Background: Peptidyl arginine deiminase 4 (PAD4) is an enzyme that converts arginine into citrulline. PAD4 is expressed in neutrophils that, when activated, can drive the formation of neutrophil ...extracellular traps (NETs). Uncontrolled activation of PAD4 and subsequent citrullination of proteins is increasingly recognized as a driver of (auto)immune diseases. Currently, our understanding of PAD4 structure–function relationships and activity control in vivo is incomplete.
Aims: To provide the current state‐of‐the‐art on PAD4 structure‐activity relationships and involvement of PAD4 in autoimmune disorders as well as in thrombo‐inflammatory disease.
Materials & Methods: Literature review and molecular modelling
Results: In this review, we used molecular modelling to generate a three‐dimensional structure of the complete PAD4 molecule. Using our model, we discuss the catalytic conversion of the arginine substrate to citrulline. Besides mechanistic insight into PAD4 function, we give an overview of biological functions of PAD4 and mechanisms that influence its activation. In addition, we discuss the crucial role of PAD4‐mediated citrullination of histones during the formation of NETs. Subsequently, we focus on the role of PAD4‐mediated NET formation and its role in pathogenesis of rheumatoid arthritis, sepsis and (immune‐)thrombosis. Finally, we summarize current efforts to design different classes of PAD4 inhibitors that are being developed for improved treatment of autoimmune disorders as well as thrombo‐inflammatory disease.
Discussion: Advances in PAD4 structure‐function are still necessary to gain a complete insight in mechanisms that control PAD4 activity in vivo. The involvement of PAD4 in several diseases signifies the need for a PAD4 inhibitor. Although progress has been made to produce an isotype specific and potent PAD4 inhibitor, currently no PAD4 inhibitor is ready for clinical use.
Conclusion: More research into PAD4 structure and function and into the regulation of its activity is required for the development of PAD4 specific inhibitors that may prove vital to combat and prevent autoimmune disorders and (thrombo)inflammatory disease.
Abstract
Coronavirus disease 19 (COVID-19) presents with disease severities of varying degree. In its most severe form, infection may lead to respiratory failure and multi-organ dysfunction. Here we ...study the levels and evolution of the damage associated molecular patterns (DAMPS) cell free DNA (cfDNA), extracellular histone H3 (H3) and neutrophil elastase (NE), and the immune modulators GAS6 and AXL in relation to clinical parameters, ICU scoring systems and mortality in patients (n = 100) with severe COVID-19. cfDNA, H3, NE, GAS6 and AXL were increased in COVID-19 patients compared to controls. These measures associated with occurrence of clinical events and intensive care unit acquired weakness (ICUAW). cfDNA and GAS6 decreased in time in patients surviving to 30 days post ICU admission. A decrease of 27.2 ng/mL cfDNA during ICU stay associated with patient survival, whereas levels of GAS6 decreasing more than 4.0 ng/mL associated with survival. The presence of H3 in plasma was a common feature of COVID-19 patients, detected in 38% of the patients at ICU admission. NETosis markers cfDNA, H3 and NE correlated well with parameters of tissue damage and neutrophil counts. Furthermore, cfDNA correlated with lowest p/f ratio and a lowering in cfDNA was observed in patients with ventilator-free days.
The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) had spread from China and, within 2 months, became a global pandemic. The infection from this disease can cause a diversity of ...symptoms ranging from asymptomatic to severe acute respiratory distress syndrome with an increased risk of vascular hyperpermeability, pulmonary inflammation, extensive lung damage, and thrombosis. One of the host defense systems against coronavirus disease 2019 (COVID‐19) is the formation of neutrophil extracellular traps (NETs). Numerous studies on this disease have revealed the presence of elevated levels of NET components, such as cell‐free DNA, extracellular histones, neutrophil elastase, and myeloperoxidase, in plasma, serum, and tracheal aspirates of severe COVID‐19 patients. Extracellular histones, a major component of NETs, are clinically very relevant as they represent promising biomarkers and drug targets, given that several studies have identified histones as key mediators in the onset and progression of various diseases, including COVID‐19. However, the role of extracellular histones in COVID‐19 per se remains relatively underexplored. Histones are nuclear proteins that can be released into the extracellular space via apoptosis, necrosis, or NET formation and are then regarded as cytotoxic damage‐associated molecular patterns that have the potential to damage tissues and impair organ function. This review will highlight the mechanisms of extracellular histone‐mediated cytotoxicity and focus on the role that histones play in COVID‐19. Thereby, this paper facilitates a bench‐to‐bedside view of extracellular histone‐mediated cytotoxicity, its role in COVID‐19, and histones as potential drug targets and biomarkers for future theranostics in the clinical treatment of COVID‐19 patients.
