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Subunit composition of the mammalian -palmitoyltransferase defines the spectrum of straight and methyl-branched long-chain bases

Museer A. Lonea,1, Andreas J. Hülsmeiera, Essa M. Saiedb,c, Gergely Karsaia, Christoph Arenzc, Arnold von Eckardsteina, and Thorsten Hornemanna,1

aInstitute of Clinical Chemistry, University Hospital Zurich, University of Zurich, Zurich-8091, Switzerland; bInstitute for Chemistry, Suez Canal University, Ismailia, Egypt; and cInstitute for Chemistry, Humboldt Universität zu Berlin, 12489 Berlin, Germany

Edited by Howard Riezman, University of Geneva, Geneva, Switzerland, and accepted by Editorial Board Member David J. Mangelsdorf, May 20, 2020 (received for review February 11, 2020) (SLs) are chemically diverse that have impor- SPT activity is metabolically controlled by negative feedback tant structural and signaling functions within mammalian cells. SLs regulation. This mechanism is well understood in yeast, where are commonly defined by the presence of a long-chain base (LCB) SPT activity is regulated by two phosphoproteins, Orm1 and that is normally formed by the conjugation of L-serine and Orm2 (9, 10). Another , Tsc3, is required for maximal palmitoyl-CoA. This pyridoxal 5-phosphate (PLP)-dependent reac- SPT activation (11). SPT together with Orm1 and Orm2, Tsc3, tion is mediated by the serine-palmitoyltransferase (SPT). and the phosphoinositide phosphatase Sac1 form a multisubunit However, SPT can also metabolize other acyl-CoAs, in the range of complex (10). Orthologs of these are also found in C14 to C18, forming a variety of LCBs that differ by structure and mammals, but their roles in regulating SL are less function. Mammalian SPT consists of three core subunits: SPTLC1, well understood. Mammalian cells express three ORM orthologs SPTLC2, and SPTLC3. Whereas SPTLC1 and SPTLC2 are ubiquitously (ORMDL1, 2, and 3) but lack the regulatory phosphorylation expressed, SPTLC3 expression is restricted to certain tissues only. sites of yeast Orm proteins (10, 12). The polypeptides ssSPTa The influence of the individual subunits on enzyme activity is not and ssSPTb are functional orthologs of Tsc3 that appear to clear. Using models deficient in SPTLC1, SPTLC2, and SPTLC3, modulate SPT activity and substrate affinity (13–15). While ssSPTa we investigated the role of each subunit on enzyme activity and promotes canonical C18 LCB synthesis, ssSPTb is associated with the LCB product spectrum. We showed that SPTLC1 is essential for increased synthesis of C20 LCBs. In mice, a single gain-of-function activity, whereas SPTLC2 and SPTLC3 are partly redundant but mutation in ssSPTb (H56L) was shown to increase C20 LCB for- differ in their enzymatic properties. SPTLC1 in combination with mation in the brain, leading to retinopathy and central neuro- SPTLC2 specifically formed C18, C19, and C20 LCBs while the com- degeneration (14). However, the role of the individual SPT subunits bination of SPTLC1 and SPTLC3 yielded a broader product spec- trum. We identified anteiso-branched-C18 SO (meC18SO) as the with respect to SPT enzyme activity and substrate affinity has not primary product of the SPTLC3 reaction. The meC18SO was syn- yet been addressed systematically. thesized from anteiso-methyl-palmitate, in turn synthesized from In the present study, using SPTLC1-, SPTLC2-, and SPTLC3- a precursor metabolite generated in the isoleucine catabolic path- deficient cell models, we demonstrated that SPTLC1 is essential way. The meC18SO is metabolized to and complex SLs and is a constituent of human low- and high-density . Significance

serine-palmitoyltransferase | long-chain base | omega-3-methyl- Sphingolipids (SLs) are complex lipids that constitute hundreds of subspecies. All SLs share a long-chain base (LCB) as a de- fining structural component. LCBs are formed by serine- phingolipids (SLs) share the presence of a long-chain base palmitoyltransferase (SPT) in the first and rate-limiting step S(LCB) backbone as a common structural element. LCBs are of SL de novo synthesis. SPT consists of three subunits that show aliphatic amino and formed in the first and rate-limiting tissue-specific expression. In presence of the SPTLC3 subunit, the step of SL de novo synthesis (SI Appendix, Fig. S1). This reaction enzyme forms a spectrum of straight and branched LCBs with is catalyzed by the enzyme serine-palmitoyltransferase (SPT). distinct biochemical and biophysical properties. This alters the composition of cellular membranes and might influence the SPT is a pyridoxal 5‐phosphate (PLP)-dependent α‐oxoamino- dynamics of membrane-related transport and signaling events. that consists of three core subunits—SPTLC1, SPTLC2, — SPTLC3 is particularly abundant in skin, and changes in SPT ac- and SPTLC3 that share a mutual homology. SPTLC2 is 68% tivity are related to dermal pathologies. Genetic variants of identical to SPTLC3 (84% similarity), whereas SPTLC1 is more SPTLC3 are associated with metabolic conditions such as distinct, sharing ∼21% identity (45% similarity) with SPTLC2 and dyslipidemia and . SPTLC3 (1). The PLP-binding motif is present in SPTLC2 and SPTLC3 but not in SPTLC1. While SPTLC1 and SPTLC2 are Author contributions: M.A.L. and T.H. designed research; M.A.L., A.J.H., and G.K. per- ubiquitously expressed, SPTLC3 expression is restricted to specific formed research; E.M.S. and C.A. contributed new reagents/analytic tools; M.A.L., A.J.H., and G.K. analyzed data; and M.A.L., A.v.E., and T.H. wrote the paper. tissues, such as placenta, skin, and some glands (2–6). LCBs vary structurally within and across species. In mammals, The authors declare no competing interest. the most abundant LCB is sphingosine (SO; d18:1), which repre- This article is a PNAS Direct Submission. H.R. is a guest editor invited by the Editorial Board. sents ∼60% of the total LCBs in human plasma (7). The remaining Published under the PNAS license. LCBs differ with respect to chain length, desaturation, and hy- 1To whom correspondence may be addressed. Email: [email protected] or droxylation (reviewed in ref. 8). Plants and fungi, including yeast, [email protected]. mostly form phytosphingosine (phytoSO; t18:0), which is also pre- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ sent at low levels in humans. Insects mainly form short-chain LCBs doi:10.1073/pnas.2002391117/-/DCSupplemental. in the range of C14 to C16 (8).

