So far some nuclear receptors for bile acids have been identified. However, no cell surface receptor for bile acids has yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile acids specifically induced receptor internalization, the activation of extracellular signal-regulated kinase mitogen-activated protein kinase, the increase of guanosine 5-O-3-thiotriphosphate binding in membrane fractions, and intracellular cAMP production in Chinese hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5 mRNA showed that it was abundantly expressed in monocytes/macrophages in human and rabbit. Treatment with bile acids was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5 originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not influence those in the original THP-1 cells, suggesting that TGR5 is implicated in the suppression of macrophage functions by bile acids.Bile acids are not simply byproducts of cholesterol metabolism but play essential roles in the absorption of dietary lipids and in the regulation of bile acid synthesis (1). Farnesoid X receptor and pregnane X receptor have been recently identified as specific nuclear receptors for bile acids (2-5). Through the activation of farnesoid X receptor bile acids repress the expression of cholesterol 7␣-hydroxylase, the rate-limiting enzyme in bile acid synthesis (2, 3). The activation of pregnane X receptor by bile acids results in both the repression of cholesterol 7␣-hydroxylase and the transcriptional induction of cytochrome P450 3a, the bile acid-metabolizing enzyme (4, 5). However, no cell surface receptor for bile acids has yet been identified. In hepatobiliary diseases including obstructive jaundice, viral hepatitis, and primary biliary cirrhosis, the mean serum concentration of bile acids exceeds 100 M (range, 70 -400 M), whereas normally this remains below 10 M (6). At such high concentrations, bile acids are known to exhibit immunosuppressive effects on cell-mediated immunity and macrophage functions (6 -8). The phagocytic capacity of the reticuloendothelial system including Kupffer cells is depressed in cholestasis or obstructive jaundice (8). Cholestatic jaundice frequently causes infectious complications and endotoxemia, which are closely related to elevated serum bile acid levels (7, 9). Furthermore, bile acids including deoxycholic acid (DCA) 1 and chenodeoxycholic acid (CDCA) have been demonstrated to have inhibitory activities on the lipopolysaccharide (LPS)-induced production of cytokines in macrophages, including interleukin (IL)-1, IL-6, and tumor necrosis factor ␣ (TNF␣) (10, 11). However, the precise mechanisms involved have remained unclear. Here we show that a novel G prot...

Two ubiquitin-like molecules, Atg12 and LC3/Atg8, are involved in autophagosome biogenesis. Atg12 is conjugated to Atg5 and forms an ϳ800-kDa protein complex with Atg16L (referred to as Atg16L complex). LC3/Atg8 is conjugated to phosphatidylethanolamine and is associated with autophagosome formation, perhaps by enabling membrane elongation. Although the Atg16L complex is required for efficient LC3 lipidation, its role is unknown. Here, we show that overexpression of Atg12 or Atg16L inhibits autophagosome formation. Mechanistically, the site of LC3 lipidation is determined by the membrane localization of the Atg16L complex as well as the interaction of Atg12 with Atg3, the E2 enzyme for the LC3 lipidation process. Forced localization of Atg16L to the plasma membrane enabled ectopic LC3 lipidation at that site. We propose that the Atg16L complex is a new type of E3-like enzyme that functions as a scaffold for LC3 lipidation by dynamically localizing to the putative source membranes for autophagosome formation.

After bacterial invasion, ubiquitin is conjugated to host endosomal proteins and recognized by the autophagic machinery independent of LC3.

