Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-01-22
2001-04-24
Brusca, John S. (Department: 1631)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
Reexamination Certificate
active
06222018
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to hypoxia-related proteins, and specifically to novel DNA-binding proteins which are induced by hypoxia.
BACKGROUND OF THE INVENTION
Mammals require molecular oxygen (O
2
) for essential metabolic processes including oxidative phosphorylation in which O
2
serves as electron acceptor during ATP formation. Systemic, local, and intracellular homeostatic responses elicited by hypoxia (the state in which O
2
demand exceeds supply) include erythropoiesis by individuals who are anemic or at high altitude (Jelkmann (1992) Physiol. Rev. 72:449-489), neovascularization in ischemic myocardium (White et al. (1992) Circ. Res. 71:1490-1500), and glycolysis in cells cultured at reduced O
2
tension (Wolfle et al. (1983) Eur. J. Biochem. 135:405-412). These adaptive responses either increase O
2
delivery or activate alternate metabolic pathways that do not require O
2
. Hypoxia-inducible gene products that participate in these responses include erythropoietin (EPO) (reviewed in Semenza (1994) Hematol. Oncol. Clinics N. Amer. 8:863-884), vascular endothelial growth factor (Shweiki et al. (1992) Nature 359:843-845; Banai et al. (1994) Cardiovasc. Res. 28:1176-1179; Goldberg & Schneider (1994) J. Biol. Chem. 269:4355-4359), and glycolytic enzymes (Firth et al. (1994) Proc. Natl. Acad. Sci. USA 91:6496-6500; Semenza et al. (1994) J. Biol. Chem. 269:23757-23763).
The molecular mechanisms that mediate genetic responses to hypoxia have been extensively investigated for the EPO gene, which encodes a growth factor that regulates erythropoiesis and thus blood O
2
-carrying capacity (Jelkmann (1992) supra; Semenza (1994) supra). Cis-acting DNA sequences required for transcriptional activation in response to hypoxia were identified in the EPO 31′-flanking region and a trans-acting factor that binds to the enhancer, hypoxia-inducible factor 1 (HIF-1), fulfilled criteria for a physiological regulator of EPO transcription: inducers of EPO expression (1% O
2
, cobalt chloride [CoCl
2
], and desferrioxamine [DFX]) also induced HIF-1 DNA binding activity with similar kinetics; inhibitors of EPO expression (actinomycin D, cycloheximide, and 2-aminopurine) blocked induction of HIF-1 activity; and mutations in the EPO 3′-flanking region that eliminated HIF-1 binding also eliminated enhancer function (Semenza (1994) supra). These results also support the hypothesis that O
2
tension is sensed by a hemoprotein (Goldberg et al. (1988) Science 242:1412-1415) and that a signal transduction pathway requiring ongoing transcription, translation, and protein phosphorylation participates in the induction of HIF-1 DNA-binding activity and EPO transcription in hypoxic cells (Semenza (1994) supra).
EPO expression is cell type specific, but induction of HIF-1 activity by 1% O
2
, CoCl
2
, or DFX was detected in many mammalian cell lines (Wang & Semenza (1993a) Proc. Natl. Acad. Sci. USA 90:4304-4308), and the EPO enhancer directed hypoxia-inducible transcription of reporter genes transfected into non-EPO-producing cells (Wang & Semenza (1993a) supra; Maxwell et al. (1993) Proc. Natl. Acad. Sci. USA 90:2423-2427). RNAs encoding several glycolytic enzymes were induced by 1% O
2
, CoCl
2
, or DFX in EPO-producing Hep3B or non-producing HeLa cells whereas cycloheximide blocked their induction and glycolytic gene sequences containing HIF-1 binding sites mediated hypoxia-inducible transcription in transfection assays (Firth et al. (1994) supra; Semenza et al. (1994) supra). These experiments support the role of HIF-1 in activating homeostatic responses to hypoxia.
SUMMARY OF THE INVENTION
The invention features a substantially purified DNA-binding protein, hypoxia-inducible factor-1 (HIF-1), characterized as activating structural gene expression where the promoter region of the structural gene contains an HIF-1 binding site. Examples of such structural genes include erythropoietin (EPO), vascular endothelial growth hormone (V-EGF), and glycolytic genes. HIF-1 is composed of two subunits, HIF-1&agr; and an isoform of HIF-1&bgr;.
The invention features a substantially purified HIF-1&agr; polypeptide, and a nucleotide sequence which encodes HIF-1&agr;.
The invention provides methods for preventing and treating hypoxia-related disorders, including tissue damage resulting from hypoxia and reperfusion, by administering a therapeutically effective amount of HIF-1 protein. Also included in the invention is gene therapy by introducing into cells a nucleotide sequence encoding HIF-1. The invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier admixed with a therapeutically effective amount of HIF-1 or nucleotide sequence encoding HIF-1.
The invention further provides a novel HIF-1&agr; variant polypeptide which functionally inactivates HIF-1 in vivo. The invention provides a method for treating an HIF-1-mediated disorder or condition by functional inactivation of HIF-1 by administration of an effective amount of the HIF-1&agr; variant of the invention.
REFERENCES:
Fijiwara et al., Expressed Sequence Tag (EST) GenBank Accession Numbers D56430, D53682, R71408, T32121, HS14513, T32145, T35966, M8743, R71117, T32012, and Q60265. GenBank. May 30, 1995 see sequence alignments.
Wang et al., Proceeding of the National Academy of Sciences USA, 92:5510-5514, 1995.
Benjamin et al., Proceeding of the National Academy of Sciences USA, 87:6263-6267, 1990.
Brusca John S.
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
The Johns Hopkins University School of Medicine
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