Biomedical subjects

J Heitman

Publications and source records attributed to J Heitman.

At least 91 records · Page 5Linked to original sources

PubMed1997

STT4 is an essential phosphatidylinositol 4-kinase that is a target of wortmannin in Saccharomyces cerevisiae.

Wortmannin is a natural product that inhibits signal transduction. One target of wortmannin in mammalian cells is the 110-kDa catalytic subunit of phosphatidylinositol 3-kinase (PI 3-kinase). We show that wortmannin is toxic to the yeast Saccharomyces cerevisiae and present genetic and biochemical evidence that a phosphatidylinositol 4-kinase (PI 4-kinase), STT4, is a target of wortmannin in yeast. In a strain background in which stt4 mutants are rescued by osmotic support with sorbitol, the toxic effects of wortmannin are similarly prevented by sorbitol. In contrast, in a different strain background, STT4 is essential under all conditions and wortmannin toxicity is not mitigated by sorbitol. Overexpression of STT4 confers wortmannin resistance, but overexpression of PIK1, a related PI 4-kinase, does not. In vitro, the PI 4-kinase activity of STT4, but not of PIK1, was potently inhibited by wortmannin. Overexpression of the phosphatidylinositol 4-phosphate 5-kinase homolog MSS4 conferred wortmannin resistance, as did deletion of phospholipase C-1. These observations support a model for a phosphatidylinositol metabolic cascade involving STT4, MSS4, and phospholipase C-1 and provide evidence that an essential product of this pathway is the lipid phosphatidylinositol 4,5-bisphosphate.

1-Phosphatidylinositol 4-Kinase↗

PubMed1997

Cyclophilin active site mutants have native prolyl isomerase activity with a protein substrate.

The prolyl isomerase activity of cyclophilins is traditionally measured by an assay in which prolyl cis/trans isomerization in a chromogenic tetrapeptide is coupled with its isomer-specific cleavage by chymotrypsin. Two variants of mitochondrial cyclophilin with substitutions in the presumed active site (R73A and H144Q) are inactive in the protease-coupled assay, but show almost wild-type activity in an assay that is based on the catalysis of a proline-limited protein folding reaction. This prolyl isomerase assay is preferable, both because coupling with proteolysis is avoided and because an intact protein instead of a short peptide is used as a substrate. Possibly, some earlier conclusions about the catalytic mechanism and the involvement of the prolyl isomerase activity in the cellular function of immunophilins may need reevaluation.

Amino Acid Isomerases↗

PubMed1997

Calcineurin is required for virulence of Cryptococcus neoformans.

Cyclosporin A (CsA) and FK506 are antimicrobial, immunosuppressive natural products that inhibit signal transduction. In T cells and Saccharomyces cerevisiae, CsA and FK506 bind to the immunophilins cyclophilin A and FKBP12 and the resulting complexes inhibit the Ca2+-regulated protein phosphatase calcineurin. We find that growth of the opportunistic fungal pathogen Cryptococcus neoformans is sensitive to CsA and FK506 at 37 degrees C but not at 24 degrees C, suggesting that CsA and FK506 inhibit a protein required for C. neoformans growth at elevated temperature. Genetic evidence supports a model in which immunophilin-drug complexes inhibit calcineurin to prevent growth at 37 degrees C. The gene encoding the C. neoformans calcineurin A catalytic subunit was cloned and disrupted by homologous recombination. Calcineurin mutant strains are viable but do not survive in vitro conditions that mimic the host environment (elevated temperature, 5% CO2 or alkaline pH) and are no longer pathogenic in an animal model of cryptococcal meningitis. Introduction of the wild-type calcineurin A gene complemented these growth defects and restored virulence. Our findings demonstrate that calcineurin is required for C. neoformans virulence and may define signal transduction elements required for fungal pathogenesis that could be targets for therapeutic intervention.

