Amyloid Conformation Research Articles

α-Synuclein Strains: Does Amyloid Conformation Explain the Heterogeneity of Synucleinopathies?

Synucleinopathies are a heterogeneous group of neurodegenerative diseases with amyloid deposits that contain the α-synuclein (SNCA/α-Syn) protein as a common hallmark. It is astonishing that aggregates of a single protein are able to give rise to a whole range of different disease manifestations. The prion strain hypothesis offers a possible explanation for this conundrum. According to this hypothesis, a single protein sequence is able to misfold into distinct amyloid structures that can cause different pathologies. In fact, a growing body of evidence suggests that conformationally distinct α-Syn assemblies might be the causative agents behind different synucleinopathies. In this review, we provide an overview of research on the strain hypothesis as it applies to synucleinopathies and discuss the potential implications for diagnostic and therapeutic purposes.

Phe-Gly motifs drive fibrillization of TDP-43's prion-like domain condensates.

Transactive response DNA-binding Protein of 43 kDa (TDP-43) assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g., muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: Salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct end point species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions and illustrate structural features that distinguishes between aggregate forms.

NMR Characterization of Angiogenin Variants and tRNAAla Products Impacting Aberrant Protein Oligomerization.

Protein oligomerization is key to countless physiological processes, but also to abnormal amyloid conformations implicated in over 25 mortal human diseases. Human Angiogenin (h-ANG), a ribonuclease A family member, produces RNA fragments that regulate ribosome formation, the creation of new blood vessels and stress granule function. Too little h-ANG activity leads to abnormal protein oligomerization, resulting in Amyotrophic Lateral Sclerosis (ALS) or Parkinson’s disease. While a score of disease linked h-ANG mutants has been studied by X-ray diffraction, some elude crystallization. There is also a debate regarding the structure that RNA fragments adopt after cleavage by h-ANG. Here, to better understand the beginning of the process that leads to aberrant protein oligomerization, the solution secondary structure and residue-level dynamics of WT h-ANG and two mutants i.e., H13A and R121C, are characterized by multidimensional heteronuclear NMR spectroscopy under near-physiological conditions. All three variants are found to adopt well folded and highly rigid structures in the solution, although the elements of secondary structure are somewhat shorter than those observed in crystallography studies. R121C alters the environment of nearby residues only. By contrast, the mutation H13A affects local residues as well as nearby active site residues K40 and H114. The conformation characterization by CD and 1D 1H NMR spectroscopies of tRNAAla before and after h-ANG cleavage reveals a retention of the duplex structure and little or no G-quadruplex formation.

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.

Volumetric multispectral optoacoustic tomography of beta‐amyloid deposits in whole mouse brain

AbstractBackgroundThe abnormal deposition of fibrillar beta‐amyloid deposits in the brain is one of the major histopathological hallmarks of Alzheimer’s disease. Currently 3D imaging using positron emission tomography for plaque visualization is of a limited resolution (1 mm) in relation to the size of mouse brain in widely used AD models.MethodWe developed a novel high‐resolution non‐invasive volumetric multi‐spectral optoacoustic tomography (vMSOT) of 100 mm resolution using amyloid probe AOI987 to visualize amyloid‐beta distribution in arcAβ and APP/PS1 transgenic mouse models of cerebral amyloidosis. Immunohistochemical staining was performed with AOI987, anti‐Aβ antibody 6E10 and fibrillar amyloid conformation antibody OC on mouse brain sections.Result In vivo vMSOT detects higher Aβ load in the cortex, hippocampus and thalamus of arcAβ mice, and in the cortex of APP/PS1 mice compared to non‐transgenic littermates, corresponding with immunohistochemical staining results in mouse brain sections. Confocal microscopy showed co‐localization of AOI987, 6E10 and OC to parenchymal and cerebral amyloid angiopathy in brain tissue sections from arcAb and APP/PS1 mice, thus verifying the specificity of the vMSOT amyloid imaging approach.ConclusionWe demonstrate a new high‐resolution in vivo 3D amyloid imaging platform across the murine brain in animal models of AD pathology, which facilitates mechanistic studies and the monitoring of putative treatments targeting Aβ.

Targeting Pathological Amyloid Aggregates with Conformation-Sensitive Antibodies.

