100 results on '"YANG, B."'
Search Results
2. Mutational analysis of residues in the nucleotide binding domain of human terminal deoxynucleotidyl transferase.
- Author
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Yang, B., primary, Gathy, K.N., additional, and Coleman, M.S., additional
- Published
- 1994
- Full Text
- View/download PDF
3. Cloning and analysis of a constitutive heat shock (cognate) protein 70 gene inducible by L-glutamine.
- Author
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LéJohn, H.B., primary, Cameron, L.E., additional, Yang, B., additional, MacBeath, G., additional, Barker, D.S., additional, and Williams, S.A., additional
- Published
- 1994
- Full Text
- View/download PDF
4. NADP(+)-activable, NAD(+)-specific glutamate dehydrogenase. Purification and immunological analysis.
- Author
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Yang, B., primary and LéJohn, H.B., additional
- Published
- 1994
- Full Text
- View/download PDF
5. Heterogeneity of the 59-kDa dystrophin-associated protein revealed by cDNA cloning and expression.
- Author
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Yang, B., primary, Ibraghimov-Beskrovnaya, O., additional, Moomaw, C.R., additional, Slaughter, C.A., additional, and Campbell, K.P., additional
- Published
- 1994
- Full Text
- View/download PDF
6. Molecular characterization of an NAD-specific glutamate dehydrogenase gene inducible by L-glutamine. Antisense gene pair arrangement with L-glutamine-inducible heat shock 70-like protein gene.
- Author
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LéJohn, H.B., primary, Cameron, L.E., additional, Yang, B., additional, and Rennie, S.L., additional
- Published
- 1994
- Full Text
- View/download PDF
7. Identification of two hyaluronan-binding domains in the hyaluronan receptor RHAMM.
- Author
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Yang, B., primary, Zhang, L., additional, and Turley, E.A., additional
- Published
- 1993
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8. Role of oligosaccharides in the processing and function of human transferrin receptors. Effect of the loss of the three N-glycosyl oligosaccharides individually or together.
- Author
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Yang, B., primary, Hoe, M.H., additional, Black, P., additional, and Hunt, R.C., additional
- Published
- 1993
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9. The G3 domain of versican inhibits mesenchymal chondrogenesis via the epidermal growth factor-like motifs.
- Author
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Zhang, Y, Cao, L, Kiani, C G, Yang, B L, and Yang, B B
- Abstract
Versican is a highly expressed proteoglycan in zones of developing tissues. To investigate whether versican plays a role in cell differentiation, we studied its role in mesenchymal condensation and chondrogenesis. Here we report that a mini-versican gene product inhibits mesenchymal chondrogenesis but not condensation. The mini-versican-treated mesenchymal cultures form fewer, smaller cartilaginous nodules and produced lower levels of link protein and type II collagen. The versican G3 domain alone, but not G1, was sufficient to inhibit mesenchymal chondrogenesis. Deletion of two epidermal growth factor (EGF)-like motifs in the G3 domain abolished the effect of versican. The G3 domain of aggrecan, which does not contain an EGF-like motif, did not inhibit mesenchymal chondrogenesis. We also generated a chimera construct containing the two EGF-like motifs of versican and the G3 domain of aggrecan, and we observed that this chimera construct inhibited chondrogenesis to a lesser extent than did the full-length versican G3 construct. Direct transfection of mesenchymal cells with different constructs produced similar results. Furthermore, treatment with versican antisense oligonucleotides and transfection with a versican antisense construct promoted chondrogenesis. Taken together, our results strongly suggest that versican inhibits mesenchymal chondrogenesis via its EGF-like motifs.
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- 1998
10. The G3 domain of versican enhances cell proliferation via epidermial growth factor-like motifs.
- Author
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Zhang, Y, Cao, L, Yang, B L, and Yang, B B
- Abstract
Versican is a member of the large aggregating chondroitin sulfate proteoglycan family. We have expressed in NIH3T3 fibroblasts a recombinant versican mini-gene comprising the G1 and G3 domains and 15% of the CS domain. We observed that expression of the mini-versican gene stimulated cell proliferation as determined by cell counting and cell cycle analysis. Addition of exogenous mini-versican protein to cultured cells produced the same result. The effects of the mini-versican were greatly reduced when the G3 domain was deleted. Expression of the G3 domain alone promotes cell proliferation, and addition of purified G3 gene products to NIH3T3 fibroblasts and cultured chicken fibroblasts enhances cell growth. Further, deletion of the epidermal growth factor (EGF)-like motifs in the versican G3 domain reduced the effects of the mini-versican on cell proliferation. In the presence of the purified mini-versican protein, antisense oligonucleotides to the EGF receptor inhibited proliferation of NIH3T3 fibroblasts, compared with control sense oligonucleotides. Taken together, these results imply that versican enhances cell proliferation, and this effect is mediated, at least in part, by the action of versican EGF-like motifs on endogenous EGF receptor.
- Published
- 1998
11. Identification of the motif in versican G3 domain that plays a dominant-negative effect on astrocytoma cell proliferation through inhibiting versican secretion and binding.
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Wu, Y, Zhang, Y, Cao, L, Chen, L, Lee, V, Zheng, P S, Kiani, C, Adams, M E, Ang, L C, Paiwand, F, and Yang, B B
- Abstract
This study was designed to investigate the mechanisms by which mutant versican constructs play a dominant-negative effect on astrocytoma cell proliferation. Although a mini-versican or a versican G3 construct promoted growth of U87 astrocytoma cells, a mini-versican lacking epidermal growth factor (EGF) motifs (versicanDeltaEGF) and a G3 mutant (G3DeltaEGF) exerted a dominant-negative effect on cell proliferation. G3DeltaEGF-transfected cells formed smaller colonies, arrested cell cycle at G(1) phase, inhibited expression of cell cycle proteins cdk4 and cyclin D1, and contained multiple nucleoli. In cell surface binding assays, G3 products expressed in COS-7 cells and bacteria bound to U87 cell surface. G3DeltaEGF products exhibited decreased binding activity, but higher levels of G3DeltaEGF products were able to inhibit the binding of G3 to the cell surface. G3DeltaEGF expression inhibited secretion of endogenous versican in astrocytoma cells and also inhibited the secretion of mini-versican in COS-7 cells co-transfected with the mini-versican and G3DeltaEGF constructs. The effect seems to depend on the expression efficiency of G3DeltaEGF, and it occurred via the carbohydrate recognition domain.
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- 2001
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12. Carbon dioxide permeability of aquaporin-1 measured in erythrocytes and lung of aquaporin-1 null mice and in reconstituted proteoliposomes.
- Author
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Yang, B, Fukuda, N, van Hoek, A, Matthay, M A, Ma, T, and Verkman, A S
- Abstract
Measurements of CO(2) permeability in oocytes and liposomes containing water channel aquaporin-1 (AQP1) have suggested that AQP1 is able to transport both water and CO(2). We studied the physiological consequences of CO(2) transport by AQP1 by comparing CO(2) permeabilities in erythrocytes and intact lung of wild-type and AQP1 null mice. Erythrocytes from wild-type mice strongly expressed AQP1 protein and had 7-fold greater osmotic water permeability than did erythrocytes from null mice. CO(2) permeability was measured from the rate of intracellular acidification in response to addition of CO(2)/HCO(3)(-) in a stopped-flow fluorometer using 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) as a cytoplasmic pH indicator. In erythrocytes from wild-type mice, acidification was rapid (t((1)/(2)), 7.3 +/- 0.4 ms, S.E., n = 11 mice) and blocked by acetazolamide and increasing external pH (to decrease CO(2)/HCO(3)(-) ratio). Apparent CO(2) permeability (P(CO(2))) was not different in erythrocytes from wild-type (0.012 +/- 0.0008 cm/s) versus null (0.011 +/- 0.001 cm/s) mice. Lung CO(2) transport was measured in anesthetized, ventilated mice subjected to a decrease in inspired CO(2) content from 5% to 0%, producing an average decrease in arterial blood pCO(2) from 77 +/- 4 to 39 +/- 3 mm Hg (14 mice) with a t((1)/(2)) of 1.4 min. The pCO(2) values and kinetics of decreasing pCO(2) were not different in wild-type versus null mice. Because AQP1 deletion did not affect CO(2) transport in erythrocytes and lung, we re-examined CO(2) permeability in AQP1-reconstituted liposomes containing carbonic anhydrase (CA) and a fluorescent pH indicator. Whereas osmotic water permeability in AQP1-reconstituted liposomes was >100-fold greater than that in control liposomes, apparent P(CO(2)) (approximately 10(-3) cm/s) did not differ. Measurements using different CA concentrations and HgCl(2) indicated that liposome P(CO(2)) is unstirred layer-limited and that HgCl(2) slows acidification because of inhibition of CA rather than AQP1. These results provide direct evidence against physiologically significant AQP1-mediated CO(2) transport and establish an upper limit to the CO(2) permeability through single AQP1 water channels.
- Published
- 2000
13. Appropriate tissue- and cell-specific expression of a single copy human angiotensinogen transgene specifically targeted upstream of the HPRT locus by homologous recombination.
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Cvetkovic, B, Yang, B, Williamson, R A, and Sigmund, C D
- Abstract
Development of experimental models by genetic manipulation in mice has proven to be very useful in determining the significance of particular genes in the development of or susceptibility to hypertension. Advances in molecular genetics, transgenic mouse technology, and physiological measurements in mice provided an opportunity to go a step further and develop models to analyze the physiological significance of specific gene variants potentially causing hypertension. In this report, we describe the development of a human angiotensinogen transgenic mouse model generated by targeting the human angiotensinogen gene upstream of the mouse HPRT locus by homologous recombination. The main benefit of this transgenic mouse model is that the human angiotensinogen gene is inserted into the mouse genome as a single copy at a predefined locus and in a specific orientation-a process that can be repeated utilizing other variants of this gene. We establish the validity of this approach by showing that the hAGT(hprt) mice have normal tissue- and cell-specific expression of the human angiotensinogen gene and normally produce and process the hAGT protein at physiological levels.
