Structural insights of an LCP protein-LytR-from through biophysical and methods.

The rise of antibiotic-resistant bacterial strains has become a critical health concern. According to the World Health Organization, the market introduction of new antibiotics is alarmingly sparse, underscoring the need for novel therapeutic targets. The LytR-CpsA-Psr (LCP) family of proteins, which facilitate the insertion of cell wall glycopolymers (CWGPs) like teichoic acids into peptidoglycan, has emerged as a promising target for antibiotic development.

Arsenite oxidase in complex with antimonite and arsenite oxyanions: Insights into the catalytic mechanism.

Arsenic contamination of groundwater is among one of the biggest health threats affecting millions of people in the world. There is an urgent need for efficient arsenic biosensors where the use of arsenic metabolizing enzymes can be explored. In this work, we have solved four crystal structures of arsenite oxidase (Aio) in complex with arsenic and antimony oxyanions and the structures determined correspond to intermediate states of the enzymatic mechanism.

Using Small-angle X-ray Scattering to Characterize Biological Systems: A General Overview and Practical Tips.

Small-angle X-ray Scattering (SAXS) is a versatile and powerful technique with applications in a wide range of fields. The continuous improvements in hardware, data analysis software, and standards for validation significantly contributed to increase its popularity and, nowadays, SAXS is a well-established method. SAXS allows to study flexible and dynamic systems (e.

Screening of Buffers and Additives for Protein Stabilization by Thermal Shift Assay: A Practical Approach.

Thermal shift assay (TSA), also commonly designed by differential scanning fluorimetry (DSF) or ThermoFluor, is a technique relatively easy to implement and perform, useful in a myriad of applications. In addition to versatility, it is also rather inexpensive, making it suitable for high-throughput approaches. TSA uses a fluorescent dye to monitor the thermal denaturation of the protein under study and determine its melting temperature (T).

Binding of V O , V OL, V OL and V O L Moieties to Proteins: X-ray/Theoretical Characterization and Biological Implications.

Vanadium compounds have frequently been proposed as therapeutics, but their application has been hampered by the lack of information on the different V-containing species that may form and how these interact with blood and cell proteins, and with enzymes. Herein, we report several resolved crystal structures of lysozyme with bound V O and V OL , where L=2,2'-bipyridine or 1,10-phenanthroline (phen), and of trypsin with V O(picolinato) and V O (phen) moieties. Computational studies complete the refinement and shed light on the relevant role of hydrophobic interactions, hydrogen bonds, and microsolvation in stabilizating the structure.

Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure.

Background: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive.

Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing.

Interrogating the Inhibition Mechanisms of Human Aldehyde Oxidase by X-ray Crystallography and NMR Spectroscopy: The Raloxifene Case.

Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors-thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition.

Improving the Anti-inflammatory Response via Gold Nanoparticle Vectorization of CO-Releasing Molecules.

CO-releasing molecules (CORMs) have been widely studied for their anti-inflammatory, antiapoptotic, and antiproliferative effects. CORM-3 is a water-soluble Ru-based metal carbonyl complex, which metallates serum proteins and readily releases CO in biological media. In this work, we evaluated the anti-inflammatory and wound-healing effects of gold nanoparticles-CORM-3 conjugates, AuNPs@PEG@BSA·Ru(CO), exploring its use as an efficient CO carrier.

Singularities of Pyogenic Streptococcal Biofilms - From Formation to Health Implication.

Biofilms are generally defined as communities of cells involved in a self-produced extracellular matrix adhered to a surface. In biofilms, the bacteria are less sensitive to host defense mechanisms and antimicrobial agents, due to multiple strategies, that involve modulation of gene expression, controlled metabolic rate, intercellular communication, composition, and 3D architecture of the extracellular matrix. These factors play a key role in streptococci pathogenesis, contributing to therapy failure and promoting persistent infections.

Multitask ATPases (NBDs) of bacterial ABC importers type I and their interspecies exchangeability.

ATP-binding cassette (ABC) type I importers are widespread in bacteria and play a crucial role in its survival and pathogenesis. They share the same modular architecture comprising two intracellular nucleotide-binding domains (NBDs), two transmembrane domains (TMDs) and a substrate-binding protein. The NBDs bind and hydrolyze ATP, thereby generating conformational changes that are coupled to the TMDs and lead to substrate translocation.

and investigations on the protein-protein interactions of glutathione S-transferases with mitogen-activated protein kinase 8 and apoptosis signal-regulating kinase 1.

Cytosolic glutathione -transferase (GST) enzymes participate in several cellular processes in addition to facilitating glutathione conjugation reactions that eliminate endogenous and exogenous toxic compounds, especially electrophiles. GSTs are thought to interact with various kinases, resulting in the modulation of apoptotic processes and cellular proliferation. The present research used a combination of and studies to investigate protein-protein interactions between the seven most abundant cytosolic GSTs-GST alpha-1 (GST-A1), GST alpha-2 (GST-A2), GST mu-1 (GST-M1), GST mu-2 (GST-M2), GST mu-5 (GST-M5), GST theta-1 (GST-T1) and GST pi-1 (GST-P1)-and Mitogen-activated protein kinase 8 (MAPK8) and Apoptosis signal-regulating kinase 1 (ASK1).

Systematic exploration of predicted destabilizing nonsynonymous single nucleotide polymorphisms (nsSNPs) of human aldehyde oxidase: A Bio-informatics study.

