Abstract
SummaryGenomic damage can feature DNA-protein crosslinks whereby their acute accumulation is utilized to treat cancer and progressive accumulation causes neurodegeneration. This is typified by tyrosyl DNA phosphodiesterase 1 (TDP1), which repairs topoisomerase-mediated chromosomal breaks. Although TDP1 levels vary in multiple clinical settings, the mechanism underpinning this variation is unknown. We reveal that TDP1 is controlled by ubiquitylation and identify UCHL3 as the deubiquitylase that controls TDP1 proteostasis. Depletion of UCHL3 increases TDP1 ubiquitylation and turnover rate and sensitizes cells to TOP1 poisons. Overexpression of UCHL3, but not a catalytically inactive mutant, suppresses TDP1 ubiquitylation and turnover rate. TDP1 overexpression in the topoisomerase therapy-resistant rhabdomyosarcoma is driven by UCHL3 overexpression. In contrast, UCHL3 is downregulated in spinocerebellar ataxia with axonal neuropathy (SCAN1), causing elevated levels of TDP1 ubiquitylation and faster turnover rate. These data establish UCHL3 as a regulator of TDP1 proteostasis and, consequently, a fine-tuner of protein-linked DNA break repair.
Highlights
Optimal protein homeostasis is essential for all aspects of cellular activities
As previously reported (Kannouche and Lehmann, 2004; Kannouche et al, 2004), a slower migrating band was observed for PCNA, a finding that is consistent with its ubiquitylation since it was dependent on UV irradiation (Figure 1A, lane 5)
A slower migrating band was observed for FlagTDP1, which was only present in samples containing His-ubiquitin, suggesting that tyrosyl DNA phosphodiesterase 1 (TDP1) is a substrate for ubiquitylation (Figure 1A, lanes 5 and 6)
Summary
Optimal protein homeostasis (proteostasis) is essential for all aspects of cellular activities It is controlled by a number of competing, but integrated, pathways including biogenesis, trafficking, and degradation (Balch et al, 2008; Meerang et al, 2011). Formaldehyde is a potent crosslinking agent generated as a metabolic by-product during de-methylation of histones and DNA (Shi et al, 2004; Trewick et al, 2002) It could be driven enzymatically, as part of physiological cycles of many DNA metabolising enzymes, such as topoisomerases, DNA glycosylases, and methyltransferases (Kiianitsa and Maizels, 2013). This linkage is generally transient and reversible, but it can become irreversible under certain physiological and pathological circumstances, causing deleterious protein-liked DNA breaks (PDBs). The most famous example of PDBs is those mediated by DNA topoisomerases (Ashour et al, 2015; Chiang et al, 2017; Pommier et al, 2016)
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