#BEN FINK 17502 SERIES#
Degradation by the 26S proteasome, which is composed of the 19S and 20S particles, is an active process that is regulated by a series of enzymes that ubiquitinate the substrate and sensitize it to degradation 21. This class of proteins can be degraded by two alternative proteasomal degradation mechanisms, which are not mutually exclusive: ubiquitin-dependent degradation by the 26S proteasome and ubiquitin-independent degradation by the 20S proteasome. In general, substrates of the 20S proteasome are composed of proteins containing unstructured or relatively unfolded regions 20. Our results revealed that DJ-1 is a regulator of the ubiquitin-independent 20S proteasomal degradation pathway. Here we combined biochemical and native mass spectrometry approaches, together with cell biology methods, to study the molecular function of DJ-1.
Overall, despite the broad interest and impressive progress that has been made in DJ-1 characterization, its key biological functions, and direct and indirect biological implications, remain unclear. DJ-1 was also shown to upregulate glutathione synthesis and heat shock proteins 16, alter dopamine receptor signalling 17 and coordinate p53 signalling 18, 19. These include roles as a cysteine protease 10, redox-regulated chaperone 11 and transcription modulator 12, as well as involvement in anti-apoptotic signalling processes 13, 14 and RNA regulation 15. To date, the molecular function of DJ-1 is still not clear, though a range of biochemical and cellular activities have been proposed for it.
In addition, oxidative stress was found to induce the rapid relocalization of DJ-1 to mitochondria 7, 9, suggesting that mitochondria could be a site for DJ-1’s neuroprotective activity. This oxidative modification is thought to enable DJ-1 to act as a sensor of cellular redox homeostasis, and to participate in cytoprotective signalling pathways in the cell. Under oxidative stress, a conserved cysteine residue in DJ-1 (Cys106) is oxidized into cysteine sulfinate and cysteine sulfonate 2. It was shown to protect cells from reactive oxygen species, reduce protein misfolding and stabilize mitochondrial potential and morphology. Models of DJ-1 deficiency or overexpression have provided evidence that the protein takes part in the oxidative stress response 7, 8. The specific role of the protein in the pathogenesis of PD is still unclear however, the diversity of disease states in which DJ-1 is implicated suggests its involvement in a fundamental biological process. A decade ago, it was realized that missense, truncation and splice site mutations in DJ-1 all lead to an autosomal recessive, early-onset familial form of Parkinson’s disease (PD reviewed in refs 2, 6). DJ-1 was also implicated in additional disorders such as ischaemic injury, amyotrophic lateral sclerosis 4, 5, fertility control and androgen receptor regulation 2. DJ-1 was originally identified as an oncogene and its expression was found to be upregulated in several malignancies including breast, lung and prostate cancer. Belonging to the ThiJ/PfpI protein superfamily, DJ-1 is highly conserved, with homologues distributed across all biological kingdoms 3. Such a tight regulation of the 20S proteasome may sustain the balance between the need to rapidly eliminate oxidatively damaged proteins and maintain the abundance of native, intrinsically unstructured proteins, which coordinate regulatory and signalling events.ĭJ-1 is a small protein of 189 amino acids that is ubiquitously expressed in almost all cells and tissues, including the brain, and exists as a homodimer that is localized to the cytoplasm, mitochondria and nucleus (for reviews, see refs 1, 2). Overall, our results suggest a regulatory circuit in which DJ-1, under conditions of oxidative stress, both upregulates and inhibits the 20S proteasome, providing a rigorous control mechanism at a time when the 20S proteasome becomes the major proteolytic machinery. Furthermore, we demonstrate that following oxidative stress, DJ-1 is involved in the Nrf2-dependent oxidative stress response that leads to the upregulation of both the 20S proteasome and its regulator, NQO1. Consequently, DJ-1 stabilizes the cellular levels of 20S proteasome substrates, as we show for α-synuclein and p53. We show that DJ-1 physically binds the 20S proteasome and inhibits its activity, rescuing partially unfolded proteins from degradation. Here we demonstrate that DJ-1 is a 20S proteasome regulator. The Parkinson’s-associated protein, DJ-1, is a highly conserved homodimer, ubiquitously expressed in cells.
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