Coordinated Regulation of the Orosomucoid-like Gene Family Expression Controls de Novo Ceramide Synthesis in Mammalian Cells

The orosomucoid-like (ORMDL) protein family is involved in the regulation of de novo sphingolipid synthesis, calcium homeostasis, and unfolded protein response. Single nucleotide polymorphisms (SNPs) that increase ORMDL3 expression have been associated with various immune/inflammatory diseases, although the pathophysiological mechanisms underlying this association are poorly understood. ORMDL proteins are claimed to be inhibitors of the serine palmitoyltransferase (SPT). However, it is not clear whether individual ORMDL expression levels have an impact on ceramide synthesis. The present study addressed the interaction with and regulation of SPT activity by ORMDLs to clarify their pathophysiological relevance. We have measured ceramide production in HEK293 cells incubated with palmitate as a direct substrate for SPT reaction. Our results showed that a coordinated overexpression of the three isoforms inhibits the enzyme completely, whereas individual ORMDLs are not as effective. Immunoprecipitation and fluorescence resonance energy transfer (FRET) studies showed that mammalian ORMDLs form oligomeric complexes that change conformation depending on cellular sphingolipid levels. Finally, using macrophages as a model, we demonstrate that mammalian cells modify ORMDL genes expression levels coordinately to regulate the de novo ceramide synthesis pathway. In conclusion, we have shown a physiological modulation of SPT activity by general ORMDL expression level regulation. Moreover, because single ORMDL3 protein alteration produces an incomplete inhibition of SPT activity, this work argues against the idea that ORMDL3 pathophysiology could be explained by a simple on/off mechanism on SPT activity.     

ORMDL/serine palmitoyltransferase stoichiometry determines effects of ORMDL3 expression on sphingolipid biosynthesis

The ORM1 (Saccharomyces cerevisiae)-like proteins (ORMDLs) and their yeast orthologs, the Orms, are negative homeostatic regulators of the initiating enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT). Genome-wide association studies have established a strong correlation between elevated expression of the endoplasmic reticulum protein ORMDL3 and risk for childhood asthma. Here we test the notion that elevated levels of ORMDL3 decrease sphingolipid biosynthesis. This was tested in cultured human bronchial epithelial cells (HBECs) (an immortalized, but untransformed, airway epithelial cell line) and in HeLa cells (a cervical adenocarcinoma cell line). Surprisingly, elevated ORMDL3 expression did not suppress de novo biosynthesis of sphingolipids. We determined that ORMDL is expressed in functional excess relative to SPT at normal levels of expression. ORMDLs and SPT form stable complexes that are not increased by elevated ORMDL3 expression. Although sphingolipid biosynthesis was not decreased by elevated ORMDL3 expression, the steady state mass levels of all major sphingolipids were marginally decreased by low level ORMDL3 over-expression in HBECs. These data indicate that the contribution of ORMDL3 to asthma risk may involve changes in sphingolipid metabolism, but that the connection is complex.     

Cell Polarity Factor Par3 Binds SPTLC1 and Modulates Monocyte Serine Palmitoyltransferase Activity and Chemotaxis

