Minimum information and guidelines for reporting a multiplexed assay of variant effect.

Multiplexed assays of variant effect (MAVEs) have emerged as a powerful approach for interrogating thousands of genetic variants in a single experiment. The flexibility and widespread adoption of these techniques across diverse disciplines have led to a heterogeneous mix of data formats and descriptions, which complicates the downstream use of the resulting datasets. To address these issues and promote reproducibility and reuse of MAVE data, we define a set of minimum information standards for MAVE data and metadata and outline a controlled vocabulary aligned with established biomedical ontologies for describing these experimental designs.

Minimum information and guidelines for reporting a Multiplexed Assay of Variant Effect.

Multiplexed Assays of Variant Effect (MAVEs) have emerged as a powerful approach for interrogating thousands of genetic variants in a single experiment. The flexibility and widespread adoption of these techniques across diverse disciplines has led to a heterogeneous mix of data formats and descriptions, which complicates the downstream use of the resulting datasets. To address these issues and promote reproducibility and reuse of MAVE data, we define a set of minimum information standards for MAVE data and metadata and outline a controlled vocabulary aligned with established biomedical ontologies for describing these experimental designs.

An atlas of amyloid aggregation: the impact of substitutions, insertions, deletions and truncations on amyloid beta fibril nucleation.

Multiplexed assays of variant effects (MAVEs) guide clinical variant interpretation and reveal disease mechanisms. To date, MAVEs have focussed on a single mutation type-amino acid (AA) substitutions-despite the diversity of coding variants that cause disease. Here we use Deep Indel Mutagenesis (DIM) to generate a comprehensive atlas of diverse variant effects for a disease protein, the amyloid beta (Aß) peptide that aggregates in Alzheimer's disease (AD) and is mutated in familial AD (fAD). The atlas identifies known fAD mutations and reveals that many variants beyond substitutions accelerate Aß aggregation and are likely to be pathogenic. Truncations, substitutions, insertions, single- and internal multi-AA deletions differ in their propensity to enhance or impair aggregation, but likely pathogenic variants from all classes are highly enriched in the polar N-terminal region of Aß. This comparative atlas highlights the importance of including diverse mutation types in MAVEs and provides important mechanistic insights into amyloid nucleation.

Understanding and evolving prions by yeast multiplexed assays.

Yeast genetics made it possible to derive the first fundamental insights into prion composition, conformation, and propagation. Fast-forward 30 years and the same model organism is now proving an extremely powerful tool to comprehensively explore the impact of mutations in prion sequences on their function, toxicity, and physical properties. Here, we provide an overview of novel multiplexed strategies where deep mutagenesis is combined to a range of tailored selection assays in yeast, which are particularly amenable for investigating prions and prion-like sequences. By mimicking evolution in a flask, these multiplexed approaches are revealing mechanistic insights on the consequences of prion self-assembly, while also reporting on the structure prion sequences adopt in vivo.

Expression of Huntingtin and TDP-43 Derivatives in Fission Yeast Can Cause Both Beneficial and Toxic Effects.

Many neurodegenerative disorders display protein aggregation as a hallmark, Huntingtin and TDP-43 aggregates being characteristic of Huntington disease and amyotrophic lateral sclerosis, respectively. However, whether these aggregates cause the diseases, are secondary by-products, or even have protective effects, is a matter of debate. Mutations in both human proteins can modulate the structure, number and type of aggregates, as well as their toxicity. To study the role of protein aggregates in cellular fitness, we have expressed in a highly tractable unicellular model different variants of Huntingtin and TDP-43. They each display specific patterns of aggregation and toxicity, even though in both cases proteins have to be very highly expressed to affect cell fitness. The aggregation properties of Huntingtin, but not of TDP-43, are affected by chaperones such as Hsp104 and the Hsp40 couple Mas5, suggesting that the TDP-43, but not Huntingtin, derivatives have intrinsic aggregation propensity. Importantly, expression of the aggregating form of Huntingtin causes a significant extension of fission yeast lifespan, probably as a consequence of kidnapping chaperones required for maintaining stress responses off. Our study demonstrates that in general these prion-like proteins do not cause toxicity under normal conditions, and in fact they can protect cells through indirect mechanisms which up-regulate cellular defense pathways.

