Pub Date : 2025-07-01Epub Date: 2025-03-27DOI: 10.1016/j.bbapap.2025.141067
Monidipa Konar , Bhavneet Kaur , Uttam Chand Saini , Sanjay K. Bhadada , Sadhna Sharma
Type 2 diabetes mellitus (T2DM) and Osteoarthritis (OA) share common risk factors like age, obesity and hypertension. Currently, 52 % of diabetic patients suffer from arthritis. Diabetes facilitates OA by altering lipid metabolism, levels of adipokines & cytokines, accumulation of advanced glycation end products, etc., which affects cartilage & bone health. However, the molecular mechanisms of the association of OA with T2DM remain unexplored. Since diabetes greatly affects the glycosylation status of proteins, the present study focused on identifying glycoproteins that could serve as diagnostic and prognostic markers for identifying osteoarthritis in diabetic individuals by LC-MS/MS. Comparative proteomic analysis revealed 20 significantly altered glycoproteins; among them, thyroxine-binding globulin (THBG), alpha-1-antitrypsin (A1AT), fibrinogen gamma chain (FGG) and angiotensinogen (AGT) were further validated. THBG, A1AT and AGT showed promising potential to identify the comorbid condition in serum and synovial fluid, however, ROC analysis identified THBG as the best candidate glycoprotein marker. Upregulation of THBG in OADM disrupts the bone remodeling cycle, degrades insulin, and promotes the expression of GLUT-1 and MMP-9. Overall, THBG could also serve as a therapeutic target for reducing the progression of osteoarthritis and alleviating pain and bone stiffness associated with the disease.
{"title":"Synovial fluid glycoproteome profiling in knee osteoarthritis: Molecular insights into type 2 diabetes-associated biomarkers and therapeutic targets","authors":"Monidipa Konar , Bhavneet Kaur , Uttam Chand Saini , Sanjay K. Bhadada , Sadhna Sharma","doi":"10.1016/j.bbapap.2025.141067","DOIUrl":"10.1016/j.bbapap.2025.141067","url":null,"abstract":"<div><div>Type 2 diabetes mellitus (T2DM) and Osteoarthritis (OA) share common risk factors like age, obesity and hypertension. Currently, 52 % of diabetic patients suffer from arthritis. Diabetes facilitates OA by altering lipid metabolism, levels of adipokines & cytokines, accumulation of advanced glycation end products, etc., which affects cartilage & bone health. However, the molecular mechanisms of the association of OA with T2DM remain unexplored. Since diabetes greatly affects the glycosylation status of proteins, the present study focused on identifying glycoproteins that could serve as diagnostic and prognostic markers for identifying osteoarthritis in diabetic individuals by LC-MS/MS. Comparative proteomic analysis revealed 20 significantly altered glycoproteins; among them, thyroxine-binding globulin (THBG), alpha-1-antitrypsin (A1AT), fibrinogen gamma chain (FGG) and angiotensinogen (AGT) were further validated. THBG, A1AT and AGT showed promising potential to identify the comorbid condition in serum and synovial fluid, however, ROC analysis identified THBG as the best candidate glycoprotein marker. Upregulation of THBG in OADM disrupts the bone remodeling cycle, degrades insulin, and promotes the expression of GLUT-1 and MMP-9. Overall, THBG could also serve as a therapeutic target for reducing the progression of osteoarthritis and alleviating pain and bone stiffness associated with the disease.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 4","pages":"Article 141067"},"PeriodicalIF":2.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742091","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 : 2025-07-01Epub Date: 2025-04-04DOI: 10.1016/j.bbapap.2025.141070
P. Ramakrishna Reddy, A. Kulandaisamy, M. Michael Gromiha
Understanding the folding and stability of transmembrane β-barrel proteins (TMBs) provides insights into their structural integrity, functional mechanisms, and implications for disease states. In this work, we have characterized the important features that influence the folding and stability of TMBs. Our results showed that lipid accessible surface area and transition energy are important for understanding the stability of TMBs. Further, this information was utilized to develop a linear regression-based method for predicting the stability of TMBs. Our method achieved a correlation and mean absolute error (MAE) of 0.96 and 0.94 kcal/mol on the jack-knife test. Moreover, we compared the stability of TMBs with globular all-β proteins and observed that long-range interactions and energetic properties are crucial for maintaining the stability of both β-barrel membrane and all-β globular proteins. On the other hand, side-chain – side-chain hydrogen bonds and lipid accessible surface area are specific to membrane proteins. These features are critical for membrane proteins because they influence a protein to embed within the membrane environment. Further, we have developed a web server, TMB Stab-pred for predicting the stability of TMBs, and it is accessible at https://web.iitm.ac.in/bioinfo2/TMBB/index.html.
