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Polypeptide inhibitor for REG gamma-20S proteasome and applications thereof

A protein and fusion protein technology, applied in the fields of cell chemistry, molecular biology and biomaterials, can solve the problems of off-target toxicity and poor bioavailability

Inactive Publication Date: 2018-05-01
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Until now, these proteasome inhibitors have not been approved for the treatment of solid tumors, mainly due to their poor bioavailability and severe "off-target" toxicity caused by broad-spectrum protein degradation

Method used

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  • Polypeptide inhibitor for REG gamma-20S proteasome and applications thereof
  • Polypeptide inhibitor for REG gamma-20S proteasome and applications thereof
  • Polypeptide inhibitor for REG gamma-20S proteasome and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Yeast two-hybrid experiment proves that REGγ is combined with NIP30

[0082] In the present invention, REGγ and NIP30 are respectively constructed on pGBK and pGAD vectors, and used in yeast two-hybrid experiments to prove the interaction between the two.

[0083] The results of yeast two-hybrid figure 1 As shown in A and 1B, yeast colonies grew on the His-Leu-Trp-Ade four-deficient yeast culture plate when pGBK-REGγ was co-transfected with pGAD-NIP30, unlike on the two-deficient plate. This proves that there is an interaction between NIP30 and REGγ.

[0084] The specific experimental method of yeast two-hybrid experiment:

[0085] 1. Burn the inoculation loop on the outer flame of an alcohol lamp to sterilize it. Then dip the yeast strain that has just been taken out from the -80°C refrigerator and inoculate it on the YPDA yeast solid medium by continuous streaking.

[0086] 2. Place the yeast culture dish in a constant temperature incubator at 30°C, and ...

Embodiment 2

[0098] Example 2 Co-immunoprecipitation experiment proves that REGγ is combined with NIP30

[0099] Example 1 demonstrated the combination of REGγ and NIP30 through yeast two-hybrid experiments. In order to further confirm this combination, the present invention further tested the combination of REGγ and NIP30 by means of co-immunoprecipitation.

[0100] Co-immunoprecipitation specific experimental method:

[0101] (1) Endogenous co-immunoprecipitation

[0102] 1. Collect 293T cells and lyse the cells with 1% NP40 cell lysate for 30 minutes on ice.

[0103] 2. Place the lysed cell lysate in a 4°C centrifuge at 12000 rpm / min, and centrifuge for 15 minutes. Take one-tenth of the cell lysate as Input, then add 20ul of protein loading to lyse, and cook in a water bath for 10 minutes.

[0104] 3. The remaining 9 / 10 cell lysate was divided into two parts, one part was added with 5ul of antibody, the other part was added with 5ul of IgG antibody, and placed in a refrigerator at 4...

Embodiment 3

[0116] Example 3 A variety of cell lines prove that NIP30 has an inhibitory effect on the degradation of P21, a representative substrate of REGγ, and its function depends on the presence of REGγ

[0117] After demonstrating the interaction between REGγ and NIP30 in Example 1 and Example 2, in order to further explore whether NIP30 has a regulatory effect on the function of REGγ, exogenously overexpress NIP30 or use siRNA to knock down NIP30, a representative substrate for REGγ The protein level and RNA level of P21 were detected.

[0118] Specific experimental methods for exogenous transiently overexpressed proteins:

[0119] 1. Transfection was carried out between 8 hours and 24 hours after the cells were fully adherent and stretched after subculture. The cell density to be transfected should preferably be controlled between 50% and 80%, so that the cells are in the logarithmic phase, so as to ensure the transfection efficiency. The main application of the present invention...

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Abstract

The invention discloses a polypeptide inhibitor having an efficient and specific inhibiting effect for the functions of REG gamma-20S proteasome. The main technical scheme is as follows: the phosphorylation / dephosphorylation modification of NIP30 is a 'molecular switch' for regulating the REG gamma-proteasome, namely, the phosphorylation modification of four sites including NIP30S226, S227, S228 and S230 has great importance for the binding of NIP30 and REG gamma and the degradation for the REG gamma substrate. According to the technical scheme, the polypeptide inhibitor is designed and synthesized based on the phosphorylation modification for the binding of NIP30 and REG gamma, and the polypeptide inhibitor can efficiently and specifically block the functions of the REG gamma-20S proteasome. Compared with the existing chemical inhibitor, the polypeptide inhibitor for the REG gamma-proteasome disclosed by the invention has the advantages of high efficiency and low toxicity, and a noveltreatment target and means are expected to be provided for the malignant tumor (such as metastatic tumor of bone with unknown primary lesion) due to overexpression of REG gamma.

Description

technical field [0001] The invention belongs to the technical fields of cytochemistry, molecular biology and biological materials, and relates to a polypeptide inhibitor capable of efficiently and specifically blocking the function of REGγ-20S proteasome and application thereof. Background technique [0002] Protein degradation plays an indispensable role in many important cell life activities and maintaining the normal function of the body. Existing evidence shows that the disorder of protein degradation can cause various diseases, such as cervical cancer (HPV-Induced Cervical Cancer), lymphoma (Lymphomas) and other malignant tumors; Alzheimer's disease (Alzheimers Disease), Huntington's disease (Huntington Disease) , Parkinson's disease (Parkinson Disease) and other neurodegenerative diseases; and some chronic inflammatory diseases, such as chronic kidney disease (Chronic Kidney Disease). [0003] In eukaryotic cells, there are mainly two types of protein degradation syst...

Claims

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Application Information

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IPC IPC(8): A61K38/17C07K14/47C07K19/00C12N5/10A61P35/00A61P35/04
Inventor 李晓涛高晓王青伟张变红李磊王晓双周星莉王莹刘江周莉张坤
Owner EAST CHINA NORMAL UNIV
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