Nitric oxide promotes cysteine N-degron proteolysis through control of oxygen availability, 2025, Kim et al.

[SNT Gatchaman]

Nitric oxide promotes cysteine N-degron proteolysis through control of oxygen availability
Kim, Haeun; Tian, Ya-Min; Ratcliffe, Peter J; Keeley, Thomas P

Selected proteins containing an N-terminal cysteine (Nt-Cys) are subjected to rapid, O2-dependent proteolysis via the Cys/Arg-branch of the N-degron pathway. Cysteine dioxygenation is catalyzed in mammalian cells by 2-aminoethanethiol dioxygenase (ADO), an enzyme that manifests extreme O2 sensitivity. The canonical substrates of this pathway in mammalia are the regulators of G-protein signaling 4, 5, and 16, as well as interleukin-32.

In addition to operating as an O2-sensing mechanism, this pathway has previously been described as a sensor of nitric oxide (NO), with robust effects on substrate stability upon modulation of NO bioavailability being widely demonstrated. Despite this, no mechanism to describe the action of NO on the Cys/Arg N-degron pathway has yet been substantiated. We demonstrate that NO can regulate the stability of Cys N-degron substrates indirectly via the regulation of ADO cosubstrate availability. Through competitive, O2-dependent inhibition of cytochrome C oxidase, NO can substantially modify cellular O2 consumption rate and, in doing so, alter the availability of O2 for Nt-Cys dioxygenation.

We show that this increase in O2 availability in response to NO exposure is sufficient to alter both dynamic and steady-state ADO substrate levels. It is likely that this mechanism operates to couple O2 supply and mitochondrial respiration with responses to G-protein-coupled receptor stimulation.

SIGNIFICANCE
Oxygen homeostasis in complex animals requires multiple sensory mechanisms to integrate cell-autonomous actions with tissue-and system-wide feedback circuits. 2-aminoethanethiol dioxygenase (ADO)-catalyzed N-degron proteolysis is rapid, highly O2 sensitive, and capable of dynamically regulating cellular function in response to changing O2 levels. A role for NO in this pathway has long been appreciated, but no clear mechanism for this action has yet been described. Here, we demonstrate that NO has a potent effect on intracellular O2 availability through inhibition of mitochondrial O2 consumption and, by doing so, strongly influences the rates of reactions catalyzed by highly O2-sensitive enzymes such as ADO, with wide-reaching implications for cell and tissue O2 homeostasis.


Web | PDF | Proceedings of the National Academy of Sciences | Open Access

 

[SNT Gatchaman]

The biochemistry and physiology of nitric oxide (NO) and oxygen (O2) are intricately linked. Physiological production of NO in mammalian cells requires O2 as a substrate, and these gases react rapidly with each other to form highly reactive nitrogen species when either or both are in excess. Given this complex biochemistry, it is not surprising that interactions with NO are commonly described in most known mechanisms of O2 sensing, including direct inhibition of hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) enzymes, competitive antagonism at cytochrome C oxidase, and hypoxic vasodilation through S-nitrosohemoglobin.

A role for NO in the proteolytic regulation of proteins bearing an N-terminal cysteine (Nt-Cys) residue has also long been appreciated. Nt-Cys dioxygenation is the second step in the regulated degradation of proteins via the Cys branch of the N-degron pathway. Following methionine cleavage, susceptible Nt-Cys are rapidly dioxygenated in the presence of O2 to cysteine sulfinic acid (-SO2H), a process catalyzed […] in animals by 2-aminoethanethiol dioxygenase (ADO).

In seminal work, it was shown that exogenous addition of NO promoted the degradation of regulators of G-protein signaling (RGS)

Here, we have systematically tested the effect of NO on each step in the mammalian Cys N-degron pathway, demonstrating that Nt-Cys dioxygenation can occur under conditions in which significant levels of NO are unlikely to be present, and confirming that NO does not influence N-degron processing downstream of ADO. Instead, we demonstrate that NO reduces cellular O2 consumption through competitive antagonism at cytochrome C oxidase and, in doing so, increases cellular O2 availability to such an extent that it manifests as changes in ADO substrate stability.

Our data reveal the importance of this process at physiological oxygen levels in cells, where a dynamic link between mitochondrial respiration and the stability of RGS proteins may represent a regulatory mechanism coupling cellular oxygenation to control of G-protein signaling. This would provide a mechanism for both cell-autonomous homeostatic control, and, in view of the diffusive paracrine properties of NO, also has the potential to contribute to the complex regulation of tissue O2 homeostasis.

See also recent review on N-degron N-degron pathways (2024, PNAS)

 

[SNT Gatchaman]

Posting in relation to Zhang et al Dissecting the genetic complexity of myalgic encephalomyelitis/chronic fatigue syndrome via deep learning-powered genome analysis (2025) —

Gene ontology (GO) analysis showed that M9 genes were associated with proteasome function and particularly degradation of ubiquitinated proteins that are targeted for turnover

Two of these, PSMB4 and PSMB5 (components of the 20S core proteasome complex), were part of the leading edge subset (i.e., the proteins that contributed the most to the enrichment signal).

we also emphasize the relevance of PSMB4, PSMB5, PSMB7, PSMD7, DNMT3A, 356 NME1, NRAS and SYNGAP1 to the pathophysiology of ME/CFS. PSMB4, PSMB5, PSMB7, and PSMD7, all proteasome subunits, are integral to antigen processing and immune regulation, making them highly relevant to ME/CFS’s immune dysfunction and persistent inflammation.

And from DecodeME —

FBXL4 (Tier 2)

• Protein: F-box/LRR-repeat protein 4. UniProt. GeneCards.

• Molecular function: Component of the mitochondria-localised SCF-FBXL4 ubiquitin E3 ligase complex. This complex restricts mitophagy by controlling the degradation of BNIP3 and NIX mitophagy receptors (26,27).