Catalytic amyloids for nucleotide hydrolysis.

The design of small peptides that assemble into catalytically active intermolecular structures has proven to be a successful strategy towards developing minimalistic catalysts that exhibit some of the unique functional features of enzymes. Among these, catalytic amyloids have emerged as a fruitful source to unravel many different activities. These assemblies can potentially have broad applications that range from biotechnology to prebiotic chemistry. Although many peptides that assemble into catalytic amyloids have been developed in recent years, the elucidation of convergent mechanistic aspects of the catalysis and the structure/function relationship is still a challenge. Novel catalytic activities are necessary to better address these issues and expand the current repertoire of applicability. In this chapter, we described a methodology to produce catalytic amyloids that are specifically active towards the hydrolysis of phosphoanhydride bonds of nucleotides. The design of potentially active amyloid-prone peptide sequences is explored using as template the active site of enzymes with nucleotidyltransferase activity. The procedures include an approach for sequence design, in vitro aggregation assays, morphological characterization of the amyloid state and a comprehensive methodology to measure activity in vitro using nucleoside and deoxynucleosides triphosphates as model substrates. The proposed strategy can also be implemented to explore different types of activities for the design of future catalytic amyloids.

Metal Ions Can Modulate the Self-Assembly and Activity of Catalytic Peptide Amyloids.

Rational design of peptides has become a powerful tool to produce self-assembled nanostructures with the ability to catalyze different chemical reactions, paving the way to develop minimalistic enzyme-like nanomaterials. Catalytic amyloid-like assemblies have emerged among the most versatile and active, but they often require additional factors for activity. Elucidating how these factors influence the structure and activity is key for the design. Here, we showed that biologically relevant metal ions can guide and modulate the self-assembly of a small peptide into diverse amyloid architectures. The morphology and catalytic activity of the resulting fibrils were tuned by the specific metal ion decorating the surface, whereas X-ray structural analysis of the amyloids showed ion-dependent shape sizes. Molecular dynamics simulations showed that the metals can strongly affect the local conformational space, which can trigger major rearrangements of the fibrils. Our results demonstrate that the conformational landscape of catalytic amyloids is broad and tunable by external factors, which can be critical for future design strategies.

IGF2 prevents dopaminergic neuronal loss and decreases intracellular alpha-synuclein accumulation in Parkinson's disease models.

Parkinson's disease (PD) is the second most common late-onset neurodegenerative disease and the predominant cause of movement problems. PD is characterized by motor control impairment by extensive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). This selective dopaminergic neuronal loss is in part triggered by intracellular protein inclusions called Lewy bodies, which are composed mainly of misfolded alpha-synuclein (α-syn) protein. We previously reported insulin-like growth factor 2 (IGF2) as a key protein downregulated in PD patients. Here we demonstrated that IGF2 treatment or IGF2 overexpression reduced the α-syn aggregates and their toxicity by IGF2 receptor (IGF2R) activation in cellular PD models. Also, we observed IGF2 and its interaction with IGF2R enhance the α-syn secretion. To determine the possible IGF2 neuroprotective effect in vivo we used a gene therapy approach in an idiopathic PD model based on α-syn preformed fibrils intracerebral injection. IGF2 gene therapy revealed a significantly preventing of motor impairment in idiopathic PD model. Moreover, IGF2 expression prevents dopaminergic neuronal loss in the SN together with a decrease in α-syn accumulation (phospho-α-syn levels) in the striatum and SN brain region. Furthermore, the IGF2 neuroprotective effect was associated with the prevention of synaptic spines loss in dopaminergic neurons in vivo. The possible mechanism of IGF2 in cell survival effect could be associated with the decrease of the intracellular accumulation of α-syn and the improvement of dopaminergic synaptic function. Our results identify to IGF2 as a relevant factor for the prevention of α-syn toxicity in both in vitro and preclinical PD models.

Catalytically Active Amyloids as Future Bionanomaterials.

