Biochemical Journal最新文献_第5页

Sulfoquinovosyl diacylglycerol is required for dimerisation of the Rhodobacter sphaeroides reaction centre-light harvesting 1 core complex. 硫代喹诺酮基二酰基甘油是水发罗杆菌 RC-LH1 核心复合物二聚化所必需的。

IF 4.4 3区生物学

Biochemical Journal Pub Date : 2024-07-03 DOI: 10.1042/BCJ20240125

Elizabeth C Martin, Adam G M Bowie, Taylor Wellfare Reid, C Neil Hunter, Andrew Hitchcock, David J K Swainsbury

{"title":"Sulfoquinovosyl diacylglycerol is required for dimerisation of the Rhodobacter sphaeroides reaction centre-light harvesting 1 core complex.","authors":"Elizabeth C Martin, Adam G M Bowie, Taylor Wellfare Reid, C Neil Hunter, Andrew Hitchcock, David J K Swainsbury","doi":"10.1042/BCJ20240125","DOIUrl":"10.1042/BCJ20240125","url":null,"abstract":"The reaction centre-light harvesting 1 (RC-LH1) core complex is indispensable for anoxygenic photosynthesis. In the purple bacterium Rhodobacter (Rba.) sphaeroides RC-LH1 is produced both as a monomer, in which 14 LH1 subunits form a C-shaped antenna around 1 RC, and as a dimer, where 28 LH1 subunits form an S-shaped antenna surrounding 2 RCs. Alongside the five RC and LH1 subunits, an additional polypeptide known as PufX provides an interface for dimerisation and also prevents LH1 ring closure, introducing a channel for quinone exchange that is essential for photoheterotrophic growth. Structures of Rba. sphaeroides RC-LH1 complexes revealed several new components; protein-Y, which helps to form the quinone channel; protein-Z, of unknown function and seemingly unique to dimers; and a tightly bound sulfoquinovosyl diacylglycerol (SQDG) lipid that interacts with two PufX arginine residues. This lipid lies at the dimer interface alongside weak density for a second molecule, previously proposed to be an ornithine lipid. In this work we have generated strains of Rba. sphaeroides lacking protein-Y, protein-Z, SQDG or ornithine lipids to assess the roles of these previously unknown components in the assembly and activity of RC-LH1. We show that whilst the removal of either protein-Y, protein-Z or ornithine lipids has only subtle effects, SQDG is essential for the formation of RC-LH1 dimers but its absence has no functional effect on the monomeric complex.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"823-838"},"PeriodicalIF":4.4,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11346425/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141080435","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

Genetically engineered human embryonic kidney cells as a novel vehicle for dual patch clamp study of human gap junction channels. 基因工程人类胚胎肾细胞作为一种新型载体，用于人类缝隙连接通道的双贴片钳研究。

IF 4.4 3区生物学

Biochemical Journal Pub Date : 2024-06-19 DOI: 10.1042/BCJ20240016

Honghong Chen, Yi X Li, Robert S Wong, Jessica L Esseltine, Donglin Bai

{"title":"Genetically engineered human embryonic kidney cells as a novel vehicle for dual patch clamp study of human gap junction channels.","authors":"Honghong Chen, Yi X Li, Robert S Wong, Jessica L Esseltine, Donglin Bai","doi":"10.1042/BCJ20240016","DOIUrl":"10.1042/BCJ20240016","url":null,"abstract":"Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"741-758"},"PeriodicalIF":4.4,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11346430/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140943786","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

Cancer-associated mutations in protein kinase C theta are loss-of-function. 与癌症相关的蛋白激酶 C Theta 基因突变是功能缺失性的。