www.pnas.org/cgi/doi/10.1073/pnas.2002391117 PNAS Latest Articles | 1of8 Downloaded by guest on October 4, 2021 for formation of an active enzyme. In contrast, SPTLC2 and the absence of SPTLC1, we observed a concomitant loss of SPTLC3 are partly redundant but differ in function and substrate SPTLC2, whereas deletion of SPTLC2 did not influence SPTLC1 specificity. Furthermore, we identified a novel, methyl-branched levels (Fig. 1A). This suggested that SPTLC2 is unstable in the LCB as a specific product of human SPTLC3. Methyl-branched absence of SPTLC1, whereas SPTLC1 is stable on its own. LCBs are the major forms in lower invertebrates, such as Cae- Next, SPT activity was measured by the time-dependent in- 15 norhabditis elegans (16), but have not been reported in humans corporation of isotope-labeled L-serine (2,3,3-D3, N) into the until now. de novo formed LCBs. As one of the deuteriums is lost during the conjugation reaction (18), the de novo synthesized LCBs had Results an additional mass of +3 Da. In HAP1 wild-type (WT) cells, we SPTLC1 Is Indispensable for De Novo SPT Function. To investigate the observed a significant formation of C18SO+3, which was com- role of individual subunits on SPT activity, we generated SPTLC1 pletely abrogated in the absence of either SPTLC1 or SPTLC2 and SPTLC2 knockout (KO) HAP1 cell lines using a CRISPR/ (Fig. 1B). Reconstitution of SPTLC1 and STPLC2 expression in Cas9 approach. HAP1 cells are haploid and derived from chronic the respective null background restored enzyme activity (Fig. myelogenous leukemia (CML) cells (17). Natively, HAP1 cells 1C). SPTLC1 expression in the SPTLC1 KO cells rescued en- express SPTLC1 and SPTLC2 but not SPTLC3 (SI Appendix, Fig. dogenous SPTLC2 expression (Fig. 1D). Expression of SPTLC3 S2A). The CRISPR/Cas9 induced loss of SPTLC1 and SPTLC2 in the absence of SPTLC2 resulted in an active SPT enzyme but was confirmed by Western blot analysis (Fig. 1A). Surprisingly, in with significantly lower formation of C18SO+3 compared with

C18SO C18SA A B 120 SPTLC2 + + - SPTLC1 + - + [kDa] 80 100 Calnexin 70 HAP1 cells SPTLC1 55 40 70 nd SPTLC2

55 pmoles/10 0 SPTLC1 + + - + SPTLC2 + + + - - + - - C D WT SPTLC1 KO 100 C18SO C18SA ** Vector + + - - - SPTLC1-V5 - - + - - SPTLC2-V5 - - - + - SPTLC3-V5 - - - - + [kDa] 100

HAP1 cells Calnexin 50 70 70 Anti-V5 55 70 nd SPTLC1 55 nd pmoles/10 70 0 SPTLC2 Vector + - - - - + - - - 55 SPTLC1 - + - - - - + - - SPTLC2 - - + - + - - + - SPTLC3 - - - + + - - - + SPTLC1 KO SPTLC2 KO E F IP V5-tag IP V5-tag 1.5 Vector + - - - + - - - C16SO SPTLC1-V5 - + - - - + - - SPTLC2-V5 C17SO - - + - - - + - C19SO SPTLC3-V5 - - - + - - - + [kDa] 1.0 70 C20SO Anti-V5 55 HAP1 cells SPTLC1 55 0.5 70 SPTLC2 55 nd ndnd pmoles/10 0 Wild type SPTLC2 KO Vector SPTLC2 SPTLC3

Fig. 1. SPT composition and activity in SPTLC1- and SPTLC2-deficient HAP1 cells. (A) Western blot showing CRISPR/Cas9-mediated loss of SPTLC1 and SPTLC2 expression in the respective HAP1 KO lines. The loss of SPTLC1 led to a concomitant loss of SPTLC2 expression. Calnexin served as a loading control. (B) SPT 15 activity in SPTLC1- and SPTLC2-2 deficient cells compared with HAP1 WT cells. SPT activity was measured by the incorporation of (2,3,3-D3, N)-L-serine into de novo formed LCBs. One deuterium is lost during the conjugation reaction, which results in a mass shift of +3 Da for the de novo formed LCBs. No LCBs were formed in presence of the SPT inhibitor myriocin. (C) Only SPTLC1 expression rescued activity in SPTLC1 KO cells, while expression of either SPTLC2 or SPTLC3 rescued activity in SPTLC2 KO cells. (D) Western blot of reconstituted HAP1 SPTLC1 KO cells. Expression of SPTLC1 restored the endogenous expression of SPTLC2. (E) Immunoprecipitation of V5-tagged SPT proteins in HAP1 WT and SPTLC2 KO cells. The precipitated proteins were separated by SDS-PAGE and analyzed by Western blotting. (F) SPTLC2 and SPTLC3 form a distinct LCB spectrum when expressed in a SPTLC2 null HAP1 background. Bars represent mean ± SEM; n = 3. **P < 0.01, unpaired t test. nd, not detected.

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.2002391117 Lone et al. Downloaded by guest on October 4, 2021 STPLC1-SPTLC2 expressing cells (Fig. 1C). These results suggested To confirm that these LCBs are generated by SPT directly and that SPTLC1–SPTLC3 interaction is independent of SPTLC2. not formed by downstream modifications, we supplemented The physical interaction between the individual SPT subunits SPTLC2 and SPTLC3 overexpressing HEK293 cells with the was confirmed by coimmunoprecipitation assays (Fig. 1E). An- respective (FA) substrates. The addition of myristate +3 tibody precipitation of V5 epitope-tagged constructs of SPTLC1 (C14:0) stimulated C16SO formation in SPTLC3 expressing (SPTLC1-V5) or SPTLC2 (SPTLC2-V5) showed coprecipitation cells only (Fig. 2B). In contrast, pentadeconate (C15:0) stimulated of the respective nontagged SPTLC1 and SPTLC2 subunits. In- C17SO+3 synthesis in both SPTLC2 and SPTLC3 cells, although terestingly, the pull-down of SPTLC3-V5 in SPTLC2 KO cells the levels were significantly higher in SPTLC3 expressing cells coprecipitated SPTLC1, while the pull-down of SPTLC3-V5 in (approximately eightfold relative to SPTLC2 cells) (Fig. 2C). +3 WT cells showed coprecipitation of both SPTLC1 and SPTLC2 The addition of palmitate (C16:0) increased C18SO levels mostly (Fig. 1E). This suggests that the mammalian SPT is a holoen- in SPTLC2- overexpressing cells (Fig. 2D), whereas supplementing +3 zyme formed by the assembly of multiple subunits, which is in heptadecanoic acid (C17:0) stimulated C19SO formation in all +3 line with earlier reports (5). cells (Fig. 2E). Stearate (C18:0) increased C20SO formation in all cell lines, but the overall levels were the highest in SPTLC3- SPT Forms a Variety of LCBs with Different Aliphatic Chain lengths. overexpressing cells, more than threefold higher compared with Next, we analyzed the spectrum of LCBs formed in SPTLC2 KO stearate-treated SPTLC2-expressing cells (Fig. 2F). HAP1 cells expressing SPTLC2 or SPTLC3. Although C18SO+3 was the dominant product formed in SPTLC1-SPTLC2–expressing ssSPTb Stimulates C20 LCB Formation. It has been previously cells, we also observed minor formation of C19SO+3 and C20SO+3 reported that the accessory SPT subunits ssSPTa and ssSPTb (Fig. 1F). In contrast, SPTLC1-SPTLC3–expressing cells formed a modulate the product spectrum of SPT (13). HAP1 cells express greater variety of LCBs ranging from C16SO+3 to C20SO+3 (Fig. ORMDL1-3 and ssSPTa but not ssSPTb (SI Appendix, Fig. S2B). 1F). To exclude that these additional LCBs are artifacts caused by Endogenous ssSPTa and b mRNA levels were not significantly SPTLC3 overexpression, we compared the LCB spectrum between influenced by the expression of SPTLC2 or SPTLC3 (SI Ap- HEK293 and Huh7 cells. Both cell types express SPTLC1 and pendix, Fig. S2B), excluding that the observed shift in the LCB SPTLC2, however Huh7 cells express SPTLC3 as well (SI Appendix, profile for SPTLC2- and SPTLC3-expressing cells is caused by a Fig. S2C). The predominantly formed LCB in either cell type was mutual change in ssSPTa or ssSPTb expression. Similarly, HEK293 C18SO+3.C20SO+3 was also formed in both cell types, although cells also expressed primarily ssSPTa, while ssSPTb expression was +3 the levels were higher in Huh7 cells (Fig. 2A). In contrast, C19SO low and not influenced by the presence of SPTLC3 (SI Appendix, BIOCHEMISTRY was primarily formed in HEK293 cells, whereas C16SO+3 and Fig. S2D). Overexpression of ssSPTa in HEK293 WT cells (SI C17SO+3 were present exclusively in Huh7 cells (Fig. 2A). Appendix,Fig.S2D) had a minor stimulatory effect on C18SO+3

