Secretory sphingomyelinase

Several physiologic and pathophysiologic processes in which sphingomyelinases (SMases) have been implicated may involve extracellular sphingomyelin (SM) hydrolysis. A candidate enzyme for these processes is a recently discovered SMase called secretory SMase, or S-SMase. S-SMase arises from the acid sphingomyelinase (ASM) gene via differential protein trafficking of a common protein precursor; this precursor can be targeted to either lysosomes or the Golgi secretory pathway. S-SMase is activated by physiologic levels of Zn2+, although the S-SMase from endothelial cells, which secrete abundant amounts of the enzyme, is partially Zn2+-independent. S-SMase functions best at acid pH but can hydrolyze certain physiologic substrates, such as atherogenic lipoproteins, at neutral pH. In endothelial cells, the secretion of S-SMase is regulated at the level of protein trafficking by inflammatory cytokines. Current work implicates a role for S-SMase in atherogenesis, and future work will be directed at understanding the potential roles of S-SMase in other processes, such as ceramide-mediated cell-signaling and the host inflammatory response.     

Caspase-containing complexes in the regulation of cell death and inflammation

Caspases are a family of cysteine proteases that are essential in the initiation and execution of apoptosis and the proteolytic maturation of inflammatory cytokines such as IL-1beta and IL-18. Caspases can be subdivided into those that have a large prodomain and those that have not. In general, apoptotic and inflammatory signalling pathways are initiated when large-prodomain caspases are recruited to large protein complexes via homotypic interactions involving death domain folds. The formation of these specialised multimeric platforms involves three major functions: (1) the sensing of cellular stress, damage, infection or inflammation; (2) multimerisation of the platform; and (3) recruitment and conformational activation of caspases. In this overview we discuss the complexes implicated in the regulation of cell death and inflammatory processes such as the death-inducing signalling complex (DISC), the apoptosome, the inflammasomes and the PIDDosome. We describe their sensing functions, compositions and functional outcomes. Inhibitory protein families such as FLIPs and CARD-only proteins prevent the recruitment of caspases in these sensing complexes, avoiding inappropriate initiation of cell death or inflammation.     

Emerging roles for TNIP1 in regulating post-receptor signaling

A vast number of cellular processes and signaling pathways are regulated by various receptors, ranging from transmembrane to nuclear receptors. These receptor-mediated processes are modulated by a diverse set of regulatory proteins. TNFα-induced protein 3-interacting protein 1 is such a protein that inhibits both transduction by transmembrane receptors, such as TNFα-receptor, EGF-R, and TLR, and nuclear receptors' PPAR and RAR activity. These receptors play key roles in regulating inflammation and inflammatory diseases. A growing number of references have implicated TNIP1 through GWAS and expression studies in chronic inflammatory diseases such as psoriasis and rheumatoid arthritis, although TNIP1s exact role has yet been determined. In this review, we aim to integrate the current knowledge of TNIP1s functions with the diseases in which it has been associated to potentially elucidate the role this regulator has in promoting or alleviating these inflammatory diseases.     

Crosstalk between TNF and glucocorticoid receptor signaling pathways

TNF is a Janus-faced protein. It possesses impressive anti-tumor activities, but it is also one of the strongest known pro-inflammatory cytokines, which hampers its use as a systemic anti-cancer agent. TNF has been shown to play a detrimental role in inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Glucocorticoids are strongly anti-inflammatory and exert their therapeutic effects through binding to their receptor, the glucocorticoid receptor. Therefore, glucocorticoids have been used for over half a century for the treatment of inflammatory diseases. However, many patients are or become resistant to the therapeutic effects of glucocorticoids. Inflammatory cytokines have been suggested to play an important role in this steroid insensitivity or glucocorticoid resistance. This review aims to highlight the mechanisms of mutual inhibition between TNF and GR signaling pathways.     

Phospholipase A2-activating protein--an important regulatory molecule in modulating cyclooxygenase-2 and tumor necrosis factor production during inflammation

Inflammation is a complex multifactorial process and a hallmark of many inflammatory diseases. Most of the tissue destruction that occurs in these diseases is the result of an aberrant or often uncontrolled immune response. Factors that play an important role in such diseases include pro-inflammatory cytokines, complement, and eicosanoids. This review focuses on eicosanoids and their regulation via phospholipase A2-activating protein, which could be targeted as a new therapeutic tool to control inflammatory diseases.     

