Lysosome dysfunction arises early and propels Alzheimer’s disease (AD). Herein, we show that amyloid precursor protein (APP), linked to early-onset AD in Down syndrome (DS), acts directly via its β-C-terminal fragment (βCTF) to disrupt lysosomal vacuolar (H
+
)–adenosine triphosphatase (v-ATPase) and acidification. In human DS fibroblasts, the phosphorylated
682
YENPTY internalization motif of APP-βCTF binds selectively within a pocket of the v-ATPase V0a1 subunit cytoplasmic domain and competitively inhibits association of the V1 subcomplex of v-ATPase, thereby reducing its activity. Lowering APP-βCTF Tyr
682
phosphorylation restores v-ATPase and lysosome function in DS fibroblasts and in vivo in brains of DS model mice. Notably, lowering APP-βCTF Tyr
682
phosphorylation below normal constitutive levels boosts v-ATPase assembly and activity, suggesting that v-ATPase may also be modulated tonically by phospho-APP-βCTF. Elevated APP-βCTF Tyr
682
phosphorylation in two mouse AD models similarly disrupts v-ATPase function. These findings offer previously unknown insight into the pathogenic mechanism underlying faulty lysosomes in all forms of AD.
Specific inhibition of a single kinase isoform is a challenging
task due to the highly conserved nature of ATP-binding sites. Casein
kinase 1 (CK1) δ and ε share 97% sequence identity in
their catalytic domains. From a comparison of the X-ray crystal structures
of CK1δ and CK1ε, we developed a potent and highly CK1ε-isoform-selective
inhibitor (SR-4133). The X-ray co-crystal structure of the CK1δ−SR-4133
complex reveals that the electrostatic surface between the naphthyl
unit of SR-4133 and CK1δ is mismatched, destabilizing the interaction
of SR-4133 with CK1δ. Conversely, the hydrophobic surface area
resulting from the Asp−Phe−Gly motif (DFG)-out conformation
of CK1ε stabilizes the binding of SR-4133 in the ATP-binding
pocket of CK1ε, leading to the selective inhibition of CK1ε.
The potent CK1ε-selective agents display nanomolar growth inhibition
of bladder cancer cells and inhibit the phosphorylation of 4E-BP1
in T24 cells, which is a direct downstream effector of CK1ε.
The specificity loop of Matrix Metalloproteinases (MMPs) is known to regulate recognition of their substrates, and the S1′−site surrounded by the loop is a unique place to address the selectivity of ligands toward each MMP. Molecular dynamics (MD) simulations of apo−MMP−13 and its complex forms with various ligands were conducted to identify the role of the specificity loop for the ligand binding to MMP−13. The MD simulations showed the dual role of T247 as a hydrogen bond donor to the ligand, as well as a contributor to the formation of the van der Waal surface area, with T245 and K249 on the S1′−site. The hydrophobic surface area mediated by T247 blocks the access of water molecules to the S1′−site of MMP−13 and stabilizes the ligand in the site. The F252 residue is flexible in order to search for the optimum location in the S1′−site of the apo−MMP−13, but once a ligand binds to the S1′−site, it can form offset π−π or edge−to−π stacking interactions with the ligand. Lastly, H222 and Y244 provide the offset π−π and π−CH(Cβ) interactions on each side of the phenyl ring of the ligand, and this sandwiched interaction could be critical for the ligand binding to MMP−13.
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