KR101493737B1 - A screening method of a material inducing or inhibiting pin1 enzyme activation - Google Patents

Abstract

The present invention relates to a method for screening a substance inducing or inhibiting activation of Pin1 enzyme using Pin1 enzyme, and more particularly, a method for screening a substance that induces or inhibits activation of Pin1 enzyme through an enzyme-substrate reaction, Inducing or activating inhibitory substance. According to the screening method of the present invention, it is possible to select the target drug efficiently by measuring the activity of the enzyme by a simple experiment and screening a large amount of substances inducing or inhibiting activation of the Pin1 enzyme.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for screening a PIN1 enzyme activation inducing or activating inhibitor,

The present invention relates to a method for screening a substance that induces activation or activation of Pin1 enzyme using Pin1 enzyme and more specifically to a method for screening a substance that induces or inhibits activation of Pin1 enzyme through an enzyme- To a method of screening for an activating or inhibiting substance.

Cystic dysplasia (CCD, OMIM 119600) is a congenital osteoarthritis of the skull, including hypoplastic cysts or aplastic clavicles, delayed suture closure, short stature, Other skeletal anomalies are characterized by them (Jarvis and Keats, 1974).

CCD is caused by haploinsufficiency of Runx2 (Mundlos et al., 1997) (Mundlos et al., 1997). Runx2 (runt related transcription factor 2) is a pentrant allele, but its allele frequency has a highly variable gene expression in terms of CCD symptoms (Golan et al., 2000; Zhou et al., 1999). However, many mutations among Runx2 alleles have been found to reduce its mRNA half-life, interfere with its transcriptional promoting domain, and decrease its DNA binding activity (Yoshida et al., 2002; Yoshida et al., 2003). However, the absence of obvious genotype-phenotypic correlations and the large number of distinct CCD phenotypes present in groups with a single Runx2 mutation remain unexplained. Nevertheless, the bone formation in CCD model Runx2 It is known to be very sensitive to dosage (Lou et al., 2009). In fact, changes in gene dosage of most Runt-related transcription factors are a common regulatory mechanism in the pathogenesis of many human diseases, including cancer (Osato et al., 1999; Song et al., 1999). This mechanism plays a role in determining Drosophila gender (Duffy and Gergen, 1991).

Mouse genetic studies have shown that Runx2 dose is an important determinant of the penetration of the CCD phenotype (Lou et al., 2009) (Lou et al., 2009). The study reported that a 70% reduction in the genotype Runx2 level could produce the CCD syndrome. This finding suggests that the range of bone phenotypes observed in CCD patients may be due to a quantitative reduction in the functional activity of Runx2. Furthermore, the genetic deficiency of Runx2 can be overcome by osteoblast-specific overexpression of structurally active MEK1 (MEK1-Ca) and exaggerated by the expression of dominant-negative MEK1 (Ge et al., 2007) Has also been reported. Taken together, these results indicate that ERK / MAPK-induced phosphorylation of Runx2 can stimulate its transcriptional activity, thereby blocking the effect of the genetic deficiency of Runx2 heterozygotes.

Pin1 (peptidylprolyl cis / trans isomerase, NIMA-interacting 1) is a member of the peptidyl-prolyl cis-trans isomer (PPIase) superfamily and forms a cis- conformations, thus altering the mode of its acquisition (Lu et al., 2007; Lu and Zhou, 2007; Yeh and Means, 2007). Pin1 specifically recognizes sequence motifs (pS / TP motif, proline-induced phosphorylation) that contain proline or phosphorothionine followed by proline among the substrates having the N-terminal WW domain (Lu et al., 2007 Lu and Zhou, 2007). A variety of Pin1 protein substrates have been identified to date and many of them are essential to organisms. The biological significance of Pin1 means that this is highly regulated. Pin1 target sequences are shared by several protein kinases including ERK, CDK and GSK3a (Hsu et al., 2001; Monje et al., 2005; Munoz et al., 2007; Patra et al., 1999; Yeh et al., 2004; ), Pin1 is closely related to cell signaling and Ser / Thr kinase activity. Thus, ERK-phosphorylated Runx2 may be the target of Pin1-mediated morphological and functional changes.

Meanwhile, the present inventors have previously shown that FGF-signaling is associated with acetylation and ubiquitination (Jeon et al., 2006; Park et al., 2010), promoting Runx2 phosphorylation and stimulating its transcriptional promoting activity (Kim et al., 2006a; Kim et al., 2003), it remains unclear how such phosphorylation can be linked to increased protein activity and stabilization.

KR 10-2006-0067895 A

The inventors of the present invention can interact with the Pin1 enzyme when the Runx2 protein is phosphorylated and the structural change of the phosphorylated phosphorylated Runx2 protein by the Pin1 enzyme increases the p300 mediated acetylation through Runx2 The activity of the protein is increased and the stability of the protein is substantially increased. Thus, even if the protein is substantially expressed in the Runx2 protein, the active regulation by the Pin1 enzyme can produce the same effect as that of the full Runx2 protein activity. In addition, since the Pin1 enzyme can interact with Runx family proteins such as Runx1 and Runx3 as well as Runx2, the activity regulating drug for Pin1 enzyme results in the role of the osteogenesis induction, hematopoiesis and anticancer activities involved in the Runx family protein . Based on these results, the present invention provides a method for screening a substance that inhibits activation or activation of Pin1 enzyme.

