CN1871360A

CN1871360A - Method of detecting regulation effect of synoviolin activity

Info

Publication number: CN1871360A
Application number: CN 200480030998
Authority: CN
Inventors: 中岛利博; 天野彻也; 张蕾; 池田理惠; 山崎聪士; 八木下尚子; 模浜广光; 小串基治
Original assignee: Eisai Co Ltd; Locomogene Inc
Current assignee: Eisai R&D Management Co Ltd; Locomogene Inc
Priority date: 2003-08-20
Filing date: 2004-08-20
Publication date: 2006-11-29

Abstract

It is intended to provide a method for evaluating an effect of controlling the activity of proline 4-hydroxyolase; a screening method with the use of the above evaluation method; and a method of screening a compound which is useful in treating or preventing fibrosis or rheumatoid arthritis. A method for detecting an effect of controlling the ubiquitination activity of proline 4-hydroxyolase alpha subunit (P4HA1) by synoviolin; and a method of screening a substance having an effect of controlling the ubiquitination activity of P4HA1 of synoviolin based on the above method. A compound found out by this screening is useful in treating or preventing diseases caused by disorders in the activity of proline 4-hydroxylase such as fibrosis and rheumatoid arthritis.

Description

Detection is to the method for the regulating effect of synovial hyperplasia factor active

Technical field

The present invention relates to detect method to the active regulating effect of the synovial hyperplasia factor (synoviolin).

And, the present invention relates to screen to treating or prevent the method for the useful compound of fibrosis and/or rheumatosis.

Background field

The synovial hyperplasia factor is a kind of protein, finds that it is a kind of membranin (referring to WO 02/052007) that is present on the synovial cell in rheumatisant source.This factor that studies show that with the animal of genetic modification is participated in the growth and the arthropathic morbidity in bone/joint directly.Therefore, think that the synovial hyperplasia factor helps the activity of normal osteogenesis or normal epiphysis growth.The protein structure prediction system shows have RING finger print preface in the synovial hyperplasia factor.Known this block is present in the E3 ubiquitin protein ligase that participates in protein degradation.In fact, confirmed that the synovial hyperplasia factor has self ubiquitin activity, this activity is that RING refers to-characteristic (seeing WO 02/052007) of type E3 ubiquitin protein ligase.

Seeking synovial hyperplasia factor binding factor is the effective way of illustrating synovial hyperplasia factor function in bone/joint development and joint disease, and this function is the intracellular signaling pathway that the synovial hyperplasia factor participates in.Especially, the substrate protein white matter of the evaluation synovial hyperplasia factor is to understand the vital task of the intracellular signaling pathway of synovial hyperplasia factor participation.

Identifying the substrate protein white matter of synovial hyperplasia factor bindin, particularly the synovial hyperplasia factor, is useful to illustrating function and the diagnosis of exploitation joint disease and the treatment novel method of the synovial hyperplasia factor in bone/joint development and joint disease.But, also do not identify the substrate of the synovial hyperplasia factor.Therefore, it is still unclear to influence the material of the synovial hyperplasia factor and its substrate interaction.

Simultaneously, collagen is important constituent and has following feature in live body:

It is the important component of reticular tissue;

Have one or more structural domains that triple-helix structure is arranged;

Form the supramolecule aggregate; With

Constitute extracellular matrix.

A main effect of collagen is that the formative tissue structure is beneficial to the cell grappling.Collagen also influences the migration of cell, differentiation, propagation and metabolic function.Inoblast is collagenic main cell.Originally, synthetic precollagen early and in rough surfaced endoplasmic reticulum, change into precollagen in cell.Propetide is connected in precollagenous N-and C-end.By precollagen peptide enzymatic lysis propetide precollagen is transformed into tropocollagen facing before emiocytosis.Excretory tropocollagen is the basic ingredient of collegen filament.The excretory tropocollagen formation collagenous fiber bundle that is cross-linked to each other.

Prolyl 4-hydroxylase is an essential enzyme during collagen synthesizes.Known the structure of prolyl 4-hydroxylase.Particularly, prolyl 4-hydroxylase has by two α subunits and two tetramer structures that the assembling of β subunit constitutes.Think that the α subunit has the function of catalysis proline(Pro) hydroxylation reaction, and the β subunit there is the disulphide isomerase activity.The α subunit that constitutes prolyl 4-hydroxylase is called P4HA1.Proline residue on prolyl 4-hydroxylase hydroxylation precollagen-X-Pro-Gly sequence, precollagen therefore form stable collagen triple-helix structure (see Pihlajanicmi, T., et al., J.Hepatol., 1991,13, S2-7).

And, reported by combination with it, prolyl 4 hydroxylases have the precollagen that prevents hydroxylation not from endoplasmic reticulum excretory function (see Walmsley, A.R, et.al., J.Biolog.Chem., 1999,274 (21), p14884-14892).The precollagen of hydroxylation is not jejune precollagen.Therefore, think that prolyl 4 hydroxylases are as the in vivo control of collagen quality.

The component that though collagen is heavy sensation of the whole body to be wanted, the biological respinse that it causes pathology is fibrosis for example.For example, collagen is piled up the fibrosis that causes organ such as lung in liver or the nephridial tissue.In rheumatosis and osteoarthropathy deformity, observe the cartilage that contains collagen in the joint and synovial cell's extracellular matrix unusual (see Masataka KUWANA, Molecular Medicine, 2001, Vol.38 (8), p900-907).

Therefore, might produce these diseases of treatment by control collagen.For example, there has been report can treat these diseases with the generation of adjusting collagen and (seen Kivirikko KI., Ann Med., 1993, Apr by inhibition stress protein HSP47 (heat shock protein(HSP) 47) activity; 25 (2): 113-26).In the exploitation of fibrosis and arthritic diagnosis and methods of treatment, the collagen of prolyl 4-hydroxylase and enzyme generation thus will become important target spot.But the mechanism of regulating prolyl 4-hydroxylase activity is still unclear.

Rheumatic arthritis (after this being called RA or rheumatosis) is the general chronic inflammation disease, observes the paraplasm that it has synovial tissue of joint.Synovial cell's (cell of synovial membrane) constitutes the fibroblast-like cells of one to six epithelial lining in synovium of joint, and thinks that it supplies protein-polysaccharide and hyaluronic acid in synovia.In RA patient's joint, observe for example synovial tissue's hyperplasia (that is, causing multilayered structure) and synovial cell are invaded symptom from profit to other tissue.The autoantibody that in the RA patients serum, has anti-self IgG Fc section.Therefore, rheumatosis is considered to autoimmune disease, but its reason is not illustrated as yet.

Summary of the invention

An object of the present invention is to illustrate the regulation mechanism of prolyl 4-hydroxylase activity.Another object of the present invention provides the method for regulating prolyl 4-hydroxylase activity of estimating.A further object of the invention provides the screening of in-service evaluation method and has the method for the compound of this function.Further purpose of the present invention provides the method for SCREENED COMPOUND, and wherein compound is useful to rheumatic arthritis or Fibrotic treatment or prevention.

Present inventors use the yeast two-hybrid method to identify the synovial hyperplasia factor-conjugated protein in screening.Using yeast two-hybrid system Clontech, MATCHMAKER Two-Hybrid System) screen in the cDNA library, human cartilage source, the synovial hyperplasia factor is used as bait.As a result, obtained prolyl 4-hydroxylase α subunit (P4HA1).Next, by the combination between the GST pulldown analysis verification P4HA1 and the synovial hyperplasia factor.

Present inventors further disclose P4HA1 by the active ubiquitinization of the ubiquitin ligase of the synovial hyperplasia factor.That is, confirm that P4HA1 is the substrate polypeptide of synovial hyperplasia factor ubiquitin ligase (E3).The synovial hyperplasia factor is induced the proteasome degraded of P4HA1 through the ubiquitinization of P4HA1.Prolyl 4-hydroxylase is the key enzyme during collagen synthesizes.Therefore, might the synovial hyperplasia factor participate in individual growth, bone/joint development and arthropathic morbidity by the activity of regulating prolyl 4-hydroxylase.

Based on these discoveries, present inventors have set up the interactional method between the synovial hyperplasia factor and the P4HA1 of disturbing of estimating.They find that also the material that uses these methods to screen this effect is possible.On the basis of these discoveries, finished the present invention.Particularly, the present invention relates to following method, it detects test-compound turns usefulness into to synovial hyperplasia factor ubiquitin adjusting activity.Perhaps, the present invention relates to utilize these method screenings that the ubiquitin of the synovial hyperplasia factor is turned into the active compound method of adjusting is arranged.

[1] a kind of test-compound that detects turns the active method of adjusting of usefulness into to synovial hyperplasia factor ubiquitin, and it comprises step:

A) under the condition that can make the substrate ubiquitinization, when test-compound exists with the synovial hyperplasia factor or its homologue and substrate insulation,

A ') with (i) substrate or (ii) the synovial hyperplasia factor or its homologue contact with test-compound, and under the condition that can make the substrate ubiquitinization with (i) and another person's insulation (ii), perhaps

A ") under the condition that can make the substrate ubiquitinization with the synovial hyperplasia factor or its homologue and substrate insulation, and they are contacted with test-compound;

B) in step a), a '), and the level of substrate ubiquitinization is measured in the arbitrary step back of a "); With

When c) the ubiquitin level that records when not having test-compound when substrate ubiquitin level is variant, detect test-compound makes the effect of substrate ubiquitinization to the synovial hyperplasia factor adjusting activity, wherein substrate is prolyl 4-hydroxylase α subunit or its homologue.

[2] method of [1] wherein coexists by following ingredients the condition that can make the substrate ubiquitinization is provided:

I) ubiquitin activating enzyme;

Ii) ubiquitin transferring enzyme;

Iii) ubiquitin; With

Iv) Triphosaden.

[3] method of [1] is wherein by providing the condition that can make the substrate ubiquitinization with the substrate ubiquitinization in the cell of expressing substrate and the synovial hyperplasia factor or its homologue.

[4] method of [1], wherein use the ubiquitin level of following any one level as the index determining substrate:

A) level of the substrate of ubiquitinization;

B) level of the substrate of ubiquitinization not; With

C) biologically active level of prolyl 4-hydroxylase α subunit.

[5] method of [1], wherein prolyl 4-hydroxylase α subunit or its homologue are following (a) to any one of (e) polypeptide:

(a) the nucleotide sequence coded polypeptide of SEQ ID NO:1;

(b) comprise the polypeptide of SEQ ID NO:2 aminoacid sequence;

(c) polypeptide, it is included in the aminoacid sequence of SEQ ID NO:2 and replaces, and deletion is inserted, and/or adds the aminoacid sequence of one or more amino acid gained, and it can be by synovial hyperplasia factor ubiquitinization;

(d) comprise the polypeptide of the aminoacid sequence of polynucleotide encoding, wherein these polynucleotide under rigorous condition with the DNA hybridization that contains SEQ ID NO:1 nucleotide sequence, and this polypeptide can be by synovial hyperplasia factor ubiquitinization; With

(e) comprise with SEQ ID NO:2 aminoacid sequence and have 70% or the polypeptide of the aminoacid sequence of above homology, and this polypeptide can be by synovial hyperplasia factor ubiquitinization.

[6] method of [1], wherein the synovial hyperplasia factor or its homologue are following (A) to any one of (E) polypeptide:

(A) the nucleotide sequence coded polypeptide of SEQ ID NO:3;

(B) comprise the polypeptide of SEQ ID NO:4 aminoacid sequence;

(C) polypeptide, it is included in the aminoacid sequence of SEQ ID NO:4 and replaces, and deletion is inserted, and/or adds the aminoacid sequence of one or more amino acid gained, and it has and will comprise the activity of the polypeptide ubiquitinization of SEQ IDNO:2 aminoacid sequence;

(D) polypeptide, its coding DNA are hybridized with the DNA that comprises SEQ ID NO:3 nucleotide sequence under rigorous condition, and it has and will comprise the activity of the polypeptide ubiquitinization of SEQ ID NO:2 aminoacid sequence; With

(E) comprise with SEQ ID NO:4 aminoacid sequence and have 70% or the polypeptide of the aminoacid sequence of above homology, and it has and will comprise the activity of the polypeptide ubiquitinization of SEQ ID NO:2 aminoacid sequence.

[7] screening turns the method for regulating active test-compound with having into to the ubiquitin of the synovial hyperplasia factor, comprises step:

A) by [1] to [6] any one method detect test-compound to synovial hyperplasia factor ubiquitin turn into usefulness the adjusting activity and

The test-compound that different substrate ubiquitin levels is arranged when b) selecting compared with the control.

[8] the substrate ubiquitin that is used to regulate the synovial hyperplasia factor turns the material of usefulness into, comprises that the compound that can obtain by the method for [7] is as activeconstituents.

[9] detect the active test kit of adjusting that synovial hyperplasia factor ubiquitin is turned into usefulness, it comprises following ingredients:

(1) the synovial hyperplasia factor or its homologue; With

(2) prolyl 4-hydroxylase α subunit or its homologue.

[10] test kit of [9] further comprises following ingredients:

(3) ubiquitin activating enzyme;

(4) ubiquitin transferring enzyme;

(5) ubiquitin; With

(6) Triphosaden.

[11] detect the active test kit of adjusting that synovial hyperplasia factor ubiquitin is turned into usefulness, comprise and express the synovial hyperplasia factor or its homologue, with the cell of prolyl 4-hydroxylase α subunit or its homologue and the instrument of detection prolyl 4-hydroxylase α subunit or its homologue ubiquitin level.

[12] a kind of test-compound that detects turns the active method of adjusting of usefulness into to synovial hyperplasia factor ubiquitin, and it comprises step:

A) can make under the synovial hyperplasia factor or its homologue and the substrate bonded condition, the insulation synovial hyperplasia factor or its homologue and substrate when test-compound exists,

A ') with (i) substrate or (ii) the synovial hyperplasia factor or its homologue contact with test-compound, and under making the synovial hyperplasia factor or its homologue and substrate bonded condition with (i) and another person's insulation (ii), perhaps

A ") can make the insulation synovial hyperplasia factor or its homologue and substrate under the substrate and the synovial hyperplasia factor or its homologue bonded condition, and they are contacted with test-compound;

B) in step a), a '), and a ") arbitrary step back measures bonded level between substrate and the synovial hyperplasia factor or its homologue; With

C) when the level that combines that records when not having test-compound in conjunction with level between substrate and the synovial hyperplasia factor or its homologue is variant, detect test-compound makes the effect of substrate ubiquitinization to the synovial hyperplasia factor adjusting activity, wherein substrate is prolyl 4-hydroxylase α subunit or its homologue.

[13] method of [12], wherein or the synovial hyperplasia factor or substrate in conjunction with solid phase or comprise can be in conjunction with the marker of solid phase.

[14] method of [12], wherein prolyl 4-hydroxylase α subunit or its homologue are following (a) to any one of (e) polypeptide:

(a) the nucleotide sequence coded polypeptide of SEQ ID NO:1;

(b) comprise the polypeptide of SEQ ID NO:2 aminoacid sequence;

(c) polypeptide, it is included in the aminoacid sequence of SEQ ID NO:2 and replaces, and deletion is inserted, and/or adds the aminoacid sequence of one or more amino acid gained, and it can be in conjunction with the synovial hyperplasia factor;

(d) comprise the polypeptide of the aminoacid sequence of polynucleotide encoding, wherein these polynucleotide under rigorous condition with the DNA hybridization that contains SEQ ID NO:1 nucleotide sequence, and this polypeptide can be in conjunction with the synovial hyperplasia factor; With

(e) comprise with SEQ ID NO:2 aminoacid sequence and have 70% or the polypeptide of the aminoacid sequence of above homology, and this polypeptide can be in conjunction with the synovial hyperplasia factor.

[15] method of [12], wherein the synovial hyperplasia factor or its homologue are following (A) to any one of (E) polypeptide:

(A) the nucleotide sequence coded polypeptide of SEQ ID NO:3;

(B) comprise the polypeptide of SEQ ID NO:4 aminoacid sequence;

(C) polypeptide, it is included in the aminoacid sequence of SEQ ID NO:4 and replaces, and deletion is inserted, and/or adds the aminoacid sequence of one or more amino acid gained, and can be in conjunction with the polypeptide that comprises SEQ IDNO:2 aminoacid sequence;

(D) polypeptide, its coding DNA are hybridized with the DNA that comprises SEQ ID NO:3 nucleotide sequence under rigorous condition, and it can be in conjunction with the polypeptide that comprises SEQ ID NO:2 aminoacid sequence; With

(E) comprise with SEQ ID NO:4 aminoacid sequence and have 70% or the polypeptide of the aminoacid sequence of above homology, and this polypeptide can be in conjunction with the polypeptide that comprises SEQ ID NO:2 aminoacid sequence.

[16] screening turns the method for regulating active test-compound with having into to the ubiquitin of the synovial hyperplasia factor, comprises step:

A) by [12] to [15] any one method detect test-compound to synovial hyperplasia factor ubiquitin turn into usefulness the adjusting activity and

B) select the substrate test-compound different with contrast with synovial hyperplasia factor bonded level.

[17] the substrate ubiquitin that is used to regulate the synovial hyperplasia factor turns the material of usefulness into, comprises that the compound that can obtain by the method for [16] is as activeconstituents.

The present inventor also finds to screen treatment or prevents Fibrotic useful compound is possible, and has therefore finished the present invention.Particularly, the present invention relates to following screening method, the test kit of this screening method and treat and/or prevent Fibrotic medicinal component it comprises that compound that available these methods obtain is as activeconstituents.

[18] a kind of method of screening treatment or preventing Fibrotic compound, it comprises step:

A) under the condition that can make the substrate ubiquitinization when test-compound exists with the synovial hyperplasia factor or its homologue and substrate insulation,

C) select test-compound as treatment or prevent Fibrotic compound, the ubiquitin level that it records when making substrate ubiquitin level be lower than this test-compound not,

Wherein substrate is prolyl 4-hydroxylase α subunit or its homologue.

[19] method of [18] wherein coexists by following ingredients the condition that can make the substrate ubiquitinization is provided:

I) ubiquitin activating enzyme;

Ii) ubiquitin transferring enzyme;

Iii) ubiquitin; With

Iv) Triphosaden.

