The acidic intrinsically disordered region of the inflammatory mediator HMGB1 mediates fuzzy interactions with CXCL12

Holehouse A.S., Kragelund B.B.

Intrinsically disordered protein regions exist in a collection of dynamic interconverting conformations that lack a stable 3D structure. These regions are structurally heterogeneous, ubiquitous and found across all kingdoms of life. Despite the absence of a defined 3D structure, disordered regions are essential for cellular processes ranging from transcriptional control and cell signalling to subcellular organization. Through their conformational malleability and adaptability, disordered regions extend the repertoire of macromolecular interactions and are readily tunable by their structural and chemical context, making them ideal responders to regulatory cues. Recent work has led to major advances in understanding the link between protein sequence and conformational behaviour in disordered regions, yet the link between sequence and molecular function is less well defined. Here we consider the biochemical and biophysical foundations that underlie how and why disordered regions can engage in productive cellular functions, provide examples of emerging concepts and discuss how protein disorder contributes to intracellular information processing and regulation of cellular function. Intrinsically disordered regions of proteins lack a defined 3D structure and exist in a collection of interconverting conformations. Recent work is shedding light on how — through their conformational malleability and adaptability — intrinsically disordered regions extend the repertoire of macromolecular interactions in the cell and contribute to key cellular functions.

Philo J.S.

Proper interpretation of analytical ultracentrifugation (AUC) data for purified proteins requires ancillary information and calculations to account for factors such as buoyancy, buffer viscosity, hydration, and temperature. The utility program SEDNTERP has been widely used by the AUC community for this purpose since its introduction in the mid-1990s. Recent extensions to this program (1) allow it to incorporate data from diffusion as well as AUC experiments; and (2) allow it to calculate the refractive index of buffer solutions (based on the solute composition of the buffer), as well as the specific refractive increment (dn/dc) of proteins based on their composition. These two extensions should be quite useful to the light scattering community as well as helpful for AUC users. The latest version also adds new terms to the partial specific volume calculations which should improve the accuracy, particularly for smaller proteins and peptides, and can calculate the viscosity of buffers containing heavy isotopes of water. It also uses newer, more accurate equations for the density of water and for the hydrodynamic properties of rods and disks. This article will summarize and review all the equations used in the current program version and the scientific background behind them. It will tabulate the values used to calculate the partial specific volume and dn/dc, as well as the polynomial coefficients used in calculating the buffer density and viscosity (most of which have not been previously published), as well as the new ones used in calculating the buffer refractive index.

Ahmed R., Forman-Kay J.D.

Abstract The spatial and temporal organization of interactions between proteins underlie the regulation of most cellular processes. The requirement for such interactions to be specific predisposes a view that protein–protein interactions are relatively static and are formed through the stable complementarity of the interacting partners. A growing body of reports indicate, however, that many interactions lead to fuzzy complexes with an ensemble of conformations in dynamic exchange accounting for the observed binding. Here, we discuss how NMR has facilitated the characterization of these discrete, dynamic complexes and how such characterization has aided the understanding of dynamic, condensed phases of phase-separating proteins with exchanging multivalent interactions.

Fuxreiter M.

Wang X., Greenblatt H.M., Bigman L.S., Yu B., Pletka C.C., Levy Y., Iwahara J.

