RESUMEN
A groin lump is not an uncommon condition in girls and female infants, and US plays a fundamental role in its exploration. The main pathologic conditions are related to the failure of obliteration of the canal of Nuck. Radiologists should gain a full understanding of the embryology and US anatomy of the inguinal canal before assessing this entity for the first time. An optimal age-adjusted US technique-including examinations at rest and during straining-is essential to help assess the canal of Nuck, diagnose a hernia, and analyze its content. The radiologist must be aware of the various types of hernial content depending on the patient's age, including intestinal, omental, ovarian, or tubouterine hernia, and the US features of each. Incarcerated hernias are common in girls and mostly contain an ovary. In such cases, it is crucial to screen for US signs suggestive of ovarian ischemic damage, thereby calling for urgent surgery. US can also depict a cyst or hydrocele of the canal of Nuck and its complications. Moreover, other rare pathologic conditions involving the inguinal area may be depicted at US, which helps guide appropriate treatment. US is the ideal modality for evaluating an inguinal lump in girls and female infants. Online supplemental material is available for this article. ©RSNA, 2022.
Asunto(s)
Quistes , Hernia Inguinal , Niño , Quistes/patología , Femenino , Hernia Inguinal/diagnóstico por imagen , Humanos , Lactante , Conducto Inguinal/patología , Masculino , Ovario , Peritoneo/patologíaRESUMEN
Placement of a central venous catheter (CVC) in the brachiocephalic vein (BCV) via the ultrasound (US)-guided supraclavicular approach was recently described in children. We aimed to determine the CVC maintenance-related complications at this site compared to the others (i.e., the femoral, the subclavian, and the jugular). We performed a retrospective data collection of prospectively registered data on CVC in young children hospitalized in a pediatric intensive care unit (PICU) during a 4-year period (May 2011 to May 2015). The primary outcome was a composite of central line-associated bloodstream infection (CLABSI) and deep-vein thrombosis (CLAT) according to the CVC site. Two hundred and twenty-five children, with respective age and weight of 7.1 (1.3-40.1) months and 7.7 (3.6-16) kg, required 257 CVCs, including 147 (57.2%) inserted in the BCV. The risk of the primary outcome was lower in the BCV than in the other sites (5.4 vs 16.4%; OR: 0.29; 95% CI: 0.12-0.70; p = 0.006). CLABSI incidence density rate (2.8 vs 8.96 per 1000 catheter days, p < 0.001) and CLAT incidence rate (2.7 vs 10%, p = 0.016) were also lower at this site. CONCLUSION: BCV catheterization via the US-guided supraclavicular approach may decrease CVC maintenance-related complications in children hospitalized in a PICU. What is Known: ⢠Placement of a central venous catheter (CVC) in children is associated with mechanical risks during insertion, and with infectious and thrombotic complications during its maintenance. ⢠Ultrasound (US)-guided supraclavicular catheterization of the brachiocephalic vein (BCV) is feasible in infants and children. What is New: ⢠This observational study suggested that BCV catheterization via the US-guided supraclavicular approach was associated with a lower risk of CVC insertion and maintenance-related complications, compared with the other catheterization sites.
