JP2011178697A - SUBSTRATE MOTIF OF RECEPTOR-TYPE TYROSINE PHOSPHATASE Ptprz, AND APPLICATION THEREOF - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for measuring activity of Ptprz, to provide a method for screening a Ptprz activity-regulating substance, to provide a Ptprz substrate motif important and indispensable for the establishment of the methods, and to provide a Ptprz substrate peptide. <P>SOLUTION: The dephosphorylation site of a substrate molecule (Git1, Magi1) of the Ptprz is clarified, and the substrate motif is discovered. As a result, the method for measuring the activity and the screening method are provided by using the substrate peptide designed based on the substrate motif. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a substrate motif of receptor tyrosine phosphatase Prprz and its use (method for measuring Ptprz activity using substrate motif and screening method for Ptprz activity promoter or activity inhibitor).

  Phosphorylation modification of tyrosine residues in intracellular proteins is involved in the control of various cellular activities such as cell proliferation, differentiation, and migration, and failure of phosphorylation control is involved in metabolism such as cancer, autoimmune diseases, diabetes, etc. As a major cause of sexually transmitted diseases and in the central nervous system, it is known to be involved in higher brain dysfunction. Phosphorylation is reversibly controlled by a protein tyrosine kinase (PTK) that adds a phosphate group to a specific tyrosine residue in the protein and a protein tyrosine phosphatase (PTP) that removes it. The PTK and PTP genes that have been identified in the human genome exceed several hundreds and are thought to be involved in specific cell functions, but the details are unknown.

  The above-mentioned PTP is largely classified into receptor type protein tyrosine phosphatase (RPTP) expressed on the cell membrane and non-receptor type tyrosine phosphatase (NR-PTP) localized in the cell. Further, the RPTP family is classified into 8 subfamilies and is composed of all 21 types of molecules (see Non-Patent Documents 1 and 2). Many RPTPs have two PTP domains on the cytoplasm side, but PTP activity is only in the first PTP domain (PTP-D1) close to the cell membrane. The second PTP domain (PTP-D2) located distal to the cell membrane does not have PTP activity, and functions as a PTP-D1 activity control and binding domain with other molecules to control the intracellular localization. Functional significance, such as, is considered (see Non-Patent Documents 1 and 2).

  Receptor type protein tyrosine phosphatase Z (also called Ptprz, or PTPζ or RPTPβ) is RPTP that is highly expressed in the central nervous system (see Non-Patent Document 3), but is also slightly expressed in peripheral tissues such as the stomach. (See Non-Patent Document 4). Among its 21 RPTPs (see Non-Patent Documents 1 and 2), only Ptprz / PTPζ and Ptprg / PTPγ belonging to the R5 subfamily have typical PDZ domain-binding motifs at their carboxyl terminus. (See Non-Patent Document 3).

The present inventors have reported that Ptprz binds to the second PDZ domain of PSD95, a major synaptic scaffold protein involved in synaptic plasticity, through its carboxyl-terminal PDZ domain-binding motif (Non-patented. Reference 4). In the adult rat brain, both Ptprz and PSD95 are distributed in the dendrites of pyramidal neurons in the hippocampus and cerebral neocortex and are detected in the post-synaptic thickening (PSD) fraction. The present inventors have Ptprz is, besides PSD95 family, membrane-associated guanylate kinase (m embrane a ssociated g uanylate kinase ) WW domain and PDZ domain-containing protein -1/3 (MAGI-1 / -3) , Veli- 3, Shinaputojanin 2 binding protein (Synj2bp: syn apto j anin 2 b inding p rotein), acidic syntrophin 1 and basic syntrophin 1 (Snta1 and Sntb1: sy ntrophin a cidic 1 and b asic 1), as well as multiple PDZ protein (Mupp1 : multiple P DZ p rotein 1), etc., it has demonstrated to interact with different PDZ domain-containing protein (see non-Patent Document 3, 5).

  The present inventors recently reported a genetic engineering method for screening a PTP substrate and named it “yeast substrate trapping method (see Non-Patent Documents 3, 5, and 6)”. This method is based on the yeast two-hybrid system, but 1) inducibly express PTK to tyrosine phosphorylate prey protein, and 2) substrate trap PTP mutant (7). Two improvements have been added: screening (see). By this method, the present inventors allowed G protein-coupled receptor interaction factor 1 (Git1: G protein-coupled receptor kinase-interactor 1), p190 RhoGAP, Golgi-related PDZ, and coiled coil motif-containing protein (GOPC / PIST: golgi- Ptprz substrate molecules such as associated PDZ and coiled-coil motif containing) and MAGI-1 were isolated and identified (see Non-Patent Documents 3 and 5).

The present inventors have produced a Ptprz knockout mouse (see Non-Patent Document 8), and in the rough cerebral synapse fraction derived from this knockout mouse, the tyrosine phosphorylation level of ErbB4 of the receptor protein tyrosine kinase (RPTK) family Is increased compared to that in an extract derived from a wild type mouse, and ErbB4 is a substrate that is directly dephosphorylated by Ptprz (see Non-Patent Document 9). However, in the case of this ErbB4, in an experiment using cultured cells, it has been found that dephosphorylation by Ptprz requires the formation of a molecular complex via a PDZ domain-containing protein such as PSD95 (non-native). Patent document 9). The carboxyl terminus of Ptprz binds to the second PDZ domain of PSD95, which is a major component of the excitatory synaptic post-hypertrophy (PSD) (see Non-Patent Document 4), while ErbB4 is the first and second of PSD95. Binds to the second PDZ domain. That is, it is considered that a molecular complex of Ptprz-ErbB4 is formed by a scaffold protein such as PSD95. Besides this ErbB4, β-catenin (see Non-Patent Document 10) and sodium channel α subunit (see Non-Patent Document 11) have been reported as substrates for Ptprz. In the yeast substrate trapping method described above, ErbB4 No positive clones containing β-catenin and sodium channel sequences have been obtained. In principle, the yeast substrate trapping method should detect only the interaction between the intracellular domain of Ptprz and the phosphorylated substrate. For this reason, it is considered that in order to dephosphorylate β-catenin and sodium channels by Ptprz, it is necessary to assist the association with the scaffold protein as in the case of ErbB4 described above (Non-patent Document 3). For example, β-catenin is known to interact with the fifth PDZ domain of epithelial MAGI-1 (see Non-Patent Document 10), and Ptprz and β-catenin are stable enzymes on MAGI-1 scaffold proteins. -It is considered possible to form a substrate complex. Similarly, the first pleckstrin homology syntrophin (PH: p leckstrin h omology) domain and thin neurotrophin Unique (SU: s yntrophin- u nique) domain interacts with the sodium channel (see Non-Patent Document 12), syntrophin It is considered that a complex can be formed because the PDZ domain of Pdprz interacts with Ptprz (see Non-Patent Documents 3 and 5).

  Physiological importance of Ptprz, which has been shown from gene knockout mouse studies, is related to memory / learning through control of neuronal synaptic plasticity (Ref. 3, 13, 14), central monoamines such as dopamine Functional control of the nervous system (Patent Document 1), contribution to the severity of experimental autoimmune encephalomyelitis (see Non-Patent Document 15), gastric mucosal damage caused by the toxin protein VacA produced by Helicobacter pylori Involvement as an essential receptor (see Non-Patent Document 16) and involvement in pain sensitivity (see Non-Patent Document 17). In addition, clinical studies strongly suggest that glioblastoma is involved in malignant transformation (see Non-Patent Document 18). These findings strongly suggest the usefulness of Ptprz as a drug discovery target. The present inventors have already proposed an assay system (Patent Document 1) for a compound having an effect of activating or inhibiting Ptprz at the individual animal level (Patent Document 1) and an assay system at a cultured cell level using ErbB4 (Patent Document 2). I have done it. However, the main use of these assay systems is to investigate in detail the pharmacology of the target compound and the presence or absence of side effects, etc., and it is a large target for thousands to hundreds of thousands of compounds intended for general drug discovery. It was an unrealistic specification in terms of labor and cost performance for use in scale screening.

