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Of-Pis1 is a potent piscidin antimicrobial peptide (AMP), recently isolated from rock bream (Oplegnathus fasciatus). This rich in histidines and glycines 24-amino acid peptide displays high and broad antimicrobial activity and no significant hemolytic toxicity against human erythrocytes, suggesting low toxicity. To better understand the mechanism of action of Of-Pis1 and its potential selectivity, using NMR and CD spectroscopies, we studied the interaction with eukaryotic and procaryotic membranes and membrane models. Anionic sodium dodecyl sulfate (SDS) and lipopolysaccharide (LPS) micelles were used to mimic procaryotic membranes, while zwitterionic dodecyl phosphocholine (DPC) was used as eukaryotic membrane surrogate. In an aqueous environment, Of-Pis1 adopts a flexible random coil conformation. In DPC and SDS instead, the N-terminal region of Of-Pis1 forms an amphipathic α-helix with the non-polar face in close contact with the micelles. Slower solvent exchange and higher pK a s of the histidine residues in SDS than in DPC suggest that Of-Pis1 interacts more tightly with SDS. Of-Pis1 also binds tightly and structurally perturbs LPS micelles. Of-Pis1 interacts with both Escherichia coli and mammalian cell membranes, but only in the presence of Escherichia coli membranes it populates the helical conformation. Furthermore, ligand-based NMR experiments support a tighter and more specific interaction with bacterial than with eukaryotic membranes. Overall, these data clearly show the selective interaction of this broadly active AMP with bacterial over eukaryotic membranes. The conformational information is discussed in terms of Of-Pis1 amino acid sequence and composition to provide insights useful to design more potent and selective AMPs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Human Sp140 protein is a leukocyte-specific member of the speckled protein (Sp) family (Sp100, Sp110, Sp140, Sp140L), a class of multi-domain nuclear proteins involved in intrinsic immunity and transcriptional regulation. Sp140 regulates macrophage transcriptional program and is implicated in several haematologic malignancies. Little is known about Sp140 structural domains and its post-translational modifications., Methods: We used mass spectrometry and biochemical experiments to investigate endogenous Sp140 SUMOylation in Burkitt's Lymphoma cells and Sp140 SUMOylation sites in HEK293T cells, FLAG-Sp140 transfected and His 6 -SUMO-1 T95K infected. NMR spectroscopy and in vitro SUMOylation reactions were applied to investigate the role of Sp140 PHD finger in the SUMOylation of the adjacent BRD., Results: Endogenous Sp140 is a SUMO-1 target, whereby FLAG-Sp140 harbors at least 13 SUMOylation sites distributed along the protein sequence, including the BRD. NMR experiments prove direct binding of the SUMO E2 ligase Ubc9 and SUMO-1 to PHD-BRD Sp140 . In vitro SUMOylation reactions show that the PHD Sp140 behaves as SUMO E3 ligase, assisting intramolecular SUMOylation of the adjacent BRD., Conclusions: Sp140 is multi-SUMOylated and its PHD finger works as versatile protein-protein interaction platform promoting intramolecular SUMOylation of the adjacent BRD. Thus, combinatorial association of Sp140 chromatin binding domains generates a multifaceted interaction scaffold, whose function goes beyond the canonical histone recognition., General Significance: The addition of Sp140 to the increasing lists of multi-SUMOylated proteins opens new perspectives for molecular studies on Sp140 transcriptional activity, where SUMOylation could represent a regulatory route and a docking surface for the recruitment and assembly of leukocyte-specific transcription regulators., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Graph theory is widely used to dissect structural communication in biomolecular systems. Here, graph theory-based approaches were applied to the headpiece of integrins, adhesion cell-surface receptors that transmit signals across the plasma membranes., Methods: Protein Structure Network (PSN) analysis incorporating dynamic information either from molecular dynamics simulations or from Elastic Network Models was applied to the β3 domains from integrins α V β 3 and α IIb β 3 in their apo and ligand-bound states., Results: Closed and open states of the β headpiece are characterized by distinct allosteric communication pathways involving highly conserved amino acids at the two different α/β interfaces in the βI domain, the closed state being prompted to the closed-to-open transition. In the closed state, pure antagonism is associated with the establishment of communication pathways that start from the ligand, pass through the β1/α3,α4 interface, and end up in the hybrid domain by involving the Y110-Q82 link, which is weakened in the agonist-bound states., Conclusions: Allosteric communication in integrins relies on highly conserved and functionally relevant amino acid residues. The αβα-sandwich architecture of integrin βI domain dictates the structural communication between ligand binding site and hybrid domain. Differently from agonists, pure antagonists are directly involved in allosteric communication pathways and exert long-distance strengthening of the βI/hybrid interface. Release of the structure network in the ligand binding site is associated with the close-to-open transition accompanying the activation process., General Significance: The study strengthens the power of graph-based analyses to decipher allosteric communication intrinsic to protein folds and modified by functionally different ligands., (Copyright © 2017 Elsevier B.V. All rights reserved.)