5.0 150 B150 C16SO A HEK293 HEK293 Control 4.0 ** Huh7 Huh7 SPTLC2 SPTLC3 *** cells cells 100 100 3.0 HEK cells 2.0 50 50 1.0 **** pmoles/10 pmoles/10

pmoles/10 ndnd 0 nd nd 0 0 C16SO C17SO C19SO C20SO [LCB] C18SO Vehicle Myristate

C D E 500 C17SO 600 C18SO 150 C19SO Control Control Control * ** 400 SPTLC2 *** SPTLC2 SPTLC2 ** SPTLC3 SPTLC3 SPTLC3 300 400 100 **

HEK cells 200 HEK cells * HEK cells 100 200 * 50 50 6 3 pmoles/10 pmoles/10 0 ndnd 0 pmoles/10 0 Vehicle Pentadecanoate Vehicle Palmitate Vehicle Heptadecanoate

F 10.0 C20SO Control SPTLC2 8.0 *** SPTLC3 6.0 HEK cells 4.0 *** 2.0 **

pmoles/10 0 Vehicle Stearate

Fig. 2. SPTLC3 forms a broader LCB spectrum than SPTLC2. (A) Profile of de novo formed LCBs in HEK293 and Huh7 cells. While SPTLC1 and 2 are expressed by both, Huh7 cells also express SPTLC3. C16SO and C17SO formation was seen only in Huh7 cells, while C18SO, C19SO, and C20SO were formed in both lines. (B–F) LCB formation in SPTLC2- and SPTLC3-overexpressing HEK293 cells after FA supplementation (50 μM): myristate (B), pentadecanoate (C), palmitate (D), 15 heptadecanoate (E), and stearate (F). De novo formed LCBs were determined by the incorporation of (2,3,3-D3, N)-L-serine. For statistical evaluation, ab- solute levels are compared between FA-treated and untreated cells (vehicle) of the same type. Bars represent mean ± SEM; n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, unpaired t test. nd, not detected.

