The MAP1B-LC1/UBE2L3 complex catalyzes degradation of cell surface CaV2.2 channels

We reported recently a new mechanism by which the neuronal N-type Ca²⁺ (Ca_V2.2) channel expression may be regulated by ubiquitination. This mechanism involves the interaction between the channel and the light chain (LC1) of the microtubule associated protein B (MAP1B). We also showed that MAP1B-LC1 could interact with the ubiquitin-conjugating E2 enzyme UBE2L3 and that the ubiquitination/degradation mechanism triggered by MAP1B-LC1 could be prevented by inhibiting the ubiquitin-proteasome proteolytic pathway. We now report that MAP1B-LC1 can interact with the 2 main variants of the Ca_V2.2 channels (Ca_V2.2e37a and Ca_V2.2e37b) and that the MAP1B-LC1-mediated regulation most likely involves an internalization of the channels via a dynamin and clathrin-dependent pathway. In addition, here we propose that this novel mechanism of Ca_V channel regulation might be conserved among N-type and P/Q-type channels.     

Mining the human autoantibody repertoire: Isolation of potent IL17A-neutralizing monoclonal antibodies from a patient with thymoma

Anti-cytokine autoantibodies have been widely reported to be present in human plasma, both in healthy subjects and in patients with underlying autoimmune conditions, such as autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) or thymic epithelial neoplasms. While often asymptomatic, they can cause or facilitate a wide range of diseases including opportunistic infections. The potential therapeutic value of specific neutralizing anti-cytokine autoantibodies has not been thoroughly investigated. Here we used mammalian cell display to isolate IL17A-specific antibodies from a thymoma patient with proven high-titer autoantibodies against the same. We identified 3 distinct clonotypes that efficiently neutralized IL17A in a cell-based in vitro assay. Their potencies were comparable to those of known neutralizing antibodies, including 2, AIN457 (secukinumab) and ixekizumab that are currently in clinical development for the treatment of various inflammatory disorders. These data clearly demonstrate that the human autoantibody repertoire can be mined for antibodies with high therapeutic potential for clinical development.     

Neurotrophic Requirements of Human Motor Neurons Defined Using Amplified and Purified Stem Cell-Derived Cultures

Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC₅₀ 1–2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.     

Calcium interacts with antifreeze proteins and chitinase from cold-acclimated winter rye

During cold acclimation, winter rye (Secale cereale) plants accumulate pathogenesis-related proteins that are also antifreeze proteins (AFPs) because they adsorb onto ice and inhibit its growth. Although they promote winter survival in planta, these dual-function AFPs proteins lose activity when stored at subzero temperatures in vitro, so we examined their stability in solutions containing CaCl2, MgCl2, or NaCl. Antifreeze activity was unaffected by salts before freezing, but decreased after freezing and thawing in CaCl2 and was recovered by adding a chelator. Ca2+ enhanced chitinase activity 3- to 5-fold in unfrozen samples, although hydrolytic activity also decreased after freezing and thawing in CaCl2. Native PAGE, circular dichroism, and Trp fluorescence experiments showed that the AFPs partially unfold after freezing and thawing, but they fold more compactly or aggregate in CaCl2. Ruthenium red, which binds to Ca(2+)-binding sites, readily stained AFPs in the absence of Ca2+, but less stain was visible after freezing and thawing AFPs in CaCl2. We conclude that the structure of AFPs changes during freezing and thawing, creating new Ca(2+)-binding sites. Once Ca2+ binds to those sites, antifreeze activity, chitinase activity and ruthenium red binding are all inhibited. Because free Ca2+ concentrations are typically low in the apoplast, antifreeze activity is probably stable to freezing and thawing in planta. Ca2+ may regulate chitinase activity if concentrations are increased locally by release from pectin or interaction with Ca(2+)-binding proteins. Furthermore, antifreeze activity can be easily maintained in vitro by including a chelator during frozen storage.     

Genetic evidence for the requirement of the endocytic pathway in the uptake of coenzyme Q6 in Saccharomyces cerevisiae

Coenzyme Q is an isoprenylated benzoquinone lipid that functions in respiratory electron transport and as a lipid antioxidant. Dietary supplementation with Q is increasingly used as a therapeutic for treatment of mitochondrial and neurodegenerative diseases, yet little is known regarding the mechanism of its uptake. As opposed to other yeast backgrounds, EG103 strains are unable to import exogenous Q₆ to the mitochondria. Furthermore, the distribution of exogenous Q₆ among endomembranes suggests an impairment of the membrane traffic at the level of the endocytic pathway. This fact was confirmed after the detection of defects in the incorporation of FM4-64 marker and CPY delivery to the vacuole. A similar effect was demonstrated in double mutant strains in Q₆ synthesis and several steps of endocytic process; those cells are unable to uptake exogenous Q₆ to the mitochondria and restore the growth on non-fermentable carbon sources. Additional data about the positive effect of peptone presence for exogenous Q₆ uptake support the hypothesis that Q₆ is transported to mitochondria through an endocytic-based system.     

