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Chemical Biology & Drug Design, 2016 Aug 20. 

A small molecule identified through an in silico screen inhibits Aurora B–INCENP interaction

  • Esra Unsal, Bahar Degirmenci, Büşra Harmanda, Burak Erman, Nurhan Ozlu

Abstract

Aurora B is a serine/threonine kinase that has a central role in the regulation of mitosis. The observation of Aurora B overexpression in cancer makes it a promising target to develop antitumoral inhibitors. We describe a new potential inhibitor that exclusively targets the interaction site of Aurora B and its activator INCENP. We performed a structure-based virtual screening and determined five potential candidates of 200 000 compounds, which selectively bind to the Aurora B::INCENP interaction site, but not to the ATP-binding site (kinase pocket) of Aurora B or other related kinases. Further characterization in vivo validated the inhibitory role of one of these five compounds in Aurora B::INCENP complex formation and exhibited hallmarks of Aurora inhibition such as chromosome congression and segregation defects that interfere with the progression into cytokinesis and result in multinuclear cells. Our results provide an alternative approach on the way of exploring specific kinase inhibitors.


 2015 Aug 13. 

Phosphoproteomic analysis of Aurora kinase inhibition in Monopolar Cytokinesis.

 
1Department of Molecular Biology and Genetics, Koç University, Istanbul, Turkey
2 Research Group Bioinformatics (NG 4), Robert Koch-Institute, Berlin, Germany.
3 Department of Genetics, Stanford University, School of Medicine, CA.
4 Department of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin Germany

Abstract

Cytokinesis is the last step of the cell cycle which requires coordinated activities of the microtubule cytoskeleton, actin cytoskeleton and membrane compartments. Aurora B kinase is one of the master regulatory kinases that orchestrate multiple events during cytokinesis. To reveal targets of the Aurora B kinase, we combined quantitative mass spectrometry with chemical genetics. Using the quantitative proteomic approach, SILAC (Stable Isotope Labeling with Amino acids in Cell culture), we analyzed the phosphoproteome of monopolar cytokinesis upon VX680 or AZD1152 mediated Aurora kinase inhibition. In total our analysis quantified over twenty thousand phosphopeptides in response to the Aurora-B kinase inhibition; 246 unique phosphopeptides were significantly down-regulated and 74 were up-regulated. Our data provide a broad analysis of downstream effectors of Aurora kinase and offer insights into how Aurora kinase regulates cytokinesis.


 2014 Dec 4. pii: e201385162. 

Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis.

 

1Department of Molecular Biology and GeneticsKoç UniversityIstanbul, Turkey

 2Max Planck Institute for Molecular Cell Biology and GeneticsDresden, Germany

 3Research Group Bioinformatics (NG 4)Robert Koch‐InstituteBerlin, Germany

 4Proteomics Center at Children's Hospital BostonBostonMA, USA

5Department of Systems BiologyHarvard Medical SchoolBostonMA, USA

 6Department of NeurobiologyHarvard Medical School and Children's Hospital Boston,BostonMA, USA 

  

 Abstract

The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy.

© 2014 The Authors.

KEYWORDS:

SILAC ; PCDH7; cell cycle; cell rounding; cell surface


Towards single-cell LC-MS phosphoproteomics.

Polat AN1Özlü N.

1Department of Molecular Biology and Genetics, Science Faculty, Koç University, Istanbul, Turkey. anpolat@ku.edu.tr nozlu@ku.edu.tr.

