JPH11326328A - Method for screening substance that inhibits bonding with xiap - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for screening physiologically active substances in a signal transmission system of a TGF-β (transformation growth factor-β), by searching for molecules involved in a process that binds a TGF-β receptor and TAB1 together and using such molecules. SOLUTION: By bringing XIAP, TAB1 (TAK1 (TGF-β-activated kinase 1 binding protein 1), and a substance to be tested into contact and examining whether the binding between the XIAP and the TAB1 is formed or not, substances that inhibit the binding between the XIAP and the TAB1 are screened. In addition. a medicinal composition containing as effective components the substances that inhibit the binding between the XIAP and the TAB1, or the XIAP and TGF-β type I and/or type II receptors obtained by this method is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for screening an inhibitor for the binding between XIAP and TAB1. The present invention also relates to a method for screening for an inhibitor for the binding between TGF-β receptor and XIAP. Further, the present invention relates to the binding of XIAP and TAB1, or TGF-β receptor and XIAP
The present invention relates to a substance obtained by a screening method for a substance that inhibits binding to a substance and its use.

[0002]

2. Description of the Related Art TGF-β (transforming growth factor-β; Transf
orming Growth Factor-β) is a multifunctional factor that controls many aspects of cellular function. As one of them, TGF-β is responsible for tissue repair and regeneration associated with various injuries (Border,
WA & Noble, NA, The New England Journal of M
edicine (1994) 331, 1286-1292).

[0003] Abnormal production of TGF-β in chronic injuries can disrupt the balance between tissue repair and regeneration, resulting in pathological fibrosis. Liver fibrosis is known as a pathological condition in which the balance of TGF-β production is lost. TGF-β in liver
Enhances the production of extracellular matrix proteins that cause fibrosis, inhibits the synthesis of extracellular matrix proteases, and induces extracellular matrix protease inhibitors to induce various organs, such as the liver. Has been shown to act as a major causative factor in human fibrosis (Border, WA & Noble, NA, The New En
gland Journal of Medicine (1994) 331, 1286-1292).

[0004] Other effects of TGF-β include cell growth inhibitory activity (Moses, HL et al., Cell (1990) 63, 245).
-247), monocyte migration activity (Wahl, SM et al., Proc. Na
USA (1987) 84, 5788-5792), bioactive substance inducing activity (Wahl, SM et al., Proc. Natl. Acad.
Sci. USA (1987) 84, 5788-5792), β-amyloid protein deposition (Wyss-Coray, T. et al., Nature (1997) 3
89, 603-606).

[0005] TGF-β transmits its signal through a heteromeric complex of type I and type II TGF-β receptors, which are transmembrane proteins containing serine- and threonine-specific kinase domains on the cytoplasmic side. (Wrana, JL et al.,
Nature (1994) 370, 341; Kingsley, DM et al., Ge
nes Dev. (1994) 8, 133). However, the signaling mechanism downstream from the TGF-β receptor into cells has not been elucidated much at the molecular level. Mitogen-Activated Protein Kinase (Mitogen-Activat) is a series of systems involved in the signaling of the TGF-β superfamily.
The ed Protein Kinase (MAPK) system is known.

[0006] The MAPK system is a conserved eukaryotic signaling system that converts receptor signals into various actions.
The MAPK system is composed of three types of protein kinases, namely MAPKKK
(Mitogen-Activated Protein Kinase Kinase Kinas
e), MAPKK (Mitogen-ActivatedProtein Kinase Kinas
e), including MAPK (Mitogen-Activated Protein Kinase). MAPK is activated by phosphorylation by MAPKK. MAPKK
Is activated by phosphorylation by MAPKKK (Nishida, E.
et al., Trends Biochem.Sci. (1993) 18, 128, Blume
r, KJ et al., Trends Biochem.Sci. (1993) 19, 2
36, David RJ et al., Trends Biochem. Sci. (1993)
19, 470, Marchall, CJ et al., Cell (1995) 80, 1
79).

[0007] TAK1 (TGF-β-Activated Kinase 1) which is one of the MAPKKK family that functions in the signal transduction system of physiologically active substances belonging to the TGF-β superfamily
Was identified by Yamaguchi, K. et al. (Yamaguchi, K. e
tal., Science (1995) 270, 2008). In addition, TAB1 (TAK1 Binding Protein 1), a protein involved in the TGF-β signal transduction system that binds to TAK1 and activates TAK1, is Shib
uya, H. et al. (Shibuya, H. et al., Sci.
ence (1996) 272, 1179-1182). TAB1 binds to TAK1
It has been shown to activate TAK1 kinase activity and transmit TGF-β signals. However, the molecular action mechanism linking TAK1 activation by TAB1 to the TGF-β receptor was not known.

[0008]

Accordingly, the present invention relates to TG
In the signaling system of F-β, TGF-β receptor and TA
An object of the present invention is to search for a molecule involved in a pathway connecting B1 and to provide a method for screening for a physiologically active substance using such a molecule. Another object of the present invention is to provide a physiologically active substance obtained by the above method and its use.

[0009]

Means for Solving the Problems In the TGF-β signal transduction system, the present inventors have proposed TAN2 (TAB1 N-terminal binding protein) as a molecule linking TGF-β and TAB1.
2) X-linked Inhibitor of Apoptosis Protein;
XIAP) was identified, confirming that this XIAP not only associates with TAB1 in mammalian cells but also with the TGF-β receptor. The present invention has been made based on the above findings.

That is, the present invention provides a method for screening a substance that inhibits the binding between XIAP and TAB1 by contacting XIAP, TAB1 and a test sample, and then examining the presence or absence of the formation of a bond between XIAP and TAB1. provide. TAB1 is Met at amino acid position 1 of the amino acid sequence shown in SEQ ID NO: 2.
An amino acid sequence consisting of Pro at amino acid position 504, or an amino acid having TAB1 biological activity and having one or more amino acids substituted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 2 It may have an array. XIAP has an amino acid sequence consisting of Met at amino acid position 1 to Ser at amino acid position 497 of the amino acid sequence shown in SEQ ID NO: 4, or has the biological activity of XIAP, and has an amino acid sequence of 1 in the amino acid sequence shown in SEQ ID NO: 4.
Alternatively, it may have an amino acid sequence in which a plurality of amino acids have been substituted, deleted and / or added. TAB1 and / or
XIAP may be fused to another peptide or polypeptide. TAB1 or XIAP may be bound to a support.
Examples of the support include beads and plates.

In the above-described screening method for a substance that inhibits the binding between XIAP and TAB1, TAB1 or XIAP is labeled, and the label is detected or measured to detect XIAP and TAB1.
The presence or absence of formation of a bond with B1 may be examined. Labels include radioisotopes, enzymes, fluorescent substances and the like.

Further, in the above-mentioned method for screening a substance that inhibits the binding between XIAP and TAB1, the XIAP bound to TAB1 is treated with a primary antibody against XIAP or a primary antibody against another peptide or polypeptide fused with XIAP. The presence or absence of formation of a bond between XIAP and TAB1 may be examined by detection or measurement. In this case, XIAP bound to TAB1 is detected or measured by a primary antibody against XIAP or a primary antibody against another peptide or polypeptide fused to XIAP and a secondary antibody against the primary antibody, whereby the binding between XIAP and TAB1 is detected. The presence or absence of formation may be checked. Alternatively, TAB1 bound to XIAP may be detected or measured by a primary antibody against TAB1 or a primary antibody against another peptide or polypeptide fused to TAB1, to determine whether or not a bond between XIAP and TAB1 is formed. . In this case, by detecting or measuring TAB1 bound to XIAP with a primary antibody against TAB1 or a primary antibody against another peptide or polypeptide fused to TAB1 and a secondary antibody against the primary antibody, the binding between XIAP and TAB1 is detected. The presence or absence of formation may be checked. The primary or secondary antibody may be labeled with a radioisotope, enzyme or fluorescent substance.

[0013] The present invention also relates to a method of introducing and / or contacting a test sample with cells expressing TAB1 and XIAP.
Or detecting a biological activity transmitted via XIAP and / or
Alternatively, the present invention provides a method for screening a substance that inhibits the binding between XIAP and TAB1 by measuring. The biological activity transmitted via TAB1 or XIAP may be that of TGF-β. Examples of the biological activity of TGF-β include enhancement of extracellular matrix protein production, inhibition of cell growth, monocyte migration, induction of bioactive substances, immunosuppression, β-amyloid protein deposition, and the like. Also, TAB1
Or, the biological activity transmitted via XIAP is TAB1 or
It may be a change in the expression level of a reporter gene activated in response to the biological activity of XIAP. Reporter genes include PAI-1, fibronectin, type I collagen, type IV collagen and the like.

Further, the present invention provides a method for bringing XIAP, TGF-β type I receptor and a test sample into contact with each other, and then contacting XIAP with TGF-β
It is intended to provide a method for screening for a substance that inhibits the binding between XIAP and TGF-β type I receptor by examining the presence or absence of formation of a binding to a type I receptor. XIAP
Has the biological activity of Met at amino acid position 1 to Ser at amino acid position 497 of the amino acid sequence shown in SEQ ID NO: 4, or XIAP, and has SEQ ID NO: 4
May have an amino acid sequence in which one or more amino acids have been substituted, deleted and / or added. The TGF-β type I receptor has an amino acid sequence consisting of Met at amino acid position 1 to Met at amino acid position 503 of the amino acid sequence shown in SEQ ID NO: 6, or T
It may have the biological activity of the GF-β type I receptor and have the amino acid sequence shown in SEQ ID NO: 6 in which one or more amino acids have been substituted, deleted and / or added. The XIAP or TGF-β type I receptor may be fused to another peptide or polypeptide. The XIAP or TGF-β type I receptor may be bound to a support. Examples of the support include beads and plates.

The above TGF-β type I receptor and X
In a method of screening for a substance that inhibits binding to IAP, XIAP or TGF-β type I receptor is labeled, and by detecting or measuring the label, the presence or absence of formation of a bond between XIAP and TGF-β type I receptor May be checked.
Labels include radioisotopes, enzymes, fluorescent substances and the like.

[0016] In the above-mentioned screening method for a substance that inhibits the binding between TGF-β type I receptor and XIAP, the method further comprises: Or TGF
The presence or absence of formation of a bond between XIAP and the TGF-β type I receptor may be determined by detecting or measuring with a primary antibody against another peptide or polypeptide fused to the -β type I receptor. In this case, the TGF-β type I receptor bound to XIAP, a primary antibody against the TGF-β type I receptor or a secondary antibody against another peptide or polypeptide fused to the TGF-β type I receptor and a secondary antibody against the primary antibody The presence or absence of the formation of a bond between XIAP and the TGF-β type I receptor may be examined by detecting or measuring the above. Alternatively, XIAP bound to TGF-β type I receptor is detected or measured by a primary antibody against XIAP or a primary antibody against another peptide or polypeptide fused to XIAP, whereby XIAP and TGF-β type I receptor The presence or absence of formation of a bond may be examined. In this case, TGF-β type
By detecting or measuring XIAP bound to the I receptor by a primary antibody against XIAP or a primary antibody against another peptide or polypeptide fused to XIAP and a secondary antibody against the primary antibody, XIAP and TGF-β type I receptor The presence or absence of the formation of a bond may be examined. The primary or secondary antibody may be labeled with a radioisotope, enzyme or fluorescent substance.

The present invention also relates to XIAP and TGF-β type I
A test sample is introduced and / or contacted with cells that express the receptor, and then the biological activity transmitted via the XIAP or TGF-β type I receptor is detected and / or measured, whereby TGF-β type I Provided is a method for screening a substance that inhibits the binding between a receptor and XIAP. The biological activity transmitted via the XIAP or TGF-β type I receptor may be the biological activity of TGF-β. Examples of the biological activity of TGF-β include enhancement of extracellular matrix protein production, inhibition of cell growth, monocyte migration, induction of bioactive substances, immunosuppression, β-amyloid protein deposition, and the like. The biological activity transmitted through the XIAP or TGF-β type I receptor is XIAP or TGF-β
It may be a change in the expression level of a reporter gene activated in response to the biological activity of the type I receptor. Reporter genes include PAI-1, fibronectin,
Type I collagen, type IV collagen and the like can be mentioned.

The present invention further relates to the steps of contacting XIAP, TGF-β type II receptor and a test sample, and then contacting XIAP with TGF-β
It is intended to provide a method for screening for a substance that inhibits the binding between XIAP and TGF-β type II receptor by examining the presence or absence of formation of a binding to a type II receptor. XIAP
Has the biological activity of Met at amino acid position 1 to Ser at amino acid position 497 of the amino acid sequence shown in SEQ ID NO: 4, or XIAP, and has SEQ ID NO: 4
May have an amino acid sequence in which one or more amino acids have been substituted, deleted and / or added. The TGF-β type II receptor has an amino acid sequence consisting of Met at amino acid position 1 to Lys at amino acid position 567 in the amino acid sequence shown in SEQ ID NO: 8, or T
It may have the biological activity of the GF-β type II receptor and have an amino acid sequence in which one or more amino acids have been substituted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 8. The XIAP or TGF-β type II receptor may be fused to another peptide or polypeptide. The XIAP or TGF-β type II receptor may be bound to a support. Examples of the support include beads and plates.

The above TGF-β type II receptor and
In a method for screening a substance that inhibits binding to XIAP, XIAP or TGF-β type II receptor is labeled, and by detecting or measuring the label, the presence or absence of formation of a bond between XIAP and TGF-β type II receptor is determined. May be checked.
Labels include radioisotopes, enzymes, fluorescent substances and the like.

Further, in the above-mentioned method for screening a substance that inhibits the binding between TGF-β type II receptor and XIAP, the method according to claim 1, wherein the TGF-β type II receptor bound to XIAP is replaced with a primary antibody against TGF-β type II receptor Or TG
The presence or absence of formation of a bond between XIAP and the TGF-β type II receptor may be determined by detecting or measuring with a primary antibody against another peptide or polypeptide fused to the F-β type II receptor. In this case, the TGF-β type II receptor bound to XIAP, a primary antibody to the TGF-β type II receptor or a secondary antibody to another peptide or polypeptide fused to the TGF-β type II receptor and a secondary antibody to the primary antibody The presence or absence of the formation of a bond between XIAP and the TGF-β type II receptor may be examined by detecting or measuring the above. Alternatively, TGF-
By detecting or measuring XIAP bound to the β type II receptor with a primary antibody against XIAP or a primary antibody against another peptide or polypeptide fused to XIAP, the formation of binding between XIAP and the TGF-β type II receptor is determined. The presence or absence may be checked. In this case, the XIAP bound to the TGF-β type II receptor is converted to a primary antibody against XIAP or
The presence or absence of formation of a bond between XIAP and the TGF-β type II receptor may be determined by detecting or measuring with a primary antibody against another peptide or polypeptide fused to XIAP and a secondary antibody against the primary antibody. The primary or secondary antibody may be labeled with a radioisotope, enzyme or fluorescent substance.

The present invention also relates to XIAP and TGF-β type II
A test sample is introduced and / or contacted with cells expressing the receptor, and then the biological activity transmitted via the XIAP or TGF-β type II receptor is detected and / or measured, thereby obtaining TGF-β type II Provided is a method for screening a substance that inhibits the binding between a receptor and XIAP. The biological activity transmitted via the XIAP or TGF-β type II receptor may be the biological activity of TGF-β. Examples of the biological activity of TGF-β include enhancement of extracellular matrix protein production, inhibition of cell growth, monocyte migration, induction of bioactive substances, immunosuppression, β-amyloid protein deposition, and the like. The biological activity transmitted via the XIAP or TGF-β type II receptor is XIAP or TGF-β
It may be a change in the expression level of a reporter gene activated in response to the biological activity of the type II receptor. Reporter genes include PAI-1, fibronectin,
Type I collagen, type IV collagen and the like can be mentioned.

The present invention also provides a substance obtained by the above-mentioned screening method. This substance may be a novel alternative to known substances that inhibit or activate TGF-β signaling, and may be either an agonist or antagonist of XIAP. The present invention also provides a pharmaceutical composition containing the substance obtained by the above-mentioned screening method as an active ingredient. This pharmaceutical composition enhances extracellular matrix protein production, inhibits cell proliferation, migrates monocytes, induces bioactive substances,
It can be used to suppress or activate various physiological actions involving signal transduction, such as immunosuppressive action and β-amyloid protein depositing action. Specifically, at least one selected from the group consisting of liver fibrosis, pulmonary fibrosis, chronic glomerulonephritis, diabetic nephropathy, vascular restenosis, keloid skin, scleroderma, autoimmune disease and Alzheimer's disease Can be used to prevent and / or treat two diseases.

The present invention also relates to a pharmaceutical composition containing XIAP as an active ingredient, which enhances extracellular matrix protein production, inhibits cell proliferation, monocyte migration, induces bioactive substances, suppresses immunology and deposits β-amyloid protein. The above-mentioned pharmaceutical composition for use in suppressing or activating at least one action selected from the group consisting of Further, the present invention relates to a pharmaceutical composition comprising XIAP as an active ingredient, comprising hepatic fibrosis, pulmonary fibrosis, glomerulonephritis, diabetic nephropathy, renal sclerosis, vascular restenosis, keloid skin, The aforementioned pharmaceutical composition for use in preventing and / or treating at least one disease selected from the group consisting of dermatosis, autoimmune disease and Alzheimer's disease.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION The TAB1 used in the present invention has an amino acid sequence consisting of Met at amino acid position 1 to Pro at amino acid position 504 in the amino acid sequence shown in SEQ ID NO: 2 and is an organism of TAB1. TA with biological activity
Anything can be used as long as it is B1. It is disclosed herein that the biological activity of TAB1 is an activity that binds to XIAP. More specifically, it is disclosed herein that the biological activity of TAB1 is an activity of binding to a polypeptide consisting of amino acid Met at position 1 to amino acid Gly at position 326 of XIAP. However, in the present invention, TAB1 only needs to have the activity of binding to XIAP. Therefore, the biological activity of TAB1 described herein may be a binding activity to XIAP.

The TAB1 used in the present invention has the biological activity of TAB1, and has one or more amino acid substitutions, deletions and / or deletions in the amino acid sequence shown in SEQ ID NO: 2.
Alternatively, it may be TAB1 having an added amino acid sequence. More specifically, TAB1 used in the present invention is TAB1
In the amino acid sequence shown in SEQ ID NO: 2, one or more, preferably 1 or 2,
It may have an amino acid sequence in which 0 or less, more preferably 1 or 10 or less amino acids are substituted. Alternatively, in the amino acid sequence shown in SEQ ID NO: 2, one or more, preferably 1 or 86 or less, more preferably 1 or 10 or less amino acid residues may be deleted. Alternatively, in the amino acid sequence shown in SEQ ID NO: 1, it may have an amino acid to which 1 or 2 or more, preferably 1 or 30 or less, more preferably 1 or 20 or less amino acids have been added. TA used in the present invention
In B1, the above amino acids may be substituted, deleted and / or added simultaneously.

TAB1 has been shown to exhibit biological activity if it has an amino acid sequence from amino acid Met at position 1 to amino acid Val at position 418 in SEQ ID NO: 2. Accordingly, TAB1 used in the present invention has an amino acid sequence from amino acid Met at position 1 to amino acid Val at position 418 in SEQ ID NO: 2, and an amino acid sequence from amino acid Asn at position 419 to amino acid Pro at position 504. May have TAB1 having an amino acid sequence in which one or more amino acid residues have been substituted, deleted and / or added. TAB1 is an amino acid sequence obtained by substituting, deleting and / or adding one or more amino acids in the amino acid sequence from amino acid Met at position 1 to amino acid Val at position 418 in SEQ ID NO: 2 as long as it has biological activity. TAB1 having the following formula:

In the amino acid sequence shown in SEQ ID NO: 2, 1
Alternatively, as TAB1 having an amino acid sequence in which a plurality of amino acids have been substituted, deleted and / or added, the amino acid Ser at position 52 can be substituted with TAB1 having Arg or amino acid Met at position 4
TAB1 having an amino acid sequence up to amino acid Val at position 18
Is mentioned.

