Search Results (224)

XYLEM CYSTEINE PROTEASE 1 (XCP1) is a cysteine protease that plays a critical role in xylem differentiation and tracheary element (TE) formation. Our previous study demonstrated that TE remodeling occurs at the graft union in the Arabidopsis thaliana (At)/Nicotiana benthamiana (Nb) interfamilial graft. Here, we identify that the AtXCP1 transcript is specifically localized in TEs at the graft interface of the incompatible At/Nb interfamilial grafts, and its expression is reduced in these incompatible grafts compared to the compatible grafts. Analysis of AtXCP1pro::GFP reporter lines revealed that AtXCP1 expression is rapidly induced by wounding at the graft interface in At/Nb interfamilial grafts during the early grafting stage. Notably, AtXCP1 expression was significantly stronger in At/Nb heterografts than in At/At homografts, and GFP fluorescence was observed in the stock xylem at 7 days after grafting (DAG) in heterografts, a dynamic process absent in At/At homografts. We found that the Atxcp1 mutant promoted the survival of At/Nb interfamilial grafts during the early grafting stage but decreased the survival after several months, indicating delayed incompatibility. Anatomical examination revealed that large cellular deposits accumulated at the graft interface in Atxcp1/Nb interfamilial grafts and exhibited abnormal TE morphology at later stages. Our findings identify AtXCP1 as a key regulator of xylem reconnection and delayed incompatibility in At/Nb interfamilial grafts. Full article

Reparative macrophage polarization and macrophage-derived reactive oxygen species (ROS) are required for ischemia-induced revascularization in peripheral artery disease (PAD). Our previous study showed that mitochondrial fission protein dynamin-related protein 1 (DRP1) promotes reparative polarization and metabolic reprogramming in macrophages and post-ischemic neovascularization. However, the redox-dependent mechanism governing DRP1 activation in this context remains elusive. Here, using a mouse hindlimb ischemia (HLI) model of PAD, we identify cysteine sulfenylation (CysOH) of DRP1 as a critical redox modification induced in ischemic bone marrow (BM)-derived cells. BM chimeric mice reconstituted with CRISPR/Cas9-generated “redox-dead” DRP1-C631A knock-in mutant (Drp1^C/A) BM exhibited markedly reduced limb perfusion recovery and CD31⁺ capillary density in ischemic muscles following HLI. These defects were associated with enhanced Ly6G⁺ neutrophil accumulation, pro-inflammatory F4/80⁺CD80⁺ M1-like macrophages and reduced anti-inflammatory F4/80⁺CD206⁺ M2-like macrophages in ischemic muscle. Mechanistically, using an in vitro PAD model, hypoxia serum starvation (HSS) rapidly induced NADPH oxidase 2-dependent cytosolic ROS production and DRP1-CysOH formation in wild-type macrophages. In contrast, Drp1^C/A macrophages failed to undergo DRP1-CysOH-dependent mitochondrial fission under HSS, resulting in aberrant metabolic reprogramming characterized by enhanced glycolysis and mitochondrial ROS, pro-inflammatory p-NF-κB and M1-genes, and suppressed anti-inflammatory p-AMPK, efferocytosis and M2-genes. Thus, our findings establish DRP1 sulfenylation as a previously unrecognized redox-sensing mechanism that links ischemia-induced ROS to reparative macrophage reprogramming and revascularization, identifying a novel therapeutic target for PAD. Full article

Cysteine biosynthesis is increasingly recognized as a critical determinant of bacterial virulence, highlighting this pathway as a promising reservoir of novel antimicrobial targets. In Pseudomonas aeruginosa, however, the molecular basis of cysteine production has only recently begun to emerge. Here, we identify PA3816 as the major P. aeruginosa serine acetyltransferase (PaCysE), the enzyme responsible for generating the activated serine intermediate that feeds O-acetylserine sulfhydrylase-mediated cysteine synthesis. Through a combination of biochemical and genetic approaches, we demonstrate that PaCysE efficiently catalyzes L-serine acetylation in vitro, and in turn, deletion mutants exhibit cysteine auxotrophy, underscoring its essential contribution to O-acetylserine production. Notably, PaCysE is less sensitive to feedback inhibition by cysteine and does not appear to form the canonical cysteine synthase complex, suggesting a regulatory architecture that diverges from well-characterized orthologs. Loss of PaCysE function has broad physiological consequences, including enhanced biofilm formation, reduced pyocyanin production, and attenuated infectivity in an animal model, linking cysteine biosynthesis directly to pathogen fitness. Finally, we identify a thiazole derivative that inhibits PaCysE activity (IC₅₀ ≈ 30 µM) and suppresses bacterial growth in a cysteine-dependent manner, providing a proof-of-concept for therapeutically targeting this pathway. Full article

