Identification of Inhibitors of the Disease-Associated Protein Phosphatase Scp1 Using Antibody Mimetic Molecules.

Protein phosphorylation is a prevalent translational modification, and its dysregulation has been implicated in various diseases, including cancer. Despite its significance, there is a lack of specific inhibitors of the FCP/SCP-type Ser/Thr protein phosphatase Scp1, characterized by high specificity and affinity. In this study, we focused on adnectin, an antibody-mimetic protein, aiming to identify Scp1-specific binding molecules with a broad binding surface that target the substrate-recognition site of Scp1. Biopanning of Scp1 was performed using an adnectin-presenting phage library with a randomized FG loop. We succeeded in identifying FG-1Adn, which showed high affinity and specificity for Scp1. Ala scanning analysis of the Scp1-binding sequence in relation to the FG-1 peptide revealed that hydrophobic residues, including aromatic amino acids, play important roles in Scp1 recognition. Furthermore, FG-1Adn was found to co-localize with Scp1 in cells, especially on the plasma membrane. In addition, Western blotting analysis showed that FG-1Adn increased the phosphorylation level of the target protein of Scp1 in cells, indicating that FG-1Adn can inhibit the function of Scp1. These results suggest that FG-1Adn can be used as a specific inhibitor of Scp1.

Bilayer Hydrogel Composed of Elastin-Mimetic Polypeptides as a Bio-Actuator with Bidirectional and Reversible Bending Behaviors.

Biologically derived hydrogels have attracted attention as promising polymers for use in biomedical applications because of their high biocompatibility, biodegradability, and low toxicity. Elastin-mimetic polypeptides (EMPs), which contain a repeated amino acid sequence derived from the hydrophobic domain of tropoelastin, exhibit reversible phase transition behavior, and thus, represent an interesting starting point for the development of biologically derived hydrogels. In this study, we succeeded in developing functional EMP-conjugated hydrogels that displayed temperature-responsive swelling/shrinking properties. The EMP-conjugated hydrogels were prepared through the polymerization of acrylated EMP with acrylamide. The EMP hydrogel swelled and shrank in response to temperature changes, and the swelling/shrinking capacity of the EMP hydrogels could be controlled by altering either the amount of EMP or the salt concentration in the buffer. The EMP hydrogels were able to select a uniform component of EMPs with a desired and specific repeat number of the EMP sequence, which could control the swelling/shrinking property of the EMP hydrogel. Moreover, we developed a smart hydrogel actuator based on EMP crosslinked hydrogels and non-crosslinked hydrogels that exhibited bidirectional curvature behavior in response to changes in temperature. These thermally responsive EMP hydrogels have potential use as bio-actuators for a number of biomedical applications.

Development of Mn2+-Specific Biosensor Using G-Quadruplex-Based DNA.

Metal ions are used in various situations in living organisms and as a part of functional materials. Since the excessive intake of metal ions can cause health hazards and environmental pollution, the development of new molecules that can monitor metal ion concentrations with high sensitivity and selectivity is strongly desired. DNA can form various structures, and these structures and their properties have been used in a wide range of fields, including materials, sensors, and drugs. Guanine-rich sequences respond to metal ions and form G-quadruplex structures and G-wires, which are the self-assembling macromolecules of G-quadruplex structures. Therefore, guanine-rich DNA can be applied to a metal ion-detection sensor and functional materials. In this study, the IRDAptamer library originally designed based on G-quadruplex structures was used to screen for Mn2+, which is known to induce neurodegenerative diseases. Circular dichroism and fluorescence analysis using Thioflavin T showed that the identified IRDAptamer sequence designated MnG4C1 forms a non-canonical G-quadruplex structure in response to low concentrations of Mn2+. A serum resistance and thermostability analysis revealed that MnG4C1 acquired stability in a Mn2+-dependent manner. A Förster resonance energy transfer (FRET) system using fluorescent molecules attached to the termini of MnG4C1 showed that FRET was effectively induced based on Mn2+-dependent conformational changes, and the limit of detection (LOD) was 0.76 µM for Mn2+. These results suggested that MnG4C1 can be used as a novel DNA-based Mn2+-detecting molecule.

