Exploring the structure and function of key enzymes involved in microbial sphingolipid biosynthesis

Sphingolipids (SLs) are a diverse class of lipid molecules derived from the amino acid L-serine and long chain fatty acids (e.g. carbon chain lengths C14-C26). When combined together these building blocks form a so-called “sphingoid”, also known as a long-chain base (LCB). Decades of research into these enigmatic molecules has revealed that SLs are essential components of eukaryotic cell membranes and control many critical cellular functions. The SL biosynthetic pathway begins in all organisms with the Claisen-like, decarboxylative condensation of L-Ser and long-chain fatty acid acyl-CoA thioester (CoASH) substrates (most commonly C16/palmitoyl) to form the intermediate 3-ketodihydrosphingosine (3-KDS). This first key irreversible reaction is catalysed by the pyridoxal 5’-phosphate (PLP)-dependent serine palmitoyltransferase (SPT). The 3-KDS product is then reduced by a NAD(P)H-dependent enzyme (KDS reductase, KDSR) to generate dihydrosphingosine (DHS). Acylation of this molecule with a long-chain fatty acid by a ceramide synthase (CerS) leads to the formation of ceramide (Cer). Downstream, phosphorylation with a phosphate group can occur on the serine-derived head group and lead to the formation of sphingosine-1-phosphate (S-1-P). The balance between Cer and S-1-P concentrations has been proposed to control the cell survival rate in the host system. Alternatively, sugars such as glucose can be added to give the family of glycosphingolipids (GSLs). In contrast to higher order species, research into SL biosynthesis in bacteria has lagged much further behind. However recent studies have revealed a number of important microbes that produce a range of SLs and Cers. Interestingly, SLs from the human microbiota including those from Bacteroides fragilis, a Gram-negative commensal bacterium from the human gut, and from the oral pathogen Porphyromonas gingivalis have recently been shown to play an essential role in host/microbe communication. Of note is that these bacterial SLs and Cers display many similar structural features to their mammalian homologues. However, there appears to be “chemical signatures” distinct to those of bacterial origin. For example, B. fragilis SLs have iso-Me branched acyl chains which are also similar to those found in the round-worm Caenorhabditis elegans. This suggests a PLP-dependent branched-chain amino acid transferase (BCAT/IlvE) transfers iso-Me chains from amino acid precursors such as L-Leu to branched-chain keto acids. To fully understand the role of bacterial SLs and their metabolism detailed investigations of the enzymes, pathways and metabolism are required. In this thesis, studies of different types of the three key enzymes (SPT, IlvE/BCAT and KDSR) involved in the core microbial SL biosynthetic pathway have been presented. Firstly, both recombinant B. fragilis SPT (BfSPT, encoded by gene BF2461) and P. gingivalis (PgSPT, gene PG1780) were expressed in E. coli, purified and studied with protein UV-vis spectrometry, enzyme kinetics, inhibition assays, mass spectrometry and protein crystallization screening. 3-KDS products were detected derived from a range of straight-chain CoA substrates (C14-C18) and amino acids (Gly, L-Ala and L-Ser) produced by BfSPT and PgSPT. Mutagenesis of a conserved loop (PAVAP) in SPT homologues was found to be associated with the catalytic efficiency of PgSPT. Also the presence of a Val353 residue in BfSPT was shown to be essential to allow the enzyme to interact with the C16-CoA substrate. Moreover, data suggested that the position (N- or C- terminus) of the 6His-affinity tag used to purify SPT influenced substrate inhibition by C16-CoA. A hypothetical 3D structural model of the PgSPT PLP:L-Ser external aldimine complex was built in order to explore the active site and residues involved in substrate binding and catalysis. In collaboration with Prof. Mary-Ellen Davey (Florida), the role on SL biosynthesis in P. gingivalis was also explored and found that SLs impact on the way this pathogen interacts with human cells. Secondly, a branched acid transaminase P. gingivalis IlvE (PgIlvE, gene PG1290) was expressed, purified and studied using UV-vis spectrometry to investigate substrate binding and enzyme activity. A multi-enzyme coupled assay for PgIlvE was developed in both the ‘forward’ and ‘reverse’ direction, studied with inhibitors such as L- and D- cycloserine (LCS/DCS), as well as x-ray crystallography. In collaboration with Dr. Jon Marles-Wright (University of Newcastle) the crystal structures of four different forms of PgIlvE; the PLP-bound, internal aldimine form, LCS ring-opened ring-closed form and the PMP form, were obtained. Two residues (F56 and Y188) were identified which played a role in substrate binding and activity. In the final chapter, recombinant KDSR from the yeast Saccharomyces cerevisiae (ScKDSR) was isolated from E. coli. Kinetic parameters for a soluble, truncated form of the enzyme were determined using the substrates KDS and NADPH. The product C18 DHS derived from C18 KDS was detected with MALDI-ToF-MS. Recent studies of human KDSR revealed that patients with point mutations in this enzyme suffered from skin disorders (erythrokeratoderma). To investigate the impact of these mutations, a series of ScKDSR mutant mimics (G176S, Y180F and G263E) were prepared. To date there has been no crystal structure of a KDSR determined but it is a member of the short-chain dehydrogenases/reductases (SDR) superfamily. A homology model of the 3D structure of ScKDSR with three possible NADPH docking positions were constructed, and residues involved in substrate binding and catalysis were suggested. The results in this thesis shed light on the key enzymes involved in the core biosynthetic pathway of microbial SLs and lay down a blueprint for future studies

