Role of a conserved arginine residue during catalysis in serine palmitoyltransferase

All sphingolipid-producing organisms require the pyridoxal 5'-phosphate (PLP)-dependent serine palmitoyltransferase (SPT) to catalyse the first reaction on the de novo sphingolipid biosynthetic pathway. SPT is a member of the alpha oxoamine synthase (AOS) family that catalyses a Claisen-like condensation of palmitoyl-CoA and L-serine to form 3-ketodihydrosphingosine (KDS). Protein sequence alignment across various species reveals an arginine residue, not involved in PLP binding, to be strictly conserved in all prokaryotic SPTs, the lcb2 subunits of eukaryotic SPTs and all members of the AOS family. Here we use UV-vis spectroscopy and site-directed mutagenesis, in combination with a substrate analogue, to show that the equivalent residue (R370) in the SPT from Sphingomonas wittichii is required to form the key PLP:L-serine quinonoid intermediate that condenses with palmitoyl-CoA and thus plays an essential role enzyme catalysis.     

Complete exon sequencing of all known Usher syndrome genes greatly improves molecular diagnosis

Background

Usher syndrome (USH) combines sensorineural deafness with blindness. It is inherited in an autosomal recessive mode. Early diagnosis is critical for adapted educational and patient management choices, and for genetic counseling. To date, nine causative genes have been identified for the three clinical subtypes (USH1, USH2 and USH3). Current diagnostic strategies make use of a genotyping microarray that is based on the previously reported mutations. The purpose of this study was to design a more accurate molecular diagnosis tool.

Methods

We sequenced the 366 coding exons and flanking regions of the nine known USH genes, in 54 USH patients (27 USH1, 21 USH2 and 6 USH3).

Results

Biallelic mutations were detected in 39 patients (72%) and monoallelic mutations in an additional 10 patients (18.5%). In addition to biallelic mutations in one of the USH genes, presumably pathogenic mutations in another USH gene were detected in seven patients (13%), and another patient carried monoallelic mutations in three different USH genes. Notably, none of the USH3 patients carried detectable mutations in the only known USH3 gene, whereas they all carried mutations in USH2 genes. Most importantly, the currently used microarray would have detected only 30 of the 81 different mutations that we found, of which 39 (48%) were novel.

Conclusions

Based on these results, complete exon sequencing of the currently known USH genes stands as a definite improvement for molecular diagnosis of this disease, which is of utmost importance in the perspective of gene therapy.     

TgPL2, a patatin‐like phospholipase domain‐containing protein,is involved in the maintenance of apicoplast lipids homeostasis in Toxoplasma

Patatin‐like phospholipases are involved in numerous cellular functions, including lipid metabolism and membranes remodeling. The patatin‐like catalytic domain, whose phospholipase activity relies on a serine‐aspartate dyad and an anion binding box, is widely spread among prokaryotes and eukaryotes. We describe TgPL2, a novel patatin‐like phospholipase domain‐containing protein from the parasitic protist Toxoplasma gondii. TgPL2 is a large protein, in which the key motifs for enzymatic activity are conserved in the patatin‐like domain. Using immunofluorescence assays and immunoelectron microscopy analysis, we have shown that TgPL2 localizes to the apicoplast, a non‐photosynthetic plastid found in most apicomplexan parasites. This plastid hosts several important biosynthetic pathways, which makes it an attractive organelle for identifying new potential drug targets. We thus addressed TgPL2 function by generating a conditional knockdown mutant and demonstrated it has an essential contribution for maintaining the integrity of the plastid. In absence of TgPL2, the organelle is rapidly lost and remaining apicoplasts appear enlarged, with an abnormal accumulation of membranous structures, suggesting a defect in lipids homeostasis. More precisely, analyses of lipid content upon TgPL2 depletion suggest this protein is important for maintaining levels of apicoplast‐generated fatty acids, and also regulating phosphatidylcholine and lysophosphatidylcholine levels in the parasite.     

