Expression, purification and characterization of 5'-nucleotidase from caterpillar fungus by efficient genome-mining.

5'- nucleotidase (5'-NT) is a key enzyme in nucleoside/nucleotide metabolic pathway, it plays an important role in the biosynthesis of cordycepin in caterpillar fungus. In this study, a 5'-NT gene was identified and mined from genomic DNA of caterpillar fungus, which was 1968 bp in length and encoded 656 amino acid residues. The recombinant 5'-NT was first time heterologously expressed in Pichia pastoris GS115, subsequently purified and functionally characterized. The optimal reaction temperature for 5'-NT was 35 °C, and it retained 52.8% of its residual activity after incubation at 50 °C for 1 h. The optimal reaction pH was 6.0 and it exhibited high activity over a neutral pH range. Furthermore, 5'-NT exhibited excellent Km (1.107 mM), Vmax (0.113 µmol/mg·min) and kcat (4.521 S-1) values compared with other typical 5'-nucleotidase. Moreover, substrate specificity analyses indicated that 5'-NT exhibited different phosphatase activity towards the substrates containing different basic groups. The work presented here could be useful to 5'-NT applications and provide more scientific basis and new ideas for the biosynthesis of artificial control cordycepin.

Transcriptome Analysis Reveals the Molecular Mechanisms Underlying Adenosine Biosynthesis in Anamorph Strain of Caterpillar Fungus.

Caterpillar fungus is a well-known fungal Chinese medicine. To reveal molecular changes during early and late stages of adenosine biosynthesis, transcriptome analysis was performed with the anamorph strain of caterpillar fungus. A total of 2,764 differentially expressed genes (DEGs) were identified (p ≤ 0.05, |log2 Ratio| ≥ 1), of which 1,737 were up-regulated and 1,027 were down-regulated. Gene expression profiling on 4-10 d revealed a distinct shift in expression of the purine metabolism pathway. Differential expression of 17 selected DEGs which involved in purine metabolism (map00230) were validated by qPCR, and the expression trends were consistent with the RNA-Seq results. Subsequently, the predicted adenosine biosynthesis pathway combined with qPCR and gene expression data of RNA-Seq indicated that the increased adenosine accumulation is a result of down-regulation of ndk, ADK, and APRT genes combined with up-regulation of AK gene. This study will be valuable for understanding the molecular mechanisms of the adenosine biosynthesis in caterpillar fungus.

Drug repurposing: Ibrutinib exhibits immunosuppressive potential in organ transplantation.

Long-term administration of classic immunosuppressants can induce severe adverse effects. The development of novel immunosuppressants confronts great challenges and opportunities. Ibrutinib, an approved drug for B-cell lineages and chronic graft versus host disease (cGVHD), exhibits immunosuppressive efficacy in autoimmune diseases. Ibrutinib's potential as an immunosuppressant in organ transplantation has not been investigated to date. In a xeno-artery patch model ex vivo, ibrutinib inhibited the proliferation of PBMCs (POD 14-42), mainly CD3+CD4+ and CD3+CD8+ T cells ex vivo. The secretion of cytokines (IL-6, IL-2 and IFN-γ) was suppressed in response to ibrutinib. In allo-skin transplantation models, ibrutinib delayed the rejection of grafted skins. Ibrutinib decreased the amount of T/B cells and lymphocyte infiltration. Altogether, ibrutinib exhibited immunosuppressive potential through cytokine regulation and T cell inhibition ex vivo and in vitro. Repositioning of ibrutinib as an immunosuppressant will greatly facilitate novel immunosuppressant development.