Dementia-related Bri2 BRICHOS is a versatile molecular chaperone that efficiently inhibits Aß42 toxicity in Drosophila.

Formation of fibrils of the amyloid-ß peptide (Aß) is suggested to play a central role in neurodegeneration in Alzheimer's disease (AD), for which no effective treatment exists. The BRICHOS domain is a part of several disease-related proproteins, the most studied ones being Bri2 associated with familial dementia and prosurfactant protein C (proSP-C) associated with lung amyloid. BRICHOS from proSP-C has been found to be an efficient inhibitor of Aß aggregation and toxicity, but its lung-specific expression makes it unsuited to target in AD. Bri2 is expressed in the brain, affects processing of Aß precursor protein, and increased levels of Bri2 are found in AD brain, but the specific role of its BRICHOS domain has not been studied in vivo Here, we find that transgenic expression of the Bri2 BRICHOS domain in the Drosophila central nervous system (CNS) or eyes efficiently inhibits Aß42 toxicity. In the presence of Bri2 BRICHOS, Aß42 is diffusely distributed throughout the mushroom bodies, a brain region involved in learning and memory, whereas Aß42 expressed alone or together with proSP-C BRICHOS forms punctuate deposits outside the mushroom bodies. Recombinant Bri2 BRICHOS domain efficiently prevents Aß42-induced reduction in γ-oscillations in hippocampal slices. Finally, Bri2 BRICHOS inhibits several steps in the Aß42 fibrillation pathway and prevents aggregation of heat-denatured proteins, indicating that it is a more versatile chaperone than proSP-C BRICHOS. These findings suggest that Bri2 BRICHOS can be a physiologically relevant chaperone for Aß in the CNS and needs to be further investigated for its potential in AD treatment.

A protease substrate profiling method that links site-specific proteolysis with antibiotic resistance.

Proteases are involved in many biological processes and have become important tools in biomedical research and industry. Technologies for engineering and characterization of, for example, proteolytic activity and specificity are essential in protease research. Here, we present a novel method for assessment of site-specific proteolysis. The assay utilizes plasmid-encoded reporters that, upon processing by a co-expressed protease, confer antibiotic resistance to bacteria in proportion to the cleavage efficiency. We have demonstrated that cells co-expressing cleavable reporters together with tobacco etch virus protease (TEVp) could be discriminated from cells with non-cleavable reporters by growth in selective media. Importantly, the resistance to antibiotics proved to correlate with the substrate processing efficiency. Thus, by applying competitive growth of a mock library in antibiotic-containing medium, we could show that the substrate preferred by TEVp was enriched relative to less-efficient substrates. We believe that this simple methodology will facilitate protease substrate identification, and hold great promise for directed evolution of proteases and protease recognition sequences towards improved or even new functionality.

Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay.

Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp). The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y) and P1 (glutamine, Q) within the substrate peptide were confirmed as being the most important specificity determinants. In position P1', glycine (G), serine (S), cysteine (C), alanine (A) and arginine (R) were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E) is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of proteases and their substrates.

Novel fluorescence-assisted whole-cell assay for engineering and characterization of proteases and their substrates.

We have developed a sensitive and highly efficient whole-cell methodology for quantitative analysis and screening of protease activity in vivo. The method is based on the ability of a genetically encoded protease to rescue a coexpressed short-lived fluorescent substrate reporter from cytoplasmic degradation and thereby confer increased whole-cell fluorescence in proportion to the protease's apparent activity in the Escherichia coli cytoplasm. We demonstrated that this system can reveal differences in the efficiency with which tobacco etch virus (TEV) protease processes different substrate peptides. In addition, when analyzing E. coli cells expressing TEV protease variants that differed in terms of their in vivo solubility, cells containing the most-soluble protease variant exhibited the highest fluorescence intensity. Furthermore, flow cytometry screening allowed for enrichment and subsequent identification of an optimal substrate peptide and protease variant from a large excess of cells expressing suboptimal variants (1:100,000). Two rounds of cell sorting resulted in a 69,000-fold enrichment and a 22,000-fold enrichment of the superior substrate peptide and protease variant, respectively. Our approach presents a new promising path forward for high-throughput substrate profiling of proteases, engineering of novel protease variants with desired properties (e.g., altered substrate specificity and improved solubility and activity), and identification of protease inhibitors.