The pathogenesis of Alzheimer's disease (AD) is not directly caused by the presence of senile plaques but rather by the detrimental effects exerted on neuronal cells by toxic soluble oligomers. Such species are formed early during the aggregation process of the Aβ1-42 peptide or can be released from mature fibrils. Nowadays, efficient tools for an early diagnosis, as well as pharmaceutical treatments targeting the harmful agents in samples of AD patients, are still missing. By integrating in vitro immunochemical assay with in vivo neuronal models of toxicity, we aim to understand and target the principles that drive toxicity in AD. We evaluated the specificity and sensitivity of A11 and OC conformational antibodies to target a range of pathologically relevant amyloid conformers and rescue their cytotoxic effects in neuronal culture models using a number of cellular readouts. We demonstrated the peculiar ability of conformational antibodies to label pathologically relevant Aβ1-42 oligomers and fibrils and to prevent their detrimental effects on neuronal cells. Our results substantially improve our knowledge on the role of toxic assemblies in neurodegenerative diseases, thus suggesting new and more effective diagnostic and therapeutic tools for AD.

Development of Conformational Antibodies to Detect Bcl-xL's Amyloid Aggregates in Metal-Induced Apoptotic Neuroblastoma Cells.

Bcl-xL, a member of the Bcl-2 family, is a pro-survival protein involved in apoptosis regulation. We have previously reported the ability of Bcl-xL to form various types of fibers, from native to amyloid conformations. Here, we have mimicked the effect of apoptosis-induced caspase activity on Bcl-xL by limited proteolysis using trypsin. We show that cleaved Bcl-xL (ΔN-Bcl-xL) forms fibers that exhibit the features of amyloid structures (BclxLcf37). Moreover, three monoclonal antibodies (mAbs), produced by mouse immunization and directed against ΔN-Bcl-xL or Bcl-xL fibers, were selected and characterized. Our results show that these mAbs specifically target ΔN-Bcl-xL in amyloid fibers in vitro. Upon metal-stress-induced apoptosis, these mAbs are able to detect the presence of Bcl-xL in amyloid aggregates in neuroblastoma SH-SY5Y cell lines. In conclusion, these specific mAbs directed against amyloidogenic conformations of Bcl-xL constitute promising tools for studying, in vitro and in cellulo, the contribution of Bcl-xL in apoptosis. These mAbs may further help in developing new diagnostics and therapies, considering Bcl-xL as a strategic target for treating brain lesions relevant to stroke and neurodegenerative diseases.

Computational Investigation of Gantenerumab and Crenezumab Recognition of Aβ Fibrils in Alzheimer's Disease Brain Tissue.

Alzheimer's disease (AD) is one of the most devastating neurodegenerative diseases without effective therapies. Immunotherapies using antibodies to lower assembled Aβ provide a promising approach and have been widely studied. Anti-amyloid antibodies are often selective to amyloid conformation, and the lack of amyloid-antibody structural information limits our understanding of these antibodies' conformation selection. Gantenerumab and crenezumab are two anti-Aβ antibodies that bind multiple forms of Aβ with different Aβ epitope preferences. Here, using molecular dynamic (MD) simulations, we study the binding of these two antibodies to the Aβ1-40 fibril, whose conformation is derived from an AD patient's brain tissue. We find that gantenerumab recognizes the Aβ1-11 monomer fragment only at slightly lower pH than the physiological environment where His6 of Aβ1-11 is protonated. Both gantenerumab and crenezumab bind with integrated Aβ fibril rather than binding to monomers within the fibril. Gantenerumab preferentially binds to the N-terminal region of the Aβ1-40 fibril, and the binding is driven by aromatic interactions. Crenezumab can recognize the N-terminal region, as well as the cross-section of the Aβ1-40 fibril, indicating its multiple binding modes in Aβ fibril recognition. These results demonstrate conformation-dependent interactions of antibody-amyloid recognition.

Structure-based machine-guided mapping of amyloid sequence space reveals uncharted sequence clusters with higher solubilities

The amyloid conformation can be adopted by a variety of sequences, but the precise boundaries of amyloid sequence space are still unclear. The currently charted amyloid sequence space is strongly biased towards hydrophobic, beta-sheet prone sequences that form the core of globular proteins and by Q/N/Y rich yeast prions. Here, we took advantage of the increasing amount of high-resolution structural information on amyloid cores currently available in the protein databank to implement a machine learning approach, named Cordax (https://cordax.switchlab.org), that explores amyloid sequence beyond its current boundaries. Clustering by t-Distributed Stochastic Neighbour Embedding (t-SNE) shows how our approach resulted in an expansion away from hydrophobic amyloid sequences towards clusters of lower aliphatic content and higher charge, or regions of helical and disordered propensities. These clusters uncouple amyloid propensity from solubility representing sequence flavours compatible with surface-exposed patches in globular proteins, functional amyloids or sequences associated to liquid-liquid phase transitions.

The protean prion protein.