• The intrinsically disordered regions (IDRs) of HMGB1 undergo fuzzy interactions. • Weak fuzzy interactions of the D/E repeats create strong autoinhibition of HMGB1. • The IDRs are mobile both in the autoinhibited and uninhibited states of HMGB1. • The release of the autoinhibition involves alteration in the fuzzy interactions. • Post-translational modifications near the D/E repeats modulate DNA-binding affinity. Highly negatively charged segments containing only aspartate or glutamate residues (“D/E repeats”) are found in many eukaryotic proteins. For example, the C-terminal 30 residues of the HMGB1 protein are entirely D/E repeats. Using nuclear magnetic resonance (NMR), fluorescence, and computational approaches, we investigated how the D/E repeats causes the autoinhibition of HMGB1 against its specific binding to cisplatin-modified DNA. By varying ionic strength in a wide range (40–900 mM), we were able to shift the conformational equilibrium between the autoinhibited and uninhibited states toward either of them to the full extent. This allowed us to determine the macroscopic and microscopic equilibrium constants for the HMGB1 autoinhibition at various ionic strengths. At a macroscopic level, a model involving the autoinhibited and uninhibited states can explain the salt concentration-dependent binding affinity data. Our data at a microscopic level show that the D/E repeats and other parts of HMGB1 undergo electrostatic fuzzy interactions, each of which is weaker than expected from the macroscopic autoinhibitory effect. This discrepancy suggests that the multivalent nature of the fuzzy interactions enables strong autoinhibition at a macroscopic level despite the relatively weak intramolecular interaction at each site. Both experimental and computational data suggest that the D/E repeats interact preferentially with other intrinsically disordered regions (IDRs) of HMGB1. We also found that mutations mimicking post-translational modifications relevant to nuclear export of HMGB1 can moderately modulate DNA-binding affinity, possibly by impacting the autoinhibition. This study illuminates a functional role of the fuzzy interactions of D/E repeats.

Mezzapelle R., De Marchis F., Passera C., Leo M., Brambilla F., Colombo F., Casalgrandi M., Preti A., Zambrano S., Castellani P., Ertassi R., Silingardi M., Caprioglio F., Basso V., Boldorini R., et. al.

Manalastas-Cantos K., Konarev P.V., Hajizadeh N.R., Kikhney A.G., Petoukhov M.V., Molodenskiy D.S., Panjkovich A., Mertens H.D., Gruzinov A., Borges C., Jeffries C.M., Svergun D.I., Franke D.

The ATSAS software suite encompasses a number of programs for the processing, visualization, analysis and modelling of small-angle scattering data, with a focus on the data measured from biological macromolecules. Here, new developments in the ATSAS 3.0 package are described. They include IMSIM, for simulating isotropic 2D scattering patterns; IMOP, to perform operations on 2D images and masks; DATRESAMPLE, a method for variance estimation of structural invariants through parametric resampling; DATFT, which computes the pair distance distribution function by a direct Fourier transform of the scattering data; PDDFFIT, to compute the scattering data from a pair distance distribution function, allowing comparison with the experimental data; a new module in DATMW for Bayesian consensus-based concentration-independent molecular weight estimation; DATMIF, an ab initio shape analysis method that optimizes the search model directly against the scattering data; DAMEMB, an application to set up the initial search volume for multiphase modelling of membrane proteins; ELLLIP, to perform quasi-atomistic modelling of liposomes with elliptical shapes; NMATOR, which models conformational changes in nucleic acid structures through normal mode analysis in torsion angle space; DAMMIX, which reconstructs the shape of an unknown intermediate in an evolving system; and LIPMIX and BILMIX, for modelling multilamellar and asymmetric lipid vesicles, respectively. In addition, technical updates were deployed to facilitate maintainability of the package, which include porting the PRIMUS graphical interface to Qt5, updating SASpy – a PyMOL plugin to run a subset of ATSAS tools – to be both Python 2 and 3 compatible, and adding utilities to facilitate mmCIF compatibility in future ATSAS releases. All these features are implemented in ATSAS 3.0, freely available for academic users at https://www.embl-hamburg.de/biosaxs/software.html.

Stringer C., Wang T., Michaelos M., Pachitariu M.

Many biological applications require the segmentation of cell bodies, membranes and nuclei from microscopy images. Deep learning has enabled great progress on this problem, but current methods are specialized for images that have large training datasets. Here we introduce a generalist, deep learning-based segmentation method called Cellpose, which can precisely segment cells from a wide range of image types and does not require model retraining or parameter adjustments. Cellpose was trained on a new dataset of highly varied images of cells, containing over 70,000 segmented objects. We also demonstrate a three-dimensional (3D) extension of Cellpose that reuses the two-dimensional (2D) model and does not require 3D-labeled data. To support community contributions to the training data, we developed software for manual labeling and for curation of the automated results. Periodically retraining the model on the community-contributed data will ensure that Cellpose improves constantly. Cellpose is a generalist, deep learning-based approach for segmenting structures in a wide range of image types. Cellpose does not require parameter adjustment or model retraining and outperforms established methods on 2D and 3D datasets.

