Pub Date : 2024-08-01Epub Date: 2024-07-09DOI: 10.1007/s10930-024-10210-3
Reabetswe Maake, Ikechukwu Achilonu
Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg2+ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg2+. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg2+, while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg2+ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg2+. Conclusively, the presence of Mg2+ induces a conformation in KpPrAT that favours nucleotide binding.
因长期住院而感染并在患者入住医疗机构两天后表现出来的感染可归类为医院获得性感染。肺炎克雷伯氏菌(K. pneumoniae)已成为一种严重的病原体,由于对碳青霉烯类耐药菌株的高化脓性发病率不断上升,已引起全球的严重关注。谷胱甘肽是一种氧化还原蛋白,能保护细胞免受氧化应激,因为它能与谷胱甘肽结合还原混合二硫化物。蛋白腺苷基转移酶(PrAT)是一种假激酶,其作用机制是将 AMP 基团从 ATP 转移到 Glutaredoxin。通过抑制 PrAT 的活性来诱导细菌产生氧化应激是一种很有前景的方法,可有效防止医院感染。因此,本研究旨在过表达、纯化和分析 ATP 与 Mg2+ 结合对肺炎克雷伯菌 PrAT(KpPrAT)的影响。pET 表达系统和镍亲和层析能有效表达和纯化 KpPrAT。远紫外 CD 光谱显示,即使在 Mg2+ 存在的情况下,该蛋白质也主要是 α-螺旋形的。用 ANS 进行的外荧光光谱分析表明,在 ATP 和 Mg2+ 存在的情况下,KpPrAT 存在一个疏水口袋,而 mant-ATP 研究则暗示了 KpPrAT 潜在的核苷酸结合能力。Mg2+ 的存在增加了蛋白质的热稳定性。通过等温滴定量热法,我们可以深入了解 ATP 与 KpPrAT(无论有无 Mg2+)结合的亲和力和热力学参数。结论是,Mg2+ 的存在会诱导 KpPrAT 形成有利于核苷酸结合的构象。
{"title":"Expression, Purification and Biophysical Characterisation of Klebsiella Pneumoniae Protein Adenylyltransferase: A Systematic Integration of Empirical and Computational Modelling Approaches.","authors":"Reabetswe Maake, Ikechukwu Achilonu","doi":"10.1007/s10930-024-10210-3","DOIUrl":"10.1007/s10930-024-10210-3","url":null,"abstract":"<p><p>Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg<sup>2+</sup> binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg<sup>2+</sup>. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg<sup>2+</sup>, while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg<sup>2+</sup> increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg<sup>2+</sup>. Conclusively, the presence of Mg<sup>2+</sup> induces a conformation in KpPrAT that favours nucleotide binding.</p>","PeriodicalId":94249,"journal":{"name":"The protein journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11345332/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141565500","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-01Epub Date: 2024-07-09DOI: 10.1007/s10930-024-10214-z
Gustavo Sganzerla Martinez, Mansi Dutt, Anuj Kumar, David J Kelvin
Determining the physicochemical properties of a protein can reveal important insights in their structure, biological functions, stability, and interactions with other molecules. Although tools for computing properties of proteins already existed, we could not find a comprehensive tool that enables the calculations of multiple properties for multiple input proteins on the proteome level at once. Facing this limitation, we developed Multiple Protein Profiler (MPP) 1.0 as an integrated tool that allows the profiling of 12 individual properties of multiple proteins in a significant manner. MPP provides a tabular and graphic visualization of properties of multiple proteins. The tool is freely accessible at https://mproteinprofiler.microbiologyandimmunology.dal.ca/ .
