1.hARD1 antiserum preparation and primary immunohistochemical analysis of hARD1 in tumor tissues.
Min YU ; Chao HUANG ; Mingjun XIANG ; Jianhua LAI ; Hui YANG ; Mingxing MA ; Deyong TAN
Chinese Journal of Biotechnology 2008;24(7):1155-1161
Human arrest defective 1 (hARD1) is an acetyltransferase; its physiological significance remains unclear. To explore the relationship between ARD1 protein and tumors, we detected the hARD1 protein in tumor tissues in vivo. We cloned hARD1 gene from Hela cell and construct recombinant plasmid pET28b-hARD1. The recombinant plasmid was transformed into E. coli BL21 (DE3)plysS. hARD1 protein was expressed by inducing with IPTG(1 mmol/L) and purified up to 95% through Ni2+ chelation affinity chromatography. We used the purified hARD1 protein as antigen immunized the Balb/c mice and obtained the hARD1 specific polyclonal antiserum. Through immunohistochemical analysis of different tumor tissues in vivo, we found that hARD1 expressed at high frequency in breast cancer, prostate cancer and lung cancer, especially, hARD1 expression frequency in breast cancer was up to 70%, which is higher than in the other tumors. These results indicate that the high expression level of hARD1 could be an indicator of the breast cancer. This new finding would be a foundation to further explore the relationship between breast tumor and hARD1.
Acetyltransferases
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analysis
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genetics
;
immunology
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Amino Acid Sequence
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Animals
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Antibodies
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blood
;
immunology
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Base Sequence
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Biomarkers, Tumor
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Breast Neoplasms
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metabolism
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pathology
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Cell Line, Tumor
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Cloning, Molecular
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Escherichia coli
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genetics
;
metabolism
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Female
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Humans
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Immune Sera
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biosynthesis
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Immunization
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Immunohistochemistry
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Lung Neoplasms
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metabolism
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pathology
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Male
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Mice
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Mice, Inbred BALB C
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Molecular Sequence Data
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N-Terminal Acetyltransferase A
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N-Terminal Acetyltransferase E
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Prostatic Neoplasms
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metabolism
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pathology
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Recombinant Proteins
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biosynthesis
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genetics
;
immunology
2.Production of anti-recombinant human arrest defective 1 protein (hARD1) monoclonal antibodies for assaying human breast cancer tissues.
Min YU ; Zehua WANG ; Junli GONG ; Mingxing MA ; Yang JIAO ; Weiwei HUANG ; Qi LÜ ; Lin LI ; Hui YANG ; Deyong TAN
Chinese Journal of Biotechnology 2010;26(1):57-62
Human arrest defective 1(hARD1) is an acetyltransferase catalyzing the N-terminal acetylation of proteins after translation. The high expression of hARD1 could be an indicator of the breast cancer. In current study, we produced an anti-hARD lp monoclonal antibody that could specifically recognize ARD1 in breast cancer tissues by using the immunohistochemical assay. The full-length His-tag hARD1 protein (1-235 aa) was over-expressed in Escherichia coli, and purified recombinant protein was injected into Balb/c mice to perform immunization procedure. Eight stable positive monoclonal cell lines were isolated. ELISA results demonstrated that all light chains of antibodies were kappa, and the heavy chains displayed three subtypes IgG1, IgG2a and IgG2b, respectively. A monoclonal antibody, which could specifically recognize hARD1 protein in breast cancer tissues, was identified by screening different cancer tissues using antibody-specificity method. Further, the specificity of the antibody was confirmed by Western blotting analysis. Our study would facilitate breast cancer diagnosis by using this ARD1 monoclonal antibody in clinic. Also, this antibody could be used as an important tool for further investigating the role of ARD1 in tumorigenesis.
Acetyltransferases
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genetics
;
immunology
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Animals
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Antibodies, Monoclonal
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biosynthesis
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genetics
;
immunology
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Biomarkers, Tumor
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biosynthesis
;
immunology
;
Breast Neoplasms
;
immunology
;
metabolism
;
pathology
;
Escherichia coli
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genetics
;
metabolism
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Female
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Humans
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Immunization
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Mice
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Mice, Inbred BALB C
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N-Terminal Acetyltransferase A
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N-Terminal Acetyltransferase E
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Recombinant Proteins
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biosynthesis
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genetics
;
immunology
3.RNPS1 stabilizes NAT10 protein to facilitate translation in cancer via tRNA ac4C modification.
