Biomedical subjects

W S Lane

Publications and source records attributed to W S Lane.

At least 55 records · Page 3Linked to original sources

PubMed1999

Troponin I is present in human cartilage and inhibits angiogenesis.

Cartilage is an avascular and relatively tumor-resistant tissue. Work from a number of laboratories, including our own, has demonstrated that cartilage is an enriched source of endogenous inhibitors of angiogenesis. In the course of a study designed to identify novel cartilage-derived inhibitors of new capillary growth, we have purified an inhibitory protein that was identified by peptide microsequencing and protein database analysis as troponin I (TnI). TnI is a subunit of the troponin complex (troponin-C and troponin-T being the other two), which, along with tropomyosin, is responsible for the calcium-dependent regulation of striated muscle contraction; independently, TnI is capable of inhibiting actomyosin ATPase. Because troponin has never previously been reported to be present in cartilage, we have cloned and expressed the cDNA of human cartilage TnI, purified this protein to apparent homogeneity, and demonstrated that it is a potent and specific inhibitor of angiogenesis in vivo and in vitro, as well as of tumor metastasis in vivo.

3T3 Cells↗

PubMed1999

A polyadenylylation-specific RNA-contact site on the surface of the bifunctional vaccinia virus RNA modifying protein VP39 that is distinct from the mRNA 5' end-binding "cleft".

VP39 is a bifunctional mRNA-modifying protein that acts as both an mRNA cap-specific 2'-O-methyltransferase and a processivity factor for VP55, the vaccinia poly(A) polymerase catalytic subunit. Although regions of the protein surface required for methyltransferase function are well defined, it has been unclear whether the protein polyadenylylation function requires direct RNA contact and, if so, where the contact site(s) might be located on the protein surface. Here, we show that the VP55-VP39 heterodimer forms a stable complex with a 50mer oligonucleotide bearing a U2-N25-U motif, as opposed to the U2-N15-U motif that is optimal for stable complex formation with VP55 alone. An oligonucleotide bearing a U2-N25-U motif in which the downstream U residue is replaced with 4thioU can be efficiently photocrosslinked to VP39, but only in the context of the VP55-VP39 heterodimer. By partial proteolysis of end-labeled VP39, the site of oligonucleotide photocrosslinking was localized to the region of VP39 between residues Lys90 and Arg122. Peptide microsequencing and confirmatory mutagenesis identified the side-chain of Arg107 as the photocrosslinking site. Substitution of this residue with lysine abolished photocrosslinking entirely, consistent with the established RNA binding role of arginine in other RNA-binding proteins. This study provides clear evidence for a polyadenylylation-specific RNA-contact site on the surface of VP39, which is distinct from the RNA-binding methyltransferase "cleft" characterized in recent crystallographic and biochemical studies.

Base Sequence↗

PubMed1999

PHA synthase from chromatium vinosum: cysteine 149 is involved in covalent catalysis.

Polyhydroxyalkanoate synthase (PHA) from Chromatium vinosum catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) and CoA. The synthase is composed of a approximately 1:1 mixture of two subunits, PhaC and PhaE. Size-exclusion chromatography indicates that in solution PhaC and PhaE exist as large molecular weight aggregates. The holo-enzyme, PhaEC, has a specific activity of 150 units/mg. Each subunit was cloned, expressed, and purified as a (His)6-tagged construct. The PhaC-(His)6 protein catalyzed polymerization with a specific activity of 0.9 unit/mg; the PhaE-(His)6 protein was inactive (specific activity <0.001 unit/mg). Addition of PhaE-(His)6 to PhaC-(His)6 increased the activity several 100-fold. To investigate the priming step of the polymerization process, the PhaEC was incubated with a trimer of HB-CoA in which the terminal hydroxyl was replaced with tritium ([3H]-sT-CoA). After Sephadex G50 chromatography, the synthase contained approximately 0.25 equiv of the labile label per PhaC. Incubation of [3H]-sT-synthase with HB-CoA resulted in production of [3H]-polymer. Digestion of [3H]-sT-synthase with trypsin and HPLC analysis resulted in isolation of three labeled peptides. Sequencing by ion trap mass spectrometry showed that they were identical and that they each contained an altered cysteine (C149). One peptide contained the [3H]-sT while the other two contained, in addition to the [3H]-sT, one and two additional monomeric HBs, respectively. Mutation of C149 to alanine gave inactive synthase. The remaining two cysteines of PhaC, 292 and 130, were also mutated to alanine. The former had wild-type (wt) activity, while the latter had 0.004 wt % activity and was capable of making polymer. A mechanism is proposed in which PhaC contains all the elements essential for catalysis and the polymerization proceeds by covalent catalysis using C149 and potentially C130.