IF 4.4 3区生物学

Biochemical Journal Pub Date : 2024-06-19 DOI: 10.1042/BCJ20240148

Stefanie J Hodapp, Nathan Gravel, Natarajan Kannan, Alexandra C Newton

{"title":"Cancer-associated mutations in protein kinase C theta are loss-of-function.","authors":"Stefanie J Hodapp, Nathan Gravel, Natarajan Kannan, Alexandra C Newton","doi":"10.1042/BCJ20240148","DOIUrl":"10.1042/BCJ20240148","url":null,"abstract":"The Ca2+-independent, but diacylglycerol-regulated, novel protein kinase C (PKC) theta (θ) is highly expressed in hematopoietic cells where it participates in immune signaling and platelet function. Mounting evidence suggests that PKCθ may be involved in cancer, particularly blood cancers, breast cancer, and gastrointestinal stromal tumors, yet how to target this kinase (as an oncogene or as a tumor suppressor) has not been established. Here, we examine the effect of four cancer-associated mutations, R145H/C in the autoinhibitory pseudosubstrate, E161K in the regulatory C1A domain, and R635W in the regulatory C-terminal tail, on the cellular activity and stability of PKCθ. Live-cell imaging studies using the genetically-encoded fluorescence resonance energy transfer-based reporter for PKC activity, C kinase activity reporter 2 (CKAR2), revealed that the pseudosubstrate and C1A domain mutations impaired autoinhibition to increase basal signaling. This impaired autoinhibition resulted in decreased stability of the protein, consistent with the well-characterized behavior of Ca2+-regulated PKC isozymes wherein mutations that impair autoinhibition are paradoxically loss-of-function because the mutant protein is degraded. In marked contrast, the C-terminal tail mutation resulted in enhanced autoinhibition and enhanced stability. Thus, the examined mutations were loss-of-function by different mechanisms: mutations that impaired autoinhibition promoted the degradation of PKC, and those that enhanced autoinhibition stabilized an inactive PKC. Supporting a general loss-of-function of PKCθ in cancer, bioinformatics analysis revealed that protein levels of PKCθ are reduced in diverse cancers, including lung, renal, head and neck, and pancreatic. Our results reveal that PKCθ function is lost in cancer.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"759-775"},"PeriodicalIF":4.4,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11346454/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140943762","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

Aflatoxin biosynthesis regulators AflR and AflS: DNA binding affinity, stoichiometry, and kinetics. 黄曲霉毒素生物合成调节剂 AflR 和 AflS：DNA 结合亲和力、化学计量学和动力学。

IF 4.4 3区生物学

Biochemical Journal Pub Date : 2024-06-19 DOI: 10.1042/BCJ20240084

Asmaa Abbas, Ranjit K Prajapati, Emil Aalto-Setälä, Alexander A Baykov, Anssi M Malinen

{"title":"Aflatoxin biosynthesis regulators AflR and AflS: DNA binding affinity, stoichiometry, and kinetics.","authors":"Asmaa Abbas, Ranjit K Prajapati, Emil Aalto-Setälä, Alexander A Baykov, Anssi M Malinen","doi":"10.1042/BCJ20240084","DOIUrl":"10.1042/BCJ20240084","url":null,"abstract":"Aflatoxins (AFs), potent foodborne carcinogens produced by Aspergillus fungi, pose significant health risks worldwide and present challenges to food safety and productivity in the food chain. Novel strategies for disrupting AF production, cultivating resilient crops, and detecting contaminated food are urgently needed. Understanding the regulatory mechanisms of AF production is pivotal for targeted interventions to mitigate toxin accumulation in food and feed. The gene cluster responsible for AF biosynthesis encodes biosynthetic enzymes and pathway-specific regulators, notably AflR and AflS. While AflR, a DNA-binding protein, activates gene transcription within the cluster, AflS enhances AF production through mechanisms that are not fully understood. In this study, we developed protocols to purify recombinant AflR and AflS proteins and utilized multiple assays to characterize their interactions with DNA. Our biophysical analysis indicated that AflR and AflS form a complex. AflS exhibited no DNA-binding capability on its own but unexpectedly reduced the DNA-binding affinity of AflR. Additionally, we found that AflR achieves its binding specificity through a mechanism in which either two copies of AflR or its complex with AflS bind to target sites on DNA in a highly cooperative manner. The estimated values of the interaction parameters of AflR, AflS and DNA target sites constitute a fundamental framework against which the function and mechanisms of other AF biosynthesis regulators can be compared.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"805-821"},"PeriodicalIF":4.4,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141199059","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

