Induction of protein aggregation in zebrafish embryos as a method for the screening of new drugs or mutations against proteinopathies.

The sustained increase in the prevalence of protein aggregation related diseases requires the development of feasible methods for the design of therapeutic alternatives. The procedure traditionally used for the search of drugs or therapeutic mutations includes in vitro experiments, designed to prevent the aggregation of model proteins, which are then complemented with cellular toxicity studies in vivo, slowing down the finding of solutions. To address this, we have developed a protocol that facilitates the search of molecules and anti-aggregation mutations since it allows to evaluate their therapeutic capabilities directly in in vivo experiments with the use of zebrafish early embryos. Avoiding the necessity of performing in vitro and in vivo procedures separately. Giving a more realistic method for the results interpretation. Zebrafish embryos were induced to produce intracellular aggregates of proteins by simple microinjections of known quantities of an aggregation prone protein previously labeled. The toxicity was evaluated by the survival of the embryos, while the formation of aggregates was quantified by fluorescence microscopy. The size distribution of the protein aggregates was revealed by means of ultracentrifuge sedimentation analysis. For the development of the present method, the human γ-tubulin protein was used as model protein, which generated intracellular aggregates in more than 60% of the injected embryos. To evaluate the method, a mutation was performed that altered the state of intracellular aggregation of γ-tubulin, obtaining a significant decrease in the amount and size of the intracellular aggregates. The present method can be used for any suitable intracellular aggregation protein model. The current method present important advantages such as: Easy induction of intracellular aggregates. Simple detection of intracellular protein aggregates through fluorescence microscopy and subcellular fractionation. Overall view of the effect of drugs or mutations by combining the toxicity, the development behavior and the size distribution of intracellular protein aggregates.

The chaperonin CCT promotes the formation of fibrillar aggregates of γ-tubulin.

The type II chaperonin CCT is involved in the prevention of the pathogenesis of numerous human misfolding disorders, as it sequesters misfolded proteins, blocks their aggregation and helps them to achieve their native state. In addition, it has been reported that CCT can prevent the toxicity of non-client amyloidogenic proteins by the induction of non-toxic aggregates, leading to new insight in chaperonin function as an aggregate remodeling factor. Here we add experimental evidence to this alternative mechanism by which CCT actively promotes the formation of conformationally different aggregates of γ-tubulin, a non-amyloidogenic CCT client protein, which are mediated by specific CCT-γ-tubulin interactions. The in vitro-induced aggregates were in some cases long fiber polymers, which compete with the amorphous aggregates. Direct injection of unfolded purified γ-tubulin into single-cell zebra fish embryos allowed us to relate this in vitro activity with the in vivo formation of intracellular aggregates. Injection of a CCT-binding deficient γ-tubulin mutant dramatically diminished the size of the intracellular aggregates, increasing the toxicity of the misfolded protein. These results point to CCT having a role in the remodeling of aggregates, constituting one of its many functions in cellular proteostasis.

Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation.

Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril's morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4'-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54-63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54-63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although with different efficiency, all formed fibrils morphologically similar to wild-type MccE492. The physiological implication of MccE492 intracellular amyloid formation is probably similar to the inactivation process observed for extracellular amyloids, and could be used as a mean of sequestering potentially toxic species inside the cell when this bacteriocin is produced in large amounts.