Kristin C. Gunsalus, Mike Boxem, David Drechsel, Maxime Devos, Kevin Drew, Lorenzo Delichtervelde, Zoltan Maliga, Na Li, Joram Mul, Niels J. Klitgord, Anthony A. Hyman, Sander Van Den Heuvel, Fabio Piano, Richard Bonneau, Mike Tipsworth, David E. Hill, Marc Vidal
[
International Worm Meeting,
2007]
Macromolecular interactions, such as protein interactions, form the basis of most biological activity. The identification of protein interaction networks leads to many novel hypotheses on the functioning of individual components, and a global understanding of their roles in vivo. However, the modular design of most proteins suggests that mapping of protein interactions at the domain level might provide deeper insights by addressing intramolecular regulation and mapping regions of binary interaction. For example, can two interactions take place simultaneously, or are they mutually exclusive because they are mediated by the same domain? C. elegans early embryogenesis forms an ideal test case for a systems approach aimed at increasing our understanding of multicellular development. The proteins involved are phenotypically related, and are likely to share many interactions between them. We used a novel application of the yeast two-hybrid system to identify the interactions involving early embryogenesis proteins and define the domains mediating these interactions. We screened 705 full length bait proteins against two libraries: a traditional cDNA library and a newly generated fragment library, which contains overlapping fragments of different sizes (average 40 fragments plus full length) of 748 early embryogenesis genes. The use of this library allowed us to identify the minimal fragments mediating interactions. Furthermore, by testing multiple fragments of each protein, we significantly increased the number of interactions that could be detected. We identified 742 interactions, of which 371 were found using the fragment library, 296 using the cDNA library, and 75 in both libraries. 446 interactions were between early embryogenesis proteins, expanding the number of such interactions from 127 to 556. Of the interactions found using the fragment library, 60% defined a minimal interaction domain smaller than half of the full length protein size, indicating that we effectively identify distinct interaction domains. We will present a detailed analysis of the interaction network generated.
[
FASEB J,
2007]
Nna1 has some sequence similarity to metallocarboxypeptidases, but the biochemical characterization of Nna1 has not previously been reported. In this work we performed a detailed genomic scan and found >100 Nna1 homologues in bacteria, Protista, and Animalia, including several paralogs in most eukaryotic species. Phylogenetic analysis of the Nna1-like sequences demonstrates a major divergence between Nna1-like peptidases and the previously known metallocarboxypeptidases subfamilies: M14A, M14B, and M14C. Conformational modeling of representative Nna1-like proteins from a variety of species indicates an unusually open active site, a property that might facilitate its action on a wide variety of peptide and protein substrates. To test this, we expressed a recombinant form of one of the Nna1-like peptidases from Caenorhabditis elegans and demonstrated that this protein is a fully functional metallocarboxypeptidase that cleaves a range of C-terminal amino acids from synthetic peptides. The enzymatic activity is activated by ATP/ADP and salt-inactivated, and is preferentially inhibited by Z-Glu-Tyr dipeptide, which is without precedent in metallocarboxypeptidases and resembles tubulin carboxypeptidase functioning; this hypothesis is strongly reinforced by the results depicted in Kalinina et al. published as accompanying paper in this journal (1). Our findings demonstrate that the M14 family of metallocarboxypeptidases is more complex and diverse than expected, and that Nna1-like peptidases are functional variants of such enzymes, representing a novel subfamily (we propose the name M14D) that contributes substantially to such diversity.--Rodriguez de la Vega, M., Sevilla, R. G., Hermoso, A., Lorenzo, J., Tanco, S., Diez, A., Fricker, L. D., Bautista, J. M., Aviles, F. X. Nna1-like proteins are active metallocarboxypeptidases of a new and diverse M14 subfamily.