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.

Immunoglobulin domain interface exchange as a platform technology for the generation of Fc heterodimers and bispecific antibodies.

Bispecific antibodies (bsAbs) are of significant importance to the development of novel antibody-based therapies, and heavy chain (Hc) heterodimers represent a major class of bispecific drug candidates. Current technologies for the generation of Hc heterodimers are suboptimal and often suffer from contamination by homodimers posing purification challenges. Here, we introduce a new technology based on biomimicry wherein the protein-protein interfaces of two different immunoglobulin (Ig) constant domain pairs are exchanged in part or fully to design new heterodimeric domains. The method can be applied across Igs to design Fc heterodimers and bsAbs. We investigated interfaces from human IgA CH3, IgD CH3, IgG1 CH3, IgM CH4, T-cell receptor (TCR) α/ß, and TCR γ/δ constant domain pairs, and we found that they successfully drive human IgG1 CH3 or IgM CH4 heterodimerization to levels similar to or above those of reference methods. A comprehensive interface exchange between the TCR α/ß constant domain pair and the IgG1 CH3 homodimer was evidenced by X-ray crystallography and used to engineer examples of bsAbs for cancer therapy. Parental antibody pairs were rapidly reformatted into scalable bsAbs that were free of homodimer traces by combining interface exchange, asymmetric Protein A binding, and the scFv × Fab format. In summary, we successfully built several new CH3- or CH4-based heterodimers that may prove useful for designing new bsAb-based therapeutics, and we anticipate that our approach could be broadly implemented across the Ig constant domain family. To our knowledge, CH4-based heterodimers have not been previously reported.

HSAN1 mutations in serine palmitoyltransferase reveal a close structure-function-phenotype relationship.

Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is a rare autosomal dominant inherited peripheral neuropathy caused by mutations in the SPTLC1 and SPTLC2 subunits of serine palmitoyltransferase (SPT). The mutations induce a permanent shift in the substrate preference from L-serine to L-alanine, which results in the pathological formation of atypical and neurotoxic 1-deoxy-sphingolipids (1-deoxySL). Here we compared the enzymatic properties of 11 SPTLC1 and six SPTLC2 mutants using a uniform isotope labelling approach. In total, eight SPT mutants (STPLC1p.C133W, p.C133Y, p.S331F, p.S331Y and SPTLC2p.A182P, p.G382V, p.S384F, p.I504F) were associated with increased 1-deoxySL synthesis. Despite earlier reports, canonical activity with l-serine was not reduced in any of the investigated SPT mutants. Three variants (SPTLC1p.S331F/Y and SPTLC2p.I505Y) showed an increased canonical activity and increased formation of C20 sphingoid bases. These three mutations are associated with an exceptionally severe HSAN1 phenotype, and increased C20 sphingosine levels were also confirmed in plasma of patients. A principal component analysis of the analysed sphingoid bases clustered the mutations into three separate entities. Each cluster was related to a distinct clinical outcome (no, mild and severe HSAN1 phenotype). A homology model based on the protein structure of the prokaryotic SPT recapitulated the same grouping on a structural level. Mutations associated with the mild form clustered around the active site, whereas mutations associated with the severe form were located on the surface of the protein. In conclusion, we showed that HSAN1 mutations in SPT have distinct biochemical properties, which allowed for the prediction of the clinical symptoms on the basis of the plasma sphingoid base profile.

Structural snapshots along the reaction pathway of ferredoxin-thioredoxin reductase.

