Molecular Biology Reports最新文献

Background: Natural products remain rich sources of anticancer agents. Fungal immunomodulatory proteins (FIPs) from medicinal mushrooms show direct in vitro tumor inhibition; we investigated a new FIP, FIP-Gre, from Ganoderma resinaceum.

Methods: FIP-Gre was identified by genome mining (LZ8 query), modeled by AlphaFold2, and assessed by docking/100-ns MD (EGFR, c-Met). Recombinant FIP-Gre was produced in E. coli BL21 (DE3)/pET-29a (+), purified by Ni-NTA (~ 12.5 kDa), and tested for hemagglutination, antibacterial activity, cytotoxicity (A549, HepG2, MCF-7; HEK-293 control), and apoptosis markers in A549 cells (8 µg/mL).

Results: FIP-Gre shares 90% identity with LZ8, contains a conserved Fve-type domain, and adopts a stable β-strand-rich fold. Docking/MD suggest strong, stable binding to EGFR and c-Met. FIP-Gre agglutinated mouse RBCs at 4 µg/mL, showed no antibacterial activity, and selectively inhibited cancer cells with IC₅₀ values of 21.27 µg/mL (A549), 29.96 µg/mL (HepG2), and 43.34 µg/mL (MCF-7) while showing no toxicity toward normal HEK-293 cells. At 8 µg/mL, FIP-Gre significantly induced apoptosis in A549 cells by upregulating Bax, Caspase-3, Caspase-9, and p53, and downregulating Bcl-2, indicating activation of the mitochondrial apoptotic pathway.

Conclusion: FIP-Gre is a structurally robust, mushroom-derived protein with selective in vitro anticancer activity and predicted RTK engagement, supporting its development, particularly against lung carcinoma.

Background: Phosphodiesterase-4B (PDE4B) regulates intracellular cAMP and drives pro-inflammatory cytokine production. Novel small-molecule PDE4B inhibitors with improved isoform selectivity are needed to broaden therapeutic options. We report the discovery and validation of SIP0401, a ChemBridge-derived small molecule prioritized via an integrated in silico pipeline combining pharmacophore modeling, molecular docking, and dynamics-based scoring.

Methods: SIP0401 was identified through virtual screening and ligand efficiency-guided filtering against the PDE4B catalytic domain. Experimental profiling included PDE-Glo™, HTRF cAMP, and differential scanning fluorimetry (DSF) with recombinant human PDE4B. Isoform preference (PDE4A/C/D) was assessed by PDE-Glo™. Aggregation, solubility, and luciferase interference were evaluated. Cellular assays included cAMP (HTRF) and TNF-α ELISA in THP-1 macrophages, and cytotoxicity (MTT) in BEAS-2B cells. Curve fitting used GraphPad Prism.

Results: SIP0401 showed high predicted binding affinity (ΔG = - 8.6 kcal/mol) and favorable interaction stability in dynamics. It inhibited PDE4B with IC₅₀ = 282 nM (PDE-Glo) and 338.4 nM (HTRF); rolipram controls gave 91.5 nM and 106.6 nM. DSF showed Tₘ shift + 4.2 °C; luciferase interference was ≤ 6%. SIP0401 was selective for PDE4B over PDE4A/C/D (IC₅₀s 1.16-1.97 µM; 5.3-7.0×). In THP-1 cells, SIP0401 increased cAMP (EC₅₀ = 2.52 µM) and inhibited TNF-α (IC₅₀ = 3.24 µM). BEAS-2B viability > 90% up to 50 µM.

Conclusion: SIP0401, identified by structure-guided virtual screening, demonstrated moderate biochemical preference for PDE4B over other PDE4 isoforms under the tested conditions. Additionally, the observed cellular activity and favorable colloidal/solubility profiles supports its further optimization and in vivo assessment.

Purpose: BOLL, a highly conserved gene crucial for meiosis in spermatogenesis, is epigenetically regulated through DNA methylation in various species. This study aimed to determine whether BOLL promoter methylation contributes to its downregulation in azoospermic men with hypospermatogenesis (HS) and to explore its potential regulatory association with human spermiogenesis.

Methods: We conducted pyrosequencing to assess methylation levels at the putative BOLL promoter on testicular samples from azoospermic men with HS versus normal spermatogenesis (NR) and evaluate correlations with spermatogenic severity and gene expression. In silico CpG island (CGI) prediction and luciferase reporter assays were used to confirm methylation-sensitive regulatory regions. Pathway enrichment analysis identified biological processes associated with BOLL. Functional assays using BOLL overexpression in HEK293T cells and GC2 cells, as well as demethylation using 5-Aza-2'-deoxycytidine (5-AZA) in GC2 cells, were conducted to explore downstream transcriptional effects.

Results: Eight CpG sites (CpGs) within the BOLL promoter CGI were significantly hypermethylated in HS samples, with seven exhibiting a significant inverse correlation with the spermatogenic score, and three with BOLL transcript levels. The region from - 1434 to + 180 was confirmed as a methylation-sensitive promoter. Gene ontology analysis indicated that spermatid development and differentiation were the top BOLL-associated pathways. BOLL overexpression by plasmid transfection in HEK293T cells and GC2 cells upregulated spermatid-specific genes (TNP2 and GAPDHS), while concurrently suppressing spermatogonia-associated markers (ID4 and PIWIL4). In GC2 cells, 5-AZA treatment enhanced Boll expression and induced downstream spermatid-specific genes, including Prm2, which was not responsive to BOLL overexpression alone.

Conclusion: We identified specific BOLL promoter CpGs that are hypermethylated in HS and associated with reduced gene expression. These findings suggest that promoter methylation may contribute to BOLL silencing and impaired spermatid differentiation, supporting its regulatory role in human spermiogenesis.

Background: Growth differentiation factor 15 (GDF15) is a biomarker for cardiovascular diseases, and its circulating levels are altered in preeclampsia (PE), which shares mechanisms with cardiovascular diseases. Several SNPs in the GDF15 locus were associated with GDF15 levels, including the GWAS lead SNP rs888663. However, no previous study had performed a functional characterization of rs888663 nor had tested the hypothesis that noncoding GDF15 SNPs affect GDF15 levels in PE or gestational hypertension (GH). We examined whether variation of rs888663 affects its enhancer activity, whether genotypes and haplotypes of rs888663 and rs1059369 are associated with PE or GH, and their effects on GDF15 levels in healthy pregnant (HP) women, and in patients with PE and GH.

Methods and results: We studied 233 HP women, 188 patients with PE, and 197 patients with GH. We performed a dual-luciferase reporter assay testing both rs888663 alleles (G/T), which found that the rs888663 region is a functional enhancer, and the T allele had a significantly higher enhancer activity. Genotypes were determined by Taqman allele discrimination assays. Plasma GDF15 levels were measured by ELISA. GDF15 levels were lower in PE and GH than in HP (P < 0.01). Patients with PE and GH carrying the TT genotype for rs1059369 and 'T, T' haplotype showed lower GDF15 levels than HP women carrying the same genotype and haplotype, respectively (P < 0.05).

Conclusion: Our findings identified a novel candidate enhancer of GDF15, with the rs888663 T allele leading to increased enhancer activity, and suggest that GDF15 SNPs affect GDF15 levels in PE or GH.