Biomedical subjects

Wanjin Hong

Publications and source records attributed to Wanjin Hong.

71 records · Page 4Linked to original sources

PubMed2003

Cidea-deficient mice have lean phenotype and are resistant to obesity.

The thermogenic activity of brown adipose tissue (BAT), important for adaptive thermogenesis and energy expenditure, is mediated by the mitochondrial uncoupling protein1 (Ucp1) that uncouples ATP generation and dissipates the energy as heat. We show here that Cidea, a protein of unknown function sharing sequence similarity with the N-terminal region of DNA fragmentation factors Dffb and Dffa, is expressed at high levels in BAT. Cidea-null mice had higher metabolic rate, lipolysis in BAT and core body temperature when subjected to cold treatment. Notably, Cidea-null mice are lean and resistant to diet-induced obesity and diabetes. Furthermore, we provide evidence that the role of Cidea in regulating thermogenesis, lipolysis and obesity may be mediated in part through its direct suppression of Ucp1 activity. Our data thus indicate a role for Cidea in regulating energy balance and adiposity.

Adipose Tissue, Brown↗

PubMed2003

Evidence for a role of SNX16 in regulating traffic between the early and later endosomal compartments.

Sorting nexins (SNXs) are a growing family of proteins characterized by the presence of a PX domain. The PX domain mediates membrane association by interaction with phosphoinositides. The SNXs are generally believed to participate in membrane trafficking, but information regarding the function of individual proteins is limited. In this report, we describe the major characteristics of one member, SNX16. SNX16 is a novel 343-amino acid protein consisting of a central PX domain followed by a potential coiled-coil domain and a C-terminal region. Like other sorting nexins, SNX16 associates with the membrane via the PX domain which interacts with the phospholipid phosphatidylinositol 3-phosphate. We show via biochemical and cellular studies that SNX16 is distributed in both early and late endosome/lysosome structures. The coiled-coil domain is necessary for localization to the later endosomal structures, as mutant SNX16 lacking this domain was found only in early endosomes. Trafficking of internalized epidermal growth factor was also delayed by this SNX16 mutant, as these cells showed a delay in the segregation of epidermal growth factor in the early endosome for its delivery to later compartments. In addition, the coiled-coil domain is shown here to be important for homo-oligomerization of SNX16. Taken together, these results suggest that SNX16 is a sorting nexin that may function in the trafficking of proteins between the early and late endosomal compartments.

Amino Acid Sequence↗

PubMed2003

PRL-3 and PRL-1 promote cell migration, invasion, and metastasis.

We demonstrate here that Chinese hamster ovary cells stably expressing PRL-3, a M(r) 20000 prenylated protein tyrosine phosphatase, or its relative, PRL-1, exhibit enhanced motility and invasive activity. A catalytically inactive PRL-3 mutant has significantly reduced migration-promoting activity. We observe that PRL-3 is associated with diverse membrane structures involved in cell movement. Furthermore, we show that PRL-3- and -1-expressing cells, but not control cells, induce metastatic tumor formation in mice. Thus, our results deliver the first evidence for a causative role of PRL-3 and -1 in promoting cell motility, invasion activity, and metastasis.

Animals↗

PubMed2003

Interaction of Arl1-GTP with GRIP domains recruits autoantigens Golgin-97 and Golgin-245/p230 onto the Golgi.

A cellular role and the mechanism of action for small GTPase Arl1 have been defined. Arl1-GTP interacts with the GRIP domains of Golgin-97 and Golgin-245, a process dependent on conserved residues of the GRIP domains that are important for Golgi targeting. The switch II region of Arl1 confers the specificity of this interaction. Arl1-GTP mediates Golgi recruitment of Golgin-97 in a switch II-dependent manner, whereas tethering Arl1-GTP onto endosomes can mediate endosomal targeting of Golgin-97. Golgin-97 and Golgin-245 are dissociated from the Golgi when Arl1 is knocked-down by its siRNA. Arl1-GTP thus functions to recruit Golgin-97 and Golgin-245 onto the Golgi via interacting with their GRIP domains.

ADP-Ribosylation Factors↗

PubMed2003

CD81 engineered with endocytotic signals mediates HCV cell entry: implications for receptor usage by HCV in vivo.

