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Background Hailey–Hailey disease (HHD), also known as familial benign chronic pemphigus, is a rare, chronic and recurrent blistering disorder, histologically characterized by suprabasal acantholysis. HHD has been linked to mutations in ATP2C1 , the gene encoding the human adenosine triphosphate (ATP)-powered calcium channel pump. Methods In this work, the genetically tractable yeast Kluyveromyces lactis has been used to study the molecular basis of Hailey-Hailey disease. The K. lactis strain depleted of PMR1 , the orthologue of the human ATP2C1 gene, was used to screen a Madin-Darby canine kidney (MDCK) cDNA library to identify genetic interactors able to suppress the oxidative stress occurring in those cells. Results We have identified the Glutathione S-transferase ϴ-subunit (GST), an important detoxifying enzyme, which restores many of the defects associated with the pmr1Δ mutant. GST overexpression in those cells suppressed the sensitivity to calcium chelating agents and partially re-established calcium (Ca 2 + ) homeostasis by decreasing the high cytosolic Ca 2 + levels in pmr1Δ strain. Moreover, we found that in the K. lactis mutant the mitochondrial dysfunction was suppressed by GST overexpression independently from calcineurin. In agreement with yeast results, a decreased expression of the human GST counterpart ( GSTT1/M1 ) was observed in lesion-derived keratinocytes from HHD patients. Conclusions These data highlighted the Glutathione S-transferase as a candidate gene associated with Hailey-Hailey disease. General significance Kluyveromyces lactis can be considered a good model to study the molecular basis of this pathology. [ABSTRACT FROM AUTHOR]

Background The construction of efficient cell factories for the production of metabolites requires the rational improvement/engineering of the metabolism of microorganisms. The subject of this paper is directed towards the quantitative understanding of the respiratory/fermentative Kluyveromyces lactis yeast metabolism and its rag8 casein kinase mutant, taken as a model for all rag gene mutations. Methods 13 C NMR spectroscopy and [1,2- 13 C 2 ]glucose were used as metabolic stable-isotope tracer to define the metabolic profiling of a K. lactis yeast and its derivative mutants. Results Rag8 showed a decrease of all 13 C glutamate fractional enrichments, except for [4- 13 C]glutamate that was higher than wild type ones. A decrease of TCA cycle flux in rag8 mutants and a contribution of a [4- 13 C]ketoglutarate pool not originating from mitochondria were suggested. 13 C lysine enrichments confirmed the presence of two compartmentalized α-ketoglutarate (α-KG) pools participating to glutamate and lysine synthesis. Moreover, an increased transaldolase, as compared to transketolase activity, was observed in the rag8 mutant by 13 C NMR isotopomer analysis of alanine. Conclusions 13 C NMR-based isotopomer analysis showed the existence of different α-KG metabolic pools for glutamate and lysine biosynthesis. In the rag8 mutant, 13 C labeled pentose phosphate intermediates participated in the synthesis of this compartmentalized α-KG pool. General significance A compartmentalization of the α-KG pools involved in lysine biosynthesis has been revealed for the first time in K. lactis . Given its great impact in metabolic engineering field, its existence should be validated/compared with other yeasts and/or fungal species. [ABSTRACT FROM AUTHOR]

Abstract: Background: In the Crabtree-negative Kluyveromyces lactis yeast the rag8 mutant is one of nineteen complementation groups constituting the fermentative-deficient model equivalent to the Saccharomyces cerevisiae respiratory petite mutants. These mutants display pleiotropic defects in membrane fatty acids and/or cell walls, osmo-sensitivity and the inability to grow under strictly anaerobic conditions (Rag− phenotype). RAG8 is an essential gene coding for the casein kinase I, an evolutionary conserved activity involved in a wide range of cellular processes coordinating morphogenesis and glycolytic flux with glucose/oxygen sensing. Methods: A metabolomic approach was performed by NMR spectroscopy to investigate how the broad physiological roles of Rag8, taken as a model for all rag mutants, coordinate cellular responses. Results: Statistical analysis of metabolomic data showed a significant increase in the level of metabolites in reactions directly involved in the reoxidation of the NAD(P)H in rag8 mutant samples with respect to the wild type ones. We also observed an increased de novo synthesis of nicotinamide adenine dinucleotide. On the contrary, the production of metabolites in pathways leading to the reduction of the cofactors was reduced. Conclusions: The changes in metabolite levels in rag8 showed a metabolic adaptation that is determined by the intracellular NAD(P)+/NAD(P)H redox balance state. General significance: The inadequate glycolytic flux of the mutant leads to a reduced/asymmetric distribution of acetyl-CoA to the different cellular compartments with loss of the fatty acid dynamic respiratory/fermentative adaptive balance response. [Copyright &y& Elsevier]