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Ceramides are bioactive sphingolipids that support the structure of the plasma membrane and mediate numerous cell-signaling events in eukaryotic cells. The finding that ceramides act as second messengers transducing cellular signals has attracted substantial attention in several fields of Biology. Since all cells contain lipid plasma membranes, the impact of various ceramides, ceramide synthases, ceramide metabolites, and other sphingolipids has been implicated in a vast range of cellular functions including, migration, proliferation, response to external stimuli, and death. The roles of lipids in these functions widely differ among the diverse cell types. Herein, we discuss the roles of ceramides and other sphingolipids in mediating the function of various immune cells; particularly dendritic cells, neutrophils, and macrophages. In addition, we highlight the main studies describing effects of ceramides in inflammation, specifically in various inflammatory settings including insulin resistance, graft-versus-host disease, immune suppression in cancer, multiple sclerosis, and inflammatory bowel disease.

Despite their relative simplicity, iron-sulfur clusters have been omnipresent as cofactors in myriad cellular processes such as oxidative phosphorylation and other respiratory pathways. Recent research advances confirm the presence of different clusters in enzymes involved in nucleic acid metabolism. Iron-sulfur clusters can therefore be considered hallmarks of cellular metabolism. Helicases, nucleases, glycosylases, DNA polymerases and transcription factors, among others, incorporate various types of clusters that serve differing roles. In this chapter, we review our current understanding of the identity and functions of iron-sulfur clusters in DNA and RNA metabolizing enzymes, highlighting their importance as regulators of cellular function.

Mitochondria and bacteria share a myriad of properties since it is believed that the powerhouses of the eukaryotic cell have evolved from a prokaryotic origin. Ribosomal RNA sequences, DNA architecture and metabolism are strikingly similar in these two entities. Proteins and nucleic acids have been a hallmark for comparison between mitochondria and prokaryotes. In this chapter, similarities (and differences) between mitochondrial and prokaryotic membranes are addressed with a focus on structure-function relationship of different lipid classes. In order to be suitable for the theme of the book, a special emphasis is reserved to the effects of bioactive sphingolipids, mainly ceramide, on mitochondrial membranes and their roles in initiating programmed cell death.

Iron-sulfur metabolism is essential for cellular function and is a key process in mitochondria. In this review, we focus on the structure and assembly of mitochondrial iron-sulfur clusters and their roles in various metabolic processes that occur in mitochondria. Iron-sulfur clusters are crucial in mitochondrial respiration, in which they are required for the assembly, stability, and function of respiratory complexes I, II, and III. They also serve important functions in the citric acid cycle, DNA metabolism, and apoptosis. Whereas the identification of iron-sulfur containing proteins and their roles in numerous aspects of cellular function has been a long-standing research area, that in mitochondria is comparatively recent, and it is likely that their roles within mitochondria have been only partially revealed. We review the status of the field and provide examples of other cellular iron-sulfur proteins to highlight their multifarious roles.

Sphingolipid research has surged in the past two decades and has produced a wide variety of evidence supporting the role of this class of molecules in mediating cellular growth, differentiation, senescence, and apoptosis. Ceramides are a subgroup of sphingolipids (SLs) that are directly involved in the process of initiation of apoptosis. We, and others, have recently shown that ceramides are capable of the formation of protein-permeable channels in mitochondrial outer membranes under physiological conditions. These pores are indeed good candidates for the pathway of release of pro-apoptotic proteins from the mitochondrial intermembrane space (IMS) into the cytosol to initiate intrinsic apoptosis. Here, we review recent findings on the regulation of ceramide channel formation and disassembly, highlighting possible implications on the initiation of the intrinsic apoptotic pathway.

The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome. It exhibits strong sequence homology with the bacteriophage T7 gene 4 protein primase-helicase (T7 gp4). Both proteins contain distinct N- and C-terminal domains separated by a flexible linker. The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain. Whereas the N-terminal domain in T7 gp4 contains a DNA primase activity, this function is lost in metazoan mtDNA helicase. Thus, although the functions of the C-terminal domain and the linker are partially understood, the role of the N-terminal region in the metazoan replicative mtDNA helicase remains elusive. Here, we show that the N-terminal domain of Drosophila melanogaster mtDNA helicase coordinates iron in a 2Fe-2S cluster that enhances protein stability in vitro. The N-terminal domain binds the cluster through conserved cysteine residues (Cys(68), Cys(71), Cys(102), and Cys(105)) that are responsible for coordinating zinc in T7 gp4. Moreover, we show that the N-terminal domain binds both single- and double-stranded DNA oligomers, with an apparent Kd of ∼120 nm. These findings suggest a possible role for the N-terminal domain of metazoan mtDNA helicase in recruiting and binding DNA at the replication fork.

Ceramide (Cer) is involved in the regulation of several cellular processes by mechanisms that depend on Cer-induced changes on membrane biophysical properties. Accumulating evidence shows that Cers with different N-acyl chain composition differentially impact cell physiology, which may in part be due to specific alterations in membrane biophysical properties. We now address how the sphingolipid (SL) N-acyl chain affects membrane properties in cultured human embryonic kidney cells by overexpressing different Cer synthases (CerSs). Our results show an increase in the order of cellular membranes in CerS2-transfected cells caused by the enrichment in very long acyl chain SLs. Formation of Cer upon treatment of cells with bacterial sphingomyelinase promoted sequential changes in the properties of the membranes: after an initial increase in the order of the fluid plasma membrane, reorganization into domains with gel-like properties whose characteristics are dependent on the acyl chain structure of the Cer was observed. Moreover, the extent of alterations of membrane properties correlates with the amount of Cer formed. These data reinforce the significance of Cer-induced changes on membrane biophysical properties as a likely molecular mechanism by which different acyl chain Cers exert their specific biological actions.

