Pub Date : 2025-02-01DOI: 10.1016/j.bbapap.2024.141061
Mikhail Matveyenka , Abid Ali , Charles L. Mitchell , Mikhail Sholukh , Dmitry Kurouski
Progressive aggregation of α-synuclein (α-Syn), a small cytosolic protein involved in cell vesicle trafficking, in the midbrain, hypothalamus, and thalamus is linked to Parkinson's disease (PD). Amyloid oligomers and fibrils formed as a result of such aggregation are highly toxic to neurons. However, it remains unclear whether amyloid-induced toxicity of neurons is the primary mechanism of the progressive neurodegeneration observed upon PD. In the current study, we investigated cytotoxicity exerted by α-Syn fibrils formed in the lipid-free environment, as well as in the presence of two phospholipids, on macrophages, dendritic cells, and microglia. We found that α-Syn fibrils are far more toxic to dendritic cells and microglia compared to neurons. We also observe low toxicity levels of such amyloids to macrophages. Real-time polymerase chain reaction (RT-PCR) results suggest that toxicity of amyloids aggregates is linked to the levels of autophagy in cells. These results suggest that a strong impairment of the immune system in the brain may be the first stop of neurodegenerative processes that are taking place upon the onset of PD.
{"title":"Elucidation of cytotoxicity of α-Synuclein fibrils on immune cells","authors":"Mikhail Matveyenka , Abid Ali , Charles L. Mitchell , Mikhail Sholukh , Dmitry Kurouski","doi":"10.1016/j.bbapap.2024.141061","DOIUrl":"10.1016/j.bbapap.2024.141061","url":null,"abstract":"<div><div>Progressive aggregation of α-synuclein (α-Syn), a small cytosolic protein involved in cell vesicle trafficking, in the midbrain, hypothalamus, and thalamus is linked to Parkinson's disease (PD). Amyloid oligomers and fibrils formed as a result of such aggregation are highly toxic to neurons. However, it remains unclear whether amyloid-induced toxicity of neurons is the primary mechanism of the progressive neurodegeneration observed upon PD. In the current study, we investigated cytotoxicity exerted by α-Syn fibrils formed in the lipid-free environment, as well as in the presence of two phospholipids, on macrophages, dendritic cells, and microglia. We found that α-Syn fibrils are far more toxic to dendritic cells and microglia compared to neurons. We also observe low toxicity levels of such amyloids to macrophages. Real-time polymerase chain reaction (RT-PCR) results suggest that toxicity of amyloids aggregates is linked to the levels of autophagy in cells. These results suggest that a strong impairment of the immune system in the brain may be the first stop of neurodegenerative processes that are taking place upon the onset of PD.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 2","pages":"Article 141061"},"PeriodicalIF":2.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142852091","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 : 2025-02-01DOI: 10.1016/j.bbapap.2024.141062
Lorieth A. Guevara Cuasapud , Pablo J. González , Félix M. Ferroni , Andrea B. Duré , Sergio D. Dalosto , Maria G. Rivas , Carlos D. Brondino
We report the molecular, biochemical and spectroscopic characterization and computational calculations of a variant of the copper-containing nitrite reductase from the rhizobial microorganism S. meliloti (SmNirK), in which the catalytic aspartate residue (AspCAT) has been replaced with serine (SerCAT, D134S) by site-directed mutagenesis. Like the wild-type enzyme, D134S is a homotrimer with the typical catalytic pocket of two-domain NirK containing two copper centers, one of type 1 (T1) and another of type 2 (T2). The T1 electron transfer center is similar to that of the wild-type enzyme but the electronic and covalent properties of T2 active site are altered by the mutation. As for the wild-type enzyme, the enzymatic activity of D134S is pH-dependent, i.e. it is higher at lower pH values, but the kcat is an order of magnitude lower. EPR studies showed a decrease in g‖ and an increase in A‖ of D134S relative to wild-type enzyme. This indicates changes in the electronic and covalent properties of T2 upon mutation, which affects the reduction potential of T2 and the T1-T2 reduction potential gap. Taken together, this evidence points to the importance of the ligands of the second coordination sphere of T2 in controlling critical parameters in catalysis. The possibility that AspCAT/SerCAT is the switch that triggers T1 → T2 electron transfer upon T2 nitrite binding and the importance of HisCAT for the pH-dependent catalytic activity of NirK are discussed.