Asunto(s)
Venas Braquiocefálicas , Infecciones Relacionadas con Catéteres/etiología , Infecciones Relacionadas con Catéteres/prevención & control , Cateterismo Venoso Central/métodos , Trombosis de la Vena/etiología , Trombosis de la Vena/prevención & control , Adolescente , Infecciones Relacionadas con Catéteres/diagnóstico , Infecciones Relacionadas con Catéteres/epidemiología , Niño , Preescolar , Femenino , Humanos , Incidencia , Lactante , Modelos Logísticos , Masculino , Estudios Retrospectivos , Resultado del Tratamiento , Ultrasonografía Intervencional , Trombosis de la Vena/diagnóstico , Trombosis de la Vena/epidemiologíaRESUMEN
Omp85 transporters mediate protein insertion into, or translocation across, membranes. They have a conserved architecture, with POTRA domains that interact with substrate proteins, a 16-stranded transmembrane ß barrel, and an extracellular loop, L6, folded back in the barrel pore. Here using electrophysiology, in vivo biochemical approaches and electron paramagnetic resonance, we show that the L6 loop of the Omp85 transporter FhaC changes conformation and modulates channel opening. Those conformational changes involve breaking the conserved interaction between the tip of L6 and the inner ß-barrel wall. The membrane-proximal POTRA domain also exchanges between several conformations, and the binding of FHA displaces this equilibrium. We further demonstrate a dynamic, physical communication between the POTRA domains and L6, which must take place via the ß barrel. Our findings thus link all three essential components of Omp85 transporters and indicate that they operate in a concerted fashion in the transport cycle.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Bordetella pertussis/genética , Bordetella pertussis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/aislamiento & purificación , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
FhaC is an integral outer membrane protein of the whooping cough agent Bordetella pertussis that mediates the transport to the cell surface of a major virulence factor, the filamentous haemagglutinin adhesin FHA. The FHA/FhaC pair is a prototypic TpsA/TpsB system of the widespread 'Two-Partner Secretion' pathway, dedicated to the transport of long extracellular proteins in various pathogenic and environmental Gram-negative bacteria. FhaC belongs to the ubiquitous Omp85 superfamily of protein transporters. The X-ray structure of FhaC shows that the transmembrane ß-barrel channel hypothesized to serve as the FHA-conducting pore is obstructed by two structural elements conserved among TpsB transporters, an N-terminal α helix and an extracellular loop. Here, we provide evidence for conformational dynamics of FhaC related to the secretion mechanism. Using paramagnetic electron resonance, electrophysiology and in vivo approaches, we showed that FhaC exchanges between open and closed conformations. The interaction with its secretory partner FHA alters this distribution of conformations. The open conformation of FhaC implies a large displacement from the channel of the N-terminal 'plug' helix, which remains in the periplasm during FHA secretion. The membrane environment favours the dynamics of the TpsB transporter.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sistemas de Secreción Bacterianos , Bordetella pertussis/metabolismo , Factores de Virulencia de Bordetella/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Conformación ProteicaRESUMEN
The chaperone/protease DegP belongs to the HtrA superfamily and is involved in protein quality control in the periplasm of Gram-negative bacteria. In Escherichia coli, typical substrates are unfolded or misfolded globular proteins that trigger the rearrangement of inactive DegP hexamers into substrate-sequestering 12- or 24-mers 'cages' for refolding or degradation. In Bordetella pertussis, DegP(Bp) facilitates, in addition, the secretion of FHA, a long ß-helical adhesin that passes through the periplasm in an extended conformation. We show that DegP(Bp) exists as soluble trimers and as a membrane-associated form. Different substrates interact differently with the distinct forms of DegP(Bp), and membrane-associated DegP(Bp) has high affinity for non-native FHA. Unlike more globular substrates, FHA does not efficiently mediate rearrangement of trimers into proteolytically active, short-lived dodecamers. In contrast to these dodecamers, membrane-associated DegP(Bp) is not committed to substrate degradation, although it is proteolytically competent. In B. pertussis, membrane-associated DegP(Bp) thus represents a specific functional form serving as a holding chaperone for client proteins including FHA. If FHA secretion is impaired, membrane-associated DegP(Bp) participates in its degradation. This form of DegP(Bp) is appropriate to handle substrates unsuitable to be sequestered in cages or non-folded, secretory proteins that must not be degraded.
