Activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in the G 2 /M cell cycle arrest induced by DNA damage, but little is known about the role of this signaling pathway in the G 1 /S transition. Upregulation of the cyclin-dependent kinase inhibitor p21Cip1 is thought to make a major contribution to the G 1 /S cell cycle arrest induced by ␥ radiation. We show here that inhibition of p38 MAPK impairs p21Cip1 accumulation and, as a result, the ability of cells to arrest in G 1 in response to ␥ radiation. We found that p38 MAPK induces p21Cip1 mRNA stabilization, without affecting its transcription or the stability of the protein. In particular, p38 MAPK phosphorylates the mRNA binding protein HuR on Thr118, which results in cytoplasmic accumulation of HuR and its enhanced binding to the p21 Cip1 mRNA. Our findings help to understand the emerging role of p38 MAPK in the cellular responses to DNA damage and reveal the existence of p53-independent networks that cooperate in modulating p21Cip1 levels at the G 1 /S checkpoint.

TDP-43 aggregation in the cytoplasm or nucleus is a key feature of the pathology of amyotrophic lateral sclerosis and frontotemporal dementia and is observed in numerous other neurodegenerative diseases, including Alzheimer's disease. Despite this fact, the inciting events leading to TDP-43 aggregation remain unclear. We observed that endogenous TDP-43 undergoes reversible aggregation in the nucleus after the heat shock and that this behavior is mediated by the C-terminal prion domain. Substitution of the prion domain from TIA-1 or an authentic yeast prion domain from RNQ1 into TDP-43 can completely recapitulate heat shock-induced aggregation. TDP-43 is constitutively bound to members of the Hsp40/Hsp70 family, and we found that heat shock-induced TDP-43 aggregation is mediated by the availability of these chaperones interacting with the inherently disordered C-terminal prion domain. Finally, we observed that the aggregation of TDP-43 during heat shock led to decreased binding to hnRNPA1, and a change in TDP-43 RNA-binding partners suggesting that TDP-43 aggregation alters its function in response to misfolded protein stress. These findings indicate that TDP-43 shares properties with physiologic prions from yeast, in that self-aggregation is mediated by a Q/N-rich disordered domain, is modulated by chaperone proteins and leads to altered function of the protein. Furthermore, they indicate that TDP-43 aggregation is regulated by chaperone availability, explaining the recurrent observation of TDP-43 aggregates in degenerative diseases of both the brain and muscle where protein homeostasis is disrupted.

Background: Mutations in the G/F domain of the human HSP40 chaperone DNAJB6 cause a protein aggregate myopathy. Results: Homologous mutations in the yeast HSP40 Sis1 alter prion propagation in a prion strain-dependent manner. Conclusion: G/F domain mutations affect chaperone-mediated processing of particular client conformers. Significance: Impaired processing of certain substrate conformations may be one mechanism that contributes to chaperonopathy pathogenesis.

BAG3 is a multi-domain hub that connects two classes of chaperones, small heat shock proteins (sHSPs) via two isoleucine-proline-valine (IPV) motifs and Hsp70 via a BAG domain. Mutations in either the IPV or BAG domain of BAG3 cause a dominant form of myopathy, characterized by protein aggregation in both skeletal and cardiac muscle tissues. Surprisingly, for both disease mutants, impaired chaperone binding is not sufficient to explain disease phenotypes. Recombinant mutants are correctly folded, show unaffected Hsp70 binding but are impaired in stimulating Hsp70-dependent client processing. As a consequence, the mutant BAG3 proteins become the node for a dominant gain of function causing aggregation of itself, Hsp70, Hsp70 clients and tiered interactors within the BAG3 interactome. Importantly, genetic and pharmaceutical interference with Hsp70 binding completely reverses stress-induced protein aggregation for both BAG3 mutations. Thus, the gain of function effects of BAG3 mutants act as Achilles heel of the HSP70 machinery.

Limb-girdle muscular dystrophy type 1D (LGMD1D) is caused by dominantly inherited missense mutations in DNAJB6, an Hsp40 co-chaperone. LGMD1D muscle has rimmed vacuoles and inclusion bodies containing DNAJB6, Z-disc proteins and TDP-43. DNAJB6 is expressed as two isoforms; DNAJB6a and DNAJB6b. Both isoforms contain LGMD1D mutant residues and are expressed in human muscle. To identify which mutant isoform confers disease pathogenesis and generate a mouse model of LGMD1D, we evaluated DNAJB6 expression and localization in skeletal muscle as well as generating DNAJB6 isoform specific expressing transgenic mice. DNAJB6a localized to myonuclei while DNAJB6b was sarcoplasmic. LGMD1D mutations in DNAJB6a or DNAJB6b did not alter this localization in mouse muscle. Transgenic mice expressing the LGMD1D mutant, F93L, in DNAJB6b under a muscle-specific promoter became weak, had early lethality and developed muscle pathology consistent with myopathy after 2 months; whereas mice expressing the same F93L mutation in DNAJB6a or overexpressing DNAJB6a or DNAJB6b wild-type transgenes remained unaffected after 1 year. DNAJB6b localized to the Z-disc and DNAJB6b-F93L expressing mouse muscle had myofibrillar disorganization and desmin inclusions. Consistent with DNAJB6 dysfunction, keratin 8/18, a DNAJB6 client also accumulated in DNAJB6b-F93L expressing mouse muscle. The RNA-binding proteins hnRNPA1 and hnRNPA2/B1 accumulated and co-localized with DNAJB6 at sarcoplasmic stress granules suggesting that these proteins maybe novel DNAJB6b clients. Similarly, hnRNPA1 and hnRNPA2/B1 formed sarcoplasmic aggregates in patients with LGMD1D. Our data support that LGMD1D mutations in DNAJB6 disrupt its sarcoplasmic function suggesting a role for DNAJB6b in Z-disc organization and stress granule kinetics.