Archives of biochemistry and biophysics最新文献_第7页

Investigating the binding of fluorescent probes to a trypanosomal-tRNA synthetase: A fluorescence spectroscopic and molecular dynamics study 研究荧光探针与锥虫- trna合成酶的结合：荧光光谱和分子动力学研究。

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-02-01 DOI: 10.1016/j.abb.2024.110263

Pratyasha Bhowal , David Jameson , Rajat Banerjee

Given the high prevalence of Chagas disease in the Americas, we targeted the unique arginyl-tRNA synthetase of its causative agent Trypanosoma cruzi. Among their many possible uses, naphthalene-derived fluorescent ligands, such as ANS and bis-ANS, may be employed in pharmacokinetic research. Although ANS and bis-ANS have become prominent fluorescent probes for protein characterization, the structural and spectroscopic characteristics of protein-ANS/bis-ANS complexes remain largely unknown. Both fluorescent dyes bind to either the folded or partially folded hydrophobic regions of proteins. Additionally, they serve to identify molten globule-like intermediates. These probes have been used to study the folding problems of protein structures and the mechanisms of protein-protein interactions. ANS and bis-ANS exhibited significant enhancement and blue shift in their emission spectra upon binding to TcArgRS, the primary enzyme responsible for attaching l-arginine to its corresponding tRNA. Through fluorescence spectroscopy and computational studies, we concluded that bis-ANS binds more tightly to TcArgRS and that ATP affects bis-ANS fluorescence signal. Thus, these probes are useful resources for studying the intricate intermolecular relationships between proteins in terms of their structure, function, and mechanism. Our study provides a framework for identifying the hydrophobic regions present in TcArgRS. The utilization of hydrophobic patches on proteins for drug targeting is noteworthy because they can assist in identifying regions on the surface of proteins that are likely to interact with ligands. These patches help identify hotspot residues that play a vital role in determining binding affinity. Drugs are mainly small and hydrophobic in nature, and they target protein surfaces which have complementary properties. In this study, we elucidated the potential of TcArgRS as a target for combating trypanosomal diseases and extending life expectancy.

鉴于美洲恰加斯病的高流行率，我们针对其病原体克氏锥虫独特的精氨酸- trna合成酶。在其许多可能的用途中，萘衍生的荧光配体，如ANS和双ANS，可用于药代动力学研究。尽管ANS和bis-ANS已经成为蛋白质表征的重要荧光探针，但蛋白质-ANS/bis-ANS复合物的结构和光谱特征在很大程度上仍然未知。两种荧光染料都与蛋白质的折叠或部分折叠的疏水区域结合。此外，它们还用于识别熔融的球状中间物。这些探针已被用于研究蛋白质结构的折叠问题和蛋白质-蛋白质相互作用的机制。在与TcArgRS结合后，ANS和bis-ANS的发射光谱明显增强和蓝移，TcArgRS是负责将l -精氨酸附着到相应tRNA上的主要酶。通过荧光光谱和计算研究，我们得出bis-ANS与TcArgRS结合更紧密，ATP影响了bis-ANS的荧光信号。因此，这些探针是研究蛋白质在结构、功能和机制方面复杂的分子间关系的有用资源。我们的研究为识别TcArgRS中存在的疏水区域提供了一个框架。疏水贴片在蛋白质上用于药物靶向是值得注意的，因为它们可以帮助识别蛋白质表面可能与配体相互作用的区域。这些补丁有助于识别在确定结合亲和力中起重要作用的热点残基。药物本质上主要是小而疏水的，它们靶向具有互补性质的蛋白质表面。在这项研究中，我们阐明了TcArgRS作为对抗锥虫病和延长预期寿命的靶点的潜力。

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引用次数: 0

Oxidative stress of mitophagy in neurodegenerative diseases: Mechanism and potential therapeutic targets 神经退行性疾病中线粒体自噬的氧化应激：机制和潜在的治疗靶点。

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-02-01 DOI: 10.1016/j.abb.2024.110283

Yixin Li, Wanying Zhang, Qihang Zhang, Yunzhe Li, Chonghui Xin, Rongze Tu, Haijing Yan

Neurodegenerative diseases are now significant chronic progressive neurological conditions that affect individuals' physical health. Oxidative stress is crucial in the development of these diseases. Among the various neurodegenerative diseases, mitochondrial damage has become a major factor in oxidative stress and disease advancement. During this process, oxidative stress and mitophagy plays an important role. In this paper, we introduced the role of mitophagy and oxidative stress in detail, and expounded the relationship between them. In addition, we summarized the pathogenesis of some neurodegenerative diseases and the mechanism of three antioxidants. The former includes AD, PD, HD and ALS, while the latter includes carnosine, adiponectin and resveratrol. Provide goals and directions for further research and treatment of neurodegenerative diseases.

