Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses. The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components. Tumour necrosis factor (TNF) is a prominent stimulus of ROS production, but the molecular mechanisms by which TNF activates NADPH oxidase are poorly understood. Here we identify riboflavin kinase (RFK, formerly known as flavokinase) as a previously unrecognized TNF-receptor-1 (TNFR1)-binding protein that physically and functionally couples TNFR1 to NADPH oxidase. In mouse and human cells, RFK binds to both the TNFR1-death domain and to p22(phox), the common subunit of NADPH oxidase isoforms. RFK-mediated bridging of TNFR1 and p22(phox) is a prerequisite for TNF-induced but not for Toll-like-receptor-induced ROS production. Exogenous flavin mononucleotide or FAD was able to substitute fully for TNF stimulation of NADPH oxidase in RFK-deficient cells. RFK is rate-limiting in the synthesis of FAD, an essential prosthetic group of NADPH oxidase. The results suggest that TNF, through the activation of RFK, enhances the incorporation of FAD in NADPH oxidase enzymes, a critical step for the assembly and activation of NADPH oxidase.

Although numerous pathogenic changes within the mitochondrial respiratory chain (RC) have been associated with an elevated occurrence of apoptosis within the affected tissues, the mechanistic insight into how mitochondrial dysfunction initiates apoptotic cell death is still unknown. In this study, we show that the specific alteration of the cytochrome c oxidase (COX), representing a common defect found in mitochondrial diseases, facilitates mitochondrial apoptosis in response to oxidative stress. Our data identified an increased ceramide synthase 6 (CerS6) activity as an important pro-apoptotic response to COX dysfunction induced either by chemical or genetic approaches. The elevated CerS6 activity resulted in accumulation of the pro-apoptotic C16 : 0 ceramide, which facilitates the mitochondrial apoptosis in response to oxidative stress. Accordingly, inhibition of CerS6 or its specific knockdown diminished the increased susceptibility of COX-deficient cells to oxidative stress. Our results provide new insights into how mitochondrial RC dysfunction mechanistically interferes with the apoptotic machinery. On the basis of its pivotal role in regulating cell death upon COX dysfunction, CerS6 might potentially represent a novel target for therapeutic intervention in mitochondrial diseases caused by COX dysfunction.

IntroductionThe proteasome, a large multicatalytic proteinase complex, is responsible for the degradation of most intracellular proteins. The proteasome has a central role in catabolism of a wide variety of proteins controlling cellular division, growth, function, and death. Numerous examples of regulatory proteins including cyclins, cyclin-dependent kinases and kinase inhibitors, oncogenes, tumor suppressor genes, and transcriptional activators and inhibitors have been found to undergo proteasomal proteolysis. Inhibition of the proteasome induces the accumulation of important regulatory intracellular proteins like cytoplasmic inhibitor of NF-B (IB␣), p53 tumor suppressor gene, and p21 and p27 cyclin-dependent kinase inhibitors, which leads to decreased NF-B activity, increased p53-mediated transcription of genes involved in apoptosis, and dysregulation of the cell cycle. [1][2][3][4] In cancer cells, the proteasome is essential to mechanisms underlying tumor cell growth, apoptosis, angiogenesis, and metastasis, thereby representing a novel target for cancer therapy. [3][4][5] Pharmacologic inhibitors of the proteasome have been shown to possess antitumor activity and have significant efficacy against a variety of malignancies. 1-2 The best characterized proteasome inhibitor, bortezomib (Velcade, previously known as PS-341; Millennium Pharmaceuticals, Cambridge, MA), is a dipeptidyl boronic acid that reversibly inhibits the chymotrypsin-like activity of the proteasome. This agent displays remarkable selectivity toward the proteasome relative to serine and cysteine proteases, and it possesses unique antitumor properties as shown in a National Cancer Institute (NCI) tumor cell line screen and in several murine xenograft models. [6][7][8][9][10][11] Bortezomib is the first proteasome inhibitor that was clinically tested in patients and became a therapeutic modality for multiple myeloma.Hodgkin lymphoma (HL) accounts for approximately 30% of all malignant lymphomas 12 with the common feature that neoplastic cells constitute only a small minority of the cells in the affected tissue, often corresponding to less than 2% of the total tumor load. Classical HL (cHL), representing approximately 95% of all HLs, is a fatal disease with 90% of untreated patients dying within 2 to 3 years. 12 The tumor cells of cHL, designated Hodgkin-ReedSternberg (H-RS) cells, are mainly derived from germinal center or post-germinal center B cells, while few (less than 2%) are derived from T cells. H-RS cells lack specific functional markers of mature B or T cells, seem to be arrested in maturation, and therefore should be physiologically prone to undergo apoptosis. [13][14] The mechanisms of apoptotic resistance in H-RS cells have been intensively investigated during the last decade. It has been shown that H-RS cells are resistant to CD95-mediated apoptosis 15 due to the constitutive expression of cFLIP. 16 In addition, H-RS cells display a defective mitochondrial apoptotic pathway 17 and uniformly show up-regulated XIAP expression, 18 whic...

Loss of skeletal muscle mass is one of the most widespread and deleterious processes in aging humans. However, the mechanistic metabolic principles remain poorly understood. In the framework of a multi‐organ investigation of age‐associated changes of ceramide species, a unique and distinctive change pattern of C16:0 and C18:0 ceramide species was detected in aged skeletal muscle. Consistently, the expression of CerS1 and CerS5 mRNA, encoding the ceramide synthases (CerS) with substrate preference for C16:0 and C18:0 acyl chains, respectively, was down‐regulated in skeletal muscle of aged mice. Similarly, an age‐dependent decline of both CerS1 and CerS5 mRNA expression was observed in skeletal muscle biopsies of humans. Moreover, CerS1 and CerS5 mRNA expression was also reduced in muscle biopsies from patients in advanced stage of chronic heart failure (CHF) suffering from muscle wasting and frailty. The possible impact of CerS1 and CerS5 on muscle function was addressed by reversed genetic analysis using CerS1Δ/Δ and CerS5Δ/Δ knockout mice. Skeletal muscle from mice deficient of either CerS1 or CerS5 showed reduced caliber sizes of both slow (type 1) and fast (type 2) muscle fibers, fiber grouping, and fiber switch to type 1 fibers. Moreover, CerS1‐ and CerS5‐deficient mice exhibited reduced twitch and tetanus forces of musculus extensor digitorum longus. The findings of this study link CerS1 and CerS5 to histopathological changes and functional impairment of skeletal muscle in mice that might also play a functional role for the aging skeletal muscle and for age‐related muscle wasting disorders in humans.