Ronen Marmorstein, Ph.D.

Professor
Gene Expression and Regulation Program
215-898-5006, Office
215-898-0381, Fax

Introduction

Ronen Marmorstein's laboratory uses biochemical, biophysical and structural techniques to understand the mechanism of gene expression and its relationship to the processes of aging and cancer. Because many cancers and other age-related diseases can be traced to defects in gene-regulatory molecules, specific mechanistic insights into their function may lead to the development of highly targeted new drugs to treat human disease.

Research Summary

The investigators in the Marmorstein laboratory use a broad range of molecular and biochemical research tools centered on determining the X-ray crystal structure of the proteins and DNA. The laboratory is studying how DNA transcription into proteins is regulated by investigating the DNA-binding proteins that directly associate with the DNA and the proteins that enzymatically modify the histone proteins that package the DNA in the nucleus of the cell. The DNA-binding proteins being studied include fungal proteins that contain a Zn₂Cys₆ region, mammalian ETS-domain proteins, and the p53 tumor suppressor. Together, these proteins form a broad model for understanding different aspects of protein-DNA recognition. The histone-modifying enzymes under study include histone acetyltransferases, deacetylases, and kinases. Several of these enzymes are misregulated in cancer and aging. The laboratory is also studying how viral oncoproteins such as human papillomavirus E6 and E7 and Adenovirus E1a mediate cancer. In addition, the researchers are studying the p53 pRb, Ink4, and p300/CBP tumor suppressor proteins. A major research goal regarding these proteins is to employ structure-based design strategies to develop protein-specific drugs for treating diseases such as cancer.

Recent Scientific Advances

Over the last year, the Marmorstein laboratory has made significant scientific advances in several areas:

Chemistry of Sir2 proteins: The Sir2 (silent information regulator 2) family of enzymes are broadly conserved from bacteria to humans, and eukaryotic organisms typically have multiple Sir2 proteins that play distinct roles in gene silencing, metabolism, cell physiology, DNA repair, genome stability and longevity. Calorie restriction has long been correlated with longevity in many organisms including yeast and mammals, and with reduced cancer risk in mammals. In yeast, the correlation of calorie restriction with longevity and genomic stability is dependent on Sir2, implicating a similar correlation in humans. Consistent with this possibility, a screen for small molecule compounds that stimulate the enzymatic activity of yeast Sir2 and human SIRT1 identified a group of plant polyphenol compounds including several currently used chemotherapeutic agents and resveratrol, a compound found in red wine that is associated with a surprising number of health benefits including the mitigation of age-related diseases such as cancer.

The Sir2 enzymes bind NAD⁺ and acetyl-lysine within protein targets and generate lysine, 2’-O-acetyl-ADP-ribose and nicotinamide products. Although, yeast Sir2 targets acetyl-lysine bearing histones to silence gene expression in vivo, other Sir2 proteins recognize non-histone targets in vivo, including a-tubulin, acetyl-CoA-syntetase and the p53 tumor suppressor protein, to mediated other cellular activities. To provide mechanistic insights into the function of Sir2 proteins, the Marmorstein laboratory has determined the X-ray crystal structures of a model Sir2 homologue from yeast, Hst2 (homologue of Sir two-2), in nascent form and in several different liganded forms, including a ternary complex of yHst2, acetylated histone H4 and an NAD⁺ mimic. The Marmorstein laboratory has also carried out extensive biochemical and enzymatic studies on the Sir2 proteins. Together, these studies have provided insights into (1) how catalysis is mediated by the Sir2 enzymes (Zhao et al., 2003a), (2) how non-conserved regions of the Sir2 enzymes mediated functions that are specific for different Sir2 enzymes (Zhao, 2003b), and (3) how different Sir2 proteins target their respective cognate substrates (Zhao et al., 2004). Together, these studies have provided novel insights into the activity of Sir2 proteins and have implications for the structure-based design of Sir2 -specific small molecule compounds that might have therapeutic applications. Future studies in the Marmorstein laboratory are directed at developing Sir2-specific inhibitors and activators and characterizing the mechanism of substrate recognition by Sir2 proteins.

