MLKL protein and application of MLKL protein as target point of cell necrosis inhibitor

A technology of inhibitors and proteins, applied in the fields of application, peptide/protein components, cells modified by introducing foreign genetic material, etc., can solve problems such as damage expansion

Inactive Publication Date: 2012-06-27
NAT INST OF BIOLOGICAL SCI BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

MLKL-involved cell necrosis can promote the inflammatory response triggered by injury, and if not controlled, it will lead to the expansion of injury

Method used

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  • MLKL protein and application of MLKL protein as target point of cell necrosis inhibitor
  • MLKL protein and application of MLKL protein as target point of cell necrosis inhibitor
  • MLKL protein and application of MLKL protein as target point of cell necrosis inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Embodiment 1, the discovery of MLKL protein

[0091] In order to obtain the regulatory protein or substrate protein that interacts with the RIP3 protein, induce cell necrosis and use NSA to prevent cell necrosis downstream of the RIP3 protein, and then use the RIP3 protein to do pull down experiments.

[0092] RIP3-HeLa cells were treated with doxycycline for 24 hours (to induce the expression of RIP3); one group of cells was treated with T / S / Z and NSA (T / S / Z+NSA), and the other group of cells was treated with DMSO (D) ;Harvest the cells and obtain the whole cell extract, perform tandem immunoprecipitation with Flag-HA, then analyze the peptides and the eluted RIP3-binding complex by SDS-PAGE, and perform silver staining. like figure 1As shown, 1 is D, 2 is T / S / Z+NSA, and the asterisk (*) marks the heavy chain of IgG. On the SDS-PAGE denaturing gel, there is a band protein shifted above the heavy chain of 55KD IgG. Therefore, it can be concluded that this protein co...

Embodiment 2

[0094] Interaction between embodiment 2, MLKL protein and RIP3 protein

[0095] 1. On the first day, spread RIP3-HT29 cells on a 96-well plate (5000 cells per well).

[0096] 2. On the second day, they were divided into three groups, and three replicate holes were set for each group, and the following treatments were performed respectively:

[0097] Group 1 (negative control treatment; D): 0.1 μL DMSO was added to each well.

[0098] Group 2 (T / S / Z): TNF-α (T), 100 nM Smacmimetic (S) and 20 μM z-VAD (Z) were added to each well at a final concentration of 20 ng / ml to induce cell necrosis;

[0099] Group 3 (T / S / Z+NSA): Add TNF-α (T), 100 nM Smacmimetic (S) and 20 μM z-VAD (Z) to each well at a final concentration of 20 ng / ml to induce cells Necrosis, while adding NSA to each well (final concentration is 0.5μM);

[0100] 3. After the cells treated in step 2 were incubated for 6 hours, the cells were harvested and lysed, and the supernatant was collected. Take 20 μl of supern...

Embodiment 3

[0103] Example 3, Domain analysis of the interaction between MLKL protein and RIP3 protein

[0104] 1. Structural domain analysis

[0105] MLKL protein consists of an N-terminal twisted-coil chain (CC) domain and a C-terminal activation-like domain. The RIP3 protein has an N-terminal kinase domain followed by a RHIM domain, and interacts with the RIP1 protein through RHIM ( Figure 3A ).

[0106] 2. Locating the MLKL protein binding domain of RIP3 protein

[0107] RIP3-FL represents the full-length RIP3 protein (as shown in sequence 3 of the sequence listing). RIP3-Kinase represents the kinase domain of the RIP3 protein (as shown in the 1st to 323rd amino acid residues from the N-terminal of the sequence 3 in the sequence listing). RIP3-K50A represents a mutant RIP3 protein without kinase activity (the full-length RIP3 protein shown in Sequence 3 of the Sequence Listing is mutated from lysine to alanine from the 50th amino acid residue at the N-terminal). MLKL-FL represen...

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Abstract

The invention discloses MLKL protein and an application of the MLKL protein as a target point of a cell necrosis inhibitor. The invention provides the protein represented by the following: (a) the protein comprises the amino acid sequence represented by the sequence 1; (b) the protein comprises the amino acid sequence represented by the sequence 13; (c) the protein is obtained through mutation of threonine on site 357 of the N terminal of the sequence 13 into alanine; (D) the protein is obtained through mutation of serine on the site 358 of the N terminal of the sequence 13 into alanine; (E) the protein comprises the amino acid sequence from the site 179 of the N terminal of the sequence 1 to the site 471 of the N terminal of the sequence 1. The protein is combined with RIP3 protein to provide an apoptosis effect, such that the protein can be used as the target point of anti-apoptosis drugs, and used for screening and developing the anti-apoptosis drugs. The invention further provides compounds for inhibitions of the nucleic acid expressed by the gene and the protein, wherein the compounds can be used as products for inhibitions of the apoptosis. The MLKL protein of the present invention provides important values for medical research and drug development.

Description

technical field [0001] The present invention relates to MLKL protein and its application as the target of cell necrosis inhibitor. Background technique [0002] In mammalian cells, receptor-interacting serine-threonine kinase 3 (RIP3) is a key signaling factor in the cell necrosis pathway. Numerous data indicate that necrosis plays an important role in many physiological and pathological conditions, such as tissue development and injury, and the body's immune response against viruses. [0003] Cell death is very important in multicellular animals. Important discoveries in this area include that cell death is programmed and carried out by very elaborate biochemical pathways in the cell. The activation of the apoptosis pathway ultimately leads to the activation of caspase-3 and caspase-7. Activation of this pair of executive proteases can cause dramatic changes in cell morphology that were later found to be characteristic of apoptosis. One of the two most well-studied apop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/63C12N5/10C12N1/00G01N33/68C12N15/113C07K7/02C07D409/12C07D241/22A61K31/635A61K38/08A61P43/00
Inventor 孙丽明王华翌汪志高何苏丹陈涉廖道红汪来刘伟龙雷晓光王晓东
Owner NAT INST OF BIOLOGICAL SCI BEIJING
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