Biomedical subjects

W S Lane

Publications and source records attributed to W S Lane.

At least 91 records · Page 5Linked to original sources

PubMed1996

Identification of a group of cellular cofactors that stimulate the binding of RNA polymerase II and TRP-185 to human immunodeficiency virus 1 TAR RNA.

A double-stranded RNA structure transcribed from the HIV-1 long terminal repeat known as TAR is critical for increasing gene expression in response to the transactivator protein Tat. Two cellular factors, RNA polymerase II and TRP-185, bind specifically to TAR RNA, but require the presence of cellular proteins known as cofactors which by themselves are unable to bind to TAR RNA. In an attempt to determine the mechanism by which these cofactors stimulate binding to TAR RNA, we purified these factors from HeLa nuclear extract and amino acid microsequence analysis performed. Three proteins were identified in the cofactor fraction including two previously described proteins, elongation factor 1alpha (EF-1alpha) and the polypyrimidine tract-binding protein (PTB), and a novel protein designated the stimulator of TAR RNA-binding proteins (SRB). SRB has a high degree of homology with a variety of cellular proteins known as chaperonins. Recombinant EF-1alpha, PTB, and SRB produced from vaccinia expression vectors stimulated the binding of RNA polymerase II and TRP-185 to TAR RNA in gel retardation analysis. These studies define a group of cellular factors that function in concert to stimulate the binding of TRP-185 and RNA polymerase II to HIV-1 TAR RNA.

Amino Acid Sequence↗

PubMed1996

Recombinant NFAT1 (NFATp) is regulated by calcineurin in T cells and mediates transcription of several cytokine genes.

Transcription factors of the NFAT family play a key role in the transcription of cytokine genes and other genes during the immune response. We have identified two new isoforms of the transcription factor NFAT1 (previously termed NFATp) that are the predominant isoforms expressed in murine and human T cells. When expressed in Jurkat T cells, recombinant NFAT1 is regulated, as expected, by the calmodulin-dependent phosphatase calcineurin, and its function is inhibited by the immunosuppressive agent cyclosporin A (CsA). Transactivation by recombinant NFAT1 in Jurkat T cells requires dual stimulation with ionomycin and phorbol 12-myristate 13-acetate; this activity is potentiated by coexpression of constitutively active calcineurin and is inhibited by CsA. Immunocytochemical analysis indicates that recombinant NFAT1 localizes in the cytoplasm of transiently transfected T cells and translocates into the nucleus in a CsA-sensitive manner following ionomycin stimulation. When expressed in COS cells, however, NFAT1 is capable of transactivation, but it is not regulated correctly: its subcellular localization and transcriptional function are not affected by stimulation of the COS cells with ionomycin and phorbol 12-myristate 13-acetate. Recombinant NFAT1 can mediate transcription of the interleukin-2, interleukin-4, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor promoters in T cells, suggesting that NFAT1 contributes to the CsA-sensitive transcription of these genes during the immune response.

Amino Acid Sequence↗

PubMed1995

The cellular factor TRP-185 regulates RNA polymerase II binding to HIV-1 TAR RNA.

Activation of HIV-1 gene expression by the transactivator Tat is dependent on an RNA regulatory element located downstream of the transcription initiation site known as TAR. To characterize cellular factors that bind to TAR RNA and are involved in the regulation of HIV-1 transcription, HeLa nuclear extract was fractionated and RNA gel-retardation analysis was performed. This analysis indicated that only two cellular factors, RNA polymerase II and the previously characterized TAR RNA loop binding protein TRP-185, were capable of binding specifically to TAR RNA. To elucidate the function of TRP-185, it was purified from HeLa nuclear extract, amino acid microsequence analysis was performed and a cDNA encoding TRP-185 was isolated. TRP-185 is a novel protein of 1621 amino acids which contains a leucine zipper and potentially a novel RNA binding motif. In gel-retardation assays, the binding of both recombinant TRP-185 and RNA polymerase II was dependent on the presence of an additional group of proteins designated cellular cofactors. Both the TAR RNA loop and bulge sequences were critical for RNA polymerase II binding, while TRP-185 binding was dependent only on TAR RNA loop sequences. Since binding of TRP-185 and RNA polymerase II to TAR RNA was found to be mutually exclusive, our results suggest that TRP-185 may function either alone or in conjunction with Tat to disengage RNA polymerase II which is stalled upon binding to nascently synthesized TAR RNA during transcriptional elongation.

Amino Acid Sequence↗

PubMed1995

Glial hyaluronate-binding protein: a product of metalloproteinase digestion of versican?

