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6 March 2003. Two research groups at Columbia University's Taub Institute have discovered new molecular partners for parkin and α-synuclein-proteins that, when mutated, cause early-onset familial forms of Parkinson's disease (PD). One of the two independent studies is published in today’s Neuron; the other is currently available in manuscript form in the Journal of Biological Chemistry.
Convincing evidence implicates parkin in the ubiquitination pathway, a complicated multi-step system for tagging specific proteins with the small peptide ubiquitin. Ubiquitination earmarks unwanted proteins for recycling, ensuring their delivery to the proteasome where they are proteolytically chewed up and spat out as small peptides. First author John Staropoli and colleagues, working in Asa Abeliovich's lab, report in Neuron that parkin's interaction with the protein hSel-10 accelerates the ubiquitination of another protein, cyclin E. This is significant, because even though cyclin E's primary role is in regulation of the cell division cycle, overproduction of cyclin E is known to induce programmed cell death, suggesting that parkin mutations may cause neurodegeneration by allowing cyclin E to accumulate.
The rationale for Staropoli's study was based on known associations of parkin homologs, which contain the so-called RING domain, a small section of amino acids that interacts with the F-box and WD domains of other proteins, such as hSel-10. When Staropoli et al. expressed parkin and hSel-10 together in cultured cells, they found the proteins bound tightly. The authors confirmed this association in extracts from human frontal cortex and demonstrated that the single amino acid mutation in parkin that causes the genetically inherited form of Parkinson’s also abolishes its binding to hSel-10.
HSel-10 is known to interact with ubiquitin ligases. These multiprotein complexes catalyze the final step in the ubiquitination pathway, namely the covalent attachment, or ligation, of ubiquitin to target proteins. HSel-10 acts as a recruiter, binding and delivering these targets, one of which is cyclin E. Staropoli and colleagues used immunoprecipitation experiments to confirm that parkin, hSel-10, and cyclin E all bind together. The authors also showed that a lack of parkin leads to accumulation of cyclin E, and that overexpression of parkin can protect neurons from cyclin E-mediated neurotoxicity. The authors suggest that this latter result points to a potential approach for developing therapeutics for PD.
In the other paper, first author Jessica Martinez and coworkers in Brett Lauring's lab report that α-synuclein interacts with the calcium-activated regulatory protein calmodulin. Martinez and colleagues found this interaction in a screen using a special form of α-synuclein, made by in-vitro protein translation, that can be activated by light. Incorporating chemically modified lysine residues, this α-synuclein reacts to light by covalently binding to other proteins, but only if they are in close proximity, because the light-activation of the lysines lasts only a few nanoseconds.
The authors spiked bovine brain extracts with this "smart" α-synuclein, zapped the mixture with light, then affinity-purified the α-synuclein along with whatever had bound to it. Analysis of the latter showed several proteins had made sufficient contact to be covalently captured, but subsequent experiments showed that one, about 17 kDa in size, was particularly abundant. Martinez et al. purified this protein and identified it as calmodulin.
When Martinez and colleagues tried similar experiments in the absence of calcium, they did not detect binding between the two proteins, suggesting that their association has a physiological role. What this could be is uncertain, but when the authors mixed α-synuclein, calmodulin, and calcium in vitro, they found that the formation of α-synuclein fibrils was accelerated. This leads them to speculate that "Ca2+/calmodulin drives the assembly of synuclein-containing multimeric complexes, or perhaps regulates the oligomerization status of synuclein."-Tom Fagan.
References:
Staropoli JF, McDermott C, Martinat C, Schulman B, Demireva E, Abeliovich A. Parkin is a component of an SCF-like ubiquitin ligase complex and protects postmitiotic neurons from kainate excitotoxicity. Neuron. 2003 Mar 6;37:735-749. Abstract
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 Feb 27. Abstract
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Comments on News and Primary Papers |
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Comment by: Mark Cookson
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Submitted 6 March 2003
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Posted 6 March 2003
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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 (
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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?
