Martinez J, Moeller I, Erdjument-Bromage H, Tempst P, Lauring B.
Parkinson's disease-associated alpha-synuclein is a calmodulin substrate.
J Biol Chem. 2003 May 9;278(19):17379-87.
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This is a great paper and an important step forward in two regards. Firstly, it makes all of us working on parkin reevaluate an important but unstated assumption, namely, that parkin acts as a single protein enzyme. The paper clearly shows that parkin can act as part of a complex in concert with additional proteins. Although previous results using recombinant parkin protein have suggested that parkin has activity as a single protein in vitro (e.g., Shimura et al., 2001; see also ARF related news story), perhaps its in-vivo activity is more complex, with adaptor proteins controlling activity towards specific proteins. Secondly, this is another example of the protective role that parkin plays in neuronal survival. Imai and colleagues demonstrated that parkin overexpression protects cells against ER stress (Imai et al., 2000) or an unfolded ER protein (Imai et al., 2001; see also ARF related news story). We have shown that parkin protects against mutant α-synuclein or proteasome inhibition (Petrucelli et al., 2002; see also ARF related news story). Although unable to confirm the protective effect of parkin on ER stress (Darios et al., 2003) showed a cell survival benefit of parkin in response to C2-ceramide, a trigger for mitochondrially mediated apoptosis. The common link between these studies is that the ability of parkin to protect cells is dependent on ubiquitination.
The mechanistic step forward that Staropoli et al. identify is that a specific parkin substrate, cyclin E, seems to be involved. By controlling cyclin E levels in the cell, parkin may prevent specific triggers of cell death, such as abortive reentry into the cell cycle. Our previous results may be related, as α-synuclein mutations impair proteasomal function, and cyclin E is a known proteasome substrate. Therefore, a key experiment now is to see if mutant α-synuclein induces cyclin E accumulation and whether this contributes to cellular toxicity. I suspect that all of these pathways converge on a relatively small number of proteasome substrates, of which cyclin E would be a great candidate. Parkin would, therefore, control cell death by ubiquitinating and/or promoting degradation of the key substrate(s), perhaps even in situations where the proteasome is partially inhibited.
Like all good papers, this study raises a few more questions for the field. Why is it that dopaminergic cells are selectively vulnerable to these processes? Is apoptosis critical here? For example, our results in postmitotic cells indicate a nonapoptotic cell death compared to the apoptosis induced in embryonic cells, which are primed for apoptosis due to their developmental stage. Finally, is cyclin E the critical substrate, or do the other suggested substrates (PaelR1, synphilin, synuclein, and others) also play a role?
The findings by Staropoli and colleagues (2003) provide strong evidence that an altered cell cycle machinery plays a crucial role in the pathogenesis of Parkinson’s disease (PD), especially the autosomal recessive, early-onset form of PD (ARPD). However, the study lacks direct evidence that accumulated cyclin E contributes to the neuronal cell death evoked by excitotoxicity. A study that demonstrates the inhibitory effect of a cyclin-dependent kinase (CDK) inhibitor on the excitotoxicity-mediated neuronal cell death in the parkin-deficient neurons would provide more solid evidence for the involvement of accumulated cyclin E in neuronal cell death. In addition, given the proposed pivotal role of parkin in the regulation of cyclin E, it is surprising that almost the same level of cyclin E is observed in sporadic PD, where parkin is intact, compared to ARPD. In any event, this paper provides a new aspect that may help us understand the mechanism of accumulation of cell-cycle markers in the vulnerable neurons, not only in PD, but also in other neurodegenerative diseases (reviewed in Bowser and Smith, 2002).
The article by Martinez and colleagues identifies the calcium-binding protein calmodulin as a new binding partner for α-synuclein. This intriguing observation adds to the growing list of proteins that bind α-synuclein. This list includes phospholipase D, 14-3-3, protein kinase C, ERK, GRK, parkin, the DA transporter, tyrosine hydroxylase, and the proteasomal protein S6’. Binding to calmodulin is particularly intriguing because of a growing body of literature suggesting that α-synuclein either binds, or is modulated by, divalent ions. Martinez cites an article by Lansbury’s group showing that calcium does not alter the CD spectrum of α-synuclein, which suggests that calcium does not induce α-helical structure or β-pleated sheet structure in α-synuclein. However, α-synuclein has four tyrosines whose physical behavior can be monitored by measuring the fluorescence emitted by these tyrosines. Both Jensen’s group and my group have shown that calcium alters this fluorescence spectrum, which suggests that calcium directly interacts with α-synuclein. Jensen’s group confirmed this interaction by examining equilibrium dialysis. Other groups have also observed that α-synuclein regulates calcium-mediated reactions. In addition, we have shown that α-synuclein interacts with a select group of other divalent cations, including zinc, magnesium and iron . Each ion appears to exhibit a different effect on α-synuclein behavior. Martinez examined whether calcium or calmodulin affects α-synuclein fibrillization, but found no effect. While this is true, magnesium does inhibit α-synuclein fibrillization, as do β and γ-synuclein. The current observation that calmodulin binds α-synuclein deepens the link between α-synuclein and divalent cations. Binding of calmodulin to calcium is known to regulate many signal transduction processes. Increasing evidence suggests that α-synuclein also regulates signal proteins, such as PKC and phospholipase D. Binding of calmodulin to α-synuclein suggests that calcium might be an important link regulating the interaction of α-synuclein with its substrates.—Benjamin Wolozin, Loyola University, Maywood, Illinois.