Sphingolipid lysosomal storage diseases: from bench to bedside.

Johann Ludwig Wilhelm Thudicum described sphingolipids (SLs) in the late nineteenth century, but it was only in the past fifty years that SL research surged in importance and applicability. Currently, sphingolipids and their metabolism are hotly debated topics in various biochemical fields. Similar to other macromolecular reactions, SL metabolism has important implications in health and disease in most cells. A plethora of SL-related genetic ailments has been described. Defects in SL catabolism can cause the accumulation of SLs, leading to many types of lysosomal storage diseases (LSDs) collectively called sphingolipidoses. These diseases mainly impact the neuronal and immune systems, but other systems can be affected as well. This review aims to present a comprehensive, up-to-date picture of the rapidly growing field of sphingolipid LSDs, their etiology, pathology, and potential therapeutic strategies. We first describe LSDs biochemically and briefly discuss their catabolism, followed by general aspects of the major diseases such as Gaucher, Krabbe, Fabry, and Farber among others. We conclude with an overview of the available and potential future therapies for many of the diseases. We strive to present the most important and recent findings from basic research and clinical applications, and to provide a valuable source for understanding these disorders.

Introduction: Enigmas of Sphingolipids.

Sphingolipid biology has enjoyed a remarkable rise to fame over the last two decades. Various molecules from this lipid family have been implicated in a variety of cellular functions in health and disease. Ceramides, which constitute the hub of sphingolipid metabolism, are apoptogenic molecules that have many proposed mechanisms of actions. Enigmas revolving around this area of research are slowly being cleared with the advent of better laboratory techniques and data analyses. In this chapter, a general introduction of the topics presented in this book is undertaken highlighting the main ideas of each chapter.

Roles of Ceramides and Other Sphingolipids in Immune Cell Function and Inflammation.

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.

Prokaryotic and Mitochondrial Lipids: A Survey of Evolutionary Origins.

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.

Very long chain ceramides interfere with C16-ceramide-induced channel formation: A plausible mechanism for regulating the initiation of intrinsic apoptosis.

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.

Iron-Sulfur Clusters in Mitochondrial Metabolism: Multifaceted Roles of a Simple Cofactor.

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.

The N-terminal domain of the Drosophila mitochondrial replicative DNA helicase contains an iron-sulfur cluster and binds DNA.

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.

Changes in membrane biophysical properties induced by sphingomyelinase depend on the sphingolipid N-acyl chain.

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.

Sphingolipids: From structural components to signaling hubs.

In late November 2019, Prof. Lina M. Obeid passed away from cancer, a disease she spent her life researching and studying its intricate molecular underpinnings. Along with her husband, Prof. Yusuf A. Hannun, Obeid laid down the foundations of sphingolipid biochemistry and oversaw its remarkable evolution over the years. Lipids are a class of macromolecules that are primarily associated with cellular architecture. In fact, lipids constitute the perimeter of the cell in such a way that without them, there cannot be cells. Hence, much of the early research on lipids identified the function of this class of biological molecules as merely structural. Nevertheless, unlike proteins, carbohydrates, and nucleic acids, lipids are elaborately diverse as they are not made up of monomers in polymeric forms. This diversity in structure is clearly mirrored by functional pleiotropy. In this chapter, we focus on a major subset of lipids, sphingolipids, and explore their historic rise from merely inert structural components of plasma membranes to lively and necessary signaling molecules that transmit various signals and control many cellular processes. We will emphasize the works of Lina Obeid since she was an integral pillar of the sphingolipid research world.

Mysterious sphingolipids: metabolic interrelationships at the center of pathophysiology.

Metabolic pathways are complex and intertwined. Deficiencies in one or more enzymes in a given pathway are directly linked with genetic diseases, most of them having devastating manifestations. The metabolic pathways undertaken by sphingolipids are diverse and elaborate with ceramide species serving as the hubs of sphingolipid intermediary metabolism and function. Sphingolipids are bioactive lipids that serve a multitude of cellular functions. Being pleiotropic in function, deficiency or overproduction of certain sphingolipids is associated with many genetic and chronic diseases. In this up-to-date review article, we strive to gather recent scientific evidence about sphingolipid metabolism, its enzymes, and regulation. We shed light on the importance of sphingolipid metabolism in a variety of genetic diseases and in nervous and immune system ailments. This is a comprehensive review of the state of the field of sphingolipid biochemistry.

