Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2001-10-04
2003-05-20
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S018000, C536S023200
Reexamination Certificate
active
06566088
ABSTRACT:
INTRODUCTION
Field of the Invention
The field of this invention is prolyl-4-hyroxylase assays.
BACKGROUND OF THE INVENTION
The ability of cells to recognize and respond to a low oxygen environment (hypoxia) is critical for the execution of a number of physiological and pathophysiological conditions (1). Almost all mammalian cells express components of a hypoxia response pathway found to be conserved in both flies (2, 3) and worms (4). The hypoxia-inducible transcription factor (HIF), lies at the heart of this pathway. HIF is a heterodimer composed of two members of the basic-Helix-Loop-Helix (bHLH)-containing PER-ARNT-SIM (PAS) domain family; HIF-1&agr; (or the closely related HIF-2&agr;/EPAS-1 or HIF-3&agr; factors) and HIF-1&bgr;, also known as the aryl hydrocarbon receptor nuclear translocator (ARNT) (5). Under normoxic conditions HIF-1a is constitutively expressed. However, this subunit is rapidly targeted for proteosome-mediated degradation (6-8) via a protein-ubiquitin ligase complex containing the product of the von Hippel Lindau tumor suppressor protein (pVHL) (9-12). pVHL recognizes the oxygen degradation domain (ODD) of HIF-1&agr; only under normoxic conditions (13-15). Following exposure to a hypoxic environment, this degradation pathway is blocked, allowing HIF-1a accumulation and subsequent movement to the nucleus where it activates hypoxia-responsive genes (reviewed in 16).
Three groups have recently reported that pVHL recognizes the ODD via a conserved proline residue that is hydroxylated exclusively under normoxic conditions (13-15). Examination of cellular extracts prepared under normoxic conditions revealed the presence of a prolyl-4-hydroxylase activity capable of modifying a proline-containing peptide derived from the ODD (13-15). This activity was greatly diminished in extracts prepared under hypoxic conditions or in the presence of “hypoxia mimics” such as CoCl
2
or the iron chelator deferoxamine mesylate (13-15). As is the case for known prolyl-4-hydroxylases, this activity was enhanced by supplementation with Fe
2+
, ascorbate and 2-oxoglutarate (14, 15).
The best-characterized prolyl-4-hydroxylase enzymes modify collagen as it matures along its exocytotic pathway (17). These enzymes are typically composed of two a and two b subunits, with the alpha subunit responsible for the prolyl hydroxylase enzymatic activity. A number of lines of evidence rule out these enzymes as the HIF prolyl hydroxylase. First, the substrate context of the modified proline residues in collagen is different from that surrounding the relevant proline residue in HIF (13-15). Second, the collagen-modifying enzymes reside within the endoplasmic reticulum rather than the cytoplasm as expected for the HIF prolyl hydroxylase (17). Finally, two recombinant isoforms of the collagen modifying enzymes expressed from baculovirus were reported to show no activity against the HIF substrate (14).
We disclose here identification of an evolutionarily and structurally conserved family of HIF prolyl hydoxylase (HPH) enzymes. Inappropriate accumulation of HIF resulting from forced expression of the HIF-1a subunit under normoxic conditions was attenuated by co-expression of HPH. Suppression of the
Drosophila melanogaster
HPH enzyme via RNA interference resulted in elevated expression of a hypoxia-inducible gene, LDH, under normoxic conditions. These findings identify an essential component of the hypoxia-response pathway and provide HPH assays for identifying selective modulators of the HPH enzymes, which modulators provide useful leads for therapeutics capable of modulating HIF prolyl hydroxylation and downstream affects, such as angiogenesis.
SUMMARY OF THE INVENTION
The invention provides methods and compositions for assaying hypoxia-inducible factor (HIF) prolyl hydroxylation. In general, the subject assays comprise the steps of: a) incubating a mixture comprising an isolated or recombinantly expressed HIF-specific prolyl hydroxylase (HPH) selected from the group consisting of a human HPH 1, HPH2 and HPH3 prolyl hydroxylase domain, and a substrate of the hydroxylase, under conditions whereby the hydroxylase prolyl hydroxylates the substrate, and b) detecting a resultant prolyl hydroxylation of the substrate. Frequently, the mixture further comprises a candidate agent which modulates the resultant prolyl hydroxylation, wherein an agent-biased prolyl hydroxylation is detected.
In particular embodiments, the hydroxylase is selected from the group consisting of human HPH 1, HPH2 and HPH3, and/or the substrate comprises LAPY (SEQ ID NO:5, residues 7-10), wherein P is hydroxylated by the hydroxylase.
The assays may be cell-based, such as wherein the hydroxylase is recombinantly expressed in the cell and the detecting step comprises detecting a transcriptional reporter of HIF dependent gene expression. Alternatively, the assay may be run in vitro, wherein the hydroxylase is isolated, and the detecting step comprises detecting a reagent which selectively binds the prolyl hydroxylated substrate.
REFERENCES:
patent: 4797471 (1989-01-01), Teetz et al.
Atreya P. Interaction of Prolyl 4-Hydroxylase with Synthetic Peptide Substrates. J of Biological Chemistry 266(5)2852-8, 1991.*
Asada S. Effect of HSP47 on Prolyl 4-Hydroxylation of Collagen Model Peptides. Cell Structure and Function 24(4)187-196, Aug. 1999.*
Takahashi Y. Hypoxic Induction of Prolyl 4-Hydroxylase I in Cultured Cells. J of Biological Chemistry 275(19)14139-46, May 12, 2000.*
Nissi R. Prolyl 4-Hydroxylase Isoenzymes I and II Have Different Expression Patterns in Several Human Tissues. J of Histochem and Cytochem 49(9)1143-1153, Sep. 2001.*
Epstein A. C. elegans EGL-9 and Mammalian Homologs . . . Cell 107(1)43-54, Oct. 5, 2001.
Bruick Richard K.
McKnight Steven L.
Board of Regents , The University of Texas System
Gitomer Ralph
Osman Richard Aron
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