Elevated sphingolipids have been associated with increased cardiovascular disease. Conversely, atherosclerosis is reduced in mice by blocking de novo synthesis of sphingolipids catalyzed by serine palmitoyltransferase (SPT). The SPT enzyme is composed of the SPTLC1 and -2 subunits, and here we describe a novel protein-protein interaction between SPTLC1 and the PDZ protein Par3 (partitioning defective protein 3). Mammalian SPTLC1 orthologs have a highly conserved C terminus that conforms to a type II PDZ protein interaction motif, and by screening PDZ domain protein arrays with an SPTLC1 C-terminal peptide, we found it bound the third PDZ domain of Par3. Overlay and immunoprecipitation assays confirmed this interaction and indicate Par3 is able to associate with the SPTLC1/2 holoenzyme by binding the C-terminal SPTLC1 PDZ motif. The physiologic existence of the SPTLC1/2-Par3 complex was detected in mouse liver and macrophages, and short interfering RNA inhibition of Par3 in human THP-1 monocytes significantly reduced SPT activity and de novo ceramide synthesis by nearly 40%. Given monocyte recruitment into inflamed vessels is thought to promote atherosclerosis, and because Par3 and sphingolipids have been associated with polarized cell migration, we tested whether the ability of THP-1 monocytes to migrate toward MCP-1 (monocyte chemoattractant protein 1) depended upon Par3 and SPTLC1 expression. Knockdown of Par3 significantly reduced MCP1-induced chemotaxis of THP-1 monocytes, as did knockdown of SPTLC1, and this Par3 effect depended upon SPT activity and was blunted by ceramide treatment. In conclusion, protein arrays were used to identify a novel SPTLC1-Par3 interaction that associates with increased monocyte serine palmitoyltransferase activity and chemotaxis toward inflammatory signals.Sphingolipids are a structurally diverse class of lipids that play correspondingly diverse roles in membrane structure, cell proliferation, immune function, and skin physiology (1–4). De novo sphingolipid synthesis is initiated by serine palmitoyltransferase (SPT),² an enzyme that condenses serine and palmitoyl-CoA forming the biosynthetic intermediate 3-ketodihydrosphingosine that is subsequently converted to ceramide, sphingomyelin, and other sphingolipids (5). SPT is a heterodimer composed of the SPTLC1 and -2 subunits (6, 7), which may form higher order multimeric structures that can include a third subunit, SPTLC3 (8, 9). Both the SPTLC2 and -3 subunits are catalytically active and contain conserved lysines that act as Schiff bases during the condensation reaction (5, 8). In contrast, SPTLC1 does not contain the conserved catalytically active lysine, but is important for stabilizing the SPTLC2 subunit and anchoring the SPT holoenyzme on the cytosolic face of the endoplasmic reticulum (10, 11).Expression and regulation of the SPTLC1/2 holoenyzme are of interest because its activity controls de novo synthesis of sphingomyelin, and increased plasma levels of this sphingolipid have been correlated with an increased incidence of cardiovascular disease in humans (12, 13). Conversely, inhibition of SPT activity with myriocin, a fungal metabolite, strongly inhibits atherosclerotic development in ApoE^−/− mice (14–18). Moreover, the increased atherosclerosis seen in ApoE^−/− mice has been associated with a post-translational increase in liver SPT activity (19). How SPT activity and sphingolipids may act to promote the progression of atherosclerosis is unclear, but the data do suggest analysis of factors that regulate SPT activity should provide mechanistic insight into the link between de novo sphingolipid synthesis and atherosclerosis. In this regard we have found that SPTLC1 can interact with the ABCA1 transporter and inhibit its ability to transfer cholesterol to apoA-I, a mechanism that would be expected to promote atherosclerosis (20). Thus, along with playing a direct role in the synthesis of sphingolipids, SPTLC1 may also have evolved as an SPT subunit whose function is to regulate SPT activity in response to the cellular demand for sphingolipids and other membrane constituents such as cholesterol. To play such a role, SPTLC1 may engage additional protein-protein interactions that integrate input from signaling pathways and allow SPTLC1 to modulate SPT activity in response to altered demand for sphingolipids.Here we have explored this hypothesis by first conducting a protein array screen for SPTLC1 interacting factors. Consistent with the potential to engage cellular factors in protein-protein interactions, sequence alignment of the SPTLC1 C terminus indicates it has been strongly conserved in mammals as a type II PDZ domain binding motif. Moreover, because topology studies indicate the SPTLC1 C terminus resides in the cytoplasm where it could be bound by PDZ proteins, we used protein arrays spotted with 123 PDZ domains from 73 different proteins to screen for interactions with the SPTLC1 C terminus. This screen indicated the SPTLC1 C terminus directly interacts with the third PDZ domain of PARD3 (partitioning defective protein 3). PARD3, also known as Par3, is a scaffolding factor that recruits signaling molecules, including atypical protein kinase C and Cdc42 into multiprotein complexes that regulate formation of membrane microdomains required for apical/basal polarity and for directed cell migration (21–25). Mutation analysis confirmed the SPTLC1-Par3 interaction depended upon the SPTLC1 C-terminal PDZ motif, and immunoprecipitation assays indicate Par3 is able to associate with the SPTLC1/2 holoenyzme by binding the SPTLC1 C-terminal PDZ motif. The Par-3 interaction with the SPTLC1/2 holoenyzme was detected in the liver, a major site of SPT activity and sphingolipid synthesis. Given SPT activity is proatheroslerotic, and because we have also detected SPTLC1 expression in macrophages, a cell type that plays a central role in the progression of atherosclerosis, we tested and found that Par3 was expressed and interacted with the SPT holoenyzme in primary mouse macrophages. Significantly, loss of Par3 expression in human THP-1 monocyte macrophages reduced SPT activity and inhibited their ability to migrate toward MCP-1 (monocyte chemotactic protein-1). Likewise, shRNA suppression of SPTLC1 reduced monocyte migration toward MCP-1, as did myriocin inhibition of SPT activity, an effect that was blunted by loss of Par3 expression. In aggregate, our work has identified a novel protein-protein interaction between SPTLC1 and Par3 that is associated with an increase in SPT activity and the promotion of polarized cell migration in response to an inflammatory signal.     

