Biochemistry Biochemistry最新文献_第4页

VPS26 Moonlights as a β-Arrestin-like Adapter for a 7-Transmembrane RGS Protein in Arabidopsis thaliana. VPS26 是拟南芥中 7 跨膜 RGS 蛋白的β-阿restin 样适配器。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-28 DOI: 10.1021/acs.biochem.4c00361

Fei Lou, Wenbin Zhou, Meral Tunc-Ozdemir, Jing Yang, Vaithish Velazhahan, Christopher G Tate, Alan M Jones

{"title":"VPS26 Moonlights as a β-Arrestin-like Adapter for a 7-Transmembrane RGS Protein in Arabidopsis thaliana.","authors":"Fei Lou, Wenbin Zhou, Meral Tunc-Ozdemir, Jing Yang, Vaithish Velazhahan, Christopher G Tate, Alan M Jones","doi":"10.1021/acs.biochem.4c00361","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00361","url":null,"abstract":"Extracellular signals perceived by 7-transmembrane (7TM)-spanning receptors initiate desensitization that involves the removal of these receptors from the plasma membrane. Agonist binding often evokes phosphorylation in the flexible C-terminal region and/or intracellular loop 3 of many 7TM G-protein-coupled receptors in animal cells, which consequently recruits a cytoplasmic intermediate adaptor, β-arrestin, resulting in clathrin-mediated endocytosis (CME) and downstream signaling such as transcriptional changes. Some 7TM receptors undergo CME without recruiting β-arrestin, but it is not clear how. Arrestins are not encoded in the Arabidopsis thaliana genome, yet Arabidopsis cells have a well-characterized signal-induced CME of a 7TM protein, designated Regulator of G Signaling 1 (AtRGS1). Here we show that a component of the retromer complex, Vacuolar Protein Sorting-Associated 26 (VPS26), binds the phosphorylated C-terminal region of AtRGS1 as a VPS26A/B heterodimer to form a complex that is required for downstream signaling. We propose that VPS26 moonlights as an arrestin-like adaptor in the CME of AtRGS1.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142520318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

Biochemical Analysis of the Regulatory Role of Gαo in the Conformational Transitions of Drosophila Pins 果蝇引脚构象转变过程中 Gαo 调控作用的生化分析

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-23 DOI: 10.1021/acs.biochem.4c0040410.1021/acs.biochem.4c00404

Yuxuan Song, Jie Ji, Chunhua Liu and Wenning Wang*,

{"title":"Biochemical Analysis of the Regulatory Role of Gαo in the Conformational Transitions of Drosophila Pins","authors":"Yuxuan Song, Jie Ji, Chunhua Liu and Wenning Wang*, ","doi":"10.1021/acs.biochem.4c0040410.1021/acs.biochem.4c00404","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00404https://doi.org/10.1021/acs.biochem.4c00404","url":null,"abstract":"Drosophila Pins (and its mammalian homologue LGN) play a crucial role in the process of asymmetric cell division (ACD). Extensive research has established that Pins/LGN functions as a conformational switch primarily through intramolecular interactions involving the N-terminal TPR repeats and the C-terminal GoLoco (GL) motifs. The GL motifs served as binding sites for the α subunit of the trimeric G protein (Gα), which facilitates the release of the autoinhibited conformation of Pins/LGN. While LGN has been observed to specifically bind to Gαi·GDP, Pins has been found to associate with both Drosophila Gαi (dGαi) and Gαo (dGαo) isoforms. Moreover, dGαo was reported to be able to bind Pins in both the GDP- and GTP-bound forms. However, the precise mechanism underlying the influence of dGαo on the conformational states of Pins remains unclear, despite extensive investigations into the Gαi·GDP-mediated regulatory conformational changes in LGN/Pins. In this study, we conducted a comprehensive characterization of the interactions between Pins-GL motifs and dGαo in both GDP- and GTP-loaded forms. Our findings reveal that Pins-GL specifically binds to GDP-loaded dGαo. Through biochemical characterization, we determined that the intramolecular interactions of Pins primarily involve the entire TPR domain and the GL23 motifs. Additionally, we observed that Pins can simultaneously bind three molecules of dGαo·GDP, leading to a partial opening of the autoinhibited conformation. Furthermore, our study presents evidence contrasting with previous observations indicating the absence of binding between dGαi and Pins-GLs, thus implying the pivotal role of dGαo as the principal participant in the ACD pathway associated with Pins.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142577769","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

