Biology subjects

Chouchani, E. T.

Publications and source records attributed to Chouchani, E. T..

9 recordsLinked to original sources

bioRxiv2026

The mechanism of cysteine sensing by LRRC58

Cysteine is essential for life but toxic in excess. Cells and tissues are exposed to elevated cysteine from endogenous sources and the environment yet maintain low intracellular concentrations to sustain life. Here we identify a mechanism for sensing cysteine to control its abundance and prevent toxicity and premature death. Using cryo-electron microscopy we determine structures of the CRL5L^RRC58 E3 ligase alone and in complex with the cysteine-catabolizing enzyme CDO1. We show that CDO1 interacts with LRRC58 in the vicinity of a zinc-coordinating cysteine motif, within which Cys296 becomes covalently modified upon cysteine exposure. Covalent modification of Cys296 and zinc ejection from this motif drive large structural rearrangements of LRRC58 relayed through Cys319, which destabilize CDO1 binding and position LRRC58 Lys111 for autoubiquitylation. This reorientation of LRRC58 permits CDO1 accumulation, thereby lowering intracellular cysteine. Mutation of Cys319 abrogates inhibitory rearrangement of LRRC58 upon cysteine modification. This renders the ligase constitutively active and prevents CDO1 stabilization, thereby driving systemic cysteine accumulation and premature death in animals. These findings establish the molecular basis of homeostatic cysteine sensing by LRRC58 and demonstrate its essential role in maintaining cysteine concentrations compatible with healthy life.

cell biology↗

bioRxiv2026

Quantitative imaging of native tissue architecture combined with deep multi-omics reveals the spatial biology of kidney aging

Aging alters tissue architecture and metabolism in spatially heterogeneous ways, yet the connections between these measurable structural and compositional phenotypes and underlying molecular changes are largely unknown. Here, we introduce NORIGAMI (NoRI-Guided AI for Multimodal Integration), a pipeline combining Normalized Raman Imaging (NoRI), deep learning, quantitative morphology, deep proteomics, and metabolomics to investigate renal aging. Unlike conventional histology, NoRI provides label-free, scattering-corrected measurements of absolute total protein and lipid mass concentrations (mg/mL). In 32 male rats aged 9, 18, 21 and 25 months, NORIGAMI predicted the chronological age of held-out rats from NoRI images (mean absolute error 0.78 months; R^2 = 0.97), and whole-tissue images outperformed models trained on isolated tubules. Quantitative imaging identified 41 age-associated morphological and compositional features (FDR < 0.05), revealing anatomically heterogeneous and non-linear changes in lipid droplet concentration, droplet size, cytoplasmic lipid concentration and tubular architecture. Deep molecular profiling quantified 11,485 proteins and 269 metabolites, revealing coordinated declines in differentiated proximal tubule (PT) identity and oxidative metabolism, selective increases in basement-membrane collagen-IV chains, and increasing molecular heterogeneity with age. A protein clock, built mainly from extracellular-matrix and heparan-sulfate proteins, predicted age with a mean absolute error of 1.6 months. Old kidneys diverged along a dominant injury-remodeling axis, seen separately in the proteome, in kidney RNA from 54 other rats and in the metabolome, in which tubular-injury and immune proteins, including the NAD+ consuming enzyme CD38, rise as differentiated-tubule proteins and NAD+ fall. NORIGAMI subsequently linked NAD+ associated molecular changes to quantitative features of tubular geometry and protein-lipid organization, while complementary KIM-1-centered analyses identified overlapping morphological associations involving epithelial injury and lipid handling and yielded additional molecular candidates for mechanistic validation. Collectively, these findings suggest that declining tubular metabolic function, altered lipid organization and injury-associated molecular programs represent interconnected features of renal aging. NORIGAMI establishes a framework for identifying the quantitative structural manifestations of molecular aging and generating spatially informed, experimentally testable hypotheses about tissue dysfunction.

