Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. ...

Universitat Autònoma de Barcelona

The aggregation of a large variety of amyloidogenic proteins is linked to the onset of devastating human disorders. Therefore, there is an urgent need for effective molecules able to modulate the aggregative properties of these polypeptides in their natural environment, in order to prevent, delay or halt the progression of such diseases. On the one hand, the complexity and cost of animal models make them inefficient at early stages of drug discovery, where large chemical libraries are usually screened. On the other hand, in vitro aggregation assays in aqueous solutions hardly reproduce (patho)physiological conditions. In this context, because the formation of insoluble aggregates in bacteria shares mechanistic and functional properties with amyloid self-assembly in higher organisms, they have emerged as a promising system to model aggregation in the cell. Here we show that bacteria provide a powerful and cost-effective system to screen for amyloid inhibitors using fluorescence spectroscopy and flow cytometry, thanks to the ability of the novel red fluorescent ProteoStat dye to detect specifically intracellular amyloid-like aggregates. We validated the approach using the Alzheimer's linked Aβ40 and Aβ42 peptides and tacrine- and huprine-based aggregation inhibitors. Overall, the present method bears the potential to replace classical in vitro anti-aggregation assays. 

A fast and specific method to screen for intracellular amyloid inhibitors using bacterial model systems.

Protein aggregation and amyloid formation is a hallmark of an increasing number of human disorders. Because protein aggregation is deleterious for the cell physiology and results in a decrease in overall cell fitness, it is thought that natural selection acts to purify aggregating proteins during evolution. This data article contains complementary figures and results related to the research article entitled "Selection against toxic aggregation-prone protein sequences in bacteria" (Navarro et al., 2014) [1]. Here, we used the AGGRESCAN3D (A3D) server, a novel in house predictor that forecasts protein aggregation properties in protein structures to illustrate a striking correlation between the structure-based predictions of aggregation propensities for Alzheimer's Aβ42 peptide variants and their previously reported deleterious effects in bacteria. 

Data on correlation between Aβ42 structural aggregation propensity and toxicity in bacteria.

Synucleinopathies are a group of progressive disorders characterized by the abnormal aggregation and accumulation of α-synuclein (aSyn), an abundant neuronal protein that can adopt different conformations and biological properties. Recently, aSyn pathology was shown to spread between neurons in a prion-like manner. Proteins like aSyn that exhibit self-propagating capacity appear to be able to adopt different stable conformational states, known as protein strains, which can be modulated both by environmental and by protein-intrinsic factors. Here, we analyzed these factors and found that the unique combination of the neurodegeneration-related metal copper and the pathological H50Q aSyn mutation induces a significant alteration in the aggregation properties of aSyn. We compared the aggregation of WT and H50Q aSyn with and without copper, and assessed the effects of the resultant protein species when applied to primary neuronal cultures. The presence of copper induces the formation of structurally different and less-damaging aSyn aggregates. Interestingly, these aggregates exhibit a stronger capacity to induce aSyn inclusion formation in recipient cells, which demonstrates that the structural features of aSyn species determine their effect in neuronal cells and supports a lack of correlation between toxicity and inclusion formation. In total, our study provides strong support in favor of the hypothesis that protein aggregation is not a primary cause of cytotoxicity.

Environmental and genetic factors support the dissociation between α-synuclein aggregation and toxicity.

α-synuclein (aSyn) is associated with both sporadic and familial forms of Parkinson's disease (PD), the second most common neurodegenerative disorder after Alzheimer's disease. In particular, multiplications and point mutations in the gene encoding for aSyn cause familial forms of PD. Moreover, the accumulation of aSyn in Lewy Bodies and Lewy neurites in disorders such as PD, dementia with Lewy bodies, or multiple system atrophy, suggests aSyn misfolding and aggregation plays an important role in these disorders, collectively known as synucleinopathies. The exact function of aSyn remains unclear, but it is known to be associated with vesicles and membranes, and to have an impact on important cellular functions such as intracellular trafficking and protein degradation systems, leading to cellular pathologies that can be readily studied in cell-based models. Thus, understanding the molecular effects of aSyn point mutations may provide important insight into the molecular mechanisms underlying disease onset.We investigated the effect of the recently identified A53E aSyn mutation. Combining in vitro studies with studies in cell models, we found that this mutation reduces aSyn aggregation and increases proteasome activity, altering normal proteostasis.We observed that, in our experimental paradigms, the A53E mutation affects specific steps of the aggregation process of aSyn and different cellular processes, providing novel ideas about the molecular mechanisms involved in synucleinopathies.

The effects of the novel A53E alpha-synuclein mutation on its oligomerization and aggregation.

Protein misfolding and aggregation have been associated with the onset of neurodegenerative disorders. Recent studies demonstrate that the aggregation process can result in a high diversity of protein conformational states, however the identity of the specific species responsible for the cellular damage is still unclear. Here, we use yeast as a model to systematically analyse the intracellular effect of expressing 21 variants of the amyloid-ß-peptide, engineered to cover a continuous range of intrinsic aggregation propensities. We demonstrate the existence of a striking negative correlation between the aggregation propensity of a given variant and the oxidative stress it elicits. Interestingly, each variant generates a specific distribution of protein assemblies in the cell. This allowed us to identify the aggregated species that remain diffusely distributed in the cytosol and are unable to coalesce into large protein inclusions as those causing the highest levels of oxidative damage. Overall, our results indicate that the formation of large insoluble aggregates may act as a protective mechanism to avoid cellular oxidative stress.

Anita Carija

Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular

Universitat Autònoma de Barcelona

Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

Anita Carija

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular

Universitat Autònoma de Barcelona

Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular