Biochimica et biophysica acta. Proteins and proteomics最新文献

Tracking heme biology with resonance Raman spectroscopy

IF 2.5 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2025-02-23 DOI: 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":"2025-02-23","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}

引用次数: 0

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

IF 2.5 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2025-02-15 DOI: 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":"2025-02-15","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}

引用次数: 0

Participation of a cysteine tetrad in the recycling mechanism of methionine sulfoxide reductase A from radiation-tolerant Deinococcus bacteria

IF 2.5 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2025-02-08 DOI: 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.

蛋氨酸氧化会形成蛋氨酸亚砜（MetO），蛋氨酸亚砜还原酶（Msrs）会将其还原成蛋氨酸。A 型 Msr（MsrA）还原 MetO 的催化机制需要一个催化半胱氨酸（Cys），并将其转化为亚硫酸。一般来说，催化半胱氨酸的再生需要两个解析半胱氨酸，形成两个连续的二硫桥，最后一个被硫代氧化还蛋白（Trx）有效还原。在这里，我们对沙漠化德氏球菌的 MsrA 进行了生化鉴定。它拥有四个 Cys，其中两个位于活性位点图案中（18 和 21），另外两个位于远端（53 和 163）。我们制作了这些半胱氨酸突变的 MsrA 变体，并分析了它们在 NADPH-Trx 还原酶/Trx 系统存在下还原 MetO 的能力、与 Trxs 形成异二聚体的能力以及与 MetO 培养后的氧化还原状态。我们发现，所有四个 Cys 都参与了 Trx 的酶活性再生过程。Cys18 还原 MetO 后，与 Cys21 形成第一个二硫桥。涉及 Cys21 与 Cys53 或 Cys163 的第二个二硫化物会被 Trx 还原，第三个 Cys53-Cys163 二硫化物也会形成并被 Trx 还原。这些发现首次强调了 Cys 四元组在 MsrA 催化和再生机制中的参与，并通过三维建模将其置于结构背景中，同时结合已知的涉及 Cys 三元组的循环机制进行了讨论。

{"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":"2025-02-08","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}

引用次数: 0

Elucidation of cytotoxicity of α-Synuclein fibrils on immune cells α-突触核蛋白原纤维对免疫细胞的细胞毒性研究。

IF 2.5 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2025-02-01 DOI: 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.

α-突触核蛋白（α-Syn）是一种参与细胞囊泡运输的小细胞质蛋白，在中脑、下丘脑和丘脑中逐渐聚集与帕金森病（PD）有关。淀粉样蛋白低聚物和原纤维是这种聚集的结果，对神经元具有高度毒性。然而，淀粉样蛋白诱导的神经元毒性是否是PD患者进行性神经变性的主要机制尚不清楚。在目前的研究中，我们研究了在无脂环境中以及两种磷脂存在下形成的α-Syn原纤维对巨噬细胞、树突状细胞和小胶质细胞的细胞毒性。我们发现α-Syn原纤维对树突细胞和小胶质细胞的毒性比神经元大得多。我们还观察到这种淀粉样蛋白对巨噬细胞的毒性很低。实时聚合酶链反应（RT-PCR）结果表明，淀粉样蛋白聚集体的毒性与细胞自噬水平有关。这些结果表明，大脑免疫系统的严重损伤可能是PD发病时发生的神经退行性过程的第一站。

{"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}

引用次数: 0

Replacement of the essential catalytic aspartate with serine leads to an active form of copper-containing nitrite reductase from the denitrifier Sinorhizobium meliloti 2011 用丝氨酸取代必需的催化天门冬氨酸导致反硝化菌Sinorhizobium meliloti 2011中含铜亚硝酸盐还原酶的活性形式。

IF 2.5 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2025-02-01 DOI: 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 (Asp_CAT) has been replaced with serine (Ser_CAT, 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 k_cat 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 Asp_CAT/Ser_CAT is the switch that triggers T1 → T2 electron transfer upon T2 nitrite binding and the importance of His_CAT for the pH-dependent catalytic activity of NirK are discussed.

我们报道了根生微生物S. meliloti （SmNirK）含铜亚硝酸盐还原酶的分子、生化和光谱表征和计算计算，其中催化的天冬氨酸残基（AspCAT）被丝氨酸（SerCAT, D134S）取代。与野生型酶一样，D134S是一种具有典型的双域NirK催化袋的同型三聚体，含有两个铜中心，一个是1型（T1），另一个是2型（T2）。T1的电子转移中心与野生型酶相似，但T2活性位点的电子和共价性质因突变而改变。对于野生型酶，D134S的酶活性是pH依赖性的，即在较低的pH值下它的酶活性较高，而kcat则低一个数量级。EPR研究表明，与野生型酶相比，D134S的g‖降低，a‖升高。这表明突变后T2的电子和共价性质发生了变化，影响了T2的还原电位和T1-T2的还原电位间隙。综上所述，这一证据表明T2的第二配位球的配体在控制催化过程中的关键参数中的重要性。讨论了AspCAT/SerCAT是触发T1 → T2亚硝酸盐结合时T2电子转移的开关的可能性以及HisCAT对ph依赖性NirK催化活性的重要性。

{"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}

引用次数: 0

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 打开伤口愈合的潜力：一个集成的硅蛋白质组学和体内分析Tacorin，一个生物活性蛋白组分，从Ananas comosus （L.）稳定。茎

IF 2.5 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2024-11-26 DOI: 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.