Oxygen-evolving photosynthetic organisms regulate carbon metabolism through a light-dependent redox signalling pathway. Electrons are shuttled from photosystem I by means of ferredoxin (Fdx) to ferredoxin-thioredoxin reductase (FTR), which catalyses the two-electron-reduction of chloroplast thioredoxins (Trxs). These modify target enzyme activities by reduction, regulating carbon flow. FTR is unique in its use of a [4Fe-4S] cluster and a proximal disulphide bridge in the conversion of a light signal into a thiol signal. We determined the structures of FTR in both its one- and its two-electron-reduced intermediate states and of four complexes in the pathway, including the ternary Fdx-FTR-Trx complex. Here we show that, in the first complex (Fdx-FTR) of the pathway, the Fdx [2Fe-2S] cluster is positioned suitably for electron transfer to the FTR [4Fe-4S] centre. After the transfer of one electron, an intermediate is formed in which one sulphur atom of the FTR active site is free to attack a disulphide bridge in Trx and the other sulphur atom forms a fifth ligand for an iron atom in the FTR [4Fe-4S] centre--a unique structure in biology. Fdx then delivers a second electron that cleaves the FTR-Trx heterodisulphide bond, which occurs in the Fdx-FTR-Trx complex. In this structure, the redox centres of the three proteins are aligned to maximize the efficiency of electron transfer from the Fdx [2Fe-2S] cluster to the active-site disulphide of Trxs. These results provide a structural framework for understanding the mechanism of disulphide reduction by an iron-sulphur enzyme and describe previously unknown interaction networks for both Fdx and Trx (refs 4-6).

SPTLC1 p.Leu38Arg, a novel mutation associated with childhood ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neuromuscular disease. Recently, several gain-of-function mutations in SPTLC1 were associated with juvenile ALS. SPTLC1 encodes for a subunit of the serine-palmitoyltransferase (SPT) - the rate-limiting enzyme in the de novo synthesis of sphingolipids (SL). SPT activity, and thus SL de novo synthesis, is tightly controlled by a homeostatic feedback mechanism mediated by ORMDL proteins. Here we report a novel SPTLC1p.L38R mutation in a young Chinese girl with a signature of juvenile ALS. The patient presented with muscular weakness and atrophy, tongue tremor and fasciculation, breathing problems and positive pyramidal signs. All SPTLC1-ALS mutations including the SPTLC1 p.L38R are located within a single membrane-spanning domain of the protein and impede the interaction with the regulatory ORMDL subunit of SPT. Pertinent to the altered homeostatic control, lipid analysis showed overall increased SL levels in the patient plasma. An increased SPT activity and SL de novo synthesis was confirmed in p.L38R expressing HEK293 cells. Particularily dihydro-sphingolipids (dhSL) were signficantly increased in patient plasma and p.L38R mutant expressing cells. Increased dhSL formation has been previously linked to neurotoxicity and may be involved in the pathomechanism of SPTLC1-ALS mutations.

Role of histidine-86 in the catalytic mechanism of ferredoxin:thioredoxin reductase.

Ferredoxin:thioredoxin reductase catalyzes the reduction of thioredoxins in plant chloroplasts using the [Fe2S2] ferredoxin as a one-electron donor and as such plays a central role in light regulation of oxygenic photosynthesis. The active-site comprises a [Fe4S4] cluster next to a redox-active disulfide that is cleaved in sequential one-electron steps and the combination of spectroscopic and crystallographic studies have revealed a catalytic mechanism involving novel site specific cluster chemistry in the oxidized, one-electron- and two-electron-reduced redox states. Histidine-86 has emerged as a potential proton donor/acceptor in the catalytic mechanism based on redox-related changes in the positioning of the imidazole ring during redox cycling and greatly decreased activity for the H86Y variant. Here we report on spectroscopic and redox characterization of the [Fe4S4] center in Synechocystis sp. PCC 6803 H86Y ferredoxin:thoredoxin reductase in the accessible redox states of both the as purified and N-ethylmaleimide-modified forms, using the combination of UV-visible absorption and variable-temperature magnetic circular dichroism, EPR, resonance Raman and Mössbauer spectroscopies. The results demonstrate that His86 is required for formation of the partially valence-localized [Fe4S4]2+ cluster that is the hallmark of two-electron-reduced intermediate. Taken together with the available structural data, the spectroscopic results indicate a functional role for His86 in protonation/deprotonation of the cluster-interacting thiol and anchoring the cluster interacting thiol in close proximity to the cluster in the two-electron-reduced intermediate.