Abstract

Protein phosphorylation is a ubiquitous posttranslational modification, which is heavily involved in signal transduction. Misregulation of protein phosphorylation is often associated with a decrease in cell viability and complex diseases such as cancer. The dynamic and low abundant nature of phosphorylated proteins makes studying phosphoproteome a challenging task. In this review, we summarize state of the art proteomic techniques to study and quantify peptide phosphorylation in biological systems and discuss their limitations. Due to its short-lived nature, the phosphorylation event cannot be precisely traced in a heterogonous cell population, which highlights the importance of analyzing phosphorylation events at the single cell level. Mainly, we focus on the methodical and instrumental developments in proteomics and nanotechnology, which will help to build more accurate and robust systems for the feasibility of phosphorylation analysis at the single cell level. We propose that an automated and miniaturized construction of analytical systems holds the key to the future of phosphoproteomics; therefore, we highlight the benchmark studies in this direction. Having advanced and automated microfluidic chip LC systems will allow us to analyze single-cell phosphoproteomics and quantitatively compare it with others. The progress in the microfluidic chip LC systems and feasibility of the single-cell phosphoproteomics will be beneficial for early diagnosis and detection of the treatment response of many crucial diseases.


 

  • Hu CK, Ozlü N, Coughlin M, Steen JJ, Mitchison TJ. Plk1 negatively regulates PRC1 to prevent premature midzone formation before cytokinesis. Mol Biol Cell. 2012 Jul;23(14):2702-11. PubMed

 

  • Özlü N, Kirchner M, Steen JJ. Measuring Phosphorylation Specific Changes in Response to Kinase Inhibitors in Mammalian Cells Using Quantitative Proteomics. Methods in Molecular Biology. 2012;795:217-31.PubMed

 

  • Özlü N, Monigatti F, Renard BY, Field CM, Steen H, Mitchison TJ, Steen JJ. Binding partner switching on microtubules and aurora-B in the mitosis to cytokinesis transition. Mol Cell Proteomics. 2010 Feb;9(2):336-50. PubMed

 

  • Timm W, Özlü N, Steen JJ, Steen H. Effect of High-Accuracy Precursor Masses on Phosphopepide Identification from MS(3) Spectra. Anal Chem. 2010 May 15;82(10):3977-80. PubMed

 

  • Özlü N, Akten B, Timm W, Haseley N, Steen H, Steen JJ. Phosphoproteomics: Wiley Interdisiplinary Reviews: Systems Biology and Medicine 2009 Oct 28; 2(3):255-276.PubMed

 

  • Liffers ST, Özlü N, Bensaddek D, Steen JJ, Steen H. Stable Isotope Labeling by Aminoacids in Cell Culture (SILAC): A Primer. Sigma-Aldrich Catalogue, 2007.

 

  • Özlü N, Srayko M, Kinoshita K, Habermann B, O'toole ET, Muller-Reichert T, Schmalz N, Desai A, Hyman AA. An essential function of the C. elegans ortholog of TPX2 is to localize activated aurora A kinase to mitotic spindles. Dev Cell. 2005 Aug;9(2):237-48. PubMed

 

  • Kurz T, Özlü N, Rudolf F, O'Rourke SM, Luke B, Hofmann K, Hyman AA, Bowerman B, Peter M. The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae. Nature. 2005 Jun 30;435(7046):1257-61. PubMed

 

  • Pelletier L, Özlü N, Hannak E, Cowan C, Habermann B, Ruer M, Muller-Reichert T, Hyman AA. The Caenorhabditis elegans centrosomal protein SPD-2 is required for both pericentriolar material recruitment and centriole duplication. Curr Biol. 2004 May 25;14(10):863-73. PubMed

 

  • L Pelletier, T Müller-Reichert, M Srayko, N Özlü, A.-L Schlaitz and AA Hyman. "The C. elegans centrosome during early embryonic development." In Centrosomes in Development and Disease, Editor EA Nigg. (2005): 474 pages.

 

  • Gonczy P, Echeverri C, Oegema K, Coulson A, Jones SJ, Copley RR, Duperon J, Oegema J, Brehm M, Cassin E, Hannak E, Kirkham M, Pichler S, Flohrs K, Goessen A, Leidel S, Alleaume AM, Martin C, Özlü N, Bork P, Hyman AA. Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature. 2000 Nov 16;408(6810):331-6. PubMed