首页 > 最新文献

Protein and Peptide Letters最新文献

英文 中文
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 Q3 Biochemistry, Genetics and 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":null,"pages":null},"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
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 Q3 Biochemistry, Genetics and 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。
{"title":"Evaluation of Cytotoxicity and Antimicrobial Activity of Experimental Composites Containing Chitosan-Silver Oxide Particles Against Two Main Pathogenic Bacteria in Periodontal Disease.","authors":"Nahid Nasrabadi, Navid Ramezanian, Parisa Ghorbanian, Ali Forouzanfar, Hamideh Sadat Mohammadipour","doi":"10.2174/0109298665240242231016103321","DOIUrl":"10.2174/0109298665240242231016103321","url":null,"abstract":"<p><strong>Introduction: </strong>Bacterial biofilm is known as the main cause of periodontal disease. Generally, the anaerobic Gram-negative, such as <i>Porphyromonas gingivalis</i> and Fusobacterium nucleatum, are considered the most identified bacteria.</p><p><strong>Objective: </strong>This study aimed to investigate the antimicrobial effect and cytotoxicity of two experimental composites containing chitosan-silver oxide (CH-Ag<sub>2</sub>O) particles.</p><p><strong>Materials and methods: </strong>Four experimental groups, including Ag2O and CH, along with two composites of CH-Ag<sub>2</sub>O 20 and CH-Ag<sub>2</sub>O 60 mg, were prepared. Antimicrobial activity was performed against <i>Porphyromonas gingivalis</i> (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.</p><p><strong>Results: </strong>The antibacterial activity of both composites was higher than both CH and Ag<sub>2</sub>O, and the greatest antibacterial properties were presented in CH-Ag<sub>2</sub>O 60. In all three measurements (24, 48, and 72 h), the greatest cytotoxicity was seen in Ag<sub>2</sub>O, followed by CH, CH-Ag<sub>2</sub>O 20, and CHAg<sub>2</sub>O 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 Ag<sub>2</sub>O 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.</p><p><strong>Conclusion: </strong>The experimental composite containing CH-Ag<sub>2</sub>O 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.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71426296","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
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。
{"title":"The GA-Hecate Peptide inhibits the ZIKV Replicative Cycle in Different Steps and can Inhibit the Flavivirus NS2B-NS3 Protease after Cell Infection.","authors":"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","doi":"10.2174/0109298665308871240703090408","DOIUrl":"10.2174/0109298665308871240703090408","url":null,"abstract":"<p><strong>Background: </strong>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.</p><p><strong>Objective: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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 IC<sub>50</sub> of 32 nM and has activity against the yellow fever virus protease.</p><p><strong>Conclusion: </strong>The results highlight the antiviral potential of the GA-Hecate bioconjugate and open the door for the development of new antivirals.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141748948","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 Q3 Biochemistry, Genetics and 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。
