JP2010195684A - REGULATION OF HIF-1 BY MTI-MMP, iFIH AND FIH-1 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for promoting or inhibiting HIF-1 activities and a method for screening a compound that promotes or inhibits HIF-1 activities. <P>SOLUTION: The method for promoting or inhibiting HIF-1α activities includes enhancing or lowering the interaction between an FHI-1 protein and an iFIH protein or MT1-MMP-CP. The method for screening a compound that promotes or inhibits HIF-1 activities includes searching a compound that enhances or lowers the interaction between the FHI-1 protein and the iFIH protein or MT1-MMP-CP. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to the activity control of HIF-1. More specifically, the present invention relates to HIF-1 activity control via FIH-1.

  HIF-1 is a major molecule that promotes transcription of genes necessary for glycolysis and genes necessary for angiogenesis and hematopoiesis in response to hypoxia (Non-patent Document 1). HIF-1 is composed of two subunits, an α subunit which is unstable under normal oxygen partial pressure and a stable β subunit. Under normal oxygen partial pressure, hydroxylation occurs on the proline residue of HIF-1α by HIF polyhydroxylases (HPHs), followed by Von Hippel Lindau (VHL) tumor suppressor protein that recognizes and binds to hydroxylated proline. HIF-1α is ubiquitinated and degraded by the proteasome. In addition to quantitative control by such protein stability, qualitative control of transcriptional activity of HIF-1α itself is also performed in an oxygen-dependent manner, and FIH (factor of inhibiting HIF) -1 enables HIF- When the asparagine residue in the 1α C-terminal activation domain (CAD) is hydroxylated, it cannot interact with the transcriptional coupling factor p300 / CBP, resulting in suppression of transcriptional activity. (Non-patent document 2, Non-patent document 3, Non-patent document 4, and Non-patent document 5). That is, HPHs controls HIF-1α negatively quantitatively and FIH-1 qualitatively. Moreover, since the Km values of HPHs and FIH-1 with respect to oxygen molecules are different, it is considered that the activity of HIF-1α is optimally adjusted at each oxygen concentration.

By the way, in many pathological situations such as inflammatory diseases and cancer, hypoxia is often observed (Non-patent Document 6, Non-patent Document 7 and Non-patent Document 8). Spherical cells and cancer cells need to exert many functions (for example, cell motility, expression of various genes, etc.), and need to adapt quickly to hypoxic situations. One factor that is thought to play an important role in such hypoxic situations is HIF-1α, which is mediated by the anaerobic glycolysis of myeloid cells under normal oxygen partial pressure. It is known that it plays an important role in ATP production, and also plays an important role in anaerobic glycolysis and angiogenesis in cancer cells (Non-patent Document 9).
Under such circumstances, in recent years, studies have been made on the possibility of suppressing the onset and progression of inflammatory diseases or cancers by suppressing the activity of HIF-1. For example, a method for searching for a functional analog of FIH-1 or a FIH-1 binding factor based on the crystal structure of FIH-1, which is an inhibitor of HIF-1, has been disclosed (Patent Document 1). However, no functional analog or FIH-1 binding factor that is actually clinically applicable has been reported so far.

Semenza et al., Curr Opin Cell Biol 13; 167-171, 2001. Kasper et al., EMBO J 24; 3846-3858, 2005 Lando et al., Genes Dev 16; 1466-1471, 2002a Mahon et al., Genes Dev 15; 2675-2686, 2001. Arany et al., Proc Natl Acad Sci USA 93; 12969-12973, 1996. Gatenby and Gillies, Nat Rev Cancer 4; 891-899, 2004 Mapp et al., Br Med Bull 51; 419-436, 1995. Mehendale et al., FASEB J 8; 1285-1295, 1994. Semenza et al., Nat Rev Cancer 3; 721-732, 2003. WO2004 / 035812

An object of the present invention is to provide a method for reducing the activity of HIF-1α.
Another object of the present invention is to provide a screening method for compounds that reduce the activity of HIF-1α.

As a result of diligent research in view of the above circumstances, the present inventors have found that iFIH is essential for the activation of HIF-1α by MT1-MMP and that intracellular peptides of FIH-1 and MT1-MMP (hereinafter referred to as “FIH-1”) And called FI) and / or the interaction between FIH-1 and iFIH (inhibitor of FIH-1) was found to be important for the activation of HIF-1α, and the present invention was completed.
Here, “MT1-MMP” is a membrane protein classified as a membrane-type matrix metalloprotease (MMP), and binds to an enzyme active domain (CAT) and a substrate necessary for protease activity and bears substrate selectivity. It is composed of an extracellular region composed of a hemopexin-like domain (HPX), a transmembrane region, and a short intracellular region (CP) composed of 20 amino acids. MT1-MMP has been reported to be expressed in monocytes / macrophages and is thought to play an important role in cell motility. “IFIH” has been known as APBA3 / Mint3. The present inventors have revealed for the first time that it is identical to a previously unknown factor, but interacts with the CP region of MT1-MMP and plays an important role in controlling HIF-1 activity.

That is, the present invention relates to the following (1) to (11).
(1) The first aspect of the present invention is “a method for enhancing or decreasing the interaction between intracellular iFIH protein and FIH-1 protein to promote or inhibit the activity of HIF-1α”.
(2) The second aspect of the present invention is “the method according to (1) above, wherein the FIH-1 protein interacts with MT1-MMP-CP”.
(3) The third aspect of the present invention is “the method according to (1) above, wherein the expression of iFIH-1 protein is reduced by RNAi method to reduce the interaction between iFIH protein and FIH-1”. is there.
(4) A fourth aspect of the present invention is a “method for enhancing or decreasing the interaction between intracellular MT1-MMP-CP and FIH-1 protein to promote or inhibit the activity of HIF-1α”. is there.
(5) According to a fifth aspect of the present invention, “the peptide having the reduced interaction between MT1-MMP-CP and FIH-1 protein represented by the amino acid sequence represented by SEQ ID NO: 12, or SEQ ID NO: 12 consisting of an amino acid sequence having one or several amino acid substitutions, deletions or insertions, interacts with FIH-1 protein, and HIF-1α by FIH-1 protein The method according to (4), which is achieved by competition with a peptide that does not inhibit activation.
(6) A sixth aspect of the present invention is “the method according to any one of (1) to (5) above, wherein the cell is a cell of an immune system or a tumor cell”.
(7) A seventh aspect of the present invention is “a method for screening a compound that promotes or inhibits HIF-1 activity,
(A) contacting FIH-1 protein with iFIH protein or MT1-MMP-CP in the presence of a test sample;
(B) detecting the interaction between FIH-1 protein and iFIH protein or MT1-MMP-CP, and (c) comparing the interaction with that in the absence of the test sample, Determining that the compound candidate is present in the test sample when the action is enhanced or decreased,
A method comprising:
(8) The eighth aspect of the present invention is as follows: “The interaction between FIH-1 protein and iFIH protein or MT1-MMP-CP is determined whether FIH-1 protein and iFIH protein or MT1-MMP-CP are bound. The method according to (7) above, which is detected as a reference.
(9) The ninth aspect of the present invention is as described in (7) or (8) above, further comprising the step of confirming that the obtained candidate compound promotes or inhibits intracellular HIF-1α activity. The method of "."
(10) The tenth aspect of the present invention is “a compound isolated by the method according to any one of (7) to (9)”.
(11) An eleventh aspect of the present invention is “the compound according to (10) above, wherein the compound is an antibody”.

  When the method of the present invention is used, the activity of HIF-1α can be inhibited.

  The method of the present invention can be used to screen for compounds that inhibit the activity of HIF-1α.

In one embodiment of the invention, a step of enhancing or decreasing the interaction of FIH-1 protein with MT1-MMP or iFIH protein is included.
In another embodiment of the present invention, a step of detecting a compound that enhances or decreases the interaction between FIH-1 protein and MT1-MMP or iFIH protein is included.
Therefore, iFIH protein, MT1-MMP and FIH-1 protein, and a method for obtaining genes encoding them are described below, and further, the interaction between FIH-1 protein and MT1-MMP or iFIH protein is enhanced or decreased. The process of detecting the compound to be performed will be described.
Here, the interaction between proteins means an action between proteins including binding between proteins, structural change, modification, stability change and the like.

“IFIH protein” is, for example, a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
The “FIH-1 protein” is a protein containing an amino acid sequence that is the same as or substantially the same as the amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 7, for example.
Furthermore, “MT1-MMP” is, for example, a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.
Here, the “protein containing substantially the same amino acid sequence” refers to “iFIH protein”, “FIH-1 protein” and “MT1-MMP”, respectively, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 About 60% or more, preferably about 70% or more, and more preferably about 70% or more with the amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 7 and the amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 Is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, most preferably about 99% amino acid sequence, and the “iFIH protein” has the ability to bind to FIH-1 protein. It has, for the "FIH-1 protein" refers to a protein that inhibits the activity of HIF-l [alpha].
Alternatively, in the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 7, and the amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 It consists of an amino acid sequence in which one or several (preferably about 1 to 30, more preferably about 1 to 10, more preferably 1 to 5) amino acids have been deleted, substituted or added, and “iFIH protein” refers to a protein that has the ability to bind to FIH-1 protein, and “FIH-1 protein” refers to a protein that inhibits the activity of HIF-1α. The amino acid deletion, addition, and substitution may be mutations originally present in the nucleic acid encoding the protein, or are newly introduced by modifying the nucleic acid by a technique known in the art. There may be. In the modification, for example, a specific amino acid residue is substituted by using a commercially available kit (for example, MutanTM-G (TaKaRa), MutanTM-K (TaKaRa)), etc. It can be carried out by substituting the base by the method of or a method analogous thereto.

“MT1-MMP-CP” refers to the intracellular region (CP) of MT1-MMP, which has the same or substantially the same amino acid sequence as the peptide represented by SEQ ID NO: 12, for example, FIH-1 A peptide having the ability to bind to a protein.
The meaning of “substantially the same” depends on the above-mentioned examples of “iFIH protein” and “FIH-1 protein”.

