RESUMO
An increasing number of cases where amyloids of different proteins are found in the same patient are being reported. This observation complicates diagnosis and clinical intervention. Amyloids of the amyloid-ß peptide or the protein α-synuclein are traditionally considered hallmarks of Alzheimer's and Parkinson's diseases, respectively. However, the co-occurrence of amyloids of these proteins has also been reported in patients diagnosed with either disease. Here, we show that soluble species containing amyloid-ß can induce the aggregation of α-synuclein. Fibrils formed under these conditions are solely composed of α-synuclein to which amyloid-ß can be found associated but not as part of the core of the fibrils. Importantly, by global kinetic analysis, we found that the aggregation of α-synuclein under these conditions occurs via heterogeneous primary nucleation, triggered by soluble aggregates containing amyloid-ß.
Assuntos
Peptídeos beta-Amiloides , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Cinética , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos , Amiloide/metabolismoRESUMO
Extracellular vesicles (EVs) are membrane-enclosed biological nanoparticles with potential as diagnostic markers and carriers for therapeutics. Characterization of EVs poses severe challenges due to their complex structure and composition, requiring the combination of orthogonal analytical techniques. Here, we demonstrate how liquid chromatography combined with multi-angle light scattering (MALS) and fluorescence detection in one single apparatus can provide multiparametric characterization of EV samples, including concentration of particles, average diameter of the particles, protein amount to particle number ratio, presence of EV surface markers and lipids, EV shape, and sample purity. The method requires a small amount of sample of approximately 107 EVs, limited handling of the sample and data analysis time in the order of minutes; it is fully automatable and can be applied to both crude and purified samples.
Assuntos
Vesículas Extracelulares , Vesículas Extracelulares/química , Cromatografia Líquida , Tamanho da PartículaRESUMO
The binding of small gas molecules such as NO and CO plays a major role in the signaling routes of the human body. The sole NO-receptor in humans is soluble guanylyl cyclase (sGC) - a histidine-ligated heme protein, which, upon NO binding, activates a downstream signaling cascade. Impairment of NO-signaling is linked, among others, to cardiovascular and inflammatory diseases. In the present work, we use a combination of theoretical tools such as MD simulations, high-level quantum chemical calculations and hybrid QM/MM methods to address various aspects of NO binding and to elucidate the most likely reaction paths and the potential intermediates of the reaction. As a model system, the H-NOX protein from Shewanella oneidensis (So H-NOX) homologous to the NO-binding domain of sGC is used. The signaling route is predicted to involve NO binding to form a six-coordinate intermediate heme-NO complex, followed by relatively facile His decoordination yielding a five-coordinate adduct with NO on the distal side with possible isomerization to the proximal side through binding of a second NO and release of the first one. MD simulations show that the His sidechain can quite easily rotate outward into solvent, with this motion being accompanied in our simulations by shifts in helix positions that are consistent with this decoordination leading to significant conformational change in the protein.
Assuntos
Química Computacional , Hemeproteínas , Heme/química , Hemeproteínas/química , Humanos , Óxido Nítrico/química , Ligação Proteica , Guanilil Ciclase Solúvel/química , Guanilil Ciclase Solúvel/metabolismoRESUMO
The synthesis of variously substituted indolo[2,3-c]quinolin-6(7H)-ones was developed via Pd-catalyzed intramolecular C-H arylation. This method highlights a strategy for preparing indoloquinoline precursors bearing versatile functional groups and provides a new approach for the synthesis of antimalarial isoneocryptolepine analogues. The plausible ring closure mechanism was examined with quantum chemical calculations, where a trigonal bipyramidal concerted metalation-deprotonation transition state is presumable.