[20] method of [18] is wherein by providing the condition that can make the substrate ubiquitinization with the substrate ubiquitinization in the cell of expressing substrate and the synovial hyperplasia factor or its homologue.

[21] method of [18], wherein use the ubiquitin level of following any one level as the index determining substrate:

A) level of the substrate of ubiquitinization;

B) level of the substrate of ubiquitinization not; With

C) biologically active level of prolyl 4-hydroxylase α subunit.

[22] method of [18], wherein prolyl 4-hydroxylase α subunit or its homologue are following (a) to any one of (e) polypeptide:

(a) the nucleotide sequence coded polypeptide of SEQ ID NO:1;

(b) comprise the polypeptide of SEQ ID NO:2 aminoacid sequence;

[23] method of [18], wherein the synovial hyperplasia factor or its homologue are following (A) to any one of (E) polypeptide:

(A) the nucleotide sequence coded polypeptide of SEQ ID NO:3;

(B) comprise the polypeptide of SEQ ID NO:4 aminoacid sequence;

(C) polypeptide, it is included in the aminoacid sequence of SEQ ID NO:4 and replaces, and deletion is inserted, and/or adds the aminoacid sequence of one or more amino acid gained, and has the activity that can ubiquitinization comprises the polypeptide of SEQ ID NO:2 aminoacid sequence;

(D) polypeptide, its coding DNA are hybridized with the DNA that comprises SEQ ID NO:3 nucleotide sequence under rigorous condition, and it has the activity that makes the polypeptide ubiquitinization that comprises SEQ ID NO:2 aminoacid sequence; With

(E) comprise with SEQ ID NO:4 aminoacid sequence and have 70% or the polypeptide of the aminoacid sequence of above homology, and it has the activity that makes the polypeptide ubiquitinization that comprises SEQ ID NO:2 aminoacid sequence.

[24] treat and/or prevent Fibrotic medicinal compositions, it comprises that the compound that can pass through [18] to [23] any one method screening is as activeconstituents.

[25] test kit of Fibrotic compound is treated or is prevented in screening, and it comprises following ingredients:

(1) the synovial hyperplasia factor or its homologue; With

(2) prolyl 4-hydroxylase α subunit or its homologue.

[26] test kit of [25] further comprises following ingredients:

(3) ubiquitin activating enzyme;

(4) ubiquitin transferring enzyme;

(5) ubiquitin; With

(6) Triphosaden.

[27] test kit of Fibrotic test-compound is treated or is prevented in screening, comprise and express the synovial hyperplasia factor or its homologue, with the cell of prolyl 4-hydroxylase α subunit or its homologue and the instrument of detection prolyl 4-hydroxylase α subunit or its homologue ubiquitin level.

[28] method of screening treatment or preventing Fibrotic compound comprises step:

C) select test-compound as treatment or prevent Fibrotic compound, wherein this compound make between substrate and the synovial hyperplasia factor or its homologue be lower than this test-compound not in conjunction with level the time record in conjunction with level,

Wherein substrate is prolyl 4-hydroxylase α subunit or its homologue.

[29] method of [28], wherein or the synovial hyperplasia factor or substrate in conjunction with solid phase or comprise can be in conjunction with the marker of solid phase.

[30] method of [28], wherein prolyl 4-hydroxylase α subunit or its homologue are following (a) to any one of (e) polypeptide:

(a) the nucleotide sequence coded polypeptide of SEQ ID NO:1;

(b) comprise the polypeptide of SEQ ID NO:2 aminoacid sequence;

[31] method of [28], wherein the synovial hyperplasia factor or its homologue are following (A) to any one of (E) polypeptide:

(A) the nucleotide sequence coded polypeptide of SEQ ID NO:3;

(B) comprise the polypeptide of SEQ ID NO:4 aminoacid sequence;

(C) polypeptide, it is included in the aminoacid sequence of SEQ ID NO:4 and replaces, and deletion is inserted, and/or adds the aminoacid sequence of one or more amino acid gained, and can be in conjunction with the polypeptide that comprises SEQ ID NO:2 aminoacid sequence;

(D) polypeptide, its coding DNA are hybridized with the DNA that comprises SEQ ID NO:3 nucleotide sequence under rigorous condition, and can be in conjunction with the polypeptide that comprises SEQ ID NO:2 aminoacid sequence; With

[32] treat and/or prevent Fibrotic medicinal compositions, comprise that the compound that can pass through [28] to [31] any one method screening is as activeconstituents.

The present inventor also finds to screen treatment or prevents the useful compound of rheumatic arthritis is possible, and has therefore finished the present invention.Particularly, the present invention relates to following screening method, the test kit of this screening method and treat and/or prevent the medicinal component of chronic rheumatic arthritis, it comprises that compound that available these methods obtain is as activeconstituents.

[33] a kind of method of screening the compound of treatment or prevention rheumatic arthritis, it comprises step:

C) select the compound of test-compound as treatment or prevention rheumatic arthritis, the ubiquitin level that it records when making substrate ubiquitin level be lower than this test-compound not,

Wherein substrate is prolyl 4-hydroxylase α subunit or its homologue.

[34] method of [33] wherein coexists by following ingredients the condition that can make the substrate ubiquitinization is provided:

I) ubiquitin activating enzyme;

Ii) ubiquitin transferring enzyme;

Iii) ubiquitin; With

Iv) Triphosaden.

[35] method of [33] is wherein by providing the condition that can make the substrate ubiquitinization with the substrate ubiquitinization in the cell of expressing substrate and the synovial hyperplasia factor or its homologue.

[36] method of [33], wherein use the ubiquitin level of following any one level as the index determining substrate:

A) level of the substrate of ubiquitinization;

B) level of the substrate of ubiquitinization not; With

C) biologically active level of prolyl 4-hydroxylase α subunit.

[37] method of [33], wherein prolyl 4-hydroxylase α subunit or its homologue are following (a) to any one of (e) polypeptide:

(a) the nucleotide sequence coded polypeptide of SEQ ID NO:1;

(b) comprise the polypeptide of SEQ ID NO:2 aminoacid sequence;

[38] method of [33], wherein the synovial hyperplasia factor or its homologue are following (A) to any one of (E) polypeptide:

(A) the nucleotide sequence coded polypeptide of SEQ ID NO:3;

(B) comprise the polypeptide of SEQ ID NO:4 aminoacid sequence;

(C) polypeptide, it is included in the aminoacid sequence of SEQ ID NO:4 and replaces, and deletion is inserted, and/or adds the aminoacid sequence of one or more amino acid gained, and has the activity that makes the polypeptide ubiquitinization that comprises SEQ IDNO:2 aminoacid sequence;

[39] treat and/or prevent the medicinal compositions of rheumatic arthritis, it comprise can be by [33] to [38] any one method screening compound as activeconstituents.

[40] test kit of the compound of screening treatment or prevention rheumatic arthritis, it comprises following ingredients:

(1) the synovial hyperplasia factor or its homologue; With

(2) prolyl 4-hydroxylase α subunit or its homologue.

[41] test kit of [39] further comprises following ingredients:

(3) ubiquitin activating enzyme;

(4) ubiquitin transferring enzyme;

(5) ubiquitin; With

(6) Triphosaden.

[42] test kit of the test-compound of screening treatment or prevention rheumatic arthritis, comprise and express the synovial hyperplasia factor or its homologue, with the cell of prolyl 4-hydroxylase α subunit or its homologue and the instrument of detection prolyl 4-hydroxylase α subunit or its homologue ubiquitin level.

[43] method of screening treatment or preventing Fibrotic compound comprises step:

A ') with (i) substrate or (ii) the synovial hyperplasia factor or its homologue contact with test-compound, and be incubated making under the synovial hyperplasia factor or its homologue and the substrate bonded condition, perhaps

C) select the compound of test-compound as treatment or prevention rheumatic arthritis, wherein this compound make between substrate and the synovial hyperplasia factor or its homologue be lower than this test-compound not in conjunction with level the time record in conjunction with level,

Wherein substrate is prolyl 4-hydroxylase α subunit or its homologue.

[44] method of [43], wherein or the synovial hyperplasia factor or substrate in conjunction with solid phase or comprise can be in conjunction with the marker of solid phase.

[45] method of [43], wherein prolyl 4-hydroxylase α subunit or its homologue are following (a) to any one of (e) polypeptide:

(a) the nucleotide sequence coded polypeptide of SEQ ID NO:1;

(b) comprise the polypeptide of SEQ ID NO:2 aminoacid sequence;

[46] method of [43], wherein the synovial hyperplasia factor or its homologue are following (A) to any one of (E) polypeptide:

(A) the nucleotide sequence coded polypeptide of SEQ ID NO:3;

(B) comprise the polypeptide of SEQ ID NO:4 aminoacid sequence;

[47] treat and/or prevent the medicinal compositions of rheumatic arthritis, comprise can be by [43] to [46] any one method screening compound as activeconstituents.

Further, present inventors have carried out the screening of test-compound with method of the present invention, and have separated the polypeptide of SEQ ID NO:5.Particularly, the present invention relates to following polypeptide, the polynucleotide of coded polypeptide and comprise that polypeptide is the medicinal compositions of activeconstituents.

[48] comprise the polypeptide of SEQ ID NO:5 aminoacid sequence.

[49] polypeptide, it is included in the aminoacid sequence of SEQ ID NO:5 and replaces, deletion, insert, and/or add the aminoacid sequence of one or more amino acid gained, wherein when with polypeptide during as the test-compound in the method for [1], the ubiquitin level of substrate is lower than the ubiquitin level that records when lacking this polypeptide.

[50] polypeptide, it is included in the aminoacid sequence of SEQ ID NO:5 and replaces, deletion, insert, and/or add the aminoacid sequence of one or more amino acid gained, wherein when with polypeptide during as the test-compound in the method for [12], substrate and the synovial hyperplasia factor or its homologue in conjunction with level be lower than record when lacking this polypeptide in conjunction with level.

[51] coding [48] is to the polynucleotide of [50] any one polypeptide.

[52] be used for the treatment of and/prevent Fibrotic medicinal compositions, it comprises that any one polypeptide of claim 48 to 50 is as activeconstituents.

[53] be used for the treatment of and/prevention rheumatic arthritis medicinal compositions, it comprises that any one polypeptide of claim 48 to 50 is as activeconstituents.

Basis of the present invention is that new discovery prolyl 4-hydroxylase activity is controlled by synovial hyperplasia factor ubiquitin prolyl 4-hydroxylase α subunit.Promptly the invention provides and detect test-compound the ubiquitin of the synovial hyperplasia factor is turned into the active method of adjusting of usefulness, comprise step:

A) under the condition that can make the substrate ubiquitinization when test-compound exists with substrate and the synovial hyperplasia factor or the insulation of its homologue,

When c) the ubiquitin level that records when not having test-compound when substrate ubiquitin level is variant, detect test-compound makes the effect of substrate ubiquitinization to the synovial hyperplasia factor adjusting activity,

Wherein substrate is P4HA1 or its homologue.

" ubiquitinization " refers in conjunction with at least one ubiquitin." ubiquitin level " refers to the quantity in conjunction with ubiquitin, or in conjunction with the site quantity of ubiquitin, perhaps both.That is, can judge the ubiquitin level in conjunction with the ubiquitin quantity of the privileged site of certain peptide species by mensuration.And when the multizone of certain peptide species during by ubiquitin, the ubiquitin level also can be judged by the number of regions of measuring ubiquitinization.

The method of judging the ubiquitin level is known.For example, have the ubiquitin affinity and can judge the ubiquitin level by use in conjunction with the material of ubiquitin.Can will resist ubiquitin antibody to be used as the material of this affinity.For example, identification specifically and can commercially obtain in conjunction with the monoclonal antibody of ubiquitin.By being marked with the material of ubiquitin affinity, can judge the ubiquitin level of substrate based on the level of the mark substance of bound substrates.Perhaps, by the ubiquitin of mark in advance is provided, can measure the ubiquitin quantity of bound substrates polypeptide based on the level of mark substance.

Can adopt normally used labeling technique to carry out the mark of this antibody and ubiquitin.Particularly, for example, can be with the material of radioactive activity, the material of enzymic activity, fluorescence activity material and luminophore are with marking.Can use combination tag indirectly mark to be combined with antibody or ubiquitin.For example, the mark of avidinization can be in conjunction with biotinylated antibody or ubiquitin.And the substrate of ubiquitinization also can be caught by the ubiquitin that combination tag has been introduced in use.By the substrate polypeptide level that mensuration is caught, can estimate the ubiquitin level.In this method, the TPPA ubiquitin level of the anti-substrate polypeptide by applying marking.The ubiquitin of following mark is can the commercial mensuration that is used for the ubiquitin level that obtains.

Fluorescein bonded ubiquitin: ubiquitin combined with fluorescent element (that is fluorescent substance).Can use fluorescence intensity to be index determining ubiquitin level.

Biotinylation ubiquitin: with biotin labeled ubiquitin (that is binding partner).Can be used the avidin post to catch by the substrate polypeptide of biotinylated ubiquitin ubiquitinization.Perhaps, use the enzyme of avidinization, fluorescent substance waits the ubiquitin level of measuring.

(His) 6-ubiquitin: with the ubiquitin of histidine-tagged mark.Can catch histidine-tagged with the nickel post.That is, can by ubiquitin with the substrate capture of ubiquitinization on post.Perhaps, be possible by anti-histidine-tagged antibody indirect mark.

When measuring the ubiquitin level, be favourable in conjunction with solid phase with substrate.For example, in advance with substrate in conjunction with solid phase.Particularly, can be with the substrate Chemical bond, or physical adsorption is in the inwall of pearl or reactor.For pearl or reactor, for example, can use polystyrene.The also known group of import feature polystyrene derivative for example amino and carboxyl is used for the Chemical bond of substrate.

Perhaps, the ubiquitin of binding affinity material is arranged, the substrate of ubiquitinization can be caught indirectly through ubiquitin by having used mark mentioned above.Vice versa, imports anti-substrate antibody or substrate is had the substrate of the material of binding affinity can be trapped on the solid phase.In any situation, the solid phase substrate from unconjugated component separating, and is measured the ubiquitin level.When ubiquitin or substrate itself were unmarked, using had the material of avidity mark can be connected thereon to ubiquitin or substrate.For example, use anti-ubiquitin antibody.At last, the level of the mark (or unconjugated mark) by measuring solid-phase capture can be judged the ubiquitin level.Measure the ubiquitin level, can judge the amount of the substrate of ubiquitinization.Perhaps, also can judge the not amount of the substrate of ubiquitinization by measuring the ubiquitin level.

In addition, the present invention shows that also the biological activity of proline-4-hydroxylase is subjected to the adjusting of P4HA1 ubiquitinization.Therefore, make index with the biologically active level of proline-4-hydroxylase and also can measure the ubiquitin level.For example, can judge the biologically active level (Methods Enzymol., Vol.82, pp.246-259,1982) of proline-4-hydroxylase by the tritium release experiment.

The synovial hyperplasia factor (or its homologue), substrate is arbitrarily with trying the order that material contacts.That is, the present invention can comprise the synovial hyperplasia factor (or its homologue), substrate with tried the step that material contacts, it is with step a) mentioned above, a '), and the random order of a ").When being tried material and exist under the situation (a), can estimate and be tried the influence of material the ubiquitin reaction of the synovial hyperplasia factor (or its homologue) with three kinds of compositions insulations.Perhaps, can with tried material be adjusted into the synovial hyperplasia factor (or its homologue) with after one of substrate contacts, can ubiquitinization (a ').In this case, estimate and to be tried material and influence or to by the influence of the substrate function of ubiquitinization synovial hyperplasia factor ubiquitin ligase is active.And, (a ") use can the condition of ubiquitinization after, tried material by contact and can be estimated the effect of material that tried the substrate of ubiquitinization.

P4HA1 or its homologue are used as substrate of the present invention.P4HA1 constitutes one of tetrameric subunit of proline-4-hydroxylase.Think that the α subunit has the function of catalysis proline(Pro) hydroxylation reaction.Its homologue is the polypeptide that has with the P4HA1 similar structures, and can be by synovial hyperplasia factor ubiquitinization.For P4HA1 of the present invention or its homologue, for example, can use following (a) polypeptide to (e):

(a) the nucleotide sequence coded polypeptide of SEQ ID NO:1;

(b) comprise the polypeptide of SEQ ID NO:2 aminoacid sequence;

(Jikken Igaku (Experimental Medicine) SupplementaryVolume in accordance with known methods, Genetic Engineering Handbook, pp 246-251, Yodosha Co., Ltd., 1991), those skilled in the art can separate the polynucleotide that include the nucleotide sequence that suddenlys change, and wherein suddenlys change in the polynucleotide that comprise SEQ ID NO:1 nucleotide sequence.For example, use SEQ ID NO:1 nucleotide sequence (or its segment), comprise that by screening the library of similar gene can will comprise the dna clone of height homologous nucleotide sequence as probe.It is the example in this library that the gene library of adding sudden change at random and the cDNA library in non-human kind source are arranged in SEQ ID NO:1 nucleotide sequence.

For example, knownly handle DNA so that base pair replaces is the method (Proc.Natl.Acad.Sci.USA., 79:7258-7260,1982) of adding sudden change to the known nucleotide sequence at random with nitrous acid.According to the method, by handling segment, the base pair replacement can be imported segment at random with nitrous acid.Perhaps, known have gapped duplex method etc. to can be used as the technology (Methods Enzymol., 154:350-367,1987) that imports target mutation to any site.The double-stranded carrier of ring-type that will become strand is hybridized at target site with comprising the synthetic oligonucleotide of sudden change, and wherein the double-stranded carrier of ring-type prepares by import sudden change to its clone gene.The complementary single-stranded dna s that is derived from carrier makes it linearizing by the restriction enzyme cracking, and anneals with the cyclic single strand carrier, and wherein the breach between carrier and synthesizing ribonucleotide is filled up with archaeal dna polymerase, and connects.Therefore, obtain double-stranded circular carrier completely.

