Previous studies using B16BL6-derived exosomes labelled with gLuc-lactadherin (gLuc-LA), a fusion protein of Gaussia luciferase (a reporter protein) and lactadherin (an exosome-tropic protein), showed that the exosomes quickly disappeared from the systemic circulation after intravenous injection in mice. In the present study, the mechanism of rapid clearance of intravenously injected B16BL6 exosomes was investigated. gLuc-LA-labelled exosomes were obtained from supernatant of B16BL6 cells after transfection with a plasmid DNA encoding gLuc-LA. Labelling was stable when the exosomes were incubated in serum. By using B16BL6 exosomes labelled with PKH26, a lipophilic fluorescent dye, it was demonstrated that PKH26-labelled B16BL6 exosomes were taken up by macrophages in the liver and spleen but not in the lung, while PKH26-labelled exosomes were taken up by the endothelial cells in the lung. Subsequently, gLuc-LA-labelled B16BL6 exosomes were injected into macrophage-depleted mice prepared by injection with clodronate-containing liposomes. The clearance of the intravenously injected B16BL6 exosomes from the blood circulation was much slower in macrophage-depleted mice than that in untreated mice. These results indicate that macrophages play important roles in the clearance of intravenously injected B16BL6 exosomes from the systemic circulation.

NUAK1 is a member of the AMP-activated protein kinase-related kinase family. Recent studies have shown that NUAK1 is involved in cellular senescence and motility in epithelial cells and fibroblasts. However, the physiological roles of NUAK1 are poorly understood because of embryonic lethality in NUAK1 null mice. The purpose of this study was to elucidate the roles of NUAK1 in adult tissues. We determined the tissue distribution of NUAK1 and generated muscle-specific NUAK1 knock-out (MNUAK1KO) mice. For phenotypic analysis, whole body glucose homeostasis and muscle glucose metabolism were examined. Quantitative phosphoproteome analysis of soleus muscle was performed to understand the molecular mechanisms underlying the knock-out phenotype. Nuak1 mRNA was preferentially expressed in highly oxidative tissues such as brain, heart, and soleus muscle. On a high fat diet, MNUAK1KO mice had a lower fasting blood glucose level, greater glucose tolerance, higher insulin sensitivity, and higher concentration of muscle glycogen than control mice. Phosphoproteome analysis revealed that phosphorylation of IRS1 Ser-1097 was markedly decreased in NUAK1-deficient muscle. Consistent with this, insulin signaling was enhanced in the soleus muscle of MNUAK1KO mice, as evidenced by increased phosphorylation of IRS1 Tyr-608, AKT Thr-308, and TBC1D4 Thr-649. These observations suggest that a physiological role of NUAK1 is to suppress glucose uptake through negative regulation of insulin signaling in oxidative muscle.

Neurofibromatosis type 1 (NF1) is caused by germline mutations in the NF1 gene and is characterized by café au lait spots and benign tumours known as neurofibromas. NF1 encodes the tumour suppressor protein neurofibromin, which negatively regulates the small GTPase Ras, with the constitutive activation of Ras signalling resulting from NF1 mutations being thought to underlie neurofibroma development. We previously showed that knockdown of neurofibromin triggers epithelial-mesenchymal transition (EMT) signalling and that such signalling is activated in NF1-associated neurofibromas. With the use of a cell-based drug screening assay, we have now identified the antiallergy drug tranilast (N-(3,4-dimethoxycinnamoyl) anthranilic acid) as an inhibitor of EMT and found that it attenuated the expression of mesenchymal markers and angiogenesis-related genes in NF1-mutated sNF96.2 cells and in neurofibroma cells from NF1 patients. Tranilast also suppressed the proliferation of neurofibromin-deficient cells in vitro more effectively than it did that of intact cells. In addition, tranilast inhibited sNF96.2 cell migration and proliferation in vivo. Knockdown of type III collagen (COL3A1) also suppressed the proliferation of neurofibroma cells, whereas expression of COL3A1 and SOX2 was increased in tranilast-resistant cells, suggesting that COL3A1 and the transcription factor SOX2 might contribute to the development of tranilast resistance.

In the retinal circuit, environmental light signals are converted into electrical signals that can be decoded properly by the brain. At the first synapse of the visual system, information flow from photoreceptors to bipolar cells is modulated by horizontal cells (HCs), however, their functional contribution to retinal output and individual visual function is not fully understood. In the current study, we investigated functional roles for HCs in retinal ganglion cell (RGC) response properties and optokinetic responses by establishing a HC-depleted mouse line. We observed that HC depletion impairs the antagonistic center-surround receptive field formation of RGCs, supporting a previously reported HC function revealed by pharmacological approaches. In addition, we found that HC loss reduces both the ON and OFF response diversities of RGCs, impairs adjustment of the sensitivity to ambient light at the retinal output level, and alters spatial frequency tuning at an individual level. Taken together, our current study suggests multiple functional aspects of HCs crucial for visual processing.

