HSP70 chaperones RNA-free TDP-43 into anisotropic intranuclear liquid spherical shells.
HSP70 chaperones RNA-free TDP-43 into anisotropic intranuclear liquid spherical shells.
复制标题
DOI：
10.1126/science.abb4309
复制
发表时间：
2021-02-05
期刊：
影响因子：
--
通讯作者：
Cleveland DW
中科院分区：
文献类型：
--
作者：
Yu H;Lu S;Gasior K;Singh D;Vazquez-Sanchez S;Tapia O;Toprani D;Beccari MS;Yates JR 3rd;Da Cruz S;Newby JM;Lafarga M;Gladfelter AS;Villa E;Cleveland DW
RNA结合蛋白TDP-43在年龄相关的神经退行性疾病中形成核内或细胞质聚集体。在这里，我们表明，RNA结合缺陷TDP-43（由神经变性引起的突变或翻译后乙酰化在其RNA识别基序）驱动TDP-43去混合成具有液体核心的核内液体球壳。我们建议将这些液滴命名为异相体，其外壳表现出双折射，这是液晶形成的证据。在数学建模的指导下，我们确定液体核心的主要成分是HSP 70家族分子伴侣，其ATP依赖性活性维持了壳和核心的流动性。体内神经元内的蛋白酶体抑制，以模拟衰老相关的蛋白酶体活性降低，诱导含TDP-43的球形壳，当ATP水平降低时，其转化为聚集体。因此，乙酰化、HSP 70和蛋白酶体活性调节TDP-43相分离和转化成凝胶/固相。RNA结合蛋白TDP-43（TAR DNA结合蛋白43）的聚集是几种年龄相关的神经退行性疾病（包括肌萎缩侧索硬化症（ALS）和额颞叶痴呆症（FTD））共有的共同病理学标志。占主导地位的核TDP-43通常经历液-液相分离（LLPS），其中均匀的溶液在两个隔室中分离，类似于醋中的油滴。核内TDP-43在生理条件下可发生相分离。细胞应激可诱导胞质TDP-43液滴，其可转变为固态，表明在神经变性中观察到的TDP-43聚集可由LLPS引发。驱动相分离和聚集的机制尚不清楚。我们确定了培养细胞和啮齿动物神经系统神经元中TDP-43相分离的关键调控机制。我们发现，RNA结合缺陷TDP-43，由ALS/FTD引起的突变或翻译后乙酰化在其RNA识别基序，相分离成异相体，液滴与对称的液体球形壳和液体核心。发现无RNA TDP-43以比周围核质高50倍的浓度富集在不等体壳中。乙酰化通过消除RNA与TDP-43的相互作用促进了异型体的形成。不等体壳表现出双折射，这是活细胞内蛋白质形成液晶区室的证据。壳是密集包装，确定与冷冻电子断层扫描，产生无膜，选择性屏障的一些核蛋白和RNA。我们的数学模型预测，各向异性体是由一个核心组件驱动的，该组件与TDP-43相互作用，弱结合，不结合RNA。在此指导下，我们使用邻近标记和定量蛋白质组学来鉴定HSP 70分子伴侣作为主要的异体型核心组分。HSP 70分子伴侣选择性结合并稳定RNA未结合的TDP-43。抑制HSP 70家族的ATP依赖性伴侣活性或降低细胞ATP水平诱导TDP-43异构体快速转化为均匀的凝胶。瞬时蛋白酶体抑制，模仿已知的蛋白酶体活性在衰老过程中的减少，引起TDP-43去混合成啮齿动物神经元中的异质体。死后ATP减少足以将异构体转化为类似于神经退行性疾病中发现的聚集体。我们确定了RNA结合蛋白TDP-43的相分离如何通过RNA结合、致病突变、翻译后修饰或细胞内的伴侣活性来调节。RNA结合缺陷型TDP-43去混合成异质体，其核心是“液体内液体内液体”。HSP 70家族的伴侣活性是维持异体型壳和核的流动性所必需的。当ATP水平下降时，各向异性体转化为蛋白质聚集体，这与患者脑组织中发现的病理性聚集体的前体一致。这些发现表明TDP-43和HSP 70分子伴侣之间在驱动RNA未结合的TDP-43相分离成异质体和防止TDP-43聚集方面的重要伙伴关系。
The RNA-binding protein TDP-43 forms intranuclear or cytoplasmic aggregates in age-related neurodegenerative diseases. Here we show that RNA-binding deficient TDP-43 (produced by neurodegeneration-causing mutations or post-translational acetylation in its RNA recognition motifs) drove TDP-43 de-mixing into intranuclear liquid spherical shells with liquid cores. We proposed the name anisosomes for these droplets, whose shells exhibited birefringence, evidence of liquid crystal formation. Guided by mathematical modeling, we identified the major components of the liquid core to be HSP70 family chaperones, whose ATP-dependent activity maintained the liquidity of shells and cores. In vivo proteasome inhibition within neurons, to mimic aging-related reduction of proteasome activity, induced TDP-43-containing spherical shells, which converted into aggregates when ATP levels were reduced. Thus, acetylation, HSP70, and proteasome activities regulate TDP-43 phase separation and conversion into a gel/solid phase. Aggregation of the RNA binding protein TDP-43 (TAR DNA-binding protein 43) is a common pathological hallmark shared by several age-related neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The predominantly nuclear TDP-43 normally undergoes Liquid-Liquid Phase Separation (LLPS), in which a homogenous solution separates in two compartments resembling oil droplets in vinegar. Intranuclear TDP-43 can phase separate under physiological conditions. Cellular stress can induce cytoplasmic TDP-43 liquid droplets, which can transition to a solid state, suggesting