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Warfare Nerve Agents and Paraoxonase-1 as a Potential Prophylactic Therapy against Intoxication. 战争神经毒剂和作为潜在预防性疗法的 Paraoxonase-1。
IF 1 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 DOI: 10.2174/0109298665284293240409045359
A R Satvik Iyengar, Prakash Y Khandave, Janek Bzdrenga, Florian Nachon, Xavier Brazzolotto, Abhay H Pande

Nerve agents are a class of lethal neurotoxic chemicals used in chemical warfare. In this review, we have discussed a brief history of chemical warfare, followed by an exploration of the historical context surrounding nerve agents. The article explores the classification of these agents, their contemporary uses, their toxicity mechanisms, and the disadvantages of the current treatment options for nerve agent poisoning. It then discusses the possible application of enzymes as prophylactics against nerve agent poisoning, outlining the benefits and drawbacks of paraoxonase- 1. Finally, the current studies on paraoxonase-1 are reviewed, highlighting that several challenges need to be addressed in the use of paraoxonase-1 in the actual field and that its potential as a prophylactic antidote against nerve agent poisoning needs to be evaluated. The literature used in this manuscript was searched using various electronic databases, such as PubMed, Google Scholar, Web of Science, Elsevier, Springer, ACS, Google Patent, and books using the keywords chemical warfare agent, butyrylcholinesterase, enzyme, nerve agent, prophylactic, and paraoxonase-1, with the time scale for the analysis of articles between 1960 to 2023. The study has suggested that concerted efforts by researchers and agencies must be made to develop effective countermeasures against NA poisoning and that paraoxonase-1 has suitable properties for the development of efficient prophylaxis against NA poisoning.

神经毒剂是化学战中使用的一类致命的神经毒性化学品。在这篇综述中,我们简要讨论了化学战的简史,随后探讨了神经毒剂的历史背景。文章探讨了这些制剂的分类、当代用途、毒性机制以及神经毒剂中毒现有治疗方案的缺点。然后,文章讨论了酶作为神经毒剂中毒预防剂的可能应用,概述了副氧合酶-1 的优点和缺点。最后,回顾了目前有关副氧杂蒽酮酶-1 的研究,强调了在实际领域使用副氧杂蒽酮酶-1 时需要应对的几个挑战,并需要评估其作为神经毒剂中毒预防性解毒剂的潜力。本手稿中使用的文献是通过PubMed、Google Scholar、Web of Science、Elsevier、Springer、ACS、Google Patent和书籍等多个电子数据库进行检索的,使用的关键词分别为化学战剂、丁酰胆碱酯酶、酶、神经毒剂、预防性、副氧自由基酶-1,分析文章的时间范围分别为1960年至2023年。研究表明,研究人员和机构必须齐心协力,开发针对NA中毒的有效对策,而PON1具有适合开发针对NA中毒的高效预防药物的特性。
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引用次数: 0
Evaluation of Cytotoxicity and Antimicrobial Activity of Experimental Composites Containing Chitosan-Silver Oxide Particles Against Two Main Pathogenic Bacteria in Periodontal Disease. 含壳聚糖-氧化银颗粒的实验复合物对牙周病中两种主要病原菌的细胞毒性和抗菌活性评价。
IF 1.6 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 DOI: 10.2174/0109298665240242231016103321
Nahid Nasrabadi, Navid Ramezanian, Parisa Ghorbanian, Ali Forouzanfar, Hamideh Sadat Mohammadipour

Introduction: Bacterial biofilm is known as the main cause of periodontal disease. Generally, the anaerobic Gram-negative, such as Porphyromonas gingivalis and Fusobacterium nucleatum, are considered the most identified bacteria.

Objective: This study aimed to investigate the antimicrobial effect and cytotoxicity of two experimental composites containing chitosan-silver oxide (CH-Ag2O) particles.

Materials and methods: Four experimental groups, including Ag2O and CH, along with two composites of CH-Ag2O 20 and CH-Ag2O 60 mg, were prepared. Antimicrobial activity was performed against Porphyromonas gingivalis (ATCC#33277) and Fusobacterium nucleatum (ATCC#25586) using the agar dilution method. Moreover, the cytotoxicity assay was performed on human gingival fibroblasts (HGF) by the use of the MTT method. The obtained data were analyzed with descriptive methods, one-way ANOVA, and Tukey's LSD tests.

Results: The antibacterial activity of both composites was higher than both CH and Ag2O, and the greatest antibacterial properties were presented in CH-Ag2O 60. In all three measurements (24, 48, and 72 h), the greatest cytotoxicity was seen in Ag2O, followed by CH, CH-Ag2O 20, and CHAg2O 60 in descending order, respectively. The cytotoxicity of these components was related to the concentration and not to the time of exposure. The results showed that Ag2O in 3.7 and 7.5 μg/ml concentrations and CH-containing groups in 250 and 500 μg/ml were toxic to the cultured HGF.

Conclusion: The experimental composite containing CH-Ag2O 60 showed the greatest antibacterial properties against two periodontal pathogens evaluated. In order to clarify the clinical significance of composite cytotoxicity, further clinical studies are necessary.

