Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-09-09
2001-07-24
Ulm, John (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S069700, C530S350000, C536S023400
Reexamination Certificate
active
06265174
ABSTRACT:
1. INTRODUCTION
The present invention relates to methods and compositions for the identification and modulation of protein-protein interactions. Specifically, the invention relates to methods and compositions for efficient, sensitive, high-throughput CadC-based screens for the identification of peptides involved in protein-protein interactions, including, but not limited to, peptides comprising amino acid sequences involved in receptor dimerization. The invention further relates to methods and compositions for efficient, sensitive, high-throughput CadC-based screens for compounds which modulate protein-protein interactions, such as, for example, modulation of interactions between protein sequences involved in receptor interactions, e.g., dimerization.
2. BACKGROUND OF THE INVENTION
All living cells possess the means to adapt to their environments. Cells rely, to a great extent, on extracellular molecules as a means by which to receive stimuli from their immediate environment. These extracellular signals are essential for the correct regulation of such diverse cellular processes as differentiation, contractility, secretion, cell growth, cell migration, contact inhibition and apoptosis. The external environmental signals received by the cell are transduced into the cell via activation of membrane-situated receptors. Activation of cell surface receptors is often dependent upon protein-protein interactions, including receptor-ligand binding, and receptor dimerization (homodimerization or heterodimerization) or oligomerization. Aberrant signalling can disrupt any of these cellular processes with detrimental results.
For reviews of signal transduction pathways see, e.g., Campbell, 1997, J. Pediat. 131:542-544; Hamilton, 1997, J. Leukoc. Biol. 62:145-155; Soede-Bobok & Touw, 1997, J. Mol. Med. 75:470-477; Heldin, 1995, Cell 80:213-223; Kishimoto et al. 1994, Cell 76:253-262; Miyajima, et al. 1992, Annu. Rev. Immunol. 10:295-331; and Cantley, et al. 1991, Cell 64:281-302.
Protein-protein interactions also play an important role in processes concerning many other cellular and viral proteins and enzymes, in addition to cell surface receptors. In some instances disruption of protein-protein interactions will lead to the loss of polypeptide function. In certain instances, loss of function of an enzyme may be therapeutically desirable. For example, the protease from HIV is a dimer (McKeever et al. 1989, J. Biol. Chem. 264:1919-1921) and the dimerization of the protein is required for function (Guenet et al. 1989, Eur. J. Pharmacol. 172:443-451, and Babe et al. 1992, Protein Sci. 10:1244-1253). Some candidate molecules (Zhang et al. 1991, J. Biol. Chem. 266:15591-15594 and Schramm et al. 1993, Biochem. Biophys. Res. Commun. 194:595-600) that block HIV protease function do so by disrupting dimerization.
Given the ubiquitous and important nature of protein-protein interactions in signal transduction pathways and in other cellular processes, compounds by which such interactions can be modulated would be very advantageous.
Attempts to identify ways to modulate such events have been reported. See, e.g., chimeric receptor studies reported by Schlessinger, and the chimeric signal transduction systems reported by Menzel et al. (utilizing heterologous Vibrio cholerae toxR in
E. coli
; U.S. Pat. No. 5,521,066), Utsumi (utilizing a chimeric
E. coli
Tar protein; Utsumi et al. 1989, Science 245:1246-1249), Riedel (utilizing different eukaryotic hormone receptors in tissue culture; Riedel et al. 1989, EMBO J. 8:2943-2954) and Moe (utilizing a bacterial aspartate binding domain and the insulin receptor (Moe et al. 1989, Proc. Natl. Acad. Sci. USA 5 86:5683-5687). See also King et al. (U.S. Pat. Nos. 5,482,835 and 5,739,029; methods for screening for agonists and antagonists for G-protein coupled receptors). Each of these methods, however, exhibits significant limitations in either specificity, broad versatility and/or sensitivity.
Despite such reports, therefore, as yet, no efficient, sensitive, versatile high throughput procaryote-based system has yet been described for identifying protein-protein interactions or for identifying compositions for modulating such interactions.
3. SUMMARY OF THE INVENTION
The present invention relates to methods and compositions for the identification and modulation of protein-protein interactions.
Specifically, the invention relates, first, to methods and compositions for efficient, sensitive, high-throughput CadC-based screens for the identification of polypeptides involved in protein-protein interactions, such as, for example, dimerizing peptides, which include, but are not limited to, peptides comprising amino acid sequences involved in receptor dimerization. Such methods can also be utilized to identify peptide ligands which enhance a protein-protein interaction of interest. Such methods can, for example, be utilized in identifying ligands for orphan receptors.
The invention further relates to methods and compositions for efficient, sensitive, high-throughput CadC-based screens for compounds which modulate protein-protein interactions, such as, for example, modulation of interactions between protein sequences involved in receptor dimerization. Compounds identified via such methods can, for example, act as agonists or antagonists of a protein-protein interaction of interest, and/or can act as mimetics of natural ligands involved in the protein-protein interaction of interest.
The compositions of the present invention include, first, CadC-fusion polypeptides, which comprise, from carboxy-terminus to amino terminus, a periplasmic domain, a transmembrane domain and a CadC transcriptional regulatory domain. Interaction of CadC-fusion polypeptides results in CadC fusion polypeptides which activate RNA polymerase-dependent transcription from a cadBA regulatory region. While not wishing to be bound by any particular mechanism, it is believed that such interaction involves dimerization or multimerization of the CadC-fusion polypeptides, such interaction being dependent upon dimerization or multimerization of the CadC-fusion polypeptide periplasmic domains, and transcriptional activation involves binding of the dimerized or multimerized CadC-fusion polypeptides to the cadBA regulatory region.
It is to be understood that while the term “dimerization” is periodically used throughout the application for purposes of clarity and ease of description, interactions between the peptides of the invention can also be involved in the formation of trimers, tetramers and higher level oligomeric or multimeric polypeptide configurations.
“CadC,” as used herein, can refer to
Escherichia coli
CadC nucleotide or amino acid sequences. Alternatively, “CadC” can refer to an Enterobacteriaceae family, e.g.,
salmonella typhimurium
, homolog of
E. coli
CadC nucleotide or amino acid sequences.
Likewise, “cadBA,” as used herein can refer to
E. coli
cadBA sequences. Alternatively, “cadBA” can refer to an Enterobacteriaceae family homologue of
E. coli
cadBA sequences.
It is also contemplated that CadC, cadBA and cells of the inventions can be derived from any procaryotic cell, other than
Vibrio cholerae
, exhibiting an inner membrane and periplasmic space.
A CadC-fusion polypeptide periplasmic domain can comprise a protein-protein interaction domain or a test domain.
A protein-protein interaction domain comprises an amino acid sequence which, under standard conditions utilized for cell (e.g.,
E. coli
) culture, interacts with, that is, binds to, a peptide or polypeptide “partner.” A test domain can comprise any amino acid sequence to be tested for an ability to exhibit such interaction.
Such interaction between two (or more) polypeptide domains is defined, identified and measured by the resultant activation of the CadC transcriptional regulatory domain and its activation of expression of cadBA-linked sequences. Such interactions can be homotypic or heterotypic interactions.
The protein-protein interaction domains of the invention can form dimers, trimers, or other oligomeric or multimeric (i.
Hsing Weihong
Menzel Rolf
Taggart Pamela A.
Corless Peter F.
Dike Bronstein Roberts & Cushman IP Group
Morphochem, Inc.
Ulm John
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