Amyloidogenesis of Bacterial Prionoid RepA-WH1 Recapitulates Dimer to Monomer Transitions of RepA in DNA Replication Initiation

Torreira, Eva; Álamo, María Moreno-del; Fuentes-Pérez, M.; Fernández, Cristina; Martín‐Benito, Jaime; Moreno‐Herrero, Fernando; Giraldo, Rafael; Llorca, Óscar

doi:10.1016/j.str.2014.11.007

Cited by 26 publications

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“…As in the full length RepA when activated to initiate DNA replication (Giraldo et al, 2003), RepA-WH1 undergoes a conformational change in vitro , coupled to dissociation of protein dimers into monomers, either on transient binding to plasmid-derived DNA sequences (Giraldo, 2007; Gasset-Rosa et al, 2008) or upon templating by RepA-WH1 aggregates themselves (Fernández-Tresguerres et al, 2010). Such process enables the monomers of the highly amyloidogenic mutant A31V of RepA-WH1 to assemble into fibers composed of intertwined tubular helical protein filaments (Giraldo, 2007; Torreira et al, 2015). RepA-WH1 fibers are of amyloid nature, as indicated by Congo red binding (Giraldo, 2007), and by a net increase in the protein β-sheet contents, according to both circular dichroism (Giraldo, 2007; Torreira et al, 2015) and surface-enhanced Raman (Fernández et al, 2016a) spectroscopies.…”

Section: Introductionmentioning

confidence: 99%

“…Such process enables the monomers of the highly amyloidogenic mutant A31V of RepA-WH1 to assemble into fibers composed of intertwined tubular helical protein filaments (Giraldo, 2007; Torreira et al, 2015). RepA-WH1 fibers are of amyloid nature, as indicated by Congo red binding (Giraldo, 2007), and by a net increase in the protein β-sheet contents, according to both circular dichroism (Giraldo, 2007; Torreira et al, 2015) and surface-enhanced Raman (Fernández et al, 2016a) spectroscopies. In our efforts to engineer a synthetic bacterial amyloid proteinopathy, we found that the amyloidogenicity of WH1(A31V) in E. coli cells can be boosted displacing its conformational equilibrium toward partial unfolding by fusing a protein to its C-terminus, distinct to the natural WH2 domain in RepA (Giraldo et al, 2003): the monomeric fluorescent protein mCherry (Fernández-Tresguerres et al, 2010; Gasset-Rosa et al, 2014; Molina-García and Giraldo, 2014).…”

Section: Introductionmentioning

confidence: 99%

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Outlining Core Pathways of Amyloid Toxicity in Bacteria with the RepA-WH1 Prionoid

et al. 2017

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The synthetic bacterial prionoid RepA-WH1 causes a vertically transmissible amyloid proteinopathy in Escherichia coli that inhibits growth and eventually kills the cells. Recent in vitro studies show that RepA-WH1 builds pores through model lipid membranes, suggesting a possible mechanism for bacterial cell death. By comparing acutely (A31V) and mildly (ΔN37) cytotoxic mutant variants of the protein, we report here that RepA-WH1(A31V) expression decreases the intracellular osmotic pressure and compromise bacterial viability under either aerobic or anaerobic conditions. Both are effects expected from threatening membrane integrity and are in agreement with findings on the impairment by RepA-WH1(A31V) of the proton motive force (PMF)-dependent transport of ions (Fe3+) and ATP synthesis. Systems approaches reveal that, in aerobiosis, the PMF-independent respiratory dehydrogenase NdhII is induced in response to the reduction in intracellular levels of iron. While NdhII is known to generate H2O2 as a by-product of the autoxidation of its FAD cofactor, key proteins in the defense against oxidative stress (OxyR, KatE), together with other stress-resistance factors, are sequestered by co-aggregation with the RepA-WH1(A31V) amyloid. Our findings suggest a route for RepA-WH1 toxicity in bacteria: a primary hit of damage to the membrane, compromising bionergetics, triggers a stroke of oxidative stress, which is exacerbated due to the aggregation-dependent inactivation of enzymes and transcription factors that enable the cellular response to such injury. The proteinopathy caused by the prion-like protein RepA-WH1 in bacteria recapitulates some of the core hallmarks of human amyloid diseases.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Outlining Core Pathways of Amyloid Toxicity in Bacteria with the RepA-WH1 Prionoid

et al. 2017

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“…TheA NTACo-functionalized AuNRs were incubated at 1:10 5 and 1:50 AuNR:H6-RepA-WH1(A31V) molar ratios,w ith protein concentrations of 20 and 0.1 mm, respectively.T his protein forms as table dimer in solution under ab road range of conditions (Scheme 1). [13a, 14,19] However,the observed 5nmred shift of the AuNRs longitudinal localized surface plasmon resonance (LSPR) at both molar ratios,i ndicates the absence of AuNR self-assembly in solution ( Figure 1A,s ee the Supporting Information). [20] This might be expected from the presence of two oppositely anchored H6 groups on the H6-RepA-WH1(A31V) dimers (Scheme 1).…”

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confidence: 94%

“…[13] This protein undergoes as ubstantial conformational change from soluble stable dimers to metastable aggregation-prone monomers that then assemble into amyloid fibers. [14] RepA-WH1, in particular its hyper-amyloidogenic mutant variant A31V,H 6-RepA-WH1(A31V), causes in bacteria at ype of amyloidosis that shares many features with mammalian neurodegenerative diseases, [15] while remaining bio-safe for humans.…”