In order to solve the above problems, the present invention provides a method for screening a substance inducing activation or inhibition of activation of a Pin1 enzyme, which comprises exposing a candidate substance to a Pin1 enzyme and measuring activation of the Pin1 enzyme.

The activation of the Pin1 enzyme is preferably measured through an enzyme-substrate reaction.

The substrate used in the enzyme-substrate reaction is preferably an oligopeptide bound with a fluorescent substance.

The fluorescent material is preferably luciferin.

The oligopeptide is preferably an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

The enzyme acting on the substrate is preferably chymotrypsin.

The screening method comprises: (a) reacting a Pin1 enzyme, a substrate bound with a fluorescent substance, and a candidate substance; (b) treating chymotrypsin; And (c) detecting fluorescence.

The Pin1 enzyme activation inducer is preferably a candidate substance for the prevention or treatment of an abnormal bone tissue disorder.

It is preferable that the abnormal bone tissue disorder is cleidocranial dysplasia (CCD) or craniosynostosis.

The Pin1 enzyme activation inducer is preferably a candidate substance for preventing or treating a hematopoietic disorder disease.

It is preferable that the substance inducing or activating activation of the Pin1 enzyme is a candidate substance for the treatment of cancer, Alzheimer's disease or demented geriatric lesion.

According to the screening method of the present invention, enzymatic activity is measured by a simple experiment to quantitatively screen substances that induce activation or activation of Pin1 enzyme. Thus, osteoporosis diseases, hematopoietic disorders, anticancer agents, Alzheimer's disease and demented geriatric lesions It is possible to select an effective target drug as a therapeutic agent.

Figure 1 shows the result of showing the appearance of damaged skeletal growth in the Pin1 mutant embryo.
Figure 2 shows the result of Pin1 interacting with the C-terminal domain of Runx2 in a phosphorylation dependent manner.
Figure 3 shows that Pin1 induces Runx2 protein into the intranuclear region.
Fig. 4 shows the result that Pin1 stabilizes Runx2.
Figure 5 shows that the Pin1 mediated structural change of phosphorylated Runx2 is a target of FGF signaling in osteoblast differentiation.
Fig. 6 shows the results showing that Pin1 is essential for the trans-activation of Runx2.
FIG. 7 is a schematic diagram showing the manner of the bidirectional regulation of Runx2 in the bone formation process. FIG.
8 is a schematic diagram showing that Pin1 is involved in phosphorylation and acetylation of Runx2.
FIG. 9 is a diagram schematically showing the enzyme-substrate reaction used in the method for screening or inhibiting Pin1 enzyme activation of the present invention. 1 indicates a peptide to be a substrate, 2 indicates succinic acid, 3 indicates aminorhprerin, 4 indicates that isomerization is changed due to introduction and reaction of isomerizing enzyme, and 5 indicates that further added chymotrypsin separates luciferin Lt; RTI ID = 0.0 > luciferin < / RTI >
FIG. 10 is a sequence chart showing a screening method for the Pin1 enzyme activation inducing or activating inhibitory substance of the present invention.

The present invention is based on the conclusion that the Pin1 enzyme structurally changes the Runx2 protein (p-Runx2) phosphorylated by the FGF2 signal to Cis-trans to increase the protein activity and stability of p-Runx2, The amino acid sequence of the C-terminus of Runx family proteins such as Runx1, Runx2 and Runx3, to which Pin1 can bind, was applied as a substrate for the enzyme-substrate reaction of the method for screening or activating the Pin1 enzyme of the present invention.

Therefore, the Pin1 enzyme increases the activity and intracellular stability of Runx1, the major gene of hematopoiesis, Runx2, the major gene of bone formation, and Runx3, which is an anticancer activity, and increases the activation of Pin1 enzyme or Runx1, Runx2, Runx3 protein The increase of the interaction between the two is possible to induce recovery to a normal state even in a pathological state in which the amount of Runx1, Runx2 and Runx3 is low. Various diseases related to bone formation and bone metabolism, diseases with hematopoietic disorders and cancer, Alzheimer's disease, It can serve as a preventive or remedy for lesions.

Accordingly, the present invention provides a screening method for activating or inhibiting activation of Pin1 enzyme, which comprises exposing a candidate substance to a Pin1 enzyme and measuring activation of the Pin1 enzyme.

The induction or inhibition of activation of the Pin1 enzyme is preferably measured through an enzyme-substrate reaction. The substrate used in the enzyme-substrate reaction is preferably an oligopeptide bound with a fluorescent substance. The fluorescent material is preferably luciferin. The oligopeptide is preferably an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: The enzyme acting on the substrate is preferably chymotrypsin. The method of the present invention comprises the steps of (a) mixing a Pin1 enzyme, a substrate bound with a fluorescent substance and a candidate substance; (b) introducing chymotrypsin; And (c) detecting fluorescence.

