J-domain proteins as modulators of amyloid protein aggregation involved in amyotrophic lateral sclerosis

Investigating the molecular mechanisms and the proteins implicated in the regulation of aggregation, protein misfolding, and the stabilization of native conformations is pertinent for prospective biological advancements. Efforts have been focused on understanding the implications of HSP70 and HSP90 chaperones in TDP-43 pathophysiology as their interplay is key in correct protein regulation in several diseases such as ALS. However, although they have recently emerged as leaders in protein chaperoning, the implications of the HSP40 family of co-chaperones in TDP-43 interactome and proteostasis in degenerating motoneurons are not well understood. Recently, we described that HSP40 or J-domain protein (JDPs) subfamilies present a distinct modulation of TDP-43 aggregation mechanism, as they either suppress or enhance the aggregation of the prion like domain (PLD), encouraging a further study to better understand this differentiation and its relevance in TDP-43 pathogenesis. This work focuses on JDP family members of subclass ¿A¿ (DNAJA2), ¿B¿ (DNAJB1) and ¿C¿ (DNAJC7) as they have been reported, together with DNAJB6, as major chaperones in TDP-43 proteostasis. We seek to provide fundamental understanding on co-chaperones mode of action and their interplay in the key molecular processes triggering pathogenesis. The deep structural characterization and dynamic study of the molecular complex formed by JDPs and TDP-43 we aim to obtain is key for the future exogenous modulation of TDP-43 aggregation mechanism and the development of new therapeutic strategies towards neurodegenerative diseases.We seek to describe human DNAJA2, DNAJB1 and DNAJC7 conformational ensembles by high-field, solution NMR spectroscopy to understand TDP-43 differential recognition. NMR spectroscopy is the only high-resolution structural technique that can characterize the disordered regions flanking the J domains, which we hypothesize are crucial in TDP-43 triaging. Moreover, we aim to determine the impact of the co-chaperones in TDP-43 aggregation, to uncouple aggregate structure and toxicity. To do so, we use kinetic binding assays to address the differential modulation of the co-chaperones, fluorescence experiments to follow TDP-43¿s liquid-to-solid transition and transmission electron microscopy to determine the morphology and polymorphism of TDP-43 amyloids We have fully assigned constructs for the three chaperones, which recapitulate the dynamic nature of the full sequences. The chaperones appear soluble and monomeric and enabled a detailed structural and dynamic study. The mechanism of recognition of TDP-43's PLD by the three chaperones differs, driven by their J-domains and adjacent disordered regions, which collectively govern both binding specificity and phase separation. In agreement with previous results from the group, we show here that the assembly of PLD amyloid fibrils is significantly enhanced in the presence of DNAJB1 and delayed in the presence of DNAJA2 and DNAJC7. High-quality TEM analysis enabled us to monitor that DNAJA2 and DNAJC7 trap TDP¿43¿s PLD into oligomeric structures, while DNAJB1 promotes PLD fibrils with significantly different morphology.This study provides crucial insights into the distinct roles of J-domain proteins (JDPs) DNAJA2, DNAJB1, and DNAJC7 in modulating TDP-43 aggregation, a key pathological hallmark in neurodegenerative diseases such as ALS. Through a combination of high-field NMR spectroscopy, kinetic binding assays, fluorescence, and transmission electron microscopy, we have uncovered that these co-chaperones exert divergent effects on the phase separation and fibrillization of TDP-43¿s prion-like domain (PLD). Notably, DNAJB1 enhances PLD aggregation and short fibril formation, while DNAJA2 and DNAJC7 suppress fibrillization by stabilizing oligomeric intermediates. These findings support a […]