This review summarizes the impact of oxidative stress on neurodegenerative diseases by regulating mitophagy, provides a deeper understanding of their pathological mechanisms, and suggests potential new therapeutic targets.

神经退行性疾病是影响个体身体健康的重要慢性进行性神经系统疾病。氧化应激在这些疾病的发展中起着至关重要的作用。在各种神经退行性疾病中，线粒体损伤已成为氧化应激和疾病进展的主要因素。在这一过程中，氧化应激和线粒体自噬起着重要作用。本文详细介绍了线粒体自噬和氧化应激的作用，并阐述了它们之间的关系。此外，我们还综述了一些神经退行性疾病的发病机制和三种抗氧化剂的作用机制。前者包括AD、PD、HD和ALS，后者包括肌肽、脂联素和白藜芦醇。为神经退行性疾病的进一步研究和治疗提供目标和方向。本文综述了氧化应激通过调节线粒体自噬对神经退行性疾病的影响，为其病理机制提供了更深入的认识，并提出了潜在的新治疗靶点。

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引用次数: 0

Conservation of mechanism in flavoprotein-catalyzed amine oxidation 黄素蛋白催化胺氧化机理的研究。

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-02-01 DOI: 10.1016/j.abb.2024.110242

Paul F. Fitzpatrick

The goals of this presentation are to summarize the present understanding of the mechanism of amine oxidation by flavoproteins and to examine the possibility that a member of the monoamine oxidase family catalyzes oxidation of a carbon-carbon bond. In the discussion of mechanism, the emphasis is on the protonation state of the amine substrate, since the once-controversial mechanism of oxidation appears to be resolved. The argument will be made that flavoproteins catalyzing amine oxidation preferentially bind the form of the substrate in which the reacting nitrogen is uncharged. The reaction of a member of L-6-hydroxynicotine oxidase, which has been proposed to oxidize a carbon-carbon bond in its substrate during nicotine catabolism, is then discussed. Analysis of the reaction product establishes that the enzyme catalyzes oxidation of a carbon-nitrogen. The effects of site-directed mutagenesis and analysis of the substrate specificity identify the key residues for substrate binding.

本报告的目的是总结目前对黄蛋白氧化胺的机制的理解，并研究单胺氧化酶家族成员催化碳-碳键氧化的可能性。在机理的讨论中，重点放在胺底物的质子化状态上，因为一度有争议的氧化机制似乎得到了解决。将提出的论点是，催化胺氧化的黄蛋白优先结合反应氮不带电的底物形式。l -6-羟基尼古丁氧化酶的一个成员在尼古丁分解代谢过程中氧化其底物中的碳-碳键，然后讨论了该反应。对反应产物的分析证实该酶催化碳氮氧化。定点诱变的效果和底物特异性分析确定了底物结合的关键残基。

引用次数: 0

The identification of insect specific iAANAT inhibitors 昆虫特异性iAANAT抑制剂的鉴定。

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-02-01 DOI: 10.1016/j.abb.2024.110282

Aidan J. Hawley, Suzeeta Bhandari , Peter W. Radulovic , Natalia Borisova , Gabrielle Henry, Tyler Holets, Christian Sabbagh, Matthew Scearbo , Gabriela Suarez , David J. Merkler

