Characterization of the amyloid bacterial inclusion bodies of the HET-s fungal prion

The formation of amyloid aggregates is related to the onset of a number of human diseases. Recent studies provide compelling evidence for the existence of related fibrillar structures in bacterial inclusion bodies (IBs). Bacteria might thus provide a biologically relevant and tuneable system to study amyloid aggregation and how to interfere with it. Particularly suited for such studies are protein models for which structural information is available in both IBs and amyloid states. The only high-resolution structure of an infectious amyloid state reported to date is that of the HET-s prion forming domain (PFD). Importantly, recent solid-state NMR data indicates that the structure of HET-s PFD in IBs closely resembles that of the infectious fibrils. Here we present an exhaustive conformational characterization of HET-s IBs in order to establish the aggregation of this prion in bacteria as a consistent cellular model in which the effect of autologous or heterologous protein quality machineries and/or anti-aggregational and anti-prionic drugs can be further studied

Yeast prions form infectious amyloid inclusion bodies in bacteria

Background: Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively. Results: Here we show that both the prion domain of Sup35 (Sup35-NM) and the Ure2 protein (Ure2p) form inclusion bodies (IBs) displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold. Conclusions: An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity

Yeast prions form infectious amyloid inclusion bodies in bacteria

Background Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively. Results Here we show that both the prion domain of Sup35 (Sup35-NM) and the Ure2 protein (Ure2p) form inclusion bodies (IBs) displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold. Conclusions An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity

Characterization of the amyloid bacterial inclusion bodies of the HET-s fungal prion

The formation of amyloid aggregates is related to the onset of a number of human diseases. Recent studies provide compelling evidence for the existence of related fibrillar structures in bacterial inclusion bodies (IBs). Bacteria might thus provide a biologically relevant and tuneable system to study amyloid aggregation and how to interfere with it. Particularly suited for such studies are protein models for which structural information is available in both IBs and amyloid states. The only high-resolution structure of an infectious amyloid state reported to date is that of the HET-s prion forming domain (PFD). Importantly, recent solid-state NMR data indicates that the structure of HET-s PFD in IBs closely resembles that of the infectious fibrils. Here we present an exhaustive conformational characterization of HET-s IBs in order to establish the aggregation of this prion in bacteria as a consistent cellular model in which the effect of autologous or heterologous protein quality machineries and/or anti-aggregational and anti-prionic drugs can be further studied

Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors

Background: The amyloid-β peptide (Aβ42) is the main component of the inter-neuronal amyloid plaques characteristic of Alzheimer's disease (AD). The mechanism by which Aβ42 and other amyloid peptides assemble into insoluble neurotoxic deposits is still not completely understood and multiple factors have been reported to trigger their formation. In particular, the presence of endogenous metal ions has been linked to the pathogenesis of AD and other neurodegenerative disorders. Results: Here we describe a rapid and high-throughput screening method to identify molecules able to modulate amyloid aggregation. The approach exploits the inclusion bodies (IBs) formed by Aβ42 when expressed in bacteria. We have shown previously that these aggregates retain amyloid structural and functional properties. In the present work, we demonstrate that their in vitro refolding is selectively sensitive to the presence of aggregation-promoting metal ions, allowing the detection of inhibitors of metal-promoted amyloid aggregation with potential therapeutic interest. Conclusions: Because IBs can be produced at high levels and easily purified, the method overcomes one of the main limitations in screens to detect amyloid modulators: the use of expensive and usually highly insoluble synthetic peptides

Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors

Background: The amyloid-β peptide (Aβ42) is the main component of the inter-neuronal amyloid plaques characteristic of Alzheimer's disease (AD). The mechanism by which Aβ42 and other amyloid peptides assemble into insoluble neurotoxic deposits is still not completely understood and multiple factors have been reported to trigger their formation. In particular, the presence of endogenous metal ions has been linked to the pathogenesis of AD and other neurodegenerative disorders. Results: Here we describe a rapid and high-throughput screening method to identify molecules able to modulate amyloid aggregation. The approach exploits the inclusion bodies (IBs) formed by Aβ42 when expressed in bacteria. We have shown previously that these aggregates retain amyloid structural and functional properties. In the present work, we demonstrate that their in vitro refolding is selectively sensitive to the presence of aggregation-promoting metal ions, allowing the detection of inhibitors of metal-promoted amyloid aggregation with potential therapeutic interest. Conclusions: Because IBs can be produced at high levels and easily purified, the method overcomes one of the main limitations in screens to detect amyloid modulators: the use of expensive and usually highly insoluble synthetic peptides

