WO2007005504A2

WO2007005504A2 - Hif-1 modulator

Info

Publication number: WO2007005504A2
Application number: PCT/US2006/025329
Authority: WO
Inventors: Frank S. Lee
Original assignee: The Trustees Of The University Of Pennsylvania
Priority date: 2005-06-30
Filing date: 2006-06-28
Publication date: 2007-01-11
Also published as: WO2007005504A3

Abstract

Described herein are compounds and methods for modulating cellular response to oxygen. Specifically, provided herein is a IOP1 molecules that stimulate, accelerate, inhibit or abrogate cellular response to oxygen exposure. The present invention also provides molecules comprising siRNA for IOP1 and PHD2, or molecules comprising peptides of IOP1 or a fragment thereof, functioning in modulating cellular response to exposure to oxygen and methods utilizing same.

Description

HIF-I MODULATOR

CROSS REFERENCE TO RELATED APPLICATIONS

FIELD OF INVENTION

[0001] This invention is directed to compounds and methods for modulating cellular response to oxygen exposure. Specifically, the invention relates to IOP1 molecules that stimulate, accelerate, inhibit or abrogate cellular response to oxygen exposure. Also provided are molecules comprising siRNA for IOP1 and PHD2, or molecules comprising peptides of IOP1 or a fragment thereof, functioning in modulating cellular response to exposure to oxygen and methods utilizing same.

BACKGROUND OF THE INVENTION

[0002] Oxygen sensing is an important function of living cells and how cells sense changes in ambient oxygen is a central problem in biology. In mammalian cells, lack of oxygen, or hypoxia, leads to the stabilization of a sequence-specific DNA-binding transcription factor called HIF (hypoxia-inducible factor), which transcriptionally activates a variety of genes linked to processes such as angiogenesis and glucose metabolism. [0003] Moreover, tissue ischemia is a major cause of morbidity and mortality. Ischemia can result from chronic hypoxia brought on by lack of blood supply to the tissue occurring from, for example, stroke, deep vein thrombosis, pulmonary embolus, and renal failure. Ischemic tissue is also found in tumors. [0004] A fundamental response of mammalian cells is the capacity to respond to low oxygen tension, and is almost universally marked by alterations in gene expression. Under low-oxygen conditions (hypoxia), a cell must respond by coordinated expression of numerous genes to ensure adaptation. Hypoxia-inducible factor 1 (HIF-I), a transcription factor that accumulates during hypoxia, stimulates genes involved in glucose metabolism, angiogenesis, and cell survival.

[0005] The transcription factor HIF is a master regulator of the transcriptional response to hypoxia. HIF consists of a heterodimeric complex between α and β subunits, both of which are members of the PAS family of proteins. The β subunit is the aryl hydrocarbon nuclear translocator protein (ARNT), while the α subunit consists of a family of proteins, the prototype of which is HIF- lα.

^' [0006] The central means by which HIF is regulated, is through oxygen-dependent degradation of the α subunit, which in turn is mediated by site-specific prolyl hydroxylation of the same subunit. Under normoxic conditions, the prolyl hydroxylase PHD (also known as HIF prolyl hydroxylase and EGLN), hydroxylates two prolyl residues in the α subunit (Pro-564 and Pro-402 in the case of HIF-I α). The modified residues provide a specific recognition motif for the von Hippel Lindau tumor suppressor protein (VHL), the substrate recognition component of an E3 ubiquitin ligase complex. This complex then polyubiquinates HIF-lα, thereby targeting it for degradation for the ubiquitin-proteasome pathway. Hence, under normoxic conditions, the HIF-I α protein is maintained at very low levels. This

5 model readily provides a mechanism by which HIF- lα can be activated by hypoxia. Specifically, the PHDs are members of the 2-oxoglutarate dependent dioxygenase family of enzymes for which molecular oxygen is an obligatory substrate ( Schofield, C. J., and Ratcliffe, P. J. (2004). Oxygen sensing by HIF hydroxylases. Nat Rev MoI Cell Biol 5, 343-354). Thus, low oxygen tension constitutes low substrate concentration, and would be expected to lead to hypohydroxylation of HIF-I α o and hence its stabilization. Following stabilization, HIF-lα translocates to the nucleus, heterodimerizes with ARNT, and then upregulates the transcription of a broad array of genes involved in angiogenesis, erythropoiesis, and metabolic adaption to hypoxia.

[0007] Factors such as HIF- lα, which stimulate or accelerate wound healing will find a variety of uses but are of particular importance in the treatment of patients with chronic wounds requiring constant and s consistent treatment, represent a substantial source of pain, may lead to life threatening infection and are a significant medical expense.

SUMMARY OF THE INVENTION

0 [0008] In one embodiment, the invention provides a method of inhibiting a cellular response to hypoxic activity, comprising contacting said cell with an Iron-Only Hydrogenase Like Protein (IOP1) peptide, wherein said peptide is represented by the formula MASPFSGALQ LTDLDDFIGP SQECIKPVKV EKRAGSGVAK IRIEDDGSYF QINQDGGTRR LEKAKVSLND CLACSGCITS AETVLITQQS HEELKKVLDA NKMAAPSQQR LVVVSVSPQS RASLAARFQL NPTDTARKLT SFFKKIGVHF s > VFDTAFSRHF SLLESQREFV RRFRGQADCR QALPLLASAC PGWICYAEKT HGSFILPHIS

TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC

VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAYGF RNIQNLVQRL KRGRCPYHYV EVMACPSGCL NGGGQLQAPD RPSRELLQHV ERLYGMVRAE APEDAPGVQE 0 LYTHWLQGTD SECAGRLLHT QYHAVEKAST GLGIRW (SEQ ID NO.1 ).

[0009] In another embodiment, the invention provides a method of modulating degradation of HIF-lα in a cell, comprising contacting said cell with Iron-Only Hydrogenase Like Protein (IOP1) peptide, wherein the Iron-Only Hydrogenase Like Protein (IOP1) peptide causes downregulating HIF-lα expression. ₅ [00010] In one embodiment, the invention provides a method of regulating the oxidative state of iron in the active site of prolyl hydroxilase 2 (PHD2)comprising contacting a cell comprising said PHD2 with Iron-Only Hydrogenase Like Protein (IOP1), or a fragment thereof that is EKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAYGF RNIQNLVQRL KRGRCPYHYV EVMACPSGCL NGGGQLQAPD RPSRELLQHV ERLYGMVRAE APEDAPGVQE LYTHWLQGTD SECAGRLLHT QYHAVEKAST GLGIRW (SEQ ID NO.21), EKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAY (SEQ ID NO.22), IYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAY (SEQ ID NO.23), EEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAAR (SEQ ID NO.24), EEEGVSLPD LEPAPLDSLC SGASA (SEQ ID NO. 25) or EEEGVSLPD LEPAPLDSL (SEQ ID NO.26), thereby altering the oxidative state of the iron in the PHD2 active site.

[oooii] In another embodiment, the invention provides a method of treating an inherited von Hippel Lindau disease in a subject, comprising administrating to said subject an effective amount of IOP1, a homologue or a fragment thereof, thereby downregulating HIF- lα. [00012] In one embodiment, the invention provides a method of increasing tissue angiogenesis in a subject, comprising administrating to said subject an effective amount of an IOP1 fragment, a short interfering RNA (siRNA) of Iron-Only Hydrogenase Like Protein (IOP1), siRNA of prolyl hydroxylase 2 (PHD2) or a combination thereof, or a molecule comprising an IOP1 fragment comprising the PHD2 binding site, wherein said administration results in elevation of HIF-I α mRNA, hypohydroxylation of HIF- lα and its stabilization, or both, thereby upregulating angiogenesis-related genes.

[00013] In another embodiment, the invention provides a method of inhibiting angiogenesis in a tumor cell, comprising contacting said tumor cell with an effective amount of Iron-Only Hydrogenase Like Protein (IOP1), an IOP1 fragment, prolyl hydroxylase 2 (PHD2)or a combination thereof, wherein said contacting results in hydroxylation of HIF- lα in said tumor cell, thereby providing a specific recognition motif for the von Hippel Lindau tumor suppressor protein (VHL) and the substrate recognition component of an E3 ubiquitin ligase complex thereby reducing expression of angiogenesis- ^" related genes.

[00014] In one embodiment, the invention provides a method of treating a pathology resulting from hypoxia in a subject, comprising administrating to said subject an effective amount of an IOP1 fragment, a short interfering RNA (siRNA) of Iron-Only Hydrogenase Like Protein (IOP1), siRNA of prolyl hydroxylase 2 (PHD2) or a combination thereof, wherein said administration results in elevation of HIF -lα mRNA, hypohydroxylation of HIF- lα and its stabilization, or both thereby upregulating angiogenesis-related genes. [00015] In another embodiment, the invention provides a method of elevating HIF-lα mRNA levels,

5 HIF-lα protein levels, or both in a subject, comprising the step of knocking down expression of IOP1 in said subject, thereby resulting in of elevating HIF-lα mRNA levels, HEF-lα protein levels, or both. [00016] In one embodiment, the invention provides a method of treating Hypoxia-induced stroke in a subject, comprising administrating to said subject an effective amount of an IOP1 fragment, siRNA of IOP1, siRNA of PHD2 or a combination thereof. o [00017] In another embodiment, the invention provides a cell expressing, or co-expressing an Iron-Only Hydrogenase Like Protein (IOP1) peptide, wherein said peptide is represented by the formula MASPFSGALQ LTDLDDFIGP SQECIKPVKV EKRAGSGVAK IRIEDDGSYF QINQDGGTRR LEKAKVSLND CLACSGCITS AETVLITQQS HEELKKVLDA NKMAAPSQQR LVVVSVSPQS RASLAARFQL NPTDTARKLT SFFKKIGVHF VFDTAFSRHF SLLESQREFV RRFRGQADCR S QALPLLASAC PGWICYAEKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAYGF RNIQNLVQRL KRGRCPYHYV EVMACPSGCL NGGGQLQAPD RPSRELLQHV ERLYGMVRAE APEDAPGVQE LYTHWLQGTD SECAGRLLHT QYHAVEKAST GLGIRW 0 (SEQ ID NO.1).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows that IOP1 is Homologous to Bacterial Iron-Only Hydrogenases. Comparison of amino ₅ acid sequences of human IOP1, human IOP2; homologues from Drosophila melanogaster (Dm;

GenBank ace NP_652122), Caenorhabditis elegans (Ce; GenBank ace AAF60782), Saccharomyces cerevesiae (Sc; GenBank ace NP_014159); and iron-only hydrogenases from Desulfovibrio vulgaris (Dv; GenBank ace CAA40970) and Clostridium pasteurianum (Cp; GenBank ace AAA23248). Numbers indicate amino acid residues. Shaded residues are identical in at least five of the seven 0 ..sequences. Closed triangles denote cysteine residues that are ligands of the H-cluster in the bacterial iron-only hydrogenases. Open triangles denote cysteine residues that are ligands of the [4Fe-4S] cluster ¹ in the ferredoxin-like domain of bacterial iron-only hydrogenases. Overlining indicates IOP1 sequence (residues 282-299) that interacts with PHD2.

Figure 2 shows that IOP1 Protein Levels Are Not Regulated by the PHD2:VHL Pathway. (A) Twenty S ng of GST, GST-IOPl (417-476), or GST-IOP2 (382-456) were subjected to Western blotting using either anti-IOPl or anti-GST antibodies. The positions of molecular mass markers (in kDa) are shown to the right. (B) Extracts (10 μg) prepared from various human (HEK293, HeIa, 786-0, Jurkat), mouse (N2a), and monkey (COS-I) cell lines were examined by Western blotting using anti-IOPl (top) or anti-β-tubulin (bottom) antibodies. Also shown is an extract prepared from COS-I cells transiently transfected with a full length IOP1 expression vector (pcDNA3-IOPl). The positions of molecular mass markers (in kDa) are shown to the right. Asterisk denotes cross-reacting band. (C) HEK293 cells were subjected to hypoxia (0.2% O₂) for the indicated periods of time, extracts prepared, and then aliquots (10 μg) examined by western blotting using antibodies against IOP1, HIF- lα, or β-tubulin. (D) COS-I cells were cotransfected with expression vectors for HA-tagged IOP1, IOP2, or HIF- lα, and ones for either Flag-tagged PHD2 or VHL. The cells were lysed, and 10 μg of extracts then examined for expression levels of the tagged proteins using either anti-HA (top) or anti-Flag (bottom) antibodies. The positions of molecular mass markers (in kDa) are shown to the right. (E) A multiple tissue Northern blot was hybridized with a ³²P-labeled antisense probe to IOP1, stripped, and then reprobed with one for γ-actin. The sizes of the messages for IOP1 and γ-actin are 2.4 and 1.9 kb, respectively. (F) Extracts (12 μg) prepared from HEK293, Hep3B, RH28, RD, and SMS-CTR cell lines were examined by Western blotting using anti-IOPl antibodies. Several of these cell lines were also exposed to 0.2% O₂ for 3 hr, and extracts examined by Western blotting using anti-HIF-lα antibodies. The positions of molecular mass markers (in kDa) are shown to the right. (G and H), COS-I cells were cotransfected with expression vectors for HA-IOPl, FlagFIH, FlagPHD2, or FlagJNKl, and maintained under normoxia (G and H) or subjected to 0.2% O₂ (H), as indicated. Cellular lysates were then incubated in the absence or presence of DSP, and anti-Flag immunoprecipitates as well as lysates then examined by western blotting.

