scholarly journals The mutational landscape of a prion-like domain

2019 ◽  
Author(s):  
Benedetta Bolognesi ◽  
Andre J. Faure ◽  
Mireia Seuma ◽  
Jörn M. Schmiedel ◽  
Gian Gaetano Tartaglia ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Rna Binding ◽  
Genetic Interactions ◽  
Disordered Proteins ◽  
Insoluble Protein ◽  
Cellular Toxicity ◽  
Intrinsically Disordered ◽  
Powerful Approach ◽  
Lateral Sclerosis

AbstractSpecific insoluble protein aggregates are the hallmarks of many neurodegenerative diseases1–5. For example, cytoplasmic aggregates of the RNA-binding protein TDP-43 are observed in 97% of cases of Amyotrophic Lateral Sclerosis (ALS)6,7. However, it is still unclear for ALS and other diseases whether it is the insoluble aggregates or other forms of the mutated proteins that cause these diseases that are actually toxic to cells8–13. Here we address this question for TDP-43 by systematically mutating14the protein and quantifying the effects on cellular toxicity. We generated >50,000 mutations in the intrinsically disordered prion-like domain (PRD) and observed that changes in hydrophobicity and aggregation potential are highly predictive of changes in toxicity. Surprisingly, however, increased hydrophobicity and cytoplasmic aggregation actually reduce cellular toxicity. Mutations have their strongest effects in a central region of the PRD, with variants that increase toxicity promoting the formation of more dynamic liquid-like condensates. The genetic interactions in double mutants reveal that specific structures exist in this ‘unstructured’ regionin vivo. Our results demonstrate that deep mutagenesis is a powerful approach for probing the sequence-function relationships of intrinsically disordered proteins as well as theirin vivostructural conformations. Moreover, we show that aggregation of TDP-43 is not harmful but actually protects cells, most likely by titrating the protein away from a toxic liquid-like phase.

scholarly journals The mutational landscape of a prion-like domain

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Benedetta Bolognesi ◽  
Andre J. Faure ◽  
Mireia Seuma ◽  
Jörn M. Schmiedel ◽  
Gian Gaetano Tartaglia ◽  
...  
Keyword(s):  
Cellular Systems ◽  
Genetic Interactions ◽  
Yeast Cells ◽  
Insoluble Protein ◽  
Cellular Toxicity ◽  
Powerful Approach ◽  
Unstructured Proteins ◽  
The Relationship ◽  
Lateral Sclerosis

Abstract 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.

scholarly journals Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Andrei Vovk ◽  
Chad Gu ◽  
Michael G Opferman ◽  
Larisa E Kapinos ◽  
Roderick YH Lim ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Intrinsically Disordered Proteins ◽  
Nucleocytoplasmic Transport ◽  
Transport Proteins ◽  
Disordered Proteins ◽  
Biophysical Model ◽  
Nuclear Pore ◽  
Intrinsically Disordered

Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function.

scholarly journals Enhancing c-MYC degradation via 20S proteasome activation induces in vivo anti-tumor efficacy

2020 ◽  
Author(s):  
Evert Njomen ◽  
Theresa A. Lansdell ◽  
Allison Vanecek ◽  
Vanessa Benham ◽  
Matt P. Bernard ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
Target Genes ◽  
Intrinsically Disordered Proteins ◽  
Therapeutic Potential ◽  
Human Cancer ◽  
Disordered Proteins ◽  
20S Proteasome ◽  
Intrinsically Disordered

SUMMARYEnhancing proteasome activity is a potential new therapeutic strategy to prevent the accumulation of aberrant high levels of protein that drive the pathogenesis of many diseases. Herein, we examine the use of small molecules to activate the 20S proteasome to reduce aberrant signaling by the undruggable oncoprotein c-MYC, to treat c-MYC driven oncogenesis. Overexpression of c-MYC is found in more than 50% of all human cancer but remains undruggable because of its highly dynamic intrinsically disordered 3-D conformation, which renders traditional therapeutic strategies largely ineffective. We demonstrate herein that small molecule activation of the 20S proteasome targets dysregulated intrinsically disordered proteins (IDPs), including c-MYC, and reduces cancer growth in vitro and in vivo models of multiple myeloma, and is even effective in bortezomib resistant cells and unresponsive patient samples. Genomic analysis of various cancer pathways showed that proteasome activation results in downregulation of many c-MYC target genes. Moreover, proteasome enhancement was well tolerated in mice and dogs. These data support the therapeutic potential of 20S proteasome activation in targeting IDP-driven proteotoxic disorders, including cancer, and demonstrate that this new therapeutic strategy is well tolerated in vivo.