简介:细菌生物膜是引起牙周病的主要原因。一般来说,厌氧革兰氏阴性菌,如牙龈卟啉单胞菌和有核梭杆菌,被认为是鉴定最多的细菌。目的:研究两种含有壳聚糖-氧化银(CH-Ag2O)颗粒的实验复合材料的抗菌效果和细胞毒性。材料和方法:制备了四个实验组,包括Ag2O和CH,以及两种CH-Ag2O 20和CH-Ag2O60mg的复合材料。使用琼脂稀释法对牙龈卟啉单胞菌(ATCC#33277)和有核梭杆菌(ATCC#25586)进行抗菌活性。此外,采用MTT法对人牙龈成纤维细胞(HGF)进行细胞毒性测定。使用描述性方法、单因素方差分析和Tukey的LSD检验对获得的数据进行分析。结果:两种复合材料的抗菌活性均高于CH和Ag2O,其中CH-Ag2O60的抗菌性能最好。在所有三次测量(24、48和72小时)中,Ag2O的细胞毒性最大,依次为CH、CH-Ag2O 20和CH-Ag2O60。这些成分的细胞毒性与浓度有关,而与暴露时间无关。结果表明,3.7和7.5μg/ml浓度的Ag2O和250和500μg/ml的含CH组对培养的HGF具有毒性。为了阐明复合细胞毒性的临床意义,有必要进行进一步的临床研究。
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引用次数: 0
The GA-Hecate Peptide inhibits the ZIKV Replicative Cycle in Different Steps and can Inhibit the Flavivirus NS2B-NS3 Protease after Cell Infection. GA-Hecate 肽在不同步骤中抑制 ZIKV 复制循环,并能在细胞感染后抑制黄病毒 NS2B-NS3 蛋白酶。
IF 1 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 DOI: 10.2174/0109298665308871240703090408
Paulo Ricardo da Silva Sanches, João Caldana Elias de Campos Faria, Cíntia Bittar, Hugo Alexandre Siqueira Guberovich Olivieri, Nathalya Cristina de Moraes Roso Mesquita, Gabriela Dias Noske, Andre Schutzer de Godoy, Glaucius Oliva, Paula Rahal, Eduardo Maffud Cilli

Background: Peptide drugs are advantageous because they are subject to rational design and exhibit highly diverse structures and broad biological activities. The NS2B-NS3 protein is a particularly promising flavivirus therapeutic target, with extensive research on the development of inhibitors as therapeutic candidates, and was used as a model in this work to determine the mechanism by which GA-Hecate inhibits ZIKV replication.

Objective: The present study aimed to evaluate the potential of GA-Hecate, a new antiviral developed by our group, against the Brazilian Zika virus and to evaluate the mechanism of action of this compound on the flavivirus NS2B-NS3 protein.

Methods: Solid-phase peptide Synthesis, High-Performance Liquid Chromatography, and Mass Spectrometry were used to obtain, purify, and characterize the synthesized compound. Real-time and enzymatic assays were used to determine the antiviral potential of GA-Hecate against ZIKV.

Results: The RT-qPCR results showed that GA-Hecate decreased the number of ZIKV RNA copies in the virucidal, pre-treatment, and post-entry assays, with 5- to 6-fold fewer RNA copies at the higher nontoxic concentration in Vero cells (HNTC: 10 μM) than in the control cells. Enzymatic and kinetic assays indicated that GA-Hecate acts as a competitive ZIKV NS2B-NS3 protease inhibitor with an IC50 of 32 nM and has activity against the yellow fever virus protease.

Conclusion: The results highlight the antiviral potential of the GA-Hecate bioconjugate and open the door for the development of new antivirals.

背景:肽类药物的优势在于可以进行合理的设计,并表现出高度多样化的结构和广泛的生物活性。NS2B-NS3蛋白是一个特别有前景的黄病毒治疗靶点,有关开发抑制剂作为治疗候选靶点的研究非常广泛,本研究以NS2B-NS3蛋白为模型,确定GA-Hecate抑制ZIKV复制的机制:本研究旨在评估本研究小组开发的新型抗病毒药物 GA-Hecate 对抗巴西寨卡病毒的潜力,并评估该化合物对黄病毒 NS2B-NS3 蛋白的作用机制:方法:采用固相肽合成、高效液相色谱法和质谱法获得、纯化和表征合成的化合物。采用实时和酶法测定 GA-Hecate 对 ZIKV 的抗病毒潜力:RT-qPCR结果显示,在杀病毒、预处理和进入后检测中,GA-猯酸都能减少ZIKV RNA拷贝数,在Vero细胞中的无毒浓度较高时(HNTC:10 μM),RNA拷贝数是对照细胞的5至6倍。酶学和动力学检测表明,GA-癸酸盐是一种竞争性 ZIKV NS2B-NS3 蛋白酶抑制剂,IC50 为 32 nM,对黄热病病毒蛋白酶也有活性:结论:研究结果凸显了 GA-Hecate 生物共轭物的抗病毒潜力,为开发新型抗病毒药物打开了大门。
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引用次数: 0
Exploring the Cell Wall and Secretory Proteins of Mycobacterium leprae as Biomarkers. 探索麻风分枝杆菌细胞壁和分泌蛋白作为生物标志物。
IF 1.6 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 DOI: 10.2174/0109298665267993231026114709
Sakshi Singh, Devesh Sharma, Sakshi Gautam, Mamta Arora, Deepa Bisht

