Is a cure for Parkinson's disease hiding inside us?

α-Synuclein (a-syn) oligomers and fibrils are behind neurodegeneration in Parkinson's disease (PD), but therapeutically targeting them is challenging. Amphipathic and cationic helical peptides inhibit amyloid formation and suppress neurotoxicity by selectively binding the solvent-accessible regions in these toxic species. Can endogenous peptides, like LL-37, constitute a new therapeutic paradigm in PD?

Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates, and delays senescence.

Stress granules (SGs) are highly conserved cytoplasmic condensates that assemble in response to stress and contribute to maintaining protein homeostasis. These membraneless organelles are dynamic, disassembling once the stress is no longer present. Persistence of SGs due to mutations or chronic stress has been often related to age-dependent protein-misfolding diseases in animals. Here, we find that the metacaspase MC1 is dynamically recruited into SGs upon proteotoxic stress in Arabidopsis (Arabidopsis thaliana). Two predicted disordered regions, the prodomain and the 360 loop, mediate MC1 recruitment to and release from SGs. Importantly, we show that MC1 has the capacity to clear toxic protein aggregates in vivo and in vitro, acting as a disaggregase. Finally, we demonstrate that overexpressing MC1 delays senescence and this phenotype is dependent on the presence of the 360 loop and an intact catalytic domain. Together, our data indicate that MC1 regulates senescence through its recruitment into SGs and this function could potentially be linked to its remarkable protein aggregate-clearing activity.

A Targetable N-Terminal Motif Orchestrates α-Synuclein Oligomer-to-Fibril Conversion.

Oligomeric species populated during α-synuclein aggregation are considered key drivers of neurodegeneration in Parkinson's disease. However, the development of oligomer-targeting therapeutics is constrained by our limited knowledge of their structure and the molecular determinants driving their conversion to fibrils. Phenol-soluble modulin α3 (PSMα3) is a nanomolar peptide binder of α-synuclein oligomers that inhibits aggregation by blocking oligomer-to-fibril conversion. Here, we investigate the binding of PSMα3 to α-synuclein oligomers to discover the mechanistic basis of this protective activity. We find that PSMα3 selectively targets an α-synuclein N-terminal motif (residues 36-61) that populates a distinct conformation in the mono- and oligomeric states. This α-synuclein region plays a pivotal role in oligomer-to-fibril conversion as its absence renders the central NAC domain insufficient to prompt this structural transition. The hereditary mutation G51D, associated with early onset Parkinson's disease, causes a conformational fluctuation in this region, leading to delayed oligomer-to-fibril conversion and an accumulation of oligomers that are resistant to remodeling by molecular chaperones. Overall, our findings unveil a new targetable region in α-synuclein oligomers, advance our comprehension of oligomer-to-amyloid fibril conversion, and reveal a new facet of α-synuclein pathogenic mutations.

Design, synthesis and structure-activity evaluation of novel 2-pyridone-based inhibitors of α-synuclein aggregation with potentially improved BBB permeability.

The treatment of Parkinson's disease (PD), the second most common neurodegenerative human disorder, continues to be symptomatic. Development of drugs able to stop or at least slowdown PD progression would benefit several million people worldwide. SynuClean-D is a low molecular weight 2-pyridone-based promising drug candidate that inhibits the aggregation of α-synuclein in human cultured cells and prevents degeneration of dopaminergic neurons in a Caenorhabditis elegans model of PD. Improving SynuClean-D pharmacokinetic/pharmacodynamic properties, performing structure/activity studies and testing its efficacy in mammalian models of PD requires the use of gr-amounts of the compound. However, not enough compound is on sale, and no synthetic route has been reported until now, which hampers the molecule progress towards clinical trials. To circumvent those problems, we describe here an efficient and economical route that enables the synthesis of SynuClean-D with good yields as well as the synthesis of SynuClean-D derivatives. Structure-activity comparison of the new compounds with SynuClean-D reveals the functional groups of the molecule that can be disposed of without activity loss and those that are crucial to interfere with α-synuclein aggregation. Several of the derivatives obtained retain the parent's compound excellent in vitro anti-aggregative activity, without compromising its low toxicity. Computational predictions and preliminary testing indicate that the blood brain barrier (BBB) permeability of SynuClean-D is low. Importantly, several of the newly designed and obtained active derivatives are predicted to display good BBB permeability. The synthetic route developed here will facilitate their synthesis for BBB permeability determination and for efficacy testing in mammalian models of PD.

