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A myzozoan-specific protein is an essential membrane-anchoring component of the succinate dehydrogenase complex in Toxoplasma parasites. 弓形虫体内的琥珀酸脱氢酶复合物中,有一种虫体特异性蛋白是必不可少的膜锚定成分。
IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-01 Epub Date: 2024-06-04 DOI: 10.1098/rsob.230463
Soraya M Zwahlen, Jenni A Hayward, Capella S Maguire, Alex R Qin, Giel G van Dooren

Succinate dehydrogenase (SDH) is a protein complex that functions in the tricarboxylic acid cycle and the electron transport chain of mitochondria. In most eukaryotes, SDH is highly conserved and comprises the following four subunits: SdhA and SdhB form the catalytic core of the complex, while SdhC and SdhD anchor the complex in the membrane. Toxoplasma gondii is an apicomplexan parasite that infects one-third of humans worldwide. The genome of T. gondii encodes homologues of the catalytic subunits SdhA and SdhB, although the physiological role of the SDH complex in the parasite and the identity of the membrane-anchoring subunits are poorly understood. Here, we show that the SDH complex contributes to optimal proliferation and O2 consumption in the disease-causing tachyzoite stage of the T. gondii life cycle. We characterize a small membrane-bound subunit of the SDH complex called mitochondrial protein ookinete developmental defect (MPODD), which is conserved among myzozoans, a phylogenetic grouping that incorporates apicomplexan parasites and their closest free-living relatives. We demonstrate that TgMPODD is essential for SDH activity and plays a key role in attaching the TgSdhA and TgSdhB proteins to the membrane anchor of the complex. Our findings highlight a unique and important feature of mitochondrial energy metabolism in apicomplexan parasites and their relatives.

琥珀酸脱氢酶(SDH)是一种蛋白质复合物,在线粒体的三羧酸循环和电子传递链中发挥作用。在大多数真核生物中,SDH 是高度保守的,由以下四个亚基组成:SdhA 和 SdhB 构成复合体的催化核心,而 SdhC 和 SdhD 则将复合体固定在膜上。弓形虫(Toxoplasma gondii)是一种 apicomplexan 寄生虫,感染了全球三分之一的人类。弓形虫的基因组编码催化亚基 SdhA 和 SdhB 的同源物,但人们对 SDH 复合物在寄生虫中的生理作用以及膜锚定亚基的特性知之甚少。在这里,我们证明了 SDH 复合物在淋球菌生命周期的致病速生阶段有助于优化增殖和氧气消耗。我们研究了 SDH 复合物的一个膜结合小亚基--线粒体蛋白畸形发育缺陷(MPODD)--的特性,该亚基在软体动物中是保守的。我们证明 TgMPODD 对 SDH 活性至关重要,并在将 TgSdhA 和 TgSdhB 蛋白吸附到复合体的膜锚上起着关键作用。我们的发现突显了类囊体寄生虫及其近缘种线粒体能量代谢的一个独特而重要的特征。
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引用次数: 0
Decoding frontotemporal and cell-type-specific vulnerabilities to neuropsychiatric disorders and psychoactive drugs. 解码额颞叶和细胞类型对神经精神疾病和精神活性药物的特异脆弱性。
IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-01 Epub Date: 2024-06-12 DOI: 10.1098/rsob.240063
Jiatong Ji, Honglu Chao, Huimei Chen, Jun Liao, Wenqian Shi, Yangfan Ye, Tian Wang, Yongping You, Ning Liu, Jing Ji, Enrico Petretto

Frontotemporal lobe abnormalities are linked to neuropsychiatric disorders and cognition, but the role of cellular heterogeneity between temporal lobe (TL) and frontal lobe (FL) in the vulnerability to genetic risk factors remains to be elucidated. We integrated single-nucleus transcriptome analysis in 'fresh' human FL and TL with genetic susceptibility, gene dysregulation in neuropsychiatric disease and psychoactive drug response data. We show how intrinsic differences between TL and FL contribute to the vulnerability of specific cell types to both genetic risk factors and psychoactive drugs. Neuronal populations, specifically PVALB neurons, were most highly vulnerable to genetic risk factors for psychiatric disease. These psychiatric disease-associated genes were mostly upregulated in the TL, and dysregulated in the brain of patients with obsessive-compulsive disorder, bipolar disorder and schizophrenia. Among these genes, GRIN2A and SLC12A5, implicated in schizophrenia and bipolar disorder, were significantly upregulated in TL PVALB neurons and in psychiatric disease patients' brain. PVALB neurons from the TL were twofold more vulnerable to psychoactive drugs than to genetic risk factors, showing the influence and specificity of frontotemporal lobe differences on cell vulnerabilities. These studies provide a cell type resolved map of the impact of brain regional differences on cell type vulnerabilities in neuropsychiatric disorders.

