Cytochrome P450 Mining for Bufadienolide Diversification.

Bufadienolides are a class of steroids with a distinctive α-pyrone ring at C17, mostly produced by toads and consisting of over 100 orthologues. They exhibit potent cardiotonic and antitumor activities and are active ingredients of the traditional Chinese medicine Chansu and Cinobufacini. Direct extraction from toads is costly, and chemical synthesis is difficult, limiting the accessibility of active bufadienolides with diverse modifications and trace content. In this work, based on the transcriptome and genome analyses, using a yeast-based screening platform, we obtained eight cytochrome P450 (CYP) enzymes from toads, which catalyze the hydroxylation of bufalin and resibufogenin at different sites. Moreover, a reported fungal CYP enzyme Sth10 was found functioning in the modification of bufalin and resibufogenin at multiple sites. A total of 15 bufadienolides were produced and structurally identified, of which six were first discovered. All of the compounds were effective in inhibiting the proliferation of tumor cells, especially 19-hydroxy-bufalin (2) and 1ß-hydroxy-bufalin (3), which were generated from bufalin hydroxylation catalyzed by CYP46A35. The catalytic efficiency of CYP46A35 was improved about six times and its substrate diversity was expanded to progesterone and testosterone, the common precursors for steroid drugs, achieving their efficient and site-specific hydroxylation. These findings elucidate the key modification process in the synthesis of bufadienolides by toads and provide an effective way for the synthesis of unavailable bufadienolides with site-specific modification and active potentials.

Identification of a 17ß-estradiol-degrading Microbacterium hominis SJTG1 with high adaptability and characterization of the genes for estrogen degradation.

Environmental estrogen contamination poses severe threat to wildlife and human. Biodegradation is an efficient strategy to remove the wide-spread natural estrogen, while strains suitable for hostile environments and fit for practical application are rare. In this work, Microbacterium hominis SJTG1 was isolated and identified with high degrading efficiency for 17ß-estradiol (E2) and great environment fitness. It could degrade nearly 100% of 10 mg/L E2 in minimal medium in 6 days, and remove 93% of 1 mg/L E2 and 74% of 10 mg/L E2 in the simulated E2-polluted solid soil in 10 days. It maintained stable E2-degrading efficiency in various harsh conditions like non-neutral pH, high salinity, stress of heavy metals and surfactants. Genome mining and comparative genome analysis revealed that there are multiple genes potentially associated with steroid degradation in strain SJTG1. One 3ß/17ß-hydroxysteroid dehydrogenase HSD-G129 induced by E2 catalyzed the 3ß/17ß-dehydrogenation of E2 and other steroids efficiently. The transcription of hsd-G129 gene was negatively regulated by the adjacent LysR-type transcriptional regulator LysR-G128, through specific binding to the conserved site. E2 can release this binding and initiate the degradation process. This work provides an efficient and adaptive E2-degrading strain and promotes the biodegrading mechanism study and actual remediation application.

Characterization of the Phenanthrene-Degrading Sphingobium yanoikuyae SJTF8 in Heavy Metal Co-Existing Liquid Medium and Analysis of Its Metabolic Pathway.

Polycyclic aromatic hydrocarbons (PAHs) are common organic pollutants with great carcinogenic threaten, and metal/PAH-contaminated environments represent one of the most difficult remedial challenges. In this work, Sphingobium yanoikuyae SJTF8 was isolated and identified with great and stable PAH-degrading efficiency even under stress conditions. It could utilize typical PAHs (naphthalene, phenanthrene, and anthracene) and heterocyclic and halogenated aromatic compounds (dibenzothiophene and 9-bromophenanthrene) as the sole carbon source. It could degrade over 98% of 500 mg/L phenanthrene in 4 days, and the cis-3,4-dihydrophenanthrene-3,4-diol was the first-step intermediate. Notably, strain SJTF8 showed great tolerance to heavy metals and acidic pH. Supplements of 0.30 mM of Cu2+, 1.15 mM of Zn2+, and 0.01 mM of Cd2+ had little effect on its cell growth and phenanthrene degradation; phenanthrene of 250 mg/L could still be degraded completely in 48 h. Further, the whole genome sequence of S. yanoikuyae SJTF8 was obtained, and three plasmids were found. The potential genes participating in stress-tolerance and PAH-degradation were annotated and were found mostly distributed in plasmids 1 and 2. Elimination of plasmid 2 resulted in the loss of the PAH-degradation ability. On the basis of genome mining results, the possible degrading pathway and the metabolites of S. yanoikuyae SJTF8 to phenanthrene were predicted.