Inhibition of protein phosphatase PPM1D enhances retinoic acid-induced differentiation in human embryonic carcinoma cell line.

The protein phosphatase PPM1D (Wip1) was originally identified as a p53 target product. Activation of PPM1D through various mechanism promotes the tumorigenic potential of various cancers by suppressing p53 and other DNA damage response proteins. New functions of PPM1D have recently been revealed in physiological processes such as cell differentiation. However, the regulatory mechanisms of signalling pathway to maintain stemness and induce cell differentiation are still unclear. Here we report that PPM1D modulates retinoic acid (RA) signalling. PPM1D knockdown resulted in decreased alkaline phosphatase activity of the human teratocarcinoma cell line NT2/D1. Inhibition of PPM1D-induced cell differentiation and decreased gene expression of the stem cell marker Oct-4 (POU5F1). RA-induced cell differentiation was promoted by reducing PPM1D activity. RA treatment elicited activation of the MEK-ERK pathway and induced rapid and transient activation of the extracellular signal-regulated kinase 1/2 (ERK-1/2). PPM1D dephosphorylated a phosphopeptide with the TEY motif in ERK-1/2 in vitro. Moreover, phosphorylation of ERK-1/2 was facilitated by PPM1D inhibition. Our study shows that PPM1D plays an important role in maintaining the undifferentiation state and a new function in RA-induced ERK regulation and cell differentiation.

Development of a Substrate Identification Method for Human Scp1 Phosphatase Using Phosphorylation Mimic Phage Display.

BACKGROUND: Protein phosphorylation is strictly regulated by protein kinases and protein phosphatases, and disordered regulation of protein phosphorylation often causes serious diseases, such as cancer. Protein phosphatases are divided into two major groups: tyrosine (Tyr) phosphatases and serine/threonine (Ser/Thr) phosphatases. Substrate trapping mutants are frequently used to characterize Tyr phosphatases and identify their substrates; however, a rapid and simple method to identify substrates for Ser/Thr phosphatases has yet to be developed. Recently it has reported that AlF4 -/AlF3 and BeF3 - form a complex with Mg2+ in the catalytic center of FCP/SCP phosphatases, and that the Mg2+-AlF4 -/AlF3 complex mimics the transition state of the hydrolysis step, while the Mg2+-BeF3 - complex mimics the aspartylphosphate intermediate. OBJECTIVES: The main objective of this study was to develop a novel methodology, termed Phosphorylation Mimic Phage Display (PMPD), to identify substrates for Ser/Thr phosphatase Scp1 using peptide phage display libraries with Mg2+ and AlF4 -. METHODS: Recombinant protein of human full-length Scp1 (rScp1) expressed in E. coli system was purified by Co2+ chelated affinity column, and then confirmed by SDS-PAGE and Western-blot analysis. The Ph.D.-C7C or M12 Phage Display Libraries (New England BioLabs, Beverly, MA) were screened using purified rScp1 immobilized on ELISA plate. Then, the plate was blocked with 0.5% (w/v) BSA in maleate buffer at 4°C for 3 h, before adding approximately 1×1010 plaque-forming units (pfu) of the phages in maleate blocking AlF4 - buffer to each well. After incubating, the wells were washed with maleate AlF4 - buffer to remove unbound phages. Then, phages were eluted with Mg2+ and AlF4 - free maleate buffer or with excess rScp1. After the third round of screening, the isolated phages were sequenced and subjected to binding analyses. RESULTS: After panning by PMPD method, 46 and 60 clones were isolated from the Ph.D. C7C and Ph.D. 12 phage libraries, respectively, as Mg2+ or/and AlF4 - -dependent binding clones. The binding analyses showed that M12-1 and Dep-3 specifically bind to Scp1 in an AlF4 --dependent manner. Notably, the Dep-3 peptide contained a Thr-Pro-Met-Ser sequence, which is similar to the Ser2-Pro3-Thr4-Ser5 (Ser/Thr-Pro-partially hydrophobic residue-Ser) sequence found in CTD, which is an endogenous substrate for Scp1. Binding analyses also showed that both BP-14 and M12-6a bound to Scp1 in a Mg2+-dependent manner. BP-14 peptide contained Ser- Thr-Tyr and Pro-Phe-Glu sequences, which are similar to the Ser-Thr-Trp and Ile-Phe-Glu sequences found in M12-6a, suggesting that one or both of these tripeptides may be the binding motif(s) recognized by Scp1. CONCLUSION: We developed a substrate identification method for the Ser/Thr phosphatase Scp1 using a novel phage display method with AlF4 -. Dep-3 showed a core sequence similar to that of the CTD of RNA polymerase II, an endogenous Scp1 substrate, suggesting that this method is applicable for identifying novel Scp1 substrate candidates. This method will also be applicable for other FCP/SCP-type phosphatases, allowing us to better understand the substrate recognition mechanisms of Ser/Thr phosphatases.

