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Biomedical subjects

W S Lane

Publications and source records attributed to W S Lane.

At least 109 records · Page 6Linked to original sources
PubMed1994

Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism.

Basal transcription of many genes in yeast is repressed by Mot1, an essential protein which is a member of the Snf2/Swi2 family of conserved nuclear factors. ADI is an ATP-dependent inhibitor of TATA-binding protein (TBP) binding to DNA that inhibits transcription in vitro. Here we demonstrate that ADI is encoded by the MOT1 gene. Mutation of MOT1 abolishes ADI activity and derepresses basal transcription in vitro and in vivo. Recombinant Mot1 removes TBP from DNA and Mot1 contains an ATPase activity which is essential for its function. Genetic interactions between Mot1 and TBP indicate that their functions are interlinked in vivo. These results provide a general model for understanding the mechanism of action of a large family of nuclear factors involved in processes such as transcription and DNA repair.

Adenosine Triphosphatases↗
PubMed1994

A mammalian protein targeted by G1-arresting rapamycin-receptor complex.

The structurally related natural products rapamycin and FK506 bind to the same intracellular receptor, FKBP12, yet the resulting complexes interfere with distinct signalling pathways. FKBP12-rapamycin inhibits progression through the G1 phase of the cell cycle in osteosarcoma, liver and T cells as well as in yeast, and interferes with mitogenic signalling pathways that are involved in G1 progression, namely with activation of the protein p70S6k (refs 5, 11-13) and cyclin-dependent kinases. Here we isolate a mammalian FKBP-rapamycin-associated protein (FRAP) whose binding to structural variants of rapamycin complexed to FKBP12 correlates with the ability of these ligands to inhibit cell-cycle progression. Peptide sequences from purified bovine FRAP were used to isolate a human cDNA clone that is highly related to the DRR1/TOR1 and DRR2/TOR2 gene products from Saccharomyces cerevisiae. Although it has not been previously demonstrated that either of the DRR/TOR gene products can bind the FKBP-rapamycin complex directly, these yeast genes have been genetically linked to a rapamycin-sensitive pathway and are thought to encode lipid kinases.

Amino Acid Sequence↗
PubMed1994

A serine kinase regulates intracellular localization of splicing factors in the cell cycle.

Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins) concentrate in 'speckles' in the nucleus of interphase cells. It is believed that nuclear speckles act as storage sites for splicing factors while splicing occurs on nascent transcripts. Splicing factors redistribute in response to transcription inhibition or viral infection, and nuclear speckles break down and reform as cells progress through mitosis. We have now identified and cloned a kinase, SRPK1, which is regulated by the cell cycle and is specific for SR proteins; this kinase is related to a Caenorhabditis elegans kinase and to the fission yeast kinase Dsk1 (ref. 7). SRPK1 specifically induces the disassembly of nuclear speckles, and a high level of SRPK1 inhibits splicing in vitro. Our results indicate that SRPK1 may have a central role in the regulatory network for splicing, controlling the intranuclear distribution of splicing factors in interphase cells, and the reorganization of nuclear speckles during mitosis.

Amino Acid Sequence↗
PubMed1994

Molecular cloning of an essential subunit of RNA polymerase II elongation factor SIII.

A transcription factor designated SIII was recently purified from mammalian cells and shown to regulate the activity of the RNA polymerase II elongation complex. SIII is a heterotrimer composed of approximately 110-, 18-, and 15-kDa polypeptides and is capable of increasing the overall rate of RNA chain elongation by RNA polymerase II by suppressing transient pausing of polymerase at multiple sites on the DNA template. Here we describe the molecular cloning and characterization of a cDNA encoding the functional 15-kDa subunit (p15) of SIII. The p15 cDNA encodes a 112-amino-acid polypeptide with a calculated molecular mass of 12,473 Da and an electrophoretic mobility indistinguishable from that of the natural p15 subunit. When combined with the 110- and 18-kDa SIII subunits, bacterially expressed p15 efficiently replaces the natural p15 subunit in reconstitution of transcriptionally active SIII. A homology search revealed that the amino-terminal half of the SIII p15 subunit shares significant sequence similarity with a portion of the RNA-binding domain of Escherichia coli transcription termination protein rho and with the E. coli NusB protein, suggesting that SIII may be evolutionarily related to proteins involved in the control of transcription elongation in eubacteria.

