Biomedical subjects

W Hong

Publications and source records attributed to W Hong.

At least 19 recordsLinked to original sources

PubMed2001

The Phox homology (PX) domain, a new player in phosphoinositide signalling.

Phosphoinositides are key regulators of diverse cellular processes. The pleckstrin homology (PH) domain mediates the action of PtdIns(3,4)P(2), PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), while the FYVE domain relays the pulse of PtdIns3P. The recent establishment that the Phox homology (PX) domain interacts with PtdIns3P and other phosphoinositides suggests another mechanism by which phosphoinositides can regulate/integrate multiple cellular events via a spectrum of PX domain-containing proteins. Together with the recent discovery that the epsin N-terminal homologue (ENTH) domain interacts with PtdIns(4,5)P(2), it is becoming clear that phosphoinositides regulate diverse cellular events through interactions with several distinct structural motifs present in many different proteins.

Amino Acid Sequence↗

PubMed2001

Lembehyne A, a spongean polyacetylene, induces neuronal differentiation in neuroblastoma cell.

Lembehyne A (LB-A), a spongean polyacetylene, induced neuronal cell differentiation in a neuroblastoma cell line, Neuro 2A. The LB-A treatment of Neuro 2A cells predominantly resulted in a morphological change with bipolar neurites. The acetylcholinesterase activity of Neuro 2A was also increased by the treatment of LB-A. Furthermore, the cell cycle of Neuro 2A cells was found to be specifically blocked at the G1 phase by LB-A. The structure-activity relationship study using the LB-A analogues revealed the importance of the terminal 1-yn-3-ol and unsaturated long-chain alkyl moieties for the neuronal differentiation activity of LB-A.

Acetylcholinesterase↗

PubMed2001

Endofin, an endosomal FYVE domain protein.

KIAA0305 is an uncharacterized member of the FYVE domain protein family. It is closely related to SARA, with about 50% identity in the carboxyl-terminal 800-amino acid region. Indirect immunofluorescence microscopy using polyclonal antibodies raised against KIAA0305 revealed that it is enriched in early endosomes. The Myc-tagged version is also faithfully targeted to the early endosome. We have tentatively called KIAA0305 endofin (for endosome-associated FYVE-domain protein). The association of endofin with endosomes is mediated by its FYVE domain because deletion mutants lacking the central FYVE finger motif are distributed in the cytoplasm. In addition, a single point mutation in the FYVE finger motif at cysteine residue 753 (C753S) is sufficient to abolish its endosomal association. Its endosomal localization is also sensitive to the phosphatidylinositol 3-kinase inhibitor, wortmannin. Using in vitro liposome binding assays, we demonstrate that Myc-tagged endofin associates preferentially with phosphatidylinositol 3-phosphate, whereas the C753S point mutant was unable to do so. We also show that endofin co-localizes with SARA but that they are not associated in a common complex because they failed to co-immunoprecipitate in co-expressing cells. Endofin also does not associate with Smad2 nor behave like SARA in affecting transforming growth factor-beta signaling. At high levels of expression, both endofin and SARA can cause an endosome aggregation/fusion effect. In COS7 cells, which can support high levels of exogenous protein expression, both proteins can also cause other structural anomalies in the endocytic pathway, as represented by enlarged vesicular structures. These endosomal aggregates/fusions accumulated endocytosed epidermal growth factor. Taken together, this report provides evidence to suggest that endofin and the highly related SARA are endosomal proteins with potential roles in regulating membrane traffic.

Amino Acid Motifs↗

PubMed2001

A membrane protein enriched in endoplasmic reticulum exit sites interacts with COPII.

