For over a dozen years, studies from Dr. Hannun's laboratory have focused on elucidating mechanisms and functions for sphingolipids in signal transduction and cell regulation. The major line of investigation has resulted in the elucidation of the sphingomyelin cycle/pathway whereby the action of several extracellular stimuli or stresses (such as TNFalpha or heat) results in activation of a neutral sphingomyelinase. This enzyme cleaves membrane sphingomyelin and causes the release and accumulation of ceramide, a candidate intracellular mediator. Multiple lines of evidence have implicated ceramide in the regulation of the stress response and in regulating cell cycle arrest, cell senescence, and apoptosis. Whereas information on the functions of ceramide and its mechanisms of action continue to accumulate, little progress has been developed concerning the regulation of neutral sphingomyelinase (N-SMase); possibly a result of the great difficulty in working with this enzyme and the lack of purified preparations. The investigator's laboratory has made important strides in the last 2-3 years in purifying the enzyme and defining several mechanisms of in vitro and cellular regulation involving glutathione (GSH) and cysteine proteases. The results have generated the following hypothesis: N-SMase is a key enzyme involved in regulating ceramide formation and in initiating ceramide-dependent pathways of cell regulation. The enzyme is regulated reversibly by GSH and irreversibly by caspases. Therefore, the specific aims are: (1) Purify, characterize, and clone neutral sphingomyelinase. (2) Determine the in vitro mechanisms of regulation of neutral sphingomyelinase by GSH, and define the mechanism and significance of this mode of regulation in cellular functions. (3) Define the in vitro and cellular regulation of neutral sphingomyelinase by the cysteine protease, caspase 7. These studies should provide a biochemical and molecular understanding of a key enzyme involved in regulated metabolism of sphingomyelin and the generation of ceramide. These studies are essential to establish and consolidate the sphingomyelin/ceramide pathway as a major mechanism in cell growth regulation. N-SMase may emerge as a key target in the understanding of the mechanisms of the stress response and in therapeutic development.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM043825-10
Application #
2828613
Study Section
Medical Biochemistry Study Section (MEDB)
Project Start
1991-01-01
Project End
2002-12-31
Budget Start
1999-01-01
Budget End
1999-12-31
Support Year
10
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Medical University of South Carolina
Department
Biochemistry
Type
Schools of Medicine
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29425
Rajagopalan, Vinodh; Canals, Daniel; Luberto, Chiara et al. (2015) Critical determinants of mitochondria-associated neutral sphingomyelinase (MA-nSMase) for mitochondrial localization. Biochim Biophys Acta 1850:628-39
Shamseddine, A A; Clarke, C J; Carroll, B et al. (2015) P53-dependent upregulation of neutral sphingomyelinase-2: role in doxorubicin-induced growth arrest. Cell Death Dis 6:e1947
Lu, Zhongyang; Li, Yanchun; Jin, Junfei et al. (2015) GPR40/FFA1 and neutral sphingomyelinase are involved in palmitate-boosted inflammatory response of microvascular endothelial cells to LPS. Atherosclerosis 240:163-73
Spincemaille, Pieter; Matmati, Nabil; Hannun, Yusuf A et al. (2014) Sphingolipids and mitochondrial function in budding yeast. Biochim Biophys Acta 1840:3131-7
Matmati, Nabil; Metelli, Alessandra; Tripathi, Kaushlendra et al. (2013) Identification of C18:1-phytoceramide as the candidate lipid mediator for hydroxyurea resistance in yeast. J Biol Chem 288:17272-84
Khavandgar, Zohreh; Poirier, Christophe; Clarke, Christopher J et al. (2011) A cell-autonomous requirement for neutral sphingomyelinase 2 in bone mineralization. J Cell Biol 194:277-89
Clarke, Christopher J; Cloessner, Emily A; Roddy, Patrick L et al. (2011) Neutral sphingomyelinase 2 (nSMase2) is the primary neutral sphingomyelinase isoform activated by tumour necrosis factor-? in MCF-7 cells. Biochem J 435:381-90
Barbosa, Antonio Daniel; Osorio, Hugo; Sims, Kellie J et al. (2011) Role for Sit4p-dependent mitochondrial dysfunction in mediating the shortened chronological lifespan and oxidative stress sensitivity of Isc1p-deficient cells. Mol Microbiol 81:515-27
Wu, Bill X; Clarke, Christopher J; Matmati, Nabil et al. (2011) Identification of novel anionic phospholipid binding domains in neutral sphingomyelinase 2 with selective binding preference. J Biol Chem 286:22362-71
Hernandez-Corbacho, Maria Jose; Jenkins, Russell W; Clarke, Christopher J et al. (2011) Accumulation of long-chain glycosphingolipids during aging is prevented by caloric restriction. PLoS One 6:e20411

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