Coordinate control of lipid composition and drug transport activities is required for normal multidrug resistance in fungi.

Pathogenic fungi present a special problem in the clinic as the range of drugs that can be used to treat these types of infections is limited. This situation is further complicated by the presence of robust inducible gene networks encoding different proteins that confer tolerance to many available antifungal drugs. The transcriptional control of these multidrug resistance systems in several key fungi will be discussed. Experiments in the non-pathogenic Saccharomyces cerevisiae have provided much of our current understanding of the molecular framework on which fungal multidrug resistance is built. More recent studies on the important pathogenic Candida species, Candida albicans and Candida glabrata, have provided new insights into the organization of the multidrug resistance systems in these organisms. We will compare the circuitry of multidrug resistance networks in these three organisms and suggest that, in addition to the well-accepted drug efflux activities, the regulation of membrane composition by multidrug resistance proteins provides an important contribution to the resistant phenotypes observed.

Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis.

A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized. The protein was extracted from the cells with 1 m NaCl, which required 1.5-fold, single-step purification to yield near-homogeneous preparations. In solution, the protein exists as homomeric aggregates, of mean diameter 21.6 nm, consisting of 22-kDa subunits. MS/MS data for peptides obtained by trypsin digestion of the thiosterase did not match any peptide from Escherichia coli thioesterases or any other thioesterases in the database. The thioesterase was associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopy and immunogold labeling. It hydrolyzed saturated and unsaturated fatty acyl-CoAs of C12 to C18 chain length with Vmax and Km of 3.58-9.73 micromol x min(-1) x (mg protein)(-1) and 2.66-4.11 microm, respectively. A catalytically important histidine residue is implicated in the active site of the enzyme. The thioesterase was active and stable over a wide range of temperature and pH. Maximum activity was observed at 65 degrees C and pH 10.5, and varied between 60% and 80% at temperatures of 25-70 degrees C and pH 6.5-10. The thioesterase also hydrolyzed p-nitrophenyl esters of C2 to C12 chain length, but substrate competition experiments demonstrated that the long-chain acyl-CoAs are better substrates for thioesterase than p-nitrophenyl esters. When assayed at 37 and 20 degrees C, the affinity and catalytic efficiency of the thioesterase for palmitoleoyl-CoA and cis-vaccenoyl-CoA were reduced approximately twofold at the lower temperature, but remained largely unaltered for palmitoyl-CoA.

High-sensitivity ST2 for prediction of adverse outcomes in chronic heart failure.

BACKGROUND: Soluble ST2 reflects activity of an interleukin-33-dependent cardioprotective signaling axis and is a diagnostic and prognostic marker in acute heart failure. The use of ST2 in chronic heart failure has not been well defined. Our objective was to determine whether plasma ST2 levels predict adverse outcomes in chronic heart failure in the context of current approaches. METHODS AND RESULTS: We determined the association between ST2 level and risk of death or transplantation in a multicenter, prospective cohort of 1141 chronic heart failure outpatients. Adjusted Cox models, receiver operating characteristic analyses, and risk reclassification metrics were used to assess the value of ST2 in predicting risk beyond currently used factors. After a median of 2.8 years, 267 patients (23%) died or underwent heart transplantation. Patients in the highest ST2 tertile (ST2 >36.3 ng/mL) had a markedly increased risk of adverse outcomes compared with the lowest tertile (ST2 ≤22.3 ng/mL), with an unadjusted hazard ratio of 3.2 (95% confidence interval [CI], 2.2 to 4.7; P<0.0001) that remained significant after multivariable adjustment (adjusted hazard ratio, 1.9; 95% CI, 1.3 to 2.9; P=0.002). In receiver operating characteristic analyses, the area under the curve for ST2 was 0.75 (95% CI, 0.69 to 0.79), which was similar to N-terminal pro-B-type natriuretic peptide (NT-proBNP) (area under the curve, 0.77; 95% CI, 0.72 to 0.81; P=0.24 versus ST2) but lower than the Seattle Heart Failure Model (area under the curve, 0.81 (95% CI, 0.77 to 0.85; P=0.014 versus ST2). Addition of ST2 and NT-proBNP to the Seattle Heart Failure Model reclassified 14.9% of patients into more appropriate risk categories (P=0.017). CONCLUSIONS: ST2 is a potent marker of risk in chronic heart failure and when used in combination with NT-proBNP offers moderate improvement in assessing prognosis beyond clinical risk scores.