An important aspect of food security is the development of innovative insecticides, particularly ones that specifically target insect pests and exhibit minimal toxicity to mammals. The insect arylalkylamine N-acyltransferases (iAANATs) could serve as targets for novel insecticides that satisfy these criteria. There exists a wealth of structural and biochemical information for the iAANATs and iAANAT knockdown experiments show that these enzymes are critical to insect health. Herein, we have expressed, purified, and characterized two new iAANATs, one from Apis mellifera (honey bee, AmNAT1) and another from Diaphorina citri (Asian citrus psyllid, DcNAT). We discovered that diminazene, a compound used to treat livestock for trypanosomiasis and babesiosis, inhibits AmNAT1, DcNAT, and D. melanogaster DmAgmNAT with modest affinity, K_i values ranging from 0.8 μM to 200 μM. We found a series of guanidines, amidines, and a hydroxamate, structurally related to diminazene, also inhibit the iAANATs, including camostat, gabexate, nafamostate, and panobinostat. Significantly, we found DmAgmNAT is far more susceptible to inhibition by four of these five of these compounds. In particular, camostat, nafamostat, and gabexate inhibit DmAgmNAT with K_i values of 0.2–30 μM, but no inhibition of AmNAT1 and DcNAT was observed at 500 μM for any of the three. These results show that a species-specific inhibitor targeted against an iAANAT is a real possibility. Also, we report that adipoyl-CoA is a substrate for AmNAT1 and DcNAT and that succinoyl-CoA is a substrate for DcNAT. These results contribute to a growing body of data suggesting that N-dicarboxyacyl-amines are metabolites in insects and other organisms.

粮食安全的一个重要方面是开发创新杀虫剂，特别是专门针对害虫且对哺乳动物毒性最小的杀虫剂。昆虫芳基烷基胺n -酰基转移酶（iAANATs）可以作为满足这些标准的新型杀虫剂的靶点。存在丰富的iAANAT结构和生化信息，iAANAT敲除实验表明，这些酶对昆虫健康至关重要。在此，我们表达、纯化并鉴定了两个新的iaanat，一个来自Apis mellifera（蜜蜂，AmNAT1），另一个来自Diaphorina citri（亚洲柑橘木虱，DcNAT）。我们发现，用于治疗家畜锥虫病和巴贝斯虫病的化合物迪米纳烯对AmNAT1、DcNAT和D. melanogaster DmAgmNAT具有适度的抑制作用，Ki值在0.8 μM ~ 200 μM之间。我们发现一系列胍类、脒类和一种羟基酸酯，结构上与咪唑烯相关，也能抑制iAANATs，包括卡莫司他、加贝酸酯、那莫司他和帕比诺司他。值得注意的是，我们发现DmAgmNAT更容易受到这五种化合物中的四种的抑制。卡莫司他、那莫司他和加贝酸酯在Ki值为0.2 ~ 30 μM时对DmAgmNAT有抑制作用，而在Ki值为500 μM时对AmNAT1和DcNAT均无抑制作用。这些结果表明，一种针对iAANAT的物种特异性抑制剂是可能的。此外，我们还报道了脂肪酰辅酶a是AmNAT1和DcNAT的底物，琥珀酰辅酶a是DcNAT的底物。这些结果有助于越来越多的数据表明，n -二羧基胺是昆虫和其他生物的代谢物。

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引用次数: 0

Exploring the structure and nucleic acid interactions of the Leishmania sp. telomerase reverse transcriptase N-terminal region 利什曼原虫端粒酶逆转录酶n端区结构与核酸相互作用的研究。

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-02-01 DOI: 10.1016/j.abb.2025.110289

Stephany C. Paiva , Guilherme Henrique Marchi Salvador , Patrick S. Barbosa , Hamine Cristina de Oliveira , Carlos Alexandre H. Fernandes , Carlos H.I. Ramos , Marcos Roberto de M. Fontes , Maria Isabel N. Cano

Leishmaniasis is a neglected tropical disease caused by protozoans of the Leishmania genus, against which no effective treatment or control is available. Like other eukaryotes, parasite telomeres are maintained by telomerase, a ribonucleoprotein complex vital for genome stability. Its protein component, TERT (telomerase reverse transcriptase), presents four structural and functional domains, with the TEN (Telomerase N-terminal) and TRBD (Telomerase RNA-binding) located at its N-terminal. The enzyme also contains an RNA component that carries the template copied by the TERT during telomere elongation. Here, we show that the tertiary structure of Leishmania major TERT (LmTERT) is conserved compared to other eukaryotes. However, the LmTERT N-terminal (LmTERT-NT) portion shows structural changes not detected in the entire protein, mainly in the TEN domain. Besides the disordered elements, the TEN gains two long β-sheets but preserves the GQ motif and the residues in β-sheet 5 that interact with the TRAP motif. In both structures, a linker flanks the TEN and TRBD. The TRBD is partially conserved in both structures and contains the canonical QFP and T motifs, invariant residues, and the putative CP and two trypanosomatid-specific motifs (TSM) besides genus-specific amino acid substitutions. Despite the structural changes, the recombinant LmTERT-NT preserves a hydrophobic cavity that binds specifically and in the picomolar range to the telomeric G-rich DNA and the TER 5′ end region. Thus, LmTERT-NT shares the canonical structural domains and motifs and is biochemically active. We discuss the importance of the TERT N-terminal region in the parasite's telomerase catalysis.

