Estudio de Hugtrel 1, un putativo transportador de udp-glucosa en células humanas
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Fecha
2012
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Profesor/a Guía
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Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
El correcto plegamiento de N-glicoproteínas ocurre al interior del Retículo Endoplásmico (RE) y depende de un riguroso control de calidad basado en las chaperonas calnexina y calreticulina. Sólo aquellas glicoproteínas completamente plegadas salen a la ruta secretoria, mientras que aquellas proteínas que permanecen con un plegamiento incompleto deben ser nuevamente glucosiladas por la proteína UDP-Glucosa glicoproteína glicosil transferasa (UGGT), antes de entrar a una nueva ronda del ciclo. Esta secuencia de reacciones requiere de moléculas de UDP-Glucosa, las cuales son sintetizadas en el citosol, por lo que deben ser internalizadas hacia el lumen del RE. Por lo tanto, es necesaria la presencia de un transportador de nucleótidos azúcar en la membrana de este organelo.
Recientemente, se ha descrito la existencia de dos transportadores de UDP-Glucosa en Arabidopsis thaliana, denominados AtUTrl y AtUTr3. AtUTrl se encuentra en el RE, mientras que AtUTr3 se encuentra tanto en RE como en el aparato de Golgi. Ambos transportadores utilizan como sustrato UDPGlucosa y ambos genes responden al mecanismo de respuesta a proteínas mal plegadas. Esto sugiere que estos transportadores pueden estar participando en
suministrar la UDP-Glucosa a la UGGT para el ciclo de calnexina/calreticulina.
En mamíferos sin embargo, no se ha descrito ninguna proteína que pueda
cumplir esta importante función.
A través del análisis de secuencia del genoma y de los ESTs, pudimos identificar un gen humano que codifica para un transportador de nucleótidos azúcar con alta identidad de secuencia a AtUTrl y AtUTr3, denominado hUGTrell. En el presente estudio se pudo comprobar la presencia de tres formas alternativas del gen, con las cuales se realizaron estudios de transporte
en levaduras mediante liposomas reconstituidos artificialmente. Además se analizó la expresión de las tres variantes de "splicing" a nivel de proteína y del transcrito en células humanas y se realizaron estudios de localización subcelular en células HeLa. Los resultados indican que, tanto las variantes hUGTrellA y hUGTrellC transportan UDP-Glucosa, y que mediante el uso de
expresión de proteínas fusionadas a GFP la variante hUGTrellA se encuentra distribuida en RE, Golgi y Compartimento Intermedio mientras que las variantes hUGTrell B y hUGTrellC se encuentran sólo en RE. Mediante el estudio de expresión de proteína y del nivel de transcrito se pudo determinar que ambas variantes se encuentran de manera endógena en células humanas, a diferencia de la variante hUGTrell B la cual no fue posible detectar. Finalmente, se analizó
la acumulación del transcrito de las distintas variantes de "splicing"de hUGTrell frente a tratamientos que inducen UPR. Los resultados muestran que las tres variantes de hUGTrell responden a este mecanismo, lo cual puede sugerir que en condiciones de una alta concentración de proteínas mal plegadas, el aumento de hUGTrell permitiría un aumento en el transporte de UDP-Glucosa hacia el interior del RE y con ello favorecer el correcto plegamiento de las glicoproteinas.
Entre las proyecciones de este trabajo se encuentran determinar cómo es la contribución de las variantes hUGTrellA y hUGTrellC en el transporte de UDP-Glucosa, ya sea de manera individual o conjunta; estudiar cómo se ve afectado el transporte de UDP-Glucosa en condiciones que inducen UPR, y determinar si existe relación entre una regulación del "splicing" de las variantes
y la acumulación de proteínas mal plegadas al interior del RE.
