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The role of mitochondrial RNA association for mitochondrial homeostasis in neurons 线粒体 RNA 关联对神经元线粒体平衡的作用
IF 4.1 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-02-07 DOI: 10.1042/bcj20230110
Segura, Inmaculada, Harbauer, Angelika
The sub-compartmentalization of cellular processes is especially important in highly polarized cells such as neurons, as their function rely on their complex morphology. The association of RNAs to the mitochondrial surface is a conserved feature from yeast to humans and it regulates several aspects of mitochondrial physiology and, hence, cellular functions. In neurons, mitochondria are emerging as platforms for RNA transport and local protein translation. In this review, we discuss how RNA localization to mitochondria helps to sustain mitochondrial function, and how this can support mitochondrial homeostasis, especially in the distal parts of the neuron, to support neuronal activity.
细胞过程的亚区化对于神经元等高度极化的细胞尤为重要,因为它们的功能依赖于其复杂的形态。RNA 与线粒体表面的结合是从酵母到人类的一个保守特征,它调控着线粒体生理学的多个方面,进而调控着细胞功能。在神经元中,线粒体正在成为 RNA 运输和局部蛋白质翻译的平台。在这篇综述中,我们将讨论 RNA 在线粒体上的定位如何有助于维持线粒体的功能,以及这如何支持线粒体的平衡,尤其是在神经元的远端部分,以支持神经元的活动。
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引用次数: 0
A highly conserved ligand-binding site for AccA transporters of antibiotic and quorum-sensing regulator in Agrobacterium leads to a different specificity. 在农杆菌中,抗生素和群体感应调节剂的AccA转运体的高度保守的配体结合位点导致了不同的特异性。
IF 4.1 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-25 DOI: 10.1042/BCJ20230273
Solange Moréra, Armelle Vigouroux, Magali Aumont-Nicaise, Mohammed Ahmar, Thibault Meyer, Abbas El Sahili, Grégory Deicsics, Almudena González-Mula, Sizhe Li, Jeanne Doré, Serena Sirigu, Pierre Legrand, Camille Penot, François André, Denis Faure, Laurent Soulère, Yves Queneau, Ludovic Vial

Plants genetically modified by the pathogenic Agrobacterium strain C58 synthesize agrocinopines A and B, whereas those modified by the pathogenic strain Bo542 produce agrocinopines C and D. The four agrocinopines (A, B, C and D) serve as nutrients by agrobacteria and signaling molecule for the dissemination of virulence genes. They share the uncommon pyranose-2-phosphate motif, represented by the l-arabinopyranose moiety in agrocinopines A/B and the d-glucopyranose moiety in agrocinopines C/D, also found in the antibiotic agrocin 84. They are imported into agrobacterial cytoplasm via the Acc transport system, including the solute-binding protein AccA coupled to an ABC transporter. We have previously shown that unexpectedly, AccA from strain C58 (AccAC58) recognizes the pyranose-2-phosphate motif present in all four agrocinopines and agrocin 84, meaning that strain C58 is able to import agrocinopines C/D, originating from the competitor strain Bo542. Here, using agrocinopine derivatives and combining crystallography, affinity and stability measurements, modeling, molecular dynamics, in vitro and vivo assays, we show that AccABo542 and AccAC58 behave differently despite 75% sequence identity and a nearly identical ligand binding site. Indeed, strain Bo542 imports only compounds containing the d-glucopyranose-2-phosphate moiety, and with a lower affinity compared with strain C58. This difference in import efficiency makes C58 more competitive than Bo542 in culture media. We can now explain why Agrobacterium/Allorhizobium vitis strain S4 is insensitive to agrocin 84, although its genome contains a conserved Acc transport system. Overall, our work highlights AccA proteins as a case study, for which stability and dynamics drive specificity.