 View all comments by Mark Cookson |
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Comment by: Mark A. Smith (Disclosure)
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Submitted 12 March 2003
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Posted 12 March 2003
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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...
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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). View all comments by Mark A. Smith |
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Comment by: Benjamin Wolozin, ARF Advisor (Disclosure)
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Submitted 12 March 2003
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Posted 12 March 2003
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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’ [1-8]. 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 [9]. Both Jensen’s group and my group have shown that calcium alters this fluorescence spectrum, which suggests that calcium directly interacts with α-synuclein [10]. Jensen’s group...
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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’ [1-8]. 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 [9]. Both Jensen’s group and my group have shown that calcium alters this fluorescence spectrum, which suggests that calcium directly interacts with α-synuclein [10]. Jensen’s group confirmed this interaction by examining equilibrium dialysis. Other groups have also observed that α-synuclein regulates calcium-mediated reactions [11, 12]. In addition, we have shown that α-synuclein interacts with a select group of other divalent cations, including zinc, magnesium and iron [9]. 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 [9, 13, 14].
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 [1, 3]. 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.
References: 1. Ostrerova N et al. alpha-Synuclein shares physical and functional homology with 14-3-3 proteins. J Neurosci. 1999 Jul 5;19(14):5782-91. Abstract
2. Jenco JM et al. Regulation of phospholipase D2: selective inhibition of mammalian phospholipase D isoenzymes by alpha- and beta-synucleins. Biochemistry. 1998 Apr 7;37(14):4901-9. Abstract
3. Ahn BH et al. alpha-Synuclein interacts with phospholipase D isozymes and inhibits pervanadate-induced phospholipase D activation in human embryonic kidney-293 cells. J Biol Chem. 2002 Apr 5;277(14):12334-42. Abstract
4. Pronin AN et al. Synucleins are a novel class of substrates for G protein-coupled receptor kinases.
J Biol Chem. 2000 Aug 25;275(34):26515-22. Abstract
5. Choi P et al. Co-association of parkin and alpha-synuclein. Neuroreport. 2001 Sep 17;12(13):2839-43. Abstract
6. Lee FJ et al. Direct binding and functional coupling of alpha-synuclein to the dopamine transporters accelerate dopamine-induced apoptosis. FASEB J. 2001 Apr;15(6):916-26. Abstract
7. Perez RG et al. A role for alpha-synuclein in the regulation of dopamine biosynthesis. J Neurosci. 2002;22(8):3090-9. (No abstract available)
8. Snyder H et al. Aggregated Synuclein binds to the S6' Proteasomal Protein and Inhibits Proteasomal Function. J. Biol. Chem. 2002. (Submitted)
9. Golts N et al. Magnesium inhibits spontaneous and iron-induced aggregation of alpha-synuclein. J Biol Chem. 2002 May 3;277(18):16116-23. Abstract
10. Nielsen MS et al. Ca2+ binding to alpha-synuclein regulates ligand binding and oligomerization. J Biol Chem. 2001 Jun 22;276(25):22680-4. Abstract
11. Lee D et al. alpha-Synuclein exhibits competitive interaction between calmodulin and synthetic membranes. J Neurochem. 2002 Sep;82(5):1007-17. Abstract
12. Park SM et al. Evidence that alpha-synuclein functions as a negative regulator of Ca(++)-dependent alpha-granule release from human platelets. Blood. 2002 Oct 1;100(7):2506-14. Abstract
13. Uversky VN et al. Biophysical properties of the synucleins and their propensities to fibrillate: inhibition of alpha-synuclein assembly by beta- and gamma-synucleins. J Biol Chem. 2002 Apr 5;277(14):11970-8. Abstract
14. Hashimoto M et al. beta-synuclein inhibits alpha-synuclein aggregation: a possible role as an anti-parkinsonian factor. Neuron. 2001 Oct 25;32(2):213-23. Abstract
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