An inhibitory mechanism of action of coiled-coil peptides against type three secretion system from enteropathogenic Escherichia coli.

Human pathogenic gram-negative bacteria, such as enteropathogenic Escherichia coli (EPEC), rely on type III secretion systems (T3SS) to translocate virulence factors directly into host cells. The coiled-coil domains present in the structural proteins of T3SS are conformed by amphipathic alpha-helical structures that play an important role in the protein-protein interaction and are essential for the assembly of the translocation complex. To investigate the inhibitory capacity of these domains on the T3SS of EPEC, we synthesized peptides between 7 and 34 amino acids based on the coiled-coil domains of proteins that make up this secretion system. This analysis was performed through in vitro hemolysis assays by assessing the reduction of T3SS-dependent red blood cell lysis in the presence of the synthesized peptides. After confirming its inhibitory capacity, we performed molecular modeling assays using combined techniques, docking-molecular dynamic simulations, and quantum-mechanic calculations of the various peptide-protein complexes, to improve the affinity of the peptides to the target proteins selected from T3SS. These techniques allowed us to demonstrate that the peptides with greater inhibitory activity, directed against the coiled-coil domain of the C-terminal region of EspA, present favorable hydrophobic and hydrogen bond molecular interactions. Particularly, the hydrogen bond component is responsible for the stabilization of the peptide-protein complex. This study demonstrates that compounds targeting T3SS from pathogenic bacteria can indeed inhibit bacterial infection by presenting a higher specificity than broad-spectrum antibiotics. In turn, these peptides could be taken as initial structures to design and synthesize new compounds that mimic their inhibitory pharmacophoric pattern.

Protein fibrillation lag times during kinetic inhibition.

Protein aggregation is linked to more than 30 human pathologies, including Alzheimer's and Parkinson's diseases. Since small oligomers that form at the beginning of the fibrillation process probably are the most toxic elements, therapeutic strategies involving fibril fragmentation could be detrimental. An alternative approach, named kinetic inhibition, aims to prevent fibril formation by using small ligands that stabilize the parent protein. The factors that govern fibrillation lag times during kinetic inhibition are largely unknown, notwithstanding their importance for designing effective long-term therapies. Inhibitor-bound species are not likely to be incorporated into the core of mature fibrils, although their presence could alter the kinetics of the fibrillation process. For instance, inhibitor-bound species may act as capping elements that impair the nucleation process and/or fibril growth. Here, we address this issue by studying the effect of two natural inhibitors on the fibrillation behavior of lysozyme at neutral pH. We analyzed a set of 79 fibrillation curves obtained in lysozyme alone and a set of 37 obtained in the presence of inhibitors. We calculated the concentrations of the relevant species at the beginning of the curves using the inhibitor-binding constants measured under the same experimental conditions. We found that inhibitor-bound protein species do not affect fibrillation onset times, which are mainly determined by the concentration of unbound protein species present in equilibrium. In this system, knowledge of the fibrillation kinetics and inhibitor affinities suffices to predict the effect of kinetic inhibitors on fibrillation lag times. In addition, we developed a new methodology to better estimate fibrillation lag times from experimental curves.

Peptides Derived From the α-Core and γ-Core Regions of a Putative Silybum marianum Flower Defensin Show Antifungal Activity Against Fusarium graminearum.

