No abstract

The 155-kDa plasma glycoprotein factor H (FH), which consists of 20 complement control protein (CCP) modules, protects self-tissue but not foreign organisms from damage by the complement cascade. Protection is achieved by selective engagement of FH, via CCPs 1–4, CCPs 6–8 and CCPs 19–20, with polyanion-rich host surfaces that bear covalently attached, activation-specific, fragments of complement component C3. The role of intervening CCPs 9–18 in this process is obscured by lack of structural knowledge. We have concatenated new high-resolution solution structures of overlapping recombinant CCP pairs, 10–11 and 11–12, to form a three-dimensional structure of CCPs 10–12 and validated it by small-angle X-ray scattering of the recombinant triple‐module fragment. Superimposing CCP 12 of this 10–12 structure with CCP 12 from the previously solved CCP 12–13 structure yielded an S-shaped structure for CCPs 10–13 in which modules are tilted by 80–110° with respect to immediate neighbors, but the bend between CCPs 10 and 11 is counter to the arc traced by CCPs 11–13. Including this four-CCP structure in interpretation of scattering data for the longer recombinant segments, CCPs 10–15 and 8–15, implied flexible attachment of CCPs 8 and 9 to CCP 10 but compact and intimate arrangements of CCP 14 with CCPs 12, 13 and 15. Taken together with difficulties in recombinant production of module pairs 13–14 and 14–15, the aberrant structure of CCP 13 and the variability of 13–14 linker sequences among orthologues, a structural dependency of CCP 14 on its neighbors is suggested; this has implications for the FH mechanism.

Protein misfolding and aggregation are associated with a many human disorders, including Alzheimer's and Parkinson's diseases. Toward increasing the effectiveness of early-stage drug discovery for these conditions, we report a bacterial platform that enables the biosynthesis of molecular libraries with expanded diversities and their direct functional screening for discovering protein aggregation inhibitors. We illustrate this approach by performing, what is to our knowledge, the largest functional screen of small-size molecular entities described to date. We generated a combinatorial library of ~200 million drug-like, cyclic peptides and rapidly screened it for aggregation inhibitors against the amyloid- peptide (A42), linked to Alzheimer's disease. Through this procedure, we identified more than 400 macrocyclic compounds that efficiently reduce A42 aggregation and toxicity in vitro and in vivo. Finally, we applied a combination of deep sequencing and mutagenesis analyses to demonstrate how this system can rapidly determine structure-activity relationships and define consensus motifs required for bioactivity.

Neurodegenerative Diseases (ND) are a major threat to the aging population and the lack of a single preventive or disease-modifying agent only serves to increase their impact. In the past few years, protein misfolding and the subsequent formation of neurotoxic oligomeric/aggregated protein species have emerged as a unifying theme underlying the pathology of these complex diseases. Recently developed microbial genetic screens and selection systems for monitoring ND-associated protein misfolding have allowed the establishment of highthroughput assays for the identification of cellular factors and processes that are important mediators of NDassociated proteotoxicities. In addition, such systems have facilitated the discovery of synthetic and natural compounds with the ability to rescue the misfolding and the associated pathogenic effects of aggregation-prone proteins associated with NDs. This review outlines such available systems in bacteria and yeast, whose usage will likely accelerate the pre-clinical discovery process for effective drugs against a variety of NDs with high socioeconomic impact.