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

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.

Computation-Guided Rational Design of Cysteine-Less Protein Variants in Engineered hCGL

“…Specifically, the mutant C229D mutation demonstrated an elevation of approximately 2 °C compared to the WT, while notably, the C109V mutant showed more than 8 °C improvement of the WT. Fernandes et al reported the T m values of cysteine to serine variants of hCGL; the T m of C84S was slightly lower than that of hCGL, and the T m of C229S was slightly higher than that of hCGL, which is consistent with our results. The T m values of other serine variants, although varying, were generally above 68 °C, while our mutants had differences from this value, which may be due to structural changes caused by the substitution of other amino acids.…”

“…Cysteine at position 84, located on the protein's surface, when mutated to the hydrophobic alanine, leads to instability in the protein, thus resulting in decreased stability of the C84A mutant. The results partially overlap with those reported by Fernandes's work, 33 cysteine to serine variants were generated and analyzed, the activities of C109S and C307F were lower than WT when L-Cys was used as a substrate. These results provided evidence that although some cysteine residual may undergo modifications, the in vitro activities were not affected by them.…”

“…The results revealed a total of 8 potential beneficial mutants (with ΔΔ G less than −1.5 or B less than −3.0) (Table ), listed as follows: C84A, C109V, C137V, C172V, C229D, C255L, C307F, and C310V. It should be noted that this rational design method ignores possible modifications on cysteine residues of hCGL, such as S-sulfhydration, S-nitrosation, and polysulfidation. − Although these reports indicated that enzyme activity is not entirely dependent on these modifications, these modifications should also be taken into account when modifying enzymes so as not to lose important regulatory sites when they function within the human body.…”

Local delivery of gaseous signaling molecules for orthopedic disease therapy

“…In parallel, NO can stimulate HO-1 gene transcription and thus increase CO production [ 158 ]. Additionally, NO inhibits CSE activity, leading to a reduction in H 2 S production [ 159 ]. Conversely, H 2 S can upregulate eNOS and iNOS expression in vascular endothelial cells [ 160 ].…”

S‐Nitroso‐l‐cysteine and ventilatory drive: A pediatric perspective

“…Though these pathways are separate from l ‐CSNO‐signaling, the two mechanisms do interact. Biochemical interactions between H 2 S and S‐nitrosothiol signaling have been recently reviewed, and the activity of H 2 S‐regulatory proteins can be inhibited by S‐nitrosylation 62,63 . The interactions between these pathways will be important to study in the future.…”