bims-indpro 2022-12-04 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2022–12–04
ten papers selected by
Sara Mingu, Johannes Gutenberg University

Fixation can change the appearance of phase separation in living cells.
Hydrophobicity of arginine leads to reentrant liquid-liquid phase separation behaviors of arginine-rich proteins.
The Mycobacterium tuberculosis PE15/PPE20 complex transports calcium across the outer membrane.
Biomolecular condensates can both accelerate and suppress aggregation of α-synuclein.
Investigating how intrinsically disordered regions contribute to protein function using HDX-MS.
DisCanVis: Visualizing integrated structural and functional annotations for better understanding of the effect of cancer mutations located within disordered proteins.
Effects of Conformational Constraint on Peptide Solubility Limits.
CLIP: accurate prediction of disordered linear interacting peptides from protein sequences using co-evolutionary information.
Come together now: Dynamic body-formation of key regulators integrates environmental cues in plant development.
Fused in sarcoma undergoes cold denaturation: Implications for phase separation.

Elife. 2022 Nov 29. pii: e79903. [Epub ahead of print]11

Fixation can change the appearance of phase separation in living cells.

Shawn Irgen-Gioro, Shawn Ryohei Yoshida, Victoria Walling, Shasha Chong.

  Fixing cells with paraformaldehyde (PFA) is an essential step in numerous biological techniques as it is thought to preserve a snapshot of biomolecular transactions in living cells. Fixed cell imaging techniques such as immunofluorescence have been widely used to detect liquid-liquid phase separation (LLPS) in vivo. Here, we compared images, before and after fixation, of cells expressing intrinsically disordered proteins that are able to undergo LLPS. Surprisingly, we found that PFA fixation can both enhance and diminish putative LLPS behaviors. For specific proteins, fixation can even cause their droplet-like puncta to artificially appear in cells that do not have any detectable puncta in the live condition. Fixing cells in the presence of glycine, a molecule that modulates fixation rates, can reverse the fixation effect from enhancing to diminishing LLPS appearance. We further established a kinetic model of fixation in the context of dynamic protein-protein interactions. Simulations based on the model suggest that protein localization in fixed cells depends on an intricate balance of protein-protein interaction dynamics, the overall rate of fixation, and notably, the difference between fixation rates of different proteins. Consistent with simulations, live-cell single-molecule imaging experiments showed that a fast overall rate of fixation relative to protein-protein interaction dynamics can minimize fixation artifacts. Our work reveals that PFA fixation changes the appearance of LLPS from living cells, presents a caveat in studying LLPS using fixation-based methods, and suggests a mechanism underlying the fixation artifact.

Keywords:  cell biology; none; physics of living systems

DOI:  https://doi.org/10.7554/eLife.79903
Nat Commun. 2022 Nov 28. 13(1): 7326

Hydrophobicity of arginine leads to reentrant liquid-liquid phase separation behaviors of arginine-rich proteins.

Yuri Hong, Saeed Najafi, Thomas Casey, Joan-Emma Shea, Song-I Han, Dong Soo Hwang.

Intrinsically disordered proteins rich in cationic amino acid groups can undergo Liquid-Liquid Phase Separation (LLPS) in the presence of charge-balancing anionic counterparts. Arginine and Lysine are the two most prevalent cationic amino acids in proteins that undergo LLPS, with arginine-rich proteins observed to undergo LLPS more readily than lysine-rich proteins, a feature commonly attributed to arginine's ability to form stronger cation-π interactions with aromatic groups. Here, we show that arginine's ability to promote LLPS is independent of the presence of aromatic partners, and that arginine-rich peptides, but not lysine-rich peptides, display re-entrant phase behavior at high salt concentrations. We further demonstrate that the hydrophobicity of arginine is the determining factor giving rise to the reentrant phase behavior and tunable viscoelastic properties of the dense LLPS phase. Controlling arginine-induced reentrant LLPS behavior using temperature and salt concentration opens avenues for the bioengineering of stress-triggered biological phenomena and drug delivery systems.

DOI: https://doi.org/10.1038/s41467-022-35001-1
PLoS Biol. 2022 Nov 28. 20(11): e3001906

The Mycobacterium tuberculosis PE15/PPE20 complex transports calcium across the outer membrane.

