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The ROGDI protein mutated in Kohlschutter-Tonz syndrome is a novel subunit of the Rabconnectin-3 complex implicated in V-ATPase assembly.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-04 DOI: 10.1016/j.jbc.2025.108381
Samuel R Winkley, Patricia M Kane
{"title":"The ROGDI protein mutated in Kohlschutter-Tonz syndrome is a novel subunit of the Rabconnectin-3 complex implicated in V-ATPase assembly.","authors":"Samuel R Winkley, Patricia M Kane","doi":"10.1016/j.jbc.2025.108381","DOIUrl":"10.1016/j.jbc.2025.108381","url":null,"abstract":"<p><p>V-ATPases are highly conserved ATP-driven rotary proton pumps found widely among eukaryotes that are composed of two subcomplexes: V<sub>1</sub> and V<sub>0</sub>. V-ATPase activity is regulated in part through reversible disassembly, during which V<sub>1</sub> physically separates from V<sub>0</sub> and both subcomplexes become inactive. Reassociation of V<sub>1</sub> to V<sub>0</sub> reactivates the complex for ATP-driven proton pumping and organelle acidification. V-ATPase reassembly in Saccharomyces cerevisiae requires the RAVE complex (Rav1, Rav2, and Skp1), and higher eukaryotes, including humans, utilize the Rabconnectin-3 complex. Mammalian Rabconnectin-3 has two subunits: Rabconnectin-3α and Rabconnectin-3β. Rabconnectin-3α isoforms are homologous to Rav1, but there is no known Rav2 homolog, and the molecular basis of the interaction between the Rabconnectin-3α and β subunits is unknown. We identified ROGDI as a Rav2 homolog and novel Rabconnectin-3 subunit. ROGDI mutations cause Kohlschutter-Tonz syndrome, an epileptic encephalopathy with amelogenesis imperfecta that has parallels to V-ATPase-related disease. ROGDI shares extensive structural homology with yeast Rav2 and can functionally replace Rav2 in yeast. ROGDI binds to the N-terminal domains of both Rabconnectin-3 α and β, similar to Rav2 binding to Rav1. Molecular modeling suggests that ROGDI may bridge the two Rabconnectin-3 subunits. ROGDI coimmunoprecipitates with Rabconnectin-3 subunits from detergent-solubilized lysates and is present with them in immunopurified lysosomes of mammalian cells. In immunofluorescence microscopy, ROGDI partially localizes with Rabconnectin-3α in acidic perinuclear lysosomes. The discovery of ROGDI as a novel Rabconnectin-3 interactor sheds new light on both Kohlschutter-Tonz syndrome and the mechanisms behind mammalian V-ATPase regulation.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108381"},"PeriodicalIF":4.0,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143572829","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Substrate recognition in Bacillus anthracis sortase B beyond its canonical pentapeptide binding motif and use in sortase-mediated ligation.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-04 DOI: 10.1016/j.jbc.2025.108382
Sophie N Jackson, Darren E Lee, Jadon M Blount, Kayla A Croney, Justin W Ibershof, Caroline M Ceravolo, Kate M Brown, Noah J Goodwin-Rice, Kyle M Whitham, James McCarty, John M Antos, Jeanine F Amacher
{"title":"Substrate recognition in Bacillus anthracis sortase B beyond its canonical pentapeptide binding motif and use in sortase-mediated ligation.","authors":"Sophie N Jackson, Darren E Lee, Jadon M Blount, Kayla A Croney, Justin W Ibershof, Caroline M Ceravolo, Kate M Brown, Noah J Goodwin-Rice, Kyle M Whitham, James McCarty, John M Antos, Jeanine F Amacher","doi":"10.1016/j.jbc.2025.108382","DOIUrl":"10.1016/j.jbc.2025.108382","url":null,"abstract":"<p><p>Sortases are critical cysteine transpeptidases that facilitate the attachment of proteins to the cell wall in Gram-positive bacteria. These enzymes are potential targets for novel antibiotic development, and versatile tools in protein engineering applications. There are six classes of sortases recognized, yet class A sortases (SrtA) are the most widely studied and utilized. SrtA enzymes endogenously recognize the amino acid sequence LPXTG, where X = any amino