Amyloid aggregation inhibitory mechanism of arginine-rich D-peptides.

It is widely believed that Alzheimer's disease pathogenesis is driven by the production and deposition of the amyloid-ß peptide (Aß) in the brain. In this study, we employ a combination of in silico and in vitro approaches to investigate the inhibitory properties of selected arginine-rich D-enantiomeric peptides (D-peptides) against amyloid aggregation. The D-peptides include D3, a 12-residue peptide with anti-amyloid potencies demonstrated in vitro and in vivo, RD2, a scrambled sequence of D3, as well as truncated RD2 variants. Using a global optimization method together with binding free energy calculations followed by molecular dynamics simulations, we perform a detailed analysis of D-peptide binding to Aß monomer and a fibrillar Aß structure. Results obtained from both molecular simulations and surface plasmon resonance experiments reveal a strong binding of D3 and RD2 to Aß, leading to a significant reduction in the amount of ß structures in both monomer and fibril, which was also demonstrated in Thioflavin T assays. The binding of the D-peptides to Aß is driven by electrostatic interactions, mostly involving the D-arginine residues and Glu11, Glu22 and Asp23 of Aß. Furthermore, we show that the anti-amyloid activities of the D-peptides depend on the length and sequence of the Dpeptide, its ability to form multiple weak hydrophobic interactions with Aß, as well as the Aß oligomer size.

Overexpression of antizyme-inhibitor in NIH3T3 fibroblasts provides growth advantage through neutralization of antizyme functions.

Antizyme inhibitor (AzI) is a homolog of ornithine decarboxylase (ODC), a key enzyme of polyamine synthesis. Antizyme inhibitor retains no enzymatic activity, but exhibits high affinity to antizyme (Az), a negative regulator of polyamine homeostasis. As polyamines are involved in maintaining cellular proliferation, and since AzI may negate Az functions, we have investigated the role of AzI in regulating cell growth. We show here that overexpression of AzI in NIH3T3 cells increased growth rate, enabled growth in low serum, and permitted anchorage-independent growth in soft agar, while reduction of AzI levels by AzI siRNA reduced cellular proliferation. Moreover, AzI overproducing cells gave rise to tumors when injected into nude mice. AzI overexpression resulted in elevation of ODC activity and of polyamine uptake. These effects of AzI are a result of its ability to neutralize Az, as overexpression of an AzI mutant with reduced Az binding failed to alter cellular polyamine metabolism and growth properties. We also demonstrate upregulation of AzI in Ras transformed cells, suggesting its relevance to some naturally occurring transformations. Finally, increased uptake activity rendered AzI overproducing and Ras-transformed cells more sensitive to toxic polyamine analogs. Our results therefore imply that AzI has a central and meaningful role in modulation of polyamine homeostasis, and in regulating cellular proliferation and transformation properties.