Bronislava Črešnar

Cytochrome P450 enzymes in the fungal kingdom.

Cytochrome P450 monooxygenases of fungi are involved in many essential cellular processes and play diverse roles. The enzymes catalyze the conversion of hydrophobic intermediates of primary and secondary metabolic pathways, detoxify natural and environmental pollutants and allow fungi to grow under different conditions. Fungal genome sequencing projects have enabled the annotation of several thousand novel cytochromes P450, many of which constitute new families. This review presents the characteristics of fungal cytochrome P450 systems and updates information on the functions of characterized fungal P450 monooxygenases as well as outlines the currently used strategies for determining the function of the many putative P450 enzymes.

Resolution and reconstitution of cytochrome P-450 containing steroid hydroxylation system of Rhizopus nigricans

11 alpha-hydroxylation of progesterone in the eucaryotic filamentous fungus Rhizopus nigricans is catalyzed by a monooxygenase. Three components of this multienzyme system, cytochrome P-450, rhizoporedoxin and a FAD containing rhizoporedoxin reductase have been separated from the postmitochondrial fraction on DEAE cellulose. Using NADPH as electron donor we showed that the presence of all three components was necessary for the reconstitution of the active electron transport chain.

Localization of the gene encoding steroid hydroxylase cytochrome P-450 from Rhizopus nigricans inside a HindIII fragment of genomic DNA

The gene encoding steroid inducible cytochrome P450 of Rhizopus nigricans ATCC 6227b has been found inside a HindIII fragment of the genomic DNA by hybridization with a partial length cDNA probe. The latter was isolated by immunoscreening a cDNA library prepared in the lambda gt11 expression system and identified on the basis of inducibility and sequence analysis. The nucleotide sequence of the cDNA probe revealed a coding sequence for the heme binding segment characteristic of the P450 gene family.

Aspects of the steroid response in fungi.

The number of fungal infections is increasing due to higher numbers of immunocompromised patients. Unfortunately, drug resistance represents a major additional problem in clinical praxis. Therefore factors contributing to infection by opportunistic pathogens, and to their growth and drug resistance are of major importance. It has been known for some time that mammalian steroid hormones are toxic to fungi. In this paper the response of fungi to the presence of steroid hormones will be discussed at different levels. First, the effect of steroid hormones on fungal growth, morphology and virulence will be considered. Processes affecting steroid intracellular concentration will be discussed; steroid uptake and, even more, steroid extrusion are currently of special interest. The role of biotransformation in the detoxification of active steroids will be taken into consideration and phases of steroid metabolism in fungal cells will be compared to phases of classical xenobiotic metabolism. Steroid signaling in fungi is presently not yet clear. It results in a global response of fungi to steroid hormones. Some of the genes differentially expressed in fungi as the result of exposure to steroid hormones may contribute to fungal drug resistance.

Resolution and reconstitution of the NADPH-cytochrome c (P-450) reductase induced by progesterone in Rhizopus nigricans.

The NADPH-cytochrome c (P-450) reductase induced in the filamentous fungus Rhizopus nigricans as a component of 11 alpha-hydroxylase of progesterone was resolved by DEAE-cellulose chromatography into two components. One of the components is an iron-sulfur protein (rhizoporedoxin), whereas the other component is a protein with reductase activity dependent on NADPH (rhizoporedoxin reductase). As shown in the reconstitution assay, the NADPH-cytochrome c (P-450) reductase activity was restored upon combination of these two proteins.

Molecular characterization of genes encoding cytosolic Hsp70s in the zygomycete fungus Rhizopus nigricans

Abstract Previous studies have shown that some stressors, including steroid hormones 21-OH progesterone and testosterone, stimulate the accumulation of heat shock protein 70 (hsp70) messenger ribonucleic acid (mRNA) population in the zygomycete filamentous fungus Rhizopus nigricans. In this study we report the cloning of 3 R nigricans hsp70 genes (Rnhsp70-1, Rnhsp70-2, and Rnhsp70-3) encoding cytosolic Hsp70s. With a Southern blot experiment under high stringency conditions we did not detect any additional highly homologous copies of the cytosolic hsp70 genes in the R nigricans genome. Sequence analyses showed that all 3 genes contain introns within the open reading frame. The dynamics of the R nigricans molecular response to progesterone, 21-OH progesterone, and testosterone, as well as to heat shock, copper ions, hydrogen peroxide, and ethanol was studied by temporal analysis of Rnhsp70-1 and Rnhsp70-2 mRNA accumulation. Northern blot experiments revealed that the Rnhsp70-2 transcript level is not affected by testosterone, whereas mRNA levels of both genes are rapidly increased with all the other stressors studied. Moreover, the decrease of transcript levels is notably delayed in ethanol stress, and a difference is observed between the profiles of Rnhsp70-1 and Rnhsp70-2 transcripts during heat stress.