CG17192 is a Phospholipase That Regulates Signaling Lipids in the Drosophila Gut upon Infection. CG17192 是一种磷脂酶，能在果蝇肠道受到感染时调节信号脂质。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-23 DOI: 10.1021/acs.biochem.4c00579

Kundan Kumar, Mrunal Pazare, Girish S Ratnaparkhi, Siddhesh S Kamat

{"title":"CG17192 is a Phospholipase That Regulates Signaling Lipids in the Drosophila Gut upon Infection.","authors":"Kundan Kumar, Mrunal Pazare, Girish S Ratnaparkhi, Siddhesh S Kamat","doi":"10.1021/acs.biochem.4c00579","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00579","url":null,"abstract":"The chemoproteomics technique, activity-based protein profiling (ABPP), has proven to be an invaluable tool in assigning functions to enzymes. The serine hydrolase (SH) enzyme superfamily, in particular, has served as an excellent example in displaying the versatility of various ABPP platforms and has resulted in a comprehensive cataloging of the biochemical activities associated within this superfamily. Besides SHs, in mammals, several other enzyme classes have been thoroughly investigated using ABPP platforms. However, the utility of ABPP platforms in fly models remains underexplored. Realizing this knowledge gap, leveraging complementary ABPP platforms, we reported the full array of SH activities during various developmental stages and adult tissues in the fruit fly (Drosophila melanogaster). Following up on this study, using ABPP, we mapped SH activities in adult fruit flies in an infection model and found that a gut-resident lipase CG17192 showed increased activity during infection. To assign a biological function to this uncharacterized lipase, we performed an untargeted lipidomics analysis and found that phosphatidylinositols were significantly elevated when CG17192 was depleted in the adult fruit fly gut. Next, we overexpressed this lipase in insect cells, and using biochemical assays, we show that CG17192 is a secreted enzyme that has phospholipase C (PLC) type activity, with phosphatidylinositol being a preferred substrate. Finally, we show during infection that heightened CG17192 regulates phosphatidylinositol levels and, by doing so, likely modulates signaling pathways in the adult fruit fly gut that might be involved in the resolution of this pathophysiological condition.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142491082","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

Context Effects on Human Milk Oligosaccharide Linkage Conformation and Dynamics Revealed by MA’AT Analysis 通过 MA'AT 分析揭示语境对人奶低聚糖连接构象和动态的影响

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-22 DOI: 10.1021/acs.biochem.4c0034810.1021/acs.biochem.4c00348

Wenhui Zhang, Reagan J. Meredith, Mi-Kyung Yoon, Ian Carmichael and Anthony S. Serianni*,

{"title":"Context Effects on Human Milk Oligosaccharide Linkage Conformation and Dynamics Revealed by MA’AT Analysis","authors":"Wenhui Zhang, Reagan J. Meredith, Mi-Kyung Yoon, Ian Carmichael and Anthony S. Serianni*, ","doi":"10.1021/acs.biochem.4c0034810.1021/acs.biochem.4c00348","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00348https://doi.org/10.1021/acs.biochem.4c00348","url":null,"abstract":"An emerging NMR method, MA’AT analysis, has been applied to investigate context effects on the conformational properties of several human milk oligosaccharides (HMOs). The MA’AT model of the β-(1→4) linkage in the disaccharide, methyl β-lactoside (MeL), was compared to those obtained for the same linkage in the HMO trisaccharides, methyl 2′-fucosyllactoside (Me2′FL) and methyl 3-fucosyllactoside (Me3FL), and in the tetrasaccharide, methyl 2′,3-difucosyllactoside (Me2′,3DFL). MA’AT analysis revealed significant context effects on the mean values and circular standard deviations (CSDs) of the psi (ψ) torsion angles in these linkages. α-Fucosylation at both O2′Gal and O3Glc of MeL to give Me2′,3DFL significantly constrained librational motion about ψ (70% reduction in the CSD) and shifted its mean value by ∼18°. α-Fucosylation at the O3Glc of MeL to give Me3FL constrained ψ more than α-fucosylation at the O2Gal to give Me2′FL. These effects can be explained by the expected solution conformation of Me3FL, which closely resembles the Lewisx trisaccharide. Comparisons of MA’AT models of ψ to those obtained by 1 μs aqueous molecular dynamics simulation (GLYCAM06) revealed identical trends, that is, MA’AT analysis was able to recapitulate molecular behavior in solution that was heretofore only available from MD simulation. The results highlight the capabilities of MA’AT analysis to determine probability distributions of molecular torsion angles in solution as well as degrees of librational averaging of these angles.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142577570","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

