Browsing by Author "Kaufman, Randal J."

Now showing 1 - 5 of 5
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    Biosynthesis, structure, and folding of the insulin precursor protein
    (Wiley, 2018-09) Liu, Ming; Weiss, Michael A.; Arunagiri, Anoop; Yong, Jing; Rege, Nischay; Sun, Jinhong; Haataja, Leena; Kaufman, Randal J.; Arvan, Peter; Biochemistry and Molecular Biology, School of Medicine
    Insulin synthesis in pancreatic β-cells is initiated as preproinsulin. Prevailing glucose concentrations, which oscillate pre- and postprandially, exert major dynamic variation in preproinsulin biosynthesis. Accompanying upregulated translation of the insulin precursor includes elements of the endoplasmic reticulum (ER) translocation apparatus linked to successful orientation of the signal peptide, translocation and signal peptide cleavage of preproinsulin-all of which are necessary to initiate the pathway of proper proinsulin folding. Evolutionary pressures on the primary structure of proinsulin itself have preserved the efficiency of folding ("foldability"), and remarkably, these evolutionary pressures are distinct from those protecting the ultimate biological activity of insulin. Proinsulin foldability is manifest in the ER, in which the local environment is designed to assist in the overall load of proinsulin folding and to favour its disulphide bond formation (while limiting misfolding), all of which is closely tuned to ER stress response pathways that have complex (beneficial, as well as potentially damaging) effects on pancreatic β-cells. Proinsulin misfolding may occur as a consequence of exuberant proinsulin biosynthetic load in the ER, proinsulin coding sequence mutations, or genetic predispositions that lead to an altered ER folding environment. Proinsulin misfolding is a phenotype that is very much linked to deficient insulin production and diabetes, as is seen in a variety of contexts: rodent models bearing proinsulin-misfolding mutants, human patients with Mutant INS-gene-induced Diabetes of Youth (MIDY), animal models and human patients bearing mutations in critical ER resident proteins, and, quite possibly, in more common variety type 2 diabetes.
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    Cellular stress and coagulation factor production: When more isn’t necessarily better
    (Elsevier, 2023-12) Chen, Zhouji; Herzog, Roland W.; Kaufman, Randal J.; Pediatrics, School of Medicine
    Remarkably, it has been 40 years since the isolation of the 2 genes involved in hemophilia A (HA) and hemophilia B (HB), encoding clotting factor (F) VIII (FVIII) and FIX, respectively. Over the years, these advances led to the development of purified recombinant protein factors that are free of contaminating viruses from human pooled plasma for hemophilia treatments, reducing the morbidity and mortality previously associated with human plasma-derived clotting factors. These discoveries also paved the way for modified factors that have increased plasma half-lives. Importantly, more recent advances have led to the development and Food and Drug Administration approval of a hepatocyte-targeted, adeno-associated viral vector-mediated gene transfer approach for HA and HB. However, major concerns regarding the durability and safety of HA gene therapy remain to be resolved. Compared with FIX, FVIII is a much larger protein that is prone to misfolding and aggregation in the endoplasmic reticulum and is poorly secreted by the mammalian cells. Due to the constraint of the packaging capacity of adeno-associated viral vector, B-domain deleted FVIII rather than the full-length protein is used for HA gene therapy. Like full-length FVIII, B-domain deleted FVIII misfolds and is inefficiently secreted. Its expression in hepatocytes activates the cellular unfolded protein response, which is deleterious for hepatocyte function and survival and has the potential to drive hepatocellular carcinoma. This review is focused on our current understanding of factors limiting FVIII secretion and the potential pathophysiological consequences upon expression in hepatocytes.
