Mechanism and regulation of the Lys6-selective deubiquitinase USP30

Abstract

Damaged mitochondria undergo a specialised form of autophagy termed mitophagy, which is initiated by the ubiquitin kinase PINK1 and the E3 ligase Parkin. Ubiquitin-specific protease USP30 antagonises Parkin-mediated ubiquitination events on mitochondria and is a key negative regulator of mitophagy. Parkin and USP30 both display an unusual preference for assembly or disassembly, respectively, of Lys6-linked polyubiquitin, a chain type that has remained poorly studied. We here report crystal structures of human USP30 bound to mono- and Lys6-linked diubiquitin, which explain how USP30 achieves Lys6-linkage preference through unique ubiquitin binding interfaces. We assess the interplay between USP30, PINK1 and Parkin, and show that distally phosphorylated ubiquitin chains impair USP30 activity. Lys6-linkage specific affimers identify numerous mitochondrial substrates of this modification, and we show that USP30 regulates Lys6-polyubiquitinated TOM20. Our work provides insights into USP30 architecture, activity, and regulation, which will aid drug design against this and related enzymes.

Dual role of USP 30 in controlling basal pexophagy and mitophagy

“…It is possible that USP30 activity is directed at a particular set of outer mitochondrial and peroxisomal membrane proteins, which collectively or individually act to nucleate locally restricted, selective autophagy. Previous work records that USP30 shows specificity for Lys6‐linked ubiquitin chains and preferentially acts on TOMM20, amongst known Parkin substrates . We have not observed significant global changes in the ubiquitylation pattern of USP30 KO lysates or purified mitochondrial fractions (Appendix Fig S2), nor have we seen any changes in TOMM20 ubiquitin status.…”

Evaluating enzyme activities and structures of DUBs

“…2). This site typically contributes the bulk of the binding energy to the ubiquitinated substrate and therefore is the most susceptible to manipulation through point mutation (e.g., Gersch et al, 2017;Keusekotten et al, 2013;Mevissen et al, 2016;Pruneda et al, 2016). Characterization of S1 site specificity can be accomplished using Ub/UBL ABPs or fluorescent substrates, as discussed above.…”

“…2). In the case of a polyUb chain, recognition of the proximal Ub moiety at the S1 0 site determines linkage specificity by orienting the ubiquitinated Lys residue into the active site, which can be manipulated through point mutations (e.g., Gersch et al, 2017;Keusekotten et al, 2013;Mevissen et al, 2016;Pruneda et al, 2016). Identification of an S1 0 site that introduces chain specificity can be performed using the panel of polyUb chains as discussed above.…”

“…In the case of Cys-based DUBs, this means mutating the active site Cys to Ala, which has been shown for a variety of tested examples to artificially enhance binding affinity to ubiquitinated substrates (Morrow et al, 2018). In DUBs where a defined S1 0 site dictates a strong preference for a particular polyUb chain type, this method has enabled crystallization of DUB-diUb complexes (Gersch et al, 2017;Keusekotten et al, 2013;Rivkin et al, 2013;Sato et al, 2015Sato et al, , 2017.…”

“…Depending on size and behavior, NMR can be a useful tool for understanding DUB dynamics and regulation in solution, as shown for the DUBs AMSH and Cezanne (Hologne et al, 2016;Mevissen et al, 2016) as well as the UBL protease SENP1 (Ambaye, Chen, Khanna, Li, & Chen, 2018). Hydrogen-deuterium exchange mass spectrometry can also be used to monitor conformational changes associated with substrate recognition (Gersch et al, 2017;Mevissen et al, 2016). Finally, single-molecule techniques can inform on more global structural parameters, such as polyUb chain conformation following DUB binding (Ye et al, 2012).…”