acid, with additional promiscuity now recognized in multiple positions for certain SrtA enzymes. Much less is known about Class B sortases (SrtB), which target a distinct sequence, typically with an N-terminal Asn, e.g., variations of NPXTG or NPQTN. Although understudied overall, two SrtB enzymes were previously shown to be specific for heme transporter proteins, and in vitro experiments with the catalytic domains of these enzymes reveal activities significantly worse than SrtA from the same organisms. Here, we use protein biochemistry, structural analyses, and computational simulations to better understand and characterize these enzymes, specifically investigating Bacillus anthracis SrtB (baSrtB) as a model SrtB protein. Structural modeling predicts a plausible enzyme-substrate complex, which is verified by mutagenesis of binding cleft residues. Furthermore, residues N- and C-terminal to the pentapeptide recognition motif are critical for observed activity. Finally, we use chimeric proteins to identify mutations that improve baSrtB activity by ∼4-fold, and demonstrate the feasibility of sortase-mediated ligation using a baSrtB enzyme variant. These studies provide insight into SrtB-target binding as well as evidence that SrtB enzymes can be modified to be of potential use in protein engineering.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108382"},"PeriodicalIF":4.0,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143572816","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Spiroplasma eriocheiris FtsZ assembles the ring-like structure assisted by SepF.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-04 DOI: 10.1016/j.jbc.2025.108373
Taishi Kasai, Yuhei O Tahara, Makoto Miyata, Daisuke Shiomi
{"title":"Spiroplasma eriocheiris FtsZ assembles the ring-like structure assisted by SepF.","authors":"Taishi Kasai, Yuhei O Tahara, Makoto Miyata, Daisuke Shiomi","doi":"10.1016/j.jbc.2025.108373","DOIUrl":"10.1016/j.jbc.2025.108373","url":null,"abstract":"<p><p>The FtsZ protein is involved in bacterial cell division. In cell-walled bacteria, such as Bacillus subtilis, FtsZ forms a ring-like structure, called the Z ring, at the cell division site and acts as a scaffold for cell wall synthesis. The inhibition of cell wall synthesis in B. subtilis has been shown to interfere with the function of the Z ring, causing a loss in cell division control. Spiroplasma, a cell wall-less bacterium, lacks most of the genes involved in cell division; however, the ftsZ gene remains conserved. The function of Spiroplasma eriocheiris FtsZ (SeFtsZ) remains to be determined. In the present study, we analyzed the biochemical characteristics of SeFtsZ. Purified SeFtsZ demonstrated lower polymerization capacity and GTPase activity than FtsZ from Escherichia coli and B. subtilis. We also investigated the relationship between SeFtsZ and SeSepF, which anchors FtsZ to the cell membrane, and found that SeSepF did not contribute to the stability of FtsZ filaments, unlike the B. subtilis SepF. SeFtsZ and SeSepF were produced in E. coli L-forms, where cell wall synthesis was inhibited. SeFtsZ formed ring-like structures in cell wall-less E. coli cells, suggesting that SeFtsZ forms Z rings and is involved in cell division independently of cell wall synthesis.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108373"},"PeriodicalIF":4.0,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143566475","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Proximal cysteine residues in proteins promote Nε-carboxyalkylation of lysine residues by α-dicarbonyl compounds.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-04 DOI: 10.1016/j.jbc.2025.108377
Sudipta Panja, Johanna Rankenberg, Cole Michel, Grace Cooksley, Marcus A Glomb, Ram H Nagaraj
