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Purpose of ribosomal protein S5 in catalyzing of protein molecules for generatation of dephosphorylation reaction

A ribosomal protein and dephosphorylation technology, which is applied to peptide/protein components, medical preparations containing active ingredients, genetic material components, etc., can solve problems such as unreported effects

Inactive Publication Date: 2013-06-19
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the effect of these compounds on RPS5

Method used

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  • Purpose of ribosomal protein S5 in catalyzing of protein molecules for generatation of dephosphorylation reaction
  • Purpose of ribosomal protein S5 in catalyzing of protein molecules for generatation of dephosphorylation reaction
  • Purpose of ribosomal protein S5 in catalyzing of protein molecules for generatation of dephosphorylation reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1 RPS5 is the protein phosphatase of Akt

[0091] Take recombinant purified phosphorylated Akt (Millipore Company) and recombinant GST-RPS5 (Abnova Biologicals Company) or self-made purified recombinant His-RPS5 in 50mM Tris-HCl (pH 7.0), 10mM MgCl 2 Incubate for 30 minutes at 30oC in the buffer. The levels of phosphorylated Akt Thr308 and Ser473 were detected by Western blot. The results showed that RPS5 concentration-dependently dephosphorylated phosphorylated Akt ( figure 1 A), showing that RPS5 is an Akt protein phosphatase.

[0092] After hepatic stellate cell HSC-T6 was stimulated with TGFb1 (2ng / ml) for 4 hours, RPS5 and Akt were immunoprecipitated with RPS5 and Akt antibodies, respectively, and then different amounts of RPS5 immunoprecipitates were co-incubated with Akt precipitates, according to the above method Detection of RPS5 protein phosphatase activity. The results showed that endogenous RPS5 also catalyzed the dephosphorylation reaction of...

Embodiment 2

[0093] Example 2 Detection of RPS5 protein phosphatase activity with phosphorylated bisamide rhodamine-110 peptide

[0094] Use phosphorylated bisamide rhodamine-110 peptide in the Ser / Thr Phosphatase assay kit (Promega, Madison) as the substrate, and the dephosphorylation reaction buffer contains 2mM MnCl 2 , detect the phosphatase activity of RPS5 according to the reaction conditions of the kit. The fluorescence intensity was measured with a Synergy 2 multifunctional microplate reader (Biotek), and the enzyme activity was expressed as % of the maximum signal. The results showed that RPS5 concentration-dependently dephosphorylated phosphorylated bisamide rhodamine-110 peptide ( figure 1 C).

Embodiment 3

[0095] Example 3 RPS5 protein phosphatase activity depends on manganese and magnesium ions, and is insensitive to broad-spectrum protein phosphatase inhibitor okadaic acid

[0096] Take recombinant purified phosphorylated Akt (Millipore Company) or self-made purified recombinant His-RPS5 in 50mM Tris-HCl (pH 7.0) solution and incubate with okadaic acid (200 nM) for 10 minutes at 4oC. The RPS5 phosphatase activity was detected according to the conditions of Example 1. The results showed that RPS5 activity was dependent on divalent metal ions Mn and Mg ( figure 1 D), but the inhibitory effect of okadaic acid on its activity is weak ( figure 1 E), These properties of RPS5 are the same as those of PP2C.

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Abstract

The invention provides a purpose of ribosomal protein S5 (RPS5) in catalyzing of protein molecules for generation of dephosphorylation reaction, the ribosomal protein S5 serves as PP2C sampling protein phosphatase, provides purposes of RPS5 or agonist or inhibitor of the RPS5, the RPS5 or the agonist or the inhibitor of the RPS5 is used for preparing compounds for treating diseases or a pathological process which an Akt and MAPK access participates in, and further provides a detection method of the RPS5 protein phosphatase activity and a method for screening the agonist or the inhibitor of the RPS5. The invention has the advantages that the RPS5 is demonstrated to be the PP2C sampling protein phosphatase for the first time, the Akt and MAPK access is regulated and controlled, and therefore the RPS5 self can be used as the protein phosphatase, can be used for preparing the compounds for treating the diseases or the pathological process which the Akt and MAPK access participates in or screening substances restraining or activating the RPS5, and is used for treating the diseases or the pathological process which the Akt and MAPK access participates in.

Description

technical field [0001] The present invention relates to a new application of ribosomal protein S5, specifically, the application of ribosomal protein S5 in catalyzing the dephosphorylation reaction of protein molecules. Background technique [0002] Ribosomal protein S5 (Ribosomal protein S5, RPS5) is an important part of eukaryotic ribosomes and plays a regulatory role in protein translation. A large number of literatures have reported that RPS5 is involved in the regulation of cell differentiation and apoptosis. Abnormal expression of RPS5 is related to the occurrence of certain tumors. For details, please refer to the following literature: (1) Tsiftsoglou AS, et al. Commitment of murine erythroleukemia (MEL) cells to terminal differentiation is associated with coordinated expression of globin and ribosomal genes. Prog Clin Biol Res, 1982; 102 pt A: 69-79; (2) Vizirianakis IS, et al. Expression of ribosomal protein S5 cloned gene during differentiation and apoptosis in mur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02A61K38/46A61K48/00A61K31/4375A61P35/00A61P29/00A61P3/10A61P9/00A61P25/18A61P25/28A61P25/24A61P39/00C12Q1/42
Inventor 张俊平李婕许维恒胥静厉建中胡宏岗田媛邱磊胡振林
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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