Non-farnesylated B-type lamin can tether chromatin inside the nucleus and its chromatin interaction requires the Ig-fold region.

Lamins are thought to direct heterochromatin to the nuclear lamina (NL); however, this function of lamin has not been clearly demonstrated in vivo. To address this, we analyzed polytene chromosome morphology when artificial lamin variants were expressed in Drosophila endoreplicating cells. We found that the CaaX-motif-deleted B-type lamin Dm0, but not A-type lamin C, was able to form a nuclear envelope-independent layer that was closely associated with chromatin. Other nuclear envelope proteins were not detected in this "ectopic lamina," and the associated chromatin showed a repressive histone modification maker but not a permissive histone modification marker nor RNA polymerase II proteins. Furthermore, deletion of the C-terminal lamin-Ig-fold domain prevents chromatin association with this ectopic lamina. Thus, non-farnesylated B-type lamin Dm0 can form an ectopic lamina and induce changes to chromatin structure and status inside the interphase nucleus.

PPM1D controls nucleolar formation by up-regulating phosphorylation of nucleophosmin.

An increase of nucleolar number and size has made nucleoli essential markers for cytology and tumour development. However, the underlying basis for their structural integrity and abundance remains unclear. Protein phosphatase PPM1D was found to be up-regulated in different carcinomas including breast cancers. Here, we demonstrate for the first time that PPM1D regulates nucleolar formation via inducing an increased phosphorylation of the nucleolar protein NPM. We show that PPM1D overexpression induces an increase in the nucleolar number regardless of p53 status. We also demonstrated that specific sequential phosphorylation of NPM is important for nucleolar formation and that PPM1D is a novel upstream regulator of this phosphorylation pathway. These results enhance our understanding of the molecular mechanisms that govern nucleoli formation by demonstrating that PPM1D regulates nucleolar formation by regulating NPM phosphorylation status through a novel signalling pathway, PPM1D-CDC25C-CDK1-PLK1.

Patterning nanofibrils through the templated growth of multiple modified amyloid peptides.

There has been considerable interest in the patterning of functionalized nanowires because of the potential applications of these materials to the construction of nanodevices. A variety of biomolecular building blocks containing amyloid peptides have been used to functionalize nanowires. However, the patterning of self-assembled nanowires can be challenging because of the difficulties associated with controlling the self-assembly of these functionalized building blocks. Herein, we present a versatile approach for the patterning of nanowires based on the combination of templated fibril growth with a versatile functionalization method using our structure-controllable amyloid peptides (SCAPs). Using this approach, we have succeeded in the formation of multi-type nanowires with tandem domain structures in high yields. Given that the mixing-SCAP method can lead to the formation of tandem fibrils, it is noteworthy that our method allowed us to control the initiation of fibril formation from the gold nanoparticles, which were attached to a short fibril as initiation points. This approach could be used to prepare a wide variety of fibril patterns, and therefore holds great potential for the development of novel self-assembled nanodevices.

Effective Cellular Morphology Analysis for Differentiation Processes by a Fluorescent 1,3a,6a-Triazapentalene Derivative Probe in Live Cells.

