Alpha-synuclein (aSyn) misfolding and aggregation are pathological features common to several neurodegenerative diseases, including Parkinson's disease (PD). Mounting evidence suggests that aSyn can be secreted and transferred from cell to cell, participating in the propagation and spreading of pathological events. Rab11, a small GTPase, is an important regulator in both endocytic and secretory pathways. Here, we show that Rab11 is involved in regulating aSyn secretion. Rab11 knockdown or overexpression of either Rab11a wild-type (Rab11a WT) or Rab11a GDP-bound mutant (Rab11a S25N) increased secretion of aSyn. Furthermore, we demonstrate that Rab11 interacts with aSyn and is present in intracellular inclusions together with aSyn. Moreover, Rab11 reduces aSyn aggregation and toxicity. Our results suggest that Rab11 is involved in modulating the processes of aSyn secretion and aggregation, both of which are important mechanisms in the progression of aSyn pathology in PD and other synucleinopathies.
Protein misfolding and aggregation into amyloid structures is linked with an increasing number of nonneuropathic (either localized or systemic) and neurodegenerative human disorders. In the present review, we compile and describe methods, which have been developed to predict, detect and characterize amyloid and amyloid-like protein deposits. We focus in the state-of-the-art methodologies to study and image amyloid aggregation in-vitro, from qualitative and low-resolution techniques to methods addressed to resolve protein structures at atomic level. We also recapitulate the most relevant literature describing approaches that have been demonstrated to be able to detect and characterize protein aggregation in cells and living organisms, as well as methodologies to report cytotoxicity associated to amyloid formation. Overall, the aim of this review is to illustrate computational and experimental methods to characterize and predict in-vitro and in-vivo amyloid aggregation, providing the readers valuable information to elect the most appropriate techniques at their convenience.
Despite genetic variation has the potential to arise new protein functions, spontaneous mutations usually destabilize the native fold. Misfolded proteins tend to form cytotoxic intracellular aggregates, decreasing cell fitness and leading to degenerative disorders in humans. Therefore, it is thought that selection against protein misfolding and aggregation constrains the evolution of protein sequences. However, obtaining experimental data to validate this hypothesis has been traditionally difficult. Here we exploit bacteria as a model organism to address this question. Using variants of the Alzheimer's related A?42 peptide designed to exhibit different in vivo aggregation propensities we show here that, in cell competition experiments, the most aggregation-prone variants are always purged out from the growing population. Flow cytometry analysis of cellular metabolism and viability demonstrates that this purifying effect responds to a clear correlation between physiological burden and intrinsic aggregation propensity. Interestingly, the fitness cost of aggregation appears to be associated with aggregation rates rather than with overall protein solubility. Accordingly, we show that, by reducing in vivo aggregation rates, the model osmolyte proline is able to buffer the metabolic impact of protein aggregation. Overall, our data provide experimental support for the role of toxic protein aggregation on the cell fitness landscape and the evolution of natural protein sequences.
Protein aggregation is linked to many pathological conditions, including several neurodegenerative diseases. The aggregation propensities of proteins are thought to be controlled to a large extent by the physicochemical properties encoded in the primary sequence. We have previously exploited a set of amyloid ? peptide (A?42) variants exhibiting a continuous gradient of intrinsic aggregation propensities to demonstrate that this rule applies in vivo in bacteria. In the present work we have characterized the behavior of these A?42 mutants when expressed in yeast. In contrast to bacteria, the intrinsic aggregation propensity is gated by yeast, in such a way that this property correlates with the formation of intracellular inclusions only above a specific aggregation threshold. Proteins displaying solubility levels above this threshold escape the inclusion formation pathway. In addition, the most aggregation-prone variants are selectively cleared by the yeast quality control degradation machinery. Thus, both inclusion formation and proteolysis target the same aggregation-prone variants and cooperate to minimize the presence of these potentially dangerous species in the cytosol. The demonstration that sorting to these pathways in eukaryotes is strongly influenced by protein primary sequence should facilitate the development of rational approaches to predict and hopefully prevent in vivo protein deposition.
Fungal prions are protein-based genetic elements. Sup35 and Ure2p constitute the best-characterized prion proteins in the yeast Saccharomyces cerevisiae. No high-resolution molecular models of the amyloid conformations adopted by the prion domains of these proteins are available yet. A quantitative description of the kinetics and thermodynamics of their self-assembly processes might provide clues on the nature of the structural changes originating their heritable and transmissible phenotypes. Here we study the temperature dependence of Sup35 and Ure2p amyloid fibril nucleation and elongation reactions at physiological pH. Both processes follow the Arrhenius law, allowing calculation of their associated thermodynamic activation parameters. Although the Gibbs energies (?G*) for the nucleation and elongation of both prions are similar, the enthalpic and entropic contributions to these two processes are dramatically different. In addition, the structural properties of the two types of prion fibrils exhibit different dependence on the polymerization temperature. Overall, we show here that the amyloidogenic pathways of Sup35 and Ure2p prions diverge significantly.
