Pub Date : 2024-04-11DOI: 10.1007/s10930-023-10178-6
Shengwang Jiang, Qingwu W. Shen
It is well known that preslaughter (antemortem) stress such as rough handling, transportation, a negative environment, physical discomfort, lack of consistent routine, and bad feed quality has a big impact on meat quality. The antemortem-induced poor meat quality is characterized by low pH, a pale and exudative appearance, and a soft texture. Previous studies indicate that antemortem stress plays a key role in regulating protein acetylation and glycolysis in postmortem (PM) muscle. However, the underlying molecular and biochemical mechanism is not clearly understood yet. In this study, we investigated the relationship between antemortem and protein acetylation and glycolysis using murine longissimus dorsi muscle isolated from ICR mice and murine muscle cell line C2C12 treated with epinephrine hydrochloride. Because adrenaline secretion increases in stressed animals, epinephrine hydrochloride was intraperitoneally injected epinephrine into mice to simulate pre-slaughter stress in this study to facilitate experimental operations and save experimental costs. Our findings demonstrated that protein acetylation in pyruvate kinase M1 (PKM1) form is significantly reduced by antemortem, and the reduced acetylation subsequently leads to an increase in PKM1 enzymatic activity which causes increased glycolysis in PM muscle. By using molecular approaches, we identified lysine 141 in PKM1 as a critical residue for acetylation. Our results in this study provide useful insight for controlling or improving meat quality in the future.
{"title":"Antemortem Stress Regulates Postmortem Glycolysis in Muscle by Deacetylation of Pyruvate Kinase M1 at K141","authors":"Shengwang Jiang, Qingwu W. Shen","doi":"10.1007/s10930-023-10178-6","DOIUrl":"https://doi.org/10.1007/s10930-023-10178-6","url":null,"abstract":"<p>It is well known that preslaughter (antemortem) stress such as rough handling, transportation, a negative environment, physical discomfort, lack of consistent routine, and bad feed quality has a big impact on meat quality. The antemortem-induced poor meat quality is characterized by low pH, a pale and exudative appearance, and a soft texture. Previous studies indicate that antemortem stress plays a key role in regulating protein acetylation and glycolysis in postmortem (PM) muscle. However, the underlying molecular and biochemical mechanism is not clearly understood yet. In this study, we investigated the relationship between antemortem and protein acetylation and glycolysis using murine longissimus dorsi muscle isolated from ICR mice and murine muscle cell line C<sub>2</sub>C<sub>12</sub> treated with epinephrine hydrochloride. Because adrenaline secretion increases in stressed animals, epinephrine hydrochloride was intraperitoneally injected epinephrine into mice to simulate pre-slaughter stress in this study to facilitate experimental operations and save experimental costs. Our findings demonstrated that protein acetylation in pyruvate kinase M1 (PKM1) form is significantly reduced by antemortem, and the reduced acetylation subsequently leads to an increase in PKM1 enzymatic activity which causes increased glycolysis in PM muscle. By using molecular approaches, we identified lysine 141 in PKM1 as a critical residue for acetylation. Our results in this study provide useful insight for controlling or improving meat quality in the future.</p>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":2.371,"publicationDate":"2024-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140569800","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}
Pub Date : 2024-04-06DOI: 10.1007/s10930-024-10194-0
Abstract
To solve the large size faultiness of Oryza sativa recombinant human serum albumin nanoparticle (OsrHSA NP), the structural discrepancies between OsrHSA and plasma-derived human serum albumin (pdHSA) were analyzed deeply in this research. It demonstrated that there were some subtle structural discrepancies located in subdomain IA and IIA between OsrHSA and pdHSA, which included peptide backbone, disulphide bridge and some amino acids. Firstly, the structural discrepancies were investigated through literature comparison, it inferred that the structural discrepancies resulted from the fatty acid (FA) binding to OsrHSA at site 2 of subdomain IA and IIA. To form a cavity for accommodation of FA molecule in OsrHSA, the peptide backbone structure of subdomain IA and IIA would change, accompanied by the conformational transition of disulphide bridges and side chain structure change of some amino acids in subdomain IA and IIA. These alterations induced the exposure of tryptophan (Trp) and tyrosine (Tyr) residues in subdomain IA and IIA and the decrease of net negative charges of molecular surface. The former would promote more OsrHSA molecules aggregate, and the latter would weaken the electrostatic repulsion. As a result, the size of OsrHSA NP was more extensive than that of pdHSA NP (175.84 ± 15.63 nm vs. 31.67 ± 1.31 nm) when the concentration of Dimethyl Sulphoxide (DMSO) was 30% (v/v). In this study, the experimental scheme of OsrHSA NP preparation was improved. There were two changes in the enhanced preparation scheme: pH 8.2 PBS buffer and 63% DMSO. It indicated that the improved OsrHSA NP carrier was comparable to the pdHSA NP carrier. The size and drug loading of paclitaxel-loaded improved OsrHSA NP were 53.57 ± 3.63 nm and 7.25 ± 0.46% (w/w), and those of docetaxel-loaded improved OsrHSA NP were 44.75 ± 2.26 nm and 8.43 ± 0.74% (w/w). Moreover, both NPs exhibited good stability for 168 h at 7.4 pH values. It is established that the improved OsrHSA NP is comparable to the pdHSA NP as a taxane delivery system.