Characterization of an 17ß-estradiol-degrading bacterium Stenotrophomonas maltophilia SJTL3 tolerant to adverse environmental factors.

Bioremediation of environmental estrogens requires microorganisms with stable degradation efficiency and great stress tolerance in complex environments. In this work, Stenotrophomonas maltophilia SJTL3 isolated from wastewater was found to be able to degrade over 90% of 10 µg/mL 17ß-estradiol (E2) in a week and the degradation dynamic was fitted by the first-order kinetic equations. Estrone was the first and major intermediate of E2 biodegradation. Strain SJTL3 exhibited strong tolerance to several adverse conditions like extreme pH (3.0-11.0), high osmolality (2%), co-existing heavy metals (6.25 µg/mL of Cu2+) and surfactants (5 CMC of Tween 80), and retained normal cell vitality and stable E2-degradaing efficiency. In solid soil, strain SJTL3 could remove nearly 100% of 1 µg/mL of E2 after the bacteria inoculation and 8-day culture. As to the contamination of 10 µg/mL E2 in soil, the biodegradation efficiency was about 90%. The further obtainment of the whole genome of strain SJTL3 and genome analysis revealed that this strain contained not only the potential genes responsible for estrogen degradation, but also the genes encoding proteins involved in stress tolerance. This work could promote the estrogen-biodegrading mechanism study and provide insights into the bioremediation application.

Characterization of an efficient estrogen-degrading bacterium Stenotrophomonas maltophilia SJTH1 in saline-, alkaline-, heavy metal-contained environments or solid soil and identification of four 17ß-estradiol-oxidizing dehydrogenases.

The efficient bioremediation of estrogen contamination in complex environments is of great concern. Here the strain Stenotrophomonas maltophilia SJTH1 was found with great and stable estrogen-degradation efficiency even under stress environments. The strain could utilize 17ß-estradiol (E2) as a carbon source and degrade 90% of 10 mg/L E2 in a week; estrone (E1) was the first degrading intermediate of E2. Notably, diverse pH conditions (3.0-11.0) and supplements of 4% salinity, 6.25 mg/L of heavy metal (Cd2+ or Cu2+), or 1 CMC of surfactant (Tween 80/ Triton X-100) had little effect on its cell growth and estrogen degradation. The addition of low concentrations of copper and Tween 80 even promoted its E2 degradation. Bioaugmentation of strain SJTH1 into solid clay soil achieved over 80% removal of E2 contamination (10 mg/kg) within two weeks. Further, the whole genome sequence of S. maltophilia SJTH1 was obtained, and a series of potential genes participating in stress-tolerance and estrogen-degradation were predicted. Four dehydrogenases similar to 17ß-hydroxysteroid dehydrogenases (17ß-HSDs) were found to be induced by E2, and the four heterogenous-expressed enzymes could oxidize E2 into E1 efficiently. This work could promote bioremediation appliance potential with microorganisms and biodegradation mechanism study of estrogens in complex real environments.

Characterization of 17ß-hydroxysteroid dehydrogenase and regulators involved in estrogen degradation in Pseudomonas putida SJTE-1.

In bacteria, the enzyme catalyzing the transformation of 17ß-estradiol is considered the key enzyme for its metabolism, whose enzymatic activity and regulatory network influence the biodegradation efficiency of this typical estrogen. In this work, a novel 17ß-hydroxysteroid dehydrogenase (17ß-HSD) was characterized from the estrogen-degrading strain Pseudomonas putida SJTE-1, and two regulators were identified. This 17ß-HSD, a member of the short-chain dehydrogenase/reductase (SDR) superfamily, could be induced by 17ß-estradiol and catalyzed the oxidization reaction at the C17 site of 17ß-estradiol efficiently. Its Km value was 0.068 mM, and its Vmax value was 56.26 µmol/min/mg; over 98% of 17ß-estradiol was oxidized into estrone in 5 min, indicating higher efficiency than other reported bacterial 17ß-HSDs. Furthermore, two genes (crgA and oxyR) adjacent to 17ß-hsd were studied which encoded the potential CrgA and OxyR regulators. Overexpression of crgA could enhance the transcription of 17ß-hsd, while that of oxyR resulted in the opposite effect. They could bind to the specific and different sites in the promoter region of 17ß-hsd gene directly, and binding of OxyR could be released by 17ß-estradiol. OxyR repressed the expression of 17ß-hsd by its specific binding to the conserved motif of GATA-N9-TATC, while CrgA activated the expression of this gene through its binding to the motif of T-N11-A. Therefore, this 17ß-HSD transformed 17ß-estradiol efficiently and the two regulators regulated its expression directly. This work could promote the study of the enzymatic mechanism and regulatory network of the estrogen biodegradation pathway in bacteria.