The genetic landscape for amyloid beta fibril nucleation accurately discriminates familial Alzheimer's disease mutations.

Plaques of the amyloid beta (Aß) peptide are a pathological hallmark of Alzheimer's disease (AD), the most common form of dementia. Mutations in Aß also cause familial forms of AD (fAD). Here, we use deep mutational scanning to quantify the effects of >14,000 mutations on the aggregation of Aß. The resulting genetic landscape reveals mechanistic insights into fibril nucleation, including the importance of charge and gatekeeper residues in the disordered region outside of the amyloid core in preventing nucleation. Strikingly, unlike computational predictors and previous measurements, the empirical nucleation scores accurately identify all known dominant fAD mutations in Aß, genetically validating that the mechanism of nucleation in a cell-based assay is likely to be very similar to the mechanism that causes the human disease. These results provide the first comprehensive atlas of how mutations alter the formation of any amyloid fibril and a resource for the interpretation of genetic variation in Aß.

Alzheimer's disease is the most common form of dementia, affecting more than 50 million people worldwide. Despite more than 400 clinical trials, there are still no effective drugs that can prevent or treat the disease. A common target in Alzheimer's disease trials is a small protein called amyloid beta. Amyloid beta proteins are 'sticky' molecules. In the brains of people with Alzheimer's disease, they join to form first small aggregates and then long chains called fibrils, a process which is toxic to neurons. Specific mutations in the gene for amyloid beta are known to cause rare, aggressive forms of Alzheimer's disease that typically affect people in their fifties or sixties. But these are not the only mutations that can occur in amyloid beta. In principle, any part of the protein could undergo mutation. And given the size of the human population, it is likely that each of these mutations exists in someone alive today. Seuma et al. reasoned that studying these mutations could help us understand the process by which amyloid beta forms new aggregates. Using an approach called deep mutational scanning, Seuma et al. mutated each point in the protein, one at a time. This produced more than 14,000 different versions of amyloid beta. Seuma et al. then measured how quickly these mutants were able to form aggregates by introducing them into yeast cells. All the mutations known to cause early-onset Alzheimer's disease accelerated amyloid beta aggregation in the yeast. But the results also revealed previously unknown properties that control how fast aggregation occurs. In addition, they highlighted a number of positions in the amyloid beta sequence that act as 'gatekeepers'. In healthy brains, these gatekeepers prevent amyloid beta proteins from sticking together. When mutated, they drive the protein to form aggregates. This comprehensive dataset will help researchers understand how proteins form toxic aggregates, which could in turn help them find ways to prevent this from happening. By providing an 'atlas' of all possible amyloid beta mutations, the dataset will also help clinicians interpret any new mutations they encounter in patients. By showing whether or not a mutation speeds up aggregation, the atlas will help clinicians predict whether that mutation increases the risk of Alzheimer's disease.

Assembling the right type of switch: Protein condensation to signal cell death.

Protein phase transitions are particularly amenable for cell signalling as these highly cooperative processes allow cells to make binary decisions in response to relatively small intracellular changes. The different processes of condensate formation and the distinct material properties of the resulting condensates provide a dictionary to modulate a range of decisions on cell fate. We argue that, on the one hand, the reversibility of liquid demixing offers a chance to arrest cell growth under specific circumstances. On the other hand, the transition to amyloids is better suited for terminal decisions such as those leading to apoptosis and necrosis. Here, we review recent examples of both scenarios, highlighting how mutations in signalling proteins affect the formation of biomolecular condensates with drastic effects on cell survival.

The mutational landscape of a prion-like domain.

