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Orm family proteins mediate sphingolipid homeostasis.

Breslow DK, Collins SR, Bodenmiller B, Aebersold R, Simons K, Shevchenko A, Ejsing CS, Weissman JS - Nature (2010)

Bottom Line: Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production.We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted.Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 1700 4th Street, San Francisco, California 94158, USA.

ABSTRACT
Despite the essential roles of sphingolipids both as structural components of membranes and critical signalling molecules, we have a limited understanding of how cells sense and regulate their levels. Here we reveal the function in sphingolipid metabolism of the ORM genes (known as ORMDL genes in humans)-a conserved gene family that includes ORMDL3, which has recently been identified as a potential risk factor for childhood asthma. Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production. We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted. Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism. Our work identifies the Orm proteins as critical mediators of sphingolipid homeostasis and raises the possibility that sphingolipid misregulation contributes to the development of childhood asthma.

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ORM gene function is conserved in human cellsa, HEK293T cells were transfected with either an empty vector or a vector encoding Ormdl3 fused to the 3×Flag epitope (N- or C-terminal fusion). Immunoprecipitations with anti-Flag agarose were analyzed by Western blot against human serine palmitoyltransferase (α-hSptlc1) and against the Flag epitope (α-Flag). b, Hela cells were transfected with siRNAs directed against the indicated genes. After 72 hr, cells were harvested and their lipids were analyzed by mass spectrometry. Ceramide levels normalized to phosphatidylcholine (PC) are shown (relative to untreated cells; average ± s.d., n = 3).
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Figure 3: ORM gene function is conserved in human cellsa, HEK293T cells were transfected with either an empty vector or a vector encoding Ormdl3 fused to the 3×Flag epitope (N- or C-terminal fusion). Immunoprecipitations with anti-Flag agarose were analyzed by Western blot against human serine palmitoyltransferase (α-hSptlc1) and against the Flag epitope (α-Flag). b, Hela cells were transfected with siRNAs directed against the indicated genes. After 72 hr, cells were harvested and their lipids were analyzed by mass spectrometry. Ceramide levels normalized to phosphatidylcholine (PC) are shown (relative to untreated cells; average ± s.d., n = 3).

Mentions: Finally, we showed that the functional complex between Orm proteins and serine palmitoyltransferase is conserved in human cells. Immunoprecipitations from HEK293T cells expressing 3×Flag-tagged Ormdl3 led to a prominent Sptlc1 band detected by Western blot (Fig. 3a). Additionally, we found that simultaneous knock-down by RNAi of ORMDL1/2/3 in Hela cells causes an approximately three-fold increase in ceramide levels (Fig. 3b, Supplementary Fig. 2 and Supplementary Data).


Orm family proteins mediate sphingolipid homeostasis.

Breslow DK, Collins SR, Bodenmiller B, Aebersold R, Simons K, Shevchenko A, Ejsing CS, Weissman JS - Nature (2010)

ORM gene function is conserved in human cellsa, HEK293T cells were transfected with either an empty vector or a vector encoding Ormdl3 fused to the 3×Flag epitope (N- or C-terminal fusion). Immunoprecipitations with anti-Flag agarose were analyzed by Western blot against human serine palmitoyltransferase (α-hSptlc1) and against the Flag epitope (α-Flag). b, Hela cells were transfected with siRNAs directed against the indicated genes. After 72 hr, cells were harvested and their lipids were analyzed by mass spectrometry. Ceramide levels normalized to phosphatidylcholine (PC) are shown (relative to untreated cells; average ± s.d., n = 3).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2877384&req=5

Figure 3: ORM gene function is conserved in human cellsa, HEK293T cells were transfected with either an empty vector or a vector encoding Ormdl3 fused to the 3×Flag epitope (N- or C-terminal fusion). Immunoprecipitations with anti-Flag agarose were analyzed by Western blot against human serine palmitoyltransferase (α-hSptlc1) and against the Flag epitope (α-Flag). b, Hela cells were transfected with siRNAs directed against the indicated genes. After 72 hr, cells were harvested and their lipids were analyzed by mass spectrometry. Ceramide levels normalized to phosphatidylcholine (PC) are shown (relative to untreated cells; average ± s.d., n = 3).
Mentions: Finally, we showed that the functional complex between Orm proteins and serine palmitoyltransferase is conserved in human cells. Immunoprecipitations from HEK293T cells expressing 3×Flag-tagged Ormdl3 led to a prominent Sptlc1 band detected by Western blot (Fig. 3a). Additionally, we found that simultaneous knock-down by RNAi of ORMDL1/2/3 in Hela cells causes an approximately three-fold increase in ceramide levels (Fig. 3b, Supplementary Fig. 2 and Supplementary Data).

Bottom Line: Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production.We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted.Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 1700 4th Street, San Francisco, California 94158, USA.

ABSTRACT
Despite the essential roles of sphingolipids both as structural components of membranes and critical signalling molecules, we have a limited understanding of how cells sense and regulate their levels. Here we reveal the function in sphingolipid metabolism of the ORM genes (known as ORMDL genes in humans)-a conserved gene family that includes ORMDL3, which has recently been identified as a potential risk factor for childhood asthma. Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production. We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted. Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism. Our work identifies the Orm proteins as critical mediators of sphingolipid homeostasis and raises the possibility that sphingolipid misregulation contributes to the development of childhood asthma.

Show MeSH
Related in: MedlinePlus