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

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.

Introduction

The 18kDa translocator protein (TSPO) [1] is a five transmembrane domain protein that resides in the outer mitochondrial membrane (OMM), at contact sites between the outer and inner mitochondrial membrane [2]. TSPO binds cholesterol with high affinity and possibly facilitates its transport across the OMM [3]. However, its physiological role in vivo remains elusive. It has previously been reported that TSPO null mice present without overt phenotypes [4].

TSPO is highly conserved across species [5] and is ubiquitously expressed [6], with higher levels in cells and organs dedicated to steroid synthesis, such as the adrenal gland cortex [7]. Interestingly, several brain conditions, including Alzheimer’s disease (AD), Parkinson’s disease (PD) and traumatic brain injury (TBI), show a significant increase in expression levels of TSPO, reflecting microglial activation that is likely to result in the production of protective neurosteroids, as a homeostatic response [8]. This has been utilized diagnostically with the development of a large number of TSPO ligands used for imaging with positron emission tomography (PET) [9, 10]. TSPO has furthermore been suggested as a drug target for neurodegenerative conditions [11] and may also be of therapeutic interest in cancer, as TSPO ligands reduce proliferation of cancer cells [12].

A single nucleotide polymorphism (rs6971) in the TSPO gene, which resulted in the substitution of alanine by threonine at position 147 (A147T) in the helical turn before the cholesterol binding motif, has hampered the use of TSPO radio-labelled ligands as an active biomarker for inflammation by in vivo visualization (PET imaging) [13]. This one point mutation is observed predominantly in Caucasian populations, in which approximately 10% are homozygous and up to 30% are heterozygous [14]. The A147T polymorphism not only decreases the binding affinities of many TSPO ligands but has also been shown to affect pregnenolone production and was found to be associated with neurological diseases like bipolar disorder [15, 16]. However, these findings were indirectly demonstrated through the actions of its ligands. To obtain insight into the functional relevance of this A147T point mutation, we asked how this mutation might impact the protein interactome of TSPO within the mitochondrial complex. Therefore, we transfected human glioma cell U87MG with full length human TSPO or its A147T variant (both with C-terminal V5 tags) and isolated mitochondria from these cells. These mitochondria enriched fraction was subsequently immunoprecipitated for the tagged proteins and further subjected to mass spectrometry (IP-MS). Selected interaction partners were further validated by co-immunoprecipitation.

Materials and methods

Mitochondria isolation

Mitochondria were isolated from U87MG cells based on a previously described protocol [17]. Briefly, cell pellets were resuspended in 1 mL aliquots of ice-cold RSB hypo buffer (10 mM NaCl, 1.5 mM MgCl2, 10 mM Tris-HCl (pH 7.5) containing complete EDTA-free protease inhibitor) and transferred to a 2-mL Dounce homogenizer. Cells were allowed to swell for 5–10 min. The progress of the swelling was checked using a phase-contrast microscope. The swollen cells were lysed with several strokes of the B pestle, pressed straight down the tube with a firm, steady pressure. Immediately 500μL of ice-cold 2.5× MS homogenization buffer (525 mM mannitol, 175 mM sucrose, 12.5 mM Tris-HCl (pH 7.5), and 2.5 mM EDTA (pH 7.5) with protease inhibitor) was added to each sample to obtain a final concentration of 1× MS homogenization buffer. Subsequently, the top of the homogenizer was covered with Parafilm and mixed by inverting twice. A portion of the homogenate was retained to perform enzyme marker assays. Then the homogenate was transferred to a centrifuge tube for density gradient centrifugation. The final volume was brought to 2 mL with ice-cold 1× MS homogenization buffer (210 mM mannitol, 70 mM sucrose, 5 mM Tris-HCl (pH 7.5), and 1 mM EDTA (pH 7.5) with protease inhibitor). Afterwards, the homogenate was centrifuged at 1300×g for 5 min to remove nuclei, unbroken cells, and large membrane fragments. The supernatant was poured into a clean centrifuge tube and centrifuged again at 1300×g for 5 min. This centrifugation step was repeated two more times. The supernatant from the third centrifugation was transferred to a clean centrifuge tube and centrifuged at 17,000×g for 15 min to obtain the mitochondria (pellet). The isolated mitochondria were washed in ice-cold 1× MS buffer and centrifuged at 17,000xg. The supernatant was discarded, and the resulting pellet (mitochondria) was resuspended in NP40 buffer containing protease inhibitor.