{"title":"Characterization of a Novel Thermostable 7α-Hydroxysteroid Dehydrogenase.","authors":"Deshuai Lou, Yangyang Cao, Hongtao Duan, Jun Tan, Binyan Li, Yuanjun Zhou, Dong Wang","doi":"10.2174/0109298665279004231229100320","DOIUrl":"10.2174/0109298665279004231229100320","url":null,"abstract":"<p><strong>Background: </strong>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).</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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 (k<sub>cat</sub>/K<sub>m</sub>) 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<sup>+</sup>, Na<sup>+</sup>, Mg<sup>2+</sup> and Cu<sup>2+</sup>-on the enzymatic activity of Sa 7α-HSDH was investigated. Only Mg<sup>2+</sup> was observed to enhance the enzyme's activity by 27% at a concentration of 300 mM. Neither K<sup>+</sup> nor Na+ had a significant influence on activity. Only Cu<sup>2+</sup> was found to reduce enzyme activity.</p><p><strong>Conclusion: </strong>We characterized the thermostable 7α-HSDH, which provides a promising biocatalyst for bioconversion of steroids at high reaction temperatures.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139576251","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
Preface. 序言
IF 1.6 4区 生物学 Q3 Biochemistry, Genetics and 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":null,"pages":null},"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
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。
{"title":"<i>UPBEAT1</i>-ROS-POD-<i>PAL</i> System under Different Xylogenesis Scenarios in Karelian Birch <i>(Betula pendula</i> Roth var. carelica (Mercl.) Hämet-Ahti).","authors":"Kseniya Mihajlovna Nikerova, Nataliya Alekseevna Galibina, Irina Nikolaevna Sofronova, Yuliya Leonidovna Moshchenskaya, Maksim Anatol'evich Korzhenevskij, Anna Vladimirovna Klimova, Tatiana Vladimirovna Tarelkina","doi":"10.2174/0109298665291781240529044444","DOIUrl":"10.2174/0109298665291781240529044444","url":null,"abstract":"<p><strong>Background: </strong>We studied <i>UPBEAT1 (UPB1)</i> which regulated superoxide radical / hydrogen peroxide ratio together with peroxidase (POD) activity and <i>PAL</i> genes expression under different ways of apical meristem development during the xylem structural elements' formation in unique woody plants <i>B. pendula</i> var. pendula with straight-grained wood and <i>B. pendula</i> var. carelica with figured wood. The differentiation process predominanced in straight-grained wood (<i>B. pendula</i> var. <i>pendula</i>) 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.</p><p><strong>Objective: </strong>The study aimed to study the xylogenesis processes occurring in the 16-year-old straight-grained silver birch (<i>Betula pendula</i> Roth) and Karelian birch (<i>Betula pendula</i> Roth var. carelica (Mercl.) Hämet-Ahti) with figured wood.</p><p><strong>Methods: </strong>Hydrogen peroxide and superoxide radical contents and peroxidase activity were determined spectrophotometrically. Gene expression for <i>PAL</i> family genes and the <i>UPBEAT1</i> gene was assessed using qRT-PCR.</p><p><strong>Results: </strong>Principal component analysis has confirmed trees with straight-grained and figured wood to be different according to UPBEAT1-ROS-POD-<i>PAL</i> system functioning.</p><p><strong>Conclusion: </strong>The higher superoxide radical/hydrogen peroxide ratio in figured Karelian birch, along with <i>UPBEAT1</i> transcription factor and <i>PAL</i> 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.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141262747","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
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 Q3 Biochemistry, Genetics and 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":null,"pages":null},"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":null,"pages":null},"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
Comparing the Soluble Form of Recombinant Human Insulin-like Growth Factor-1 (rhIGF-1) in Escherichia coli Using Thioredoxin as Fused and Co-expressed Protein. 利用硫氧还蛋白作为融合蛋白和共表达蛋白比较大肠杆菌中重组人胰岛素样生长因子-1(rhIGF-1)的可溶性形式。
IF 1 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-01-01 DOI: 10.2174/0109298665314267240624091046
Sara Hemmati, Parvaneh Maghami, Javad Ranjbari, Maryam Tabarzad