Although all mammalian COPII components have now been cloned, little is known of their interactions with other regulatory proteins involved in exit from the endoplasmic reticulum (ER). We report here that a mammalian protein (Yip1A) that is about 31% identical to S. cerevisiae and which interacts with and modulates COPII-mediated ER-Golgi transport. Yip1A transcripts are ubiquitously expressed. Transcripts of a related mammalian homologue, Yip1B, are found specifically in the heart. Indirect immunofluorescence microscopy revealed that Yip1A is localized to vesicular structures that are concentrated at the perinuclear region. The structures marked by Yip1A co-localized with Sec31A and Sec13, components of the COPII coat protein complex. Immunoelectron microscopy also showed that Yip1A co-localizes with Sec13 at ER exit sites. Overexpression of the hydrophilic N terminus of Yip1A arrests ER-Golgi transport of the vesicular stomatitis G protein and causes fragmentation and dispersion of the Golgi apparatus. A glutathione S-transferase fusion protein with the hydrophilic N terminus of Yip1A (GST-Yip1A) is able to bind to and deplete vital components from rat liver cytosol that is essential for in vitro vesicular stomatitis G transport. Peptide sequence analysis of cytosolic proteins that are specifically bound to GST-Yip1A revealed, among other proteins, mammalian COPII components Sec23 and Sec24. A highly conserved domain at the N terminus of Yip1A is required for Sec23/Sec24 interaction. Our results suggest that Yip1A is involved in the regulation of ER-Golgi traffic at the level of ER exit sites.

Amino Acid Sequence↗

PubMed2001

Interaction of farnesylated PRL-2, a protein-tyrosine phosphatase, with the beta-subunit of geranylgeranyltransferase II.

Protein of regenerating liver (PRL)-1, -2, and -3 comprise a subgroup of closely related protein-tyrosine phosphatases featuring a C-terminal prenylation motif conforming to either the consensus sequence for farnesylation, CAAX, or geranylgeranylation, CCXX. Yeast two-hybrid screening for PRL-2-interacting proteins identified the beta-subunit of Rab geranylgeranyltransferase II (betaGGT II). The specific interaction of betaGGT II with PRL-2 but not with PRL-1 or -3 occurred in yeast and HeLa cells. Chimeric PRL-1/-2 molecules were tested for their interaction with betaGGT II, and revealed that the C-terminal region of PRL-2 is required for interaction, possibly the PRL variable region immediately preceeding the CAAX box. Additionally, PRL-2 prenylation is prequisite for betaGGT II binding. As prenylated PRL-2 is localized to the early endosome, we propose that this is where the interaction occurs. PRL-2 is not a substrate for betaGGT II, as isoprenoid analysis showed that PRL-2 was solely farnesylated in vivo. Co-expression of the alpha-subunit (alpha) of GGT II, betaGGT II, and PRL-2 resulted in alpha/betaGGT II heterodimer formation and prevented PRL-2 binding. Expression of PRL-2 alone inhibited the endogenous alpha/betaGGT II activity in HeLa cells. Together, these results indicate that the binding of alphaGGT II and PRL-2 to betaGGT II is mutually exclusive, and suggest that PRL-2 may function as a regulator of GGT II activity.

Amino Acid Sequence↗

PubMed2001

Lack of effects by sigma ligands on neuropeptide Y-induced G-protein activation in rat hippocampus and cerebellum.

It has been suggested that neuropeptide Y (NPY) and sigma (sigma) receptor ligands may share a putative NPY/sigma receptor in rat brain. To study whether NPY and sigma receptor ligands have an inverse agonism at this putative NPY/sigma receptor, we measured their effects on G-protein activity in rat brain. Using [35S]GTPgammaS autoradiography, we found that NPY-induced G-protein activation exhibited a discrete distribution pattern in rat brain. G-protein activation in superficial cortical layers and hippocampal CA1-3 region was mainly attributed to Y1 and Y2 receptors, respectively. In the presence of 10 microM sigma-receptor agonist BD737 or 10 microM sigma-receptor antagonist haloperidol, the distribution and density of [35S]GTPgammaS binding stimulated by 10 nM NPY was not significantly altered. In rat cerebellar membranes, NPY stimulated high-affinity GTPase activity in a dose-related manner, with maximal effects of 29% increase over basal level seen at 500 nM. This NPY-elicited GTPase activity was not significantly affected by micromolar concentrations of the sigma-receptor antagonists Dup734 or haloperidol. Since no significant effects by sigma-receptor ligands on NPY-induced G-protein activation were observed, we did not see an inverse agonism of NPY and sigma-receptor ligands at the putative NPY/sigma receptor measured at the level of G-protein activation, suggesting that sigma receptors and NPY receptors do not represent a common population in rat hippocampus and cerebellum. It is also suggested that G-protein activation is not a convergent point for the signal transduction mechanisms of NPY receptors and sigma receptors.