The perpetuation of inflammation is an important pathophysiological contributor to the global medical burden. Chronic inflammation is promoted by non-programmed cell death
; however, how inflammation ...is instigated, its cellular and molecular mediators, and its therapeutic value are poorly defined. Here we use mouse models of atherosclerosis-a major underlying cause of mortality worldwide-to demonstrate that extracellular histone H4-mediated membrane lysis of smooth muscle cells (SMCs) triggers arterial tissue damage and inflammation. We show that activated lesional SMCs attract neutrophils, triggering the ejection of neutrophil extracellular traps that contain nuclear proteins. Among them, histone H4 binds to and lyses SMCs, leading to the destabilization of plaques; conversely, the neutralization of histone H4 prevents cell death of SMCs and stabilizes atherosclerotic lesions. Our data identify a form of cell death found at the core of chronic vascular disease that is instigated by leukocytes and can be targeted therapeutically.
Trafficking of leukocytes and their local activity profile are of pivotal importance for many (patho)physiological processes. Fittingly, microenvironments are complex by nature, with multiple ...mediators originating from diverse cell types and playing roles in an intimately regulated manner. To dissect aspects of this complexity, effectors are initially identified and structurally characterized, thus prompting familial classification and establishing foci of research activity. In this regard, chemokines present themselves as role models to illustrate the diversification and fine-tuning of inflammatory processes. This in turn discloses the interplay among chemokines, their cell receptors and cognate glycosaminoglycans, as well as their capacity to engage in new molecular interactions that form hetero-oligomers between themselves and other classes of effector molecules. The growing realization of versatility of adhesion/growth-regulatory galectins that bind to glycans and proteins and their presence at sites of inflammation led to testing the hypothesis that chemokines and galectins can interact with each other by protein–protein interactions. In this review, we present some background on chemokines and galectins, as well as experimental validation of this chemokine–galectin heterodimer concept exemplified with CXCL12 and galectin-3 as proof-of-principle, as well as sketch out some emerging perspectives in this arena.
MicroRNAs (miRNAs) are versatile regulators of gene expression with profound implications for human disease including atherosclerosis, but whether they can exert posttranslational functions to ...control cell adaptation and whether such noncanonical features harbor pathophysiological relevance is unknown. Here, we show that miR-126-5p sustains endothelial integrity in the context of high shear stress and autophagy. Bound to argonaute-2 (Ago2), miR-126-5p forms a complex with Mex3a, which occurs on the surface of autophagic vesicles and guides its transport into the nucleus. Mutational studies and biophysical measurements demonstrate that Mex3a binds to the central U- and G-rich regions of miR-126-5p with nanomolar affinity via its two K homology domains. In the nucleus, miR-126-5p dissociates from Ago2 and binds to caspase-3 in an aptamer-like fashion with its seed sequence, preventing dimerization of the caspase and inhibiting its activity to limit apoptosis. The antiapoptotic effect of miR-126-5p outside of the RNA-induced silencing complex is important for endothelial integrity under conditions of high shear stress promoting autophagy: ablation of Mex3a or ATG5 in vivo attenuates nuclear import of miR-126-5p, aggravates endothelial apoptosis, and exacerbates atherosclerosis. In human plaques, we found reduced nuclear miR-126-5p and active caspase-3 in areas of disturbed flow. The direct inhibition of caspase-3 by nuclear miR-126-5p reveals a noncanonical mechanism by which miRNAs can modulate protein function.
Acute infection is a well-established risk factor of cardiovascular inflammation increasing the risk for a cardiovascular complication within the first weeks after infection. However, the nature of ...the processes underlying such aggravation remains unclear. Lipopolysaccharide derived from Gram-negative bacteria is a potent activator of circulating immune cells including neutrophils, which foster inflammation through discharge of neutrophil extracellular traps (NETs). Here, we use a model of endotoxinemia to link acute infection and subsequent neutrophil activation with acceleration of vascular inflammation Methods: Acute infection was mimicked by injection of a single dose of lipopolysaccharide into hypercholesterolemic mice. Atherosclerosis burden was studied by histomorphometric analysis of the aortic root. Arterial myeloid cell adhesion was quantified by intravital microscopy.
