Biochemistry Biochemistry最新文献_第4页

Redox Homeostasis within the Drug-Resistant Malarial Parasite Digestive Vacuole. 耐药疟原虫消化液泡内的氧化还原稳态。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-05-20 Epub Date: 2025-05-01 DOI: 10.1021/acs.biochem.4c00750

Andreas Willems, Therese Oertel, Paul D Roepe

{"title":"Redox Homeostasis within the Drug-Resistant Malarial Parasite Digestive Vacuole.","authors":"Andreas Willems, Therese Oertel, Paul D Roepe","doi":"10.1021/acs.biochem.4c00750","DOIUrl":"10.1021/acs.biochem.4c00750","url":null,"abstract":"We have developed a cost-effective strategy for the complete synthesis of azetidinyl coumarin fluorophore derivatives that report changes in physiologic levels of glutathione (GSH), which includes a more cost- effective synthesis of the probe precursor hydroxyl derivative and its subsequent derivatization to promote subcellular localization. We functionalize coumarin derivatives with a cyano side chain similar to a previous strategy (Jiang X. et al., Nature Communications 2017, 8; 16087) and validate the 7-azetidinyl conformation as an explanation for enhanced GSH-dependent coumarin fluorescence. We couple the azetidinyl probe to different mass dextrans using either no linker or a 6C linker and also synthesize a morpholino derivative. We titrate the fluorescence of the different functionalized probes vs [GSH] in vitro. We load one dextran-conjugated probe within the digestive vacuole (DV) of live intraerythrocytic P. falciparum malarial parasites and also measure cytosolic localization of the morpholino probe. Using significantly improved single-cell photometry (SCP) methods, we show that the morpholino probe faithfully reports [GSH] from the live parasite cytosol, while the 70 kDa dextran-conjugated probe reports DV redox homeostasis for control chloroquine-sensitive (CQS) and artemisinin-sensitive (ARTS) transfectant parasites vs their genetically matched chloroquine-resistant (CQR)/artemisinin-sensitive (CQR/ARTS) and CQR artemisinin-resistant (CQR/ARTR) strains, respectively. We quantify rapid changes in DV redox homeostasis for these parasites ± drug pulses under live-cell perfusion conditions. The results are important for understanding the pharmacology of antimalarial drugs and the molecular mechanisms underlying CQR and ARTR phenomena.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"2247-2261"},"PeriodicalIF":2.9,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12096432/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143953400","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

Identification and Functional Characterization of the Polymerizing Glycosyltransferase Required for the Transfer of d-Ribose to the d-GalfNAc Moiety of the Capsular Polysaccharide of Campylobacter jejuni. 空肠弯曲杆菌荚膜多糖d-核糖向d-GalfNAc部分转移所需的聚合糖基转移酶的鉴定和功能表征

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-05-20 Epub Date: 2025-05-01 DOI: 10.1021/acs.biochem.5c00052

Dao Feng Xiang, Tamari Narindoshvili, Frank M Raushel

{"title":"Identification and Functional Characterization of the Polymerizing Glycosyltransferase Required for the Transfer of d-Ribose to the d-GalfNAc Moiety of the Capsular Polysaccharide of Campylobacter jejuni.","authors":"Dao Feng Xiang, Tamari Narindoshvili, Frank M Raushel","doi":"10.1021/acs.biochem.5c00052","DOIUrl":"10.1021/acs.biochem.5c00052","url":null,"abstract":"Campylobacter jejuni is the leading cause of food poisoning in the United States. The exterior surface of this bacterium is coated with a capsular polysaccharide (CPS) that helps protect the organism from the host immune system. In the HS:2 serotype of strain C. jejuni NCTC 11168, the minimal repeating trisaccharide consist of d-ribose, N-acetyl-d-galactosamine (GalNAc) and the serinol amide of d-glucuronic acid. Here we demonstrate that the C-terminal domain of Cj1432 (residues 574-914) is responsible for the transfer of d-ribose-5-P from phosphoribosyl pyrophosphate (PRPP) to C5 of the d-GalfNAc moiety of the growing polysaccharide chain. In the next step the middle domain of Cj1432 (residues 357-573) catalyzes the hydrolysis of phosphate from this product. The N-terminal domain of Cj1432 (residues 1-356) catalyzes the transfer of d-GlcA from UDP-d-GlcA to C2 of the d-ribose moiety and thus Cj1432 catalyzes three consecutive reactions during the biosynthesis of the capsular polysaccharide of C. jejuni. We have previously shown that the remaining three reactions required for the polymerization of the CPS are catalyzed by the bifunctional enzyme Cj1438 and Cj1435. We have now demonstrated that the minimal repeating trisaccharide of the CPS of C. jejuni NCTC 11168 requires six enzyme-catalyzed reactions with six intermediate structures. This accomplishment will now enable the large-scale cell-free enzyme-catalyzed synthesis of well-defined oligomers of the CPS that can potentially be used in the production of glycoconjugate vaccines for the prevention of infections by C. jejuni.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"2226-2236"},"PeriodicalIF":2.9,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12096443/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143955680","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