The number of modified amino acid is generally 50 amino acid or still less, preferred 30 amino acid or still less, more preferably 5 amino acid or still less (for example, an amino acid), or several amino acid.When any substituted amino acid, by may be easy to keep the activity of original protein with another aminoacid replacement that similar characteristics is arranged.Except that above-mentioned aminoacid replacement, albumen of the present invention comprises the polypeptide of the conservative replacement that contains, and is equivalent to P4HA1 on its function (SEQ ID NO:2).Think that conservative the replacement is important, for example, when substituted amino acid in the structural domain essential to protein-active.This amino acid whose conservative replacement is well known to those skilled in the art.

Being fit to the conservative amino acid whose example that replaces is for example Methionin of basic aminoacids, arginine and Histidine; Acidic amino acid is aspartic acid and L-glutamic acid for example; Uncharged polare Aminosaeren is glycine for example, l-asparagine, glutamine, Serine, Threonine, tyrosine and halfcystine; Nonpolar amino acid is L-Ala for example, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met) and tryptophane; β-branched-chain amino acid is Threonine for example, Xie Ansuan and Isoleucine; With die aromatischen Aminosaeuren for example tyrosine, phenylalanine, tryptophane and Histidine.

Protein-active replaces by non-conservation can further be increased (for example, the albumen of stabilizing active) or to reduce (for example, dominant negative).

Polypeptide comprises synthetic and polypeptide natural generation, wherein this polypeptide is included in the aminoacid sequence of SEQ ID NO:2 and replaces, deletion, insert, and/or add the aminoacid sequence of one or more amino acid gained, and be equivalent to the polypeptide that comprises SEQ ID NO:2 aminoacid sequence on the function." a plurality of amino acid " refers to general 50 amino acid or still less here, preferred 30 amino acid or still less, more preferably 5 amino acid or still less (for example, 1 amino acid) and several amino acid.As in the interferon gene, eukaryotic gene generally has polymorphism.The nucleotide sequence variation that is caused by polymorphism can cause replacing, and deletion is inserted, and/or added one or more amino acid.The polypeptide of natural generation can be used for the present invention, this polypeptide is included in the aminoacid sequence of SEQ ID NO:2 and replaces, and deletion is inserted, and/or add the aminoacid sequence of one or more amino acid gained, and be equivalent to the polypeptide that comprises SEQ ID NO:2 aminoacid sequence on the function.

Both made by polymorphism to cause that nucleotide sequence changes, but in aminoacid sequence, do not had any variation.Such nucleotide sequence sudden change is called silent mutation.Also the gene that the nucleotide sequence that silent mutation is arranged can be constituted is used for the present invention." polymorphism " used herein meaning is that certain gene contains differentiated nucleotide sequence between individual in population and individuality.Polymorphism does not relate to the ratio of the differential gene of being found.

In addition, can will use the method for hybridization as the method that obtains P4HA1 albumen or its homologue.Particularly, the P4HA1-coding nucleotide of SEQ ID NO:1 or the representative of its fragment can be used as the polynucleotide of separation energy and probe hybridization.By under rigorous condition, hybridizing, can filter out the polynucleotide of height nucleotide sequence homology.The protein isolate of gained more may comprise P4HA1 albumen or its homologue." height nucleotide sequence homology " refer to, for example, 70% or above identical, and preferred 90% or more than.

Particularly, rigorous condition comprises, condition: 6 * SSC for example, 40% methane amide, 25 ℃ of hybridization and with 1 * SSC in 55 ℃ of washings.Preciseness is according to condition salt concn for example, methane amide concentration and temperature variation.Regulating these conditions is tangible to reach required preciseness to those skilled in the art.

For example, use hybridization the polynucleotide of coding non-human P4HA1 homologue can be separated.Constitute the albumen of function equivalent among the present invention by the P4HA1 homologue that is derived from non-human animal's polynucleotide encoding, non-human animal mouse for example wherein, rat, rabbit, pig, or goat.

Polynucleotide of the present invention can have any origin.That is, they can obtain from cDNA or genomic dna, or synthetic preparation.In addition, the present invention also comprises the polynucleotide that have based on any nucleotide sequence of genetic code degeneracy, the albumen of the present invention as long as it can be encoded.

Albumen by importing albumen that sudden change obtains and from the isolating polynucleotide encoding of above-mentioned hybridization technique to people P4HA1 (SEQ ID NO:2) is general to have highly amino acid sequence homology with people P4HA1 (SEQ ID NO:2)." high homology " refers to sequence 30% or above identical, preferred 50% or more than, and more preferably 80% or above (for example, 95% or more than).Judge nucleotide sequence or aminoacid sequence homogeny can internet usage homology search site [for example, can use homology to search for for example FASTA, BLAST, PSI-BLAST, with SSEARCH in the Japanese DNA database (DDBJ) (for example, at website http://www.ddbj.nig.ac/jp/E-mail/homology-j.html, the homology of Japanese DNA database (DDBJ) search (search and analysis) page or leaf).In addition, can finish search (for example, the BLAST page or leaf of NCBI website with BLAST at National Center forBiotechnology Information (NCBI); Http:// www.ncbi.nlm.nih.gov/BLAST/; Altschul, S.F.et al., J.MolBiol., 1990,215 (3): 403-10; Altschul, S.F.﹠amp; Gish, W., Meth.Enzymol., 1996,266:460-480; Altschul, S.F.et al., Nucleic Acids Res., 1997,25:3389-3402)].

The synovial hyperplasia factor or its homologue are used as ubiquitin ligase of the present invention (E3).The synovial hyperplasia factor is the polypeptide that comprises fourth finger (RING finger) motif.Identified the nucleotide sequence (SEQ ID NO:3) of its aminoacid sequence (SEQ ID NO:4) and encoding amino acid sequence.And " its homologue " is similar to the synovial hyperplasia factor structure and the polypeptide of energy ubiquitin P4HA1.For example, following polypeptide (A) to (E) can be used as " the synovial hyperplasia factor or its homologue " of the present invention:

(A) the nucleotide sequence coded polypeptide of SEQ ID NO:3;

(B) comprise the polypeptide of SEQ ID NO:4 aminoacid sequence;

(C) polypeptide, it is included in the aminoacid sequence of SEQ ID NO:4 and replaces, and deletion is inserted, and/or adds the aminoacid sequence of one or more amino acid gained, and it has and will comprise the activity of the polypeptide ubiquitinization of SEQ ID NO:2 aminoacid sequence;

Method based on known amino acid sequence information and known nucleotide sequence information acquisition function equivalent polypeptide has above been described.As mentioned above to the structural condition that the hope of homologue reaches.Can be with this obtainable homologue as the synovial hyperplasia factor of the present invention.For example, WO02/052007 discloses various synovial hyperplasia factor homologs.As long as have the function of ubiquitin P4HA1, these homologues can be used for method of the present invention.

Polypeptide comprises synthetic and polypeptide natural formation, wherein this polypeptide is included in the aminoacid sequence of SEQ ID NO:4 and replaces, deletion, insert, and/or add the aminoacid sequence of one or more amino acid gained, and it is functionally equivalent to the polypeptide that comprises SEQ ID NO:2 aminoacid sequence." a plurality of amino acid " refers to general 50 amino acid or still less here, preferred 30 amino acid or still less, more preferably 5 amino acid or still less (for example, 1 amino acid) and several amino acid.As in the interferon gene, eukaryotic gene generally has polymorphism.The nucleotide sequence variation that is caused by polymorphism can cause one or more amino acid whose replacements, and deletion is inserted, and/or adds.The protein of natural generation can be used for the present invention, this albumen is included in the aminoacid sequence of SEQ ID NO:4 one or more aminoacid replacement, and deletion is inserted, and/or the aminoacid sequence that adds, and be equivalent to the albumen that comprises SEQ ID NO:4 aminoacid sequence on the function.

Both made by polymorphism to cause that nucleotide sequence changes, but in aminoacid sequence, do not had any variation.Such nucleotide sequence sudden change is called silent mutation.Also the gene that the nucleotide sequence that silent mutation is arranged can be constituted is used for the present invention." polymorphism " used herein meaning is that certain gene contains the difference nucleotide sequence between individual in population and individuality.Polymorphism does not relate to the ratio of the differential gene of being found.

In addition, can will use the method for hybridization as the method that obtains synovial hyperplasia factor protein or its homologue.Particularly, the synovial hyperplasia factor-coding nucleotide of SEQ ID NO:3 among the present invention or the representative of its fragment can be used as the polynucleotide of separation energy and probe hybridization.By under rigorous condition, hybridizing, can filter out the polynucleotide of height nucleotide sequence homology.The protein isolate of gained more may comprise synovial hyperplasia factor protein or its homologue." height nucleotide sequence homology " refer to, for example, 70% or above identical, and preferred 90% or more than.

For example, use hybridization can separate the polynucleotide of coding non-human synovial hyperplasia factor homologs.Constitute the albumen of function equivalent among the present invention by the synovial hyperplasia factor homologs that is derived from non-human animal's polynucleotide encoding, non-human animal mouse for example wherein, rat, rabbit, pig, or goat.

Albumen by importing albumen that sudden change obtains and from the isolating polynucleotide encoding of above-mentioned hybridization technique to people's synovial hyperplasia factor (SEQ ID NO:4) is general to have highly amino acid sequence homology with people's synovial hyperplasia factor (SEQ ID NO:4)." high homology " refers to sequence 30% or above identical, preferred 50% or more than, and more preferably 80% or above (for example, 95% or more than).Judge nucleotide sequence or aminoacid sequence homogeny can internet usage homology search site [for example, can use homology to search for for example FASTA, BLAST, PSI-BLAST, with SSEARCH in the Japanese DNA database (DDBJ) (for example, at website http://www.ddbj.nig.ac.jp/E-mail/homology-j.html, the homology of Japanese DNA database (DDBJ) search (search and analysis) page or leaf).In addition, can finish search (for example, the BLAST page or leaf of NCBI website with BLAST at National Center for Biotechnology Information (NCBI); Http:// www.ncbi.nlm.nih.gov/BLAST/; Altschul, S.F.et al., J.Mol.Biol., 1990,215 (3): 403-10; Altschul, S.F.﹠amp; Gish, W., MethEnzymol., 1996,266:460-480; Altschul, S.F.et al., Nucleic Acids Res., 1997,25:3389-3402).

In fact, the present inventor discloses the clone that an amino acid deletion is arranged in the aminoacid sequence that constitutes the synovial hyperplasia factor in WO02/052007.As long as it has function required for the present invention, the albumen that contains sudden change in aminoacid sequence is also included within the synovial hyperplasia factor homologs of the present invention.Confirmed to have the sudden change of an amino acid deletion to lack gca in the 1293-1295 place in being equivalent to SEQ ID NO:3 by inventors, therefore 412 positions are L-Ala in SEQ ID NO:4.

In the present invention, a kind of proteic P4HA1 ubiquitin function can be by contacting this albumen under above-mentioned ubiquitin condition with substrate, and detect the substrate ubiquitinization and confirm.

Next, phrase " can make the condition of substrate or its homologue ubiquitinization " and refers to that in the present invention P4HA1 can be by synovial hyperplasia factor ubiquitinization under this condition.This condition can comprise in external or the body.For example, coexist by following ingredients the condition that can make the substrate ubiquitinization be provided:

I) ubiquitin activating enzyme (E1);

Ii) ubiquitin transferring enzyme (E2);

Iii) ubiquitin; With

Iv) Triphosaden.

It is generally acknowledged that proteinic ubiquitinization is carried out in the following manner in live body.Except four kinds of compositions mentioned above, proteinic ubiquitinization needs ubiquitin ligase (ligase enzyme/E3).Ubiquitin activating enzyme (E1), ubiquitin transferring enzyme (E2), and ubiquitin ligase (ligase enzyme/E3) is formed the polyenzyme system that is known as the ubiquitin system.Some substrates are by the E2 ubiquitinization, but P4HA1 is by the synovial hyperplasia factor (E3) ubiquitinization.

Ubiquitin activating enzyme (E1) activates ubiquitin transferring enzyme (E2).And (substrate protein is discerned and caught to ligase enzyme/E3) to ubiquitin ligase, and the ubiquitin that utilizes activated ubiquitin transferring enzyme (E2) is with the substrate protein ubiquitinization of catching.In the ubiquitin system, (ligase enzyme/E3) has the vital role of identification substrate protein to ubiquitin ligase.That is, (ligase enzyme/E3) means the termination of substrate protein to ubiquitin ligase to the selection of substrate.

In the ubiquitin system, (ligase enzyme/E3) combine is important to the ubiquitin ligase of substrate and identification substrate.Therefore, the substrate among the present invention must be P4HA1 or its homologue.Simultaneously, (ligase enzyme/E3) must be the synovial hyperplasia factor or its homologue to ubiquitin ligase.But other enzyme can be any enzyme that can make the substrate ubiquitinization.

For example, the enzyme with the yeast source is used as ubiquitin activating enzyme (E1) in an embodiment, and people source UbcH5c is as ubiquitin ligase (E2).But ubiquitin activating enzyme (E1) is not limited to the enzyme in this primary yeast source in the present invention.For example, rat source ubiquitin activating enzyme (E1) can be used for the present invention.Similarly, ubiquitin transferring enzyme (E2) is not limited to the enzyme in people source.E1 and/or E2 can be arbitrarily the enzyme or the recombinant chous of natural generation.As long as they can keep the activity of E1 or E2, these recombinant chous can structurally be modified.Structural modification comprises the replacement of aminoacid sequence, and deletion is inserted and interpolation.For example, E2 is used for the following examples, wherein E2 prepares by in colon bacillus people source UbcH5c being expressed as the GST-fusion rotein.This recombinant chou is also included among the E2 of the present invention.

When using composition (i) to (iv) providing substrate can be by the condition of the synovial hyperplasia factor ubiquitinization time, those skilled in the art can design other condition that ubiquitinization needs.Particularly, those skilled in the art can determine the heat-retaining condition that uses and the amount of every kind of composition.The example of these conditions is represented with composition, for example, represents with following concentration:

The synovial hyperplasia factor (E3): 500ng

GST-P4HA1：800ng

(i) ubiquitin activating enzyme (E1): 60ng

(ii) ubiquitin transferring enzyme (E2): 03mg

(iii) ubiquitin ( ³²The Histidine ubiquitin of P-mark): 075 μ g

The condition that can following foundation can make the substrate ubiquitinization: in following reaction buffer, add composition mentioned above producing 30 μ l reaction mixtures, and with this mixture 37 ℃ of insulations 1 hour.When needing, can suitably regulate the concentration of composition.For example, can in 1/10th to ten times of scopes of amount mentioned above, adjust concentration.

The composition of reaction buffer

50mM Tris-HCl pH of buffer 7.4

5mM?MgCl ₂

2mM?NaF

10nM?Okadaic?acid

2mM?ATP(iv)

0.6mM dithiothreitol (DTT) (DTT)

Perhaps, can make up the condition that can make the substrate ubiquitinization of the present invention in vivo.That is, provide the condition that can make the substrate ubiquitinization can pass through ubiquitin substrate in the cell of expressing substrate and the synovial hyperplasia factor or its homologue.For example, can be with the synovial cell as the cell of expressing the synovial hyperplasia factor and substrate.

Perhaps, the condition of substrate ubiquitinization that can make of the present invention can be provided by cell, wherein forces the expression synovial hyperplasia factor (or its homologue) and substrate by genetic engineering in this cell.In the cell of gene as the foreign gene importing with the coding synovial hyperplasia factor (or its homologue) or substrate, use derivable expression system, the expression time of the control synovial hyperplasia factor or substrate is possible.Viable cell has the ubiquitin system.That is, exist composition (i) extremely (iv) in the cell.Therefore, exist, can make up condition that can ubiquitinization of the present invention by allowing the synovial hyperplasia factor and substrate.

The nucleotide sequence of the coding synovial hyperplasia factor and P4HA1 (its substrate) is known.Those skilled in the art can design the expression system based on known genetic information.Can be with zooblast, insect cell, yeast waits as the host cell that forces expression.

The substrate of ubiquitinization can reclaim by destroying cell in cell.And, can be with the separating substances target substrate that ubiquitin or substrate is had avidity.The protein that except the target substrate, has many ubiquitinization in the cell.Therefore, when ubiquitin substrate in cell, the ubiquitinization of preferred special judgement target substrate protein.

For example, can be the method for catching substrate protein with the antibody of anti-substrate protein specifically as this purpose.Perhaps, for example histidine-tagged by substrate protein being expressed as the fusion rotein of labelled peptide, and making sign with label, to separate substrate protein be possible.Judge so the ubiquitin level of the substrate protein that reclaims from cell then with method mentioned above.

For example, the ubiquitin condition can have the ubiquitin of label and the synovial hyperplasia factor (or its homologue) cotransformation P4HA1 (or its homologue) express cell to make up by using.Judge that the ubiquitin level can be by carrying out immunoprecipitation with anti-tag antibody, and measure precipitation partly in the level of P4HA1.Can measure P4HA1 level in the precipitation part with the antibody mediated immunity of anti-P4HA1.Perhaps, judge that the substrate of ubiquitinization can be by the level of unprecipitated P4HA1 behind the mensuration immunoprecipitation.

And the ubiquitin condition can have the ubiquitin of label and the synovial hyperplasia factor (or its homologue) cotransformation P4HA1 express cell to make up by using.With the antibody mediated immunity post precipitation of anti-P4HA1, can judge the ubiquitin level with the antibody of anti-label or the antibody of anti-ubiquitin.For example, can be with the synovial cell as the P4HA1 express cell.

The ubiquitin that the present invention also relates to screen the synovial hyperplasia factor turns the method for regulating active test-compound with having into, comprises the following steps:

A) use the present invention to detect the active method of adjusting that synovial hyperplasia factor ubiquitin is turned into usefulness, to detect test-compound turns usefulness into to synovial hyperplasia factor ubiquitin adjusting activity; With

Be described below example more specifically according to screening method of the present invention.

The most normally, screening the material that influences the synovial hyperplasia factor and P4HA1 interphase interaction can be by when test-compound exists, with the amount of the synovial hyperplasia factor (enzyme) and P4HA1 (its substrate) reaction and assaying reaction.In this case, more objective screening is possible, its by with the control group comparing result of the synovial hyperplasia factor and P4HA1 reaction when lacking test-compound.

