Kazuishi Kubota

Platelet-derived growth factor BB secreted from osteoclasts acts as an osteoblastogenesis inhibitory factor.

Osteoclasts and osteoblasts are responsible for strict bone maintenance with a balance between bone formation and resorption by interacting with each other. Recently, it has been revealed that osteoblasts/stromal cells regulate differentiation of osteoclasts/hematopoietic cells by two factors, the receptor activator of nuclear factor‐κB (NF‐κB) ligand (RANKL) on the plasma membrane, and secreted osteoprotegerin (OPG). However, no factors have yet been reported by which osteoclasts/hematopoietic cells regulate osteoblasts/stromal cells. To elucidate the possibility of signal transduction from osteoclasts to osteoblasts, we studied the conditioned medium of mouse osteoclast‐like myeloma cell line RAW264.7 treated with RANKL. We found that this medium contains a factor that inhibits differentiation of mouse osteoblast precursor‐like cell line MC3T3‐E1 to osteoblasts induced by bone morphogenetic protein 4 (BMP‐4) and named this factor osteoblastogenesis inhibitory factor (OBIF). OBIF was purified by successive three‐step chromatography by heparin affinity, anion exchange, and reversed‐phase columns. Osteoblastogenesis inhibitory activity made one peak in each chromatography step, showing the factor is a single entity. Active fractions were loaded on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and bands of proteins were excised, digested by trypsin, and analyzed by liquid chromatography equipped with tandem mass spectrometry (LC/MS/MS). Consequently, we have identified this factor to be platelet‐derived growth factor BB (PDGF BB) homodimer. Furthermore, this identification of PDGF BB as OBIF was confirmed by neutralization of the inhibitory activity of the medium with anti‐PDGF antibody. These results show, for the first time, that osteoclasts regulate osteoblasts directly and suggest that PDGF BB is a key factor in bone remodeling.

Proteomic analysis of the SIRT6 interactome: novel links to genome maintenance and cellular stress signaling.

The chromatin regulatory factor SIRT6 plays pivotal roles in metabolism, tumor suppression, and aging biology. Despite the fundamental roles of SIRT6 in physiology and disease, only a handful of molecular and functional interactions of SIRT6 have been reported. Here, we characterize the SIRT6 interactome and identify 80+ novel SIRT6-interacting proteins. The discovery of these SIRT6-associations considerably expands knowledge of the SIRT6 interaction network, and suggests previously unknown functional interactions of SIRT6 in fundamental cellular processes. These include chromatin remodeling, mitotic chromosome segregation, protein homeostasis, and transcriptional elongation. Extended analysis of the SIRT6 interaction with G3BP1, a master stress response factor, uncovers an unexpected role and mechanism of SIRT6 in regulating stress granule assembly and cellular stress resistance.

Neural cell adhesion molecule 2 as a target molecule for prostate and breast cancer gene therapy

In adenovirus‐derived gene therapy, one of the problems is the difficulty in specific targeting. We have recently demonstrated that monoclonal antibody (mAb) libraries screened by fiber‐modified adenovirus vector (Adv‐FZ33), which is capable of binding to immunoglobulin‐G (IgG), provide a powerful approach for the identification of suitable target antigens for prostate cancer therapy. Hybridoma libraries from mice immunized with androgen‐dependent prostate cancer cell line LNCaP were screened and mAb were selected. Through this screening, we obtained one mAb, designated LNI‐29, that recognizes a glycoprotein with an apparent molecular mass of 100 kD. It was identified as neural cell adhesion molecule 2 (NCAM2). Some prostate and breast cancer cell lines highly expressed NCAM2 whereas normal prostate cell lines expressed NCAM2 at low levels. In contrast to the low efficiency of gene transduction by Adv‐FZ33 with a control antibody, LNI‐29‐mediated Adv‐FZ33 infection induces high rates of gene delivery in NCAM2‐positive cancers. NCAM2‐mediated therapeutic gene transduction of uracil phosphoribosyltransferase (UPRT) had a highly effective cytotoxic effect on NCAM2‐positive cancer cells, whereas it had less of an effect in cases with a control antibody. In conclusion, NCAM2 should be a novel gene therapy target for the treatment of prostate and breast cancer. (Cancer Sci 2011; 102: 808–814)

IQGAP1 selectively interacts with K-Ras but not with H-Ras and modulates K-Ras function

K-Ras is frequently mutated and activated especially in pancreatic cancers. To analyze K-Ras function, we have searched for K-Ras interacting proteins and found IQ motif containing GTPase activating protein 1 (IQGAP1) as a novel K-Ras binding protein. IQGAP1 has been known as a scaffold protein for B-Raf, MEK1/2 and ERK1/2. Here we showed that IQGAP1 selectively formed a complex with K-Ras but not with H-Ras, and recruited B-Raf to K-Ras. We found that IQ motif region of IQGAP1 interacted with K-Ras. Both active and inactive K-Ras interacted with IQGAP1, and effector domain mutants of K-Ras also associated with IQGAP1, indicating that IQGAP1 interacts with K-Ras irrespective of Ras-effectors like B-Raf. We also found that overexpression or knock-down of IQGAP1 affected the interaction between K-Ras and B-Raf, and IQGAP1 overexpression increased ERK1/2 phosphorylation in K-Ras dependent manner in PANC1 cells. Our data suggest that IQGAP1 has a novel mechanism to modulate K-Ras pathway.