Although CD81 has been shown to bind HCV E2 protein, its role as a receptor for HCV remains controversial. In this study, we constructed two CD81 chimeras by linking the cytoplasmic domains of recycling surface receptors, low-density lipoprotein receptor (LDLR), and transferrin receptor (TfR), respectively, to CD81 and compared their internalization properties to wild-type CD81. Binding experiments with anti-hCD81 antibody showed that cell-surface CD81 chimeric receptors were internalized much more efficiently than wild-type CD81. In addition, CD81 chimeras, but not wild-type CD81, could internalize recombinant E2 protein and E2-enveloped viral particles from the serum of HCV-infected patients into Huh7 liver cells. The latter resulted in persistent positive-strand viral RNA and accumulation of replication intermediates, negative-strand viral RNA, in the infected cells, suggesting that the internalized viruses have undergone replication. Therefore, it appeared that CD81, possibly in association with a liver-specific endocytotic protein(s), represents one of the pathways by which HCV can infect hepatocytes.

Antigens, CD↗

PubMed2003

The cytoplasmic domain of Vamp4 and Vamp5 is responsible for their correct subcellular targeting: the N-terminal extenSion of VAMP4 contains a dominant autonomous targeting signal for the trans-Golgi network.

SNAREs represent a superfamily of proteins responsible for the last stage of docking and subsequent fusion in diverse intracellular membrane transport events. The Vamp subfamily of SNAREs contains 7 members (Vamp1, Vamp2, Vamp3/cellubrevin, Vamp4, Vamp5, Vamp7/Ti-Vamp, and Vamp8/endobrevin) that are distributed in various post-Golgi structures. Vamp4 and Vamp5 are distributed predominantly in the trans-Golgi network (TGN) and the plasma membrane, respectively. When C-terminally tagged with enhanced green fluorescent protein, the majority of Vamp4 and Vamp5 is correctly targeted to the TGN and plasma membrane, respectively. Swapping the N-terminal cytoplasmic region and the C-terminal membrane anchor domain between Vamp4 and Vamp5 demonstrates that the N-terminal cytoplasmic region of these two SNAREs contains the correct subcellular targeting information. As compared with Vamp5, Vamp4 contains an N-terminal extension of 51 residues. Appending this 51-residue N-terminal extension onto the N terminus of Vamp5 results in targeting of the chimeric protein to the TGN, suggesting that this N-terminal extension of Vamp4 contains a dominant and autonomous targeting signal for the TGN. Analysis of deletion mutants of this N-terminal region suggests that this TGN-targeting signal is encompassed within a smaller region consisting of a di-Leu motif followed by two acidic clusters. The essential role of the di-Leu motif and the second acidic cluster was then established by site-directed mutagenesis.

Adaptor Proteins, Vesicular Transport↗

PubMed2003

Syntaxin 6 regulates Glut4 trafficking in 3T3-L1 adipocytes.

Insulin stimulates the movement of glucose transporter-4 (Glut4)-containing vesicles to the plasma membrane of adipose cells. We investigated the role of post-Golgi t-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the trafficking of Glut4 in 3T3-L1 adipocytes. Greater than 85% of syntaxin 6 was found in Glut4-containing vesicles, and this t-SNARE exhibited insulin-stimulated movement to the plasma membrane. In contrast, the colocalization of Glut4 with syntaxin 7, 8, or 12/13 was limited and these molecules did not translocate to the plasma membrane. We used adenovirus to overexpress the cytosolic domain of these syntaxin's and studied their effects on Glut4 traffic. Overexpression of the cytosolic domain of syntaxin 6 did not affect insulin-stimulated glucose transport, but increased basal deGlc transport and cell surface Glut4 levels. Moreover, the syntaxin 6 cytosolic domain significantly reduced the rate of Glut4 reinternalization after insulin withdrawal and perturbed subendosomal Glut4 sorting; the corresponding domains of syntaxins 8 and 12 were without effect. Our data suggest that syntaxin 6 is involved in a membrane-trafficking step that sequesters Glut4 away from traffic destined for the plasma membrane. We speculate that this is at the level of traffic of Glut4 into its unique storage compartment and that syntaxin 16 may be involved.

3T3-L1 Cells↗

PubMed2003

GLUT4 recycles via a trans-Golgi network (TGN) subdomain enriched in Syntaxins 6 and 16 but not TGN38: involvement of an acidic targeting motif.

Insulin stimulates glucose transport in fat and muscle cells by triggering exocytosis of the glucose transporter GLUT4. To define the intracellular trafficking of GLUT4, we have studied the internalization of an epitope-tagged version of GLUT4 from the cell surface. GLUT4 rapidly traversed the endosomal system en route to a perinuclear location. This perinuclear GLUT4 compartment did not colocalize with endosomal markers (endosomal antigen 1 protein, transferrin) or TGN38, but showed significant overlap with the TGN target (t)-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Syntaxins 6 and 16. These results were confirmed by vesicle immunoisolation. Consistent with a role for Syntaxins 6 and 16 in GLUT4 trafficking we found that their expression was up-regulated significantly during adipocyte differentiation and insulin stimulated their movement to the cell surface. GLUT4 trafficking between endosomes and trans-Golgi network was regulated via an acidic targeting motif in the carboxy terminus of GLUT4, because a mutant lacking this motif was retained in endosomes. We conclude that GLUT4 is rapidly transported from the cell surface to a subdomain of the trans-Golgi network that is enriched in the t-SNAREs Syntaxins 6 and 16 and that an acidic targeting motif in the C-terminal tail of GLUT4 plays an important role in this process.