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- 2000
14. SNARE interactions are not selective. Implications for membrane fusion specificity.
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Yang, B, Gonzalez, L, Prekeris, R, Steegmaier, M, Advani, R J, and Scheller, R H
- Abstract
The SNARE hypothesis proposes that membrane trafficking specificity is mediated by preferential high affinity interactions between particular v (vesicle membrane)- and t (target membrane)-SNARE combinations. The specificity of interactions among a diverse set of SNAREs, however, is unknown. We have tested the SNARE hypothesis by analyzing potential SNARE complexes between five proteins of the vesicle-associated membrane protein (VAMP) family, three members of the synaptosome-associated protein-25 (SNAP-25) family and three members of the syntaxin family. All of the 21 combinations of SNAREs tested formed stable complexes. Sixteen were resistant to SDS denaturation, and most complexes thermally denatured between 70 and 90 degreesC. These results suggest that the specificity of membrane fusion is not encoded by the interactions between SNAREs.
- Published
- 1999
15. Misfolding of mutant aquaporin-2 water channels in nephrogenic diabetes insipidus.
- Author
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Tamarappoo, B K, Yang, B, and Verkman, A S
- Abstract
We reported that several aquaporin-2 (AQP2) point mutants that cause nephrogenic diabetes insipidus (NDI) are retained in the endoplasmic reticulum (ER) of transfected mammalian cells and degraded but can be rescued by chemical chaperones to function as plasma membrane water channels (Tamarappoo, B. K., and Verkman, A. S. (1998) J. Clin. Invest. 101, 2257-2267). To test whether mutant AQP2 proteins are misfolded, AQP2 folding was assessed by comparative detergent extractability and limited proteolysis, and AQP2 degradation kinetics was measured by label-pulse-chase and immunoprecipitation. In ER membranes from transfected CHO cells containing [(35)S]methionine-labeled AQP2, mutants T126M and A147T were remarkably detergent-resistant; for example wild-type AQP2 was >95% solubilized by 0.5% CHAPS whereas T126M was <10% solubilized. E258K, an NDI-causing AQP2 mutant which is retained in the Golgi, is highly detergent soluble like wild-type AQP2. The mutants and wild-type AQP2 were equally susceptible to digestion by trypsin, thermolysin, and proteinase K. Stopped-flow light scattering measurements indicated that T126M AQP2 at the ER was fully functional as a water channel. Pulse-chase studies indicated that the increased degradation rates for T126M (t((1)/(2)) 2.5 h) and A147T (2 h) compared with wild-type AQP2 (4 h) involve a brefeldin A-resistant, ER-dependent degradation mechanism. After growth of cells for 48 h in the chemical chaperone glycerol, AQP2 mutants T126M and A147T became properly targeted and relatively detergent-soluble. These results provide evidence that NDI-causing mutant AQP2 proteins are misfolded, but functional, and that chemical chaperones both correct the trafficking and folding defects. Strategies to facilitate protein folding might thus have therapeutic efficacy in NDI.
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- 1999
16. Severely impaired urinary concentrating ability in transgenic mice lacking aquaporin-1 water channels.
- Author
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Ma, T, Yang, B, Gillespie, A, Carlson, E J, Epstein, C J, and Verkman, A S
- Abstract
Water channel aquaporin-1 (AQP1) is strongly expressed in kidney in proximal tubule and descending limb of Henle epithelia and in vasa recta endothelia. The grossly normal phenotype in human subjects deficient in AQP1 (Colton null blood group) and in AQP4 knockout mice has suggested that aquaporins (other than the vasopressin-regulated water channel AQP2) may not be important in mammalian physiology. We have generated transgenic mice lacking detectable AQP1 by targeted gene disruption. In kidney proximal tubule membrane vesicles from knockout mice, osmotic water permeability was reduced 8-fold compared with vesicles from wild-type mice. Although the knockout mice were grossly normal in terms of survival, physical appearance, and organ morphology, they became severely dehydrated and lethargic after water deprivation for 36 h. Body weight decreased by 35 +/- 2%, serum osmolality increased to >500 mOsm, and urinary osmolality (657 +/- 59 mOsm) did not change from that before water deprivation. In contrast, wild-type and heterozygous mice remained active after water deprivation, body weight decreased by 20-22%, serum osmolality remained normal (310-330 mOsm), and urine osmolality rose to >2500 mOsm. Urine [Na+] in water-deprived knockout mice was <10 mM, and urine osmolality was not increased by the V2 agonist DDAVP. The results suggest that AQP1 knockout mice are unable to create a hypertonic medullary interstitium by countercurrent multiplication. AQP1 is thus required for the formation of a concentrated urine by the kidney.
- Published
- 1998
17. Urea transporter UT3 functions as an efficient water channel. Direct evidence for a common water/urea pathway.
- Author
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Yang, B and Verkman, A S
- Abstract
A family of molecular urea transporters (UTs) has been identified whose members appear to have an exceptionally high transport turnover rate. To test the hypothesis that urea transport involves passage through an aqueous channel, osmotic water permeability was measured in Xenopus oocytes expressing UTs. The UT3 class of urea transporters functioned as efficient water channels. Quantitative measurement of single channel water permeability (pf) using epitope-tagged rat UTs gave pf (in cm3/s x 10(-14)) of 0.14 +/- 0.11 (UT2) and 1.4 +/- 0.2 (UT3), compared with 6.0 and 2.3 for water channels AQP1 and AQP3, respectively. Relative single channel urea permeabilities (purea) were 1.0 (UT2), 0.44 (UT3), and 0.0 (AQP1). UT3-mediated water and urea transport were weakly temperature-dependent (activation energy <4 kcal/mol), inhibited > 75% by the urea transport inhibitor 1,3-dimethylthiourea, but not inhibited by the water transport inhibitor HgCl2. To test for a common water/urea pore, the urea reflection coefficient (sigmaurea) was measured by independent induced osmosis and solvent drag methods. In UT3-expressing oocytes, the time course of oocyte volume in response to different urea gradients (induced osmosis) gave sigmaurea approximately 0.3 for the UT3 pathway, in agreement with sigmaurea determined by the increase in uptake of [14C]urea during osmotic gradient-induced oocyte swelling (solvent drag). In oocytes of comparable water and urea permeability coexpressing AQP1 (permeable to water, not urea) and UT2 (permeable to urea, not water), sigmaurea = 1. These results indicate that UT3 functions as a urea/water channel utilizing a common aqueous pathway. The water transporting function and low urea reflection coefficient of UT3 in vasa recta may be important for the formation of a concentrated urine by countercurrent exchange in the kidney.
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- 1998
18. Water and glycerol permeabilities of aquaporins 1-5 and MIP determined quantitatively by expression of epitope-tagged constructs in Xenopus oocytes.
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Yang, B and Verkman, A S
- Abstract
The goal of this study was to compare single channel water and glycerol permeabilities of mammalian aquaporins (AQP) 1-5 and the major intrinsic protein of lens fiber (MIP). Each of the six cloned cDNAs from rat was left untagged or was epitope-tagged with c-Myc or FLAG at either the N or C terminus so that results would not depend on epitope identity or location. The constructs were expressed in Xenopus oocytes for measurement of osmotic water permeability (Pf), [3H]glycerol uptake, and protein expression. Each of the 30 epitope-tagged constructs was expressed strongly at the oocyte plasma membrane. The 10-min uptake of [3H]glycerol was increased significantly (range of 4.5-8-fold over control) in oocytes expressing untagged AQP3 (GLIP) and each of the four tagged AQP3 constructs; [3H]glycerol uptake was not increased in oocytes expressing AQP1, AQP2, AQP4, AQP5, or MIP. In oocytes microinjected with 5 ng of cRNA, average Pf values (in cm/s x 10(-3)) were 0.67 +/- 0.06 (control), 19 +/- 2 (AQP1), 10 +/- 1 (AQP2), 8 +/- 2 (AQP3), 29 +/- 1 (AQP4), 10 +/- 1 (AQP5), and 1.3 +/- 0.2 (MIP), and they were relatively insensitive to the presence, identity, or location of the epitope tag. Pf values were not affected by protein kinase A or C activation. After normalization for plasma membrane expression by immunoprecipitation of microdissected plasma membranes, single channel water permeabilities (pf, referenced to the AQP1 pf of 6 x 10(-14) cm3/s) were (in cm3/s x 10(-14)) 3.3 +/- 0.2 (AQP2), 2.1 +/- 0.3 (AQP3), 24 +/- 0.6 (AQP4), 5.0 +/- 0.4 (AQP5), and 0.25 +/- 0.05 (MIP); pf values were insensitive to epitope identity and location. These results indicate very different intrinsic water permeabilities for the mammalian aquaporin homologs, with the pf value for AQP4 remarkably higher than those for the others. The pf values establish limits on aquaporin tissue densities required for physiological function and suggest significant structural and functional differences among the aquaporins.
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- 1997
19. Three novel proteins of the syntaxin/SNAP-25 family.
- Author
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Steegmaier, M, Yang, B, Yoo, J S, Huang, B, Shen, M, Yu, S, Luo, Y, and Scheller, R H
- Abstract
Intracellular membrane traffic is thought to be regulated in part by soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) through the formation of complexes between these proteins present on vesicle and target membranes. All known SNARE-mediated fusion events involve members of the syntaxin and vesicle-associated membrane protein families. The diversity of mammalian membrane compartments predicts the existence of a large number of different syntaxin and vesicle-associated membrane protein genes. To further investigate the spectrum of SNAREs and their roles in membrane trafficking we characterized three novel members of the syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) subfamilies. The proteins are broadly expressed, suggesting a general role in vesicle trafficking, and localize to distinct membrane compartments. Syntaxin 8 co-localizes with markers of the endoplasmic reticulum. Syntaxin 17, a divergent member of the syntaxin family, partially overlaps with endoplasmic reticulum markers, and SNAP-29 is broadly localized on multiple membranes. SNAP-29 does not contain a predicted membrane anchor characteristic of other SNAREs. In vitro studies established that SNAP-29 is capable of binding to a broad range of syntaxins.
- Published
- 1998
20. cDNA cloning, gene organization, and chromosomal localization of a human mercurial insensitive water channel. Evidence for distinct transcriptional units.