Fusarium graminearum is the etiological agent of Fusarium head blight (FHB), a disease that produces a significant decrease in wheat crop yield and it is further aggravated by the presence of mycotoxins in the affected grains that may cause health problems to humans and animals. Plant defensins and defensin-like proteins are antimicrobial peptides (AMPs); they are small basic, cysteine-rich peptides (CRPs) ubiquitously expressed in the plant kingdom and mostly involved in host defence. They present a highly variable sequence but a conserved structure. The γ-core located in the C-terminal region of plant defensins has a conserved ß-hairpin structure and is a well-known determinant of the antimicrobial activity among disulphide-containing AMPs. Another conserved motif of plant defensins is the α-core located in the N-terminal region, not conserved among the disulphide-containing AMPs, it has not been yet extensively studied. In this report, we have cloned the putative antimicrobial protein DefSm2, expressed in flowers of the wild plant Silybum marianum. The cDNA encodes a protein with two fused basic domains of an N-terminal defensin domain (DefSm2-D) and a C-terminal Arg-rich and Lys-rich domain. To further characterize the DefSm2-D domain, we built a 3D template-based model that will serve to support the design of novel antifungal peptides. We have designed four potential antifungal peptides: two from the DefSm2-D α-core region (SmAPα1-21 and SmAPα10-21) and two from the γ-core region (SmAPγ27-44 and SmAPγ29-35). We have chemically synthesized and purified the peptides and further characterized them by electrospray ionization mass spectrometry (ESI-MS) and Circular dichroism (CD) spectroscopy. SmAPα1-21, SmAPα10-21, and SmAPγ27-44 inhibited the growth of the phytopathogen F. graminearum at low micromolar concentrations. Conidia exposure to the fungicidal concentration of the peptides caused membrane permeabilization to the fluorescent probe propidium iodide (PI), suggesting that this is one of the main contributing factors in fungal cell killing. Furthermore, conidia treated for 0.5h showed cytoplasmic disorganization as observed by transmission electron microscopy (TEM). Remarkably, the peptides derived from the α-core induced morphological changes on the conidia cell wall, which is a promising target since its distinctive biochemical and structural organization is absent in plant and mammalian cells.

A metabolic control analysis approach to introduce the study of systems in biochemistry: the glycolytic pathway in the red blood cell.

Metabolic control analysis (MCA) is a promising approach in biochemistry aimed at understanding processes in a quantitative fashion. Here the contribution of enzymes and transporters to the control of a given pathway flux and metabolite concentrations is determined and expressed quantitatively by means of numerical coefficients. Metabolic flux can be influenced by a wide variety of modulators acting on one or more metabolic steps along the pathway. We describe a laboratory exercise to study metabolic regulation of human erythrocytes (RBCs). Within the framework of MCA, students use these cells to determine the sensitivity of the glycolytic flux to two inhibitors (iodoacetic acid: IA, and iodoacetamide: IAA) known to act on the enzyme glyceraldehyde-3-phosphate-dehydrogenase. Glycolytic flux was estimated by determining the concentration of extracellular lactate, the end product of RBC glycolysis. A low-cost colorimetric assay was implemented, that takes advantage of the straightforward quantification of the absorbance signal from the photographic image of the multi-well plate taken with a standard digital camera. Students estimate flux response coefficients for each inhibitor by fitting an empirical function to the experimental data, followed by analytical derivation of this function. IA and IAA exhibit qualitatively different patterns, which are thoroughly analyzed in terms of the physicochemical properties influencing their action on the target enzyme. IA causes highest glycolytic flux inhibition at lower concentration than IAA. This work illustrates the feasibility of using the MCA approach to study key variables of a simple metabolic system, in the context of an upper level biochemistry course. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):502-515, 2018.

N-glycosylation Triggers a Dual Selection Pressure in Eukaryotic Secretory Proteins.

Nearly one third of the eukaryotic proteome traverses the secretory pathway and most of these proteins are N-glycosylated in the lumen of the endoplasmic reticulum. N-glycans fulfill multiple structural and biological functions, and are crucial for productive folding of many glycoproteins. N-glycosylation involves the attachment of an oligosaccharide to selected asparagine residues in the sequence N-X-S/T (X ≠ P), a motif known as an N-glycosylation'sequon'. Mutations that create novel sequons can cause disease due to the destabilizing effect of a bulky N-glycan. Thus, an analogous process must have occurred during evolution, whenever ancestrally cytosolic proteins were recruited to the secretory pathway. Here, we show that during evolution N-glycosylation triggered a dual selection pressure on secretory pathway proteins: while sequons were positively selected in solvent exposed regions, they were almost completely eliminated from buried sites. This process is one of the sharpest evolutionary signatures of secretory pathway proteins, and was therefore critical for the evolution of an efficient secretory pathway.

Exploring protein interfaces with a general photochemical reagent.

