CHMP7

引言 DNA 修复和自噬是维持细胞生存的两个关键生物学过程。DNA修复负责修复DNA损伤，确保基因组稳定；而自噬则负责清除细胞内多余的、受损的、错误折叠或聚集的蛋白质和细胞器，维持细胞稳态。拓扑异构酶 I (TOP1) 在DNA复制和转录中起着至关重要的作用，它通过切断DNA形成超螺旋结构，释放扭转应力。然而，TOP1与DNA形成的共价键——TOP1cc是一种有害的DNA损伤，会导致基因组不稳定和细胞死亡。TOP1cc修复主要依赖于酪氨酸DNA磷酸二酯酶 1 (TDP1) 介导的切除修复或MRE11核酶介导的DNA切割和切除修复【1】。然而，TOP1cc的修复过程与蛋白质降解途径的关系尚不清楚。自噬因子在DNA复制叉附近富集，表明自噬可能在DNA损伤修复中发挥作用。TEX264是一种保守的蛋白质，既是自噬受体，也与TOP1cc修复有关【2】。然而，TEX264是否通过自噬介导TOP1cc修复，目前尚不清楚。 近日，来自英国牛津大学拉德克利夫医院的Kristijan Ramadan研究团队在Cell杂志上发表题为TEX264 drives selective autophagy of DNA lesions to promote DNA repair and cell survival的研究论文，该研究发现了TEX264介导的核吞噬作用在维持基因组稳定性和细胞存活中发挥关键作用。 首先通过免疫共沉淀和蛋白质组学分析，研究人员发现自噬相关蛋白（如Beclin-1、TEX264、CHMP7、ATG7、SNAP29）在DNA复制叉处富集，并且这种富集在CPT（诱导DNA复制压力的化合物）处理后被显著增强，这表明自噬可能在响应 DNA 复制压力时发挥作用。接着，研究人员使用LysoIP技术分离溶酶体，并通过质谱分析其蛋白质组成。结果表明，CPT 处理后，许多DNA复制和核蛋白（如DNA聚合酶、MCM复合物蛋白、核孔复合物）被运送到溶酶体，表明溶酶体可能直接参与 DNA 损伤的清除。 进一步，研究人员发现用CPT处理后，TOP1cc水平的恢复受到mTOR抑制剂Torin的促进，而自噬抑制剂ATG7的缺失则完全阻止了TOP1cc的修复，证明自噬是TOP1cc修复的关键途径。其他自噬相关蛋白，如syntaxin 17和RB1CC1，也参与了TOP1cc的降解和细胞对CPT的存活，说明自噬修复TOP1cc的过程是一个复杂的网络，涉及多个自噬相关蛋白。使用mCherry-TOP1-GFP报告系统证实了TOP1在低剂量CPT处理后进入溶酶体，表明 TOP1cc可以直接被溶酶体降解。 通过分析γ-H2AX 和53BP1的定位，研究人员发现低剂量CPT诱导的DNA损伤主要表现为复制压力，而不是DNA 双链断裂。此外，研究人员发现ATR抑制剂VE-822强烈抑制了TOP1cc的运送，而ATM抑制剂KU-55933没有影响，这表明ATR在响应DNA复制压力和促进 TOP1cc 降解中起着关键作用。自噬抑制剂ATG7和syntaxin 17的缺失加剧了低剂量 CPT 处理后蛋白质聚集物的形成，说明自噬在防止蛋白质聚集物形成中发挥作用。 最后，研究人员发现MRE11核酶的缺失强烈抑制了TOP1cc的运送，而MUS81和TDP1的缺失没有影响，表明MRE11核酶在将TOP1cc从核内运送到溶酶体中起着关键作用。使用 LysoIP-Seq技术分离溶酶体中的DNA片段，并通过测序分析其序列特征。结果表明，溶酶体中分离的DNA片段主要来自核内 DNA，且大部分来自内含子和着丝粒区域，表明溶酶体可以清除整个TOP1cc损伤，包括其蛋白质部分和相关的DNA片段。使用 TOP1cc 特异性抗体和LysoIP等技术证实了TOP1cc在低剂量CPT处理后在细胞质中的存在。这表明 TOP1cc可以从核内被运送到细胞质，并被溶酶体降解。 图1 TEX264驱动的选择性自噬促进DNA修复和细胞存活（Credit: Cell） 总之，这项研究证明了选择性自噬在DNA修复中的进化保守作用，并表明它对维持基因组稳定性和细胞存活至关重要。此外，该发现还表明，在治疗结直肠癌患者时， TEX264的表达水平与对伊立替康等TOP1抑制剂的化疗反应相关。 参考文献 1. Pommier, Y. (2006). Topoisomerase I inhibitors: camptothecins and beyond. Nat. Rev. Cancer 6, 789–802. https://doi-org.libproxy1.nus.edu.sg/10.1038/nrc1977.7. Stingele, J., Bellelli, R., and Boulton, S.J. (2017). Mechanisms of DNA–protein crosslink repair. Nat. Rev. Mol. Cell Biol. 18, 563–573. 2. Fielden, J., Wiseman, K., Torrecilla, I., Li, S., Hume, S., Chiang, S.-C., Ruggiano, A., Narayan Singh, A., Freire, R., Hassanieh, S., et al. (2020). TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adducts. Nat. Commun. 11, 1274. https://www-cell-com.libproxy1.nus.edu.sg/cell/fulltext/S0092-8674(24)00911-5 责编|探索君 排版|探索君 文章来源|“BioArt” End 往期精选 围观 一文读透细胞死亡(Cell Death) | 24年Cell重磅综述（长文收藏版） 热文 Cell | 是什么决定了细胞的大小？ 热文 Nature | 2024年值得关注的七项技术 热文 Nature | 自身免疫性疾病能被治愈吗？科学家们终于看到了希望 热文 CRISPR技术进化史 | 24年Cell综述