{"title":"Replacement of the essential catalytic aspartate with serine leads to an active form of copper-containing nitrite reductase from the denitrifier Sinorhizobium meliloti 2011","authors":"Lorieth A. Guevara Cuasapud , Pablo J. González , Félix M. Ferroni , Andrea B. Duré , Sergio D. Dalosto , Maria G. Rivas , Carlos D. Brondino","doi":"10.1016/j.bbapap.2024.141062","DOIUrl":"10.1016/j.bbapap.2024.141062","url":null,"abstract":"<div><div>We report the molecular, biochemical and spectroscopic characterization and computational calculations of a variant of the copper-containing nitrite reductase from the rhizobial microorganism <em>S. meliloti</em> (<em>Sm</em>NirK), in which the catalytic aspartate residue (Asp<sub>CAT</sub>) has been replaced with serine (Ser<sub>CAT</sub>, D134S) by site-directed mutagenesis. Like the wild-type enzyme, D134S is a homotrimer with the typical catalytic pocket of two-domain NirK containing two copper centers, one of type 1 (T1) and another of type 2 (T2). The T1 electron transfer center is similar to that of the wild-type enzyme but the electronic and covalent properties of T2 active site are altered by the mutation. As for the wild-type enzyme, the enzymatic activity of D134S is pH-dependent, i.e. it is higher at lower pH values, but the <em>k</em><sub>cat</sub> is an order of magnitude lower. EPR studies showed a decrease in <em>g</em><sub>‖</sub> and an increase in <em>A</em><sub>‖</sub> of D134S relative to wild-type enzyme. This indicates changes in the electronic and covalent properties of T2 upon mutation, which affects the reduction potential of T2 and the T1-T2 reduction potential gap. Taken together, this evidence points to the importance of the ligands of the second coordination sphere of T2 in controlling critical parameters in catalysis. The possibility that Asp<sub>CAT</sub>/Ser<sub>CAT</sub> is the switch that triggers T1 → T2 electron transfer upon T2 nitrite binding and the importance of His<sub>CAT</sub> for the pH-dependent catalytic activity of NirK are discussed.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 2","pages":"Article 141062"},"PeriodicalIF":2.5,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142913782","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 : 2024-11-26DOI: 10.1016/j.bbapap.2024.141060
Puji Rahayu , Doni Dermawan , Florensia Nailufar , Erna Sulistyaningrum , Raymond R. Tjandrawinata
Tacorin, a bioactive protein fraction derived from pineapple stem (Ananas comosus), has emerged as a promising therapeutic agent for wound healing. This study employs an integrated approach, combining in silico proteomics and in vivo investigations, to unravel the molecular mechanisms underlying Tacorin's wound healing properties. In the domain of in silico proteomics, the composition of Tacorin is elucidated through LC/MS-MS protein sequencing, revealing ananain (23.77 kDa) and Jacalin-like lectin (14.99 kDa) as its predominant constituents. Molecular protein-protein docking simulations unveil favorable interactions between Tacorin's components and key regulators of wound healing, including TGF-β, TNF-α, and MMP-2. The calculated free binding energies indicate strong binding affinities between Tacorin proteins and their target receptors. Specifically, ananain demonstrates a binding affinity of −12.2 kcal/mol with TGF-β, suggesting its potential as a potent activator of TGF-β-mediated signaling, while Jacalin-like lectin exhibits the most favorable binding affinity of −8.7 kcal/mol with TNF-α. Subsequent 100 ns molecular dynamics (MD) simulations provide insights into the dynamic behavior and stability of Tacorin-receptor complexes, shedding light on the molecular determinants of Tacorin's therapeutic effects. Complementing the in silico analyses, in vivo studies evaluate Tacorin's efficacy in wound healing using skin and uterine incision models. Tacorin treatment accelerates wound closure and promotes tissue repair in both models, as evidenced by macroscopic observations and histological assessments. Overall, this study provides compelling evidence of Tacorin's therapeutic potential in wound healing and underscores the importance of elucidating its molecular mechanisms for further development and clinical translation.
{"title":"Unlocking the wound-healing potential: An integrative in silico proteomics and in vivo analysis of Tacorin, a bioactive protein fraction from Ananas comosus (L.) Merr. Stem","authors":"Puji Rahayu , Doni Dermawan , Florensia Nailufar , Erna Sulistyaningrum , Raymond R. Tjandrawinata","doi":"10.1016/j.bbapap.2024.141060","DOIUrl":"10.1016/j.bbapap.2024.141060","url":null,"abstract":"<div><div>Tacorin, a bioactive protein fraction derived from pineapple stem (<em>Ananas comosus</em>), has emerged as a promising therapeutic agent for wound healing. This study employs an integrated approach, combining <em>in silico</em> proteomics and <em>in vivo</em> investigations, to unravel the molecular mechanisms underlying Tacorin's wound healing properties. In the domain of <em>in silico</em> proteomics, the composition of Tacorin is elucidated through LC/MS-MS protein sequencing, revealing ananain (23.77 kDa) and Jacalin-like lectin (14.99 kDa) as its predominant constituents. Molecular protein-protein docking simulations unveil favorable interactions between Tacorin's components and key regulators of wound healing, including TGF-β, TNF-α, and MMP-2. The calculated free binding energies indicate strong binding affinities between Tacorin proteins and their target receptors. Specifically, ananain demonstrates a binding affinity of −12.2 kcal/mol with TGF-β, suggesting its potential as a potent activator of TGF-β-mediated signaling, while Jacalin-like lectin exhibits the most favorable binding affinity of −8.7 kcal/mol with TNF-α. Subsequent 100 ns molecular dynamics (MD) simulations provide insights into the dynamic behavior and stability of Tacorin-receptor complexes, shedding light on the molecular determinants of Tacorin's therapeutic effects. Complementing the <em>in silico</em> analyses, <em>in vivo</em> studies evaluate Tacorin's efficacy in wound healing using skin and uterine incision models. Tacorin treatment accelerates wound closure and promotes tissue repair in both models, as evidenced by macroscopic observations and histological assessments. Overall, this study provides compelling evidence of Tacorin's therapeutic potential in wound healing and underscores the importance of elucidating its molecular mechanisms for further development and clinical translation.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 1","pages":"Article 141060"},"PeriodicalIF":2.5,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142748237","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 : 2024-10-28DOI: 10.1016/j.bbapap.2024.141059