Analgesics↗

PubMed2001

Retinoblastoma-binding protein 2 (Rbp2) potentiates nuclear hormone receptor-mediated transcription.

Retinoblastoma-binding protein 2 (Rbp2) was originally identified as a retinoblastoma protein (RB) pocket domain-binding protein. Although Rbp2 has been shown to interact with RB, p107, TATA-binding protein, and T-cell oncogene rhombotin-2, the physiological function of Rbp2 remains unclear. Here we demonstrate that Rbp2 not only binds to nuclear receptors (NRs) but also enhances the transcription mediated by them. Rbp2 interacts with the DNA-binding domains of NRs and potentiates NR-mediated transcription in an AF-2-dependent manner. Both the N-terminal and C-terminal domains of Rbp2 are critical for the transactivation activity of Rbp2 on NRs. The C terminus is the NR-interacting region. In addition, RB functions in maximizing the effect of Rbp2 on the transcription by NRs. These results suggest that Rbp2 is a coregulator of NRs and define a potential role for Rbp2 in NR-mediated transcription.

Amino Acid Motifs↗

PubMed2001

Ykt6 forms a SNARE complex with syntaxin 5, GS28, and Bet1 and participates in a late stage in endoplasmic reticulum-Golgi transport.

The yeast SNARE Ykt6p has been implicated in several trafficking steps, including vesicular transport from the endoplasmic reticulum (ER) to the Golgi, intra-Golgi transport, and homotypic vacuole fusion. The functional role of its mammalian homologue (Ykt6) has not been established. Using antibodies specific for mammalian Ykt6, it is revealed that it is found mainly in Golgi-enriched membranes. Three SNAREs, syntaxin 5, GS28, and Bet1, are specifically associated with Ykt6 as revealed by co-immunoprecipitation, suggesting that these four SNAREs form a SNARE complex. Double labeling of Ykt6 and the Golgi marker mannosidase II or the ER-Golgi recycling marker KDEL receptor suggests that Ykt6 is primarily associated with the Golgi apparatus. Unlike the KDEL receptor, Ykt6 does not cycle back to the peripheral ER exit sites. Antibodies against Ykt6 inhibit in vitro ER-Golgi transport of vesicular stomatitis virus envelope glycoprotein (VSVG) only when they are added before the EGTA-sensitive stage. ER-Golgi transport of VSVG in vitro is also inhibited by recombinant Ykt6. In the presence of antibodies against Ykt6, VSVG accumulates in peri-Golgi vesicular structures and is prevented from entering the mannosidase II compartment, suggesting that Ykt6 functions at a late stage in ER-Golgi transport. Golgi apparatus marked by mannosidase II is fragmented into vesicular structures in cells microinjected with Ykt6 antibodies. It is concluded that Ykt6 functions in a late step of ER-Golgi transport, and this role may be important for the integrity of the Golgi complex.

Base Sequence↗

PubMed2001

Retinoblastoma protein is functionally distinct from its homologues in affecting glucocorticoid receptor-mediated transcription and apoptosis.

