bims-indpro 2022-10-16 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2022–10–16
23 papers selected by
Sara Mingu, Johannes Gutenberg University

Connecting sequence features within the disordered C-terminal linker of Bacillus subtilis FtsZ to functions and bacterial cell division.
New insights into disordered proteins and regions according to the FOD-M model.
Intrinsic Protein Disorder and Conditional Folding in AlphaFoldDB.
Structure and Transition Dynamics of Intrinsically Disordered Proteins Probed by Single-Molecule Spectroscopy.
Numerical Techniques for Applications of Analytical Theories to Sequence-Dependent Phase Separations of Intrinsically Disordered Proteins.
Targeting disordered-structured domain interactions in Galectin-3 based on NMR and enhanced MD.
REMD Simulations of Full-Length Alpha-Synuclein Together with Ligands Reveal Binding Region and Effect on Amyloid Conversion.
eIF4G1 N-terminal intrinsically disordered domain is a multi-docking station for RNA, Pab1, Pub1, and self-assembly.
Distinct binding interactions trigger opposite conformational modulations on pathogenic and wildtype Huntingtin exon 1 proteins.
AMBRA1 and its role as a target for anticancer therapy.
Selenoprotein S: A versatile disordered protein.
Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.
Germline immortality relies on TRIM32-mediated turnover of a maternal mRNA activator in C. elegans.
Fluorescence Lifetime Imaging Microscopy of Biomolecular Condensates.
Quasi-analytical resolution-correction of elastic neutron scattering from proteins.
Fluorescence Correlation Spectroscopy and Phase Separation.
Non-catalytic allostery in α-TAT1 by a phospho-switch drives dynamic microtubule acetylation.
An Introduction to the Stickers-and-Spacers Framework as Applied to Biomolecular Condensates.
Intracellular mRNA phase separation induced by cationic polymers for tumor immunotherapy.
Effects of Charged Polyelectrolytes on Amyloid Fibril Formation of a Tau Fragment.
Balanced Force Field ff03CMAP Improving the Dynamics Conformation Sampling of Phosphorylation Site.
Insights into the role of Nup62 and Nup93 in assembling cytoplasmic ring and central transport channel of the nuclear pore complex.
Cryo-Electron Tomography of Reconstituted Biomolecular Condensates.

Proc Natl Acad Sci U S A. 2022 Oct 18. 119(42): e2211178119

Connecting sequence features within the disordered C-terminal linker of Bacillus subtilis FtsZ to functions and bacterial cell division.

Min Kyung Shinn, Megan C Cohan, Jessie L Bullock, Kiersten M Ruff, Petra A Levin, Rohit V Pappu.

  Intrinsically disordered regions (IDRs) can function as autoregulators of folded enzymes to which they are tethered. One example is the bacterial cell division protein FtsZ. This includes a folded core and a C-terminal tail (CTT) that encompasses a poorly conserved, disordered C-terminal linker (CTL) and a well-conserved 17-residue C-terminal peptide (CT17). Sites for GTPase activity of FtsZs are formed at the interface between GTP binding sites and T7 loops on cores of adjacent subunits within dimers. Here, we explore the basis of autoregulatory functions of the CTT in Bacillus subtilis FtsZ (Bs-FtsZ). Molecular simulations show that the CT17 of Bs-FtsZ makes statistically significant CTL-mediated contacts with the T7 loop. Statistical coupling analysis of more than 1,000 sequences from FtsZ orthologs reveals clear covariation of the T7 loop and the CT17 with most of the core domain, whereas the CTL is under independent selection. Despite this, we discover the conservation of nonrandom sequence patterns within CTLs across orthologs. To test how the nonrandom patterns of CTLs mediate CTT-core interactions and modulate FtsZ functionalities, we designed Bs-FtsZ variants by altering the patterning of oppositely charged residues within the CTL. Such alterations disrupt the core-CTT interactions, lead to anomalous assembly and inefficient GTP hydrolysis in vitro and protein degradation, aberrant assembly, and disruption of cell division in vivo. Our findings suggest that viable CTLs in FtsZs are likely to be IDRs that encompass nonrandom, functionally relevant sequence patterns that also preserve three-way covariation of the CT17, the T7 loop, and core domain.

Keywords:  autoinhibition; autoregulation; covariation; intrinsically disordered proteins; polymerization

DOI:  https://doi.org/10.1073/pnas.2211178119
PLoS One. 2022 ;17(10): e0275300

New insights into disordered proteins and regions according to the FOD-M model.

Irena Roterman, Katarzyna Stapor, Piotr Fabian, Leszek Konieczny.

