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Post-translational modifications of Tau are emerging as key players in determining the onset and progression of different tauopathies such as Alzheimer's disease, and are recognized to mediate the structural diversity of the disease-specific Tau amyloids. Here we show that the E3 ligase CHIP catalyzes the site-specific ubiquitination of Tau filaments both in vitro and in cellular models, proving that also Tau amyloid aggregates are direct substrate of PTMs. Transmission electron microscopy and mass spectrometry analysis on ubiquitin-modified Tau amyloids revealed that the conformation of the filaments restricts CHIP-mediated ubiquitination to specific positions of the repeat domain, while only minor alterations in the structure of the fibril core were inferred using seeding experiments in vitro and in a cell-based tauopathy model. Overexpression of CHIP significantly increased the ubiquitination of exogenous PHF, proving that the ligase can interact and modify Tau aggregates also in a complex cellular environment., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Water-soluble melanin-protein-Fe/Cu conjugates derived from norepinephrine and fibrillar β-lactoglobulin are reliable models for neuromelanin (NM) of human brain locus coeruleus. Both iron and copper promote catecholamine oxidation and exhibit strong tendency to remain coupled in oligonuclear aggregates. The Fe-Cu clusters are EPR silent and affect the 1 H NMR spectra of the conjugates through a specific sequence of signals. Derivatives containing only Fe or Cu exhibit different NMR patterns. The EPR spectra show weak signals of paramagnetic Fe III in conjugates containing Fe or mixed Fe-Cu sites due to small amounts of mononuclear centers. The latter derivatives exhibit EPR signals for isolated Cu II centers. These features parallel the EPR behavior of NM from locus coeruleus. The spectral data indicate that Fe III is bound to the melanic fraction, whereas Cu II is bound on the protein fibrils, suggesting that the Fe-Cu clusters occur at the interface between the two components of the synthetic NMs., (© 2022 Wiley-VCH GmbH.)

Tyrosinase enzymes (Tys) are involved in the key steps of melanin (protective pigments) biosynthesis and molecules targeting the binuclear copper active site on tyrosinases represent a relevant strategy to regulate enzyme activities. In this work, the possible synergic effect generated by a combination of known inhibitors is studied. For this, derivatives containing kojic acid (KA) and 2-hydroxypyridine-N-oxide (HOPNO) combined with a thiosemicarbazone (TSC) moiety were synthetized. Their inhibition activities were evaluated on purified tyrosinases from different sources (mushroom, bacterial, and human) as well as on melanin production by lysates from the human melanoma MNT-1 cell line. Results showed significant enhancement of the inhibitory effects compared with the parent compounds, in particular for HOPNO-TSC. To elucidate the interaction mode with the dicopper(II) active site, binding studies with a tyrosinase bio-inspired model of the dicopper(II) center were investigated. The structure of the isolated adduct between one ditopic inhibitor (KA-TSC) and the model complex reveals that the binding to a dicopper center can occur with both chelating sites. Computational studies on model complexes and docking studies on enzymes led to the identification of KA and HOPNO moieties as interacting groups with the dicopper active site., (© 2020 Wiley-VCH GmbH.)

Proteins reconfigure their 3D-structure, and consequently their function, under the control of specific molecular interactions that sense, process and transmit information from the surrounding environment. When this fundamental process is hampered, many pathologies occur as in the case of protein misfolding diseases. In this work, we follow the early steps of α-synuclein (aS) aggregation, a process associated with Parkinson's disease etiopathogenesis, that is promptly promoted by a light-mediated binding between the protein and a photoactive foldamer. The latter can switch between two conformations, one of which generates supramolecular fibrillar seeds that act as molecular templates able to induce a fast β-sheet transition for aS monomers that successively undergo fibrillar polymerization. The proposed method represents a powerful tool to study protein aggregation relevant to misfolding diseases in a controlled and inducible system., (© 2020 Wiley-VCH GmbH.)

