Masafumi Tsujimoto

Function of nucleophosmin/B23, a nucleolar acidic protein, as a histone chaperone

We previously identified and purified a nucleolar phosphoprotein, nucleophosmin/B23, as a stimulatory factor for replication from the adenovirus chromatin. We show here that nucleophosmin/B23 functions as a histone chaperone protein such as nucleoplasmin, TAF‐I, and NAP‐I. Nucleophosmin/B23 was shown to bind to histones, preferentially to histone H3, to mediate formation of nucleosome, and to decondense sperm chromatin. These activities of B23 were dependent on its acidic regions as other histone chaperones, suggesting that B23/nucleophosmin is a member of histone chaperone proteins.

Cutting Edge: Coding Single Nucleotide Polymorphisms of Endoplasmic Reticulum Aminopeptidase 1 Can Affect Antigenic Peptide Generation In Vitro by Influencing Basic Enzymatic Properties of the Enzyme

ER aminopeptidase 1 (ERAP1) customizes antigenic peptide precursors for MHC class I presentation and edits the antigenic peptide repertoire. Coding single nucleotide polymorphisms (SNPs) in ERAP1 were recently linked with predisposition to autoimmune disease, suggesting a link between pathogenesis of autoimmunity and ERAP1-mediated Ag processing. To investigate this possibility, we analyzed the effect that disease-linked SNPs have on Ag processing by ERAP1 in vitro. Michaelis–Menten analysis revealed that the presence of SNPs affects the Michaelis constant and turnover number of the enzyme. Strikingly, specific ERAP1 allele-substrate combinations deviate from standard Michaelis–Menten behavior, demonstrating substrate-inhibition kinetics; to our knowledge, this phenomenon has not been described for this enzyme. Cell-based Ag-presentation analysis was consistent with changes in the substrate inhibition constant Ki, further supporting that ERAP1 allelic composition may affect Ag processing in vivo. We propose that these phenomena should be taken into account when evaluating the possible link between Ag processing and autoimmunity.

Placental leucine aminopeptidase/oxytocinase in maternal serum and placenta during normal pregnancy

Placental leucine aminopeptidase (P-LAP), which is identical with cystine aminopeptidase as oxytocinase, was found to be homologous with rat insulin-regulated membrane aminopeptidase (IRAP) by sequence comparison. In the current study, we determined the P-LAP levels in maternal serum and placenta during healthy pregnancy. P-LAP activities in maternal serum increased with gestation and rose to the peak of 80 IU/ml at 38 weeks of gestation. Northern blot analysis revealed the increase of P-LAP mRNA levels in placenta in the third trimester compared to the first trimester. P-LAP protein and related activities could be detected in the conditioned medium of placental tissue, while they could not be detected in that of human umbilical vein endothelial cells. Immunohistochemically P-LAP was positively stained in the apical membrane of syncytiotrophoblast cells throughout the gestation. These results established the normal range of serum and tissue P-LAP levels during pregnancy and the possible source of serum P-LAP, which will be helpful to elucidate the physiological and clinical roles of P-LAP/oxytocinase/IRAP.

SPERM CHROMATIN DECONDENSATION BY TEMPLATE ACTIVATING FACTOR I THROUGH DIRECT INTERACTION WITH BASIC PROTEINS

ABSTRACT Template activating factor I (TAF-I) was originally identified as a host factor required for DNA replication and transcription of adenovirus genome complexed with viral basic proteins. Purified TAF-I was shown to bind to core histones and stimulate transcription from nucleosomal templates. Human TAF-I consists of two acidic proteins, TAF-Iα and TAF-Iβ, which differ from each other only in their amino-terminal regions. Here, we report that TAF-I decondenses demembraned Xenopus sperm chromatin. Human TAF-Iβ has a chromatin decondensation activity comparable to that of NAP-I, another histone binding protein, whereas TAF-Iα has only a weak activity. Analysis of molecular mechanisms underlying the chromatin decondensation by TAF-I revealed that TAF-I interacts directly with sperm basic proteins. Deletion of the TAF-I carboxyl-terminal acidic region abolishes the decondensation activity. Interestingly, the acidic region itself is not sufficient for decondensation, since an amino acid substitution mutant in the dimerization domain of TAF-I which has the intact acidic region does not support chromatin decondensation. We detected the β form of TAF-I in Xenopus oocytes and eggs by immunoblotting, and the cloning of its cDNA led us to conclude thatXenopus TAF-Iβ also decondenses sperm chromatin. These results suggest that TAF-I plays a role in remodeling higher-order chromatin structure as well as nucleosomal structure through direct interaction with chromatin basic proteins.

