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Abstract: Fungal laccases have great potential as biocatalysts oxidizing a variety of aromatic compounds using oxygen as co-substrate. Here, the crystal structure of 7D5 laccase (PDB 6H5Y), developed in Saccharomyces cerevisiae and overproduced in Aspergillus oryzae, is compared with that of the wild type produced by basidiomycete PM1 (Coriolopsis sp.), PDB 5ANH. SAXS showed both enzymes form monomers in solution, 7D5 laccase with a more oblate geometric structure due to heavier and more heterogeneous glycosylation. The enzyme presents superior catalytic constants towards all tested substrates, with no significant change in optimal pH or redox potential. It shows noticeable high catalytic efficiency with ABTS and dimethyl-4-phenylenediamine, 7 and 32 times better than the wild type, respectively. Computational simulations demonstrated a more favorable binding and electron transfer from the substrate to the T1 copper due to the introduced mutations. PM1 laccase is exceptionally stable to thermal inactivation (t1/2 70 °C = 1.2 h). Yet, both enzymes display outstanding structural robustness at high temperature. They keep folded during 2 h at 100 °C though, thereafter, 7D5 laccase unfolds faster. Rigidification of certain loops due to the mutations added on the protein surface would diminish the capability to absorb temperature fluctuations leading to earlier protein unfolding.

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Abstract: Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells’ limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2’s cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with μ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.

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Abstract: The emerging pharmacological target soluble epoxide hydrolase (sEH) is a bifunctional enzyme exhibiting two different catalytic activities, which are located in two distinct domains. Although the physiological role of the C-terminal hydrolase domain is well-investigated, little is known about its phosphatase activity located in the N-terminal domain of the sEH (sEH-P). Herein, we report the discovery and optimization of the first inhibitor of human and rat sEH-P, applicable in vivo. X-ray structure analysis of the sEH phosphatase domain complexed with an inhibitor provides insights in the molecular basis of small-molecule sEH-P inhibition and helps to rationalize the structure-activity relationships. 4-(4-(3,4-Dichlorophenyl)-5-phenyloxazol-2-yl)butanoic acid (22b, SWE101) has an excellent pharmacokinetic and pharmacodynamic profile in rats and enables the investigation of the physiological and pathophysiological role of sEH-P in vivo.

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Abstract: Copper (Cu) is required for maturation of cuproenzymes, cell proliferation, and angiogenesis, and its transport entails highly specific protein–protein interactions. In humans, the Cu chaperone Atox1 mediates Cu(I) delivery to P-type ATPases Atp7a and Atp7b (the Menkes and Wilson disease proteins, respectively), which are responsible for Cu release to the secretory pathway and excess Cu efflux. Cu(I) handover is believed to occur through the formation of three-coordinate intermediates where the metal ion is simultaneously linked to Atox1 and to a soluble domain of Cu–ATPases, both sharing a CxxC dithiol motif. The ultrahigh thermodynamic stability of chelating S-donor ligands secures the redox-active and potentially toxic Cu(I) ion, while their kinetic lability allows facile metal transfer. The same CxxC motifs can interact with and mediate the biological response to antitumor platinum drugs, which are among the most used chemotherapeutics. We show that cisplatin and an oxaliplatin analogue can specifically bind to the heterodimeric complex Atox1–Cu(I)–Mnk1 (Mnk1 is the first soluble domain of Atp7a), thus leading to a kinetically stable adduct that has been structurally characterized by solution NMR and X-ray crystallography. Of the two possible binding configurations of the Cu(I) ion in the cage made by the CxxC motifs of the two proteins, one (bidentate Atox1 and monodentate Mnk1) is less stable and more reactive toward cis-Pt(II) compounds, as shown by using mutated proteins. A Cu(I) ion can be retained at the Pt(II) coordination site but can be released to glutathione (a physiological thiol) or to other complexing agents. The Pt(II)-supported heterodimeric complex does not form if Zn(II) is used in place of Cu(I) and transplatin instead of cisplatin. The results indicate that Pt(II) drugs can specifically affect Cu(I) homeostasis by interfering with the rapid exchange of Cu(I) between Atox1 and Cu–ATPases with consequences on cancer cell viability and migration.

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Abstract: Copper (Cu) is essential to the growth and the morphological development of Streptomyces lividans. Understanding how Cu regulates the key development switches in this Gram-positive bacterium has been an area of extensive research. In particular, how Cu homeostasis and regulation are controlled and how metalation of enzymes important for morphological development is achieved have been previously investigated. To this end, this thesis reports the discovery of a cytosolic copper storage protein in S. lividans and offers new insight into intracellular Cu regulation, which is not under direct control of the Cu regulator protein CsoR (copper sensitive operon repressor). This copper storage protein belongs to a family of recently discovered cytosolic proteins known as Csp3. These members are exclusively found in the bacterial cytosol and comprise of a four-helix bundle that assemble into homotetramers and can bind between 70-80 Cu(I) ions through mainly Cys thiolate coordination. In Chapter 2 bioinformatic analyses reveals the phylogenetic distribution of Csp3 across Bacteria and Archaea and confirms the presence of Csp3 in S. lividans. Furthermore, the Csp3 in S. lividans is located in a gene environment that is sensitive to elevated Cu levels. Taxonomic distribution of these genes reveals a possible link to a novel transmembrane Cu export system that could facilitate removal of Cu from Csp3. X-ray structures of the apo and Cu(I) bound forms of the Csp3 from S. lividans have been determined and confirm a homotetramer assembly that can bind up to 80 Cu(I) ions (Chapter 3). The binding of Cu(I) ions in Csp3 is found to be cooperative with a Hill coefficient of 1.9 and Cu(I) can be transferred to Csp3 from a CopZ-like Cu(I) chaperone (Chapter 3). A Δcsp3 null-mutant in S. lividans reveals that Csp3 is operable at high Cu levels and this suggests it acts to provide an additional level of protection against Cu toxicity once the CsoR system becomes saturated (Chapter 3). The mechanism of Cu(I)-loading to Csp3 has also been investigated through X-ray crystallography, site-directed mutagenesis and stopped-flow reaction kinetics using aqueous Cu(I) and Cu(I) chelated by a donor. A clear role for a His residue (His107) leading to the formation of a tetranuclear [Cu4(μ2-S-Cys)4(Nδ1-His)] cluster is observed, followed by the loading of Cu(I) in a fluxional and dynamic manner (Chapters 4 and 5). Finally, over-expression studies of a putative transmembrane protein (SLI_RS17250) that is encoded by a neighbouring gene to the S. lividans Csp3 gene and could be part of a novel Cu export system, identified in Chapter 2, is described (Chapter 6).