scholarly journals Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

2020 ◽  
Vol 117 (21) ◽  
pp. 11421-11431 ◽  
Author(s):  
Benjamin S. Schuster ◽  
Gregory L. Dignon ◽  
Wai Shing Tang ◽  
Fleurie M. Kelley ◽  
Aishwarya Kanchi Ranganath ◽  
...  
Keyword(s):  
Phase Separation ◽  
Intrinsically Disordered Proteins ◽  
Lipid Membrane ◽  
Coarse Grained ◽  
Disordered Proteins ◽  
Sequence Features ◽  
Intrinsically Disordered ◽  
Arginine Residues

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.

scholarly journals Interplay of Structural Disorder and Short Binding Elements in the Cellular Chaperone Function of Plant Dehydrin ERD14

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1856
Author(s):  
Nikoletta Murvai ◽  
Lajos Kalmar ◽  
Bianka Szalaine Agoston ◽  
Beata Szabo ◽  
Agnes Tantos ◽  
...  
Keyword(s):  
Heat Stress ◽  
Intrinsically Disordered Proteins ◽  
Stress Protein ◽  
Structural Disorder ◽  
Disordered Proteins ◽  
Sequence Motifs ◽  
E Coli ◽  
Intrinsically Disordered

Details of the functional mechanisms of intrinsically disordered proteins (IDPs) in living cells is an area not frequently investigated. Here, we dissect the molecular mechanism of action of an IDP in cells by detailed structural analyses based on an in-cell nuclear magnetic resonance experiment. We show that the ID stress protein (IDSP) A. thaliana Early Response to Dehydration (ERD14) is capable of protecting E. coli cells under heat stress. The overexpression of ERD14 increases the viability of E. coli cells from 38.9% to 73.9% following heat stress (50 °C × 15 min). We also provide evidence that the protection is mainly achieved by protecting the proteome of the cells. In-cell NMR experiments performed in E. coli cells show that the protective activity is associated with a largely disordered structural state with conserved, short sequence motifs (K- and H-segments), which transiently sample helical conformations in vitro and engage in partner binding in vivo. Other regions of the protein, such as its S segment and its regions linking and flanking the binding motifs, remain unbound and disordered in the cell. Our data suggest that the cellular function of ERD14 is compatible with its residual structural disorder in vivo.

scholarly journals Genetically tunable frustration controls allostery in an intrinsically disordered transcription factor

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jing Li ◽  
Jordan T White ◽  
Harry Saavedra ◽  
James O Wrabl ◽  
Hesam N Motlagh ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Structural Changes ◽  
Intrinsically Disordered Proteins ◽  
Tertiary Structure ◽  
Control Function ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Novel Strategy ◽  
Structured Proteins

Intrinsically disordered proteins (IDPs) present a functional paradox because they lack stable tertiary structure, but nonetheless play a central role in signaling, utilizing a process known as allostery. Historically, allostery in structured proteins has been interpreted in terms of propagated structural changes that are induced by effector binding. Thus, it is not clear how IDPs, lacking such well-defined structures, can allosterically affect function. Here, we show a mechanism by which an IDP can allosterically control function by simultaneously tuning transcriptional activation and repression, using a novel strategy that relies on the principle of ‘energetic frustration’. We demonstrate that human glucocorticoid receptor tunes this signaling in vivo by producing translational isoforms differing only in the length of the disordered region, which modulates the degree of frustration. We expect this frustration-based model of allostery will prove to be generally important in explaining signaling in other IDPs.

scholarly journals Single-embryo phosphoproteomics reveals the importance of intrinsic disorder in cell cycle dynamics

2021 ◽  
Author(s):  
Juan Manuel Valverde ◽  
Geronimo Dubra ◽  
Henk van den Toorn ◽  
Guido van Mierlo ◽  
Michiel Vermeulen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Intrinsically Disordered Proteins ◽  
Protein Complexes ◽  
Disordered Proteins ◽  
Cell Cycles ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Egg Extracts ◽  
Cell Cycle Dynamics

