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Contact Details

Phone
+64 3 479 7870
Email
adam.middleton@otago.ac.nz
Position
Senior Research Fellow
Department
Department of Biochemistry
Qualifications
PhD
Research summary
Protein 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 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.

Publications

Middleton, A. J., Barzak, F. M., Fokkens, T. J., Nguyen, K., & Day, C. L. (2023). Zinc finger 1 of the RING E3 ligase, RNF125, interacts with the E2 to enhance ubiquitylation. Structure. Advance online publication. doi: 10.1016/j.str.2023.07.007

Middleton, A. J., & Day, C. L. (2023). From seeds to trees: How E2 enzymes grow ubiquitin chains. Biochemical Society Transactions. Advance online publication. doi: 10.1042/BST20220880

Paluda, A., Middleton, A. J., Rossig, C., Mace, P. D., & Day, C. L. (2022). Ubiquitin and a charged loop regulate the ubiquitin E3 ligase activity of Ark2C. Nature Communications, 13, 1181. doi: 10.1038/s41467-022-28782-y

Middleton, A. J., Teyra, J., Zhu, J., Sidhu, S. S., & Day, C. L. (2021). Identification of ubiquitin variants that inhibit the E2 ubiquitin conjugating enzyme, Ube2k. ACS Chemical Biology, 16, 1745-1756. doi: 10.1021/acschembio.1c00445

Das, A., Middleton, A. J., Padala, P., Ledgerwood, E. C., Mace, P. D., & Day, C. L. (2021). The structure and ubiquitin binding properties of TRAF RING heterodimers. Journal of Molecular Biology, 433, 166844. doi: 10.1016/j.jmb.2021.166844

Bredow, M., Tomalty, H. E., Graham, L. A., Gruneberg, A. K., Middleton, A. J., Vanderbeld, B., … Walker, V. K. (2020). 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. 2156). (2nd ed.) (pp. 303-332). New York, NY: Humana Press. doi: 10.1007/978-1-0716-0660-5_21

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

Chapter in Book - Research

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

Chapter in Book - Research

Middleton, A. J., Barzak, F. M., Fokkens, T. J., Nguyen, K., & Day, C. L. (2023). Zinc finger 1 of the RING E3 ligase, RNF125, interacts with the E2 to enhance ubiquitylation. Structure. Advance online publication. doi: 10.1016/j.str.2023.07.007

Journal - Research Article

Paluda, A., Middleton, A. J., Rossig, C., Mace, P. D., & Day, C. L. (2022). Ubiquitin and a charged loop regulate the ubiquitin E3 ligase activity of Ark2C. Nature Communications, 13, 1181. doi: 10.1038/s41467-022-28782-y

Journal - Research Article

Das, A., Middleton, A. J., Padala, P., Ledgerwood, E. C., Mace, P. D., & Day, C. L. (2021). The structure and ubiquitin binding properties of TRAF RING heterodimers. Journal of Molecular Biology, 433, 166844. doi: 10.1016/j.jmb.2021.166844

Journal - Research Article

Middleton, A. J., Teyra, J., Zhu, J., Sidhu, S. S., & Day, C. L. (2021). Identification of ubiquitin variants that inhibit the E2 ubiquitin conjugating enzyme, Ube2k. ACS Chemical Biology, 16, 1745-1756. doi: 10.1021/acschembio.1c00445

Journal - Research Article

Middleton, A. J., Zhu, J., & Day, C. L. (2020). The RING domain of RING Finger 12 efficiently builds degradative ubiquitin chains. Journal of Molecular Biology, 432, 3790-3801. doi: 10.1016/j.jmb.2020.05.001

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, 592(8), 1434-1444. doi: 10.1002/1873-3468.13029

Journal - Research Article

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

Journal - Research Article

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

Journal - Research Article

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

Journal - Research Article

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

Journal - Research Article

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

Journal - Research Article

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

Journal - Research Article

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

Journal - Research Article

Middleton, A. J., & Day, C. L. (2023). From seeds to trees: How E2 enzymes grow ubiquitin chains. Biochemical Society Transactions. Advance online publication. doi: 10.1042/BST20220880

Journal - Research Other

Middleton, A. J., & Day, C. L. (2019). Ubiquitin variant inhibitors meet the deubiquitinase USP15. Structure, 27(4), 564-565. doi: 10.1016/j.str.2019.03.011

Journal - Research Other

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