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Ubiquitin-interacting motif-like structural domain of mycobacterium tuberculosis secretory protein

A Mycobacterium tuberculosis, binding domain technology, applied in the field of cell biology, can solve the problem of unclear influence of PtpA protein on cell signaling pathways

Active Publication Date: 2015-08-26
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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  • Ubiquitin-interacting motif-like structural domain of mycobacterium tuberculosis secretory protein
  • Ubiquitin-interacting motif-like structural domain of mycobacterium tuberculosis secretory protein
  • Ubiquitin-interacting motif-like structural domain of mycobacterium tuberculosis secretory protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0022] The domain of interaction between Mycobacterium tuberculosis secretory protein PtpA and ubiquitin, its amino acid sequence is:

[0023] PRSGTHALDVEDPYYGDHSDFEEVFAVIESALPGLHDWVDERLARNG. The UIML domain and its structural analogues can be used in the screening of anti-tuberculosis drugs.

[0024] The preparation method of the PtpA UIML domain protein is to construct the recombinant plasmid pGEX-6P-1-PtpA-UIML containing the PtpA UIML domain gene, and transform the plasmid into Escherichia coli BL21, and use IPTG to induce protein expression at 30°C; Sonicate the bacteria, 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 Elute the protein; add the collected eluate to a 10KD protein conce...

Embodiment 2

[0025] Example 2 Mycobacterium tuberculosis secreted protein PtpAUIML domain directly interacts with ubiquitin

[0026] Through the crystal structure analysis of the known PtpA and Ub proteins, it was found that there is 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 believe 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 reported eukaryotic UIM domain, which is our first discovery 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.

[0027] Through in vitro experiments, the A140 site mutated PtpA (A140E) protein and the wild-type PtpA protein were subj...

Embodiment 3

[0030] Example 3 Ubiquitin loses the ability to regulate the enzyme activity of the phosphatase PtpA (A140E) mutant

[0031] 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.

[0032] 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 an ubiquitin-interacting motif-like (UIML) structural domain of mycobacterium tuberculosis secretory protein, and belongs to the field of cytobiology. The ubiquitin-interacting motif-like structural domain of PtpA and a key binding site (A140) thereof are determined for the first time; the ubiquitin-interacting motif-like structural domain comprises the 115th amino acid of PtpA protein to the 161th amino acid of the PtpA protein, wherein hydrophobic interaction of hydrophobic amino acid sequence EEVFAVIESA (136-145) with ubiquitin (Ub) can be realized; and mutation of the A140 site of the UIML structural domain is capable of resulting in loss of binding capacity of Ub with PtpA, and loss of enzyme activity regulatory functions of Ub. The ubiquitin-interacting motif-like (UIML) structural domain is capable of providing specific sequences for screening of PtpA molecule-based antituberculous drugs and vaccine development, providing treatment of a plurality of diseases in clinic with novel tools and ideas, possesses promising prospect in development and clinical application of a plurality of drugs such as antitumor drugs, and can be directly used for scientific research field or for guiding development of preparations used for reversible control of JNK and P38 signal channels.

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 (Serine / threonine protein kinase, STPK) and two eukaryotic-like tyrosine phosphatases (PtpA and PtpB), which in Mycobacterium tuberculosis During the process of infecting host macrophages, they can be secreted into the cytoplasm of host cells. These proteins interact with different protein molecules in host cells, and interfere with the true effect by regulating the phosphorylation level of each target protein in host cells. The purpose of signaling pathways in nuclear bi...

Claims

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Application Information

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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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