An Alkaliphilic Chitinase Unveils Environment-Dependent Variation in the Canonical Catalytic Machinery of Family-18 Glycoside Hydrolases. 一种亲碱几丁质酶揭示了18家族糖苷水解酶典型催化机制的环境依赖性变异。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-05-20 Epub Date: 2025-05-02 DOI: 10.1021/acs.biochem.5c00082

Jogi Madhuprakash, Bjørn Dalhus, Bastien Bissaro, Lal Duhsaki, Gustav Vaaje-Kolstad, Morten Sørlie, Åsmund K Røhr, Vincent G H Eijsink

{"title":"An Alkaliphilic Chitinase Unveils Environment-Dependent Variation in the Canonical Catalytic Machinery of Family-18 Glycoside Hydrolases.","authors":"Jogi Madhuprakash, Bjørn Dalhus, Bastien Bissaro, Lal Duhsaki, Gustav Vaaje-Kolstad, Morten Sørlie, Åsmund K Røhr, Vincent G H Eijsink","doi":"10.1021/acs.biochem.5c00082","DOIUrl":"10.1021/acs.biochem.5c00082","url":null,"abstract":"Chitinases belonging to glycoside hydrolase family-18 (GH18) employ substrate-assisted catalysis and typically have neutral/acidic pH-optima. We describe the structural and functional analysis of CaChiA, a chitinase from the anaerobic alkaliphilic bacterium Chitinivibrio alkaliphilus with an alkaline pH optimum (8.8) and unique active site features, including a noncanonical catalytic HxxExDxE motif, which is DxxDxDxE in other chitinases. Propka calculations indicated a significantly higher pKa for the catalytic acid/base, Glu148, in CaGH18, compared to other GH18 enzymes, aligning with its alkaline pH optimum. Both Propka calculations and functional studies of enzyme variants with mutations in the catalytic center suggested that not the change in the catalytic motif, but rather a unique glutamine, Gln57, modulating the properties of this motif, enables activity at alkaline pH. Further characterization of CaChiA unveiled additional peculiar enzyme properties, such as a unique ability to convert chitin to chitotriose. Thus, CaChiA adds novel catalytic capabilities to the widespread family of GH18 chitinases, made possible by adaptation of an intricate catalytic center.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"2291-2305"},"PeriodicalIF":2.9,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143954047","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

Emergent and Convergent Features in the Laboratory Evolution of Polymerase Ribozymes

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-05-19 DOI: 10.1021/acs.biochem.5c0016010.1021/acs.biochem.5c00160

David P. Horning*,

{"title":"Emergent and Convergent Features in the Laboratory Evolution of Polymerase Ribozymes","authors":"David P. Horning*, ","doi":"10.1021/acs.biochem.5c0016010.1021/acs.biochem.5c00160","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00160https://doi.org/10.1021/acs.biochem.5c00160","url":null,"abstract":"In modern biology, molecular heredity is established by polymerase proteins that copy genetic information encoded in the sequence of nucleic acids. Prior to the emergence of coded protein synthesis, this role may have been filled by RNA polymerase ribozymes. Although such enzymes can no longer be found in extant life, ribozymes first evolved from random sequence populations have been progressively engineered in the laboratory to function as general RNA-dependent RNA polymerases. Polymerase ribozymes discovered in the past ten years can catalyze hundreds of sequential RNA synthesis reactions, match the complexity and catalytic sophistication of biological RNA enzymes, and employ many of the same strategies used by polymerase proteins to copy nucleic acids. This review describes the approaches to directed in vitro evolution that have led to the discovery of RNA enzymes that copy RNA molecules processively and accurately, and surveys how laboratory evolution has shaped biochemical and structural adaptations in these enzymes. The review then considers the challenges and opportunities that remain in the effort to propagate and evolve RNA genes with RNA catalysts alone.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 11","pages":"2364–2375 2364–2375"},"PeriodicalIF":2.9,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144194231","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

