RESUMEN

The HtrA proteins due to their proteolytic, and in many cases chaperone activity, efficiently counteract consequences of stressful conditions. In the environmental bacterium and nosocomial pathogen Stenotrophomonas maltophilia HtrA (HtrASm) is induced as a part of adaptive response to host temperature (37°C). We examined the biochemical properties of HtrASm and compared them with those of model HtrAEc from Escherichia coli. We found that HtrASm is a protease and chaperone that operates over a wide range of pH and is highly active at temperatures between 35 and 37°C. The temperature-sensitive activity corresponded well with the lower thermal stability of the protein and weaker stability of the oligomer. Interestingly, the enzyme shows slightly different substrate cleavage specificity when compared to other bacterial HtrAs. A computational model of the three-dimensional structure of HtrASm indicates differences in the S1 substrate specificity pocket and suggests weaker inter-trimer interactions when compared to HtrAEc. The observed features of HtrASm suggest that this protein may play a protective role under stressful conditions acting both as a protease and a chaperone. The optimal temperatures for the protein activity may reflect the evolutionary adaptation of S. maltophilia to life in soil or aqueous environments, where the temperatures are usually much below 37°C.

Asunto(s)

Proteínas Bacterianas/química , Fenómenos Bioquímicos , Serina Endopeptidasas/química , Stenotrophomonas maltophilia/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Biología Computacional , Activación Enzimática , Estabilidad de Enzimas , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Proteolisis , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/metabolismo , Especificidad por Sustrato

RESUMEN

AIM: The measurements of semiconductor diode detector response as a function of field size and beam angle of high-energy photons. BACKGROUND: In vivo dosimetry plays an important role in the therapeutic process of the patient. Because of the different orientation of the beam relative to the patient and different sizes of irradiation fields, it is extremely important to take into account the response of the detector depending on the angle and the size of the beam. MATERIALS AND METHODS: In this study we used a 30 cm × 30 cm × 25 cm PMMA slab phantom. On the surface of the phantom, various semiconductor detectors were placed sequentially in two configurations, angle and tilt. RESULTS: For the measurements of the calibration factor based on the different value of the angle, the correction coefficient value was close to 1.00 for smaller values of the angle for all the detectors used in the energy range of 6-12 MV. For the measurements, the calibration factor based on the size of the field of irradiation to the value of the correction coefficient is 1.00 for the field of 8 cm × 8 cm and 10 cm × 10 cm. With the increase field size, the correction factor shows a linear relationship in the direction of value less than 1.00. CONCLUSION: Flat Detectors - used for both photon beams generated by the accelerating potential of 6 MV and 20 MV show a greater angular dependence than the cylindrical detectors. Also, the repeatability of measurements made using the flat detector is less as evidenced by larger standard deviations for the results.

RESUMEN

High-temperature requirement A (HtrA; DegP) from Escherichia coli, an important element of the extracytoplasmic protein quality-control system, is a member of the evolutionarily conserved family of serine proteases. The characteristic feature of this protein is its allosteric mode of activation. The regulatory loops, L3, L2, L1 and LD, play a crucial role in the transmission of the allosteric signal. Yet, the role of LD has not been fully elucidated. Therefore, we undertook a study to explain the role of the individual LD residues in inducing and maintaining the proteolytic activity of HtrA. We investigated the influence of amino acid substitutions located within the LD loop on the kinetics of a model substrate cleavage as well as on the dynamics of the oligomeric structure of HtrA. We found that the mutations that were expected to disturb the loop's structure and/or interactions with the remaining regulatory loops severely diminished the proteolytic activity of HtrA. The opposite effect, that is, increased activity, was observed for G174S substitution, which was predicted to strengthen the interactions mediated by LD. HtrAG174S protein had an equilibrium shifted toward the active enzyme and formed preferentially high-order oligomeric forms.

Asunto(s)

Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Proteínas de Choque Térmico/química , Proteínas Periplasmáticas/química , Serina Endopeptidasas/química , Sitio Alostérico/genética , Sustitución de Aminoácidos , Estabilidad de Enzimas , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/metabolismo , Desnaturalización Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo

RESUMEN

Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically.

Asunto(s)

Escherichia coli/enzimología , Proteínas de Choque Térmico/metabolismo , Proteínas Periplasmáticas/metabolismo , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Dicroismo Circular , Disulfuros , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Histidina/genética , Histidina/metabolismo , Cinética , Datos de Secuencia Molecular , Mutagénesis , Oligopéptidos/genética , Oligopéptidos/metabolismo , Oxidación-Reducción , Proteínas Periplasmáticas/química , Proteínas Periplasmáticas/genética , Proteolisis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Alineación de Secuencia , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Especificidad por Sustrato , Resonancia por Plasmón de Superficie , Temperatura