Japanese Patent No. 3785460 JP 2009-1521 A

Mol. Cell. Biol. 21, 2001, 7117-7136 Nat. Rev. Mol. Cell Biol. 7, 2006, 833-846 Biochemistry Volume 77, 2005, 1255-1268 Brain Res. Mol. Brain Res. 72, 1999, 47-54 Methods 35, 2005, 54-63 Proc. Natl. Acad. Sci. USA 98, 2001, 6593-6598 Proc. Natl. Acad. Sci. USA 94, 1997, 1680-1685 Neurosci. Lett. 247, 1998, 135-138 J. Biochem. 142, 2007, 343-350 Biochem. Biophys. Res. Commun. 270, 2000, 903-909 Nat. Neurosci. 3, 2000, 437-444 J. Neurosci. 18, 1998, 128-137 J. Neurosci. 2005, 25, 1081-1088 Neurosci. Lett. 2006, 399, 33-38 Nat. Genet. 2002, 32, 411-414 Nat. Genet. 33, 2003, 375-381 Behav. Brain Res. 2009, 201, 29-40 Diagn. Mol. Pathol. 2009, 18, 206-218

  In the field of drug discovery, it is often performed to search for substances that act on specific target molecules by large-scale screening, but this requires a great deal of labor and cost. Therefore, an assay system that is the most important point for determining the success or failure of screening is required not only to have a technical advantage based on scientific evidence, but also to be excellent in simplicity and cost performance. An object of the present invention is to provide and implement a screening method effective for efficiently searching and identifying candidate substances in order to obtain substances that can enhance or attenuate the phosphatase activity possessed by Ptprz, that is, Ptprz activity modulators. Providing necessary Ptprz substrate motif and Ptprz substrate peptide, and providing optimal assay conditions for measuring Ptprz activity using Ptprz substrate peptide.

  In order to realize a method for measuring the activity of Ptprz and a screening method for a Ptprz activity modulator, it is important to find the structural characteristics (substrate motif) of the phosphorylation site of the substrate that Ptprz efficiently dephosphorylates. As shown in the Examples described later, in the present invention, intensive studies were conducted focusing on Git1 and Magi1, which are Ptprz substrate molecules. As a result, the inventors succeeded in finding a substrate peptide containing a substrate motif of Ptprz and an optimal substrate motif for in vitro activity evaluation. In addition, optimum assay conditions such as pH were found. These results have made it possible to achieve the desired activity measurement and screening. These methods are expected to greatly contribute to the establishment of prevention and treatment methods for various diseases caused by Ptprz dysfunction.

（但し、式中の各アミノ酸残基の上の数字は基準位置のアミノ酸残基Y（チロシン）からの相対位置を表し、D/EはD（アスパラギン酸）又はE（グルタミン酸）を表し、Xは任意のアミノ酸残基を表し、I/VはI（イソロイシン）又はV（バリン）を表し、S/I/VはS(セリン)、I（イソロイシン）又はV（バリン）を表す）
からなる、Ptprzの基質モチーフ。
［２］-2位のアミノ酸残基がD（アスパラギン酸）及びE（グルタミン酸）以外のアミノ酸残基である、［１］に記載の基質モチーフ。
［３］-2位のアミノ酸残基がA（アラニン）、N（アスパラギン）、P（プロリン）又はV（バリン）である、［１］に記載の基質モチーフ。
［４］以下の配列（配列番号２）、即ち

（但し、式中の各アミノ酸残基の上の数字は基準位置Zからの相対位置を表し、Zはリン酸化チロシン又はリン酸化チロシンのアナログであり、D/EはD（アスパラギン酸）又はE（グルタミン酸）を表し、Xは任意のアミノ酸残基を表し、I/VはI（イソロイシン）又はV（バリン）を表し、S/I/VはS(セリン)、I（イソロイシン）又はV（バリン）を表す）
を含む、Ptprzの基質ペプチド。
［５］-2位のアミノ酸残基がD（アスパラギン酸）及びE（グルタミン酸）以外のアミノ酸残基である、［４］に記載の基質ペプチド。
［６］-2位のアミノ酸残基がA（アラニン）、N（アスパラギン）、P（プロリン）又はV（バリン）である、［４］に記載の基質ペプチド。
［７］+1位のアミノ酸残基がS（セリン）である、［４］〜［６］のいずれか一項に記載の基質ペプチド。
［８］前記配列として、配列番号３〜６のいずれかの配列を含む、［４］に記載の基質ペプチド。
［９］前記配列のカルボキシ末端に少なくとも一つのアミノ酸残基が連結されている、［４］〜［８］のいずれか一項に記載の基質ペプチド。
［１０］カルボキシ末端に連結されているアミノ酸残基がD（アスパラギン酸）、E（グルタミン酸）、G（グリシン）又はF（フェニルアラニン）である、［９］に記載の基質ペプチド。
［１１］前記配列のアミノ末端に少なくとも一つのアミノ酸残基が連結されている、［４］〜［１０］のいずれか一項に記載の基質ペプチド。
［１２］アミノ末端に連結されているアミノ酸残基がV（バリン）、G（グリシン）又はA（アラニン）である、［１１］に記載の基質ペプチド。
［１３］５個〜１２個の残基からなる、［４］〜［１２］のいずれか一項に記載の基質ペプチド。
［１４］５個〜９個の残基からなる、［４］〜［１２］のいずれか一項に記載の基質ペプチド。
［１５］配列番号３〜３８のいずれかのアミノ酸配列からなる、［４］に記載の基質ペプチド。
［１６］リン酸化チロシンのアナログが、クマリン誘導体である、［４］〜［１５］のいずれか一項に記載の基質ペプチド。
［１７］リン酸化チロシンのアナログがホスホクマリン−アミノ−プロピオン酸（phosphocoumaryl-amino-propionic acid）である、［４］〜［１５］のいずれか一項に記載の基質ペプチド。
［１８］以下のステップ（１）及び（２）を含む、Ptprz活性測定法：
（１）［４］〜［１７］のいずれか一項に記載の基質ペプチドに対して試料を作用させるステップ；
（２）前記基質ペプチドの脱リン酸化を検出するステップ。
［１９］試料がPtprz又はPtprz類似分子である、［１８］に記載のPtprz活性測定法。
［２０］以下のステップ（ｉ）及び（ｉｉ）を含む、Ptprz活性調節物質のスクリーニング法：
（ｉ）被験物質の存在下、［４］〜［１７］のいずれか一項に記載の基質ペプチドに対してPtprzを作用させるステップ；
（ｉｉ）前記基質ペプチドの脱リン酸化を検出し、検出結果に基づき被験物質の有効性を判定するステップ。
［２１］被験物質の非存在下でPtprzを作用させた場合と比較して前記基質ペプチドの脱リン酸化レベルが高いときに、被験物質がPtprzの脱リン酸化作用を促進すると判断し、被験物質がPtprzの活性促進に有効であると判定する、［２０］に記載のスクリーニング法。
［２２］被験物質の非存在下でPtprzを作用させた場合と比較して前記基質ペプチドの脱リン酸化レベルが低いときに、被験物質がPtprzの脱リン酸化作用を抑制すると判断し、被験物質がPtprzの活性抑制に有効であると判定する、［２０］に記載のスクリーニング法。 The present invention listed below is based on the above-mentioned results.
[1] The following sequence (SEQ ID NO: 1),

