Biomedical subjects

Zhiying Zou

Publications and source records attributed to Zhiying Zou.

6 recordsLinked to original sources

PubMed2006

Structure-function analysis reveals discrete beta3 integrin inside-out and outside-in signaling pathways in platelets.

A unique aspect of integrin receptor function is the transmission of bidirectional signals. In platelets alphaIIbbeta3 integrins require "inside-out" signals to bind fibrinogen and form thrombi. Following ligand binding, alphaIIbbeta3 integrins generate "outside-in" signals that contribute to thrombus stability. Because integrin cytoplasmic tails are short and lack enzymatic activity, bidirectional signals are believed to be mediated by interactions with intracellular proteins, but the molecular basis for integrin signal transduction remains poorly understood. In the present study we have used retroviral vectors to express alphaIIbbeta3 integrins with mutant beta3 tails in mouse platelets and test mechanisms of bidirectional signaling. Using this approach we identify mutations (eg, beta3Y747A) that confer loss of signaling in both directions and others (eg, beta3T762A) that confer a selective loss of outside-in signals. These results reveal the presence of discrete bidirectional signaling pathways controlled by integrin beta subunits in platelets and describe a high-throughput means of further investigating these pathways in vivo.

Animals↗

PubMed2006

In vivo beta1 integrin function requires phosphorylation-independent regulation by cytoplasmic tyrosines.

Integrins are heterodimeric adhesion receptors associated with bidirectional signaling. In vitro studies support a role for the binding of evolutionarily conserved tyrosine motifs (NPxY) in the beta integrin cytoplasmic tail to phosphotyrosine-binding (PTB) domain-containing proteins, an interaction proposed to be dynamically regulated by tyrosine phosphorylation. Here we show that replacement of both beta1 integrin cytoplasmic tyrosines with alanines, resulting in the loss of all PTB domain interaction, causes complete loss of beta1 integrin function in vivo. In contrast, replacement of beta1 integrin cytoplasmic tyrosines with phenylalanines, a mutation that prevents tyrosine phosphorylation, conserves in vivo integrin function. These results have important implications for the molecular mechanism and regulation of integrin function.

Amino Acid Motifs↗

PubMed2005

Identification and characterization of a second fibronectin gene in zebrafish.

Fibronectin (FN) is a highly conserved extracellular matrix protein that plays crucial roles in vertebrate embryogenesis. Previously, it was reported that zebrafish possess a single FN gene (fn1a). Here we report the presence of a second zebrafish FN gene (fn1b) that encodes a protein with a predicted primary structure that is similar to FNs identified in other vertebrates possessing 12 type I, 2 type II and 17 type III repeats including two alternative splice sites (EIIIA and EIIIB) and a variable region (V). Zebrafish FN1b exhibits 62.0% amino acid identity with zebrafish FN1a, 54% with human and 55% with Xenopus laevis FNs respectively. Employing RT-PCR analysis, we demonstrate that EIIIB- and V- isoforms are produced by alternative splicing of a single fn1b transcript. The FN1b EIIIA- isoform was not detected in zebrafish embryos or adult tissues nor were EIIIA, EIIIB or V region splice variants of fn1a found. FN1b mRNA was detected by RT-PCR in embryos at the gastrula-stage (8hpf) through 72hpf and in various adult tissues. EIIIB- and V+ are the predominant forms of FN1b present in the zebrafish embryo. Unlike FN1a, which is present at a relatively high amount at the embryonic stages before gastrulation, the FN1b isoforms are present at very low amounts at the early cleavage stage. The presence of multiple isoforms of FN1b along with a different pattern of expression compared to FN1a indicates that the two fn genes have separate roles in zebrafish development.

Alternative Splicing↗

PubMed2004

Linking receptor-mediated endocytosis and cell signaling: evidence for regulated intramembrane proteolysis of megalin in proximal tubule.

Megalin, a member of the low density lipoprotein receptor gene family, is required for efficient protein absorption in the proximal tubule. Recent studies have shown that the low density lipoprotein receptor-related protein, another member of this gene family, is proteolytically processed by gamma-secretase implying a role for low density lipoprotein receptor-related protein in a Notchlike signaling pathway. This pathway has been shown to involve: 1) metalloprotease-mediated ectodomain shedding and gamma-secretase-mediated intramembrane proteolysis of some receptors. Experiments were performed to determine whether megalin undergoes similar processing. By immunocytochemistry, immunoblotting, and a fluorogenic enzyme assay presenilin-1 (required for gamma-secretase activity) and gamma-secretase activity were found in the brush border of proximal kidney tubules where megalin is localized. Using a fluorogenic peptide containing an amyloid precursor protein gamma-secretase cleavage site and Compound E, a specific gamma-secretase inhibitor, we found high levels of gamma-secretase activity in renal brush border membrane vesicles. Immunoblotting analysis of renal microsomes and opossum kidney proximal tubule (OKP) cells using antibodies directed to the cytosolic domain of megalin showed a 35-40-kDa, membrane-associated, carboxyl-terminal fragment of megalin (MCTF). When cells were incubated with 200 nm phorbol 12-myristate 13-acetate, the appearance of the MCTF increased 2.5-fold and was blocked by metalloprotease inhibitors. When the cells were incubated with gamma-secretase inhibitor Compound E, it caused a 2-fold increase in MCTF. Finally, incubating the cells with 1 microm vitamin D-binding protein resulted in a 25% increase in the appearance of the MCTF. In summary, the MCTF is produced by protein kinase C regulated, metalloprotease-mediated ectodomain shedding and is the substrate for gamma-secretase. We postulate that the enzymatic processing of megalin represents part of a novel ligand-dependent signaling pathway in the proximal tubule that links receptor-mediated endocytosis with cell signaling.