RESUMEN

Bacterial HtrAs are serine proteases engaged in extracytoplasmic protein quality control and are required for the virulence of several pathogenic species. The proteolytic activity of HtrA (DegP) from Escherichia coli, a model prokaryotic HtrA, is stimulated by stressful conditions; the regulation of this process is mediated by the LA, LD, L1, L2, and L3 loops. The precise mechanism of action of the LA loop is not known due to a lack of data concerning its three-dimensional structure as well as its mode of interaction with other regulatory elements. To address these issues we generated a theoretical model of the three-dimensional structure of the LA loop as per the resting state of HtrA and subsequently verified its correctness experimentally. We identified intra- and intersubunit contacts that formed with the LA loops; these played an important role in maintaining HtrA in its inactive conformation. The most significant proved to be the hydrophobic interactions connecting the LA loops of the hexamer and polar contacts between the LA' (the LA loop on an opposite subunit) and L1 loops on opposite subunits. Disturbance of these interactions caused the stimulation of HtrA proteolytic activity. We also demonstrated that LA loops contribute to the preservation of the integrity of the HtrA oligomer and to the stability of the monomer. The model presented in this work explains the regulatory role of the LA loop well; it should also be applicable to numerous Enterobacteriaceae pathogenic species as the amino acid sequences of the members of this bacterial family are highly conserved.

Asunto(s)

Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Proteínas de Choque Térmico/química , Modelos Moleculares , Proteínas Periplasmáticas/química , Serina Endopeptidasas/química , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Mutación , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/metabolismo , Estabilidad Proteica , Estructura Terciaria de Proteína , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Relación Estructura-Actividad

RESUMEN

The HtrA proteases degrade damaged proteins and thus control the quality of proteins and protect cells against the consequences of various stresses; they also recognize specific protein substrates and in this way participate in regulation of many pathways. In many pathogenic bacteria strains lacking the HtrA function lose virulence or their virulence is decreased. This is due to an increased vulnerability of bacteria to stresses or to a decrease in secretion of virulence factors. In some cases HtrA is secreted outside the cell, where it promotes the pathogen's invasiveness. Thus, the HtrA proteases of bacterial pathogens are attractive targets for new therapeutic approaches aimed at inhibiting their proteolytic activity. The exported HtrAs are considered as especially promising targets for chemical inhibitors. In this review, we characterize the model prokaryotic HtrAs and HtrAs of pathogenic bacteria, focusing on their role in virulence. In humans HtrA1, HtrA2(Omi) and HtrA3 are best characterized. We describe their role in promoting cell death in stress conditions and present evidence indicating that HtrA1 and HtrA2 function as tumor suppressors, while HtrA2 stimulates cancer cell death induced by chemotherapeutic agents. We characterize the HtrA2 involvement in pathogenesis of Parkinson's and Alzheimer's diseases, and briefly describe the involvement of human HtrAs in other diseases. We hypothesize that stimulation of the HtrA's proteolytic activity might be beneficial in therapies of cancer and neurodegenerative disorders, and discuss the possibilities of modulating HtrA proteolytic activity considering the present knowledge about their structure and regulation.

Asunto(s)

Enfermedad de Alzheimer/tratamiento farmacológico , Proteínas Mitocondriales/antagonistas & inhibidores , Enfermedad de Parkinson/tratamiento farmacológico , Inhibidores de Proteasas/uso terapéutico , Serina Endopeptidasas/química , Enfermedad de Alzheimer/enzimología , Animales , Serina Peptidasa A2 que Requiere Temperaturas Altas , Humanos , Proteínas Mitocondriales/metabolismo , Enfermedad de Parkinson/enzimología , Proteolisis , Serina Endopeptidasas/metabolismo

RESUMEN

The aim of this study was to characterize the role of particular elements of the regulatory loop L2 in the activation process and maintaining the proteolytic activity of HtrA (DegP) from Escherichia coli. We measured the effects of various mutations introduced to the L2 loop's region (residues 228-238) on the stability of HtrA molecule and its proteolytic activity. We demonstrated that most mutations affected the activity of HtrA. In the case of the following substitutions: L229N, N235I, I238N, the proteolytic activity was undetectable. Thus, the majority of interactions mediated by the studied amino-acid residues seem to play important role in maintaining the active conformation. Formation of contacts between the apical parts (residues 231-234) of the L2 loops within the HtrA trimer, in particular the residues D232, was shown to play a crucial role in the activation process of HtrA. Stabilization of these intermolecular interactions by substitution of D232 with valine caused a stimulation of proteolytic activity whereas deletion of this region abolished the activity. Since the pathogenic E. coli strains require active HtrA for virulence, the apical part of L2 is of particular interest in terms of structure-based drug design for treatment E. coli infections.

Asunto(s)

Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas Periplasmáticas/química , Proteínas Periplasmáticas/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Sustitución de Aminoácidos , Secuencia de Bases , Dominio Catalítico/genética , Cartilla de ADN/genética , ADN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/patogenicidad , Proteínas de Escherichia coli/genética , Proteínas de Choque Térmico/genética , Calor , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Periplasmáticas/genética , Plásmidos/genética , Conformación Proteica , Desnaturalización Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Serina Endopeptidasas/genética , Espectroscopía Infrarroja por Transformada de Fourier , Virulencia/genética , Virulencia/fisiología