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Proteomics Core FacilityPublications

CHD4 is a RanGTP-dependent MAP that stabilizes microtubules and regulates bipolar spindle formation.
Yokoyama, H., Nakos, K., Santarella-Mellwig, R., Rybina, S., Krijgsveld, J., Koffa, M.D. & Mattaj, I.W.
Curr Biol. 2013 Dec 16;23(24):2443-51. doi: 10.1016/j.cub.2013.09.062. Epub 2013Nov 21.
BACKGROUND: Production of the GTP-bound form of the Ran GTPase (RanGTP) around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)-containing proteins. Several NLS proteins have been identified as spindle assembly factors, but the complexity of the process led us to search for additional proteins with distinct roles in spindle assembly. RESULTS: We identify a chromatin-remodeling ATPase, CHD4, as a RanGTP-dependent microtubule (MT)-associated protein (MAP). MT binding occurs via the region containing an NLS and chromatin-binding domains. In Xenopus egg extracts and cultured cells, CHD4 largely dissociates from mitotic chromosomes and partially localizes to the spindle. Immunodepletion of CHD4 from egg extracts significantly reduces the quantity of MTs produced around chromatin and prevents spindle assembly. CHD4 RNAi in both HeLa and Drosophila S2 cells induces defects in spindle assembly and chromosome alignment in early mitosis, leading to chromosome missegregation. Further analysis in egg extracts and in HeLa cells reveals that CHD4 is a RanGTP-dependent MT stabilizer. Moreover, the CHD4-containing NuRD complex promotes organization of MTs into bipolar spindles in egg extracts. Importantly, this function of CHD4 is independent of chromatin remodeling. CONCLUSIONS: Our results uncover a new role for CHD4 as a MAP required for MT stabilization and involved in generating spindle bipolarity.
Europe PMC

Comprehensive histone phosphorylation analysis and identification of pf14-3-3 protein as a histone h3 phosphorylation reader in malaria parasites.
Dastidar, E.G., Dzeyk, K., Krijgsveld, J., Malmquist, N.A., Doerig, C., Scherf, A. & Lopez-Rubio, J.J.
PLoS One. 2013;8(1):e53179. doi: 10.1371/journal.pone.0053179. Epub 2013 Jan 7.
The important role of histone posttranslational modifications, particularly methylation and acetylation, in Plasmodium falciparum gene regulation has been established. However, the role of histone phosphorylation remains understudied. Here, we investigate histone phosphorylation utilizing liquid chromatography and tandem mass spectrometry to analyze histones extracted from asexual blood stages using two improved protocols to enhance preservation of PTMs. Enrichment for phosphopeptides lead to the detection of 14 histone phospho-modifications in P. falciparum. The majority of phosphorylation sites were observed at the N-terminal regions of various histones and were frequently observed adjacent to acetylated lysines. We also report the identification of one novel member of the P. falciparum histone phosphosite binding protein repertoire, Pf14-3-3I. Recombinant Pf14-3-3I protein bound to purified parasite histones. In silico structural analysis of Pf14-3-3 proteins revealed that residues responsible for binding to histone H3 S10ph and/or S28ph are conserved at the primary and the tertiary structure levels. Using a battery of H3 specific phosphopeptides, we demonstrate that Pf14-3-3I preferentially binds to H3S28ph over H3S10ph, independent of modification of neighbouring residues like H3S10phK14ac and H3S28phS32ph. Our data provide key insight into histone phosphorylation sites. The identification of a second member of the histone modification reading machinery suggests a widespread use of histone phosphorylation in the control of various nuclear processes in malaria parasites.
Europe PMC

Anticancer compound ABT-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice.
Kakkola, L., Denisova, O.V., Tynell, J., Viiliainen, J., Ysenbaert, T., Matos, R.C., Nagaraj, A., Ohman, T., Kuivanen, S., Paavilainen, H., Feng, L., Yadav, B., Julkunen, I., Vapalahti, O., Hukkanen, V., Stenman, J., Aittokallio, T., Verschuren, E.W., Ojala, P.M., Nyman, T., Saelens, X., Dzeyk, K. & Kainov, D.E.
Cell Death Dis. 2013 Jul 25;4:e742. doi: 10.1038/cddis.2013.267.
ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9-mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections.
Europe PMC