a Glucose-infusion Rat Model of Non-Insulin-dependent Diabetes Mellitus
2016Co-Authors: Cristina Alarcn, Jack L. Leahy, Christopher J. RhodesAbstract:HyperProinsulinemia in non-insulin-dependent diabetes mellitus (NIDDM) is due to an increased release of proinsu-lin from pancreatic 8 cells. This could reside in increased secretory demand placed on the 13 cell by hyperglycemia or in the Proinsulin conversion mechanism. In this study, biosynthesis of the Proinsulin conversion enzymes (PC2, PC3, and carboxypeptidase-H [CP-H]) and Proinsulin, were examined in islets isolated from 48-h infused rats with 50 % (wt/vol) glucose (hyperglycemic, hyperinsulinemic, and increased pancreatic Proinsulin to insulin ratio), 20% (wt/vol) glucose (normoglycemic but hyperinsulinemic), and 0.45 % (wt/vol) saline (controls). A decrease in the islet content of PC2, PC3, and CP-H from hyperglycemic rats was observed. This reduction did not correlate with any deficiency in mRNA levels or biosynthesis of PC2, PC3, CP-H, or Proinsulin. Furthermore, Proinsulin conversion rate was comparable in islets from hyperglycemic and control rats. However, in islets from hyperglycemic rats an abnor-mal increased proportion of Proinsulin was secreted, that was accompanied by an augmented release of PC2, PC3 and CP-H. Stimulation of the 18 cell's secretory pathway by hyperglycemia, resulted in Proinsulin being prematurely secreted from islets before its conversion could be com-pleted. Thus, hyperProinsulinemia induced by chronic hy-perglycemia likely results from increased 1 cell secretory demand, rather than a defect in the Proinsulin processin
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glucose induced translational control of Proinsulin biosynthesis is proportional to preProinsulin mrna levels in islet β cells but not regulated via a positive feedback of secreted insulin
Journal of Biological Chemistry, 2003Co-Authors: Barton Luke Wicksteed, Cristina Alarcon, Isabelle Briaud, Melissa K Lingohr, Christopher J. RhodesAbstract:Abstract Proinsulin biosynthesis is regulated in response to nutrients, most notably glucose. In the short term (≤2h) this is due to increases in the translation of pre-existing mRNA. However, prolonging glucose stimulation (24 h) also increases preProinsulin mRNA levels. It has been proposed that secreted insulin from the pancreatic β-cell regulates its own synthesis through a positive autocrine feedback mechanism. Here the comparative contributions of translation and mRNA levels on the levels of Proinsulin biosynthesis were examined in isolated pancreatic islets. Also, the autocrine role of insulin upon four β-cell functions (insulin secretion, Proinsulin translation, preProinsulin mRNA levels, and total protein synthesis) was investigated in parallel. The results showed that Proinsulin biosynthesis is regulated, in the short term (1 h), solely at the level of translation, through an ∼6-fold increase in response to glucose (2.8 mm versus 16.7 mm glucose). In the longer term, when preProinsulin mRNA levels have increased ∼2-fold, a corresponding increase was observed in the fold response of Proinsulin translation to a stimulatory glucose concentration (≥10-fold). Importantly, neither exogenously added nor secreted insulin were found to play any role in regulating insulin secretion, Proinsulin translation, preProinsulin mRNA levels, or total protein synthesis. The results presented here indicate that long term nutritional state sets the preProinsulin mRNA level in the β-cell at which translation control regulates short term changes in rates of Proinsulin biosynthesis in response to glucose, but this is not mediated by any autocrine effect of insulin.
