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Interaction and cleavage of cell and plasma proteins by the platelet-aggregating serine protease PA-BJ of Bothrops jararaca venom.
Pub Date : 2025-01-27 DOI: 10.1016/j.biochi.2025.01.007
Daniela Cajado-Carvalho, Mariana S L C Morone, Nancy da Rós, Solange M T Serrano

PA-BJ is a serine protease present in Bothrops jararaca venom that triggers platelet aggregation and granule secretion by activating the protease-activated receptors PAR-1 and PAR-4, without clotting fibrinogen. These receptors also have a relevant role in endothelial cells, however, the interaction of PA-BJ with other membrane-bound or soluble targets is not known. Here we explored the activity of PA-BJ on endothelial cell receptor, cytoskeleton, and coagulation proteins in vitro, and show the degradation of fibrinogen and protein C, and the limited proteolysis of actin, EPCR, PAR-1, and thrombomodulin. Antithrombin, factors XI and XIII and protein S were not cleaved by PA-BJ. Moreover, using surface plasmon resonance PA-BJ was demonstrated to bind to actin, EPCR, fibrinogen, PAR-1, and thrombomodulin, with dissociation constants (KD) in the micromolar range. Considering that these proteins play critical roles in pathways of blood coagulation and maintenance of endothelium integrity, their binding and cleavage by PA-BJ could contribute to the alterations in hemostasis and cell permeability observed in B. jararaca envenomation process.

{"title":"Interaction and cleavage of cell and plasma proteins by the platelet-aggregating serine protease PA-BJ of Bothrops jararaca venom.","authors":"Daniela Cajado-Carvalho, Mariana S L C Morone, Nancy da Rós, Solange M T Serrano","doi":"10.1016/j.biochi.2025.01.007","DOIUrl":"10.1016/j.biochi.2025.01.007","url":null,"abstract":"<p><p>PA-BJ is a serine protease present in Bothrops jararaca venom that triggers platelet aggregation and granule secretion by activating the protease-activated receptors PAR-1 and PAR-4, without clotting fibrinogen. These receptors also have a relevant role in endothelial cells, however, the interaction of PA-BJ with other membrane-bound or soluble targets is not known. Here we explored the activity of PA-BJ on endothelial cell receptor, cytoskeleton, and coagulation proteins in vitro, and show the degradation of fibrinogen and protein C, and the limited proteolysis of actin, EPCR, PAR-1, and thrombomodulin. Antithrombin, factors XI and XIII and protein S were not cleaved by PA-BJ. Moreover, using surface plasmon resonance PA-BJ was demonstrated to bind to actin, EPCR, fibrinogen, PAR-1, and thrombomodulin, with dissociation constants (K<sub>D</sub>) in the micromolar range. Considering that these proteins play critical roles in pathways of blood coagulation and maintenance of endothelium integrity, their binding and cleavage by PA-BJ could contribute to the alterations in hemostasis and cell permeability observed in B. jararaca envenomation process.</p>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143069804","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
The modulation of low molecular weight sulfur compounds levels in visceral adipose tissue of TLR2-deficient mice on a high-fat diet.
Pub Date : 2025-01-25 DOI: 10.1016/j.biochi.2025.01.008
Patrycja Bronowicka-Adamska, Dominika Szlęzak, Anna Bentke-Imiolek, Kinga Kaszuba, Monika Majewska-Szczepanik

