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Inhibition of binding of Hox and homeodomain containing proteins and uses thereof

A technology of protein interaction and protein, which is applied in the field of inducing the gene encoding bone matrix protein and inducing the gene encoding osteopontin, which can solve problems such as deficiencies

Inactive Publication Date: 2004-06-23
亚拉巴马大学伯明翰分校研究基金会
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The prior art is also deficient in methods for regulating transcription by Hox proteins and / or homeobox-containing proteins

Method used

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  • Inhibition of binding of Hox and homeodomain containing proteins and uses thereof
  • Inhibition of binding of Hox and homeodomain containing proteins and uses thereof
  • Inhibition of binding of Hox and homeodomain containing proteins and uses thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Two-hybrid library screening

[0056] The Smad1 cDNA was cloned into the SaH / PstI site of the pBGT9 vector to generate the pGBT9 / Smad1 bait plasmid. Using this bait plasmid, a human U2 OS osteoblast-like pACT cDNA library was screened according to the procedure provided by the manufacturer (Clontech, CA). To determine the interaction between Hoxc-8 and Smad1, a full-length mouse Hoxc-8 cDNA was subcloned into the XhoI and RcoRI sites of the pACT vector. pACT / Hoxc-8 was co-transformed into Y190 with pBGT9 / Smad1, and colonies were analyzed for β-galactosidase expression by colony transfer filtration assay and liquid assay.

Embodiment 2

[0058] Expression and purification of glutathione S-transferase (GST) fusion protein

[0059] GST fusions of GST-Smad1 and -Smad3 were generated by restriction digestion of pGBT-Smad1(SalI / HindIII) and pCMV5-Smad3(BamHI / SalI) and then inserted into the SalI / HindIII and BamHI / SalI sites of the pGEX-KG vector, respectively constructs. GST-Smad2 and -Smad4 obtained from pCMV-Smad2 and pCMV5-Smad4 were digested with EcoRI / SalI and then inserted into the EcoRI / SalI sites of pGEX-5X-2 and pGEX-5X-1 vectors (Amersham Pharmacia Biotech), respectively. GST-Hoxc-8 and GST-Hoxa-9 were PCR amplified with high-fidelity Pfu-Turbo DNA polymerase (Stratagene) and cloned into the BamHI / EcoRI and BamHI / XbaI sites of pGEX-KG vector, respectively. Dr. C. Abate-Shen (Center of Advanced Biotechnology and Medicine, Piscataway, NJ) provided GST-Msx-1 and -Msx-2 expression plasmids. The above-mentioned GST construct was transformed into BL21, and the fusion protein was expressed and purified.

Embodiment 3

[0061] GST folding test

[0062] exist[ 35 S] In the presence of methionine, Smad1 or Hoxc-8 were translated with linearized Smad1 or Hoxc-8 pBluescript (SK) plasmids, respectively, using the TNT-coupled reticulocyte lysis system according to the manufacturer's (Promega) protocol. The tagged Smad1 protein was confirmed by SDS-PAGE.

[0063] Smad1-containing lysates (5 μl) were mixed with equivalent amounts (1 μg) of GST or GST-Hoxc-8. Alternatively, Hoxc-8-containing lysates were mixed with GST alone or GST-Smad1. The samples were incubated on ice for 30 minutes, and then GST-agarose diluted in NENT buffer (50 µl) was added to each sample, followed by incubation at 4°C for 30 minutes. Sepharose beads were washed four times in PBS / 0.1% TritonX-100 solution, and bound proteins were eluted by incubation in 2×SDS buffer at 10° C. for 5 minutes. As injections, in vitro translated labeled Smad1 protein lysates (1 microgram) were loaded on 12.5% ​​SDS-PAGE along with eluted sam...

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Abstract

The present invention demonstrates that BMP-2 / 4 activates osteopontin protein gene transcription by removing Hoxc-8 binding through Smad1 interaction with the Hoxc-8 DNA binding domain. Since the DNA binding domain is conserved in all Hox and homeodomain-containing proteins, Smad1 likely interacts with all Hox or homeodomain-containing proteins. Furthermore, the present invention reveals the Smad1-mediated transcriptional mechanism in the BMP-2 / 4 signaling pathway and also provides information about the transcriptional roles of the Hox genes during embryonic development.

Description

Background technique [0001] Cross References to Related Applications [0002] This application claims the benefit of Provisional Patent Application US Serial No. 60 / 080,859 (filed April 6, 1998, now abandoned). [0003] Federal Funds Instructions [0004] This invention was made in part using funding obtained under grant DK53757 from the National Institutes of Health. Accordingly, the Federal Government has certain rights in this invention. [0005] field of invention [0006] The present invention relates generally to the field of transcriptional regulation. More specifically, the present invention relates to the inhibition of Hox binding to homeodomain-containing proteins and uses thereof. [0007] Description of related technologies [0008] Bone morphogenetic proteins (BMPs), a subclass of TGFβ, are powerful growth factors that regulate embryonic development, vertebrate patterning, and mesenchymal cell differentiation (10, 11). BMP-2 / 4, identified as osteoind...

Claims

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Application Information

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IPC IPC(8): C12Q1/02A61K31/00A61K38/00A61K39/395A61K45/00A61P9/00A61P19/08A61P19/10A61P25/00A61P35/00A61P43/00C12Q1/04C12Q1/68
CPCA61K31/00A61P19/08A61P19/10A61P25/00A61P35/00A61P43/00A61P9/00
Inventor 曹旭史杏明常志杰
Owner 亚拉巴马大学伯明翰分校研究基金会
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