Histone- and chromatin-binding activity of template activating factor-I.

Template activating factor‐I (TAF‐I) is a histone‐binding chromatin remodeling factor. We recently found that TAF‐I is capable of mediating decondensation of Xenopus sperm chromatin by releasing sperm‐specific basic proteins. Here we present evidence that TAF‐I preferentially binds to histone H3 among four core histones. Immunofluorescent staining revealed that TAF‐I binds to the decondensed sperm chromatin, of which protein components predominantly consist of histones H3 and H4.

The Arabidopsis aminopeptidase LAP2 regulates plant growth, leaf longevity and stress response

Peptidases are known to play key roles in multiple biological processes in all living organisms. In higher plants, the vast majority of putative aminopeptidases remain uncharacterized. In this study, we performed functional and expression analyses of the Arabidopsis LAP2 through cDNA cloning, isolation of T-DNA insertional mutants, characterization of the enzymatic activity, characterization of gene expression and transcriptomics and metabolomics analyses of the mutants. Loss of function of LAP2, one of the 28 aminopeptidases in Arabidopsis, reduced vegetative growth, accelerated leaf senescence and rendered plants more sensitive to various stresses. LAP2 is highly expressed in the leaf vascular tissue and the quiescent center region. Integration of global gene expression and metabolite analyses suggest that LAP2 controlled intracellular amino acid turnover. The mutant maintained free leucine by up-regulating key genes for leucine biosynthesis. However, this influenced the flux of glutamate strikingly. As a result, γ-aminobutyric acid, a metabolite that is derived from glutamate, was diminished in the mutant. Decrements in these nitrogen-rich compounds are associated with morphological alterations and stress sensitivity of the mutant. The results indicate that LAP2 is indeed an enzymatically active aminopeptidase and plays key roles in senescence, stress response and amino acid turnover.

The Paired-box Homeodomain Transcription Factor Pax6 Binds to the Upstream Region of the TRAP Gene Promoter and Suppresses Receptor Activator of NF-κB Ligand (RANKL)-induced Osteoclast Differentiation

Background: Negative regulation of osteoclast differentiation is critical for suppression of pathological bone destruction. Results: Pax6 is induced by RANKL in osteoclasts and attenuates osteoclast differentiation via blocking TRAP gene expression. Conclusion: Pax6 functions together with its co-receptor to suppress TRAP gene expression and osteoclastogenesis. Significance: This study provides a new aspect for investigating the molecular targets linked to physiological bone resorption. Osteoclast formation is regulated by balancing between the receptor activator of nuclear factor-κB ligand (RANKL) expressed in osteoblasts and extracellular negative regulatory cytokines such as interferon-γ (IFN-γ) and interferon-β (IFN-β), which can suppress excessive bone destruction. However, relatively little is known about intrinsic negative regulatory factors in RANKL-mediated osteoclast differentiation. Here, we show the paired-box homeodomain transcription factor Pax6 acts as a negative regulator of RANKL-mediated osteoclast differentiation. Electrophoretic mobility shift and reporter assays found that Pax6 binds endogenously to the proximal region of the tartrate acid phosphatase (TRAP) gene promoter and suppresses nuclear factor of activated T cells c1 (NFATc1)-induced TRAP gene expression. Introduction of Pax6 retrovirally into bone marrow macrophages attenuates RANKL-induced osteoclast formation. Moreover, we found that the Groucho family member co-repressor Grg6 contributes to Pax6-mediated suppression of the TRAP gene expression induced by NFATc1. These results suggest that Pax6 interferes with RANKL-mediated osteoclast differentiation together with Grg6. Our results demonstrate that the Pax6 pathway constitutes a new aspect of the negative regulatory circuit of RANKL-RANK signaling in osteoclastogenesis and that the augmentation of Pax6 might therefore represent a novel target to block pathological bone resorption.