经致病性农杆菌菌株C58基因修饰的植物合成agrocinopines A和B,而经致病性菌株Bo542基因修饰的植物产生agrocinopines C和D。这四种agrocinopines (A、B、C和D)通过农杆菌和信号分子作为营养物质传播毒力基因。它们共享罕见的吡喃糖-2-磷酸基序,以agrocinopines A/B中的L-arabinopyranose片段和agrocinopines C/D中的D-glucopyranose片段为代表,也在抗生素agrocin 84中发现。它们通过Acc转运系统(包括与ABC转运蛋白偶联的溶质结合蛋白AccA)进入农杆菌细胞质。我们之前意外地发现,来自菌株C58 (AccAC58)的AccA识别了所有四种agrocinopines和agrocin84中存在的pyranose-2-phosphate基序,这意味着菌株C58能够进口源自竞争菌株Bo542的agrocinopines C/D。在这里,我们使用农用罂粟碱衍生物,结合晶体学、亲和力和稳定性测量、建模、分子动力学、体外和体内实验,我们发现AccABo542和AccAC58在75%的序列相同和几乎相同的配体结合位点的情况下表现不同。事实上,菌株Bo542只输入含有d -葡萄糖吡喃糖-2-磷酸片段的化合物,与菌株C58相比,亲和力较低。这种进口效率的差异使得C58在培养基上比Bo542更具竞争力。我们现在可以解释为什么农杆菌/葡萄异根菌菌株S4对agrocin 84不敏感,尽管它的基因组包含一个保守的Acc转运系统。总的来说,我们的工作突出了AccA蛋白作为一个案例研究,其稳定性和动力学驱动特异性。
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引用次数: 0
The mTORC2 signaling network: targets and cross-talks. mTORC2 信号网络:目标和交叉联系。
IF 4.1 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-25 DOI: 10.1042/BCJ20220325
Aparna Ragupathi, Christian Kim, Estela Jacinto

The mechanistic target of rapamycin, mTOR, controls cell metabolism in response to growth signals and stress stimuli. The cellular functions of mTOR are mediated by two distinct protein complexes, mTOR complex 1 (mTORC1) and mTORC2. Rapamycin and its analogs are currently used in the clinic to treat a variety of diseases and have been instrumental in delineating the functions of its direct target, mTORC1. Despite the lack of a specific mTORC2 inhibitor, genetic studies that disrupt mTORC2 expression unravel the functions of this more elusive mTOR complex. Like mTORC1 which responds to growth signals, mTORC2 is also activated by anabolic signals but is additionally triggered by stress. mTORC2 mediates signals from growth factor receptors and G-protein coupled receptors. How stress conditions such as nutrient limitation modulate mTORC2 activation to allow metabolic reprogramming and ensure cell survival remains poorly understood. A variety of downstream effectors of mTORC2 have been identified but the most well-characterized mTORC2 substrates include Akt, PKC, and SGK, which are members of the AGC protein kinase family. Here, we review how mTORC2 is regulated by cellular stimuli including how compartmentalization and modulation of complex components affect mTORC2 signaling. We elaborate on how phosphorylation of its substrates, particularly the AGC kinases, mediates its diverse functions in growth, proliferation, survival, and differentiation. We discuss other signaling and metabolic components that cross-talk with mTORC2 and the cellular output of these signals. Lastly, we consider how to more effectively target the mTORC2 pathway to treat diseases that have deregulated mTOR signaling.

雷帕霉素的机制靶标(mTOR)控制着细胞的新陈代谢,以应对生长信号和应激刺激。mTOR 的细胞功能由两个不同的蛋白复合物介导,即 mTOR 复合物 1(mTORC1)和 mTORC2。雷帕霉素及其类似物目前在临床上用于治疗多种疾病,并在阐明其直接靶标 mTORC1 的功能方面发挥了重要作用。尽管缺乏特异性的 mTORC2 抑制剂,但通过基因研究破坏 mTORC2 的表达,可以揭示这一更难以捉摸的 mTOR 复合物的功能。与 mTORC1 响应生长信号一样,mTORC2 也会被合成代谢信号激活,但也会被压力触发。人们对营养限制等应激条件如何调节 mTORC2 的激活以实现代谢重编程并确保细胞存活仍知之甚少。mTORC2的下游效应因子种类繁多,但表征最明确的mTORC2底物包括AGC蛋白激酶家族成员Akt、PKC和SGK。在此,我们回顾了 mTORC2 是如何受细胞刺激调控的,包括复杂成分的区隔和调节是如何影响 mTORC2 信号传导的。我们详细阐述了其底物(尤其是 AGC 激酶)的磷酸化如何介导其在生长、增殖、存活和分化中的各种功能。我们还讨论了与 mTORC2 交叉作用的其他信号和代谢成分,以及这些信号的细胞输出。最后,我们将探讨如何更有效地针对 mTORC2 通路治疗 mTOR 信号失调的疾病。
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引用次数: 0
The role of phosphorylation in calmodulin-mediated gating of human AQP0. 磷酸化在钙调素介导的人AQP0门控中的作用。
IF 4.1 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-10 DOI: 10.1042/BCJ20230158
Stefan Kreida, Jennifer Virginia Roche, Julie Winkel Missel, Tamim Al-Jubair, Carl Johan Hagströmer, Veronika Wittenbecher, Sara Linse, Pontus Gourdon, Susanna Törnroth-Horsefield