{"title":"The Investigation of the Subtle Structural Discrepancies between Oryza Sativa Recombinant and Plasma-Derived Human Serum Albumins to Design a Novel Nanoparticle as a Taxane Delivery System","authors":"","doi":"10.1007/s10930-024-10194-0","DOIUrl":"https://doi.org/10.1007/s10930-024-10194-0","url":null,"abstract":"<h3>Abstract</h3> <p>To solve the large size faultiness of <em>Oryza sativa</em> recombinant human serum albumin nanoparticle (OsrHSA NP), the structural discrepancies between OsrHSA and plasma-derived human serum albumin (pdHSA) were analyzed deeply in this research. It demonstrated that there were some subtle structural discrepancies located in subdomain IA and IIA between OsrHSA and pdHSA, which included peptide backbone, disulphide bridge and some amino acids. Firstly, the structural discrepancies were investigated through literature comparison, it inferred that the structural discrepancies resulted from the fatty acid (FA) binding to OsrHSA at site 2 of subdomain IA and IIA. To form a cavity for accommodation of FA molecule in OsrHSA, the peptide backbone structure of subdomain IA and IIA would change, accompanied by the conformational transition of disulphide bridges and side chain structure change of some amino acids in subdomain IA and IIA. These alterations induced the exposure of tryptophan (Trp) and tyrosine (Tyr) residues in subdomain IA and IIA and the decrease of net negative charges of molecular surface. The former would promote more OsrHSA molecules aggregate, and the latter would weaken the electrostatic repulsion. As a result, the size of OsrHSA NP was more extensive than that of pdHSA NP (175.84 ± 15.63 nm vs. 31.67 ± 1.31 nm) when the concentration of Dimethyl Sulphoxide (DMSO) was 30% (v/v). In this study, the experimental scheme of OsrHSA NP preparation was improved. There were two changes in the enhanced preparation scheme: pH 8.2 PBS buffer and 63% DMSO. It indicated that the improved OsrHSA NP carrier was comparable to the pdHSA NP carrier. The size and drug loading of paclitaxel-loaded improved OsrHSA NP were 53.57 ± 3.63 nm and 7.25 ± 0.46% (w/w), and those of docetaxel-loaded improved OsrHSA NP were 44.75 ± 2.26 nm and 8.43 ± 0.74% (w/w). Moreover, both NPs exhibited good stability for 168 h at 7.4 pH values. It is established that the improved OsrHSA NP is comparable to the pdHSA NP as a taxane delivery system.</p>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":2.371,"publicationDate":"2024-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140569367","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}
Pub Date : 2024-04-01Epub Date: 2023-08-25DOI: 10.1007/s10930-023-10141-5
Pavel Buslaev, Gerrit Groenhof
Molecular dynamics (MD) simulations are routinely performed of biomolecules in solution, because this is their native environment. However, the structures used in such simulations are often obtained with X-ray crystallography, which provides the atomic coordinates of the biomolecule in a crystal environment. With the advent of free electron lasers and time-resolved techniques, X-ray crystallography can now also access metastable states that are intermediates in a biochemical process. Such experiments provide additional data, which can be used, for example, to optimize MD force fields. Doing so requires that the simulation of the biomolecule is also performed in the crystal environment. However, in contrast to simulations of biomolecules in solution, setting up a crystal is challenging. In particular, because not all solvent molecules are resolved in X-ray crystallography, adding a suitable number of solvent molecules, such that the properties of the crystallographic unit cell are preserved in the simulation, can be difficult and typically is a trial-and-error based procedure requiring manual interventions. Such interventions preclude high throughput applications. To overcome this bottleneck, we introduce gmXtal, a tool for setting up crystal simulations for MD simulations with GROMACS. With the information from the protein data bank (rcsb.org) gmXtal automatically (i) builds the crystallographic unit cell; (ii) sets the protonation of titratable residues; (iii) builds missing residues that were not resolved experimentally; and (iv) adds an appropriate number of solvent molecules to the system. gmXtal is available as a standalone tool https://gitlab.com/pbuslaev/gmxtal .