利什曼病是一种被忽视的热带病，由利什曼原虫属的原生动物引起，目前尚无有效的治疗或控制方法。像其他真核生物一样，寄生虫的端粒是由端粒酶维持的，端粒酶是一种对基因组稳定至关重要的核糖核蛋白复合物。它的蛋白质成分TERT（端粒酶逆转录酶）具有四个结构域和功能域，其中TEN（端粒酶n端）和TRBD（端粒酶rna结合）位于其n端。这种酶还含有一种RNA成分，它携带TERT在端粒延伸过程中复制的模板。在这里，我们发现利什曼原虫主要TERT （LmTERT）的三级结构与其他真核生物相比是保守的。然而，LmTERT n端（LmTERT- nt）部分显示了整个蛋白质中未检测到的结构变化，主要是在TEN结构域。除了无序元件外，TEN获得了两个长β-片，但保留了GQ基序和β-片5中与TRAP基序相互作用的残基。在这两种结构中，一个连接体位于TEN和TRBD的两侧。TRBD在这两个结构中都是部分保守的，除了属特异性氨基酸替换外，还包含典型的QFP和T基序、不变残基、推测的CP和两个锥虫特异性基序（TSM）。尽管结构发生了变化，重组LmTERT-NT保留了一个疏水腔，可以在皮摩尔范围内特异性结合富含g的端粒DNA和ter5 '端区。因此，LmTERT-NT共享规范结构域和基序，具有生物化学活性。我们讨论了TERT n端区域在寄生虫端粒酶催化中的重要性。

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引用次数: 0

Structural and functional snapshots of a broad-specificity endoglucanase from Thermogutta terrifontis for biomass saccharification 用于生物质糖化的 Thermogutta terrifontis 广特异性内切葡聚糖酶的结构和功能快照

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-02-01 DOI: 10.1016/j.abb.2024.110274

Naveed Hussain , Halina Mikolajek , Peter J. Harrison , Neil Paterson , Muhammad W. Akhtar , Saima Sadaf , James H. Naismith

Multifunctionality, processivity, and thermostability are critical for the cost-effective enzymatic saccharification of non-food plant biomass polymers such as β-glucans, celluloses, and xylans to generate biofuels and other valuable products. We present molecular insights into a processive multifunctional endo-1,3-1,4-β-d-glucanase (Tt_End5A) from the hyperthermophilic bacterium Thermogutta terrifontis. Tt_End5A demonstrated activities against a broad spectrum of β-polysaccharides, including barley glucan, lichenan, carboxymethyl cellulose, regenerated amorphous cellulose (RAC), Avicel, xylan, laminarin, mannan, curdlan, xanthan, and various chromogenic substrates at pH 7 and temperatures ranging from 70 to 80°C. The enzyme exhibited a high level of processivity on RAC and retained over 90% activity at 80°C for an extended period, indicating exceptional thermal stability. The 1.20 Å crystal structure of the Tt_End5A catalytic domain revealed an archetypal glycoside hydrolase family 5 (GH5) catalytic TIM-(β/α)₈-barrel, supplemented with additional β-strands, elongated α-helices, and a rare cis-non-Pro (His481-cis-Ala482) peptide. A large central cleft was observed in the 3D structure, which is likely related to the enzyme's multifunctionality and processivity. The catalytic domain is preceded by a novel N-terminal multivalent carbohydrate-binding module (CBM) that enhances the enzymatic degradation of insoluble polysaccharides. Mutagenesis studies, ligand interaction analyses, and the structurally conserved positions of E329 and E448 in Tt_End5A suggest that these residues function as the proton donor and nucleophile in the catalytic mechanism. Owing to its multifunctionality and processivity, Tt_End5A can reduce the need for multiple saccharification enzymes to generate fermentable sugars from plant biomass for bioethanol production. Additionally, it holds promise for applications in the pharmaceutical, feed, and food industries.

