Glycosphingolipids (GSL) are components of the outer leaflet of the plasma membrane (PM) of vertebrate tissues. Our current knowledge of GSL metabolism and their intracellular traffic has been derived from metabolic studies but the exact mechanisms by which GSLs are transported from sites of synthesis (endoplasmic reticulum and Golgi) to the sites of residence (PM) and degradation (lysosomes) have not been clearly defined. It is now established that components of the PM reach the lysosomal compartment mainly by endocytic membrane flow. According to a new model, GSLs derived from the PM are thought to end up in intra-endosomal vesicles which could be delivered, by successive processes of membrane fission and fusion, along the endocytic pathway directly into the lumen of the lysosomes. Here the GSLs are degraded in a step-wise manner by exohydrolases. However, the catabolism of membrane-bound GSLs with short hydrophilic head groups needs the assistance of sphingolipid activator proteins (SAPs), which lift the GSLs from the plane of the membrane and present them for degradation to the lysosomal exohydrolases, which are usually water-soluble. The inherited deficiency of one of these enzymes or SAPs causes the lysosomal storage of their respective GSL substrates. In the case of the simultaneous deficiency of all 4 different SAPs the storage of all GSLs with short hydrophilic head groups occurs within multivesicular bodies and/or intra-lysosomal vesicles.

Sphingolipidoses are inherited diseases belonging to the class of lysosomal storage diseases (LSDs), which are characterized by the accumulation of indigestible material in the lysosome caused by specific defects in the lysosomal degradation machinery. While some LSDs can be efficiently treated by enzyme replacement therapy (ERT), this is not possible if the nervous system is affected due to the presence of the blood-brain barrier. Sphingolipidoses in particular often present as severe, untreatable forms of LSDs with massive sphingolipid and membrane accumulation in lysosomes, neurodegeneration and very short life expectancy. The digestion of intralumenal membranes within lysosomes is facilitated by lysosomal sphingolipid activator proteins (saposins), which are cleaved from a prosaposin precursor. Prosaposin mutations cause some of the severest forms of sphingolipidoses, and are associated with perinatal lethality in mice, hampering studies on disease progression. We identify the Drosophila prosaposin orthologue Saposin-related (Sap-r) as a key regulator of lysosomal lipid homeostasis in the fly. Its mutation leads to a typical spingolipidosis phenotype with an enlarged endolysosomal compartment and sphingolipid accumulation as shown by mass spectrometry and thin layer chromatography. Sap-r mutants show reduced viability with ∼50% survival to adulthood, allowing us to study progressive neurodegeneration and analyze their lipid profile in young and aged flies. Additionally, we observe a defect in sterol homeostasis with local sterol depletion at the plasma membrane. Furthermore, we find that autophagy is increased, resulting in the accumulation of mitochondria in lysosomes, concomitant with increased oxidative stress. Together, we establish Drosophila Sap-r mutants as a lysosomal storage disease model suitable for studying the age-dependent progression of lysosomal dysfunction associated with lipid accumulation and the resulting pathological signaling events.

Prosaposin deficiency (pSap-d) and saposin B deficiency (SapB-d) are both lipid storage disorders caused by mutations in the PSAP gene that codes for the 65-70 kDa prosaposin protein, which is the precursor for four sphingolipid activator proteins, saposins A-D. We report on two new patients with PSAP gene defects; one, with pSap-d, who had a severe neurovisceral dystrophy and died as a neonate, and the other with SapB-d, who presented with a metachromatic leukodystrophy-like disorder but had normal arylsulfatase activity. Screening for urinary sphingolipids was crucial to the diagnosis of both patients, with electrospray ionization tandem mass spectrometry also providing quantification. The pSap-d patient is the first case with this condition where urinary sphingolipids have been investigated. Multiple sphingolipids were elevated, with globotriaosylceramide showing the greatest increase. Both patients had novel mutations in the PSAP gene. The pSap-d patient was homozygous for a splice-acceptor site mutation two bases upstream of exon 10. This mutation led to a premature stop codon and yielded low levels of transcript. The SapB-d patient was a compound heterozygote with a splice-acceptor site variant exclusively affecting the SapB domain on one allele, and a 2 bp deletion leading to a null, that is, pSap-d mutation, on the other allele. Phenotypically, pSap-d is a relatively uniform disease of the neonate, whereas SapB-d is heterogeneous with a spectrum similar to that in metachromatic leukodystrophy. The possible existence of genotypes and phenotypes intermediate between those of pSap-d and the single saposin deficiencies is speculated.