Acyltransferases↗

PubMed1999

latheo encodes a subunit of the origin recognition complex and disrupts neuronal proliferation and adult olfactory memory when mutant.

The Drosophila latheo (lat) gene was identified in a behavioral screen for olfactory memory mutants. The original hypomorphic latP1 mutant (Boynton and Tully, 1992) shows a structural defect in adult brain. Homozygous lethal lat mutants lack imaginal discs, show little cell proliferation in the CNS of third instar larvae, and die as early pupae. latP1 was cloned, and all of the above mentioned defects of hypomorphic or homozygous lethal lat mutants were rescued with a lat+ transgene. lat encodes a novel protein with homology to a subunit of the origin recognition complex (ORC). Human and Drosophila LAT both associate with ORC2 and are related to yeast ORC3, suggesting that LAT functions in DNA replication during cell proliferation.

Amino Acid Sequence↗

PubMed1999

The identification of myriocin-binding proteins.

BACKGROUND: Myriocin is a natural product that potently induces apoptosis of a murine cytotoxic T lymphocyte cell line (CTLL-2) and inhibits a serine palmitoyltransferase (SPT) activity that has been detected in cell extracts and is thought to initiate sphingolipid biosynthesis. Because SPT has never been biochemically purified and a comprehensive appraisal of myriocin-binding proteins has not been conducted, we isolated specific targets using myriocin-based affinity chromatography. RESULTS: Myriocin derivatives were synthesized and evaluated using CTLL-2 proliferation and SPT activity assays. Guided by these results, affinity chromatography matrices were prepared and two specific myriocin-binding proteins were isolated from CTLL-2 lysates. Analyses of these polypeptides establish conclusively that they are murine LCB1 and LCB2, mammalian homologs of two yeast proteins that have been genetically linked to sphingolipid biosynthesis. CONCLUSION: The ability of myriocin-containing matrices to bind factors that have SPT activity and the exclusive isolation of LCB1 and LCB2 as myriocin-binding proteins demonstrates that the two proteins are directly responsible for SPT activity and that myriocin acts directly upon these polypeptides.

Acyltransferases↗

PubMed1999

Interaction of the Ski oncoprotein with Smad3 regulates TGF-beta signaling.

TGF-beta treatment of cells induces a variety of physiologic responses, including growth inhibition, differentiation, and induction of apoptosis. TGF-beta induces phosphorylation and nuclear translocation of Smad3. We describe here the association of Smad3 with the nuclear protooncogene protein Ski in response to the activation of TGF-beta signaling. Association with Ski represses transcriptional activation by Smad3, and overexpression of Ski renders cells resistant to the growth-inhibitory effects of TGF-beta. The transcriptional repression as well as the growth resistance to TGF-beta by overexpression of Ski can be overcome by overexpression of Smad3. These results demonstrate that Ski is a novel component of the TGF-beta signaling pathway and shed light on the mechanism of action of the Ski oncoprotein.

Animals↗

PubMed1999

Activator-specific requirement of yeast mediator proteins for RNA polymerase II transcriptional activation.