As a preferable example, the method for screening the activation or inhibition of Pin1 enzyme of the present invention is a substance for measuring the activity of Pin1, which is isomerase, and the part which becomes the substrate of the isomerization enzyme before the fluorescent substance (luciferin) When the substance X capable of inducing the activity of the isomerization enzyme is added, the structure of the substance is changed by the Cis-Trans isomerization so that the fluorescent substance is expressed, whereby the substance X is converted into the isomerization enzyme The activity of the substance can be visually and quantitatively confirmed.

The amino acid sequence of the fluorophore-linked oligopeptide used as the substrate may be Suc-Ala-Xaa-Pro-Phe- (4 -) - luciferin. Here, Suc represents succinic acid and is intended to protect the amino acid sequence that follows. The four amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 5 are Ala-Xaa-Pro-Phe wherein Xaa is alanine, leucine, phenylalanine, glutamic acid, or aspartic acid acid. luciferin is a substrate of luciferase that reacts to fluorescence and is a fluorescent substance widely used as a common assay tool.

Isomerase is an enzyme that catalyzes the process of converting one molecule (compound, protein, etc.) into an isomer of the same chemical formula but structurally different. In the present invention, specifically, Pin 1 isomerase (Peptidyl prolyl cis / trans isomerase 1 ), But it can also be used to measure the activity of other isomerases by substituting the specific ligand site of the enzyme.

The Pin1 enzyme is known to be involved in the degeneration of tau protein, a pathological target of Alzheimer's disease, and alleviates its pathological progress. It induces structural changes of various proteins involved in cell death and proliferation, And is an isomerase that is continuously increasing in interest. The present inventors have also recognized that it is important to screen for a substance capable of increasing or inhibiting the activity of Pin1 enzyme based on various results that bone formation can be promoted by the Pin1 enzyme, Design.

The isomerase mainly interacts with a protein having the sequence of a specific amino acid sequence as its substrate and reversibly changes its structure into a Cis-Trans optical isomer form. The sequence that becomes the substrate varies depending on the isomerization enzymes, but the Pin1 enzyme has fewer than 10 short peptide sequences, and Alanine (A) -Glutamic Acid (E) -Prolin (P) -Phenylalanine , It is known that Prolin and Phnylalanine have a Cis structure, and when this structure is reacted by isomerizing enzyme, it only induces isomeric changes in the trans structure without changing the other residues. In addition, the sequence of AAAPF, in which the number of Alanin (A) -Leucine (L) -Proline (P) -Phenylalanine (F), AD (Aspartic acid) -PF, AA Mechanism is known.

A variety of screening methods for drugs targeting a specific protein are used, but representative methods include a method in which a marker is separated or activated when the protein is activated by a drug. These markers can be used to express specific colors through enzymatic reactions or to absorb fluorescence by absorbing light. Among them, luciferin can easily measure the activity of proteins through the presence or absence of fluorescence, and the intensity of fluorescence , It is possible to measure the degree of activity of the protein to a very sensitive level, and it can be easily assayed on a 96-well plate, so that it is advantageously used for analyzing and comparing various kinds of drugs at once.

In the method for screening or activating the Pin1 enzyme of the present invention, luciferin is linked to a Cis structure followed by a peptide sequence serving as a substrate, a chimotrypsin as a Pin1 enzyme and a peptide degrading enzyme is prepared in a single well, The experiment is completed by treating the drug candidate (X), which is expected to induce or inhibit activation of the Pin1 enzyme, to effect the reaction. When the drug candidate X has the ability to activate the Pin1 enzyme, the Pin1 enzyme activated by X binds to the peptide sequence of the substrate bound to the fluorescent substance to change the Cis structure to the Trans structure, The ligation site of chymotrypsin which is hidden by the fluorescent moiety becomes accessible in the Trans structure, and luciferin, which is a fluorescent substance, is separated from the substrate bound to the fluorescent substance. The luciferase assay kit is used to measure the fluorescence intensity according to the amount of luciferin. In addition, when the drug candidate X has the ability to inhibit the activation of the Pin1 enzyme, it can be confirmed that the activity of the enzyme is lowered and the fluorescence decreases as compared with the control group of the drug candidate X-treated.

Hereinafter, the present invention will be described in more detail with reference to specific examples. An embodiment of the present invention is schematically illustrated in Fig.

[ Example ]

One. Runx2 In the stabilization of Pin1 And Runx2 Wow Pin1 Identification of interaction

1-1. Materials and methods

1-1-1. Mouse system

Pin1 and Runx2 deficient mice were derived from the same background system, as described in the prior art (Fujimori et al., 1999; Komori et al., 1997). Runx2 + / + Pin1 +/- and Runx2 +/- Pin1 +/- mice were obtained from heterozygous Runx2 and Pin1 mice and Runx2 + / + Pin1 +/- and Runx2 +/- Pin1 +/- mice were obtained from Runx2 and Pin1 mice for Runx2 and Pin1 genetic interaction analysis. - The offspring were successively produced from the crossbreeding between the mice. All animal experiments were conducted with approval from Seoul National University Institute of Animal Resources and Use Committee.

