Molecular mechanism for the synchronized electrostatic coacervation and co-aggregation of a-synuclein and tau

Amyloid aggregation of α-synuclein (αS) is the hallmark of Parkinson’s disease and other synucleinopathies. Recently, Tau protein, generally associated with Alzheimer’s disease, has been linked to αS pathology and observed to co- localize in αS-rich disease inclusions, although the molecular mechanisms for the co-aggregation of both proteins remain elusive. We report here that αS phase-separates into liquid condensates by electrostatic complex coacerva- tion with positively charged polypeptides such as Tau. Condensates undergo either fast gelation or coalescence followed by slow amyloid aggregation depending on the affinity of αS for the poly-cation and the rate of valence exhaustion of the condensate network. By combining a set of advanced bio- physical techniques, we have been able to characterize αS/Tau liquid-liquid phase separation and identified key factors that lead to the formation of hetero-aggregates containing both proteins in the interior of the liquid protein condensates

Interrogating peroxidase intermediate I : a rapid freeze quench \u2013 EPR approach

Abstract: This work is focused on the in-depth spectroscopic analysis of horseradish peroxidase (HRP) and chloroperoxidase (CPO), proteins selected for their scientific and industrial relevance. HRP, being extensively employed in immunoassays, has a crucial biochemical role, while CPO's structural and functional resemblance to cytochrome P450 emphasizes its importance in pharmacology. The enzymatic cycle of both peroxidases goes via an oxidized intermediate state, called Compound I. It stores two oxidizing equivalents, one in an iron(IV)-oxo moiety, and one as a free radical on the porphyrin ring or on an amino acid in the protein. Its investigation forms a central aspect of this research. Although abundantly studied, the exact description of the difference in the electronic structure of Compound I of various heme proteins remains challenging. The employed methodologies consist of established techniques, notably Continuous-Wave and Pulsed Electron Paramagnetic Resonance (EPR), UV-Vis spectroscopy, and stopped-flow spectrophotometry, alongside novel methods devised by the author. UV-Vis spectroscopy and stopped-flow spectrophotometry serve as standard tools in biotechnology, allowing the basic characterization and reaction kinetics of enzyme resting states and intermediates involved in enzyme turnover. Pulsed EPR, utilised in an attempt to elucidate the electron spin distribution of the resting state and Compound I forms of HRP and CPO, offers advantages in examining hyperfine interaction between unpaired electron(s) and surrounding nuclei. With the intention of enhance CPO production, an innovative solid substrate method has been explored, promising potential for scalability. Caldariomyces fumago-derived CPO production presents specific challenges due to cultivation and purification demands. Yet, the solid substrate method offers advantages, including increased yields, higher purity and cost-effectiveness, crucial for industrial applications. Furthermore, the author made crucial contributions to the developmental stages of three different rapid freeze quench apparatus used for trapping Compound I from CPO immediately after the initiation of the catalytic cycle. This involvement spanned from mechanical and technical creation to test, improvement and calibration of all three systems. EPR is a fundamental tool in exploring the electronic state of paramagnetic centers in proteins, such as the resting state and Compound I in HRP and CPO. M. Bracci ix Pulsed EPR techniques, such as HYSCORE and ENDOR, were utilized to investigate the resting state of both enzymes, providing insights into the electronic structure of their active centers and highlight their differences. To aid in the interpretation of HYSCORE spectra, a user-friendly graphical interface was developed, based on MATLAB and EasySpin, enabling researchers to manipulate experimental data and conduct simulations, even with minimal coding expertise. A theoretical model of the electronic structure of Compound I, previously proposed and based on the crystal field theory, was revisited. Starting from the energy of the t2g levels of Fe(IV) and a model for the magnetic interaction between the ion and the radical, this theory is suitable for interpreting the features of continuous wave EPR spectra, as demonstrated by the tests performed to literature data on the Compound I of different heme proteins. Subsequently, it was effectively used to simulate the CW EPR spectra of the intermediate of the proteins under investigation in this study. Finally, HYSCORE and ENDOR of the Compound I form of HRP and CPO revealed crucial differences in their 14N and 1H hyperfine couplings, which could be related to the difference in location of the radical

Molecular mechanism for the synchronized electrostatic coacervation and co-aggregation of alpha-synuclein and tau

16 pags., 8 figs.Amyloid aggregation of α-synuclein (αS) is the hallmark of Parkinson’s disease and other synucleinopathies. Recently, Tau protein, generally associated with Alzheimer’s disease, has been linked to αS pathology and observed to co-localize in αS-rich disease inclusions, although the molecular mechanisms for the co-aggregation of both proteins remain elusive. We report here that αS phase-separates into liquid condensates by electrostatic complex coacervation with positively charged polypeptides such as Tau. Condensates undergo either fast gelation or coalescence followed by slow amyloid aggregation depending on the affinity of αS for the poly-cation and the rate of valence exhaustion of the condensate network. By combining a set of advanced biophysical techniques, we have been able to characterize αS/Tau liquid-liquid phase separation and identified key factors that lead to the formation of hetero-aggregates containing both proteins in the interior of the liquid protein condensates.This work has been funded by MCIN/AEI/ 10.13039/501100011033 and “ERDF A way of making Europe”, by the “European Union” (grants: PGC2018-096335-B100 to N.C., PID2019-109276RA-I00 to J.O. and PID2019-109306RB-I00 to D.V.L.) and the Centro Universitario de la Defensa de Zaragoza-University of Zaragoza (grants: UZCUD2019-BIO-01 and UZCUD2020‐BIO‐01 to N.C. and I.G.), and is also part of a project that has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 813209 (I.G.). J.O. is a Ramón y Cajal Fellow (grant RYC2018-026042-I funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”). NMR experiments were performed in the “Manuel Rico” NMR Laboratory (LMR) of the Spanish National Research Council (CSIC), a node of the Spanish Large-Scale National Facility (ICTS R-LRB)