Correction: The chaperonin CCT interacts with and mediates the correct folding and activity of three subunits of translation initiation factor eIF3: b, i and h. 更正：伴侣素 CCT 与翻译起始因子 eIF3 的三个亚基（b、i 和 h）相互作用，并介导其正确折叠和活性。

IF 4.1 3区生物学

Biochemical Journal Pub Date : 2024-06-19 DOI: 10.1042/BJ20130979_COR

Anne Roobol, Jo Roobol, Martin J Carden, Matthew E Smith, John W B Hershey, Amandine Bastide, John R P Knight, Anne E Willis, C Mark Smales

引用次数: 0

RNA processing by the CRISPR-associated NYN ribonuclease. CRISPR 相关 NYN 核糖核酸酶对 RNA 的处理。

IF 4.4 3区生物学

Biochemical Journal Pub Date : 2024-06-19 DOI: 10.1042/BCJ20240151

Haotian Chi, Malcolm F White

{"title":"RNA processing by the CRISPR-associated NYN ribonuclease.","authors":"Haotian Chi, Malcolm F White","doi":"10.1042/BCJ20240151","DOIUrl":"10.1042/BCJ20240151","url":null,"abstract":"CRISPR-Cas systems confer adaptive immunity in prokaryotes, facilitating the recognition and destruction of invasive nucleic acids. Type III CRISPR systems comprise large, multisubunit ribonucleoprotein complexes with a catalytic Cas10 subunit. When activated by the detection of foreign RNA, Cas10 generates nucleotide signalling molecules that elicit an immune response by activating ancillary effector proteins. Among these systems, the Bacteroides fragilis type III CRISPR system was recently shown to produce a novel signal molecule, SAM-AMP, by conjugating ATP and SAM. SAM-AMP regulates a membrane effector of the CorA family to provide immunity. Here, we focus on NYN, a ribonuclease encoded within this system, probing its potential involvement in crRNA maturation. Structural modelling and in vitro ribonuclease assays reveal that NYN displays robust sequence-nonspecific, Mn2+-dependent ssRNA-cleavage activity. Our findings suggest a role for NYN in trimming crRNA intermediates into mature crRNAs, which is necessary for type III CRISPR antiviral defence. This study sheds light on the functional relevance of CRISPR-associated NYN proteins and highlights the complexity of CRISPR-mediated defence strategies in bacteria.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"793-804"},"PeriodicalIF":4.4,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11346440/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141086703","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

Mechanisms and pathologies of human mitochondrial DNA replication and deletion formation. 人类线粒体 DNA 复制和缺失形成的机制和病理。

IF 4.4 3区生物学

Biochemical Journal Pub Date : 2024-06-05 DOI: 10.1042/BCJ20230262

Tiago M Bernardino Gomes, Amy E Vincent, Katja E Menger, James B Stewart, Thomas J Nicholls

