Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

Severe anaphylactoid reactions to cuprammonium cellulose hemodialyzers.

Twenty-one severe reactions to hemodialysis occurred in approximately 260,000 dialysis treatments at three centers within a 10 1/2-year period. Reactions typically appeared within minutes of initiating dialysis, and were characterized by cardiopulmonary, mucocutaneous, and/or gastrointestinal tract symptoms highly suggestive of anaphylaxis. Four respiratory arrests and one death resulted. Analysis of dialyzer use patterns and of each patient's dialyzer exposure history strongly implicated hollow-fiber dialyzers made of cuprammonium cellulose (CC) as a cause of these reactions. No obvious factors could be found to identify predisposed patients. Less than optimal rinsing of the CC hollow-fiber dialyzers prior to use may have been responsible for some, but not all, of these reactions.

Yeast TOR (DRR) proteins: amino-acid sequence alignment and identification of structural motifs.

The yeast TOR1 (DRR1) and TOR2 (DRR2) proteins are putative targets of the immunosuppressive drug rapamycin (Rm), defined by dominant drug-resistance mutations. They share a large C-terminal domain that exhibits sequence similarity to the 110-kDa subunit of phosphatidylinositol (PI) 3-kinases. In this report, we present an amino acid (aa) sequence alignment of TOR1 (DRR1) and TOR2 (DRR2) and identify conserved and nonconserved motifs within the N-terminal domain that are indicative of possible nuclear localization. We also show that the mutations responsible for Rm resistance in four independent drr2dom alleles alter the identical aa (Ser1975-->Arg) previously identified in drr1dom mutants (Ser1972-->Arg or Asn). Models for TOR (DRR) protein function are discussed.

The yeast FKS1 gene encodes a novel membrane protein, mutations in which confer FK506 and cyclosporin A hypersensitivity and calcineurin-dependent growth.

FK506 and cyclosporin A (CsA) are potent immunosuppressive agents that display antifungal activity. They act by blocking a Ca(2+)-dependent signal transduction pathway leading to interleukin-2 transcription. Each drug forms a complex with its cognate cytosolic immunophilin receptor (i.e., FKBP12-FK506 and cyclophilin-CsA) which acts to inhibit the Ca2+/calmodulin-dependent protein phosphatase 2B, or calcineurin (CN). We and others have defined the Saccharomyces cerevisiae FKS1 gene by recessive mutations resulting in 100-1000-fold hypersensitivity to FK506 and CsA (as compared to wild type), but which do not affect sensitivity to a variety of other antifungal drugs. The fks1 mutant also exhibits a slow-growth phenotype that can be partially alleviated by exogenously added Ca2+ [Parent et al., J. Gen. Microbiol. 139 (1993) 2973-2984]. We have cloned FKS1 by complementation of the drug-hypersensitive phenotype. It contains a long open reading frame encoding a novel 1876-amino-acid (215 kDa) protein which shows no similarity to CN or to other protein phosphatases. The FKS1 protein is predicted to contain 10 to 12 transmembrane domains with a structure resembling integral membrane transporter proteins. Genomic disruption experiments indicate that FKS1 encodes a nonessential function; fks1::LEU2 cells exhibit the same growth and recessive drug-hypersensitive phenotypes observed in the original fks1 mutants. Furthermore, the fks1::LEU2 allele is synthetically lethal in combination with disruptions of both of the nonessential genes encoding the alternative forms of the catalytic A subunit of CN (CNA1 and CNA2). These data suggest that FKS1 provides a unique cellular function which, when absent, increases FK506 and CsA sensitivity by making the CNs (or a CN-dependent function) essential.

The yeast cyclophilin multigene family: purification, cloning and characterization of a new isoform.