Nuclear and cytoplasmic morphological changes provide important information about cell differentiation processes, cell functions, and signal responses. There is a strong desire to develop a rapid and simple method for visualizing cytoplasmic and nuclear morphology. Here, we developed a novel and rapid method for probing cellular morphological changes of live cell differentiation process by a fluorescent probe, TAP-4PH, a 1,3a,6a-triazapentalene derivative. TAP-4PH showed high fluorescence in cytoplasmic area, and visualized cytoplasmic and nuclear morphological changes of live cells during differentiation. We demonstrated that TAP-4PH visualized dendritic axon and spine formation in neuronal differentiation, and nuclear structural changes during neutrophilic differentiation. We also showed that the utility of TAP-4PH for visualization of cytoplasmic and nuclear morphologies of various type of live cells. Our visualizing method has no toxicity and no influence on the cellular differentiation and function. The cell morphology can be rapidly observed after addition of TAP-4PH and can continue to be observed in the presence of TAP-4PH in cell culture medium. Moreover, TAP-4PH can be easily removed after observation by washing for subsequent biological assay. Taken together, these results demonstrate that our visualization method is a powerful tool to probe differentiation processes before subsequent biological assay in live cells.

Novel inhibitors targeting PPM1D phosphatase potently suppress cancer cell proliferation.

Protein phosphatase magnesium-dependent 1δ (PPM1D, Wip1) is a p53 inducible serine/threonine phosphatase. PPM1D is a promising target protein in cancer therapy since overexpression, missense mutations, truncating mutations, and gene amplification of PPM1D are reported in many tumors, including breast cancer and neuroblastoma. Herein, we report that a specific inhibitor, SL-176 that can be readily synthesized in 10 steps, significantly inhibits proliferation of a breast cancer cell line overexpressing PPM1D and induces G2/M arrest and apoptosis. SL-176 decreases PPM1D enzyme activity potently and specifically in vitro. These results demonstrate that SL-176 could be a useful lead compound in the development of effective anti-cancer agents.

Synthesis of yellow and red fluorescent 1,3a,6a-triazapentalenes and the theoretical investigation of their optical properties.

To expand the originally developed fluorescent 1,3a,6a-triazapentalenes as fluorescent labelling reagents, the fluorescence wavelength of the 1,3a,6a-triazapentalenes was extended to the red color region. Based on the noteworthy correlation of the fluorescence wavelength with the inductive effect of the 2-substituent, electron-deficient 2-(2-cyano-4-methoxycarbonylphenyl)-1,3a,6a-triazapentalene and 2-(2,6-dicyano-4-methoxycarbonylphenyl)-1,3a,6a-triazapentalene were synthesized. The former exhibited yellow fluorescence and the latter exhibited red fluorescence, and both compounds exhibited large Stokes shifts, and the 1,3a,6a-triazapentalene system enabled the same fluorescent chromophore to cover the entire region of visible wavelengths. The potential applications of the 1,3a,6a-triazapentalenes as fluorescent probes in the fields of the life sciences were investigated, and the 1,3a,6a-triazapentalene system was clearly proven to be useful as a fluorescent reagent for live cell imaging. Quantum chemical calculations were performed to investigate the optical properties of the 1,3a,6a-triazapentalenes. These calculations revealed that the excitation involves a significant charge-transfer from the 1,3a,6a-triazapentalene skeleton to the 2-substituent. The calculated absorption and fluorescence wavelengths showed a good correlation with the experimental ones, and thus the system could enable the theoretical design of substituents with the desired optical properties.

Secreted Frizzled-related protein potentiation versus inhibition of Wnt3a/ß-catenin signaling.

Wnt signaling regulates a variety of cellular processes during embryonic development and in the adult. Many of these activities are mediated by the Frizzled family of seven-pass transmembrane receptors, which bind Wnts via a conserved cysteine-rich domain (CRD). Secreted Frizzled-related proteins (sFRPs) contain an amino-terminal, Frizzled-like CRD and a carboxyl-terminal, heparin-binding netrin-like domain. Previous studies identified sFRPs as soluble Wnt antagonists that bind directly to Wnts and prevent their interaction with Frizzleds. However, subsequent observations suggested that sFRPs and Frizzleds form homodimers and heterodimers via their respective CRDs, and that sFRPs can stimulate signal transduction. Here, we present evidence that sFRP1 either inhibits or enhances signaling in the Wnt3a/ß-catenin pathway, depending on its concentration and the cellular context. Nanomolar concentrations of sFRP1 increased Wnt3a signaling, while higher concentrations blocked it in HEK293 cells expressing a SuperTopFlash reporter. sFRP1 primarily augmented Wnt3a/ß-catenin signaling in C57MG cells, but it behaved as an antagonist in L929 fibroblasts. sFRP1 enhanced reporter activity in L cells that were engineered to stably express Frizzled 5, though not Frizzled 2. This implied that the Frizzled expression pattern could determine the response to sFRP1. Similar results were obtained with sFRP2 in HEK293, C57MG and L cell reporter assays. CRDsFRP1 mimicked the potentiating effect of sFRP1 in multiple settings, contradicting initial expectations that this domain would inhibit Wnt signaling. Moreover, CRDsFRP1 showed little avidity for Wnt3a compared to sFRP1, implying that the mechanism for potentiation by CRDsFRP1 probably does not require an interaction with Wnt protein. Together, these findings demonstrate that sFRPs can either promote or suppress Wnt/ß-catenin signaling, depending on cellular context, concentration and most likely the expression pattern of Fzd receptors.