Identification of a metabolizing enzyme in human kidney by proteomic correlation profiling

Molecular identification of endogenous enzymes and biologically active substances from complex biological sources remains a challenging task, and although traditional biochemical purification is sometimes regarded as outdated, it remains one of the most powerful methodologies for this purpose. While biochemical purification usually requires large amounts of starting material and many separation steps, we developed an advanced method named “proteomic correlation profiling” in our previous study. In proteomic correlation profiling, we first fractionated biological material by column chromatography, and then calculated each proteins correlation coefficient between the enzyme activity profile and protein abundance profile determined by proteomics technology toward fractions. Thereafter, we could choose possible candidates for the enzyme among proteins with a high correlation value by domain predictions using informatics tools. Ultimately, this streamlined procedure requires fewer purification steps and reduces starting materials dramatically due to low required purity compared with conventional approaches. To demonstrate the generality of this approach, we have now applied an improved workflow of proteomic correlation profiling to a drug metabolizing enzyme and successfully identified alkaline phosphatase, tissue-nonspecific isozyme (ALPL) as a phosphatase of CS-0777 phosphate (CS-0777-P), a selective sphingosine 1-phosphate receptor 1 modulator with potential benefits in the treatment of autoimmune diseases including multiple sclerosis, from human kidney extract. We identified ALPL as a candidate protein only by the 200-fold purification and only from 1 g of human kidney. The identification of ALPL as CS-0777-P phosphatase was strongly supported by a recombinant protein, and contribution of the enzyme in human kidney extract was validated by immunodepletion and a specific inhibitor. This approach can be applied to any kind of enzyme class and biologically active substance; therefore, we believe that we have provided a fast and practical option by combination of traditional biochemistry and state-of-the-art proteomic technology.

Identification of bioactivating enzymes involved in the hydrolysis of laninamivir octanoate, a long-acting neuraminidase inhibitor, in human pulmonary tissue.

Laninamivir octanoate (LO) is an octanoyl ester prodrug of the neuraminidase inhibitor laninamivir. After inhaled administration, LO exhibits clinical efficacy for both treatment and prophylaxis of influenza virus infection, resulting from hydrolytic bioactivation into its pharmacologically active metabolite laninamivir in the pulmonary tissue. In this study, we focused on the identification of LO-hydrolyzing enzymes from human pulmonary tissue extract using proteomic correlation profiling—a technology integration of traditional biochemistry and proteomics. In a single elution step by gel-filtration chromatography, LO-hydrolyzing activity was separated into two distinct peaks, designated as peak I and peak II. By mass spectrometry, 1160 and 1003 proteins were identified and quantitated for peak I and peak II, respectively, and enzyme candidates were ranked based on the correlation coefficient between the enzyme activity and the proteomic profiles. Among proteins with a high correlation value, S-formylglutathione hydrolase (esterase D; ESD) and acyl-protein thioesterase 1 (APT1) were selected as the most likely candidates for peak I and peak II, respectively, which was confirmed by LO-hydrolyzing activity of recombinant proteins. In the case of peak II, LO-hydrolyzing activity was completely inhibited by treatment with a specific APT1 inhibitor, palmostatin B. Moreover, immunohistochemical analysis revealed that both enzymes were mainly localized in the pulmonary epithelia, a primary site of influenza virus infection. These findings demonstrate that ESD and APT1 are key enzymes responsible for the bioactivation of LO in human pulmonary tissue.

