In vivo interaction between the human dehydrodolichyl diphosphate synthase and the Niemann-Pick C2 protein revealed by a yeast two-hybrid system

Dehydrodolichyl diphosphate (DedolPP) synthase catalyzes the sequential condensation of isopentenyl diphosphate with farnesyl diphosphate to synthesize DedolPP, a biosynthetic precursor for dolichol which plays an important role as a sugar-carrier lipid in the biosynthesis of glycoprotein in eukaryotic cells. During certain pathological processes like Alzheimer's disease or some neurological disorders, dolichol has been shown to accumulate in human brain. In order to understand the regulatory mechanism of dolichol in eukaryotes, we performed a yeast two-hybrid screen using full length human DedolPP synthase gene [Endo et al. BBA 1625 (2003) 291] as a bait to find some proteins specifically interacting with the enzyme. We identified Niemann-Pick Type C2 protein (NPC2) to show a specific interaction with human DedolPP synthase. This interaction was further confirmed by in vitro co-immunoprecipitation experiment, indicating the possible physiological interaction between NPC2 and DedolPP synthase proteins in human.     

Synthesis and biological evaluation of sphingosine kinase substrates as sphingosine-1-phosphate receptor prodrugs

In the search for bioactive sphingosine 1-phosphate (S1P) receptor ligands, a series of 2-amino-2-heterocyclic-propanols were synthesized. These molecules were discovered to be substrates of human-sphingosine kinases 1 and 2 (SPHK1 and SPHK2). When phosphorylated, the resultant phosphates showed varied activities at the five sphingosine-1-phosphate (S1P) receptors (S1P_1–5). Agonism at S1P₁ was displayed in vivo by induction of lymphopenia. A stereochemical preference of the quaternary carbon was crucial for phosphorylation by the kinases and alters binding affinities at the S1P receptors. Oxazole and oxadiazole compounds are superior kinase substrates to FTY720, the prototypical prodrug immunomodulator, fingolimod (FTY720). The oxazole-derived structure was the most active for human SPHK2. Imidazole analogues were less active substrates for SPHKs, but more potent and selective agonists of the S1P₁ receptor; additionally, the imidazole class of compounds rendered mice lymphopenic.     

The ribostamycin biosynthetic gene cluster in Streptomyces ribosidificus: comparison with butirosin biosynthesis

A cluster of genes for ribostamycin (Rbm) biosynthesis was isolated from Streptomyces ribosidificus ATCC 21294. Sequencing of 31.892 kb of the genomic DNA of S. ribosidificus revealed 26 open reading frames (ORFs) encoding putative Rbm biosynthetic genes as well as resistance and other genes. One of ten putative Rbm biosynthetic genes, rbmA, was expressed in S. lividans TK24, and shown to encode 2-deoxy-scyllo-inosose (DOI) synthase. Acetylation of various aminoglycoside-aminocyclitol (AmAcs) by RbmI confirmed it to be an aminoglycoside 3-N-acetyltransferase. Comparison of the genetic control of ribostamycin and butirosin biosynthesis pointed to a common biosynthetic route for these compounds, despite the considerable differences between them in genetic organization.     

Antinociceptive effect of amitriptyline in mice of acute pain models

Tricyclic antidepressant drugs induce antinociceptive effect and suggest that their analgesic action could be related to the monoaminergic activity of the drugs. The analgesic activity of amitriptyline was observed in mouse models of acute pain. Mice were divided into different groups and were given amitriptyline in different doses alone and in combination with morphine. Reaction time in Hot-Plate and Tail-Flick tests was observed. Results showed that amitriptyline had antinociceptive effect in acute pain state in experimental models. Amitriptyline in combination with morphine had better analgesic effect than the morphine alone in Hot-Plate test.     

Renewed interests in the discovery of bioactive actinomycete metabolites driven by emerging technologies

Actinomycetes are prolific sources of bioactive molecules. Traditional workflows including bacterial isolation, fermentation, metabolite identification and structure elucidation have resulted in high rates of natural product rediscovery in recent years. Recent advancements in multi-omics techniques have uncovered cryptic gene clusters within the genomes of actinomycetes, potentially introducing vast resources for the investigation of bioactive molecules. While developments in culture techniques have allowed for the fermentation of difficult-to-culture actinomycetes, high-throughput metabolite screening has offered plenary tools to accelerate hits discovery. A variety of new bioactive molecules have been isolated from actinomycetes of unique environmental origins, such as endophytic and symbiotic actinomycetes. Synthetic biology and genome mining have also emerged as new frontiers for the discovery of bioactive molecules. This review covers the highlights of recent developments in actinomycete-derived natural product drug discovery.     

Characterization of L-glutamine:2-deoxy-scyllo-inosose aminotransferase (tbmB) from Streptomyces tenebrarius

2-Deoxystreptamine (DOS)-containing aminoglycoside-aminocyclitol (AmAc) antibiotics represent the majority of clinically important AmAcs. Biosynthetic investigations of formation of DOS in actinomycetes are limited to the characterization of 2-deoxy-scyllo-inosose synthase, the first step enzyme of the DOS biosynthetic pathway. A gene encoding L-glutamine:2-deoxy-scyllo-inosose aminotransferase (tbmB) from the tobramycin producer Streptomyces tenebrarius was expressed heterologously in Escherichia coli. The conversions of 2-deoxy-scyllo-inosose to 2-deoxy-scyllo-inosamine and scyllo-inosose to scyllo-inosamine with the activity of TbmB were determined in vitro. The results indicate that tbmB catalyzes the second step of the DOS biosynthetic pathway during the biosynthesis of 2-deoxystreptamine, a subunit of tobramycin, in S. tenebrarius.     

Sphingosine kinase 2 is required for modulation of lymphocyte traffic by FTY720

Immunotherapeutic drugs that mimic sphingosine 1-phosphate (S1P) disrupt lymphocyte trafficking and cause T helper and T effector cells to be retained in secondary lymphoid tissue and away from sites of inflammation. The prototypical therapeutic agent, 2-alkyl-2-amino-1,3-propanediol (FTY720), stimulates S1P signaling pathways only after it is phosphorylated by one or more unknown kinases. We generated sphingosine kinase 2 (SPHK2) null mice to demonstrate that this kinase is responsible for FTY720 phosphorylation and thereby its subsequent actions on the immune system. Both systemic and lymphocyte-localized sources of SPHK2 contributed to FTY720 induced lymphopenia. Although FTY720 was selectively activated in vivo by SPHK2, other S1P pro-drugs can be phosphorylated to cause lymphopenia through the action of additional sphingosine kinases. Our results emphasize the importance of SPHK2 expression in both lymphocytes and other tissues for immune modulation and drug metabolism.