Vishant Boradia, Andrew Frando, Christoph Grundner.

The mechanisms by which nutrients traverse the Mycobacterium tuberculosis (Mtb) outer membrane remain mostly unknown and, in the absence of classical porins, likely involve specialized transport systems. Calcium ions (Ca2+) are an important nutrient and serve as a second messenger in eukaryotes, but whether bacteria have similar Ca2+ signaling systems is not well understood. To understand the basis for Ca2+ transport and signaling in Mtb, we determined Mtb's transcriptional response to Ca2+. Overall, only few genes changed expression, suggesting a limited role of Ca2+ as a transcriptional regulator. However, 2 of the most strongly down-regulated genes were the pe15 and ppe20 genes that code for members of a large family of proteins that localize to the outer membrane and comprise many intrinsically disordered proteins. PE15 and PPE20 formed a complex and PPE20 directly bound Ca2+. Ca2+-associated phenotypes such as increased ATP consumption and biofilm formation were reversed in a pe15/ppe20 knockout (KO) strain, suggesting a direct role in Ca2+ homeostasis. To test whether the PE15/PPE20 complex has a role in Ca2+ transport across the outer membrane, we created a fluorescence resonance energy transfer (FRET)-based Ca2+ reporter strain. A pe15/ppe20 KO in the FRET background showed a specific and selective loss of Ca2+ influx that was dependent on the presence of an intact outer cell wall. These data show that PE15/PPE20 form a Ca2+-binding protein complex that selectively imports Ca2+, show a distinct transport function for an intrinsically disordered protein, and support the emerging idea of a general family-wide role of PE/PPE proteins as idiosyncratic transporters across the outer membrane.

DOI: https://doi.org/10.1371/journal.pbio.3001906
Sci Adv. 2022 Dec 02. 8(48): eabq6495

Biomolecular condensates can both accelerate and suppress aggregation of α-synuclein.

Wojciech P Lipiński, Brent S Visser, Irina Robu, Mohammad A A Fakhree, Saskia Lindhoud, Mireille M A E Claessens, Evan Spruijt.

Biomolecular condensates present in cells can fundamentally affect the aggregation of amyloidogenic proteins and play a role in the regulation of this process. While liquid-liquid phase separation of amyloidogenic proteins by themselves can act as an alternative nucleation pathway, interaction of partly disordered aggregation-prone proteins with preexisting condensates that act as localization centers could be a far more general mechanism of altering their aggregation behavior. Here, we show that so-called host biomolecular condensates can both accelerate and slow down amyloid formation. We study the amyloidogenic protein α-synuclein and two truncated α-synuclein variants in the presence of three types of condensates composed of nonaggregating peptides, RNA, or ATP. Our results demonstrate that condensates can markedly speed up amyloid formation when proteins localize to their interface. However, condensates can also significantly suppress aggregation by sequestering and stabilizing amyloidogenic proteins, thereby providing living cells with a possible protection mechanism against amyloid formation.

DOI: https://doi.org/10.1126/sciadv.abq6495
Biochem Soc Trans. 2022 Dec 01. pii: BST20220206. [Epub ahead of print]

Investigating how intrinsically disordered regions contribute to protein function using HDX-MS.

Matthew A H Parson, Meredith L Jenkins, John E Burke.

  A large amount of the human proteome is composed of highly dynamic regions that do not adopt a single static conformation. These regions are defined as intrinsically disordered, and they are found in a third of all eukaryotic proteins. They play instrumental roles in many aspects of protein signaling, but can be challenging to characterize by biophysical methods. Intriguingly, many of these regions can adopt stable secondary structure upon interaction with a variety of binding partners, including proteins, lipids, and ligands. This review will discuss the application of Hydrogen-deuterium exchange mass spectrometry (HDX-MS) as a powerful biophysical tool that is particularly well suited for structural and functional characterization of intrinsically disordered regions in proteins. A focus will be on the theory of hydrogen exchange, and its practical application to identify disordered regions, as well as characterize how they participate in protein-protein and protein-membrane interfaces. A particular emphasis will be on how HDX-MS data can be presented specifically tailored for analysis of intrinsically disordered regions, as well as the technical aspects that are critical to consider when designing HDX-MS experiments for proteins containing intrinsically disordered regions.

