Once activated, the E3 ligase activity of Parkin (PRKN) catalyzes the assembly of mono ubiquitin (Ub) or polyUb chains on a broad range of substrates including mitofusin 1 (MFN1) (Tnaka A et al., 2010), MFN2 (Rakovic A et al., 2011; McLelland GL et al., 2018; Vranas M et al., 2022), mitochondrial import receptor subunit TOM20 homolog (TOMM20) (Koyano F et al., 2019; Zittlau KI et al., 2022), and voltage dependent anion selective channel proteins (VDAC1) (Geisler S et al., 2010; Ordureau A et al., 2018; Ham SJ et al., 2020), VDAC2, VDAC3 (Sun Y et al., 2012). Other PRKN substrates identified by the various proteomic studies: Sarraf SA et al., 2013; Ordureau A et al., 2014, 2018; Rose CM et al., 2016; Antico O et al., 2021; Zittlau KI et al., 2022. PRKN catalyzes the formation of different types of ubiquitin linkages on its substrates, including K6, K27, K48, and K63 linkages (Harper JW et al., 2018; Antico O et al., 2021; Buneeva O & Medvedev A 2022). Multiple E2 enzymes, including UBE2N, UBE2L3 or UBE2D2/3, are implicated in mediating the ubiquitin ligase activity of PRKN (Fiesel FC et al., 2014; Geisler S et al., 2014).
Conjugated Ub chains on mitochondrial proteins mark them either for degradation via the proteasomal pathway or recruit cargo receptors such as SQSTM1 and OPTN to initiate mitophagy (Heo LM et al. 2015; Lazarou M et al. 2015). Deubiquitinases such as USP15 and USP30 remove Ub moieties from the mitochondrial substrates to counteract PRKN-mediated ubiquitination and suppress mitophagy (Gersch M et al., 2017; Cornelissen T et al., 2014; reviewed by Onishi M et al., 2021).
PRKN is a member of the RINGinBetweenRING family of E3 Ub ligases, which mediate Ub transfer to a substrate from an E2 ubiquitin-conjugating enzyme through the formation of a thioester intermediate on a reactive cysteine (C431 in PRKN) located in the RING2 catalytic domain (Wenzel et al., 2011; reviewed by Wang XS et al., 2023). Structural and biochemical studies reveal that at steady state PRKN is maintained in an autoinhibited conformation (Trempe JF et al., 2013; Kumar A et al., 2015, 2017; Tang MY et al., 2017). PRKN binding to phosphorylated Ub moieties at MOM followed by PINK1-mediated phosphorylation of PRKN at S65 release the inhibitory interdomain interactions thus promoting the E3 ligase activity of PRKN (Iguchi M et al., 2013; Kumar A et al., 2017; Gladkova C et al., 2018; Condos TE et al., 2018). In particular, phosphoUb binding induces conformational changes in PRKN that lead to the release of the ubiquitin-like (UBL) domain allowing PINK1 to phosphorylate PRKN at S65 within the ULB domain (Kumar A et al., 2015, 2017; Kazlauskaite A et al., 2015). This phosphorylation of PRKN, in turn, liberates the RING1 domain from the repressor element of Parkin (REP), enabling recruitment of E2 enzyme. Ub conjugates to the binding site on RING1, and enhances reactivity of the catalytic C431 residue in RING2 (Iguchi M et al., 2013; Caulfield TR et al., 2014; Ordureau A et al., 2015; Sauve V et al., 2015, 2018; Tang MY et al., 2017; Gladkova C et al., 2018). PRKN-mediated ubiquitination of MOM proteins generates more Ub moieties for PINK1-mediated phosphorylation of Ub thus leading to a feedforward loop in the PINK1:PRKN pathway (Ordureau A et al., 2015; Sauve V et al., 2022).
This Reactome event shows PRKN-mediated K63-linked and K48-linked ubiquitination of the selected MOM proteins such as MFN1, MFN2, TOMM20, VDAC13 in the presence of Ub-conjugating E2 enzymes.