In another embodiment of the present invention, since the step of expressing iFIH protein, FIH-1 protein or MT1-MMP (or MT1-MMP-CP) extracellularly or intracellularly is included, these proteins or peptides A nucleic acid for expressing the protein is required.
Nucleotide sequences of nucleic acids encoding human iFIH protein, FIH-1 protein and MT1-MMP are DNA consisting of nucleotide sequences known as SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 8, respectively.
As for the nucleic acid encoding MT1-MMP-CP, for example, any nucleic acid encoding the peptide of SEQ ID NO: 12 can be used.

A nucleic acid encoding iFIH protein, a nucleic acid encoding FIH-1, and a nucleic acid encoding MT1-MMP are obtained using a suitable library by preparing a probe based on the nucleotide sequence represented by the above SEQ ID NO. Alternatively, it can also be obtained by PCR using PCR primers prepared based on the nucleotide sequence. The source of these nucleic acids is not limited to human cells, but may be cells or tissues of mammals other than humans, such as monkeys, mice, rats, rabbits, and cows.
When nucleic acids are obtained by a hybridization method using a probe, all conditions that can be selected based on ordinary knowledge of those skilled in the art can be employed as hybridization conditions.
In general, by performing screening under highly stringent conditions, nucleic acids having high homology with the nucleic acids represented by SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 8 can be obtained.

Here, stringent conditions are hybridization conditions that are easily determined by those skilled in the art, and are generally empirical experimental conditions that depend on the probe length, washing temperature, and salt concentration. In general, the longer the probe, the higher the temperature for proper annealing, and the shorter the probe, the lower the temperature. Hybridization generally depends on the ability to reanneal in an environment where the complementary strand is slightly below its melting point.
Specifically, for example, as a low stringency condition, in a filter washing step after hybridization, washing is performed in a 0.1 × SSC, 0.1% SDS solution at a temperature of 37 ° C. to 42 ° C. Can be raised. In addition, examples of highly stringent conditions include washing in 65 ° C., 5 × SSC and 0.1% SDS in the washing step.

Proteins or peptides used in the present invention (including, but not limited to, MT1-MMP, MT1-MMP-CP, HFI-1 protein, iHFI protein, HIF-1α, etc.) The term “peptide” includes not only a naturally occurring protein but also a fragment or other peptide or protein fused form.
Here, the peptide fused with the natural protein is not particularly limited, and examples thereof include FLAG, His tag, c-myc fragment, T7-tag, and E-tag. Examples of the fused protein include GST (glutathione S-transferase) and HA (influenza agglutinin).

The recombinant of the protein or peptide of the present invention is used to detect a variation in the activity of the protein or peptide of the present invention or a variation in the interaction between the protein or peptide of the present invention outside or inside the cell. Can do.
In order to prepare a recombinant of the protein or peptide of the present invention, it is necessary to prepare an expression vector incorporating a nucleic acid encoding the protein or peptide of the present invention (hereinafter referred to as the nucleic acid of the present invention). Such an expression vector can be obtained by ligating the nucleic acid of the present invention to an appropriate vector so that it can be expressed.
Examples of vectors that can be used here include plasmid DNA and phage DNA. Plasmid DNA includes plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pCBD-C, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194, etc.), yeast-derived plasmids (eg, YEp13, YEp24, YCp50, YIp30, etc.) and the like, and examples of the phage DNA include λ phage. Furthermore, animal viruses such as retroviruses and vaccinia viruses, and insect virus vectors such as baculoviruses and togaviruses can also be used.

In addition, the promoter that can be used is not particularly limited as long as it is appropriate for the host used for gene expression.
For example, when animal cells are used as the host, SRα promoter, CMV promoter, SV40 promoter, LTR promoter, HSV-TK promoter, EF-1α promoter and the like can be mentioned.
When the host is Escherichia coli, tac promoter, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, etc. are mentioned. When the host is Bacillus subtilis, SPO1 promoter, SPO2 promoter, penP promoter, etc. are mentioned. It is done.
When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like can be mentioned.
When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.

In addition to the promoter sequence, a selection marker, terminator, enhancer, splicing signal, poly A addition signal, ribosome binding sequence (SD sequence), SV40 origin of replication (SV40ori) and the like are appropriately linked to the expression vector of the present invention. Can do.
The selection markers include, but are not limited to, hygromycin resistance marker ^(Hyg r), dihydrofolate reductase gene (dhfr), the ampicillin resistance gene ^(Amp r), the kanamycin resistance gene ^(Kan r), neomycin resistant gene (Neo ^r , G418) can be used.
For the purpose of facilitating the isolation and purification of the recombinant protein, an appropriate signal sequence may be added to the N-terminal side of the polypeptide of the present invention.
When the host is Escherichia coli, alkaline phosphatase signal, OmpA signal, etc. can be used. When the host is Bacillus subtilis, α-amylase signal sequence, subtilis signal sequence, etc. can be used. In some cases, α-factor signal sequence, invertase signal sequence, etc. can be used. When the host is an animal cell, for example, insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used. It is.

Inserting the nucleic acid of the present invention into the above-described vector means that the cloned DNA is digested as it is or if desired with a restriction enzyme, a linker is added, and inserted into a restriction enzyme site or a multicloning site of the vector DNA. This can be done. The DNA to be ligated may have ATG as a translation initiation codon on the 5 ′ end side, and may have TAA, TGA or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter. The DNA to be ligated needs to be incorporated into a vector so that the polypeptide of the present invention encoded in the DNA is expressed in the host cell.
By the above method, an expression vector containing the nucleic acid of the present invention can be constructed.

  The expression vector prepared as described above needs to introduce the protein or peptide of the present invention into the host. Here, the host is not particularly limited as long as it can express the protein of the present invention. For example, Escherichia such as Escherichia coli, Bacillus such as Bacillus subtilis, Pseudomonas putida such as Pseudomonas, Rhizobium meliloti and other bacteria belonging to Rhizobium genus such as Rhizobium meliloti, Yeast such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, monkey cell COS-7, Vero, Chinese hamster ovary cell (CHO cell), or Sf9, Sf9 Insect cells such as

  As a method for introducing a vector into E. coli, a method using calcium ions, an electroporation method, or the like can be used. As a method for introducing a recombinant vector into yeast, an electroporation method, a spheroplast method, a lithium acetate method, or the like can be used. Examples of methods for introducing a recombinant vector into animal cells or animal cells include the DEAE dextran method, the electroporation method, the calcium phosphate method, and a method using a cationic lipid.

  As a medium for culturing transformants obtained using microorganisms such as Escherichia coli and yeast as a host, the medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms. As long as the medium can be used, either a natural medium or a synthetic medium may be used. As the carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. As the nitrogen source, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor and the like are used. As the inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.

  Culturing is performed under conditions suitable for the host cell. For example, as the medium for culturing Escherichia coli, LB medium, M9 medium and the like are preferable. Agents such as isopropyl-1-thio-β-D-galactoside, 3β-indolylacrylic acid can be added to make the promoter work efficiently if desired. In the case of Escherichia coli, the culture is usually carried out at about 15 to 37 ° C. for about 3 to 24 hours, and if necessary, aeration or stirring can be added. When the host is Bacillus subtilis, the culture is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and aeration and agitation can be added as necessary.

  Examples of the medium for culturing yeast include SD medium and YPD medium. The pH of the medium is preferably adjusted to about 5-8. The culture is usually performed at about 20 to 35 ° C. for about 24 to 72 hours, and aeration and agitation are added as necessary. When cultivating a transformant whose host is an insect cell or an insect, examples of the medium include a grace insect medium containing bovine serum. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary.

When cultivating a transformant whose host is an animal cell, for example, a MEM medium, DMEM medium, RPMI-1640 medium or the like containing about 5 to 20% fetal calf serum is used as the medium. The pH is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C. for about 15 to 60 hours, and aeration and agitation are added as necessary.
The method for preparing a transformant using an animal cell as a host can be used for gene therapy for a disease associated with the function of HIF-1. The nucleic acid that can be expressed in the cells to be treated for the purpose of gene therapy is not limited, for example, a factor that inhibits the interaction between iFIH and FIH-1 (for example, but not limited to, shRNA, siRNA , A partial peptide of FIH-1 that binds to iFIH, etc.), factors that inhibit the interaction between MT1-MMP-CP and FIH-1 (for example, but not limited to, MT1-MMP-CP, MT1-MMP-CP) FIH-1 partial peptides, etc. that bind to A vector used for the purpose of gene therapy can be easily selected by those skilled in the art. For example, an adenovirus vector, a retrovirus vector, a lentivirus vector, and the like can be used. For in vivo administration to cells, methods such as ex vivo and in vivo can be used.

  When the recombinant of the protein or peptide of the present invention is used extracellularly, the host cells expressing the recombinant are collected, suspended in an appropriate buffer, and the cells are destroyed by ultrasound, lysozyme, and / or freeze-thawing. Thereafter, a method of obtaining a crude extract containing the protein or peptide of the present invention by centrifugation or filtration is appropriately used. The buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100. When the protein or peptide of the present invention is secreted into the culture solution, the cells or cells are separated from the supernatant by a method known per se after completion of the culture, and the supernatant is collected. Purification of the protein or peptide of the present invention contained in the culture supernatant or extract thus obtained can be performed by appropriately combining known separation / purification methods. As these known separation and purification methods, methods utilizing solubility such as salting out and solvent precipitation methods, dialysis methods, ultrafiltration methods, gel filtration methods, and mainly using differences in molecular weight such as SDS-PAGE. Method, method utilizing the difference in charge such as ion exchange chromatography, method utilizing specific affinity such as affinity chromatography, method utilizing difference in hydrophobicity such as reverse phase high performance liquid chromatography, isoelectric point A method using the difference in isoelectric point such as electrophoresis is used.