额颞叶异常与神经精神疾病和认知能力有关,但颞叶(TL)和额叶(FL)之间的细胞异质性在易受遗传风险因素影响方面的作用仍有待阐明。我们将 "新鲜 "人类 FL 和 TL 的单核转录组分析与遗传易感性、神经精神疾病中的基因失调以及精神活性药物反应数据相结合。我们展示了 TL 和 FL 之间的内在差异如何导致特定细胞类型易受遗传风险因素和精神活性药物的影响。神经元群体,特别是 PVALB 神经元,最容易受到精神疾病遗传风险因素的影响。在强迫症、双相情感障碍和精神分裂症患者的大脑中,这些精神疾病相关基因大多在TL中上调,在TL中失调。在这些基因中,与精神分裂症和双相情感障碍有关的GRIN2A和SLC12A5在TL PVALB神经元和精神疾病患者大脑中明显上调。TL的PVALB神经元对精神活性药物的易损性是遗传风险因素的两倍,这表明额颞叶差异对细胞易损性的影响和特异性。这些研究为神经精神疾病患者大脑区域差异对细胞类型脆弱性的影响提供了一个细胞类型解析图。
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引用次数: 0
Identification of the modulatory Ca2+-binding sites of acid-sensing ion channel 1a. 鉴定酸感应离子通道 1a 的 Ca2+ 结合调节位点
IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-01 Epub Date: 2024-06-19 DOI: 10.1098/rsob.240028
Ophélie Molton, Olivier Bignucolo, Stephan Kellenberger

Acid-sensing ion channels (ASICs) are neuronal Na+-permeable ion channels activated by extracellular acidification. ASICs are involved in learning, fear sensing, pain sensation and neurodegeneration. Increasing the extracellular Ca2+ concentration decreases the H+ sensitivity of ASIC1a, suggesting a competition for binding sites between H+ and Ca2+ ions. Here, we predicted candidate residues for Ca2+ binding on ASIC1a, based on available structural information and our molecular dynamics simulations. With functional measurements, we identified several residues in cavities previously associated with pH-dependent gating, whose mutation reduced the modulation by extracellular Ca2+ of the ASIC1a pH dependence of activation and desensitization. This occurred likely owing to a disruption of Ca2+ binding. Our results link one of the two predicted Ca2+-binding sites in each ASIC1a acidic pocket to the modulation of channel activation. Mg2+ regulates ASICs in a similar way as does Ca2+. We show that Mg2+ shares some of the binding sites with Ca2+. Finally, we provide evidence that some of the ASIC1a Ca2+-binding sites are functionally conserved in the splice variant ASIC1b. Our identification of divalent cation-binding sites in ASIC1a shows how Ca2+ affects ASIC1a gating, elucidating a regulatory mechanism present in many ion channels.

酸感应离子通道(ASIC)是一种神经元Na+渗透离子通道,可被细胞外酸化激活。ASIC参与学习、恐惧感、痛觉和神经变性。增加细胞外 Ca2+ 浓度会降低 ASIC1a 对 H+ 的敏感性,这表明 H+ 和 Ca2+ 离子会竞争结合位点。在此,我们根据现有的结构信息和分子动力学模拟,预测了 ASIC1a 上与 Ca2+ 结合的候选残基。通过功能测量,我们确定了以前与 pH 依赖性门控相关的空腔中的几个残基,这些残基的突变降低了细胞外 Ca2+ 对 ASIC1a 激活和脱敏的 pH 依赖性的调节。出现这种情况的原因可能是 Ca2+ 的结合受到了破坏。我们的研究结果将每个 ASIC1a 酸性口袋中两个预测的 Ca2+ 结合位点中的一个与通道激活的调节联系起来。Mg2+ 调节 ASIC 的方式与 Ca2+ 相似。我们发现 Mg2+ 与 Ca2+ 共享一些结合位点。最后,我们提供的证据表明,ASIC1a 的某些 Ca2+ 结合位点在剪接变体 ASIC1b 中具有功能上的一致性。我们对 ASIC1a 中二价阳离子结合位点的鉴定显示了 Ca2+ 如何影响 ASIC1a 的门控,阐明了许多离子通道中都存在的调控机制。
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引用次数: 0
Terminal regions of a protein are a hotspot for low complexity regions and selection. 蛋白质的末端区域是低复杂性区域和选择的热点。
IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-01 Epub Date: 2024-06-12 DOI: 10.1098/rsob.230439
Lokdeep Teekas, Sandhya Sharma, Nagarjun Vijay