The bacterial cell wall is composed of a wide variety of intricate proteins in addition to lipids, glycolipids, and polymers. Given the diversity of cell wall proteins among bacterial species, they are a feasible target for biomarker identification and characterization in clinical research and diagnosis of the disease. The slow growth rate of Mycobacterium leprae poses a major hurdle in the accurate diagnosis of leprosy before the onset of peripheral neuropathy. The use of biomarker- based diagnostic methods can help in preventing the spread and manifestation of leprosy. Despite many advances in research methods and techniques, there remains a knowledge gap regarding the cell wall proteomes of M. leprae that can be used as biomarkers. The cell wall and secretory proteins of M. leprae are the major focus of this review article. This article enfolds the characteristics and functions of M. leprae cell wall proteins and gives an insight into those cell wall proteins that are yet to be established as biomarkers. Tools and techniques used in cell wall extraction and biomarker identification can also be explored in this article.

细菌细胞壁除了脂质、糖脂质和聚合物外,还由多种复杂的蛋白质组成。鉴于细菌种类之间细胞壁蛋白的多样性,它们是临床研究和疾病诊断中生物标志物鉴定和表征的可行靶点。麻风分枝杆菌生长速度缓慢,这是在周围神经病变发病前准确诊断麻风的主要障碍。使用基于生物标志物的诊断方法可以帮助预防麻风病的传播和表现。尽管在研究方法和技术方面取得了许多进展,但关于麻风分枝杆菌细胞壁蛋白质组学可作为生物标志物的知识差距仍然存在。本文就麻风分枝杆菌的细胞壁和分泌蛋白作一综述。本文介绍了麻风分枝杆菌细胞壁蛋白的特征和功能,并对尚未确定为生物标志物的细胞壁蛋白进行了深入研究。用于细胞壁提取和生物标志物鉴定的工具和技术也可以在本文中进行探讨。
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引用次数: 0
Improving Photocleavage Efficiency of Photocleavable Protein for Antimicrobial Peptide Histatin 1 Expression. 提高光可裂解蛋白的光裂解效率,用于抗菌肽组蛋白 1 的表达。
IF 1.6 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 DOI: 10.2174/0109298665276722231212053009
Nana Zhou, Tai An, Yuan Zhang, Guomiao Zhao, Chao Wei, Xuemei Shen, Fan Li, Xiaoyan Wang

Background: Antimicrobial peptides (AMPs) are promising alternative agents for antibiotics to overcome antibiotic resistance problems. But, it is difficult to produce large-scale antimicrobial research due to the toxicity towards expression hosts or degradation by peptidases in the host. Therefore, heterologous recombinant expression of antimicrobial peptides has always been a challenging issue.

Objectives: To overcome toxicity to the expression host and low expression level, a new photocleavable protein fusion expression method for antimicrobial peptides is provided.3 Methods: Through directed evolution and high throughput screening, a photocleavable protein mutant R6-2-6-4 with a higher photocleavage efficiency was obtained. The DNA coding sequence of antimicrobial peptide Histatin 1 was fused within the sequence of R6-2-6-4 gene. The fusion gene was successfully expressed in Escherichia coli expression system.

Results: Antimicrobial peptide Histatin 1 could be successfully expressed and purified by fusing within PhoCl mutant R6-2-6-4. The antimicrobial activity was rarely affected, and the MIC value was 33 ug/mL, which was basically equivalent to 32 ug/mL of the chemically synthesized Histatin 1. After amplification in a 5 L fermenter, the expression of PhoCl mutant (R6-2-6-4)-Histatin1 improved up to 87.6 mg/L in fermenter, and Histatin1 obtained by photocleavage also could up to 11 mg/L. The prepared Histatin1 powder remained stable when stored at 4oC for up to 4 months without any degradation. In addition, the expression and photocleavage of β -Defensin105 and Lysostaphin verified the certain universality of the PhoCl mutant fusion expression system.

Conclusion: Antimicrobial peptides Histatin 1, β -Defensin 105 and Lysostaphin were successfully expressed and purified by photocleavable protein mutant. This may provide a novel strategy to express and purify antimicrobial peptides in the Escherichia coli expression system.