Amyloid Precursor Protein Secretases↗

PubMed2003

Vectorial acylation in Saccharomyces cerevisiae. Fat1p and fatty acyl-CoA synthetase are interacting components of a fatty acid import complex.

In Saccharomyces cerevisiae Fat1p and fatty acyl-CoA synthetase (FACS) are hypothesized to couple import and activation of exogenous fatty acids by a process called vectorial acylation. Molecular genetic and biochemical studies were used to define further the functional and physical interactions between these proteins. Multicopy extragenic suppressors were selected in strains carrying deletions in FAA1 and FAA4 or FAA1 and FAT1. Each strain is unable to grow under synthetic lethal conditions when exogenous long-chain fatty acids are required, and neither strain accumulates the fluorescent long-chain fatty acid C(1)-BODIPY-C(12) indicating a fatty acid transport defect. By using these phenotypes as selective screens, plasmids were identified encoding FAA1, FAT1, and FAA4 in the faa1Delta faa4Delta strain and encoding FAA1 and FAT1 in the faa1Delta fat1Delta strain. Multicopy FAA4 could not suppress the growth defect in the faa1Delta fat1Delta strain indicating some essential functions of Fat1p cannot be performed by Faa4p. Chromosomally encoded FAA1 and FAT1 are not able to suppress the growth deficiencies of the fat1Delta faa1Delta and faa1Delta faa4Delta strains, respectively, indicating Faa1p and Fat1p play distinct roles in the fatty acid import process. When expressed from a 2-mu plasmid, Fat1p contributes significant oleoyl-CoA synthetase activity, which indicates vectorial esterification and metabolic trapping are the driving forces behind import. Evidence of a physical interaction between Fat1p and FACS was provided using three independent biochemical approaches. First, a C-terminal peptide of Fat1p deficient in fatty acid transport exerted a dominant negative effect against long-chain acyl-CoA synthetase activity. Second, protein fusions employing Faa1p as bait and portions of Fat1p as trap were active when tested using the yeast two-hybrid system. Third, co-expressed, differentially tagged Fat1p and Faa1p or Faa4p were co-immunoprecipitated. Collectively, these data support the hypothesis that fatty acid import by vectorial acylation in yeast requires a multiprotein complex, which consists of Fat1p and Faa1p or Faa4p.

Acyl Coenzyme A↗

PubMed2002

Fatty acid transport in Saccharomyces cerevisiae. Directed mutagenesis of FAT1 distinguishes the biochemical activities associated with Fat1p.

The fatty acid transport protein Fat1p functions as a component of the long-chain fatty acid transport apparatus in the yeast Saccharomyces cerevisiae. Fat1p has significant homologies to the mammalian fatty acid transport proteins (FATP) and the very long-chain acyl-CoA synthetases (VLACS). In order to further understand the functional roles intrinsic to Fat1p (fatty acid transport and VLACS activities), a series of 16 alleles carrying site-directed mutations within FAT1 were constructed and analyzed. Sites chosen for the construction of amino acid substitutions were based on conservation between Fat1p and the mammalian FATP orthologues and included the ATP/AMP and FATP/VLACS signature motifs. Centromeric and 2 mu plasmids encoding mutant forms of Fat1p were transformed into a yeast strain containing a deletion in FAT1 (fat1Delta). For selected subsets of FAT1 mutant alleles, we observed differences between the wild type and mutants in 1) growth rates when fatty acid synthase was inhibited with 45 microm cerulenin in the presence of 100 microm oleate (C(18:1)), 2) levels of fatty acid import monitored using the accumulation of the fluorescent fatty acid 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-S-indacene-3-dodecanoic acid and [(3)H]oleate, 3) levels of lignoceryl (C(24:0)) CoA synthetase activities, and 4) fatty acid profiles monitored using gas chromatography/mass spectrometry. In most cases, there was a correlation between growth on fatty acid/cerulenin plates, the levels of fatty acid accumulation, very long-chain fatty acyl-CoA synthetase activities, and the fatty acid profiles in the different FAT1 mutants. For several notable exceptions, the fatty acid transport and very long-chain fatty acyl-CoA synthetase activities were distinguishable. The characterization of these novel mutants provides a platform to more completely understand the role of Fat1p in the linkage between fatty acid import and activation to CoA thioesters.

Acyl Coenzyme A↗