Locomotion creates various patterns of optic flow on the retina, which provide the observer with information about their movement relative to the environment. However, it is unclear how these optic flow patterns are encoded by the cortex. Here, we use two-photon calcium imaging in awake mice to systematically map monocular and binocular responses to horizontal motion in four areas of the visual cortex. We find that neurons selective to translational or rotational optic flow are abundant in higher visual areas, whereas neurons suppressed by binocular motion are more common in the primary visual cortex. Disruption of retinal direction selectivity in Frmd7 mutant mice reduces the number of translation-selective neurons in the primary visual cortex and translation- and rotation-selective neurons as well as binocular direction-selective neurons in the rostrolateral and anterior visual cortex, blurring the functional distinction between primary and higher visual areas. Thus, optic flow representations in specific areas of the visual cortex rely on binocular integration of motion information from the retina.

Biliary tract cancer (BTC) arises from biliary epithelial cells (BECs) and includes intrahepatic cholangiocarcinoma (IHCC), gallbladder cancer (GC), and extrahepatic cholangiocarcinoma (EHCC). Although frequent KRAS mutations and epigenetic changes at the INK4A/ARF locus have been identified, the molecular pathogenesis of BTC is unclear and the development of corresponding anticancer agents remains inadequate. We isolated epithelial cell adhesion molecule (EpCAM)-positive BECs from the mouse intrahepatic bile duct, gallbladder, and extrahepatic bile duct, and established organoids derived from these cells. Introduction of activated KRAS and homozygous deletion of Ink4a/Arf in the cells of each organoid type conferred the ability to form lethal metastatic adenocarcinoma with differentiated components and a pronounced desmoplastic reaction on cell transplantation into syngeneic mice, indicating that the manipulated cells correspond to BTC-initiating cells. The syngeneic mouse models recapitulate the pathological features of human IHCC, GC, and EHCC, and they should therefore prove useful for the investigation of BTC carcinogenesis and the development of new therapeutic strategies. Tumor cells isolated from primary tumors formed organoids in three-dimensional culture, and serial syngeneic transplantation of these cells revealed that their cancer stem cell properties were supported by organoid culture, but not by adherent culture. Adherent culture thus attenuated tumorigenic activity as well as the expression of both epithelial and stem cell markers, whereas the expression of epithelial-mesenchymal transition (EMT)-related transcription factor genes and mesenchymal cell markers was induced. Our data show that organoid culture is important for maintenance of epithelial cell characteristics, stemness, and tumorigenic activity of BTC-initiating cells.

In many parts of the central nervous system, including the retina, it is unclear whether cholinergic transmission is mediated by rapid, point-to-point synaptic mechanisms, or slower, broad-scale 'non-synaptic' mechanisms. Here, we characterized the ultrastructural features of cholinergic connections between direction-selective starburst amacrine cells and downstream ganglion cells in an existing serial electron microscopy data set, as well as their functional properties using electrophysiology and two-photon acetylcholine (ACh) imaging. Correlative results demonstrate that a 'tripartite' structure facilitates a 'multi-directed' form of transmission, in which ACh released from a single vesicle rapidly (~1 ms) co-activates receptors expressed in multiple neurons located within ~1 µm of the release site. Cholinergic signals are direction-selective at a local, but not global scale, and facilitate the transfer of information from starburst to ganglion cell dendrites. These results suggest a distinct operational framework for cholinergic signaling that bears the hallmarks of synaptic and non-synaptic forms of transmission.

Small extracellular vesicles (sEVs) are important mediators of intercellular communication with respect to diverse pathophysiological processes. Here, we determined novel phosphatidylserine (PS)-deficient sEV subpopulations as a major somatic cell-derived sEV subpopulation in blood because of long blood circulation half-life through escape from macrophage uptake. PS(-)-sEVs were identified in various cultured cells as a minor population. However, as a result of rapid uptake of PS(+)-sEVs by macrophages, circulating somatic cell-derived sEVs in the blood were found to be mainly PS(-)-sEVs. These results suggest that endogenous PS(-)-sEVs could indeed be the key player in sEV-mediated intercellular communication, a good target for sEV-based diagnosis, and a potent candidate for sEV-based drug delivery. Our findings bring a paradigm shift in the understanding of the biology and translational applications of sEVs.