A collection of intrinsically disordered proteins (IDPs) having regions with the status of intrinsically disordered (IDR) according to the Disprot database was analyzed from the point of view of the structure of hydrophobic core in the structural unit (chain / domain). The analysis includes all the Homo Sapiens as well as Mus Musculus proteins present in the DisProt database for which the structure is available. In the analysis, the fuzzy oil drop modified model (FOD-M) was used, taking into account the external force field, modified by the presence of other factors apart from polar water, influencing protein structuring. The paper presents an alternative to secondary-structure-based classification of intrinsically disordered regions (IDR). The basis of our classification is the ordering of hydrophobic core as calculated by the FOD-M model resulting in FOD-ordered or FOD-unordered IDRs.

DOI: https://doi.org/10.1371/journal.pone.0275300
Protein Sci. 2022 Oct 10. e4466

Intrinsic Protein Disorder and Conditional Folding in AlphaFoldDB.

Damiano Piovesan, Alexander Miguel Monzon, Silvio C E Tosatto.

Intrinsically disordered regions (IDRs) defying the traditional protein structure-function paradigm have been difficult to analyze. The availability of accurate structure predictions on a large scale in AlphaFoldDB offers a fresh perspective on IDR prediction. Here we establish three baselines for IDR prediction from AlphaFoldDB models based on the recent CAID dataset. Surprisingly, AlphaFoldDB is highly competitive for predicting both IDRs and conditionally folded binding regions, demonstrating the plasticity of the disorder to structure continuum. This article is protected by copyright. All rights reserved.

DOI: https://doi.org/10.1002/pro.4466
Langmuir. 2022 Oct 10.

Structure and Transition Dynamics of Intrinsically Disordered Proteins Probed by Single-Molecule Spectroscopy.

Ushasi Pramanik, Atanu Nandy, Laxmikanta Khamari, Saptarshi Mukherjee.

Intrinsically disordered proteins (IDPs) are a class of proteins that do not follow the unanimated perspective of the structure-function paradigm. IDPs enunciate the dynamics of motions which are often difficult to characterize by a particular experimental or theoretical approach. The chameleon nature of the IDPs is a result of an alteration or transition in their conformation upon binding with ligands. Experimental investigations via ensemble-average approaches to probe this randomness are often difficult to synchronize. Thus, to sense the substates of different conformational ensembles of IDPs, researchers have often targeted approaches based on single-molecule measurements. In this Perspective, we will discuss various single-molecule approaches to explore the conformational transitions of IDPs in different scenarios, the outcome, challenges, and future prospects.

DOI: https://doi.org/10.1021/acs.langmuir.2c02409
Methods Mol Biol. 2023 ;2563 51-94

Numerical Techniques for Applications of Analytical Theories to Sequence-Dependent Phase Separations of Intrinsically Disordered Proteins.

Yi-Hsuan Lin, Jonas Wessén, Tanmoy Pal, Suman Das, Hue Sun Chan.

  Biomolecular condensates, physically underpinned to a significant extent by liquid-liquid phase separation (LLPS), are now widely recognized by numerous experimental studies to be of fundamental biological, biomedical, and biophysical importance. In the face of experimental discoveries, analytical formulations emerged as a powerful yet tractable tool in recent theoretical investigations of the role of LLPS in the assembly and dissociation of these condensates. The pertinent LLPS often involves, though not exclusively, intrinsically disordered proteins engaging in multivalent interactions that are governed by their amino acid sequences. For researchers interested in applying these theoretical methods, here we provide a practical guide to a set of computational techniques devised for extracting sequence-dependent LLPS properties from analytical formulations. The numerical procedures covered include those for the determination of spinodal and binodal phase boundaries from a general free energy function with examples based on the random phase approximation in polymer theory, construction of tie lines for multiple-component LLPS, and field-theoretic simulation of multiple-chain heteropolymeric systems using complex Langevin dynamics. Since a more accurate physical picture often requires comparing analytical theory against explicit-chain model predictions, a commonly utilized methodology for coarse-grained molecular dynamics simulations of sequence-specific LLPS is also briefly outlined.

Keywords:  Biomolecular condensates; Coarse-grained molecular dynamics; Complex-Langevin dynamics; Field-theoretical simulation; Flory-Huggins theory; Intrinsically disordered proteins; Membraneless organelles; Polymer theory; Random phase approximation; Sequence charge pattern

DOI:  https://doi.org/10.1007/978-1-0716-2663-4_3
Biophys J. 2022 Oct 07. pii: S0006-3495(22)00828-1. [Epub ahead of print]

Targeting disordered-structured domain interactions in Galectin-3 based on NMR and enhanced MD.

Supriyo Bhattacharya, Mingfeng Zhang, Weidong Hu, Tong Qi, Nora Heisterkamp.

