Stress- and ubiquitylation-dependent phase separation of the proteasome.

The proteasome is a major proteolytic machine that regulates cellular proteostasis through selective degradation of ubiquitylated proteins1,2. A number of ubiquitin-related molecules have recently been found to be involved in the regulation of biomolecular condensates or membraneless organelles, which arise by liquid-liquid phase separation of specific biomolecules, including stress granules, nuclear speckles and autophagosomes3-8, but it remains unclear whether the proteasome also participates in such regulation. Here we reveal that proteasome-containing nuclear foci form under acute hyperosmotic stress. These foci are transient structures that contain ubiquitylated proteins, p97 (also known as valosin-containing protein (VCP)) and multiple proteasome-interacting proteins, which collectively constitute a proteolytic centre. The major substrates for degradation by these foci were ribosomal proteins that failed to properly assemble. Notably, the proteasome foci exhibited properties of liquid droplets. RAD23B, a substrate-shuttling factor for the proteasome, and ubiquitylated proteins were necessary for formation of proteasome foci. In mechanistic terms, a liquid-liquid phase separation was triggered by multivalent interactions of two ubiquitin-associated domains of RAD23B and ubiquitin chains consisting of four or more ubiquitin molecules. Collectively, our results suggest that ubiquitin-chain-dependent phase separation induces the formation of a nuclear proteolytic compartment that promotes proteasomal degradation.

Stepwise multipolyubiquitination of p53 by the E6AP-E6 ubiquitin ligase complex.

The human papillomavirus (HPV) oncoprotein E6 specifically binds to E6AP (E6-associated protein), a HECT (homologous to the E6AP C terminus)-type ubiquitin ligase, and directs its ligase activity toward the tumor suppressor p53. To examine the biochemical reaction in vitro, we established an efficient reconstitution system for the polyubiquitination of p53 by the E6AP-E6 complex. We demonstrate that E6AP-E6 formed a stable ternary complex with p53, which underwent extensive polyubiquitination when the isolated ternary complex was incubated with E1, E2, and ubiquitin. Mass spectrometry and biochemical analysis of the reaction products identified lysine residues as p53 ubiquitination sites. A p53 mutant with arginine substitutions of its 18 lysine residues was not ubiquitinated. Analysis of additional p53 mutants retaining only one or two intact ubiquitination sites revealed that chain elongation at each of these sites was limited to 5-6-mers. We also determined the size distribution of ubiquitin chains released by en bloc cleavage from polyubiquitinated p53 to be 2-6-mers. Taken together, these results strongly suggest that p53 is multipolyubiquitinated with short chains by E6AP-E6. In addition, analysis of growing chains provided strong evidence for step-by-step chain elongation. Thus, we hypothesize that p53 is polyubiquitinated in a stepwise manner through the back-and-forth movement of the C-lobe, and the permissive distance for the movement of the C-lobe restricts the length of the chains in the E6AP-E6-p53 ternary complex. Finally, we show that multipolyubiquitination at different sites provides a signal for proteasomal degradation.

HERC2 Facilitates BLM and WRN Helicase Complex Interaction with RPA to Suppress G-Quadruplex DNA.

BLM and WRN are RecQ DNA helicasesessential for genomic stability. Here, we demonstrate that HERC2, a HECT E3 ligase, is critical for their functions to suppress G-quadruplex (G4) DNA. HERC2 interacted with BLM, WRN, and replication protein A (RPA) complexes during the S-phase of the cell cycle. Depletion of HERC2 dissociated RPA from BLM and WRN complexes and significantly increased G4 formation. Triple depletion revealed that HERC2 has an epistatic relationship with BLM and WRN in their G4-suppressing function. In vitro, HERC2 released RPA onto single-stranded DNA (ssDNA) rather than anchoring onto RPA-coated ssDNA. CRISPR/Cas9-mediated deletion of the catalytic ubiquitin-binding site of HERC2 inhibited ubiquitination of RPA2, caused RPA accumulation in the helicase complexes, and increased G4, indicating an essential role for E3 activity in the suppression of G4. Both depletion of HERC2 and inactivation of E3 sensitized cells to the G4-interacting compounds telomestatin and pyridostatin. Overall, these results indicate that HERC2 is a master regulator of G4 suppression that affects the sensitivity of cells to G4 stabilizers. Given that HERC2 expression is frequently reduced in many types of cancers, G4 accumulation as a result of HERC2 deficiency may provide a therapeutic target for G4 stabilizers.Significance: HERC2 is revealed as a master regulator of G-quadruplex, a DNA secondary structure that triggers genomic instability and may serve as a potential molecular target in cancer therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/22/6371/F1.large.jpg Cancer Res; 78(22); 6371-85. ©2018 AACR.

