Autoinhibition of Mint1 adaptor protein regulates amyloid precursor protein binding and processing

Abstract

Mint adaptor proteins bind to the amyloid precursor protein (APP) and regulate APP processing associated with Alzheimer's disease; however, the molecular mechanisms underlying Mint regulation in APP binding and processing remain unclear. Biochemical, biophysical, and cellular experiments now show that the Mint1 phosphotyrosine binding (PTB) domain that binds to APP is intramolecularly inhibited by the adjacent C-terminal linker region. The crystal structure of a C-terminally extended Mint1 PTB fragment reveals that the linker region forms a short α-helix that folds back onto the PTB domain and sterically hinders APP binding. This intramolecular interaction is disrupted by mutation of Tyr633 within the Mint1 autoinhibitory helix leading to enhanced APP binding and β-amyloid production. Our findings suggest that an autoinhibitory mechanism in Mint1 is important for regulating APP processing and may provide novel therapies for Alzheimer's disease.M ints [also known as X11s or amyloid precursor protein (APP) binding, family A] are a family of adaptor proteins encoded by three genes: neuron-specific Mint1 and Mint2, and the ubiquitously expressed Mint3 (1, 2). Mints consist of a divergent N-terminal region and conserved C-terminal sequences composed of a phosphotyrosine binding (PTB) domain and two tandem PDZ (postsynaptic density-95/discs large/zona occludens-1) domains. The PTB domain of all Mints interacts directly with a conserved YENPTY cytoplasmic motif of the APP that is essential for regulating APP trafficking and processing (3-6). APP is a type I transmembrane protein that is sequentially cleaved by site-specific proteases (7-10). Abnormal processing of APP generates Aβ peptides leading to the accumulation of extracellular plaques that are characteristic of Alzheimer's disease (AD). β-Secretase is the first amyloidogenic cleavage, generating a soluble APP ectodomain (sAPPβ) and a C-terminal fragment (CTFβ). CTFβ is subsequently cleaved by γ-secretase (a protein complex of anterior pharyx defective 1, nicastrin, presenilin, and presenilin enhancer 2) to release the small peptides Aβ40 and Aβ42, the latter having an increased propensity to form amyloid aggregates found in the brains of AD patients (11)(12)(13)(14). Alternatively, APP is sequentially cleaved by α-secretase within the Aβ region then γ-secretase, which initiates the nonamyloidogenic or nontoxic pathway (9, 10).Considerable evidence indicates that the APP-Mint interaction is biologically significant because loss of each individual Mint protein delays age-dependent production of amyloid plaques in transgenic AD mouse models (15). Past studies have indicated that the N-and C-terminal regions of Mints play an important role in regulating APP binding and cleavage events based on deletion analysis of Mints in a heterologous expression system (6, 16). The Mint N terminus was shown to promote APP binding and Aβ production, whereas the C-terminal region disrupted PTB-mediated binding to APP and decreased Aβ peptides (6, 16). However, the structural basis ...

Mint Proteins Are Required for Synaptic Activity-dependent Amyloid Precursor Protein (APP) Trafficking and Amyloid β Generation

“…However, it is now clearly recognized that they play a dynamic role in finely tuning cellular information and responses (29,30). Mint1 and Mint2 play more sophisticated roles in achieving functional regulation through autoinhibitory mechanisms (31,32). We have shown previously that Mint1 undergoes a conformational switch between a "closed" state that does not bind APP and an "open" state involved in APP binding and that this switch plays a central role in regulating A␤ production (31).…”

“…Mint1 and Mint2 play more sophisticated roles in achieving functional regulation through autoinhibitory mechanisms (31,32). We have shown previously that Mint1 undergoes a conformational switch between a "closed" state that does not bind APP and an "open" state involved in APP binding and that this switch plays a central role in regulating A␤ production (31). In addition, previous NMR studies observed intramolecular contacts between the Mint1 C-terminal tail and the PDZ1 domain, which blocks it ability to bind exogenous targets such as PS1 (33).…”

A New Mint1 Isoform, but Not the Conventional Mint1, Interacts with the Small GTPase Rab6

“…Several groups have already studied the regulation of the Mint adaptor proteins and the impact on APP processing. It has been shown that Mint1 activity is controlled by autoinhibitory mechanisms [56]. In the autoinhibited state the C-terminus of Mint1 binds to the PTB domain and so undergoes a conformational change, which leads to the loss of its APP binding affinity.…”

Intracellular Amyloid Precursor Protein Sorting and Amyloid- Secretion Are Regulated by Src-Mediated Phosphorylation of Mint2

“…For lentivirus production, Mint2-WT, Mint2-Y3E or Mint2-Y3F was inserted into the pLitmusIRES shuttle vector with a 5′ IRES and subsequently inserted into pFUW-NLS-EGFP- cre to generate pFUW-EGFP- cre -IRES-Mint2-WT, -Mint2-Y3E and -Mint2-Y3F plasmids. To generate GST-Mint2 peptides for in vitro phosphorylation analysis, PCR was used to amplify the rat Mint2 N-terminal region (Mint2-N; residues 1–399), the Mint2 N-terminal region containing Y3F mutations (Mint2-N-Y3F; residues 1–399), the Mint2-PTB domain (residues 363–546; Matos et al, 2012), or the Mint2 C-terminal region (Mint2-C; residues 546–750) which were then inserted into pGEX-KG. APPΔCT15 (residues 1–680) was amplified by PCR using human APP695 as a template and inserted into the pCMV5 mammalian expression vector.…”