Cells in multicellular organisms sense and respond to their environments is integral to the formation of tissues and organs in development, adaptation to changes in nutrient levels and hormone signals, and regeneration of damaged tissues in adults. Lesions in these sensing systems result in disease states such as birth defects, cancer, and degenerative diseases. Thus, understanding the molecular mechanisms behind cell sensing will not only provide a better understanding of biological processes, it will provide insights into the causes of human diseases, and will aid diagnosis, treatment, and cure. My laboratory has been studying one such pathway, the Notch signaling pathway. In Notch signaling, local information from neighboring cells is sensed through a transmembrane receptor encoded by the Notch gene(s), and is transduced into the nucleus to activate the expression of downstream genes, leading to cell differentiation. One unique feature of the Notch pathway is that upon activation, the receptor is converted to a transcription factor through cleavage of the Notch intracellular domain (NICD) from the membrane. Following cleavage, NICD activates transcription by binding a factor called CSL. This interaction involves bivalent contacts between distant regions of NICD and CSL, and is mediated by a ~100 residue RAM linker segment, which though largely disordered, plays a key role in orchestrating displacement of co-repressors, and recruitment of coactivators. NICD activity is also strongly modulated by a number of cytosolic proteins such as Deltex, although the mechanistic details are murky. In this proposal, we use traditional biophysical, structural methods, and solution thermodynamics to characterize these aspects of Notch signaling, but also combine these methods with functional assays for Notch signaling using mammalian cell culture. This second approach is a new direction for my laboratory, but results so far have been rewarding, giving results from biophysical studies a functional context, and focusing cell culture studies on quantitative, sharply focused mechanistic questions. We are also putting greater emphasis on NMR and computational simulations. Specifically, we are determining the structural basis of interaction between the Notch receptor and Deltex, are identifying surface substitutions that disrupt binding, and are determining how disrupting this interaction perturbs ubiqutination, transcriptional activation, receptor endocytosis, and interaction with a putative deubiquitinase. In addition, we are characterizing the RAM region of NICD using NMR, AUC, simulation, and mutagenesis. Finally, we are determining how RAM enhances the conserved bivalent interaction between NICD and CSL, and how this bivalent interaction thermodynamically couples corepressor dissociation with coactivator binding. The system under study here is ideal for understanding how intrinsically disordered regions influence protein structure and function.

Public Health Relevance

The proposed research will determine how proteins in the Notch signaling pathway work together to form complex tissues and cells with specialized functions, like those in the immune system, nervous system, and organs. Disrupting this pathway results in diseases such as cancer, leukemia, and birth defects. By better understanding how the Notch pathway works, and what goes wrong when it is disrupted, we can better diagnose and treat these Notch-associated diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060001-11
Application #
8518359
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Flicker, Paula F
Project Start
2001-04-01
Project End
2016-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
11
Fiscal Year
2013
Total Cost
$302,953
Indirect Cost
$109,953
Name
Johns Hopkins University
Department
Physiology
Type
Schools of Arts and Sciences
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Allgood, Andrea Gayle; Barrick, Doug (2011) Mapping the Deltex-binding surface on the notch ankyrin domain using analytical ultracentrifugation. J Mol Biol 414:243-59
Johnson, Scott E; Ilagan, M Xenia G; Kopan, Raphael et al. (2010) Thermodynamic analysis of the CSL x Notch interaction: distribution of binding energy of the Notch RAM region to the CSL beta-trefoil domain and the mode of competition with the viral transactivator EBNA2. J Biol Chem 285:6681-92
Barrick, Doug (2009) Biological regulation via ankyrin repeat folding. ACS Chem Biol 4:19-22
Bertagna, Angela; Toptygin, Dima; Brand, Ludwig et al. (2008) The effects of conformational heterogeneity on the binding of the Notch intracellular domain to effector proteins: a case of biologically tuned disorder. Biochem Soc Trans 36:157-66
Lubman, Olga Y; Ilagan, Ma Xenia G; Kopan, Raphael et al. (2007) Quantitative dissection of the Notch:CSL interaction: insights into the Notch-mediated transcriptional switch. J Mol Biol 365:577-89
Bradley, Christina Marchetti; Barrick, Doug (2006) The notch ankyrin domain folds via a discrete, centralized pathway. Structure 14:1303-12
Zweifel, Mark E; Leahy, Daniel J; Barrick, Doug (2005) Structure and Notch receptor binding of the tandem WWE domain of Deltex. Structure (Camb) 13:1599-611
Bradley, Christina Marchetti; Barrick, Doug (2005) Effect of multiple prolyl isomerization reactions on the stability and folding kinetics of the notch ankyrin domain: experiment and theory. J Mol Biol 352:253-65
Street, Timothy O; Bradley, Christina Marchetti; Barrick, Doug (2005) An improved experimental system for determining small folding entropy changes resulting from proline to alanine substitutions. Protein Sci 14:2429-35
Mello, Cecilia C; Bradley, Christina Marchetti; Tripp, Katherine W et al. (2005) Experimental characterization of the folding kinetics of the notch ankyrin domain. J Mol Biol 352:266-81

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