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Methods and compositions for regulating cellular signaling

a technology of cellular signaling and composition, applied in the direction of instruments, transferases, genetic material ingredients, etc., can solve the problems of not previously addressed parts of the pkk function of the signaling pathway

Inactive Publication Date: 2003-10-23
RGT UNIV OF MICHIGAN
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  • Abstract
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Benefits of technology

[1991]). The PROMOTERFINDER kit (Clontech) uses PCR, nested primers and special libraries to "walk in" genomic DNA. This process avoids the need to screen libraries and is useful in finding intron / exon junctions.
[0244] Still other embodiments of the present invention provide PKK / DIK and RICK3 variants that have intracellular half-lives dramatically different than the corresponding wild-type protein. For example, the altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular process that result in destruction of, or otherwise inactivate PKK / DIK and RICK3. Such variants, and the genes which encode them, can be utilized to alter the location of PKK / DIK and RICK3 expression by modulating the half-life of the protein. For instance, a short half-life can give rise to more transient PKK / DIK and RICK3 biological effects and, when part of an inducible expression system, can allow tighter control of PKK / DIK and RICK3 levels within the cell. As above, such proteins, and particularly their recombinant nucleic acid constructs, can be used in gene therapy protocols.
[0252] A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations, and for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis or recombination of PKK / DIK and / or RICK3 homologs or variants. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
[1989]) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for PKK / DIK or RICK3.
[0272] Preferably, for in vivo administration, an appropriate immunosuppressive treatment is employed in conjunction with the viral vector (e.g., adenovirus vector), to avoid immunodeactivation of the viral vector and transfected cells. For example, immunosuppressive cytokines, such as interleukin-12 (IL-12), interferon-gamma (IFN-.gamma.), or anti-CD4 antibody, can be administered to block humoral or cellular immune responses to the viral vectors. In addition, it is advantageous to employ a viral vector that is engineered to express a minimal number of antigens.
[0320] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Problems solved by technology

However, the particular signaling pathway in which PKK functions has not been previously addressed.

Method used

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  • Methods and compositions for regulating cellular signaling
  • Methods and compositions for regulating cellular signaling
  • Methods and compositions for regulating cellular signaling

Examples

Experimental program
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Effect test

example 2

[0342] PKK is Highly Related to RICK

[0343] To identify novel RICK-like molecules, public protein and nucleotide databases were searched for homologous proteins using the entire RICK sequence (Inohara et al., J. Biol. Chem. 273:12296 [1998]). The search identified RIP (E values; 4.times.10.sup.-29 and 3.times.10.sup.-29 for human and mouse RIP, respectively) and its homologue RIP3 (E values; 1.times.10.sup.-31 and 5.times.10.sup.-30 for human and mouse RIP3, respectively) as molecules with significant homology to RICK (FIG. 1). In addition, the search identified PKK, a kinase of unknown function, as the most homologous protein to RICK in available databases (E=4.times.10.sup.-51 for mouse PKK and 4.times.10.sup.-10 for human PKK). The search also identified zebrafish orthologues of PKK and RICK. The domain structure of the fish PKK and RICK was identical to that of their mammalian orthologues (FIG. 1A). Zebrafish PKK was more homologous to human RICK (E=5.times.10.sup.-50) than human...

example 3

[0344] PKK Activates NF-.kappa.B and AP-1

[0345] This example describes that the expression of PKK activates NF-.kappa.B. Transfection of the wild-type PKK cDNA into HEK293T cells induced activation of NF-.kappa.B in a dose-dependent manner, as measured with a reporter luciferase construct (FIG. 2A). The induction of NF-.kappa.B by PKK was specific in that transfection of the PKK cDNA did not induce transactivation of NF-AT, NF-IL6, p53, IRF-1 and class II MHC-dependent promoters (FIG. 2B). In control experiments, the transcriptional activity of the reporter constructs was stimulated by expression of proteins known to induce their activation (FIG. 2B). Expression of PKK induced significant activation of AP-1 (FIG. 2B) as did expression of MEKK1, a known activator of AP-1 (Xu et al., Proc. Natl. Acad. Sci. USA. 93:5291 [1996]).

example 4

[0346] The Kinase Domain of PKK is Essential for NF-.kappa.B Activation

[0347] To identify the domains of PKK that are required for NF-.kappa.B activation, a series of deletion mutants carrying each domain alone or in combination were constructed (FIG. 3A). Expression of PKK mutants containing the kinase domain alone (SEQ ID NO: 27) or a combination with other domains such as the IM domain (SEQ ID NO: 28) resulted in NF-.kappa.B activation, while mutants containing the IM region (SEQ ID NO: 29) and / or ankyrin repeats-containing domain (ARD) alone (SEQ ID NO: 30) were inactive (FIG. 3C). Immunoblotting analysis showed that the lack of activity of the mutants could not be explained by-different expression levels of the mutant proteins (FIG. 3C, inset). Thus, the kinase domain of PKK is necessary and sufficient for NF-.kappa.B activation. The present invention is not limited to a particular mechanism. Indeed an understanding of the mechanism is not necessary to practice the present inve...

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Abstract

The present invention related to methods and compositions for modulating cellular signaling. In particular, the present invention relates to PKK and RICK3 proteins. The present invention further relates the to use of PKK and RICK3 proteins in modulating NF-kappaB signaling. The present invention thus provides novel targets for drug screening and therapeutics.

Description

[0002] The present invention related to methods and compositions for modulating cellular signaling. In particular, the present invention relates to PKK and RICK3 proteins. The present invention further relates to the use of PKK and RICK3 proteins in modulating NF-.kappa.B signaling.[0003] All forms of diabetes are characterized by chronic hyperglycemia and the development of diabetes-specific microvascular pathology in the retina, renal glomerulus and peripheral nerve. As a consequence of its microvascular pathology, diabetes is a leading cause of blindness, end-stage renal disease and a variety of debilitating neuropathies. Diabetes is also associated with accelerated atherosclerotic macrovascular disease affecting arteries that supply the heart, brain and lower extremities. As a result, patients with diabetes have a much higher risk of myocardial infarction, stroke and limb amputation. Large prospective clinical studies show a strong relationship between glycaemia and diabetic mic...

Claims

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Application Information

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IPC IPC(8): A61K48/00C12N9/12G01N33/68
CPCA61K48/00C12N9/1205G01N33/5008G01N33/5041G01N2500/00G01N33/6848G01N33/6851G01N33/6872G01N33/6893G01N33/68
Inventor NUNEZ, GABRIELINOHARA, NAOHIROMUTO, AKIHIRO
Owner RGT UNIV OF MICHIGAN
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