The prion protein, PrP, can adopt at least 2 conformations, the overwhelmingly prevalent cellular conformation (PrPC) and the scrapie conformation (PrPSc). PrPC features a globular C-terminal domain containing 3 α-helices and a short β-sheet and a long flexible N-terminal tail whose exact conformation in vivo is not yet known and a metastable subdomain with β-strand propensity has been identified within it. The PrPSc conformation is very rare and has the characteristics of an amyloid. Furthermore, PrPSc is a prion, i.e., it is infectious. This involves 2 steps: (1) PrPSc can template PrPC and coerce it to adopt the PrPSc conformation and (2) PrPSc can be transmitted between individuals, by oral, parenteral, and other routes and thus propagate as an infectious agent. However, this is a simplification: On the one hand, PrPSc is not a single conformation, but rather, a set of alternative similar but distinct conformations. Furthermore, other amyloid conformations of PrP exist with different biochemical and propagative properties. In this issue of PLOS Biology, Asante and colleagues describe the first murine model of familial human prion disease and demonstrate the emergence and propagation of 2 PrP amyloid conformers. Of these, one causes neurodegeneration, whereas the other does not. With its many conformers, PrP is a truly protean protein.

Tuning Functional Amyloid Formation Through Disulfide Engineering.

Many organisms produce “functional” amyloid fibers, which are stable protein polymers that serve many roles in cellular biology. Certain Enterobacteriaceae assemble functional amyloid fibers called curli that are the main protein component of the biofilm extracellular matrix. CsgA is the major protein subunit of curli and will rapidly adopt the polymeric amyloid conformation in vitro. The rapid and irreversible nature of CsgA amyloid formation makes it challenging to study in vitro. Here, we engineered CsgA so that amyloid formation could be tuned to the redox state of the protein. A double cysteine variant of CsgA called CsgACC was created and characterized for its ability to form amyloid. When kept under oxidizing conditions, CsgACC did not adopt a β-sheet rich structure or form detectable amyloid-like aggregates. Oxidized CsgACC remained in a soluble, non-amyloid state for at least 90 days. The addition of reducing agents to CsgACC resulted in amyloid formation within hours. The amyloid fibers formed by CsgACC were indistinguishable from the fibers made by CsgA WT. When measured by thioflavin T fluorescence the amyloid formation by CsgACC in the reduced form displayed the same lag, fast, and plateau phases as CsgA WT. Amyloid formation by CsgACC could be halted by the addition of oxidizing agents. Therefore, CsgACC serves as a proof-of-concept for capitalizing on the convertible nature of disulfide bonds to control the aggregation of amyloidogenic proteins.

Intercellular Transmission of a Synthetic Bacterial Cytotoxic Prion-Like Protein in Mammalian Cells.

RepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of plasmid DNA replication. When carrying a mutation enhancing its amyloidogenesis (A31V), the N-terminal domain (WH1) generates cytosolic amyloid particles that are inheritable within a bacterial lineage. Such amyloids trigger in bacteria a lethal cascade reminiscent of mitochondrial impairment in human cells affected by neurodegeneration. To fulfill all the criteria to qualify as a prion-like protein, horizontal (intercellular) transmissibility remains to be demonstrated for RepA-WH1. Since this is experimentally intractable in bacteria, here we transiently expressed in a murine neuroblastoma cell line the soluble, barely cytotoxic RepA-WH1 wild type [RepA-WH1(WT)] and assayed its response to exposure to in vitro-assembled RepA-WH1(A31V) amyloid fibers. In parallel, murine cells releasing RepA-WH1(A31V) aggregates were cocultured with human neuroblastoma cells expressing RepA-WH1(WT). Both the assembled fibers and donor-derived RepA-WH1(A31V) aggregates induced, in the cytosol of recipient cells, the formation of cytotoxic amyloid particles. Mass spectrometry analyses of the proteomes of both types of injured cells pointed to alterations in mitochondria, protein quality triage, signaling, and intracellular traffic. Thus, a synthetic prion-like protein can be propagated to, and become cytotoxic to, cells of organisms placed at such distant branches of the tree of life as bacteria and mammalia, suggesting that mechanisms of protein aggregate spreading and toxicity follow default pathways.IMPORTANCE Proteotoxic amyloid seeds can be transmitted between mammalian cells, arguing that the intercellular exchange of prion-like protein aggregates can be a common phenomenon. RepA-WH1 is derived from a bacterial intracellular functional amyloid protein, engineered to become cytotoxic in Escherichia coli Here, we have studied if such bacterial aggregates can also be transmitted to, and become cytotoxic to, mammalian cells. We demonstrate that RepA-WH1 is capable of entering naive cells, thereby inducing the cytotoxic aggregation of a soluble RepA-WH1 variant expressed in the cytosol, following the same trend that had been described in bacteria. These findings highlight the universality of one of the central principles underlying prion biology: No matter the biological origin of a given prion-like protein, it can be transmitted to a phylogenetically unrelated recipient cell, provided that the latter expresses a soluble protein onto which the incoming protein can readily template its amyloid conformation.

