Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-06-17
2001-01-30
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S091200, C435S252300, C435S254110, C435S320100, C435S325000, C435S410000, C435S195000, C536S023200, C536S023500
Reexamination Certificate
active
06180342
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of vacuolar proton ATPase subunits and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative and neurological disorders.
BACKGROUND OF THE INVENTION
Proton-translocating adenosine triphosphatases (proton ATPases) are a large class of membrane-proteins that use the energy of ATP hydrolysis to generate an electrochemical proton gradient across a membrane. The resultant gradient may be used to transport other ions across the membrane (Na
+
, K
+
, or Cl
−
) or to maintain an acidic environment important to the function of many cellular vesicles (Mellman, I. et al. (1986) Ann. Rev. Biochem. 55:663-700). Proton ATPases are further subdivided into the mitochondrial F-ATPases, the plasma membrane ATPases, and the vacuolar ATPases.
The vacuolar proton ATPases (vp-ATPases) provide most of the energy required for transport processes in the vacuolar system in eukaryotic cells. vp-ATPases establish and maintain an acidic pH within various vesicles involved in the processes of endocytosis and exocytosis. Such vesicles include phagosomes, lysosomes, endosomes, and secretory vesicles. Endocytosis is the process in cells of internalizing nutrients, solutes or small particles (pinocytosis), or large particles such as internalized receptors, viruses, bacteria, or bacterial toxins (phagocytosis). Exocytosis is the process of transporting molecules to the cell surface. Exocytosis facilitates the placement or localization of membrane-bound receptors or other membrane proteins and the secretion of hormones, neurotransmitters, digestive enzymes, and wastes. Endocytosis and exocytosis are fundamental to the function of all types of cells.
Alterations in both endocytosis and exocytosis play a role in a variety of disorders. For example, synaptic vesicles play a major role in neural transmission at nerve terminals through storage and controlled release of neurotransmitters. Neurotransmitter uptake into synaptic vesicles is driven by the electrochemical proton gradient generated by vp-ATPase. Inactivation of vp-ATPase has been shown to inhibit glutamate uptake by synaptic vesicles, decreasing neurotransmitter release during episodes of oxidative stress or in response to second messenger signaling. Additionally, inactivation of vp-ATPase has been shown to trigger apoptosis in a variety of immortalized and primary cell lines. Activation of vp-ATPases has been found to delay apoptosis (Wang, Y. et al. (1998) J. Neurochem. 70:646-652; Nishihara, T. et al. (1995) Biochem. Biophys. Res. Commun. 212: 255-262; and Niessen, H. et al. (1997) Blood 90: 4598-4601.)
The vp-ATPases and the F-ATPases, which function in ATP synthesis and hydrolysis in mitochondria, are related in both their subunit structure and evolutionary origin. Both contain distinct catalytic and membrane sectors, and each sector contains multiple subunits. The catalytic sector of vp-ATPase consists of five subunits designated A (72 kDa), B (57 kDa), C (41 kDa), D (34 kDa), and E (33 kDa) (Nelson, H. et al. (1995) Proc. Natl. Acad. Sci. 92:497-501). Three subunits, AC115, AC39, and a proteolipid component, have been identified in the membrane sector of vp-ATPase from various sources. The proteolipid subunit has been implicated in the mechanism of energy transfer in the enzyme. The membrane sector has several functions including proton conduction across the membrane, energy coupling with the catalytic sector, communication with the lumen, and modulation of enzyme activity. Mutational studies in yeast have shown that, while the membrane sector may be assembled independently of the catalytic sector, assembly of the catalytic sector is absolutely dependent on previous assembly of the membrane sector (Noumi, T. et al. (1991) Proc. Natl. Acad. Sci. 88: 1938-42; Ludwig, J. et al. (1998) J. Biol. Chem. 273: 10939-10947; and Supekova, L. et al. (1996)199:1147-1156). Thus, expression and assembly of the membrane sector subunits control the overall activity of the enzyme complex.
The discovery of new vacuolar proton ATPase subunits 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 cell proliferative and neurological disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, vacuolar proton ATPase subunits, referred to collectively as “VATPS” and individually as “VATPS-1” and “VATPS-2.” In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO: 2.
The invention further provides a substantially purified variant having at least 90% amino acid identity to the amino acid sequences of SEQ ID NO: 1 or SEQ ID NO: 2, or to a fragment of either of these sequences. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO: 2. The invention also includes an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO: 2.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, a fragment of SEQ ID NO: 1 and a fragment of SEQ ID NO: 2, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO: 2.
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, a fragment of SEQ ID NO:
and a fragment of SEQ ID NO: 4. The invention further provides an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, a fragment of SEQ ID NO: 3, and a fragment of SEQ ID NO: 4, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, a fragment of SEQ ID NO: 3, and a fragment of SEQ ID NO: 4.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO: 2. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO: 2, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide 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 polypeptide having the amino acid sequence selected from the group consisting
Bandman Olga
Corley Neil C.
Guegler Karl J.
Lal Preeti
Patterson Chandra
Incyte Pharmaceuticals Inc.
Incyte Pharmaceuticals, Inc.
Slobodyansky Elizabeth
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