Protein folding, natural conformational changes, or interaction between partners involved in recognition phenomena brings about differences in the solvent-accessible surface area (SASA) of the polypeptide chain. This primary event can be monitored by the differential chemical reactivity of functional groups along the protein sequence. Diazirine (DZN), a photoreactive gas similar in size to water, generates methylene carbene (:CH(2)). The extreme chemical reactivity of this species allows the almost instantaneous and indiscriminate modification of its immediate molecular cage. (3)H-DZN was successfully used in our laboratory for studying protein structure and folding. Here we address for the first time the usefulness of this probe to examine the area of interaction in protein-protein complexes. For this purpose we chose the complex formed between hen egg white lysozyme (HEWL) and the monoclonal antibody IgG(1) D1.3. :CH(2) labeling of free HEWL or complexed with IgG(1) D1.3 yields 2.76 and 2.32 mmol CH(2) per mole protein at 1 mM DZN concentration, respectively. This reduction (15%) becomes consistent with the expected decrement in the SASA of HEWL occurring upon complexation derived from crystallographic data (11%), in agreement with the known unspecific surface labeling reaction of :CH(2). Further comparative analysis at the level of tryptic peptides led to the identification of the sites involved in the interaction. Remarkably, those peptides implicated in the contact area show the highest differential labeling: H(15)GLDNYR(21), G(117)TDVQAWIR(125), andG(22)YSLGNWVCAAK(33). Thus, protein footprinting with DZN emerges as a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.

El clima laboral desde las percepciones del profesional de enfermería de instituciones hospitalarias públicas de Corrientes capital / The work environment from the perceptions of the nursing professional of public hospitals in Corrientes capital

El clima laboral no es otra cosa que el medio en el que se desarrolla el trabajo cotidiano. La calidad de éste influye directamente en la satisfacción de los trabajadores y por ende en la productividad; se encuentra íntimamente relacionado con el manejo social de los directivos, las ventajas y desventajas del liderazgo, comportamientos de los trabajadores, manera de trabajar y de relacionarse, los recursos materiales que se utilizan y las características propias de la actividad que desarrollan. Como objetivo se propuso describir las percepciones sobre el clima laboral que tienen los profesionales de enfermería de planta permanente de los hospitales públicos Escuela "Gral. José Francisco de San Martín", "Juan Ramón Vidal" y Pediátrico "Juan Pablo II", de Corrientes Capital, a través de un estudio cuantitativo, descriptivo, observacional y transversal realizado de Agosto a Octubre de 2018. Se diseñó una encuesta para la medición de las variables en las que incluían tres dimensiones, de la organización y el puesto de trabajo, los datos sociodemográficos y el entorno y condiciones laborales. La población total en estudio comprendió a 145, del cual se extrajo un tamaño muestral de los tres conglomerados de 123. Los resultados permitieron indagar las diferentes percepciones sobre clima laboral identificando las características relacionadas con los estilos de liderazgo preponderante, las habilidades comunicacionales entre pares y otros, disponibilidad de recursos y condiciones del espacio en que se desenvuelven y las expectativas personales. Se detectaron condiciones satisfactorias respecto a factores que influyen y determinan aspectos positivos para un entorno laboral saludable como el estilo de liderazgo democrático reconocido por la mayoría; así también como opiniones positivas respecto a la confiabilidad de la información impartida por sus superiores. Sin embargo algunos determinantes resultaron desfavorables para un buen clima laboral como los recursos materiales insuficientes, distorsiones comunicativas e inconvenientes en las relaciones interpersonales. Las dimensiones estudiadas permitieron detectar puntos débiles sobre los cuales se hace imprescindible reforzar y acompañar al trabajador. El conocimiento del clima laboral en las diferentes instituciones de salud pública proporcionó información acerca de los procesos que determinan los comportamientos organizacionales, permitiendo describir actitudes y conductas de los miembros y conformación de la estructura institucional. Así también destacar características favorables a fin de promover políticas organizacionales para su afianzamiento y consolidación

