A Promising Antiprion Trimethoxychalcone Binds to the Globular Domain of the Cellular Prion Protein and Changes Its Cellular Location.

The search for antiprion compounds has been encouraged by the fact that transmissible spongiform encephalopathies (TSEs) share molecular mechanisms with more prevalent neurodegenerative pathologies, such as Parkinson's and Alzheimer's diseases. Cellular prion protein (PrPC) conversion into protease-resistant forms (protease-resistant PrP [PrPRes] or the scrapie form of PrP [PrPSc]) is a critical step in the development of TSEs and is thus one of the main targets in the screening for antiprion compounds. In this work, three trimethoxychalcones (compounds J1, J8, and J20) and one oxadiazole (compound Y17), previously identified in vitro to be potential antiprion compounds, were evaluated through different approaches in order to gain inferences about their mechanisms of action. None of them changed PrPC mRNA levels in N2a cells, as shown by reverse transcription-quantitative real-time PCR. Among them, J8 and Y17 were effective in real-time quaking-induced conversion reactions using rodent recombinant PrP (rPrP) from residues 23 to 231 (rPrP23-231) as the substrate and PrPSc seeds from hamster and human brain. However, when rPrP from residues 90 to 231 (rPrP90-231), which lacks the N-terminal domain, was used as the substrate, only J8 remained effective, indicating that this region is important for Y17 activity, while J8 seems to interact with the PrPC globular domain. J8 also reduced the fibrillation of mouse rPrP23-231 seeded with in vitro-produced fibrils. Furthermore, most of the compounds decreased the amount of PrPC on the N2a cell surface by trapping this protein in the endoplasmic reticulum. On the basis of these results, we hypothesize that J8, a nontoxic compound previously shown to be a promising antiprion agent, may act by different mechanisms, since its efficacy is attributable not only to PrP conversion inhibition but also to a reduction of the PrPC content on the cell surface.

Analysis of protein structure in intact cells: crosslinking in vivo between introduced cysteines in the transmembrane domain of a bacterial chemoreceptor.

Oxidative crosslinking of cysteines introduced by site-specific mutagenesis is a powerful tool for structural analysis of proteins, but the approach has been limited to studies in vitro. We recently reported that intact cells of Escherichia coli could be treated with Cu(II)-(o-phenanthroline)3 or molecular iodine in a way that left unperturbed flagellar function or general chemotactic response, yet crosslinks were quantitatively formed between select cysteines in adjoining transmembrane helices of chemoreceptor Trg. This suggested that oxidative crosslinking might be utilized for structural analysis in vivo. Thus, we used our comprehensive collection of Trg derivatives, each containing a single cysteine at one of the 54 positions in the two transmembrane segments of the receptor monomer to characterize patterns of crosslinking in vivo and in vitro for this homodimeric protein. We found that in vivo crosslinking compared favorably as a technique for structural analysis with the more conventional in vitro approach. Patterns of crosslinking generated by oxidation treatments of intact cells indicated extensive interaction of transmembrane segment 1 (TM1) with its homologous partner (TM1') in the other subunit and a more distant placement of TM2 and TM2', the same relationships identified by crosslinking in isolated membranes. In addition, the same helical faces for TM1-TM1' interaction and TM2-TM2' orientation were identified in vivo and in vitro. The correspondence of the patterns also indicates that structural features identified by analysis of in vitro crosslinking are relevant to the organization of the chemoreceptor in its native environment, the intact, functional cell. It appears that the different features of the two functionally benign treatments used for in vivo oxidations can provide insights into protein dynamics.

Detecting the conformational change of transmembrane signaling in a bacterial chemoreceptor by measuring effects on disulfide cross-linking in vivo.

Transmembrane signaling by bacterial chemoreceptors is thought to involve relative movement among the four transmembrane helices of the homodimer. We assayed that movement by measuring effects of ligand occupancy on rates of oxidative cross-linking between cysteines introduced into neighboring helices of the transmembrane domain of chemoreceptor Trg from Escherichia coli. Measurements were done on chemoreceptors in their native environment, intact cells that were motile and chemotactically responsive. Receptor occupancy did not appear to cause drastic rearrangement of the four-helix structure since, among 67 cysteine pairs tested, the same 19 exhibited oxidative cross-linking in the presence or absence of saturating chemoattractant. However, occupancy did cause subtle changes that were detected as effects on rates of cross-linking. Among the seven disulfides appropriate for measurements of initial rates of formation, ligand occupancy had significant and different effects on all three cross-links that connected the two helices within a subunit but had minimal effects on the four that spanned the packing interface between subunits. This constitutes direct evidence that the conformational change of transmembrane signaling involves significant movement within a subunit and minimal movement between subunits, a pattern deduced from several previous studies and now documented directly. Among possible modes of movement between the two helices of a subunit, axial sliding of one helix relative to the other was the conformational change that best accounted for the observed effects on cross-linking.