{"title":"Proximal cysteine residues in proteins promote N<sup>ε</sup>-carboxyalkylation of lysine residues by α-dicarbonyl compounds.","authors":"Sudipta Panja, Johanna Rankenberg, Cole Michel, Grace Cooksley, Marcus A Glomb, Ram H Nagaraj","doi":"10.1016/j.jbc.2025.108377","DOIUrl":"10.1016/j.jbc.2025.108377","url":null,"abstract":"<p><p>Advanced glycation end products (AGEs) are protein modifications resulting from the chemical reaction between lysine and arginine residues in proteins, and carbonyl compounds, including glyoxal (GO) and methylglyoxal (MGO). N<sup>ε</sup>-carboxymethyllysine (CML) and N<sup>ε</sup>-carboxyethyllysine (CEL), formed by glycation from GO and MGO, are among the major AGEs in tissue proteins. Incubation with GO or MGO resulted in higher CML and CEL formation in the two cysteine residues containing αA-crystallin (αAC) than in the cysteine lacking αB-crystallin (αBC). Mass spectrometric data showed K70 and K166 to be heavily modified with CML and CEL in GO- and MGO-modified αAC. In silico analysis of the structure of αAC showed K70 and K166 to be proximal to C142. Mutation or reductive alkylation of cysteine residues in αAC significantly reduced CML and CEL formation. The addition of GSH or N-acetylcysteine enhanced CML and CEL formation in αBC. The introduction of a cysteine residue proximal to a lysine residue in αBC increased the CML and CEL accumulation. Our data showed that CML and CEL formation occurs through a hemithioacetal intermediate formed from the reaction between thiols and GO or MGO. Together, these results highlight a mechanism by which thiols influence protein AGE levels. In addition, CML and CEL are ligands for RAGE, a receptor for AGEs, which has been implicated in several aging and diabetes-associated diseases. Therefore, further understanding of the biosynthesis of CML and CEL could lead to the development of new therapies against those diseases.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108377"},"PeriodicalIF":4.0,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143572345","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Dentin sialoprotein promotes endothelial differentiation of dental pulp stem cells through DSPaa34-50-endoglin-AKT1 axis.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-04 DOI: 10.1016/j.jbc.2025.108380
Ximin Xu, Jing Fu, Guobin Yang, Zhi Chen, Shuo Chen, Guohua Yuan
{"title":"Dentin sialoprotein promotes endothelial differentiation of dental pulp stem cells through DSP<sub>aa34-50</sub>-endoglin-AKT1 axis.","authors":"Ximin Xu, Jing Fu, Guobin Yang, Zhi Chen, Shuo Chen, Guohua Yuan","doi":"10.1016/j.jbc.2025.108380","DOIUrl":"10.1016/j.jbc.2025.108380","url":null,"abstract":"<p><p>Dentin sialoprotein (DSP), a major dentin extracellular matrix noncollagenous protein, is well recognized as an important regulator for dentinogenesis. DSP as a secreted protein can interact with membrane receptors, activate intracellular signaling, and initiate the odontoblastic differentiation of dental papilla cells. In a recent study, we have demonstrated that DSP can induce the endothelial differentiation of dental pulp stem cells (DPSCs), a type of tooth pulp-derived multipotent stem cells, dependent on membrane receptor endoglin (ENG). However, the intimate mechanisms by which DSP-ENG association facilitates the endothelial differentiation of DPSCs remain enigmatic. Here, we find that the amino acid (aa) residues 34-50 of DSP (DSP<sub>aa34-50</sub>) is responsible for its association with ENG using a series of co-immunoprecipitation assays. Immunofluorescent staining and in situ proximity ligation assay demonstrate that overexpressed ENG in human embryonic kidney 293T cells shows codistribution and proximity ligation assay signals to the supplemented DSP<sub>aa34-50</sub> protein but not to DSP without aa34-50 (DSP<sub>Δ34-50</sub>) on cell surfaces. Moreover, the zona pellucida domain of ENG mediates its association with DSP<sub>aa34-50</sub>. Further experiments indicate that DSP<sub>aa34-50</sub> exhibits equivalent effects to the full-length DSP on the migration and endothelial differentiation of DPSCs dependent on ENG but DSP<sub>Δ34-50</sub> does not. Mechanistically, DSP<sub>aa34-50</sub> activates AKT1 and triggers the expression of blood vessel development-related genes in DPSCs. Multiple experiments demonstrate that AKT1 inhibition suppresses the DSP<sub>aa34-50</sub>-induced migration and endothelial differentiation of DPSCs. Thus, AKT1 mediates the cellular and molecular functions of DSP<sub>aa34-50</sub>-ENG association. Collectively, these findings identify that DSP promotes the endothelial differentiation of DPSCs through the DSP<sub>aa34-50</sub>-ENG-AKT1 signaling axis.