Exploring the role of the CXXC motif in the regulation of some enzymes involved in redox signaling.

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The CXXC-motif refers to a protein sequence where two cysteine residues are separated by intervening residues, denoted by 'X' (X=any amino acid). This motif is found in many enzyme active-sites i.e. thioredoxin and protein disulfide isomease (PDI). In addition, the CXXC-motif is found peripheral to the active sites, yet performing crucial functional and regulatory roles. Studies have also shown that variation in the XX residues or those N- or C- terminal to the CXXC-motif can modulate enzyme activity. In the short term, we will determine the role of the CXXC-motif and flanking residues in the regulation of PDI, S-nitrosoglutathione reductase (GSNOR) and cystathionine-gamma-lyase (CSE), some key enzymes involved in thiol redox, nitric oxide (NO) and hydrogen sulfide (H2S) signaling. The work on PDI and GSNOR will indicate whether a lysine (K) residue at the C-terminal end of the CXXC-motif or other lysine residues within PDI and GSNOR are the sites for post translational modification by acetylation. We will assess the consequences of lysine-acetylation on the regulation, secretion and extra-cellular and intra-cellular localization of PDI and GSNOR. CSE contains 2 CXXC-motifs. We will test CSE's CXXC-motifs for: a) a previously undiscovered PDI-like thiol reductase activity role; and b) thiol-disulfide mediated regulatory role of its activity. In addition, we will use CSE-generated (homo)cysteine persulfides to determine whether H2S directly participates in sulfide signaling. Our long term goals are to investigate the cross-talk between enzymes that participate in thiol-mediated signaling.In terms of impact to the field, the research program will introduce acetylation as a newly discovered mode of regulation for PDI and GSNOR, key enzymes involved in thiol-mediated signaling and implicated in several pathologies. PDI-work will yield information on the regulation of its secretion, cell surface attachment and thiol reductase and chaperone activities. GSNOR will be characterized with respect to: a new allosteric site; the role of acetylation on its catalytic/allosteric behaviour; interaction with eNOS; and intracellular localization. The work on CSE will yield information on its redox regulation and a new PDI-like thiol reductase activity. The CSE-generated homocysteine persulfides will be used to answer the fundamental question of whether sulfide participates in protein signaling or is a bystander molecule. Furthermore, information gained with respect to the control of the activities of these enzymes will be invaluable for the development of pharmaceutical agents to control these enzymes and the signaling pathways they modulate.