Protein S-nitrosation occurs throughout biology and plays a regulatory role in all major diseases, including cardiovascular disease, diabetes, asthma and cancer. Yet, the chemical and structural biology underlying these events remains undiscovered. We will bridge this gap through crystallographic, biochemical and molecular genetics investigations of three key proteins: thioredoxin, soluble guanylate cyclase and glutathione S-nitroso reductase (GSNOR). Together, these proteins perform much of the RSNO-based chemistry thought to occur in the cell and will allow us to investigate the structure and dynamics of these activities in normal and disease states.
Specific Aim 1 : Transnitrosation reactions involving human thioredoxin. Thioredoxin has emerged as a key intermediate in protein transnitrosation and is also implicated in several diseases, including cancer and heart disease. We have achieved a crystal structure of S-nitrosated human thioredoxin through transnitrosation by S-nitrosogluatathione (GSNO), the first such structure to be described. We are now in a position to explore the structure and biochemistry of this modification in vitro and in a model cancer cell line.
Specific Aim 2 : GSNO stimulation of soluble guanylate cyclase. The best understood pathway for nitric oxide signaling is through binding to heme in soluble guanylate cyclase (sGC), which initiates cGMP production to regulate physiological activities such as blood pressure and memory formation. We have discovered that GSNO is also a potent stimulant of sGC. We will undertake biochemical and molecular genetic experiments to reveal the role of GSNO in sGC function Specific Aim 3: GSNO metabolism by GSNO reductase. Much of the nitric oxide produced in vivo is initially captured by glutathione as GSNO, which participates in SNO chemistry and is metabolized by GSNOR. We have determined a high resolution structure for GSNOR and discovered an inhibitor of the protein. We now propose a program in drug discovery and biochemistry targeted to GSNOR.
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|Weichsel, Andrzej; Kem, Michelle; Montfort, William R (2010) Crystal structure of human thioredoxin revealing an unraveled helix and exposed S-nitrosation site. Protein Sci 19:1801-6|
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