Noncovalent Inhibition and Covalent Inactivation of Proline Dehydrogenase by Analogs of N-Propargylglycine N-丙炔基甘氨酸类似物对脯氨酸脱氢酶的非共价抑制和共价失活作用

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-22 DOI: 10.1021/acs.biochem.4c0042910.1021/acs.biochem.4c00429

John J. Tanner*, Juan Ji, Alexandra N. Bogner, Gary K. Scott, Sagar M. Patel, Javier Seravalli, Kent S. Gates, Christopher C. Benz and Donald F. Becker,

{"title":"Noncovalent Inhibition and Covalent Inactivation of Proline Dehydrogenase by Analogs of N-Propargylglycine","authors":"John J. Tanner*, Juan Ji, Alexandra N. Bogner, Gary K. Scott, Sagar M. Patel, Javier Seravalli, Kent S. Gates, Christopher C. Benz and Donald F. Becker, ","doi":"10.1021/acs.biochem.4c0042910.1021/acs.biochem.4c00429","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00429https://doi.org/10.1021/acs.biochem.4c00429","url":null,"abstract":"The flavoenzyme proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the oxidation of l-proline to Δ1-pyrroline-5-carboxylate. The enzyme is a target for chemical probe discovery because of its role in the metabolism of certain cancer cells. N-propargylglycine is the first and best characterized mechanism-based covalent inactivator of PRODH. This compound consists of a recognition module (glycine) that directs the inactivator to the active site and an alkyne warhead that reacts with the FAD after oxidative activation, leading to covalent modification of the FAD N5 atom. Here we report structural and kinetic data on analogs of N-propargylglycine with the goals of understanding the initial docking step of the inactivation mechanism and to test the allyl group as a warhead. The crystal structures of PRODH complexed with unreactive analogs in which N is replaced by S show how the recognition module mimics the substrate proline by forming ion pairs with conserved arginine and lysine residues. Further, the C atom adjacent to the alkyne warhead is optimally positioned for hydride transfer to the FAD, providing the structural basis for the first bond-breaking step of the inactivation mechanism. The structures also suggest new strategies for designing improved N-propargylglycine analogs. N-allylglycine, which consists of a glycine recognition module and allyl warhead, is shown to be a covalent inactivator; however, it is less efficient than N-propargylglycine in both enzyme inactivation and cellular assays. Crystal structures of the N-allylglycine-inactivated enzyme are consistent with covalent modification of the N5 by propanal.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142577766","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

Characterization of the Flavin-Dependent Monooxygenase Involved in the Biosynthesis of the Nocardiosis-Associated Polyketide† 参与诺卡氏菌相关多酮† 生物合成的黄素依赖性单加氧酶的特征描述

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-21 DOI: 10.1021/acs.biochem.4c0048010.1021/acs.biochem.4c00480

Antonio Del Rio Flores, and , Chaitan Khosla*,

{"title":"Characterization of the Flavin-Dependent Monooxygenase Involved in the Biosynthesis of the Nocardiosis-Associated Polyketide†","authors":"Antonio Del Rio Flores,  and , Chaitan Khosla*, ","doi":"10.1021/acs.biochem.4c0048010.1021/acs.biochem.4c00480","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00480https://doi.org/10.1021/acs.biochem.4c00480","url":null,"abstract":"Some species of the Nocardia genus harbor a highly conserved biosynthetic gene cluster designated as the NOCardiosis-Associated Polyketide (NOCAP) synthase that produces a unique glycolipid natural product. The NOCAP glycolipid is composed of a fully substituted benzaldehyde headgroup linked to a polyfunctional alkyl tail and an O-linked disaccharide composed of 3-α-epimycarose and 2-O-methyl-α-rhamnose. Incorporation of the disaccharide unit is preceded by a critical step involving hydroxylation by NocapM, a flavin monooxygenase. In this study, we employed biochemical, spectroscopic, and kinetic analyses to explore the substrate scope of NocapM. Our findings indicate that NocapM catalyzes hydroxylation of diverse aromatic substrates, although the observed coupling between NADPH oxidation and substrate hydroxylation varies widely from substrate to substrate. Our in-depth biochemical characterization of NocapM provides a solid foundation for future mechanistic studies of this enzyme as well as its utilization as a practical biocatalyst.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142577659","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

