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Rheb protein activator and application thereof

An activator and protein technology, applied in peptide/protein components, medical preparations containing active ingredients, metabolic diseases, etc., can solve problems such as difficult to find, and achieve high specificity, great medicinal value, and excellent medicinal value. Effect

Active Publication Date: 2022-06-03
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, because the binding of Rheb to its GEF is relatively transient, it is difficult to detect by traditional means of detecting protein interaction.

Method used

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  • Rheb protein activator and application thereof
  • Rheb protein activator and application thereof
  • Rheb protein activator and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, a kind of method of screening Rheb protein activator

[0042] A method for screening Rheb protein activators, comprising the following steps:

[0043] 1) Construct the fusion expression vector (HAFlag-xxID-Rheb) of biotin ligase (xxID) and Rheb, transfer the vector into HeLa cells, screen the HeLa-xxID-Rheb stable expression cell line, and construct the control cell line HeLa- xxID stably expressing cell lines were cultured in a medium containing 10% dialysis serum; 16 hours before the experiment, a serum-free medium was used to avoid the interference of insulin.

[0044] 2) Then, insulin (0.9 μM) and biotin (50 μM) were added to the HeLa-xxID-Rheb stable expression cell line for stimulation, stimulated for 15 min, and biotin (50 μM) was added to the HeLa-xxID stable expression cell line for stimulation. Insulin was added, stimulated for 15 minutes, the sample was cleaved, and after protein extraction, streptavidin was used to pull down to enrich the biot...

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Abstract

The invention relates to the technical field of biology, and particularly discloses a Rheb protein activator and application thereof. A novel proximity labeling technology is used for screening to obtain a Rhb protein activator-ATP6AP1 protein as a GEF (guanine nucleotide exchange factor) of Rhb, 30 amino acids at the C terminal of the ATP6AP1 protein can directly activate the Rhb in a targeting manner, so that Rheb-GDP is converted into a GTP form, the curative effect is further exerted, and the screened ATP6AP1 protein has a relatively large medicinal value, and can be used for preparing a drug for preventing and treating diseases. Particularly, the pharmaceutical composition has a better medicinal value in the field of treating diabetes mellitus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Rheb protein activator and its application. Background technique [0002] Rheb is a small GTPase of the Ras superfamily, with Rheb-GDP and Rheb-GTP forms, and is an upstream positive regulator of mammalian target of rapamycin complex 1 (mTORC1) signaling pathway It regulates the process of cell growth, proliferation and differentiation together with the mTORC1 signaling pathway. [0003] GDP-GTP exchange requires the participation of specific factors, namely: Rheb-GTP is hydrolyzed to GDP form under the catalysis of GAP, and Rheb-GDP can be reactivated by GEF (guanine nucleotide exchange factor) to convert to GTP form. TSC2 is currently known to function as a GAP (G protein regulator) of Rheb, but no known GEF of Rheb has been found so far. At the same time, no small molecule drugs or short peptide drugs that can directly target and activate Rheb have been found in vitro and in v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47G01N27/62A61K38/17A61P3/10
CPCC07K14/4705G01N27/62A61P3/10A61K38/00Y02A50/30
Inventor 松阳洲冯然刘峰吴苏周志芬李若菲
Owner SUN YAT SEN UNIV