Chukwuemeka S. Adindu , Katie Tombrello , Luke A. Martz , Tonya N. Zeczycki , Holly R. Ellis
Oxidation and assimilation of persulfides in bacteria is often catalyzed by a persulfide dioxygenase and sulfurtransferase in consecutive reactions. Enzymes responsible for the oxidation of persulfides have not been clearly defined in Pseudomonas aeruginosa PAO1. The characterized mercaptopropionate dioxygenase (MDO) in P. aeruginosa PAO1 has been proposed to catalyze the oxidation of 3-mercaptopropionate. However, the physiological role of MDO is uncertain given the expression of a sulfurtransferase (ST) enzyme on the same operon as the thiol dioxygenase. The st gene had a co-occurrence frequency with mdo of 0.94 demonstrating the co-expression and physiological link of the two genes. There are four tandem rhodanese domains in the ST enzyme with two of the domains containing potential catalytic Cys residues (Cys191 and Cys435) capable of forming a persulfide. Only Cys435 was accessible in thiol quantification assays, and results from H/D-X MS analyses further established the accessibility of the domain containing Cys435. Both thiosulfate and mercaptopyruvate served as sulfur donors to the ST enzyme, with Cys435 forming the persulfide intermediate. Kinetic investigations of MDO suggested the enzyme had a broader substrate specificity than previously identified, oxidizing both mercaptopropionate and mercaptopyruvate thiol and persulfide substrates. The results obtained from these investigations provide insight into the overall mechanism and physiological role of the mdo operon in sulfide oxidation and assimilation.
{"title":"A distinct co-expressed sulfurtransferase extends the physiological role of mercaptopropionate dioxygenase in Pseudomonas aeruginosa PAO1","authors":"Chukwuemeka S. Adindu , Katie Tombrello , Luke A. Martz , Tonya N. Zeczycki , Holly R. Ellis","doi":"10.1016/j.bbapap.2024.141059","DOIUrl":"10.1016/j.bbapap.2024.141059","url":null,"abstract":"<div><div>Oxidation and assimilation of persulfides in bacteria is often catalyzed by a persulfide dioxygenase and sulfurtransferase in consecutive reactions. Enzymes responsible for the oxidation of persulfides have not been clearly defined in <em>Pseudomonas aeruginosa</em> PAO1. The characterized mercaptopropionate dioxygenase (MDO) in <em>P. aeruginosa</em> PAO1 has been proposed to catalyze the oxidation of 3-mercaptopropionate. However, the physiological role of MDO is uncertain given the expression of a sulfurtransferase (ST) enzyme on the same operon as the thiol dioxygenase. The <em>st</em> gene had a co-occurrence frequency with <em>mdo</em> of 0.94 demonstrating the co-expression and physiological link of the two genes. There are four tandem rhodanese domains in the ST enzyme with two of the domains containing potential catalytic Cys residues (Cys191 and Cys435) capable of forming a persulfide. Only Cys435 was accessible in thiol quantification assays, and results from H/D-X MS analyses further established the accessibility of the domain containing Cys435. Both thiosulfate and mercaptopyruvate served as sulfur donors to the ST enzyme, with Cys435 forming the persulfide intermediate. Kinetic investigations of MDO suggested the enzyme had a broader substrate specificity than previously identified, oxidizing both mercaptopropionate and mercaptopyruvate thiol and persulfide substrates. The results obtained from these investigations provide insight into the overall mechanism and physiological role of the <em>mdo</em> operon in sulfide oxidation and assimilation.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 1","pages":"Article 141059"},"PeriodicalIF":2.5,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142543387","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 : 2024-10-23DOI: 10.1016/j.bbapap.2024.141057
Mengxue Wang , Xinyi Sun , Shijiang Peng , Feifan Wang , Kangli Zhao , Dang Wang
Coronaviruses replicate by using the 3C-like protease (3CLpro) to cleave polyprotein precursors and host proteins. However, current tools for identifying 3CLpro cleavage sites are limited, particularly in Gammacoronaviruses (GammaCoV) and Deltacoronaviruses (DeltaCoV). This study aims to fill this gap by identifying 3CLpro cleavage sites in these viruses to provide deeper insights into their pathogenic mechanisms. By integrating sequence alignments and structural model comparisons, we developed a position-specific scoring matrix (PSSM) based on self-cleavage motifs, revealing specific preferences for each residue. Utilizing AlphaFold2's predicted alignment error (PAE) and predicted local distance difference test (pLDDT), we found that most cleavage sequences are located in regions with high PAE and low pLDDT values. KEGG pathway analysis showed that potential host protein cleavage targets are mainly concentrated in pathways related to nucleo-cytoplasmic transport and endocytosis. Through in vitro cleavage experiments and mutational analysis, we identified and validated three high-scoring proteins—nucleoporin 58 (NUP58), cell division cycle 73 (CDC73), and signal transducing adaptor molecule 2 (STAM2). These findings suggest that 3CLpro not only plays a vital role in viral replication but may also influence host cell functions by cleaving host proteins. This study provides an effective tool for identifying 3CLpro cleavage sites, revealing the pathogenic mechanisms of coronaviruses, and offering new insights for developing potential therapeutic targets.
