Masaaki Oyama

Unc93B1 biases Toll-like receptor responses to nucleic acid in dendritic cells toward DNA- but against RNA-sensing

Toll-like receptors (TLRs) 3, 7, and 9 recognize microbial nucleic acids in endolysosomes and initiate innate and adaptive immune responses. TLR7/9 in dendritic cells (DCs) also respond to self-derived RNA/DNA, respectively, and drive autoantibody production. Remarkably, TLR7 and 9 appear to have mutually opposing, pathogenic or protective, impacts on lupus nephritis in MRL/lpr mice. Little is known, however, about the contrasting relationship between TLR7 and 9. We show that TLR7 and 9 are inversely linked by Unc93B1, a multiple membrane-spanning endoplasmic reticulum (ER) protein. Complementation cloning with a TLR7-unresponsive but TLR9-responsive cell line revealed that amino acid D34 in Unc93B1 repressed TLR7-mediated responses. D34A mutation rendered Unc93B1-deficient DCs hyperresponsive to TLR7 ligand but hyporesponsive to TLR9 ligand, with TLR3 responses unaltered. Unc93B1 associates with and delivers TLR7/9 from the ER to endolysosomes for ligand recognition. The D34A mutation up-regulates Unc93B1 association with endogenous TLR7 in DCs, whereas Unc93B1 association with TLR9 was down-regulated by the D34A mutation. Consistently, the D34A mutation up-regulated ligand-induced trafficking of TLR7 but down-regulated that of TLR9. Collectively, TLR response to nucleic acids in DCs is biased toward DNA-sensing by Unc93B1.

Non-muscle myosin IIA is a functional entry receptor for herpes simplex virus-1

Herpes simplex virus-1 (HSV-1), the prototype of the α-herpesvirus family, causes life-long infections in humans. Although generally associated with various mucocutaneous diseases, HSV-1 is also involved in lethal encephalitis. HSV-1 entry into host cells requires cellular receptors for both envelope glycoproteins B (gB) and D (gD). However, the gB receptors responsible for its broad host range in vitro and infection of critical targets in vivo remain unknown. Here we show that non-muscle myosin heavy chain IIA (NMHC-IIA), a subunit of non-muscle myosin IIA (NM-IIA), functions as an HSV-1 entry receptor by interacting with gB. A cell line that is relatively resistant to HSV-1 infection became highly susceptible to infection by this virus when NMHC-IIA was overexpressed. Antibody to NMHC-IIA blocked HSV-1 infection in naturally permissive target cells. Furthermore, knockdown of NMHC-IIA in the permissive cells inhibited HSV-1 infection as well as cell–cell fusion when gB, gD, gH and gL were coexpressed. Cell-surface expression of NMHC-IIA was markedly and rapidly induced during the initiation of HSV-1 entry. A specific inhibitor of myosin light chain kinase, which regulates NM-IIA by phosphorylation, reduced the redistribution of NMHC-IIA as well as HSV-1 infection in cell culture and in a murine model for herpes stromal keratitis. NMHC-IIA is ubiquitously expressed in various human tissues and cell types and, therefore, is implicated as a functional gB receptor that mediates broad HSV-1 infectivity both in vitro and in vivo. The identification of NMHC-IIA as an HSV-1 entry receptor and the involvement of NM-IIA regulation in HSV-1 infection provide an insight into HSV-1 entry and identify new targets for antiviral drug development.

FBXL21 Regulates Oscillation of the Circadian Clock through Ubiquitination and Stabilization of Cryptochromes

In the mammalian circadian clockwork, CRY1 and CRY2 repressor proteins are regulated by posttranslational modifications for temporally coordinated transcription of clock genes. Previous studies revealed that FBXL3, an F-box-type E3 ligase, ubiquitinates CRYs and mediates their degradation. Here, we found that FBXL21 also ubiquitinates CRYs but counteracts FBXL3. Fbxl21(-/-) mice exhibited normal periodicity of wheel-running rhythms with compromised organization of daily activities, while an extremely long-period phenotype of Fbxl3(-/-) mice was attenuated in Fbxl3/Fbxl21 double-knockout mice. The double knockout destabilized the behavioral rhythms progressively and sometimes elicited arrhythmicity. Surprisingly, FBXL21 stabilized CRYs and antagonized the destabilizing action by FBXL3. Predominantly cytosolic distribution of FBXL21 contrasts with nuclear localization of FBXL3. These results emphasize the physiological importance of antagonizing actions between FBXL21 and FBXL3 on CRYs, and their combined actions at different subcellular locations stabilize oscillation of the circadian clock.

The initial phase of chromosome condensation requires Cdk1-mediated phosphorylation of the CAP-D3 subunit of condensin II

The cell cycle transition from interphase into mitosis is best characterized by the appearance of condensed chromosomes that become microscopically visible as thread-like structures in nuclei. Biochemically, launching the mitotic program requires the activation of the mitotic cyclin-dependent kinase Cdk1 (cyclin-dependent kinase 1), but whether and how Cdk1 triggers chromosome assembly at mitotic entry are not well understood. Here we report that mitotic chromosome assembly in prophase depends on Cdk1-mediated phosphorylation of the condensin II complex. We identified Thr 1415 of the CAP-D3 subunit as a Cdk1 phosphorylation site, which proved crucial as it was required for the Polo kinase Plk1 (Polo-like kinase 1) to localize to chromosome axes through binding to CAP-D3 and thereby hyperphosphorylate the condensin II complex. Live-cell imaging analysis of cells carrying nonphosphorylatable CAP-D3 mutants in place of endogenous protein suggested that phosphorylation of Thr 1415 is required for timely chromosome condensation during prophase, and that the Plk1-mediated phosphorylation of condensin II facilitates its ability to assemble chromosomes properly. These observations provide an explanation for how Cdk1 induces chromosome assembly in cells entering mitosis, and underscore the significance of the cooperative action of Plk1 with Cdk1.

