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The attachment of ubiquitin to target proteins plays important regulatory tasks in eukaryotic cellular processes.  The most studied after effect of ubiquitination is the subsequent degradation of the target protein by the 26S proteasome.  Degradation of cellular proteins has many regulatory effects including cell cycle progression, stress response, signal transduction, transcriptional activation and DNA repair. Protein ubiquitination is both dynamic and reversible. The ubiquitination of target proteins can be reversed by the action of deubiquitinating enzymes. Deubiquitinating enzymes play important roles in the salvage of proteins from destruction by the 26S proteasome. Since this is a relatively new topic of research, the biochemistry and role of the enzymes involved in deubiquitination is not well understood. A crucial piece of information lacking about USPs is the identification of their target proteins. It will allow a more complete picture of the ubiquitination/deubiquitination pathways and the biological processes that they govern to be drawn.  Also missing however is functional data resulting from the removal of these proteins from the cell.

One class of deubiquitinating enzymes are known as ubiquitin specific proteases (USPs). USPs are presumed to be involved in the removal of ubiquitin molecules from specific target proteins. There are several examples in the literature that demonstrate the importance of USPs in cellular functions.  However, the most notable is USP7-p53-mdm2.  USP7 is able to regulate the levels of p53 and mdm2 proteins by deubiquitinating these proteins once they have been targeted for degradation by ubiquitination. 

Projects

Structural and Functional Characterization of human USP7.

The USP7 protein is found in a large number of eukaryotes ranging from yeast to humans.  Human USP7 is a multidomain protein with 1102 residues. The N-terminal domain is responsible for binding ubiquitinated substrates including p53 and Mdm2, the catalytic domain is located in the middle domain, however, the function of the C-terminal domain is still unknown.  The crystal structures of the N-terminal and catalytic domains have been determined. I would like to continue working on crystal structures of human USP7 and gain insight into its biological function.

Structural and Functional Characterization of the yeast USP7 homolog, UBP15.

There is about 70 % sequence conservation between yeast and human USP7 proteins. I will focus on the crystallization and structure determination of yeast UBP15.  This will be an opportune starting point for the functional characterization of yeast UBP15. We have identified the important residues in human USP7 that are involved in peptide binding (164DWGF167) and these residues are absolutely conserved in all organisms whose USP7 sequences are known, indicating that a similar mode of peptide binding may also be occurring in UBP15.

Selected Publications:

Sheng Y, Saridakis V, Sarkari F, Duan S, Wu T, Arrowsmith CH, Frappier L. (2006) Molecular recognition of p53 and MDM2 by USP7/HAUSP.  Nature Structural and Molecular Biology, 13, 285-291.

Saridakis V, Sheng Y, Sarkari F, Holowaty MN, Shire K, Nguyen T, Zhang RG, Liao J, Lee W, Edwards AM, Arrowsmith CH and Frappier L (2005).  Structure of the p53-binding domain of HAUSP/USP7 bound             to Epstein-Barr Nuclear Antigen 1 (EBNA1): Implications for EBV-mediated immortalization.  Molecular Cell, 18, 25-36.

Saridakis V, Yakunin A, Xu X, Anandakumar P, Pennycooke M, Gu J, Cheung F, Lew JM, Sanishvili R, Joachimiak A, Arrowsmith CH, Christendat D, Edwards AM (2004). The structural basis for methylmalonic aciduria. The crystal structure of archaeal ATP:cobalamin adenosyltransferase.  Journal of Biological Chemistry, 279, 23646-23653.

Lalor DJ, Schnyder T, Saridakis V, Pilloff DE, Dong A, Tang H, Leyh TS, Pai EF (2003).  Structural and       functional analysis of a truncated form of Saccharomyces cerevisiae ATP sulfurylase: C-terminal domain essential for oligomer formation but not for activity.  Protein Engineering, 16, 1071-1079.