{"title":"Multiple Protein Profiler 1.0 (MPP): A Webserver for Predicting and Visualizing Physiochemical Properties of Proteins at the Proteome Level.","authors":"Gustavo Sganzerla Martinez, Mansi Dutt, Anuj Kumar, David J Kelvin","doi":"10.1007/s10930-024-10214-z","DOIUrl":"10.1007/s10930-024-10214-z","url":null,"abstract":"<p><p>Determining the physicochemical properties of a protein can reveal important insights in their structure, biological functions, stability, and interactions with other molecules. Although tools for computing properties of proteins already existed, we could not find a comprehensive tool that enables the calculations of multiple properties for multiple input proteins on the proteome level at once. Facing this limitation, we developed Multiple Protein Profiler (MPP) 1.0 as an integrated tool that allows the profiling of 12 individual properties of multiple proteins in a significant manner. MPP provides a tabular and graphic visualization of properties of multiple proteins. The tool is freely accessible at https://mproteinprofiler.microbiologyandimmunology.dal.ca/ .</p>","PeriodicalId":94249,"journal":{"name":"The protein journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11345329/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141560708","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Renilla luciferase catalyzes the oxidation of coelenterazine to coelenteramide and results in the emission of a photon of light. Although Renilla luciferase has various applications in biotechnology, its low thermal stability limits the development of its applications. Arginine is a well-known stabilizing amino acid that plays a key role in protein stabilization against inactivation. However, its impact on enzyme properties is unpredictable. This study investigates the impact of arginine on the kinetics and thermal stability of Renilla luciferase. The enzyme's performance was significantly enhanced in the presence of arginine, with catalytic efficiency increasing by 3.31-fold and 3.08-fold when exposed to 0.2 M and 0.3 M arginine, respectively. Additionally, arginine improved the thermal stability of Renilla luciferase. Molecular dynamics simulation showed that the addition of 0.2 M arginine reduced the binding of coelenteramide, the reaction product and an enzyme inhibitor, to the active site of the Renilla luciferase. Therefore, the release of the product was accelerated, and the affinity of Renilla luciferase for coelenterazine increased. Furthermore, Molecular dynamics studies indicated an increased network of water molecules surrounding Renilla luciferase in the presence of 0.2 M arginine. This network potentially enhances the hydrophobic effect on the protein structure, ultimately improving enzyme stability. The findings of this study hold promise for the development of commercial kits incorporating Renilla luciferase.
瑞宁拉荧光素酶催化腔肠素氧化成腔肠酰胺,从而发出光子。尽管瑞宁拉荧光素酶在生物技术中有多种应用,但其热稳定性较低,限制了其应用的发展。精氨酸是一种众所周知的稳定氨基酸,在稳定蛋白质防止失活方面起着关键作用。然而,精氨酸对酶特性的影响却难以预测。本研究探讨了精氨酸对雷尼拉荧光素酶动力学和热稳定性的影响。在精氨酸存在的情况下,酶的性能显著提高,当暴露于 0.2 M 和 0.3 M 精氨酸时,催化效率分别提高了 3.31 倍和 3.08 倍。此外,精氨酸还提高了雷尼拉荧光素酶的热稳定性。分子动力学模拟显示,加入 0.2 M 精氨酸后,反应产物和酶抑制剂腔肠酰胺与雷尼拉荧光素酶活性位点的结合减少。因此,产物的释放速度加快,而雷尼拉荧光素酶对腔肠素的亲和力增加。此外,分子动力学研究表明,在 0.2 M 精氨酸存在的情况下,Renilla 荧光素酶周围的水分子网络增加。这种网络可能会增强蛋白质结构的疏水效应,最终提高酶的稳定性。这项研究的结果为开发含有雷尼拉荧光素酶的商业试剂盒带来了希望。
{"title":"Exploring the Potential of Arginine to Increase Coelenterazine-Renilla Luciferase Affinity and Enzyme Stability: Kinetic and Molecular Dynamics Studies.","authors":"Maryam Salehian, Rahman Emamzadeh, Mahboobeh Nazari","doi":"10.1007/s10930-024-10208-x","DOIUrl":"10.1007/s10930-024-10208-x","url":null,"abstract":"<p><p>Renilla luciferase catalyzes the oxidation of coelenterazine to coelenteramide and results in the emission of a photon of light. Although Renilla luciferase has various applications in biotechnology, its low thermal stability limits the development of its applications. Arginine is a well-known stabilizing amino acid that plays a key role in protein stabilization against inactivation. However, its impact on enzyme properties is unpredictable. This study investigates the impact of arginine on the kinetics and thermal stability of Renilla luciferase. The enzyme's performance was significantly enhanced in the presence of arginine, with catalytic efficiency increasing by 3.31-fold and 3.08-fold when exposed to 0.2 M and 0.3 M arginine, respectively. Additionally, arginine