Amyloidogenic Motif Research Articles

Spiders Use Structural Conversion of Globular Amyloidogenic Domains to Make Strong Silk Fibers

AbstractSpider silk—an environmentally friendly protein‐based material—is widely recognized for its extraordinary mechanical properties. Biomimetic spider silk‐like fibers made from recombinant spider silk proteins (spidroins) currently falls short compared to natural silks in terms of mechanical performance. In this study, it is discovered that spiders use structural conversion of molecular enhancers—conserved globular 127‐residue spacer domains—to make strong silk fibers. This domain lacks poly‐Ala motifs but interestingly contains motifs that are similar to human amyloidogenic motifs, and that it self‐assembles into amyloid‐like fibrils through a non‐nucleation‐dependent pathway, likely to avoid the formation of cytotoxic intermediates. Incorporating this spacer domain into a recombinant chimeric spidroin facilitates self‐assembly into silk‐like fibers, increases fiber molecular homogeneity, and markedly enhances fiber mechanical strength. These findings highlight that spiders employ diverse strategies to produce silk with exceptional mechanical properties. The spacer domain offers a way to enhance the properties of recombinant spider silk‐like fibers and other functional materials.

Local structural preferences in shaping tau amyloid polymorphism

Tauopathies encompass a group of neurodegenerative disorders characterised by diverse tau amyloid fibril structures. The persistence of polymorphism across tauopathies suggests that distinct pathological conditions dictate the adopted polymorph for each disease. However, the extent to which intrinsic structural tendencies of tau amyloid cores contribute to fibril polymorphism remains uncertain. Using a combination of experimental approaches, we here identify a new amyloidogenic motif, PAM4 (Polymorphic Amyloid Motif of Repeat 4), as a significant contributor to tau polymorphism. Calculation of per-residue contributions to the stability of the fibril cores of different pathologic tau structures suggests that PAM4 plays a central role in preserving structural integrity across amyloid polymorphs. Consistent with this, cryo-EM structural analysis of fibrils formed from a synthetic PAM4 peptide shows that the sequence adopts alternative structures that closely correspond to distinct disease-associated tau strains. Furthermore, in-cell experiments revealed that PAM4 deletion hampers the cellular seeding efficiency of tau aggregates extracted from Alzheimer’s disease, corticobasal degeneration, and progressive supranuclear palsy patients, underscoring PAM4’s pivotal role in these tauopathies. Together, our results highlight the importance of the intrinsic structural propensity of amyloid core segments to determine the structure of tau in cells, and in propagating amyloid structures in disease.

Disease-associated patterns of acetylation stabilize tau fibril formation

Assembly of tau into beta-sheet-rich amyloids dictates the pathology of a diversity of diseases. Lysine acetylation has been proposed to drive tau amyloid assembly, but no direct mechanism has emerged. Using tau fragments, we identify patterns of acetylation that flank amyloidogenic motifs on the tau fragments that promote rapid fibril assembly. We determined a 3.9Å cryo-EM amyloid fibril structure assembled from an acetylated tau fragment uncovering how lysine acetylation can mediate gain-of-function interactions. Comparison of the structure to an exvivo tauopathy fibril reveals regions of structural similarity. Finally, we show that fibrils encoding disease-associated patterns of acetylation are active in cell-based tau aggregation assays. Our data uncover the dual role of lysine residues in limiting tau aggregation while their acetylation leads to stabilizing pro-aggregation interactions. Design of tau sequence with specific acetylation patterns may lead to controllable tau aggregation to direct folding of tau into distinct amyloid folds.

Molecular basis of amyloid deposition in myocardium: not only ATTR and AL. Case report

Alterations in the proteostasis network and accumulation of misfolded protein aggregates — is one of the new pathogenesis concepts of chronic heart failure. We hypothesis in addition to well-known transthyretin (ATTR) and AL-amyloidosis some patients may represent amyloid lesion in myocardium came from undescribed amyloidogenic precursors due to misfolding of myocardial structural proteins.Here, we report on the case of patient with hypertrophic and restrictive phenotype of cardiomyopathy, biventricular heart failure, considered for heart transplant, and excluded known types of amyloidosis. Genetic testing revealed extended deletion in the gene of giant protein titin (TTN).We present with the use of bioinformatic analysis and molecular modeling how this mutation could lead to unfolding of corresponding protein and open its amyloidogenic motifs for intermolecular interactions, therefore, provide amyloidogenic ability. This data enables in more detail to decipher the pathogenesis of chronic heart failure on the background of cardiomyopathy, planning further studies for development of personalized risk profiling in different types of amyloidosis and elaborate more personalized treatment approach for such patients in the future.