Structural analysis of a natural apolipoprotein A-I variant (L60R) associated with amyloidosis

The reason that determines the pathological deposition of human apolipoprotein A-I variants inducing organ failure has been under research since the early description of natural mutations in patients. To shed light into the events associated with protein aggregation, we studied the structural perturbations that may occur in the natural variant that shows a substitution of a Leucine by an Arginine in position 60 (L60R). Circular dichroism, intrinsic fluorescence measurements, and proteolysis analysis indicated that L60R was more unstable, more sensitive to cleavage and the N-terminus was more disorganized than the protein with the native sequence (Wt). A higher tendency to aggregate was also detected when L60R was incubated at physiological pH. In addition, the small structural rearrangement observed for the freshly folded variant led to the release of tumor necrosis factor-α and interleukin-1β from a model of macrophages. However, the mutant preserved both its dimeric conformation and its lipid-binding capacity. Our results strongly suggest that the chronic disease may be a consequence of the native conformation loss which elicits the release of protein conformations that could be either cytotoxic or precursors of amyloid conformations.

Deep mutational scanning reveals the structural basis for α-synuclein activity.

Defining the biologically active structures of proteins in their cellular environments remains challenging for proteins with multiple conformations and functions, where only a minor conformer might be associated with a given function. Here, we use deep mutational scanning to probe the structure and dynamics of α-synuclein, a protein known to adopt disordered, helical, and amyloid conformations. We examined the effects of 2,600 single-residue substitutions on the ability of intracellularly expressed α-synuclein to slow the growth of yeast. Computational analysis of the data showed that the conformation responsible for this phenotype is a long, uninterrupted, amphiphilic helix with increasing dynamics toward the C terminus. Deep mutational scanning can therefore determine biologically active conformations in cellular environments, even for a highly dynamic multi-conformational protein.

Near-Infrared Light-Powered Janus Nanomotor Significantly Facilitates Inhibition of Amyloid-β Fibrillogenesis.

Inspired by the natural motors, artificial nanomotors (NMs) have emerged as intelligent, advanced, and multifunctional nanoplatforms that can perform complex tasks in living environments. However, the functionalization of these fantastic materials is in its infancy, hindering the success of this booming field. Herein, an inhibitor-conjugated near-infrared (NIR) laser-propelled Janus nanomotor (JNM-I) was constructed and first applied in the modulation of amyloid-β protein (Aβ) aggregation which is highly associated with Alzheimer's disease (AD). Under NIR light illumination, JNM-I exhibited efficient propulsion through the "self-thermophoresis" effect, and the active motion of JNM-I increased the opportunity of the contacts between the immobilized inhibitors and Aβ species, leading to an intensification of JNM-I on modulating the on-pathway Aβ aggregation, as evidenced by the distinct changes of the amyloid morphology, conformation, and cytotoxicity. For example, with a NIR irradiation, 200 μg/mL of JNM-I increased the cultured SH-SY5Y cell viability from 68% to nearly 100%, but it only protected the cells to 89% viability without an NIR irradiation. Meanwhile, the NIR irradiation effectively improved the blood-brain barrier (BBB) penetration of JNM-I. Such a JNM-I has connected artificial nanomotors with protein aggregation and provided new insight into the potential applications of various nanomotors in the prevention and treatment of AD.

Amyloid formation by intrinsically disordered trans-activation domain of cMyb

The cMyb trans-activation domain is one of the model systems to understand the folding and binding mechanisms in intrinsically disordered proteins. cMyb (291–315) TAD (cMyb TAD) upon interaction with KIX plays a crucial role in transcriptional regulation. However, nothing is known regarding its aggregation behaviour on change of buffer conditions or stressed environment. Notably, most of the disease-associated amyloid-forming proteins such as Aβ, Tau, α-synuclein, and amylin are natively unstructured. Nevertheless, to date, very fewer evidence on aggregation behaviours on TAD domains are available. Therefore, this is necessary to investigate the aggregation propensity of intrinsically disordered cMyb TAD domain in isolation. As an essential step in that direction, we have extensively studied the aggregation behaviour of cMyb TAD using the standard approaches for aggregation studies and systematically probed the amyloid conformations. These aggregates are ThT and ANS-positive whose amyloid nature was also confirmed by Far-UV CD spectroscopic studies suggesting that cMyb TAD fibrils are rich in β-sheet secondary structure, transmission electron microscopy revealed the formation of characteristic long branched amyloid fibrils of 6–16 nm diameter, and MTT assay in SH-SY5Y neuroblastoma cells suggest that these aggregates are cytotoxic. This amyloid nature of cMyb TAD may affect its binding with KIX and alter cMyb function (transcriptional regulation) under acidic/stressed conditions.

