Emerging evidence suggests the crucial roles of protein misfolding in the pathogenesis of amyotrophic lateral sclerosis (ALS). In particular, TAR DNA-binding protein 43kDa (TDP-43) mediates diverse upstream cascades, leading to motor neuron degeneration in ALS. Recent evidence strongly suggests that the mishandlings of the proteins turnover and RNA function are involved in the pathogenesis of ALS. In this sense, we have focused on the conformation dynamics of two RNA recognition motifs (RRM1 and RRM2) in TDP-43, attempting to explore a toxic conversion toward TDP43 proteinopathy. To identify the pathogenic cores of RRM1, a dominant RNA processing domain, we performed NMR monitoring of RRM1 protein subjected to high pressure stress. We found three crucial core regions in RRM1 misfolding, and its conformation is tightly regulated by the free state of cysteines (173 and 175) located in one core. Strikingly, the substitution of these cysteines showed structural and functional features of TDP-43 proteinopathy. Cysteine residues in RRM2 (C198 and C244) had no impact on its conformation. Interestingly, using our new monoclonal antibody, we found that D247 in RRM2 is exposed in the misfolded and unfolded conditions of TDP-43.
Using reversible covalent linked immunoprecipitation (ReCLIP) in the in vitro ubiquitination mixture, we identified von Hippel-Lindau (VHL), an adaptor protein in Culin2 (CUL2) E3 complex, as a TDP-43-interacting protein. VHL preferentially interacted misfolded forms of TDP-43, through the partial recognition of E246/D247 in RRM2 domain of TDP-43. However, VHL-CUL2 did not promote the ubiquitination or degradation of TDP-43. The overexpression of misfolded forms of TDP-43 sequestered VHL to form round inclusions, showing features of juxtanuclear quality control compartment (JUNQ). Our finding clearly indicates that the aberrant compartmentization of misfolded TDP-43 into JUNQ is associated with its interaction with VHL, possibly affecting the protein quality control system. The aberrantly exposed cores in misfolding TDP-43 may induce VHL-mediated inclusion formation in ALS proteinopathy.