To assess the role of sortilin in the sorting and trafficking of sphingolipid activator proteins (SAPs) the function of sortilin was abolished by a dominant-negative mutant and by the use of RNAi. Mutant sortilin lacking the carboxyl-terminal region that contains the sorting signal abolished the trafficking of SAPs to the lysosomes. Both sortilin and SAPs were retained in the Golgi apparatus. The use of chemically synthesized siRNA effectively blocked the trafficking of SAPs to the lysosomes as well. Additionally, we created a stable COS-7 cell line transfected with the pSilencer 3.1 H1 neo vector containing a selected siRNA template oligonucleotide (small hairpin interference RNA) where the levels of sortilin were greatly suppressed. The elimination of sortilin by this method will permit to determine whether or not sortilin is involved in a general mechanism of lysosomal sorting that involves the trafficking of various soluble lysosomal proteins other than SAPs.

Ceramide is a sphingolipid which regulates a variety of signaling pathways in eukaryotic cells. Exogenous ceramide has been shown to induce cellular apoptosis. In this study, we observed that exogenous ceramide induced two distinct morphologies of cell fate following C2-ceramide treatment between the two breast cancer cell lines MCF-7 (wild type p53) and MDA-MB-231 (mutant p53) cells. The growth assessment showed that C2-ceramide caused significant growth inhibition and apoptosis in MDA-MB-231 cells through down-regulating the expression of mutant p53 whereas up-regulating the expression of pro-apoptotic Bad, and the proteolytic activation of caspase-3. However, senescence-associated (SA)-β-galactosidase (β-gal) was regulated in MCF-7 cells after C2-ceramide treatment. The results of proliferation and apoptosis assays showed that MCF-7 cells were more resistant to C2-ceramide treatment compared to MDA-MB-231 cells. Furthermore, C2-ceramide treatment induced a time-responsive increase in Rb protein, a key regulator of senescence accompanied with the upregulation of both mRNA level and protein level of SA-genes PAI-1 and TGaseII in MCF-7 but not in MDA-MB-231 cells, suggesting that some cancer cells escape apoptosis through modulating senescence-like phenotype. The results of our present study depicted the mechanism of C2-ceramide-resistant breast cancer cells, which might benefit the strategic development of ceramide-based chemotherapeutics against cancer in the future.

Aflatoxins are immunosuppressive and carcinogenic secondary metabolites, produced by the filamentous ascomycete Aspergillus flavus, that are hazardous to animal and human health. In this study, we show that multiplexed host-induced gene silencing (HIGS) of Aspergillus flavus genes essential for fungal sporulation and aflatoxin production (nsdC, veA, aflR, and aflM) confers enhanced resistance to Aspergillus infection and aflatoxin contamination in groundnut (<20 ppb). Comparative proteomic analysis of contrasting groundnut genotypes (WT and near-isogenic HIGS lines) supported a better understanding of the molecular processes underlying the induced resistance and identified several groundnut metabolites that might play a significant role in resistance to Aspergillus infection and aflatoxin contamination. Fungal differentiation and pathogenicity proteins, including calmodulin, transcriptional activator-HacA, kynurenine 3-monooxygenase 2, VeA, VelC, and several aflatoxin pathway biosynthetic enzymes, were downregulated in Aspergillus infecting the HIGS lines. Additionally, in the resistant HIGS lines, a number of host resistance proteins associated with fatty acid metabolism were strongly induced, including phosphatidylinositol phosphate kinase, lysophosphatidic acyltransferase-5, palmitoyl-monogalactosyldiacylglycerol Δ-7 desaturase, ceramide kinase-related protein, sphingolipid Δ-8 desaturase, and phospholipase-D. Combined, this knowledge can be used for groundnut pre-breeding and breeding programs to provide a safe and secure food supply.

