Comprehensively Surveying Structure and Function of RING Domains from Drosophila melanogaster

Using a complete set of RING domains from Drosophila melanogaster, all the solved RING domains and cocrystal structures of RING-containing ubiquitin-ligases (RING-E3) and ubiquitin-conjugating enzyme (E2) pairs, we analyzed RING domains structures from their primary to quarternary structures. The results showed that: i) putative orthologs of RING domains between Drosophila melanogaster and the human largely occur (118/139, 84.9%); ii) of the 118 orthologous pairs from Drosophila melanogaster and the human, 117 pairs (117/118, 99.2%) were found to retain entirely uniform domain architectures, only Iap2/Diap2 experienced evolutionary expansion of domain architecture; iii) 4 evolutionary structurally conserved regions (SCRs) are responsible for homologous folding of RING domains at the superfamily level; iv) besides the conserved Cys/His chelating zinc ions, 6 equivalent residues (4 hydrophobic and 2 polar residues) in the SCRs possess good-consensus and conservation- these 4 SCRs function in the structural positioning of 6 equivalent residues as determinants for RING-E3 catalysis; v) members of these RING proteins located nucleus, multiple subcellular compartments, membrane protein and mitochondrion are respectively 42 (42/139, 30.2%), 71 (71/139, 51.1%), 22 (22/139, 15.8%) and 4 (4/139, 2.9%); vi) CG15104 (Topors) and CG1134 (Mul1) in C3HC4, and CG3929 (Deltex) in C3H2C3 seem to display broader E2s binding profiles than other RING-E3s; vii) analyzing intermolecular interfaces of E2/RING-E3 complexes indicate that residues directly interacting with E2s are all from the SCRs in RING domains. Of the 6 residues, 2 hydrophobic ones contribute to constructing the conserved hydrophobic core, while the 2 hydrophobic and 2 polar residues directly participate in E2/RING-E3 interactions. Based on sequence and structural data, SCRs, conserved equivalent residues and features of intermolecular interfaces were extracted, highlighting the presence of a nucleus for RING domain fold and formation of catalytic core in which related residues and regions exhibit preferential evolutionary conservation

Novel Control of S Phase of the Cell Cycle by Ubiquitin-conjugating Enzyme H7

Timely degradation of regulatory proteins by the ubiquitin proteolytic pathway (UPP) is an established paradigm of cell cycle regulation during the G2/M and G1/S transitions. Less is known about roles for the UPP during S phase. Here we present evidence that dynamic cell cycle–dependent changes in levels of UbcH7 regulate entrance into and progression through S phase. In diverse cell lines, UbcH7 protein levels are dramatically reduced in S phase but are fully restored by G2. Knockdown of UbcH7 increases the proportion of cells in S phase and doubles the time to traverse S phase, whereas UbcH7 overexpression reduces the proportion of cells in S phase. These data suggest a role for UbcH7 targets in the completion of S phase and entry into G2. Notably, UbcH7 knockdown was coincident with elevated levels of the checkpoint kinase Chk1 but not Chk2. These results argue that UbcH7 promotes S phase progression to G2 by modulating the intra-S phase checkpoint mediated by Chk1. Furthermore, UbcH7 levels appear to be regulated by a UPP. Together the data identify novel roles for the UPP, specifically UbcH7 in the regulation of S phase transit time as well as in cell proliferation