A pyrene-inhibitor fluorescent probe with large Stokes shift for the staining of Aß1-42, α-synuclein, and amylin amyloid fibrils as well as amyloid-containing Staphylococcus aureus biofilms.

Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer's disease, Parkinson's disease, and type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity toward that diversity of amyloid fibrils or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here we show the feasibility of the approach by designing, synthesizing, and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aß1-42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The most soluble derivative, N-acetyl-2-(2-methyl-4-oxo-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-d][1,3]oxazin-7-yl)-N-(pyren-1-ylmethyl)acetamide (compound 1D), stains similarly well amyloid fibrils formed by Aß1-42, α-synuclein, or amylin, provides a sensitivity only slightly lower than that of thioflavin T, displays a large Stokes shift, allows efficient excitation in the UV spectral region, and is not cytotoxic. Compound 1D can also stain amyloid fibrils formed by staphylococcal peptides present in biofilm matrices and can be used to distinguish, by direct staining, Staphylococcus aureus biofilms containing amyloid-forming phenol-soluble modulins from those lacking them. Graphical abstract ᅟ.

The Disordered C-Terminus of Yeast Hsf1 Contains a Cryptic Low-Complexity Amyloidogenic Region.

Response mechanisms to external stress rely on networks of proteins able to activate specific signaling pathways to ensure the maintenance of cell proteostasis. Many of the proteins mediating this kind of response contain intrinsically disordered regions, which lack a defined structure, but still are able to interact with a wide range of clients that modulate the protein function. Some of these interactions are mediated by specific short sequences embedded in the longer disordered regions. Because the physicochemical properties that promote functional and abnormal interactions are similar, it has been shown that, in globular proteins, aggregation-prone and binding regions tend to overlap. It could be that the same principle applies for disordered protein regions. In this context, we show here that a predicted low-complexity interacting region in the disordered C-terminus of the stress response master regulator heat shock factor 1 (Hsf1) protein corresponds to a cryptic amyloid region able to self-assemble into fibrillary structures resembling those found in neurodegenerative disorders.

Understanding and predicting protein misfolding and aggregation: Insights from proteomics.

Protein misfolding and aggregation are being found to be associated with an increasing number of human diseases and premature aging, either because they promote a loss of protein function or, more frequently, because the aggregated species gain a toxic activity. Despite potentially harmful, aggregation seems to be a generic property of polypeptide chains and aggregation-prone protein sequences seem to be ubiquitous, which, counterintuitively, suggests that they serve evolutionary conserved functions. The in vitro study of individual aggregation reactions of a large number of proteins has provided important insights on the structural and sequential determinants of this process. However, it is clear that understanding the role played by protein aggregation and its regulation in health and disease at the cellular, developmental, and evolutionary levels require more global approaches. The use of model organisms and their proteomic analysis hold the power to provide answers to such issues. In the present review, we address how, initially, computational large-scale analysis and, more recently, experimental proteomics are helping us to rationalize how, why and when proteins aggregate, as well as to decipher the strategies organisms have developed to control proteins aggregation propensities.

Structure of activated thrombin-activatable fibrinolysis inhibitor, a molecular link between coagulation and fibrinolysis.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a metallocarboxypeptidase (MCP) that links blood coagulation and fibrinolysis. TAFI hampers fibrin-clot lysis and is a pharmacological target for the treatment of thrombotic conditions. TAFI is transformed through removal of its prodomain by thrombin-thrombomodulin into TAFIa, which is intrinsically unstable and has a short half-life in vivo. Here we show that purified bovine TAFI activated in the presence of a proteinaceous inhibitor renders a stable enzyme-inhibitor complex. Its crystal structure reveals that TAFIa conforms to the alpha/beta-hydrolase fold of MCPs and displays two unique flexible loops on the molecular surface, accounting for structural instability and susceptibility to proteolysis. In addition, point mutations reported to enhance protein stability in vivo are mainly located in the first loop and in another surface region, which is a potential heparin-binding site. The protein inhibitor contacts both the TAFIa active site and an exosite, thus contributing to high inhibitory efficiency.

An α-helical peptide-based plasmonic biosensor for highly specific detection of α-synuclein toxic oligomers.

