Fe65 is a brain enriched adaptor protein involved in various cellular processes, including actin cytoskeleton regulation, DNA repair and transcription. A well-studied interacting partner of Fe65 is the transmembrane amyloid-beta precursor protein (APP), which can undergo regulated intramembrane proteolysis (RIP). Following beta and gamma-secretase-mediated RIP, the released APP intracellular domain (AICD) together with Fe65 can translocate to the nucleus and regulate transcription. In this study, we investigated if Fe65 nuclear localization can also be regulated by different alpha-secretases, also known to participate in RIP of APP and other transmembrane proteins. We found that in both Phorbol 12-myristate 13-acetate and all-trans retinoic acid differentiated neuroblastoma cells a strong negative impact on Fe65 nuclear localization, equal to the effect observed upon gamma-secretase inhibition, could be detected following inhibition of all three (ADAM9, ADAM10 and ADAM17) alpha-secretases. Moreover, using the comet assay and analysis of Fe65 dependent DNA repair associated posttranslational modifications of histones, we could show that inhibition of alpha-secretase-mediated Fe65 nuclear translocation resulted in impaired capacity of the cells to repair DNA damage. Taken together this suggests that alpha-secretase processing of APP and/or other Fe65 interacting transmembrane proteins play an important role in regulating Fe65 nuclear translocation and DNA repair.

Influenza neuraminidase (NA) is a sialidase that contributes to viral mobility by removing the extracellular receptors for the haemagglutinin (HA) glycoprotein. However, it remains unclear why influenza NAs evolved to function as Ca2+-dependent tetramers that display variable stability. Here, we show that the Ca2+ ion located at the centre of the NA tetramer is a major stability determinant, as this Ca2+ ion is required for catalysis and its binding affinity varies between NAs. By examining NAs from 2009 pandemic-like H1N1 viruses, we traced the affinity variation to local substitutions that cause residues in the central Ca2+-binding pocket to reposition. A temporal analysis revealed that these local substitutions predictably alter the stability of the 2009 pandemic-like NAs and contribute to the tendency for the stability to vary up and down over time. In addition to the changes in stability, the structural plasticity of NA was also shown to support the formation of heterotetramers, which creates a mechanism for NA to obtain hybrid properties and propagate suboptimal mutants. Together, these results demonstrate how the structural restrictions for activity provide influenza NA with several mechanisms for adaptation and diversification.

Influenza viruses replicate within the nucleus of the host cell. This uncommon RNA virus trait provides influenza with the advantage of access to the nuclear machinery during replication. However, it also increases the complexity of the intracellular trafficking that is required for the viral components to establish a productive infection. The segmentation of the influenza genome makes these additional trafficking requirements especially challenging, as each viral RNA (vRNA) gene segment must navigate the network of cellular membrane barriers during the processes of entry and assembly. To accomplish this goal, influenza A viruses (IAVs) utilize a combination of viral and cellular mechanisms to coordinate the transport of their proteins and the eight vRNA gene segments in and out of the cell. The aim of this review is to present the current mechanistic understanding for how IAVs facilitate cell entry, replication, virion assembly, and intercellular movement, in an effort to highlight some of the unanswered questions regarding the coordination of the IAV infection process.

Alzheimer’s disease is a neurodegenerative disease characterized by aberrant proteolysis of the transmembrane protein APP. The brain enriched adaptor protein Fe65 interacts with APP and participates together with APP and/or APP fragments in a number of cytoplasmic and nuclear functions. However, how the Fe65 subcellular localization, interaction with APP/APP fragments are regulated, as well as how Fe65 influences APP processing, is still not fully understood. In this study, we investigated the effect of Fe65 Ser-228 phosphorylation on Fe65 nuclear localization, APP interaction and APP processing. We show that although a Ser-228 phosphomimetic variant of Fe65 (Fe65-S2285E) was not excluded from the nucleus, a clear reduction of the nuclear level and the nuclear/cytoplasmic ratio of Fe65-S228E could be observed, suggesting that phosphorylation of Ser-288 could participate in regulation of the Fe65 subcellular localization. Interestingly, we found that not only Fe65-S2285E, but also mutation of Ser-228 to alanine (Fe65-S228A) resulted in a similar and dramatic increase of the Fe65 interaction with full-length APP. Moreover, we found that this increased APP interaction resulted in reduced α-secretase processing of APP and thus less generation of the neuroprotective sAPPα fragment. This suggest that the N-terminal domain of Fe65 may have a more prominent role in mediating the Fe65-APP interaction and regulating APP processing than previously thought.

Fe65 is a brain enriched adaptor protein involved in various cellular processes, including actincytoskeleton regulation, DNA repair and transcription. A well-studied interacting partner of Fe65 is the transmembrane amyloid-β precursor protein (APP), which can undergo regulatedintramembrane proteolysis (RIP). Binding of Fe65 to APP is thought to “activate” Fe65 andfollowing β- and γ-secretase mediated RIP, the released APP intracellular domain (AICD)-Fe65 complex is believed to translocate to the nucleus and regulate transcription. In this study, we investigated if and to what extent Fe65 nuclear localization can also be regulated by different α-secretases, also known to participate in RIP of APP and other transmembrane proteins. We found that in both PMA and RA differentiated neuroblastoma cells, a strong negative impact on Fe65 nuclear localization, equal to the effect observed upon γ-secretase inhibition, could be observed following inhibition of all three ADAM9, ADAM10 and ADAM17 α-secretases. Consistent with our previous study showing that α-secretase processing regulate Fe65 nuclear localization in undifferentiated cells. However, in contrast to what we found in undifferentiated cells, the major constitutive APP α-secretase ADAM10, had little or no role in differentiated neuroblastoma cells. Instead, other α-secretases, likely ADAM17, played a more important role. Taken together this suggest that α-secretase processing of APP or other Fe65 interacting transmembrane proteins play an important role in regulating Fe65 nuclear translocation and functions.