1-1-2. Cell culture, transfection and reagents

MC3T3-E1 cells were maintained in α-minimal essential medium (MEM) and HEK-293 and C2C12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM / 10% FBS) with 10% heat-inactivated fetal serum supplemented with antibiotics Respectively. Mouse embryonic fibroblasts (MEFs) were isolated from E13.5 embryos with the Pin1 + / +, Pin1 +/- and Pin1 - / - genotypes as described in the prior art (Fujimori et al., 1999). MEFs were cultured in DMEM / 10% FBS and 3-5 cells were used. For osteogenic cultures of MC3T3-E1, the medium was augmented with ascorbic acid and beta -glycerophosphate.

1-1-3. Transfection

Neon transfection systems (Invitrogen, San Diego, CA) were used for siRNA and DNA transfection into MC3T3-E1 or C2C12 cells. Co-transfection experiments were performed using D-Fection reagent (Lugen Sci, Korea) and transfection of HEK-293 cells was performed according to the manufacturer's description using Polyget reagent (SignaGen Laboratory) .

1-1-4. Pin1 Knockdown

SMART pool siRNAs, a mixture of four rodent Pin1 siRNAs, were designed and purchased from Dharmacon (Chicago, IL). Non-targeted siRNA was also purchased from Dharmacon and used as a negative control.

1-1-5. RNA  Extraction and Quantitative Real-time PCR ( qPCR ) Expression analysis

Total RNA was extracted from the entire femurs of the newborn mice using an easy-BLUE (TM) total RNA extraction kit (homogenized by shredding in liquid nitrogen). SuperScript II First Strand Synthesis System cDNAs were synthesized from 1 g of total RNA using a reverse transcription kit (Invitrogen). Approximately 100 ng of RNA was used for each reaction in the relative qPCR.

1-1-6. GST  Full-down analysis, Immune sedimentation  ( IP ) US Immunoblot  analysis

Cell extracts were resuspended in 50 mM HEPES (pH 7.5), 150 mM NaCl, 100 mM NaF, 1 mM DTT, 1 mM EDTA, 0.25% Na-deoxycholate supplemented with protease and phosphatase inhibitors containing Na ₃ VO ₄ , 0.25% CHAPS, 1% NP-40 and 10% glycerol lysis buffer. For acetylation and protein stabilization assays, the buffer was supplemented with 1 mM NaB or 1 M MG132. For immunoblot analyzes, cells were lysed in a buffer containing 0.2% SDS. Recombinant GST or GST-Pin1 protein (2 [mu] g) was incubated with MC3T3-E1 or lysate containing Runx2 proteins from transiently transfected HEK-293 cells for 2 hours at 4 [deg.] C. Then, glutathione agarose beads were added and the mixture was incubated for 30 minutes. To examine phosphorylation-dependent interactions, cells were lysed in binding buffer without phosphatase inhibitor and the resulting cell extracts were incubated with lambda phosphatase for 30 minutes at room temperature prior to GST-Pin1 pull-down analysis. The precipitated protein was then analyzed by immunoblot detection using specific antibodies. Runx2 and MEK1-Ca were transiently expressed in HEK-293 cells in combination with the co-vector Pin1-WT or Pin1-W34A for co-IP of Runx2 and Pin1. Cell lysates were incubated with anti-Pin1 antibody for 3 hours, agarose beads were added, and the entire mixture was incubated for 1 hour. The co-purified protein was then subjected to immunoblot analysis using specific antibodies. Detection of acetylated or phosphorylated Runx2 was performed using the same IP conditions.

1-1-7. Runx2 of Ubiquitination  And Acetylation

For the analysis of ubiquitinated and acetylated Runx2 in whole cell or subcellular fraction of MEF cells, analysis was carried out under denaturing conditions using urea lysis buffer. Briefly, whole cells or nuclear fractions expressing the His-Runx2 protein were completely dissolved in the urea lysis buffer and subsequently added to the NTA purification column as described in the prior art (Li et al., 2008). For the specific detection of ubiquitin-conjugates, FLAG-Ub constructs were used. Affinity-purified proteins were analyzed by immunoblotting with anti-FLAG or anti-acetyl lysine antibody.

1-1-8. Runx2 Promoting activity of

The transcriptional activity of Runx2 was measured using a 6xOSE2-Luc reporter plasmid, as described in the prior art (Kim et al., 2003). MC3T3-E1 cells were transiently transfected with the Runx2 and 6xOSE2-Luc reporter plasmids using either the empty vector or the MEK1-Ca expression vector with or without Pin1 overexpression. After 24 hours of transfection, luciferase activity was measured. Alternatively, C2C12 cells stably transfected with the 6xOSE2-Luc reporter vector were transiently transfected using a blank vector or using Pin1-WT or Pin1-Y23A expression vectors. After 18 hours of transfection, cells were stimulated with 20 ng / ml FGF2 for 24 hours and the resulting luciferase activity was then measured.

