Proteins associated with apoptosis

Details Proteins associated with apoptosis Proteins associated with apoptosis

1999-09-30

2001-08-28

Huff, Sheela (Department: 1642)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C435S004000, C435S007100

Reexamination Certificate

active

06281334

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of three human proteins associated with cell proliferation and to the use of these sequences in the diagnosis. prevention, and treatment of disorders associated with abnormal cell proliferation and apoptosis.
BACKGROUND OF THE INVENTION
Differentiation, growth, and function of eukaryotic cells are coordinated with various mechanisms which regulate DNA replication and prevent excessive proliferation. While uncoordinated cell proliferation can cause cancer, autoimmune diseases, and inflammatory diseases, programmed cell death or apoptosis removes excessive or damaged cells without causing tissue destruction and inflammatory response. A plethora of genes, named oncogenes, have been identified with cancer. Several highly conserved processes have been shown to regulate apoptosis.
bcl-2 proto-oncogene is a repressor of apoptosis and functions downstream in the regulation of cell-death processes. The C. elegans homolog of Bcl-2, CED-9, represses the apoptosis of 131 cells during the nematode development (Hengartner, M. O. and Horvitz, H. R. (1994) Cell 76: 665-676). Several Bcl-2-related proteins, such as Bax, Bcl-X
L
, and Bad, share homology with Bcl-2 mostly within two conserved regions, named BH1 and BH2. Bcl-X
L
and Bcl-2 are shown to repress apoptosis, while Bax and Bad promote apoptosis. Specifically, Bax is capable of dimerizing with Bcl-2 and Bcl-X
L,
whereas Bad is able to dimerize with Bcl-X
L
but not with Bcl-2. When Bax dimerizes with Bcl-2, the apoptosis-inhibiting function of Bcl-2 is suppressed (Oltvai, Z. N. et al. (1993) Cell 74: 609-619; Yin, X.-M. et al. (1994) Nature 369: 321-323). Similarly, when Bad displaces Bax and dimerizes with BCl-X
L,
the apoptosis-repressive activity of Bcl-X
L
is inhibited (Yang, E. et al. (1995) Cell 80: 285-291).
Leucine-rich repeat (LRR) is a structural motif of about 20 to 29 amino acid residues in length associated with protein-protein interactions. The motif contains leucine or other aliphatic residues at positions
2
,
5
,
7
,
12
,
16
,
21
, and
24
and asparagine, cysteine or threonine at position
10
. X-ray structure determination of LRR motifs suggests that each LRR is composed of a &bgr;-sheet and an &agr;-helix.
p37NB is a 37 kDa LRR protein identified in human neuroblastoma cells (Kim, D. et al. (1996) Biochim. Biophys. Acta 1309: 183-188). Northern blot hybridization and RT-PCR studies show that p37NB is differentially expressed in several neuroblastoma cell lines. A related LRR protein, PRELP, is characterized as a 42 kDa secreted protein (Bengtsson, E. et al. (1995) J. Biol. Chem. 270: 25639-25644). PRELP consists of 10 LRR motifs ranging in length from 20 to 26 residues with asparagine at position
10
. Northern analysis shows differential expression of PRELP in various tissues.
MA-3 or TIS is a mouse protein associated with apoptosis (Shibahara, K. et al. (1995) Gene 166: 297-301; Onishi, Y. and Kizaki, H. (1996) Biochim. Biophys. Res. Commun. 228: 7-13). The nucleotide sequence of the mouse proteins predicts an amino acid sequence of 469 residues. MA-3 is highly expressed in thymus and is present in all apoptosis-inducible cell lines including thymocytes, T cells, B cells, and pheochromocytoma (Shibahara et al, supra). TIS expression is down-regulated in the RVC lymphoma cells incubated with an topoisomerase I inhibitor, an antitumor drug, and the low expression level of TIS may be a contributing factor to the cytotoxicity of the topoisomerase inhibitors (Onishi and Kizaki, supra).
The discovery of three new human proteins associated with cell proliferation 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 disorders associated with abnormal cell proliferation and apoptosis.
SUMMARY OF THE INVENTION
The invention features three substantially purified polypeptides, proteins associated with cell proliferation, referred to collectively as “APOP” and individually as “APOP-1”, “APOP-2”, and “APOP-3.” In one aspect, the invention provides a substantially purified polypeptide, APOP, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQ ID NO: 1, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:5.
The invention further provides a substantially purified variant of APOP having at least 90% amino acid identity to the amino acid sequences of SEQ ID NO: 1, SEQ ID NO:3, or SEQ ID NO:5, or to a fragment of either of these sequences. The invention also provides an isolated and purified polynucleotide sequence encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQ ID NO: 1, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:5. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide identity to the polynucleotide sequence encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQ ID NO: 1, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:5.
Additionally, the invention provides a composition comprising a polynucleotide sequence encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQ ID NO: 1, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:5. The invention further provides an isolated and purified polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQ ID NO: 1, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:5, as well as an isolated and purified polynucleotide sequence which is complementary to the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQ ID NO: 1, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:5.
The invention also provides an isolated and purified polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:4, and a fragment of SEQ ID NO:6. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide identity to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:4, and a fragment of SEQ ID NO:6, as well as an isolated and purified polynucleotide sequence which is complementary to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:4, and a fragment of SEQ ID NO:6.
The invention further provides an expression vector containing at least a fragment of the polynucleotide sequence encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQ ID NO: 1, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:5. 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, SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQ ID NO: 1, a fragment of SEQ ID NO:3, or a fragment of SEQ ID NO:5, 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 APOP under conditions suitable for

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