The paracaspase MALT1 mediates T cell antigen receptor-induced signaling to the transcription factor NF-kappaB and is indispensable for T cell activation and proliferation. Enhanced expression of MALT1 or aberrant expression of a fusion protein of the apoptosis inhibitor API2 and MALT1 has been linked to mucosa-associated lymphoid tissue lymphoma. Despite the presence of a caspase-like domain, MALT1 proteolytic activity has not yet been demonstrated. Here we show that T cell antigen receptor stimulation induced recruitment of the NF-kappaB inhibitor A20 into a complex of MALT1 and the adaptor protein Bcl-10, leading to MALT1-mediated processing of A20. API2-MALT1 expression likewise resulted in cleavage of A20. MALT1 cleaved human A20 after arginine 439 and impaired its NF-kappaB-inhibitory function. Our studies identify A20 as a substrate of MALT1 and emphasize the importance of MALT1 proteolytic activity in the 'fine tuning' of T cell antigen receptor signaling.

Inappropriate functioning of the immune system is linked to immune deficiency, autoimmune disease, and cancer. It is therefore not surprising that intracellular immune signaling pathways are tightly controlled. One of the best studied transcription factors in immune signaling is NF-B, which is activated by multiple receptors and regulates the expression of a wide variety of proteins that control innate and adaptive immunity. A20 is an early NF-B-responsive gene that encodes a ubiquitin-editing protein that is involved in the negative feedback regulation of NF-B signaling. Here, we discuss the mechanism of action of A20 and its role in the regulation of inflammation and immunity. A20 (also known as TNFAIP3) was originally identified as a TNF 2 -inducible gene in human umbilical vein endothelial cells (1). Subsequent research demonstrated that A20 is also induced in many other cell types and by a wide range of other stimuli (reviewed in Ref. 2). Although A20 was originally characterized as an inhibitor of TNF-induced apoptosis (3), it has been most intensively studied as an inhibitor of NF-B activation. NF-B is a dimeric transcription factor that plays a key role in inflammation and immunity. A deregulated NF-B response has been associated with several autoimmune diseases and some cancers (4). The activity of NF-B is tightly regulated by interaction with inhibitory IB (inhibitor of B) proteins, which are regulated by IKK-mediated IB phosphorylation, followed by their ubiquitination and proteolysis, enabling the entry of NF-B into the nucleus. In most cases, the activation of NF-B is transient and cyclic upon continuous stimulation, which is due to specific negative feedback control systems such as the NF-Binducible synthesis of IB and A20 proteins (5). NF-B activation pathways are broadly classified as either canonical or noncanonical, depending on whether activation involves IB degradation or processing of the p100 NF-B precursor (4).The canonical pathway, which is the predominant NF-B signaling pathway, is activated by pro-inflammatory cytokines such as TNF and IL-1 and microbial components that activate, for example, TLRs or antigen receptors. The non-canonical pathway of NF-B activation operates mainly in B cells in response to a subset of TNFR family members, including the lymphotoxin-␤ receptor.Initial evidence for the NF-B inhibitory function of A20 came from several studies in which overexpression of A20 was shown to prevent NF-B activation in response to TNF and several other pro-inflammatory stimuli (reviewed in Ref.2). The observation that A20 expression is itself under the control of NF-B suggested its involvement in the negative feedback regulation of NF-B activation (6). This was eventually confirmed by the generation of A20-deficient mice, which show a sustained NF-B response and severe inflammation (7). The mechanism by which A20 inhibits NF-B activation remained a mystery for several years until it was recently found that A20 can act as a dual ubiquitin-editing enzyme.

The unfolded protein response (UPR) or endoplasmic reticulum (ER) stress response is a physiological process enabling cells to cope with altered protein synthesis demands. However, under conditions of obesity, prolonged activation of the UPR has been shown to have deteriorating effects on different metabolic pathways. Here we identify Bax inhibitor-1 (BI-1), an evolutionary conserved ER-membrane protein, as a novel modulator of the obesity-associated alteration of the UPR. BI-1 partially inhibits the UPR by interacting with IRE1␣ and inhibiting IRE1␣ endonuclease activity as seen on the splicing of the transcription factor Xbp-1. Because we observed a down-regulation of BI-1 expression in liver and muscle of genetically obese ob/ob and db/db mice as well as in mice with diet-induced obesity in vivo, we investigated the effect of restoring BI-1 expression on metabolic processes in these mice. Importantly, BI-1 overexpression by adenoviral gene transfer dramatically improved glucose metabolism in both standard diet-fed mice as well as in mice with diet-induced obesity and, critically, reversed hyperglycemia in db/db mice. This improvement in whole body glucose metabolism and insulin sensitivity was due to dramatically reduced gluconeogenesis as shown by reduction of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase expression. Taken together, these results identify BI-1 as a critical regulator of ER stress responses in the development of obesityassociated insulin resistance and provide proof of concept evidence that gene transfer-mediated elevations in hepatic BI-1 may represent a promising approach for the treatment of type 2 diabetes.

TNF is a major proinflammatory cytokine, which exerts its effects through two different receptors known as TNF-R1 and TNF-R2. Whereas TNF-R1 induced signaling pathways have been very well characterized during the past years, TNF-R2 has been much less well studied. Nevertheless, the function of TNF-R2 should not be underestimated, the more because this receptor shows a much more restricted but inducible expression. Several inflammatory diseases and cancers show upregulated levels of soluble TNF-R2 or are associated with TNF-R2 polymorphisms, implicating an important role for TNF-R2 as a therapeutic target. Here we will review the mechanisms that regulate TNF-R2 expression, as well as discuss TNF-R2 induced signal transduction and cross-talk with TNF-R1.

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Although Toll-like receptor (TLR)-induced expression of several proinflammatory genes is required to provoke an efficient immune response, excessive or prolonged activation of TLR signaling can contribute to the development of septic shock and several inflammatory diseases. Given this inherent danger of unrestrained TLR signaling to the organism, it is not surprising that many negative feedback mechanisms have evolved to hold TLR signaling in check. In this context, TLR stimulation induces several negative regulators of TLR-induced signaling to nuclear factor (NF)-kappaB dependent gene expression. Here we describe the use of Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) to study respectively the cellular protein and mRNA expression levels of the NF-kappaB inhibitory proteins A20 and ABIN-3 in response to TLR4 stimulation by lipopolysaccharide (LPS).