class="head no_bottom_margin" id="sec1title">IntroductionOptimal 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 (, ). Adjusting these processes to the demand of individual proteins and pathways is essential to maintain cellular function. Perturbation in proteostasis causes human disease. For example, failure to degrade and subsequently clear proteins that are covalently linked to DNA cause human neurological disease and premature aging (, , , , ). The linkage of proteins to DNA could be non-enzymatically driven by the vicinity of proteins to DNA in the presence of endogenous crosslinking agents, such as aldehydes. Formaldehyde is a potent crosslinking agent generated as a metabolic by-product during de-methylation of histones and DNA (, ). It could also be driven enzymatically, as part of physiological cycles of many DNA metabolising enzymes, such as topoisomerases, DNA glycosylases, and methyltransferases (). 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 (, , ).Topoisomerases are elegant biological tools that overcome topological entanglements inherent to the intertwined and compact nature of DNA. Their function is important for many aspects of DNA metabolism, such as gene transcription, DNA replication, recombination, and repair (). Topoisomerases achieve this by transiently cleaving one or two strands of the DNA, thereby allowing the swiveling or rotation of the other strand or duplex around the break. Topoisomerase I (TOP1) generates intermediates in which the TOP1 is linked to the 3′ terminus of a single-strand break (SSB), whereas TOP2 intermediates are linked to the 5′ termini of a DNA double-strand break (DSB). Accumulation of TOP1- or TOP2-mediated PDBs cause neurological disease in humans (, href="#bib1" rid="bib1" class=" bibr popnode">Alagoz et al., 2013, href="#bib20" rid="bib20" class=" bibr popnode">El-Khamisy et al., 2005, href="#bib25" rid="bib25" class=" bibr popnode">Gómez-Herreros et al., 2014, href="#bib65" rid="bib65" class=" bibr popnode">Walker and El-Khamisy, 2018) and has been widely exploited in cancer chemotherapy (href="#bib2" rid="bib2" class=" bibr popnode">Alagoz et al., 2014, href="#bib5" rid="bib5" class=" bibr popnode">Ashour et al., 2015, href="#bib15" rid="bib15" class=" bibr popnode">Das et al., 2014, href="#bib46" rid="bib46" class=" bibr popnode">Meisenberg et al., 2017, href="#bib52" rid="bib52" class=" bibr popnode">Rehman et al., 2018). Accumulation of PDBs is counteracted by a number of PDB repair activities that constantly monitor and precisely disjoin the stalled topoisomerase from DNA termini or nucleolytically cut the DNA to release the stalled topoisomerase and a fragment of DNA. The former mode of repair spares the loss of genetic information and is conducted by a class of enzymes that specifically cleave the covalent linkage between the stalled topoisomerase and DNA called “tyrosyl DNA phosphodiesterases” (TDPs). TDP1 primarily disjoins stalled TOP1 peptides from PDBs, whereas TDP2 exhibits greater preference toward TOP2-mediated PDBs (href="#bib20" rid="bib20" class=" bibr popnode">El-Khamisy et al., 2005, href="#bib12" rid="bib12" class=" bibr popnode">Cortes Ledesma et al., 2009, href="#bib56" rid="bib56" class=" bibr popnode">Schellenberg et al., 2017). Defects in either TDP1 or TDP2 cause accumulation of PDBs and interfere with transcription, leading to neuronal cell death (href="#bib25" rid="bib25" class=" bibr popnode">Gómez-Herreros et al., 2014, href="#bib26" rid="bib26" class=" bibr popnode">Hudson et al., 2012, href="#bib33" rid="bib33" class=" bibr popnode">Katyal et al., 2007). In contrast, their overexpression has been linked to the resistance of cancer cells to topoisomerase targeting therapies (href="#bib7" rid="bib7" class=" bibr popnode">Barthelmes et al., 2004, href="#bib16" rid="bib16" class=" bibr popnode">Do et al., 2012, href="#bib18" rid="bib18" class=" bibr popnode">Duffy et al., 2016, href="#bib39" rid="bib39" class=" bibr popnode">Liu et al., 2007, href="#bib45" rid="bib45" class=" bibr popnode">Meisenberg et al., 2014). Despite their roles in many aspects of cellular activities and their implication in multiple clinical settings, the mechanisms that maintain TDPs proteostasis are not known.TDP1 proteostasis is particularly attractive since a specific mutation in its active site that substitutes histidine 493 to arginine perturbs the completion of its catalytic cycle and additionally produces a TDP1-mediated PDB (href="#bib13" rid="bib13" class=" bibr popnode">Cuya et al., 2016, href="#bib28" rid="bib28" class=" bibr popnode">Interthal et al., 2005). Bi-allelic TDP1H493R mutation is associated with ∼70% reduction of TDP1 protein level and leads to the accumulation of both TOP1- and TDP1-mediated PDBs, causing neurodegeration in spinocerebellar ataxia with axonal neuropathy 1 (SCAN1). Cells derived from SCAN1 patients exhibit marked sensitivity to TOP1 poisons, such as camptothecin and irinotecan (href="#bib20" rid="bib20" class=" bibr popnode">El-Khamisy et al., 2005, href="#bib28" rid="bib28" class=" bibr popnode">Interthal et al., 2005, href="#bib48" rid="bib48" class=" bibr popnode">Miao et al., 2006, href="#bib71" rid="bib71" class=" bibr popnode">Zhou et al., 2005). Although much is known about TDP1 biology, little is known about the mechanisms that control its steady-state level and why levels are markedly reduced in SCAN1 remains unexplained.Post-translational modifications have been shown to control the steady-state level and modulate the function of several DNA repair factors (href="#bib21" rid="bib21" class=" bibr popnode">Elserafy and El-Khamisy, 2018, href="#bib44" rid="bib44" class=" bibr popnode">Meisenberg et al., 2012, href="#bib49" rid="bib49" class=" bibr popnode">Parsons et al., 2008). Protein ubiquitylation plays an important role in controlling the half-life of proteins via the ubiquitin-proteasome system (href="#bib3" rid="bib3" class=" bibr popnode">Amm et al., 2014). It offers a reversible mode of control for a myriad of cellular processes such as protein sorting, signal transduction and DNA repair (href="#bib10" rid="bib10" class=" bibr popnode">Chen and Chen, 2013, href="#bib22" rid="bib22" class=" bibr popnode">Francisco et al., 2014, href="#bib44" rid="bib44" class=" bibr popnode">Meisenberg et al., 2012, href="#bib49" rid="bib49" class=" bibr popnode">Parsons et al., 2008, href="#bib50" rid="bib50" class=" bibr popnode">Parsons et al., 2009, href="#bib63" rid="bib63" class=" bibr popnode">van Cuijk et al., 2014). The transient nature and fine tuning of ubiquitylation is achieved by an intricate balance of two opposing activities: the ubiquitin-conjugating cascade, which is a complex set of enzymes that conjugate ubiquitin to proteins, and deubiquitylase enzymes (DUBs), which remove ubiquitin molecules from the modified proteins (href="#bib38" rid="bib38" class=" bibr popnode">Komander et al., 2009, href="#bib47" rid="bib47" class=" bibr popnode">Metzger et al., 2014). Here, we report that TDP1 levels are regulated by ubiquitylation and identify UCHL3 as the deubiquitylase enzyme controlling TDP1 proteostasis. A low level of UCHL3 is associated with reduced TDP1 protein levels, causing neurological disease, whereas its overexpression causes higher TDP1 levels in cancer.
展开▼