XIAP (X-linked Inhib) used in the present invention
As an itor of Apoptosis Protein, XIAP having an amino acid sequence consisting of Met at amino acid position 1 to Ser at amino acid position 497 in the amino acid sequence shown in SEQ ID NO: 4 and having the biological activity of XIAP , Any (Duckett, CS, et al., EMB
O J. (1996) 15, 2685-2694). It is disclosed herein that the biological activity of XIAP binds to TAB1 and transmits the biological activity through TAB1. It also directly inhibits activation of caspases 3 and 7 by various stimuli (ultraviolet irradiation, anti-Fas antibody stimulation, TNFa stimulation, etc.) (Deverau
x, QL, et al., Nature (1997) 388, 300-304) and have been shown to inhibit apoptosis induced by those stimuli (Duckett,
CS, et al., Mol. Cell. Biol. (1998) 18, 608-61.
Five). However, in the present invention, XIAP only needs to have an activity of binding to TAB1, and may be XIAP which has lost the anti-apoptotic activity of XIAP. Therefore, the biological activity of XIAP described herein may be a binding activity to TAB1.

XIAP has been shown to exhibit biological activity if it has an amino acid sequence from amino acid Met at position 1 to amino acid Gly at position 326 in SEQ ID NO: 4. Therefore, XIAP used in the present invention has an amino acid sequence from amino acid Met at position 1 to amino acid Gly at position 326 in SEQ ID NO: 4 and an amino acid sequence from amino acid Cys at position 327 to Ser at position 497 in SEQ ID NO: 4. It may be XIAP having an amino acid sequence in which one or more amino acids have been substituted, deleted and / or added. XIAP
Is an amino acid sequence in which one or more amino acids have been substituted, deleted and / or added in the amino acid sequence from amino acid Met at position 1 to amino acid Gly at position 326 in SEQ ID NO: 4 as long as it has the biological activity. May be provided.

The XIAP used in the present invention has the biological activity of XIAP and has one or more amino acid substitutions, deletions and / or deletions in the amino acid sequence shown in SEQ ID NO: 4.
Alternatively, it may be XIAP having an added amino acid sequence. More specifically, XIAP used in the present invention is XIAP
In the amino acid sequence shown in SEQ ID NO: 4, 1 or 2 or more, preferably 1 or 2
It may have an amino acid sequence in which 0 or less, more preferably 1 or 10 or less amino acids are substituted.

Alternatively, XIAP has an amino acid sequence shown in SEQ ID NO: 4 in which 1 or 2 or more, preferably 1 or 171 or less, more preferably 1 or 10 or less amino acid residues have been deleted. You may. Alternatively, in the amino acid sequence shown in SEQ ID NO: 4, 1 or 2
More than one, preferably 1 or 30 or less, more preferably 1 or 20 or less amino acid residues may have an added amino acid.

The TGF-β type I receptor used in the present invention has an amino acid sequence consisting of Met at amino acid position 1 to Met at amino acid position 503 in the amino acid sequence shown in SEQ ID NO: 6; Any TGF-β type I receptor having the biological activity of the type I receptor may be used. TGF-β type I
The biological activity of the receptor is to bind to TGF-β and transmit its biological activity via TAB1. More specifically, after binding of TGF-β and TGF-β type II receptor, it forms a heterotetramer with TGF-β type II receptor via TGF-β and transmits TGF-β signal. It is clear that there is. However, in the present invention, the TGF-β type I receptor only needs to have the activity of binding to XIAP, and the TGF-β type I receptor has the activity of binding to TGF-β, and the TGF-β type II receptor has It may be a TGF-β type I receptor that has lost the activity of forming a heterotetramer or the activity of transmitting a TGF-β signal. Therefore, the biological activity of the TGF-β type I receptor described herein may be a binding activity to XIAP.

The TGF-β type I used in the present invention
The TGF-β receptor has the biological activity of the TGF-β type I receptor, and has an amino acid sequence shown in SEQ ID NO: 6 having one or more amino acid substitutions, deletions and / or additions. It may be a beta type I receptor. More specifically, TGF- used in the present invention
As long as the β type I receptor has the biological activity of the TGF-β type I receptor, in the amino acid sequence shown in SEQ ID NO: 6, 1 or more, preferably 1 or 20 or less, more preferably 1 or 10 It may have an amino acid sequence in which not more than three amino acids have been substituted.

Alternatively, the TGF-β type I receptor has one or more, preferably one or less, more preferably one or less in the amino acid sequence shown in SEQ ID NO: 6.
Alternatively, it may have an amino acid sequence in which 10 or less amino acids have been deleted. Alternatively, the amino acid sequence shown in SEQ ID NO: 6 may have an amino acid sequence in which 1 or more, preferably 1 or 30 or less, more preferably 1 or 20 or less amino acids are added.

The TGF-β type II receptor used in the present invention has an amino acid sequence consisting of Met at amino acid position 1 to Lys at amino acid position 567 in the amino acid sequence shown in SEQ ID NO: 8; Any TGF-β type II receptor having the biological activity of the type II receptor may be used. TGF-β type II
The biological activity of the receptor binds to TGF-β,
Activating the type I receptor and transmitting its biological activity via TAB1. For more details, see TGF-
It binds to β, forms a heterotetramer with TGF-β type I receptor, activates the kinase activity of TGF-β type I receptor, and reveals an activity to transmit TGF-β signal. I have. However, in the present invention, the TGF-β type II receptor only needs to have the activity of binding to XIAP, and the TGF-β type II receptor TG
Loss F-β binding activity, TGF-β type I receptor heterotetramer formation activity, TGF-β type I receptor kinase activation activity, or TGF-β signal transduction activity TGF-β type II receptor. Therefore, the TGF-β type II described here
The biological activity of the receptor may be the binding activity to XIAP.

The TGF-β type II used in the present invention
The receptor has the biological activity of a TGF-β type II receptor, and has an amino acid sequence shown in SEQ ID NO: 8 having one or more amino acid substitutions, deletions and / or additions. It may be a beta type II receptor. More specifically, TGF- used in the present invention
The β type II receptor has one or more, preferably 1 or 20 or more amino acids in the amino acid sequence shown in SEQ ID NO: 8 as long as it has the biological activity of the TGF-β type II receptor.
It may have an amino acid sequence in which not more than 1, more preferably not more than 1 or 10 amino acids have been substituted.

Alternatively, the TGF-β type II receptor has one or more, preferably no more than 1 or 20, more preferably no more than 1 in the amino acid sequence shown in SEQ ID NO: 8.
Alternatively, it may have an amino acid sequence in which 10 or less amino acids have been deleted. Alternatively, the amino acid sequence shown in SEQ ID NO: 8 may have an amino acid sequence in which 1 or 2 or more, preferably 1 or 30 or less, more preferably 1 or 20 or less amino acids are added.

It is already known that a polypeptide having an amino acid sequence in which one or more amino acid residues have been substituted, deleted and / or added in a certain amino acid sequence maintains its biological activity (Mark). , DF et al.,
Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zo
ller, MJ & Smith, M. Nucleic Acids Research (198
2) 10, 6487-6500, Wang, A. et al., Science 224, 14
31-1433, Dalbadie-McFarland, G. et al., Proc. Nat
l. Acad. Sci. USA (1982) 79, 6409-6413).

The TAB1, XIAP, TGF-β type I or type II receptor used in the present invention may have different amino acid sequences, molecular weights, isoelectric points, sugars, etc. depending on the species of origin, the host producing them and / or the purification method. The presence or absence of the addition of the chain, the position of the addition of the sugar chain, the structure of the sugar chain, the phosphorylation state and / or the presence or absence of the disulfide bond are different. However, as long as it can be suitably used in the present invention, it may have any structure, but human is preferred as a species derived therefrom.

The TAB1, XIAP, TGF-β type I or type II receptor used in the present invention is also the above TAB1, XIAP, TGF-β type I or type II receptor fused to another peptide or polypeptide. May be. Known methods can be used for preparing these fusion polypeptides. The other peptide or polypeptide to be fused to the TAB1, XIAP, TGF-β type I or type II receptor may be any peptide or polypeptide as long as it is effectively used in the present invention. For example, as a peptide, FLAG (Hopp, TP et al., Bi
oTechnology (1988) 6, 1204-1210), 6 × His, 10 × His consisting of 6 His (histidine) residues, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, p18
HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, Protein C
A known peptide such as a fragment of is used. Examples of the polypeptide include GST (glutathione S. transferase), HA (influenza agglutinin), immunoglobulin constant region, β-galactosidase, MBP (maltose binding protein) and the like. These can use what is marketed.

DNA encoding TAB1 used in the present invention
Examples include a base sequence consisting of base A at position 7 to base G at position 1518 in the base sequence shown in SEQ ID NO: 1. The DNA encoding XIAP used in the present invention includes a base at position 82 in the base sequence shown in SEQ ID NO: 3.
And a base sequence consisting of base T at position 1572 from A. Examples of the DNA encoding the TGF-β type I receptor used in the present invention include a base sequence consisting of base A at base position 77 to base G at position 1585 in the base sequence shown in SEQ ID NO: 5. TGF-β type used in the present invention
The DNA encoding the II receptor includes the nucleotides from base A at position 336 to 2036 in the base sequence shown in SEQ ID NO: 7.
And a base sequence consisting of base A at the position.

The DNA encoding the TAB1, XIAP, TGF-β type I or type II receptor used in the present invention may be any DNA having the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, or 7, respectively. It may be of any origin. Such DNA includes, for example, genomic DNA, cDNA, synthetic
DNA. These may be cDNA libraries obtained from various cells, tissues or organs or species other than humans, DNA obtained from genomic libraries, or they may be commercially available DNA libraries. The vectors used for these libraries may be any plasmids, bacteriophages, YAC vectors, and the like.

The TAB1, XIAP, TGF-β type I or type II receptor used in the present invention includes TAB1, XIAP, TGF-β
Using a DNA encoding a type I or type II receptor, it can be obtained as a recombinant protein produced using a gene recombination technique as described below. For example, a recombinant protein can be obtained by cloning the nucleotide sequence of the TAB1, XIAP, TGF-β type I or type II receptor gene described in the present specification from a cell, tissue, or organ that expresses them, and using an appropriate vector. And introduced into a host for production. This recombinant protein can be used in the present invention.

Specifically, mRNA encoding the gene is isolated from cells, tissues or organs expressing the protein used in the present invention. mRNA can be isolated by known methods, for example, guanidine ultracentrifugation (Chirgwin, JM e
t al., Biochemistry (1979) 18, 5294-5299), AGP
Method C (Chomczynski, P. and Sacchi, N., Anal. Bioche
m. (1987) 162, 156-159)
Total R using mRNA Purification Kit (Pharmacia) etc.
Purify mRNA from NA. Also, QuickPrep mRNA P
By using urification Kit (Pharmacia)
NA can also be prepared directly.

A gene cDNA is synthesized from the obtained mRNA using reverse transcriptase. AMV synthesis
Reverse Transcriptase First-strand cDNA Synthesis
Kit (Seikagaku Corporation) or the like can also be used. To perform cDNA synthesis and amplification, use Marathon cDN
5'-RACE method using A Amplification kit (manufactured by CLONTECH) and polymerase chain reaction (PCR) (Frohman, MA et al., Proc.
Natl. Acad. Sci. USA (1988) 85, 8998-9002; Bely
avsky, A. et al., Nucleic Acids Res. (1989) 17, 29
19-2932) can be used.

From the obtained PCR product, the target DNA
Fragments are prepared and ligated with vector DNA. Further, a recombinant vector is prepared from this, introduced into E. coli, etc., and colonies are selected to prepare a desired recombinant vector. The base sequence of the target DNA is confirmed by a known method, for example, a dideoxynucleotide chain termination method. Once the desired DNA is obtained, it is incorporated into an expression vector.

The DNA encoding the TAB1, XIAP, TGF-β type I or type II receptor used in the present invention also hybridizes to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5 or 7, respectively. And has its biological activity
It may be DNA encoding TAB1, XIAP, TGF-β type I or type II receptor. The conditions under which the DNA encoding the TAB1, XIAP, TGF-β type I or type II receptor hybridizes may be any suitable stringency conditions.

[0048] Such hybridization conditions include:
For example, low stringency conditions can be mentioned. Low stringency conditions include, for example, 42 ° C., 5 ×
Wash conditions provided by SSC, 0.1% sodium dodecyl sulfate, 50% formamide. More preferably, the conditions are high stringency. High stringency conditions include, for example, 60 ° C., 0.1 × SSC, 0.1% s
Cleaning conditions provided by odium dodecyl sulfate. It is already known that a protein encoded by a DNA that hybridizes under appropriate conditions to a nucleotide sequence encoding a certain protein has the same biological activity as that protein.

Human TAB1 having an amino acid sequence consisting of amino acid Met at amino acid position 1 to amino acid Pro at amino acid position 504 of the amino acid sequence shown in SEQ ID NO: 2
Escherichia coli harboring plasmid TABI-f-4 containing DNA encoding Escherichia coli DH5a (TABI-f-4) was named on July 19, 1996 by the National Institute of Advanced Industrial Science and Technology Deposited at the research institute (1-3 1-3 Higashi, Tsukuba, Ibaraki Prefecture) under the Budapest Treaty under the deposit number FERM BP-5599.

The amino acid sequence shown in SEQ ID NO: 2 from the amino acid position Met at position 1 to the amino acid position 504
Escherichia coli carrying the plasmid pBS-TAB1 containing the DNA encoding the human TAB1 wherein the amino acid Ser at position 52 is Arg and the amino acid Ser at position 52 is Escherichi
a. HB101 (pBS-TAB1), 1996 (1996)
On April 19, it was deposited internationally under the Budapest Treaty with the Research Institute of Biotechnology and Industrial Technology (1-3 1-3 Higashi, Tsukuba, Ibaraki Prefecture) under the deposit number FERM BP-5508.

The DNA encoding TAB1, XIAP, TGF-β type I or type II receptor used in the present invention may be a cDNA library or genomic live obtained from various cells, tissues or organs, or a species other than human. Rally, or they may be commercially available DNA libraries. Vectors used for these libraries include plasmids, bacteriophages, YA
Any material such as a C vector may be used. The DNA encoding TAB1, XIAP, TGF-β type I or type II receptor used in the present invention can be constructed from the above-mentioned DNA by a commercially available kit or a known method. Examples include digestion with a restriction enzyme, addition of a linker, insertion of an initiation codon (ATG) and / or a termination codon (ATT, TGA or TAG), and the like.

The TAB1, XIAP, TGF-β type I or type II receptor expression vector used in the present invention may be any expression vector suitably used in the present invention. Examples of the expression vector include mammal-derived expression vectors such as pEF, pCDM8, insect cell-derived expression vectors such as pBacPAK8, plant-derived expression vectors such as pMH1, pMH2, and animal virus-derived expression vectors such as pHSV and pMV. Expression vectors derived from yeast, such as pNV11, expression vectors derived from Bacillus subtilis, such as p
PL608, pKTH50, Escherichia coli-derived expression vector such as pGE
X, pGEMEX, pMALp2.

The expression vector for the TAB1, XIAP, TGF-β type I or type II receptor used in the present invention includes, for example, DNA encoding the TAB1, XIAP, TGF-β type I or type II receptor. Can be produced by ligating downstream of the promoter.
As the promoter / enhancer, a promoter / enhancer derived from a mammal, for example, an EF1-α promoter / enhancer, a γ-actin promoter / enhancer, a promoter / enhancer derived from an insect virus, for example, a polynuclear (polyhedrin) virus promoter / enhancer, a plant-derived enhancer Promoter / enhancer, for example, tobacco mosaic virus promoter / enhancer, promoter / enhancer derived from animal virus, for example, SV40 promoter / enhancer, human
CMV promoter / enhancer, promoter / enhancer derived from yeast, for example, alcohol dehydrogenase promoter / enhancer, promoter derived from Escherichia coli /
Enhancers, for example, Lac promoter / enhancer, Trp promoter / enhancer, Tac promoter / enhancer.

For example, when using the SV40 promoter / enhancer, the method of Mulligan et al. (Nature (1979)
277, 108) and when using the HEF1α promoter / enhancer, the method of Mizushima et al. (Nucleic Acids R
es. (1990) 18, 5322). In the case of Escherichia coli, expression can be carried out by operably linking a commonly used useful promoter, a signal sequence for secretion, and a gene to be expressed. For example, examples of the promoter include a lacZ promoter and an araB promoter. When using the lacZ promoter, the method of Ward et al. (Nature (1098) 341, 544-546; FA)
SEB J. (1992) 6, 2422-2427), when using the araB promoter, the method of Better et al. (Science (1988) 240, 10
41-1043).

For expression of TAB1, XIAP, TGF-β type I or type II receptor used in the present invention, a signal sequence suitable for a host used for expression may be added. Examples of the signal sequence include a signal sequence of a secretory protein. As the signal sequence of the secretory protein, for example, a signal sequence of a mammalian-derived secretory protein,
An example is an immunoglobulin signal sequence.
As the signal sequence of the secretory protein, a signal sequence of a secretory protein derived from Escherichia coli, for example, OmpA, pelB (Lei, S. et al.
P. et al J. Bacteriol. (1987) 169, 4379) and the like.

The expression vector thus prepared can be introduced into a host by a known method. As a method for introduction into a host, for example, electroporation (EM
BO J. (1982) 1, 841-845), calcium phosphate method (Vir
ology (1973) 52, 456-467), and the liposome method. The replication origin is SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV)
And the like can be used. Furthermore, in order to amplify the gene copy number in the host cell system, the expression vector is selected as an aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine guanine phosphoribosyltransferase (HPRT) gene, dihydrofolate reductase (HPH) gene as a selection marker. DHFR) gene and the like. Furthermore, a sequence with higher expression efficiency can be designed in consideration of the codon usage of the host used for expression (Grantham, R. et al., Nucleic Acid).
ic Acids Research (1981) 9, r43-r74).

In the present invention, any production system can be used for producing a recombinant protein. Production systems for producing recombinant proteins include in vitro and in vivo production systems. Examples of the in vitro production system include a production system using eukaryotic cells and a production system using prokaryotic cells.

When eukaryotic cells are used, there are production systems using animal cells, plant cells, and fungal cells. As animal cells, (1) mammalian cells, for example, CHO (J. Exp. Med. (1995)
108,945), COS, Myeloma, BHK (baby hamster kidn
ey), HeLa, Vero, (2) Amphibian cells such as Xenopus oocytes (Valle, et al., Nature (1981) 29
1, 358-340), or (3) insect cells such as sf9, sf2
1, Tn5 is known. As CHO cells, dhfr ^- CHO (Proc. Natl.
cad. Sci. USA (1980) 77, 4216-4220) and CHO K-1 (Pro
Natl. Acad. Sci. USA (1968) 60, 1275) can be suitably used.

As plant cells, cells derived from Nicotiana tabacum are known, which may be callus cultured. As fungal cells, yeasts such as the genus Saccharomyces, such as Saccharomyces cerevisiae, and filamentous fungi, such as the genus Aspergillus, such as Aspergillus niger, are known.
When using prokaryotic cells, there is a production system using bacterial cells. Escherichia coli (E. coli) and Bacillus subtilis are known as bacterial cells.

[0060] These cells are transformed with the desired DNA, and the transformed cells are cultured in vitro to obtain a recombinant protein. Culture is performed according to a known method. For example, DMEM, MEM, RPMI1
640, IMDM can be used. At that time, a serum replacement solution such as fetal calf serum (FCS) may be used in combination, or serum-free culture may be performed. The pH during the culturing is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C for about 15 to 200
Perform the operation for a time, and change the medium, aerate, and agitate as necessary. The cells into which the gene has been introduced are made to produce a recombinant protein and collected.

On the other hand, examples of an in vivo production system include a production system using animals and a production system using plants.
The desired DNA is introduced into these animals or plants, and recombinant proteins are produced and recovered in the animals or plants. When using animals, there are production systems using mammals and insects. Goats, pigs, sheep, mice, and cows can be used as mammals (Vicki Glase
r, SPECTRUM Biotechnology Applications, 1993). When a mammal is used, a transgenic animal can be used.

For example, a target DNA is inserted into a gene such as goat β-casein, which encodes a protein uniquely produced in milk, to prepare a fusion gene.
A DNA fragment containing the fusion gene into which the DNA has been inserted is injected into a goat embryo, and the embryo is introduced into a female goat. A recombinant protein is obtained from milk produced by a transgenic goat born from a goat that has received an embryo or a progeny thereof. Hormones may optionally be used in transgenic goats to increase the amount of milk containing recombinant protein produced from the transgenic goats (Ebert, KM et al., Bi
o / Technology (1994) 12, 699-702).

