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Pepper catalase: a broad analysis of its modulation during fruit ripening and by nitric oxide. 辣椒过氧化氢酶:果实成熟期一氧化氮对其调节作用的广泛分析。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-03 DOI: 10.1042/BCJ20240247
Salvador González-Gordo, Javier López-Jaramillo, Marta Rodríguez-Ruiz, Jorge Taboada, José M Palma, Francisco J Corpas

Catalase is a major antioxidant enzyme located in plant peroxisomes that catalyzes the decomposition of H2O2. Based on our previous transcriptomic (RNA-Seq) and proteomic (iTRAQ) data at different stages of pepper (Capsicum annuum L.) fruit ripening and after exposure to nitric oxide (NO) enriched atmosphere, a broad analysis has allowed us to characterize the functioning of this enzyme. Three genes were identified, and their expression was differentially modulated during ripening and by NO gas treatment. A dissimilar behavior was observed in the protein expression of the encoded protein catalases (CaCat1-CaCat3). Total catalase activity was down-regulated by 50% in ripe (red) fruits concerning immature green fruits. This was corroborated by non-denaturing polyacrylamide gel electrophoresis, where only a single catalase isozyme was identified. In vitro analyses of the recombinant CaCat3 protein exposed to peroxynitrite (ONOO-) confirmed, by immunoblot assay, that catalase underwent a nitration process. Mass spectrometric analysis identified that Tyr348 and Tyr360 were nitrated by ONOO-, occurring near the active center of catalase. The data indicate the complex regulation at gene and protein levels of catalase during the ripening of pepper fruits, with activity significantly down-regulated in ripe fruits. Nitration seems to play a key role in this down-regulation, favoring an increase in H2O2 content during ripening. This pattern can be reversed by the exogenous NO application. While plant catalases are generally reported to be tetrameric, the analysis of the protein structure supports that pepper catalase has a favored quaternary homodimer nature. Taken together, data show that pepper catalase is down-regulated during fruit ripening, becoming a target of tyrosine nitration, which provokes its inhibition.

过氧化氢酶是植物过氧物酶体中的一种主要抗氧化酶,能催化 H2O2 的分解。根据我们之前在辣椒果实成熟的不同阶段以及暴露于富含氮氧化物的环境后所获得的转录组(RNA-Seq)和蛋白质组(iTRAQ)数据,我们进行了广泛的分析,从而确定了这种酶的功能特征。研究发现了三个基因,它们的表达在果实成熟期和氮氧化物气体处理过程中受到不同程度的调节。在编码的蛋白过氧化氢酶(CaCat1-CaCat3)的蛋白质表达中观察到了不同的行为。与未成熟的绿色果实相比,成熟果实中过氧化氢酶的总活性降低了 50%。非变性聚丙烯酰胺凝胶电泳证实了这一点,在电泳中只发现了一种过氧化氢酶同工酶。通过免疫印迹分析,体外分析暴露于过亚硝酸的重组 CaCat3 蛋白证实过氧化氢酶发生了硝化过程。质谱分析发现,Tyr348 和 Tyr360 被过氧化亚硝酸盐硝化,发生在过氧化氢酶活性中心附近。这些数据表明,在果实成熟过程中,过氧化氢酶在基因和蛋白质水平上的调控十分复杂,成熟果实的过氧化氢酶活性显著降低。硝化作用在这种下调中起着关键作用,有利于成熟过程中 H2O2 的增加。施用外源 NO 可以逆转这种模式。据报道,植物过氧化氢酶一般是四聚体,而蛋白质结构分析表明,辣椒过氧化氢酶具有四元同二聚体的性质。综上所述,数据表明辣椒过氧化氢酶在果实成熟过程中会被下调,成为酪氨酸硝化的靶标,从而导致其受到抑制。
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引用次数: 0
Integrating bacterial molecular genetics with chemical biology for renewed antibacterial drug discovery. 将细菌分子遗传学与化学生物学相结合,重新发现抗菌药物。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-03 DOI: 10.1042/BCJ20220062
Susannah L Parkhill, Eachan O Johnson

