Stress granules (SGs) are dynamic condensates associated with protein misfolding diseases. They sequester stalled mRNAs and signaling factors, such as the mTORC1 subunit raptor, suggesting that SGs coordinate cell growth during and after stress. However, the molecular mechanisms linking SG dynamics and signaling remain undefined. We report that the chaperone Hsp90 is required for SG dissolution. Hsp90 binds and stabilizes the dual-specificity tyrosine-phosphorylation-regulated kinase 3 (DYRK3) in the cytosol. Upon Hsp90 inhibition, DYRK3 dissociates from Hsp90 and becomes inactive. Inactive DYRK3 is subjected to two different fates: it either partitions into SGs, where it is protected from irreversible aggregation, or it is degraded. In the presence of Hsp90, DYRK3 is active and promotes SG disassembly, restoring mTORC1 signaling and translation. Thus, Hsp90 links stress adaptation and cell growth by regulating the activity of a key kinase involved in condensate disassembly and translation restoration.

Three missense mutations targeting the same proline 209 (Pro209) codon in the co-chaperone Bcl2associated athanogene 3 (BAG3) have been reported to cause distal myopathy, dilated cardiomyopathy or Charcot-Marie-Tooth type 2 neuropathy. Yet, it is unclear whether distinct molecular mechanisms underlie the variable clinical spectrum of the rare patients carrying these three heterozygous Pro209 mutations in BAG3. Here, we studied all three variants and compared them to the BAG3_Glu455Lys mutant, which causes dilated cardiomyopathy. We found that all BAG3_Pro209 mutants have acquired a toxic gain-of-function, which causes these variants to accumulate in the form of insoluble HDAC6-and vimentin-positive aggresomes. The aggresomes formed by mutant BAG3 led to a relocation of other chaperones such as HSPB8 and Hsp70, which, together with BAG3, promote the so-called chaperoneassisted selective autophagy (CASA). As a consequence of their increased aggregation-proneness, mutant BAG3 trapped ubiquitinylated client proteins at the aggresome, preventing their efficient clearance. Combined, these data show that all BAG3_Pro209 mutants, irrespective of their different clinical phenotypes, are characterized by a gain-of-function that contributes to the gradual loss of protein homeostasis. Protein homeostasis is maintained by a complex network of molecular chaperones and co-chaperones providing protection to client proteins at every stage of their lifetime 1. As soon as a nascent polypeptide leaves the ribosomal exit tunnel, chaperones interact with exposed domains to facilitate protein folding 2. In case of protein misfolding, chaperones will either try to refold or guide the polypeptide towards degradation by proteasomes or the autophagy-lysosomal pathway 1. The activity of many chaperones is critically dependent on co-chaperones. One family of co-chaperones is represented by the Bcl2-associated athanogene (BAG) family of proteins, which in humans includes six members, encoded by 6 different genes 3. All six family members share a conserved BAG-domain, which is essential for their binding to the Hsp70 chaperones 4. BAG3 is a well-characterized family member that contains a number of additional protein domains besides the conserved BAG-domain, including two Ile-Pro-Val (IPV)-motifs, a PxxP domain and a WW-domain (Fig. 1a). Each of these domains is known to have specific interacting partners. For instance, the BAG-domain is known to mediate the interaction with Hsp70/Hsc70 or Bcl2 5-7. The IPV-motifs have been shown to be indispensable for binding to small heat shock proteins (sHSPs) 8 , the WW-domain binds LATS1 9 , and the PxxP domain is necessary for the interaction with dynein and PLC-γ 10,11. The sHSP with the highest affinity for the IPV-motifs of BAG3 is HSPB8 (Hsp22) 12,13. In fact, the protein stability of HSPB8 is critically dependent on BAG3, as it is rapidly degraded in its absence 14. As also other members of the HSPB family are capable of binding to BAG3, it is thought that in case HSPB8 would be unable to fulfil its

One of the critical events that regulates muscle cell differentiation is the replacement of the lamin B receptor (LBR)-tether with the lamin A/C (LMNA)-tether to remodel transcription and induce differentiation-specific genes. Here, we report that localization and activity of the LBR-tether are crucially dependent on the muscle-specific chaperone HSPB3 and that depletion of HSPB3 prevents muscle cell differentiation. We further show that HSPB3 binds to LBR in the nucleoplasm and maintains it in a dynamic state, thus promoting the transcription of myogenic genes, including the genes to remodel the extracellular matrix. Remarkably, HSPB3 overexpression alone is sufficient to induce the differentiation of two human muscle cell lines, LHCNM2 cells, and rhabdomyosarcoma cells. We also show that mutant R116P-HSPB3 from a myopathy patient with chromatin alterations and muscle fiber disorganization, forms nuclear aggregates that immobilize LBR. We find that R116P-HSPB3 is unable to induce myoblast differentiation and instead activates the unfolded protein response. We propose that HSPB3 is a specialized chaperone engaged in muscle cell differentiation and that dysfunctional HSPB3 causes neuromuscular disease by deregulating LBR.

Background and aims: Sphingosine-1 phosphate (S1P) is a lysosphingolipid present in the ovarian follicular fluid. The role of the lysosphingolipid in gonads of the female is widely unclear. At nanomolar concentrations, S1P binds and activates five specific G protein-coupled receptors (GPCRs), known as S1P 1-5 , modulating different signaling pathways. S1P 1 and S1P 3 are highly expressed in human primary granulosa lutein cells (hGLC), as well as in the immortalized human primary granulosa cell line hGL5. In this study, we evaluated the signaling cascade activated by S1P and its synthetic analogues in hGLC and hGL5 cells, exploring the biological relevance of S1PRstimulation in this context. METHODS AND RESULTS. hGLC and hGL5 cells were treated with a fixed dose (0.1 μM) of S1P, or by S1P 1 -and S1P 3 -specific agonists SEW2871 and CYM5541. In granulosa cells, S1P and, at a lesser extent, SEW2871 and CYM5541, potently induced CREB phosphorylation. No cAMP production was detected and pCREB activation occurred even in the presence of the PKA inhibitor H-89. Moreover, S1Pdependent CREB phosphorylation was dampened by the mitogen-activate protein kinase (MEK) inhibitor U0126 and by the L-type Ca 2+ channel blocker verapamil. The complete inhibition of CREB phosphorylation occurred by blocking either S1P 2 or S1P 3 with the specific receptor antagonists JTE-013 and TY52156, or under PLC/PI3K depletion. S1P-dependent CREB phosphorylation induced FOXO1 and the EGF-like epiregulinencoding gene (EREG), confirming the exclusive role of gonadotropins and interleukins in this process, but did not affect steroidogenesis. However, S1P or agonists did not modulate granulosa cell viability and proliferation in our conditions. Conclusions: This study demonstrates for the first time that S1P may induce a cAMP-independent activation of pCREB in granulosa cells, although this is not sufficient to induce intracellular steroidogenic signals and progesterone synthesis. S1P-induced FOXO1 and EREG gene expression suggests that the activation of S1P-S1PR axis may cooperate with gonadotropins in modulating follicle development.

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