Aspects of the progesterone response in Hortaea werneckii : Steroid detoxification, protein induction and remodelling of the cell wall

Progesterone in sublethal concentrations temporarily inhibits growth of Hortaea werneckii. This study investigates some of the compensatory mechanisms which are activated in the presence of progesterone and are most probably contributing to escape from growth inhibition. These mechanisms lead on the one hand to progesterone biotransformation/detoxification but, on the other, are suggested to increase the resistance of H. werneckii to the steroid. Biotransformation can detoxify progesterone efficiently in the early logarithmic phase, with mostly inducible steroid transforming enzymes, while progesterone biotransformation/detoxification in the late logarithmic and stationary phases of growth is not very efficient. The relative contribution of constitutive steroid transforming enzymes to progesterone biotransformation is increased in these latter phases of growth. In the presence of progesterone, activation of the cell wall integrity pathway is suggested by the overexpression of Pck2 which was detected in the stationary as well as the logarithmic phase of growth of the yeast. Progesterone treated H. werneckii cells were found to be more resistant to cell lysis than mock treated cells, indicating for the first time changes in the yeast cell wall as a result of treatment with progesterone.

Isolation, partial length sequence and expression of steroid inducible hps 70 gene from Rhizopus nigricans.

Previous studies have shown that filamentous fungus Rhizopus nigricans responds to addition of different steroids into growth medium with induction of hydroxylation system and that some steroids provoke stress response. The purpose of this study was to investigate whether those steroids provoke induction of Hsp70 gene(s), well studied markers of stress response in different cells and organisms. The expression studies of fungal Hsp70 gene(s) using Northern blot analysis showed that fungal hsp70 mRNA was upregulated after treatment of mycelia with deoxycorticosterone and testosterone, but not after exposure to progesterone. In addition, expression of fungal Hsp70 mRNA was elevated after exposure of mycelia to heat shock (32° C), ethanol, heavy metal (CuSO4), and oxidative stressor (H2O2), whereas treatment of mycelia with osmotic stressor (KCl) didn’t have any influence on stress protein expression. The partial nucleotide sequence and deduced amino acid sequence homology search revealed that the cDNA clone (λ hs20/2), isolated from cDNA library prepared from heat shock treated fungal mycelia, contained Hsp70 gene of DnaK subfamily.

Identification and partial characterization of Rhizopus nigricans Gβ proteins and their expression in the presence of progesterone

The mammalian steroid hormone progesterone actuates a signalling pathway in the zygomycete Rhizopus nigricans which includes heterotrimeric G proteins. To investigate the possibility that the Gβ subunit of these proteins is involved in the signalling, a cDNA library from R. nigricans exposed to progesterone was prepared and a sequence coding for a Gβ subunit was searched for. Using degenerate primers, two sequences, RnGPB1 and RnGPB2, were identified that exhibited a high degree of identity with those for Gβ from other filamentous fungi, but not from yeast. The presence of more than one Gβ subunit is very rare among the fungi, and it has been to date reported only for Rhizopus oryzae. We have shown that progesterone increases the expression of RnGPB1, but has no influence on the expression of RnGPB2. Therefore, our studies imply the involvement of Gβ subunit 1 in the response of R. nigricans to progesterone. Moreover, the Gβ subunit is subjected to endogenous ADP-ribosylation in the presence of NAD, which could be important in some, as yet unknown, cell process. Article from a special issue on steroids and microorganisms.

Thermodynamics of the isothermal interaction of human immunoglobulin G with guanidinium chloride

A thermodynamic study of the isothermal interaction of human immunoglobulin G with guanidinium chloride, a strong denaturant, has been performed. Free energies of interaction were calculated using preferential binding data obtained by measuring densities at constant chemical potential and constant composition, respectively. Enthalpies of interaction were determined calorimetrically. The values of both thermodynamic parameters as well as those of entropies of interaction have been found to depend crucially on the extent of denaturant binding.