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Function of actin 84 lysine monomethylation in cytokinesis and cell proliferation as well as application thereof to drug development

A technology of actin and lysine, applied in peptide/protein components, medical preparations containing active ingredients, applications, etc., can solve problems such as unclear structure and function

Active Publication Date: 2013-02-06
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The height change of actin and myosin at the cleavage groove is regulated by protein post-translational modifications (PTMs), and studies have shown that the recruitment of myosin to the cleavage groove in the early and late stages of mitosis is It is accomplished by phosphorylation modification, and the degradation of phosphorylated kinases will lead to myosin localization and actin depolymerization at the cleavage groove. Actin depolymerization and polymerization are also regulated by various post-translational modification signaling pathways, such as Oxidation, arginylation, sumoylation and S-nitrosylation, which have important effects on actin structure, distribution and function, however whether actin on the contractile ring and midbody have similar modifications, these How the modification affects the structure and function of the contractile-ring midbody is unclear

Method used

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  • Function of actin 84 lysine monomethylation in cytokinesis and cell proliferation as well as application thereof to drug development
  • Function of actin 84 lysine monomethylation in cytokinesis and cell proliferation as well as application thereof to drug development
  • Function of actin 84 lysine monomethylation in cytokinesis and cell proliferation as well as application thereof to drug development

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: Experimental methods and materials applied in the embodiments of the present invention

[0045] Experimental Materials:

[0046] 1. Information on the cell lines used

[0047]

[0048] 2. Antibody information used

[0049]

[0050]

[0051] 3. Plasmid information used

[0052]

[0053] 4. Chemical reagents used

[0054] Nocodazole (Sigma, M1404), Cytochalasin D (Sigma, C8273) and Blebbistatin (Sigma, B0560), taxol (Invitrogen P3456) and phalloidin (Invitrogen P3457), Lipofectamine 2000 (Invitrogen), Lipofectamine RNAiMAX (Invitrogen), PI-stained kit (Becton-Dickinson), Annexin V-FITC & PI Detection Kit (Jiamay Biotech, Beijing, China), DAPI (Vector Laboratories, Burlingame, CA), anti-flag M2 affinity gel (sigma, A2220), anti-HA affinity gel (sigma), GFP-Trap-A beads (ChromoTek, gta-20), anti-Rabbit Ig IP Beads (eBioscience), anti-Mouse Ig IP Beads (eBioscience), ECL Western Blotting Detection Kit (GE)

[0055] experimental method:

[00...

Embodiment 2

[0200] Example 2: Purification of ALKBH4 fusion protein to prepare rabbit-derived antibody against ALKBH4 protein

[0201] 1. ALKBH4 protein domain and possible activity mechanism

[0202] Escherichia coli AlkB protein as mononuclear non-heme Fe 2+ And α-ketoglutarate-dependent dioxygenase superfamily members, which use metal ions as prosthetic groups and oxygen molecules as co-substrates, activate dioxygen molecules to catalyze the oxidation of methyl groups that are in contact with nitrogen atoms on stable heterocycles group, causing a conformational change that removes the methyl group by releasing formaldehyde. ALKBH protein is a member of the human dioxygenase AlkB protein family. The commonality of this family protein is that it has the functional domain of Escherichia coli AlkB protein: the ferrous ion binding domain is HxDx n The H domain, α-ketoglutarate and the substrate binding domain, the RxxxxxR domain. The ALKBH4 protein also has such domains: iron ion binding...

Embodiment 3

[0216] Example 3: Verification of the localization and expression of the ALKBH4 gene in cells

[0217] 1. The ALKBH4 protein is localized on the centrosome.

[0218] In this experiment, immunofluorescence technique was used to verify the localization of ALKBH4 in cells. MRC5 cells were inoculated into six-well plates covered with coverslips at a seeding density of 30-40%, and treated with Nocodazole at a final concentration of 0.04 μg / ml the next night. 16 hours, the purpose of adding Nocodazole is to arrest the cells in mitosis. After 16 hours, wash once with PBS, replace with fresh complete medium to continue culturing for 1 hour, wash twice with PBS, fix and osmotically drill holes, the specific method is the same as in Example 1. The centrosome marker γ-tubulin and the spindle marker α-tubulin were hybridized with ALKBH4 antibody as the primary antibody, and the secondary antibody was hybridized with FITC-conjugated anti-mouse secondary antibody and Cy3-conjugated anti-ra...

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PUM

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Abstract

The invention discloses function of actin new methylation, namely 84 lysine monomethylation (actin K84mel), in cytokinesis and cell proliferation as well as potential application value in allusion to actin K84mel anti-cancer drug development. Only after demethylation is carried out on the actin K84mel by the ALKBH4 protein, the NMII is capable of sliding along actin fibers. The actin K84mel is capable of suppressing the interference of the contraction of contractile rings on the cytokinesis. Through the ALKBH4 gene silencing, the expression index of the actin K84mel can be increased; and through the over-expression of the actin K84mel, the mitotic time retardation and the cytokinesis failure are caused, the cell apoptosis and the multinuclear cell generation are finally caused, and the cell proliferation and migration defects are caused and the cells finally die. The expression index of the actin K84mel can be used for developing anti-tumor drugs.

Description

technical field [0001] The invention relates to the function of actin 84-position lysine monomethylation modification (actin K84me1) in cytokinesis and cell proliferation and its potential application value in the research and development of anticancer drugs targeting actin K84me1. Background technique [0002] Cytokinesis usually goes through four main stages: cleavage plane specification, cleavage furrow ingression, midbody formation, and abscission. Failure of cytokinesis often leads to centrosome overduplication and genomic instability, which can lead to malignant proliferation and migration. To ensure that cytokinesis occurs correctly in space and time, many components of cytokinesis and other cellular pathway processes are involved in this tightly regulated process. Cytokinesis is generally considered to be accomplished by the contractile ring (contractile ring) formed by actin fibers (F-actin) and type II non-muscle myosin (non-muscle myosin II or NM II). Actin Fibe...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K38/17A61P35/00C12N15/12C07K14/47
Inventor 杨运桂李明明史悦
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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