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Compositions and methods for inhibiting G protein signaling

A G protein and protein technology, applied in the field of compositions and methods for inhibiting G protein signal transduction, can solve problems such as inability to promote disaggregation of heterotrimers

Inactive Publication Date: 2008-04-30
UNIVERSITY OF ROCHESTER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other Gβγ-binding peptides, such as QEHA from adenylate cyclase II (Weng, et al. (1996) J. Biol. Chem. 271:26445-26448; Chen, et al. (1997) Proc. Natl .Acad.Sci.USA94:2711-2714) and the 643-670 amino acids of the C-terminal region of βARK (GRK2) (Koch, et al. (1993) see above) although competitively combined with the Gα subunit, but does not promote disaggregation of heterotrimers (Ghosh, et al. (2003) supra)

Method used

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  • Compositions and methods for inhibiting G protein signaling
  • Compositions and methods for inhibiting G protein signaling
  • Compositions and methods for inhibiting G protein signaling

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1: Materials

[0113] Peptides were purchased from Alpha Diagnostic International (San Antonio, TX) or - Genosys (St. Louis, MO), HPLC purified to >90% and molecular weight determined by mass spectrometry. Ni-NTA agarose was purchased from (Valencia, CA). Streptavidin-coated polystyrene beads were purchased from Spherotec (Libertyville, IL). HRP-conjugated anti-M13 antibody was purchased from Amersham Biosciences (Piscataway, NJ). HRP-conjugated neutravidin was purchased from Pierce (Rockford, IL). Unless otherwise specified, all molecular biology reagents were purchased from INVITROGEN TM (Carlsbad, CA).

Embodiment 2

[0114] Example 2: Gβ 1 gamma 2 and SIGK peptide expression and purification

[0115] wild-type bovine Gβ 1 cDNA and N-terminal (His) 6 -labeled bovine Gγ 2 The cDNA of the baculovirus was used to produce the corresponding protein. with high titer of Gβ 1 and Gγ 2 Baculovirus infection of High-5 cells (INVITROGEN TM , Carlsbad, CA; 2×10 6 cells / mL). Gβ was purified using a modification of the standard method (Kozaza and Gilman (1995) J. Biol. Chem. 270:1734-41) 1 gamma 2 . All steps were done at 4 °C. The cells were harvested by centrifugation at 2600g 60 hours after infection, and then 50mL of lysis buffer (20mM HEPES, pH 8, 150mM NaCl, 5mM β-ME, 1mM EDTA, 1mL Protease Inhibitor Cocktail P-2714) to resuspend the cells. Cells were lysed by sonication and centrifuged at 2600 g to pellet membranes. Membranes were triturated to resuspend and homogenized in 100 mL lysis buffer. Add 1% of Luburol (C12E10, St. Louis, MO) and stirred to dissolve the membrane, and th...

Embodiment 3

[0117] Example 3: Crystallography

[0118] A 1.5 molar excess of peptide SIGK was added to Gβ 1 gamma 2 In, Gβ for crystallization 1 gamma 2 • The concentration of the SIGK complex was 7 mg / mL. Crystals were grown by vapor diffusion at 20°C using an equal volume (2 μL) of protein and crystallization solution (15-17% PEG 4000, 100 mM HEPES, pH 7.5, 0.01-0.05M sodium acetate, 10% glycerol). The diameter of the crystal reaches 150 μm×50 μm×20 μm within one week. Crystals were cryoprotected with 15% glycerol and frozen in liquid nitrogen.

[0119] Gβ 1 gamma 2 · SIGK’s Advanced Light Source (ALS) beamlines 8.2.1 and 8.2.2 (Berkeley, CA) and Advanced Photon Source (APS) photon beamline BM-19 (Chicago, IL) for natural crystals to filter. Data sets obtained from ALS 8.2.2 were used to determine structure. More than 100 crystals screened, diffraction limited to 7 for structure determination to 2.7 data set. The diffraction data were indexed, integrated and scaled (Table...

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Abstract

The present invention relates to methods for identifying agents which bind to specific amino acid residues of the protein interaction site of G protein Beta subunit. Compounds identified in accordance with the assay of the invention and methods for using the compound for modulating at least one activity of a G protein are also provided.

Description

[0001] introduction [0002] This invention was made during the course of research funded by the National Institutes of Health (Grant Numbers: GM60286 and DK46371). The US Government has certain rights in this invention. Background technique [0003] Five mammalian isoforms of the G protein β subunit (37 kDa) and 12 isoforms of the G protein γ subunit (7.8 kDa) have been discovered (Offermanns (2003) Prog. Biophys. Mol. Biol 83: 101-30). The obligate heterodimer (Gβγ) composed of G protein β subunit and γ subunit plays a regulatory role in many pathways of eukaryotic cells (Neves, et al. (2002) Science 296: 1636- 9; Clapham and Neer (1997) Annu. Rev. Pharmacol. Toxicol. 37:167-203). Gβγ was originally identified as a guanylate dissociation inhibitor (GDI), which binds tightly to the GDP-bound G protein α subunit (Gα), thereby constituting the basic form of the G protein heterotrimer, in which Neither Gα nor Gβγ are active in signaling. G protein-coupled receptors (GPCRs) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/00C07K1/00
CPCG01N2333/4719G01N2500/00G01N33/6872A61P25/36A61P29/00A61P39/02A61P43/00A61P9/04A61K31/352
Inventor A·V·斯马卡J·方特T·博纳斯
Owner UNIVERSITY OF ROCHESTER
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