Figure 3 shows the mapping of PHD2 Interaction Site in IOP1. (A) Schematic diagram of IOP1, with shaded area denoting predicted [4Fe-4S] ferredoxin-like domain, and hatched area denoting domain with homology to iron-only hydrogenases. Numbers at the top and in the left column indicate amino acid residues. The ability of various IOP1 fragments to interact with PHD2 is indicated to the right. (B) In vivo coimmunoprecipitation studies. COS-I cells were cotransfected with an expression vector for FlagPHD2 and ones for the GAL4 DNA binding domain fused to the indicated IOP1 residues. The former was then immunoprecipitated using anti-Flag antibodies, and the immunoprecipitates (IP) then ^examined for the absence or presence of the IOP1 fusion proteins by Western blotting using anti-GAL4 antibodies. Aliquots of lysates were also examined by Western blotting (WB) using the same antibodies, or anti-Flag antibodies. (C) In vitro coimmunoprecipitation studies. S-labeled, in vitro translated GAL4-IOP1 (198-476), GAL4-IOP1 (198-368), or GAL4-IOP1 (238-368) was incubated in the absence or presence of 5 μg of recombinant (His)₆FlagPHD2. The latter was then immunoprecipitated with anti-Flag antibodies, and the immunoprecipitates examined for the absence or presence of the IOP fusion proteins by SDS-PAGE followed by autoradiography. In designates 10% of total input, and asterisks indicate in vitro translation products. (D) GST pulldown studies. ³⁵S- labeled, in vitro translated PHD2 was incubated with the indicated GST fusion proteins immobilized on GSH-agarose resin. The resins were then washed and examined for the absence or presence of PHD2 by SDS-PAGE followed by autoradiography. Input (10% of the total) is shown. Positions of molecular mass markers (in kDa) are shown to the right. Results shown in (B-D) are representative of two to four independent experiments.

Figure 4 shows the mapping of IOP 1 Interaction Site in PHD2. (A) Schematic diagram of PHD2, with shaded area denoting predicted MYND-type zinc finger, and hatched area denoting prolyl hydroxylase domain. Numbers at the top and in the left column indicate amino acid residues. The ability of various PHD2 fragments to interact with IOP1 (282-305) is indicated to the right. (B) In vitro translated, ³⁵S- labeled PHD2 (lanes 1-8, 12-14), PHDl (lanes 9-11), or PHD3 (lanes 15-17), or fragments of PHD2 (lanes 18-32), were assayed for binding to the indicated GST fusion proteins. Input (10% of total) is shown. In lanes 20 and 26, asterisks designate in vitro translation products. Results shown are representative of two to four independent experiments. (C) Comparison of MYND-type zinc finger domains from human PHD2 (residues 21 to 58), human MTG8, Drosophila melanogaster Nervy, Drosophila melanogaster Deaf, and Caenorhabditis elegans hypothetical protein R07E5.10. Shaded residues are identical in at least three of the five sequences. Asterisks denote cysteine or histidine residues that are predicted to be zinc ligands. Triangles denote residues mutated (Cys-36 and Cys-42) in the PHD2 fragment examined in lanes 30-32 of (B).

Figure 5 shows the regulation of HRE activity by IOP1. (A, B, and E) COS cells were cotransfected with an expression vector for HA-tagged proteins, and ones for short hairpin loops or the pShagl vector (V). Two days postransfection, cellular extracts examined by western blotting using anti-HA antibodies. (C, D, F, and H) The indicated cells were cotransfected with an (eHRE)₃-Luc reporter gene, pRL-TK, and short haiipin loop expression vectors encoding the indicated siRNAs. Two days later, cells were maintained under 21% O₂ (C, D, F, and H), or subjected to 0.2% O₂ (C, D, F, and H) or 1% O₂ (F) for 18 hr, and harvested. Firefly luciferase activities were normalized to that of Renilla luciferase. Insets of (F) and (H) show results for 21% O₂, with the columns corresponding to the first three columns of (F) or the first two columns in each panel of (H), and with a magnified Y-axis. Each experiment shows data performed in duplicate, and is representative of two to four independent

.experiments. (G) Northern analyses. Hep3B cells were transfected with the indicated siRNAs. Three days postransfection, cells were exposed to 0.2% O₂ for 8 hr and harvested. Poly A (+) RNA (0.2 μg) was subjected to agarose gel electrophoresis and Northern blotting then performed with the indicated probes. Figure 6 shows the regulation of HIF- lα protein level by IOP1. Hep3B (A-C) or HEK293 (D) cells were transfected with the indicated synthetic siRNAs. Seventy-two hrs postransfection, some cells were exposed to 0.2% or 1% O₂ for 3 hr (A, C, D), or for the indicated times (B). For a given experiment, equal quantities of cellular lysates were subjected to western blotting using the indicated antibodies. In the left half of (C), the top panel shows a single exposure of an anti-HIF-lα western blot. Additional exposures of each half are shown immediately beneath this panel. In the right half of (C), cells were exposed to 1% O₂ for 3 hr and then reoxygenated (Reox) in 21% O₂ for the indicated times.

Figure 7 shows that IOP1 modulates HIF hydroxylation._(A) HEK293 or 768-0 cells were left unstimulated or treated with 100 μM desferrioximine for 2 hr as indicated, and cellular lysates prepared. Hydroxylated HIF-2α was then isolated by first incubating with recombinant His-tagged VBC complex, then with anti-His antibodies, and subsequently with Protein G-agarose. Eluates from the immunoprecipitates or aliquots of lysates were then subjected to western blotting using anti-HIF-2α antibodies. Hyp-HIF-2α denotes hydroxylated HIF-2α. (B) 786-O cells were transfected with the indicated synthetic siRNAs. Four days postransfection, cell lysates were prepared and hydroxylated HIF-2α was then isolated from 150 μg of lysate as described in (A), except that the VBC complex was omitted from the first sample. Eluates from the immunoprecipitates, or aliquots of lysates (8 μg) were then subjected to western blotting using the indicated antibodies. This experiment was performed four times, and representative results from one experiment are shown. Figure 8 shows cellular localization of IOP1.JA) COS-I cells were cotransfected with expression vectors for HA-IOPl and FlagPHD2, and then fixed, permeablized, and stained with DAPI, or with anti-HA or anti-Flag antibodies. Confocal images of single colors and an overlay of colors (Merged) are shown. (B) Cytoplasmic and mitochondrial fractions (C and M, respectively) from HeIa, Hep3B, and HEK293 cells were prepared and examined by Western blotting using antibodies against IOP1, cytochrome C, or PHD2, as indicated. NS denotes nonspecific band. (C) SlOO cytoplasmic extract (20 μg) from HeIa S3 cells was examined by Western blotting using anti-IOPl antibodies. Also shown (left lane) is an extract prepared from COS-I cells transiently transfected with a full length IOP1 expression vector (pcDNA3-IOPl). The positions of molecular mass markers (in kDa) are shown to the right. NS denotes nonspecific band. Figure 9 shows the regulation of HIF-I α mRNA and HIF target genes by IOP1. Hep3B cells were transfected with siCONTROL Non-Targeting siRNA #1 (CON), IOP1-A siRNA (IOP1), or PHD2-A siRNA (PHD2). Seventy-two hrs postransfection, some cells were exposed to 1 % O₂ for 8 hr. RNA was extracted from the cells and reverse transcribed. Real-Time PCR was then performed using TaqMan probes for (A) HBF-I α, (B) HIF-2α, (C) PHDl, (D) PHD2, (E) PHD3, (F) IOP1, (G) Glutl, or (H) OS-9. RQ indicates relative quantification. * indicates P < 0.05, ** indicates P < 0.01, using the Student's t test DETAILED DESCRIPTION OF THE INVENTION

[00018] In one embodiment, IOP1 modulates HIF hydroxylation, in addition to the intrinsic, graded use by PHD of molecular oxygen as an obligatory substrate. Under normoxic condition in one embodiment and hypoxic condition in another embodiment, IOP1 knockdown upregulates HEF-lα, implying that IOP1 serves to downregulate HIF activity. In one embodiment, IOP1 is a component of an enlarging network of proteins regulating HIF stability, which include PHD, VHL, Siahla/2, ARDl, Jabl/CSN5, and OS-9. [00019] It is to be understood that the use of the term "modulates" is to refer to stimulating, enhancing, inhibiting or abrogating, as defined herein. Modulating HIF hydroxylation refers to HIF expression. In another embodiment, HIF 1-α activity is modulated, as described. In another embodiment, effects of HIF expression and/or activity are via HIF-lα ubiquitination and/or degradation. [00020] In another embodiment, the terms "normoxic" or "hypoxic" refer to the concentration of dissolved oxygen in the cell. Under normoxic condition, the partial pressure of oxygen in the inspired gas is equal to that of air at sea level, about 150 mm Hg (0.197 Atm or 20%). Under hypoxic condition, the partial pressure drops to 1-2%, or 0.01-0.02 Atm.

[00021] The central means by which HIF is regulated in one embodiment, is through oxygen-dependent degradation of the α subunit, which in turn is mediated by site-specific prolyl hydroxylation of this subunit. Under normoxic conditions and in another embodiment, the prolyl hydroxylase PHD (also known as HIF prolyl hydroxylase and EGLN) hydroxylates two prolyl residues in the α subunit (Pro- 564 and Pro-402 in the case of HIF-lα). The modified residues provide in one embodiment, a specific recognition motif for the von Hippel Lindau tumor suppressor protein (VHL) and the substrate recognition component of an E3 ubiquitin ligase complex in another embodiment. In one embodiment, this complex then polyubiquinates HIF- lα, thereby targeting it for degradation for the ubiquitin- proteasome pathway. Hence, under normoxic conditions, the HIF-lα protein is maintained at very low levels.

[00022] The initiation of cellular responses to fluctuations in the partial pressure and consequently the concentrations of dissolved oxygen can be rapid and involve transcriptional and posttranscriptional mechanisms. In one embodiment hypoxia stimulates reactive oxygen species (ROS) release from the mitochondria that in another embodiment regulates the transcriptional and posttranslational response to low-oxygen conditions. In one embodiment, hypoxia inhibits macromolecule synthesis in an oxygen- dependent manner to conserve energy utilization. [00023] Accordingly and in one embodiment, the invention provides a method of inhibiting a cellular response to hypoxic activity, comprising contacting said cell with IOP1 peptide, thereby downregulating HIF activity in the cell and modulating the cellular response to hypoxic activity. [00024] In one embodiment, such cellular responses are important clinically in the healing or repair of several critical target tissues and organs, such as bone, connective tissue, eye, heart, liver, skin, the vascular system, and the endocrine system. In another embodiment, the term " cellular response" refers to any genotypic, phenotypic, and/or morphologic change to a cell, cell line, tissue, tissue culture, organ or patient that is induced by an IOP1 peptide. Thus, in one embodiment, the present invention is directed to a method for potentiating a cellular response to an IOP1 peptide, comprising contacting a cell which expresses HIF-lα an effective amount of an IOP1 peptide to enhance the cellular response elicited by the IOP1 peptide, wherein the IOP1 peptide enhances the cellular response by downregulating HIF-lα expression

[00025] In another embodiment, the term "peptide", when in reference to any peptide of this invention, is meant to include native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. , Such modifications include, but are not limited to N terminal, C terminal or peptide bond modification, including, but not limited to, backbone modifications, and residue modification, each of which represents an additional embodiment of the invention. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).

[00026] It is to be understood that any amino acid sequence whether obtained naturally or synthetically, by any means, exhibiting sequence, structural, or functional homology to the peptides described herein are considered as part of this invention. [00027] In another embodiment, the invention provides a method of inhibiting a cellualr response to hypoxic activity, comprising contacting said cell with IOP1 peptide, wherein wherein the peptide comprises the amino acid sequence: MASPFSGALQ LTDLDDFIGP SQECIKPVKV EKRAGSGV AK IRIEDDGSYF QINQDGGTRR LEKAKVSLND CLACSGCITS AETVLITQQS HEELKKVLDA NKMAAPSQQR LVVVSVSPQS RASLAARFQL NPTDTARKLT SFFKKIGVHF VFDTAFSRHF SLLESQREFV RRFRGQADCR QALPLLASAC PGWICYAEKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT

YKPLRNKDFQ EVTLEKEGQV LLHFAMAYGF RNIQNLVQRL KRGRCPYHYV EVMACPSGCL NGGGQLQAPD RPSRELLQHV ERLYGMVRAE APEDAPGVQE LYTHWLQGTD SECAGRLLHT QYHAVEKAST GLGIRW (SEQ ID NO.L). [00028] The minimal IOP1 binding site in PHD2 resides in one embodiment, in an N-terminal domain encoding a MYND-type zinc finger, which in another embodiment, is implicated in protein-protein interactions, as opposed to protein-DNA interactions.