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glycerol stimulated Proinsulin biosynthesis in isolated pancreatic rat islets via adenoviral induced expression of glycerol kinase is mediated via mitochondrial metabolism
Diabetes, 2001Co-Authors: Robert H Skelly, Christopher J. Rhodes, Barton Wicksteed, Peter A AntinozziAbstract:In this study, we examined whether adenoviral-mediated glycerol kinase (AdV-CMV-GlyK) expression in isolated rat pancreatic islets could introduce glycerol-induced Proinsulin biosynthesis. In AdV-CMV-GlyK-infected islets, specific glycerol-induced Proinsulin biosynthesis translation and insulin secretion were observed in parallel from the same islets. The threshold concentration of glycerol required to stimulate Proinsulin biosynthesis was lower (0.25-0.5 mmol/l) than that for insulin secretion (1.0-1.5 mmol/l), reminiscent of threshold differences for glucose-stimulated Proinsulin biosynthesis versus insulin secretion. The dose-dependent glycerol-induced Proinsulin biosynthesis correlated with the rate of glycerol oxidation in AdV-CMV-GlyK-infected islets, indicating that glycerol metabolism was required for the response. However, glycerol did not significantly increase lactate output from AdV-CMV-GlyK-infected islets, but the dihydroxyacetone phosphate (DHAP) to alpha-glycerophosphate (alpha-GP) ratio significantly increased in AdV-CMV-GlyK-infected islets incubated at 2 mmol/l glycerol compared with that at a basal level of 2.8 mmol/l glucose (P 75%; P = 0.05), similarly to glucose-induced Proinsulin biosynthesis and insulin secretion in AdV-CMV-GlyK-infected and control islets. These data indicated that in AdV-CMV-GlyK-infected islets, the importance of mitochondrial metabolism of glycerol was required to generate stimulus-response coupling signals to induce Proinsulin biosynthesis and insulin secretion.
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a distinct difference in the metabolic stimulus response coupling pathways for regulating Proinsulin biosynthesis and insulin secretion that lies at the level of a requirement for fatty acyl moieties
Biochemical Journal, 1998Co-Authors: R H Skelly, L C Bollheimer, B L Wicksteed, Barbara E Corkey, Christopher J. RhodesAbstract:The regulation of Proinsulin biosynthesis in pancreatic β-cells is vital for maintaining optimal insulin stores for glucose-induced insulin release. The majority of nutrient fuels that induce insulin release also stimulate Proinsulin biosynthesis, but since insulin exocytosis and Proinsulin synthesis involve different cellular mechanisms, a point of divergence in the respective metabolic stimulus–response coupling pathways must exist. A parallel examination of the metabolic regulation of Proinsulin biosynthesis and insulin secretion was undertaken in the same β-cells. In MIN6 cells, glucose-induced Proinsulin biosynthesis and insulin release shared a requirement for glycolysis to generate stimulus-coupling signals. Pyruvate stimulated both Proinsulin synthesis (threshold 0.13–0.2 mM) and insulin release (threshold 0.2–0.3 mM) in MIN6 cells, which was eliminated by an inhibitor of pyruvate transport (1 mM α-cyano-4-hydroxycinnamate). A combination of α-oxoisohexanoate and glutamine also stimulated Proinsulin biosynthesis and insulin release in MIN6 cells, which, together with the effect of pyruvate, indicated that anaplerosis was necessary for instigating secondary metabolic stimulus-coupling signals in the β-cell. A consequence of increased anaplerosis in β-cells is a marked increase in malonyl-CoA, which in turn inhibits β-oxidation and elevates cytosolic fatty acyl-CoA levels. In the β-cell, long-chain fatty acyl moieties have been strongly implicated as metabolic stimulus-coupling signals for regulating insulin exocytosis. Indeed, it was found in MIN6 cells and isolated rat pancreatic islets that exogenous oleate, palmitate and 2-bromopalmitate all markedly potentiated glucose-induced insulin release. However, in the very same β-cells, these fatty acids in contrast inhibited glucose-induced Proinsulin biosynthesis. This implies that neither fatty acyl moieties nor β-oxidation are required for the metabolic stimulus–response coupling pathway specific for Proinsulin biosynthesis, and represent an early point of divergence of the two signalling pathways for metabolic regulation of Proinsulin biosynthesis and insulin release. Therefore alternative metabolic stimulus-coupling factors for the specific control of Proinsulin biosynthesis at the translational level were considered. One possibility examined was an increase in glycerophosphate shuttle activity and change in cytosolic redox state of the β-cell, as reflected by changes in the ratio of α-glycerophosphate to dihydroxyacetone phosphate. Although 16.7 mM glucose produced a significant rise in the α-glycerophosphate/dihydroxyacetone phosphate ratio, 1 mM pyruvate did not. It follows that the cytosolic redox state and fatty acyl moieties are not necessarily involved as secondary metabolic stimulus-coupling factors for regulation of Proinsulin biosynthesis. However, the results indicate that glycolysis and the subsequent increase in anaplerosis are indeed necessary for this signalling pathway, and therefore an extramitochondrial product of β-cell pyruvate metabolism (that is upstream of the increased cytosolic fatty acyl-CoA) acts as a key intracellular secondary signal for specific control of Proinsulin biosynthesis by glucose at the level of translation.
