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Chimeric peptides for the regulation of GTPases

a technology of gtpase and chimeric peptides, which is applied in the direction of peptide sources, antibody medical ingredients, drug compositions, etc., can solve the problems of preventing effective cycling between and negatively affecting the activity of rho protein, and achieve the effects of modulating gtpase activity, inhibiting gtpase activity, and reducing gtpase activity

Inactive Publication Date: 2005-03-31
CHILDRENS HOSPITAL MEDICAL CENT CINCINNATI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0013] A further embodiment is a method of treating a disease related to up-regulation of GTPase activity in a mammal, by administering to the mammal a composition as disclosed above in an amount effective to reduce the GTPase activity. In one aspect, the disease is a GTP hydrolysis-related disorder.
[0014] One embodiment is a method for modulating GTPase activity in a cell, by: contacting said ce

Problems solved by technology

A third class of regulators of Rho GTPases, the Rho GDP-dissociation inhibitors, can negatively impact Rho protein activities by sequestering them in the GDP-bound state and preventing effective cycling between the two conformational states.

Method used

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  • Chimeric peptides for the regulation of GTPases
  • Chimeric peptides for the regulation of GTPases
  • Chimeric peptides for the regulation of GTPases

Examples

Experimental program
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example 1

Distinct Roles of RhoA, RhoB and RhoC in Cell Transformation and Migration

[0274] Among the three closely related Rho proteins, RhoA, RhoB and RhoC, RhoA is the best characterized one and has been shown to regulate actin stress fiber and focal complex formation, to promote cell growth and to transform NIH 3T3 fibroblasts. By contrast, the function of RhoB or RhoC in fibroblasts has not been examined in detail and has not been directly compared with that of RhoA. To make comparisons of the cellular roles of RhoA, RhoB and RhoC, two sets of activating mutants were generated for each of the Rho proteins: the fast-cycling Rho-F30L and the GTPase-defective Rho-Q63L. Both types of mutants result in the net enhancement of the active Rho-GTP species in cells but involved distinct mechanisms: Rho-F30L proteins contained significantly increased intrinsic GDP / GTP exchange activity and remained responsive to RhoGAP stimulation to cycle between the GDP- and GTP-bound states, while the Rho-Q63L m...

example 2

Targeting Individual Rho GTPase Activities by p190-Rho Chimeras

[0283] The commonly used biochemical tools to implicate the involvement of a Rho protein in a particular signaling pathway include the dominant negative mutant of the Rho protein and certain bacterial toxins that can modify the Rho protein function. These reagents are limited by their non-specific nature in interfering with Rho GTPase functions (Geig, et al. 1999, Nat. Cell. Biol. 1, E25-E27, herein incorporated by reference in its entirety) and can have limited therapeutic value in targeting specific Rho proteins. In order to specifically inhibit individual Rho protein function, we hypothesize that the negative regulatory role of RhoGAPs, the RhoGAP domain in particular, could be exploited to downregulate Rho protein activity if it is directed to where the active Rho GTPase substrates reside in cells. The RhoGAP domain of p190 has previously been demonstrated as a catalyst to specifically stimulate GTP-hydrolysis of Rh...

example 3

Comparison of GAP Activities of Fusion Proteins In Vitro

[0285] To ensure that the C-terminal sequences of various Rho proteins fused to the GAP domain of p190 do not interfere with the GAP function of p190, we expressed the GST-tagged p190 and p190-Rho chimeras in Cos-7 cells, purified them by glutathione-agarose affinity beads and compared their GAP activities in vitro. Under similar doses, p190-RhoA-C, p190-RhoB-C and p190-RhoC-C displayed comparable GAP activities as p190 GAP domain alone on the RhoA substrate to stimulate [γ32P]GTP-hydrolysis in a time dependent manner, indicating that all four p190 and p190-Rho chimera proteins are functional. To evaluate the efficacy and specificity of the chimeric constructs in cells, we employed the above described fast-cycling mutant Rho-F30L and GTPase-defective mutant Rho-Q63L expressing cells as testing systems, taking advantage of the facts that the F30L mutant form of the Rho GTPases is fully responsive to GAP stimulation while the GT...

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Abstract

Chimeric peptides or fusion proteins are disclosed that include a RhoGAP activity domain and at least one specificity domain that targets a specific Rho protein. The fusion proteins can be used to inhibit any GTPase activity within a cell. The fusion proteins are particularly advantageous for the treatment of cancer. The present invention generally relates to The present invention generally relates to chimeric peptides capable of regulating GTPases, and more particularly, to methods of targeting individual GTPases by using GTPase-activating proteins. Such proteins may be used for the treatment of cancers and other GTPase-related diseases. This invention relates to nucleic acid molecules and the encoded GTPase activating proteins, and variants thereof, and to the use of these molecules in the characterization, diagnosis, prevention, and treatment of cell signaling, immune, and cell proliferative disorders, particularly cancer. Disclosed herein are compounds and methods for regulating transcription of a selected gene. Methods are provided for constructing and for using such compounds to regulate transcription of a selected gene.

Description

RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional application 60 / 494,719, filed Aug. 13, 2003, herein incorporated by reference in its entirety.[0002] This invention was made in part with Government support under Grant No. R01 GM60523, awarded by the National Institutes of Health. The Government may have certain rights in this invention.FIELD OF THE INVENTION [0003] The present invention generally relates to chimeric peptides capable of regulating GTPases, and more particularly, to methods of targeting individual GTPases by using GTPase-activating proteins. Such proteins may be used for the treatment of cancers and other GTPase-related diseases. BACKGROUND OF THE INVENTION [0004] The Ras-related Rho family of small GTPases is involved in a wide variety of cellular processes, including the regulation of the actin cytoskeletal organization, cell-to-cell or cell-to-extracellular matrix adhesion, intracellular membrane trafficking, gene transcription, apop...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N9/14
CPCC07K14/4706C07K2319/01C12Y306/05002C07K2319/02C07K2319/03C12N9/14A61P35/00A61P43/00
Inventor ZHENG, YIWILLIAMS, DAVID
Owner CHILDRENS HOSPITAL MEDICAL CENT CINCINNATI
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