Substrate targeting specificity by histone acetyltransferases: Histone acetyltransferase (HAT) enzymes mediate gene activation by acetylating specific lysine residues within the N-terminal tails of the histone proteins that serve to package DNA in the cell nucleus. Interestingly, although HAT proteins harbor very homologous activities, they fall into distinct families that show low sequence homology and have distinct histone substrate specificities. Earlier studies from the Marmorstein laboratory demonstrated that GCN5/PCAF and MYST subfamilies mediate catalysis by distinct mechanisms and this information was used to design and structurally characterize GCN5/PCAF-specific inhibitors.

More recently, the Marmorstein laboratory has used the GCN5/PCAF enzymes as a model to understand how HAT proteins are directed to their cognate targets (Poux et al., 2003) and how this direction is modulated by other histone modifications such as phosphorylation. (Clements et al., 2003). Together, these studies have implications for how other HAT proteins might recognize their respective cognate targets and how other subunits of multisubunit HAT complexes might modulate substrate specificity as well as catalysis. Future studies in this are directed at designing improved HAT specific Inhibitors and characterizing the structure and mechanism of action of multisubunit HAT complexes.

Mechanism of action of tumor suppressors and viral oncoproteins: The retinoblastoma protein (pRb) plays a key role in the G1 to S transition of the cell cycle by binding to and inhibiting the E2F transcription factor. This factor, when released from pRb, functions to stimulate the expression of S-phase specific genes. pRb is mutated in several cancers including breast carcinomas. pRb is also a target of several known DNA viral oncoproteins, including human papillomavirus (HPV) E7 and adenovirus (Ad) E1A. The Marmorstein laboratory has characterized the binding properties of pRb to HPV-E7 and Ad-E1a and more recently characterized the binding of pRb with E2F using both solution binding studies and structural studies (Xiao et al., 2003). These experiments reveal that the transactivation domain of E2F, the region of E2F responsible for turning on the transcription of target genes, and the LXCXE motif of E7 binds to independent regions of pRb, and that other regions of both proteins are involved in competitive binding. The research team is currently characterizing the biochemical and structural properties of these other E2F and E7 regions both alone and in complex with pRb.

The p53 protein activates transcription of genes that induce apoptosis in response to cellular or genotoxic stress such as DNA damage or hypoxia. p53 is the most mutated gene in human cancer and these mutations are correlated with more than 50% of all human cancer. The majority of tumor derived p53 mutations map to the core DNA binding domain and disrupt p53 function by either distabilizing protein-DNA contacts or by lowering the thermostabilty of the core domain. The Marmorstein laboratory has recently completed the high resolution crystal structure of the p53 core domain and have used this a structure-based scaffold to design small molecule compounds that might stabilize tumor derived p53 stability mutants (Ho et al., submitted). Future studies in the Marmorstein laboratory are directed at using these small molecule scaffolds for the further development of p53 stabilizing compounds that might have therapeutic applications for the treatment of p53-mediated cancers.

Selected Publications

Xiao, B., Spencer, J., Clements, A., Ali-Khan, N., Burghammer, M. Perrakis, A., Marmorstein, R. and Gamblin, S.J. “Crystal structure of the retinoblastoma tumor suppressor protein bound to E2F and the molecular basis of its regulation” (2003) Proc. Natl. Acad. Sci. USA,100, 2363-2368.

Clements, A, Poux, A, Lo, S., Pillus, L., Berger, S. L. and Marmorstein, R. “Structural basis for histone and phospho-histone binding by the Gcn5 histone acetyltransferase.” (2003) Mol. Cell, 12, 461-473.

Zhao, K., Xiaomei, C. and Marmorstein, R. “Structure of the yeast Hst2 histone deactylase in ternary complex with 2’-O-acetyl ADP ribose and histone peptide” (2003a) Structure, 11, 1403-1411.

Zhao, K., Xiaomei, C., Clements, A. and Marmorstein, R. “Structure and autoregulation of a yeast Hst2 homolog of Sir2” (2003b) Nature Structural Biology., 10, 864-871.

Poux, A. N. and Marmorstein, R. “Molecular basis for Gcn5/PCAF histone acetyltransferase selectivity for histone and non-histone substrates” (2003) Biochemistry, 42, 14366-14372.

Zhao, K., Chai, X. and Marmorstein, R. “Structure and substrate binding properties of CobB, a Sir2 homolog protein deacetylase from Eschericia coli” (2004) J. Mol. Biol., 337, 731-741.