Kazuyasu Sakaguchi

Hokkaido University | Hokudai · Department of Chemistry

Protein phosphatases and kinases control multiple cellular events including proliferation, differentiation, and stress responses through regulating reversible protein phosphorylation, the most important post-translational modification. Members of metal-dependent protein phosphatase (PPM) family, also known as PP2C phosphatases, are Ser/Thr phosphat...

The human PIAS proteins are small ubiquitin-like modifier (SUMO) E3 ligases that participate in important cellular functions. Several of these functions depend on a conserved SUMO-interacting motif (SIM) located in the central region of all PIAS proteins (SIM1). Recently, it was determined that Siz2, a yeast homolog of PIAS proteins, possesses a se...

The interactions between SUMO proteins and SUMO-interacting motif (SIM) in nuclear bodies formed by the promyelocytic leukemia (PML) protein (PML-NBs) have been shown to be modulated by either phosphorylation of the SIMs or acetylation of SUMO proteins. However, little is known about how this occurs at the atomic level. In this work, we examined th...

The p53 protein plays a number of roles in protecting organisms from different genotoxic stresses and this includes DNA damage induced by acetaldehyde, a metabolite of alcohol. Since the common tree shrew ingests high levels of alcohol as part of its normal diet, this suggests that its p53 protein may possess unique properties. Using a combination...

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 differentiat...

Obesity is a worldwide public health problem, which is associated with various severe diseases including diabetes, hypertension, atherosclerosis, and cancer. Recent studies have revealed that combination treatment of several different compounds using low doses is effective to reduce side effects. Thus, there is a need to develop an efficient inhibi...

Imaging of lipid droplets by Monodansylpentane. 3T3-L1 cells were differentiated for 8 days with 15 μM SL-104. (TIF)

Inhibitory activity of SL-176 for His-mousePPM1D (1–413) in vitro. Phosphatase activity of His-mousePPM1D (1–413) for p53-derived peptide was measured. (TIF)

Quantification of lipid droplets with 10 μM SL-104 and SL-188. Amount of lipid droplets was quantified by Oil Red O staining. (TIF)

Epigenetic memory for signal-dependent transcription has remained elusive. So far, the concept of epigenetic memory has been largely limited to cell-autonomous, preprogrammed processes such as development and metabolism. Here we show that IFNβ stimulation creates transcriptional memory in fibroblasts, conferring faster and greater transcription upo...

Peptide – mediated biomineralization is an emerging and promising biomimetic approach for the synthesis of nanomaterials. This nature – inspired technique of producing inorganic nanostructures depends on the biomineralization peptide to control the shape and morphology of the prevailing inorganic nanostructure. One of the challenges in peptide – me...

p53 is a tumor suppressor protein, and its missense mutations are frequently found in human cancers. During the multi-step progression of cancer, p53 mutations generally accumulate at the mid or late stage, but not in the early stage, and the underlying mechanism is still unclear. In this study, using mammalian cell culture and mouse ex vivo system...

Introduction: Peptide-mediated biomineralization is a promising bioinspired technique of nanostructure formation. The biomineralization peptide is responsible for the regulation of the growth and morphology of the inorganic nanostructure. The 3D properties of the biomineralization peptide is a crucial factor in which the success of creating functi...

p53 acts as a transcriptional factor for tumor suppression via tetramerization. Although the dominant-negative effect occurs by hetero-oligomerization between wild-type and mutant p53, the precise mechanism remains unclear. Here, we report an analysis of the transcriptional ability of each hetero-tetramer p53 at the physiological protein expression...

Binding affinity and specificity are crucial factors that influence nanostructure control by biomineralization peptides. In this paper, we analysed the role that the oligomeric state of a silver biomineralization peptide plays in regulating the morphology of silver nanostructure formation. Oligomerization was achieved by conjugating the silver spec...

Background: The most common cytogenetic lesions in the embryonal neural tumors medulloblastoma (MB) and neuroblastoma (NB) affect chromosome 17, with 17q+ or isochromosome 17q, in approximately one-third of MB with these aberrations being a significant indicator of poor clinical outcome. Similarly, in NB gain of 17q is the most powerful genetic pre...

Protein phosphatase Magnesium-dependent 1, Delta (PPM1D) is a wild-type p53-inducible Ser/Thr phosphatase that acts as a negative regulator of the p53 tumor suppressor. Gene amplification and overexpression of PPM1D have been reported in various cancers including leukemia and neuroblastoma. Therefore, PPM1D is a promising target in cancer therapy....