Cyclophilins (Cyps) constitute a highly conserved family of proteins present in a wide variety of organisms. Historically, Cyps were first identified by their ability to bind the immunosuppressive agent cyclosporin A (CsA) with high affinity; they later were found to have peptidyl-prolyl cis-trans isomerase (PPIase) activity, which catalyzes the folding of oligopeptides at proline-peptide bonds in vitro and may be important for protein folding in vivo. Cells of Saccharomyces cerevisiae contain at least two distinct Cyp-related PPIases encoded by the genes CYP1 and CYP2. A yeast strain (GL81) containing genomic disruptions of three known yeast PPIase-encoding genes [CYP1, CYP2 and RBP1 (for rapamycin-binding protein); Koltin et al., Mol. Cell. Biol. 11 (1991) 1718-1723] was previously constructed and found to be viable. Soluble fractions of these cells possess residual CsA-sensitive PPIase activity (2-5% of that present in wild-type cells as assayed in vitro). We have purified an approx. 18-kDa protein exhibiting PPIase activity from a soluble fraction of GL81 cells and determined that its N-terminal amino acid (aa) sequence exhibits significant homology (but nonidentity) to the Cyp1 and Cyp2 proteins. We designate the gene for this new protein, CYP3. Using a degenerate oligodeoxyribonucleotide (oligo) based on the N-terminal aa sequence, plus an internal oligo homologous to a conserved region within the portion of CYP1 and CYP2 that had been deleted in the genome, a CYP3-specific DNA fragment was generated by the polymerase chain reaction (PCR) using GL81 genomic DNA as a substrate. This PCR fragment was used as a probe to isolate CYP3 genomic and cDNA clones.(ABSTRACT TRUNCATED AT 250 WORDS)

The tyrosine89 residue of yeast FKBP12 is required for rapamycin binding.

Rapamycin (Rm) is a macrolide antifungal agent related to FK506 that exhibits potent immunosuppressive properties which are mediated through interaction with specific cytoplasmic receptors (FKBPs or RBPs, for FK506- and Rm-binding proteins, respectively). These proteins possess peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro which is inhibited by the binding of Rm and FK506. In Saccharomyces cerevisiae, Rm sensitivity (Rms) is mediated by binding of the drug to RBP1, a homolog of the 12-kDa human FK506-binding protein (FKBP12); null mutations in the yeast RBP1 gene result in a recessive drug resistance phenotype. To identify missense mutations that define amino acid (aa) residues in RBP1 involved in drug sensitivity, we selected and genetically characterized over 250 independent RmR rbp1 mutants and screened them for both RBP1-specific mRNA and protein expression. Whereas all rbp1 mutants expressed abundant levels of RBP1 mRNA, stable RBP1 protein production was detected in only one mutant strain. The RBP1 gene was PCR-generated (in triplicate) from several rbp1 mutants and independent clones were sequenced. Most of the immunoblot-negative alleles were found to contain various types of null mutations; however, some alleles contained specific missense mutations that apparently affect protein stability in vivo. The single immunoblot-positive allele was found to contain a mutation altering a specific residue (Tyr89) which is conserved among the known FKBPs, and which, based on the solution and x-ray structures of human FKBP12, has been proposed to be part of a hydrophobic drug-binding pocket for FK506 and Rm.(ABSTRACT TRUNCATED AT 250 WORDS)

The CYP2 gene of Saccharomyces cerevisiae encodes a cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase with an N-terminal signal sequence.

Cells of Saccharomyces cerevisiae contain a major cytosolic cyclophilin (Cyp)-related peptidyl-prolyl cis-trans isomerase (PPIase) which is the target for cyclosporin A (CsA) cytotoxicity and which is encoded by the CYP1 gene [Haendler et al., Gene 83 (1989) 39-46]. We recently identified a second Cyp-related gene in yeast, CYP2 [Koser et al., Nucleic Acids Res. 18 (1990) 1643] which predicts a protein with a hydrophobic leader sequence. A sequence lacking 33 codons from the 5'-end of the CYP2 open reading frame was generated by the polymerase chain reaction and engineered for expression in Escherichia coli. The corresponding recombinant truncated protein was purified and found to exhibit PPIase activity which was inhibited by CsA. The CYP2 gene is genetically unlinked to CYP1. As with CYP1, genomic disruption of CYP2 had no effect on haploid cell viability. Disruption of all three of the known yeast PPIase-encoding genes [CYP1, CYP2, and RBP1 for rapamycin-binding protein; Koltin et al., Mol. Cell. Biol. 11 (1991) 1718-1723] in the same haploid cell also resulted in no apparent cellular phenotype, suggesting either that none of these enzymes have an essential function or that additional PPIases can compensate for their specific absence. Whereas cells containing a genomic disruption of CYP1 exhibited a CsA-resistant phenotype, genomic disruption of CYP2 had no effect on CsA sensitivity. This suggests that the CYP1 gene product is the primary cellular target for CsA toxicity in yeast. Since both purified Cyps display CsA sensitivity in vitro, our data suggest that Cyp1 and Cyp2 differ in terms of their cellular function and/or localization.

