Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-09-20
2004-01-06
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S252300, C435S325000, C536S023100, C530S350000
Reexamination Certificate
active
06673570
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acids and encoded polypeptides which interact with Smad proteins. The invention also relates to agents which bind the nucleic acids or polypeptides. The invention further relates to methods of using such nucleic acids and polypeptides in the treatment and/or diagnosis of disease.
BACKGROUND OF THE INVENTION
Members of the transforming growth factor-&bgr; (TGF-&bgr;) family are multifunctional cytokines with elicit a wide range of cellular effects, including growth inhibition, differentiation and apoptosis (Heldin et al.,
Nature
390:465-471, 1997). The signaling induced by TGF-&bgr; family members are initiated through a heteromeric transmembrane kinase complex that consists of type I and type II receptors. The activated type I receptor induces the phosphorylation of receptor-activated Smads (R-Smads) which heteromerize with Smad4. These complexes translocate from the cytoplasm to the nucleus to direct transcriptional regulation of responsive genes (Heldin et al., 1997).
Recently, Smad6 and Smad7 were isolated, which form a subfamily among the Smads and function to inhibit the intracellular signaling by R-Smad/Smad4 complexes. Smad6 and Smad7 constitutively associate with type I receptor by blocking association and phosphorylation of R-Smads (Hayashi et al.,
Cell
89:1165-1173, 1997; Imamura et al.,
Nature
389:622-626, 1997; Nakao et al.,
Nature
389:631-635, 1997). Smad6 and Smad7 are rapidly induced by members of the TGF-&bgr; family (Afrakhte et al.,
Biochem. Biophys. Res. Commun
. 249:505-511, 1998), suggesting that inhibitory Smads may take part in a negative feedback control mechanism to modulate the signaling induced by members of TGF-&bgr; family.
The central role of Smads and TGF-&bgr; in cellular processes presents a need for additional factors to modulate Smads and TGF-&bgr; interactions with signal transduction pathways.
SUMMARY OF THE INVENTION
Using the yeast two hybrid system, proteins that specifically bind with Smad6 and Smad7 have been isolated. The invention provides these isolated Smad associating proteins (SAPs) and fragments of those molecules, as well as agents which bind such polypeptides, including antibodies. The invention also provides nucleic acid molecules encoding SAPs, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The foregoing can be used in the diagnosis or treatment of conditions characterized by the expression of a Smad associating protein, or in the treatment of conditions characterized by the expression of a SAP, or in the treatment of a condition characterized by the expression of a Smad nucleic acid or polypeptide, or by the inadequate or excessive activity of a Smad polypeptide. The invention also provides methods for identifying pharmacological agents useful in the diagnosis or treatment of such conditions. Here, the identification of several SAPs is presented. The SAPs bind to Smad polypeptides including Smad6 and Smad7 and thus are components of TGF-&bgr; superfamily signaling pathways.
According to one aspect of the invention, isolated nucleic acid molecules are provided. The isolated nucleic acid molecules are nucleic acid molecules which hybridize under stringent conditions to a molecule consisting of the nucleic acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:5 and which code for a polypeptide which binds Smad6, or nucleic acid molecules that differ from the foregoing nucleic acid molecules in codon sequence due to the degeneracy of the genetic code, or complements of the foregoing nucleic acid molecules. Preferably the isolated nucleic acid molecule consists of SEQ ID NO:3 or SEQ ID NO:5.
According to another aspect of the invention, isolated nucleic acid molecules are provided which are unique fragments of nucleotides 1-2399 of SEQ ID NO:3 between 12 and 2398 nucleotides in length or of nucleotides 1-855 of SEQ ID NO:5 between 12 and 854 nucleotides in length. Also provided are complements of the foregoing unique fragments provided that the nucleic acid molecule excludes sequences consisting of GenBank accession numbers AF176069, AF293384, AA305358, AI219112, N33797 and AB030502. In certain embodiments, the isolated nucleic acid molecule consists of at least 22, 25, 30, 40, 50, 75 or 100 contiguous nucleotides. In other embodiments, the isolated nucleic acid molecule consists of between 20 and 32 contiguous nucleotides.
According to still another aspect of the invention, expression vectors including any of the foregoing isolated nucleic acid molecules operably linked to a promoter are provided. Also provided are host cells transformed or transfected with the expression vectors, as well as transgenic non-human animals including the expression vectors.
According to yet another aspect of the invention, methods for producing a polypeptide are provided. The methods include culturing the foregoing host cells under conditions which permit the expression of polypeptide. Preferably the methods include isolating the polypeptide.
In another aspect of the invention, isolated polypeptides are provided which are encoded by the foregoing isolated nucleic acid molecules. Preferred isolated polypeptides include molecules comprising the amino acid sequences of SEQ ID NO:4, SEQ ID NO:6, fragments or functional variants of SEQ ID NO:4, and a fragments or functional variants of SEQ ID NO:6.
According to still another aspect of the invention, isolated polypeptides are provided which include a fragment or functional variant of SEQ ID NO:2. In certain embodiments the fragment of SEQ ID NO:2 consists of amino acids 1-101+234-424, 106-424 or 234-424.
According to yet another aspect of the invention, an isolated complex of polypeptides is provided. The complex includes one of the foregoing polypeptide bound to a polypeptide selected from the group consisting of Smad6, Smad7 and fragments thereof.
Also included as an aspect of the invention are isolated polypeptides which bind selectively a polypeptide encoded by the foregoing isolated nucleic acid molecules, provided that the isolated polypeptide is not a Smad, STAM or cyclin polypeptide. In certain embodiments, the isolated polypeptide binds to an epitope defined by a polypeptide consisting of the sequence of SEQ ID NOs:2, 4 or 6. In other embodiments, the isolated polypeptide is an antibody fragment selected from the group consisting of a Fab fragment, a F(ab)
2
fragment or a fragment including a CDR3 region selective for a SAP polypeptide. In still other embodiments the isolated polypeptide is a monoclonal antibody, a humanized antibody or a chimeric antibody.
According to still another aspect of the invention, methods for modulating TGF-&bgr; superfamily signal transduction in a mammalian cell are provided. The methods include contacting the mammalian cell with an amount of an agent which increases the amount of a Smad associating protein selected from the group consisting of SAP1/AMSH (SEQ ID NO:2), SAP2 (SEQ ID NO:4), SAP3 (SEQ ID NO:6), Hsp40 homolog (U40992; SEQ ID NO:8), Uba80 (X63237; SEQ ID NO:10), Tax-1 binding protein (U33822; SEQ ID NO:12), rabaptin-5 (NM
—
004703; SEQ ID NO:14), and 26S proteinase S5a (U51007; SEQ ID NO:16) or a fragment thereof in the cell effective to reduce TGF-&bgr; superfamily signal transduction in the mammalian cell. In certain embodiments, the agent is a nucleic acid molecule encoding one of the foregoing polypeptides.
According to another aspect of the invention, methods for regulating the cell cycle in a mammalian cell are provided. The methods include contacting the mammalian cell with an amount of an agent which increases the amount of SAP2 (SEQ ID NO:4), or a fragment thereof, in the cell effective to bind a cyclin and regulate the cell cycle in the mammalian cell.
In further aspects of the invention, methods for identifying lead compounds for a pharmacological agent are provided. In certain embodiments, the methods include forming a mixture comprising a Smad6 or Smad7 polypeptide, a SAP polypeptide, and
Dijke Peter ten
Heldin Carl-Henrik
Itoh Fumiko
Itoh Susumu
Carlson Karen Cochrane
Ludwig Institute for Cancer Research
Wolf Greenfield & Sacks P.C.
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