塔可林是一种从菠萝茎（Ananas comosus）中提取的生物活性蛋白，已成为一种有前景的伤口愈合治疗剂。本研究采用综合方法，结合硅蛋白质组学和体内研究，揭示他可林伤口愈合特性的分子机制。在硅蛋白组学领域，通过LC/MS-MS蛋白测序对Tacorin的组成进行了分析，发现其主要成分为ananain （23.77 kDa）和jacalin样凝集素（14.99 kDa）。分子蛋白对接模拟揭示了Tacorin成分与伤口愈合关键调节因子（包括TGF-β、TNF-α和MMP-2）之间有利的相互作用。计算的自由结合能表明Tacorin蛋白与其靶受体之间具有很强的结合亲和力。具体而言，ananain与TGF-β的结合亲和力为- 12.2 kcal/mol，表明其可能是TGF-β介导的信号传导的有效激活剂，而jacalin样凝集素与TNF-α的结合亲和力为- 8.7 kcal/mol。随后的100ns分子动力学（MD）模拟提供了对他可林受体复合物的动态行为和稳定性的深入了解，揭示了他可林治疗效果的分子决定因素。作为计算机分析的补充，体内研究通过皮肤和子宫切口模型评估了他可林在伤口愈合中的功效。宏观观察和组织学评估证明，他可林治疗在两种模型中都能加速伤口愈合并促进组织修复。总的来说，这项研究提供了令人信服的证据，证明了他可林在伤口愈合中的治疗潜力，并强调了阐明其分子机制对进一步开发和临床转化的重要性。

{"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}

引用次数: 0

A distinct co-expressed sulfurtransferase extends the physiological role of mercaptopropionate dioxygenase in Pseudomonas aeruginosa PAO1 铜绿假单胞菌 PAO1 中一种独特的共表达硫基转移酶扩展了巯丙酸二氧酶的生理作用。

IF 2.5 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2024-10-28 DOI: 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.

细菌中过硫化物的氧化和同化通常是由过硫化物二氧化酶和硫转移酶在连续反应中催化的。铜绿假单胞菌 PAO1 中负责氧化过硫化物的酶尚未明确定义。有人提出，铜绿假单胞菌 PAO1 中特征性的巯基丙酸二氧酶（MDO）可催化 3-巯基丙酸的氧化。然而，由于硫醇二氧酶在同一操作子上表达硫转移酶（ST），MDO 的生理作用尚不确定。ST 基因与 mdo 基因的共现频率为 0.94，表明这两个基因的共同表达和生理联系。ST 酶中有四个串联的菱形结构域，其中两个结构域含有能形成过硫化物的潜在催化 Cys 残基（Cys191 和 Cys435）。在硫醇定量分析中，只有 Cys435 是可触及的，H/D-X MS 分析结果进一步确定了含有 Cys435 的结构域的可触及性。硫代硫酸盐和巯基丙酮酸都是 ST 酶的硫供体，Cys435 形成过硫化物中间体。对 MDO 的动力学研究表明，该酶的底物特异性比以前发现的更广，可氧化巯基丙酸盐和巯基丙酮酸盐硫醇和过硫化物底物。这些研究结果有助于深入了解 mdo 操作子在硫化物氧化和同化过程中的总体机制和生理作用。

{"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}

引用次数: 0

Deciphering the cleavage sites of 3C-like protease in Gammacoronaviruses and Deltacoronaviruses 解密伽马冠状病毒和德尔塔冠状病毒中 3C 样蛋白酶的裂解位点。

IF 2.5 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2024-10-23 DOI: 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 (3CL^pro) to cleave polyprotein precursors and host proteins. However, current tools for identifying 3CL^pro cleavage sites are limited, particularly in Gammacoronaviruses (GammaCoV) and Deltacoronaviruses (DeltaCoV). This study aims to fill this gap by identifying 3CL^pro 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 3CL^pro 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^pro cleavage sites, revealing the pathogenic mechanisms of coronaviruses, and offering new insights for developing potential therapeutic targets.