*仅供医学专业人士阅读参考染色体不稳定性（CIN）是癌细胞的一大特征。染色体的不稳定性会导致包裹碎裂DNA的微核产生。在很长的一段时间里，科学家认为微核仅仅是染色体不稳定的副产物。然而，近年来，科学家逐渐发现微核没那么简单。他们注意到，微核有个特点，与真正的细胞核相比，微核不仅非常容易破裂，而且一旦破裂还不能像细胞核那样完成修复。如此以来，微核中包裹的DNA碎片就会释放出来，再次造成癌细胞的DNA损伤和染色体重排，从而加剧癌细胞基因组的不稳定性，帮助癌细胞进化，最终促进癌症耐药和进展。此外，微核释放到细胞质中的DNA，还会激活cGAS-STING通路，引发炎症反应，促进癌症转移。不难看出，微核是癌细胞促进自身不断进化，让自己变得更强大的重要工具。不得不说，癌细胞为了生存，对自己下手也真是有点儿狠了。虽然微核促进细胞进化升级的原因差不多搞清楚了，但是微核为啥那么容易破裂，目前科学家还知之甚少。近日，由意大利欧洲癌症研究所Stefano Santaguida领衔的团队[1]，以及美国纪念斯隆-凯特琳癌症中心Samuel F. Bakhoum领衔的研究团队[2]，以封面论文的形式，在顶级期刊《科学》上背靠背发表了两篇重磅研究成果，从两个方面解开了谜题。他们发现，癌细胞里微核附近的线粒体产生的活性氧，一方面会促进微核包膜的破裂，另一方面还会通过另一套机制，促进微核包膜修复蛋白的降解，加速微核的破裂。既然发现了微核易碎裂的原因，那么染色体不稳定性特征明显的癌症就有望迎来新治疗靶点了。▲ 论文封面：微核附近线粒体（金色卵形体）释放活性氧（金色光芒），招募自噬受体p62（红色），促加速微核包膜（灰色）破裂在开始介绍这两项研究成果之前，要简单交代一下细胞核膜的修复机制。在很多生理或病理背景下，细胞核膜常常会破裂，因此要维持细胞的正常功能的话，细胞核膜破裂之后就得立即修复。在漫长的进化过程中，哺乳动物体内已经有了一套细胞核膜修复的机制。这个修复过程设计三个重要蛋白：介导核膜修复的关键蛋白ESCRT-III复合物，把ESCRT-III复合物锚定在破损部位的两个重要支架蛋白CHMP7和LEMD2。三者互相配合，是细胞核膜修复的关键。接下来，咱们先来看看纪念斯隆-凯特琳癌症中心Samuel F. Bakhoum团队的研究成果。▲ Bakhoum团队论文首页截图他们在观察癌细胞的时候发现，微核和线粒体所在的位置存在重叠，而且与线粒体长时间接触的微核更容易破裂。这让他们想到，微核的破裂可能与线粒体产生的活性氧有关。后续实验证明，事实确实如此，直接加入活性氧会促进微核的破裂，而抑制活性氧产生的物质（NAC等）会抑制微核的破裂。究其背后原因，Bakhoum团队发现，在缺氧的条件下，癌细胞线粒体释放的活性氧会促进CHMP7在微核包膜中的异常定位/聚集，并抑制微核包膜中CHMP7的磷酸化，促进CHMP7与LEMD2通过“病理性相互作用”机制形成寡聚体，从而使微核包膜变形、塌陷。▲ Bakhoum团队发现的机制示意图欧洲癌症研究所Stefano Santaguida团队的出发点跟Bakhoum团队不一样。他们先在人类细胞系中诱导出微核，然后分离出微核和细胞核，并分析二者的蛋白组成。在分析对比之后，他们发现在自噬途径中识别泛素化结构的自噬受体p62/SQSTM1（后文简称p62），在微核中显著上调。随后，基于多种成像手段，Santaguida团队证实，p62定位于微核腔内。▲ Santaguida团队论文首页截图随后的机制研究表明，微核在靠近线粒体之后，活性氧会促进p62同源寡聚化，紧接着p62会介导膜修复蛋白ESCRT-III复合物的自噬降解，抑制已经破损的微核包膜修复，进而加速微核的破裂。▲ Santaguida团队发现的机制示意图同期发表在《科学》杂志上的评论文章指出，这两项研究表明，损害微核包膜完整性和破坏ESCRT-III复合体的机制是并存的，二者都会导致灾难性的微核包膜破损[3]。值得注意的是，这两种机制都是由位于微核附近线粒体产生的活性氧触发的。总的来说，Stefano Santaguida团队和Samuel F. Bakhoum团队的这两项研究成果，让我们对微核促进癌症进展有了新的认知，有望为癌症的治疗提供新靶点。奇点糕近期推出了《2024ASCO年会深度盘点》课程，为大家系统盘点了2024ASCO年会上实体瘤领域的重磅进展，让您在100分钟内迅速深入把握前沿。长按识别下图中的二维码即可购买，购买后您可以在「奇点网小程序」收听，或下载奇点网「医学奇点」苹果客户端收听。如果遇到任何技术问题，大家可以戳我们下图中的客服小伙伴来解决~~参考文献：[1].Sara Martin et al., A p62-dependent rheostat dictates micronuclei catastrophe and chromosome rearrangements. Science 385, eadj7446. 2024. doi:10.1126/science.adj7446[2].Melody Di Bona et al.,Micronuclear collapse from oxidative damage. Science 385, eadj8691. 2024. doi:10.1126/science.adj8691[3].Marianna Maddaluno, Carmine Settembre ,Micronuclear collapse mechanisms in cancer. Science 385,930-931. 2024. doi:10.1126/science.adr7417本文作者丨BioTalker

A hallmark of cancer cells is aneuploidy, when a cell has too few or too many chromosomes. This happens during cell division, when chromosomes can get left behind and not incorporated into the nucleus of a new daughter cell. In 2022, on the alpine shores of Lake Maggiore in northern Italy, longtime friends Samuel Bakhoum, M.D., Ph.D., and Stefano Santaguida, Ph.D., were catching up. Pandemic restrictions had prevented the two from seeing each other for years, but, at a small conference in the lakeside town of Stresa, they seized the opportunity to share what they’d been working on recently—the collapse of micronuclei in cancer cells.