Characterization and functional evidence for Orf2 of Streptomyces sp. 139 as a novel dipeptidase E.

Aspartyl dipeptidase (dipeptidase E) can hydrolyze Asp-X dipeptides (where X is any amino acid), and the enzyme plays a key role in the degradation of peptides as nutrient sources. Dipeptidase E remains uncharacterized in Streptomyces. Orf2 from Streptomyces sp. 139 is located in the exopolysaccharide biosynthesis gene cluster, which may be a novel dipeptidase E with "S134-H170-D198" catalytic triad by sequence and structure comparison. Herein, recombinant Orf2 was expressed in E. coli and characterized dipeptidase E activity using the Asp-ρNA substrate. The optimal pH and temperature for Orf2 are 7.5 and 40 ℃; Vmax and Km of Orf2 are 0.0787 mM·min-1 and 1.709 mM, respectively. Orf2 exhibits significant degradation activities to Asp-Gly-Gly, Asp-Leu, Asp-His, and isoAsp-Leu and minimal activities to Asp-Pro and Asp-Ala. Orf2 contains a Ser-His-Asp catalytic triad characterized by point mutation. In addition, the Asp147 residue of Orf2 is also proven to be critical for the enzyme's activity through molecular docking and point mutation. Transcriptome analysis reveals the upregulation of genes associated with ribosomes, amino acid biosynthesis, and aminoacyl-tRNA biosynthesis in the orf2 mutant strain. Compared with the orf2 mutant strain and WT, the yield of crude polysaccharide does not change significantly. However, crude polysaccharides from the orf2 mutant strain exhibit a wider range of molecular weight distribution. The results indicate that the Orf2 links nutrient stress to secondary metabolism as a novel dipeptidase E. KEY POINTS: • A novel dipeptidase E with a Ser-His-Asp catalytic triad was characterized from Streptomyces sp. 139. • Orf2 was involved in peptide metabolism both in vitro and in vivo. • Orf2 linked nutrient stress to mycelia formation and secondary metabolism in Streptomyces.

Screening of Microbial Natural Products and Biological Evaluation of Trichomicin as Potential Anti-Cytokine Storm Agents.

COVID-19 has remained an uncontained, worldwide pandemic. Most of the infected people had mild symptoms in the early stage, and suddenly worsened or even died in the later stage which made the cytokine release syndrome (CRS) once again aroused people's attention. CRS is an excessive immunity of the body to external stimuli such as viruses, bacteria, and nanomaterials, which can cause tissue damage, local necrosis or even death. Lipopolysaccharide (LPS) is one of the most effective CRS inducers, which can activate macrophages to release cytokines, including tumor necrosis factor (TNF-α), interleukin-1ß (IL-1ß), IL- 6 and chemokines. We used RT-PCR to detect the expression of representative cytokines in mouse and human cells at different concentrations of Trichomicin, Ebosin, and 1487B after LPS stimulation. The results showed that the expression of TNF-α, IL-1ß, IL-6, and CXCL10 all increased after LPS stimulation. Among the various drugs, Trichomicin had the most obvious inhibitory effect on cytokine expression in vitro, and it was further verified in vivo that Trichomicin can improve the survival rate of mice stimulated with LPS. Finally, it was proved that Trichomicin inhibited the Stat3 and NF-κB pathways and reduced the phosphorylation of Stat3 and p65 after LPS stimulation, thereby inhibiting the response of macrophages to pro-inflammatory stimuli. The article clarified the inhibitory activity and mechanism of action of Trichomicin on CRS, and laid the foundation for the research on the anti-cytokine storm activity of microbial natural products.

Ebosin Ameliorates Psoriasis-Like Inflammation of Mice via miR-155 Targeting tnfaip3 on IL-17 Pathway.

Psoriasis is a recurrent autoimmune skin disease with aberrant regulation of keratinocytes and immunocytes. There is no universally accepted single treatment available for psoriasis, and the establishment of a common treatment option to control its signs and symptoms is urgently needed. Here, we found Ebosin, a novel exopolysaccharide isolated from Streptomyces sp. 139 by our lab, not only could ameliorate inflammation in LPS-induced keratinocytes through IKK/NF-kapaB pathway, but also attenuate psoriatic skin lesions and reduce inflammatory factors expression in imiquimod (IMQ)-mediated psoriatic mice. Except for inhibiting the expression of epidermal differentiation related proteins, Ebosin significantly increased the percentage of CD4+Foxp3+CD25+ Tregs and decreased CD4+IL17A+ Th17 cells in psoriatic mice. Furthermore, we demonstrate that Ebosin significantly suppressed the IL-17 signaling pathway via A20 (encoded by tnfaip3) in vivo. As the direct binding of tnfaip3 to miR-155 has been demonstrated by luciferase reporter assay, and Ebosin has been demonstrated to inhibit miR-155 level in vitro and in vivo, our study first indicates that Ebosin reduces inflammation through the miR-155-tnfaip3-IL-17 axis and T cell differentiation in a psoriasis-like model. Thus, we conclude that Ebosin can act as a promising therapeutic candidate for the treatment of psoriasis.

ACBP suppresses the proliferation, migration, and invasion of colorectal cancer via targeting Wnt/beta-catenin signaling pathway.

