Methionine aminopeptidase type 3

Details Methionine aminopeptidase type 3 Methionine aminopeptidase type 3

2000-03-10

2003-10-28

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S252300, C435S320100, C536S023200, C536S023600, C530S300000, C530S350000

Reexamination Certificate

active

06638750

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a human cDNA encoding a methionine aminopeptidase type-3 (MetAP-3) protein. The invention also relates to nucleic acid molecules associated with or derived from this cDNA including complements, homologues and fragments thereof, and methods of using these nucleic acid molecules, to generate, for example, polypeptides and fragments thereof. The invention also provides methods of using the nucleic acids, for example, to produce a protein and fragments thereof and to screen for compounds or compositions that preferentially or specifically effect the activity of a MetAP-3 protein.
BACKGROUND
Angiogenesis, the process of new blood vessel formation, is essential for the exponential growth of solid tumors and tumor metastasis. Radiological and cytocidal treatments, combined with regimens involving selective inhibitors of angiogenesis should lead to dramatic reductions in tumor growth. One angiogenesis inhibitor was first discovered as a fungal contaminant of bovine endothelial cell cultures that inhibited cell proliferation (Ingber et al.
Nature
348:555-557, 1990). This product was subsequently isolated from
A. fumagatus
and identified as fumagillin, a well-known amebicide and antibiotic (McCowen et al.,
Science
113:202-203 (1951)). Fumagillin was found to be a potent inhibitor of endothelial cell proliferation, but its therapeutic window was insufficient for further clinical advancement. TNP-470, a fumagillin-like derivative with 50-fold higher potency, was subsequently developed from a directed chemical approach (Ingber et al.,
Nature
348:555-557 (1990), Kusaka et al.,
Biochem. Biophys. Res. Commun
. 174:1070-1076 (1991)). This compound's therapeutic use is limited, however, by its lack of oral availability and dose-limiting neurotoxicity.
Until recently, the molecular target for fumagillin or TNP-470 was unknown. In 1997, the target protein was isolated, purified, and identified by mass spectrometry as the type-2 methionine aminopeptidase (MetAP-2). Both fumagillin and TNP-470 are now known as potent inhibitors of MetAP-2, but not the type-1 enzyme. This result identified MetAP-2 as an anti-angiogenesis target (Sin et al.
Proc. Natl. Acad. Sci
. (
U.S.A
.) 94:6099-6103 (1997), Griffith et al.
Chem. Biol
. 4:461-471 (1997).
The methionine aminopeptidases were first isolated from eubacteria and shown to be cobalt-containing enzymes with molecular masses of about 30 kDa (Ben-Bassat et al.,
J. Bacteriol
. 169:751-757 (1987), Suh et al.,
Gene
169:17-23 (1996), and Miller et al.,
Proc. Natl. Acad. Sci
. (
U.S.A
.) 91:2473-2477, 1987). The structure of these enzymes consists of a novel protease fold with pseudosymmetry around a pair of cobalt ions (Roderick and Matthews,
Biochemistry
32:3907-3912, 1993).
Enzymes with the same substrate specificity, but with larger molecular masses, were isolated from yeast and pig. Highly homologous regions at the C-terminal domain (~30 kDa) of the eukaryotic and the prokaryotic forms were discovered, although the N-terminal domain of the eukaryote enzymes was found to be unique (Kendall and Bradshaw,
J. Biol. Chem
. 267:20667-20673 (1992)). The N-terminal domain of the yeast enzyme contained sequences consistent with two zinc-finger structures, indicating a potential site of nucleic acid interaction. This class of enzyme was designated methionine peptidase Type I (MetAP-1). The porcine enzyme lacked the zinc-binding domains, but contained a block of polylysine and aspartic residues within the N-terminal domain, and was described as Type II (MetAP-2). Both isozymes have been found from Archebacteria to man, indicating a critical metabolic function (Arfin et al.
Proc. Natl. Acad. Sci
. (
U.S.A
.) 92:7714-7718 (1995), Bradshaw et al.,
TIBS
23: 2.63-267 (1998)).
