A20 and ABIN-1 cooperate in balancing CBM complex-triggered NF-κB signaling in activated T cells.

T cell activation initiates protective adaptive immunity, but counterbalancing mechanisms are critical to prevent overshooting responses and to maintain immune homeostasis. The CARD11-BCL10-MALT1 (CBM) complex bridges T cell receptor engagement to NF-κB signaling and MALT1 protease activation. Here, we show that ABIN-1 is modulating the suppressive function of A20 in T cells. Using quantitative mass spectrometry, we identified ABIN-1 as an interactor of the CBM signalosome in activated T cells. A20 and ABIN-1 counteract inducible activation of human primary CD4 and Jurkat T cells. While A20 overexpression is able to silence CBM complex-triggered NF-κB and MALT1 protease activation independent of ABIN-1, the negative regulatory function of ABIN-1 depends on A20. The suppressive function of A20 in T cells relies on ubiquitin binding through the C-terminal zinc finger (ZnF)4/7 motifs, but does not involve the deubiquitinating activity of the OTU domain. Our mechanistic studies reveal that the A20/ABIN-1 module is recruited to the CBM complex via A20 ZnF4/7 and that proteasomal degradation of A20 and ABIN-1 releases the CBM complex from the negative impact of both regulators. Ubiquitin binding to A20 ZnF4/7 promotes destructive K48-polyubiquitination to itself and to ABIN-1. Further, after prolonged T cell stimulation, ABIN-1 antagonizes MALT1-catalyzed cleavage of re-synthesized A20 and thereby diminishes sustained CBM complex signaling. Taken together, interdependent post-translational mechanisms are tightly controlling expression and activity of the A20/ABIN-1 silencing module and the cooperative action of both negative regulators is critical to balance CBM complex signaling and T cell activation.

Synthesis of GPR40 targeting 3 H- and 18 F-probes towards selective beta cell imaging.

Diabetes affects an increasing number of patients worldwide and is responsible for a significant rise in healthcare expenses. Imaging of ß-cells in vivo is expected to contribute to an improved understanding of the underlying pathophysiology, improved diagnosis, and development of new treatment options for diabetes. Here, we describe the first radiosyntheses of [3 H]-TAK875 and [18 F]-TAK875 derivatives to be used as ß-cell imaging probes addressing the free fatty acid receptor 1 (FFAR1/GPR40). The fluorine-labeled derivative showed similar agonistic activity as TAK875 in a functional assay. The radiosynthesis of the 18 F-labelled tracer 2a was achieved with 16.7 ± 5.7% radiochemical yield in a total synthesis time of 60-70 min.

Adverse bioenergetic effects of N-acyl amino acids in human adipocytes overshadow beneficial mitochondrial uncoupling.

OBJECTIVE: Enhancing energy turnover via uncoupled mitochondrial respiration in adipose tissue has great potential to improve human obesity and other metabolic complications. However, the amount of human brown adipose tissue and its uncoupling protein 1 (UCP1) is low in obese patients. Recently, a class of endogenous molecules, N-acyl amino acids (NAAs), was identified as mitochondrial uncouplers in murine adipocytes, presumably acting via the adenine nucleotide translocator (ANT). Given the translational potential, we investigated the bioenergetic effects of NAAs in human adipocytes, characterizing beneficial and adverse effects, dose ranges, amino acid derivatives and underlying mechanisms. METHOD: NAAs with neutral (phenylalanine, leucine, isoleucine) and polar (lysine) residues were synthetized and assessed in intact and permeabilized human adipocytes using plate-based respirometry. The Seahorse technology was applied to measure bioenergetic parameters, dose-dependency, interference with UCP1 and adenine nucleotide translocase (ANT) activity, as well as differences to the established chemical uncouplers niclosamide ethanolamine (NEN) and 2,4-dinitrophenol (DNP). RESULT: NAAs with neutral amino acid residues potently induce uncoupled respiration in human adipocytes in a dose-dependent manner, even in the presence of the UCP1-inhibitor guanosine diphosphate (GDP) and the ANT-inhibitor carboxyatractylate (CAT). However, neutral NAAs significantly reduce maximal oxidation rates, mitochondrial ATP-production, coupling efficiency and reduce adipocyte viability at concentrations above 25 µM. The in vitro therapeutic index (using induced proton leak and viability as determinants) of NAAs is lower than that of NEN and DNP. CONCLUSION: NAAs are potent mitochondrial uncouplers in human adipocytes, independent of UCP1 and ANT. However, previously unnoticed adverse effects harm adipocyte functionality, reduce the therapeutic index of NAAs in vitro and therefore question their suitability as anti-obesity agents without further chemical modifications.