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108380"},"PeriodicalIF":4.0,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143573082","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Covalent inhibition of the SARS-CoV-2 NiRAN domain via an active-site cysteine.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-04 DOI: 10.1016/j.jbc.2025.108378
Genaro Hernandez, Adam Osinski, Abir Majumdar, Jennifer L Eitson, Monika Antczak, Krzysztof Pawłowski, Hanspeter Niederstrasser, Kelly A Servage, Bruce Posner, John W Schoggins, Joseph M Ready, Vincent S Tagliabracci
{"title":"Covalent inhibition of the SARS-CoV-2 NiRAN domain via an active-site cysteine.","authors":"Genaro Hernandez, Adam Osinski, Abir Majumdar, Jennifer L Eitson, Monika Antczak, Krzysztof Pawłowski, Hanspeter Niederstrasser, Kelly A Servage, Bruce Posner, John W Schoggins, Joseph M Ready, Vincent S Tagliabracci","doi":"10.1016/j.jbc.2025.108378","DOIUrl":"10.1016/j.jbc.2025.108378","url":null,"abstract":"<p><p>The kinase-like NiRAN domain of nsp12 in SARS-CoV-2 catalyzes the formation of the 5' RNA cap structure. This activity is required for viral replication, offering a new target for the development of antivirals. Here, we develop a high-throughput assay to screen for small molecule inhibitors targeting the SARS-CoV-2 NiRAN domain. We identified NCI-2, a compound with a reactive chloromethyl group that covalently binds to an active site cysteine (Cys53) in the NiRAN domain, inhibiting its activity. NCI-2 can enter cells, bind to, and inactivate ectopically expressed nsp12. A cryo-EM reconstruction of the SARS-CoV-2 replication-transcription complex (RTC) bound to NCI-2 offers a detailed structural blueprint for rational drug design. Although NCI-2 showed limited potency against SARS-CoV-2 replication in cells, our work lays the groundwork for developing more potent and selective inhibitors targeting the NiRAN domain. This approach presents a promising therapeutic strategy for effectively combating COVID-19 and potentially mitigating future coronavirus outbreaks.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108378"},"PeriodicalIF":4.0,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143573079","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Structure-function studies of a nucleoplasmin isoform from Plasmodium falciparum.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-04 DOI: 10.1016/j.jbc.2025.108379
Ketul Saharan, Somanath Baral, Surajit Gandhi, Ajit Kumar Singh, Sourav Ghosh, Rahul Das, Viswanathan Arun Nagaraj, Dileep Vasudevan
{"title":"Structure-function studies of a nucleoplasmin isoform from Plasmodium falciparum.","authors":"Ketul Saharan, Somanath Baral, Surajit Gandhi, Ajit Kumar Singh, Sourav Ghosh, Rahul Das, Viswanathan Arun Nagaraj, Dileep Vasudevan","doi":"10.1016/j.jbc.2025.108379","DOIUrl":"10.1016/j.jbc.2025.108379","url":null,"abstract":"<p><p>An organized regulation of gene expression and DNA replication is vital for the progression of the complex life cycle of Plasmodium falciparum (Pf), involving multiple hosts and various stages. These attributes rely on the dynamic architecture of chromatin governed by several factors, including histone chaperones. Nucleoplasmin class of histone chaperones perform histone chaperoning function and participate in various developmental processes in eukaryotes. Here, our crystal structure confirmed that Pf indeed possesses a nucleoplasmin isoform (PfNPM), and the N-terminal core domain (NTD) adopts the characteristic pentameric doughnut