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Oligopeptide inhibitor for targeting calcineurin and substrate nuclear factor of activated T cells thereof and application of oligopeptide inhibitor

A calmodulin and phosphatase technology, applied in the field of short peptide inhibitors, can solve problems such as increasing the risk of obesity, cardiovascular system diseases, and failure

Active Publication Date: 2020-12-29
BEIJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has many unresolvable toxic and side effects, the main adverse reactions are nephrotoxicity, neurotoxicity, and liver toxicity, which may lead to long-term failure of transplanted kidneys and normal kidneys
It can also aggravate high blood pressure and high blood fat, so it will cause lesions in the cardiovascular system and increase the risk of obesity

Method used

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  • Oligopeptide inhibitor for targeting calcineurin and substrate nuclear factor of activated T cells thereof and application of oligopeptide inhibitor
  • Oligopeptide inhibitor for targeting calcineurin and substrate nuclear factor of activated T cells thereof and application of oligopeptide inhibitor
  • Oligopeptide inhibitor for targeting calcineurin and substrate nuclear factor of activated T cells thereof and application of oligopeptide inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Design of pep3 short peptide targeting calmodulin phosphatase and its substrate T cell activation nuclear factor and synthesis of gene sequence

[0059] The present invention aims at the binding site between CN and its substrate—PxIxIT site and LxVP site, and uses 17 amino acids of the short peptide A238L as Linker to link the EV motif (pep1) of RCAN1 with the LxVP motif (pep2) of NFATc1, An active polypeptide with two binding sites with CN was synthesized, named peptide3 (pep3 for short) ( figure 1 ). The amino acid sequences of the three pep short peptides and Linker peptides are shown in Table 1.

[0060] Table 1 Amino acid sequence of each short peptide involved in the present invention

[0061]

[0062] According to the codon preference of mammals, press figure 1 The amino acid sequence shown is a nucleic acid sequence synthesized at Huada Gene Biotechnology Co., Ltd. The nucleic acid sequence encoding the short peptide of Pep1 is shown in SEQ ID ...

Embodiment 2

[0063] Example 2. The binding ability of pep3 short peptide to CN is much stronger than that of pep1 and pep2—GST Pull-Down experiment

[0064] 1. Construction of recombinant expression vector

[0065] The coding genes of the optimized Pep1, Pep2 and Pep3 short peptides in Example 1 were respectively cloned into the multiple cloning site of the pGEX-4T-1 vector fused to express the GST tag protein, so that the short peptide and the GST tag protein could be fused and expressed, Three recombinant expression vectors were obtained.

[0066] The correct recombinant expression vector for expressing Pep1 short peptide verified by sequencing was named pGEX-4T-1-Pep1; the correct recombinant expression vector for expressing Pep2 short peptide verified by sequencing was named pGEX-4T-1-Pep2; The correct recombinant expression vector for expressing Pep3 short peptide verified by sequencing was named pGEX-4T-1-Pep3.

[0067] 2. Transform Escherichia coli

[0068] The above-mentioned th...

Embodiment 3

[0094] Example 3. Inhibition of CN activity by pep3 short peptide—using RII short peptide as a substrate to measure the activity of malachite green

[0095] The pep3 short peptide used in this implementation was synthesized by Zhongke Yaguang Biotechnology Co., Ltd., and its amino acid sequence is shown in SEQ ID No.4.

[0096] 1. Determination of proenzyme activity

[0097] The proenzyme activity of CN catalytic subunit (catalytic subunit, A subunit, CNA) on CN physiological substrate RII short peptide (RII subunit of protein phosphokinase, amino acid sequence is shown in Table 1) was determined. The experimental methods are shown in Table 2.

[0098] Table 2 CNA proenzyme activity assay scheme

[0099]

[0100] Note: The enzyme solution in the table is the original enzyme solution of CNA (enzyme solution with higher concentration of CN obtained by lysing the mouse brain). The enzyme diluent is the diluent (recipe: 50mM (pH=7.4) Tris-HCl; 0.1mM EDTA; 0.1mMEGTA; 0.2% (v / ...

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Abstract

The invention discloses an oligopeptide inhibitor for targeting calcineurin and a substrate nuclear factor of activated T cells (NFAT) thereof and application of the oligopeptide inhibitor. The invention provides a fusion polypeptide, and the fusion polypeptide is formed by connecting an EV motif of a calcineurin regulatory factor 1 and an LxVP motif of the nuclear factor 1 of activated T cells through an A238L connecting peptide, or is obtained by connecting a cell-penetrating peptide to an amino terminal and / or a carboxyl terminal of the fusion polypeptide. The pep3 oligopeptide constructedby the invention shows relatively high capability of being bonded with calcineurin (CN) and inhibiting enzymatic activity, and exerts an immunosuppressive effect in an intracellular CN / NFAT signal path; the pep3 oligopeptide can destroy CN / NFAT interaction more specifically than a traditional inhibitor cyclosporin A, and has an immunosuppressive function at an animal level; and the pep3 oligopeptide can be used as a tool drug for local immunosuppression or can be used for developing other immunosuppressive drugs.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a short peptide inhibitor targeting calmodulin phosphatase and its substrate T cell activation nuclear factor and application thereof. Background technique [0002] Calmodulin phosphatase (calcineurin, CN) is dependent on Ca 2+ It is a serine / threonine protein phosphatase with calmodulin (CaM), which can interact with substrates and target proteins in vivo. CN is a heterodimer composed of catalytic subunit (catalytic subunit, A subunit, CNA) and regulatory subunit (regulatory subunit, Bsubunit, CNB). The two subunits are tightly bound and only denaturation can separate them. Only when CNA is present, its activity is very low, and only when CNB is present at the same time, it can show high specificity phosphatase activity. When Ca 2+ When bound to CNB, it has an important effect on the activity of CN, but the more dominant effect is due to the regulation of CaM. [0003] T cell a...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K38/17A61K47/64A61P37/06A61P37/08A61P11/06
CPCC07K14/47A61P37/06A61P37/08A61P11/06C07K2319/10C07K2319/00A61K38/00
Inventor 骆静王璐王萍程娜魏群
Owner BEIJING NORMAL UNIVERSITY
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