PLP-dependent enzymes as entry and exit gates of sphingolipid metabolism.

Sphingolipids are membrane constituents as well as signaling molecules involved in many essential cellular processes. Serine palmitoyltransferase (SPT) and sphingosine-1-phosphate lyase (SPL), both PLP (pyridoxal 5'-phosphate)-dependent enzymes, function as entry and exit gates of the sphingolipid metabolism. SPT catalyzes the condensation of serine and a fatty acid into 3-keto-dihydrosphingosine, whereas SPL degrades sphingosine-1-phosphate (S1P) into phosphoethanolamine and a long-chain aldehyde. The recently solved X-ray structures of prokaryotic homologs of SPT and SPL combined with functional studies provide insight into the structure-function relationship of the two enzymes. Despite carrying out different reactions, the two enzymes reveal striking similarities in the overall fold, topology, and residues crucial for activity. Unlike their eukaryotic counterparts, bacterial SPT and SPL lack a transmembrane helix, making them targets of choice for biochemical characterization because the use of detergents can be avoided. Both human enzymes are linked to severe diseases or disorders and might therefore serve as targets for the development of therapeutics aiming at the modulation of their activity. This review gives an overview of the sphingolipid metabolism and of the available biochemical studies of prokaryotic SPT and SPL, and discusses the major similarities and differences to the corresponding eukaryotic enzymes.

A prokaryotic S1P lyase degrades extracellular S1P in vitro and in vivo: implication for treating hyperproliferative disorders.

Sphingosine-1-phosphate (S1P) regulates a broad spectrum of fundamental cellular processes like proliferation, death, migration and cytokine production. Therefore, elevated levels of S1P may be causal to various pathologic conditions including cancer, fibrosis, inflammation, autoimmune diseases and aberrant angiogenesis. Here we report that S1P lyase from the prokaryote Symbiobacterium thermophilum (StSPL) degrades extracellular S1P in vitro and in blood. Moreover, we investigated its effect on cellular responses typical of fibrosis, cancer and aberrant angiogenesis using renal mesangial cells, endothelial cells, breast (MCF-7) and colon (HCT 116) carcinoma cells as disease models. In all cell types, wild-type StSPL, but not an inactive mutant, disrupted MAPK phosphorylation stimulated by exogenous S1P. Functionally, disruption of S1P receptor signaling by S1P depletion inhibited proliferation and expression of connective tissue growth factor in mesangial cells, proliferation, migration and VEGF expression in carcinoma cells, and proliferation and migration of endothelial cells. Upon intravenous injection of StSPL in mice, plasma S1P levels rapidly declined by 70% within 1 h and then recovered to normal 6 h after injection. Using the chicken chorioallantoic membrane model we further demonstrate that also under in vivo conditions StSPL, but not the inactive mutant, inhibited tumor cell-induced angiogenesis as an S1P-dependent process. Our data demonstrate that recombinant StSPL is active under extracellular conditions and holds promise as a new enzyme therapeutic for diseases associated with increased levels of S1P and S1P receptor signaling.

Structure and function of sphingosine-1-phosphate lyase, a key enzyme of sphingolipid metabolism.

Sphingosine-1-phosphate lyase (SPL), a key enzyme of sphingolipid metabolism, catalyzes the irreversible degradation of sphingoid base phosphates. Its main substrate sphingosine-1-phosphate (S1P) acts both extracellularly, by binding G protein-coupled receptors of the lysophospholipid receptor family, and inside the cell, as a second messenger. There, S1P takes part in regulating various cellular processes and its levels are tightly regulated. SPL is a pivotal enzyme regulating S1P intracellular concentrations and a promising drug target for the design of immunosuppressants. We structurally and functionally characterized yeast SPL (Dpl1p) and its first prokaryotic homolog, from Symbiobacterium thermophilum. The Dpl1p structure served as a basis for a very reliable model of Homo sapiens SPL. The above results, together with in vitro and in vivo studies of SPL mutants, reveal which residues are involved in activity and substrate binding and pave the way to studies aimed at controlling the activity of this pivotal enzyme.

