Caracterización de la apoptosis inducida por el sistema 2-5a-RNAsa L

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 15-02-2008The interferons (IFNs) are cytokines that are released from animal cells in response to a variety of stimuli including viral infections. IFN regulates central processes in animal cells such as proliferation and differentiation, modulation of the immune response and inhibition of virus replication by the induction of different genes. Among these IFN-induced genes, the best characterized are the genes that code for the 2-5A/RNase L system, the 68 KDa protein kinase dsRNA-dependent (PKR), and proteins that belong to the Mx family. The 2-5A/RNase L system is composed of the 2´-5´oligoadenylate synthetase (2´-5´OAS) and 2-5A-dependent RNase (RNase L), enzymes that constitute a powerful system in the antiviral defence. The 2-5A/RNase L system is activated by double stranded RNA (dsRNA) produced by the viral infection. Once activated, RNase L is the effector protein of the 2-5A/RNase L system, which degrades ribosomal, viral and messenger RNAs dealing to the inhibition of protein synthesis and therefore blocking viral replication. Activation of RNase L also induces apoptosis in mammalian infected cells. To obtain further information into the molecular mechanisms by which RNase L induces apoptosis, we expressed human RNase L and 2´-5´OAS in HeLa cells using recombinant vaccinia viruses as vectors and we analysed in detail different biochemical markers of apoptosis as well as the relation of RNA degradation and apoptosis induction. Furthermore, we carried out microarray experiments after activation of the 2-5A/RNase L system, in order to analyze the expression of specific host genes. Our findings provide evidence for the implication of different caspases (2, 8 and 9) in the apoptosis induced by the 2-5A/RNase L system, particularly, we have defined the role of the intrinsic pathway. The 2-5A/RNase L system induces the release of cytochrome c from mitochondria to cytosol in a caspase dependent manner, principally, dependent of caspase 2 activation. The onset of apoptosis elicits the disruption of mitochondrial membrane potential (ΔΨm), as well as the generation of reactive oxygen species (ROS) which are caspase dependent processes. Moreover, the activation of RNase L induces morphological alterations in the mitochondria. Apoptosis induced by the 2-5A/RNase L system involves mitochondrial proteins, such as the human anti-apoptotic protein Bcl-2, which blocks both the apoptosis and the change of ΔΨm induced by the activation of RNase L. RNase L and 2´-5´OAS are localized in the mitochondria and cytosol fractions at the beginning of apoptosis (24 hpi), while at the onset of apoptosis both enzymes are largely in mitochondria (48 hpi). The results presented in this work demonstrate that activation of RNase L produces degradation of rRNA which is previous and necessary for the induction of apoptosis. Moreover, activation of RNase L produces degradation of the mitochondrial rRNA 16S, which confirms the localization of the 2-5A/RNase L system in this organelle and brings out a new function of RNase L in mitocondrial RNA metabolism. The microarray analysis identified genes which are significantly increased or repressed by the expression of the 2-5A/RNase L system. Overall, our findings demonstrate the biological significance of the 2-5A/RNase L system in the induction of apoptosis through the mitochondrial pathway and define molecular mechanisms involved