{"title":"Mechanisms and pathologies of human mitochondrial DNA replication and deletion formation.","authors":"Tiago M Bernardino Gomes, Amy E Vincent, Katja E Menger, James B Stewart, Thomas J Nicholls","doi":"10.1042/BCJ20230262","DOIUrl":"10.1042/BCJ20230262","url":null,"abstract":"Human mitochondria possess a multi-copy circular genome, mitochondrial DNA (mtDNA), that is essential for cellular energy metabolism. The number of copies of mtDNA per cell, and their integrity, are maintained by nuclear-encoded mtDNA replication and repair machineries. Aberrant mtDNA replication and mtDNA breakage are believed to cause deletions within mtDNA. The genomic location and breakpoint sequences of these deletions show similar patterns across various inherited and acquired diseases, and are also observed during normal ageing, suggesting a common mechanism of deletion formation. However, an ongoing debate over the mechanism by which mtDNA replicates has made it difficult to develop clear and testable models for how mtDNA rearrangements arise and propagate at a molecular and cellular level. These deletions may impair energy metabolism if present in a high proportion of the mtDNA copies within the cell, and can be seen in primary mitochondrial diseases, either in sporadic cases or caused by autosomal variants in nuclear-encoded mtDNA maintenance genes. These mitochondrial diseases have diverse genetic causes and multiple modes of inheritance, and show notoriously broad clinical heterogeneity with complex tissue specificities, which further makes establishing genotype-phenotype relationships challenging. In this review, we aim to cover our current understanding of how the human mitochondrial genome is replicated, the mechanisms by which mtDNA replication and repair can lead to mtDNA instability in the form of large-scale rearrangements, how rearranged mtDNAs subsequently accumulate within cells, and the pathological consequences when this occurs.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"481 11","pages":"683-715"},"PeriodicalIF":4.4,"publicationDate":"2024-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11346376/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141157280","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

The monodomain Kunitz protein EgKU-7 from the dog tapeworm Echinococcus granulosus is a high-affinity trypsin inhibitor with two interaction sites. 来自犬带绦虫棘球蚴的单链库尼茨蛋白 EgKU-7 是一种高亲和力胰蛋白酶抑制剂，具有两个相互作用位点。

IF 4.4 3区生物学

Biochemical Journal Pub Date : 2024-06-05 DOI: 10.1042/BCJ20230514

Martín Fló, Leonardo Pellizza, Rosario Durán, Beatriz Alvarez, Cecilia Fernández

{"title":"The monodomain Kunitz protein EgKU-7 from the dog tapeworm Echinococcus granulosus is a high-affinity trypsin inhibitor with two interaction sites.","authors":"Martín Fló, Leonardo Pellizza, Rosario Durán, Beatriz Alvarez, Cecilia Fernández","doi":"10.1042/BCJ20230514","DOIUrl":"10.1042/BCJ20230514","url":null,"abstract":"Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of ∼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"717-739"},"PeriodicalIF":4.4,"publicationDate":"2024-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140943788","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

Efficient overexpression and purification of severe acute respiratory syndrome coronavirus 2 nucleocapsid proteins in Escherichia coli. 在大肠杆菌中高效过表达和纯化 SARS-CoV-2 核壳蛋白。

IF 4.4 3区生物学

Biochemical Journal Pub Date : 2024-06-05 DOI: 10.1042/BCJ20240019

Emma L Brudenell, Manoj B Pohare, Domen Zafred, Janine Phipps, Hailey R Hornsby, John F Darby, Junxiao Dai, Ellen Liggett, Kathleen M Cain, Perdita E Barran, Thushan I de Silva, Jon R Sayers

{"title":"Efficient overexpression and purification of severe acute respiratory syndrome coronavirus 2 nucleocapsid proteins in Escherichia coli.","authors":"Emma L Brudenell, Manoj B Pohare, Domen Zafred, Janine Phipps, Hailey R Hornsby, John F Darby, Junxiao Dai, Ellen Liggett, Kathleen M Cain, Perdita E Barran, Thushan I de Silva, Jon R Sayers","doi":"10.1042/BCJ20240019","DOIUrl":"10.1042/BCJ20240019","url":null,"abstract":"The fundamental biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"669-682"},"PeriodicalIF":4.4,"publicationDate":"2024-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11346444/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140847091","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

Validation of GCN5L1/BLOC1S1/BLOS1 antibodies using knockout cells and tissue. 利用基因敲除细胞和组织验证 GCN5L1/BLOC1S1/BLOS1 抗体。

IF 4.4 3区生物学

Biochemical Journal Pub Date : 2024-05-22 DOI: 10.1042/BCJ20230302

Paramesha Bugga, Michael W Stoner, Janet R Manning, Bellina A S Mushala, Nisha Bhattarai, Maryam Sharifi-Sanjani, Bradley R Webster, Dharendra Thapa, Iain Scott

引用次数: 0