Hedgehog (Hh) proteins are secreted morphogens that regulate normal cell differentiation as well as malignant cell growth. Covalent attachment of the fatty acid palmitate to the N-terminus of Hh is critical for Hh function. Unlike nearly all other palmitoylated proteins, that contain thioester linked palmitate, palmitate is linked to Hh via amide (N-) bond. The overall goals of this research are to elucidate the enzymology and biochemical mechanism of N-palmitoylation of developmentally important signaling proteins, and to understand how expression and function of N-palmitoyl transferases is regulated. We will use Hh proteins as model systems to address the following issues: 1. To reconstitute N-palmitoylation using purified Hedgehog and Rasp proteins Genetic experiments in flies and mice indicate that Rasp, a multipass membrane protein, is required for Hh palmitoylation. To date, there is no biochemical evidence that Rasp, or its mammalian homolog Mart-2, functions independently and catalytically as a palmitoyl transferase. We have now succeeded in purifying Mart -2 to homogeneity in active form. The biochemical parameters and enzymatic mechanism of Shh and Hh N-palmitoylation will be determined. 2. Structure/Function analysis of Rasp/Mart-2 and Hh/Shh How do MBOAT proteins work and how do they recognize their substrates? Using deletion analysis and chimeric proteins formed between Rasp and another MBOAT family member Porcupine, we will identify the transmembrane and/or cytoplasmic loop regions of Rasp and Mart-2 that comprise the active site and are important for catalysis. We will identify the minimum N-palmitoylation sequence motif within Hh/Shh and use this information to identify other substrates for Rasp and Mart-2. 3. To determine how N-Palmitoylation by Rasp/Mart-2 is regulated within the cell Subcellular localization and trafficking experiments will be performed to determine when and where Hh is palmitoylated. Methods to inhibit Mart-2 mediated palmitoylation will be devised. A high throughput screen will be exploited to identify novel small molecular inhibitors of Hh palmitoylation. These reagents could potentially be clinically useful as anti-tumor agents in Hh driven malignancies. The effects of Mart-2 inhibition on growth of Shh-dependent pancreatic cancer cells will be assayed.

Public Health Relevance

Hh signaling has been shown to drive the growth of many human cancers, including medulloblastoma, melanoma, and pancreatic tumors. The proposed studies will help us understand how Hh proteins work in normal and malignant cells and will aim to develop Hh inhibitors that could potentially be clinically useful as anti-tumor agents in Hh driven malignancies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057966-12
Application #
8030423
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Chin, Jean
Project Start
1998-08-01
Project End
2013-02-28
Budget Start
2011-03-01
Budget End
2013-02-28
Support Year
12
Fiscal Year
2011
Total Cost
$344,564
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Matevossian, Armine; Resh, Marilyn D (2015) Membrane topology of hedgehog acyltransferase. J Biol Chem 290:2235-43
Matevossian, Armine; Resh, Marilyn D (2015) Hedgehog Acyltransferase as a target in estrogen receptor positive, HER2 amplified, and tamoxifen resistant breast cancer cells. Mol Cancer 14:72
Petrova, E; Matevossian, A; Resh, M D (2015) Hedgehog acyltransferase as a target in pancreatic ductal adenocarcinoma. Oncogene 34:263-8
Callier, Patrick; Calvel, Pierre; Matevossian, Armine et al. (2014) Loss of function mutation in the palmitoyl-transferase HHAT leads to syndromic 46,XY disorder of sex development by impeding Hedgehog protein palmitoylation and signaling. PLoS Genet 10:e1004340
Rios-Esteves, Jessica; Haugen, Brittany; Resh, Marilyn D (2014) Identification of key residues and regions important for porcupine-mediated Wnt acylation. J Biol Chem 289:17009-19
Resh, Marilyn D (2013) Covalent lipid modifications of proteins. Curr Biol 23:R431-5
Rios-Esteves, Jessica; Resh, Marilyn D (2013) Stearoyl CoA desaturase is required to produce active, lipid-modified Wnt proteins. Cell Rep 4:1072-81
Petrova, Elissaveta; Rios-Esteves, Jessica; Ouerfelli, Ouathek et al. (2013) Inhibitors of Hedgehog acyltransferase block Sonic Hedgehog signaling. Nat Chem Biol 9:247-9
Buglino, John A; Resh, Marilyn D (2012) Palmitoylation of Hedgehog proteins. Vitam Horm 88:229-52
Resh, Marilyn D (2012) Targeting protein lipidation in disease. Trends Mol Med 18:206-14

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