The application of dyes to understanding the aetiology of infection inspired antimicrobial chemotherapy and the first wave of antibacterial drugs. The second wave of antibacterial drug discovery was driven by rapid discovery of natural products, now making up 69% of current antibacterial drugs. But now with the most prevalent natural products already discovered, ∼107 new soil-dwelling bacterial species must be screened to discover one new class of natural product. Therefore, instead of a third wave of antibacterial drug discovery, there is now a discovery bottleneck. Unlike natural products which are curated by billions of years of microbial antagonism, the vast synthetic chemical space still requires artificial curation through the therapeutics science of antibacterial drugs - a systematic understanding of how small molecules interact with bacterial physiology, effect desired phenotypes, and benefit the host. Bacterial molecular genetics can elucidate pathogen biology relevant to therapeutics development, but it can also be applied directly to understanding mechanisms and liabilities of new chemical agents with new mechanisms of action. Therefore, the next phase of antibacterial drug discovery could be enabled by integrating chemical expertise with systematic dissection of bacterial infection biology. Facing the ambitious endeavour to find new molecules from nature or new-to-nature which cure bacterial infections, the capabilities furnished by modern chemical biology and molecular genetics can be applied to prospecting for chemical modulators of new targets which circumvent prevalent resistance mechanisms.

应用染料来了解感染的病因激发了抗菌化疗和第一波抗菌药物的出现。天然产物的快速发现推动了抗菌药物发现的第二波浪潮,目前天然产物已占到现有抗菌药物的 69%。但是,现在已经发现了最普遍的天然产物,要发现一类新的天然产物,必须筛选出 107 种新的土栖细菌。因此,抗菌药物发现并没有出现第三次浪潮,而是出现了发现瓶颈。与经过数十亿年微生物拮抗作用整理的天然产物不同,广阔的合成化学空间仍然需要通过抗菌药物治疗学进行人工整理--系统地了解小分子如何与细菌生理相互作用、产生理想的表型并使宿主受益。细菌分子遗传学可以阐明与疗法开发相关的病原体生物学,也可以直接用于了解具有新作用机制的新化学制剂的机制和责任。因此,将化学专业知识与细菌感染生物学的系统分析相结合,可以促进下一阶段抗菌药物的发现。面对从自然界或从新到自然界寻找治疗细菌感染的新分子的宏伟目标,现代化学生物学和分子遗传学所提供的能力可用于寻找新靶点的化学调节剂,以规避普遍存在的抗药性机制。
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引用次数: 0
Sulfoquinovosyl diacylglycerol is required for dimerisation of the Rhodobacter sphaeroides reaction centre-light harvesting 1 core complex. 硫代喹诺酮基二酰基甘油是水发罗杆菌 RC-LH1 核心复合物二聚化所必需的。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-07-03 DOI: 10.1042/BCJ20240125
Elizabeth C Martin, Adam G M Bowie, Taylor Wellfare Reid, C Neil Hunter, Andrew Hitchcock, David J K Swainsbury

The reaction centre-light harvesting 1 (RC-LH1) core complex is indispensable for anoxygenic photosynthesis. In the purple bacterium Rhodobacter (Rba.) sphaeroides RC-LH1 is produced both as a monomer, in which 14 LH1 subunits form a C-shaped antenna around 1 RC, and as a dimer, where 28 LH1 subunits form an S-shaped antenna surrounding 2 RCs. Alongside the five RC and LH1 subunits, an additional polypeptide known as PufX provides an interface for dimerisation and also prevents LH1 ring closure, introducing a channel for quinone exchange that is essential for photoheterotrophic growth. Structures of Rba. sphaeroides RC-LH1 complexes revealed several new components; protein-Y, which helps to form the quinone channel; protein-Z, of unknown function and seemingly unique to dimers; and a tightly bound sulfoquinovosyl diacylglycerol (SQDG) lipid that interacts with two PufX arginine residues. This lipid lies at the dimer interface alongside weak density for a second molecule, previously proposed to be an ornithine lipid. In this work we have generated strains of Rba. sphaeroides lacking protein-Y, protein-Z, SQDG or ornithine lipids to assess the roles of these previously unknown components in the assembly and activity of RC-LH1. We show that whilst the removal of either protein-Y, protein-Z or ornithine lipids has only subtle effects, SQDG is essential for the formation of RC-LH1 dimers but its absence has no functional effect on the monomeric complex.