The present invention includes a method of enhancing or decreasing the interaction between iFIH protein and FIH-1 protein to promote or inhibit the activity of HIF-1α.
As a method for enhancing the interaction between the iFIH protein and the FIH-1 protein, one that promotes the interaction, for example, an MT1-MMP mutant TM-CP (see, for example, FIG. 1a) is added. To do.
On the other hand, the method for reducing the interaction between iFIH protein and FIH-1 protein includes a method for losing or reducing iFIH expression and losing or reducing the interaction with FIH-1. Examples of such a method include a method (RNAi method or the like) that inhibits iFIH transcription by introducing an antisense nucleotide or antisense oligonucleotide to iFIH into a target cell. Introduction of antisense nucleotides or antisense oligonucleotides can be easily carried out by those skilled in the art. For example, antisense nucleotides or antisense oligonucleotides can be directly introduced into cells even if they are expressed in cells. However, any method can be used. Moreover, these derivatives can also be used instead of such an antisense nucleotide or antisense oligonucleotide.

Further, the present invention includes a method for inhibiting or promoting the activity of HIF-1α by enhancing or decreasing the interaction between MT1-MMP-CP and FIH-1 protein.
Examples of a method for enhancing the interaction between the iFIH protein and the FIH-1 protein include adding a substance that promotes the interaction.
On the other hand, as a method for reducing the interaction between MT1-MMP-CP and FIH-1 protein, in the peptide represented by the amino acid sequence represented by SEQ ID NO: 12, or the amino acid sequence represented by SEQ ID NO: 12, A peptide comprising an amino acid sequence having one or several amino acid substitutions, deletions or insertions, interacting with FIH-1 protein, and does not inhibit activation of HIF-1α by FIH-1 protein Examples thereof include a method of competitively inhibiting the interaction between MT1-MMP-CP and FIH-1 protein in the cell (or expressing the peptide in the target cell).
Moreover, a mutation can be introduced into MT1-MMP-CP. In addition to destroying the whole, the mutation includes a method of substituting or deleting amino acids that play an important role in the interaction with the FIH-1 protein. A method for searching for amino acids important for interaction with the FIH-1 protein can be easily selected by those skilled in the art. For example, a method such as an alanine scan can also be used. For example, as an amino acid suitable for introducing a mutation, the 14th arginine of MT1-MMP-CP (SEQ ID NO: 12) is preferable.

Further, the present invention includes a compound that enhances or decreases the interaction between iFIH protein and FIH-1 protein to promote or inhibit the activity of HIF-1α, and MT1-MMP-CP and FIH-1 protein. Compounds that enhance or decrease the interaction of HIF-1α to inhibit or promote the activity of HIF-1α are included.
In the present invention, compounds that lose or reduce the interaction between iFIH protein and FHI-1, or between MT1-MMP-CP and FHI-1 protein include, for example, iFIH protein or MT1-MMP-CP. Mention may also be made of antibodies. These antibodies include, for example, an antibody against a partial region (for example, N-terminal region) of iFIH protein that interacts with FHI-1 protein, or an antibody against intracellular peptide (CP) of MT1-MMP.
Examples of the antibody include a partial region (for example, N-terminal region) of iFIH protein that interacts with FHI-1 protein, a monoepitope specific antibody, a polyepitope specific antibody, a single epitope against intracellular peptide (CP) of MT1-MMP. Single chain antibodies and fragments thereof are included. These antibodies include, for example, monoclonal antibodies, polyclonal antibodies, humanized antibodies and the like.
Polyclonal antibodies can be prepared, for example, by injecting a mixture of immunogen and adjuvant into a mammalian host animal. Usually, the immunogen and / or adjuvant is injected multiple times subcutaneously or intraperitoneally into the host animal. As the immunogen, the soluble Spo11 protein retaining the DNA binding ability of the present invention can be used. Examples of adjuvants include complete Freud and monophosphoryl lipid A synthesis-trehalose dicorynomycolate (MPL-TDM).

A monoclonal antibody can be prepared using, for example, a hybridoma method.
This method includes the following four steps: (i) immunizing a host animal or lymphocytes from the host animal, (ii) monoclonal antibody-secreting (or potentially secreting) lymphocytes Harvest, (iii) fuse lymphocytes to immortalized cells, (iv) select cells that secrete the desired monoclonal antibody.
A mouse, rat, guinea pig, hamster, or other suitable host animal is selected as the immunized animal and the immunogen is injected. Alternatively, lymphocytes obtained from immunized animals may be immunized in vitro. If human cells are desired, peripheral blood lymphocytes (PBLs) are generally used. However, spleen cells or lymphocytes from other mammals are more common and preferred.

  After immunization, lymphocytes obtained from the host animal are fused with an immortalized cell line using a fusing agent such as polyethylene glycol in order to establish hybridoma cells. As fusion cells, rodent, bovine, or human myeloma cells immortalized by transformation are used, or rat or mouse myeloma cell lines are used. After cell fusion, the cells are grown in a suitable medium containing one or more substrates that inhibit the growth or survival of unfused lymphocytes and immortalized cell lines. Conventional techniques use parent cells that lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT). In this case, hypoxanthine, aminopterin and thymidine are added to a medium (HAT medium) that inhibits the growth of HGPRT-deficient cells and allows the growth of hybridomas. A hybridoma that produces a desired antibody is selected from the hybridomas thus obtained, and the target monoclonal antibody can be obtained from the medium in which the hybridoma grows according to a conventional method.

The present invention provides a screening method for a compound that promotes or inhibits HIF-1 activity,
(A) contacting FIH-1 protein with iFIH protein or MT1-MMP-CP in the presence of a test sample;
(B) detecting the interaction between FIH-1 protein and iFIH protein or MT1-MMP-CP, and (c) comparing the interaction with that in the absence of the test sample, Determining that the compound candidate is present in the test sample when the action is enhanced or decreased,
Including the method. In the present embodiment, the FIH-1 protein and iFIH protein, or the FIH-1 protein and MT1-MMP-CP interact to increase or decrease the HIF-1α activity inhibitory effect of the FIH-1 protein. Based on this, the present invention provides a method for inhibiting the activity of HIF-1α using a compound that enhances or decreases these interactions.

Such screening can be performed using in vitro or intracellular systems. When using an intracellular system, for example, incubating cells expressing FIH-1 protein and MT1-MMP and / or iFIH protein in the presence of a test sample, and detecting fluctuations in HIF-1α activity Thus, screening can be performed. Changes in the activity of HIF-1α can be detected by a reporter assay (see, for example, the “Examples” section below).
In addition, a two-hybrid system (Two-hybrid method) may be used to detect a compound that reduces the interaction between FIH-1 protein and iFIH protein, or FIH-1 protein and MT1-MMP-CP. In this case, the candidate compound obtained by the two-hybrid method can be further screened again in the above-described cell system for detecting fluctuations in the activity of HIF-1α.

The screening of the present invention can also be performed using, for example, immunoprecipitation. In the presence of a test sample, a cell expressing FIH-1 protein and iFIH protein and / or MT1-MMP (or MT1-MMP-CP) is prepared, and after culturing, for example, an antibody is extracted from the cell extract. The complex containing FIH-1 protein is recovered and whether or not iFIH protein or MT1-MMP (or MT1-MMP-CP) is contained in this complex is confirmed. Presence or absence of iFIH protein or MT1-MMP (or MT1-MMP-CP) can be confirmed by an immunoblotting method using an antibody against these. As a result, the presence of iFIH protein or MT1-MMP (or MT1-MMP-CP) is not confirmed, or compared to the case where a similar experiment is performed in the absence of the test sample, iFIH protein or MT1- When the abundance of MMP (or MT1-MMP-CP) is significantly reduced, the interaction between FIH-1 protein and iFIH protein and / or MT1-MMP (or MT1-MMP-CP) is a test sample. It can be judged that it was inhibited by.
Other than the immunoprecipitation method, for example, screening can also be performed by an immunological technique using an antibody column or the like.
In addition, when expressing the protein or peptide of the present invention, when expressed as a fusion with an epitope tag or the like, an antibody against the tag can also be used.

  Furthermore, the above immunological technique can be performed in a complete in vitro system without using cells. In this case, in vitro, the FIH-1 protein and iFIH protein or MT1-MMP (or MT1-MMP-CP) are incubated in the presence of the test sample, for example, an antibody against the FIH-1 protein (or an antibody against the tag) ), The complex containing FIH-1 protein is recovered by pull-down or the like, and the presence or absence of iFIH protein or MT1-MMP (or MT1-MMP-CP) in the complex is confirmed. In addition to the pull-down method, an antibody column can also be used.

Compounds obtained by the method of the present invention (for example, high molecular compounds other than low molecular compounds are also included) are expected to promote or inhibit the activity of HIF-1α. Therefore, these compounds are used in, for example, promotion of angiogenesis in ischemic diseases (for example, infarcts), immunotherapy by activation of macrophages, or diseases associated with a high activity state of HIF-1α, It can be used in the form of a pharmaceutical composition that is effective for treating disorders and the like and does not adversely affect the living body. Such compositions typically include a pharmaceutically acceptable carrier in addition to the compound obtained by the present invention.
“Pharmaceutically acceptable carriers” are those suitable for pharmaceutical administration, including solvents, dispersion media, coatings, antibacterial and antifungal agents, agents that act isotonically to delay adsorption and the like. Examples of preferred for diluting the carrier and the carrier include, but are not limited to, water, saline, finger solution, dextrose solution, collagen, human serum albumin, organic solvent, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, Carboxyvinyl polymer, sodium alginate, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petroleum jelly, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol , Lactose and the like. Non-aqueous media such as liposomes and non-volatile oils are also used. In addition, certain compounds that protect or promote the activity of the compounds of the present invention may be included in the composition.