Inhibition of tumor suppressor protein p53-dependent transcription by a tetramerization domain peptide via hetero-oligomerization.

Tumor suppressor protein p53 induces cell cycle arrest, apoptosis, and senescence in response to cellular stresses. The p53 tetramer formation is essential for its functions. Despite of these crucial functions of p53 for integrity of genome, activation of the p53 signal pathway causes low induced pluripotent stem (iPS) cell generation efficiency. In this study, we report transient inhibition of p53-dependent transcription using a p53 tetramerization domain peptide that contains cell penetrating and nuclear localization signals. The peptide was efficiently introduced into cells and inhibited p21 expression via hetero-tetramerization with endogenous p53 protein. This method can be applied towards safe and efficient iPS cell generation.

A small molecule inhibitor of p53-inducible protein phosphatase PPM1D.

PPM1D is a p53-inducible Ser/Thr protein phosphatase. PPM1D gene amplification and overexpression have been reported in a variety of human tumors, including breast cancer and neuroblastoma. Because the phosphatase activity of PPM1D is essential for its oncogenic role, PPM1D inhibitors should be viable anti-cancer agents. In our current study, we showed that SPI-001 was a potent and specific PPM1D inhibitor. SPI-001 inhibited PPM1D phosphatase activity in PPM1D-overexpressing human breast cancer cells and increased phosphorylation of p53. Furthermore, SPI-001 suppressed cell proliferation by inducing apoptosis. Our present study suggested that SPI-001 was a potential lead compound in developing anti-cancer drugs.

Phosphatase assay for multi-phosphorylated substrates using phosphatase specific-motif antibody.

Protein phosphorylation plays central roles in a wide variety of signal transduction pathways and most phosphorylated proteins contain multi-phosphorylated sites. PPM1 type Ser/Thr protein phosphatase family is known to show rigid substrate specificity unlike other Ser/Thr phosphatase PPP family including PP1, PP2A and PP2B. PPM1 type phosphatases are reported to play important roles in growth regulation and in cellular stress signalling. In this study, we developed a phosphatase assay of PPM1D using phosphatase motif-specific antibody. PPM1D is a member of PPM1 type Ser/Thr phosphatase and known to dephosphorylate Ser(P)-Gln sequence. The gene amplification and overexpression of PPM1D were reported in many human cancers. We generated the monoclonal antibody specific for the Ser(P)-Gln sequence, named 3G9-H11. The specificity of this method using ELISA enables the convenient measurement of the dephosphorylation level of only PPM1D target residues of substrate peptides with multiple phosphorylated sites in the presence of multiple phosphatases. In addition, the antibody was applicable to immunoblotting assay for PPM1D function analysis. These results suggested that this method should be very useful for the PPM1D phosphatase assay, including high-throughput analysis and screening of specific inhibitors as anti-cancer drugs. The method using phosphatase motif-specific antibody can be applied to other PPM1 phosphatase family.

sFRP-1 binds via its netrin-related motif to the N-module of thrombospondin-1 and blocks thrombospondin-1 stimulation of MDA-MB-231 breast carcinoma cell adhesion and migration.

