Research Project
10299521
The role of reactive nitrogen species in over eighty human diseases including atherosclerosis, cancer, neurodegeneration, and stroke is well demonstrated by the accumulation of the biomarker 3-nitrotyrosine (nitroTyr). NitroTyr is not randomly distributed across the proteome as might be expected, but rather is found on specific tyrosines on specific proteins. In response to these observations, the PI has greatly advanced this field by developing genetic code expansion (GCE) technologies enabling site-specific incorporation of nitroTyr into recombinant proteins in bacteria and mammalian cells. Collaborative work using these tools has now firmly established that nitroTyr-proteins are causative agents in amyotrophic lateral sclerosis, atherosclerosis, and cancer, supporting our central hypothesis that nitroTyr-modified proteins are key players in human disease and that understanding the basis for their accumulation and removal, as well as their mechanistic roles in pathology will lead to new opportunities for therapeutic intervention. Further support comes from the breakthrough discovery of a denitrase enzyme that is a tumor suppressor: the ?D2? pseudo-phosphatase domain of the protein tyrosine phosphatase receptor T (PTPRTD2) is a tyrosine denitrase that when knocked out promotes cancer growth. This upends the paradigm that nitroTyr-proteins are an unregulated by-product of stress and makes possible a new research strategy that should accelerate progress. Instead of identifying specific diseases and associated nitroTyr modified proteins one at a time, under the hypothesis that this denitrase represents a new enzyme family involved in regulating the impact of nitroTyr, characterizing these denitrases and the breadth of their substrates should speed the identification of physiologically relevant nitroTyr modifications and also provide new avenues to define their impact. This will be done through pursuing two aims that encompass: (1) defining the denitrase substrate scope and the structure-function relationships critical for substrate recognition, and (2) converting denitrases and their substrates into traps and inhibitors which will be used to identify denitrase/substrate pairs and aid studies of their physiological/pathological impacts in cells. Preliminary work demonstrating feasibility has already identified two additional denitrase substrates, which have altered function upon site-specific nitration. The proposed work to define what nitroTyr proteins are substrates of denitrases will also help resolve why nitrated proteins accumulate in disease, and for every case in which it is discovered that a denitrase/nitroTyr-substrate pair contribute to pathology development, the mapping of that process will open up a new avenue for therapeutic intervention. As (i) the developer of existing nitroTyr GCE technologies, (ii) an enzymologist and (iii) acting director of the Unnatural Protein Facility, the PI is superbly qualified to lead this work and all needed facilities are available. Furthermore, key collaborators are already engaged who bring the expertise in structural biology and cell biology needed for the breadth of work proposed.
