{'fraction_non_alphanumeric': 0.03189368770764119, 'fraction_numerical': 0.017940199335548173, 'mean_word_length': 5.693333333333333, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Ubiquitination of Neuronal Nitric Oxide Synthase in the P450 Oxygenase and Calmodulin‐binding Domain

Nitric oxide synthase (NOS), a cytochrome P450‐like hemoprotein enzyme, catalyzes the synthesis of nitric oxide, a critical signaling molecule in a variety of physiological processes. Our lab has discovered that certain drugs suicide‐inactivate neuronal NOS (nNOS) and lead to the preferential ubiquitination of the inactivated nNOS by an Hsp70‐ and CHIP (C‐terminus of Hsp70‐interacting protein)‐dependent process. Conversely, calmodulin and certain reversible inhibitors stabilize the nNOS. We wish to understand the process by which altered nNOS is recognized and ubiquitinated. We developed an in vitro ubiquitination system that contains purified E1, E2 (UbcH5a), and CHIP that recapitulate the cells’ ability to selectively recognize and ubiquitinate the altered forms of nNOS. For example, the slowly reversible inhibitor, NG‐nitro‐L‐arginine, but not the D‐isomer, protects nNOS from ubiquitination. We identified 12 ubiquitination sites through LC‐MS/ MS analysis of the tryptic peptides obtained from the ubiquitinated nNOS. Nine sites are located in the oxygenase domain, two in the calmodulin‐binding domain, and one in the reductase domain. Mutational analyses of residues 739 and 743 in the calmodulin‐binding domain are underway. The sites of ubiquitination are consistent with the importance of the Oxy‐CaM domain in regulating stability of nNOS. Supported in part by NIH grant GM077430.