physiology↗

bioRxiv2026

Redox activation of ATG5 licenses autophagy upon nutrient restriction

Dietary restriction (DR) protects against metabolic disease, extends lifespan, and is associated with remodeling of tissue reactive oxygen species (ROS). ROS control biological adaptation through reversible oxidation of protein cysteines, yet the targets of DR-initiated redox signaling are unknown. Here we generate OxiDR, a tissue-resolved atlas of the cysteine redox proteome that quantifies oxidation state under DR. Rather than oxidizing the proteome broadly, DR selectively targets a high-amplitude set of cysteines in a tissue-specific manner, allowing systematic classification of biological processes subject to DR-mediated redox regulation. Among the cysteines most highly oxidized upon DR is Cys19 of the core autophagy protein ATG5. We show oxidation of Cys19 is required for ATG5-mediated autophagosome formation and for autophagy triggered by nutrient restriction in human cells and mice. Reversible oxidation of this cysteine promotes ATG5 binding to ATG10, thus forming the ATG5-ATG12 conjugate that lipidates LC3B/ATG8 and matures the autophagosome. In mice, loss of this redox switch prevents effective initiation of autophagy upon nutrient restriction, resulting in gross tissue pathology and rapid onset of mortality. The autophagic response to nutrient restriction is thus gated by oxidation of a single cysteine.

cell biology↗

bioRxiv2026

A quantitative redox proteome of the human muscle response to exercise

Reactive oxygen species (ROS) regulate protein function through reversible cysteine oxidation. In human skeletal muscle, exercise-induced ROS initiates adaptations such as mitochondrial biogenesis, increased insulin sensitivity, and hypertrophy. However, specific protein targets of ROS regulation during exercise remain unclear owing to longstanding challenges in analyzing redox proteomes in vivo. We applied cysteine derivatization and multiplexed proteomics to map muscle protein cysteine oxidation in humans during exercise. The OxiMuscle dataset quantifies reversible modifications across 9,177 unique cysteine sites on 2,782 proteins, comprising 17,492 individual cysteine site measurements in young men undergoing three types of exercise, providing the first comprehensive, site-resolved and quantitative analysis of the exercise-regulated redox cysteine proteome in humans. We systematically define cysteine oxidation targets regulated by at least one form of exercise, many of which reside in proteins with established roles in muscle physiology. Among these sites is a redox-regulated cysteine on the autophagy receptor protein p62. We demonstrate that reversible oxidation of this cysteine regulates p62-mediated autophagy upon myotube contraction and mouse muscle adaptation to exercise in vivo. Together, these results define a redox-driven mechanism linking exercise-induced autophagy to muscle adaptation. More broadly, our findings offer a comprehensive resource on redox-signaling networks in human muscle, accessible at http://oximuscle-alb-1899330623.us-east-1.elb.amazonaws.com/.

cell biology↗

bioRxiv2026

Multi-omic characterization of axolotl perilymph-cerebrospinal fluid reveals shifts in composition during limb regeneration

The axolotl salamander can fully regenerate amputated limbs, yet the systemic consequences underlying this process remain largely understudied. Cerebrospinal fluid is an emerging signaling medium capable of communicating with both the central and peripheral nervous systems, but its composition and potential role in salamander limb regeneration have not yet been examined using modern multi-omics techniques. Here, we developed a protocol for extracting mixed perilymph-cerebrospinal fluid (P-CSF) from axolotl and provided the first proteomic and metabolomic characterization of this biological fluid. We identified 2,626 unique proteins and 173 high-confidence metabolites and quantified them across four time points of early limb regeneration. We demonstrated that limb amputation drives progressive shifts in P-CSF proteins, including an elevation of sarcomeric muscle proteins, regeneration-associated factors, and protease/extracellular matrix proteins. We observed shifts in metabolites involved in oxidative stress, polyunsaturated fatty acid oxidation, and histamine metabolism. Injury-comparison experiments revealed that the observed proteomic changes as a result of limb amputation are different than crush injury, denervation, or tail amputation. This study proposes axolotl P-CSF as a reservoir for limb amputation-associated systemic signaling and as a potential conduit of signals involved in limb regeneration.

systems biology↗

bioRxiv2024

A widespread family of viral sponge proteins reveals specific inhibition of nucleotide signals in anti-phage defense