(However, the number above each amino acid residue in the formula represents the relative position from the amino acid residue Y (tyrosine) at the reference position, D / E represents D (aspartic acid) or E (glutamic acid), X Represents any amino acid residue, I / V represents I (isoleucine) or V (valine), and S / I / V represents S (serine), I (isoleucine) or V (valine))
A substrate motif of Ptprz consisting of
[2] The substrate motif according to [1], wherein the amino acid residue at position -2 is an amino acid residue other than D (aspartic acid) and E (glutamic acid).
[3] The substrate motif according to [1], wherein the amino acid residue at position -2 is A (alanine), N (asparagine), P (proline), or V (valine).
[4] The following sequence (SEQ ID NO: 2),

(However, the number above each amino acid residue in the formula represents a relative position from the reference position Z, Z is phosphorylated tyrosine or a phosphorylated tyrosine analog, and D / E is D (aspartic acid) or E (Glutamic acid), X represents any amino acid residue, I / V represents I (isoleucine) or V (valine), and S / I / V represents S (serine), I (isoleucine) or V ( Represents valine))
A substrate peptide of Ptprz.
[5] The substrate peptide according to [4], wherein the amino acid residue at position -2 is an amino acid residue other than D (aspartic acid) and E (glutamic acid).
[6] The substrate peptide according to [4], wherein the amino acid residue at position -2 is A (alanine), N (asparagine), P (proline) or V (valine).
[7] The substrate peptide according to any one of [4] to [6], wherein the amino acid residue at position +1 is S (serine).
[8] The substrate peptide according to [4], including any one of SEQ ID NOs: 3 to 6 as the sequence.
[9] The substrate peptide according to any one of [4] to [8], wherein at least one amino acid residue is linked to the carboxy terminus of the sequence.
[10] The substrate peptide according to [9], wherein the amino acid residue linked to the carboxy terminus is D (aspartic acid), E (glutamic acid), G (glycine) or F (phenylalanine).
[11] The substrate peptide according to any one of [4] to [10], wherein at least one amino acid residue is linked to the amino terminus of the sequence.
[12] The substrate peptide according to [11], wherein the amino acid residue linked to the amino terminus is V (valine), G (glycine) or A (alanine).
[13] The substrate peptide according to any one of [4] to [12], consisting of 5 to 12 residues.
[14] The substrate peptide according to any one of [4] to [12], consisting of 5 to 9 residues.
[15] The substrate peptide according to [4], comprising the amino acid sequence of any one of SEQ ID NOs: 3 to 38.
[16] The substrate peptide according to any one of [4] to [15], wherein the phosphorylated tyrosine analog is a coumarin derivative.
[17] The substrate peptide according to any one of [4] to [15], wherein the phosphorylated tyrosine analog is phosphocoumaryl-amino-propionic acid.
[18] A method for measuring Ptprz activity, comprising the following steps (1) and (2):
(1) A step of causing a sample to act on the substrate peptide according to any one of [4] to [17];
(2) A step of detecting dephosphorylation of the substrate peptide.
[19] The method for measuring Ptprz activity according to [18], wherein the sample is Ptprz or a Ptprz-like molecule.
[20] A screening method for a Ptprz activity modulator comprising the following steps (i) and (ii):
(I) a step of allowing Ptprz to act on the substrate peptide according to any one of [4] to [17] in the presence of a test substance;
(Ii) detecting the dephosphorylation of the substrate peptide and determining the effectiveness of the test substance based on the detection result.
[21] When the dephosphorylation level of the substrate peptide is higher than when Ptprz is allowed to act in the absence of the test substance, it is determined that the test substance promotes the dephosphorylation action of Ptprz; The screening method according to [20], wherein it is determined that is effective for promoting the activity of Ptprz.
[22] When the dephosphorylation level of the substrate peptide is lower than when Ptprz is allowed to act in the absence of the test substance, the test substance is judged to suppress the dephosphorylation action of Ptprz, and the test substance The screening method according to [20], wherein it is determined that is effective for suppressing the activity of Ptprz.

Identification of the dephosphorylation site of Ptprz in Git1. A: Schematic diagram of Git1 (770 amino acid residues). The arrowhead indicates the position of the tyrosine residue (relationship). GAP represents a GTPase activation domain, ANK represents an ankyrin repeat region, and SHD1 represents Spa2 homology domain 1. Git1-WT is wild-type Git1, and the Git11Y9F series is obtained by substituting the tyrosine residue (Y) at each position with phenylalanine (F). B: Git1 with a FLAG tag at the N-terminus and a mutant thereof were expressed in HEK cells. These cultured cells were treated with 100 μM vanadic acid (a nonspecific inhibitor of endogenous tyrosine phosphatase activity) for 20 minutes to induce a state in which intracellular proteins were forcibly tyrosine phosphorylated. Git1 was immunoprecipitated from the protein extract of the cultured cells using an antibody against the FLAG tag, and the tyrosine phosphorylation level was evaluated by Western blotting using a phosphorylated tyrosine antibody (PY) (upper side). The amount of Git1 protein was evaluated with the FLAG antibody (lower row). Phosphorylation was observed at three sites, 392, 554, and 607, and these were determined to be the main tyrosine phosphorylation sites of Git1. C: Recombinant Ptprz (GST-PtprzICR) was added to Git1 and its mutants isolated by immunoprecipitation in vitro and allowed to stand at 37 ° C. for 20 minutes. The degree of Git1 dephosphorylation by Ptprz was analyzed by Western blot in the same manner as described above. GST is a negative control and AP (alkaline phosphatase) is a positive control. The Git1 mutant that is most dephosphorylated by Ptprz is Git1-Y9F-Y554, and from this, the 554th tyrosine residue was determined to be the substrate site of Ptprz. On the other hand, when AP is used as an enzyme, all are dephosphorylated. Identification of Ptprz dephosphorylation sites in Magi. A: Schematic diagram of Magi1 (1248 amino acid residues). The arrowhead indicates the position of the tyrosine residue (relevant part). GK represents a guanylate kinase domain, WW represents a motif in which two tryptophans are conserved, and PDZ represents a PSD-95 / Dlg-A / Zo-1 domain. Magi-WT is wild-type Magi, Magi1-Y829F and Magi1-Y858F are mutants in which the tyrosine residue of Magi1 is substituted with phenylalanine, Magi1-ΔC1, Magi1-ΔC2, and Magi1-ΔC3 are C1-terminal deletion mutations in Magi1 Is the body. B: Magi1 with a FLAG tag at the N-terminus and its mutant were expressed in HEK cells, and phosphorylated state was induced by vanadate treatment. Recombinant Ptprz (GST-PtprzICR) was added in vitro to Magi1 and C-terminal deletion mutants isolated by immunoprecipitation and allowed to stand at 37 ° C for 20 minutes. The degree of dephosphorylation by Ptprz was determined by Western blotting. Analyzed. For comparison, half the amount (0.5 lane) and only the solvent (0 lane) were simultaneously performed with respect to the standard addition amount of Ptprz (1 lane). As a result, since only the Magi1-ΔC3 mutant was not dephosphorylated by Ptprz, it was determined that the substrate site of Ptprz was present in the 789-983 region on Magi1. C: Dephosphorylation by Ptprz was evaluated in the same manner as described above using mutant Magi1 in which the 829th and 858th tyrosine residues that can be phosphorylated in the 789-983 region on Magi1 were substituted with phenylalanine. As a result, the Magi1-Y829F mutant in which only the 858th tyrosine residue was left was dephosphorylated well by Ptprz, and thus the 858th phosphorylated tyrosine residue was determined to be the substrate site of Ptprz. Identification of Ptprz substrate motif. A: Examination of optimum pH. A phosphorylated pseudopeptide (7 amino acid residues) synthesized based on the sequence near the 554th tyrosine residue of Git1 was used as a substrate to evaluate and examine the optimum pH conditions for a dephosphorylation assay using recombinant Ptprz. . The optimum pH was estimated as pH 6.5. B: Using phosphorylated pseudopeptide (7 amino acid residues) synthesized based on the sequence near the 554th tyrosine residue of Git1 as a substrate, evaluating nearby amino acid residues important for dephosphorylation using recombinant Ptprz ·investigated. The graph shows the efficiency of dephosphorylation of the mutant substrate peptide of Git1 as a relative value with respect to the wild-type Git1 peptide (refer to the horizontal axis for mutation sites and mutant residues). As a result, as the substrate of Ptprz, acidic amino acid residues such as aspartic acid and glutamic acid are good at relative positions -4 and -3, whereas acidic amino acid residues are inconvenient at -1 and +1, isoleucine, valine, Serine was found to be good. C: Substrate motif of Ptprz presented from the above results. D: Sequences near phosphorylation sites for Git1 (554th tyrosine residue), Magi1 (858th tyrosine residue) and p190RhoGAP (1105th tyrosine residue) listed as representative examples of good substrate motifs for Ptprz A comparison. It was found that there was homology in the boxed area. Based on the Ptprz substrate motif sequence found this time (see Fig. 3C), a PIST sequence that was known to be dephosphorylated by Ptprz but whose dephosphorylation site was unknown was searched. It was found that there was a well-matched sequence in PIST. Amino acid residues are written in one letter, and the numbers at the top of the table indicate the relative positions from the tyrosine residue as a substrate.