3T3 Cells↗

PubMed2002

Expression of a full-length hepatitis C virus cDNA up-regulates the expression of CC chemokines MCP-1 and RANTES.

We had previously reported the cloning of the complete genome of an isolate of hepatitis C virus (HCV), HCV-S1, of genotype 1b. We have constructed a full-length complementary DNA (cDNA) clone of HCV-S1 using nine overlapping cDNA clones that encompassed its entire genome. HCV core, E1, E2, NS-3, -4B, -5A, and -5B proteins were detected in 293T cells by immunoblot analyses when expression of the full-length HCV-S1 was driven under a CMV promoter. Expression of full-length HCV-S1 led to induction of the CC chemokines RANTES and MCP-1 at both the mRNA and the protein levels in HeLa, Huh7, and HepG2 cells. Reporter gene assays showed that a minimal MCP-1 promoter construct containing 128 nucleotides upstream of its translational start site was sufficient for optimal HCV-mediated activation. HCV induced AP-1 binding activities to this region, as determined from electrophoretic mobility shift assays and supershifts with anti-AP-1 antibodies. Transfection of full-length HCV-S1 up-regulated both AP-1 binding activities as well as c-jun transcripts. A minimal promoter construct containing 181 nucleotides upstream of the RANTES translational start site was sufficient for maximal HCV-mediated induction. Gel mobility shift and supershift assays showed that HCV induced NF-kappaB and other unknown binding activities to the A/B-site within this region. In HeLa cells, HCV core and NS5A could separately augment promoter activities of both MCP-1 and RANTES. In Huh7 cells, only NS5A produced a similar effect, while rather surprisingly, HCV core induced a dramatic reduction in promoter activities of these two genes. This study provides the first direct evidence for the induction of CC chemokines in HCV infection and draws attention to their roles in affecting the progress and outcome of HCV-associated liver diseases.

Chemokine CCL2↗

PubMed2002

Ultrastructural characterization of endoplasmic reticulum--Golgi transport containers (EGTC).

Recent observations made in live cells expressing green fluorescent protein (GFP)-tagged cargo markers have demonstrated the existence of large, mobile transport intermediates linking peripheral ER exit sites (ERES) to the perinuclear Golgi. Using a procedure of rapid ethane freezing, we examined ultrastructurally the intermediates involved in ER-Golgi transport of the vesicular stomatitis virus (VSV) G protein. When released at the permissive temperature of 32 degrees C, VSVG is first found to be concentrated in pleiomorphic, membrane-bound structures (of about 0.4 to 1 microm in diameter) with extensive budding profiles. These structures are devoid of COPII components and Golgi markers, but are enriched in COPI, the retrograde cargo ERGIC53, and the tethering protein p115. The structures appear to be able to undergo fusion with the Golgi stack and are tentatively referred to as ER-Golgi transport containers, or EGTCs. VSVG protein exiting the ERES at 15 degrees C is first found in clusters or strings of COPII-containing small vesicles, and morphological analysis indicates that these clusters and strings of COPII vesicles may coalesce by homotypic fusion to form the EGTCs. Together with the large transport containers mediating transport from the trans-Golgi network to the plasma membrane, EGTCs represents an emerging class of large membranous structures mediating anterograde transport between the major stations of the exocytic pathway.

Animals↗

PubMed2002

Sec34 is implicated in traffic from the endoplasmic reticulum to the Golgi and exists in a complex with GTC-90 and ldlBp.