背景:抗菌肽(AMPs)是抗生素的替代药物,有望克服抗生素耐药性问题。但是,由于抗菌肽对表达宿主的毒性或被宿主体内的肽酶降解,很难进行大规模的抗菌研究。因此,抗菌肽的异源重组表达一直是一个具有挑战性的问题:目的:为克服对表达宿主的毒性和低表达水平,提供一种新的抗菌肽光可溶性蛋白融合表达方法:方法:通过定向进化和高通量筛选,获得了具有更高光裂解效率的光裂解蛋白突变体 R6-2-6-4。在 R6-2-6-4 基因序列中融合了抗菌肽 Histatin 1 的 DNA 编码序列。融合基因在大肠杆菌表达系统中成功表达:结果:在 PhoCl 突变体 R6-2-6-4 中融合抗菌肽 Histatin 1 可成功表达和纯化。在5 L发酵罐中扩增后,PhoCl突变体(R6-2-6-4)-Histatin1在发酵罐中的表达量提高到87.6 mg/L,光裂解得到的Histatin1也可达到11 mg/L。制备的 Histatin1 粉末在 4oC 温度下保存 4 个月仍保持稳定,未发生任何降解。此外,β -Defensin105和Lysostaphin的表达和光裂解验证了PhoCl突变体融合表达系统具有一定的通用性:结论:抗菌肽Histatin 1、β -Defensin105和Lysostaphin通过光裂解蛋白突变体成功表达和纯化。这为在大肠杆菌表达系统中表达和纯化抗菌肽提供了一种新策略。
{"title":"Improving Photocleavage Efficiency of Photocleavable Protein for Antimicrobial Peptide Histatin 1 Expression.","authors":"Nana Zhou, Tai An, Yuan Zhang, Guomiao Zhao, Chao Wei, Xuemei Shen, Fan Li, Xiaoyan Wang","doi":"10.2174/0109298665276722231212053009","DOIUrl":"10.2174/0109298665276722231212053009","url":null,"abstract":"<p><strong>Background: </strong>Antimicrobial peptides (AMPs) are promising alternative agents for antibiotics to overcome antibiotic resistance problems. But, it is difficult to produce large-scale antimicrobial research due to the toxicity towards expression hosts or degradation by peptidases in the host. Therefore, heterologous recombinant expression of antimicrobial peptides has always been a challenging issue.</p><p><strong>Objectives: </strong>To overcome toxicity to the expression host and low expression level, a new photocleavable protein fusion expression method for antimicrobial peptides is provided.3 Methods: Through directed evolution and high throughput screening, a photocleavable protein mutant R6-2-6-4 with a higher photocleavage efficiency was obtained. The DNA coding sequence of antimicrobial peptide Histatin 1 was fused within the sequence of R6-2-6-4 gene. The fusion gene was successfully expressed in <i>Escherichia coli</i> expression system.</p><p><strong>Results: </strong>Antimicrobial peptide Histatin 1 could be successfully expressed and purified by fusing within PhoCl mutant R6-2-6-4. The antimicrobial activity was rarely affected, and the MIC value was 33 ug/mL, which was basically equivalent to 32 ug/mL of the chemically synthesized Histatin 1. After amplification in a 5 L fermenter, the expression of PhoCl mutant (R6-2-6-4)-Histatin1 improved up to 87.6 mg/L in fermenter, and Histatin1 obtained by photocleavage also could up to 11 mg/L. The prepared Histatin1 powder remained stable when stored at 4oC for up to 4 months without any degradation. In addition, the expression and photocleavage of β -Defensin105 and Lysostaphin verified the certain universality of the PhoCl mutant fusion expression system.</p><p><strong>Conclusion: </strong>Antimicrobial peptides Histatin 1, β -Defensin 105 and Lysostaphin were successfully expressed and purified by photocleavable protein mutant. This may provide a novel strategy to express and purify antimicrobial peptides in the <i>Escherichia coli</i> expression system.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":" ","pages":"141-152"},"PeriodicalIF":1.6,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139513364","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
UPBEAT1-ROS-POD-PAL System under Different Xylogenesis Scenarios in Karelian Birch (Betula pendula Roth var. carelica (Mercl.) Hämet-Ahti). 卡累利阿桦木[Betula pendula Roth var. carelica (Mercl.) Hämet-Ahti]不同木质部发生情况下的 UPBEAT1-ROS-POD-PAL 系统。
IF 1 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 DOI: 10.2174/0109298665291781240529044444
Kseniya Mihajlovna Nikerova, Nataliya Alekseevna Galibina, Irina Nikolaevna Sofronova, Yuliya Leonidovna Moshchenskaya, Maksim Anatol'evich Korzhenevskij, Anna Vladimirovna Klimova, Tatiana Vladimirovna Tarelkina

Background: We studied UPBEAT1 (UPB1) which regulated superoxide radical / hydrogen peroxide ratio together with peroxidase (POD) activity and PAL genes expression under different ways of apical meristem development during the xylem structural elements' formation in unique woody plants B. pendula var. pendula with straight-grained wood and B. pendula var. carelica with figured wood. The differentiation process predominanced in straight-grained wood (B. pendula var. pendula) or proliferation - in the figured wood. The investigation was conducted in the radial row (cambial zone - differentiating xylem - mature xylem) during the active cambial growth period.

Objective: The study aimed to study the xylogenesis processes occurring in the 16-year-old straight-grained silver birch (Betula pendula Roth) and Karelian birch (Betula pendula Roth var. carelica (Mercl.) Hämet-Ahti) with figured wood.