  Intrinsically disordered regions (IDRs) are common and important functional domains in many proteins. However, IDRs are difficult to target for drug development due to the lack of defined structures which would facilitate the identification of possible drug-binding pockets. Galectin-3 is a carbohydrate-binding protein of which overexpression has been implicated in a wide variety of disorders including cancer and inflammation. Apart from its carbohydrate recognition/binding domain (CRD), Galectin-3 also contains a functionally important disordered N-terminal domain (NTD) that contacts the C-terminal domain (CTD) and could be a target for drug development. To overcome challenges involved in inhibitor design due to lack of structure and the highly dynamic nature of the NTD, we used a protocol combining nuclear magnetic resonance data from recombinant Galectin-3 with accelerated molecular dynamics (MD) simulations. This approach identified a pocket in the CTD with which the NTD makes frequent contact. In accordance with this model, mutation of residues L131 and L203 in this pocket caused loss of Galectin-3 agglutination ability, signifying the functional relevance of the cavity. In-silico screening was used to design candidate inhibitory peptides targeting the newly discovered cavity and experimental testing of only 3 of these yielded one peptide that inhibits the agglutination promoted by wild type Galectin-3. NMR experiments further confirmed that this peptide indeed binds to a cavity in the CTD not within the actual CRD. Our results show that it is possible to apply a combination of MD simulations and NMR experiments to precisely predict the binding interface of a disordered domain with a structured domain, and furthermore use this predicted interface for designing inhibitors. This procedure can be potentially extended to many other targets in which similar IDR interactions play a vital functional role.

Keywords:  Intrinsically disordered; acute lymphoblastic leukemia; agglutination; carbohydrate recognition domain; carbohydrate-binding; lectin

DOI:  https://doi.org/10.1016/j.bpj.2022.10.008
Int J Mol Sci. 2022 Sep 30. pii: 11545. [Epub ahead of print]23(19):

REMD Simulations of Full-Length Alpha-Synuclein Together with Ligands Reveal Binding Region and Effect on Amyloid Conversion.

Pavel I Semenyuk.

  Alpha-synuclein is a key protein involved in the development and progression of Parkinson's disease and other synucleinopathies. The intrinsically disordered nature of alpha-synuclein hinders the computational screening of new drug candidates for the treatment of these neurodegenerative diseases. In the present work, replica exchange molecular dynamics simulations of the full-length alpha-synuclein together with low-molecular ligands were utilized to predict the binding site and effect on the amyloid-like conversion of the protein. This approach enabled an accurate prediction of the binding sites for three tested compounds (fasudil, phthalocyanine tetrasulfonate, and spermine), giving good agreement with data from experiments by other groups. Lots of information about the binding and protein conformational ensemble enabled the suggestion of a putative effect of the ligands on the amyloid-like conversion of alpha-synuclein and the mechanism of anti- and pro-amyloid activity of the tested compounds. Therefore, this approach looks promising for testing new drug candidates for binding with alpha-synuclein or other intrinsically disordered proteins and at the same time the estimation of the effect on protein behavior, including amyloid-like aggregation.

Keywords:  aggregation inhibitor; alpha-synuclein; amyloid conversion; binding site prediction; intrinsically disordered proteins; molecular modeling

DOI:  https://doi.org/10.3390/ijms231911545
Front Mol Biosci. 2022 ;9 986121

eIF4G1 N-terminal intrinsically disordered domain is a multi-docking station for RNA, Pab1, Pub1, and self-assembly.

Belén Chaves-Arquero, Santiago Martínez-Lumbreras, Nathalie Sibille, Sergio Camero, Pau Bernadó, M Ángeles Jiménez, Silvia Zorrilla, José Manuel Pérez-Cañadillas.

  Yeast eIF4G1 interacts with RNA binding proteins (RBPs) like Pab1 and Pub1 affecting its function in translation initiation and stress granules formation. We present an NMR and SAXS study of the N-terminal intrinsically disordered region of eIF4G1 (residues 1-249) and its interactions with Pub1, Pab1 and RNA. The conformational ensemble of eIF4G11-249 shows an α-helix within the BOX3 conserved element and a dynamic network of fuzzy π-π and π-cation interactions involving arginine and aromatic residues. The Pab1 RRM2 domain interacts with eIF4G1 BOX3, the canonical interaction site, but also with BOX2, a conserved element of unknown function to date. The RNA1 region interacts with RNA through a new RNA interaction motif and with the Pub1 RRM3 domain. This later also interacts with eIF4G1 BOX1 modulating its intrinsic self-assembly properties. The description of the biomolecular interactions involving eIF4G1 to the residue detail increases our knowledge about biological processes involving this key translation initiation factor.