As an insect, for example, a silkworm can be used. When using silkworm, target DN
B. Infect the silkworm with the baculovirus into which A has been inserted, and obtain a desired recombinant protein from the body fluid of the silkworm (Maeda,
S. et al., Nature (1985) 315, 592-594). Furthermore, when using a plant, for example, tobacco can be used. When tobacco is used, the DNA of interest is inserted into a plant expression vector, for example, pMON530, and this vector is
Introduce into bacteria such as Agrobacterium tumefaciens. The bacteria are transferred to tobacco, such as Nicotiana taba
cum, and the desired recombinant protein is obtained from the leaves of this tobacco (Julian, K.-C. Ma et al., Eur. J. Immunol.
(1994) 24, 131-138).

The genes are introduced into these animals or plants as described above, and the recombinant proteins are produced and recovered in the animals or plants. The recombinant protein expressed and produced as described above can be separated from the host inside and outside the cell and from the host and purified to homogeneity. The separation and purification of the protein used in the present invention may be performed by a commonly used separation and purification method, and is not limited at all.

For example, proteins can be separated by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc. Can be purified (Antibodies: A La
boratory Manual.Ed Harlow and David Lane, Cold Sp
ring Harbor Laboratory, 1988).

Examples of the chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography and the like (Strategies for Protein Purification and Chassis).
racterization: A Laboratory Course Manual. Ed Dan
iel R. Marshak et al., Cold Spring Harbor Laborato
ry Press, 1996). These chromatographs are HP
It can be performed using liquid phase chromatography such as LC and FPLC.

The concentration of the protein can be measured by a known method. For example, measurement of absorbance or Bradford
Method may be used. In a screening method for a substance that inhibits the binding between XIAP and TAB1, XIAP, TAB1 and a test sample are contacted, and then the presence or absence of the formation of a bond between XIAP and TAB1 is examined.

Further, in a screening method for a substance that inhibits the binding between TGF-β type I receptor and XIAP, TGF-β type I receptor, XIAP and a test sample are contacted, and then TGF-β type I receptor and XIAP The presence or absence of the formation of a bond with is examined. Further, in a screening method for a substance that inhibits the binding between TGF-β type II receptor and XIAP, a TGF-β type II receptor, XIAP and a test sample are contacted, and then TGF-β type II receptor and
The presence or absence of formation of a bond with XIAP is examined. The screening system provided in the present invention is performed as an in vitro assay system.

One embodiment of an in vitro assay system is performed in a non-cellular system. Specifically, one of TAB1 or XIAP or TGF-β type I or type II receptor was bound to the support, the other and the test sample were added thereto, incubated, washed, and then bound to the support. What is necessary is just to detect or measure the binding of the other protein to the protein.

TAB1, XIAP, TGF-β type I or type II
Receptors are cells that uniquely express them, TAB1, XIAP, TGF-β used in the present invention, cells transfected with DNA encoding type I or type II receptor, TAB1, XIAP, TGF used in the present invention. A protein produced from an animal or a plant into which DNA encoding the -β type I or type II receptor has been introduced can be used in a purified state or in a partially purified state.

The purified or partially purified protein (TAB
1, one of XIAP, TGF-β type I or type II receptor) is bound to the support. In order to bind the protein to the support, the protein may be immobilized on the support by a standard method. As a support for binding proteins,
For example, insoluble polysaccharides, such as agarose, dextran, cellulose, and synthetic resins, such as polystyrene, polyacrylamide, and silicon, may be used. More specifically, commercially available beads and plates manufactured using them as raw materials are used. In the case of beads, a column or the like filled with these may be used. In the case of a plate, a multi-well plate (eg, a 96-well multi-well plate) or a biosensor chip can be used. In order to bind the protein to the support, a commonly used method utilizing chemical bonding, physical adsorption and the like may be used. Alternatively, an antibody that specifically recognizes a protein can be bound to a support in advance, and the antibody can be bound to the protein. Avidin /
It can be attached via biotin.

The binding between TAB1 and XIAP and between XIAP and TGF-β type I or type II receptor are usually carried out in a buffer. As the buffer, for example, a phosphate buffer, a Tris buffer, or the like is used. Incubation conditions include conditions that have already been used, for example, incubation at 4 ° C. to room temperature for 1 hour to 24 hours. After incubation, wash TAB1 and XIAP, XIAP and TG
Any substance that does not prevent binding to the F-β type I or type II receptor can be used. For example, a buffer containing a surfactant is used. As a surfactant, for example, 0.05% T
Ween 20 is used.

The test samples to be screened include, for example, peptides, polypeptides, synthetic compounds, fermented microorganisms, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts or animal cell extracts or These libraries are mentioned. The substances contained in these test samples are substances that are expected to act in an inhibitory manner on the binding between TAB1 and XIAP and between XIAP and TGF-β type I or type II receptor. These inhibitors act on TAB1
XIAP binding or TGF-β type I or type I to XIAP
Inhibits binding to the I receptor. These inhibitors suppress or activate TGF-β signaling.

[0074] The TAB1 contained in these test samples was
XIAP binding or TGF-β type I or type I to XIAP
To select a substance that inhibits the binding of the I receptor, by incubating and washing under appropriate conditions,
Specific and non-specific binding can be separated. Then, the binding of XIAP to TAB1 or the binding state of TGF-β type I or type II receptor to XIAP used in the present invention may be evaluated.

In the screening method of the present invention, a control group may be provided together with a group in which a test sample is brought into contact with a protein. As a control group, a negative control group containing no test sample or TAB1 and XIAP or XIAP
A positive control group containing a substance that clearly inhibits binding to the TGF-β type I or type II receptor or both groups can be set.

When detecting or measuring the bound protein in the present invention, the bound protein may be simply detected, or the bound protein may be quantitatively measured.
In these cases, compare the results obtained with the negative control group without the test sample, the results obtained with the group containing the test sample and / or the results obtained with the positive control group containing an apparent binding inhibitor. The target TAB1 and XI
An inhibitor of the binding between AP or XIAP and a TGF-β type I or type II receptor can be detected.

Further, these results are obtained as numerical values, and by comparing those numerical values, TAB1 and XIAP or XIAP
The activity of a substance that inhibits binding to a TGF-β type I or type II receptor can also be quantitatively measured. When measuring quantitatively, the target TAB1 and XIAP or XIAP are compared by comparing the numerical value obtained in the negative control group without the test sample with the numerical value obtained in the group to which the test sample is applied.
And a substance that inhibits the binding between the TGF-β and the TGF-β type I or type II receptor. If the test sample contains a substance that inhibits the binding between the target TAB1 and XIAP or XIAP and the TGF-β type I or type II receptor, the obtained value decreases, and the sample containing the binding inhibitor is reduced. Can be determined.

For quantitative measurement, TAB1 and XIAP
Alternatively, it can be quantified based on a standard curve created by numerical values obtained in a positive control group containing a known amount of a substance known to inhibit binding of XIAP to TGF-β type I or type II receptor. . When the amount of bound protein is large, TAB1 and XIAP or XIAP contained in the test sample
If the activity of the substance that inhibits the binding to the TGF-β type I or type II receptor is low, while the amount of the bound protein is small, the TAB1 and XIAP or XIAP and XIAP and TGF-
It is presumed that the substance that inhibits binding to the β type I or type II receptor has a strong binding inhibitory activity.

In the method of the present invention for screening an inhibitor of the binding between TAB1 and XIAP or XIAP and TGF-β type I or type II receptor, a bioassay utilizing surface plasmon resonance as a means for detecting or measuring the bound protein is used. Sensors can be used. A biosensor using the surface plasmon resonance phenomenon can observe a protein-protein interaction as a surface plasmon resonance signal in real time using a small amount of protein and without labeling (for example, BIAcore; Phacore).
rmacia). Therefore, by using a biosensor such as BIAcore, TAB1 and XIAP or XIAP and TGF-β type
It is possible to evaluate binding to the I or type II receptor. That is, the other protein is brought into contact with a sensor chip on which one of TAB1, XIAP, TGF-β type I or type II receptor is immobilized, and the immobilized TAB
1. It is intended to detect a protein binding to XIAP, TGF-β type I or type II receptor as a change in resonance signal.

More specifically, the operation may be performed as follows. First, activate the sensor chip CM5 (Biosensor) and start TAB
1. Immobilize XIAP, TGF-β type I or type II receptor on a sensor chip. That is, EDC / NHS aqueous solution (200 mM EDC (N-ethyl-N '-(3-dimethylaminopropyl)
carbonate hydrochloride), 50mM NHS (N-hydroxysucc
After activating the sensor chip with inimide)), HBS
Buffer (10mM HEPES pH7.4, 150mM NaCl, 3.4mM ED
Wash the sensor chip with TA, 0.05% Tween20).

Next, an appropriate amount of the interacting protein dissolved in the HBS buffer is brought into contact with the sensor chip to be immobilized. After washing the sensor chip with HBS buffer, ethanolamine solution (1M ethanolamine hydrochl
oride, pH 8.5) to block remaining active groups on the sensor chip. Wash the sensor chip again with HBS buffer and use it for binding evaluation. Next, an appropriate amount of the protein dissolved in the HBS buffer is injected. At this time, the amount of the protein having an interaction binding to TAB1, XIAP, TGF-β type I or type II receptor immobilized on the sensor chip is observed as an increase in resonance signal value.

Further, in the above binding evaluation system, TAB1,
A test sample is injected following the protein interacting with XIAP, TGF-β type I or type II receptor. A control group may be provided together with the group into which the test sample is injected. As the control group, a negative control group containing no test sample, a positive control group containing an apparent binding inhibitor, or both groups can be used. The bound protein can be quantitatively measured as a change in resonance signal value. In this case, by comparing the results obtained in the negative control group without the test sample, the results obtained in the group with the test sample and / or the results obtained in the positive control group with an apparent binding inhibitor, In addition, the target binding inhibitor can be detected and determined.

In the method for screening an inhibitor of the binding of TAB1 to XIAP or XIAP to TGF-β type I or type II receptor of the present invention, any of the proteins is labeled as a means for detecting or measuring the bound protein. ,
The label of the bound protein can be detected or measured. For example, in the above-described screening method, the other protein to be brought into contact with one protein together with the test sample is labeled in advance, and after incubation with the test sample, the protein bound by washing is detected or measured by the label. I do. That is, the test sample and the other labeled protein are preferably brought into contact with one of the proteins bound to the support. After incubation, washing may be performed to detect or measure the label of the bound protein.

TAB1, XIAP, TGF-β type I or type II
The receptor can be labeled by a commonly known method. Labeling substances include, for example, radioisotopes,
Enzymes, fluorescent substances, biotin / avidin and the like.
As these labeling substances, commercially available labeling substances can be used. As radioisotopes, for example, ³² P, ³³ P,
^{131 I, 125 I, 3 H} , 14 C, 35 S and the like. Examples of the enzyme include alkaline phosphatase, horseradish peroxidase, β-galactosidase, β-glucosidase and the like. Examples of the fluorescent substance include fluorescein isothiocyanate (FITC) and rhodamine. These can be obtained commercially and labeled by a known method.

Specifically, it can be performed as follows. That is, a solution containing any of the proteins is added to the plate and left overnight. After washing, the plate is blocked with, for example, BSA to prevent non-specific binding of proteins. Wash again and add the test sample and the other labeled protein to the plate. At the same time, a group not containing a test sample (negative control) and / or a group to which a known concentration of a binding inhibitor was added (positive control) are placed and incubated. After incubation, the cells are washed and the bound protein is detected or measured. For detection or measurement, in the case of a radioisotope, detection or measurement is performed by liquid scintillation. In the case of an enzyme, its substrate is added, and the enzymatic change of the substrate, eg, color development, is detected or measured by an absorptiometer. In the case of a fluorescent substance, it is detected or measured by a fluorometer. By comparing these results with the values obtained in the control group, the test sample containing the inhibitor can be determined.

In the method of the present invention for screening for an inhibitor of the binding of TAB1 to XIAP or XIAP to TGF-β type I or type II receptor, one of the proteins is specifically used as a means for detecting or measuring the bound protein. A primary antibody that recognizes can be used.

For example, in the above-mentioned screening method, one protein is brought into contact with a test sample together with another protein, and incubated with the test sample.
After washing, the bound protein is detected or measured with a primary antibody that specifically recognizes the protein. That is, the test sample and the other protein are preferably brought into contact with one of the proteins bound to the support. After incubation, washing may be performed, and the bound protein may be detected or measured with a primary antibody that specifically recognizes the protein.
The primary antibody is preferably labeled with a labeling substance.

Specifically, it can be performed as follows. That is, a solution containing any of the proteins is added to the plate and left overnight. After washing, the plate is blocked with, for example, BSA to prevent non-specific binding of proteins. Wash again and add the test sample and the other protein to the plate. At the same time, a group not containing a test sample (negative control) and / or a group to which a known concentration of a binding inhibitor was added (positive control) are placed and incubated.

After the incubation, the antibody is washed and an antibody against the protein added together with the test sample is added. After an appropriate incubation, the plate is washed and the protein is detected or measured by a primary antibody that specifically recognizes the protein.
For detection or measurement, in the case of a radioisotope, detection or measurement is performed by liquid scintillation. In the case of an enzyme, its substrate is added, and the enzymatic change of the substrate, eg, color development, is detected or measured by an absorptiometer. In the case of a fluorescent substance, it is detected or measured by a fluorometer. By comparing these results with the values obtained in the control group, the test sample containing the inhibitor can be determined.

The TAB1 of the present invention and XIAP, or XIAP and TGF-β
In a method for screening an inhibitor for binding to a type I or type II receptor, as a means for detecting or measuring the bound protein, TAB1, XIAP, another peptide or polypeptide fused to a TGF-β type I or type II receptor A primary antibody that specifically recognizes can be used.

For example, in the above-mentioned screening method, one of the proteins is brought into contact with the other sample together with the test sample, incubated with the test sample, washed, and then the bound protein is fused with the other protein. Detection or measurement is performed using a primary antibody that specifically recognizes a peptide or protein. That is, the test sample and the other protein are preferably brought into contact with one of the proteins bound to the support. After incubation, washing is performed, and the bound protein may be detected or measured with a primary antibody that specifically recognizes another peptide or polypeptide fused to the protein. The primary antibody is preferably labeled with a labeling substance.

Specifically, it can be performed as follows. That is, a solution containing any of the proteins is added to the plate and left overnight. After washing, the plate is blocked with, for example, BSA to prevent non-specific binding of proteins. Wash again and add the test sample and the other protein fused to the other peptide or polypeptide to the plate. Group not containing test sample at the same time (negative control)
And / or a group to which a known concentration of a binding inhibitor was added (positive control), and these were incubated.

After the incubation, an antibody against another peptide or polypeptide fused with the protein added to the washed sample together with the test sample is added. After an appropriate incubation, the plate is washed and the protein is detected or measured by a primary antibody that specifically recognizes another peptide or polypeptide fused to the protein. For detection or measurement, in the case of a radioisotope, detection or measurement is performed by liquid scintillation. In the case of an enzyme, its substrate is added, and the enzymatic change of the substrate, eg, color development, is detected or measured by an absorptiometer. In the case of a fluorescent substance, it is detected or measured by a fluorometer. By comparing these results with the values obtained in the control group, the test sample containing the inhibitor can be determined.

In the method for screening an inhibitor of the binding of TAB1 to XIAP or XIAP to TGF-β type I or type II receptor of the present invention, TAB1, XIAP, TGF- β type
A primary antibody that specifically recognizes the I or type II receptor and a secondary antibody that specifically recognizes the primary antibody can be used.

For example, in the above-described screening method, one of the proteins is brought into contact with another protein together with a test sample, incubated with the test sample, and then washed to specifically recognize the bound protein. Detection or measurement is performed using a primary antibody and a secondary antibody that specifically recognizes the primary antibody. That is, the test sample and the other protein are preferably brought into contact with one of the proteins bound to the support. After incubation, washing is performed, and the bound protein may be detected or measured with a primary antibody that specifically recognizes the protein and a secondary antibody that specifically recognizes the primary antibody. The secondary antibody is preferably labeled with a labeling substance.

Specifically, it can be performed as follows. That is, a solution containing any of the proteins is added to the plate and left overnight. After washing, the plate is blocked with, for example, BSA to prevent non-specific binding of proteins. Wash again and add the test sample and the other protein to the plate. At the same time, a group not containing a test sample (negative control) and / or a group to which a known concentration of a binding inhibitor was added (positive control) are placed and incubated.

After incubation, a primary antibody against another peptide or polypeptide fused to the protein added to the washed and washed sample is added. After a suitable incubation, the plate is washed and then a secondary antibody that specifically recognizes the primary antibody is added. After appropriate incubation, washing is performed, and the protein is detected or measured by a secondary antibody that specifically recognizes a primary antibody that specifically recognizes the protein. For detection or measurement, in the case of a radioisotope, detection or measurement is performed by liquid scintillation. In the case of an enzyme, its substrate is added, and the enzymatic change of the substrate, eg, color development, is detected or measured by an absorptiometer. In the case of a fluorescent substance, it is detected or measured by a fluorometer. By comparing these results with the values obtained in the control group, the test sample containing the inhibitor can be determined.

In the method for screening an inhibitor of the binding of TAB1 to XIAP or XIAP to TGF-β type I or type II receptor according to the present invention, TAB1, XIAP, TGF-β Type I
Alternatively, a primary antibody that specifically recognizes another peptide or polypeptide fused to the type II receptor and a secondary antibody that specifically recognizes the primary antibody can be used.

For example, in the above-mentioned screening method, one of the proteins is brought into contact with another protein together with a test sample, incubated with the test sample, washed, and then the bound protein is fused with the other protein. Detection or measurement is performed using a primary antibody that specifically recognizes the peptide or polypeptide and a secondary antibody that specifically recognizes the primary antibody. That is, the test sample and the other protein are preferably brought into contact with one of the proteins bound to the support. After incubation, washing is performed, and the bound protein is detected or measured by a primary antibody that specifically recognizes another peptide or polypeptide fused to the protein and a secondary antibody that specifically recognizes the primary antibody. I just need. The secondary antibody is preferably labeled with a labeling substance.

Specifically, it can be performed as follows. That is, a solution containing any of the proteins is added to the plate and left overnight. After washing, the plate is blocked with, for example, BSA to prevent non-specific binding of proteins. Wash again and add the test sample and the other protein fused to the other peptide or polypeptide to the plate. Group not containing test sample at the same time (negative control)
And / or a group to which a known concentration of a binding inhibitor was added (positive control), and these were incubated.

After incubation, a primary antibody to another peptide or polypeptide fused to the protein added to the wash and test sample is added. After a suitable incubation, the plate is washed and then a secondary antibody that specifically recognizes the primary antibody is added. After an appropriate incubation, washing is performed, and the protein is detected or measured by a secondary antibody that specifically recognizes a primary antibody that specifically recognizes another peptide or polypeptide fused to the protein. For detection or measurement, in the case of a radioisotope, detection or measurement is performed by liquid scintillation. In the case of an enzyme, its substrate is added, and the enzymatic change of the substrate, eg, color development, is detected or measured by an absorptiometer. In the case of a fluorescent substance, it is detected or measured by a fluorometer. By comparing these results with the values obtained in the control group, the test sample containing the inhibitor can be determined.

More specifically, the present invention is particularly preferably applied to EL
ISA (Enzyme-linked Immunosorbent Assay) can be performed as follows. That is, XIAP fused with another peptide or polypeptide, for example, 6 × His, is diluted with an immobilization buffer (0.1 M NaHCO 3, 0.02% NaN 3, pH 9.6). An appropriate amount of the diluted aqueous solution is added to each well of a 96-well immunoplate (manufactured by Nunc), and the mixture is incubated at 4 ° C overnight.

After washing each well three times with a washing buffer (prepared with 0.05% Tween 20 in PBS), 200 ml of a 5% BSA (manufactured by SIGMA) solution in PBS is added, and blocking is performed at 4 ° C. overnight. .

Next, each well was washed three times with a washing buffer.
An appropriate amount of a test sample is added to another peptide or polypeptide diluted with a dilution buffer (1% BSA, 0.5% Tween 20, PBS), for example, TAB1 fused with FLAG and a test sample, and incubated at room temperature for 1 hour. Wash each well three times with wash buffer, add 100 ml mouse anti-FLAG M2 antibody (manufactured by IBI) diluted to 3 mg / ml with dilution buffer to each well, and incubate at room temperature for 1 hour.

Each well is washed three times with a washing buffer, 100 ml of an alkaline phosphatase-labeled goat anti-mouse IgG antibody (manufactured by ZYMED) diluted 1000-fold with a dilution buffer is added to each well, and the mixture is incubated at room temperature for 1 hour. Wash each well 5 times with a washing buffer, and add a coloring solution (substrate buffer; 50 mM NaHCO3, 10 mM MgCl2, p-phenylphosphate dissolved at a concentration of 1 mg / ml in pH 9.8; SIGMA).
After adding 100 ml to each well and reacting at room temperature, the absorbance at 405 nm is measured using a microplate reader (Model 3550, BIO-R
AD). By comparing these results with the values obtained in the negative control group and / or the positive control group, the test sample containing the inhibitor can be determined.