{"title":"TMB Stab-pred: Predicting the stability of transmembrane β-barrel proteins using their sequence and structural signatures","authors":"P. Ramakrishna Reddy, A. Kulandaisamy, M. Michael Gromiha","doi":"10.1016/j.bbapap.2025.141070","DOIUrl":"10.1016/j.bbapap.2025.141070","url":null,"abstract":"<div><div>Understanding the folding and stability of transmembrane β-barrel proteins (TMBs) provides insights into their structural integrity, functional mechanisms, and implications for disease states. In this work, we have characterized the important features that influence the folding and stability of TMBs. Our results showed that lipid accessible surface area and transition energy are important for understanding the stability of TMBs. Further, this information was utilized to develop a linear regression-based method for predicting the stability of TMBs. Our method achieved a correlation and mean absolute error (MAE) of 0.96 and 0.94 kcal/mol on the jack-knife test. Moreover, we compared the stability of TMBs with globular all-β proteins and observed that long-range interactions and energetic properties are crucial for maintaining the stability of both β-barrel membrane and all-β globular proteins. On the other hand, side-chain – side-chain hydrogen bonds and lipid accessible surface area are specific to membrane proteins. These features are critical for membrane proteins because they influence a protein to embed within the membrane environment. Further, we have developed a web server, TMB Stab-pred for predicting the stability of TMBs, and it is accessible at <span><span>https://web.iitm.ac.in/bioinfo2/TMBB/index.html</span><svg><path></path></svg></span>.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 4","pages":"Article 141070"},"PeriodicalIF":2.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143794638","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}
Some species of microalgae, including Chlorella, can utilize a variety of dissolved organic carbon sources, such as sugars and organic acids, for growth. In the presence of both light and these organic substrates, they exhibit mixotrophic metabolism, combining heterotrophic assimilation of organic carbon with photosynthetic carbon fixation. In this study, we analyzed using proteomic and transcriptomic approaches the ability of Chlorella sp. to shift its metabolism when it was grown in photoautotrophy in BG-11 medium and mixotrophy in 30 % diluted olive mill wastewaters (OMWWs) pretreated with laccases. Using UniProt, InterPro, KEGG Pathway, and Gene Ontology databasesproteomic and transcriptomic data have been analyzed. In mixotrophy, over-expression of cell cycle, signaling, and transport proteins, as well as chaperone proteins, were identified and associated with an overall decrease in photosynthesis and carbohydrate/lipid metabolic pathways. In addition, the expression of light-independent protochlorophyllide reductase, malate synthase, acetyl-CoA carboxylase and pyruvate kinase were modulated. Surprisingly an upregulation of the ammonium transporter protein, which could play a role in OMWWs detoxification was detected. Homology modeling investigation of the three-dimensional structure of ammonium transporter protein revealed that it holds a functional trimeric structure with a lengthy C-terminal region that may be involved in the regulation and activation of ammonium transport.