Mutations in the SPTLC2 subunit of serine palmitoyltransferase cause hereditary sensory and autonomic neuropathy type I

Hereditary sensory and autonomic neuropathy type I (HSAN-I) is an axonal peripheral neuropathy associated with progressive distal sensory loss and severe ulcerations. Mutations in the first subunit of the enzyme serine palmitoyltransferase (SPT) have been associated with HSAN-I. The SPT enzyme catalyzes the first and rate-limiting step in the de novo sphingolipid synthesis pathway. However, different studies suggest the implication of other genes in the pathology of HSAN-I. Therefore, we screened the two other known subunits of SPT, SPTLC2 and SPTLC3, in a cohort of 78 HSAN patients. No mutations were found in SPTLC3, but we identified three heterozygous missense mutations in the SPTLC2 subunit of SPT in four families presenting with a typical HSAN-I phenotype. We demonstrate that these mutations result in a partial to complete loss of SPT activity in vitro and in vivo. Moreover, they cause the accumulation of the atypical and neurotoxic sphingoid metabolite 1-deoxy-sphinganine. Our findings extend the genetic heterogeneity in HSAN-I and enlarge the group of HSAN neuropathies associated with SPT defects. We further show that HSAN-I is consistently associated with an increased formation of the neurotoxic 1-deoxysphinganine, suggesting a common pathomechanism for HSAN-I.     

Cloning and initial characterization of a new subunit for mammalian serine-palmitoyltransferase

Serine-palmitoyltransferase (SPT) catalyzes the rate-limiting step of the de novo synthesis of sphingolipids. SPT is considered to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Here we report the identification of a novel, third, SPT subunit (SPTLC3) that shows 68% homology to the SPTLC2 subunit. Quantitative real-time PCR revealed that SPTLC3 expression is highly variable between different human tissues and cell lines. The highest expression was observed in placenta tissue and human trophoblast cell lines. The overexpression of SPTLC3 in Hek293 cells, which otherwise have very little endogenous SPTLC3, led to a 2- to 3-fold increase in cellular SPT activity. Silencing of SPTLC3 expression in HepG2 cells or human trophoblast cells by transfecting SPTLC3-specific siRNA resulted in a significant reduction of cellular SPT activity. The expression of two SPT isoforms could be a cellular mechanism to adjust SPT activity to tissue-specific requirements of sphingolipid synthesis.     

Is the mammalian serine palmitoyltransferase a high-molecular-mass complex?