The cell cycle regulator, retinoblastoma protein, is known to potentiate glucocorticoid receptor-activated transcription through the interaction of its pocket domain with the transcription coactivator, hBRM. We now show that glucocorticoid receptor-induced apoptosis is also dependent on both the retinoblastoma protein and hBRM. p107 and p130, which share extensive sequence homology with the pocket domain of the retinoblastoma protein but not its N-terminal region, also interact with hBRM but do not support either glucocorticoid receptor-dependent activity. This difference arises from the divergent N-terminal domain of the retinoblastoma protein, which, when fused to the pocket domains, confers upon p107 and p130 the ability to influence glucocorticoid receptor activities. This effect probably results from the promotion of glucocorticoid receptor-targeted chromatin remodeling by the hBRM-containing SWI/SNF complex because the N-terminal domain of the retinoblastoma protein enhances glucocorticoid receptor-hBRM interactions. These results highlight that, besides the interaction between hBRM and the pocket domain of RB, the N-terminal region of the retinoblastoma protein is also essential for glucocorticoid receptor-induced apoptosis and the potentiation of glucocorticoid receptor-mediated transcription and provide a basis for functional distinction between the retinoblastoma protein and its homologues.

Apoptosis↗

PubMed2001

Stimulation of NF-E2 DNA binding by CREB-binding protein (CBP)-mediated acetylation.

The hematopoietic transcription factor NF-E2 is an important regulator of erythroid and megakaryocytic gene expression. The transcription cofactor cAMP-response element-binding protein (CREB)-binding protein (CBP) has previously been implicated in mediating NF-E2 function. In this report, we examined the role of CBP, a coactivator with intrinsic acetyltransferase activity, in the regulation of NF-E2. We found that both the hematopoietic-specific subunit of NF-E2, p45, and the widely expressed small subunit, MafG, interact with CBP in vitro and in vivo. CBP acetylates MafG, but not p45, predominantly in the basic region of MafG. Immunoprecipitation experiments with anti-acetyl lysine antibodies demonstrate that MafG is acetylated in vivo in erythroid cells. Transfection experiments further show that CBP stimulates MafG acetylation in intact cells in an E1A-sensitive manner. Acetylation of MafG augments DNA binding activity of NF-E2, and mutations at the major acetylation sites markedly reduce DNA binding and transcriptional activation by NF-E2. Together, these results suggest that recruitment of CBP by NF-E2 to specific erythroid/megakaryocytic promoters might regulate transcription by at least two mechanisms involving both modification of chromatin structure and modulation of transcription factor activity.

Acetylation↗

PubMed2001

Targets of a protease inhibitor, KNI-272, in HIV-1-infected cells.

The targets of a protease inhibitor, KNI-272, in the HIV-1 life cycle were investigated in this study. Neither expression of HIV-1 Gag proteins nor production of virus particles was detected in cells infected acutely with HIV-1 cultured in the presence of KNI-272. Although HIV-1 proviral DNA was detected in the cells by PCR, the inhibitor depressed the amount of the proviral DNA in a concentration dependent manner. These results indicate that one of the targets of KNI-272 occurs in the stage before the expression of viral structural proteins. No direct inhibition of reverse transcription was found with the inhibitor. To confirm the inhibition of viral protease, persistently HIV-1-infected cells were cultured in the presence of the inhibitor and examined by electron microscopy for the morphology of HIV-1 particles. Doughnut-shaped immature particles were observed in the extracellular space of the cells, and disrupted semicircular shaped particles were also seen at the higher concentration of KNI-272. A bioassay for infectivity showed that the virus particles were not infectious, and immunofluorescent assay using anti-p17 antibody, that does not react with the precursor of Gag protein, revealed that Gag precursor p55 protein in the cells was not processed. Thus, KNI-272 blocked the maturation of viral particles. Consequently, KNI-272 has at least two inhibition targets in the stages of the HIV-1 life cycle.

Anti-HIV Agents↗

PubMed2001

Evidence for prebudding arrest of ER export in animal cell mitosis and its role in generating Golgi partitioning intermediates.