{"title":"Evolution of Chlorella sp. RCC288 proteome and transcriptome during its adaptation to oil mill wastewater","authors":"Bouthaina Menaa , Rihab Hachicha , Pascal Dubessay , Slim Abdelkafi , Imen Fendri , Philippe Michaud","doi":"10.1016/j.bbapap.2025.141077","DOIUrl":"10.1016/j.bbapap.2025.141077","url":null,"abstract":"<div><div>Some species of microalgae, including <em>Chlorella</em>, can utilize a variety of dissolved organic carbon sources, such as sugars and organic acids, for growth. In the presence of both light and these organic substrates, they exhibit mixotrophic metabolism, combining heterotrophic assimilation of organic carbon with photosynthetic carbon fixation. In this study, we analyzed using proteomic and transcriptomic approaches the ability of <em>Chlorella</em> sp. to shift its metabolism when it was grown in photoautotrophy in BG-11 medium and mixotrophy in 30 % diluted olive mill wastewaters (OMWWs) pretreated with laccases. Using UniProt, InterPro, KEGG Pathway, and Gene Ontology databasesproteomic and transcriptomic data have been analyzed. In mixotrophy, over-expression of cell cycle, signaling, and transport proteins, as well as chaperone proteins, were identified and associated with an overall decrease in photosynthesis and carbohydrate/lipid metabolic pathways. In addition, the expression of light-independent protochlorophyllide reductase, malate synthase, acetyl-CoA carboxylase and pyruvate kinase were modulated. Surprisingly an upregulation of the ammonium transporter protein, which could play a role in OMWWs detoxification was detected. Homology modeling investigation of the three-dimensional structure of ammonium transporter protein revealed that it holds a functional trimeric structure with a lengthy C-terminal region that may be involved in the regulation and activation of ammonium transport.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 4","pages":"Article 141077"},"PeriodicalIF":2.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143941554","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 : 2025-07-01Epub Date: 2025-04-26DOI: 10.1016/j.bbapap.2025.141075
Jéssica Fernandes Scortecci , Adriano de Freitas Fernandes , Vitor Hugo Balasco Serrão , Marinônio Lopes Cornélio , Mario de Oliveira Neto , Otavio Henrique Thiemann
The Selenocysteine (Sec - U) biosynthesis pathway is present in eukaryotes and prokaryotes, in which its incorporation is directed by the stop codon UGA and the structural mRNA element named Sec Insertion Sequence (SECIS) that contain an essential kink-turn motif recognized by specific RNA-binding proteins. SBP2 is the key player in the interaction with the SECIS element in eukaryotes, and it is essential for the biosynthesis pathway. Free-living amoebas are part of the Heterolobosea phylum, and several species, including Naegleria fowleri, are known human pathogens. In 2013 it was reported that Naegleria gruberi (NgSBP2), which is non-pathogenic, had a divergent SBP2 sequence and all the genes essential for Sec synthesis. The identity of NgSBP2 is confirmed experimentally and its binding affinity to the SECIS element is demonstrated. The N-terminal and the C-terminal domains (NgSBP2-NT and NgSBP2-CT, respectively) of NgSBP2 contain disordered regions, particularly in the N-terminal domain. The SECIS element is bonded to NgSBP2-CT, which results in a decrease in the disordered sequence of the domain, and the NgSBP2-NT domain interacts with NgSBP2-CT.SECIS complex, as we present here. The findings reveal the molecular interaction patterns underlying the selenocysteine incorporation pathway in an early-branching eukaryote, which is influenced by multiple protein-RNA interactions.