Amino Acid Sequence↗
PubMed1994

GTP-dependent binding of the antiproliferative agent didemnin to elongation factor 1 alpha.

The marine natural product, didemnin B, is a 7-amino acid, cyclic depsipeptide that inhibits G1 cell cycle progression at nanomolar concentrations by undefined mechanisms. It has been reported to exhibit immunosuppressive activities in animals and is undergoing clinical trials as a potential antineoplastic drug. In addition, at higher concentrations, didemnin B has been shown to inhibit in vivo and in vitro protein synthesis. However, the mechanisms by which inhibition is achieved are unknown. To investigate didemnin's various modes of action, an affinity column was synthesized and used to purify didemnin-binding proteins. The major retained protein was the 49-kDa guanine nucleotide-binding elongation factor, EF-1 alpha, which was identified by peptide sequence analysis. Moreover, didemnin binds EF-1 alpha only in the presence of GTP but does not inhibit the GTPase activity of EF-1 alpha. Therefore, EF-1 alpha is likely to be the intracellular target responsible for didemnin B's ability to inhibit protein synthesis. Furthermore, this specificity of didemnin affinity for the GTP-bound conformation of a guanine nucleotide-binding protein with homology to the Ras superfamily suggests a possible mode of action for didemnin's antiproliferative activity.

Amino Acid Sequence↗
PubMed1994

A subset of HLA-B27 molecules contains peptides much longer than nonamers.

An unusual monoclonal antibody (MARB4) directed against HLA-B27 that reacts with only approximately 5-20% of the cell surface HLA-B27 was used for large-scale purification of these molecules. Subsequent mass spectrometry of HLA-B27-bound peptides showed that the minor MARB4-reactive population contained peptides primarily from 900 to 4000 Da in size (approximately 8-33 amino acid residues), whereas the major HLA-B27 population contained peptides in the mass range of 900-1400 Da (approximately 8-12 amino acid residues). Thus, a subset of HLA-B27 molecules binds to peptides much longer than nonamers. Typical HLA-B27-binding peptides contain arginine in position 2. Further analysis by Edman sequencing of the pooled bound peptides revealed that the major population contained substantial amounts of arginine at positions 1 and 9 (40-50%) and exclusively arginine at position 2, as expected. The minor population of peptides also contained detectable amounts of arginine at these positions, but at the level of only approximately 10%; no marked enrichment at any position was observed. These long HLA-B27-bound peptides could represent either intermediates in the formation of nonamers or adventitiously bound peptides. Lastly, in the TAP2 mutant cell line BM36.1 transfected with HLA-B*2705, MARB4-reactive HLA-B27 molecules were absent from the cell surface, indicating that the peptide transporter was required for delivery of the long peptides. Thus, during the folding of class I heavy chains, peptides of diverse lengths are available and participating.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Mouse serum amyloid A protein (SAA5) structure and expression.

A novel member of the mouse serum amyloid A protein family, SAA5, has been identified as a normal apolipoprotein component of non-acute-phase high density lipoprotein (HDL). The structure of SAA5 was derived from a clone isolated from a normal Balb/c liver cDNA library. The clone predicts a pre-SAA5 molecule of 130 residues from which an 18-residue leader peptide is cleaved. The mature molecule has an octapeptide insert spanning from position 70 to 77. Similar inserts are found in human C-SAA and, paradoxically, in acute-phase SAA molecules of a number of other species. There is 48% amino acid identity between apo-SAA5 and the other mouse SAA proteins and 57% identity between the human C-SAA and apo-SAA5. The SAA5 mRNA is three times larger than previously identified SAA mRNAs. Although SAA5 is constitutively expressed in the liver, it has a rapid albeit muted response to inflammatory stimuli. The increase of SAA5 mRNA is due to increased transcription rather than mRNA stabilization. Plasma SAA5 levels during the acute phase are biphasic, either because of translational control or displacement from HDL and rapid clearance. We propose that constitutive SAAs (SAA5) on normal HDL contribute to its normal physiological role, whereas the dramatically inducible family members (SAA1, SAA2, SAA3) equip this particle for an altered functional role during inflammation.

