Activation of Munc13-1 by Diacylglycerol (DAG)-Lactones.

Munc13-1 is a key protein necessary for vesicle fusion and neurotransmitter release in the brain. Diacylglycerol (DAG)/phorbol ester binds to its C1 domain in the plasma membrane and activates it. The C1 domain of Munc13-1 and protein kinase C (PKC) are homologous in terms of sequence and structure. In order to identify small-molecule modulators of Munc13-1 targeting the C1 domain, we studied the effect of three DAG-lactones, (R,Z)-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-yl)methyl pivalate (JH-131e-153), (E)-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-yl)methyl pivalate (AJH-836), and (E)-(2-(hydroxymethyl)-4-(4-nitrobenzylidene)-5-oxotetrahydrofuran-2-yl)methyl 4-(dimethylamino)benzoate (130C037), on Munc13-1 activation using the ligand-induced membrane translocation assay. JH-131e-153 showed higher activation than AJH-836, and 130C037 was not able to activate Munc13-1. To understand the role of the ligand-binding site residues in the activation process, three alanine mutants were generated. For AJH-836, the order of activation was wild-type (WT) Munc13-1 > R592A > W588A > I590A. For JH-131e-153, the order of activation was WT > I590 ≈ R592A ≈ W588A. Overall, the Z isomer of DAG-lactones showed higher potency than the E isomer and Trp-588, Ile-590, and Arg-592 were important for its binding. When comparing the activation of Munc13-1 and PKC, the order of activation for JH-131e-153 was PKCα > Munc13-1 > PKCε and for AJH-836, the order of activation was PKCε > PKCα > Munc13-1. Molecular docking supported higher binding of JH-131e-153 than AJH-836 with the Munc13-1 C1 domain. Our results suggest that DAG-lactones have the potential to modulate neuronal processes via Munc13-1 and can be further developed for therapeutic intervention for neurodegenerative diseases.

Targeted regulation of TAK1 counteracts dystrophinopathy in a DMD mouse model.

Muscular dystrophies make up a group of genetic neuromuscular disorders that involve severe muscle wasting. TGF-ß-activated kinase 1 (TAK1) is an important signaling protein that regulates cell survival, growth, and inflammation. TAK1 has been recently found to promote myofiber growth in the skeletal muscle of adult mice. However, the role of TAK1 in muscle diseases remains poorly understood. In the present study, we have investigated how TAK1 affects the progression of dystrophic phenotype in the mdx mouse model of Duchenne muscular dystrophy (DMD). TAK1 is highly activated in the dystrophic muscle of mdx mice during the peak necrotic phase. While targeted inducible inactivation of TAK1 inhibits myofiber injury in young mdx mice, it results in reduced muscle mass and contractile function. TAK1 inactivation also causes loss of muscle mass in adult mdx mice. By contrast, forced activation of TAK1 through overexpression of TAK1 and TAB1 induces myofiber growth without having any deleterious effect on muscle histopathology. Collectively, our results suggest that TAK1 is a positive regulator of skeletal muscle mass and that targeted regulation of TAK1 can suppress myonecrosis and ameliorate disease progression in DMD.

Comparison of the ligand binding site of C1 domains: a molecular dynamics simulation study of the C1 domain-phorbol 13-acetate-membrane system.

C1 domains are lipid-binding structural units of about 50 residues. Typical C1 domains associate with the plasma membrane and bind to diacylglycerol/phorbol ester during the activation of the proteins containing these domains. Although the overall structure of the C1 domains are similar, there are differences in their primary sequence and in the orientation of the ligand/lipid binding residues. To gain structural insights into the ligand/lipid binding, we performed molecular docking of phorbol 13-acetate into the C1 domain and 1.0 µs molecular dynamics simulation on the C1 domain-ligand-lipid ternary system for PKCθ C1A, PKCδ C1B, PKCßII C1B, PKCθ C1B, Munc13-1 C1, and ßII-Chimaerin C1. We divided these C1 domains into three types based on the orientations of Gln-27 and Trp/Tyr-22. In type 1, Trp/Tyr-22 is outside and Gln-27 is inside the ligand binding pocket. In type 2, both Trp/Tyr-22 and Gln-27 are outside the ligand binding pocket, and in type 3, Trp/Tyr-22 is inside and Gln-27 is outside the pocket. The type 1 C1 domains showed higher ligand binding and higher membrane binding with a shorter distance between the C1 domain and the membrane than the type 2 and type 3. For ligand binding, Pro-11 plays a major role in the type 1 and 2, and Gly-23 in the type 1 and type 3 C1 domains. This study elucidates the role of Gln-27, Trp-22, Pro-11 and Gly-23 in ligand/lipid binding in typical C1 domains and bears significance in developing selective modulators of C1 domain-containing proteins.Communicated by Ramaswamy H. Sarma.

Targeted ablation of Fn14 receptor improves exercise capacity and inhibits neurogenic muscle atrophy.

Skeletal muscle atrophy is a prevalent complication in multiple chronic diseases and disuse conditions. Fibroblast growth factor-inducible 14 (Fn14) is a member of the TNF receptor superfamily and a bona fide receptor of the TWEAK cytokine. Accumulating evidence suggests that Fn14 levels are increased in catabolic conditions as well as during exercise. However, the role of Fn14 in the regulation of skeletal muscle mass and function remains poorly understood. In this study, through the generation of novel skeletal muscle-specific Fn14-knockout mice, we have investigated the muscle role of Fn14 in the regulation of exercise capacity and denervation-induced muscle atrophy. Our results demonstrate that there was no difference in skeletal muscle mass between control and muscle-specific Fn14-knockout mice. Nevertheless, the deletion of Fn14 in skeletal muscle significantly improved exercise capacity and resistance to fatigue. This effect of Fn14 deletion is associated with an increased proportion of oxidative myofibers and higher capillaries number per myofiber in skeletal muscle. Furthermore, our results demonstrate that targeted deletion of Fn14 inhibits denervation-induced muscle atrophy in adult mice. Deletion of Fn14 reduced the expression of components of the ubiquitin-proteasome system and non-canonical NF-kappa B signaling in denervated skeletal muscle, as well as increased the phosphorylation of Akt kinase and FoxO3a transcription factor. Collectively, our results demonstrate that targeted inhibition of Fn14 improves exercise tolerance and inhibits denervation-induced muscle atrophy in adult mice.