Endoglin Is Essential for the Maintenance of Self-Renewal and Chemoresistance in Renal Cancer Stem Cells.

Renal cell carcinoma (RCC) is a deadly malignancy due to its tendency to metastasize and resistance to chemotherapy. Stem-like tumor cells often confer these aggressive behaviors. We discovered an endoglin (CD105)-expressing subpopulation in human RCC xenografts and patient samples with a greater capability to form spheres in vitro and tumors in mice at low dilutions than parental cells. Knockdown of CD105 by short hairpin RNA and CRISPR/cas9 reduced stemness markers and sphere-formation ability while accelerating senescence in vitro. Importantly, downregulation of CD105 significantly decreased the tumorigenicity and gemcitabine resistance. This loss of stem-like properties can be rescued by CDA, MYC, or NANOG, and CDA might act as a demethylase maintaining MYC and NANOG. In this study, we showed that Endoglin (CD105) expression not only demarcates a cancer stem cell subpopulation but also confers self-renewal ability and contributes to chemoresistance in RCC

Snapshots of the reaction coordinate of a thermophilic 2'-deoxyribonucleoside/ribonucleoside transferase

Funding: P.T. is funded by IBioIC (IBioIC 2020-2-1), and C.M.C. is funded by the Wellcome Trust (217078/Z/19/Z). C.M.C. and D.H. are funded by research grants from NuCana plc..Nucleosides are ubiquitous to life and are required for the synthesis of DNA, RNA, and other molecules crucial for cell survival. Despite the notoriously difficult organic synthesis of nucleosides, 2′-deoxynucleoside analogues can interfere with natural DNA replication and repair and are successfully employed as anticancer, antiviral, and antimicrobial compounds. Nucleoside 2′-deoxyribosyltransferase (dNDT) enzymes catalyze transglycosylation via a covalent 2′-deoxyribosylated enzyme intermediate with retention of configuration, having applications in the biocatalytic synthesis of 2′-deoxynucleoside analogues in a single step. Here, we characterize the structure and function of a thermophilic dNDT, the protein from Chroococcidiopsis thermalis (CtNDT). We combined enzyme kinetics with structural and biophysical studies to dissect mechanistic features in the reaction coordinate, leading to product formation. Bell-shaped pH-rate profiles demonstrate activity in a broad pH range of 5.5–9.5, with two very distinct pKa values. A pronounced viscosity effect on the turnover rate indicates a diffusional step, likely product (nucleobase1) release, to be rate-limiting. Temperature studies revealed an extremely curved profile, suggesting a large negative activation heat capacity. We trapped a 2′-fluoro-2′-deoxyarabinosyl-enzyme intermediate by mass spectrometry and determined high-resolution structures of the protein in its unliganded, substrate-bound, ribosylated, 2′-difluoro-2′-deoxyribosylated, and in complex with probable transition-state analogues. We reveal key features underlying (2′-deoxy)ribonucleoside selection, as CtNDT can also use ribonucleosides as substrates, albeit with a lower efficiency. Ribonucleosides are the building blocks of RNA and other key intracellular metabolites participating in energy and metabolism, expanding the scope of use of CtNDT in biocatalysis.Peer reviewe

Monitoring land cover change and disturbance of the Mount Wutai World Cultural Landscape Heritage Protected Area based on Remote Sensing time-series image from 1987 to 2018

