Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
1998-05-28
2002-04-02
Romeo, David (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C530S350000
Reexamination Certificate
active
06365711
ABSTRACT:
BACKGROUND OF THE INVENTION
TGF-&bgr; superfamily members signal through activation of transmembrane serine-threonine kinase receptors. These receptors phosphorylate and activate Smads, a novel class of signal transducers. Signals initiated by TGF-&bgr; superfamily members are important for regulating cellular processes, including cell division, survival, differentiation, and specification of developmental fate throughout the growth and development of diverse organisms.
During early embryogenesis of the frog
Xenopus laevis
, the TGF-&bgr; growth factor family plays a central role in the specification and patterning of various tissues: TGF-&bgr; superfamily members activin, Vg-1, and TGF-&bgr; all induce a full range of dorsal and ventral mesodermal markers in early embryonic tissue, whereas other TGF-&bgr; superfamily members specify axial pattern or epidermal, as opposed to neural, tissue. Almost all the critical patterning events in early Xenopus embryogenesis appear to involve members of the TGF-&bgr; superfamily.
The transforming growth factor &bgr; (TGF-&bgr;) superfamily of cytokines, which includes bone morphogenic proteins (BMPs), activin, TGF-&bgr;, and Vg-1, regulate a wide range of normal and pathological biological processes. These processes include cell specification during development, terminal differentiation of many cell types, fibrosis during wound healing or organ damage (e.g., cirrhosis), proliferation and invasiveness of normal and transformed cells, and angiogenesis and immune suppression induced by tumors (Roberts and Sporn,
Peptide growth factors and their receptors I
, eds. Sporn and Roberts, Berlin, Springer-Verlage, 419-473, 1990; Sporn et al., Science 33: 532-534, 1986). For example, one member of the family, TGF-&bgr;, is secreted by a wide variety of tumors and has a wide variety of immunosuppressive effects, including the ability to induce apoptosis in B and T lymphocytes (Brabletz et al., Mol. Cell Biol. 13: 1155-1162, 1993; Cahouchi et al., Oncogene 11: 1615-1622, 1995; Weller et al., Exp. Cell Res. 221: 395-403, 1995). The ability to manipulate specific aspects of TGF-&bgr; superfamily signalling in vivo would be a powerful tool both for understanding the role of these factors in normal embryonic patterning and for controlling a broad range of pathological processes.
SUMMARY OF THE INVENTION
We have discovered methods and reagents for identifying compounds that modulate TGF-&bgr; superfamily signalling. These methods and compounds are useful for the detection and treatment of conditions involving abnormal TGF-&bgr; superfamily signalling.
In the first four aspects, the invention provides methods for detecting compounds capable of modulating TGF-&bgr; superfamily signalling. The methods include the steps of providing a cell having a reporter gene operably linked to a DNA-binding-protein recognition site, in addition to having either:
a) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of Smad2 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a gene activating moiety,
b) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein comprising a polypeptide fragment of Smad2 covalently bonded to a gene activating moiety,
c) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of Smad3 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a gene activating moiety, or
d) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein comprising a polypeptide fragment of Smad3 covalently bonded to a gene activating moiety; exposing the cell to the compound; and measuring reporter gene expression in the cell, where a change in the reporter gene expression indicates that the compound is capable of modulating TGF-&bgr; superfamily signalling.
In the fifth, sixth, seventh, and eighth aspects, the invention features a cell useful for detecting a compound capable of modulating TGF-&bgr; superfamily signalling, the cell having a reporter gene operably linked to a DNA-binding-protein recognition site in addition to having either:
a) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of Smad2 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein, the second fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a gene activating moiety,
b) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein, the second fusion protein comprising a polypeptide fragment of Smad2 covalently bonded to a gene activating moiety,
c) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of Smad3 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein, the second fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a gene activating moiety, or
d) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein, the second fusion protein comprising a polypeptide fragment of Smad3 covalently bonded to a gene activating moiety.
In preferred embodiments of the first eight aspects of the invention, a decrease in reporter gene expression indicates a compound that is capable of inhibiting TGF-&bgr; superfamily signalling, and an increase in reporter gene expression indicates a compound that is capable of enhancing TGF-&bgr; superfamily signalling. In other embodiments of these aspects of the invention, reporter gene expression may be assayed by a color reaction or assayed by cell viability. In still another embodiment of the first eight aspects of the invention, the cell may be a yeast cell.
In the ninth, tenth, eleventh, and twelfth aspects, the invention provides a method for detecting a compound capable of modulating TGF-&bgr; superfamily signalling. The method comprises the steps of providing a first polypeptide comprising a polypeptide fragment of FAST-1, providing a second polypeptide, the second polypeptide comprising a polypeptide fragment of either Smad2 or Smad3 (or alternatively, providing a first polypeptide comprising a polypeptide fragment of Smad2 or Smad3, and providing a second polypeptide comprising a polypeptide fragment of FAST-1), exposing the first polypeptide to the second polypeptide and to the compound, and measuring the level of interaction between the first polypeptide and the second polypeptide, wherein an alteration in the level of interaction indicates that the compound is capable of modulating TGF-&bgr; superfamily signalling.
In one preferred embodiment of the ni
Chen Xin
Whitman Malcolm
Medlen & Carroll LLP
President and Fellows of Harvard College
Romeo David
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