Phenyl-N-tert-butylnitrone demonstrates broad-spectrum inhibition of apoptosis-associated gene expression in endotoxin-treated rats

Systemic exposure to gram-negative bacterial substances such as lipopolysaccharide (LPS, or endotoxin) induces an uncontrolled, massive inflammatory reaction which culminates in multiple system organ failure and death. Septic shock often does not respond to corticosteroids; however, certain low-molecular-weight antioxidant compounds have been discovered to possess potent anti-inflammatory action, and some of these novel compounds can rescue animals from experimentally induced septic shock. Phenyl-N-tert-butylnitrone (PBN) is the archetype of the nitrone class of antioxidants which we have previously shown to suppress LPS-induced cytokine biosynthesis in vivo. Using a multiprobe ribonuclease protection assay, we now demonstrate the ability of PBN to suppress proapoptotic gene expression in the LPS-induced model of endotoxic shock. The broad-spectrum gene-suppressive affects of PBN are discussed in the context of inflammatory signal transduction and models are proposed to explain why certain antioxidants may also possess anti-inflammatory and antiapoptotic properties.     

Refolding of therapeutic proteins produced in Escherichia coli as inclusion bodies

Overexpression of cloned or synthetic genes in Escherichia coli often results in the formation of insoluble protein inclusion bodies. Within the last decade, specific methods and strategies have been developed for preparing active recombinant proteins from these inclusion bodies. Usually, the inclusion bodies can be separated easily from other cell components by centrifugation, solubilized by denaturants such as guanidine hydrochloride (Gdn-HCl) or urea, and then renatured through a refolding process such as dilution or dialysis. Recent improvements in renaturation procedures have included the inhibition of aggregation during refolding by application of low molecular weight additives and matrix-bound renaturation. These methods have made it possible to obtain high yields of biologically active proteins by taking into account process parameters such as protein concentration, redox conditions, temperature, pH, and ionic strength.     

Functional improvement of antibody fragments using a novel phage coat protein III fusion system

Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria constitute an easy and inexpensive method compared to hybridoma cultures. Such approaches have, however, often suffered from low yields and poor functionality. A general method is described here which enables expressions of functional antibody fragments when fused to the amino-terminal domain(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity.     

In vitro responses to a 65-kilodalton mycobacterial protein by synovial T cells from inflammatory arthritis patients

Bacterial Ag, especially those of mycobacteria, have been implicated in the pathogenesis of experimental inflammatory arthritis in rodents, while in man, reactive arthritis has a clear temporal relationship to infection with particular bacteria. To investigate the role of immune responses to bacterial Ag in inflammatory arthritis, we have examined the proliferative responses of paired synovial fluid and PBMC when stimulated with 1) suspensions of irradiated or heat-killed bacteria associated with reactive arthritis (ReA), 2) purified protein derivative, 3) a recombinant 65-kDa heat shock protein of Mycobacterium leprae. The 65-kDa Ag was stimulatory to synovial fluid mononuclear cells, but not PBMC, from patients with different arthropathies, including most of those with ReA, but also some with rheumatoid arthritis. Furthermore, the magnitude of these responses correlated more closely with responses to ReA-associated bacteria (such as Salmonella), than with responses to the mycobacterial Ag represented in purified protein derivative. These results suggest that the 65-kDa molecule, which is common to a wide range of bacteria, may be an important immunogen for the T cell-mediated immune responses within the joint in different clinically defined inflammatory arthropathies.     

