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Lacritin Cleavage-Potentiated Targeting of Iron - Respiratory Reciprocity Promotes Bacterial Death.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-26 DOI: 10.1016/j.jbc.2025.108455
Mohammad Sharifian Gh, Fatemeh Norouzi, Mirco Sorci, Tanweer S Zaidi, Gerald B Pier, Alecia Achimovich, George M Ongwae, Binyong Liang, Margaret Ryan, Michael Lemke, Georges Belfort, Mihaela Gadjeva, Andreas Gahlmann, Marcos M Pires, Henrietta Venter, Thurl E Harris, Gordon W Laurie
{"title":"Lacritin Cleavage-Potentiated Targeting of Iron - Respiratory Reciprocity Promotes Bacterial Death.","authors":"Mohammad Sharifian Gh, Fatemeh Norouzi, Mirco Sorci, Tanweer S Zaidi, Gerald B Pier, Alecia Achimovich, George M Ongwae, Binyong Liang, Margaret Ryan, Michael Lemke, Georges Belfort, Mihaela Gadjeva, Andreas Gahlmann, Marcos M Pires, Henrietta Venter, Thurl E Harris, Gordon W Laurie","doi":"10.1016/j.jbc.2025.108455","DOIUrl":"10.1016/j.jbc.2025.108455","url":null,"abstract":"<p><p>Discovering new bacterial signaling pathways offers unique antibiotic strategies. With current antibiotic classes targeting cell wall synthesis or depolarizing the inner-membrane or altering the bacterial metabolome or inhibiting replication or transcription pathways, manipulation of transporters to limit bacterial respiration and thereby pathogenesis has been a decades long quest. Here we report an inhibitor of multiple bacterial transporters. The inhibitor is the bactericidal N-104 endogenous cleavage fragment of the prosecretory mitogen lacritin. Lacritin is now known to be widely distributed in plasma, cerebral spinal fluid, tears and saliva. With the bactericidal mechanism determined to be nonlytic by surface plasmon resonance as confirmed by lack of SYTOX Orange entry, we performed an unbiased resistance screen of 3,884 E. coli gene knockout strains revealing a complex N-104 polypharmacology. Validation in the virulent P. aeruginosa strain PA14 - one of three WHO Priority 1: Critical list species focused on an approach that sequentially couples three transporters and downstream transcription to lethally suppress respiration. By targeting outer membrane YaiW, cationic N-104 translocates into the periplasm where it ligates inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. With FeoB favoring an anaerobic environment, N-104 promotes the expression of genes regulating anaerobic respiration while largely suppressing those involved in aerobic respiration - a strategy counterproductive under aerobic conditions. This mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with tear thrombin fragment GKY20 as tested on antibiotic resistant clinical isolates.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108455"},"PeriodicalIF":4.0,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742879","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
Molecular basis underlying the specificity of an antagonist AA92593 for mammalian melanopsins.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-26 DOI: 10.1016/j.jbc.2025.108461
Kohei Obayashi, Ruisi Zou, Tomoki Kawaguchi, Toshifumi Mori, Hisao Tsukamoto
{"title":"Molecular basis underlying the specificity of an antagonist AA92593 for mammalian melanopsins.","authors":"Kohei Obayashi, Ruisi Zou, Tomoki Kawaguchi, Toshifumi Mori, Hisao Tsukamoto","doi":"10.1016/j.jbc.2025.108461","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108461","url":null,"abstract":"<p><p>Melanopsin functions in intrinsically photosensitive retinal ganglion cells of mammals to regulate circadian clock and pupil constriction. The opsinamide AA92593 has been reported to specifically inhibit mouse and human melanopsin functions as a competitive antagonist against retinal; however, the molecular mechanisms underlying its specificity have not been resolved. In this study, we attempted to identify amino acid residues responsible for the susceptibility of mammalian melanopsins to AA92593. Our cell-based assays confirmed that AA92593 effectively inhibited the light-induced cellular responses of mammalian melanopsins, but not those of non-mammalian vertebrate and invertebrate melanopsins. These results suggest that amino acid residues specifically conserved among mammalian melanopsins are important for the antagonistic effect of AA92593, and we noticed Phe-94<sup>2.61</sup>, Ser-188<sup>ECL2</sup>, and Ser-269<sup>6.52</sup> as candidate residues. Substitutions of these residues reduced the antagonistic effect of AA92593. We conducted docking and molecular dynamics simulations based on the AlphaFold-predicted melanopsin structure. The simulations indicated that Phe-94<sup>2.61</sup>, Ser-188<sup>ECL2</sup>, and Ser-269<sup>6.52</sup> are located at the AA92593-binding site, and additionally identified Trp-189<sup>ECL2</sup> and Leu-207<sup>5.42</sup> interacting with the antagonist. Substitutions of Trp-189<sup>ECL2</sup> and Leu-207<sup>5.42</sup> affected the antagonistic effect of AA92593. Furthermore, substitutions of these amino acid residues converted the AA92593-insensitive non-mammalian melanopsins susceptible to the antagonist. Based on experiments and molecular simulations, five amino acid residues, at positions 94<sup>2.61</sup>, 188<sup>ECL2</sup>, 189<sup>ECL2</sup>, 207<sup>5.42</sup>, and 269<sup>6.52</sup>, were found to be responsible for the specific susceptibility of mammalian melanopsins to AA92593.