The screening method of the present invention comprises a high Thr
It can also be used for oughput screening (HTS).
Specifically, up to blocking is manually performed, and the subsequent reaction is performed by a robot to automate the process, thereby realizing High Throughput screening. That is, XIAP fused with another peptide or polypeptide such as 6 × His is immobilized on a solid phase buffer (0.1 M Na
Dilute with HCO ₃ , 0.02% NaN ₃ , pH 9.6). An appropriate amount of the diluted aqueous solution is added to each well of a 96-well immunoplate (manufactured by Nunc), and the mixture is incubated at 4 ° C overnight.

After washing each well three times with a washing buffer (prepared with 0.05% Tween20 in PBS), 200 ml of a 5% BSA (manufactured by SIGMA) solution in PBS was added, and blocking was performed at 4 ° C. overnight. I do. Next, for example, Biomek2000 HTS sys
Set the blocked immunoplate on the tem (manufactured by Beckman) and execute the system control program. At this time, Biomek 2000 dispenser (Beckma
n) or a Multipipette 96-well simultaneous dispenser (manufactured by Sagian) can be used to dispense the solution into each hole of the immunoplate and to remove the solution. For washing each hole of the immunoplate, use an EL404 microplate washer (Bio
Tek). Also, to measure the absorbance
SPECTRAmax250 plate reader (Molecular Devices)
Can be used.

The program is set to perform the following operations. That is, wash each well three times with wash buffer,
Test sample and dilution buffer (1% BSA, 0.5% Tween20, PB
Other peptides or polypeptides diluted in S), such as M
An appropriate amount of TAB1 fused with BP (maltose binding protein) is added. Group not containing test sample at the same time (negative control)
And a group to which a known concentration of a binding inhibitor was added (positive control), and these were incubated at room temperature for 1 hour.

Each well was washed three times with the washing buffer, and the rabbit anti-MBP antiserum diluted 5,000-fold with the dilution buffer (New
England Biolabs) is added to each well and incubated for 1 hour at room temperature. Wash each well three times with wash buffer, and add alkaline phosphatase-labeled goat anti-rabbit IgG antibody (manufactured by TAGO) diluted 5000 times with dilution buffer.
Add 00 ml to each well and incubate for 1 hour at room temperature.

Each well was washed 5 times with a washing buffer, and a coloring solution (substrate buffer; 50 mM NaHCO ₃ , 10 mM MgCl ₂ , pH
100 ml of p-nitrophenyl phosphate dissolved at a concentration of 1 mg / ml in 9.8 was added to each well, and the mixture was reacted at room temperature. / Molecul
arDevices). By comparing these results with the values obtained in the control group, the test sample containing the inhibitor can be identified.

As the antibody used in the present invention, a commercially available antibody or an antibody contained in a commercially available kit can be used, or a monoclonal antibody or a polyclonal antibody obtained by a known means can be used. A monoclonal antibody is immunized with a desired sensitizing antigen according to a usual immunization method, and the obtained immunocytes are fused with a known parent cell by a usual cell fusion method, and by a usual screening method, It can be produced by screening monoclonal antibody producing cells.

Specifically, a monoclonal or polyclonal antibody may be prepared as follows. For example, the sensitizing antigen for obtaining the antibody is not limited to the animal species from which it is derived, but is preferably derived from a mammal, such as a human, mouse or rat, from which the peptide or polypeptide used in the present invention is actually derived. . Of these, human-derived sensitizing antigens are particularly preferred. For example, when human TAB1, human XIAP, TGF-β type I or type II receptor is used as a sensitizing antigen, their nucleotide sequence and amino acid sequence can be obtained using the gene sequence disclosed herein. . In addition, human TAB1, human XIAP,
When using other peptides or polypeptides to be fused to TGF-β type I or type II receptor as sensitizing antigens, those peptides or polypeptides are chemically synthesized or obtained by genetic engineering techniques. be able to.

As the protein, peptide or polypeptide used as a sensitizing antigen, its full length may be used, or its fragment may also be used. Examples of the fragment include a C-terminal fragment and an N-terminal fragment. The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion, and is generally rodent, Lagomorphs and primates are used.

As rodent animals, for example, mice, rats, hamsters and the like are used. As an animal of the order Lagomorpha, for example, a rabbit is used. As the primate animal, for example, a monkey is used. As a monkey,
Monkeys (Old World monkeys) of the lower nose are used, for example, cynomolgus monkeys, rhesus monkeys, baboons, chimpanzees, and the like. Immunization of an animal with a sensitizing antigen is performed according to a known method. For example, as a general method, the sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen is PBS (Phosphate-
Buffered Saline) or an appropriate amount diluted with physiological saline or the like, and suspended in an ordinary adjuvant, if desired.
After mixing and emulsifying an appropriate amount of Freund's complete adjuvant, it is preferable to administer to the mammal several times every 4 to 21 days after emulsification. In addition, a suitable carrier can be used at the time of immunization of the sensitizing antigen. Immunization is performed in this manner, and an increase in a desired antibody level in serum is confirmed by a conventional method.

Here, in order to obtain a polyclonal antibody,
After confirming that the level of the desired antibody in the serum has increased, the blood of the mammal sensitized with the antigen is removed. The serum is separated from the blood by a known method. A serum containing the polyclonal antibody may be used as the polyclonal antibody, and if necessary, a fraction containing the polyclonal antibody may be further isolated from the serum.

In order to obtain a monoclonal antibody, after confirming that the level of the desired antibody is increased in the serum of a mammal sensitized with the antigen, immune cells may be removed from the mammal and subjected to cell fusion. . At this time, spleen cells are particularly preferable as the immune cells used for cell fusion. As the mammalian myeloma cell as the other parent cell to be fused with the immunocyte, various known cell lines are suitably used.

Cell fusion of the above-mentioned immune cells and myeloma cells is basically performed by a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, C., Methods Enzymol.
(1981) 73, 3-46). More specifically, the cell fusion is carried out, for example, in a normal nutrient culture in the presence of a cell fusion promoter. Examples of the fusion promoter include polyethylene glycol (PEG),
Sendai virus (HVJ) or the like is used, and if necessary, an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency.

The ratio of the use of the immune cells to the myeloma cells is, for example, 1 to 1 for the myeloma cells.
It is preferably set to 0 times. As the culture solution used for the cell fusion, for example, RPMI 1640 culture solution suitable for the growth of the myeloma cell line, MEM culture solution, and other ordinary culture solutions used for this type of cell culture can be used. A serum replacement solution such as fetal calf serum (FCS) can also be used in combination.

In the cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution, and 37
PEG solution heated to about ° C, for example, having an average molecular weight of 10
A PEG solution of about 00 to 6000 is usually 30 to 60%
By adding and mixing at a concentration of (w / v), a desired fused cell (hybridoma) is formed. Subsequently, by repeatedly adding an appropriate culture solution and centrifuging to remove the supernatant, a cell fusion agent or the like unfavorable for hybridoma growth can be removed.

The hybridoma is selected by culturing it in an ordinary selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). Culture in the HAT culture solution is continued for a period of time sufficient to kill cells (non-fused cells) other than the target hybridoma, usually several days to several weeks. Then
The usual limiting dilution method is performed, and screening and cloning of the hybridoma producing the desired antibody are performed.

In addition to obtaining the above hybridoma by immunizing an animal other than human with an antigen, human lymphocytes such as EB
Virus-infected human lymphocytes are sensitized in vitro with proteins, peptides or polypeptides or their expression cells or lysates thereof, and the sensitized lymphocytes are fused with human-derived myeloma cells capable of permanent division, such as U266. As a result, a hybridoma producing a desired human antibody having a binding activity to a peptide or polypeptide can also be obtained (JP-A-63-17688).

Further, a transgenic animal having a human antibody gene repertoire is immunized with a protein, peptide or polypeptide serving as an antigen, a cell expressing the same or a lysate thereof to obtain antibody-producing cells, which are called myeloma cells. A human antibody against the protein, peptide or polypeptide used in the present invention may be obtained using the fused hybridoma (International Patent Application Publication Nos. WO92-03918, WO93-2227, W
O94-02602, WO94-25585, WO96
-33735 and WO96-34096). The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen.

In order to obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a conventional method, and a method of obtaining the culture supernatant is used. And a method for obtaining the ascites. The former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies. In addition to producing antibodies using hybridomas, immunized cells such as sensitized lymphocytes that produce antibodies are converted to oncogenes (on
cogene) may be used.

[0124] The thus obtained monoclonal antibody can also be obtained as a recombinant antibody produced using a gene recombination technique. For example, a recombinant antibody is
The antibody gene is cloned from an immune cell such as a hybridoma or a sensitized lymphocyte producing the antibody, inserted into an appropriate vector, and introduced into a host to produce. This recombinant antibody can be used in the present invention (for example, Borrebaeck, CAK and Larrick, JW, THERA
PEUTIC MONOCLONAL ANTIBODIES, Published in the Unit
(See ed Kingdom by MACMILLAN PUBLISHERS LTD, 1990)
.

The antibodies used in the present invention may be antibody fragments or modified antibodies thereof as long as they have the desired binding activity. For example, antibody fragments include Fab, F (ab ')
2. Single chain Fv (scFv) in which Fv or H chain and L chain Fv are linked by an appropriate linker. Specifically, an antibody is treated with an enzyme, for example, papain or pepsin, to generate an antibody fragment, or a gene encoding these antibody fragment is constructed and introduced into an expression vector. Is expressed.

The antibody expressed and produced as described above can be separated from the host inside and outside the cell and from the host and purified to homogeneity. The separation and purification of the antibody used in the present invention may be carried out by using the separation and purification methods used for ordinary proteins, and is not limited at all. For example, if appropriate selection and combination of chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc., antibodies can be separated and purified. (Antibodies: A Laboratory Manual. Ed Harlow and
David Lane, Cold Spring Harbor Laboratory, 1988)
.

The columns used for affinity chromatography include a protein A column and a protein G column.
Columns. For example, as a column using a protein A column, Hyper D, POROS, Sepharose FF
(Pharmacia) and the like. Examples of chromatography other than affinity chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography and the like (Strategies for Prot.
ein Purification and Characterization: A Laborato
ry Course Manual. Ed Daniel R. Marshak et al., Col
d Spring Harbor Laboratory Press, 1996). These chromatographys can be performed using liquid phase chromatography such as HPLC and FPLC.

For the concentration measurement or activity confirmation of the antibody obtained above, known methods, for example, ELISA, EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent antibody method can be used. The primary antibody or secondary antibody obtained as described above can be labeled by a generally known method. Examples of the labeling substance include a radioisotope, an enzyme, and a fluorescent substance. As these labeling substances, commercially available labeling substances can be used. As the radioisotope, for example, ³² P, ³³ P, ¹³¹ I, ¹²⁵ I, ³ H,
¹⁴ C and ³⁵ S. Examples of the enzyme include alkaline phosphatase, horseradish peroxidase, β-galactosidase, β-glucosidase and the like. Examples of the fluorescent substance include fluorescein isothiocyanate (FITC) and rhodamine. Commercially available products may be obtained as these labeling substances, and labeling may be performed by a known method.

[0129] Another specific example of the screening system provided in the present invention is performed in an in vitro assay system using cells. In a method for screening a substance that inhibits the binding between TAB1 and XIAP, TAB1 and XIAP
Introducing and / or contacting a test sample with cells expressing
The biological activity transmitted via TAB1 or XIAP may then be detected and / or measured.

In a screening method for a substance that inhibits the binding between XIAP and TGF-β type I receptor, a test sample is introduced and / or contacted with cells expressing XIAP and TGF-β type I receptor. XIAP or
Biological activity transmitted via the TGF-β type I receptor may be detected and / or measured. XIAP and TG
In a method for screening a substance that inhibits binding to F-β type II receptor, a test sample is introduced and / or contacted with cells expressing XIAP and TGF-β type II receptor, and then XIAP or TGF-β type II The biological activity transmitted via the receptor may be detected and / or measured.

In the above method, TAB1 and XIAP or XIAP
And TGF-β type I or type II receptors are expressed in the cells used. In the examples described below, TAB1
And XIAP were found to enhance TAB1 and TAK1-mediated biological activities. Also, XIAP
And TGF-β type I and type II receptors were found to bind. Therefore, TAB1 and XIAP or XIAP
And TGF-β type I or type II receptor inhibitors inhibit signaling through XIAP biological activity. Via TAB1 and TGF-β receptor
It is already known to transmit the biological activity of TGF-β. Therefore, by inhibiting the binding between XIAP and TAB1 and XIAP or TGF-β type I or type II receptor, and by inhibiting the signaling of TGF-β through XIAP, TGF-β
The biological activity of β is inhibited.

TAB1 and XIAP and TGF-β type I and type I
Cells expressing the I receptor are preferably TAB1 and XIAP and TGF-β types
It can be prepared by introducing DNAs encoding I and type II receptors. Also, TGF-β type I
Alternatively, the gene encoding TAB1 and the gene expressing XIAP may be introduced into cells expressing the type II receptor by a commonly used method.

Furthermore, TAB1 and / or XIAP and / or TGF-
Cells expressing β type I and type II receptors are
It can be produced by genetic engineering. For example, TGF-β
TAB1 in cells expressing type I and type II receptors
May be introduced by a commonly used method. Alternatively, the genes encoding TAB1 or TAB1 and XIAP may be introduced into cells that do not express the TGF-β type I and type II receptors by a commonly used method. In the examples described later, since the biological activity of TGF-β is transmitted via XIAP, TAB1 and XIAP and TGF-β
It becomes possible to detect or measure TGF-β signal transduction via type I and type II receptors. TAB
1, Genes encoding XIAP, TGF-β type I or type II receptors can be obtained by the methods described herein above.

As the cells expressing TAB1, XIAP and / or TGF-β receptor or TAB1 and TGF-β receptor, any cells can be used as long as they can be used in the present invention. Cells that can be used in the present invention include prokaryotic cells and eukaryotic cells. Prokaryotic cells include bacterial cells and the like. Eukaryotic cells include mammalian cells, insect cells, yeast cells, and the like. The biological activity of TGF-β is enhanced by extracellular matrix protein production, cell growth inhibition,
Monocyte migration, physiologically active substance-inducing, immunosuppressive,
β-amyloid protein deposition. Therefore, TG
To detect the biological activity of F-β, these effects may be detected or measured.

In order to detect or measure these effects in order to detect the biological activity of TGF-β, it is possible to use a cell line or the like which is known to express these effects. it can. For example, human fibrosarcoma cell line HT1080 cells or the like can be used to detect or measure the effect of enhancing extracellular matrix protein production. To detect or measure the cell growth inhibitory effect, the mink lung epithelial cell line Mv1L
u cells and the like can be used. Monocytes and the like can be used to detect or measure monocyte migration. For detecting or measuring the physiologically active substance-inducing action, mononuclear cells or the like can be used. For detecting or measuring the immunosuppressive action, mononuclear cells and the like can be used. To detect or measure the action of depositing β-amyloid protein, use TGF-β
And transgenic mice that overexpress β-amyloid (primary cultured cells derived therefrom).

[0136] These cell lines are labeled with TAB1, XIAP or XIAP.
A gene encoding TAB1, TGF-β type I and / or type II receptor may be introduced, a test sample may be added, and the effect reflecting the biological activity of TGF-β may be detected or measured. The introduction and contact of the test sample may be performed by adding the test sample to the cell culture solution.

Signal transduction via TAB1 can be detected and / or measured by increasing the expression level of a reporter gene that is activated in response to the biological activity of TAB1.
As a reporter gene activated in response to the biological activity of TAB1, PAI-1, fibronectin, type I collagen and type IV collagen can be used. When, for example, fibronectin is used as a reporter gene, Northern analysis may be performed to detect and / or measure its expression level.

The TAB1, XIAP, TGF-β type I or type II receptor expressed in cells may be a fusion protein with another polypeptide. TAB1, XIAP, TGF-β type I or other polypeptide to be subjected to fusion with the type II receptor may be any polypeptide as long as it can be used in the screening method of the present invention, preferably a transcriptional regulator It is.

For example, each subunit of a transcriptional regulator consisting of a heterodimer known to activate transcription of a reporter gene bound to DNA and TAB1
And XIAP are fused, and these are included in an expression vector and introduced into cells. If the test sample does not contain a substance that inhibits the binding between TAB1 and XIAP,
The subunit fused with the AP forms a heterodimer,
And transcription factor consisting of the heterodimer is DNA
Activates the reporter gene. Also, TAB1
If the test sample contains a substance that inhibits the binding of XIAP to TAB1, the binding between TAB1 and XIAP is inhibited, resulting in the inability of the transcriptional regulator subunit to form a heterodimer and activation of the reporter gene. do not do. By examining the change in the expression level of the reporter gene, the target inhibitor can be detected or measured. When examining changes in the expression level of a reporter gene in such a system, two
hybrid system (Fields, S., and Sternglanz, R., Tre
nds. Genet. (1994) 10, 286-292) can be used.

More specifically, the following may be performed. That is, the gene encoding TAB1 and the gene encoding the DNA binding domain of LexA are linked to prepare an expression vector. The gene encoding TAB1 consisting of full length or 1 to 418 positions was transformed into a yeast two-hybrid expression plasmid pBTM116.
(Vojtek, AB, et al., Cell (1993) 74, 205-214)
Insert in such a way that the frame matches, and construct an expression plasmid.

Next, in the full length or SEQ ID NO: 4, 1
An expression vector is prepared by linking a gene encoding XIAP consisting of amino acids from position 326 to a gene encoding GAL4 transcription activation domain. The gene encoding XIAP can be constructed by inserting it into the yeast two-hybrid expression plasmid pGAD10 (manufactured by CLONTECH) so that the frames match.

Yeast L4 incorporating the HIS3 gene whose transcription is regulated by a promoter having a LexA binding motif
After the 0 strain is transformed with each two-hybrid expression plasmid and incubated on a histidine-free synthetic medium, yeast growth is observed only when protein interaction is observed. As described above, the increase in the expression level of the reporter gene can be examined depending on the degree of growth of the transformant, and an inhibitor for the binding between TAB1 and XIAP can be screened.

In Mv1Lu cells, PAI-1 (Plasminogen activator inhibitor type) was stimulated by TGF-β1.
The expression of 1) increases. XIAP and TAB1 and / or TAK1 to Mv1
What is necessary is just to make it express in a Lu cell, and examine the influence of TGF-β1 stimulation on the expression of PAI-1. XIAP expression vector, TAB1
And / or TAK1 expression vector is EcoRI and No. of pCOS1.
A gene encoding XIAP, TAB1, and / or TAK1 may be inserted into the tI restriction enzyme site to construct an expression plasmid.

A reporter gene construct containing a luciferase gene controlled by a TGF-β responsive element derived from the PAI-1 gene was added to cells into which each expression plasmid was introduced into Mv1Lu cells and control cells into which pCOS1 containing no inserted gene had been introduced. p3TP-Lux; Carcamo, J. et al.,
Mol. Cell. Biol., (1994) 14, 3810-3821)
Culture in 10 ng / ml TGF-β1 or medium without TGF-β1, respectively. The luciferase activity in the cell extract is measured.

In cells to which no test sample was added, luciferase activity was increased by the addition of TGF-β1, whereas in cells to which the test sample was added, luciferase activity was not increased. That is, the inhibitor for the binding between TAB1 and XIAP contained in the test sample inhibits the increase in the expression level of PAI-1 induced by TGF-β1 stimulation. In this manner, an increase in the expression level of the reporter gene can be examined, and an inhibitor for the binding between TAB1 and XIAP can be screened.

TAB1 and XIAP obtained by using the screening method of the present invention, or XIAP and TGF-β type I or type I
A binding inhibitor for binding to the I receptor is a test compound using a screening method, such as a peptide, protein, non-peptidic compound, synthetic compound, microbial fermentation product, marine organism extract, plant extract, cell extract,
It can be obtained by screening an animal tissue extract. These test compounds may be novel compounds or known compounds.

These binding inhibitors are compounds that inhibit the binding of TAB1 to XIAP or XIAP to TGF-β type I and / or type II receptor. TAB1 and XIAP obtained using the screening method of the present invention, or XIAP and TGF-
TAB1 and XIAP, which are obtained by using the screening method of the present invention, may also be compounds obtained by adding, deleting or substituting a part of the structural formula of a binding inhibitor for binding to β type I and / or type II receptor, Or XIAP and TGF-β
Included in binding inhibitors for binding to type I and / or type II receptors.