分子动力学(MD)模拟通常是对溶液中的生物分子进行模拟,因为这是生物分子的原生环境。然而,此类模拟中使用的结构通常是通过 X 射线晶体学获得的,X 射线晶体学提供了晶体环境中生物大分子的原子坐标。随着自由电子激光器和时间分辨技术的出现,X 射线晶体学现在还能获取作为生化过程中间产物的蜕变态。这些实验提供了额外的数据,可用于优化 MD 力场等。这样做需要在晶体环境中对生物分子进行模拟。然而,与溶液中的生物分子模拟相比,晶体的建立具有挑战性。特别是,由于并非所有的溶剂分子都能在 X 射线晶体学中解析,因此要添加适当数量的溶剂分子,从而在模拟中保留晶体学单元格的特性,可能会非常困难,而且通常是一个需要人工干预的试错过程。这种干预排除了高通量应用。为了克服这一瓶颈,我们推出了 gmXtal,这是一种利用 GROMACS 进行 MD 模拟的晶体模拟设置工具。利用蛋白质数据库(rcsb.org)中的信息,gmXtal 可自动:(i) 建立晶体学单元格;(ii) 设置可滴定残基的质子化;(iii) 建立实验中未解析的缺失残基;(iv) 为系统添加适当数量的溶剂分子。
{"title":"gmXtal: Cooking Crystals with GROMACS.","authors":"Pavel Buslaev, Gerrit Groenhof","doi":"10.1007/s10930-023-10141-5","DOIUrl":"10.1007/s10930-023-10141-5","url":null,"abstract":"<p><p>Molecular dynamics (MD) simulations are routinely performed of biomolecules in solution, because this is their native environment. However, the structures used in such simulations are often obtained with X-ray crystallography, which provides the atomic coordinates of the biomolecule in a crystal environment. With the advent of free electron lasers and time-resolved techniques, X-ray crystallography can now also access metastable states that are intermediates in a biochemical process. Such experiments provide additional data, which can be used, for example, to optimize MD force fields. Doing so requires that the simulation of the biomolecule is also performed in the crystal environment. However, in contrast to simulations of biomolecules in solution, setting up a crystal is challenging. In particular, because not all solvent molecules are resolved in X-ray crystallography, adding a suitable number of solvent molecules, such that the properties of the crystallographic unit cell are preserved in the simulation, can be difficult and typically is a trial-and-error based procedure requiring manual interventions. Such interventions preclude high throughput applications. To overcome this bottleneck, we introduce gmXtal, a tool for setting up crystal simulations for MD simulations with GROMACS. With the information from the protein data bank (rcsb.org) gmXtal automatically (i) builds the crystallographic unit cell; (ii) sets the protonation of titratable residues; (iii) builds missing residues that were not resolved experimentally; and (iv) adds an appropriate number of solvent molecules to the system. gmXtal is available as a standalone tool https://gitlab.com/pbuslaev/gmxtal .</p>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11058868/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10058074","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-20DOI: 10.1007/s10930-024-10193-1
Jose Carlos Santos Salgado, Robson Carlos Alnoch, Maria de Lourdes Teixeira de Moraes Polizeli, Richard John Ward
Biological macromolecules are found in different shapes and sizes. Among these, enzymes catalyze biochemical reactions and are essential in all organisms, but is there a limit size for them to function properly? Large enzymes such as catalases have hundreds of kDa and are formed by multiple subunits, whereas most enzymes are smaller, with molecular weights of 20–60 kDa. Enzymes smaller than 10 kDa could be called microenzymes and the present literature review brings together evidence of their occurrence in nature. Additionally, bioactive peptides could be a natural source for novel microenzymes hidden in larger peptides and molecular downsizing could be useful to engineer artificial enzymes with low molecular weight improving their stability and heterologous expression. An integrative approach is crucial to discover and determine the amino acid sequences of novel microenzymes, together with their genomic identification and their biochemical biological and evolutionary functions.