Amino Acid Sequence↗
PubMed1994

Evidence for a non-alpha-helical DNA-binding motif in the Rel homology region.

The Rel family of transcription factors serve as terminal messengers in a variety of developmental and receptor-mediated signaling pathways. These proteins are related by a domain of approximately 280 amino acids, the Rel homology region, which mediates dimerization and sequence-specific binding to DNA. Here we report the use of photocrosslinking and site-directed mutagenesis to identify specific contact partners in a Rel protein-DNA interface. Within the Rel homology region of NF-kappa B p50 (also known as KBF1), two amino acid residues were identified by photocrosslinking to adjacent bases in a beta-interferon regulatory element. Secondary structure analysis suggests that the DNA-binding motif of the Rel homology region comprises a beta-turn-beta structure, in contrast to the alpha-helical motifs so commonly observed in transcription factors.

Amino Acid Sequence↗
PubMed1994

Molecular cloning of gefiltin (ON1): serial expression of two new neurofilament mRNAs during optic nerve regeneration.

The goldfish visual pathway displays a remarkable capacity for continued development and plasticity. The intermediate filament proteins of this pathway do not match the intermediate filament protein composition of adult higher vertebrate neurons, which lack the capacity for growth and development. Using a goldfish retina lambda gt10 library we isolated cDNA clones representing the predominant goldfish optic nerve neurofilament protein, ON1. The mRNA for this protein is abundant in retinal ganglion cells, and its level increases slowly during optic nerve regeneration. The rate of ON1 mRNA accumulation after optic nerve crush was compared with that of plasticin, a previously described novel type III neurofilament from goldfish retinal ganglion cells. Plasticin mRNA is normally expressed at low steady state levels, but accumulates dramatically and rapidly, preceding gefiltin mRNA, in response to optic nerve crush. The predicted amino acid sequence for ON1 indicates that it is a novel intermediate filament protein. We have named it gefiltin, for goldfish eye intermediate filament protein. The serial expression of plasticin and gefiltin is discussed with respect to the diversity of neurofilament proteins during neurogenesis.

Amino Acid Sequence↗
PubMed1994

DNA binding by an amino acid residue in the C-terminal half of the Rel homology region.

BACKGROUND: Members of the Rel family of transcription factors are important in regulating the inflammatory, acute phase, and immune responses in mammals. The structural basis for sequence-specific binding by Rel proteins is poorly understood, however. In the studies reported here, a new photoaffinity labeling procedure has been used to probe DNA contacts established by a Rel protein, the p50 homodimer of NF-kappa B. RESULTS: Using a novel post-synthetic modification method, 8-azido-2'-deoxyadenosine (N3dA), a photoactive analog of 2'-deoxyadenosine, was introduced at a specific site within a consensus DNA binding site for the NF-kappa B p50 homodimer. Upon irradiation with ultraviolet light, the N3dA-substituted DNA was efficiently photocrosslinked to p50. The crosslinked amino acid of p50 was identified as K244, which lies in the carboxy-terminal half of the Rel homology region (RHR). Mutation of K244 exerts strong effects on DNA binding, confirming the importance of this residue for p50-DNA interactions. CONCLUSIONS: We have used N3dA-containing DNA to identify a residue of NF-kappa B p50 that contacts DNA illustrating the value of this approach in studies of protein-DNA interactions. K244 appears not to contact a DNA base, but rather a phosphate moiety that lies nearby. The segment of protein sequence in which K244 resides has been implicated in dimerization. The results presented here suggest that the DNA-binding domain extends farther toward the carboxy-terminus than previously thought.

