The conformational plasticity of structurally unrelated lipid transport proteins correlates with their mode of action.

Lipid transfer proteins (LTPs) are key players in cellular homeostasis and regulation, as they coordinate the exchange of lipids between different cellular organelles. Despite their importance, our mechanistic understanding of how LTPs function at the molecular level is still in its infancy, mostly due to the large number of existing LTPs and to the low degree of conservation at the sequence and structural level. In this work, we use molecular simulations to characterize a representative dataset of lipid transport domains (LTDs) of 12 LTPs that belong to 8 distinct families. We find that despite no sequence homology nor structural conservation, the conformational landscape of LTDs displays common features, characterized by the presence of at least 2 main conformations whose populations are modulated by the presence of the bound lipid. These conformational properties correlate with their mechanistic mode of action, allowing for the interpretation and design of experimental strategies to further dissect their mechanism. Our findings indicate the existence of a conserved, fold-independent mechanism of lipid transfer across LTPs of various families and offer a general framework for understanding their functional mechanism.

Recurrent de-novo gain-of-function mutation in SPTLC2 confirms dysregulated sphingolipid production to cause juvenile amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) leads to paralysis and death by progressive degeneration of motor neurons. Recently, specific gain-of-function mutations in SPTLC1 were identified in patients with juvenile form of ALS. SPTLC2 encodes the second catalytic subunit of the serine-palmitoyltransferase (SPT) complex. METHODS: We used the GENESIS platform to screen 700 ALS whole-genome and whole-exome data sets for variants in SPTLC2. The de-novo status was confirmed by Sanger sequencing. Sphingolipidomics was performed using liquid chromatography and high-resolution mass spectrometry. RESULTS: Two unrelated patients presented with early-onset progressive proximal and distal muscle weakness, oral fasciculations, and pyramidal signs. Both patients carried the novel de-novo SPTLC2 mutation, c.203T>G, p.Met68Arg. This variant lies within a single short transmembrane domain of SPTLC2, suggesting that the mutation renders the SPT complex irresponsive to regulation through ORMDL3. Confirming this hypothesis, ceramide and complex sphingolipid levels were significantly increased in patient plasma. Accordingly, excessive sphingolipid production was shown in mutant-expressing human embryonic kindney (HEK) cells. CONCLUSIONS: Specific gain-of-function mutations in both core subunits affect the homoeostatic control of SPT. SPTLC2 represents a new Mendelian ALS gene, highlighting a key role of dysregulated sphingolipid synthesis in the pathogenesis of juvenile ALS. Given the direct interaction of SPTLC1 and SPTLC2, this knowledge might open new therapeutic avenues for motor neuron diseases.

Recurrent de novo SPTLC2 variant causes childhood-onset amyotrophic lateral sclerosis (ALS) by excess sphingolipid synthesis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the upper and lower motor neurons with varying ages of onset, progression and pathomechanisms. Monogenic childhood-onset ALS, although rare, forms an important subgroup of ALS. We recently reported specific SPTLC1 variants resulting in sphingolipid overproduction as a cause for juvenile ALS. Here, we report six patients from six independent families with a recurrent, de novo, heterozygous variant in SPTLC2 c.778G>A [p.Glu260Lys] manifesting with juvenile ALS. METHODS: Clinical examination of the patients along with ancillary and genetic testing, followed by biochemical investigation of patients' blood and fibroblasts, was performed. RESULTS: All patients presented with early-childhood-onset progressive weakness, with signs and symptoms of upper and lower motor neuron degeneration in multiple myotomes, without sensory neuropathy. These findings were supported on ancillary testing including nerve conduction studies and electromyography, muscle biopsies and muscle ultrasound studies. Biochemical investigations in plasma and fibroblasts showed elevated levels of ceramides and unrestrained de novo sphingolipid synthesis. Our studies indicate that SPTLC2 variant [c.778G>A, p.Glu260Lys] acts distinctly from hereditary sensory and autonomic neuropathy (HSAN)-causing SPTLC2 variants by causing excess canonical sphingolipid biosynthesis, similar to the recently reported SPTLC1 ALS associated pathogenic variants. Our studies also indicate that serine supplementation, which is a therapeutic in SPTLC1 and SPTCL2-associated HSAN, is expected to exacerbate the excess sphingolipid synthesis in serine palmitoyltransferase (SPT)-associated ALS. CONCLUSIONS: SPTLC2 is the second SPT-associated gene that underlies monogenic, juvenile ALS and further establishes alterations of sphingolipid metabolism in motor neuron disease pathogenesis. Our findings also have important therapeutic implications: serine supplementation must be avoided in SPT-associated ALS, as it is expected to drive pathogenesis further.