BACKGROUND: α-Synuclein (αS) aggregation is the main neurological hallmark of a group of neurodegenerative disorders, collectively referred to as synucleinopathies, of which Parkinson's disease (PD) is the most prevalent. αS oligomers are elevated in the cerebrospinal fluid (CSF) of PD patients, standing as a biomarker for disease diagnosis. However, methods for early PD detection are still lacking. We have recently identified the amphipathic 22-residue peptide PSMα3 as a high-affinity binder of αS toxic oligomers. PSMα3 displayed excellent selectivity and reproducibility, binding to αS toxic oligomers with affinities in the low nanomolar range and without detectable cross-reactivity with functional monomeric αS. RESULTS: In this work, we leveraged these PSMα3 unique properties to design a plasmonic-based biosensor for the direct detection of toxic oligomers under label-free conditions. SIGNIFICANCE AND NOVELTY: We describe the integration of the peptide in a lab-on-a-chip plasmonic platform suitable for point-of-care measurements of αS toxic oligomers in CSF samples in real-time and at an affordable cost, providing an innovative biosensor for PD early diagnosis in the clinic.

A functional and structural study of the major metalloprotease secreted by the pathogenic fungus Aspergillus fumigatus.

Fungalysins are secreted fungal peptidases with the ability to degrade the extracellular matrix proteins elastin and collagen and are thought to act as virulence factors in diseases caused by fungi. Fungalysins constitute a unique family among zinc-dependent peptidases that bears low sequence similarity to known bacterial peptidases of the thermolysin family. The crystal structure of the archetype of the fungalysin family, Aspergillus fumigatus metalloprotease (AfuMep), has been obtained for the first time. The 1.8 Šresolution structure of AfuMep corresponds to that of an autoproteolyzed proenzyme with separate polypeptide chains corresponding to the N-terminal prodomain in a binary complex with the C-terminal zinc-bound catalytic domain. The prodomain consists of a tandem of cystatin-like folds whose C-terminal end is buried into the active-site cleft of the catalytic domain. The catalytic domain harbouring the key catalytic zinc ion and its ligands, two histidines and one glutamic acid, undergoes a conspicuous rearrangement of its N-terminal end during maturation. One key positively charged amino-acid residue and the C-terminal disulfide bridge appear to contribute to its structural-functional properties. Thus, structural, biophysical and biochemical analysis were combined to provide a deeper comprehension of the underlying properties of A. fumigatus fungalysin, serving as a framework for the as yet poorly known metallopeptidases from pathogenic fungi.

A glimpse into the structural properties of α-synuclein oligomers.

α-Synuclein (αS) aggregation is the main neurological hallmark of a group of debilitating neurodegenerative disorders, collectively referred to as synucleinopathies, of which Parkinson's disease is the most prevalent. αS oligomers formed during the initial stages of aggregation are considered key pathogenic drivers of disease onset and progression, standing as privileged targets for therapeutic intervention and diagnosis. However, the structure of αS oligomers and the mechanistic basis of oligomer to fibril conversion are yet poorly understood, thereby precluding the rational formulation of strategies aimed at targeting oligomeric species. In this review, we delve into the recent advances in the structural and mechanistic characterization of αS oligomers. We also discuss how these advances are transforming our understanding of these elusive species and paving the way for oligomer-targeting therapeutics and diagnosis.

aSynPEP-DB: a database of biogenic peptides for inhibiting α-synuclein aggregation.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder, yet effective treatments able to stop or delay disease progression remain elusive. The aggregation of a presynaptic protein, α-synuclein (aSyn), is the primary neurological hallmark of PD and, thus, a promising target for therapeutic intervention. However, the lack of consensus on the molecular properties required to specifically bind the toxic species formed during aSyn aggregation has hindered the development of therapeutic molecules. Recently, we defined and experimentally validated a peptide architecture that demonstrated high affinity and selectivity in binding to aSyn toxic oligomers and fibrils, effectively preventing aSyn pathogenic aggregation. Human peptides with such properties may have neuroprotective activities and hold a huge therapeutic interest. Driven by this idea, here, we developed a discriminative algorithm for the screening of human endogenous neuropeptides, antimicrobial peptides and diet-derived bioactive peptides with the potential to inhibit aSyn aggregation. We identified over 100 unique biogenic peptide candidates and ensembled a comprehensive database (aSynPEP-DB) that collects their physicochemical features, source datasets and additional therapeutic-relevant information, including their sites of expression and associated pathways. Besides, we provide access to the discriminative algorithm to extend its application to the screening of artificial peptides or new peptide datasets. aSynPEP-DB is a unique repository of peptides with the potential to modulate aSyn aggregation, serving as a platform for the identification of previously unexplored therapeutic agents. Database URL:  https://asynpepdb.ppmclab.com/.