Ultra rapid in vivo screening for anti-Alzheimer anti-amyloid drugs

More than 46 million people worldwide suffer from Alzheimer's disease. A large number of potential treatments have been proposed; among these, the inhibition of the aggregation of amyloid β-peptide (Aβ), considered one of the main culprits in Alzheimer's disease. Limitations in monitoring the aggregation of Aβ in cells and tissues restrict the screening of anti-amyloid drugs to in vitro studies in most cases. We have developed a simple but powerful method to track Aβ aggregation in vivo in realtime, using bacteria as in vivo amyloid reservoir. We use the specific amyloid dye Thioflavin-S (Th-S) to stain bacterial inclusion bodies (IBs), in this case mainly formed of Aβ in amyloid conformation. Th-S binding to amyloids leads to an increment of fluorescence that can be monitored. The quantification of the Th-S fluorescence along the time allows tracking Aβ aggregation and the effect of potential antiaggregating agents

Thioflavin-S staining of bacterial inclusion bodies for the fast, simple, and inexpensive screening of amyloid aggregation inhibitors

Amyloid aggregation is linked to a large number of human disorders, from neurodegenerative diseases as Alzheimer"s disease (AD) or spongiform encephalopathies to non-neuropathic localized diseases as type II diabetes and cataracts. Because the formation of insoluble inclusion bodies (IBs) during recombinant protein production in bacteria has been recently shown to share mechanistic features with amyloid self-assembly, bacteria have emerged as a tool to study amyloid aggregation. Herein we present a fast, simple, inexpensive and quantitative method for the screening of potential anti-aggregating drugs. This method is based on monitoring the changes in the binding of thioflavin-S to intracellular IBs in intact Eschericchia coli cells in the presence of small chemical compounds. This in vivo technique fairly recapitulates previous in vitro data. Here we mainly use the Alzheimer"s related beta-amyloid peptide as a model system, but the technique can be easily implemented for screening inhibitors relevant for other conformational diseases simply by changing the recombinant amyloid protein target. Indeed, we show that this methodology can be also applied to the evaluation of inhibitors of the aggregation of tau protein, another amyloidogenic protein with a key role in AD

On the Binding of Congo Red to Amyloid Fibrils

Amyloids are characterized by their capacity to bind Congo red (CR), one of the most used amyloid‐specific dyes. The structural features of CR binding were unknown for years, mainly because of the lack of amyloid structures solved at high resolution. In the last few years, solid‐state NMR spectroscopy enabled the determination of the structural features of amyloids, such as the HET‐s prion forming domain (HET‐s PFD), which also has recently been used to determine the amyloid-CR interface at atomic resolution. Herein, we combine spectroscopic data with molecular docking, molecular dynamics, and excitonic quantum/molecular mechanics calculations to examine and rationalize CR binding to amyloids. In contrast to a previous assumption on the binding mode, our results suggest that CR binding to the HET‐s PFD involves a cooperative process entailing the formation of a complex with 1:1 stoichiometry. This provides a molecular basis to explain the bathochromic shift in the maximal absorbance wavelength when CR is bound to amyloids

