Mechanism and regulation of the Lys6-selective deubiquitinase USP30

Area of Science:

• Cell Biology
• Biochemistry
• Structural Biology

Background:

• Mitophagy, a crucial cellular process for removing damaged mitochondria, is regulated by PINK1 and Parkin.
• Ubiquitin-specific protease USP30 acts as a negative regulator of mitophagy by counteracting Parkin's ubiquitination activity.
• USP30 and Parkin exhibit a specific preference for Lys6-linked polyubiquitin chains, a modification type that remains understudied.

Purpose of the Study:

• To elucidate the structural basis for USP30's preference for Lys6-linked ubiquitin chains.
• To investigate the regulatory interplay between USP30, PINK1, and Parkin in mitophagy.
• To identify mitochondrial substrates ubiquitinated with Lys6-linked chains and understand USP30's role in their regulation.

Main Methods:

• X-ray crystallography to determine the structures of human USP30 bound to monoubiquitin and Lys6-linked diubiquitin.
• Biochemical assays to assess the interplay between USP30, PINK1, and Parkin.
• Development and application of Lys6-linkage-specific affimers to identify mitochondrial substrates.

Main Results:

• Crystal structures reveal unique ubiquitin-binding interfaces enabling USP30's Lys6-linkage specificity.
• Distally phosphorylated ubiquitin chains were found to inhibit USP30 activity.
• USP30 was identified as a regulator of Lys6-polyubiquitinated TOM20 on mitochondria.

Conclusions:

• The study provides structural and functional insights into USP30's unique mechanism for processing Lys6-linked ubiquitin chains.
• Understanding USP30's regulation, particularly its interaction with PINK1 and Parkin, is key to controlling mitophagy.
• These findings offer a foundation for designing targeted therapeutics for diseases involving mitochondrial dysfunction.