Data di Pubblicazione:
2015
Citazione:
ROLE OF TRNA MODIFYING ENZYMES IN PANCREATIC BETA CELL DEMISE / C. Cosentino ; co-tutor: M. Cnop, M. Igoillo-Esteve ; tutore: C. Battaglia ; coordinatore: M. Clerici. DIPARTIMENTO DI BIOTECNOLOGIE MEDICHE E MEDICINA TRASLAZIONALE, 2015 Dec 17. 28. ciclo, Anno Accademico 2015. [10.13130/c-cosentino_phd2015-12-17].
Abstract:
Transfer RNAs (tRNAs) are small molecules of 70-80 nucleotides with a crucial role in protein synthesis. tRNAs once transcribed are highly modified and the methylation is the most common modification. Several enzymes are responsible of tRNA modification and their function is necessary to regulate the stability, the aminoacylation and the rigidity of the structure of tRNAs. De-aminoacylated or degraded tRNAs can act as important signal molecules in the cells, activating different pathways of stress response. For this reason is not surprising that mutations in genes codifying for tRNA modifying enzymes have been associated to many human diseases. Polymorphisms in the gene CDKAL1, codifying the Cdk5 regulatory associated protein 1, have been linked to the development of type 2 diabetes (T2D) in human. CDKAL1 is a methyl-thio transferase that modifies the residue in position 37 of tRNAs, which recognize the codon UUU for lysine. The absence of CDKAL1, and consequently of the modification catalyzed by the enzyme, was shown to induces a decrease of incorporation of lysine residues in proinsulin at the level of pancreatic beta cells. Lysine residues are crucial for the correct maturation of proinsulin. It was shown that the absence of CDKLA1 mediated modifications leads to defects in the processing of proinsulin to produce insulin and c-peptide and to impaired glucose-stimulated insulin secretion. Furthermore in CDKAL1 knock out beta cells it’s observed an increase of markers of endoplasmic reticulum (ER) stress. The chronic activation of ER stress processes decreases the general protein synthesis and the chronic activations triggers pro-apoptotic pathways. These events have been linked to the development of T2D.
The aim of the present work is to study the role of tRNA modifying enzymes in pancreatic beta cells and to investigate the consequences of mutations in these genes on cell function and survival.
A Whole Exome Sequencing study performed previously from my group produced a list of candidate genes for Congenital Hyperinsulinism (CHI). CHI is a rare disease, characterized by inappropriate insulin secretion leading to hypoglycemia. Mutations in nine genes are already known to be causative of the disease, but in 50% of patients the genetic cause is unknown. Using bioinformatics tool I identified CDKAL1 as one of the most promising candidate genes. The mutation identified leads to the substitution of a Serine with a Phenylalanine in position 561, with probable consequences on the transmembrane domain that ensures the correct localization of the protein in the membrane of the ER. In order to study the consequences of S561F CDKAL1 variant in beta cells, I used molecular biology techniques inducing the overexpression of wild type and mutated gene in INS-1E cell line, derived from rat insulinoma. The localization of CDKAL1 was analyzed by immunofluorescence microscopy: the S561F variant affect the localization of the protein that, although still inserted in the ER, tends to accumulate in vesicular structures in some regions of the ER membrane. I also studied the impact of S561F CDKAL1 overexpression on the beta cell function, by measuring the content and the release of insulin in basal growing conditions. I observed an increase of insulin content induced by the overexpression of the wild type protein while the insulin release was not changed. On the other hand, the S561F variant doesn’t affect the insulin content that doesn’t change compared to not-transfected cells, but induces an increase in insulin release. These preliminary results suggest that the S561F CDKAL1 variant could have a role in the development of beta cell dysfunction leading to an inappropriate insulin secretion.
The second part of my project regards the methyl-
Tipologia IRIS:
Tesi di dottorato
Keywords:
tRNA modifying enzymes; Diabetes; Congenital Hyperinsulinism; Apoptosis; Beta cell
Elenco autori:
C. Cosentino
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