Pentadecanoic Acid-Releasing PDMS: Towards a New Material to Prevent Biofilm Formation.

Microbial biofilm formation on medical devices paves the way for device-associated infections. is one of the most common strains involved in such infections as it is able to colonize numerous devices, such as intravenous catheters, prosthetic joints, and heart valves. We previously reported the antibiofilm activity against of pentadecanoic acid (PDA) deposited by drop-casting on the silicon-based polymer poly(dimethyl)siloxane (PDMS).

Chemical Probes to Investigate Central Nervous System Disorders: Design, Synthesis and Mechanism of Action of a Potent Human Serine Racemase Inhibitor.

The intricate signaling network within the central nervous system (CNS) involving -methyl-d-aspartate receptors (NMDARs) has been recognized as a key player in severe neurodegenerative diseases. The indirect modulation of NMDAR-mediated neurotransmission through inhibition of serine racemase (SR)-the enzyme responsible for the synthesis of the NMDAR coagonist d-serine-has been suggested as a therapeutic strategy to treat these conditions. Despite the inherent challenges posed by SR conformational flexibility, a ligand-based drug design strategy has successfully produced a series of potent covalent inhibitors structurally related to amino acid analogues.

Identification of small molecules affecting the interaction between human hemoglobin and Staphylococcus aureus IsdB hemophore.

Human hemoglobin (Hb) is the preferred iron source of Staphylococcus aureus. This pathogenic bacterium exploits a sophisticated protein machinery called Iron-regulated surface determinant (Isd) system to bind Hb, extract and internalize heme, and finally degrade it to complete iron acquisition. IsdB, the surface exposed Hb receptor, is a proven virulence factor of S.

Methionine gamma lyase: Structure-activity relationships and therapeutic applications.

Methionine gamma lyase (MGL) is a bacterial and plant enzyme that catalyzes the conversion of methionine in methanthiol, 2-oxobutanoate and ammonia. The enzyme belongs to fold type I of the pyridoxal 5'-dependent family. The catalytic mechanism and the structure of wild type MGL and variants were determined in the presence of the natural substrate as well as of many sulfur-containing derivatives.

Human Serine Racemase Weakly Binds the Third PDZ Domain of PSD-95.

Human serine racemase (hSR) is a pyridoxal-5'-phosphate (PLP)-dependent dimer that catalyzes the formation of D-serine from L-serine, as well as the dehydration of both L- and D-serine to pyruvate and ammonia. As D-serine is a co-agonist of N-methyl-D-aspartate receptors (NMDARs), hSR is a key enzyme in glutamatergic neurotransmission. hSR activity is finely regulated by Mg, ATP, post-translational modifications, and the interaction with protein partners.

From hemoglobin allostery to hemoglobin-based oxygen carriers.

Hemoglobin (Hb) plays its vital role through structural and functional properties evolutionarily optimized to work within red blood cells, i.e., the tetrameric assembly, well-defined oxygen affinity, positive cooperativity, and heterotropic allosteric regulation by protons, chloride and 2,3-diphosphoglycerate.

Enzyme Replacement Therapy for Genetic Disorders Associated with Enzyme Deficiency.

Mutations in human genes might lead to the loss of functional proteins, causing diseases. Among these genetic disorders, a large class is associated with the deficiency in metabolic enzymes, resulting in both an increase in the concentration of substrates and a loss in the metabolites produced by the catalyzed reactions. The identification of therapeutic actions based on small molecules represents a challenge to medicinal chemists because the target is missing.

The allosteric interplay between S-nitrosylation and glycine binding controls the activity of human serine racemase.

Human serine racemase (hSR) catalyzes the biosynthesis of D-serine, an obligatory co-agonist of the NMDA receptors. It was previously found that the reversible S-nitrosylation of Cys113 reduces hSR activity. Here, we show by site-directed mutagenesis, fluorescence spectroscopy, mass spectrometry, and molecular dynamics that S-nitrosylation stabilizes an open, less-active conformation of the enzyme.

