As an in vitro model system, patient-derived epidermolysis bullosa simplex keratinocytes have had an immense impact on what we know today about keratin filament function and their role in disease development. In the absence of gene therapy, screening com pound libraries for new or better drug s is another approach to improve existing treatments for genodennatoses. However inthis study, we report of the potential pitfalls when using this type of celllines as a "reporter" system. When cell lines with different genetic backgrounds are being used in cellbased assays, the greatest obstacle is to determine the most appropriate culture conditions (i.e., the composition of medium, number of cells plated and number of day s in culture). We demonstratehow culture conditions can greatly interfere with the cellular response in cell-based assays (cell proliferation, metabolic activity and migration), potentially al so giving rise to misleading data.
COBISS.SI-ID: 30115545
We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain ofE. coli, a conventional host organism, and keratin 14 (K14) as an example ofa complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments
COBISS.SI-ID: 5014298
Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused by mutations in keratin genes (KRT5 or KRT14), with no existing therapies. Aggregates of misfolded mutant keratins are seen in cultured keratinocytes from severe EBS patients. In other protein-folding disorders, involvement of molecular chaperones and the ubiquitin-proteasome system may modify disease severity. In this study, the effects of heat stress on keratin aggregation in immortalized cells from two patients with EBS (KRT5) and a healthy control were examined with and without addition of various test compounds. Heat-induced (43 °C, 30 minutes) aggregates were observed in all cell lines, the amount of which correlated with the donor phenotype. In EBS cells pre-exposed to proteasome inhibitor, MG132, and p38-mitogen-activated protein kinase (MAPK) inhibitor, SB203580, the proportion of aggregate-positive cells increased, suggesting a role of proteasomes and phosphorylation in removing mutated keratin. In contrast, aggregates were reduced by pretreatment with twochemical chaperones, trimethylamine N-oxide (TMAO) and 4-phenylbutyrate (4-PBA). TMAO also modulated stress-induced p38/c-jun N-terminal kinase (JNK) activation and expression of heat shock protein (HSPA1A), the latter of which colocalized with phosphorylated keratin 5 in EBS cells. Taken together, our findings suggest therapeutic targets for EBS and other keratinopathies.
COBISS.SI-ID: 28273369