Methods: Hydrogen peroxide and superoxide radical contents and peroxidase activity were determined spectrophotometrically. Gene expression for PAL family genes and the UPBEAT1 gene was assessed using qRT-PCR.

Results: Principal component analysis has confirmed trees with straight-grained and figured wood to be different according to UPBEAT1-ROS-POD-PAL system functioning.

Conclusion: The higher superoxide radical/hydrogen peroxide ratio in figured Karelian birch, along with UPBEAT1 transcription factor and PAL genes upregulation, distinguished it from straight-grained silver birch. This metabolic picture confirmed the shift of Karelian birch xylogenesis towards proliferation processes, accompanied by ROS and phenolic compounds' flow and POD activity.

研究背景本研究对具有直纹木质部的B. pendula变种和具有花纹木质部的B. pendula变种进行了研究,研究发现UPBEAT1(UPB1)在直纹木质部(B. pendula var.研究发现,分化过程在直纹木(B. pendula var.调查是在木质部生长活跃期对径向行(木质部分化区-木质部分化-成熟木质部)进行的:本研究旨在研究 16 年生直纹银桦(Betula pendula Roth)和卡累利阿桦(Betula pendula Roth var. carelica (Mercl.) Hämet-Ahti)花纹木的木质部生成过程:方法:用分光光度法测定过氧化氢和超氧自由基的含量以及过氧化物酶的活性。使用 qRT-PCR 评估 PAL 家族基因和 UPBEAT1 基因的表达:结果:主成分分析证实,根据 UPBEAT1-ROS-POD-PAL 系统的功能,直纹木和花纹木的树木是不同的:结论:花纹卡累利阿桦木的超氧自由基/过氧化氢比率较高,同时 UPBEAT1 转录因子和 PAL 基因上调,使其与直纹银桦区分开来。这种新陈代谢情况证实了卡累利阿桦木木质化过程转向增殖过程,同时伴随着 ROS 和酚类化合物的流动以及 POD 活性。
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引用次数: 0
Preface. 序言
IF 1.6 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 DOI: 10.2174/092986653101240120234231
Ben M Dunn
{"title":"Preface.","authors":"Ben M Dunn","doi":"10.2174/092986653101240120234231","DOIUrl":"10.2174/092986653101240120234231","url":null,"abstract":"","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":"31 1","pages":"2"},"PeriodicalIF":1.6,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140040194","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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 a Novel Thermostable 7α-Hydroxysteroid Dehydrogenase. 一种新型恒温 7α- 羟类固醇脱氢酶的特性。
IF 1.6 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 DOI: 10.2174/0109298665279004231229100320
Deshuai Lou, Yangyang Cao, Hongtao Duan, Jun Tan, Binyan Li, Yuanjun Zhou, Dong Wang

Background: 7α-Hydroxysteroid dehydrogenase (7α-HSDH) plays a pivotal role in vivo in the biotransformation of secondary bile acids and has great potential in industrial biosynthesis due to its broad substrate specificity. In this study, we expressed and characterized a novel thermostable 7α-HSDH (named Sa 7α-HSDH).

Methods: The DNA sequence was derived from the black bear gut microbiome metagenomic sequencing data, and the coding sequence of Sa 7α-HSDH was chemically synthesized. The heterologous expression of the enzyme was carried out using the pGEX-6p-1 vector. Subsequently, the activity of the purified enzyme was studied by measuring the absorbance change at 340 nm. Finally, the three-dimensional structure was predicted with AlphaFold2.

Results: Coenzyme screening results confirmed it to be NAD(H) dependent. Substrate specificity test revealed that Sa 7α-HSDH could catalyze taurochenodeoxycholic acid (TCDCA) with catalytic efficiency (kcat/Km) 3.81 S-1 mM-1. The optimum temperature of Sa 7α-HSDH was measured to be 75°C, confirming that it belongs to thermophilic enzymes. Additionally, its thermostability was assessed using an accelerated stability test over 32 hours. The catalytic activity of Sa 7α-HSDH remained largely unchanged for the first 24 hours and retained over 90% of its functionality after 32 hours at 50°C. Sa 7α-HSDH exhibited maximal activity at pH 10. The effect of metal ions-K+, Na+, Mg2+ and Cu2+-on the enzymatic activity of Sa 7α-HSDH was investigated. Only Mg2+ was observed to enhance the enzyme's activity by 27% at a concentration of 300 mM. Neither K+ nor Na+ had a significant influence on activity. Only Cu2+ was found to reduce enzyme activity.

Conclusion: We characterized the thermostable 7α-HSDH, which provides a promising biocatalyst for bioconversion of steroids at high reaction temperatures.