Ub-ProT reveals global length and composition of protein ubiquitylation in cells.

Protein ubiquitylation regulates diverse cellular processes via distinct ubiquitin chains that differ by linkage type and length. However, a comprehensive method for measuring these properties has not been developed. Here we describe a method for assessing the length of substrate-attached polyubiquitin chains, "ubiquitin chain protection from trypsinization (Ub-ProT)." Using Ub-ProT, we found that most ubiquitylated substrates in yeast-soluble lysate are attached to chains of up to seven ubiquitin molecules. Inactivation of the ubiquitin-selective chaperone Cdc48 caused a dramatic increase in chain lengths on substrate proteins, suggesting that Cdc48 complex terminates chain elongation by substrate extraction. In mammalian cells, we found that ligand-activated epidermal growth factor receptor (EGFR) is rapidly modified with K63-linked tetra- to hexa-ubiquitin chains following EGF treatment in human cells. Thus, the Ub-ProT method can contribute to our understanding of mechanisms regulating physiological ubiquitin chain lengths and composition.

Structure of the Dnmt1 Reader Module Complexed with a Unique Two-Mono-Ubiquitin Mark on Histone H3 Reveals the Basis for DNA Methylation Maintenance.

The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.

In Vivo Ubiquitin Linkage-type Analysis Reveals that the Cdc48-Rad23/Dsk2 Axis Contributes to K48-Linked Chain Specificity of the Proteasome.

Ubiquitin-binding domain (UBD) proteins regulate numerous cellular processes, but their specificities toward ubiquitin chain types in cells remain obscure. Here, we perform a quantitative proteomic analysis of ubiquitin linkage-type selectivity of 14 UBD proteins and the proteasome in yeast. We find that K48-linked chains are directed to proteasomal degradation through selectivity of the Cdc48 cofactor Npl4. Mutating Cdc48 results in decreased selectivity, and lacking Rad23/Dsk2 abolishes interactions between ubiquitylated substrates and the proteasome. Among them, only Npl4 has K48 chain specificity in vitro. Thus, the Cdc48 complex functions as a K48 linkage-specifying factor upstream of Rad23/Dsk2 for proteasomal degradation. On the other hand, K63 chains are utilized in endocytic pathways, whereas both K48 and K63 chains are found in the MVB and autophagic pathways. Collectively, our results provide an overall picture of the ubiquitin network via UBD proteins and identify the Cdc48-Rad23/Dsk2 axis as a major route to the proteasome.

Cytoplasmic proteasomes are not indispensable for cell growth in Saccharomyces cerevisiae.

The 26S proteasome is an essential protease complex responsible for the degradation of ubiquitinated proteins in eukaryotic cells. In rapidly proliferating yeast cells, proteasomes are mainly localized in the nucleus, but the biological significance of the proteasome localization is still unclear. In this study, we investigated the relationship between the proteasome localization and the functions by the anchor-away technique, a ligand-dependent sequestration of a target protein into specific compartment(s). Anchoring of the proteasome to the plasma membrane or the ribosome resulted in conditional depletion of the nuclear proteasomes, whereas anchoring to histone resulted in the proteasome sequestration into the nucleus. We observed that the accumulation of ubiquitinated proteins in all the proteasome-targeted cells, suggesting that both the nuclear and cytoplasmic proteasomes have proteolytic functions and that the ubiquitinated proteins are produced and degraded in each compartment. Consistent with previous studies, the nuclear proteasome-depleted cells exhibited a lethal phenotype. In contrast, the nuclear sequestration of the proteasome resulted only in a mild growth defect, suggesting that the cytoplasmic proteasomes are not basically indispensable for cell growth in rapidly growing yeast cells.