Introduction: Insulin-like growth factor-1 (IGF-1) is a single-chain polypeptide with various physiological functions. Escherichia coli is one of the most desirable hosts for recombinant protein production, especially for human proteins whose post-translation modifications are not essential for their bioactivity, such as hIGF-1.

Objectives: In this study, bacterial thioredoxin (Trx) was studied as a fused and non-fused protein to convert the insoluble form of recombinant human IGF-1 (rhIGF-1) to its soluble form in E. coli.

Methods: The rhIGF-1 was expressed in the E. coli Origami strain in the form of fused-Trx. It was co-expressed with Trx and then purified and quantified. In the next step, the biological activity of rhIGF-1 was evaluated by alkaline phosphatase (ALP) activity assay in human adipose- derived stem cells (hASCs) regarding the differentiation enhancement effect of IGF-1 through the osteogenic process.

Results: Results showed that Trx in both the fused and non-fused forms had a positive effect on the production of the soluble form of rhIGF-1. A significant increase in ALP activity in hASCs after rhIGF-1 treatment was observed, confirming protein bioactivity.

Conclusion: It was strongly suggested that the overproduction of Trx could increase the solubility of co-expressed recombinant proteins by changing the redox state in E. coli cells.

简介胰岛素样生长因子-1(IGF-1)是一种单链多肽,具有多种生理功能。大肠杆菌是生产重组蛋白质最理想的宿主之一,尤其是那些翻译后修饰对其生物活性并不重要的人类蛋白质,如 hIGF-1:本研究研究了细菌硫氧还蛋白(Trx)作为融合蛋白和非融合蛋白在大肠杆菌中将重组人 IGF-1 (rhIGF-1)的不溶性形式转化为可溶性形式:方法:在大肠杆菌 Origami 菌株中以融合-Trx 的形式表达 rhIGF-1。方法:在大肠杆菌 Origami 菌株中以融合-Trx 的形式表达 rhIGF-1,并与 Trx 共同表达,然后进行纯化和定量。下一步,通过碱性磷酸酶(ALP)活性测定评估了rhIGF-1在人脂肪来源干细胞(hASCs)中的生物活性,以了解IGF-1通过成骨过程增强分化的作用:结果表明,融合型和非融合型Trx对可溶性rhIGF-1的产生有积极影响。rhIGF-1处理后,hASCs的ALP活性明显增加,证实了蛋白质的生物活性:结论:这强烈表明,Trx 的过度产生可通过改变大肠杆菌细胞中的氧化还原状态来增加共表达重组蛋白的溶解度。
{"title":"Comparing the Soluble Form of Recombinant Human Insulin-like Growth Factor-1 (rhIGF-1) in <i>Escherichia coli</i> Using Thioredoxin as Fused and Co-expressed Protein.","authors":"Sara Hemmati, Parvaneh Maghami, Javad Ranjbari, Maryam Tabarzad","doi":"10.2174/0109298665314267240624091046","DOIUrl":"10.2174/0109298665314267240624091046","url":null,"abstract":"<p><strong>Introduction: </strong>Insulin-like growth factor-1 (IGF-1) is a single-chain polypeptide with various physiological functions. <i>Escherichia coli</i> is one of the most desirable hosts for recombinant protein production, especially for human proteins whose post-translation modifications are not essential for their bioactivity, such as hIGF-1.</p><p><strong>Objectives: </strong>In this study, bacterial thioredoxin (Trx) was studied as a fused and non-fused protein to convert the insoluble form of recombinant human IGF-1 (rhIGF-1) to its soluble form in E. coli.</p><p><strong>Methods: </strong>The rhIGF-1 was expressed in the <i>E. coli</i> Origami strain in the form of fused-Trx. It was co-expressed with Trx and then purified and quantified. In the next step, the biological activity of rhIGF-1 was evaluated by alkaline phosphatase (ALP) activity assay in human adipose- derived stem cells (hASCs) regarding the differentiation enhancement effect of IGF-1 through the osteogenic process.</p><p><strong>Results: </strong>Results showed that Trx in both the fused and non-fused forms had a positive effect on the production of the soluble form of rhIGF-1. A significant increase in ALP activity in hASCs after rhIGF-1 treatment was observed, confirming protein bioactivity.</p><p><strong>Conclusion: </strong>It was strongly suggested that the overproduction of Trx could increase the solubility of co-expressed recombinant proteins by changing the redox state in <i>E. coli</i> cells.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141498757","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
Clay-Polymer Nanocomposites Mediated Inhibition of Protein Aggregation: Possible Role in the Prevention of Proteinopathies. 粘土聚合物纳米复合材料介导的蛋白质聚集抑制:在预防蛋白质病中的可能作用。
IF 1.6 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-10-04 DOI: 10.2174/0109298665274059231002071951
Romana Parveen, Sher Ali, Sadaf Fatima

Background: The transformation of proteins from their native conformation into highly ordered fibrillar structures due to their misfolding and aggregation under particular conditions are described as beta-sheet enriched amyloid fibrils. The accumulation of these fibrils in different body parts is the major cause of several neurological and non-neurological conditions (proteinopathies).

