Catechol-induced covalent modifications modulate the aggregation tendency of α-synuclein: An in-solution and in-silico study.

Parkinson's disease (PD) stands as a challenging neurodegenerative condition characterized by the emergence of Lewy Bodies (LBs), intracellular inclusions within dopaminergic neurons. These LBs harbor various proteins, prominently including α-Synuclein (Syn) aggregates, implicated in disease pathology. A promising avenue in PD treatment involves targeting Syn aggregation. Recent findings from our research have shown that 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET) possess the ability to impede the formation of Syn fibrils by disrupting the aggregation process. Notably, these compounds primarily engage in noncovalent interactions with the protein, leading to the formation of off-pathway oligomers that deter fibril growth. Through proteolysis studies and mass spectrometry (MS) analysis, we have identified potential covalent modifications of Syn in the presence of DOPAC, although the exact site remains elusive. Employing molecular dynamics simulations, we delved into how DOPAC-induced covalent alterations might affect the mechanism of Syn aggregation. Our findings indicate that the addition of a covalent adduct on certain residues enhances fibril flexibility without compromising its secondary structure stability. Furthermore, in the monomeric state, the modified residue fosters novel bonding interactions, thereby influencing long-range interactions between the N- and C-termini of the protein.

Assessing the physicochemical stability and intracellular trafficking of mRNA-based COVID-19 vaccines.

The emergence of SARS-CoV-2 in Wuhan, China in 2019 has had a profound impact on humanity in every facet. While vaccines against this viral pathogen have been approved a year later, limitations to this therapeutic intervention persist, such as drug sensitivity to transportation and storage conditions, as well as significant financial losses from non-injected resuspended vials. Our research delves into the effects of thermal denaturation (4 - 40 °C) and light irradiation (720 and 10460 kJ/m2) on the mRNA-based vaccines BNT162b2 from BioNTech/Pfizer and mRNA-1273 from Moderna. We also investigated vaccine stability following incubation in syringes to simulate potential interactions with silicon oil. By assaying the effects of these stressors via biochemical and biophysical methods, we aim to elucidate the physicochemical properties, integrity, and stability of these mRNA-based vaccines. Furthermore, the incorporation of a fluorophore into both vaccines allowed us to monitor their localization within cells and assess their capacity to evade vesicular transport mechanisms, thus evaluating the differences between the two formulations. A comprehensive understanding of the aforementioned attributes can enable the establishment of optimal storage and manipulation conditions for these vaccines, thereby ensuring their safe and efficacious application while minimizing the waste of functional and safe therapeutic agents.

Environmental Signals Act as a Driving Force for Metabolic and Defense Responses in the Antarctic Plant Colobanthus quitensis.

During evolution, plants have faced countless stresses of both biotic and abiotic nature developing very effective mechanisms able to perceive and counteract adverse signals. The biggest challenge is the ability to fine-tune the trade-off between plant growth and stress resistance. The Antarctic plant Colobanthus quitensis has managed to survive the adverse environmental conditions of the white continent and can be considered a wonderful example of adaptation to prohibitive conditions for millions of other plant species. Due to the progressive environmental change that the Antarctic Peninsula has undergone over time, a more comprehensive overview of the metabolic features of C. quitensis becomes particularly interesting to assess its ability to respond to environmental stresses. To this end, a differential proteomic approach was used to study the response of C. quitensis to different environmental cues. Many differentially expressed proteins were identified highlighting the rewiring of metabolic pathways as well as defense responses. Finally, a different modulation of oxidative stress response between different environmental sites was observed. The data collected in this paper add knowledge on the impact of environmental stimuli on plant metabolism and stress response by providing useful information on the trade-off between plant growth and defense mechanisms.

Biodiversity and Bioprospecting of Fungal Endophytes from the Antarctic Plant Colobanthus quitensis.

Microorganisms from extreme environments are considered as a new and valuable reservoir of bioactive molecules of biotechnological interest and are also utilized as tools for enhancing tolerance to (a)biotic stresses in crops. In this study, the fungal endophytic community associated with the leaves of the Antarctic angiosperm Colobanthus quitensis was investigated as a new source of bioactive molecules. We isolated 132 fungal strains and taxonomically annotated 26 representative isolates, which mainly belonged to the Basidiomycota division. Selected isolates of Trametes sp., Lenzites sp., Sistotrema sp., and Peniophora sp. displayed broad extracellular enzymatic profiles; fungal extracts from some of them showed dose-dependent antitumor activity and inhibited the formation of amyloid fibrils of α-synuclein and its pathological mutant E46K. Selected fungal isolates were also able to promote secondary root development and fresh weight increase in Arabidopsis and tomato and antagonize the growth of pathogenic fungi harmful to crops. This study emphasizes the ecological and biotechnological relevance of fungi from the Antarctic ecosystem and provides clues to the bioprospecting of Antarctic Basidiomycetes fungi for industrial, agricultural, and medical applications.

