Transmissibility, cross-aggregation and toxicity of bacterial prion-like protein RepA-Wh1 in cultured mammalian cells
Author
Revilla Garcia, AídaAdvisor
Giraldo Suárez, RafaelEntity
UAM. Departamento de Biología Molecular; CSIC. Centro de Investigaciones Biológicas (CIB)Date
2019-06-14Subjects
Proteínas - Tesis doctorales; Enfermedades degenerativas - Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-06-2019Esta tesis tiene embargado el acceso al texto completo hasta el 14-12-2020
Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Abstract
Over the last few years, amyloid proteins have become a matter of major concern in Biomedicine.
Albeit several amyloidogenic proteins are functional (Fowler, DM et al. 2007), most are cytotoxic,
leading to human diseases specifically referred to as amyloid proteinopathies or amyloidoses.
These include neurodegenerative (Alzheimer´s, Parkinson´s, Huntington´s or prion diseases among
others) and systemic (such as dialysis related amyloidosis) proteinopathies (Chiti, F et al. 2017).
Pathogenesis of amyloidoses is directly linked to the inherent ability of some proteins to misfold
and self-assemble into amyloid fibrils (Eisenberg, D et al. 2012). The templated assembly of an
otherwise soluble protein into fibrillary amyloid aggregates contributes to the conformational
propagation of neurodegeneration. Although in origin self-replicative ability was exclusively
related to PrP prion, many other amyloid-related proteins can also be transmitted through a similar
mechanism, thus being called prionoids or prion-like proteins (Scheckel, C et al. 2018).
The intrincate complexity of amyloidoses has empowered searching for suitable model systems
providing further insight into the molecular and cellular basis underlying protein amyloidogenesis.
In our laboratory, we engineered de novo, through Synthetic Biology, a model system based on the
WH1 domain in RepA, a replication protein encoded by the Pseudomonas savastanoi pPS10
plasmid (Giraldo, R et al. 2004). Upon binding to plasmid-specific DNA sequences, RepA-WH1
experiences a natural structural transformation, analogue to the mammalian prion protein PrP, by
undergoing an increase in β-sheet conformation which leads to its assembly into amyloid fibrils
(Giraldo, R. 2007). This enabled engineering RepA-WH1 as a synthetic amyloidogenic device to
control protein amyloidogenesis both in vitro and in Escherichia coli (Giraldo, R et al. 2016).
Previous work in bacteria have demonstrated that RepA-WH1, when expressed in E. coli,
aggregates as inheritable cytotoxic particles in cytoplasm (Gasset-Rosa, F et al. 2014; Molina-
Garcia, L et al. 2014). This qualifies RepA-WH1 as the first entirely bacterial synthetic prion-like
protein. Furthermore, transcriptomic and proteomic approaches have contributed to outline
pathways for amyloid toxicity (Molina-Garcia, L et al. 2017).
As a proof of concept of the bacterial RepA-WH1 prion-like protein as a feasible minimalist
model system for human neurodegenerative amyloidoses, in this Thesis we have explored its
amyloid cytotoxicity upon expression in cultured mammalian cells. We have also addressed the
ability of RepA-WH1 to cross-seed intra and inter-cellular aggregation then contributing to the
transmission of the amyloid phenotype. The results shown in this Thesis recapitulate both in
murine and human mammalian cells a prion-like phenotype, thus contributing to unravel the complexity of human neurodegenerative diseases.
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