Expression of the double-stranded RNA-dependent kinase PKR influences osteosarcoma attachment independent growth,migration, and invasion

Osteosarcoma (OS) is a rare, insidious tumor of mesenchymal origin that most often affects children, adolescents, and young adults. While the primary tumor can be controlled with chemotherapy and surgery, it is the lung metastases that are eventually fatal. Multiple studies into the initial drivers of OS development have been undertaken, but few of these have examined innate immune/inflammatory signaling. A central figure in inflammatory signaling is the innate immune/stress-activated kinase double-stranded RNA-dependent protein kinase (PKR). To characterize the role of PKR in OS, U2OS, and SaOS-2 osteosarcoma cell lines were stably transfected with wild-type or dominant-negative (DN) PKR. Overexpression of PKR enhanced colony formation in soft agar (U2OS and SaOS-2), enhanced cellular migration (U2OS), and invasive migration (SaOS-2). In contrast, overexpression of DN-PKR inhibited attachment-independent growth, migration and/or invasion. These data demonstrate a role for inflammatory signaling in OS formation and migration/invasion and suggest the status of PKR expression/activation may have prognostic value.     

Effects of arachidonic acid analogs on FcepsilonRI-mediated activation of mast cells

Polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA) have been shown to modulate a number of inflammatory disorders. Mast cells play a critical role in the initiation and maintenance of inflammatory responses. However, the effects of PUFAs on mast cell functions have not been fully addressed. We here-in examined the effects of PUFAs on the high affinity IgE receptor (FcepsilonRI)-mediated mast cell activation using RBL-2H3 cells, a rat mast cell line, that were cultured in the medium containing palmitic acid (PA), AA, or the AA analogs mead acid (MA) and eicosapentaenoic acid (EPA). In AA-supplemented cells, the FcepsilonRI-mediated beta-hexosamidase and TNF-alpha release, calcium (Ca(2+)) influx, and some protein tyrosine phosphorylations including Syk and linker for activation of T cells (LAT) were enhanced, whereas, in MA- or PA-supplemented cells, they were not changed when compared with cells cultured in control medium. In EPA-supplemented cells, the enhancements of beta-hexosamidase release and protein tyrosine phosphorylations were observed. Furthermore, in AA- or EPA-supplemented cells, FcepsilonRI-mediated intracellular production of reactive oxygen species (ROS) that is required for the tyrosine phosphorylation of LAT and Ca(2+) influx were enhanced when compared with the other cells. Thus, preincubation of AA or EPA augmented FcepsilonRI-mediated degranulation in mast cells by affecting early events of FcepsilonRI signal transduction, which might be associated with the change of fatty acid composition of the cell membrane and enhanced production of ROS. The results suggest that some PUFAs can modulate FcepsilonRI-mediated mast cell activation and might affect FcepsilonRI/mast cell-mediated inflammation, such as allergic reaction.     

Regulation of inflammation by extracellular acidification and proton-sensing GPCRs

Under ischemic and inflammatory circumstances, such as allergic airway asthma, rheumatoid arthritis, atherosclerosis, and tumors, extracellular acidification occurs due to the stimulation of anaerobic glycolysis. An acidic microenvironment has been shown to modulate pro-inflammatory or anti-inflammatory responses, including cyclooxygenase-2 (COX-2) expression, prostaglandin synthesis, and cytokine expression, in a variety of cell types, and thereby to exacerbate or ameliorate inflammation. However, molecular mechanisms underlying extracellular acidic pH-induced actions have not been fully understood. Recent studies have shown that ovarian cancer G protein-coupled receptor 1 (OGR1)-family G protein-coupled receptors (GPCRs) can sense extracellular pH or protons, which in turn stimulates intracellular signaling pathways and subsequent diverse cellular responses. In the present review, I discuss extracellular acidic pH-induced inflammatory responses and related responses in inflammatory cells, such as macrophages and neutrophils, and non-inflammatory cells, such as smooth muscle cells and endothelial cells, focusing especially on proton-sensing GPCRs.     

