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THE MOLECULAR MECHANISM UNDERLYING THE AUTOIMMUNE-ASSOCIATED
PTPN22 R620W VARIATION AND THE QUEST FOR THERAPEUTICS
by
Stephanie Michele Stanford
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR, AND CELLULAR BIOLOGY)
August 2010
Copyright 2010 Stephanie Michele Stanford
Object Description
| Title | The molecular mechanism underlying the autoimmune-associated PTPN22 R620W variation and the quest for therapeutics |
| Author | Stanford, Stephanie Michele |
| Author email | sbond@usc.edu; stephanie@liai.org |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Genetic, Molecular & Cellular Biology |
| School | Keck School of Medicine |
| Date defended/completed | 2010-04-26 |
| Date submitted | 2010 |
| Restricted until | Unrestricted |
| Date published | 2010-08-10 |
| Advisor (committee chair) | Bottini, Nunzio |
| Advisor (committee member) |
Stallcup, Michael Hacia, Joseph Allayee, Hooman |
| Abstract | PTPs involved in modulation of signal transduction through the T cell receptor (TCR) are promising targets for human autoimmunity. Here we will focus on the lymphoid tyrosine phosphatase LYP, a critical negative modulator of TCR signaling encoded by the PTPN22 gene. A missense C1858T single nucleotide polymorphism in the PTPN22 gene recently emerged as a major risk factor for multiple human autoimmune diseases. In T cells, LYP forms a complex with the negative regulatory kinase Csk and is a critical negative regulator of signaling through the T cell receptor. The C1858T SNP results in the LYP-R620W variation within the LYP-Csk interaction motif. LYP-W620 exhibits reduced interaction with Csk and is a gain-of-function inhibitor of TCR signaling. While strong genetic and functional evidence has suggested that LYP is a promising candidate drug target for treatment of human autoimmunity, the molecular mechanism of the autoimmune-associated R620W variation remains unknown.; We hypothesize that an inhibitor of LYP could potentially revert the gain-of-function effect and restore normal TCR signaling levels in carriers of LYP-W620. While a small molecule inhibitor would be useful for both understanding the role of LYP in the immune system and for validating LYP as a drug target, the development of specific, cell-permeable PTP inhibitors is currently hampered by the lack of methods for high-throughput screening (HTS) to identify cell-permeable leads.; Here we present the mechanism by which the pathogenic R620W polymorphism of LYP causes a gain-of-function form of the phosphatase. We also propose two approaches to identify candidate cell-permeable inhibitors of PTPs. We show that through a novel screen of a library of drug-like small-molecule compounds, we were able to identify a non-phospho-mimetic cell-permeable inhibitor of LYP which increases TCR signaling and T cell activation. We also propose the first cell-based assay to directly monitor PTP activity at the single-cell level. Our assay can be adapted to screening of inhibitor libraries for any PTP of interest to identify cell-permeable inhibitors. |
| Keyword | PTPN22; lymphoid phosphatase; PTP; Csk; tyrosine phosphatase; autoimmunity; TCR signaling; cell-based assay |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m3388 |
| Rights | Stanford, Stephanie Michele |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-Stanford-3949 |
| Archival file | uscthesesreloadpub_Volume14/etd-Stanford-3949.pdf |
Description
| Title | Page 1 |
| Full text | i THE MOLECULAR MECHANISM UNDERLYING THE AUTOIMMUNE-ASSOCIATED PTPN22 R620W VARIATION AND THE QUEST FOR THERAPEUTICS by Stephanie Michele Stanford A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (GENETIC, MOLECULAR, AND CELLULAR BIOLOGY) August 2010 Copyright 2010 Stephanie Michele Stanford |
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