Subunit composition of the mammalian serine-palmitoyltransferase defines the spectrum of straight and methyl-branched long-chain bases.

Sphingolipids (SLs) are chemically diverse lipids that have important structural and signaling functions within mammalian cells. SLs are commonly defined by the presence of a long-chain base (LCB) that is normally formed by the conjugation of l-serine and palmitoyl-CoA. This pyridoxal 5-phosphate (PLP)-dependent reaction is mediated by the enzyme serine-palmitoyltransferase (SPT). However, SPT can also metabolize other acyl-CoAs, in the range of C14 to C18, forming a variety of LCBs that differ by structure and function. Mammalian SPT consists of three core subunits: SPTLC1, SPTLC2, and SPTLC3. Whereas SPTLC1 and SPTLC2 are ubiquitously expressed, SPTLC3 expression is restricted to certain tissues only. The influence of the individual subunits on enzyme activity is not clear. Using cell models deficient in SPTLC1, SPTLC2, and SPTLC3, we investigated the role of each subunit on enzyme activity and the LCB product spectrum. We showed that SPTLC1 is essential for activity, whereas SPTLC2 and SPTLC3 are partly redundant but differ in their enzymatic properties. SPTLC1 in combination with SPTLC2 specifically formed C18, C19, and C20 LCBs while the combination of SPTLC1 and SPTLC3 yielded a broader product spectrum. We identified anteiso-branched-C18 SO (meC18SO) as the primary product of the SPTLC3 reaction. The meC18SO was synthesized from anteiso-methyl-palmitate, in turn synthesized from a precursor metabolite generated in the isoleucine catabolic pathway. The meC18SO is metabolized to ceramides and complex SLs and is a constituent of human low- and high-density lipoproteins.

Precision mouse models of Yars/dominant intermediate Charcot-Marie-Tooth disease type C and Sptlc1/hereditary sensory and autonomic neuropathy type 1.

Animal models of neurodegenerative diseases such as inherited peripheral neuropathies sometimes accurately recreate the pathophysiology of the human disease, and sometimes accurately recreate the genetic perturbations found in patients. Ideally, models achieve both, but this is not always possible; nonetheless, such models are informative. Here we describe two animal models of inherited peripheral neuropathy: mice with a mutation in tyrosyl tRNA-synthetase, YarsE196K , modeling dominant intermediate Charcot-Marie-Tooth disease type C (diCMTC), and mice with a mutation in serine palmitoyltransferase long chain 1, Sptlc1C133W , modeling hereditary sensory and autonomic neuropathy type 1 (HSAN1). YarsE196K mice develop disease-relevant phenotypes including reduced motor performance and reduced nerve conduction velocities by 4 months of age. Peripheral motor axons are reduced in size, but there is no reduction in axon number and plasma neurofilament light chain levels are not increased. Unlike the dominant human mutations, the YarsE196K mice only show these phenotypes as homozygotes, or as compound heterozygotes with a null allele, and no phenotype is observed in E196K or null heterozygotes. The Sptlc1C133W mice carry a knockin allele and show the anticipated increase in 1-deoxysphingolipids in circulation and in a variety of tissues. They also have mild behavioral defects consistent with HSAN1, but do not show neurophysiological defects or axon loss in peripheral nerves or in the epidermis of the hind paw or tail. Thus, despite the biochemical phenotype, the Sptlc1C133W mice do not show a strong neuropathy phenotype. Surprisingly, these mice were lethal as homozygotes, but the heterozygous genotype studied corresponds to the dominant genetics seen in humans. Thus, YarsE196K homozygous mice have a relevant phenotype, but imprecisely reproduce the human genetics, whereas the Sptlc1C133W mice precisely reproduce the human genetics, but do not recreate the disease phenotype. Despite these shortcomings, both models are informative and will be useful for future research.