Human serine racemase is inhibited by glyceraldehyde 3-phosphate, but not by glyceraldehyde 3-phosphate dehydrogenase.

Murine serine racemase (SR), the enzyme responsible for the biosynthesis of the neuromodulator d-serine, was reported to form a complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting in SR inhibition. In this work, we investigated the interaction between the two human orthologues. We were not able to observe neither the inhibition nor the formation of the SR-GAPDH complex.

The Energy Landscape of Human Serine Racemase.

Human serine racemase is a pyridoxal 5'-phosphate (PLP)-dependent dimeric enzyme that catalyzes the reversible racemization of L-serine and D-serine and their dehydration to pyruvate and ammonia. As D-serine is the co-agonist of the N-methyl-D-aspartate receptors for glutamate, the most abundant excitatory neurotransmitter in the brain, the structure, dynamics, function, regulation and cellular localization of serine racemase have been investigated in detail. Serine racemase belongs to the fold-type II of the PLP-dependent enzyme family and structural models from several orthologs are available.

ADIFAB fluorescence data used for the quantification of free fatty acids in media at different pH.

We investigated the pH dependence of the fluorescence spectra of ADIFAB (FFA Sciences), a probe used for the quantification of free fatty acids (FFA). Data reports the change in the emission peak of ADIFAB and in the affinity for FFA as a function of pH. An algorithm based on spectral deconvolution allowed to correct ADIFAB fluorescence spectra for the spectroscopic effect caused by pH.

Phospholipid components of the synthetic pulmonary surfactant CHF5633 probed by fluorescence spectroscopy.

CHF5633 (Chiesi Farmaceutici, Italy) is a synthetic pulmonary surfactant currently under clinical development for the treatment of Respiratory Distress Syndrome in premature infants. The product is composed of phospholipids in liposomal organization, together with two peptide analogues of human surfactant proteins B and C. Phospholipids in liposomes can undergo oxidation of unsaturated lipids and hydrolysis, with formation of fatty acids and lysolipids, both affecting the physico-chemical properties of the formulation.

Engineering methionine γ-lyase from Citrobacter freundii for anticancer activity.

Methionine deprivation of cancer cells, which are deficient in methionine biosynthesis, has been envisioned as a therapeutic strategy to reduce cancer cell viability. Methionine γ-lyase (MGL), an enzyme that degrades methionine, has been exploited to selectively remove the amino acid from cancer cell environment. In order to increase MGL catalytic activity, we performed sequence and structure conservation analysis of MGLs from various microorganisms.

Glutamine 89 is a key residue in the allosteric modulation of human serine racemase activity by ATP.

Serine racemase (SR) catalyses two reactions: the reversible racemisation of L-serine and the irreversible dehydration of L- and D-serine to pyruvate and ammonia. SRs are evolutionarily related to serine dehydratases (SDH) and degradative threonine deaminases (TdcB). Most SRs and TdcBs - but not SDHs - are regulated by nucleotides.

Soluble and Nanoporous Silica Gel-Entrapped Methionine -Lyase.

Methionine γ-lyase is a pyridoxal 5'-phosphate dependent tetramer that catalyzes the α,γ-elimination of methionine in ammonia, methanethiol and α-ketobutyrate. MGL catalytic power has been exploited as a therapeutic strategy to reduce the viability of cancer cells or bacteria. In order to obtain a stable enzyme to be delivered at the site of action, MGL can be encapsulated in a variety of matrices.

Gene cloning, characterization, and cytotoxic activity of methionine γ-lyase from Clostridium novyi.

The exploitation of methionine-depleting enzyme methionine γ-lyase (MGL) is a promising strategy against specific cancer cells that are strongly dependent on methionine. To identify MGL from different sources with high catalytic activity and efficient anticancer action, we have expressed and characterized MGL from Clostridium novyi and compared its catalytic efficiency with the previously studied MGL from Citrobacter freundii. The purified recombinant MGL exhibits k and k /K for methionine γ-elimination reaction that are 2.

Magnesium and calcium ions differentially affect human serine racemase activity and modulate its quaternary equilibrium toward a tetrameric form.