Amino Acid Sequence↗

PubMed1997

Functions of FKBP12 and mitochondrial cyclophilin active site residues in vitro and in vivo in Saccharomyces cerevisiae.

Cyclophilin and FK506 binding protein (FKBP) accelerate cis-trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5-20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 "active-site" mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.

Binding Sites↗

PubMed1997

The immunosuppressant FK506 and its nonimmunosuppressive analog L-685,818 are toxic to Cryptococcus neoformans by inhibition of a common target protein.

The immunosuppressant FK506 (tacrolimus) is an antifungal natural product macrolide that suppresses the immune system by blocking T-cell activation. In complex with the intracellular protein FKBP12, FK506 inhibits calcineurin, a Ca(2+)-calmodulin-dependent serine-threonine protein phosphatase. We recently reported that growth of the opportunistic fungal pathogen Cryptococcus neoformans is resistant to FK506 at 24 degrees C but sensitive at 37 degrees C and that calcineurin, the target of FKBP12-FK506, is required for growth at 37 degrees C in vitro and pathogenicity in vivo. These findings identify calcineurin as a potential antifungal drug target. In previous studies the calcineurin inhibitor cyclosporin A (CsA) was effective against murine pulmonary infections but exacerbated cryptococcal meningitis in rabbits and mice, likely because CsA does not cross the blood-brain barrier. Although we find that FK506 penetrates the CNS, FK506 also exacerbates cryptococcal meningitis in rabbits. Thus, FK506 immunosuppression outweighs antifungal action in vivo. Like FK506, the nonimmunosuppressive FK506 analog L-685,818 is toxic to C. neoformans in vitro at 37 degrees C but not at 24 degrees C, and FK506-resistant mutants are resistant to L-685,818, indicating a similar mechanism of action. Fluconazole-resistant C. neoformans clinical isolates were also found to be susceptible to both FK506 and L-685,818. Our findings identify calcineurin as a novel antifungal drug target and suggest the nonimmunosuppressive FK506 analog L-685,818 or other congeners warrant further consideration as antifungal drugs for C. neoformans.

Animals↗

PubMed1997

FKBP12 physically and functionally interacts with aspartokinase in Saccharomyces cerevisiae.

The peptidyl-prolyl isomerase FKBP12 was originally identified as the intracellular receptor for the immunosuppressive drugs FK506 (tacrolimus) and rapamycin (sirolimus). Although peptidyl-prolyl isomerases have been implicated in catalyzing protein folding, the cellular functions of FKBP12 in Saccharomyces cerevisiae and other organisms are largely unknown. Using the yeast two-hybrid system, we identified aspartokinase, an enzyme that catalyzes an intermediate step in threonine and methionine biosynthesis, as an in vivo binding target of FKBP12. Aspartokinase also binds FKBP12 in vitro, and drugs that bind the FKBP12 active site, or mutations in FKBP12 surface and active site residues, disrupt the FKBP12-aspartokinase complex in vivo and in vitro.fpr1 mutants lacking FKBP12 are viable, are not threonine or methionine auxotrophs, and express wild-type levels of aspartokinase protein and activity; thus, FKBP12 is not essential for aspartokinase activity. The activity of aspartokinase is regulated by feedback inhibition by product, and genetic analyses reveal that FKBP12 is important for this feedback inhibition, possibly by catalyzing aspartokinase conformational changes in response to product binding.

Aspartate Kinase↗

PubMed1996

Immunosuppressant target protein FKBP12 is required for P-glycoprotein function in yeast.

