Hsp40 proteins phase separate to chaperone the assembly and maintenance of membraneless organelles

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.

HspB8 prevents aberrant phase transitions of FUS by chaperoning its folded RNA-binding domain

“…Recent studies have provided evidence for a link between stress granules and small heat shock proteins (sHSPs) ( Mateju et al, 2017 ; Ganassi et al, 2016 ; Liu et al, 2020 ; Yu et al, 2021 ; Gu et al, 2020 ). These ATP-independent chaperones hold unfolded proteins in a refolding-competent state and both their intrinsically disordered region (IDR) and folded α-crystallin domain (αCD) contribute to this activity ( Haslbeck et al, 2019 ).…”

Liquid-liquid phase separation: Orchestrating cell signaling through time and space

“…Conversely, LLPS is also linked to dysregulated signaling in disease. LLPS of both PKA (Zhang et al, 2020) and the chaperone DNAJB1 (Gu et al, 2020) is required for proper signaling, whereas a fusion oncoprotein combining the J domain of DNAJB1 with PKA C disrupts RIa LLPS, which may underlie a rare form of liver cancer (Zhang et al, 2020). Similarly, the loss of LLPS by a mutant of the RNA-binding protein TDP43 (Conicella et al, 2020) may underlie abnormal activation of inflammatory signaling in amyotrophic lateral sclerosis (Yu et al, 2020).…”

Mechanisms and regulation underlying membraneless organelle plasticity control

“…This co-phase separation prevents FUS to carry out disease-associated amyloid aggregation. The co-LLPS among Tyr-rich FUS-LC, Arg-rich FUS-RGG, and Arg of N-terminal domain of Hdj1 is attributed to cation–π interactions ( Gu et al, 2020 ). Moreover, FUS, TATA-box binding protein associated factor 15 (TAF15), hnRNPA2, Ewing sarcoma (EWS) and cold-inducible RNA-binding protein can be assembled into hydrogels in vitro ( Kato et al, 2012 ; Kwon et al, 2013 ; Elbaum-Garfinkle et al, 2015 ), nonetheless, some of these proteins have to undergo LLPS first.…”