Protein Arginine Deiminase (PAD) activity is aberrantly upregulated in multiple human diseases, including rheumatoid arthritis, colitis, and cancer. Thus, these enzymes are potential therapeutic targets. One isozyme, PAD4, helps control a number of physiological processes, including gene transcription, apoptosis, cell growth, and neutrophil extracellular trap formation. However, the specific roles of PAD4 in each of these processes are incompletely defined. Additionally, its mode of regulation and its contribution kinase signaling are only beginning to be understood and appreciated. The physiological roles of the other human PADs (i.e., PADs 1, 2, 3, and 6) are even less well understood. Building on previous work that described the development of PAD- targeted irreversible inhibitors and activity based proteomic probes (ABPPs), we propose to develop isozyme specific PAD inactivators. Additionally, we will use these compounds to identify and characterize the factors that regulate PAD activity, focusing initially on PAD4. Additionally, we will examine crosstalk between serine phosphorylation and arginine citrullination at both the molecular and cell biology levels.
Specific aims are: (1) The first aim of this proposal is focused on developing isozyme specific PAD inhibitors with improved potency and bioavailability.
This aim builds on our exciting discovery that haloacetamidine bearing compounds act as irreversible PAD inhibitors. Two strategies are proposed: (i) the replacement of the backbone amides in Cl-amidine, an inhibitor developed by the PI's lab, with libraries of triazoles and tetrazoles;and (i) a cyclic peptoid library approach. We will also identify novel warheads with altered reactivity tha overcome the limitations of the fluoro- and chloroacetamidine warheads. These compounds will ultimately serve as useful chemical probes of PAD function both directly and when converted into ABPPs to discover the factors that regulate the activity of a particular isozyme. (2) The second aim focuses on characterizing the post-translational modifications (PTMs) that regulate PAD4 activity. This work builds on our prior efforts showing that PAD4 is proteolyzed, acetylated, and ubiquitinated in vivo and these PTMs correlate with different activity states. Specifically, we describe an integrated chemical biology approach to examine the effects of these PTMs on PAD4 activity both in vitro and in vivo. (3) The third aim will study crosstalk between citrullination and serine phosphorylation. We are focused on these studies because we hypothesize that such crosstalk exists to 'fine-tune'cell signaling. Specifically, we demonstrate that crosstalk plays a role in regulating the phosphorylation of ELK1. Additionally, we will take candidate and proteomic approaches to determine the scope of crosstalk between these two PTMs. Once complete, the proposed studies will not only increase our understanding of PAD biology but will provide a suite of chemical probes that can be used to study protein citrullination.

Public Health Relevance

The project focuses on the development and use of chemical probes targeting the Protein Arginine Deiminases (PADs), a group of enzymes whose activity is aberrantly increased in rheumatoid arthritis, colitis, and cancer. These probes will be used in an integrated chemical biology approach to characterize the factors that regulate PAD activity and explore crosstalk between protein phosphorylation and protein citrullination. In total, these studies will increase our understanding of PAD biology, which is critical for understanding how these enzymes contribute to human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM079357-09
Application #
8897493
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Fabian, Miles
Project Start
2007-04-01
Project End
2016-11-30
Budget Start
2014-08-01
Budget End
2014-11-30
Support Year
9
Fiscal Year
2014
Total Cost
$131,164
Indirect Cost
$52,857
Name
University of Massachusetts Medical School Worcester
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Clancy, Kathleen W; Weerapana, Eranthie; Thompson, Paul R (2016) Detection and identification of protein citrullination in complex biological systems. Curr Opin Chem Biol 30:1-6
Ghari, Fatemeh; Quirke, Anne-Marie; Munro, Shonagh et al. (2016) Citrullination-acetylation interplay guides E2F-1 activity during the inflammatory response. Sci Adv 2:e1501257
Li, Guangyuan; Hayward, Isaac N; Jenkins, Brittany R et al. (2016) Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland. PLoS One 11:e0147503
Fuhrmann, Jakob; Thompson, Paul R (2016) Protein Arginine Methylation and Citrullination in Epigenetic Regulation. ACS Chem Biol 11:654-68
Kawalkowska, Joanna; Quirke, Anne-Marie; Ghari, Fatemeh et al. (2016) Abrogation of collagen-induced arthritis by a peptidyl arginine deiminase inhibitor is associated with modulation of T cell-mediated immune responses. Sci Rep 6:26430
Bawadekar, Mandar; Gendron-Fitzpatrick, Annette; Rebernick, Ryan et al. (2016) Tumor necrosis factor alpha, citrullination, and peptidylarginine deiminase 4 in lung and joint inflammation. Arthritis Res Ther 18:173
Fuhrmann, Jakob; Subramanian, Venkataraman; Kojetin, Douglas J et al. (2016) Activity-Based Profiling Reveals a Regulatory Link between Oxidative Stress and Protein Arginine Phosphorylation. Cell Chem Biol 23:967-977
Khan, Shaihla A; Edwards, Brian S; Muth, Aaron et al. (2016) GnRH Stimulates Peptidylarginine Deiminase Catalyzed Histone Citrullination in Gonadotrope Cells. Mol Endocrinol 30:1081-1091
Knight, Jason S; Subramanian, Venkataraman; O'Dell, Alexander A et al. (2015) Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice. Ann Rheum Dis 74:2199-206
Fuhrmann, Jakob; Clancy, Kathleen W; Thompson, Paul R (2015) Chemical biology of protein arginine modifications in epigenetic regulation. Chem Rev 115:5413-61

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