Insoluble protein aggregates are the hallmarks of many neurodegenerative diseases. For example, aggregates of TDP-43 occur in nearly all cases of amyotrophic lateral sclerosis (ALS). However, whether aggregates cause cellular toxicity is still not clear, even in simpler cellular systems. We reasoned that deep mutagenesis might be a powerful approach to disentangle the relationship between aggregation and toxicity. We generated >50,000 mutations in the prion-like domain (PRD) of TDP-43 and quantified their toxicity in yeast cells. Surprisingly, mutations that increase hydrophobicity and aggregation strongly decrease toxicity. In contrast, toxic variants promote the formation of dynamic liquid-like condensates. Mutations have their strongest effects in a hotspot that genetic interactions reveal to be structured in vivo, illustrating how mutagenesis can probe the in vivo structures of unstructured proteins. Our results show that aggregation of TDP-43 is not harmful but protects cells, most likely by titrating the protein away from a toxic liquid-like phase.

Fibril-induced glutamine-/asparagine-rich prions recruit stress granule proteins in mammalian cells.

Prions of lower eukaryotes are self-templating protein aggregates that replicate by converting homotypic proteins into stable, tightly packed beta-sheet-rich protein assemblies. Propagation is mediated by prion domains, low-complexity regions enriched in polar and devoid of charged amino acid residues. In mammals, compositionally similar domains modulate the assembly of dynamic stress granules (SGs) that associate via multivalent weak interactions. Dysregulation of SGs composed of proteins with prion-like domains has been proposed to underlie the formation of pathological inclusions in several neurodegenerative diseases. The events that drive prion-like domains into transient or solid assemblies are not well understood. We studied the interactors of the prototype prion domain NM of Saccharomyces cerevisiae Sup35 in its soluble or fibril-induced prion conformation in the mammalian cytosol. We show that the interactomes of soluble and prionized NM overlap with that of SGs. Prion induction by exogenous seeds does not cause SG assembly, demonstrating that colocalization of aberrant protein inclusions with SG components does not necessarily reveal SGs as initial sites of protein misfolding.

An Integrative Study of Protein-RNA Condensates Identifies Scaffolding RNAs and Reveals Players in Fragile X-Associated Tremor/Ataxia Syndrome.

Recent evidence indicates that specific RNAs promote the formation of ribonucleoprotein condensates by acting as scaffolds for RNA-binding proteins (RBPs). We systematically investigated RNA-RBP interaction networks to understand ribonucleoprotein assembly. We found that highly contacted RNAs are structured, have long UTRs, and contain nucleotide repeat expansions. Among the RNAs with such properties, we identified the FMR1 3' UTR that harbors CGG expansions implicated in fragile X-associated tremor/ataxia syndrome (FXTAS). We studied FMR1 binding partners in silico and in vitro and prioritized the splicing regulator TRA2A for further characterization. In a FXTAS cellular model, we validated the TRA2A-FMR1 interaction and investigated implications of its sequestration at both transcriptomic and post-transcriptomic levels. We found that TRA2A co-aggregates with FMR1 in a FXTAS mouse model and in post-mortem human samples. Our integrative study identifies key components of ribonucleoprotein aggregates, providing links to neurodegenerative disease and allowing the discovery of therapeutic targets.

Reaching the limit.

How many copies of a protein can be made before it becomes toxic to the cell?

A Concentration-Dependent Liquid Phase Separation Can Cause Toxicity upon Increased Protein Expression.

Multiple human diseases are associated with a liquid-to-solid phase transition resulting in the formation of amyloid fibers or protein aggregates. Here, we present an alternative mechanism for cellular toxicity based on a concentration-dependent liquid-liquid demixing. Analyzing proteins that are toxic when their concentration is increased in yeast reveals that they share physicochemical properties with proteins that participate in physiological liquid-liquid demixing in the cell. Increasing the concentration of one of these proteins indeed results in the formation of cytoplasmic foci with liquid properties. Demixing occurs at the onset of toxicity and titrates proteins and mRNAs from the cytoplasm. Focus formation is reversible, and resumption of growth occurs as the foci dissolve as protein concentration falls. Preventing demixing abolishes the dosage sensitivity of the protein. We propose that triggering inappropriate liquid phase separation may be an important cause of dosage sensitivity and a determinant of human disease.