- Author
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Yang, B, Ma, T, and Verkman, A S
- Abstract
Two distinct cDNAs encoding a human mercurial insensitive water channel (hMIWC) were cloned from a fetal brain cDNA library. The longest open reading frame of cDNA clone hMIWC1 encoded 301 amino acids with 94% identity to rat MIWC (Hasegawa, H., Ma, T., Skach, W., Matthay, M. M., and Verkman, A. S. (1994) J. Biol. Chem. 269, 5497-5500). A second cDNA (hMIWC2) had a distinct 5'-sequence upstream from base pair (bp) -34 in clone hMIWC1 and contained two additional inframe translation start codons. Expression of hMIWC cRNAs in Xenopus oocytes increased osmotic water permeability by 10-20-fold in a mercurial insensitive manner. Cell-free translation in a reticulocyte lysate/microsome system generated single protein bands at 30 kDa (hMIWC1) and 32-34 kDa (hMIWC2) without glycosylation. Northern blot and polymerase chain reaction/Southern blot analysis showed expression of mRNA encoding hMIWC in human brain - muscle >> heart, kidney, lung, and trachea. Analysis of hMIWC genomic clones indicated two distinct but overlapping transcription units from which multiple hMIWC mRNAs are transcribed. The promoter region of hMIWC1 was identified and contained TATA, CAAT, AP-1, and other regulatory elements. Primer extension revealed hMIWC1 transcription initiation at 46 bp downstream from the TATA box. There were three introns (lengths 0.9, 0.2, and 6 kilobases) in the hMIWC1 coding sequence at bp 381, 546, and 627. A distinct 5'-sequence in clone hMIWC2 suggested an alternative upstream transcription initiation site. Two alternatively spliced, nonfunctional hMIWC transcripts with exon 3 deletion and partial exon 4 deletion were identified. A poly(A)+ signal sequence was identified at 138 bp downstream of the translation stop codon. Genomic Southern blot analysis indicated the presence of a single copy hMIWC gene; chromosome-specific polymerase chain reaction and in situ hybridization localized hMIWC to human chromosome 18q22. The structural organization of the hMIWC gene represents a first step in definition of hMIWC differential expression, regulation, and possible role in human disease.
- Published
- 1995
21. The mercurial insensitive water channel (AQP-4) forms orthogonal arrays in stably transfected Chinese hamster ovary cells.
- Author
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Yang, B, Brown, D, and Verkman, A S
- Abstract
The mercurial insensitive water channel (MIWC, AQP-4) is a water-selective transporter expressed at the basolateral plasma membrane of principal cells in kidney collecting duct, airway epithelium, and gastric parietal cells, as well as in astrocytes and skeletal muscle plasmalemma. Because these sites correspond to membranes where orthogonal arrays of particles (OAPs) have been observed by freeze-fracture electron microscopy, we tested the hypothesis that MIWC forms OAPs. Chinese hamster ovary cells were stably transfected with the coding sequence of rat MIWC under a cytomegalovirus promoter. Immunostaining of clonal cell populations showed MIWC expression at the plasma membrane. A single band at 31 kDa was detected on immunoblot. Cell fractionation by sucrose gradient centrifugation indicated strong MIWC expression in plasma membrane fractions with lesser expression in Golgi. Functional analysis by stopped-flow light scattering showed high mercurial insensitive water permeability in plasma membrane vesicles. Freeze-fracture electron microscopy showed distinct OAPs on the plasma membrane P-face of MIWC-expressing cells with morphology indistinguishable from that in basolateral membrane of kidney collecting duct; the E-face showed corresponding linear grooves (spacing, approximately 8 nm) in transfected cells and collecting duct. OAPs were not observed in control (empty vector-transfected) cells or CHIP28 (AQP1)-transfected cells in which disorganized intramembrane particle aggregates were found. These results provide direct evidence that a molecular water channel can spontaneously assemble in regular arrays.
- Published
- 1996
22. SH3 domain-mediated interaction of dystroglycan and Grb2.
- Author
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Yang, B, Jung, D, Motto, D, Meyer, J, Koretzky, G, and Campbell, K P
- Abstract
Dystroglycan is a novel laminin receptor that links the extracellular matrix and sarcolemma in skeletal muscle. The dystroglycan complex containing alpha- and beta-dystroglycan also serves as an agrin receptor in muscle, where it may regulate agrin-induced acetylcholine receptor clustering at the neuromuscular junction. beta-Dystroglycan has now been expressed in vitro and shown to directly interact with Grb2, an adapter protein involved in signal transduction and cytoskeletal organization. Protein binding assays with two Grb2 mutants, Grb2/P49L and Grb2/G203R, which correspond to the loss-of-function mutants in the Caenorhabditis elegans sem-5, demonstrated that the dystroglycan-Grb2 association is through beta-dystroglycan C-terminal proline-rich domains and Grb2 Src homology 3 domains. Affinity chromatography has also shown endogenous skeletal muscle Grb2 interacts with beta-dystroglycan. Immunoprecipitation experiments have demonstrated that Grb2 associates with alpha/beta-dystroglycan in vivo in both skeletal muscle and brain. The specific dystroglycan-Grb2 interaction may play an important role in extracellular matrix-mediated signal transduction and/or cytoskeleton organization in skeletal muscle that may be essential for muscle cell viability.
- Published
- 1995
23. Identification of alpha-syntrophin binding to syntrophin triplet, dystrophin, and utrophin.
- Author
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Yang, B, Jung, D, Rafael, J A, Chamberlain, J S, and Campbell, K P
- Abstract
Syntrophin represents three cytoplasmic components of the dystrophin-glycoprotein complex that links the cytoskeleton to the extracellular matrix in skeletal muscle. alpha-Syntrophin has now been translated in vitro and shown to associate directly with all three components of the syntrophin triplet and with dystrophin. The in vitro translated 71-kDa non-muscle dystrophin isoform, containing the cystein-rich/C-terminal domain, can also interact with the syntrophin triplet. The syntrophin binding motif in dystrophin was localized to exons 73 and 74 including amino acids 3447-3481 by comparing the interactions of alpha-syntrophin and seven overlapping human dystrophin fusion proteins. More than one syntrophin interaction site in this binding motif was suggested. alpha-Syntrophin also interacts directly with a C-terminal utrophin fusion protein. alpha-Syntrophin is localized to the muscle sarcolemma as well as to the neuromuscular junction in control mouse muscle. However, similar to utrophin, alpha-syntrophin is only present at the neuromuscular junction in mdx mouse muscle in which dystrophin is absent. Our data suggest that alpha-syntrophin binds all syntrophin isoforms, and syntrophin directly interacts with dystrophin through more than one binding site in dystrophin exons 73 and 74 including amino acids 3447-3481.
- Published
- 1995
24. Identification and characterization of the dystrophin anchoring site on beta-dystroglycan.
- Author
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Jung, D, Yang, B, Meyer, J, Chamberlain, J S, and Campbell, K P
- Abstract
Dystrophin, the product of the Duchenne muscular dystrophy gene, is tightly associated with the sarcolemmal membrane to a large glycoprotein complex. One function of the dystrophin-glycoprotein complex is to link the cytoskeleton to the extracellular matrix in skeletal muscle. However, the molecular interactions of dystrophin with the membrane components of the dystrophin-glycoprotein complex are still elusive. Here, we demonstrate and characterize a specific interaction between beta-dystroglycan and dystrophin. We show that skeletal muscle and brain dystrophin as well as brain dystrophin isoforms specifically bind to beta-dystroglycan. To localize and characterize the dystrophin and beta-dystroglycan interaction domains, we reconstituted the interaction in vitro using dystrophin fusion proteins and in vitro translated beta-dystroglycan. We demonstrated that the 15 C-terminal amino acids of beta-dystroglycan constituted a unique binding site for the second half of the hinge 4 and the cysteine-rich domain of dystrophin (amino acids 3054-3271). This dystrophin binding site is located in a proline-rich environment of beta-dystroglycan within amino acids 880-895. The identification of the interaction sites in dystrophin and beta-dystroglycan provides further insight into the structure and the molecular organization of the dystrophin-glycoprotein complex at the sarcolemma membrane and will be helpful for studying the pathogenesis of Duchenne muscular dystrophy.
- Published
- 1995
25. Rapid conversion of porcine pluripotent stem cells into macrophages with chemically defined conditions.
- Author
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Wu X, Ni Y, Li W, Yang B, Yang X, Zhu Z, Zhang J, Wu X, Shen Q, Liao Z, Yuan L, Chen Y, Du Q, Wang C, Liu P, Miao Y, Li N, Zhang S, Liao M, and Hua J
- Subjects
- Animals, Endocytosis, Hematopoiesis drug effects, Lipopolysaccharides pharmacology, Mesoderm metabolism, Porcine respiratory and reproductive syndrome virus physiology, Signal Transduction drug effects, Transcription Factors metabolism, Transforming Growth Factor beta metabolism, Time Factors, Macrophages, Alveolar cytology, Macrophages, Alveolar drug effects, Macrophages, Alveolar metabolism, Macrophages, Alveolar virology, Pluripotent Stem Cells cytology, Pluripotent Stem Cells drug effects, Swine virology
- Abstract
A renewable source of porcine macrophages derived from pluripotent stem cells (PSCs) would be a valuable alternative to primary porcine alveolar macrophages (PAMs) in the research of host-pathogen interaction mechanisms. We developed an efficient and rapid protocol, within 11 days, to derive macrophages from porcine PSCs (pPSCs). The pPSC-derived macrophages (pPSCdMs) exhibited molecular and functional characteristics of primary macrophages. The pPSCdMs showed macrophage-specific surface protein expression and macrophage-specific transcription factors, similar to PAMs. The pPSCdMs also exhibited the functional characteristics of macrophages, such as endocytosis, phagocytosis, porcine respiratory and reproductive syndrome virus infection and the response to lipopolysaccharide stimulation. Furthermore, we performed transcriptome sequencing of the whole differentiation process to track the fate transitions of porcine PSCs involved in the signaling pathway. The activation of transforming growth factor beta signaling was required for the formation of mesoderm and the inhibition of the transforming growth factor beta signaling pathway at the hematopoietic endothelium stage could enhance the fate transformation of hematopoiesis. In summary, we developed an efficient and rapid protocol to generate pPSCdMs that showed aspects of functional maturity comparable with PAMs. pPSCdMs could provide a broad prospect for the platforms of host-pathogen interaction mechanisms., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
26. The abscisic acid-responsive element binding factors MAPKKK18 module regulates abscisic acid-induced leaf senescence in Arabidopsis.
