Key role of integrin αIIbβ3 signaling to Syk kinase in tissue factor-induced thrombin generation

The fibrin(ogen) receptor, integrin α(IIb)β(3), has a well-established role in platelet spreading, aggregation and clot retraction. How α(IIb)β(3) contributes to platelet-dependent coagulation is less well resolved. Here, we demonstrate that the potent suppressing effect of clinically used α(IIb)β(3) blockers on tissue factor-induced thrombin generation is linked to diminished platelet Ca(2+) responses and phosphatidylserine (PS) exposure. The same blockers suppress these responses in platelets stimulated with collagen and thrombin receptor agonists, whereas added fibrinogen potentiates these responses. In platelets spreading on fibrinogen, outside-in α(IIb)β(3) signaling similarly enhances thrombin-induced Ca(2+) rises and PS exposure. These responses are reduced in α(IIb)β(3)-deficient platelets from patients with Glanzmann's thrombasthenia. Furthermore, the contribution of α(IIb)β(3) to tissue factor-induced platelet Ca(2+) rises, PS exposure and thrombin generation in plasma are fully dependent on Syk kinase activity. Tyrosine phosphorylation analysis confirms a key role of Syk activation, which is largely but not exclusively dependent on α(IIb)β(3) activation. It is concluded that the majority of tissue factor-induced procoagulant activity of platelets relies on Syk activation and ensuing Ca(2+) signal generation, and furthermore that a considerable part of Syk activation relies on α(IIb)β(3) signaling. These results hence point to a novel role of Syk in integrin-dependent thrombin generation.     

Nesprin interchain associations control nuclear size

Nesprins-1/-2/-3/-4 are nuclear envelope proteins, which connect nuclei to the cytoskeleton. The largest nesprin-1/-2 isoforms (termed giant) tether F-actin through their N-terminal actin binding domain (ABD). Nesprin-3, however, lacks an ABD and associates instead to plectin, which binds intermediate filaments. Nesprins are integrated into the outer nuclear membrane via their C-terminal KASH-domain. Here, we show that nesprin-1/-2 ABDs physically and functionally interact with nesprin-3. Thus, both ends of nesprin-1/-2 giant are integrated at the nuclear surface: via the C-terminal KASH-domain and the N-terminal ABD-nesprin-3 association. Interestingly, nesprin-2 ABD or KASH-domain overexpression leads to increased nuclear areas. Conversely, nesprin-2 mini (contains the ABD and KASH-domain but lacks the massive nesprin-2 giant rod segment) expression yields smaller nuclei. Nuclear shrinkage is further enhanced upon nesprin-3 co-expression or microfilament depolymerization. Our findings suggest that multivariate intermolecular nesprin interactions with the cytoskeleton form a lattice-like filamentous network covering the outer nuclear membrane, which determines nuclear size.     

The NOX toolbox: validating the role of NADPH oxidases in physiology and disease

Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis.     

An eicosanoid-centric view of atherothrombotic risk factors

Cardiovascular disease is the foremost cause of morbidity and mortality in the Western world. Atherosclerosis followed by thrombosis (atherothrombosis) is the pathological process underlying most myocardial, cerebral, and peripheral vascular events. Atherothrombosis is a complex and heterogeneous inflammatory process that involves interactions between many cell types (including vascular smooth muscle cells, endothelial cells, macrophages, and platelets) and processes (including migration, proliferation, and activation). Despite a wealth of knowledge from many recent studies using knockout mouse and human genetic studies (GWAS and candidate approach) identifying genes and proteins directly involved in these processes, traditional cardiovascular risk factors (hyperlipidemia, hypertension, smoking, diabetes mellitus, sex, and age) remain the most useful predictor of disease. Eicosanoids (20 carbon polyunsaturated fatty acid derivatives of arachidonic acid and other essential fatty acids) are emerging as important regulators of cardiovascular disease processes. Drugs indirectly modulating these signals, including COX-1/COX-2 inhibitors, have proven to play major roles in the atherothrombotic process. However, the complexity of their roles and regulation by opposing eicosanoid signaling, have contributed to the lack of therapies directed at the eicosanoid receptors themselves. This is likely to change, as our understanding of the structure, signaling, and function of the eicosanoid receptors improves. Indeed, a major advance is emerging from the characterization of dysfunctional naturally occurring mutations of the eicosanoid receptors. In light of the proven and continuing importance of risk factors, we have elected to focus on the relationship between eicosanoids and cardiovascular risk factors.     