Switch-like cyclin-dependent kinase (CDK)-1 activation is thought to underlie the abruptness of mitotic onset, but how CDKs can simultaneously phosphorylate many diverse substrates is unknown, and direct evidence for such phosphorylation dynamics in vivo is lacking. Here, we analysed protein phosphorylation states in single Xenopus embryos throughout synchronous cell cycles. Over a thousand phosphosites were dynamic in vivo, and assignment of cell cycle phases using egg extracts revealed hundreds of S-phase phosphorylations. Targeted phosphoproteomics in single embryos showed switch-like mitotic phosphorylation of diverse protein complexes. The majority of cell cycle-regulated phosphosites occurred in CDK consensus motifs, and 72% located to intrinsically disordered regions. Dynamically phosphorylated proteins, and documented substrates of cell cycle kinases, are significantly more disordered than phosphoproteins in general. Furthermore, 30-50% are components of membraneless organelles. Our results suggest that phosphorylation of intrinsically disordered proteins by cell cycle kinases, particularly CDKs, allows switch-like mitotic cellular reorganisation.

scholarly journals Computational Prediction of Intrinsically Disordered Proteins Based on Protein Sequences and Convolutional Neural Networks

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Hao He ◽  
Yong Yang
Keyword(s):  
Neural Networks ◽  
Convolutional Neural Networks ◽  
Intrinsically Disordered Proteins ◽  
Prediction Method ◽  
Computational Prediction ◽  
Disordered Proteins ◽  
Biological Functions ◽  
Intrinsically Disordered ◽  
Central Residue

Intrinsically disordered proteins (IDPs) possess at least one region that lacks a single stable structure in vivo, which makes them play an important role in a variety of biological functions. We propose a prediction method for IDPs based on convolutional neural networks (CNNs) and feature selection. The combination of sequence and evolutionary properties is used to describe the differences between disordered and ordered regions. Especially, to highlight the correlation between the target residue and adjacent residues, multiple windows are selected to preprocess the protein sequence through the selected properties. The shorter windows reflect the characteristics of the central residue, and the longer windows reflect the characteristics of the surroundings around the central residue. Moreover, to highlight the specificity of sequence and evolutionary properties, they are preprocessed, respectively. After that, the preprocessed properties are combined into feature matrices as the input of the constructed CNN. Our method is training as well as testing based on the DisProt database. The simulation results show that the proposed method can predict IDPs effectively, and the performance is competitive in comparison with IsUnstruct and ESpritz.

scholarly journals PETISCO is a novel protein complex required for 21U RNA biogenesis and embryonic viability

2018 ◽  
Author(s):  
Ricardo J. Cordeiro Rodrigues ◽  
António Miguel de Jesus Domingues ◽  
Svenja Hellmann ◽  
Sabrina Dietz ◽  
Bruno F. M. de Albuquerque ◽  
...  
Keyword(s):  
Protein Complex ◽  
Intrinsically Disordered Proteins ◽  
Rna Binding ◽  
Disordered Proteins ◽  
Sequence Motif ◽  
C Elegans ◽  
Conserved Sequence ◽  
Intrinsically Disordered ◽  
Rna Biogenesis ◽  
Novel Protein

AbstractPiwi proteins are important for germ cell development in almost all animals studied thus far. These proteins are guided to specific targets, such as transposable elements, by small guide RNAs, often referred to as piRNAs, or 21U RNAs in C. elegans. In this organism, even though genetic screens have uncovered a number of potential 21U RNA biogenesis factors, little is known about how these factors interact or what they do. Based on the previously identified 21U biogenesis factor PID-1, we here define a novel protein complex, PETISCO, that is required for 21U RNA biogenesis. PETISCO contains both potential 5’-cap and 5’-phosphate RNA binding domains, suggesting involvement in 5’ end processing. We define the interaction architecture of PETISCO and reveal a second function for PETISCO in embryonic development. This essential function of PETISCO is not mediated by PID-1, but by TOST-1. Vice versa, TOST-1 is not involved in 21U RNA biogenesis. Both PID-1 and TOST-1 are small, intrinsically disordered proteins that interact directly with the PETISCO protein ERH-2 (enhancer of rudimentary homolog 2) using a conserved sequence motif. Finally, our data suggest an important role for TOST-1:PETISCO in SL1 homeostasis in the early embryo. Our work describes the first molecular platform for 21U RNA production in C. elegans, and strengthens the view that 21U RNA biogenesis is built upon a much more widely used, snRNA-related pathway.

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