improved the thermal stability of Renilla luciferase. Molecular dynamics simulation showed that the addition of 0.2 M arginine reduced the binding of coelenteramide, the reaction product and an enzyme inhibitor, to the active site of the Renilla luciferase. Therefore, the release of the product was accelerated, and the affinity of Renilla luciferase for coelenterazine increased. Furthermore, Molecular dynamics studies indicated an increased network of water molecules surrounding Renilla luciferase in the presence of 0.2 M arginine. This network potentially enhances the hydrophobic effect on the protein structure, ultimately improving enzyme stability. The findings of this study hold promise for the development of commercial kits incorporating Renilla luciferase.</p>","PeriodicalId":94249,"journal":{"name":"The protein journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141187227","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}
HSPB8 is a heat shock protein belonging to a family of ATP-independent stress proteins called HSPB which are present far and wide in the cells of various organisms. They are committed to protein quality control (PQC) and strive to avert protein aggregation and to procreate a pool of non-native proteins that can be swiftly folded. Their fundamental expression or stress inducibility is regulated by various cis-elements localized in the HSPB regulatory regions. In the current study we have predicted and confirmed two alternatively spliced novel transcripts of HSPB8 gene in liver, brain, and heart. These spliced variants have smaller sizes owing to smaller N terminal regions and showed remarkable changes in their cellular localization. Novel isoform (HSPB8-N1) was predicted to be majorly localized to nuclear region while the reported isoform (HSPB8) and one of the novel isoforms (HSPB8-N2) were predicted to be cytoplasmic in nature. There were many changes observed in the phosphorylation sites of the novel isoforms as well. The newly reported isoforms lack several structural motifs that are essential for various functional endeavors of the HSPB8 protein. In silico analysis of the conceptually translated protein was carried out using various bioinformatics tools to gain an understanding of their properties in order to explore their possible potential in therapeutics.
HSPB8 是一种热休克蛋白,属于称为 HSPB 的不依赖 ATP 的应激蛋白家族。它们致力于蛋白质质量控制(PQC),努力避免蛋白质聚集,并生成可快速折叠的非原生蛋白质池。它们的基本表达或应激诱导性受 HSPB 调控区域中的各种顺式元素调控。在目前的研究中,我们预测并证实了肝脏、大脑和心脏中 HSPB8 基因的两种交替剪接的新型转录本。由于 N 端区域较小,这些剪接变体的体积也较小,而且它们的细胞定位也发生了显著变化。据预测,新型异构体(HSPB8-N1)主要定位于核区,而已报道的异构体(HSPB8)和新型异构体之一(HSPB8-N2)则定位于细胞质。新型异构体的磷酸化位点也发生了许多变化。新报道的异构体缺乏几个对 HSPB8 蛋白的各种功能至关重要的结构基序。我们利用各种生物信息学工具对概念翻译蛋白进行了硅学分析,以了解它们的特性,从而探索它们在治疗方面的潜力。
{"title":"Identification of Alternatively Spliced Novel Isoforms of Human HSPB8 Gene.","authors":"Naira Rashid, Pallavi Juneja, Akshat Rathi, Insha Sultan, Sayeed Ur Rehman","doi":"10.1007/s10930-024-10215-y","DOIUrl":"10.1007/s10930-024-10215-y","url":null,"abstract":"<p><p>HSPB8 is a heat shock protein belonging to a family of ATP-independent stress proteins called HSPB which are present far and wide in the cells of various organisms. They are committed to protein quality control (PQC) and strive to avert protein aggregation and to procreate a pool of non-native proteins that can be swiftly folded. Their fundamental expression or stress inducibility is regulated by various cis-elements localized in the HSPB regulatory regions. In the current study we have predicted and confirmed two alternatively spliced novel transcripts of HSPB8 gene in liver, brain, and heart. These spliced variants have smaller sizes owing to smaller N terminal regions and showed remarkable changes in their cellular localization. Novel isoform (HSPB8-N1) was predicted to be majorly localized to nuclear region while the reported isoform (HSPB8) and one of the novel isoforms (HSPB8-N2) were predicted to be cytoplasmic in nature. There were many changes observed in the phosphorylation sites of the novel isoforms as well. The newly reported isoforms lack several structural motifs that are essential for various functional endeavors of the HSPB8 protein. In silico analysis of the conceptually translated protein was carried out using various bioinformatics tools to gain an understanding of their properties in order to explore their possible potential in therapeutics.</p>","PeriodicalId":94249,"journal":{"name":"The protein journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141560707","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}