Lone et al. PNAS Latest Articles | 3of8 Downloaded by guest on October 4, 2021 synthesis (Fig. 3A); however, this effect was not observed in novel metabolite as 16-(omega-3-) methyl-branched sphingosine, SPTLC2- or SPTLC3- overexpressing cells that already showed which was confirmed by comparison to a chemically synthesized increased C18SO+3 formation relative to vector-transfected con- 16-methyl-C18SO standard (SI Appendix,Fig.S6). The RT of the trols (Fig. 3A). Overexpressing ssSPTa or ssSPTb (SI Appendix, synthetic standard was identical to that of the endogenously formed Fig. S2D) did not induce synthesis of C16SO+3 or C17SO+3 in LCB, confirming it as an omega-3-methyl-C18SO (meC18SO) SPTLC1-SPTLC2–expressing cells (Fig. 3 B and C). However, by (Fig. 4C). Similar to C16SO and C17SO, the synthesis of meC18SO trend, their formation in SPTLC3-expressing cells appeared to be in SPTLC3-expressing HEK293 cells was only marginally decreased slightly reduced in the presence of ssSPTa and b (Fig. 3 B and C). by ssSPTa or ssSPTb expression (SI Appendix,Fig.S4B). However, +3 On the other hand, ssSPTb expression significantly stimulated the the 80- to 100-fold increase in meC18SO observed in these cells formation of C20SO+3 (Fig. 3D). Although this effect was also after supplementation with ante-mePA (SI Appendix,Fig.S4C) seen in ssSPTb-transfected HEK293 WT cells, it was even stronger indicated that primarily substrate availability limits the formation of for cells coexpressing SPTLC2 or SPTLC3 (Fig. 3D). Over- this branched LCB. expression of ssSPTa had no effect on C20SO+3 formation. In- BCFAs are synthesized from branched-chain amino acids dependent of ssSPTb expression, C20SO+3 formation was generally (BCAAs) such as valine (Val), leucine (Leu), and isoleucine (Ile) higher in SPTLC3-expressing cells, indicating that the presence of (19). Therefore, we supplemented SPTLC3 expressing HEK293 cells with increasing amounts of stable isotope labeled 6[13C]- ssSPTb is not essential, but has a stimulatory effect on the formation 13 of C20 LCBs. isoleucine and 6[ C]-leucine. Since ante-mePA is synthesized from 2-methyl-butyryl-CoA generated early in the Ile catabolic 13 C19 LCBs Are Formed as Two Isomeric Species. In addition to the pathway (20), 5[ C] from the Ile label are expected to be in- LCBs described above, we also observed the formation of an corporated into meC18SO (Fig. 4D). We observed a dose-dependent additional metabolite that was present only in SPTLC3-expressing incorporation of label from Ile, but not from Leu, into the de novo formed meC18SO (Fig. 4E and SI Appendix,Fig.S4D). We also cells (Fig. 4A and SI Appendix, Figs. S3 and S4A). This LCB was 13 13 15 isotope-labeled with D - N-L-serine, and its formation was observed the de novo formation of 3[ C]C17SO in the 6[ C]- 3 – blocked by the SPT inhibitor myriocin (SI Appendix, Fig. S4A). isoleucine supplemented cells (Fig. 4E). This may be explained by This novel LCB was isobaric to C19SO but differed in retention the fact that Ile is terminally catabolized to propionyl-CoA, which time (RT) by 0.42 min (SI Appendix,Fig.S3). Despite the dif- then acts as a three-carbon precursor for the synthesis of odd ference in RT, the fragmentation pattern of this LCB was identical chain fatty acids (reviewed in ref. 19). These results lend further to that of C19SO+3 (SI Appendix,Fig.S5A–C); therefore, we support to the idea that C17SO is formed directly from the odd hypothesized that this metabolite could be a methylated C18 LCB. chain FA pentadecanoate (C15:0). The hypothesis was tested by supplementing SPTLC3-expressing Branched meC18 LCBs Are Incorporated into Ceramides and Complex HEK293 cells with the branched-chain fatty acid (BCFA) iso-or SLs. The de novo formed LCBs are metabolized to ceramides anteiso-methyl-palmitate (iso-mePA; ante-mePA). The addition of (Cer) and further to complex SLs, such as (SM) ante-mePA, but not of iso-mePA or heptadecanoate (C17:0), and . To determine whether meC18SO is also stimulated the synthesis of this LCB (Fig. 4B). This identified the incorporated into complex SLs, we performed a sphingolipido- 15 mic analysis of D3- N-L-serine–supplemented SPTLC2- and SPTLC3-overexpressing HEK293 cells. The shift in RT between +3 +3 C18SO C16SO C19SO and meC18SO was retained for the complex forms, A500 B30 vector vector * which allowed for the chromatographic differentiation among the ssSPTa ssSPTa isobaric SL species. C18SO+3 in Cer and SM was primarily con- 400 ssSPTb ssSPTb 20 jugated to C16:0,C22:0,C24:0,andC24:1 FAs (SI Appendix, Fig. S7 A 300 HEK cells HEK cells and B). For Cer, the relative species abundance increased with * 200 longer N-acyl chains (C24:0 or C24:1; SI Appendix,Fig.S7A); 10 however, for SM, the C16:0 N-acylated species was the most 100 abundant (SI Appendix,Fig.S7B). In contrast, meC18SO+3-based pmoles/10 pmoles/10 0 0 nd Cer (meCer) and SM (meSM) preferentially contained longer N-acyl chains (C22:0,C24:0,andC24:1), while the levels with a WT SPTLC2 SPTLC3 WT SPTLC2 SPTLC3 conjugated C16:0 FA were very low (Fig. 5A). Supplementation with ante-mePA significantly increased meC18SO+3-based SL C D levels in SPTLC3-expressing HEK293 cells, but the N-acylation C17SO * C20SO 16 36 pattern was conserved (SI Appendix,Fig.S7C and D). Surpris- vector * vector *** ssSPTa 30 ssSPTa ingly, among C19SO-derived SLs, C22:0 N-acyl Cer(d19:1/22:0) ssSPTb ssSPTb 12 24 *** was the most abundant (SI Appendix,Fig.S7E and F). Compar- 18 ative analysis (mol%) of detected Cer and SM species revealed HEK cells HEK cells 8 12 *** conspicuous differences in N-acylation patterns of C18SO, 6 meC18SO, and C19SO (Fig. 5B). In addition, for C18SO- and 4 3 meC18SO-based SLs, the total de novo produced SM was three- 1.5 pmoles/10 pmoles/10 fold higher than the total Cer produced from either LCB 0 0 (Fig. 5C); however, for C19SO- based SLs, total Cer were more WT SPTLC2 SPTLC3 WT SPTLC2 SPTLC3 abundant than SM containing this LCB (Fig. 5C).

Fig. 3. LCB formation by SPT is modulated through ssSPTa and ssSPTb. (A) Branched-Chain LCBs in Plasma. Finally, we wanted to examine the ssSPTa induces the formation of C18SO in HEK293 WT but has no effect on extent to which meC18SO is present in plasma. To do so, we synthesis in SPTLC2- and SPTLC3-overexpressing cells. (B and C) The de novo compared the LCB profiles in plasma from mice and humans. formation of C16SO and C17SO is independent of ssSPTa and ssSPTb ex- pression. (D) ssSPTb stimulates the formation of C20SO in SPTLC2- and Total SL levels were significantly lower in mice than in humans. SPTLC3-expressing cells. This stimulatory effect is higher in SPTLC3-expressing In both species, C18SO was the most abundant LCB, followed by cells. Bars represent mean ± SEM; n = 3. *P < 0.05; ***P < 0.001, unpaired C18SAdienine, a dienic downstream product of SO (7) (Fig. 6A). t test. nd, not detected. Among the list of SPTLC3-specific LCBs, C16SO and C17SO

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.2002391117 Lone et al. Downloaded by guest on October 4, 2021 AB5 meC18SO 400 meC18SO C 100 **** 4 vehicle 300 ante-mePA 75 3 iso-mePA HEK cells HAP1 cells 200 heptadecanoate 50 20 2 me18SO

10 Intensity [%] 25 1 C19SO pmoles/10 pmoles/10 0 nd nd 0 0 me18SO Vector +-- 8.07.57.06.56.0 SPTLC2 - +- SPTLC3 SPTLC3 --+ Retention time

DE5[ C]-meC18SO O 4.0 3[ C]-C17SO (CH ) OH OH C 3.0 H N O

6[ C]-Isoleucine 5[ C]-ante-mePA HEK cells 2.0

1.0 OH

CH CH OH pmoles/10 0 CH 0 0.5 2.51.0 [mM] H N 6[ C]-Isoleucine 5[ C]-16-methyl sphingosine

Fig. 4. SPTLC3 activity induces formation of omega-3-methylated LCB (meC18SO). (A) meC18SO is exclusively formed by SPTLC3-expressing SPTLC2 KO HAP1 cells. (B) Formation of meC18SO in SPTLC3-expressing HEK293 cells is specifically enhanced by supplementation (10 μM FA) with ante-mePA and not by iso- mePA or heptadecanoate. (C) De novo formed meC18SO+3 and the chemically synthesized 16-methyl-C18SO standard have the same retention time. (D) Scheme showing the formation of ante-mePA from isotope-labeled 6[13C] isoleucine and its incorporation in omega-3 branched LCB. Filled circles reflect the position of labeled [13C]. (E) Dose-dependent incorporation of isotope-labeled isoleucine in meC18SO in SPTLC3-expressing HEK293 cells. Besides the for- BIOCHEMISTRY mation of 5[13C]-meC18SO, we also observed the formation of 3[13C]-C17SO due to the metabolic conversion of isoleucine into propionyl-CoA. Bars represent mean ± SEM; n = 3. ****P < 0.0001, ANOVA followed by Bonferroni correction. nd, not detected.

