NUPR1L, A NEW P53-TARGET GENE WHICH NEGATIVELY REGULATES THE PROTUMORAL FACTOR NUPR1.

LOPEZ-MILLAN MB; GARCIA MARÍA NOÉ; DANIEL GRASSO; MOLEJON MI; NEIRA JL; IOVANNA JL

Granada

Congreso; XXXVII Congreso de la Sociedad Española de Ciencias Fisiológicas (SECF); 2014

Sociedad Española de Ciencias Fisiológicas (SECF)

Introduction Nupr1 gene has been identified in pancreas through the activation of its expression in acute pancreatitis. Nupr1 is a stress protein with nuclear localization and is highly expressed in PDAC (pancreatic ductal adenocarcinoma). In fact, our lab has demonstrated an essential role in the PanIN (pancreatic intraepithelial neoplasia) formation and in the tumor progression, revealing a protumoral role. We have identified by database analysis a sequence with 57% homology to the Nupr1, which we named Nupr1L (L=like) (Fig. 1). Our aim is study the role of this novel protein as well as the possible interaction with the tumor protein Nupr1. Material and methods According to the theoretical sequences of the NUPR1L, we have designed a RT-PCR-based strategy to cloning the human Nupr1L transcript. The promoter region of Nupr1L was identified from the human genome sequence and it was amplified by PCR and subcloned. Site-directed mutagenesis to the p53-sequences was performed. The transactivation activity of the promoter or its mutants was evaluated by a Luciferase reporter assay system. Subnuclear localization was determinate by IF of the Flag-tagged NUPR1L protein. mRNA expression of Nupr1 and Nupr1L was measured by RT-qPCR. The viability and cell cycle assays were carried on by standard methods. Results The NUPR1L human gene encodes for a protein of 97 aa with amolecular weight of 11.4 KDa and a isoelectric point of 10.5. The primary structure analysis reveals the presence of a nuclear localization site (NLS) and a cyclin recognition site (RxL) (Fig. 2). Accordingly, Nupr1L is localized on the euchromatin. Nupr1L mRNA is overexpressed in response to metabolic stress such as glucose deprivation. Two p53-response elements were identified on its promoter and its functionality validated by a transactivation approach showing a dose-dependent response to p53. Likewise, we confirmed the specificity of these sites through a directed mutagenesis. Since Nupr1L is a typical p53-target gene we studied the effect of its expression on cell cycle and cell death. We found that overexpression of Nupr1L induces a G1 cell cycle arrest with a significant decreases of S and on cell death. Surprisingly, overexpression of Nupr1L is accompanied of a significant down-regulation of Nupr1 transcript. In this way, we demonstrated a significant repression activity of the Nupr1L on the Nupr1promoter. Congruently, effects of Nupr1L, such as decreasing of cell viability and cell cycle, are abolished by the concomitant overexpression of Nupr1, indicating a Nupr1-mediated role. Conclusions: Nupr1L is a new p53-target gene which has an inhibitor effect on the proliferation that is at least partially mediated by the inhibition of the expression of its structural-related protein Nupr1. Nupr1Lcould be a novel antitumor gene regulated by p53andregulatingits downstream and sequence-related gene Nupr1. Fig. 1. Homology between Nupr1 and Nupr1L proteins in the context of its primary structure. Residues labeled with a (*) indicate 100% identity, residues labeled with (:) indicate positions with conservative substitutions, residues labeled with (.) indicate substitutions less conservative. Fig. 2. Structure of Nupr1L. Schematic representation of Nupr1L, which contains a Nuclear Localization Signal (NLS) and a cyclin recognition site (RxL).