Macroautophagy is a mechanism of degradation of cytoplasmic components in all eukaryotic cells. In macroautophagy, cytoplasmic components are wrapped by double-membrane structures called autophagosomes, whose formation involves unique membrane dynamics, i.e., de novo formation of a double-membrane sac called the isolation membrane and its elongation. However, the precise regulatory mechanism of isolation membrane formation and elongation remains unknown. In this study, we showed that Golgi-resident small GTPase Rab33B (and Rab33A) specifically interacts with Atg16L, an essential factor in isolation membrane formation, in a guanosine triphosphate-dependent manner. Expression of a GTPase-deficient mutant Rab33B (Rab33B-Q92L) induced the lipidation of LC3, which is an essential process in autophagosome formation, even under nutrient-rich conditions, and attenuated macroautophagy, as judged by the degradation of p62/sequestosome 1. In addition, overexpression of the Rab33B binding domain of Atg16L suppressed autophagosome formation. Our findings suggest that Rab33 modulates autophagosome formation through interaction with Atg16L. INTRODUCTIONMacroautophagy is a well-conserved mechanism in all eukaryotic cells, and it is important for cells to rid the cell of injured or unwanted cytoplasmic material and to degrade normal cellular components in response to energy needs (Levine and Klionsky, 2004). Macroautophagy (simply referred to as autophagy hereafter) is thought to be the major autophagic pathway, and functions not only as a nutrient supply but also as a defense against stresses, including bacterial intrusion, unfolded protein accumulation, and so on (Mizushima, 2007).In mammalian autophagy, a precursor structure, a crescent-shaped small membrane compartment called isolation membrane core, is present in the initial step in autophagosome formation and it elongates to form characteristic double-membrane structures, called autophagosomes. Finally, autophagosomes fuse with lysosomes to degrade its contents (Yoshimori, 2004).During the past decade, many genes essential for autophagy (i.e., ATG genes) have been identified by genetic analysis of Saccharomyces cerevisiae (Thumm et al., 1994;Tsukada and Ohsumi, 1993; reviewed in Klionsky et al., 2003), and because most of the yeast ATG genes have mammalian counterparts, the fundamental molecular machinery (or proteinprotein interaction cascade) for autophagy is thought to be conserved in all eukaryotic cells (Ohsumi, 2001;Mizushima et al., 2002).Atg5 interacts with Atg12, a ubiquitin-like protein (Ubl) covalently conjugated to Atg5, and with Atg16L, and these proteins form ϳ800-kDa complex through homooligomerization of Atg16L (referred to as Atg5-12/16L complex hereafter). The complex is specifically present on isolation membranes and never present on mature autophagosomes (Mizushima et al., 1998(Mizushima et al., , 2001(Mizushima et al., , 2003. LC3, another Ubl, is conjugated to phosphatidylethanolamine (PE) and localized at elongating isolation membranes and mature aut...

Small GTPase Rab is generally thought to control intracellular membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs have never been identified, and the Rab binding specificity of the Rab effectors previously reported has never been thoroughly investigated. In this study we systematically screened for novel Rab effectors by a yeast two-hybrid assay with 28 different mouse or human Rabs (Rab1-30) as bait and identified 27 Rab-binding proteins, including 19 novel ones. We further investigated their Rab binding specificity by a yeast twohybrid assay with a panel of 60 different GTP-locked mouse or human Rabs. Unexpectedly most (17 of 27) of the Rab-binding proteins we identified exhibited broad Rab binding specificity and bound multiple Rab isoforms. As an example, inositol-polyphosphate 5-phosphatase OCRL (oculocerebrorenal syndrome of Lowe) bound the greatest number of Rabs (i.e. 16 distinct Rabs). Others, however, specifically recognized only a single Rab isoform or only two closely related Rab isoforms. The interaction of eight of the novel Rab-binding proteins identified (e.g. INPP5E and Cog4) with a specific Rab isoform was confirmed by co-immunoprecipitation assay and/or colocalization analysis in mammalian cell cultures, and the novel Rab2B-binding domain of Golgi-associated Rab2B interactor (GARI) and GARI-like proteins was identified by deletion and homology search analyses. The findings suggest that most Rab effectors (or Rab-binding proteins) regulate intracellular membrane trafficking through interaction with several Rab isoforms rather than through a single Rab isoform.