- Author
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Zhao G, Cheng Q, Zhao Y, Wu F, Mu B, Gao J, Yang L, Yan J, Zhang H, Cui X, Chen Q, Lu F, Ao Q, Amdouni A, Jiang YQ, and Yang B
- Subjects
- Basic-Leucine Zipper Transcription Factors metabolism, MAP Kinase Kinase Kinases metabolism, Plants, Genetically Modified metabolism, Transcription Factors metabolism, Abscisic Acid metabolism, Abscisic Acid pharmacology, Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Gene Expression Regulation, Plant, Plant Senescence genetics, Plant Senescence physiology, Plant Leaves genetics, Plant Leaves physiology, Regulatory Elements, Transcriptional
- Abstract
The mitogen-activated protein kinase kinase kinase 18 (MAPKKK18) has been reported to play a role in abiotic stress priming in long-term abscisic acid (ABA) response including drought tolerance and leaf senescence. However, the upstream transcriptional regulators of MAPKKK18 remain to be determined. Here, we report ABA-responsive element binding factors (ABFs) as upstream transcription factors of MAPKKK18 expression. Mutants of abf2, abf3, abf4, and abf2abf3abf4 dramatically reduced the transcription of MAPKKK18. Our electrophoresis mobility shift assay and dual-luciferase reporter assay demonstrated that ABF2, ABF3, and ABF4 bound to ABA-responsive element cis-elements within the promoter of MAPKKK18 to transactivate its expression. Furthermore, enrichments of the promoter region of MAPKKK18 by ABF2, ABF3, and ABF4 were confirmed by in vivo chromatin immunoprecipitation coupled with quantitative PCR. In addition, we found that mutants of mapkkk18 exhibited obvious delayed leaf senescence. Moreover, a genetic study showed that overexpression of ABF2, ABF3, and ABF4 in the background of mapkkk18 mostly phenocopied the stay-green phenotype of mapkkk18 and, expression levels of five target genes of ABFs, that is, NYE1, NYE2, NYC1, PAO, and SAG29, were attenuated as a result of MAPKKK18 mutation. These findings demonstrate that ABF2, ABF3, and ABF4 act as transcription regulators of MAPKKK18 and also suggest that, at least in part, ABA acts in priming leaf senescence via ABF-induced expression of MAPKKK18., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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27. Glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) interacts with the band 3 receptor to promote erythrocyte invasion by malaria parasites.
- Author
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Lu J, Chu R, Yin Y, Yu H, Xu Q, Yang B, Sun Y, Song J, Wang Q, Xu J, Lu F, and Cheng Y
- Subjects
- Animals, Anion Exchange Protein 1, Erythrocyte metabolism, Ankyrins metabolism, Humans, Plasmodium vivax metabolism, Erythrocytes parasitology, Malaria, Falciparum metabolism, Plasmodium falciparum metabolism, Protozoan Proteins metabolism
- Abstract
Glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) is an erythrocyte binding protein known to be involved in malarial parasite invasion. Although anti-GAMA antibodies have been shown to block GAMA attachment to the erythrocyte surface and subsequently inhibit parasite invasion, little is known about the molecular mechanisms by which GAMA promotes the invasion process. In this study, LC-MS analysis was performed on the erythrocyte membrane to identify the specific receptor that interacts with GAMA. We found that ankyrin 1 and the band 3 membrane protein showed affinity for GAMA, and characterization of their binding specificity indicated that both Plasmodium falciparum and Plasmodium vivax GAMA bound to the same extracellular loop of band 3 (loop 5). In addition, we show the interaction between GAMA and band 3 was sensitive to chymotrypsin. Furthermore, antibodies against band 3 loop 5 were able to reduce the binding activity of GAMA to erythrocytes and inhibit the invasion of P. falciparum merozoites into human erythrocytes, whereas antibodies against P. falciparum GAMA (PfGAMA)-Tr3 only slightly reduced P. falciparum invasion. The identification and characterization of the erythrocyte GAMA receptor is a novel finding that identifies an essential mechanism of parasite invasion of host erythrocytes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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28. Calreticulin enhances the secretory trafficking of a misfolded α-1-antitrypsin.
- Author
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Mohan HM, Yang B, Dean NA, and Raghavan M
- Subjects
- Animals, Binding Sites, Calnexin deficiency, Calnexin genetics, Calnexin metabolism, Calreticulin deficiency, Calreticulin genetics, Cell Line, Endoplasmic Reticulum metabolism, Humans, Inclusion Bodies metabolism, Mice, Mutagenesis, Site-Directed, Polysaccharides chemistry, Polysaccharides metabolism, Protein Binding, Protein Folding, Vesicular Transport Proteins genetics, Vesicular Transport Proteins metabolism, alpha 1-Antitrypsin chemistry, alpha 1-Antitrypsin genetics, Calreticulin metabolism, Protein Transport physiology, alpha 1-Antitrypsin metabolism
- Abstract
α1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A mutant of AAT (E342K) called ATZ forms polymers that are present at only low levels in the serum and induce intracellular protein inclusions, causing lung emphysema and liver cirrhosis. An understanding of factors that can reduce the intracellular accumulation of ATZ is of great interest. We now show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media:cell ratio. This effect is more pronounced for ATZ than with AAT and is only partially dependent on the glycan-binding site of CRT, which is generally relevant to substrate recruitment and folding by CRT. The CRT-related chaperone calnexin does not enhance ATZ secretory trafficking, despite the higher cellular abundance of calnexin-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body formation and reducing ATZ interactions with components required for ER-Golgi trafficking, coincident with reduced levels of the protein transport protein Sec31A in CRT-deficient cells. These findings indicate a novel role for CRT in promoting the secretory trafficking of a protein that forms polymers and large intracellular inclusions. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ inclusion bodies. Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung and liver diseases linked to AAT deficiency., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Mohan et al.)
- Published
- 2020
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29. The urea transporter UT-A1 plays a predominant role in a urea-dependent urine-concentrating mechanism.
- Author
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Geng X, Zhang S, He J, Ma A, Li Y, Li M, Zhou H, Chen G, and Yang B
- Subjects
- Animals, Male, Membrane Transport Proteins genetics, Mice, Mice, Knockout, Urea Transporters, Kidney metabolism, Membrane Transport Proteins metabolism, Urea metabolism, Urine
- Abstract
Urea transporters are a family of urea-selective channel proteins expressed in multiple tissues that play an important role in the urine-concentrating mechanism of the mammalian kidney. Previous studies have shown that knockout of urea transporter (UT)-B, UT-A1/A3, or all UTs leads to urea-selective diuresis, indicating that urea transporters have important roles in urine concentration. Here, we sought to determine the role of UT-A1 in the urine-concentrating mechanism in a newly developed UT-A1-knockout mouse model. Phenotypically, daily urine output in UT-A1-knockout mice was nearly 3-fold that of WT mice and 82% of all-UT-knockout mice, and the UT-A1-knockout mice had significantly lower urine osmolality than WT mice. After 24-h water restriction, acute urea loading, or high-protein (40%) intake, UT-A1-knockout mice were unable to increase urine-concentrating ability. Compared with all-UT-knockout mice, the UT-A1-knockout mice exhibited similarly elevated daily urine output and decreased urine osmolality, indicating impaired urea-selective urine concentration. Our experimental findings reveal that UT-A1 has a predominant role in urea-dependent urine-concentrating mechanisms, suggesting that UT-A1 represents a promising diuretic target., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Geng et al.)
- Published
- 2020
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30. The microtubule-associated protein EML3 regulates mitotic spindle assembly by recruiting the Augmin complex to spindle microtubules.
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Luo J, Yang B, Xin G, Sun M, Zhang B, Guo X, Jiang Q, and Zhang C
- Subjects
- Amino Acid Substitution, Cell Cycle Proteins genetics, Gene Knockdown Techniques, HEK293 Cells, HeLa Cells, Humans, Microtubule-Associated Proteins genetics, Microtubules genetics, Mutation, Missense, Spindle Apparatus genetics, Tubulin genetics, Tubulin metabolism, Cell Cycle Proteins metabolism, Kinetochores metabolism, Microtubule-Associated Proteins metabolism, Microtubules metabolism, Spindle Apparatus metabolism
- Abstract
In all eukaryotes, a functional mitotic spindle is essential for distributing duplicated chromosomes into daughter cells. Mitotic spindle assembly involves highly ordered arrangement of microtubules (MTs). The Augmin protein complex recruits γ-tubulin ring complex (γ-TuRC) to MTs and thereby promotes MT-based MT nucleation and mitotic spindle assembly. However, several factors that may promote Augmin recruitment to MTs remain unknown. Here, we show that echinoderm microtubule-associated protein-like 3 (EML3), an MT-associated protein, facilitates binding between MTs and Augmin/γ-TuRC and recruiting the latter to MTs for proper mitotic spindle assembly and kinetochore-MT connections. Using immunofluorescence microscopy, live-cell imaging, and immunoprecipitation assays, we found that EML3 recruits Augmin/γ-TuRC to the MTs to enhance MT-based MT nucleation in both spindle and small acentrosomal asters. We also noted that the EML3-mediated recruitment is controlled by cyclin-dependent kinase 1 (CDK1), which phosphorylated EML3 at Thr-881 and promoted its binding to Augmin/γ-TuRC. RNAi-mediated EML3 knockdown in HeLa cells reduced spindle localization of Augmin/γ-TuRC, which resulted in abnormal spindle assembly and caused kinetochore-MT misconnection. The introduction of exogenous WT or a Thr-881 phosphorylation mimic EML3 variant into the EML3 knockdown cells restored normal Augmin/γ-TuRC localization and spindle assembly. The EML3 knockdown also affected the spindle assembly checkpoint, delaying chromosome congression and cell division. Taken together, our results indicate that EML3 regulates mitotic spindle assembly and the kinetochore-MT connection by regulating MT-based MT nucleation and recruiting Augmin/γ-TuRC to MTs., (© 2019 Luo et al.)
- Published
- 2019
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31. FTY720/Fingolimod Reduces Synucleinopathy and Improves Gut Motility in A53T Mice: CONTRIBUTIONS OF PRO-BRAIN-DERIVED NEUROTROPHIC FACTOR (PRO-BDNF) AND MATURE BDNF.