Keywords:  HDX-MS; PI4K; hydrogen exchange; hydrogen-deuterium exchange mass spectrometry; intrinsically disordered proteins; protein–protein interactions

DOI:  https://doi.org/10.1042/BST20220206
Protein Sci. 2022 Nov 30. e4522

DisCanVis: Visualizing integrated structural and functional annotations for better understanding of the effect of cancer mutations located within disordered proteins.

Norbert Deutsch, Mátyás Pajkos, Gábor Erdős, Zsuzsanna Dosztányi.

  Intrinsically disordered proteins (IDPs) play important roles in a wide range of biological processes and have been associated with various diseases, including cancer. In the last few years, cancer genome projects have systematically collected genetic variations underlying multiple cancer types. In parallel, the number and different types of disordered proteins characterized by experimental methods have also significantly increased. Nevertheless, the role of IDPs in various types of cancer is still not well understood. In this work we present DisCanVis, a novel visualization tool for cancer mutations with a special focus on intrinsically disordered proteins. In order to aid the interpretation of observed mutations, genome level information is combined with information about the structural and functional properties of proteins. The web server enables users to inspect individual proteins, collect examples with existing annotations of protein disorder and associated function or to discover currently uncharacterized examples with likely disease relevance. Through a REST API interface and pre-compiled tables the analysis can be extended to a group of proteins. The web server is freely available for academic researchers at https://discanvis.elte.hu/. This article is protected by copyright. All rights reserved.

Keywords:  cancer genome projects; intrinsically disordered proteins; mutations; sequence predictions; sequence variations; visualization; web server

DOI:  https://doi.org/10.1002/pro.4522
J Phys Chem B. 2022 Nov 30.

Effects of Conformational Constraint on Peptide Solubility Limits.

Riley J Workman, Suresh Gorle, B Montgomery Pettitt.

Liquid-liquid phase separation of proteins preferentially involves intrinsically disordered proteins or disordered regions. Understanding the solution chemistry of these phase separations is key to learning how to quantify and manipulate systems that involve such processes. Here, we investigate the effect of cyclization on the liquid-liquid phase separation of short polyglycine peptides. We simulated separate aqueous systems of supersaturated cyclic and linear GGGGG and observed spontaneous liquid-liquid phase separation in each of the solutions. The cyclic GGGGG phase separates less robustly than linear GGGGG and has a higher aqueous solubility, even though linear GGGGG has a more favorable single molecule solvation free energy. The versatile and abundant interpeptide contacts formed by the linear GGGGG stabilize the condensed droplet phase, driving the phase separation in this system. In particular, we find that van der Waals close contact interactions are enriched in the droplet phase as opposed to electrostatic interactions. An analysis of the change in backbone conformational entropy that accompanies the phase transition revealed that cyclic peptides lose significantly less entropy in this process as expected. However, we find that the enhanced interaction enthalpy of linear GGGGG in the droplet phase is enough to compensate for a larger decrease in conformational entropy.

DOI: https://doi.org/10.1021/acs.jpcb.2c06458
Brief Bioinform. 2022 Dec 01. pii: bbac502. [Epub ahead of print]

CLIP: accurate prediction of disordered linear interacting peptides from protein sequences using co-evolutionary information.

Zhenling Peng, Zixia Li, Qiaozhen Meng, Bi Zhao, Lukasz Kurgan.

