| Abstract: | Membraneless organelles contain a wide spectrum of molecular chaperones, indicating their important roles in modulating the metastable conformation and biological function of membraneless organelles. Here we report that class I and II Hsp40 (DNAJ) proteins possess a high ability of phase separation rendered by the flexible G/F-rich region. Different Hsp40 proteins localize in different membraneless organelles. Specifically, human Hdj1 (DNAJB1), a class II Hsp40 protein, condenses in ubiquitin (Ub)-rich nuclear bodies, while Hdj2 (DNAJA1), a class I Hsp40 protein, condenses in nucleoli. Upon stress, both Hsp40 proteins incorporate into stress granules (SGs). Mutations of the G/F-rich region not only markedly impaired Hdj1 phase separation and SG involvement and disrupted the synergistic phase separation and colocalization of Hdj1 and fused in sarcoma (FUS) in cells. Being cophase separated with FUS, Hdj1 stabilized the liquid phase of FUS against proceeding into amyloid aggregation in vitro and alleviated abnormal FUS aggregation in cells. Moreover, Hdj1 uses different domains to chaperone FUS phase separation and amyloid aggregation. This paper suggests that phase separation is an intrinsic property of Hsp40 proteins, which enables efficient incorporation and function of Hsp40 in membraneless organelles and may further mediate the buildup of chaperone network in membraneless organelles.Membraneless organelles in cells are highly diverse and fulfill important biological functions under both normal and stress conditions (1, 2). Protein phase separation can drive the assembly of membraneless organelles, which incorporates hundreds of different proteins and nucleic acids (3–6). Membraneless organelles feature a physical property in between liquid and solid and present as a metastable state of complex supermolecular assembly (7). The maintenance of membraneless organelles in the liquidlike state rather than spontaneous solidification or dissociation is essential for membraneless organelles to fulfill their biological functions (8, 9). Failure of the conformational maintenance of membraneless organelles results in pathological protein aggregation, which is closely associated with various neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (10–14).Molecular chaperones are key components of the quality control system in cells to maintain protein homeostasis (proteostasis). They assist client proteins in folding and translocation and maintain clients in the native conformation avoiding misfolding and aggregation (15). Proteomic analysis of SGs has identified a wide spectrum of molecular chaperones, including Hsp40 (DNAJ), Hsp70, Hsp90, and small Hsps (5, 16), which indicate the importance of chaperones in the assembly and maintenance of membraneless organelles. Studies on yeast Hsp40 proteins (e.g., Sis1 and Ydj1) reveal that they accumulate in SGs and directly influence the assembly, dynamics and clearance of SGs (17). Human Hsp40 proteins, e.g., Hdj1 (DNAJB1) and Hdj2 (DNAJA1), cannot only inhibit the aggregation of a variety of amyloid proteins including α-synuclein, Τau, and polyQ in vitro (18–20) and rescue the cytotoxicity of polyQ and SG-associated RNA-binding proteins including fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43) in cellular and animal disease models (21–23). However, the mechanism of how Hsp40s rescue the cytotoxicity of these proteins remains unknown. Moreover, genetic defects of Hsp40 proteins have been identified in neurodegenerative diseases including ALS (24), Parkinson’s disease (25), cerebellar ataxia (26), and distal hereditary motor neuron neuropathy (27). These evidences suggest that Hsp40 proteins play an important role in maintaining the neuronal proteostasis by regulating protein assembly and preventing pathological amyloid aggregation.In this paper, we find that human class I and II Hsp40 proteins—Hdj2 and Hdj1 localize differently in nucleoli and Ub-rich nuclear bodies, respectively, under normal conditions. Under stress, both chaperones condense in SGs in the cytoplasm. We show that the close association of Hdj1 and Hdj2 with various membraneless organelles attributes to their phase separation property via the flexible G/F-rich region, which we show is a common property shared by class I and II Hsp40 proteins in different organisms. Furthermore, we find that Hdj1 synergistically phase separates with FUS and maintains the phase-separated state of FUS from proceeding to amyloid aggregation. Interestingly, Hdj1 employs distinctive mechanisms to chaperone the different states (i.e., soluble and phase-separated states) of FUS. This paper suggests that phase separation renders the localization and function of Hsp40 in various membraneless organelles, which may further recruit cochaperones (e.g., Hsp70) for the function and regulation of membraneless organelles. |