Ultrastructural localization of aminopeptidase A/angiotensinase and placental leucine aminopeptidase/oxytocinase in chorionic villi of human placenta.

AIMS Membrane-bound aminopeptidases in human placenta are thought to be involved in maintaining homeostasis during pregnancy by metabolizing bioactive peptides such as oxytocin and angiotensin at the interface between the fetus and mother. Because determining the precise localization of these enzymes is required to support this notion, we investigated the ultrastructural localization of two principal enzymes, aminopeptidase A (APA; EC 3.4.11.7)/angiotensinase and placental leucine aminopeptidase (P-LAP; EC 3.4.11.3)/oxytocinase in human first trimester and full-term placenta. METHODS Immunohistochemical analysis using anti-P-LAP and anti-APA antibodies was performed on ultrathin frozen sections of fixed human placental villi. RESULTS Transmission immunoelectron microscopy revealed that both enzymes were expressed on the surface of apical microvilli of syncytiotrophoblast cells and, to a lesser extent, on the basal infoldings. The location of the two enzymes did not vary between the first trimester and full-term placenta sections, while the staining intensities were slightly enhanced in full-term villi. CONCLUSIONS Our observation that P-LAP and APA are present on the microvilli, which is a site of interaction between the mother and fetus, suggests possible involvement of these enzymes in cleaving peptide hormones from the fetus and mother in order to regulate bioactivity.

Next-Generation Sequencing of Protein-Coding and Long Non-protein-Coding RNAs in Two Types of Exosomes Derived from Human Whole Saliva

Exosomes are small extracellular vesicles containing microRNAs and mRNAs that are produced by various types of cells. We previously used ultrafiltration and size-exclusion chromatography to isolate two types of human salivary exosomes (exosomes I, II) that are different in size and proteomes. We showed that salivary exosomes contain large repertoires of small RNAs. However, precise information regarding long RNAs in salivary exosomes has not been fully determined. In this study, we investigated the compositions of protein-coding RNAs (pcRNAs) and long non-protein-coding RNAs (lncRNAs) of exosome I, exosome II and whole saliva (WS) by next-generation sequencing technology. Although 11% of all RNAs were commonly detected among the three samples, the compositions of reads mapping to known RNAs were similar. The most abundant pcRNA is ribosomal RNA protein, and pcRNAs of some salivary proteins such as S100 calcium-binding protein A8 (protein S100-A8) were present in salivary exosomes. Interestingly, lncRNAs of pseudogenes (presumably, processed pseudogenes) were abundant in exosome I, exosome II and WS. Translationally controlled tumor protein gene, which plays an important role in cell proliferation, cell death and immune responses, was highly expressed as pcRNA and pseudogenes in salivary exosomes. Our results show that salivary exosomes contain various types of RNAs such as pseudogenes and small RNAs, and may mediate intercellular communication by transferring these RNAs to target cells as gene expression regulators.

Characterization of Membrane Integrity and Morphological Stability of Human Salivary Exosomes

Exosomes are derived from various sources, including primary and cultured cell lines and body fluids. It is now evident that they are important for communication between cells. They have, therefore, been proposed as potential carriers to deliver drugs to specific sites. In this study, we examined stability of exosomes derived from human saliva. Exosomes were stored at 4°C for up to 20 months and their membrane integrity assessed. Several exosomal markers, such as dipeptidyl peptidase IV (DPP IV; membrane marker) and programmed cell death 6-interacting protein (Alix, lumen marker), were retained intact after 20 months storage at 4°C. Moreover, intact exosomes could be isolated from whole saliva that had been stored at 4°C. Membrane disruption with detergents such as Triton X-100 and Nonidet P-40 caused partial solubilization of DPP IV and release of Alix into the supernatant. In contrast, sodium dodecyl sulfate treatment caused a complete disruption of the membrane. In addition, membrane stability was maintained after freezing and thawing. These results indicated that human saliva-derived exosomes are stable, maintaining their membrane integrity over a long storage period.