Cyclic oligonucleotide-based antiviral signaling systems (CBASS) are bacterial anti-phage defense operons that use nucleotide signals to control immune activation. Here we biochemically screen 57 diverse E. coli and Bacillus phages for the ability to disrupt CBASS immunity and discover anti-CBASS 4 (Acb4) from the Bacillus phage SPO1 as the founding member of a large family of >1,300 immune evasion proteins. A 2.1 [A] crystal structure of Acb4 in complex with 3'3'-cGAMP reveals a tetrameric assembly that functions as a sponge to sequester CBASS signals and inhibit immune activation. We demonstrate Acb4 alone is sufficient to disrupt CBASS activation in vitro and enable immune evasion in vivo. Analyzing phages that infect diverse bacteria, we explain how Acb4 selectively targets nucleotide signals in host defense and avoids disruption of cellular homeostasis. Together, our results reveal principles of immune evasion protein evolution and explain a major mechanism phages use to inhibit host immunity.

microbiology↗

bioRxiv2024

Mitochondrial complex III-derived ROS amplify immunometabolic changes in astrocytes and promote dementia pathology

Neurodegenerative disorders alter mitochondrial functions, including the production of reactive oxygen species (ROS). Mitochondrial complex III (CIII) generates ROS implicated in redox signaling, but its triggers, targets, and disease relevance are not clear. Using site-selective suppressors and genetic manipulations together with mitochondrial ROS imaging and multiomic profiling, we found that CIII is the dominant source of ROS production in astrocytes exposed to neuropathology-related stimuli. Astrocytic CIII-ROS production was dependent on nuclear factor-{kappa}B (NF-{kappa}B) and the mitochondrial sodium-calcium exchanger (NCLX) and caused oxidation of select cysteines within immune and metabolism-associated proteins linked to neurological disease. CIII-ROS amplified metabolomic and pathology-associated transcriptional changes in astrocytes, with STAT3 activity as a major mediator, and facilitated neuronal toxicity in a non-cell- autonomous manner. As proof-of-concept, suppression of CIII-ROS in mice decreased dementia-linked tauopathy and neuroimmune cascades and extended lifespan. Our findings establish CIII-ROS as an important immunometabolic signal transducer and tractable therapeutic target in neurodegenerative disease.

neuroscience↗

bioRxiv2024

Ergothioneine boosts mitochondrial respiration and exercise performance via direct activation of MPST

Ergothioneine (EGT) is a diet-derived, atypical amino acid that accumulates to high levels in human tissues. Reduced EGT levels have been linked to age-related disorders, including neurodegenerative and cardiovascular diseases, while EGT supplementation is protective in a broad range of disease and aging models in mice. Despite these promising data, the direct and physiologically relevant molecular target of EGT has remained elusive. Here we use a systematic approach to identify how mitochondria remodel their metabolome in response to exercise training. From this data, we find that EGT accumulates in muscle mitochondria upon exercise training. Proteome-wide thermal stability studies identify 3-mercaptopyruvate sulfurtransferase (MPST) as a direct molecular target of EGT; EGT binds to and activates MPST, thereby boosting mitochondrial respiration and exercise training performance in mice. Together, these data identify the first physiologically relevant EGT target and establish the EGT-MPST axis as a molecular mechanism for regulating mitochondrial function and exercise performance.

molecular biology↗

bioRxiv2024

A comprehensive landscape of the zinc-regulated human proteome

Zinc is an essential micronutrient that regulates a wide range of physiological processes, principally through Zn2+ binding to protein cysteine residues. Despite being critical for modulation of protein function, for the vast majority of the human proteome the cysteine sites subject to regulation by Zn2+ binding remain undefined. Here we develop ZnCPT, a comprehensive and quantitative mapping of the zinc-regulated cysteine proteome. We define 4807 zinc-regulated protein cysteines, uncovering protein families across major domains of biology that are subject to either constitutive or inducible modification by zinc. ZnCPT enables systematic discovery of zinc-regulated structural, enzymatic, and allosteric functional domains. On this basis, we identify 52 cancer genetic dependencies subject to zinc regulation, and nominate malignancies sensitive to zinc-induced cytotoxicity. In doing so, we discover a mechanism of zinc regulation over Glutathione Reductase (GSR) that drives cell death in GSR-dependent lung cancers. We provide ZnCPT as a resource for understanding mechanisms of zinc regulation over protein function.

cell biology↗