{"title":"Deciphering the cleavage sites of 3C-like protease in Gammacoronaviruses and Deltacoronaviruses","authors":"Mengxue Wang , Xinyi Sun , Shijiang Peng , Feifan Wang , Kangli Zhao , Dang Wang","doi":"10.1016/j.bbapap.2024.141057","DOIUrl":"10.1016/j.bbapap.2024.141057","url":null,"abstract":"<div><div>Coronaviruses replicate by using the 3C-like protease (3CL<sup>pro</sup>) to cleave polyprotein precursors and host proteins. However, current tools for identifying 3CL<sup>pro</sup> cleavage sites are limited, particularly in <em>Gammacoronaviruses</em> (GammaCoV) and <em>Deltacoronaviruses</em> (DeltaCoV). This study aims to fill this gap by identifying 3CL<sup>pro</sup> cleavage sites in these viruses to provide deeper insights into their pathogenic mechanisms. By integrating sequence alignments and structural model comparisons, we developed a position-specific scoring matrix (PSSM) based on self-cleavage motifs, revealing specific preferences for each residue. Utilizing AlphaFold2's predicted alignment error (PAE) and predicted local distance difference test (pLDDT), we found that most cleavage sequences are located in regions with high PAE and low pLDDT values. KEGG pathway analysis showed that potential host protein cleavage targets are mainly concentrated in pathways related to nucleo-cytoplasmic transport and endocytosis. Through <em>in vitro</em> cleavage experiments and mutational analysis, we identified and validated three high-scoring proteins—nucleoporin 58 (NUP58), cell division cycle 73 (CDC73), and signal transducing adaptor molecule 2 (STAM2). These findings suggest that 3CL<sup>pro</sup> not only plays a vital role in viral replication but may also influence host cell functions by cleaving host proteins. This study provides an effective tool for identifying 3CL<sup>pro</sup> cleavage sites, revealing the pathogenic mechanisms of coronaviruses, and offering new insights for developing potential therapeutic targets.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 1","pages":"Article 141057"},"PeriodicalIF":2.5,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142493886","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}
In-house generated mAbs to apolipoprotein B-100 (apoB-100) clones hLDL-E8, hLDL-2D8 and hLDL-F5 were extensively studied to determine their complementarity-determining regions (CDRs), binding epitopes and affinity. RT-PCR revealed that all mAbs consisted of kappa light chains and gamma heavy chains. DNA sequencing and bioinformatic analysis showed that the variable gene and protein sequences of their CDRs shared over 50 % identity with the existing databases. The 3D structures of the mAb variable fragments (Fv) with a QSQE score above 0.7 were constructed using the SWISS-MODEL platform. The structural accuracy was confirmed by Ramachandran plots, with 99 % of amino acid residues falling within acceptable regions. Thrombolytic cleavage of apoB-100 and Western blot analysis demonstrated that hLDL-E8 and hLDL-F5 specifically bind to the T3 fragment (aa 1297–3249), whereas hLDL-2D8 binds to the T4 fragment (aa 1–1297). These findings were supported with epitope-binding assays using inhibition ELISA, which indicated that hLDL-E8 binds at different epitopes from hLDL-2D8 and has some overlap with hLDL-F5. Lastly, the binding affinity of the mAbs was examined by indirect ELISA. The average affinity constants (Kaff) for mAbs hLDL-2D8, hLDL-E8 and hLDL-F5 are 1.51 ± 0.69 × 109 Mol−1, 7.25 ± 3.56 × 108 Mol−1 and 4.39 ± 2.63 × 106 Mol−1, respectively. Additionally, the behavior of the antibodies in the dose-response curve revealed that hLDL-F5 may recognize two epitopes of apoB-100 or have very low binding affinity. In contrast, hLDL-2D8 and hLDL-E8 each recognize a single epitope. These findings provide information that will be useful when selecting mAbs for both laboratory and clinical research purposes.