Aldehyde Oxidase (hAOX1) is a cytosolic enzyme involved in the metabolism of drugs and xenobiotic compounds. The enzyme belongs to the xanthine oxidase (XO) family of Mo containing enzyme and is a homo-dimer of two 150 kDa monomers. Nonsynonymous Single Nucleotide Polymorphisms (nsSNPs) of hAOX1 have been reported as affecting the ability of the enzyme to metabolize different substrates.

Human aldehyde oxidase (hAOX1): structure determination of the Moco-free form of the natural variant G1269R and biophysical studies of single nucleotide polymorphisms.

Human aldehyde oxidase (hAOX1) is a molybdenum enzyme with high toxicological importance, but its physiological role is still unknown. hAOX1 metabolizes different classes of xenobiotics and is one of the main drug-metabolizing enzymes in the liver, along with cytochrome P450. hAOX1 oxidizes and inactivates a large number of drug molecules and has been responsible for the failure of several phase I clinical trials.

Biofilm development and computational screening for new putative inhibitors of a homolog of the regulatory protein BrpA in Streptococcus dysgalactiae subsp. dysgalactiae.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), a Lancefield group C streptococci (GCS), is a frequent cause of bovine mastitis. This highly prevalent disease is the costliest in dairy industry.

Shedding Light on the Interaction of Human Anti-Apoptotic Bcl-2 Protein with Ligands through Biophysical and in Silico Studies.

Bcl-2 protein is involved in cell apoptosis and is considered an interesting target for anti-cancer therapy. The present study aims to understand the stability and conformational changes of Bcl-2 upon interaction with the inhibitor venetoclax, and to explore other drug-target regions. We combined biophysical and in silico approaches to understand the mechanism of ligand binding to Bcl-2.

First insights of peptidoglycan amidation in Gram-positive bacteria - the high-resolution crystal structure of Staphylococcus aureus glutamine amidotransferase GatD.

Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.

Binding of vanadium to human serum transferrin - voltammetric and spectrometric studies.

Previous studies generally agree that in the blood serum vanadium is transported mainly by human serum transferrin (hTF). In this work through the combined use of electrochemical techniques, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and small-angle X-ray scattering (SAXS) data it is confirmed that both V and V bind to apo-hTF and holo-hTF. The electrochemical behavior of solutions containing vanadate(V) solutions at pH=7.

Electron transfer through arsenite oxidase: Insights into Rieske interaction with cytochrome c.

Arsenic is a widely distributed environmental toxin whose presence in drinking water poses a threat to >140 million people worldwide. The respiratory enzyme arsenite oxidase from various bacteria catalyses the oxidation of arsenite to arsenate and is being developed as a biosensor for arsenite. The arsenite oxidase from Rhizobium sp.

Highly selective tungstate transporter protein TupA from Desulfovibrio alaskensis G20.

Molybdenum and tungsten are taken up by bacteria and archaea as their soluble oxyanions through high affinity transport systems belonging to the ATP-binding cassette (ABC) transporters. The component A (ModA/TupA) of these transporters is the first selection gate from which the cell differentiates between MoO, WO and other similar oxyanions. We report the biochemical characterization and the crystal structure of the apo-TupA from Desulfovibrio desulfuricans G20, at 1.

Interaction of [V O(acac) ] with Human Serum Transferrin and Albumin.

[VO(acac) ] is a remarkable vanadium compound and has potential as a therapeutic drug. It is important to clarify how it is transported in blood, but the reports addressing its binding to serum proteins have been contradictory. We use several spectroscopic and mass spectrometric techniques (ESI and MALDI-TOF), small-angle X-ray scattering and size exclusion chromatography (SEC) to characterize solutions containing [VO(acac) ] and either human serum apotransferrin (apoHTF) or albumin (HSA).

Structural basis for the role of mammalian aldehyde oxidases in the metabolism of drugs and xenobiotics.

Aldehyde oxidases (AOXs) are molybdo-flavoenzymes characterized by broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into the corresponding carboxylic acids and hydroxylating various heteroaromatic rings. Mammals are characterized by a complement of species-specific AOX isoenzymes, that varies from one in humans (AOX1) to four in rodents (AOX1, AOX2, AOX3 and AOX4). The physiological function of mammalian AOX isoenzymes is unknown, although human AOX1 is an emerging enzyme in phase-I drug metabolism.

The Escherichia coli Periplasmic Aldehyde Oxidoreductase Is an Exceptional Member of the Xanthine Oxidase Family of Molybdoenzymes.

The xanthine oxidase (XO) family comprises molybdenum-dependent enzymes that usually form homodimers (or dimers of heterodimers/trimers) organized in three domains that harbor two [2Fe-2S] clusters, one FAD, and a Mo cofactor. In this work, we crystallized an unusual member of the family, the periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli. This is the first example of an E.

Structure and function of mammalian aldehyde oxidases.

Mammalian aldehyde oxidases (AOXs; EC1.2.3.

Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms.

Aldehyde oxidase (AOX1) is an enzyme with broad substrate specificity, catalyzing the oxidation of a wide range of endogenous and exogenous aldehydes as well as N-heterocyclic aromatic compounds. In humans, the enzyme's role in phase I drug metabolism has been established and its importance is now emerging. However, the true physiologic function of AOX1 in mammals is still unknown.