Serine Palmitoyltransferase Subunit 3 and Metabolic Diseases.

Sphingolipids (SL) are a class of chemically diverse lipids that have important structural and physiological functions in eukaryotic cells. SL entail a long chain base (LCB) as the common structural element, which is typically formed by the condensation of L-serine and long chain acyl-CoA. This condensation is the first and the rate-limiting step in the de novo SL synthesis and catalyzed by the enzyme serine palmitoyltransferase (SPT). Although palmitoyl-CoA is the preferred substrate, SPT can also metabolize other acyl-CoAs, thereby forming a variety of LCBs, which differ in structures and functions. The mammalian SPT enzyme is composed of three core subunits: SPTLC1, SPTLC2, and SPTLC3. Whereas SPTLC1 and SPTLC2 are ubiquitously expressed, SPTLC3 expression is restricted to a few specific tissues. The SPTLC1 subunit is essential and can associate with either SPTLC2 or SPTLC3 to form an active enzyme. Depending on the stoichiometry of the SPTLC2 and SPTLC3 subunits, the spectrum of SPT products varies. While SPTLC1 and SPTLC2 primarily form C18 and C20 LCBs, the combination of SPTLC1 and SPTLC3 produces a broader spectrum of LCBs. Genetic and population based studies have shown that SPTLC3 expression and function are associated with an altered plasma SL profile and an increased risk for cardio-metabolic diseases. Animal and in vitro studies showed that SPTLC3 might be involved in hepatic and cardiac pathology and could be a therapeutic target for these conditions.Here we present an overview of the current data on the role of SPTLC3 in normal and pathological conditions.

Metabolism of HSAN1- and T2DM-associated 1-deoxy-sphingolipids inhibits the migration of fibroblasts.

Hereditary sensory neuropathy type 1 (HSAN1) is a rare axonopathy, characterized by a progressive loss of sensation (pain, temperature, and vibration), neuropathic pain, and wound healing defects. HSAN1 is caused by several missense mutations in the serine palmitoyltransferase long-chain base subunit 1 and serine palmitoyltransferase long-chain base subunit 2 of the enzyme serine palmitoyltransferase-the key enzyme for the synthesis of sphingolipids. The mutations change the substrate specificity of serine palmitoyltransferase, which then forms an atypical class of 1-deoxy-sphinglipids (1-deoxySLs). Similarly, patients with type 2 diabetes mellitus also present with elevated 1-deoxySLs and a comparable clinical phenotype. The effect of 1-deoxySLs on neuronal cells was investigated in detail, but their impact on other cell types remains elusive. Here, we investigated the consequences of externally added 1-deoxySLs on the migration of fibroblasts in a scratch assay as a simplified cellular wound-healing model. We showed that 1-deoxy-sphinganine (1-deoxySA) inhibits the migration of NIH-3T3 fibroblasts in a dose- and time-dependent manner. This was not seen for a non-native, L-threo stereoisomer. Supplemented 1-deoxySA was metabolized to 1-deoxy-(dihydro)ceramide and downstream to 1-deoxy-sphingosine. Inhibiting downstream metabolism by blocking N-acylation rescued the migration phenotype. In contrast, adding 1-deoxy-sphingosine had a lesser effect on cell migration but caused the massive formation of intracellular vacuoles. Further experiments showed that the effect on cell migration was primarily mediated by 1-deoxy-dihydroceramides rather than by the free base or 1-deoxyceramides. Based on these findings, we suggest that limiting the N-acylation of 1-deoxySA could be a therapeutic approach to improve cell migration and wound healing in patients with HSAN1 and type 2 diabetes mellitus.

