Our Science – Appella Website
Ettore Appella, M.D.
The p53 Tumor Suppressor Protein
The human p53 tumor suppressor protein is normally present in a latent state at low levels, 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 post-translational modifications to p53 itself and possibly to other proteins with which p53 interacts. We have prepared a suite of antibodies that recognize specific post-translational modifications of p53 and have used these to characterize the responses to DNA damaging agents. Our analysis of p53 modified at individual sites has revealed a complex and unexpected interdependency in the modifications of p53. However, as regulation may be lost or altered in tissue culture experiments, we are also analyzing the effects and patterns of p53 modifications in tissues from knock-in mutant mice. We have used quantitative mass spectrometry to explore the roles of p53 post-translational modifications in the response to ionizing radiation (IR) by determining the global effects of IR on the levels of proteins in thymocytes of the p53 knock-in mice. These studies are enabling us to better understand the widespread roles of p53 in cells, including its effects on various signaling pathways, and how specific modifications of p53 modulate its functions. In addition to the global analyses, we are also using biophysical, biochemical, and structural methods to explore the modulation of p53 protein-protein interactions by post-translational modifications. For example, we have examined the increased affinity of the N-terminal transactivation domain of p53 for the Taz2 domain of its co-activator p300. Such interactions are critical for the recruitment of transcriptional coactivators to gene promoters for transcriptional activation by p53.
The Wip1 (PPM1D) Phosphatase and its Roles in the Cell
The Wip1 phosphatase (PPM1D) is a member of the PP2C family of evolutionarily conserved protein phosphatases. We initially discovered Wip1 in a screen for proteins up-regulated by p53 after IR; subsequently, Wip1 has been implicated as a negative regulator of p53 function through its ability to attenuate the activity of p38 MAPK, Chk2 and ATM kinases, major effectors of p53 stabilization and activation after stress. The importance of this negative-feedback loop was demonstrated by our observations that Wip1 complements several oncogenes during in vitro cellular transformation assays and that PPM1D is amplified or overexpressed in several types of cancers, including human primary breast cancer, neuroblastoma and ovarian clear cell adenocarcinoma. 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. To better understand the connection between Wip1 activity and the control of cellular proliferation, we are identifying novel substrates of Wip1 phosphatase activity and determining the functional effects. Since Wip1 has been shown to be amplified in tumors, we are developing specific inhibitors of its activity that would provide selective targeting either when given alone or in combination with standard cancer chemo- or radio-therapy.
Design of Antiviral Drugs against the HIV Virus
Development of drug-resistant HIV strains in response to nucleoside, non-nucleoside, and reverse transcriptase and protease inhibitor therapies has necessitated the search for novel antiretroviral agents that target new structures for treatment of HIV and prevention of its transmission. The involvement of HIV-1 NCp7 zinc fingers in multiple phases of the HIV-1 replication cycle and their resistance to mutation have indicated that they may be a good target for antiretroviral therapy. We have synthesized novel uncharged, S-acyl, 2-mercaptobenzamide thioesters (SAMTs) characterized by potent antiviral activity with low cellular toxicity. The overall goal of our research is to understand how these compounds block the transmission of HIV-1 by targeting the highly conserved zinc-binding domains of NCp7. Our studies are currently focused on elucidating the molecular mechanism of several lead compounds. Also, we are evaluating formulation of the inhibitors for use as topical microbicides and using mass spectrometry to identify metabolites of the SAMTs when applied topically in a non-human primate system. The identification of potentially safe and efficacious single or combination candidate microbicides in non-human primates, and the elucidation of their pharmacokinetics, should lead to studies necessary for preclinical evaluation of these compounds.
This page was last updated on 6/7/2013.