[00029] In one embodiment, IOP1 knockdown decreases HIF hydroxylation (Figure 7). In another embodiment, IOP1 might regulate the oxidation state of the active site iron in PHD2, which is subject to redox regulation, thereby identifying the active site iron as a regulatory locus in this enzyme. In another embodiment, IOP1, through its interaction with the N-terminal MYND zinc finger, allosterically regulates the catalytic activity of PHD2. In one embodiment, IOP1 promotes the assembly of iron into PHD2, analogous to the essential role of its yeast homologue Narlp in the assembly of cytosolic and nuclear iron-sulfur cluster proteins. [00030] In one embodiment, the term "IOP1 knockdown" refers to a genetically modified organism that carries one or more genes in its chromosomes that has been made less active or had its "expression" reduced or is the use of a reagent such as an antisense oligo to decrease expression of a specific gene, copying the effects of such a genetic modification in other embodiments. In another embodiment, the term "knockdown" refers to gene silencing using double stranded RNA, known as RNA interference (RNAi) in one embodiment, and the development of gene knockdown using morpholino oligos. In one embodiment, the term "knockdown" is used since the phenotype is rarely due to a complete loss of function.

[00031] In one embodiment, the term "morpholino oligos" refers to synthetic molecules which are the product of a radical redesign of natural nucleic acid structure that can block access of other molecules to small (-25 base) regions of ribonucleic acid (RNA). In one embodiment, Morpholinos, are about 25 bases in length, bind to complementary sequences of RNA by standard nucleic acid base-pairing. In another embodiment, Morpholinos do not degrade their target RNA molecules, but rather act by "steric blocking", binding to a target sequence within an RNA and simply getting in the way of molecules which might otherwise interact with the RNA. In one embodiment, morpholino oligos that are specifically capable of interacting with mRNA of IOP1 are used as the reagent to knckdown IOP1 expression.

[00032] In one embodiment, the terms "homologue", or "homologous", when used in reference to IOP1- derived peptides, indicates a percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions nor insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known in the art . In another embodiment, when in reference to polynucleotides encoding for siRNA derived peptides, similarly indicates a percentage of nucleotides in a candidate sequence that are identical with the nucleotides of a corresponding native nucleic acid sequence. [00033] Homology may be determined in the latter case by computer algorithm for sequence alignment, by methods well described in the art. For example, computer algorithm analysis of nucleic acid sequence homology may include the utilization of any number of software packages available, such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT and TREMBL packages.

[00034] An additional means of determining homology is via determination of candidate sequence hybridization, methods of which are well described in the art (See, for example, "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., Eds. (1985); Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, (Volumes 1-3) Cold Spring Harbor Press, N. Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. Y). For example methods of hybridization may be carried out under moderate to stringent conditions, to the complement of a DNA encoding a native IOP1 peptide. Hybridization conditions being, for example, overnight incubation at 42 ⁰C in a solution comprising: 10-20 % formamide, 5 X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7. 6), 5 X Denhardt's solution, 10 % dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.

[00035] As used herein, the terms "homology", "homologue" or "homologous", in any instance, indicate that the sequence referred to, whether an amino acid sequence, or a nucleic acid sequence, exhibits, in one embodiment at least 70 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 72 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 75 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 80 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 82 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 85 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 87 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 90 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 92 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 95 % or more correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 97% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 99 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits 95 % - 100 % correspondence with the indicated sequence. Similarly, as used herein, the reference to a correspondence to a particular sequence includes both direct correspondence, as well as homology to that sequence as herein defined.

[00036] Homology, as used herein, may refer to sequence identity, or may refer to structural identity, or functional identity. By using the term "homology" and other like forms, it is to be understood that any molecule, whether nucleic acid or peptide, that functions similarly, and/or contains sequence identity, and/or is conserved structurally so that it approximates the reference sequence, is to be considered as part of this invention.

[00037] Protein and/or peptide homology for any peptide sequence listed herein may be determined by immunoblot analysis, or via computer algorithm analysis of amino acid sequences, utilizing any of a number of software packages available, via methods well known to one skilled in the art. Some of these packages may include the FASTA, BLAST, MPsrch or Scanps packages, and may employ the use of the Smith and Waterman algorithms, and/or global/local or BLOCKS alignments for analysis, for example. [00038] According to this aspect of the invention and in one embodiment, the invention provides a method of inhibiting a cellular response to hypoxic activity, comprising contacting said cell with IOP1 peptide, and PHD2.

[00039] In one embodiment, the term "contacting a cell", refers to any exposure of a cell to a peptide, nucleic acid, or composition of this invention. Cells may be in direct contact with compounds and compositions of the invention, or exposed indirectly, through methods well described in the art. For example, cells grown in media in vitro, wherein the media is supplemented with any of the IOP1- derived peptides, IOP1 siRNA nucleic acids, compounds or compositions would be an example of a method of contacting a cell, considered a part of this invention. Another example would be oral or parenteral administration of a peptide, nucleic acid, compound or composition, whose administration results in vivo cellular exposure to these compounds, within specific sites within a body. Such administration is also considered as part of this invention, as part of what is meant by the phrase "contacting a cell".

[00040] Dosing is dependent on the cellular responsiveness to the administered IOPl-derived peptides, IOP1 siRNA nucleic acids, compounds or compositions comprising same. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates. [00041] In another embodiment, IOP1 functions under both normoxia and hypoxia, in contrast to bacterial iron-only hydrogenases, which function under anoxia. In one embodiment, the activity of IOP1 shows differences depending on the oxygen concentration. In one embodiment, the PHD-HIF pathway plays a central role in the cellular response to hypoxia and in another embodiment, is an attractive pharmacologic target, particularly with regard to ischemic and neoplastic diseases. In one embodiment, IOP1 provides other opportunities for therapeutic manipulation of this pathway. [00042] According to this aspect of the invention and in one embodiment, the invention provides a method of modulating degradation of HIFl-α in a cell, comprising contacting said cell with the peptide represented by the amino acide sequence of SEQ ID NO.s 1, 22, 23, 24, 25 or a combination thereof. In another embodiment, the cell is under normoxic condition, or in one embodiment, under hypoxic conditions.

[00043] In another embodiment, the invention provides a method of regulating the oxidative state of iron in the active site of PHD2 comprising contacting a cell comprising said PHD2 with IOP1, or a fragment thereof that is EKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAYGF RNIQNLVQRL KRGRCPYHYV EVMACPSGCL NGGGQLQAPD RPSRELLQHV ERLYGMVRAE APEDAPGVQE LYTHWLQGTD SECAGRLLHT QYHAVEKAST GLGIRW (SEQ ID NO.21), EKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAY (SEQ ID NO.22), IYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAY (SEQ ID NO.23), EEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAAR (SEQ ID NO.24), EEEGVSLPD LEPAPLDSLC SGASA (SEQ ID NO. 25) or EEEGVSLPD LEPAPLDSL (SEQ ID NO.26), thereby altering the oxidative state of the iron in the PHD2 active site.

[00044] The PHDs comprise a family of three related proteins, PHDl, PHD2, and PHD3. siRNA used in one embodiment, in a variety of mammalian cell lines implicate PHD2 as the primary isoform responsible for maintaining low HIF- lα levels under normoxia. In another embodiment, the mRNAs for PHD2 and PHD3 are themselves upregulated by hypoxia, and provide a mechanism for the poststimulation downregulation of HIF- lα activity. PHDs serve in one embodiment as in-vitro oxygen sensors. In another embodiment, recombinant PHD expressed as either an in vitro translated protein in reticulocyte lysates, or from E. coli shows a progressively increasing capacity to hydroxylate HIF- lα in response to differing oxygen concentrations. In one embodiment, kinetic measurements performed with insect cell extracts overexpressing PHD show a weak K_m for oxygen (230-250 μM, which is approximately six-fold weaker than that of the collagen prolyl hydroxylases). Thus, PHD performs in one embodiment, a graded hydroxylation reaction on HIF- lα in a manner dependent on molecular oxygen, directly coupling oxygen concentration in another embodiment, to an enzymatic modification. In another embodiment, other in-vitro oxygen sensing mechanisms exist, implicating a role for mitochondria in the HIF activation pathway. [00045] Tumor development is characterized in one embodiment by an initial phase of rapid expansion, followed by a period of slowed growth as the proliferating malignant cells outstrip the local supply of oxygen and nutrients. In another embodiment, in the absence of a dedicated blood supply, early-stage tumors attain steady-state volumes of a few cubic millimeters. To resume growth, these micro tumors adapt in one embodiment, to hypoxic stress through alterations in cellular metabolism and the stimulation of neovascularization, which provides the additional blood needed to sustain cellular proliferation. In another embodiment, cellular adaptation to growth during hypoxic stress contributes to malignant progression and is correlated with a poor clinical outcome in several types of cancer. In one embodiment, hypoxic adaptation increases rates of anaerobic glycolysis and the secretion of proangiogenic factors, such as in another embodiment, vascular endothelial growth factors (VEGFs). [00046] In one embodiment, hypoxia is an indicator of therapeutic response to radiotherapy, chemotherapy, and surgery. In another embodiment, it decreases the effectiveness of radiation that requires free radicals to kill cells, makes them refractory to killing by chemotherapy agents that require rapidly proliferating cells to be effective, and selects for tumor cells that are highly aggressive. [00047] In another embodiment, the invention provides a method of inhibiting a cellular response to hypoxic activity, comprising contacting said cell with IOP1 peptide, wherein the cell is a preneoplastic cell, an inflammatory cell or an infected cell.

[00048] In one embodiment, VHL disease refers to a cancer syndrome. VHL patients develop in another embodiment, cysts or tumors in the kidneys, retinas, brain, spinal cord, adrenals, pancreas, inner ear, or the scrotum. VHL syndrome is caused by germline mutations in the VHL tumour suppressor, and VHL tumours are associated with loss or mutation of the remaining wild-type allele. VHL is inactivated in 80% of sporadic clear cell renal carcinomas (RCC), the predominant form of kidney cancer. The ability of RCC cells to form tumours in one embodiment, is abrogated by introduction of wild-type VHL. In another embodiment, VHL-associated tumours are highly vascularized, indicating VHL negatively regulates the production of hypoxia-inducible factors such as in one embodiment, the angiogenic vascular endothelial growth factor (VEGF). VHL^'7" tumour cells have high levels of these factors, and reintroduction of VHL down-regulates them under normoxic conditions.

[00049] According to this aspect of the invention and in another embodiment, the invention provides a method of treating an inherited VHL disease in a subject, comprising administrating to said subject an effective amount of IOP1, a homologue or a fragment thereof.

[00050] In one embodiment, the fragment of IOP1 used in treating an inherited VHL disease in a subject, comprising administrating to said subject an effective amount of IOP1, a homologue or a fragment thereof, is EKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAYGF RNIQNLVQRL KRGRCPYHYV EVMACPSGCL NGGGQLQAPD RPSRELLQHV ERLYGMVRAE APEDAPGVQE LYTHWLQGTD SECAGRLLHT QYHAVEKAST GLGIRW (SEQ ID NO.21), EKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAY (SEQ ID NO.22), IYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAY (SEQ ID NO.23), EEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAAR (SEQ ID NO.24), EEEGVSLPD LEPAPLDSLC SGASA (SEQ ID NO. 25), EEEGVSLPD LEPAPLDSL (SEQ ID NO.26) or a combination thereof.

[00051] As will be appreciated by one skilled in the art, a fragment or derivative of a nucleic acid sequence or gene that encodes for a protein or peptide can still function in the same manner as the entire, wild type gene or sequence. Likewise, forms of nucleic acid sequences can have variations as compared to wild type sequences, nevertheless encoding a protein or peptide, or fragments thereof,, retaining wild type function exhibiting the same biological effect, despite these variations. Each of these represents a separate embodiment of this present.

[00052] The nucleic acids of the present invention can be produced by any synthetic or recombinant process such as is well known in the art. Nucleic acids according to the invention can further be modified to alter biophysical or biological properties by means of techniques known in the art. For example, the nucleic acid can be modified to increase its stability against nucleases (e.g., "end- capping"), or to modify its lipophilicity, solubility, or binding affinity to complementary sequences. [00053] DNA according to the invention can also be chemically synthesized by methods known in the art. For example, the DNA can be synthesized chemically from the four nucleotides in whole or in part by methods known in the art. Such methods include those described in Caruthers (1985). DNA can also be synthesized by preparing overlapping double-stranded oligonucleotides, filling in the gaps, and ligating the ends together (see, generally, Sambrook et al. (1989) and Glover et al. (1995)). DNA expressing functional homologs of the protein can be prepared from wild-type DNA by site-directed mutagenesis (see, for example, Zoller et al. (1982); Zoller (1983); and Zoller (1984); McPherson (1991)). The DNA obtained can be amplified by methods known in the ail. One suitable method is the polymerase chain reaction (PCR) method described in Saiki et al. (1988), Mullis et al., U.S. Pat. No.4,683,195, and Sambrook et al. (1989).