Kelch-like ECH-associated protein 1 (Keap1) acts as a repressor of nuclear factor-erythroid 2-related factor 2 (Nrf2), a major transcription factor regulating cellular antioxidant response. Keap1 is the substrate adaptor subunit of the cullin 3-RING E3 ubiquitin ligase complex that specifically facilitates Nrf2 ubiquitination and its proteasomal degradation. Keap1 is rich in cysteine residues, and several of them undergo various modifications, such as sulphydration, nitrosylation and glutathionylation under cellular stress conditions. Some of these modifications alter the conformation of Keap1, preventing Nrf2 from ubiquitination and subsequent proteasome-mediated degradation. As a result, newly synthesised Nrf2 translocates to the nucleus to induce the expression of diverse genes involved in protecting cells against oxidative stress. Protein CoAlation is a reversible redox-dependent post-translational modification (PTM) in which coenzyme A (CoA) forms disulphide bonds with oxidised cysteine residues under oxidative or metabolic stress. In this study, we demonstrate for the first time that disulphide Keap1 dimer undergoes CoAlation in cellular response to oxidative stress induced by various oxidising compounds. Furthermore, glucose deprivation also induces CoAlation of the disulphide Keap1 dimer in HEK293/Pank1β cells. We also demonstrate that the Keap1^{11 Cys-less} mutant is not CoAlated in response to diamide treatment or glucose deprivation. In summary, this study uncovers a novel PTM of Keap1 by the key metabolic integrator CoA, which provides new insights into the regulation of the Keap1-Nrf2 antioxidant pathway under oxidative and metabolic stress. Full article

Gram-positive bacteria covalently anchor specific proteins to the peptidoglycan cell wall via sortase, a cysteine transpeptidase that targets proteins with a cell wall sorting signal. Sortase enzymes are critical for bacterial pathogenesis, and their inhibitors have become promising therapeutic targets for infection management. Filifactor alocis, a Gram-positive anaerobic bacterium, is now proposed as a diagnostic indicator of periodontal disease. Unlike other bacteria, F. alocis encodes a single putative sortase, FA1364. In this study, we functionally characterized the putative sortase FA1364 and found that it belongs to the class A family (SrtA). The SrtA-anchored surface proteins (FA1006, FA1336, FA1424, and FA1750) were identified, and MS/MS analysis confirmed that SrtA is required for their cell-surface localization. The recombinant SrtA protein could recognize and cleave the LPKTG sorting motif with cysteine 191 and arginine 200 as essential catalytic residues. F. alocis FLL101 (ΔFA1364::ermF) showed reduced ability to coaggregate and form biofilm, along with decreased collagen binding and survival in epithelial cells. Additionally, the FA1364-defective mutant exhibited increased sensitivity to air exposure. Collectively, these results suggest that the F. alocis SrtA protein is an important virulence factor and may represent a novel therapeutic target for the control of periodontal diseases. Full article

Protein turnover is essential for cellular metabolism, organelle biogenesis, stress adaptation, and ultimately the viability of cells and tissues. Papain-like cysteine proteases (PLCPs) are one of the vital components in protein degradation. PLCPs have been reported to act in senescence-associated proteolysis, but their roles in vegetative growth remain unclear. We identified PtCP1, an AALP-like PLCP in Populus tomentosa, localized to the vacuole and acid-triggered activated. CRISPR/Cas9-generated loss-of-function mutant (d7) showed dwarfism and non-stomatal photosynthetic limitations. On the other hand, the gain-of-function line (EM, deleted ERFNIN domain) exhibited accelerated growth and enhanced photosynthetic parameters. We showed d7 had the accumulation of Rubisco, which was the most important protein in photosynthetic carbon fixation. Transcriptomics revealed dysregulated carbon metabolism in d7. This data supported PtCP1-mediated proteolysis regulated photosynthetic carbon assimilation via altered Rubisco turnover, and then it increased the biomass accumulation during vegetative growth in woody plants. Full article

