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Título

Desarrollo de nuevas herramientas para la detección e inhibición de la amiloidosis causada por el prionoide RepA-WH1 en Escherichia coli y caracterización de su toxicidad.

AutorMolina-García, Laura CSIC ORCID
DirectorGiraldo, R. CSIC ORCID
Palabras claveAmiloidosis
Escherichia coli
Prionoide RepA-WH1
Fecha de publicación30-jun-2015
ResumenIntroduction: The correct biological function of cells depends on the proper folding of thousands of proteins. Amyloid proteinopathies are neurodegenerative and systemic diseases that share a common etiology: the accumulation of insoluble aggregates of a particular protein, either misfolded or post-­‐translationally processed, which are made of crossed β-­‐sheet assemblies, the signature 3D structure of amyloids. In the crossed-­‐β structure, multiple copies of a peptide sequence, each provided by a different molecule of the same protein, assemble as strands in β-­‐sheets of indefinite extension which then stack and twist as fibres (Chiti & Dobson, 2006). Amyloid aggregates are able to replicate by acting as templates for the structural transformation of other soluble molecules of the same protein into the aggregated state. This propagate the amyloid conformation among cells and tissues as a self-­‐replicative macromolecular assembly. However, amyloids are not always related to disease. The aggregated amyloid states of a number of proteins have been reported to have physiological roles, including CsgA-­‐curlin (which holds bacterial biofilms), Pmel17 (a scaffolding protein for enzymes involved in melanin synthesis in vertebrates), CPEB (involved in memory storage in the molusk Aplysia. sp) and yeast prions, among others (Otzen & Nielsen, 2008). In terms of toxicity, oligomers of an amyloidogenic protein, in their process toward assembling as fibres, seem to be the most toxic species. These oligomers act either directly by targeting membranes, or by depleting essential cell factors through coaggregation, or indirectly by contributing to generate reactive oxygen species (ROS) (Chiti & Dobson, 2006). Their involvement in a number of serious, often fatal, diseases such as Parkinson, Alzheimer or prion disorders among others, has positioned amyloid biology as a hot spot in protein science research, currently focused on the search for small molecules that could inhibit amyloidogenesis. To do so, apart from studies carried out in human cell cultures and mice, which have uncovered many pathways possibly linked to disease, simpler model systems that provide further insights into the essentials of the process are needed. These include Drosophila and Caenorhabditis animal models and, among microorganisms, yeast (Narayan et al., 2014).
Fungal prions have been crucial to unravel the molecular basis of amyloid assembly and propagation, but they are notoptimal models for disease related studies due to their nature as epigenetic determinants that confer beneficial phenotypes to their carriers. The unsurpassed potential of Escherichia coli as a model system in Biology have also been exploited to study the heterologous aggregation of amyloidogenic proteins, such as the Saccharomyces cerevisiae prion Sup35p or the Alzheimer Aβ peptides, which aggregate as intracellular inclusion bodies (IBs). In our group, we work with the RepA protein, encoded within the replicon module of the Pseudomonas plasmid pPS10 (Nieto et al., 1992). As a dimer, RepA binds to an inversely repeated operator DNA sequence to repress its own transcription (García de Viedma et al., 1995). Upon binding to directly repeated DNA sequences found at the plasmid replication origin, RepA dissociates into monomers to initiate DNA replication (Giraldo, 2003; Díaz-­‐López et al., 2003, 2006). RepA is made of two winged-­‐helix (WH) domains (Giraldo et al., 1998): the N-­‐terminal domain (RepA-­‐WH1) acts as a dimerization module, whereas the C-­‐terminal domain (RepA-­‐WH2) is the main DNA binding interface (Giraldo & Fernández-­‐Tresguerres, 2004). Similarly to the mammalian prion protein PrP and α-­‐synuclein (involved in the aggregates found in Parkinson’s disease), the RepA-­‐WH1 domain undergoes a large α-­‐helix to β-­‐sheet conformational transition upon sequence-­‐ specific binding to double-­‐stranded (ds) DNA (Giraldo, 2003; Díaz-­‐López et al., 2003, 2006). According to predictions made in silico, a peptide of RepA-­‐WH1 (L26VLCAASLID34) has exhibited a high potential to aggregate as β-­‐strands. The score for the aggregation prediction was higher in a single point mutant in this sequence (A31V), which