Molecular Determinants of Fibrillation in a Viral Amyloidogenic Domain from Combined Biochemical and Biophysical Studies.

The Nipah and Hendra viruses (NiV and HeV) are biosafety level 4 human pathogens classified within the Henipavirus genus of the Paramyxoviridae family. In both NiV and HeV, the gene encoding the Phosphoprotein (P protein), an essential polymerase cofactor, also encodes the V and W proteins. These three proteins, which share an intrinsically disordered N-terminal domain (NTD) and have unique C-terminal domains (CTD), are all known to counteract the host innate immune response, with V and W acting by either counteracting or inhibiting Interferon (IFN) signaling. Recently, the ability of a short region within the shared NTD (i.e., PNT3) to form amyloid-like structures was reported. Here, we evaluated the relevance of each of three contiguous tyrosine residues located in a previously identified amyloidogenic motif (EYYY) within HeV PNT3 to the fibrillation process. Our results indicate that removal of a single tyrosine in this motif significantly decreases the ability to form fibrils independently of position, mainly affecting the elongation phase. In addition, we show that the C-terminal half of PNT3 has an inhibitory effect on fibril formation that may act as a molecular shield and could thus be a key domain in the regulation of PNT3 fibrillation. Finally, the kinetics of fibril formation for the two PNT3 variants with the highest and the lowest fibrillation propensity were studied by Taylor Dispersion Analysis (TDA). The results herein presented shed light onto the molecular mechanisms involved in fibril formation.

Modifying Amyloid Motif Aggregation Through Local Structure.

Protein assembly into β-sheet-rich amyloid structures is a general biophysical phenomenon that has significant biological consequences, most notable for their prominent association with neurodegenerative diseases, including Alzheimer's, Huntington's, or Parkinson's diseases. The assembly of amyloid structures is driven by short sequences called amyloid motifs. In many neurodegenerative diseases, intrinsically disordered proteins (IDPs) self-assemble through amyloid motifs, but these motifs are present in all proteins, including folded globular proteins. Importantly, mechanistic knowledge is lacking for how IDPs, which do not adopt a stable tertiary structure, mask these amyloidogenic motifs to mitigate or slow the formation of β-sheet-rich amyloid structures that cause disease. Our recent work has shown that local structural elements can modify the aggregation propensity of amyloid motifs in the intrinsically disordered microtubule-associated protein tau by adopting metastable β-hairpin-like structures that shield the amyloid motif, and disease-causing mutations change the conformation, thus increase aggregation propensity (Chen, Nat Commun 10:2493, 2019). Here we describe a protocol that correlates experimentally determined aggregation propensities for peptides measured by the Thioflavin T (ThT) fluorescence aggregation assay with their conformational ensembles derived from Groningen machine chemical simulations (GROMACS). Integration of experiment and simulation will help uncover structural rules behind changes in conformation that modulate protein aggregation. We anticipate that our general protocol will help identify key interactions in local structures that engage amyloid-forming motifs in IDPs which influence aggregation behavior.

Identification of a Region in the Common Amino-terminal Domain of Hendra Virus P, V, and W Proteins Responsible for Phase Transition and Amyloid Formation.

Henipaviruses are BSL-4 zoonotic pathogens responsible in humans for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain (NTD) and a β-enriched C-terminal domain (CTD). These terminals are critical for V binding to DDB1, which is a cellular protein that is a component of the ubiquitin ligase E3 complex, as well as binding to MDA5 and LGP2, which are two host sensors of viral RNA. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquid-to-hydrogel phase transition and identified the V region responsible for this phenomenon. This region, referred to as PNT3 and encompassing residues 200–310, was further investigated using a combination of biophysical and structural approaches. Congo red binding assays, together with negative-staining transmisison electron microscopy (TEM) studies, show that PNT3 forms amyloid-like fibrils. Fibrillation abilities are dramatically reduced in a rationally designed PNT3 variant in which a stretch of three contiguous tyrosines, falling within an amyloidogenic motif, were replaced by three alanines. Worthy to note, Congo red staining experiments provided hints that these amyloid-like fibrils form not only in vitro but also in cellula after transfection or infection. The present results set the stage for further investigations aimed at assessing the functional role of phase separation and fibrillation by the Henipavirus V proteins.