Obesity treatment requires an individualized approach, emphasizing the need to identify metabolic pathways of diagnostic relevance. Toll-like receptors (TLRs), particularly TLR2 and TLR4, play a crucial role in metabolic disorders, as receptor deficiencies improves insulin sensitivity and reduces obesity-related inflammation. Additionally, hydrogen sulfide (H2S) influences lipolysis, adipogenesis, and adipose tissue browning through persulfidation. This study investigates the impact of a high-fat diet (HFD) on low molecular weight sulfur compounds in the visceral adipose tissue (VAT) of C57BL/6 and TLR2-deficient mice. It focuses on key enzymes involved in H2S metabolism: cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CGL), 3-mercaptopyruvate sulfurtransferase (MPST), and thiosulfate sulfurtransferase (TST). In C57BL/6 mice on HFD, MPST activity decreased, while CBS level increased, potentially compensating for H2S production. In contrast, TLR2-deficient mice on HFD exhibited higher MPST activity but reduced level of CBS and CGL activity, suggesting that TLR2 deficiency mitigates HFD-induced changes in sulfur metabolism. TST activity was lower in TLR2-deficient mice, indicating an independent regulatory role of TLR2 in TST activity. Elevated oxidative stress, reflected by increased glutathione levels, was observed in wild-type mice. Interestingly, cysteine and cystine were detectable only in the VAT of the C57BL/6 ND group and were absent in all other groups. The capacity for hydrogen sulfide production in tissues from TLR2-/-B6 HFD group was significantly lower than in the C57BL/6 HFD group. In conclusion, TLR2 modulates sulfur metabolism, oxidative stress, and inflammation in obesity. TLR2 deficiency disrupts H2S production and redox balance, potentially contributing to metabolic dysfunction, highlighting TLR2 as a potential therapeutic target for obesity-related metabolic disorders.

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引用次数: 0
Neutrophils and purinergic signaling: Partners in the crime against Leishmania parasites?
Pub Date : 2025-01-23 DOI: 10.1016/j.biochi.2025.01.004
Mariana M Chaves

The parasite of the genus Leishmania is the causative agent of diseases that affect humans called leishmaniasis. These diseases affect millions of people worldwide and the currently existing drugs are either very toxic or the parasites acquire resistance. Therefore, new elimination mechanisms need to be elucidated so that new therapeutic strategies can be developed. Much has already been discussed about the role of neutrophils in Leishmania infection, and their participation is still controversial. A recent study showed that receptors present in the neutrophil membrane, the purinergic receptors, can control the infection when activated, but the triggering mechanism has not been elucidated. In this review, we will address the possible participation of purinergic receptors expressed in the neutrophil extracellular membrane that may be participating in the detection of Leishmania infection and their possible effects during parasitism.

{"title":"Neutrophils and purinergic signaling: Partners in the crime against Leishmania parasites?","authors":"Mariana M Chaves","doi":"10.1016/j.biochi.2025.01.004","DOIUrl":"10.1016/j.biochi.2025.01.004","url":null,"abstract":"<p><p>The parasite of the genus Leishmania is the causative agent of diseases that affect humans called leishmaniasis. These diseases affect millions of people worldwide and the currently existing drugs are either very toxic or the parasites acquire resistance. Therefore, new elimination mechanisms need to be elucidated so that new therapeutic strategies can be developed. Much has already been discussed about the role of neutrophils in Leishmania infection, and their participation is still controversial. A recent study showed that receptors present in the neutrophil membrane, the purinergic receptors, can control the infection when activated, but the triggering mechanism has not been elucidated. In this review, we will address the possible participation of purinergic receptors expressed in the neutrophil extracellular membrane that may be participating in the detection of Leishmania infection and their possible effects during parasitism.</p>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143043750","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Macrolactin a is an inhibitor of protein biosynthesis in bacteria. 大泌乳素A是细菌中蛋白质生物合成的抑制剂。
Pub Date : 2025-01-16 DOI: 10.1016/j.biochi.2025.01.003
Alexey S Vasilchenko, Dmitry A Lukyanov, Diana S Dilbaryan, Konstantin S Usachev, Darya V Poshvina, Amir Kh Taldaev, Arina A Nikandrova, Arina N Imamutdinova, Natalia S Garaeva, Aydar G Bikmullin, Evelina A Klochkova, Alexander L Rusanov, Daniil D Romashin, Natalia G Luzgina, Ilya A Osterman, Petr V Sergiev, Anastasia V Teslya

