The Journal of Biological Chemistry最新文献_第9页

Characterization of Rab32- and Rab38-positive lysosome-related organelles in osteoclasts and macrophages. 破骨细胞和巨噬细胞中Rab32-和Rab38阳性溶酶体相关细胞器的特征。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-08-23 DOI: 10.1016/j.jbc.2023.105191

Kazuya Noda, Shiou-Ling Lu, Siyu Chen, Kanako Tokuda, Yangjie Li, Feike Hao, Yoh Wada, Ge-Hong Sun-Wada, Shinya Murakami, Mitsunori Fukuda, Takashi Itoh, Takeshi Noda

{"title":"Characterization of Rab32- and Rab38-positive lysosome-related organelles in osteoclasts and macrophages.","authors":"Kazuya Noda, Shiou-Ling Lu, Siyu Chen, Kanako Tokuda, Yangjie Li, Feike Hao, Yoh Wada, Ge-Hong Sun-Wada, Shinya Murakami, Mitsunori Fukuda, Takashi Itoh, Takeshi Noda","doi":"10.1016/j.jbc.2023.105191","DOIUrl":"10.1016/j.jbc.2023.105191","url":null,"abstract":"Both the biogenesis and functions of osteoclasts and macrophages involves dynamic membrane traffic. We screened transcript levels for Rab family small GTPases related to osteoclasts and identified Rab38. Rab38 expression is upregulated during osteoclast differentiation and maturation. In osteoclasts, both Rab38 and its paralog, Rab32, colocalize to lysosome-related organelles (LROs). In macrophages, Rab32 is also found in LROs. LROs are part of the endocytic pathway but are distinct from lysosomes. After receptor activator of NF-κB ligand stimulation, LROs contain cathepsin K and tartrate-resistant acid phosphatase inside and help both proteins to accumulate around bone resorption pits. After osteoclast maturation, these enzymes are hardly found within LROs. In macrophages derived from Rab32 and Rab38 double knockout mice, both acidification and V-ATPase a3 localization were severely compromised. Both the double knockout macrophage and bafilomycin-treated wildtype macrophage show an increase in Lamp1-positive organelles, implying that biogenesis of lysosomes and LROs are related. These results indicate that Rab32 and Rab38 both play a crucial role in LRO biogenesis in macrophages and in osteoclasts.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":"299 10","pages":"105191"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10518718/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10356835","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Mathematical models disentangle the role of IL-10 feedbacks in human monocytes upon proinflammatory activation. 数学模型阐明了IL-10在人类单核细胞促炎激活中的作用。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-09-01 DOI: 10.1016/j.jbc.2023.105205

Niloofar Nikaein, Kedeye Tuerxun, Gunnar Cedersund, Daniel Eklund, Robert Kruse, Eva Särndahl, Eewa Nånberg, Antje Thonig, Dirk Repsilber, Alexander Persson, Elin Nyman

{"title":"Mathematical models disentangle the role of IL-10 feedbacks in human monocytes upon proinflammatory activation.","authors":"Niloofar Nikaein, Kedeye Tuerxun, Gunnar Cedersund, Daniel Eklund, Robert Kruse, Eva Särndahl, Eewa Nånberg, Antje Thonig, Dirk Repsilber, Alexander Persson, Elin Nyman","doi":"10.1016/j.jbc.2023.105205","DOIUrl":"10.1016/j.jbc.2023.105205","url":null,"abstract":"Inflammation is one of the vital mechanisms through which the immune system responds to harmful stimuli. During inflammation, proinflammatory and anti-inflammatory cytokines interplay to orchestrate fine-tuned and dynamic immune responses. The cytokine interplay governs switches in the inflammatory response and dictates the propagation and development of the inflammatory response. Molecular pathways underlying the interplay are complex, and time-resolved monitoring of mediators and cytokines is necessary as a basis to study them in detail. Our understanding can be advanced by mathematical models that enable to analyze the system of interactions and their dynamical interplay in detail. We, therefore, used a mathematical modeling approach to study the interplay between prominent proinflammatory and anti-inflammatory cytokines with a focus on tumor necrosis factor and interleukin 10 (IL-10) in lipopolysaccharide-primed primary human monocytes. Relevant time-resolved data were generated by experimentally adding or blocking IL-10 at different time points. The model was successfully trained and could predict independent validation data and was further used to perform simulations to disentangle the role of IL-10 feedbacks during an acute inflammatory event. We used the insight to obtain a reduced predictive model including only the necessary IL-10-mediated feedbacks. Finally, the validated reduced model was used to predict early IL-10-tumor necrosis factor switches in the inflammatory response. Overall, we gained detailed insights into fine-tuning of inflammatory responses in human monocytes and present a model for further use in studying the complex and dynamic process of cytokine-regulated acute inflammation.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105205"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/c1/cc/main.PMC10556785.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10499780","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Development and validation of a purification system for functional full-length human SR-B1 and CD36. 功能性全长人SR-B1和CD36纯化系统的开发和验证。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-08-23 DOI: 10.1016/j.jbc.2023.105187

