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In JoVE (1)
Other Publications (3)
Articles by Andrea L. Ambrosio in JoVE
JoVE General
In vivo and in vitro Studies of Adaptor-clathrin Interaction
Department of Biochemistry and Molecular Biology, Colorado State University
Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.
Other articles by Andrea L. Ambrosio on PubMed
Isolation of Two Novel Mannan- and L-fucose-binding Lectins from the Green Alga Enteromorpha Prolifera: Biochemical Characterization of EPL-2
EPL-1 and EPL-2 represent lectins isolated from the green alga Enteromorpha prolifera. Both lectins are 20- to 22-kDa single-chain, nonglycosylated proteins. N-terminal sequence analysis of peptides representing over 70% of their primary structures shows that EPL-1 and EPL-2 represent novel proteins. Sedimentation-diffusion equilibrium experiments showed that EPL-1 and EPL-2 had average apparent molecular masses of 60000+/-6000 Da (EPL-1) and 59500+/-3000 Da (EPL-2), indicating that EPL-1 and EPL-2 have a tendency to self-associate into higher order aggregates, possibly homodimers and homotetramers, in equilibrium. The carbohydrate-binding specificity of EPL-2 was studied by enzyme-linked lectin assay and intrinsic fluorescence measurements. The results show that the combining site of EPL-2 is capable of accommodating both D-mannose and L-fucose, which share the conformation of the hydroxyl groups at positions 2 (axial) and 4 (equatorial), and includes subsites for the substituents at O1 and for branched mannose residues.
Embryonic Dorsal-ventral Signaling: Secreted Frizzled-related Proteins As Inhibitors of Tolloid Proteinases
Here we report an unexpected role for the secreted Frizzled-related protein (sFRP) Sizzled/Ogon as an inhibitor of the extracellular proteolytic reaction that controls BMP signaling during Xenopus gastrulation. Microinjection experiments suggest that the Frizzled domain of Sizzled regulates the activity of Xolloid-related (Xlr), a metalloproteinase that degrades Chordin, through the following molecular pathway: Szl -| Xlr -| Chd -| BMP --> P-Smad1 --> Szl. In biochemical assays, the Xlr proteinase has similar affinities for its endogenous substrate Chordin and for its competitive inhibitor Sizzled, which is resistant to enzyme digestion. Extracellular levels of Sizzled and Chordin in the gastrula embryo and enzyme reaction constants were all in the 10(-8) M range, consistent with a physiological role in the regulation of dorsal-ventral patterning. Sizzled is also a natural inhibitor of BMP1, a Tolloid metalloproteinase of medical interest. Furthermore, mouse sFRP2 inhibited Xlr, suggesting a wider role for this molecular mechanism.
Crossveinless-2 Is a BMP Feedback Inhibitor That Binds Chordin/BMP to Regulate Xenopus Embryonic Patterning
Vertebrate Crossveinless-2 (CV2) is a secreted protein that can potentiate or antagonize BMP signaling. Through embryological and biochemical experiments we find that: (1) CV2 functions as a BMP4 feedback inhibitor in ventral regions of the Xenopus embryo; (2) CV2 complexes with Twisted gastrulation and BMP4; (3) CV2 is not a substrate for tolloid proteinases; (4) CV2 binds to purified Chordin protein with high affinity (K(D) in the 1 nM range); (5) CV2 binds even more strongly to Chordin proteolytic fragments resulting from Tolloid digestion or to full-length Chordin/BMP complexes; (6) CV2 depletion causes the Xenopus embryo to become hypersensitive to the anti-BMP effects of Chordin overexpression or tolloid inhibition. We propose that the CV2/Chordin interaction may help coordinate BMP diffusion to the ventral side of the embryo, ensuring that BMPs liberated from Chordin inhibition by tolloid proteolysis cause peak signaling levels.