High-Throughput Algorithmic Optimization of In Vitro Transcription for SARS-CoV-2 mRNA Vaccine Production 用于 SARS-CoV-2 mRNA 疫苗生产的体外转录高通量算法优化

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-20 DOI: 10.1021/acs.biochem.4c0018810.1021/acs.biochem.4c00188

Spencer E. McMinn*, Danielle V. Miller*, Daniel Yur, Kevin Stone, Yuting Xu, Ajit Vikram, Shashank Murali, Jessica Raffaele, David Holland, Sheng-Ching Wang and Joseph P. Smith,

{"title":"High-Throughput Algorithmic Optimization of In Vitro Transcription for SARS-CoV-2 mRNA Vaccine Production","authors":"Spencer E. McMinn*, Danielle V. Miller*, Daniel Yur, Kevin Stone, Yuting Xu, Ajit Vikram, Shashank Murali, Jessica Raffaele, David Holland, Sheng-Ching Wang and Joseph P. Smith, ","doi":"10.1021/acs.biochem.4c0018810.1021/acs.biochem.4c00188","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00188https://doi.org/10.1021/acs.biochem.4c00188","url":null,"abstract":"The in vitro transcription (IVT) of messenger ribonucleic acid (mRNA) from the linearized deoxyribonucleic acid (DNA) template of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant (B.1.617.2) was optimized for total mRNA yield and purity (by percent intact mRNA) utilizing machine learning in conjunction with automated, high-throughput liquid handling technology. An iterative Bayesian optimization approach successfully optimized 11 critical process parameters in 42 reactions across 5 experimental rounds. Once the optimized conditions were achieved, an automated, high-throughput screen was conducted to evaluate commercially available T7 RNA polymerases for rate and quality of mRNA production. Final conditions showed a 12% yield improvement and a 50% reduction in reaction time, while simultaneously significantly decreasing (up to 44% reduction) the use of expensive reagents. This novel platform offers a powerful new approach for optimizing IVT reactions for mRNA production.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142577549","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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 Characterization of Ancient Prosaposin-like Proteins from the Protist Dictyostelium discoideum 盘基原生动物中古老的原肠蛋白样蛋白的分子特征描述

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-18 DOI: 10.1021/acs.biochem.4c0047910.1021/acs.biochem.4c00479

Marius Ortjohann, and , Matthias Leippe*,

{"title":"Molecular Characterization of Ancient Prosaposin-like Proteins from the Protist Dictyostelium discoideum","authors":"Marius Ortjohann,  and , Matthias Leippe*, ","doi":"10.1021/acs.biochem.4c0047910.1021/acs.biochem.4c00479","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00479https://doi.org/10.1021/acs.biochem.4c00479","url":null,"abstract":"To combat the permanent exposure to potential pathogens every organism relies on an immune system. Important factors in innate immunity are antimicrobial peptides (AMPs) that are structurally highly diverse. Some AMPs are known to belong to the saposin-like proteins (SAPLIPs), a group of polypeptides with a broad functional spectrum. The model organism Dictyostelium discoideum possesses a remarkably large arsenal of potential SAPLIPs, which are termed amoebapore-like peptides (Apls), but the knowledge about these proteins is very limited. Here, we report about the biochemical characterization of AplE1, AplE2, AplK1, and AplK2, which are derived from the two precursor proteins AplE and AplK, thereby resembling prosaposins of vertebrates. We produced these Apls as recombinant polypeptides in Escherichia coli using a self-splicing intein to remove an affinity tag used for purification. All recombinant Apls exhibited pore-forming activity in a pH-dependent manner, as evidenced by liposome depolarization, showing higher activities the more acidic the setting was. Lipid preference was detected for negatively charged phospholipids and in particular for cardiolipin. Antimicrobial activity against various bacteria was found to be inferior in classical microdilution assays. However, all of the Apls studied permeabilized the cytoplasmic membrane of live Bacillus subtilis. Collectively, we assume that the selected Apls interact by their cationic charge with negatively charged bacterial membranes in acidic environments such as phagolysosomes and eventually lyse the target cells by pore formation.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00479","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142577647","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Factor XIII Activation Peptide Residues Play Important Roles in Stability, Activation, and Transglutaminase Activity 因子 XIII 活化肽基在稳定性、激活和转谷氨酰胺酶活性中发挥重要作用

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-18 DOI: 10.1021/acs.biochem.4c0031810.1021/acs.biochem.4c00318