PITB: A high affinity transthyretin aggregation inhibitor with optimal pharmacokinetic properties.

The aggregation of wild-type transthyretin (TTR) and over 130 genetic TTR variants underlies a group of lethal disorders named TTR amyloidosis (ATTR). TTR chemical chaperones are molecules that hold great promise to modify the course of ATTR progression. In previous studies, we combined rational design and molecular dynamics simulations to generate a series of TTR selective kinetic stabilizers displaying exceptionally high affinities. In an effort to endorse the previously developed molecules with optimal pharmacokinetic properties, we conducted structural design optimization, leading to the development of PITB. PITB binds with high affinity to TTR, effectively inhibiting tetramer dissociation and aggregation of both the wild-type protein and the two most prevalent disease-associated TTR variants. Importantly, PITB selectively binds and stabilizes TTR in plasma, outperforming tolcapone, a drug currently undergoing clinical trials for ATTR. Pharmacokinetic studies conducted on mice confirmed that PITB exhibits encouraging pharmacokinetic properties, as originally intended. Furthermore, PITB demonstrates excellent oral bioavailability and lack of toxicity. These combined attributes position PITB as a lead compound for future clinical trials as a disease-modifying therapy for ATTR.

Exploring cryptic amyloidogenic regions in prion-like proteins from plants.

Prion-like domains (PrLDs) are intrinsically disordered regions (IDRs) of low sequence complexity with a similar composition to yeast prion domains. PrLDs-containing proteins have been involved in different organisms' regulatory processes. Regions of moderate amyloid propensity within IDRs have been shown to assemble autonomously into amyloid fibrils. These sequences tend to be rich in polar amino acids and often escape from the detection of classical bioinformatics screenings that look for highly aggregation-prone hydrophobic sequence stretches. We defined them as cryptic amyloidogenic regions (CARs) and recently developed an integrated database that collects thousands of predicted CARs in IDRs. CARs seem to be evolutionary conserved among disordered regions because of their potential to stablish functional contacts with other biomolecules. Here we have focused on identifying and characterizing CARs in prion-like proteins (pCARs) from plants, a lineage that has been poorly studied in comparison with other prionomes. We confirmed the intrinsic amyloid potential for a selected pCAR from Arabidopsis thaliana and explored functional enrichments and compositional bias of pCARs in plant prion-like proteins.

CARs-DB: A Database of Cryptic Amyloidogenic Regions in Intrinsically Disordered Proteins.

Proteome-wide analyses suggest that most globular proteins contain at least one amyloidogenic region, whereas these aggregation-prone segments are thought to be underrepresented in intrinsically disordered proteins (IDPs). In recent work, we reported that intrinsically disordered regions (IDRs) indeed sustain a significant amyloid load in the form of cryptic amyloidogenic regions (CARs). CARs are widespread in IDRs, but they are necessarily exposed to solvent, and thus they should be more polar and have a milder aggregation potential than conventional amyloid regions protected inside globular proteins. CARs are connected with IDPs function and, in particular, with the establishment of protein-protein interactions through their IDRs. However, their presence also appears associated with pathologies like cancer or Alzheimer's disease. Given the relevance of CARs for both IDPs function and malfunction, we developed CARs-DB, a database containing precomputed predictions for all CARs present in the IDPs deposited in the DisProt database. This web tool allows for the fast and comprehensive exploration of previously unnoticed amyloidogenic regions embedded within IDRs sequences and might turn helpful in identifying disordered interacting regions. It contains >8,900 unique CARs identified in a total of 1711 IDRs. CARs-DB is freely available for users and can be accessed at http://carsdb.ppmclab.com. To validate CARs-DB, we demonstrate that two previously undescribed CARs selected from the database display full amyloidogenic potential. Overall, CARs-DB allows easy access to a previously unexplored amyloid sequence space.

Development of a Highly Potent Transthyretin Amyloidogenesis Inhibitor: Design, Synthesis, and Evaluation.

Transthyretin amyloidosis (ATTR) is a group of fatal diseases described by the misfolding and amyloid deposition of transthyretin (TTR). Discovering small molecules that bind and stabilize the TTR tetramer, preventing its dissociation and subsequent aggregation, is a therapeutic strategy for these pathologies. Departing from the crystal structure of TTR in complex with tolcapone, a potent binder in clinical trials for ATTR, we combined rational design and molecular dynamics (MD) simulations to generate a series of novel halogenated kinetic stabilizers. Among them, M-23 displays one of the highest affinities for TTR described so far. The TTR/M-23 crystal structure confirmed the formation of unprecedented protein-ligand contacts, as predicted by MD simulations, leading to an enhanced tetramer stability both in vitro and in whole serum. We demonstrate that MD-assisted design of TTR ligands constitutes a new avenue for discovering molecules that, like M-23, hold the potential to become highly potent drugs to treat ATTR.

