Engineered geranyl diphosphate methyltransferase produces 2-methyl-dimethylallyl diphosphate as a noncanonical C6 unit for terpenoid biosynthesis.

Terpenoids constitute the largest class of natural products with complex structures, essential functions, and versatile applications. Creation of new building blocks beyond the conventional five-carbon (C5) units, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate, expands significantly the chemical space of terpenoids. Structure-guided engineering of an S-adenosylmethionine-dependent geranyl diphosphate (GPP) C2-methyltransferase from Streptomyces coelicolor yielded variants converting DMAPP to a new C6 unit, 2-methyl-DMAPP. Mutation of the Gly residue at the position 202 resulted in a smaller substrate-binding pocket to fit DMAPP instead of its native substrate GPP. Replacement of Phe residue at the position 222 with a Tyr residue contributed to DMAPP binding via hydrogen bond. Furthermore, using Escherichia coli as the chassis, we demonstrated that 2-methyl-DMAPP was accepted as a start unit to generate noncanonical trans- and cis-prenyl diphosphates (C5n+1) and terpenoids. This work provides insights into substrate recognition of prenyl diphosphate methyltransferases, and strategies to diversify terpenoids by expanding the building block portfolio.

Irf1- and Egr1-activated transcription plays a key role in macrophage polarization: A multiomics sequencing study with partial validation.

BACKGROUND: Macrophage polarization has a causal role in the pathogenesis and resolution of various clinical diseases. DNA-binding transcription factors (TFs) have been identified as essential factors during gene transcription. Better insight into the TFs that regulate macrophage polarization could provide novel therapeutic targets. METHODS: IFN-γ (50 ng/mL) or IL4 (20 ng/mL) was utilized to stimulate bone marrow-derived macrophages from mice for 24 h for M1- and M2-polarized macrophage model construction, respectively. First, ATAC-seq (Assay for Targeting Accessible-Chromatin with high throughout sequencing) and motif analysis were conducted to identify potential transcription factors (TFs) involved in M1 and M2 macrophage polarization. Second, essential TFs were identified through RNA-seq, after which, their expression was compared between M0-polarized and M1/M2-polarized macrophages. Furthermore, a multiomic analysis of RNA-seq (siRNA knock down of the identified TFs), ChIP-seq and ATAC-seq was utilized to explore the TF-regulated molecular network. GO and KEGG analyses were used to expound the main functions of the TF-regulated molecular network. Finally, the top 5 TF-regulated genes were validated through flow cytometry, ELISA and qPCR. The cut-off values for high-throughput sequencing and qPCR were FDR < 0.05 and P < 0.05, respectively. RESULTS: Compared with M0 macrophages, 10,771 and 4,848 peaks were identified by ATAC-seq during M1 and M2 macrophage polarization, respectively (FDR < 0.05). Fifty and 62 TF binding motifs were identified for the TFs that participate in M1 and M2 macrophage polarization, respectively. The most significantly highly expressed TFs in M1 and M2 macrophages were identified by RNA-seq as Irf1 and Egr1, with LogFC values of 3.2 and 2.8, respectively. Multiomic analyses further found that Irf1 regulated the transcription of 90 genes and that Egr1 regulated the transcription of 116 genes. The Irf1-regulated molecular network played a key role in the inflammatory response and viral defence of M1 macrophages, and 116 Egr1-regulated genes included anti-inflammatory and cell proliferation genes. Validation experiments indicated that IFN-γ-induced Gbp5, Nos2, CD86, Cxcl10 and Cxcl5 expression was significantly downregulated in siIrf1-BMDMs, and IL4-induced Itgax, Nipal1, Bhlhe40, CD206 and Ffar4 expression was significantly downregulated in siEgr1-BMDMs (P < 0.05). CONCLUSIONS: Through multiomic analyses of epigenetic sequencing and RNA-seq with partial validation, the current study found that Irf1- and Egr1-induced transcription plays key roles in M1 and M2 macrophage polarization, respectively.

Suppression of HELLS by miR-451a represses mTOR pathway to hinder aggressiveness of SCLC.

BACKGROUND: Uncovering molecular pathogenesis and mechanisms of small cell lung cancer (SCLC) will contribute to SCLC therapy. Multiple studies demonstrated that miR-451a acts as an anti-tumor miRNA in non-small cell lung cancer. However, the mechanism of miR-451a in SCLC was ambiguous. OBJECTIVE: We aimed to explore the function of miR-451a in SCLC and decipher the underlying mechanisms. METHODS: TargetScan and dual-luciferase reporter assays were used to analyze the target genes of miR-451a. Cell counting kit-8 and colony formation assays were performed to assess the roles of miR-451a on cell growth. Gene set enrichment analysis (GSEA) was utilized to enrich biological pathways. Western blot was used to measure protein expression. RESULTS: MiR-451a expression was reduced dramatically in SCLC tissues and cell lines (NCI-H1688 and NCI-H446). Helicase, Lymphoid Specific (HELLS) was proved to be a target gene of miR-451a. In addition, cell proliferation assays in SCLC cells transfected with miR-451a mimic and/or HELLS revealed that miR-451a inhibited cell proliferation via targeting HELLS. Moreover, the roles of miR-451a/HELLS in expression of key proteins in mTOR and apoptosis signaling pathways suggested that miR-451a inactivated mTOR and activated apoptosis signaling pathway via directly silencing HELLS. CONCLUSIONS: Our study indicated that miR-451a hinders SCLC cell proliferation in vitro through regulating mTOR and apoptosis signaling pathways via silencing HELLS, suggesting that miR-451a could be a promising tumor suppressor in SCLC. And there is a potential for miR-451a to be a drug target and biomarker for SCLC.

Pathway crosstalk analysis of non-small cell lung cancer based on microarray gene expression profiling.

AIMS AND BACKGROUND: Lung cancer is characterized by uncontrolled cell growth in the lung tissue. A major challenge in cancer research is the biological interpretation of the complexity of cancer somatic mutation profiles. This study examines the role of pathway crosstalk in the metastatic process of lung cancer cells based on DNA microarray analysis. METHODS: We downloaded the gene expression profile GSE10096 from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified and the gene functions of selected DEGs were further analyzed. After KEGG pathway analysis, dysfunctional pathways and dysfunctional crosstalk between pathways in two types of lung cancer cells (low metastasis, M1, and high metastasis, M5) were examined. RESULTS: A total of 13433 genes were filtered as DEGs, and after pathway analysis, 108 signaling pathways related to cancer signaling pathways were screened, including a host pathway hsa05223 and 79 neighbor pathways. Dysfunctional crosstalk analysis of pathways revealed that pathway crosstalk dysfunction of M1 and M5 cells mainly occurred between hsa05223 (non-small cell lung cancer) and hsa04310 (Wnt signaling pathway), and between non-small cell lung cancer and hsa04520 (adherens junction), respectively. Significant pathway crosstalk dysfunction also existed between adherens junction and other classical signaling pathways such as hsa04110 (cell cycle), hsa04310 (Wnt signaling pathway), hsa04350 (TGF-beta signaling pathway), and hsa04630 (Jak-STAT signaling pathway). CONCLUSIONS: Our discovery will help to elucidate the molecular mechanisms of the high carcinogenic and metastatic potential of lung cancer cells. In addition, it will pave the way to developing effective therapies for lung cancer.