Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site engineering of sphingomyelin synthases

Kol, Matthijs; Panatala, Radhakrishnan; Nordmann, Mirjana; Swart, Leoni; Suijlekom, Leonie van; Cabukusta, Birol; Hilderink, Angelika; Grabietz, Tanja; Mina, John G.; Somerharju, Pentti; Korneev, Sergei M.; Tafesse, Fikadu G.; Holthuis, Joost C. M.

doi:10.1194/jlr.m068692

Cited by 9 publications

(4 citation statements)

References 48 publications

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“…9. Topological analysis using the N-glycosylation gel-shift assay showed that the N-terminal SAMD is cytosolic (30). DGK␦2 is also cytosolic (21,31).…”

Section: Interaction and Functional Linkage Between Dgk␦ And Smsrmentioning

confidence: 99%

Diacylglycerol kinase δ and sphingomyelin synthase–related protein functionally interact via their sterile α motif domains

Murakami

Hoshino

Sakai

et al. 2020

Journal of Biological Chemistry

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Introduction Diacyglycerol kinase δ isozyme (DGKδ) plays critical roles in lipid signaling by phosphorylating diacylglycerol (DG) into phosphatidic acid (PA). DGKδ regulates a wide variety of physiological and pathological events, such as type II diabetes and obsessive compulsive disorder. Because DGK is one of the components of phosphatidylinositol (PI) turnover, it is thought that DGKδ also utilizes mainly 18:0/20:4‐DG (X:Y; the total number of carbon atoms: the total number of double bonds) derived from PI turnover. Interestingly, we recently demonstrated that DGKδ preferably metabolized palmitic acid (16:0)‐containing DG molecular species, but not arachidonic acid (20:4)‐containing DG species, in response to high glucose stimulation. However, it is still unclear what kind of DG‐generating enzyme provides palmitic acid‐containing DG species. Sphingomyelin synthase (SMS) 1, SMS2 and SMS‐related protein (SMSr) are DG‐generating enzymes utilizing phosphatidylcholine/phosphatidylethanolamine and ceramide. SMS1 and SMSr contain a sterile α motif domain (SAM), which is a protein‐protein interaction module, at their N‐termini. DGKδ also possesses SAM at its C‐terminus, and DGKδ‐SAM is highly homologous to SMSr‐SAM. Therefore, we hypothesised that DGKδ interacts with SMSr through their SAMs. In the present study, we investigated the interaction between DGKδ‐SAM and SMSr‐SAM. Results We first examined whether DGKδ‐SAM interacts with SMSr‐SAM by immunoprecipitation analysis. We found that SMSr‐SAM, but not SMS1‐SAM, was co‐immunoprecipitated with DGKδ‐SAM. Full‐length DGKδ (DGKδ‐FL) was also co‐immunoprecipitated with SMSr‐FL more strongly than with SMS1‐FL and with SMS2‐FL. To examine whether SMSr‐SAM contribute to the interaction, we performed immunoprecipitation analysis using SMSr‐FL and a SAM‐deletion mutant (SMSr‐DSAM). DGKδ‐FL was strongly co‐immunoprecipitated with SMSr, whereas SMSr‐DSAM only weakly interacted with DGKδ‐FL. Immunostaining analysis demonstrated that DGKδ‐FL co‐localized partly with SMSr‐FL in COS‐7 cells overexpressing these proteins. These results strongly suggest that DGKδ interacts with SMSr through their SAMs. Conclusion In summary, the present study for the first time showed that DGKδ interacted with SMSr mainly through their SAMs. It is possible that SMSr is one of the candidates of up stream DG‐providing enzymes of DGKδ, which composes a new pathway independent of PI turnover. Support or Funding Information This work was supported by JSPS KAKENHI (Grant Number JP18J20003) and supported in part by Venture Business Laboratory in Chiba University (Nanohana Competition 2018 Award). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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“…9. Topological analysis using the N-glycosylation gel-shift assay showed that the N-terminal SAMD is cytosolic (30). DGK␦2 is also cytosolic (21,31).…”

