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Dual Proximity Labeling Strategy Identifies TrxR1 as a Cuproprotein that Regulates Apoptosis via Caspase-3 S-Nitrosylation

ABSTRACT: Copper (Cu) performs essential structural and functional roles in all aerobic organisms, but identifying natural cuproproteins through traditional affinity methods is hindered by the variety of copper ligand combinations and binding locations in proteins. Here, using ATOX1 as a bait, fused with the APEX2 or TurboID proximity labeling enzymes, with quantitative proteomics in cancer and normal cells (H1299 and HEK293T), we predicted 682 highly enriched candidate Cu-binding proteins. Among them, we functionally validated the novel cuproprotein, thioredoxin reductase 1 (TrxR1). Through cryo-electron microscopy (cryo-EM), we resolved three copper-binding sites across its N- and C-terminal redox centers. Functional assays demonstrated that copper inhibits TrxR1 reductase activity and impairs the denitrosylation function of its substrate, thioredoxin 1 (Trx1), consequently inhibiting caspase-3 cleavage and apoptosis. Beyond expanding the scope of annotated cuproproteins, this study provides a versatile strategy for cuproprotein discovery and defines the copper functions in modulating apoptosis through the TrxR1/Trx1 regulatory module.

ORGANISM(S): Homo Sapiens

SUBMITTER: Jing Wang

PROVIDER: PXD068622 | iProX | Mon Sep 22 00:00:00 BST 2025

REPOSITORIES: iProX

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Dual -Proximity Labeling Strategy Identifies TrxR1 as a Cuproprotein that Regulates Apoptosis via Caspase-3 S-Nitrosylation

Project description:Copper (Cu) performs essential structural and functional roles in all aerobic organisms, but identifying natural cuproproteins through traditional affinity methods is hindered by the variety of copper ligand combinations and binding locations in proteins. Here, using ATOX1 as a bait, fused with the APEX2 or TurboID proximity labeling enzymes, with quantitative proteomics in cancer and normal cells (H1299 and HEK293T), we predicted 682 highly enriched candidate Cu-binding proteins. Among them, we functionally validated the novel cuproprotein, thioredoxin reductase 1 (TrxR1). Through cryo-electron microscopy (cryo-EM), we resolved three copper-binding sites across its N- and C-terminal redox centers. Functional assays demonstrated that copper inhibits TrxR1 reductase activity and impairs the denitrosylation function of its substrate, thioredoxin 1 (Trx1), consequently inhibiting caspase-3 cleavage and apoptosis. Beyond expanding the scope of annotated cuproproteins, this study provides a versatile strategy for cuproprotein discovery and defines the copper functions in modulating apoptosis through the TrxR1/Trx1 regulatory module.

2025-09-22 | PXD068649 |

The thioredoxin-1 system is essential for fueling DNA synthesis during T cell metabolic reprogramming and proliferation

Project description:The thioredoxin-1 (Trx1) system is a key player of the cellular redox balance and a sensor of energy and glucose metabolism. Here we report critical c-Myc-dependent activation of the Trx1 system during glucose-driven T cell expansion during development and immune responses but endogenous Trx1-repression during quiescence. Thioredoxin-reductase-1 (Txnrd1)-deletion in CD4-CD8- thymocytes prevented their expansion, while deletion in CD4+CD8+ thymocytes did not affect further maturation and peripheral homeostasis of T-cells. Moreover, B cell development was Txnrd1-independent. Txnrd1 was critical for expansion of activated T cells. Unbiased metabolomics revealed that Txnrd1 is necessary for donating reducing equivalent to ribonucleotide reductase (RNR) at the last step of nucleotide biosynthesis. Impaired availability of 2’-deoxyribonucleotides induced the DNA damage response and cell cycle arrest of Txnrd1-deficient T cells. These results uncover a pivotal role of the Trx1 system in metabolic reprograming of thymic and peripheral T cells and provide a rationale for targeting of Txnrd1 in T-cell leukemia.

