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Structure-Function Studies of the TRIM Family of Ubiquitin E3 Ligases

Final Report Summary - TRIM (Structure-Function Studies of the TRIM Family of Ubiquitin E3 Ligases)

Ubiquitin (Ub) and ubiquitin-like proteins (UBLs) play a role in a wide range of cellular processes including protein degradation, cell signaling, transcription regulation and DNA damage response. The regulation of numerous cellular processes by these proteins is ascribed to their ability to form a large spectrum of modifications. To date, besides ubiquitin that can modify a target protein with mono or poly-ubiquitin chains with varying linkages and length, more than dozen different UBLs are encoded in human genome, thereby significantly increase the repertoire of ubiquitin modifications. Not surprisingly, improper regulation of these modifications has been observed in many human diseases including cancer, Alzheimer’s disease, Parkinson’s disease and viral infections. My lab is interested in the molecular mechanisms governing Ub and UBL conjugation to cellular proteins. More than 900 enzymes are involved in deposition or removal of Ub/UBL from target proteins, and only little is known about their specificity, activity and interaction network. We use X-ray crystallography, enzymology, cell biology, biochemistry and a variety of biophysical methods in order to provide structural and functional data required for understanding the molecular mechanisms underlying the function of these enzymes.

In this work we focused on a group of enzymes known as TRIM proteins that are involved in ubiquitin assembly. This family of proteins plays pivotal roles in many physiological and pathophysiological processes such as immune signaling, transcriptional regulation, apoptosis, cancer and developmental and genetic diseases. Despite the numerous vital roles that TRIM proteins play in the cell, important aspects of their mechanism remain unknown mainly due to their multi-domain architecture, their ability to self-associate into dimers and higher order complexes, and their ability to function not only with ubiquitin but also with ubiquitin-like proteins. To gain biochemical understanding on TRIM25 E3 ligase activity we have successfully purified this protein. We then showed that this protein functions as E3 ligase in an in vitro system comprising pure proteins. Currently we are attempting to generate crystal structure of TRIM25 in complex with E2 enzymes in order to understand, at the atomic level, the mechanism of TRIM25 E3 ligase activity.

As noted, my interest is in the regulation of protein ubiquitination in the cell, which is accomplished by the balance between ubiquitin deposition and removal. While the TRIM project focuses on the assembly of ubiquitin chains we also investigated how the ubiquitin chains are disassembly by a deubiquitinating enzyme called AMSH. Specifically, we were interested how the length of the ubiquitin chains is regulated and whether the DUB has a preferential cleavage of ubiquitin chains based on their length. Ubiquitin chains that we have generated for the TRIM project served as a substrate for the DUB assay. Initially we cloned and purified the DUB AMSH and generated several constructs possessing different AMSH domains. Then we purified another protein called STAM that binds AMSH and stimulates the DUB activity. We have found that AMSH in the presence of SATM possesses preferential cleavage of ubiquitin chains based on their length. Moreover, we have shown that the VHS domain of STAM is required for this preferential cleavage. This work which shows for the first time how a DUB is affected by the length of the ubiquitin chain, thereby suggesting that not only the ubiquitin linkage is regulated but also the length of the chain. we have published this work in the journal of biological chemistry.

Besides understanding the contribution of the ubiquitin chain’s length on the DUB activity we also focused on mixed ubiquitin chains. To that end we solved the crystal structure of mixed tri-ubiquitin chain possessing K48 and K63 linkages. Our structure reveals that the two ubiquitins that are connected via a K48 linkage adopt a compact structure whereas the two ubiquitins connected with K63 linkage possess an extended structure. We then complemented the crystal structure with small angle x-ray scattering (SAXS) data to understand how the mixed chain behaves in solution. Unexpectedly, we also found that the order of the linkages within the mixed chain (i.e. whether the K48 linkage is at the proximal or distal end) affects the susceptibility of the linkage to cleavage by a K48 specific DUB. Our results suggest that in mixed chains, not only the type of the linkages but also their sequence determine the structural and functional properties of the chain. We recently published this work in JMB.

Taken together, in this research proposal we have provided significant data for understanding the mechanism of ubiquitin chain formation by the TRIM E3 ligases. We have successfully generated an in vitro system to characterize these proteins, and we are now in the process of understanding their structure at the atomic level. In parallel we have deciphered that the length of the ubiquitin chain as well as the order of the linkages within the chain are critical factors that determine the functionality of these ubiquitin chains. Our research therefore significantly advances our understanding of protein modification by ubiquitin and opens new directions to investigate this complex modification. Specifically, our results can lead to the development of new drugs that can interfere with protein modifications by ubiquitin.

The Marie Curie Career Integration Grant not only enabled me to perform the above research but also to establish myself as an independent researcher in the field of protein modification by ubiquitin. Moreover, currently I am evaluated for tenure via a review process that includes scientific excellence, teaching quality and the ability to attain external funding. For me having the Marie Curie Career Integration was a crucial step towards tenure and scientific independence. I would like to take this opportunity and to thank CIG for being a wonderful platform for young PI. The grant enabled me to hire very talented students and postdocs. But more important, it allowed me to focus on my research and to publish our work in a high profile journals.