The ubiquitin reaction of synovial hyperplasia factor catalysis P4HA1.Correspondingly, judge that the amount of reacting can be by reacting and measure the amount of the P4HA1 of ubiquitinization under the situation about existing at ubiquitin.Can measure the amount of the P4HA1 of ubiquitinization, promptly in conjunction with the amount of the ubiquitin of P4HA1, the method for the material by using the specific combination ubiquitin for example is as anti-ubiquitin antibody.Can obtain anti-ubiquitin antibody according to the conventional method.For example, ubiquitin is injected to immune animal as immunogen, and from the blood collecting serum of animal.Can use mouse, rat, rabbit is waited as the immunne response animal.Can be with monoclonal antibody as antibody.It is known obtaining this monoclonal antibody method.

And, the ubiquitin of the mark of ubiquitin own and detection or mensuration mark can be measured ubiquitin.For mark used herein, can use the general labelling thing.Particularly, the example of marker comprises the radioactivity marker, enzyme labelling thing, vitamin H etc.For example, when ubiquitin usefulness ³²During the P mark, can detect with radioautograph. ³²The radioactivity of P also can be measured with liquid scintillation counter.Biotin labeled ubiquitin can detect with the avidin of mark.

Concrete method can illustrate in the following method.

Method 1

Preparation has the P4HA1 of label, and with itself and E1, E2, ATP, the synovial hyperplasia factor, ubiquitin and test-compound insulation are to form P4HA1-ubiquitin mixture.Next, with the antibody response of anti-P4HA1 label, mixture is separated with unreacted ubiquitin through immunoprecipitation.And, detect or measure the ubiquitin that forms mixture with anti-ubiquitin antibody.Detect or measure the reactive system that does not comprise test-compound in the same way as described above, and the contrast test-compound exists and the amount of bonded ubiquitin when lacking.

Method 2

With P4HA1, E1, E2, ATP, the synovial hyperplasia factor, [ ³²P]-ubiquitin of mark and test-compound be incubated together to form P4HA1-ubiquitin mixture.Next, mixture and unreacted ubiquitin is separated from one another by electrophoresis etc.And, detect or measure the ubiquitin that forms mixture with radioautograph.Detect or measure the reactive system that does not comprise test-compound in the same way as described above, and the contrast test-compound exists and the amount of bonded ubiquitin when lacking.

In method mentioned above, can use biotin labeled ubiquitin to replace ³²The ubiquitin of P-mark.In this case, the avidin with mark detects or measures ubiquitin.Can use horseradish peroxidase, alkaline phosphatase, fluorescence etc. the thing that serves as a mark.

Method 3

With the synovial hyperplasia factor, E1, E2, ATP, P4HA1, be connected in contain liquid dodge body pearl [ ³³P]-or [ ³²P]-ubiquitin of mark and test-compound reaction, the exciting light that the ubiquitin of mark is launched with combining of P4HA1 detects or mensuration with liquid scintillation counter.To detect or to measure the reactive system that does not comprise test-compound with above-mentioned same mode, the amount of bonded ubiquitin when contrast existence and shortage test-compound.

In method 1 to 3, when test-compound is when suppressing the material (antagonist) of synovial hyperplasia factor active, promptly in the synovial hyperplasia factor and P4HA1 interphase interaction, the amount of bonded ubiquitin is lower than shortage test-compound person when having test-compound.When test-compound is that the result is opposite when activating the material (agonist) of the synovial hyperplasia factor and P4HA1 interphase interaction.Therefore, above-mentioned result of experiment can be used for judging that test-compound is agonist (activator) or antagonist (inhibitor).

Present inventors find combination of the synovial hyperplasia factor and ubiquitin P4HA1.Therefore, can influence the adjusting of estimating synovial hyperplasia factor ubiquitin P4HA1 to bonded between the two by detecting.That is, the present invention relates to detect test-compound synovial hyperplasia factor ubiquitin turned into the adjusting activity of usefulness, comprise step:

The synovial hyperplasia factor (or its homologue), substrate is arbitrarily with trying the order that material contacts.That is, the present invention can comprise the synovial hyperplasia factor (or its homologue), substrate with tried the step that material contacts, it is with step a) mentioned above, a '), and the random order of a ").When being tried material and exist, during with three kinds of compositions insulations (a), can estimate and be tried material the synovial hyperplasia factor (or its homologue) and the influence of substrate bonded.Perhaps, can contact back (a ') with the synovial hyperplasia factor (or its homologue) or substrate will being tried material, and provide the synovial hyperplasia factor (or its homologue) and the substrate can the bonded condition.In this case, evaluation is tried the influence of material to the synovial hyperplasia factor or its substrate binding ability.And, tried material by provide the synovial hyperplasia factor (or its homologue) and substrate to contact after can the bonded condition at (a "), can estimate and be tried the influence of material the synovial hyperplasia factor and substrate complex.

Preferably with (i) substrate or (ii) the synovial hyperplasia factor or its homologue be connected in solid phase, or preferably include can be in conjunction with the mark of solid phase.For example, can binding partner in conjunction with the mark of solid phase.Binding partner comprises vitamin H etc.Vitamin H can be captured on the solid phase, as avidin-dextran.By in conjunction with solid phase, both is separated easily in conjunction with product.

Be easy detection, can mark not by immobilised other composition.Can be according to the method for traditional labelled protein with the synovial hyperplasia factor, its homologue or substrate mark.Particularly, the material of available radioactivity, the material of enzymic activity, the material of fluorescence activity, radiative material etc. the thing that serves as a mark.Can use combination tag to allow the indirect conjugated protein of marker.For example, can be with the marker of avidinization in conjunction with biotinylated protein.

In the method for the invention, can with any have combine active albumen with the synovial hyperplasia factor as P4HA1 or its homologue.Particularly, P4HA1 or its homologue comprise that following (a) is to any one of (e) polypeptide:

(a) the nucleotide sequence coded polypeptide of SEQ ID NO:1;

(b) comprise the polypeptide of SEQ ID NO:2 aminoacid sequence;

In the method for the invention, can with any have combine active protein with P4HA1 as the synovial hyperplasia factor or its homologue.Particularly, the synovial hyperplasia factor or its homologue comprise any one of following polypeptide (A) to (E):

(A) the nucleotide sequence coded polypeptide of SEQ ID NO:3;

(B) comprise the polypeptide of SEQ ID NO:4 aminoacid sequence;

(C) polypeptide, it is included in the aminoacid sequence of SEQ ID NO:4 and replaces, and deletion is inserted, and/or adds the aminoacid sequence of one or more amino acid gained, and it can be in conjunction with the polypeptide that comprises SEQID NO:2 aminoacid sequence;

(E) comprise with SEQ ID NO:4 aminoacid sequence and have 70% or the polypeptide of the aminoacid sequence of above homology, and it can be in conjunction with the polypeptide that comprises SEQ ID NO:2 aminoacid sequence.

Import any method of suddenling change as mentioned above to known amino acid sequence.From the polynucleotide of the artificial or natural generation sudden change of encoding, the method for using hybridization or PCR to separate the polynucleotide of coding structure similar polypeptide also is known.Those skilled in the art can select to combine active polypeptide with the synovial hyperplasia factor or P4HA1 from the various mutant of acquisition like this.For example, when the synovial hyperplasia factor that is immobilized when certain albumen is caught, conclude that this albumen has the activity in conjunction with the synovial hyperplasia factor.When the P4HA1 that is immobilized when certain albumen catches, conclude that this albumen has the activity in conjunction with P4HA1.

For example, be integrated into suitable expression vector and express this DNA, can obtain the protein fragments of P4HA1 by dna fragmentation with P4HA1 cDNA.Active by verifying above-mentioned protein fragments storehouse with combining of the synovial hyperplasia factor, can judge the synovial hyperplasia factor in conjunction with essential structural domain.In the method for the invention, the synovial hyperplasia factor fragment that comprises the structural domain of judgement like this can be used as the homologue of the synovial hyperplasia factor.And, come from another kind and its and constitute the conservative P4HA1 copy of the aminoacid sequence of structural domain and comprise that the polypeptide of its fragment sequence also can be used as the homologue of P4HA1.

In the present invention, by influencing the synovial hyperplasia factor and substrate in conjunction with regulating the method that synovial hyperplasia factor ubiquitin turns the compound of usefulness into, detecting test-compound is useful to the active method of adjusting that synovial hyperplasia factor ubiquitin turns usefulness into as screening.That is, the ubiquitin that the present invention relates to screen the synovial hyperplasia factor turns the method for regulating active test-compound with having into, comprises step:

A) by using above-mentioned test-compound that synovial hyperplasia factor ubiquitin is turned into the active detection method of adjusting of usefulness, detect test-compound turns usefulness into to synovial hyperplasia factor ubiquitin adjusting activity; With

B) select compared with the control, the synovial hyperplasia factor has different test-compounds with substrate bonded level.

According to the present invention's detection the active method of adjusting that synovial hyperplasia factor ubiquitin turns usefulness into is described as mentioned.Use these methods, by estimate the active of various test-compounds and with the control group specific activity, can screen and have the active test-compound of target.Any compound with target activity level of checking can be used as contrast of the present invention.For example, can be with the active compound of known no target as negative control.Also the ubiquitin level of measuring under the shortage test-compound situation can be used the negative control in making comparisons.Have certain active material to compare by use, can screen has the more material of high reactivity level.

The test-compound that is used for the present invention's screening is not specifically limited.For example can be used as test-compound: mineral compound, organic compound, peptide, protein, natural generation or the synthetic low-molecular weight compound, natural generation or the synthetic high-molecular weight compounds, the tissue or cell extract, the natural compounds in culture supernatant of microorganism and plant or halobios source.The cDNA library of the expression product of gene library or expression also can be used as test-compound.

According to screening method of the present invention, can screen synovial hyperplasia factor ubiquitin turned into having and regulate active compound.This compound comprises treatment rheumatic arthritis or Fibrotic material." ubiquitin is turned into the adjusting activity of usefulness " and comprise stimulating activity in the present invention and suppress active.The proteasome degradation that proteinic ubiquitin induced protein is relied on by ATP-.Infer that ubiquitin reduces paraprotein and unessential albumen in live body.Therefore, by regulating the activity of synovial hyperplasia factor ubiquitin P4HA1 control prolyl 4-hydroxylase.

Be beneficial to the activity level of P4HA1 ubiquitin increase enzyme.For example, increase the expression of the synovial hyperplasia factor among the rheumatic arthritis patient synovial cell.As a result, promoted the ubiquitinization of P4HA1, and the activity level of enzyme has increased.Predict that its mechanism is because the result of P4HA1 ubiquitinization makes the degraded of unusual molecular specific ground, and the ratio of normal protein increases.The example of paraprotein is following albumen:

-unusual the albumen of modifying

-unusual folding albumen.

Vice versa, and when the ubiquitinization of P4HA1 was suppressed, the accumulation increase of paraprotein and prolyl 4-hydroxylase activity descended.The decline that paraprotein causes is not only because their enzymic activity, and because their effects are to suppress the contact of normal protein and other subunit or the contact of enzyme molecule and substrate.

Synovial hyperplasia factor ubiquitin P4HA1 had regulate active compound and turn into being useful material for the substrate ubiquitin of regulating the synovial hyperplasia factor.That is, the invention provides the material that the substrate ubiquitin of regulating the synovial hyperplasia factor turns usefulness into, wherein comprise can be with the compound of the inventive method screening as activeconstituents for material.The material that turns usefulness according to adjusting ubiquitin of the present invention into is useful for treating and/or preventing the disease that is caused unusually by prolyl 4-hydroxylase activity.Prolyl 4-hydroxylase is an enzyme of being responsible for collagenic supersession.Therefore, be subjected to the ubiquitinization of the synovial hyperplasia factor can treat or prevent disease by adjusting with the collagen resulting anomaly.

In fact, the experimental affirmation of present inventor is compared with normal cell, and the mouse cell of having deleted the synovial hyperplasia factor by genetic engineering shows the prolyl 4-hydroxylase activity (Fig. 6) of reduction, and has reduced collagen generation (Fig. 5).The protein content of P4HA1 does not have significant difference (Fig. 4) between these cells and normal cell.That is, think that P4HA1 is induced prolyl 4-hydroxylase activity to increase by synovial hyperplasia factor ubiquitinization.Therefore, the effect that suppresses the synovial hyperplasia factor can suppress too much collagen generation.Perhaps, the effect of the enhancing synovial hyperplasia factor can stimulate the generation of collagen.

More specifically, use the influence to the P4HA1 ubiquitinization to be index, the synovial hyperplasia factor inhibitors of discovery is useful to treating and/or preventing the disease or the pathological state that are caused by the fibrocyte accumulation.For example, can classify pulmonary fibrosis or hepatic fibrosis as cause disease or pathological state (Kivirikko KI., Ann Med., 1993, Apr by the fibrocyte accumulation; 25 (2): 113-26).

Fibrosis refers to have the disease of pathological state in the present invention, wherein as to repairing and the replying of disorganization, and the formation of fiber and pile up increase.Pulmonary fibrosis mentioned above and hepatic fibrosis are Fibrotic representative diseases.Pointed out that various mechanism participate in the possibility of these disease causes.To two kinds of situation tissue fibrosis damaging tissue functions is general.For example, pulmonary fibrosis infringement respiratory function.Hepatic fibrosis suppresses liver blood stream and causes dysfunction of liver.Therefore, if can improve fibrosis, the symptom of these diseases can be treated, cure, or prevention.

The known fiberization for example development of hepatic fibrosis can suppress (Kivirikko KI., Ann Med., 1993 Apr by suppressing the collagen generation; 25 (2): 113-26).

The method that is used for the treatment of or prevents Fibrotic compound according to the present invention screening can be undertaken by the same step of method that the compound of synovial hyperplasia factor ubiquitin P4HA1 is regulated in screening mentioned above.

Therefore, the invention provides the method that screening is used for the treatment of or prevents Fibrotic compound, comprise step:

C) select a kind of compound as treatment or prevent Fibrotic compound, the ubiquitin level that it records when making substrate ubiquitin level be lower than this test-compound not,

Wherein substrate is prolyl 4-hydroxylase α subunit or its homologue.

Perhaps, the invention provides the method that screening is used for the treatment of or prevents Fibrotic compound, comprise step:

A) insulation when test-compound exists under the synovial hyperplasia factor or its homologue and substrate can the bonded conditions,

A ') with (i) substrate or (ii) the synovial hyperplasia factor or its homologue contact with test-compound, and can be incubated under the bonded condition, perhaps in the synovial hyperplasia factor and substrate

A ") can be incubated under the bonded condition at the synovial hyperplasia factor or its homologue and substrate, and they are contacted with test-compound.

B) in step a), a '), and the arbitrary step back of a ") measure substrate and the synovial hyperplasia factor or its homologue in conjunction with level; With

C) select a kind of test-compound as treatment or prevent Fibrotic compound, its record when making substrate and the synovial hyperplasia factor be lower than this test-compound not in conjunction with level in conjunction with level,

Wherein substrate is prolyl 4-hydroxylase α subunit or its homologue.

The excessive generation of collagen is one of rheumatismal representative pathological state in the rheumatosis.Therefore, the active adjusting of control synovial hyperplasia factor pair P4HA1, and the generation of inhibition collagen is effective (Kivirikko KI.Ann Med.1993 Apr as rheumatismal therapeutic strategy; 25 (2): 113-26).

Therefore, the invention provides the method that screening is used for the treatment of or prevents the compound of rheumatic arthritis, comprise step:

C) select the compound of a kind of compound as treatment or prevention rheumatic arthritis, the ubiquitin level that it records when making substrate ubiquitin level be lower than this test-compound not,

Wherein substrate is prolyl 4-hydroxylase α subunit or its homologue.

The present invention relates to screen the method that is used for the treatment of or prevents the rheumatic arthritis compound, comprise step:

A ") can be incubated under the bonded condition at the synovial hyperplasia factor or its homologue and substrate, and they are contacted with test-compound,

C) select the compound of a kind of test-compound as treatment or prevention rheumatic arthritis, its record when making substrate and the synovial hyperplasia factor be lower than this test-compound not in conjunction with level in conjunction with level,

Wherein substrate is prolyl 4-hydroxylase α subunit or its homologue.

Simultaneously, use the P4HA1 ubiquitinization is act as index, the synovial hyperplasia factor activator agent of discovery is useful in the collagen supplement therapy.For example, with the age, uviolizing stress wait the collagen of reduction to replenish by giving the agent of synovial hyperplasia factor activator.

In addition, the present invention relates to detect synovial hyperplasia factor ubiquitin is turned into the active test kit of adjusting of usefulness and the test kit of screening treatment or prevention fibrosis and rheumatic arthritis, every kind comprises following ingredients.Can obtain the essential following ingredients of test kit of the present invention (1) and (2) with mode mentioned above.

(1) the synovial hyperplasia factor or its homologue; With

(2) P4HA1 or its homologue.

P4HA1 or its homologue in the mark test kit of the present invention in advance.Perhaps, test kit may further include marking method.For example, test kit may further include the antibody of mark, its identification and in conjunction with P4HA1 or its homologue.

Test kit of the present invention may further include following ingredients.These compositions also are mentioned above.

(3) ubiquitin activating enzyme;

(4) ubiquitin transferring enzyme;

(5) ubiquitin; With

(6) Triphosaden (ATP).

In the above-mentioned moiety, can be with the prior mark of ubiquitin.Perhaps, test kit may further include the method for mark ubiquitin.For example, can use identification and in conjunction with the antibody of the mark of ubiquitin.

Can be with the P4HA1 that constitutes test kit of the present invention in conjunction with solid phase pearl for example.Especially, comprise that the pearl of liquid sudden strain of a muscle body is favourable, because can easily detect ³²The ubiquitin of P-mark and the combination of P4HA1.Therefore, preferably will comprise in conjunction with containing liquid and dodge the test kit of the P4HA1 of body pearl and radiolabeled ubiquitin as test kit of the present invention.

Test kit of the present invention may further include the pipe and/or the medium of culturing cell.Test kit of the present invention can be used to realize method of the present invention mentioned above.