p21-activated kinase 1 (Pak1) is a member of the highly conserved family of serine/threonine protein kinases regulated by Ras-related small G-proteins, Cdc42/Rac1. It has been previously demonstrated to be involved in cardiac protection. Based on recent studies, this review provides an overview of the role of Pak1 in cardiac diseases including disrupted Ca2+ homoeostasis-related cardiac arrhythmias, adrenergic stress- and pressure overload-induced hypertrophy, and ischaemia/reperfusion injury. These findings demonstrate the important role of Pak1 mediated through the phosphorylation and transcriptional modification of hypertrophy and/or arrhythmia-related genes. This review also discusses the anti-arrhythmic and anti-hypertrophic, protective function of Pak1 and the beneficial effects of fingolimod (an FDA-approved sphingolipid drug), a Pak1 activator, and its ability to prevent arrhythmias and cardiac hypertrophy. These findings also highlight the therapeutic potential of Pak1 signalling in the treatment and prevention of cardiac diseases.

Saposin deficiency is a childhood neurodegenerative lysosomal storage disorder (LSD) that can cause premature death within three months of life. Saposins are activator proteins that promote the function of lysosomal hydrolases that mediate the degradation of sphingolipids. There are four saposin proteins in humans, which are encoded by the prosaposin gene. Mutations causing an absence or impaired function of individual saposins or the whole prosaposin gene lead to distinct LSDs due to the storage of different classes of sphingolipids. The pathological events leading to neuronal dysfunction induced by lysosomal storage of sphingolipids are as yet poorly defined. We have generated and characterised a Drosophila model of saposin deficiency that shows striking similarities to the human diseases. Drosophila saposin-related (dSap-r) mutants show a reduced longevity, progressive neurodegeneration, lysosomal storage, dramatic swelling of neuronal soma, perturbations in sphingolipid catabolism, and sensory physiological deterioration. Our data suggests a genetic interaction with a calcium exchanger (Calx) pointing to a possible calcium homeostasis deficit in dSap-r mutants. Together these findings support the use of dSap-r mutants in advancing our understanding of the cellular pathology implicated in saposin deficiency and related LSDs.

Neuronal Ceroid Lipofuscinoses (NCLs) are progressive degenerative diseases mainly affect brain and retina. They are characterized by accumulation of autofluorescent storage material, mitochondrial ATPase subunit C, or sphingolipid activator proteins A and D in lysosomes of most cells. Heterogenous storage material in NCLs is not completely disease-specific. Most of CLN proteins and their natural substrates are not well-characterized. Studies have suggested variants of Late-Infantile NCLs (LINCLs) include the major type CLN2 and minor types CLN5, CLN6, CLN7, and CLN8. Therefore, combination of clinical and molecular analysis has become a more effective diagnosis method. We studied 4 late-infantile NCL siblings characterized by seizures, ataxia as early symptoms, followed by progressive regression in intelligence and behavior, but mutations are located in different genes. Symptoms and progression of 4 types of LINCLs are compared. Pathology of LINCLs is also discussed. We performed Nest-Generation Sequencing on these phenotypically similar families. Three novel variants c.1551+1insTGAT in TPP1, c.244G>T in CLN6, c.554-5A>G in MFSD8 were identified. Potential outcome of the mutations in structure and function of proteins are studied. In addition, we observed some common and unique clinical features of Chinese LINCL patient as compared with those of Western patients, which greatly improved our understanding of the LINCLs.

The neuronal ceroid-lipofuscinoses (NCLs) are inherited lysosomal storage diseases and constitute the most common group of children's progressive encephalopathies. Most childhood forms of NCL are clinically characterized by progressive loss of vision as well as mental and motor deterioration, epileptic seizures, and premature death, while the rare adult forms are dominated by dementia. All forms of NCL share common pathomorphological features. Autofluorescent, periodic acid-Schiff- and Sudan black B-positive granules, resistant to lipid solvents, accumulate in the cytoplasm of most nerve cells, and there is progressive and remarkably selective neuronal degeneration and loss. For a long time, the NCLs were grouped under the heading of the "amaurotic family idiocies" and conceived as lipidoses. However, in the late 1980s and 1990s the NCL storage cytosomes were shown to consist largely of two hydrophobic proteins: either subunit c of mitochondrial ATP synthase or sphingolipid activator proteins A and D. Since 1995 numerous mutations in at least seven different genes have been shown to underlie the multiple human and animal forms of NCL. This review discusses the historical evolution of the NCL concept and the impact of the recent biochemical and molecular genetic findings on our views on the classification and pathogenesis of these devastating brain disorders.

