Aggregating sequences that occur in many proteins constitute weak spots of bacterial proteostasis

Aggregation is a sequence-specific process, nucleated by short aggregation-prone regions (APRs) that can be exploited to induce aggregation of proteins containing the same APR. Here, we find that most APRs are unique within a proteome, but that a small minority of APRs occur in many proteins. When aggregation is nucleated in bacteria by such frequently occurring APRs, it leads to massive and lethal inclusion body formation containing a large number of proteins. Buildup of bacterial resistance against these peptides is slow. In addition, the approach is effective against drug-resistant clinical isolates of Escherichiacoli and Acinetobacterbaumannii, reducing bacterial load in a murine bladder infection model. Our results indicate that redundant APRs are weak points of bacterial protein homeostasis and that targeting these may be an attractive antibacterial strategy

The prion-like RNA-processing protein HNRPDL forms inherently toxic amyloid-like inclusion bodies in bacteria

BACKGROUND: The formation of protein inclusions is connected to the onset of many human diseases. Human RNA binding proteins containing intrinsically disordered regions with an amino acid composition resembling those of yeast prion domains, like TDP-43 or FUS, are being found to aggregate in different neurodegenerative disorders. The structure of the intracellular inclusions formed by these proteins is still unclear and whether these deposits have an amyloid nature or not is a matter of debate. Recently, the aggregation of TDP-43 has been modelled in bacteria, showing that TDP-43 inclusion bodies (IBs) are amorphous but intrinsically neurotoxic. This observation raises the question of whether it is indeed the lack of an ordered structure in these human prion-like protein aggregates the underlying cause of their toxicity in different pathological states. RESULTS: Here we characterize the IBs formed by the human prion-like RNA-processing protein HNRPDL. HNRPDL is linked to the development of limb-girdle muscular dystrophy 1G and shares domain architecture with TDP-43. We show that HNRPDL IBs display characteristic amyloid hallmarks, since these aggregates bind to amyloid dyes in vitro and inside the cell, they are enriched in intermolecular β-sheet conformation and contain inner amyloid-like fibrillar structure. In addition, despite their ordered structure, HNRPDL IBs are highly neurotoxic. CONCLUSIONS: Our results suggest that at least some of the disorders caused by the aggregation of human prion-like proteins would rely on the formation of classical amyloid assemblies rather than being caused by amorphous aggregates. They also illustrate the power of microbial cell factories to model amyloid aggregation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0284-7) contains supplementary material, which is available to authorized users

Influence of the Stability of a Fused Protein and Its Distance to the Amyloidogenic Segment on Fibril Formation

Conversion of native proteins into amyloid fibrils is irreversible and therefore it is difficult to study the interdependence of conformational stability and fibrillation by thermodynamic analyses. Here we approached this problem by fusing amyloidogenic poly-alanine segments derived from the N-terminal domain of the nuclear poly (A) binding protein PABPN1 with a well studied, reversibly unfolding protein, CspB from Bacillus subtilis. Earlier studies had indicated that CspB could maintain its folded structure in fibrils, when it was separated from the amyloidogenic segment by a long linker. When CspB is directly fused with the amyloidogenic segment, it unfolds because its N-terminal chain region becomes integrated into the fibrillar core, as shown by protease mapping experiments. Spacers of either 3 or 16 residues between CspB and the amyloidogenic segment were not sufficient to prevent this loss of CspB structure. Since the low thermodynamic stability of CspB (ΔGD = 12.4 kJ/mol) might be responsible for unfolding and integration of CspB into fibrils, fusions with a CspB mutant with enhanced thermodynamic stability (ΔGD = 26.9 kJ/mol) were studied. This strongly stabilized CspB remained folded and prevented fibril formation in all fusions. Our data show that the conformational stability of a linked, independently structured protein domain can control fibril formation

Energy barriers for HET-s prion forming . domain amyloid formation

International audienceThe prion-forming domain comprising residues 218-289 of the fungal prion HET-s forms infectious amyloid fibrils at physiological pH. Because a high-resolution molecular model for the structure of these fibrils exists, it constitutes an attractive system with which to study the mechanism of amyloid assembly. Understanding aggregation under specific conditions requires a quantitative knowledge of the kinetics and thermodynamics of the self-assembly process. We report here the study of the temperature and agitation dependence of the HET-s(218-289) fibril nucleation (k(n)) and elongation (k(e)) rate constants at physiological pH. Over our temperature and agitation range, k(n) and k(e) increased 30-fold and three-fold, respectively. Both processes followed the Arrhenius law, allowing calculation of the thermodynamic activation parameters associated with them. The data confirm the nucleation reaction as the rate-limiting step of amyloid fibril formation. The formation of the nucleus appears to depend mainly on enthalpic factors, whereas both enthalpic and entropic effects contribute similarly to the energy barrier to fibril elongation. A kinetic model is proposed in which nucleation depends on the presence of an initially collapsed, but poorly structured, HET-s(218-289) state and in which the fibril tip models the conformation of the incoming monomers without substantial disorganization of its structure during the elongation process

Aggregation of the neuroblastoma-associated mutant (S120G) of the nucleoside diphosphate kinase A-NM23-H1 into mayloid fibrils

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A novel class of multitarget anti-Alzheimer benzohomoadamantane\u2012chlorotacrine hybrids modulating cholinesterases and glutamate NMDA receptors

The development of multitarget compounds against multifactorial diseases, such as Alzheimer's disease, is an area of very intensive research, due to the expected superior therapeutic efficacy that should arise from the simultaneous modulation of several key targets of the complex pathological network. Here we describe the synthesis and multitarget biological profiling of a new class of compounds designed by molecular hybridization of an NMDA receptor antagonist fluorobenzohomoadamantanamine with the potent acetylcholinesterase (AChE) inhibitor 6-chlorotacrine, using two different linker lengths and linkage positions, to preserve or not the memantine-like polycyclic unsubstituted primary amine. The best hybrids exhibit greater potencies than parent compounds against AChE (IC50 0.33 nM in the best case, 44-fold increased potency over 6-chlorotacrine), butyrylcholinesterase (IC50 21 nM in the best case, 24-fold increased potency over 6-chlorotacrine), and NMDA receptors (IC50 0.89 \u3bcM in the best case, 2-fold increased potency over the parent benzohomoadamantanamine and memantine), which suggests an additive effect of both pharmacophoric moieties in the interaction with the primary targets. Moreover, most of these compounds have been predicted to be brain permeable. This set of biological properties makes them promising leads for further anti-Alzheimer drug development