The mitochondrial kinase PTEN-induced kinase 1 (PINK1) and cytosolic ubiquitin ligase (E3) Parkin/PRKN are involved in mitochondrial quality control responses. PINK1 phosphorylates ubiquitin and the Parkin ubiquitin-like (Ubl) domain at serine 65 and promotes Parkin activation and translocation to damaged mitochondria. Upon Parkin activation, the Ubl domain is ubiquitinated at lysine (K) 27 and K48 residues. However, the contribution of K27/K48 ubiquitination toward Parkin activity remains unclear. In this study, ubiquitination of K56 (corresponding to K27 in the human), K77 (K48 in the human) or both was blocked by generating Drosophila Parkin (dParkin) mutants to examine the effects of Parkin Ubl domain ubiquitination on Parkin activation in Drosophila. The dParkin, in which K56 was replaced with arginine (dParkin K56R), rescued pupal lethality in flies by co-expression with PINK1, whereas dParkin K77R could not. The dParkin K56R exhibited reduced abilities of mitochondrial fragmentation and motility arrest, which are mediated by degrading Parkin E3 substrates Mitofusin and Miro, respectively. Pathogenic dParkin K56N, unlike dParkin K56R, destabilized the protein, suggesting that not only was dParkin K56N non-ubiquitin-modified at K56, but also the structure of the Ubl domain for activation was largely affected. Ubiquitin attached to K27 of the Ubl domain during PINK1-mediated Parkin activation was likely to be phosphorylated because human Parkin K27R weakened Parkin self-binding and activation in trans. Therefore, our findings suggest a new mechanism of Parkin activation, where an activation complex is formed through phospho-ubiquitin attachment on the K27 residue of the Parkin Ubl domain.

Kinase networks are important for cellular signal transduction. Despite tremendous efforts to uncover these signaling pathways, huge numbers of uncharacterized phosphosites still remain in the human proteome. Because of the transient nature of kinase-substrate interactions in vivo, it is almost impossible to identify direct substrates. Here, we present a strategy for the rapid, accurate and high-throughput discovery of in vitro kinase substrates using quantitative proteomics. Using 385 purified kinases (354 wild-type protein kinases, 21 mutants and 10 lipid kinases), we identified a total of 175,574 potential direct kinase substrates. In addition, we identified novel kinase groups, such as one group containing 30 threonine-directed kinases and another containing 15 serine/threonine/tyrosine kinases. Surprisingly, we observed that the diversity of substrates for tyrosine kinases was much higher than that for serine-threonine kinases.

Following recent advances in high-throughput mass spectrometry (MS)-based proteomics, the numbers of identified phosphoproteins and their phosphosites have greatly increased in a wide variety of organisms. Although a critical role of phosphorylation is control of protein signaling, our understanding of the phosphoproteome remains limited. Here, we report unexpected, large-scale connections revealed between the phosphoproteome and protein interactome by integrative data-mining of yeast multi-omics data. First, new phosphoproteome data on yeast cells were obtained by MS-based proteomics and unified with publicly available yeast phosphoproteome data. This revealed that nearly 60% of ∼6,000 yeast genes encode phosphoproteins. We mapped these unified phosphoproteome data on a yeast protein-protein interaction (PPI) network with other yeast multi-omics datasets containing information about proteome abundance, proteome disorders, literature-derived signaling reactomes, and in vitro substratomes of kinases. In the phospho-PPI, phosphoproteins had more interacting partners than nonphosphoproteins, implying that a large fraction of intracellular protein interaction patterns (including those of protein complex formation) is affected by reversible and alternative phosphorylation reactions. Although highly abundant or unstructured proteins have a high chance of both interacting with other proteins and being phosphorylated within cells, the difference between the number counts of interacting partners of phosphoproteins and nonphosphoproteins was significant independently of protein abundance and disorder level. Moreover, analysis of the phospho-PPI and yeast signaling reactome data suggested that co-phosphorylation of interacting proteins by single kinases is common within cells. These multi-omics analyses illuminate how wide-ranging intracellular phosphorylation events and the diversity of physical protein interactions are largely affected by each other.

