SBIR-STTR Award

Well-defined and novel ubiquitin derivatives would be invaluable tools for basic research and drug discovery efforts, but these are commercially unavailable. We propose in this Phase I SBIR grant to develop a comprehensive panel of ubiquitin variants, making all combinations of lysine mutations and N-terminal modifications, as well as a collection of N-terminal epitope and hapten tags. Each ubiquitin will be tested for competence in E1 activation and E2 conjugation assays. The panel of unmodified and modified ubiquitin proteins will be used to produce unanchored polyubiquitin chains with unique lysine linkages, using various ubiquitin conjugation enzymes systems. Methods to synthesize, scale-up, purify to homogeneity and characterize alternate chain linkages in vitro will be developed. These polyubiquitin chain substrates will be extensively characterized in relevant and novel biochemical assays. They will finally be tested for hydrolysis against a battery of deubiquitinating enzymes, including Isopeptidase T, UCH-L1, UCH-L3, BAP1, and USP14, to characterize these hydrolytic specificities of these enzymes. Once fully characterized, these novel Ubiquitin proteins and polyubiquitin chains will be commercialized in Phase II and made available to the entire research community as both free standing reagents, and as part of a broader assay platform for designing inhibitors of any deubiquitination enzyme. This work will also result in the ability to produce, at commercial scale, ubiquitin chains using comprehensive linkages and modification types. This defined product suite creates invaluable options for new research tools and assays, which will facilitate and accelerate discovery and target research efforts. Such a panel of proteins would revolutionize the speed and ease of ubiquitin-related research endeavors, facilitate the further understanding of fundamental questions about misdirected or abnormal ubiquitination processes, and aid in the discovery of new therapeutics, especially as regards to deubiquitination and the proteasome

Deubiquitinating enzymes play critical regulatory roles in the ubiquitin-proteasome pathway (UPP). Despite their relevance to human disease and interest by pharma as targets for therapeutic development, this diverse class of enzymes is understudied and detailed biochemical data is lacking. Highly purified DUB enzymes and appropriate substrates for sensitive and homogeneous assays are not currently commercially available. In Phase I we made new ubiquitin (Ub) proteins and poly-ubiquitin chains which are successful DUB substrates. This project was successful and generated a comprehensive tool set with wide-ranging that facilitated the identification and characterization of new DUBs and other UPP components. In this proposal we build on that progress and focus on two major topics: new deubiquitinating enzymes and novel ubiquitin chain substrates. Our specific aims are to: i) develop new DUBs to expand our current range of commercially available enzymes; ii) test the complete set of DUBs against the Ub substrates and chains made in Phase I; and iii) use new modification on Ub chains to develop a continuous flour-based assay system for DUB proteolysis. We will generate the most comprehensive panel of such proteins that are commercially available. We will characterize these DUBs using our existing Phase I generated substrates to understand their biology and possibly shed light on their function in the cell. The Phase I poly-ubiquitin chain prototypes will be the starting point for modified chains, creating a universal DUB substrate that is optimal for fluorescent-based assay formats such as fluorescent resonance energy transfer (FRET). The substrate will also contain relevant isopeptide bond linkages at lysine positions 48 and 63, in order to dissect DUB linkage preferences. These reagents will be commercialized to further advance DUB research and drug discovery in protein catabolism, signal transduction, cell cycle, apoptosis, and neurodegeneration.

Public Health Relevance:
SBIR research project is to support the design, development, and production of biological products and diagnostic laboratory analysis kits. Specifically it relates to the molecular biochemistry of the human protein ""ubiquitin"" which has roles in numerous disease states such as cancer, inflammation, infection and neurodegeneration. The research data and useful kit products generated by this project will enable new research and drug development in the ubiquitin pathway for various human diseases.

Public Health Relevance:
This Public Health Relevance is not available.

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