Keywords:  NMR; Pab1; Pub1; SAXS (small-angle X-ray scattering); eIF4G1; intrinsically disordered proteins (IDP); paramagnetic relaxation enhancement (PRE); structural ensemble

DOI:  https://doi.org/10.3389/fmolb.2022.986121
Phys Chem Chem Phys. 2022 Oct 10.

Distinct binding interactions trigger opposite conformational modulations on pathogenic and wildtype Huntingtin exon 1 proteins.

Jiaming Guan, Zhijian Song, Guanghong Wei, Qin Qiao.

Abnormal elongation of the polyglutamine tract transforms exon 1 of the Huntingtin protein (Htt-exon-1) from wildtype to pathogenic form, and causes Huntington's disease. As an intrinsically disordered protein, the structural ensemble of Htt-exon-1 is highly heterogeneous and the detailed conformation of toxic species is still under debate. Ispinesib, a potential small-molecule drug, has been identified to selectively link the pathogenic Htt-exon-1 into the autophagosome to degrade, thus opening an innovative therapeutic direction. However, the molecular mechanisms behind this selectivity remain largely elusive. Herein, we carry out extensive molecular dynamics simulations with an enhanced sampling method to investigate the ispinesib inducing conformational changes of pathogenic and wildtype Htt-exon-1 and the corresponding binding mechanisms. Our simulations reveal that the ispinesib binding induces opposite conformational changes in pathogenic and wildtype Htt-exon-1, i.e., the 'entropy collapse' with significant reduction of β-sheets versus the 'entropy expansion' with a slight increase of α-helices. Network analyses further reveal that there are two stable binding sites in the pathogenic Htt-exon-1, while the binding on the wildtype Htt-exon-1 is highly dynamic and non-specific. These dramatic differences originate from the underlying distinct binding interactions. More specifically, stronger hydrogen bonds serve as the specific binding anchors in pathogenic Htt-exon-1, while stronger hydrophobic interactions dominate in the dynamic binding with wildtype Htt-exon-1. Our simulations provide an atomistic mechanism for the ispinesib selective binding on the pathogenic Htt-exon-1, and further shed light on the general mechanisms of small molecule modulation on intrinsically disordered proteins.

DOI: https://doi.org/10.1039/d2cp03173f
Front Oncol. 2022 ;12 946086

AMBRA1 and its role as a target for anticancer therapy.

Xiang Li, Yuan Lyu, Junqi Li, Xinjun Wang.

  The activating molecule in Beclin1-regulated autophagy protein 1 (AMBRA1) is an intrinsically disordered protein that regulates the survival and death of cancer cells by modulating autophagy. Although the roles of autophagy in cancer are controversial and context-dependent, inhibition of autophagy under some circumstances can be a useful strategy for cancer therapy. As AMBRA1 is a pivotal autophagy-associated protein, targeting AMBRA1 similarly may be an underlying strategy for cancer therapy. Emerging evidence indicates that AMBRA1 can also inhibit cancer formation, maintenance, and progression by regulating c-MYC and cyclins, which are frequently deregulated in human cancer cells. Therefore, AMBRA1 is at the crossroad of autophagy, tumorigenesis, proliferation, and cell cycle. In this review, we focus on discussing the mechanisms of AMBRA1 in autophagy, mitophagy, and apoptosis, and particularly the roles of AMBRA1 in tumorigenesis and targeted therapy.

Keywords:  AMBRA1; autophagy; mitophagy; targeted therapy; tumorigenesis

DOI:  https://doi.org/10.3389/fonc.2022.946086
Arch Biochem Biophys. 2022 Oct 11. pii: S0003-9861(22)00312-5. [Epub ahead of print] 109427

Selenoprotein S: A versatile disordered protein.

Farid Ghelichkhani, Fabio A Gonzalez, Mariia A Kapitonova, Stephanie A Schaefer-Ramadan, Jun Liu, Rujin Cheng, Sharon Rozovsky.

  Selenoprotein S (selenos) is a small, intrinsically disordered membrane protein that is associated with various cellular functions, such as inflammatory processes, cellular stress response, protein quality control, and signaling pathways. It is primarily known for its contribution to the ER-associated degradation (ERAD) pathway, which governs the extraction of misfolded proteins or misassembled protein complexes from the ER to the cytosol for degradation by the proteasome. However, selenos's other cellular roles in signaling are equally vital, including the control of transcription factors and cytokine levels. Consequently, genetic polymorphisms of selenos are associated with increased risk for diabetes, dyslipidemia, and cardiovascular diseases, while high expression levels correlate with poor prognosis in several cancers. Its inhibitory role in cytokine secretion is also exploited by viruses. Since selenos binds multiple protein complexes, however, its specific contributions to various cellular pathways and diseases have been difficult to establish. Thus, the precise cellular functions of selenos and their interconnectivity have only recently begun to emerge. This review aims to summarize recent insights into the structure, interactome, and cellular roles of selenos.1. The molecular biology and biochemistry of selenos.