During mitosis the interconnected Golgi complex of animal cells breaks down to produce both finely dispersed elements and discrete vesiculotubular structures. The endoplasmic reticulum (ER) plays a controversial role in generating these partitioning intermediates and here we highlight the importance of mitotic ER export arrest in this process. We show that experimental inhibition of ER export (by microinjecting dominant negative Sar1 mutant proteins) is sufficient to induce and maintain transformation of Golgi cisternae to vesiculotubular remnants during interphase and telophase, respectively. We also show that buds on the ER, ER exit sites and COPII vesicles are markedly depleted in mitotic cells and COPII components Sec23p, Sec24p, Sec13p and Sec31p redistribute into the cytosol, indicating ER export is inhibited at an early stage. Finally, we find a markedly uneven distribution of Golgi residents over residual exit sites of metaphase cells, consistent with tubulovesicular Golgi remnants arising by fragmentation rather than redistribution via the ER. Together, these results suggest selective recycling of Golgi residents, combined with prebudding cessation of ER export, induces transformation of Golgi cisternae to vesiculotubular remnants in mitotic cells. The vesiculotubular Golgi remnants, containing populations of slow or nonrecycling Golgi components, arise by fragmentation of a depleted Golgi ribbon independently from the ER.

COP-Coated Vesicles↗

PubMed2001

SNX3 regulates endosomal function through its PX-domain-mediated interaction with PtdIns(3)P.

The sorting nexin (SNX) protein family is implicated in regulating membrane traffic, but the mechanism is still unknown. We show that SNX3 is associated with the early endosome through a novel motif (PX domain) capable of interaction with phosphatidylinositol-3-phosphate (PtdIns(3)P). Overexpression of SNX3 alters endosomal morphology and delays transport to the lysosome. Transport from the early to the recycling endosome is affected upon microinjection of SNX3 antibodies. Our results highlight a novel mechanism by which SNX proteins regulate traffic and uncover a novel class of effectors for PtdIns(3)P.

Androstadienes↗

PubMed2001

Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex.

Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway.

Animals↗

PubMed2001

Regulation of Golgi structure and function by ARF-like protein 1 (Arl1).

Arl1 is a member of the ARF-like protein (Arl) subfamily of small GTPases. Nothing is known about the function of Arl1 except for the fact that it is essential for normal development in Drosophila and that it is associated with the Golgi apparatus. In this study, we first demonstrate that Arl1 is enriched at the trans side of the Golgi, marked by AP-1. Association of Arl1 with the Golgi is saturable in intact cells and depends on N-terminal myristoylation. Over-expression of Arl1(T31N), which is expected to be restricted to the GDP-bound form and thus function as a dominant-negative mutant, causes the disappearance of the Golgi apparatus (marked by Golgi SNARE GS28), suggesting that Arl1 is necessary for maintaining normal Golgi structure. Overexpression of Arl1(Q71L), a mutant restricted primarily to the activated GTP-bound form, causes an expansion of the Golgi apparatus with massive and stable Golgi association of COPI and AP-1 coats. Interestingly, Golgi ARFs also become stably associated with the expanded Golgi. Transport of the envelope protein of vesicular stomatitis virus (VSV-G) along the secretory pathway is arrested at the expanded Golgi upon expression of Arl1(Q71L). The structure of stacked cisternae of the Golgi is disrupted in cells expressing Arl1(Q71L), resulting in the transformation of the Golgi into an extensive vesicule-tubule network. In addition, the GTP form of Arl1 interacts with arfaptin-2/POR1 but not GGA1, both of which interact with GTP-restricted ARF1, suggesting that Arl1 and ARF1 share some common effectors in regulating cellular events. On the basis of these observations, we propose that one of the mechanisms for the cell to regulate the structure and function of the Golgi apparatus is through the action of Arl1.

ADP-Ribosylation Factors↗

PubMed2001

[Genotype polymorphism and its implications of mannose-binding protein allele in 5 Chinese nationalities].