The pharmaceutical composition according to the present invention includes intravenous, intradermal, subcutaneous, oral (including inhalation, etc.), transdermal and transmucosal administration, and is formulated to be suitable for a therapeutically appropriate route of administration. It becomes. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application include, but are not limited to, sterile diluents such as water for injection, saline solutions, non-volatile oils, polyethylene glycols, Glycerin, propylene glycol, or other synthetic solvents, benzyl alcohol or other preservatives such as methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, soothing agents such as benzalkonium chloride, procaine hydrochloride, ethylenediaminetetraacetic acid Chelating agents such as (EDTA), buffering agents such as acetate, citrate, or phosphate, and agents for osmotic pressure adjustment such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations are contained in ampoules, glass or plastic disposable syringes or multiple dose vials.

  Pharmaceutical compositions suitable for injection include sterile aqueous solutions (water soluble) or dispersion media and sterile powders for the preparation of sterile injectable solutions or dispersion media at the time of use. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). When used as an injection, the composition must be sterile and must be fluid enough to be administered with a syringe. The composition must be stable to chemical changes, corrosion, and the like during formulation and storage, and must prevent contamination from microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures. For example, using a coating agent such as lectin, maintaining a required particle size in the dispersion medium, and maintaining a proper fluidity by using a surfactant. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal can be used to prevent microbial contamination. In addition, polyalcohols such as sugar, mannitol, sorbitol, and agents that maintain isotonicity such as sodium chloride may be included in the composition. Compositions that can delay adsorption include agents such as aluminum monostearate and gelatin.

  Sterile injectable solutions are prepared by adding the required ingredients alone or in combination with other ingredients to the required amount of the active compound in a suitable solvent and sterilizing. Generally, a dispersion medium is prepared by incorporating the active compound into a sterile medium that contains a basic dispersion medium and the other necessary ingredients described above. Methods for preparing a sterile powder for preparing a sterile injectable solution include vacuum drying and lyophilization to prepare a powder containing the active ingredient and any desired ingredients derived from the sterile solution. .

Oral compositions include inert diluents or carriers that are not harmful when incorporated into the body. Oral compositions are, for example, contained in gelatin capsules or compressed into tablets. For oral treatment, the active compound is incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a flowable carrier, and the composition in the flowable carrier is applied orally. In addition, pharmaceutically compatible binding agents, and / or adjuvant materials may be included.
Tablets, pills, capsules, troches and the like may contain any of the following components or compounds with similar properties: excipients such as microcrystalline cellulose, binding such as gum arabic, tragacanth or gelatin Agents; swelling agents such as alginic acid, PRIMOGEL, or corn starch such as starch or lactose; lubricants such as magnesium stearate or STRROTES; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or peppermint, methyl A fragrance additive such as salicylic acid or orange flavor.

  The compounds of the present invention can be prepared as sustained release formulations such as delivery systems encapsulated in implantable tablets and microcapsules using a carrier that can prevent immediate removal from the body. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such materials can be readily prepared by those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. Useful liposomes are prepared as a lipid composition comprising, but not limited to, phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanol (PEG-PE) through a filter of appropriate pore size to obtain a size suitable for use, and reverse phase. Purified by evaporation.

In the prevention or treatment of a specific disease with the compound of the present invention, the appropriate dosage level depends on the condition of the patient to be administered, the administration method, etc., but can be easily optimized by those skilled in the art. It is.
In the case of injection administration, for example, it is preferable to administer about 0.1 μg / kg to about 500 mg / kg of the patient's body weight per day, and it will generally be possible to administer a single dose or divided into multiple doses. Preferably, the dosage level is about 0.1 μg / kg to about 250 mg / kg per day, more preferably about 0.5 μg to about 100 mg / kg per day.
For oral administration, the composition is preferably provided in the form of a tablet containing 1.0 to 1000 mg of active ingredient, preferably 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750. 0, 800.0, 900.0 and 1000.0 mg. The compounds are administered on a regimen of 1 to 4 times daily, preferably once or twice daily.

  A pharmaceutical composition or formulation must consist of uniform unit doses to ensure a constant dose. A unit dose is a unit formulated with a pharmaceutically acceptable carrier, including a single dose effective for treating a patient. The unit dosage of the present invention is determined by the physical and chemical characteristics of the compound to be formulated, the expected therapeutic effect, and the formulation considerations specific to the compound. .

  The pharmaceutical composition of the present invention can be included in the form of a kit in a container or pack together with instructions for administration. When the pharmaceutical composition according to the present invention is supplied as a kit, different constituents of the pharmaceutical composition are packaged in separate containers and mixed immediately before use. The reason why the components are packaged separately in this way is to enable long-term storage without losing the function of the active component.

  Reagents contained in the kit are supplied in any type of container in which the components remain active for an extended period of time, are not adsorbed by the container material, and are not subject to alteration. For example, a sealed glass ampoule includes a buffer packaged under a neutral and non-reactive gas such as nitrogen gas. Ampoules are composed of glass, polycarbonate, organic polymers such as polystyrene, ceramics, metals, or any other suitable material commonly used to hold reagents. Examples of other suitable containers include simple bottles made from similar materials such as ampoules, and packaging materials that are internally lined with foil such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, or the like. The container has a sterile access port such as a bottle having a stopper that can be penetrated by a hypodermic needle.

  The kit also includes instructions for use. Instructions for using the kit comprising the pharmaceutical composition are printed on paper or other material and / or floppy disk, CD-ROM, DVD-ROM, Zip disk, video tape, audio tape, etc. It may be supplied as an electrically or electromagnetically readable medium. Detailed instructions for use may be actually attached to the kit, or may be posted on a website designated by the manufacturer or distributor of the kit or notified by e-mail or the like.

  Examples are shown below, but the present invention is not limited to the following examples.

1. Experimental materials and experimental methods 1-1. Plasmid
Mouse FIH-1 cDNA was isolated from macrophages by RT-PCR. Human FIH-1 and APBA3 / iFIH cDNA were isolated from HEK293 cells by RT-PCR. Each cDNA was cloned into pENTR / D TOPO vector (Invitrogen). FIH-1N (1-302aa, SEQ ID NO: 6 and 7), APBA3-N / iFIH-N (1-214aa, SEQ ID NO: 2, 3, and 4) and APBA3-C / iFIH-C (215-575aa, SEQ ID NO: 2, 3 and 4) were each prepared by PCR based on the isolated cDNA and cloned into the pENTR / D TOPO vector. Myr-APBA3-N / Myr-iFIH-N inserts a myristoylated sequence derived from v-src (SEQ ID NO: 13) (Cross et al., 1984) using PCR before the start codon, and similarly pENTR / D Cloned into vector. Mouse and human MT1-MMP were used in which a FLAG tag or Myc tag was inserted downstream of the Furin cleavage site prepared in the classroom. MT1-MMP domain-deficient mutants (CAT (112-284aa), HPX (319-507aa), CP (562-581aa), extracellular region (112-507aa)) and alanine-substituted mutants were prepared using PCR. And cloned into the pENTR / D TOPO vector. Those cloned into these pENTR / D TOPO vectors were inserted into pcDNA3.1, pDEST15, pDEST17, pLenti6, and pAd vectors (all Invitrogen) using LR recombinase to prepare expression vectors.

1-2. Mice MT1-MMP-deficient mice and wild-type mice were produced by crossing heterozygous mice produced by the inventors according to a standard method and backcrossed 12 times with C57BL / 6 mice. Wild-type and MT1-MMP-deficient mice used in this experiment were maintained in an SPF environment, and were used for experiments 10-18 days after birth.

1-3. Immunohistochemical staining Frozen sections were prepared with a thickness of 10 μm. The cells were fixed with 4% paraformaldehyde / PBS for 5 minutes, and reacted with 0.03% H2O2 / PBS for 15 minutes to inactivate endogenous peroxidase. After reacting with PBS containing 5% goat serum and 3% BSA for 1 hour at room temperature, anti-F4 / 80 antibody (BMA Biomedicals) or anti-CD45 antibody (Bechman Coulter) was reacted overnight at 4 ° C. After washing with PBS, the sections were reacted with Histofine HRP-added anti-rat IgG antibody (Nichirei) for 30 minutes, washed with PBS, and then developed with DAB solution.
1-4. Preparation of bone marrow derived macrophages Bone marrow derived macrophages were performed based on previous reports (Celada et al., 1984; Kobayashi et al., 2002). Both ends of the femur, tibia, and humerus of the mouse were cut, and the bone marrow cells were washed with PBS. After washing three times with PBS, bone marrow cells were washed in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 30% L929 culture supernatant and 20% FBS in a 5% CO ₂ atmosphere in a humid environment. Cultured at 7 ° C. for 7 days to differentiate into macrophages. The differentiated macrophages were used in the experiment after being cultured for 24 hours in a culture solution excluding the L929 culture supernatant. In hypoxic experiments, differentiated macrophages were cultured in a Model 9200 incubator (Wakken) set in 1% O ₂ and 5% CO ₂ atmosphere and used for the experiment.

1-5. Analysis of invasive ability and motility Analysis of invasive ability against Matrigel and motility using transwell were performed based on previous reports (Nonaka et al., 2005; Ueda et al., 2003). An 8-μm pore-sized transwell (Corning) coated with Matrigel (Becton Dickinson) (invasive ability) or uncoated (motility) was loaded into a 24-well plate. 500 μl of DMEM containing 10 ng / ml MCP-1 (R & D Systems) was placed in the lower chamber and 200 μl of cell suspension (2 × 10 ⁵ cells) was placed in the upper chamber. After culturing the cells for 2 hours with motility and 6 hours with invasive ability at 37 ° C., the cells that migrated to the lower chamber were stained with Giemsa staining solution, and the cells were counted under a microscope.
1-6. Measurement of Chemokinesis (Random Movement) Macrophages were seeded on a 35 mm glass dish and cultured overnight. 10 ng / ml of MCP-1 was added, and after 30 minutes, cell images were obtained using a Leica AS MDW time-lapse system (Leica Microsystems) every 30 minutes. The total of the linear movement distances of the centroids of the cells at each time was analyzed by ImageJ software (NIH) and used as the cell movement distance.

