Vikram Narayan

Publications (9) View all

• Article: DNA-binding regulates site-specific ubiquitination of IRF-1.

  Vivien Landré, Emmanuelle Pion, Vikram Narayan, Dimitris P Xirodimas, Kathryn L Ball
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  ABSTRACT: Understanding the determinants for site-specific ubiquitination by E3-ligase components of the ubiquitin-machinery is proving to be a challenge. Here we investigate the role of an E3-ligase docking site (Mf2-domain) in an intrinsically disordered domain of IRF-1, a short-lived IFN-γ regulated transcription factor, in ubiquitination of the protein. Ubiquitin modification of full-length IRF-1 by E3-ligases like CHIP and MDM2, which dock to the Mf2-domain was specific for lysine residues found predominantly in loop structures that extend from the DNA-binding domain, whereas no modification was detected in the more conformationally flexible C-terminal half of the protein. The E3-docking site was not available when IRF-1 was in its DNA bound conformation and cognate DNA-binding sequences strongly suppressed ubiquitination highlighting a strict relationship between ligase binding and site-specific modification at residues in the DNA-binding domain. Hyperubiquitination of a non-DNA binding mutant supports a mechanism where an active DNA-bound pool of IRF-1 is protected from polyubiquitination and degradation.
  Biochemical Journal 11/2012; · 4.90 Impact Factor
• Article: ALDH2 mediates 5-nitrofuran activity in multiple species.

  Linna Zhou, Hironori Ishizaki, Michaela Spitzer, Kerrie L Taylor, Nicholas D Temperley, Stephen L Johnson, Paul Brear, Philippe Gautier, Zhiqiang Zeng, Amy Mitchell, Vikram Narayan, Ewan M McNeil, David W Melton, Terry K Smith, Mike Tyers, Nicholas J Westwood, E Elizabeth Patton
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  ABSTRACT: Understanding how drugs work in vivo is critical for drug design and for maximizing the potential of currently available drugs. 5-nitrofurans are a class of prodrugs widely used to treat bacterial and trypanosome infections, but despite relative specificity, 5-nitrofurans often cause serious toxic side effects in people. Here, we use yeast and zebrafish, as well as human in vitro systems, to assess the biological activity of 5-nitrofurans, and we identify a conserved interaction between aldehyde dehydrogenase (ALDH) 2 and 5-nitrofurans across these species. In addition, we show that the activity of nifurtimox, a 5-nitrofuran anti-trypanosome prodrug, is dependent on zebrafish Aldh2 and is a substrate for human ALDH2. This study reveals a conserved and biologically relevant ALDH2-5-nitrofuran interaction that may have important implications for managing the toxicity of 5-nitrofuran treatment.
  Chemistry & biology 07/2012; 19(7):883-92. · 6.52 Impact Factor
• Article: Exploiting the MDM2-CK1α protein-protein interface to develop novel biologics that induce UBL-kinase-modification and inhibit cell growth.

  Anne-Sophie Huart, Nicola J MacLaine, Vikram Narayan, Ted R Hupp
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  ABSTRACT: Protein-protein interactions forming dominant signalling events are providing ever-growing platforms for the development of novel Biologic tools for controlling cell growth. Casein Kinase 1 α (CK1α) forms a genetic and physical interaction with the murine double minute chromosome 2 (MDM2) oncoprotein resulting in degradation of the p53 tumour suppressor. Pharmacological inhibition of CK1 increases p53 protein level and induces cell death, whilst small interfering RNA-mediated depletion of CK1α stabilizes p53 and induces growth arrest. We mapped the dominant protein-protein interface that stabilizes the MDM2 and CK1α complex in order to determine whether a peptide derived from the core CK1α-MDM2 interface form novel Biologics that can be used to probe the contribution of the CK1-MDM2 protein-protein interaction to p53 activation and cell viability. Overlapping peptides derived from CK1α were screened for dominant MDM2 binding sites using (i) ELISA with recombinant MDM2; (ii) cell lysate pull-down towards endogenous MDM2; (iii) MDM2-CK1α complex-based competition ELISA; and (iv) MDM2-mediated ubiquitination. One dominant peptide, peptide 35 was bioactive in all four assays and its transfection induced cell death/growth arrest in a p53-independent manner. Ectopic expression of flag-tagged peptide 35 induced a novel ubiquitin and NEDD8 modification of CK1α, providing one of the first examples whereby NEDDylation of a protein kinase can be induced. These data identify an MDM2 binding motif in CK1α which when isolated as a small peptide can (i) function as a dominant negative inhibitor of the CK1α-MDM2 interface, (ii) be used as a tool to study NEDDylation of CK1α, and (iii) reduce cell growth. Further, this approach provides a technological blueprint, complementing siRNA and chemical biology approaches, by exploiting protein-protein interactions in order to develop Biologics to manipulate novel types of signalling pathways such as cross-talk between NEDDylation, protein kinase signalling, and cell survival.
  PLoS ONE 01/2012; 7(8):e43391. · 4.09 Impact Factor
• Chapter: p53 and Immunity

  Vikram Narayan, Sarah E. Meek, Kathryn L. Ball, Ayeda Ayed, Theodore Hupp
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  ABSTRACT: Since its discovery in 1979, many different roles for the tumor suppressor protein p53 in tumorigenesis have been described. Correct p53 function is required for proper regulation of cell division, apoptosis, senescence, and the responses to cellular stresses such as DNA damage and hypoxia. Indeed, mutations in p53 are observed in as many as 50% of human cancers.1 However, recent reports have highlighted an emerging role for p53 in anti-viral immunity. This chapter reviews the available literature on p53 and the body’s immune response, and how p53 may link immunity and cancer.
  07/2011: pages 178-186;
• Article: A multiprotein binding interface in an intrinsically disordered region of the tumor suppressor protein interferon regulatory factor-1.

  Vikram Narayan, Petr Halada, Lenka Hernychová, Yuh Ping Chong, Jitka Žáková, Ted R Hupp, Borivoj Vojtesek, Kathryn L Ball
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  ABSTRACT: The interferon-regulated transcription factor and tumor suppressor protein IRF-1 is predicted to be largely disordered outside of the DNA-binding domain. One of the advantages of intrinsically disordered protein domains is thought to be their ability to take part in multiple, specific but low affinity protein interactions; however, relatively few IRF-1-interacting proteins have been described. The recent identification of a functional binding interface for the E3-ubiquitin ligase CHIP within the major disordered domain of IRF-1 led us to ask whether this region might be employed more widely by regulators of IRF-1 function. Here we describe the use of peptide aptamer-based affinity chromatography coupled with mass spectrometry to define a multiprotein binding interface on IRF-1 (Mf2 domain; amino acids 106-140) and to identify Mf2-binding proteins from A375 cells. Based on their function as known transcriptional regulators, a selection of the Mf2 domain-binding proteins (NPM1, TRIM28, and YB-1) have been validated using in vitro and cell-based assays. Interestingly, although NPM1, TRIM28, and YB-1 all bind to the Mf2 domain, they have differing amino acid specificities, demonstrating the degree of combinatorial diversity and specificity available through linear interaction motifs.
  Journal of Biological Chemistry 01/2011; 286(16):14291-303. · 4.77 Impact Factor