Detailed Mechanism of ORMDLs Function

Abstract

Sphingolipids (SLs) are one of the major lipid types present in eukaryotic cells. SLs not only serve as an important constituent of the plasma membrane but also as bioactive lipids which regulate several signal transduction processes such as cell growth and differentiation. The de novo biosynthesis begins in the endoplasmic reticulum (ER) with the condensation of serine and palmitoyl Co-A by the rate-limiting enzyme, serine palmitoyltransferase (SPT). SPT is homeostatically regulated by small ER proteins: ORMDLs in mammalian cells and Orms in yeast. The ORMDLs stereo-specifically senses D-erythro ceramide to regulate SPT activity in response to the cellular SL levels. Mammalian cells have three ORMDL isoforms that show high amino acid sequence similarity, but why cells need three ORMDLs is still unknown. Here we are investigating the role of different ORMDL isoforms and the amino-acid regions of ORMDL involved in SPT regulation. We are also looking at regulation of individual ORMDL isoform protein turnover, as an additional mechanism by which SLs are maintained in the cells. The successful generation of ORMDL2/3, ORMDL1/2, and ORMDL1/3 double-knockout cell lines by CRISPR-Cas9 reveals that cells are compensating for the missing ORMDLs by increasing the protein levels of the remaining ORMDL. Surprisingly, we do not see any changes in the mRNA levels of the endogenous ORMDL in double-knockout cell lines, suggesting that ORMDL protein levels are post-transcriptionally regulated. We find an important clue to regulation by finding that ORMDLs are short-lived proteins and that their degradation is regulated by p62 receptor-mediated selective autophagy. Characterization of the ORMDL isoforms shows that individual ORMDL isoforms are alone sufficient to regulate SPT activity. Lipidomic results show that there is no significant change in total ceramide levels in the ORMDL double-knockout cell line, confirming that each individual ORMDL isoform is sufficient to control ceramide or SL levels in cells. Based on our findings, we still do not know why cells need three different ORMDL isoforms. We hypothesize that each ORMDL isoforms respond to different ceramide levels in cell. To test this hypothesis, we used a cell-free system to measure the response of each isoform to different levels of ceramide. We found that ORMDL isoforms response to different ceramide levels, to downregulate the activity of SPT. To investigate which region of ORMDL is important for its function, we have generated three deletion mutant constructs on cytosolic N- terminus & C-terminus and two polyA substitution mutant constructs on the cytosolic loop of mORMDL1 by using site-directed mutagenesis approach. We have confirmed the co-localization of mutant constructs in the ER by immunofluorescence staining. Further, we have established a platform to test our mutant constructs and we are in the process of testing the mutant constructs. These data strongly suggest that cells require an optimal amount of ORMDL protein and sense the different ceramide levels to control overall SL levels in cells by regulating SPT enzyme activity.