Asunto(s)
Bordetella pertussis/enzimología , Membrana Celular/enzimología , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Periplasmáticas/metabolismo , Serina Endopeptidasas/metabolismo , Bordetella pertussis/química , Bordetella pertussis/genética , Bordetella pertussis/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Proteínas Periplasmáticas/química , Proteínas Periplasmáticas/genética , Estructura Terciaria de Proteína , Transporte de Proteínas , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Especificidad por Sustrato , Factores de Virulencia de Bordetella/química , Factores de Virulencia de Bordetella/genética , Factores de Virulencia de Bordetella/metabolismoRESUMEN
US plays a main role in evaluating neonatal gastrointestinal disorders. The authors describe the US appearance of normal neonatal bowel, midgut volvulus, small bowel atresia, meconium ileus, meconium peritonitis, different meconium plug syndromes, Hirschsprung disease, intestinal duplication cysts, some other abdominal cysts, some intraperitoneal infections, excluding necrotizing enterocolitis, and anorectal anomalies. The use of sonography in the imaging diagnostic strategies of these congenital and acquired anomalies is emphasized.
Asunto(s)
Enfermedades Intestinales/diagnóstico por imagen , Enfermedades Intestinales/embriología , Ultrasonografía Prenatal/métodos , Enterocolitis Necrotizante/diagnóstico por imagen , HumanosRESUMEN
Par27 from Bordetella pertussis belongs to a newly discovered class of dimeric peptidyl-prolyl isomerase (PPIase)/chaperones from the parvulin family. It is a tripartite protein with a central PPIase domain surrounded by N- and C-terminal sub-domains (NTD and CTD). Here, the Par27 structure was characterized by X-ray crystallography, small-angle X-ray scattering and template-based modeling. In the crystal lattice, Par27 consists of alternating well ordered and poorly ordered domains. The PPIase domains gave rise to diffuse scattering and could not be solved, whereas a 2.2A resolution crystal structure was obtained for the NTD and CTD, revealing a cradle-shaped dimeric platform. Despite a lack of sequence similarity with corresponding sub-domains, the topology of the peptide chain in the NTD/CTD core is similar to that of other monomeric PPIase/chaperones such as SurA and trigger factor from Escherichia coli. In Par27, dimerization occurs by sub-domain swapping. Because of the strong amino acid sequence similarity to other parvulin domains, a model for the Par27 PPIase domain was built by template-based modeling and validated against small-angle X-ray scattering (SAXS) data. A model of the full-length dimeric Par27 structure was built by rigid-body modeling and filtering against SAXS data using the partial crystal structure of the NTD/CTD core and the template-based PPIase model. The flexibility of protein was accounted for by representing the structure as an ensemble of different conformations that collectively reproduce the scattering data. The refined models exhibit a cradle-like shape reminiscent of other PPIase/chaperones, and the variability in the orientation of the PPIase domains relative to the NTD/CTD core platform observed in the different models suggests inter-domain flexibility that could be important for the biological activity of this protein.
Asunto(s)
Proteínas Bacterianas/química , Bordetella pertussis/enzimología , Isomerasa de Peptidilprolil/química , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Simulación de Dinámica Molecular , Isomerasa de Peptidilprolil/genética , Isomerasa de Peptidilprolil/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Menkes disease is an X-linked recessive disorder affecting copper metabolism due to an inactivating mutation of ATP7A gene. This result in loss of copper intestinal absorption, tissue deficiency and failure in multiple essential copper-enzyme systems such as the cytochrome c oxidase. Symptoms usually occur during the first months of life with neurological signs such as epilepsy associated to other signs among them typical hair appearance. We report the case of a 3 month-old infant hospitalized due to partial tonic-clonic seizures. Laboratory findings showed increased of lactates in blood and in cerebrospinal fluid. First screenings for infectious, metabolic and genetic causes were negative. After recurrence of multifocal seizures further investigations are made according to the presence of thick and tortuous hair. Low levels of ceruloplasmin and copper in plasma are in agreement with the suspected diagnosis of Menkes disease. Molecular analysis of the ATP7A gene confirmed the diagnosis with a non-sens mutation.