Macrolactin A (McA) is a secondary metabolite produced by Bacillus species. It has been known for its antimicrobial properties since the late 1980s, although the exact mechanism of its antibacterial activity remains unknown. In this study, we have found that McA is an inhibitor of protein synthesis in bacteria. Our conclusion is based on the results obtained by in vivo and in vitro bioreporter systems. We demonstrated that the inhibitory activity of McA is independent of bacterial species. However, the concentration of McA required to inhibit protein synthesis in the E. coli cell-free translational model was found to be 50 times lower than the concentration required in the S. aureus cell-free translational model. To investigate the mechanism of McA's inhibitory activity, we conducted a toe-printing assay, sequenced and annotated the genomes of McA-resistant Bacillus pumilus McAR and its parental strain. The results showed that McA inhibits the initial step of the elongation phase of protein synthesis. We identified single and multiple nucleotide polymorphisms in the gene encoding the translation elongation factor Tu (EF-Tu). Molecular modeling showed that the McA molecule can form non-covalent bonds with amino acids at the interface of domains 1 and 2 of EF-Tu. A cross-resistance assay was conducted using kirromycin on B. pumilus McAR. The results confirmed the assumption that McA has a mode of action similar to that of other elfamycin-like antibiotics (targeting EF-Tu). Overall, our study addresses a significant gap in our understanding of the mechanism of action of McA, a representative member of the macrolide antibiotics.

大泌乳素A (McA)是芽孢杆菌产生的次生代谢物。自20世纪80年代末以来,它一直以其抗菌特性而闻名,尽管其抗菌活性的确切机制尚不清楚。在这项研究中,我们发现McA是细菌中蛋白质合成的抑制剂。我们的结论是基于体内和体外生物报告系统获得的结果。我们证明了McA的抑制活性与细菌种类无关。然而,在大肠杆菌无细胞翻译模型中,抑制蛋白质合成所需的McA浓度比金黄色葡萄球菌无细胞翻译模型中所需的浓度低50倍。为了研究McA抑制活性的机制,我们进行了足印试验,对耐McA的短小芽孢杆菌McAR及其亲本菌株进行了基因组测序和注释。结果表明,McA抑制了蛋白质合成延伸阶段的第一步。我们鉴定了翻译延伸因子Tu (EF-Tu)基因的单核苷酸和多核苷酸多态性。分子模拟表明,McA分子可以在EF-Tu结构域1和2的界面上与氨基酸形成非共价键。用克罗霉素对小分枝杆菌mccar进行了交叉耐药试验。结果证实了McA的作用模式类似于其他elfamycin类抗生素(靶向EF-Tu)的假设。总的来说,我们的研究解决了我们对大环内酯家族的代表成员McA的作用机制的理解上的重大差距。
{"title":"Macrolactin a is an inhibitor of protein biosynthesis in bacteria.","authors":"Alexey S Vasilchenko, Dmitry A Lukyanov, Diana S Dilbaryan, Konstantin S Usachev, Darya V Poshvina, Amir Kh Taldaev, Arina A Nikandrova, Arina N Imamutdinova, Natalia S Garaeva, Aydar G Bikmullin, Evelina A Klochkova, Alexander L Rusanov, Daniil D Romashin, Natalia G Luzgina, Ilya A Osterman, Petr V Sergiev, Anastasia V Teslya","doi":"10.1016/j.biochi.2025.01.003","DOIUrl":"10.1016/j.biochi.2025.01.003","url":null,"abstract":"<p><p>Macrolactin A (McA) is a secondary metabolite produced by Bacillus species. It has been known for its antimicrobial properties since the late 1980s, although the exact mechanism of its antibacterial activity remains unknown. In this study, we have found that McA is an inhibitor of protein synthesis in bacteria. Our conclusion is based on the results obtained by in vivo and in vitro bioreporter systems. We demonstrated that the inhibitory activity of McA is independent of bacterial species. However, the concentration of McA required to inhibit protein synthesis in the E. coli cell-free translational model was found to be 50 times lower than the concentration required in the S. aureus cell-free translational model. To investigate the mechanism of McA's inhibitory activity, we conducted a toe-printing assay, sequenced and annotated the genomes of McA-resistant Bacillus pumilus McA<sup>R</sup> and its parental strain. The results showed that McA inhibits the initial step of the elongation phase of protein synthesis. We identified single and multiple nucleotide polymorphisms in the gene encoding the translation elongation factor Tu (EF-Tu). Molecular modeling showed that the McA molecule can form non-covalent bonds with amino acids at the interface of domains 1 and 2 of EF-Tu. A cross-resistance assay was conducted using kirromycin on B. pumilus McA<sup>R</sup>. The results confirmed the assumption that McA has a mode of action similar to that of other elfamycin-like antibiotics (targeting EF-Tu). Overall, our study addresses a significant gap in our understanding of the mechanism of action of McA, a representative member of the macrolide antibiotics.</p>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143018181","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Redesigning methionine γ-lyase for improved stability and catalytic activity in the β-elimination reaction for the synthesis of thiosulfinates. 重新设计蛋氨酸γ-裂解酶,提高β-消除反应合成硫代亚硫酸盐的稳定性和催化活性。
Pub Date : 2025-01-15 DOI: 10.1016/j.biochi.2025.01.002
Vitalia Kulikova, Natalya Anufrieva, Elena Morozova, Kseniya Levshina, Svetlana Revtovich, Pavel Solyev

