RESUMEN
Protein glycosylation is a complex process that depends not only on the activities of several enzymes and transporters but also on a subtle balance between vesicular Golgi trafficking, compartmental pH, and ion homeostasis. Through a combination of autozygosity mapping and expression analysis in two siblings with an abnormal serum-transferrin isoelectric focusing test (type 2) and a peculiar skeletal phenotype with epiphyseal, metaphyseal, and diaphyseal dysplasia, we identified TMEM165 (also named TPARL) as a gene involved in congenital disorders of glycosylation (CDG). The affected individuals are homozygous for a deep intronic splice mutation in TMEM165. In our cohort of unsolved CDG-II cases, we found another individual with the same mutation and two unrelated individuals with missense mutations in TMEM165. TMEM165 encodes a putative transmembrane 324 amino acid protein whose cellular functions are unknown. Using a siRNA strategy, we showed that TMEM165 deficiency causes Golgi glycosylation defects in HEK cells.
Asunto(s)
Trastornos Congénitos de Glicosilación/genética , Proteínas de la Membrana/genética , Mutación , Adolescente , Antiportadores , Proteínas de Transporte de Catión , Células Cultivadas , Niño , Preescolar , Enanismo/genética , Femenino , Fibroblastos , Aparato de Golgi/metabolismo , Humanos , Lactante , Recién Nacido , Masculino , Linaje , Piel/citologíaRESUMEN
Pharmacological inhibitors that block amyloid precursor protein (APP) cleavage and the formation of senile plaques are under development for the treatment of familial Alzheimer's disease. Unfortunately, many inhibitors that block gamma-secretase-mediated cleavage of APP also have immunosuppressive side effects. In addition to APP, numerous other proteins undergo gamma-secretase-mediated cleavage. In order to develop safer inhibitors, it is necessary to determine which of the gamma-secretase substrates contribute to the immunosuppressive effects. Because APP family members are widely expressed and are reported to influence calcium flux, transcription and apoptosis, they could be important for normal lymphocyte maturation. We find that APP and amyloid precursor-like protein 2 are expressed by stromal cells of thymus and lymph nodes, but not by lymphocytes. Although signals provided by thymic stromal cells are critical for normal T cell differentiation, lymphocyte development proceeds unperturbed in mice deficient for these APP family members.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Ganglios Linfáticos/inmunología , Linfocitos/inmunología , Timo/inmunología , Secretasas de la Proteína Precursora del Amiloide/inmunología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Diferenciación Celular/inmunología , Ganglios Linfáticos/metabolismo , Linfocitos/metabolismo , Ratones , Ratones Noqueados , Células del Estroma/inmunología , Células del Estroma/metabolismo , Timo/citología , Timo/metabolismoRESUMEN
The hetero-octameric conserved oligomeric Golgi (COG) complex is essential for the structure/function of the Golgi apparatus through regulation of membrane trafficking. Here, we describe a patient with a mild form of a congenital disorder of glycosylation type II (CDG-II), which is caused by a homozygous nonsense mutation in the hCOG8 gene. This leads to a premature stop codon resulting in a truncated Cog8 subunit lacking the 76 C-terminal amino acids. Mass spectrometric analysis of the N- and O-glycan structures identified a mild sialylation deficiency. We showed that the molecular basis of this defect in N- and O-glycosylation is caused by the disruption of the Cog1-Cog8 interaction due to truncation. As a result, Cog1 deficiency accompanies the Cog8 deficiency, preventing assembly of the intact, stable complex and resulting in the appearance of smaller subcomplexes. Moreover, levels of beta1,4-galactosytransferase were significantly reduced. The defects in O-glycosylation could be fully restored by transfecting the patient's fibroblasts with full-length Cog8. The Cog8 defect described here represents a novel type of CDG-II, which we propose to name as CDG-IIh or CDG caused by Cog8 deficiency (CDG-II/Cog8).
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Errores Innatos del Metabolismo de los Carbohidratos/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Brefeldino A/farmacología , Errores Innatos del Metabolismo de los Carbohidratos/genética , Niño , Codón sin Sentido , Análisis Mutacional de ADN , Femenino , Fibroblastos/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilación , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Humanos , Immunoblotting , Inmunoprecipitación , Polisacáridos/metabolismo , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TransfecciónRESUMEN
Processing of the amyloid precursor protein (APP) leads to the production of amyloid-beta (Abeta), the major component of extracellular plaques in the brains of Alzheimer's disease (AD) patients. Presenilin-1 (PS-1) plays a key role in the final step of Abeta formation, the gamma-secretase cleavage. Previously, we showed that PS-1 is retained in pre-Golgi compartments by incorporation into COPI-coated membranes of the vesicular tubular clusters (VTCs) between endoplasmic reticulum (ER) and Golgi complex. Here, we show that PS-1 also mediates the retention of the beta-cleavage-derived APP-C-terminal fragment (CTFbeta) and/or Abeta in pre-Golgi membranes. Overexpression of PS-1 increased the percentage of CTFbeta and/or Abeta in VTCs as well as their distribution to COPI-coated VTC membranes. By contrast, overexpression of the dominant-negative aspartate mutant PS-1(D257A) or PS-knockout decreased incorporation of these APP derivatives into COPI-coated membranes. Sorting of APP derivatives to COPI-coated VTC membranes was not depending on the APP cytosolic tail. In post-Golgi compartments, PS-1 expression enhanced the association of full-length APP/APPs with endosomal compartments at the expense of plasma membrane-bound APP. We conclude that PS-1, in addition to its role in gamma-secretase cleavage, is also required for the subcellular routing of APP and its derivatives. Malfunctioning of PS-1 in this role may have important consequences for the progress of AD.