Percutaneous aspiration biopsy of nodular lung lesions.

Over a 24 month period, 39 patients with nodular lung leasions suspected of being malignant on chest x-ray study underwent transthoracic needle aspiration biopsy. An accuracy rate of nearly 100 per cent was obtained in 34 of the lesions subsequently proved to be malignant. Achieving these results requires very close cooperation between the departments of radiology, cardiology, and cytopathology.

High risk of drug-resistant tuberculosis when first-line therapy fails in a high HIV prevalence setting.

SETTING: Lesotho national multidrug-resistant tuberculosis (MDR-TB) program. OBJECTIVE: To determine the prevalence of drug-resistant TB (DR-TB) among patients registered for MDR-TB treatment after failure or suspected failure of the standard 6-month regimen for new TB patients (Category I). DESIGN: We conducted a retrospective cohort study of patients registered for MDR-TB treatment following failure or suspected failure of Category I. RESULTS: A total of 76 patients were included in the analysis, including 51 Category I treatment failures and 25 suspected Category I treatment failures. The prevalence of resistance to any drug was 92% among the treatment failures and 72% among the suspected failures. The proportion of MDR-TB was respectively 78% and 28% among the treatment failures and suspected failures. Among the subgroup of human immunodeficiency virus (HIV) positive patients, the proportion of MDR-TB was 84% among failures and 23% among suspected failures. CONCLUSION: DR-TB and MDR-TB were common among patients in whom Category I failed. Early initiation of empiric second-line anti-tuberculosis treatment while awaiting culture and drug susceptibility testing (DST) results should be considered for HIV-negative and -positive patients who have failed first-line anti-tuberculosis treatment; patients suspected to be failing a first-line regimen should undergo DST at the end of the intensive phase.

High rate of hypothyroidism among patients treated for multidrug-resistant tuberculosis in Lesotho.

BACKGROUND: Hypothyroidism is a known side effect of treatment for multidrug-resistant tuberculosis (MDR-TB), but it is considered to be rare. Hypothyroidism has vague and non-specific symptoms, and can be easily missed by clinicians. OBJECTIVE: To report the high rate of hypothyroidism in a cohort of MDR-TB patients in Lesotho and to describe our approach to diagnosis and management. DESIGN: A retrospective study of 212 patients who initiated treatment for MDR-TB in Lesotho between 27 July 2007 and 24 March 2009 was performed. RESULTS: Among 186 patients screened, 129 (69%) had hypothyroidism, defined as at least one documented thyroid-stimulating hormone (TSH) result > 10.0 mIU/l; 100 (54%) patients had a maximum TSH > 20.0 mIU/l. At 93 days after starting MDR-TB treatment, half of the patients had developed hypothyroidism. CONCLUSION: Hypothyroidism may be more common during MDR-TB treatment than previously recognized. Screening all patients, even those without symptoms, for hypothyroidism within 2-3 months of starting MDR-TB treatment should be considered until prospective studies can inform screening guidelines.

A pilot study to evaluate the development of resistance to nevirapine in asymptomatic human immunodeficiency virus-infected patients with CD4 cell counts of > 500/mm3: AIDS Clinical Trials Group Protocol 208.

Treatment of human immunodeficiency virus (HIV) infection with nevirapine in patients with < 400 CD4 cells/mm3 rapidly selects for virus with reduced susceptibility to nevirapine. To test whether resistance would develop less quickly in patients with a lower virus burden, nevirapine was studied in asymptomatic patients with > 500 CD4 cells/mm3. With 400 mg of nevirapine daily, the median reduction in HIV RNA was 0.51 log10 copies/mL, and all isolates recovered by 12 weeks were resistant to nevirapine. As in patients with lower CD4 cell counts, some patients experienced sustained reduction in plasma HIV RNA despite the presence of resistant virus. These results suggest that lower levels of HIV RNA and immunosuppression did not retard the rate of emergence of nevirapine-resistant virus; also, a polymerase chain reaction-based HIV RNA assay is sufficiently sensitive to evaluate the antiviral effect of a drug in patients with > 500 CD4 cells/mm3.