Recent genome-wide association studies suggest that genetic factors contribute to primary biliary cirrhosis (PBC) susceptibility. Although several reports have demonstrated that the interleukin (IL) 12 signaling pathway is involved in PBC pathogenesis, its precise genetic factors have not been fully clarified. Here, we performed an association analysis between IL12A, IL12RB, and signal transducer and activator of transcription 4 (STAT4) genetic variations and susceptibility to PBC. Single nucleotide polymorphisms (SNPs) were genotyped in 395 PBC patients and 458 healthy subjects of Japanese ethnicity and evaluated for associations with PBC susceptibility, anti-nuclear antibody (ANA) status, and anti-mitochondrial antibody (AMA) status. We detected significant associations with PBC susceptibility for several STAT4 SNPs (rs10168266; P = 9.4 × 10(-3), rs11889341; P = 1.2 × 10(-3), rs7574865; P = 4.0 × 10(-4), rs8179673; P = 2.0 × 10(-4), and rs10181656; P = 4.2 × 10(-5)). Three risk alleles (rs7574865; P = 0.040, rs8179673; P = 0.032, and rs10181656; P = 0.031) were associated with ANA status, but not with AMA positivity. Our findings confirm that STAT4 is involved in PBC susceptibility and may play a role in ANA status in the Japanese population.

Killer cell immunoglobulin-like receptors (KIRs) are involved in the activation and inhibition of natural killer cells. Although combinations of KIRs and HLA have been associated with spontaneous and treatment-induced clearance of hepatitis C virus (HCV) infection, their roles are not fully understood in the context of hepatocellular carcinoma (HCC) development. We enrolled 787 consecutive patients with chronic HCV infection, which included 174 cases of HCC, and 325 healthy subjects to clarify the involvement of HLA-Bw and C, KIRs, and major histocompatibility complex class I chain-related gene A (MICA) gene polymorphisms (rs2596542 and rs1051792) in chronic HCV infection and HCV-related HCC. We observed a significant association with chronic hepatitis C susceptibility for HLA-Bw4 (P = 0.00012; odds ratio [OR] = 1.66) and significant protective associations for HLA-C2 and KIR2DL1-HLA-C2 (both P = 0.00099; OR = 0.57). When HCC patients were stratified into younger (<65 years) and older (≥65 years) groups, the frequencies of KIR2DL2-HLA-C1 and KIR2DS2-HLA-C1 (P = 0.008; OR = 2.89 and P = 0.015; OR = 2.79, respectively) as well as rs2596542 and rs1051792 (P = 0.020; OR = 2.17 and P = 0.038; OR = 2.01, respectively) were significantly higher in younger patients. KIR2DL2-HLA-C1 (OR = 2.75; 95% confidence interval: 1.21-6.21, P = 0.015) and rs1051792 (OR = 2.48; 95% confidence interval: 1.23-4.98, P = 0.011) were independently associated with HCC development in younger patients. These results suggest that KIR2DL2-HLA-C1 and rs1051792 may represent molecular biomarkers to identify early onset HCV-related HCC.

The clinical significance of polymorphisms in the interleukin-28B gene encoding interferon (IFN)-λ3, which has antiviral effects, is known in chronic HCV but not in HBV infection. Thus, we measured IFN-λ3 levels in patients with HBV and investigated its clinical significance and association with nucleos(t)ide (NUC) analogue administration.

The insertion of ion mobility spectrometry (IMS) between LC and MS can improve peptide identification in both proteomics and phosphoproteomics by providing structural information that is complementary to LC and MS, because IMS separates ions on the basis of differences in their shapes and charge states. However, it is necessary to know how phosphate groups affect the peptide collision cross sections (CCS) in order to accurately predict phosphopeptide CCS values and to maximize the usefulness of IMS. In this work, we systematically characterized the CCS values of 4,433 pairs of mono-phosphopeptide and corresponding unphosphorylated peptide ions using trapped ion mobility spectrometry (TIMS). Nearly one-third of the mono-phosphopeptide ions evaluated here showed smaller CCS values than their unphosphorylated counterparts, even though phosphorylation results in a mass increase of 80 Da. Significant changes of CCS upon phosphorylation occurred mainly in structurally extended peptides with large numbers of basic groups, possibly reflecting intramolecular interactions between phosphate and basic groups.

We present a motif-targeting phosphoproteome analysis workflow utilizing in vitro kinase reaction to enrich a subset of peptides with specific primary sequence motifs. Phosphopeptides are enriched and dephosphorylated with alkaline phosphatase, followed by in vitro kinase reaction to phosphorylate substrate peptides with specific primary-sequence motifs. These phosphopeptides are enriched again, TMT-labeled, dephosphorylated to enhance MS-detectability, and analyzed by LC/MS/MS. We applied this approach to inhibitor-treated cancer cells, and successfully profiled the inhibitory spectra of multiple kinase inhibitors. We anticipate this approach will be applicable to target specific subsets of the phosphoproteome using the wide variety of available recombinant protein kinases.

