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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
Ovarian tachykinin signaling system induces the growth of secondary follicles during the gonadotropin-independent process.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-03 DOI: 10.1016/j.jbc.2025.108375
Tsuyoshi Kawada, Masato Aoyama, Shin Matsubara, Tomohiro Osugi, Tsubasa Sakai, Shinji Kirimoto, Satsuki Nakaoka, Yuki Sugiura, Keiko Yasuda, Honoo Satake
{"title":"Ovarian tachykinin signaling system induces the growth of secondary follicles during the gonadotropin-independent process.","authors":"Tsuyoshi Kawada, Masato Aoyama, Shin Matsubara, Tomohiro Osugi, Tsubasa Sakai, Shinji Kirimoto, Satsuki Nakaoka, Yuki Sugiura, Keiko Yasuda, Honoo Satake","doi":"10.1016/j.jbc.2025.108375","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108375","url":null,"abstract":"<p><p>Mammalian follicle growth development is mainly regulated by the hypothalamus-pituitary-gonadal (HPG) axis after puberty. Although pituitary hormones, gonadotropins, are involved in HPG axis signaling, they are not responsible for the growth of early-stage follicles, namely, primordial follicles, primary follicles, and secondary follicles, in both sexually immature and mature individuals. Unlike those of gonadotropin-dependent follicle growth, the specific regulatory factors of gonadotropin-independent follicle growth have yet to be identified. Here, we identified tachykinins (TKs) as inducers of gonadotropin-independent secondary follicle growth. TKs play various roles as neuropeptides or hormones in a wide variety of biological events both in the central nervous system and in peripheral tissues, but a direct effect of TKs on ovarian follicles has yet to investigated. Follicle development was suppressed in sexually immature 3-week-old knockout mice of Tac1 gene encoding TKs (substance P and neurokinin A), which is independent of gonadotropins. TKs and their receptors are specifically localized to granulosa cells in mouse secondary follicles. Furthermore, TKs upregulate the prostaglandin (PG) synthase COX-2 via the JAK1-STAT3 signaling cascade. We also demonstrated that PGE2 and PGF2α are major PGs in the immature ovary, and the secondary follicle growth was enhanced by interaction between PGE2 / PGF2α and their receptors, PGE2 receptor (EP2) localized in the oocyte membrane and PGF2α receptor (FP) localized in the oocyte membrane, the granulosa cells, and theca cells. Consequently, this study paves the way for exploring gonadotropin-independent early-stage follicle growth systems and relevant dysfunctions, including pediatric endocrinological diseases.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108375"},"PeriodicalIF":4.0,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143567263","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
Requirements for nuclear GRP78 transcriptional regulatory activities and interaction with nuclear GRP94.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-02-28 DOI: 10.1016/j.jbc.2025.108369
Ze Liu, Dat P Ha, Liangguang Leo Lin, Ling Qi, Amy S Lee
{"title":"Requirements for nuclear GRP78 transcriptional regulatory activities and interaction with nuclear GRP94.","authors":"Ze Liu, Dat P Ha, Liangguang Leo Lin, Ling Qi, Amy S Lee","doi":"10.1016/j.jbc.2025.108369","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108369","url":null,"abstract":"<p><p>GRP78, a molecular chaperone primarily located in the endoplasmic reticulum (ER), has recently been discovered to translocate into the nucleus of stressed and cancer cells where it assumes a new function reprogramming the transcriptome. This study explores the requirements of GRP78 nuclear translocation and its transcriptional activity and investigates the role of ER-associated degradation (ERAD) in the process. We show that the ER-processed, mature form of GRP78 is the major form of nuclear GRP78 and is the form with transcriptional regulatory activity. In contrast, exogenously expressed GRP78 designed to lack its ER signal peptide, thus preventing it from entering the ER or undergoing any ER-related processing/modification, while able to enter the nucleus, lacks transcriptional regulatory activity towards E-Box containing target genes. Additionally, the ATP-binding and substrate-binding activities of GRP78 are critical for this transcriptional regulatory function. We further discover that GRP94, an ER chaperone that acts in concert with GRP78 on protein folding, can translocate to the nucleus and co-localize with nuclear GRP78 upon ER stress. These findings suggest that some form of ER processing of GRP78, in addition to cleavage of the ER signal peptide, is critical for its nuclear activity and that in stressed cells, ER chaperones may assume new functions in the nucleus yet to be explored.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108369"},"PeriodicalIF":4.0,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143536831","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