1-1-9. Systemic skeletal staining and immunostaining

Systemic skeletal staining of cartilage and bone was performed as described in the prior art (McLeod, 1980) using alcian blue and alizarin red S. For histological evaluation, bones were fixed in 4% formaldehyde in PBS, de-calcined in 10% EDTA, dehydrated and embedded in paraffin. Sections of 5-10 mu m were prepared, deparaffinized and rehydrated. The tissue sections were then stained with H & E or used for immunostaining fluorescence.

Detection of co-localized Pin1 and Runx2 required antigen retrieval. Briefly, this included immobilizing the samples in 4% formaldehyde and boiling them in Tris-EDTA buffer with 5% urea for 10 minutes.

1-1-10. Statistical analysis

All quantitative data are expressed as means ± SD. Each experiment was performed at least 4 times and shows the results from one representative experiment. Statistical differences were analyzed using Student's t test. Values of p <0.05 were considered statistically significant. Gene data were evaluated using the chi-square (χ ² ) test and IBP SPSS Statistics 19.0 software.

1-2. result

1-2-1. Pin1 By mutation of Runx2 -relation CCD  Phenotype

Pin1 +/- and Pin1 - / - mice exhibit a wide range of bone phenotypes at birth. Pin1 follows normal Mendelian trait rules and exhibits normal skeletal patterning just prior to birth, but these mice are irregular and undergo significant postnatal mortality (<35% survival in the case of Pin1 - / - mice). Mutant mice were taller (FIG. IA) and exhibited variable skeletal phenotypes associated with hypo-mineralization across the skeleton (FIG. 1B). The bones of the calvarial bone (FIG. 1C), collarbone (FIG. 1D) and paw (FIG. 1E) and hind paw (FIG. 1F) were obviously affected. Bone phenotypes observed in Pin1 +/- and Pin1 - / - mice were similar to those typically observed in human patients with Runx2 +/- mice and CCD (Table 1).

The majority of homozygous and heterozygous Pin1 mutant mice had widely open fontanels and / or dysplastic clavicles. The epiphyseal growth plate of the neonatal Pin1-deficient mice exhibited spatial irregularity in the chondrocyte column and the proliferative and hypertrophic zones were short (Fig. 1G). In tibial metaphysis, Pin1-deficient mice were observed to have reduced histological staining of the extracellular matrix (Fig. 1H, pink) and loss of immune fluorescent staining of type I collagen (Fig. 1I). Similarities in craniofacial defects observed in Pin1-deficient and Runx2-deficient mutant mice suggest that these two proteins are functionally linked during the bone development process. Immunofluorescent staining of Runx2 protein has indeed demonstrated dramatic reduction of Runx2 levels in the developing bone of Pin1 null mice (Fig. 1J), suggesting that Pin1 is required for Runx2 protein expression. In addition, mRNA levels of four typical bone markers and Runx2 target genes, Col1A1 , alkaline phosphatase ( Alp ), osteocalcin ( Oc ) and bone-forming transcription factors, Dlx5 , decreased dramatically in Pin1-bearing mouse tibia . However, the reduced Runx2 in Pin1 member mutations mRNA levels were not sufficient to account for the observed CCD phenotype (Figure 1K), suggesting that the phenotype of Pin1-bearing mice is the result of translation or post-translational modification of Runx2.

1-2-2. Phosphorylation-dependent Pin1  For binding Target Runx2  C-terminus

Pin1 is known to interact directly with proteins containing the phosphopercoline-proline motif (Lu et al., 1999; Ranganathan et al., 1997; Yaffe et al., 1997). Runx2 is phosphorylated at that position (Ge et al., 2009; Ge et al., 2011; Kim et al., 2006a; Rajgopal et al., 2007; Wee et al., 2002; Xiao et al., 2002) Respectively. As expected, the endogenous Runx2 protein from differentiated MC3T3-E1 osteoblasts was pulled down using recombinant glutathione-S-transferase (GST) -Pin1 fusion protein, which resulted in Runx2 and Pin1 Complexes. Runx2 was shown to be phosphorylated by activation of the FGF receptor (FGFR) and the effect of FGF2 / FGFR signaling on the Runx2-Pin1 interaction (Kim et al., 2003) was tested. As shown in Figure 2B, administration of FGF2 stimulated the binding of Pin1 to Runx2 (Figure 2B). Similarly, overexpression of MEK, a component of the structurally activated FGFR signaling pathway, also increased the binding of Pin1 to Runx2 with an increase in p-Erk levels (data not shown). Conversely, dephosphorylation of Runx2 by lambda-phosphatase (lambda-P) strongly attenuated the interaction between the two proteins (Fig. 2C), indicating that this interaction occurs in a phosphorylation-dependent manner . Co-expression and immunoprecipitation assays of epitope-tagged wild-type Pin1 (WT) and Runx2 proteins revealed that they acted in vivo in mammalian cells. The Pin1-W34A mutation (WW domain mutation) failed to interact with Runx2 (Figure 2D), suggesting that the WW domain of Pin1 is critical to its interaction with Runx2, as in most other Pin1 interactions do.