SPT (serine palmitoyltransferase) catalyses the rate-limiting step for the de novo synthesis of sphingolipids. Mammalian SPT is believed to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. We reported previously the identification of a new third SPT subunit, SPTLC3. In the present study, we have investigated the structure of the SPT complex in more detail. Pull-down assays with antibodies against SPTLC3 concomitantly co-precipitated SPTLC1 and SPTLC2 in human placenta extracts and SPTLC3 overexpressing human embryonic kidney-293 cells. By size exclusion chromatography, we determined the molecular mass of the functional SPT complex to be approx. 480 kDa. By Blue-native-PAGE experiments we demonstrated that all three SPT subunits (SPTLC1-3) are co-localized within a single SPT complex. On the basis of these results we conclude that the functional SPT is not a dimer, but a higher organized complex, composed of three distinct subunits (SPTLC1, SPTLC2 and SPTLC3) with a molecular mass of 480 kDa. The stoichiometry of SPTLC2 and SPTLC3 in this complex seems not to be fixed and is probably changed dynamically in dependence of the tissue specific SPTLC2 and SPTLC3 expression levels. Based on our own and earlier published data we propose a model of an octameric SPT structure. The observed dynamic composition of the SPT complex could provide a cellular mechanism to adjust SPT activity to tissue specific requirements in sphingolipid synthesis.     

Serine palmitoyltransferase subunit 1 is present in the endoplasmic reticulum,nucleus and focal adhesions,and functions in cell morphology

Serine palmitoyltransferase (SPT) has been localized to the endoplasmic reticulum (ER) by subcellular fractionation and enzymatic assays, and fluorescence microscopy of epitope-tagged SPT; however, our studies have suggested that SPT subunit 1 might be present also in focal adhesions and the nucleus. These additional locations have been confirmed by confocal microscopy using HEK293 and HeLa cells, and for focal adhesions by the demonstration that SPT1 co-immunoprecipitates with vinculin, a focal adhesion marker protein. The focal adhesion localization of SPT1 is associated with cell morphology, and possibly cell migration, because it is seen in most cells before they reach confluence but disappears when they become confluent, and is restored by a standard scratch-wound healing assay. Conversely, elimination of SPT1 using SPTLC1 siRNA causes cell rounding. Thus, in addition to its “traditional” localization in the ER for de novo sphingolipid biosynthesis, SPT1 is present in other cellular compartments, including focal adhesions where it is associated with cell morphology.     

1-Deoxysphingolipid synthesis compromises anchorage-independent growth and plasma membrane endocytosis in cancer cells

Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty acyl-CoAs into diverse sphingolipids (SLs) that serve as structural components of membranes and signaling molecules within or amongst cells. However, SPT also uses alanine as a substrate in the contexts of low serine availability, alanine accumulation, or disease-causing mutations in hereditary sensory neuropathy type I, resulting in the synthesis and accumulation of 1-deoxysphingolipids (deoxySLs). These species promote cytotoxicity in neurons and impact diverse cellular phenotypes, including suppression of anchorage-independent cancer cell growth. While altered serine and alanine levels can promote 1-deoxySL synthesis, they impact numerous other metabolic pathways important for cancer cells. Here, we combined isotope tracing, quantitative metabolomics, and functional studies to better understand the mechanistic drivers of 1-deoxySL toxicity in cancer cells. We determined that both alanine treatment and SPTLC1^C133W expression induce 1-deoxy(dihydro)ceramide synthesis and accumulation but fail to broadly impact intermediary metabolism, abundances of other lipids, or growth of adherent cells. However, we found that spheroid culture and soft agar colony formation were compromised when endogenous 1-deoxySL synthesis was induced via SPTLC1^C133W expression. Consistent with these impacts on anchorage-independent cell growth, we observed that 1-deoxySL synthesis reduced plasma membrane endocytosis. These results highlight a potential role for SPT promiscuity in linking altered amino acid metabolism to plasma membrane endocytosis.     

