Biochimica et biophysica acta. General subjects最新文献

KANK1 was found as a tumor suppressor gene based on frequent deletions in renal cell carcinoma and the inhibitory activity of tumor cell proliferation. Previously, we reported that knockdown of KANK1 induced centrosomal amplification, leading to abnormal cell division, through the hyperactivation of RhoA small GTPase. Here, we investigated the loss of KANK1 function by performing CRISPR/Cas9-based genome editing to knockout the gene. After several rounds of genome editing, however, there were no cell lines with complete loss of KANK1, and the less the wild-type KANK1 dosage, the greater the number of cells with abnormal numbers of centrosomes and rates of cell-doubling and apoptosis, suggesting the involvement of KANK1 haploinsufficiency in centrosome aberrations. The rescue of KANK1-knockdown cells with a KANK1-expressing plasmid restored the rates of cells exhibiting centrosomal amplification to the control level. RNA-sequencing analysis of the cells with reduced dosages of functional KANK1 revealed potential involvement of other cell proliferation-related genes, such as EGR1, MDGA2, and BMP3, which have been reported to show haploinsufficiency when they function. When EGR1 protein expression was reduced by siRNA technology, the number of cells exhibiting centrosomal amplification increased, along with the reduction of KANK1 protein expression, suggesting their functional relationship. Thus, KANK1 haploinsufficiency may contribute to centrosome aberrations through the network of haploinsufficiency-related genes.

Cannabidiol (CBD) has antioxidant and anti-inflammatory activities. However, the anti-tumor effect of CBD on hepatocellular carcinoma (HCC) remains unclear. Here, we investigated whether CBD displays anti-tumorigenic effects in HCC cells and whether it could reduce tumorigenesis and metastases in vivo. First, this study treated HCC cells with different concentrations of CBD, followed by analyzing the changes in the proliferative, apoptotic, migratory and invasive abilities. The effects of CBD on the growth and metastasis of HCC cells in vivo were verified by tumorigenesis and metastasis assays. Subsequently, the target genes of CBD were predicted through the SwissTarget website and the genes differentially expressed in cells after CBD treatment were analyzed by microarray for intersection. The enrichment of the pathways after CBD treatment was analyzed by KEGG enrichment analysis, followed by western blot validation. Finally, rescue assays were used to validate the functions of genes as well as pathways in the growth and metastasis of HCC cells. A significant weakening of the ability of HCC cells to grow and metastasize in vitro and in vivo was observed upon CBD treatment. Mechanistically, CBD reduced GRP55 expression in HCC cells, along with increased TP53 expression and blocked MAPK signaling activation. In CBD-treated cells, the anti-tumor of HCC cells was restored after overexpression of GRP55 or deletion of TP53. CBD inhibits the MAPK signaling activation and increases the TP53 expression by downregulating GRP55 in HCC cells, thereby suppressing the growth and metastasis of HCC cells.

The phosphoinositide 3-kinase (PI3K) is involved in regulation of multiple intracellular processes. Although the inhibitory analysis is generally employed for validating a physiological role of PI3K, increasing body of evidence suggests that PI3K inhibitors can exhibit PI3K-unrelated activity as well. Here we studied Ca²⁺ signaling initiated by aminergic agonists in a variety of different cells and analyzed effects of the PI3K inhibitor PI828 on cell responsiveness. It turned out that PI828 inhibited Ca²⁺ transients elicited by acetylcholine (ACh), histamine, and serotonin, but did not affect Ca²⁺ responses to norepinephrine and ATP. Another PI3K inhibitor wortmannin negligibly affected Ca²⁺ signaling initiated by any one of the tested agonists. Using the genetically encoded PIP₃ sensor PH(Akt)-Venus, we confirmed that both PI828 and wortmannin effectively inhibited PI3K and ascertained that this kinase negligibly contributed to ACh transduction. These findings suggested that PI828 inhibited Ca²⁺ responses to aminergic agonists tested, involving an unknown cellular mechanism unrelated to the PI3K inhibition. Complementary physiological experiments provided evidence that PI828 could inhibit Ca²⁺ signals induced by certain agonists, by acting extracellularly, presumably, through their surface receptors. For the muscarinic M3 receptor, this possibility was verified with molecular docking and molecular dynamics. As demonstrated with these tools, wortmannin could be bound in the extracellular vestibule at the muscarinic M3 receptor but this did not preclude binding of ACh to the M3 receptor followed by its activation. In contrast, PI828 could sterically block the passage of ACh into the allosteric site, preventing activation of the muscarinic M3 receptor.

