Proinsulin Trafficking through the Secretory Pathway.

Proinsulin, the insulin precursor protein, is synthesized in the Endoplasmic Reticulum (ER) of pancreatic beta cells, where it folds to the native state, involving the formation of three evolutionarily conserved disulfide bonds. Once folded, proinsulin exits the ER, traverses the secretory pathway to the trans-Golgi Network (TGN) and into budding secretory granules. Proinsulin is then processed by endopeptidases that excise the connecting C-peptide that links the B- and A-chains, leading to the creation of mature, two-chain insulin. Upon secretion to the bloodstream, insulin binds to cell surface insulin receptors on target tissues, resulting in the activation of signaling cascades that promote metabolic homeostasis. This thesis aims to look at two distinct aspects of the proinsulin maturation process. In the first part of my thesis work, I have designed a proinsulin with a shortened linker peptide with the intent to create a bioengineered protein that acts as a single-chain insulin (SCI), i.e., without a requirement for cleavage by endopeptidases. SCIs expressed via gene therapy have been found to be effective in reversing diabetes in rodent models, obviating the need for exogenous insulin injection. However, to date, very little structure-function analysis of SCIs has been performed. In Chapter 2, I have examined the structural features of the linker peptide that would allow for mammalian expression, secretion, and bioactivity of SCIs for development into future diabetes therapeutics. In the second part of my thesis work, I have been attempting to identify the ER oxidoreductase(s) that promote(s) formation of the three disulfide bonds of proinsulin, which heretofore are unknown. In Chapter 3, I present results that point to two key members of the family of PDI-like ER oxidoreductases: Protein Disulfide Isomerase itself, and Endoplasmic Reticulum protein 72 (ERp72), which may both play critical, yet opposing, roles in this process. As the misfolding of proinsulin is implicated in the progression of various forms of diabetes, understanding the key factors that control the balance of proinsulin folding and misfolding (by regulating proinsulin disulfide bond formation) could also provide potential benefit for designing therapies that increase insulin production.Ph.D.Cellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91500/1/grajpal_1.pd

Single-Chain Insulins as Receptor Agonists

Single-chain insulins (SCIs) are single polypeptide chains in which the insulin B-chain links contiguously with the insulin A-chain via an uncleaved connecting peptide. Although direct linkage of insulin B- and A-chains produces SCIs with little insulin receptor binding, biologists have been interested in bioengineering linker peptides that form a flexible reverse turn, allowing SCIs to activate insulin receptors. In this report, we have investigated a series of cDNAs intended to explore the significance of linker length, cleavability, and the impact of certain site-dependent residues for the bioactivity of recombinant SCIs on insulin receptors. SCI concentration is readily measured by RIA with a (proinsulin plus insulin)-specific polyclonal antibody. Although dibasic flanking residues may result in potential endoproteolytic susceptibility, a linker with -Gln-Arg- flanking sequences resisted cleavage even in secretory granules, ensuring single-chain behavior. Effective SCIs exhibit favorable and specific binding with insulin receptors. SCIs with linkers bearing an Arg residue immediately preceding the A-chain were most bioactive, although efficient receptor interaction was inhibited as SCI linker length increased, approaching that observed for proinsulin. SCIs activate downstream metabolic signaling, stimulating glucose uptake into adipocytes and suppressing gluconeogenic enzyme biosynthesis in hepatocytes, with only limited cross-reactivity on IGF-I receptors. SCIs might theoretically have utility either in immunotherapy or gene therapy in insulin-deficient diabetes

Evaluation of Antisense Oligonucleotides Targeting ATXN3 in SCA3 Mouse Models

The most common dominantly inherited ataxia, spinocerebellar ataxia type 3 (SCA3), is an incurable neurodegenerative disorder caused by a CAG repeat expansion in the ATXN3 gene that encodes an abnormally long polyglutamine tract in the disease protein, ATXN3. Mice lacking ATXN3 are phenotypically normal; hence, disease gene suppression offers a compelling approach to slow the neurodegenerative cascade in SCA3. Here we tested antisense oligonucleotides (ASOs) that target human ATXN3 in two complementary mouse models of SCA3: yeast artificial chromosome (YAC) MJD-Q84.2 (Q84) mice expressing the full-length human ATXN3 gene and cytomegalovirus (CMV) MJD-Q135 (Q135) mice expressing a human ATXN3 cDNA. Intracerebroventricular injection of ASOs resulted in widespread delivery to the most vulnerable brain regions in SCA3. In treated Q84 mice, three of five tested ASOs reduced disease protein levels by >50% in the diencephalon, cerebellum, and cervical spinal cord. Two ASOs also significantly reduced mutant ATXN3 in the mouse forebrain and resulted in no signs of astrogliosis or microgliosis. In Q135 mice expressing a single ATXN3 isoform via a cDNA transgene, ASOs did not result in similar robust ATXN3 silencing. Our results indicate that ASOs targeting full-length human ATXN3 would likely be well tolerated and could lead to a preventative therapy for SCA3

Abstracts of National Conference on Research and Developments in Material Processing, Modelling and Characterization 2020

This book presents the abstracts of the papers presented to the Online National Conference on Research and Developments in Material Processing, Modelling and Characterization 2020 (RDMPMC-2020) held on 26th and 27th August 2020 organized by the Department of Metallurgical and Materials Science in Association with the Department of Production and Industrial Engineering, National Institute of Technology Jamshedpur, Jharkhand, India. Conference Title: National Conference on Research and Developments in Material Processing, Modelling and Characterization 2020Conference Acronym: RDMPMC-2020Conference Date: 26–27 August 2020Conference Location: Online (Virtual Mode)Conference Organizer: Department of Metallurgical and Materials Engineering, National Institute of Technology JamshedpurCo-organizer: Department of Production and Industrial Engineering, National Institute of Technology Jamshedpur, Jharkhand, IndiaConference Sponsor: TEQIP-