Biochemical and biophysical research communications最新文献

PIK3CA, which encodes protein p110α, is one of the most frequently mutated oncogenes and a promising drug-target for human cancer. Previously, we demonstrate that p85β is released from PI3K complex which contain PIK3CA helical domain mutations and translocates into nucleus to regulate tri-methylation of H3K27, thereby promoting tumorigenicity. Here, we identify DIRAS2 and SOWAHB as target genes of nuclear p85β in PIK3CA-helical-domain-mutant tumors. DIRAS2 and SOWAHB are tumor suppressive genes, whose expression are repressed by nuclear p85β through histone methyltransferase EZH2. More importantly, combination of PI3K inhibitor and importin-β inhibitor effectively inhibits the growth of PIK3CA-helical-domain-mutant tumors by synchronously blocking both AKT signaling and nuclear p85β/DIRAS2 and SOWAHB axis. In this study, we evaluate the combination effect of Alpelisib and Importazole for PIK3CA helical domain mutant tumors and demonstrate its underlying mechanism.

WBP1L is a broadly expressed transmembrane adaptor protein involved in regulating hematopoietic stem cell function and T cell development. It interacts with NEDD4-family E3 ubiquitin ligases and regulates important chemokine receptor CXCR4. Using tandem affinity purification coupled with mass spectrometry, we identified novel WBP1L interactions with the IFNγ receptor and the Cullin-RING ubiquitin ligases CRL1^β-TrCP1/2. We found that WBP1L interaction with the IFNγ receptor serves to downregulate proximal IFNγ receptor signaling in female macrophages, while the interaction with CRL1^β-TrCP1/2 ubiquitin ligases regulates WBP1L protein levels. Disrupting this interaction, as well as inhibiting proteasome activity or neddylation, increased WBP1L protein levels, demonstrating that CRL1^β-TrCP1/2 ubiquitin ligases regulate WBP1L protein abundance. These data provide important insights into the mechanisms controlling WBP1L function.

Atrial fibrillation (AF) is a prevalent arrhythmia closely associated with atrial fibrosis, posing significant challenges to cardiovascular health. Recent studies have identified the sodium-glucose co-transporter 1 (SGLT1) as a potential key player in the pathophysiology of heart diseases, particularly in the context of AF and atrial fibrosis. This review aims to synthesize current knowledge regarding the mechanisms by which SGLT1 influences the development of AF and atrial fibrosis, with a specific focus on its relationship with endothelial-mesenchymal transition (EMT). By analyzing the latest research findings, this paper discusses how SGLT1 may modulate structural and functional changes in the atria, thereby enhancing our understanding of the underlying mechanisms driving AF.

Technological innovation can drive scientific inquiry by allowing researchers to answer questions that were once out of reach. Eukaryotic mRNA synthesis was not so long ago thought of as a deterministic, sequential process in which transcriptional regulators and general transcription factors assemble in an orderly fashion into chromatin to, ultimately, activate RNA polymerase II. Advances in fluorescence microscopy techniques have revealed a much more complex scenario, wherein transcriptional regulators dynamically engage with chromatin in a more stochastic, probabilistic way. In this review, we will concentrate on what fluorescence fluctuation methods have taught us about the journey of transcription factors within live cells. Specifically, we summarized how these techniques have contributed to reshaping our understanding of the mechanism(s) of action of the glucocorticoid receptor, a ligand-regulated transcription factor involved in many physiological and pathological processes. This receptor regulates a variety of gene networks in a context-specific manner and its activity can be quickly and easily controlled by the addition of specific ligands. Thus, it is widely used as a model to study the mechanisms of transcription factors through live-cell imaging.

Colorectal cancer (CRC) is a fatal cancer prevalent worldwide, and epithelial-mesenchymal transition (EMT) is a key factor in tumor invasion and metastasis. Piperine, a natural alkaloid known for its antitumor properties, faces limitations in clinical use due to its moderate potency. To address this, our team synthesized and validated a new derivative, HJJ_3_5, which has shown potent antitumor activity against CRC cells. We assessed HJJ_3_5's inhibitory effects on the colon cancer cell line HCT116 through MTT, colony formation, and assays for cell migration and invasion. To uncover HJJ_3_5's molecular mechanisms, we utilized transcriptomics, weighted gene co-expression network analysis (WGCNA), and machine learning to identify key EMT-related genes. Western blot and immunofluorescence experiments confirmed the expression changes of these key proteins. Our findings indicate that HJJ_3_5 significantly suppressed the proliferation, migration, and invasion of HCT116 cells in vitro, outperforming piperine. We discovered that HJJ_3_5 downregulated the expression of COL12A1, PJA2, VCAN, MEF2C, DPYD, and DDR2 genes in HCT116 cells, which resulted in a decrease in the EMT regulator SNAI1, thus inhibiting EMT in these cells. In summary, this study presents novel evidence that the piperine derivative HJJ_3_5 inhibits the migration and invasion of colorectal cancer cells through SNAI1-mediated EMT.

Maltose-binding protein (MBP) and glutathione S-transferase (GST) are widely used solubility-enhancing protein tags, typically employed to address various issues related to protein expression and purification. The detection of these tags are usually achieved through binding of corresponding antibodies. Designing low-cost binders as alternatives to antibodies is of great significance. This study employed a de novo design approach, starting with a large number of protein scaffolds and screening out 6 candidate binders targeting MBP and 4 candidate binders targeting GST based on scoring functions. Flow cytometry low-affinity selection and biolayer interferometry (BLI) quantitative results showed that MBP and GST can interact strongly with one or several binders, exhibiting nanomolar binding. Among them, LZMB3 has a binding dissociation constant (K_D) of 54.05 ± 1.46 nM, while LJGB3 and LJGB4 have K_D values of 105.4 ± 1.812 nM and 437.9 ± 17.69 nM, respectively.