Hayley R Powers, Shawn E Jenjak, Brian F Volkman, Daisy Sahoo

{"title":"Development and validation of a purification system for functional full-length human SR-B1 and CD36.","authors":"Hayley R Powers, Shawn E Jenjak, Brian F Volkman, Daisy Sahoo","doi":"10.1016/j.jbc.2023.105187","DOIUrl":"10.1016/j.jbc.2023.105187","url":null,"abstract":"Scavenger receptor class B type 1 (SR-B1) and CD36 are both members of the class B scavenger receptor family that play important roles in lipoprotein metabolism and atherosclerotic disease. SR-B1 is the primary receptor for high-density lipoproteins, while CD36 is the receptor responsible for the internalization of oxidized low-density lipoproteins. Despite their importance, class B scavenger receptor structure has only been studied by functional domain or peptide fragments-there are currently no reports of utilizing purified full-length protein. Here we report the successful expression and purification of full-length human SR-B1 and CD36 using an Spodoptera frugiperda insect cell system. We demonstrate that both SR-B1 and CD36 retained their normal functions in Spodoptera frugiperda cells, including lipoprotein binding, lipid transport, and the formation of higher order oligomers in the plasma membrane. Purification schemes for both scavenger receptors were optimized and their purity was confirmed by SDS-PAGE. Both purified scavenger receptors were assessed for stability by thermal shift assay and shown to maintain stable melting temperatures up to 6 weeks post-purification. Microscale thermophoresis was used to demonstrate that purified SR-B1 and CD36 were able to bind their native lipoprotein ligands. Further, there was no difference in affinity of SR-B1 for high-density lipoprotein or CD36 for oxidized low-density lipoprotein, when comparing glycosylated and deglycosylated receptors. These studies mark a significant step forward in creating physiologically relevant tools to study scavenger receptor function and lay the groundwork for future functional studies and determination of receptor structure.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":"299 10","pages":"105187"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10509710/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10633609","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Measurement of gluconeogenesis by ²H₂O labeling and mass isotopomer distribution analysis. 通过2H2O标记和质量等位异构体分布分析测量糖异生。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-09-01 DOI: 10.1016/j.jbc.2023.105206

Naveed Ziari, Marc Hellerstein

{"title":"Measurement of gluconeogenesis by 2H2O labeling and mass isotopomer distribution analysis.","authors":"Naveed Ziari, Marc Hellerstein","doi":"10.1016/j.jbc.2023.105206","DOIUrl":"10.1016/j.jbc.2023.105206","url":null,"abstract":"The gluconeogenesis pathway, which converts nonsugar molecules into glucose, is critical for maintaining glucose homeostasis. Techniques that measure flux through this pathway are invaluable for studying metabolic diseases such as diabetes that are associated with dysregulation of this pathway. We introduce a new method that measures fractional gluconeogenesis by heavy water labeling and gas chromatographic-mass spectrometric analysis. This technique circumvents cumbersome benchwork or inference of positionality from mass spectra. The enrichment and pattern of deuterium label on glucose is quantified by use of mass isotopomer distribution analysis, which informs on how much of glucose-6-phosphate-derived glucose comes from the gluconeogenesis (GNG) pathway. We use an in vivo model of the GNG pathway that is based on previously published models but offers a new approach to calculating GNG pathway and subpathway contributions using combinatorial probabilities. We demonstrated that this method accurately quantifies fractional GNG through experiments that perturb flux through the pathway and by probing analytical sensitivity. While this method was developed in mice, the results suggest that it is translatable to humans in a clinical setting.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105206"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10539955/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10518911","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Structural analysis of a bacterial UDP-sugar 2-epimerase reveals the active site architecture before and after catalysis. 细菌UDP糖2-二聚酶的结构分析揭示了催化前后的活性位点结构。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-09-03 DOI: 10.1016/j.jbc.2023.105200