The mammalian P-glycoprotein (Pgp) is a approximately 170-kDa membrane protein that mediates multidrug resistance in many chemotherapy-resistant tumors by effluxing toxic compounds from the cell. Pgp homologs are expressed in many organisms, from bacteria to yeast and mammals. Previous studies established a model system to analyze the function of murine, human, and Plasmodium falciparum Pgp by heterologous expression in the yeast Saccharomyces cerevisiae. However, such studies have been hampered by the inherent resistance of yeast cells to chemotherapeutic agents. We find that an erg6 mutation, which blocks the final synthetic step of the membrane sterol ergosterol, renders yeast sensitive to anthracyclines and dactinomycin, clinically relevant Pgp substrates. We demonstrate that expression of the murine mdr3 gene confers dactinomycin resistance in both the erg6 mutant yeast strain and in an erg6 rad52 DNA repair mutant yeast strain. Similarly, murine mdr3 expression confers resistance to the immunosuppressants cyclosporin A (CsA) and FK506 in a CsA-FK506-sensitive vph6 mutant yeast strain. CsA and FK506 are known to partially overcome Pgp-mediated drug resistance, suggesting the targets of these drugs might regulate Pgp function. We find that both murine mdr3 and the yeast Pgp homolog STE6 function in yeast mutants lacking the CsA target proteins cyclophilin A and calcineurin. In contrast, murine mdr3 function was severely compromised in yeast mutants lacking the FK506/rapamycin target protein FKBP12. Both wild-type FKBP12 and an F43Y FKBP12 mutant with reduced prolyl isomerase activity supported mdr3 function. Our results support the model that immunosuppressants reverse multidrug resistance by competing with other Pgp substrates but reveal that inhibition of FKBP12-dependent Pgp function may also contribute to reversal of multidrug resistance by FK506 and rapamycin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Functional expression of the multidrug resistance-associated protein in the yeast Saccharomyces cerevisiae.

The multidrug resistance-associated protein (MRP) is a member of the ATP binding cassette superfamily of transporters which includes the mammalian P-glycoproteins (P-gp) family. In order to facilitate the biochemical and genetic analyses of MRP, we have expressed human MRP in the yeast Saccharomyces cerevisiae and have compared its functional properties to those of the mouse Mdr3 P-gp isoform. Expression of both MRP and Mdr3 in the anthracycline hypersensitive mutant VASY2563 restored cellular resistance to Adriamycin in this mutant. MRP and Mdr3 expression produced pleiotropic effects on drug resistance in this mutant, as corresponding VASY2563 transformants also acquired resistance to the anti-fungal agent FK506 and to the K+/H+ ionophore valinomycin. The appearance of increased cellular resistance to the toxic effect of Adriamycin (ADM) in MRP and Mdr3 transformants was concomitant with a reduced intracellular accumulation of [14C]ADM in spheroplasts prepared from these cells. Moreover, MRP and Mdr3, but not control spheroplasts, could mediate a time-dependent reduction in the overall cell-associated [14C]ADM from preloaded cells, suggesting the presence of an active ADM transport mechanism in MRP and Mdr3 transformants. Finally, human MRP was found to complement the biological activity of the yeast peptide pheromone transporter Ste6 and partially restored mating in a sterile ste6 null mutant. These findings suggest that despite their relatively low level of structural homology, MRP and P-gp share similar functional aspects, since both proteins can mediate transport of chemotherapeutic drugs and the a mating peptide pheromone in yeast.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1996

Mammalian RAFT1 kinase domain provides rapamycin-sensitive TOR function in yeast.

In complex with the prolyl isomerase FKBP12, the natural product rapamycin blocks signal transduction in organisms as diverse as yeast and man. The yeast targets of FKBP12-rapamycin, TOR1 and TOR2, are large proteins with homology to lipid and protein kinases. A mammalian FKBP12-rapamycin binding protein, RAFT1, shares 39% and 43% identity with TOR1 and TOR2 proteins, respectively but has not been linked to rapamycin action in vivo. We find that when expressed in yeast, neither wild-type nor mutant RAFT1 complemented tor mutations or conferred rapamycin resistance. In contrast, TOR1-RAFT1 and TOR1-RAFT1 hybrid proteins containing the carboxy-terminal RAFT1 kinase domain complemented tor2 and tor1 mutant strains, respectively. Moreover, TOR2-RAFT1 and TOR1-RAFT1 hybrid proteins mutated at the position corresponding to rapamycin-resistant TOR mutants (S20351) conferred rapamycin resistance. Like the TOR2 protein, the TOR2-RAFT1 proteins were stably expressed, localized to the vacuolar surface, and associated with a phosphatidylinositol-4 kinase activity. These findings directly link the mammalian TOR homolog RAFT1 to rapamycin action in vivo and indicate that the TOR/RAFT1 kinase domain has been functionally conserved from yeast to man.