TLR7 mediated viral recognition results in focal type I interferon secretion by dendritic cells

Plasmacytoid dendritic cells (pDC) sense viral RNA through toll-like receptor 7 (TLR7), form self-adhesive pDC–pDC clusters, and produce type I interferons. This cell adhesion enhances type I interferon production, but little is known about the underlying mechanisms. Here we show that MyD88-dependent TLR7 signaling activates CD11a/CD18 integrin to induce microtubule elongation. TLR7+ lysosomes then become linked with these microtubules through the GTPase Arl8b and its effector SKIP/Plekhm2, resulting in perinuclear to peripheral relocalization of TLR7. The type I interferon signaling molecules TRAF3, IKKα, and mTORC1 are constitutively associated in pDCs. TLR7 localizes to mTORC1 and induces association of TRAF3 with the upstream molecule TRAF6. Finally, type I interferons are secreted in the vicinity of cell–cell contacts between clustered pDCs. These results suggest that TLR7 needs to move to the cell periphery to induce robust type I interferon responses in pDCs.Antiviral immune responses involve clustering of plasmacytoid dendritic cells (pDC) in response to endosomal TLR7-mediated sensing of viral RNA. Here the authors show the GTPase Arl8b controls translocation of TLR7+ endosomes to the periphery of the cell via microtubule interactions, thus enabling pDC clustering and type I interferon production.

SMARCAD1 is an ATP-dependent stimulator of nucleosomal H2A acetylation via CBP, resulting in transcriptional regulation

Histone acetylation plays a pivotal role in transcriptional regulation, and ATP-dependent nucleosome remodeling activity is required for optimal transcription from chromatin. While these two activities have been well characterized, how they are coordinated remains to be determined. We discovered ATP-dependent histone H2A acetylation activity in Drosophila nuclear extracts. This activity was column purified and demonstrated to be composed of the enzymatic activities of CREB-binding protein (CBP) and SMARCAD1, which belongs to the Etl1 subfamily of the Snf2 family of helicase-related proteins. SMARCAD1 enhanced acetylation by CBP of H2A K5 and K8 in nucleosomes in an ATP-dependent fashion. Expression array analysis of S2 cells having ectopically expressed SMARCAD1 revealed up-regulated genes. Using native genome templates of these up-regulated genes, we found that SMARCAD1 activates their transcription in vitro. Knockdown analysis of SMARCAD1 and CBP indicated overlapping gene control, and ChIP-seq analysis of these commonly controlled genes showed that CBP is recruited to the promoter prior to SMARCAD1. Moreover, Drosophila genetic experiments demonstrated interaction between SMARCAD1/Etl1 and CBP/nej during development. The interplay between the remodeling activity of SMARCAD1 and histone acetylation by CBP sheds light on the function of chromatin and the genome-integrity network.

Identification of activating enzymes of a novel FBPase inhibitor prodrug, CS‐917

CS‐917 (MB06322) is a selective small compound inhibitor of fructose 1,6‐bisphosphatase (FBPase), which is expected to be a novel drug for the treatment of type 2 diabetes by inhibiting gluconeogenesis. CS‐917 is a bisamidate prodrug and activation of CS‐917 requires a two‐step enzyme catalyzed reaction. The first‐step enzyme, esterase, catalyzes the conversion of CS‐917 into the intermediate form (R‐134450) and the second‐step enzyme, phosphoramidase, catalyzes the conversion of R‐134450 into the active form (R‐125338). In this study, we biochemically purified the CS‐917 esterase activity in monkey small intestine and liver. We identified cathepsin A (CTSA) and elastase 3B (ELA3B) as CS‐917 esterases in the small intestine by mass spectrometry, whereas we found CTSA and carboxylesterase 1 (CES1) in monkey liver. We also purified R‐134450 phosphoramidase activity in monkey liver and identified sphingomyelin phosphodiesterase, acid‐like 3A (SMPADL3A), as an R‐134450 phosphoramidase, which has not been reported to have any enzyme activity. Recombinant human CTSA, ELA3B, and CES1 showed CS‐917 esterase activity and recombinant human SMPDL3A showed R‐134450 phosphoramidase activity, which confirmed the identification of those enzymes. Identification of metabolic enzymes responsible for the activation process is the requisite first step to understanding the activation process, pharmacodynamics and pharmacokinetics of CS‐917 at the molecular level. This is the first identification of a phosphoramidase other than histidine triad nucleotide‐binding protein (HINT) family enzymes and SMPDL3A might generally contribute to activation of the other bisamidate prodrugs.

Shotgun Protein Analysis by Liquid Chromatography-Tandem Mass Spectrometry

Two-dimensional electrophoresis (2DE) is an excellent technology for the analysis of complex protein mixtures, but it has drawbacks, such as hardly detecting very hydrophobic proteins. Shotgun protein analysis is one of the major technologies used to compensate for the weaknesses of 2DE. In this approach, total proteins are digested as a mixture and the digested peptides are separated by one-dimensional or multidimensional chromatography and introduced into a tandem mass spectrometer. Since the shotgun approach is the primary strategy in proteomics besides 2DE, a great number of related methodologies have been developed. In this chapter, we would like to introduce the simplest protocol, in which proteins are digested in solution and the digested peptides are analyzed by one-dimensional reversed-phase liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), as a starting point for shotgun protein analysis.