- Author
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Vidal-Martínez G, Vargas-Medrano J, Gil-Tommee C, Medina D, Garza NT, Yang B, Segura-Ulate I, Dominguez SJ, and Perez RG
- Subjects
- Aging drug effects, Aging genetics, Aging pathology, Animals, Brain-Derived Neurotrophic Factor genetics, Gastrointestinal Motility genetics, Humans, Mice, Mice, Transgenic, Parkinson Disease genetics, Parkinson Disease metabolism, Parkinson Disease pathology, Protein Precursors genetics, alpha-Synuclein genetics, Aging metabolism, Brain-Derived Neurotrophic Factor metabolism, Fingolimod Hydrochloride pharmacology, Gastrointestinal Motility drug effects, Parkinson Disease drug therapy, Protein Precursors metabolism, alpha-Synuclein metabolism
- Abstract
Patients with Parkinson's disease (PD) often have aggregated α-synuclein (aSyn) in enteric nervous system (ENS) neurons, which may be associated with the development of constipation. This occurs well before the onset of classic PD motor symptoms. We previously found that aging A53T transgenic (Tg) mice closely model PD-like ENS aSyn pathology, making them appropriate for testing potential PD therapies. Here we show that Tg mice overexpressing mutant human aSyn develop ENS pathology by 4 months. We then evaluated the responses of Tg mice and their WT littermates to the Food and Drug Administration-approved drug FTY720 (fingolimod, Gilenya) or vehicle control solution from 5 months of age. Long term oral FTY720 in Tg mice reduced ENS aSyn aggregation and constipation, enhanced gut motility, and increased levels of brain-derived neurotrophic factor (BDNF) but produced no significant change in WT littermates. A role for BDNF was directly assessed in a cohort of young A53T mice given vehicle, FTY720, the Trk-B receptor inhibitor ANA-12, or FTY720 + ANA-12 from 1 to 4 months of age. ANA-12-treated Tg mice developed more gut aSyn aggregation as well as constipation, whereas FTY720-treated Tg mice had reduced aSyn aggregation and less constipation, occurring in part by increasing both pro-BDNF and mature BDNF levels. The data from young and old Tg mice revealed FTY720-associated neuroprotection and reduced aSyn pathology, suggesting that FTY720 may also benefit PD patients and others with synucleinopathy. Another finding was a loss of tyrosine hydroxylase immunoreactivity in gut neurons with aggregated aSyn, comparable with our prior findings in the CNS., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
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32. Genetic Deficiency of Mtdh Gene in Mice Causes Male Infertility via Impaired Spermatogenesis and Alterations in the Expression of Small Non-coding RNAs.
- Author
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Meng X, Yang S, Zhang Y, Wang X, Goodfellow RX, Jia Y, Thiel KW, Reyes HD, Yang B, and Leslie KK
- Subjects
- Animals, DNA Damage, DNA Repair, Exons, Gene Deletion, Genotype, Homozygote, In Situ Hybridization, Fluorescence, Male, Mice, Mice, Knockout, MicroRNAs metabolism, RNA-Binding Proteins, Spermatocytes metabolism, Spermatozoa physiology, Testis metabolism, Gene Expression Regulation, Infertility, Male genetics, Membrane Proteins genetics, Membrane Proteins physiology, RNA, Small Untranslated metabolism, Spermatogenesis genetics
- Abstract
Increased expression of metadherin (MTDH, also known as AEG-1 and 3D3/LYRIC) has been associated with drug resistance, metastasis, and angiogenesis in a variety of cancers. However, the specific mechanisms through which MTDH is involved in these processes remain unclear. To uncover these mechanisms, we generated Mtdh knock-out mice via a targeted disruption of exon 3. Homozygous Mtdh knock-out mice are viable, but males are infertile. The homozygous male mice present with massive loss of spermatozoa as a consequence of meiotic failure. Accumulation of γ-H2AX in spermatocytes of homozygous Mtdh knock-out mice confirms an increase in unrepaired DNA breaks. We also examined expression of the DNA repair protein Rad18, which is regulated by MTDH at the post-transcriptional level. In testes from Mtdh exon 3-deficient mice, Rad18 foci were increased in the lumina of the seminiferous tubules. The Piwi-interacting RNA (piRNA)-interacting protein Mili was expressed at high levels in testes from Mtdh knock-out mice. Accordingly, genome-wide small RNA deep sequencing demonstrated altered expression of piRNAs in the testes of Mtdh knock-out mice as compared with wild type mice. In addition, we observed significantly reduced expression of microRNAs (miRNAs) including miR-16 and miR-19b, which are known to be significantly reduced in the semen of infertile men. In sum, our observations indicate a crucial role for MTDH in male fertility and the DNA repair mechanisms required for normal spermatogenesis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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33. Biochemical characterization and structural analysis of a bifunctional cellulase/xylanase from Clostridium thermocellum.
- Author
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Yuan SF, Wu TH, Lee HL, Hsieh HY, Lin WL, Yang B, Chang CK, Li Q, Gao J, Huang CH, Ho MC, Guo RT, and Liang PH
- Subjects
- Amino Acid Sequence, Amino Acids chemistry, Amino Acids genetics, Amino Acids metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Binding Sites genetics, Catalytic Domain, Cellobiose chemistry, Cellobiose metabolism, Cellulase genetics, Cellulase metabolism, Clostridium thermocellum genetics, Crystallography, X-Ray, Disaccharides chemistry, Disaccharides metabolism, Endo-1,4-beta Xylanases genetics, Endo-1,4-beta Xylanases metabolism, Enzyme Assays, Kinetics, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Binding, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Substrate Specificity, Thermotoga maritima enzymology, Thermotoga maritima genetics, Bacterial Proteins chemistry, Cellulase chemistry, Clostridium thermocellum enzymology, Endo-1,4-beta Xylanases chemistry, Protein Structure, Tertiary
- Abstract
We expressed an active form of CtCel5E (a bifunctional cellulase/xylanase from Clostridium thermocellum), performed biochemical characterization, and determined its apo- and ligand-bound crystal structures. From the structures, Asn-93, His-168, His-169, Asn-208, Trp-347, and Asn-349 were shown to provide hydrogen-bonding/hydrophobic interactions with both ligands. Compared with the structures of TmCel5A, a bifunctional cellulase/mannanase homolog from Thermotoga maritima, a flexible loop region in CtCel5E is the key for discriminating substrates. Moreover, site-directed mutagenesis data confirmed that His-168 is essential for xylanase activity, and His-169 is more important for xylanase activity, whereas Asn-93, Asn-208, Tyr-270, Trp-347, and Asn-349 are critical for both activities. In contrast, F267A improves enzyme activities., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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34. Characterization of the Raptor/4E-BP1 interaction by chemical cross-linking coupled with mass spectrometry analysis.
- Author
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Coffman K, Yang B, Lu J, Tetlow AL, Pelliccio E, Lu S, Guo DC, Tang C, Dong MQ, and Tamanoi F
- Subjects
- Adaptor Proteins, Signal Transducing chemistry, Amino Acid Sequence, Animals, Carrier Proteins chemistry, Carrier Proteins genetics, Conserved Sequence, HEK293 Cells, Humans, Intracellular Signaling Peptides and Proteins, Lysine metabolism, Mechanistic Target of Rapamycin Complex 1, Molecular Sequence Data, Multiprotein Complexes metabolism, Mutation genetics, Peptides metabolism, Phosphoproteins chemistry, Phosphoproteins genetics, Protein Binding drug effects, Protein Structure, Tertiary, Rats, Regulatory-Associated Protein of mTOR, Substrate Specificity drug effects, TOR Serine-Threonine Kinases metabolism, Adaptor Proteins, Signal Transducing metabolism, Carrier Proteins metabolism, Cross-Linking Reagents pharmacology, Mass Spectrometry methods, Phosphoproteins metabolism
- Abstract
mTORC1 plays critical roles in the regulation of protein synthesis, growth, and proliferation in response to nutrients, growth factors, and energy conditions. One of the substrates of mTORC1 is 4E-BP1, whose phosphorylation by mTORC1 reverses its inhibitory action on eIF4E, resulting in the promotion of protein synthesis. Raptor in mTOR complex 1 is believed to recruit 4E-BP1, facilitating phosphorylation of 4E-BP1 by the kinase mTOR. We applied chemical cross-linking coupled with mass spectrometry analysis to gain insight into interactions between mTORC1 and 4E-BP1. Using the cross-linking reagent bis[sulfosuccinimidyl] suberate, we showed that Raptor can be cross-linked with 4E-BP1. Mass spectrometric analysis of cross-linked Raptor-4E-BP1 led to the identification of several cross-linked peptide pairs. Compilation of these peptides revealed that the most N-terminal Raptor N-terminal conserved domain (in particular residues from 89 to 180) of Raptor is the major site of interaction with 4E-BP1. On 4E-BP1, we found that cross-links with Raptor were clustered in the central region (amino acid residues 56-72) we call RCR (Raptor cross-linking region). Intramolecular cross-links of Raptor suggest the presence of two structured regions of Raptor: one in the N-terminal region and the other in the C-terminal region. In support of the idea that the Raptor N-terminal conserved domain and the 4E-BP1 central region are closely located, we found that peptides that encompass the RCR of 4E-BP1 inhibit cross-linking and interaction of 4E-BP1 with Raptor. Furthermore, mutations of residues in the RCR decrease the ability of 4E-BP1 to serve as a substrate for mTORC1 in vitro and in vivo.
- Published
- 2014
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35. Self-renewal and differentiation of muscle satellite cells are regulated by the Fas-associated death domain.
- Author
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Cheng W, Wang L, Yang B, Zhang R, Yao C, He L, Liu Z, Du P, Hammache K, Wen J, Li H, Xu Q, and Hua Z
- Subjects
- Animals, Cell Cycle, Cell Lineage, Cell Proliferation, Cell Separation, Hindlimb, Mice, Mice, Inbred C57BL, Mitosis, Phosphorylation, Receptors, Notch metabolism, Signal Transduction, Cell Differentiation, Fas-Associated Death Domain Protein metabolism, Satellite Cells, Skeletal Muscle cytology, Satellite Cells, Skeletal Muscle metabolism
- Abstract
Making the decision between self-renewal and differentiation of adult stem cells is critical for tissue repair and homeostasis. Here we show that the apoptotic adaptor Fas-associated death domain (FADD) regulates the fate decisions of muscle satellite cells (SCs). FADD phosphorylation was specifically induced in cycling SCs, which was high in metaphase and declined in later anaphase. Furthermore, phosphorylated FADD at Ser-191 accumulated in the uncommitted cycling SCs and was asymmetrically localized in the self-renewing daughter SCs. SCs containing a phosphoryl-mimicking mutation at Ser-191 of FADD (FADD-D) expressed higher levels of stem-like markers and reduced commitment-associated markers. Moreover, a phosphoryl-mimicking mutation at Ser-191 of FADD suppressed SC activation and differentiation, which promoted the cycling SCs into a reversible quiescent state. Therefore, these data indicate that FADD regulates the fate determination of cycling SCs.
- Published
- 2014
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36. Bioengineered Chinese hamster ovary cells with Golgi-targeted 3-O-sulfotransferase-1 biosynthesize heparan sulfate with an antithrombin-binding site.