The microRNA miR-23b suppresses IL-17-associated autoimmune inflammation by targeting TAB2, TAB3 and IKK-α

Inflammatory cytokines such as interleukin-17 (IL-17) promote inflammatory autoimmune diseases. Although several microRNAs (miRNAs) have been shown to regulate autoimmune pathogenesis by affecting lymphocyte development and function, the role of miRNAs in resident cells present in inflammatory lesions remains unclear. Here we show that miR-23b is downregulated in inflammatory lesions of humans with lupus or rheumatoid arthritis, as well as in the mouse models of lupus, rheumatoid arthritis or multiple sclerosis. IL-17 downregulates miR-23b expression in human fibroblast-like synoviocytes, mouse primary kidney cells and astrocytes and is essential for the downregulation of miR-23b during autoimmune pathogenesis. In turn, miR-23b suppresses IL-17-, tumor necrosis factor α (TNF-α)- or IL-1β-induced NF-κB activation and inflammatory cytokine expression by targeting TGF-β-activated kinase 1/MAP3K7 binding protein 2 (TAB2), TAB3 and inhibitor of nuclear factor κ-B kinase subunit α (IKK-α) and, consequently, represses autoimmune inflammation. Thus, IL-17 contributes to autoimmune pathogenesis by suppressing miR-23b expression in radio-resident cells and promoting proinflammatory cytokine expression.     

Monocyte chemotactic protein-induced protein (MCPIP) promotes inflammatory angiogenesis via sequential induction of oxidative stress, endoplasmic reticulum stress and autophagy

Major diseases such as cardiovascular diseases, rheumatoid arthritis, diabetes, obesity and tumor growth are known to involve inflammation. Inflammatory molecules such as MCP-1, TNF-α, IL-1β and IL-8 are known to promote angiogenesis. MCP-induced protein (MCPIP), originally discovered as a novel zinc finger protein induced by MCP-1, is also induced by other inflammatory agents. MCPIP was shown to mediate MCP-1-induced angiogenesis. Whether angiogenesis induced by other inflammatory agents is mediated via MCPIP is unknown and the molecular mechanisms involved in angiogenesis induced by MCPIP have not been elucidated. The aim of this study was to bridge this gap and delineate the sequential processes involved in angiogenesis mediated via MCPIP. siRNA knockdown of MCPIP was used to determine whether different inflammatory agents, MCP-1, TNF-α, IL-1β and IL-8, mediate angiogenesis via MCPIP in human umbilical vein endothelial cells (HUVECs). Chemical inhibitors and specific gene knockdown approach were used to inhibit each process postulated. Oxidative stress was inhibited by apocynin or cerium oxide nanoparticles or knockdown of NADPH oxidase subunit, phox47. Endoplasmic reticulum (ER) stress was blocked by tauroursodeoxycholate or knockdown of ER stress signaling protein IRE-1 and autophagy was inhibited by the use of 3'methyl adenine, or LY 294002 or by specific knockdown of beclin1. Matrigel assay was used as a tool to study angiogenic differentiation induced by inflammatory agents or MCPIP overexpression in HUVECs. Tube formation induced by inflammatory agents, TNF-α, IL-1β, IL-8 and MCP-1 was inhibited by knockdown of MCPIP. Forced MCPIP-expression induced oxidative stress, ER stress, autophagy and angiogenic differentiation in HUVECs. Inhibition of each step caused inhibition of each subsequent step postulated. The results reveal that angiogenesis induced by inflammatory agents is mediated via induction of MCPIP that causes oxidative and nitrosative stress resulting in ER stress leading to autophagy required for angiogenesis. The sequence of events suggested to be involved in inflammatory angiogenesis by MCPIP could serve as possible targets for therapeutic intervention of angiogenesis-related disorders.     

Allergy influences the inflammatory status of the brain and enhances tau-phosphorylation

Despite the existing knowledge regarding the neuropathology of Alzheimer's disease (AD), the cause of sporadic forms of the disease is unknown. It has been suggested that systemic inflammation may have a role, but the exact mechanisms through which inflammatory processes influence the pathogenesis and progress of AD are not obvious. Allergy is a chronic inflammatory disease affecting more than 20% of the Western population, but the effects of allergic conditions on brain functions are largely unknown. The aim of this study was to investigate whether or not chronic peripheral inflammation associated with allergy affects the expression of AD-related proteins and inflammatory markers in the brain. On the basis of previously described models for allergy in mice we developed a model of chronic airway allergy in mouse, with ovalbumin as allergen. The validity of the chronic allergy model was confirmed by a consistent and reproducible eosinophilia in the bronchoalveolar lavage (BAL) fluid of allergic animals. Allergic mice were shown to have increased brain levels of both immunoglobulin (Ig) G and IgE with a widespread distribution. Allergy was also found to increase phosphorylation of tau protein in the brain. The present data support the notion that allergy-dependent chronic peripheral inflammation modifies the brain inflammatory status, and influences phosphorylation of an AD-related protein, indicating that allergy may be yet another factor to be considered for the development and/or progression of neurodegenerative diseases such as AD.     