Nebulized Liposomal Amphotericin B for Treatment of Pulmonary Infection Caused by <Emphasis Type="Italic">Hormographiella aspergillata</Emphasis>: Case Report and Literature Review

Invasive fungal infection is a serious complication following allogeneic hematopoietic stem cell transplantation. Pulmonary infection due to Hormographiella aspergillata is an uncommon condition associated with a high mortality rate. The susceptibility of H. aspergillata to available antifungal agents is not well established. We report for the first time a case of H. aspergillata lung infection that responded poorly to conventional treatment with liposomal amphotericin B (LAmB; 3 mg kg^?1 of body weight per day) with renal damage at higher posology (5 mg kg^?1 of body weight per day), but improved rapidly after addition of nebulized LAmB to intravenous LAmB (3 mg kg^?1 of body weight per day). Successful treatment of our patient using nebulized LAmB would be worth evaluating in cases refractory to standard treatment or when the reference treatment may not be extended due to interaction or side effects.     

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection

Background  

The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv) that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings.     

The External Aldimine Form of Serine Palmitoyltransferase: STRUCTURAL, KINETIC, AND SPECTROSCOPIC ANALYSIS OF THE WILD-TYPE ENZYME AND HSAN1 MUTANT MIMICS*

Sphingolipid biosynthesis begins with the condensation of l-serine and palmitoyl-CoA catalyzed by the PLP-dependent enzyme serine palmitoyltransferase (SPT). Mutations in human SPT cause hereditary sensory autonomic neuropathy type 1, a disease characterized by loss of feeling in extremities and severe pain. The human enzyme is a membrane-bound hetereodimer, and the most common mutations are located in the enzymatically incompetent monomer, suggesting a “dominant” or regulatory effect. The molecular basis of how these mutations perturb SPT activity is subtle and is not simply loss of activity. To further explore the structure and mechanism of SPT, we have studied the homodimeric bacterial enzyme from Sphingomonas paucimobilis. We have analyzed two mutants (N100Y and N100W) engineered to mimic the mutations seen in hereditary sensory autonomic neuropathy type 1 as well as a third mutant N100C designed to mimic the wild-type human SPT. The N100C mutant appears fully active, whereas both N100Y and N100W are significantly compromised. The structures of the holoenzymes reveal differences around the active site and in neighboring secondary structure that transmit across the dimeric interface in both N100Y and N100W. Comparison of the l-Ser external aldimine structures of both native and N100Y reveals significant differences that hinder the movement of a catalytically important Arg³⁷⁸ residue into the active site. Spectroscopic analysis confirms that both N100Y and N100W mutants subtly affect the chemistry of the PLP. Furthermore, the N100Y and R378A mutants appear less able to stabilize a quinonoid intermediate. These data provide the first experimental insight into how the most common disease-associated mutations of human SPT may lead to perturbation of enzyme activity.Sphingolipids are ubiquitous constituents of eukaryotic cells, where they play important roles in signaling, differentiation, and apoptosis (1–4). Defects in sphingolipid catabolism have been linked to several human diseases, such as hypertension, cancer, and disorders of the peripheral nervous system. The most common inherited peripheral neuropathy is hereditary sensory autonomic neuropathy type 1 (HSAN1).⁶ The disease leads to progressive loss of sensation in extremities and is often associated with searing pain (5–8). Genetic studies by two independent groups mapped the disease-associated mutations to the lcb1 (long chain base 1) gene on chromosome 9q22, which encodes the SPT1 subunit of serine palmitoyltransferase (SPT; EC 2.3.1.50) (9–11). SPT catalyzes the first and rate-limiting step of the sphingolipid biosynthetic pathway in all organisms studied to date (12). The reaction is a pyridoxal 5′-phosphate (PLP)-dependent, decarboxylative, Claisen condensation of the amino acid l-serine and the long chain (C16) fatty acid palmitoyl-CoA, which produces the sphingolipid precursor, 3-ketodihydrosphingosine (KDS).SPT belongs to the α-oxoamine synthase subfamily of PLP-dependent enzymes, which contains three other well characterized members: 8-amino-7-oxononanoate synthase (AONS) (13, 14), 5-aminolevulinate synthase (ALAS) (15–17), and 2-amino-3-ketobutyrate-CoA ligase (KBL) (18). These enzymes catalyze the Claisen-like condensation between an amino acid and an acyl-coenzyme A thioester (19). A common mechanism has been proposed (12, 14, 20–23) comprising the following steps: formation of an external aldimine via displacement of the lysine-PLP internal aldimine (holo-SPT) by the incoming amino acid substrate; formation of a quinonoid intermediate by abstraction of the α-proton from