The SPTLC3 Subunit of Serine Palmitoyltransferase Generates Short Chain Sphingoid Bases

The enzyme serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in the de novo synthesis of sphingolipids. Previously the mammalian SPT was described as a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Recently we identified a novel third SPT subunit (SPTLC3). SPTLC3 shows about 68% identity to SPTLC2 and also includes a pyridoxal phosphate consensus motif. Here we report that the overexpression of SPTLC3 in HEK293 cells leads to the formation of two new sphingoid base metabolites, namely C₁₆-sphinganine and C₁₆-sphingosine. SPTLC3-expressing cells have higher in vitro SPT activities with lauryl- and myristoyl-CoA than SPTLC2-expressing cells, and SPTLC3 mRNA expression levels correlate closely with the C₁₆-sphinganine synthesis rates in various human and murine cell lines. Approximately 15% of the total sphingolipids in human plasma contain a C₁₆ backbone and are found in the high density and low density but not the very low density lipoprotein fraction. In conclusion, we show that the SPTLC3 subunit generates C₁₆-sphingoid bases and that sphingolipids with a C₁₆ backbone constitute a significant proportion of human plasma sphingolipids.Sphingolipids comprise a class of bioactive lipids that contribute to plasma membrane and plasma lipoprotein formation and exert a broad range of cellular signaling functions such as cell proliferation, endocytosis, and the response of cells to inflammatory and apoptotic stress signals (1–4).Sphingolipids are derived from the aliphatic amino alcohol sphingosine, which is formed from the precursors l-serine and palmitoyl-CoA. The condensation of serine with palmitoyl-CoA is catalyzed by the enzyme serine palmitoyltransferase (SPT)³ (EC 2.3.1.50) and leads to the intermediate 3-ketodihydrosphingosine. 3-Ketodihydrosphingosine is then rapidly converted to dihydrosphingosine (sphinganine) and dihydroceramide. The desaturation of dihydroceramide generates ceramide, and the breakdown of ceramide by ceramidase finally forms sphingosine. The sphingosine backbone of ceramide is usually O-linked to a polar head group such as phosphocholine or carbohydrates and amide-linked to an acyl group. The combination of the sphingosine backbone with different head groups, in particular with various oligosaccharides, leads to a complex variety of different sphingolipid metabolites (5, 6). Moreover, it was shown recently that SPT is also able to use l-alanine as an alternative substrate, thereby generating the atypical sphingoid base 1-deoxysphinganine (7).SPT belongs to the family of pyridoxal phosphate-dependent α-oxoamine synthases. Other members of this family include 5-aminolevulinic acid synthase, 2-amino-3 ketobutyrate ligase, and 8-amino-7-oxononanoate synthase (8). SPT is ubiquitously expressed, and enzyme activity has been detected in all tissues tested so far including brain, lung, liver, kidney, and muscle (9). SPT is essential for embryonic development, and homozygous SPT knock-out mice are not viable (10). SPT has been believed to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. The two subunits SPTLC1 and SPTLC2 show a similarity at AA level of ∼20% and are highly conserved among species. Although both subunits seem to be required for enzyme activity, only the SPTLC2 subunit contains a pyridoxal phosphate binding motif (8, 11).Recently, we identified and cloned a novel third SPT subunit (SPTLC3) (12). The SPTLC3 sequence shows 68% homology to the SPTLC2 subunit and also includes a pyridoxal phosphate consensus motive. The SPTLC3 gene is present in mammals, birds, and some lower vertebrates like fish (Danio rerio) and frog (Xenopus laevis) but not in invertebrate lineages. The SPTLC3 mRNA has been detected in most human tissues with a particularly high expression in placenta (12), indicating a special role for SPTLC3 during development and pregnancy. By using immunoprecipitation, native gel analysis, cross-linking studies, and size exclusion chromatography, it was demonstrated that the native SPT enzyme contains all three subunits and forms a protein complex with a molecular mass of about 460 kDa (13). However, because SPTLC2 and SPTLC3 are encoded by two distinct genes and expressed within the same cell types, we assume a distinct function for the two subunits. One of these differences might be altered substrate affinity or enzymatic activity. This issue is addressed in the present study.     

