Human MAD proteins and uses thereof

Details Human MAD proteins and uses thereof Human MAD proteins and uses thereof

1998-10-30

2001-10-30

Huff, Sheela (Department: 1642)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C530S350000, C536S023100, C536S023500

Reexamination Certificate

active

06309856

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. The polypeptides of the present invention has been putatively identified as human MAD homologs. More particularly, one polypeptide of the present invention has been putatively identified as a novel human MAD protein, sometimes hereafter referred to as “MADr3”. The invention also relates to inhibiting or stimulating the actions of such polypeptides.
BACKGROUND OF THE INVENTION
Cellular growth and differentiation appear to be initiated, promoted, maintained and regulated by a multiplicity of stimulatory, inhibitory and synergistic factors and hormones. The alteration and/or breakdown of the cellular homeostasis mechanism seems to be a fundamental cause of growth related diseases. Growth modular factors are implicated in a wide variety of pathological and physiological processes including signal transduction, cell communication, growth and development, embryogenesis, immune response, hematopoiesis cell survival and differentiation, inflammation, tissue repair and remodeling, atherosclerosis and cancer.
The transforming growth factor &bgr; (TGF-&bgr;) family of cytokines/growth factors regulates cell proliferation, differentiation, recognition and death, and figures in the control of development, tissue recycling and repair. Included in this family are Drosophila decapentaplegic (DPP) [Sekelsky et al,
Proc. Natl. Acad. Sci., USA
, 139:1347-1358 (1995)], TGF&bgr;-1, TGF&bgr;-2 and TGF&bgr;-3; and bone morphogenetic proteins, BMP2/BMP4 [Hoodless et al,
Cell
, 85:489-500 (1996); Graff et al,
Cell
, 85:479-487 (1996); Liu et al,
Nature
, 381:620-623 (1996); and Vhang et al,
Nature
, 383:168-1782 (1996)].
TGF-&bgr; family members signal by simultaneously contacting two transmembrane serine/threonine kinases known as the type I and type II receptors. Certain proteins mediate the complex signalling cascade of the TGF-&bgr; family. It has been suggested that different TGF-&bgr; family members may signal through different MAD isoforms. MAD (Mothers against DPP) protein was discovered in Drosophila to be required for the signal transduction of DPP. Other MAD proteins in other species, such as Xenopus, mouse, and human, have been found to transduce signaling of BMP2/BMP4.
Several reports suggest that subsequent to ligand activation, the TGF-&bgr; type I receptor phosphorylates MAD [Hoodless et al, cited above; and Liu et al, cited above]. The phosphorylated MAD then translocates into the nucleus and effects gene expression of selected early intermediate genes specific to the MAD isoform subtype [J. Massague,
Cell
, 85:947-950 (June 28, 1996)].
Therefore, selective antagonists of the MAD isoforms are anticipated to be beneficial in many diseases where selective interruption of TGF-&bgr; or BMP signaling are indicated [see, e.g., Eppert et al,
Cell
, 86:543-552 (Aug. 23, 1996)]. Such disorders include, without limitation, chronic renal failure, scarring, colorectal carcinoma, and cardiovascular disease.
There, thus, exists a need in the art for a variety of TGF-&bgr; signalling proteins, antagonists and agonists thereof, as well as compositions and methods for the use of same.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides polypeptides, inter alia, that have been identified as novel MAD polypeptides of human origin, as well as biologically active and diagnostically or therapeutically useful fragments, variants, analogs and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing. In one embodiment, the polypeptide is human MADr3. In another embodiment, the polypeptide is human MADr4. These polypeptides have been identified as MAD polypeptides, e.g., by homology between the amino acid sequence or nucleotide sequence of known MAD polypeptides.
Among the particularly preferred embodiments of this aspect of the invention are variants of human MADr3 and MADr4 encoded by naturally occurring alleles of the human genes for same.
In another aspect of the present invention, there are provided non-naturally occurring synthetic, isolated and/or recombinant MADr3 or MADr4 polypeptides, fragments, consensus fragments and/or sequences having conservative amino acid substitutions of the MADr3 or MADr4 of the present invention. These polypeptides may bind MADr3 or MADr4 ligands, or may also modulate, quantitatively or qualitatively, MADr3 or MADr4 ligand binding.
In another aspect, the present invention provides synthetic, isolated or recombinant polypeptides which are designed to inhibit or mimic various MADr3 or MADr4 or fragments thereof.
In another aspect of the invention, there are provided isolated nucleic acid molecules encoding MAD polypeptides, particularly human MADr3 and MADr4. Such molecules include polynucleotides, mRNAs, DNAs, cDNAs, genomic DNAs and fragments thereof, as well as analogs and biologically active and diagnostically or therapeutically useful variants, analogs or derivatives thereof, including fragments of the variants, analogs and derivatives.
In a particularly preferred embodiment of this aspect of the invention, the polynucleotide comprises the region encoding human MADr3 in the sequence set out in
FIGS. 1A-1D
[SEQ ID NOS: 1 and 2]. In another particularly preferred embodiment of this aspect of the invention, the polynucleotide comprises the region of human MADr4 in the sequence set out in
FIGS. 2 and 3
[SEQ ID NOS: 3 and 4].
Among the particularly preferred embodiments of this aspect of the invention are naturally occurring allelic variants of human MADr3 and MADr4.
In yet another aspect of the present invention, there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible from the human cDNA contained in plasmid pHSBHI91.
In still another aspect, this invention provides nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to nucleic acid sequences of the present invention, e.g., human MADr3 or MADr4 sequences.
In another aspect, the invention provides a process for producing the aforementioned polypeptides, polypeptide fragments, variants and derivatives, fragments of the variants and derivatives, and analogs of the foregoing. In a preferred embodiment, the invention provides methods for producing the aforementioned MADr3 and MADr4 polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing (i.e., having expressibly incorporated therein) a nucleic acid sequence encoding a polypeptide of the present invention under conditions for expression of human MAD polypeptide in the host and then recovering the expressed polypeptide.
In still another aspect, the invention provides products, compositions, processes and methods that utilize the aforementioned polypeptides and polynucleotides for research, biological, clinical and therapeutic purposes, inter alia. MAD polypeptides, particularly human MADr3 and MADr4 polypeptides, may be employed for therapeutic purposes, including, but not limited to, treatments to stimulate wound healing, to restore normal neurological functioning after trauma or AIDS dementia, to treat ocular disorders, to target certain cells, to treat kidney and liver disorders, to prevent scarring; to treat ulcers and corneal incisions, and to treat cancers.
Also provided are compositions and methods that can be useful as potential modulators of MADr3 or MADr4 function, by binding to ligands or modulating ligand binding, due to their expected biological properties, which may be used in diagnostic, therapeutic and/or research applications.
In another aspect, the invention provides a method for utilizing these polypeptides and proteins for the screening of chemical or natural compounds or ligands thereof which inhibit or stimulate the phosphorylatio

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