Acute phase mediator oncostatin M regulates affinity of alpha1-protease inhibitor for concanavalin A in hepatoma-derived but not lung-derived epithelial cells

Quantitative changes in plasma protein concentrations during tissue injury or inflammation (acute phase response) are often accompanied by specific alterations in the carbohydrate moieties of these proteins. The glycosylation changes comprise alterations in the type of branching of the carbohydrate structures as revealed by modulated reactivity of acute phase glycoproteins with the lectin concanavalin A. Interestingly, inflammation-induced changes in the glycosylation of acute phase proteins have been shown to affect the functional properties of these proteins. In this study we demonstrate that synthesis of acute phase protein alpha(1)-PI, the controlling inhibitor of neutrophil elastase, is significantly up-regulated in hepatic and lung-derived epithelial cells by the inflammatory mediator oncostatin M. Although oncostatin M markedly altered the concanavalin A reactivity of hepatic alpha(1)-PI, lung-derived epithelial cells did not change the pattern of alpha(1)-PI glycan branching upon stimulation with oncostatin M. These results indicate that inflammation-induced changes in glycosylation of alpha(1)-PI may have different impacts on functional properties of liver and lung-synthesized alpha(1)-PI.     

Src protein tyrosine kinase family and acute inflammatory responses

Acute inflammatory responses are one of the major underlying mechanisms for tissue damage of multiple diseases, such as ischemia-reperfusion injury, sepsis, and acute lung injury. By use of cellular and molecular approaches and transgenic animals, Src protein tyrosine kinase (PTK) family members have been identified to be essential for the recruitment and activation of monocytes, macrophages, neutrophils, and other immune cells. Src PTKs also play a critical role in the regulation of vascular permeability and inflammatory responses in tissue cells. Importantly, animal studies have demonstrated that small chemical inhibitors for Src PTKs attenuate tissue injury and improve survival from a variety of pathological conditions related to acute inflammatory responses. Further investigation may lead to the clinical application of these inhibitors as drugs for ischemia-reperfusion injury (such as stroke and myocardial infarction), sepsis, acute lung injury, and multiple organ dysfunction syndrome.     

On the temperature and pressure dependence of a range of properties of a type of water model commonly used in high-temperature protein unfolding simulations

Molecular dynamics simulations of protein folding and unfolding are often carried out at temperatures (400-600 K) that are much higher than physiological or room temperature to speed up the (un)folding process. Use of such high temperatures changes both the protein and solvent properties considerably, compared to physiological or room temperature. Water models designed for use in conjunction with biomolecules, such as the simple point charge (SPC) model, have generally been calibrated at room temperature and pressure. To determine the distortive effect of high simulation temperatures on the behavior of such "room temperature" water models, the structural, dynamic, and thermodynamic properties of the much-used SPC water model are investigated in the temperature range from 300 to 500 K. Both constant pressure and constant volume conditions, as used in protein simulations, were analyzed. We found that all properties analyzed change markedly with increasing temperature, but no phase transition in this temperature range was observed.     

Protein Misfolding and Aggregation in Ageing and Disease

Although intensively researched, the fundamental mechanism of protein misfolding that leads to protein aggregation and associated diseases remains somewhat enigmatic. The failure of a protein to correctly fold de novo or to remain correctly folded can have profound consequences on a living system especially when the cellular quality control processes fail to eliminate the rogue proteins. Over 20 different human diseases have now been designated as ‘conformational diseases’ and include neurodegenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD) and Creutzfeldt Jakob disease (CJD) that are becoming increasingly prevalent in an ageing human population. Such diseases are usually characterised by the deposition of specific misfolded proteins as amyloid fibrils and hence are often referred to as the amyloidoses.