By experiment of the present invention or screening method compounds identified is the material standed for that is used for the medicinal substance of disease, and can be used to its prevention or treatment, and wherein this disease has the synovial hyperplasia factor and prolyl 4-hydroxylase to participate in.For example, fibrosis and rheumatic arthritis are the examples of these diseases.Except that activeconstituents, these compound formulations can be become pharmaceutically useful composition with solvent by suitably mixing other solute.When being used as medicine, not only can directly giving the patient, and can isolated compound be prepared into the medicinal compositions administration according to known pharmaceutical methods with isolated compound with screening method isolated compound of the present invention.

Suitably pharmaceutically useful carrier of associating or for example sterilized water, physiological saline, vegetables oil, emulsifying agent and outstanding agent of carrier when for example, the administration of compound can be passed through preparation.Medicinal compositions of the present invention can be any form, the aqueous solution for example, sheet, capsule, ingot, lozenge, elixir, suspensoid, syrup, nose drops, and inhalation.Can suitably determine the content of compound.Generally can give the patient according to technology well known by persons skilled in the art, for example, intra-arterial injection, intravenous injection, subcutaneous injection, oral and intra-articular injection.

Dosage is according to patient's body weight and age, administering mode, and changes such as symptom, but those skilled in the art can suitably select proper dosage.General dosage changes according to effective haemoconcentration of material and metabolic rate, thinks the about 0.1mg/kg of maintenance dose every day to about 1.0g/kg, and preferably about 0.1mg/kg is about 10mg/kg extremely, and 0.1mg/kg about 1.0mg/kg extremely more preferably from about.Can be once to carrying out administration several times.If compound can be mixed polynucleotide and can be carried out gene therapy so by polynucleotide encoding in gene therapy vector.

The former technical information of quoting among the present invention is all introduced it with for referencial use at this.

The accompanying drawing summary

The formation structure of the bait that the graphic presentation of Fig. 1 is used in screening with yeast two-hybrid system.

Fig. 2 is radioautograph, show [ ³²S]-the Flag-P4HA1 fusion rotein of mark and the combination of GST-SynodTM and GST-Syno RING fusion rotein.

Fig. 3 is radioautograph, the P4HA1 albumen that shows GST albumen or GST-fusion is by the measurement result of synovial hyperplasia factor protein ubiquitinization, and wherein the synovial hyperplasia factor protein comprises: Syno dTM, Syno dTM (C307S), Syno dTM (dRNG) and Syno RING.

Fig. 4 is a photo, shows to use the Western trace to judge P4HA1 expression amount in different mouse embryo fibroblasts.

Collagen content in the culture supernatant of the different mouse embryo fibroblasts of the graphic presentation of Fig. 5, Z-axis shows the ratio (0.09 μ g collagen/mg albumen is defined as 1) of collagen content, transverse axis shows the type of mouse cell.

Prolyl 4-hydroxylase activity in the different mouse embryo fibroblasts of the graphic presentation of Fig. 6,, Z-axis shows ³H-H ₂The ratio of O output, wherein Syno+ /+actual value be defined as 1, transverse axis shows the type of mouse cell.

The Photomicrograph of Fig. 7 (200 times) shows synovial hyperplasia factor expression in the liver cirrhosis pathological tissue.

A last left side: with the dyeing of antiskid film hyperplasia factor antibody

Last right: the phenodin nuclear staining

Bottom left: with mouse IgG (contrast) dyeing

Synovial hyperplasia factor expression in the pathological tissue of the Photomicrograph of Fig. 8 (200 times/right and 400 a times/left side) demonstration nodular fasciitis.Begin the dyeing that Photomicrograph shows in order and be from top: use mouse IgG, antiskid film hyperplasia factor antibody and used the antiskid film hyperplasia factor antibody of blocking peptide.

Finish the preferred plan of invention

Next, reference example specifically describes the present invention.

Embodiment 1 is by yeast two-hybrid method screening P4HA1

Use Clontech MATCHMAKER Two-Hybrid System as yeast two-hybrid system, by yeast conversion method (Pro.Natl.Aca.Sci.USA., 88:9578-9582,1991) screening synovial hyperplasia factor substrate protein.The end of synovial hyperplasia factor cDNA is modified with EcoR I/Xho I at 706bp to 1854bp and 805bp to 1260bp, and the EcoR I/Xho I site of cDNA being inserted the pGBT9 carrier.Hereinafter the synovial hyperplasia factor fragment with 706bp to 1854bp and 805bp to 1260bp is called Syno dTM and Syno RING (Fig. 1) respectively.

Human cartilage source cDNA is inserted pACT2 preparing carriers library (pACT2-Y), and this library is used for the screening of synovial hyperplasia factor substrate protein.After keeping 15 minutes with 42 ℃ of heat shock protein(HSP)s, with pGBT9-Syno dTM or pGBT9-Syno RING (2.0 μ g), and pACT2-Y (20 μ g) imports yeast Y190 bacterial strain.With the yeast Y190 that imports carrier with Tris-EDTA (pH 7.5) damping fluid (TE damping fluid) washing after, will be coated on the SD-Trp-Leu-His flat board and with the yeast Y190 solution of TE damping fluid dilution and keep 10 days in 30 ℃.On the bacterium colony that grows, carry out the analysis of beta-galactosidase enzymes filter membrane as substrate, and detect positive colony with 0.5mg/ml X-gal.

With positive colony in the SD-Leu-His substratum in 30 ℃ of shaking culture 10 days, and extract cerevisiae dna (Methods Enzymol., 194:169-182,1991) with alkali-SDS method.The cerevisiae dna that extracts is transformed in the colon bacillus HB101 bacterial strain, is coated on the M9 flat board and (Leu), and in 37C cultivated 2 days.The bacterium colony that grows is cultivated in the LB substratum that has added 20 μ g/ml penbritins, in 37 ℃ of shaking culture 16 hours, and with alkali-SDS method extraction plasmid DNA.Analyze the human cartilage source cDNA fragment in the plasmid DNA that has been inserted into extraction with the BigDye Terminator Cycle Sequencing system of AppliedBiosystems.

With BLAST ( Http:// www.ncbi.nlm.nih.gov/BLAST/) result of sequential analysis shows a α subunit that has obtained prolyl 4-hydroxylase, be α polypeptide I (accession number XM_032511P4HA1), it is the key enzyme (precollagen-proline(Pro), 2-oxoglutarate 4-dioxygenase (proline-4-hydroxylase)) of precollagenous proline residue hydroxylation during collagen produces.

The combination of the embodiment 2 synovial hyperplasia factors and P4HA1

With TNT-link coupled translation system (Promega Corporation) and pcDNA3-Flag-P4HA1 preparation [ ³⁵S]-the Flag-P4HA1 fusion rotein of mark.With fusion rotein and GST-Syno dTM, GST-Syno RING and GST albumen add in the binding buffer liquid together, and in 4 ℃ of reactions 16 hours, wherein binding buffer liquid contains 20mM HEPES (pH 7.9), 100mM NaCl, 1mM EDTA, 005% tween, 5% glycerine, 1mM DTT, 0.2mM NaVO ₄, 5mM NaF, and 1mMPMSF.The reaction product of gained is carried out SDS-PAGE and (BAS2000 Fujix) detects its radioactivity with image analyzer.Observe GST-Syno dTM and GST-Syno RING fusion rotein with [ ³⁵S]-combination of the pcDNA3-Flag-P4HA1 of mark.And between contrast GST albumen and pcDNA3-Flag-P4HA1, do not find in conjunction with (Fig. 2).These results show that the synovial hyperplasia factor is in conjunction with P4HA1.

The substrate that embodiment 3 identifies as the synovial hyperplasia factor of ubiquitin ligase (E3)

By the effect of ubiquitin involved enzyme system, ubiquitin covalent attachment target protein (substrate), wherein this enzyme system comprises activating enzyme (E1), desmoenzyme (E2) and ligase enzyme (E3).By repeating this reaction, formed many ubiquitin chain, this is as the sign of substrate protein enzyme body degraded.E3 is a most important enzyme of selecting substrate.Evaluation to the substrate protein of the synovial hyperplasia factor is considered to judging that the signal path that the synovial hyperplasia factor participates in is essential.The albumen in conjunction with the synovial hyperplasia factor that uses the screening of yeast two-hybrid method to obtain is candidate's substrate of synovial hyperplasia factor ubiquitin ligase (E3).

Therefore, use the ubiquitin ligase activity determining system to carry out in vitro study.CDNA is inserted GST-fusion rotein carrier (pGEX 4T-1), and in colon bacillus, produce fusion rotein, wherein cDNA comprises Syno dTM, Syno RING, the 307th halfcystine is substituted by the SynodTM (C307S) of Serine gained, the Syno dTM (dRING) of the deleted back of 291-329 amino acids gained and all zones of P4HA1.The fusion rotein that will obtain from the dissolving supernatant of colon bacillus is in conjunction with Glutathione Sepharose ^TM4B pearl (Amersham Biosciences).

Next, the GST of the every kind of synovial hyperplasia factor-fusion rotein pearl partly uses zymoplasm (a kind of proteolytic enzyme) to remove, and therefore produces the synovial hyperplasia factor protein.With the front insert the Histidine in PKA site-ubiquitin protein with [ ³²P-γ] the ATP mark.With E1 (the yeast source), E2 (UbcH5c), ATP, [ ³²P-γ]-ubiquitin of mark, the P4HA1 albumen pearl that GST-merges and every kind of synovial hyperplasia factor protein mix and are incorporated in 37 ℃ of reactions 60 minutes.After the reaction, each sample is mixed with 4x SDS-PAGE damping fluid, boiled 5 minutes, and carry out 10%SDS-PAGE.

(BAS2000 Fujix) carries out image analysis, and detects the band of ubiquitinization with image analyzer with the glue that developed.The result shows that P4HA1 is by Syno dTM and Syno RING ubiquitinization (Fig. 3).But P4HA1 is not by Syno dTM (C307S) and Syno dTM (dRING) ubiquitinization.This explanation P4HA1 is the substrate of the synovial hyperplasia factor.

The embodiment 4 synovial hyperplasia factors and collagen produce

P4HA1 expression level and collagen produce level in the various mouse embryo fibroblasts (MEFs)

With embryo fibroblast (MEFs) with 2 * 10 ⁵Cell inoculation was cultivated 3 days, reclaimed then.

Use anti-P4HA1 antibody (to resist-hPH (α) IgG of purifying, Daiichi Fine Chemical Co., Ltd) expression level of usefulness Western trace quantitative judgement P4HA1.Simultaneously, (monoclonal antibody is 10Da) with anti--beta-actin antibody (monoclonal resisting-beta-actin, CloneAC-15, Sigma) expression level of the contrast synovial hyperplasia factor and beta-actin with antiskid film hyperplasia factor antibody.Use Sircol ^TMSoluble collagen is measured test kit (Biocolor Ltd.) detection by quantitative collagen.By being defined as 1, collagen content (0.09 μ g collagen/mg protein) in MEF (syno+ /+) cell carries out data processing.

As a result, the P4HA1 protein level of the mouse of the deletion synovial hyperplasia factor (syno-/-) MEFs basically with identical (Fig. 4) of wild-type mice (syno+ /+) MEFs.Contrast, find syno-/-collagen content in the MEFs culture supernatant be lower than syno+ /+(Fig. 5).These two discoveries show that the synovial hyperplasia factor controls the possibility of the quality of prolyl 4-hydroxylase protein in the cell by the P4HA1 ubiquitinization.

The comparison of prolyl 4-hydroxylase activity among the various MEFs

Measure prolyl 4-hydroxylase activity with reference to following article.

1)Gribble?TJ，Comstock?JP，Udenfriend?S.，“Collagen?chain?formation?andPeptidyl?proline?hydroxylation?in?monolayer?tissue?cultures?of?L-929fibroblasts.”，Arch?Biochem?Biophys.，1969，Jan；129(1)：308-16

2)Margolis?RL，Lukens?LN.，“The?role?of?hydroxylation?in?the?secretion?ofcollagen?by?mouse?fibroblasts?in?culture.Arch?Biochem?Biophys.”，1971，Dec；147(2)：612-8.

3)Methods?enzymol.，Vol.82(1982)p247-265.

To normal synovial cell (that is, collagenic cell add α，α′-Lian Biding in) the substratum to stop the hydroxylation reaction of hydroxylase in the cell, in culture supernatant, add then [2,3- ³H] proline(Pro), and extract synthetic ³H-tropocollagen.From syno-/-MEF and syno+ /+the MEF culturing cell reclaims the lysate that comprises P4H, and measure and make ³The activity of H-tropocollagen hydroxylation ( ³H-H ₂The output of O).

As a result, ³H-H ₂The output of O Syno+ /+be the total cell extract of 3928cpm/ μ g, Syno-/-be the total cell extract of 27.58cpm/ μ g.With Syno+ /+actual value be defined as 1, ³H-H ₂Ratio such as Fig. 6 of O output show, show syno-/-MEFs in the enzymic activity of P4H be lower than syno+ /+MEFs person (Fig. 6).

Synovial hyperplasia factor expression in the pathological tissue of embodiment 5 liver cirrhosis and nodular fasciitis

For identifying the expressive site of the synovial hyperplasia factor in Fibrotic disease, use the pathological tissue of liver cirrhosis and nodular fasciitis to carry out histogenic immunity dyeing with antiskid film hyperplasia factor antibody.

Under the situation of informed consent, obtain the pathological tissue of liver cirrhosis and nodular fasciitis.To organize according to traditional method and to use formalin fixed, paraffin embedding, the slicing machine section, and as tissue sample.To handle with dimethylbenzene (three times, each 5 minutes) immerse in the serial spirituous solution of 6 reduction levels gradually (each reduces by 10%) with the tissue slice of removing paraffin fully, its from 100% ethanol to 50% ethanolic soln, and with 1% bovine serum albumin (BSA) sealing 30 minutes.

After sealing is finished, with the anti-solution (antiskid film hyperplasia factor monoclonal antibodies) of tissue and the PBS dilution that contains 1%BSA in room temperature reaction 60 minutes.Subsequently, will be organized among the PBS washing 5 times, each 5 minutes, then with the anti-mouse immuning ball protein antibody of two anti-HRP-marks of 1%BSA solution dilution in room temperature reaction 30 minutes.After reaction is finished, will be organized among the PBS washing 5 times, each 5 minutes, and further with colouring reagents (3,3 '-diaminobenzidine four hydrochloric acid) reaction.After reaching suitable colour developing, tissue is washed with water and examines under a microscope.

In addition, tissue being carried out the bush sperm nucleus redyes and examines under a microscope.And, in contrast, examine under a microscope the sample that is used for the reaction of painted and blocking peptide, wherein blocking peptide comprises epi-position aminoacid sequence of antiskid film hyperplasia factor monoclonal antibodies.

As a result, on the hepatic parenchymal cells of cirrhotic tissue, observe synovial hyperplasia factor high expression level, particularly in the part (Fig. 7) of height cell proliferation.At manadesma inoblast tumor-like hyperplasia is in the nodular fasciitis tissue of feature, also observes synovial hyperplasia factor high expression level (Fig. 8) in the tubercle part of height hyperplasia.

Embodiment 6 screenings suppress the method for the synovial hyperplasia factor and P4HA1 bonded compound

Research suppresses high flux screening (HTS) method of the synovial hyperplasia factor and its substrate P4HA1 bonded compound.Design a system, wherein the synovial hyperplasia factor expression is the albumen of histidine-tagged fusion and P4HA1 is expressed as the GST-fusion rotein.In this system, when the synovial hyperplasia factor during in conjunction with P4HA1, between anti--GST antibody of the anti-Histidine antibody of XL665-mark and Eu (K)-mark energy takes place and shift, detect fluorescence then.

1) preparation of MBP-Syno dTM-His

MBP synovial hyperplasia factor dTM-Hisx ₁₂Comprise its disappearance transmembrane segment (TM) (synovial hyperplasia factor dTM) of the synovial hyperplasia factor that MBP-merges.MBP synovial hyperplasia factor dTM-Hisx ₁₂Prepare by in colon bacillus, producing fusion rotein, and with Ni-NTA post (QIAGEN) purifying.

2) preparation of His-E2

His-E2 inserts the protein carrier (pET) of histidine-tagged fusion by the cDNA that will comprise the UbcH5c complete area, produces fusion rotein and obtain in colon bacillus, the fusion rotein of gained Ni-NTA post (QIAGEN) purifying.

3) preparation of GST-P4HA1

The cDNA that will comprise the P4HA1 complete area inserts GST-fusion rotein carrier (pGEX 4T-1), and produces this fusion rotein in colon bacillus.GST-P4HA1 prepares by obtaining this fusion rotein from colon bacillus dissolving supernatant, and uses Glutathione Sepharose ^TM4B pearl (Amersham Bioscience) purifying.

4) to suppressing the synovial hyperplasia factor and the active mensuration of P4HA1 bonded

With 384-orifice plate preparation standard reaction mixture, it comprises the 60ng MBP-synovial hyperplasia factor-Histidine, 10ng GST-P4HA1,1mM DTT, 1mM EDTA, 50mM NaCl, 0.1%BSA, 0.3%DMSO and the test-compound in 15 μ L 50mM HEPES (pH 7.5) solution.The 5 μ L 50mM HEPES solution that contain GST-P4HA1 by adding start reaction.Behind the room temperature reaction 30 minutes, add 5 μ L 50mM HEPES solution, it comprises anti--GST antibody (the 20000 B counts of Eu (K)-mark, CIS bio intemational), anti-Histidine antibody of XL665-mark (0.025 μ g, CIS bio international) and 1M KF.Mixture is spent the night in the room temperature placement, use RUBYstar ^TMDetect fluorescence.

According to suppressing active research, the partial peptide (SEQ ID NO:5) of the local synovial hyperplasia factor of modifying is with the IC of 167 μ g/ml ₅₀Suppress the synovial hyperplasia factor and P4HA1 combination.