OBJECTIVE: To detect the genotypes and sequences of the exon 1 of human mannose-binding protein (MBP) allele in 5 Chinese nationalities. METHODS: The genotypes of MBP gene of 5 Chinese nationalities were detected by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). The exon 1 of the MBP gene of 22 Chinese Hans was analyzed by using ABI 310 genetic analyzer. RESULTS: The DNA sequences of exon 1 of Chinese MBP gene were acquired. The allele frequencies of the codon 54 of the MBP gene (MBP-54) of 5 Chinese nationalities were 0.181(Hans), 0.128(Uygurs), 0.181(Mongols), 0.179(Tibetans) and 0.181(Yis). The allele distribution for MBP-54 mutation of 5 Chinese nationalities was in good agreement with Hardy-Weinberg equilibrium. Compared with the Hans, Uygurs had a lower MBP-54 mutation rate. There were no differences in the allele frequencies between the chronic hepatitis B patients and health controls in Chinese Hans. The mutations of the codons 52 and 57 were not detected in this study. CONCLUSION: A higher prevalence of MBP-54 mutation was found in 5 Chinese nationalities, MBP-54 mutation was not associated with the persistence of hepatitis B.

Adolescent↗

PubMed2001

[Genotyping of HIV resistant alleles in indigenous Tibetan ethnic group of China: low frequency of CCR5triangle32 and high frequency of CCR2b-64I alleles].

OBJECTIVE: To investigate the allelic polymorphism of CCR5triangle32, CCR5m303, CCR2b-64I and SDF1-3'A in Tibetan population in Lasa area of China. METHODS: The genomic DNA samples from 330 Tibetan subjects' whole blood samples were purified by use of QIAgen Blood Kit and identified by PCR or PCR-RFLP analyses. RESULTS: The mutation frequencies of CCR5triangle32 and CCR5m303 alleles were lower than 0.15%, and those of CCR2 b-64I and SDF1-3'A alleles were 29.42% and 19.24% respectively in the study samples. The allelic polymorphisms of the four alleles of Tibetan population were similar to those of Chinese Han population. Genotype distribution of the four alleles was in accordance with Hardy-Weinberg equilibrium. The above results suggest that Tibetan population may be relatively susceptive to HIV-1. CONCLUSION: The Chinese Tibetan may have a lower frequency of CCR5triangle32 and a higher frequency of CCR2b-64I allele, compared with Caucasian.

Adolescent↗

PubMed2001

Genotypes and polymorphisms of mutant CCR5-delta 32, CCR2-64I and SDF1-3' a HIV-1 resistance alleles in indigenous Han Chinese.

OBJECTIVE: To evaluate the frequencies and polymorphisms of CCR5-delta 32, CCR2-64I and SDF1-3' A alleles conferring resistance to HIV-1 infection in Chinese population from Han ethnic origin. METHODS: This cohort was comprised of 1251 subjects (915 men and 336 women) aged 15-80 years and none was HIV-1 positive. Genotyping of allelic CCR5-delta 32, CCR2-64I and SDF1-3' A variants was performed using PCR or PCR/RFLP assay, and further confirmed by direct DNA sequencing. RESULTS: Our finding shows that the delta 32 deletion mutation in the CCR5 gene does occur in this population and can be inherited in a Mendelian fashion in indigenous Han Chinese at a very low frequency of 0.00119 (n = 1254). The frequencies of mutant CCR2-64I and SDF1-3' A alleles were 0.20023 (n = 1251) and 0.2873 (n = 893), in this population, which are higher than those found in American Caucasians. Furthermore the polymorphisms of CCR2-64I and SDF1-3' A alleles in the Han Chinese population were different from those in American Caucasians. Statistical analysis showed that the genotype distribution of CCR5-delta 32, CCR2-64I and SDF1-3' A alleles was in equilibrium according to the Hardy-Weinberg equation. CONCLUSION: The CCR5-delta 32 mutation may not be a major resistant factor against HIV-1 infection in indigenous Han Chinese. The significance of higher frequencies of CCR2-641 and SDF1-3' A alleles (0.20023 and 0.2791) in the Han population remains to be clarified in HIV-1-positive carriers and AIDS patients.

Acquired Immunodeficiency Syndrome↗