Volatile low complexity regions (LCRs) are a novel source of adaptive variation, functional diversification and evolutionary novelty. An interplay of selection and mutation governs the composition and length of low complexity regions. High %GC and mutations provide length variability because of mechanisms like replication slippage. Owing to the complex dynamics between selection and mutation, we need a better understanding of their coexistence. Our findings underscore that positively selected sites (PSS) and low complexity regions prefer the terminal regions of genes, co-occurring in most Tetrapoda clades. We observed that positively selected sites within a gene have position-specific roles. Central-positively selected site genes primarily participate in defence responses, whereas terminal-positively selected site genes exhibit non-specific functions. Low complexity region-containing genes in the Tetrapoda clade exhibit a significantly higher %GC and lower ω (dN/dS: non-synonymous substitution rate/synonymous substitution rate) compared with genes without low complexity regions. This lower ω implies that despite providing rapid functional diversity, low complexity region-containing genes are subjected to intense purifying selection. Furthermore, we observe that low complexity regions consistently display ubiquitous prevalence at lower purity levels, but exhibit a preference for specific positions within a gene as the purity of the low complexity region stretch increases, implying a composition-dependent evolutionary role. Our findings collectively contribute to the understanding of how genetic diversity and adaptation are shaped by the interplay of selection and low complexity regions in the Tetrapoda clade.

易变低复杂性区域(LCR)是适应性变异、功能多样化和进化新颖性的新来源。选择和突变的相互作用决定了低复杂性区域的组成和长度。由于复制滑动等机制,高%GC和突变提供了长度变异性。由于选择和突变之间存在复杂的动态关系,我们需要更好地了解它们之间的共存关系。我们的研究结果表明,正向选择位点(PSS)和低复杂性区域更倾向于基因的末端区域,在大多数四足动物支系中同时存在。我们观察到,基因内的正选位点具有位置特异性。中央阳性选择位点基因主要参与防御反应,而末端阳性选择位点基因则表现出非特异性功能。与不含低复杂性区域的基因相比,四足纲中含低复杂性区域的基因表现出明显较高的%GC和较低的ω(dN/dS:非同义替换率/同义替换率)。较低的ω意味着,尽管提供了快速的功能多样性,但含有低复杂性区域的基因受到了强烈的纯化选择。此外,我们还观察到,低复杂性区域在较低的纯度水平下始终显示出无处不在的普遍性,但随着低复杂性区域伸展纯度的增加,低复杂性区域在基因内的特定位置表现出偏好性,这意味着低复杂性区域在进化过程中起着依赖于组成的作用。我们的研究结果共同有助于人们理解遗传多样性和适应性是如何通过选择和低复杂性区域的相互作用而在四足纲支系中形成的。
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引用次数: 0
The cytosolic form of dual localized BolA family protein Bol3 is important for adaptation to iron starvation in Aspergillus fumigatus. 双重定位的 BolA 家族蛋白 Bol3 的胞浆形式对于曲霉适应铁饥饿非常重要。
IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-01 Epub Date: 2024-06-26 DOI: 10.1098/rsob.240033
Simon Oberegger, Matthias Misslinger, Klaus Faserl, Bettina Sarg, Hesso Farhan, Hubertus Haas