Billy Tsai - One of the best experts on this subject based on the ideXlab platform.
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distinct states of Proinsulin misfolding in midy
Cellular and Molecular Life Sciences, 2021Co-Authors: Leena Haataja, Ming Liu, Billy Tsai, Balamurugan Dhayalan, Anoop Arunagiri, Michael A Weiss, Anis Hassan, Kaitlin Regan, Peter ArvanAbstract:A precondition for efficient Proinsulin export from the endoplasmic reticulum (ER) is that Proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) "interchain" disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third Proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a "lose-A6/A11" mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of Proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed Proinsulin disulfide bond formation. Here, we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep + 2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic β-cells. Three of these mutants, however, must disrupt the Cys(A6)-Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of Proinsulin folding and ER export, because when introduced into the Proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type Proinsulin. A growing molecular understanding of Proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.
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distinct states of Proinsulin misfolding in midy
bioRxiv, 2021Co-Authors: Leena Haataja, Ming Liu, Billy Tsai, Balamurugan Dhayalan, Anoop Arunagiri, Michael A Weiss, Anis Hassan, Kaitlin Regan, Peter ArvanAbstract:A precondition for efficient Proinsulin export from the endoplasmic reticulum (ER) is that Proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) "interchain" disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third Proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a "lose-A6/A11" mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of Proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed Proinsulin disulfide bond formation. Here we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep+2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic {beta}-cells. Three of these mutants, however, must disrupt the Cys(A6)-Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of Proinsulin folding and ER export, because when introduced into the Proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type Proinsulin. A growing molecular understanding of Proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.
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cells deploy a two pronged strategy to rectify misfolded Proinsulin aggregates
Molecular Cell, 2019Co-Authors: Corey N Cunningham, Peter Arvan, Anoop Arunagiri, Jeffrey M Williams, Jeffrey Knupp, Billy TsaiAbstract:Insulin gene coding sequence mutations are known to cause mutant INS-gene-induced diabetes of youth (MIDY), yet the cellular pathways needed to prevent misfolded Proinsulin accumulation remain incompletely understood. Here, we report that Akita mutant Proinsulin forms detergent-insoluble aggregates that entrap wild-type (WT) Proinsulin in the endoplasmic reticulum (ER), thereby blocking insulin production. Two distinct quality-control mechanisms operate together to combat this insult: the ER luminal chaperone Grp170 prevents Proinsulin aggregation, while the ER membrane morphogenic protein reticulon-3 (RTN3) disposes of aggregates via ER-coupled autophagy (ER-phagy). We show that enhanced RTN-dependent clearance of aggregated Akita Proinsulin helps to restore ER export of WT Proinsulin, which can promote WT insulin production, potentially alleviating MIDY. We also find that RTN3 participates in the clearance of other mutant prohormone aggregates. Together, these results identify a series of substrates of RTN3-mediated ER-phagy, highlighting RTN3 in the disposal of pathogenic prohormone aggregates.