D’Agostino G., Artinger M., Locati M., Perez L., Legler D.F., Bianchi M.E., Rüegg C., Thelen M., Marchese A., Rocchi M.B., Cecchinato V., Uguccioni M.

The chemokine receptor CXCR4 plays a fundamental role in homeostasis and pathology by orchestrating recruitment and positioning of immune cells, under the guidance of a CXCL12 gradient. The ability of chemokines to form heterocomplexes, enhancing their function, represents an additional level of regulation on their cognate receptors. In particular, the multi-faceted activity of the heterocomplex formed between CXCL12 and the alarmin HMGB1 is emerging as an unexpected player able to modulate a variety of cell responses, spanning from tissue regeneration to chronic inflammation. Nowadays, little is known on the selective signaling pathways activated when CXCR4 is triggered by the CXCL12/HMGB1 heterocomplex. In the present work, we demonstrate that this heterocomplex acts as a CXCR4 balanced agonist, activating both G protein and β-arrestins-mediated signaling pathways to sustain chemotaxis. We generated β-arrestins knock out HeLa cells by CRISPR/Cas9 technology and show that the CXCL12/HMGB1 heterocomplex-mediated actin polymerization is primarily β arrestin1 dependent, while chemotaxis requires both β arrestin1 and β arrestin2. Triggering of CXCR4 with the CXCL12/HMGB1 heterocomplex leads to an unexpected receptor retention on the cell surface, which depends on β-arrestin2. In conclusion, the CXCL12/HMGB1 heterocomplex engages the β arrestin proteins differently from CXCL12, promoting a prompt availability of CXCR4 on the cell surface, and enhancing directional cell migration. These data unveil the signaling induced by the CXCL12/HMGB1 heterocomplex in view of identifying biased CXCR4 antagonists or agonists targeting the variety of functions it exerts.

Bianchi M.E., Mezzapelle R.

The CXCR4 receptor upon binding its ligands triggers multiple signaling pathways that orchestrate cell migration, hematopoiesis and cell homing and retention in the bone marrow. However, CXCR4 also directly controls cell proliferation of non-hematopoietic cells. This review focuses on recent reports pointing to its pivotal role in tissue regeneration and stem cell activation, and discusses the connection to the known role of CXCR4 in promoting tumor growth. The mechanisms may be similar in all cases, since regeneration often recapitulates developmental processes, and cancer often exploits developmental pathways. Moreover, cell migration and cell proliferation appear to be downstream of the same signaling pathways. A deeper understanding of the complex signaling originating from CXCR4 is needed to exploit the opportunities to repair damaged organs safely and effectively.

Bugge K., Brakti I., Fernandes C.B., Dreier J.E., Lundsgaard J.E., Olsen J.G., Skriver K., Kragelund B.B.

Living organisms depend on timely and organized interactions between proteins linked in interactomes of high complexity. The recent increased precision by which protein interactions can be studied, and the enclosure of intrinsic structural disorder, suggest that it is time to zoom out and embrace protein interactions beyond the most central points of physical encounter. The present paper discusses protein-protein interactions in the view of structural disorder with an emphasis on flanking regions and contexts of disorder-based interactions. Context constitutes an overarching concept being of physicochemical, biomolecular, and physiological nature, but it also includes the immediate molecular context of the interaction. For intrinsically disordered proteins, which often function by exploiting short linear motifs, context contributes in highly regulatory and decisive manners and constitute a yet largely unrecognized source of interaction potential in a multitude of biological processes. Through selected examples, this review emphasizes how multivalency, charges and charge clusters, hydrophobic patches, dynamics, energetic frustration, and ensemble redistribution of flanking regions or disordered contexts are emerging as important contributors to allosteric regulation, positive and negative cooperativity, feed-back regulation and negative selection in binding. The review emphasises that understanding context, and in particular the role the molecular disordered context and flanking regions take on in protein interactions, constitute an untapped well of energetic modulation potential, also of relevance to drug discovery and development.