{"title":"Microenzymes: Is There Anybody Out There?","authors":"Jose Carlos Santos Salgado, Robson Carlos Alnoch, Maria de Lourdes Teixeira de Moraes Polizeli, Richard John Ward","doi":"10.1007/s10930-024-10193-1","DOIUrl":"https://doi.org/10.1007/s10930-024-10193-1","url":null,"abstract":"<p>Biological macromolecules are found in different shapes and sizes. Among these, enzymes catalyze biochemical reactions and are essential in all organisms, but is there a limit size for them to function properly? Large enzymes such as catalases have hundreds of kDa and are formed by multiple subunits, whereas most enzymes are smaller, with molecular weights of 20–60 kDa. Enzymes smaller than 10 kDa could be called microenzymes and the present literature review brings together evidence of their occurrence in nature. Additionally, bioactive peptides could be a natural source for novel microenzymes hidden in larger peptides and molecular downsizing could be useful to engineer artificial enzymes with low molecular weight improving their stability and heterologous expression. An integrative approach is crucial to discover and determine the amino acid sequences of novel microenzymes, together with their genomic identification and their biochemical biological and evolutionary functions.</p>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":2.371,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140166986","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}
Pub Date : 2024-03-02DOI: 10.1007/s10930-024-10187-z
Neha Kausar Ansari, Amaan Rais, Aabgeena Naeem
Protein aggregation is related to numerous pathological conditions like Alzheimer’s and Parkinson’s disease. In our study, we have shown that an already existing FDA-approved drug; methotrexate (MTX) can be reprofiled on preformed α-chymotrypsinogen A (α-Cgn A) aggregates. The zymogen showed formation of aggregates upon interaction with mercuric ions, with increasing concentration of Hg2Cl2 (0-150 µM). The hike in ThT and ANS fluorescence concomitant with blue shift, bathochromic shift and the hyperchromic effect in the CR absorbance, RLS and turbidity measurements, substantiate the zymogen β-rich aggregate formation. The secondary structural alterations of α- Cgn A as analyzed by CD measurements, FTIR and Raman spectra showed the transformation of native β-barrel conformation to β-inter-molecular rich aggregates. The native α- Cgn A have about 30% α-helical content which was found to be about 3% in presence of mercuric ions suggesting the formation of aggregates. The amorphous aggregates were visualized by SEM. On incubation of Hg2Cl2 treated α- Cgn A with increasing concentration of the MTX resulted in reversing aggregates to the native-like structure. These results were supported by remarkable decrease in ThT and ANS fluorescence intensities and CR absorbance and also consistent with CD, FTIR, and Raman spectroscopy data. MTX was found to increase the α-helical content of the zymogen from 3 to 15% proposing that drug is efficient in disrupting the β-inter-molecular rich aggregates and reverting it to native like structure. The SEM images are in accordance with CD data showing the disintegration of aggregates. The most effective concentration of the drug was found to be 120 µM. Molecular docking analysis showed that MTX molecule was surrounded by the hydrophobic residues including Phe39, His40, Arg145, Tyr146, Thr151, Gly193, Ser195, and Gly216 and conventional hydrogen bonds, including Gln73 (bond length: 2.67Å), Gly142 (2.59Å), Thr144 (2.81Å), Asn150 (2.73Å), Asp153 (2.71Å), and Cys191 (2.53Å). This investigation will help to find the use of already existing drugs to cure protein misfolding-related abnormalities.