Peptides and proteins can aggregate into highly ordered and structured conformations called amyloids. These supramolecular structures generally have convergent features, such as the formation of intermolecular beta sheets, that lead to fibrillary architectures. The resulting fibrils have unique mechanical properties that can be exploited to develop novel nanomaterials. In recent years, sequences of small peptides have been rationally designed to self-assemble into amyloids that catalyze several chemical reactions. These amyloids exhibit reactive surfaces that can mimic the active sites of enzymes. In this review, I provide a state-of-the-art summary of the development of catalytically active amyloids. I will focus especially on catalytic activities mediated by hydrolysis, which are the most studied examples to date, as well as novel types of recently reported activities that promise to expand the possible repertoires. The combination of mechanical properties with catalytic activity in an amyloid scaffold has great potential for the development of future bionanomaterials aimed at specific applications.

Design and Testing of Synthetic Catalytic Amyloids Based on the Active Site of Enzymes.

The amyloid fold is nowadays recognized as an alternative conformation accessible to different proteins and peptides. The highly stable and ordered structural organization of amyloid fibrils can be exploited for the design of novel nanomaterials with emergent properties. Recent works have demonstrated that the functional features of the active site of enzymes can be partially recreated using this fold as a scaffold to develop catalytically active amyloids. We describe in this chapter a protocol to design functionally active amyloids that emerge from the self-assembly in vitro of synthetic peptides with sequences based on the active site of enzymes. Using this protocol, we show the development of amyloids that catalyze the metal-dependent hydrolysis of the phosphoanhydride bonds of nucleoside triphosphates.

Recent High-Resolution Structures of Amyloids Involved in Neurodegenerative Diseases.

Amyloids are highly ordered aggregates composed of proteins or peptides. They are involved in several pathologies, including hallmark neurodegenerative disorders such as Alzheimer's (AD) and Parkinson's (PD). Individuals affected by these diseases accumulate in their brains amyloids inclusions composed of misfolded forms of a peptide (Aß) and a protein (Tau) in AD and α-synuclein protein (α-Sn) in PD. Tau and α-Sn aggregates are also present in other neurodegenerative diseases. The insoluble nature and heterogeneity of amyloids have hampered their study at the molecular level. However, the use of solid state NMR and Cryogenic-electron microscopy along with fine-tuned modulation of the aggregation in vitro and improved isolation methods of brain-derived amyloids has allowed the elucidation of these elusive conformations at high resolution. In this work, we review the latest progress on the recent amyloid structures reported for Aß, Tau, and α-Sn. The two-fold symmetry emerges as a convergent feature in the tridimensional arrangement of the protofilaments in the fibrillary structure of these pathological amyloids, with many of them exhibiting a Greek-key topology as part of their overall architecture. These specific features can serve as novel guides to seek potential molecular targets in drug design efforts.

Catalytic Amyloids as Novel Synthetic Hydrolases.

Amyloids are supramolecular assemblies composed of polypeptides stabilized by an intermolecular beta-sheet core. These misfolded conformations have been traditionally associated with pathological conditions such as Alzheimer's and Parkinson´s diseases. However, this classical paradigm has changed in the last decade since the discovery that the amyloid state represents a universal alternative fold accessible to virtually any polypeptide chain. Moreover, recent findings have demonstrated that the amyloid fold can serve as catalytic scaffolds, creating new opportunities for the design of novel active bionanomaterials. Here, we review the latest advances in this area, with particular emphasis on the design and development of catalytic amyloids that exhibit hydrolytic activities. To date, three different types of activities have been demonstrated: esterase, phosphoesterase and di-phosphohydrolase. These artificial hydrolases emerge upon the self-assembly of small peptides into amyloids, giving rise to catalytically active surfaces. The highly stable nature of the amyloid fold can provide an attractive alternative for the design of future synthetic hydrolases with diverse applications in the industry, such as the in situ decontamination of xenobiotics.

Functional characterization of the ATPase-like activity displayed by a catalytic amyloid.

