Relative Sensitivity of Parkin and Other Cysteine-containing Enzymes to Stress-induced Solubility Alterations

Abstract

Loss of parkin function is a predominant cause of familial Parkinsonism. Emerging evidence also suggests that parkin expression variability may confer a risk for sporadic Parkinson disease. We have recently demonstrated that a wide variety of Parkinson disease-linked stressors, including dopamine (DA), induce parkin solubility alterations and promote its aggregation within the cell, a phenomenon that may underlie the progressive susceptibility of the brain to degeneration. The vulnerability of parkin to stress-induced modification is likely due to its abundance of cysteine residues. Here, we performed a comprehensive mutational analysis and demonstrate that Cys residues residing both within and outside of the RING-IBR (in between RING fingers)-RING domain of parkin are important in maintaining its solubility. The majority of these Cys residues are highly conserved in parkin across different species and potentially fulfil important structural roles. Further, we found that both parkin and HHARI (human homologue of Drosophila ariadne), another RING-IBR-RING-type ubiquitin ligase, are comparably more susceptible to solubility alterations induced by oxidative and nitrosative stress when compared with other non-RING-IBR-RING Cys-containing enzymes. However, parkin appears to be uniquely sensitive to DA-mediated stress, the specificity of which is likely due to DA modification of 2 Cys residues on parkin (Cys-268 and Cys-323) that are distinct from other RING-IBR-RING members. Parkinson disease (PD)4 is the most common neurodegenerative movement disorder characterized pathologically by the rather selective loss of midbrain dopaminergic neurons in the substantia nigra pars compacta and the presence of intraneuronal protein inclusions known as Lewy bodies. Although most cases of PD occur in a sporadic manner, a subset of PD cases is inheritable and attributable to mutations in specific genes. These familial PD-linked genes include ␣-synuclein, parkin, DJ-1, PINK1, and LRRK2 (1, 2). Of these, mutations in the parkin gene are currently recognized to be a predominant cause of familial, early onset PD (3-5). Further, emerging evidence also suggests that parkin expression variability may confer a risk for the development of the more common, sporadic form of PD (6, 7).The importance of functional parkin to dopaminergic neuronal survival is probably related to the multitude of neuroprotective roles it serves (8, 9). Parkin functions as a ubiquitin ligase associated with protein homeostasis and apparently confers protection to neurons against a diverse range of cellular insults (8,9). Recently, we have demonstrated that a wide variety of PD-linked stressors, including dopamine (DA), induce parkin solubility alterations and promote its aggregation within the cell (10). Our observations corroborated with a similar study conducted by LaVoie et al. (11), who further showed that DA covalently modifies parkin via its Cys residues, although the number and location of the Cys targeted by DA remain unknown. Since parkin functions as a br...

Aberrant Folding of Pathogenic Parkin Mutants

“…We found that HHARI is sensitive to oxidative stress-induced misfolding similarly to Parkin, which is in line with recent observations (33,36). However, the propensity to misfold upon C-terminal truncations was specific for Parkin.…”

“…Misfolding of Parkin induced by pathogenic mutations or cellular stress has been established as a major mechanism of Parkin inactivation, accentuating a possible pathological role of Parkin even in sporadic PD (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(33)(34)(35). It is obvious that the high cysteine content found in the RBR domain predisposes Parkin to oxidative stress-induced inactivation and misfolding, a phenomenon that has recently been shown experimentally (36). Interestingly, in comparison to other RBR proteins Parkin seems to be uniquely sensitive to dopamine-induced inactivation (33,36).…”

“…It is obvious that the high cysteine content found in the RBR domain predisposes Parkin to oxidative stress-induced inactivation and misfolding, a phenomenon that has recently been shown experimentally (36). Interestingly, in comparison to other RBR proteins Parkin seems to be uniquely sensitive to dopamine-induced inactivation (33,36).…”

Identification of a Novel Zn2+-binding Domain in the Autosomal Recessive Juvenile Parkinson-related E3 Ligase Parkin

“…A corollary of this conclusion is that the ARJP mutations C289G, C431F, and C441R found in the parkin RING1 and RING2 domains are also expected to be important zinc coordinating residues because our work shows that these domains each bind two zinc ions. These proteins and several other cysteine substituted parkin derivatives also form insoluble aggresomes consistent with disruption of zinc binding to the RING1, IBR, and RING2 domains (55,57). In particular, a C166A substitution caused increased aggregation and insolubility compared with wild type parkin consistent with a structural zinc-binding role in RING0 (57).…”

“…These proteins and several other cysteine substituted parkin derivatives also form insoluble aggresomes consistent with disruption of zinc binding to the RING1, IBR, and RING2 domains (55,57). In particular, a C166A substitution caused increased aggregation and insolubility compared with wild type parkin consistent with a structural zinc-binding role in RING0 (57).…”