Regulation of A-type lamins by the Msp58-EDD complex
Laminopathies are an exceptional group of diseases commonly associated with dysfunctional nuclear lamina, a perinuclear meshwork of intermediate filaments lining the nucleoplasmic side of nuclear envelope (118,119,100). Autosomal-dominant Emery-Dreifuss muscular dystrophy (EDMD), are caused by mutations in the LMNA gene that encodes lamin A/C. A common feature of these mutant cells is the abnormal shape of the nuclear envelope. This is suggested as an underlying reason of the weakening of the nuclear structure observed in fibroblasts from patients with laminopathies. Therefore, elucidating the molecular mechanisms that regulate the expression and localization of lamin A/C is relevant and necessary to understand these rare diseases and to design efficient treatment approaches. Msp58 and EDD ubiquitin ligase are nuclear proteins that are involved in a variety of essential cellular processes. We demonstrated not only that these two proteins colocalize in the nucleus, but also that EDD is a negative regulator of Msp58, probably via the ubiquitin-proteasome pathway. This regulatory protein association plays an important role in the normal progression throughout the mammalian cell cycle. Interestingly, we observed that when the expression levels of Msp58 increase (either by knocking down EDD or by Msp58 overexpression), a high proportion of transfected cells have abnormal nuclei, represented by blebbing of the nuclear envelope. This phenotype is similar to the one observed in cells from patients with laminopathies. To elucidate whether there is a relation between this abnormal morphology with the expression levels of lamin A/C and/or Msp58, we downregulated lamin A/C and analyzed its effect on Msp58 protein levels by immunoblotting. Our results confirm that the depletion of lamin A/C leads to a significant reduction of Msp58, suggesting an uncharacterized association between these two proteins. However the molecular and functional mechanism underlying the latest observations remain to be elucidated. Given these results, it is possible that Msp58 and EDD play an important function in regulating either the expression or localization of lamin A/C in mammalian cells. To test this hypothesis, we propose to analyze the effect of silencing EDD and/or Msp58 on the expression (by immunoblotting) and localization (by immunofluorescence microscopy) of lamin A/C. It has been shown that downregulation of lamin A/C results in activation and nuclear translocation of ERK kinase, which can be used as an additional experimental readout for the proposed approaches.