Aspergillus fumigatus is the predominant mould pathogen for humans. Adaption to host-imposed iron limitation has previously been demonstrated to be essential for its virulence. [2Fe-2S] clusters are crucial as cofactors of several metabolic pathways and mediate cytosolic/nuclear iron sensing in fungi including A. fumigatus. [2Fe-2S] cluster trafficking has been shown to involve BolA family proteins in both mitochondria and the cytosol/nucleus. Interestingly, both A. fumigatus homologues, termed Bol1 and Bol3, possess mitochondrial targeting sequences, suggesting the lack of cytosolic/nuclear versions. Here, we show by the combination of mutational, proteomic and fluorescence microscopic analyses that expression of the Bol3 encoding gene leads to dual localization of gene products to mitochondria and the cytosol/nucleus via alternative translation initiation downstream of the mitochondrial targeting sequence, which appears to be highly conserved in various Aspergillus species. Lack of either mitochondrial Bol1 or Bol3 was phenotypically inconspicuous while lack of cytosolic/nuclear Bol3 impaired growth during iron limitation but not iron sensing which indicates a particular importance of [2Fe-2S] cluster trafficking during iron limitation. Remarkably, cytosolic/nuclear Bol3 differs from the mitochondrial version only by N-terminal acetylation, a finding that was only possible by mutational hypothesis testing.

烟曲霉是人类最主要的霉菌病原体。以前的研究表明,适应宿主施加的铁限制对其毒力至关重要。[2Fe-2S]簇是包括烟曲霉在内的真菌中几种代谢途径和介导胞质/核铁感应的关键辅助因子。研究表明,[2Fe-2S]簇的运输涉及线粒体和细胞质/细胞核中的 BolA 家族蛋白。有趣的是,被称为 Bol1 和 Bol3 的烟曲霉同源物都具有线粒体靶向序列,这表明缺乏细胞质/核版本。在这里,我们通过突变、蛋白质组和荧光显微镜分析表明,Bol3 编码基因的表达通过线粒体靶向序列下游的替代翻译起始,导致基因产物在线粒体和细胞质/核中的双重定位,而线粒体靶向序列在不同曲霉菌种中似乎是高度保守的。线粒体 Bol1 或 Bol3 的缺失在表型上并不明显,而细胞质/核 Bol3 的缺失会影响铁限制过程中的生长,但不会影响铁感应,这表明[2Fe-2S]簇的贩运在铁限制过程中尤为重要。值得注意的是,细胞质/核 Bol3 与线粒体 Bol3 的区别仅在于 N 端乙酰化,这一发现只能通过突变假设检验来实现。
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引用次数: 0
Structural and functional characterization of nanobodies that neutralize Omicron variants of SARS-CoV-2. 中和 SARS-CoV-2 Omicron 变体的纳米抗体的结构和功能特征。
IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-01 Epub Date: 2024-06-04 DOI: 10.1098/rsob.230252
Katy Cornish, Jiandong Huo, Luke Jones, Parul Sharma, Joseph W Thrush, Sahar Abdelkarim, Anja Kipar, Siva Ramadurai, Miriam Weckener, Halina Mikolajek, Sai Liu, Imogen Buckle, Eleanor Bentley, Adam Kirby, Ximeng Han, Stephen M Laidlaw, Michelle Hill, Lauren Eyssen, Chelsea Norman, Audrey Le Bas, John Clarke, William James, James P Stewart, Miles Carroll, James H Naismith, Raymond J Owens

The Omicron strains of SARS-CoV-2 pose a significant challenge to the development of effective antibody-based treatments as immune evasion has compromised most available immune therapeutics. Therefore, in the 'arms race' with the virus, there is a continuing need to identify new biologics for the prevention or treatment of SARS-CoV-2 infections. Here, we report the isolation of nanobodies that bind to the Omicron BA.1 spike protein by screening nanobody phage display libraries previously generated from llamas immunized with either the Wuhan or Beta spike proteins. The structure and binding properties of three of these nanobodies (A8, H6 and B5-5) have been characterized in detail providing insight into their binding epitopes on the Omicron spike protein. Trimeric versions of H6 and B5-5 neutralized the SARS-CoV-2 variant of concern BA.5 both in vitro and in the hamster model of COVID-19 following nasal administration. Thus, either alone or in combination could serve as starting points for the development of new anti-viral immunotherapeutics.

