Research
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Arvan Lab Overview

Secretory Protein Folding, Sorting, and Targeting

Research Areas

We are investigating proinsulin folding in the endoplasmic reticulum (ER). Recent evidence strongly implicates that proinsulin misfolding takes place in various genetic forms of diabetes mellitus. As a flagship example, we are investigating mutations in the coding sequence of preproinsulin, which (depending upon the particular mutant allele) trigger autosomal dominant diabetes that can begin as early as the neonatal period or as late as young adulthood. We have named this disease as Mutant INS gene-induced Diabetes of Youth (MIDY). We have been characterizing the misfolding of various MIDY mutants as well as exploring the molecular mechanisms by which the mutant proinsulin impairs production of insulin derived from the nonmutant INS allele. As a model, we examine mice bearing one of several MIDY mutations in just one or two of the four alleles encoding mouse insulin. 

We are interested in the most common form of diabetes known as "type 2" diabetes. We have identified misfolded isoforms of proinsulin by virtue of mispaired disulfide bonds, and examined interactions of these misfolded forms with endoplasmic reticulum (ER) molecular chaperones. Our evidence suggest that the formation of intermolecular disulfide-linked proinsulin complexes occurs even before the initial onset of diabetes in the db/db (leptin receptor-deficient) diabetic mouse. The misfolding appears to occur within the ER, and is linked to an increase in markers of ‘ER stress’.  

We are interested in early aspects of preproinsulin biosynthesis, including initial translation of the INS mRNA, leading to the translocation of newly-synthesized preproinsulin from the cytosol across the ER membrane to enter the secretory pathway. We have been defining the role of various accessory proteins of the ER membrane that assist in preproinsulin translocation.

We are concentrating on ways to modulate the ER environment to impact on proinsulin folding, in the hope of developing small molecules that may be of benefit to insulin production, as well as manipulation of ER oxidoreductase activity. We have also developed a fluorescent proinsulin (called hPro-CpepSfGFP) and mice expressing this fluorescent proinsulin exclusively in pancreatic beta cells. The hPro-CpepSfGFP allows for production of authentic human insulin as well as stoichiometric quantities of fluorescent C-peptide, which can be followed by fluorescence microscopy in live mice as a quantifiable measure of pancreatic insulin content. The animals can then be mated to various genetic models of diabetes.   

We have been concentrating on humans and mice with congenital hypothyroidism caused by an Endoplasmic Reticulum Storage Disease as a consequence of expression of misfolded mutant thyroglobulin (Tg). Many human families have been described with the disease, and the thyroglobulin mutations in these families are known. We cloned the mutation causing congenital goiter in cog/cog mice, and this involves a single amino acid change contained within the carboxy-terminal cholinesterase-like (ChEL) domain of Tg. This same domain is also involved in cases of human hypothyroidism, as it functions as an intramolecular chaperone for the upstream part of the Tg protein. 

Congenital hypothyroidism in the rat dwarf (rdw/rdw) is also caused by a single point mutation in the ChEL domain. However, the dwarf rat develops an unusually hypoplastic (small) thyroid gland despite an increased in the blood levels of the growth promoting hormone known as "thyroid stimulating hormone" (TSH).  We have been concentrating on the hypothesis that, as a consequence of expression of the mutant Tg protein, thyroid cell death from ER stress occurs in mice, rats, and humans with the disease. 

Congenital hypothyroidism from homozygous mutant TG gene is uncommon but heterozygotes are quite common in the human population although they routinely go undiagnosed. We find that heterozygous expression of mutant mouse Tg protein already triggers dramatic ER stress from the accumulated misfolded secretory protein. Studies indicate that the mutant Tg protein is not so easy to completely degrade. The degradation process known as ERAD (ER-associated degradation), is under active investigation in our mouse models.   

Each individual monomer of Tg is stabilized by a large number of intramolecular disulfide bonds. Tg is ultimately a homodimeric protein. Our recent studies suggest that Tg secretion, and some thyroid hormone synthesis, depends upon a dimeric structure. We are particularly interested in further understanding the formation and importance of various specific Tg disulfide bonds.

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