Spectroscopic characterization of site-specific [Fe(4)S(4)] cluster chemistry in ferredoxin:thioredoxin reductase: implications for the catalytic mechanism.

Light regulation of enzyme activities in oxygenic photosynthesis is mediated by ferredoxin:thioredoxin reductase (FTR), a novel class of disulfide reductase with an active site comprising a [Fe(4)S(4)](2+) cluster and an adjacent disulfide, that catalyzes reduction of the thioredoxin disulfide in two sequential one-electron steps using a [Fe(2)S(2)](2+/+) ferredoxin as the electron donor. In this work, we report on spectroscopic (EPR, VTMCD, resonance Raman, and Mössbauer) and redox characterization of the active site of FTR in various forms of the enzyme, including wild-type FTR, point-mutation variants at each of the active-site cysteine residues, and stable analogues of the one-electron-reduced FTR-Trx heterodisulfide intermediate. The results reveal novel site-specific Fe(4)S(4)-cluster chemistry in oxidized, one-electron-reduced, and two-electron-reduced forms of FTR. In the resting enzyme, a weak interaction between the Fe(4)S(4) cluster and the active-site disulfide promotes charge buildup at a unique Fe site and primes the active site to accept an electron from ferredoxin to break the disulfide bond. In one-electron-reduced analogues, cleavage of the active-site disulfide is accompanied by coordination of one of the cysteine residues that form the active-site disulfide to yield a [Fe(4)S(4)](3+) cluster with two cysteinate ligands at a unique Fe site. The most intriguing result is that two-electron-reduced FTR in which the disulfide is reduced to a dithiol contains an unprecedented electron-rich [Fe(4)S(4)](2+) cluster comprising both valence-delocalized and valence-localized Fe(2+)Fe(3+) pairs. These results provide molecular level insights into the catalytic mechanism of FTR, and two viable mechanisms are proposed.

Characterization of ferredoxin:thioredoxin reductase modified by site-directed mutagenesis.

Ferredoxin:thioredoxin reductase (FTR) is a key regulatory enzyme of oxygenic photosynthetic cells involved in the reductive regulation of important target enzymes. It catalyzes the two-electron reduction of the disulfide of thioredoxins with electrons from ferredoxin involving a 4Fe-4S cluster and an adjacent active-site disulfide. We replaced Cys-57, Cys-87, and His-86 in the active site of Synechocystis FTR by site-directed mutagenesis and studied the properties of the mutated proteins. Mutation of either of the active-site cysteines yields inactive enzymes, which have different spectral properties, indicating a reduced Fe-S cluster when the inaccessible Cys-87 is replaced and an oxidized cluster when the accessible Cys-57 is replaced. The oxidized cluster in the latter mutant can be reversibly reduced with dithionite showing that it is functional. The C57S mutant is a very stable protein, whereas the C87A mutant is more labile because of the missing interaction with the cluster. The replacement of His-86 greatly reduces its catalytic activity supporting the proposal that His-86 increases the nucleophilicity of the neighboring cysteine. Ferredoxin forms non-covalent complexes with wild type (WT) and mutant FTRs, which are stable except with the C87A mutant. WT and mutant FTRs form stable covalent heteroduplexes with active-site modified thioredoxins. In particular, heteroduplexes formed with WT FTR represent interesting one-electron-reduced reaction intermediates, which can be split by reduction of the Fe-S cluster. Heteroduplexes form non-covalent complexes with ferredoxin demonstrating the ability of FTR to simultaneously dock thioredoxin and ferredoxin, which is in accord with the proposed reaction mechanism and the structural analyses.