Structural analysis of full-length lysine acetyltransferase 7 by cryo-electron microscopy

Objective·To analyze the full-length protein structure of human-derived lysine acetyltransferase 7 (KAT7) using cryo-electron microscopy (Cryo-EM) and to obtain the profile information of human-derived KAT7.Methods·The recombinant protein expression plasmid pGEX-4T1-GST-KAT7 was constructed by using the pGEX-4T1 vector and the full-length gene of human-derived KAT7, and the KAT7 protein was expressed in the prokaryotic protein expression system BL21 (DE3). The GST-KAT7 recombinant protein was obtained by using GST affinity chromatography. After removing the GST protein tag with TEV protease, KAT7 was further isolated and purified by HiLoad 16/600 Superdex 75 pg volume exclusion chromatography. The obtained protein samples were identified by Western blotting, and the samples were screened. The protein morphology was observed under negative-stain electron microscopy, and data were collected by using Cryo-EM. The protein particles were selected and the spatial structure of the full-length KAT7 was analyzed with the Cryo-EM analysis software CryoSparc. The MYST structural domain model (5GK9) in the Protein Data Bank (PDB) and AlphaFold prediction model of KAT7 were matched with the generated structural model by UCSF Chimera software.Results·The full-length protein of human-derived KAT7 was successfully purified by affinity chromatography, and high purity KAT7 was obtained by volume exclusion chromatography. After identifying KAT7 by Western blotting, the spatial structure of KAT7 full-length protein was initially resolved by Cryo-EM and single-particle reconstruction techniques, and a preliminary three-dimensional structure model with a resolution of about 10 Å was obtained by three-dimensional optimization. The spatial structure of KAT7 full-length protein was irregular and semi-loop-shaped, and the existing MYST domain model (PDB: 5GK9) can be matched into the C-terminal part of the KAT7 full-length model. The adjusted AlphaFold prediction model can also match the KAT7 full-length structure model.Conclusion·A preliminary analysis of the spatial structure model of full-length protein of human-derived KAT7 is performed by using Cryo-EM

Microarray-assisted pathway analysis identifies MT1X & NFκB as mediators of TCRP1-associated resistance to cisplatin in oral squamous cell carcinoma.

We recently reported that TCRP1, a novel multidrug-resistance associated human gene, can mediate cisplatin resistance in OSCC cells. However, the molecular mechanism underlying this role of TCRP1 remained to be elucidated. In this study, by using Human Toxicology and Drug Resistance Microarray, we identified 30 genes with significantly different expression levels between Tca/PYM and TCRP1 knockdown cell lines. Co-immunoprecipitation experiments and GST-pull down assays showed that metallothionein1X (MT1X) and Akt interact with TCRP1. siRNA-mediated knockdown of TCRP1 and MT1X was found to sensitize cells to cisplatin, leading to increased apoptosis and inhibition of cell proliferation. These functions of TCRP1 may be caused at least in part via activation of the PI3K/Akt/NF-κB signaling pathway. Taken together, our findings indicate that TCRP1 may be an important drug target for improvement of the treatment and survival of patients with oral squamous cell carcinoma

MicroRNA-493 suppresses tumor growth, invasion and metastasis of lung cancer by regulating E2F1.

miRNAs have been proposed to be key regulators of progression and metastasis in cancer. However, an understanding of their roles and molecular mechanisms is needed to provide deeper insights for better therapeutic opportunities. In this study we investigated the role and mechanism of miR-493 in the development and progression of nonsmall-cell lung cancer (NSCLC). Our data indicated that the expression of miR-493 was markedly reduced in pulmonary carcinoma. The ectopic expression of miR-493 impaired cell growth and invasion in vitro and in vivo. Mechanically, miR-493 commonly directly targeted E2F1, which resulted in a robust reduction of the expression of mRNA and protein. This effect, in turn, decreased the growth, invasion and metastasis of lung cancer cells. Our findings highlight the importance of miR-493 dysfunction in promoting tumor progression, and implicate miR-493 as a potential therapeutic target in lung cancer

Genes with downregulated expression in Tca/PYM-siTCRP1 cells.

a<p>Function: 1. Apoptosis genes; 2. Cell cycle genes; 3. Cell growth, proliferation and differentiation genes; 4. Transporters; 5. Response to stress; 6. Chaperones/heat shock proteins; 7. Transcription factors and regulators; 8. Drug metabolizing enzymes.</p

Diallyl disulfide suppresses SRC/Ras/ERK signaling-mediated proliferation and metastasis in human breast cancer by up-regulating miR-34a.

Diallyl disulfide (DADS) is one of the major volatile components of garlic oil. DADS has various biological properties, including anticancer, antiangiogenic, and antioxidant effects. However, the anticancer mechanisms of DADS in human breast cancer have not been elucidated, particularly in vivo. In this study, we demonstrated that the expression of miR-34a was up-regulated in DADS-treated MDA-MB-231 cells. miR-34a not only inhibited breast cancer growth but also enhanced the antitumor effect of DADS, both in vitro and in vivo. Furthermore, Src was identified as a target of miR-34a, with miR-34a inhibiting SRC expression and consequently triggering the suppression of the SRC/Ras/ERK pathway. These results suggest that DADS could be a promising anticancer agent for breast cancer. miR-34a may also demonstrate a potential gene therapy agent that could enhance the antitumor effects of DADS

<i>In vitro</i> effects of TCRP1 interfering RNA on human OSCC.

<p><b>A.</b> Real-time PCR analysis of TCRP1 mRNA expression. Treatment of Tca/PYM cells with TCRP1 interfering plasmid (pAU-siTCRP1) led to a significant decrease in TCRP1 mRNA levels. No reduction in expression was observed for untreated cells or cells treated with scrambled interfering plasmid. <b>B.</b> TCRP1 expression in Tca/PYM, Tca/PYM-Con, and Tca/PYM-siTCRP1 cells were examined by Western blot. Decreased expression of TCRP1 was observed after transfection with TCRP1 interfering plasmid in Tca/PYM cells. No reduction in expression was observed for untreated cells or cells treated with scrambled interfering plasmid. β-actin was used as the loading control. <b>C.</b> Responses of Tca/PYM and Tca/PYM-siTCRP1 cells to PYM. Tca8113/PYM cells were more resistant to PYM. <b>D.</b> Responses of Tca/PYM and Tca/PYM-siTCRP1 cells to DDP. Tca8113/PYM-siTCRP1 cells were more sensitive to DDP.</p

Identification of genes potentially involved in TCRP1-mediated multidrug-resistance phenotype.

<p><b>A.</b> Differentially expressed genes in the Tca/PYM-Con (left) and Tca/PYM-siTCRP1 (right) cells were analyzed by Human Toxicology and Drug Resistance Microarray (OHS-401). This microarray included 263 key genes critical in drug metabolism and resistance. <b>B.</b> A heat map generated from the microarray shows gene expression as Tca/PYM-siTCRP1 over Tca/PYM-Con cells for the genes whose expressions had increased or decreased by more than 1.5-fold in TCRP1 knockdown cells. <b>C.</b> GO analysis of functional gene grouping of the differentially expressed genes involved in TCRP1-associated multidrug-resistance phenotype.</p