S-Nitrosation: Molecular Mechanisms and Biological Implications

S-Nitrosation, a nitric oxide (NO)-driven post-translational modification, modulates protein functionality, cellular signaling cascades, and oxidative stress modulation by transforming cysteine thiol groups (-SH) into S-nitrosothiol (-SNO) adducts. This redox-sensitive modification is integral to the physiological regulation of cardiovascular, neurological, and immune systems, while its dysregulation is implicated in numerous pathological conditions. By dynamically altering protein conformation and interaction networks, S-nitrosation serves as a critical mediator of cellular homeostasis and disease progression. This review comprehensively examines the molecular underpinnings, regulatory pathways, and physiological implications of S-nitrosation, bridging its biochemical basis to functional outcomes in health and disease.

Nitric Oxide Dynamics: Biosynthesis, Transport, and Stabilization in S-Nitrosation

Generation and delivery of NO: the starting basis of S- nitrosation

Nitric Oxide Synthase (NOS) and NO Synthesis

• Enzyme type:
  • Endothelial type (eNOS): regulates vasodilation and continuously releases low concentration of NO.
  • Neuronal type (nNOS): It participates in nerve signal conduction and is activated by calcium ions.
  • Inducible type (iNOS): It is expressed in large quantities during inflammation or infection, resulting in high concentration of NO.
  • Substrate and cofactors: L- arginine, NADPH, tetrahydrobiopterin (BH4).
• NO delivery and stabilization:
  • Direct diffusion: NO, as a fat-soluble gas, freely passes through the cell membrane, but its half-life is short (seconds).
  • Vector-mediated:
    • S- nitrosoglutathine (GSNO): Glutathione (GSH) combines with NO to prolong half-life and promote long-distance transportation.
    • Metalloproteins: Hemoglobin and ceruloplasmin reversibly bind/release NO through the metal center.

S-nitrosation of hTrx in a cell context (Almeida VS et al., 2022).

Chemical Mechanisms of S-Nitrosation: From Reactivity to Specificity

1. Molecular Pathways of Chemical Modification

Metal Ion-Mediated Direct S-Nitrosation

Transition metal ions, such as Cu²⁺ and Fe²⁺, facilitate direct S-nitrosation by forming transient nitrosyl-metal complexes, which lower the energy barrier for cysteine thiol modification. For instance, Cu²⁺ in superoxide dismutase (SOD1) catalyzes S-nitrosation of neuronal proteins, modulating redox signaling. Similarly, heme-bound Fe²⁺ in cytochrome c oxidase mediates mitochondrial protein modifications, exemplified by complex I Cys39 nitrosation.

Kinetic Influences

Reaction efficiency under metal catalysis depends on environmental factors: acidic conditions (pH<6.0) enhance Cu²⁺ activity tenfold, while Zn²⁺ competitively inhibits Cu²⁺ binding, impairing S-nitrosation in neurodegenerative contexts.

Oxidative Intermediate-Driven Indirect Pathways

Reactive nitrogen species (RNS) like dinitrogen trioxide (N₂O₃) and peroxynitrite (ONOO⁻) act as secondary mediators. N₂O₃ forms via a tri-molecular reaction (neutral pH) and preferentially modifies hydrophobic cysteine residues (e.g., hemoglobin Cys93), stabilizing S-nitrosothiols (RSNOs). In contrast, ONOO⁻, generated rapidly in high ROS environments, induces dual post-translational modifications (e.g., tyrosine nitration and cysteine nitrosation), disrupting mitochondrial complex I function during inflammation.

2. Determinants of Modification Specificity

Microenvironmental Regulation

• Acidic pH: Lysosomal acidity (pH 4.5–5.0) promotes NO protonation, enhancing S-nitrosation of targets like LAMP-2 Cys76, critical for autophagy. Proton-coupled electron transfer (PCET) mechanisms further modulate thiol reactivity.
• Hydrophobic Shielding: Hydrophobic pockets (e.g., hemoglobin β-chain Cys93) sequester RSNOs from aqueous hydrolysis, extending half-life from minutes to hours.