E2F1 induces TINCR transcriptional activity and accelerates gastric cancer progression via activation of TINCR/STAU1/CDKN2B signaling axis.

Recent evidence indicates that E2F1 transcription factor have pivotal roles in the regulation of cellular processes, and is found to be dysregulated in a variety of cancers. Long non-coding RNAs (lncRNAs) are also reported to exert important effect on tumorigenesis. E2F1 is aberrantly expressed in gastric cancer (GC), and biology functions of E2F1 in GC are controversial. The biological characteristics of E2F1 and correlation between E2F1 and lncRNAs in GC remain to be found. In this study, integrated analysis revealed that E2F1 expression was significantly increased in GC cases and its expression was positively correlated with the poor pathologic stage, large tumor size and poor prognosis. Forced E2F1 expression promotes proliferation, whereas loss of E2F1 function decreased cell proliferation by blocking of cell cycle in GC cells. Mechanistic analyses indicated that E2F1 accelerates GC growth partly through induces TINCR transcription. TINCR could bind to STAU1 (staufen1) protein, and influence CDKN2B mRNA stability and expression, thereby affecting the proliferation of GC cells. Together, our findings suggest that E2F1/TINCR/STAU1/CDKN2B signaling axis contributes to the oncogenic potential of GC and may constitute a potential therapeutic target in this disease.

MicroRNA-509-3p increases the sensitivity of epithelial ovarian cancer cells to cisplatin-induced apoptosis.

AIMS: XIAP is upregulated in chemoresistant epithelial ovarian cancer (EOC). However, the molecular mechanism of this dysregulation remains unclear. MATERIALS & METHODS: The regulation of XIAP by miR-509-3p was investigated by luciferase reporter assay, real-time PCR and immunobloting, and the roles of miR-509-3p in cellular proliferation and apoptosis were accessed through MTT and DAPI assays. RESULTS: miR-509-3p, a downregulated miRNA in chemoresistant EOC, can directly target the XIAP via its 3'UTR. Overexpression of miR-509-3p can not only downregulate the expression of XIAP in ovarian cancer cells but also inhibit the proliferation of EOC cells and increase their sensitivity to cisplatin-induced apoptosis. CONCLUSIONS: Our data suggest that restoring certain dysregulated miRNAs to their normal levels could increase the therapeutic effects of anticancer drugs.

Phylogenetic and biological analysis of a laboratory-generated gammaretrovirus xenotropic murine leukemia virus-related virus (XMRV).

A xenotropic murine leukemia virus-related virus (XMRV) has been reported to be an emerging pathogen associated with prostate cancer (PC) and chronic fatigue syndrome (CFS). However, recent studies have demonstrated that XMRV is a laboratory-derived virus resulting from genetic recombination between two mouse viral genomes during serial xenograft tissue transplantation. This study describes a phylogenetic analysis that compared XMRV with the ecotropic murine leukemia viruses (E-MLV), xenotropic MLV (X-MLV), and other retroviruses, including HTLV-1 and HIV-1. We found that sequences corresponding to three XMRV structural proteins (Env, Gag, and Pol) exhibited high degrees of homology with X-MLV (>91 %) and E-MLV (67-96 %), but not HTLV-1 (13-16 %) or HIV-1 (10-15 %), indicating that XMRV was derived from X-MLV and/or E-MLV. We then compared the infectivity of XMRV and E-MLV for human and murine lymphocytes, respectively. Results showed that human PBMCs were not susceptible to XMRV infection, suggesting that XMRV exhibits host cell tropism similar to E-MLV that only infects murine PBMCs. These data suggest that it is unlikely that this laboratory-generated retrovirus could cause disease in humans.