1-7. Measurement of ATP concentration
The ATP concentration of macrophages was measured using ATP Bioluminescence Assay Kit CLS II (Roche Applied Science). The amount of protein in the sample was measured using a Bradford assay kit (Bio-Rad), and the amount of ATP was averaged by the amount of protein. In an experiment using a glycolytic inhibitor, 0.2 M 2-deoxyglucose (SIGMA) or ultrapure water was added to the culture solution, and ATP was measured after 3 hours of culture.

1-8. Detection of HIF-1α and p300 / CBP in macrophage nucleoprotein
Macrophage nucleoprotein was collected using Nuclear Extract Kit (ActiveMotif). 10 μg of nucleoprotein was analyzed using TransAM HIF-1 kit (ActiveMotif). The kit includes a plate coated with an HRE oligonucleotide to which HIF-1 binds. The binding of HIF-1 in the sample was detected using an anti-HIF-1α antibody. Anti-p300 / CBP antibody (Upstate) was used for detection of p300 / CBP.
1-9. Reporter assay
The reporter assay for analyzing the transcriptional activity of HIF-1α was performed with modification based on the report of Lando et al. (Lando et al., 2002b). As a reporter plasmid, four upstream activation sequences were arranged in series in front of the firefly luciferase gene (4 × UAS), and a pGL3 Basic plasmid (Promega) into which a TATA box derived from thymidine kinase (TK) was inserted was prepared. A pRL vector (Promega) expressing Renilla luciferase was used as an internal control. HEK293 cells (5 × 10 ⁴ cells / well) were seeded in a 24-well plate, and a reporter plasmid was introduced using Lipofectamine ^™ 2000 (Invitrogen) under the following conditions. Reporter plasmid (100 ng), internal control vector (10 ng), Gal4BD-CAD plasmid (50 ng), MT1-MMP and mutant, or APBA3 / iFIH and mutant expression plasmid (200 ng). In experiments for analyzing the effects of MT1-MMP and APBA3 / iFIH mutants on HIF-1α CAD activity, 50 ng of each gene was introduced. After 24 hours after gene transfer, the cells were washed with PBS, passive cell lysis buffer (Promega) was added, and the cells were further disrupted by freeze-thawing. The luciferase activity of the lysate was measured with a TD20 / 20 luminometer (Promega) using a Dual-Luciferase Reporter Assay System (Promega).

1-10. Production of recombinant protein
For the production of GST, GST-CP, GST-CPR / A, GST-CP5, GST-CAD, GST-FIH-1, GST-FIH-1N, BL21 Gold (DE3) pLysS E. coli strain (Stratagene) is expressed in pDEST15 After transforming with a vector (Invitrogen) and culturing in LB medium, 0.5 mM isopropyl-D-thiogalactoside (IPTG) was added and culturing at 37 ° C. for 3 hours. After the cells were removed by centrifugation, the cells were suspended in 1% Triton-X / PBS to which protease inhibitor cocktail III (EMD Biosciences) was added, and then subjected to ultrasonic disruption. Glutathione Sepharose 4B (GE Healthcare) was added to the supernatant after centrifugation and reacted at 4 ° C. for 1 hour. Washed 5 times with 1% Triton-X / PBS and stored.
(His) 6 tag For the production of mouse FIH-1, APBA3, APBA3N, BL21 Gold (DE3) pLysS E. coli strain was transformed with pDEST17 expression vector (Invitrogen), cultured in LB medium, 0.1 mM IPTG In addition, induction was performed at 20 ° C. for 16 hours. The cells were resuspended in TBS to which protease inhibitor cocktail III was added and subjected to ultrasonic disruption. Then, 10 mM imidazole and Ni 2 ⁺ -treated chelation sepharose (GE Healthcare) was added to the supernatant and reacted at 4 ° C. for 1 hour. After the beads were washed 5 times with 50 mM imidazole, the bound protein was eluted with 500 mM imidazole, dialyzed against TBS, and used for the experiment.

1-11. GST pull-down assay About 10 μg of GST fusion protein bound to glutathione sepharose 4B (GE Healthcare), lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP) containing 0.5 mg / ml BSA -40) for 30 minutes at 4 ° C. Thereafter, 2 μg of (His) 6-labeled FIH-1 protein, 0.5 mg / ml BSA, and in the peptide competition experiment, biotin-labeled CP peptide or control peptide shuffled with the CP sequence (SEQ ID NO: 14) (synthesis is Invitrogen) Was added to the beads. After mixing at 4 ° C. for 2 hours using a rotator, the beads were washed 4 times with a lysis buffer, Laemmli sample buffer and β mercaptoethanol were added and heated to elute the protein. The eluted protein was subjected to SDS-PAGE and detected using anti- (His) 6 antibody (Roche Applied Science) or anti-GST antibody (GeneTex).

1-12. Production of Lentiviral Vector A lentiviral vector was produced according to the specifications using ViraPower ^™ Lenticular Expression System (Invitrogen). Lentivirus concentration was based on previous reports (Pfeifer et al., 2000). The culture supernatant containing lentivirus was collected, centrifuged at 1,500 g for 10 minutes, and the supernatant was collected. After filtration using a 0.45 μm filter (Millipore), the virus solution was centrifuged at 70,000 g for 2 hours at 21 ° C., and the supernatant was removed. Suspend the virus in 200 μl of DMEM to prepare a 10-fold dilution series, and then infect HeLa cells plated with 1 × 10 ⁵ cells / well in a 6-well plate. The price was calculated. When introducing MT1-MMP and mutants into macrophages, they were infected with 3 MOI, and gene expression was confirmed by RT-PCR using the following specific primers.
MT1-MMP (sense); 5-atgtctccccgcccctcgacc-3 ′ (SEQ ID NO: 15)
MT1-MMP (antisense); 5'-actattggccttgactcagt-3 '(SEQ ID NO: 16)
β-actin (sense); 5′-gccaacacagtgctgtctgg-3 ′ (SEQ ID NO: 17)
β-actin (antisense); 5′-atctgctggaaggtggacag-3 ′. (SEQ ID NO: 18)

1-13. Preparation of Adenovirus Vector An adenovirus vector was prepared using ViraPower ™ Adenoviral Expression System (Invitrogen). The virus titer was measured according to the instructions, and the virus titer was determined from the number of plaques produced after preparing a 10-fold dilution series of virus solution and infecting 293A cells.
1-14. Immunoblotting
The cells were thawed with 1 ml of lysis buffer (1% NP-40, 50 mM Tris pH 8.0, 150 mM NaCl) and centrifuged at 20,000 g for 15 minutes at 4 ° C. The supernatant was collected and the protein concentration was measured with Bradford assay kit (Bio-Rad). Laemmli sample buffer and mercaptoethanol were added to the cell lysate and heated, and then subjected to SDS-PAGE. After transferring the protein from the gel to a PVDF membrane (Millipore), anti-MT1-MMP mouse antibody (Daiichi Fine Chemical), anti-transferrin receptor mouse antibody (Invitrogen), anti-p300 / CBP antibody (Upstate), anti-lamin A / C mouse antibody (BD Biosciences), anti-FIH-1 goat antibody (Santa Cruz Biotechnology), anti-actin mouse antibody (CHEMICON), anti-FLAG M2 antibody (Sigma), anti-Myc mouse antibody (Roche), anti-V5 mouse antibody (Invitrogen), anti-vitrogen The reaction was carried out overnight with APBA3 mouse antibody (BD Biosciences) at 4 ° C. After washing 3 times with PBST, it was reacted with HRP-added anti-mouse IgG antibody (GE Healthcare) and HRP-added anti-goat IgG antibody (SIGMA) for 1 hour, washed 5 times with PBST, and then allowed to emit light with ECL plus (GE Healthcare). A band was detected.

1-15. Yeast-Two-Hybrid Screening Using a commercial service from Hybridigens, FIH-1 was baited using a human placenta cDNA library.
1-16. Expression suppression experiment by shRNA
The shRNA sequences used are as follows.
Mouse FIH-1; 5'-caccggaccctcgaataccctcaagacgaatctttgcaggttattcgaggtcctttt-3 '(SEQ ID NO: 19)
Human FIH-1 # 1; 5'-caccgctgaccgacacaacaatctttgtcgaaaaacagattttgtgtggtcagcttttt-3 '(SEQ ID NO: 20)
Human FIH-1 # 2; 5'-caccggaagagtgtcatggactctcgaaagaagatccatgacaatctttcctttt-3 '(SEQ ID NO: 21)
Mouse APBA3 / iFIH; 5'-caccgccacttcctacagggagaacacagagagtgtctcctgtgagaactggc-3 '(SEQ ID NO: 22)
These sequences were cloned into pENTR / U6 TOPO and subcloned into pLenti6 BLOCKiT lentiviral vector (Invitrogen) with LR recombination. The shRNA expression lentiviral vector was prepared as described above, and the shRNA expression lentiviral vector was introduced into each cell at 40 MOI. Macrophages were used for experiments 72 hours after introduction. HEK293 cells were subjected to drug selection with blasticidin for 1 week 72 hours after introduction and then used for experiments.

1-17. Expression suppression experiment using siRNA The siRNA designed and synthesized by B-bridge was used. This siRNA is three different siRNA cocktails for one gene. The sequence is as follows.
Human APBA3 / iFIH:
5′-gauggaacuguugaugagua-3 ′ (SEQ ID NO: 23);
5'-gggaggguccaccucgagaa-3 '(SEQ ID NO: 24);
5′-gguucukuguguccugauga-3 ′ (SEQ ID NO: 25).
Control siRNAs:
5′-auccgcgcgauaguacgua-3 ′ (SEQ ID NO: 26)
5′-uuacgcguagcguauaacg-3 ′ (SEQ ID NO: 27)
5′-uauucgcgcguauagcggu-3 ′ (SEQ ID NO: 28)
HEK293 cells were seeded at 1 × 10 ⁴ / well in a 24-well plate, and 10 nM siRNA cocktail (including three target sequences) was introduced using Lipofectamine ^™ RNAiMAX (Invitrogen) as specified. A reporter assay was performed 48 hours after the introduction.