Human glutathione transferases catalyze the reaction between glutathione and nitrooleic acid.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-02-28 DOI: 10.1016/j.jbc.2025.108362
Martina Steglich, Nicole Larrieux, Ari Zeida, Joaquín Dalla Rizza, Sonia R Salvatore, Mariana Bonilla, Matías N Möller, Alejandro Buschiazzo, Beatriz Alvarez, Francisco J Schopfer, Lucía Turell
{"title":"Human glutathione transferases catalyze the reaction between glutathione and nitrooleic acid.","authors":"Martina Steglich, Nicole Larrieux, Ari Zeida, Joaquín Dalla Rizza, Sonia R Salvatore, Mariana Bonilla, Matías N Möller, Alejandro Buschiazzo, Beatriz Alvarez, Francisco J Schopfer, Lucía Turell","doi":"10.1016/j.jbc.2025.108362","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108362","url":null,"abstract":"<p><p>Nitroalkene fatty acids (NO<sub>2</sub>-FAs) are formed endogenously. They regulate cell signaling pathways and are being developed clinically to treat inflammatory diseases. NO<sub>2</sub>-FAs are electrophilic and form thioether adducts with glutathione (GSH), which are exported from cells. Glutathione transferases (GSTs), a superfamily of enzymes, contribute to the cellular detoxification of hydrophobic electrophiles by catalyzing their conjugation to GSH. Herein, we evaluated the capacity of five human GSTs (M1-1, M2-2, M4-4, A4-4, and P1-1) to catalyze the reaction between nitrooleic acid (NO<sub>2</sub>-OA) and GSH. The reaction was monitored by HPLC-ESI-MS/MS and catalytic activity was detected with hGSTs M1-1 and A4-4. Using stopped-flow spectrophotometry, a 1400 and 7500-fold increase in the apparent second-order rate constant was observed for hGST M1-1 and hGST A4-4, respectively, compared to the uncatalyzed reaction (pH 7.4, 25 °C), in part due to a higher availability of the thiolate. The crystal structure of hGST M1-1 in complex with the adduct was solved at 2.55 Å resolution, revealing that the ligand was bound within the reaction center, and establishing a foundation to build a model of hGST A4-4 in complex with the adduct. A larger number of interactions between the enzyme and the fatty acid were observed for hGST A4-4 compared to hGST M1-1, probably contributing to the increased catalysis. Altogether, these results show, for the first time, that hGSTs can catalyze the reaction between GSH and NO<sub>2</sub>-FAs, likely affecting the signaling actions of these metabolites and expanding the repertoire of GST reactions.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108362"},"PeriodicalIF":4.0,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143537189","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
In vitro characterization of the yeast DEAH/RHA RNA helicase Dhr1.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-02-28 DOI: 10.1016/j.jbc.2025.108366
Ran Lin, Ezzeddine Elmir, Madison J Reynolds, Arlen W Johnson
{"title":"In vitro characterization of the yeast DEAH/RHA RNA helicase Dhr1.","authors":"Ran Lin, Ezzeddine Elmir, Madison J Reynolds, Arlen W Johnson","doi":"10.1016/j.jbc.2025.108366","DOIUrl":"10.1016/j.jbc.2025.108366","url":null,"abstract":"<p><p>In eukaryotic ribosome biogenesis, the small subunit (SSU) processome is a metastable intermediate in the assembly of the small (40S) subunit. In the SSU processome, the ribosomal RNA domains are splayed open by the intervention of assembly factors as well as U3 snoRNA. A critical step during the transition from the SSU processome to the nearly mature pre-40S particle is the removal of the U3 snoRNA to allow the formation of the central pseudoknot, a universally conserved structure which connects all domains of the subunit and contributes to its dynamic nature during translation. We previously identified the DEAH/RHA RNA helicase Dhr1 as the enzyme responsible for displacing the U3 snoRNA and the SSU processome factor Utp14 as an activator of Dhr1. Here, we have utilized biochemical and yeast genetic methods to further characterize Dhr1. We show that the N terminus as well as an internal loop within the RecA2 domain are autoinhibitory. We found that Utp14 can activate the ATPase activity of Dhr1 lacking the autoinhibitory N-terminal loop but not full-length Dhr1. We considered the possibility that Utp14 activates Dhr1 by relieving the autoinhibition of the loop within the RecA2 domain. However, our results are more consistent with Utp14 activating Dhr1 by binding to the surface of the RecA1 and RecA2 domains rather than displacing the inhibitory loop. This position of Utp14 is distinct from how G-patch proteins activate other DEXH/RHA helicases and is consistent with our previous conclusion that Utp14 is not a canonical G-patch protein.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108366"},"PeriodicalIF":4.0,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143536720","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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