were the most abundant in humans and mice, respectively (Fig. of the three SPT core subunits. Expression of SPTLC2 and 6 B and C). Although C20SO was present in both, it was the least SPTLC3 in the SPTLC1 null background did not result in an abundant of the LCBs detected in human plasma (Fig. 6 B and active enzyme, whereas both generated a functional SPT enzyme C). Interestingly, meC18SO was detected only in human plasma in presence of SPTLC1 (Fig. 1C). Interestingly, the deletion of and was absent in mouse plasma (Fig. 6 B and C). SPTLC1 caused a concomitant loss of SPTLC2, whereas SPTLC1 SLs are mostly carried by low- and high-density lipoproteins levels were essentially unchanged when SPTLC2 was deleted (LDL and HDL, respectively) (21); therefore, we determined the (Fig. 1A). This confirms earlier reports showing that SPTLC2 is LCB profiles in purified human LDL and HDL fractions (Fig. degraded in the absence of SPTLC1 even though mRNA levels 6D). LDL contained >70% of C18SO and SAdienine (Fig. 6E). are not altered (22). Immunoprecipitation showed that both Moreover, C19 and C20SO, as well as the SPTLC3-specific SPTLC2 and SPTLC3 interact with SPTLC1 independently. LCBs C16, C17, and meC18SO, were relatively more abundant SPTLC3 coprecipitated with SPTLC1 and SPTLC2 in WT HAP1 in LDL (Fig. 6F) but were also present in HDL. cells but also precipitated with SPTLC1 in the absence of SPTLC2 (Fig. 1E). Similarly, antibody precipitation of ectopically expressed Discussion SPTLC2-V5 coeluted with untagged (endogenous) SPTLC2 (Fig. SPT is an essential enzyme in mammals, and its deficiency is 1E). This points to a higher-order structure for the SPT complex embryonically lethal. Here we investigated the function of each and suggests that the individual SPTLC2 and SPTLC3 subunits

A meC18SO based SL B C 15 **** 120 C24:1 5 **** 100 C24:0 **** C22:0 4 **** 10 80 C16:0 ** 3 HEK cells 60 **** 2 5 ** 40

* Ratio [Cer/SM] 20 1 pmoles/10 0 N -acyl species [mol%] 0 0 [N-acyl [LCB] [LCB] 16:0 22:0 24:0 24:1 16:0 22:0 24:0 24:1 C C C C C C C C length] C18SOC19SO C18SOC19SO C19SO C18SO Cer SM meC18SO meC18SO meC18SO Cer SM

Fig. 5. meC18SO is efficiently incorporated into ceramides and sphingomyelins. (A) The profile of de novo formed Cer and SM species containing meC18SO. The LCB is preferably N-acylated with longer FA chains. (B) The relative N-acyl distribution for Cer and SM differs for meC18SO-, C19SO-, and C18SO-based SL species. The de novo formed species are compared (mol%) between WT and SPTLC3-expressing HEK293 cells. (C) Total Cer:SM ratio indicating that C19SO is retained in Cer, whereas meC18SO and C18SO are efficiently converted to SM. Bars represent mean ± SEM; n = 3. *P < 0.05; **P < 0.01; ****P < 0.0001, ANOVA followed by Bonferroni correction.

Lone et al. PNAS Latest Articles | 5of8 Downloaded by guest on October 4, 2021 Human Mice ABC80 Human 15 1.5 Mice 60 *** 10 1.0 40 *** 5 0.5 20 LCB levels [µM] LCB levels [µM] LCB levels [µM] 0 0 0 nd

C18SO C16SOC17SOC19SO C20SO C16SOC17SOC19SO C20SO me18SO SAdienine meC18SO

D E F 75 LDL 15 **** LDL **** [kDa] LDL HDL HDL HDL *** ApoB 250 50 10

70 *** 25 **** 5 **** 25 ApoA *** µmoles/µgram protein 0 µmoles/µgram protein 0

C18SO C16SO C17SO C19SO C20SO SAdienine meC18SO

Fig. 6. LCB profile in human and mouse plasma and in isolated human fractions. (A–C) The most abundant LCB in mouse and human plasma was C18SO, followed by SAdienine. meC18SO was detected in human plasma. (D) SDS gel of the purified LDL and HDL fractions (Coomassie blue-stained). (E and F) LCB analysis from purified LDL and HDL fractions. Bars represent mean ± SEM; n = 4. ***P < 0.001; ****P < 0.0001, multiple t test using Bonferroni–Dunn correction. nd, not detected.

can replace each other within this structure. In this respect, In addition to the LCBs formed from straight-chain saturated SPTLC1 might be important to anchor the enzyme to the endo- fatty acids, we identified a previously undescribed methyl- plasmic reticulum through its N-terminal membrane-binding do- branched LCB exclusively formed by SPTLC3 (Fig. 4A and SI main. In yeast, the N-terminal domain of SPTLC1 mediates Appendix,S4A and B). Through MS fragmentation, metabolic binding to Orm proteins, which are integral membrane proteins as labeling, and comparison with a chemically synthesized standard, well (23, 24). As SPTLC3 expression is specific to certain tissues, the LCB was identified as meC18SO (SI Appendix, Fig. S5 and the composition of the SPT complex might be altered in response Fig. 4 B–E). We showed that this branched LCB is metabolized to the relative tissue expression levels of SPTLC1, 2, and 3. Such to complex SLs, although we observed significant differences in concentration-dependent substitutions within complexes have also the N-acyl profiles for C18SO-, C19SO-, and meC18SO-containing been described for some sequence-neutral DNA-binding proteins (25). Cer and SM (Fig. 5 A and B and SI Appendix,Fig.S7A–F). All In addition to C18 LCBs, SPTLC1-2–expressing cells also lipids were formed within the same cell type and thus on exactly synthesize C19 and C20 LCBs (Fig. 1F). However, in the pres- the same background of synthase (CerS) isoforms. This ence of SPTLC3, cells formed a broader spectrum of odd and suggests that not only the spectrum of expressed CerS , but even LCBs, ranging from C16 to C20 (Figs. 1F and 2 A–F). The alsothetypeofLCBmightinfluencetheN-acyl spectrum of Cer overexpression of ssSPTa mildly stimulated C18 LCB synthesis in and complex SLs. WT cells but had no effect in SPTLC2- or SPTLC3-overexpressing Interestingly, the LCBs formed by either SPTLC2 or SPTLC3 cells, which already show increased SPT activity compared with do not follow a clear continuum. SPTLC2 showed the highest WT cells (Fig. 3A). However, ssSPTb overexpression had a sig- activity with palmitate (C16:0), followed by heptadecanoate (C17:0), nificant effect on the synthesis of C20SO. Compared with WT cells, and only minor activity was seen with stearate (C18:0)(Fig.2D–F). basal C20SO synthesis was increased in SPTLC2-overexpressing In contrast, SPTLC3 had the highest activity with ante-mePA, and even more so in SPTLC3-overexpressing HEK293 cells, followed by pentadecanoate (C17:0) and myristate (C14:0)(SI Ap- while coexpression of ssSPTb had an additional stimulatory effect pendix, Fig. S4C and Fig. 2 C and B). This differs from earlier on C20SO synthesis (Fig. 3D). The stimulation of C20SO synthesis in vitro data indicating a direct association of SPT activity with the by ssSPTb expression was more pronounced than stearate sup- length of the FA substrate (relative to palmitate) (3). Therefore, plementation (Figs. 2F and 3D). This indicates that C20SO for- cellular SPT activity seems to depend on additional factors, such mation is regulated beyond the level of substrate availability. As as the microenvironment, lost when structural integrity of the cell stearate is one of the most abundant FAs, and accumulation of is destroyed. C20SO (or of SL derived from it) is neurotoxic (14), such regu- Among all tested FA substrates, SPTLC3 showed the highest lation might be necessary to maintain physiological levels of this activity with the BCFA ante-mePA (Fig. 2 B–F and SI Appendix, important LCB. Fig. S4C). BCFAs are formed through the of BCAAs,