Objectives: To prevent these proteinopathies, inhibition of protein aggregation is considered a promising strategy. Therefore, in this study, we synthesized montmorillonite (MMT) based poly- orthophenylenediamine (PoPD) nanocomposites (NCs) and characterized their size and morphology due to their remarkable biological properties. Further, the effect of these nanocomposites on inhibition of fibril formation was assessed.

Methods: These nanocomposites were evaluated for their anti-amyloidogenic potential on two model proteins of amyloidopathies, i.e., human lysozyme and human serum albumin (HL & HSA), by using several biophysical methods, such as Thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) fluorescence, congo red dye binding assay (CR). Secondary structural content was evaluated by Circular dichroism (CD) spectroscopy.

Results: Results demonstrated that synthesized nanocomposites significantly inhibited fibril formation in dose-dependent manner that corresponds to their ability to arrest fibrillation. It is suggested that they may adsorb proteins to protect them against aggregation when they are subjected to aggregating conditions.

Conclusion: This study offers an opportunity to understand the mechanism of inhibition of fibril formation by nanocomposites, showing that they inhibit amyloid formation and amyloid diseases. Thus, the study concludes that these nanocomposites are promising candidates as therapeutic molecules for proteinopathies and are envisaged to enrich the area of personalized medicine, augmenting the human healthcare system.

