Notch1 signaling is mediated by importins alpha 3, 4, and 7

The Notch signaling pathway is an important regulation system for the development and self-renewal of different tissues. A specific feature of this signaling cascade is the function of Notch as a surface receptor and regulator of gene expression. Hence, Notch activation and signal transduction requires the proteolytic release of the Notch intracellular domain (NICD), which activates the transcription of cell-specific genes after its transport into the nucleus. To date, little is known about the mechanisms that mediate NICD nuclear import. We here show that transport of NICD into the nucleus is mediated by the canonical importin α/β1 pathway. GST pull-down experiments revealed that NICD binds via one of its four potential nuclear localization signals to importins α3, α4, and α7, but not to α1 and α5. siRNA-mediated knockdown experiments showed that importins α3, α4 (and to a lesser extent, α7) mediate nuclear import of NICD and thus are directly involved in Notch signaling

Structural determinants for interaction of inwardly rectifying potassium channel proteins with phosphatidylinositol 4,5-Bisphosphate

This study concerns the investigations of direct interaction of the various proteins forming inwardly rectifying potassium channels in mammalian cells (Kir proteins) with phosphoinositol phospholipids, especially phospahtidylinositol-4,5 bisphosphate (PI(4,5)P2)

Structural determinants of Kvβ1.3-induced channel inactivation: a hairpin modulated by PIP2

Inactivation of voltage-gated Kv1 channels can be altered by Kvβ subunits, which block the ion-conducting pore to induce a rapid (‘N-type') inactivation. Here, we investigate the mechanisms and structural basis of Kvβ1.3 interaction with the pore domain of Kv1.5 channels. Inactivation induced by Kvβ1.3 was antagonized by intracellular PIP2. Mutations of R5 or T6 in Kvβ1.3 enhanced Kv1.5 inactivation and markedly reduced the effects of PIP2. R5C or T6C Kvβ1.3 also exhibited diminished binding of PIP2 compared with wild-type channels in an in vitro lipid-binding assay. Further, scanning mutagenesis of the N terminus of Kvβ1.3 revealed that mutations of L2 and A3 eliminated N-type inactivation. Double-mutant cycle analysis indicates that R5 interacts with A501 and T480 of Kv1.5, residues located deep within the pore of the channel. These interactions indicate that Kvβ1.3, in contrast to Kvβ1.1, assumes a hairpin structure to inactivate Kv1 channels. Taken together, our findings indicate that inactivation of Kv1.5 is mediated by an equilibrium binding of the N terminus of Kvβ1.3 between phosphoinositides (PIPs) and the inner pore region of the channel

Mechanism of amyloid plaque formation suggests an intracellular basis of Aβ pathogenicity

The formation of extracellular amyloid plaques is a common patho-biochemical event underlying several debilitating human conditions, including Alzheimer’s disease (AD). Considerable evidence implies that AD damage arises primarily from small oligomeric amyloid forms of Aβ peptide, but the precise mechanism of pathogenicity remains to be established. Using a cell culture system that reproducibly leads to the formation of Alzheimer’s Aβ amyloid plaques, we show here that the formation of a single amyloid plaque represents a template-dependent process that critically involves the presence of endocytosis- or phagocytosis-competent cells. Internalized Aβ peptide becomes sorted to multivesicular bodies where fibrils grow out, thus penetrating the vesicular membrane. Upon plaque formation, cells undergo cell death and intracellular amyloid structures become released into the extracellular space. These data imply a mechanism where the pathogenic activity of Aβ is attributed, at least in part, to intracellular aggregates