Glial hyaluronate-binding protein (GHAP) is a 60 kDa glycoprotein with an amino acid sequence identical to that of the hyaluronate-binding region of versican, a large fibroblast aggregating proteoglycan found in the brain. Both GHAP and versican were identified by immunoblot in bovine brain extracts prepared only minutes after death. Human recombinant collagenase, stromelysin, mouse gelatinase and gelatinases isolated from human brain by affinity chromatography digest versican and give rise to a polypeptide with electrophoretic mobility identical to GHAP. Immunoblot analysis, peptide mapping and C-terminal amino acid sequencing indicate that the polypeptide generated by digestion with human brain gelatinases is identical to GHAP. We suggest that GHAP is a naturally occurring versican degradation product.

Amino Acid Sequence↗

PubMed1995

Yeast global transcriptional regulators Sin4 and Rgr1 are components of mediator complex/RNA polymerase II holoenzyme.

Sin4 and Rgr1 proteins, previously shown by genetic studies to play both positive and negative roles in the transcriptional regulation of many genes, are identified here as components of mediator and RNA polymerase II holoenzyme complexes. Results with Sin4 deletion and Rgr1 truncation strains indicate the association of these proteins in a subcomplex comprising Sin4, Rgr1, Gal11, and a 50-kDa polypeptide. Taken together with the previous genetic evidence, our findings point to a role of the mediator in repression as well as in transcriptional activation.

Base Sequence↗

PubMed1995

Role of IRS-2 in insulin and cytokine signalling.

The protein IRS-1 acts as an interface between signalling proteins with Src-homology-2 domains (SH2 proteins) and the receptors for insulin, IGF-1, growth hormone, several interleukins (IL-4, IL-9, IL-13) and other cytokines. It regulates gene expression and stimulates mitogenesis, and appears to mediate insulin/IGF-1-stimulated glucose transport. Thus, survival of the IRS-1-/- mouse with only mild resistance to insulin was surprising. This dilemma is provisionally resolved with our discovery of a second IRS-signalling protein. We purified and cloned a likely candidate called 4PS from myeloid progenitor cells and, because of its resemblance to IRS-1, we designate it IRS-2. Alignment of the sequences of IRS-2 and IRS-1 revealed a highly conserved amino terminus containing a pleckstrin-homology domain and a phosphotyrosine-binding domain, and a poorly conserved carboxy terminus containing several tyrosine phosphorylation motifs. IRS-2 is expressed in many cells, including tissues from IRS-1-/- mice, and may be essential for signalling by several receptor systems.

Amino Acid Sequence↗

PubMed1995

Elongin (SIII): a multisubunit regulator of elongation by RNA polymerase II.

The Elongin (SIII) complex activates elongation by mammalian RNA polymerase II by suppressing transient pausing of the polymerase at many sites within transcription units. Elongin is a heterotrimer composed of A, B, and C subunits of 110, 18, and 15 kilodaltons, respectively. Here, the mammalian Elongin A gene was isolated and expressed, and the Elongin (SIII) complex reconstituted with recombinant subunits. Elongin A is shown to function as the transcriptionally active component of Elongin (SIII) and Elongin B and C as regulatory subunits. Whereas Elongin C assembles with Elongin A to form an AC complex with increased specific activity, Elongin B, a member of the ubiquitin-homology gene family, appears to serve a chaperone-like function, facilitating assembly and enhancing stability of the Elongin (SIII) complex.

Amino Acid Sequence↗

PubMed1995

Positive regulation of general transcription factor SIII by a tailed ubiquitin homolog.

General transcription factor SIII, a heterotrimer composed of 110-kDa (p110), 18-kDa (p18), and 15-kDa (p15) subunits, increases the catalytic rate of transcribing RNA polymerase II by suppressing transient pausing by polymerase at multiple sites on DNA templates. Here we report molecular cloning and biochemical characterization of the SIII p18 subunit, which is found to be a member of the ubiquitin homology (UbH) gene family and functions as a positive regulatory subunit of SIII. p18 is a 118-amino acid protein composed of an 84-residue N-terminal UbH domain fused to a 34-residue C-terminal tail. Mechanistic studies indicate that p18 activates SIII transcriptional activity above a basal level inherent in the SIII p110 and p15 subunits. Taken together, these findings establish a role for p18 in regulating the activity of the RNA polymerase II elongation complex, and they bring to light a function for a UbH domain protein in transcriptional regulation.

Amino Acid Sequence↗

PubMed1995

Naturally processed peptides from two disease-resistance-associated HLA-DR13 alleles show related sequence motifs and the effects of the dimorphism at position 86 of the HLA-DR beta chain.