{"title":"Biophysical analysis of SECIS binding protein 2 (SBP2) from Naegleria gruberi","authors":"Jéssica Fernandes Scortecci , Adriano de Freitas Fernandes , Vitor Hugo Balasco Serrão , Marinônio Lopes Cornélio , Mario de Oliveira Neto , Otavio Henrique Thiemann","doi":"10.1016/j.bbapap.2025.141075","DOIUrl":"10.1016/j.bbapap.2025.141075","url":null,"abstract":"<div><div>The Selenocysteine (Sec - U) biosynthesis pathway is present in eukaryotes and prokaryotes, in which its incorporation is directed by the stop codon UGA and the structural mRNA element named Sec Insertion Sequence (SECIS) that contain an essential kink-turn motif recognized by specific RNA-binding proteins. SBP2 is the key player in the interaction with the SECIS element in eukaryotes, and it is essential for the biosynthesis pathway. Free-living amoebas are part of the Heterolobosea phylum, and several species, including <em>Naegleria fowleri</em>, are known human pathogens. In 2013 it was reported that <em>Naegleria gruberi</em> (<em>Ng</em>SBP2), which is non-pathogenic, had a divergent SBP2 sequence and all the genes essential for Sec synthesis. The identity of <em>Ng</em>SBP2 is confirmed experimentally and its binding affinity to the SECIS element is demonstrated. The N-terminal and the C-terminal domains (<em>Ng</em>SBP2-NT and <em>Ng</em>SBP2-CT, respectively) of <em>Ng</em>SBP2 contain disordered regions, particularly in the N-terminal domain. The SECIS element is bonded to NgSBP2-CT, which results in a decrease in the disordered sequence of the domain, and the NgSBP2-NT domain interacts with NgSBP2-CT.SECIS complex, as we present here. The findings reveal the molecular interaction patterns underlying the selenocysteine incorporation pathway in an early-branching eukaryote, which is influenced by multiple protein-RNA interactions.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 4","pages":"Article 141075"},"PeriodicalIF":2.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143898770","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 : 2025-05-01Epub Date: 2025-02-08DOI: 10.1016/j.bbapap.2025.141063
Pascal Rey , Nicolas Rouhier , Chloé Carassus , Arjan de Groot , Laurence Blanchard
Methionine oxidation leads to the formation of methionine sulfoxide (MetO), which is reduced back to Met by methionine sulfoxide reductases (Msrs). The catalytic mechanism used by A-type Msr (MsrA) for MetO reduction requires a catalytic cysteine (Cys), which is converted to a sulfenic acid. In general, two resolving Cys are required for the regeneration of the catalytic Cys forming two consecutive disulfide bridges, the last one being efficiently reduced by thioredoxin (Trx). Here, we performed the biochemical characterization of MsrA from Deinococcus deserti. It possesses four Cys, two present in the active site motif (18 and 21) and two distal ones (53 and 163). We produced MsrA variants mutated for these cysteines and analyzed their capacity to reduce MetO in the presence of the NADPH-Trx reductase/Trx system, their ability to form heterodimers with Trxs, and their redox status after incubation with MetO. We show that all four Cys are involved in the regeneration process of enzyme activity by Trx. After MetO reduction by Cys18, a first disulfide bridge is formed with Cys21. A second disulfide involving Cys21 with either Cys53 or Cys163 is reduced by Trx, and a third Cys53-Cys163 disulfide can be formed and also reduced by Trx. These findings highlighting for the first time the involvement of a Cys tetrad in the catalytic and regeneration mechanisms for a MsrA are placed in a structural context by performing 3D modelling and discussed in relation to the known recycling mechanisms involving a Cys triad.