Ngo T., Stephens B.S., Gustavsson M., Holden L.G., Abagyan R., Handel T.M., Kufareva I.

Chemokines and their receptors are orchestrators of cell migration in humans. Because dysregulation of the receptor-chemokine system leads to inflammation and cancer, both chemokines and receptors are highly sought therapeutic targets. Yet one of the barriers for their therapeutic targeting is the limited understanding of the structural principles behind receptor-chemokine recognition and selectivity. The existing structures do not include CXC subfamily complexes and lack information about the receptor distal N-termini, despite the importance of the latter in signaling, regulation, and bias. Here, we report the discovery of the geometry of the complex between full-length CXCR4, a prototypical CXC receptor and driver of cancer metastasis, and its endogenous ligand CXCL12. By comprehensive disulfide cross-linking, we establish the existence and the structure of a novel interface between the CXCR4 distal N-terminus and CXCL12 β1-strand, while also recapitulating earlier findings from nuclear magnetic resonance, modeling and crystallography of homologous receptors. A cross-linking–informed high-resolution model of the CXCR4-CXCL12 complex pinpoints the interaction determinants and reveals the occupancy of the receptor major subpocket by the CXCL12 proximal N terminus. This newly found positioning of the chemokine proximal N-terminus provides a structural explanation of CXC receptor-chemokine selectivity against other subfamilies. Our findings challenge the traditional two-site understanding of receptor-chemokine recognition, suggest the possibility of new affinity and signaling determinants, and fill a critical void on the structural map of an important class of therapeutic targets. These results will aid the rational design of selective chemokine-receptor targeting small molecules and biologics with novel pharmacology.

Eckardt V., Miller M.C., Blanchet X., Duan R., Leberzammer J., Duchene J., Soehnlein O., Megens R.T., Ludwig A., Dregni A., Faussner A., Wichapong K., Ippel H., Dijkgraaf I., Kaltner H., et. al.

Clouser A.F., Baughman H.E., Basanta B., Guttman M., Nath A., Klevit R.E.

Small heat shock proteins (sHSPs) are nature’s ‘first responders’ to cellular stress, interacting with affected proteins to prevent their aggregation. Little is known about sHSP structure beyond its structured α-crystallin domain (ACD), which is flanked by disordered regions. In the human sHSP HSPB1, the disordered N-terminal region (NTR) represents nearly 50% of the sequence. Here, we present a hybrid approach involving NMR, hydrogen-deuterium exchange mass spectrometry, and modeling to provide the first residue-level characterization of the NTR. The results support a model in which multiple grooves on the ACD interact with specific NTR regions, creating an ensemble of ‘quasi-ordered’ NTR states that can give rise to the known heterogeneity and plasticity of HSPB1. Phosphorylation-dependent interactions inform a mechanism by which HSPB1 is activated under stress conditions. Additionally, we examine the effects of disease-associated NTR mutations on HSPB1 structure and dynamics, leveraging our emerging structural insights.

De Leo F., Quilici G., Tirone M., De Marchis F., Mannella V., Zucchelli C., Preti A., Gori A., Casalgrandi M., Mezzapelle R., Bianchi M.E., Musco G.

Wang L., Brasnett C., Borges-Araújo L., Souza P.C., Marrink S.J.

Fassi E.M., Pirani E., Cecchinato V., Cavalli A., Roda G., Uguccioni M., Sgrignani J., Grazioso G.

AbstractInflammation is a vital defense mechanism activated in response to injury or infection, which induces the release of cytokines and chemokines to promote tissue repair. However, persistent inflammation may result in the development of autoimmune diseases.During pathological conditions such as rheumatoid arthritis, HMGB1 is kept in its reduced isoform and can complex with CXCL12 enhancing cell migration and exacerbating the immune responses. Small organic compounds selective for HMGB1 have been previously reported to be able to disrupt the CXCL12/HMGB1 heterocomplex, but due to their low affinity, they are unsuitable for further development as novel anti-inflammatory drugs.We previously reported a peptide (HBP08) that binds to HMGB1 with high affinity (Kd= 0.8 µM), and blocks the activity of the heterocomplex, in line with a wide literature that supports the use of peptides to design protein-protein interaction inhibitors. In the present work, we computationally optimized the HBP08 peptide sequence, finding new analogues endowed with improved affinity for HMGB1. In particular, HBP08-2 inhibited the activity of the CXCL12/HMGB1 heterocomplex with an IC5010-fold lower (3.31 µM) and displayed a Kd28-fold lower (28.1 ± 7.0 nM) than the parent peptide HBP08.