Aquaporin-0 (AQP0) is the main water channel in the mammalian lens and is involved in accommodation and maintaining lens transparency. AQP0 binds the Ca2+-sensing protein calmodulin (CaM) and this interaction is believed to gate its water permeability by closing the water-conducting pore. Here, we express recombinant and functional human AQP0 in Pichia pastoris and investigate how phosphorylation affects the interaction with CaM in vitro as well as the CaM-dependent water permeability of AQP0 in proteoliposomes. Using microscale thermophoresis and surface plasmon resonance technology we show that the introduction of the single phospho-mimicking mutations S229D and S235D in AQP0 reduces CaM binding. In contrast, CaM interacts with S231D with similar affinity as wild type, but in a different manner. Permeability studies of wild-type AQP0 showed that the water conductance was significantly reduced by CaM in a Ca2+-dependent manner, whereas AQP0 S229D, S231D and S235D were all locked in an open state, insensitive to CaM. We propose a model in which phosphorylation of AQP0 control CaM-mediated gating in two different ways (1) phosphorylation of S229 or S235 abolishes binding (the pore remains open) and (2) phosphorylation of S231 results in CaM binding without causing pore closure, the functional role of which remains to be elucidated. Our results suggest that site-dependent phosphorylation of AQP0 dynamically controls its CaM-mediated gating. Since the level of phosphorylation increases towards the lens inner cortex, AQP0 may become insensitive to CaM-dependent gating along this axis.

水通道蛋白-0 (AQP0)是哺乳动物晶状体中的主要通道,参与调节和维持晶状体的透明度。AQP0结合Ca2+感应蛋白钙调蛋白(CaM),这种相互作用被认为通过关闭导水孔来控制其水渗透性。本研究在帕斯德酵母中表达重组的功能性人类AQP0,并研究磷酸化如何影响体外与CaM的相互作用以及AQP0在蛋白脂质体中依赖CaM的透水性。利用微尺度热泳(MST)和表面等离子体共振(SPR)技术,我们发现在AQP0中引入单磷酸模拟突变S229D和S235D减少了cam结合。相比之下,CaM与S231D的相互作用与野生型具有相似的亲和力,但方式不同。对野生型AQP0的渗透性研究表明,CaM以Ca2+依赖的方式显著降低了AQP0的水电导,而AQP0 S229D、S231D和S235D均锁定在开放状态,对CaM不敏感。我们提出了一个模型,其中AQP0的磷酸化以两种不同的方式控制cam介导的门控(1)S229或S235的磷酸化消除了结合(孔保持开放),(2)S231的磷酸化导致cam结合而不导致孔关闭,其功能作用仍有待阐明。我们的研究结果表明,AQP0的位点依赖性磷酸化动态控制其cam介导的门控。由于磷酸化水平向晶状体内皮层方向增加,AQP0可能对沿该轴的cam依赖性门控变得不敏感。
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引用次数: 0
Proximity labelling reveals effects of disease-causing mutation on the DNAJC5/cysteine string protein α interactome. 近距离标记揭示了致病突变对DNAJC5/半胱氨酸串联蛋白α相互作用组的影响。
IF 4.1 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-09 DOI: 10.1042/BCJ20230319
Eleanor Barker, Amy Milburn, Nordine Helassa, Dean Hammond, Natalia Sanchez-Soriano, Alan Morgan, Jeff Barclay

Cysteine string protein α (CSPα), also known as DNAJC5, is a member of the DnaJ/Hsp40 family of co-chaperones. The name derives from a cysteine-rich domain, palmitoylation of which enables localization to intracellular membranes, notably neuronal synaptic vesicles. Mutations in the DNAJC5 gene that encodes CSPα cause autosomal dominant, adult-onset neuronal ceroid lipofuscinosis (ANCL), a rare neurodegenerative disease. As null mutations in CSP-encoding genes in flies, worms and mice similarly result in neurodegeneration, CSP is evidently an evolutionarily conserved neuroprotective protein. However, the client proteins that CSP chaperones to prevent neurodegeneration remain unclear. Traditional methods for identifying protein-protein interactions such as yeast 2-hybrid and affinity purification approaches are poorly suited to CSP, due to its requirement for membrane anchoring and its tendency to aggregate after cell lysis. Therefore, we employed proximity labelling, which enables identification of interacting proteins in situ in living cells via biotinylation. Neuroendocrine PC12 cell lines stably expressing wild type or L115R ANCL mutant CSP constructs fused to miniTurbo were generated; then the biotinylated proteomes were analysed by liquid chromatographymass spectrometry (LCMS) and validated by western blotting. This confirmed several known CSP-interacting proteins, such as Hsc70 and SNAP-25, but also revealed novel binding proteins, including STXBP1/Munc18-1. Interestingly, some protein interactions (such as Hsc70) were unaffected by the L115R mutation, whereas others (including SNAP-25 and STXBP1/Munc18-1) were inhibited. These results define the CSP interactome in a neuronal model cell line and reveal interactions that are affected by ANCL mutation and hence may contribute to the neurodegeneration seen in patients.

