Differential mitochondrial protein interaction profile between human translocator protein and its A147T polymorphism variant

Abstract

The translocator protein (TSPO) has been implicated in mitochondrial transmembrane cholesterol transport, brain inflammation, and other mitochondrial functions. It is upregulated in glial cells during neuroinflammation in Alzheimer's disease. High affinity TSPO imaging radioligands are utilized to visualize neuroinflammation. However, this is hampered by the common A147T polymorphism which compromises ligand binding. Furthermore, this polymorphism has been linked to increased risk of neuropsychiatric disorders, and possibly reduces TSPO protein stability. Here, we used immunoprecipitation coupled to mass-spectrometry (IP-MS) to establish a mitochondrial protein binding profile of wild-type (WT) TSPO and the A147T polymorphism variant. Using mitochondria from human glial cells expressing either WT or A147T TSPO, we identified 30 WT TSPO binding partners, yet only 23 for A147T TSPO. Confirming that A147T polymorphism of the TSPO might confer loss of function, we found that one of the identified interactors of WT TSPO, 14-3-3 theta (YWHAQ), a protein involved in regulating mitochondrial membrane proteins, interacts much less with A147T TSPO. Our data presents a network of mitochondrial interactions of TSPO and its A147T polymorphism variant in human glial cells and indicate functional relevance of A147T in mitochondrial protein networks.

Fig 1. Optimization of sample preparation for the identification of binding partners between human TSPO and its mutant A147T by IP-MS.

A) Transfection efficiency of V5-tagged TSPO^WT and TSPO^A147T in U87MG cells (42.6% ± 5.93), indicating equal protein expression levels; red, anti-V5 immunofluorescence and blue, DAPI indicating cell nuclei. n = 3. B) Mitochondrial enrichment of U87MG cells as assessed by Western blot for VDAC as a mitochondrial marker protein and hnRNP as a nuclear marker protein, indicating that purified mitochondria were isolated, n = 2. C) Representative immunoblot of mitochondrial lysates from transfected U87MG cells with V5-tagged TSPO^WT and TSPO^A147T. This confirms equal expression levels of V5 in both TSPO^WT and TSPO^A147T extracts. D) Immunoprecipitation of TSPO^WT and TSPO^A147T from transfected U87MG cells with increasing amount of mouse V5 antibody, with 2μg per 400 μg of total protein chosen for subsequent experiments. Non-specific mouse IgG antibody was used as a negative control. Immunoblot of input (10% of the total lysates) confirms comparable expression levels of V5 tag between TSPO^WT and TSPO^A147T. Immunoblot of unbound V5 tag shows the amount of non-precipitated TSPO. E) Immunoprecipitation of TSPO^WT and TSPO^A147T from transfected U87MG cells. Precipitated proteins were visualized by silver staining of SDS-PAGE.

Fig 2. Identification of binding partners of hTSPO^WT and hTSPO^A147T by label free semiquantitative proteome analysis.

A) Diagram of the immunoprecipitation-mass spectrometry approach to identify the TSPO^WT and TSPO^A147T interactomes. We identified 56 proteins for V5-tagged human TSPO^WT and 47 proteins for human TSPO^A147T, of which two thirds comprised of mitochondrial proteins and the remainder were cytoplasmic and nuclear proteins. Of these, 23 selected candidates interacted with both TSPO^WT and TSPO^A147T and 7 candidates interacted only with TSPO^WT, after further selection criteria with protein fold difference of p<0.01–0.1 B) A functional protein association network generated using STRING (v10.5) and Cytoscape (v6.3.0) bioinformatics software. Network edges represent protein-protein associations and represent confidence, with the strength of the association represented by line thickness. The circles (nodes) depict specific proteins, and the color represents sub-cellular localization of each protein, as shown in the legend. The 7 protein candidates which interact solely with TSPO^WT are indicated on nodes with red outlines. Nodes with no network edges represent proteins that have never previously been reported as TSPO interaction partners.

Fig 3. Validated interactions of hTSPO^WT and hTSPO^A147T.

A) Co-immunoprecipitation (IP) of voltage-dependent anion channel 1 (VDAC1) and hTSPO^WT or hTSPO^A147T with VDAC antibody and detected by immunoblotting for V5 and VDAC1. GAPDH was used as loading control and did not co-precipitate with VDAC1. A comparable amount of V5-TSPO was co-purified with VDAC1 with each of TSPO^WT and TSPO^A147T. B) Co-IP of heat shock protein HSP90-alpha (Hsp90AA1) and hTSPO^WT or hTSPO^A147T with Hsp90AA1 antibody and detected by immunoblotting for V5 and Hsp90AA1. A comparable amount of V5-TSPO was co-purified with Hsp90AA1 with each of TSPO^WT and TSPO^A147T. C-D) 14-3-3 θ and sequestosome 1 (SQSTM1) were expressed in U87MG cells together with V5-tagged human TSPO^WT or TSPO^A147T. C) Co-IP of 14-3-3 θ and hTSPO^WT or hTSPO^A147T with V5 antibody and detected by immunoblotting for myc and V5. 14-3-3 θ was co-purified with V5 and weaker interaction was observed in TSPO^A147T compare with TSPO^WT. D) Co-IP of SQSTM1 and hTSPO^WT or hTSPO^A147T with V5 antibody and detected by immunoblotting for myc and V5. Comparable amount of SQSTM1 was co-purified with TSPO^WT and TSPO^A147T. Results from 3 independent experiments were quantified by densitometry using Image J and represented as mean ± S.D. (Student t test) * p <0.05.