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Nucleation of Amyloid Oligomers by RepA‐WH1‐Prionoid‐Functionalized Gold Nanorods

et al. 2016

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Understanding protein amyloidogenesis is an important topic in protein science,f ueled by the role of amyloid aggregates,especially oligomers,inthe etiology of anumber of devastating human degenerative diseases.H owever,t he mechanisms that determine the formation of amyloid oligomers remain elusive due to the high complexity of the amyloidogenesis process.F or instance,g old nanoparticles promote or inhibit amyloid fibrillation. We have functionalized gold nanorods with am etal-chelating group to selectively immobilize soluble RepA-WH1, amodel synthetic bacterial prionoid, using ah exa-histidine tag (H6). H6-RepA-WH1 undergoes stable amyloid oligomerization in the presence of catalytic concentrations of anisotropic nanoparticles.T hen, in ap hysically separated event, such oligomers promote the growth of amyloid fibers of untagged RepA-WH1. SERS spectral changes of H6-RepA-WH1 on spherical citrate-AuNP substrates provide evidence for structural modifications in the protein, whicha re compatible with ag radual increase in bsheet structure,asexpected in amyloid oligomerization.

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“…The WH1 domain in RepA forms stable dimers in solution. Upon allosteric binding to distinct ligands, dimers dissociate into metastable monomers that subsequently assemble as amyloid oligomers and fibres in vitro (Giraldo, 2007; Torreira et al, 2015). When expressed in E. coli , the hyper-amyloidogenic domain variant RepA-WH1(A31V) forms particles of various sizes, distributed across the bacterial cytosol (Fernández-Tresguerres et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Intercellular transmission of a bacterial cytotoxic prion-like protein in mammalian cells

Revilla-García

Fernández

Álamo

et al. 2019

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1RepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of 2 plasmid DNA replication. When carrying a mutation enhancing its amyloidogenesis (A31V), the N-2007; Torreira et al., 2015). When expressed in E. coli, the hyper-amyloidogenic domain variant RepA-1 WH1(A31V) forms particles of various sizes, distributed across the bacterial cytosol (Fernández-2 Tresguerres et al., 2010). These particles are transmissible vertically (during cell division, i.e. from 3 mother to daughter cells) as two distinct aggregate strains with remarkable appearances and phenotypes: 4 elongated and mildly cytotoxic, and compact and acutely cytotoxic, respectively (Gasset-Rosa et al., 5 2014). Co-expression of soluble and aggregation-prone RepA-WH1 variants in E. coli demonstrated that 6 the A31V variant can template its conformation on the parental WT protein (Molina-García and 7 Giraldo, 2014). Systems analyses (Molina-García et al., 2017), together with in vitro reconstruction in 8 cytomimetic lipid vesicles (Fernández et al., 2016b; Fernández and Giraldo, 2018), suggest that RepA-9 WH1(A31V) oligomers target the internal bacterial membrane, hampering proton motive force and thus 10 ATP synthesis and transport through membranes, and enhance oxidative stress. In parallel, protein 11 factors mounting the defence against stress and envelope damage co-aggregate with RepA-WH1(A31V) 12 amyloids (Molina-García et al., 2017). Altogether, bacteria viability is severely compromised by RepA-13 WH1 amyloidosis, in a way resembling some of the central mitochondrial routes found in human 14 amyloidoses (Haelterman et al., 2014; Norambuena et al., 2018; Mathys et al., 2019; Wang et al., 2019).15 However, E. coli is not suitable for addressing the issues of cell-to-cell transmissibility of protein 16 aggregates and the subsequent intracellular amyloid cross-aggregation, since this Gram-negative 17 bacterium does not uptake large protein particles due to the insurmountable obstacle of its three-layered 18 cell envelope. 19To explore the capability of the prion-like protein RepA-WH1 to propagate in a heterologous host, 20 here we exposed murine neuroblastoma cells, transiently expressing mCherry-tagged soluble RepA-21 WH1(WT), to in vitro assembled RepA-WH1(A31V) amyloid fibres. In addition, we studied the 22 intercellular induction of protein aggregation by co-culturing murine cells releasing RepA-WH1(A31V) 23 aggregates with human neuroblastoma cells stably expressing soluble RepA-WH1(WT). Confocal 24 microscopy and biochemical studies showed that the mammalian cells can take up RepA-WH1(A31V) 25 amyloid fibres, that cross-seed the cytosolic aggregation of the endogenous RepA-WH1(WT) in the 26 recipient cells. Moreover, co-culture of cells releasing the RepA-WH1(A31V) variant also induced the 27 aggregation of RepA-WH1(WT) in bystander cells, suggesting intercellular transmission of RepA-28 5 WH1(A31V) seeds. In both setups of experimental transmission, the induced RepA-WH1(WT) 1 aggregates were cytoto...

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Amyloidogenesis of Bacterial Prionoid RepA-WH1 Recapitulates Dimer to Monomer Transitions of RepA in DNA Replication Initiation

Cited by 26 publications

References 32 publications

Outlining Core Pathways of Amyloid Toxicity in Bacteria with the RepA-WH1 Prionoid

Outlining Core Pathways of Amyloid Toxicity in Bacteria with the RepA-WH1 Prionoid

Nucleation of Amyloid Oligomers by RepA‐WH1‐Prionoid‐Functionalized Gold Nanorods

Intercellular transmission of a bacterial cytotoxic prion-like protein in mammalian cells

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