The first aspect of the present invention relates to a substrate motif of Ptprz. The substrate motif of the present invention is useful for elucidating the function of Ptprz or for drug discovery research targeting Ptprz. In particular, it is useful in designing a substrate peptide of Ptprz (see the following description). Here, the term “motif” refers to a characteristic common sequence having a specific function. The “substrate motif” of the present invention has a characteristic sequence in functioning as a substrate of Ptprz. The substrate motif of the present invention consists of the following sequence (SEQ ID NO: 1).

  In the above sequence, the number above each amino acid residue represents the relative position from amino acid residue Y (tyrosine) at the reference position. D / E represents D (aspartic acid) or E (glutamic acid), X represents any amino acid residue, I / V represents I (isoleucine) or V (valine), and S / I / V Represents S (serine), I (isoleucine) or V (valine).

  The amino acid residue at each position was determined by comparison of the amino acid residues at the corresponding positions of the substrate molecules (Git1, p190, Mag1 and PIST) and the results of the amino acid substitution experiment (see the example below, see FIG. 3B). . In determining the amino acid residue at position -3, the degree of dephosphorylation was greatly reduced when the acidic amino acid residue aspartic acid (D) was changed to the nonpolar amino acid residue alanine (A). For the amino acid residue at the -2 position, a slight decrease in activity was observed by substituting the acidic amino acid D (aspartic acid) with A (alanine) (FIG. 3B). Is an amino acid residue other than D (aspartic acid) and E (glutamic acid). When further embodied, it is A (alanine), N (asparagine), P (proline) or V (valine) which is the amino acid residue at the corresponding position in each substrate molecule.

  The second aspect of the present invention relates to a substrate peptide of Ptprz. The “substrate peptide of Ptprz” refers to a peptide that functions as a substrate of Ptprz. Therefore, it contains a site that is dephosphorylated by Ptprz. As used herein, the term “peptide” is clearly distinguished from the term “protein”. That is, Ptprz substrate proteins Git1, p190, Magi1, PIST, etc. are distinguished from the substrate peptides of the present invention in that they have a large molecular weight or form specific higher-order structures to exhibit specific functions. (The substrate peptide concept of the present invention does not include these substrate proteins).

The substrate peptide of the present invention is designed based on the sequence of the substrate motif and includes the following sequence (SEQ ID NO: 2).

  In the above sequence, the number above each amino acid residue represents a relative position from the reference position Z. Z is phosphorylated tyrosine or a phosphorylated tyrosine analog. D / E represents D (aspartic acid) or E (glutamic acid), X represents any amino acid residue, I / V represents I (isoleucine) or V (valine), and S / I / V Represents S (serine), I (isoleucine) or V (valine).

  The amino acid residue at position -2 showed a slight decrease in activity due to substitution of A (alanine) to D (aspartic acid), which is an acidic amino acid (FIG. 3B), and the corresponding position in each substrate molecule Since it is not an acidic amino acid residue, amino acid residues other than D (aspartic acid) and E (glutamic acid) are preferred. More preferably, it is A (alanine), N (asparagine), P (proline) or V (valine) which is an amino acid residue at the corresponding position in each substrate molecule.

  On the other hand, the amino acid residue at position +1 does not contain a PDZ domain that can bind to the carboxy terminus of Ptprz when it is recognized as a Ptprz substrate, and its substrate specificity is determined by phosphotyrosine and its neighboring sequences. The type of amino acid residue in Git1 and p190, which is thought to be considered, is emphasized, and S (serine) is preferred.

The example of the specific arrangement | sequence contained in the said arrangement | sequence (sequence number 2) is shown below. These sequences correspond to the amino acid sequences of the substrate molecules Git1, p190, Magi1 and PIST and can function as particularly excellent substrates.
DAIZS (SEQ ID NO: 3. Z is phosphorylated tyrosine or an analog of phosphorylated tyrosine)
ENIZS (SEQ ID NO: 4, Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
EPIZI (SEQ ID NO: 5. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
EVVZV (SEQ ID NO: 6, Z is phosphorylated tyrosine or phosphorylated tyrosine analog)

  One embodiment of the substrate peptide of the present invention includes a sequence in which at least one amino acid residue is linked to the amino terminus of the above sequence (SEQ ID NO: 2). Specific examples of amino acid residues directly linked to the amino terminus are D (aspartic acid), E (glutamic acid), G (glycine) and F (phenylalanine). The sequence of the substrate peptide in this example corresponds to the amino acid sequences of the substrate molecules Git1, p190, Magi1 and PIST (see FIG. 3D). If the number of amino acid residues linked to the amino terminus is too large, it may have an unexpected effect on the substrate recognition by Ptprz and cause an increase in preparation cost. ~ 3.

  Another embodiment of the substrate peptide of the present invention includes the above sequence (SEQ ID NO: 2) or a sequence in which at least one amino acid residue is linked to the carboxy terminus of the sequence of the above embodiment. Specific examples of amino acid residues directly linked to the carboxy terminus are V (valine), G (glycine) or A (alanine). The sequence of the substrate peptide in this example corresponds to the amino acid sequences of the substrate molecules Git1, p190, Mag1 and PIST (see FIG. 3D). The number of amino acid residues linked to the carboxy terminus is preferably 1 to 5, and more preferably 1 to 3, since there is a possibility of unexpected influence on substrate recognition by Ptprz if it is too large.