The H50Q mutation induces a 10-fold decrease in the solubility of α-synuclein.

The conversion of α-synuclein from its intrinsically disordered monomeric state into the fibrillar cross-ß aggregates characteristically present in Lewy bodies is largely unknown. The investigation of α-synuclein variants causative of familial forms of Parkinson disease can provide unique insights into the conditions that promote or inhibit aggregate formation. It has been shown recently that a newly identified pathogenic mutation of α-synuclein, H50Q, aggregates faster than the wild-type. We investigate here its aggregation propensity by using a sequence-based prediction algorithm, NMR chemical shift analysis of secondary structure populations in the monomeric state, and determination of thermodynamic stability of the fibrils. Our data show that the H50Q mutation induces only a small increment in polyproline II structure around the site of the mutation and a slight increase in the overall aggregation propensity. We also find, however, that the H50Q mutation strongly stabilizes α-synuclein fibrils by 5.0 ± 1.0 kJ mol(-1), thus increasing the supersaturation of monomeric α-synuclein within the cell, and strongly favors its aggregation process. We further show that wild-type α-synuclein can decelerate the aggregation kinetics of the H50Q variant in a dose-dependent manner when coaggregating with it. These last findings suggest that the precise balance of α-synuclein synthesized from the wild-type and mutant alleles may influence the natural history and heterogeneous clinical phenotype of Parkinson disease.

The cleverSuite approach for protein characterization: predictions of structural properties, solubility, chaperone requirements and RNA-binding abilities.

MOTIVATION: The recent shift towards high-throughput screening is posing new challenges for the interpretation of experimental results. Here we propose the cleverSuite approach for large-scale characterization of protein groups. DESCRIPTION: The central part of the cleverSuite is the cleverMachine (CM), an algorithm that performs statistics on protein sequences by comparing their physico-chemical propensities. The second element is called cleverClassifier and builds on top of the models generated by the CM to allow classification of new datasets. RESULTS: We applied the cleverSuite to predict secondary structure properties, solubility, chaperone requirements and RNA-binding abilities. Using cross-validation and independent datasets, the cleverSuite reproduces experimental findings with great accuracy and provides models that can be used for future investigations. AVAILABILITY: The intuitive interface for dataset exploration, analysis and prediction is available at http://s.tartaglialab.com/clever_suite.

Single point mutations induce a switch in the molecular mechanism of the aggregation of the Alzheimer's disease associated Aß42 peptide.

Single point mutations in the Alzheimer's disease associated Aß42 peptide are found to alter significantly its neurotoxic properties in vivo and have been associated with early onset forms of this devastating condition. We show that such mutations can induce structural changes in Aß42 fibrils and are associated with a dramatic switch in the fibril-dependent mechanism by which Aß42 aggregates. These observations reveal how subtle perturbations to the physicochemical properties of the Aß peptide, and the structural properties of fibrils that it forms, can have profound effects on the mechanism of its aggregation and pathogenicity.

Principles of self-organization in biological pathways: a hypothesis on the autogenous association of alpha-synuclein.

Previous evidence indicates that a number of proteins are able to interact with cognate mRNAs. These autogenous associations represent important regulatory mechanisms that control gene expression at the translational level. Using the catRAPID approach to predict the propensity of proteins to bind to RNA, we investigated the occurrence of autogenous associations in the human proteome. Our algorithm correctly identified binding sites in well-known cases such as thymidylate synthase, tumor suppressor P53, synaptotagmin-1, serine/ariginine-rich splicing factor 2, heat shock 70 kDa, ribonucleic particle-specific U1A and ribosomal protein S13. In addition, we found that several other proteins are able to bind to their own mRNAs. A large-scale analysis of biological pathways revealed that aggregation-prone and structurally disordered proteins have the highest propensity to interact with cognate RNAs. These findings are substantiated by experimental evidence on amyloidogenic proteins such as TAR DNA-binding protein 43 and fragile X mental retardation protein. Among the amyloidogenic proteins, we predicted that Parkinson's disease-related α-synuclein is highly prone to interact with cognate transcripts, which suggests the existence of RNA-dependent factors in its function and dysfunction. Indeed, as aggregation is intrinsically concentration dependent, it is possible that autogenous interactions play a crucial role in controlling protein homeostasis.