The formation of aggregates by misfolded proteins is thought to be inherently toxic, affecting cell fitness. This observation has led to the suggestion that selection against protein aggregation might be a major constraint on protein evolution. The precise fitness cost associated with protein aggregation has been traditionally difficult to evaluate. Moreover, it is not known if the detrimental effect of aggregates on cell physiology is generic or depends on the specific structural features of the protein deposit. In bacteria, the accumulation of intracellular protein aggregates reduces cell reproductive ability, promoting cellular aging. Here, we exploit the cell division defects promoted by the intracellular aggregation of Alzheimers-disease-related amyloid ? peptide in bacteria to demonstrate that the fitness cost associated with protein misfolding and aggregation is connected to the protein sequence, which controls both the in vivo aggregation rates and the conformational properties of the aggregates. We also show that the deleterious impact of protein aggregation on bacterial division can be buffered by molecular chaperones, likely broadening the sequential space on which natural selection can act. Overall, the results in the present work have potential implications for the evolution of proteins and provide a robust system to experimentally model and quantify the impact of protein aggregation on cell fitness.
The formation of insoluble protein deposits in human tissues is linked to the onset of more than 40 different disorders, ranging from dementia to diabetes. In these diseases, the proteins usually self-assemble into ordered ?-sheet enriched aggregates known as amyloid fibrils. Here we study the structure of the inclusions formed by maize transglutaminase (TGZ) in the chloroplasts of tobacco transplastomic plants and demonstrate that they have an amyloid-like nature. Together with the evidence of amyloid structures in bacteria and fungi our data argue that amyloid formation is likely a ubiquitous process occurring across the different kingdoms of life. The discovery of amyloid conformations inside inclusions of genetically modified plants might have implications regarding their use for human applications.
Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. Here, we report an evaluation of fructan synthesis in three Lactobacillus gasseri strains, identification of the fructansucrase-encoding genes and characterization of the recombinant proteins and fructan (oligosaccharide) products. High-performance anion-exchange chromatography and nuclear magnetic resonance analysis of the fructo-oligosaccharides (FOS) and polymers produced by the L. gasseri strains and the recombinant enzymes revealed that, in situ, L. gasseri strains DSM 20604 and 20077 synthesize inulin (and oligosaccharides) and levan products, respectively. L. gasseri DSM 20604 is only the second Lactobacillus strain shown to produce inulin polymer and FOS in situ, and is unique in its distribution of FOS synthesized, ranging from DP2 to DP13. The probiotic bacterium L. gasseri DSM 20243 did not produce any fructan, although we identified a fructansucrase-encoding gene in its genome sequence. Further studies showed that this L. gasseri DSM 20243 gene was prematurely terminated by a stop codon. Exchanging the stop codon for a glutamine codon resulted in a recombinant enzyme producing inulin and FOS. The three recombinant fructansucrase enzymes characterized from three different L. gasseri strains have very similar primary protein structures, yet synthesize different fructan products. An interesting feature of the L. gasseri strains is that they were unable to ferment raffinose, whereas their respective recombinant enzymes converted raffinose into fructan and FOS.
An increasing number of proteins are being shown to assemble into amyloid structures, self-seeding fibrillar aggregates that may lead to pathological states or play essential biological functions in organisms. Bacterial cell factories have raised as privileged model systems to understand the mechanisms behind amyloid assembly and the cellular fitness cost associated to the formation of these aggregates. In the near future, these bacterial systems will allow implementing high-throughput screening approaches to identify effective modulators of amyloid aggregation.
Protein aggregation is being found to be associated with an increasing number of human diseases. Aggregation can lead to a loss of function (lack of active protein) or to a toxic gain of function (cytotoxicity associated with protein aggregates). Although potentially harmful, protein sequences predisposed to aggregation seem to be ubiquitous in all kingdoms of life, which suggests an evolutionary advantage to having such segments in polypeptide sequences. In fact, aggregation-prone segments are essential for protein folding and for mediating certain protein-protein interactions. Moreover, cells use protein aggregates for a wide range of functions. Against this background, life has adapted to tolerate the presence of potentially dangerous aggregation-prone sequences by constraining and counteracting the aggregation process. In the present review, we summarize the current knowledge of the advantages associated with aggregation-prone stretches in proteomes and the strategies that cellular systems have developed to control the aggregation process.
Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively.
The amyloid-? peptide (A?42) is the main component of the inter-neuronal amyloid plaques characteristic of Alzheimers disease (AD). The mechanism by which A?42 and other amyloid peptides assemble into insoluble neurotoxic deposits is still not completely understood and multiple factors have been reported to trigger their formation. In particular, the presence of endogenous metal ions has been linked to the pathogenesis of AD and other neurodegenerative disorders.
Limited data exists on midterm results concerning paediatric interventions on stenotic or occluded systemic veins following indwelling lines, cardiac surgery, or catheterisations. The purpose of this study was to report our acute and intermediate results concerning patients with (Group A) and without (Group B) congenital heart diseases (CHD) over a 10-year period.
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