First report on invasion of yellow fever mosquito, Aedes aegypti, at Narita International Airport, Japan in August 2012.

The invasion of the yellow fever mosquito Aedes aegypti at Narita International Airport, Japan was detected for the first time. During the course of routine vector surveillance at Narita International Airport, 27 Ae. aegypti adults emerged from larvae and pupae collected from a single larvitrap placed near No. 88 spot at passenger terminal 2 on August 8, 2012. After the appearance of Ae. aegypti in the larvitrap, we defined a 400-m buffer zone and started an intensive vector survey using an additional 34 larvitraps and 15 CO2 traps. International aircraft and passenger terminal 2 were also inspected, and one Ae. aegypti male was collected from the cargo space of an international aircraft from Darwin via Manila on August 28, 2012. Larvicide treatment with 1.5% fenitrothion was conducted in 64 catch basins and one ditch in the 400-m buffer zone. Twenty-four large water tanks were also treated at least once with 0.5% pyriproxyfen, an insect growth regulator. No Ae. aegypti eggs or adults were found during the 1-month intensive vector survey after finding larvae and pupae in the larvitrap. We concluded that Ae. aegypti had failed to establish a population at Narita International Airport.

Clinical characteristics of norovirus gastroenteritis among hospitalized children in Japan.

Acute diarrhea is one of commonest pediatric illnesses worldwide. Although the importance of norovirus as a cause of gastroenteritis outbreaks is well documented, its role in sporadic acute gastroenteritis is not well characterized. The aim of this study was to clarify the prevalence and clinical characteristics of norovirus gastroenteritis among hospitalized children. Between November 2007 and April 2008, inpatients under 12 years of age with acute gastroenteritis in a single hospital in Japan were investigated. A stool sample from each patient was screened for enteropathogenic bacteria and tested by reverse transcription polymerase reaction for norovirus and by an immunochromatographic method for rotavirus and enteric adenoviruses. The clinical features of children with norovirus gastroenteritis were compared with those of children with rotavirus and children without noro- or rotovirus infections. Among 107 patients included in this study, norovirus and rotavirus were detected in 36 (34%) and 37 (35%) patients, respectively. Compared with rotavirus enteritis, the duration of vomiting and diarrhea was significantly longer, and serum C-reactive protein concentrations were higher, in patients with norovirus enteritis. Norovirus was detected as frequently as rotavirus in hospitalized pediatric gastroenteritis patients. Our results suggest that norovirus gastroenteritis among hospitalized young children is not less severe than rotavirus gastroenteritis.

Molecular mechanism of activation of human Cdc7 kinase: bipartite interaction with Dbf4/activator of S phase kinase (ASK) activation subunit stimulates ATP binding and substrate recognition.

Cdc7 is a serine/threonine kinase conserved from yeasts to human and is known to play a key role in the regulation of initiation at each replication origin. Its catalytic function is activated via association with the activation subunit Dbf4/activator of S phase kinase (ASK). It is known that two conserved motifs of Dbf4/ASK are involved in binding to Cdc7, and both are required for maximum activation of Cdc7 kinase. Cdc7 kinases possess unique kinase insert sequences (kinase insert I-III) that are inserted at defined locations among the conserved kinase domains. However, precise mechanisms of Cdc7 kinase activation are largely unknown. We have identified two segments on Cdc7, DAM-1 (Dbf4/ASK interacting motif-1; amino acids 448-457 near the N terminus of kinase insert III) and DAM-2 (C-terminal 10-amino acid segment), that interact with motif-M and motif-C of ASK, respectively, and are essential for kinase activation by ASK. The C-terminal 143-amino acid polypeptide (432-574) containing DAM-1 and DAM-2 can interact with Dbf4/ASK. Characterization of the purified ASK-free Cdc7 and Cdc7-ASK complex shows that ATP binding of the Cdc7 catalytic subunit requires Dbf4/ASK. However, the "minimum" Cdc7, lacking the entire kinase insert II and half of kinase insert III, binds to ATP and shows autophosphorylation activity in the absence of ASK. However, ASK is still required for phosphorylation of exogenous substrates by the minimum Cdc7. These results indicate bipartite interaction between Cdc7 and Dbf4/ASK subunits facilitates ATP binding and substrate recognition by the Cdc7 kinase.