TAB1 and XIAP, or XIAP and TGF-β type I or type II, obtained by the screening method of the present invention.
The binding inhibitor for binding to the receptor can also be a substance that activates TGF-β signaling, or TG
It may be a substance that suppresses F-β signaling. TGF-
β is known to have an extracellular matrix protein production enhancing action, a cell growth inhibiting action, a monocyte migration action, a physiologically active substance-inducing action, and an immunosuppressive action. TGF-β receptor and XIAP and TAB1 bind to each other,
Responsible for β signaling system. Therefore, TAB1 and XIAP obtained by the screening method of the present invention, or XIAP and
A substance that inhibits binding to the TGF-β type I or type II receptor can be obtained as a substance that activates or suppresses TGF-β signal transduction.

TAB1 and XIAP obtained by using the screening method of the present invention, or XIAP and TGF-β type I or type I
Human and mammals, such as mice, rats, guinea pigs, rabbits,
When used as a medicament for chickens, cats, dogs, sheep, pigs, cows, monkeys, baboons, and chimpanzees, it can be carried out according to generally known methods.

Parenteral, for example, orally, if necessary, as capsules or microcapsules, or as a sterile solution or suspension in water or other pharmaceutically acceptable liquid. Can be used For example, TAB1
XIAP or TGF-β alone or physiologically recognized binding inhibitor for binding to type I or type II receptor,
It can be prepared by mixing with excipients, vehicles, preservatives, stabilizers and anti-adsorption agents in the unit dosage form required for generally accepted formulations. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.

Examples of additives that can be mixed with tablets and capsules include gelatin, HSA (human serum albumin), crystalline cellulose, alginic acid, magnesium stearate, sucrose, and lactose. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol and D-sorbitol.
Mannose, D-mannitol, sodium chloride, and suitable solubilizers, for example, alcohols, specifically, ethanol, polyalcohols, for example, propylene glycol, polyethylene glycol, nonionic surfactants, for example, polysorbate 80 ^™ , HCO -50, benzyl benzoate, benzyl alcohol, phosphate buffer,
It may be used in combination with sodium acetate buffer, benzalkonium chloride, procaine hydrochloride, benzyl alcohol, and phenol.

TAB1 and XIAP or TGF-β type I or type I
The dose of the binding inhibitor for the binding of the I receptor is
Although there is a difference depending on the symptoms, in the case of oral administration, in general, for an adult (assuming a body weight of 60 kg), about 0.1 mg / day is used.
1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, and administration method. 0.01 to 30 mg, preferably about 0.1 to 20
Conveniently, mg, more preferably on the order of about 0.1 to 10 mg, will be administered by intravenous injection. For other animals,
The dose can be administered in terms of weight per 60 kg. Hereinafter, the present invention will be described in more detail with reference to Examples, but is not intended to limit the scope of the present invention.

[0153]

[Example] TAB1-binding molecule involved in TGF-β signaling
Example 1 Identification and Analysis of XIAP Example 1 Isolation of TAB1 Binding Molecule XIAP Each factor belonging to the TGF-β superfamily exerts various activities in cell proliferation, differentiation and morphogenesis. Each member of the TGF-β superfamily transmits signals through a heteromeric receptor complex consisting of type I and type II Ser / Thr kinase receptors.
After the ligand binds to the type II receptor, the type II receptor phosphorylates the type I receptor and activates the type I receptor to transmit a signal (Wrana, JL, et al., Natur
e, 370, 341-347, 1994).

Recently, two proteins that function in the TGF-β superfamily signaling pathway, TAK1 (Yamag
uchi, K., et al., Science, 270, 2008-2011, 1995)
And TAB1 were identified (Shibuya, H., et al., Scien
ce, 272, 1179-1182, 1996). TAK1 is a member of the MAP kinase kinase kinase (MAPKKK) family,
TAB1 functions as an activator of TAK1.

However, the mechanism of action that links the TGF-β receptor with TAK1 activation is still unclear, and it is presumed that further signaling molecules are involved. TA
The C-terminal 68 amino acid portion of B1 [TAB1 (437-504)] is more than sufficient to bind to and activate TAK1 (Shib
uya, H., et al., Science, 272, 1179 -1182, 199
6) It is possible that the N-terminal region of TAB1 interacts with other signal transduction system components and regulates signal transduction. Furthermore, truncated TAB1 [TAB1 (1-418)], which lacks the TAK1 binding domain at the C-terminus of TAB1, functions as a dominant negative inhibitor of TGF-β response,
It has been shown to inhibit β-induced gene expression and TAK1 activation (Shibuya H., et al., Science, 272, 1179-118).
2, 1996; Shirakabe, K., et al., J. Biol. Chem. 27
2, 8141-8144, 1997). Therefore, it is considered that the N-terminal region of TAB1 is involved in TGF-β signal transduction via protein-protein interaction.

Therefore, in order to identify a protein that functions upstream of TAB1-TAK1 in the TGF-β signal transduction pathway, a molecule that interacts with TAB1 was isolated using the yeast two-hybrid method. Using TAB1 (1-418) lacking the C-terminal as a bait, a human brain cDNA library (CLONTECH
Was screened. That is, the TAB1 (1-418) expression vector is an expression vector pBTM116 (Vojtek, A. et al.) Containing a gene encoding the DNA binding domain (DBD) of LexA.
B., et al., Cell, 74, 205-214, 1993) by inserting the gene encoding TAB1 (1-418) in frame.
It is constructed to be expressed as a fusion protein with the GAL4 transcription activation domain (GAD). These TAB1 (1-418)
The yeast L40 strain was transformed using an expression vector and a cDNA library containing 1 × 10 ⁶ clones, and cultured on a histidine-deficient medium. The L40 strain contains the HIS3 gene downstream of the promoter containing the LexA binding site, and can grow on a histidine-free medium only when the interaction of the two molecules is observed. As a result, 15 positive clones were obtained. Was.
Of these 15 clones, 5 belong to the IAP family, XIAP (also known as MIHA or ILP (Lis
ten, P., et al., Nature 379, 349-353, 1996; Uren,
AG, et al., Proc. Natl. Acad. Sci. USA 93, 4974-
4978, 1996; Duckett, CS, et al., EMBO J. 15, 268
5 1996).

Example 2 Confirmation of Binding Specificity between TAB1 and IAP Protein The IAP protein was first identified in baculovirus, and is conserved between eukaryotic cells during evolution.
Baculovirus IAP repeat (BIR) characteristic of N-terminal
And a RING zinc finger domain at the C-terminus.
IAP proteins in animal cells include c-IAP1 and c-IAP in addition to XIAP.
2 are known, all of which have three BIR motifs and one RING finger motif (Clem, RJ
and Duckett, CS TIBTECH, 7, 337-339, 1997) (Fig.
1). c-IAP1 and c-IAP2 have 73% amino acid identity, whereas XIAP shows only 43% identity to either c-IAP1 or c-IAP2. Also cI
AP1 and c-IAP2 have been shown to bind to tumor necrosis factor (TNF) receptor binding factor 1 (TRAF1) and TRAF2 via the BIR motif, but XIAP does not interact with either (Rothe, M., etal., Cell, 83, 1243-1252 1995;
Roy, N., et al., EMBO J., 16, 6914-6925, 1997). Therefore, the binding specificity between TAB1 and IAP protein was clarified using a two-hybrid method.

As a TAB1 expression vector, pAL expression as a fusion protein with the transcriptional activation domain (GAD) of GAL4
GAD-TAB1 (Shibuya, H., et al., Science, 272, 1179-
1181, 1996) and c-IAP1 (MIHB), c-IAP2
(MIHC), and XIAP (MIHA) and DNA binding domain (DB
D) fusion protein expression vector (Uren, A., et a
l., Proc. Natl. Acad. Sci. USA, 93, 4974-4978, 199
6) was used. The yeast HF7c strain was transformed with pGAD-TAB1 and one of the IAP expression vectors, and growth on a histidine-deficient medium was examined to confirm the presence or absence of each interaction.

As a result, only the yeast transformed with each of the expression vectors of TAB1 and XIAP grew on a histidine-deficient medium, and an interaction between TAB1 and XIAP was observed. However, no interaction was observed between TAB1 and c-IAP1 or c-IAP2 (FIG. 2). Therefore, it was shown that TAB1 specifically interacts only with XIAP. Moreover, XIAP (1-P) lacking the C-terminal RING finger domain among the clones isolated in the two-hybrid screening
326), the N-terminal region of XIAP, probably B
It was suggested that TAB1 interacts via IR motif. Similarly, c-IAP1 and c-IAP2 have their N-terminal BIR
It is known to associate with TRAF1 and TRAF2 via a motif, suggesting a functional difference between the BIR domains of the IAP protein. Above, the BIR motif is IAP
It probably plays an important role in regulating signal transduction through the.

Example 3 Interaction between TAB1 and XIAP in Animal Cells Next, whether or not XIAP could interact with TAB1 in animal cells was examined. N-terminal Flag tag (SEQ ID NO: 9) and TAB
1 fusion protein (Flag-TAB1) expression vector
The cDNA encoding TAB1 was transformed into the vector pFLAG-CMV-2 (Kodak
Was constructed. In addition, M
The XIAP (Myc-XIAP) expression vector to which the yc tag (SEQ ID NO: 10) is fused is a vector that encodes a full-length XIAP-encoding cDNA.
pCS2MT (Rupp, RA et al., Genes Dev. (1994) 8, 13
11-1323). The Myc-XIAP and Flag-TAB1 expression vectors were each introduced into human embryonic kidney-derived cell line 293 cells, and the respective fusion proteins were transiently expressed. Next, a cell extract was prepared from the cells, and immunoprecipitated using an anti-Flag monoclonal antibody M2 (manufactured by Kodak). After washing the immune complex, it was separated by SDS polyacrylamide gel electrophoresis and transferred to a PVDF membrane. Myc-XIAP contained in the immunoprecipitate was detected using an anti-Myc monoclonal antibody 9E10 (Boehringer Mannheim). The expression of Myc-XIAP and Flag-TAB1 was confirmed by immunoblotting the cell extract directly with the 9E10 antibody and immunoblotting the immunoprecipitate with the M2 antibody with the M2 antibody, respectively. As a result, XIAP was co-precipitated with TAB1, and it was revealed that XIAP interacts with TAB1 in animal cells as well as in yeast (FIG. 3).

Example 4 Confirmation of Interaction between XIAP and TGF-β Receptor Signaling of TGF-β was confirmed by TGF-β type I (TβR-I) and type II (TβR-I) capable of forming a heteromeric complex. TβR-
II) It was known that the receptor was mediated, and it was examined whether XIAP could associate with these TGF-β receptors. Influenza agglutinin (HA) tag fused
TβR-I (HA-TβR-I) (Hoodless, PA, Cell, 85, 489
-500, 1996), TβR-II fused to Myc tag (Myc-TβR-I
I) (Chen, RH, et al., Nature, 377, 548-552, 199
5) The expression vector encoding XIAP and / or XIAP was introduced into 293 cells, and the respective proteins were transiently expressed. Then, the cells were cultured in a medium containing or not containing 3 ng / ml human TGF-β1.
Treated for 0 minutes. A cell extract was prepared from these cells, and an antibody (X) against XIAP or control mouse I
Immunoprecipitation was performed using gG (C). The monoclonal antibody against XIAP was composed of full-length XIAP and glutathione-S
-A fusion protein with transferase was prepared by immunization. After washing, the immune complex was separated by SDS polyacrylamide gel electrophoresis. This was transferred to a PVDF membrane, and then immunoblotted using an anti-HA polyclonal antibody Y11 (manufactured by Santa Cruz) and an anti-Myc antibody 9E11 to detect co-precipitated HA-TβRI and TβRII, respectively. In addition, the cell extract was directly subjected to immunoblotting, and the presence or absence of expression of each protein was confirmed. In the absence of the XIAP expression vector, no interaction between XIAP and the TGF-β receptor was detected. In contrast, in cells into which the XIAP expression vector has been introduced, TβR-I and TβR-II have X
Although co-precipitation with IAP was clearly detected, no difference was observed between cells treated with TGF-β and cells not treated (FIG. 4). Therefore, under overexpression conditions, XIAP was thought to interact constitutively with the TGF-β receptor complex. In addition, from the above results, XIAP binds to TGF-β receptor and TAB1-TAK1,
It may be involved in -β signaling pathway.

Example 5 X in the TGF-β signaling system
Examination of the effects of IAP Plasminogen activator inhibitor 1 (PAI-
1) Reporter gene construct (p3TP-Lux) containing luciferase gene controlled by TGF-β responsive element derived from gene promoter region (Carcamo, J., et.
al., Mol. Cell. Biol., 14, 3810-3821, 1994) was used to examine the effect of XIAP on TGF-β signaling in animal cells.

First, p3TP-Lux and TAK1, TAB1 and XIAP expression vectors were combined in various combinations with the mink lung epithelial cell line Mv1L.
u cells were introduced. The total amount of DNA used for transfection with an empty expression vector containing no cDNA was 5 mg.
And constant. These cells were then transformed with 2 ng / ml human TG.
The cells were cultured for 20 hours in the presence or absence of F-β1. afterwards,
A cell extract was prepared, and the luciferase activity contained therein was measured. The transfection efficiency in each experiment was standardized by the expression amount of β-galactosidase in an expression vector (Act-LacZ) in which a LacZ gene was linked downstream of the β-actin promoter.

In Mv1Lu cells, only a low basal level of luciferase activity was observed in the absence of TGF-β, and luciferase activity was strongly induced by the addition of TGF-β. Further, even when the TAB1 and XIAP expression vectors were introduced, the transcription level from p3TP-Lux did not change at all with Mv1Lu cells. In contrast, TAK1 and TAB1
Alternatively, in cells into which the XIAP expression vector was simultaneously introduced, TG
An increase in luciferase activity in the absence of F-β was confirmed. Furthermore, co-expression with TAK1, TAB1, and XIAP induced strong luciferase independent of TGF-β (FIG. 5). Furthermore, the human skin keratinocyte cell line HaCat
A similar ligand-independent response was clearly observed in the cells.

Next, R-1B / Mv1Lu-derived R-1B /
L17 cells (Carcamo, J., et al., Mol. Cell. Biol., 1
4, 3810-3821, 1994) to investigate the effect of XIAP on TGF-β signaling. As described above, p3TP-Lux and each expression plasmid were introduced into R-1B / L17 cells, and their luciferase activities were measured. First, active or inactive TβR-I was expressed in R-1B / L17 cells, and luciferase activity in those cells was measured. T at position 204 of TβR-I
It is known that the mutant TβR-I (T204D) in which hr is replaced with Asp exhibits constitutive kinase activity and can transmit its signal even in the absence of TGF-β (Weiser, R., et al.,
EMBO J., 14, 2199-2208, 1995), TβR in R-1B / L17 cells
-I (T204D) expression vector was introduced, and TβR-I (T2
When 04D) was expressed, transcription of the p3TP-Lux reporter gene was induced, and luciferase activity was enhanced (FIG. 6A). In addition, the mutant TβR-I (K232R) in which Lys at position 232 of TβR-I was substituted with Arg did not show kinase activity (Weiser,
R., et al., EMBO J., 14, 2199-2208, 1995), TβR-I
Even when the (K232R) expression vector was introduced (FIG. 6KR), only the same level of luciferase activity was exhibited as when the empty expression vector was introduced (FIG. 6V). Thus, TβR
It is possible to transmit a TGF-β signal by artificially introducing a signal into R-1B / L17 cells lacking -I. Next, as a result of introducing the XIAP expression vector together with TAK1 and TAB1, these proteins were expressed simultaneously.
It was found to induce 3TP promoter activity. These results suggest that XIAP functions as a positive regulator of the TGF-β response.

As described above, XIAP was identified as a binding molecule to the TGF-β receptor and TAB1. The above results indicate that XIAP is TGF-
This suggests that XIAP acts as an adapter protein that promotes the recruitment of TAB1 to the β receptor.
Function in the TGF-β signaling pathway, and
It has been clarified that the molecule acts as a mediator connecting the receptor and TAB1-TAK1.

[0167]

According to the screening method of the present invention, TAB1
And XIAP, or XIAP and TGF-β type I and / or type II
It has been clarified that screening for a receptor binding inhibitor is possible. The screening method of the present invention is useful for screening for a binding inhibitor between TAB1 and XIAP or between XIAP and TGF-β type I and / or type II receptor. Obtained by the screening method of the present invention
An inhibitor of the binding between TAB1 and XIAP or XIAP and TGF-β type I and / or type II receptor is useful as a medicament.

[0168]