Xiaochen WANG ; Rongsong LING ; Yurong PENG ; Weiqiong QIU ; Demeng CHEN
International Journal of Oral Science 2024;16(1):6-6
Existing studies have underscored the pivotal role of N-acetyltransferase 10 (NAT10) in various cancers. However, the outcomes of protein-protein interactions between NAT10 and its protein partners in head and neck squamous cell carcinoma (HNSCC) remain unexplored. In this study, we identified a significant upregulation of RNA-binding protein with serine-rich domain 1 (RNPS1) in HNSCC, where RNPS1 inhibits the ubiquitination degradation of NAT10 by E3 ubiquitin ligase, zinc finger SWIM domain-containing protein 6 (ZSWIM6), through direct protein interaction, thereby promoting high NAT10 expression in HNSCC. This upregulated NAT10 stability mediates the enhancement of specific tRNA ac4C modifications, subsequently boosting the translation process of genes involved in pathways such as IL-6 signaling, IL-8 signaling, and PTEN signaling that play roles in regulating HNSCC malignant progression, ultimately influencing the survival and prognosis of HNSCC patients. Additionally, we pioneered the development of TRMC-seq, leading to the discovery of novel tRNA-ac4C modification sites, thereby providing a potent sequencing tool for tRNA-ac4C research. Our findings expand the repertoire of tRNA ac4C modifications and identify a role of tRNA ac4C in the regulation of mRNA translation in HNSCC.
Humans
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DNA-Binding Proteins
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Head and Neck Neoplasms/genetics*
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N-Terminal Acetyltransferases
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RNA, Transfer
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Serine
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Signal Transduction
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Squamous Cell Carcinoma of Head and Neck
4.Overexpression of NAT10 induced platinum drugs resistance in breast cancer cell.
Pan QI ; Ya Ke CHEN ; Rui Li CUI ; Rui Juan HENG ; Sheng XU ; Xiao Ying HE ; Ai Min YUE ; Jiang Kun KANG ; Hao Han LI ; Yong Xin ZHU ; Cong WANG ; Yu Lu CHEN ; Kua HU ; Yan Yan YIN ; Li Xue XUAN ; Yu SONG
Chinese Journal of Oncology 2022;44(6):540-549
Objective:b> To observe the platinum drugs resistance effect of N-acetyltransferase 10 (NAT10) overexpression in breast cancer cell line and elucidate the underlining mechanisms. Methods:b> The experiment was divided into wild-type (MCF-7 wild-type cells without any treatment) group, NAT10 overexpression group (H-NAT10 plasmid transfected into MCF-7 cells) and NAT10 knockdown group (SH-NAT10 plasmid transfected into MCF-7 cells). The invasion was detected by Transwell array, the interaction between NAT10 and PARP1 was detected by co-immunoprecipitation. The impact of NAT10 overexpression or knockdown on the acetylation level of PARP1 and its half-life was also determined. Immunostaining and IP array were used to detect the recruitment of DNA damage repair protein by acetylated PARP1. Flow cytometry was used to detect the cell apoptosis. Results:b> Transwell invasion assay showed that the number of cell invasion was 483.00±46.90 in the NAT10 overexpression group, 469.00±40.50 in the NAT10 knockdown group, and 445.00±35.50 in the MCF-7 wild-type cells, and the differences were not statistically significant (P>0.05). In the presence of 10 μmol/L oxaliplatin, the number of cell invasion was 502.00±45.60 in the NAT10 overexpression group and 105.00±20.50 in the NAT10 knockdown group, both statistically significant (P<0.05) compared with 219.00±31.50 in wild-type cells. In the presence of 10 μmol/L oxaliplatin, NAT10 overexpression enhanced the binding of PARP1 to NAT10 compared with wild-type cells, whereas the use of the NAT10 inhibitor Remodelin inhibited the mutual binding of the two. Overexpression of NAT10 induced PARP1 acetylation followed by increased PARP1 binding to XRCC1, and knockdown of NAT10 expression reduced PARP1 binding to XRCC1. Overexpression of NAT10 enhanced PARP1 binding to LIG3, while knockdown of NAT10 expression decreased PARP1 binding to LIG3. In 10 μmol/L oxaliplatin-treated cells, the γH2AX expression level was 0.38±0.02 in NAT10 overexpressing cells and 1.36±0.15 in NAT10 knockdown cells, both statistically significant (P<0.05) compared with 1.00±0.00 in wild-type cells. In 10 μmol/L oxaliplatin treated cells, the apoptosis rate was (6.54±0.68)% in the NAT10 overexpression group and (12.98±2.54)% in the NAT10 knockdown group, both of which were statistically significant (P<0.05) compared with (9.67±0.37)% in wild-type cells. Conclusion:b> NAT10 overexpression enhances the binding of NAT10 to PARP1 and promotes the acetylation of PARP1, which in turn prolongs the half-life of PARP1, thus enhancing PARP1 recruitment of DNA damage repair related proteins to the damage sites, promoting DNA damage repair and ultimately the survival of breast cancer cells.
Breast Neoplasms/enzymology*
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Cell Line, Tumor
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Drug Resistance, Neoplasm
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Female
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Humans
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MCF-7 Cells
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N-Terminal Acetyltransferases/metabolism*
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Organoplatinum Compounds/pharmacology*
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Oxaliplatin/pharmacology*
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X-ray Repair Cross Complementing Protein 1