Plasmacytoid dendritic cells sense skin injury and promote wound healing through type I interferons.

Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-α/ß)-producing cells that express intracellular toll-like receptor (TLR) 7 and TLR9 and recognize viral nucleic acids in the context of infections. We show that pDCs also have the ability to sense host-derived nucleic acids released in common skin wounds. pDCs were found to rapidly infiltrate both murine and human skin wounds and to transiently produce type I IFNs via TLR7- and TLR9-dependent recognition of nucleic acids. This process was critical for the induction of early inflammatory responses and reepithelization of injured skin. Cathelicidin peptides, which facilitate immune recognition of released nucleic acids by promoting their access to intracellular TLR compartments, were rapidly induced in skin wounds and were sufficient but not necessary to stimulate pDC activation and type I IFN production. These data uncover a new role of pDCs in sensing tissue damage and promoting wound repair at skin surfaces.

Regulation of TLR7/9 responses in plasmacytoid dendritic cells by BST2 and ILT7 receptor interaction.

Plasmacytoid dendritic cells (pDCs) produce copious type I interferon (IFN) upon sensing nucleic acids through Toll-like receptor (TLR) 7 and TLR9. Uncontrolled pDC activation and IFN production are implicated in lymphopenia and autoimmune diseases; therefore, a mechanism controlling pDC IFN production is essential. Human pDCs specifically express an orphan receptor, immunoglobulin-like transcript 7 (ILT7). Here, we discovered an ILT7 ligand expressed by human cell lines and identified it as bone marrow stromal cell antigen 2 (BST2; CD317). BST2 directly binds to purified ILT7 protein, initiates signaling via the ILT7-FcepsilonRIgamma complex, and strongly inhibits production of IFN and proinflammatory cytokines by pDCs. Readily induced by IFN and other proinflammatory cytokines, BST2 may modulate the human pDC's IFN responses through ILT7 in a negative feedback fashion.

Suppression of allergic diarrhea in murine ovalbumin-induced allergic diarrhea model by PG102, a water-soluble extract prepared from Actinidia arguta.

BACKGROUND: Allergic reactions to food can involve diarrhea, vomiting, nausea and abnormal pain. PG102 has previously been shown to control various factors involved in allergy pathogenesis, including IgE and various Th1 and Th2 cytokines, in vivo as well as in vitro [Park EJ, et al.: J Allergy Clin Immunol 2005;116:1151-1157; Park EJ, et al.: J Invest Dermatol 2007;127:1154-1160]. These data indicate that PG102 might have antiallergic effects on allergic diarrhea. Here, we investigated whether PG102 could prevent allergic diarrhea in the murine ovalbumin (OVA)-induced allergic diarrhea model. METHODS: BALB/c mice were orally treated with PG102, dexamethasone or water for 9 days on a daily basis, followed by subcutaneous injection with OVA on day 0. Animals were orally administrated with OVA from day 7, 3 times a week, over a period of approximately 20 days. Incidence of diarrhea, serum, OVA-restimulated splenocytes and lamina propria lymphocytes were analyzed. RESULTS: Oral administration of PG102 could suppress the incidence of diarrhea in a murine allergic diarrhea model. The amelioration of allergic diarrhea by PG102 was accompanied with the inhibition of mast cell infiltration into the large intestine. The serum level of IgE, IL-6 and MCP-1 was decreased in PG102-treated mice. When splenocytes were isolated from respective groups and cultured in the presence of OVA, cells from PG102-administrated animals produced lesser amounts of IL-6 and MCP-1. CONCLUSIONS: PG102 has the potential to be used as a preventive for food allergic diseases.