Background

Sialic acids are essential monosaccharides influencing several biological processes and disease states. The sialyltransferases catalyze the transfer of Sia residues to glycoconjugates playing critical roles in cellular recognition and signaling. Despite their importance, the molecular mechanisms underlying their substrate specificity, especially between different organisms, remain poorly understood. Recently, the human ST8Sia IV, a key enzyme in the synthesis of polysialic acids, was found to accept only CMP-Neu5Ac as a sugar-donor, whereas the whitefish Coregonus maraena enzyme showed a wider donor substrate specificity, accepting CMP-Neu5Ac, CMP-Neu5Gc, and CMP-Kdn. However, what causes these differences in donor substrate specificity is unknown.

Methods

Computational approaches were used to investigate the structural and biochemical determinants of the donor substrate specificity in ST8Sia IV. Accurate structural models of the human and fish ST8Sia IV catalytic domains and their complexes with three sialic acid donors (CMP-Neu5Ac, CMP-Neu5Gc, and CMP-Kdn) were generated. Subsequently, molecular dynamics simulations were conducted to analyze the stability and interactions within these complexes and identify differences in complex stability and substrate binding sites between the two ST8Sia IV.

Results

Our MD simulations revealed that the human enzyme effectively stabilizes CMP-Neu5Ac, whereas CMP-Neu5Gc and CMP-Kdn are unstable and explore different conformations. In contrast, the fish ST8Sia IV stabilizes all three donor substrates. Based on these data, we identified the key interacting residues for the different Sias parts of the substrate donors.

General significance

This work advances our knowledge of the enzymatic mechanisms governing sialic acid transfer, shedding light on the evolutionary adaptations of sialyltransferases.

Nanocarrier surface functionalization has been widely regarded as a promising approach for achieving precise and targeted drug delivery systems. In this work, the fabrication of functionalized-Ag-decorated Fe₃O₄@SiO₂ (Fe₃O₄@SiO₂-Ag) nanocarriers with folic acid (FA) and β-cyclodextrin (BCD) exhibit a remarkable capacity for delivering two types of anticancer drugs, i.e., doxorubicin (DOX) and epirubicin (EPI), into cancer cells. The effective functionalization of Fe₃O₄@SiO₂-Ag nanoparticles has been achieved through the use of cysteine (Cys) as an anchor for attaching FA and BCD via EDC-NHS coupling and Steglich esterification methods, respectively. The findings indicate that surface functionalization had no significant impact on the physicochemical characteristics of the nanoparticles. However, it notably affected DOX and EPI loading and release efficiency. The electrostatic conjugation of DOX/EPI onto the surface of Fe₃O₄@SiO₂-Ag/Cys/FA and Fe₃O₄@SiO₂-Ag/Cys/BCD exhibited maximum loading efficiency of 50–60% at concentration ratio of DOX/EPI to nanoparticles of 1:14. These nanocarriers also achieved an 40–47% DOX/EPI release over 36 days. Furthermore, the drug-loaded functionalized-nanocarrier showed cytotoxic effects on SK-MEL-2 cells, as demonstrated by an in vitro MTT assay. This suggests that the as-prepared functionalized-nanoparticles have promise as a carrier for the efficient anticancer drugs.

Under certain stress conditions, astrocytes operate in aerobic glycolysis, a process controlled by pyruvate dehydrogenase (PDH) inhibition through its E1 α subunit (Pda1) phosphorylation. This supplies lactate to neurons, which save glucose to obtain NADPH to, among other roles, counteract reactive oxygen species. A failure in this metabolic cooperation causes severe damage to neurons. In this work, using humanized Saccharomyces cerevisiae cells in which its endogenous Cu/Zn Superoxide Dismutase (SOD1) was replaced by human ortholog, we investigated the role of human SOD1 (hSOD1) in aerobic glycolysis regulation and its implications to amyotrophic lateral sclerosis (ALS), a neurodegenerative disease. Yeast cells ferment glucose even in the presence of oxygen and switch to respiratory metabolism after glucose exhaustion. However, like cells of SOD1-knockout strain, cells expressing A4V mutant of hSOD1 growing on glucose showed a respiratory phenotype, i.e., low glucose and high oxygen consumptions and low intracellular oxidation levels in response to peroxide stress, contrary to cells expressing wild-type (WT) SOD1 (yeast or human). The A4V mutation in hSOD1 is linked to ALS. In contrast to WT SOD1 strains, PDH activity of both sod1Δ and A4V hSOD1 cells did not change in response to a metabolic shift toward oxidative metabolism, which was associated to lower Pda1 phosphorylation levels under growth on glucose. Taken together, our results suggest that A4V mutant cannot regulate aerobic glycolysis via Pda1 phosphorylation the same way WT hSOD1, which might be linked to problems observed in the motor neurons of ALS patients with the SOD1 A4V mutation.