was recurrently isolated in blind genetic searches for RepA variants with more efficient plasmid replication activity (Fernández-­‐Tresguerres et al., 1995; Maestro et al., 2003). A synthetic peptide containing this sequence assembles into fibres, visualized by TEM. These fibres also exhibit the characteristic amyloid pattern showed by X-­‐ray diffraction (Giraldo, 2007). Not only this peptide but also the whole RepA-­‐WH1(A31V) domain assembles into amyloid fibres in vitro (Giraldo, 2007) whose structure have recently been solved by TEM (Torreira et al., 2015). Furthermore, fusions of RepA-­‐WH1(A31V) to a fluorescence protein tag allow us to follow the aggregation of the protein in the E.coli cytoplasm, revealing a drastic reduction in cell proliferation upon formation of aggregates (Fernández-­‐Tresguerres et al., 2010). Those inclusions, purified from cell extracts, also enable conformational templating of soluble RepA-­‐WH1(A31V) in vitro. In addition to this toxicity, RepA-­‐WH1(A31V) aggregates differ from conventional IBs in their higher affinity for the amyloidotropic fluorophore BTA-­‐1 and in exhibiting dynamic interconversion between two distinct amyloid species, which resemble prion strains. We have also shownthat the latter process is modulated by DnaK (the E.coli Hsp70 chaperone) that generates oligomeric RepA-­‐WH1 particles, which are readily transferred to the progeny during bacterial cell division (Gasset-­‐Rosa et al., 2014). Being vertically transmissible from mother to daughter cells, but not infectious, RepA-­‐WH1 qualifies as the first entirely bacterial prionoid.
Objectives & Results: Considering the precedents described above, the objectives of this thesis were: 1. To explore the in vivo ability of different variants of the RepA-­‐WH1 prionoid to cross-­‐ seed their aggregation in Escherichia coli. 2. To identify and characterize the cellular pathways involved in the toxicity produced by the RepA-­‐WH1(A31V) prionoid in E. coli. 3. To develop an in vivo screening system based on chimeras between repeats of the amyloidogenic sequence in WH1 and the E. coli translation terminator factor RF1, for identification of protein sequences with amyloid potential and its validation through compounds that inhibits the amyloidogenic process. 1. The co-­‐aggregation ability of RepA-­‐WH1 variants was studied in vivo by means of pairwise coexpression of repA-­‐WH1 alleles coding for protein variants with diverse amyloidogenic properties: the hyperamiloidogenic variant A31V, which aggregates as multiple globular foci, the mildly amyloidogenic WT variant, which remains soluble in the E. coli cytoplasm, and the ΔN37 variant, which lacks the amyloidogenic N-­‐terminal stretch of RepA and aggregates as conventional mono or bipolar IBs (Giraldo et al., 1998; Gasset-­‐ Rosa et al., 2014). All these three variants were tracked via epitope tags and fusions to different colored fluorescent proteins to address how they mutually influence their aggregation in vivo. We found that the interplay between their intrinsic aggregation tendencies, expression levels and intermolecular contacts determines the solubility vs. aggregation balance for each RepA-­‐WH1 variant. Diffused fluorescence was only observed in cells simultaneously expressing the WT variants fused to both fluorescent tags (Figure 19A). However, both aggregation-­‐prone variants, A31V and ΔN37, dominated over the WT, which became aggregated following the pattern of its coexpressed partner (Figure 19A). The amount of protein present in the soluble or aggregated fraction of cell lysates was analysed by Western-­‐blot confirming the same results (Figure 19B) (Molina-­‐García & Giraldo, 2014). 2. The pathways of amyloid toxicity of RepA-­‐WH1 were studied combining transcriptomic, proteomic and physiological approaches. We have analyzed throughmicroarrays the gene expression pattern of cells expressing RepA-­‐WH1 and through mass spectrometry the fraction of the E. coli proteome co-­‐aggregated with RepA-­‐WH1 amyloid inclusions. Combining both types of approaches, we identified 47 potential cellular targets grouped in 32 functional clusters, comprising 5 fundamental biological processes that we also validated by studying membrane integrity, oxygen consumption rates, iron and ATP levels and sensitivity to oxygen reactive species (ROS). All of these parameters were lower in cells expressing the hyperamyloidogenic variant A31V (Figures 26-­‐28), except for sensitivity to ROS which was higher due to the increase in the expression levels of Ndh-­‐II dehydrogenase in that mutant (Figures 29-­‐31).