Use of a silicone catheter with a Dacron cuff for dialysis short-term vascular access.

Polyurethane and Teflon subclavian vein catheters have been widely used for temporary vascular access for hemodialysis, but their use has been associated with a significant complication rate. A silicone dual-lumen catheter with a Dacron cuff placed in the internal jugular or subclavian vein was evaluated as a means of obtaining short-term vascular access. Sixty-two catheters in 54 patients provided a cumulative experience of 206 patient-months. Blood flow rates greater than or equal to 200 mL/min were achieved, with a mean recirculation of 2.1%. Catheter function was better with placement on the right side. Exit-site infections developed in nine patients, for a rate of 5.3 episodes per 100 patient-months; all resolved with antibiotics. Catheter-related bacteremia occurred in one patient, for a rate of 0.49 episodes per 100 patient-months, a rate much lower than rates reported for polyurethane and Teflon catheters. Clotting occurred in 24.5% of catheters, and thrombolytic therapy was always successful in restoring function. Because of the lower rate of complications, the silicone dual-lumen catheter with a Dacron cuff provides a safer alternative for short-term hemodialysis vascular access than the Teflon and polyurethane catheters.

Mapping the functional domains of human recombinant phosphodiesterase 4A: structural requirements for catalytic activity and rolipram binding.

To identify functional domains of the 886-amino acid human recombinant cAMP-specific phosphodiesterase (PDE) subtype A (rhPDE4A), we engineered the expression of seven mutant proteins containing both NH2- and COOH-terminal truncations. The level of rhPDE4A protein expression in yeast was monitored by immunoblotting using enzyme-specific antisera. Biochemical profiles of the mutant proteins were compared with those of the full-length protein or a fully active truncated form of the enzyme (rhPDE4A Met265-886), lacking the first 264 amino acids. The smallest catalytically active fragment generated was Met332-722, which at 45 kDa is less than half the mass of the full-length enzyme (approximately 110 kDa) but spans the most highly conserved region of the PDE superfamily. Two prototypical PDE4 inhibitors, rolipram and RP 73401, inhibited cAMP hydrolyzing activity of all truncated forms of the enzyme, with IC50 values of 70-2000 nM and 0.2-0.6 nM, respectively. [3H](R)-Rolipram bound to two sites on Met265-886, a high affinity site (Kd1 = 0.7 +/- 0.3 nM) and a low affinity site (Kd2 = 34 +/- 10 nM). Interestingly, [3H](R)-rolipram failed to bind to Met332-886 with high affinity, indicating that high affinity binding is not required for inhibition of enzyme activity. Low affinity rolipram binding was still present in Met332-886 (Kd = 101 +/- 7 nM). In contrast to [3H](R)-rolipram, [3H]RP 73401 bound to a single class of high affinity sites on Met265-886 (Kd = 0.4 +/- 0.1 nM). Further truncation of the enzyme to Met332-886 had no effect on [3H]RP 73401 binding (Kd = 0.2 +/- 0.03 nM). We conclude that the catalytic center of rhPDE4A lies between amino acids 332 and 722. Furthermore, amino acids 265-332 may form a high affinity binding site for rolipram that is outside of the catalytic domain. As a more likely alternative, these amino acids may not form a distinct binding site but instead may be required for the recombinant enzyme to assume a conformation that binds rolipram at the catalytic domain with a high affinity.

A low-Km, rolipram-sensitive, cAMP-specific phosphodiesterase from human brain. Cloning and expression of cDNA, biochemical characterization of recombinant protein, and tissue distribution of mRNA.

