Revealing the dynamic allosteric changes required for formation of the cysteine synthase complex by hydrogen-deuterium exchange MS

CysE and CysK, the last two enzymes of the cysteine biosynthetic pathway, engage in a bienzyme complex, cysteine synthase, with yet incompletely characterized three-dimensional structure and regulatory function. Being absent in mammals, the two enzymes and their complex are attractive targets for antibacterial drugs. We have used hydrogen/deuterium exchange MS to unveil how complex formation affects the conformational dynamics of CysK and CysE. Our results support a model where CysE is present in solution as a dimer of trimers, and each trimer can bind one CysK homodimer. When CysK binds to one CysE monomer, intratrimer allosteric communication ensures conformational and dynamic symmetry within the trimer. Furthermore, a long-range allosteric signal propagates through CysE to induce stabilization of the interface between the two CysE trimers, preparing the second trimer for binding the second CysK with a nonrandom orientation. These results provide new molecular insights into the allosteric formation of the cysteine synthase complex and could help guide antibacterial drug design

A novel assay for phosphoserine phosphatase exploiting serine acetyltransferase as the coupling enzyme

Phosphoserine phosphatase (PSP) catalyzes the final step of de novo L-serine biosynthesis— the hydrolysis of phosphoserine to serine and inorganic phosphate—in humans, bacteria, and plants. In published works, the reaction is typically monitored through the discontinuous malachite green phosphate assay or, more rarely, through a continuous assay that couples phosphate release to the phosphorolysis of a chromogenic nucleoside by the enzyme purine nucleoside phosphorylase (PNP). These assays suffer from numerous drawbacks, and both rely on the detection of phosphate. We describe a new continuous assay that monitors the release of serine by exploiting bacterial serine acetyltransferase (SAT) as a reporter enzyme. SAT acetylates serine, consuming acetyl-CoA and releasing CoA-SH. CoA-SH spontaneously reacts with Ellman’s reagent to produce a chromophore that absorbs light at 412 nm. The catalytic parameters estimated through the SAT-coupled assay are fully consistent with those obtained with the published methods, but the new assay exhibits several advantages. Particularly, it depletes L-serine, thus allowing more prolonged linearity in the kinetics. Moreover, as the SAT-coupled assay does not rely on phosphate detection, it can be used to investigate the inhibitory effect of phosphate on PSP

Iron metabolism at the interface between host and pathogen: From nutritional immunity to antibacterial development

Nutritional immunity is a form of innate immunity widespread in both vertebrates and invertebrates. The term refers to a rich repertoire of mechanisms set up by the host to inhibit bacterial proliferation by sequestering trace minerals (mainly iron, but also zinc and manganese). This strategy, selected by evolution, represents an effective front-line defense against pathogens and has thus inspired the exploitation of iron restriction in the development of innovative antimicrobials or enhancers of antimicrobial therapy. This review focuses on the mechanisms of nutritional immunity, the strategies adopted by opportunistic human pathogen Staphylococcus aureus to circumvent it, and the impact of deletion mutants on the fitness, infectivity, and persistence inside the host. This information finally converges in an overview of the current development of inhibitors targeting the different stages of iron uptake, an as-yet unexploited target in the field of antistaphylococcal drug discovery

Immobilization of proteins in silica gel: Biochemical and biophysical properties

The development of silica-based sol-gel techniques compatible with the retention of protein structure and function started more than 20 years ago, mainly for the design of biotechnological devices or biomedical applications. Silica gels are optically transparent, exhibit good mechanical stability, are manufactured with different geometries, and are easily separated from the reaction media. Biomolecules encapsulated in silica gel normally retain their structural and functional properties, are stabilized with respect to chemical and physical insults, and can sometimes exhibit enhanced activity in comparison to the soluble form. This review briefly describes the chemistry of protein encapsulation within the pores of a silica gel three-dimensional network, the mechanism of interaction between the protein and the gel matrix, and its effects on protein structure, function, stability and dynamics. The main applications in the field of biosensor design are described. Special emphasis is devoted to silica gel encapsulation as a tool to selectively stabilize subsets of protein conformations for biochemical and biophysical studies, an application where silica-based encapsulation demonstrated superior performance with respect to other immobilization techniques

Iron metabolism at the interface between host and pathogen: From nutritional immunity to antibacterial development

Nutritional immunity is a form of innate immunity widespread in both vertebrates and invertebrates. The term refers to a rich repertoire of mechanisms set up by the host to inhibit bacterial proliferation by sequestering trace minerals (mainly iron, but also zinc and manganese). This strategy, selected by evolution, represents an effective front-line defense against pathogens and has thus inspired the exploitation of iron restriction in the development of innovative antimicrobials or enhancers of antimicrobial therapy. This review focuses on the mechanisms of nutritional immunity, the strategies adopted by opportunistic human pathogen Staphylococcus aureus to circumvent it, and the impact of deletion mutants on the fitness, infectivity, and persistence inside the host. This information finally converges in an overview of the current development of inhibitors targeting the different stages of iron uptake, an as-yet unexploited target in the field of antistaphylococcal drug discovery

Manual de implantação do treino e visita (T&V).