Anticancer bioactive peptide (ACBP), a novel bioactive peptide isolated from spleens of goats immunized with tumor extracts in our lab, can inhibit the proliferation of CRC in vitro and vivo. However, it remains unclear how the proliferation of CRC is inhibited by ACBP at the molecular level. Here, we provide evidences showing that ACBP significantly inhibits the expression of Wnt/ß-catenin related genes (cyclin D1, met and c-myc) through pharmacotranscriptomic and qRT-PCR analysis in CRCs. Active ß-catenin, a key protein within Wnt pathway, was compromised remarkably by ACBP in three CRCs, including HCT116, RKO and HT29. Thus nuclear accumulation of active ß-catenin was retarded and finally lead to the decreased expression of oncogenes cyclin D1, met, and c-myc. In addition, we proved that active ß-catenin reduction was mainly due to the inhibition of phospho-LRP6 and stimulation of phospho-ß-catenin by ACBP. Based on the detection of Met and C-Myc in CRC tumor tissue without prior radiotherapy or chemotherapy, our results demonstrated that ACBP can act as a promising anticancer agent for CRC by targeting Wnt/ß-catenin pathway, especially active ß-catenin.

Trichomicin Suppresses Colorectal Cancer via Comprehensive Regulation of IL-6 and TNFα in Tumor Cells, TAMs, and CAFs.

Trichomicin, a small-molecule compound isolated from fungi, has been identified with bioactivity of antitumor. In this study, a colon cancer subcutaneous mice model was used to evaluate the antitumor effects of Trichomicin in vivo. Treatment with Trichomicin significantly inhibited tumor growth in a xenograft mouse colon cancer model. The underlying molecular mechanism has also been investigated through the quantification of relevant proteins. The expression levels of IL-6 and TNFα were reduced in tumor tissues of mice treated with Trichomicin, which was consistent with results of in vitro experiments in which Trichomicin suppressed the expression of IL-6 and TNFα in tumor and stromal cells. In addition, Trichomicin inhibited TNFα-induced activation of NF-κB and basal Stat3 signaling in vitro, which resulted in reduced expression of the immune checkpoint protein PD-L1 in tumor and stromal cells. Conclusively, Trichomicin, a promising new drug candidate with antitumor activity, exerted antitumor effects against colon cancer through inhibition of the IL-6 and TNFα signaling pathways.

Synergistic Effect between Human Papillomavirus 18 and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone on Malignant Transformation of Immortalized SHEE Cells.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific nitrosamine (TSNA) that induces malignant tumors in rodents. High-risk human papillomavirus (hr-HPV) infection is an important cause of several human cancers. Epidemiological evidence has shown that HPV cooperatively induces carcinogenesis with tobacco smoke. In the present study, the synergistic carcinogenesis of NNK and HPV18 was investigated. Immortalized human esophageal epithelial SHEE cells containing the HPV18 E6E7 gene were constructed by lentiviral transfection. SHEE-E6E7 cells were exposed to NNK along with SHEE-V cells without HPV18 E6E7 as a negative control. The cooperation of NNK and HPV was examined by wound-healing, transwell, and colony-forming assays. The results showed that NNK exposure promoted the migration, invasion, and proliferation abilities of both SHEE-E6E7 and SHEE-V cells; however, the changes in these phenotypic features were remarkably stronger in SHEE-E6E7 cells than those in SHEE-V cells. Our findings indicate that NNK promotes malignant transformation of human esophageal epithelial cells and suggest a synergistic carcinogenesis with the HPV18 E6E7 oncogene. As reported previously, the formation of pyridyloxybutylated DNA adducts is a crucial step in NNK-mediated carcinogenesis. In order to clarify the influence of HPV on the formation of NNK-induced DNA adducts, the amounts of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts were determined using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. We observed that the levels of HPB-releasing adducts in SHEE-E6E7 cells were significantly higher (p < 0.01) than those of SHEE-V cells, which was in line with results of the phenotypic assays. In conclusion, this study provides direct evidence that NNK and HPV18 exhibit a synergistic effect on formation of DNA adducts, resulting in malignant transformation of esophageal epithelial cells. Such knowledge on the interaction between infection and smoking habits in the development of cancers informs cancer-prevention strategies. Further studies to delineate the molecular mechanism and to identify specific intervention targets are worthwhile.

NBGNU: a hypoxia-activated tripartite combi-nitrosourea prodrug overcoming AGT-mediated chemoresistance.

Aim: A hypoxia-activated combi-nitrosourea prodrug, N-(2-chloroethyl)-N'-2-(2-(4-nitrobenzylcarbamate)-O6-benzyl-9-guanine)ethyl-N-nitrosourea (NBGNU), was synthesized and evaluated for its hypoxic selectivity and anticancer activity in vitro. Results: The prodrug was designed as a tripartite molecule consisting of a chloroethylnitrosourea pharmacophore to induce DNA interstrand crosslinks (ICLs) and an O6-benzylguanine analog moiety masked by a 4-nitrobenzylcarbamate group to induce hypoxia-activated inhibition of O6-alkylguanine-DNA alkyltransferase. NBGNU was tested for hypoxic selectivity, cytotoxicity and DNA ICLs ability. The reduction product amounts, cell death rates and DNA ICL levels induced by NBGNU under hypoxic conditions were all significantly higher than those induced by NBGNU under normoxic conditions. Conclusion: The tripartite combi-nitrosourea prodrug exhibits desirable tumor-hypoxia targeting ability and abolished chemoresistance compared with the conventional chloroethylnitrosoureas.