Acid sphingomyelinase (ASM) is a lysosomal phosphodiesterase that catalyzes the hydrolysis of sphingomyelin to produce ceramide and phosphocholine. While other lysosomal sphingolipid hydrolases require a saposin activator protein for full activity, the ASM polypeptide incorporates a built-in N-terminal saposin domain and does not require an external activator protein. Here, we report the crystal structure of human ASM and describe the organization of the three main regions of the enzyme: the N-terminal saposin domain, the proline-rich connector, and the catalytic domain. The saposin domain is tightly associated along an edge of the large, bowl-shaped catalytic domain and adopts an open form that exposes a hydrophobic concave surface approximately 30Å from the catalytic center. The calculated electrostatic potential of the enzyme is electropositive at the acidic pH of the lysosome, consistent with the strict requirement for the presence of acidic lipids in target membranes. Docking studies indicate that sphingomyelin binds with the ceramide-phosphate group positioned at the binuclear zinc center and molecular dynamic simulations indicate that the intrinsic flexibility of the saposin domain is important for monomer-dimer exchange and for membrane interactions. Overall, ASM uses a combination of electrostatic and hydrophobic interactions to cause local disruptions of target bilayers in order to bring the lipid headgroup to the catalytic center in a membrane-bound reaction.

Abnormal/cancerous cells within healthy epithelial tissues undergo apical extrusion to protect against carcinogenesis, although they acquire invasive capacity once carcinogenesis progresses. However, the molecular mechanisms by which cancer cells escape from apical extrusion and invade surrounding tissues remain elusive. In this study, we demonstrate a molecular mechanism for cell fate switching during epithelial cell competition. We found that during competition within epithelial cell layers, Src transformation promotes maturation of focal adhesions and degradation of extracellular matrix. Src-transformed cells underwent basal delamination by Src activation within sphingolipid/cholesterol-enriched membrane microdomains/lipid rafts, whereas they were apically extruded when Src was outside of lipid rafts. A comparative analysis of contrasting phenotypes revealed that activation of the Src-STAT3-MMP axis through lipid rafts was required for basal delamination. CUB-domain-containing protein 1 (CDCP1) was identified as an Src-activating scaffold and as a Met regulator in lipid rafts, and its overexpression induced basal delamination. In renal cancer models, CDCP1 promoted epithelial-mesenchymal transition-mediated invasive behavior by activating the Src-STAT3-MMP axis through Met activation. Overall, these results suggest that spatial activation of Src signaling in lipid rafts confers resistance to apical extrusion and invasive potential on epithelial cells to promote carcinogenesis.

Four sphingolipid activator proteins (i.e., saposins A-D) are synthesized from a single precursor protein, prosaposin (PS), which exerts exogenous neurotrophic effects in vivo and in vitro. Kainic acid (KA) injection in rodents is a good model in which to study neurotrophic factor elevation; PS and its mRNA are increased in neurons and the choroid plexus in this animal model. An 18-mer peptide (LSELIINNATEELLIKGL; PS18) derived from the PS neurotrophic region prevents neuronal damage after ischemia, and PS18 is a potent candidate molecule for use in alleviating ischemia-induced learning disabilities and neuronal loss. KA is a glutamate analog that stimulates excitatory neurotransmitter release and induces ischemia-like neuronal degeneration; it has been used to define mechanisms involved in neurodegeneration and neuroprotection. In the present study, we demonstrate that a subcutaneous injection of 0.2 and 2.0 mg/kg PS18 significantly improved behavioral deficits of Wistar rats (n = 6 per group), and enhanced the survival of hippocampal and cortical neurons against neurotoxicity induced by 12 mg/kg KA compared with control animals. PS18 significantly protected hippocampal synapses against KA-induced destruction. To evaluate the extent of PS18- and KA-induced effects in these hippocampal regions, we performed histological evaluations using semithin sections stained with toluidine blue, as well as ordinal sections stained with hematoxylin and eosin. We revealed a distinctive feature of KA-induced brain injury, which reportedly mimics ischemia, but affects a much wider area than ischemia-induced injury: KA induced neuronal degeneration not only in the CA1 region, where neurons degenerate following ischemia, but also in the CA2, CA3, and CA4 hippocampal regions.

The sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that is now appreciated as key regulatory factor for various cellular functions in the kidney, including matrix remodeling. It is generated by two sphingosine kinases (Sphk), Sphk1 and Sphk2, which are ubiquitously expressed, but have distinct enzymatic activities and subcellular localizations. In this study, we have investigated the role of Sphk2 in podocyte function and its contribution to diabetic nephropathy. We show that streptozotocin (STZ)-induced nephropathy and albuminuria in mice is prevented by genetic depletion of Sphk2. This protection correlated with an increased protein expression of the transcription factor Wilm's tumor suppressor gene 1 (WT1) and its target gene nephrin, and a reduced macrophage infiltration in immunohistochemical renal sections of STZ-treated Sphk2-/- mice compared to STZ-treated wildtype mice. To investigate changes on the cellular level, we used an immortalized human podocyte cell line and generated a stable knockdown of Sphk2 (Sphk2-kd) by a lentiviral transduction method. These Sphk2-kd cells accumulated sphingosine as a consequence of the knockdown, and showed enhanced nephrin and WT1 mRNA and protein expressions similar to the finding in Sphk2 knockout mice. Treatment of wildtype podocytes with the highly selective Sphk2 inhibitor SLM6031434 caused a similar upregulation of nephrin and WT1 expression. Furthermore, exposing cells to the profibrotic mediator transforming growth factor β (TGFβ) resulted on the one side in reduced nephrin and WT1 expression, but on the other side, in upregulation of various profibrotic marker proteins, including connective tissue growth factor (CTGF), fibronectin (FN) and plasminogen activator inhibitor (PAI) 1. All these effects were reverted by Sphk2-kd and SLM6031434. Mechanistically, the protection by Sphk2-kd may depend on accumulated sphingosine and inhibited PKC activity, since treatment of cells with exogenous sphingosine not only reduced the phosphorylation pattern of PKC substrates, but also increased WT1 protein expression. Moreover, the selective stable knockdown of PKCδ increased WT1 expression, suggesting the involvement of this PKC isoenzyme in WT1 regulation. The glucocorticoid dexamethasone, which is a treatment option in many glomerular diseases and is known to mediate a nephroprotection, not only downregulated Sphk2 and enhanced cellular sphingosine, but also enhanced WT1 and nephrin expressions, thus, suggesting that parts of the nephroprotective effect of dexamethasone is mediated by Sphk2 downregulation. Altogether, our data demonstrated that loss of Sphk2 is protective in diabetes-induced podocytopathy and can prevent proteinuria, which is a hallmark of many glomerular diseases. Thus, Sphk2 could serve as a new attractive pharmacological target to treat proteinuric kidney diseases.

Glutaredoxin 6 (Grx6) of Saccharomyces cerevisiae is an integral thiol oxidoreductase protein of the endoplasmic reticulum/Golgi vesicles. Its absence alters the redox equilibrium of the reticulum lumen toward a more oxidized state, thus compensating the defects in protein folding/secretion and cell growth caused by low levels of the oxidase Ero1. In addition, null mutants in GRX6 display a more intense unfolded protein response than wild-type cells upon treatment with inducers of this pathway. These observations support a role of Grx6 in regulating the glutathionylation of thiols of endoplasmic reticulum/Golgi target proteins and consequently the equilibrium between reduced and oxidized glutathione in the lumen of these compartments. A specific function influenced by Grx6 activity is the homeostasis of intracellular calcium. Grx6-deficient mutants have reduced levels of calcium in the ER lumen, whereas accumulation occurs at the cytosol from extracellular sources. This results in permanent activation of the calcineurin-dependent pathway in these cells. Some but not all the phenotypes of the mutant are coincident with those of mutants deficient in intracellular calcium transporters, such as the Golgi Pmr1 protein. The results presented in this study provide evidence for redox regulation of calcium homeostasis in yeast cells.