Abstract
Members of the bcl-2 protein family share regions of sequence similarity, the bcl-2 homology (BH) domains. Bcl-2, the most studied member of this family, has four BH domains, BH1–4, and has a critical role in resistance to antineoplastic drugs by regulating the mitochondrial apoptotic pathway. Moreover, it is also involved in other relevant cellular processes such as tumor progression, angiogenesis and autophagy. Deciphering the network of bcl-2-interacting factors should provide a critical advance in understanding the different functions of bcl-2. Here, we characterized bcl-2 interactome by mass spectrometry in human lung adenocarcinoma cells. In silico functional analysis associated most part of the identified proteins to mitochondrial functions. Among them we identified SRA stem–loop interacting RNA-binding protein, SLIRP, a mitochondrial protein with a relevant role in regulating mitochondrial messenger RNA (mRNA) homeostasis. We validated bcl-2/SLIRP interaction by immunoprecipitation and immunofluorescence experiments in cancer cell lines from different histotypes. We showed that, although SLIRP is not involved in mediating bcl-2 ability to protect from apoptosis and oxidative damage, bcl-2 binds and stabilizes SLIRP protein and regulates mitochondrial mRNA levels. Moreover, we demonstrated that the BH4 domain of bcl-2 has a role in maintaining this binding.
Highlights
In this work, we investigated the network of bcl-2-interacting factors in order to identify novel putative bcl-2-binding proteins, which in turn should provide critical advances in Received 06.8.15; revised 04.11.15; accepted 04.11.15; Edited by J Chipuk
We demonstrated that removal of or mutations at the BH4 domain abrogate the ability of bcl-2 to induce Vascular Endothelial Growth Factor expression and transcriptional activity,[12] reduce the interaction between bcl-2 and Hypoxia Inducible Factor-1α proteins and the capability of exogenous bcl-2 protein to localize in the nucleus[13] and mediate inhibition of autophagy.[14]
A total of 210 proteins were identified with false discovery rate o1% in FLAG-bcl-2 IM after subtracting the proteins found in the IM
Summary
We investigated the network of bcl-2-interacting factors in order to identify novel putative bcl-2-binding proteins, which in turn should provide critical advances in Received 06.8.15; revised 04.11.15; accepted 04.11.15; Edited by J Chipuk. In accordance with this evidence, the level of SLIRP protein was not increased in H1299 FLAG bcl-2del extract with respect to that of controls cells (Figure 6b).
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