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108461"},"PeriodicalIF":4.0,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742884","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
A cluster of inhibitory residues in the regulatory domain prevents activation of the cystic fibrosis transmembrane conductance regulator.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-26 DOI: 10.1016/j.jbc.2025.108460
Min Wu, Yawei Xiong, Mengyuan Cao, Yunqi Zhi, Yan Jin, Yizhen Huang, Jeng-Haur Chen
{"title":"A cluster of inhibitory residues in the regulatory domain prevents activation of the cystic fibrosis transmembrane conductance regulator.","authors":"Min Wu, Yawei Xiong, Mengyuan Cao, Yunqi Zhi, Yan Jin, Yizhen Huang, Jeng-Haur Chen","doi":"10.1016/j.jbc.2025.108460","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108460","url":null,"abstract":"<p><p>Activation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl<sup>‒</sup> channel requires PKA phosphorylation at the regulatory (R) domain to relieve inhibition of ATP-dependent channel activity. This study aimed to identify the primary inhibitory site that prevents channel activation. CFTR mutants with deletion of residues 760-783 (ΔR<sub>760-783</sub>) elicited constitutive macroscopic and single-channel Cl<sup>‒</sup> currents in the presence of ATP before PKA phosphorylation, suggesting that protein segment R<sub>760-783</sub> in the R domain blocks CFTR activation. With the background of ΔR<sub>760-835</sub>, further deletion of R<sub>708-759</sub> led to fully active channels in the presence of ATP, but absence of PKA, suggesting that R<sub>708-759</sub> prevents the activation of ΔR<sub>760-835</sub>-CFTR. R<sub>760-783</sub> peptides were unstructured in buffered solutions in circular dichroism spectroscopy and the N771P mutation that interrupts the α-helix formation induced no apparent constitutive current before PKA phosphorylation. These data suggest that interpeptide interactions by α-helices likely contribute trivially to the blocking effect of R<sub>760-783</sub>. CFTR mutants with small deletions or alanine replacements containing any one of residues R<sup>766</sup> and S<sup>768</sup> in a PKA consensus sequence and M<sup>773</sup> and T<sup>774</sup> generated PKA-independent CFTR Cl<sup>‒</sup> currents. Similarly, introducing the mutations Q767C or T774C into a control CFTR construct produced constitutive CFTR Cl<sup>‒</sup> currents by positively charged MTSET modification of target cysteines. Moreover, PKA-independent single-channel activity was evidently observed in R766K-, S768K- and T774K-CFTR mutants. Therefore, the four residues R<sup>766</sup>, S<sup>768</sup>, M<sup>773</sup> and T<sup>774</sup> may form an inhibitory module that precludes CFTR activation through side-chain interactions. This inhibitory mechanism might be emulated by other PKA-dependent proteins.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108460"},"PeriodicalIF":4.0,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742890","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
Determining T-cell Receptor Binding Orientation and Peptide-HLA Interactions Using Cross-Linking Mass Spectrometry.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-26 DOI: 10.1016/j.jbc.2025.108445
Thomas Powell, Vijaykumar Karuppiah, Saher Afshan Shaikh, Robert Pengelly, Nicole Mai, Keir Barnbrook, Amit Sharma, Stephen Harper, Martin Ebner, Andrew J Creese
{"title":"Determining T-cell Receptor Binding Orientation and Peptide-HLA Interactions Using Cross-Linking Mass Spectrometry.","authors":"Thomas Powell, Vijaykumar Karuppiah, Saher Afshan Shaikh, Robert Pengelly, Nicole Mai, Keir Barnbrook, Amit Sharma, Stephen Harper, Martin Ebner, Andrew J Creese","doi":"10.1016/j.jbc.2025.108445","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108445","url":null,"abstract":"<p><p>T cell receptors (TCRs) recognize specific peptides presented by human leukocyte antigens (HLAs) on the surface of antigen presenting cells and are involved in fighting pathogens and cancer surveillance. Canonical docking orientation of TCRs to their target peptide-HLAs (pHLAs) is essential for T cell activation, with reverse binding TCRs lacking functionality. TCR binding geometry and molecular interaction footprint with pHLAs is typically obtained by determining the crystal structure. Here, we describe the use of a cross-linking tandem mass spectrometry (XL-MS/MS) method to decipher the binding orientation of several TCRs to their target pHLAs. Cross-linking sites were localized to specific residues and their molecular interactions showed differentiation between TCRs binding in canonical or reverse orientations. Structural prediction and crystal structure determination of two TCR-pHLA complexes validated these findings. The XL-MS/MS method described herein offers a faster and simpler approach for elucidating TCR-pHLA binding orientation and interactions.