反应中心-采光 1(RC-LH1)核心复合体是无氧光合作用所不可或缺的。在紫色芽孢杆菌(Rhodobacter (Rba.) sphaeroides)中,RC-LH1 既可以单体形式产生,其中 14 个 LH1 亚基围绕一个 RC 形成 C 形天线;也可以二聚体形式产生,其中 28 个 LH1 亚基围绕两个 RC 形成 S 形天线。除了五个 RC 和 LH1 亚基外,还有一个名为 PufX 的多肽为二聚化提供了一个界面,同时也防止了 LH1 环的闭合,为光异养生长提供了一个必不可少的醌交换通道。Rba. sphaeroides RC-LH1 复合物的结构揭示了几种新的成分:有助于形成醌通道的蛋白质-Y;功能未知且似乎是二聚体特有的蛋白质-Z;以及与两个 PufX 精氨酸残基相互作用的紧密结合的磺基喹诺酮二酰甘油(SQDG)脂质。这种脂质位于二聚体界面上,与密度较弱的第二种分子并存,以前曾被认为是鸟氨酸脂质。在这项工作中,我们生成了缺乏蛋白-Y、蛋白-Z、SQDG 或鸟氨酸脂质的 Rba.sphaeroides 菌株,以评估这些以前未知的成分在 RC-LH1 组装和活性中的作用。我们发现,虽然去除蛋白-Y、蛋白-Z 或鸟氨酸脂质只会产生微小的影响,但 SQDG 对 RC-LH1 二聚体的形成至关重要,但其缺失对单体复合物没有功能性影响。
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引用次数: 0
Genetically engineered human embryonic kidney cells as a novel vehicle for dual patch clamp study of human gap junction channels. 基因工程人类胚胎肾细胞作为一种新型载体,用于人类缝隙连接通道的双贴片钳研究。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-19 DOI: 10.1042/BCJ20240016
Honghong Chen, Yi X Li, Robert S Wong, Jessica L Esseltine, Donglin Bai

Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels.

半数以上编码缝隙连接亚基的人类连接蛋白基因突变与人类遗传性疾病有关。人类间隙连接(GJ)通道的功能研究对于揭示与疾病相关的附件素突变体的病因机制至关重要。然而,常用的爪蟾卵母细胞、N2A、HeLa 和其他重组表达人类连接蛋白的模型细胞都有不同的显著局限性。在这里,我们利用 CRISPR-Cas9 系统开发了一种内源连接蛋白(Cx43 和 Cx45)均被敲除的人类细胞系(HEK293)。双基因敲除的 HEK293 细胞无背景 GJ 耦合,可轻松转染多个人类连接蛋白基因(如编码 Cx46、Cx50、Cx37、Cx45、Cx26 和 Cx36 的基因),这些基因可成功形成功能性 GJ,并可随时用于双膜片钳分析。单个基因敲除的 Cx43 或 Cx45 HEK 细胞系也可用于表征分别由 Cx45 或 Cx43 形成的人类 GJ 通道,其表达水平适合研究宏观和单通道 GJ 通道特性。与心律失常有关的 Cx45 突变体 R184G 在 DKO HEK293 细胞中不能形成功能性 GJ,且定位受损。这些基因工程 HEK293 细胞非常适合用于人类 GJ 通道的膜片钳研究。
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引用次数: 0
Cancer-associated mutations in protein kinase C theta are loss-of-function. 与癌症相关的蛋白激酶 C Theta 基因突变是功能缺失性的。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-19 DOI: 10.1042/BCJ20240148
Stefanie J Hodapp, Nathan Gravel, Natarajan Kannan, Alexandra C Newton

