Hsp70 and Hsp40 chaperones work synergistically in a wide range of biological processes including protein synthesis, membrane translocation, and folding. We used nuclear magnetic resonance spectroscopy to determine the solution structure and dynamic features of an Hsp40 in complex with an unfolded client protein. Atomic structures of the various binding sites in the client complexed to the binding domains of the Hsp40 reveal the recognition pattern. Hsp40 engages the client in a highly dynamic fashion using a multivalent binding mechanism that alters the folding properties of the client. Different Hsp40 family members have different numbers of client-binding sites with distinct sequence selectivity, providing additional mechanisms for activity regulation and function modification. Hsp70 binding to Hsp40 displaces the unfolded client. The activity of Hsp40 is altered in its complex with Hsp70, further regulating client binding and release.

TAR DNA binding protein of 43 kDa (TDP-43) is a nuclear factor functioning in RNA processing. It is also a major deposited protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin (FTLD-U). To understand the mechanism underlying the inclusion body formation and possible functional alteration, we studied some TDP-43 fragments and their effects on RNA processing in cell models. The results show that the 35-kDa fragment of TDP-43 (namely TDP-35, residues 90-414), but not TDP-25A (184-414), is capable of forming cytoplasmic inclusion bodies and altering pre-mRNA splicing. The inclusions formed by TDP-35 can also recruit full-length TDP-43 to cytoplasmic deposition from functionally nuclear localization. The in vitro studies demonstrate that TDP-35, rather than TDP-43 and TDP-25A, is prone to aggregation, and it further serves as a seed to facilitate aggregation of full-length TDP-43. This suggests that fragmentation of TDP-43 leads to cellular redistribution, inclusion body formation, and altered RNA processing, which are implicated in the molecular pathogenesis of ALS and FTLD.

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