Sec34p/Grd20p has been implicated in endoplasmic reticulum (ER)-to-Golgi transport and/or post-Golgi trafficking events and exists in a protein complex consisting of at least eight subunits in yeast. Although the mammalian counterpart (Sec34) of Sec34p has been molecularly identified, its role and interacting partners remain undefined. In this study, we have prepared antibodies specifically against the recombinant N-terminal fragment of Sec34 that recognize a polypeptide of about 93 kDa and label the Golgi apparatus. In a well-characterized semi-intact cell assay that reconstitutes transport of the envelope glycoprotein (VSVG) of vesicular stomatitis virus from the ER to the Golgi apparatus, anti-Sec34 antibodies inhibited the transport in a dose-dependent manner. The inhibition by anti-Sec34 antibodies could be neutralized by a noninhibitory amount of the antigen. Large-scale immunoprecipitation of rat liver cytosol with immobilized anti-Sec34 antibodies has co-immunoprecipitated GTC-90 and ldlBp, two peripheral Golgi proteins previously shown to exist in separate protein complexes. Two mammalian homologues (Dor1 and Cod1) of the yeast Sec34 complex were similarly recovered in the Sec34 immunoprecipitates. When expressed in transfected cells, epitope-tagged ldlCp and Cod2 were co-immunoprecipitated with anti-Sec34 antibodies with efficiencies comparable to that observed for tagged ldlBp, Dor1, and Cod1. Direct interactions of Sec34 with ldlBp and ldlCp were further demonstrated in vitro. These results suggest that Sec34, GTC-90, and ldlBp/ldlCp are part of the same protein complex(es) that regulates diverse aspects of Golgi function, including transport from the ER to the Golgi apparatus.

ATP-Binding Cassette Transporters↗

PubMed2002

Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform.

The molecular mechanisms underlying early/recycling endosomes-to-TGN transport are still not understood. We identified interactions between the TGN-localized putative t-SNAREs syntaxin 6, syntaxin 16, and Vti1a, and two early/recycling endosomal v-SNAREs, VAMP3/cellubrevin, and VAMP4. Using a novel permeabilized cell system, these proteins were functionally implicated in the post-Golgi retrograde transport step. The function of Rab6a' was also required, whereas its closely related isoform, Rab6a, has previously been implicated in Golgi-to-endoplasmic reticulum transport. Thus, our study shows that membrane exchange between the early endocytic and the biosynthetic/secretory pathways involves specific components of the Rab and SNARE machinery, and suggests that retrograde transport between early/recycling endosomes and the endoplasmic reticulum is critically dependent on the sequential action of two members of the Rab6 subfamily.

Animals↗

PubMed2002

GS15 forms a SNARE complex with syntaxin 5, GS28, and Ykt6 and is implicated in traffic in the early cisternae of the Golgi apparatus.

The subcellular localization, interacting partners, and function of GS15, a Golgi SNARE, remain to be established. In our present study, it is revealed that unlike proteins (Bet1 and the KDEL receptor) cycling between the Golgi and the intermediate compartment (IC, inclusive of the ER exit sites), GS15 is not redistributed into the IC upon incubation at 15 degrees C or when cells are treated with brefeldin A. Immuno-electron microscopy (immuno-EM) reveals that GS15 is mainly found in the medial-cisternae of the Golgi apparatus and adjacent tubulo-vesicular elements. Coimmunoprecipitation experiments suggest that GS15 exists in a distinct SNARE complex that contains SNAREs (syntaxin5, GS28, and Ykt6) that are implicated in both ER-to-Golgi and intra-Golgi transport but not with SNAREs involved exclusively in ER-to-Golgi traffic. Furthermore, components of COPI coat can be selectively coimmunoprecipitated with GS15 from Golgi extracts. Overexpression of mutant forms of GS15 affects the normal distribution of cis- and medial-Golgi proteins (GS28, syntaxin 5, and Golgi mannosidase II), whereas proteins of the trans-Golgi and TGN (Vti1-rp2/Vti1a and syntaxin 6) and Golgi matrix/scaffold (GM130 and p115) are less affected. When the level of GS15 is reduced by duplex 21-nt small interfering RNA (siRNA)-mediated knockdown approach, diverse markers of the Golgi apparatus are redistributed into small dotty and diffuse labeling, suggesting an essential role of GS15 in the Golgi apparatus.

Animals↗

PubMed2002

Interorganellar regulation of lysosome positioning by the Golgi apparatus through Rab34 interaction with Rab-interacting lysosomal protein.

We present evidence to suggest the existence of a regulatory pathway for the Golgi apparatus to modulate the spatial positioning of otherwise distantly located lysosomes. Rab34, a new member of the Rab GTPase family, is associated primarily with the Golgi apparatus. Expression of wild-type or GTP-restricted but not GDP-restricted versions of Rab34 causes spatial redistribution of lysosomes from the periphery to the peri-Golgi region. The regulation of lysosomal positioning by Rab34 depends on its association with the membrane mediated by prenylation and its direct interaction with Rab-interacting lysosomal protein (RILP). This biological activity, mediated by Rab34-RILP interaction, is dependent on Lys82 in the switch I region. Our results have uncovered a novel mechanism for the Golgi apparatus to regulate the spatial distribution of another organelle.