Pub Date : 2024-07-29DOI: 10.1007/s10930-024-10227-8
Benjamin P Todd, Kevin M Downard
It is demonstrated, for the first time, that a mass spectrometry approach (known as phylonumerics) can be successfully implemented for structural phylogenetics investigations to chart the evolution of a protein's structure and function. Illustrated for the compact globular protein myoglobin, peptide masses produced from the proteolytic digestion of the protein across animal species generate trees congruent to the sequence tree counterparts. Single point mutations calculated during the same mass tree building step can be followed along interconnected branches of the tree and represent a viable structural metric. A mass tree built for 15 diverse animal species, easily resolve the birds from mammal species, and the ruminant mammals from the remainder of the animals. Mutations within helix-spanning peptide segments alter both the mass and structure of the protein in these segments. Greater evolution is found in the B-helix over the A, E, F, G and H helices. A further mass tree study, of six more closely related primate species, resolves gorilla from the other primates based on a P22S mutation within the B-helix. The remaining five primates are resolved into two groups based on whether they contain a glycine or serine at position 23 in the same helix. The orangutan is resolved from the gibbon and siamang by its G-helix C110S mutation, while homo sapiens are resolved from chimpanzee based on the Q116H mutation. All are associated with structural perturbations in such helices. These structure altering mutations can be tracked along interconnecting branches of a mass tree, to follow the protein's structure and evolution, and ultimately the evolution of the species in which the proteins are expressed. Those that have the greatest impact on a protein's structure, its function, and ultimately the evolution of the species, can be selectively tracked or monitored.
该研究首次证明,质谱方法(称为 "系统数值学")可成功应用于结构系统学研究,以绘制蛋白质结构和功能的进化图谱。以结构紧凑的球状蛋白质肌红蛋白为例,通过对不同动物物种的蛋白质进行蛋白酶解产生的肽质量生成了与序列树对应的树。在同一质量树构建步骤中计算出的单点突变可沿着质量树相互连接的分支进行追踪,是一种可行的结构度量方法。为 15 个不同动物物种构建的质量树可以很容易地将鸟类与哺乳动物物种区分开来,并将反刍哺乳动物与其他动物区分开来。跨越螺旋的肽段内的突变会改变这些肽段内蛋白质的质量和结构。与 A、E、F、G 和 H 螺旋相比,B 螺旋的进化程度更高。对六种亲缘关系更近的灵长类动物进行的进一步质量树研究,根据 B 螺旋中的 P22S 突变,将大猩猩与其他灵长类动物区分开来。其余五种灵长类动物则根据它们在同一螺旋的第 23 位含有甘氨酸还是丝氨酸分成了两组。猩猩因其 G 螺旋 C110S 突变而与长臂猿和暹罗猿区分开来,而智人则因 Q116H 突变而与黑猩猩区分开来。所有这些都与这些螺旋的结构扰动有关。这些改变结构的突变可以沿着质量树相互连接的分支进行追踪,以了解蛋白质的结构和进化,并最终了解表达蛋白质的物种的进化。可以有选择地跟踪或监测那些对蛋白质结构、功能以及物种进化影响最大的突变。
{"title":"Structural Phylogenetics with Protein Mass Spectrometry: A Proof-of-Concept.","authors":"Benjamin P Todd, Kevin M Downard","doi":"10.1007/s10930-024-10227-8","DOIUrl":"https://doi.org/10.1007/s10930-024-10227-8","url":null,"abstract":"<p><p>It is demonstrated, for the first time, that a mass spectrometry approach (known as phylonumerics) can be successfully implemented for structural phylogenetics investigations to chart the evolution of a protein's structure and function. Illustrated for the compact globular protein myoglobin, peptide masses produced from the proteolytic digestion of the protein across animal species generate trees congruent to the sequence tree counterparts. Single point mutations calculated during the same mass tree building step can be followed along interconnected branches of the tree and represent a viable structural metric. A mass tree built for 15 diverse animal species, easily resolve the birds from mammal species, and the ruminant mammals from the remainder of the animals. Mutations within helix-spanning peptide segments alter both the mass and structure of the protein in these segments. Greater evolution is found in the B-helix over the A, E, F, G and H helices. A further mass tree study, of six more closely related primate species, resolves gorilla from the other primates based on a P22S mutation within the B-helix. The remaining five primates are resolved into two groups based on whether they contain a glycine or serine at position 23 in the same helix. The orangutan is resolved from the gibbon and siamang by its G-helix C110S mutation, while homo sapiens are resolved from chimpanzee based on the Q116H mutation. All are associated with structural perturbations in such helices. These structure altering mutations can be tracked along interconnecting branches of a mass tree, to follow the protein's structure and evolution, and ultimately the evolution of the species in which the proteins are expressed. Those that have the greatest impact on a protein's structure, its function, and ultimately the evolution of the species, can be selectively tracked or monitored.</p>","PeriodicalId":94249,"journal":{"name":"The protein journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141794471","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}
