Calpain2 is a conventional member of the non-lysosomal calpain protease family that has been shown to affect the dynamics of focal and cell-cell adhesions by proteolyzing the components of adhesion complexes. Here, we inactivated calpain2 using CRISPR/Cas9 in epithelial MDCK cells. We show that depletion of calpain2 has multiple effects on cell morphology and function. Calpain2-depleted cells develop epithelial shape, however, they cover a smaller area, and cell clusters are more compact. Inactivation of calpain2 enhanced restoration of transepithelial electrical resistance after calcium switch, decreased cell migration, and delayed cell scattering induced by HGF/SF. In addition, calpain2 depletion prevented morphological changes induced by ERK2 overexpression. Interestingly, proteolysis of several calpain2 targets, including E-cadherin, β-catenin, talin, FAK, and paxillin, was not discernibly affected by calpain2 depletion. Taken together, these data suggest that calpain2 regulates the stability of cell-cell and cell-substratum adhesions indirectly without affecting the proteolysis of these adhesion complexes.

• Klíčová slova
• Actin, Adherens junctions, Calpains, Cell scattering, ERK, Epithelial polarity, Focal adhesions, HGF/SF, Migration, Proteases, Tight junctions, Transepithelial electrical resistance,
• MeSH
• beta-katenin metabolismus   MeSH
• buněčná adheze * MeSH
• buňky MDCK MeSH
• CRISPR-Cas systémy MeSH
• epitelové buňky * metabolismus   cytologie   MeSH
• hepatocytární růstový faktor metabolismus   MeSH
• kadheriny metabolismus   MeSH
• kalpain * metabolismus   MeSH
• mitogenem aktivovaná proteinkinasa 1 metabolismus   MeSH
• pohyb buněk MeSH
• proteolýza MeSH
• psi MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta-katenin MeSH
• hepatocytární růstový faktor MeSH
• kadheriny MeSH
• kalpain * MeSH
• mitogenem aktivovaná proteinkinasa 1 MeSH
• vápník MeSH

Epilepsy is a disorder that affects around 1% of the population. Approximately one third of patients do not respond to anti-convulsant drugs treatment. To understand the underlying biological processes involved in drug resistant epilepsy (DRE), a combination of proteomics strategies was used to compare molecular differences and enzymatic activities in tissue implicated in seizure onset to tissue with no abnormal activity within patients. Label free quantitation identified 17 proteins with altered abundance in the seizure onset zone as compared to tissue with normal activity. Assessment of oxidative protein damage by protein carbonylation identified additional 11 proteins with potentially altered function in the seizure onset zone. Pathway analysis revealed that most of the affected proteins are involved in energy metabolism and redox balance. Further, enzymatic assays showed significantly decreased activity of transketolase indicating a disruption of the Pentose Phosphate Pathway and diversion of intermediates into purine metabolic pathway, resulting in the generation of the potentially pro-convulsant metabolites. Altogether, these findings suggest that imbalance in energy metabolism and redox balance, pathways critical to proper neuronal function, play important roles in neuronal network hyperexcitability and can be used as a primary target for potential therapeutic strategies to combat DRE. SIGNIFICANCE: Epileptic seizures are some of the most difficult to treat neurological disorders. Up to 40% of patients with epilepsy are resistant to first- and second-line anticonvulsant therapy, a condition that has been classified as refractory epilepsy. One potential therapy for this patient population is the ketogenic diet (KD), which has been proven effective against multiple refractory seizure types However, compliance with the KD is extremely difficult, and carries severe risks, including ketoacidosis, renal failure, and dangerous electrolyte imbalances. Therefore, identification of pathways disruptions or shortages can potentially uncover cellular targets for anticonvulsants, leading to a personalized treatment approach depending on a patient's individual metabolic signature.

• Klíčová slova
• Drug resistant epilepsy, Protein carbonylation, Proteomics, Seizure,
• MeSH
• antikonvulziva terapeutické užití   MeSH
• energetický metabolismus MeSH
• epilepsie * farmakoterapie   MeSH
• lidé MeSH
• oxidace-redukce MeSH
• záchvaty * farmakoterapie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• antikonvulziva MeSH