Embodiment 7 screenings suppress the method for the active compound of P4HA1 ubiquitinization of the synovial hyperplasia factor

1) utilize XL665-Eu (K) energy to shift the method (HTRF method) that screening suppresses the active compound of ubiquitinization

Research suppresses high flux screening (HTS) method of the active compound of P4HA1 ubiquitinization of the synovial hyperplasia factor.Design a system, wherein carry out the ubiquitinization of the ubiquitin of Eu (K)-mark as substrate with the P4HA1 that is expressed as the GST-fusion rotein, when the ubiquitin of Eu (K)-mark takes place in P4HA1, energy taking place between the ubiquitin of the anti-GST antibody of XL665-mark and Eu (K)-mark shift, detects fluorescence then.

With 384-orifice plate preparation standard reaction mixture, it comprises the 60ng MBP-synovial hyperplasia factor-His, 15ng GST-P4HA1,15ng E1 (Boston Biochem), 200ng His-E2, the ubiquitin of 6.25nM Eu (K)-mark (CIS bio international), 5mM MgCl ₂, 2mM ATP, 1mM DTT, 0.1%BSA, 0.3%DMSO and the test-compound in 15 μ L 20mM HEPES (pH 75) solution.After 30 minutes, add 5 μ L 0.5M EDTA termination reactions in 37 ℃ of reactions.To wherein adding 5 μ L20mM HEPES solution, it comprises the anti-his antibody (0.025 μ g) of XL665-mark, and 1MKF.Mixture is spent the night in the room temperature placement, use RUBYstar ^TMDetect fluorescence.

To hang down 20 times concentration than suppressing bonded concentration, the partial peptide (SEQ ID NO:5) of the local synovial hyperplasia factor of modifying suppresses the ubiquitinization of P4HA1.

2) screen the method that suppresses the active compound of ubiquitinization with ELISA

96-hole elisa plate is spent the night in 4 ℃ of absorption with anti-GST antibody, seal until use in room temperature with 5%BSA/PBS solution then.Plate is used 300 μ L PBS (PBSA) washed twice that contain 0.3%BSA before use.

Preparation standard reaction mixture in reaction tubes, it contains the 80ng MBP-synovial hyperplasia factor-His, 40ng GST-P4HA1,15ng E1,200ng His-E2,5mM MgCl ₂, 2mM ATP, 1mM DTT, 0.1%BSA, 0.3%DMSO and the test-compound in 30 μ L 50mM HEPES (pH7.5) solution., add and contain the solution 70 μ L of 70mM EDTA and 50mMHEPES after 30 minutes in room temperature reaction with termination reaction.Add its (30 μ l) in the plate contain 70 μ L PBSA in advance and in room temperature insulation 1 hour.After 300 μ L PBSA washing three times, add mouse anti-ubiquitin antibody solution 100 μ L, and mixture is incubated 1 hour in room temperature with PBSA dilution in 1: 5000.After 300 μ L PBSA washing three times, add the anti-mouse IgG solution of 100 μ L, and mixture is incubated 1 hour in room temperature with the horseradish peroxidase-labeled of PBSA dilution in 1: 10000.After 300 μ L PBSA washing three times, add 100 μ L TMB solution and mixture is incubated in room temperature.When observing enough colors, add 1M phosphoric acid with termination reaction, measure optical density(OD) in 450nm.

The partial peptide (SEQ ID NO:5) of the local synovial hyperplasia factor of modifying is with the IC of 55 μ g/ml ₅₀The ubiquitinization that suppresses P4HA1.

Industrial usability

Can be according to evaluation of the present invention to synovial hyperplasia factor ubiquitin prolyl 4 hydroxylase α subunit Regulate active. In addition, provide based on the inventive method and screen synovial hyperplasia factor ubiquitin dried meat ammonia Acyl 4-hydroxylase α subunit has the method for regulating active compound. And, the invention provides screening and use In the method for the treatment of or preventing the compound of rheumatic arthritis.

The fact that the synovial hyperplasia factor is regulated the prolyl 4 hydroxylase activity by ubiquitin α subunit is this The new discovery that inventors disclose for the first time. Prolyl 4 hydroxylase is the synthetic key enzyme of collagen in the body. Therefore, can be with the compound of method of the present invention screening to treating and/or preventing the hydroxylation by prolyl 4-The disease that enzymatic activity causes unusually is useful. For example, pile up fibrillatable or the rheumatism that causes by collagen The property arthritis can use compounds for treating or the prevention that obtains according to the present invention.

Sequence table

＜110〉Locomogene Inc. (LOCOMOGENE, INC.)

Eisai Co., Ltd (Eisai Co., Ltd.)

＜120〉detection is to the method for the regulating effect of synovial hyperplasia factor active

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<150>JP?2003-295951

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<151>2003-08-20

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<400>1

atg?atc?tgg?tat?ata?tta?att?ata?gga?att?ctg?ctt?ccc?cag?tct?ttg 48

Met?Ile?Trp?Tyr?Ile?Leu?Ile?Ile?Gly?Ile?Leu?Leu?Pro?Gln?Ser?Leu

1 5 10 15

gct?cat?cca?ggc?ttt?ttt?act?tca?att?ggt?cag?atg?act?gat?ttg?atc 96

Ala?His?Pro?Gly?Phe?Phe?Thr?Ser?Ile?Gly?Gln?Met?Thr?Asp?Leu?Ile

20 25 30

cat?act?gag?aaa?gat?ctg?gtg?act?tct?ctg?aaa?gat?tat?att?aag?gca 144

His?Thr?Glu?Lys?Asp?Leu?Val?Thr?Ser?Leu?Lys?Asp?Tyr?Ile?Lys?Ala

35 40 45

gaa?gag?gac?aag?tta?gaa?caa?ata?aaa?aaa?tgg?gca?gag?aag?tta?gat 192

Glu?Glu?Asp?Lys?Leu?Glu?Gln?Ile?Lys?Lys?Trp?Ala?Glu?Lys?Leu?Asp

50 55 60

cgg?cta?act?agt?aca?gcg?aca?aaa?gat?cca?gaa?gga?ttt?gtt?ggg?cat 240

Arg?Leu?Thr?Ser?Thr?Ala?Thr?Lys?Asp?Pro?Glu?Gly?Phe?Val?Gly?His

65 70 75 80

cca?gta?aat?gca?ttc?aaa?tta?atg?aaa?cgt?ctg?aat?act?gag?tgg?agt 288

Pro?Val?Asn?Ala?Phe?Lys?Leu?Met?Lys?Arg?Leu?Asn?Thr?Glu?Trp?Ser

85 90 95

gag?ttg?gag?aat?ctg?gtc?ctt?aag?gat?atg?tca?gat?ggc?ttt?atc?tct 336

Glu?Leu?Glu?Asn?Leu?Val?Leu?Lys?Asp?Met?Ser?Asp?Gly?Phe?Ile?Ser

100 105 110

aac?cta?acc?att?cag?aga?cag?tac?ttt?cct?aat?gat?gaa?gat?cag?gtt 384

Asn?Leu?Thr?Ile?Gln?Arg?Gln?Tyr?Phe?Pro?Asn?Asp?Glu?Asp?Gln?Val

115 120 125

ggg?gca?gcc?aaa?gct?ctg?tta?cgt?ctc?cag?gat?acc?tac?aat?ttg?gat 432

Gly?Ala?Ala?Lys?Ala?Leu?Leu?Arg?Leu?Gln?Asp?Thr?Tyr?Asn?Leu?Asp

130 135 140

aca?gat?acc?atc?tca?aag?ggt?aat?ctt?cca?gga?gtg?aaa?cac?aaa?tct 480

Thr?Asp?Thr?Ile?Ser?Lys?Gly?Asn?Leu?Pro?Gly?Val?Lys?His?Lys?Ser

145 150 155 160

ttt?cta?acg?gct?gag?gac?tgc?ttt?gag?ttg?ggc?aaa?gtg?gcc?tat?aca 528

Phe?Leu?Thr?Ala?Glu?Asp?Cys?Phe?Glu?Leu?Gly?Lys?Val?Ala?Tyr?Thr

165 170 175

gaa?gca?gat?tat?tac?cat?acg?gaa?ctg?tgg?atg?gaa?caa?gcc?cta?agg 576

Glu?Ala?Asp?Tyr?Tyr?His?Thr?Glu?Leu?Trp?Met?Glu?Gln?Ala?Leu?Arg

180 185 190

caa?ctg?gat?gaa?ggc?gag?att?tct?acc?ata?gat?aaa?gtc?tct?gtt?cta 624

Gln?Leu?Asp?Glu?Gly?Glu?Ile?Ser?Thr?Ile?Asp?Lys?Val?Ser?Val?Leu

195 200 205

gat?tat?ttg?agc?tat?gcg?gta?tat?cag?cag?gga?gac?ctg?gat?aag?gca 672

Asp?Tyr?Leu?Ser?Tyr?Ala?Val?Tyr?Gln?Gln?Gly?Asp?Leu?Asp?Lys?Ala

210 215 220

ctt?ttg?ctc?aca?aag?aag?ctt?ctt?gaa?cta?gat?cct?gaa?cat?cag?aga 720

Leu?Leu?Leu?Thr?Lys?Lys?Leu?Leu?Glu?Leu?Asp?Pro?Glu?His?Gln?Arg

225 230 235 240

gct?aat?ggt?aac?tta?aaa?tat?ttt?gag?tat?ata?atg?gct?aaa?gaa?aaa 768

Ala?Asn?Gly?Asn?Leu?Lys?Tyr?Phe?Glu?Tyr?Ile?Met?Ala?Lys?Glu?Lys

245 250 255

gat?gtc?aat?aag?tct?gct?tca?gat?gac?caa?tct?gat?cag?aaa?act?aca 816

Asp?Val?Asn?Lys?Ser?Ala?Ser?Asp?Asp?Gln?Ser?Asp?Gln?Lys?Thr?Thr

260 265 270

cca?aag?aaa?aaa?ggg?gtt?gct?gtg?gat?tac?ctg?cca?gag?aga?cag?aag 864

Pro?Lys?Lys?Lys?Gly?Val?Ala?Val?Asp?Tyr?Leu?Pro?Glu?Arg?Gln?Lys

275 280 285

tac?gaa?atg?ctg?tgc?cgt?ggg?gag?ggt?atc?aaa?atg?acc?cct?cgg?aga 912

Tyr?Glu?Met?Leu?Cys?Arg?Gly?Glu?Gly?Ile?Lys?Met?Thr?Pro?Arg?Arg

290 295 300

cag?aaa?aaa?ctc?ttt?tgc?cgc?tac?cat?gat?gga?aac?cgt?aat?cct?aaa 960

Gln?Lys?Lys?Leu?Phe?Cys?Arg?Tyr?His?Asp?Gly?Asn?Arg?Asn?Pro?Lys

305 310 315 320

ttt?att?ctg?gct?cca?gct?aaa?cag?gag?gat?gaa?tgg?gac?aag?cct?cgt 1008

Phe?Ile?Leu?Ala?Pro?Ala?Lys?Gln?Glu?Asp?Glu?Trp?Asp?Lys?Pro?Arg

325 330 335

att?att?cgc?ttc?cat?gat?att?att?tct?gat?gca?gaa?att?gaa?atc?gtc 1056

Ile?Ile?Arg?Phe?His?Asp?Ile?Ile?Ser?Asp?Ala?Glu?Ile?Glu?Ile?Val

340 345 350

aaa?gac?cta?gca?aaa?cca?agg?ctg?agc?cga?gct?aca?gta?cat?gac?cct 1104

Lys?Asp?Leu?Ala?Lys?Pro?Arg?Leu?Ser?Arg?Ala?Thr?Val?His?Asp?Pro

355 360 365

gag?act?gga?aaa?ttg?acc?aca?gca?cag?tac?aga?gta?tct?aag?agt?gcc 1152

Glu?Thr?Gly?Lys?Leu?Thr?Thr?Ala?Gln?Tyr?Arg?Val?Ser?Lys?Ser?Ala

370 375 380

tgg?ctc?tct?ggc?tat?gaa?aat?cct?gtg?gtg?tct?cga?att?aat?atg?aga 1200

Trp?Leu?Ser?Gly?Tyr?Glu?Asn?Pro?Val?Val?Ser?Arg?Ile?Asn?Met?Arg

385 390 395 400

ata?caa?gat?cta?aca?gga?cta?gat?gtt?tcc?aca?gca?gag?gaa?tta?cag 1248

Ile?Gln?Asp?Leu?Thr?Gly?Leu?Asp?Val?Ser?Thr?Ala?Glu?Glu?Leu?Gln

405 410 415

gta?gca?aat?tat?gga?gtt?gga?gga?cag?tat?gaa?ccc?cat?ttt?gac?ttt 1296

Val?Ala?Asn?Tyr?Gly?Val?Gly?Gly?Gln?Tyr?Glu?Pro?His?Phe?Asp?Phe

420 425 430

gca?cgg?aaa?gat?gag?cca?gat?gct?ttc?aaa?gag?ctg?ggg?aca?gga?aat 1344

Ala?Arg?Lys?Asp?Glu?Pro?Asp?Ala?Phe?Lys?Glu?Leu?Gly?Thr?Gly?Asn

435 440 445

aga?att?gct?aca?tgg?ctg?ttt?tat?atg?agt?gat?gtg?tct?gca?gga?gga 1392

Arg?Ile?Ala?Thr?Trp?Leu?Phe?Tyr?Met?Ser?Asp?Val?Ser?Ala?Gly?Gly

450 455 460

gcc?act?gtt?ttt?cct?gaa?gtt?gga?gct?agt?gtt?tgg?ccc?aaa?aaa?gga 1440

Ala?Thr?Val?Phe?Pro?Glu?Val?Gly?Ala?Ser?Val?Trp?Pro?Lys?Lys?Gly

465 470 475 480

act?gct?gtt?ttc?tgg?tat?aat?ctg?ttt?gcc?agt?gga?gaa?gga?gat?tat 1488

Thr?Ala?Val?Phe?Trp?Tyr?Asn?Leu?Phe?Ala?Ser?Gly?Glu?Gly?Asp?Tyr

485 490 495

agt?aca?cgg?cat?gca?gcc?tgt?cca?gtg?cta?gtt?ggc?aac?aaa?tgg?gta 1536

Ser?Thr?Arg?His?Ala?Ala?Cys?Pro?Val?Leu?Val?Gly?Asn?Lys?Trp?Val

500 505 510

tcc?aat?aaa?tgg?ctc?cat?gaa?cgt?gga?caa?gaa?ttt?cga?aga?cct?tgt 1584

Ser?Asn?Lys?Trp?Leu?His?Glu?Arg?Gly?Gln?Glu?Phe?Arg?Arg?Pro?Cys

515 520 525

acg?ttg?tca?gaa?ttg?gaa?tga 1605

Thr?Leu?Ser?Glu?Leu?Glu

530

<210>2

<211>534

<212>PRT

＜213〉people (Homo sapiens)

<400>2

Met?Ile?Trp?Tyr?Ile?Leu?Ile?Ile?Gly?Ile?Leu?Leu?Pro?Gln?Ser?Leu

1 5 10 15

Ala?His?Pro?Gly?Phe?Phe?Thr?Ser?Ile?Gly?Gln?Met?Thr?Asp?Leu?Ile

20 25 30

His?Thr?Glu?Lys?Asp?Leu?Val?Thr?Ser?Leu?Lys?Asp?Tyr?Ile?Lys?Ala

35 40 45

Glu?Glu?Asp?Lys?Leu?Glu?Gln?Ile?Lys?Lys?Trp?Ala?Glu?Lys?Leu?Asp

50 55 60

Arg?Leu?Thr?Ser?Thr?Ala?Thr?Lys?Asp?Pro?Glu?Gly?Phe?Val?Gly?His

65 70 75 80

Pro?Val?Asn?Ala?Phe?Lys?Leu?Met?Lys?Arg?Leu?Asn?Thr?Glu?Trp?Ser

85 90 95

Glu?Leu?Glu?Asn?Leu?Val?Leu?Lys?Asp?Met?Ser?Asp?Gly?Phe?Ile?Ser

100 105 110

Asn?Leu?Thr?Ile?Gln?Arg?Gln?Tyr?Phe?Pro?Asn?Asp?Glu?Asp?Gln?Val

115 120 125

Gly?Ala?Ala?Lys?Ala?Leu?Leu?Arg?Leu?Gln?Asp?Thr?Tyr?Asn?Leu?Asp

130 135 140

Thr?Asp?Thr?Ile?Ser?Lys?Gly?Asn?Leu?Pro?Gly?Val?Lys?His?Lys?Ser

145 150 155 160

Phe?Leu?Thr?Ala?Glu?Asp?Cys?Phe?Glu?Leu?Gly?Lys?Val?Ala?Tyr?Thr

165 170 175

Glu?Ala?Asp?Tyr?Tyr?His?Thr?Glu?Leu?Trp?Met?Glu?Gln?Ala?Leu?Arg

180 185 190

Gln?Leu?Asp?Glu?Gly?Glu?Ile?Ser?Thr?Ile?Asp?Lys?Val?Ser?Val?Leu

195 200 205

Asp?Tyr?Leu?Ser?Tyr?Ala?Val?Tyr?Gln?Gln?Gly?Asp?Leu?Asp?Lys?Ala

210 215 220

Leu?Leu?Leu?Thr?Lys?Lys?Leu?Leu?Glu?Leu?Asp?Pro?Glu?His?Gln?Arg

225 230 235 240

Ala?Asn?Gly?Asn?Leu?Lys?Tyr?Phe?Glu?Tyr?Ile?Met?Ala?Lys?Glu?Lys

245 250 255

Asp?Val?Asn?Lys?Ser?Ala?Ser?Asp?Asp?Gln?Ser?Asp?Gln?Lys?Thr?Thr

260 265 270

Pro?Lys?Lys?Lys?Gly?Val?Ala?Val?Asp?Tyr?Leu?Pro?Glu?Arg?Gln?Lys

275 280 285

Tyr?Glu?Met?Leu?Cys?Arg?Gly?Glu?Gly?Ile?Lys?Met?Thr?Pro?Arg?Arg

290 295 300

Gln?Lys?Lys?Leu?Phe?Cys?Arg?Tyr?His?Asp?Gly?Asn?Arg?Asn?Pro?Lys

305 310 315 320

Phe?Ile?Leu?Ala?Pro?Ala?Lys?Gln?Glu?Asp?Glu?Trp?Asp?Lys?Pro?Arg

325 330 335

Ile?Ile?Arg?Phe?His?Asp?Ile?Ile?Ser?Asp?Ala?Glu?Ile?Glu?Ile?Val

340 345 350

Lys?Asp?Leu?Ala?Lys?Pro?Arg?Leu?Ser?Arg?Ala?Thr?Val?His?Asp?Pro

355 360 365

Glu?Thr?Gly?Lys?Leu?Thr?Thr?Ala?Gln?Tyr?Arg?Val?Ser?Lys?Ser?Ala

370 375 380

Trp?Leu?Ser?Gly?Tyr?Glu?Asn?Pro?Val?Val?Ser?Arg?Ile?Asn?Met?Arg

385 390 395 400

Ile?Gln?Asp?Leu?Thr?Gly?Leu?Asp?Val?Ser?Thr?Ala?Glu?Glu?Leu?Gln

405 4l0 415

Val?Ala?Asn?Tyr?Gly?Val?Gly?Gly?Gln?Tyr?Glu?Pro?His?Phe?Asp?Phe

420 425 430

Ala?Arg?Lys?Asp?Glu?Pro?Asp?Ala?Phe?Lys?Glu?Leu?Gly?Thr?Gly?Asn

435 440 445

Arg?Ile?Ala?Thr?Trp?Leu?Phe?Tyr?Met?Ser?Asp?Val?Ser?Ala?Gly?Gly

450 455 460

Ala?Thr?Val?Phe?Pro?Glu?Val?Gly?Ala?Ser?Val?Trp?Pro?Lys?Lys?Gly

465 470 475 480

Thr?Ala?Val?Phe?Trp?Tyr?Asn?Leu?Phe?Ala?Ser?Gly?Glu?Gly?Asp?Tyr

485 490 495

Ser?Thr?Arg?His?Ala?Ala?Cys?Pro?Val?Leu?Val?Gly?Asn?Lys?Trp?Val

500 505 510

Ser?Asn?Lys?Trp?Leu?His?Glu?Arg?Gly?Gln?Glu?Phe?Arg?Arg?Pro?Cys

515 520 525

Thr?Leu?Ser?Glu?Leu?Glu

530

<210>3

<211>3374

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(403)..(2256)