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Proinsulin misfolding is an early event in the progression to type 2 diabetes
eLife, 2019Co-Authors: Anoop Arunagiri, Leena Haataja, Billy Tsai, Adrienne W Paton, James C Paton, Anita Pottekat, Fawnnie Pamenan, Soohyun Kim, Lori M Zeltser, Pamela ItkinansariAbstract:Our body fine-tunes the amount of sugar in our blood thanks to specialized ‘beta cells’ in the pancreas, which can release a hormone called insulin. To produce insulin, the beta cells first need to build an early version of the molecule – known as Proinsulin – inside a cellular compartment called the endoplasmic reticulum. This process involves the formation of internal staples that keep the molecule of Proinsulin folded correctly. Individuals developing type 2 diabetes have spikes of sugar in their blood, and so their bodies often respond by trying to make large amounts of insulin. After a while, the beta cells can fail to keep up, which brings on the full-blown disease. However, scientists have discovered that early in type 2 diabetes, the endoplasmic reticulum of beta cells can already show signs of stress; yet, the exact causes of this early damage are still unknown. To investigate this, Arunagiri et al. looked into whether Proinsulin folds correctly during the earliest stages of type 2 diabetes. Biochemical experiments showed that even healthy beta cells contained some misfolded Proinsulin molecules, where the molecular staples that should fold Proinsulin internally were instead abnormally linking Proinsulin molecules together. Further work revealed that the misfolded Proinsulin was accumulating inside the endoplasmic reticulum. Finally, obese mice that were in the earliest stages of type 2 diabetes had the highest levels of abnormal Proinsulin in their beta cells. Overall, the work by Arunagiri et al. suggests that large amounts of Proinsulin molecules stapling themselves to each other in the endoplasmic reticulum of beta cells could be an early hallmark of the disease, and could make it get worse. A separate study by Jang et al. also shows that a protein that limits the misfolding of Proinsulin is key to maintain successful insulin production in animals eating a Western-style, high fat diet. Hundreds of millions of people around the world have type 2 diabetes, and this number is rising quickly. Detecting and then fixing early problems associated with the condition may help to stop the disease in its track.
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misfolded Proinsulin in the endoplasmic reticulum during development of beta cell failure in diabetes
Annals of the New York Academy of Sciences, 2018Co-Authors: Anoop Arunagiri, Ming Liu, Leena Haataja, Billy Tsai, Corey N Cunningham, Neha Shrestha, Peter ArvanAbstract:The endoplasmic reticulum (ER) is broadly distributed throughout the cytoplasm of pancreatic beta cells, and this is where all Proinsulin is initially made. Healthy beta cells can synthesize 6000 Proinsulin molecules per second. Ordinarily, nascent Proinsulin entering the ER rapidly folds via the formation of three evolutionarily conserved disulfide bonds (B7-A7, B19-A20, and A6-A11). A modest amount of Proinsulin misfolding, including both intramolecular disulfide mispairing and intermolecular disulfide-linked protein complexes, is a natural by-product of Proinsulin biosynthesis, as is the case for many proteins. The steady-state level of misfolded Proinsulin-a potential ER stressor-is linked to (1) production rate, (2) ER environment, (3) presence or absence of naturally occurring (mutational) defects in Proinsulin, and (4) clearance of misfolded Proinsulin molecules. Accumulation of misfolded Proinsulin beyond a certain threshold begins to interfere with the normal intracellular transport of bystander Proinsulin, leading to diminished insulin production and hyperglycemia, as well as exacerbating ER stress. This is most obvious in mutant INS gene-induced Diabetes of Youth (MIDY; an autosomal dominant disease) but also likely to occur in type 2 diabetes owing to dysregulation in Proinsulin synthesis, ER folding environment, or clearance.