The Ca2+-independent, but diacylglycerol-regulated, novel protein kinase C (PKC) theta (θ) is highly expressed in hematopoietic cells where it participates in immune signaling and platelet function. Mounting evidence suggests that PKCθ may be involved in cancer, particularly blood cancers, breast cancer, and gastrointestinal stromal tumors, yet how to target this kinase (as an oncogene or as a tumor suppressor) has not been established. Here, we examine the effect of four cancer-associated mutations, R145H/C in the autoinhibitory pseudosubstrate, E161K in the regulatory C1A domain, and R635W in the regulatory C-terminal tail, on the cellular activity and stability of PKCθ. Live-cell imaging studies using the genetically-encoded fluorescence resonance energy transfer-based reporter for PKC activity, C kinase activity reporter 2 (CKAR2), revealed that the pseudosubstrate and C1A domain mutations impaired autoinhibition to increase basal signaling. This impaired autoinhibition resulted in decreased stability of the protein, consistent with the well-characterized behavior of Ca2+-regulated PKC isozymes wherein mutations that impair autoinhibition are paradoxically loss-of-function because the mutant protein is degraded. In marked contrast, the C-terminal tail mutation resulted in enhanced autoinhibition and enhanced stability. Thus, the examined mutations were loss-of-function by different mechanisms: mutations that impaired autoinhibition promoted the degradation of PKC, and those that enhanced autoinhibition stabilized an inactive PKC. Supporting a general loss-of-function of PKCθ in cancer, bioinformatics analysis revealed that protein levels of PKCθ are reduced in diverse cancers, including lung, renal, head and neck, and pancreatic. Our results reveal that PKCθ function is lost in cancer.

不受 Ca2+ 影响、但受二酰甘油调控的新型蛋白激酶 C (PKC) theta (θ)在造血细胞中高度表达,参与免疫信号转导和血小板功能。越来越多的证据表明,PKCθ可能与癌症有关,尤其是血癌、乳腺癌和胃肠道间质瘤(GISTs),但如何靶向这种激酶(作为致癌基因或肿瘤抑制因子)尚未确定。在这里,我们研究了四种癌症相关突变(自体抑制假底物中的 R145H/C、调控 C1A 结构域中的 E161K 和调控 C 端尾中的 R635W)对 PKCθ 细胞活性和稳定性的影响。使用基因编码的基于 FRET 的 PKC 活性报告器--C 激酶活性报告器 2(CKAR2)进行的活细胞成像研究发现,伪底物和 C1A 结构域突变削弱了自身抑制作用,从而增加了基础信号传导。自抑功能受损导致蛋白质稳定性下降,这与钙离子调控的 PKC 同工酶的典型行为一致,即自抑功能受损的突变会导致功能丧失,因为突变蛋白质会被降解。与此形成鲜明对比的是,C 端尾部突变会增强自身抑制作用并提高稳定性。因此,所研究的突变是通过不同的机制导致功能缺失的:削弱自身抑制作用的突变促进了 PKC 的降解,而增强自身抑制作用的突变则稳定了无活性的 PKC。生物信息学分析表明,在肺癌、肾癌、头颈癌和胰腺癌等多种癌症中,PKCθ的蛋白水平都有所降低,这证明PKCθ在癌症中普遍丧失功能。我们的研究结果表明,PKCθ的功能在癌症中丧失。
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引用次数: 0
Aflatoxin biosynthesis regulators AflR and AflS: DNA binding affinity, stoichiometry, and kinetics. 黄曲霉毒素生物合成调节剂 AflR 和 AflS:DNA 结合亲和力、化学计量学和动力学。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-19 DOI: 10.1042/BCJ20240084
Asmaa Abbas, Ranjit K Prajapati, Emil Aalto-Setälä, Alexander A Baykov, Anssi M Malinen