{"title":"CDR identification, epitope mapping and binding affinity determination of novel monoclonal antibodies generated against human apolipoprotein B-100","authors":"Tariga Sritrakarn , Kanokwan Lowhalidanon , Panida Khunkaewla","doi":"10.1016/j.bbapap.2024.141058","DOIUrl":"10.1016/j.bbapap.2024.141058","url":null,"abstract":"<div><div>In-house generated mAbs to apolipoprotein B-100 (apoB-100) clones hLDL-E8, hLDL-2D8 and hLDL-F5 were extensively studied to determine their complementarity-determining regions (CDRs), binding epitopes and affinity. RT-PCR revealed that all mAbs consisted of kappa light chains and gamma heavy chains. DNA sequencing and bioinformatic analysis showed that the variable gene and protein sequences of their CDRs shared over 50 % identity with the existing databases. The 3D structures of the mAb variable fragments (Fv) with a QSQE score above 0.7 were constructed using the SWISS-MODEL platform. The structural accuracy was confirmed by Ramachandran plots, with 99 % of amino acid residues falling within acceptable regions. Thrombolytic cleavage of apoB-100 and Western blot analysis demonstrated that hLDL-E8 and hLDL-F5 specifically bind to the T3 fragment (aa 1297–3249), whereas hLDL-2D8 binds to the T4 fragment (aa 1–1297). These findings were supported with epitope-binding assays using inhibition ELISA, which indicated that hLDL-E8 binds at different epitopes from hLDL-2D8 and has some overlap with hLDL-F5. Lastly, the binding affinity of the mAbs was examined by indirect ELISA. The average affinity constants (K<sub>aff</sub>) for mAbs hLDL-2D8, hLDL-E8 and hLDL-F5 are 1.51 ± 0.69 × 10<sup>9</sup> Mol<sup>−1</sup>, 7.25 ± 3.56 × 10<sup>8</sup> Mol<sup>−1</sup> and 4.39 ± 2.63 × 10<sup>6</sup> Mol<sup>−1</sup>, respectively. Additionally, the behavior of the antibodies in the dose-response curve revealed that hLDL-F5 may recognize two epitopes of apoB-100 or have very low binding affinity. In contrast, hLDL-2D8 and hLDL-E8 each recognize a single epitope. These findings provide information that will be useful when selecting mAbs for both laboratory and clinical research purposes.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 1","pages":"Article 141058"},"PeriodicalIF":2.5,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142493885","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 : 2024-10-16DOI: 10.1016/j.bbapap.2024.141053
Mst Sharmin Aktar , Nikhila Kashyap Dhanvantari Madhuresh , Reza A. Ghiladi, Stefan Franzen
At least two of the six methionine (Met) residues in dehaloperoxidase (DHP) are shown to act as electron donors in both autoreduction and protein-heme crosslinking. Autoreduction observed in the two isozymes, DHP-A and DHP-B, is explained by the high heme reduction potential and an endogenous source of electrons from methionine (Met) or cysteine (Cys). This study provides evidence of a connection to protein-heme crosslinking that occurs when DHP is activated by H2O2 in competition with substrate oxidation and autoreduction. The autoreduction yields of DHP-A and DHP-B are comparable and both are inversely proportional to DHP concentration. Both isoenzymes show an anti-cooperative effect on autoreduction kinetics associated with protein dimerization. Despite the presence of five tyrosine (Tyr) amino acids in DHP-A and four Tyr in DHP-B, the mass spectral evidence does not support a Tyr-heme or interprotein Tyr-Tyr crosslinking event as observed in some mammalian myoglobins. LC-MS and tandem MS/MS studies revealed three amino acids that were involved in the heme-protein crosslink, Cys73, Met63 and Met64. Cys73 facilitates dimer formation in DHP-A which also appears to slow the rate of autoreduction, but is not involved in covalent protein-heme crosslinking. Based on mutational studies, Met63 and 64 are involved in both covalent heme crosslinking and autoreduction. Proton-coupled electron transfer and crosslinking by Met to the heme may serve to regulate DHP function and protect it from uncontrolled oxidative damage.
{"title":"The role of proton-coupled electron transfer from protein to heme in dehaloperoxidase","authors":"Mst Sharmin Aktar , Nikhila Kashyap Dhanvantari Madhuresh , Reza A. Ghiladi, Stefan Franzen","doi":"10.1016/j.bbapap.2024.141053","DOIUrl":"10.1016/j.bbapap.2024.141053","url":null,"abstract":"<div><div>At least two of the six methionine (Met) residues in dehaloperoxidase (DHP) are shown to act as electron donors in both autoreduction and protein-heme crosslinking. Autoreduction observed in the two isozymes, DHP-A and DHP-B, is explained by the high heme reduction potential and an endogenous source of electrons from methionine (Met) or cysteine (Cys). This study provides evidence of a connection to protein-heme crosslinking that occurs when DHP is activated by H<sub>2</sub>O<sub>2</sub> in competition with substrate oxidation and autoreduction. The autoreduction yields of DHP-A and DHP-B are comparable and both are inversely proportional to DHP concentration. Both isoenzymes show an anti-cooperative effect on autoreduction kinetics associated with protein dimerization. Despite the presence of five tyrosine (Tyr) amino acids in DHP-A and four Tyr in DHP-B, the mass spectral evidence does not support a Tyr-heme or interprotein Tyr-Tyr crosslinking event as observed in some mammalian myoglobins. LC-MS and tandem MS/MS studies revealed three amino acids that were involved in the heme-protein crosslink, Cys73, Met63 and Met64. Cys73 facilitates dimer formation in DHP-A which also appears to slow the rate of autoreduction, but is not involved in covalent protein-heme crosslinking. Based on mutational studies, Met63 and 64 are involved in both covalent heme crosslinking and autoreduction. Proton-coupled electron transfer and crosslinking by Met to the heme may serve to regulate DHP function and protect it from uncontrolled oxidative damage.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 1","pages":"Article 141053"},"PeriodicalIF":2.5,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142456983","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 : 2024-10-13DOI: 10.1016/j.bbapap.2024.141056
