Characterizing the function of SUMO-targeted ubiquitin networks in coordinating nuclear and cytosolic protein quality control pathways

在有膜或无膜细胞器中划分细胞过程，如核PML小体（NBs）和副斑或胞质应激颗粒（SGs），是生化途径的一个关键概念。无膜细胞器具有RNA或蛋白质质量控制中心的功能，其形成、分解和组成对细胞应激高度敏感。一个例子是胞质应激颗粒，它由未翻译的mrna和rna结合蛋白组成，在热或氧化还原应激下在胞质中组装。核rna结合蛋白，包括FUS和TDP-43，被招募到SGs，但在应激释放和SG分解后，被重新安置到细胞核中。在神经退行性疾病，如肌萎缩性侧索硬化症（ALS）和额颞叶变性（FTLD）中，TDP-43或FUS的致病突变可诱导持续应激颗粒的形成。蛋白毒性应激和TDP-43或FUS突变体的表达也会触发核颗粒中这两种蛋白的核池富集，这表明细胞核和细胞质膜细胞器的动力学与细胞应激反应密切相关。然而，许多方面，如翻译后修饰在这些过程中的参与，仍然不清楚。此外，胞质应力颗粒与核凝聚物的相互联系正在出现，但这些相互联系在正常和病理条件下的潜在分子基础仍然是谜。我们证明了sumo靶向的泛素连接酶（StUbL）途径在应激释放时参与了应激颗粒的分解。在响应细胞应激诱导的StUbL途径中，蛋白质最初被聚合SUMO链标记，从而触发Ub连接酶RNF4的泛素化。该通路被认为是一个与PML NBs相关的核蛋白质量控制系统。我们现在发现，在释放胁迫时，SGs的适当分解需要SUMO途径的关键成分，如修饰剂SUMO2、SUMO连接酶PIAS1、StUbL RNF4以及NB支架蛋白PML。这些数据揭示了迄今为止未知的SUMO信号与SG清除的相互作用，以及SG和PML NBs之间前所未有的相互作用。此外，我们证明了StUbL信号控制FUS和TDP-43的ALS/ ftdl相关突变体的泛素化和区隔化。在这里，我们的目标是(i)剖析SUMO/StUbL调控的SGs和PML NB的动力学；（ii）了解SGs和NB在蛋白质静止网络中SUMO- rnf4依赖的串扰；（iii）定义StUbL通路中SUMO和Ub脱jujugs的应激调节；（iv）揭示SUMO信号传导对致病性TDP-43突变体的影响。更好地了解这些过程将提高我们对神经退行性疾病中蛋白质平衡紊乱的理解，并为治疗干预开辟新的途径。

Compartmentalizing cellular processes in membranous or membrane-less organelles, such as nuclear PML bodies (NBs) and paraspeckles or cytosolic stress granules (SGs), is a key concept of biochemical pathways. Membrane-less organelles function as RNA or protein quality control centers and their formation, disassembly and composition are highly sensitive to cellular stress. An example are cytosolic stress granules, which are composed of untranslated mRNAs and RNA-binding proteins and assemble in the cytosol in response to heat or redox stress. Nuclear RNA-binding proteins, including FUS and TDP-43, are recruited to SGs, but upon stress release and SG disassembly are relocated to the nucleus. In neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), pathogenic mutants of TDP-43 or FUS can induce formation of persistant stress granules. Proteotoxic stress and expression of TDP-43 or FUS mutants also trigger enrichment of the nuclear pool of both proteins in nuclear granules demonstrating that the dynamics of both nuclear and cytosolic membrane less-organelles is tightly connected to the cellular stress response. However, many aspects, such as involvement of post-translational modifications in these processes, have remained unclear. Further, interconnections of cytosolic stress granules with nuclear condensates are emerging, but the underlying molecular basis of these interconnections under normal and pathological conditions have remained enigmatic. We demonstrated that the SUMO-targeted ubiquitin ligase (StUbL) pathway is critically involved in the disassembly of stress granules upon stress release. In the StUbL pathway, which is induced in response to cellular stress, proteins are initially marked with polymeric SUMO chains thereby triggering their subsequent ubiquitylation by the Ub ligase RNF4. The pathway is considered as a nuclear protein quality control system, which is associated with PML NBs. We now found that the proper disassembly of SGs upon release from stress requires key component of the SUMO pathway, such as the modifier SUMO2, the SUMO ligase PIAS1, the StUbL RNF4 as well as the NB scaffold protein PML. The data reveal a so far unknown interconnection of SUMO signaling with SG clearance and an unprecedented interplay between SGs and PML NBs. Additionally, we demonstrate that StUbL signaling controls ubiquitylation and compartmentalization of ALS/FTDL-associated mutants of FUS and TDP-43. Here we aim to (i) dissect the SUMO/StUbL-regulated dynamics of SGs and PML NB (ii) understand the SUMO-RNF4-dependent crosstalk of SGs and NBs in proteostasis networks (iii) Define stress regulation of SUMO and Ub deconjugases in the StUbL pathway (iv) unveil the impact of SUMO signaling on pathogenic TDP-43 mutants.A better knowledge of these processes will improve our understanding of disrupted proteostasis in neurodegeneration and open up new avenues for therapeutic intervention.