Importin α is a crucial player in the nucleocytoplasmic transport of nuclear localization signal (NLS)-containing cargo proteins and is suggested to bind to DNA directly. We hypothesized that importin α, after binding to DNA, may move along DNA via sliding or hopping. We investigated the movement dynamics of importin αs fused to AcGFP along DNA using single-molecule fluorescence microscopy and single-tethered DNA arrays. Single-molecule data demonstrated importin α diffuses along DNA in fast and slow mobility modes. The diffusion by importin α in the fast mobility mode did not depend on salt concentration, suggesting sliding motion with continuous contact with DNA. The sliding was supported by restricted diffusion of importin α in Cas9 obstacles bound to DNA. Next, we tested whether importin α can transport a cargo molecule along DNA. Two-color imaging data established that importin α co-slides along DNA with SV40 TAg-NLS as a model cargo. We found that importin β1 together with RanGTP significantly enhanced the DNA binding of importin α and the recruitment of a model cargo TRIM28 to DNA, suggesting that importin β1/RanGTP are involved in the switching of importin α/cargo from the nuclear transport pathway to DNA sliding. Single-molecule and in vivo immunofluorescence assay demonstrates importin α assists in accumulating TRIM28 within nuclear chromatin regions. Thus, we present novel findings on the sliding dynamics and the cargo transport of importin α along DNA. The relatively faster sliding by importin α allows efficient delivery of cargo proteins to their target sites, even on long genomic DNA.

Polyamines within the cell are tightly regulated by spermidine/spermine N-acetyltransferase (SSAT) enzymes. While several SSATs have been investigated in different bacterial species, there is still a significant gap in knowledge about which proteins are functional SSATs in many organisms. For example, while it is known that Pseudomonas aeruginosa synthesizes the polyamine spermidine, the SSAT that acetylates this molecule and its importance in regulating intracellular polyamines remains unknown. We previously identified a candidate Gcn5-related N-acetyltransferase (GNAT) protein from P. aeruginosa (PA2271) that could fulfill this role since it acetylates spermidine, but no further studies were conducted. Here, we explored the structure/function relationship of the PA2271 protein by determining its X-ray crystal structure and performing enzyme kinetics assays. We also identified active site residues that are essential for catalysis and substrate binding. As the study progressed, we encountered results that led us to explore the importance of four cysteine residues on enzyme activity and disulfide bond formation or modification of cysteine residues. We found these cysteine residues in PA2271 are important for protein solubility and activity, and there is an interrelationship between cysteine residues that contribute to these effects. Furthermore, we also found disulfide bonds could form between C₁₂₁ and C₁₆₅ and speculate that these residues may contribute to redox regulation of PA2271 protein activity.

This study investigated time-dependent changes in intracellular Ca²⁺ levels in T cells, regulatory mechanisms, and functional effects after acute exercise. Male C57BL/6 mice were assigned to control and exercise groups, with the latter sacrificed at different intervals post-exercise. Murine splenic lymphocytes were isolated, and cytosolic Ca²⁺ levels were measured using Fluo-3/AM. T-cell proliferation was assessed by flow cytometry and CFSE labeling, apoptosis by Annexin V/PI staining, and cytokine levels by CBA. RNA sequencing results were validated by qRT-PCR. The findings revealed that exercise significantly altered intracellular calcium oscillations in CD3⁺ cells, leading to reduced mitogen-stimulated proliferation, increased IL-6, IL-5, and IL-13 production, and decreased IL-2 secretion. Additionally, there was an increase in the apoptotic fraction of CD3⁺ cells, with upregulated expression of Cav1.1, Cav3.2, Cav3.3, SERCA2B, PKCθ, Bcl-xL, and FADD, and downregulated Ryr3 (p < 0.05). Transcriptomic analysis identified 607 differentially expressed genes involved in calcium ion binding and related pathways, including calcium signaling and cytokine-cytokine receptor interactions. Thus, acute exercise induces specific calcium oscillation patterns in T cells, mediated by PKCθ, affecting proliferation, apoptosis, and cytokine production. These changes are attributed to increased calcium influx through Cav1.1, Cav3.2, and Cav3.3 channels, decreased calcium reuptake via SERCA2B, and reduced calcium release through Ryr3. This research provides novel insights into how exercise modulates immune cell function by altering calcium levels, potential implications for enhancing immune responses or reducing inflammation.

Osteoporosis, a significant bone disease predominantly affecting elderly and postmenopausal women, leads to increased bone fragility and fracture risk, presenting a major public health concern with substantial socioeconomic implications. This study investigated the therapeutic potential of Lactobacillus strains, known for their immunomodulatory properties, in an ovariectomy-induced osteoporosis mouse model. Among three tested strains Lactobacillus casei GKC1, Lactobacillus rhamnosus GKLC1, and Lactobacillus johnsonii GKJ2, GKC1 demonstrated superior efficacy in promoting osteogenesis-related gene expression, including alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP2) and runt-related transcription factor 2 (RUNX2). In ovariectomized mice (n = 8/group), both live and heat-inactivated GKC1 (57 mg/kg) and fermented GKC1 (1000 mg/kg) were administered orally for 28 days, with alendronate (2.5 mg/kg) serving as a positive control. The treatment significantly improved bone mineral density and femoral microstructure parameters compared to the ovariectomized control group. For the first time, heat-inactivated GKC1 exhibited superior anti-inflammatory effects through reduction of IL-17A and enhanced bone microstructural integrity, suggesting its potential as a safe and effective therapeutic agent for postmenopausal osteoporosis management. These findings provide compelling evidence for the development of postbiotic-based interventions in osteoporosis treatment, offering a promising alternative to conventional therapeutics.