RESUMO
Caspases play important roles in apoptotic cell death and in some other functions, such as cytokine maturation, inflammation, or differentiation. We show here that the 5'-flanking region of the human CASP-2 gene contains three functional response elements for sterol regulatory element binding proteins (SREBPs), proteins that mediate the transcriptional activation of genes involved in cholesterol, triacylglycerol, and fatty acid synthesis. Exposure of several human cell lines to statins, lipid-lowering drugs that drive SREBP proteolytic activation, induced the CASP-2 gene to an extent similar to that for known targets of SREBP proteins. Adenoviral vector-mediated transfer of active SREBP-2 also induced expression of the CASP-2 gene and the caspase-2 protein and increased the cholesterol and triacylglycerol cellular content. These rises in lipids were strongly impaired following small interfering RNA-mediated silencing of the CASP-2 gene. Taken together, our results identify the human CASP-2 gene as a member of the SREBP-responsive gene battery that senses lipid levels in cells and raise the possibility that caspase-2 participates in the control of cholesterol and triacylglycerol levels.
Assuntos
Cisteína Endopeptidases/fisiologia , Proteína de Ligação a Elemento Regulador de Esterol 2/fisiologia , Região 5'-Flanqueadora , Sítios de Ligação , Caspase 2 , Linhagem Celular Tumoral , Colesterol/biossíntese , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/genética , Regulação da Expressão Gênica , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , RNA Interferente Pequeno/genética , Elementos de Resposta , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Triglicerídeos/biossínteseRESUMO
Sterol Regulatory Element Binding Proteins (SREBP) are transcription factors that regulate lipid synthesis. We have shown recently that the human CASP-2 gene, encoding procaspase-2, was under the positive control of SREBP-2 that transactivates several responsive elements in the promoter region. We describe here the function of an additional SREBP-responsive element located in the first intron. Remarkably, this site is uniquely responsive to SREBP-1c that is mainly involved in fatty acids synthesis. This observation, together with our recent findings, strengthens the notion that the CASP-2 gene belongs to the SREBP-responsive gene battery in human cells.
Assuntos
Caspases/genética , Íntrons , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Sítios de Ligação , Caspase 2 , Caspases/metabolismo , Células Cultivadas , DNA/metabolismo , Humanos , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Ativação TranscricionalRESUMO
In response to DNA damage, p53-induced protein with a death domain (PIDD) forms a complex called the PIDDosome, which either consists of PIDD, RIP-associated protein with a death domain and caspase-2, forming a platform for the activation of caspase-2, or contains PIDD, RIP1 and NEMO, important for NF-κB activation. PIDDosome activation is dependent on auto-processing of PIDD at two different sites, generating the fragments PIDD-C and PIDD-CC. Despite constitutive cleavage, endogenous PIDD remains inactive. In this study, we screened for novel PIDD regulators and identified heat shock protein 90 (Hsp90) as a major effector in both PIDD protein maturation and activation. Hsp90, together with p23, binds PIDD and inhibition of Hsp90 activity with geldanamycin efficiently disrupts this association and impairs PIDD auto-processing. Consequently, both PIDD-mediated NF-κB and caspase-2 activation are abrogated. Interestingly, PIDDosome formation itself is associated with Hsp90 release. Characterisation of cytoplasmic and nuclear pools of PIDD showed that active PIDD accumulates in the nucleus and that only cytoplasmic PIDD is bound to Hsp90. Finally, heat shock induces Hsp90 release from PIDD and PIDD nuclear translocation. Thus, Hsp90 has a major role in controlling PIDD functional activity.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Processamento de Proteína Pós-Traducional , Benzoquinonas/farmacologia , Proteínas de Transporte/química , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Células HEK293 , Células HeLa , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Lactamas Macrocíclicas/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismoRESUMO
PIDD has been implicated in survival and apoptotic pathways in response to DNA damage, and a role for PIDD was recently identified in non-homologous end-joining (NHEJ) repair induced by γ-irradiation. Here, we present an interaction of PIDD with PCNA, first identified in a proteomics screen. PCNA has essential functions in DNA replication and repair following UV irradiation. Translesion synthesis (TLS) is a process that prevents UV irradiation-induced replication blockage and is characterized by PCNA monoubiquitination and interaction with the TLS polymerase eta (polη). Both of these processes are inhibited by p21. We report that PIDD modulates p21-PCNA dissociation, and promotes PCNA monoubiquitination and interaction with polη in response to UV irradiation. Furthermore, PIDD deficiency leads to a defect in TLS that is associated, both in vitro and in vivo, with cellular sensitization to UV-induced apoptosis. Thus, PIDD performs key functions upon UV irradiation, including TLS, NHEJ, NF-κB activation and cell death.