Redox Dynamics

• Glutathione Balance: Elevated GSH levels (>5 mM) suppress S-nitrosation by reducing RNS, whereas oxidative stress (GSH/GSSG<1) promotes sulfinic acid formation, temporarily permitting nitrosation during ischemia-reperfusion.
• Thioredoxin Modulation: Cytoplasmic Trx1 reverses RSNOs (e.g., de-nitrosating Caspase-3), while mitochondrial Trx2 regulates complex I Cys39 to balance ROS and energy production.

Regulatory mechanisms of GSNOR in protein denitrosation on the intersection of signaling pathways of ROSs and RNSs (Jahnová J et al., 2019)

Structural and Sequence Determinants

• Conformational Exposure: Structural shifts expose buried cysteines (e.g., p53 Cys124 during DNA binding), enabling nitrosation-linked functional inactivation in cancer.
• Residue Synergy: Adjacent basic residues (e.g., iNOS Arg266/Lys269) electrostatically attract RNS, while hydrogen bonds (e.g., hemoglobin His92-Cys93) stabilize RSNOs.

3. Advanced Analytical and Experimental Approaches

Probe-Based Labeling

• Biotin-Switch Refinement: Substituting methylthiosulfonate with iodoacetamide minimizes nonspecific thiol blocking, improving specificity in complex matrices.
• Click Chemistry: Alkyne-functionalized NO donors enable spatially resolved RSNO tagging in live cells via copper-catalyzed azide cycloaddition.

Structural and Dynamic Profiling

• Cryo-EM/X-ray Crystallography: Resolve S-nitrosation-induced conformational changes (e.g., SNO-Hb quaternary restructuring) and atomic-level modification sites (e.g., Caspase-3 Cys163).
• Real-Time Monitoring: FRET-based probes track nitrosation-driven structural dynamics, while SPR quantifies binding affinity alterations (e.g., SNO-p53 and MDM2 interactions).

4. Emerging Frontiers and Innovations

• Targeted Modulation: Design pH-sensitive small molecules to selectively induce S-nitrosation in acidic tumor microenvironments, enhancing therapeutic precision.
• Systems-Level Mapping: Integrate single-cell transcriptomics with spatiotemporal mass spectrometry to delineate dynamic S-nitrosation networks across disease progression.
• Computational Prediction: Develop AI models (e.g., AlphaFold-SNO variants) to predict cysteine nitrosation propensity and functional impacts, accelerating mechanistic discovery.

Dynamic Regulation of S-Nitrosation: Enzymatic Control and Redox Networks

1. Enzymatic Systems Governing S-Nitrosation Reversibility

Thioredoxin (Trx) System

• Catalytic Mechanism:
  • The Trx active site, featuring a conserved CXXC motif (Cys32/Cys35), directly reduces S-nitrosothiols (RSNOs) to free thiols (-SH), with NADPH sustaining reduction via thioredoxin reductase (TrxR).
  • Reaction:RSNO+Trx-(SH)₂→RSH+Trx-S₂+HNO
• Compartment-Specific Roles:
  • Cytosolic Trx1: Regulates nitrosation of apoptotic mediators (e.g., ASK1, NF-κB), suppressing cell death pathways.
  • Mitochondrial Trx2: Maintains redox balance by dynamically modulating S-nitrosation of complex I (NDUFS2), linking ROS production to energy metabolism.
• Disease Relevance:
  • Cancer: Elevated Trx1 levels enhance tumor cell survival by reversing pro-apoptotic S-nitrosation (e.g., Caspase-3 reactivation).
  • Neurodegeneration: Trx2 dysfunction in Parkinson's disease leads to mitochondrial RSNO accumulation, exacerbating oxidative stress and α-synuclein pathology.

