Systematic approaches to identify E3 ligase substrates

Abstract

Protein ubiquitylation is a widespread post-translational modification, regulating cellular signalling with many outcomes, such as protein degradation, endocytosis, cell cycle progression, DNA repair and transcription. E3 ligases are a critical component of the ubiquitin proteasome system (UPS), determining the substrate specificity of the cascade by the covalent attachment of ubiquitin to substrate proteins. Currently, there are over 600 putative E3 ligases, but many are poorly characterized, particularly with respect to individual protein substrates. Here, we highlight systematic approaches to identify and validate UPS targets and discuss how they are underpinning rapid advances in our understanding of the biochemistry and biology of the UPS. The integration of novel tools, model systems and methods for target identification is driving significant interest in drug development, targeting various aspects of UPS function and advancing the understanding of a diverse range of disease processes.

Enzyme–substrate relationships in the ubiquitin system: approaches for identifying substrates of ubiquitin ligases

“…Additional recent perspectives on these issues are also available [110, 111]. The development of approaches that take unique advantage of ubiquitin and Ubl biochemistry (e.g., UBAITs, Neddylators, Ubiquitin Ligase Substrate Traps) may improve the depth of coverage and help identify less abundant substrates for E3 ligases.…”

A rice really interesting new gene H2‐type E3 ligase, OsSIRH2‐14, enhances salinity tolerance via ubiquitin/26S proteasome‐mediated degradation of salt‐related proteins

“…RING E3 ligases can interact and regulate multiple substrate proteins in a diverse functional network (Iconomou & Saunders, ; Lim et al, ; Lim et al, ). OsSIRH2‐14 E3 ligase interacts with multiple substrates, including OsSalT, OsPRF2, and OsHKT2;1 (Lim et al, ; Figures and S7).…”

“…These results suggest that the reduced Na + accumulation in OsSIRH2-14-overexpressing plants might lead to decreased oxidative damage and increased capacity for proline and soluble sugars. RING E3 ligases can interact and regulate multiple substrate proteins in a diverse functional network (Iconomou & Saunders, 2016;Lim et al, 2013;Lim et al, 2015). OsSIRH2-14 E3 ligase interacts with multiple substrates, including OsSalT, OsPRF2, and OsHKT2;1 (Lim et al, 2013;Figures 4 and S7).…”

TNF receptor associated factor 6 interacts with ALS-linked misfolded superoxide dismutase 1 and promotes aggregation

“…Importantly, TRAF6 has been previously implicated in neurodegenerative diseases, including Parkinson's disease (36)(37)(38), Huntington's disease (39), and Alzheimer's disease (40)(41)(42), where it stimulates the ubiquitination of disease-associated proteins and exerts a regulatory role on their turnover and aggregation (43). The detection of TRAF6 as a binding partner of misfolded SOD1 was additionally intriguing, since physiological interactions between E3 ubiquitin ligases and their substrates are generally known to be weak, transient, and not readily detected by IP-MS based techniques (44)(45)(46). We speculated that the interaction is unnaturally stable and of possible pathological relevance.…”