Physicochemical principles of protein aggregation.

This chapter provides a theoretical framework on the quantitative description of protein aggregation. The reader is provided with an overview of the fundamental theory of linear and helical polymers, as well as an introduction on the parameters governing evolution of aggregates over time. The models presented for the interpretation of the protein aggregation process take into account the contributions of different physicochemical parameters such as charge, hydrophobicity, and secondary structure propensity. Finally, we discuss our current understanding of how prediction of aggregation rates and identification of aggregation-prone protein regions are predicted from the information contained in the primary amino acid sequence.

X-inactivation: quantitative predictions of protein interactions in the Xist network.

The transcriptional silencing of one of the female X-chromosomes is a finely regulated process that requires accumulation in cis of the long non-coding RNA X-inactive-specific transcript (Xist) followed by a series of epigenetic modifications. Little is known about the molecular machinery regulating initiation and maintenance of chromosomal silencing. Here, we introduce a new version of our algorithm catRAPID to investigate Xist associations with a number of proteins involved in epigenetic regulation, nuclear scaffolding, transcription and splicing processes. Our method correctly identifies binding regions and affinities of protein interactions, providing a powerful theoretical framework for the study of X-chromosome inactivation and other events mediated by ribonucleoprotein associations.

Neurodegenerative diseases: quantitative predictions of protein-RNA interactions.

Increasing evidence indicates that RNA plays an active role in a number of neurodegenerative diseases. We recently introduced a theoretical framework, catRAPID, to predict the binding ability of protein and RNA molecules. Here, we use catRAPID to investigate ribonucleoprotein interactions linked to inherited intellectual disability, amyotrophic lateral sclerosis, Creutzfeuld-Jakob, Alzheimer's, and Parkinson's diseases. We specifically focus on (1) RNA interactions with fragile X mental retardation protein FMRP; (2) protein sequestration caused by CGG repeats; (3) noncoding transcripts regulated by TAR DNA-binding protein 43 TDP-43; (4) autogenous regulation of TDP-43 and FMRP; (5) iron-mediated expression of amyloid precursor protein APP and α-synuclein; (6) interactions between prions and RNA aptamers. Our results are in striking agreement with experimental evidence and provide new insights in processes associated with neuronal function and misfunction.

Selenium-enhanced electron microscopic imaging of different aggregate forms of a segment of the amyloid ß peptide in cells.

The aggregation of misfolded proteins is a common feature underlying a wide range of age-related degenerative disorders, including Alzheimer's and Parkinson's diseases. A key aspect of understanding the molecular origins of these conditions is to define the manner in which specific types of protein aggregates influence disease pathogenesis through their interactions with cells. We demonstrate how selenium-enhanced electron microscopy (SE-EM), combined with tomographic reconstruction methods, can be used to image, here at a resolution of 5-10 nm, the interaction with human macrophage cells of amyloid aggregates formed from Aß(25-36), a fragment of the Aß peptide whose self-assembly is associated with Alzheimer's disease. We find that prefibrillar aggregates and mature fibrils are distributed into distinct subcellular compartments and undergo varying degrees of morphological change over time, observations that shed new light on the origins of their differential toxicity and the mechanisms of their clearance. In addition, the results show that SE-EM provides a powerful and potentially widely applicable means to define the nature and location of protein assemblies in situ and to provide detailed and specific information about their partitioning and processing.