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and oncogenesis [1]. The common denominator of these processes is the initial C-terminal activation of ubiquitin The University of Alberta Edmonton, Alberta T6G 2H7 (Ub) by the activating enzyme (E1) followed by its subsequent transfer to a Ub-conjugating enzyme (E2) as a Canada 3 Department of Biochemistry and R.S. covalent E2-Ub thiolester intermediate. At this point, the mechanism of ubiquitination appears to diverge along McLaughlin Macromolecular Structure Facility either of two lines. In one case, Ub is transferred directly from the E2 to the lysine of a target protein in a reaction The University of Western Ontario London, Ontario N6A 5C1 that is facilitated by a Ub protein ligase (E3). In the other case, Ub is first transferred from the E2 to the active site Canada cysteine of an E3 as an E3-Ub thiolester intermediate, whereupon it is then transferred to the target protein. In either event, degradation of the target by the 26S Summary proteasome is facilitated by the assembly of a multi-Ub chain on the target in which the C terminus of each Ub Background: Ubiquitin-conjugating enzymes (E2s) are is linked to Lys48 (K48) of its neighbor. central enzymes involved in ubiquitin-mediated protein The ability of E2 proteins to orchestrate ubiquitination degradation. During this process, ubiquitin (Ub) and the through their interactions with Ub, E1, E3, and target E2 protein form an unstable E2-Ub thiolester intermediproteins makes them central players of the ubiquitin casate prior to the transfer of ubiquitin to an E3-ligase procade. All E2 proteins consist of a catalytic domain (150 tein and the labeling of a substrate for degradation. A residues) that includes the active site cysteine used to series of complex interactions occur among the target form the E2-Ub thiolester complex. Furthermore, X-ray substrate, ubiquitin, E2, and E3 in order to efficiently crystallographic structures of the catalytic domains from facilitate the transfer of the ubiquitin molecule. However, the E2 proteins Ubc1 (vide infra), Ubc2 [2], Ubc7 [3], and due to the inherent instability of the E2-Ub thiolester, Ubc9 [4] from Saccharomyces cerevisiae have shown the structural details of this complex intermediate are that this region is structurally conserved. Four ␣ helices not known. (␣1-␣4) essentially form one face of the protein, while a 4 strand antiparallel ␤ sheet (␤1-␤4) is found on the Results: A three-dimensional model of the E2-Ub thibackside of the enzyme between helices ␣1 and ␣2 in olester intermediate has been determined for the catathe sequence. The thiolester-forming cysteine is located lytic domain of the E2 protein Ubc1 (Ubc1 ⌬450 ) and ubion a relatively unstructured region (L2) ranging from 20 quitin from S. cerevisiae. The interface of the E2-Ub to 30 residues in length and linking ␤4 and ␣2. Recent intermediate was determined by kinetically monitoring X-ray crystallographic studies have shown that two thiolester formation by 1 H-15 N HSQC spectra by using structurally unrelated E3 proteins, E6AP and cCbl, intercombin...

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SUMMARY Several gastropod molluscs produce glues that are interesting because they are dilute gels and yet they produce strong adhesion. Specific glue proteins have been identified that play a central role in this adhesion, possibly by crosslinking other polymers in the gel. This study investigates the role of metals in the action of these glue proteins. Atomic absorption spectrometry showed that glue from the slug Arion subfuscus contains substantial quantities of zinc (46±7 p.p.m. and 189±80 p.p.m. in two different sets of experiments) and also iron, copper and manganese (2–7 p.p.m.). Iron-specific staining demonstrates that iron is bound specifically to the 15 kDa glue protein. Several approaches were used to show that these metals have important functional effects. Adding iron or copper to dissolved glue causes the proteins to precipitate rapidly, although zinc has no effect. Removing iron and related transition metals with a chelator during secretion of the glue causes a sixfold increase in the solubility of the glue. Once the glue has set, however, removing these metals has no effect. Finally, the gel-stiffening activity of the glue proteins was measured in the presence and absence of the chelator. The chelator eliminated the gel-stiffening effect of the proteins, suggesting that transition metals were necessary for the proteins to act on the gel. Thus, the glue contains transition metals and these metals play an essential role in glue function.

This study profiles the evidence base relative to inclusive employment for people with intellectual disabilities. The lack of evidence on the degree to which social inclusion is being achieved through community-based employment highlights a critical area requiring attention.