{"title":"Methotrexate for Drug Repurposing as an Anti-Aggregatory Agent to Mercuric Treated α-Chymotrypsinogen-A","authors":"Neha Kausar Ansari, Amaan Rais, Aabgeena Naeem","doi":"10.1007/s10930-024-10187-z","DOIUrl":"https://doi.org/10.1007/s10930-024-10187-z","url":null,"abstract":"<p>Protein aggregation is related to numerous pathological conditions like Alzheimer’s and Parkinson’s disease. In our study, we have shown that an already existing FDA-approved drug; methotrexate (MTX) can be reprofiled on preformed α-chymotrypsinogen A (α-Cgn A) aggregates. The zymogen showed formation of aggregates upon interaction with mercuric ions, with increasing concentration of Hg<sub>2</sub>Cl<sub>2</sub> (0-150 µM). The hike in ThT and ANS fluorescence concomitant with blue shift, bathochromic shift and the hyperchromic effect in the CR absorbance, RLS and turbidity measurements, substantiate the zymogen β-rich aggregate formation. The secondary structural alterations of α- Cgn A as analyzed by CD measurements, FTIR and Raman spectra showed the transformation of native β-barrel conformation to β-inter-molecular rich aggregates. The native α- Cgn A have about 30% α-helical content which was found to be about 3% in presence of mercuric ions suggesting the formation of aggregates. The amorphous aggregates were visualized by SEM. On incubation of Hg<sub>2</sub>Cl<sub>2</sub> treated α- Cgn A with increasing concentration of the MTX resulted in reversing aggregates to the native-like structure. These results were supported by remarkable decrease in ThT and ANS fluorescence intensities and CR absorbance and also consistent with CD, FTIR, and Raman spectroscopy data. MTX was found to increase the α-helical content of the zymogen from 3 to 15% proposing that drug is efficient in disrupting the β-inter-molecular rich aggregates and reverting it to native like structure. The SEM images are in accordance with CD data showing the disintegration of aggregates. The most effective concentration of the drug was found to be 120 µM. Molecular docking analysis showed that MTX molecule was surrounded by the hydrophobic residues including Phe39, His40, Arg145, Tyr146, Thr151, Gly193, Ser195, and Gly216 and conventional hydrogen bonds, including Gln73 (bond length: 2.67Å), Gly142 (2.59Å), Thr144 (2.81Å), Asn150 (2.73Å), Asp153 (2.71Å), and Cys191 (2.53Å). This investigation will help to find the use of already existing drugs to cure protein misfolding-related abnormalities.</p>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-03-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140017404","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}
S100A8 and S100A9 belong to the calcium-binding, damage associated molecular pattern (DAMP) proteins shown to aggravate the pathogenesis of rheumatoid arthritis (RA) through their interaction with the TLR4, RAGE and CD36 receptors. S100A8 and S100A9 proteins tend to exist in monomeric, homo and heterodimeric forms, which have been implicated in the pathogenesis of RA, via interacting with Pattern Recognition receptors (PRRs). The study aims to assess the influence of changes in the structure and biological assembly of S100A8 and S100A9 proteins as well as their interaction with significant receptors in RA through computational methods and surface plasmon resonance (SPR) analysis. Molecular docking analysis revealed that the S100A9 homodimer and S100A8/A9 heterodimer showed higher binding affinity towards the target receptors. Most S100 proteins showed good binding affinity towards TLR4 compared to other receptors. Based on the 50 ns MD simulations, TLR4, RAGE, and CD36 formed stable complexes with the monomeric and dimeric forms of S100A8 and S100A9 proteins. However, SPR analysis showed that the S100A8/A9 heterodimers formed stable complexes and exhibited high binding affinity towards the receptors. SPR data also indicated that TLR4 and its interactions with S100A8/A9 proteins may play a primary role in the pathogenesis of RA, with additional contributions from CD36 and RAGE interactions. Subsequent in vitro and in vivo investigations are warranted to corroborate the involvement of S100A8/A9 and the expression of TLR4, RAGE, and CD36 in the pathophysiology of RA.