Assuntos
Antimaláricos , Paládio , Alcaloides , Catálise , Estrutura Molecular , QuinolinasRESUMO
Nitrogen reduction reaction (N2RR) carried out on biomimetic catalytic systems is considered to be a promising alternative for the traditional Haber-Bosch ammonia synthesis. Unfortunately, the selectivity of the currently known biomimetic catalysts is poor, as they also catalyze the unproductive hydrogen evolution reaction (HER). In the present computational study, we examine the HER activity of early N2RR intermediates in EP3 (E = B, Si) ligated single-site biomimetic iron complexes by calculating and comparing the activation Gibbs free energies of HER and N2RR elementary steps. We find that, in contrast to previous suggestions, early N2RR intermediates are not likely sources of HER under turnover conditions, as the barriers of the competing N2RR steps are significantly lower. Consequently, future research should focus on preventing other potential HER mechanisms, e.g., hydride formation, rather than accelerating the consumption of early N2RR intermediates as proposed earlier to design more efficient biomimetic catalysts.
RESUMO
Biomimetic nitrogen fixation provides an attractive alternative for the century-old Haber-Bosch process; however, the performance of the currently available molecular biomimetic catalysts is very limited. In this work, we are aiming to understand the catalytic cycle of one of the most promising biomimetic complex families that can be the cornerstone of future computer-aided rational design of biomimetic complexes. We calculate the Gibbs free energy of all elementary reaction steps of homogeneous dinitrogen reduction to NH3 on single-site iron complexes with EPPP tetradentate ligands (E = B, Si). We examine all possible mechanisms and identify the dominant pathways and the critical elementary steps that can be rate-determining in the catalytic cycle of nitrogen fixation. We find that the catalytic mechanism depends on the applied ligand and that the distal pathway observed with E = B is the most favorable route regarding the catalytic performance. Our calculations also reveal the lack of thermodynamic driving force in the last steps of the catalytic cycle that can be responsible for the low catalytic activity of the studied biomimetic catalysts. Our results can serve as a starting point for the rational design of biomimetic complexes, which should focus on establishing a steadily decreasing Gibbs free energy profile, as suggested by the Sabatier principle.
RESUMO
Several RNA binding proteins involved in membraneless organelles can form pathological amyloids associated with neurodegenerative diseases, but the mechanisms of how this aggregation is modulated remain elusive. Here we investigate how heterotypic protein-RNA interactions modulate the condensation and the liquid to amyloid transition of hnRNPA1A, a protein involved in amyothropic lateral sclerosis. In the absence of RNA, formation of condensates promotes hnRNPA1A aggregation and fibrils are localized at the interface of the condensates. Addition of RNA modulates the soluble to amyloid transition of hnRNPA1A according to different pathways depending on RNA/protein stoichiometry. At low RNA concentrations, RNA promotes both condensation and amyloid formation, and the catalytic effect of RNA adds to the role of the interface between the dense and dilute phases. At higher RNA concentrations, condensation is suppressed according to re-entrant phase behaviour but formation of hnRNPA1A amyloids is observed over longer incubation times. Our findings show how heterotypic nucleic acid-protein interactions affect the kinetics and molecular pathways of amyloid formation.
Assuntos
Amiloide , Ribonucleoproteína Nuclear Heterogênea A1 , RNA , Ribonucleoproteína Nuclear Heterogênea A1/química , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Amiloide/química , Amiloide/metabolismo , RNA/química , RNA/metabolismo , Humanos , Condensados Biomoleculares/química , Condensados Biomoleculares/metabolismo , CinéticaRESUMO
The mechanisms that underlie the regulation of enzymatic reactions by biomolecular condensates and how they scale with compartment size remain poorly understood. Here we use intrinsically disordered domains as building blocks to generate programmable enzymatic condensates of NADH-oxidase (NOX) with different sizes spanning from nanometers to microns. These disordered domains, derived from three distinct RNA-binding proteins, each possessing different net charge, result in the formation of condensates characterized by a comparable high local concentration of the enzyme yet within distinct environments. We show that only condensates with the highest recruitment of substrate and cofactor exhibit an increase in enzymatic activity. Notably, we observe an enhancement in enzymatic rate across a wide range of condensate sizes, from nanometers to microns, indicating that emergent properties of condensates can arise within assemblies as small as nanometers. Furthermore, we show a larger rate enhancement in smaller condensates. Our findings demonstrate the ability of condensates to modulate enzymatic reactions by creating distinct effective solvent environments compared to the surrounding solution, with implications for the design of protein-based heterogeneous biocatalysts.