“We were so happy and excited to be together in real life,” Santaguida, a molecular biologist at the European Institute of Oncology and the University of Milan, told Fierce Biotech in an interview. “We were working on two separate aspects at the very beginning, without knowing of each other’s work.”Teaming up to tackle the problem has now resulted in two papers published Aug. 29 in the journal Science. The studies explain in mechanistic detail how micronuclei rupture and collapse in cancer cells, an important process that drives cancer progression. By getting tangled with mitochondria, micronuclei are exposed to reactive oxygen species that alter the behavior of a key repair protein and over-activate a protein-destroying pathway. A hallmark of cancer cells is aneuploidy, when a cell has too few or too many chromosomes. This happens during cell division, when chromosomes can get left behind and not incorporated into the nucleus of a new daughter cell. A stranded chromosome forms a shoddy nuclear membrane around itself, which is much smaller and less functional than the main nuclear envelope that surrounds the nucleus. These micronuclei can then break apart, leaving the chromosome exposed to the cellular elements.“That rupture process underlies a lot of the means by which micronuclei seem to drive cancer progression,” Bakhoum, a cell biologist at Memorial Sloan Kettering Cancer Center in New York City, told Fierce in an interview.Enzymes in the cytoplasm break chunks off the free-floating DNA, leading to mutations that can spur new cancer traits or alter how genes are expressed. And the cell can also confuse the loose chromosome for a virus, sparking inflammation that can lead the tumor to evolve immune resistance.Melody Di Bona, Ph.D., a cancer biologist working in Bakhoum’s group, was observing cervical cancer cells under the microscope when she noticed that micronuclei tended to get tangled up with mitochondria. “My hypothesis when I started to look at cells was that mitochondria were actually entering the micronucleus,” Di Bona told Fierce in a joint interview with Santaguida.Investigating further, she instead found that the chance of rupture greatly increased as the micronucleus was exposed to more and more toxic forms of oxygen-containing molecules leaking from the mitochondria, called reactive oxygen species (ROS). Through a series of experiments, she pieced together how the membrane unraveling unfolds.Tumors tend to be low on oxygen, which the mitochondria normally need to function; when oxygen