Keywords:  SELENOS; SEPS1; SelS; TANIS; VIMP

DOI:  https://doi.org/10.1016/j.abb.2022.109427
Microb Cell Fact. 2022 Oct 14. 21(1): 211

Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

Andrea Colarusso, Concetta Lauro, Marzia Calvanese, Ermenegilda Parrilli, Maria Luisa Tutino.

   BACKGROUND: A significant fraction of the human proteome is still inaccessible to in vitro studies since the recombinant production of several proteins failed in conventional cell factories. Eukaryotic protein kinases are difficult-to-express in heterologous hosts due to folding issues both related to their catalytic and regulatory domains. Human CDKL5 belongs to this category. It is a serine/threonine protein kinase whose mutations are involved in CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental pathology still lacking a therapeutic intervention. The lack of successful CDKL5 manufacture hampered the exploitation of the otherwise highly promising enzyme replacement therapy. As almost two-thirds of the enzyme sequence is predicted to be intrinsically disordered, the recombinant product is either subjected to a massive proteolytic attack by host-encoded proteases or tends to form aggregates. Therefore, the use of an unconventional expression system can constitute a valid alternative to solve these issues.
RESULTS: Using a multiparametric approach we managed to optimize the transcription of the CDKL5 gene and the synthesis of the recombinant protein in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 applying a bicistronic expression strategy, whose generalization for recombinant expression in the cold has been here confirmed with the use of a fluorescent reporter. The recombinant protein largely accumulated as a full-length product in the soluble cell lysate. We also demonstrated for the first time that full-length CDKL5 produced in Antarctic bacteria is catalytically active by using two independent assays, making feasible its recovery in native conditions from bacterial lysates as an active product, a result unmet in other bacteria so far. Finally, the setup of an in cellulo kinase assay allowed us to measure the impact of several CDD missense mutations on the kinase activity, providing new information towards a better understanding of CDD pathophysiology.
CONCLUSIONS: Collectively, our data indicate that P. haloplanktis TAC125 can be a valuable platform for both the preparation of soluble active human CDKL5 and the study of structural-functional relationships in wild type and mutant CDKL5 forms. Furthermore, this paper further confirms the more general potentialities of exploitation of Antarctic bacteria to produce "intractable" proteins, especially those containing large intrinsically disordered regions.

Keywords:  Antarctic bacterium; Bicistronic design; In cellulo kinase assay; Intrinsically disordered protein (IDP); Pseudoalteromonas haloplanktis TAC125; Psychrophilic gene expression system; Recombinant protein aggregation; Recombinant protein condensation; Tricistronic design

DOI:  https://doi.org/10.1186/s12934-022-01939-6
Sci Adv. 2022 Oct 14. 8(41): eabn0897

Germline immortality relies on TRIM32-mediated turnover of a maternal mRNA activator in C. elegans.

Tosin D Oyewale, Christian R Eckmann.

How the germ line achieves a clean transition from maternal to zygotic gene expression control is a fundamental problem in sexually reproducing organisms. Whereas several mechanisms terminate the maternal program in the soma, this combined molecular reset and handover are poorly understood for primordial germ cells (PGCs). Here, we show that GRIF-1, a TRIM32-related and presumed E3 ubiquitin ligase in Caenorhabditis elegans, eliminates the maternal cytoplasmic poly(A) polymerase (cytoPAP) complex by targeting the germline-specific intrinsically disordered region of its enzymatic subunit, GLD-2, for proteasome-mediated degradation. Interference with cytoPAP turnover in PGCs causes frequent transgenerational sterility and, eventually, germline mortality. Hence, positively acting maternal RNA regulators are cleared via the proteasome system to avoid likely interference between maternal and zygotic gene expression programs to maintain transgenerational fertility and acquire germline immortality. This strategy is likely used in all animals that preform their immortal germ line via maternally inherited germplasm determinants.

DOI: https://doi.org/10.1126/sciadv.abn0897
Methods Mol Biol. 2023 ;2563 135-148

Fluorescence Lifetime Imaging Microscopy of Biomolecular Condensates.

My Diem Quan, Shih-Chu Jeff Liao, Josephine C Ferreon, Allan Chris M Ferreon.

