Xiwei Wu

Abstract

Specific intracellular autoantigens targeted by autoantibodies in systemic autoimmune and chronic inflammatory diseases undergo posttranslational modifications, such as cleavage, during cell death that could potentially enhance their immunogenicity. In light of the increasing interest in the immunological consequences of defective clearance of apoptotic cells, we explored whether autoantigens cleaved during apoptosis undergo an additional wave of proteolysis as apoptosis progresses to secondary necrosis in the absence of phagocytosis. Jurkat T cells treated with different apoptosis inducers underwent a rapid apoptosis that gradually progressed to secondary necrosis. During the initial apoptotic stages, several nuclear autoantigens, including PARP, Topo I, and LEDGF/p75 were cleaved into their signature apoptotic fragments. Progression of apoptosis to secondary necrosis was associated with additional proteolysis of these and other autoantigens in a caspase-independent manner. Our data indicated that in the absence of phagocytosis, apoptotic cells may undergo secondary necrosis associated with additional proteolytic degradation of specific autoantigens. Secondary necrosis may occur in vivo in autoimmune disorders associated with impaired clearance of apoptotic cells and serve as a source of modified forms of specific autoantigens that might stimulate autoantibody responses under proinflammatory conditions.

One of the autoantigens cleaved during apoptosis and necrosis was LEDGF/p75, a major target of autoantibodies in atopic and chronic inflammatory disorders. LEDGF/p75 is a survival protein that protects mammalian cells against various environmental stresses, including oxidative stress and serum starvation. We observed that LEDGF/p75 was cleaved during apoptosis into fragments of 65 and 58 kD. The 65 kD fragment was generated by caspases-3 and -7 cleaving simultaneously at DEVPD30G and DAQD486G, whereas the 58 kD fragment was generated by subsequent cleavage at WEID85N. Sequence analysis revealed that the DEVPD30G and WEID85N sites conserved among LEDGF/p75 proteins from different species and lie within the highly conserved PWWP domain. Expression of LEDGF/p75 significantly protected HepG2 cells from serum starvation-induced death, whereas expression of the 65 kD fragment not only failed to protect cells, but also promote cell death by possible dominant negative effect. The apoptotic cleavage of LEDGF/p75 may contribute to the pathogenesis of atopic disorders by abolishing its pro-survival function and enhancing its immunogenicity.

LLU Discipline

Microbiology and Molecular Genetics

School

Graduate School

First Advisor

Carlos A. Casiano

Second Advisor

Daisy D. De Leon

Third Advisor

Alan P. Escher

Fourth Advisor

Mark S. Johnson

Fifth Advisor

Michael B. Lilly

Sixth Advisor

Anthony J. Zuccarelli

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

2002

Date (Title Page)

6-2002

Language

English

Library of Congress/MESH Subject Headings

Apoptosis -- immunology; Autoantigens -- physiology; Autoimmunity. Cell Aging

Type

Dissertation

Page Count

xv; 251

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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