Asunto(s)
Epilepsia/diagnóstico , Hiperlactatemia/diagnóstico , Síndrome del Pelo Ensortijado/diagnóstico , Diagnóstico Diferencial , Epilepsia/etiología , Humanos , Hiperlactatemia/etiología , Lactante , Masculino , Síndrome del Pelo Ensortijado/complicaciones , Índice de Severidad de la EnfermedadRESUMEN
Dysregulation of the Amyloid Precursor Protein (APP) processing leading to toxic species of amyloid ß peptides (Aß) is central to Alzheimer's disease (AD) etiology. Aß peptides are produced by sequential cleavage of APP by ß-secretase (BACE-1) and γ-secretase. Lysosomotropic agent, chloroquine (CQ), has been reported to inhibit Aß peptide production. However, this effect is accompanied by an inhibition of lysosome-mediated degradation pathways. Following on from the promising activity of two series of APP metabolism modulators derived from CQ, we sought to develop new series of compounds that would retain the inhibitory effects on Aß production without altering lysosome functions. Herein, we applied a ligand-based pharmacophore modeling approach coupled with de novo design that led to the discovery of a series of biaryl compounds. Structure-activity relationship studies revealed that minor modifications like replacing a piperidine moiety of compound 30 by a cyclohexyl (compound 31) allowed for the identification of compounds with the desired profile. Further studies have demonstrated that compounds 30 and 31 act through an indirect mechanism to inhibit ß-secretase activity. This work shows that it is possible to dissociate the inhibitory effect on Aß peptide secretion of CQ-derived compounds from the lysosome-mediated degradation effect, providing a new profile of indirect ß-secretase inhibitors.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/metabolismo , Descubrimiento de Drogas , Inhibidores de Proteasas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Fenotipo , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The small Tims are chaperones that facilitate insertion of hydrophobic precursors into the inner mitochondrial membrane. We purified Tim12 and found it forms dimers that bind to Tim9. In this interaction, Tim12 undergoes structural changes that may be important for transport of its substrates in the mitochondrial carrier import pathway.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Dimerización , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/aislamiento & purificación , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/aislamiento & purificación , Membranas Mitocondriales/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/química , Proteínas Mitocondriales/aislamiento & purificación , Chaperonas Moleculares/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificaciónRESUMEN
Tim9 and Tim10 belong to the small Tim family of mitochondrial ATP-independent chaperones. They are organised in a specific hetero-oligomeric complex (TIM10) that escorts polytopic proteins into the mitochondrial inner membrane. The contributions of the individual subunits to the assembly and function of the TIM10 complex remain poorly understood. Here, we show that substrate recognition and assembly of the complex are mediated by distinct domains of the subunits. These are unrelated to the characteristic "twin CX3C" motif that is present in all small Tims and ensures proper folding of the unassembled subunits. Specifically, we show that substrate recognition is achieved by the Tim10 subunit, whilst Tim9 serves a more structural role. The N-terminal domain of Tim10 is a substrate sensor whilst its C-terminal part is essential for complex formation. By contrast, both N and C-terminal domains of Tim9 are involved in the stability of the complex.
Asunto(s)
Proteínas de la Membrana/química , Proteínas de Transporte de Membrana/química , Proteínas Mitocondriales/química , Proteínas de Saccharomyces cerevisiae/química , Secuencia de Bases , ADN de Hongos/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Complejos Multiproteicos , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Subunidades de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
OBJECTIVE: Congenital giant hydronephrosis due to ureteropelvic junction obstruction is exceptional and treatment often requires nephrectomy of a poorly functioning kidney. However, a more conservative approach by nephroplication is also possible. The objective of this study was to evaluate the long-term functional outcome of these kidneys treated by nephroplication during the neonatal period. MATERIAL AND METHODS: From 1996 to 1998, 5 neonates with congenital giant hydronephrosis were managed in our department (3 antenatal diagnoses). The mean anteroposterior pelvic dilatation was 145 mm (range: 110-180 mm). All infants were treated by pyeloplasty with nephroplication, regardless of the preoperative ultrasound and scintigraphic appearance of the renal parenchyma. RESULTS: The mean follow-up was 9 years (range: 8-10 years). A marked initial reduction of global kidney volume was constantly observed (more than 50% of the initial volume). The mean relative renal function was 32.4% (range: 10-42%) without any residual obstruction. Renal growth was satisfactory in 4 cases (40 mm at 1 year, 72 mm at 6 years) with an atrophic kidney in 1 case. No child developed hypertension or elevated serum creatinine. CONCLUSION: Congenital giant hydronephrosis does not systematically justify primary nephrectomy even in the presence of a healthy contralateral kidney. Despite the pejorative initial ultrasound and scintigraphic appearance, which is always difficult to interpret, early nephroplication improves drainage of pyelocaliceal cavities, decreases the residual dilatation and allows significant nephron sparing in most of these children.
