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Verification of ACAP4 activity inhibiting protein and functions of ACAP4 activity inhibiting protein in cell migration

A technology of activity inhibition and protein, which is applied in the field of protein and its coding gene and application, and can solve problems such as imperfections

Inactive Publication Date: 2015-03-11
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, a variety of proteins in cells have been found to effectively regulate ARF6 activity (Inoue and Randazzo, 2007), but the research on how ACAP4 dynamically regulates ARF6 activity in space and time is still incomplete, so looking for related proteins that regulate ACAP4 function is an important work

Method used

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  • Verification of ACAP4 activity inhibiting protein and functions of ACAP4 activity inhibiting protein in cell migration
  • Verification of ACAP4 activity inhibiting protein and functions of ACAP4 activity inhibiting protein in cell migration
  • Verification of ACAP4 activity inhibiting protein and functions of ACAP4 activity inhibiting protein in cell migration

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1. Discovery and preliminary identification of ACAP4 activity inhibitory proteins:

[0054] 1. Discovery of ACAP4 activity inhibitory protein:

[0055] ACAP4 is an ARF6-GTPase activating protein named and patented by us (Ding et al., 2010; Fang et al., 2006). In order to further understand its function and molecular mechanism in cells and organisms, we use The yeast two-hybrid method was used to screen for interacting proteins. First, we use acap4cDNA (patent authorization number: ZL200610007868.X) as a template, apply primers acap4-f (upstream primer, SEQ ID NO: 3) and acap4-r (downstream primer, SEQ ID NO: 4), use polymerase chain The coding region of the acap4 gene was amplified by PCR. PCR was catalyzed by LA Taq polymerase (Takara Company), hot start at 94°C, annealing temperature at 60°C, and extension at 72°C for 3 minutes. After the reaction, the PCR amplification product was directly digested with NdeI and NcoI endonucleases (Takara Company), and the...

Embodiment 2

[0067] The detection analysis of embodiment 2, Acapin and ACAP4 interaction relationship:

[0068] 1. Detection of the interaction between Acapin and ACAP4 in cells:

[0069] Using the method in Example 1 to insert the Acapin gene into p3xFLAG-myc-CMV respectively TM In the -24 vector (Sigma company), the ACAP4 gene was inserted into the p-TRE-HA vector (BD company). HEK293T cells (purchased from ATCC) were subcultured into 10 cm culture dishes with a cell density of about 60%, and placed in a cell culture incubator for culture and growth. After 24 hours, HA-ACAP4 was single-transfected or co-transfected with FLAG-Acapin into HEK293T cells according to the method of calcium phosphate transfection cells in the "Molecular Cloning Experiment Guide", and the cells were collected 24 hours later for co-immunoprecipitation experiments . Cells were first lysed with cell lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM DTT, 0.5% TritionX-100, 1 mMPMSF). After centrif...

Embodiment 3

[0072] Example 3, Acapin analysis of ACAP4GAP activity inhibitory function:

[0073] 1. Acapin binds to the GAP functional domain of ACAP4:

[0074] Using the method in Example 1, the different functional domain fragments of ACAP4, ACAP4 1-402 , ACAP4 402-903 , ACAP4 1-303 , ACAP4 303-402 , ACAP4 402-560 , ACAP4 560-660 , ACAP4 660-903 Inserted into the pGEX-6P-1 vector (Amershanm Bioscience Company), such as image 3 .A shown.

[0075] Using the method in Example 2, ACAP4 proteins expressing and purifying different functional domains were incubated with FLAG-Acapin expressed in HEK293T cells. In vitro pull down results are as follows image 3 .B shows that the GAP functional domain of ACAP4 has the strongest binding ability to Acapin, while the BAR functional domain has a weaker binding ability to Acapin.

[0076] 2. Acapin and ARF6 competitively bind ACAP4:

[0077] Using the method in Example 1, Acapin and ARF6 genes were inserted into the p-TRE-HA vector (BD com...

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Abstract

The invention discloses verification of an ACAP4 activity inhibiting protein and functions of the ACAP4 activity inhibiting protein in cell migration. The invention aims to provide an ACAP4 activity inhibiting protein and a coding gene thereof, and application of the ACAP4 activity inhibiting protein in preparing drugs for inhibiting tumor metastasis. The protein is one of the following amino acid sequences: 1) SEQ ID NO:1 in the sequence table; and 2) protein with function of regulating ACAP4 and ARF6 activity subjected to substitution, deletion or addition of one to ten amino acid residues on the amino acid residue sequence disclosed as SEQ ID NO:1 in the sequence table. The protein is an ACAP4-activity-inhibiting protein, and can display the signal passage function and regulate the migration function of tumor cells by regulating the combining capacity with ACAP4 and the activity of the substrate ARF6. Therefore, the protein can be used as a drug target to perform screening on small-molecule compounds, thereby detecting possible drugs for inhibiting tumor cell metastasis. The protein and coding gene thereof can perform important functions in the fields of medicine and pharmacy, and have wide prospects.

Description

technical field [0001] The present invention relates to protein and its coding gene and application, especially relates to ACAP4 activity inhibiting protein and its coding gene and its function in tumor cell migration. Background technique [0002] ACAP4 was discovered by the Cell Dynamics Laboratory of the University of Science and Technology of China by incubating ARF6 dominant activating mutants with HeLa cell lysates, and was identified by mass spectrometry and biochemical experiments (Fang et al., 2006). The protein has a GTPase Activation of proteolytic activity (patent authorization number: ZL200610007868.X, patent name: an ARF6 activation protein and its coding gene and application). Now more and more evidence shows that ACAP4 is involved in the regulation of downstream signaling pathways mediated by epidermal growth factor (EGF), and plays an important role in the occurrence and development of related malignant tumors (Yu et al.,2011; Zhao et al. , 2013). ARF6 pla...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K16/18C12N15/12C12N15/113C12N15/11A61K38/17A61K31/7088A61P35/00
CPCA61K31/7088A61K38/00C07K14/47C07K16/18C12N15/1135
Inventor 姚雪彪周佳佳王峰松赵玄女王冬梅丁霞
Owner UNIV OF SCI & TECH OF CHINA
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