- Author
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Datta P, Li G, Yang B, Zhao X, Baik JY, Gemmill TR, Sharfstein ST, and Linhardt RJ
- Subjects
- Amidohydrolases biosynthesis, Amidohydrolases genetics, Animals, CHO Cells, Cricetinae, Cricetulus, Golgi Apparatus genetics, Heparin genetics, Heparitin Sulfate genetics, Humans, Mice, Sulfotransferases genetics, Golgi Apparatus enzymology, Heparin biosynthesis, Heparitin Sulfate biosynthesis, Metabolic Engineering, Sulfotransferases biosynthesis
- Abstract
HS3st1 (heparan sulfate 3-O-sulfotransferase isoform-1) is a critical enzyme involved in the biosynthesis of the antithrombin III (AT)-binding site in the biopharmaceutical drug heparin. Heparin is a highly sulfated glycosaminoglycan that shares a common biosynthetic pathway with heparan sulfate (HS). Although only granulated cells, such as mast cells, biosynthesize heparin, all animal cells are capable of biosynthesizing HS. As part of an effort to bioengineer CHO cells to produce heparin, we previously showed that the introduction of both HS3st1 and NDST2 (N-deacetylase/N-sulfotransferase isoform-2) afforded HS with a very low level of anticoagulant activity. This study demonstrated that untargeted HS3st1 is broadly distributed throughout CHO cells and forms no detectable AT-binding sites, whereas Golgi-targeted HS3st1 localizes in the Golgi and results in the formation of a single type of AT-binding site and high anti-factor Xa activity (137 ± 36 units/mg). Moreover, stable overexpression of HS3st1 also results in up-regulation of 2-O-, 6-O-, and N-sulfo group-containing disaccharides, further emphasizing a previously unknown concerted interplay between the HS biosynthetic enzymes and suggesting the need to control the expression level of all of the biosynthetic enzymes to produce heparin in CHO cells.
- Published
- 2013
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37. Dragon (repulsive guidance molecule RGMb) inhibits E-cadherin expression and induces apoptosis in renal tubular epithelial cells.
- Author
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Liu W, Li X, Zhao Y, Meng XM, Wan C, Yang B, Lan HY, Lin HY, and Xia Y
- Subjects
- Animals, Cadherins genetics, Caspase 3 genetics, Caspase 3 metabolism, Cell Adhesion Molecules, Neuronal, Cell Hypoxia genetics, Cell Line, Epithelial Cells pathology, GPI-Linked Proteins, Kidney Tubules, Proximal pathology, Mice, Mice, Knockout, Nerve Tissue Proteins genetics, RNA, Messenger biosynthesis, RNA, Messenger genetics, Ureteral Obstruction genetics, Ureteral Obstruction pathology, Apoptosis, Cadherins biosynthesis, Epithelial Cells metabolism, Gene Expression Regulation, Kidney Tubules, Proximal metabolism, Nerve Tissue Proteins biosynthesis, Ureteral Obstruction metabolism
- Abstract
Dragon is one of the three members of the repulsive guidance molecule (RGM) family, i.e. RGMa, RGMb (Dragon), and RGMc (hemojuvelin). We previously identified the RGM members as bone morphogenetic protein (BMP) co-receptors that enhance BMP signaling. Our previous studies found that Dragon is highly expressed in the tubular epithelial cells of mouse kidneys. However, the roles of Dragon in renal epithelial cells are yet to be defined. We now show that overexpression of Dragon increased cell death induced by hypoxia in association with increased cleaved poly(ADP-ribose) polymerase and cleaved caspase-3 levels in mouse inner medullary collecting duct (IMCD3) cells. Dragon also inhibited E-cadherin expression but did not affect epithelial-to-mesenchymal transition induced by TGF-β in IMCD3 cells. Previous studies suggest that the three RGM members can function as ligands for the receptor neogenin. Interestingly, our present study demonstrates that the Dragon actions on apoptosis and E-cadherin expression in IMCD3 cells were mediated by the neogenin receptor but not through the BMP pathway. Dragon expression in the kidney was up-regulated by unilateral ureteral obstruction in mice. Compared with wild-type mice, heterozygous Dragon knock-out mice exhibited 45-66% reduction in Dragon mRNA expression, decreased epithelial apoptosis, and increased tubular E-cadherin expression and had attenuated tubular injury after unilateral ureteral obstruction. Our results suggest that Dragon may impair tubular epithelial integrity and induce epithelial apoptosis both in vitro and in vivo.
- Published
- 2013
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38. Hepatitis B viral RNA directly mediates down-regulation of the tumor suppressor microRNA miR-15a/miR-16-1 in hepatocytes.
- Author
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Wang Y, Jiang L, Ji X, Yang B, Zhang Y, and Fu XD
- Subjects
- Argonaute Proteins genetics, Argonaute Proteins metabolism, Blotting, Western, Genes, Tumor Suppressor, Hep G2 Cells, Hepatitis B virus physiology, Hepatocytes pathology, Hepatocytes virology, Host-Pathogen Interactions genetics, Humans, RNA Interference, Reverse Transcriptase Polymerase Chain Reaction, Trans-Activators genetics, Viral Regulatory and Accessory Proteins, Virus Replication genetics, Down-Regulation, Hepatitis B virus genetics, Hepatocytes metabolism, MicroRNAs genetics, RNA, Viral genetics
- Abstract
Hepatitis B virus (HBV) is a key risk factor for the development of hepatocellular carcinoma (HCC). Recent work suggests a functional link between HCC and microRNA expression, but the mechanism underlying the functional interaction between microRNA and HBV infection has remained largely elusive. Here we present evidence that the microRNA machinery serves as a defense system against HBV infection, which, in turn, reprograms the expression of specific microRNAs. We demonstrate a critical role of miR-15a/miR-16-1 in this functional interplay between microRNA and HBV infection, but in contrast to various indirect mechanisms mediated by viral proteins, we unexpectedly found that the HBx transcript directly triggers the down-regulation of miR-15a/miR-16-1 via the microRNA targeting sequences in the viral RNA. Because miR-15a and miR-16-1 are well known tumor suppressor microRNAs in multiple human cancers, our findings raise the intriguing possibility that viral RNA-mediated down-regulation of specific tumor suppressor microRNAs may contribute to HCC development in HBV-infected cells.
- Published
- 2013
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39. Sequence analysis and domain motifs in the porcine skin decorin glycosaminoglycan chain.
- Author
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Zhao X, Yang B, Solakyildirim K, Joo EJ, Toida T, Higashi K, Linhardt RJ, and Li L
- Subjects
- Amino Acid Motifs, Animals, Chondroitin Sulfates chemistry, Chromatography methods, Computational Biology methods, Dermatan Sulfate chemistry, Disaccharides chemistry, Electrophoresis, Polyacrylamide Gel, HEK293 Cells, Humans, Magnetic Resonance Spectroscopy methods, Mass Spectrometry methods, Protein Structure, Tertiary, Skin metabolism, Swine, Tandem Mass Spectrometry methods, Decorin chemistry, Glycosaminoglycans chemistry
- Abstract
Decorin proteoglycan is comprised of a core protein containing a single O-linked dermatan sulfate/chondroitin sulfate glycosaminoglycan (GAG) chain. Although the sequence of the decorin core protein is determined by the gene encoding its structure, the structure of its GAG chain is determined in the Golgi. The recent application of modern MS to bikunin, a far simpler chondroitin sulfate proteoglycans, suggests that it has a single or small number of defined sequences. On this basis, a similar approach to sequence the decorin of porcine skin much larger and more structurally complex dermatan sulfate/chondroitin sulfate GAG chain was undertaken. This approach resulted in information on the consistency/variability of its linkage region at the reducing end of the GAG chain, its iduronic acid-rich domain, glucuronic acid-rich domain, and non-reducing end. A general motif for the porcine skin decorin GAG chain was established. A single small decorin GAG chain was sequenced using MS/MS analysis. The data obtained in the study suggest that the decorin GAG chain has a small or a limited number of sequences.
- Published
- 2013
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40. APOBEC3G inhibits microRNA-mediated repression of translation by interfering with the interaction between Argonaute-2 and MOV10.
- Author
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Liu C, Zhang X, Huang F, Yang B, Li J, Liu B, Luo H, Zhang P, and Zhang H
- Subjects
- APOBEC-3G Deaminase, Argonaute Proteins genetics, Cytidine Deaminase genetics, HIV-1 genetics, HIV-1 metabolism, Humans, MicroRNAs genetics, RNA Helicases genetics, RNA, Small Cytoplasmic genetics, Signal Recognition Particle genetics, Argonaute Proteins metabolism, Cytidine Deaminase metabolism, MicroRNAs metabolism, Protein Biosynthesis physiology, RNA Helicases metabolism, RNA, Small Cytoplasmic metabolism, Signal Recognition Particle metabolism
- Abstract
The apolipoprotein-B-mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) is a potent restrictive factor for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. It belongs to the cytidine deaminase family. Recent studies have shown that A3G significantly inhibits microRNA (miRNA)-mediated repression of translation. However, the mechanism underlying this action must be clarified. In this report, we have demonstrated that A3G counteracts miRNA-mediated repression of translation by inhibiting the interaction between moloney leukemia virus 10 (MOV10) protein and Argonaute-2 (AGO2), causing either abnormal assembly or abnormal maturation of miRNA-inducing silencing complex (miRISC). Through a series of MOV10 deletions, we found that A3G binds to a domain at the C terminus in MOV10, where it competitively inhibits the binding of AGO2 to that same domain. The interaction between A3G and MOV10 relies on its association with a small RNA named 7SL RNA. The A3G mutant W127L, which is unable to bind to 7SL RNA, shows significantly incapability to counteract the miRNA-mediated repression of translation. Our data demonstrate a novel mechanism involved in the regulation of miRISC activity. Although both A3G and MOV10 belong to the interferon antiviral system and inhibit HIV-1 and other retroviruses, their opposing effects on the cellular miRNA activity suggest that they play much more complicated regulatory roles in various cellular functions.
- Published
- 2012
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41. Regulation of protein kinase C inactivation by Fas-associated protein with death domain.
