Molecular chirality mediated amyloid formation on phospholipid surfaces.

One of the neuropathological features of Alzheimer's disease (AD) is the misfolding of amyloid-ß to form amyloid aggregates, a process highly associated with biological membranes. However, how molecular chirality affects the amyloid formation on phospholipid surfaces has seldom been reported. Here, l- and d-aspartic acid-modified 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (l-/d-Asp-DPPE) is synthesized to construct chiral phospholipid bilayers. We discover that the l-Asp-DPPE liposomes slightly inhibit the Aß(1-40) nucleation process but cannot affect the oligomer elongation process. By contrast, the d-Asp-DPPE liposomes strongly inhibit both nucleation and elongation of the peptide. Notably, l- and d-Asp-DPPE liposomes not only have good biocompatibility but can also rescue Aß(1-40)-aggregation induced cytotoxicity with significant chiral discrimination, in which the cell viability is higher in the presence of d-Asp-DPPE liposomes. Mechanism analysis and molecular dynamics simulation clearly demonstrate that differential electrostatic interactions of Lys16 in Aß(1-40) with l- or d-Asp on the phospholipid contribute to the remarkable chiral discrimination. This study provides a deeper understanding of the crucial amyloidosis process from the perspective of the chiral interface and reveals that the convergence of d-amino acids with the liposomes might be a feasible route for AD prevention.

Effects of naturally occurring charged mutations on the structure, stability, and binding of the Pin1 WW domain.

Pin1 is a peptidyl-prolyl cis-trans isomerase, whose WW domain specifically recognizes the pSer/Thr-Pro motif. Pin1 is involved in multiple phosphorylation events that regulate the activities of various substrates, and Pin1 deregulation has been reported in various diseases, including cancer and Alzheimer's disease. The WW domain of Pin1 has been used as a small model protein to investigate the folding mechanisms of the ß-sheet structure by studying the effect of mutations or its naturally occurring variants. However, only a few studies have investigated the structure and binding of Pin1 WW mutants. In the present work, two naturally occurring Pin1 WW variants, namely, G20D and S16R, derived from the cynomolgus monkey and African green monkey, respectively, were selected to investigate the influence of charge mutation on the structure, stability, and binding properties of the Pin1 WW domain. Analysis using a combination of nuclear magnetic resonance (NMR) and chemical shift-based calculations revealed that the G20D and S16R mutants had high structural similarity to the wild-type Pin1 WW domain. However, the presence of a charge mutation significantly decreased the stability of the Pin1 WW domain. Both the wild-type and G20D forms of the Pin1 WW domain utilized a three-site mode to bind to a phosphorylated Tau peptide, pT231, whereas the S16R mutant binds to the pT231 peptide either in a non-specific manner or through a totally different binding mechanism. Correspondingly, the wild-type and two mutant Pin1 WW domains showed different binding affinities to the Tau phosphopeptide. Considering that the WW domain participates in the catalytic activity of the Pin1 isomerase, our study represents a novel approach for studying Pin1 function through the analysis of its naturally occurring mutants.

Substrate-activated conformational switch on chaperones encodes a targeting signal in type III secretion.

The targeting of type III secretion (TTS) proteins at the injectisome is an important process in bacterial virulence. Nevertheless, how the injectisome specifically recognizes TTS substrates among all bacterial proteins is unknown. A TTS peripheral membrane ATPase protein located at the base of the injectisome has been implicated in the targeting process. We have investigated the targeting of the EspA filament protein and its cognate chaperone, CesAB, to the EscN ATPase of the enteropathogenic E. coli (EPEC). We show that EscN selectively engages the EspA-loaded CesAB but not the unliganded CesAB. Structure analysis revealed that the targeting signal is encoded in a disorder-order structural transition in CesAB that is elicited only upon the binding of its physiological substrate, EspA. Abrogation of the interaction between the CesAB-EspA complex and EscN resulted in severe secretion and infection defects. Additionally, we show that the targeting and secretion signals are distinct and that the two processes are likely regulated by different mechanisms.

The intrinsically disordered TC-1 interacts with Chibby via regions with high helical propensity.

Thyroid cancer 1 (TC-1) is a 106-residue naturally disordered protein that has been found to associate with thyroid, gastric, and breast cancers. Recent studies showed that the protein functions as a positive regulator in the Wnt/beta-catenin signaling pathway, a pathway that is known to play essential roles in developmental processes and causes tumor formation when misregulated. By competing with beta-catenin for binding to Chibby (Cby), a conserved nuclear protein that antagonizes the beta-catenin-mediated transcriptions, TC-1 up-regulates a number of beta-catenin target genes that are known to be involved in the aggressive behavior of cancers. In order to gain a molecular understanding of the role TC-1 plays in regulating the Wnt/beta-catenin signaling pathway, detailed structural studies of the protein and its interaction with Cby are essential. In this work, we used nuclear magnetic resonance (NMR) spectroscopy to elucidate the structure of TC-1 and its interaction with Cby. Our results indicate that even though TC-1 is naturally disordered, the protein adopts fairly compact conformations under nondenaturing conditions. Chemical shift analysis and relaxation measurements show that three regions (D44-R53, K58-A64, and D73-T88) with high-helical propensity are present in the C-terminal portion of TC-1. Upon addition of Cby, significant broadening of resonance signals derived from these helical regions of TC-1 was observed. The result indicates that the intrinsically disordered TC-1 interacts with Cby via its transient helical structure.