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are involved in many cellular processes and possess unequalled catalytic versatility. Rational design through site-directed mutagenesis is a powerful strategy for creating tailor-made enzymes for a wide range of biocatalytic applications. PLP-dependent methionine γ-lyase (MGL), which degrades sulfur-containing amino acids, is an encouraging enzyme for many therapeutic purposes - from combating bacterial resistant strains and fungi to antitumor activity. A two-component biosystem MGL/S-alk(en)yl-l-cysteine sulfoxide (an uncommon substrate for this enzyme) produces antimicrobial thiosulfinates during the β-elimination reaction. SH-groups of the enzyme are modified by the products of this reaction, which leads to the inactivation of the enzyme. Successful and efficient rational stabilization of MGL from Clostridium novyi can be achieved using site-directed mutagenesis. We have managed to obtain an improved version of the enzyme better than the natural one regarding the β-elimination reaction. Cys118, Cys184 and Cys273 of MGL from Clostridium novyi were substituted by His and two Ala, respectively. The resulting Cys-del variant had 2-3-fold improved kcat/Km value for conventional β-eliminating substrates and up to 10-fold increase in catalytic efficiency with S-substituted-l-cysteine sulfoxides compared to the wild-type MGL. The Cys-del MGL remained active under the thiosulfinates, which are products of β-elimination reaction of S-alk(en)yl-l-cysteine sulfoxides. Moreover, Cys-del variant proved to be more stable during shelf storage. Thus, we have created an effective enzyme component of the biocatalytic system that is capable of generating antimicrobial drugs - thiosulfinates.

吡哆醛5'-磷酸(PLP)依赖酶参与许多细胞过程,并具有无与伦比的催化多功能性。通过位点定向诱变的合理设计是为广泛的生物催化应用创造定制酶的有力策略。plp依赖性蛋氨酸γ-裂解酶(MGL)可以降解含硫氨基酸,是一种令人鼓舞的酶,用于许多治疗目的-从对抗细菌耐药菌株和真菌到抗肿瘤活性。双组分生物系统MGL/S-alk(en)yl- l-半胱氨酸亚砜(该酶的一种罕见底物)在β-消除反应中产生抗菌硫代亚硫酸盐。酶的sh基团被该反应的产物修饰,导致酶失活。利用定点诱变技术可以成功、高效、合理地稳定新梭菌的MGL。我们已经设法获得了一种改良版的酶,在β消除反应方面比天然酶更好。新梭菌MGL的Cys118、Cys184和Cys273分别被His和两个Ala取代。与野生型MGL相比,得到的Cys-del变体对传统β-消除底物的kcat/Km值提高了2-3倍,对s取代- l -半胱氨酸亚砜的催化效率提高了10倍。Cys-del MGL在巯基亚砜(S-alk(en)yl- l-半胱氨酸亚砜的β-消除反应产物)作用下保持活性。此外,Cys-del变体在货架储存期间被证明更稳定。因此,我们已经创建了一个有效的酶组分的生物催化系统,能够产生抗菌药物-硫代亚硫酸盐。
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引用次数: 0
Interaction of small heat shock proteins with BAG3. 小热休克蛋白与bag3的相互作用。
Pub Date : 2025-01-13 DOI: 10.1016/j.biochi.2025.01.001
Maria A Zamotina, Lydia K Muranova, Arthur I Zabolotskii, Nikolai B Gusev

