NSMB paper uses Affimer® binders for new insights into the control of mitophagy

Following last week’s publication in Molecular Cell, David Komander’s group at the MRC Laboratory of Molecular Biology have authored another exciting paper utilising Affimer binders in the field of ubiquitin research. Published today in Nature Structural and Molecular Biology (link is external), their latest study examines a deubiquitinase, USP30, that plays an important role in mitophagy and is considered a drug target against forms of Parkinson’s disease. Their work shows how USP30 structurally recognises its preferred diubiquitin linkage and acts upon the substrate TOM20, to form a potential upstream regulator of mitophagy.

Crystals of the Lys6-diubiquitin selective deubiquitinase USP30
Crystals of the Lys6-diubiquitin selective deubiquitinase USP30

One of the major novel features of this work is the investigation of the atypical lysine-6 (K6) ubiquitin chain. Polyubiquitin chains can form through eight different ubiquitin linkages, involving either the N-terminal methionine residue or one of seven different lysine residues within the ubiquitin protein. The most studied linkages have been K48 and K63, while the lack of effective antibodies developed to the other atypical chain types have largely prevented their investigation. To overcome this limitation posed by antibody technology, David Komander’s research group adopted Affimer binders specific to the K6 diubiquitin linkage and used these to explore the biology of this atypical linkage in mitophagy.

Mitochondria undergo mitophagy following excessive damage. This process is initiated by PINK1 and Parkin and involves high levels of ubiquitination of substrates on the mitochondrial outer membrane, including the K6 ubiquitin linkage, to signal mitochondrial turnover. This is an important process in maintaining cellular function, with loss of function mutations in PINK1 and Parkin representing major causes of early onset autosomal recessive juvenile Parkinson’s disease.

In this study, the researchers examined USP30, a deubiquitinase that acts as a negative regulator of mitophagy by antagonising Parkin-mediated ubiquitination events. The crystal structure of the human enzyme bound to K6 diubiquitin demonstrated how it achieves its preference for the hydrolysis of the K6 chain type over the other ubiquitin linkages, using a different triad of amino acids in the active site compared to the majority of USP along with a hydrophobic patch underneath the triad.

Though it is known that Parkin assembles the K6 linkage, among others, when ubiquitinating substrates on the mitochondrial outer membrane, the precise substrates that are polyubiquitinated in this way are not. Using the K6-specific Affimer binders the researchers were able to enrich the K6 ubiquitinated proteins from a cell extract and identify a number of Parkin substrates, including the mitochondrial import receptor, TOM20. Inhibiting ubiquitination of TOM20 has previously been shown to impede mitophagy, thus the researchers suggest that the K6 polyubiquitination of TOM20 may act as an upstream regulator for mitophagy, with USP30 acting as a potential brake on this process.

Pharmacological inhibition of USP30 holds the potential for increased mitochondrial turnover. “The structural differences identified here between USP30 and other known deubiquitinases allow the possibility of developing specific inhibitors to target this process”, Malte Gersch, the first author of the study, explains. The importance of further research into the atypical ubiquitin linkages and USP30 is reflected in that any potential treatments may offer hope for patients of early onset Parkinson’s disease and other mitochondrial dysfunction diseases.