Aflatoxins (AFs), potent foodborne carcinogens produced by Aspergillus fungi, pose significant health risks worldwide and present challenges to food safety and productivity in the food chain. Novel strategies for disrupting AF production, cultivating resilient crops, and detecting contaminated food are urgently needed. Understanding the regulatory mechanisms of AF production is pivotal for targeted interventions to mitigate toxin accumulation in food and feed. The gene cluster responsible for AF biosynthesis encodes biosynthetic enzymes and pathway-specific regulators, notably AflR and AflS. While AflR, a DNA-binding protein, activates gene transcription within the cluster, AflS enhances AF production through mechanisms that are not fully understood. In this study, we developed protocols to purify recombinant AflR and AflS proteins and utilized multiple assays to characterize their interactions with DNA. Our biophysical analysis indicated that AflR and AflS form a complex. AflS exhibited no DNA-binding capability on its own but unexpectedly reduced the DNA-binding affinity of AflR. Additionally, we found that AflR achieves its binding specificity through a mechanism in which either two copies of AflR or its complex with AflS bind to target sites on DNA in a highly cooperative manner. The estimated values of the interaction parameters of AflR, AflS and DNA target sites constitute a fundamental framework against which the function and mechanisms of other AF biosynthesis regulators can be compared.

黄曲霉毒素是由曲霉菌产生的强效食源性致癌物质,在全球范围内对健康构成重大威胁,并对食物链中的食品安全和生产率构成挑战。目前迫切需要新的策略来干扰黄曲霉毒素的生产、培育抗逆性作物和检测受污染的食物。了解黄曲霉毒素产生的调控机制对于采取有针对性的干预措施以减少毒素在食品和饲料中的积累至关重要。负责黄曲霉毒素生物合成的基因簇编码生物合成酶和途径特异性调控因子,特别是 AflR 和 AflS。AflR 是一种 DNA 结合蛋白,可激活基因簇内的基因转录,而 AflS 则通过尚未完全清楚的机制提高黄曲霉毒素的产量。在这项研究中,我们制定了纯化重组 AflR 和 AflS 蛋白的方案,并利用多种检测方法来鉴定它们与 DNA 的相互作用。我们的生物物理分析表明,AflR 和 AflS 形成了一个复合物。AflS 本身没有 DNA 结合能力,但却意外地降低了 AflR 的 DNA 结合亲和力。此外,我们还发现,AflR 是通过一种机制实现其结合特异性的,在这种机制中,两个拷贝的 AflR 或其与 AflS 的复合物以高度合作的方式结合到 DNA 上的目标位点。AflR、AflS 和 DNA 目标位点相互作用参数的估计值构成了一个基本框架,可以据此比较其他黄曲霉毒素生物合成调节剂的功能和机制。
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引用次数: 0
Correction: The chaperonin CCT interacts with and mediates the correct folding and activity of three subunits of translation initiation factor eIF3: b, i and h. 更正:伴侣素 CCT 与翻译起始因子 eIF3 的三个亚基(b、i 和 h)相互作用,并介导其正确折叠和活性。
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-19 DOI: 10.1042/BJ20130979_COR
Anne Roobol, Jo Roobol, Martin J Carden, Matthew E Smith, John W B Hershey, Amandine Bastide, John R P Knight, Anne E Willis, C Mark Smales
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引用次数: 0
RNA processing by the CRISPR-associated NYN ribonuclease. CRISPR 相关 NYN 核糖核酸酶对 RNA 的处理。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-19 DOI: 10.1042/BCJ20240151
Haotian Chi, Malcolm F White

