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
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- 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. PubMed.
- 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 postmitotic neurons from kainate excitotoxicity. Neuron. 2003 Mar 6;37(5):735-49. PubMed.