Abstract

To evade the host response to infection, viruses have developed means to survive and propagate. HPV 16, a causative agent of cervical cancer and of some cases of oropharyngeal cancers, is one example. We have reported that the early viral protein E6 binds to proteins necessary for propagation of the apoptotic signal following receptor/ligand interactions, such as those mediated by FADD DED and procaspase 8 DED. E6 expression leads to the dose-dependent accelerated degradation of FADD and the protection of E6-expressing cells from Fas-induced apoptosis. Surprisingly, the splice isoforms of E6, E6large and E6*, affect the stability of procaspase 8 DED differentially, resulting in either protection or sensitization to apoptosis-inducing stimuli, respectively.

In this study, we sought to localize the E6 binding sites on FADD and procaspase 8 through the creation of deletion and site-directed mutants, followed by the evaluation of these mutants in biological assays. We found that a novel binding domain characterized by serine 16, serine 18, and leucine 20 mediates binding of E6 to FADD DED. Peptides created to mimic this domain blocked E6/FADD interaction in vitro, and over-expressing the proposed binding domain in E6-expressing U2OS osteosarcoma cells and in the HPV positive cervical carcinoma cell line SiHa, restored sensitivity to Fas-induced apoptosis.

A sequence alignment between the DEDs of FADD and procaspase 8 suggested that E6 may bind to these proteins in a similar manner. Mutations/deletions in the proposed E6 binding domain on procaspase 8 were introduced and the resulting constructs tested for their ability to bind E6. As predicted, some of the amino acids in the N-terminus of the protein are involved in binding. Interestingly, a peptide that obstructs E6/FADD binding also blocks E6/procaspase 8 binding. Screening of a small molecule library identified chemicals that can impede E6 binding, suggesting the possibility that druglike molecules that target these interactions can be identified.

Our findings demonstrate the existence of a novel E6 binding domain and contribute to the literature regarding the use of peptides and small molecules to inhibit protein-protein interaction. These findings also support the possible development of novel therapeutic approaches for HR-HPV induced cancers.

LLU Discipline

Microbiology and Molecular Genetics

School

Graduate Studies

First Advisor

Penelope J. Duerksen-Hughes

Second Advisor

Danilo S. Boskovic

Third Advisor

Carlos A. Casiano

Fourth Advisor

Daisy De Leon

Fifth Advisor

Jonathan W. Neidigh

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

2008

Date (Title Page)

6-2008

Language

English

Library of Congress/MESH Subject Headings

Papillomavirus, Human -- dissertations; Fas-Associated Death Domain Protein -- metabolism; Protein Binding; Apoptosis; Antigens -- immunology

Type

Dissertation

Page Count

xii; 177

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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