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.2002391117 Lone et al. Downloaded by guest on October 4, 2021 and we found that de novo formed meC18SO incorporated Immunoprecipitation Assays. Cells were harvested by scraping in ice-cold isotope-labeled Ile but not Leu (Fig. 4E and SI Appendix, Fig. extraction buffer (Hepes-NaOH 50 mM, pH 7.4) containing potassium ace- S4D). Suppression of BCAA catabolism drives the development tate (150 mM), magnesium acetate (2 mM), calcium chloride (1 mM), and and progression of hepatic , and their hepatic accumula- glycerol (15%) without detergent. After centrifugation, cell pellets were tion correlates with tumor multiplicity and size in mice (26). A resuspended in extraction buffer containing Triton-X-100 (0.5%) and in- cubated on ice for 45 min, followed by centrifugation at 13,000 rpm at 4 °C. high- diet induces hepatic expression of SPTLC3, but not of Buffer-equilibrated Sepharose beads (40 μL) conjugated to mouse anti-V5 SPTLC1 or SPTLC2 in mice, which is correlated with the de- antibody were added, followed by incubation for 1.5 h at 4 °C with agita- velopment of hepatocarcinoma (27, 28). High plasma BCAA tion. Beads were collected by centrifugation and washed three times with levels are also found in other metabolic conditions and consid- extraction buffer (1 mL). Finally, the bound protein was specifically eluted by ered to be predictive markers for and type 2 competition with V5-peptide (Sigma-Aldrich, 2 μg/mL) for 15 min at room (T2DM) (29, 30). Given the presence of meC18SO in temperature, and the beads removed by centrifugation (1,800 rpm for human plasma (Fig. 6B), it would be interesting to test whether 5 min). After addition of 5× SDS loading buffer to the supernatant, proteins plasma meC18SO levels are altered in patients with hepatic were separated by SDS-PAGE, and coprecipitating proteins were detected by cancer or T2DM. Western blot analysis with anti-V5 and protein-specific antibodies. The noncanonical SPTLC3-derived LCBs are relatively low in plasma (Fig. 6 A and B). We cannot exclude the possibility that Isotope Labeling Assay. The L-serine labeling assay and SPT activity mea- surements were performed as described previously (38). Cells were grown to diet and microbiota contribute to the LCB profile in plasma as 70% confluence in DMEM growth media. For labeling, the media was ex- well. However, the strong stimulatory effect of FA supplemen- changed to L-serine–free DMEM (Genaxxon Bioscience) containing 10% FBS, 15 tation on LCBs formed by SPTLC3 (in particular for meC18SO) 1% penicillin/streptomycin, and isotope-labeled D3- N-L-serine (1 mM) indicates that primarily substrate availability limits the formation (Cambridge Isotope Laboratories). Cells were grown for another 16 h in the of these LCBs in SPTLC3-expressing cells. Several genome-wide labeling media. Inhibitors (when used) were added together with the la- association studies have implicated variants in the SPTLC3 ge- beling media. Fatty acid supplementation was performed by adding the nomic locus in altered plasma lipids (31–33) and also in LDL respective FA (10 or 50, μM as indicated) in labeling media for the duration levels (34). Polymorphisms at an intergenic locus in of the assay. For lipid analysis, cells were harvested on ice and frozen after SPTLC3 influence plasma Cer levels and the risk for cardio- counting (Z2 Coulter Counter; Beckman Coulter). vascular disease (35). We also detected SPTLC3-derived SLs in LDL and HDL fractions isolated from human plasma (Fig. 6F), Lipid Analysis. Sphingoid base extraction from frozen cell pellets or plasma (100 μL) was performed as described previously (38). Hydrolyzed lipids were even though the physiological relevance of this association re- resuspended in 200 μL of reconstitution buffer (70% methanol, 10 mM BIOCHEMISTRY mains unclear. Furthermore, the greatest variety of LCBs have ammonium acetate, pH 8.5). LCBs were separated via a reverse-phase C18 been reported from skin (36), which is also one of the tissues column (Uptisphere, 120 Å, 5 μm, 125 × 2 mm; Interchim) connected to a with the highest SPTLC3 expression (1). Here SPTLC3 and FA QTRAP 6500+ LC-MS/MS System (Sciex). For chromatography, a binary sol- metabolism might have coevolved to maintain skin-specific func- vent system consisting of solvent A (50% methanol, 10 mM ammonium tions, such as the water permeability barrier. In addition, Ile is formate, 0.2% formic acid) and solvent B (100% methanol) at a constant efficiently metabolized to anteiso-fatty acids in skin (37), indicating flow rate of 0.6 mL/min was used. The column was equilibrated with 30% a possible role for meC18SO-derived SLs in this tissue. solvent B, and a linear gradient to 50% B was run over 9 min. The concen- In conclusion, we have shown that SPT forms a variety of tration of solvent B was increased to 100% over 0.5 min. After 2 min at different LCB structures, particularly when the SPTLC3 subunit 100% solvent B, the column was equilibrated using 30% solvent B for 1.5 min. Sample ionization was achieved via electrospray ionization in positive is present. This, in combination with the variability of the N-acyl ion mode. chain and the diversity of conjugated head group structures, in- Alternatively, dried lipid extracts were dissolved in 75 μL of derivatization creases the already vast number of potential SL species in mam- mix (methanol/ethanol/H2O, 85:55:15 [vol/vol/vol]), and 5 μL of ortho- mals. To obtain a comprehensive picture of SL metabolism, the phthalaldehyde (OPA) working solution (990 μL of boric acid, 10 μLof inclusion of noncanonical SL will be important for future lip- OPA [50 mg/mL in EtOH] and 1.5 μLofβ-mercaptoethanol) was added. idomic and metabolic studies. This will help unveil the physio- Samples were separated via the reverse-phase C18 column (Uptisphere logical and pathophysiological relevance of SPTLC3 and of 120 Å, 5 μm, 125 × 2 mm) connected to a Q-Exactive MS analyzer (Thermo noncanonical SL species in mammals. Fisher Scientific). Solvent A (50% methanol, 2.5 mM ammonium acetate) and solvent B (100% methanol) at a constant flow rate of 0.3 mL per min were Materials and Methods used. The column was equilibrated in 50% solvent B, and lipids were eluted Cell Lines, Cell Culture, and Transfections. HAP1 cells were cultured in Iscove’s with a linear gradient to 100% solvent B (25 min), followed by 100% solvent modified Dulbecco’s medium (Thermo Fisher Scientific) supplemented with B (5 min) and then reequilibration at 50% solvent B (5 min). Atmospheric 10% FBS (Thermo Fisher Scientific; FSA15-043), 4 mM L-, and 1% pressure chemical ionization was in positive ion mode (39, 40). penicillin/streptomycin. HEK293 and Huh7 cells were cultured in Dulbecco’s For lipidomics profiling, frozen cell pellets were resuspended in 50 μLof modified Eagle’s medium (DMEM; Sigma-Aldrich) with 10% FCS. Cells were PBS and extracted with 1 mL of methanol/methyl-tert-butyl ether/chloro-

