Insulin degrading enzyme (IDE) is an evolutionarily conserved, 110 kDa metalloprotease that is involved in the clearance of insulin and amyloid ? (A?). Accumulating genetic evidence in rodents and humans strongly support the role of IDE in the progression of type 2 diabetes mellitus and Alzheimer's disease. Thus, it is vital to understand the functions, catalytic mechanism, and regulation of IDE from a molecular perspective to develop viable IDE-based therapeutic strategies. We have solved the structures of human IDE in complex with insulin, A?, and other functionally relevant substrates such as natriuretic peptides and proinflammatory chemokines, CCL3/CCL4. IDE has two 55 kDa domains, which form an enclosed catalytic chamber to entrap its substrates. Our structural and biochemical analyses reveal how IDE uses an enclosed catalytic chamber to selectively recognize the global features of its substrates. The long-term goal of this research is (1) to delineate the function(s) of IDE and the mechanism(s) of its regulation and (2) to elucidate the role of IDE in human diseases. The objectives of this application are to understand (1) the molecular basis for the open-closed conformational switch and dynamics of IDE during catalysis and (2) the molecular mechanism for the regulation of IDE. In addition, we will develop potent chemical modulators of IDE to be used as potential therapeutic agents and as tools to address the biological functions of this enzyme. The central hypothesis is that the open-closed conformational switch of IDE is the key regulatory step of IDE that is subject to allosteric regulation by dimerization, posttranslational modifications, cellular factors, and chemical modulators. The rationale for the proposed research is that understanding the regulation and functions of IDE and developing small chemical modulators of IDE will ultimately allow us to better design IDE-based therapeutic strategies specific to certain human diseases such as diabetes, Alzheimer's disease, and inflammation. Guided by our preliminary data, we will study the regulation and functions of IDE in three specific aims:
in Aim 1, we will use single molecule Forster resonance energy transfer analyses to address the conformational switches and dynamics that occur during catalysis of IDE and to determine how the catalysis of IDE is regulated.
Aim 2 is to use two distinct screening methods to develop potent small molecule compounds that can modulate the activity of IDE and use such compounds to address the biological functions of IDE.
Aim 3 is to combine structural, biochemical, and mutational studies to address the molecular basis for the regulation of IDE by dimerization, physiologically relevant cellular factors such as intermediate filament proteins, nestin and vimentin, and by posttranslational modifications such as phosphorylation and acetylation. The proposed research is significant because it will generate new insights in the dynamics and regulation of a key enzyme involved in diabetes and Alzheimer's disease and because it will also lead to the discovery of new chemical leads that can potently modulate this enzyme. The proposed research is innovative because it employs biophysical, biochemical, cellular, and medicinal chemical approaches to investigate the regulation and functions of IDE.

Public Health Relevance

The catalytic activity of IDE is controlled by the open-closed conformational switch, which is regulated by the interaction of IDE with itself (dimerization) and with cellular factors as well as through posttranslational modifications. We propose to use smFRET, biochemical, structural, chemical, and cellular approaches to better understand how IDE is regulated and what its biological functions are. The success of our studies will offer new insights in the regulation of IDE and new tools to explore the therapeutic potential of IDE.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM081539-07
Application #
8537935
Study Section
Special Emphasis Panel (ZRG1-BCMB-R (02))
Program Officer
Flicker, Paula F
Project Start
2007-07-15
Project End
2015-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
7
Fiscal Year
2013
Total Cost
$252,779
Indirect Cost
$62,416
Name
University of Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Charton, Julie; Gauriot, Marion; Guo, Qing et al. (2014) Imidazole-derived 2-[N-carbamoylmethyl-alkylamino]acetic acids, substrate-dependent modulators of insulin-degrading enzyme in amyloid-? hydrolysis. Eur J Med Chem 79:184-93
King, John V; Liang, Wenguang G; Scherpelz, Kathryn P et al. (2014) Molecular basis of substrate recognition and degradation by human presequence protease. Structure 22:996-1007
Maianti, Juan Pablo; McFedries, Amanda; Foda, Zachariah H et al. (2014) Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by multiple hormones. Nature 511:94-8
McCord, Lauren A; Liang, Wenguang G; Dowdell, Evan et al. (2013) Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme. Proc Natl Acad Sci U S A 110:13827-32
Tripathi, Abhishek; Saini, Vikas; Marchese, Adriano et al. (2013) Modulation of the CXC chemokine receptor 4 agonist activity of ubiquitin through C-terminal protein modification. Biochemistry 52:4184-92
Ralat, Luis A; Kalas, Vasilios; Zheng, Zhongzhou et al. (2011) Ubiquitin is a novel substrate for human insulin-degrading enzyme. J Mol Biol 406:454-66
Saini, Vikas; Marchese, Adriano; Tang, Wei-Jen et al. (2011) Structural determinants of ubiquitin-CXC chemokine receptor 4 interaction. J Biol Chem 286:44145-52
Ralat, Luis A; Guo, Qing; Ren, Min et al. (2011) Insulin-degrading enzyme modulates the natriuretic peptide-mediated signaling response. J Biol Chem 286:4670-9
Ren, Min; Guo, Qing; Guo, Liang et al. (2010) Polymerization of MIP-1 chemokine (CCL3 and CCL4) and clearance of MIP-1 by insulin-degrading enzyme. EMBO J 29:3952-66
Guo, Qing; Manolopoulou, Marika; Bian, Yao et al. (2010) Molecular basis for the recognition and cleavages of IGF-II, TGF-alpha, and amylin by human insulin-degrading enzyme. J Mol Biol 395:430-43

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