Asunto(s)
Hidronefrosis/congénito , Hidronefrosis/cirugía , Femenino , Humanos , Recién Nacido , Masculino , Factores de Tiempo , Procedimientos Quirúrgicos Urológicos/métodosRESUMEN
TpsB proteins are Omp85 superfamily members that mediate protein translocation across the outer membrane of Gram-negative bacteria. Omp85 transporters are composed of N-terminal POTRA domains and a C-terminal transmembrane ß-barrel. In this work, we track the in vivo secretion path of the Bordetella pertussis filamentous haemagglutinin (FHA), the substrate of the model TpsB transporter FhaC, using site-specific crosslinking. The conserved secretion domain of FHA interacts with the POTRA domains, specific extracellular loops and strands of FhaC and the inner ß-barrel surface. The interaction map indicates a funnel-like pathway, with conformationally flexible FHA entering the channel in a non-exclusive manner and exiting along a four-stranded ß-sheet at the surface of the FhaC barrel. This sheet of FhaC guides the secretion domain of FHA along discrete steps of translocation and folding. This work demonstrates that the Omp85 barrel serves as a channel for translocation of substrate proteins.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bordetella pertussis/metabolismo , Fragmentos de Péptidos/metabolismo , Factores de Virulencia de Bordetella/metabolismo , Reactivos de Enlaces Cruzados/química , Cisteína/química , Escherichia coli/metabolismo , Citometría de Flujo , Regulación Bacteriana de la Expresión Génica , Hemaglutininas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Plásmidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Especificidad por SustratoRESUMEN
The small Tims chaperone hydrophobic precursors across the mitochondrial intermembrane space. Tim9 and Tim10 form the soluble TIM10 complex that binds precursors exiting from the outer membrane. Tim12 functions downstream, as the only small Tim peripherally attached on the inner membrane. We show that Tim12 has an intrinsic affinity for inner mitochondrial membrane lipids, in contrast to the other small Tims. We find that the C-terminal end of Tim12 is essential in vivo. Its deletion crucially abolishes assembly of Tim12 in complexes with the other Tims. The N-terminal end contains targeting information and also mediates direct binding of Tim12 to the transmembrane segments of the carrier substrates. These results provide a molecular basis for the concept that the essential role of Tim12 relies on its unique assembly properties that allow this subunit to bridge the soluble and membrane-embedded translocases in the carrier import pathway.
Asunto(s)
Proteínas de la Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Estructura Terciaria de Proteína/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de SecuenciaRESUMEN
Brain ultrasonography plays a central role in the detection and management of neonatal disease in the preterm infant. Although morphological study, using high-frequency transducers, remains the cornerstone of imaging, pulsed and colour Doppler scans provide additional information and improve the diagnostic and prognostic accuracy of ultrasonography. Particular features of normal brain US in the extremely preterm infant are reported. Cerebral haemorrhage and its different patterns (intraventricular haemorrhage and periventricular hemorrhagic infarction) are described. The value of Doppler techniques is emphasized, e.g. demonstration of coloured signal within the aqueduct of Sylvius, visualization of patency of the terminal veins, demonstration of Doppler spectrum fluctuations, recognition of low blood flow, and the detection of vasodilatation. The sonographic diagnosis of periventricular leucomalacia and its difficulties are documented. Some uncommon brain lesions of the premature infant are illustrated, e.g. gangliothalamic ischaemic damage, cortical necrosis, focal infarcts, etc. The importance of repeating the US examinations until near term is highlighted.