The heat shock protein Hsp27 controls mitochondrial function by modulating ceramide generation.

Sphingolipids have key functions in membrane structure and cellular signaling. Ceramide is the central molecule of the sphingolipid metabolism and is generated by ceramide synthases (CerS) in the de novo pathway. Despite their critical function, mechanisms regulating CerS remain largely unknown. Using an unbiased proteomics approach, we find that the small heat shock protein 27 (Hsp27) interacts specifically with CerS1 but not other CerS. Functionally, our data show that Hsp27 acts as an endogenous inhibitor of CerS1. Wild-type Hsp27, but not a mutant deficient in CerS1 binding, inhibits CerS1 activity. Additionally, silencing of Hsp27 enhances CerS1-generated ceramide accumulation in cells. Moreover, phosphorylation of Hsp27 modulates Hsp27-CerS1 interaction and CerS1 activity in acute stress-response conditions. Biologically, we show that Hsp27 knockdown impedes mitochondrial function and induces lethal mitophagy in a CerS1-dependent manner. Overall, we identify an important mode of CerS1 regulation and CerS1-mediated mitophagy through protein-protein interaction with Hsp27.

Ablation of ceramide synthase 2 strongly affects biophysical properties of membranes.

Little is known about the effects of altering sphingolipid (SL) acyl chain structure and composition on the biophysical properties of biological membranes. We explored the biophysical consequences of depleting very long acyl chain (VLC) SLs in membranes prepared from lipid fractions isolated from a ceramide synthase 2 (CerS2)-null mouse, which is unable to synthesize C22-C24 ceramides. We demonstrate that ablation of CerS2 has different effects on liver and brain, causing a significant alteration in the fluidity of the membrane and affecting the type and/or extent of the phases present in the membrane. These changes are a consequence of the depletion of VLC and unsaturated SLs, which occurs to a different extent in liver and brain. In addition, ablation of CerS2 causes changes in intrinsic membrane curvature, leading to strong morphological alterations that promote vesicle adhesion, membrane fusion, and tubule formation. Together, these results show that depletion of VLC-SLs strongly affects membrane biophysical properties, which may compromise cellular processes that critically depend on membrane structure, such as trafficking and sorting.

Visualization of ceramide channels by transmission electron microscopy.

Functional studies have shown that the sphingolipid ceramide, self-assembles in phospholipid membranes to form large channels capable of allowing proteins to cross the membrane. Here these channels are visualized by negative stain transmission electron microscopy. The images contain features consistent with stain-filled pores having a roughly circular profile. There is no indication of tilt, and the results are consistent with the formation of right cylinders. The sizes of the pores range from 5 to 40nm in diameter with an asymmetric distribution indicating no apparent upper size limit. The size distribution matches well with the distribution of sizes calculated from electrophysiological measurements.

A critical role for ceramide synthase 2 in liver homeostasis: I. alterations in lipid metabolic pathways.

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.

Implications of Membrane Binding by the Fe-S Cluster-Containing N-Terminal Domain in the Drosophila Mitochondrial Replicative DNA Helicase.

Recent evidence suggests that iron-sulfur clusters (ISCs) in DNA replicative proteins sense DNA-mediated charge transfer to modulate nuclear DNA replication. In the mitochondrial DNA replisome, only the replicative DNA helicase (mtDNA helicase) from Drosophila melanogaster (Dm) has been shown to contain an ISC in its N-terminal, primase-like domain (NTD). In this report, we confirm the presence of the ISC and demonstrate the importance of a metal cofactor in the structural stability of the Dm mtDNA helicase. Further, we show that the NTD also serves a role in membrane binding. We demonstrate that the NTD binds to asolectin liposomes, which mimic phospholipid membranes, through electrostatic interactions. Notably, membrane binding is more specific with increasing cardiolipin content, which is characteristically high in the mitochondrial inner membrane (MIM). We suggest that the N-terminal domain of the mtDNA helicase interacts with the MIM to recruit mtDNA and initiate mtDNA replication. Furthermore, Dm NUBPL, the known ISC donor for respiratory complex I and a putative donor for Dm mtDNA helicase, was identified as a peripheral membrane protein that is likely to execute membrane-mediated ISC delivery to its target proteins.