SUMMARY The working environment is nothing other than the medium in which daily work is carried out. The quality of this directly affects the satisfaction of workers and productivity; it is closely related to the social management of managers, the advantages and disadvantages of leadership, the behavior of workers, the way of working and the relationship, the resources used and the characteristics of the corresponding activity. What is the objective of describing the perceptions about the work climate that the nursing professionals of the permanent plant of public hospitals have? José Francisco de San Martín "," Juan Ramón Vidal "and Pediatric" Juan Pablo II ", of Corrientes Capital, through a quantitative, descriptive, observational and transversal study carried out from August to October 2018. A survey was designed to measure the variables that include the dimensions, the organization and the job, the sociodemographic data and the environment and working conditions. The total population in the study comprised 145, from which a sample size of the three conglomerates of 123 was extracted.The results allowed the different perceptions about the work climate identifying the characteristics related to the predominant styles of leadership, the communication skills between peers and others, the availability of resources and the conditions of the space in which they develop and personal expectations. Satisfactory conditions were detected regarding factors that influence and determine positive aspects for a work environment such as the style of democratic leadership recognized by the majority; As well as positive opinions regarding the reliability of the information imparted by their superiors. However, some determinants were unfavorable for a good working environment as insufficient resources, communicative distortions and inconveniences in interpersonal relationships. The dimensions studied allowed to detect the weak points on which it is essential and accompany the worker. The knowledge of the work environment in the different health institutions provided information about the processes that determine the organizational behaviors, the descriptors and behaviors of the members and the conformation of the institutional structure. Also highlight favorable characteristics in order to promote organizational policies for its consolidation and consolidation

RESUMO O ambiente de trabalho nada mais é do que o meio em que o trabalho diário é realizado. A qualidade disso afeta diretamente a satisfação dos trabalhadores e, portanto, a produtividade; Ele está intimamente relacionado com a gestão social dos gestores, as vantagens e desvantagens de comportamentos de liderança dos trabalhadores, maneira de trabalhar e se relacionar, recursos materiais são utilizados e as características das atividades realizadas. Como objetivo, foi proposto descrever as percepções sobre o clima de trabalho que os profissionais de enfermagem permanente dos hospitais públicos possuem. "Gral. José Francisco de San Martin "" Juan Ramón Vidal "e Pediátrica" Juan Pablo II", Corrientes Capital, através de um estudo quantitativo, descritivo, observacional transversal realizado de agosto a outubro 2018. Uma pesquisa foi desenvolvida para medir as variáveis em que eles incluíam três dimensões, a organização e o trabalho, os dados sociodemográficos e o ambiente e condições de trabalho. A população total em estudo compreendeu 145, dos quais foi extraído um tamanho de amostra dos três conglomerados de 123. Os resultados permitiram investigar as diferentes percepções do ambiente de trabalho, identificando as características relacionadas com os estilos predominantes de liderança, habilidades de comunicação entre pares e outros, disponibilidade de recursos e condições de espaço em que vivem e expectativas pessoais. Foram detectadas condições satisfatórias quanto aos fatores que influenciam e determinam aspectos positivos para um ambiente de trabalho saudável, como o estilo de liderança democrática reconhecido pela maioria; bem como opiniões positivas sobre a confiabilidade das informações transmitidas por seus superiores. No entanto, alguns determinantes foram desfavoráveis para um bom ambiente de trabalho, como recursos materiais insuficientes, distorções de comunicação e inconveniências nas relações interpessoais. As dimensões estudadas permitiram detectar pontos fracos sobre os quais é essencial reforçar e acompanhar o trabalhador. O conhecimento do ambiente de trabalho nas diferentes instituições de saúde pública forneceu informações sobre os processos que determinam os comportamentos organizacionais, permitindo descrever as atitudes e comportamentos dos membros e a conformação da estrutura institucional. Destacar também características favoráveis para promover políticas organizacionais para sua consolidação e consolidação

Probing protein surface with a solvent mimetic carbene coupled to detection by mass spectrometry.