We have isolated cDNA clones from human frontal cortex cDNA libraries that encode a unique subtype of the low-Km, cAMP-specific phosphodiesterases (PDEs IV). The 564-amino acid sequence of the protein (human brain PDE IV (hPDE IVB)) shows significant homology to a PDE IV subtype expressed in human monocytes (hPDE IVA), particularly within the approximately 300-amino acid PDE IV catalytic domain. The degree of protein sequence identity is much greater between hPDE IVB and a homolog derived from rat brain (92% over 562 amino acids) than between hPDE IVB and hPDE IVA (76% over 538 amino acids), suggesting a greater subtype-specific versus species-specific conservation of protein sequence. Analysis of the distribution of hPDE IVB mRNA expression revealed a restricted pattern, with an approximately 4-kilobase mRNA detected in brain, heart, lung, and skeletal muscle and not in placenta, liver, kidney, or pancreas. An additional approximately 5-kilobase hPDE IVB-related mRNA species was detected in brain tissue. Recombinant hPDE IVB displayed all of the expected kinetic characteristics for a PDE IV, including sensitivity to the isozyme-selective inhibitor rolipram (Ki = 0.085 microM). Scatchard analysis of (R)-[3H]rolipram binding data suggested the presence of two noninteracting high affinity rolipram-binding sites (Kd = 0.4 and 6 nM) or a negatively cooperative interaction among multiple binding sites.

Role of conserved histidines in catalytic activity and inhibitor binding of human recombinant phosphodiesterase 4A.

To identify critical amino acids within the central conserved region of recombinant human cAMP-specific phosphodiesterase 4 subtype A (rhPDE4A), we engineered the expression of point mutants in a fully active rhPDE4A/Met201-886. When histidine residues at positions 433, 437, 473, and 477, which are highly conserved among all PDE families, were changed independently to serine residues, cAMP hydrolyzing activities were substantially reduced or abolished. The ability of these mutants to bind prototypical PDE4 inhibitors [3H]-(R)-rolipram or [3H]RP 73401 was also decreased in parallel with the loss of catalytic activity. The parallel loss of catalytic activity and inhibitor binding suggests that these changes resulted from non-localized perturbations in the structure of the enzyme. More interesting results were obtained when histidine residues at positions 505 and 506 were changed independently to aspar agines. The K(m) value for cAMP increased 3-fold in H505N (K(m) = 11 +/- 3 microM) and 11-fold in H506N (K(m) = 44 +/- 6 microM) compared with the wild-type protein (K(m) = 4 +/- 1 microM). These mutant proteins bound [3H]-(R)-rolipram and [3H]RP 73401 with K(d) values of 1.8 +/- 0.4 and 0.3 +/- 0.1 nM, respectively, for H505N, and 3.9 +/- 0.9 and 0.5 +/- 0.1 nM, respectively, for H506N. These values are nearly identical to those obtained with the wild-type rhPDE4A/Met201-886. In contrast, the IC50 values for cAMP competition with either [3H]-(R)-rolipram or [3H]RP 73401 binding increased approximately 2-fold in H505N and approximately 13-fold in H506N compared with the wild type protein. These increases are virtually identical to the changes in the K(m) value for cAMP in these mutants. We conclude that His506 and, perhaps, His505 are involved in binding of cAMP to PDE4A/Met201-886 but not in binding of PDE4-selective inhibitors.

Human mitogen-activated protein kinase CSBP1, but not CSBP2, complements a hog1 deletion in yeast.

CSBP1 and CSBP2 are human homologues of the Saccharomyces cerevisiae Hog1 mitogen-activated protein kinase which is required for growth in high osmolarity media. Expression of CSBP1, but not CSBP2, complemented a hog1 delta phenotype. A CSBP2 mutant (A34V) that complements hog1 delta was isolated and found to have approximately 3-fold lower kinase activity than the wild-type CSBP2. Further analysis revealed that both the kinase activity and tyrosine phosphorylation of CSBP1 and CSBP2 (A34V) is regulated by salt. In contrast, wild-type CSBP2 is constitutively active but dependent on the upstream kinase, Pbs2. Mutagenesis studies showed that reduction or elimination of CSBP2 kinase activity restores salt responsiveness as measured by tyrosine phosphorylation suggesting that too high a level of kinase activity can result in desensitization of the host cell and inability to grow in high salt.