Coiled coils, consisting of at least two α-helices, have important roles in the regulation of transcription, cell differentiation, and cell growth. Peptides composed of d-amino acids (d-peptides) have received great attention for their potential in biomedical applications, because they give large diversity for the design of peptidyl drug and are mo...

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...

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 ch...

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 chang...

Temperature-dependent cellular uptake of TAP-4PH. HL-60 cells were incubated with 50 μM TAP-4PH for 30 min at 37 or 4°C. Cellular uptake of TAP-4PH was measured by flow cytometric analysis. Data represent the mean ± S.D., n = 3. **p < 0.005, Student’s t-test. (TIF)

TAP-4PH visualization of PC-12 cells. Merged bright-field and TAP-4PH fluorescent (cyan) images of PC-12 cells. (TIF)

Co-staining with Golgi apparatus and ER probes in A549 cells. A549 cells were stained with a Golgi apparatus probe and ER probe, followed by treatment with 50 μM TAP-4PH for 30 min. and then observed by fluorescence microscopy. Merged image was constructed with images of TAP-4PH (cyan), Golgi apparatus probe (yellow), and ER probe (magenta). Scale...

Co-staining with the nuclear staining probe acridine orange and TAP-4PH in A549 cells. Left panel, TAP-4PH; middle panel, acridine orange; right panel, merged images of TAP-4PH (cyan) and acridine orange (magenta). Scale bar: 20 μm. (TIF)

TAP-4PH visualization of HL-60 cells. (a) HL-60 cells before and after differentiation into neutrophil-like cells by ATRA for 5 days. After differentiation, the cells were co-stained with Nuclear Green LSC1 for nucleus and TAP-4PH. Left panels, TAP-4PH; middle panels, Nuclear Green LSC1; right panels, merged images of TAP-4PH (cyan) and Nuclear Gre...

Cell cycle analysis of HL-60 cells after incubation for 48 h in the presence (left) or absence (right) of 10 or 50 μM TAP-4PH. Data shown are representative of three independent experiments. (TIF)

TAP-4PH visualization of differentiated 3T3-L1 adipocytes. 3T3-L1 cells were induced to differentiate into adipocytes for 8 days. The cells were then treated with 50 μM TAP-4PH for 30 min and observed by fluorescence microscopy. Scale bar: 10 μm. (TIF)

Fluorescence emission spectra of 10 μM TAP-4PH with the indicated concentrations of DNA in PBS. (TIF)

Protein expression using Escherichia coli is a common and important method for recombinant protein production. Herein, we quantitatively analyzed the correlation between protein expression in vivo and thermodynamic structure stability in vitro using the tetramerization domain of tumor suppressor protein p53. We found a strong positive relationship...

Tetramer formation of p53 is essential for its tumor suppressor function. p53 not only acts as a tumor suppressor protein by inducing cell cycle arrest and apoptosis in response to genotoxic stress, but it also regulates other cellular processes, including autophagy, stem cell self-renewal and reprogramming of differentiated cells into stem cells,...

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...

Background: The most common cytogenetic lesions in the embryonal neural tumors medulloblastoma (MB) and neuroblastoma (NB) affect chromosome 17, with 17q+ or isochromosome 17q, in approximately one-third of MB with these aberrations being a significant indicator of poor clinical outcome. Similarly, in NB gain of 17q is the most powerful genetic pre...

The physical and chemical properties of silver nanomaterials are highly dependent on their shape and size. Recently, Ag nanoparticles have been reported to be useful for medicinal and pharmaceutical applications including anticancer activity. In this study, we showed that Ag nanoplates possess significantly higher antiproliferative activity on huma...

PML and several other proteins localizing in PML-nuclear bodies (PML-NB) contain phosphoSIMs (SUMO-interacting motifs), and phosphorylation of this motif plays a key role in their interaction with SUMO family proteins. We examined the role that phosphorylation plays in the binding of the phosphoSIMs of PML and Daxx to SUMO1 at the atomic level. The...

PPM1D is a p53-inducible Ser/Thr phosphatase. One of the main functions of PPM1D in normal cells is to act as a negative regulator of the p53 tumor suppressor by dephosphorylating p53 and several kinases. PPM1D is considered an oncoprotein owing to both its functions and the fact that gene amplification and overexpression of PPM1D are reported in s...