Lipopolysaccharide treatment rapidly enhanced atherosclerotic lesion size by expansion of the lesional myeloid cell accumulation. Lipopolysaccharide treatment led to the deposition of NETs along the arterial lumen, and inhibition of NET release annulled lesion expansion during endotoxinemia, thus suggesting that NETs regulate myeloid cell recruitment. To study the mechanism of monocyte adhesion to NETs, we used in vitro adhesion assays and biophysical approaches. In these experiments, NET-resident histone H2a attracted monocytes in a receptor-independent, surface charge-dependent fashion. Therapeutic neutralization of histone H2a by antibodies or by in silico designed cyclic peptides enables us to reduce luminal monocyte adhesion and lesion expansion during endotoxinemia.
Our study shows that NET-associated histone H2a mediates charge-dependent monocyte adhesion to NETs and accelerates atherosclerosis during endotoxinemia.
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The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has posed a serious threat to global health and the economy for over two years, prompting the ...need for development of antiviral inhibitors. Due to its vital role in viral replication, RNA-dependent RNA polymerase (RdRp) is a promising therapeutic target. Herein, we analyzed amino acid sequence conservation of RdRp across coronaviruses. The conserved amino acids at the catalytic binding site served as the ligand-contacting residues for in silico screening to elucidate possible resistant mutation. Molecular docking was employed to screen inhibitors of SARS-CoV-2 from the ZINC ChemDiv database. The top-ranked compounds selected from GOLD docking were further investigated for binding modes at the conserved residues of RdRp, and ten compounds were selected for experimental validation. Of which, three compounds exhibited promising antiviral activity. The most promising candidate showed ahalf-maximal effective concentration (EC50) of 5.04 µM. Molecular dynamics simulations, binding free-energy calculation and hydrogen bond analysis were performed to elucidate the critical interactions providing a foundation for developing lead compounds effective against SARS-CoV-2.
Chemokines and galectins are simultaneously upregulated and mediate leukocyte recruitment during inflammation. Until now, these effector molecules have been considered to function independently. ...Here, we tested the hypothesis that they form molecular hybrids. By systematically screening chemokines for their ability to bind galectin‐1 and galectin‐3, we identified several interacting pairs, such as CXCL12 and galectin‐3. Based on NMR and MD studies of the CXCL12/galectin‐3 heterodimer, we identified contact sites between CXCL12 β‐strand 1 and Gal‐3 F‐face residues. Mutagenesis of galectin‐3 residues involved in heterodimer formation resulted in reduced binding to CXCL12, enabling testing of functional activity comparatively. Galectin‐3, but not its mutants, inhibited CXCL12‐induced chemotaxis of leukocytes and their recruitment into the mouse peritoneum. Moreover, galectin‐3 attenuated CXCL12‐stimulated signaling via its receptor CXCR4 in a ternary complex with the chemokine and receptor, consistent with our structural model. This first report of heterodimerization between chemokines and galectins reveals a new type of interaction between inflammatory mediators that can underlie a novel immunoregulatory mechanism in inflammation. Thus, further exploration of the chemokine/galectin interactome is warranted.
Synopsis
Chemokines and galectins are simultaneously upregulated during inflammation and mediate leukocyte recruitment. A systematic screen now demonstrates their physical interaction as heterodimers, identifying several novel interacting pairs.
Chemokines and galectins can engage in cross talk by pairing, as exemplified by galectin‐3 and CXCL12.
The association of CXCL12 with galectin‐3 appears to have potential for modulating chemokine activity.
Galectin‐3 inhibits CXCL12‐induced chemotaxis of leukocytes and their recruitment to inflammation sites.
Galectin‐3 attenuates CXCL12‐stimulated signaling via its receptor CXCR4 in a ternary complex.
Chemokines and galectins are simultaneously upregulated during inflammation and mediate leukocyte recruitment. A systematic screen now demonstrates their physical interaction as heterodimers, identifying several novel interacting pairs.
Cardiovascular disease is the major cause of death worldwide. The success of medication and other preventive measures introduced in the last century have not yet halted the epidemic of cardiovascular ...disease. Although the molecular mechanisms of the pathophysiology of the heart and vessels have been extensively studied, the burden of ischemic cardiovascular conditions has risen to become a top cause of morbidity and mortality. Calcium has important functions in the cardiovascular system. Calcium is involved in the mechanism of excitation-contraction coupling that regulates numerous events, ranging from the production of action potentials to the contraction of cardiomyocytes and vascular smooth muscle cells. Both in the heart and vessels, the rise of intracellular calcium is sensed by calmodulin, a protein that regulates and activates downstream kinases involved in regulating calcium signalling. Among them is the calcium calmodulin kinase family, which is involved in the regulation of cardiac functions. In this review, we present the current literature regarding the role of calcium/calmodulin pathways in the heart and vessels with the aim to summarize our mechanistic understanding of this process and to open novel avenues for research.
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