Short peptide inhibitor targeting calmodulin phosphatase and its substrate T-cell activation nuclear factor and its application

A calmodulin and phosphatase technology, applied in the field of short peptide inhibitors, can solve problems such as exhaustion, cardiovascular system lesions, aggravated hypertension, and hyperlipidemia

Active Publication Date: 2022-04-05
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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  • Short peptide inhibitor targeting calmodulin phosphatase and its substrate T-cell activation nuclear factor and its application
  • Short peptide inhibitor targeting calmodulin phosphatase and its substrate T-cell activation nuclear factor and its application
  • Short peptide inhibitor targeting calmodulin phosphatase and its substrate T-cell activation nuclear factor and its application

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 a short peptide inhibitor targeting calmodulin phosphatase and its substrate T cell activation nuclear factor and application thereof. The present invention provides a fusion polypeptide, which is formed by linking the EV motif of calmodulin phosphatase regulatory factor 1 and the LxVP motif of T cell activation nuclear factor 1 through an A238L linking peptide, or at its amino terminal and / or carboxyl terminal Obtained after linking with a penetrating peptide. The pep3 short peptide constructed by the present invention has a strong ability to bind to CN and inhibit enzyme activity, and exerts an immunosuppressive effect in the intracellular CN / NFAT signaling pathway, which is better than the traditional inhibitor environmental protection bacteria A more specifically destroys the CN / NFAT interaction, and has immunosuppressive function at the animal level, and can be used as a tool drug for local immunosuppression or other immunosuppression.

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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Patent Type & Authority Patents(China)
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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