conformation. Furthermore, PfNPM exists as a pentamer in solution, and the N-terminal core domain exhibits thermal and chemical stability. PfNPM interacts individually with assembled H2A/H2B and H3/H4 with an equimolar stoichiometry, wherein the acidic tracts of PfNPM were found to be necessary for these interactions. Further, H3/H4 displays a higher binding affinity for PfNPM than H2A/H2B, potentially due to stronger electrostatic interactions. The interaction studies also suggested that H2A/H2B and H3/H4 might share the same binding site on the PfNPM distal face, wherein H3/H4 could substitute H2A/H2B due to a higher binding affinity. Intriguingly, PfNPM neither demonstrated direct interaction with the nucleosome core particles nor displayed nucleosome assembly function, suggesting it may not be directly associated with histone deposition on the parasite genomic DNA. Furthermore, our immunofluorescence results suggested that PfNPM predominantly localizes in the nucleus and exhibits expression only in the early blood stages, such as ring and trophozoite. Altogether, we provide the first report on the structural and functional characterization of PfNPM.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108379"},"PeriodicalIF":4.0,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143572814","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
The filopodial myosin DdMyo7 is a slow, calcium regulated motor.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-03 DOI: 10.1016/j.jbc.2025.108371
Casey Eddington, Margaret A Titus
{"title":"The filopodial myosin DdMyo7 is a slow, calcium regulated motor.","authors":"Casey Eddington, Margaret A Titus","doi":"10.1016/j.jbc.2025.108371","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108371","url":null,"abstract":"<p><p>MyTH4-FERM (MF) myosins are a family of molecular motors with critical roles in the formation and organization of thin membrane protrusions supported by parallel bundles of actin - filopodia, microvilli and stereocilia. The amoeboid MF myosin DdMyo7 is essential for filopodia formation but its mechanism of action is unknown. The motor properties of a forced dimer of the DdMyo7 motor were characterized using an in vitro motility assay to address this question. The DdMyo7 motor associates with two different light chains, the Dictyostelium calmodulins CalA and CalB, whose binding is shown to be sensitive to the presence of calcium. TIRF motility assays of the dimerized DdMyo7 motor reveal that it is a slow, processive motor that moves along actin at ∼ 40 nm/sec, and the activity of the motor is significantly reduced in the presence of Ca<sup>2+</sup>. The speed of DdMyo7 is similar to that of other Myo7 familiy members such as human Myo7A and fly DmMyo7A, but is at least 10-fold slower than the mammalian filopodial MF myosin, Myo10. The results show that evolutionarily distant native filopodial myosins can promote filopodia elongation using motors with distinct properties, revealing diverse mechanisms of myosin-based filopodia formation.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108371"},"PeriodicalIF":4.0,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143566490","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Pseudomonas aeruginosa PfpI is a methylglyoxalase.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-03 DOI: 10.1016/j.jbc.2025.108374
Larson Grimm, Andre Wijaya, Isabel Askenasy, Rahan Rudland Nazeer, Hikaru Seki, Paul D Brear, Wendy Figueroa, David R Spring, Martin Welch