Cross-talk between Remodeling and de Novo Pathways Maintains Phospholipid Balance through Ubiquitination

Phosphatidylcholine (PtdCho), the major phospholipid of animal membranes, is generated by its remodeling and de novo synthesis. Overexpression of the remodeling enzyme, LPCAT1 (acyl-CoA:lysophosphatidylcholine acyltransferase) in epithelia decreased de novo PtdCho synthesis without significantly altering cellular PtdCho mass. Overexpression of LPCAT1 increased degradation of CPT1 (cholinephosphotransferase), a resident Golgi enzyme that catalyzes the terminal step for de novo PtdCho synthesis. CPT1 degradation involved its multiubiquitination and processing via the lysosomal pathway. CPT1 mutants harboring arginine substitutions at multiple carboxyl-terminal lysines exhibited proteolytic resistance to effects of LPCAT1 overexpression in cells and restored de novo PtdCho synthesis. Thus, cross-talk between phospholipid remodeling and de novo pathways involves ubiquitin-lysosomal processing of a key molecular target that mechanistically provides homeostatic control of cellular PtdCho content.     

De Novo Mutations in Ataxin-2 Gene and ALS Risk

Pathogenic CAG repeat expansion in the ataxin-2 gene (ATXN2) is the genetic cause of spinocerebellar ataxia type 2 (SCA2). Recently, it has been associated with Parkinsonism and increased genetic risk for amyotrophic lateral sclerosis (ALS). Here we report the association of de novo mutations in ATXN2 with autosomal dominant ALS. These findings support our previous conjectures based on population studies on the role of large normal ATXN2 alleles as the source for new mutations being involved in neurodegenerative pathologies associated with CAG expansions. The de novo mutations expanded from ALS/SCA2 non-risk alleles as proven by meta-analysis method. The ALS risk was associated with SCA2 alleles as well as with intermediate CAG lengths in the ATXN2. Higher risk for ALS was associated with pathogenic CAG repeat as revealed by meta-analysis.     

Mutations in the yeast LCB1 and LCB2 genes, including those corresponding to the hereditary sensory neuropathy type I mutations, dominantly inactivate serine palmitoyltransferase.

It was recently demonstrated that mutations in the human SPTLC1 gene, encoding the Lcb1p subunit of serine palmitoyltransferase (SPT), cause hereditary sensory neuropathy type I . As a member of the subfamily of pyridoxal 5'-phosphate enzymes known as the alpha-oxoamine synthases, serine palmitoyltransferase catalyzes the committed step of sphingolipid synthesis. The residues that are mutated to cause hereditary sensory neuropathy type I reside in a highly conserved region of Lcb1p that is predicted to be a catalytic domain of Lcb1p on the basis of alignments with other members of the alpha-oxoamine synthase family. We found that the corresponding mutations in the LCB1 gene of Saccharomyces cerevisiae reduce serine palmitoyltransferase activity. These mutations are dominant and decrease serine palmitoyltransferase activity by 50% when the wild-type and mutant LCB1 alleles are coexpressed. We also show that serine palmitoyltransferase is an Lcb1p small middle dotLcb2p heterodimer and that the mutated Lcb1p proteins retain their ability to interact with Lcb2p. Modeling studies suggest that serine palmitoyltransferase is likely to have a single active site that lies at the Lcb1p small middle dotLcb2p interface and that the mutations in Lcb1p reside near the lysine in Lcb2p that is expected to form the Schiff's base with the pyridoxal 5'-phosphate cofactor. Furthermore, mutations in this lysine and in a histidine residue that is also predicted to be important for pyridoxal 5'-phosphate binding to Lcb2p also dominantly inactivate SPT similar to the hereditary sensory neuropathy type 1-like mutations in Lcb1p.     