Direct interaction of Plk4 with STIL ensures formation of a single procentriole per parental centriole

Formation of one procentriole next to each pre-existing centriole is essential for centrosome duplication, robust bipolar spindle assembly and maintenance of genome integrity. However, the mechanisms maintaining strict control over centriole copy number are incompletely understood. Here we show that Plk4 and STIL, the key regulators of centriole formation, form a protein complex that provides a scaffold for recruiting HsSAS-6, a major component of the centriolar cartwheel, at the onset of procentriole formation. Furthermore, we demonstrate that phosphorylation of STIL by Plk4 facilitates the STIL/HsSAS-6 interaction and centriolar loading of HsSAS-6. We also provide evidence that negative feedback by centriolar STIL regulates bimodal centriolar distribution of Plk4 and seemingly restricts occurrence of procentriole formation to one site on each parental centriole. Overall, these findings suggest a mechanism whereby coordinated action of three critical factors ensures formation of a single procentriole per parental centriole.

Diversity of Translation Start Sites May Define Increased Complexity of the Human Short ORFeome

Our previous proteomics analysis of small proteins expressed in human K562 cells provided the first direct evidence of translation of upstream ORFs in human full-length cDNAs (Oyama, M., Itagaki, C., Hata, H., Suzuki, Y., Izumi, T., Natsume, T., Isobe, T., and Sugano, S. (2004) Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs. Genome Res. 14, 2048–2052). In the present study, we performed an in-depth proteomics analysis of human K562 and HEK293 cells using a two-dimensional nano-liquid chromatography-tandem mass spectrometry system. The results led to the identification of eight protein-coding regions besides 197 small proteins with a theoretical mass less than 20 kDa that were already annotated coding sequences in the curated mRNA database. In addition to the upstream ORFs in the presumed 5′-untranslated regions of mRNAs, bioinformatics analysis based on accumulated 5′-end cDNA sequence data provided evidence of novel short coding regions that were likely to be translated from the upstream non-AUG start site or from the new short transcript variants generated by utilization of downstream alternative promoters. Protein expression analysis of the GRINL1A gene revealed that translation from the most upstream start site occurred on the minor alternative splicing transcript, whereas this initiation site was not utilized on the major mRNA, resulting in translation of the downstream ORF from the second initiation codon. These findings reveal a novel post-transcriptional system that can augment the human proteome via the alternative use of diverse translation start sites coupled with transcriptional regulation through alternative promoters or splicing, leading to increased complexity of short protein-coding regions defined by the human transcriptome.

Temporal Perturbation of Tyrosine Phosphoproteome Dynamics Reveals the System-wide Regulatory Networks

Signal transduction systems are known to widely regulate complex biological events such as cell proliferation and differentiation. Because phosphotyrosine-dependent networks play a key role in transmitting signals, a comprehensive and fine description of their dynamic behavior can lead us to systematically analyze the regulatory mechanisms that result in each biological effect. Here we established a mass spectrometry-based framework for analyzing tyrosine phosphoproteome dynamics through temporal network perturbation. A highly time-resolved description of the epidermal growth factor-dependent signaling pathways in human A431 cells revealed a global view of their multiphase network activation, comprising a spike signal transmission within 1 min of ligand stimulation followed by the prolonged activation of multiple Src-related molecules. Temporal perturbation of Src family kinases with the corresponding inhibitor PP2 in the prolonged activation phase led to the down-regulation of the molecules related to cell adhesion and receptor degradation, whereas the canonical cascades as well as the epidermal growth factor receptor relatively maintained their activities. Our methodology provides a system-wide view of the regulatory network clusters involved in signal transduction that is essential to refine the literature-based network structures for a systems biology analysis.

Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein.

Tardigrades, also known as water bears, are small aquatic animals. Some tardigrade species tolerate almost complete dehydration and exhibit extraordinary tolerance to various physical extremes in the dehydrated state. Here we determine a high-quality genome sequence of Ramazzottius varieornatus, one of the most stress-tolerant tardigrade species. Precise gene repertoire analyses reveal the presence of a small proportion (1.2% or less) of putative foreign genes, loss of gene pathways that promote stress damage, expansion of gene families related to ameliorating damage, and evolution and high expression of novel tardigrade-unique proteins. Minor changes in the gene expression profiles during dehydration and rehydration suggest constitutive expression of tolerance-related genes. Using human cultured cells, we demonstrate that a tardigrade-unique DNA-associating protein suppresses X-ray-induced DNA damage by ∼40% and improves radiotolerance. These findings indicate the relevance of tardigrade-unique proteins to tolerability and tardigrades could be a bountiful source of new protection genes and mechanisms.

Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection

Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections.

Tks5-dependent formation of circumferential podosomes/invadopodia mediates cell–cell fusion

Tks5, a master regulator of invadopodia in cancer cells, is also crucial for osteoclast cell–cell fusion.