<223>

<400>3

gccctttctt?atgagcatgc?ctgtgttggg?ttgacagtga?gggtaataat?gacttgttgg 60

ttgattgtag?atatagggct?ctcccttgca?aggtaattag?gctccttaaa?ttacctgtaa 120

gattttcttg?ccacagcatc?cattctggtt?aggctggtga?tcttctgagt?agtgatagat 180

tggttggtgg?tgaggtttac?aggtgttccc?ttctcttact?cctggtgttg?gctacaatca 240

ggtggcgtct?agagcagcat?gggacaggtg?ggtaagggga?gtcttctcat?tatgcagaag 300

tgatcaactt?aaatctctgt?cagatctacc?tttatgtagc?ccggcagtcg?cgcggattga 360

gcgggctcgc?ggcgctgggt?tcctggtctc?cgggccaggg?ca?atg?ttc?cgc?acg 414

Met?Phe?Arg?Thr

1

gca?gtg?atg?atg?gcg?gcc?agc?ctg?gcg?ctg?acc?ggg?gct?gtg?gtg?gct 462

Ala?Val?Met?Met?Ala?Ala?Ser?Leu?Ala?Leu?Thr?Gly?Ala?Val?Val?Ala

5 10 15 20

cac?gcc?tac?tac?ctc?aaa?cac?cag?ttc?tac?ccc?act?gtg?gtg?tac?ctg 510

His?Ala?Tyr?Tyr?Leu?Lys?His?Gln?Phe?Tyr?Pro?Thr?Val?Val?Tyr?Leu

25 30 35

acc?aag?tcc?agc?ccc?agc?atg?gca?gtc?ctg?tac?atc?cag?gcc?ttt?gtc 558

Thr?Lys?Ser?Ser?Pro?Ser?Met?Ala?Val?Leu?Tyr?Ile?Gln?Ala?Phe?Val

40 45 50

ctt?gtc?ttc?ctt?ctg?ggc?aag?gtg?atg?ggc?aag?gtg?ttc?ttt?ggg?caa 606

Leu?Val?Phe?Leu?Leu?Gly?Lys?Val?Met?Gly?Lys?Val?Phe?Phe?Gly?Gln

55 60 65

ctg?agg?gca?gca gag?atg?gag?cac?ctt?ctg?gaa?cgt?tcc?tgg?tac?gcc 654

Leu?Arg?Ala?Ala?Glu?Met?Glu?Hi?s?Leu?Leu?Glu?Arg?Ser?Trp?Tyr?Ala

70 75 80

gtc?aca?gag?act?tgt?ctg?gcc?ttc?acc?gtt?ttt?cgg?gat?gac?ttc?agc 702

Val?Thr?Glu?Thr?Cys?Leu?Ala?Phe?Thr?Val?Phe?Arg?Asp?Asp?Phe?Ser

85 90 95 100

ccc?cgc?ttt?gtt?gca?ctc?ttc?act?ctt?ctt?ctc?ttc?ctc?aaa?tgt?ttc 750

Pro?Arg?Phe?Val?Ala?Leu?Phe?Thr?Leu?Leu?Leu?Phe?Leu?Lys?Cys?Phe

105 110 115

cac?tgg?ctg?gct?gag?gac?cgt?gtg?gac?ttt?atg?gaa?cgc?agc?ccc?aac 798

His?Trp?Leu?Ala?Glu?Asp?Arg?Val?Asp?Phe?Met?Glu?Arg?Ser?Pro?Asn

120 125 130

atc?tcc?tgg?ctc?ttt?cac?tgc?cgc?att?gtc?tct?ctt?atg?ttc?ctc?ctg 846

Ile?Ser?Trp?Leu?Phe?His?Cys?Arg?Ile?Val?Ser?Leu?Met?Phe?Leu?Leu

135 140 145

ggc?atc?ctg?gac?ttc?ctc?ttc?gtc?agc?cac?gcc?tat?cac?agc?atc?ctg 894

Gly?Ile?Leu?Asp?Phe?Leu?Phe?Val?Ser?His?Ala?Tyr?His?Ser?Ile?Leu

150 155 160

acc?cgt?ggg?gcc?tct?gtg?cag?ctg?gtg?ttt?ggc?ttt?gag?tat?gcc?atc 942

Thr?Arg?Gly?Ala?Ser?Val?Gln?Leu?Val?Phe?Gly?Phe?Glu?Tyr?Ala?Ile

165 170 175 180

ctg?atg?acg?atg?gtg?ctc?acc?atc?ttc?atc?aag?tat?gtg?ctg?cac?tcc 990

Leu?Met?Thr?Met?Val?Leu?Thr?Ile?Phe?Ile?Lys?Tyr?Val?Leu?His?Ser

185 190 195

gtg?gac?ctc?cag?agt?gag?aac?ccc?tgg?gac?aac?aag?gct?gtg?tac?atg 1038

Val?Asp?Leu?Gln?Ser?Glu?Asn?Pro?Trp?Asp?Asn?Lys?Ala?Val?Tyr?Met

200 205 210

ctc?tac?aca?gag?ctg?ttt?aca?ggc?ttc?atc?aag?gtt?ctg?ctg?tac?atg 1086

Leu?Tyr?Thr?Glu?Leu?Phe?Thr?Gly?Phe?Ile?Lys?Val?Leu?Leu?Tyr?Met

215 220 225

gcc?ttc?atg?acc?atc?atg?atc?aag?gtg?cac?acc?ttc?cca?ctc?ttt?gcc 1134

Ala?Phe?Met?Thr?Ile?Met?Ile?Lys?Val?His?Thr?Phe?Pro?Leu?Phe?Ala

230 235 240

atc?cgg?ccc?atg?tac?ctg?gcc?atg?aga?cag?ttc?aag?aaa?gct?gtg?aca 1182

Ile?Arg?Pro?Met?Tyr?Leu?Ala?Met?Arg?Gln?Phe?Lys?Lys?Ala?Val?Thr

245 250 255 260

gat?gcc?atc?atg?tct?cgc?cga?gcc?atc?cgc?aac?atg?aac?acc?ctg?tat 1230

Asp?Ala?Ile?Met?Ser?Arg?Arg?Ala?Ile?Arg?Asn?Met?Asn?Thr?Leu?Tyr

265 270 275

cca?gat?gcc?acc?cca?gag?gag?ctc?cag?gca?atg?gac?aat?gtc?tgc?atc 1278

Pro?Asp?Ala?Thr?Pro?Glu?Glu?Leu?Gln?Ala?Met?Asp?Asn?Val?Cys?Ile

280 285 290

atc?tgc?cga?gaa?gag?atg?gtg?act?ggt?gcc?aag?aga?ctg?ccc?tgc?aac 1326

Ile?Cys?Arg?Glu?Glu?Met?Val?Thr?Gly?Ala?Lys?Arg?Leu?Pro?Cys?Asn

295 300 305

cac?att?ttc?cat?acc?agc?tgc?ctg?cgc?tcc?tgg?ttc?cag?cgg?cag?cag 1374

His?Ile?Phe?His?Thr?Ser?Cys?Leu?Arg?Ser?Trp?Phe?Gln?Arg?Gln?Gln

310 315 320

acc?tgc?ccc?acc?tgc?cgt?atg?gat?gtc?ctt?cgt?gca?tcg?ctg?cca?gcg 1422

Thr?Cys?Pro?Thr?Cys?Arg?Met?Asp?Val?Leu?Arg?Ala?Ser?Leu?Pro?Ala

325 330 335 340

cag?tca?cca?cca?ccc?ccg?gag?cct?gcg?gat?cag?ggg?cca?ccc?cct?gcc 1470

Gln?Ser?Pro?Pro?Pro?Pro?Glu?Pro?Ala?Asp?Gln?Gly?Pro?Pro?Pro?Ala

345 350 355

ccc?cac?ccc?cca?cca?ctc?ttg?cct?cag?ccc?ccc?aac?ttc?ccc?cag?ggc 1518

Pro?His?Pro?Pro?Pro?Leu?Leu?Pro?Gln?Pro?Pro?Asn?Phe?Pro?Gln?Gly

360 365 370

ctc?ctg?cct?cct?ttt?cct?cca?ggc?atg?ttc?cca?ctg?tgg?ccc?ccc?atg 1566

Leu?Leu?Pro?Pro?Phe?Pro?Pro?Gly?Met?Phe?Pro?Leu?Trp?Pro?Pro?Met

375 380 385

ggc?ccc?ttt?cca?cct?gtc?ccg?cct?ccc?ccc?agc?tca?gga?gag?gct?gtg 1614

Gly?Pro?Phe?Pro?Pro?Val?Pro?Pro?Pro?Pro?Ser?Ser?Gly?Glu?Ala?Val

390 395 400

gct?cct?cca?tcc?acc?agt?gca?gca?gcc?ctt?tct?cgg?ccc?agt?gga?gca 1662

Ala?Pro?Pro?Ser?Thr?Ser?Ala?Ala?Ala?Leu?Ser?Arg?Pro?Ser?Gly?Ala

405 410 415 420

gct?aca?acc?aca?gct?gct?ggc?acc?agt?gct?act?gct?gct?tct?gcc?aca 1710

Ala?Thr?Thr?Thr?Ala?Ala?Gly?Thr?Ser?Ala?Thr?Ala?Ala?Ser?Ala?Thr

425 430 435

gca?tct?ggc?cca?ggc?tct?ggc?tct?gcc?cca?gag?gct?ggc?cct?gcc?cct 1758

Ala?Ser?Gly?Pro?Gly?Ser?Gly?Ser?Ala?Pro?Glu?Ala?Gly?Pro?Ala?Pro

440 445 450

ggt?ttc?ccc?ttc?cct?cct?ccc?tgg?atg?ggt?atg?ccc?ctg?cct?cca?ccc 1806

Gly?Phe?Pro?Phe?Pro?Pro?Pro?Trp?Met?Gly?Met?Pro?Leu?Pro?Pro?Pro

455 460 465

ttt?gcc?ttc?ccc?cca?atg?cct?gtg?ccc?cct?gcg?ggc?ttt?gct?ggg?ctg 1854

Phe?Ala?Phe?Pro?Pro?Met?Pro?Val?Pro?Pro?Ala?Gly?Phe?Ala?Gly?Leu

470 475 480

acc?cca?gag?gag?cta?cga?gct?ctg?gag?ggc?cat?gag?cgg?cag?cac?ctg 1902

Thr?Pro?Glu?Glu?Leu?Arg?Ala?Leu?Glu?Gly?His?Glu?Arg?Gln?His?Leu

485 490 495 500

gag?gcc?cgg?ctg?cag?agc?ctg?cgt?aac?atc?cac?aca?ctg?ctg?gac?gcc 1950

Glu?Ala?Arg?Leu?Gln?Ser?Leu?Arg?Asn?Ile?His?Thr?Leu?Leu?Asp?Ala

505 510 515

gcc?atg?ctg?cag?atc?aac?cag?tac?ctc?acc?gtg?ctg?gcc?tcc?ttg?ggg 1998

Ala?Met?Leu?Gln?Ile?Asn?Gln?Tyr?Leu?Thr?Val?Leu?Ala?Ser?Leu?Gly

520 525 530

ccc?ccc?cgg?cct?gcc?act?tca?gtc?aac?tcc?act?gag?ggg?act?gcc?act 2046

Pro?Pro?Arg?Pro?Ala?Thr?Ser?Val?Asn?Ser?Thr?Glu?Gly?Thr?Ala?Thr

535 540 545

aca?gtt?gtt?gct?gct?gcc?tcc?tcc?acc?agc?atc?cct?agc?tca?gag?gcc 2094

Thr?Val?Val?Ala?Ala?Ala?Ser?Ser?Thr?Ser?Ile?Pro?Ser?Ser?Glu?Ala

550 555 560

acg?acc?cca?acc?cca?gga?gcc?tcc?cca?cca?gcc?cct?gaa?atg?gaa?agg 2142

Thr?Thr?Pro?Thr?Pro?Gly?Ala?Ser?Pro?Pro?Ala?Pro?Glu?Met?Glu?Arg

565 570 575 580

cct?cca?gct?cct?gag?tca?gtg?ggc?aca?gag?gag?atg?cct?gag?gat?gga 2190

Pro?Pro?Ala?Pro?Glu?Ser?Val?Gly?Thr?Glu?Glu?Met?Pro?Glu?Asp?Gly

585 590 595

gag?ccc?gat?gca?gca?gag?ctc?cgc?cgg?cgc?cgc?ctg?cag?aag?ctg?gag 2238

Glu?Pro?Asp?Ala?Ala?Glu?Leu?Arg?Arg?Arg?Arg?Leu?Gln?Lys?Leu?Glu

600 605 610

tct?cct?gtt?gcc?cac?tgacactgcc?ccagcccagc?cccagcctct?gctcttttga 2293

Ser?Pro?Val?Ala?His

615

gcagccctcg?ctggaacatg?tcctgccacc?aagtgccagc?tccctctctg?tctgcaccag 2353

ggagtagtac?ccccagctct?gagaaagagg?cggcatcccc?taggccaagt?ggaaagaggc 2413

tggggttccc?atttgactcc?agtcccaggc?agccatgggg?atctcgggtc?agttccagcc 2473

ttcctctcca?actcttcagc?cctgtgttct?gctggggcca?tgaaggcaga?aggtttagcc 2533

tctgagaagc?cctcttcttc?ccccacccct?ttccaggaga?aggggctgcc?cctccaagcc 2593

ctacttgtat?gtgcggagtc?acactgcagt?gccgaacagt?attagctccc?gttcccaagt 2653

gtggactcca?gaggggctgg?aggcaagcta?tgaacttgct?cgctggccca?cccctaagac 2713

tggtacccat?ttccttttct?taccctgatc?tccccagaag?cctcttgtgg?tggtggctgt 2773

gccccctatg?ccctgtggca?tttctgcgtc?ttactggcaa?ccacacaact?cagggaaagg 2833

aatgcctggg?agtgggggtg?caggcgggca?gcactgaggg?accctgcccc?gcccctcccc 2893

ccaggcccct?ttcccctgca?gcttctcaag?tgagactgac?ctgtctcacc?cagcagccac 2953

tgcccagccg?cactccaggc?aagggccagt?gcgcctgctc?ctgaccactg?caatcccagc 3013

gcccaaggaa?ggccacttct?caactggcag?aacttctgaa?gtttagaatt?ggaattactt 3073

ccttactagt?gtcttttggc?ttaaattttg?tcttttgaag?ttgaatgctt?aatcccggga 3133

aagaggaaca?ggagtgccag?actcctggtc?tttccagttt?agaaaaggct?ctgtgccaag 3193

gagggaccac?aggagctggg?acctgcctgc?ccctgtcctt?tccccttggt?tttgtgttac 3253

aagagttgtt?ggagacagtt?tcagatgatt?atttaatttg?taaatattgt?acaaatttta 3313

atagcttaaa?ttgtatatac?agccaaataa?aaacttgcat?taacaaaaaa?aaaaaaaaaa 3373

a 3374

<210>4

<211>617

<212>PRT

＜213〉people (Homo sapiens)

