Rapid in vitro quantification of TDP-43 and FUS mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral sclerosis.

Identified genetic mutations cause 20% of frontotemporal dementia (FTD) and 5-10% of amyotrophic lateral sclerosis (ALS) cases: however, for the remainder of patients the origin of disease is uncertain. The overlap in genetic, clinical and pathological presentation of FTD and ALS suggests these two diseases are related. Post-mortem, ~ 95% of ALS and ~ 50% of FTD patients show redistribution of the nuclear protein TDP-43 to the cytoplasm within affected neurons, while ~ 5% ALS and ~ 10% FTD show mislocalisation of FUS protein. We exploited these neuropathological features to develop an unbiased method for the in vitro quantification of cytoplasmic TDP-43 and FUS. Utilising fluorescently-tagged cDNA constructs and immunocytochemistry, the fluorescence intensity of TDP-43 or FUS was measured in the nucleus and cytoplasm of cells, using the freely available software CellProfiler. Significant increases in the amount of cytoplasmic TDP-43 and FUS were detectable in cells expressing known FTD/ALS-causative TARDBP and FUS gene mutations. Pharmacological intervention with the apoptosis inducer staurosporine and mutation in a secondary gene (CYLD) also induced measurable cytoplasmic mislocalisation of endogenous FUS and TDP-43, respectively. These findings validate this methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used for initial prioritisation of predicted FTD/ALS-causative mutations.

Important role of a LAL regulator StaR in the staurosporine biosynthesis and high-production of Streptomyces fradiae CGMCC 4.576.

Staurosporine, belonging to indolocarbazole compounds, is regarded as an excellent lead compound for synthesizing antitumor agents as a potent inhibitor against various protein kinases. In this study, two separate clusters (cluster A and cluster B), corresponding to biosyntheses of K-252c (staurosporine aglycone) and sugar moiety, were identified in Streptomyces fradiae CGMCC 4.576 and heterologously expressed in Streptomyces coelicolor M1146 separately or together. StaR, a cluster-situated LAL family regulator, activates staurosporine biosynthesis by binding to the promoter regions of staO-staC and staG-staN. The conserved sequences GGGGG and GCGCG were found through gradually truncating promoters of staO and staG, and further determined by mutational experiments. Overexpression of staR with the supplementation of 0.01 g L-1 FeSO4 increased staurosporine production to 5.2-fold compared with that of the parental strain Streptomyces fradiae CGMCC 4.576 in GYM medium. Our results provided an approach for improvement of staurosporine production mediated by a positive regulator and established the basis for dissecting the regulatory mechanisms of other indolocarbazole compounds with clinical application value.

Characterization and functional modification of StaC and RebC, which are involved in the pyrrole oxidation of indolocarbazole biosynthesis.

The diversity of indolocarbazole natural products results from the differences in oxidation states of the pyrroline ring moiety. In the biosynthetic pathways for staurosporine and rebeccamycin, two homologous enzymes having 64% identity, StaC and RebC, are responsible for the selective production of K252c, which has one oxo group at the pyrroline ring, and arcyriaflavin A, which has two. Although StaC has a FAD-binding motif, most StaC molecules do not contain FAD, and the protein cannot be reconstituted with FAD in vitro. In this study, we mutated Ala-118 in StaC by replacing a glutamine that is conserved in FAD monooxygenases, resulting in increased FAD content as well as catalytic activity. In addition, mutations around the substrate-binding sites of StaC and RebC can change the product selectivity. Specifically, StaC-N244R-V246T and RebC-F216V-R239N mutants produced substantial amounts of arcyriaflavin A and K252c, respectively.

Engineering biosynthetic pathways to generate antitumor indolocarbazole derivatives.

The indolocarbazole family of natural products is a source of lead compounds with potential therapeutic applications in the treatment of cancer and neurodegenerative disorders. Rebeccamycin and staurosporine are two members of this family, which are produced by different actinomycete strains. Although both compounds display antitumor activity, their distinct structural features determine different modes of action: rebeccamycin targets DNA topoisomerase I, while staurosporine is a protein kinase inhibitor. Here we examine the biosyntheses of rebeccamycin and staurosporine while we summarize our recent work concerning (a) identification and characterization of genes involved in the biosynthesis of indolocarbazoles in actinomycetes, and (b) generation of novel indolocarbazole derivatives in microorganisms by combinatorial biosynthesis.

Deciphering the late steps in the biosynthesis of the anti-tumour indolocarbazole staurosporine: sugar donor substrate flexibility of the StaG glycosyltransferase.

The indolocarbazole staurosporine is a potent inhibitor of a variety of protein kinases. It contains a sugar moiety attached through C-N linkages to both indole nitrogen atoms of the indolocarbazole core. Staurosporine biosynthesis was reconstituted in vivo in a heterologous host Streptomyces albus by using two different plasmids: the 'aglycone vector' expressing a set of genes involved in indolocarbazole biosynthesis together with staG (encoding a glycosyltransferase) and/or staN (coding for a P450 oxygenase), and the 'sugar vector' expressing a set of genes responsible for the biosynthesis of the sugar moiety. Attachment of the sugar to the two indole nitrogens of the indolocarbazole core was dependent on the combined action of StaG and StaN. When StaN was absent, the sugar was attached only to one of the nitrogen atoms, through an N-glycosidic linkage, as in the indolocarbazole rebeccamycin. The StaG glycosyltransferase showed flexibility with respect to the sugar donor. When the 'sugar vector' was substituted by constructs directing the biosynthesis of l-rhamnose, L-digitoxose, L-olivose and D-olivose, respectively, StaG and StaN were able to transfer and attach all of these sugars to the indolocarbazole aglycone.