Structural analysis of human FANCL, the E3 ligase in the Fanconi anemia pathway

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links. At the heart of this pathway is the monoubiquitination of the FANCI-FANCD2 (ID) complex by the multiprotein "core complex" containing the E3 ubiquitin ligase FANCL. Vertebrate organisms have the eight-protein core complex, whereas invertebrates apparently do not. We report here the structure of the central domain of human FANCL in comparison with the recently solved Drosophila melanogaster FANCL. Our data represent the first structural detail into the catalytic core of the human system and reveal that the central fold of FANCL is conserved between species. However, there are macromolecular differences between the FANCL proteins that may account for the apparent distinctions in core complex requirements between the vertebrate and invertebrate FA pathways. In addition, we characterize the binding of human FANCL with its partners, Ube2t, FANCD2, and FANCI. Mutational analysis reveals which residues are required for substrate binding, and we also show the domain required for E2 binding.     

Determination of the new anthelmintic monepantel and its sulfone metabolite in milk and muscle using a UHPLC-MS/MS and QuEChERS method

This is the first paper to report a method for the detection of the new anthelmintic monepantel and its sulfone metabolite in goat's milk and ovine muscle. Samples were extracted and purified using a modified QuEChERS method. A concentration step was included when analyzing in the low μg kg(-1) range. Analysis was carried out by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in a 13min run time using atmospheric pressure electrospray ionisation in the negative mode (ESI(-)) and multiple reaction monitoring (MRM) scanning. Monepantel (m/z 472) and monepantel-sulfone (m/z 504) both had product ions at m/z 186 and m/z 166. The method has been single-laboratory validated according to the 2002/657/EC guidelines. The mean recovery in milk was 108 and 106% for monepantel and monepantel-sulfone, respectively. The mean recovery in muscle was 109 and 108% for monepantel and monepantel-sulfone, respectively. The coefficients of variation for the within laboratory repeatability and reproducibility were ≤6.4% in milk and ≤14.2% in muscle. The decision limits (CCα) in milk were 2.20 and 2.08 μg kg(-1) for monepantel and monepantel-sulfone, respectively. The decision limits (CCα) in muscle were 771 and 746 μg kg(-1) for monepantel and monepantel-sulfone, respectively.     

The autophagy regulator Rubicon is a feedback inhibitor of CARD9-mediated host innate immunity

Assembly of a scaffold consisting of CARD9, BCL10, and MALT1 (CBM complex) is critical for effective signaling by multiple pattern recognition receptors (PRRs) including Dectin and RIG-I. The RUN domain Beclin-1-interacting cysteine-rich-containing Rubicon protein associates constitutively with the Beclin-UVRAG-Vps34 complex under normal conditions to regulate autophagy. Rubicon also interacts with the phagocytic NADPH-oxidase complex upon TLR stimulation to induce potent antimicrobial responses. Here, we show Rubicon is a physiological feedback inhibitor of CBM-mediated PRR signaling, preventing unbalanced proinflammatory responses. Upon Dectin-1- or RIG-I-mediated activation, Rubicon dynamically exchanges binding partners from 14-3-3β to CARD9 in a stimulation-specific and phosphorylation-dependent manner, disassembling the CBM signaling complex and ultimately terminating PRR-induced cytokine production. Remarkably, Rubicon's actions in the autophagy complex, phagocytosis complex, and CBM complex are functionally and genetically separable. Rubicon thus differentially targets signaling complexes, depending on environmental stimuli, and may function to coordinate various immune responses against microbial infection.     

Hypothalamic Fkbp51 is induced by fasting, and elevated hypothalamic expression promotes obese phenotypes

To discover hypothalamic genes that might play a role in regulating energy balance, we carried out a microarray screen for genes induced by a 48-h fast in male C57Bl/6J mouse hypothalamus. One such gene was Fkbp51 (FK506 binding protein 5; Locus NP_034350). The product of this gene is of interest because it blocks glucocorticoid action, suggesting that fasting-induced elevation of this gene in the hypothalamus may reduce glucocorticoid negative feedback, leading to elevated glucocorticoid levels, thus promoting obese phenotypes. Subsequent analysis demonstrated that a 48-h fast induces Fkbp51 in ventromedial, paraventricular, and arcuate hypothalamic nuclei of mice and rats. To assess if hypothalamic Fkbp51 promotes obesity, the gene was transferred to the hypothalamus via an adeno-associated virus vector. Within 2 wk following Fkbp51 overexpression, mice on a high-fat diet exhibited elevated body weight, without hyperphagia, relative to mice receiving the control mCherry vector. Body weight remained elevated for more than 8 wk and was associated with elevated corticosterone and impaired glucose tolerance. These studies suggest that elevated hypothalamic Fkbp51 promotes obese phenotypes.