2. Result 2-1. MT1-MMP increases the transcriptional activity of HIF-1α depending on the intracellular region (CP).
Since CAD of HIF-1α transmits transcriptional activity by binding to p300 / CBP, it was examined whether the transcriptional activity of CAD is controlled by MT1-MMP. In order to analyze the transcription activity of CAD itself, a vector expressing a chimeric protein in which a Gal4 DNA binding region and a CAD fragment are combined is constructed, and when the CAD fusion protein is bound, luciferase is transcribed and the activity of luciferase is measured. A reporter system (Lando et al., 2002b) that can measure the transcriptional activity of CAD was used (FIG. 1a). It was revealed that when a reporter plasmid and an MT1-MMP expression plasmid were introduced into HEK293 cells, the transcriptional activity of CAD increased about 2.3 times compared to the control (FIG. 1b). Furthermore, in order to determine which domain of MT1-MMP is involved in the control of CAD of HIF-1α, a reporter assay was performed by introducing an MT1-MMP deletion mutant expression vector. As a result, the enzyme active domain-deficient (dCAT) and hemopexin-like domain-deficient (dHPX) mutants activated CAD to the same extent as wild-type MT1-MMP, but the intracellular domain-deficient (dCP) mutants showed CAD activity. Conversion was not seen (FIG. 1b). Moreover, the mutant (TM-CP) from the transmembrane domain at the C-terminal of MT1-MMP to CP was sufficient for the activation of CAD (FIG. 1b, TM-CP).
These results revealed that MT1-MMP activated HIF-1α CAD in an intracellular region-dependent manner. Furthermore, when these MT1-MMP mutants were introduced into a lentiviral vector into MT1-MMP-deficient macrophages, the ATP amount of MT1-MMP-deficient macrophages with mutants other than dCP (FIG. 1c) and Cell motility (FIG. 1d) was restored.

  The MT1-MMP CP is composed of 20 amino acids (SEQ ID NO: 12). Therefore, in order to analyze which amino acid residue is necessary for the control of CAD, a mutant was prepared in which the C-terminal of MT1-MMP was deleted by 4, 8, 12, 16, 20 amino acids. As a result of introduction of these mutants and a reporter plasmid into HEK293 cells and analysis, the deletion of the first 4 amino acids (dCP17-20) did not affect the activation of CAD (FIG. 2a). However, the activation of CAD did not occur as in the case of dCP having no intracellular region due to the deletion of the next 4 amino acids (dCP13-20) (FIG. 2a). That is, it was considered that the amino acid CP13-16 plays an important role in the activation of CAD by MT1-MMP. Therefore, as a result of preparing a mutant in which all four amino acids of CP13-16 were substituted with alanine and conducting a reporter assay, activation of CAD by MT1-MMP did not occur in the alanine-substituted mutant (FIG. 2b). Furthermore, when a mutant in which each amino acid of CP13-16 was substituted with alanine was prepared and analyzed, the ability to activate CAD was reduced to a different extent for each amino acid substitution. Among them, the R / A substitution of CP14 had the greatest effect, and the CAD activation ability of MT1-MMP was almost completely lost (FIG. 2b, R / A). That is, it was revealed that arginine of CP14 is important for CAD activation.

2-2. FIH-1 mediates MT1-MMP-dependent HIF-1α regulation.
FIH-1 is the only negative regulator of HIF-1α CAD currently reported (Lando et al., 2002a; Mahon et al., 2001). Therefore, in order to verify whether FIH-1 is involved in the activation of CAD by MT1-MMP in HEK293 cells, HEK293 cells in which the expression level of endogenous FIH-1 was reduced using shRNA were prepared. (FIG. 3a, shFIH-1 # 1 and # 2). The decrease in the expression level of FIH-1 increased the activity of CAD by about 5 times in HEK293 (FIG. 3b, mock), and in this situation, no activation of CAD by MT1-MMP was detected (FIG. 3b, MT1F, dCP). Similarly, when FIH-1 expression in wild-type and MT1-MMP-deficient macrophages is reduced using a shRNA-expressing lentiviral vector (FIG. 3c), the amount of ATP in MT1-MMP-deficient macrophages to the same extent as wild-type macrophages. Recovered (FIG. 3d). From these results, it was suggested that MT1-MMP suppressed the function of FIH-1 in a CP-dependent manner, and as a result, the transcriptional activity of HIF-1α was increased.
Subsequently, in order to analyze whether there is a direct interaction between CP and FIH-1 of MT1-MMP, recombinant protein (GST-CP) in which CP peptide is bound to glutathione S-transferase (GST) ( (His) A pull-down assay using 6-labeled FIH-1 was performed. As a result, FIH-1 bound to GST-CP but not to GST (FIG. 4a). Further, the binding between GST-CP and FIH-1 was competed in a dose-dependent manner with the CP peptide, but not with the control peptide (FIG. 4a, CP peptide / GST-CP; control peptide / GST-CP). Subsequently, the interaction between FIH-1 and GST-CP (GST-R / A) in which arginine of CP14, which is important for CAD activation, was clarified in the experiment of FIG. As a result, the binding ability of GST-R / A to FIH-1 was significantly reduced as compared with GST-CP (FIG. 4b). Moreover, when the MT1-MMP mutant with R / A substitution was introduced into MT1-MMP-deficient macrophages, an increase in ATP amount was observed in wild-type MT1-MMP-introduced macrophages, but the R / A mutant was introduced. Even so, ATP production could not be increased (FIG. 4c).
From the above results, it was revealed that FIH-1 is a mediator when MT1-MMP controls HIF-1α in a CP-dependent manner.

2-3. APBA3 / Mint3 is a novel binding factor for FIH-1.
Preliminary experiments showed that MT1-MMP CP peptide did not affect FIH-1 function at least in vitro, so MT1-MMP alone did not directly inhibit FIH-1, but other molecules We hypothesized that the presence of the protein may be required, and searched for the binding factor of FIH-1 by yeast-two hybrid screening. As a result of analyzing the binding between the candidate protein obtained by the yeast-two hybrid screening and FIH-1 by immunoprecipitation, only APBA3 / Mint-3 showed stable binding to FIH-1 (FIG. 5b). . APBA3 is a homologue of APBA1 and is known to bind to the intracellular region of amyloid beta precursor protein (Tanahashi and Tabira, 1999). APBA1 and 2 are mainly expressed in the nervous system, whereas APBA3 has been confirmed to be expressed in a wide range of tissues (Okamoto et al., 2001; Okamoto and Sudhof, 1998; Tanahashi and Tabira, 1999). Its biological role is unclear. The C-terminal region of APBA3 consists of one PTB domain and two PDZ domains having homology with APBA1 and 2, but the N-terminal region has a unique sequence that does not show homology with other APBA (FIG. 7a). . APBA3 has been reported to bind to the intracellular region of MT5-MMP via the PDZ domain (Wang et al., 2004) and did not show clear binding to the intracellular region of MT1-MMP (FIG. 6). .
Subsequently, in order to analyze to which region of APBA3 FIH-1 binds, the N-terminal and C-terminal of FLAG-tagged APBA3 were co-expressed in Myc-tagged FIH-1 and HEK293 cells, respectively. It was subjected to immunoprecipitation. As a result, FIH-1 was co-precipitated at the full length and N-terminus of APBA3, but co-precipitation did not occur at the C-terminus (FIG. 7b). From the above, it was revealed that FIH-1 binds to the N-terminal region of APBA3, which is a unique region among families.

2-4. APBA3 enhances the transcriptional activity of HIF-1α by competitively inhibiting the binding between FIH-1 and HIF-1αCAD.
Subsequently, which region of FIH-1 was required for binding to APBA3 was analyzed by pull-down assay. FIH-1 has an enzyme active domain having a Cupin-like structure and a dimerization domain on the C-terminal side (FIG. 7a). FIH-1 binds to HIF-1α at the C-terminal dimer binding site and hydroxylates the asparagine residue of HIF-1α in the enzyme active domain (Dann et al., 2002; Elkins et al., 2003; Hewitson et al. 2002; Lancaster et al., 2004). GST-fused FIH-1 (GST-FIH-1) bound to the N-terminal region fragment of APBA3 as well as the CAD fragment of HIF-1α (FIG. 7c). In contrast, GST-FIH-1 lacking the C-terminal dimerization domain (GST-FIH-1N) did not bind to either CAD of HIF-1α or the N-terminal region of APBA3 (FIG. 7c). On the other hand, Von Hippel-Lindau protein (VHL) (Lee et al., 2003), which is known to bind to the N-terminal side of FIH-1, binds to both GST-FIH-1 lacking full-length and C-terminal. Was confirmed (FIG. 7c, VHL). The above results revealed that the FIH-1 C-terminal dimerization domain is required for binding to APBA3 and HIF-1α CAD. Interestingly, pull-down of CAD fragments by GST-FIH-1 was competitively inhibited by APBA3 N-terminal fragment in a dose-dependent manner (FIG. 7d), but such competitive inhibition did not occur in VHL (FIG. 7e). ). This result was attributed to the fact that both the N-terminal fragment of HIF-1α CAD and APBA3 required a dimerization domain for binding to FIH-1.