To expand the originally developed fluorescent 1,3a,6a-triazapentalenes as fluorescent labeling reagents, the fluorescence wavelength of 1,3a,6a-triazapentalene was extended to the red color region. Based on the noteworthy correlation of the fluorescence wavelength with the inductive effect of the 2-substituent, further electron-deficient 2-(2-cyan...

Effects of a buffer on the structure and catalytic activity of Pd nanostructures obtained from biomineralization were evaluated. Significant structural differences attributed to the buffer were observed. In addition, the Pd nanostructures prepared in buffered solutions had enhanced catalytic activity in the reduction of aminonitrophenol isomers. Ou...

Highly branched, coral-like Pd nanostructures were formed using a biomineralization peptide conjugated to an oligomeric peptide that simultaneously controls the spatial orientation, arrangement and valency. The Pd nanocoral showed very high catalytic activity in the reduction of nitrophenol. The results highlight the importance of topological arran...

Provided is a novel protein phosphatase inhibitor. The protein phosphatase inhibitor contains, as an active ingredient, a silicon compound represented by the following general formula (1) or a salt thereof, wherein R1, R2 and R3 are the same or different and represent a hydrocarbon group having 1 to 12 carbon atoms; X represents an optionally subst...

Amyloid peptides have great potential as building blocks in the creation of functional nanowires due to their natural ability to self-assemble into nanofibrillar structures and because they can be easily modified with various functional groups. However, significant modifications of an amyloid peptide generally alter its self-assembly property, maki...

Histone deacetylase (HDAC) inhibitors belong to a new class of potential anticancer agents. It may be possible to reduce some of the toxicity by specifically targeting only the HDAC isoform. Here, stereoisomeric HDAC inhibitors containing fluoroalkene were analyzed for their specificity toward HDAC isoforms. Z-Form 1(Z) showed high affinity to HDAC...

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. I...

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 sh...

The formation process and structure of a self-assembled monolayer (SAM) of lipoic-acid-terminated polyproline on a gold surface in aqueous solution were investigated by several techniques. The amount of polyproline molecules on the gold surface was determined from the area of the reductive desorption peak, and orientation and thickness of the polyp...

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 effect...

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...

The tumor suppressor p53, a 393-amino acid transcription factor, induces cell cycle arrest and apoptosis in response to genotoxic stress. Its inactivation via the mutation of its gene is a key step in tumor progression, and tetramer formation is critical for p53 post-translational modification and its ability to activate or repress the transcriptio...

The tumor suppressor p53, a 393-amino acid transcription factor, induces cell cycle arrest and apoptosis in response to genotoxic stress. Its inactivation via the mutation of its gene is a key step in tumor progression, and tetramer formation is critical for p53 post-translational modification and its ability to activate or repress the transcriptio...

We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera pept...

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...

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.

Inhibitors of histone deacetylases (HDAC) are emerging as a promising class of anti-cancer agents. The mercaptoacetoamide-based inhibitors are reported to be less toxic than hydroxamate and are worthy of further consideration. Therefore, we have designed a series of analogs as potential inhibitors of HDACs, in which the mercaptoacetamide group was...

Inactivation of the tumor suppressor p53 is central to carcinogenesis and acquisition of resistance to drug-induced apoptosis. The majority of alterations are missense mutations and occur within the DNA-binding domain. However, little is known about the point mutations in the tetramerization domain (TD). Here we investigated the properties of a new...

To estimate the transcriptional activity of p53 in individual living mammalian cells, we constructed the enhanced green fluorescent protein-red fluorescent protein (EGFP-DsRed) reporter system with the EGFP-p53 expression vector and the reporter plasmid, which carried a p53-dependent promoter. The expression level and transcriptional activity of EG...

The intra-cellular level of tumor suppressor protein p53 is tightly controlled by an autoregulatory feedback loop between the protein and its negative regulator MDM2. The role of MDM2 in down-regulating the p53 response in unstressed conditions and in the post-stress recovery phase is well documented. However, interplay between the N-terminal phosp...

Unsaturated gramicidin S analog, [ΔPhe4,4′]gramicidin S, was synthesized by conventional solution method in order to evaluate the role of the dehydrophenylalanine residues replacing d-phenylalanine4,4′ in stabilizing the bioactive β-shect conformation. The dehydrophenylalanine (ΔPhe) moiety was introduced by dehydroazlactonization of the β-phenylse...