Secreted frizzled-related protein (sFRP)-1 is a Wnt antagonist that inhibits breast carcinoma cell motility, whereas the secreted glycoprotein thrombospondin-1 stimulates adhesion and motility of the same cells. We examined whether thrombospondin-1 and sFRP-1 interact directly or indirectly to modulate cell behavior. Thrombospondin-1 bound sFRP-1 with an apparent K(d)=48nM and the related sFRP-2 with a K(d)=95nM. Thrombospondin-1 did not bind to the more distantly related sFRP-3. The association of thrombospondin-1 and sFRP-1 is primarily mediated by the amino-terminal N-module of thrombospondin-1 and the netrin domain of sFRP-1. sFRP-1 inhibited α3ß1 integrin-mediated adhesion of MDA-MB-231 breast carcinoma cells to a surface coated with thrombospondin-1 or recombinant N-module, but not adhesion of the cells on immobilized fibronectin or type I collagen. sFRP-1 also inhibited thrombospondin-1-mediated migration of MDA-MB-231 and MDA-MB-468 breast carcinoma cells. Although sFRP-2 binds similarly to thrombospondin-1, it did not inhibit thrombospondin-1-stimulated adhesion. Thus, sFRP-1 binds to thrombospondin-1 and antagonizes stimulatory effects of thrombospondin-1 on breast carcinoma cell adhesion and motility. These results demonstrate that sFRP-1 can modulate breast cancer cell responses by interacting with thrombospondin-1 in addition to its known effects on Wnt signaling.

Probing phenylalanine environments in oligomeric structures with pentafluorophenylalanine and cyclohexylalanine.

Stabilization of protein structures and protein-protein interactions are critical in the engineering of industrially useful enzymes and in the design of pharmaceutically valuable ligands. Hydrophobic interactions involving phenylalanine residues play crucial roles in protein stability and protein-protein/peptide interactions. To establish an effective method to explore the hydrophobic environments of phenylalanine residues, we present a strategy that uses pentafluorophenylalanine (F5Phe) and cyclohexylalanine (Cha). In this study, substitution of F5Phe or Cha for three Phe residues at positions 328, 338, and 341 in the tetramerization domain of the tumor suppressor protein p53 was performed. These residues are located at the interfaces of p53-p53 interactions and are important in the stabilization of the tetrameric structure. The stability of the p53 tetrameric structure did not change significantly when F5Phe-containing peptides at positions Phe328 or Phe338 were used. In contrast, the substitution of Cha for Phe341 in the hydrophobic core enhanced the stability of the tetrameric structure with a T(m) value of 100 degrees C. Phe328 and Phe338 interact with each other through pi-interactions, whereas Phe341 is buried in the surrounding alkyl side-chains of the hydrophobic core of the p53 tetramerization domain. Furthermore, high pressure-assisted denaturation analysis indicated improvement in the occupancy of the hydrophobic core. Considerable stabilization of the p53 tetramer was achieved by filling the identified cavity in the hydrophobic core of the p53 tetramer. The results indicate the status of the Phe residues, indicating that the "pair substitution" of Cha and F5Phe is highly suitable for probing the environments of Phe residues.

Enhancement of transcriptional activity of mutant p53 tumor suppressor protein through stabilization of tetramer formation by calix[6]arene derivatives.

Li-Fraumeni syndrome, a hereditary disorder characterized by familial clusters of early-onset multiple tumors, is caused by mutation of the TP53 gene, which encodes the p53 tumor suppressor protein. Mutation of Arg337 to histidine in the tetramerization domain of p53 is most frequently observed in Li-Fraumeni syndrome. This mutation is reported to destabilize the tetrameric structure of p53. We designed and synthesized calix[6]arene derivatives, which have six imidazole or pyrazole groups at the upper rim. In this study, we report, for the first time, the enhancement of the in vivo transcriptional activity of the most common Li-Fraumeni p53 mutant by imidazole-calix[6]arene through stabilization of the oligomer formation.

Drastic effects on fibril formation of amyloid-beta peptides by the addition of amino acid residue units to the termini.

We report that the addition of amino acids to the amyloid peptide dramatically affected the structure and the rate of formation of amyloid fibrils. The attachment of three lysines to Abeta(10-35) gave the formation of remarkably long fibrils, while three glutamates resulted in a faster formation rate of the fibrils.