Rafael Giraldo
Group Leader
Amyloids are stable protein assemblies that regulate phenotypes and enable their epigenetic inheritance. However, when they are the outcome of protein misfolding, amyloids can trigger diseases (i.e., human neurodegenerative and systemic amyloidosis). We create, through bottom-up Synthetic Biology, bio-resources based on bacterial amyloids with two major aims: i) understanding the molecular determinants of the shift between function and toxicity in natural amyloids; and ii) generating new devices based on amyloids as constructive resources in Biotechnology and Biomedicine.
Publications
Fernández C, Núñez-Ramírez R, Jiménez M, Rivas G, Giraldo R RepA-WH1, the agent of an amyloid proteinopathy in bacteria, builds oligomeric pores through lipid vesicles. Sci Rep 2016; 6: 23144
Molina-García L, Moreno-del Álamo M, Botias P, Martín-Moldes Z, Fernández M, Sánchez-Gorostiaga A, Alonso-del Valle A, Nogales J, García-Cantalejo J, Giraldo R Outlining core pathways of amyloid toxicity in bacteria with the RepA-WH1 prionoid. Front Microbiol 2017; 8: 539
Molina-García L, Giraldo R. Enabling stop codon read-through translation in bacteria as a probe for amyloid aggregation. Sci Rep 2017; 7: 11908
Fernández C, Giraldo R Modulation of the aggregation of the prion-like protein RepA-WH1 by chaperones in a cell-free expression system and in cytomimetic lipid vesicles..ACS Synth Biol 2018; 7: 2087-2093
Giraldo R. Optogenetic navigation of routes leading to protein amyloidogenesis in bacteria . J Mol Biol 2019; doi: 10.1016/j.jmb.2019.01.037
We engineer RepA, a manifold protein from a plasmid in Gram-negative bacteria whose WH1 domain undergoes conformational changes capacitating it either as a transcriptional repressor, as a DNA replication initiator or, through its assembly as amyloid oligomers, to hinder premature re-replication rounds. Although very stable in solution, RepA-WH1 dimers become metastable monomers upon allosteric binding to plasmid-specific dsDNA sequences, or acidic phospholipids, thus paving the way towards amyloidogenesis (Fig. 1A). The fusion of a hyper-amyloidogenic mutant variant of RepA-WH1 to the fluorescent protein mCherry generates a prion-like protein (prionoid) that is epigenetically transmitted from mother-to-daughter Escherichia coli cells, causing a synthetic ‘generic’ amyloid proteinopathy. In the bacterial cytosol, RepA-WH1 aggregates propagate as two distinct strains, with the appearance of multiple globular, dense foci or a single comet-shaped, fluidized particle that exhibit, respectively, acute or mild cytotoxicity. The Hsp70 chaperone DnaK drives the transformation of the globular into the comet-shaped particles. RepA-WH1 amyloidosis recapitulates in bacteria some of the hallmarks of the mitochondrial route associated with human amyloid diseases, including the formation of oligomeric pores at the internal membrane, the generation of reactive oxygen species and the loss of function of stress-responsive cell factors, due to co-aggregation (Fig. 1B). We have used RepA-WH1 as a benchmark for the design of synthetic tools to probe protein amyloidogenesis, including gold nanoparticles-based sensors, screening devices exploiting amyloid-promoted overriding of translation termination, both in yeast or in bacteria, and in vitro expression devices that address amyloidosis within cytomimetic lipid vesicles. We have recently achieved control on RepA-WH1 amyloidogenesis through optogenetics, i.e., the fusion of a blue light-responsive plant domain (LOV2) to the N-terminus of WH1. Expressing LOV2-WH1-mCherry fusions in E. coli under blue light illumination leads to the assembly of oligomers that are cytotoxic, hampering bacterial growth. In the darkness, the protein assembles as relatively inert amyloid fibrils and hydrogels (Fig. 1C). We are now exploring different venues of application of these devices as novel antimicrobials (‘optobiotics’).
The prion-like protein RepA-WH1 as a SynBio resource. A. RepA-WH1 amyloidogenesis, as characterized in vitro. B. Expression of the A31V variant of RepA-WH1-mCherry in E. coli results in the appearance of amyloid aggregates (red), whose oligomeric precursors drill membranes, thus generating empty bacterial sheaths (arrows). C. An optogenetic device to control amyloid assembly and bacterial growth (‘optobiotics’). Blue light illumination of a photosensor LOV2 domain (green) fused to RepA-WH1-mCherry alters the conformation of WH1 and forms cytotoxic oligomers (bottom: spot assay of E. coli growth).