[Sequence List] SEQ ID NO: 1 Sequence length: 1515 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA Sequence characteristics Characteristic symbol: CDS Location: 1. .1512 Sequence ATG GCG GCG CAG AGG AGG AGC TTG CTG CAG AGT GAG CAG CAG CCA AGC 48 Met Ala Ala Gln Arg Arg Ser Leu Leu Gln Ser Glu Gln Gln Pro Ser 1 5 10 15 TGG ACA GAT GAC CTG CCT CTC TGC CAC CTC TCT GGG GTT GGC TCA GCC 96 Trp Thr Asp Asp Leu Pro Leu Cys His Leu Ser Gly Val Gly Ser Ala 20 25 30 TCC AAC CGC AGC TAC TCT GCT GAT GGC AAG GGC ACT GAG AGC CAC CCG 144 Ser Asn Arg Ser Tyr Ser Ala Asp Gly Lys Gly Thr Glu Ser His Pro 35 40 45 CCA GAG GAC AGC TGG CTC AAG TTC AGG AGT GAG AAC AAC TGC TTC CTG 192 Pro Glu Asp Ser Trp Leu Lys Phe Arg Ser Glu Asn Asn Cys Phe Leu 50 55 60 TAT GGG GTC TTC AAC GGC TAT GAT GGC AAC CGA GTG ACC AAC TTC GTG 240 Tyr Gly Val Phe Asn Gly Tyr Asp Gly Asn Arg Val Thr Asn Phe Val 65 70 75 80 GCC CAG CGG CTG TCC GCA GAG CTC CTG CTG GGC CAG CTG AAT GCC GAG 288 Ala Gln Arg Leu Ser Ala Glu Leu Leu Leu Gly Gln Leu Asn Ala Glu 85 90 95 CAC GCC GAG GCC GAT GTG CGG CGT GTG CTG CTG CAG GCC TTC GAT GTG 336 His Ala Glu Ala Asp Val Arg Arg Val Leu Leu Gln Ala Phe Asp Val 100 105 110 GTG GAG AGG AGC TTC CTG GAG TCC ATT GAC GAC GCC TTG GCT GAG AAG 384 Val Glu Arg Ser Phe Leu Glu Ser Ile Asp Asp Ala Leu Ala Glu Lys 115 120 125 GCA AGC CTC CAG TCG CAA TTG CCA GAG GGA GTC CCT CAG CAC CAG CTG 432 Ala Ser Leu Gln Ser Gln Leu Pro Glu Gly Val Pro Gln His Gln Leu 130 135 140 CCT CCT CAG TAT CAG AAG ATC CTT GAG AGA CTC AAG ACG TTA GAG AGG 480 Pro Pro Gln Tyr Gln Lys Ile Leu Glu Arg Leu Lys Thr Leu Glu Arg 145 150 155 160 GAA ATT TCG GGA GGG GCC ATG GCC GTT GTG GCG GTC CTT CTC AAC AAC 528 Glu Ile Ser Gly Gly Ala Met Ala Val Val Ala Val Leu Leu Asn Asn 165 170 175 AAG CTC TAC GTC GCC AAT GTC GGT ACA AAC CGT GCA CTT TTA TGC AAA 576 Lys Leu Tyr Val Ala Asn Val Gly Thr Asn Arg Ala Leu Leu Cys Lys 180 185 190 TCG ACA GTG GAT GGG TTG CAG GTG ACA CAG CTG AAC GTG GAC CAC ACC 624 Ser Thr Val Asp Gly Leu Gln Val Thr Gln Leu Asn Val Asp His Thr 195 200 205 ACA GAG AAC GAG GAT GAG CTC TTC CGT CTT TCG CAG CTG GGC TTG GAT 672 Thr Glu Asn Glu Asp Glu Leu Phe Arg Leu Ser Gln Leu Gly Leu Asp 210 215 220 GCT GGA AAG ATC AAG CAG GTG GGG ATC ATC TGT GGG CAG GAG AGC ACC 720 Ala Gly Lys Ile Lys Gln Val Gly Ile Ile Cys Gly Gln Glu Ser Thr 225 230 235 240 CGG CGG ATC GGG GAT TAC AAG GTT AAA TAT GGC TAC ACG GAC ATT GAC 768 Arg Arg Ile Gly Asp Tyr Lys Val Lys Tyr Gly Tyr Thr Asp Ile Asp 245 250 255 CTT CTC AGC GCT GCC AAG TCC AAA CCA ATC ATC GCA GAG CCA GAA ATC 816 Leu Leu Ser Ala Ala Lys Ser Lys Pro Ile Ile Ala Glu Pro Glu Ile 260 265 270 CAT GGG GCA CAG CCG CTG GAT GGG GTG ACG GGC TTC TTG GTG CTG ATG 864 His Gly Ala Gln Pro Leu Asp Gly Val Thr Gly Phe Leu Val Leu Met 275 280 285 TCG GAG GGG TTG TAC AAG GCC CTA GAG GCA GCC CAT GGG CCT GGG CAG 912 Ser Glu Gly Leu Tyr Lys Ala Leu Glu Ala Ala His Gly Pro Gly Gln 290 295 300 GCC AAC CAG GAG ATT GCT GCG ATG ATT GAC ACT GAG TTT GCC AAG CAG 960 Ala Asn Gln Glu Ile Ala Ala Met Ile Asp Thr Glu Phe Ala Lys Gln 305 310 315 320 ACC TCC CTG GAC GCA GTG GCC CAG GCC GTC GTG GAC CGG GTG AAG CGC 1008 Thr Ser Leu Asp Ala Val Ala Gln Ala Val Val Asp Arg Val Lys Arg 325 330 335 ATC CAC AGC GAC ACC TTC GCC AGT GGT GGG GAG CGT GCC AGG TTC TGC 1056 Ile His Ser Asp Thr Phe Ala Ser Gly Gly Glu Arg Ala Arg Phe Cys 340 345 350 CCC CGG CAC GAG GAC ATG ACC CTG CTA GTG AGG AAC TTT GGC TAC CCG 1104 Pro Arg His Glu Asp Met Thr Leu Leu Val Arg Asn Phe Gly Tyr Pro 355 360 365 CTG GGC GAA ATG AGC CAG CCC ACA CCG AGC CCA GCC CCA GCT GCA GGA 1152 Leu Gly Glu Met Ser Gln Pro Thr Pro Ser Pro Ala Pro Ala Ala Gly 370 375 380 GGA CGA GTG TAC CCT GTG TCT GTG CCA TAC TCC AGC GCC CAG AGC ACC 1200 Gly Arg Val Tyr Pro Val Ser Val Pro Tyr Ser Ser Ala Gln Ser Thr 385 390 395 400 AGC AAG ACC AGC GTG ACC CTC TCC CTT GTC ATG CCC TCC CAG GGC CAG 1248 Ser Lys Thr Ser Val Thr Leu Ser Leu Val Met Pro Ser Gln Gly Gln 405 410 415 ATG GTC AAC GGG GCT C AC AGT GCT TCC ACC CTG GAC GAA GCC ACC CCC 1296 Met Val Asn Gly Ala His Ser Ala Ser Thr Leu Asp Glu Ala Thr Pro 420 425 430 ACC CTC ACC AAC CAA AGC CCG ACC TTA ACC CTG CAG TCC ACC AAC ACG 1344 Thr Leu Thr Asn Gln Ser Pro Thr Leu Thr Leu Gln Ser Thr Asn Thr 435 440 445 CAC ACG CAG AGC AGC AGC TCC AGC TCT GAC GGA GGC CTC TTC CGC TCC 1392 His Thr Gln Ser Ser Ser Ser Ser Ser Asp Gly Gly Leu Phe Arg Ser 450 455 460 CGG CCC GCC CAC TCG CTC CCG CCT GGC GAG GAC GGT CGT GTT GAG CCC 1440 Arg Pro Ala His Ser Leu Pro Pro Gly Glu Asp Gly Arg Val Glu Pro 465 470 470 475 480 TAT GTG GAC TTT GCT GAG TTT TAC CGC CTC TGG AGC GTG GAC CAT GGC 1488 Tyr Val Asp Phe Ala Glu Phe Tyr Arg Leu Trp Ser Val Asp His Gly 485 490 495 GAG CAG AGC GTG GTG ACA GCA CCG TAG 1515 Glu Gln Ser Val Val Thr Ala Pro 500

SEQ ID NO: 2 Sequence Length: 504 Sequence Type: Amino Acid Topology: Linear Sequence Type: Protein Sequence Met Ala Ala Gln Arg Arg Ser Leu Leu Gln Ser Glu Gln Gln Pro Ser 1 5 10 15 Trp Thr Asp Asp Leu Pro Leu Cys His Leu Ser Gly Val Gly Ser Ala 20 25 30 Ser Asn Arg Ser Tyr Ser Ala Asp Gly Lys Gly Thr Glu Ser His Pro 35 40 45 Pro Glu Asp Ser Trp Leu Lys Phe Arg Ser Glu Asn Asn Cys Phe Leu 50 55 60 Tyr Gly Val Phe Asn Gly Tyr Asp Gly Asn Arg Val Thr Asn Phe Val 65 70 75 80 Ala Gln Arg Leu Ser Ala Glu Leu Leu Leu Gly Gln Leu Asn Ala Glu 85 90 95 His Ala Glu Ala Asp Val Arg Arg Val Leu Leu Gln Ala Phe Asp Val 100 105 110 Val Glu Arg Ser Phe Leu Glu Ser Ile Asp Asp Ala Leu Ala Glu Lys 115 120 125 Ala Ser Leu Gln Ser Gln Leu Pro Glu Gly Val Pro Gln His Gln Leu 130 135 140 Pro Pro Gln Tyr Gln Lys Ile Leu Glu Arg Leu Lys Thr Leu Glu Arg 145 150 155 160 Glu Ile Ser Gly Gly Ala Met Ala Val Val Ala Val Leu Leu Asn Asn 165 170 175 Lys Leu Tyr Val Ala Asn Val Gly Thr Asn Arg Ala Leu Leu Cys Lys 180 185 190 Ser Thr Val Asp Gly Leu Gln Val Thr Gln Leu Asn Val Asp His Thr 195 200 205 Thr Glu Asn Glu Asp Glu Leu Phe Arg Leu Ser Gln Leu Gly Leu Asp 210 215 220 Ala Gly Lys Ile Lys Gln Val Gly Ile Ile Cys Gly Gln Glu Ser Thr 225 230 235 240 Arg Arg Ile Gly Asp Tyr Lys Val Lys Tyr Gly Tyr Thr Asp Ile Asp 245 250 255 Leu Leu Ser Ala Ala Lys Ser Lys Pro Ile Ile Ala Glu Pro Glu Ile 260 265 270 270 His Gly Ala Gln Pro Leu Asp Gly Val Thr Gly Phe Leu Val Leu Met 275 280 285 Ser Glu Gly Leu Tyr Lys Ala Leu Glu Ala Ala His Gly Pro Gly Gln 290 295 300 Ala Asn Gln Glu Ile Ala Ala Met Ile Asp Thr Glu Phe Ala Lys Gln 305 310 315 320 Thr Ser Leu Asp Ala Val Ala Gln Ala Val Val Asp Arg Val Lys Arg 325 330 335 Ile His Ser Asp Thr Phe Ala Ser Gly Gly Glu Arg Ala Arg Phe Cys 340 345 350 Pro Arg His Glu Asp Met Thr Leu Leu Val Arg Asn Phe Gly Tyr Pro 355 360 365 Leu Gly Glu Met Ser Gln Pro Thr Pro Ser Pro Ala Pro Ala Ala Gly 370 375 380 380 Gly Arg Val Tyr Pro Val Ser Val Pro Tyr Ser Ser Ala Gln Ser Thr 385 390 395 400 400 Ser Lys Thr Ser Val Thr Leu Ser Leu Val Met Pro Ser Gln Gly Gln 405 410 415 Met Val Asn Gly Ala His Ser Ala Ser Thr Leu Asp Glu Ala Thr Pro 420 425 430 Thr Leu Thr Asn Gln Ser Pro Thr Leu Thr Leu Gln Ser Thr Asn Thr 435 440 445 His Thr Gln Ser Ser Ser Ser Ser Ser Asp Gly Gly Leu Phe Arg Ser 450 455 460 Arg Pro Ala His Ser Leu Pro Pro Gly Glu Asp Gly Arg Val Glu Pro 465 470 475 480 Tyr Val Asp Phe Ala Glu Phe Tyr Arg Leu Trp Ser Val Asp His Gly 485 490 495 Glu Gln Ser Val Val Thr Ala Pro 500

SEQ ID NO: 3 Sequence length: 1659 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA Sequence characteristics Characteristic symbol: CDS Location: 82 .. 1572 Sequence CGGCCGAGCC GATCGCCGCG GGGCAGTTCG GGCCGGCTGT CCTGGCGCGA AAAGGTGGAC 60 AAGTCCTATT TTCAAGAGAA G ATG ACT TTT AAC AGT TTT GAA GGA TCT AAA 111 Met Thr Phe Asn Ser Phe Glu Gly Ser Lys 1 5 10 ACT TGT GTA GATC AGA GTC AGA TGA TGA TGA GAA GAG 159 Thr Cys Val Pro Ala Asp Ile Asn Lys Glu Glu Glu Phe Val Glu Glu 15 20 25 TTT AAT AGA TTA AAA ACT TTT GCT AAT TTT CCA AGT GGT AGT CCT GTT 207 Phe Asn Arg Leu Lys Thr Phe Ala Asn Phe Pro Ser Gly Ser Pro Val 30 35 40 TCA GCA TCA ACA CTG GCA CGA GCA GGG TTT CTT TAT ACT GGT GAA GGA 255 Ser Ala Ser Thr Leu Ala Arg Ala Gly Phe Leu Tyr Thr Gly Glu Gly 45 50 55 GAT ACC GTG CGG TGC TTT AGT TGT CAT GCA GCT GTA GAT AGA TGG CAA 303 Asp Thr Val Arg Cys Phe Ser Cys His Ala Ala Val Asp Arg Trp Gln 60 65 70 TAT GGA GAC TCA GCA G TT GGA AGA CAC AGG AAA GTA TCC CCA AAT TGC 351 Tyr Gly Asp Ser Ala Val Gly Arg His Arg Lys Val Ser Pro Asn Cys 75 80 85 90 AGA TTT ATC AAC GGC TTT TAT CTT GAA AAT AGT GCC ACG CAG TCT ACA 399 Arg Phe Ile Asn Gly Phe Tyr Leu Glu Asn Ser Ala Thr Gln Ser Thr 95 100 105 AAT TCT GGT ATC CAG AAT GGT CAG TAC AAA GTT GAA AAC TAT CTG GGA 447 Asn Ser Gly Ile Gle Asn Gly Gln Tyr Lys Val Glu Asn Tyr Leu Gly 110 115 120 AGC AGA GAT CAT TTT GCC TTA GAC AGG CCA TCT GAG ACA CAT GCA 495 Ser Arg Asp His Phe Ala Leu Asp Arg Pro Ser Glu Thr His Ala Asp 125 130 135 TAT CTT TTG AGA ACT GGG CAG GTT GTA GAT ATA TCA GAC ACC ATA TAC 543 Tyr Leu Leu Arg Thr Gly Gln Val Val Asp Ile Ser Asp Thr Ile Tyr 140 145 150 CCG AGG AAC CCT GCC ATG TAT AGT GAA GAA GCT AGA TTA AAG TCC TTT 591 Pro Arg Asn Pro Ala Met Tyr Ser Glu Glu Ala Arg Leu Lys Ser Phe 155 160 165 170 CAG AAC TGG CCA GAC TAT GCT CAC CTA ACC CCA AGA GAG TTA GCA AGT 639 Gln Asn Trp Pro Asp Tyr Ala His Leu Thr Pro Arg Glu Leu Ala Ser 175 180 185 GCT GGA C TC TAC TAC ACA GGT ATT GGT GAC CAA GTG CAG TGC TTT TGT 687 Ala Gly Leu Tyr Tyr Thr Gly Ile Gly Asp Gln Val Gln Cys Phe Cys 190 195 200 TGT GGT GGA AAA CTG AAA AAT TGG GAA CCT TGT GAT CGT GCC TGG TCA 735 Cys Gly Gly Lys Leu Lys Asn Trp Glu Pro Cys Asp Arg Ala Trp Ser 205 210 215 GAA CAC AGG CGA CAC TTT CCT AAT TGC TTC TTT GTT TTG GGC CGG AAT 783 Glu His Arg Arg His Phe Pro Asn Cys Phe Phe Val Leu Gly Arg Asn 220 225 230 CTT AAT ATT CGA AGT GAA TCT GAT GCT GTG AGT TCT GAT AGG AAT TTC 831 Leu Asn Ile Arg Ser Glu Ser Asp Ala Val Ser Ser Asp Arg Asn Phe 235 240 245 250 CCA AAT TCA ACA AAT CTT CCA AGA AAT CCA TCC ATG GCA GAT TAT GAA 879 Pro Asn Ser Thr Asn Leu Pro Arg Asn Pro Ser Met Ala Asp Tyr Glu 255 260 265 GCA CGG ATC TTT ACT TTT GGG ACA TGG ATA TAC TCA GTT AAC AAG GAG 927 Ala Arg Ile Phe Thr Phe Gly Thr Trp Ile Tyr Ser Val Asn Lys Glu 270 275 280 CAG CTT GCA AGA GCT GGA TTT TAT GCT TTA GGT GAA GGT GAT AAA GTA 975 Gln Leu Ala Arg Ala Gly Phe Tyr Ala Leu Gly Glu Gly Asp Lys Val 285 290 Two 95 AAG TGC TTT CAC TGT GGA GGA GGG CTA ACT GAT TGG AAG CCC AGT GAA 1023 Lys Cys Phe His Cys Gly Gly Gly Leu Thr Asp Trp Lys Pro Ser Glu 300 305 310 GAC CCT TGG GAA CAA CAT GCT AAA TGG TAT CCA GGG TGC AAA TAT CTG 1071 Asp Pro Trp Glu Gln His Ala Lys Trp Tyr Pro Gly Cys Lys Tyr Leu 315 320 325 330 TTA GAA CAG AAG GGA CAA GAA TAT ATA AAC AAT ATT CAT TTA ACT CAT 1119 Leu Glu Gln Lys Gly Gln Glu Tyr Ile Asn Asn Ile His Leu Thr His 335 340 345 TCA CTT GAG GAG TGT CTG GTA AGA ACT ACT GAG AAA ACA CCA TCA CTA 1167 Ser Leu Glu Glu Cys Leu Val Arg Thr Thr Glu Lys Thr Pro Ser Leu 350 355 360 ACT AGA AGA ATT GAT GAT ACC ATC TTC CAA AAT CCT ATG GTA CAA GAA 1215 Thr Arg Arg Ile Asp Asp Thr Ile Phe Gln Asn Pro Met Val Gln Glu 365 370 375 GCT ATA CGA ATG GGG TTC AGT TTC AAG GAC ATT AAG AAA ATA ATG GAG 1263 Ala Ile Arg Met Gly Phe Ser Phe Lys Asp Ile Lys Lys Ile Met Glu 380 385 390 GAA AAA ATT CAG ATA TCT GGG AGC AAC TAT AAA TCA CTT GAG GTT CTG 1311 Glu Lys Ile Gln Ile Ser Gly Ser Asn Tyr Lys Ser Leu Gl u Val Leu 395 400 405 410 GTT GCA GAT CTA GTG AAT GCT CAG AAA GAC AGT ATG CCA GAT GAG TCA 1359 Val Ala Asp Leu Val Asn Ala Gln Lys Asp Ser Met Pro Asp Glu Ser 415 420 425 AGT CAG ACT TCA TTA CAG AAA GAG ATT AGT ACT GAA GAG CAG CTA AGG 1407 Ser Gln Thr Ser Leu Gln Lys Glu Ile Ser Thr Glu Glu Gln Leu Arg 430 435 440 CGC CTG CAA GAG GAG AAG CTT TGC AAA ATC TGT ATG GAT AGA AAT ATT 1455 Arg Leu Gln Glu Glu Lys Leu Cys Lys Ile Cys Met Asp Arg Asn Ile 445 450 455 GCT ATC GTT TTT GTT CCT TGT GGA CAT CTA GTC ACT TGT AAA CAA TGT 1503 Ala Ile Val Phe Val Pro Cys Gly His Leu Val Thr Cys Lys Gln Cys 460 465 470 GCT GAA GCA GTT GAC AAG TGT CCC ATG TGC TAC ACA GTC ATT ACT TTC 1551 Ala Glu Ala Val Asp Lys Cys Pro Met Cys Tyr Thr Val Ile Thr Phe 475 480 485 490 AAG CAA AAA ATT TTT ATG TCT TAATCTAACT CTATAGTAGG CATTAT Gln Lys Ile Phe Met Ser 495 TGTTCTTATT ACCCTGATTG AATGTGTGAT GTGAACTGAC TTTAAGTAAT CAGGATT 1659

SEQ ID NO: 4 Sequence Length: 497 Sequence Type: Amino Acid Topology: Linear Sequence Type: Protein Sequence Met Thr Phe Asn Ser Phe Glu Gly Ser Lys Thr Cys Val Pro Ala Asp 1 5 10 15 Ile Asn Lys Glu Glu Glu Phe Val Glu Glu Phe Asn Arg Leu Lys Thr 20 25 30 Phe Ala Asn Phe Pro Ser Gly Ser Pro Val Ser Ala Ser Thr Leu Ala 35 40 45 Arg Ala Gly Phe Leu Tyr Thr Gly Glu Gly Asp Thr Val Arg Cys Phe 50 55 60 Ser Cys His Ala Ala Val Asp Arg Trp Gln Tyr Gly Asp Ser Ala Val 65 70 75 80 Gly Arg His Arg Lys Val Ser Pro Asn Cys Arg Phe Ile Asn Gly Phe 85 90 95 Tyr Leu Glu Asn Ser Ala Thr Gln Ser Thr Asn Ser Gly Ile Gln Asn 100 105 110 Gly Gln Tyr Lys Val Glu Asn Tyr Leu Gly Ser Arg Asp His Phe Ala 115 120 125 Leu Asp Arg Pro Ser Glu Thr His Ala Asp Tyr Leu Leu Arg Thr Gly 130 135 140 Gln Val Val Asp Ile Ser Asp Thr Ile Tyr Pro Arg Asn Pro Ala Met 145 150 155 160 Tyr Ser Glu Glu Ala Arg Leu Lys Ser Phe Gln Asn Trp Pro Asp Tyr 165 170 175 Ala His Leu Thr Pro Arg Glu Leu Ala Ser Ala Gly Leu Tyr Tyr Thr 180 185 190 Gly Ile Gly Asp Gln Val Gln Cys Phe Cys Cys Gly Gly Lys Leu Lys 195 200 205 Asn Trp Glu Pro Cys Asp Arg Ala Trp Ser Glu His Arg Arg His Phe 210 215 220 Pro Asn Cys Phe Phe Val Leu Gly Arg Asn Leu Asn Ile Arg Ser Glu 225 230 235 240 Ser Asp Ala Val Ser Ser Asp Arg Asn Phe Pro Asn Ser Thr Asn Leu 245 250 255 Pro Arg Asn Pro Ser Met Ala Asp Tyr Glu Ala Arg Ile Phe Thr Phe 260 265 270 270 Gly Thr Trp Ile Tyr Ser Val Asn Lys Glu Gln Leu Ala Arg Ala Gly 275 280 285 285 Phe Tyr Ala Leu Gly Glu Gly Asp Lys Val Lys Cys Phe His Cys Gly 290 295 295 Gly Gly Leu Thr Asp Trp Lys Pro Ser Glu Asp Pro Trp Glu Gln His 305 310 315 320 Ala Lys Trp Tyr Pro Gly Cys Lys Tyr Leu Leu Glu Gln Lys Gly Gln 325 330 335 Glu Tyr Ile Asn Asn Ile His Leu Thr His Ser Leu Glu Glu Cys Leu 340 345 350 Val Arg Thr Thr Glu Lys Thr Pro Ser Leu Thr Arg Arg Ile Asp Asp 355 360 365 Thr Ile Phe Gln Asn Pro Met Val Gln Glu Ala Ile Arg Met Gly Phe 370 375 380 380 Ser Phe Lys Asp IleLys Lys Ile Met Glu Glu Lys Ile Gln Ile Ser 385 390 395 400 400 Gly Ser Asn Tyr Lys Ser Leu Glu Val Leu Val Ala Asp Leu Val Asn 405 410 415 Ala Gln Lys Asp Ser Met Pro Asp Glu Ser Ser Gln Thr Ser Leu Gln 420 425 430 Lys Glu Ile Ser Thr Glu Glu Gln Leu Arg Arg Leu Gln Glu Glu Lys 435 440 445 Leu Cys Lys Ile Cys Met Asp Arg Asn Ile Ala Ile Val Phe Val Pro 450 455 460 Cys Gly His Leu Val Thr Cys Lys Gln Cys Ala Glu Ala Val Asp Lys 465 470 475 480 Cys Pro Met Cys Tyr Thr Val Ile Thr Phe Lys Gln Lys Ile Phe Met 485 490 495 Ser