SARS-CoV-2 的 Omicron 株对开发基于抗体的有效治疗方法构成了巨大挑战,因为免疫逃避已经破坏了大多数可用的免疫疗法。因此,在与该病毒的 "军备竞赛 "中,需要不断发现新的生物制剂来预防或治疗 SARS-CoV-2 感染。在这里,我们报告了通过筛选以前从用武汉或贝塔尖峰蛋白免疫的骆驼身上产生的纳米抗体噬菌体展示文库,分离出与 Omicron BA.1 尖峰蛋白结合的纳米抗体。对其中三个纳米抗体(A8、H6 和 B5-5)的结构和结合特性进行了详细表征,从而深入了解了它们在欧米茄尖峰蛋白上的结合表位。H6和B5-5的三聚体版本在体外和仓鼠COVID-19模型中鼻给药后都能中和关注BA.5的SARS-CoV-2变体。因此,无论是单独使用还是联合使用,都可以作为开发新型抗病毒免疫疗法的起点。
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引用次数: 0
Plasmodium LCCL domain-containing modular proteins have their origins in the ancestral alveolate. 疟原虫含 LCCL 结构域的模块蛋白起源于祖先的肺泡蛋白。
IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-01 Epub Date: 2024-06-12 DOI: 10.1098/rsob.230451
Callum De Hoest-Thompson, Virginia Marugan-Hernandez, Johannes T Dessens

Plasmodium species encode a unique set of six modular proteins named LCCL lectin domain adhesive-like proteins (LAPs) that operate as a complex and that are essential for malaria parasite transmission from mosquito to vertebrate. LAPs possess complex architectures obtained through unique assemblies of conserved domains associated with lipid, protein and carbohydrate interactions, including the name-defining LCCL domain. Here, we assessed the prevalence of Plasmodium LAP orthologues across eukaryotic life. Our findings show orthologous conservation in all apicomplexans, with lineage-specific repertoires acquired through differential lap gene loss and duplication. Besides Apicomplexa, LAPs are found in their closest relatives: the photosynthetic chromerids, which encode the broadest repertoire including a novel membrane-bound LCCL protein. LAPs are notably absent from other alveolate lineages (dinoflagellates, perkinsids and ciliates), but are encoded by predatory colponemids, a sister group to the alveolates. These results reveal that the LAPs are much older than previously thought and pre-date not only the Apicomplexa but the Alveolata altogether.

疟原虫物种编码一套独特的六种模块蛋白,名为 LCCL 凝集素结构域粘附样蛋白(LAPs),它们作为一个复合体运作,是疟原虫从蚊子传播到脊椎动物的关键。LAPs 具有复杂的结构,通过与脂质、蛋白质和碳水化合物相互作用相关的保守结构域(包括名称定义的 LCCL 结构域)的独特组合而获得。在这里,我们评估了真核生物中疟原虫 LAP 直向同源物的普遍性。我们的研究结果表明,在所有类囊体中都存在直向同源物保护现象,通过不同圈层基因的缺失和复制获得了特定世系的同源物。除了类囊体之外,LAPs 还存在于它们的近亲:光合染色体,光合染色体编码最广泛的序列,包括一种新型膜结合 LCCL 蛋白。LAPs 明显不存在于其他瓣膜虫系(甲藻、倒鞭毛虫和纤毛虫)中,但在瓣膜虫的姊妹类--捕食性疣藻中却有编码。这些结果表明,LAPs 的历史比以前认为的要早得多,不仅早于甲壳纲,而且早于齿孔纲。
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引用次数: 0
Greatwall-Endos-PP2A/B55Twins network regulates translation and stability of maternal transcripts in the Drosophila oocyte-to-embryo transition. Greatwall-Endos-PP2A/B55Twins 网络调节果蝇卵母细胞向胚胎转化过程中母体转录本的翻译和稳定性。
IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-01 Epub Date: 2024-06-19 DOI: 10.1098/rsob.240065
Hélène Rangone, Laura Bond, Timothy T Weil, David M Glover

The transition from oocyte to embryo requires translation of maternally provided transcripts that in Drosophila is activated by Pan Gu kinase to release a rapid succession of 13 mitotic cycles. Mitotic entry is promoted by several protein kinases that include Greatwall/Mastl, whose Endosulfine substrates antagonize Protein Phosphatase 2A (PP2A), facilitating mitotic Cyclin-dependent kinase 1/Cyclin B kinase activity. Here we show that hyperactive greatwallScant can not only be suppressed by mutants in its Endos substrate but also by mutants in Pan Gu kinase subunits. Conversely, mutants in me31B or trailer hitch, which encode a complex that represses hundreds of maternal mRNAs, enhance greatwallScant . Me31B and Trailer Hitch proteins, known substrates of Pan Gu kinase, copurify with Endos. This echoes findings that budding yeast Dhh1, orthologue of Me31B, associates with Igo1/2, orthologues of Endos and substrates of the Rim15, orthologue of Greatwall. endos-derived mutant embryos show reduced Me31B and elevated transcripts for the mitotic activators Cyclin B, Polo and Twine/Cdc25. Together, our findings demonstrate a previously unappreciated conservation of the Greatwall-Endosulfine pathway in regulating translational repressors and its interactions with the Pan Gu kinase pathway to regulate translation and/or stability of maternal mRNAs upon egg activation.