The multisubunit Mediator complex of Saccharomyces cerevisiae is required for most RNA polymerase II (Pol II) transcription. The Mediator complex is composed of two subcomplexes, the Rgr1 and Srb4 subcomplexes, which appear to function in the reception of activator signals and the subsequent modulation of Pol II activity, respectively. In order to determine the precise composition of the Mediator complex and to explore the specific role of each Mediator protein, our goal was to identify all of the Mediator components. To this end, we cloned three previously unidentified Mediator subunits, Med9/Cse2, Med10/Nut2, and Med11, and isolated mutant forms of each of them to analyze their transcriptional defects. Differential display and Northern analyses of mRNAs from wild-type and Mediator mutant cells demonstrated an activator-specific requirement for each Mediator subunit. Med9/Cse2 and Med10/Nut2 were required, respectively, for Bas1/Bas2- and Gcn4-mediated transcription of amino acid biosynthetic genes. Gal11 was required for Gal4- and Rap1-mediated transcriptional activation. Med11 was also required specifically for MFalpha1 transcription. On the other hand, Med6 was required for all of these transcriptional activation processes. These results suggest that distinct Mediator proteins in the Rgr1 subcomplex are required for activator-specific transcriptional activation and that the activation signals mediated by these Mediator proteins converge on Med6 (or the Srb4 subcomplex) to modulate Pol II activity.

Amino Acid Sequence↗

PubMed1998

Requirement of RSF and FACT for transcription of chromatin templates in vitro.

Transcription of naked DNA in vitro requires the general transcription factors and RNA polymerase II. However, this minimal set of factors is not sufficient for transcription when the DNA template is packaged into chromatin. Here, a factor that facilitates activator-dependent transcription initiation on chromatin templates was purified. This factor, remodeling and spacing factor (RSF), has adenosine triphosphate-dependent nucleosome-remodeling and spacing activities. Polymerases that initiate transcription with RSF can only extend their transcripts in the presence of FACT (facilitates chromatin transcription). Thus, the minimal factor requirements for activator-dependent transcription on chromatin templates in vitro have been defined.

Adenosine Triphosphate↗

PubMed1998

The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities.

Histone acetylation and deacetylation were found to be catalyzed by structurally distinct, multisubunit complexes that mediate, respectively, activation and repression of transcription. ATP-dependent nucleosome remodeling, mediated by different multisubunit complexes, was thought to be involved only in transcription activation. Here we report the isolation of a protein complex that contains both histone deacetylation and ATP-dependent nucleosome remodeling activities. The complex contains the histone deacetylases HDAC1/2, histone-binding proteins, the dermatomyositis-specific autoantigen Mi2beta, a polypeptide related to the metastasis-associated protein 1, and a novel polypeptide of 32 kDa. Patients with dermatomyositis have a high rate of malignancy. The finding that Mi2beta exists in a complex containing histone deacetylase and nucleosome remodeling activities suggests a role for chromatin reorganization in cancer metastasis.

Adenosine Triphosphatases↗

PubMed1998

Affinity-purification and characterization of caveolins from the brain: differential expression of caveolin-1, -2, and -3 in brain endothelial and astroglial cell types.

Caveolins 1, 2 and 3 are the principal protein components of caveolae organelles. It has been proposed that caveolae play a vital role in a number of essential cellular functions including signal transduction, lipid metabolism, cellular growth control and apoptotic cell death. Thus, a major focus of caveolae-related research has been the identification of novel caveolins, caveolae-associated proteins and caveolin-interacting proteins. However, virtually nothing is known about the expression of caveolins in brain tissue. Here, we report the purification and characterization of caveolins from brain tissue under non-denaturing conditions. As a final step in the purification, we employed immuno-affinity chromatography using rabbit polyclonal anti-caveolin IgG and specific elution at alkaline pH. The final purified brain caveolin fractions contained three bands with molecular masses of 52 kDa, 24 kDa and 22 kDa as visualized by silver staining. Sequencing by ion trap mass spectrometry directly identified the major 24-kDa component of this hetero-oligomeric complex as caveolin 1. Further immunocyto- and histochemical analyses demonstrated that caveolin 1 was primarily expressed in brain endothelial cells. Caveolins 2 and 3 were also detected in purified caveolin fractions and brain cells. The cellular distribution of caveolin 2 was similar to that of caveolin 1. In striking contrast, caveolin 3 was predominantly expressed in brain astroglial cells. This finding was surprising as our previous studies have suggested that the expression of caveolin 3 is confined to striated (cardiac and skeletal) and smooth muscle cells. Electron-microscopic analysis revealed that astrocytes possess numerous caveolar invaginations of the plasma membrane. Our results provide the first biochemical and histochemical evidence that caveolins 1, 2 and 3 are expressed in brain endothelial and astroglial cells.