Runx1 and Runx3, closely related Runt-domain transcription factors such as Runx2, also bind to Pin1 (Figure 2E). Virtual Experiment (In silico analysis was performed to identify the conserved pS / TP motifs in Runx proteins. Twelve putative WW domain target motifs were identified in Runx2, seven of which were well conserved across all Runx proteins (Figure 2F and Table 2).

These seven conserved target positions were then examined to identify the common motifs required for the interaction of Runx proteins with Pin1. Four of the conserved pS / T-P motifs were removed, while one C-terminal deletion preserving the other three conserved motifs showed strongly reduced Pin1 binding (FIG. 2G). Similarly, of these four motifs, substitution of all four serine or threonine residues with alanine residues (Runx2-4AP) completely abolished the interaction between Runx2 and Pin1 (Figure 2H).

1-2-3. Pin1 On by Runx2 of Into the nucleus  move

In early studies, this region has been shown to play a crucial role in bone physiology, as mice lacking the C-terminal region of Runx2 exhibit similar bone phenotypes to those of Runx2 knockout mice (Choi et al., 2001) . Here we have also observed that Pin1 controls the nuclear accumulation of Runx2, particularly at the speckle-like nucleus centers of Runx2. Over time observation of the distribution of both proteins through viable cell imaging showed that nuclear complexes positive for Runx2 and Pin1 were clearly co-localized (Fig. 3A), furthermore, Pin1 staining was observed at the same centers Accelerated accumulation of Runx2. This finding demonstrates that localization at the nucleus of a particular Pin1 region determines the pattern of Runx2 centers accumulation. Previously, Pin1 was thought to be localized to the nucleus almost exclusively and focused on distinct nuclear staining structures in the intrinsic domain, which were either less compacted in the absence of catalytic activity or completely absent in the absence of the WW domain (Rippmann et al., 2000). Thus, our data indicate that Pin1 activity promotes nuclear accumulation of Runx2 protein. We will now discuss the question of whether the accumulated Runx2 proteins require Pin1 activity. As shown in Figure 3B, wild-type Pin1 clearly induced the focal accumulation of Runx2 in transiently transfected MC3T3-E1 cells expressing fluorescently-tagged Runx2 and Pin1. In contrast, either induces nuclear accumulation of Runx2 through overexpression of either catalytically inactive forms of Pin1 (Pin1-C113A, Figure 3C) or Pin1 (Pin1-Y23A, Figure 3D) with a binding motif mutation I could not.

1-2-4. Pin1 - dependency Ploll Isomerization  by Runx2  stabilize

Inhibition of Pin1 enzyme activity by a potent inhibitor of dipentamethylenetriamine monosulphide (DTM) or hypoglycemia (Tatara et al., 2009) significantly reduces endogenous Runx2 levels in MC3T3-E1 cells 4A), supporting the hypothesis that loss of Pin1 activity decreases Runx2 protein expression (see FIG. 1J). Expression of mouse embryonic fibroblast (MEFs) epitope-tagged Runx2 from Pin1 + / +, Pin1 +/- and Pin1 - / - mice showed that Runx2 expression decreased with Pin1 expression, which decreased Runx2 protein accumulation (FIG. 4B). Pin1 has been found to be an enzyme that determines the fate of Runx2 accumulation, which is a transient transfection of Pin1 siRNA that nearly destroys endogenous Pin1 protein expression and strongly down-regulates the normal Myc-Runx2 level (Fig. 4C). Furthermore, co-expression of wild-type Pin1 (Pin1-WT) with Runx2 could increase Runx2 protein levels (Figure 4D). Thus, the dielectric loss of Pin1 reduces Runx2 levels (Fig. 1J), which indicates that the pyrrole isomerization activity of Pin1 modifies the fate-determining step of Runx2. Pin1 overexpression increased Runx2 steady state protein levels by inhibiting Runx2 degradation after the new Runx2 protein synthesis was blocked by the protein synthesis inhibitor cycloheximide (CHX, FIG. 4E). The decay curves shown by the epitope-labeled Runx2 protein expressed before protein synthesis inhibition showed a Runx2 half-life of approximately 4 to 6 hours, consistent with previous reports (Jeon et al., 2006; Shen et al., 2006) . The co-expression of Pin1 prevented Runx2 protein degradation, since Runx2 protein levels were not changed after 6 hours (Fig. 4E). Furthermore, mutations of the four Pin1 interaction sites (Rx2-4AP; see Figure 2H) at the Runx2 C-terminus greatly reduced Runx2 stability (Figure 4F). Confirming these key findings, co-expression of wild-type Pin1 inhibited ubiquitination of Runx2, while expression of catalytically inactive Pin1 (C113A) did not inhibit ubiquitination (Fig. 4G). Thus, Pin1 stabilizes Runx2 by reducing its ubiquitination and degradation.