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    Ectopic clotting factor VIII expression and misfolding in hepatocytes as a cause for hepatocellular carcinoma
    (Elsevier, 2022-12-07) Kapelanski-Lamoureux, Audrey; Chen, Zhouji; Gao, Zu-Hua; Deng, Ruishu; Lazaris, Anthoula; Lebeaupin, Cynthia; Giles, Lisa; Malhotra, Jyoti; Yong, Jing; Zou, Chenhui; de Jong, Ype P.; Metrakos, Peter; Herzog, Roland W.; Kaufman, Randal J.; Pediatrics, School of Medicine
    Hemophilia A gene therapy targets hepatocytes to express B domain deleted (BDD) clotting factor VIII (FVIII) to permit viral encapsidation. Since BDD is prone to misfolding in the endoplasmic reticulum (ER) and ER protein misfolding in hepatocytes followed by high-fat diet (HFD) can cause hepatocellular carcinoma (HCC), we studied how FVIII misfolding impacts HCC development using hepatocyte DNA delivery to express three proteins from the same parental vector: (1) well-folded cytosolic dihydrofolate reductase (DHFR); (2) BDD-FVIII, which is prone to misfolding in the ER; and (3) N6-FVIII, which folds more efficiently than BDD-FVIII. One week after DNA delivery, when FVIII expression was undetectable, mice were fed HFD for 65 weeks. Remarkably, all mice that received BDD-FVIII vector developed liver tumors, whereas only 58% of mice that received N6 and no mice that received DHFR vector developed liver tumors, suggesting that the degree of protein misfolding in the ER increases predisposition to HCC in the context of an HFD and in the absence of viral transduction. Our findings raise concerns of ectopic BDD-FVIII expression in hepatocytes in the clinic, which poses risks independent of viral vector integration. Limited expression per hepatocyte and/or use of proteins that avoid misfolding may enhance safety.
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    IL-15 blockade and rapamycin rescue multifactorial loss of factor VIII from AAV-transduced hepatocytes in hemophilia A mice
    (Elsevier, 2022-12-07) Butterfield, John S. S.; Yamada, Kentaro; Bertolini, Thais B.; Syed, Farooq; Kumar, Sandeep R. P.; Li, Xin; Arisa, Sreevani; Piñeros, Annie R.; Tapia, Alejandro; Rogers, Christopher A.; Li, Ning; Rana, Jyoti; Biswas, Moanaro; Terhorst, Cox; Kaufman, Randal J.; de Jong, Ype P.; Herzog, Roland W.; Pediatrics, School of Medicine
    Hepatic adeno-associated viral (AAV) gene transfer has the potential to cure the X-linked bleeding disorder hemophilia A. However, declining therapeutic coagulation factor VIII (FVIII) expression has plagued clinical trials. To assess the mechanistic underpinnings of this loss of FVIII expression, we developed a hemophilia A mouse model that shares key features observed in clinical trials. Following liver-directed AAV8 gene transfer in the presence of rapamycin, initial FVIII protein expression declines over time in the absence of antibody formation. Surprisingly, loss of FVIII protein production occurs despite persistence of transgene and mRNA, suggesting a translational shutdown rather than a loss of transduced hepatocytes. Some of the animals develop ER stress, which may be linked to hepatic inflammatory cytokine expression. FVIII protein expression is preserved by interleukin-15/interleukin-15 receptor blockade, which suppresses CD8+ T and natural killer cell responses. Interestingly, mice with initial FVIII levels >100% of normal had diminishing expression while still under immune suppression. Taken together, our findings of interanimal variability of the response, and the ability of the immune system to shut down transgene expression without utilizing cytolytic or antibody-mediated mechanisms, illustrate the challenges associated with FVIII gene transfer. Our protocols based upon cytokine blockade should help to maintain efficient FVIII expression.
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    Role of Proinsulin Self-Association in Mutant INS Gene–Induced Diabetes of Youth
    (American Diabetes Association, 2020-05) Sun, Jinhong; Xiong, Yi; Li, Xin; Haataja, Leena; Chen, Wei; Mir, Saiful A.; Lv, Li; Madley, Rachel; Larkin, Dennis; Anjum, Arfah; Dhayalan, Balamurugan; Rege, Nischay; Wickramasinghe, Nalinda P.; Weiss, Michael A.; Itkin-Ansari, Pamela; Kaufman, Randal J.; Ostrov, David A.; Arvan, Peter; Liu, Ming; Biochemistry and Molecular Biology, School of Medicine
    Abnormal interactions between misfolded mutant and wild-type (WT) proinsulin (PI) 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, PI-WT dimerizes in the ER. Here, we suggest that the normal PI-PI contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that PI B-chain tyrosine-16 (Tyr-B16), which is a key residue in normal PI dimerization, helps confer dominant-negative behavior of MIDY mutant PI-C(A7)Y. Substitutions of Tyr-B16 with either Ala, Asp, or Pro in PI-C(A7)Y decrease the abnormal interactions between the MIDY mutant and PI-WT, rescuing PI-WT export, limiting ER stress, and increasing insulin production in β-cells and human islets. This study reveals the first evidence indicating that noncovalent PI-PI contact initiates dominant-negative behavior of misfolded PI, pointing to a novel therapeutic target to enhance PI-WT export and increase insulin production.