grown at 37 °C in a 5% CO2 atmosphere. HAP1 SPTLC1/SPTLC2 KO cells were form (MMC) (4:3:3 [vol/vol/vol]) containing D7SA (d18:0), D7SO (d18:1), dhCer generated by a commercial service (Horizon Discovery). The introduced (d18:0/12:0), ceramide (d18:1/12:0), glucosylceramide (d18:1/8:0), SM (d18:1/ frame-shift mutation resulting in a premature stop was confirmed by 18:1 [D9]), and D7-S1P. After brief vortexing, the samples were mixed con- sequencing. tinuously (1,400 rpm for 30 min) in a Thermomixer (Eppendorf) at 37 °C. The Standard molecular biology techniques were used for generation of all single-phase supernatant was collected, dried under N2, and dissolved in plasmid constructs used in the study. Transient plasmid transfections were 100 μL of methanol/isopropanol (1/1). Untargeted lipid analysis was per- performed with Lipofectamine 3000 (Thermo Fisher Scientific) for HAP1 WT formed on a high-resolution Q-Exactive MS analyzer (Thermo Scientific) as and SPTLC1 and 2 deletion cells or Viromer yellow (Lipocalyx) for HEK293 described previously (41). cells, according to the supplier’s protocol. Transgenic HEK293 cell lines were selected for growth in DMEM containing 400 μg/mL Geneticin (Thermo Chemical Synthesis. The synthesis of meC18SO was carried out in eight con- Fisher Scientific). secutive steps (SI Appendix, Fig. S6) from a previously described intermediate (42) and in analogy to the synthetic schemes for deoxy- (43). Western Blot Analysis. Total protein was extracted from frozen cell pellets Full synthetic details will be soon reported elsewhere. using HET lysis buffer (50 mM Hepes pH 8.0, 1 mM EDTA, 0.2% Triton-X-100). Lysates containing 50 μg of protein were separated by sodium dodecyl Isolation of LDL and HDL. LDL (1.006 < d <1.063 g/mL) and HDL (1.063 < d < sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and, after blotting 1.21 g/mL) were isolated from fresh human plasma as described previously onto a PVDF membrane, detected with either tag-specific or protein-specific (44, 45). Isolated fractions were quantified for protein amounts and aliquots (SPTLC1, SPTLC2) polyclonal antibodies. separated by SDS-PAGE (10%) for quality assurance. The fractions were

Lone et al. PNAS Latest Articles | 7of8 Downloaded by guest on October 4, 2021 stored at −20 °C. Lipoprotein particle fractions amounting to 200 μgof Data Availability Statement. All data discussed in this study are included in the protein were aliquoted and processed for lipid extraction and LCB analysis. main text and SI Appendix.

Statistical Analysis. Data are expressed as mean ± SEM. Statistical evaluation ACKNOWLEDGMENTS. We thank Jeannette Fries and Irina Alecu for was performed using Student’s t test for unpaired data, a multiple t test critically reviewing the manuscript. Financial support for this work was using Bonferroni–Dunn correction, or one-way ANOVA with Bonferroni provided by the Swiss National Foundation (Projects 31003A_153390 and correction. P values < 0.05 were considered statistically significant. Statistical 31003A_179371), the Novartis Foundation (Project 18B081), and the Funda- analyses were performed with Prism 8.0 (GraphPad Software). ção para a Ciência e Tecnologia (Project PTDC/BBB-BQB/3710/2014).