多功能性、加工能力和热稳定性对于具有成本效益的非食用植物生物质聚合物（如β-葡聚糖、纤维素和木聚糖）酶糖化生产生物燃料和其他有价值的产品至关重要。我们介绍了来自超嗜热细菌Thermogutta terrifontis的一种过程多功能内切-1,3-1,4-β- d -葡聚糖酶（Tt_End5A）的分子见解。Tt_End5A在pH值为7、温度为70-80℃的条件下，对广谱β-多糖（包括大麦葡聚糖、地衣聚糖、羧甲基纤维素、再生无定形纤维素（RAC）、Avicel、木聚糖、层粘连素、甘露聚糖、凝乳聚糖、黄原胶和各种显色底物）具有抑制活性。该酶在RAC上表现出高水平的加工能力，并在80℃下长时间保持90%以上的活性，表明了优异的热稳定性。Tt_End5A催化结构域的1.20 Å晶体结构揭示了一个典型的糖苷水解酶家族5 （GH5）催化TIM-(β/α)8桶，补充了额外的β-链，细长的α-螺旋和罕见的顺式-非pro （His481-cis-Ala482）肽。在三维结构中观察到一个大的中央裂缝，这可能与酶的多功能性和加工性有关。催化结构域之前是一个新型的n端多价碳水化合物结合模块（CBM），可以增强不溶性多糖的酶降解。突变研究、配体相互作用分析以及E329和E448在Tt_End5A中的结构保守位置表明，这些残基在催化机制中起到质子供体和亲核试剂的作用。由于Tt_End5A的多功能性和加工性，它可以减少对多种糖化酶的需求，从而从植物生物质中产生可发酵糖用于生物乙醇生产。此外，它还有望应用于制药、饲料和食品工业。

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引用次数: 0

Light regulation of flavin reduction by NAD(P)H: Activation of 2-haloacrylate hydratase NAD(P)H对黄素还原的轻度调控：2-卤代丙烯酸水合酶的激活。

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-02-01 DOI: 10.1016/j.abb.2024.110285

Karina Kizjakina , Yumin Dai , Pablo Sobrado

We report a novel light-dependent activation mechanism for 2-haloacrylate hydratase (2HAH), a flavin-dependent dehalogenase. Initial assays revealed inconsistent enzyme activity, stabilized only after chemical reduction or exposure to bright light. Spectroscopic analysis showed that light accelerates flavin reduction by NAD(P)H, completing in 30 s under bright light versus slow reduction in the dark. Blue light specifically triggered full activation, while red light had no effect. Sequence and structural analyses indicate that 2HAH does not share homology with known light-sensitive flavoproteins, suggesting an uncharacterized regulatory mechanism. These findings advance our understanding of flavin enzyme regulation and introduce light as a potential tool for modulating 2HAH activity.

我们报告了一种新的光依赖性 2-卤丙烯酸酯氢化酶（2HAH）的活化机制，这是一种黄素-脱氧内酯脱卤酶。最初的测定显示酶活性不稳定，只有在化学还原或暴露在强光下后才会稳定。光谱分析显示，光能加速 NAD(P)H 对黄素的还原，在强光下 30 秒就能完成还原，而在黑暗中还原速度较慢。蓝光能特异性地引发完全活化，而红光则没有影响。序列和结构分析表明，2HAH 与已知的光敏黄素蛋白不具有同源性，这表明存在一种未定性的调控机制。这些发现加深了我们对黄素酶调控的理解，并将光作为调节2HAH活性的一种潜在工具。

引用次数: 0

Modulating vascular smooth muscle cell phenotype via Wnt-Independent FRZB pathways 通过不依赖wnt的FRZB通路调节血管平滑肌细胞表型。

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-02-01 DOI: 10.1016/j.abb.2025.110290

Hyomin Kim , Eun Kyoung Kim , Yeuni Yu , Hye Jin Heo , Dokyoung Kim , Su-Yeon Cho , Yujin Kwon , Won Kyu Kim , Kihun Kim , Dai Sik Ko , Yun Hak Kim

Background and aims

Vascular smooth muscle cells are pivotal in atherosclerosis, transitioning from a contractile to a synthetic phenotype, which is associated with increased proliferation and inflammation. FRZB, a Wnt signaling modulator, has been implicated in vascular pathology, but its specific role in vascular smooth muscle cell phenotype modulation is not well understood. This study investigates the role of FRZB in regulating vascular smooth muscle cell phenotypes.