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108445"},"PeriodicalIF":4.0,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742862","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
CONNECTING THE DOTS: EPIGENETIC REGULATION OF EXTRACHROMOSOMAL AND INHERITED DNA AMPLIFICATIONS.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-26 DOI: 10.1016/j.jbc.2025.108454
Chloe Azadegan, John Santoro, Johnathan R Whetstine
{"title":"CONNECTING THE DOTS: EPIGENETIC REGULATION OF EXTRACHROMOSOMAL AND INHERITED DNA AMPLIFICATIONS.","authors":"Chloe Azadegan, John Santoro, Johnathan R Whetstine","doi":"10.1016/j.jbc.2025.108454","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108454","url":null,"abstract":"<p><p>DNA amplification has intrigued scientists for decades. Since its discovery, significant progress has been made in understanding the mechanisms promoting DNA amplification and their associated function(s). While DNA copy gains were once thought to be regulated purely by stochastic processes, recent findings have revealed the important role of epigenetic modifications in driving these amplifications and their integration into the genome. Furthermore, advances in genomic technology have enabled detailed characterization of these genomic events in terms of size, structure, formation, and regulation. This review highlights how our understanding of DNA amplifications has evolved over time, tracing its trajectory from initial discovery to the contemporary landscape. We describe how recent discoveries have started to uncover how these genomic events occur by controlled biological processes rather than stochastic mechanisms, presenting opportunities for therapeutic modulation.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108454"},"PeriodicalIF":4.0,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742857","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
Conversion of glutamate into proline by the leucine analog BCH enhances biphasic insulin secretion in pancreatic β-cells.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-26 DOI: 10.1016/j.jbc.2025.108449
Sevda Gheibi, Luis Rodrigo Cataldo, Hamidreza Ardalani, Lisa Nocquet, Peter Spégel, Susanne G Straub, Geoffrey W G Sharp, Malin Fex, Hindrik Mulder
{"title":"Conversion of glutamate into proline by the leucine analog BCH enhances biphasic insulin secretion in pancreatic β-cells.","authors":"Sevda Gheibi, Luis Rodrigo Cataldo, Hamidreza Ardalani, Lisa Nocquet, Peter Spégel, Susanne G Straub, Geoffrey W G Sharp, Malin Fex, Hindrik Mulder","doi":"10.1016/j.jbc.2025.108449","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108449","url":null,"abstract":"<p><p>Biphasic insulin secretion, which fails in Type 2 Diabetes, can be provoked by various nutrient stimuli, glucose being the superior physiological one. To identify pathways that may play a role in β-cell stimulus-secretion coupling, we compared β-cell and islet functional, secretory, and metabolic responses to glucose and 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), a leucine analog, that acts as an allosteric activator of glutamate dehydrogenase (GDH). We employed a range of techniques, including insulin secretion assays, mitochondrial activity measurements, ATP/ADP ratio assessments, and cytosolic Ca<sup>2+</sup> level quantifications. Metabolomics was used to analyze cellular metabolite profiles in response to glucose and BCH. Additionally, we investigated the role of proline synthesis by silencing ALDH18A1, encoding proline 5-carboxylate (P5C) synthase, in both clonal β-cells and human islets. BCH and glucose similarly induced a biphasic insulin response in INS-1 832/13 cells, parallelled by increased mitochondrial activity and raised ATP/ADP ratios, plasma membrane depolarization, and elevated cytosolic Ca<sup>2+</sup> levels. Metabolomics revealed that proline levels increased significantly only in BCH-stimulated β-cells. Silencing ALDH18A1 disrupted insulin secretion in response to both glucose and BCH, accompanied by reduced cytosolic Ca<sup>2+</sup> levels, ATP/ADP ratios, and mitochondrial activity. Our findings demonstrated that BCH-induced activation of GDH leads to the conversion of glutamate into proline, which apparently enhances β-cell stimulus-secretion coupling. This work identifies a previously unrecognized role of proline metabolism in β-cell function and provides novel insights into the complex regulation of insulin secretion.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108449"},"PeriodicalIF":4.0,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742859","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