Serine racemase is the pyridoxal 5'-phosphate dependent enzyme that catalyzes both production and catabolism of d-serine, a co-agonist of the NMDA glutamate receptors. Mg, or, alternatively, Ca, activate human serine racemase by binding both at a specific site and - as ATP-metal complexes - at a distinct ATP binding site. We show that Mg and Ca bind at the metal binding site with a 4.

Human serine racemase is allosterically modulated by NADH and reduced nicotinamide derivatives.

Serine racemase catalyzes both the synthesis and the degradation of d-serine, an obligatory co-agonist of the glutamatergic NMDA receptors. It is allosterically controlled by adenosine triphosphate (ATP), which increases its activity around 7-fold through a co-operative binding mechanism. Serine racemase has been proposed as a drug target for the treatment of several neuropathologies but, so far, the search has been directed only toward the active site, with the identification of a few, low-affinity inhibitors.

The Ball and Chain of Polyubiquitin Structures.

Ubiquitylation is a post-translational modification implicated in several different cellular pathways. The possibility of forming chains through covalent crosslinking between any of the seven lysines, or the initial methionine, and the C terminus of another moiety provides ubiquitin (Ub) with special flexibility in its function in signalling. Here, we review the knowledge accumulated over the past several years about the functions and structural features of polyUb chains.

The missing links to link ubiquitin: Methods for the enzymatic production of polyubiquitin chains.

Attachment of ubiquitin (Ub) as monoUb and polyUb chains of different lengths and linkages to proteins plays a dominant role in very different regulatory mechanisms. Therefore, the study of polyUb chains has assumed a central interest in biochemistry and structural biology. An essential step necessary to allow in vitro biochemical and structural studies of polyUbs is the production of their chains in high quantities and purity.

Allosteric regulation of deubiquitylase activity through ubiquitination.

Ataxin-3, the protein responsible for spinocerebellar ataxia type-3, is a cysteine protease that specifically cleaves poly-ubiquitin chains and participates in the ubiquitin proteasome pathway. The enzymatic activity resides in the N-terminal Josephin domain. An unusual feature of ataxin-3 is its low enzymatic activity especially for mono-ubiquitinated substrates and short ubiquitin chains.

Characterization of the conformational fluctuations in the Josephin domain of ataxin-3.

As for a variety of other molecular recognition processes, conformational fluctuations play an important role in the cleavage of polyubiquitin chains by the Josephin domain of ataxin-3. The interaction between Josephin and ubiquitin appears to be mediated by the motions of α-helical hairpin that is unusual among deubiquitinating enzymes. Here, we characterized the conformational fluctuations of the helical hairpin by incorporating NMR measurements as replica-averaged restraints in molecular dynamics simulations, and by validating the results by small-angle x-ray scattering measurements.

The challenge of producing ubiquitinated proteins for structural studies.

Protein ubiquitination is an important post-translational modification involved in several essential signalling pathways. It has different effects on the target protein substrate, i.e.

Enzymatic production of mono-ubiquitinated proteins for structural studies: The example of the Josephin domain of ataxin-3.

Protein ubiquitination occurs through formation of an isopeptide bond between the C-terminal glycine of ubiquitin (Ub) and the ɛ-amino group of a substrate lysine residue. This post-translational modification, which occurs through the attachment of single and/or multiple copies of mono-ubiquitin and poly-ubiquitin chains, is involved in crucial cellular events such as protein degradation, cell-cycle regulation and DNA repair. The abnormal functioning of ubiquitin pathways is also implicated in the pathogenesis of several human diseases ranging from cancer to neurodegeneration.

The role of interruptions in polyQ in the pathology of SCA1.

At least nine dominant neurodegenerative diseases are caused by expansion of CAG repeats in coding regions of specific genes that result in abnormal elongation of polyglutamine (polyQ) tracts in the corresponding gene products. When above a threshold that is specific for each disease the expanded polyQ repeats promote protein aggregation, misfolding and neuronal cell death. The length of the polyQ tract inversely correlates with the age at disease onset.