Pub Date : 2024-06-01Epub Date: 2024-05-14DOI: 10.1007/s10930-024-10204-1
Handan Açelya Kapkaç, Muhittin Arslanyolu
Glutathione-S-transferase enzymes (GSTs) are essential components of the phase II detoxification system and protect organisms from oxidative stress induced by xenobiotics and harmful toxins such as 1-chloro-2,4-dinitrobenzene (CDNB). In Tetrahymena thermophila, the TtGSTm34 gene was previously reported to be one of the most responsive GST genes to CDNB treatment (LD50 = 0.079 mM). This study aimed to determine the kinetic features of recombinantly expressed and purified TtGSTm34 with CDNB and glutathione (GSH). TtGSTm34-8xHis was recombinantly produced in T. thermophila as a 25-kDa protein after the cloning of the 660-bp full-length ORF of TtGSTm34 into the pIGF-1 vector. A three-dimensional model of the TtGSTm34 protein constructed by the AlphaFold and PyMOL programs confirmed that it has structurally conserved and folded GST domains. The recombinant production of TtGSTm34-8xHis was confirmed by SDS‒PAGE and Western blot analysis. A dual-affinity chromatography strategy helped to purify TtGSTm34-8xHis approximately 3166-fold. The purified recombinant TtGSTm34-8xHis exhibited significantly high enzyme activity with CDNB (190 µmol/min/mg) as substrate. Enzyme kinetic analysis revealed Km values of 0.68 mM with GSH and 0.40 mM with CDNB as substrates, confirming its expected high affinity for CDNB. The optimum pH and temperature were determined to be 7.0 and 25 °C, respectively. Ethacrynic acid inhibited fully TtGSTm34-8xHis enzyme activity. These results imply that TtGSTm34 of T. thermophila plays a major role in the detoxification of xenobiotics, such as CDNB, as a first line of defense in aquatic protists against oxidative damage.
{"title":"Molecular Cloning, Expression and Enzymatic Characterization of Tetrahymena thermophila Glutathione-S-Transferase Mu 34.","authors":"Handan Açelya Kapkaç, Muhittin Arslanyolu","doi":"10.1007/s10930-024-10204-1","DOIUrl":"10.1007/s10930-024-10204-1","url":null,"abstract":"<p><p>Glutathione-S-transferase enzymes (GSTs) are essential components of the phase II detoxification system and protect organisms from oxidative stress induced by xenobiotics and harmful toxins such as 1-chloro-2,4-dinitrobenzene (CDNB). In Tetrahymena thermophila, the TtGSTm34 gene was previously reported to be one of the most responsive GST genes to CDNB treatment (LD50 = 0.079 mM). This study aimed to determine the kinetic features of recombinantly expressed and purified TtGSTm34 with CDNB and glutathione (GSH). TtGSTm34-8xHis was recombinantly produced in T. thermophila as a 25-kDa protein after the cloning of the 660-bp full-length ORF of TtGSTm34 into the pIGF-1 vector. A three-dimensional model of the TtGSTm34 protein constructed by the AlphaFold and PyMOL programs confirmed that it has structurally conserved and folded GST domains. The recombinant production of TtGSTm34-8xHis was confirmed by SDS‒PAGE and Western blot analysis. A dual-affinity chromatography strategy helped to purify TtGSTm34-8xHis approximately 3166-fold. The purified recombinant TtGSTm34-8xHis exhibited significantly high enzyme activity with CDNB (190 µmol/min/mg) as substrate. Enzyme kinetic analysis revealed K<sub>m</sub> values of 0.68 mM with GSH and 0.40 mM with CDNB as substrates, confirming its expected high affinity for CDNB. The optimum pH and temperature were determined to be 7.0 and 25 °C, respectively. Ethacrynic acid inhibited fully TtGSTm34-8xHis enzyme activity. These results imply that TtGSTm34 of T. thermophila plays a major role in the detoxification of xenobiotics, such as CDNB, as a first line of defense in aquatic protists against oxidative damage.</p>","PeriodicalId":94249,"journal":{"name":"The protein journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140924242","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}