BACKGROUND: Amyloids are highly ordered polypeptide aggregates stabilized by a beta-sheet structural core. Though classically associated to pathology, reports on novel functional roles of these proteins have increasingly emerged in the past decade. Moreover, the recent discovery that amyloids formed with rationally designed small peptides can exhibit catalytic reactivity has opened up new opportunities in both biology and biotechnology. The observed activities typically require the binding of divalent metals, giving rise to active metal-amyloid complexes. METHODS: Peptide (SDIDVFI) was aggregated in vitro. The structure of the self-assembled species was analyzed using fluorescence, transmission electron microscopy, circular dichroism and computational modeling. A kinetic characterization of the emerging catalytic activity was performed. RESULTS: The peptide self-assembled into canonical amyloids that exhibited catalytic activity towards hydrolysis of the phosphoanhydride bonds of adenosine triphosphate (ATP), partially mimicking an ATPase-like enzyme. Both amyloid formation and activity are shown to depend on manganese (Mn2+) binding. The activity was not restricted to ATP but also affected all other ribonucleotides (GTP, CTP and UTP). Peptides carrying a single aspartate exhibited a similar activity. CONCLUSIONS: The phosphoanhydride bonds appear as the main specificity target of the Mn2+-amyloid complex. A single aspartate per peptide is sufficient to enable the hydrolytic activity. GENERAL SIGNIFICANCE: Catalytic amyloids are shown for the first time to catalyze the hydrolysis of all four ribonucleotides. Our results should contribute towards understanding the biological implications of amyloid-mediated reactivity as well as in the design of future catalytic amyloids for biotechnological applications.

Prion-like characteristics of the bacterial protein Microcin E492.

Microcin E492 (Mcc) is a pore-forming bacteriotoxin. Mcc activity is inhibited at the stationary phase by formation of amyloid-like aggregates in the culture. Here we report that, in a similar manner as prions, Mcc naturally exists as two conformers: a ß-sheet-rich, protease-resistant, aggregated, inactive form (Mccia), and a soluble, protease-sensitive, active form (Mcca). The exogenous addition of culture medium containing Mccia or purified in vitro-generated Mccia into the culture induces the rapid and efficient conversion of Mcca into Mccia, which is maintained indefinitely after passaging, changing the bacterial phenotype. Mccia prion-like activity is conformation-dependent and could be reduced by immunodepleting Mccia. Interestingly, an internal region of Mcc shares sequence similarity with the central domain of the prion protein, which is key to the formation of mammalian prions. A synthetic peptide spanning this sequence forms amyloid-like fibrils in vitro and is capable of inducing the conversion of Mcca into Mccia in vivo, suggesting that this region corresponds to the prion domain of Mcc. Our findings suggest that Mcc is the first prokaryotic protein with prion properties which harnesses prion-like transmission to regulate protein function, suggesting that propagation of biological information using a prion-based conformational switch is an evolutionary conserved mechanism.

Overcoming electrostatic repulsions during amyloid assembly: Effect of pH and interaction with divalent metals using model peptides.

Amyloids are polypeptide aggregates involved in many pathologies including Alzheimer's disease. Amyloid assembly is a complex process affected by different interactions including hydrogen bonding, van der Waals forces and electrostatic interactions. The highly regular amyloid structure allows for an arrangement of residues that forces side chains to be closely positioned, giving rise to potentially unfavorable interactions such as electrostatic repulsions. In these cases, amyloid assembly will depend on a balance between stabilizing versus unfavorable interactions. In this study, we rationally designed several amyloid-prone model peptides that had two acidic groups and tested their assembly into amyloids under different conditions. We found that at low pH (pH 4.0), most peptides spontaneously formed amyloids whereas no or little aggregation was observed at higher pHs (pH 8.0). When divalent metals with affinity for carboxylate groups were added at millimolar concentrations, most peptides exhibited a metal-dependent switch to the amyloid state at pH 8.0. Our results show that electrostatic repulsion between amyloid-prone sequences can be overcome in conditions that affect protonation of residue side chains. Moreover, the presence of divalent metals can contribute to electrostatic shielding through specific coordination with acidic groups and thus promote amyloid assembly.

Development of a novel catalytic amyloid displaying a metal-dependent ATPase-like activity.