冠状病毒通过使用 3C 样蛋白酶（3CLpro）裂解多聚蛋白前体和宿主蛋白进行复制。然而，目前用于识别 3CLpro 裂解位点的工具非常有限，尤其是在伽马冠状病毒（GammaCoV）和德尔塔冠状病毒（DeltaCoV）中。本研究旨在通过确定这些病毒中的 3CLpro 裂解位点来填补这一空白，从而更深入地了解其致病机制。通过整合序列比对和结构模型比较，我们开发了基于自裂解模式的特定位置评分矩阵（PSSM），揭示了每个残基的特定偏好。利用 AlphaFold2 的预测配位误差（PAE）和预测局部距离差异测试（pLDDT），我们发现大多数裂解序列位于 PAE 值高而 pLDDT 值低的区域。KEGG通路分析表明，潜在的宿主蛋白裂解目标主要集中在与核-胞质转运和内吞有关的通路上。通过体外裂解实验和突变分析，我们发现并验证了三个高得分蛋白--核疏松蛋白 58（NUP58）、细胞分裂周期 73（CDC73）和信号转导适配分子 2（STAM2）。这些发现表明，3CLpro 不仅在病毒复制中发挥着重要作用，还可能通过裂解宿主蛋白影响宿主细胞的功能。这项研究为确定 3CLpro 的裂解位点、揭示冠状病毒的致病机制提供了有效工具，并为开发潜在的治疗靶点提供了新的见解。

{"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}

引用次数: 0

CDR identification, epitope mapping and binding affinity determination of novel monoclonal antibodies generated against human apolipoprotein B-100 针对人类脂蛋白 B-100 生成的新型单克隆抗体的 CDR 识别、表位图绘制和结合亲和力测定。

IF 2.5 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2024-10-23 DOI: 10.1016/j.bbapap.2024.141058

Tariga Sritrakarn , Kanokwan Lowhalidanon , Panida Khunkaewla

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_aff) 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.

对内部生成的载脂蛋白 B-100（apoB-100）克隆 hLDL-E8、hLDL-2D8 和 hLDL-F5 mAbs 进行了广泛研究，以确定它们的互补决定区（CDR）、结合表位和亲和力。RT-PCR 显示，所有 mAbs 都由 kappa 轻链和 gamma 重链组成。DNA 测序和生物信息学分析表明，其 CDR 的可变基因和蛋白质序列与现有数据库的一致性超过 50%。利用 SWISS-MODEL 平台构建了 QSQE 分数高于 0.7 的 mAb 可变片段（Fv）的三维结构。拉马钱德兰图证实了结构的准确性，99% 的氨基酸残基位于可接受的区域内。apoB-100的溶栓裂解和Western印迹分析表明，hLDL-E8和hLDL-F5与T3片段（aa 1297-3249）特异性结合，而hLDL-2D8则与T4片段（aa 1-1297）结合。利用抑制酶联免疫吸附法进行的表位结合试验证实了这些发现，结果表明 hLDL-E8 与 hLDL-2D8 结合的表位不同，与 hLDL-F5 有一些重叠。最后，通过间接 ELISA 检测了 mAbs 的结合亲和力。mAbs hLDL-2D8、hLDL-E8 和 hLDL-F5 的平均亲和力常数（Kaff）分别为 1.51 ± 0.69 × 109 Mol-1、7.25 ± 3.56 × 108 Mol-1 和 4.39 ± 2.63 × 106 Mol-1。此外，抗体在剂量-反应曲线中的表现显示，hLDL-F5 可能识别载脂蛋白 100 的两个表位或具有极低的结合亲和力。相比之下，hLDL-2D8 和 hLDL-E8 只能识别一个表位。这些发现为实验室和临床研究人员选择 mAbs 提供了有用的信息。

{"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}

引用次数: 0

The role of proton-coupled electron transfer from protein to heme in dehaloperoxidase 脱卤过氧化物酶中从蛋白质到血红素的质子耦合电子传递的作用。

IF 2.5 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2024-10-16 DOI: 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.

在脱卤过氧化物酶（DHP）的六个蛋氨酸（Met）残基中，至少有两个残基在自动还原和蛋白质-血红素交联过程中充当电子供体。在 DHP-A 和 DHP-B 两种同工酶中观察到的自动还原现象可通过高血红素还原电位和来自蛋氨酸（Met）或半胱氨酸（Cys）的内源性电子源来解释。本研究提供的证据表明，当 DHP 被 H2O2 激活与底物氧化和自动还原竞争时，会发生与蛋白质血红素交联的联系。DHP-A 和 DHP-B 的自动还原产率相当，且都与 DHP 浓度成反比。这两种同工酶在与蛋白质二聚化相关的自动还原动力学上都显示出反合作效应。尽管在 DHP-A 中存在五个酪氨酸（Tyr）氨基酸，在 DHP-B 中存在四个酪氨酸，但质谱证据并不支持在某些哺乳动物肌红蛋白中观察到的酪氨酸-血红素或蛋白质间酪氨酸-酪氨酸交联事件。LC-MS 和串联 MS/MS 研究揭示了参与血红素-蛋白质交联的三个氨基酸：Cys73、Met63 和 Met64。Cys73 促进了 DHP-A 中二聚体的形成，这似乎也减缓了自动还原的速度，但并不参与共价蛋白质-血红素交联。根据突变研究，Met63 和 64 既参与共价血红素交联，也参与自动还原。质子耦合电子传递和 Met 与血红素的交联可能有助于调节 DHP 的功能，保护其免受失控的氧化损伤。

{"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}

引用次数: 0