Research in my laboratory aims at developing mechanistic understanding of how ubiquitin signaling works. Specifically, we are focused on understanding structural and biochemical aspects of enzymes that make ubiquitination a reversible process, ultimately regulating ubiquitin signaling. About 100 such enzymes, collectively called deubiquitinases (DUBs), have been discovered in human genome, but with the exception of a very few, their biological function and mechanism of action remain obscure. Using protein crystallography combined with mutational analysis and biophysical studies, we are trying to uncover how DUBs select their substrates and how their catalytic activity is regulated. Many of these enzymes appear to become activated upon binding to larger protein assemblies, such as proteasome and ESCRT (endosomal sorting complex required for transport). Our goal is to define the mechanism of recruitment and activation of DUBs in these macromolecular assemblies. Additionally, we have taken initiative directed at discovering small-molecule modulators of these enzymes with potential therapeutic values.
My current projects involve kinetic and structural studies of human BRCA-1 associating protein (BAP1), a ubiquitin C-terminal hydrolase (UCH) that has been shown to interact with oncogenic (BRCA-1), transcriptional regulator (HCF-1), and HOX gene repressor (ASXL1) proteins. Complementary to our investigation of human BAP1 are structural studies of Calypso, the Drosophila melanogaster homolog, which has been shown to deubiqitinate histones when in the polycomb recessive deubiquitination (PR-DUB) complex. Primarily, we hope to learn more about how BAP1, which contains a UCHL5-like domain, processes polyubiquitin chains, but I would also like to pursue studies of BAP1’s association with ASXL1 and its role in activation and silencing of HOX genes.
My research is focused on the structural and functional study of UCH37 and its interaction with Rpn13 and Rpn2. UCH37 is a deubiquitinating enzyme (DUB), which consists of a ubiquitin carboxy-terminal hydrolase (UCH) domain and a C-terminal domain. Its C-terminal domain has KEKE motif that can mediate interaction with Rpn13. Also, Rpn13 interacts with Rpn2, a proteasome subunit. However, the mechanism of how UCH37 is activated by Rpn13 when it is associated with proteasome is not known. My research goal is to gain structural insights into this mechanism by using X-ray crystallography.
The endosomal sorting complexes required for transport (ESCRT) machinery is a series of protein assemblies that facilitates the down-regulation of cell surface receptors. This is a ubiquitin dependent process in which receptors targeted for degradation are ubiquitinated and then shuttled through the complexes, from there the ubiquitin tag is removed, and the receptor is endocytosed and sent off to the lysosome. The ESCRT machinery is regulated by two deubiquitinating enzymes, AMSH and USP8. My project aims to structurally and functionally characterize AMSH in hopes to understand its biochemical role within the ESCRT machinery.
I am currently working on determining the mechanism and specificity of ubiquitin binding to ubiquitin specific protease 28 (USP28) and ubiquitin specific protease 25 (USP25). USP28 and USP25 are human proteins that both possess tissue specific isoforms. To do this I am employing biophysical techniques such as analytical ultracentrifugation and isothermal titration calorimetry. From this, I am hoping to determine the binding affinity of each protein as well as the polyubiquitin chain specificity. Along with my work on USP28 and USP25 I am also investigating the cross reactivity of Nedd8, a ubiqutin-like protein (Ubl), with deubiquitinating enzymes.
My first project is the Activation of UCHL5 for Polyubiquitin Cleavage. UCHL5 is a 329 amino acid DUB of the UCH family. Its specific function is to cleave polyubiquitin chains, presumably from the distal end on the 19S proteasome. It has been found to dock to the 19S regulatory particle through the interactions with the subunit Rpn13. It is not yet understood how UCHL5 is activated once bound to the proteasome. The goal of my project is to investigate UCHL5's activation for polyubiquitin chain cleavage through structural and biochemical studies.
My second project is the Importance of Oxyanion Hole in UCH enzymes. The putative oxyanion hole residue, glutamine, is conserved in UCH enzymes, but its contribution to transition-state stabilization is only 2 kca/mol, inconsistent with its role as an oxyanion stabilizer. It is possible that this residue is contributing to rate enhancement through other means, such as a CH…O hydrogen bond with the catalytic histidine, which we are exploring through mutagenesis and kinetic analysis.
The focus of my research is on a metalloprotein, phenylalanine hydroxylase (PAH), which originates from a family of pterin-dependent enzymes known as aromatic amino acid hydroxylases (AAAHs). PAH catalyzes the conversion of L-Phenylalanine to L-Tyrosine in the presence of ferrous iron, tetrahydrobiopterin (BH4), and molecular oxygen. The disease phenylketonuria (PKU) arises in humans when dysfunctional PAH impairs metabolism of phenylalanine, resulting in accumulation of abnormal levels of phenylalanine and its toxic derivatives in the blood. Our current interest is to examine binding of both substrates (pterin and phenylalanine) in the active site of cPAH and extrapolate the role that residues constituting the secondary coordination sphere of cPAH play in activity of the enzyme. Furthermore the insights gained from these studies should help to advance our understanding of the hydroxylation mechanism catalyzed not only by PAH, but also by other members of the AAH family, which additionally could be of use to those involved in catalyst design.
My research interest includes understanding the role of reversible ubiquitination in endosomal sorting and trafficking. Signal-receiving molecules (receptors) on the surface of cells are degraded intracellularly to terminate the signal at appropriate times preventing catastrophic turned-on-forever state of the cell, which can lead to uncontrolled cell growth. This degradative process of signal attenuation is executed by reversible modification of the receptor by ubiquitin, executed by coordinated action of several proteins, including deubiquitinases (DUBs). Mammalian systems have two endosome-associated DUBs (AMSH and USP8) that seem to have distinct function, but what exactly they do is not known. Using X-ray crystallography, biochemical and biophysical studies, I hope to understand the molecular nature of USP8's role in receptor down regulation. Additionally, I am also interested in developing molecular probes for better understanding of ubiquitin signaling.
The oxyanions of Chlorine have diverse uses. However, the contamination of water by these chemicals is of environmental concern. At the microbial level, two enzymes, namely, Perchlorate reductase (PerR) and Chlorite dismutase (Cld) get rid of Perchlorate (ClO4-), one of the major toxic oxychlorine species contaminating water. PerR reduces perchlorate to chlorate and chlorate to chlorite; Cld catalyzes the dismutation of chlorite to Cl- and O2. In order to understand the enzyme chemistry and to develop useful catalysts for environmental chlorine remediation, water-soluble iron porphyrins have been synthesized and studied as catalysts for chlorite dismutation. Along with porphyrins, synthetic systems such as water-soluble salen complexes are being synthesized to study the dismutation of chlorite. Water soluble Schiff base complexes have been synthesized to study DNA cleavage and epoxidation of olefins. Different Mn-salen complexes modeled on the above known complexes are being prepared and the dismutation reaction will be investigated using techniques like UV-Vis Absorption Spectroscopy and Ion Chromatography.