Designed Two- and Three-Dimensional Protein Nanocage Networks Driven by Hydrophobic Interactions Contributed by Amyloidogenic Motifs.

Precise manipulation of protein self-assembly by noncovalent interactions into programmed networks to mimic naturally occurring nanoarchitectures in living organisms is a challenge due to its structural heterogeneity, flexibility, and complexity. Herein, by taking advantage of both the hydrophobic forces contributed by the "GLMVG" motif, a kind of amyloidogenic motif (AM), and the high symmetry of protein nanocages, we have built an effective protein self-assembly strategy for the construction of two-dimensional (2D) or three-dimensional (3D) protein nanocage arrays. According to this strategy, "GLMVG" AMs from β-amyloid 42 were grafted onto the outer surface of a 24-mer ferritin nanocage close to its C4 symmetry channels, initially resulting in the production of subgrade 2D nanocage arrays and ultimately generating 3D highly ordered arrays with a simple cubic packing pattern as the reaction time increases. More importantly, the reversibility and the formation rate of these protein arrays can be modulated by pH. This work provides a de novo design strategy for accurate control over 2D or 3D protein self-assemblies.

Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms

Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.

Amyloidogenic motifs revealed by n-gram analysis

Amyloids are proteins associated with several clinical disorders, including Alzheimer’s, and Creutzfeldt-Jakob’s. Despite their diversity, all amyloid proteins can undergo aggregation initiated by short segments called hot spots. To find the patterns defining the hot spots, we trained predictors of amyloidogenicity, using n-grams and random forest classifiers. Since the amyloidogenicity may not depend on the exact sequence of amino acids but on their more general properties, we tested 524,284 reduced amino acid alphabets of different lengths (three to six letters) to find the alphabet providing the best performance in cross-validation. The predictor based on this alphabet, called AmyloGram, was benchmarked against the most popular tools for the detection of amyloid peptides using an external data set and obtained the highest values of performance measures (AUC: 0.90, MCC: 0.63). Our results showed sequential patterns in the amyloids which are strongly correlated with hydrophobicity, a tendency to form β-sheets, and lower flexibility of amino acid residues. Among the most informative n-grams of AmyloGram we identified 15 that were previously confirmed experimentally. AmyloGram is available as the web-server: http://smorfland.uni.wroc.pl/shiny/AmyloGram/ and as the R package AmyloGram. R scripts and data used to produce the results of this manuscript are available at http://github.com/michbur/AmyloGramAnalysis.

β-Hairpin of Islet Amyloid Polypeptide Bound to an Aggregation Inhibitor.

In type 2 diabetes, the formation of islet amyloid consisting of islet amyloid polypeptide (IAPP) is associated with reduction in β-cell mass and contributes to the failure of islet cell transplantation. Rational design of inhibitors of IAPP amyloid formation has therapeutic potential, but is hampered by the lack of structural information on inhibitor complexes of the conformationally flexible, aggregation-prone IAPP. Here we characterize a β-hairpin conformation of IAPP in complex with the engineered binding protein β-wrapin HI18. The β-strands correspond to two amyloidogenic motifs, 12-LANFLVH-18 and 22-NFGAILS-28, which are connected by a turn established around Ser-20. Besides backbone hydrogen bonding, the IAPP:HI18 interaction surface is dominated by non-polar contacts involving hydrophobic side chains of the IAPP β-strands. Apart from monomers, HI18 binds oligomers and fibrils and inhibits IAPP aggregation and toxicity at low substoichiometric concentrations. The IAPP β-hairpin can serve as a molecular recognition motif enabling control of IAPP aggregation.

Cryptic Amyloidogenic Elements in the 3′ UTRs of Neurofilament Genes Trigger Axonal Neuropathy

Abnormal protein aggregation is observed in an expanding number of neurodegenerative diseases. Here, we describe a mechanism for intracellular toxic protein aggregation induced by an unusual mutation event in families affected by axonal neuropathy. These families carry distinct frameshift variants in NEFH (neurofilament heavy), leading to a loss of the terminating codon and translation of the 3' UTR into an extra 40 amino acids. In silico aggregation prediction suggested the terminal 20 residues of the altered NEFH to be amyloidogenic, which we confirmed experimentally by serial deletion analysis. The presence of this amyloidogenic motif fused to NEFH caused prominent and toxic protein aggregates in transfected cells and disrupted motor neurons in zebrafish. We identified a similar aggregation-inducing mechanism in NEFL (neurofilament light) and FUS (fused in sarcoma), in which mutations are known to cause aggregation in Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis, respectively. In summary, we present a protein-aggregation-triggering mechanism that should be taken into consideration during the evaluation of stop-loss variants.