The contextual-based multi-source time-series remote sensing and proposed Comprehensive Heritage Area Threats Index (CHATI) index are used to analyze the spatiotemporal land use/land cover (LULC) and threats to the Mount Wutai World Heritage Area. The results show disturbances, such as forest coverage, vegetation conditions, mining area, and built-up area, in the research area changed dramatically. According to the CHATI, although different disturbances have positive or negative influences on environment, as an integrated system it kept stable from 1987 to 2018. Finally, this research uses linear regression and the F-test to mark the remarkable spatial-temporal variation. In consequence, the threats on Mount Wutai be addressed from the macro level and the micro level. Although there still have some drawbacks, the effectiveness of threat identification has been tested using field validation and the results are a reliable tool to raise the public awareness of WHA protection and governance

The novel anti-cancer fluoropyrimidine NUC-3373 is a potent inhibitor of thymidylate synthase and an effective DNA-damaging agent

Funding: GMZ and CMC are funded by research grant from NuCana plc. PT is funded by IBioIC, CMC is funded by the Wellcome trust (217078/Z/19/Z).Introduction Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU. Methods Human colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC–MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage. Results We demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU. Conclusion Taken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.Publisher PDFPeer reviewe

Characterization of inositol lipid metabolism in gut-associated Bacteroidetes

Inositol lipids are ubiquitous in eukaryotes and have finely tuned roles in cellular signalling and membrane homoeostasis. In Bacteria, however, inositol lipid production is relatively rare. Recently, the prominent human gut bacterium Bacteroides thetaiotaomicron (BT) was reported to produce inositol lipids and sphingolipids, but the pathways remain ambiguous and their prevalence unclear. Here, using genomic and biochemical approaches, we investigated the gene cluster for inositol lipid synthesis in BT using a previously undescribed strain with inducible control of sphingolipid synthesis. We characterized the biosynthetic pathway from myo-inositol-phosphate (MIP) synthesis to phosphoinositol dihydroceramide, determined the crystal structure of the recombinant BT MIP synthase enzyme and identified the phosphatase responsible for the conversion of bacterially-derived phosphatidylinositol phosphate (PIP-DAG) to phosphatidylinositol (PI-DAG). In vitro, loss of inositol lipid production altered BT capsule expression and antimicrobial peptide resistance. In vivo, loss of inositol lipids decreased bacterial fitness in a gnotobiotic mouse model. We identified a second putative, previously undescribed pathway for bacterial PI-DAG synthesis without a PIP-DAG intermediate, common in Prevotella. Our results indicate that inositol sphingolipid production is widespread in host-associated Bacteroidetes and has implications for symbiosis

An intramolecular salt bridge drives the soluble domain of GTP-bound atlastin into the postfusion conformation

Before ER tubule fusion, the atlastin GTPase undergoes a “prefusion” to “postfusion” conformational change that is mediated by an intramolecular salt bridge

Investigation of hospital discharge cases and SARS-CoV-2 introduction into Lothian care homes

Background The first epidemic wave of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Scotland resulted in high case numbers and mortality in care homes. In Lothian, over one-third of care homes reported an outbreak, while there was limited testing of hospital patients discharged to care homes. Aim To investigate patients discharged from hospitals as a source of SARS-CoV-2 introduction into care homes during the first epidemic wave. Methods A clinical review was performed for all patients discharges from hospitals to care homes from 1st March 2020 to 31st May 2020. Episodes were ruled out based on coronavirus disease 2019 (COVID-19) test history, clinical assessment at discharge, whole-genome sequencing (WGS) data and an infectious period of 14 days. Clinical samples were processed for WGS, and consensus genomes generated were used for analysis using Cluster Investigation and Virus Epidemiological Tool software. Patient timelines were obtained using electronic hospital records. Findings In total, 787 patients discharged from hospitals to care homes were identified. Of these, 776 (99%) were ruled out for subsequent introduction of SARS-CoV-2 into care homes. However, for 10 episodes, the results were inconclusive as there was low genomic diversity in consensus genomes or no sequencing data were available. Only one discharge episode had a genomic, time and location link to positive cases during hospital admission, leading to 10 positive cases in their care home. Conclusion The majority of patients discharged from hospitals were ruled out for introduction of SARS-CoV-2 into care homes, highlighting the importance of screening all new admissions when faced with a novel emerging virus and no available vaccine

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant