Human Cystathionine γ-Lyase Is Inhibited by s-Nitrosation: A New Crosstalk Mechanism between NO and H2S.

Dalila G. F. Fernandes,Catarina S. Tomé,João B. Vicente,João Nunes,Karim Zuhra,Alexandra M. M. Antunes,Alessandro Giuffrè,João M. F. Costa

doi:10.3390/antiox10091391

Abstract

The ‘gasotransmitters’ hydrogen sulfide (H2S), nitric oxide (NO), and carbon monoxide (CO) act as second messengers in human physiology, mediating signal transduction via interaction with or chemical modification of protein targets, thereby regulating processes such as neurotransmission, blood flow, immunomodulation, or energy metabolism. Due to their broad reactivity and potential toxicity, the biosynthesis and breakdown of H2S, NO, and CO are tightly regulated. Growing evidence highlights the active role of gasotransmitters in their mutual cross-regulation. In human physiology, the transsulfuration enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are prominent H2S enzymatic sources. While CBS is known to be inhibited by NO and CO, little is known about CSE regulation by gasotransmitters. Herein, we investigated the effect of s-nitrosation on CSE catalytic activity. H2S production by recombinant human CSE was found to be inhibited by the physiological nitrosating agent s-nitrosoglutathione (GSNO), while reduced glutathione had no effect. GSNO-induced inhibition was partially reverted by ascorbate and accompanied by the disappearance of one solvent accessible protein thiol. By combining differential derivatization procedures and mass spectrometry-based analysis with functional assays, seven out of the ten protein cysteine residues, namely Cys84, Cys109, Cys137, Cys172, Cys229, Cys307, and Cys310, were identified as targets of s-nitrosation. By generating conservative Cys-to-Ser variants of the identified s-nitrosated cysteines, Cys137 was identified as most significantly contributing to the GSNO-mediated CSE inhibition. These results highlight a new mechanism of crosstalk between gasotransmitters.

Highlights

The H2S-generating activity of human cystathionine γ-lyase (CSE) was measured prior to and after incubatResults ing the enzyme with S3.-nitrosoglutathione (GSNO) (Figure 1)
We evaluated the H2S-synthesizing activity of each variant
Despite growing interest in the role of gasotransmitters in human pathophysiology, Despite growing interest in the role of gasotransmitters inreactive human species pathophysiology, the regulatory networks that tightly control the levels of these in different the regulatory networks that tightly control the levels of these reactive species in different physiological contexts are slowly but surely unravelled

Summary

Introduction

Hydrogen sulfide (H2 S), nitric oxide (NO), and carbon monoxide (CO), the three gasotransmitters in mammalian physiology are gaseous molecules endogenously produced to act as second messengers and mediate signal transduction [1,2,3]. This signaling function is achieved by targeting proteins, namely via binding to metal centers and post-translational modification of specific residues (reviewed e.g., in [4,5]). The ability of gasotransmitters to interact with proteins underlies both their physiological roles and potential toxicity.

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Results

Conclusion