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Biomedical subjects

M N Hall

Publications and source records attributed to M N Hall.

15 recordsLinked to original sources
PubMed1992

A yeast cyclophilin gene essential for lactate metabolism at high temperature.

The cyclophilins are a family of ubiquitous eukaryotic proteins first identified by high affinity for cyclosporin A (CsA). The immunosuppressant and cytotoxic effects of CsA are thought to result from formation of a toxic complex between cyclophilin and CsA rather than from inhibition of cyclophilin function. The physiological role(s) of the cyclophilins is unknown. Cyclophilins have in vitro peptidylprolyl cistrans isomerase (PPIase) activity, and thus may be involved in protein folding in vivo. We have isolated a yeast cyclophilin gene, CPR3, which encodes a presumptive mitochondrial isoform. While CPR3 disruption mutants lack any phenotype at 30 degrees C, they are unable to grow on L-lactate at 37 degrees C. Disruptions of two other cyclophilin genes (CPR1, CPR2) and of FPR1, the gene encoding an FK506 binding protein with PPIase activity, do not affect growth on L-lactate at 37 degrees C. L-Lactate metabolism requires transcriptional induction of CYB2, the gene encoding flavocytochrome b2; cpr3 mutants induce transcription of this gene normally. This result demonstrates a conditional lethal phenotype for a cyclophilin mutation and presents a system for genetic and biochemical analysis of cyclophilin function.

Amino Acid Isomerases↗
PubMed1992

The T-DNA-linked VirD2 protein contains two distinct functional nuclear localization signals.

Agrobacterium tumefaciens causes neoplastic growth in plants by transferring a piece of DNA, called T-DNA, into the nucleus of the plant cell. The virulence protein VirD2 of A. tumefaciens is tightly linked to the T-DNA and is thought to direct it to the plant genome. Here we show that the VirD2 protein contains two nuclear localization signals that are functional both in yeast and in plant cells. One signal is located in the N-terminal part of the protein and resembles a single-cluster-type nuclear localization signal. The second signal is near the C terminus and is a bipartite-type nuclear localization signal. The involvement of these sequences in the entry of the T-DNA into the nucleus is discussed.

Amino Acid Sequence↗
PubMed1992

Proline isomerases at the crossroads of protein folding, signal transduction, and immunosuppression.

The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin block T-cell activation by interfering with signal transduction. The institution of CsA therapy for prophylaxis against graft rejection revolutionized human organ transplants, and clinical trials with FK506 and rapamycin are in progress. The targets for these drugs, cyclophilin for CsA and FKBP for FK506 and rapamycin, are members of two unrelated families of ubiquitous, highly conserved, abundant proteins. Although unrelated, both cyclophilin and FKBP catalyze proline isomerization and may fold proteins. The structures of both cyclophilin and FKBP have been determined, in some cases in complex with drugs or substrates. The cyclophilin-CsA and FKBP-FK506 complexes prevent T-cell response to antigen, bind and modulate the activity of the protein phosphatase calcineurin, and prevent nuclear import of a subunit of NF-AT, a T-cell activation transcription factor. In contrast, rapamycin blocks T-cell responses to IL-2. Yeast genetic studies suggest that the FKBP-rapamycin target is a protein complex involved in cell cycle progression. Further studies should provide fundamental insights into T-cell activation, signal transduction, and protein folding, and hold the promise of more specific immunosuppressive therapies.

Amino Acid Isomerases↗
PubMed1991

Nuclear import substrates compete for a limited number of binding sites. Evidence for different classes of yeast nuclear import receptors.

A nuclear receptor likely involved in nuclear protein import is described. Purified ATP-depleted yeast nuclei show saturable high-affinity binding of the yeast nuclear protein Mcm1. The dissociation constant for the binding is 0.5 microM, and the number of binding sites is approximately 3,500 per nucleus, equivalent to 10-30 binding sites per nuclear pore. Mcm1 competes with other yeast nuclear proteins Ste12 and Swi5, but not with Rap1 or Nop1, indicating that there may be different types of import receptors. Bound Mcm1 is resistant to extraction by nucleases, salt, and non-ionic detergent, but can be released by 5 M urea, suggesting that Mcm1 binds to a yeast equivalent of the nuclear pore complex-lamina fraction of higher eukaryotes.

