Quantitative Middle-Down MS Analysis of Parkin-Mediated Ubiquitin Chain Assembly

Abstract

Misregulation of the E3 ubiquitin ligase Parkin and the kinase PINK1 underlie both inherited and idiopathic Parkinson's disease-associated neurodegeneration. Parkin and PINK1 work together to catalyze the assembly of ubiquitin chains on substrates located on the outer mitochondrial membrane to facilitate autophagic removal of damaged mitochondria through a process termed mitophagy. Quantitative measurements of Parkin-mediated chain assembly, both in vitro and on mitochondria, have revealed that chains are composed of Lys6, Lys11, Lys48, and Lys63 linkages. The combinatorial nature of these chains is further expanded by the ability of PINK1 to phosphorylate individual subunits. The precise architecture of chains produced by the coordinated action of PINK1 and Parkin, however, are unknown. Here, we demonstrate that quantitative middle-down mass spectrometry using uniformly 15 N-labeled ubiquitin variants as internal standards informs on the extent of chain branching. We find that Parkin is a prolific branching enzyme in vitro. Quantitative middle-down mass spectrometry also reveals that phospho-Ser65ubiquitin (pSer65-Ub)-a key activator of Parkin-is not incorporated into chains to a significant extent. Our results suggest that Parkin-mediated chain branching is "on-pathway", and branch points are the principal targets of the deubiquitinase USP30.

Emerging functions of branched ubiquitin chains

“…The HECT E3 WWP1 has been demonstrated to synthesize branched chains containing K48 and K63 linkages in the presence of a single E2, UBE2L3 32 , whereas UBE3C and the bacterial HECT-like E3 NleL have been reported to assemble branched K29/K48 and K6/K48 chains, respectively, also in the presence of a single E2 17 , 18 , 33 . The RBR E3 Parkin, which is often mutated in early-onset Parkinson’s disease, has recently been shown to synthesize branched K6/K48 chains 34 , consistent with previous reports that Parkin forms chains of complex topology including multiple linkages 35 – 37 .…”

“…The rationale for investing in these technologies is considerable because of the direct link that these signals have to human health and disease. Branched polymers have been implicated in the degradation of aggregation-prone proteins that are directly responsible for causing neurodegenerative diseases 11 , and Parkin, a ubiquitin ligase that is often mutated in early-onset Parkinson’s disease, was recently reported to synthesize branched chains 34 , 37 . Ultimately, the “ubiquitin code” will need to be expanded to include branched polymers, and cracking this expanded code will undoubtedly be a major challenge for the field.…”

Regulation of mitochondrial cargo-selective autophagy by posttranslational modifications

“…Moreover, K27 linkages were detected ( 52 , 86 ). Indeed, parkin is able to build other types of ubiquitin-linked chains (K6, K11, and K27) apart from K48 and K63, not only linear but also branched poly-ubiquitin chains ( 77 , 82 , 112 ). The timing of parkin target selection and the linkage specificity of ubiquitin chain elongation are likely regulated by PTMs and regulatory factors that need to be further elucidated.…”

Mitochondrial Quality Control: A Pathophysiological Mechanism and Therapeutic Target for Stroke

“…The overexpression of PINK1 and Parkin was suggested to promote mitochondria collapse and trafficking to the perinuclear area, an autophagyassociated subcellular region (Vives-Bauza et al, 2010). The activated Parkin then catalyzes the ubiquitylation of OMM proteins such as Mfn1, Mfn2, Fis1, voltage-dependent anionselective channel (VDAC), and CDGSH iron-sulfur domaincontaining protein 1 (CISD1) (Deol et al, 2020). These substrates of Parkin lack specificity, but the ubiquitin chains assembling on the substrates are varied in density, structure, linkage, and ubiquitin-binding domains (UBDs) (Harper et al, 2018;Deol et al, 2020).…”

“…The activated Parkin then catalyzes the ubiquitylation of OMM proteins such as Mfn1, Mfn2, Fis1, voltage-dependent anionselective channel (VDAC), and CDGSH iron-sulfur domaincontaining protein 1 (CISD1) (Deol et al, 2020). These substrates of Parkin lack specificity, but the ubiquitin chains assembling on the substrates are varied in density, structure, linkage, and ubiquitin-binding domains (UBDs) (Harper et al, 2018;Deol et al, 2020). Diverse ubiquitin chains are able to bind specific mitophagy receptors and consequently accurately regulate autophagosomal capture (Harper et al, 2018).…”