The number of residues constituting the substrate peptide of the present invention is not particularly limited, but is preferably 5 to 12, more preferably 5 to 9. Hereinafter, specific examples of sequences constituting the substrate peptide of the present invention are shown.
DAIZS (corresponding to the sequence from position −3 to position +1 of Git1 shown in FIG. 3D. SEQ ID NO: 7. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
DDAIZS (corresponding to the sequence of positions −4 to +1 of Git1 shown in FIG. 3D. SEQ ID NO: 8, Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
EDDAIZS (corresponding to the sequence of -5 to +1 of Git1 shown in FIG. 3D. SEQ ID NO: 9, Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
LEDDAIZS (corresponding to the sequence from position 6 to position +1 of Git1 shown in FIG. 3D. SEQ ID NO: 10. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
DAIZSV (corresponding to the sequence of positions −3 to +2 of Git1 shown in FIG. 3D. SEQ ID NO: 11. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
DDAIZSV (corresponding to the sequence of positions −4 to +2 of Git1 shown in FIG. 3D. SEQ ID NO: 12, Z is phosphorylated tyrosine or an analog of phosphorylated tyrosine)
EDDAIZSV (corresponds to the sequence from position −5 to position +2 of Git1 shown in FIG. 3D. SEQ ID NO: 13. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
LEDDAIZSV (corresponding to the sequence of positions −6 to +2 of Git1 shown in FIG. 3D. SEQ ID NO: 14. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
ENIZS (corresponding to the sequence of positions −3 to +1 of p190 shown in FIG. 3D. SEQ ID NO: 15. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
EENIZS (corresponding to the sequence of positions −4 to +1 of p190 shown in FIG. 3D. SEQ ID NO: 16, Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
EEENIZS (corresponding to the sequence of positions −5 to +1 of p190 shown in FIG. 3D. SEQ ID NO: 17. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
NEEENIZS (corresponding to the sequence of positions −6 to +1 of p190 shown in FIG. 3D. SEQ ID NO: 18. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
ENIZSV (corresponding to the sequence from position −3 to position +2 of p190 shown in FIG. 3D. SEQ ID NO: 19, Z is phosphorylated tyrosine or an analog of phosphorylated tyrosine)
EENIZSV (corresponds to the sequence of positions −4 to +2 of p190 shown in FIG. 3D. SEQ ID NO: 20; Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
EEENIZSV (corresponds to the sequence of positions −5 to +2 of p190 shown in FIG. 3D. SEQ ID NO: 21. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
NEEENIZSV (corresponding to the sequence from position −6 to position +2 of p190 shown in FIG. 3D. SEQ ID NO: 22. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
EPIZI (corresponding to the sequence of positions −3 to +1 of Magi1 shown in FIG. 3D. SEQ ID NO: 23. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
GEPIZI (corresponding to the sequence of positions −4 to +1 of Magi1 shown in FIG. 3D. SEQ ID NO: 24. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
PGEPIZI (corresponding to the sequence of positions -5 to +1 of Magi1 shown in FIG. 3D. SEQ ID NO: 25. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
EPGEPIZI (corresponding to the sequence of positions -6 to +1 of Magi1 shown in FIG. 3D. SEQ ID NO: 26. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
EPIZIG (corresponding to the sequence of positions −3 to +2 of Magi1 shown in FIG. 3D. SEQ ID NO: 27. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
GEPIZIG (corresponding to the sequence of positions −4 to +2 of Magi1 shown in FIG. 3D. SEQ ID NO: 28. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
PGEPIZIG (corresponding to the sequence of positions −5 to +2 of Magi1 shown in FIG. 3D. SEQ ID NO: 29. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
EPGEPIZIG (corresponding to the sequence of positions −6 to +2 of Magi1 shown in FIG. 3D. SEQ ID NO: 30. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
EVVZV (corresponding to the sequence of positions −3 to +1 of PIST shown in FIG. 3D. SEQ ID NO: 31. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
FEVVZV (corresponds to the sequence of positions −4 to +1 of PIST shown in FIG. 3D. SEQ ID NO: 32. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
EFEVVZV (corresponding to the sequence of positions −5 to +1 of PIST shown in FIG. 3D. SEQ ID NO: 33. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
IEFEVVZV (corresponding to the sequence of positions −6 to +1 of PIST shown in FIG. 3D. SEQ ID NO: 34. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
EVVZVA (corresponds to the sequence of positions −3 to +2 of PIST shown in FIG. 3D. SEQ ID NO: 35. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
FEVVZVA (corresponding to the sequence of positions −4 to +2 of PIST shown in FIG. 3D. SEQ ID NO: 36, Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
EFEVVZVA (corresponds to the sequence of positions −5 to +2 of PIST shown in FIG. 3D. SEQ ID NO: 37. Z is phosphorylated tyrosine or phosphorylated tyrosine analog)
IEFEVVZVA (corresponding to the sequence of positions −6 to +2 of PIST shown in FIG. 3D. SEQ ID NO: 38, Z is phosphorylated tyrosine or phosphorylated tyrosine analog)

The residue at the reference position in the substrate peptide of the present invention is phosphorylated tyrosine or a phosphorylated tyrosine analog. An example of an analog of phosphorylated tyrosine is a coumarin derivative. Specific examples of the coumarin derivative that can constitute the substrate peptide of the present invention are the following compounds (see WO 2007/014326).

  This compound is called pCAP (phosphocoumaryl-amino-propionic acid). With a substrate peptide containing the compound, its dephosphorylation can be detected by fluorescence. Therefore, a simple and efficient assay is possible.

  Various modifications can also be made on condition that it functions as a substrate for Ptprz. In other words, the substrate peptide of the present invention may be a modified peptide. The “modified peptide” in the present invention is a partial replacement of a basic structure (a site that exhibits the function of a substrate peptide. Typically, a site consisting of any of the sequences of SEQ ID NOs: 2 to 6) and other molecules. A compound having a structure different from the basic structure at least in part by the addition of Those skilled in the art can design modifications such as substitutions using well-known or conventional means. Moreover, it is also easy for those skilled in the art to prepare the target modified body using well-known thru | or a usual means based on this design, and to investigate the property and effect | action.

  Examples of peptide modifications are those in which the functional group in the constituent amino acid residue is protected by an appropriate protecting group (acyl group, alkyl group, monosaccharide, oligosaccharide, polysaccharide, etc.), or a sugar chain is added. Various peptide derivatives and labels classified as alkylamines, alkylamides, sulfinyl, sulfonylamides, halides, amides, aminoalcohols, esters, aminoaldehydes, etc. by replacing the N-terminal or C-terminal with other atoms, etc. (For example, a peptide labeled with biotin or FITC at the N-terminus, a peptide labeled with a fluorescent dye, etc.). The protecting group is linked by an amide bond, an ester bond, a urethane bond, a urea bond, or the like according to the peptide site to which the protecting group is bound, the kind of the protecting group to be used, or the like.

  The substrate peptide of the present invention can be produced using a known peptide synthesis method (for example, solid phase synthesis method, liquid phase synthesis method). When pCAP is included as a phosphorylated tyrosine analog, a substrate peptide can be prepared with reference to Bioorg. Med. Chem. Lett .. 15, 5142-5145, 2005. In addition, when it exists in nature, the peptide of the present invention can also be prepared by operations such as extraction and purification. As an acquisition source of the peptide of the present invention, for example, animal cells (including humans), plant cells, body fluids (blood, urine, etc.) are assumed.

  The peptides of the present invention may be prepared using genetic engineering techniques. That is, the peptide of the present invention can be prepared by introducing a nucleic acid encoding the peptide of the present invention into a suitable host cell and recovering the peptide expressed in the transformant. The recovered peptide is purified as necessary. The recovered peptide can be subjected to an appropriate substitution reaction and converted into a desired modified peptide.