Binding Sites↗
PubMed1991

Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast.

FK506 and rapamycin are related immunosuppressive compounds that block helper T cell activation by interfering with signal transduction. In vitro, both drugs bind and inhibit the FK506-binding protein (FKBP) proline rotamase. Saccharomyces cerevisiae cells treated with rapamycin irreversibly arrested in the G1 phase of the cell cycle. An FKBP-rapamycin complex is concluded to be the toxic agent because (i) strains that lack FKBP proline rotamase, encoded by FPR1, were viable and fully resistant to rapamycin and (ii) FK506 antagonized rapamycin toxicity in vivo. Mutations that conferred rapamycin resistance altered conserved residues in FKBP that are critical for drug binding. Two genes other than FPR1, named TOR1 and TOR2, that participate in rapamycin toxicity were identified. Nonallelic noncomplementation between FPR1, TOR1, and TOR2 alleles suggests that the products of these genes may interact as subunits of a protein complex. Such a complex may mediate nuclear entry of signals required for progression through the cell cycle.

Amino Acid Sequence↗
PubMed1991

FK 506-binding protein proline rotamase is a target for the immunosuppressive agent FK 506 in Saccharomyces cerevisiae.

FK 506 and cyclosporin A are potent immunosuppressive compounds that inhibit T-cell activation by interfering with signal transduction. In vitro, FK 506 binds and inhibits the activity of FK 506-binding protein (FKBP), a peptidylprolyl rotamase (cis-trans isomerase). Cyclosporin A acts similarly on a different proline rotamase, cyclophilin. Experiments described here demonstrate genetically that FKBP is a target for FK 506 in vivo. We have isolated the gene encoding the FKBP proline rotamase (FPR1) from Saccharomyces cerevisiae. The encoded yeast protein is highly homologous with bovine and human FKBP and shares no homology with cyclophilin. Disruption of FPR1 and CPR1 (encoding cyclophilin) individually or in combination is not lethal; thus, either enzymatic proline rotamerization is not essential for life or an unknown proline rotamase can substitute for the missing enzymes. Overexpression or disruption of FPR1 confers resistance to growth inhibition by FK 506, suggesting that FKBP is a target for FK 506 in yeast. However, FKBP is only one of at least two targets because strains lacking FKBP are only partially resistant to FK 506.

Amino Acid Sequence↗
PubMed1991

Yeast cell-free nuclear protein import requires ATP hydrolysis.

Saccharomyces cerevisiae nuclear proteins are shown to localize specifically to isolated yeast nuclei under conditions selective for nuclear proteins. Nuclear association is time- and temperature-dependent, requires ATP hydrolysis, and is abolished by protease pretreatment of nuclei. The nucleus-localized protein is translocated across the nuclear envelope as determined by inaccessibility to externally added immobilized protease. This cell-free system, consisting of components from an organism amenable to genetic analysis, will facilitate the study of the poorly understood mechanism of nuclear protein localization. The finding that ATP hydrolysis is required for nuclear import is the most direct evidence that nuclear localization is energy-dependent.

Adenosine Triphosphate↗
PubMed1990

Active transport of proteins into the nucleus.

Nuclear proteins are actively and posttranslationally transported across the nuclear envelope. This transport is a highly selective process that can be divided into two steps, receptor-binding followed by translocation through the nuclear envelope. Receptor-binding is mediated by nuclear localization signals that have been identified in many nuclear proteins. Translocation is energy-dependent and occurs through the nuclear pore complex.

Amino Acid Sequence↗
PubMed1990

Homeodomain of yeast repressor alpha 2 contains a nuclear localization signal.

