Notch signaling is an evolutionarily conserved pathway that regulates processes as diverse as cell fate specification, stem cell proliferation and maintenance, cell death, compartment boundary formation and cortical neurite outgrowth. Also, aberrant Notch signaling is involved in a variety of human diseases including cerebrovascular dementia, cancer and developmental disorders affecting liver, heart, skeleton, eye, and kidney. Although a lot of effort has been devoted to understanding the function of the genes with all-or-none effects in this pathway, much less is known about how animals fine-tune signaling, an issue of potentially high relevance to human disease pathogenesis and therapeutics. The focus of this proposal is on the characterization of a novel Notch regulator rumi, which we have identified in an unbiased chemical mutagenesis screen in Drosophila. Unlike other regulators of Notch, null mutations of rumi exhibit a dramatically temperature-sensitive Notch loss-of- function phenotype. In vivo analysis indicates that Rumi functions in the endoplasmic reticulum of the signal-receiving cell and is required upstream of the Presenilin function. Biochemical and cell culture studies have shown that Rumi is able to add glucose residues to specific EGF repeats of Notch. In this proposal we will use a combination of genetic, cell biological and biochemical analyses to identify the mechanism underlying the temperature-sensitivity and reversibility of the rumi phenotype, to test the hypothesis that Rumi regulates Notch signaling via altering the glycosylation pattern of the Notch protein in vivo, and to identify other Notch "fine-tuning" genes by screening for genetic modifiers of rumi. Rumi is a highly conserved protein, as transgenic expression of human Rumi rescues rumi mutations in flies. Moreover, vertebrate studies have shown that manipulation of the Notch pathway can be of potential therapeutic value in several disease contexts, including inner ear hair cell loss, muscle injury and demyelination. Therefore, our hope is that by shedding light on the interface of cell biology and development, the research proposed in this grant will not only unravel some of the strategies used by animals to regulate signaling, but might also contribute to efforts aimed at altering the outcome of human diseases. Since to our knowledge Rumi is the first protein O- glucosyltransferase identified in animals, our studies will also establish a framework for understanding the role of this highly conserved modification in metazoan biology. PUBLIC HEALTH REVELANCE: Alterations in Notch signaling causes a variety of human diseases including cancer, cardiovascular, skeletal and neurological disorders. Notch signaling is also involved in the regulation of stem cell division and differentiation. In this proposal we will characterize how addition of glucose residues to the Notch protein fine-tunes signaling mediated by this important pathway.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM084135-05S1
Application #
8718761
Study Section
Neurogenesis and Cell Fate Study Section (NCF)
Program Officer
Gaillard, Shawn R
Project Start
2008-04-01
Project End
2014-01-16
Budget Start
2012-03-01
Budget End
2014-01-16
Support Year
5
Fiscal Year
2013
Total Cost
$92,031
Indirect Cost
$33,225
Name
Baylor College of Medicine
Department
Genetics
Type
Schools of Medicine
DUNS #
051113330
City
Houston
State
TX
Country
United States
Zip Code
77030
LeBon, Lauren; Lee, Tom V; Sprinzak, David et al. (2014) Fringe proteins modulate Notch-ligand cis and trans interactions to specify signaling states. Elife 3:e02950
Leonardi, Jessica; Jafar-Nejad, Hamed (2014) Structure-function analysis of Drosophila Notch using genomic rescue transgenes. Methods Mol Biol 1187:29-46
LeBon, Lauren; Lee, Tom V; Sprinzak, David et al. (2014) Correction: Fringe proteins modulate Notch-ligand cis and trans interactions to specify signaling states. Elife 3:
Haltom, Amanda R; Lee, Tom V; Harvey, Beth M et al. (2014) The protein O-glucosyltransferase Rumi modifies eyes shut to promote rhabdomere separation in Drosophila. PLoS Genet 10:e1004795
Lee, Tom V; Sethi, Maya K; Leonardi, Jessica et al. (2013) Negative regulation of notch signaling by xylose. PLoS Genet 9:e1003547
Leonardi, Jessica; Fernandez-Valdivia, Rodrigo; Li, Yi-Dong et al. (2011) Multiple O-glucosylation sites on Notch function as a buffer against temperature-dependent loss of signaling. Development 138:3569-78
Takeuchi, Hideyuki; Fernandez-Valdivia, Rodrigo C; Caswell, Devin S et al. (2011) Rumi functions as both a protein O-glucosyltransferase and a protein O-xylosyltransferase. Proc Natl Acad Sci U S A 108:16600-5
Fernandez-Valdivia, Rodrigo; Takeuchi, Hideyuki; Samarghandi, Amin et al. (2011) Regulation of mammalian Notch signaling and embryonic development by the protein O-glucosyltransferase Rumi. Development 138:1925-34
Jafar-Nejad, Hamed; Leonardi, Jessica; Fernandez-Valdivia, Rodrigo (2010) Role of glycans and glycosyltransferases in the regulation of Notch signaling. Glycobiology 20:931-49
Lee, Tom V; Takeuchi, Hideyuki; Jafar-Nejad, Hamed (2010) Regulation of notch signaling via O-glucosylation insights from Drosophila studies. Methods Enzymol 480:375-98

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