Amyloids are protein aggregates of highly regular structure that are involved in diverse pathologies such as Alzheimer's and Parkinson's disease. Recent evidence has shown that under certain conditions, small peptides can self-assemble into amyloids that exhibit catalytic reactivity towards certain compounds. Here we report a novel peptide with a sequence derived from the active site of RNA polymerase that displays hydrolytic activity towards ATP. The catalytic reaction proceeds in the presence of the divalent metal manganese and the products are ADP and AMP. The kinetic data shows a substrate-dependent saturation of the activity with a maximum rate achieved at around 1 mM ATP. At higher ATP concentrations, we also observed substrate inhibition of the activity. The self-assembly of the peptide into amyloids is strictly metal-dependent and required for the catalysis. Our results show that aspartate-containing amyloids can also be catalysts under conditions that include interactions with metals. Moreover, we show for the first time an amyloid that exerts reactivity towards a biologically essential molecule.

Strain-dependent profile of misfolded prion protein aggregates.

Prions are composed of the misfolded prion protein (PrP(Sc)) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrP(Sc) aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrP(Sc) aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrP(Sc) aggregates distribute in a wide range of arrangements and the relative proportion of each species depends on the prion strain. We observed a direct correlation between the density of the predominant PrP(Sc) aggregates and the incubation periods for the strains studied. The relative presence of PrP(Sc) in fractions of different sucrose densities was indicative of the protein deposits present in the brain as analyzed by histology. Interestingly, no association was found between sensitivity to proteolytic degradation and aggregation profiles. Therefore, the organization of PrP molecules in terms of the density of aggregates generated may determine some of the particular strain properties, whereas others are independent from it. Our findings may contribute to understand the mechanisms of strain variation and the role of PrP(Sc) aggregates in prion-induced neurodegeneration.

The extent of protease resistance of misfolded prion protein is highly dependent on the salt concentration.

Transmissible spongiform encephalopathies are neurodegenerative diseases caused by prions in mammals. An aberrantly folded protein (PrP(Sc)) is the main component of these proteinaceous infectious particles. Prions exhibit strong resistance to protease digestion, which is typically exploited for biochemical discrimination from its native cellular form (PrP(C)). This classical feature has been partially challenged by the isolation of sizeable amounts of protease-sensitive PrP(Sc) isoforms that self-propagate in vivo. Here, we report that the degree of PrP(Sc) protease resistance is highly dependent on the concentration of salt in the solution. Similar changes were observed in PrP(Sc) obtained from different strains and species. Strikingly, the effect of salt is reversible and is associated with changes on the size of PrP(Sc) particles, but surprisingly, the more protease-sensitive species consists of a larger size. These findings shed light on the mechanistic aspects of prion proteolysis and should be considered when assessing samples of biomedical relevance.

Protein misfolding cyclic amplification of infectious prions.

Prions are proteinaceous infectious agents responsible for the transmission of prion diseases. The lack of a procedure for cultivating prions in the laboratory has been a major limitation to the study of the unorthodox nature of this infectious agent and the molecular mechanism by which the normal prion protein (PrP(C)) is converted into the abnormal isoform (PrP(Sc)). Protein misfolding cyclic amplification (PMCA), described in detail in this protocol, is a simple, fast and efficient methodology to mimic prion replication in the test tube. PMCA involves incubating materials containing minute amounts of infectious prions with an excess of PrP(C) and boosting the conversion by cycles of sonication to fragment the converting units, thereby leading to accelerated prion replication. PMCA is able to detect the equivalent of a single molecule of infectious PrP(Sc) and propagate prions that maintain high infectivity, strain properties and species specificity. A single PMCA assay takes little more than 3 d to replicate a large amount of prions, which could take years in an in vivo situation. Since its invention 10 years ago, PMCA has helped to answer fundamental questions about this intriguing infectious agent and has been broadly applied in research areas that include the food industry, blood bank safety and human and veterinary disease diagnosis.

High-resolution structure of infectious prion protein: the final frontier.