Amino Acid Sequence↗

PubMed1998

Rabbit beta-globin is extended beyond its UGA stop codon by multiple suppressions and translational reading gaps.

Translational reading gaps occur when genetic information encoded in mRNA is not translated during the normal course of protein synthesis. This phenomenon has been observed thus far only in prokaryotes and is a mechanism for extending the reading frame by circumventing the normal stop codon. Reading frames of proteins may also be extended by suppression of the stop codon mediated by a suppressor tRNA. The rabbit beta-globin read-through protein, the only known, naturally occurring read-through protein in eukaryotes, was sequenced by ion trap mass spectrometry to determine how the reading frame is extended. Seven different proteolytic peptide fragments decoded by the same sequence that spans the UGA stop codon of rabbit beta-globin mRNA were detected. Three of these peptides contain translational reading gaps of one to three amino acids that correspond to the UGA stop codon site and/or one or two of the immediate downstream codons. To our knowledge, this is the first reported example of the occurrence of reading gaps in protein synthesis in eukaryotes. This event is unique in that it is associated with bypasses involving staggered lengths of untranslated information. Four of the seven peptides contain serine, tryptophan, cysteine, and arginine decoded by UGA and thus arise by suppression. Serine is donated by selenocysteine tRNA, and it, like the other tRNAs, has previously been shown to suppress UGA in vitro in mammals, but not in vivo.

Amino Acid Sequence↗

PubMed1998

Purification and characterization of the nuclear RNase P holoenzyme complex reveals extensive subunit overlap with RNase MRP.

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that cleaves precursor tRNA transcripts to give mature 5' ends. RNase P in eubacteria has a large, catalytic RNA subunit and a small protein subunit that are required for precursor tRNA cleavage in vivo. Although the eukaryotic holoenzymes have similar, large RNA subunits, previous work in a number of systems has suggested that the eukaryotic enzymes require a greater protein content. We have purified the Saccharomyces cerevisiae nuclear RNase P to apparent homogeneity, allowing the first comprehensive analysis of an unexpectedly complex subunit composition. Peptide sequencing by ion trap mass spectrometry identifies nine proteins that copurify with the nuclear RNase P RNA subunit, totaling 20-fold more protein than in the bacterial enzyme. All of these proteins are encoded by genes essential for RNase P activity and for cell viability. Previous genetic studies suggested that four proteins might be subunits of both RNase P and RNase MRP, the related rRNA processing enzyme. We demonstrate that all four of these proteins, Pop1p, Pop3p, Pop4p, and Rpp1p, are integral subunits of RNase P. In addition, four of the five newly identified protein subunits, Pop5p, Pop6p, Pop7p, and Pop8p, also appear to be shared between RNase P and RNase MRP. Only one polypeptide, Rpr2p, is unique to the RNase P holoenzyme by genetic depletion and immunoprecipitation studies. The large increase in the number of protein subunits over eubacterial RNase P is consistent with an increase in functional complexity in eukaryotes. The degree of structural similarity between nuclear RNase P and RNase MRP suggests that some aspects of their functions in pre-tRNA and pre-rRNA processing pathways might overlap or be coordinated.

Amino Acid Sequence↗

PubMed1998

Role of the human homolog of the yeast transcription factor SPT5 in HIV-1 Tat-activation.

