ROLE OF TRNA MODIFYING ENZYMES IN PANCREATIC BETA CELL DEMISE
Creators: name: COSENTINO, CRISTINA
Publisher: name: Universit� degli Studi di Milano
Year: 2015

description: 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-transferase TRMT10A. A mutation in this gene - the insertion of a stop codon and consequent absence of mature protein - was identified in patients affected by microcephaly and young onset diabetes. It was demonstrated that TRMT10A modifies guanine residues, but its role in tRNA modification in human is still not demonstrated. The absence of the protein leads to an increase of cell death in basal conditions and sensitizes cells to ER stress-induced apoptosis. My work aimed at the characterization of the consequences of TRMT10A deficiency on tRNA modification and stability. I used lymphoblast cells derived from controls subjects and patients to investigate this tRNAs molecules specific for glutamine and methionine were identified to be modified by the enzyme, and the development of a northern blot technique allowed me to obtain preliminary on the aminoacylation and stability of these molecules in TRMT10A deficiency conditions. Furthermore I investigated the mechanisms that lead to beta cell death, triggered by the absence of the protein. With this purpose I induced the silencing of the gene in two different cell lines: INS-1E and EndoC- ?H1 (human beta cell line). Results obtained demonstrated that TRMT10A deficiency triggers the activation of the intrinsic pathway of apoptosis through the modulation of Bim expression, a proapoptotic protein of the BH3-only family. The results obtained highlighted the importance of TRMT10A for the survival of the beta cells. Furthermore the activation of the intrinsic pathway of apoptosis is one of the events observed in the development of type 2 diabetes. These findings can give additional proves that the monogenic forms of diabetes can be used as model for the study of mechanisms involved in type 2 diabetes. Even if further investigations on the complex processes involved are needed, the present work provides important evidences of the role of tRNA modifying enzyme in beta cell homeostasis. Moreover recent reports about the role of tRNAs in signalling pathways support the hypothesis that these molecules can be important mediators of stress response in beta cells, and the tRNA modifying enzymes may act as activators or inhibitors of these responses.