High Mobility Group Box 1 protein was discovered as a nuclear protein, but it has a "second life" outside the cell where it acts as a damage-associated molecular pattern. HMGB1 is passively released or actively secreted in a number of diseases, including trauma, chronic inflammatory disorders, autoimmune diseases and cancer. Extracellular HMGB1 triggers and sustains the inflammatory response by inducing cytokine release and by recruiting leucocytes. These characteristics make extracellular HMGB1 a key molecular target in multiple diseases. A number of strategies have been used to prevent HMGB1 release or to inhibit its activities. Current pharmacological strategies include antibodies, peptides, decoy receptors and small molecules. Noteworthy, salicylic acid, a metabolite of aspirin, has been recently found to inhibit HMGB1. HMGB1 undergoes extensive post-translational modifications, in particular acetylation and oxidation, which modulate its functions. Notably, high levels of serum HMGB1, in particular of the hyper-acetylated and disulfide isoforms, are sensitive disease biomarkers and are associated with different disease stages. In the future, the development of isoform-specific HMGB1 inhibitors may potentiate and fine-tune the pharmacological control of inflammation. We review here the current therapeutic strategies targeting HMGB1, in particular the emerging and relatively unexplored small molecules-based approach., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

CpG-DNA or its synthetic analog CpG-ODN activates innate immunity through Toll-like receptor 9 (TLR9). However, the mechanism of TLR9 activation by CpG-DNA remains elusive. Here we have identified HMGB1 as a CpG-ODN-binding protein. HMGB1 interacts and preassociates with TLR9 in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), and hastens TLR9's redistribution to early endosomes in response to CpG-ODN. CpG-ODN stimulates macrophages and dendritic cells to secrete HMGB1; in turn, extracellular HMGB1 accelerates the delivery of CpG-ODNs to its receptor, leading to a TLR9-dependent augmentation of IL-6, IL-12, and TNFalpha secretion. Loss of HMGB1 leads to a defect in the IL-6, IL-12, TNFalpha, and iNOS response to CpG-ODN. However, lack of intracellular TLR9-associated HMGB1 can be compensated by extracellular HMGB1. Thus, the DNA-binding protein HMGB1 shuttles in and out of immune cells and regulates inflammatory responses to CpG-DNA.

High-mobility group box 1 protein (HMGB1) is a nuclear component, but extracellularly it serves as a signaling molecule involved in acute and chronic inflammation, for example in sepsis and arthritis. The identification of HMGB1 inhibitors is therefore of significant experimental and clinical interest. We show that glycyrrhizin, a natural anti-inflammatory and antiviral triterpene in clinical use, inhibits HMGB1 chemoattractant and mitogenic activities, and has a weak inhibitory effect on its intranuclear DNA-binding function. NMR and fluorescence studies indicate that glycyrrhizin binds directly to HMGB1 (K(d) approximately 150 microM), interacting with two shallow concave surfaces formed by the two arms of both HMG boxes. Our results explain in part the anti-inflammatory properties of glycyrrhizin, and might direct the design of new derivatives with improved HMGB1-binding properties.