Artesunate enhances the efficacy of Enzalutamide in advanced prostate cancer.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-26 DOI: 10.1016/j.jbc.2025.108458
Xinyi Wang, Jinghui Liu, Fengyi Mao, Yifan Kong, Qiongsi Zhang, Chaohao Li, Daheng He, Chi Wang, Yanquan Zhang, Ruixin Wang, Sally R Ellingson, Qiou Wei, Zhiguo Li, Xiaoqi Liu
{"title":"Artesunate enhances the efficacy of Enzalutamide in advanced prostate cancer.","authors":"Xinyi Wang, Jinghui Liu, Fengyi Mao, Yifan Kong, Qiongsi Zhang, Chaohao Li, Daheng He, Chi Wang, Yanquan Zhang, Ruixin Wang, Sally R Ellingson, Qiou Wei, Zhiguo Li, Xiaoqi Liu","doi":"10.1016/j.jbc.2025.108458","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108458","url":null,"abstract":"<p><p>Prostate cancer (PCa) is one of the leading causes of death among men worldwide. Treatments targeting the androgen receptor (AR) pathway remain the standard therapy for PCa patients. Enzalutamide (ENZ), a second-generation AR inhibitor, was developed to treat castration-resistant prostate cancer (CRPC). However, while patients initially respond to ENZ, drug resistance typically develops within a few months. Artesunate (ART), a semi-synthetic derivative of the Artemisinin plant, is approved for anti-malaria treatment. In this study, we conducted an FDA-approved drug screening and identified Artesunate as a potential candidate for overcoming ENZ resistance in prostate cancer (ENZ-R PCa). Mechanistically, ART induces the degradation of c-Myc, enhancing the efficacy of ENZ. Additionally, patient dataset analysis revealed that c-Myc plays a significant role in developing ENZ resistance. To summarize, these findings suggest a novel therapeutic strategy for ENZ-resistant prostate cancer.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108458"},"PeriodicalIF":4.0,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742894","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
Flavin transferase ApbE: from discovery to applications.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-26 DOI: 10.1016/j.jbc.2025.108453
Xiaoman Fan, Marco W Fraaije
{"title":"Flavin transferase ApbE: from discovery to applications.","authors":"Xiaoman Fan, Marco W Fraaije","doi":"10.1016/j.jbc.2025.108453","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108453","url":null,"abstract":"<p><p>ApbE is a unique, membrane-bound enzyme which covalently attaches a flavin cofactor to specific target proteins. This irreversible post-translational modification is crucial for proper functioning of various bacterial proteins. ApbEs have also been identified in archaea and eukaryotes. This review summarizes current knowledge on the structural and mechanistic properties of this unique protein-modifying enzyme and its recent applications. The relatively small flavin transferase is typically anchored to the outer membrane of bacteria and possesses a conserved flavin-binding domain and a catalytic domain. It recognizes a specific sequence motif of its target proteins, resulting in flavinylation of a threonine or serine. For flavinylation, it depends on magnesium and utilizes FAD as substrate to attach the FMN moiety to the target protein, analogous to phosphorylation. ApbE-mediated flavinylation supports critical bacterial respiratory and metabolic pathways. Recently, ApbE was also shown to be a versatile tool for selectively modifying proteins. Using the flavin-tagging approach, proteins can be decorated with FMN or other flavins. Furthermore, it was demonstrated that ApbE can be employed to turn natural noncovalent flavoproteins into covalent flavoproteins. In summary, ApbE is crucial for the maturation of various flavoproteins by catalyzing covalent flavinylation. While great progress has been made in understanding the role and mode of action of ApbE, there are still many bacterial proteins predicted to be flavinylated by ApbE for which their role is enigmatic. Also, exploration of the potential of ApbE as protein modification tool has just begun. Clearly, future research will generate new ApbE-related insights and applications.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108453"},"PeriodicalIF":4.0,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742864","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
A major Toxoplasma Serine Protease Inhibitor protects the parasite against gut-derived serine proteases and NETosis damage.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-26 DOI: 10.1016/j.jbc.2025.108457