The correct folding of N-glycoproteins occurs within the Endoplasmic Reticulum (ER) and depends on a strict quality control based on chaperone proteins calnexin and calreticulin. Only completely folded glycoproteins exit to the secretory pathway, while those proteins that remains unfolded must be glucosylated again by the protein UDP-Glucose glycoprotein glycosyl transferase (UGGT), before entry to a new round cycle. This sequence of reactions requires UDP-Glucose molecules, which are synthesized in the cytosol, so they must be internalized into the ER lumen. Therefore, the presence of a nucleotide sugar transporter in the membrane of this organelle is necessary. Recently, the existence of two UDP-Glucose transporters in Arabidopsis thaliana, called AtUTr1 and AtUTr3, has been reported. AtUTr1 is located in the ER, while AtUTr3 is found in ER as well as in the Golgi apparatus. Both transporters use UDP-Glucose as substrate and both genes respond to the mechanism of unfolded proteins response. This suggests that these transporters may be involved in providing UDP-Glucose to the UGGT for the calnexin/ calreticulin cycle. Nevertheless, to date in mammals no protein that can fulfil this important function has been described. Through the analysis of genome sequences and ESTs, we identified a human gene encoding for a nucleotide sugar transporter with high sequence identity to AtUTr1 and AtUTr3, called hUGTre11. In this study we could prove the the presence of three alternative forms of the gene, which were undertaken transport studies in yeast using artificially reconstituted liposomes. Also, we analysed the expression of the three splicing variants at protein and transcript level in human cells and we performed subcellular localisation studies in HeLa cells. Results indicate that, both hUGTre11A and hUGTre11C variants transport UDP-Glucose, and through the use of GFP fusion proteins, we found that hUGTre11A variant is located in ER, Golgi and Intermediate Compartment while hUGTre11B and hUGTre11C variants are located in ER only. Through the study of expression of protein and transcript level we could determine that both variants are found endogenously in human cells, unlike hUGTre11B variant which was not detectable. Finally, we analysed the transcript accumulation for the three hUGTre11 splicing variants subject to UPR-inducing treatments. The results show that three hUGTre11 variants respond to this mechanism, suggesting that in conditions with a high unfolded proteins concentration, the increase of hUGTre11 would allow a higher UDP-Glucose transport into the ER lumen and thereby favouring the glycoprotein correct folding. Projections of this work includes to determine how is the contribution of each splicing variant hUGTre11A and hUGTre11 C for the UDP-Glucose transport, either individually or jointly; to study how is affected the UDP-Glucose transport in UPR-induced conditions, and to determine if there is a relation between a splicing regulation of hUGTre11 variants and the accumulation of unfolded proteins in the ER.
The correct folding of N-glycoproteins occurs within the Endoplasmic Reticulum (ER) and depends on a strict quality control based on chaperone proteins calnexin and calreticulin. Only completely folded glycoproteins exit to the secretory pathway, while those proteins that remains unfolded must be glucosylated again by the protein UDP-Glucose glycoprotein glycosyl transferase (UGGT), before entry to a new round cycle. This sequence of reactions requires UDP-Glucose molecules, which are synthesized in the cytosol, so they must be internalized into the ER lumen. Therefore, the presence of a nucleotide sugar transporter in the membrane of this organelle is necessary. Recently, the existence of two UDP-Glucose transporters in Arabidopsis thaliana, called AtUTr1 and AtUTr3, has been reported. AtUTr1 is located in the ER, while AtUTr3 is found in ER as well as in the Golgi apparatus. Both transporters use UDP-Glucose as substrate and both genes respond to the mechanism of unfolded proteins response. This suggests that these transporters may be involved in providing UDP-Glucose to the UGGT for the calnexin/ calreticulin cycle. Nevertheless, to date in mammals no protein that can fulfil this important function has been described. Through the analysis of genome sequences and ESTs, we identified a human gene encoding for a nucleotide sugar transporter with high sequence identity to AtUTr1 and AtUTr3, called hUGTre11. In this study we could prove the the presence of three alternative forms of the gene, which were undertaken transport studies in yeast using artificially reconstituted liposomes. Also, we analysed the expression of the three splicing variants at protein and transcript level in human cells and we performed subcellular localisation studies in HeLa cells. Results indicate that, both hUGTre11A and hUGTre11C variants transport UDP-Glucose, and through the use of GFP fusion proteins, we found that hUGTre11A variant is located in ER, Golgi and Intermediate Compartment while hUGTre11B and hUGTre11C variants are located in ER only. Through the study of expression of protein and transcript level we could determine that both variants are found endogenously in human cells, unlike hUGTre11B variant which was not detectable. Finally, we analysed the transcript accumulation for the three hUGTre11 splicing variants subject to UPR-inducing treatments. The results show that three hUGTre11 variants respond to this mechanism, suggesting that in conditions with a high unfolded proteins concentration, the increase of hUGTre11 would allow a higher UDP-Glucose transport into the ER lumen and thereby favouring the glycoprotein correct folding. Projections of this work includes to determine how is the contribution of each splicing variant hUGTre11A and hUGTre11 C for the UDP-Glucose transport, either individually or jointly; to study how is affected the UDP-Glucose transport in UPR-induced conditions, and to determine if there is a relation between a splicing regulation of hUGTre11 variants and the accumulation of unfolded proteins in the ER.
Notas
Tesis (Doctor en Biotecnología)
Palabras clave
Células, Glucosa, Transporte