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The ubiquitin-binding domain of a secreted protein from Mycobacterium tuberculosis

A technology of Mycobacterium tuberculosis and binding domain, applied in the field of cell biology, can solve the problem that the influence of PtpA protein on cell signaling pathway is not clear.

Active Publication Date: 2018-04-06
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the influence of PtpA protein on cell signaling pathways is not yet clear.

Method used

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  • The ubiquitin-binding domain of a secreted protein from Mycobacterium tuberculosis
  • The ubiquitin-binding domain of a secreted protein from Mycobacterium tuberculosis
  • The ubiquitin-binding domain of a secreted protein from Mycobacterium tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 The Ubiquitin Binding Domain (UIML Domain) of Mycobacterium Tuberculosis Secretory Protein PtpA

[0022] The domain of the interaction between Mycobacterium tuberculosis secreted protein PtpA and ubiquitin, its amino acid sequence is: PRSGTHALDVEDPYYGDHSDFEEVFAVIESALPGLHDWVDERLARNG. The UIML domain and its structural analogues can be used in the screening of anti-tuberculosis drugs.

[0023] The preparation method of the PtpAUIML domain protein is to construct the recombinant plasmid pGEX-6P-1-PtpA-UIML containing the PtpAUIML domain gene, and transform the plasmid into Escherichia coli BL21, and use IPTG to induce protein expression at 30°C; Collect the supernatant after high-speed centrifugation; slowly flow the supernatant through the filled Glutathione-Sepharose beads (GE), then add column washing buffer to fully wash the column, and finally add 2-3ml eluent to elute Protein; add the collected eluate into a 10KD protein concentrator tube (millipore), cent...

Embodiment 2

[0024] Example 2 Mycobacterium tuberculosis secretory protein PtpAUIML domain directly interacts with ubiquitin

[0025] Through the crystal structure analysis of the known PtpA and Ub proteins, it was found that there was an amino acid sequence (UIML domain) similar to the eukaryotic UIM domain in the PtpA protein ( figure 1 ), because there is a hydrophobic amino acid sequence similar to the eukaryotic UIM domain in this sequence, we think that this domain may be related to the interaction of ubiquitin, and it is the first prokaryotic UIM domain we discovered. The UIML domain is not consistent with the eukaryotic UIM domain that has been reported so far, and it is the first time we have discovered it in a prokaryotic protein. At the same time, through crystal structure analysis, we think that the A140 site may be the key site of the hydrophobic interaction between PtpA protein and ubiquitin.

[0026] Through in vitro experiments, the PtpA (A140E) protein with A140 site muta...

Embodiment 3

[0029]Example 3 Ubiquitin loses the ability to regulate the enzyme activity of the phosphatase PtpA (A140E) mutant

[0030] In the Chinese invention patent application 201310466907.2, it is recorded that Ub has a strong activation effect on the phosphatase activity of PtpA, thereby enhancing its activity to dephosphorylate the substrates p-JNK and p-P38.

[0031] After performing the same enzyme activity experiment, we found that because PtpA (A140E) lost the ability to interact with ubiquitin, its phosphatase activity was no longer regulated by ubiquitin ( image 3 ). Also in the dephosphorylation reaction in vitro, we also obtained the same result, that is, the dephosphorylation activity of PtpA (A140E) on the substrates p-JNK and p-P38 was no longer regulated by ubiquitin ( Figure 4 ).

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Abstract

The invention discloses a ubiquitin binding domain of a secretory protein of mycobacterium tuberculosis, belonging to the field of cell biology. The present invention determines for the first time the ubiquitin binding domain of PtpA and its key binding site (A140). The ubiquitin binding domain is from the 115th amino acid to the 161st amino acid of the PtpA protein, wherein the hydrophobic amino acid sequence EEVFAVIESA (136‑145) is capable of hydrophobic interactions with ubiquitin. When the A140 site of the UIML domain is mutated, the binding ability of Ub to PtpA and the regulation of its enzyme activity will be lost. The achievements of the present invention provide specific sequences for anti-tuberculosis drug screening and vaccine development based on PtpA molecules, and provide new tools and ideas for clinical treatment of various diseases, especially in the development and clinical application of various anti-tumor drugs. It will have broad prospects, and can also be directly applied to the field of scientific research or guide the development of preparations that reversibly regulate JNK and P38 signaling pathways.

Description

technical field [0001] The invention relates to a ubiquitin binding domain of a secretory protein of mycobacterium tuberculosis, belonging to the field of cell biology. Background technique [0002] Tuberculosis (TB) is an ancient disease caused by Mycobacterium tuberculosis. It, together with AIDS and malaria, is known as the three major infectious diseases threatening human health in the world today. Studies have shown that the Mycobacterium tuberculosis genome encodes 11 serine / threonine protein kinases (STPK) and two eukaryotic-like tyrosine phosphatases (PtpA and PtpB), which are involved in Mycobacterium tuberculosis infection. The process of host macrophages can be secreted into the cytoplasm of host cells, these proteins interact with different protein molecules in host cells, and interfere with eukaryotic cells by regulating the phosphorylation level of each target protein in host cells The purpose of biological signaling pathways. These pathogen-host interaction ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/70G01N33/68A61K45/00A61P31/06C12R1/32
Inventor 刘翠华汪静李冰曦鲁燕
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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