The topology of the Lcb1p subunit of yeast serine palmitoyltransferase

The structural organization and topology of the Lcb1p subunit of yeast and mammalian serine palmitoyltransferases (SPT) were investigated. In the yeast protein, three membrane-spanning domains were identified by insertion of glycosylation and factor Xa cleavage sites at various positions. The first domain of the yeast protein, located between residues 50 and 84, was not required for the stability, membrane association, interaction with Lcb2p, or enzymatic activity. Deletion of the comparable domain of the mammalian protein SPTLC1 also had little effect on its function, demonstrating that this region is not required for membrane localization or heterodimerization with SPTLC2. The second and third membrane-spanning domains of yeast Lcb1p, located between residues 342 and 371 and residues 425 and 457, respectively, create a luminal loop of approximately 60 residues. In contrast to the first membrane-spanning domain, the second and third membrane-spanning domains were both required for Lcb1p stability. In addition, mutations in the luminal loop destabilized the SPT heterodimer indicating that this region of the protein is important for SPT structure and function. Mutations in the extreme carboxyl-terminal region of Lcb1p also disrupted heterodimer formation. Taken together, these data suggest that in contrast to other members of the alpha-oxoamine synthases that are soluble homodimers, the Lcb1p and Lcb2p subunits of the SPT heterodimer may interact in the cytosol, as well as within the membrane and/or the lumen of the endoplasmic reticulum.     

Ribonucleotide reductase subunit p53R2 regulates mitochondria homeostasis and function in KB and PC-3 cancer cells

Ribonucleotide reductase (RR) is a rate-limiting enzyme that catalyzes de novo conversion of ribonucleotide 5′-diphosphates to the corresponding 2′-deoxynucleotide, essential for DNA synthesis and replication. The mutations or knockout of RR small subunit, p53R2, results in the depletion of mitochondrial DNA (mtDNA) in human, implying that p53R2 might play a critical role for maintaining mitochondrial homeostasis. In this study, siRNA against p53R2 knockdown approach is utilized to examine the impact of p53R2 depletion on mitochondria and to derive underlying mechanism in KB and PC-3 cancer cells. Our results reveal that the p53R2 expression not only positively correlates with mtDNA content, but also partakes in the proper mitochondria function, such as ATP synthesis, cytochrome c oxidase activity and membrane potential maintenance. Furthermore, overexpression of p53R2 reduces intracellular ROS and protects the mitochondrial membrane potential against oxidative stress. Unexpectedly, knockdown of p53R2 has a modest, if any, effect on mitochondrial and total cellular dNTP pools. Taken together, our study provides functional evidence that mitochondria is one of p53R2-targeted organelles and suggests an unexpected function of p53R2, which is beyond known RR function on dNTP synthesis, in mitochondrial homeostatic control.     

Anti-inflammatory action of lipid nanocarrier-delivered myriocin: therapeutic potential in cystic fibrosis

Background

Sphingolipids take part in immune response and can initiate and/or sustain inflammation. Various inflammatory diseases have been associated with increased ceramide content, and pharmacological reduction of ceramide diminishes inflammation damage in vivo. Inflammation and susceptibility to microbial infection are two elements in a vicious circle. Recently, sphingolipid metabolism inhibitors were used to reduce infection. Cystic fibrosis (CF) is characterized by a hyper-inflammation and an excessive innate immune response, which fails to evolve into adaptive immunity and to eradicate infection. Chronic infections result in lung damage and patient morbidity. Notably, ceramide content in mucosa airways is higher in CF mouse models and in patients than in control mice or healthy subjects.

Methods

The therapeutic potential of myriocin, an inhibitor of the sphingolipid de novo synthesis rate limiting enzyme (Serine Palmitoyl Transferase, SPT),was investigated in CF cells and mice models.

Results

We treated CF human respiratory epithelial cells with myriocin, This treatment resulted in reduced basal, as well as TNFα-stimulated, inflammation. In turn, TNFα induced an increase in SPT in these cells, linking de novo synthesis of ceramide to inflammation. Furthermore, myriocin-loaded nanocarrier, injected intratrachea prior to P. aeruginosa challenge, enabled a significant reduction of lung infection and reduced inflammation.

Conclusions

The presented data suggest that de novo ceramide synthesis is constitutively enhanced in CF mucosa and that it can be envisaged as pharmacological target for modulating inflammation and restoring effective innate immunity against acute infection.

