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1.
Acta Trop ; 225: 106176, 2022 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-34627755

RESUMEN

The expression of antigens in their immunologically-active form remains a challenge, both in the analysis of regulatory pathways exploited by parasitic nematodes or in the development of vaccines. Despite the success of native proteins to induce protective immunity, recombinant proteins expressed in bacteria, yeast or insect cells offer only limited protective capacities, presumably due to incorrect folding or missing complex posttranslational modifications. The present study investigates the feasibility of using the free-living nematode Caenorhabditis elegans as an alternative expression system for proteins found in the secretome of parasitic nematodes. Exemplified by the expression of the extracellular superoxide dismutase from Haemonchus contortus (HcSODe) and the extracellular and glycosylated glutathione S-transferase from the filarial parasite Onchocerca volvulus (OvGST1), we continue our efforts to improve production and purification of recombinant proteins expressed in C. elegans. We demonstrate that sufficient quantities of functional proteins can be expressed in C. elegans for subsequent immunological and biochemical studies.


Asunto(s)
Haemonchus , Nematodos , Onchocerca volvulus , Animales , Caenorhabditis elegans/genética , Haemonchus/genética , Proteínas Recombinantes/genética
2.
Hum Mutat ; 39(7): 934-938, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29663568

RESUMEN

Early myoclonic epilepsy (EME) or Aicardi syndrome is one of the most severe epileptic syndromes affecting neonates. We performed whole exome sequencing in a sporadic case affected by EME and his parents. In the proband, we identified a homozygous missense variant in the ubiquitin-like modifier activating enzyme 5 (UBA5) gene, encoding a protein involved in post-translational modifications. Functional analysis of the UBA5 variant protein reveals that it is almost completely unable to perform its trans-thiolation activity. Although recessive variants in UBA5 have recently been associated with epileptic encephalopathy, variants in this gene have never been reported to cause EME. Our results further demonstrate the importance of post-translational modifications such as the addition of an ubiquitin-fold modifier 1 (UFM1) to target proteins (ufmylation) for normal neuronal networks activity, and reveal that the dysfunction of the ubiquitous UBA5 protein is a cause of EME.


Asunto(s)
Epilepsias Mioclónicas/genética , Predisposición Genética a la Enfermedad , Espasmos Infantiles/genética , Enzimas Activadoras de Ubiquitina/genética , Adulto , Consanguinidad , Epilepsias Mioclónicas/fisiopatología , Síndromes Epilépticos/genética , Síndromes Epilépticos/fisiopatología , Femenino , Homocigoto , Humanos , Recién Nacido , Masculino , Mutación Missense/genética , Espasmos Infantiles/fisiopatología , Secuenciación del Exoma
3.
Am J Hum Genet ; 99(3): 695-703, 2016 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-27545681

RESUMEN

Via whole-exome sequencing, we identified rare autosomal-recessive variants in UBA5 in five children from four unrelated families affected with a similar pattern of severe intellectual deficiency, microcephaly, movement disorders, and/or early-onset intractable epilepsy. UBA5 encodes the E1-activating enzyme of ubiquitin-fold modifier 1 (UFM1), a recently identified ubiquitin-like protein. Biochemical studies of mutant UBA5 proteins and studies in fibroblasts from affected individuals revealed that UBA5 mutations impair the process of ufmylation, resulting in an abnormal endoplasmic reticulum structure. In Caenorhabditis elegans, knockout of uba-5 and of human orthologous genes in the UFM1 cascade alter cholinergic, but not glutamatergic, neurotransmission. In addition, uba5 silencing in zebrafish decreased motility while inducing abnormal movements suggestive of seizures. These clinical, biochemical, and experimental findings support our finding of UBA5 mutations as a pathophysiological cause for early-onset encephalopathies due to abnormal protein ufmylation.


Asunto(s)
Alelos , Encefalopatías/genética , Mutación/genética , Proteínas/metabolismo , Enzimas Activadoras de Ubiquitina/genética , Edad de Inicio , Animales , Mapeo Encefálico , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Niño , Preescolar , Neuronas Colinérgicas/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Epilepsia/genética , Exoma/genética , Femenino , Fibroblastos , Genes Recesivos/genética , Humanos , Discapacidad Intelectual/genética , Imagen por Resonancia Magnética , Masculino , Microcefalia/genética , Trastornos del Movimiento , Proteínas/genética , Transmisión Sináptica/genética , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/deficiencia , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
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