Modelos proteicos para el estudio de la agregación amiloide in vitro e in vivo

Durante los últimos años la agregación proteica se ha convertido en un tema de elevada importancia en biología, biotecnología y medicina. Un número creciente de evidencias demuestran fehacientemente que el mal plegamiento de proteínas y su agregación, muchas veces en forma de fibras amiloides, conlleva la formación de depósitos celulares insolubles que son los responsables finales de un creciente número de enfermedades humanas. Este tipo de enfermedades, agrupadas bajo el concepto de enfermedades conformacionales, engloban una gran diversidad de afecciones tanto neurodegenerativas como sistémicas de las que cabria destacar enfermedades con una gran relevancia socioeconómica como pueden ser la enfermedad de Alzheimer, Parkinson, Huntington o la diabetes tipo II entre otras. La producción recombinante en células bacterianas de las proteínas implicadas en este tipo de enfermedades da lugar, muchas veces, a la formación de agregados proteicos, denominados cuerpos de inclusión (IBs), que obstaculizan la obtención de éstas en su forma nativa. Aunque inicialmente se creyó que estos IBs eran simplemente agregados de proteínas plegadas de forma amorfa a causa de interacciones básicamente hidrofóbicas, recientes estudios han demostrado que éstos están compuestos mayoritariamente por proteínas recombinantes producidas que agregan adquiriendo conformaciones amiloides similares a las obtenidas en humanos. Este hecho hace que el estudio de estos IBs bacterianos pueda ser de vital importancia para la comprensión de las enfermedades conformacionales en humanos. Esta tesis está centrada en el estudio de los procesos de agregación proteica y en la caracterización de los agregados formados tanto in vitro como in vivo utilizando como modelo varias proteínas y péptidos sin ningún tipo de homología secuencial ni estructural en un intento de abarcar un amplio abanico conformacional que ilustre el universo conformacional de las proteínas. Son muchos los factores, tanto intrínsecos como extrínsecos a la cadena polipeptídica, que pueden influir en el proceso de formación de fibras amiloides in vitro. El estudio de la influencia de estos determinantes nos permite conocer las interacciones que dirigen la deposición proteica y proponer posibles mecanismos de agregación. Los resultados obtenidos nos han permitido estudiar en detalle el efecto que tienen algunos de estos factores esenciales sobre la agregación proteica, y como su efecto puede variar dependiendo de las características conformacionales de las proteínas. El estudio del efecto de estos determinantes nos ha permitido obtener información sobre las interacciones moleculares que dirigen la formación de las fibras amiloides y de los posibles mecanismos que pueden seguir las proteínas, inicialmente solubles, para adquirir la estructura común en hoja β altamente ordenada. Puesto que las células procariotas se han convertido en sistemas sencillos pero fisiológicamente relevantes para el estudio de la formación de estos agregados, la segunda parte de esta tesis se ha centrado en el estudio biofísico in vivo de los agregados formados en el interior celular utilizando células procariotas como modelo. Los resultados obtenidos demuestran que las proteínas presentes en IBs bacterianos muestran estructuras amiloides comparables a las obtenidas tanto in vitro como en organismos eucariotas, y que los factores estudiados in vitro también pueden afectar, de forma similar, a la formación amiloide in vivo.In recent years, protein aggregation has become a topic of great importance in biology, biotechnology and medicine areas. A growing body of evidences show conclusively that the protein misfolding and aggregation, often in the form of amyloid fibrils, leading to the formation of insoluble cellular deposits that are ultimately responsible for an increasing number of human diseases. Such diseases, grouped under the concept of conformational diseases, encompass a wide variety of neurodegenerative and systemic disorders which can be highlighted with a large socioeconomic importance such as Alzheimer's disease, Parkinson's diseases, Huntington's diseases and type II diabetes among others. The recombinant production of proteins involved in these diseases in bacterial cells leads often to the formation of protein aggregates, called inclusion bodies (IBs), hindering the obtaining of these in their native form. Although it was initially believed that these IBs were simply folded protein aggregates amorphous basically because of hydrophobic interactions, recent studies have shown that these IBs are mainly composed of recombinant proteins acquiring amyloid conformations similar to those in humans. This fact makes the study of these bacterial IBs can be of vital importance for the understanding of conformational diseases in humans. This thesis is based on the study of protein aggregation processes and in the characterization of the aggregates formed in vitro and in vivo using as model various proteins and peptides without any structural or sequence homology. Many factors, both intrinsic and extrinsic to the polypeptide chain, can influence the process of amyloid fibril formation in vitro. The study of the influence of these determinants allows us to understand the interactions that direct protein deposition and suggest possible mechanisms of aggregation. The results obtained have allowed us to study in detail the effect of some of these factors in protein aggregation, and their effect may vary depending on the conformational characteristics of proteins. The study of the effect of these determinants has allowed us to obtain information about the molecular interactions that direct the formation of amyloid fibers and possible mechanisms that may follow the protein, soluble initially to acquire the common structure in highly ordered β sheet. Since prokaryotic cells have become simple but physiologically relevant systems for the study of the formation of these aggregates, the second part of this thesis has focused on biophysical study in vivo of the aggregates formed within the cell using prokaryotic as a model. The results demonstrate that the proteins in bacterial IBs show amyloid-like structures comparable to those obtained in vitro and in eukaryotes, and in vitro study factors can also affect similarly to amyloid formation in vivo