{"title":"Participation of a cysteine tetrad in the recycling mechanism of methionine sulfoxide reductase A from radiation-tolerant Deinococcus bacteria","authors":"Pascal Rey , Nicolas Rouhier , Chloé Carassus , Arjan de Groot , Laurence Blanchard","doi":"10.1016/j.bbapap.2025.141063","DOIUrl":"10.1016/j.bbapap.2025.141063","url":null,"abstract":"<div><div>Methionine oxidation leads to the formation of methionine sulfoxide (MetO), which is reduced back to Met by methionine sulfoxide reductases (Msrs). The catalytic mechanism used by A-type Msr (MsrA) for MetO reduction requires a catalytic cysteine (Cys), which is converted to a sulfenic acid. In general, two resolving Cys are required for the regeneration of the catalytic Cys forming two consecutive disulfide bridges, the last one being efficiently reduced by thioredoxin (Trx). Here, we performed the biochemical characterization of MsrA from <em>Deinococcus deserti</em>. It possesses four Cys, two present in the active site motif (18 and 21) and two distal ones (53 and 163). We produced MsrA variants mutated for these cysteines and analyzed their capacity to reduce MetO in the presence of the NADPH-Trx reductase/Trx system, their ability to form heterodimers with Trxs, and their redox status after incubation with MetO. We show that all four Cys are involved in the regeneration process of enzyme activity by Trx. After MetO reduction by Cys18, a first disulfide bridge is formed with Cys21. A second disulfide involving Cys21 with either Cys53 or Cys163 is reduced by Trx, and a third Cys53-Cys163 disulfide can be formed and also reduced by Trx. These findings highlighting for the first time the involvement of a Cys tetrad in the catalytic and regeneration mechanisms for a MsrA are placed in a structural context by performing 3D modelling and discussed in relation to the known recycling mechanisms involving a Cys triad.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 3","pages":"Article 141063"},"PeriodicalIF":2.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143389955","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 : 2025-05-01Epub Date: 2025-02-15DOI: 10.1016/j.bbapap.2025.141064
Sk Alim , Sudheer K. Cheppali , Sonali S. Pawar, Musti J. Swamy
Fibronectin type-II (FnII) proteins are major constituents in the seminal plasma of many mammals and play a crucial role in sperm capacitation. Additionally, the seminal FnII proteins from bull and horse exhibit chaperone-like activity (CLA), by acting as small heat shock proteins (shsps). The present work demonstrates that the major FnII protein of donkey seminal plasma, DSP-1 exhibits CLA with broad specificity and protects various client proteins such as alcohol dehydrogenase, lactate dehydrogenase and enolase against thermal and oxidative stress. Binding of phosphorylcholine (PrC) – the head group moiety of choline phospholipids, which are the physiological ligands of DSP-1 – decreased the CLA whereas binding of 1,2-dioleoyl-sn-glycero-3-phospholcholine (DOPC) increased the CLA. Biophysical studies suggested that these contrasting effects on the CLA by phosphorylcholine and diacyl phosphatidylcholine could be attributed to changes in the surface hydrophobicity of DSP-1 upon binding to these ligands. Interestingly, binding of PrC reduced DSP-1 tetramers to monomers with lower surface hydrophobicity, whereas binding to DOPC liposomes increased its surface hydrophobicity. These results, which demonstrate that DSP-1 exhibits CLA and functions as a molecular chaperone, expand the family of mammalian seminal FnII proteins that function as shsps.
{"title":"DSP-1, the major fibronectin type-II protein of donkey seminal plasma is a small heat-shock protein and exhibits chaperone-like activity against thermal and oxidative stress","authors":"Sk Alim , Sudheer K. Cheppali , Sonali S. Pawar, Musti J. Swamy","doi":"10.1016/j.bbapap.2025.141064","DOIUrl":"10.1016/j.bbapap.2025.141064","url":null,"abstract":"<div><div>Fibronectin type-II (FnII) proteins are major constituents in the seminal plasma of many mammals and play a crucial role in sperm capacitation. Additionally, the seminal FnII proteins from bull and horse exhibit chaperone-like activity (CLA), by acting as small heat shock proteins (<em>shsp</em>s). The present work demonstrates that the major FnII protein of donkey seminal plasma, DSP-1 exhibits CLA with broad specificity and protects various client proteins such as alcohol dehydrogenase, lactate dehydrogenase and enolase against thermal and oxidative stress. Binding of phosphorylcholine (PrC) – the head group moiety of choline phospholipids, which are the physiological ligands of DSP-1 – decreased the CLA whereas binding of 1,2-dioleoyl-<em>sn</em>-glycero-3-phospholcholine (DOPC) increased the CLA. Biophysical studies suggested that these contrasting effects on the CLA by phosphorylcholine and diacyl phosphatidylcholine could be attributed to changes in the surface hydrophobicity of DSP-1 upon binding to these ligands. Interestingly, binding of PrC reduced DSP-1 tetramers to monomers with lower surface hydrophobicity, whereas binding to DOPC liposomes increased its surface hydrophobicity. These results, which demonstrate that DSP-1 exhibits CLA and functions as a molecular chaperone, expand the family of mammalian seminal FnII proteins that function as <em>shsp</em>s.