  Biomolecular condensates of ribonucleoproteins (RNPs) such as the transactivation response element (TAR) DNA-binding protein 43 (TDP-43) arise from liquid-liquid phase separation (LLPS) and play vital roles in various biological processes including the formation-dissolution of stress granules (SGs). These condensates are thought to be directly linked to neurodegenerative diseases, providing a depot of aggregation-prone proteins and serving as a cauldron of protein aggregation and fibrillation. Despite recent research efforts, biochemical processes and rearrangements within biomolecular condensates that trigger subsequent protein misfolding and aggregation remain to be elucidated. Fluorescence lifetime imaging microscopy (FLIM) provides a minimally intrusive high-sensitivity and high-resolution imaging method to monitor in-droplet spatiotemporal changes that initiate and lead to protein aggregation. In this chapter, we describe a FLIM application for characterizing chemical chaperone-assisted decoupling of TDP-43 liquid-liquid phase separation and aggregation/fibrillation, highlighting potential therapeutic strategies to combat pathological RNP-associated aggregates without compromising cellular stress responses.

Keywords:  Droplet maturation; Fluorescence lifetime imaging microscopy; Intrinsically disordered proteins; Liquid-liquid phase separation; Neurodegenerative diseases; Protein aggregation; TDP-43

DOI:  https://doi.org/10.1007/978-1-0716-2663-4_6
J Chem Phys. 2022 Oct 07. 157(13): 134103

Quasi-analytical resolution-correction of elastic neutron scattering from proteins.

Abir N Hassani, Andreas M Stadler, Gerald R Kneller.

Elastic neutron scattering from proteins reflects the motional amplitudes resulting from their internal collective and single-atom dynamics and is observable if the global diffusion of whole molecules is either blocked or cannot be resolved by the spectrometer under consideration. Due to finite instrumental resolution, the measured elastic scattering amplitude always contains contaminations from quasielastic neutron scattering and some model must be assumed to extract the resolution-corrected counterpart from corresponding experimental spectra. Here, we derive a quasi-analytical method for that purpose, assuming that the intermediate scattering function relaxes with a "stretched" Mittag-Leffler function, Eα(-(t/τ)α) (0 < α < 1), toward the elastic amplitude and that the instrumental resolution function has Gaussian form. The corresponding function can be integrated into a fitting procedure and allows for eliminating the elastic intensity as a fit parameter. We illustrate the method for the analysis of two proteins in solution, the intrinsically disordered Myelin Basic Protein, confirming recently published results [Hassani et al., J. Chem. Phys. 156, 025102 (2022)], and the well-folded globular protein myoglobin. We also briefly discuss the consequences of our findings for the extraction of mean square position fluctuations from elastic scans.

DOI: https://doi.org/10.1063/5.0103960
Methods Mol Biol. 2023 ;2563 161-198

Fluorescence Correlation Spectroscopy and Phase Separation.

Juan Jeremías Incicco, Debjit Roy, Melissa D Stuchell-Brereton, Andrea Soranno.

  A quantitative understanding of the forces controlling the assembly and functioning of biomolecular condensates requires the identification of phase boundaries at which condensates form as well as the determination of tie-lines. Here, we describe in detail how Fluorescence Correlation Spectroscopy (FCS) provides a versatile approach to estimate phase boundaries of single-component and multicomponent solutions as well as insights about the transport properties of the condensate.

Keywords:  Biomolecular condensates; Fluorescence Correlation Spectroscopy; Intrinsically disordered proteins; Phase separation

DOI:  https://doi.org/10.1007/978-1-0716-2663-4_8
J Cell Biol. 2022 Nov 07. pii: e202202100. [Epub ahead of print]221(11):

Non-catalytic allostery in α-TAT1 by a phospho-switch drives dynamic microtubule acetylation.

Abhijit Deb Roy, Evan G Gross, Gayatri S Pillai, Shailaja Seetharaman, Sandrine Etienne-Manneville, Takanari Inoue.

Spatiotemporally dynamic microtubule acetylation underlies diverse physiological and pathological events. Despite its ubiquity, the molecular mechanisms that regulate the sole microtubule acetylating agent, α-tubulin-N-acetyltransferase-1 (α-TAT1), remain obscure. Here, we report that dynamic intracellular localization of α-TAT1 along with its catalytic activity determines efficiency of microtubule acetylation. Specifically, we newly identified a conserved signal motif in the intrinsically disordered C-terminus of α-TAT1, consisting of three competing regulatory elements-nuclear export, nuclear import, and cytosolic retention. Their balance is tuned via phosphorylation by CDK1, PKA, and CK2, and dephosphorylation by PP2A. While the unphosphorylated form binds to importins and resides both in cytosol and nucleus, the phosphorylated form binds to specific 14-3-3 adapters and accumulates in the cytosol for maximal substrate access. Unlike other molecules with a similar phospho-regulated signal motif, α-TAT1 uniquely uses the nucleus as a hideout. This allosteric spatial regulation of α-TAT1 function may help uncover a spatiotemporal code of microtubule acetylation in normal and aberrant cell behavior.

DOI: https://doi.org/10.1083/jcb.202202100
Methods Mol Biol. 2023 ;2563 95-116

An Introduction to the Stickers-and-Spacers Framework as Applied to Biomolecular Condensates.