背景:7α-羟类固醇脱氢酶(7α-HSDH)在体内仲胆汁酸的生物转化中发挥着关键作用,由于其广泛的底物特异性,在工业生物合成中具有巨大潜力。在本研究中,我们表达并鉴定了一种新型恒温 7α-HSDH (命名为 Sa 7α-HSDH):方法:DNA序列来自黑熊肠道微生物组元基因组测序数据,Sa 7α-HSDH的编码序列由化学合成。利用 pGEX-6p-1 载体对该酶进行了异源表达。随后,通过测量 340 纳米波长处的吸光度变化研究了纯化酶的活性。最后,利用 AlphaFold2.Results 预测了酶的三维结构:结果:辅酶筛选结果表明该酶依赖于 NAD(H)。底物特异性测试表明,Sa 7α-HSDH 可催化牛磺鹅去氧胆酸(TCDCA),催化效率(kcat/Km)为 3.81 S-1 mM-1。测得Sa 7α-HSDH的最适温度为75℃,证实它属于嗜热型酶。此外,还通过 32 小时的加速稳定性测试评估了它的热稳定性。在最初的 24 小时内,Sa 7α-HSDH 的催化活性基本保持不变,在 50°C 温度下 32 小时后,其功能保持了 90% 以上。Sa 7α-HSDH 在 pH 值为 10 时表现出最大活性。研究了金属离子-K+、Na+、Mg2+ 和 Cu2+ 对 Sa 7α-HSDH 酶活性的影响。在浓度为 300 mM 时,只有 Mg2+ 能使酶的活性提高 27%。K+ 和 Na+ 对酶活性都没有显著影响。只有 Cu2+ 会降低酶的活性:我们鉴定了恒温 7α-HSDH 的特性,它为类固醇在高温反应条件下的生物转化提供了一种前景广阔的生物催化剂。
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引用次数: 0
In Silico Analysis of Natural Plant-Derived Cyclotides with Antifungal Activity against Pathogenic Fungi. 具有抗致病真菌活性的天然植物环肽的硅学分析
IF 1.6 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 DOI: 10.2174/0109298665295545240223114346
Akshita Sharma, Bisma Butool, Pallavi Sahu, Reema Mishra, Aparajita Mohanty

Background: Fungal infections in plants, animals, and humans are widespread across the world. Limited classes of antifungal drugs to treat fungal infections and loss of drug efficacy due to rapidly evolving fungal strains pose a challenge in the agriculture and health sectors. Hence, the search for a new class of antifungal agents is imperative. Cyclotides are cyclic plant peptides with multiple bioactivities, including antifungal activity. They have six conserved cysteine residues forming three disulfide linkages (CI-CIV, CII-CV, CIII-CVI) that establish a Cyclic Cystine Knot (CCK) structure, making them extremely resistant to chemical, enzymatic, and thermal attacks.

Aim: This in silico analysis of natural, plant-derived cyclotides aimed to assess the parameters that can assist and hasten the process of selecting the cyclotides with potent antifungal activity and prioritize them for in vivo/ in vitro experiments.

Objective: The objective of this study was to conduct in silico studies to compare the physicochemical parameters, sequence diversity, surface structures, and membrane-cyclotide interactions of experimentally screened (from literature survey) potent (MIC ≤ 20 μM) and non-potent (MIC > 20 μM) cyclotides for antifungal activity.

Methodology: Cyclotide sequences assessed for antifungal activity were retrieved from the database (Cybase). Various online and offline tools were used for sequence-based studies, such as physicochemical parameters, sequence diversity, and neighbor-joining trees. Structure-based studies involving surface structure analysis and membrane-cyclotide interaction were also carried out. All investigations were conducted in silico.

Results: Physicochemical parameter values, viz. isoelectric point, net charge, and the number of basic amino acids, were significantly higher in potent cyclotides compared to non-potent cyclotides. The surface structure of potent cyclotides showed a larger hydrophobic patch with a higher number of hydrophobic amino acids. Furthermore, the membrane-cyclotide interaction studies of potent cyclotides revealed lower transfer free energy (ΔG transfer) and higher penetration depth into fungal membranes, indicating higher binding stability and membrane-disruption ability.

Conclusion: These in silico studies can be applied for rapidly identifying putatively potent antifungal cyclotides for in vivo and in vitro experiments, which will ultimately be relevant in the agriculture and pharmaceutical sectors.