Another aspect of the present invention provides a method for measuring Ptprz activity based on the achievement of clarifying the substrate motif of Ptprz. The method for measuring Ptprz activity of the present invention can be used for evaluation of Ptprz activity, and is useful as a research tool for Ptprz or Ptprz-related molecules (substrate molecules and binding molecules of Ptprz). Specifically, for example, the Ptprz activity measurement method of the present invention can be used for elucidating the function of Ptprz. In the Ptprz activity measurement method of the present invention, the following steps (1) and (2) are performed.
(1) A step of causing a sample to act on the substrate peptide of the present invention (2) A step of detecting dephosphorylation of the substrate peptide

  In step (1), first, a substrate peptide of the present invention and a sample are prepared. In order to cause the sample to act on the substrate peptide, both are usually placed in the same container (such as a well of a multiwell plate) and brought into contact with each other. One of them may be immobilized in advance. The solid phase can be formed by binding or adsorption to an insoluble support. As reaction conditions, conditions suitable for the enzymatic reaction of Ptprz are employed. Optimum conditions may vary depending on the type of sample, but those skilled in the art can set optimal conditions through preliminary experiments. As a result of the study by the present inventors, pH 6.5 to 7.0 was found as the optimum reaction pH for Ptprz (see Examples described later). Therefore, it is preferable to adopt the pH condition unless there are special circumstances. The temperature condition is preferably around 30 ° C.

  Various samples are applicable. For example, a sample derived from a living body (human or non-human animal / plant, microorganism) (for example, blood, serum, lymph, spinal fluid, bone marrow, tissue extract, plant extract, cell extract, etc.) can be employed. . Alternatively, Ptprz itself (or a part thereof) or a Ptprz-like molecule (a molecule that is expected or expected to have Ptprz-like enzyme activity) can be used as a sample. Typically, Ptprz (which may be a recombinant) is adopted as a sample. The amino acid sequence of human Ptprz and the base sequence encoding it are shown in SEQ ID NO: 43 and SEQ ID NO: 44, respectively.

  In step (2) following step (1), dephosphorylation of the substrate peptide is detected. The detection result is used to calculate the Ptprz enzyme activity of the sample. In this way, the Ptprz enzyme activity of the sample is evaluated using the dephosphorylation level of the substrate peptide. A specific method for detecting the dephosphorylation of the substrate peptide may be appropriately selected according to the type of the substrate peptide to be used. When a substrate peptide containing a phosphorylated tyrosine residue is used, for example, dephosphorylation of the substrate peptide can be detected by an immunological technique using a phosphorylated tyrosine-specific antibody. In addition, when a substrate peptide containing pCAP is used, dephosphorylation of the substrate peptide can be detected easily and rapidly by fluorescence.

A further aspect of the present invention provides a screening method for a Ptprz activity modulator, which is an application or use of a Ptprz activity measurement method. The screening method of the present invention is useful as a means for searching for and identifying a Ptprz activity promoter (typically an agonist) or an activity inhibitor (typically an antagonist). In the screening method of the present invention, the following steps (i) and (ii) are performed.
(I) a step of causing Ptprz to act on the substrate peptide of the present invention in the presence of a test substance (ii) a step of detecting the dephosphorylation of the substrate peptide and determining the effectiveness of the test substance based on the detection result

  In step (i), a test substance, a substrate peptide, and Ptprz are prepared, and Ptprz is allowed to act on the substrate peptide under conditions where the test substance exists. For example, step (i) is performed by placing all of the test substance, substrate peptide and Ptprz in the same container (such as a well of a multiwell plate). However, as long as a state in which Ptprz can act on the substrate peptide is formed, the container to be used, the order of addition of each element, and the like can be arbitrarily selected and set. The reaction conditions in step (i) are the same as those in step (1) of the above Ptprz enzyme activity measurement method.

  As a test substance, organic compounds or inorganic compounds having various molecular sizes can be used. Examples of organic compounds include nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosylglycerides, cerebrosides, etc.), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.) The test substance may be derived from a natural product or may be synthetic. In some cases, an efficient screening system can be constructed using, for example, a combinatorial synthesis technique, and plant extracts, cell extracts, culture supernatants, etc. may be used as test substances. These drugs may be used as test substances.

  In step (ii), the dephosphorylation of the substrate peptide is detected, and the effectiveness of the test substance is determined based on the detection result. It is preferable to determine the effectiveness of the test substance by comparison with a control (reference). As a control, the detection result when Ptprz is allowed to act on the substrate peptide of the present invention in the absence of the test substance can be employed. When the target substance exists as a solution or a mixed solution, the objectivity, reliability, accuracy, etc. of the determination result are improved by making a control using only the solvent.

Specific examples (a) and (b) of determination criteria in step (ii) are shown below.
(A) When the dephosphorylation level of the substrate peptide is higher than when Ptprz is allowed to act in the absence of the test substance, the test substance is judged to promote the dephosphorylation action of Ptprz, and the test substance Is effective in promoting the activity of Ptprz.
(B) When the dephosphorylation level of the substrate peptide is lower than when Ptprz is allowed to act in the absence of the test substance, the test substance is judged to suppress the dephosphorylation action of Ptprz, and the test substance Is determined to be effective in suppressing Ptprz activity.

  When the determination criterion (a) is adopted, the test substance can be evaluated for the Ptprz activity promoting action. Therefore, it is effective when it is intended to screen for a Ptprz activity promoter. On the other hand, when the determination criterion (b) is adopted, the Ptprz activity inhibitory action of the test substance will be evaluated, and the Ptprz activity inhibitor can be screened.

  Regarding the evaluation method for substances that promote Ptprz activity or substances that inhibit Ptprz activity, if a standard accelerator or inhibitor is available in advance, its concentration (abundance) and dephosphorization It is also possible to obtain a relationship with the detection result of oxidation and compare the detection result of a new test substance against this. In this case, the activity of the substance that promotes the activity of Ptprz or the substance that suppresses the activity of Ptprz is evaluated as a relative value with respect to the reference substance.

  Detection of dephosphorylation of the substrate peptide may be performed over time to obtain a plurality of detection results having different elapsed times from the start of the test. In this case, the effectiveness of the test substance is determined based on the obtained plurality of detection results. Such detection and determination over time can provide a lot of useful information regarding the action of the test substance (for example, information useful for understanding the duration and action mechanism).

  Step (i) may be performed by setting a plurality of test groups having different test substance addition concentrations. In this case, in step (ii), the detection result of each test group is compared to determine the effectiveness of the test substance. In this way, more detailed information on the effectiveness of the test substance, such as concentration dependence, can be obtained.

  Examples of usefulness of the substance obtained by the screening method of the present invention are as follows: (1) Since Ptprz is involved in memory / learning through the control of neuronal synaptic plasticity, for example, an effect of improving memory / learning, (2) Since Ptprz is involved in monoamine neuronal function control, for example, depression, schizophrenia, drug dependence, (3) Ptprz's contribution to the severity of experimental autoimmune encephalomyelitis (4) Ptprz is involved as an essential receptor for gastric mucosal damage by the toxin protein VacA produced by Helicobacter pylori, for example, gastric mucosal protective action, (5) glioblastoma Since Ptprz contributes to malignant transformation, it has an antitumor inhibitory action, and (6) Ptprz is involved in pain sensitivity, and thus it is an analgesic action. When the selected substance has a sufficient medicinal effect, the substance can be used as it is as an active ingredient of a medicine. On the other hand, when it does not have a sufficient medicinal effect, it can be used as an active ingredient of a medicine after it has been modified by chemical modification to enhance its medicinal effect. Of course, even if it has a sufficient medicinal effect, the same modification may be applied for the purpose of further increasing the medicinal effect.

  The receptor tyrosine phosphatase Ptprz recognizes multiple protein molecules as substrates. Substrate molecules usually have multiple phosphorylated tyrosines. The following studies were conducted with the aim of identifying the dephosphorylation site of Ptprz.