Primary cilia elongation in response to interleukin-1 mediates the inflammatory response

Primary cilia are singular, cytoskeletal organelles present in the majority of mammalian cell types where they function as coordinating centres for mechanotransduction, Wnt and hedgehog signalling. The length of the primary cilium is proposed to modulate cilia function, governed in part by the activity of intraflagellar transport (IFT). In articular cartilage, primary cilia length is increased and hedgehog signaling activated in osteoarthritis (OA). Here, we examine primary cilia length with exposure to the quintessential inflammatory cytokine interleukin-1 (IL-1), which is up-regulated in OA. We then test the hypothesis that the cilium is involved in mediating the downstream inflammatory response. Primary chondrocytes treated with IL-1 exhibited a 50% increase in cilia length after 3 h exposure. IL-1-induced cilia elongation was also observed in human fibroblasts. In chondrocytes, this elongation occurred via a protein kinase A (PKA)-dependent mechanism. G-protein coupled adenylate cyclase also regulated the length of chondrocyte primary cilia but not downstream of IL-1. Chondrocytes treated with IL-1 exhibit a characteristic increase in the release of the inflammatory chemokines, nitric oxide and prostaglandin E2. However, in cells with a mutation in IFT88 whereby the cilia structure is lost, this response to IL-1 was significantly attenuated and, in the case of nitric oxide, completely abolished. Inhibition of IL-1-induced cilia elongation by PKA inhibition also attenuated the chemokine response. These results suggest that cilia assembly regulates the response to inflammatory cytokines. Therefore, the cilia proteome may provide a novel therapeutic target for the treatment of inflammatory pathologies, including OA.     

Metabolic syndrome as a risk factor for neurological disorders

The metabolic syndrome is a cluster of common pathologies: abdominal obesity linked to an excess of visceral fat, insulin resistance, dyslipidemia and hypertension. At the molecular level, metabolic syndrome is accompanied not only by dysregulation in the expression of adipokines (cytokines and chemokines), but also by alterations in levels of leptin, a peptide hormone released by white adipose tissue. These changes modulate immune response and inflammation that lead to alterations in the hypothalamic ‘bodyweight/appetite/satiety set point,’ resulting in the initiation and development of metabolic syndrome. Metabolic syndrome is a risk factor for neurological disorders such as stroke, depression and Alzheimer’s disease. The molecular mechanism underlying the mirror relationship between metabolic syndrome and neurological disorders is not fully understood. However, it is becoming increasingly evident that all cellular and biochemical alterations observed in metabolic syndrome like impairment of endothelial cell function, abnormality in essential fatty acid metabolism and alterations in lipid mediators along with abnormal insulin/leptin signaling may represent a pathological bridge between metabolic syndrome and neurological disorders such as stroke, Alzheimer’s disease and depression. The purpose of this review is not only to describe the involvement of brain in the pathogenesis of metabolic syndrome, but also to link the pathogenesis of metabolic syndrome with neurochemical changes in stroke, Alzheimer’s disease and depression to a wider audience of neuroscientists with the hope that this discussion will initiate more studies on the relationship between metabolic syndrome and neurological disorders.     

Spectrin-based skeleton as an actor in cell signaling

This review focuses on the recent advances in functions of spectrins in non-erythroid cells. We discuss new data concerning the commonly known role of the spectrin-based skeleton in control of membrane organization, stability and shape, and tethering protein mosaics to the cellular motors and to all major filament systems. Particular effort has been undertaken to highlight recent advances linking spectrin to cell signaling phenomena and its participation in signal transduction pathways in many cell types.     

Protein intrinsic disorder as a flexible armor and a weapon of HIV-1

Many proteins and protein regions are disordered in their native, biologically active states. These proteins/regions are abundant in different organisms and carry out important biological functions that complement the functional repertoire of ordered proteins. Viruses, with their highly compact genomes, small proteomes, and high adaptability for fast change in their biological and physical environment utilize many of the advantages of intrinsic disorder. In fact, viral proteins are generally rich in intrinsic disorder, and intrinsically disordered regions are commonly used by viruses to invade the host organisms, to hijack various host systems, and to help viruses in accommodation to their hostile habitats and to manage their economic usage of genetic material. In this review, we focus on the structural peculiarities of HIV-1 proteins, on the abundance of intrinsic disorder in viral proteins, and on the role of intrinsic disorder in their functions.     

Mutations in the PCNA-binding domain of CDKN1C cause IMAGe syndrome

IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital anomalies) is an undergrowth developmental disorder with life-threatening consequences. An identity-by-descent analysis in a family with IMAGe syndrome identified a 17.2-Mb locus on chromosome 11p15 that segregated in the affected family members. Targeted exon array capture of the disease locus, followed by high-throughput genomic sequencing and validation by dideoxy sequencing, identified missense mutations in the imprinted gene CDKN1C (also known as P57KIP2) in two familial and four unrelated patients. A familial analysis showed an imprinted mode of inheritance in which only maternal transmission of the mutation resulted in IMAGe syndrome. CDKN1C inhibits cell-cycle progression, and we found that targeted expression of IMAGe-associated CDKN1C mutations in Drosophila caused severe eye growth defects compared to wild-type CDKN1C, suggesting a gain-of-function mechanism. All IMAGe-associated mutations clustered in the PCNA-binding domain of CDKN1C and resulted in loss of PCNA binding, distinguishing them from the mutations of CDKN1C that cause Beckwith-Wiedemann syndrome, an overgrowth syndrome.