Krishna A.A., Abhirami B.L., Kumaran A.

Gao Y., Liu J., Wu M., Zhang Y., Wang M., Lyu Q., Zhang W., Zhou Y., Cheuk Y.C., Wang X., Liu Y., Wang W., Tu W.

Shen P., Zhang L., Jiang X., Yu B., Zhang J.

Ruggieri E., Domenico E.D., Locatelli A.G., Isopo F., Damanti S., Lorenzo R.D., Milan E., Musco G., Rovere-Querini P., Cenci S., Vénéreau E.

Maugeri N., Manfredi A.A.

Platelets navigate the fine balance between homeostasis and injury. They regulate vascular homeostasis and drive repair post-injury amidst leukocyte extravasation. Crucially, platelets initiate extracellular traps generation and promote immunothrombosis. In chronic human diseases, platelet action often extends beyond its normative role, sparking sustained reciprocal activation of leukocytes and mural cells, culminating in adverse vascular remodeling. Studies in the last decade have spotlighted a novel key player in platelet activation, the high mobility group box 1 (HMGB1) protein. Despite its initial characterization as a chromatin molecule, anucleated platelets express abundant HMGB1, which has emerged as a linchpin in thromboinflammatory risks and microvascular remodeling. We propose that a comprehensive assessment of platelet HMGB1, spanning quantification of content, membrane localization, and accumulation of HMGB1-expressing vesicles in biological fluids should be integral to dissecting and quantifying platelet activation. This review provides evidence supporting this claim and underscores the significance of platelet HMGB1 as a biomarker in conditions associated with heightened thrombotic risks and systemic microvascular involvement, spanning cardiovascular, autoimmune and infectious diseases.

Kumar V., Kumar P.

Nucleocytoplasmic translocation of HMGB1 (high mobility group box-1) plays a significant role in disease progression. Several methods contribute to the translocation of HMGB1 from the nucleus to the cytoplasm, including inflammasome activation, TNF-α signaling, CRM1-mediated transport, reactive oxygen species (ROS), JAK/STAT pathway, RIP3-mediated p53 involvement, XPO-1-mediated transport, and calcium-dependent mechanisms. Due to its diverse functions at various subcellular locations, HMGB1 has been identified as a crucial factor in several Central Nervous System (CNS) disorders, including Huntington’s disease (HD), Parkinson’s disease (PD), and Alzheimer’s disease (AD). HMGB1 displays a wide array of roles in the extracellular environment as it interacts with several receptors, including CXCR4, TLR2, TLR4, TLR8, and RAGE, by engaging in these connections, HMGB1 can effectively regulate subsequent signaling pathways, hence exerting an impact on the progression of brain disorders through neuroinflammation. Therefore, focusing on treating neuroinflammation could offer a common therapeutic strategy for several disorders. The objective of the current literature is to demonstrate the pathological role of HMGB1 in various neurological disorders. This review also offers insights into numerous therapeutic targets that promise to advance multiple treatments intended to alleviate brain illnesses.

Vinals Guitart A., Lee C., Espirito Santo A., Ruan J., Redfield C., Yue W., Burgess-Brown N., Fedorov O., Nanchahal J.