A patent review of MALT1 inhibitors (2013-present).

INTRODUCTION: MALT1 is the only human paracaspase, a protease with unique cleavage activity and substrate specificity. As a key regulator of immune responses, MALT1 has attracted attention as an immune modulatory target for the treatment of autoimmune/inflammatory diseases. Further, chronic MALT1 protease activation drives survival of lymphomas, suggesting that MALT1 is a suitable drug target for lymphoid malignancies. Recent studies have indicated that MALT1 inhibition impairs immune suppressive function of regulatory T cells in the tumor microenvironment, suggesting that MALT1 inhibitors may boost anti-tumor immunity in the treatment of solid cancers. AREAS COVERED: This review summarizes the literature on MALT1 patents and applications. We discuss the potential therapeutic uses for MALT1 inhibitors based on patents and scientific literature. EXPERT OPINION: There has been a steep increase in MALT1 inhibitor patents. Compounds with high selectivity and good bioavailability have been developed. An allosteric binding pocket is the preferred site for potent and selective MALT1 targeting. MALT1 inhibitors have moved to early clinical trials, but toxicological studies indicate that long-term MALT1 inhibition can disrupt immune homeostasis and lead to autoimmunity. Even though this poses risks, preventing immune suppression may favor the use of MALT1 inhibitors in cancer immunotherapies.

TRAF6 prevents fatal inflammation by homeostatic suppression of MALT1 protease.

Balanced control of T cell signaling is critical for adaptive immunity and protection from autoimmunity. By combining genetically engineered mouse models, biochemical analyses and pharmacological interventions, we describe an unexpected dual role of the tumor necrosis factor receptor­associated factor 6 (TRAF6) E3 ligase as both a positive and negative regulator of mucosa-associated lymphoid tissue 1 (MALT1) paracaspase. Although MALT1-TRAF6 recruitment is indispensable for nuclear factor κB signaling in activated T cells, TRAF6 counteracts basal MALT1 protease activity in resting T cells. In mice, loss of TRAF6-mediated homeostatic suppression of MALT1 protease leads to severe autoimmune inflammation, which is completely reverted by genetic or therapeutic inactivation of MALT1 protease function. Thus, TRAF6 functions as a molecular brake for MALT1 protease in resting T cells and a signaling accelerator for MALT1 scaffolding in activated T cells, revealing that TRAF6 controls T cell activation in a switch-like manner. Our findings have important implications for development and treatment of autoimmune diseases.

Long-Term Cold Adaptation Does Not Require FGF21 or UCP1.

Brown adipose tissue (BAT)-dependent thermogenesis and its suggested augmenting hormone, FGF21, are potential therapeutic targets in current obesity and diabetes research. Here, we studied the role of UCP1 and FGF21 for metabolic homeostasis in the cold and dissected underlying molecular mechanisms using UCP1-FGF21 double-knockout mice. We report that neither UCP1 nor FGF21, nor even compensatory increases of FGF21 serum levels in UCP1 knockout mice, are required for defense of body temperature or for maintenance of energy metabolism and body weight. Remarkably, cold-induced browning of inguinal white adipose tissue (iWAT) is FGF21 independent. Global RNA sequencing reveals major changes in response to UCP1- but not FGF21-ablation in BAT, iWAT, and muscle. Markers of mitochondrial failure and inflammation are observed in BAT, but in particular the enhanced metabolic reprogramming in iWAT supports the thermogenic role of UCP1 and excludes an important thermogenic role of endogenous FGF21 in normal cold acclimation.