Profiling of Parkin-Binding Partners Using Tandem Affinity Purification

<div><p>Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting approximately 1–2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the <i>Parkin</i> gene (<i>PARK2</i>) are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells), we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from <i>Drosophila melanogaster</i>. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function.</p></div

Results from genome-wide analyses of two traits.

<p>Plots show the likelihood ratio test (LRT) and regional, genomic and total heritabilities across the genome from analyses of data from Croatian and Italian populations: a) serum uric acid concentration and b) height. Vertical axis is the LRT and heritability (%) and horizontal axis is window number across the genome. RG h2, WG h2 and total h2 are regional heritability, residual whole genome heritability and total (sum of genomic and regional) heritability, respectively.</p

Direct protein interactions of two ParkinTAP candidates selected as exemplary proteins: LRPPRC (A) and TOMM70A (B).

<p>Proteins are represented as nodes and interactions as edges; the edges are drawn as solid and dashed lines for binary and complex interactions, respectively. Binary interactions to the selected candidates are represented by thicker edges. ParkinTAP ND X are ParkinTAP candidates at network distance X of MonogenicPD, where ParkinTAP ND 1 are direct MonogenicPD interactors. A: LRPPRC. There are many interactors of LRPPRC in iRefIndex, resulting in a dense network of complex interactions. LRPPRC interacts with MonogenicPD PARK7, as well as with 48 other ParkinTAP candidates, and the network includes 14 ParkinIP and 77 MonogenicPDIP. B: TOMM70A. Only eight proteins interact directly with TOMM70A, including one ParkinTAP candidate (HSP90AA1), two ParkinIP (TOMM20, UBC) and one MonogenicPDIP (VDAC1).</p

Comparison of single marker analysis and regional heritability analyses of serum uric acid concentration in a population from Orkney.

<p>-log P values are plotted against position in the genome. Points represent individual markers and circles results from regional heritability analysis with 100 marker windows. Genome-wide significance thresholds are represented by dashed lines (red for single marker analysis, blue for regional heritability analysis). Alternating shades represent the separate chromosomes. Results surpassing the genome-wide significance threshold are solid red for single marker analysis and solid blue for regional heritability analysis.</p

GO biological processes enriched in ParkinTAP and RelatedPD datasets.

<p>GO biological processes enriched in ParkinTAP and RelatedPD datasets.</p

Regional heritability using single and multiple regional relationship matrices (100 SNPs) for Height.

<p>(LRT from all windows were significant at suggestive level).</p>1<p>Likelihood ratio test for regional heritability >0;</p>2<p>Estimated regional heritability in model with that single region and genomic effects;</p>3<p>Minimum heritability for that region from models with sets of 5 regional effects and genomic effect;</p>4<p>Maximum heritability for that region from models with sets of 5 regional effects and genomic effect;</p>5<p>Average heritability for that region over models with sets of 5 regional effects and genomic effect.</p

Functional similarity network FunSimPDsub.

<p>This network includes ParkinTAP candidates and MonogenicPD proteins with a functional similarity score ≥0.7; six significant clusters were identified (P≤0.05).</p

Comparisons of regional heritability and GWAS results for two most significant windows for uric acid concentration.

<p>Comparisons shown on –log₁₀ P basis by conversion of likelihood ratio test (LRT) statistic assuming is distribution is a mixture of half χ₁² and half zero. Lines indicate results for regional heritability for three window sizes (100 SNP = green; 20 SNP = red; 10 SNP = blue). Crosses show results for individual SNPs in GWAS with grey line showing moving average of 10 adjacent SNPs. <b>a</b>) Results for chromosome 4, window 21 (SLC2A9 region). Additional dashed green line is result for 100 SNP window with relationships estimated omitting 14 most significant individual SNPs (in red). <b>b</b>) Results for chromosome 5, window 277. Additional dashed green line is result for 100 SNP window with relationships estimated omitting 3 most significant individual SNPs (indicated by red crosses).</p

Regional heritabilities for uric acid concentration for three most significant windows.

<p>Heritabilities are estimated in a model with both regional and genomic genetic effects using all 100 SNPs in the window to derive regional relationships and fitting 0, 1 or 3 SNPs with the highest –Log₁₀ P value from the GWAS in that window as covariates in the analysis.</p

Summary of results for ParkinTAP candidates passing the first seven selection levels.

<p>Summary of results for ParkinTAP candidates passing the first seven selection levels.</p