Glutaredoxin (Grx) System

• GSH-Dependent Reduction:
  • Grx utilizes glutathione (GSH) via mono-/dithiol mechanisms to degrade RSNOs, critical for mitochondrial integrity.
  • Reaction:RSNO+2GSH→RSH+GSSG+HNO
• Pathophysiological Impact:
  • Cardiac Injury: Grx2 deficiency in ischemia-reperfusion models causes mitochondrial RSNO overload, impairing complex I and worsening tissue damage.

S-Nitrosoglutathione Reductase (GSNOR)

• Function: Degrades GSNO to glutathione disulfide (GSSG), indirectly suppressing global S-nitrosation.
  • Reaction:GSNO+NADH→GSSH+NAD++NH
• Therapeutic Targeting:
  • Inflammatory Disorders: GSNOR inhibitors (e.g., N6025) elevate GSNO, attenuating NF-κB-driven inflammation in COPD clinical trials.

2. Redox Equilibrium and Thiol State Dynamics

NADPH Oxidase (NOX)-Mediated Regulation

• ROS Generation: NOX enzymes (e.g., NOX2/4) produce superoxide (O₂⁻), which transforms into H₂O₂or peroxynitrite (ONOO⁻), modulating nitrosation.
• Redox Disruption:
  • Trx Inactivation: ROS oxidize Trx's catalytic cysteines (Cys32/Cys35), impairing RSNO reduction.
  • Glutathione Reductase (GR) Suppression: Oxidative GR inactivation reduces GSH regeneration, exacerbating oxidative stress (e.g., atherosclerotic endothelial dysfunction).

Thiol Oxidation Hierarchy

• Functional States:
  • -SNO: Transient signaling (e.g., vasodilation via SNO-Hb).
  • -SSG: Stress-adaptive modification (e.g., HSP70 Cys267-SSG enhances chaperone activity).
  • -SOH/SO₂H: Irreversible oxidative damage markers (e.g., Tau Cys322-SOH in Alzheimer's neurofibrillary tangles).

3. Emerging Therapeutic and Analytical Frontiers

Targeted Interventions

• Small Molecules:
  • Trx Inhibitors (PX-12): Amplify oxidative stress in tumors, sensitizing them to chemotherapy (Phase II trials in pancreatic cancer).
  • NOX Inhibitors (GKT137831): Mitigate diabetic nephropathy by preserving Trx activity.
• Gene Therapy: AAV-mediated Trx2 delivery rescues mitochondrial function in Parkinson's models, reducing α-synuclein aggregation.

Real-Time Monitoring Tools

• Fluorescent Probes:
  • HyPer-SNO: Genetically encoded sensor for live-cell S-nitrosation imaging.
  • Grx2-roGFP: Tracks mitochondrial Grx2 activity dynamically, linking redox shifts to metabolic states.
• Computational Integration: AI-driven models predict cysteine nitrosation hotspots and functional outcomes, accelerating drug discovery and mechanistic studies.

Detection Methods and Technological Advancements

1. Biochemical Methodologies

• Biotin Switch Technique (BST) Optimization:
  • Enhanced Specificity: Substitution of methyl methanethiosulfonate (MMTS) with iodoacetamide (IAM) minimizes nonspecific thiol blocking, effectively reducing interference from glutathione adducts.
  • Click Chemistry Integration: Alkyne-functionalized HPDP-Biotin enables copper-catalyzed azide-alkyne cycloaddition (CuAAC), significantly improving enrichment efficiency for low-abundance S-nitrosylated (SNO) proteins.
• Mass Spectrometry Innovations:
  • High-Resolution Mass Spectrometry (HRMS): Coupling with Orbitrap Fusion Lumos allows precise identification of S-nitrosylated peptides (+28.99 Da) while resolving isotopic interferences.
  • Multiplexed Quantification: Integration of tandem mass tags (TMT) or isobaric tags (iTRAQ) with ¹⁵N metabolic labeling enables dynamic profiling of SNO modifications under diverse conditions (e.g., inflammatory responses).