General significance

Myriocin stands as a powerful immunomodulatory agent for inflammatory and infectious diseases.     

Two de novo mutations of MFN2 associated with early-onset Charcot-Marie-Tooth disease type 2A neuropathy

Charcot-Marie-Tooth disease type 2A (CMT2A) is one of the subdivisions of CMT2, an axonal defective form of peripheral neuropathy. Different mutations in the mitochondrial GTPase mitofusin 2 (MFN2) gene produce various degrees of severity of CMT2A phenotype or CMT2A related hereditary motor and sensory neuropathy VI (HMSN VI). The occurrence of de novo mutations in MFN2 is by far the most frequent as compared to other CMT genes. About 26% of the pathogenic MFN2 mutations reported in the Inherited Peripheral Neuropathies Mutations Database are de novo. This study identified two de novo mutations of MFN2, c.1048T>C (S350P) and c.310C>T (R104W), from two Korean CMT2A patients with early onset severe clinical symptoms. The comparative genotype-phenotype correlations of these mutations according to a previously reported case were also viewed. The R104W mutation has been reported recurrently, outspread over different ethnic backgrounds as de novo. The re-occurrence of the same pathogenic de novo variants within and amongst different ethnic groups clearly suggests a susceptible hot spot for mutation in the MFN2 gene. If the deleterious mutations discourage fitness and reproduction, this negative selection factor should ultimately reduce the prevalence of the disease. It appears that spontaneous de novo mutations in turn seem to be maintaining the disease phenotype??s prevalence.     

Mitochondrial protein alterations in a familial peripheral neuropathy caused by the V144D amino acid mutation in the sphingolipid protein,SPTLC1

Axonal degeneration is the final common path in many neurological disorders. Subsets of neuropathies involving the sensory neuron are known as hereditary sensory neuropathies (HSNs). Hereditary sensory neuropathy type I (HSN-I) is the most common subtype of HSN with autosomal dominant inheritance. It is characterized by the progressive degeneration of the dorsal root ganglion (DRG) with clinical symptom onset between the second or third decade of life. Heterozygous mutations in the serine palmitoyltransferase (SPT) long chain subunit 1 (SPTLC1) gene were identified as the pathogenic cause of HSN-I. Ultrastructural analysis of mitochondria from HSN-I patient cells has displayed unique morphological abnormalities that are clustered to the perinucleus where they are wrapped by the endoplasmic reticulum (ER). This investigation defines a small subset of proteins with major alterations in abundance in mitochondria harvested from HSN-I mutant SPTLC1 cells. Using mitochondrial protein isolates from control and patient lymphoblasts, and a combination of 2D gel electrophoresis, immunoblotting and mass spectrometry, we have shown the increased abundance of ubiquinol-cytochrome c reductase core protein 1, an electron transport chain protein, as well as the immunoglobulin, Ig kappa chain C. The regulation of these proteins may provide a new route to understanding the cellular and molecular mechanisms underlying HSN-I.     

Infantile-onset ascending hereditary spastic paraplegia with bulbar involvement due to the novel ALS2 mutation c.2761C > T

Recessive mutations in the alsin gene cause three clinically distinct motor neuron diseases: juvenile amyotrophic lateral sclerosis (ALS2), juvenile primary lateral sclerosis (JPLS) and infantile-onset ascending hereditary spastic paraplegia (IAHSP). A total of 23 different ALS2 mutations have been described for the three disorders so far. Most of these mutations result in a frameshift leading to a premature truncation of the alsin protein. We report the novel ALS2 truncating mutation c.2761C > T; p.R921X detected by homozygosity mapping and sequencing in two infants affected by IAHSP with bulbar involvement. The mutation c.2761C > T resides in the pleckstrin domain, a characteristic segment of guanine nucleotide exchange factors of the Rho GTPase family, which is involved in the overall neuronal development or maintenance. This study highlights the importance of using homozygosity mapping combined with candidate gene analysis to identify the underlying genetic defect as in this Saudi consanguineous family.