Analysis of the role of the secretory pathway Ca2+/Mn2+- ATPase, PMR1/ATP2C1, in calcium homeostasis: from oxidative stress to skin disease

During my PhD, the genetically tractable Kluyveromyces lactis yeast has been used to study the molecular basis of Hailey–Hailey disease (HHD). This pathology, also known as familial benign chronic pemphigus, is a rare, chronic and recurrent blistering disorder, characterized by acantholysis. The genetics and pathophysiology of HHD have been linked to mutations in ATP2C1. The gene encodes for an adenosine triphosphate (ATP)-powered calcium channel pump and PMR1 is the yeast orthologue. Similarly to human HHD-cells, in yeast the loss of PMR1 promotes cellular toxicity caused by increased oxidative stress linked to the alteration of calcium homeostasis. By a functional suppression screening of yeast mutant with a cDNA library, we found that Glutathione S-transferase (GST), an important detoxifying enzyme, could be a candidate gene associated with Hailey-Hailey disease. Expression of mammalian GST in Klpmr1 alleviated several defects; these include oxidative-stress toxicity, calcium alteration and mitochondrial dysfunction. Additionally, the discoveries made in yeast were validated in HHD-derived cells. Indeed, HHD-lesional derived keratinocytes showed a decreased expression of GST gene when compared to non-lesional skin derived cells from the same patients. In parallel to the genetic screening, a drug screen was performed to find compounds with potential therapeutic effects for HHD treatment. Indeed, currently, there is no reported cure for HHD. For this reason, 131 FDA-approved natural compounds were screened for their ability to alleviate the calcium alterations and the oxidative stress of Kluyveromyces lactis pmr1cells. The drug collection includes inhibitors, activators and antagonists acting on molecular targets involved in different signaling pathways. Initially, for the sake of simplicity, the initial screening system was based on the cell wall alterations of the pmr1∆ mutant, utilizing the chitin binding stilbene fluorescent brightener calcofluor white. In the first part of the screening 12 molecules resulted toxic, while 71% of the library molecules were ineffective and the remaining appeared to have a partial effect. Therefore, in a second round of the screening, these last compounds were evaluated at higher concentrations. Based on this, six molecules were selected for further analysis. Their effects on other defects of pmr1 mutant strain were also evaluated, ranging from alteration of Ca2+ homeostasis to sensitivity to the ROS-generator menadione, as well as the mitochondrial functionality.