{"title":"Computational Deciphering of the Role of S100A8 and S100A9 Proteins and Their Changes in the Structure Assembly Influences Their Interaction with TLR4, RAGE, and CD36","authors":"Sivasakthi Paramasivam, Senthamil Selvan Perumal, Sanmuga Priya Ekambaram","doi":"10.1007/s10930-024-10186-0","DOIUrl":"https://doi.org/10.1007/s10930-024-10186-0","url":null,"abstract":"<p>S100A8 and S100A9 belong to the calcium-binding, damage associated molecular pattern (DAMP) proteins shown to aggravate the pathogenesis of rheumatoid arthritis (RA) through their interaction with the TLR4, RAGE and CD36 receptors. S100A8 and S100A9 proteins tend to exist in monomeric, homo and heterodimeric forms, which have been implicated in the pathogenesis of RA, via interacting with Pattern Recognition receptors (PRRs). The study aims to assess the influence of changes in the structure and biological assembly of S100A8 and S100A9 proteins as well as their interaction with significant receptors in RA through computational methods and surface plasmon resonance (SPR) analysis. Molecular docking analysis revealed that the S100A9 homodimer and S100A8/A9 heterodimer showed higher binding affinity towards the target receptors. Most S100 proteins showed good binding affinity towards TLR4 compared to other receptors. Based on the 50 ns MD simulations, TLR4, RAGE, and CD36 formed stable complexes with the monomeric and dimeric forms of S100A8 and S100A9 proteins. However, SPR analysis showed that the S100A8/A9 heterodimers formed stable complexes and exhibited high binding affinity towards the receptors. SPR data also indicated that TLR4 and its interactions with S100A8/A9 proteins may play a primary role in the pathogenesis of RA, with additional contributions from CD36 and RAGE interactions. Subsequent in vitro and in vivo investigations are warranted to corroborate the involvement of S100A8/A9 and the expression of TLR4, RAGE, and CD36 in the pathophysiology of RA.</p>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-03-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140017551","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}
Pub Date : 2023-12-19DOI: 10.1007/s10930-023-10168-8
Abstract
Protein–protein interactions are crucial for the entry of viruses into the cell. Understanding the mechanism of interactions is essential in studying human-virus association, developing new biologics and drug candidates, as well as viral infections and antiviral responses. Experimental methods to analyze human-virus protein–protein interactions based on protein sequence data are time-consuming and labor-intensive, so machine learning models are being developed to predict interactions and determine large-scale interactomes between species. The present work highlights the importance of sequence features in classifying interacting and non-interacting proteins from the protein sequence data. Higher dimensional amino acid sequence features such as Amino Acid Composition (AAC), Dipeptide Composition (DPC), Grouped Amino Acid Composition (GAAC), Pseudo-Amino Acid Composition (PAAC) etc., are extracted. Following feature extraction, three datasets were created: Dataset 1 contains all of the extracted features. While Datasets 2 and 3 contain the most relevant features obtained through dimensionality reduction. To analyze the importance of high-dimensional features and their participation in protein–protein interactions, a random forest classifier is trained on three datasets. With dimensionality reduction, the model exhibited exceptional accuracy, indicating that dimensionality reduction fails to capture the complexity of interactions and the underlying relationships between human and viral proteins. As a result of retaining high-dimensional features, it is possible to capture all the characteristics of protein–protein interactions that resemble host–pathogen associations, leading to the development of biologically meaningful models. Our proposed approach is a more realistic and comprehensive classification model, leading to deeper insights and better applications in virology and drug development.