CRISPR-Cas systems confer adaptive immunity in prokaryotes, facilitating the recognition and destruction of invasive nucleic acids. Type III CRISPR systems comprise large, multisubunit ribonucleoprotein complexes with a catalytic Cas10 subunit. When activated by the detection of foreign RNA, Cas10 generates nucleotide signalling molecules that elicit an immune response by activating ancillary effector proteins. Among these systems, the Bacteroides fragilis type III CRISPR system was recently shown to produce a novel signal molecule, SAM-AMP, by conjugating ATP and SAM. SAM-AMP regulates a membrane effector of the CorA family to provide immunity. Here, we focus on NYN, a ribonuclease encoded within this system, probing its potential involvement in crRNA maturation. Structural modelling and in vitro ribonuclease assays reveal that NYN displays robust sequence-nonspecific, Mn2+-dependent ssRNA-cleavage activity. Our findings suggest a role for NYN in trimming crRNA intermediates into mature crRNAs, which is necessary for type III CRISPR antiviral defence. This study sheds light on the functional relevance of CRISPR-associated NYN proteins and highlights the complexity of CRISPR-mediated defence strategies in bacteria.

CRISPR-Cas系统赋予原核生物适应性免疫力,有助于识别和破坏入侵的核酸。III 型 CRISPR 系统由大型多亚基核糖核蛋白复合物和催化 Cas10 亚基组成。当检测到外来 RNA 被激活时,Cas10 会产生核苷酸信号分子,通过激活辅助效应蛋白引起免疫反应。在这些系统中,最近发现脆弱拟杆菌 III 型 CRISPR 系统通过结合 ATP 和 SAM 产生一种新型信号分子 SAM-AMP。SAM-AMP能调节CorA家族的膜效应器,从而提供免疫力。在此,我们重点研究该系统中编码的核糖核酸酶 NYN,探究其参与 crRNA 成熟的可能性。结构建模和体外核糖核酸酶测定显示,NYN 具有强大的序列非特异性、Mn2+ 依赖性 ssRNA 切断活性。我们的研究结果表明,NYN在将crRNA中间体修剪成成熟的crRNA方面发挥作用,而这是III型CRISPR抗病毒防御所必需的。这项研究揭示了 CRISPR 相关 NYN 蛋白的功能相关性,并凸显了 CRISPR 介导的细菌防御策略的复杂性。
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引用次数: 0
Mechanisms and pathologies of human mitochondrial DNA replication and deletion formation. 人类线粒体 DNA 复制和缺失形成的机制和病理。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-05 DOI: 10.1042/BCJ20230262
Tiago M Bernardino Gomes, Amy E Vincent, Katja E Menger, James B Stewart, Thomas J Nicholls

Human mitochondria possess a multi-copy circular genome, mitochondrial DNA (mtDNA), that is essential for cellular energy metabolism. The number of copies of mtDNA per cell, and their integrity, are maintained by nuclear-encoded mtDNA replication and repair machineries. Aberrant mtDNA replication and mtDNA breakage are believed to cause deletions within mtDNA. The genomic location and breakpoint sequences of these deletions show similar patterns across various inherited and acquired diseases, and are also observed during normal ageing, suggesting a common mechanism of deletion formation. However, an ongoing debate over the mechanism by which mtDNA replicates has made it difficult to develop clear and testable models for how mtDNA rearrangements arise and propagate at a molecular and cellular level. These deletions may impair energy metabolism if present in a high proportion of the mtDNA copies within the cell, and can be seen in primary mitochondrial diseases, either in sporadic cases or caused by autosomal variants in nuclear-encoded mtDNA maintenance genes. These mitochondrial diseases have diverse genetic causes and multiple modes of inheritance, and show notoriously broad clinical heterogeneity with complex tissue specificities, which further makes establishing genotype-phenotype relationships challenging. In this review, we aim to cover our current understanding of how the human mitochondrial genome is replicated, the mechanisms by which mtDNA replication and repair can lead to mtDNA instability in the form of large-scale rearrangements, how rearranged mtDNAs subsequently accumulate within cells, and the pathological consequences when this occurs.