Pub Date : 2024-06-01Epub Date: 2024-03-07DOI: 10.1007/s10930-024-10182-4
Anabella L Origone, Esteban G Vega Hissi, Constanza S Liggieri, Gerardo E Camí, Andrés Illanes, Sonia E Barberis
The present study aims at understanding the effect of organic solvents on the specific proteolytic activity and operational stability of asclepain cI in aqueous-organic media, using correlations between geometrical and structural parameters of asclepain cI. These correlations were determined by molecular dynamics (MD) simulations and the secondary structure of the enzyme validated by Fourier-transform Infrared (FTIR) spectroscopy. Asclepain cI exhibited significantly higher catalytic potential in 29 of the 42 aqueous-organic media tested, composed by 0.1 mM TRIS hydrochloride buffer pH 8 (TCB) and an organic solvent, than in buffer alone. Asclepain cI in water-organic miscible systems showed high FTIR spectral similarity with that obtained in TCB, while in immiscible systems the enzyme acquired different secondary structures than in buffer. Among the conditions studied, asclepain cI showed the highest catalytic potential in 50% v/v ethyl acetate in TCB. According to MD simulations, that medium elicited solvation and flexibility changes around the active center of asclepain cI and conducted to a new secondary structure with the active center preserved. These results provide valuable insights into the elucidation of the molecular mechanism of asclepain cI tolerance to organic solvents and pave the way for its future application for the synthesis of peptides in aqueous-organic media.
本研究旨在利用 Asclepain cI 的几何和结构参数之间的相关性,了解有机溶剂对 asclepain cI 在水有机介质中的特定蛋白水解活性和操作稳定性的影响。这些相关性由分子动力学(MD)模拟确定,酶的二级结构由傅立叶变换红外光谱(FTIR)验证。在由 0.1 mM TRIS hydrochloride buffer pH 8 (TCB) 和有机溶剂组成的 42 种测试水有机介质中,Asclepain cI 在 29 种介质中表现出的催化潜能明显高于仅在缓冲液中的催化潜能。水-有机混溶体系中的 Asclepain cI 与在 TCB 中获得的傅立叶变换红外光谱具有很高的相似性,而在不混溶体系中,酶获得的二级结构与缓冲液中的不同。在所研究的条件中,asclepain cI 在 TCB 中的 50% v/v 乙酸乙酯中显示出最高的催化潜能。根据 MD 模拟,该介质引起了 asclepain cI 活性中心周围的溶解和柔性变化,并在保留活性中心的情况下形成了新的二级结构。这些结果为阐明 Asclepain cI 耐有机溶剂的分子机理提供了有价值的见解,并为其今后在水有机介质中合成多肽的应用铺平了道路。
{"title":"Effect of Organic Solvents on the Activity, Stability and Secondary Structure of asclepain cI, Using FTIR and Molecular Dynamics Simulations.","authors":"Anabella L Origone, Esteban G Vega Hissi, Constanza S Liggieri, Gerardo E Camí, Andrés Illanes, Sonia E Barberis","doi":"10.1007/s10930-024-10182-4","DOIUrl":"10.1007/s10930-024-10182-4","url":null,"abstract":"<p><p>The present study aims at understanding the effect of organic solvents on the specific proteolytic activity and operational stability of asclepain cI in aqueous-organic media, using correlations between geometrical and structural parameters of asclepain cI. These correlations were determined by molecular dynamics (MD) simulations and the secondary structure of the enzyme validated by Fourier-transform Infrared (FTIR) spectroscopy. Asclepain cI exhibited significantly higher catalytic potential in 29 of the 42 aqueous-organic media tested, composed by 0.1 mM TRIS hydrochloride buffer pH 8 (TCB) and an organic solvent, than in buffer alone. Asclepain cI in water-organic miscible systems showed high FTIR spectral similarity with that obtained in TCB, while in immiscible systems the enzyme acquired different secondary structures than in buffer. Among the conditions studied, asclepain cI showed the highest catalytic potential in 50% v/v ethyl acetate in TCB. According to MD simulations, that medium elicited solvation and flexibility changes around the active center of asclepain cI and conducted to a new secondary structure with the active center preserved. These results provide valuable insights into the elucidation of the molecular mechanism of asclepain cI tolerance to organic solvents and pave the way for its future application for the synthesis of peptides in aqueous-organic media.</p>","PeriodicalId":94249,"journal":{"name":"The protein journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140061705","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}
Pub Date : 2024-06-01Epub Date: 2024-03-16DOI: 10.1007/s10930-024-10191-3
Hamda Khan, Afreen Khanam, Adnan Ahmad Khan, Rizwan Ahmad, Arbab Husain, Safia Habib, Saheem Ahmad, Moinuddin