Emergent and Convergent Features in the Laboratory Evolution of Polymerase Ribozymes. 聚合酶核酶实验室进化中的突现与趋同特征。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-05-19 DOI: 10.1021/acs.biochem.5c00160

David P Horning

{"title":"Emergent and Convergent Features in the Laboratory Evolution of Polymerase Ribozymes.","authors":"David P Horning","doi":"10.1021/acs.biochem.5c00160","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00160","url":null,"abstract":"In modern biology, molecular heredity is established by polymerase proteins that copy genetic information encoded in the sequence of nucleic acids. Prior to the emergence of coded protein synthesis, this role may have been filled by RNA polymerase ribozymes. Although such enzymes can no longer be found in extant life, ribozymes first evolved from random sequence populations have been progressively engineered in the laboratory to function as general RNA-dependent RNA polymerases. Polymerase ribozymes discovered in the past ten years can catalyze hundreds of sequential RNA synthesis reactions, match the complexity and catalytic sophistication of biological RNA enzymes, and employ many of the same strategies used by polymerase proteins to copy nucleic acids. This review describes the approaches to directed in vitro evolution that have led to the discovery of RNA enzymes that copy RNA molecules processively and accurately, and surveys how laboratory evolution has shaped biochemical and structural adaptations in these enzymes. The review then considers the challenges and opportunities that remain in the effort to propagate and evolve RNA genes with RNA catalysts alone.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144100967","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

Predicting Protein Function in the AI and Big Data Era. 人工智能和大数据时代的蛋白质功能预测。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-05-17 DOI: 10.1021/acs.biochem.5c00186

Riccardo Percudani, Carlo De Rito

引用次数: 0

Predicting Protein Function in the AI and Big Data Era

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-05-17 DOI: 10.1021/acs.biochem.5c0018610.1021/acs.biochem.5c00186

Riccardo Percudani*, and , Carlo De Rito,

引用次数: 0

Mu Opioid Receptor Positive Allosteric Modulator BMS-986122 Confers Agonist-Dependent G Protein Subtype Signaling Bias. Mu阿片受体阳性变构调节剂BMS-986122引起激动剂依赖的G蛋白亚型信号偏倚。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-05-16 DOI: 10.1021/acs.biochem.5c00022

Grant M Grieble, Brian I Knapp, Jean M Bidlack

{"title":"Mu Opioid Receptor Positive Allosteric Modulator BMS-986122 Confers Agonist-Dependent G Protein Subtype Signaling Bias.","authors":"Grant M Grieble, Brian I Knapp, Jean M Bidlack","doi":"10.1021/acs.biochem.5c00022","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00022","url":null,"abstract":"The mu opioid receptor (MOR) is a G protein-coupled receptor (GPCR) and is responsible for the effects of all medically used opioids. Most opioids activate all inhibitory Gαi/o/z proteins through MOR, initiating signaling events that culminate in a variety of physiological effects such as analgesia, euphoria, and respiratory depression. Gaining a better understanding of how the chemical structure of opioids influences the functional activation profiles of G protein subtypes by MOR is critical for disentangling the multitude of opioid effects and the development of safer analgesics. A recent development in opioid pharmacology has been the discovery of positive allosteric modulators (PAMs) for opioid receptors, such as BMS-986122, which act at the MOR to increase the potency of full agonists and the efficacy of partial agonists. Here, we utilized a nanoBRET-based functional assay system in live HEK 293T cells to study how the pharmacological properties of opioids were uniquely affected by BMS-986122 when the MOR signaled through specific inhibitory Gα subunits. We report that BMS-986122 differentially enhanced opioid activity when the MOR signaled through different Gα subunits with the greatest difference observed with partial agonists. Additionally, the binding affinity of BMS-986122 to the MOR was significantly altered by the co-binding Gα subunit. Site-directed mutagenesis experiments revealed key amino acid residue differences on Gαi/o subunits involved in the differential effects observed. This study sheds light on the molecular features of biased signaling for both opioid ligands and G proteins, which may prove useful for the further development of biased agonists or allosteric modulators at the MOR.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2025-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144074908","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

Mu Opioid Receptor Positive Allosteric Modulator BMS-986122 Confers Agonist-Dependent G Protein Subtype Signaling Bias

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-05-16 DOI: 10.1021/acs.biochem.5c0002210.1021/acs.biochem.5c00022

