A computer-simulated mechanism of familial Alzheimer's disease: Mutations enhance thermal dynamics and favor looser substrate-binding to γ-secretase.

The 4-subunit intramembrane protease complex γ-secretase cleaves many substrates including fragments of the ß-amyloid precursor protein (APP), leading to formation of Aß peptides, and Notch. Mutations in APP and the catalytic subunit of γ-secretase, presenilin, cause familial Alzheimer's disease (fAD). Mutations are assumed to change the substrate-binding and cleavage and thereby the Aß formed. Whereas a wild-type structure of substrate-bound γ-secretase became recently available from cryogenic electron microscopy (6IYC), the structure and dynamics of mutant proteins remain obscure. Here, we studied five prominent mutants of substrate-bound γ-secretase by explicit all-atom molecular dynamics in a phospholipid membrane model at physiological temperature using the experimental structure as template: The presenilin 1 mutants E280A, G384A, A434C, and L435F and the V717I mutant of APP. Our structures and dynamics provide the first atomic detail into how fAD-causing mutations affect substrate binding to γ-secretase. The pathogenic mutations tend to increase the space and variability in the substrate binding site, as seen e.g. from the distance from catalytic aspartate to substrate cleavage sites. We suggest that we have identified the molecular cause of the "imprecise cleavage" that leads to two trimming pathways in γ-secretase, consistent with the FIST model, which may rationalize the experimental Aß42/Aß40 ratios as a molecular basis for fAD.

The Pathogenic A2V Mutant Exhibits Distinct Aggregation Kinetics, Metal Site Structure, and Metal Exchange of the Cu2+ -Aß Complex.

A prominent current hypothesis is that impaired metal ion homeostasis may contribute to Alzheimer's disease (AD). We elucidate the interaction of Cu2+ with wild-type (WT) Aß1-40 and the genetic variants A2T and A2V which display increasing pathogenicity as A2T

Discovery of multiple interacting partners of gankyrin, a proteasomal chaperone and an oncoprotein--evidence for a common hot spot site at the interface and its functional relevance.

Gankyrin, a non-ATPase component of the proteasome and a chaperone of proteasome assembly, is also an oncoprotein. Gankyrin regulates a variety of oncogenic signaling pathways in cancer cells and accelerates degradation of tumor suppressor proteins p53 and Rb. Therefore gankyrin may be a unique hub integrating signaling networks with the degradation pathway. To identify new interactions that may be crucial in consolidating its role as an oncogenic hub, crystal structure of gankyrin-proteasome ATPase complex was used to predict novel interacting partners. EEVD, a four amino acid linear sequence seems a hot spot site at this interface. By searching for EEVD in exposed regions of human proteins in PDB database, we predicted 34 novel interactions. Eight proteins were tested and seven of them were found to interact with gankyrin. Affinity of four interactions is high enough for endogenous detection. Others require gankyrin overexpression in HEK 293 cells or occur endogenously in breast cancer cell line- MDA-MB-435, reflecting lower affinity or presence of a deregulated network. Mutagenesis and peptide inhibition confirm that EEVD is the common hot spot site at these interfaces and therefore a potential polypharmacological drug target. In MDA-MB-231 cells in which the endogenous CLIC1 is silenced, trans-expression of Wt protein (CLIC1_EEVD) and not the hot spot site mutant (CLIC1_AAVA) resulted in significant rescue of the migratory potential. Our approach can be extended to identify novel functionally relevant protein-protein interactions, in expansion of oncogenic networks and in identifying potential therapeutic targets.