Search PubMed⌕ Search

Biomedical subjects

J Heitman

Publications and source records attributed to J Heitman.

At least 109 records · Page 6Linked to original sources
PubMed1995

vph6 mutants of Saccharomyces cerevisiae require calcineurin for growth and are defective in vacuolar H(+)-ATPase assembly.

We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B). A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype. The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain. We demonstrate that cev1 is an allele of the VPH6 gene and that vph6 mutant strains fail to assemble the vacuolar H(+)-ATPase (V-ATPase). The VPH6 gene was mapped on chromosome VIII and is predicted to encode a 181-amino acid (21 kD) protein with no identity to other known proteins. We find that calcineurin is essential for viability in many mutant strains with defects in V-ATPase function or vacuolar acidification. In addition, we find that calcineurin modulates extracellular acidification in response to glucose, which we propose occurs via calcineurin regulation of the plasma membrane H(+)-ATPase PMA1. Taken together, our findings suggest calcineurin plays a general role in the regulation of cation transport and homeostasis.

Base Sequence↗
PubMed1995

Molecular mechanisms of immunosuppression by cyclosporine, FK506, and rapamycin.

The immunosuppressant cyclosporine A revolutionized treatment of graft rejection. Two newer agents, FK506 and rapamycin, show great clinical potential. These drugs suppress the immune system by forming protein-drug complexes that interact with and inhibit key components of the signal transduction pathways required for T-cell activation. The target of the cyclophilin A-cyclosporine A and FKBP12-FK506 complexes is calcineurin, a protein phosphatase required for signaling via the T-cell receptor. Cyclosporine A and FK506 nephrotoxicity may reflect renal-specific functions of calcineurin. The target of the FKBP12-rapamycin complex is TOR, a lipid and protein kinase homolog that is likely to be required for T-cell proliferation in response to interleukin-2. The identification of cyclosporine A, FK506, and rapamycin targets reveals much concerning T-cell signaling and provides the means to design novel immunosuppressants with reduced toxicity.

Animals↗
PubMed1994

Immunophilins interact with calcineurin in the absence of exogenous immunosuppressive ligands.

The peptidyl-prolyl isomerases FKBP12 and cyclophilin A (immunophilins) form complexes with the immunosuppressants FK506 and cyclosporin A that inhibit the phosphatase calcineurin. With the yeast two hybrid system, we detect complexes between FKBP12 and the calcineurin A catalytic subunit in both the presence and absence of FK506. Mutations in FKBP12 surface residues or the absence of the calcineurin B regulatory subunit perturb the FK506-dependent, but not the ligand-independent, FKBP12-calcineurin complex. By affinity chromatography, both FKBP12 and cyclophilin A bind calcineurin A in the absence of ligand, and FK506 and cyclosporin A respectively potentiate these interactions. Both in vivo and in vitro, the peptidyl-prolyl isomerase active sites are dispensable for ligand-independent immunophilin-calcineurin complexes. Lastly, by genetic analyses we demonstrate that FKBP12 modulates calcineurin functions in vivo. These findings reveal that immunophilins interact with calcineurin in the absence of exogenous ligands and suggest that immunosuppressants may take advantage of the inherent ability of immunophilins to interact with calcineurin.

Amino Acid Isomerases↗
PubMed1994

Calcineurin is essential in cyclosporin A- and FK506-sensitive yeast strains.

The immunophilin-immunosuppressant complexes cyclophilin-cyclosporin A (CsA) and FKBP12-FK506 inhibit the phosphatase calcineurin to block T-cell activation. Although cyclophilin A, FKBP12, and calcineurin are highly conserved from yeast to man, none had previously been shown to be essential for viability. We find that CsA-sensitive yeast strains are FK506 hypersensitive and demonstrate that calcineurin is required for viability in these strains. Mutants lacking cyclophilin A or FKBP12 are resistant to CsA or FK506, respectively. Thus, both the immunosuppressive and the antifungal actions of CsA and FK506 result from calcineurin inhibition by immunophilin-drug complexes. In yeast strains in which calcineurin is not essential, calcineurin inhibition or mutation of calcineurin confers hypersensitivity to LiCl or NaCl, suggesting that calcineurin regulates cation transport.

Amino Acid Isomerases↗
PubMed1993

Rapid freezing of whole blood or buffy coat samples for polymerase chain reaction and cell culture analysis: application to detection of human immunodeficiency virus in blood donor and recipient repositories. The Transfusion Safety Study Group.

