Modulators of actin

Details Modulators of actin Modulators of actin

1999-06-01

2001-03-06

Prouty, Rebecca F. (Department: 1652)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C424S185100, C436S086000

Reexamination Certificate

active

06197932

ABSTRACT:

INTRODUCTION
1. Field of the Invention
The invention relates to a class of polypeptides involved in actin stabilization.
2. Background of the Invention
The actin cytoskeleton plays a central role in defining cellular structure and effecting dynamic changes in morphology. By selectively stabilizing and destabilizing actin polymerization, the cell is able to effect a wide range of structural reorganization and effect phenomena such as cell motility, phagocytosis, cytokinesis, mitosis, etc. Because these phenomenon are involved in myriad medically significant physiologies and pathologies, e.g. the progress of many pathogenic infections, invasion and metastisis of neoplasia, fertilization, clotting and wound repair, etc., the stability of actin polymerization is a choice target for therapuetic intervention. In fact, potent a drugs effecting actin filament destabilization and stabilization such as fungal-derived alkaloids including the cytochalasins and phalloidins are well known. Here we disclose a new family of modulators of actin polymer stabilization derived from a novel human diaphanous protein and gene.
Relevant Literature
Lynch ED, et al. (1997) Science 278(5341): 1315-1318 disclose nonsyndromic deafness DFNA1 associated with mutation of a human homolog of the Drosophila gene diaphanous.
Watanabe N, et al. (1997) EMBO J 16:3044-3056, disclose a mouse gene with sequence similarity to the disclosed human gene. Bione S, et al. (1998) Am J Hum Genet 62(3): 533-541, report that a human homologue of the Drosophila melanogaster diaphanous gene is disrupted in premature ovarian failure. Vahava O, et al. (1998) Science 279(5358): 1950-1954. Mutation in transcription factor POU4F3 associated with inherited progressive hearing loss in humans.
SUMMARY OF THE INVENTION
The invention provides methods and compositions which find use, inter alia, for modulating the stabilization of actin filaments. The compositions may comprise one or more polypeptide moieties derived from a novel human diaphanous polypeptide and/or one or more nucleic acid moieties derived from a novel human diaphanous gene or gene transcript. The invention also provides agents which specifically modif the binding of a natural human diaphanous gene or gene product with a natural binding target thereof. Polypeptide components of subject compositions provide human diaphanous-specific structure and activity and may be produced recombinantly from transformed host cells from the subject human diaphanous polypeptide encoding nucleic acids. The invention provides isolated human diaphanous hybridization probes and primers capable of specifically hybridizing with the disclosed human diaphanous genes, human diaphanous-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for human diaphanous transcripts), therapy (e.g. modulating a cellular function such as auditory signal transduction by introducing into the cell a subject modulator) and in the biopharmaceutical industry (e.g. as immunogens, reagents for isolating additional natural human diaphanous genes and alleles, reagents for screening bio/chemical libraries for ligands and lead and/or pharmacologically active agents, etc.).
DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION
In one embodiment, the modulators of the invention comprise a human diaphanous polypeptide (a plurality of amino acids linearly joined through peptide bonds) having a natural human diaphanous polypeptide-specific sequence and bioactivity (i.e. distinguished from natural murine and drosophila diaphanous sequences and bioactivities). SEQ ID NO: 1 depicts an exemplary natural cDNA encoding a human diaphanous polypeptide and SEQ ID NO: 2 depicts the corresponding encoded natural human diaphanous polypeptide. The subject polypeptides comprise at least a 6, preferably at least a 12, more preferably at least a 18, most preferably at least a 24 residue domain of SEQ ID NO:2, not found in natural mouse or drosophila diaphanous polypeptides. Human specific sequences are readily identified by aligning the respectivel sequences. In a particular embodiment, the subject polypeptides comprise at least a 36, preferably at least a 72, more preferably at least a 144, most preferably at least a 288 residue domain of SEQ ID NO:2.
The polypeptides provide natural human diaphanous polypeptide specific bioactivity or function, such as specific ligand binding or binding inhibition, antigenicity, immunogenicity, etc. Human diaphanous polypeptide-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the molecular interaction of a human diaphanous polypeptide with a binding target is evaluated. The binding target may be a natural intracellular binding target such as a human diaphanous polypeptide regulating protein, effector or other regulator that directly modulates a human diaphanous polypeptide activity or its localization; or non-natural binding target such a specific immune protein such as an antibody, or an human diaphanous polypeptide specific agent such as those identified in bio/chemical screening assays. Exemplary binding targets include human prolifin and Rho polypeptides. Human diaphanous polypeptide-binding specificity may assayed by functional assays described below, binding equilibrium constants (usually at least about 10
7
M
−1
, preferably at least about 10
8
M
−1
, more preferably at least about 10
9
M
−1
), by the ability of the subject polypeptides to function as negative mutants in a human diaphanous polypeptide-expressing cells, to elicit a human diaphanous polypeptide specific antibody in a heterologous host (e.g. a rodent or rabbit), etc. The human diaphanous polypeptide binding specificity of the human diaphanous polypeptides necessarily distinguishes that of natural murine and drosophila homologs. In a particular embodiment, the sequence and function also distinguishes those of the natural human diaphanous 2 polypeptide.
In particular embodiments, modulators comprising human diaphanous polypeptides are isolated, pure or recombinant: an “isolated” polypeptide is unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, and more preferably at least about 5% by weight of the total polypeptide in a given sample and a pure polypeptide constitutes at least about 90%, and preferably at least about 99% by weight of the total polypeptide in a given sample. A recombinant polypeptide comprises a non-natural terminus residue or is joined to other than an amino acid which it is joined to in a natural polypeptide. The polypeptides may be synthesized, produced by recombinant technology, or purified from cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g.
Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) or that are otherwise known in the art. Material and methods for the expression of heterologous recombinant polypeptides in bacterial cells (e.g.
E. coli
), yeast (e.g.
S. Cerevisiae
), animal cells (e.g. CHO, 3T3, BHK, baculovirus-compatible insect cells, etc.). The polypeptides may be provided uncomplexed with other moieties including other polypeptide moieties, complexed in a wide variety of covalent and/or non-covalent associations and binding complexes, etc., which may provide enhanced activity, stability, availability, targeting, etc.
Exemplary active modulators comprising human diaphanous polypeptides moieties include (using N→C nomenclature convention):
hDia1-del-1: MRG—residues 121-151 of SEQ ID NO:2 fusion polypeptide
hDia1-del-2: GFP—re

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