Maryam Saffarian, Julia D Romano, Michael E Grigg, Isabelle Coppens
{"title":"A major Toxoplasma Serine Protease Inhibitor protects the parasite against gut-derived serine proteases and NETosis damage.","authors":"Maryam Saffarian, Julia D Romano, Michael E Grigg, Isabelle Coppens","doi":"10.1016/j.jbc.2025.108457","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108457","url":null,"abstract":"<p><p>Toxoplasmosis is a life-threatening opportunistic infection in immunocompromised patients, caused by the parasite Toxoplasma gondii. Infection is initiated through oral ingestion of Toxoplasma cysts that must survive the harsh environment of the gut to undergo excystation. Released parasites invade intestinal epithelial cells, and then disseminate throughout tissues for encystation, mainly in the brain. How Toxoplasma escapes destruction mediated by gastrointestinal proteases is poorly understood. T. gondii has nine genes encoding serine protease inhibitor proteins (TgPIs). TgPI-1 is highly expressed across all Toxoplasma strains and developmental stages and contains three domains for binding to various serine proteases. Here, we explore the role of TgPI-1 in protecting Toxoplasma against serine proteases in the gut and neutrophil-derived proteases in the lamina propria. TgPI-1 localizes to the parasite plasma membrane and cyst wall. We generated ΔTgPI-1 parasites, and the mutant is more sensitive to neutrophil elastase (NE), trypsin and chymotrypsin than WT. Neutrophils exposed to Toxoplasma release Neutrophil Extracellular Traps (NET) with strain-dependent morphologies, ranging from spiky to extended cloudy. TgPI-1 was detected on NET containing NE, and ΔTgPI-1 parasites are more susceptible to destruction by NETosis. In mice, ΔTgPI-1 parasites exhibit reduced infectivity, poor dissemination to abdominal organs, and lower cyst burden in the brain. These findings shed light on a strategy employed by Toxoplasma to counteract enzymatic antimicrobial defenses in gut tissues, highlighting potential avenues for controlling tissue dissemination of this medically significant parasite.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108457"},"PeriodicalIF":4.0,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742893","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
Signal termination of the chemokine receptor CCR9 is governed by an arrestin-independent phosphorylation mechanism.
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-03-26 DOI: 10.1016/j.jbc.2025.108462
Thomas D Lamme, Martine J Smit, Christopher T Schafer
{"title":"Signal termination of the chemokine receptor CCR9 is governed by an arrestin-independent phosphorylation mechanism.","authors":"Thomas D Lamme, Martine J Smit, Christopher T Schafer","doi":"10.1016/j.jbc.2025.108462","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108462","url":null,"abstract":"<p><p>The chemokine receptor CCR9 coordinates immune cell migration from the thymus to the small intestine along gradients of the chemokine CCL25. Receptor dysregulation is associated with a variety of inflammatory bowel diseases such as Crohn's and ulcerative colitis, while aberrant CCR9 overexpression correlates with tumor metastasis. Despite being an attractive therapeutic target, attempts to clinically antagonize CCR9 have been unsuccessful. This highlights the need for a deeper understanding of its specific regulatory mechanisms and signaling pathways. CCR9 is a G protein-coupled receptor (GPCR) and activates G<sub>i</sub> and G<sub>q</sub> pathways. Unexpectedly, live-cell BRET assays reveal only limited G protein activation and signaling is rapidly terminated. Truncating the receptor C-terminus significantly enhanced G protein coupling, highlighting a regulatory role of this domain. Signal suppression was not due to canonical arrestin-coordinated desensitization. Rather, removal of GPCR kinase (GRK) phosphorylation led to sustained and robust G protein activation by CCR9. Using site-directed mutagenesis, we identified specific phosphorylation motifs that attenuate G protein coupling. Receptor internalization did not correlate with G protein activation capabilities. Instead, CCR9 phosphorylation disrupted the interaction of G protein heterotrimers with the receptor. This interference may lead to rapid loss of productive coupling and downstream signaling as phosphorylation would effectively render the receptor incapable of G protein coupling. An arrestin-independent, phosphorylation-driven deactivation mechanism could complement arrestin-dependent regulation of other GPCRs and have consequences for therapeutically targeting these receptors.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108462"},"PeriodicalIF":4.0,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742950","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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