Anemia and sarcopenia associated with renal dysfunction caused by cytokine imbalance can contribute to decreased quality of life for older individuals. Growth differentiation factor-15 (GDF-15) is associated with renal dysfunction, although whether it is related to anemia or sarcopenia is unclear. In this study we examined the association of GDF-15 with renal function, hemoglobin and sarcopenia in healthy community-dwelling older females in Japan.

Regnase-1 is an endoribonuclease crucial for controlling inflammation by degrading mRNAs encoding cytokines and inflammatory mediators in mammals. However, it is unclear how Regnase-1-mediated mRNA decay is controlled in interleukin (IL)-1β- or Toll-like receptor (TLR) ligand-stimulated cells. Here, by analyzing the Regnase-1 interactome, we found that IL-1β or TLR stimulus dynamically induced the formation of Regnase-1-β-transducin repeat-containing protein (βTRCP) complex. Importantly, we also uncovered a novel interaction between Regnase-1 and 14-3-3 in both mouse and human cells. In IL-1R/TLR-stimulated cells, the Regnase-1-14-3-3 interaction is mediated by IRAK1 through a previously uncharacterized C-terminal structural domain. Phosphorylation of Regnase-1 at S494 and S513 is critical for Regnase-1-14-3-3 interaction, while a different set of phosphorylation sites of Regnase-1 is known to be required for the recognition by βTRCP and proteasome-mediated degradation. We found that Regnase-1-14-3-3 and Regnase-1-βTRCP interactions are not sequential events. Rather, 14-3-3 protects Regnase-1 from βTRCP-mediated degradation. On the other hand, 14-3-3 abolishes Regnase-1-mediated mRNA decay by inhibiting Regnase-1-mRNA association. In addition, nuclear-cytoplasmic shuttling of Regnase-1 is abrogated by 14-3-3 interaction. Taken together, the results suggest that a novel inflammation-induced interaction of 14-3-3 with Regnase-1 stabilizes inflammatory mRNAs by sequestering Regnase-1 in the cytoplasm to prevent mRNA recognition.

Mass-spectrometry-based phosphoproteomics can identify more than 10,000 phosphorylated sites in a single experiment. But, despite the fact that enormous phosphosite information has been accumulated in public repositories, protein kinase-substrate relationships remain largely unknown. Here, we describe a method to identify endogenous substrates of kinases by using a combination of a proximity-dependent biotin identification method, called BioID, with two other independent methods, kinase-perturbed phosphoproteomics and phosphorylation motif matching. For proof of concept, this approach was applied to casein kinase 2 (CK2) and protein kinase A (PKA), and we identified 24 and 35 putative substrates, respectively. We also show that known cancer-associated missense mutations near phosphosites of substrates affect phosphorylation by CK2 or PKA and thus might alter downstream signaling in cancer cells bearing these mutations. This approach extends our ability to probe physiological kinase-substrate networks by providing new methodology for large-scale identification of endogenous substrates of kinases.

Neuropeptide Y (NPY) is expressed in the developing central nervous system, however, its role in the brain development remains unclear. In this study, C57/B6 mice were intraperitoneally administered 1 nmol/capita/day of NPY, 10 nmol/capita/day of an NPY-receptor 1-specific antagonist (Y1R-A), or NPY and Y1R-A simultaneously (NPY+Y1R-A) from postnatal day (P) 7 to P14. Recombinant NPY reached the P14 cerebrum in 1 hour. These treatments didn't significantly affect body weight gain or P14 brain weight. The ratio of myelinated axons to total axons in the parietal cerebrum was significantly higher in the NPY group than in the control group. The expression of myelin basic protein (MBP)-mRNA in the cerebrum was significantly higher in the NPY group than in the control group and was significantly lower in the NPY+Y1R-A group than in the NPY group, while it was significantly higher in the NPY+Y1R-A group than in the control group. In cultured oligodendroglioma-derived B12 cells, NPY didn't influence the MBP-mRNA expression, while neurotrophin 3 (NT3) increased MBP mRNA via receptor-type tyrosine kinase type C (Trk C). NPY administration significantly increased NT3-mRNA expression in the P14 cerebrum as deduced by quantitative real-time PCR. The change in phosphorylated Trk C (P-Trk C) was proportional to that of the NT3-mRNA expression, and the proportion of P-Trk C was higher in the NPY group than in the control group. These results suggest that NPY, partially via Y1R, induces NT3 which, via Trk C phosphorylation, accelerates myelination by oligodendrocytes in the mouse brain during the neonatal period.