BAG3 is a universal adapter protein involved in various cellular processes, including the regulation of apoptosis, chaperone-assisted selective autophagy, and heat shock protein function. The interaction between small heat shock proteins (sHsps) and their α-crystallin domains (Acds) with full-length BAG3 protein and its IPV domain was analyzed using size-exclusion chromatography, native gel electrophoresis, and chemical cross-linking. HspB7 and the 3D mutant of HspB1 (which mimics phosphorylation) showed no interaction, HspB6 weakly interacted, and HspB8 strongly interacted with full-length BAG3. In contrast to the full-length sHsps, their α-crystallin domains (AcdB1, AcdB5, and AcdB6) were able to interact with BAG3, with AcdB8 again being the strongest interactor. Among all the full-length sHsps analyzed, only HspB8 bound to the IPV domain of BAG3. AcdB1, AcdB5, AcdB6, and AcdB8 interacted with the IPV domain of BAG3, with AcdB8 displaying the highest binding efficiency. The stoichiometry of crosslinked complexes formed by HspB8 (or its Acd) and the IPV domain of BAG3 was 2:1, whereas for the other sHsps and their Acds, it was 1:1. These findings suggest that while the IPV domain of BAG3 and the Acds of sHsps play an important role in binding, other structural regions significantly contribute to this interaction. The unique binding efficiency between BAG3 and HspB8 may be attributed to the intrinsic disorder and simple oligomeric structure of HspB8.

BAG3是一种通用的适配蛋白,参与多种细胞过程,包括调节细胞凋亡、伴侣辅助的选择性自噬和热休克蛋白功能。采用粒径排除色谱、天然凝胶电泳和化学交联等方法分析了小热休克蛋白(sHsps)及其α-结晶蛋白结构域(Acds)与全长BAG3蛋白及其IPV结构域的相互作用。HspB7与HspB1三维突变体(模拟磷酸化)无相互作用,HspB6与全长BAG3弱相互作用,HspB8与全长BAG3强相互作用。与全长sHsps相比,它们的α-结晶蛋白结构域(AcdB1、AcdB5和AcdB6)能够与BAG3相互作用,其中AcdB8仍然是最强的相互作用体。在分析的所有全长sHsps中,只有HspB8与BAG3的IPV结构域结合。AcdB1、AcdB5、AcdB6和AcdB8与BAG3的IPV结构域相互作用,其中AcdB8的结合效率最高。HspB8(或其Acd)与BAG3的IPV结构域形成的交联配合物的化学计量比为2:1,而其他sHsps及其Acds形成的交联配合物的化学计量比为1:1。这些发现表明,虽然BAG3的IPV结构域和sHsps的Acds在结合中发挥重要作用,但其他结构区域也对这种相互作用起着重要作用。BAG3与HspB8之间独特的结合效率可能归因于HspB8固有的无序性和简单的寡聚结构。
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引用次数: 0
Elucidating on the quaternary structure of viper venom phospholipase A2 enzymes in aqueous solution. 蛇毒磷脂酶A2在水溶液中的四级元结构研究。
Pub Date : 2025-01-10 DOI: 10.1016/j.biochi.2024.12.015
Joana R da Silva, Maria João Ramos, Pedro A Fernandes