Rameesa D. Syed Mohammed, Lianay Gutierrez Luque and Muriel C. Maurer*,

{"title":"Factor XIII Activation Peptide Residues Play Important Roles in Stability, Activation, and Transglutaminase Activity","authors":"Rameesa D. Syed Mohammed, Lianay Gutierrez Luque and Muriel C. Maurer*, ","doi":"10.1021/acs.biochem.4c0031810.1021/acs.biochem.4c00318","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00318https://doi.org/10.1021/acs.biochem.4c00318","url":null,"abstract":"A subunit of factor XIII (FXIII-A) contains a unique activation peptide (AP) that protects the catalytic triad and prevents degradation. In plasma, FXIII is activated proteolytically (FXIII-A*) by thrombin and Ca2+ cleaving AP, while in cytoplasm, it is activated nonproteolytically (FXIII-A°) with increased Ca2+ concentrations. This study aimed to elucidate the role of individual parts of the FXIII-A AP in protein stability, thrombin activation, and transglutaminase activity. Recombinant FXIII-A AP variants were expressed, and SDS-PAGE was used to monitor thrombin hydrolysis at the AP cleavage sites R37–G38. Transglutaminase activities were assessed by cross-linking lysine mimics to Fbg αC (233–425, glutamine–substrate) and monitoring reactions by mass spectrometry and in-gel fluorescence assays. FXIII-A AP variants, S19P, E23K, and D24V, degraded during purification, indicating their vital role in FXIII-A2 stability. Mutation of P36 to L36/F36 abolished the proteolytic cleavage of AP and thus prevented activation. FXIII-A N20S and P27L exhibited slower thrombin activation, likely due to the loss of key interdomain H-bonding interactions. Except N20S and P15L/P16L, all activatable FXIII-A* variants (P15L, P16L, S19A, and P27L) showed similar cross-linking activity to WT. By contrast, FXIII-A° P15L, P16L, and P15L/P16L had significantly lower cross-linking activity than FXIII-A° WT, suggesting that loss of these prolines had a greater structural impact. In conclusion, FXIII-A AP residues that play crucial roles in FXIII-A stability, activation, and activity were identified. The interactions between these AP amino acid residues and other domains control the stability and activity of FXIII.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142577548","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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

Reaction Mechanism of the Terminal Plastoquinone QB in Photosystem II as Revealed by Time-Resolved Infrared Spectroscopy 通过时间分辨红外光谱揭示光系统 II 中末端重醌 QB 的反应机制

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-16 DOI: 10.1021/acs.biochem.4c0050910.1021/acs.biochem.4c00509

Yuki Kato*, Honami Ito and Takumi Noguchi*,

{"title":"Reaction Mechanism of the Terminal Plastoquinone QB in Photosystem II as Revealed by Time-Resolved Infrared Spectroscopy","authors":"Yuki Kato*, Honami Ito and Takumi Noguchi*, ","doi":"10.1021/acs.biochem.4c0050910.1021/acs.biochem.4c00509","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00509https://doi.org/10.1021/acs.biochem.4c00509","url":null,"abstract":"The secondary plastoquinone (PQ) electron acceptor QB in photosystem II (PSII) undergoes a two-step photoreaction through electron transfer from the primary PQ electron acceptor QA, converting into plastoquinol (PQH2). However, the detailed mechanism of the QB reactions remains elusive. Here, we investigated the reaction mechanism of QB in cyanobacterial PSII core complexes using two time-revolved infrared (TRIR) methods: dispersive-type TRIR spectroscopy and rapid-scan Fourier transform infrared spectroscopy. Upon the first flash, the ∼140 μs phase is attributed to electron transfer from QA•– to QB, while the ∼2.2 and ∼440 ms phases are assigned to the binding of an internal PQ in a nearby cavity to the vacant QB site and an external PQ traveling to the QB site through channels, respectively, followed by immediate electron transfer. The resultant QB•– is suggested to be in equilibrium with QBH•, which is protonated at the distal oxygen. Upon the second flash, the ∼130 μs and ∼3.3 ms phases are attributed to electron transfer to QBH• and the protonation of QB•– followed by electron transfer, respectively, forming QBH–, which then immediately accepts a proton from D1-H215 at the proximal oxygen to become QBH2. The resultant D1-H215 anion is reprotonated in ∼22 ms via a pathway involving the bicarbonate ligand. The final ∼490 ms phase may reflect the release of PQH2 and its replacement with PQ. The present results highlight the importance of time-resolved infrared spectroscopy in elucidating the mechanism of QB reactions in PSII.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142577579","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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