AbstractHigh Mobility Group Box 1 protein in its fully-reduced state (FR-HMGB1) binds to CXC Ligand 12 (CXCL12) and signals through CXC Receptor 4 (CXCR4) to transition stem and progenitor cells from the quiescent G0state to GAlert. On exposure to tissue activating factors, cells in GAlertreadily enter G1to effect tissue repair. However, conversion of FR-HMGB1 to the disulfide form may result in deleterious inflammation through signaling via Toll-Like Receptors 2 (TLR-2) and 4 (TLR-4), and the Receptor for Advanced Glycation End products (RAGE). Using peptide arrays, biolayer interferometry and nuclear magnetic resonance, we mapped the residues and motifs of HMGB1 that interact with CXCL12, TLR-2, TLR-4, and RAGE. Identification of repeating sequences across the Box A and Box B domains that interact with CXCL12 enabled us to design a construct comprising two HMG B Boxes in tandem (dBB12L), which promotes tissue repair as effectively as wild type FR-HMGB1 but is unable to signal through TLR-2, TLR-4 or RAGE.

Bianchi M.E., Rubartelli A., Sitia R.

Li Y., Feng Y., Geng S., Xu F., Guo H.

Cancer EMT is a pivotal process that drives carcinogenesis, metastasis, and cancer recurrence, with its initiation and regulation intricately governed by biochemical pathways in a precise spatiotemporal manner. Recently, the membrane-less biomolecular condensates formed via liquid-liquid phase separation (LLPS) have emerged as a universal mechanism underlying the spatiotemporal collaboration of biological activities in cancer EMT. In this review, we first elucidate the current understanding of LLPS formation and its cellular functions, followed by an overview of valuable tools for investigating LLPS. Secondly, we examine in detail the LLPS-mediated biological processes crucial for the initiation and regulation of cancer EMT. Lastly, we address current challenges in advancing LLPS research and explore the potential modulation of LLPS using therapeutic agents.

Young V.L., McSweeney A.M., Edwards M.J., Ward V.K.

An intrinsically disordered protein (IDP) or region (IDR) lacks or has little protein structure but still maintains function. This lack of structure creates flexibility and fluidity, allowing multiple protein conformations and potentially transient interactions with more than one partner. Caliciviruses are positive-sense ssRNA viruses, containing a relatively small genome of 7.6–8.6 kb and have a broad host range. Many viral proteins are known to contain IDRs, which benefit smaller viral genomes by expanding the functional proteome through the multifunctional nature of the IDR. The percentage of intrinsically disordered residues within the total proteome for each calicivirus type species can range between 8 and 23%, and IDRs have been experimentally identified in NS1-2, VPg and RdRP proteins. The IDRs within a protein are not well conserved across the genera, and whether this correlates to different activities or increased tolerance to mutations, driving virus adaptation to new selection pressures, is unknown. The function of norovirus NS1-2 has not yet been fully elucidated but includes involvement in host cell tropism, the promotion of viral spread and the suppression of host interferon-λ responses. These functions and the presence of host cell-like linear motifs that interact with host cell caspases and VAPA/B are all found or affected by the disordered region of norovirus NS1-2. The IDRs of calicivirus VPg are involved in viral transcription and translation, RNA binding, nucleotidylylation and cell cycle arrest, and the N-terminal IDR within the human norovirus RdRP could potentially drive liquid–liquid phase separation. This review identifies and summarises the IDRs of proteins within the Caliciviridae family and their importance during viral replication and subsequent host interactions.

Jiang J., Sun M., Wang Y., Huang W., Xia L.

The high-mobility group box (HMGB) family consists of four DNA-binding proteins that regulate chromatin structure and function. In addition to their intracellular functions, recent studies have revealed their involvement as extracellular damage-associated molecular patterns (DAMPs), contributing to immune responses and tumor development. The HMGB family promotes tumorigenesis by modulating multiple processes including proliferation, metabolic reprogramming, metastasis, immune evasion, and drug resistance. Due to the predominant focus on HMGB1 in the literature, little is known about the remaining members of this family. This review summarizes the structural, distributional, as well as functional similarities and distinctions among members of the HMGB family, followed by a comprehensive exploration of their roles in tumor development. We emphasize the distributional and functional hierarchy of the HMGB family at both the organizational and subcellular levels, with a focus on their relationship with the tumor immune microenvironment (TIME), aiming to prospect potential strategies for anticancer therapy.

Yu B., Bolik-Coulon N., Rangadurai A.K., Kay L.E., Iwahara J.

Kumar P., Tacke F.