Since APBA3 competed with the binding between HIF-1α and FIH-1, it was considered that APBA3 might inhibit the function of FIH-1 that suppresses the transcriptional activity of HIF-1α. Therefore, the effect of APBA3 on the transcriptional activity of HIF-1α was analyzed using a reporter assay. When APBA3 was expressed in HEK293 cells, the activity of HIF-1α was dramatically increased compared to the control group (FIG. 8a). Subsequently, in order to confirm whether the activation of HIF-1α CAD by APBA3 is mediated by FIH-1, a reporter assay was performed using HEK293 cells in which FIH-1 expression was reduced by two shRNA expression vectors. (FIG. 8b). As a result, the activity of CAD in the control group increased according to the decrease in the expression level of FIH-1, and was almost the same as that of the group into which APBA3 was introduced (FIG. 8a). From this, it became clear that the activation of HIF-1α CAD by APBA3 is mediated by FIH-1. Moreover, as a result of analyzing which region of APBA3 is related to activation of HIF-1α CAD, the N-terminal fragment of APBA3 that binds to FIH-1 activated HIF-1α CAD, but bound to FIH-1. The non-C-terminal fragment did not affect the activity of CAD (FIG. 8c).
The above results supported the idea that APBA3 acts competitively on the binding between HIF-1α and FIH-1 and prevents the inactivation of HIF-1α by FIH-1. Therefore, APBA3 was newly named “inhibitor of FIH-1 (iFIH)” based on its function.

2-5. iFIH is essential for the activation of HIF-1α by MT1-MMP.
Since APBA is known to localize to the cell membrane by binding to the PDZ binding sequence frequently found in the intracellular domain of the membrane protein via the PDZ domain (Tanahashi and Tabira, 1999), iFIH is MT1-MMP. It was considered to be a key to explain the mechanism of FIH-1 suppression by. Therefore, it was examined whether iFIH is necessary for the activation of CAD by MT1-MMP. First, MT1-MMP was expressed in HEK293 cells, and it was confirmed by a reporter assay that the activity of HIF-1α was increased (FIG. 9a). In order to analyze whether iFIH is involved in this process, the expression of iFIH was decreased using siRNA (FIG. 9b). The effect of decreased expression of iFIH was not clear in the control group, suggesting that endogenous iFIH alone is not sufficient to control the activity of HIF-1α (FIG. 9a, mock). However, the decrease in iFIH expression canceled the activation of HIF-1α by MT1-MMP (FIG. 9a, MT1F). This revealed that iFIH plays an important role in the activation of HIF-1α CAD by MT1-MMP. However, overexpression of iFIH in HEK293 cells increased HIF-1α activity in the absence of MT1-MMP (FIG. 8), whereas the different results show that endogenous iFIH has little effect on HIF-1α activity. was gotten. Since the difference in the expression level was considered as the cause, the relationship between iFIH and MT1-MMP in the activation of HIF-1α was analyzed using a reporter assay under the condition of low expression level.

First, a condition was set in which MT1-MMP or iFIH alone hardly increased the activity of HIF-1α (FIG. 10a, iFIH + MT1). Under these conditions, co-expression of iFIH and MT1-MMP increased the activity of HIF-1α synergistically (FIG. 10a, iFIH + MT1). As shown in FIG. 8c, the N-terminal region of iFIH is sufficient to bind to FIH-1 and cancel the inhibition of HIF-1α activity. Interestingly, however, MT1-MMP did not affect the activation of HIF-1α by iFIH-N (FIG. 10a, iFIH-N + MT1). Since the C-terminal region lacking iFIH-N is important for localization to the cell membrane (Okamoto et al., 2001), a myristoylation signal (Cross et al., 1984) is added to iFIH-N (Myr-iFIH- N) It was made possible to localize to the cell membrane (FIG. 10b). Then, Myr-iFIH-N cooperated with MT1-MMP to increase the activity of CAD (FIG. 10a, Myr-iFIH-N).
From the above results, under the conditions of low expression close to the endogenous expression level, iFIH alone hardly activates CAD, but it can be activated by the presence of MT1-MMP, and also depends on MT1-MMP. It was revealed that iFIH needs to be localized in the cell membrane for effective iFIH control.

The experiment using macrophages described above showed that MT1-MMP promoted ATP production by suppressing FIH-1 (FIG. 3). Therefore, it was analyzed whether iFIH is related to this process. When the iFIH expression level was decreased using the shRNA expression lentiviral vector, the ATP level of wild-type macrophages decreased to about 50% (FIG. 11, WT, shiFIH). On the other hand, the amount of ATP (about 40% of the wild type) in MT1-MMP-deficient macrophages was not affected by the decreased expression of iFIH-1 (FIG. 11, MT1-/-, shiFIH).
From the above results, it was revealed that iFIH plays an important role in the control of ATP production by MT1-MMP in macrophages.

2-6. MT1-MMP promotes the binding between iFIH and FIH-1 in an intracellular region-dependent manner.
From the results so far, it is clear that the binding between iFIH and FIH-1 is important for the suppression of FIH-1 (FIGS. 7 and 8), and that FIFI-1 is required for the suppression of FIH-1 by MT1-MMP. (FIGS. 10 and 11). Therefore, suppression of FIH-1 by MT1-MMP at the cellular level was thought to be due to promotion of the binding between iFIH and FIH-1. In order to verify this possibility, HEK293 cells that stably express FLAG-tagged FIH-1 were first prepared, and then a Myc-tagged MT1-MMP and a mutant lacking an intracellular region (dCP) A cell in which was stably expressed was prepared. FIH-1 was immunoprecipitated from the cell lysate using anti-FLAG antibody beads, and iFIH in the precipitate was analyzed by immunoblotting. As a result, endogenous FIH-1 has been co-precipitated to the same extent in all cell groups, whereas endogenous FIFIH has been clearly co-precipitated by MT1-MMP expression compared to control EGFP. (FIG. 12). In addition, the intracellular region of MT1-MMP was important for the promotion of coprecipitation (FIG. 12, dCP). On the other hand, MT1-MMP was not detected in this sediment. These results revealed a mechanism in which MT1-MMP suppresses FIH-1 activity and activates HIF-1α by promoting the binding of FIH-1 and iFIH in an intracellular region-dependent manner.

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  The present invention provides a method for controlling the activity of HIF-1 and a screening method for compounds that regulate the activity of HIF-1. Therefore, it greatly contributes to the development of therapeutic methods and therapeutic agents for diseases related to HIF-1.