T serotypes and antimicrobial susceptibilities of group A streptococcus isolates from pediatric pharyngotonsillitis.

Group A streptococcus (GAS) is a major cause of pediatric pharyngotonsillitis. In this study we determined the T serotype and antimicrobial susceptibility of GAS isolates from Japanese children. From January to December 2006, a total of 438 isolates of GAS were obtained from pharyngeal swabs of 438 children with pharyngotonsillitis. The commonest T serotype was type 1 (110 strains, 25.1%), followed by type 12 (107, 24.4%) and type 4 (77, 17.6%). All GAS isolated from pharyngeal swabs were susceptible to beta-lactams (benzylpenicillin, amoxicillin, cefotaxime, ceftriaxone, imipenem, panipenem, and cefditoren) and vancomycin, but 19.6, 19.6, 3.2, 11.6, and 27.6% were resistant to erythromycin, clarithromycin, clindamycin, minocycline, and norfloxacin, respectively. Resistance varied considerably with the T serotype. In particular, type 4 isolates had the highest resistance (67.5, 67.5, 26.0, and 53.2% were resistant to erythromycin, clarithromycin, minocycline, and norfloxacin, respectively).

The role of CD11b in phagocytosis and dendritic cell development.

Activation of resting T cells is highly dependent on dendritic cells (DCs), which take up antigens and present antigenic peptides to T cells in the context of the major histocompatibility complex (MHC). In this study, we generated a monoclonal antibody, which we call 1C4 that recognizes integrin alpha(M)beta(2) (CD11b/CD18) on the surface of conventional DCs (cDCs) and is internalized after binding. Addition of 1C4 inhibited the ability of immature DCs to phagocytose apoptotic cells. 1C4 treatment also partially inhibited the generation of cDCs from bone marrow in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF). Our findings suggest that not only CD11b is involved in the phagocytosis of apoptotic cells, but also that mAb such as 1C4 may be a useful tool for the delivery of specific proteins into the cytoplasm of immature DCs.

Cdc7 as a potential new target for cancer therapy.

Cdc7 kinase plays crucial roles in firing of replication origins and in proper maintenance of replication forks, which are the sites of DNA replication. The inactivation of Cdc7 causes destabilization of replication forks leading to acute genomic instability and induces massive cell death preferentially in cancer cells. Thus, Cdc7 kinase may be a promising novel target for cancer therapy. Indeed, the first classes of Cdc7 inhibitors have been reported and have been shown to be effective in delaying tumor growth in animal models.

SAGE library screening reveals ILT7 as a specific plasmacytoid dendritic cell marker that regulates type I IFN production.

Plasmacytoid dendritic cells (pDCs) link innate to acquired immune responses by producing high levels of type I IFN upon infection. In order to identify the specific genes that control pDC, we compared serial analysis of gene expression libraries from human pDCs, herpes simplex virus-stimulated pDCs and monocytes. We found that Ig-like transcript ILT7 is specifically expressed on pDC cell surfaces and is down-regulated when pDC mature in response to viral or bacterial stimulation. ILT7 expression on the cell surface required association with the Fc epsilon RI gamma adaptor molecule. Although treatment with one anti-ILT7-specific mAb suppressed type I IFN production in response to cytosine-phosphate-guanosice (CpG) stimulation, another anti-ILT7 mAb up-regulated type I IFN production. We conclude that ILT7 is a key regulator of human pDC function.

Ly49Q defines 2 pDC subsets in mice.