背景:植物、动物和人类中的真菌感染在全球广泛存在。用于治疗真菌感染的抗真菌药物种类有限,而且由于真菌菌株的快速进化而导致药效丧失,这给农业和卫生部门带来了挑战。因此,寻找一类新的抗真菌药物势在必行。环肽是具有多种生物活性(包括抗真菌活性)的环状植物肽。它们有六个保守的半胱氨酸残基,形成三个二硫键(CI-CIV、CII-CV、CIII-CVI),建立了环状胱氨酸结(CCK)结构,使其具有极强的抗化学、酶和热攻击能力。目的:本研究对天然植物环肽进行了硅学分析,旨在评估可帮助和加速筛选具有强效抗真菌活性的环肽的参数,并将其优先用于体内/体外实验:本研究的目的是进行硅学研究,比较实验筛选(来自文献调查)的强效(MIC ≤ 20 μM)和非强效(MIC > 20 μM)环苷酸的理化参数、序列多样性、表面结构和膜-环苷酸相互作用的抗真菌活性:从数据库(Cybase)中检索抗真菌活性评估的环肽序列。基于序列的研究使用了各种在线和离线工具,如理化参数、序列多样性和邻接树。此外,还进行了基于结构的研究,包括表面结构分析和膜-环肽相互作用。所有研究都是在硅学中进行的:结果:与非强效环化苷酸相比,强效环化苷酸的理化参数值,即等电点、净电荷和碱性氨基酸的数量明显较高。强效环苷酸的表面结构显示出更大的疏水斑块和更多的疏水氨基酸。此外,强效环苷酸的膜-环苷酸相互作用研究显示,其转移自由能(ΔG转移)较低,对真菌膜的穿透深度较高,这表明其具有较高的结合稳定性和膜破坏能力:结论:硅学研究可用于快速鉴定体内和体外实验中的潜在强效抗真菌环化物,最终将应用于农业和医药领域。
{"title":"<i>In Silico</i> Analysis of Natural Plant-Derived Cyclotides with Antifungal Activity against Pathogenic Fungi.","authors":"Akshita Sharma, Bisma Butool, Pallavi Sahu, Reema Mishra, Aparajita Mohanty","doi":"10.2174/0109298665295545240223114346","DOIUrl":"10.2174/0109298665295545240223114346","url":null,"abstract":"<p><strong>Background: </strong>Fungal infections in plants, animals, and humans are widespread across the world. Limited classes of antifungal drugs to treat fungal infections and loss of drug efficacy due to rapidly evolving fungal strains pose a challenge in the agriculture and health sectors. Hence, the search for a new class of antifungal agents is imperative. Cyclotides are cyclic plant peptides with multiple bioactivities, including antifungal activity. They have six conserved cysteine residues forming three disulfide linkages (C<sup>I</sup>-C<sup>IV</sup>, C<sup>II</sup>-C<sup>V</sup>, C<sup>III</sup>-C<sup>VI</sup>) that establish a Cyclic Cystine Knot (CCK) structure, making them extremely resistant to chemical, enzymatic, and thermal attacks.</p><p><strong>Aim: </strong>This <i>in silico</i> analysis of natural, plant-derived cyclotides aimed to assess the parameters that can assist and hasten the process of selecting the cyclotides with potent antifungal activity and prioritize them for <i>in vivo</i>/ <i>in vitro</i> experiments.</p><p><strong>Objective: </strong>The objective of this study was to conduct <i>in silico</i> studies to compare the physicochemical parameters, sequence diversity, surface structures, and membrane-cyclotide interactions of experimentally screened (from literature survey) potent (MIC ≤ 20 μM) and non-potent (MIC > 20 μM) cyclotides for antifungal activity.</p><p><strong>Methodology: </strong>Cyclotide sequences assessed for antifungal activity were retrieved from the database (Cybase). Various online and offline tools were used for sequence-based studies, such as physicochemical parameters, sequence diversity, and neighbor-joining trees. Structure-based studies involving surface structure analysis and membrane-cyclotide interaction were also carried out. All investigations were conducted <i>in silico</i>.</p><p><strong>Results: </strong>Physicochemical parameter values, <i>viz.</i> isoelectric point, net charge, and the number of basic amino acids, were significantly higher in potent cyclotides compared to non-potent cyclotides. The surface structure of potent cyclotides showed a larger hydrophobic patch with a higher number of hydrophobic amino acids. Furthermore, the membrane-cyclotide interaction studies of potent cyclotides revealed lower transfer free energy (ΔG transfer) and higher penetration depth into fungal membranes, indicating higher binding stability and membrane-disruption ability.</p><p><strong>Conclusion: </strong>These <i>in silico</i> studies can be applied for rapidly identifying putatively potent antifungal cyclotides for <i>in vivo</i> and <i>in vitro</i> experiments, which will ultimately be relevant in the agriculture and pharmaceutical sectors.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":" ","pages":"247-260"},"PeriodicalIF":1.6,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140040193","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
Design of Artificial C-Peptides as Potential Anti-HIV-1 Inhibitors Based on 6-HB Formation Mechanism. 基于 6-HB 形成机制设计人工 C 肽作为潜在的抗 HIV-1 抑制剂
IF 1 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 DOI: 10.2174/0109298665312274240530060233
Hui Luo, Yan Zhao, Yuheng Ma, Guodong Liang, Lu Ga, Zhao Meng

Background: The six-helix bundle (6-HB) is a core structure formed during the membrane fusion process of viruses with the Class I envelope proteins. Peptide inhibitors, including the marketed Enfuvirtide, blocking the membrane fusion to exert inhibitory activity were designed based on the heptads repeat interactions in 6-HB. However, the drawbacks of Enfuvirtide, such as drug resistance and short half-life in vivo, have been confirmed in clinical applications. Therefore, novel design strategies are pivotal in the development of next-generation peptide-based fusion inhibitors.

Objective: The de novo design of α-helical peptides against MERS-CoV and IAVs has successfully expedited the development of fusion inhibitors. The reported sequences were completely nonhomologous with natural peptides, which can provide some inspirations for the antiviral design against other pathogenic viruses with class I fusion proteins. Here, we design a series of artificial C-peptides based on the similar mechanism of 6-HB formation and general rules of heptads repeat interaction.