Methionine aminopeptidase-2 is bi-functional. One action is the removal of the N-terminal methionine residues from their protein substrates. MetAP-2 can also bind to and prevent phosphorylation of the &agr;-subunit of the peptide change initiation factor eIF-2 by one or more eIF-2 kinases (Datta et al.,
Proc. Natl. Acad. Sci. USA
85: 3324-2238 1(1988), Wu et al.,
J. Biol. Chem
. 268:10796-10781 (1993)). This action promotes protein synthesis within the cell. The eIF-2 phosphorylation inhibitory activity of MetAP-2 is unaffected by TNP-470 binding, indicating that the loss of aminopeptidase activity is involved in the anti-angiogenic activity of TNP-470 (Griffith et al.,
Chem. Biol
. 4:461-471 (1997)). The function of methionine peptidase activity in endothelial cell proliferation during tumorigenesis is unclear, although inhibition of MetAP-2 may play a role in altering the stability of one or more protein(s) whose abnormal presence or absence results in endothelial cell dysregulation. Several signaling proteins also appear to be modified by the covalent attachment of myristic acid to a glycine residue which occurs only after the initial amino-terminal methionine removal by MetAP-2 (Peseckis et al.,
J. Biol. Chem
. 267:5107-5114 (1993)). Inhibition of methionine aminopeptidase activity may prevent this covalent attachment, resulting in improper functioning of a signal component specific to endothelial cell cycle regulation (Sin et al.
Proc. Natl. Acad. Sci
. (
U.S.A
.) 94:6099-6103 (1997)).
N-terminal processing agents, such as the methionine aminopeptidases, also function to initiate post-translational peptide or protein modifications which may control or induce activation, translocation, or protein turnover (Bradshaw et al.,
TIBS
23: 263-267 (1998)). Because this initial processing is important for normal protein functioning, it is possible that alteration of methionine aminopeptidase activity is a factor in a variety of diseases, including angiogenesis. Therapies can thus be developed which can modify methionine aminopeptidase activity to restore proper protein processing.
Methionine aminopeptidase activity can also be used to modify recombinant proteins expressed and harvested from
E. coli
or other expression systems. Recombinant proteins that retain the N-terminal methionine, in some cases, have biological characteristics that differ from the native species that retain the N-terminal methionine, including the induction of undesireable antibodies. Using a methionine aminopeptidase for recombinant protein modification provides a low-cost method of generating potentially life-saving therapeutic proteins and to mimic the structure of native protein species which are used to combat or eliminate the causes of various diseases (Sandman et al., Biotechnology (N Y) 13:504-6 (1995)).
Clearly, an understanding of methionine aminopeptidase activity and its role in various tissues can provide useful therapeutic and diagnostic insight into angiogenesis and tumor metastasis. The known MetAP-2 inhibitors are not good candidates for clinical use as angiogenesis inhibitors due to their neurotoxic effects. Differential expression of mammalian MetAP-1, MetAP-2, or other unidentified methionine aminopeptidases may partially or totally account for the observed variation in sensitivity of different cell types to inhibition by TNP-470 and other MetAP-2 inhibitors, and thus account for the observed toxicity of these drugs.
SUMMARY OF THE INVENTION
One aspect of the present invention is to provide a novel methionine aminopeptidase, MetAP-3, and its nucleic acids, proteins, peptides, fragments, and homologues.
Another aspect of the invention is to provide new and advantageous targets to screen for diagnostic and therapeutic agents and compositions useful for diagnosis or treatment of angiogenesis-related diseases.
The invention provides a substantially-pure nucleic acid comprising a nucleic acid sequence selected from the group consisting of: SEQ NO: 7 or complements thereof; nucleic acid sequences that specifically hybridize to SEQ NO: 7 or complements thereof, especially those that hybridize under stringent conditions; nucleic acid sequences encoding a MetAP-3 protein or fragment thereof, or complement of these nucleic acid sequ

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