The transactivator protein Tat stimulates transcriptional elongation from the HIV-1 LTR. One mechanism by which Tat increases HIV-1 transcription is by interacting with RNA polymerase II and TFIIH to increase phosphorylation of the polymerase C-terminal domain. Recent studies indicate that specific elongation factors may also be required to modulate Tat function. Here, we used biochemical analysis and in vitro transcription assays to identify cellular factors required for Tat activation. This analysis resulted in the purification of a cellular factor Tat-CT1 which is a human homolog of the yeast transcription factor SPT5. Immunodepletion of Tat-CTl from HeLa extract demonstrated that this factor was involved in transcriptional activation by Tat. However, the absence of this factor from HeLa extract did not prevent transcriptional activation by VP16. These findings are consistent with a model in which Tat-mediated effects on transcriptional elongation are mediated in part by the action of the human homolog of the yeast transcription factor SPT5.

Amino Acid Sequence↗

PubMed1998

Repair of oxidatively damaged guanine in Saccharomyces cerevisiae by an alternative pathway.

BACKGROUND: Transversion mutations are caused by 8-oxoguanine (OG), a DNA lesion produced by the spontaneous oxidation of guanine nucleotides, which mis-pairs with adenine during replication. Resistance to this mutagenic threat is mediated by the GO system, the components of which are functionally conserved in bacteria and mammals. To date, only one of three GO system components has been identified in the budding yeast Saccharomyces cerevisiae, namely the OG:C-specific glycosylase/lyase yOgg1. Furthermore, S. cerevisiae has been reported to contain a unique glycosylase/lyase activity, yOgg2, which excises OG residues opposite adenines. Paradoxically, according to the currently accepted model, yOgg2 activity should increase the mutagenicity of OG lesions. Here we report the isolation of yOgg2 and the elucidation of its role in oxidative mutagenesis. RESULTS: Borohydride-dependent cross-linking using an OG-containing oligonucleotide substrate led to the isolation of yOgg1 and a second protein, Ntg1, which had previously been shown to process oxidized pyrimidines in DNA. We demonstrate that Ntg1 has OG-specific glycosylase/lyase activity indistinguishable from that of yOgg2. Targeted disruption of the NTG1 gene resulted in complete loss of yOgg2 activity and yeast lacking NTG1 had an elevated rate of A:T to C:G transversions. CONCLUSIONS: The Ntg1 and yOgg2 activities are encoded by a single gene. We propose that yOgg2 has evolved to process OG:A mis-pairs that have arisen through mis-incorporation of 8-oxo-dGTP during replication. Thus, the GO system in S. cerevisiae is fundamentally distinct from that in bacteria and mammals.

Affinity Labels↗

PubMed1998

Tyrosylprotein sulfotransferase: purification and molecular cloning of an enzyme that catalyzes tyrosine O-sulfation, a common posttranslational modification of eukaryotic proteins.

Tyrosine O-sulfation is a common posttranslational modification of proteins in all multicellular organisms. This reaction is mediated by a Golgi enzyme activity called tyrosylprotein sulfotransferase (TPST) that catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to tyrosine residues within acidic motifs of polypeptides. Tyrosine O-sulfation has been shown to be important in protein-protein interactions in several systems. For example, sulfation of tyrosine residues in the leukocyte adhesion molecule P-selectin glycoprotein ligand 1 (PSGL-1) is required for binding to P-selectin on activated endothelium. In this report we describe the purification of TPST from rat liver microsomes based on its affinity for the N-terminal 15 amino acids of PSGL-1. We have isolated human and mouse TPST cDNAs that predict type II transmembrane proteins of 370 amino acid residues with almost identical primary structure. The human cDNA encodes a fully functional N-glycosylated enzyme with an apparent molecular mass of approximately 54 kDa when expressed in mammalian cells. This enzyme defines a new class of Golgi sulfotransferases that may catalyze tyrosine O-sulfation of PSGL-1 and other protein substrates involved in diverse physiologic functions including inflammation and hemostasis.

Amino Acid Sequence↗

PubMed1998

Association of acyl-CoA synthetase-1 with GLUT4-containing vesicles.