Methods: The inhibitory activity of peptides against HIV-1 was assessed by HIV-1 Env-mediated cell-cell fusion assays. Interaction between artificial C-peptides and target peptides was evaluated by circular dichroism, polyacrylamide gel electrophoresis, size-exclusion chromatography, and sedimentation velocity analysis. Molecular docking studies were performed by using Schrödinger molecular modelling software.

Results: The best-performing artificial C-peptide, 1SR, was highly active against HIV-1 env-mediated cell-cell fusion. 1SR binds to the gp41 NHR region, assembling polymer to prevent endogenous 6-HB formation.

Conclusion: We have found an artificial C-lipopeptide lead compound with inhibitory activity against HIV-1. Also, this paper enriched both N- and C-teminal heptads repeat interaction rules in 6-HB and provided an effective idea for next-generation peptide-based fusion inhibitors against HIV-1.

背景:六螺旋束(6-HB)是病毒与 I 类包膜蛋白膜融合过程中形成的核心结构。根据 6-HB 中的七元重复相互作用设计出了阻断膜融合以发挥抑制活性的肽抑制剂,包括已上市的恩夫韦肽。然而,恩夫韦肽的耐药性和体内半衰期短等缺点已在临床应用中得到证实。因此,新颖的设计策略对于开发基于多肽的下一代融合抑制剂至关重要:目的:针对 MERS-CoV 和 IAV 的 α 螺旋多肽的全新设计成功加快了融合抑制剂的开发。所报道的序列与天然肽完全非同源,这可以为针对其他具有 I 类融合蛋白的致病病毒的抗病毒设计提供一些启发。在此,我们根据 6-HB 形成的相似机制和七联重复相互作用的一般规则设计了一系列人工 C 肽:方法:通过 HIV-1 Env 介导的细胞-细胞融合试验评估肽对 HIV-1 的抑制活性。通过圆二色性、聚丙烯酰胺凝胶电泳、尺寸排阻色谱和沉降速度分析评估了人工 C 肽和目标肽之间的相互作用。使用薛定谔分子建模软件进行了分子对接研究:结果:性能最好的人工 C 肽 1SR 对 HIV-1 env 介导的细胞-细胞融合具有高度活性。1SR 与 gp41 NHR 区域结合,形成聚合物,阻止内源性 6-HB 的形成:结论:我们发现了一种具有抑制 HIV-1 活性的人工 C-脂肽先导化合物。结论:我们发现了一种对 HIV-1 具有抑制活性的人工 C 脂肽先导化合物,并丰富了 6-HB 中 N 端和 C 端七肽重复相互作用的规则,为下一代基于多肽的 HIV-1 融合抑制剂提供了有效的思路。
{"title":"Design of Artificial C-Peptides as Potential Anti-HIV-1 Inhibitors Based on 6-HB Formation Mechanism.","authors":"Hui Luo, Yan Zhao, Yuheng Ma, Guodong Liang, Lu Ga, Zhao Meng","doi":"10.2174/0109298665312274240530060233","DOIUrl":"10.2174/0109298665312274240530060233","url":null,"abstract":"<p><strong>Background: </strong>The six-helix bundle (6-HB) is a core structure formed during the membrane fusion process of viruses with the Class I envelope proteins. Peptide inhibitors, including the marketed Enfuvirtide, blocking the membrane fusion to exert inhibitory activity were designed based on the heptads repeat interactions in 6-HB. However, the drawbacks of Enfuvirtide, such as drug resistance and short half-life <i>in vivo</i>, have been confirmed in clinical applications. Therefore, novel design strategies are pivotal in the development of next-generation peptide-based fusion inhibitors.</p><p><strong>Objective: </strong>The de novo design of α-helical peptides against MERS-CoV and IAVs has successfully expedited the development of fusion inhibitors. The reported sequences were completely nonhomologous with natural peptides, which can provide some inspirations for the antiviral design against other pathogenic viruses with class I fusion proteins. Here, we design a series of artificial C-peptides based on the similar mechanism of 6-HB formation and general rules of heptads repeat interaction.</p><p><strong>Methods: </strong>The inhibitory activity of peptides against HIV-1 was assessed by HIV-1 Env-mediated cell-cell fusion assays. Interaction between artificial C-peptides and target peptides was evaluated by circular dichroism, polyacrylamide gel electrophoresis, size-exclusion chromatography, and sedimentation velocity analysis. Molecular docking studies were performed by using Schrödinger molecular modelling software.</p><p><strong>Results: </strong>The best-performing artificial C-peptide, 1SR, was highly active against HIV-1 env-mediated cell-cell fusion. 1SR binds to the gp41 NHR region, assembling polymer to prevent endogenous 6-HB formation.</p><p><strong>Conclusion: </strong>We have found an artificial C-lipopeptide lead compound with inhibitory activity against HIV-1. Also, this paper enriched both N- and C-teminal heptads repeat interaction rules in 6-HB and provided an effective idea for next-generation peptide-based fusion inhibitors against HIV-1.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":" ","pages":"447-457"},"PeriodicalIF":1.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141443271","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
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Protein and Peptide Letters
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