Grant M. Grieble, Brian I. Knapp and Jean M. Bidlack*,

{"title":"Mu Opioid Receptor Positive Allosteric Modulator BMS-986122 Confers Agonist-Dependent G Protein Subtype Signaling Bias","authors":"Grant M. Grieble, Brian I. Knapp and Jean M. Bidlack*, ","doi":"10.1021/acs.biochem.5c0002210.1021/acs.biochem.5c00022","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00022https://doi.org/10.1021/acs.biochem.5c00022","url":null,"abstract":"The mu opioid receptor (MOR) is a G protein-coupled receptor (GPCR) and is responsible for the effects of all medically used opioids. Most opioids activate all inhibitory Gαi/o/z proteins through MOR, initiating signaling events that culminate in a variety of physiological effects such as analgesia, euphoria, and respiratory depression. Gaining a better understanding of how the chemical structure of opioids influences the functional activation profiles of G protein subtypes by MOR is critical for disentangling the multitude of opioid effects and the development of safer analgesics. A recent development in opioid pharmacology has been the discovery of positive allosteric modulators (PAMs) for opioid receptors, such as BMS-986122, which act at the MOR to increase the potency of full agonists and the efficacy of partial agonists. Here, we utilized a nanoBRET-based functional assay system in live HEK 293T cells to study how the pharmacological properties of opioids were uniquely affected by BMS-986122 when the MOR signaled through specific inhibitory Gα subunits. We report that BMS-986122 differentially enhanced opioid activity when the MOR signaled through different Gα subunits with the greatest difference observed with partial agonists. Additionally, the binding affinity of BMS-986122 to the MOR was significantly altered by the co-binding Gα subunit. Site-directed mutagenesis experiments revealed key amino acid residue differences on Gαi/o subunits involved in the differential effects observed. This study sheds light on the molecular features of biased signaling for both opioid ligands and G proteins, which may prove useful for the further development of biased agonists or allosteric modulators at the MOR.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 11","pages":"2376–2393 2376–2393"},"PeriodicalIF":2.9,"publicationDate":"2025-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.5c00022","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144194279","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

Expanding the Riboglow-FLIM Toolbox with Different Fluorescence Lifetime-Producing RNA Tags. 扩展具有不同荧光寿命产生RNA标签的Riboglow-FLIM工具箱。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2025-05-15 DOI: 10.1021/acs.biochem.4c00567

Zachary Stickelman, Nadia Sarfraz, Morgan K Rice, Ben J Lambeck, Sonja Milkovich, Esther Braselmann

{"title":"Expanding the Riboglow-FLIM Toolbox with Different Fluorescence Lifetime-Producing RNA Tags.","authors":"Zachary Stickelman, Nadia Sarfraz, Morgan K Rice, Ben J Lambeck, Sonja Milkovich, Esther Braselmann","doi":"10.1021/acs.biochem.4c00567","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00567","url":null,"abstract":"RNAs are essential elements of biology with subcellular localizations critical for function. Genetically tagging fluorescent RNA reporters to an RNA of interest allows for investigating RNA spatiotemporal dynamics. We previously developed a fluorescence lifetime imaging microscopy (FLIM)-based RNA-tagging platform, Riboglow-FLIM. Here, a genetically encoded RNA tag binds a fluorescent probe, causing an increase in both fluorescence intensity and fluorescence lifetime. Importantly, the Riboglow platform is derived from a bacterial riboswitch RNA family and different riboswitch sequences from nature may build the basis for multiplexing capabilities. We previously observed fluorescence lifetime differences for two RNA tags in vitro and in live mammalian cells as a proof-of-concept demonstration. As an in-depth expansion for multiplexing capabilities, here we evaluate the performance of different RNA sequences in vitro for systematically expanding the RNA tag sequence space of Riboglow-FLIM. We use two methods of varying the genetic tag to evaluate multiplexing capabilities, a literature-guided and a rational design approach. The literature-guided approach includes riboswitch sequences with both indirect and direct evidence of probe binding. For this, a phylogenetic tree of riboswitch-derived tags from indirect binding results was constructed, and RNA members from different branches were characterized. We also designed RNA mutations rationally based on insights from established Riboglow RNA tags. Together, nine different RNA tags yielded a wide range of fluorescence lifetimes for the Riboglow-FLIM platform, building the foundation to tag and track several different RNAs simultaneously. These findings will serve as the basis for achieving multiplexed RNA imaging in live cells using a fluorescence lifetime sensor.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144074903","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