2018-03-15 | GSE107090 | GEO

Oxidized thioredoxin-1 restrains the NLRP1 inflammasome [mBMDM]

Project description:The danger signals that activate the NLRP1 inflammasome have yet to be firmly established. NLRP1 undergoes autoproteolysis to generate N-terminal (NT) and C-terminal (CT) fragment, which importantly, is a necessary step for its check-point regulation by the DPP9 ternary complex and the mechanistic activation of NLRP1 through functional degradation. Here, we report an added layer of regulatory complexity to NLRP1 activity, in the form of a repressive interaction that NLRP1 forms with the oxidized, but not reduced, form of thioredoxin-1 (TRX1). Loss of TRX1 destabilizes the NT fragment of NLRP1 and promotes enhanced inflammasome activation. The TRX1 interaction occurs through the NACHT-LRR of NLRP1 and requires nucleotide binding in its ATPase domain. In addition, we found that several patient-derived and ATPase-inactivating mutations in the NACHT-LRR region hyperactive the inflammasome by destabilize protein folding and are also shown to abrogate TRX1 binding. Thus, NLRP1 appears to detect intracellular reductive stress through a decrease in the fraction of intracellular oxidized TRX1, which enhances protein disorder, leading to inflammasome signaling. These findings link the cellular redox environment to NLRP1-mediated innate immunity.

2022-11-04 | GSE215035 | GEO

Oxidized thioredoxin-1 restrains the NLRP1 inflammasome [NTERT-1]

2022-11-04 | GSE215034 | GEO

A new structural paradigm in copper resistance in a human pathogen

Project description:Copper is essential for both innate and adaptive immune function and copper resistance has emerged as an important determinant of virulence of microbial pathogens. In the human pathogen Streptococcus pneumoniae (Spn), cytoplasmic copper resistance is mediated by an operon encoding the copper-responsive repressor CopY, CupA, of unknown function, and CopA, a copper effluxing P1B-type ATPase. We show that CupA is a novel cell membrane-anchored Cu(I) chaperone for CopA, and that a Cu(I)-binding competent, membrane-localized CupA, like CopA, is obligatory for copper resistance.

2013-05-24 | GSE39176 | GEO

Mycobacterium tuberculosis Rv0102-regulated copper uptake facilitates bacterial escape from phagolysosome

Project description:The host restricts Mycobacterium tuberculosis (Mtb) access of transition metal ions to inhibit its growth. Cu is essential for the survival of Mtb, but Cu excess is toxic. During co-evolution, Mtb has developed various mechanisms to maintain copper homeostasis. In this report, we firstly found Mtb Rv0102 encoded membrane protein is critical for Mycobacterium Cu uptake. The intracellular Cu level of M. smegmatis (Ms) Rv0102 homolog MSMEG_4702 knockout strain decreased threefold than the wild type, the deletion mutant was more tolerant to higher Cu level. This indicates that Rv0102 is significant for Cu homeostasis and resistance to Cu toxicity. Knocking out the homologous gene MMAR_0267 in M. marinum (Mm) also enhanced its virulence in zebrafish and improved its survival within macrophages. In THP-1 cells infected with Mm, Mm MMAR_0267 deletion mutants’ intracellular survival increased fourfolds and accompanied by less cell apoptosis. Cu deficiency down-regulates the transcription of the M. marinum virulence factor CFP-10, dampening the STING cytosolic signaling in macrophages, fewer IFN-β and less cell apoptosis. These results suggest that Cu is an important factor in inducing cell apoptosis. In summary, mycobacteria can effectively counteract the host's early key nutritional immune mechanisms by regulating copper metabolism, to increase its virulence.

2023-06-30 | GSE235754 | GEO

64Cu DOTA-Trastuzumab PET/CT in Studying Patients With Gastric Cancer

Project description:This pilot clinical trial studies copper Cu 64 (64Cu) tetra-azacyclododecanetetra-acetic acid (DOTA)-trastuzumab positron emission tomography (PET)/computed tomography (CT) in studying patients with gastric, or stomach cancer. Diagnostic procedures, such as copper Cu 64-DOTA-trastuzumab PET/CT, may help doctors study the characteristics of tumors and choose the best treatment.

| 2151356 | ecrin-mdr-crc

Thioredoxin-1 promotes the restoration of alveolar bone in periodontitis with diabetes