HLA-DR13 has been associated with resistance to two major infectious diseases of humans. To investigate the peptide binding specificity of two HLA-DR13 molecules and the effects of the Gly/Val dimorphism at position 86 of the HLA-DR beta chain on natural peptide ligands, these peptides were acid-eluted from immunoaffinity-purified HLA-DRB1*1301 and -DRB1*1302, molecules that differ only at this position. The eluted peptides were subjected to pool sequencing or individual peptide sequencing by tandem MS or Edman microsequencing. Sequences were obtained for 23 peptides from nine source proteins. Three pool sequences for each allele and the sequences of individual peptides were used to define binding motifs for each allele. Binding specificities varied only at the primary hydrophobic anchor residue, the differences being a preference for the aromatic amino acids Tyr and Phe in DRB1*1302 and a preference for Val in DRB1*1301. Synthetic analogues of the eluted peptides showed allele specificity in their binding to purified HLA-DR, and Ala-substituted peptides were used to identify the primary anchor residues for binding. The failure of some peptides eluted from DRB1*1302 (those that use aromatic amino acids as primary anchors) to bind to DRB1*1301 confirmed the different preferences for peptide anchor residues conferred by the Gly-->Val change at position 86. These data suggest a molecular basis for the differential associations of HLA-DRB1*1301 and DRB1*1302 with resistance to severe malaria and clearance of hepatitis B virus infection.

Alleles↗

PubMed1995

Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin.

Lactacystin is a Streptomyces metabolite that inhibits cell cycle progression and induces neurite outgrowth in a murine neuroblastoma cell line. Tritium-labeled lactacystin was used to identify the 20S proteasome as its specific cellular target. Three distinct peptidase activities of this enzyme complex (trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing activities) were inhibited by lactacystin, the first two irreversibly and all at different rates. None of five other proteases were inhibited, and the ability of lactacystin analogs to inhibit cell cycle progression and induce neurite outgrowth correlated with their ability to inhibit the proteasome. Lactacystin appears to modify covalently the highly conserved amino-terminal threonine of the mammalian proteasome subunit X (also called MB1), a close homolog of the LMP7 proteasome subunit encoded by the major histocompatibility complex. This threonine residue may therefore have a catalytic role, and subunit X/MB1 may be a core component of an amino-terminal-threonine protease activity of the proteasome.

Acetylcysteine↗

PubMed1995

The MHC class I homolog encoded by human cytomegalovirus binds endogenous peptides.

The ability of a human cytomegalovirus-encoded homolog of MHC class I molecules to serve as a peptide receptor was investigated. Sequencing of peptide material eluted from the purified viral protein revealed a mixture of endogenous peptides with characteristics similar to those eluted from conventional class I molecules, that is, anchor residues, and a predominance of short peptides derived from cytoplasmic proteins. The possible function(s) of this viral MHC homolog are discussed in light of the finding that it binds endogenous peptides.

Amino Acid Sequence↗

PubMed1995

The pancornulins: a group of small proline rich-related cornified envelope precursors with bifunctional capabilities in isopeptide bond formation.

In this report, the pancornulins are identified as members of the spr (small, proline-rich) multigene family by amino acid sequence and mass spectrometry analyses. One of the pancornulins (14.9 kDa) is identical to the protein predicted by spr-1 clone 128. The other pancornulins (16.9 kDa and 22 kDa) are novel members of the spr family. Immunoelectron microscopy of purified cornified envelopes with a pancornulin-specific antibody established these proteins more definitively as cornified envelope precursors. In addition, two-dimensional electrophoretic analyses of keratinocyte extracts labeled enzymatically with dansylcadaverine (to identify amine acceptors) or dansylPGGQQIV (to identify amine donors) showed that both glutamine and lysine residues within the pancornulins participate in the isopeptide linkage characteristic of cornified envelope formation. These results contrasted with those obtained using involucrin, a prominent cornified envelope protein shown capable of acting only as an amine acceptor in this system. Novel partial cDNAs obtained after reverse transcription and polymerase chain reaction amplification of total messenger RNA with pancornulin-specific primers suggest that the spr multigene family may be even larger than previously described. The bifunctional reactivity of the pancornulins in cross-linking and the large number of family members identified to date suggest that the pancornulins and other spr-1-related proteins may be more important in cornified envelope formation than previously considered, perhaps functioning as "bridge" molecules during the early phases of cornified envelope assembly.

Amino Acid Sequence↗

PubMed1995

MG-160, a membrane sialoglycoprotein of the medial cisternae of the rat Golgi apparatus, binds basic fibroblast growth factor and exhibits a high level of sequence identity to a chicken fibroblast growth factor receptor.