is low, nitric oxide binds in its place instead, which produces ROS as a byproduct. ROS are unstable and can steal electrons from other molecules, messing with their forms and functions. Di Bona found that ROS take electrons away from amino acids called cysteines in the protein CHMP7, changing its form so that it abnormally binds to another protein called LEMD2 that sits inside the micronuclear membrane. What’s more, ROS damage also prevents CHMP7 from being kicked out of the cell.“LEMD2 is already in the membrane,” Bakhoum said. “And now imagine a protein that's aggregating inside, right underneath, and pulling it away.” The force of CHMP7 tugging at LEMD2 causes the micronucleus’s membrane to collapse and rupture.Di Bona first looked at CHMP7 because it’s normally a key part of a nuclear membrane repair enzyme called ESCRT-III. But this enzyme is prevented from patching up the rupturing micronucleus due to the actions of another protein called p62.“p62 is a protein that has its own life,” Santaguida said. “It works in the cells by recognizing proteins or organelles that have to be degraded,” targeting them for a process called autophagy.Santaguida’s team found that ROS from mitochondria also alter p62, ramping up its activity to send proteins down the autophagy pathway. This overreactive p62 degrades the repair enzyme ESCRT-III, further dooming the micronuclear membrane to collapse. To connect p62 with cancer prognosis, the team analyzed data from the tumors of 875 gastric cancer patients and found that higher levels of p62 were correlated with shortened survival.“The higher the levels of p62, the higher the likelihood to get a worse prognosis,” Santaguida said. “It's a prognostic marker that can be employed in the clinic tomorrow.” In addition to the prognostic power of p62, the collapse mechanism presents several other potential targets for new drugs. Therapies could target cysteines in CHMP7 and push them away from a pathological path, Bakhoum said. And numerous other molecules play bit parts in the process too, like kinases in the nuclear factor κB pathway—targeting them could reduce the odds of micronuclei rupturing and causing cancer progression.Chromosomes in cancer cells are starting to attract more attention from biotechs. Bakhoum is a co-founder and scientific adviser of Volastra Therapeutics—a biotech focused on chromosomal instability—and also serves on the scientific advisory board of precision oncology company Meliora Therapeutics.“We can't suppress chromosome instability [in cancer] because the cat's out of the bag, but can we at least kneecap it?” Bakhoum said. “Can we intervene in a way that prevents all the bad consequences and sequela of micronuclear rupture?”