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.     

Direct evidence that the N-terminal extensions of the TAP complex act as autonomous interaction scaffolds for the assembly of the MHC I peptide-loading complex

The loading of antigenic peptides onto major histocompatibility complex class I (MHC I) molecules is an essential step in the adaptive immune response against virally or malignantly transformed cells. The ER-resident peptide-loading complex (PLC) consists of the transporter associated with antigen processing (TAP1 and TAP2), assembled with the auxiliary factors tapasin and MHC I. Here, we demonstrated that the N-terminal extension of each TAP subunit represents an autonomous domain, named TMD(0), which is correctly targeted to and inserted into the ER membrane. In the absence of coreTAP, each TMD(0) recruits tapasin in a 1:1 stoichiometry. Although the TMD(0)s lack known ER retention/retrieval signals, they are localized to the ER membrane even in tapasin-deficient cells. We conclude that the TMD(0)s of TAP form autonomous interaction hubs linking antigen translocation into the ER with peptide loading onto MHC I, hence ensuring a major function in the integrity of the antigen-processing machinery.     

The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation

Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5^KD cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5^KD cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5^KD compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5^KD cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5^KD cells is due to a role of Cdk5 in other pathways or the altered polymer levels.     

Emerging roles of immunoproteasomes beyond MHC class I antigen processing

The proteasome is a multi-catalytic protein complex whose primary function is the degradation of abnormal or foreign proteins. Upon exposure of cells to interferons (IFNs), the β1i/LMP2, β2i/MECL-1, and β5i/LMP7 subunits are induced and incorporated into newly synthesized immunoproteasomes (IP), which are thought to function solely as critical players in the optimization of the CD8(+) T-cell response. However, the observation that IP are present in several non-immune tissues under normal conditions and/or following pathological events militates against the view that its role is limited to MHC class I presentation. In support of this concept, the recent use of genetic models deficient for β1i/LMP2, β2i/MECL-1, or β5i/LMP7 has uncovered unanticipated functions for IP in innate immunity and non-immune processes. Herein, we review recent data in an attempt to clarify the role of IP beyond MHC class I epitope presentation with emphasis on its involvement in the regulation of protein homeostasis, cell proliferation, and cytokine gene expression.     

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.     

CD24 interacts with and promotes the activity of c-src within lipid rafts in breast cancer cells, thereby increasing integrin-dependent adhesion

Expression of the glycosylphosphatidylinositol-anchored membrane protein CD24 correlates with a poor prognosis for many human cancers, and in experimental tumors can promote metastasis. However, the mechanism by which CD24 contributes to tumor progression remains unclear. Here we report that in MTLy breast cancer cells CD24 interacts with and augments the kinase activity of c-src, a protein strongly implicated in promoting invasion and metastasis. This occurs within and is dependent upon intact lipid rafts. CD24-augmented c-src kinase activity increased formation of focal adhesion complexes, accelerated phosphorylation of FAK and paxillin and consequently enhanced integrin-mediated adhesion. Loss and gain of function approaches showed that c-src activity is necessary and sufficient to mediate the effects of CD24 on integrin-dependent adhesion and cell spreading, as well as on invasion. Together these results indicate that c-src is a CD24-activated mediator that promotes integrin-mediated adhesion and invasion, and suggest a mechanism by which CD24 might contribute to tumor progression through stimulating the activity of c-src or another member of the Src family.     

Immune aging and autoimmunity

Age is an important risk for autoimmunity, and many autoimmune diseases preferentially occur in the second half of adulthood when immune competence has declined and thymic T cell generation has ceased. Many tolerance checkpoints have to fail for an autoimmune disease to develop, and several of those are susceptible to the immune aging process. Homeostatic T cell proliferation which is mainly responsible for T cell replenishment during adulthood can lead to the selection of T cells with increased affinity to self- or neoantigens and enhanced growth and survival properties. These cells can acquire a memory-like phenotype, in particular under lymphopenic conditions. Accumulation of end-differentiated effector T cells, either specific for self-antigen or for latent viruses, have a low activation threshold due to the expression of signaling and regulatory molecules and generate an inflammatory environment with their ability to be cytotoxic and to produce excessive amounts of cytokines and thereby inducing or amplifying autoimmune responses.