Alina K. Bakunova , Ilya O. Matyuta , Mikhail E. Minyaev , Konstantin M. Boyko , Vladimir O. Popov , Ekaterina Yu. Bezsudnova
Pyridoxal-5′-phosphate (PLP)-dependent transaminases are key enzymes of amino acid metabolism in cells and remarkable biocatalysts of stereoselective amination for process chemistry applications. As cofactor-dependent enzymes, transaminases are prone to cofactor leakage. Here we discuss the holoenzyme-apoenzyme interconversion and the kinetics of PLP incorporation into the apo form of a PLP-dependent transaminase from Haliscomenobacter hydrossis. PLP binding to the apoenzyme was slow in buffer, but was accelerated in the presence of substrates. Two crystal structures of the apoenzyme were obtained: the directly obtained apoenzyme (PDB ID: 7P8O) and the one obtained by soaking crystals of the holoenzyme in a phenylhydrazine solution (PDB ID: 8YRU). The mechanism of PLP association with the apoenzyme was proposed on the basis of structural analysis of these apo forms. Three rearrangement steps, including (I) anchoring of the PLP via the phosphate group, (II) displacement of two loops, and (III) Schiff-bonding between the PLP and the ε-amino group of the catalytic lysine residue, reconstituted the active holo form of the transaminase from H. hydrossis. The results obtained allowed us to determine in the active site a permanent part and elements that are assembled by PLP, these findings may be useful for transaminase engineering for biocatalysis.
{"title":"Incorporation of pyridoxal-5′-phosphate into the apoenzyme: A structural study of D-amino acid transaminase from Haliscomenobacter hydrossis","authors":"Alina K. Bakunova , Ilya O. Matyuta , Mikhail E. Minyaev , Konstantin M. Boyko , Vladimir O. Popov , Ekaterina Yu. Bezsudnova","doi":"10.1016/j.bbapap.2024.141056","DOIUrl":"10.1016/j.bbapap.2024.141056","url":null,"abstract":"<div><div>Pyridoxal-5′-phosphate (PLP)-dependent transaminases are key enzymes of amino acid metabolism in cells and remarkable biocatalysts of stereoselective amination for process chemistry applications. As cofactor-dependent enzymes, transaminases are prone to cofactor leakage. Here we discuss the holoenzyme-apoenzyme interconversion and the kinetics of PLP incorporation into the apo form of a PLP-dependent transaminase from <em>Haliscomenobacter hydrossis</em>. PLP binding to the apoenzyme was slow in buffer, but was accelerated in the presence of substrates. Two crystal structures of the apoenzyme were obtained: the directly obtained apoenzyme (PDB ID: <span><span>7P8O</span><svg><path></path></svg></span>) and the one obtained by soaking crystals of the holoenzyme in a phenylhydrazine solution (PDB ID: <span><span>8YRU</span><svg><path></path></svg></span>). The mechanism of PLP association with the apoenzyme was proposed on the basis of structural analysis of these apo forms. Three rearrangement steps, including (I) anchoring of the PLP via the phosphate group, (II) displacement of two loops, and (III) Schiff-bonding between the PLP and the ε-amino group of the catalytic lysine residue, reconstituted the active holo form of the transaminase from <em>H. hydrossis</em>. The results obtained allowed us to determine in the active site a permanent part and elements that are assembled by PLP, these findings may be useful for transaminase engineering for biocatalysis.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 1","pages":"Article 141056"},"PeriodicalIF":2.5,"publicationDate":"2024-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142456984","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 : 2024-10-11DOI: 10.1016/j.bbapap.2024.141055
Melissa A.E. van de Wal , Cenna Doornbos , Janne M. Bibbe , Judith R. Homberg , Clara van Karnebeek , Martijn A. Huynen , Jaap Keijer , Evert M. van Schothorst , Peter A.C. 't Hoen , Mirian C.H. Janssen , Merel J.W. Adjobo-Hermans , Mariusz R. Wieckowski , Werner J.H. Koopman
Paediatric Leigh syndrome (LS) is an early-onset and fatal neurodegenerative disorder lacking treatment options. LS is frequently caused by mutations in the NDUFS4 gene, encoding an accessory subunit of mitochondrial complex I (CI), the first complex of the oxidative phosphorylation (OXPHOS) system. Whole-body Ndufs4 knockout (KO) mice (WB-KO mice) are widely used to study isolated CI deficiency, LS pathology and interventions. These animals develop a brain-specific phenotype via an incompletely understood pathomechanism. Here we performed a quantitative analysis of the sub-brain proteome in six-weeks old WB-KO mice vs. wildtype (WT) mice. Brain regions comprised of a brain slice (BrSl), cerebellum (CB), cerebral cortex (CC), hippocampus (HC), inferior colliculus (IC), and superior colliculus (SC). Proteome analysis demonstrated similarities between CC/HC, and between IC/SC, whereas BrSl and CB differed from these two groups and each other. All brain regions displayed greatly reduced levels of two CI structural subunits (NDUFS4, NDUFA12) and an increased level of the CI assembly factor NDUFAF2. The level of CI-Q module subunits was significantly more reduced in IC/SC than in BrSl/CB/CC/HC, whereas other OXPHOS complex levels were not reduced. Gene ontology and pathway analysis demonstrated specific and common proteome changes between brain regions.