SEQ ID NO: 5 Sequence length: 2308 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA Sequence characteristics Characteristic symbol: CDS Location: 77. . 1585 sequence GGCGGAGGCGA GGTTTGCTGG GGTGAGGCAG CGG
CGCGGGCC GGGCCGGGC GGGCCACAGGG 60 CGGTGGCGGC GGGACC ATG GAG GCG GCG GT
C GCT GCT CCG CGT CCC CGG 109 Met Glu Ala Ala Va
l Ala Ala Pro Arg Pro Arg 1
5 10 CTG CTC CTC CTC GTG CTG GCG GCG GCG
GCG GCG GCG GCG GCG GCG CTG 157 Leu Leu Leu Leu Val Leu Ala Ala Ala
Ala Ala Ala Ala Ala Ala Leu 15 20
25 CTC CCG GGG GCG ACG GCG TTA CAG TGT
TTC TGC CAC CTC TGT ACA AAA 205 Leu Pro Gly Ala Thr Ala Leu Gln Cys
Phe Cys His Leu Cys Thr Lys 30 35
40 GAC AAT TTT ACT TGT GTG ACA GAT GGG
CTC TGC TTT GTC TCT GTC ACA 253 Asp Asn Phe Thr Cys Val Thr Asp Gly
Leu Cys Phe Val Ser Val Thr 4550
55 GAG ACC ACA GAC AAA GTT ATA CAC AAC
AGC ATG TGT ATA GCT GAA ATT 301 Glu Thr Thr Asp Lys Val Ile His Asn
Ser Met Cys Ile Ala Glu Ile 60 65
70 75 GAC TTA ATT CCT CGA GAT AGG CCG TTT
GTA TGT GCA CCC TCT TCA AAA 349 Asp Leu Ile Pro Arg Asp Arg Pro Phe
Val Cys Ala Pro Ser Ser Lys 80
85 90 ACT GGG TCT GTG ACT ACA ACA TAT TGC
TGC AAT CAG GAC CAT TGC AAT 397 Thr Gly Ser Val Thr Thr Thr Tyr Cys
Cys Asn Gln Asp His Cys Asn 95 100
105 AAA ATA GAA CTT CCA ACT ACT GTA AAG
TCA TCA CCT GGC CTT GGT CCT 445 Lys Ile Glu Leu Pro Thr Thr Val Lys
Ser Ser Pro Gly Leu Gly Pro 110 115
120 GTG GAA CTG GCA GCT GTC ATT GCT GGA
CCA GTG TGC TTC GTC TGC ATC 493 Val Glu Leu Ala Ala Val Ile Ala Gly
Pro Val Cys Phe Val Cys Ile 125 130
135 TCA CTC ATG TTG ATG GTC TAT ATC TGC
CAC AAC CGC ACT GTC ATT CAC 541 Ser Leu Met Leu Met Val Tyr Ile Cys
His Asn Arg Thr Val Ile His 140 145
150 155 CAT CGA GTG CCA AAT GAA GAG GAC CCT
TCA TTA GAT CGC CCT TTT ATT 589 His Arg Val Pro Asn Glu Glu Asp Pro
Ser Leu Asp Arg Pro Phe Ile 160
165 170 TCA GAG GGT ACT ACG TTG AAA GAC TTA
ATT TAT GAT ATG ACA ACG TCA 637 Ser Glu Gly Thr Thr Leu Lys Asp Leu
Ile Tyr Asp Met Thr Thr Ser 175 180
185 GGT TCT GGC TCA GGT TTA CCA TTG CTT
GTT CAG AGA ACA ATT GCG AGA 685 Gly Ser Gly Ser Gly Leu Pro Leu Leu
Val Gln Arg Thr Ile Ala Arg 190 195
200 ACT ATT GTG TTA CAA GAA AGC ATT GGC
AAA GGT CGA TTT GGA GAA GTT 733 Thr Ile Val Leu Gln Glu Ser Ile Gly
Lys Gly Arg Phe Gly Glu Val 205 210
215 TGG AGA GGA AAG TGG CGG GGA GAA GAA
GTT GCT GTT AAG ATA TTC TCC 781 Trp Arg Gly Lys Trp Arg Gly Glu Glu
Val Ala Val Lys Ile Phe Ser 220 225
230 235 TCT AGA GAA GAA CGT TCG TGG TTC CGT
GAG GCA GAG ATT TAT CAA ACT 829 Ser Arg Glu Glu Arg Ser Trp Phe Arg
Glu Ala Glu Ile Tyr Gln Thr 240
245 250 GTA ATG TTA CGT CAT GAA AAC ATC CTG
GGA TTT ATA GCA GCA GAC AAT 877 Val Met Leu Arg His Glu Asn Ile Leu
Gly Phe Ile Ala Ala Asp Asn 255 260
265 AAA GAC AAT GGT ACT TGG ACT CAG CTC
TGG TTG GTG TCA GAT TAT CAT 925 Lys Asp Asn Gly Thr Trp Thr Gln Leu
Trp Leu Val Ser Asp Tyr His 270 275
280 GAG CAT GGA TCC CTT TTT GAT TAC TTA
AAC AGA TAC ACA GTT ACT GTG 973 Glu His Gly Ser Leu Phe Asp Tyr Leu
Asn Arg Tyr Thr Val Thr Thr 285 290
295 GAA GGA ATG ATA AAA CTT GCT CTG TCC
ACG GCG AGC GGT CTT GCC CAT 1021 Glu Gly Met Ile Lys Leu Ala Leu Ser
Thr Ala Ser Gly Leu Ala His 300 305
310 315 CTT CAC ATG GAG ATT GTT GGT ACC CAA
GGA AAG CCA GCC ATT GCT CAT 1069 Leu His Met Glu Ile Val Gly Thr Gln
Gly Lys Pro Ala Ile Ala His 320
325 330 AGA GAT TTG AAA TCA AAG AAT AAT ATC TTG
GTA AAG AAG AAT GGA ACT TGC 1117 Arg Asp Leu Lys Ser Lys Asn Ile Leu
Val Lys Lys Asn Gly Thr Cys 335 340
345 TGT ATT GCA GAC TTA GGA CTG GCA GTA
AGA CAT GAT TCA GCC ACA GAT 1165 Cys Ile Ala Asp Leu Gly Leu Ala Val
Arg His Asp Ser Ala Thr Asp 350 355
360 ACC ATT GAT ATT GCT CCA AAC CAC AGA
GTG GGA ACA AAA AGG TAC ATG 1213 Thr Ile Asp Ile Ala Pro Asn His Arg
Val Gly Thr Lys Arg Tyr Met 365 370
375 GCC CCT GAA GTT CTC GAT GAT TCC ATA
AAT ATG AAA CAT TTT GAA TCC 1261 Ala Pro Glu Val Leu Asp Asp Ser Ile
Asn Met Lys His Phe Glu Ser 380 385
390 395 TTC AAA CGT GCT GAC ATC TAT GCA ATG
GGC TTA GTA TTC TGG GAA ATT 1309 Phe Lys Arg Ala Asp Ile Tyr Ala Met
Gly Leu Val Phe Trp Glu Ile 400
405 410 GCT CGA CGA TGT TCC ATT GGT GGA ATT
CAT GAA GAT TAC CAA CTG CCT 1357 Ala Arg Arg Cys Ser Ile Gly Gly Ile
His Glu Asp Tyr Gln Leu Pro 415 420
425 TAT TAT GAT CTT GTA CCT TCT GAC CCA
TCA GTT GAA GAA ATG AGA AAA 1405 Tyr Tyr Asp Leu Val Pro Ser Asp Pro
Ser Val Glu Glu Met Arg Lys 430 435
440 GTT GTT TGT GAA CAG AAG TTA AGG CCA
AAT ATC CCA AAC AGA TGG CAG 1453 Val Val Cys Glu Gln Lys Leu Arg Pro
Asn Ile Pro Asn Arg Trp Gln 445 450
455 AGC TGT GAA GCC TTG AGA GTA ATG GCT
AAA ATT ATG AGA GAA TGT TGG 1501 Ser Cys Glu Ala Leu Arg Val Met Ala
Lys Ile Met Arg Glu Cys Trp 460 465
470 475 TAT GCC AAT GGA GCA GCT AGG CTT ACA
GCA TTG CGG ATT AAG AAA ACA 1549 Tyr Ala Asn Gly Ala Ala Arg Leu Thr
Ala Leu Arg Ile Lys Lys Thr 480
485 490 TTA TCG CAA CTC AGT CAA CAG GAA GGC
ATC AAA ATG TAATTCTACA 1595 Leu Ser Gln Leu Ser Gln Gln Glu Gly
Ile Lys Met 495 500 GCTTTGCCTG AACTTCCCTT TTTTCTTCAG ATC
TGCTCCT GGGTTTTAAT TTGGGAGGTC 1655 AGTTGTTCTA CCTCACTGAG AGGGAACAGA AGG
ATATTGC TTCCTTTTGC AGCAGTGTAA 1715 TAAAGTCAAT TAAAACTTCCCAGGATTTC TTT
GGACCCA GGAAACAGCC AGTTGGGTCC 1775 TTCTGTGCA CTATGAACGC TTCTTTCCCA GGA
CAGAAAA TGTGTAGTCT ACCTTTATTTT 1835 TTTATTAACA AAACTTGTTTT TTTAAAAAGA TGA
TTGCTGG TCTTAACTTT AGGTAACTCT 1895 GCTGTGCTGG AGATCATCTT TAAGGGCAAA GGA
GTTGGAT TGCTGAATTA CAATGAAACA 1955 TGTCTTATTA CTAAAGAAAAG TGATTTACTC CTG
GTTAGTA CATCTCAGA GGATTCTGAA 2015 CCACTAGAGT TTCCTTGATT CAGACTTTGA ATG
TACTGTTT CTATAGTTTT TCAGGATCTT 2075 AAAACTAACCA CTTATAAAAC TCTTATCTTG AGT
CTAAAAA TGACCTCATA TAGTAGTGAG 2135 GAACATATT CATGCAATTG TATTTTGTAT ACT
ATTATTG TTCTTCACT TATTCGAAC 2195 ATTCATGCC TTCAAAATGG GATTGTACTA TAC
CAGTAAG TGCACTCTCT GTGTCTTTCT 2255 AATGGAATG AGTAGAATTG CTGAAAGTCT CTA
TGTTAAA ACCTATATG TTT 2308

SEQ ID NO: 6 Sequence length: 503 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence Met Glu Ala Ala Val Ala Ala Pro Arg Pro Arg Leu Leu Leu Leu Val 1 5 10 15 Leu Ala Ala Ala Ala Ala Ala Ala Ala Ala Leu Leu Pro Gly Ala Thr 20 25 30 Ala Leu Gln Cys Phe Cys His Leu Cys Thr Lys Asp Asn Phe Thr Cys 35 40 45 Val Thr Asp Gly Leu Cys Phe Val Ser Val Thr Glu Thr Thr Asp Lys 50 55 60 Val Ile His Asn Ser Met Cys Ile Ala Glu Ile Asp Leu Ile Pro Arg 65 70 75 80 Asp Arg Pro Phe Val Cys Ala Pro Ser Ser Lys Thr Gly Ser Val Thr 85 90 95 Thr Thr Tyr Cys Cys Asn Gln Asp His Cys Asn Lys Ile Glu Leu Pro 100 105 110 Thr Thr Val Lys Ser Ser Pro Gly Leu Gly Pro Val Glu Leu Ala Ala 115 120 125 Val Ile Ala Gly Pro Val Cys Phe Val Cys Ile Ser Leu Met Leu Met 130 135 140 Val Tyr Ile Cys His Asn Arg Thr Val Ile His His Arg Val Pro Asn 145 150 155 160 Glu Glu Asp Pro Ser Leu Asp Arg Pro Phe Ile Ser Glu Gly Thr Thr 165 170 175 Leu Lys Asp Leu Ile Tyr Asp Met Thr Thr Ser Gly Ser Gly Ser Gly 180 185 190 Leu Pro Leu Leu Val Gln Arg Thr Ile Ala Arg Thr Ile Val Leu Gln 195 200 205 Glu Ser Ile Gly Lys Gly Arg Phe Gly Glu Val Trp Arg Gly Lys Trp 210 215 220 Arg Gly Glu Glu Val Ala Val Lys Ile Phe Ser Ser Arg Glu Glu Arg 225 230 235 240 Ser Trp Phe Arg Glu Ala Glu Ile Tyr Gln Thr Val Met Leu Arg His 245 250 255 Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp Asn Lys Asp Asn Gly Thr 260 265 270 270 Trp Thr Gln Leu Trp Leu Val Ser Asp Tyr His Glu His Gly Ser Leu 275 280 285 Phe Asp Tyr Leu Asn Arg Tyr Thr Val Val Thr Glu Gly Met Ile Lys 290 295 300 Leu Ala Leu Ser Thr Ala Ser Gly Leu Ala His Leu His Met Glu Ile 305 310 315 320 Val Gly Thr Gly Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser 325 330 335 Lys Asn Ile Leu Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp Leu 340 345 350 Gly Leu Ala Val Arg His Asp Ser Ala Thr Asp Thr Ile Asp Ile Ala 355 360 365 Pro Asn His Arg Val Gly Thr Lys Arg Tyr Met Ala Pro Glu Val Leu 370 375 380 380 Asp Asp Ser Ile AsnMet Lys His Phe Glu Ser Phe Lys Arg Ala Asp 385 390 395 400 400 Ile Tyr Ala Met Gly Leu Val Phe Trp Glu Ile Ala Arg Arg Cys Ser 405 410 415 Ile Gly Gly Ile His Glu Asp Tyr Gln Leu Pro Tyr Tyr Asp Leu Val 420 425 430 Pro Ser Asp Pro Ser Val Glu Glu Met Arg Lys Val Val Cys Glu Gln 435 440 445 Lys Leu Arg Pro Asn Ile Pro Asn Arg Trp Gln Ser Cys Glu Ala Leu 450 455 460 Arg Val Met Ala Lys Ile Met Arg Glu Cys Trp Tyr Ala Asn Gly Ala 465 470 475 480 Ala Arg Leu Thr Ala Leu Arg Ile Lys Lys Thr Leu Ser Gln Leu Ser 485 490 495 Gln Gln Glu Gly Ile Lys Met 500

SEQ ID NO: 7 Sequence length: 2090 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA Sequence characteristics Symbol indicating characteristics: CDS Location: 336 .. 2036 SEQ GTTGGCGAGG AGTTTCCTGT TTCCCCCGCA GCGCTGAGTT GAAGTTGAGT GAGTCACTCG 60 CGCGCACGGA GCGACGACAC CCCCGCGCGT GCACCCGCTC GGGACAGGAG CCGGACTCCT 120 GTGCAGCTTC CCTCGGCCGC CGGGGGCCTC CCCGCGCCTC GCCGGCCTCC AGGCCCCTCC 180 TGGCTGGCGA GCGGGCGCCA CATCTGGCCC GCACATCTGC GCTGCCGGCC CGGCGCGGGG 240 TCCGGAGAGG GCGCGGCGCG GAGCGCAGCC AGGGGTCCGG GAAGGCGCCG TCCGTGCGCT 300 GGGGGCTCGG TCTATGACGA GCAGCGGGGT CTGCC ATG GGT CGG GGG CTG CTC 353 Met Gly Arg Gly Leu Leu 1 5 AGG GGC CTG TGG CCG CTG CAC ATC GTC CTG TGG ACG CGT ATC GCC AGC 401 Arg Gly Leu Trp Pro Leu His Ile Val Leu Trp Thr Arg Ile Ala Ser 10 15 20 ACG ATC CCA CCG CAC GTT CAG AAG TCG GTT AAT AAC GAC ATG ATA GTC 449 Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val 25 30 35 ACT GAC AAC AAC GGT GCA GTC AAG TTT CCA CA A CTG TGT AAA TTT TGT 497 Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys 40 45 50 GAT GTG AGA TTT TCC ACC TGT GAC AAC CAG AAA TCC TGC ATG AGC AAC 545 Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn 55 60 65 70 TGC AGC ATC ACC TCC ATC TGT GAG AAG CCA CAG GAA GTC TGT GTG GCT 593 Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala 75 80 85 GTA TGG AGA AAG AAT GAC GAG AAC ATA ACA CTA GAG ACA GTT TGC CAT 641 Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His 90 95 100 GAC CCC AAG CTC CCC TAC CAT GAC TTT ATT CTG GAA GAT GCT GCT TCT 689 Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser 105 110 115 CCA AAG TGC ATT ATG AAG GAA AAA AAA AAG CCT GGT GAG ACT TTC TTC 737 Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe 120 125 130 ATG TGT TCC TGT AGC TCT GAT GAG TGC AAT GAC AAC ATC ATC TTC TCA 785 Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser 135 140 145 150 GAA GAA TAT AAC ACC AGC AAT CCT GAC TT G TTG CTA GTC ATA TTT CAA 833 Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu Leu Val Ile Phe Gln 155 160 165 GTG ACA GGC ATC AGC CTC CTG CCA CCA CTG GGA GTT GCC ATA TCT GTC 881 Val Thr Gly Ile Ser Leu Leu Pro Pro Leu Gly Val Ala Ile Ser Val 170 175 180 ATC ATC ATC TTC TAC TGC TAC CGC GTT AAC CGG CAG CAG AAG CTG AGT 929 Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn Arg Gln Gln Lys Leu Ser 185 190 195 TCA ACC TGG GAA ACC GGC AAG ACG CGG AAG CTC ATG GAG TTC AGC GAG 977 Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys Leu Met Glu Phe Ser Glu 200 205 210 CAC TGT GCC ATC ATC CTG GAA GAT GAC CGC TCT GAC ATC AGC TCC ACG 1025 His Cys Ala Ile Ile Leu Glu Asp Asp Arg Ser Asp Ile Ser Ser Thr 215 220 225 230 TGT GCC AAC AAC ATC AAC CAC AAC ACA GAG CTG CTG CCC ATT GAG CTG 1073 Cys Ala Asn Asn Ile Asn His Asn Thr Glu Leu Leu Pro Ile Glu Leu 235 240 245 GAC ACC CTG GTG GGG AAA GGT CGC TTT GCT GAG GTC TAT AAG GCC AAG 1121 Asp Thr Leu Val Gly Lys Gly Arg Phe Ala Glu Val Tyr Lys Ala Lys 250 255 260 CTG AAG CAG AAC ACT TC A GAG CAG TTT GAG ACA GTG GCA GTC AAG ATC 1169 Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu Thr Val Ala Val Lys Ile 265 270 270 275 TTT CCC TAT GAG GAG TAT GCC TCT TGG AAG ACA GAG AAG GAC ATC TTC 1217 Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys Thr Glu Lys Asp Ile Phe 280 285 290 TCA GAC ATC AAT CTG AAG CAT GAG AAC ATA CTC CAG TTC CTG ACG GCT 1265 Ser Asp Ile Asn Leu Lys His Glu Asn Ile Leu Gln Phe Leu Thr Ala 295 300 305 310 GAG GAG CGG AAG ACG GAG TTG GGG AAA CAA TAC TGG CTG ATC ACC GCC 1313 Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln Tyr Trp Leu Ile Thr Ala 315 320 325 TTC CAC GCC AAG GGC AAC CTA CAG GAG TAC CTG ACG CGG CAT GTC ATC 1361 Phe His Ala Lys Gly Asn Leu Gln Glu Tyr Leu Thr Arg His Val Ile 330 335 340 AGC TGG GAG GAC CTG CGC AAG CTG GGC AGC TCC CTC GCC CGG GGG ATT 1409 Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser Ser Leu Ala Arg Gly Ile 345 350 355 GCT CAC CTC CAC AGT GAT CAC ACT CCA TGT GGG AGG CCC AAG ATG CCC 1457 Ala His Leu His Ser Asp His Thr Pro Cys Gly Arg Pro Lys Met Pro 360 365 370 ATC GTG CAC AGG GAC CTC AAG AGC TCC AAT ATC CTC GTG AAG AAC GAC 1505 Ile Val His Arg Asp Leu Lys Ser Ser Asn Ile Leu Val Lys Asn Asp 375 380 385 390 CTA ACC TGC TGC CTG TGT GAC TTT GGG CTT TCC CTG CGT CTG GAC CCT 1553 Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu Ser Leu Arg Leu Asp Pro 395 400 405 ACT CTG TCT GTG GAT GAC CTG GCT AAC AGT GGG CAG GTG GGA ACT GCA 1601 Thr Leu Ser Val Asp Asp Leu Ala Asn Ser Gly Gln Val Gly Thr Ala 410 415 420 AGA TAC ATG GCT CCA GAA GTC CTA GAA TCC AGG ATG AAT TTG GAG AAT 1649 Arg Tyr Met Ala Pro Glu Val Leu Glu Ser Arg Met Asn Leu Glu Asn 425 430 435 GCT GAG TCC TTC AAG CAG ACC GAT GTC TAC TCC ATG GCT CTG GTG CTC 1697 Ala Glu Ser Phe Lys Gln Thr Asp Val Tyr Ser Met Ala Leu Val Leu 440 445 450 TGG GAA ATG ACA TCT CGC TGT AAT GCA GTG GGA GAA GTA AAA GAT TAT 1745 Trp Glu Met Thr Ser Arg Cys Asn Ala Val Gly Glu Val Lys Asp Tyr 455 460 465 470 GAG CCT CCA TTT GGT TCC AAG GTG CGG GAG CAC CCC TGT GTC GAA AGC 1793 Glu Pro Pro Phe Gly Ser Lys Val Arg Glu His Pro Cys Val Glu Ser 475 480 485 ATG AAG GAC AAC GTG TTG AGA GAT CGA GGG CGA CCA GAA ATT CCC AGC 1841 Met Lys Asp Asn Val Leu Arg Asp Arg Gly Arg Pro Glu Ile Pro Ser 490 495 500 TTC TGG CTC AAC CAC CAG GGC ATC CAG ATG GTG TGT GAG ACG TTG ACT 1889 Phe Trp Leu Asn His Gln Gly Ile Gln Met Val Cys Glu Thr Leu Thr 505 510 515 GAG TGC TGG GAC CAC GAC CCA GAG GCC CGT CTC ACA GCC CAG TGT GTG 1937 Glu Cys Trp Asp His Asp Pro Glu Ala Arg Leu Thr Ala Gln Cys Val 520 525 530 GCA GAA CGC TTC AGT GAG CTG GAG CAT CTG GAC AGG CTC TCG GGG AGG 1985 Ala Glu Arg Phe Ser Glu Leu Glu His Leu Asp Arg Leu Ser Gly Arg 535 540 545 550 AGC TGC TCG GAG GAG AAG ATT CCT GAA GAC GGC TCC CTA AAC ACT ACC 2033 Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp Gly Ser Leu Asn Thr Thr 555 560 565 565 AAA TAGCTCTTAT GGGGCAGGCT GGGCATGTCC AAAGAGGCTG CCCCTCTCCACA20