Adaptor Proteins, Signal Transducing↗

PubMedDate not supplied

Mutually exclusive interactions of EHD1 with GS32 and syndapin II.

GS32/SNAP-29 is a SNAP-25-like SNARE and has been shown to interact with syntaxin 6. Using immobilized recombinant GS32, we have recovered EHD1 as a major GS32-interacting protein from total HeLa cell extracts. This interaction is mediated by the EH domain of EHD1 and the N-terminal NPF-containing 17-residue region of GS32. Co-immunoprecipitation suggests that GS32 could also interact with EHD1 in intact cells. When immobilized GST-EHD1 was used to fish out interacting proteins from total brain extracts, syndapin II was identified as a major interacting partner. Similar to the GS32-EHD1 interaction, syndapin II also interacts with the EH domain of EHD1 via its NPF repeat region. Interaction of endogenous EHD1 and syndapin II was also established by co-immunoprecipitation. Furthermore, interaction of GS32 and syndapin II with EHD1 was shown to be mutually exclusive, suggesting that EHD1 may regulate/participate in the functional pathways of both GS32 and syndapin II in a mutual exclusive manner. Opposing roles of GS32 and syndapin II in regulating the surface level of transferrin receptor (TfR) were observed.

Amino Acid Motifs↗

PubMedDate not supplied

Trans-Golgi network syntaxin 10 functions distinctly from syntaxins 6 and 16.

Syntaxin 10 is a soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein localized to the trans-Golgi network (TGN), where two other members of the syntaxin family, syntaxins 6 and 16, also reside. The role of syntaxin 10 in regulating TGN protein traffic is not yet defined. Syntaxin 10 co-localizes well with syntaxins 6 and 16 at the TGN in interphase cells, and appears to interact with both syntaxin 6 and 16 as evidenced by co-immunoprecipitation analyses. However, unlike syntaxin 6 and 16, neither syntaxin 10 antibodies nor its cytosolic domain inhibits endosome-TGN transport of shiga toxin. Syntaxin 16 knockdown with small interfering RNA (siRNA) affects the TGN localization of syntaxin 6 but not syntaxin 10, and clearly inhibits endosome-TGN transport. On the other hand, knockdown of syntaxin 10 expressions had no significant effect on the TGN localization of syntaxin 6 and 16, and did not inhibit endosome-TGN transport. Unlike syntaxin 16, syntaxin 10 does not interact specifically with Vps45, the Sec1/Munc18 (SM) family member at the TGN. On the other hand, syntaxin 10 reciprocally co-immunoprecipitated endosomal syntaxin 12/13, and knockdown of syntaxin 10 expressions affects the surface expression of transferrin receptor (TfR) and seems to induce the formation of an immobile TfR pool. Therefore, in spite of its co-localization and possible interaction with syntaxin 16, syntaxin 10 is not part of the syntaxin 16-based SNARE complex involved in endosome-TGN transport, and may have a hitherto unrecognized function in the TGN-endosome boundary.

Animals↗

PubMedDate not supplied

Knockout of Arfrp1 leads to disruption of ARF-like1 (ARL1) targeting to the trans-Golgi in mouse embryos and HeLa cells.

ADP-ribosylation factor related protein 1 (ARFRP1) is a member of the ARF-family of GTPases which operate as molecular switches in the regulation of intracellular protein traffic. Deletion of the mouse Arfrp1 gene leads to embryonic lethality during early gastrulation, suggesting that ARFRP1 is required for cell adhesion-related processes. Here we show that ARFRP1 specifically controls targeting of ARL1 and its effector Golgin-245 to the trans-Golgi. GTP-bound ARFRP1 (ARFRP1-Q79L mutant) is associated with Golgi membranes and co-localized with the GTPase ARL1. In contrast, the guanine nucleotide exchange defective ARFRP1 mutant (ARFRP1-T31N) clusters within the cytosol. ARFRP1-T31N or depletion of endogenous ARFRP1 by RNA interference disrupts the Golgi association of ARL1 and of the GRIP-domain protein Golgin-245 and alters the distribution of a trans-Golgi network marker, syntaxin 6. In contrast, the targeting of two other Golgi-associated proteins, GM130 and giantin, was unaffected. Furthermore, in Arfrp1-/ - embryos ARL1 dislocated from Golgi membranes whereas it was associated with intracellular membranes in wild-type embryos. These data suggest that lethality of Arfrp1 knockout embryos is due to a specific disruption of protein targeting, e.g., of ARL1 and Golgin-245, to the Golgi.

ADP-Ribosylation Factor 1↗