The yeast repressor alpha 2 is shown, by analysis of deletion-bearing alpha 2-beta-galactosidase hybrid proteins, to have two structurally distinct nuclear localization signals. The cellular location of hybrid proteins was determined by indirect immunofluorescence and optical sectioning of whole fixed yeast cells. The two nuclear localization signals are far apart in the alpha 2 primary structure and do not have any sequence homology. One signal is, as reported previously, within the aminoterminal 13 amino acids of alpha 2. Deletion of only this aminoterminal signal has no evident effect on nuclear localization. The second signal is in a central portion of alpha 2, within the alpha 2 homeodomain. Since this signal is within the amino terminus of the alpha 2 homeodomain, the homeodomain mediates nuclear localization in addition to, and independently of, DNA binding. Deletion of only this second signal results in inefficient localization and accumulation of mutant protein at discrete sites on the nuclear envelope assumed to be nuclear pores. We propose that the two signals in alpha 2 are functionally distinct and act at different steps in a localization pathway.

Base Sequence↗
PubMed1987

Homeo domain of the yeast repressor alpha 2 is a sequence-specific DNA-binding domain but is not sufficient for repression.

The alpha 2 protein, the product of the MAT alpha 2 gene, is a regulator of cell type in the yeast Saccharomyces cerevisiae. It represses transcription of a group of cell type-specific genes by binding to an operator located upstream of each target gene. Fifteen in-frame deletions within the coding region of the MAT alpha 2 gene were constructed. The deletion alleles were examined for phenotypes conferred in vivo, and the encoded mutant proteins were assayed for ability to bind specifically to the operator in vitro. This analysis has revealed that the sequence-specific DNA-binding domain of alpha 2 is located within a region of 68 amino acids. This region of alpha 2 has significant homology with the homeo domain, a conserved sequence found in the products of several Drosophila homeotic and segmentation genes. In addition, there is a class of mutant alpha 2 proteins that binds tightly and specifically to the operator in vitro, but fails to repress transcription in vivo.

Base Sequence↗
PubMed1987

Signal sequence mutations that alter coupling of secretion and translation of an Escherichia coli outer membrane protein.

The lamB701-708 signal sequence mutation reduces expression of LamB, an outer membrane protein of Escherichia coli. To investigate the possibility that synthesis and export of LamB are coupled, as suggested by the expression defect of the lamB701-708 mutation, we isolated intragenic suppressors of the lamB701-708 mutation. The expression defect imposed by the lamB701-708 mutation is suppressed by an export-defective signal sequence mutation, suggesting that translation and export are coupled. The additional observation that not all export-defective signal sequence mutations suppressed the lamB701-708 expression defect suggests that translational arrest can be uncoupled from export.

Amino Acid Sequence↗
PubMed1979

Transcriptional regulation of Escherichia coli K-12 major outer membrane protein 1b.

Eleven independent insertion mutations were isolated that prevented expression of major outer membrane protein 1b. Seven of the mutations were Mucts insertions located at ombP. These ompB::Mucts strains fell into two phenotypic classes with regard to expression of proteins 1a and 1b. The remaining four mutants were comprised of one Tn5 and three Mucts insertions mapping at par. The Mucts insertions at par were used to construct fusions of the lac operon to the par promoter. Expression of beta-galactosidase in these fusion strains reflected known regulatory properties of protein 1b. When an ompB allele was introduced into the par-lac fusion strains, beta-galactosidase activity was reduced 14- to 31-fold. Transcriptional regulation of the par gene and the existence of two functions at ompB are discussed. The results suggest that par is the structural gene for protein 1b and that an ompB gene product is a diffusible, positive regulatory element controlling expression of par.

Bacterial Proteins↗
PubMedDate not supplied

Penetration of the pericardium by a gastric ulcer--survival after pericardiocentesis.

Elderly patients often have unusual manifestations of common illnesses. We describe a geriatric patient having a pneumopericardium from pericardial penetration by a gastric ulcer. Benign gastric ulceration causing perforation of the pericardium or other cardiac structures was recognized as early as 1854; however, until 1964, the condition was invariably fatal. Our patient survived the episode after early pericardiocentesis and medical treatment. Aspects of this uncommon clinical entity and a brief review of the literature are described.

Aged↗