Gallein is known as an inhibitor of Gβγ subunits, but roles of gallein in bone metabolism have not been reported. Fibroblast growth factor 2 (FGF-2) increases angiogenesis and promotes bone regeneration during the early stages of fracture healing. Osteoprotegerin (OPG) secreted by osteoblasts, binds to the receptor activator of nuclear factor-κB (RANK) ligand (RANKL) as a decoy receptor and prevents RANKL from binding to RANK, resulting in the suppression of bone resorption. Our previous report demonstrated that FGF-2 activates the phosphorylation of p38 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (JNK), and p44/p42 MAPK in osteoblast-like MC3T3-E1 cells. Additionally, FGF-2-activated phosphorylation of p38 MAPK and JNK but not p44/p42 MAPK is positively involved in OPG synthesis in these cells. This work aimed to investigate the effects of gallein on the FGF-2-elicited OPG synthesis in osteoblast-like MC3T3-E1 cells and the mechanism. Our findings demonstrated that gallein significantly increased the FGF-2-elicited OPG synthesis in MC3T3-E1 cells. By contrast, fluorescein, gallein-like compound that does not bind Gβγ, did not affect the FGF-2-elicited OPG synthesis. Gallein significantly enhanced the FGF-2-induced OPG mRNA expression levels. Gallein did not affect the FGF-2-activated phosphorylation of p38 MAPK and p44/p42 MAPK, but significantly increased the FGF-2-activated phosphorylation of JNK, while fluorescein did not affect JNK phosphorylation. SP600125, a specific JNK inhibitor, strongly inhibited gallein-induced enhancement of FGF-2-induced OPG synthesis and mRNA expression levels. Our results indicated that gallein increases the FGF-2-induced OPG synthesis due to the JNK activation in the osteoblast.

Background

Drought and water stress impose major limitations to crops, including Maize, as they affect the plant biology at multiple levels. Drought activates the cellular signalling machinery to maintain the osmotic and ROS homeostasis for controlling plant response and adaptation to stress. Molecular priming of seeds plays a significant role in imparting stress tolerance by helping plants to remember the stress, which improves their response when they encounter stress again.

Methods

In this study, we examined the effect of priming maize seeds with H₂O₂ and proline, individually or in combination, on response to drought stress. We investigated the role of molecular priming on the physiological, biochemical and molecular response of maize seedlings during drought stress.

Results

We observed that seed-priming played a significant role in mediating stress tolerance of seedlings under drought stress as indicated by changes in growth, biochemical properties, pigment and osmolyte accumulation, antioxidant enzyme activities, gas exchange parameters and gene expression. Seed-priming resulted in reduced expression of specific miRNAs to increase target transcripts associated with synthesis of osmolytes and maintenance of ROS homeostasis for reducing potential damage to the cellular components.

Conclusions

Seed-priming induced changes in the growth, biochemical properties, pigment and osmolyte accumulation, antioxidant enzyme activities, gas exchange parameters and gene expression, though the response was dependent on the genotype, as well as concentration and combination of the priming agents.

Background

Vascular endothelial growth factor (VEGF) is overexpressed in most malignant tumors, which has important impact on tumor angiogenesis and development. Its gene promoter i-motif structure formed by C-rich sequence can regulate gene expression, which is a promising new target for anti-tumor therapy.

Methods

We screened various compounds and studied their effects on VEGF through extensive experiments, including SPR, MST, TO displacement, FRET, CD, ESI-MS, NMR, MTT, clone formation, qPCR, Western blot, dual-luciferase reporter assay, immunofluorescence, cell scrape, apoptosis, transwell assay, and animal model.

Results

After extensive screening, bisacridine derivative B09 was found to have selective binding and stabilization to VEGF promoter i-motif, which could down-regulate VEGF gene expression. B09 showed potent inhibition on MCF-7 and HGC-27 cell proliferation and metastasis. B09 significantly inhibited tumor growth in xenograft mice model with HGC-27 cells, showing decreased VEGF expression analyzed through immunohistochemistry.

Conclusion

B09 could specifically regulate VEGF gene expression, possibly through interacting with promoter i-motif structure. As a lead compound, B09 could be further developed for innovative anti-cancer agent targeting VEGF.