The versatility of the fungal cytochrome P450 monooxygenase system is instrumental in xenobiotic detoxification.

Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.

Virtual screening yields inhibitors of novel antifungal drug target, benzoate 4-monooxygenase.

Fungal CYP53 enzymes are highly conserved proteins, involved in phenolic detoxification, and have no homologues in higher eukaryotes, rendering them favorable drug targets. Aiming to discover novel CYP53 inhibitors, we employed two parallel virtual screening protocols and evaluated highest scoring hit compounds by analyzing the spectral binding interactions, by surveying the antifungal activity, and assessing the inhibition of catalytic activity. On the basis of combined results, we selected 3-methyl-4-(1H-pyrrol-1-yl)benzoic acid (compound 2) as the best candidate for hit-to-lead follow-up in the antifungal drug discovery process.

Four novel ATP2A2 mutations in Slovenian patients with Darier disease.

BACKGROUND: Darier disease (DD) is an autosomal dominant genodermatosis caused by mutations in the ATP2A2 gene. It has been reported that depletion of Ca(2+) stores within the endoplasmic reticulum of keratinocytes is associated with impaired cell cycle regulation and terminal differentiation. Mechanical stress, heat, or UV irradiation might delay cell cycle exit and permit progression into the quiescent stage without repair. When there is associated DNA damage, this can lead to an accumulation of secondary somatic mutations and possible clonal proliferation of damaged keratinocyes within keratotic papules and plaques. OBJECTIVE: We sought to present clinical, demographic, and genetic analysis of the cohort of Slovenian patients with DD, which represents 52% of DD patients in the country. METHODS: We examined 28 Slovenians with DD and screened genomic DNA for ATP2A2 mutations and RNA for splice site mutations. RESULTS: The estimated prevalence of the disease in Slovenia is 2.7/100.000. We identified 7 different ATP2A2 mutations, 4 of which are novel: A516P, R559G, 463-6del6, and 1762-6del18. We also found two previously described polymorphisms in intron XVIII (2741 + 54 G>A) and in exon 15 (2172 G>A; A724A), with allele frequencies of 64.15% and 11.32%, respectively. There was a history of perceptive deafness in two DD patients from two families. LIMITATIONS: Analysis of SERCA2 expression, measurements of Ca(2+) uptake and their influence on desmosomal assembly in vitro would add additional value to the study. Although single-stranded conformational analysis (SSCP) is a common and accepted method for screening for the presence of mutations, it does miss 10% to 20% of mutations. CONCLUSIONS: We identified 4 novel ATP2A2 mutations in Slovenian patients with DD. Deafness seems to be a new phenotypic characteristic of DD patients.

Darier disease in Slovenia: spectrum of ATP2A2 mutations and relation to patients' phenotypes.

ATP2A2 encodes the sarco/endoplasmic reticulum Ca2+- ATPase (SERCA2) and has been identified as a defective gene in Darier disease (DD). It is an autosomal dominant genodermatosis, which is characterized by loss of adhesion between suprabasal epidermal keratinocytes (acantholysis) and abnormal keratinization (dyskeratosis). We examined 28 Slovenian patients with DD (the cohort of patients represents over 50% of all DD patients in Slovenia) and screened genomic DNA for ATP2A2 mutations and RNA for splice site mutations. We identified 7 different ATP2A2 mutations, 4 of which are novel: A516P, R559G, 544+1del6, and 1762-6del18. We also found two previously described polymorphisms 2741+54 G>A in intron XVIII and 2172 G>A (A724A) in exon 15, with allele frequencies of 64.2% and 11.3%, respectively. The mutations are scattered throughout the gene and affect the actuator, phosphorylation, stalk and transmembrane domains of SERCA2. A P160L mutation in a Slovene patient with severe DD and a history of deafness is another consistent genotype-phenotype correlation. It seems that mutations of the ATP2A2 gene may also play a role in the pathogenesis of deafness, which seems to be a new phenotypic characteristic of DD patients.

Alterations in genes encoding sarcoplasmic-endoplasmic reticulum Ca(2+) pumps in association with head and neck squamous cell carcinoma.

Recent studies have suggested that a perturbation of intracellular Ca(2+) homeostasis or signaling could contribute to cancer development. The purpose of this study was to evaluate whether germline variants of the ATP2A2 and ATP2A3 genes might act as susceptibility alleles in head and neck squamous cell carcinoma. In both genes, we identified eight different alterations in 11 patients with head and neck squamous cell carcinoma (11/79; P = 0.0002, odds ratio = 0.054, 95% confidence interval = 0.0069-0.4236). We also detected low expression level of both genes in connection with some of alterations, but could not correlate low expression level with methylation in the promoter region of either gene. The results suggest that Ca(2+) pumps of sarcoplasmic-endoplasmic reticulum are involved in an increased susceptibility to develop head and neck squamous cell carcinoma in humans.

Alterations in the ATP2A2 gene in correlation with colon and lung cancer.