Across brain regions, upregulation of cold-shock-associated proteins, mitochondrial fatty acid (FA) oxidation and synthesis (mtFAS) were the most prominent. FA-related pathways were predominantly upregulated in CB and HC. Based upon these results, we argue that stimulation of these pathways is futile and pro-pathological and discuss alternative strategies for therapeutic intervention in LS.
Significance
The Ndufs4 knockout mouse model is currently the most relevant and most widely used animal model to study the brain-linked pathophysiology of human Leigh Syndrome (LS) and intervention strategies. We demonstrate that the Ndufs4 knockout brain engages futile and pro-pathological responses. These responses explain both negative and positive outcomes of intervention studies in Leigh Syndrome mice and patients, thereby guiding novel intervention opportunities.
小儿利氏综合征（LS）是一种发病较早且致命的神经退行性疾病，目前尚无治疗方法。LS常由NDUFS4基因突变引起，该基因编码线粒体复合体I（CI）的一个附属亚基，而线粒体复合体I是氧化磷酸化（OXPHOS）系统的第一个复合体。全身 Ndufs4 基因敲除（KO）小鼠（WB-KO 小鼠）被广泛用于研究孤立的 CI 缺乏、LS 病理和干预措施。这些动物通过不完全清楚的病理机制形成了大脑特异性表型。在这里，我们对六周大的 WB-KO 小鼠与野生型小鼠的脑下蛋白质组进行了定量分析。脑区包括大脑切片（BrSl）、小脑（CB）、大脑皮层（CC）、海马（HC）、下丘（IC）和上丘（SC）。蛋白质组分析表明，CC/HC 之间以及 IC/SC 之间存在相似性，而 BrSl 和 CB 则与这两组和其他组不同。所有脑区的两个 CI 结构亚基（NDUFS4 和 NDUFA12）的水平都大大降低，而 CI 组装因子 NDUFAF2 的水平则有所提高。与 BrSl/CB/CC/HC 相比，IC/SC 中 CI-Q 模块亚基的水平明显降低，而其他 OXPHOS 复合物的水平并未降低。基因本体和通路分析表明了不同脑区之间蛋白质组的特殊和共同变化。在各个脑区，冷休克相关蛋白、线粒体脂肪酸氧化和合成（mtFAS）的上调最为显著。与脂肪酸相关的通路主要在 CB 和 HC 中上调。基于这些结果，我们认为刺激这些通路是徒劳的，而且会导致病理变化，并讨论了 LS 治疗干预的替代策略。意义：Ndufs4基因敲除小鼠模型是目前研究人类利氏综合征（LS）脑相关病理生理学和干预策略最相关、应用最广泛的动物模型。我们证明，Ndufs4基因敲除的大脑会产生徒劳的和有利于病理的反应。这些反应解释了在莱氏综合征小鼠和患者中进行的干预研究的消极和积极结果，从而为新的干预机会提供了指导。
{"title":"Ndufs4 knockout mice with isolated complex I deficiency engage a futile adaptive brain response","authors":"Melissa A.E. van de Wal , Cenna Doornbos , Janne M. Bibbe , Judith R. Homberg , Clara van Karnebeek , Martijn A. Huynen , Jaap Keijer , Evert M. van Schothorst , Peter A.C. 't Hoen , Mirian C.H. Janssen , Merel J.W. Adjobo-Hermans , Mariusz R. Wieckowski , Werner J.H. Koopman","doi":"10.1016/j.bbapap.2024.141055","DOIUrl":"10.1016/j.bbapap.2024.141055","url":null,"abstract":"<div><div>Paediatric Leigh syndrome (LS) is an early-onset and fatal neurodegenerative disorder lacking treatment options. LS is frequently caused by mutations in the <em>NDUFS4</em> gene, encoding an accessory subunit of mitochondrial complex I (CI), the first complex of the oxidative phosphorylation (OXPHOS) system. Whole-body <em>Ndufs4</em> knockout (KO) mice (WB-KO mice) are widely used to study isolated CI deficiency, LS pathology and interventions. These animals develop a brain-specific phenotype <em>via</em> an incompletely understood pathomechanism. Here we performed a quantitative analysis of the sub-brain proteome in six-weeks old WB-KO mice <em>vs.</em> wildtype (WT) mice. Brain regions comprised of a brain slice (BrSl), cerebellum (CB), cerebral cortex (CC), hippocampus (HC), inferior colliculus (IC), and superior colliculus (SC). Proteome analysis demonstrated similarities between CC/HC, and between IC/SC, whereas BrSl and CB differed from these two groups and each other. All brain regions displayed greatly reduced levels of two CI structural subunits (NDUFS4, NDUFA12) and an increased level of the CI assembly factor NDUFAF2. The level of CI-Q module subunits was significantly more reduced in IC/SC than in BrSl/CB/CC/HC, whereas other OXPHOS complex levels were not reduced. Gene ontology and pathway analysis demonstrated specific and common proteome changes between brain regions.</div><div>Across brain regions, upregulation of cold-shock-associated proteins, mitochondrial fatty acid (FA) oxidation and synthesis (mtFAS) were the most prominent. FA-related pathways were predominantly upregulated in CB and HC. Based upon these results, we argue that stimulation of these pathways is futile and pro-pathological and discuss alternative strategies for therapeutic intervention in LS.</div></div><div><h3>Significance</h3><div>The <em>Ndufs4</em> knockout mouse model is currently the most relevant and most widely used animal model to study the brain-linked pathophysiology of human Leigh Syndrome (LS) and intervention strategies. We demonstrate that the <em>Ndufs4</em> knockout brain engages futile and pro-pathological responses. These responses explain both negative and positive outcomes of intervention studies in Leigh Syndrome mice and patients, thereby guiding novel intervention opportunities.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 1","pages":"Article 141055"},"PeriodicalIF":2.5,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142456982","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 : 2024-10-09DOI: 10.1016/j.bbapap.2024.141054