人类线粒体拥有多拷贝环状基因组--线粒体 DNA(mtDNA),它对细胞能量代谢至关重要。每个细胞的 mtDNA 副本数量及其完整性由核编码的 mtDNA 复制和修复机制维持。异常的 mtDNA 复制和 mtDNA 断裂被认为会导致 mtDNA 的缺失。这些缺失的基因组位置和断点序列在各种遗传性和获得性疾病中显示出相似的模式,在正常衰老过程中也能观察到,这表明缺失的形成有一个共同的机制。然而,由于对 mtDNA 复制机制的争论不休,因此很难建立清晰、可检验的模型来说明 mtDNA 重排是如何在分子和细胞水平上产生和传播的。如果细胞内的 mtDNA 副本比例过高,这些缺失就会损害能量代谢,原发性线粒体疾病就会出现这种情况,这些疾病可能是偶发病例,也可能是由核编码的 mtDNA 维护基因中的常染色体变异引起的。这些线粒体疾病具有不同的遗传原因和多种遗传方式,临床表现出广泛的异质性和复杂的组织特异性,这使得建立基因型与表型之间的关系变得更具挑战性。在这篇综述中,我们旨在介绍我们目前对人类线粒体基因组如何复制、mtDNA 复制和修复导致大规模重排形式的 mtDNA 不稳定性的机制、重排的 mtDNA 随后如何在细胞内积累以及发生这种情况时的病理后果的理解。
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引用次数: 0
The monodomain Kunitz protein EgKU-7 from the dog tapeworm Echinococcus granulosus is a high-affinity trypsin inhibitor with two interaction sites. 来自犬带绦虫棘球蚴的单链库尼茨蛋白 EgKU-7 是一种高亲和力胰蛋白酶抑制剂,具有两个相互作用位点。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-05 DOI: 10.1042/BCJ20230514
Martín Fló, Leonardo Pellizza, Rosario Durán, Beatriz Alvarez, Cecilia Fernández

Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of ∼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.

典型的库尼茨蛋白(MEROPS 数据库中的 I2 家族,库尼茨-A 家族)是丝氨酸肽酶的元虫竞争性抑制剂,能模仿底物形成 1:1 的紧密复合物。在人类和家畜中引起囊性棘球蚴病的犬带绦虫--棘球蚴粒球绦虫--编码一个扩大的单域 Kunitz 蛋白家族,其中一些蛋白被分泌到犬宿主界面。库尼茨蛋白 EgKU-7 除了包含由关键精氨酸组成的抗肽酶环的库尼茨结构域外,还含有一个约 20 个氨基酸的 C 端延伸。使用 EgKU-7、一个 C 端截短的变体和一个关键精氨酸被丙氨酸取代的突变体进行的动力学、电泳和质谱研究表明,EgKU-7 是牛和犬胰蛋白酶的紧密抑制剂,它具有与肽酶相互作用的两个而不是一个位点的不寻常特性。其中一个位点位于抗肽酶环,可被牛胰蛋白酶部分水解,但不能被犬胰蛋白酶水解,这表明它对目标酶具有特异性。另一个位点位于 C 端延伸部分。阳离子牛胰蛋白酶和犬胰蛋白酶能水解该延伸部分的特定精氨酸,但阴离子犬胰蛋白酶却不能。据我们所知,这是首次报道单结构域的 Kunitz-A 蛋白与其靶标有两个相互作用位点。考虑到 EgKU-7 的推测直向同源物存在于其他绦虫中,我们的发现揭开了抑肽酶相互作用的新篇章,为宿主-寄生虫进化协奏曲增添了新的音符。
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