This study focuses on the quaternary structure of the viper-secreted phospholipase A2 (PLA2), a central toxin in viper envenomation. PLA2 enzymes catalyze the hydrolysis of the sn-2 ester bond of membrane phospholipids. Small-molecule inhibitors that act as snakebite antidotes, such as varespladib, are currently in clinical trials. These inhibitors likely bind to the enzyme in the aqueous cytosol prior to membrane-binding. Thus, understanding its controversial solution structure is key for drug design. Crystal structures of PLA2 in the PDB show at least four different dimeric conformations, the most well-known being "extended" and "compact". This variability among enzymes with >50 % sequence identity raises questions about their transferability to aqueous solution. Therefore, we performed extensive molecular dynamics (MD) simulations of several PLA2 enzymes in water to determine their quaternary structure under physiological conditions. The MD simulations strongly indicate that PLA2 enzymes adopt a "semi-compact" conformation in cytosol, a hybrid between extended and compact conformations. To our knowledge, this is the first study that determines the most favorable dimeric conformation of PLA2 enzymes in solution, providing a basis for advancements in snakebite envenoming treatment. Recognizing snakebite envenoming as a neglected tropical disease has driven the search for efficient, affordable alternatives to the current antivenoms. Therefore, understanding the main drug targets within snake venom is crucial to this achievement.

本研究的重点是毒蛇分泌的磷脂酶A2 (PLA2)的四级结构,这是毒蛇中毒的中心毒素。PLA2酶催化膜磷脂的sn-2酯键的水解。作为毒蛇咬伤解毒剂的小分子抑制剂,如varespladib,目前正处于临床试验阶段。这些抑制剂可能在膜结合之前先与含水细胞质中的酶结合。因此,了解其有争议的溶液结构是药物设计的关键。PDB中PLA2的晶体结构表现出至少四种不同的二聚体构象,最著名的是“延伸”和“紧致”。具有bbb50 %序列同一性的酶之间的这种可变性提出了它们在水溶液中的可转移性问题。因此,我们对水中几种PLA2酶进行了广泛的分子动力学(MD)模拟,以确定它们在生理条件下的四级结构。MD模拟强烈表明PLA2酶在细胞质中采用“半紧实”构象,介于扩展构象和紧实构象之间。据我们所知,这是第一次确定PLA2酶在溶液中最有利的二聚体构象的研究,为蛇咬伤的治疗提供了基础。认识到蛇咬伤是一种被忽视的热带病,促使人们寻找有效、负担得起的抗蛇毒血清替代品。因此,了解蛇毒中的主要药物靶点对这一成就至关重要。
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引用次数: 0
Structural and functional significance of two conserved lysine residues in acylated sites of Kingella kingae RtxA cytotoxin. Kingella kingae RtxA细胞毒素酰化位点两个保守赖氨酸残基的结构和功能意义。
Pub Date : 2024-12-31 DOI: 10.1016/j.biochi.2024.12.016
Humaira Khaliq, Adriana Osickova, Michaela Lichvarova, Miroslav Sulc, Kevin Munoz Navarrete, Carlos Espinosa-Vinals, Jiri Masin, Radim Osicka

Kingella kingae, an emerging pediatric pathogen, secretes the pore-forming toxin RtxA, which has been implicated in the development of various invasive infections. RtxA is synthesized as a protoxin (proRtxA), which gains its biological activity by fatty acylation of two lysine residues (K558 and K689) by the acyltransferase RtxC. The low acylation level of RtxA at K558 (2-23 %) suggests that the complete acylation at K689 is crucial for toxin activity. Using a bacterial two-hybrid system, we show that substitutions of K558, but not K689, partially reduce the interaction of proRtxA with RtxC and that the acyltransferase interacts independently with each acylated site in vivo. While substitutions of K558 had no effect on the acylation of K689, substitutions of K689 resulted in an average 40 % increase in the acylation of K558. RtxA mutants monoacylated at either K558 or K689 irreversibly bound to erythrocyte membranes, with binding efficiency corresponding to the extent of lysine acylation. However, these mutants lysed erythrocytes with similarly low efficiency as nonacylated proRtxA and showed only residual overall membrane activity in planar lipid bilayers. Interestingly, despite forming fewer pores, the monoacylated mutants exhibited single-pore characteristics, such as conductance and lifetime, similar to those of intact RtxA. These findings indicate that the acylation at either K558 or K689 is sufficient for the irreversible insertion of RtxA into the membrane, but not for the efficient formation of membrane pores. Alternatively, K558 and K689 per se may play a crucial structural role in pore formation, regardless of their acylation status.