1-Phosphatidylinositol 4-Kinase↗

PubMed1996

Regional bivalent-univalent pairing versus trivalent pairing of a trisomic chromosome in Saccharomyces cerevisiae.

In meiosis I, homologous chromosomes pair, recombine and segregate to opposite poles. These events and subsequent meiosis II ensure that each of the four meiotic products has one complete set of chromosomes. In this study, the meiotic pairing and segregation of a trisomic chromosome in a diploid (2n + 1) yeast strain was examined. We find that trivalent pairing and segregation is the favored arrangement. However, insertions near the centromere in one of the trisomic chromosomes leads to preferential pairing and segregation of the "like" centromeres of the remaining two chromosomes, suggesting that bivalent-univalent pairing and segregation is favored for this region.

Chromosomes, Fungal↗

PubMed1996

Calcineurin mutants render T lymphocytes resistant to cyclosporin A.

The immunosuppressants cyclosporin A (CsA) and FK506 have been widely used to prevent and treat graft rejection after human organ and tissue transplantations. CsA and FK506 associate with intracellular binding proteins (i.e., CsA with cyclophilin A and FK506 with FKBP12) to form protein/drug complexes that suppress the immune system by preventing activation of T cells in response to antigen presentation. The common target of CsA and FK506 is calcineurin, a Ca2+/calmodulin-regulated, serine/threonine-specific protein phosphatase that regulates the nuclear import of a transcription factor, NF-AT, required for expression of T cell activation genes. In previous studies, we identified calcineurin mutations that block binding by the cyclophilin A/CsA or FKBP12/FK506 complexes and thereby render yeast cells resistant to the antifungal effects of CsA or FK506. In this report, we demonstrate that the corresponding mutations in murine calcineurin render the T cell receptor signal transduction cascade CsA resistant in human Jurkat T cells. Our findings support the recently determined calcineurin X-ray crystal structure, provide evidence that calcineurin is the only CsA-sensitive component limiting signaling from the T cell receptor to the nucleus, and suggest a means to render cells and tissues resistant to the toxic side effects of CsA and FK506.

Amino Acid Sequence↗

PubMed1995

FKBP12-rapamycin target TOR2 is a vacuolar protein with an associated phosphatidylinositol-4 kinase activity.

In complex with the immunophilin FKBP12, the natural product rapamycin inhibits signal transduction events required for G1 to S phase cell cycle progression in yeast and mammalian cells. Genetic studies in yeast first implicated the TOR1 and TOR2 proteins as targets of the FKBP12-rapamycin complex. We report here that the TOR2 protein is membrane associated and localized to the surface of the yeast vacuole. Immunoprecipitated TOR2 protein contains readily detectable phosphatidylinositol-4 (PI-4) kinase activity attributable to either a TOR2 intrinsic activity or to a PI-4 kinase tightly associated with TOR2. Importantly, we find that rapamycin stimulates FKBP12 binding to wild-type TOR2 but not to a rapamycin-resistant TOR2-1 mutant protein. Surprisingly, FKBP12-rapamycin binding does not markedly inhibit the PI kinase activity associated with TOR2, but does cause a delocalization of TOR2 from the vacuolar surface, which may deprive the TOR2-associated PI-4 kinase activity of its in vivo substrate. Several additional findings indicate that vacuolar localization is important for TOR2 function and, conversely, that TOR2 modulates vacuolar morphology and segregation. These studies demonstrate that TOR2 is an essential, highly conserved component of a signal transduction pathway regulating cell cycle progression conserved from yeast to man.