1. Materials and Methods (1) Plasmid Rat-derived Git1 (G protein-coupled receptor kinase-interactor 1), mouse-derived Magi1 (membrane associated guanylate kinase, WW and PDZ domain containing 1) and mouse-derived PIST (protein interacting specifically with Tc10) Plasmids pFLAG-Git1 and pFLAG-Magi1 for expressing each molecule in mammalian cells were prepared according to the method described in the literature (Methods, 35, 54-63, 2005). Moreover, using these plasmids as templates, expression plasmids in which specific tyrosine residues were substituted with phenylalanine were prepared using QuickChange Multi Site-Directed Mutagenesis Kit (Stratagene).

(2) Cell culture and plasmid introduction
HEK293T cells were cultured in Dulbecco (DMEM) medium containing 10% normal bovine serum under conditions of 37 ° C. and 5% CO ₂ . The expression plasmid was introduced into HEK293T cells by a conventional calcium phosphate method.

(3) Vanadic acid treatment
36 hours after introducing the plasmid into HEK293T cells, the medium was changed to OPTI-MEM (Invitrogen), which is a serum-free medium, and Na ₃ VO ₄ (sodium vanadate, Sigma) was added at a final concentration of 100 μM, and 20 It was allowed to stand for minutes to induce phosphorylation of the substrate molecule. Vanadic acid inhibits tyrosine phosphatase nonspecifically.

(4) Protein extraction and immunoprecipitation Cell culture buffer (10 mM Tris-HCl, pH 7.4, 150) containing protease inhibitor cocktail (complete EDTA-free, Roche) in cultured cells after removal of the culture solution after vanadic acid treatment mM NaCl, 1% TritonX100, 1 mM Na ₃ VO ₄ , 10 mM NaF) was added, dissolved on ice for 30 minutes, and centrifuged at 10,000 g for 15 minutes to obtain a cell extract. Anti-FLAG antibody-binding beads (ANTI-FLAG M2 agarose, Sigma) were added to this cell extract, and mixed at 4 ° C. for 1 hour to adsorb the FLAG tag protein onto the beads. Nonspecific adsorption to the beads was thoroughly washed with TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.01% Tween 20). In the expressed protein derived from pFLAG-Git1 and pFLAG-Magi1, a FLAG tag is added to the N-terminus.

(5) Western blot analysis
The degree of tyrosine phosphorylation of FLAG-Git1 protein and FLAG-Magi1 protein was analyzed by Western blotting. Sample buffer for SDS-PAGE was added to the beads adsorbed with FLAG protein and eluted by heat treatment at 100 ° C. for 5 minutes. After separation by SDS-PAGE, it was transferred onto a PVDF membrane by a semi-dry method. This transcription membrane was blocked with TBST buffer containing 1% BSA for 1 hour, and then the protein retained on the PVDF membrane was detected by binding horseradish peroxidase (HRP) to PY20 monoclonal antibody that recognizes phosphorylated tyrosine. It was made to react with the antibody for use (GE healthcare). For detection of FLAG protein, FLAG M2 monoclonal antibody (Sigma) and HRP-conjugated anti-mouse antibody (secondary antibody for detection, GE Healthcare) were used. ECL plus (GE Healthcare) was used as the chromogenic substrate for HRP. An X-ray film (Kodak) was used for detection of chemiluminescence.

(6) Dephosphorylation assay of Git1 protein and Magi1 protein GST-PtprzICR, which is a fusion protein of glutathione S transferase (GST) and rat Ptprz in the entire intracellular region, was published in the literature (Proc. Natl. Acad. Sci. USA, 98, 2001, 6593-6598). The above immunoprecipitated bead sample was equilibrated with a buffer for PTP assay (25 mM HEPES, pH 6.8, 50 mM NaCl, 5 mM EDTA, 1 mM DTT, 50 μg / ml bovine serum albumin), and then a constant phosphorus was detected by Western blot. The amount of beads giving an oxidation signal was calculated and adjusted. 1 μg of GST-PtprzICR or GST (negative control) was added to 20 μl of this immunoprecipitation bead suspension, and a dephosphorylation reaction was performed at 37 ° C. for 20 minutes. The reaction was stopped by adding SDS-PAGE sample buffer. As a positive control for the dephosphorylation reaction, 1 mU alkaline phosphatase (AP, Toyobo) was used. When AP was used as an enzyme, immunoprecipitation bead samples were equilibrated with an AP assay buffer (100 mM Tris-HCl, pH 9.5, 00 mM NaCl, 50 mM MgCl ₂ , 50 μg / ml bovine serum albumin).

(7) Synthesis of artificial substrate peptide
From the 554th tyrosine residue (relative position 0) of Git1 identified as the dephosphorylation site of Ptprz, 4 residues (relative position -4 to -1) on the amino group (N) terminal side, carboxyl (C) An artificial peptide (7 amino acid residues) consisting of two residues on the terminal side (relative positions +1 to +2) was synthesized. In order to monitor dephosphorylation, an artificial peptide (pCAP peptide) into which an amino acid derivative phosphocoumaryl-amino-propionic acid (pCAP) mimicking this was substituted in place of phosphorylated tyrosine was synthesized. pCAP is a compound that generates fluorescence when dephosphorylated (Mitara. S., and Barrios, AM., Bioorg. Med. Chem. Lett .. 15, 5142-5145, 2005, WO 2007/014326). Synthesis of pCAP derivatives and pCAP-introduced peptides were performed based on literature (Bioorg. Med. Chem. Lett .. 15, 5142-5145, 2005). All synthesized peptides were purified by reverse phase chromatography (purity 90% or more).

(8) In vitro peptide dephosphorylation assay A three-component buffer (50 mM, Tris 50 mM Bis-Tris, 100 mM acetate) capable of examining pH conditions without changing salt strength was used as a basic buffer (J. Bio. Chem. 275, 18201-18209, 2001 and Methods in Enzumology, 87, 405-426). On the day of the measurement, DTT (5 mM) and Briji35 (0.01%) were added to the basic buffer adjusted to the target pH at the final concentration in parentheses to obtain an assay buffer. Each pCAP peptide was added to this at a final concentration of 50 μM, and stored on light and ice until use. This substrate solution was placed in a quartz cuvette for fluorescence measurement, set in a fluorescence spectrophotometer (F-4500, Hitachi), and allowed to stand for 10 minutes or more for equilibration with the temperature (30 ° C.) in the measurement chamber. After the temperature was equilibrated sufficiently, the aforementioned GST-PtprzICR was added at a final concentration of 5 nM and mixed by pipetting to start the dephosphorylation reaction. The fluorescence measurement was performed at an excitation wavelength (334 nm) and an emission wavelength (460 nm) based on the literature (AM., Bioorg. Med. Chem. Lett .. 15, 5142-5145, 2005). The dephosphorylation level was evaluated by the rate of increase in fluorescence intensity accompanying dephosphorylation of pCAP residues.

2. Results and Discussion (1) Identification of Ptprz dephosphorylation sites in Git1 and Magi1
Git1, Magi and PIST were isolated as Ptprz substrates using a yeast screening system named yeast Substrate-trapping system (Methods, 35, 54-63, 2005). There are multiple tyrosine phosphorylation sites in these molecules (Phospho Site Plus, http://www.phosphosite.org), but the dephosphorylation site of Ptprz was unknown. A plasmid expressing a YF mutant in which 10 tyrosine residues (Y) that may undergo phosphorylation modification in Git1 were substituted with phenylalanine (F) so as not to undergo phosphorylation modification was prepared (FIG. 1A). ). These plasmids were introduced into HEK cells to express Git1 and YF mutants, and phosphorylation was induced by vanadate treatment. As a result, under conditions where wild-type Git1 (Git1-WT) is significantly phosphorylated, phenylalanine substitution of all 10 tyrosine residues (Git1Y10F) does not detect phosphorylation, and positions 392, 554, or 607 It was found that reversion to tyrosine residues was phosphorylated (FIG. 1B). From this result, the main phosphorylation sites of Git1 were determined to be the 392rd, 554th, and 607th tyrosine residues.

