Samples of A with and without peptides were incubated at 37 C for 1 h prior to cross-linking. bind to A, inhibit aggregation, and reduce its toxicity. Previously, we recognized strand G of transthyretin as a specific A binding domain name. In this work we further explore and define the necessary features of this binding domain name. We demonstrate that peptides derived from transthyretin bind A and inhibit its toxicity. We also show that, although both transthyretin and transthyretin-derived peptides bind A and inhibit toxicity, they differ significantly in their effect on A aggregation. 0.05). Taken together, we hypothesize that G16 oligomers (observed both in PICUP and TEM) scavenge A monomers and/or small A oligomers, creating larger soluble globular oligomeric assemblies. G16 reduces or eliminates further growth of A fibrils, while having little or no effect on pre-existing A fibrils. This explanation is consistent with the increase in the molecular excess weight of cross-linked A aggregates in the presence of G16, the large increase in aggregate size and scattering intensity detected by light scattering, the shift in morphology observed by TEM, the decrease in the formation of precipitable aggregates, and the small decrease in thioflavin T fluorescence. Comparison to TTR mTTR is an designed transthyretin mutant that is SH-4-54 stable as a monomer;51 solvent exposure of strand G is much higher in monomeric than tetrameric TTR (Determine ?(Determine1)1) . Like G16, mTTR reduced ThT fluorescence of A (Physique ?(Figure7).7). In sharp contrast to G16s effect, mTTR inhibited rather than enhanced A aggregation (Physique ?(Physique5).5). This result is usually consistent with our previous statement that mTTR decreased A aggregation, as measured by both arrest of growth of aggregate size as well as inhibition of formation of new aggregates.33 Previously we showed by TEM that A fibrils were shorter Rabbit polyclonal to SQSTM1.The chronic focal skeletal disorder, Pagets disease of bone, affects 2-3% of the population overthe age of 60 years. Pagets disease is characterized by increased bone resorption by osteoclasts,followed by abundant new bone formation that is of poor quality. The disease leads to severalcomplications including bone pain and deformities, as well as fissures and fractures. Mutations inthe ubiquitin-associated (UBA) domain of the Sequestosome 1 protein (SQSTM1), also designatedp62 or ZIP, commonly cause Pagets disease since the UBA is necessary for aggregatesequestration and cell survival in the presence of mTTR, but there was no switch in the morphology.34 Thus, although both mTTR and G16 bind to A, presumably via similar binding domains, the outcome of that binding conversation is quite different. mTTR binds to A aggregates and prevents their continued growth, but does not cause significant conformational changes. In contrast, remodeling of A to large globular aggregates is usually a consequence of SH-4-54 G16 binding to A. There are several possible explanations for differences between G16 and mTTR in their effect on A aggregation. One possibility is that the oligomeric nature of G16 facilitates multivalent binding to A and subsequent formation of clusters of oligomers. Since mTTR does not self-associate under our experimental conditions, it also does not coalesce A oligomers into larger aggregates. Another possibility is usually that the greater conformational flexibility of the G16 binding surface may facilitate its adaptation to and remodeling of A, while steric restrictions from the nonbinding scaffold of mTTR prevent remodeling. Effect of TTR-Derived Peptides on A Toxicity Given that G16 bound to A but displayed different effects on A aggregation than did TTR and mTTR, we tested whether G16 was effective at inhibiting A toxicity. Since A oligomers are widely believed to be more harmful than fibrils, 35 and since our data indicated that G16 greatly increased the appearance of soluble globules in A, we were concerned that G16 might actually enhance toxicity. Using an MTS assay, we observed that 10 M A was harmful to main neuronal cultures and that G16 inhibited A toxicity in a dose-dependent manner (Physique ?(Physique8,8, top). No inhibition of toxicity was observed for Gsc (Physique ?(Physique8,8, SH-4-54 top). Neither G16 nor Gsc alone was harmful (data not shown.) The results from MTS assay were confirmed by TUNEL staining (Physique ?(Physique8,8, bottom). We conclude that G16 inhibits A toxicity at substoichiometric ratio, as a consequence of its binding. The fact that both G16 and TTR inhibit toxicity, although they have very different effects on A aggregation, suggest that it is the binding conversation per se that is the relevant measure for impact on toxicity rather than the A aggregation state. It has been hypothesized that A toxicity is not linked to the of prefibrillar aggregate(s), but rather to the of their growth into fibrils.52 This might help to explain why both TTR and G16 prevent A induced toxicity even though they have different SH-4-54 effects on A aggregation. Open in a separate window Physique 8 (top) Toxicity was measured in.