[00054] In one embodiment, the invention provides a method of increasing angiogenesis in a subject, comprising administrating to said subject an effective amount of siRNA of IOP1, siRNA of PHD2 or a combination thereof. [00055] In one embodiment, the term "siRNA" refers to RNA interference, which in one embodiment refers to the process of sequence-specific post-transcriptional gene silencing in animals, mediated by short interfering RNAs (siRNAs). In another embodiment, the process of post-transcriptional gene silencing is an evolutionarily-conserved cellular defense mechanism used to prevent the expression of

5 foreign genes. Such protection from foreign gene expression evolved one embodiment, in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or in another embodiment, from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA of viral genomic RNA. In one embodiment, the presence of dsRNA in cells triggers the RNAi response. Q [00056] By the term "conserved", amino acid sequences comprising the peptides of this invention remain in one embodiment, essentially unchanged throughout evolution, and exhibit homology among various species producing the protein

[00057] The presence of long dsRNAs in cells stimulates in another embodiment, the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in one embodiment, in the processing of s the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs). Short interfering RNAs derived from dicer activity are in another embodiment about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. Small RNAs function in one embodiment, by base-pairing to complementary RNA or DNA target sequences. When bound to RNA, small RNAs trigger RNA cleavage in another embodiment, or translational inhibition of the target sequence in another Q embodiment. When bound to DNA target sequences, small interfering RNAs mediate in one embodiment, DNA methylation of the target sequence. The consequence of these events, in one embodiment, is the inhibition of gene expression.

[00058] In one embodiment, the siRNA of IOP1 used in the method of increasing angiogenesis in a subject, comprising administrating to said subject an effective amount of siRNA of IOP1, siRNA of 5 PHD2 or a combination thereof , is represented by the sequences of SEQ ID NO.13 and SEQ ID NO.14, or SEQ ID NO.15 and SEQ ID NO.16, which are GACGGGAGCU ACUUCCAAAU U and UUU GGA AGU AGC UCC CGU CUU or GCA UCA AGC CUG UCA AAG UUU and ACU UUG ACA GGC UUG AUG CUU respectively. [00059] In another embodiment, the siRNA of IOP1 is derived from a short hairpin loop transcribed from Q a plasmid vector containing the following two oligonucleotides: 5'- TAT CTG TAG GAT TCA GCT GAA ACC GTG CGA AGC TTG GCA CGG TTT CAG CTG AAT CCT ACA GAT ACT GTT TTT T-3' (SEQ ID NO.5) and 5'- GAT CAA AAA ACA GTA TCT GTA GGA TTC AGC TGA AAC CGT GCC AAG CTT CGC ACG GTT TCA GCT GAA TCC TAC AGA TAC G-3' (SEQ ID NO.6). [00060] In another embodiment, the siRNA of PHD2 used in the method of increasing angiogenesis in a ₅ subject, comprising administrating to said subject an effective amount of siRNA of IOP1, siRNA of PHD2 or a combination thereof , is represented by the sequences described by SEQ ID NO 17 and SEQ ID NO.18, or SEQ ID NO. 19 and SEQ ID NO.20, which are CAA GGU AAG UGG AGG UAU AdTdT and UAU ACC UCC ACU UAC CUU GdTdT or UGU ACG UCA UGU UGA UAA UdTdT and AUU AUC AAC AUG ACG UAC AdTdT respectively. [00061] In one embodiment, provided herein, is a method of increasing tissue angiogenesis in a subject, comprising administrating to said subject an effective amount of an IOP1 fragment, a short interfering RNA (siRNA) of Iron-Only Hydrogenase Like Protein (IOP1), siRNA of prolyl hydroxylase 2 (PHD2) or a combination thereof, or a molecule comprising an IOP1 fragment comprising the PHD2 binding site, wherein said administration results in elevation of HIF -Ia mRNA, hypohydroxylation of HIF- lα and its stabilization, or both, thereby upregulating angiogenesis-related genes.

[00062] In one embodiment, the invention provides a method of inhibiting angiogenesis in a tumor cell, comprising contacting said tumor cell with an effective amount of IOP1, an IOP1 fragment, PHD2 or a combination thereof. [00063] In another embodiment, the invention provides a method of inhibiting angiogenesis in a tumor cell, comprising contacting said tumor cell with an effective amount of IOP1, an IOP1 fragment, PHD2 or a combination thereof, wherein the IOP1, is a fragment or a homologue thereof, or in another embodiment, the IOP1 comprises fragments of the peptide corresponding to the amino acid represented by SEQ ID NO.l, that are EKT HGSFHJPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAYGF RNIQNLVQRL KRGRCPYHYV EVMACPSGCL NGGGQLQAPD RPSRELLQHV ERLYGMVRAE APEDAPGVQE LYTHWLQGTD SECAGRLLHT QYHAVEKAST GLGIRW (SEQ ID NO.21), EKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHV AEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAY (SEQ ID NO.22), IYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAY (SEQ ID NO.23), EEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAAR (SEQ ID NO.24), EEEGVSLPD LEPAPLDSLC SGASA (SEQ ID NO. 25), EEEGVSLPD LEPAPLDSL (SEQ ID NO.26) or a combination thereof.

[00064] In one embodiment, the term "Ischemia" refers to a low oxygen state, due in another embodiment to obstruction of the arterial blood supply or in one embodiment, to an inadequate blood flow, leading to hypoxia in the tissue. In one embodiment, Ischemia refers to a condition where the oxygen-rich blood flow to a part of the body is restricted. Cardiac ischemia refers in another embodiment to lack of blood flow and oxygen to the heart muscle.

[00065] Cardiac ischemia occurs in one embodiment when an artery becomes narrowed or blocked for a short time, preventing oxygen-rich blood from reaching the heart. If ischemia is severe or lasts too long, in another embodiment, a heart attack (myocardial infarction) results, leading in one embodiment to heart tissue death. In another embodiment, a temporary blood shortage to the heart causes a pain in the angina pectoris. In one embodiment, there is no pain, wherein these cases are referred to as silent ischemia.

[00066] According to this aspect of the invention, and in one embodiment, the invention provides a method of treating ischemia in a subject, or in another embodiment, hypoxia induced stroke, comprising administrating to said subject an effective amount of siRNA of IOP1, siRNA of PHD2 or a combination thereof.

[00067] In another embodiment, the siRNA of IOP1 used for the method of treating ischemia in a subject, or in another embodimet, hypoxia induced stroke, comprising administrating to said subject an effective amount of siRNA of IOP1, siRNA of PHD2 or a combination thereof, is represented by the sequences of SEQ ID NO.13 and SEQ ID NO.14, or SEQ ID NO.15 and SEQ ID NO.16, which are GACGGGAGCU ACUUCCAAAU U and UUU GGA AGU AGC UCC CGU CUU or GCA UCA AGC CUG UCA AAG UUU and ACU UUG ACA GGC UUG AUG CUU respectively. [00068] In another embodiment, the siRNA of IOP1 is derived from a short hairpin loop transcribed from a plasmid vector containing the following two oligonucleotides: 5'- TAT CTG TAG GAT TCA GCT GAA ACC GTG CGA AGC TTG GCA CGG TTT CAG CTG AAT CCT ACA GAT ACT GTT TTT T-3' (SEQ ID NO.5) and 5'- GAT CAA AAA ACA GTA TCT GTA GGA TTC AGC TGA AAC CGT GCC AAG CTT CGC ACG GTT TCA GCT GAA TCC TAC AGA TAC G-3' (SEQ ID NO.6) [00069] In another embodiment, the siRNA of PHD2 used for the method of treating ischemia in a subject, or in another embodiment, hypoxia induced stroke, comprising administrating to said subject an effective amount of siRNA of IOP1, siRNA of PHD2 or a combination thereof, is represented by the sequences described by SEQ ID NO 17 and SEQ ID NO.18, or SEQ ID NO. 19 and SEQ ID NO.20, which are CAA GGU AAG UGG AGG UAU AdTdT and UAU ACC UCC ACU UAC CUU GdTdT or UGU ACG UCA UGU UGA UAA UdTdT and AUU AUC AAC AUG ACG UAC AdTdT respectively. [00070] In one embodiment, knockdown of IOP1 elevates HIF- lα mRNA levels and HIF- lα protein levels, and augments hypoxia-induced HIF target gene expression. In another embodiment, IOP1 serves to maintain HIF-lα mRNA levels at low steady state levels. The function of IOP1 cannot be substituted for by IOP2/Narf, which like IOP1, is homologous to the iron-only hydrogenases. The function of IOP2/Narf is binding to prelamin A in another embodiment, a component of the nuclear envelope. [00071] In contrast to green algal iron-only hydrogenases, IOP1 is not induced at either the mRNA or protein level by hypoxia (Fig. 2C and 9F). In one embodiment, the knockdown studies as shown in Example 2 herein, provide strong evidence that IOP1 is functional under both hypoxic in one embodiment or normoxic conditions in another embodiment, in contrast to bacterial iron-only hydrogenases, which are typically inactivated by exposure to oxygen. In one embodiment, IOP1 function displays a more subtle oxygen dependence.

[00072] In one embodiment, by western blotting (Fig. 6), reporter gene assays (Fig. 5), and real-time PCR analyses (Fig. 9), the functional effects of IOP1 knockdown consistently appear to be more significant under hypoxic than normoxic conditions. In one embodiment, IOP1 regulates HIF-lα transcription, or HIF-lα mRNA stability, or both in other embodiments. Mammalian cells poessess two iron-only hydrogenase homologues, IOP1 and IOP2, whereas yeast possesses one, Narlp. In one embodiment, IOP1 is a hydrogenase homologue of Narlp. In another embodiment, IOP1, IOP2, or both, constitute functional homologues of Narlp in the assembly of cytoplasmic iron-sulfur clusters. [00073] In one embodiment, the effects of IOP1 on HIF- lα levels are mediated in a manner dependent or independent of the cytoplasmic iron-sulfur cluster assembly pathway.

[00074] In one embodiment, provided herein, is a method of treating a pathology resulting from hypoxia in a subject, comprising administrating to said subject an effective amount of an IOP1 fragment, a short interfering RNA (siRNA) of Iron-Only Hydrogenase Like Protein (IOP1), siRNA of prolyl hydroxylase 2 (PHD2) or a combination thereof, wherein said administration results in elevation of HIF -Ia mRNA, hypohydroxylation of HIF- lα and its stabilization, or both thereby upregulating angiogenesis-related genes.

[00075] In one embodiment, the invention provides a method of elevating HIF-lα mRNA levels, HIF- lα protein levels, or both in a subject, comprising the step of knocking down expression of IOP1 in said subject, thereby resulting in of elevating HIF-lα mRNA levels, HIF-lα protein levels, or both [00076] In one embodiment, the invention provides a method of increasing HIF-lα mRNA levels, or HIF- lα protein levels, or both in other embodiments, comprising the step of knocking down expression of IOP1 in said subject. In one embodiment, the IOP1 knockdown as described herein, results in augmenting hypoxia-induced HIF target gene expression. In another embodiment, the IOP1 knockdown results in a about a 2 to about 3 fold increase in HIF- lα mRNA levels under normoxia conditions, or between about 3 to about 4 fold increase in HIF- lα mRNA levels under hypoxia conditions. In one embodiment, the subject is under normoxia or hypoxia condition. In one embodiment, the IOP1 knockdown results in a 2.3 fold increase in HIF-lα mRNA levels under normoxia conditions, or 3.1 fold increase in HIF- lα mRNA levels under hypoxia conditions (see Figure 9). [00077] In one embodiment, the hypoxia-induced HIF target gene whose expression is augmented as the result of IOP1 knockdown according to the methods described herein, is Glutl, or PHD2, PHD3, or a combination thereof in other embodiments.

[00078] In one embodiment, siRNA for IOP1, or in another embodiment Morpholino oligos for IOP1 mRNA, or both are used to knockdown the expression of IOP1 in the subject. In one embodiment, the siRNA of IOP1 is derived from a short hairpin loop transcribed from a plasmid vector. In another embodiment, the short hairpin loop is transcribed from a plasmid vector containing an oligonucleotide represented by the sequence: 5'- TAT CTG TAG GAT TCA GCT GAA ACC GTG CGA AGC TTG GCA CGG TTT CAG CTG AAT CCT ACA GAT ACT GTT TTT T-3' (SEQ ID NO.5), or by the sequence: 5'- GAT CAA AAA ACA GTA TCT GTA GGA TTC AGC TGA AAC CGT GCC AAG CTT CGC ACG GTT TCA GCT GAA TCC TAC AGA TAC G-3' (SEQ ID NO.6), or both in another embodiment.

[00079] Hypoxia-induced stroke refers in one embodiment, to inadequate delivery of oxygen to the brain, and ischemia results from insufficient cerebral blood flow (CBF). Normal CBF is 50-55 ml/lOOgm/min. If the CBF drops below 18, electrical activity ceases. If the CBF dips below 12, neuronal metabolism stops. The consequences of cerebral ischemia depend in another embodiment, on the degree and duration of reduced CBF. In one embodiment, neurons tolerate ischemia for 30-60 minutes. Perfusion must be reestablished before 3-6 hours of ischemia have elapsed or before the CBF drops to 10. In one embodiment, the term the "ischemic penumbra" refers to the time the range of CBF is between 12 and 18, because the neuronal damage is mild and reversible if flow is restored within a few hours. Clinically, a window of opportunity is available to intervene therapeutic and to prevent the ischemic brain tissue from going on to infarction.