<400>4

Met?Phe?Arg?Thr?Ala?Val?Met?Met?Ala?Ala?Ser?Leu?Ala?Leu?Thr?Gly

1 5 10 15

Ala?Val?Val?Ala?His?Ala?Tyr?Tyr?Leu?Lys?His?Gln?Phe?Tyr?Pro?Thr

20 25 30

Val?Val?Tyr?Leu?Thr?Lys?Ser?Ser?Pro?Ser?Met?Ala?Val?Leu?Tyr?Ile

35 40 45

Gln?Ala?Phe?Val?Leu?Val?Phe?Leu?Leu?Gly?Lys?Val?Met?Gly?Lys?Val

50 55 60

Phe?Phe?Gly?Gln?Leu?Arg?Ala?Ala?Glu?Met?Glu?His?Leu?Leu?Glu?Arg

65 70 75 80

Ser?Trp?Tyr?Ala?Val?Thr?Glu?Thr?Cys?Leu?Ala?Phe?Thr?Val?Phe?Arg

85 90 95

Asp?Asp?Phe?Ser?Pro?Arg?Phe?Val?Ala?Leu?Phe?Thr?Leu?Leu?Leu?Phe

100 105 110

Leu?Lys?Cys?Phe?His?Trp?Leu?Ala?Glu?Asp?Arg?Val?Asp?Phe?Met?Glu

115 120 125

Arg?Ser?Pro?Asn?Ile?Ser?Trp?Leu?Phe?His?Cys?Arg?Ile?Val?Ser?Leu

130 135 140

Met?Phe?Leu?Leu?Gly?Ile?Leu?Asp?Phe?Leu?Phe?Val?Ser?His?Ala?Tyr

145 150 155 160

His?Ser?Ile?Leu?Thr?Arg?Gly?Ala?Ser?Val?Gln?Leu?Val?Phe?Gly?Phe

165 170 175

Glu?Tyr?Ala?Ile?Leu?Met?Thr?Met?Val?Leu?Thr?Ile?Phe?Ile?Lys?Tyr

180 185 190

Val?Leu?His?Ser?Val?Asp?Leu?Gln?Ser?Glu?Asn?Pro?Trp?Asp?Asn?Lys

195 200 205

Ala?Val?Tyr?Met?Leu?Tyr?Thr?Glu?Leu?Phe?Thr?Gly?Phe?Ile?Lys?Val

210 215 220

Leu?Leu?Tyr?Met?Ala?Phe?Met?Thr?Ile?Met?Ile?Lys?Val?His?Thr?Phe

225 230 235 240

Pro?Leu?Phe?Ala?Ile?Arg?Pro?Met?Tyr?Leu?Ala?Met?Arg?Gln?Phe?Lys

245 250 255

Lys?Ala?Val?Thr?Asp?Ala?Ile?Met?Ser?Arg?Arg?Ala?Ile?Arg?Asn?Met

260 265 270

Asn?Thr?Leu?Tyr?Pro?Asp?Ala?Thr?Pro?Glu?Glu?Leu?Gln?Ala?Met?Asp

275 280 285

Asn?Val?Cys?Ile?Ile?Cys?Arg?Glu?Glu?Met?Val?Thr?Gly?Ala?Lys?Arg

290 295 300

Leu?Pro?Cys?Asn?His?Ile?Phe?His?Thr?Ser?Cys?Leu?Arg?Ser?Trp?Phe

305 310 315 320

Gln?Arg?Gln?Gln?Thr?Cys?Pro?Thr?Cys?Arg?Met?Asp?Val?Leu?Arg?Ala

325 330 335

Ser?Leu?Pro?Ala?Gln?Ser?Pro?Pro?Pro?Pro?Glu?Pro?Ala?Asp?Gln?Gly

340 345 350

Pro?Pro?Pro?Ala?Pro?His?Pro?Pro?Pro?Leu?Leu?Pro?Gln?Pro?Pro?Asn

355 360 365

Phe?Pro?Gln?Gly?Leu?Leu?Pro?Pro?Phe?Pro?Pro?Gly?Met?Phe?Pro?Leu

370 375 380

Trp?Pro?Pro?Met?Gly?Pro?Phe?Pro?Pro?Val?Pro?Pro?Pro?Pro?Ser?Ser

385 390 395 400

Gly?Glu?Ala?Val?Ala?Pro?Pro?Ser?Thr?Ser?Ala?Ala?Ala?Leu?Ser?Arg

405 410 415

Pro?Ser?Gly?Ala?Ala?Thr?Thr?Thr?Ala?Ala?Gly?Thr?Ser?Ala?Thr?Ala

420 425 430

Ala?Ser?Ala?Thr?Ala?Ser?Gly?Pro?Gly?Ser?Gly?Ser?Ala?Pro?Glu?Ala

435 440 445

Gly?Pro?Ala?Pro?Gly?Phe?Pro?Phe?Pro?Pro?Pro?Trp?Met?Gly?Met?Pro

450 455 460

Leu?Pro?Pro?Pro?Phe?Ala?Phe?Pro?Pro?Met?Pro?Val?Pro?Pro?Ala?Gly

465 470 475 480

Phe?Ala?Gly?Leu?Thr?Pro?Glu?Glu?Leu?Arg?Ala?Leu?Glu?Gly?His?Glu

485 490 495

Arg?Gln?His?Leu?Glu?Ala?Arg?Leu?Gln?Ser?Leu?Arg?Asn?Ile?His?Thr

500 505 510

Leu?Leu?Asp?Ala?Ala?Met?Leu?Gln?Ile?Asn?Gln?Tyr?Leu?Thr?Val?Leu

515 520 525

Ala?Ser?Leu?Gly?Pro?Pro?Arg?Pro?Ala?Thr?Ser?Val?Asn?Ser?Thr?Glu

530 535 540

Gly?Thr?Ala?Thr?Thr?Val?Val?Ala?Ala?Ala?Ser?Ser?Thr?Ser?Ile?Pro

545 550 555 560

Ser?Ser?Glu?Ala?Thr?Thr?Pro?Thr?Pro?Gly?Ala?Ser?Pro?Pro?Ala?Pro

565 570 575

Glu?Met?Glu?Arg?Pro?Pro?Ala?Pro?Glu?Ser?Val?Gly?Thr?Glu?Glu?Met

580 585 590

Pro?Glu?Asp?Gly?Glu?Pro?Asp?Ala?Ala?Glu?Leu?Arg?Arg?Arg?Arg?Leu

595 600 605

Gln?Lys?Leu?Glu?Ser?Pro?Val?Ala?His

610 615

<210>5

<211>21

<212>PRT

＜213〉human sequence

<400>5

Asp?Asn?Val?Cys?Ile?Ile?Cys?Arg?Glu?Glu?Met?Val?Thr?Gly?Ala?Lys

1 5 10 15

Arg?Leu?Asp?Cys?Asn

20

Claims

1. one kind is detected test-compound synovial hyperplasia factor ubiquitin is turned into the active method of adjusting of usefulness, and it comprises step:

2. the process of claim 1 wherein coexists by following ingredients provides the condition that can make the substrate ubiquitinization:

I) ubiquitin activating enzyme;

Ii) ubiquitin transferring enzyme;

Iii) ubiquitin; With

Iv) Triphosaden.

3. the process of claim 1 wherein by in the cell of expressing substrate and the synovial hyperplasia factor or its homologue, providing the condition that can make the substrate ubiquitinization with the substrate ubiquitinization.

4. the process of claim 1 wherein with the ubiquitin level of following any one level as the index determining substrate:

A) level of the substrate of ubiquitinization;

B) level of the substrate of ubiquitinization not; With

C) biologically active level of prolyl 4-hydroxylase α subunit.

5. the process of claim 1 wherein that prolyl 4-hydroxylase α subunit or its homologue are following (a) to any one of (e) polypeptide:

(a) the nucleotide sequence coded polypeptide of SEQ ID NO:1;

(b) comprise the polypeptide of SEQ ID NO:2 aminoacid sequence;

6. the process of claim 1 wherein that the synovial hyperplasia factor or its homologue are following (A) to any one of (E) polypeptide:

(A) the nucleotide sequence coded polypeptide of SEQ ID NO:3;

(B) comprise the polypeptide of SEQ ID NO:4 aminoacid sequence;

7. screening turns the method for regulating active test-compound with having into to the ubiquitin of the synovial hyperplasia factor, comprises step:

A) by any one method of claim 1 to 6 detect test-compound to synovial hyperplasia factor ubiquitin turn into usefulness the adjusting activity and

8. the substrate ubiquitin that is used to regulate the synovial hyperplasia factor turns the material of usefulness into, comprises that the compound that can obtain by the method for claim 7 is as activeconstituents.

9. detect the active test kit of adjusting that synovial hyperplasia factor ubiquitin is turned into usefulness, it comprises following ingredients:

(1) the synovial hyperplasia factor or its homologue; With

(2) prolyl 4-hydroxylase α subunit or its homologue.

10. the test kit of claim 9 further comprises following ingredients:

(3) ubiquitin activating enzyme;

(4) ubiquitin transferring enzyme;

(5) ubiquitin; With

(6) Triphosaden.

11. detect the active test kit of adjusting that synovial hyperplasia factor ubiquitin is turned into usefulness, comprise and express the synovial hyperplasia factor or its homologue, with the cell of prolyl 4-hydroxylase α subunit or its homologue and the instrument of detection prolyl 4-hydroxylase α subunit or its homologue ubiquitin level.

12. one kind is detected test-compound synovial hyperplasia factor ubiquitin is turned into the active method of adjusting of usefulness, it comprises step:

13. the method for claim 12, wherein or the synovial hyperplasia factor or substrate in conjunction with solid phase or comprise can be in conjunction with the marker of solid phase.

14. the method for claim 12, wherein prolyl 4-hydroxylase α subunit or its homologue are following (a) to any one of (e) polypeptide:

(a) the nucleotide sequence coded polypeptide of SEQ ID NO:1;

(b) comprise the polypeptide of SEQ ID NO:2 aminoacid sequence;

15. the method for claim 12, wherein the synovial hyperplasia factor or its homologue are following (A) to any one of (E) polypeptide:

(A) the nucleotide sequence coded polypeptide of SEQ ID NO:3;

(B) comprise the polypeptide of SEQ ID NO:4 aminoacid sequence;

(C) polypeptide, it is included in the aminoacid sequence of SEQ ID NO:4 and replaces, and deletion is inserted, and/or adds the aminoacid sequence of one or more amino acid gained, and it can be in conjunction with the polypeptide that comprises SEQ IDNO:2 aminoacid sequence;

16. screening turns the method for regulating active test-compound with having into to the ubiquitin of the synovial hyperplasia factor, comprises step:

A) by any one method of claim 12 to 15 detect test-compound to synovial hyperplasia factor ubiquitin turn into usefulness the adjusting activity and

B) select the test-compound that substrate and synovial hyperplasia factor bonded level are different from contrast.

17. the substrate ubiquitin that is used to regulate the synovial hyperplasia factor turns the material of usefulness into, comprises that the compound that can obtain by the method for claim 16 is as activeconstituents.

18. the method for screening treatment or preventing Fibrotic compound, it comprises step:

C) select a kind of test-compound as treatment or prevent Fibrotic compound, the ubiquitin level that it records when making substrate ubiquitin level be lower than this test-compound not,

Wherein substrate is prolyl 4-hydroxylase α subunit or its homologue.

19. the method for claim 18 wherein coexists by following ingredients the condition that can make the substrate ubiquitinization is provided:

I) ubiquitin activating enzyme;

Ii) ubiquitin transferring enzyme;

Iii) ubiquitin; With

Iv) Triphosaden.

20. the method for claim 18 is wherein by providing the condition that can make the substrate ubiquitinization with the substrate ubiquitinization in the cell of expressing substrate and the synovial hyperplasia factor or its homologue.

21. the method for claim 18 is wherein used the ubiquitin level of following any one level as the index determining substrate:

A) level of the substrate of ubiquitinization;

B) level of the substrate of ubiquitinization not; With

C) biologically active level of prolyl 4-hydroxylase α subunit.

22. the method for claim 18, wherein prolyl 4-hydroxylase α subunit or its homologue are following (a) to any one of (e) polypeptide:

(a) the nucleotide sequence coded polypeptide of SEQ ID NO:1;

(b) comprise the polypeptide of SEQ ID NO:2 aminoacid sequence;

23. the method for claim 18, wherein the synovial hyperplasia factor or its homologue are following (A) to any one of (E) polypeptide:

(A) the nucleotide sequence coded polypeptide of SEQ ID NO:3;

(B) comprise the polypeptide of SEQ ID NO:4 aminoacid sequence;

24. treat and/or prevent Fibrotic medicinal compositions, it comprises that the compound that can pass through any one method screening of claim 18 to 23 is as activeconstituents.

25. the test kit of Fibrotic compound is treated or prevented in screening, it comprises following ingredients:

(1) the synovial hyperplasia factor or its homologue; With

(2) prolyl 4-hydroxylase α subunit or its homologue.

26. the test kit of claim 25 further comprises following ingredients:

(3) ubiquitin activating enzyme;

(4) ubiquitin transferring enzyme;

(5) ubiquitin; With

(6) Triphosaden.

27. the test kit of Fibrotic test-compound is treated or is prevented in screening, comprise and express the synovial hyperplasia factor or its homologue, with the cell of prolyl 4-hydroxylase α subunit or its homologue and the instrument of detection prolyl 4-hydroxylase α subunit or its homologue ubiquitin level.

28. screening treatment or the method for preventing Fibrotic compound comprise step:

Wherein substrate is prolyl 4-hydroxylase α subunit or its homologue.

29. the method for claim 28, wherein the synovial hyperplasia factor or substrate are in conjunction with solid phase or comprise can be in conjunction with the marker of solid phase.

30. the method for claim 28, wherein prolyl 4-hydroxylase α subunit or its homologue are following (a) to any one of (e) polypeptide:

(a) the nucleotide sequence coded polypeptide of SEQ ID NO:1;

(b) comprise the polypeptide of SEQ ID NO:2 aminoacid sequence;

31. the method for claim 28, wherein the synovial hyperplasia factor or its homologue are following (A) to any one of (E) polypeptide:

(A) the nucleotide sequence coded polypeptide of SEQ ID NO:3;

(B) comprise the polypeptide of SEQ ID NO:4 aminoacid sequence;

(E) comprise with SEQ ID NO:4 aminoacid sequence and have 70% or the polypeptide of the aminoacid sequence of above homology, and can be in conjunction with the polypeptide that comprises SEQ ID NO:2 aminoacid sequence.

32. treat and/or prevent Fibrotic medicinal compositions, comprise that the compound that can pass through any one method screening of claim 28 to 31 is as activeconstituents.

33. the method for the compound of screening treatment or prevention rheumatic arthritis, it comprises step:

Wherein substrate is prolyl 4-hydroxylase α subunit or its homologue.

34. the method for claim 33 wherein coexists by following ingredients the condition that can make the substrate ubiquitinization is provided:

I) ubiquitin activating enzyme;

Ii) ubiquitin transferring enzyme;

Iii) ubiquitin; With

Iv) Triphosaden.

35. the method for claim 33 is wherein by providing the condition that can make the substrate ubiquitinization with the substrate ubiquitinization in the cell of expressing substrate and the synovial hyperplasia factor or its homologue.

36. the method for claim 33 is wherein used the ubiquitin level of following any one level as the index determining substrate:

A) level of the substrate of ubiquitinization;

B) level of the substrate of ubiquitinization not; With

C) biologically active level of prolyl 4-hydroxylase α subunit.

37. the method for claim 33, wherein prolyl 4-hydroxylase α subunit or its homologue are following (a) to any one of (e) polypeptide:

(a) the nucleotide sequence coded polypeptide of SEQ ID NO:1;

(b) comprise the polypeptide of SEQ ID NO:2 aminoacid sequence;

38. the method for claim 33, wherein the synovial hyperplasia factor or its homologue are following (A) to any one of (E) polypeptide:

(A) the nucleotide sequence coded polypeptide of SEQ ID NO:3;

(B) comprise the polypeptide of SEQ ID NO:4 aminoacid sequence;

39. treat and/or prevent the medicinal compositions of rheumatic arthritis, it comprises that the compound that can pass through any one method screening of claim 33 to 38 is as activeconstituents.

40. the test kit of the compound of screening treatment or prevention rheumatic arthritis, it comprises following ingredients:

(1) the synovial hyperplasia factor or its homologue; With

(2) prolyl 4-hydroxylase α subunit or its homologue.

41. the test kit of claim 39 further comprises following ingredients:

(3) ubiquitin activating enzyme;

(4) ubiquitin transferring enzyme;

(5) ubiquitin; With

(6) Triphosaden.

42. the test kit of the test-compound of screening treatment or prevention rheumatic arthritis, comprise and express the synovial hyperplasia factor or its homologue, with the cell of prolyl 4-hydroxylase α subunit or its homologue and the instrument of detection prolyl 4-hydroxylase α subunit or its homologue ubiquitin level.

43. screening treatment or the method for preventing Fibrotic compound comprise step:

Wherein substrate is prolyl 4-hydroxylase α subunit or its homologue.

44. the method for claim 43, wherein or the synovial hyperplasia factor or substrate in conjunction with solid phase or comprise can be in conjunction with the marker of solid phase.

45. the method for claim 43, wherein prolyl 4-hydroxylase α subunit or its homologue are following (a) to any one of (e) polypeptide:

(a) the nucleotide sequence coded polypeptide of SEQ ID NO:1;

(b) comprise the polypeptide of SEQ ID NO:2 aminoacid sequence;

46. the method for claim 43, wherein the synovial hyperplasia factor or its homologue are following (A) to any one of (E) polypeptide:

(A) the nucleotide sequence coded polypeptide of SEQ ID NO:3;

(B) comprise the polypeptide of SEQ ID NO:4 aminoacid sequence;

47. treat and/or prevent the medicinal compositions of rheumatic arthritis, comprise that the compound that can pass through any one method screening of claim 43 to 46 is as activeconstituents.

48. comprise the polypeptide of SEQ ID NO:5 aminoacid sequence.

49. polypeptide, it is included in the aminoacid sequence of SEQ ID NO:5 and replaces, deletion, insert, and/or add the aminoacid sequence of one or more amino acid gained, wherein when with polypeptide during as the test-compound in the method for claim 1, the ubiquitin level of substrate is lower than the ubiquitin level that records when lacking this polypeptide.

50. polypeptide, it is included in the aminoacid sequence of SEQ ID NO:5 and replaces, deletion, insert, and/or add the aminoacid sequence of one or more amino acid gained, wherein when with polypeptide during as the test-compound in the method for claim 12, substrate and the synovial hyperplasia factor or its homologue in conjunction with level be lower than record when lacking this polypeptide in conjunction with level.

51. the polynucleotide of any one polypeptide of coding claim 48 to 50.

52. be used for the treatment of and/prevent Fibrotic medicinal compositions, it comprises that any one polypeptide of claim 48 to 50 is as activeconstituents.

53. be used for the treatment of and/prevention rheumatic arthritis medicinal compositions, it comprises that any one polypeptide of claim 48 to 50 is as activeconstituents.