CRISPR/Cas9 deletion of ORMDLs reveals complexity in sphingolipid metabolism
| Autor: | Christopher Green, Deanna L. Davis, Sarah Spiegel, Michael Maceyka, Usha Mahawar, Clement Oyeniran, Binks W. Wattenberg, Cynthia Weigel, Jason Newton, Briana N. James |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Ceramide QD415-436 030204 cardiovascular system & hematology Biochemistry sphingolipid biosynthesis sphingomyelin HDM house dust mite serine palmitoyltransferase PI propidium iodide 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Endocrinology ROS reactive oxygen species Downregulation and upregulation Ormdl GluCer glucosylceramide Sphingosine-1-phosphate ceramide sphingoid bases CRISPR/Cas9 S1P sphingosine-1-phosphate Sphingosine Serine C-palmitoyltransferase Cell Biology GalCer galactosylceramides gRNA guide RNA Sphingolipid SPT serine palmitoyltransferase Cell biology 030104 developmental biology chemistry SPL S1P lyase sphingosine-1-phosphate Lactosylceramides CRISPR-Cas Systems Sphingomyelin Research Article |
| Zdroj: | Journal of Lipid Research Journal of Lipid Research, Vol 62, Iss, Pp 100082-(2021) |
| ISSN: | 1539-7262 0022-2275 |
| Popis: | The serine palmitoyltransferase (SPT) complex catalyzes the rate-limiting step in the de novo biosynthesis of ceramides, the precursors of sphingolipids. The mammalian ORMDL isoforms (ORMDL1-3) are negative regulators of SPT. However, the roles of individual ORMDL isoforms are unclear. Using siRNA against individual ORMDLs, only single siORMDL3 had modest effects on dihydroceramide and ceramide levels, whereas downregulation of all three ORMDLs induced more pronounced increases. With the CRISPR/Cas9-based genome-editing strategy, we established stable single ORMDL3 KO (ORMDL3-KO) and ORMDL1/2/3 triple-KO (ORMDL-TKO) cell lines to further understand the roles of ORMDL proteins in sphingolipid biosynthesis. While ORMDL3-KO modestly increased dihydroceramide and ceramide levels, ORMDL-TKO cells had dramatic increases in the accumulation of these sphingolipid precursors. SPT activity was increased only in ORMDL-TKO cells. In addition, ORMDL-TKO but not ORMDL3-KO dramatically increased levels of galactosylceramides, glucosylceramides, and lactosylceramides, the elevated N-acyl chain distributions of which broadly correlated with the increases in ceramide species. Surprisingly, although C16:0 is the major sphingomyelin species, it was only increased in ORMDL3-KO, whereas all other N-acyl chain sphingomyelin species were significantly increased in ORMDL-TKO cells. Analysis of sphingoid bases revealed that although sphingosine was only increased 2-fold in ORMDL-TKO cells, levels of dihydrosphingosine, dihydrosphingosine-1-phosphate, and sphingosine-1-phosphate were hugely increased in ORMDL-TKO cells and not in ORMDL3-KO cells. Thus, ORMDL proteins may have a complex, multifaceted role in the biosynthesis and regulation of cellular sphingolipids. |
| Databáze: | OpenAIRE |
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