The Effect of Octapeptide Repeats on Prion Folding and Misfolding.

Misfolding of prion protein (PrP) into amyloid aggregates is the central feature of prion diseases. PrP has an amyloidogenic C-terminal domain with three α-helices and a flexible tail in the N-terminal domain in which multiple octapeptide repeats are present in most mammals. The role of the octapeptides in prion diseases has previously been underestimated because the octapeptides are not located in the amyloidogenic domain. Correlation between the number of octapeptide repeats and age of onset suggests the critical role of octapeptide repeats in prion diseases. In this study, we have investigated four PrP variants without any octapeptides and with 1, 5 and 8 octapeptide repeats. From the comparison of the protein structure and the thermal stability of these proteins, as well as the characterization of amyloids converted from these PrP variants, we found that octapeptide repeats affect both folding and misfolding of PrP creating amyloid fibrils with distinct structures. Deletion of octapeptides forms fewer twisted fibrils and weakens the cytotoxicity. Insertion of octapeptides enhances the formation of typical silk-like fibrils but it does not increase the cytotoxicity. There might be some threshold effect and increasing the number of peptides beyond a certain limit has no further effect on the cell viability, though the reasons are unclear at this stage. Overall, the results of this study elucidate the molecular mechanism of octapeptides at the onset of prion diseases.

Quantitative phosphoproteomic analysis identifies the potential therapeutic target EphA2 for overcoming sorafenib resistance in hepatocellular carcinoma cells.

Limited therapeutic options are available for advanced-stage hepatocellular carcinoma owing to its poor diagnosis. Drug resistance to sorafenib, the only available targeted agent, is commonly reported. The comprehensive elucidation of the mechanisms underlying sorafenib resistance may thus aid in the development of more efficacious therapeutic agents. To clarify the signaling changes contributing to resistance, we applied quantitative phosphoproteomics to analyze the differential phosphorylation changes between parental and sorafenib-resistant HuH-7 cells. Consequently, an average of ~1500 differential phosphoproteins were identified and quantified, among which 533 were significantly upregulated in resistant cells. Further bioinformatic integration via functional categorization annotation, pathway enrichment and interaction linkage analysis led to the discovery of alterations in pathways associated with cell adhesion and motility, cell survival and cell growth and the identification of a novel target, EphA2, in resistant HuH-7R cells. In vitro functional analysis indicated that the suppression of EphA2 function impairs cell proliferation and motility and, most importantly, overcomes sorafenib resistance. The attenuation of sorafenib resistance may be achieved prior to its development through the modulation of EphA2 and the subsequent inhibition of Akt activity. Binding analyses and in silico modeling revealed a ligand mimic lead compound, prazosin, that could abate the ligand-independent oncogenic activity of EphA2. Finally, data obtained from in vivo animal models verified that the simultaneous inhibition of EphA2 with sorafenib treatment can effectively overcome sorafenib resistance and extend the projected survival of resistant tumor-bearing mice. Thus our findings regarding the targeting of EphA2 may provide an effective approach for overcoming sorafenib resistance and may contribute to the management of advanced hepatocellular carcinoma.