Unveiling the membrane bound dihydroorotate: Quinone oxidoreductase from Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen and one of the most frequent causes for community acquired and nosocomial bacterial infections. Even so, its energy metabolism is still under explored and its respiratory enzymes have been vastly overlooked. In this work, we unveil the dihydroorotate:quinone oxidoreductase (DHOQO) from S. aureus, the first example of a DHOQO from a Gram-positive organism. This protein was shown to be a FMN containing menaquinone reducing enzyme, presenting a Michaelis-Menten behaviour towards the two substrates, which was inhibited by Brequinar, Leflunomide, Lapachol, HQNO, Atovaquone and TFFA with different degrees of effectiveness. Deletion of the DHOQO coding gene (Δdhoqo) led to lower bacterial growth rates, and effected in cell morphology and metabolism, most importantly in the pyrimidine biosynthesis, here systematized for S. aureus MW2 for the first time. This work unveils the existence of a functional DHOQO in the respiratory chain of the pathogenic bacterium S. aureus, enlarging the understanding of its energy metabolism.

Dynamic interactions and Ca2+-binding modulate the holdase-type chaperone activity of S100B preventing tau aggregation and seeding.

The microtubule-associated protein tau is implicated in the formation of oligomers and fibrillar aggregates that evade proteostasis control and spread from cell-to-cell. Tau pathology is accompanied by sustained neuroinflammation and, while the release of alarmin mediators aggravates disease at late stages, early inflammatory responses encompass protective functions. This is the case of the Ca2+-binding S100B protein, an astrocytic alarmin which is augmented in AD and which has been recently implicated as a proteostasis regulator, acting over amyloid ß aggregation. Here we report the activity of S100B as a suppressor of tau aggregation and seeding, operating at sub-stoichiometric conditions. We show that S100B interacts with tau in living cells even in microtubule-destabilizing conditions. Structural analysis revealed that tau undergoes dynamic interactions with S100B, in a Ca2+-dependent manner, notably with the aggregation prone repeat segments at the microtubule binding regions. This interaction involves contacts of tau with a cleft formed at the interface of the S100B dimer. Kinetic and mechanistic analysis revealed that S100B inhibits the aggregation of both full-length tau and of the microtubule binding domain, and that this proceeds through effects over primary and secondary nucleation, as confirmed by seeding assays and direct observation of S100B binding to tau oligomers and fibrils. In agreement with a role as an extracellular chaperone and its accumulation near tau positive inclusions, we show that S100B blocks proteopathic tau seeding. Together, our findings establish tau as a client of the S100B chaperone, providing evidence for neuro-protective functions of this inflammatory mediator across different tauopathies.

Cu2+-binding to S100B triggers polymerization of disulfide cross-linked tetramers with enhanced chaperone activity against amyloid-ß aggregation.

S100B is an extracellular protein implicated in Alzheimer's Disease and a suppressor of amyloid-ß aggregation. Herein we report a mechanism tying Cu2+ binding to a change in assembly state yielding disulfide cross-linked oligomers with higher anti-aggregation activity. This chemical control of chaperone function illustrates a regulatory process relevant under metal and proteostasis dysfunction as in neurodegeneration.

The S100B Alarmin Is a Dual-Function Chaperone Suppressing Amyloid-ß Oligomerization through Combined Zinc Chelation and Inhibition of Protein Aggregation.

Amyloid beta (Aß) aggregation and imbalance of metal ions are major hallmarks of Alzheimer's disease (AD). Indeed, amyloid plaques of AD patients are enriched in zinc and Aß42, and AD related-cognitive decline is dependent on extracellular zinc concentration. In vitro, zinc induces the formation of polymorphic Aß42 oligomers that delay the formation of amyloid fibers at the expense of increased cellular toxicity. S100B is an inflammatory alarmin and one of the most abundant proteins in the brain and is upregulated in AD and associated with amyloid plaques, where it exerts extracellular functions. Recent findings have uncovered novel neuroprotective functions for S100B as a suppressor of Aß aggregation and toxicity and in the regulation of zinc homeostasis in neurons. Here we combine biophysical and kinetic approaches to demonstrate that such S100B protective functions converge, making the protein a dual-function chaperone capable of suppressing the formation of toxic Aß oligomers through both chelation of zinc and inhibition of protein aggregation. From detailed kinetic analysis of Aß42 aggregation monitoring ThT fluorescence, we show that substoichiometric S100B prevents the formation of toxic off-pathway oligomers that are formed by monomeric Aß42 in the presence of zinc. Indeed, S100B is effective when added during the lag and transition phases of Aß42 aggregation, and its action under these circumstances results from its ability to buffer zinc, as it perfectly mimics the effect obtained with the chelating agent EDTA. Further, bioimaging analysis combining transmission electron microscopy and atomic force microscopy confirms that catalytic amounts of S100B partly revert the formation of toxic oligomers. Taken together these results indicate a new role for S100B as a dual chaperone whose distinct functions are interrelated and depend on the relative levels of zinc, S100B, and Aß, which dynamically evolve during AD.