从卵母细胞到胚胎的转变需要母体提供的转录本的翻译,在果蝇中,Pan Gu 激酶激活转录本的翻译,从而快速完成 13 个有丝分裂周期。有丝分裂的进入是由包括 Greatwall/Mastl 在内的几种蛋白激酶促进的,Greatwall/Mastl 的内硫酸底物可拮抗蛋白磷酸酶 2A(PP2A),从而促进有丝分裂周期性细胞周期蛋白依赖性激酶 1/Cyclin B 激酶的活性。在这里,我们发现亢进的 GreatwallScant 不仅能被其 Endos 底物的突变体抑制,还能被 Pan Gu 激酶亚基的突变体抑制。相反,编码抑制数百种母体 mRNA 的复合物的 me31B 或 trailer hitch 的突变体会增强 greatwallScant 的活性。Me31B和Trailer Hitch蛋白是已知的Pan Gu激酶底物,它们与Endos共聚。这与芽殖酵母 Dhh1(Me31B 的直系同源物)与 Igo1/2(Endos 的直系同源物和 Rim15(Greatwall 的直系同源物)的底物)结合的发现相呼应。总之,我们的研究结果表明,在调节翻译抑制因子方面,Greatwall-Endosulfine 通路及其与 Pan Gu 激酶通路之间的相互作用,在卵子活化时调节母体 mRNA 的翻译和/或稳定性方面,具有以前未被认识到的保护作用。
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引用次数: 0
MYH7 R453C induced cardiac remodelling via activating TGF-β/Smad2/3, ERK1/2 and Nox4/ROS/NF-κB signalling pathways. MYH7 R453C通过激活TGF-β/Smad2/3、ERK1/2和Nox4/ROS/NF-κB信号通路诱导心脏重塑。
IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-01 Epub Date: 2024-06-12 DOI: 10.1098/rsob.230427
Lingyu Wang, Linquan Li, Dazhong Zhao, Hongming Yuan, Huanyu Zhang, Jiahuan Chen, Daxin Pang, Yi Lu, Hongsheng Ouyang

Hypertrophic cardiomyopathy (HCM) is a monogenic cardiac disorder commonly induced by sarcomere gene mutations. However, the mechanism for HCM is not well defined. Here, we generated transgenic MYH7 R453C and MYH6 R453C piglets and found both developed typical cardiac hypertrophy. Unexpectedly, we found serious fibrosis and cardiomyocyte loss in the ventricular of MYH7 R453C, not MYH6 R453C piglets, similar to HCM patients. Then, RNA-seq analysis and western blotting identified the activation of ERK1/2 and PI3K-Akt pathways in MYH7 R453C. Moreover, we observed an increased expression of fetal genes and an excess of reactive oxygen species (ROS) in MYH7 R453C piglet models, which was produced by Nox4 and subsequently induced inflammatory response. Additionally, the phosphorylation levels of Smad2/3, ERK1/2 and NF-kB p65 proteins were elevated in cardiomyocytes with the MYH7 R453C mutation. Furthermore, epigallocatechin gallate, a natural bioactive compound, could be used as a drug to reduce cell death by adjusting significant downregulation of the protein expression of Bax and upregulated Bcl-2 levels in the H9C2 models with MYH7 R453C mutation. In conclusion, our study illustrated that TGF-β/Smad2/3, ERK1/2 and Nox4/ROS pathways have synergistic effects on cardiac remodelling and inflammation in MYH7 R453C mutation.