Section: Interaction and Functional Linkage Between Dgk␦ And Smsrmentioning

confidence: 99%

Diacylglycerol kinase δ and sphingomyelin synthase–related protein functionally interact via their sterile α motif domains

Murakami

Hoshino

Sakai

et al. 2020

Journal of Biological Chemistry

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“…Next, catalytic activities of the heterologously expressed enzymes were determined in cell lysates using a recently established quantitative CPE synthase activity assay31. Whereas the D348E mutation completely abolished SMSr-mediated CPE production, there was no significant difference between the catalytic activities of SMSr and SMSr OD , indicating that the oligomeric state of the enzyme does not critically influence its catalytic capacity (Fig.…”

Section: Resultsmentioning

confidence: 99%

ER residency of the ceramide phosphoethanolamine synthase SMSr relies on homotypic oligomerization mediated by its SAM domain

Cabukusta

Kol

Kneller

et al. 2017

Sci Rep

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SMSr/SAMD8 is an ER-resident ceramide phosphoethanolamine synthase with a critical role in controlling ER ceramides and suppressing ceramide-induced apoptosis in cultured cells. SMSr-mediated ceramide homeostasis relies on the enzyme’s catalytic activity as well as on its N-terminal sterile α-motif or SAM domain. Here we report that SMSr-SAM is structurally and functionally related to the SAM domain of diacylglycerol kinase DGKδ, a central regulator of lipid signaling at the plasma membrane. Native gel electrophoresis indicates that both SAM domains form homotypic oligomers. Chemical crosslinking studies show that SMSr self-associates into ER-resident trimers and hexamers that resemble the helical oligomers formed by DGKδ-SAM. Residues critical for DGKδ-SAM oligomerization are conserved in SMSr-SAM and their substitution causes a dissociation of SMSr oligomers as well as a partial redistribution of the enzyme to the Golgi. Conversely, treatment of cells with curcumin, a drug disrupting ceramide and Ca2+ homeostasis in the ER, stabilizes SMSr oligomers and promotes retention of the enzyme in the ER. Our data provide first demonstration of a multi-pass membrane protein that undergoes homotypic oligomerization via its SAM domain and indicate that SAM-mediated self-assembly of SMSr is required for efficient retention of the enzyme in the ER.

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“…Compared to protein-protein interactions, it remains challenging to elucidate lipid-protein interactions. [60] However, lipoproteins are not only fundamental components of biological membranes, they also participate in numerous biological processes including cell signaling, transcriptional regulation, and protein trafficking, among others. Traditional methods used to investigate lipid-protein interactions include X-ray crystallography, NMR, and atomic force microscopy (AFM).…”

Section: Lipid-protein Interactionmentioning

confidence: 99%

Recent Developments and Applications of Photoconjugation Chemistry

Fan

Zhang

Lei³

2018

Chimia

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Understanding of biological systems has always been the general interest in chemical biology, and chemical modifications of biomolecules are often required to elucidate their functions and properties. There has thus been a rapid development of covalent chemistries for the modification of macromolecules. Among these strategies, photochemistry provides the advantage of using biocompatible light as an energy source to trigger bioconjugation reactions; this circumvents the use of toxic reagents (e.g. metal catalysts and chelating ligands). Light-induced chemistry can achieve precise spatial and temporal conjugation of biomolecules in their native environment.

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Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site engineering of sphingomyelin synthases

Cited by 9 publications

References 48 publications

Diacylglycerol kinase δ and sphingomyelin synthase–related protein functionally interact via their sterile α motif domains

Diacylglycerol kinase δ and sphingomyelin synthase–related protein functionally interact via their sterile α motif domains

ER residency of the ceramide phosphoethanolamine synthase SMSr relies on homotypic oligomerization mediated by its SAM domain

Recent Developments and Applications of Photoconjugation Chemistry

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