GLUT4, the glucose transporter present in insulin-sensitive tissues, resides in intracellular vesicular structures and translocates to the cell surface in response to insulin. In an attempt to identify proteins present in these structures, GLUT4-enriched vesicles prepared from rat adipocytes treated with or without insulin were prepared by sucrose velocity gradient centrifugation and immunoadsorbed with anti-GLUT4 antibody. We report here the sequence identification by high performance liquid chromatography-ion trap mass spectrometry of a p75 protein band, long chain acyl-CoA synthetase-1, specifically present in immunoadsorbed GLUT4-containing vesicles but not in vesicles adsorbed by nonimmune serum. Acyl-CoA synthetase activity detected in GLUT4-enriched vesicles prepared by gradient centrifugation from insulin-treated adipocytes was decreased to about the same extent as GLUT4 protein. Additionally, immunoadsorbed GLUT4 vesicles were found to catalyze palmitoylation of proteins when incubated with labeled palmitate, a pathway that requires palmitate esterification with CoA. These data indicate that the insulin-sensitive membrane compartment that sequesters GLUT4 in fat cells contains long chain acyl-CoA synthetase-1 and its product fatty acyl-CoA, shown previously to be required for budding and fusion in membrane trafficking processes.

Acyl Coenzyme A↗

PubMed1998

Sortilin is the major 110-kDa protein in GLUT4 vesicles from adipocytes.

Vesicles containing the glucose transporter GLUT4 from rat adipocytes contain a major protein of 110 kDa. We have isolated this protein, obtained the sequences of peptides, and cloned a large portion of its cDNA. This revealed that the protein is sortilin, a novel membrane protein that was cloned in another context from a human source while this work was in progress. Subcellular fractionation of rat and 3T3-L1 adipocytes, together with GLUT4 vesicle isolation, showed that sortilin was primarily located in the low density microsomes in vesicles containing GLUT4. Insulin caused a 1.7-fold increase in the amount of sortilin at the plasma membranes of 3T3-L1 adipocytes, as assessed by cell surface biotinylation. The expression of sortilin in 3T3-L1 cells occurred only upon differentiation. Previous characterization of sortilin has led to the suggestion that it functions to sort lumenal proteins from the trans Golgi. The significance of its insulin-stimulated increase at the cell surface and of its expression upon differentiation will require definitive delineation of its function.

3T3 Cells↗

PubMed1998

DNA-dependent protein kinase phosphorylation of IkappaB alpha and IkappaB beta regulates NF-kappaB DNA binding properties.

Regulation of the IkappaB alpha and IkappaB beta proteins is critical for modulating NF-kappaB-directed gene expression. Both IkappaB alpha and IkappaB beta are substrates for cellular kinases that phosphorylate the amino and carboxy termini of these proteins and regulate their function. In this study, we utilized a biochemical fractionation scheme to purify a kinase activity which phosphorylates residues in the amino and carboxy termini of both IkappaB alpha and IkappaB beta. Peptide microsequence analysis by capillary high-performance liquid chromatography ion trap mass spectroscopy revealed that this kinase was the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PK phosphorylates serine residue 36 but not serine residue 32 in the amino terminus of IkappaB alpha and also phosphorylates threonine residue 273 in the carboxy terminus of this protein. To determine the biological relevance of DNA-PK phosphorylation of IkappaB alpha, murine severe combined immunodeficiency (SCID) cell lines which lack the DNA-PKcs gene were analyzed. Gel retardation analysis using extract prepared from these cells demonstrated constitutive nuclear NF-kappaB DNA binding activity, which was not detected in extracts prepared from SCID cells complemented with the human DNA-PKcs gene. Furthermore, IkappaB alpha that was phosphorylated by DNA-PK was a more potent inhibitor of NF-kappaB binding than nonphosphorylated IkappaB alpha. These results suggest that DNA-PK phosphorylation of IkappaB alpha increases its interaction with NF-kappaB to reduce NF-kappaB DNA binding properties.

3T3 Cells↗