Amino Acid Sequence↗

PubMed1995

TOR mutations confer rapamycin resistance by preventing interaction with FKBP12-rapamycin.

The antifungal, immunosuppressive compound rapamycin arrests the cell cycle in G1 in both yeast cells and T-lymphocytes. Previous genetic studies in yeast identified mutations in three genes, FPR1 (FKBP12), TOR1, and TOR2, which confer rapamycin resistance, and genetic findings implicated the TOR proteins as direct targets of FKBP12-rapamycin. Consistent with this model, we find that modulating TOR1 and TOR2 expression alters rapamycin sensitivity. We describe several TOR2 mutations that confer rapamycin resistance. These mutations prevent FKBP12-rapamycin binding to TOR2, as assayed with the two-hybrid system. We find that TOR1 and the mammalian TOR homologue (mTOR) also bind FKBP12-rapamycin, and mutations corresponding to those in TOR2 similarly block FKBP12-rapamycin binding. We demonstrate that FKBP12 prolyl isomerase activity is not required for FKBP12-rapamycin binding to TOR and that a composite protein-drug surface contacts the TOR proteins. These studies confirm that the TOR proteins are direct targets of FKBP12-rapamycin, reveal that drug-resistant mutations prevent this association, and define structural features of these complexes.

Alleles↗

PubMed1995

Myristoylation of calcineurin B is not required for function or interaction with immunophilin-immunosuppressant complexes in the yeast Saccharomyces cerevisiae.

Calcineurin is a heterodimeric Ca2+/calmodulin-dependent protein phosphatase that regulates signal transduction and is the target of immunophilin-immunosuppressive drug complexes in T-lymphocytes and in yeast. Calcineurin is composed of a catalytic A subunit and a regulatory B subunit that is myristoylated at its amino terminus. We employed genetic and biochemical approaches to investigate the functional roles of myristoylation of calcineurin B (CNB1) in Saccharomyces cerevisiae. A calcineurin B mutant in which glycine 2 was substituted by alanine (CNB1-G2A) did not incorporate [3H]myristate when expressed in yeast. Both wild-type calcineurin B and the CNB1-G2A mutant protein are partially associated with membranes and cytoskeletal structures; hence, myristoylation is not required for these associations. In several independent genetic assays of calcineurin functions (recovery from alpha-factor arrest, survival during cation stress, and viability of a calcineurin-dependent strain), the nonmyristoylated CNB1-G2A mutant protein exhibited full biological activity. In vitro, both wild-type and CNB1-G2A mutant proteins formed complexes with both cyclophilin A-cyclosporin A (CsA) and FKBP12-FK506 that contained calcineurin A. Interestingly, expression of the nonmyristoylated CNB1-G2A mutant protein rendered yeast cells partially resistant to the immunosuppressant CsA, but not to FK506. This study demonstrates that calcineurin B myristoylation is not required for function, but may participate in inhibition by the cyclophilin A-CsA complex.

Amino Acid Isomerases↗

PubMed1995

Mutations that perturb cyclophilin A ligand binding pocket confer cyclosporin A resistance in Saccharomyces cerevisiae.

In complex with the peptidyl-prolyl isomerase cyclophilin A, the immunosuppressive antifungal drug cyclosporin A (CsA) inhibits a Ca2+/calmodulin-dependent protein phosphatase, calcineurin, which regulates signal transduction. We isolated and characterized cyclophilin A mutations that confer CsA resistance in a Saccharomyces cerevisiae strain whose growth is CsA-sensitive. Three mutations (G70S, H90Y, and G102A) alter single amino acids conserved between yeast and human cyclophilin A, which structural analyses implicate in CsA binding to human cyclophilin A. By Western analysis, all three mutant proteins are expressed in yeast. In vitro, two purified mutant cyclophilins (G70S, G102A) retain prolyl isomerase activity and have moderately reduced affinity for CsA and calcineurin but, when bound to CsA, do bind and inhibit calcineurin phosphatase activity. In contrast, the purified H90Y mutant cyclophilin is dramatically decreased in prolyl isomerase activity, CsA affinity, and calcineurin binding and inhibition. These studies identify conserved cyclophilin A residues that participate in CsA binding and catalysis.

