Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-04-19
2001-12-04
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C530S350000, C536S023500
Reexamination Certificate
active
06326158
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of two SH3-containing proteins and to the use of these sequences in the diagnosis, prevention, and treatment of cancer and immune and developmental disorders.
BACKGROUND OF THE INVENTION
Physical interactions between proteins are critical in executing various cellular processes. Recently, several small protein domains have been identified that appear to function in these various protein-protein interactions. Two such domains are Src homology 2 (SH2) and Src homology 3 (SH3) (Musacchio, A. et al. (1994) Prog. Biophys. Mol. Biol. 61: 283-297).
SH3 domains, defined by their homology to a region of the proto-oncogene c-Src, are small protein domains of 50 to 60 amino acids found in a diverse group of proteins. SH3 domains bind to proline-rich ligands and are involved in a number of cellular processes including subcellular localization, G-protein signaling, and tyrosine kinase regulation. Proteins which contain SH3 domains are important for cellular organization and the control of cellular morphology. Several proteins associated with the cytoskeleton, including &agr;-spectrin and myosin-1, contain SH3 domains (Pawson, T. et al. (1992) Cell 71:359-362). The small GTP-binding protein Rho binds with high affinity to SH3 domains, is involved in actin bundling, and regulates the assembly of focal adhesions (Ridley, A. J. et al. (1992) Cell 70:389-399).
Formins are a small group of proline-rich phosphoproteins which are localized largely in the nucleus and are required for proper limb and kidney development in the mouse (Chan, D. C. et al. (1996) EMBO J. 15: 1045-54). Formins appear to function by interacting with certain SH3-containing proteins (formin binding proteins, FBP) that are expressed during mouse embryogenesis. In addition to three SH3-containing FBPs (FBP1, FBP17, and FBP27), five FBPs containing an “SH3-like” binding motif have also been identified. The new motif, termed WWP/WW because of highly conserved tryptophan and proline residues, compete with the SH3-containing FBPs for binding to proline-rich regions of formin. Chan et al. (supra) suggest that this competition may serve to regulate the function of SH3 domains in these interactions.
SH3 domains are characterized by a conserved structure composed of two antiparallel, B-pleated sheets packed against each other at rights angles. This packing forms a hydrophobic pocket lined with residues that are highly conserved between different SH3 domains. The hydrophobic pocket makes critical hydrophobic contacts with proline residues in the ligand binding protein (Feng, S. et al. (1994) Science 266: 1241-47). Electrostatic interactions are also sometimes important for specificity. In FBP17, for example, the sequence ALYTF is similar to the highly conserved ALYDY sequence in many SH3 domains. The Y or F in position five of this sequence is thought to be part of the hydrophobic binding pocket of SH3. W
204
and Y
221
in FBP17 are also thought to be important for ligand binding.
The regulation of protein interactions by SH3-containing proteins has implications in various diseases. Regulation of protein tyrosine kinase activity by SH3-containing proteins may be important in controlling some types of cancer. It is well known that cellular transformation (oncogenesis) is often accompanied by increased tyrosine kinase activity (Charbonneau, H. and N. K. Tonks (1992) Annu. Rev. Cell Biol. 8:463-93). SH3-containing proteins are important in the immune response and immune disorders. In phagoeytes, the NADPH oxidase mutiprotein complex is activated by inflammatory stimuli to produce superoxide, a precursor for antimicrobial oxidants. This activation is dependent on the interaction of SH3-containing oxidase proteins p47-phox, p67-phox, and p40-phox with other proteins of the oxidase complex (McPhail, L. C. (1994) J. Exp. Med. 180:2011-2015). The SH3 domains of p47-phox and p67-phox may be responsible for assembly of the functional oxidase complex (Pawson, supra).
The discovery of new SH3-containing proteins and the polynucleotides encoding them satisfy a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of cancer, and immune and developmental disorders.
SUMMARY OF THE INVENTION
The invention features two substantially purified polypeptides, SH3-containing proteins (referred to collectively as “HS3C” and individually as “HS3C-1” and “HS3C-2”), having the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO:3, or fragments thereof
The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or fragments thereof and a composition comprising said polynucleotide sequence. The invention also provides an isolated and purified polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO:1, or fragments of the polynucleotide sequence. The invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, or fragments or variants of the polynucleotide sequence.
The invention also provides an isolated and purified sequence comprising SEQ ID NO:2 or variants thereof. The invention also provides an isolated and purified polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof.
The present invention fisher provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences. In yet another aspect, the expression vector containing the polynucleotide sequence is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding HS3C under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified HS3C-1 having the amino acid sequence of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention also provides a purified antagonist of the polypeptide of SEQ ID NO:1. In one aspect the invention provides a purified antibody which binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:1.
Still further, the invention provides a purified agonist of the polypeptide of SEQ ID NO:1.
The invention also provides a method for treating or preventing cancer, the method comprising administering to a subject in need of such treatment an effective amount of a purified antagonist of HS3C-1.
The invention also provides a method for treating or preventing an immune disorder, the method comprising administering to a subject in need of such treatment an effective amount of a purified antagonist of HS3C-1.
The invention also provides a method for detecting a polynucleotide which encodes HS3C-1 in a biological sample containing nucleic cacid material, the method comprising the steps of: a) hybridizing the complement of the polynucleotide sequence which encodes SEQ ID NO:1 to the nucleic acid material of the biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding HS3C-1 in the biological sample. In one aspect the nucleic acid material of the biological sample is amplified by the polymerase chain reaction prior to the hybridizing step.
The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:3 or fragments thereof and a composition comprising said polynucleotide sequence. The invention
Bandman Olga
Guegler Karl J.
Lal Preeti
Fronda Christian L.
Incyte Genomics Inc.
Incyte Genomics, Inc.
Nashed Nashaat T.
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