the PLP-amino acid external aldimine; a Claisen condensation with the fatty acid-CoA thioester substrate, followed by displacement of the CoASH to form a β-ketoacid intermediate; decarboxylation of this species to form a product quinonoid; protonation of this quinonoid to form the product external aldimine; and finally release of the α-oxoamine product and regeneration of the enzyme PLP-internal aldimine (Fig. 1). Structural studies have shown AONS, ALAS, and KBL isoforms from various species to be homodimers (13, 17, 18). In marked contrast, eukaryotic SPTs are heterodimeric, membrane-bound proteins consisting of two subunits, SPT1 and SPT2 (encoded by the lcb1 and lcb2 gene, respectively) (24).Open in a separate windowFIGURE 1.Proposed catalytic mechanism of SPT. a, the internal aldimine (holo-SPT) is displaced by l-serine to form the external adlimine; b, binding of second substrate palmitoyl-CoA causes conformational change to give the Dunathan intermediate; c, formation of the quinonoid by deprotonation of Cα hydrogen; d, thioester bond hydrolysis and release of CoASH to give β-keto acid intermediate; e, decarboxylation to form KDS product quinonoid; f, reprotonation to form KDS product external aldimine; g, KDS product release and reformation of the holo-form. The l-serine external aldimine has been trapped in the current study. The dotted line shows that steps e and f may be bypassed by an alternative mechanism that does not proceed via a product quinonoid (see Ref. 21).Analysis of a number of HSAN1 patients has revealed the four most common mutations to be C133W, C133Y, V144D, and G387A, with the cysteine mutations appearing to be the most prevalent in populations (10, 25). The impact that these mutations have on SPT activity and sphingolipid metabolism has been the focus of attention by a number of groups. Most surprisingly, these disease-associated mutations occur in the SPT1 monomer, a protein that must be inactive, since it lacks the key lysine, histidine, and aspartate residues necessary to bind and stabilize the PLP cofactor. It is the SPT2 protein that contains the conserved, active site lysine residue that forms a Schiff base internal aldimine with the PLP. However, both subunits are essential to produce functionally active SPT heterodimer (26–29), indicating that the inactive subunit has a crucial role in function. Dunn and co-workers (30) created several mutations in the yeast lcb1 and lcb2 genes, including those corresponding to the HSAN1 SPT1 mutations, and found that they dominantly inhibit SPT activity. A transgenic mouse model also revealed that the LCB1 C133W mutation led to mice with decreased tissue SPT activity and HSAN1 symptoms despite unaltered ceramide concentrations (31). A different study found that SPT activity was decreased in the tissues of HSAN1 patients, and in a Chinese hamster ovary model, hamster LCB1 C133Y and C133W mutations could not rescue cells lacking endogenous LCB1 (32). These combined studies revealed that the HSAN1 mutations act directly on the SPT enzyme but the pathological consequence on sphingolipid metabolism is unclear. Very recently, Dunn and colleagues discovered a small 80-amino acid protein (TSC3p) in yeast that stimulated yeast SPT activity, but its role is still unclear (33). Recently, a third eukaryotic subunit (SPT3) has been characterized and appears to be required for optimum SPT activity, although it is expressed most highly in placental tissue and human trophoblasts (34). It has been suggested that these three subunits could form a higher order SPT complex (35). The instability and hydrophobic nature of eukaryotic SPTs has made their isolation and therefore biophysical characterization particularly challenging (24, 36–38).We have targeted homodimeric SPT from Sphingomonas paucimobilis EY2395 to provide molecular insight into the enzymatic properties of SPT (39, 40). Recently, we reported the first high resolution x-ray crystal structure of the S. paucimobilis holo-SPT (41) and showed that the active site containing the PLP cofactor is at the dimer interface. We used this structure to model the human enzyme and map the human cysteine residue (Cys¹³³) of SPT1 onto Asn¹⁰⁰ of the bacterial SPT (41). We found that Asn¹⁰⁰ is proximal to the PLP binding site and lies at the dimer interface. To explore the effects of the HSAN1 mutations on human SPT activity and structure, we have studied mutants of the bacterial enzyme. We made the N100C mutant to better mimic native human SPT and N100W and N100Y to mimic the most common mutations in human SPT. We have characterized the mutations using kinetics, spectroscopy, and structural biology, including structures of the external l-Ser aldimine. These data provide molecular insights into the effects of HSAN1 mutations. Also, as part of these studies, we identify residues that play a role in the stabilization of intermediates in the SPT reaction and have been able to generate new insights into the substrate specificity and mechanism of the α-oxamine synthase family.     