肥厚型心肌病(HCM)是一种单基因心脏疾病,通常由肌节基因突变诱发。然而,HCM 的发病机制尚未明确。在这里,我们生成了转基因 MYH7 R453C 和 MYH6 R453C 仔猪,发现它们都出现了典型的心脏肥大。出乎意料的是,我们发现 MYH7 R453C 仔猪的心室出现了严重的纤维化和心肌细胞丢失,而 MYH6 R453C 仔猪则没有,这与 HCM 患者相似。然后,RNA-seq 分析和 Western 印迹确定了 MYH7 R453C 中 ERK1/2 和 PI3K-Akt 通路的激活。此外,我们还观察到在 MYH7 R453C 小猪模型中胎儿基因表达增加,活性氧(ROS)过量,由 Nox4 产生,随后诱发炎症反应。此外,MYH7 R453C 突变的心肌细胞中 Smad2/3、ERK1/2 和 NF-kB p65 蛋白的磷酸化水平升高。此外,表没食子儿茶素没食子酸酯作为一种天然生物活性化合物,可通过调节MYH7 R453C突变的H9C2模型中Bax蛋白表达的显著下调和Bcl-2水平的上调来减少细胞死亡。总之,我们的研究表明,TGF-β/Smad2/3、ERK1/2和Nox4/ROS通路对MYH7 R453C突变的心脏重塑和炎症具有协同作用。
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引用次数: 0
Structural and functional insights into the C-terminal signal domain of the Bacteroidetes type-IX secretion system. 类杆菌 IX 型分泌系统 C 端信号域的结构和功能研究。
IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-06-01 Epub Date: 2024-06-12 DOI: 10.1098/rsob.230448
Danuta Mizgalska, Arturo Rodríguez-Banqueri, Florian Veillard, Mirosław Książęk, Theodoros Goulas, Tibisay Guevara, Ulrich Eckhard, Jan Potempa, F Xavier Gomis-Rüth

Gram-negative bacteria from the Bacteroidota phylum possess a type-IX secretion system (T9SS) for protein secretion, which requires cargoes to have a C-terminal domain (CTD). Structurally analysed CTDs are from Porphyromonas gingivalis proteins RgpB, HBP35, PorU and PorZ, which share a compact immunoglobulin-like antiparallel 3+4 β-sandwich (β1-β7). This architecture is essential as a P. gingivalis strain with a single-point mutant of RgpB disrupting the interaction of the CTD with its preceding domain prevented secretion of the protein. Next, we identified the C-terminus ('motif C-t.') and the loop connecting strands β3 and β4 ('motif Lβ3β4') as conserved. We generated two strains with insertion and replacement mutants of PorU, as well as three strains with ablation and point mutants of RgpB, which revealed both motifs to be relevant for T9SS function. Furthermore, we determined the crystal structure of the CTD of mirolase, a cargo of the Tannerella forsythia T9SS, which shares the same general topology as in Porphyromonas CTDs. However, motif Lβ3β4 was not conserved. Consistently, P. gingivalis could not properly secrete a chimaeric protein with the CTD of peptidylarginine deiminase replaced with this foreign CTD. Thus, the incompatibility of the CTDs between these species prevents potential interference between their T9SSs.

细菌门的革兰氏阴性细菌拥有一种用于分泌蛋白质的 IX 型分泌系统(T9SS),该系统要求货物具有一个 C 端结构域(CTD)。对牙龈卟啉单胞菌蛋白 RgpB、HBP35、PorU 和 PorZ 的 CTD 进行了结构分析,它们共享一个紧凑的免疫球蛋白样反平行 3+4 β-三明治(β1-β7)。这种结构非常重要,因为一株牙龈脓疱病菌的 RgpB 单点突变体破坏了 CTD 与其前端结构域的相互作用,从而阻止了蛋白质的分泌。接下来,我们确定了 C 端("motif C-t.")和连接 β3 和 β4 链的环路("motif Lβ3β4")是保守的。我们生成了两株 PorU 的插入突变体和置换突变体,以及三株 RgpB 的消融突变体和点突变体,结果表明这两个图案与 T9SS 的功能有关。此外,我们还测定了连翘丹菌 T9SS 的载体 mirolase CTD 的晶体结构,该结构与卟啉单胞菌 CTD 的一般拓扑结构相同。然而,图案 Lβ3β4 并不保守。同样,牙龈卟啉菌不能正常分泌肽精氨酸脱氨酶 CTD 被这种外来 CTD 取代的嵌合蛋白。因此,这些物种之间 CTD 的不兼容性阻止了它们的 T9SS 之间的潜在干扰。
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