Pin1 and Alzheimer's Disease
Pin1 is a peptidyl-prolyl isomerase (PPIase) which catalyzes the cis-trans isomerization in many proteins, including the amyloid precursor protein (APP) from which the pathogenic amyloid-beta peptide (Aβ) is proteolytically derived. The accumulation of Aβ peptides in the brain, leading to the formation of neuritic plaques, is one of the major hallmarks of Alzheimer’s Disease (AD). Uncatalyzed cis-trans isomerization can be thought of as a molecular switch where a peptide bond alternates slowly between two conformations (cis and trans) and a large energy barrier between them prohibits rapid “switching” between the states. Pin1 lowers this energy barrier and allows for fast “switching” between cis and trans conformations. Pin1 has been shown to bind APP, to catalyze isomerization of the peptide bond at a conserved phosphorylated Thr668-Pro669 motif and to protect against amyloidogenic processing of APP. Additionally, Pin1 has been shown to be down-regulated or inhibited in AD neurons. Taken together, these results indicate that Pin1-catalyzed acceleration of the APP cis-trans isomerization rate represents a molecular timer that regulates APP processing and Aβ production.
An advantageous approach in elucidating the structural and functional interactions between Pin1 and APP is to utilize Pin1’s individual domains, WW and PPIase (Fig. 1), to examine the enzyme-substrate interactions using NMR spectroscopy and isothermal titration calorimetry (ITC). We examine both the binding of substrate to the WW domain of Pin1and the binding and catalysis of substrate to/by the PPIase domain. In both cases, a smaller form of APP (a 21-residue phosphopeptide of APP, G659-Q679, referred to herein as pT20) is utilized as the substrate for Pin1; pT20 has been shown to display structural and dynamic features indistinguishable from the native intracellular C-terminal domain of APP (AICD). Further, we take advantage of isotopic labeling of either the substrate or the Pin1 domains to observe the same binding or catalytic event from two perspectives. The substrate perspective allows the distinct cis and trans isomers to be tracked separately, yielding isomer-specific interactions, while the protein perspective reveals residue-specific changes, suggesting structure-based mechanisms for binding/catalysis (Fig. 1).
Fig. 1: Protein vs. substrate perspective: independent views of the same binding reaction stepsinteraction. using the isolated WW domain. A) Catalytic (PPIase) and binding (WW) domains have separate substrate interaction surfaces, shown occupied by space-filled atoms. B) The complete proposed mechanism for the Pin1-pT20 interaction. States A and B represent free substrate in the trans and cis conformers, respectively. F and C states represent those conformers bound to the WW domain, and E and D states represent those conformers bound to the PPIase domain. The kij’s are the microscopic rates for each proposed reaction step.
We have performed NMR titration experiments and subsequent lineshape analysis on pT20 and the individual Pin1 domains. More than 50% of the residues in the WW domain show substantial chemical shift changes upon substrate binding, suggesting a global response (within the domain) to binding. 15N-labeled substrate also displays substantial chemical shift changes upon binding to WW. From lineshape analysis of the titration data, we extracted off-rates for substrate binding, and binding constants were determined from ITC. Similar experiments were performed on the PPIase domain, which senses changes due to substrate binding and catalysis. The overall goal of these experiments is to comprehensively determine the microscopic rates for each step in the Pin1-catalyzed pT20 isomerization mechanism. Binding constants determined from independent ITC experiments confirm these rates. Finally, results from both ITC and NMR titration experiments provide thermodynamic information on the free energy landscape of the Pin1-pT20 interaction.
This work is supported by the National Institutes of Health (NIH).