Biotin switch based methods for SNO detection (Wang H et al., 2011).

2. Imaging Technology Breakthroughs

• Fluorescent Probe Development:
  • Genetically Encoded Sensors (GeNOps): Engineered cpGFP-based probes fused to NO-binding domains permit real-time tracking of S-nitrosylation dynamics in subcellular compartments (e.g., mitochondrial vs. cytosolic pools).
  • Near-Infrared Probes: Cy7-NEM derivatives exhibit deep tissue penetration (>5 mm), facilitating noninvasive SNO imaging in tumor xenograft models for therapeutic guidance.
• Advanced Chemiluminescence Platforms:
  • Microfluidics-Enhanced Detection: Integration of microfluidic chips with chemiluminescent sensors achieves ultrasensitive quantification of NO metabolites (detection limit: 1 nM NO₂⁻) at single-cell resolution.

3. Integration of Cutting-Edge Technologies

• Spatial Omics Applications:
  • MALDI Mass Spectrometry Imaging (MSI): BST-coupled MSI maps spatial heterogeneity of S-nitrosylation in tissue sections, revealing organ- or region-specific modification patterns (e.g., brain region-specific SNO profiles).
• Single-Cell Proteomic Profiling:
  • Nanodroplet Processing (nPOP): High-throughput single-cell mass spectrometry deciphers RSNO modification heterogeneity across cellular subpopulations, identifying drug-resistant tumor cell signatures.

Biological function and application

• Regulating plant stress response: This study demonstrates that nitric oxide (NO) in plants modulates hormone signaling by mediating S-nitrosylation of critical E3 ubiquitin ligase components. Specifically, post-translational modification of Cys140 in TIR1 (an auxin receptor) and Cys118 in ASK1 facilitates the assembly of SCFTIR1 and SCFCOI1 complexes, thereby enhancing transcriptional activation of auxin- and jasmonate (JA)-responsive genes. Mutants lacking S-nitrosylation capacity exhibit impaired protein interactions and compromised hormonal signaling. These findings reveal that redox-driven S-nitrosylation governs spatiotemporal regulation of SCF complex activity, positioning NO as a central hub integrating oxidative signaling with hormonal pathways to orchestrate developmental plasticity and stress adaptation in plants (Terrile MC et al., 2022).
• Participate in cell aging pathway: This study elucidates endothelial cells (ECs) modulate cellular fate determination through S-nitrosylation under impaired Nrf2 signaling. Both Nrf2-deficient ECs and murine aortic tissues exhibited accelerated senescence without classical oxidative damage, demonstrating that Nrf2 loss directly orchestrates a redox-independent senescence pathway. This work identifies the Keap1/GAPDH/NOS triad as a previously unrecognized mammalian S-nitrosylation catalytic machinery. By delineating how dysregulated Keap1 drives senescence through S-nitrosylation, our findings provide a molecular framework for targeting endothelial dysfunction in cardiovascular pathologies (Kopacz A et al., 2020).
• Related to disease: This study elucidates a novel mechanism through which the transcription factor Krüppel-like factor 4 (KLF4) modulates endothelial cell (EC) function via S-nitrosylation and contributes to the pathogenesis of pulmonary arterial hypertension (PAH). We identified that KLF4 undergoes site-selective S-nitrosylation at Cys437 under nitrosative stress in ECs, resulting in diminished nuclear localization and transcriptional repression. Functionally, S-nitrosylated KLF4 (SNO-KLF4) impairs KLF4-mediated vasodilation. Our findings establish S-nitrosylation as a critical post-translational regulatory mechanism for KLF4, offering new insights into redox-dependent vascular dysfunction and therapeutic targeting in PAH (Ban Y et al., 2019).

References

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