Insulin-like growth factors (IGFs) mediate differentiation decisions during embryonic development, cell growth signaling and responses to metabolic stress. We found that the production of these important growth factors is dependent on the activity of the endoplasmic reticulum chaperone glucose regulate protein 94 (GRP94), which is essential for their biosynthesis. GRP94 is different from other molecular chaperones in its selectivity towards only a small number of client proteins, preference for late folding intermediates and the absence of any known co-factors. All these aspects suggest a unique mode of action for GRP94. This project will elucidate the enigmatic action cycle of GRP94 by taking advantage of cells whose growth depends on the GRP94-IGF axis. In our first aim we will use a novel cell-based assay to screen for mutants of GRP94 that do not support IGF-II production and thus define amino acids that are essential for this chaperone function.
In Aim 2, we will determine whether these mutants are defective in general mechanistic aspects, such as ATP binding and hydrolysis or calcium binding, or whether the mutations define a client binding domain. We will also test the ability of GRP94 mutants to support clients other than IGF, to discover whether there are client-specific mutants.
Our third aim i s to understand how GRP94 recognizes IGF, by exploiting the structural similarities and differences among a set of insulin-like proteins. These studies will inform on the client selectivity of GRP94, which is currently not understood. Finally, we will take advantage of the clinical consequences of IGF deficiency to search for functionally important human GRP94 variants. Following on our finding that at least one such variant affects IGF production, we will genotype and sequence IGF-deficient patient populations to seek allelic GRP94 variants that may explain low IGF production. Together, these results will lead to a new understanding of how GRP94 chaperones client proteins.

Public Health Relevance

This project aims to decipher how GRP94, representing the family of HSP90 chaperones, selects its client proteins and affects their proper production. The work will use a novel cell-based assay, with insulin-like growth factor as the client, to determine the functionality and characterize biochemically both natural and site-directed mutations in GRP94.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM077480-03
Application #
8322048
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2010-06-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
3
Fiscal Year
2012
Total Cost
$317,567
Indirect Cost
$124,517
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Eletto, Daniela; Eletto, Davide; Boyle, Sarah et al. (2016) PDIA6 regulates insulin secretion by selectively inhibiting the RIDD activity of IRE1. FASEB J 30:653-65
Marzec, Michal; Hawkes, Colin P; Eletto, Davide et al. (2016) A Human Variant of Glucose-Regulated Protein 94 That Inefficiently Supports IGF Production. Endocrinology 157:1914-28
Dersh, Devin; Iwamoto, Yuichiro; Argon, Yair (2016) Tay-Sachs disease mutations in HEXA target the α chain of hexosaminidase A to endoplasmic reticulum-associated degradation. Mol Biol Cell 27:3813-3827
Dersh, Devin; Jones, Stephanie M; Eletto, Davide et al. (2014) OS-9 facilitates turnover of nonnative GRP94 marked by hyperglycosylation. Mol Biol Cell 25:2220-34
Eletto, Davide; Eletto, Daniela; Dersh, Devin et al. (2014) Protein disulfide isomerase A6 controls the decay of IRE1α signaling via disulfide-dependent association. Mol Cell 53:562-76
Gidalevitz, Tali; Stevens, Fred; Argon, Yair (2013) Orchestration of secretory protein folding by ER chaperones. Biochim Biophys Acta 1833:2410-24
Duerfeldt, Adam S; Peterson, Laura B; Maynard, Jason C et al. (2012) Development of a Grp94 inhibitor. J Am Chem Soc 134:9796-804
Barton, Elisabeth R; Park, SooHyun; James, Jose K et al. (2012) Deletion of muscle GRP94 impairs both muscle and body growth by inhibiting local IGF production. FASEB J 26:3691-702
Eletto, Davide; Maganty, Avinash; Eletto, Daniela et al. (2012) Limitation of individual folding resources in the ER leads to outcomes distinct from the unfolded protein response. J Cell Sci 125:4865-75
Marzec, Michal; Eletto, Davide; Argon, Yair (2012) GRP94: An HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum. Biochim Biophys Acta 1823:774-87

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