Mechanical Properties of Human Bronchial Epithelial Cells Expressing Wt- and Mutant CFTR.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). A single recessive mutation, the deletion of phenylalanine 508 (F508del), causes severe CF and resides on 70% of mutant chromosomes. Disorganization of the actin cytoskeleton has been previously reported in relation to the CF phenotype. In this work, we aimed to understand this alteration by means of Atomic Force Microscopy and Force Feedback Microscopy investigation of mechanical properties of cystic fibrosis bronchial epithelial (CFBE) cells stably transduced with either wild type (wt-) or F508del-CFTR. We show here that the expression of mutant CFTR causes a decrease in the cell's apparent Young modulus as compared to the expression of the wt protein.

Zinc Binding to Tau Influences Aggregation Kinetics and Oligomer Distribution.

Metal ions are well known modulators of protein aggregation and are key players in Alzheimer's Disease, being found to be associated to pathologic protein deposits in diseased brains. Therefore, understanding how metals influence amyloid aggregation is critical in establishing molecular mechanisms that underlie disease onset and progression. Here, we report data on the interaction of full-length human Tau protein with calcium and zinc ions, evidencing that Tau self-assembly is differently regulated, depending on the type of bound metal ion. We established that Tau binds 4 Zn2+ and 1 Ca2+ per monomer while using native mass spectrometry analysis, without inducing order or substantial conformational changes in the intrinsically disordered Tau, as determined by structural analysis using circular dichroism and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopies. However, Tau aggregation is found to proceed differently in the calcium- and -zinc bound forms. While the rate of aggregation, as determined from thioflavin-T (ThT) fluorescence kinetics, is highly increased in both cases, the reaction proceeds via different mechanisms, as evidenced by the absence of the lag phase in the reaction of zinc-bound Tau. Monitoring Tau aggregation using native mass spectrometry indeed evidenced a distinct distribution of Tau conformers along the reaction, as confirmed by dynamic light scattering analysis. We propose that such differences arise from zinc binding at distinct locations within the Tau sequence that prompt both the rapid formation of seeding oligomers through interactions at high affinity sites within the repeat domains, as well as amorphous aggregation, through low affinity interactions with residues elsewhere in the sequence, including at the fuzzy coat domain.

Facile functionalization of cotton with nanostructured silver/titania for visible-light plasmonic photocatalysis.

In the present work, a simple, reliable and cost-effective approach to functionalize cotton fabrics with Ag-TiO2 nanoparticles strongly bound to the fibres and with visible-light-responsive photo-activity is presented. The hybrid cotton-Ag-TiO2 fabrics were characterized by Raman, AFM, FE-SEM, TGA, XPS GSDR, and LIL to confirm the generation of metallic Ag nanoparticles and crystalline TiO2 and investigate how the concentration of Ag and TiO2 precursors affected the morphology and the luminescence properties of the nanostructured layer grafted on the cotton fibres. The photocatalytic activity of the cotton-Ag-TiO2 hybrid systems was evaluated by the discoloration of Remazol Brilliant Blue R in water under a xenon lamp irradiation (sunlight simulator) equipped with selective filters. The extended photocatalytic activity to the visible is here explained by a synergistic effect of both the excitation of the Ag NPs plasmon resonance by visible light and a delayed electron-hole recombination rate caused by Ag NPs, as it can be observed by UV absorption.

Cellulose films: designing template-free nanoporous cellulose films on semiconducting surfaces.

In this work, we report the preparation of ultrathin submicro- and nanoporous cellulose films onto Si (100). The effect of different experimental conditions of preparation on the film surface morphology was studied, namely the role of the film casting method (spin- versus dip-coating), solvent (toluene or tetrahydrofuran), substrate pretreatment (hydrophilicity degree), and regeneration procedure with HCl vapors (two consecutive dips followed by regeneration or regeneration after each dip). The surface morphological structures presented in this work were never obtained before without the use of templates. A rather regular two-dimensional pore network was obtained onto the less hydrophilic Si substrate (contact angle≅68°), after two consecutive dips (with an intercalary rotation of 180º) in trimethylsilyl cellulose diluted in toluene and regeneration at the end. All the surfaces were characterized by atomic force microscopy.