Hyperglycaemia is a life-threatening risk factor that occurs in both chronic and acute phases and has been linked to causing injury to many organs. Protein modification was triggered by hyperglycaemic stress, which resulted in pathogenic alterations such as impaired cellular function and tissue damage. Dysregulation in cellular function increases the condition associated with metabolic disorders, including cardiovascular diseases, nephropathy, retinopathy, and neuropathy. Hyperglycaemic stress also increases the proliferation of cancer cells. The major areas of experimental biomedical research have focused on the underlying mechanisms involved in the cellular signalling systems involved in diabetes-associated chronic hyperglycaemia. Reactive oxygen species and oxidative stress generated by hyperglycaemia modify many intracellular signalling pathways that result in insulin resistance and β-cell function degradation. The dysregulation of post translational modification in β cells is clinically associated with the development of diabetes mellitus and its associated diseases. This review will discuss the effect of hyperglycaemic stress on protein modification and the cellular signalling involved in it. The focus will be on the significant molecular changes associated with severe metabolic disorders.
{"title":"The complex landscape of intracellular signalling in protein modification under hyperglycaemic stress leading to metabolic disorders.","authors":"Hamda Khan, Afreen Khanam, Adnan Ahmad Khan, Rizwan Ahmad, Arbab Husain, Safia Habib, Saheem Ahmad, Moinuddin","doi":"10.1007/s10930-024-10191-3","DOIUrl":"10.1007/s10930-024-10191-3","url":null,"abstract":"<p><p>Hyperglycaemia is a life-threatening risk factor that occurs in both chronic and acute phases and has been linked to causing injury to many organs. Protein modification was triggered by hyperglycaemic stress, which resulted in pathogenic alterations such as impaired cellular function and tissue damage. Dysregulation in cellular function increases the condition associated with metabolic disorders, including cardiovascular diseases, nephropathy, retinopathy, and neuropathy. Hyperglycaemic stress also increases the proliferation of cancer cells. The major areas of experimental biomedical research have focused on the underlying mechanisms involved in the cellular signalling systems involved in diabetes-associated chronic hyperglycaemia. Reactive oxygen species and oxidative stress generated by hyperglycaemia modify many intracellular signalling pathways that result in insulin resistance and β-cell function degradation. The dysregulation of post translational modification in β cells is clinically associated with the development of diabetes mellitus and its associated diseases. This review will discuss the effect of hyperglycaemic stress on protein modification and the cellular signalling involved in it. The focus will be on the significant molecular changes associated with severe metabolic disorders.</p>","PeriodicalId":94249,"journal":{"name":"The protein journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140141295","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}
Universal stress proteins (USPs) are widely distributed and play crucial roles in cellular responses to biotic and abiotic stresses. These roles include regulating cell growth and development, cell motility, hypoxia responses, and ion sequestration. With the increasing frequency and intensity of extreme weather events due to climate change, pathogens have developed different strategies to withstand environmental stresses, in which USPs play a significant role in their survival and virulence. In this study, we analyzed the importance of USPs in various organisms, such as archaea, plants, and fungi, as a parameter that influences their survival. We discussed the different types Of USPs and their role, aiming to carry out fundamental research in this field to identify significant constraints for better understanding of USP functions at molecular level. Additionally, we discussed concepts and research techniques that could help overcome these hurdles and facilitate new molecular approaches to better understand and target USPs as important stress adaptation and survival regulators. Although the precise characteristics of USPs are still unclear, numerous innovative uses have already been developed, tested, and implemented. Complementary approaches to basic research and applications, as well as new technology and analytical techniques, may offer insights into the cryptic but crucial activities of USPs in various living systems.
