RESUMEN
Williams syndrome (WS) is a rare genetic disorder caused by a microdeletion of chromosome 7q11.23. Although the mortality rate of patients with WS is not very high, sudden cardiac death can occur, particularly in cases complicated by coronary artery stenosis. A 3-month-old female infant with supravalvular aortic stenosis and peripheral pulmonary stenosis was discovered unconscious in bed by her mother. She was immediately transferred to an emergency hospital but succumbed despite multiple attempts as resuscitation. DNA microarray analysis revealed microdeletions of 7q11.23 and 16p11.2, confirming WS and unexpectedly identifying 16p11.2 deletion syndrome which is known to be associated with neurodevelopmental disorders. Postmortem computed tomography revealed a severely enlarged heart, indicative of cardiac dysfunction. External examination revealed moderate-to-severe developmental delays in height and body weight. The heart, on internal examination, revealed whitish-discolored lesions; histologically severe fibrotic changes and thickening of the intima in the coronary arteries and aorta. In the brain, the dentate gyrus of the hippocampus appeared malformed. Taken together, these findings suggest that the cause of death was cardiac dysfunction due to WS. In addition, it could be possible that 16p11.2 deletion syndrome and dentate gyrus malformation contributed to her death. Future autopsy studies are warranted to clarify the precise role of microdeletion disorders in sudden death to reduce future preventable deaths in children.
Asunto(s)
Trastorno Autístico , Trastornos de los Cromosomas , Estenosis Coronaria , Discapacidad Intelectual , Síndrome de Williams , Humanos , Niño , Lactante , Femenino , Síndrome de Williams/complicaciones , Síndrome de Williams/genética , Deleción Cromosómica , Muerte Súbita Cardíaca/etiología , Cromosomas Humanos Par 16RESUMEN
BACKGROUND: Ganciclovir is a therapeutic choice for extremely premature infants with severe postnatal cytomegalovirus disease, but little is known about its optimal dose size and dosing interval for them. CASE PRESENTATION: We treated an extremely premature female infant with postnatal cytomegalovirus infection with intravenous administration of ganciclovir since 49 days of life (postmenstrual age of 31 weeks). After ganciclovir treatment was initiated at a dose of 5 mg/kg every 12 h, cytomegalovirus loads in the peripheral blood were markedly decreased. However, since plasma ganciclovir trough level was too high, the interval was extended to every 24 h. Subsequently, the trough level and the estimated 12-h area under the concentration-time curve (AUC0-12) were decreased from 3.5 mg/L to 0.3 mg/L and 53.9 mg · h/L to 19.2 mg · h/L, respectively, resulting in an exacerbation of viremia and clinical condition. Adjustment of dosing interval from 24 h to 12 h led to a peak level of 4.2 mg/L, trough level of 1.1 mg/L, and AUC0-12 of 31.8 mg · h/L, resulting in a marked suppression of viral load. CONCLUSIONS: Monitoring the therapeutic drug levels and cytomegalovirus loads is useful in obtaining a proper treatment effect and preventing overdosage during ganciclovir therapy in premature infants with postnatal cytomegalovirus infection.
Asunto(s)
Antivirales/administración & dosificación , Infecciones por Citomegalovirus/tratamiento farmacológico , Monitoreo de Drogas , Ganciclovir/administración & dosificación , Recien Nacido con Peso al Nacer Extremadamente Bajo , Recien Nacido Extremadamente Prematuro , Enfermedades del Prematuro/tratamiento farmacológico , Antivirales/uso terapéutico , Área Bajo la Curva , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Ganciclovir/uso terapéutico , Humanos , Recién Nacido , Carga ViralRESUMEN
Recent studies demonstrated that endosomal transport played important roles in various plant functions. The RAB GTPase regulates the tethering and fusion steps of vesicle trafficking to target membranes in each trafficking pathway by acting as a molecular switch. RAB GTPase activation is catalyzed by specific guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP on the RAB GTPase with GTP. RAB5 is a key regulator of endosomal trafficking and is uniquely diversified in plants; the plant-unique RAB5 group ARA6 was acquired in addition to conventional RAB5 during evolution. In Arabidopsis thaliana, conventional RAB5, ARA7 and RHA1 regulate the endosomal/vacuolar trafficking pathways, whereas ARA6 acts in the pathway from the endosome to the plasma membrane. Despite their distinct functions, all RAB5 members are activated by the common GEF VACUOLAR PROTEIN SORTING 9a (VPS9a). VPS9a consists of an N-terminal conserved domain and C-terminal region (CTR) with no similarity to known functional domains. In this study, we investigated the function of the CTR by generating truncated versions of VPS9a and found that it was specifically responsible for ARA6 regulation; moreover, the CTR was required for the oligomerization and correct localization of VPS9a. The oligomerization of VPS9a was mediated by a distinctive region consisting of 36 amino acids in the CTR that was conserved in plant RAB5 GEFs. Thus the VPS9a CTR plays an important role in the regulation of the two RAB5 groups in plants.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Factores de Intercambio de Guanina Nucleótido/genética , Proteínas de Unión al GTP rab/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Transporte de Proteínas , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Rice glutelin polypeptides are initially synthesized on the endoplasmic reticulum (ER) membrane as a proglutelin, which are then transported to the protein storage vacuole (PSV) via the Golgi apparatus. Rab5 and its cognate activator guanine nucleotide exchange factor (GEF) are essential for the intracellular transport of proglutelin from the Golgi apparatus to the PSV. Results from previous studies showed that the double recessive type of glup4/rab5a and glup6/gef mutant accumulated much higher amounts of proglutelin than either parent line. The present study demonstrates that the double recessive type of glup4/rab5a and glup6/gef mutant showed not only elevated proglutelin levels and much larger paramural bodies but also reduced the number and size of PSVs, indicating a synergistic mutation effect. These observations led us to the hypothesis that other isoforms of Rab5 and GEF also participate in the intracellular transport of rice glutelin. A database search identified a novel guanine nucleotide exchange factor, Rab5-GEF2. Like GLUP6/GEF, Rab5-GEF2 was capable of activating Rab5a and two other Rab5 isoforms in in vitro GTP/GDP exchange assays. GEF proteins consist of the helical bundle (HB) domain at the N-terminus, Vps9 domain, and a C-terminal region. By the deletion analysis of GEFs, the HB domain was found essential for the activation of Rab5 proteins.
