About the Project
The aim is to understand multiple aspects of the structure and function of HACE1. This includes understanding two potentially inter-related aspects of HACE1 structure and function: the functional relationship between the domains of HACE1, and how HACE1 captures its substrates. These aims will be achieved using a combination of structural and functional studies employing a range of molecular and cellular techniques that probe protein conformations and interactions. The techniques will include protein expression and purification, protein crystallization, mutagenesis, calorimetry, surface plasmon resonance, small angle X-ray scattering, NMR spectroscopy, and ubiquitination assays.
HACE1 is a HECT-type ubiquitin ligase comprising an N-terminal ankyrin repeat region and a C-terminal HECT domain that catalyses ubiquitination, with an intervening 250 amino acid region of unknown function. As a ubiquitin ligase, HACE1 brings together ubiquitin and protein substrate and then catalyses transfer of ubiquitin to substrate, thereby acting as “molecular matchmaker and catalyst”(1). Known ubiquitination substrate proteins of HACE1 include Rac1, Rab GTPases, the autophagy receptors optineurin and p62, and TRAF2. Via these substrates, plus ligase-independent protein-protein interactions, HACE1 is a key player in numerous processes, including:
1. HACE1 is a tumour suppressor involved in multiple cancers(2), including prevention of the key step in breast cancer progression(3) and suppression of tumourigenicity of human lung cancer cells(4)
2. Via Rac1 ubiquitination, HACE1 participates in host defence against pathogens, controls cell migration, and confers cellular protection against reactive oxygen species-induced damage(5,6)
3. HACE1 protects the heart under pressure stress by controlling autophagic clearance of protein aggregates(7), and also protects against neurodegeneration by enhancing function of the oxidative stress response master regulator Nrf2(8), both independent of E3 ligase activity
4. Via TRAF2 ubiquitination, HACE1 is a critical regulator of TNFR1-mediated cell fate, exerting tumour suppressor and anti-inflammatory activities(9)
As mentioned in 3. above, HACE1 can act as an adaptor protein, independent of its ubiquitin ligase capacity. Hence the “dual-function” in the project title. In the adaptor role of HACE1, the HECT domain is reportedly involved in protein-protein interactions(8,10); this led us to hypothesise that in its ubiquitin ligase role, the HECT domain of HACE1 is involved not only in catalysing ubiquitination of substrates, but also in capturing substrates. The conventional view of substrate recognition by HECT ligases, however, is that the domains N-terminal of the HECT domain mediate substrate recognition - in HACE1, probably the ankyrin repeat domain. The student will test the validity of the conventional view of substrate recognition by HECT ligases by assessing whether both ankyrin repeat and HECT domains of HACE1, and indeed the 250-residue sequence between them, are involved in recognition of key substrates Rac1 and optineurin. Having established which parts of HACE1 are responsible for substrate capture, there is potential to undertake a multiplexed two-hybrid assay to generate HACE1 variants with increased specificity for a particular substrate.
The student will also tackle the functional relationship between the domains of HACE1, dealing with questions such as (i) Do the N- and C-terminal domains of HACE1 interact? (ii) Does substrate binding to HACE1 affect inter-domain association, and vice versa? (iii) How does ubiquitination activity differ between full length HACE1 and HECT domain alone, and how does this correlate with data on N-terminal and HECT domain interaction?
Cellular aspects of the project will involve collaboration with the groups of Josef Penninger, Scientific Director of IMBA in Vienna; Makoto Furutani-Seiki, Yamaguchi University, Japan; and Giordano Pula, University of Bath.
References
1. Berndsen, C. E. & Wolberger, C. New insights into ubiquitin E3 ligase mechanism. Nat Rev Nephrol 21, 301–307 (2014).
2. Zhang, L. et al. The E3 ligase HACE1 is a critical chromosome 6q21 tumor suppressor involved in multiple cancers. Nat Med 13, 1060–1069 (2007).
3. Goka, E. T. & Lippman, M. E. Loss of the E3 ubiquitin ligase HACE1 results in enhanced Rac1 signaling contributing to breast cancer progression. Oncogene 1–11 (2015). doi:10.1038/onc.2014.468
4. Liu, Z. et al. Ubiquitylation of autophagy receptor Optineurin by HACE1 activates selective autophagy for tumor suppression. Cancer Cell 26, 106–120 (2014).
5. Torrino, S. et al. The E3 ubiquitin-ligase HACE1 catalyzes the ubiquitylation of active Rac1. Developmental Cell 21, 959–965 (2011).
6. Cetinbas, N. et al. Loss of the tumour suppressor Hace1 leads to ROS-dependent glutamine addiction. Oncogene 34, 4005–4010 (2015).
7. Zhang, L. et al. HACE1-dependent protein degradation provides cardiac protection in response to haemodynamic stress. Nature Comm. 5, 1–14 (2014).
8. Rotblat, B. et al. HACE1 reduces oxidative stress and mutant Huntingtin toxicity by promoting the NRF2 response. Proc. Natl. Acad. Sci. U.S.A. 111, 3032–3037 (2014).
9. Tortola, L. et al. The Tumor Suppressor Hace1 Is a Critical Regulator of TNFR1-Mediated Cell Fate. Cell Rep. 15, 1481–1492 (2016).
10. Zhao, J., Zhang, Z., Vucetic, Z., Soprano, K. J. & Soprano, D. R. HACE1: A novel repressor of RAR transcriptional activity. J. Cell. Biochem. 107, 482–493 (2009).
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