Amino Acids↗
PubMed1994

Self-peptides bound to the type I diabetes associated class II MHC molecules HLA-DQ1 and HLA-DQ8.

Genetic susceptibility to several autoimmune disorders is associated with the expression of certain MHC class II alleles. Insight into the etiology of such diseases awaits the identification of the class II restriction elements and the possible pathogenic peptides. Towards these aims, self-peptides bound to HLA-DQ1 and HLA-DQ8, allotypes considered to be neutral and permissive respectively towards the development of insulin-dependent diabetes mellitus, are reported. These naturally processed peptides were isolated from immunoaffinity purified HLA-DQ molecules expressed in cultured B lymphocytes. The chromatographic profiles of the peptide repertoires are unique, whereas the size distributions exhibit general similarity to those reported for naturally processed self-peptides bound to HLA-DR. Twenty-eight individual peptides representing 10 nested sets were identified by combined Edman microsequencing and mass spectrometry. Peptide length varied from 13 to 74 amino acids. Source proteins included MHC molecules and other integral membrane proteins, as well as secretory, cytosolic and mitochondrial proteins. Promiscuous invariant chain peptides were identified among the self-peptides bound to HLA-DQ8. No dominant amino acid markers suggestive of particular enzymatic processing events were detected. Some structural features of DQ1 and DQ8 that may relate to the bound peptides are discussed. Peptide specificity was confirmed in binding assays with purified HLA-DQ and HLA-DR protein.

Amino Acid Sequence↗
PubMed1994

Brassica napus plastid and mitochondrial chaperonin-60 proteins contain multiple distinct polypeptides.

Plastid chaperonin-60 protein was purified to apparent homogeneity from Brassica napus using a novel protocol. The purified protein, which migrated as a single species by nondenaturing polyacrylamide gel electrophoresis, contained four polypeptides: three variants of p60cpn60 alpha and p60cpn60 beta. Partial amino acid sequence determination demonstrated that each variant of p60cpn60 alpha is a distinct translation product. During this study, additional chaperonin-60 proteins were purified. These proteins, which were free from contaminating plastid chaperonin-60, were separated into at least two high molecular weight species that were resolved only by nondenaturing polyacrylamide gel electrophoresis. These proteins contained three 60-kD polypeptides. Two of these polypeptides were recognized by existing antisera, whereas the third was not. Partial amino acid sequence data revealed that each of these, including the immunologically distinct polypeptide, is a chaperonin-60 subunit of putative mitochondrial origin. The behavior of chaperonin-60 proteins during blue A Dyematrex chromatography suggests that this matrix may be generally useful for the identification of chaperonin-60 proteins.

Amino Acid Sequence↗
PubMed1994

pCyP B: a chloroplast-localized, heat shock-responsive cyclophilin from fava bean.

When the immunosuppressants cyclosporin A (CsA) and FK506 bind to their intracellular receptors (immunophillins), they form complexes that bind to calcineurin and block calcineurin-dependent signaling pathways in immune cells. Previously, we reported that higher plants also express immunophilins and have a Ca(2+)-dependent signaling pathway sensitive to immunophilin-ligand complexes. Based on an N-terminal peptide sequence of a chloroplast-localized cyclophilin (pCyP B), we isolated a cDNA clone encoding the preprotein of the cyclophilin. The deduced amino acid sequence of this cDNA starts with a putative transit sequence for chloroplast targeting. The mature pCyP B protein has rotamase activity with low-substrate specificity. Enzyme activity was inhibited by CsA with an inhibition constant of 3.9 nM. Similar to other CyPs from mammalian cells, pCyP B, when complexed with CsA, inhibited the phosphatase activity of bovine calcineurin. The mRNA level of pCyP B was high in leaf tissue but was not detectable in roots. Expression of the transcript in the leaf tissues was regulated by light and induced by heat shock. These findings illustrate the conserved nature of cyclophilin proteins among all of the eukaryotes and suggest that cyclophilins have a unique mode of regulation in higher plants.