Compartment-specific synthesis of phosphatidylethanolamine is required for normal heavy metal resistance.

Control of lipid composition of membranes is crucial to ensure normal cellular functions. Saccharomyces cerevisiae has two different phosphatidylserine decarboxylase enzymes (Psd1 and Psd2) that catalyze formation of phosphatidylethanolamine. The mitochondrial Psd1 provides roughly 70% of the phosphatidylethanolamine (PE) biosynthesis in the cell with Psd2 carrying out the remainder. Here, we demonstrate that loss of Psd2 causes cells to acquire sensitivity to cadmium even though Psd1 remains intact. This cadmium sensitivity results from loss of normal activity of a vacuolar ATP-binding cassette transporter protein called Ycf1. Measurement of phospholipid levels indicates that loss of Psd2 causes a specific reduction in vacuolar membrane PE levels, whereas total PE levels are not significantly affected. The presence of a phosphatidylinositol transfer protein called Pdr17 is required for Psd2 function and normal cadmium tolerance. We demonstrate that Pdr17 and Psd2 form a complex in vivo that seems essential for maintenance of vacuolar PE levels. Finally, we refine the localization of Psd2 to the endosome arguing that this enzyme controls vacuolar membrane phospholipid content by regulating phospholipids in compartments that will eventually give rise to the vacuole. Disturbance of this regulation of intracellular phospholipid balance leads to selective loss of membrane protein function in the vacuole.

Differential roles of transcriptional mediator subunits in regulation of multidrug resistance gene expression in Saccharomyces cerevisiae.

The multiprotein transcriptional Mediator complex provides a key link between RNA polymerase II and upstream transcriptional activator proteins. Previous work has established that the multidrug resistance transcription factors Pdr1 and Pdr3 interact with the Mediator component Med15/Gal11 to drive normal levels of expression of the ATP-binding cassette transporter-encoding gene PDR5 in Saccharomyces cerevisiae. PDR5 transcription is induced upon loss of the mitochondrial genome (rho(0) cells) and here we provide evidence that this rho(0) induction is Med15 independent. A search through other known Mediator components determined that Med12/Srb8, a member of the CDK8 Mediator submodule, is required for rho(0) activation of PDR5 transcription. The CDK8 submodule contains the cyclin C homologue (CycC/Srb11), cyclin-dependent kinase Cdk8/Srb10, and the large Med13/Srb9 protein. Loss of these other proteins did not lead to the same block in PDR5 induction. Chromatin immunoprecipitation analyses demonstrated that Med15 is associated with the PDR5 promoter in both rho(+) and rho(0), whereas Med12 recruitment to this target promoter is highly responsive to loss of the mitochondrial genome. Coimmunoprecipitation experiments revealed that association of Pdr3 with Med12 can only be detected in rho(0) cells. These experiments uncover the unique importance of Med12 in activated transcription of PDR5 seen in rho(0) cells.

Evidence for the bifunctional nature of mitochondrial phosphatidylserine decarboxylase: role in Pdr3-dependent retrograde regulation of PDR5 expression.