Application of yeast to studying amyloid and prion diseases.

Amyloids are fibrous cross-β protein aggregates that are capable of proliferation via nucleated polymerization. Amyloid conformation likely represents an ancient protein fold and is linked to various biological or pathological manifestations. Self-perpetuating amyloid-based protein conformers provide a molecular basis for transmissible (infectious or heritable) protein isoforms, termed prions. Amyloids and prions, as well as other types of misfolded aggregated proteins are associated with a variety of devastating mammalian and human diseases, such as Alzheimer's, Parkinson's and Huntington's diseases, transmissible spongiform encephalopathies (TSEs), amyotrophic lateral sclerosis (ALS) and transthyretinopathies. In yeast and fungi, amyloid-based prions control phenotypically detectable heritable traits. Simplicity of cultivation requirements and availability of powerful genetic approaches makes yeast Saccharomyces cerevisiae an excellent model system for studying molecular and cellular mechanisms governing amyloid formation and propagation. Genetic techniques allowing for the expression of mammalian or human amyloidogenic and prionogenic proteins in yeast enable researchers to capitalize on yeast advantages for characterization of the properties of disease-related proteins. Chimeric constructs employing mammalian and human aggregation-prone proteins or domains, fused to fluorophores or to endogenous yeast proteins allow for cytological or phenotypic detection of disease-related protein aggregation in yeast cells. Yeast systems are amenable to high-throughput screening for antagonists of amyloid formation, propagation and/or toxicity. This review summarizes up to date achievements of yeast assays in application to studying mammalian and human disease-related aggregating proteins, and discusses both limitations and further perspectives of yeast-based strategies.

Nanoscopic insights into the surface conformation of neurotoxic amyloid β oligomers.

Raman spectroscopy assisted by localized plasmon resonances generating effective hot spots at the gaps between intertwined silver nanowires is herein adopted to unravel characteristic molecular motifs on the surface of Aβ42 misfolded oligomers that are critical in driving intermolecular interactions in neurodegeneration.

Concentration-dependent polymorphism of insulin amyloid fibrils.

Protein aggregation into highly structured fibrils has long been associated with several neurodegenerative disorders, such as Alzheimer’s or Parkinson’s disease. Polymorphism of amyloid fibrils increases the complexity of disease mechanisms and may be one of the reasons for the slow progress in drug research. Here we report protein concentration as another factor leading to polymorphism of insulin amyloid fibrils. Moreover, our data suggests that insulin amyloid conformation can self-replicate only via elongation, while seed-induced nucleation will lead to environment-defined conformation of fibrils. As similar observations were already described for a couple of other amyloid proteins, we suggest it to be a generic mechanism for self-replication of different amyloid fibril conformations.

A KLVFFAE-Derived Peptide Probe for Detection of Alpha-Synuclein Fibrils.

Aggregation of an amyloid protein, α-synuclein (αS), is a critical step in the neurodegenerative pathway of Parkinson's diseases (PD). Specific detection of amyloid conformers (i.e., monomers, oligomers, and fibrils) produced during αS aggregation is critical in better understanding a molecular basis of PD and developing a diagnostic tool. While various molecular probes are available for detection of αS fibrils, which may serve as a reservoir of toxic αS aggregate forms, these probes suffer from limited conformer-specificity and operational flexibility. In the present study, we explored the potential of non-self-aggregating peptides derived from the highly aggregation-prone KLVFFAE region of an amyloid protein, β-amyloid, as molecular probes for αS aggregates. We show that of the four peptides tested (KLVFWAK, ELVFWAE, and their C-terminal capping variants, all of which were attached with fluorescein isothiocyanate at their respective N-termini), KLVFWAK with C-terminal capping was selectively bound to αS fibrils over monomers and oligomers and readily used for monitoring αS fibrilization. Our analyses suggest that binding of the peptide to αS fibrils is mediated by both electrostatic and hydrophobic interactions. We anticipate that our peptide can readily be optimized for conformer-specificity and operational flexibility. Overall, this study presents the creation of a KLVFFAE-based molecular probe for αS fibrils and demonstrates fine-tuning of its conformer-specificity by terminal mutations and capping.