{"title":"Significance of Sequence Features in Classification of Protein–Protein Interactions Using Machine Learning","authors":"","doi":"10.1007/s10930-023-10168-8","DOIUrl":"https://doi.org/10.1007/s10930-023-10168-8","url":null,"abstract":"<h3>Abstract</h3> <p>Protein–protein interactions are crucial for the entry of viruses into the cell. Understanding the mechanism of interactions is essential in studying human-virus association, developing new biologics and drug candidates, as well as viral infections and antiviral responses. Experimental methods to analyze human-virus protein–protein interactions based on protein sequence data are time-consuming and labor-intensive, so machine learning models are being developed to predict interactions and determine large-scale interactomes between species. The present work highlights the importance of sequence features in classifying interacting and non-interacting proteins from the protein sequence data. Higher dimensional amino acid sequence features such as Amino Acid Composition (AAC), Dipeptide Composition (DPC), Grouped Amino Acid Composition (GAAC), Pseudo-Amino Acid Composition (PAAC) etc., are extracted. Following feature extraction, three datasets were created: Dataset 1 contains all of the extracted features. While Datasets 2 and 3 contain the most relevant features obtained through dimensionality reduction. To analyze the importance of high-dimensional features and their participation in protein–protein interactions, a random forest classifier is trained on three datasets. With dimensionality reduction, the model exhibited exceptional accuracy, indicating that dimensionality reduction fails to capture the complexity of interactions and the underlying relationships between human and viral proteins. As a result of retaining high-dimensional features, it is possible to capture all the characteristics of protein–protein interactions that resemble host–pathogen associations, leading to the development of biologically meaningful models. Our proposed approach is a more realistic and comprehensive classification model, leading to deeper insights and better applications in virology and drug development.</p>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2023-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138816585","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}
Pub Date : 2023-12-08DOI: 10.1007/s10930-023-10162-0
Nicholas E. Burgis, Kandise VanWormer, Devin Robbins, Jonathan Smith
Recent clinical data have identified infant patients with lethal ITPA deficiencies. ITPA is known to modulate ITP concentrations in cells and has a critical function in neural development which is not understood. Polymorphism of the ITPA gene affects outcomes for both ribavirin and thiopurine based therapies and nearly one third of the human population is thought to harbor ITPA polymorphism. In a previous site-directed mutagenesis alanine screen of the ITPA substrate selectivity pocket, we identified the ITPA mutant, E22A, as a gain-of function mutant with enhanced ITP hydrolysis activity. Here we report a rational enzyme engineering experiment to investigate the biochemical properties of position 22 ITPA mutants and find that the E22D ITPA has two- and four-fold improved substrate selectivity for ITP over the canonical purine triphosphates ATP and GTP, respectively, while maintaining biological activity. The novel E22D ITPA should be considered as a platform for further development of ITPA therapies.
{"title":"An ITPA Enzyme with Improved Substrate Selectivity","authors":"Nicholas E. Burgis, Kandise VanWormer, Devin Robbins, Jonathan Smith","doi":"10.1007/s10930-023-10162-0","DOIUrl":"https://doi.org/10.1007/s10930-023-10162-0","url":null,"abstract":"<p>Recent clinical data have identified infant patients with lethal ITPA deficiencies. ITPA is known to modulate ITP concentrations in cells and has a critical function in neural development which is not understood. Polymorphism of the <i>ITPA</i> gene affects outcomes for both ribavirin and thiopurine based therapies and nearly one third of the human population is thought to harbor <i>ITPA</i> polymorphism. In a previous site-directed mutagenesis alanine screen of the ITPA substrate selectivity pocket, we identified the ITPA mutant, E22A, as a gain-of function mutant with enhanced ITP hydrolysis activity. Here we report a rational enzyme engineering experiment to investigate the biochemical properties of position 22 ITPA mutants and find that the E22D ITPA has two- and four-fold improved substrate selectivity for ITP over the canonical purine triphosphates ATP and GTP, respectively, while maintaining biological activity. The novel E22D ITPA should be considered as a platform for further development of ITPA therapies.</p>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2023-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138561658","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}
Pub Date : 2023-11-28DOI: 10.21203/rs.3.rs-3121889/v1
M. Ghahramani, Mohammad Bagher Shahsavani, S. H. Khaleghinejad, Ali Niazi, A. Moosavi-Movahedi, Reza Yousefi
Angiotensin-converting enzyme 2 (ACE2) has a specific interaction with the coronavirus spike protein, enabling its entry into human cells. This membrane enzyme converts angiotensin II into angiotensin 1-7, which has an essential role in protecting the heart and improving lung function. Many therapeutic properties have been attributed to the human recombinant ACE2 (hrACE2), especially in combating complications related to diabetes mellitus and hypertension, as well as, preventing the coronavirus from entering the target tissues. In the current study, we designed an appropriate gene construct for the hybrid protein containing the ACE2 catalytic subunit and the B subunit of cholera toxin (CTB-ACE2). This structural feature will probably help the recombinant hybrid protein enter the mucosal tissues, including the lung tissue. Optimization of this hybrid protein expression was investigated in BL21 bacterial host cells. Also, the hybrid protein was identified with an appropriate antibody using the ELISA method. A large amount of the hybrid protein (molecular weight of ~ 100 kDa) was expressed as the inclusion body when the induction was performed in the presence of 0.25 mM IPTG and 1% sucrose for 10 h. Finally, the protein structural features were assessed using several biophysical methods. The fluorescence emission intensity and oligomeric size distribution of the CTB-ACE2 suggested a temperature-dependent alteration. The β-sheet and α-helix were also dominant in the hybrid protein structure, and this protein also displays acceptable chemical stability. In overall, according to our results, the efficient expression and successful purification of the CTB-ACE2 protein may pave the path for its therapeutic applications against diseases such as covid-19, diabetes mellitus and hypertension.
