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Otago Medical School staff profiles

Dr Adam Middleton

PositionResearch Fellow
DepartmentDepartment of Biochemistry
QualificationsPhD
Research summaryProtein Biochemistry, Structural Biology, Apoptosis

Research

Transfer of the small protein ubiquitin to substrates underlies almost all aspects of eukaryotic cells. Maintaining the balance between the addition and removal of ubiquitin is critical, and disruptions to transfer are associated with many diseases, such as cancer and neurological disorders. Importantly, ubiquitin can be added to a substrate protein as a single molecule, or it can modify itself and form ubiquitin chains. This code or language directs the fate of the substrate proteins.

In our lab we investigate the regulation of ubiquitin transfer by E2 enzymes and E3 ligases. To perform these studies, the laboratory employs ubiquitin transfer assays, binding studies, X-ray crystallography, and other biophysical techniques.

How are different ubiquitin chains produced?

Ubiquitin molecules bound to E2

Ubiquitin chains linked by Lys48 direct substrate proteins to be degraded by the proteasome, and have a critical role in regulating protein abundance. The central players in the ubiquitylation cascade are the E2 enzymes, and they typically determine the type of ubiquitin chain built. For construction of a ubiquitin chain, the E2 enzyme must bind to at least two ubiquitin moieties: the donor ubiquitin (UbD) and the acceptor ubiquitin (UbA) (see model in figure). We want to understand more about how E2 enzymes interact with UbD and UbA, and how this binding can specify the chain topology. One of our approaches is to use ubiquitin variants (UbVs) - which are modified versions of ubiquitin - that can bind tightly to enzymes of the ubiquitin cascade.

To date, 100+ UbVs have been isolated that bind to various parts of the ubiquitin machinery. Our current goal is to assess the influence of the UbVs on the in vitro activity of the E2 an E3 enzymes. We are employing high-throughput techniques in order to effectively analyse the relatively large number of UbVs isolated. Those that are of interest those that either inhibit or accelerate ubiquitin transfer are being assessed in cell-based systems.

Can E2s produce mixed chains?

Some E2 enzymes only produce chains with one topology (homotypic chains). When E2 enzymes are elongating ubiquitin chains, do they have a mechanism to ensure they are using the right kind of a chain as a template? If so, there may be a UbA-1 binding site that is linkage-type specific. An analysis of all the crystal structures of E2~Ub conjugates in the PDB (https://www.rcsb.org/) has allowed us to map some potential binding sites for UbA and a putative UbA-1 site. These need to be experimentally verified by site-directed mutagenesis and sensitive ubiquitin transfer assays. Together, these two sites could provide some E2s with the ability to only elongate particular chain types.

Ubiquitin molecules bound to E2

Publications

Budhidarmo, R., Zhu, J., Middleton, A. J., & Day, C. L. (2018). The RING domain of RING Finger 11 (RNF11) protein binds Ubc13 and inhibits formation of polyubiquitin chains. FEBS Letters. Advance online publication. doi: 10.1002/1873-3468.13029

Foglizzo, M., Middleton, A. J., Burgess, A. E., Crowther, J. M., Dobson, R. C. J., Murphy, J. M., Day, C. L., & Mace, P. D. (2018). A bidentate Polycomb Repressive-Deubiquitinase complex is required for efficient activity on nucleosomes. Nature Communications, 9, 3932. doi: 10.1038/s41467-018-06186-1

Groves, M. R., Schroer, C. F. E., Middleton, A. J., Lunev, S., Danda, N., Ali, A. M., … Williams, C. (2018). Structural insights into K48-linked ubiquitin chain formation by the Pex4p-Pex22p complex. Biochemical & Biophysical Research Communications, 496(2), 562-567. doi: 10.1016/j.bbrc.2017.12.150

Middleton, A. J., Budhidarmo, R., Das, A., Zhu, J., Foglizzo, M., Mace, P. D., & Day, C. L. (2017). The activity of TRAF RING homo- and heterodimers is regulated by zinc finger 1. Nature Communications, 8, 1788. doi: 10.1038/s41467-017-01665-3

Middleton, A. J., Wright, J. D., & Day, C. L. (2017). Regulation of E2s: A role for additional ubiquitin binding sites? Journal of Molecular Biology, 429(22), 3430-3440. doi: 10.1016/j.jmb.2017.06.008

Chapter in Book - Research

Middleton, A. J., Budhidarmo, R., & Day, C. L. (2014). Use of E2~ubiquitin conjugates for the characterization of ubiquitin transfer by RING E3 ligases such as the inhibitor of apoptosis proteins. In A. Ashkenazi, J. A. Wells & J. Yuan (Eds.), Methods in enzymology (Vol. 545). (pp. 243-263). San Diego, CA: Elsevier. doi: 10.1016/B978-0-12-801430-1.00010-X

Middleton, A. J., Vanderbeld, B., Bredow, M., Tomalty, H., Davies, P. L., & Walker, V. K. (2014). Isolation and characterization of ice-binding proteins from higher plants. In D. K. Hincha & E. Zuther (Eds.), Plant cold acclimation: Methods and Protocols (Methods in Molecular Biology: Vol. 1166). (pp. 255-277). New York: Springer. doi: 10.1007/978-1-4939-0844-8_19

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Journal - Research Article

Budhidarmo, R., Zhu, J., Middleton, A. J., & Day, C. L. (2018). The RING domain of RING Finger 11 (RNF11) protein binds Ubc13 and inhibits formation of polyubiquitin chains. FEBS Letters. Advance online publication. doi: 10.1002/1873-3468.13029

Groves, M. R., Schroer, C. F. E., Middleton, A. J., Lunev, S., Danda, N., Ali, A. M., … Williams, C. (2018). Structural insights into K48-linked ubiquitin chain formation by the Pex4p-Pex22p complex. Biochemical & Biophysical Research Communications, 496(2), 562-567. doi: 10.1016/j.bbrc.2017.12.150

Foglizzo, M., Middleton, A. J., Burgess, A. E., Crowther, J. M., Dobson, R. C. J., Murphy, J. M., Day, C. L., & Mace, P. D. (2018). A bidentate Polycomb Repressive-Deubiquitinase complex is required for efficient activity on nucleosomes. Nature Communications, 9, 3932. doi: 10.1038/s41467-018-06186-1

Middleton, A. J., Budhidarmo, R., Das, A., Zhu, J., Foglizzo, M., Mace, P. D., & Day, C. L. (2017). The activity of TRAF RING homo- and heterodimers is regulated by zinc finger 1. Nature Communications, 8, 1788. doi: 10.1038/s41467-017-01665-3

Middleton, A. J., Wright, J. D., & Day, C. L. (2017). Regulation of E2s: A role for additional ubiquitin binding sites? Journal of Molecular Biology, 429(22), 3430-3440. doi: 10.1016/j.jmb.2017.06.008

Foglizzo, M., Middleton, A. J., & Day, C. L. (2016). Structure and function of the RING domains of RNF20 and RNF40, dimeric E3 ligases that monoubiquitylate histone H2B. Journal of Molecular Biology, 428(20), 4073-4086. doi: 10.1016/j.jmb.2016.07.025

Middleton, A. J., & Day, C. L. (2015). The molecular basis of lysine 48 ubiquitin chain synthesis by Ube2K. Scientific Reports, 5, 16793. doi: 10.1038/srep16793

Middleton, A. J., Marshall, C. B., Faucher, F., Bar-Dolev, M., Braslavsky, I., Campbell, R. L., … Davies, P. L. (2012). Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site. Journal of Molecular Biology, 416, 713-724. doi: 10.1016/j.jmb.2012.01.032

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