Asunto(s)
Glútenes/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Endospermo , Aparato de Golgi/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Datos de Secuencia Molecular , Mutación , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Transporte de Proteínas/genética , Análisis de Secuencia de ADN , Vacuolas/metabolismo , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as a precursor, which are then transported via the Golgi to protein storage vacuoles (PSVs), where they are proteolytically processed into acidic and basic subunits. The glutelin precursor mutant6 (glup6) accumulates abnormally large amounts of proglutelin. Map-base cloning studies showed that glup6 was a loss-of-function mutant of guanine nucleotide exchange factor (GEF), which activates Rab GTPase, a key regulator of membrane trafficking. Immunofluorescence studies showed that the transport of proglutelins and α-globulins to PSV was disrupted in glup6 endosperm. Secreted granules of glutelin and α-globulin were readily observed in young glup6 endosperm, followed by the formation of large dilated paramural bodies (PMBs) containing both proteins as the endosperm matures. The PMBs also contained membrane biomarkers for the Golgi and prevacuolar compartment as well as the cell wall component, ß-glucan. Direct evidence was gathered showing that GLUP6/GEF activated in vitro GLUP4/Rab5 as well as several Arabidopsis (Arabidopsis thaliana) Rab5 isoforms to the GTP-bound form. Therefore, loss-of-function mutations in GEF or Rab5 disrupt the normal transport of proglutelin from the Golgi to PSVs, resulting in the initial extracellular secretion of these proteins followed, in turn, by the formation of PMBs. Overall, our results indicate that GLUP6/GEF is the activator of Rab5 GTPase and that the cycling of GTP- and GDP-bound forms of this regulatory protein is essential for the intracellular transport of proglutelin and α-globulin from the Golgi to PSVs and in the maintenance of the general structural organization of the endomembrane system in rice seeds.
Asunto(s)
Endospermo/metabolismo , Glútenes/metabolismo , Aparato de Golgi/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Oryza/metabolismo , Vacuolas/metabolismo , Mapeo Cromosómico , Endospermo/genética , Endospermo/ultraestructura , Prueba de Complementación Genética , Glútenes/genética , Aparato de Golgi/genética , Factores de Intercambio de Guanina Nucleótido/genética , Microscopía Electrónica de Transmisión , Mutación , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Transporte de Proteínas/genética , Vacuolas/genética , Proteínas de Unión al GTP rab5RESUMEN
Rab small GTPases regulate vesicle transport in eukaryotes by interacting with various effectors. Guanine nucleotide-exchange factor (GEF) catalyzes the transition from inactive GDP-bound Rab to active GTP-bound Rab. The existence of several GDP-bound intermediates containing the Arabidopsis thaliana Rab5 homologue ARA7 and the GEF VPS9a prior to the formation of a nucleotide-free binary complex has been proposed [Uejima et al. (2010), J. Biol. Chem. 285, 36689-36697]. During this process, VPS9a directly interacts with the ß-phosphate of GDP and the P-loop lysine of ARA7 via a catalytically important aspartate finger, which promotes the release of GDP from ARA7. However, it is unclear how VPS9a removes Mg2+ from ARA7 before forming the GDP-bound ternary complex. Here, the structure of the ARA7-GDP-Ca2+-VPS9a complex is reported, in which the aspartate finger directly coordinates the divalent metal ion. Ca2+ is bound to the canonical Mg2+-binding site, coordinated by the ß-phosphate of GDP and the P-loop serine of ARA7. Unexpectedly, Ca2+ is further coordinated by the aspartate finger and the main chain of VPS9a. This structure may represent the earliest intermediate step in the GEF-catalyzed nucleotide-exchange reaction of ARA7 before the metal-free GDP-bound intermediates are created.
Asunto(s)
Proteínas de Arabidopsis/química , Calcio/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Proteínas de Unión al GTP rab/química , Arabidopsis/enzimología , Proteínas de Arabidopsis/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Calcio/química , Cristalografía por Rayos X , Factores de Intercambio de Guanina Nucleótido/metabolismo , Unión Proteica , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP ß-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP ß-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.