Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-09-16
2001-10-09
Huff, Sheela (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S387100, C530S387900, C530S389100, C514S012200
Reexamination Certificate
active
06300472
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of three new human Rab proteins and to the use of these sequences in the diagnosis, prevention, and treatment of inflammation and disorders associated with cell proliferation and apoptosis.
BACKGROUND OF THE INVENTION
Vesicle trafficking is defined as the vesicular transport of materials between different subcellular compartments of eukaryotic cells. Vesicles bud from a donor membrane and fuse with a recipient one carrying internalized materials from one site to another. Rab proteins, low molecular weight (LMW) guanidine triphosphatases (GTPases), belong to the Ras superfamily; they help regulate vesicular transport by directing the vesicles to and from the correct membrane surfaces (Novick, P. and Brennwald, P. (1993) Cell 75: 597-601).
Rab proteins assist the binding of a transport vesicle to its proper acceptor membrane and initiate the membrane fusion process using the energy derived from the hydrolysis of GTP. Rab proteins have a highly variable amino terminus and a prenylated carboxy terminus. The amino terminus contains signal sequences, and the carboxy terminus determines the target membrane to which the Rab protein binds. The targeting process is assisted by a series of escort proteins (Khosravi-Far, R. et al. (1991) Proc. Natl. Acad. Sci. 88: 6264-6268).
In the medial Golgi, it has been shown that GTP-bound Rab proteins initiate the binding of VA-MP-like proteins of the transport vesicle to syntaxin-like proteins on the acceptor membrane triggering membrane fusion events. After transport, GTPase-activating proteins in the target membrane convert the Rab proteins to their GDP-bound state, and guanine-nucleotide dissociation inhibitor helps return the GDP-bound proteins to their membrane of origin.
To date, more than 30 Rab proteins have been identified, and each may have a characteristic intracellular location where it functions in distinct, tissue-specific transport events. For example, Rab2 is important in ER-to-Golgi transport; Rab1 and Rab6 are localized to the Golgi apparatus; Rab3 transports secretory vesicles to the extracellular membrane; Rab5 and Rab7 are localized to the early and late endosomal fusion events, respectively; and Rab 10 mediates vesicle fusion from the medial Golgi to the trans Golgi.
Structurally, the Rab proteins display features characteristic of LMW GTP-binding proteins. Four sequence regions, motifs I-IV, are conserved in the Rab proteins. Motif I, the most variable region among the four, has a signature of GXXXXGK, and the terminal lysine residue interacts with the &bgr;- and &ggr;-phosphates of GTP. Motifs II, III, and IV are highly conserved and function in regulating the binding of &ggr;-phosphate, GTP, and the guanine base of GTP, respectively.
In addition to the conserved motifs, the arginine residue following the second GTP-binding domain, the phenylalanine residue adjacent to the fourth GTP-binding domain, and the carboxy terminal cysteines are highly conserved. The cysteines are particularly important in that they are essential for membrane localization. The Rab proteins also have an effector region located in between Motif I and Motif II which has been characterized as the interaction site for GAP, a regulatory protein which stimulates the intrinsic GTPase activity.
Experimental evidence has established the essential role of Rab in vesicle trafficking, cell function, and cell differentiation. Human Rab1, Rab2, Rab3B, Rab4, Rab5, and Rab6 genes isolated from a human pheochromocytoma cDNA library exhibit GTPase activities when produced in
E. Coli
(Zahraoui, A. et al. (1989) 264: 12394-12401). Although differentially expressed, two isoforms of Rab28, hRab28S and hRab28L, exhibit comparable GTPase-related activities in rat tissues (Brauers, A. et al. (1996) Eur. J. Biochem. 237: 833-840). Localization of murine Rab24 in endoplasmic reticulum/cis-Golgi region of Semliki Forest virus and the vaccinia T7 vector systems indicates that Rab24 may be involved in autophagy-related processes (Olkkonen, V. M. et al. (1993) J. Cell. Sci. 106: 1249-1261). Over expression of Rab proteins significantly enhances the function of Rev, a viral gene essential for processing HIV-1 (Fridell, R.A. et al. (1996) Proc. Natl. Acad. Sci. 93: 4421-4424). A deficiency in the prenylation of one particular Rab is associated with choroideremia, a form of retinal degeneration that causes blindness (Seabra, M. et al. (1996) J. Biol. Chem. 270:24420-24427). Interaction between Rab protein and Cdc2 protein kinase in vitro inhibited vesicle fusion and implicated Rab protein function in mediating cell cycle events (Toumikoski, T. et al. (1989, Nature 342: 942-945). Thus, Rab proteins appear to be involved in the complex and critical processes of vesicle trafficking for the directed release of various molecules.
The discovery of three new human Rab proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of inflammation and disorders associated with cell proliferation and apoptosis.
SUMMARY OF THE INVENTION
The invention features three substantially purified polypeptides, designated individually as RABP-1, RABP-2 and RABP-3 and collectively as RABP having the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.
The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide RAPB-1, comprising the amino acid sequence of SEQ ID NO: 1 or fragments thereof and compositions comprising said polynucleotide sequence. The invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence 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 or 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 RABP-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 RABP-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 stimulating cell proliferation comprising administering to a cell an effective amount of a pharmaceutical composition comprising purified RABP-1.
The invention also provides a method for treating a disorder associated with an increase in apoptosis comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical compos
Corley Neil C.
Hillman Jennifer L.
Lal Preeti
Shah Purvi
Huff Sheela
Incyte Genomics Inc.
Incyte Genomics, Inc.
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