Project description:Treatment of periodontitis in people with diabetes remains challenging. Diabetes-enhanced oxidative stress is a primary cause of aggravation of periodontitis. The present study aimed to investigate the therapeutic potential of thioredoxin-1 (TRX1), an essential endogenous antioxidant protein, in the management of periodontitis with diabetes, as well as its role in modulating osteogenic differentiation. Our findings indicated that the production of reactive oxygen species (ROS) was elevated, while the expression of TRX1 was significantly reduced in the periodontal tissues of periodontitis mice with diabetes. Furthermore, knockdown of TRX1 in periodontal ligament stem cells (PDLSCs) resulted in the inhibition of osteogenic differentiation through disrupting Wnt/β-catenin signal pathway. However, this inhibition was restored upon administration of recombinant human TRX1 (rhTRX1). Importantly, rhTRX1 treatment decreased ROS generation, activated Wnt/β-catenin signal pathway and considerably promoted the alveolar bone repair of periodontitis mice with diabetes. These findings highlighted the crucial protective role of TRX1 in periodontitis with diabetes and suggested that it may serve as a potential therapeutic target for refractory periodontitis associated with oxidative stress.

2023-09-06 | GSE235930 | GEO

Novel Genetic and Pharmacological Modulators of Copper Homeostasis

Project description:Copper (Cu) homeostasis is critical to the health of most organisms, and is thus tightly regulated by several highly conserved processes. Using the unique genomic tools available in yeast, we have expanded current knowledge of these processes by identifying 237 genes important for Cu-dependent growth. 116 of these genes, when deleted, decreased Cu-dependent respiratory growth. The remaining 121 genes, when deleted, produced respiratory growth defects that were specifically rescued by addition of Cu. Among the genes identified by our screen are known regulators of copper homeostasis, genes required to maintain low vacuolar pH, and genes where evidence supporting a functional link with Cu has been heretofore lacking. Many genes identified are not only conserved in man, but also associated with diseases spanning a variety of clinical phenotypes. We demonstrate that the approved drug disulfiram rescues Cu-deficiencies of both environmental and genetic origin, thus underscoring a potential paradigm for treating the broad spectrum of Cu-related disease in man.

2013-05-19 | E-MEXP-3884 | biostudies-arrayexpress

Systems analysis of Chlamydomonas Cu nutrition reveals connections between Cu and multiple O2-dependent metabolic steps

Project description:We used digital gene expression (NlaIII sequence tags) and RNA-Seq to compare the transcriptomes of Cu-replete vs. Cu–deficient Chlamydomonas wild-type cells to reveal dozens of mRNAs whose abundance is modified. Half of the corresponding genes are targets of CRR1, a master regulator of nutritional copper sensing, and are associated with candidate CRR1 binding sites. The targets include many plastid-localized proteins, like FDX5 encoding a ferredoxin isoform, and CGL78, encoding a protein conserved in the green lineage, indicative of modified plastid metabolism. Immunoblot analysis and proteome profiles recapitulate the transcriptome profiles. New evidence for Cu sparing is suggested by up-regulation of AOF1 encoding a copper-independent but flavin-dependent amine oxidase and down-regulation of two metal- binding proteins. Genes encoding redox proteins, many of which function in lipid metabolism, are over-represented, which is compatible with the role of Cu in biology. Lipid profiles indicate a CRR1-dependent increase in Cu-deficient cells in the proportion of unsaturated (16:2, 16:3, 16:4, 18:2) fatty acids at the expense of the more saturated (16:0, 16:1, 18:0) precursors, especially on plastid galactolipids, which validates the increased expression of acyl-ACP and plastid-localized w-6 desaturases. CRR1-independent changes in the transcriptome suggest a role for Cu in oxygen sensing in Chlamydomonas. Sampling of Chlamydomonas CC-1021 (2137) and crr1-2, crr1:CRR1 mutant cells (the mutant is knock-down for the transcription factor crr1, which plays a key role in the transcriptional response to copper levels) cultivated in TAP or minimal medium under Cu-sufficient (control) and Cu-defficient conditions. poly-A purification, NlaIII digestion/random fragmentation

2011-10-23 | E-GEOD-25124 | biostudies-arrayexpress

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