[00080] In one embodiment cytotoxic edema results from failure of the Na/K ion pump. In another embodiment, this stage is reversible. Prolonged ischemia leads in one embodiment, to cell death and coagulation necrosis. After 3-6 hours of ischemia, irreversible damage occurs to the capillary endothelium. The blood-brain barrier becomes dysfunctional and serum proteins and water leak into the interstitial space. In one embodiment, reperfusion is required to produce vasogenic edema. In another embodiment, vasogenic edema is maximal when residual CBF is between 5 and 10. In one embodiment, there is an influx of macrophages to resorb nonviable tissue. Capillary proliferation begins in another embodiment, near the end of the first week. The end result of cerebral infarction in one embodiment, is an area of encephalomalacia with some surrounding gliosis. The amount of gliosis depends on the number of surviving astrocytes.

[00081] A "conditionally inducible element" is an element of the expression vector that confers positive regulation on transcription of a downstream expressed region under inducing conditions. It may be obtained from enhancer regions that are also conditionally inducible, but constitutively active enhancers that increase basal transcription under most or all conditions are not preferred sources for conditionally inducible elements.

[00082] A "transcription factor" is a protein that specifically binds a cognate sequence found in conditionally inducible elements. Binding of a positively-acting transcription factor to its cognate site in a conditionally inducible element will increase expression; binding of a negatively-acting transcription factor to its cognate site in a silencer element will decrease expression. Such increases or decreases can be measured relative to the presence or absence of the transcription factor, or the presence or absence of an element in the expressed vector, under controlled reaction conditions. The presence or activity of the transcription factor may be dependent on the type of host cell or organism or the conditions under which that host is kept. [00083] In one embodiment, provided herein is a cell expressing, or co-expressing an Iron-Only Hydrogenase Like Protein (IOP1) peptide, wherein said peptide is represented by the formula MASPFSGALQ LTDLDDFIGP SQECIKPVKV EKRAGSGVAK IRIEDDGSYF QINQDGGTRR LEKAKVSLND CLACSGCITS AETVLITQQS HEELKKVLDA NKMAAPSQQR LVVVSVSPQS RASLAARFQL NPTDTARKLT SFFKKIGVHF VFDTAFSRHF SLLESQREFV RRFRGQADCR QALPLLASAC PGWICYAEKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAYGF RNIQNLVQRL KRGRCPYHYV EVMACPSGCL NGGGQLQAPD RPSRELLQHV ERLYGMVRAE APEDAPGVQE LYTHWLQGTD SECAGRLLHT QYHAVEKAST GLGIRW (SEQ ID NO.1).

[00084] The term "about" as used herein means in quantitative terms plus or minus 5%, or in another embodiment plus or minus 10%, or in another embodiment plus or minus 15%, or in another embodiment plus or minus 20%. [00085] The term "subject" refers in one embodiment to a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae. The subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice and humans. The term "subject" does not exclude an individual that is normal in all respects.

[00086] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention

EXAMPLES

Experimental Procedures

Yeast Two-Hybrid Screen [00087] The yeast two-hybrid screen ( Fields, S., and Song, O. (1989). A novel genetic system to detect protein-protein interactions. Nature 340, 245-246) was performed with a Matchmaker GAL4 Two-Hybrid System 3 kit (BD Biosciences). The bait vector, pGBKT7-PHD2 R383A was constructed from pGBKT7 and pcDNA3-FlagPHD2 R383A ( Huang, J., Zhao, Q., Mooney, S. M., and Lee, F. S. (2002). Sequence determinants in hypoxia-inducible factor-1 alpha for hydroxylation by the prolyl hydroxylases PHDl, PHD2, and PHD3. J Biol Chem 277, 39792-39800). S. cerevesiae strain AH109 transformed with pGBKT7-PHD2 R383A was mated with S. cerevesiae strain Y187 pretransformed with either human adult kidney or brain Matchmaker Libraries (BD Biosciences). Following mating, positives were selected on -His/-Leu/-Trp/+ X-α-Gal media and subjected to two additional rounds of selection on -Ade/-His/-Leu/-Trp/+ X-α-Gal media. A total of 3.4 x 10⁶ clones were screened (2.6 x 10⁶ from the kidney library; 0.8 x 10⁶ from the brain library).

Plasmids

[00088] pGEX-IOPl was constructed by subcloning the 1.8 kb EcoR I/Sma I fragment of IMAGE clone 4660895 (ATCC) into the EcoR I/Sma I site of pGEX-5X-l. Results from 5'-RACE performed on HeIa cell total RNA are consistent with the first ATG within this clone being the physiologic initiator codon. pcDNA3-IOPl and pcDNA3 -HA-IOPl were constructed by subcloning the 1.8 kb BamH I/Xho I fragment of pGEX-IOPl into the BamH I/Xho I sites of pcDNA3 and pcDNA3-HA, respectively. pcDNA3-GAL4-IOPl and pGEX-IOPl fusion protein vectors were constructed by subcloning restriction enzyme fragments of positive clone K22 (residues 198-476) into pcDNA3-GAL4 or pGEX- 5X- 1, respectively. The restriction enzymes employed (and the residues in IOP1 that correspond to the restriction enzyme sites) were BgI II (residue 237-238), Ear I (residue 281), Pst I (residues 305-306), Sac I (residues 329-332), BsiW I (residues 368-369) and Sac I (residues 414-417). pGEX-IOPl (282- 299) was constructed by subcloning an oligonucleotide duplex comprised of the following sequences into the Bam HI/Xho I site of pGEX-5X-l : 5'-GAT CCA AAG CTT CCATGGAGAGGAAGAGGGCGTAAGCTTGCCAGATCTCGAGCCAGCCCCTCTAGACAGCCT CTAGACTAGTCTCGAGTAA-3' (SEQ ID NO.l) and 5'- TCG ATT ACT CGA GAC TAG TCT AGA GGC TGT CTA GAG GGG CTG GCT CGA GAT CTG GCA AGC TTA CGCCCTCTTCCTCTCCATGGAAGCTTTG-3' (SEQ ID NO.2). [00089] pENTR-IOP2 was constructed by first subcloning the 1.1 kb EcoR I/Xho I fragment of IMAGE clone 3447710 (ATCC) into the EcoR I/Xho I site of pENTR3C. Then into the BamH I(blunt)/Xma I site of the product was subcloned the 0.6 kb Sfo I/Xma I fragment of IMAGE clone 2820621, yielding pENTR-IOP2. pDEST-HA-IOP2 was prepared by GATEWAY LR Clonase reactions (Invitrogen) using pENTR-IOP2 as the entry vector and pDEST-HA as the destination vector. pGEX-IOP2 (264- 287) was constructed by subcloning an oligonucleotide duplex comprised of the following sequences into the Bam HI/Xho I site of pGEX-5X-l: 5'- GAT CCA AGA GCA AGG TGA CCT CTC AGT GAG AGATGCTGCAGTCGACACTCTGTTTGGAGACTTGAAGGAGGACAAAGTGTAGC-S' (SEQ ID NO.3) and 5'- TCG AGC TAC ACT TTG TCC TCC TTC AAG TCT CCA AAC AGA GTG TCG ACT GCA GCA TCT CTC ACT GAG AGG TCA CCT TGC TCT TG-3' (SEQ ID NO.4). [00090] pcDNA3-FlagPHD2 (1-195) was constructed by subcloning the 0.6 kb Hind Ill/Xho I fragment of pcDNA3-FlagPHD2 (Huang et al., 2002) into the Hind III/Xho I site of pcDNA3-Flag. pcDNA3- FlagPHD2 (196-426) was constructed by subcloning the 0.7 kb Xho I/Xba I fragment of pcDNA3- FlagPHD2 into the Xho I/Xba I site of pcDNA3-Flag. pcDNA3-PHD2 (1-124) was constructed by digesting pcDNA3-FlagPHD2 (1-195) with Not I, followed by self-ligation. pcDNA3-FlagPHD2 (130- 194) was constructed by first digesting pcDNA3-FlagPHD2 (1-195) with Xho I/Xba I followed by treatment with Klenow and self-ligation; then digesting the product with AfI II/Not I followed by treatment with Klenow and self-ligation. pcDNA3-FlagPHD2 (1-63) was constructed by standard recombinant DNA methods. pcDNA3-FlagPHD2 (1-195) C36S/C42S was constructed by PCR. pcDNA3-FlagFIH was constructed by subcloning the FIH coding sequence from IMAGE clone 4138066 (ATCC) into pcDNA3-Flag. The sources of all other plasmids have been described (Huang et al., 2002; Lee et al., 1998; Yu et al., 2001a; Yu et al., 2001b).

Antibodies

[00091] The IOP1 (417-476) coding sequence was subcloned into pMAL-c2X (New England Biolabs) or pGEX-5X-l (Amersham Biosciences), and subsequently employed for purification of the corresponding MBP or GST fusion proteins, respectively, from E. coli using affinity chromatography. Polyclonal antibodies to MBP-IOPl (417-476) were raised in rabbits and then affinity purified on GST- IOP1 (417-476) coupled to agarose (Alpha Diagnostic International, Inc). Additional antibodies included anti-HA (F-7, Santa Cruz Biotechnology), anti-Flag (M2, Sigma), anti-GST (Z-5, Santa Cruz), anti-HIF-lα (Clone 54, BD Biosciences), anti-PHD2 (NB-137, Novus Biologicals), anti-cytochrome C (7HB, Santa Cruz), anti-His (H-15, Santa Cruz), anti-HIF-2α (190b, Santa Cruz), anti-α-tubulin (B-7, Santa Cruz), anti-β-tubulin (D-10, Santa Cruz), anti-IκBα (C-21, Santa Cruz), anti-JNKl (FL, Santa Cruz), and anti-GAL4 (RK5C1, Santa Cruz).

Northern blotting and Real-Time PCR

[00092] pBS-IOPl (359 - 714) was constructed by subcloning the 0.4 kb Xba I/Bgl II fragment of pcDNA3-HA-IOPl containing the indicated nucleotides of the IOP1 cDNA into the Xba I/BamH I site of pBS-SK. A radiolabeled antisense RNA probe for IOP1 was prepared from linearized pBS-IOPl (359 - 714) with T7 RNA polymerase and 50 μCi of [α-³²P]ATP using a Strip-EZ RNA probe synthesis kit (Ambion). The probe was then hybridized to a Multiple Tissue Northern Blot membrane (BD Biosciences) for 18 h at 65 ⁰C, washed with low and high stringency wash solutions (Ambion Northern Max-Gly kit), exposed for autoradiography, stripped, and then reprobed with a radiolabeled antisense RNA probe for γ-actin (Tu et al., 2001). A probe for PHD2 was prepared by subcloning the 0.7 kb Xho I/Xba I fragment of pcDNA3-FlagPHD2 (Huang et al., 2002) into the Xho I/Xba I site of pBS KS. Probes for Glutl and PGK (gifts of Drs. Celeste Simon and Cheng- Jun Hu, University of Pennsylvania School of Medicine) were subcloned into pBS KS. For these probes, radiolabeled antisense probes were prepared using a Maxiscript T7 kit (Ambion). Poly A (+) RNA was isolated from cells using an Oligotex Direct kit (Qiagen). For Real-Time PCR, total RNA was harvested from cells using Trizol reagent (Invitrogen). Reverse transcription reactions were performed using TaqMan Reverse Transcription Reagents (ABI). Real-Time PCR reactions were performed on 20 ng equivalents of cDNA using an ABI 7300 Real-Time PCR machine and TaqMan probes (ABI). Relative quantification was performed employing the ΔΔQ method and 18S RNA as the endogenous control.