A visão sobre transferência de tecnologia na Embrapa; A metodologia treino e visita (T&V); O treino e visita em sua forma original; Adaptação do treino e visita para o Brasil; Consolidação do T&V soja/grãos - a experiência da Embrapa Soja; Programa de profissionalização do produtor rural (PPRR) da COPACOL; Projeto grãos da EMATER-Paraná; A aplicação da metodologia em diferentes projetos; Projeto T&V café; Projeto T&V saúde, alimentação e geração de renda; Projeto T&V girassol; Projeto T&V pecuária de corte e de leite no Paraná; Projeto T&V sementes; Projeto T&V grãos no Rio Grande do Sul; Estratégias para a implantação do T&V; Definição do foco; Criação do grupo gestor; Criação do comitê técnico; Pré-requisitos para a implantação do T&V; Anexo 1 - Modelo de relatório do T&V grãos - safra 2002/03; Anexo 2 - Modelo de relatório do T&V - saúde, alimentação e geração de renda; Anexo 3 - Modelo de marco zero do Projeto Grãos/EMATER-PR.bitstream/item/80261/1/Manual-de-implantacao-do-treino-e-visita-V-T.pd

L-serine biosynthesis in the human central nervous system: Structure and function of phosphoserine aminotransferase

Organisms from all kingdoms of life synthesize L-serine (L-Ser) from 3-phosphoglycerate through the phosphorylated pathway, a three-step diversion of glycolysis. Phosphoserine aminotransferase (PSAT) catalyzes the intermediate step, the pyridoxal 5′-phosphate-dependent transamination of 3-phosphohydroxypyruvate and L-glutamate to O-phosphoserine (OPS) and α-ketoglutarate. PSAT is particularly relevant in the central nervous system of mammals because L-Ser is the metabolic precursor of D-serine, cysteine, phospholipids, and nucleotides. Several mutations in the human psat gene have been linked to serine deficiency disorders, characterized by severe neurological symptoms. Furthermore, PSAT is overexpressed in many tumors and this overexpression has been associated with poor clinical outcomes. Here, we report the detailed functional and structural characterization of the recombinant human PSAT. The reaction catalyzed by PSAT is reversible, with an equilibrium constant of about 10, and the enzyme is very efficient, with a kcat/Km of 5.9 × 106 M−1 s−1, thus contributing in driving the pathway towards the products despite the extremely unfavorable first step catalyzed by 3-phosphoglycerate dehydrogenase. The 3D X-ray crystal structure of PSAT was solved in the substrate-free as well as in the OPS-bound forms. Both structures contain eight protein molecules in the asymmetric unit, arranged in four dimers, with a bound cofactor in each subunit. In the substrate-free form, the active site of PSAT contains a sulfate ion that, in the substrate-bound form, is replaced by the phosphate group of OPS. Interestingly, fast crystal soaking used to produce the substrate-bound form allowed the trapping of different intermediates along the catalytic cycle

Management of Hypothyroidism in Internal Medicine: Patient Profile and Effects of an Educational Programme in the Cluster-Randomized FADOI TIAMO Study.

Background and aims: There is still limited knowledge regarding the clinical profile and appropriateness of treatment in patients with hypothyroidism hospitalized in Internal Medicine (IM) Departments in Italy. The aim of this study is to evaluate: 1) the characteristics of patients and possible deviations from national and international clinical practice recommendations (CPRs) in evidence-based guidelines (EBGs); 2) the improvement of patient management by means of a standardized educational programme (EP). Methods: A nationwide multicentre study, comprising two replications of a retrospective survey (phases 1 and 3) with an intervening EP (phase 2) in half of the centres and no EP in the other half, was conducted. The EP was based on outreach visits. Centres were assigned to the two arms of the study, labelled the training group (TG) and control group (CG) respectively, by cluster randomization. Four EBGs and 39 CPRs provided the basis on which 22 treatment management indicators were identified (7 referring to the time of hospital admission, 15 to post-admission). Results: The 21 participating centres recruited 587 hospitalized patients with hypothyroidism, 421 of which were females (71.7%, mean age 74.1 + 14.4 yrs): 318 in phase 1 and 269 in phase 3. The cause of hypothyroidism was unknown in 282 patients (48%). Evaluation at the time of admission identified satisfactory adherence to CPRs (>50%) for 63.6% of the indicators. In the phase 3, TG centres showed significant improvement vs CG in 4 of the 15 post-admission indicators, while 1 out of 15 was significantly worse. Conclusions: The EP based on outreach visits significantly improved some indicators in the management of patients with hypothyroidism, with specific reference to appropriateness of TSH dosage and levothyroxine (LT4) treatment modality. Clinical trial registration: ClinicalTrials.gov, identifier NCT05314790

Hydrophobic and ionic-interactions in bulk and confined water with implications for collapse and folding of proteins

Water and water-mediated interactions determine thermodynamic and kinetics of protein folding, protein aggregation and self-assembly in confined spaces. To obtain insights into the role of water in the context of folding problems, we describe computer simulations of a few related model systems. The dynamics of collapse of eicosane shows that upon expulsion of water the linear hydrocarbon chain adopts an ordered helical hairpin structure with 1.5 turns. The structure of dimer of eicosane molecules has two well ordered helical hairpins that are stacked perpendicular to each other. As a prelude to studying folding in confined spaces we used simulations to understand changes in hydrophobic and ionic interactions in nano droplets. Solvation of hydrophobic and charged species change drastically in nano water droplets. Hydrophobic species are localized at the boundary. The tendency of ions to be at the boundary where water density is low increases as the charge density decreases. Interaction between hydrophobic, polar, and charged residue are also profoundly altered in confined spaces. Using the results of computer simulations and accounting for loss of chain entropy upon confinement we argue and then demonstrate, using simulations in explicit water, that ordered states of generic amphiphilic peptide sequences should be stabilized in cylindrical nanopores