James B Thoden, James O McKnight, Charles W Kroft, Joshua D T Jast, Hazel M Holden

{"title":"Structural analysis of a bacterial UDP-sugar 2-epimerase reveals the active site architecture before and after catalysis.","authors":"James B Thoden, James O McKnight, Charles W Kroft, Joshua D T Jast, Hazel M Holden","doi":"10.1016/j.jbc.2023.105200","DOIUrl":"10.1016/j.jbc.2023.105200","url":null,"abstract":"The sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, was first identified ∼40 years ago in the O-antigen of Pseudomonas aeruginosa O:3,a,d. Since then, it has been observed on the O-antigens of various pathogenic Gram-negative bacteria including Bordetella pertussis, Escherichia albertii, and Pseudomonas mediterranea. Previous studies have established that five enzymes are required for its biosynthesis beginning with uridine dinucleotide (UDP)-N-acetyl-d-glucosamine (UDP-GlcNAc). The final step in the pathway is catalyzed by a 2-epimerase, which utilizes UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid as its substrate. Curious as to whether this biochemical pathway is found in extreme thermophiles, we examined the published genome sequence for Thermus thermophilus HB27 and identified five ORFs that could possibly encode for the required enzymes. The focus of this investigation is on the ORF WP_011172736, which we demonstrate encodes for a 2-epimerase. For this investigation, ten high resolution X-ray crystallographic structures were determined to resolutions of 2.3 Å or higher. The models have revealed the manner in which the 2-epimerase anchors its UDP-sugar substrate as well as its UDP-sugar product into the active site. In addition, this study reveals for the first time the manner in which any sugar 2-epimerase can simultaneously bind UDP-sugars in both the active site and the allosteric binding region. We have also demonstrated that the T. thermophilus enzyme is allosterically regulated by UDP-GlcNAc. Whereas the sugar 2-epimerases that function on UDP-GlcNAc have been the focus of past biochemical and structural analyses, this is the first detailed investigation of a 2-epimerase that specifically utilizes UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid as its substrate.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105200"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10622841/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10518912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The RavA-ViaA chaperone complex modulates bacterial persistence through its association with the fumarate reductase enzyme. RavA-ViaA伴侣复合物通过与富马酸还原酶的结合调节细菌的持久性。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-09-03 DOI: 10.1016/j.jbc.2023.105199

Vaibhav Bhandari, Sean E Reichheld, Scott Houliston, Alexander Lemak, Cheryl H Arrowsmith, Simon Sharpe, Walid A Houry

{"title":"The RavA-ViaA chaperone complex modulates bacterial persistence through its association with the fumarate reductase enzyme.","authors":"Vaibhav Bhandari, Sean E Reichheld, Scott Houliston, Alexander Lemak, Cheryl H Arrowsmith, Simon Sharpe, Walid A Houry","doi":"10.1016/j.jbc.2023.105199","DOIUrl":"10.1016/j.jbc.2023.105199","url":null,"abstract":"Regulatory ATPase variant A (RavA) is a MoxR AAA+ protein that functions together with a partner protein termed von Willebrand factor type A interacting with AAA+ ATPase (ViaA). RavA-ViaA are functionally associated with anaerobic respiration in Escherichia coli through interactions with the fumarate reductase (Frd) electron transport complex. Through this association, RavA and ViaA modulate the activity of the Frd complex and, hence, are proposed to have chaperone-like activity. However, the functional role of RavA-ViaA in the cell is not yet well established. We had demonstrated that RavA-ViaA can sensitize E. coli cells to sublethal concentrations of the aminoglycoside class of antibiotics. Since Frd has been associated with bacterial persistence against antibiotics, the relationship of RavA-ViaA and Frd was explored within this context. Experiments performed here reveal a function of RavA-ViaA in bacterial persistence upon treatment with antibiotics through the association of the chaperone complex with Frd. As part of this work, the NMR structure of the N-terminal domain of ViaA was solved. The structure reveals a novel alpha helical fold, which we name the VAN fold, that has not been observed before. We show that this domain is required for the function of the chaperone complex. We propose that modulating the levels of RavA-ViaA could enhance the susceptibility of Gram-negative bacteria to antibiotics.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105199"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10585395/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10499779","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Endogenous Rab38 regulates LRRK2's membrane recruitment and substrate Rab phosphorylation in melanocytes. 内源性Rab38调节黑素细胞中LRRK2的膜募集和底物Rab磷酸化。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-08-23 DOI: 10.1016/j.jbc.2023.105192