背景:蛋白质在特定条件下由于错误折叠和聚集而从天然构象转变为高度有序的原纤维结构,被描述为富含β片的淀粉样蛋白原纤维。这些原纤维在身体不同部位的积聚是几种神经和非神经疾病(蛋白质病)的主要原因。目的:为了预防这些蛋白质病,抑制蛋白质聚集被认为是一种很有前途的策略。因此,在本研究中,我们合成了基于蒙脱石(MMT)的聚邻苯二胺(PoPD)纳米复合材料(NCs),并对其尺寸和形态进行了表征。此外,评估了这些纳米复合材料对原纤维形成的抑制作用。方法:采用硫黄素T(ThT)和1-苯胺基-8-萘磺酸盐(ANS)荧光、刚果红染料结合分析(CR)等生物物理方法,评价了这些纳米复合材料对淀粉样变性的两种模型蛋白,即人溶菌酶和人血清白蛋白(HL&HSA)的抗淀粉样变性潜力。二级结构含量通过圆二色性(CD)光谱进行评估。结果:结果表明,合成的纳米复合材料以剂量依赖的方式显著抑制原纤维的形成,这与它们阻止纤颤的能力相对应。有人认为,当它们受到聚集条件时,它们可以吸附蛋白质以保护它们免受聚集。结论:本研究为了解纳米复合材料抑制原纤维形成的机制提供了机会,表明它们可以抑制淀粉样蛋白的形成和淀粉样蛋白疾病。因此,该研究得出结论,这些纳米复合材料是蛋白质疾病治疗分子的有前途的候选者,有望丰富个性化医学领域,增强人类医疗保健系统。
{"title":"Clay-Polymer Nanocomposites Mediated Inhibition of Protein Aggregation: Possible Role in the Prevention of Proteinopathies.","authors":"Romana Parveen,&nbsp;Sher Ali,&nbsp;Sadaf Fatima","doi":"10.2174/0109298665274059231002071951","DOIUrl":"https://doi.org/10.2174/0109298665274059231002071951","url":null,"abstract":"<p><strong>Background: </strong>The transformation of proteins from their native conformation into highly ordered fibrillar structures due to their misfolding and aggregation under particular conditions are described as beta-sheet enriched amyloid fibrils. The accumulation of these fibrils in different body parts is the major cause of several neurological and non-neurological conditions (proteinopathies).</p><p><strong>Objectives: </strong>To prevent these proteinopathies, inhibition of protein aggregation is considered a promising strategy. Therefore, in this study, we synthesized montmorillonite (MMT) based poly- orthophenylenediamine (PoPD) nanocomposites (NCs) and characterized their size and morphology due to their remarkable biological properties. Further, the effect of these nanocomposites on inhibition of fibril formation was assessed.</p><p><strong>Methods: </strong>These nanocomposites were evaluated for their anti-amyloidogenic potential on two model proteins of amyloidopathies, i.e., human lysozyme and human serum albumin (HL & HSA), by using several biophysical methods, such as Thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) fluorescence, congo red dye binding assay (CR). Secondary structural content was evaluated by Circular dichroism (CD) spectroscopy.</p><p><strong>Results: </strong>Results demonstrated that synthesized nanocomposites significantly inhibited fibril formation in dose-dependent manner that corresponds to their ability to arrest fibrillation. It is suggested that they may adsorb proteins to protect them against aggregation when they are subjected to aggregating conditions.</p><p><strong>Conclusion: </strong>This study offers an opportunity to understand the mechanism of inhibition of fibril formation by nanocomposites, showing that they inhibit amyloid formation and amyloid diseases. Thus, the study concludes that these nanocomposites are promising candidates as therapeutic molecules for proteinopathies and are envisaged to enrich the area of personalized medicine, augmenting the human healthcare system.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2023-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49681633","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
期刊
Protein and Peptide Letters
全部 Acc. Chem. Res. ACS Applied Bio Materials ACS Appl. Electron. Mater. ACS Appl. Energy Mater. ACS Appl. Mater. Interfaces ACS Appl. Nano Mater. ACS Appl. Polym. Mater. ACS BIOMATER-SCI ENG ACS Catal. ACS Cent. Sci. ACS Chem. Biol. ACS Chemical Health & Safety ACS Chem. Neurosci. ACS Comb. Sci. ACS Earth Space Chem. ACS Energy Lett. ACS Infect. Dis. ACS Macro Lett. ACS Mater. Lett. ACS Med. Chem. Lett. ACS Nano ACS Omega ACS Photonics ACS Sens. ACS Sustainable Chem. Eng. ACS Synth. Biol. Anal. Chem. BIOCHEMISTRY-US Bioconjugate Chem. BIOMACROMOLECULES Chem. Res. Toxicol. Chem. Rev. Chem. Mater. CRYST GROWTH DES ENERG FUEL Environ. Sci. Technol. Environ. Sci. Technol. Lett. Eur. J. Inorg. Chem. IND ENG CHEM RES Inorg. Chem. J. Agric. Food. Chem. J. Chem. Eng. Data J. Chem. Educ. J. Chem. Inf. Model. J. Chem. Theory Comput. J. Med. Chem. J. Nat. Prod. J PROTEOME RES J. Am. Chem. Soc. LANGMUIR MACROMOLECULES Mol. Pharmaceutics Nano Lett. Org. Lett. ORG PROCESS RES DEV ORGANOMETALLICS J. Org. Chem. J. Phys. Chem. J. Phys. Chem. A J. Phys. Chem. B J. Phys. Chem. C J. Phys. Chem. Lett. Analyst Anal. Methods Biomater. Sci. Catal. Sci. Technol. Chem. Commun. Chem. Soc. Rev. CHEM EDUC RES PRACT CRYSTENGCOMM Dalton Trans. Energy Environ. Sci. ENVIRON SCI-NANO ENVIRON SCI-PROC IMP ENVIRON SCI-WAT RES Faraday Discuss. Food Funct. Green Chem. Inorg. Chem. Front. Integr. Biol. J. Anal. At. Spectrom. J. Mater. Chem. A J. Mater. Chem. B J. Mater. Chem. C Lab Chip Mater. Chem. Front. Mater. Horiz. MEDCHEMCOMM Metallomics Mol. Biosyst. Mol. Syst. Des. Eng. Nanoscale Nanoscale Horiz. Nat. Prod. Rep. New J. Chem. Org. Biomol. Chem. Org. Chem. Front. PHOTOCH PHOTOBIO SCI PCCP Polym. Chem.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1