Asunto(s)
Encefalopatías/diagnóstico por imagen , Ecoencefalografía/métodos , Enfermedades del Prematuro/diagnóstico por imagen , Encéfalo/irrigación sanguínea , Encéfalo/patología , Encefalopatías/patología , Diagnóstico Diferencial , Humanos , Recién Nacido , Recien Nacido Prematuro , Enfermedades del Prematuro/patologíaRESUMEN
The phytopathogenic bacterium Ralstonia solanacearum encodes a family of seven type III secretion system (T3SS) effectors that contain both a leucine-rich repeat and an F-box domain. This structure is reminiscent of a class of typical eukaryotic proteins called F-box proteins. The latter, together with Skp1 and Cullin1 subunits, constitute the SCF-type E3 ubiquitin ligase complex and control specific protein ubiquitinylation. In the eukaryotic cell, depending on the nature of the polyubiquitin chain, the ubiquitin-tagged proteins either see their properties modified or are doomed for degradation by the 26S proteasome. This pathway is essential to many developmental processes in plants, ranging from hormone signaling and flower development to stress responses. Here, we show that these previously undescribed T3SS effectors are putative bacterial F-box proteins capable of interacting with a subset of the 19 different Arabidopsis Skp1-like proteins like bona fide Arabidopsis F-box proteins. A R. solanacearum strain in which all of the seven GALA effector genes have been deleted or mutated was no longer pathogenic on Arabidopsis and less virulent on tomato. Furthermore, we found that GALA7 is a host-specificity factor, required for disease on Medicago truncatula plants. Our results indicate that the GALA T3SS effectors are essential to R. solanacearum to control disease. Because the F-box domain is essential to the virulence function of GALA7, we hypothesize that these effectors act by hijacking their host SCF-type E3 ubiquitin ligases to interfere with their host ubiquitin/proteasome pathway to promote disease.
Asunto(s)
Arabidopsis/microbiología , Proteínas Bacterianas/metabolismo , Proteínas F-Box/metabolismo , Medicago truncatula/microbiología , Enfermedades de las Plantas/microbiología , Ralstonia solanacearum/patogenicidad , Solanum lycopersicum/microbiología , Secuencia de Aminoácidos , Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas F-Box/química , Proteínas F-Box/genética , Genes Bacterianos/genética , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Ralstonia solanacearum/genética , Ralstonia solanacearum/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , VirulenciaRESUMEN
Most of the bacterial proteins that are active in extracytoplasmic locations are translocated through the inner membrane by the Sec translocase. Translocase comprises a membrane "pore" and the peripheral ATPase SecA. Where preproteins bind to SecA and how they activate translocation ATPase remains elusive. To address this central question we have purified to homogeneity the mature and preprotein parts of an exported protein (pCH5EE). pCH5EE satisfies a minimal size required for protein translocation and its membrane insertion is SecA-dependent. Purified pCH5EE and CH5EE can form physical complexes with SecA and can functionally suppress the elevated ATPase of a constitutively activated mutant. These properties render pCH5EE and CH5EE unique tools for the biochemical mapping of the preprotein binding site on SecA.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Clonación Molecular/métodos , Proteínas de Transporte de Membrana/metabolismo , Precursores de Proteínas/metabolismo , Sitios de Unión , Escherichia coli/genética , Unión Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/aislamiento & purificación , Señales de Clasificación de Proteína , Transporte de Proteínas , Canales de Translocación SEC , Proteína SecARESUMEN