Kingella kingae是一种新兴的儿科病原体,分泌成孔毒素RtxA,这与各种侵袭性感染的发展有关。RtxA是一种原蛋白(protxa),它通过酰基转移酶RtxC对两个赖氨酸残基(K558和K689)的脂肪酰化而获得生物活性。RtxA在K558位点的低酰化水平(2-23%)表明K689位点的完全酰化对毒素活性至关重要。利用细菌双杂交系统,我们发现K558而不是K689的取代部分减少了protxa与RtxC的相互作用,并且酰基转移酶在体内独立地与每个酰基化位点相互作用。K558的取代对K689的酰化没有影响,而K689的取代导致K558的酰化平均增加40%。在K558或K689位点单酰化的RtxA突变体不可逆地与红细胞膜结合,其结合效率与赖氨酸酰化的程度相对应。然而,这些突变体裂解红细胞的效率与非酰化的protxa相似,并且仅在平面脂质双分子层中显示残余的整体膜活性。有趣的是,尽管形成较少的孔隙,单酰化突变体表现出单孔特征,如电导和寿命,类似于完整的RtxA。这些发现表明,K558或K689的酰化足以使RtxA不可逆地插入膜中,但不能有效地形成膜孔。另外,K558和K689本身可能在孔隙形成中发挥关键的结构作用,而不管它们的酰化状态如何。
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引用次数: 0
Highly specific aptamer trap for extremophilic RNA polymerases. 针对嗜极 RNA 聚合酶的高特异性适配体陷阱。
Pub Date : 2024-05-15 DOI: 10.1016/j.biochi.2024.05.014
Ivan Petushkov, Andrey Feklistov, Andrey Kulbachinskiy

During transcription initiation, the holoenzyme of bacterial RNA polymerase (RNAP) specifically recognizes promoters using a dedicated σ factor. During transcription elongation, the core enzyme of RNAP interacts with nucleic acids mainly nonspecifically, by stably locking the DNA template and RNA transcript inside the main cleft. Here, we present a synthetic DNA aptamer that is specifically recognized by both core and holoenzyme RNAPs from extremophilic bacteria of the Deinococcus-Thermus lineage. The aptamer binds RNAP with subnanomolar affinities, forming extremely stable complexes even at high ionic strength conditions, blocks RNAP interactions with the DNA template and inhibits RNAP activity during transcription elongation. We propose that the aptamer binds at a conserved site within the downstream DNA-binding cleft of RNAP and traps it in an inactive conformation. The aptamer can potentially be used for structural studies to reveal RNAP conformational states, affinity binding of RNAP and associated factors, and screening of transcriptional inhibitors.

在转录启动过程中,细菌 RNA 聚合酶(RNAP)的全酶利用专用的 σ 因子特异性地识别启动子。在转录延伸过程中,RNAP 的核心酶主要通过将 DNA 模板和 RNA 转录本稳定地锁定在主裂隙内与核酸进行非特异性相互作用。在这里,我们展示了一种合成的DNA适配体,它能被来自嗜极细菌(Deinococcus-Thermus lineage)的核心和全酶RNAP特异性识别。这种适配体能以亚纳摩尔级的亲和力与 RNAP 结合,即使在高离子强度条件下也能形成极其稳定的复合物,阻断 RNAP 与 DNA 模板的相互作用,并在转录延伸过程中抑制 RNAP 的活性。我们认为,该配合物与 RNAP 下游 DNA 结合裂隙中的一个保守位点结合,并使其处于非活性构象。这种适配体可用于结构研究以揭示 RNAP 的构象状态、RNAP 与相关因子的亲和结合以及转录抑制剂的筛选。
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引用次数: 0
Inside front cover-EDB 内侧前盖EDB
Pub Date : 2023-10-17 DOI: 10.1016/S0300-9084(23)00260-2
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引用次数: 0
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