DNA vaccination can break immunological tolerance to PrP in wild-type mice and attenuates prion disease after intracerebral challenge

Transmissible spongiform encephalopathies (TSEs) can be ameliorated by prion protein (PrP)-specific antibodies, but active immunization is complicated by immune tolerance to the normal cellular host protein (PrP(C)). Here, we show that DNA immunization of wild-type mice can break immune tolerance against the prion protein, resulting in the induction of PrP-specific antibody and T-cell responses. PrP immunogenicity was increased by fusion to the lysosomal targeting signal from LIMPII (lysosomal integral membrane protein type II). Although mice immunized with a PrP-LIMPII DNA vaccine showed a dramatic delay in the onset of early disease signs after intracerebral challenge, immunization against PrP also had some deleterious effects. These results clearly confirm the feasibility of using active immunization to protect against TSEs and, in the absence of effective treatments, indicate a suitable alternative for combating the spread of these diseases.     

Structural and kinetic analysis of two covalent sialosyl-enzyme intermediates on Trypanosoma rangeli sialidase

Trypanosoma rangeli sialidase is a glycoside hydrolase (family GH33) that catalyzes the cleavage of alpha-2-->3-linked sialic acid residues from sialoglycoconjugates with overall retention of anomeric configuration. Retaining glycosidases usually operate through a ping-pong mechanism, wherein a covalent intermediate is formed between the carbohydrate and an active site carboxylic acid of the enzyme. Sialidases, instead, appear to use a tyrosine as the catalytic nucleophile, leaving the possibility of an essentially different catalytic mechanism. Indeed, a direct nucleophilic role for a tyrosine was shown for the homologous trans-sialidase from Trypanosoma cruzi, although itself not a typical sialidase. Here we present the three-dimensional structures of the covalent glycosyl-enzyme complexes formed by the T. rangeli sialidase with two different mechanism-based inactivators at 1.9 and 1.7 Angstroms resolution. To our knowledge, these are the first reported structures of enzymatically competent covalent intermediates for a strictly hydrolytic sialidase. Kinetic analyses have been carried out on the formation and turnover of both intermediates, showing that structural modifications to these inactivators can be used to modify the lifetimes of covalent intermediates. These results provide further evidence that all sialidases likely operate through a similar mechanism involving the transient formation of a covalently sialylated enzyme. Furthermore, we believe that the ability to "tune" the inactivation and reactivation rates of mechanism-based inactivators toward specific enzymes represents an important step toward developing this class of inactivators into therapeutically useful compounds.     

Trypanosoma rangeli sialidase: cloning, expression and similarity to T.cruzi trans-sialidase

Sialidases are hydrolytic enzymes present from virus to highereukaryotes, catalyzing the removal of sialic acid from glycoconjugates.Some protozoa Trypanosomatidae secrete high levels of sialidaseinto the medium. We have now purified the secreted sialidasefrom Trypanosoma rangeli Its N-terminal sequence reveals 100%identity with the corresponding region of the trans-sialidasefrom T.cruzi Trans-sialidase, although homologous to viral andbacterial sialidases, displays a novel sialyltransferase activityand is involved in host cell invasion. Several homologous trans-sialidase-likegenes were cloned from genomic DNA of T.rangeli, and groupedin three subfamilies. Active siali-dase-encoding genes werefound in one of them. The re-combinant sialidase shows similarproperties to those of the native enzyme, including undetectabletrans-sialidase activity. Nevertheless, it has an overall identityof 68.9% with the catalytic domain of T.cruzi trans-sialidase,increasing to 86.7% admitting conservative substitutions. Onlythree other eukaryotic sialidases have been previously cloned,none of them showing significant homology to trans-sialidase.The isolation of a highly similar sialidase is relevant to furtheridentify the molecular determinants allowing trans-sialidaseactivity. As a first approach, chimeric constructs between sialidaseand trans-sialidase were generated, one of them rendering asialidase with three times lower K_m than the natural enzyme. eukaryotic sialidase gene family glycosidase parasite sialic acid