半胱氨酸串联蛋白α(CSPα)又称 DNAJC5,是 DnaJ/Hsp40 协同伴侣蛋白家族的成员。其名称源于一个富含半胱氨酸的结构域,该结构域的棕榈酰化作用可使其定位到细胞内膜,特别是神经元突触囊泡。编码 CSPα 的 DNAJC5 基因发生突变会导致常染色体显性、成人发病型神经细胞类脂膜炎(ANCL),这是一种罕见的神经退行性疾病。在苍蝇、蠕虫和小鼠中,CSP编码基因的无效突变同样会导致神经退行性病变,因此CSP显然是一种进化保守的神经保护蛋白。然而,目前仍不清楚 CSP 可通过与哪些客户蛋白结合来防止神经退行性变。传统的蛋白质-蛋白质相互作用鉴定方法,如酵母双杂交和亲和纯化方法,都不太适合 CSP,因为它需要膜锚定,而且细胞裂解后容易聚集。因此,我们采用了近距离标记法,通过生物素化在活细胞中原位鉴定相互作用的蛋白质。我们生成了稳定表达与 miniTurbo 融合的野生型或 L115R ANCL 突变型 CSP 构建物的神经内分泌 PC12 细胞系;然后用液相色谱-质谱联用仪(LCMS)分析了生物素化的蛋白质组,并用 Western 印迹法进行了验证。这证实了几种已知的 CSP 相互作用蛋白,如 Hsc70 和 SNAP-25,但也发现了新的结合蛋白,包括 STXBP1/Munc18-1。有趣的是,一些蛋白质的相互作用(如 Hsc70)不受 L115R 突变的影响,而另一些(包括 SNAP-25 和 STXBP1/Munc18-1)则受到抑制。这些结果确定了神经元模型细胞系中的CSP相互作用组,揭示了受ANCL突变影响的相互作用,因此可能导致患者的神经退行性变。
{"title":"Proximity labelling reveals effects of disease-causing mutation on the DNAJC5/cysteine string protein α interactome.","authors":"Eleanor Barker, Amy Milburn, Nordine Helassa, Dean Hammond, Natalia Sanchez-Soriano, Alan Morgan, Jeff Barclay","doi":"10.1042/BCJ20230319","DOIUrl":"10.1042/BCJ20230319","url":null,"abstract":"<p><p>Cysteine string protein α (CSPα), also known as DNAJC5, is a member of the DnaJ/Hsp40 family of co-chaperones. The name derives from a cysteine-rich domain, palmitoylation of which enables localization to intracellular membranes, notably neuronal synaptic vesicles. Mutations in the DNAJC5 gene that encodes CSPα cause autosomal dominant, adult-onset neuronal ceroid lipofuscinosis (ANCL), a rare neurodegenerative disease. As null mutations in CSP-encoding genes in flies, worms and mice similarly result in neurodegeneration, CSP is evidently an evolutionarily conserved neuroprotective protein. However, the client proteins that CSP chaperones to prevent neurodegeneration remain unclear. Traditional methods for identifying protein-protein interactions such as yeast 2-hybrid and affinity purification approaches are poorly suited to CSP, due to its requirement for membrane anchoring and its tendency to aggregate after cell lysis. Therefore, we employed proximity labelling, which enables identification of interacting proteins in situ in living cells via biotinylation. Neuroendocrine PC12 cell lines stably expressing wild type or L115R ANCL mutant CSP constructs fused to miniTurbo were generated; then the biotinylated proteomes were analysed by liquid chromatographymass spectrometry (LCMS) and validated by western blotting. This confirmed several known CSP-interacting proteins, such as Hsc70 and SNAP-25, but also revealed novel binding proteins, including STXBP1/Munc18-1. Interestingly, some protein interactions (such as Hsc70) were unaffected by the L115R mutation, whereas others (including SNAP-25 and STXBP1/Munc18-1) were inhibited. These results define the CSP interactome in a neuronal model cell line and reveal interactions that are affected by ANCL mutation and hence may contribute to the neurodegeneration seen in patients.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10903463/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139401634","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"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
The consequence of ATP synthase dimer angle on mitochondrial morphology studied by cryo-electron tomography. 利用低温电子断层扫描技术研究 ATP 合酶二聚体角度对线粒体形态的影响。
IF 4.1 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-01-02 DOI: 10.1042/BCJ20230450