Methods

Vascular smooth muscle cell regions were categorized based on FRZB expression levels, and various analyses, including differential gene expression, KEGG pathway analysis, and Disease Ontology analysis, were conducted. FRZB knockdown in human aortic vascular smooth muscle cell was performed using siRNA, followed by assessments of cell migration, proliferation, and phenotype marker expression.

Results

FRZB expression was significantly reduced in synthetic type compared to contractile type in both mouse models and human samples. FRZB knockdown in human vascular smooth muscle cells led to increased cell migration and proliferation, alongside decreased expression of contractile markers and increased synthetic markers. Unexpectedly, FRZB knockdown suppressed Wnt signaling. Pathway analysis revealed associations with the PI3K-Akt signaling pathway, focal adhesion, and ECM interactions.

Conclusions

Our study highlights FRZB's role in Vascular smooth muscle cell phenotype modulation, showing that reduced FRZB expression correlates with a synthetic phenotype and increased disease markers. FRZB does not enhance Wnt signaling but may regulate vascular smooth muscle cell behavior through alternative pathways. These findings suggest FRZB as a potential therapeutic target for stabilizing vascular smooth muscle cells and managing atherosclerosis.

背景和目的：血管平滑肌细胞在动脉粥样硬化中起关键作用，从收缩型向合成型转变，这与增殖和炎症增加有关。FRZB是一种Wnt信号调节剂，与血管病理有关，但其在血管平滑肌细胞表型调节中的具体作用尚不清楚。本研究探讨FRZB在调节血管平滑肌细胞表型中的作用。方法：根据FRZB表达水平对血管平滑肌细胞区域进行分类，并进行差异基因表达、KEGG通路分析、疾病本体分析等多种分析。在人主动脉血管平滑肌细胞中使用siRNA进行FRZB敲除，随后评估细胞迁移、增殖和表型标记表达。结果：与收缩型相比，合成型FRZB在小鼠模型和人样本中的表达均显著降低。FRZB在人血管平滑肌细胞中敲低导致细胞迁移和增殖增加，同时收缩标记物表达减少，合成标记物表达增加。出乎意料的是，FRZB敲除抑制了Wnt信号。通路分析显示与PI3K-Akt信号通路、局灶黏附和ECM相互作用有关。结论：我们的研究强调了FRZB在血管平滑肌细胞表型调节中的作用，表明FRZB表达的减少与合成表型和疾病标志物的增加相关。FRZB不增强Wnt信号，但可能通过其他途径调节血管平滑肌细胞的行为。这些发现表明FRZB是稳定血管平滑肌细胞和控制动脉粥样硬化的潜在治疗靶点。

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引用次数: 0

Oxidation of CaMKIIα cysteines inhibits autonomous activation induced by phosphorylation CaMKIIα 半胱氨酸的氧化抑制了磷酸化诱导的自主激活。

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-02-01 DOI: 10.1016/j.abb.2024.110268

Nathália Rocco-Machado , Max Deng , Yi He , Rodney L. Levine

Ca²⁺/calmodulin-dependent protein kinase II α (CaMKIIα) “autonomous” activation induced by Thr286 phosphorylation has a crucial role in synaptic plasticity. Previous studies showed that in Alzheimer's disease brain, CaMKIIα autophosphorylation at Thr286 is reduced while the level of cysteine-oxidized CAMKIIα is elevated. We performed tryptic mapping of the oxidized CaMKIIα and discovered the formation of a disulfide between the N-terminal Cys6 and the regulatory domain Cys280. The apparent pK_a values of Cys6 and Cys280 are 7.1 and 7.7, respectively, lower than the 8.5 for free Cys. The low apparent pK_a of Cys6 facilitates the oxidation of its thiol to the sulfenic acid at physiological pH. The thiolate of Cys280 can then attack the sulfenic acid to form a disulfide. Using an antibody against phosphorylated Thr286, we showed that disulfide formation prevents Thr286 phosphorylation. CaMKIIα autonomous activation induced by disulfide formation is much lower than the autonomous activation induced by phosphorylation. The decreased autonomous activation may contribute to the synaptic impairment of Alzheimer's disease. We also generated a CaMKIIα mutant in which Cys6 was mutated to Ser6. This mutation prevented disulfide formation and restored autonomous activation induced by phosphorylation. Our findings provide insight into the mechanistic details of CaMKIIα autonomous activation induced by disulfide formation that may contribute to the impairment of long-term potentiation in Alzheimer's disease.