SEQ ID NO: 8 Sequence length: 567 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val 20 25 30 Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 35 40 45 Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 50 55 60 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 65 70 75 80 Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 85 90 95 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 100 105 110 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 115 120 125 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 130 135 140 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu 145 150 155 160 Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu 165 170 175 Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn 180 185 190 Arg Gln Gln Lys Leu Ser Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys 195 200 205 Leu Met Glu Phe Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg 210 215 220 Ser Asp Ile Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu 225 230 235 240 Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gly Lys Gly Arg Phe Ala 245 250 255 Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu 260 265 270 Thr Thr Ala Val Lys Ile Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys 275 280 285 Thr Glu Lys Asp Ile Phe Ser Asp Ile Asn Leu Lys His Glu Asn Ile 290 295 300 Leu Gln Phe Leu Thr Ala Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln 305 310 315 320 Tyr Trp Leu Ile Thr Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr 325 330 335 Leu Thr Arg His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser 340 345 350 Ser Leu Ala Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro Cys 355 360 365 Gly Arg Pro Lys Met Pro Ile Val His Arg Asp Leu Lys Ser Ser Asn 370 375 380 Ile Leu Val Lys AsnAsp Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu 385 390 395 400 400 Ser Leu Arg Leu Asp Pro Thr Leu Ser Val Asp Asp Leu Ala Asn Ser 405 410 415 Gly Gln Val Gly Thr Ala Arg Tyr Met Ala Pro Glu Val Leu Glu Ser 420 425 430 Arg Met Asn Leu Glu Asn Ala Glu Ser Phe Lys Gln Thr Asp Val Tyr 435 440 445 Ser Met Ala Leu Val Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val 450 455 460 Gly Glu Val Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu 465 470 475 480 His Pro Cys Val Glu Ser Met Lys Asp Asn Val Leu Arg Asp Arg Gly 485 490 495 Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn His Gln Gly Ile Gln Met 500 505 510 Val Cys Glu Thr Leu Thr Glu Cys Trp Asp His Asp Pro Glu Ala Arg 515 520 525 Leu Thr Ala Gln Cys Val Ala Glu Arg Phe Ser Glu Leu Glu His Leu 530 535 540 Asp Arg Leu Ser Gly Arg Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp 545 550 555 560 Gly Ser Leu Asn Thr Thr Lys 565

SEQ ID NO: 9 Sequence length: 9 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Met Asp Tyr Lys Asp Asp Asp Asp Lys 15 SEQ ID NO: 10 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Leu Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn 1 5 10

[Brief description of the drawings]

FIG. 1 schematically shows a cellular IAP. The positions of the BIR motif (grey squares) and the RING zinc finger domain (black squares) are shown in the schematic diagram of each protein.

FIG. 2 shows the interaction of TAB1 with cellular members of the IAP family in yeast, particularly the protein interaction in a two-hybrid system.

FIG. 3 shows the interaction between XIAP and TAB1 in animal cells.

FIG. 4 shows the interaction between XIAP and TGF-β receptor in animal cells.

FIG. 5 shows the effect of XIAP on TGF-β signaling, particularly the effect of XIAP on 3TP promoter activity. Values are expressed as fold increase compared to luciferase activity obtained in unstimulated cells into which the empty vector was introduced. Values are the average of one representative experiment in which each transfection was performed twice. Standard error is less than 5%.

FIG. 6 shows the effect of XIAP on TGF-β signaling, particularly the effect of XIAP on 3TP promoter activity in R-1B / 17 cells. Values are expressed as fold increase compared to luciferase activity obtained in cells into which the empty vector has been introduced. Values are the average of one representative experiment when each transfection was performed twice. Standard error is less than 5%.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 38/22 ACV C07K 7/06 39/395 ABR 7/08 45/00 AAM 14/47 ABB G01N 33/536 B ADA C C07K 7/06 D 7/08 C12P 21/08 14/47 A61K 37/02 ACD G01N 33/536 ACS ADS AED // C12N 15/09 ZNA 37/24 ACV C12P 21/08 C12N 15/00 ZNAA

Claims (63)

[Claims] 1. A method for screening a substance that inhibits the binding between XIAP and TAB1, by contacting XIAP, TAB1, and a test substance, and then examining the presence or absence of formation of a bond between XIAP and TAB1. 2. TAB1 is a protein having the amino acid sequence shown in SEQ ID NO: 2 from Met at amino acid position 1 to P at amino acid position 504.
An amino acid sequence comprising ro or a biological activity of TAB1, and having an amino acid sequence in which one or more amino acid residues are substituted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 1. 2. The screening method according to 1. 3. XIAP is selected from Met at amino acid position 1 to S at amino acid position 497 of the amino acid sequence shown in SEQ ID NO: 4.
er or an amino acid sequence having the biological activity of XIAP and having an amino acid sequence in which one or more amino acid residues have been substituted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 4. 2. The screening method according to 1. 4. The screening method according to claim 1, wherein TAB1 and / or XIAP is fused to another peptide or polypeptide. 5. The screening method according to claim 1, wherein TAB1 or XIAP is bound to a support. 6. The screening method according to claim 5, wherein the support is a bead or a plate. 7. The screening method according to claim 1, wherein TAB1 or XIAP is labeled, and the presence or absence of a bond between XIAP and TAB1 is determined by detecting or measuring the label. 8. The screening method according to claim 7, wherein the label is a radioisotope, an enzyme or a fluorescent substance. 9. XIAP bound to TAB1 is detected or measured by a primary antibody against XIAP or a primary antibody against another peptide or polypeptide fused to XIAP to examine the presence or absence of formation of a bond between XIAP and TAB1. The screening method according to claim 1. 10. XIAP bound to TAB1 is detected or measured by a primary antibody against XIAP or a primary antibody against another peptide or polypeptide fused with XIAP and a secondary antibody against the primary antibody, whereby XIAP and TAB1 are bound to each other. 10. The screening method according to claim 9, wherein the presence or absence of a bond is examined. 11. The presence or absence of formation of a bond between XIAP and TAB1 by detecting or measuring TAB1 bound to XIAP with a primary antibody against TAB1 or a primary antibody against another peptide or polypeptide fused to TAB1. The screening method according to claim 1. 12. Detecting or measuring TAB1 bound to XIAP with a primary antibody against TAB1 or a primary antibody against another peptide or polypeptide fused to TAB1 and a secondary antibody against the primary antibody, whereby XIAP and TAB1 The screening method according to claim 11, wherein the presence or absence of the formation of a bond is examined. 13. The method according to claim 9, wherein the primary antibody or the secondary antibody is labeled with a radioisotope, an enzyme or a fluorescent substance.
13. The screening method according to any one of claims 12 to 12. 14. A test sample is introduced and / or brought into contact with cells expressing TAB1 and XIAP, and then the biological activity transmitted via TAB1 or XIAP is detected and / or measured, whereby XIAP and TAB1 are detected. A method for screening for a substance that inhibits the binding to a substance. 15. The screening method according to claim 14, wherein the biological activity transmitted via TAB1 or XIAP is TGF-β biological activity. 16. The biological activity of TGF-β is at least one selected from the group consisting of enhancement of extracellular matrix protein production, inhibition of cell proliferation, monocyte migration, induction of bioactive substances, immunosuppression, and β-amyloid protein deposition. The screening method according to claim 15, which has one effect. 17. The biological activity transmitted via TAB1 or XIAP is a change in the expression level of a reporter gene activated in response to the biological activity of TAB1 or XIAP.
5. The screening method according to 4. 18. The screening method according to claim 17, wherein the reporter gene is PAI-1, fibronectin, type I collagen or type IV collagen. 19. Contacting XIAP, TGF-β type I receptor and a test sample, and then examining the presence or absence of the formation of a bond between XIAP and TGF-β type I receptor,
A method for screening for a substance that inhibits the binding between a β type I receptor and XIAP. 20. XIAP has an amino acid sequence consisting of Met at amino acid position 1 to Ser at amino acid position 497 of the amino acid sequence shown in SEQ ID NO: 4, or has a biological activity of XIAP, and In the amino acid sequence shown, 1
20. The screening method according to claim 19, wherein the plurality of amino acids have substitution, deletion and / or addition amino acid sequences. 21. The TGF-β type I receptor is an amino acid sequence consisting of Met at amino acid position 1 to Met at amino acid position 503 of the amino acid sequence shown in SEQ ID NO: 6, or a biological sequence of TGF-β type I receptor. The screening method according to claim 19, which has an activity and has an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 6. 22. The screening method according to claim 19, wherein the XIAP or TGF-β type I receptor is fused with another peptide or polypeptide. 23. The screening method according to claim 19, wherein the XIAP or TGF-β type I receptor is bound to a support. 24. The screening method according to claim 23, wherein the support is a bead or a plate. 25. XIAP or TGF-β type I receptor is labeled, and the label is detected or measured.
25. The screening method according to any one of claims 19 to 24, wherein the presence or absence of formation of a bond with a TGF-β type I receptor is examined. 26. The screening method according to claim 25, wherein the label is a radioisotope, an enzyme, or a fluorescent substance. 27. The TGF-β type I receptor bound to XIAP is detected or measured by a primary antibody against the TGF-β type I receptor or a primary antibody against another peptide or polypeptide fused to the TGF-β type I receptor. The screening method according to any one of claims 19 to 26, wherein the presence or absence of formation of a bond between XIAP and the TGF-β type I receptor is determined. 28. A primary antibody against a TGF-β type I receptor bound to XIAP or a primary antibody against another peptide or polypeptide fused to the TGF-β type I receptor and a secondary antibody against the primary antibody against the primary antibody against the TGF-β type I receptor. XIAP by detecting or measuring with the next antibody
28. The screening method according to claim 27, wherein the presence or absence of binding between the TGF-β type I receptor and TGF-β is examined. 29. X bound to a TGF-β type I receptor
The presence or absence of the formation of a bond between XIAP and a TGF-β type I receptor by detecting or measuring IAP with a primary antibody against XIAP or a primary antibody against another peptide or polypeptide fused to XIAP. 2
7. The screening method according to any one of 6. 30. X bound to a TGF-β type I receptor
By detecting or measuring IAP with a primary antibody to XIAP or a primary antibody to another peptide or polypeptide fused to XIAP and a secondary antibody to the primary antibody, the formation of binding between XIAP and the TGF-β type I receptor is determined. The screening method according to claim 29, wherein the presence or absence is checked. 31. The primary antibody or the secondary antibody is labeled with a radioisotope, an enzyme, or a fluorescent substance.
31. The screening method according to any one of 7 to 30. 32. A test sample is introduced and / or contacted with cells expressing XIAP and TGF-β type I receptor, and then the biological activity transmitted via XIAP or TGF-β type I receptor is detected and A method of screening for a substance that inhibits the binding between TGF-β type I receptor and XIAP by measuring it. 33. The screening method according to claim 32, wherein the biological activity transmitted via XIAP or TGF-β type I receptor is TGF-β biological activity. 34. At least one of the biological activities of TGF-β selected from the group consisting of enhancement of extracellular matrix protein production, inhibition of cell proliferation, monocyte migration, induction of bioactive substances, immunosuppression and β-amyloid protein deposition. 34. The screening method according to claim 33, which has one effect. 35. An expression level of a reporter gene whose biological activity transmitted via XIAP or TGF-β type I receptor is activated in response to the biological activity of XIAP or TGF-β type I receptor 33. The screening method according to claim 32, which is a change. 36. The screening method according to claim 35, wherein the reporter gene is PAI-1, fibronectin, type I collagen, or type IV collagen. 37. Contacting XIAP, TGF-β type II receptor and a test sample, and then examining the presence or absence of formation of a bond between XIAP and TGF-β type II receptor,
A method of screening for a substance that inhibits the binding between β type II receptor and XIAP. 38. XIAP has an amino acid sequence consisting of Met at amino acid position 1 to Ser at amino acid position 497 of the amino acid sequence shown in SEQ ID NO: 4, or has a biological activity of XIAP, and In the amino acid sequence shown, 1
38. The screening method according to claim 37, wherein a plurality of amino acids have a substituted, deleted and / or added amino acid sequence. 39. The TGF-β type II receptor is an amino acid sequence consisting of Met at amino acid position 1 to Lys at amino acid position 567 of the amino acid sequence shown in SEQ ID NO: 8, or a biological sequence of TGF-β type II receptor. 38. The screening method according to claim 37, which has an activity and has an amino acid sequence in which one or more amino acids have been substituted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 6. 40. The screening method according to any one of claims 37 to 39, wherein the XIAP or TGF-β type II receptor is fused with another peptide or polypeptide. 41. The screening method according to any one of claims 37 to 40, wherein the XIAP or TGF-β type II receptor is bound to a support. 42. The screening method according to claim 41, wherein the support is a bead or a plate. 43. XIAP or TGF-β type II receptor is labeled, and the label is detected or measured to form XIAP or TGF-β type II receptor.
43. The screening method according to any one of claims 37 to 42, wherein the presence or absence of binding to a TGF-β type II receptor is examined. 44. The screening method according to claim 43, wherein the label is a radioisotope, an enzyme or a fluorescent substance. 45. The TGF-β type II receptor bound to XIAP is detected or measured by a primary antibody against the TGF-β type II receptor or a primary antibody against another peptide or polypeptide fused to the TGF-β type II receptor. The screening method according to any one of claims 37 to 44, wherein the presence or absence of formation of a bond between XIAP and a TGF-β type II receptor is determined. 46. A TGF-β type II receptor bound to XIAP, which is a primary antibody against the TGF-β type II receptor or a secondary antibody against another peptide or polypeptide fused to the TGF-β type II receptor. XIAP by detecting or measuring with the next antibody
46. The screening method according to claim 45, wherein the presence or absence of formation of a bond between TGF-β type II receptor and TGF-β is examined. 47. A binding to the TGF-β type II receptor
XIAP is detected or measured by a primary antibody against XIAP or a primary antibody against another peptide or polypeptide fused with XIAP, thereby examining the presence or absence of formation of a bond between XIAP and TGF-β type II receptor. 4
5. The screening method according to any one of 4. 48. Bound to the TGF-β type II receptor
XIAP is detected or measured by a primary antibody to XIAP or a primary antibody to another peptide or polypeptide fused to XIAP and a secondary antibody to the primary antibody to form a bond between XIAP and a TGF-β type II receptor. The screening method according to claim 47, wherein the presence or absence is checked. 49. The primary antibody or the secondary antibody is labeled with a radioisotope, an enzyme or a fluorescent substance.
49. The screening method according to any one of 5 to 48. 50. A test sample is introduced and / or contacted with cells expressing XIAP and TGF-β type II receptor, and then the biological activity transmitted via XIAP or TGF-β type II receptor is detected and A method of screening for a substance that inhibits the binding between TGF-β type II receptor and XIAP by measuring it. 51. The screening method according to claim 50, wherein the biological activity transmitted via XIAP or TGF-β type II receptor is TGF-β biological activity. 52. The biological activity of TGF-β is at least one selected from the group consisting of enhancement of extracellular matrix protein production, inhibition of cell proliferation, monocyte migration, induction of bioactive substances, immunosuppression, and β-amyloid protein deposition. 52. The screening method according to claim 51, which has one effect. 53. An expression level of a reporter gene which activates a biological activity transmitted via the XIAP or TGF-β type II receptor in response to the biological activity of the XIAP or TGF-β type II receptor. 51. The screening method of claim 50, wherein the method is a change. 54. The screening method according to claim 53, wherein the reporter gene is PAI-1, fibronectin, type I collagen or type IV collagen. 55. A substance obtained by the screening method according to any one of claims 1 to 54. 56. The substance according to claim 55, which is a novel alternative to a known substance that inhibits or activates TGF-β signal transduction. 57. The substance according to claim 55, which is an agonist of XIAP. 58. The method of claim 5, which is an antagonist of XIAP.
5. The substance according to 5. 59. A pharmaceutical composition comprising a substance obtained by the screening method according to any one of claims 1 to 54 as an active ingredient. 60. Enhanced production of extracellular matrix protein,
60. The pharmaceutical composition according to claim 59, which is used for suppressing or activating at least one action selected from the group consisting of cell growth inhibition, monocyte migration, bioactive substance induction, immunosuppression, and β-amyloid protein deposition. . 61. Liver fibrosis, pulmonary fibrosis, glomerulonephritis, diabetic nephropathy, renal sclerosis, vascular restenosis, keloid skin,
60. The pharmaceutical composition according to claim 59, which is used for preventing and / or treating at least one disease selected from the group consisting of scleroderma, autoimmune disease and Alzheimer's disease. 62. A pharmaceutical composition comprising XIAP as an active ingredient, which is selected from the group consisting of enhancement of extracellular matrix protein production, inhibition of cell proliferation, monocyte migration, induction of a physiologically active substance, immunosuppression and β-amyloid protein deposition. The aforementioned pharmaceutical composition used for inhibiting or activating at least one effect selected. 63. A pharmaceutical composition comprising XIAP as an active ingredient, comprising hepatic fibrosis, pulmonary fibrosis, glomerulonephritis, diabetic nephropathy, renal sclerosis, vascular restenosis, skin keloid, scleroderma The above-mentioned pharmaceutical composition used for preventing and / or treating at least one disease selected from the group consisting of disease, autoimmune disease and Alzheimer's disease.