{"title":"Pseudomonas aeruginosa PfpI is a methylglyoxalase.","authors":"Larson Grimm, Andre Wijaya, Isabel Askenasy, Rahan Rudland Nazeer, Hikaru Seki, Paul D Brear, Wendy Figueroa, David R Spring, Martin Welch","doi":"10.1016/j.jbc.2025.108374","DOIUrl":"10.1016/j.jbc.2025.108374","url":null,"abstract":"<p><p>Pseudomonas aeruginosa is an opportunistic pathogen, commonly associated with human airway infections. Based on its amino acid sequence similarity with Pyrococcus furiosus protease I, P. aeruginosa PfpI was originally annotated as an intracellular protease. In this work, we show that PfpI is a methylglyoxalase. The X-ray crystal structure of the purified protein was solved to 1.4 Å resolution. The structural data indicated that PfpI shares the same constellation of active site residues (including the catalytic Cys112 and His113) as those seen in a well-characterized bacterial methylglyoxalase from Escherichia coli, YhbO. Using NMR, we confirmed that PfpI qualitatively converted methylglyoxal into lactic acid. Quantitation of lactate produced by the methylglyoxalase activity of PfpI yielded a k<sub>cat</sub> of 102 min<sup>-1</sup> and a K<sub>M</sub> of 369 μM. Mutation of Cys112 and His113 in PfpI led to complete loss of methylglyoxalase activity. To investigate the functional impact of PfpI in vivo, a ΔpfpI deletion mutant was made. Quantitative proteomic analyses revealed a pattern of changes consistent with perturbation of ribosomal function, Zn<sup>2+</sup> limitation, C1 metabolism, and glutathione metabolism. These findings are consistent with PfpI being a glutathione-independent methylglyoxalase. Previously, transposon insertion (pfpI::Tn) mutants have been reported to exhibit phenotypes associated with antibiotic resistance, motility, and the response to oxidative stress. However, the ΔpfpI mutant generated in this study displayed none of these phenotypes. Whole-genome sequencing of the previously described pfpI::Tn mutants revealed that they also contain a variety of other genetic changes that likely account for their observed phenotypes.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108374"},"PeriodicalIF":4.0,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143567256","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
The contribution of the Golgi and the endoplasmic reticulum to calcium and pH homeostasis in Toxoplasma gondii.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-03 DOI: 10.1016/j.jbc.2025.108372
Abigail Calixto, Katherine Moen, Silvia Nj Moreno
{"title":"The contribution of the Golgi and the endoplasmic reticulum to calcium and pH homeostasis in Toxoplasma gondii.","authors":"Abigail Calixto, Katherine Moen, Silvia Nj Moreno","doi":"10.1016/j.jbc.2025.108372","DOIUrl":"10.1016/j.jbc.2025.108372","url":null,"abstract":"<p><p>The cytosolic Ca<sup>2+</sup> concentration of all cells is highly regulated demanding the coordinated operation of Ca<sup>2+</sup> pumps, channels, exchangers and binding proteins. In the protozoan parasite Toxoplasma gondii calcium homeostasis, essential for signaling, governs critical virulence traits. However, the identity of most molecular players involved in signaling and homeostasis in T. gondii are unknown or poorly characterized. In this work we studied a putative calcium proton exchanger, TgGT1_319550 (TgCAXL1), which belongs to a family of Ca<sup>2+</sup>/proton exchangers that localize to the Golgi apparatus. We localized TgCAXL1 to the Golgi and the endoplasmic reticulum (ER) of T. gondii and validated its role as a Ca<sup>2+</sup>/proton exchanger by yeast complementation. Characterization of a knock-out mutant for TgCAXL1 (Δcaxl) underscored the role of TgCAXL1 in Ca<sup>2+</sup> storage by the ER and acidic stores, most likely the Golgi. Most interestingly, TgCAXL1 function is linked to the Ca<sup>2+</sup> pumping activity of the Sarcoplasmic Reticulum Ca<sup>2+</sup>-ATPase (TgSERCA). TgCAXL1 functions in cytosolic pH regulation and recovery from acidic stress. Our data showed for the first time the role of the Golgi in storing and modulating Ca<sup>2+</sup> signaling in T. gondii and the potential link between pH regulation and TgSERCA activity, which is essential for filling intracellular stores with Ca<sup>2+</sup>.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108372"},"PeriodicalIF":4.0,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143566476","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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