血管紧张素转换酶 2(ACE2)与冠状病毒尖峰蛋白有特殊的相互作用,使其能够进入人体细胞。这种膜酶可将血管紧张素 II 转化为血管紧张素 1-7,后者在保护心脏和改善肺功能方面发挥着重要作用。人重组 ACE2(hrACE2)具有许多治疗特性,特别是在防治糖尿病和高血压相关并发症以及阻止冠状病毒进入靶组织方面。在本研究中,我们设计了一种合适的基因构建体,用于构建含有 ACE2 催化亚基和霍乱毒素 B 亚基的杂交蛋白(CTB-ACE2)。这一结构特征可能有助于重组杂交蛋白进入包括肺组织在内的粘膜组织。研究人员在 BL21 细菌宿主细胞中优化了这种杂交蛋白的表达。此外,还使用 ELISA 方法用适当的抗体对杂交蛋白进行了鉴定。在 0.25 mM IPTG 和 1% 蔗糖存在下诱导 10 小时后,大量杂交蛋白(分子量约为 100 kDa)以包涵体形式表达。CTB-ACE2 的荧光发射强度和寡聚体大小分布表明其变化与温度有关。在杂交蛋白结构中,β-片层和α-螺旋也占主导地位,而且这种蛋白还显示出可接受的化学稳定性。总之,根据我们的研究结果,CTB-ACE2 蛋白的高效表达和成功纯化可能会为其治疗应用铺平道路,如用于治疗covid-19、糖尿病和高血压等疾病。
{"title":"Efficient Expression in the Prokaryotic Host System, Purification and Structural Analyses of the Recombinant Human ACE2 Catalytic Subunit as a Hybrid Protein with the B Subunit of Cholera Toxin (CTB-ACE2).","authors":"M. Ghahramani, Mohammad Bagher Shahsavani, S. H. Khaleghinejad, Ali Niazi, A. Moosavi-Movahedi, Reza Yousefi","doi":"10.21203/rs.3.rs-3121889/v1","DOIUrl":"https://doi.org/10.21203/rs.3.rs-3121889/v1","url":null,"abstract":"Angiotensin-converting enzyme 2 (ACE2) has a specific interaction with the coronavirus spike protein, enabling its entry into human cells. This membrane enzyme converts angiotensin II into angiotensin 1-7, which has an essential role in protecting the heart and improving lung function. Many therapeutic properties have been attributed to the human recombinant ACE2 (hrACE2), especially in combating complications related to diabetes mellitus and hypertension, as well as, preventing the coronavirus from entering the target tissues. In the current study, we designed an appropriate gene construct for the hybrid protein containing the ACE2 catalytic subunit and the B subunit of cholera toxin (CTB-ACE2). This structural feature will probably help the recombinant hybrid protein enter the mucosal tissues, including the lung tissue. Optimization of this hybrid protein expression was investigated in BL21 bacterial host cells. Also, the hybrid protein was identified with an appropriate antibody using the ELISA method. A large amount of the hybrid protein (molecular weight of ~ 100 kDa) was expressed as the inclusion body when the induction was performed in the presence of 0.25 mM IPTG and 1% sucrose for 10 h. Finally, the protein structural features were assessed using several biophysical methods. The fluorescence emission intensity and oligomeric size distribution of the CTB-ACE2 suggested a temperature-dependent alteration. The β-sheet and α-helix were also dominant in the hybrid protein structure, and this protein also displays acceptable chemical stability. In overall, according to our results, the efficient expression and successful purification of the CTB-ACE2 protein may pave the path for its therapeutic applications against diseases such as covid-19, diabetes mellitus and hypertension.","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2023-11-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139226518","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}