Functional amyloid (FA) is widespread in bacteria and serves multiple purposes such as strengthening of biofilm and contact with eukaryotic hosts. Unlike pathological amyloid, FA has been subjected to evolutionary optimization which is likely to be reflected in the aggregation mechanism. FA from different bacteria, including Escherichia coli (CsgA) and Pseudomonas (FapC), contains a number of imperfect repeats which may be key to efficient aggregation. Here we report on the aggregative behavior of FapC constructs which represent all single, double, and triple deletions of the protein's three imperfect repeats. Analysis of the fibrillation kinetics by the program Amylofit reveals that the removal of these repeats increases the tendency of the growing fibrils to fragment and also generally increases aggregation half‐times. Remarkably, even the mutant lacking all three repeats was able to fibrillate, although fibrillation was much more irregular and led to significantly altered and destabilized fibrils. We conclude that imperfect repeats can promote fibrillation efficiency thanks to their modular design, though the context of the imperfect repeats also plays a significant role.

Several human proteins cause disease by misfolding and aggregating into amyloid fibril deposits affecting the surrounding tissues. Multiple other proteins co-associate with the diseased deposits but little is known about how this association is influenced by the nature of the amyloid aggregate and the properties of the amyloid-forming protein. In this study, we investigated the co-aggregation of plasma and cerebrospinal proteins in the presence of pre-formed amyloid fibrils. We evaluated the fibril-associated proteome across multiple amyloid fibril types that differ in their amino acid sequences, ultrastructural morphologies, and recognition by amyloid-binding dyes. The fibril types included aggregates formed by Amyloid β, α-synuclein, and FAS4 that are associated with pathological disorders, and aggregates formed by the glucagon and C-36 peptides, currently not linked to any human disease. Our results highlighted a highly similar response to the amyloid fold within the body fluid of interest. Fibrils with diverse primary sequences and ultrastructural morphologies only differed slightly in the composition of the co-aggregated proteins but were clearly distinct from less fibrillar and amorphous aggregates. The type of body fluid greatly affected the resulting amyloid interactome, underlining the role of the in vivo environment. We conclude that protein fibrils lead to a specific response in protein co-aggregation and discuss the effects hereof in the context of amyloid deposition.

The serine protease high-temperature requirement protein A1 (HtrA1) is associated with protein-misfolding disorders such as Alzheimer's disease and transforming growth factor β–induced protein (TGFBIp)–linked corneal dystrophy. In this study, using several biochemical and biophysical approaches, including recombinant protein expression, LC-MS/MS and 2DE analyses, and thioflavin T (ThT) fluorescence assays for amyloid fibril detection, and FTIR assays, we investigated the role of HtrA1 both in normal TGFBIp turnover and in corneal amyloid formation. We show that HtrA1 can cleave WT TGFBIp but prefers amyloidogenic variants. Corneal TGFBIp is extensively processed in healthy people, resulting in C-terminal degradation products spanning the FAS1-4 domain of TGFBIp. We show here that HtrA1 cleaves the WT FAS1-4 domain only inefficiently, whereas the amyloidogenic FAS1-4 mutations transform this domain into a considerably better HTRA1 substrate. Moreover, HtrA1 cleavage of the mutant FAS1-4 domains generated peptides capable of forming in vitro amyloid aggregates. Significantly, these peptides have been previously identified in amyloid deposits in vivo , supporting the idea that HtrA1 is a causative agent for TGFBIp-associated amyloidosis in corneal dystrophy. In summary, our results indicate that TGFBIp is an HtrA1 substrate and that some mutations in the gene encoding TGFBIp cause aberrant HtrA1-mediated processing that results in amyloidogenesis in corneal dystrophies.

No abstract

The enzymes of microorganisms that live in cold environments must be able to function at ambient temperatures. Cold-adapted enzymes generally have less ordered structures that convey a higher catalytic rate, but at the cost of lower thermodynamic stability. In this study, we characterized P355, a novel intracellular subtilisin protease (ISP) derived from the genome of Planococcus halocryophilus Or1, which is a bacterium metabolically active down to −25°C. P355′s stability and activity at varying pH values, temperatures, and salt concentrations, as well as its temperature-dependent kinetics, were determined and compared to an uncharacterized thermophilic ISP (T0099) from Parageobacillus thermoglucosidasius, a previously characterized ISP (T0034) from Planococcus sp. AW02J18, and Subtilisin Carlsberg (SC). The results showed that P355 was the most heat-labile of these enzymes, closely followed by T0034. P355 and T0034 exhibited catalytic constants (kcat) that were much higher than those of T0099 and SC. Thus, both P355 and T0034 demonstrate the characteristics of the stability-activity trade-off that has been widely observed in cold-adapted proteases.