Biochemistry and Cell Biology最新文献

Doxorubicin is a chemotherapeutic drug for cancer that intercalates into nucleosome-free regions at promoters. Doxorubicin was reported to result in loss of histone H3 trimethylated lysine 4 (H3K4me3). To further explore doxorubicin's mechanism of action, we determined the genomic location of the binding sites of doxorubicin in leukemic cells. The effect of doxorubicin intercalation into the chromatin of leukemic cells on histone modifications was also determined. We show that doxorubicin binding sites were present in the nucleosome-free regions associated with regulatory regions (promoters, enhancers, and super-enhancers) and in the gene body (introns). Doxorubicin treatment did not alter the levels of H3K4me3 and many other histone modifications but significantly lowered H2B ubiquitinated at lysine 120 (H2BK120ub), an elongation-dependent modification. Lastly, we demonstrate that doxorubicin results in the degradation of the largest subunit (RPB1) of RNA polymerase II.

G protein-coupled receptor (GPCR) signaling regulates a wide range of pathophysiological cell functions via G protein α and βγ subunits. Small molecules targeting the subunits of Gα and Gβγ have been developed as cancer therapeutics. We have previously reported that transforming growth factor-α (TGF-α) induces the migration of human hepatocellular carcinoma (HCC) HuH7 cells through the activation of AKT, p38 mitogen-activated protein kinase (MAPK), Rho-kinase, and c-Jun N-terminal kinase (JNK). This study aims to determine whether Gβγ subunits regulate the TGF-α-induced migration of HCC HuH7 cells using gallein, a Gβγ subunits inhibitor. The Janus family of tyrosine kinase/signal transducer and activator of transcription 3 (STAT3) signaling pathway was also involved in the regulation of the migration. Gallein significantly reduced the TGF-α-induced cell migration. In contrast, fluorescein, a gallein-related compound that has no effect on Gβγ subunits, failed to affect the cell migration. Gallein suppressed the TGF-α-stimulated phosphorylation of JNK without affecting the phosphorylation of epidermal growth factor receptor, AKT, p38 MAPK, target protein of Rho-kinase, and STAT3. Conversely, fluorescein did not attenuate the phosphorylation of JNK. These results strongly suggest that Gβγ subunits act as positive regulators in TGF-α-induced migration of HCC cells via the JNK signaling pathway.

This study used a modified flapless (FLT) version of tannase from Lactobacillus plantarum, (LpTan) to explore the effects of "stacking" site mutations predicted by Protein Repair One Stop Shop (PROSS) to increase stability. Four different LpTan structural-state models (including apo, substrate- and product- bound as well as FLT) were comparatively applied, yielding 143 predicted mutations. Of these, eight mutations (including Q63T, A65D, A184Y, A257D, V276Y, T321G, G421D, and G439D (FLT numbering)) were selected to stack, based on conservation of the prediction across all four structural states. Combinatorial screening of the arising 256-member library yielded a selection of possible hits, of which four were further characterized. Variant P6H7 contained 7 of the 8 mutations (excluding V276Y) and showed the highest significant k_cat, 17% higher than FLT and 30% higher than LpTan, and a 4.5 °C increase in T_m . Variant P8E5 with 6 of 8 mutations (excluding A257D and G439D), yielded a 6.5 °C increase in T_m compared to FLT. The two other variants showed more moderate increases, albeit still greater than FLT or LpTan. Overall, the ability to design thermal stabilized versions of a tannase is emphasized. Putative mechanisms underlying the stabilization imparted by the highlighted variations are discussed.

Recruitment of faculty members in academic departments shapes the department for decades in research and teaching arenas. A diverse department is beneficial for all students as representation of underrepresented minority groups in the professoriate can inspire a greater diversity of students to pursue higher levels of education or research-focused careers. Increased diversity benefits research directly as diverse teams have been shown to have better ideas and outcomes. In 2020, our department had lower gender diversity than expected based on the pool of qualified personnel in Canada. Therefore, we altered our hiring process, primarily by redacting applications, for recruitment into entry-level tenure-track faculty positions. This resulted in the increased hiring of women (17% to 80%) with no substantial change in hiring of racially diverse individuals (50% to 40%). Overall, combined with retirements, the percentage of women faculty in the department went from 25% to 50% and the percentage of racialized faculty went from 38% to 44%. Thus, our intervention was successful in increasing the diversity of our department within a short timeframe. Our experience could provide other departments with a template for making substantive change, even in the absence of internal expertise in the area.