1. T. Hornemann, S. Richard, M. F. Rütti, Y. Wei, A. von Eckardstein, Cloning and initial 22. K. Hanada, Serine palmitoyltransferase, a key enzyme of metabolism. characterization of a new subunit for mammalian serine-palmitoyltransferase. J. Biol. Biochim. Biophys. Acta 1632,16–30 (2003). Chem. 281, 37275–37281 (2006). 23. G. Han et al., The ORMs interact with transmembrane domain 1 of Lcb1 and regulate 2. B. Weiss, W. Stoffel, Human and murine serine-palmitoyl-CoA transferase: Cloning, serine palmitoyltransferase oligomerization, activity, and localization. Biochim. Biophys. expression and characterization of the key enzyme in sphingolipid synthesis. Eur. Acta Mol. Cell Biol. Lipids 1864,245–259 (2019). J. Biochem. 249, 239–247 (1997). 24. L. Hjelmqvist et al., ORMDL proteins are a conserved new family of endoplasmic re- 3. A. H. Merrill Jr., D. W. Nixon, R. D. Williams, Activities of serine palmitoyltransferase ticulum membrane proteins. Genome Biol. 3, RESEARCH0027 (2002). (3-ketosphinganine synthase) in microsomes from different rat tissues. J. Lipid Res. 26, 25. J. S. Graham, R. C. Johnson, J. F. Marko, Concentration-dependent exchange accel- 617–622 (1985). erates turnover of proteins bound to double-stranded DNA. Nucleic Acids Res. 39, 4. T. Hornemann et al., The SPTLC3 subunit of serine palmitoyltransferase generates 2249–2259 (2011). short-chain sphingoid bases. J. Biol. Chem. 284, 26322–26330 (2009). 26. R. E. Ericksen et al., Loss of BCAA catabolism during carcinogenesis enhances mTORC1 5. T. Hornemann, Y. Wei, A. von Eckardstein, Is the mammalian serine palmitoyl- activity and promotes tumor development and progression. Cell Metab. 29, – transferase a high-molecular-mass complex? Biochem. J. 405, 157–164 (2007). 1151 1165 e6 (2019). 6. S. B. Russo, R. Tidhar, A. H. Futerman, L. A. Cowart, Myristate-derived d16:0 sphin- 27. R. Cinar et al., Hepatic -1 receptors mediate diet-induced insulin re- golipids constitute a cardiac sphingolipid pool with distinct synthetic routes and sistance by increasing de novo synthesis of long-chain ceramides. Hepatology 59, – functional properties. J. Biol. Chem. 288, 13397–13409 (2013). 143 153 (2014). 7. G. Karsai et al., FADS3 is a delta14Z sphingoid base desaturase that contributes to 28. Y. Yoshimine et al., Hepatic expression of the Sptlc3 subunit of serine palmitoyl- gender differences to the human plasma sphingolipidome. J. Biol. Chem. 295,1889–1897 transferase is associated with the development of hepatocellular carcinoma in a – (2019). mouse model of nonalcoholic steatohepatitis. Oncol. Rep. 33, 1657 1666 (2015). 8. S. T. Pruett et al., Biodiversity of sphingoid bases (“sphingosines”) and related amino 29. S. Gancheva, T. Jelenik, E. Álvarez-Hernández, M. Roden, Interorgan metabolic – alcohols. J. Lipid Res. 49, 1621–1639 (2008). crosstalk in human insulin resistance. Physiol. Rev. 98, 1371 1415 (2018). 9. S. Han, M. A. Lone, R. Schneiter, A. Chang, Orm1 and Orm2 are conserved endo- 30. C. J. Lynch, S. H. Adams, Branched-chain amino acids in metabolic signalling and in- sulin resistance. Nat. Rev. Endocrinol. 10, 723–736 (2014). plasmic reticulum membrane proteins regulating lipid homeostasis and protein 31. A. A. Hicks et al., Genetic determinants of circulating sphingolipid concentrations in quality control. Proc. Natl. Acad. Sci. U.S.A. 107, 5851–5856 (2010). European populations. PLoS Genet. 5, e1000672 (2009). 10. D. K. Breslow et al., Orm family proteins mediate sphingolipid homeostasis. Nature 32. T. Illig et al., A genome-wide perspective of genetic variation in human metabolism. 463, 1048–1053 (2010). Nat. Genet. 42, 137–141 (2010). 11. K. Gable, H. Slife, D. Bacikova, E. Monaghan, T. M. Dunn, Tsc3p is an 80- 33. A. Demirkan et al.; DIAGRAM Consortium; CARDIoGRAM Consortium; CHARGE protein associated with serine palmitoyltransferase and required for optimal enzyme Consortium; EUROSPAN consortium, Genome-wide association study identifies novel activity. J. Biol. Chem. 275, 7597–7603 (2000). loci associated with circulating phospho- and sphingolipid concentrations. PLoS 12. F. M. Roelants, D. K. Breslow, A. Muir, J. S. Weissman, J. Thorner, Protein kinase Ypk1 Genet. 8, e1002490 (2012). phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeo- 34. C. J. Willer et al.; Global Lipids Genetics Consortium, Discovery and refinement of loci stasis in . Proc. Natl. Acad. Sci. U.S.A. 108, 19222–19227 associated with lipid levels. Nat. Genet. 45, 1274–1283 (2013). (2011). 35. R. Tabassum et al.; FinnGen Project, Genetic architecture of human plasma lipidome 13. G. Han et al., Identification of small subunits of mammalian serine palmitoyl- and its link to cardiovascular disease. Nat. Commun. 10, 4329 (2019). transferase that confer distinct acyl-CoA substrate specificities. Proc. Natl. Acad. Sci. 36. M. K. Lai et al., Biological effects of naturally occurring sphingolipids, uncommon – U.S.A. 106, 8186 8191 (2009). variants, and their analogs. Neuromolecular Med. 18 , 396–414 (2016). 14. L. Zhao et al., Elevation of 20-carbon long chain bases due to a mutation in serine 37. H. Oku, N. Yagi, J. Nagata, I. Chinen, Precursor role of branched-chain amino acids in palmitoyltransferase small subunit b results in . Proc. Natl. Acad. the of iso and anteiso fatty acids in rat skin. Biochim. Biophys. Acta 1214, – Sci. U.S.A. 112, 12962 12967 (2015). 279–287 (1994). 15. A. N. Kimberlin et al., Arabidopsis 56-amino acid serine palmitoyltransferase- 38. A. Zhakupova et al., ORMDL3 expression levels have no influence on the activity of interacting proteins stimulate sphingolipid synthesis, are essential, and affect myco- serine palmitoyltransferase. FASEB J. 30, 4289–4300 (2016). – toxin sensitivity. Plant Cell 25, 4627 4639 (2013). 39. A. Othman et al., Plasma deoxysphingolipids: A novel class of biomarkers for the 16. J. T. Hannich, D. Mellal, S. Feng, A. Zumbuehl, H. Riezman, Structure and conserved metabolic syndrome? Diabetologia 55, 421–431 (2012). function of iso-branched sphingoid bases from the nematode Caenorhabditis ele- 40. H. Bode et al., HSAN1 mutations in serine palmitoyltransferase reveal a close gans. Chem. Sci. 8, 3676–3686 (2017). structure-function-phenotype relationship. Hum. Mol. Genet. 25, 853–865 (2016). 17. P. Essletzbichler et al., Megabase-scale deletion using CRISPR/Cas9 to generate a fully 41. G. Karsai et al., DEGS1-associated aberrant sphingolipid metabolism impairs nervous haploid human cell line. Genome Res. 24, 2059–2065 (2014). system function in humans. J. Clin. Invest. 129, 1229–1239 (2019). 18. H. Ikushiro, S. Fujii, Y. Shiraiwa, H. Hayashi, Acceleration of the substrate Calpha 42. E. M. Saied, S. Banhart, S. E. Bürkle, D. Heuer, C. Arenz, A series of ceramide analogs deprotonation by an analogue of the second substrate palmitoyl-CoA in serine pal- modified at the 1-position with potent activity against the intracellular growth of mitoyltransferase. J. Biol. Chem. 283, 7542–7553 (2008). Chlamydia trachomatis. Future Med. Chem. 7, 1971–1980 (2015). 19. M. M. Adeva-Andany, L. López-Maside, C. Donapetry-García, C. Fernández-Fernández, C. 43. E. M. Saied, T. L. Le, T. Hornemann, C. Arenz, Synthesis and characterization of some Sixto-Leal, Enzymes involved in branched-chain amino acid metabolism in humans. atypical sphingoid bases. Bioorg. Med. Chem. 26, 4047–4057 (2018). Amino Acids 49,1005–1028 (2017). 44. M. C. Camus, M. J. Chapman, P. Forgez, P. M. Laplaud, Distribution and character- 20. M. Wallace et al., Enzyme promiscuity drives branched-chain in ization of the serum lipoproteins and apoproteins in the mouse, Mus musculus. adipose tissues. Nat. Chem. Biol. 14, 1021–1031 (2018). J. Lipid Res. 24, 1210–1228 (1983). 21. J. Iqbal, M. T. Walsh, S. M. Hammad, M. M. Hussain, Sphingolipids and lipoproteins in 45. L. Rohrer et al., Binding, internalization and transport of apolipoprotein A-I by vas- health and metabolic disorders. Trends Endocrinol. Metab. 28, 506–518 (2017). cular endothelial cells. Biochim. Biophys. Acta 1761, 186–194 (2006).

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