Sarcoendoplasmic reticulum calcium transport ATPases (SERCA-type calcium pumps), proteins that accumulate calcium in the endoplasmic reticulum, play an important role in numerous signaling pathways controlling tumor growth, differentiation, and cell death. Reports that Atp2a2 (Serca2) haploinsufficient mice often developed cancer prompted us to study the involvement of the ATP2A2 gene in human cancer development. We found 13 different novel alterations of the ATP2A2 gene in 27 of 416 alleles of patients with two different types of cancer. Changes in ATP2A2 were significantly more common in patients with colon cancer (P < 0.0001, odds ratio OR = 25.3) or lung cancer (P = 0.046, OR = 8.05). The 13 alterations were missense mutations (2), intronic deletions (2), intronic insertions (1), and single-nucleotide alterations (8: two in the coding region, three in the intronic region, and three in the promoter region). We detected lost or reduced expression of ATP2A2 in all patients with alterations in the promoter region, as well as in patients with a combination of gene alterations. Our results suggest that germline alterations of ATP2A2 may predispose to lung and colon cancer and that an impaired ATP2A2 gene might be involved, directly or indirectly, as an early event in carcinogenesis.

Hereditary medullary thyroid cancer in Slovenia--genotype-phenotype correlations.

BACKGROUND: Medullary thyroid cancer (MTC) is a rare endocrine tumor that may be sporadic or inherited in settings of MEN2A, MEN2B and FMTC. Germline point mutations in the RET proto-oncogene are responsible for tumor occurrence, inheritance and great clinical variability. The aim of this study was to correlate the genotype and phenotype of patients with hereditary MTC (age at diagnosis, sex, TNM classification and clinical features). PATIENTS: Between 1997 and 2003 genetic testing was performed in 69 out of 98 patients with "sporadic" MTC. Carriage of mutation was found in 14 (20.2%) patients (index patients) and in 16 out of 31 (51.6%) of their relatives. One patient with MEN2B and codon 918 mutation was excluded from further analysis. METHODS: Genomic DNA was isolated from peripheral blood leukocytes. Exons 10, 11, 13, 14, 15 and 16 of the RET proto-oncogene were amplified in polymerase chain reactions. Point mutations of the RET gene were detected with single-strand conformation analysis and DNA sequencing. Detected mutations were confirmed with restriction enzyme analysis. RESULTS: Codon 634 mutations were detected in 15 patients (50%; aged 18-76 years; 6 families), codon 618 in nine patients (30%; aged 12-65 years; 4 families) and codon 790 in five patients (16.6%; aged 16-74 years; 3 families). The median age at diagnosis was 31 +/- 17.3, 33 +/- 15.9 and 36 +/- 23.8 years for patients with codon 618, 634 and 790 mutations. Selected by sex, females with codon mutations 618 and 634 versus 790 had median age at diagnosis of 34.5 +/- 15.6 years and 43.5 +/- 22.9 years, whereas the inverse result was observed in males (26.5 +/- 18.0 versus 16 years). The male/female ratio was 1:2 for patients with codon 618 and 634 mutations and 1:4 for patients with codon 790 mutations. Some of the data suggested correlation between specific genotypes, tumor size, stage of MTC and age at diagnosis. Pheochromocytoma (12 out of 15 patients) and primary hyperparathyroidism (6 out of 15 patients) were diagnosed solely in patients with codon 634 mutations. One patient with FMTC and Hirschprung disease was found in a family with codon 618 mutations. CONCLUSION: Correlation between tumor size, stage of MTC at diagnosis in view of patient's age, and specific genotype were indicated in our limited series and were more evident in female patients with codon 790 mutations. Later onset and a probably less aggressive course of MTC in these patients than in patients with other mutations should be considered in planning prophylactic thyroid surgery. MEN2A syndrome was related solely to codon 634 mutations.

ATP2A3 gene is involved in cancer susceptibility.

The sarco/endoplasmatic reticulum calcium-ATPase (SERCA) translocates Ca(2+) from cytosol to the lumen of the ER and thus regulates Ca(2+) homeostasis, perturbations of which have been suggested to contribute to cancer. We have previously detected an increased number of alterations in the ATP2A2 gene in various cancer types and in the ATP2A3 gene in head and neck squamous cell carcinoma. Here, we further analyzed the ATP2A3 gene in colon, lung, and CNS cancers. We identified a statistically significant increase of alterations in each (colon cancer, p=0.0052, lung cancer, p=0.0026, CNS tumors, p=0.0045) cancer type, and all 3 types together (p=0.0016). Epigenetic study of the ATP2A3 gene indicated an unchanged methylation status, whereas expression of the ATP2A3 gene was normal for exon 14 mutations and reduced in connection with a nucleotide change in intron VI in all studied cancer types. Identification of a significant number of alterations in cancer patients suggests that ATP2A3 is involved in increased cancer susceptibility in humans. The mostly normal expression and methylation status of the ATP2A3 gene, as well as the absence of somatic alterations, further suggest that the ATP2A3 gene may not act as a classical tumor suppressor gene, but rather haplo-insufficiency of this gene may be enough to change the cell and tissue environment in such a way to predispose to cancer development.

P160L mutation in the Ca(2+) ATPase 2A domain in a patient with severe Darier disease.

Darier disease (DD) is caused by mutations of the ATP2A2 gene, which encodes the sarco/endoplasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2). The mutations affect protein expression, degradation and activity. We report a patient with severe sporadic DD, who did not respond adequately to repeated courses of orally administered acitretin and isotretinoin. He was found to harbor the missense P160L mutation of the ATP2A2 gene in a heterozygous state in the A domain of SERCA2 and polymorphism in intron 18 (2741 + 54 G --> A). The A domain plays a key role in translocation of Ca(2+) from cytoplasm to endoplasmic reticulum lumen, thus establishing a low intracellular Ca(2+) concentration.