The hexosamine biosynthetic pathway (HBP) is upregulated in many cancer cell types leading to upregulation of post-translational modification of proteins by β-N-acetylglucosamine (O-GlcNAc), the product of HBP. However, our knowledge of the identity of proteins that undergo O-GlcNAcylation in cancer cells and consequently their roles is very limited. We investigated the O-GlcNAcylation of epidermal growth factor receptor (EGFR) and glucose transporter 1 (GLUT1) in T47D and MDA-MB-231 breast cancer cell models. We examined the effect of the loss of putative O-GlcNAcylation sites in EGFR and GLUT1 on cell-signaling pathways and their functional consequences on cell cycle progression and cell metabolism using fluorescence-activated cell sorting analysis and in vitro assays. EGFR and GLUT1 undergo O-GlcNAcylation in T47D and MDA-MB-231 breast cancer cells, which enhances their functions and prevents their intrinsic downregulation. This appears to involve an interplay between phosphorylation, O-GlcNAcylation, and ubiquitination in both proteins. Importantly, perturbing the putative O-GlcNAcylation sites in both proteins adversely affected their stability, functions, and metabolic status of breast cancer cells, including glucose uptake and lactate production. In conclusion, the reprogrammed metabolism in cancer cells extends beyond energy and macromolecule requirements and contributes to cell-signaling events that support the stability and function of cancer promoting proteins.

The XVIth International Conference on Lactoferrin, held in Rome in November 2023, showcased cutting-edge research on the multifunctional glycoprotein lactoferrin (Lf). Known for its broad antimicrobial, anti-inflammatory, and immunomodulatory properties, Lf continues to challenge the one-protein-one-function paradigm. Presentations highlighted its evolving therapeutic applications, including anti-biofilm strategies, modulation of immune responses, iron homeostasis, gut health, and cancer inhibition. Studies emphasized the importance of Lf source, iron saturation, and formulation, including recombinant and peptide derivatives. The collection underscores the expanding biomedical relevance of Lf and sets the stage for continued exploration at the upcoming XVIIth Conference in Mazatlán, Mexico.

Filamins are dimeric actin-binding protein that play a critical role in mechanical signaling. They contain a mechanosensory region (MSR) that naturally folds into a globular closed conformation. Under mechanical stress, the MSR unfolds into an open conformation, exposing binding sites for numerous proteins. Filamins are involved in diverse cellular functions, and their mechanical binding targets are highly context-dependent. In this study, we employed AlphaFold2 modelling for screening proteins that specifically recognize the open conformation of filamins. We focused on the Drosophila melanogaster filamin, Cheerio, and conducted a biased screen to identify mechanical binding proteins. We selected the top 132 hits from the initial screening for further characterization. All identified binding proteins specifically recognize the open conformation of the MSR and not the closed conformation. Interestingly, the binding regions of these proteins lack obvious sequence similarity. While some false positives were identified, they could be effectively filtered out based on the secondary structure formed at the binding interface. This study provides a framework for identifying specifically filamin interactions in mechanosignaling.

Transcription factors (TFs) are specialized proteins that bind DNA in a sequence-specific manner and modulate RNA polymerase II (Pol II) in multiple steps of the transcription process. Phase separation is a spontaneous or driven process that can form membrane-less organelles called condensates. By creating different liquid phases at active transcription sites, the formation of transcription condensates can reduce the water content of the condensate and lower the dielectric constant in biological systems, which in turn alters the structure and function of proteins and nucleic acids in the condensate. In RNA Pol II transcription, phase separation formation shortens the time at which TFs bind to target DNA sites and promotes transcriptional bursting. RNA Pol II transcription is engaged in developing several diseases, such as cardiovascular disease, by regulating different TFs and mediating the occurrence of phase separation. This review aims to summarize the advances in the molecular mechanisms of RNA Pol II transcriptional regulation, in particular the effect of TFs and phase separation. The role of RNA Pol II transcriptional regulation in cardiovascular disease will be elucidated, providing potential therapeutic targets for the management and treatment of cardiovascular disease.