RESUMEN
The serine protease high-temperature requirement protein A1 (HtrA1) is associated with protein-misfolding disorders such as Alzheimer's disease and transforming growth factor ß-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.
Asunto(s)
Amiloide/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Serina Peptidasa A1 que Requiere Temperaturas Altas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Anciano de 80 o más Años , Cromatografía Líquida de Alta Presión , Córnea/metabolismo , Enfermedades de la Córnea/metabolismo , Enfermedades de la Córnea/patología , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Serina Peptidasa A1 que Requiere Temperaturas Altas/genética , Humanos , Mutagénesis Sitio-Dirigida , Péptidos/análisis , Péptidos/metabolismo , Dominios Proteicos , Pliegue de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Masas en Tándem , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/genéticaRESUMEN
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.