1-2-5. Pin1 - Mediated Ploll Isomerization  by FGF2 / MAPK In signaling Runx2  stabilize

Activated FGF receptor stimulation of FGF2 or Erk-MAP kinase is crucial to the pathogenesis of cingulate anatomy (Shukla et al., 2007) and clavicle skull formation abnormalities (Ge et al., 2007). Here, immunofluorescence studies demonstrate that co-localization of endogenous Pin1 and Runx2 is highly inducible in the nuclei of MC3T3-E1 cells, followed by FGF2 (FIG. 5A). FGF2 treatment of MEFs from Pin1 + / + or Pin1 - / - mice stimulated Erk phosphorylation (FIG. 5B), indicating that Erk activation was not affected by the absence of Pin1. In contrast, the expression of Runx2 downstream gene osteocalcin was strongly enhanced by FGF2 treatment in Pin1 + / + cells, but attenuated by FGF2 treatment among Pin1 - / - cells (Fig. 5C). These results suggest that the FGF2-stimulated Erk pathway and Runx2 phosphorylation are intact in Pin member mice, indicating that phosphorylation occurs prior to prolyl isomerization and MAPK-dependent phosphorylation of the S / TP 2 peptide is the target of Pin1 Hsu et al., 2001; Monje et al., 2005; Yeh et al., 2004). Therefore, the present inventors have tested the ability of MEK1 to increase the level of Runx2 protein, and it is known that MEK1 acts downstream of FGF2 and stimulates ERK / MAP kinase. Co-expression of MEK1-Ca increased both Erk phosphorylation and Runx2 expression (Fig. 5D). Based on the results shown in Figures 5D (lanes 2 to 3) and 5E (lanes 3 to 3), endogenous Pin1 was sufficient to support Runx2 stabilization after its phosphorylation. Expression of MEK1-Ca with Pin1-C113A, but not wild-type Pin1, was found to eliminate MEK-dependence at Runx2 protein levels. The Pin1-C113A mutation reduces the level of Runx2 below the level observed in the control cells, indicating that this mutation interferes with the stabilizing activity of the endogenous Pin1 (Fig. 5E).

The ubiquitination of Runx2 is inhibited by p300-mediated acetylation and promoted by HDAC-dependent deacetylation (Jeon et al., 2006). Since Pin1 inhibits Runx2 ubiquitination (see FIG. 4G), we tested the importance of Runx2 acetylation through the activity of the pyrroloisotrophic agent of Pin1. The low glone-induced inhibition of Pin1 isomerization activity strongly reduced the level of acetylated Runx2 (Fig. 5F), supporting the hypothesis that Pin1 function loss can destabilize Runx2 (see Fig. 4C). Runx2 acetylation levels are dramatically upregulated by wild-type Pin1, but not by Pin1-C113A (Fig. 5G). Pin1-dependent acetylation has also been demonstrated by Runx1 and Runx3, both of which are conserved lysine and S / T-P 2 peptides. Expression of wild-type Pin1, not Pin1-C113A, increased acetylation of all three Runx proteins (Fig. 5G).

Skull development and suture closure involve FGF-signaling, Erk activation, and Runx2 activity, and we tested the occurrence of crossover in FGFR2 mutations caused by changes in Runx2 acetylation. While overexpression of wild type FGFR2 did not affect Runx2 acetylation, overexpression of the structurally active FGFR2 mutation (S354C) significantly increased Runx2 acetylation. However, treatment with the Pin1 inhibitor DTM attenuated this FGFR2 mutation-mediated increase in Runx2 acetylation (Figure 5H). We also found that Runx2 expression levels reflect their acetylation (data not shown). Taken together, these results indicate that Pin1-mediated regulation and stabilization of Runx2 acetylation is controlled by multiple post-translational cascade reactions that occur in the order of phosphorylation, prolyl isomerization and acetylation 5J).

1-2-6. Pin1 On by Runx2 Promoting transcriptional activity

It has previously been known that osteoblast-specific component 2 (OSE2) selectively and / or synergistically responds to FGF stimuli that are located within the promoter region of the osteocalcin (Geoffroy et al., 1995) (Boguslawski et al., 2000; Boudreaux And Towler, 1996; Newberry et al., 1996; Xiao et al., 2002). Such a reaction requires precise control of phosphorylation and cooperation with various proteins including common transcription factors, co-activators and chromatin altering enzymes (Lee et al., 1999; Montecino et al., 1999; Sierra et al., 2003 ). These findings suggested that Pin1-mediated activation of Runx2 and nuclear localization may be linked to its transcriptional promoting activity. Indeed, overexpression of MEK1-Ca or Runx2 independently enhanced 6xOSE2-Luc reporter activity in C2C12 cells, and co-expression of these two components synergistically stimulated Luc reporter activity (Fig. 6A). The low glone-induced inhibition of Pin1 markedly inhibited the transcriptional promoting activity of Runx2 from MEK1-Ca overexpression (Figure 6A). In addition, FGF2 treatment stimulated 5x10-fold activation of 6xOSE2-Luc reporter and overexpression of wild-type Pin1 synergistically enhanced the transcriptional activity of FGF2-stimulated Runx2. However, overexpression of the mutant Pin1 inhibited FGF2-stimulated Runx2 transcriptional activity (Fig. 6B), indicating that Pin1 was indispensable to FGF2-stimulated Runx2 transcriptional activity.