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 3","pages":"Article 141064"},"PeriodicalIF":2.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143432278","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 : 2025-05-01Epub Date: 2025-02-23DOI: 10.1016/j.bbapap.2025.141065
Amanda Bartkowiak, Ewa Szczesny-Malysiak, Jakub Dybas
Heme proteins are a large group of biomolecules with heme incorporated as a prosthetic group. Apart from cytochromes present in almost all cell types, many other specific heme proteins are expressed in different kinds of cells, e.g. hemoglobin in the erythrocytes, myoglobin (skeletal and vascular smooth muscle cells), cytoglobin (fibroblasts) and neuroglobin (neurons and retina). Among their wide and diverse biological functions, the most important is their unique ability to bind, store, and transport gaseous molecules, such as oxygen, carbon monoxide, and nitric oxide. Resonance Raman (RR) spectroscopy is an exceptional analytical tool that allows for qualitative and quantitative characterization of heme proteins in biological systems. Due to its high sensitivity, even subtle structural alterations of the heme group can be monitored and tracked during cellular processes. Resonance Raman excitation within the Soret absorption band (390–440 nm) provides rich information on the environment of heme's active site, allowing differentiation of the iron ion oxidation and spin states, and tracking the movement of the porphyrin ring plane in response to the changes in oxygenation status. Herein, we summarize and discuss recent developments in RR applications aimed to link the structure-function relationship of heme proteins within biological systems, connected, e.g., with the formation of hemoglobin (Hb) adducts (nitrosylhemoglobin, cyanhemoglobin, sulfhemoglobin), irreversible Hb alterations deteriorating oxygen binding and differentiation of heme proteins oxidation state within live cells in situ.
{"title":"Tracking heme biology with resonance Raman spectroscopy","authors":"Amanda Bartkowiak, Ewa Szczesny-Malysiak, Jakub Dybas","doi":"10.1016/j.bbapap.2025.141065","DOIUrl":"10.1016/j.bbapap.2025.141065","url":null,"abstract":"<div><div>Heme proteins are a large group of biomolecules with heme incorporated as a prosthetic group. Apart from cytochromes present in almost all cell types, many other specific heme proteins are expressed in different kinds of cells, e.g. hemoglobin in the erythrocytes, myoglobin (skeletal and vascular smooth muscle cells), cytoglobin (fibroblasts) and neuroglobin (neurons and retina). Among their wide and diverse biological functions, the most important is their unique ability to bind, store, and transport gaseous molecules, such as oxygen, carbon monoxide, and nitric oxide. Resonance Raman (RR) spectroscopy is an exceptional analytical tool that allows for qualitative and quantitative characterization of heme proteins in biological systems. Due to its high sensitivity, even subtle structural alterations of the heme group can be monitored and tracked during cellular processes. Resonance Raman excitation within the Soret absorption band (390–440 nm) provides rich information on the environment of heme's active site, allowing differentiation of the iron ion oxidation and spin states, and tracking the movement of the porphyrin ring plane in response to the changes in oxygenation status. Herein, we summarize and discuss recent developments in RR applications aimed to link the structure-function relationship of heme proteins within biological systems, connected, e.g., with the formation of hemoglobin (Hb) adducts (nitrosylhemoglobin, cyanhemoglobin, sulfhemoglobin), irreversible Hb alterations deteriorating oxygen binding and differentiation of heme proteins oxidation state within live cells in situ.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 3","pages":"Article 141065"},"PeriodicalIF":2.5,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143497595","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 : 2025-02-01Epub Date: 2024-12-16DOI: 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 : 2025-02-01Epub Date: 2024-12-30DOI: 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 : 2025-01-01Epub 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":"2025-01-01","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}
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