Amino Acid Isomerases↗
PubMed1993

Isolation of the cyclosporin-sensitive T cell transcription factor NFATp.

Nuclear factor of activated T cells (NFAT) is a transcription factor that regulates expression of the cytokine interleukin-2 (IL-2) in activated T cells. The DNA-binding specificity of NFAT is conferred by NFATp, a phosphoprotein that is a target for the immunosuppressive compounds cyclosporin A and FK506. Here, the purification of NFATp from murine T cells and the isolation of a complementary DNA clone encoding NFATp are reported. A truncated form of NFATp, expressed as a recombinant protein in bacteria, binds specifically to the NFAT site of the murine IL-2 promoter and forms a transcriptionally active complex with recombinant protein fragment react with T cell NFATp. The molecular cloning of NFATp should allow detailed analysis of a T cell transcription factor that is central to initiation of the immune response.

Amino Acid Sequence↗
PubMed1993

DNA topoisomerase I is involved in both repression and activation of transcription.

Reconstituted transcription reactions containing the seven general transcription factors, in addition to RNA polymerase II, respond poorly to transcriptional activators. Two factors, Dr2 and ACF, necessary for high levels of transcription in response to an activator have been identified. ACF can enhance basal and activated transcription. Dr2 represses basal transcription, but this can be overcome by transcriptional activators or TFIIA. Dr2 is human DNA topoisomerase I. The DNA relaxation activity of topoisomerase I is dispensable for transcriptional repression. The effect of Dr2 is specific for TATA-box-containing promoters and is mediated by the TATA-binding protein.

Amino Acid Sequence↗
PubMed1993

Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles.

Naturally processed peptides were acid extracted from immunoaffinity-purified HLA-DR2, DR3, DR4, DR7, and DR8. Using the complementary techniques of mass spectrometry and Edman microsequencing, > 200 unique peptide masses were identified from each allele, ranging from 1,200 to 4,000 daltons (10-34 residues in length), and a total of 201 peptide sequences were obtained. These peptides were derived from 66 different source proteins and represented sets nested at both the amino- and carboxy-terminal ends with an average length of 15-18 amino acids. Strikingly, most of the peptides (> 85%) were derived from endogenous proteins that intersect the endocytic/class II pathway, even though class II molecules are thought to function mainly in the presentation of exogenous foreign peptide antigens. The predominant endogenous peptides were derived from major histocompatibility complex-related molecules. A few peptides derived from exogenous bovine serum proteins were also bound to every allele. Four prominent promiscuous self-peptide sets (capable of binding to multiple HLA-DR alleles) as well as 84 allele-specific peptide sets were identified. Binding experiments confirmed that the promiscuous peptides have high affinity for the binding groove of all HLA-DR alleles examined. A potential physiologic role for these endogenous self-peptides as immunomodulators of the cellular immune response is discussed.

Alleles↗
PubMed1993

Identification, by protein sequencing and gene transfection, of sgp-60 as the murine homologue of CD48.

We have recently described a murine lymphocyte protein, provisionally termed sgp-60, which is expressed on virtually all lymphocytes of both T- and B-cell origin. A hamster monoclonal antibody to sgp-60 can inhibit interleukin-2 (IL-2) production, IL-2-receptor expression, and T-cell proliferation, events normally observed after stimulation of T cells with an antibody to the T-cell receptor/CD3 complex or with the lectin concanavalin A. Our previous studies did not reveal the molecular nature of the sgp-60 antigen. Purification of sgp-60 and protein sequencing demonstrate that sgp-60 is identical to the CD48 antigen, a ligand for the CD2 antigen, which is also called Blast-1 in humans, BCM1 in mice, and OX-45 in rats. The identity of sgp-60 and CD48 was independently confirmed in gene transfection experiments. The anti-sgp-60 monoclonal antibody was selectively reactive with COS-7 cells transfected with a BCM1 cDNA clone but not with Kb-transfected controls. The results of the present report, together with our previous functional studies, may have implications for the role of CD2 and CD48 in murine T-cell activation.

Amino Acid Sequence↗