In Vitro Binding Assays [00093] Five μl of ³⁵S-labelled, in vitro translated protein (prepared using pcDNA3-FlagPHDl, pcDNA3-FlagPHD2, pcDNA3-FlagPHD3, or the appropriate pcDNA3-FlagPHD2 fragment or pcDNA3-GAL4 fusion protein template, and a TnTQuick T7 reticulocyte lysate kit) was incubated with GST fusion protein (typically 20 μg) immobilized on 10 μl of GSH-agarose or 5 μg of (His)₆Flag- PHD2 purified from baculovirus-infected insect cells (Huang et al., 2002) in 500 μl of buffer A (20 mM Hepes, pH 7.9, 100 mM KCl, 1 mM DTT) supplemented with 0.2% NP-40 and 0.2% BSA with rocking for 1 hr at 4 ⁰C. In the latter case, the (His)₆Flag-PHD2 was then immunoprecipitated by incubation with 10 μl of M2-agarose (Sigma) for 1 hr at 4 ⁰C. The resins were washed three times with buffer A supplemented with 0.2% NP-40, eluted with 2x SDS-PAGE loading buffer followed by heating at 100 ⁰C for 3 min, and the eluates then subjected to SDS-PAGE followed by autoradiography.

siRNA

[00094] Short hairpin loop vectors were prepared in pShagl (Paddison et al., 2002). Shag-IOPl-7, which targets bp 376-404 of the IOP1 coding sequence, was constructed by subcloning into the BseR I/Bam HI site of pShagl a duplex consisting of the following two oligonucleotides: 5'- TAT CTG TAG GAT TCA GCT GAA ACC GTG CGA AGC TTG GCA CGG TTT CAG CTG AAT CCT ACA GAT ACT GTT TTT T-3' (SEQ ID NO.5) and 5^!- GAT CAA AAA ACA GTA TCT GTA GGA TTC AGC TGA AAC CGT GCC AAG CTT CGC ACG GTT TCA GCT GAA TCC TAC AGA TAC G-3' (SEQ ID NO.6). pShag-mIOPl-7 was constructed using the following two oligonucleotides: 5'- TGT CTG TGG GGT_CCA GQC GAA ACC TGG CGA AGC TTG GCC AGG TTT CGG CTG GAC CCC ACA GAC ACT GTT TTT T-3' (SEQ ID NO.7, relevant mismatches to IOP1-7 sequence are highlighted) and 5'- GAT CAA AAA ACA GTG TCT GTG GGG TCC AGC CGA AAC CTG GCC AAG CTT CGC CAG GTT TCG GCT GGA CCC CAC AGA CAC G-3' (SEQ ID NO.8). pShag- IOP2-4, which targets bp 751-779 of the IOP2 coding sequence, was constructed in an analogous manner with the following two oligonucleotides: 5'- CAA TTT CAC CTG ATG TTA ACA CGC AGT CGA AGC TTG GAC TGC GTG TTA ACA TCA GGT GAA ATT CCT CTT TTT T-3' (SEQ ID NO.9) and 5'- GAT CAA AAA AGA GGA ATT TCA CCT GAT GTT AAC ACG CAG TCC AAG CTT CGA CTG CGT GTT AAC ATC AGG TGA AAT TGC G-3¹ (SEQ ID NO.10). BLAST searches using the siRNA sequences fail to reveal any evidence of off-site targets. The IOP1 nucleotide sequences targeted by the other short hairpin loop vectors were as follows: 1, 1175-1203; 2, 557-585; 3, 710-738; 4, 793-821; 5, 995-1023; 6, 1088-1115; 8, 1032-1060; 9, 65-87; 10, 281-303. The IOP2 nucleotide sequences targeted by the other short hairpin loop vectors were as follows: 1, 1115-1143; 2, 501-529; 3, 662-690; 5, 905-933; 6, 1041-1069. pShag-PHD2 was constructed based on published PHD2 siRNA sequence information (Berra et al., 2003) using the following two oligonucleotides: 5'- ATA ACA AGC AAC CAT GGC TTT CGT CCG GGA AGC TTG CCG GAC GAA AGC CAT GGT TGC TTG TTA TCC GTT TTT T-3' (SEQ ID NO.l1) and 5'- GAT CAAAAAACG GAT AAC AAG CAA CCA TGG CTT TCG TCC GGC AAG CTT CCC GGA CGA AAG CCA TGG TTG CTT GTT ATCG-3'(SEQIDN0.12).

[00095] Synthetic double stranded siRNA was synthesized by Dharmacon. The sequences for synthetic siRNA duplexes were as follows. IOP1-A: GAC GGG AGC UAC UUC CAA AUU (SEQ ID NO.13) and UUU GGA AGU AGC UCC CGU CUU (SEQ ID NO.14). IOP1-B: GCA UCA AGC CUG UCA AAG UUU (SEQ ID NO.15) and ACU UUG ACA GGC UUG AUG CUU (SEQ ID NO.16). PHD2- A: CAA GGU AAG UGG AGG UAU AdTdT (SEQ ID NO.17) and UAU ACC UCC ACU UAC CUU GdTdT (SEQ ID NO.18). PHD2-B: UGU ACG UCA UGU UGA UAA UdTdT (SEQ ID NO.19) and AUU AUC AAC AUG ACG UAC AdTdT (SEQ ID NO.20). Control-A: Non-specific Control Duplex IX (Dharmacon Cat # D001206-09-05). Control-B: siCONTROL Non-Targeting siRNA #1 (Dharmacon Cat # D-001210-01-05).

Cell Culture, Transfection, Immunoprecipitations, and Immunofluorescence

[00096] HeIa, COS, 786-0 and Hep3B cells were obtained from ATCC. RH28, RD, and SMS-CTR cells were gifts of Dr. Frederick Barr (University of Pennsylvania School of Medicine). The source of N2a and HEK293 cells has been described (Zhao and Lee, 2003). Cells were grown in Dulbecco's

Modified Eagle's Medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. For normoxic conditions, cells were maintained in a humidified 5% CO₂ tissue culture incubator. For experiments involving 1% O₂, cells were either placed in either a Billups- Rothenberg modular incubator or an In Vivo 200 hypoxia workstation (Ruskinn Technology) perfused with 1% O₂/5% CO₂/balance N₂. For other experiments, cells were placed in an In Vivo 200 hypoxia workstation perfused with 10% H₂/5% CO₂/balance N₂ in the presence of a palladium catalyst. In this case, measurements using gas chromatography reveal an oxygen concentration of 0.2%. [00097] Cells were typically seeded in 6-well (for coimmunoprecipitation studies), 12- well (for synthetic siRNA studies) or 24-well (for luciferase assays) plates, or in 10 cm dishes (for Northern analyses) and transfected using FuGENEό (plasmid DNA) or Lipofectamine2000 (synthetic siRNA). For siRNA transfections, the final siRNA concentration was 20 nM. For coimmunoprecipitations, cells were harvested 24 hr postransfection and were performed as described (Yu et al., 2001a), except that EDTA was omitted from the coimmunoprecipitation buffer. Crosslinking studies were performed as described (Kim et al., 2002) with the following modifications. The buffer employed was 40 mM Hepes, pH 7.5, 120 mM NaCl, 10 mM sodium pyrophosphate, 10 mM β-glyecerolphosphate, 1.5 mM Na₃VO₄, 1% Triton X-100, 10 μg/ml leupeptin and 1 mM PMSF. DSP was added to a final concentration of 1 mg/ml within 10 sec after lysis. The lysates were incubated for 30 min at 20 ⁰C, then the crosslinking quenched by the addition of Tris, pH 7.4, to a final concentration of 75 mM for 30 min at 20 ⁰C. For cells grown under hypoxic conditions, the lysis buffer was degassed prior to use, and crosslinking performed under hypoxic conditions. Following immunoprecipitation using 20 μl anti-Flag (M2)- agarose, crosslinks were reversed by the addition of SDS loading buffer containing 0.5 M DTT followed by incubation at 37 ⁰C for 1 hr. [00098] For Northern analyses, cells were transfected on two consecutive days with siRNA and harvested three days after the initial transfection. For immunoprecipitation of hydroxylated HIF, cells were transfected on two consecutive days with siRNA and harvested four days after the initial transfection. Cell lysates were prepared as described (Yu et al., 2001a), except that DTT was omitted. Equal quantities of lysates were incubated first with 0.1 μg of recombinant (His)₆- FlagVHL:ElonginB:ElonginC (VBC) complex (Huang et al., 2002), then with 10 μl of anti-His antibodies, and subsequently with 10 μl of Protein G- Agarose (Invitrogen), each for 1 hr at 4 ⁰C. Immunoprecipitates were washed and subjected to western blotting with anti-HIF-2α antibodies. [00099] Cytoplasmic and mitochondrial fractions were prepared using a Mitochondrial Fractionation Kit (Active Motif). SlOO cytoplasmic fractions were also prepared by the method of Dignam et al. (Dignam et al., 1983). Confocal immunofluorescence microscopy was performed as previously described (Huang et al., 2002).

Luciferase Assays and Western Blotting

[000100] For luciferase assays employing short hairpin loops, cells were stimulated 48 hr postransfection and then harvested 18 hr later. Typically, 100 ng of pShag vector, 50 ng of pRL-TK (internal transfection control), and 25 to 100 ng of (eHRE)₃-Luc were transfected. Luciferase activities were measured using a Dual-Luciferase Reporter Assay System (Promega) and a Wallac LB9507 luminometer. For synthetic siRNA transfections, media was replaced 24 hr postransfection and cells were stimulated 72 hr postransfection. For western blotting following synthetic siRNA treatment, cellular lysates were prepared as described (Yu et al., 2001a) except that DTT was omitted. Protein concentrations of extracts were determined using a BioRad DC protein assay kit. For a given experiment, equal quantities of cellular lysates (typically 10 μg) were subjected to western blotting.

Gas analysis

[000101] Hydrogen and certain oxygen measurements were performed on an Agilent 2890A micro gas chromatograph equipped with a MolSieve 5A column and a thermal conductivity detector, and argon as the carrier gas. Samples were prepared in either 25 cm² tissue culture flasks sealed with

Teflon-coated septa held in place with screw thread closures, or in 20 mm diameter headspace vials fitted with Teflon-coated septa held in place with crimp seals. Gases were analyzed directly from the head space of flasks containing transiently transfected mammalian cells, or headspace vials containing extracts prepared under hypoxic conditions and supplemented with 20 mM sodium dithionate and 5 mM methyl viologen as the electron donor. Cultures of Desulfovibrio desulfurans (ATCC # 7757) cultured in modified Baar's media were employed as a positive control for hydrogen production. To assay for hydrogen-dependent reducing activity, cell extracts were measured for their capacity to reduce

5 mM methyl viologen in the presence of 10% hydrogen (90% nitrogen). Using these methods, we have thus far been unable to demonstrate hydrogenase activity using recombinant IOP1 or IOP2 expressed from transiently transfected mammalian cells, under either normoxic or hypoxic conditions.

RESULTS

Example 1. IQPl is a Novel Hydrogenase-like Protein that Modulates Hypoxia Inducible Factor

Activity

[000102] In this example, a novel protein, IOP1, with ancestral origins in anaerobic bacteria is described. This protein interacts with the N-terminal, noncatalytic domain of PHD2. Knockdown of this protein in mammalian cells dysregulates HIF- lα and HIF- lα dependent transcription, indicating that IOP1 is part of the network of proteins that allows cells to respond appropriately to oxygen concentration in the cell.

[000103] The yeast two-hybrid approach using catalytically inactive (R383A) PHD2 was employed as a bait to screen human adult brain and kidney cDNA libraries. From the former, two independent clones of HIF- lα (residues 357-826 and 395-826) were obtained, both of which contain the oxygen- dependent degradation domain shown to bind to PHD. From the latter library, two independent partial clones (residues 171-476 and 198-476) of a novel protein corresponding to Unigene Hs.513247 (Figure 1) were isolated. This protein is homologous to the iron-only hydrogenases, a family of oxygen-labile proteins found in anaerobic organisms that catalyzes the reversible reduction of protons to molecular hydrogen gas, a reaction critical for anaerobic metabolism. Within the hydrogenase domain, it is 36% and 30% identical with iron-only hydrogenases from Desulfovibrio vulgaris and Clostridium pasteurianum, respectively (P < 10^~18 and < 10^"14, respectively). In addition, this protein is 47% identical in this domain to Narf, a human protein found to bind prelamin A. [000104] In one embodiment, the factor was named Iron-Only Hydrogenase-like Protein 1 (IOP1), and propose renaming Narf as IOP2. Bacterial iron-only hydrogenases contain a distinctive active site iron-sulfur cluster, termed the H-cluster and an additional iron-sulfur cluster present in a ferredoxin-like domain. The H-cluster contains six iron atoms, four of which are present as a [4Fe-4S] cubane subcluster that is bridged to a [2Fe] subcluster. The eight cysteine residues involved in chelating the H- cluster and the ferredoxin Fe cluster in bacterial hydrogenases are conserved in IOP1 and IOP2, as well as in homologues from other species (Figure 1).

[000105] Antibodies that react with IOP1 but not IOP2. (Figure 2A) were prepared. Western blotting using these antibodies reveals the presence of a 55 kDa protein in a variety of cell lines that comigrates with that obtained from COS-I cells overexpressing IOP1 (Figure 2B). Hypoxic exposure of HEK293 cells, which leads to elevated HIF-lα protein levels, failed to reveal any appreciable change in that of IOP1 (Figure 2C). Consistent with this, coexpression in COS-I cells of either PHD2 or VHL with IOP1, or for that matter IOP2, did not diminish the expression level of either IOP (Figure 2D, top panel, lanes 1-3 and 7-9), in contrast to the level of coexpressed HIF-lα, which is markedly decreased by both (top panel, lanes 4-6). Northern blotting reveals an IOP1 message of 2.4 kb with a wide tissue distribution, particularly high in heart and skeletal muscle (Figure 2E). The distribution is similar to that seen for PHD2 on Northern analysis of human tissues, and is similar to that of IOP2/Narf as well. Western blotting of extracts from a number of skeletal muscle cell lines, including RH28, RD, and SMS-CTR (Figure 2F), reveals higher levels of expression of IOP1 protein than in Hep3B cells, and comparable to that in HEK293 cells, which in turn display relatively high levels of IOP1 protein (Figure 2B). These muscle cell lines display robust induction of HIF-lα upon exposure to hypoxia (Figure 2F).

