Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-08-04
2001-03-13
Achutamurthy, Ponnathapau (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C530S350000, C435S004000, C435S007100, C435S091500, C435S091500
Reexamination Certificate
active
06200790
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of three vesicle transport associated proteins and to the use of these sequences in the diagnosis, prevention, and treatment of cancer, and immune, reproductive, and developmental disorders.
BACKGROUND OF THE INVENTION
Vesicle transport is the general process in eukaryotic cells by which proteins synthesized in the endoplasmic reticulum (ER) are transported via the Golgi network to the various compartments in the cell where they will function. Other proteins are transported to the cell surface by this process where they may be secreted (exocytosis). Such proteins include membrane bound receptors or other membrane proteins, neurotransmitters, hormones, and digestive enzymes. The transport process uses a series of transport vesicles that shuttle a protein from one membrane-bound compartment (donor compartment) to another (acceptor compartment) until the protein reaches its proper destination (Rothman, J. E. and Wieland, F. T. (1996) Science 272:227-34).
The process begins with the budding of a vesicle out of the donor membrane. The vesicle contains the protein to be transported and is surrounded by a protective coat made up of protein subunits recruited from the cytosol. The initial budding process and coating processes are controlled by cytosolic GTP-binding proteins (GTPB). When GTP binds and activates the GTPB, the GTP-GTPB complex binds to the donor membrane and initiates the vesicle assembly process. The coated vesicle containing the GTP-GTPB complex detaches from the donor compartment and is transported through the cytosol. During the transport process, the GTP is hydrolyzed to GDP, and the inactivated GTPB dissociates from the transport vesicle and is recycled. At this point, the protective coat of the vesicle becomes unstable and dissociates from the enclosed vesicle. The uncoated vesicle is recognized by its acceptor compartment through exposed surface identifiers (v-SNAREs) which bind with corresponding molecules on the acceptor compartment membrane (t-SNAREs). The transport process ends when the vesicle fuses with the target membrane.
Many of the proteins involved in synaptic vesicle transport have been identified and the biochemical interactions between them have been characterized. Interestingly, many of these proteins are homologous to yeast proteins involved in yeast secretory pathways. For example, BET1, BOS1, and SEC22 form a set of interacting yeast genes required in endoplasmic reticulum (ER) to Golgi protein transport (Newman, A. P. et al. (1992) EMBO Journal 11: 3609-17). Each of these genes encodes a small integral membrane protein with hydrophilic, cytoplasmic N-terminal and central regions, and a C-terminal hydrophobic transmembrane anchor. Mammalian counterparts of yeast bet1 and sec22 have been identified (Hay, J. C. et al. (1996) J. Biol. Chem. 271: 5671-79). Both proteins are widely expressed in mammalian tissues, as would be expected of proteins involved in fundamental membrane trafficking reactions. Mammalian bet1 and sec22 are localized to the ER membrane and the Golgi membrane, respectively, indicating that they may participate in vesicle transport in opposite directions between the ER and the Golgi apparatus (Hay et al. supra).
GTP-binding/GTPase proteins are likewise essential in both yeast and mammalian secretory pathways. At least four genes encoding GTPB's have been shown to be involved in transport between ER and the plasma membrane. These include SEC4, YPT1, SAR1, and ARF1 (Dascher, C. et al. (1991) Mol. Cell. Biol. 11:872-85). Yeast sec4 GTPB, essential for late stages of vesicle secretion, is homologous to mammalian Ra3a GTPase. The Rho proteins are another small family of GTPBs found in both yeast and mammals (Nakano, K. and Issei, M. (1995) Gene 155: 119-22). Rho proteins regulate the actin cytoskeleton during cell division and control signal transduction by linking receptors of growth factors to actin polymerization. RhoD is a member of the mammalian Rho family that provides a link between membrane trafficking and cytoskeleton regulation. RhoD causes rearrangements of the actin cytoskeleton and cell surface, and governs endosome motility and distribution (Murphy, C. et al. (1996) Nature 384: 427-32).
GTP-binding proteins share several amino acid sequence motifs, termed motifs I-IV. Motif I has the signature, GXXXXGK. The lysine residue is essential in interacting with the &bgr;- and &ggr;-phosphates of GTP. Motif II, III, and IV are highly conserved, with DTAGQ, NKXD, EXSAK/L as their respective signatures. These motifs regulate the binding of &ggr;-phosphate, GTP, and the guanine base of GTP, respectively. In addition, Rho proteins have a Cys-aliphatic residue-aliphatic residue-X (CAAX) box for the binding of a prenyl group and either a palmitoylation site or a basic amino acid-rich region, suggesting their role in membrane-associated functions.
The discovery of new vesicle transport associated proteins and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of cancer, and immune, reproductive, and developmental disorders.
SUMMARY OF THE INVENTION
The invention features three substantially purified polypeptides, vesicle transport associated proteins VTAP-1, VTAP-2, and VTAP-3 (referred to collectively as VTAP), having the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, 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 a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequences SEQ ID NO:1, or fragments of said 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 said polynucleotide sequence.
The invention also provides an isolated and purified sequence comprising SEQ ID NO:2 or variants thereof. In addition, the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2. The invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments of variants thereof.
The present invention further 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 VTAP-1 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 VTAP-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 comprising administering to a subject in need of such treatment an effective amount of a purified antagonist of VTAP-1.
The invention also provides a method for treating or preventing a reproductive disorder comprising ad
Corley Neil C.
Hillman Jennifer L.
Lal Preeti
Shah Purvi
Achutamurthy Ponnathapau
Incyte Genomics, Inc.
Incyte Pharmaceuticals Inc.
Mayhew Bradley S.
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