Our results indicate that cell damage is produced by a ROS-­‐dependent mechanism, most likely involving Fenton chemistry. These findings support a role for free iron in amyloid cyto-­‐toxicity, possibly due to membrane damage by amyloid oligomers as has been proposed for the general cyto-­‐toxicity in human proteinopathies induced by distinct amyloids. 3. The design of the WH1(Rn)-­‐RF1 chimeras screening system was based on the molecular basis of the yeast prion Sup35p [PSI+]. Sup35p is the translation release factor 3 in eukaryotes (eRF3) and drives the final stage of polypeptide synthesis after stop codon recognition by its partner Sup45p (eRF1). In normal situations (non prion phenotype, [psi-­‐]), Sup35p is soluble and functional. However, when it aggregates (prion phenotype, [PSI+]) the reduced levels of the soluble factor make it unable to finish translation efficiently, promoting stop codon readthrough and the generation of longer polypeptides (Figure 9; Tessier & Lindquist 2009). This feature has enabled the development of a widely used amyloid aggregation reporter system, in which a premature stop codon, placed in one of the genes involved in the adenine biosynthetic pathway, acts as a sensor of Sup35p aggregation: when the protein remains soluble, it finishes the translation at the premature stop codon and the previous metabolite in the pathway is accumulated, causing a red coloration in the yeast cells. On the contrary, when Sup35p is aggregated, it ignores the premature stop codon and ribosomes complete the synthesis of the whole protein making the cells exhibit their natural white coloration. Previous work of our laboratory had shown that the substitution of the amyloid repeats present in Sup35p with tandem repeats of the amiloidogenic peptide of RepA-­‐WH1 allowed the recovery of the prion phenotype (Gasset-­‐Rosa & Giraldo, 2015). Based on all of these precedents, we have developed here a system in which the aggregation of the bacterial translation release factor 1 (RF1), promoted by its fusion to the same repeats of the RepA-­‐ WH1 amyloid peptide that were functional in yeast, produces the readthrough of a
Conclusions 1.1. The hyperamyloidogenic RepA-­‐WH1(A31V) variant induces cross-­‐aggregation in the cytoplasm of E. coli, enhancing the growth of amyloid particles by a different variant of the same protein with reduced amyloidogenicity. 1.2. The colocalization assay performed in this Thesis easily discriminates bona fide amyloid aggregation from formation of IBs. 2.1. The expression of different variants of RepA-­‐WH1 prionoid, A31V or ΔN37, induces a different gene expression pattern in E. coli cells that allowed us to identify the cellular response specifically due to amyloid aggregation. Both proteins variants also sequester different cellular factors that coaggregate with them. 2.2. The initial event in the cyto-­‐toxicity cascade of RepA-­‐WH1 prionoid seems to be membrane damage. 2.3. The cellular toxicity of RepA-­‐WH1 prionoid is produced by a ROS-­‐dependent mechanism, most likely involving Fenton chemistry. 3.1. The screening system based on WH1(Rn)-­‐RF1 chimeras developed in this Thesis is useful to identify protein sequences with amyloidogenic potential in vivo. 3.2. The amyloid aggregation of the WH1(Rn)-­‐RF1 chimeras depends on having two or more repeats of the RepA-­‐WH1(A31V) amyloidogenic peptide. 3.3. Resveratrol treatment reverse the colored and aggregated phenotype of the WH1(Rn)-­‐ RF1 prionoids, provided they carry 2 or more repeats of the WH1(A31V) amyloidogenic peptide. Thus, the WH1(Rn)-­‐RF1 chimeras are useful to monitor the activity of potential inhibitors of amyloidosis. 3.4. The chimeric WH1(Rn)-­‐RF1 screening system is the first designed to monitor the amyloidogenic potential of peptide sequences based on biotechnological manipulation of the bacterial translation process.
Descripción168p.- 42 fig.- 6 tab.-
URIhttp://hdl.handle.net/10261/121036
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