  One of key features of intrinsically disordered regions (IDRs) is facilitation of protein-protein and protein-nucleic acids interactions. These disordered binding regions include molecular recognition features (MoRFs), short linear motifs (SLiMs) and longer binding domains. Vast majority of current predictors of disordered binding regions target MoRFs, with a handful of methods that predict SLiMs and disordered protein-binding domains. A new and broader class of disordered binding regions, linear interacting peptides (LIPs), was introduced recently and applied in the MobiDB resource. LIPs are segments in protein sequences that undergo disorder-to-order transition upon binding to a protein or a nucleic acid, and they cover MoRFs, SLiMs and disordered protein-binding domains. Although current predictors of MoRFs and disordered protein-binding regions could be used to identify some LIPs, there are no dedicated sequence-based predictors of LIPs. To this end, we introduce CLIP, a new predictor of LIPs that utilizes robust logistic regression model to combine three complementary types of inputs: co-evolutionary information derived from multiple sequence alignments, physicochemical profiles and disorder predictions. Ablation analysis suggests that the co-evolutionary information is particularly useful for this prediction and that combining the three inputs provides substantial improvements when compared to using these inputs individually. Comparative empirical assessments using low-similarity test datasets reveal that CLIP secures area under receiver operating characteristic curve (AUC) of 0.8 and substantially improves over the results produced by the closest current tools that predict MoRFs and disordered protein-binding regions. The webserver of CLIP is freely available at http://biomine.cs.vcu.edu/servers/CLIP/ and the standalone code can be downloaded from http://yanglab.qd.sdu.edu.cn/download/CLIP/.

Keywords:  intrinsic disorder; linear interacting peptides; molecular recognition features; protein function; protein–nucleic acids interactions; protein–protein interactions

DOI:  https://doi.org/10.1093/bib/bbac502
Front Plant Sci. 2022 ;13 1052107

Come together now: Dynamic body-formation of key regulators integrates environmental cues in plant development.

Rebecca C Burkart, Ali Eljebbawi, Yvonne Stahl.

  Plants as sessile organisms are constantly exposed to changing environmental conditions, challenging their growth and development. Indeed, not only above-ground organs but also the underground root system must adapt accordingly. Consequently, plants respond to these constraints at a gene-regulatory level to ensure their survival and well-being through key transcriptional regulators involved in different developmental processes. Recently, intrinsically disordered domains within these regulators are emerging as central nodes necessary not only for interactions with other factors but also for their partitioning into biomolecular condensates, so-called bodies, possibly driven by phase separation. Here, we summarize the current knowledge about body-forming transcriptional regulators important for plant development and highlight their functions in a possible environmental context. In this perspective article, we discuss potential mechanisms for the formation of membrane-less bodies as an efficient and dynamic program needed for the adaptation to external cues with a particular focus on the Arabidopsis root. Hereby, we aim to provide a perspective for future research on transcriptional regulators to investigate body formation as an expeditious mechanism of plant-environment interactions.

Keywords:  adaptation; body-formation; development; environmental stimuli; phase separation; plant memory; transcription factor; transcriptional regulator

DOI:  https://doi.org/10.3389/fpls.2022.1052107
Protein Sci. 2022 Nov 30. e4521

Fused in sarcoma undergoes cold denaturation: Implications for phase separation.

Sara S Félix, Douglas V Laurents, Javier Oroz, Eurico J Cabrita.

  The mediation of liquid-liquid phase separation (LLPS) for FUS protein is generally attributed to the low-complexity, disordered domains and is enhanced at low temperature. The role of FUS folded domains on the LLPS process remains relatively unknown since most studies are mainly based on fragmented FUS domains. Here, we investigate the effect of metabolites on full-length FUS LLPS using turbidity assays and differential interference contrast (DIC) microscopy, and explore the behavior of the folded domains by nuclear magnetic resonance (NMR) spectroscopy. Full-length FUS LLPS is maximal at low concentrations of glucose and glutamate, moderate concentrations of NaCl, Zn2+ and Ca2+ and at the isoelectric pH. The FUS RNA-recognition motif (RRM) and zinc-finger (ZnF) domains are found to undergo cold denaturation above 0°C at a temperature that is determined by the conformational stability of the ZnF domain. Cold unfolding exposes buried nonpolar residues that can participate in LLPS-promoting hydrophobic interactions. Therefore, these findings constitute the first evidence that FUS globular domains may have an active role in LLPS under cold stress conditions and in the assembly of stress granules, providing further insight into the environmental regulation of LLPS. This article is protected by copyright. All rights reserved.

Keywords:  cold denaturation; cold stress; fused in sarcoma (FUS); liquid-liquid phase separation (LLPS); nuclear magnetic resonance (NMR)

DOI:  https://doi.org/10.1002/pro.4521