UCSF DIABETES, ENDOCRINOLOGY & METABOLISM TRAINING PROGRAM FACULTY RESEARCH SUMMARIES

My research focus is on the mechanisms and biology of intracellular protein misfolding, with the ultimate goal of understanding the molecular bases of—and eventually developing novel therapeutics for—the protein misfolding diseases. Specifically, my efforts center on type 2 diabetes, for which evidence is accruing that it represents an example of a protein misfolding disease.

To study intracellular protein misfolding, I will focus on the endoplasmic reticulum (ER), which in eukaryotic cells is a protein-folding factory and a way station for proteins destined for the cell exterior or other distal destinations. These secretory and transmembrane proteins are injected during their biogenesis in an unfolded state into the ER, where they must complete folding without aggregating. A plethora of ER-resident activities (the protein-folding machinery) facilitates this process. Insufficient ER protein-folding capacity leads to an accumulation of unfolded proteins in the ER lumen (referred to as ER stress) and triggers an adaptive mechanism called the unfolded protein response (UPR), which in S. cerevisiae causes induction of a major transcriptional program to alleviate ER stress. Transcriptionally-upregulated genes in the UPR include those encoding chaperones, oxido-reductases, phospholipid biosynthetic enzymes, ER-associated degradation (ERAD) components, and many proteins functioning downstream in the secretory pathway. These activities act in concert to override and extinguish ER stress.

ER stress is sensed by a bifunctional ER transmembrane kinase/RNase, called Ire1. Upon accumulation of ER misfolded proteins, Ire1 is activated and initiates non-conventional splicing of the mRNA encoding the Hac1 transcriptional activator. This splicing event relieves translational attenuation of HAC1 mRNA; consequently the accumulation of Hac1 protein causes upregulation of UPR targets. Using a chemical-genetic approach, I have recently discovered that upon ER unfolded protein accumulation, Ire1's kinase domain engages adenosine nucleotide, throwing a conformational switch to activate its HAC1 mRNA-specific RNase. In the course of this work, I developed novel tools to manipulate the yeast UPR using cell-permeable drugs which target Ire1's kinase domain. These tools will be exploited further to dissect the molecular mechanism of signaling in the mammalian UPR by Ire1.

For mammalian cells, cellular survival during ER stress requires the UPR. The mammalian UPR remodels the environment of the ER to respond to ER stress through two mechanisms: (1) as with yeast, a massive transcriptional up-regulation of UPR target genes, and (2) a global translational down-regulation, which reduces the load of secretory and transmembrane proteins delivered to the ER. The transcriptional arm of the UPR is controlled by the mammalian Ire1 ortholog, Ire1a, and the translational arm is controlled by the ER-transmembrane eIF-2a kinase, Perk.

I plan to study the biological consequences of unchecked ER stresses on mammalian cells and in living mice, using type 2 diabetes as a model disease. Evidence is mounting that a serious physiological consequence of unchecked ER stress on pancreatic islet ß-cells is apoptosis, leading to type 2 diabetes. (1) Perk -/- mice accumulate unfolded proteins in the ER of several professional secretory tissues and develop infantile diabetes due to rapid ß -cell death early in life. (2) The Akita diabetic mouse mutant misfolds a variant proinsulin, leading to proteotoxicity in the ER. (3) The human islet amyloid polypeptide (hIAPP) is a common amyloidogenic ß-cell proteotoxin, known to cause ß -cell ER damage, which may lead to diabetes in humans. Together, the implications of these studies lead to a unified hypothesis wherein misfolded protein buildup in the ER of pancreatic islet ß-cells is deleterious for their long-term function and survival.

Building on these studies, I will investigate specifically what role is played by the mammalian UPR in containing the various ER stresses that cause ß-cell attrition, and which could lead to diabetes. A classical genetic (gene “knockout”) approach to this question is complicated by the fact that IRE1a is essential for mouse development, and perk -/- mice have pleiotropic defects in several organ systems, which lead to death early in life. As an alternative approach I am developing systems for conditionally manipulating the two arms of the mammalian UPR so as to observe the specific effects of unchecked ER stress on pancreatic islet ß -cells in the living mouse. To this end, I will use chemical-genetic approaches, which permit highly-specific drug targeting of modified Ire1a and Perk kinase domains, using cell-permeable ATP-competitive drugs.

As I have successfully validated this approach for conditional control of the yeast UPR—it is now possible to switch off the UPR even during ER stress, and switch on the UPR even in the absence of ER stress—conditional manipulation of the two arms of the mammalian UPR is being developed. This will be accomplished by gene targeting of the IRE1 a and PERK kinase domain-coding regions in mouse embryonic stem (ES) cells to generate “knock-in” mice whose Ire1 a and Perk gene products are sensitized to these ATP-competitive drugs. The biological consequences of these manipulations on b -cells will be studied in these knock-in mice mutants and also in the genetic backgrounds of Akita and hIAPP to test the hypothesis that a crippled UPR promotes diabetes.

Selected Reference

Papa FR, Zhang C, Shokat K, Walter P. Bypassing a kinase activity with an ATP-competitive drug.
Science. 2003 Nov 28;302(5650):1533-7.

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