{"title":"Molecular, Structural, and Functional Diversity of Universal Stress Proteins (USPs) in Bacteria, Plants, and Their Biotechnological Applications.","authors":"Bilkees Nabi, Manoj Kumawat, Neeraj Ahlawat, Sushma Ahlawat","doi":"10.1007/s10930-024-10192-2","DOIUrl":"10.1007/s10930-024-10192-2","url":null,"abstract":"<p><p>Universal stress proteins (USPs) are widely distributed and play crucial roles in cellular responses to biotic and abiotic stresses. These roles include regulating cell growth and development, cell motility, hypoxia responses, and ion sequestration. With the increasing frequency and intensity of extreme weather events due to climate change, pathogens have developed different strategies to withstand environmental stresses, in which USPs play a significant role in their survival and virulence. In this study, we analyzed the importance of USPs in various organisms, such as archaea, plants, and fungi, as a parameter that influences their survival. We discussed the different types Of USPs and their role, aiming to carry out fundamental research in this field to identify significant constraints for better understanding of USP functions at molecular level. Additionally, we discussed concepts and research techniques that could help overcome these hurdles and facilitate new molecular approaches to better understand and target USPs as important stress adaptation and survival regulators. Although the precise characteristics of USPs are still unclear, numerous innovative uses have already been developed, tested, and implemented. Complementary approaches to basic research and applications, as well as new technology and analytical techniques, may offer insights into the cryptic but crucial activities of USPs in various living systems.</p>","PeriodicalId":94249,"journal":{"name":"The protein journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140141294","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}
Metallothioneins are a group of cysteine-rich proteins that play an important role in the homeostasis and detoxification of heavy metals. The objective of this research was to explore the significance of metallothionein in Trichoderma harzianum tolerance to zinc. At the inhibitory concentration of 1000 ppm, the fungus adsorbed 16.7 ± 0.4 mg/g of metal. The HPLC and SDS-PAGE electrophoresis data suggested that the fungus production of metallothionein was twice as high in the presence of zinc as in the control group. The examination of the genes; metallothionein expression activator (MEA) and Cu fist revealed that the MEA, with a C2H2 zinc finger domain, increased significantly in the presence of zinc. It was observed that in T. harzianum, the enhanced expression of the metallothionein gene was managed by the metallothionein activator under zinc overload conditions. According to our knowledge, this is the first report on the role of metallothionein in the resistance of T. harzianum to zinc.
{"title":"A Potential Involvement of Metallothionein in the Zinc Tolerance of Trichoderma harzianum: Experimental Findings.","authors":"Negin Ejmalian, Soheila Mirzaei, Asghar Mirzaie-Asl, Mehrdad Chaichi","doi":"10.1007/s10930-024-10185-1","DOIUrl":"10.1007/s10930-024-10185-1","url":null,"abstract":"<p><p>Metallothioneins are a group of cysteine-rich proteins that play an important role in the homeostasis and detoxification of heavy metals. The objective of this research was to explore the significance of metallothionein in Trichoderma harzianum tolerance to zinc. At the inhibitory concentration of 1000 ppm, the fungus adsorbed 16.7 ± 0.4 mg/g of metal. The HPLC and SDS-PAGE electrophoresis data suggested that the fungus production of metallothionein was twice as high in the presence of zinc as in the control group. The examination of the genes; metallothionein expression activator (MEA) and Cu fist revealed that the MEA, with a C<sub>2</sub>H<sub>2</sub> zinc finger domain, increased significantly in the presence of zinc. It was observed that in T. harzianum, the enhanced expression of the metallothionein gene was managed by the metallothionein activator under zinc overload conditions. According to our knowledge, this is the first report on the role of metallothionein in the resistance of T. harzianum to zinc.</p>","PeriodicalId":94249,"journal":{"name":"The protein journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140137648","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}