We report the primary structure of MG-160, a 160 kDa membrane sialoglycoprotein residing in the medial cisternae of the Golgi apparatus of rat neurons, pheochromocytoma (PC-12), and several other cells. The cDNA encodes a polypeptide of 1,171 amino acids with an M(r) of 133,403. An intralumenal cleavable signal peptide is followed by a Pro-Gln-rich segment and 16 contiguous, approx. 60-residue-long, regularly spaced cysteine-rich segments showing sequence identities ranging from 15 to 35%. The lumenal domain is followed by a single membrane spanning domain and a short carboxy-terminal cytoplasmic tail. The protein contains 5 potential NXT glycosylation sites. The sequence of MG-160 shows no homologies with enzymes and other membrane proteins of the Golgi apparatus. MG-160 displays a so far unique feature for a membrane protein of the Golgi apparatus: namely, an upstream, open reading frame (uORF), encoding 58 amino acids, located in front of the major open reading frame (ORF). Most vertebrate mRNAs containing uORF or AUG codons in front of the major ORF encode growth factors and cell surface receptors (Geballe and Morris 1994). In that regard a 90% identity between the primary structure of MG-160 and a receptor for acidic and basic fibroblast growth factors (CFR), isolated from chicken embryos (Burrus et. al., 1992), may be relevant. Immunoreactivity for MG-160 has been detected in the Golgi apparatus of neural and other cells of 2-day-old chicken embryos and adult chicken; furthermore, recombinant human basic fibroblast growth factor (bFGF) binds MG-160 purified from rat brain. MG-160 shows no sequence similarity with members of the family of fibroblast growth factor receptors (FGFR) involved in signal transduction. These findings are consistent with the hypothesis that MG-160 is involved in the traffic and processing of endogenous or autocrine FGFs. This is the first example of an intrinsic membrane protein of the Golgi apparatus which binds a growth factor and may be involved in its regulation.

Amino Acid Sequence↗

PubMed1995

Neuron-specific enolase: a neuronal survival factor in the retinal extracellular matrix?

To identify soluble proteins of the retinal interphotoreceptor matrix (IPM), we isolated IPM from the bovine eye by gentle lavage and subjected it to SDS-PAGE. In the resultant gel, a 46 kDa band was particularly prominent and appeared to be a single protein. This protein was electroblotted to nitrocellulose membrane, digested with trypsin, and selected peptides were isolated by HPLC and subjected to Edman microsequencing. The amino acid sequences of the peptides were found to be virtually identical to that of human neuron-specific enolase (NSE). A monoclonal antibody specific for human NSE confirmed the presence of this enzyme in the bovine IPM by both Western blotting and immunocytochemical analysis. Immunofluorescence microscopy demonstrated that NSE is mainly localized to the basal domain of the IPM surrounding photoreceptor cells but is also prominent in the inner segments of the cone photoreceptor neurons. When NSE was added to cultures of human retinoblastoma cells, no effect on morphology was observed. However, a positive effect on cell growth and/or survival was readily apparent. It thus seems that not only is NSE a significant component of the retinal extracellular matrix, but that it could function as a survival (neuronotrophic) factor for photoreceptor neurons.

Amino Acid Sequence↗

PubMed1994

Active-site labeling of an aminoglycoside antibiotic phosphotransferase (APH(3')-IIIa).

The aminoglycoside antibiotics are inactivated by modifying enzymes that are now widely distributed in many pathogenic bacteria. This situation threatens the continued use of these clinically important drugs. We have undertaken studies to understand the molecular mechanism of aminoglycoside resistance, and we report the affinity labeling of the enterococcal aminoglycoside 3'-phosphotransferase, APH(3')-IIIa, with an electrophilic ATP analogue, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA). Incubation of purified APH(3')-IIIa with FSBA resulted in time-dependent irreversible inactivation of enzyme activity with a binding constant, Ki, of 0.406 mM and a rate of maximal inactivation, kmax, of 0.086 min-1. Addition of ATP completely protected the enzyme from inactivation, consistent with labeling of the ATP binding site. Reaction of APH(3')-IIIa with [14C]FSBA showed that inactivated APH(3')-IIIa incorporates 1 mol of FSBA/mol of enzyme. Peptide mapping of FSBA-inactivated APH(3')-IIIa resulted in the identification of two peptide peaks with highly increased absorbance at 260 nm, indicative of covalent labeling with FSBA. Analysis by electrospray ionization mass spectrometry and Edman degradation revealed two tryptic peptides, Val31-Lys44 and Leu34-Arg49, which incorporated the FSBA label at Lys33 and Lys44, respectively. This establishes the importance of the N-terminal region of APHs in ATP binding, a region of these enzymes which has heretofore not been considered for involvement in substrate binding.