Emma Buzzard, Mathew McLaren, Piotr Bragoszewski, Andrea Brancaccio, Holly Ford, Bertram Daum, Patricia Kuwabara, Ian Collinson, Vicki Gold

Mitochondrial ATP synthases form rows of dimers, which induce membrane curvature to give cristae their characteristic lamellar or tubular morphology. The angle formed between the central stalks of ATP synthase dimers varies between species. Using cryo-electron tomography and sub-tomogram averaging, we determined the structure of the ATP synthase dimer from the nematode worm C. elegans and show that the dimer angle differs from previously determined structures. The consequences of this species-specific difference at the dimer interface were investigated by comparing C. elegans and S. cerevisiae mitochondrial morphology. We reveal that C. elegans has a larger ATP synthase dimer angle with more lamellar (flatter) cristae when compared to yeast. The underlying cause of this difference was investigated by generating an atomic model of the C. elegans ATP synthase dimer by homology modelling. A comparison of our C. elegans model to an existing S. cerevisiae structure reveals the presence of extensions and rearrangements in C. elegans subunits associated with maintaining the dimer interface. We speculate that increasing dimer angles could provide an advantage for species that inhabit variable-oxygen environments by forming flatter more energetically efficient cristae.

线粒体 ATP 合成酶形成成排的二聚体,这些二聚体促使膜弯曲,从而使嵴呈现出特有的片状或管状形态。ATP 合成酶二聚体中心柄之间形成的角度因物种而异。利用低温电子断层扫描和子图平均法,我们确定了线虫C. elegans的ATP合成酶二聚体的结构,并表明二聚体的角度与之前确定的结构不同。通过比较线虫和麦角虫的线粒体形态,我们研究了二聚体界面上这种物种特异性差异的后果。我们发现,与酵母相比,秀丽隐杆线粒体的 ATP 合酶二聚体角度更大,嵴的片状(扁平)更多。我们通过同源建模生成了优雅子 ATP 合酶二聚体的原子模型,从而研究了造成这种差异的根本原因。将我们的秀丽隐杆线虫模型与现有的 S. cerevisiae 结构进行比较后发现,秀丽隐杆线虫亚基中存在与维持二聚体界面相关的延伸和重排。我们推测,增加二聚体角度可以形成更扁平、能量效率更高的嵴,从而为栖息在变氧环境中的物种提供优势。
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引用次数: 0
Up-regulation of SLC7A11/xCT creates a vulnerability to selenocystine-induced cytotoxicity SLC7A11/xCT 的上调使人容易受到硒胱氨酸诱导的细胞毒性的影响
IF 4.1 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-12-20 DOI: 10.1042/bcj20230317
Tan, Shawn Lu Wen, Tan, Hui Min, Israeli, Erez, Fatihah, Indah, Ramachandran, Vignesh, Ali, Shamsia Bte, Goh, Shane Jun An, Wee, Jillian, Tan, Alicia Qian Ler, Tam, Wai Leong, Han, Weiping
The SLC7A11/xCT cystine and glutamate antiporter has emerged as an attractive target for cancer therapy due to its selective overexpression in multiple cancers and its role in preventing ferroptosis. Utilizing pharmacological and genetic approaches in hepatocellular carcinoma cell lines, we demonstrate that overexpression of SLC7A11 engenders hypersensitivity towards l-selenocystine, a naturally occurring diselenide that bears close structural similarity to l-cystine. We find that the abundance of SLC7A11 positively correlates with sensitivity to l-selenocystine, but surprisingly, not to Erastin, an inhibitor of SLC7A11 activity. Our data indicate that SLC7A11 acts as a transport channel for l-selenocystine, which preferentially incites acute oxidative stress and damage eventuating to cell death in cells that highly express SLC7A11. Hence, our findings raise the prospect of l-selenocystine administration as a novel strategy for targeting cancers that up-regulate SLC7A11 expression.