Plasmacytoid dendritic cells (pDCs) play an important primary role for antiviral innate immunity by rapidly producing large amounts of type 1 interferon (IFN) upon viral infection. To study pDC biology, we generated a monoclonal antibody, termed 2E6, that recognizes pDCs. Molecular cloning of a cDNA encoding the 2E6 antigen revealed that it is a type II C-type lectin, Ly49Q, that consists of 247 amino acids with high homology to the natural killer (NK) receptor family Ly49, with an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic domain. Ly49Q is expressed on pDCs but not on NK cells or myeloid dendritic cells. B220+, CD11c+, CD11b- pDCs in bone marrow were divided into Ly49Q+ and Ly49Q- subsets. While both subsets produced IFN-alpha upon cytosine-phosphate-guanosine (CpG) and herpes simplex virus stimulation, Ly49Q- pDCs responded poorly to influenza virus. In addition, Ly49Q- pDCs produced inflammatory cytokines such as interleukin 6 (IL-6), IL-12, and tumor necrosis factor alpha (TNF-alpha) upon stimulation at lower levels than those produced by Ly49Q+ pDCs. In contrast to bone marrow, Ly49Q+ pDCs were only found in peripheral blood, lymph nodes, and spleen. These results indicate that Ly49Q is a specific marker for peripheral pDCs and that expression of Ly49Q defines 2 subsets of pDCs in bone marrow.

Role of NFATx (NFAT4/NFATc3) in expression of immunoregulatory genes in murine peripheral CD4+ T cells.

Ca(2+)-regulated NFAT family members are transcription factors crucial for the expression of various cytokine genes and other immunoregulatory genes. Analyses of mice defective in one or two NFAT family members have revealed functions specific to each NFAT gene. However, the redundant functions of several family members limit the usefulness of gene disruption analysis. For example, CD4(+) T cells isolated from NFATx-disrupted mice do not show any modulation in cytokine gene expression, perhaps because other family members compensate for its absence. To analyze the role of NFATx in the regulation of immunoregulatory genes in T cells, we made a gain-of-function mutant by creating transgenic mice expressing a constitutively nuclear form of NFATx in T cell lineages. In naive CD4(+) T cells, NFATx up-regulated the expression of several cytokine genes and activation markers and suppressed the expression of CD154. In Th1 cells, NFATx enhanced the expression of the Th1 cytokine genes, IFN-gamma and TNF-alpha. In contrast, NFATx suppressed Th2 cytokine genes such as IL-4 and IL-5 in Th2 cells. It has been reported that both NFAT1 and NFATx are required to maintain the homeostasis of the immune system. Our results suggest that NFATx exerts this function by inhibiting the expression of some critical immunoregulatory genes.

A constitutively nuclear form of NFATx shows efficient transactivation activity and induces differentiation of CD4(+)CD8(+) T cells.

The Ca(2+) signal facilitates nuclear translocation of NFAT through the dephosphorylation of clustered serine residues in the calcium regulatory domain by the Ca(2+)/calmodulin-dependent phosphatase calcineurin. The conformation of dephosphorylated NFAT exposes the nuclear localization signal for translocation into the nucleus and masks the nuclear export sequence to keep the protein in the nucleus. It has been reported that deletion of some serine-rich motifs masking the nuclear localization signal results in the translocation of NFAT into the nucleus, but that the nuclear export sequence located at the N terminus also needs to be deleted for NFATx (NFAT4/NFATc3) to exert efficient transactivation function. Here, we report that deletion of the critical serine-rich motifs of NFATx leads to a conformation that efficiently exposes the nuclear localization signal and that has stronger transcription activity compared with the fully activated wild-type protein in the presence of the nuclear export sequence. This also suggests that the regulation of the transactivation domain by phosphorylation observed in NFAT1 may not contribute significantly to the transcription activity of NFATx. The expression of this constitutively nuclear form of NFATx in the CD4(+)CD8(+) T cell line facilitates differentiation into the CD4 single-positive stage upon stimulation with phorbol ester. Our data suggest that NFATx is involved in the regulation of co-receptor expression during differentiation into the CD4 single-positive stage.