Computational design and molecular dynamics simulations suggest the mode of substrate binding in ceramide synthases.

Until now, membrane-protein stabilization has relied on iterations of mutations and screening. We now validate a one-step algorithm, mPROSS, for stabilizing membrane proteins directly from an AlphaFold2 model structure. Applied to the lipid-generating enzyme, ceramide synthase, 37 designed mutations lead to a more stable form of human CerS2. Together with molecular dynamics simulations, we propose a pathway by which substrates might be delivered to the ceramide synthases.

The Complex Tail of Circulating Sphingolipids in Atherosclerosis and Cardiovascular Disease.

Sphingolipids (SLs) are critical players in a number of cellular processes and have recently been implicated in a large number of human diseases, including atherosclerosis and cardiovascular disease (CVD). SLs are generated intracellularly in a stepwise manner, starting with the generation of the sphingoid long chain base (LCB), followed by N-acylation of the LCB to form ceramide, which can be subsequently metabolized to sphingomyelin and glycosphingolipids. Fatty acids, which are taken up by cells prior to their activation to fatty acyl-CoAs, are used in 2 of these enzymatic steps, including by ceramide synthases, which use fatty acyl-CoAs of different chain lengths to generate ceramides with different N-acyl chain lengths. Recently, alterations in plasma ceramides with specific N-acyl chain lengths and degrees of saturation have emerged as novel biomarkers for the prediction of atherosclerosis and overall cardiovascular risk in the general population. We briefly review the sources of plasma SLs in atherosclerosis, the roles of SLs in CVD, and the possible use of the "ceramide score" as a prognostic marker for CVD.

Ceramide synthase 2 deletion decreases the infectivity of HIV-1.

The lipid composition of HIV-1 virions is enriched in sphingomyelin (SM), but the roles that SM or other sphingolipids (SLs) might play in the HIV-1 replication pathway have not been elucidated. In human cells, SL levels are regulated by ceramide synthase (CerS) enzymes that produce ceramides, which can be converted to SMs, hexosylceramides, and other SLs. In many cell types, CerS2, which catalyzes the synthesis of very long chain ceramides, is the major CerS. We have examined how CerS2 deficiency affects the assembly and infectivity of HIV-1. As expected, we observed that very long chain ceramide, hexosylceramide, and SM were reduced in CerS2 knockout cells. CerS2 deficiency did not affect HIV-1 assembly or the incorporation of the HIV-1 envelope (Env) protein into virus particles, but it reduced the infectivites of viruses produced in the CerS2-deficient cells. The reduced viral infection levels were dependent on HIV-1 Env, since HIV-1 particles that were pseudotyped with the vesicular stomatitis virus glycoprotein did not exhibit reductions in infectivity. Moreover, cell-cell fusion assays demonstrated that the functional defect of HIV-1 Env in CerS2-deficient cells was independent of other viral proteins. Overall, our results indicate that the altered lipid composition of CerS2-deficient cells specifically inhibit the HIV-1 Env receptor binding and/or fusion processes.

Different rates of flux through the biosynthetic pathway for long-chain versus very-long-chain sphingolipids.

The backbone of all sphingolipids (SLs) is a sphingoid long-chain base (LCB) to which a fatty acid is N-acylated. Considerable variability exists in the chain length and degree of saturation of both of these hydrophobic chains, and recent work has implicated ceramides with different LCBs and N-acyl chains in distinct biological processes; moreover, they may play different roles in disease states and possibly even act as prognostic markers. We now demonstrate that the half-life, or turnover rate, of ceramides containing diverse N-acyl chains is different. By means of a pulse-labeling protocol using stable-isotope, deuterated free fatty acids, and following their incorporation into ceramide and downstream SLs, we show that very-long-chain (VLC) ceramides containing C24:0 or C24:1 fatty acids turn over much more rapidly than long-chain (LC) ceramides containing C16:0 or C18:0 fatty acids due to the more rapid metabolism of the former into VLC sphingomyelin and VLC hexosylceramide. In contrast, d16:1 and d18:1 ceramides show similar rates of turnover, indicating that the length of the sphingoid LCB does not influence the flux of ceramides through the biosynthetic pathway. Together, these data demonstrate that the N-acyl chain length of SLs may not only affect membrane biophysical properties but also influence the rate of metabolism of SLs so as to regulate their levels and perhaps their biological functions.

The role of ceramide in regulating endoplasmic reticulum function.

Sphingolipids (SLs) are an important class of membrane lipids containing a long chain sphingoid base backbone. SL synthesis is compartmentalized between two major cell organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. The initial steps of sphingolipid synthesis take place in the ER, where the simplest SL, ceramide, is synthesized. Although ceramide is a critical membrane component, an imbalance of ceramide levels can have significant deleterious effects on cell properties leading to events such as apoptosis. For this reason and others, ER ceramide levels must be tightly regulated. Here, we describe the biological and biophysical properties of ceramide and discuss how this might impact the ER membrane. This article is part of a special issue entitled: ER Platforms for Membrane Lipid Dynamics.

A Stroll Down the CerS Lane.

The majority of enzymes in the sphingolipid (SL) biosynthetic pathway have been identified over the past couple of decades. Despite significant work, and despite their crucial and central roles in SL synthesis, significant information is still lacking concerning the enzymes that catalyze the N-acylation of sphingoid long chain bases, namely the ceramide synthases (CerS), a family of six mammalian genes originally named longevity assurance (Lass) genes. Each of these six endoplasmic reticulum (ER) membrane-bound enzymes utilizes a relatively restricted sub-set of fatty acyl-CoAs for N-acylation, but are far more promiscuous about the use of long chain bases. The reason that mammals and other species have multiple CerS, generating a specific subset of ceramides, is not yet known, but implies an important role for ceramides containing specific fatty acids in cell physiology. In this brief chapter, we will stroll down the CerS lane and discuss what is known, and what is not known, about this important enzyme family.

Eleven residues determine the acyl chain specificity of ceramide synthases.

Lipids display large structural complexity, with ∼40,000 different lipids identified to date, ∼4000 of which are sphingolipids. A critical factor determining the biological activities of the sphingolipid, ceramide, and of more complex sphingolipids is their N-acyl chain length, which in mammals is determined by a family of six ceramide synthases (CerS). Little information is available about the CerS regions that determine specificity toward different acyl-CoA substrates. We previously demonstrated that substrate specificity resides in a region of ∼150 residues in the Tram-Lag-CLN8 domain. Using site-directed mutagenesis and biochemical analyses, we now narrow specificity down to an 11-residue sequence in a loop located between the last two putative transmembrane domains (TMDs) of the CerS. The specificity of a chimeric protein, CerS5(299-309→CerS2), based on the backbone of CerS5 (which generates C16-ceramide), but containing 11 residues from CerS2 (which generates C22-C24-ceramides), was altered such that it generated C22-C24 and other ceramides. Moreover, a chimeric protein, CerS4(291-301→CerS2), based on CerS4 (which normally generates C18-C22 ceramides) displayed significant activity toward C24:1-CoA. Additional data supported the notion that substitutions of these 11 residues alter the specificities of the CerS toward their cognate acyl-CoAs. Our findings may suggest that this short loop may restrict adjacent TMDs, leading to a more open conformation in the membrane, and that the CerS acting on shorter acyl-CoAs may have a longer, more flexible loop, permitting TMD flexibility. In summary, we have identified an 11-residue region that determines the acyl-CoA specificity of CerS.