由于 SLC7A11/xCT 胱氨酸和谷氨酸拮抗剂在多种癌症中的选择性过表达及其在防止铁变态反应中的作用,它已成为一个有吸引力的癌症治疗靶点。我们在肝癌细胞系中利用药理学和遗传学方法证明,过表达 SLC7A11 会导致对 l-硒代胱氨酸(一种天然存在的与 l-胱氨酸结构相似的二硒化物)过敏。我们发现 SLC7A11 的丰度与对 l-硒代胱氨酸的敏感性呈正相关,但令人惊讶的是,对 SLC7A11 活性抑制剂 Erastin 却不敏感。我们的数据表明,SLC7A11 是 l-硒代胱氨酸的转运通道,在高表达 SLC7A11 的细胞中,l-硒代胱氨酸会优先引发急性氧化应激和损伤,最终导致细胞死亡。因此,我们的研究结果提出了将施用 l-硒代胱氨酸作为靶向 SLC7A11 表达上调的癌症的新策略的前景。
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引用次数: 0
MicroRNA, miR-501 regulate the V(D)J recombination in B cells 微RNA、miR-501调控B细胞中的V(D)J重组
IF 4.1 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-12-20 DOI: 10.1042/bcj20230250
Kumari, Rupa, Roy, Urbi, Desai, Sagar, Mondal, Arannya S., Nair, Rajshree R., Nilavar, Namrata, Choudhary, Bibha, Raghavan, Sathees C.
The stringent regulation of RAGs (Recombination activating genes), the site-specific endonuclease responsible for V(D)J recombination, is important to prevent genomic rearrangements and chromosomal translocations in lymphoid cells. In the present study, we identify a microRNA, miR-501, which can regulate the expression of RAG1 in lymphoid cells. Overexpression of the pre-miRNA construct led to the generation of mature miRNAs and a concomitant reduction in RAG1 expression, whereas inhibition using anti-miRs resulted in its enhanced expression. The direct interaction of the 3′UTR of miR-501 with RAG1 was confirmed by the reporter assay. Importantly, overexpression of miRNAs led to inhibition of V(D)J recombination in B cells, revealing their impact on the physiological function of RAGs. Of interest is the inverse correlation observed for miR-501 with RAG1 in various leukemia patients and lymphoid cell lines, suggesting its possible use in cancer therapy. Thus, our results reveal the regulation of RAG1 by miR-501-3p in B cells and thus V(D)J recombination and its possible implications on immunoglobulin leukemogenesis.
RAGs(重组激活基因)是负责 V(D)J 重组的位点特异性内切酶,严格调控 RAGs 对防止淋巴细胞中的基因组重排和染色体易位非常重要。在本研究中,我们发现了一种能调节淋巴细胞中 RAG1 表达的 microRNA,即 miR-501。过量表达前 miRNA 构建物会导致成熟 miRNA 的产生,同时降低 RAG1 的表达,而使用抗 miRs 抑制则会导致其表达增强。报告实验证实了 miR-501 的 3′UTR 与 RAG1 的直接相互作用。重要的是,miRNAs 的过度表达导致抑制了 B 细胞中的 V(D)J 重组,揭示了它们对 RAGs 生理功能的影响。值得关注的是,在各种白血病患者和淋巴细胞系中观察到 miR-501 与 RAG1 呈反相关,这表明它可能用于癌症治疗。因此,我们的研究结果揭示了 miR-501-3p 在 B 细胞中对 RAG1 的调控,从而揭示了 V(D)J 重组及其对免疫球蛋白白血病发生的可能影响。
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引用次数: 0
Into the fold: advances in understanding aPKC membrane dynamics 进入折叠:了解 aPKC 膜动力学的进展
IF 4.1 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-12-20 DOI: 10.1042/bcj20230390
Cobbaut, Mathias, Parker, Peter J., McDonald, Neil Q.
