Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-02-01
2001-11-06
Huff, Sheela (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S387100, C530S387900
Reexamination Certificate
active
06313266
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of a human nucleolin-like protein and to the use of these sequences in the diagnosis, treatment, and prevention of cancer, autoimmune disorders, and Alzheimer's disease.
BACKGROUND OF THE INVENTION
The nuclei of eukaryotic cells contain a subcompartment termed the nucleolus, which is the site of ribosome production. Ribosomes are comprised of ribosomal RNA and various protein components imported from the cytoplasm. Ribosomes are essential scaffolding and catalytic elements of the protein translation machinery found in the cytosol.
The main protein component in the nucleolus of eukaryotic cells is nucleolin. Nucleolin is an essential component of ribosome biogenesis. The carboxy-terminus of nucleolin is a glycine-rich region that contains RNA binding domains, which interact specifically with stem-loop structures typical of ribosomal RNA. The amino-terminus of nucleolin contains a binding site for histone H1, and binding of nucleolin to H1 promotes the decondensation of chromatin necessary to begin DNA transcription. Nucleolin also contains binding sites for nuclear localization sequences, and has been shown to shuttle between the nucleus and the cytoplasm. Thus, nucleolin also plays a role in importing proteins into the nucleus, and is a crucial element of ribosome biogenesis. (Lapeyre, B. et al. (1987) Proc. Natl. Acad. Sci.84:1472-1476; Erard, M. S. et al. (1988) Eur. J. Biochem. 175:525-530.) Elevated levels of autoantibodies to nucleolin are implicated in autoimmune diseases such as systemic sclerosis, systemic lupus erythematosus, and certain cases of chronic graft-versus-host disease. (Deng, J. S. et al. (1996) Mol. Biol. Rep. 23:191-195) In addition, there is a clinical correlation between disease severity and levels of autoantibodies to nucleolin. (Bell, S. A. et al. (1996) Br. J. Dermatol. 134:848-854.) Thus, overexpression of nucleolin and the mislocalization of nucleolin outside the nucleolus may mediate various autoimmune disorders.
One characteristic of Alzheimer's disease is the deposition of senile, or neuritic, plaques throughout the brains of affected individuals. These senile plaques contain &bgr;A4 amyloid protein, which is produced from &bgr; amyloid precursor protein (APP). Messenger RNA for APP is elevated in the brains of Alzheimer's disease patients, and nucleolin has been shown to bind and stabilize APP MRNA, increasing the message's half-life and thereby increasing the level of APP. Thus, nucleolin regulates APP expression, and therefore is involved in overproduction of &bgr;A4 amyloid protein and the deposition of senile plaques in Alzheimer's disease. (Zaidi, S. H. E. and Malter, J. S. (1995) J Biol Chem 270:17292-17298.)
The discovery of a new human nucleolin-like protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of cancer, autoimmune disorders, and Alzheimer's disease.
SUMMARY OF THE INVENTION
The invention features a substantially purified polypeptide, human nucleolin-like protein (HNLP), comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides a substantially purified variant of HNLP having at least 90% amino acid identity to the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide identity to the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
Additionally, the invention provides a composition comprising a polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention further provides an isolated and purified polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purified polynucleotide sequence which is complementary to the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide sequence comprising SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 90% polynucleotide identity to the polynucleotide sequence comprising SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides an isolated and purified polynucleotide sequence which is complementary to the polynucleotide sequence comprising SEQ ID NO:2 or a fragment of SEQ ID NO:2.
The invention further provides an expression vector containing at least a fragment of the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. 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 of SEQ ID NO:1 or a fragment of SEQ ID NO:1, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide sequence encoding HNLP 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 HNLP having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist and a purified antagonist of the polypeptide.
The invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of HNLP.
The invention also provides a method for treating or preventing an autoimmune disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of HNLP.
The invention also provides a method for treating or preventing Alzheimer's disease, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of HNLP.
The invention also provides a method for detecting a polynucleotide encoding HNLP in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence encoding the polypeptide comprising SEQ ID NO:1 or a fragment of SEQ ID NO:1 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding HNLP in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.
REFERENCES:
Lapeyre, B. et al., “Nucleolin, the major nucleolar protein of growing eukaryotic cells: An unusual protein structure revealed by the nucleotide sequence”,Proc. Natl. Acad. Sci. USA, 84: 1472-1476 (1987).
Erard, M.S. et al., “A major nucleolar protein, nucleolin, induces chromatin decondensation by binding to histone H1”,Eur. J. Biochem., 175: 525-530 (1988).
Deng, J.S. et al., “Internalization of anti-nucleolin antibody into viable HEp-2 cells”,Mol. Biol. Rep., 23: 191-195 (1996).
Bell, S.A. et
Bandman Olga
Corley Neil C.
Shah Purvi
Yue Henry
Huff Sheela
Incyte Genomics Inc.
Incyte Genomics, Inc.
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