Ceramide is an important lipid signaling molecule that plays critical roles in regulating cell behavior. Ceramide synthesis is surprisingly complex and is orchestrated by six mammalian ceramide synthases, each of which produces ceramides with restricted acyl chain lengths. We have generated a CerS2 null mouse and characterized the changes in the long chain base and sphingolipid composition of livers from these mice. Ceramide and downstream sphingolipids were devoid of very long (C22–C24) acyl chains, consistent with the substrate specificity of CerS2 toward acyl-CoAs. Unexpectedly, C16-ceramide levels were elevated, and as a result, total ceramide levels were unaltered; however, C16-ceramide synthesis in vitro was not increased. Levels of sphinganine were also significantly elevated, by up to 50-fold, reminiscent of the effect of the ceramide synthase inhibitor, fumonisin B1. With the exceptions of glucosylceramide synthase and neutral sphingomyelinase 2, none of the other enzymes tested in either the sphingolipid biosynthetic or degradative pathways were significantly changed. Total glycerophospholipid and cholesterol levels were unaltered, although there was a marked elevation in C18:1 and C18:2 fatty acids in phosphatidylethanolamine, concomitant with a reduction in C18:0 and C20:4 fatty acids. Finally, differences were observed in the biophysical properties of lipid extracts isolated from liver microsomes, with membranes from CerS2 null mice displaying higher membrane fluidity and showing morphological changes. Together, these results demonstrate novel modes of cross-talk and regulation between the various branches of lipid metabolic pathways upon inhibition of very long acyl chain ceramide synthesis.

Mitochondria mediate both cell survival and death. The intrinsic apoptotic pathway is initiated by the permeabilization of the mitochondrial outer membrane to pro-apoptotic inter-membrane space (IMS) proteins. Many pathways cause the egress of IMS proteins. Of particular interest is the ability of ceramide to self-assemble into dynamic water-filled channels. The formation of ceramide channels is regulated extensively by Bcl-2 family proteins and dihydroceramide. Here, we show that the chain length of biologically active ceramides serves as an important regulatory factor. Ceramides are synthesized by a family of six mammalian ceramide synthases (CerS) each of which produces a subset of ceramides that differ in their fatty acyl chain length. Various ceramides permeabilize mitochondria differentially. Interestingly, the presence of very long chain ceramides reduces the potency of C16-mediated mitochondrial permeabilization indicating that the intercalation of the lipids in the dynamic channel has a destabilizing effect, reminiscent of dihydroceramide inhibition of ceramide channel formation (Stiban et al., 2006). Moreover, mitochondria isolated from cells overexpressing the ceramide synthase responsible for the production of C16-ceramide (CerS5) are permeabilized faster upon the exogenous addition of C16-ceramide whereas they are resistant to permeabilization with added C24-ceramide. On the other hand mitochondria isolated from CerS2-overexpressing cells show the opposite pattern, indicating that the product of CerS2 inhibits C16-channel formation ex vivo and vice versa. This interplay between different ceramide metabolic enzymes and their products adds a new dimension to the complexity of mitochondrial-mediated apoptosis, and emphasizes its role as a key regulatory step that commits cells to life or death.

Mitochondrial DNA (mtDNA) helicase is an essential part of the mtDNA replisome. The mtDNA helicase shows high amino acid sequence homology with bacteriophage T7 gene 4 protein (T7gp4), which has both DNA primase and DNA helicase activities in its N-terminal domain (NTD) and C-terminal domain (CTD), respectively. Although the NTD of mtDNA helicase in metazoa does not have detectable primase activity, retention of extensive amino acid sequence homology throughout most eukaryotic lineages suggests other important roles, such as in protein stability and/ or protein-protein interactions. Here, we investigate the structural and biochemical properties of the NTD of Drosophila mtDNA helicase by constructing two N-terminal truncated variants: a full-length NTD (including both the zinc binding domain (ZBD) and the RNA polymerase domain (RPD)), and a ZBD only construct. We show that NTD and ZBD constructs of Drosophila mtDNA helicase contain an iron-sulfur cluster, most likely a 2Fe-2S, as determined by mass spectro...

Ceramide synthases (CerS) are integral membrane proteins of the endoplasmic reticulum. Six mammalian CerS have been described, with each utilizing fatty acyl CoAs of relatively defined chain lengths for N-acylation of the sphingoid long chain base. In this chapter, we review the main functional features of the CerS proteins, discuss their fatty acid specificity, kinetics, tissue distribution and mode of inhibition, as well as possible posttranslational modifications. We then address the reason that mammals contain six distinct CerS, whereas most other enzymes in the sphingolipid biosynthetic pathway only occur in one or two isoforms. Finally, we discuss the putative roles of CerS and the ceramide derived from the CerS, in signaling pathways and in development of disease.