Garrett M Ginell, Alex S Holehouse.

  Cellular organization is determined by a combination of membrane-bound and membrane-less biomolecular assemblies that range from clusters of tens of molecules to micrometer-sized cellular bodies. Over the last decade, membrane-less assemblies have come to be referred to as biomolecular condensates, reflecting their ability to condense specific molecules with respect to the remainder of the cell. In many cases, the physics of phase transitions provides a conceptual framework and a mathematical toolkit to describe the assembly, maintenance, and dissolution of biomolecular condensates. Among the various quantitative and qualitative models applied to understand intracellular phase transitions, the stickers-and-spacers framework offers an intuitive yet rigorous means to map biomolecular sequences and structure to the driving forces needed for higher-order assembly. This chapter introduces the fundamental concepts behind the stickers-and-spacers model, considers its application to different biological systems, and discusses limitations and misconceptions around the model.

Keywords:  Biomolecular condensates; Intrinsically disordered proteins; Phase separation; Phase transition

DOI:  https://doi.org/10.1007/978-1-0716-2663-4_4
J Nanobiotechnology. 2022 Oct 08. 20(1): 442

Intracellular mRNA phase separation induced by cationic polymers for tumor immunotherapy.

Zhen Xing, Jing Xue, Xindian Ma, Congwei Han, Zhenzhen Wang, Shunhuang Luo, Chunming Wang, Lei Dong, Junfeng Zhang.

  The formation of biomolecular condensates via liquid‒liquid phase separation (LLPS) is an advantageous strategy for cells to organize their subcellular compartments for diverse functions. Recent findings suggest that RNA or RNA-related LLPS techniques have potential for the development of new cellular regulation strategies. However, manipulating RNA LLPS in living cells has great challenges. Herein, we report that cationic polymers (CPs) have strong RNA LLPS-inducing activity. By introducing CPs into living cells or RNA solutions, significant RNA LLPS was verified through confocal imaging, turbidity assays, and fluorescence recovery after photobleaching (FRAP) tests. Among them, turbidity kinetics determinations indicated that the hydrophilic positively charged amino groups on the CPs play essential roles in RNA phase separation. Moreover, the LLPS induced by the cationic polymers dramatically changed the gene expression patterns in the cells. Interestingly, we found that TGFβ1 mRNA was highly encapsulated in the RNA droplets, which lowered the immunosuppressive capability of the tumor cells and triggered marked antitumor reactions in a mouse breast cancer model. Thus, we present here the CP-based modulation of RNA LLPS as a novel transcriptional manipulation method with potential for cancer immunotherapy drug development.

Keywords:  Cationic polymers; Immunotherapy; TGFβ1; Tumor microenvironment; mRNA phase separation

DOI:  https://doi.org/10.1186/s12951-022-01647-8
ACS Chem Neurosci. 2022 Oct 11.

Effects of Charged Polyelectrolytes on Amyloid Fibril Formation of a Tau Fragment.

Majedul Islam, Emily Argueta, Ewa P Wojcikiewicz, Deguo Du.

  The microtubule-associated protein tau is involved in more than 20 different neurological disorders characterized by aberrant intracellular aggregation of tau in the brain. Here, we investigated the aggregation of a novel 20-residue model peptide, tau298-317, which is derived from the key microtubule binding domain of the full sequence tau. Our results show that tau298-317 highly mimics the physical and aggregation properties of tau. Under normal physiological conditions, the peptide maintains a disordered random coil without aggregation. The presence of polyanionic heparin (Hep) significantly promotes the aggregation of this peptide to form amyloid fibrils. The Hep-induced aggregation is sensitive to the ionic strength of the solution and the introduction of the negatively charged phosphate group on a serine (Ser305) residue in the sequence, suggesting an important role of electrostatic interactions in the mechanism of Hep-mediated aggregation. In addition, two positively charged polysaccharides, chitosan (CHT) and its quaternary derivative N-trimethyl chitosan (TMC), were found to effectively inhibit Hep-induced aggregation of tau298-317 in a concentration-dependent manner. Attractive electrostatic interactions between the positively charged moieties in CHT/TMC and the negatively charged residues of Hep play a critical role in inhibiting Hep-peptide interactions and suppressing peptide aggregation. Our results suggest that positively charged polyelectrolytes with optimized charged groups and charge distribution patterns can serve as effective molecular candidates to block tau-Hep interactions and prevent aggregation of tau induced by Hep and other polyanions.

Keywords:  aggregation; chitosan; electrostatic interaction; heparin; polyelectrolytes; tau

DOI:  https://doi.org/10.1021/acschemneuro.2c00374
Int J Mol Sci. 2022 Sep 25. pii: 11285. [Epub ahead of print]23(19):

Balanced Force Field ff03CMAP Improving the Dynamics Conformation Sampling of Phosphorylation Site.