Prions are the proteinaceous infectious agents responsible for the transmission of prion diseases. The main or sole component of prions is the misfolded prion protein (PrP(Sc)), which is able to template the conversion of the host's natively folded form of the protein (PrP(C)). The detailed mechanism of prion replication and the high-resolution structure of PrP(Sc) are unknown. The currently available information on PrP(Sc) structure comes mostly from low-resolution biophysical techniques, which have resulted in quite divergent models. Recent advances in the production of infectious prions, using very pure recombinant protein, offer new hope for PrP(Sc) structural studies. This review highlights the importance of, challenges for and recent progress toward elucidating the elusive structure of PrP(Sc), arguably the major pending milestone to reach in understanding prions.

Kosmotropic anions promote conversion of recombinant prion protein into a PrPSc-like misfolded form.

Prions are self-propagating proteins involved in transmissible spongiform encephalopaties in mammals. An aberrant conformation with amyloid-like features of a cell surface protein, termed prion protein (PrP), is thought to be the essential component of the infectious particle, though accessory co-factor molecules such as lipids and nucleotides may be involved. The cellular co-factors and environmental conditions implicated in PrP misfolding are not completely understood. To address this issue, several studies have been done inducing misfolding of recombinant PrP (recPrP) into classical amyloid structures using partially denaturing conditions. In this work, we report that misfolding of recPrP into PrP(Sc)-like aggregates can be induced by simply incubating the protein in the presence of kosmotropic salts at concentrations that are known to retain or increase the stability of the protein. We used a simple experimental reaction (protein, buffer and salts) submitted to agitation/incubation cycles at physiological temperature and pH. The formation of protease resistant-recPrP was time and salt-concentration dependent and required the presence of kosmotropic anions such as F(-) or SO(4)(-2). The molecular weights of the protease resistant recPrP fragments are reminiscent of those found in degradation assays of bona fide PrP(Sc). The aggregates also exhibited PrP(Sc)-like ultrastructural features including rod-shape morphology under electron microscope, high beta-sheet content and thioflavin-T positive signal. The formation of recPrP aggregates with PrP(Sc) biochemical features under conditions closer to physiological in the absence of organic co-factor molecules provides a simple setup that may prove helpful to understand the molecular mechanism of PrP misfolding.

Generation of prions in vitro and the protein-only hypothesis.

Prions are self-propagating proteinaceous infectious agents capable of transmitting disease in the absence of nucleic acids. The nature of the infectious agent in prion diseases has been at the center of passionate debate for the past 30 years. However, recent reports on the in vitro generation of prions have settled all doubts that the misfolded prion protein (PrP(Sc)) is the key component in propagating infectivity. However, we still do not understand completely the mechanism of prion replication and whether or not other cellular factors besides PrP(Sc) are required for infectivity. In this article, we discuss these recent reports under the context of the protein-only hypothesis and their implications.

Molecular cross talk between misfolded proteins in animal models of Alzheimer's and prion diseases.

The central event in protein misfolding disorders (PMDs) is the accumulation of a misfolded form of a naturally expressed protein. Despite the diversity of clinical symptoms associated with different PMDs, many similarities in their mechanism suggest that distinct pathologies may cross talk at the molecular level. The main goal of this study was to analyze the interaction of the protein misfolding processes implicated in Alzheimer's and prion diseases. For this purpose, we inoculated prions in an Alzheimer's transgenic mouse model that develop typical amyloid plaques and followed the progression of pathological changes over time. Our findings show a dramatic acceleration and exacerbation of both pathologies. The onset of prion disease symptoms in transgenic mice appeared significantly faster with a concomitant increase on the level of misfolded prion protein in the brain. A striking increase in amyloid plaque deposition was observed in prion-infected mice compared with their noninoculated counterparts. Histological and biochemical studies showed the association of the two misfolded proteins in the brain and in vitro experiments showed that protein misfolding can be enhanced by a cross-seeding mechanism. These results suggest a profound interaction between Alzheimer's and prion pathologies, indicating that one protein misfolding process may be an important risk factor for the development of a second one. Our findings may have important implications to understand the origin and progression of PMDs.