The tumor suppressor protein p53 is a tetrameric phosphoprotein that induces cell cycle, development, and differentiation by regulating the expression of target genes. The tetramerization of p53 is essential for its tumor suppressor functions. It has been known that oxidation of proteins affects their structure and function. A methionine residue (M...

PPM1D is a PPM1 type protein phosphatase and is induced in response to DNA damage. PPM1D-deficient mice show defects in spermatogenesis and lymphoid cell functions but the mechanisms underlying these phenotypes remain unknown. In our current study, we identify and characterize an alternative splicing variant (denoted PPM1D430) of human PPM1D at bot...

Amyroid fibril, which is known to cause BSE and Alzheimer disease, is a solid and stable fiber of several tens of nm wide and several mu m long and has a potential to be used as nano-materials because functional molecules and metal and semiconductor nano-particles can be attached. However, it is not yet possible to align the Amyroid fibrils on a so...

Histone acetyltransferases form crucial links in transducing extrinsic signals to actual initiation of transcription. A multitude of stress signal integrations occur through the interaction of p300 with p53 phosphorylated at different residues of the transactivation domain. How such interactions activate different gene expression programs remains l...

Protein phosphatase magnesium-dependent 1, delta (PPM1D) is a member of the PPM1 (formerly PP2C) protein phosphatase family, and is induced in response to DNA damage. The overexpression of PPM1D is thought to exert oncogenic effects through the inhibition of tumor suppressor proteins. PPM1D shows high selectivity for the primary sequence in its sub...

Pigment-dispersing factor (PDF) is an 18-mer peptide that acts as a principal neurotransmitter of the insect circadian clock. Our previous study, utilizing anti-Uca beta-PDH polyclonal antibody (pAb) to immunolabel the optic lobe of the cricket Gryllus bimaculatus, suggested the existence of an alternative PDF-like peptide in the outer cells of the...

Pig kidney Na/K-ATPase preparations showed a positive cooperative effect for pNPP in Na-pNPPase activity. Measurements of the Na-pNPPase activity, Na-ATPase activity and the accumulation of phosphoenzyme (EP) under conditions of pNPP saturation showed several different ATP affinities. The presence of pNPP reduced both the maximum amount of EP and N...

The p53 tumor suppressor is a tetrameric transcriptional enhancer, and its activity is compromised by mutations that cause amino acid substitutions in its tetramerization domain. Here we analyze the biochemical and biophysical properties of peptides corresponding to amino acids 319-358 of wild-type human p53, which includes the tetramerization doma...

The present invention provides methods for generating phosphorylation site-specific immunological reagents. More specifically, a phosphopeptide mimetic is incorporated into a polypeptide in place of a phosphorylated amino acid. The polypeptide is used as antigen by standard methods to generate either monoclonal or polyclonal antibodies which cross-...

Activity-oligomeric assembly relationships using octaethylene glycol dodecyl ether (C12E8) solubilized pig gastric H/K-ATPase (unmodified H/K-ATPase) or H/K-ATPase modified with Fluorescein 5'-isothiocyanate (FITC-H/K-ATPase) were examined. The amount of oligomeric species in FITC-H/K-ATPase, which retained little H/K-ATPase activity was estimated...

The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in the transmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 and Glu-954 in M9, the side chains of which appear to be in close proximity, were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K, V920E and Q923N/E/D/L, were expressed in HeLa cells. Ouabain-resistan...

The S-3-nitro-2-pyridinesulfenyl (SNpys) group in an affinity ligand can bind to a free thiol group of a cysteine residue in a target receptor molecule, forming a disulfide bond via the thiol-disulfide exchange reaction. SNpys-containing Leu-enkephalin analogues of [-Ala2, Leu5]-enkephalyl-Cys(Npys)6 and [-Ala2,Leu(CH2SNpys)5]enkephalin, and dynorp...

TheS-3-nitro-2-pyridinesulfenyl (SNpys) group in an affinity ligand can bind to a free thiol group of a cysteine residue in a target receptor molecule, forming a disulfide bond via the thiol-disulfide exchange reaction. SNpys-containing Leu-enkephalin analogues of [d-Ala2, Leu5]-enkephalyl-Cys(Npys)6 and [d-Ala2,Leu(CH2SNpys)5]enkephalin, and dynor...