A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for 13C labeling of the C-terminal domain of angiopoietin-2, 15N labeling of ubiquitin and for 2H/13C/15N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.

A common single-nucleotide polymorphism in the human brain-derived neurotrophic factor (BDNF) gene results in a Val66Met substitution in the BDNF prodomain region. This single-nucleotide polymorphism is associated with alterations in memory and with enhanced risk to develop depression and anxiety disorders in humans. Here we show that the isolated BDNF prodomain is detected in the hippocampus and that it can be secreted from neurons in an activity-dependent manner. Using nuclear magnetic resonance spectroscopy and circular dichroism we find that the prodomain is intrinsically disordered, and the Val66Met substitution induces structural changes. Surprisingly, application of Met66 (but not Val66) BDNF prodomain induces acute growth cone retraction and a decrease in Rac activity in hippocampal neurons. Expression of p75NTR and differential engagement of the Met66 prodomain to the SorCS2 receptor are required for this effect. These results identify the Met66 prodomain as a new active ligand which modulates neuronal morphology.

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BACKGROUND AND PURPOSECardiolipin plays an important role in mitochondrial respiration and cardiolipin peroxidation is associated with age-related diseases. Hydrophobic interactions between cytochrome c and cardiolipin converts cytochrome c from an electron carrier to a peroxidase. In addition to cardiolipin peroxidation, this impedes electron flux and inhibits mitochondrial ATP synthesis. SS-31 (D-Arg-dimethylTyr-Lys-Phe-NH2) selectively binds to cardiolipin and inhibits cytochrome c peroxidase activity. Here, we examined whether SS-31 also protected the electron carrier function of cytochrome c. EXPERIMENTAL APPROACHInteractions of SS-31 with cardiolipin were studied using liposomes and bicelles containing phosphatidylcholine alone or with cardiolipin. Structural interactions were assessed by fluorescence spectroscopy, turbidity and nuclear magnetic resonance. Effects of cardiolipin on electron transfer kinetics of cytochrome c were determined by cytochrome c reduction in vitro and oxygen consumption using mitoplasts, frozen and fresh mitochondria.

SUMMARY The CARMA1/Bcl10/MALT1 (CBM) signalosome mediates antigen receptor-induced NF-κB signaling to regulate multiple lymphocyte functions. While CARMA1 and Bcl10 contain caspase recruitment domains (CARDs), MALT1 is a paracaspase with structural similarity to caspases. Here we show that the reconstituted CBM signalosome is a helical filamentous assembly in which substoichiometric CARMA1 nucleates Bcl10 filaments. Bcl10 filament formation is a highly cooperative process whose threshold is sensitized by oligomerized CARMA1 upon receptor activation. In cells, both cotransfected CARMA1/Bcl10 complex and the endogenous CBM signalosome are filamentous morphologically. Combining crystallography, nuclear magnetic resonance, and electron microscopy, we reveal the structure of the Bcl10 CARD filament and the mode of interaction between CARMA1 and Bcl10. Structure-guided mutagenesis confirmed the observed interfaces in Bcl10 filament assembly and MALT1 activation in vitro and NF-κB activation in cells. These data support a paradigm of nucleation-induced signal transduction with threshold response due to cooperativity and signal amplification by polymerization.