Transforming growth factor beta 3 (TGF-β3) is a homodimeric cytokine with potential therapeutic applications in wound healing, tissue engineering and regenerative medicine. Production of recombinant TGF-β3 in Escherichia coli faces significant challenges due to TGF-β3’s propensity for misfolding and aggregation, driven by a high disulfide bond content and low aqueous solubility. To address these limitations, the impacts of substituting non-conserved cysteine residues C7, C16 and C77 with serine on TGF-β3 folding, dimerization and activity were investigated. Whilst C7 and C16 form an intra-chain disulfide bond, C77 forms an inter-chain disulfide bond stabilizing dimer formation. Our results showed that the C7S, C16S double cysteine mutant protein exhibited reduced aggregation, increased dimer formation, and maintained wild-type biological activity in nano-luciferase reporter gene assay. In contrast, both C77S single and C7S, C16S, C77S triple mutants were purified predominantly in monomeric forms and displayed about 2.5-fold reduced activities. Our findings highlight the roles of the non-conserved C7, C16 and C77 cysteine residues in TGF-β3 folding and aggregation. The identification of the C7S, C16S mutant as a more soluble protein with wild-type TGF-β3 activity offers a promising strategy for improving recombinant TGF-β3 production to facilitate therapeutic applications. This study underscores the importance of targeted cysteine engineering to overcome the inherent challenges associated with the production of TGF-β3 and related complex disulfide-rich proteins. Full article

Human neuroglobin (Ngb) is a globin featuring a disulfide bond (Cys46–Cys55) and a redox-active cysteine residue (Cys120) and plays a dual role in cellular stress responses. In this study, we investigated how wild-type (WT) Ngb and its two mutants, C120S Ngb, in which Cys120 is replaced by serine, and A15C Ngb, which contains an engineered Cys15–Cys120 disulfide bridge, modulate oxidative stress in triple-negative breast cancer (MDAMB231) and hormone receptor-positive breast cancer (MCF-7) cells. In both cell lines, WT Ngb enhanced cell survival under H₂O₂-induced oxidative stress by scavenging reactive oxygen species (ROS) through oxidation of Cys120. In contrast, the C120S and A15C mutants lost this protective capacity and instead promoted apoptosis. Mass spectrometry analysis confirmed the oxidation of Cys120 to sulfenic acid in WT Ngb, whereas both mutants exhibited impaired redox activity, leading to elevated ROS levels, lipid peroxidation, and activation of caspase-9/3. AO/EB staining further revealed that WT Ngb attenuated DNA damage, while the mutants exacerbated apoptosis in both MDAMB231 and MCF-7 cells. These results demonstrate that Cys120 acts as a critical redox switch, dictating whether Ngb exerts cytoprotective or pro-apoptotic effects across different breast cancer cell types. Our findings suggest that WT Ngb may help protect normal tissues during cancer therapy, whereas engineered Ngb mutants could be used to selectively sensitize both triple-negative and hormone receptor-positive breast cancer cells to oxidative damage, offering a novel redox-targeted therapeutic strategy. Full article

This study systematically characterized the L-cysteine biosynthetic pathway in Bacillus licheniformis and demonstrated that exogenous serine supplementation significantly upregulated the expression of pathway-associated genes, confirming serine as the primary precursor driving L-cysteine synthesis. Through targeted gene deletions, we generated knockout strains BL2ΔglyA, BL2ΔsdaAA, BL2ΔmetC, BL2Δ2, and BL2Δ3 to minimize precursor diversion and product degradation. Combinatorial overexpression of the feedback-resistant mutant cysE^f and the transporter eamA yielded an engineered strain achieving 1.075 g/L L-cysteine in shake-flask fermentation with an 18.69% molar conversion yield. These findings highlight the potential of B. licheniformis as a platform for sulfur metabolic engineering and provide a sustainable fermentation strategy to replace traditional high-pollution hydrolysis-based L-cysteine production. Additionally, this work reveals fundamental differences in sulfur metabolism networks between Gram-positive and Gram-negative bacteria, elucidating microbial metabolic diversity and the cross-regulatory mechanisms linking sulfur, carbon, and nitrogen metabolism. Full article

