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Polypeptide inhibitor and application thereof

A technology of polypeptide inhibitors and uses, applied in the field of bioengineering, can solve the problems of poor treatment effect of human glioblastoma, achieve good enzyme activity inhibition effect, broaden the design, and improve the effect of mitophagy ability

Active Publication Date: 2021-02-12
PEKING UNIV SHENZHEN GRADUATE SCHOOL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a polypeptide inhibitor and its use. Solve the technical problem that the drugs in the prior art are not effective in the treatment of human glioblastoma

Method used

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  • Polypeptide inhibitor and application thereof
  • Polypeptide inhibitor and application thereof
  • Polypeptide inhibitor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Preliminary truncation screening of polypeptide inhibitors

[0029] The present invention designs more effective USP30 polypeptide inhibitors based on the USP30 transmembrane domain. Such as figure 1 As shown, previous studies have shown (Lee, J.G., Kim, W., Gygi, S. & Ye, Y. Characterization of the deubiquitinating activity of USP19 and its role in endoplasmic reticulum-associated degradation. J Biol Chem 289, 3510-3517 ( 2014).) USP19 has a transmembrane domain of self-inhibitory activity. By comparing the sequences of the transmembrane domains of USP19 and USP30, we found that the transmembrane domains of the two have a high similarity ( figure 1 a, 1b). Therefore, we speculate that the transmembrane domain sequence of USP30 has a similar function of inhibiting autocatalytic activity.

[0030] The present invention synthesizes a polypeptide Q1 containing a transmembrane domain (TM) and a mitochondrial outer membrane localization sequence (MLS) based o...

Embodiment 2

[0034] Embodiment 2: the alanine screening of polypeptide inhibitor

[0035] As shown above, the Q14 polypeptide in the transmembrane domain (TM) and mitochondrial outer membrane localization sequence (MLS) of USP30, which is rich in hydrophobic N-terminus and C-terminus rich in basic amino acids, can significantly inhibit the activity of USP30. The present invention intends to explore which residues on the polypeptide play an important role in inhibiting the function of USP30. Such as figure 2 As shown, the present invention has carried out alanine mutations to 18 amino acids of the polypeptide to detect the impact of amino acids on the inhibition of USP30 ( figure 2 a). From the results of enzyme activity, it can be seen that the mutation of the amino acid on the Q14 polypeptide has a greater impact on the catalytic activity of Q14 to inhibit the USP30 protein, and the mutation of the N-terminal of the polypeptide has a smaller impact than the C-terminal, especially MT6 ...

Embodiment 3

[0037] Previous studies have shown that USP30 protein is a special USP-type deubiquitinase, which is more inclined to catalyze the hydrolysis of ubiquitin chains with a compact structure, and the hydrolysis speed of Lys 6-type ubiquitin chains is the fastest. Therefore, the present invention pre-incubated Q14 polypeptides with different concentrations and 2 μg USP30 protein for 30 minutes, then added the tetraubiquitin chain of Lys 6, stopped the reaction after 15 minutes in a 37-degree water bath, and detected the hydrolyzed protein by silver staining experiment. The ubiquitin chain was used to evaluate the inhibitory effect of polypeptide Q14 on the catalytic activity of USP30. Consistent with the results of the Ub-AMC enzyme digestion assay, with the increase of Q14 concentration, compared with the control, the monoubiquitin chains hydrolyzed gradually decreased ( image 3 a). This indicated that Q14 gradually inhibited the catalytic activity of USP30 with increasing conce...

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Abstract

The amino acid sequence of the polypeptide inhibitor is as shown in SEQ ID NO. 1. The invention also provides application of the polypeptide inhibitor in preparation of a medicine for inhibiting the activity of USP30 protease. An enzyme activity inhibition experiment proves that the polypeptide provided by the invention can effectively inhibit the deubiquitination enzyme activity of USP30. Ubiquitin chain hydrolysis and mitochondrial deubiquitination experiments prove that the polypeptide provided by the invention can effectively inhibit the catalytic activity of deubiquitination enzyme USP30.A coprecipitation experiment, a cell thermal migration experiment and an immunofluorescence co-localization experiment prove that the polypeptide can interact with USP30 protein in cells. The Westernblot experiment of mitochondrial autophagy related protein and the detection experiment of mitochondrial ATP content and mitochondrial mtDNA content prove that the polypeptide can inhibit the catalytic activity of USP30 in cells and improve the mitochondrial autophagy level.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to a polypeptide, specifically a polypeptide inhibitor and its application. Background technique [0002] With the acceleration of population aging, the morbidity and mortality of Parkinson's disease are increasing worldwide, and Parkinson's disease has become one of the main threats to human health. Previous studies have shown that the exact etiology of Parkinson's disease is still unclear, and genetic factors, environmental factors, aging, oxidative stress, etc. may all be involved in the degeneration and death of dopaminergic neurons in Parkinson's disease. At present, the pathological mechanism of Parkinson's disease needs to be further studied, and at the same time, there is a lack of specific drugs to cure Parkinson's disease. Therefore, further elucidation of the pathological mechanism of Parkinson's disease and the search for more effective drug targets will provide a basis for th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/48A61K38/48A61P35/00
CPCC12N9/485C12Y304/19012A61P35/00A61K38/00
Inventor 李子刚尹丰秦璇
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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