Loss of autocrine endothelial-derived VEGF significantly reduces hemangiosarcoma development in conditional p53-deficient mice

Malignant transformation of the endothelium is rare, and hemangiosarcomas comprise only 1% of all sarcomas. For this reason and due to the lack of appropriate mouse models, the genetic mechanisms of malignant endothelial transformation are poorly understood. Here, we describe a hemangiosarcoma mouse model generated by deleting p53 specifically in the endothelial and hematopoietic lineages. This strategy led to a high incidence of hemangiosarcoma, with an average latency of 25 weeks. To study the in vivo roles of autocrine or endothelial cell autonomous VEGF signaling in the initiation and/or progression of hemangiosarcomas, we genetically deleted autocrine endothelial sources of VEGF in this mouse model. We found that loss of even a single conditional VEGF allele results in substantial rescue from endothelial cell transformation. These findings highlight the important role of threshold levels of autocrine VEGF signaling in endothelial malignancies and suggest a new approach for hemangiosarcoma treatment using targeted autocrine VEGF inhibition.     

Inner structural organization of the distal humerus in Paranthropus and Homo

The taxonomic attribution of isolated hominin distal humeri has been a matter of uncertainty and disagreement notwithstanding their relative abundance in the fossil record. Four taxonomically-based morphotypes, respectively representing P. boisei, P. robustus, non-erectus early Homo and H. erectus, have been identified based on the cross-sectional outer shape variation of an assemblage of Plio-Pleistocene eastern and southern African specimens (Lague, 2015). However, the existence of possible differences between Paranthropus and Homo in the inner structural organisation at this skeletal site remains unexplored. We used noninvasive imaging techniques to tentatively characterize the endostructural organization of five early Pleistocene distal humeri from South Africa (TM 1517g, SK 24600, SKX 10924, SKX 34805) and Ethiopia (Gombore IB), which have been variably attributed to Paranthropus or Homo. While the investigated specimens reveal diverse degrees of inner preservation related to their taphonomic and diagenetic history, in all but SK 24600 from Swartkrans we could comparatively assess some geometric properties at the most distal cross-sectional level (%CA, I_x/I_y, I_max/I_min) and quantify cortical bone thickness topographic variation across the preserved shaft portions by means of a 2-3D Relative Cortical Thickness index. Whenever possible, we also provided details about the site-specific organization of the cancellous network and measured the same parameters in a comparative sample of twelve adult extant humans. For most features, our results indicate two main patterns: the first includes the specimens TM 1517g, SKX 10924 and SKX 34805, while the second endostructural morphotype sets apart the robust Homo aff. erectus Gombore IB specimen from Melka Kunture, which more closely resembles the condition displayed by our comparative human sample. Notably, marked differences in the amount and pattern of proximodistal cortical bone distribution have been detected between Gombore IB and SKX 34805 from Swartkrans. Given its discordant outer and inner signatures, we conclude that the taxonomic status of SKX 34805 deserves further investigations.