  Recombinant PTP enzyme expressing and purifying the entire intracellular region of Ptprz was added to each phosphorylated Git1 protein obtained by immunoprecipitation. As a result, regarding the Git1-Y9F-Y554 protein, a marked decrease in phosphorylation level was observed (FIG. 1C), and the 554th tyrosine residue was determined to be the substrate site of Ptprz.

  For Magi1, a mutant lacking the region at the carboxy terminus was first prepared (FIG. 2A), and the dephosphorylation region of Ptprz was narrowed down. As a result, it was found that the mutant lacking the 789th and subsequent mutants (Magi1-ΔC3) was not subjected to dephosphorylation by the Ptprz enzyme despite being tyrosine phosphorylated (FIG. 2B). Furthermore, when a YF mutant was prepared for the 829th and 858th positions that could be phosphorylated in this region, the degree of dephosphorylation by Ptprz was significantly reduced when the 858th tyrosine residue was replaced. The 858th tyrosine residue was determined to be the dephosphorylation site of Ptprz.

(2) Identification of Ptprz substrate motif
An artificial substrate peptide was prepared based on the Git1 sequence, and a Ptprz enzyme assay system was constructed. As a result of examining the assay conditions, it was found that the optimum pH of Ptprz as an enzyme was very narrow as pH 6.5-7.0 (FIG. 3A). This indicates that pH 6.5, which has the highest enzyme activity, is desirable for the enzyme assay system of Ptprz, and that compounds and operations that cause pH changes have a strong influence on the enzyme activity of Ptprz.

  An attempt was made to find a good substrate motif of Ptprz using the identified Git1 dephosphorylation site and an artificial peptide containing the neighboring sequence (pCAP peptide). Degree of dephosphorylation of each substituted peptide (with -4 to -1, +1 amino acid substitution), based on the degree of dephosphorylation of the substrate peptide having the original sequence of Git1 (100) Was evaluated, the dephosphorylation level decreased in the peptides substituted with amino acids having different properties (FIG. 3B). Further, from this result, a good substrate peptide sequence of Ptprz was derived (FIG. 3C). In addition, for the -2 position, a slight decrease in activity was observed due to the substitution of A (alanine) to acidic amino acid D (aspartic acid), so other than D (aspartic acid) and E (glutamic acid) Amino acid residues are preferred. The wild-type Git1 sequence (top), where a good substrate site of Ptprz has been identified, the dephosphorylation site of Magi1, and the p190 RhoGAP already reported in the literature (Neurosci letts, 399, 33-38, 2006) When the dephosphorylation sites were aligned, high homology was observed among the three members (FIG. 3D). The substrate motif presented in the present invention includes the sequences of these plurality of substrate molecules, and is judged to scientifically support the usefulness of this substrate motif as a substrate peptide sequence for evaluating Ptprz activity. It was also found that PIST, which was known to be dephosphorylated by Ptprz but whose dephosphorylation site was unknown, contained a sequence that matched the Ptprz substrate site in the sequence (FIG. 3D). ).

3. Summary As described above, a motif that is a good substrate for Ptprz could be determined. The information on the substrate motif is useful for the construction of an assay method for measuring the activity of Ptprz and a screening method targeting an activity promoter or active male inhibitor of Ptprz.

  The activity measurement method and screening method of the present invention are useful as a means for searching and identifying a prophylactic / therapeutic agent for various diseases caused by Ptprz dysfunction. It is also useful as a tool for Ptprz research (for example, for function elucidation).

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (22)

The following sequence (SEQ ID NO: 1):

(However, the number above each amino acid residue in the formula represents the relative position from the amino acid residue Y (tyrosine) at the reference position, D / E represents D (aspartic acid) or E (glutamic acid), X Represents any amino acid residue, I / V represents I (isoleucine) or V (valine), and S / I / V represents S (serine), I (isoleucine) or V (valine))
A substrate motif of Ptprz consisting of   The substrate motif according to claim 1, wherein the amino acid residue at position -2 is an amino acid residue other than D (aspartic acid) and E (glutamic acid).   The substrate motif according to claim 1, wherein the amino acid residue at position -2 is A (alanine), N (asparagine), P (proline) or V (valine). The following sequence (SEQ ID NO: 2), ie

(However, the number above each amino acid residue in the formula represents a relative position from the reference position Z, Z is phosphorylated tyrosine or a phosphorylated tyrosine analog, and D / E is D (aspartic acid) or E (Glutamic acid), X represents any amino acid residue, I / V represents I (isoleucine) or V (valine), and S / I / V represents S (serine), I (isoleucine) or V ( Represents valine))
A substrate peptide of Ptprz.   The substrate peptide according to claim 4, wherein the amino acid residue at position -2 is an amino acid residue other than D (aspartic acid) and E (glutamic acid).   The substrate peptide according to claim 4, wherein the amino acid residue at position -2 is A (alanine), N (asparagine), P (proline) or V (valine).   The substrate peptide according to any one of claims 4 to 6, wherein the amino acid residue at position +1 is S (serine).   The substrate peptide according to claim 4, comprising any one of SEQ ID NOs: 3 to 6 as the sequence.   The substrate peptide according to any one of claims 4 to 8, wherein at least one amino acid residue is linked to the carboxy terminus of the sequence.   The substrate peptide according to claim 9, wherein the amino acid residue linked to the carboxy terminus is D (aspartic acid), E (glutamic acid), G (glycine) or F (phenylalanine).   The substrate peptide according to any one of claims 4 to 10, wherein at least one amino acid residue is linked to the amino terminus of the sequence.   The substrate peptide according to claim 11, wherein the amino acid residue linked to the amino terminus is V (valine), G (glycine) or A (alanine).   The substrate peptide according to any one of claims 4 to 12, comprising 5 to 12 residues.   The substrate peptide according to any one of claims 4 to 12, comprising 5 to 9 residues.   The substrate peptide according to claim 4, comprising the amino acid sequence of any one of SEQ ID NOs: 3 to 38.   The substrate peptide according to any one of claims 4 to 15, wherein the phosphorylated tyrosine analog is a coumarin derivative.   The substrate peptide according to any one of claims 4 to 15, wherein the phosphorylated tyrosine analog is phosphocoumaryl-amino-propionic acid. Ptprz activity measurement method comprising the following steps (1) and (2):
(1) A step of causing a sample to act on the substrate peptide according to any one of claims 4 to 17;
(2) A step of detecting dephosphorylation of the substrate peptide.   The method for measuring Ptprz activity according to claim 18, wherein the sample is Ptprz or a Ptprz-like molecule. A screening method for a Ptprz activity modulator comprising the following steps (i) and (ii):
(I) a step of causing Ptprz to act on the substrate peptide according to any one of claims 4 to 17 in the presence of a test substance;
(Ii) detecting the dephosphorylation of the substrate peptide and determining the effectiveness of the test substance based on the detection result.   When the dephosphorylation level of the substrate peptide is high compared to the case where Ptprz is allowed to act in the absence of the test substance, it is determined that the test substance promotes the dephosphorylation action of Ptprz, and the test substance is Ptprz The screening method according to claim 20, wherein the screening method is determined to be effective for promoting activity.   When the dephosphorylation level of the substrate peptide is low compared to the case where Ptprz is allowed to act in the absence of the test substance, it is determined that the test substance suppresses the dephosphorylation action of Ptprz, and the test substance is Ptprz The screening method according to claim 20, wherein the screening method is determined to be effective for activity suppression.

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