Anurag Kirti , Hema Rajaram
Nucleoside diphosphate kinase (Ndk/NDK/NDPK) is known to possess pleiotropic functions, one of which is that as a protein kinase, and has been shown to be involved in stress tolerance in plants. To assess its role in the cyanobacterium Nostoc PCC 7120, which is hitherto unreported, recombinant strain overexpressing Ndk, Anndk+ was generated. Phosphoproteomic analysis of Anndk+ and its comparison with that of the vector control, AnpAM, revealed differential phosphorylation at S/T/Y sites of proteins belonging to varied functional groups, with over 17 % phosphoproteins involved in photosynthesis. A total of 177 phosphopeptides and 117 phosphoproteins were identified, including newly identified phosphopeptides in any cyanobacteria. Compared to AnpAM, the Anndk+ cells exhibited (i) lower photosynthetic efficiency and electron transport rate at low PAR (photosynthetically active radiation), (ii) no change in photochemical quenching across PAR, (iii) but distinct non-photochemical quenching [zero Y(NPQ) and high Y(NO) in Anndk+ and high Y(NPQ) and low (NO) in AnpAM], and (iv) increased tolerance to γ-radiation, oxidative, salt and DCMU stresses. The observed modulation of phosphoproteome linked to physiological changes upon overexpression of Ndk in Nostoc could be a combination of direct protein kinase activity of Ndk and/or indirectly through other protein kinases and phosphatases whose phosphorylation status is mediated by Ndk. This is the first report on a direct correlation between Ndk levels, phosphorylation status of proteins and stress tolerance in any cyanobacteria.
{"title":"Phosphoproteome modulation by nucleoside diphosphate kinase affects photosynthesis & stress tolerance of Nostoc PCC 7120","authors":"Anurag Kirti , Hema Rajaram","doi":"10.1016/j.bbapap.2024.141054","DOIUrl":"10.1016/j.bbapap.2024.141054","url":null,"abstract":"<div><div>Nucleoside diphosphate kinase (Ndk/NDK/NDPK) is known to possess pleiotropic functions, one of which is that as a protein kinase, and has been shown to be involved in stress tolerance in plants. To assess its role in the cyanobacterium <em>Nostoc</em> PCC 7120, which is hitherto unreported, recombinant strain overexpressing Ndk, An<em>ndk</em><sup><em>+</em></sup> was generated. Phosphoproteomic analysis of An<em>ndk</em><sup>+</sup> and its comparison with that of the vector control, AnpAM, revealed differential phosphorylation at S/T/Y sites of proteins belonging to varied functional groups, with over 17 % phosphoproteins involved in photosynthesis. A total of 177 phosphopeptides and 117 phosphoproteins were identified, including newly identified phosphopeptides in any cyanobacteria. Compared to AnpAM, the An<em>ndk</em><sup>+</sup> cells exhibited (i) lower photosynthetic efficiency and electron transport rate at low PAR (photosynthetically active radiation), (ii) no change in photochemical quenching across PAR, (iii) but distinct non-photochemical quenching [zero Y(NPQ) and high Y(NO) in An<em>ndk</em><sup>+</sup> and high Y(NPQ) and low (NO) in AnpAM], and (iv) increased tolerance to γ-radiation, oxidative, salt and DCMU stresses. The observed modulation of phosphoproteome linked to physiological changes upon overexpression of Ndk in <em>Nostoc</em> could be a combination of direct protein kinase activity of Ndk and/or indirectly through other protein kinases and phosphatases whose phosphorylation status is mediated by Ndk. This is the first report on a direct correlation between Ndk levels, phosphorylation status of proteins and stress tolerance in any cyanobacteria.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 1","pages":"Article 141054"},"PeriodicalIF":2.5,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142399168","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}
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