Amyloid-like conformation and interaction for the self-assembly in barnacle underwater cement.

Barnacles are unique marine sessile crustaceans and permanently attach to various foreign surfaces during most of their lifespan. The protein complex secreted from their body and used to attach their calcareous shell to almost all surfaces in water has long fascinated us because we have limited technology with which to attach materials in water. Unraveling the mechanism of underwater attachment by barnacles is thus important for interface science, for the understanding of the biology and physiology of barnacles, and for the development of technology to prevent fouling. Previous studies have indicated that the intermolecular interactions optimized by conformations of the adhesive proteins are crucial in the self-assembly and/or curing of the adhesive. This study aimed to identify the possible structural determinants responsible for the self-assembly. Thioflavin T binding screening of peptides designed on the basis of the primary structure of a bulk 52 kDa cement protein indicated the presence of some amyloidogenic motifs in the protein. The conformation of the peptide was transformed to a β-sheet by an increase in either pH or ionic strength, resulting in its self-assembly. Thioflavin T binding was inhibited by small polyphenolic molecules, suggesting the contribution of aromatic interactions during self-assembly. The occurrence of amyloid-like units in the protein implies that the protein conformation is an important factor contributing to the self-assembly of the cement, the first event of the curing, as the adhesive material is secreted into the seawater out of the animal's body.

Novel multimeric IL-1 receptor antagonist for the treatment of rheumatoid arthritis

Protein therapeutics targeting inflammatory mediators have shown great promise for the treatment of autoimmunities such as rheumatoid arthritis (RA). However, a significant challenge in this area has been their low in vivo stability and consequently their severely compromised therapeutic efficacy. One such therapeutic molecule IL-1 receptor antagonist (IL-1ra), used in the treatment of rheumatoid arthritis, has displayed only modest efficacy in human clinical trials owing to its short biological half-life. Herein, we report a novel approach to conglomerate individual protein entities into a drug depot by incorporation of an amyloidogenic motif Lys-Phe-Phe-Glu (KFFE) thereby dramatically improving their systemic persistence and in turn their therapeutic efficacy in a mice model of autoimmune arthritis.

Prion protein self-peptides modulate prion interactions and conversion

BackgroundMolecular mechanisms underlying prion agent replication, converting host-encoded cellular prion protein (PrPC) into the scrapie associated isoform (PrPSc), are poorly understood. Selective self-interaction between PrP molecules forms a basis underlying the observed differences of the PrPC into PrPSc conversion process (agent replication). The importance of previously peptide-scanning mapped ovine PrP self-interaction domains on this conversion was investigated by studying the ability of six of these ovine PrP based peptides to modulate two processes; PrP self-interaction and conversion.ResultsThree peptides (octarepeat, binding domain 2 -and C-terminal) were capable of inhibiting self-interaction of PrP in a solid-phase PrP peptide array. Three peptides (N-terminal, binding domain 2, and amyloidogenic motif) modulated prion conversion when added before or after initiation of the prion protein misfolding cyclic amplification (PMCA) reaction using brain homogenates. The C-terminal peptides (core region and C-terminal) only affected conversion (increased PrPres formation) when added before mixing PrPC and PrPSc, whereas the octarepeat peptide only affected conversion when added after this mixing.ConclusionThis study identified the putative PrP core binding domain that facilitates the PrPC-PrPSc interaction (not conversion), corroborating evidence that the region of PrP containing this domain is important in the species-barrier and/or scrapie susceptibility. The octarepeats can be involved in PrPC-PrPSc stabilization, whereas the N-terminal glycosaminoglycan binding motif and the amyloidogenic motif indirectly affected conversion. Binding domain 2 and the C-terminal domain are directly implicated in PrPC self-interaction during the conversion process and may prove to be prime targets in new therapeutic strategy development, potentially retaining PrPC function. These results emphasize the importance of probable PrPC-PrPC and required PrPC-PrPSc interactions during PrP conversion. All interactions are probably part of the complex process in which polymorphisms and species barriers affect TSE transmission and susceptibility.