Multidrug resistance in the yeast Saccharomyces cerevisiae is sensitive to the mitochondrial genome status of cells. Cells that lose their organellar genome ([rho(0)] cells) dramatically induce transcription of multiple or pleiotropic drug resistance genes via increased expression of a zinc cluster-containing transcription factor designated Pdr3. A major Pdr3 target gene is the ATP-binding cassette transporter-encoding gene PDR5. Pdr5 has been demonstrated to act as a phospholipid floppase catalyzing the net outward movement of phosphatidylethanolamine (PE). Since the mitochondrially localized Psd1 enzyme provides a major route of PE biosynthesis, we evaluated the potential linkage between Psd1 function and PDR5 regulation. Overproduction of Psd1 in wild-type ([rho(+)]) cells was found to induce PDR5 transcription and drug resistance in a Pdr3-dependent manner. Loss of the PSD1 gene from [rho(0)] cells prevented the normal activation of PDR5 expression. Surprisingly, expression of a catalytically inactive form of Psd1 still supported PDR5 transcriptional activation, suggesting that PE levels were not the signal triggering PDR5 induction. Expression of green fluorescent protein fusions mapped the region required to induce PDR5 expression to the noncatalytic amino-terminal portion of Psd1. Psd1 is a novel bifunctional protein required both for PE biosynthesis and regulation of multidrug resistance.

Negative transcriptional regulation of multidrug resistance gene expression by an Hsp70 protein.

One of the most common origins of multidrug resistance occurs via the overproduction of ATP-binding cassette (ABC) transporter proteins. These ABC transporters then act as broad specificity drug pumps and efflux a wide range of toxic agents out of the cell. The yeast Saccharomyces cerevisiae exhibits multiple or pleiotropic drug resistance (Pdr) often through the over-production of a plasma membrane-localized ABC transporter protein called Pdr5p. Expression of the PDR5 gene is controlled by two zinc cluster-containing transcription factors called Pdr1p and Pdr3p. Cells that lack their mitochondrial genome (rho(0) cells) strongly induce PDR5 transcription in a Pdr3p-dependent fashion. To identify proteins associated with Pdr3p that might act to regulate this factor, a tandem affinity purification (TAP) moiety was fused to Pdr3p, and this recombinant protein was purified from yeast cells. The cytosolic Hsp70 chaperone Ssa1p co-purified with TAP-Pdr3p. Overexpression of Ssa1p repressed expression of PDR5 but had no effect on expression of other genes involved in the Pdr phenotype. This Ssa1p-mediated repression required the presence of Pdr3p and did not influence Pdr1p-dependent gene expression. Loss of the nucleotide exchange factor Fes1p mimicked Ssa1p-mediated repression of PDR5. Co-immunoprecipitation experiments indicated that Ssa1p was associated with Pdr3p but not Pdr1p in yeast cells. Finally, rho(0) cells had less Ssa1p bound to Pdr3p than rho(+) cells, consistent with Ssa1p-mediated repression of Pdr3p activity serving as a key regulatory step in control of multidrug resistance in yeast.

Formation of amyloid fibrils via longitudinal growth of oligomers.

Mature amyloid fibrils are believed to be formed by the lateral association of discrete structural units designated as protofibrils, but this lateral association of protofibrils has never been directly observed. We have recently characterized a thioesterase from Alcaligenes faecalis, which was shown to exist as homomeric oligomers with an average diameter of 21.6 nm consisting of 22 kDa subunits in predominantly beta-sheet structure. In this study, we have shown that upon incubation in a 75% ethanol solution, the oligomeric particles of protein were transformed into amyloid-like fibrils. TEM pictures obtained at various stages during fibril growth helped us to understand to a certain extent the early events in the fibrillization process. When incubated in 75% ethanol, oligomeric particles of protein grew to approximately 35-40 nm in diameter before fusion. Fusion of two oligomers of 35-40 nm resulted in the formation of a fibril. Fibril formation was accompanied by a reduction in the diameter of the particle to approximately 20-25 nm along with concomitant elongation to approximately 110 nm, indicating reorganization and strengthening of the structure. The elongation process continued by sequential addition of oligomeric units to give fibers 500-1000 nm in length with a further reduction in diameter to 17-20 nm. Further elongation resulted in the formation of fibers that were more than 4000 nm in length; the diameter, however, remained constant at 17-20 nm. These data clearly show that the mature fibrils have assembled via longitudinal growth of oligomers and not via lateral association of protofibrils.