Farnesoid X receptor activation induces the degradation of hepatotoxic 1-deoxysphingolipids in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Patients with non-alcoholic fatty liver disease (NAFLD) exhibit higher levels of plasma 1-deoxysphingolipids than healthy individuals. The aim of this study was to investigate the role of farnesoid X receptor (FXR) in 1-deoxysphingolipid de novo synthesis and degradation. METHODS: Mice were fed with a high-fat diet (HFD) to induce obesity and NAFLD, and then treated with the FXR ligand obeticholic acid (OCA). Histology and gene expression analysis were performed on liver tissue. Sphingolipid patterns from NAFLD patients and mouse models were assessed by liquid chromatography-mass spectrometry. The molecular mechanism underlying the effect of FXR activation on sphingolipid metabolism was studied in Huh7 cells and primary cultured hepatocytes, as well as in a 1-deoxysphinganine-treated mouse model. RESULTS: 1-deoxysphingolipids were increased in both NAFLD patients and mouse models. FXR activation by OCA protected the liver against oxidative stress, apoptosis, and reduced 1-deoxysphingolipid levels, both in a HFD-induced mouse model of obesity and in 1-deoxysphinganine-treated mice. In vitro, FXR activation lowered intracellular 1-deoxysphingolipid levels by inducing Cyp4f-mediated degradation, but not by inhibiting de novo synthesis, thereby protecting hepatocytes against doxSA-induced cytotoxicity, mitochondrial damage, and apoptosis. Overexpression of Cyp4f13 in cells was sufficient to ameliorate doxSA-induced cytotoxicity. Treatment with the Cyp4f pan-inhibitor HET0016 or FXR knock-down fully abolished the protective effect of OCA, indicating that OCA-mediated 1-deoxysphingolipid degradation is FXR and Cyp4f dependent. CONCLUSIONS: Our study identifies FXR-Cyp4f as a novel regulatory pathway for 1-deoxysphingolipid metabolism. FXR activation represents a promising therapeutic strategy for patients with metabolic syndrome and NAFLD.

Accumulation of long-chain bases in yeast promotes their conversion to a long-chain base vinyl ether.

Long-chain bases (LCBs) are the precursors to ceramide and sphingolipids in eukaryotic cells. They are formed by the action of serine palmitoyl-CoA transferase (SPT), a complex of integral membrane proteins located in the endoplasmic reticulum. SPT activity is negatively regulated by Orm proteins to prevent the toxic overaccumulation of LCBs. Here we show that overaccumulation of LCBs in yeast results in their conversion to a hitherto undescribed LCB derivative, an LCB vinyl ether. The LCB vinyl ether is predominantly formed from phytosphingosine (PHS) as revealed by conversion of odd chain length tracers C17-dihydrosphingosine and C17-PHS into the corresponding LCB vinyl ether derivative. PHS vinyl ether formation depends on ongoing acetyl-CoA synthesis, and its levels are elevated when the LCB degradative pathway is blocked by deletion of the major LCB kinase, LCB4, or the LCB phosphate lyase, DPL1. PHS vinyl ether formation thus appears to constitute a shunt for the LCB phosphate- and lyase-dependent degradation of LCBs. Consistent with a role of PHS vinyl ether formation in LCB detoxification, the lipid is efficiently exported from the cells.

Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis.

Proper functioning of intracellular membranes is critical for many cellular processes. A key feature of membranes is their ability to adapt to changes in environmental conditions by adjusting their composition so as to maintain constant biophysical properties, including fluidity and flexibility. Similar changes in the biophysical properties of membranes likely occur when intracellular processes, such as vesicle formation and fusion, require dramatic changes in membrane curvature. Similar modifications must also be made when nuclear pore complexes (NPCs) are constructed within the existing nuclear membrane, as occurs during interphase in all eukaryotes. Here we report on the role of the essential nuclear envelope/endoplasmic reticulum (NE/ER) protein Brl1 in regulating the membrane composition of the NE/ER. We show that Brl1 and two other proteins characterized previously-Brr6, which is closely related to Brl1, and Apq12-function together and are required for lipid homeostasis. All three transmembrane proteins are localized to the NE and can be coprecipitated. As has been shown for mutations affecting Brr6 and Apq12, mutations in Brl1 lead to defects in lipid metabolism, increased sensitivity to drugs that inhibit enzymes involved in lipid synthesis, and strong genetic interactions with mutations affecting lipid metabolism. Mutations affecting Brl1 or Brr6 or the absence of Apq12 leads to hyperfluid membranes, because mutant cells are hypersensitive to agents that increase membrane fluidity. We suggest that the defects in nuclear pore complex biogenesis and mRNA export seen in these mutants are consequences of defects in maintaining the biophysical properties of the NE.