Storage of lymphocytes for later use in prospective epidemiologic studies of blood donors and transfusion recipients has been limited by the cost of separating peripheral blood mononuclear cells (PBMCs). When the Transfusion Safety Study began in 1985, it was decided to establish a cell repository of cryopreserved buffy coat (BC) samples, and thus far over 20,000 samples have been accumulated from enrolled subjects. To determine if these specimens could be used for polymerase chain reaction, a simple thawing and pelleting technique for recovering hemoglobin-free total white cells (WBCs) was developed. To validate the technique, parallel analysis was conducted of BCs, whole blood (WB), and PBMC samples from human immunodeficiency virus type 1 (HIV-1)-seropositive subjects. Immediate postthaw cell courts of 29 frozen-thawed (F-T) WB and BC samples averaged 90 percent of the prefreeze (input) values. Representative WBC populations were obtained by immediate pelleting. Amplification of HIV-1 gag sequences from F-T BCs and F-T WB was 94 and 75 percent, respectively, which is as sensitive as that obtained with freshly separated PBMC lysates. Quantitative HIV-1 proviral load analysis by serial dilution of 23 F-T BCs and 8 WB lysates showed results comparable to those obtained with lysates of fresh PBMCs. Values for WBC differential and immunophenotyping could be applied to express viral load relative to total WBCs, PBMCs, or CD4+ cells. These results establish the basis for simplified virologic analysis of cryopreserved BC or WB specimens.

Blood Donors↗
PubMed1993

Tn5-mediated bleomycin resistance in Escherichia coli requires the expression of host genes.

The transposon Tn5 expresses a gene, ble, whose product increases the viability of Escherichia coli and also confers resistance to the DNA-cleaving antibiotic bleomycin and the DNA-alkylating agent ethylmethanesulphonate. We find that the Ble protein induces expression of an alkylation inducible gene, aidC, and that both the AidC gene product and DNA polymerase I are required for Ble to confer bleomycin resistance. These findings support models in which Ble enhances DNA repair and suggest that Tn5 confers a fitness advantage to the host bacterium by increasing the repair of spontaneous DNA lesions. Such co-operation between a transposon and its host suggests that Tn5 is a symbiotic rather than a selfish DNA element.

Bacterial Proteins↗
PubMed1993

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae.

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

Amino Acid Sequence↗
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

Allogeneic leukocytes but not therapeutic blood elements induce reactivation and dissemination of latent human immunodeficiency virus type 1 infection: implications for transfusion support of infected patients.

Various immunologic stimuli and heterologous viral regulatory elements have been shown to increase susceptibility to, and replication of, human immunodeficiency virus type 1 (HIV-1) in lymphocytes and monocytes in vitro. Transfusion of allogeneic blood components from heterologous donors constitutes a profound immunologic stimulus to the recipient, in addition to being a potential route of transmission of lymphotropic viral infections. To investigate the hypothesis that transfusions, and particularly those containing leukocytes, activate HIV-1 replication in infected recipient cells, we cocultured peripheral blood mononuclear cells (PBMC) from three anti-HIV-1-positive individuals with allogeneic donor PBMC, as well as partially purified populations of donor lymphocytes, monocytes, granulocytes, platelets, and red blood cells (RBC) and allogeneic cell-free plasma. Allogeneic PBMC induced a dose-related activation of HIV-1 expression in in vivo infected cells, followed by dissemination of HIV-1 to previously uninfected patient cells. Activation of HIV-1 replication was observed with donor lymphocytes, monocytes, and granulocytes, whereas no effect was seen with leukocyte-depleted RBC, platelets, or plasma (ie, therapeutic blood constituents). Allogeneic donor PBMC were also shown to upregulate HIV-1 expression in a "latently" infected cell line, and to increase susceptibility of heterologous donor PBMC to acute HIV-1 infection. Studies should be performed to evaluate whether transfusions of leukocyte-containing blood components accelerate HIV-1 dissemination and disease progression in vivo. If so, HIV-1-infected patients should be transfused as infrequently as possible and leukocyte-depleted (filtered) blood components should be used to avoid this complication.

Acquired Immunodeficiency Syndrome↗
PubMed1992

How the EcoRI endonuclease recognizes and cleaves DNA.

One popular recombinant DNA tool is the EcoRI endonuclease, which cleaves DNA at GAATTC sites and serves as a paradigm for sequence specific DNA-enzyme interactions. The recently revised X-ray crystal structure of an EcoRI-DNA complex reveals EcoRI employs novel DNA recognition motifs, a four alpha-helix bundle and two extended chains, which project into the major groove to contact substrate purines and pyrimidines. Interestingly, pyrimidine contacts had been predicted based on genetic and biochemical studies. Current work focuses on the EcoRI active site structure, enzyme and substrate conformational changes during catalysis, and host-restriction system interactions.

Allosteric Regulation↗
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↗