Adenosine↗

PubMed1994

Interaction of poliovirus polypeptide 3CDpro with the 5' and 3' termini of the poliovirus genome. Identification of viral and cellular cofactors needed for efficient binding.

Poliovirus proteinase 3CDpro by itself is not an RNA-binding protein. Two cellular proteins have been purified from HeLa cells (p50 and p36) which interact with purified 3CDpro but only p36-3CDpro bind to the 5'-terminal 110 nucleotides of polioviral RNA genome, an RNA segment whose secondary structure resembles a cloverleaf. The identity of these factors was determined by microsequencing tryptic digests of the purified proteins. Host protein p50 is the eukaryotic elongation factor EF-1 alpha, and p36 an N-terminal fragment thereof. p36, referred to as host factor, did not appear to interact with purified 3Cpro or 3Dpol. Significantly, the formation of a 3CDpro-cloverleaf complex was also observed in the presence of purified poliovirus polypeptide 3AB, the precursor of VPg. 3AB by itself does not stably bind to the cloverleaf. Competition experiments have demonstrated that the RNA-protein interactions are specific for the full-length cloverleaf. UV cross-linking studies were employed to examine the protein components of the cloverleaf ribonucleoproteins. RNA footprinting was used to determine the site on the cloverleaf where the viral and cellular factors bind. Finally, we have discovered that 3AB-3CDpro also interacts with the 3'-terminal sequence of poliovirus RNA. In contrast to the 5'-terminal cloverleaf, the 3'-terminal RNA can bind 3AB in the absence of other proteins. A model for initiation of poliovirus RNA synthesis is presented.

3C Viral Proteases↗

PubMed1994

Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma.

The phenomenon of inhibition of tumor growth by tumor mass has been repeatedly studied, but without elucidation of a satisfactory mechanism. In our animal model, a primary tumor inhibits its remote metastases. After tumor removal, metastases neovascularize and grow. When the primary tumor is present, metastatic growth is suppressed by a circulating angiogenesis inhibitor. Serum and urine from tumor-bearing mice, but not from controls, specifically inhibit endothelial cell proliferation. The activity copurifies with a 38 kDa plasminogen fragment that we have sequenced and named angiostatin. A corresponding fragment of human plasminogen has similar activity. Systemic administration of angiostatin, but not intact plasminogen, potently blocks neovascularization and growth of metastases. We here show that the inhibition of metastases by a primary mouse tumor is mediated, at least in part, by angiostatin.

Amino Acid Sequence↗

PubMed1994

Kinetics, stoichiometry, and identification of the reactive thiolate in the inactivation of UDP-GlcNAc enolpyruvoyl transferase by the antibiotic fosfomycin.

Fosfomycin [(1R,2S)-1,2-epoxypropylphosphonic acid] has been shown to exert its antibiotic effect through the inhibition of UDP-GlcNAc enolpyruvoyl transferase [Kahan, F. M., et al. (1974) Ann. N.Y. Acad. Sci. 235, 364], the enzyme responsible for catalyzing the first committed step in bacterial cell wall biosynthesis. Time-dependent inactivation of MurZ by fosfomycin was found to be greatly accelerated by the presence of cosubstrate UDP-GlcNAc but could also be speeded appreciably by the unreactive substrate analog 3-deoxy-UDP-GlcNAc. These results argue against a reaction-based participation of the cosubstrate and suggest that UDP-GlcNAc has a role in influencing active site conformation critical to the inactivation event. A study of the influence of UDP-GlcNAc and fosfomycin on the kinetics of inactivation allowed the determination of dissociation constants for fosfomycin (KF = 8.6 microM) and UDP-GlcNAc (KS = 14 microM), in addition to a limiting inactivation rate constant (k(inact) = 7.4 min-1) at saturating UDP-GlcNAc and fosfomycin concentrations. Mass spectrometry of inactivated MurZ demonstrated an increase in molecular weight of 138, consistent with the covalent addition of a molar equivalent of fosfomycin (136 kDa). Titration of MurZ with fosfomycin revealed a stoichiometry of 1 molecule of inhibitor per active site when assessed using either enzyme activity or mass spectrometry as an index of modification. Peptide mapping of tryptic digests of fosfomycin-inactivated MurZ revealed modification of a unique 41-mer, the sequence of which revealed that Cys115 was the site of attachment of fosfomycin.

Alkyl and Aryl Transferases↗