Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-11-24
2001-11-06
Priebe, Scott D. (Department: 1632)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S252300, C435S325000
Reexamination Certificate
active
06313280
ABSTRACT:
TECHNICAL FIELD
The present invention relates to SMAD—interacting polypeptides (“SIP's”) such as cofactors for SMAD proteins and the use thereof.
BACKGROUND
The development from a single cell to a fully organized organism is a complex process wherein cell division and differentiation are involved. Certain proteins play a central role in this process. These proteins are divided into different families of which the transforming growth factor &bgr; (“TGF-&bgr;”) family of ligands, their serine/threonine kinase (“STK”) receptors and their signalling components are undoubtedly key regulatory polypeptides. Members of the TGF-&bgr; superfamily have been documented to play crucial roles in early developmental events such as mesoderm formation and gastrulation, but also at later stages in processes such as neurogenesis, organogenesis, apoptosis and establishment of left-right asymmetry. In addition, TGF-&bgr; ligands and components of their signal transduction pathway have been identified as putative tumor suppressors in the adult organism.
Recently, “SMAD proteins” have been identified as downstream targets of the STK receptors (Massagué,1996,
Cell,
85, p. 947-950). These SMAD proteins are signal transducers which become phosphorylated by activated type I receptors and thereupon accumulate in the nucleus where they may be involved in transcriptional activation. SMAD proteins comprise a family of at least 5 subgroups which show high cross-species homology. They are generally proteins of about 450 amino acids (50-60 kDa) with highly conserved N-terminal and C-terminal domains, linked by a variable, proline-rich, middle region. On the basis of experiments carried out in cell lines or in Xenopus embryos, it has been suggested that the subgroups define distinct signalling pathways: SMAD1 mediates BMP2/4 pathways, while SMAD2 and SMAD3 act in TGF-&bgr;/activin signal transduction cascades. It has been demonstrated that these SMADs act in a complex with SMAD4 (dpc-4) to elicit certain activin, bone morphogenetic protein (BMP) or TGF-&bgr; responses (Lagna et al., 1996,
Nature,
383, p.832-836 and Zhang et al., 1996,
Nature,
383, p. 168-172).
SMAD proteins have a three-domain structure and their highly conserved carboxyl domain (C-domain) is necessary and sufficient for SMAD function in the nucleus. The concept that this domain of SMAD proteins might interact with transcription factors in order to regulate transcription of target genes has previously been put forth (Meersseman et al, 1997,
Mech.Dev.,
61, p. 127-140). This hypothesis has been supported by the recent identification of a new winged-helix transcription factor (“FAST1”) which forms an activin-dependent complex with SMAD2 and binds to an activin responsive element in the Mix-2 promoter (Chen et al.,
Nature
383, p. 691-696, 1996). However, cofactors for SMAD proteins other than FAST 1 have not yet been identified.
Beyond the determination of the mechanism of activation of STK receptors and SMAD, and the heteromerization of the latter, little is known about other downstream components in the signal transduction machinery. Thus, understanding how cells respond to TGF-&bgr; related ligands remains a crucial central question in this field.
In order to clearly demonstrate that SMAD proteins might have a function in transcriptional regulation—either directly or indirectly—it is necessary to identify putative co-factors of SMAD proteins, response elements in target genes for these SMAD proteins and/or co-factors, and to investigate the ligand-dependency of these activities.
To understand those interactions molecular and developmental biology research on (i) functional aspects of the ligands, receptors and signaling components (in particular members of the SMAD family), in embryogenesis and disease, (ii) structure-function analysis of the ligands and the receptors, (iii) the elucidation of signal transduction, (iv) the identification of cofactors for SMAD (related) proteins, and (v) ligand-responsive genes in cultured cell and the Drosophila, amphibian, fish and murine embryo are all of utmost importance.
DISCLOSURE OF THE INVENTION
We have found that by carrying out a two hybrid screening assay, SMAD interacting protein(s) are obtainable where SMAD C-domain fused to a DNA-binding domain as “bait” and a vertebrate cDNA library as “prey” respectively are used. It is evident to those of skill in the art that other appropriate cDNA libraries can be used as well. By using, for example, SMAD1 C-domain fused to GAL4 DNA-binding domain and a mouse embryo cDNA as bait and prey respectively, a partial SMAD4 and other SMAD-interacting protein (SIP) cDNAs, including SIP1, were obtained.
Surprisingly, it has been found that at least four SMAD interacting proteins thus obtained contain a DNA binding zinc finger domain. One of these proteins, SIP1, is a novel member of the family of zinc finger/homeodomain proteins containing &dgr;-crystallin enhancer binding protein and certain Drosophila zfh-1, the former of which has been identified as a DNA-binding repressor. It has been shown that one DNA binding domain of SIP1 (the C-terminal zinc finger cluster or SIP1
czf
) binds to E2 box regulatory sequences and to the Brachyury protein binding site. It has been demonstrated in cells that SIP1 interferes with E2 box and Brachyury-mediated transcription activation. SIP1 fails to interact with full-size SMAD in yeast. We have shown for the first time that SMAD proteins can interact with a DNA-binding repressor and, as such, appear to be directly involved in TGF-&bgr; ligand-controlled repression of target genes which are involved in the strict regulation of normal early development.
In summary, characteristics of SIP 1 include the following:
a) it fails to interact with full size XSMAD1 in yeast,
b) it is a new member of the family of zinc finger/homeodomain proteins including &dgr;-crystallin enhancer binding protein and/or Drosophila zfh-1,
c) SIP1
czf
binds to E2 box sites,
d) SIP1
czf
binds to the Brachyury protein binding site,
e) it interferes with Brachyury-mediated transcription activation in cells, and
f) it interacts with C-domain of SMAD 1, 2 and/or 5.
As used herein, “E2 box sites” means a -CACCTG- regulatory conserved nucleotide sequence which contains the binding site CACCT for &dgr;-crystallin enhancer binding proteins as described in Sekido et al, 1996,
Gene,
173, p.227-232. These E2 box sites are known targets for important basic helix-loop-helix (bHLH factors such as MyoD, a transcription factor in embryogenesis and myogenesis.
So, the SIP1 according to the invention (a zinc finger/homeodomain protein) binds to specific sites in the promoter region of a number of genes which are relevant for the immune response and early embryogenesis and as such may be involved in transcriptional regulation of important differentiation genes in significant biological processes such as cell growth and differentiation, embryogenesis, and abnormal cell growth including cancer.
The invention also includes an isolated nucleic acid sequence including the nucleotide sequence as provided in SEQ ID NO 1 coding for a SMAD interacting protein or a functional fragment thereof.
Furthermore, a recombinant expression vector including the isolated nucleic acid sequence (in sense or anti-sense orientation) operably linked to a suitable control sequence belongs to the present invention and cells transfected or transduced with a recombinant expression vector as well.
Another aspect of the invention is a polypeptide including the amino acid sequence according to SEQ.ID.NO 2 or a functional fragment thereof. The present invention also includes variants or homologues of amino acids enclosed in the disclosed polypeptides wherein the amino acids are modified and/or substituted by other amino acids obvious for a person skilled in the art. For example, post-expression modifications of the polypeptide such as phosphorylations are not excluded from the scope of the current invention.
A pharmaceutical composition including the previously identified nucleic acid(s) or a p
Huylebroeck Danny
Remacle Jacques
Verschueren Kristin
Priebe Scott D.
Schnizer Richard
TraskBritt
Vlaams Interuniversitair Instituut Voor Biotechnologie
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