that TDP-43 aggregation observed in neurodegeneration could be initiated by LLPS. Mechanisms driving phase separation and aggregation were not known. We identified key regulatory mechanisms of TDP-43 phase separation in cultured cells and in neurons in the rodent nervous system. We found that RNA-binding deficient TDP-43, produced by ALS/FTD-causing mutations or post-translational acetylation in its RNA recognition motifs, phase-separated into anisosomes, droplets with symmetrical liquid spherical shells and liquid cores. RNA-free TDP-43 was found to be enriched in anisosomal shells at concentrations 50-fold higher than surrounding nucleoplasm. Acetylation promoted anisosomal formation by abolishing RNA interaction with TDP-43. Anisosomal shells exhibited birefringence, evidence of a liquid crystal compartment formed from proteins within living cells. Shells were densely packed, as determined with cryo-electron tomography, producing a membraneless, selective barrier to some nuclear proteins and RNAs. Our mathematical modeling predicted that anisosomes were driven by a core component that self-interacted, weakly bound TDP-43, and did not bind RNA. Guided by this, we used proximity labeling and quantitative proteomics to identify HSP70 chaperones as the major anisosomal core components. HSP70 chaperones selectively bound to and stabilized RNA-unbound TDP-43. Inhibiting ATP-dependent chaperone activity of the HSP70 family or reducing cellular ATP levels induced rapid conversion of TDP-43 anisosomes into uniform gels. Transient proteasome inhibition, mimicking the known reduction in proteasome activity during aging, provoked TDP-43 de-mixing into anisosomes in neurons in rodents. Postmortem ATP reduction was sufficient to convert anisosomes into aggregates similar to those found in neurodegenerative disease. We identified how phase separation of the RNA-binding protein TDP-43 can be regulated through RNA binding, disease-causing mutation, post-translational modification, or chaperone activity inside cells. RNA binding deficient TDP-43 de-mixed into anisosomes whose cores were a “liquid-inside-a-liquid-inside-a-liquid”. Chaperone activity of the HSP70 family was required to maintain liquidity of anisosomal shells and cores. When ATP levels fell, anisosomes converted into protein aggregates, consistent with being precursors of the pathological aggregates found in patient brain tissues. These findings suggest an essential partnership between TDP-43 and HSP70 chaperones in driving RNA-unbound TDP-43 phase separation into anisosomes and preventing TDP-43 aggregation.
登录
查看更多内容
DOI：
10.1016/j.mcn.2013.07.007
发表时间：
2013-09
期刊：
Molecular and cellular neurosciences
影响因子：
--
作者：
Burkhardt MF;Martinez FJ;Wright S;Ramos C;Volfson D;Mason M;Garnes J;Dang V;Lievers J;Shoukat-Mumtaz U;Martinez R;Gai H;Blake R;Vaisberg E;Grskovic M;Johnson C;Irion S;Bright J;Cooper B;Nguyen L;Griswold-Prenner I;Javaherian A
通讯作者：
Javaherian A
影响因子：
8.8
作者：
Flores, Brittany N.;Li, Xingli;Barmada, Sami J.
通讯作者：
Barmada, Sami J.
影响因子：
16.6
作者：
Cohen, Todd J.;Hwang, Andrew W.;Restrepo, Clark R.;Yuan, Chao-Xing;Trojanowski, John Q.;Lee, Virginia M. Y.
通讯作者：
Lee, Virginia M. Y.
影响因子：
64.8
作者：
Gallego, Laura D.;Schneider, Maren;Koehler, Alwin
通讯作者：
Koehler, Alwin
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.