[000106] IOP1 was coexpressed with PHD2 in COS-I cells, cellular extracts were prepared in the presence of the crosslinking reagent dithiobis-sulfosuccinimidylpropionate (DSP), and then the PHD2 was immunoprecipitated. As shown in Figure 2G (top panel, lane 9), IOP1 can be crosslinked to PHD2. In the absence of DSP, coimmunoprecipitation is not observed (lane 4), indicating that the interaction is either weak or transient. The crosslinking is observed under both normoxic and hypoxic conditions (Figure 2H, top panel, lanes 4 and 8). As negative controls, IOP1 is not present in either anti-Factor Inhibiting HIF (FIH) or anti-JNKl immunoprecipitates, regardless of whether or not DSP is present (Figure 2G). [000107] Studies were conducted to examine the intracellular localization of IOP1. In one series of experiments, HA-IOPl and Flag-PHD2 were coexpressed in COS-I cells. As shown in Figure 8A, overexpressed HA-IOPl yields both a cytoplasmic and punctate appearance, with a substantial portion colocalizing with Flag-PHD2, which is predominantly cytoplasmic. Whether the punctate staining of HA-IOPl reflects overexpression-associated aggregation, or a specific subcellular localization will require further investigation. Cytoplasmic and mitochondrial fractions were also prepared from HeIa, Hep3B, and HEK293 cells and these fractions were examined for the presence of endogenous proteins. Western blotting of these extracts provides additional evidence that both IOP1 and PHD2 are largely cytoplasmic proteins (Figure 8B, lanes 1, 3, and 5). Relatively small amounts of each appear to be present in the mitochondrial fraction (lanes 2, 4, and 6). IOP1 is also readily detected in HeIa cell SlOO cytoplasmic extracts (Figure 8C). The predominantly cytoplasmic localization of IOP1 contrasts with that of IOP2/Narf, which is nuclear.

[000108] PHD2 binding site in IOP1 (Figure 3A) was mapped. Initially, various IOP1 fragments were coexpressed with PHD2 in COS cells, PHD2 was immunoprecipitated, and then immunoprecipitates were examined for the absence or presence of IOP1. The IOP1 fragment originally identified in the yeast two-hybrid screen, IOP1 (198-476), binds to PHD2 (Figure 3B, top panel, lane 6), as do smaller IOP1 fragments comprising residues 198-368 and 238-368 (top panel, lanes 12 and 18, respectively). Separately, in vitro translated versions of the same three fragments (IOP1 (198-476), IOP1 (198-368) and IOP1 (238-368 ) were found to also bind to recombinant, baculovirus-expressed PHD2 (Figure 3C, lanes 3, 6, and 9). GST pulldown assays reveal that in vitro translated PHD2 binds to IOP1 (282-329) (Figure 3D, lane 4) as well as to progressively smaller segments of IOP1 comprising residues 282-305 and 282-299 (lanes 10 and 11, respectively), the latter of which consists of only eighteen amino acids. The specificity of the IOP1 :PHD2 interaction is supported by the fact that the latter does not bind to the comparable amino acid sequence of IOP2 (residues 264-287) (Figure 3D, lane 12). [000109] IOP1 binding site in PHD2 was likewise mapped, using GST pulldown assays (Figure 4A). IOP1 (282-305) is shown to bind the N-terminal, noncatalytic domain of PHD2 (residues 1-195) but not the C-terminal catalytic domain (residues 196-426) (Figure 4B, compare lanes 4 and 8). This is in contrast to HIF-l α (531-575), which binds the latter but not the former (lanes 7 and 3, respectively). The noncatalytic domain of PHD2 is not conserved in either PHDl or PHD3, and in contrast to PHD2 (lane 14), neither binds IOP1 (282-305) (lanes 11 and 17). The IOP1 (282-305) binding site resides in PHD2 residues 1-124 (lane 20) and more specifically, residues 1-63 (lane 26), a domain predicted to contain a MYND-type zinc finger (Figure 4C). Mutation of two PHD2 cysteine residues predicted to be zinc ligands, Cys-36 and Cys-42, abolishes the interaction (Figure 4B, compare lanes 29 and 32), indicating, that the integrity of such a zinc finger is essential for this interaction. [000110] IOP1 is not regulated at the protein level in a HIF-like manner by the PHD2:VHL pathway (Figure 2C, D) and interacts with non-catalytic domain of PHD2 (Figure 4B), collectively suggesting that IOP1 may not be a downstream target of PHD2. To examine the converse possibility, namely that IOP1 might regulate PHD2 and hence HIF activity, IOP1 was initially overexpressed, however, no consistent effects on HIF-dependent reporter gene activity were found. siRNA approaches wrere then employed, to knock down IOP1. A series of short hairpin loop-derived siRNAs were first screened, for their capacity to reduced coexpressed IOP1 protein levels, and one that was particularly potent (designated IOP1-7, Figure 5A) was identified. A comparably effective siRNA was found for IOP2 (IOP2-4, Figure 5A). The specificity of these siRNA vectors is supported by the fact that they do not affect the levels of coexpressed VHL, JNKl, nor that of the other IOP isoform (Figure 5A, B). HeIa cells were transfected next with these constructs and a hypoxia response element (HRE) reporter gene, and as shown in Figure 5C, the IOP1-7 siRNA was found to significantly enhance hypoxia- induced activation of an HRE reporter gene (sixth column). IOP2-4 siRNA or a mutant version of IOP1-7 siRNA (mIOPl-7), which differs at six nucleotides compared to wild type, failed to produce this effect (Figure 5C, eighth and seventh columns, respectively). The mutant siRNA is, as expected, defective in silencing human IOP1. The effect of the IOP1 siRNA was also seen with HEK293 and Hep3B cells subjected to hypoxia (Figure 5D, left and right panels, respectively).

[000111] siRNA to PHD2 increases HIFl-α protein levels in many mammalian cell lines, thereby implicating PHD2 as the isoform that maintains HIF-I α at low levels under normoxia. A siRNA vector directed against PHD2 that was effective in decreasing coexpressed PHD2 protein levels was prepared (Figure 5E). In Hep3B cells, this siRNA augmented HRE reporter gene activity under normoxia (Figure 5F, inset). Importantly, the effects of IOP1 siRNA was found to be comparable to that of PHD2 siRNA under both moderate (1 % 02) and severe (0.2% O₂) hypoxia (Figure 5F), as well as under normoxia (Figure 5F, inset). The relatively modest effects seen under normoxic conditions, are assumed to be due to inhibition of the transcriptional activity of HIF- lα by Factor Inhibiting HIF (FIH). IOP1 is expressed at readily detectable levels in a number of skeletal muscle cell lines (Figure 2F), and the effects of IOP1 knockdown on HRE reporter gene activity in these cell lines were found to be comparable to that seen in Hep3B cells (Figure 5H).

[000U2] HIF target gene expression were examined by Northern blotting. Synthetic siRNA and a sequence that targeted an IOP1 sequence were used, distinct from that targeted by the IOP1-7 short hairpin loop just described. As shown in Figure 5G, knockdown of either IOP1 or PHD2 increases Glut 1 and PGK message. IOP1 and PHD2 Northern blots confirm knockdown, and the Northern for PHD2 also provides evidence that IOP1 knockdown augments PHD2 message expression (second panel from bottom), validating PHD2 gene as a HIF target.

[000113] The possibility that IOP1 siRNA-induced potentiation of HRE reporter gene activity might be due to increased HIF protein levels was examined. Two different synthetic IOP1 siRNAs were employed in this analysis. As negative controls, two nonspecific siRNAs were used, and as a positive control, PHD2 siRNA were used. As shown in Figure 6A, both IOP1 siRNAs were effective in decreasing IOP1 protein levels in Hep3B cells (left, second panel from top, lanes 2 and 4), just as a PHD2 siRNA was in decreasing PHD2 protein levels (left, third panel from top, lane 5). These results confirm the authenticity of the 55 kDa band recognized by the anti-IOPl antibodies. Importantly, under conditions in which PHD2 knockdown increases hypoxia-induced HIF-I α levels (lane 5), IOP1 knockdown was found to augment it as well (lanes 2 and 4). These effects are also seen under normoxia (Figure 6A, middle), conditions under which siRNA to PHD2 augments HIF- lα levels (lane 5) as seen previously (Appelhoff et al., 2004; Berra et al., 2003). As additional controls, IOP1 knockdown does not affect the protein levels of either IκBα or JNKl (Figure 6A, right). [000114] The effect of IOP1 knockdown on HIF- lα levels is seen throughout the time-dependent induction of HIF-lα in response to 0.2% O₂ (Figure 6B, left to right). This augmentation is also seen under milder hypoxic conditions, 1% O₂ (Figure 6C, left), conditions under which comparable effects are seen with PHD2 knockdown. Cells treated with siRNA, were subjected to 1% O₂, and then reoxygenated them, resulting in the rapid degradation of HIF-I α (Figure 6C, two sets of panels on the right), thus indicating as unlikely, the possibility that IOP1 knockdown simply abolishes the HIF- lα degradation pathway. The effects of both IOP1 and PHD2 siRNA are found to be similar in HEK293 cells, under normoxia as well as both modest (1% O₂) and severe (0.2% O₂) hypoxia (Figure 6D). Modest increases in PHD2 protein level upon IOP1 knockdown is noted in both cell types, as well as Hep3B cells (for example, compare lanes 1 and 2, third panel from top, Figure 6A), consistent with the Northern blot analysis (Figure 5G), indicating that PHD2 is a HIF gene target.

[000115] This approach was extended to VHL-deficient 786-0 cells, which are predicted to express significant quantities of hydroxylated HIF. This was verified experimentally by incubating 786-0 extracts with recombinant VHL, immunoprecipitating the latter, and then examining the immunoprecipitates for the absence or presence of HIF-2α (the HIF isoform selectively expressed in these cells. As shown in Figure 7 A (top panel, lane 3), HIF-2α is readily detectable in the VHL immunoprecipitates. To confirm that this represents hydroxylated HIF-2α, the same experiment were performed using non-hydroxylated HIF-2α prepared by treating Hep3B cells with desferrioximine (bottom panel, lane 2). In contrast to the hydroxylated HIF-2α obtained from 786-0 cells, this non- hydroxylated HIF-2α does not coimmunoprecipitate with VHL (top panel, lane 2). [000116] IOP1 or PHD2 in 786-0 cells were then knocked down, and the same approach was used to determine the amount of hydroxylated HIF-2α in these cells. As shown in Figure 7B, siRNA to PHD2 is found to reduce the levels of both total and hydroxylated HIF-2α (top panel, compare lanes 2 and 4). Significantly, knockdown of IOP1 produces essentially the same result (top panel, lane 3). Results indicate that IOP1 modulates levels of HIF hydroxylation, most likely through PHD2 regulation. The origin of the residual hydroxylation seen upon IOP1 or PHD2 knockdown is assumed to be a reflection of hydroxylation by other PHD isoforms.

Example 2. IQPl regulates HIF-lα mRNA levels

[000117] While IOP1 acts directly on PHD2, the data prompts the consideration of the possibility that IOP1 regulates other aspects of HIF- lα, such as mRNA levels. To test this, Hep3B cells were treated with IOP1, PHD2, or control siRNA, subjected some cells to hypoxia, isolated total RNA from these cells, and then performed Real-Time PCR to examine mRNA levels of select genes. Examination of the results for Glutl and PHD3, for example, confirms the known hypoxia-inducibility of these genes (Fig. 9G and E, respectively, compare first and fourth columns). Moreover, examination of the results for IOP1 and PHD2 establishes that messages for both are substantially diminished by their respective siRNA treatments (to levels of 7% and 9%, respectively, under normoxia, and 7% and 23% under hypoxia, respectively; Fig. 9F and D). [000118] Importantly, IOP1 knockdown was found to significantly upregulate HIF-lα message under both normoxia and hypoxia (2.3-fold and 3.1-fold over the control siRNA, respectively; Fig. 9A). This effect is selective because it is not seen with HIF-2α, which is not significantly affected or even diminished by either IOP1 or PHD2 knockdown (Fig. 9B). OS-9 message is not affected either, indicating that the effects of IOP1 knockdown on HIF-lα protein levels are not mediated through changes in OS-9 (Fig. 9FH). PHDl message provides an additional negative control (Fig. 9C). Conversely, both IOP1 and PHD2 knockdown augment hypoxia-induced induction of the known HIF target gene Glutl (Fig. 9G). Moreover, IOP1 knockdown augments hypoxia induction of PHD2 and PHD3 message (Fig. 9D and E, respectively), both of which are known HIF target genes. It is concluded therefore, that augmentation of HIF- lα message provides a mechanism by which IOP1 knockdown increases HIF-lα activity.

[000119] The foregoing has been a description of certain non-limiting preferred embodiments of the invention. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

Claims

What is claimed is:

1. A method of inhibiting a cellular response to hypoxic activity, comprising contacting said cell with an Iron-Only Hydrogenase Like Protein (IOP1) peptide.

2. The method of claim 1, wherein said peptide comprises the amino acid sequence: MASPFSGALQ

LTDLDDFIGP SQECIKPVKV EKRAGSGVAK IRIEDDGSYF QINQDGGTRR LEKAKVSLND CLACSGCITS AETVLITQQS HEELKKVLDA NKMAAPSQQR LVVVSVSPQS RASLAARFQL NPTDTARKLT SFFKKIGVHF VFDTAFSRHF

SLLESQREFV RRFRGQADCR QALPLLASAC PGWICYAEKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAYGF RNIQNLVQRL KRGRCPYHYV EVMACPSGCL NGGGQLQAPD RPSRELLQHV ERLYGMVRAE APEDAPGVQE LYTHWLQGTD SECAGRLLHT QYHAVEKAST GLGIRW (SEQ ID NO.l).