Amino Acid Isomerases↗

PubMed1995

Targets of immunophilin-immunosuppressant complexes are distinct highly conserved regions of calcineurin A.

The immunosuppressive complexes cyclophilin A-cyclosporin A (CsA) and FKBP12-FK506 inhibit calcineurin, a heterodimeric Ca(2+)-calmodulin-dependent protein phosphatase that regulates signal transduction. We have characterized CsA- or FK506-resistant mutants isolated from a CsA-FK506-sensitive Saccharomyces cerevisiae strain. Three mutations that confer dominant CsA resistance are single amino acid substitutions (T350K, T350R, Y377F) in the calcineurin A catalytic subunit CMP1. One mutation that confers dominant FK506 resistance alters a single residue (W430C) in the calcineurin A catalytic subunit CMP2. In vitro and in vivo, the CsA-resistant calcineurin mutants bind FKBP12-FK506 but have reduced affinity for cyclophilin A-CsA. When introduced into the CMP1 subunit, the FK506 resistance mutation (W388C) blocks binding by FKBP12-FK506, but not by cyclophilin A-CsA. Co-expression of CsA-resistant and FK506-resistant calcineurin A subunits confers resistance to CsA and to FK506 but not to CsA plus FK506. Double mutant calcineurin A subunits (Y377F, W388C CMP1 and Y419F, W430C CMP2) confer resistance to CsA, to FK506 and to CsA plus FK506. These studies identify cyclophilin A-CsA and FKBP12-FK506 binding targets as distinct, highly conserved regions of calcineurin A that overlap the binding domain for the calcineurin B regulatory subunit.

Amino Acid Isomerases↗

PubMed1995

Gene disruption with PCR products in Saccharomyces cerevisiae.

We describe here the generation of gene disruption constructs using PCR amplification of selectable markers with primers that provide homology to the target gene of interest. We find that regions of homology as short as 38 to 50 bp suffice to mediate homologous recombination in yeast. We describe applications of this technology to three specific yeast genes that would have been difficult to disrupt with current methods. By dispensing with the need to either clone the gene of interest or engineer a standard disruption construct, this method should facilitate analysis of sequenced genes of unknown function, which will soon include the entire yeast genome.

Base Sequence↗

PubMed1995

Saturation mutagenesis of His114 of EcoRI reveals relaxed-specificity mutants.

EcoRI recognizes and cleaves DNA at GAATTC sites and is one of the best characterized sequence-specific restriction endonucleases (ENases). In previous studies, an EcoRI mutant, which exhibited relaxed substrate specificity and cleaved both canonical and EcoRI star sites, was isolated. This mutant enzyme has Tyr instead of His114. Here, we subjected residue 114 of the EcoRI ENase to saturation mutagenesis. The resulting mutant enzymes were characterized both in vivo and in vitro, resulting in the identification of mutants with canonical (H114K, Q, D, I) or relaxed (H114Y, F, S, T) specificity, as well as one mutant with severely impaired activity (H114P). In the X-ray structure of an EcoRI-substrate complex, His114 is located between the catalytic and recognition regions of EcoRI and may directly contact the DNA phosphate backbone. Based on our genetic and biochemical findings and the X-ray structure, we propose that His114 participates in substrate recognition and catalysis, either directly, via protein-DNA interactions, or indirectly, by mediating conformational changes that trigger DNA cleavage in response to substrate recognition.

Amino Acid Sequence↗