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  • Even though Nb was raised against WT gelsolin

    2019-07-21

    Even though Nb11 was raised against WT gelsolin, it was shown to bind the D187N variant as well, with a slightly lower affinity [19]. Crystallographic structures of N184K and G167R variants, as isolated G2 as well, are also available [30,31]. N184K neither impairs calcium binding nor it significantly destabilizes the C-terminal tail. Loss of conformational stability of N184K-mutated G2 comes from the rearrangement of the polar contacts, which the mutated residue is part of, in the core of the domain. As for the D187N/Y case, N184K A 887826 eventually leads to furin proteolysis. Contrarily, the pathological mechanism underlying the G167R-dependent disease is still to be fully elucidated. An alternative amyloidogenic pathway has in fact been proposed based on the observation that this variant dimerizes via a domain-swap mechanism [31]. At the same time, even the G167R mutant is prone to aberrant proteolysis and shows impaired thermal stability. The WTG2 and D187N- and N184K-mutated variants share a high structural conservation. The Nb11 binding interface of the G167RG2 variant is not affected by its dimerization. As a consequence, it comes as no surprise that Nb11 binds the renal variants with similar efficiency. On the contrary, the protection from proteolysis of the N184K and G167R mutants would have been difficult to predict because the impact of these substitutions on the G2 dynamics as well as the mechanism of destabilization is significantly different. We tested Nb11 protection of the renal variants in standard furin assays and observed indirect inhibition of the protease activity comparable to that of the D187N variant. To further investigate this aspect, we aimed at testing Nb11's activity on mutated G2 in a more biological context. As neither an animal nor a cell model of renal AGel amyloidosis is currently available, we employed a nematode-based assay already developed and validated by our group and already successfully used to determine the toxicity of different amyloidogenic proteins and investigate the mechanisms underlying their proteotoxic effect [[24], [25], [26], [27],29]. This is based on the knowledge that the rhythmic contraction and relaxation of the C. elegans pharynx, the organ fundamental for the worm's feeding and survival, is sensitive to molecules that can act as “chemical stressors” such as some proteins induced in stress conditions. We here showed for the first time that all isolated G2, at concentrations similar to those observed as circulating in humans [55] can cause a biologically relevant toxic effect recognized by C. elegans. Whereas WTG2 only induced a transient reduction of the pharyngeal pumping, all the pathological mutants caused an impairment which persisted still after 24 h, suggesting that these proteins caused a permanent tissue damage. Interestingly, Nb11, which was able to bind and chaperone all the disease-causing mutant forms of GSN and prevent their aberrant proteolysis, completely abolished the proteotoxic effects induced by G2 domains in worms. Although the mechanisms underlying the ability of C. elegans to recognize G2 domain as toxic remains to be elucidated, these findings are consistent with the hypothesis that the toxicity can be ascribed to the partially unfolded status of the proteins and that Nb11 hides the structural determinant of toxicity. The involvement of the folding status in G2 toxicity was further assessed by using a protein totally unfolded due to the truncation of residues 151–167. The toxic effect caused by WTΔG2 protein on the pharynx of worms was greater and more persistent than that observed with the folded WTG2. Additional studies are required to fully elucidate the mechanisms underlying the toxicity of G2 domains on the C. elegans pharynx. The knowledge of high-resolution determinants of D187N toxicity and the therapeutic action of Nb11 may contribute to the ongoing efforts in the expansion of the currently limited landscape of therapeutic interventions against FAF and other amyloidosis-related diseases. The use of the C. elegans-based assay for the evaluation of the proteotoxic potential of the G2 domains offers unprecedented opportunities to investigate the molecular mechanisms underlying the AGel forms caused by different GSN variants. This model can also be employed for rapidly screen the protective effect of novel or repurposed drugs thus accelerating the identification of an effective therapy against AGel amyloidosis.