Alexandra Unapanta, Farbod Shavarebi, Jacob Porath, Yiyi Shen, Carson Balen, Albert Nguyen, Josh Tseng, Weng Si Leong, Michelle Liu, Pawel Lis, Santiago M Di Pietro, Annie Hiniker

{"title":"Endogenous Rab38 regulates LRRK2's membrane recruitment and substrate Rab phosphorylation in melanocytes.","authors":"Alexandra Unapanta, Farbod Shavarebi, Jacob Porath, Yiyi Shen, Carson Balen, Albert Nguyen, Josh Tseng, Weng Si Leong, Michelle Liu, Pawel Lis, Santiago M Di Pietro, Annie Hiniker","doi":"10.1016/j.jbc.2023.105192","DOIUrl":"10.1016/j.jbc.2023.105192","url":null,"abstract":"Point mutations in leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease and augment LRRK2's kinase activity. However, cellular pathways that endogenously enhance LRRK2 kinase function have not been identified. While overexpressed Rab29 draws LRRK2 to Golgi membranes to increase LRRK2 kinase activity, there is little evidence that endogenous Rab29 performs this function under physiological conditions. Here, we identify Rab38 as a novel physiologic regulator of LRRK2 in melanocytes. In mouse melanocytes, which express high levels of Rab38, Rab32, and Rab29, knockdown (or CRISPR knockout) of Rab38, but not Rab32 or Rab29, decreases phosphorylation of multiple LRRK2 substrates, including Rab10 and Rab12, by both endogenous LRRK2 and exogenous Parkinson's disease-mutant LRRK2. In B16-F10 mouse melanoma cells, Rab38 drives LRRK2 membrane association and overexpressed kinase-active LRRK2 shows striking pericentriolar recruitment, which is dependent on the presence of endogenous Rab38 but not Rab32 or Rab29. Consistently, knockdown or mutation of BLOC-3, the guanine nucleotide exchange factor for Rab38 and Rab32, inhibits Rab38's regulation of LRRK2. Deletion or mutation of LRRK2's Rab38-binding site in the N-terminal armadillo domain decreases LRRK2 membrane association, pericentriolar recruitment, and ability to phosphorylate Rab10. In sum, our data identify Rab38 as a physiologic regulator of LRRK2 function and lend support to a model in which LRRK2 plays a central role in Rab GTPase coordination of vesicular trafficking.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105192"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10551901/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10062978","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Binding of the nuclear ribonucleoprotein family member FUS to RNA prevents R-loop RNA:DNA hybrid structures. 核核糖核蛋白家族成员FUS与RNA的结合阻止了R环RNA:DNA杂交结构。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-09-09 DOI: 10.1016/j.jbc.2023.105237

Valery F Thompson, Daniel R Wieland, Vivian Mendoza-Leon, Helen I Janis, Michelle A Lay, Lucas M Harrell, Jacob C Schwartz

{"title":"Binding of the nuclear ribonucleoprotein family member FUS to RNA prevents R-loop RNA:DNA hybrid structures.","authors":"Valery F Thompson, Daniel R Wieland, Vivian Mendoza-Leon, Helen I Janis, Michelle A Lay, Lucas M Harrell, Jacob C Schwartz","doi":"10.1016/j.jbc.2023.105237","DOIUrl":"10.1016/j.jbc.2023.105237","url":null,"abstract":"The protein FUS (FUSed in sarcoma) is a metazoan RNA-binding protein that influences RNA production by all three nuclear polymerases. FUS also binds nascent transcripts, RNA processing factors, RNA polymerases, and transcription machinery. Here, we explored the role of FUS binding interactions for activity during transcription. In vitro run-off transcription assays revealed FUS-enhanced RNA produced by a non-eukaryote polymerase. The activity also reduced the formation of R-loops between RNA products and their DNA template. Analysis by domain mutation and deletion indicated RNA-binding was required for activity. We interpret that FUS binds and sequesters nascent transcripts to prevent R-loops from forming with nearby DNA. DRIP-seq analysis showed that a knockdown of FUS increased R-loop enrichment near expressed genes. Prevention of R-loops by FUS binding to nascent transcripts has the potential to affect transcription by any RNA polymerase, highlighting the broad impact FUS can have on RNA metabolism in cells and disease.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105237"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10556777/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10204910","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Hemes on a string: insights on the functional mechanisms of PgcA from Geobacter sulfurreducens. Hemes on a string：硫还原地理杆菌PgcA功能机制的见解。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-08-16 DOI: 10.1016/j.jbc.2023.105167