3,4-Dihydroxyphenylethanol and 3,4-dihydroxyphenylacetic acid affect the aggregation process of E46K variant of α-synuclein at different extent: Insights into the interplay between protein dynamics and catechol effect.

Parkinson's disease (PD) is a chronic multifactorial disease, whose etiology is not completely understood. The amyloid aggregation of α-synuclein (Syn) is considered a major cause in the development of the disease. The presence of genetic mutations can boost the aggregation of the protein and the likelihood to develop PD. These mutations can lead to early onset (A30P, E46K, and A53T) or late-onset (H50Q) forms of PD. The disease is also linked to an increase in oxidative stress and altered levels of dopamine metabolites. The molecular interaction of these molecules with Syn has been previously studied, while their effect on the pathological mutant structure and function is not completely clarified. By using biochemical and biophysical approaches, here we have studied the interaction of the familial variant E46K with two dopamine-derived catechols, 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylethanol. We show that the presence of these catechols causes a decrease in the formation of amyloid fibrils in a dose-dependent manner. Native- and Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) provide evidence that this effect is strongly conformation dependent. Indeed, these molecules interact differently with the interconverting conformers of Syn and its familial variant E46K in solution, selecting the most prone-to-aggregation one, confining it into an off-pathway oligomer. These findings suggest that catechols could be a molecular scaffold for the design of compounds potentially useful in the treatment of Parkinson's disease and related conditions.

Managing antibody stability: Effects of stressors on Ipilimumab from the commercial formulation to diluted solutions.

The stability of the monoclonal antibody Ipilimumab, the active ingredient of Yervoy®, used for the treatment of different types of cancer, has been investigated. Shaking/temperature, light exposure and dilution, protein drug renowned stressors, were applied on a 30-45-day series of experiments to observe the physicochemical and biological behavior of the molecule. Ipilimumab demonstrated stability under shaking and heat up to 45 days, without any unfolding during the induced combined stressors. Under artificial sunlight, the mAb showed to be sensitive even under the minimum dose tested (720 kJ/m2) with formation of aggregates, particularly when diluted in glucose solution. The light-induced soluble aggregates were higher in the case of diluted samples irradiated with much higher light doses (10460 kJ/m2). The aggregation of Ipilimumab took place also by irradiating the non-diluted formulation, indicating that the excipients did not protect completely the drug from photodegradation. Amino acid oxidation and deamidation were found. Anyway, after irradiation with both light doses, soluble Ipilimumab maintained its typical ß-sheets structure, and the tertiary structure was nearly maintained compared to the dark. As an additional stressor test, the effect of dilution on the formulation was monitored by using a saline solution (1 mg/mL Ipilimumab) applied during hospital infusion. After two days from dilution, the protein exhibited aggregation and chemical modifications including oxidation and deamidation. When stability conditions were compromised, the viability of human cell lines treated with the stressed formulation slight decreased suggesting low potential biological toxicity of the modified mAb. As this study has demonstrated the susceptibility of Ipilimumab to light, specific solutions, and excipients as well as the use of safe light in manufacturing, handling, and storage of this drug should be promoted. Moreover, the use of proper primary and secondary packaging should be indicated to avoid the detrimental effect of light on the mAb structure and efficacy. A detailed understanding of Ipilimumab physicochemical properties, integrity, and stability could assure the best storage and manipulation conditions for its safe and successful application in cancer therapy.

Structural Features and Toxicity of α-Synuclein Oligomers Grown in the Presence of DOPAC.

The interplay between α-synuclein and dopamine derivatives is associated with oxidative stress-dependent neurodegeneration in Parkinson's disease (PD). The formation in the dopaminergic neurons of intraneuronal inclusions containing aggregates of α-synuclein is a typical hallmark of PD. Even though the biochemical events underlying the aberrant aggregation of α-synuclein are not completely understood, strong evidence correlates this process with the levels of dopamine metabolites. In vitro, 3,4-dihydroxyphenylacetaldehyde (DOPAL) and the other two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET), share the property to inhibit the growth of mature amyloid fibrils of α-synuclein. Although this effect occurs with the formation of differently toxic products, the molecular basis of this inhibition is still unclear. Here, we provide information on the effect of DOPAC on the aggregation properties of α-synuclein and its ability to interact with membranes. DOPAC inhibits α-synuclein aggregation, stabilizing monomer and inducing the formation of dimers and trimers. DOPAC-induced oligomers did not undergo conformational transition in the presence of membranes, and penetrated the cell, where they triggered autophagic processes. Cellular assays showed that DOPAC reduced cytotoxicity and ROS production induced by α-synuclein aggregates. Our findings show that the early radicals resulting from DOPAC autoxidation produced covalent modifications of the protein, which were not by themselves a primary cause of either fibrillation or membrane binding inhibition. These findings are discussed in the light of the potential mechanism of DOPAC protection against the toxicity of α-synuclein aggregates to better understand protein and catecholamine biology and to eventually suggest a scaffold that can help in the design of candidate molecules able to interfere in α-synuclein aggregation.