Bozitao Zhong, Ge Song, Hai-Feng Chen.

  Phosphorylation plays a key role in plant biology, such as the accumulation of plant cells to form the observed proteome. Statistical analysis found that many phosphorylation sites are located in disordered regions. However, current force fields are mainly trained for structural proteins, which might not have the capacity to perfectly capture the dynamic conformation of the phosphorylated proteins. Therefore, we evaluated the performance of ff03CMAP, a balanced force field between structural and disordered proteins, for the sampling of the phosphorylated proteins. The test results of 11 different phosphorylated systems, including dipeptides, disordered proteins, folded proteins, and their complex, indicate that the ff03CMAP force field can better sample the conformations of phosphorylation sites for disordered proteins and disordered regions than ff03. For the solvent model, the results strongly suggest that the ff03CMAP force field with the TIP4PD water model is the best combination for the conformer sampling. Additional tests of CHARMM36m and FB18 force fields on two phosphorylated systems suggest that the overall performance of ff03CMAP is similar to that of FB18 and better than that of CHARMM36m. These results can help other researchers to choose suitable force field and solvent models to investigate the dynamic properties of phosphorylation proteins.

Keywords:  disordered proteins; ff03CMAP; force field; phosphorylation

DOI:  https://doi.org/10.3390/ijms231911285
Mol Biol Cell. 2022 Oct 12. mbcE22010027

Insights into the role of Nup62 and Nup93 in assembling cytoplasmic ring and central transport channel of the nuclear pore complex.

Pankaj K Madheshiya, Ekta Shukla, Jyotsana Singh, Shrankhla Bawaria, Mohammed Yousuf Ansari, Radha Chauhan.

The nuclear pore complex (NPC) is a highly modular assembly of 34 distinct nucleoporins (Nups), to form a versatile transport channel between the nucleus and cytoplasm. Among them, Nup62 is known as an essential component for nuclear transport while, Nup93 for the proper nuclear envelope assembly. These Nups constitute various NPC subcomplexes: such as central transport channel (CTC), cytoplasmic ring (CR) and inner ring (IR). However, how they play their role in the NPC assembly and transport activity is not clear. Here we delineated the interacting regions, conducted biochemical reconstitution and structural characterization of the mammalian CR complex to reveal its intrinsic dynamic behaviour and a distinct '4' shaped architecture resembling the CTC complex. Our in vitro reconstitution data demonstrate that Nup62 coiled-coil domain is critical to form both Nup62322-525•Nup88517-742 and Nup62322-525•Nup88517-742•Nup214693-926 heterotrimers and both can bind to the Nup931-150. We therefore propose that the Nup93 act as a 'sensor' to bind to Nup62 shared heterotrimers including Nup62•Nup54 heterotrimer of the CTC, which was not shown previously as an interacting partner. Altogether, our biochemical study suggests that the Nup62 via its coiled-coil domain is central to form compositionally distinct yet structurally similar heterotrimers, and the Nup93 binds these diverse heterotrimers non-selectively.

DOI: https://doi.org/10.1091/mbc.E22-01-0027
Methods Mol Biol. 2023 ;2563 297-324

Cryo-Electron Tomography of Reconstituted Biomolecular Condensates.

Fergus Tollervey, Xiaojie Zhang, Mainak Bose, Jenny Sachweh, Jeffrey B Woodruff, Titus M Franzmann, Julia Mahamid.

  The assembly of membraneless compartments by phase separation has recently been recognized as a mechanism for spatial and temporal organization of biomolecules within the cell. The functions of such mesoscale assemblies, termed biomolecular condensates, depend on networks of multivalent interactions between proteins, their structured and disordered domains, and commonly also include nucleic acids. Cryo-electron tomography is an ideal tool to investigate the three-dimensional architecture of such pleomorphic interaction networks at nanometer resolution and thus form inferences about function. However, preparation of suitable cryo-electron microscopy samples of condensates may be prone to protein denaturation, low retention of material on the sample carrier, and contamination associated with cryo-sample preparation and transfers. Here, we describe a series of protocols designed to obtain high-quality cryo-electron tomography data of biomolecular condensates reconstituted in vitro. These include critical screening by light microscopy, cryo-fixation by plunge freezing, sample loading into an electron microscope operated at liquid nitrogen temperature, data collection, processing of the data into three-dimensional tomograms, and their interpretation.

Keywords:  3D tomograms; Molecular architecture; Phase diagrams; Phase separation; Plunge freezing; Protein/RNA condensates

DOI:  https://doi.org/10.1007/978-1-0716-2663-4_15