Tomás M Fernandes, Marta A Silva, Leonor Morgado, Carlos A Salgueiro

{"title":"Hemes on a string: insights on the functional mechanisms of PgcA from Geobacter sulfurreducens.","authors":"Tomás M Fernandes, Marta A Silva, Leonor Morgado, Carlos A Salgueiro","doi":"10.1016/j.jbc.2023.105167","DOIUrl":"10.1016/j.jbc.2023.105167","url":null,"abstract":"Microbial extracellular reduction of insoluble compounds requires soluble electron shuttles that diffuse in the environment, freely diffusing cytochromes, or direct contact with cellular conductive appendages that release or harvest electrons to assure a continuous balance between cellular requirements and environmental conditions. In this work, we produced and characterized the three cytochrome domains of PgcA, an extracellular triheme cytochrome that contributes to Fe(III) and Mn(IV) oxides reduction in Geobacter sulfurreducens. The three monoheme domains are structurally homologous, but their heme groups show variable axial coordination and reduction potential values. Electron transfer experiments monitored by NMR and visible spectroscopy show the variable extent to which the domains promiscuously exchange electrons while reducing different electron acceptors. The results suggest that PgcA is part of a new class of cytochromes - microbial heme-tethered redox strings - that use low-complexity protein stretches to bind metals and promote intra- and intermolecular electron transfer events through its cytochrome domains.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105167"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/bc/b5/main.PMC10570954.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10017048","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Both chloride-binding sites are required for KCC2-mediated transport. KCC2介导的转运需要两个氯化物结合位点。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-08-23 DOI: 10.1016/j.jbc.2023.105190

Lisa Becker, Jens Hausmann, Anna-Maria Hartmann

{"title":"Both chloride-binding sites are required for KCC2-mediated transport.","authors":"Lisa Becker, Jens Hausmann, Anna-Maria Hartmann","doi":"10.1016/j.jbc.2023.105190","DOIUrl":"10.1016/j.jbc.2023.105190","url":null,"abstract":"The K+-Cl- cotransporter 2 (KCC2) plays an important role in inhibitory neurotransmission, and its impairment is associated with neurological and psychiatric disorders, including epilepsy, schizophrenia, and autism. Although KCCs transport K+ and Cl- in a 1:1 stoichiometry, two Cl- coordination sites were indicated via cryo-EM. In a comprehensive analysis, we analyzed the consequences of point mutations of residues coordinating Cl- in Cl1 and Cl2. Individual mutations of residues in Cl1 and Cl2 reduce or abolish KCC2WT function, indicating a crucial role of both Cl- coordination sites for KCC2 function. Structural changes in the extracellular loop 2 by inserting a 3xHA tag switches the K+ coordination site to another position. To investigate, whether the extension of the extracellular loop 2 with the 3xHA tag also affects the coordination of the two Cl- coordination sites, we carried out the analogous experiments for both Cl- coordinating sites in the KCC2HA construct. These analyses showed that most of the individual mutation of residues in Cl1 and Cl2 in the KCC2HA construct reduces or abolishes KCC2 function, indicating that the coordination of Cl- remains at the same position. However, the coupling of K+ and Cl- in Cl1 is still apparent in the KCC2HA construct, indicating a mutual dependence of both ions. In addition, the coordination residue Tyr569 in Cl2 shifted in KCC2HA. Thus, conformational changes in the extracellular domain affect K+ and Cl--binding sites. However, the effect on the Cl--binding sites is subtler.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105190"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10518353/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10062976","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0