The next problem to be addressed is whether the deletion (? C) of the Pin1 target domain or the mutations in the Pin1 target components (2AP # 1, 2AP # 2 and 4AP; see legend) affect the transcriptional activity of Runx2 (Fig. 6C). The C-terminal deletion of Runx2 abolished FGF2-stimulated Runx2 transcriptional activity, indicating that Pin1-binding to the C-terminal region of Runx2 is required for its activation by FGF2 (Figure 6C). In addition, the KR mutation of Runx2 (Jeon et al., 2006) was not activated by FGF2, indicating that acetylation of these four lysine residues is crucial for FGF2-mediated transcriptional activity of Runx2 (Fig. 6C). Finally, inhibition of p300 (HAT inhibitor) by anacardic acid (AA) treatment dramatically inhibited FGF2-stimulated Runx2 activity, whereas administration of TSA (HDAC inhibitor) increased Runx2 activity (Fig. 6D). These findings demonstrate that Runx2 activation by FGF2 is closely related to p300 or HDAC-induced lysine acetylation. Taken together, these data demonstrate that post-phosphorylation cases, including prolyl isomerization and subsequent acetylation, are crucial for Runx2 activity. This finding may explain our previous observations that phosphorylation-dependent transcriptional stimulation of Runx2 target genes is promoted (Park et al., 2010).

2. Assay  process

2-1. Assay For carrying out the Before experiment  Ready

(1) Preparation of a peptide conjugated aminoluciferin

(2) Prepare Lucifein detection reagent (Promega # V859A or V859B)

(3) Preparation of Resuspension Buffer (buffer capable of protease activation at pH 6.5 to 8.5, final MgSO ₄ 50 mM)

2-2. Assay Steps

(1) Luciferin detection reagent and resuspension buffer were kept at room temperature

(2) Mix 10 ml of Resuspension Buffer containing protease (chymotrypsin) and 50 ml of Luciferin detection reagent

(3) 10 μM each of the Pin1 enzyme, the fluorescent substance-bound substrate and the target substance (X) was dissolved in Lucifein detection reagent

(4) The dissolved compound was added to the solution of (2), and the reaction was carried out at room temperature for 30 minutes

(5) Incubate the same amount of samples in a 96-well plate and measure the enzyme activity by measuring with a fluorescence meter

<110> SNU R & DB FOUNDATION <120> A SCREENING METHOD OF A MATERIAL INDUCING OR INHIBITING PIN1          ENZYME ACTIVATION <130> <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 4 <212> PRT <213> Runx2 C-terminal motif <400> 1 Ala Ala Pro Phe   One <210> 2 <211> 4 <212> PRT <213> Runx2 C-terminal motif <400> 2 Ala Leu Pro Phe   One <210> 3 <211> 4 <212> PRT <213> Runx2 C-terminal motif <400> 3 Ala Phe Pro Phe   One <210> 4 <211> 4 <212> PRT <213> Runx2 C-terminal motif <400> 4 Ala Glu Pro Phe   One <210> 5 <211> 4 <212> PRT <213> Runx2 C-terminal motif <400> 5 Ala Asp Pro Phe   One

Claims (11)

The candidate substance is exposed to the Pin1 enzyme and the activation of the Pin1 enzyme is measured through an enzyme-substrate reaction, wherein the substrate used in the enzyme-substrate reaction is selected from the group consisting of SEQ ID NOS: 1 to 5 Wherein the oligonucleotide is an oligopeptide selected from the group consisting of SEQ ID NO: delete delete The method according to claim 1,
Wherein the fluorescent substance is luciferin. 2. The method of claim 1, wherein the fluorescent substance is luciferin. delete The method according to claim 1,
Wherein the enzyme acting on the substrate is chymotrypsin. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method according to claim 1,
(a) reacting a Pin1 enzyme, a substrate bound with a fluorescent substance and a candidate substance;
(b) treating chymotrypsin; And
(c) detecting fluorescence. The method for screening a substance that inhibits activation or activation of Pin1 enzyme. The method according to any one of claims 1, 4, 6, and 7,
Wherein the Pin1 enzyme activation inducer is a candidate substance for prevention or treatment of abnormal bone tissue disorder. 9. The method of claim 8,
Wherein the bone disorder disorder is cleidocranial dysplasia (CCD) or craniosynostosis. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method according to any one of claims 1, 4, 6, and 7,
Wherein the Pin1 enzyme activation inducer is a candidate substance for prevention or treatment of an abnormal hematopoietic function. The method according to any one of claims 1, 4, 6, and 7,
Wherein the Pin1 enzyme inducing or activating inhibitor is a candidate substance for the treatment of cancer, Alzheimer's disease or demented elderly lesion.

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