Ca2+/钙调素依赖性蛋白激酶IIα （CaMKIIα）Thr286磷酸化诱导的“自主”激活在突触可塑性中起着至关重要的作用。先前的研究表明，在阿尔茨海默病的大脑中，CaMKIIα Thr286的自磷酸化水平降低，而半胱氨酸氧化的CaMKIIα水平升高。我们对氧化CaMKIIα进行了色氨酸定位，发现在n端Cys6和调节结构域Cys280之间形成了二硫化物。Cys6和Cys280的表观pKa值分别为7.1和7.7，低于游离Cys的8.5。Cys280的低表观pKa有利于其硫醇在生理ph下氧化为硫酸，然后Cys280的硫醇可以攻击硫酸形成二硫化物。利用抗磷酸化Thr286的抗体，我们发现二硫化物的形成阻止了Thr286的磷酸化。CaMKIIα由二硫化物形成诱导的自主激活远低于磷酸化诱导的自主激活。自主激活的减少可能与阿尔茨海默病的突触损伤有关。我们还生成了CaMKIIα突变体，其中Cys6突变为Ser6。这种突变阻止了二硫化物的形成，并恢复了磷酸化诱导的自主激活。我们的研究结果提供了对CaMKIIα自主激活的机制细节的深入了解，CaMKIIα自主激活可能导致阿尔茨海默病的长期增强功能受损。

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引用次数: 0

Tools to investigate oxygen-related challenges with flavin-dependent enzymes 利用黄素依赖酶研究氧相关挑战的工具。

IF 3.8 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Archives of biochemistry and biophysics

Pub Date : 2025-02-01 DOI: 10.1016/j.abb.2024.110246

Ariadna Pié Porta, Elif Erdem, John M. Woodley

Enzymes have multiple applications in medicine but during the past decades interest in the application of enzymes as (bio)catalysts to produce a wide range of valuable molecules in various industries has increased. Many chemical compounds (from pharmaceuticals to bulk commodities) can be produced by a series of enzymatically-catalysed chemical steps, and in many cases one of these steps is an oxidation.

The use of molecular oxygen as an oxidising agent in biocatalytic processes is a double-edged approach. From one side, the oxygen is supplied to the reactor in the form of air bubbling, which is cheap, highly available and non-toxic. From the other side, bubbling air into the reaction media creates a gas-liquid interface which adsorbs enzymes and compromises their stability. Moreover, the oxygen is quite insoluble in water, which often results in oxygen-limited reactions.

These aspects are the main limiting factors for the stability and kinetics of enzymes that perform oxidative biocatalysis and prevent the reaction from happening at a rate that is high/competitive enough for industrial feasibility. Therefore, we need systems to mimic and understand better these factors to try and mitigate their effects upon scale-up.

In this review, we present two complementary systems to study these factors: one apparatus that ensures a constant gas-liquid interface and another one that maintains a constant oxygen partial pressure. Both can provide highly valuable information regarding the maximum rate of reaction and about the deactivation profiles of enzymes in the presence of bubbles.

酶在医学上有多种应用，但在过去的几十年里，人们对酶作为（生物）催化剂在各种工业中生产各种有价值分子的应用越来越感兴趣。许多化合物（从药品到大宗商品）可以通过一系列酶催化的化学步骤生产，在许多情况下，其中一个步骤是氧化。在生物催化过程中使用分子氧作为氧化剂是一种双刃剑的方法。从一侧，氧气以鼓泡空气的形式供给反应器，这种方式廉价、易得且无毒。从另一边，冒泡的空气进入反应介质，形成气液界面，吸附酶，破坏酶的稳定性。此外，氧不溶于水，这常常导致限氧反应。这些方面是进行氧化生物催化的酶的稳定性和动力学的主要限制因素，并阻止反应以足够高/有竞争力的工业可行性的速度发生。因此，我们需要系统来模拟和更好地理解这些因素，以试图减轻它们在扩大规模时的影响。在这篇综述中，我们提出了两个互补的系统来研究这些因素：一个确保气液界面恒定的装置，另一个保持恒定的氧分压。两者都可以提供有关最大反应速率和气泡存在时酶的失活概况的非常有价值的信息。

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引用次数: 0