FIG. 1 shows the analysis result of the domain of MT1-MMP necessary for the activation of HIF-1α CAD. (A): Upper panel: Reporter system for monitoring the transcriptional activity of HIF-1α. CAD: HIF-1α C-terminal transcriptional activation domain: Gal4BD, Gal4 DNA binding domain: UAS, Gal4 binding site; TATA, thymidine kinase minimal promoter. Lower panel: MT1-MMP deficient mutant construct. SP, signal peptide; Pro, propeptide; CAT, enzyme active domain; HPX, hemopexin-like domain; TM, transmembrane domain; CP, intracellular region. A FLAG tag was inserted between Pro and CAT. SP and Pro are separated while being transported to the cell membrane. (B): Upper panel: Effect of MT1-MMP construct on HIF-1α CAD reporter activity in HEK293 cells. Lower panel: MT1-MMP construct expression was confirmed by immunoblotting. (C, d): Analysis of effects on ATP amount (c) and motility (d) when MT1-MMP-deficient mutant is introduced into MT1-/-macrophages. MT1-MMP macrophages were introduced with MT1-MMP-deficient mutant expression gene using a lentiviral vector, and the expression of MT1-MMP mutant was confirmed by RT-PCR (c, lower panel). The amount of ATP was measured 72 hours after gene introduction (c, upper panel). For motility, cells were seeded on a transwell not coated with Matrigel, and cells that migrated to the lower chamber after 2 hours were counted (d). (Bd) Data are shown as mean ± standard deviation and analyzed by Student's t test. * P <0.05, ** p <0.01. FIG. 2 shows the analysis result of the ability to activate HIF-1α CAD of the MT1-MMP intracellular region mutant. (A, b): Analysis of the CAD activation ability of the intracellular region (CP) mutant of MT1-MMP. The effects of the MT1-MMP-CP continuous deletion mutant (a) and the CP13-16 alanine substitution mutant (b) on the CAD reporter were analyzed. The construct is shown in the left panel and the effect on the CAD reporter is shown in the right panel. The expression of each mutant was confirmed by immunoblotting (lower panel). Data are shown as mean ± standard deviation and analyzed by Student's t test. * P <0.05, ** p <0.01. FIG. 3 shows that the increase in transcriptional activity of HIF-1α by MT1-MMP is mediated by FIH-1. (A): FIH-1 expression suppression in HEK293 cells. Two different shRNAs (# 1 and # 2 sequences) were expressed, and the expression level of FIH-1 was analyzed by immunoblotting. (B): Effect of shRNA on CAD reporter activity. (C): FIH-1 expression suppression in WT and MT1 − / − macrophages. ShRNA was expressed in macrophages using a lentiviral vector, and the amount of FIH-1 mRNA was analyzed by real-time PCR. (D): Influence of FIH-1 expression suppression on WT and MT1-/-macrophage ATP levels. By reducing the expression level of FIH-1, there was no change in the ATP level of WT and MT1 − / − macrophages. (B, d) Data are shown as mean ± standard deviation and analyzed by Student's t test. ** p <0.01. FIG. 4 shows that the intracellular region of MT1-MMP binds directly to FIH-1. (A): Analysis of direct binding between intracellular region (CP) of MT1-MMP and FIH-1. In vitro pull-down assay was performed using GST-CP fusion protein and (His) 6-labeled FIH-1 protein. FIH-1 bound to CP was detected by immunoblotting (IB). The binding of FIH-1 and GST-CP was competed with the CP peptide but not with the control peptide. (B): The effect of R / A substitution at CP14 on CP-FIH-1 binding was analyzed using a pull-down assay. (C) Effect on the amount of ATP when R / A-substituted MT1-MMP is introduced into MT1-/-macrophages. The construct shown in the figure was introduced into macrophages using a lentiviral vector. Upper panel: confirmation of MT1-MMP mRNA expression in MT1-/-macrophages by RT-PCR. Lower panel: ATP amount. Data are shown as mean ± standard deviation and analyzed by Student's t test. * P <0.05. FIG. 5 shows that APBA3 / iFIH binds to FIH-1. (A): After yeast two hybrid screening using human placenta cDNA library, eight proteins were selected as candidates for binding to FIH-1. A FLAG tag was added to these proteins and expressed in HEK293 cells, and the binding ability to Myc-tagged FIH-1 was analyzed. The candidate protein was immunoprecipitated (IP) from the cell lysate using an anti-FLAG antibody (upper panel), and the candidate protein in the precipitate was detected by immunoblotting (IB) using the anti-FLAG antibody. FIH-1 in immunoprecipitate (IP) and FIH-1 in cell lysate (WCL) were detected by immunoblotting (lower panel). FIH-1, factor inhibiting HIF-1; mock, mock-transfected; APBA3, amyloid precursor protein-binding member 3, CCM2, cerebral cavernous malformation; mitochondrial ribosomal protein L38; NFKBIA, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor ; PAFAH1B2, Homo sapiens platelet-activating factor acetylhydrolase isoform Ib beta subunit 30kDa; SH3BLG1, SH3-domain GRB2-like endophilin B1. FIH-1 co-precipitated only with APBA3 / iFIH. (B): VBA-tagged APBA3 / iFIH and FLAG-tagged FIH-1 were expressed in HEK293 cells. Immunoprecipitation (IP) was performed as described above, and immunoblotting (IB) was performed using the antibodies shown in the figure. APBA3 / iFIH co-precipitated with FIH-1. FIG. 6 shows the results of an APBA3 / iFIH pull-down assay using GST fusion proteins in the intracellular region of MT1-MMP and MT5-MMP. His6-labeled APBA3 / iFIH was analyzed by pull-down assay using GST fusion protein (GST, GST-MT1CP, GST-MT5CP, GST-FIH-1) -added Sepharose beads. GST, glutathione S transferase, GST-MT1CP, MT1-MMP intracellular region-GST fusion protein; GST-MT5CP, MT5-MMP intracellular region-GST fusion protein; GST-FIH-1, FIH-1-GST fusion protein. APBA3 / iFIH bound to the beads was detected by immunoblotting using an anti-His6 antibody (upper panel). The GST protein added to the beads was detected with an anti-GST antibody. GST-FIH-1 bound best with APBA3 / iFIH, and GST-MT5CP bound less. Under these experimental conditions, the binding between GST-MT1CP and APBA3 / iFIH could not be detected. FIG. 7 shows that APBA3 and HIF-1α bind competitively with FIH-1. (A): Structure of APBA3 and each fragment: full length (full), N-terminal (N) and C-terminal (C) regions (upper panel). The N-terminal region (N) does not include a known domain structure. The C-terminal region (C) contains one PTB domain and two PDZ domains. The numbers shown in the figure represent amino acid numbers. FIH-1 and N-terminal fragment structure (bottom panel): CUPIN; cupin-like domain responsible for enzyme activity, black box; dimerization domain; FIH-1 N, N-terminal fragment. (B): InHyc-tagged FIH-1 and FLAG-tagged APBA3 full length (full), N-terminal fragment (N) or C-terminal fragment (C) were expressed in HEK293 cells. FLAG-tagged protein was immunoprecipitated (IP) and detected by immunoblotting (IB) using an anti-FLAG antibody (upper panel). FIH-1 in the precipitate (IP) and cell lysate (WCL) was detected by immunoblotting (IB) using an anti-Myc antibody (lower panel). (C): Pull-down assay using beads to which GST, GST-FIH-1, and GST-FIH-1N have been added. A His6-tagged APBA3N fragment, HIF-1α CAD, or VHL was detected by immunoblotting. (D): A pull-down assay of His6-tagged HIF-1α CAD using beads added with GST-FIH-1 was carried out in the presence of the amount of His6-labeled APBA3N fragment shown in the figure. (E): Pull-down assay of His6-tagged VHL protein using beads added with GST-FIH-1 was performed in the presence of the amount of His6-labeled APBA3N fragment shown in the figure. FIG. 8 shows that APBA3 inhibits the transcriptional activity suppression function of FIH-1 against HIF-1α. (A): APK3 expression construct (APBA3, white box) or control vector (mock, black box) was introduced into HEK293, and relative luciferase activity was measured. Endogenous FIH-1 expression was suppressed using the shRNA sequences shFIH # 1 and shFIH # 2. As a negative control, shRNA (shLacZ) against a gene (LacZ) encoding β-galactosidase was used. The relative luciferase activity was shown with 100% of cells into which the Mock vector and shLacZ were introduced. (B): FIH-1 expression of shLacZ, shFIH # 1, and shFIH # 2 expressing HEK293 cells was analyzed by immunoblotting. (C): Expression of luciferase was used to measure the effect of full-length APBA3, N-terminal and C-terminal fragments on the transcriptional activity of HIF-1α CAD. Relative luciferase activity was shown with 100% of cells transfected with the control vector. Data are shown as mean ± standard deviation (n = 3) and analyzed by Student's t test. ** p <0.01. FIG. 9 shows that iFIH / APBA3 is required for the suppression of FIH-1 by MT1-MMP. (A): siRNA against iFIH (iFIH siRNA; open box) or siRNA for control (control siRNA; black box) is introduced into HEK293 cells, and then MT1-MMP (MT1F) or control vector (mock) and reporter plasmid are introduced into the gene. did. The relative luciferase activity was determined with the luciferase activity of the control siRNA and mock introduced cells as 100%. Data are shown as mean ± standard deviation (n = 3) and analyzed by Student's t test. ** p <0.01. (B): iFIH, MT1-MMP, and actin (loading control) were detected by immunoblotting. FIG. 10 shows that membrane localization is required for iFIH / APBA3 to work cooperatively with MT1-MMP. (A): MT1-MMP, iFIH, iFIH N-terminal fragment (iFIH-N) was co-expressed in HEK293 cells. The activity of HIF-1αCAD was monitored by luciferase activity, and relative luciferase activity was shown with the luciferase activity of the cells into which the mock vector had been introduced as 100% (upper panel). The expression of MT1-MMP or iFIH was confirmed by immunoblotting (lower panel). Arrow is endogenous iFIH. Data are shown as mean ± standard deviation (n = 3) and analyzed by Student's t test. ** p <0.01. (B): Localization of APBA3 / iFIH, APBA3-N / iFIH-N and Myr-APBA3-N / Myr-iFIH-N. FLAG-tagged iFIH, iFIH-N and Myr-iFIH-N were expressed in HEK293 cells. The intracellular localization of these proteins was analyzed using an anti-FLAG antibody. Nuclei were counterstained with Hoechst 33342. Compared to iFIH-N, Myr-iFIH-N is localized in the periphery of the cell. FIG. 11 shows that iFIH is required for promotion of ATP production by MT1-MMP in macrophages. shRNA was used to suppress iFIH expression of WT and MT1 − / − macrophages. ShRNA against the LacZ gene (shLacZ) was used as a negative control. The amount of intracellular ATP was measured (upper panel), and protein expression was confirmed by immunoblotting using the antibodies shown in the figure (lower panel). Data are shown as mean ± standard deviation (n = 3) and analyzed by Student's t test. ** p <0.01. FIG. 12 shows that MT1-MMP promotes the binding of FIH-1 and iFIH in an intracellular region-dependent manner. HEK293 cells were co-expressed with FLAG-tagged FIH-1 and negative control (EGFP), MT1-MMP (MT1), or MT1-MMP-CP deficient mutant (dCP). Cell lysate (WCL) and anti-FLAG antibody immunoprecipitate (IP) were analyzed by immunoblotting using the antibodies shown in the figure. It is observed that endogenous FIH-1 (arrow) forms a homodimer with FLAG-labeled FIH-1.

Claims (11)

  A method of promoting or inhibiting the activity of HIF-1α by enhancing or reducing the interaction between intracellular iFIH protein and FIH-1 protein.   The method of claim 1, wherein the FIH-1 protein interacts with MT1-MMP-CP.   The method according to claim 1, wherein the expression of iFIH-1 protein is reduced by RNAi method to reduce the interaction between iFIH protein and FIH-1.   A method of promoting or inhibiting the activity of HIF-1α by enhancing or decreasing the interaction between intracellular MT1-MMP-CP and FIH-1 protein.   One or several reductions in the interaction between the MT1-MMP-CP and the FIH-1 protein in the peptide represented by the amino acid sequence represented by SEQ ID NO: 12 or the amino acid sequence represented by SEQ ID NO: 12 Claims that are achieved by competition with a peptide that comprises an amino acid sequence having amino acid substitutions, deletions or insertions, interacts with FIH-1 protein and does not inhibit activation of HIF-1α by FIH-1 protein Item 5. The method according to Item 4.   The method according to any one of claims 1 to 5, wherein the cell is a cell of an immune system or a tumor cell. A method of screening for compounds that promote or inhibit HIF-1 activity comprising:
(A) contacting FIH-1 protein with iFIH protein or MT1-MMP-CP in the presence of a test sample;
(B) detecting the interaction between FIH-1 protein and iFIH protein or MT1-MMP-CP, and (c) comparing the interaction with that in the absence of the test sample, Determining that the compound candidate is present in the test sample when the action is enhanced or decreased,
Including methods.   The method according to claim 7, wherein the interaction between the FIH-1 protein and the iFIH protein or MT1-MMP-CP is detected based on the presence or absence of binding between the FIH-1 protein and the iFIH protein or MT1-MMP-CP.   The method according to claim 7 or 8, further comprising the step of confirming that the obtained candidate compound promotes or inhibits intracellular HIF-1α activity.   A compound isolated by the method according to any one of claims 7 to 9.   The compound according to claim 10, wherein the compound is an antibody.