- Author
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Cheng W, Wang L, Zhang R, Du P, Yang B, Zhuang H, Tang B, Yao C, Yu M, Wang Y, Zhang J, Yin W, Li J, Zheng W, Lu M, and Hua Z
- Subjects
- Amino Acid Motifs, Animals, Cell Movement, Cytoskeleton metabolism, Enzyme Stability, Fas-Associated Death Domain Protein genetics, HEK293 Cells, Humans, Isoenzymes metabolism, Mice, Mice, Knockout, Phosphorylation, Protein Binding, Protein Phosphatase 2 metabolism, Proteolysis, Signal Transduction, Fas-Associated Death Domain Protein metabolism, Protein Kinase C metabolism, Protein Processing, Post-Translational
- Abstract
Protein kinase C (PKC) plays important roles in diverse cellular processes. PKC has been implicated in regulating Fas-associated protein with death domain (FADD), an important adaptor protein involved in regulating death receptor-mediated apoptosis. FADD also plays an important role in non-apoptosis processes. The functional interaction of PKC and FADD in non-apoptotic processes has not been examined. In this study, we show that FADD is involved in maintaining the phosphorylation of the turn motif and hydrophobic motif in the activated conventional PKC (cPKC). A phosphoryl-mimicking mutation (S191D) in FADD (FADD-D) abolished the function of FADD in the facilitation of the turn motif and hydrophobic motif dephosphorylation of cPKC, suggesting that phosphorylation of Ser-191 negatively regulates FADD. We show that FADD interacts with PP2A, which is a major phosphatase involved in dephosphorylation of activated cPKC and FADD deficiency abolished PP2A mediated dephosphorylation of cPKC. We show that FADD deficiency leads to increased stability and activity of cPKC, which, in turn, promotes cytoskeleton reorganization, cell motility, and chemotaxis. Collectively, these results reveal a novel function of FADD in a non-apoptotic process by modulating cPKC dephosphorylation, stability, and signaling termination.
- Published
- 2012
- Full Text
- View/download PDF
42. Hindgut innate immunity and regulation of fecal microbiota through melanization in insects.
- Author
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Shao Q, Yang B, Xu Q, Li X, Lu Z, Wang C, Huang Y, Söderhäll K, and Ling E
- Subjects
- Animals, Bombyx, Catechol Oxidase chemistry, Enzyme Precursors chemistry, Feces, Immune System, Insecta, Laccase chemistry, Microscopy, Fluorescence methods, Models, Biological, Muramidase chemistry, Time Factors, Wound Healing, Immunity, Innate physiology, Intestinal Mucosa metabolism, Melanins chemistry, Metagenome physiology
- Abstract
Many insects eat the green leaves of plants but excrete black feces in an as yet unknown mechanism. Insects cannot avoid ingesting pathogens with food that will be specifically detected by the midgut immune system. However, just as in mammals, many pathogens can still escape the insect midgut immune system and arrive in the hindgut, where they are excreted out with the feces. Here we show that the melanization of hindgut content induced by prophenoloxidase, a key enzyme that induces the production of melanin around invaders and at wound sites, is the last line of immune defense to clear bacteria before feces excretion. We used the silkworm Bombyx mori as a model and found that prophenoloxidase produced by hindgut cells is secreted into the hindgut contents. Several experiments were done to clearly demonstrate that the blackening of the insect feces was due to activated phenoloxidase, which served to regulate the number of bacteria in the hindgut. Our analysis of the silkworm hindgut prophenoloxidase discloses the natural secret of why the phytophagous insect feces is black and provides insight into hindgut innate immunity, which is still rather unclear in mammals.
- Published
- 2012
- Full Text
- View/download PDF
43. Lipocalin-2 induces cardiomyocyte apoptosis by increasing intracellular iron accumulation.
- Author
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Xu G, Ahn J, Chang S, Eguchi M, Ogier A, Han S, Park Y, Shim C, Jang Y, Yang B, Xu A, Wang Y, and Sweeney G
- Subjects
- Acute-Phase Proteins pharmacology, Animals, Annexin A5 metabolism, Apoptosis drug effects, Blotting, Western, Caspase 3 metabolism, Cell Line, DNA Fragmentation drug effects, In Situ Nick-End Labeling, Lipocalin-2, Lipocalins pharmacology, Mice, Myocytes, Cardiac cytology, Oncogene Proteins pharmacology, Phosphatidylserines pharmacology, Protein Transport drug effects, Protein Transport physiology, Rats, bcl-2-Associated X Protein metabolism, Acute-Phase Proteins metabolism, Apoptosis physiology, Iron metabolism, Lipocalins metabolism, Myocytes, Cardiac metabolism, Oncogene Proteins metabolism
- Abstract
Our objective was to determine whether lipocalin-2 (Lcn2) regulates cardiomyocyte apoptosis, the mechanisms involved, and the functional significance. Emerging evidence suggests that Lcn2 is a proinflammatory adipokine associated with insulin resistance and obesity-related complications, such as heart failure. Here, we used both primary neonatal rat cardiomyocytes and H9c2 cells and demonstrated for the first time that Lcn2 directly induced cardiomyocyte apoptosis, an important component of cardiac remodeling leading to heart failure. This was shown by detection of DNA fragmentation using TUNEL assay, phosphatidylserine exposure using flow cytometry to detect annexin V-positive cells, caspase-3 activity using enzymatic assay and immunofluorescence, and Western blotting for the detection of cleaved caspase-3. We also observed that Lcn2 caused translocation of the proapoptotic protein Bax to mitochondria and disruption of mitochondrial membrane potential. Using transient transfection of GFP-Bax, we confirmed that Lcn2 induced co-localization of Bax with MitoTracker® dye. Importantly, we used the fluorescent probe Phen Green SK to demonstrate an increase in intracellular iron in response to Lcn2, and depleting intracellular iron using an iron chelator prevented Lcn2-induced cardiomyocyte apoptosis. Administration of recombinant Lcn2 to mice for 14 days increased cardiomyocyte apoptosis as well as an acute inflammatory response with compensatory changes in cardiac functional parameters. In conclusion, Lcn2-induced cardiomyocyte apoptosis is of physiological significance and occurs via a mechanism involving elevated intracellular iron levels and Bax translocation.
- Published
- 2012
- Full Text
- View/download PDF
44. Distinct involvement of the Gab1 and Grb2 adaptor proteins in signal transduction by the related receptor tyrosine kinases RON and MET.
- Author
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Chaudhuri A, Xie MH, Yang B, Mahapatra K, Liu J, Marsters S, Bodepudi S, and Ashkenazi A
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Animals, Binding Sites, Cell Line, Tumor, GRB2 Adaptor Protein genetics, HEK293 Cells, Humans, Mice, NIH 3T3 Cells, Phosphoproteins genetics, Phosphorylation physiology, Proto-Oncogene Proteins c-met genetics, Receptor Protein-Tyrosine Kinases genetics, Adaptor Proteins, Signal Transducing metabolism, GRB2 Adaptor Protein metabolism, Phosphoproteins metabolism, Proto-Oncogene Proteins c-met metabolism, Receptor Protein-Tyrosine Kinases metabolism, Signal Transduction physiology
- Abstract
Although the signal transduction mechanisms of the receptor tyrosine kinase MET are well defined, less is known about its close relative RON. MET initiates intracellular signaling by autophosphorylation on specific cytoplasmic tyrosines that form docking sites for the adaptor proteins Grb2 and Gab1. Grb2 binds directly and is essential for all of the biological activities of MET. Gab1 docks either directly or indirectly via Grb2 and controls only a subset of MET functions. Because MET and RON possess similar adaptor binding sites, it was anticipated that their adaptor interactions would be conserved. Here we show that in contrast to MET, RON relies primarily on Gab1 for signal transmission. Surprisingly, disruption of the Grb2 docking site of RON or Grb2 depletion augments activity, whereas enhancement of Grb2 binding attenuates Gab1 recruitment and signaling. Hence, RON and MET differ in their adaptor interactions; furthermore, Grb2 performs a novel antagonistic role in the context of RON signaling.
- Published
- 2011
- Full Text
- View/download PDF
45. WSS25 inhibits growth of xenografted hepatocellular cancer cells in nude mice by disrupting angiogenesis via blocking bone morphogenetic protein (BMP)/Smad/Id1 signaling.
- Author
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Qiu H, Yang B, Pei ZC, Zhang Z, and Ding K
- Subjects
- Animals, Cell Line, Tumor, Glucans chemistry, Humans, Male, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Nude, Molecular Structure, Neoplasm Transplantation, Neoplasms, Experimental, Quartz Crystal Microbalance Techniques, Transplantation, Heterologous, Bone Morphogenetic Proteins metabolism, Carcinoma, Hepatocellular metabolism, Carcinoma, Hepatocellular pathology, Glucans pharmacology, Heparitin Sulfate analogs & derivatives, Heparitin Sulfate pharmacology, Inhibitor of Differentiation Protein 1 metabolism, Liver Neoplasms metabolism, Liver Neoplasms pathology, Neovascularization, Pathologic metabolism, Signal Transduction drug effects, Smad Proteins metabolism
- Abstract
The highly expressed Id1 (inhibitor of DNA binding/differentiation) protein promotes angiogenesis in HCC and is a well established target for anti-angiogenesis therapeutic strategies. Heparan sulfate (HS) mimetics such as PI-88 can abrogate HS-protein interactions to inhibit angiogenesis. Id1 is the direct downstream effector of bone morphogenetic proteins (BMPs), which are angiogenic and HS-binding proteins. Thus, targeting BMPs by HS mimetics may inhibit angiogenesis via attenuating Id1 expression. We report here that a HS mimetic WSS25 potently inhibited the tube formation of HMEC-1 cells on Matrigel and their migration. Meanwhile, WSS25 (25 μg/ml) nearly completely blocked Id1 expression in the HMEC-1 cells as demonstrated by oligo-angiogenesis microarray analysis and further confirmed by RT-PCR and Western blotting. BMP/Smad/Id1 signaling also was blocked by WSS25 treatment in HMEC-1 cells. Importantly, Id1 knockdown in HMEC-1 cells caused the disruption of their tube formation on Matrigel. By employing quartz crystal microbalance analysis, we found that WSS25 strongly bound to BMP2. Moreover, WSS25 impaired BMP2-induced tube formation of HMEC-1 cells on Matrigel and angiogenesis in Matrigel transplanted into C57BL6 mice. Furthermore, WSS25 (100 mg/kg) abrogated the growth of HCC cells xenografted in male nude mice. Immunohistochemical analysis showed that both the expression of Id1 and the endothelial cell marker CD31 were lower in the WSS25-treated tumor tissue than in the control. Therefore, WSS25 is a potential drug candidate for HCC therapy as a tumor angiogenesis inhibitor.
- Published
- 2010
- Full Text
- View/download PDF
46. Molecular basis of the acceleration of the GDP-GTP exchange of human ras homolog enriched in brain by human translationally controlled tumor protein.