3. The method of claim 1 , comprising contacting the cell with prolyl hydroxylase 2 (PHD2).

4. A method of modulating degradation of HIF- lα in a cell, comprising contacting said cell with Iron-Only Hydrogenase Like Protein (IOP1) peptide, wherein the Iron-Only Hydrogenase Like Protein (IOP1) peptide causes downregulating HIF- lα expression.

5. The method of claim 1, wherein said peptide comprises an amino acid sequence corresponding to, homologous to or a fragment of SEQ ID NO. 1.

6. The method of claim 1, wherein said peptide comprises fragments of the peptide corresponding to the amino acid represented by SEQ ID NO.l, that are EKT HGSFDLPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC

VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL

EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAYGF

RNIQNLVQRL KRGRCPYHYV EVMACPSGCL NGGGQLQAPD RPSRELLQHV

ERLYGMVRAE APEDAPGVQE LYTHWLQGTD SECAGRLLHT QYHAVEKAST GLGIRW (SEQ ID NO.21), EKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAY (SEQ ID NO.22), IYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC s SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ

EVTLEKEGQV LLHFAMAY (SEQ ID NO.23), EEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAAR (SEQ ID NO.24), EEEGVSLPD LEPAPLDSLC SGASA (SEQ ID NO. 25) or EEEGVSLPD LEPAPLDSL (SEQ ID NO.26).

o

7. A method of regulating the oxidative state of iron in the active site of prolyl hydroxilase 2 (PHD2)comprising contacting a cell comprising said PHD2 with Iron-Only Hydrogenase Like Protein (IOP1), or a fragment thereof that is EKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL s EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAYGF

RNIQNLVQRL KRGRCPYHYV EVMACPSGCL NGGGQLQAPD RPSRELLQHV ERLYGMVRAE APEDAPGVQE LYTHWLQGTD SECAGRLLHT QYHAVEKAST GLGIRW (SEQ ID NO.21), EKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD 0 LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT

YKPLRNKDFQ EVTLEKEGQV LLHFAMAY (SEQ ID NO.22), IYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAY (SEQ ID NO.23), EEEGVSLPD LEPAPLDSLC SGASAEEPTS 5 HRGGGSGGYL EHVFRHAAR (SEQ ID NO.24), EEEGVSLPD LEPAPLDSLC SGASA (SEQ

ID NO. 25) or EEEGVSLPD LEPAPLDSL (SEQ ID NO.26), thereby altering the oxidative state of the iron in the PHD2 active siteprolyl hydroxilase 2 (PHD2).

8. The method of claim 7, wherein said fragment is represented by the amino acid formula: 0 EEEGVSLPD LEPAPLDSL (SEQ ID NO.26), its homologue or combination thereof.

9. The method of claim 4, wherein said cell is under normoxic conditions.

10. The method of claim 4, wherein said cell is under hypoxic conditions.

5 11. The method of claim 1, wherein said cell is a preneoplastic cell, an inflammatory cell or an infected cell.

12. A method of treating an inherited von Hippel Lindau disease in a subject, comprising administrating to said subject an effective amount of IOP1, a homologue or a fragment thereof,

5 thereby downregulating HIF- lα.

13. The method of claim 12, wherein said fragment is EKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL

I₀ EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAYGF

RNIQNLVQRL KRGRCPYHYV EVMACPSGCL NGGGQLQAPD RPSRELLQHV ERLYGMVRAE APEDAPGVQE LYTHWLQGTD SECAGRLLHT QYHAVEKAST GLGIRW (SEQ ID NO.21), EKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD is LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT

YKPLRNKDFQ EVTLEKEGQV LLHFAMAY (SEQ ID NO.22), IYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAY (SEQ ID NO.23), EEEGVSLPD LEPAPLDSLC SGASAEEPTS

20 HRGGGSGGYL EHVFRHAAR (SEQ ID NO.24), EEEGVSLPD LEPAPLDSLC SGASA (SEQ

ID NO. 25) or EEEGVSLPD LEPAPLDSL (SEQ ID NO.26).

14. A method of increasing tissue angiogenesis in a subject, comprising administrating to said subject an effective amount of an IOP1 fragment, a short interfering RNA (siRNA) of Iron-Only

25 Hydrogenase Like Protein (IOP1), siRNA of prolyl hydroxylase 2 (PHD2) or a combination thereof, or a molecule comprising an IOP1 fragment comprising the PHD2 binding site, wherein said administration results in elevation of HIF -lα mRNA, hypohydroxylation of HIF-I α and its stabilization, or both, thereby upregulating angiogenesis-related genes.

30 15. The method of claim 14, wherein said IOP1 fragment comprises a PHD2 binding site.

16. The method of claim 14, wherein said siRNA of IOP1 is represented by the sequences of SEQ ID NO.13 and SEQ ID NO.14, or SEQ ID NO.15 and SEQ ID NO.16.

35 17. The method of claim 14, wherein said siRNA of PHD2 is represented by the sequence described by SEQ ID NO 17 and SEQ ID NO.18, or SEQ ID NO. 19 and SEQ ID NO.20.

18. The method of claim 14, wherein said siRNA of IOP1 is represented by a nucleotide sequence: GACGGGAGCU ACUUCCAAAU U and UUU GGA AGU AGC UCC CGU CUU or GCA s UCA AGC CUGUCAAAGUUU andACU UUGACA GGC UUG AUG CUU.

19. The method of claim 14, wherein said siRNA of IOP1 is derived from a short hairpin loop transcribed from a plasmid vector comprising: 5'- TAT CTG TAG GAT TCA GCT GAA ACC GTG CGA AGC TTG GCA CGG TTT CAG CTG AAT CCT ACA GAT ACT GTT TTT T-3' o (SEQ ID NO.5); and 5'- GAT CAA AAA ACA GTA TCT GTA GGA TTC AGC TGA AAC CGT GCC AAG CTT CGC ACG GTT TCA GCT GAA TCC TAC AGA TAC G-3' (SEQ ID NO.6)

20. The method of claim 14, wherein said siRNA of PHD2 is represented by a nucleotide sequence: s CAA GGU AAG UGG AGG UAU AdTdT and UAU ACC UCC ACU UAC CUU GdTdT or

UGU ACG UCA UGU UGA UAA UdTdT and AUU AUC AAC AUG ACG UAC AdTdT.

21. A method of inhibiting angiogenesis in a tumor cell, comprising contacting said tumor cell with an effective amount of Iron-Only Hydrogenase Like Protein (IOP1), an IOP1 fragment, prolyl 0 hydroxylase 2 (PHD2)or a combination thereof, wherein said contacting results in hydroxylation of HIF- lα in said tumor cell, thereby providing a specific recognition motif for the von Hippel Lindau tumor suppressor protein (VHL) and the substrate recognition component of an E3 ubiquitin ligase complex thereby reducing expression of angiogenesis-related genes.

5 22. The method of claim 21, wherein said IOP1, is a fragment or a homologue thereof.

23. The method of claim 22, wherein said IOP1 comprises fragments of the peptide corresponding to the amino acid represented by SEQ ID NO.l, that are EKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC 0 VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL

EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAYGF RNIQNLVQRL KRGRCPYHYV EVMACPSGCL NGGGQLQAPD RPSRELLQHV ERLYGMVRAE APEDAPGVQE LYTHWLQGTD SECAGRLLHT QYHAVEKAST GLGIRW (SEQ ID NO.21), EKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH 5 VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLc SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT

YKPLRNKDFQ EVTLEKEGQV LLHFAMAY (SEQ ID NO.22), IYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ s EVTLEKEGQV LLHFAMAY (SEQ ID NO.23), EEEGVSLPD LEPAPLDSLC SGASAEEPTS

HRGGGSGGYL EHVFRHAAR (SEQ ID NO.24), EEEGVSLPD LEPAPLDSLC SGASA (SEQ ID NO. 25), EEEGVSLPD LEPAPLDSL (SEQ ID NO.26) or a combination thereof.

24. A method of treating a pathology resulting from hypoxia in a subject, comprising administrating to o said subject an effective amount of an IOP1 fragment, a short interfering RNA (siRNA) of Iron-

Only Hydrogenase Like Protein (IOP1), siRNA of prolyl hydroxylase 2 (PHD2) or a combination thereof, wherein said administration results in elevation of HIF -lα mRNA, hypohydroxylation of HIF- lα and its stabilization, or both, thereby upregulating angiogenesis-related genes.

s 25. The method of claim 24, wherein said IOP1 fragment comprises a PHD2 binding site.

26. The method of claim 24, wherein said siRNA of IOP1 is represented by a nucleotide sequence: GACGGGAGCU ACUUCCAAAU U and UUU GGA AGU AGC UCC CGU CUU or GCA UCA AGC CUG UCA AAG UUU and ACU UUG ACA GGC UUG AUG CUU. 0

27. The method of claim 24, wherein said siRNA of IOP1 is derived from a short hairpin loop transcribed from a plasmid vector containing the following two oligonucleotides: 5'- TAT CTG TAG GAT TCA GCT GAA ACC GTG CGA AGC TTG GCA CGG TTT CAG CTG AAT CCT ACA GAT ACT GTT TTT T-3' (SEQ ID NO.5) and 5'- GAT CAA AAA ACA GTA TCT GTA s GGA TTC AGC TGA AAC CGT GCC AAG CTT CGC ACG GTT TCA GCT GAA TCC TAC AGA TAC G-3' (SEQ ID NO.6).

28. The method of claim 24, wherein said siRNA of PHD2 is represented by a nucleotide sequence: CAA GGU AAG UGG AGG UAU AdTdT and UAU ACC UCC ACU UAC CUU GdTdT or 0 UGU ACG UCA UGU UGA UAA UdTdT and AUU AUC AAC AUG ACG UAC AdTdT.

29. The method of claim 24, wherein said pathology is ischemia, hypoxia-induced stroke, myocardial infarction, brain annurism or a combination thereof.

₅ 30. A method of elevating HIF-lα mRNA levels, HEF-lα protein levels, or both in a subject, comprising the step of knocking down expression of IOP1 in said subject, thereby resulting in of elevating HIF-lα mRNA levels, HIF-lα protein levels, or both.

31. The method of claim 30, wherein knocking down expression of IOP1 results in augmenting hypoxia-induced HIF target gene expression.

32. The method of claim 31, wherein the hypoxia-induced HIF target gene is Glutl, PHD2, PHD3, or a combination thereof.

33. The method of claim 30, wherein the subject is under normoxia or hypoxia condition.

34. The method of claim 30, wherein the step of knocking down IOP1 expression is done using a siRNA of IOP1, Morpholino oligos for IOP1 mRNA, or a combination thereof.

35. The method of claim 34, wherein said siRNA of IOP1 is derived from a short hairpin loop transcribed from a plasmid vector.

36. The method of claim 35, wherein the short hairpin loop is transcribed from a plasmid vector containing an oligonucleotide represented by the sequence: 5'- TAT CTG TAG GAT TCA GCT GAA ACC GTG CGA AGC TTG GCA CGG TTT CAG CTG AAT CCT ACA GAT ACT GTT TTTT-3' (SEQID NO.5).

37. The method of claim 35, wherein the short hairpin loop is transcribed from a plasmid vector containing an oligonucleotide represented by the sequence: 5'- GAT CAA AAA ACA GTA TCT GTA GGA TTC AGC TGA AAC CGT GCC AAG CTT CGC ACG GTT TCA GCT GAA TCC

TAC AGA TAC G-3' (SEQ ID NO.6).

38. The method of claim 35, wherein the siRNA of IOP1 is derived from a short hairpin loop transcribed from a plasmid vector containing the following two oligonucleotides: 5'- TAT CTG TAG GAT TCA GCT GAA ACC GTG CGA AGC TTG GCA CGG TTT CAG CTG AAT CCT ACA GAT ACT GTT TTT T-3' (SEQ ID NO.5) and 5'- GAT CAA AAA ACA GTA TCT GTA GGA TTC AGC TGA AAC CGT GCC AAG CTT CGC ACG GTT TCA GCT GAA TCC TAC AGATAC G-3' (SEQ IDNO.6).

39. A cell expressing, or co-expressing an Iron-Only Hydrogenase Like Protein (IOP1) peptide, wherein said peptide is represented by the formula MASPFSGALQ LTDLDDFIGP SQECIKPVKV EKRAGSGVAK IRIEDDGSYF QINQDGGTRR LEKAKVSLND

CLACSGCITS AETVLITQQS HEELKKVLDA NKMAAPSQQR LVVVSVSPQS RASLAARFQL NPTDTARKLT SFFKKIGVHF VFDTAFSRHF SLLESQREFV

RRFRGQADCR QALPLLASAC PGWICYAEKT HGSFILPHIS TARSPQQVMG SLVKDFFAQQ QHLTPDKIYH VTVMPCYDKK LEASRPDFFN QEHQTRDVDC VLTTGEVFRL LEEEGVSLPD LEPAPLDSLC SGASAEEPTS HRGGGSGGYL EHVFRHAARE LFGIHVAEVT YKPLRNKDFQ EVTLEKEGQV LLHFAMAYGF RNIQNLVQRL KRGRCPYHYV EVMACPSGCL NGGGQLQAPD RPSRELLQHV ERLYGMVRAE APEDAPGVQE LYTHWLQGTD SECAGRLLHT QYHAVEKAST GLGIRW

(SEQ ID NO.l).