Ceramide synthases: roles in cell physiology and signaling.

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.

Iron-sulfur clusters in nucleic acid metabolism: Varying roles of ancient cofactors.

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.

Time-related changes in hepatic and colonic mitochondrial oxygen consumption after abdominal infection in rats.

BACKGROUND: Evidence suggests that early adaptive responses of hepatic mitochondria occur in experimentally induced sepsis. Little is known about both colonic mitochondrial function during abdominal infection and long-term changes in mitochondrial function under inflammatory conditions. We hypothesize that hepatic and colonic mitochondrial oxygen consumption changes time-dependently after sterile laparotomy and in the course of abdominal infection. The aim of the present study was to investigate the hepatic and colonic mitochondrial respiration after sterile laparotomy and abdominal infection over up to 96 h. METHODS: After approval of the local Animal Care and Use Committee, 95 Wistar rats were randomized into 8 groups (n = 11-12): 1-4 sham (laparotomy only) and 5-8 colon ascendens stent peritonitis (CASP). Healthy, unoperated animals served as controls (n = 9). The mitochondrial respiration in colon and liver homogenates was assessed 24, 48, 72, and 96 h after surgery. Mitochondrial oxygen consumption was determined using a Clark-type electrode. State 2 (oxygen consumption in the presence of the substrates for complexes I and II) and state 3 respiration (ADP dependent) were assessed. The respiratory control ratio (RCR state 3/state 2) and ADP/O ratio (ADP added/oxygen consumed) were calculated for both complexes. Data are presented as means ± SD, two-way ANOVA followed by Tukey's post hoc test. RESULTS: Hepatic RCR was initially (after 24 h) elevated in both operated groups; after 48 h only, the septic group was elevated compared to controls. In CASP groups, the hepatic ADP/O ratio for complex I was elevated after 24 h (vs. controls) and after 48 h (vs. sham) but declined after 72 h (vs. controls). The ADP/O ratio for complex II stayed unchanged over the time period until 96 h. The colonic RCR and ADP/O did not change over time after sham or CASP operation. CONCLUSION: Hepatic, but not colonic, mitochondrial respiration is increased in the initial phase (until 48 h) and normalizes in the longer course of time (until 96 h) of abdominal infection.

Carbon dioxide in carbonated beverages induces ghrelin release and increased food consumption in male rats: Implications on the onset of obesity.

BACKGROUND: The dangerous health risks associated with obesity makes it a very serious public health issue. Numerous studies verified a correlation between the increase in obesity and the parallel increase in soft drink consumption among world populations. The effects of one main component in soft drinks namely the carbon dioxide gas has not been studied thoroughly in any previous research. METHODS: Male rats were subjected to different categories of drinks and evaluated for over a year. Stomach ex vivo experiments were undertaken to evaluate the amount of ghrelin upon different beverage treatments. Moreover, 20 male students were tested for their ghrelin levels after ingestion of different beverages. RESULTS: Here, we show that rats consuming gaseous beverages over a period of around 1 year gain weight at a faster rate than controls on regular degassed carbonated beverage or tap water. This is due to elevated levels of the hunger hormone ghrelin and thus greater food intake in rats drinking carbonated drinks compared to control rats. Moreover, an increase in liver lipid accumulation of rats treated with gaseous drinks is shown opposed to control rats treated with degassed beverage or tap water. In a parallel study, the levels of ghrelin hormone were increased in 20 healthy human males upon drinking carbonated beverages compared to controls. CONCLUSIONS: These results implicate a major role for carbon dioxide gas in soft drinks in inducing weight gain and the onset of obesity via ghrelin release and stimulation of the hunger response in male mammals.

Regulation of ceramide channel formation and disassembly: Insights on the initiation of apoptosis.

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.