In the brain of individuals with Alzheimer's disease, the regulatory protein ubiquitin is found conjugated to different lysine residues of tau protein assembled into pathological paired helical filaments. To shed light on the hitherto unexplored ubiquitination-linked conformational transitions of tau, the availability of in vitro ubiquitin conjugation methods is of primary importance. In our work, we focused on the four-repeat domain of tau and assembled an enzymatic machinery formed by UBE1, Ubc13, and CHIP enzymes. The enzymatic reaction resulted in monoubiquitination at multiple sites, reminiscent of the ubiquitination pattern observed in vivo. We further exploited chemoselective disulfide coupling reactions to construct three tau regioisomers with site-specific monoubiquitination. Protein aggregation experiments revealed that the multiple enzyme-derived products were unable to convert into amyloid fibrils, while the semisynthetic conjugates exhibited diverse capability to form filaments. This study contributes novel insight into the effects of a key post-translational modification on aberrant protein self-assembly., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The deciphering of the binding mode of tyrosinase (Ty) inhibitors is essential to understand how to regulate the tyrosinase activity. In this paper, by combining experimental and theoretical methods, we studied an unsymmetrical tyrosinase functional model and its interaction with 2-hydroxypyridine-N-oxide (HOPNO), a new and efficient competitive inhibitor for bacterial Ty. The tyrosinase model was a dinuclear copper complex bridged by a chelated ring with two different complexing arms (namely (bis(2-ethylpyridyl)amino)methyl and (bis(2-methylpyridyl)amino)methyl). The geometrical asymmetry of the complex induces an unsymmetrical binding of HOPNO. Comparisons have been made with the binding modes obtained on similar symmetrical complexes. Finally, by using quantum mechanics/molecular mechanics (QM/MM) calculations, we studied the binding mode in tyrosinase from a bacterial source. A new unsymmetrical binding mode was obtained, which was linked to the second coordination sphere of the enzyme., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A fluorescence-based system to sense oxygen in solution is described. The method exploits the sensitivity of the endogenous fluorescence of type-3 copper proteins towards the presence of oxygen by translating the near-UV emission of the protein to label fluorescence in the visible range through a FRET mechanism. The main protein in this study, a recombinant tyrosinase from the soil bacterium Streptomyces antibioticus, has been covalently labeled with a variety of fluorescent dye molecules with emission maxima spanning the whole visible wavelength range. In all cases, the emission of the label varied considerably between O2-bound and O2-free protein with a contrast exceeding that of the Trp emission for some labels. It is shown that different constructs may be simultaneously observed using a single excitation wavelength. Next to the described application in oxygen sensing, the method may be applicable to any protein showing variations in tryptophan fluorescence, for example as a function of ligand binding or catalysis.

The (1)H NMR relaxation characteristics of the histidines in the oxidised type-3 copper site of tyrosinase (Ty(met)) from the bacterium Streptomyces antibioticus in the halide-bound forms (Ty(met)X with X = F(-), Cl(-), Br(-)) have been determined and analysed. The (1)H NMR spectra of the Ty(met)X species display remarkably sharp, well-resolved, paramagnetically shifted (1)H signals, which originate from the protons of the six His residues coordinated to the two Cu(II) ions in the type-3 centre. From the temperature-dependence of the (1)H paramagnetic shifts the following values for the exchange-coupling parameter -2J were determined: 260 (Ty(met)F), 200 (Ty(met)Cl) and 162 cm(-1) (Ty(met)Br). The (1)H T(1) relaxation is dipolar in origin and correlates with the Cu--H distances. Electronic relaxation times tau(S) derived from the (1)H T(1) data amount to about 10(-11) s and follow the order Ty(met)F>Ty(met)Cl>Ty(met)Br. They are two orders of magnitude shorter than the tau(S) values reported for mononuclear copper systems, in accordance with the sharpness of the (1)H signals. The results corroborate the Cu(2) bridging mode of the halide ions. On the basis of the measured hyperfine interaction constants for the ligand histidine nuclei, it is concluded that 70-80 % of the spin density in the excited triplet state resides on the two copper ions and the bridging atoms.

The activity of mushroom tyrosinase towards a representative series of phenolic and diphenolic substrates structurally related to tyrosine has been investigated in a mixed solvent of 34.4% methanol-glycerol (7:1, v/v) and 65.6% (v/v) aqueous 50 mM Hepes buffer at pH 6.8 at various temperatures. The kinetic activation parameters controlling the enzymatic reactions and the thermodynamic parameters associated with the process of substrate binding to the enzyme active species have been deduced from the temperature variation of the kcat and KM parameters. The activation free energy is dominated by the enthalpic term, the value of which lies in the relatively narrow range of 61+/-9 kJ mol(-1) irrespective of substrate or reaction type (monophenolase or diphenolase). The activation entropies are small and generally negative and contribute no more than 10% to the activation free energy. The substrate binding parameters are characterized by large and negative enthalpy and entropy contributions, which are typically dictated by polar protein-substrate interactions. The substrate 4-hydroxyphenylpropionic acid exhibits a strikingly anomalous temperature dependence of the enzymatic oxidation rate, with deltaH(double dagger) approximately = 150 kJ mol(-1) and deltaS(double dagger) approximately = 280 J K(-1) mol(-1), due to the fact that it can competitively bind to the enzyme through the phenol group, like the other substrates, or the carboxylate group, like carboxylic acid inhibitors. A kinetic model that takes into account the dual substrate/inhibitor nature of this compound enables rationalization of this anomalous behavior.