Rab small GTPases are involved in the transport of vesicles between different membranous organelles. RAB-3 is an exocytic Rab that plays a modulatory role in synaptic transmission. Unexpectedly, mutations in the Caenorhabditis elegans RAB-3 exchange factor homologue, aex-3, cause a more severe synaptic transmission defect as well as a defecation defect not seen in rab-3 mutants. We hypothesized that AEX-3 may regulate a second Rab that regulates these processes with RAB-3. We found that AEX-3 regulates another exocytic Rab, RAB-27. Here, we show that C. elegans RAB-27 is localized to synapse-rich regions pan-neuronally and is also expressed in intestinal cells. We identify aex-6 alleles as containing mutations in rab-27. Interestingly, aex-6 mutants exhibit the same defecation defect as aex-3 mutants. aex-6; rab-3 double mutants have behavioral and pharmacological defects similar to aex-3 mutants. In addition, we demonstrate that RBF-1 (rabphilin) is an effector of RAB-27. Therefore, our work demonstrates that AEX-3 regulates both RAB-3 and RAB-27, that both RAB-3 and RAB-27 regulate synaptic transmission, and that RAB-27 potentially acts through its effector RBF-1 to promote soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function. INTRODUCTIONNeurotransmitter release is accomplished by the fusion of neurotransmitter-filled synaptic vesicles at the presynaptic nerve terminal. This process occurs through a series of highly regulated steps that include synaptic vesicle transport, docking/tethering, priming, fusion, endocytosis, recycling, and neurotransmitter refilling (Sudhof, 2004). Several genes have been assigned roles in the various steps of the synaptic vesicle cycle. Rab3 (termed RAB-3 in Caenorhabditis elegans), a member of the Rab family of small GTPases, regulates synaptic transmission, possibly through the docking, priming, or fusion steps Schluter et al., 2004).Rabs act in a variety of cell types and regulate vesicular transport between organelles. Rabs cycle on and off membranes via a GTP-dependent mechanism (Zerial and McBride, 2001). Rab activity is regulated by two proteins, which act in an antagonistic manner. The guanine nucleotide exchange factor (GEF) exchanges GTP for GDP, and the GTPase activating protein activates the intrinsic GTPase activity of a Rab (Bernards, 2003). GDP bound Rabs are held off of the membrane by a GDP dissociation inhibitor (Wu et al., 1996). The GTP/membrane-bound form of Rabs typically binds to a variety of effectors that regulate particular steps of membrane transport and cell signaling (Zerial and McBride, 2001;Spang, 2004).Rab3 was once thought to play a central role in regulating release. However, recent work has shown that a quadruple knockout of all four isoforms of Rab3 in mice only results in a 30% reduction in evoked synaptic response (Schluter et al., 2004). This is consistent with work in C. elegans showing that mutations in the single rab-3 gene cause only mild defects in synaptic transmission . Surprisingly, more dramatic phe...

This is the first report of impedance technique run on single particle LiCoO 2 electrodes with the aim of clarifying its electronic and ionic transport properties. Measurements were successfully conducted on a LiCoO 2 particle of 15 m diam resulting in impedance magnitude on the order of M⍀. The impedance spectra exhibited ͑i͒ one semicircle in the high frequency region, ͑ii͒ Warburg impedance in low frequencies, and finally, ͑iii͒ a limiting capacitance in the very low frequencies. The spectra were analyzed using a modified Randles-Ershler circuit, so that the reaction kinetics could be precisely evaluated. The charge transfer resistance decreased as the potential increased, whereas the double layer capacitance was almost invariant with the potential. Thus, the apparent chemical diffusion coefficient (D app ) of lithium ions was determined to be 10 Ϫ11 to 10 Ϫ7 cm 2 /s as function of electrode potential. These results are in agreement with those obtained by potential step chronoamperometry technique.

It has recently been proposed that the TBC (Tre2/Bub2/Cdc16) domain functions as a GAP (GTPase-activating protein) domain for small GTPase Rab. Because of the large number of Rab proteins in mammals, however, most TBC domains have never been investigated for Rab-GAP activity. In this study we established panels of the GTP-fixed form of 60 different Rabs constructed in pGAD-C1, a yeast two-hybrid bait vector. We also constructed a yeast two-hybrid prey vector (pGBDU-C1) that harbors the cDNA of 40 distinct TBC proteins. Systematic investigation of 2400 combinations of 60 GTP-fixed Rabs and 40 TBC proteins by yeast two-hybrid screening revealed that seven TBC proteins specifically and differentially interact with specific Rabs (e.g. OATL1 interacts with Rab2A; FLJ12085 with Rab5A/B/C; and Evi5-like with Rab10). Measurement of in vitro Rab-GAP activity revealed that OATL1 and Evi5-like actually possess significant Rab2A-and Rab10-GAP activity, respectively, but that FLJ12085 do not display Rab5A-GAP activity at all. These results indicate that specific interaction between TBC protein and Rab would be a useful indicator for screening for the target Rabs of some TBC/Rab-GAP domains, but that there is little correlation between the Rab-binding activity and Rab-GAP activity of other TBC proteins.