Our Science – Appella Website

Ettore Appella, M.D.

Laboratory of Cell Biology Head, Chemical Immunology Section Senior Investigator Building 37, Room 2140A National Cancer Institute 37 Convent Drive Bethesda, MD 20892-4256 Phone:   301-402-4177 Fax:   301-496-7220 E-Mail:   appellae@mail.nih.gov

Biography

Dr. Appella obtained his M.D. from the University of Rome, Italy, and continued his research at Johns Hopkins and the NIH (National Institute of Diabetes and Digestive and Kidney Diseases) on dehydrogenases. Since 1965, he has been in the Laboratory of Cell Biology where he continues his research on tumor immunology, the p53 tumor suppressor protein, and the design of antiviral drugs against HIV.

Research

MHC/Peptide Complexes Structure and Function Analysis

Class I major histocompatibility complex (MHC) molecules are polymorphic cell surface proteins that assemble with peptides derived from the degradation of intracellular proteins. Cell surface class I/peptide complexes are ligands for T cell receptors (TCR) on CD8+ T lymphocytes. Our research is aimed toward understanding the biochemical and functional details of MHC-peptide and MHC-peptide-TCR interactions; these characterizations reveal fupof the immune response and are a step toward principle-based development of immunotherapies. We anticipate that a detailed understanding of structure-activity relationships among immunologic ligands and receptors may lead to the effective use of peptides or peptidomimetics as specific modulators of immune responses. We are particularly interested in studying the factors that govern class I-restricted TCR specificity and cross-reactivity, as well as the qualitative responses observed to peptides that behave as agonists, or partial-full antagonists. Our approach features the extensive use of peptide-epitope analogs to probe recognition in model systems involving human and murine class I molecules. Our ongoing work is presently concentrated in three major areas: (1) analysis of epitope cross-recognition; (2) characterization of the CTL response to peptides derived from tumor-related proteins; and (3) application of mass spectrometric techniques to the analysis of immunologically relevant proteins and peptides.


The p53 Tumor Suppressor Protein and Wip1 (PPM1D) phosphatase
The human p53 tumor suppressor protein normally is present in a latent state and at a low level, but a variety of cellular stresses, including DNA damage, activate signaling pathways that transiently stabilize the p53 protein, cause it to accumulate in the nucleus, and activate it as a transcription factor. Activation leads either to growth arrest at the G₁/S or G₂/M transitions of the cell cycle or to apoptosis. The molecular mechanisms by which stabilization and activation occur are incompletely understood but are believed to be mediated by multiple posttranslational modifications to p53 itself and possibly to other proteins with which p53 interacts. We have prepared antibodies that recognize p53 only when it has been modified at a particular site. These antibodies were then used to characterize the responses to DNA damaging agents. The analysis of p53 modified at individual sites revealed a complex and unexpected interdependency in site phosphorylation. However, regulation may be lost or altered in tissue culture experiments mainly due to genetic changes that occur upon prolonged passage in culture. Most of these problems can be ameliorated by working with mice; therefore, we are analyzing modification patterns in different mouse tissues after generating knock-in mutations for each site, especially for those tissues that show increased tumor development.
The Wip1 phosphatase (PPMD1) is a member of the PP2C family of evolutionarily conserved protein phosphatases. Our initial research publications on Wip1 described it as a p53-regulated gene; subsequently, however, Wip1 has been implicated as a negative regulator of p53 functions through its ability to attenuate p38 MAPK activity, thereby mediating the inactivation of p53 after various cellular stresses. The importance of this negative-feedback loop was further illustrated by our findings that Wip1 phosphatase complements several oncogenes for cell transformation in vitro and that PPM1D is amplified and overexpressed in certain types of cancers, including human primary breast cancer, neuroblastoma and ovarian clear cell adenocarcinoma. Whether or not p38 MAPK is the only target for the Wip1 phosphatase, several recent studies suggest that Wip1 positively regulates cell proliferation and behaves as an oncogene, whereas depletion of Wip1 significantly reduces cell proliferation rates and activates apoptosis. These findings indicate that Wip1 may be a promising new target for treating certain types of tumors.

Design of Antiviral Drugs Against the HIV Virus
Development of drug-resistant HIV strains in response to nucleoside, nonnucleoside, reverse transcriptase and protease inhibitors has necessitated the search for novel antiretroviral agents that target new structures for therapy of acquired immunodeficiency syndrome (AIDS). The involvement of HIV-1 NCp7 Zn fingers in multiple phases of the HIV-1 replication cycle and their mutationally nonpermissive nature have provided incentives for choosing this structure as a target for antiretroviral therapy. We have synthesized novel unchanged, S-acyl 2 mercaptobenzamide thioesters that overcame the cellular toxicity associated with disulfide benzamides and showed superior antiviral activity and minimal cytotoxicity. Our ongoing work is concentrated on determining the mechanism of action of these compounds and determining the sites of modification on the NCp7 protein. For this purpose, a fluorescence-based assay using a zinc-specific fluorophore, Newport Green (NPG), mass spectrometry, and NMR are used to follow the kinetics of zinc ejection and to determine the site(s) of covalent modification on the target protein. Target selectivity, toxicology, and efficacy in a murine and monkey HIV model are also being studied in order to assess their potential for in vivo antiviral activity.

This page was last updated on 6/11/2008.