Two critical transitions in mitosis are regulated by targeted proteolysis. For entry into anaphase, the proteins blocking sister chromatid separation must be destroyed. For exit from telophase, cyclins in the mitiotic cyclin/cdk complexes must be destroyed. The destruction occurs through the ubiquitin/proteasome system. Three types of enzymes, designated E1, E2, and E3 function sequentially to attach polyubiquitin chains to the targeted proteins. The proteasome recognized these chains and hydrolyzes the covalently attached protein. The long-term objective of our research is to understand at a structural level the regulation of mitotic progression by targeted proteolysis.
The specific aims are to determine the x-ray crystal structures of essential components in this system and to correlate the structures to the rapidly growing body of relevant biochemical and genetic data. The insights gained from this work will provide a valuable framework for proposing further biochemical, biophysical and genetic experiments. The particular components we are studying are: 1) the calm E2 enzyme (E2C) involved in the destruction events leading both to entry into anaphase and exit from mitosis. We already have solved the structure to 2.0 A and are in the final stages of refinement. 2) the human homolog of E2C, UbcH10. Comparison of UbcH10 and E2C with each other and with the known non-cell cycle E2 structures will be instrumental in delineating the structural features important for functional specificity. 3) the N- terminal fragment of cyclin B. This fragment contains the structural features necessary to elicit mitotic-specific destruction. 4) an analog of the ubiquitin-E2 catalytic thiol ester intermediate. X-ray analysis will provide structural insight into the molecular species that interacts with the E3 ligase and/or target protein. This work represents the first, necessary steps for understanding at a structural level the pivotal proteolytic events in cell cycle control. This work also will have broader ramifications. Targeted proteolysis by the ubiquitin/proteasome system has proven to be an essential regulation mechanism in diverse areas of cellular function and has been implicated in common virus-induced cancers. The proposed project will contribute to the understanding of these diverse processes by helping to elucidate the common proteolytic mechanism.
| Petri, Edward T; Errico, Alessia; Escobedo, Lourdes et al. (2007) The crystal structure of human cyclin B. Cell Cycle 6:1342-9 |
| Tolbert, Blanton S; Tajc, Stephen G; Webb, Helen et al. (2005) The active site cysteine of ubiquitin-conjugating enzymes has a significantly elevated pKa: functional implications. Biochemistry 44:16385-91 |
