Sphingolipids are a diverse but metabolically related group of lipids with unique functional and physical properties that are critical for cell signaling and membrane architecture. Misregulation of particular sphingolipids has been implicated in such diverse diseases as cancer, inflammatory disease, and cardiovascular disease. In particular, as outlined below, genome-wide association studies have strongly implicated the subject of this proposal, the sphingolipid metabolic regulator ORMDL, in the risk for childhood asthma. The overall level of cellular sphingolipid is determined by the initiating, and rate limiting enzyme in the biosynthetic pathway, serine palmitoyltransferase (SPT). Not surprisingly, SPT activity is homeostatically controlled-i.e. SPT activity is strongly inhibited by elevated levels of cellular sphingolipid. We have demonstrated that this homeostatic control is mediated by a set of small membrane proteins found in the endoplasmic reticulum, the ORMDLs. The goal of the studies proposed here is to establish the mechanism by which the ORMDLs mediate SPT inhibition in response to elevated sphingolipid levels and to begin to uncover how those mechanisms impact on risk for asthma. These studies will answer basic questions concerning how ORMDLs sense cellular sphingolipid levels and how changes in sphingolipid levels are transmitted through ORMDL to affect SPT activity. We will test the hypothesis that ceramide directly binds ORMDL to trigger SPT regulation. We will determine the structural elements in ORMDL that are essential for mediating sphingolipid regulation of SPT, will use a recently constructed cell-free system to quantitate the levels of sphingolipid sensed by the ORMDL system, and will test the hypothesis that elevation of sphingolipid induces changes in ORMDL topology. Finally we will construct a reconstituted system with purified ORMDL and SPT to test the hypothesis that the ORMDLs and SPT are the only required components of this regulatory system. In addition we propose to address an essential question concerning the genetics of ORMDL expression that has emerged from genome-wide association studies in asthma. Single nucleotide polymorphisms (SNPs) adjacent to the gene of one of the ORMDL isoforms, ORMDL3, are strongly and consistently associated with the risk for childhood asthma and with elevated expression of ORMDL3. Although the population-based studies are intriguing and suggestive, a detailed examination of this phenomenon has been hamstrung by the inability to directly test the effect of these SNPs on ORMDL3 expression. It has not been possible to directly measure the degree to which these SNPs affect ORMDL3 expression nor in what cell types and conditions this elevated expression occurs. Here we propose to use emerging Crispr/Cas9 genome editing technology to produce paired cell lines, in asthma-relevant cell types, containing either a risk or non-risk genotype at the most prominent risk-associated SNP. These will be used to examine the extent, cell type, and stimuli specificity of elevation of ORMDL3 expression induced by the risk allele and how this affects sphingolipid metabolism.

Public Health Relevance

Certain lipids (fatty substances) called sphingolipids are important in a number of diseases, including asthma. Current therapies for asthma are insufficient and to date there is no cure. The proposed research will determine how levels of these lipids are controlled in cells. This knowledge will identify novel targets for the treatment of asthma and other diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL131340-02
Application #
9323552
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Noel, Patricia
Project Start
2016-08-01
Project End
2020-05-31
Budget Start
2017-06-01
Budget End
2018-05-31
Support Year
2
Fiscal Year
2017
Total Cost
$381,250
Indirect Cost
$131,250
Name
Virginia Commonwealth University
Department
Biochemistry
Type
Domestic Higher Education
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
Wattenberg, Binks W (2018) The long and the short of ceramides. J Biol Chem 293:9922-9923
Davis, Deanna; Kannan, Muthukumar; Wattenberg, Binks (2018) Orm/ORMDL proteins: Gate guardians and master regulators. Adv Biol Regul 70:3-18