ORMDL/serine palmitoyltransferase stoichiometry determines effects of ORMDL3 expression on sphingolipid biosynthesis

Abstract

The ORM1 ( Saccharomyces cerevisiae )-like proteins (ORMDLs) and their yeast orthologs, the ORMs, regulate the initiating and rate-limiting enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT) ( 1-4 ). The three ORMDL isoforms (ORMDL1-3) are small (17.5 kDa) hydrophobic membrane proteins that are situated in the endoplasmic reticulum along with SPT. The three isoforms are highly homologous and their functions appear to be redundant at the cellular level ( 3, 4 ). The ORMDLs are negative regulators of SPT and mediate the homeostatic regulation of SPT in response to cellular sphingolipid levels. siRNA depletion of the ORMDLs results in elevated sphingolipid synthesis under control conditions and reverses the homeostatic inhibition of sphingolipid synthesis resulting from elevated cellular sphingolipid content ( 1,3,4 ). The Orms and ORMDLs form stable physical complexes with SPT ( 1 ), even under conditions in which the ORMDLs are minimally inhibiting SPT. In yeast, the Orm regulatory activity is controlled by Orm phosphorylation. This may not be the case for the mammalian ORMDLs, which lack the sequences that are phosphorylated in the yeast Orms. Clinical interest in ORMDL function has been sparked by the observation that SNPs adjacent to the ORMDL3 gene are highly correlated with Abstract The ORM1 ( Saccharomyces cerevisiae )-like proteins (ORMDLs) and their yeast orthologs, the Orms, are negative homeostatic regulators of the initiating enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT). Genome-wide association studies have established a strong correlation between elevated expression of the endoplasmic reticulum protein ORMDL3 and risk for childhood asthma. Here we test the notion that elevated levels of ORMDL3 decrease sphingolipid biosynthesis. This was tested in cultured human bronchial epithelial cells (HBECs) (an immortalized, but untransformed, airway epithelial cell line) and in HeLa cells (a cervical adenocarcinoma cell line). Surprisingly, elevated ORMDL3 expression did not suppress de novo biosynthesis of sphingolipids. We determined that ORMDL is expressed in functional excess relative to SPT at normal levels of expression. ORMDLs and SPT form stable complexes that are not increased by elevated ORMDL3 expression. Although sphingolipid biosynthesis was not decreased by elevated ORMDL3 expression, the steady state mass levels of all major sphingolipids were marginally decreased by low level ORMDL3 over-expression in HBECs. These data indicate that the contribution of ORMDL3 to asthma risk may involve changes in sphingolipid metabolism, but that the connection is complex. 3 February 2015. Published, JLR Papers in Press, February 17, 2015 DOI 10.1194 Abbreviations: C6 , ceramide-N -hexanoyl-D-erythro-sphingosine; HBEC, human bronchial epithelial cell; IP, immunoprecipitation; ORMDL, ORM1 ( Saccharomyces cerevisiae )-like protein; scSPT, single-chain serine palmitoyltransferase; SPT, serine palmitoyltransferase; SPTLC1, serine palmitoyltransferase long chain base ...

The role of ORMDL proteins, guardians of cellular sphingolipids, in asthma

“…These findings suggested that yeast ORM proteins are negative regulators of sphingolipid biosynthesis and act through the inhibition of SPT. This conclusion was later confirmed in other studies using cells of various origin, including rice (66), mice (67), and human cell lines (68)(69)(70)(71).…”

“…Furthermore, when expression of all ORMDLs was decreased and ORMDL3 had been re-introduced, the production of ceramides was lowered in comparison with ORMDLs siRNA (67). This contradicts previous findings (68,70) and supports the theory that it is ORMDL3-mediated inhibition of sphingolipid metabolism that stays behind the ORMDL3-associated asthma phenotype. The levels of ORMDL proteins could also be regulated by the speed of their degradation.…”

“…They induced de novo synthesis of ceramides by palmitate. Changes in ceramide production were observed only when expression of all three ORMDL proteins was reduced (68,70). Cells exposed simultaneously to ORMDL1, ORMDL2, ORMDL3 siRNAs showed increased production of ceramides in comparison with wild-type cells in both studies.…”

“…In cells overexpressing ORMDL3 in mid-levels, inhibition of SPT activity was observed (67). On the other hand, when strong ORMDL3 overexpression was induced, the levels of the studied sphingolipids remained unchanged (68) or even elevated (67). As normal inhibition of de novo biosynthesis of sphingolipids was observed, increased recycling of complex ceramides was behind the observed higher levels of sphingolipids in the cell.…”

“…De novo ceramide synthesis is initiated by exposure of cells to different cell stressors including inductors of unfolded protein responses (68), inhibitors of TORC signaling, and various chemotherapeutic agents (94), and cell activators (70). Better understanding of the regulation of sphingolipid biosynthesis pathways is needed to comprehend the origin of diseases in which these pathways are deregulated.…”