Much knowledge into protein folding, ligand binding, and complex formation can be derived from the examination of the nature and size of the accessible surface area (SASA) of the polypeptide chain, a key parameter in protein science not directly measurable in an experimental fashion. To this end, an ideal chemical approach should aim at exerting solvent mimicry and achieving minimal selectivity to probe the protein surface regardless of its chemical nature. The choice of the photoreagent diazirine to fulfill these goals arises from its size comparable to water and from being a convenient source of the extremely reactive methylene carbene (:CH(2)). The ensuing methylation depends primarily on the solvent accessibility of the polypeptide chain, turning it into a valuable signal to address experimentally the measurement of SASA in proteins. The superb sensitivity and high resolution of modern mass spectrometry techniques allows us to derive a quantitative signal proportional to the extent of modification (EM) of the sample. Thus, diazirine labeling coupled to electrospray mass spectrometry (ESI-MS) detection can shed light on conformational features of the native as well as non-native states, not easily addressable by other methods. Enzymatic fragmentation of the polypeptide chain at the level of small peptides allows us to locate the covalent tag along the amino acid sequence, therefore enabling the construction of a map of solvent accessibility. Moreover, by subsequent MS/MS analysis of peptides, we demonstrate here the feasibility of attaining amino acid resolution in defining the target sites.

Experimentally approaching the solvent-accessible surface area of a protein: insights into the acid molten globule of bovine alpha-lactalbumin.

Each conformational state of a protein is inextricably related to a defined extent of solvent exposure that plays a key role in protein folding and protein interactions. However, accurate measurement of the solvent-accessible surface area (ASA) is difficult for any state other than the native (N) state. We address this fundamental physicochemical parameter through a new experimental approach based on the reaction of the photochemical reagent diazirine (DZN) with the polypeptide chain. By virtue of its size, DZN is a reasonable molecular mimic of aqueous solvent. Here, we structurally characterize nonnative states of the paradigmatic protein alpha-lactalbumin. Covalent tagging resulting from unspecific methylene (:CH(2)) reaction allows one to obtain a global estimate of ASA and to map out solvent accessibility along the amino acid sequence. By its mild apolar nature, DZN also reveals a hydrophobic phase in the acid-stabilized state of alpha-lactalbumin, in which there is clustering of core residues accessible to the solvent. In a fashion reminiscent of the N state, this acid-stabilized state also exhibits local regions where increased :CH(2) labeling indicates its nonhomogenous nature, likely pointing to the existence of packing defects. By contrast, the virtual absence of a defined long-range organization brings about a featureless labeling pattern for the unfolded state. Overall, :CH(2) labeling emerges as a fruitful technique that is able to quantify the ASA of the polypeptide chain, thus probing conformational features such as the outer exposed surface and inner cavities, as well as revealing the existence of noncompact apolar phases in nonnative states.

Assessing native and non-native conformational states of a protein by methylene carbene labeling: the case of Bacillus licheniformis beta-lactamase.

Much knowledge of protein folding can be derived from the examination of the nature and size of solvent-exposed surfaces along conformational transitions. We exploit here a general photochemical modification with methylene carbene of the accessible surface area (ASA) of the polypeptide chain. Labeling of Bacillus licheniformis beta-lactamase (BL-betaL) with 1 mM 3H-diazirine yielded 8.3 x 10(-3) mol CH2/mol protein, in agreement with the prediction for an unspecific surface labeling phenomenon. The unfolded state U in 7 M urea was labeled 60% more than the native state N. This result lies well below the increment of ASA expected from theoretical estimates and points to the presence of residual organization in state U and/or of cavities or crevices favoring the partition of the reagent in state N. A partially folded state I was demonstrated from two sequential transitions occurring at 1.5-3.0 M and 3.5-6.5 M urea. This technique shows a close correlation with optical probes most sensitive to changes in tertiary structure, a statement supported by the fact that the largest change occurs along the N-I portion of the N-I-U transition and along the acid pH-induced N-A transition. In the latter case, state A is labeled 70% more than state N, an increment consistent with the loosening of tight interactions in the core of the protein. Fragmentation of labeled BL-betaL into peptides provides a sequential map of solvent accessibility. Thus, amino acid residues pertaining to the Omega-loop and to helices alpha5 and alpha6 line the major cavity of the protein, that is big enough to lodge the diazirine reagent. Methylene labeling, by introducing an original (and perhaps unique) experimental measurement of ASA, enlightens subtle aspects of complex transitions and makes possible a comparative structural characterization of the native as well as non-native states.