The spotted-leaf mutant, characterized by spontaneous lesion formation resembling pathogen-induced hypersensitive cell death, serves as an ideal model for studying the molecular mechanisms behind rice (Oryza sativa) disease resistance and programmed cell death, as these plants display hypersensitive responses that mimic those triggered by pathogen infection. In this study, we generated a knockout line using CRISPR/Cas9 technology in homologous mutant HM113-induced calli. LOC_Os07g30510 encodes a cysteine-rich receptor kinase with a DUF26 domain, consisting of 688 amino acids. HM113 was localized to the cytosol and expressed in most rice tissues at various growth stages. A single nucleotide substitution from A to T was observed at the 847th base of LOC_Os07g30510, causing an amino acid change from serine to cysteine. Our results demonstrated that the A847T mutation was responsible for the spotted-leaf phenotype in the HM113 mutant through gene editing technology, as new frameshift mutations were introduced upstream of the A847T site in the HM113 gene. The mutation phenotype of HM113 was eliminated and resistance to bacterial blight was also lost, indicating that it is a gain-of-function gene. Full article

Tumor necrosis factor α (TNF-α) activates the nuclear factor κB (NF-κB) signaling pathway, which promotes the expression of NF-κB-responsive genes, including intercellular adhesion molecule 1 (ICAM-1). We previously reported that cardamonin, a chalcone-type flavonoid, inhibited TNF-α-induced ICAM-1 expression in human lung adenocarcinoma A549 cells. However, the mechanisms by which cardamonin inhibits the TNF-α-induced NF-κB signaling pathway have yet to be elucidated. Therefore, we herein investigated the effects of cardamonin on TNF-α-induced gene expression and the NF-κB-dependent signaling pathway. Cardamonin reduced TNF-α-induced ICAM-1 mRNA expression and NF-κB reporter activity. It did not affect the inhibitor of NF-κB α (IκBα) degradation, but prevented RelA nuclear translocation and binding to the ICAM-1 promoter. Consistent with this result, three other chalcone derivatives (4′-hydroxychalcone, isoliquiritigenin, and xanthohumol) did not affect the degradation of IκBα, but inhibited nuclear RelA translocation. Cardamonin exhibited the same inhibitory profiles in human breast cancer MCF-7 cells and human fibrosarcoma HT-1080 cells. Cysteine 38 (C38) of RelA was not a primary target site of cardamonin because cardamonin inhibited the nuclear translocation of the RelA C38S mutant. An in silico molecular docking analysis confirmed that cardamonin was not positioned close enough to RelA C38 to mediate covalent binding, and also that cardamonin interacted with RelA at different sites. Mutations in these interaction sites abrogated the nuclear translocation of RelA in response to a TNF-α stimulation. The present results demonstrate that cardamonin inhibited the nuclear translocation of RelA and its DNA binding in the NF-κB signaling pathway in response to a TNF-α stimulation. Full article

Leaves represent an important organ in plant photosynthesis, and moderately rolled leaves would be beneficial in establishing an ideal plant architecture and thereby increasing rice yields. In this study, a stable inherited rolled leaf mutant was obtained via ethyl methanesulfonate (EMS) mutagenesis from japonica variety WYJ27, which was named rll2 (rolling leaf 2). rll2 showed a leaf-rolling phenotype at the seedling stage, which increased with growth. Compared with the wild type, the leaves at all levels of rll2 were significantly shorter and narrower, and the leaf-rolling index gradually decreased from the highest leaf to the third-highest leaf. Semi-thin sections showed that the bulliform cells of rll2 were significantly larger than those of the wild type, and the number of cells was significantly higher than that of the wild type. Genetic analysis showed that rll2 is controlled by a pair of recessive nuclear genes. Map-based cloning revealed that RLL2 encodes a conserved and plant-specific calpain-like cysteine proteinase. RLL2 was mainly expressed in young roots, shoots, spikelets, and panicles. Transcriptome sequencing showed that a total of 104 genes were differentially expressed in the wild type and rll2. Moreover, several transcription factor genes were significantly altered in the rll2 mutant. Taken together, our findings indicate that RLL2 plays an important role in leaf rolling by regulating bulliform cells, which may be useful in breeding rice with an ideal plant architecture. Full article