SecA, the preprotein translocase ATPase, has a helicase DEAD motor. To catalyze protein translocation, SecA possesses two additional flexible domains absent from other helicases. Here we demonstrate that one of these "specificity domains" is a preprotein binding domain (PBD). PBD is essential for viability and protein translocation. PBD mutations do not abrogate the basal enzymatic properties of SecA (nucleotide binding and hydrolysis), nor do they prevent SecA binding to the SecYEG protein conducting channel. However, SecA PBD mutants fail to load preproteins onto SecYEG, and their translocation ATPase activity does not become stimulated by preproteins. Bulb and Stem, the two sterically proximal PBD substructures, are physically separable and have distinct roles. Stem binds signal peptides, whereas the Bulb binds mature preprotein regions as short as 25 amino acids. Binding of signal or mature region peptides or full-length preproteins causes distinct conformational changes to PBD and to the DEAD motor. We propose that (a) PBD is a preprotein receptor and a physical bridge connecting bound preproteins to the DEAD motor, and (b) preproteins control the ATPase cycle via PBD.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Proteínas Bacterianas/fisiología , Proteínas de Transporte de Membrana/fisiología , Adenosina Trifosfatasas/química , Bacillus subtilis/metabolismo , Técnicas Biosensibles , Catálisis , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Hidrólisis , Cinética , Espectroscopía de Resonancia Magnética , Proteínas de la Membrana/metabolismo , Modelos Genéticos , Modelos Moleculares , Mutación , Péptidos/química , Análisis por Matrices de Proteínas , Unión Proteica , Conformación Proteica , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Canales de Translocación SEC , Proteína SecA , Especificidad por Sustrato , TemperaturaRESUMEN
BACKGROUND: Pelvi-ureteric junction (PUJ) obstruction is caused by the presence of an aperistaltic dysplastic segment at the PUJ. Besides this intrinsic aetiology, extrinsic abnormalities, mainly crossing vessels, may be an associated factor. OBJECTIVE: To determine the reliability of colour Doppler US in the detection of crossing vessels in children with surgically proven PUJ obstruction. MATERIALS AND METHODS: Forty-eight patients (50 kidneys) with PUJ obstruction, surgically treated from 1998 to 2001, were prospectively studied by colour Doppler US prior to open pyeloplasty. There were 33 boys and 15 girls (age 2 months-12 years; median 3.5 years). There were 24 right kidneys and 26 left kidneys. The indication for surgery was according to the usual criteria. The crossing vessel was considered as present when depicted on colour Doppler US at the PUJ. RESULTS: Colour Doppler assessment of the crossing vessels was correct in all but two patients. At surgery, a crossing vessel was found in 14 kidneys (i.e. 28%). Colour Doppler US results were correlated with intra-operative findings in 50 renal units. Surgically proven vessels in 14 kidneys were identified by colour Doppler US in 13, and not shown in 1. In PUJ obstruction without a crossing vessel ( n=36), US findings were concordant in 26, discordant in 8 and inconclusive in 2 (with kidney malrotation) cases. Colour Doppler US had a sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), and accuracy of 92.8%, 76.5%, 62%, 96.3% and 78%, respectively. The reliability of colour Doppler US was greatly improved with the increasing experience of the sonographer, as shown by the results of the last 2 years (26 kidneys):Se=100%, Sp=87.5%, PPV=81.8%, NPV=100%, accuracy=88.5%. CONCLUSIONS: Colour Doppler US is reliable in the detection of crossing vessels at the PUJ.
Asunto(s)
Ultrasonografía Doppler en Color , Obstrucción Ureteral/diagnóstico por imagen , Preescolar , Femenino , Humanos , Pelvis Renal/irrigación sanguínea , Pelvis Renal/diagnóstico por imagen , Masculino , Uréter/irrigación sanguínea , Uréter/diagnóstico por imagenRESUMEN
SecA, the dimeric ATPase subunit of protein translocase, contains a DEAD helicase catalytic core that binds to a regulatory C-terminal domain. We now demonstrate that IRA1, a conserved helix-loop-helix structure in the C-domain, controls C-domain conformation through direct interdomain contacts. C-domain conformational changes are transmitted to the DEAD motor and alter its conformation. These interactions establish DEAD motor/C-domain conformational cross-talk that requires a functional IRA1. IRA1-controlled binding/release cycles of the C-domain to the DEAD motor couple this cross-talk to protein translocation chemistries, i.e. DEAD motor affinities for ligands (nucleotides, preprotein signal peptides, and SecYEG, the integral membrane component of translocase) and ATP turnover. IRA1-mediated global co-ordination of SecA catalysis is essential for protein translocation.