Cryptic amyloidogenic regions in intrinsically disordered proteins: Function and disease association.

The amyloid conformation is considered a fundamental state of proteins and the propensity to populate it a generic property of polypeptides. Multiple proteome-wide analyses addressed the presence of amyloidogenic regions in proteins, nurturing our understanding of their nature and biological implications. However, these analyses focused on highly aggregation-prone and hydrophobic stretches that are only marginally found in intrinsically disordered regions (IDRs). Here, we explore the prevalence of cryptic amyloidogenic regions (CARs) of polar nature in IDRs. CARs are widespread in IDRs and associated with IDPs function, with particular involvement in protein-protein interactions, but their presence is also connected to a risk of malfunction. By exploring this function/malfunction dichotomy, we speculate that ancestral CARs might have evolved into functional interacting regions playing a significant role in protein evolution at the origins of life.

Prion-like proteins: from computational approaches to proteome-wide analysis.

Prions are self-perpetuating proteins able to switch between a soluble state and an aggregated-and-transmissible conformation. These proteinaceous entities have been widely studied in yeast, where they are involved in hereditable phenotypic adaptations. The notion that such proteins could play functional roles and be positively selected by evolution has triggered the development of computational tools to identify prion-like proteins in different kingdoms of life. These algorithms have succeeded in screening multiple proteomes, allowing the identification of prion-like proteins in a diversity of unrelated organisms, evidencing that the prion phenomenon is well conserved among species. Interestingly enough, prion-like proteins are not only connected with the formation of functional membraneless protein-nucleic acid coacervates, but are also linked to human diseases. This review addresses state-of-the-art computational approaches to identify prion-like proteins, describes proteome-wide analysis efforts, discusses these unique proteins' functional role, and illustrates recently validated examples in different domains of life.

Tolcapone, a potent aggregation inhibitor for the treatment of familial leptomeningeal amyloidosis.

Hereditary transthyretin amyloidosis (ATTR) is a disease characterized by the extracellular deposition of transthyretin (TTR) amyloid fibrils. Highly destabilizing TTR mutations cause leptomeningeal amyloidosis, a rare, but fatal, disorder in which TTR aggregates in the brain. The disease remains intractable, since liver transplantation, the reference therapy for systemic ATTR, does not stop mutant TTR production in the brain. In addition, despite current pharmacological strategies have shown to be effective against in vivo TTR aggregation by stabilizing the tetramer native structure and precluding its dissociation, they display low brain permeability. Recently, we have repurposed tolcapone as a molecule to treat systemic ATTR. Crystal structures and biophysical analysis converge to demonstrate that tolcapone binds with high affinity and specificity to three unstable leptomeningeal TTR variants, stabilizing them and, consequently, inhibiting their aggregation. Because tolcapone is an FDA-approved drug that crosses the blood-brain barrier, our results suggest that it can translate into a first disease-modifying therapy for leptomeningeal amyloidosis. DATABASES: PDB codes for A25T-TTR, V30G-TTR, and Y114C-TTR bound to tolcapone are 6TXV, 6TXW, and 6XTK, respectively.

α-Helical peptidic scaffolds to target α-synuclein toxic species with nanomolar affinity.

α-Synuclein aggregation is a key driver of neurodegeneration in Parkinson's disease and related syndromes. Accordingly, obtaining a molecule that targets α-synuclein toxic assemblies with high affinity is a long-pursued objective. Here, we exploit the biophysical properties of toxic oligomers and amyloid fibrils to identify a family of α-helical peptides that bind to these α-synuclein species with low nanomolar affinity, without interfering with the monomeric functional protein. This activity is translated into a high anti-aggregation potency and the ability to abrogate oligomer-induced cell damage. Using a structure-guided search we identify a human peptide expressed in the brain and the gastrointestinal tract with analogous binding, anti-aggregation, and detoxifying properties. The chemical entities we describe here may represent a therapeutic avenue for the synucleinopathies and are promising tools to assist diagnosis by discriminating between native and toxic α-synuclein species.