- Author
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Dong X, Yang B, Li Y, Zhong C, and Ding J
- Subjects
- Binding Sites, Biomarkers, Tumor chemistry, Biomarkers, Tumor genetics, Cell Line, Humans, Mechanistic Target of Rapamycin Complex 1, Molecular Conformation, Monomeric GTP-Binding Proteins chemistry, Monomeric GTP-Binding Proteins genetics, Multiprotein Complexes, Neuropeptides chemistry, Neuropeptides genetics, Protein Binding, Protein Structure, Secondary, Proteins, Ras Homolog Enriched in Brain Protein, Signal Transduction, TOR Serine-Threonine Kinases, Transcription Factors genetics, Transcription Factors metabolism, Tumor Protein, Translationally-Controlled 1, Biomarkers, Tumor metabolism, Guanosine Diphosphate metabolism, Guanosine Triphosphate metabolism, Monomeric GTP-Binding Proteins metabolism, Neuropeptides metabolism
- Abstract
Ras homolog enriched in brain (Rheb), a small GTPase, positively regulates the mTORC1 pathway. The GDP-GTP exchange of Rheb has been suggested to be facilitated by translationally controlled tumor protein (TCTP). Here we demonstrate that human TCTP (hTCTP) interacts with human Rheb (hRheb) and accelerates its GDP release in vitro and that hTCTP activates the mTORC1 pathway in vivo. To investigate the underlying mechanism, we built structure models of GDP- and GTP-bound hRheb in complexes with hTCTP and performed molecular dynamics simulations of the models, which predict key residues involved in the interactions and region of hRheb undergoing conformational change during the GDP-GTP exchange. These results are verified with site-directed mutagenesis and in vitro biochemical and in vivo cell biological analyses. Furthermore, a crystal structure of the E12V mutant hTCTP, which lacks the guanine nucleotide exchange factor activity, shows that the deficiency appears to be caused by loss of a salt-bridging interaction with Lys-45 of hRheb. These data collectively provide insights into the molecular mechanisms of how hTCTP interacts with hRheb and activates the mTORC1 pathway.
- Published
- 2009
- Full Text
- View/download PDF
47. Down-regulation of miR-1/miR-133 contributes to re-expression of pacemaker channel genes HCN2 and HCN4 in hypertrophic heart.
- Author
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Luo X, Lin H, Pan Z, Xiao J, Zhang Y, Lu Y, Yang B, and Wang Z
- Subjects
- Animals, Animals, Newborn, Base Sequence, Cells, Cultured, Heart Ventricles metabolism, Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels, Male, Rats, Rats, Sprague-Dawley, Cardiomegaly genetics, Cyclic Nucleotide-Gated Cation Channels genetics, Down-Regulation, Ion Channels genetics, MicroRNAs genetics, Muscle Proteins genetics, Potassium Channels genetics
- Abstract
Cardiac hypertrophy is characterized by electrical remolding with increased risk of arrhythmogenesis. Enhanced abnormal automaticity of ventricular cells contributes critically to hypertrophic arrhythmias. The pacemaker current I(f), carried by the hyperpolarization-activated channels encoded mainly by the HCN2 and HCN4 genes in the heart, plays an important role in determining cardiac automaticity. Their expressions reportedly increase in hypertrophic and failing hearts, contributing to arrhythmogenesis under these conditions. We performed a study on post-transcriptional regulation of expression of HCN2 and HCN4 genes by microRNAs. We experimentally established HCN2 as a target for repression by the muscle-specific microRNAs miR-1 and miR-133 and established HCN4 as a target for miR-1 only. We unraveled robust increases in HCN2 and HCN4 protein levels in a rat model of left ventricular hypertrophy and in angiotensin II-induced neonatal ventricular hypertrophy. The up-regulation of HCN2/HCN4 was accompanied by pronounced reduction of miR-1/miR-133 levels. Forced expression of miR-1/miR-133 by transfection prevented overexpression of HCN2/HCN4 in hypertrophic cardiomyocytes. The serum-responsive factor protein level was found significantly decreased in hypertrophic hearts, and silencing of this protein by RNA interference resulted in increased levels of miR-1/miR-133 and concomitant increases in HCN2 and HCN4 protein levels. We conclude that down-regulation of miR-1 and miR-133 expression contributes to re-expression of HCN2/HCN4 and thereby the electrical remodeling process in hypertrophic hearts. Our study also sheds new light on the cellular function and pathological role of miR-1/miR-133 in the heart.
- Published
- 2008
- Full Text
- View/download PDF
48. Crystal structure of HAb18G/CD147: implications for immunoglobulin superfamily homophilic adhesion.
- Author
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Yu XL, Hu T, Du JM, Ding JP, Yang XM, Zhang J, Yang B, Shen X, Zhang Z, Zhong WD, Wen N, Jiang H, Zhu P, and Chen ZN
- Subjects
- Amino Acid Sequence, Biomarkers, Tumor, Cell Line, Tumor, Crystallography, X-Ray, Dimerization, Humans, Models, Molecular, Molecular Conformation, Molecular Sequence Data, Neoplasm Metastasis, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Thermodynamics, Basigin chemistry, Immunoglobulins chemistry
- Abstract
CD147, a member of the immunoglobulin superfamily (IgSF), plays fundamental roles in intercellular interactions in numerous pathological and physiological processes. Importantly, our previous studies have demonstrated that HAb18G/CD147 is a novel hepatocellular carcinoma (HCC)-associated antigen, and HAb18G/CD147 stimulates adjacent fibroblasts and HCC cells to produce elevated levels of several matrix metalloproteinases, facilitating invasion and metastasis of HCC cells. In addition, HAb18G/CD147 has also been shown to be a novel universal cancer biomarker for diagnosis and prognostic assessment of a wide range of cancers. However, the structural basis underlying the multifunctional character of CD147 remains unresolved. We report here the crystal structure of the extracellular portion of HAb18G/CD147 at 2.8A resolution. The structure comprises an N-terminal IgC2 domain and a C-terminal IgI domain, which are connected by a 5-residue flexible linker. This unique C2-I domain organization is distinct from those of other IgSF members. Four homophilic dimers exist in the crystal and adopt C2-C2 and C2-I dimerization rather than V-V dimerization commonly found in other IgSF members. This type of homophilic association thus presents a novel model for homophilic interaction between C2 domains of IgSF members. Moreover, the crystal structure of HAb18G/CD147 provides a good structural explanation for the established multifunction of CD147 mediated by homo/hetero-oligomerizations and should represent a general architecture of other CD147 family members.
- Published
- 2008
- Full Text
- View/download PDF
49. Glial cell aquaporin-4 overexpression in transgenic mice accelerates cytotoxic brain swelling.
- Author
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Yang B, Zador Z, and Verkman AS
- Subjects
- Animals, Aquaporin 4 genetics, Biological Transport genetics, Blood-Brain Barrier pathology, Brain Edema genetics, Brain Edema pathology, Cell Membrane genetics, Cell Membrane metabolism, Cell Membrane pathology, Female, Gene Deletion, Gene Expression Regulation genetics, Glial Fibrillary Acidic Protein genetics, Glial Fibrillary Acidic Protein metabolism, Intracranial Hypertension genetics, Intracranial Hypertension pathology, Mice, Mice, Knockout, Neuroglia pathology, Time Factors, Water Intoxication genetics, Water Intoxication pathology, Water-Electrolyte Balance genetics, Aquaporin 4 biosynthesis, Blood-Brain Barrier metabolism, Brain Edema metabolism, Intracranial Hypertension metabolism, Neuroglia metabolism, Water Intoxication metabolism
- Abstract
Aquaporin-4 (AQP4) is a water transport protein expressed in glial cell plasma membranes, including glial cell foot processes lining the blood-brain barrier. AQP4 deletion in mice reduces cytotoxic brain edema produced by different pathologies. To determine whether AQP4 is rate-limiting for brain water accumulation and whether altered AQP4 expression, as occurs in various pathologies, could have functional importance, we generated mice that overexpressed AQP4 in brain glial cells by a transgenic approach using the glial fibrillary acid protein promoter. Overexpression of AQP4 protein in brain by approximately 2.3-fold did not affect mouse survival, appearance, or behavior, nor did it affect brain anatomy or intracranial pressure (ICP). However, following acute water intoxication produced by intraperitoneal water injection, AQP4-overexpressing mice had an accelerated progression of cytotoxic brain swelling, with ICP elevation of 20 +/- 2 mmHg at 10 min, often producing brain herniation and death. In contrast, ICP elevation was 14 +/- 2 mmHg at 10 min in control mice and 9.8 +/- 2 mmHg in AQP4 knock-out mice. The deduced increase in brain water content correlated linearly with brain AQP4 protein expression. We conclude that AQP4 expression is rate-limiting for brain water accumulation, and thus, that altered AQP4 expression can be functionally significant.
- Published
- 2008
- Full Text
- View/download PDF
50. Derepression of microRNA-mediated protein translation inhibition by apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) and its family members.
- Author
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Huang J, Liang Z, Yang B, Tian H, Ma J, and Zhang H
- Subjects
- APOBEC-3G Deaminase, Base Sequence, CD4-Positive T-Lymphocytes metabolism, Cytidine Deaminase chemistry, Flow Cytometry, Genes, Reporter, Humans, Models, Biological, Molecular Sequence Data, Mutation, RNA-Binding Proteins chemistry, Ribonuclease, Pancreatic metabolism, Apolipoproteins B genetics, Cytidine Deaminase metabolism, MicroRNAs metabolism, Protein Biosynthesis
- Abstract
The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. A3G interacts with a vast spectrum of RNA-binding proteins and is located in processing bodies and stress granules. However, its cellular function remains to be further clarified. Using a luciferase reporter gene and green fluorescent protein reporter gene, we demonstrate that A3G and other APOBEC family members can counteract the inhibition of protein synthesis by various microRNAs (miRNAs) such as mir-10b, mir-16, mir-25, and let-7a. A3G could also enhance the expression level of miRNA-targeted mRNA. Further, A3G facilitated the association of microRNA-targeted mRNA with polysomes rather than with processing bodies. Intriguingly, experiments with a C288A/C291A A3G mutant indicated that this function of A3G is separable from its cytidine deaminase activity. Our findings suggest that the major cellular function of A3G, in addition to inhibiting the mobility of retrotransposons and replication of endogenous retroviruses, is most likely to prevent the decay of miRNA-targeted mRNA in processing bodies.
- Published
- 2007
- Full Text
- View/download PDF
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