Atypical protein kinase Cs (aPKCs) are part of the PKC family of protein kinases and are atypical because they don't respond to the canonical PKC activators diacylglycerol (DAG) and Ca2+. They are central to the organization of polarized cells and are deregulated in several cancers. aPKC recruitment to the plasma membrane compartment is crucial to their encounter with substrates associated with polarizing functions. However, in contrast with other PKCs, the mechanism by which atypical PKCs are recruited there has remained elusive until recently. Here, we bring aPKC into the fold, summarizing recent reports on the direct recruitment of aPKC to membranes, providing insight into seemingly discrepant findings and integrating them with existing literature.
非典型蛋白激酶 C(aPKC)是蛋白激酶 PKC 家族的一部分,之所以是非典型的,是因为它们对典型 PKC 激活剂二酰甘油(DAG)和 Ca2+ 没有反应。它们是极化细胞组织的核心,在几种癌症中都出现了失调。将 aPKC 招募到质膜区是它们遇到与极化功能相关的底物的关键。然而,与其他 PKCs 不同的是,非典型 PKCs 被招募到质膜区的机制直到最近仍不为人所知。在此,我们将 aPKC 纳入研究范围,总结了最近有关 aPKC 直接招募到膜上的报道,深入分析了看似不一致的发现,并将其与现有文献进行了整合。
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引用次数: 0
Sperm induce a secondary increase in ATP levels in mouse eggs that is independent of Ca2+ oscillations. 精子诱导小鼠卵子中ATP水平的二次增加,这与Ca2+振荡无关。
IF 4.1 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-12-20 DOI: 10.1042/BCJ20230065
Cindy Ikie-Eshalomi, Elnur Aliyev, Sven Hoehn, Tomasz P Jurkowski, Karl Swann

Egg activation at fertilization in mouse eggs is caused by a series of cytosolic Ca2+ oscillations that are associated with an increase in ATP concentrations driven by increased mitochondrial activity. We have investigated the role of Ca2+ oscillations in these changes in ATP at fertilization by measuring the dynamics of ATP and Ca2+ in mouse eggs. An initial ATP increase started with the first Ca2+ transient at fertilization and then a secondary increase in ATP occurred ∼1 h later and this preceded a small and temporary increase in the frequency of Ca2+ oscillations. Other stimuli that caused Ca2+ oscillations such as PLCz1 or thimerosal, caused smaller or slower changes in ATP that failed to show the distinct secondary rise. Sperm-induced Ca2+ oscillations in the egg also triggered changes in the fluorescence of NADH which followed the pattern of Ca2+ spikes in a similar pattern to oscillations triggered by PLCz1 or thimerosal. When eggs were loaded with low concentrations of the Ca2+ chelator BAPTA, sperm triggered one small Ca2+ increase, but there were still extra phases of ATP increase that were similar to control fertilized eggs. Singular Ca2+ increases caused by thapsigargin were much less effective in elevating ATP levels. Together these data suggest that the secondary ATP increase at fertilization in mouse eggs is not caused by increases in cytosolic Ca2+. The fertilizing sperm may stimulate ATP production in eggs via both Ca2+ and by another mechanism that is independent of PLCz1 or Ca2+ oscillations.

小鼠卵子受精时的卵子激活是由一系列细胞质Ca2+振荡引起的,该振荡与线粒体活性增加引起的ATP浓度增加有关。我们通过测量小鼠卵中ATP和Ca2+的动态,研究了Ca2+振荡在受精时ATP变化中的作用。初始的ATP增加始于受精时的第一次Ca2+瞬态,然后在大约1小时后发生ATP的二次增加,这先于Ca2+振荡频率的小而暂时的增加。其他刺激引起Ca2+振荡,如PLCz1或硫柳汞,引起ATP的较小或较慢的变化,未能显示出明显的继发性上升。精子诱导的卵子内Ca2+振荡也触发了NADH荧光的变化,这种变化遵循Ca2+峰值的模式,与PLCz1或硫柳汞引发的振荡模式相似。当卵子中含有低浓度的Ca2+螯合剂BAPTA时,精子触发了一个小的Ca2+增加,但仍然有与对照受精卵相似的额外阶段的ATP增加。由thapsigarin引起的单一Ca2+增加在提高ATP水平方面的效果要小得多。综上所述,这些数据表明,在小鼠卵子受精时,次级ATP的增加不是由细胞质Ca2+的增加引起的。受精精子可能通过Ca2+和另一种独立于PLCz1或Ca2+振荡的机制刺激卵子中ATP的产生。
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