Glycogen metabolism plays a key role in bacterial adaptation. In Streptococcus pneumoniae, the glycogen-degrading enzyme SpuA is widely conserved, but its physiological significance remains unclear. In this study, we investigated how SpuA affects bacterial growth and response to acid stress. We found that the spuA deletion strain (ΔspuA) produced more acidic metabolites under anaerobic conditions than the wild-type strain. In a mouse infection model, bronchoalveolar lavage fluid (BALF) from ΔspuA-infected mice was more acidic on day 1 post-infection, showing a lower bacterial load than wild-type infection—a finding consistent with the early growth delay observed in vitro—but the mutant later exhibited enhanced persistence at 72 h. ΔspuA strains also showed greater tolerance to formic acid and higher intake of serum amyloid A1 (SAA1), which may further contribute to their survival in acidic environments. Transcriptomic analysis revealed reduced utilization of certain amino acids, particularly cysteine, in ΔspuA strains. However, the addition of 0.05% (v/v) formic acid restored amino acid utilization in ΔspuA strains, and co-supplementation with formic acid and cysteine significantly enhanced ΔspuA growth in vitro. These findings suggest that in the absence of SpuA, S. pneumoniae shifts its metabolism toward formic acid production, which may act both as a metabolic signal and a stressor that influences bacterial gene expression. This shift is accompanied by increased expression of tRNAs and growth rescue, suggesting enhanced amino acid utilization capacity. Although our findings reveal a potential link between formic acid metabolism and amino acid utilization through tRNA regulation, further validation using metabolic flux analyses or targeted metabolomics will be required to confirm this relationship. These observations imply a metabolic adaptation that facilitates bacterial growth under low-oxygen, acidic conditions during infection. Our results also raise the possibility that SpuA plays a role in restraining bacterial overgrowth in the host, thereby promoting a more balanced coexistence between pathogen and host. Full article

Protein S-palmitoylation is a reversible lipid modification that regulates various aspects of protein function, including membrane association, subcellular localization, trafficking, stability, and activity. The depalmitoylase ABHD17A removes palmitate from multiple substrates, but its cellular positioning and the role of its own palmitoylation in regulating its function remain unclear. This study identifies a palmitoylation code within the conserved N-terminal cysteine cluster of ABHD17A, which governs its intracellular distribution and plasma membrane (PM) targeting. N-terminal palmitoylation is essential for PM localization. Through the use of code-restricted mutants, we found that modifications in the middle region (C14, C15) are critical for PM targeting and catalytic activity, while modifications at the front (C10, C11) and rear (C18) influence endosomal routing and delivery to the PM. Alanine scanning revealed that adjacent hydrophobic residues, particularly L9 and F13, are crucial for initial engagement with endomembranes. Sequence analysis and mutagenesis identified two tyrosine-based YXXØ motifs within the alpha/beta hydrolase fold; disruption of the proximal motif (L115A) decreased surface abundance and redirected ABHD17A to autophagosomes, indicating a need for YXXØ-dependent endosomal sorting, likely at the trans-Golgi network. Biochemical assays demonstrated a continuum of acylation states influenced by the palmitoylation code. This requirement for the middle region was conserved in ABHD17B and ABHD17C. Overall, our findings suggest a stepwise mechanism for ABHD17A delivery to the PM, enabling its depalmitoylase activity on membrane-bound substrates. Full article

Class 1 phytoglobins (Pgbs) are known for their multifunctional roles in plant stress responses, with recent studies suggesting broader interactions involving metabolic and transcriptional regulation. Interestingly, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) moonlights in many roles in colocalized spaces during cellular stress that are strikingly suitable for supporting Pgb function. This study investigates the molecular interactions of class 1 Pgb from sugar beet (Beta vulgaris), BvPgb 1.2, and an alanine-substituted mutant (C86A), focusing on their ability to bind GAPDH and DNA. Using dual-emission isothermal spectral shift (SpS) analysis, we report strong binding interactions with GAPDH, with dissociation constants (K_D) of 260 ± 50 nM for the recombinant wild-type protein (rWT) and a significantly stronger affinity for C86A (120 ± 40 nM), suggesting that the cysteine residue limits the interaction. Remarkably strong DNA-binding affinities were also observed for both variants, displaying biphasic binding. This behavior is characteristic of hexacoordinated globins and reflects the presence of two distinct species: a fast-reacting open pentacoordinated form and a slow-reacting closed hexacoordinated form with high apparent affinity. Here, the K_D in the open configuration was 120 ± 50 nm and 50 ± 20 nM for rWT and C86A, respectively. In the closed configuration, however, the cysteine appears to support the interaction, as the K_D was measured at 100 ± 10 pM and 230 ± 60 pM for rWT and C86A, respectively. Protein–protein docking studies reinforced these findings, revealing electrostatically driven interactions between BvPgb 1.2 and GAPDH, characterized by a substantial buried surface area indicative of a stable, biologically relevant complex. Protein–DNA docking similarly confirmed energetically favorable binding near the heme pocket without obstructing ligand accessibility. Together, these findings indicate a potential regulatory role for BvPgb 1.2 through its interaction with GAPDH and DNA. Full article