Machado-Oliveira Gisela, Guerreiro Eduarda, Matias Ana Catarina, Facucho-Oliveira João, Pacheco-Leyva Ivette, Bragança José
Department of Biomedical Sciences and Medicine, University of Algarve, Portugal; CBMR - Centre for Biomedical Research, University of Algarve, Campus of Gambelas, 8005-139 Faro, Portugal.
Department of Biomedical Sciences and Medicine, University of Algarve, Portugal.
Arch Biochem Biophys. 2015 Jun 15;576:61-72. doi: 10.1016/j.abb.2015.04.012. Epub 2015 May 5.
CITED2 is a ubiquitously expressed nuclear protein exhibiting a high affinity for the cysteine-histidine-rich domain 1 (CH1) of the transcriptional co-activators CBP/p300. CITED2 is particularly efficient in the inhibition of the hypoxia-inducible factor-1α (HIF-1α) dependent transcription by competing with it for the interaction with the CH1 domain. Here we report a direct and specific interaction between CITED2 and the F-box and leucine rich repeat protein 5 (FBXL5), a substrate adaptor protein which is part of E3 ubiquitin ligase complexes mediating protein degradation by the proteasome. We demonstrated that depletion of FBXL5 by RNA interference led to an increase of CITED2 protein levels. Conversely, overexpression of FBXL5 caused the decrease of CITED2 protein levels in a proteasome-dependent manner, and impaired the interaction between CITED2 and the CH1 domain of p300 in living cells. In undifferentiated mouse embryonic stem cells, the overexpression of FBXL5 also reduced Cited2 protein levels. Finally, we evidenced that FBXL5 overexpression and the consequent degradation of CITED2 enabled the transcriptional activity of the N-terminal transactivation domain of HIF-1α. Collectively, our results highlighted a novel molecular interaction between CITED2 and FBXL5, which might regulate the steady state CITED2 protein levels and contribute to the modulation of gene expression by HIF-1α.
CITED2是一种广泛表达的核蛋白，对转录共激活因子CBP/p300的富含半胱氨酸-组氨酸结构域1（CH1）具有高亲和力。CITED2通过与缺氧诱导因子-1α（HIF-1α）竞争与CH1结构域的相互作用，在抑制HIF-1α依赖性转录方面特别有效。在此，我们报告了CITED2与F-box和富含亮氨酸重复蛋白5（FBXL5）之间直接且特异性的相互作用，FBXL5是一种底物衔接蛋白，是介导蛋白酶体蛋白降解的E3泛素连接酶复合物的一部分。我们证明，通过RNA干扰耗尽FBXL5会导致CITED2蛋白水平升高。相反，FBXL5的过表达以蛋白酶体依赖性方式导致CITED2蛋白水平降低，并损害活细胞中CITED2与p300的CH1结构域之间的相互作用。在未分化的小鼠胚胎干细胞中，FBXL5的过表达也降低了Cited2蛋白水平。最后，我们证明FBXL5的过表达以及随之而来的CITED2降解能够激活HIF-1α N端反式激活结构域的转录活性。总的来说，我们的结果突出了CITED2与FBXL5之间一种新的分子相互作用，这可能调节CITED2蛋白的稳态水平，并有助于HIF-1α对基因表达的调控。