Immunoprecipitation

Immunoprecipitation (IP) of transfected cells was carried out as previously described [18]. Following harvesting the transfected cells and isolating the mitochondria, the sample(s) were pre-incubated with protein G-coupled magnetic beads (Invitrogen) for 1 h. Samples were then incubated with antibodies against V5 overnight at 4°C. The following day, the samples were incubated with protein G-coupled magnetic beads (1 h at 4°C on a rotating platform) and then washed 4 times with IP buffer (50mM HEPES (pH 7.5), 140mM sodium chloride, 0.2% (v/v) NP40 with protease inhibitors). The precipitate was recovered from the beads by boiling in hot (95°C) sample buffer (1M Tris-HCl (pH 8.0), 20% (v/v) β-mercaptoethanol, 40% (v/v) glycerol, 9.2% (w/v) SDS and 0.2% (w/v) bromophenol blue) and proteins were separated by SDS-PAGE, Western blot transferred to PVDF membrane, and probed for Myc (1:5000) and V5 (1:5000). The second approach of IP of transfected cells was carried out by immunoprecipitation with antibodies against respective interacting proteins. The subsequent procedure was similar to that outlined above, and immunoblotting was carried out with V5 (1:5000).

Immunoblotting

For SDS PAGE and Western blot analysis of TSPO transfected cells, 10μg protein was loaded onto SDS PAGE gels as the input and the entire immunoprecipitate for each sample was loaded in successive lanes. Proteins were subsequently electrophoretically transferred onto nitrocellulose membranes (GE Healthcare) in a Trans-Blot SD Semidry Transfer Cell (BioRad) at 25V for 45 min. Membranes were subsequently blocked in 5% (w/v) BSA in Tris-buffered saline containing 0.1% (v/v) Tween20 (TBS-T) for 1 h at ambient temperature, followed by incubation with the primary antibody diluted in 5% (w/v) BSA/TBS-T (overnight at 4°C on a shaking platform). For IP experiments using TSPO-transfected cells, the primary antibodies used were to V5 (1:5000), myc (1:5000), VDAC1 (1:1000), Hsp90AA1 (1:2000), YWHAE (1:1000), YWHAZ (1:1000), or YWHAG (1:1000). The following day the primary antibody was removed, the membranes washed three times with TBS-T and incubated with alkaline phosphatase-coupled secondary antibodies (1:10,000, Sigma) in 1% (w/v) BSA/TBS-T for 45 min at room temperature. Protein bands were visualized with Immobilon Chemiluminescent Alkaline Phosphatase substrate (Millipore) and detected in a VersaDoc Model 4000 CCD camera system (BioRad). To determine equal loading and for normalization, membranes were stripped by washing in distilled water for 5 min, followed by 0.2M sodium hydroxide for 10 min and then distilled water for 5 min after which they were reprobed with antibodies against GAPDH.

Expression plasmids

To obtain expression constructs for transient transfection of U87MG cells, the coding sequence encoding human TSPO (hTSPO) was amplified from cDNA previously generated by reverse transcription from total HEK293 mRNA isolated using Trizol (Life Technologies) and cloned into either a pENTR/SD/TOPO vector (Invitrogen) or pGEM-T-Easy vector (Promega). For cloning into pGEM-T-Easy vector, hTSPO was amplified by PCR using PfuTurbo High-Fidelity DNA Polymerase (Agilent Technologies). A polyA tail was added to this blunt-end PCR product and ligated into the pGEM-T-Easy vector overnight at 4°C. Ligated vectors are transformed into One Shot Top10 E. coli for pENTR/SD/TOPO, DH5αE. Coli for pGEM-T-Easy vectors and plated out onto plates containing 50 μg/mL kanamycin (pENTR/SD/TOPO) and 100 μg/mL ampicillin (pGEM-T-Easy). Positive clones were identified via restriction digest and confirmed via DNA sequencing.

Cloning and site-directed mutagenesis

Site-directed mutagenesis was carried out to introduce the A147T point mutation into the hTSPO cDNA according to the Agilent Technologies’ Quikchange XL Site-directed Mutagenesis Kit instructions. Briefly, mutagenic oligonucleotide primers incorporating the A147T point mutation were used to introduce the mutation into hTSPO in pGEM-T-Easy. Twenty-five cycles of PCR were carried out in a final volume of 50μl, containing 1ng of hTSPO in pGEM-T-Easy as template, 10pmol of forward and reverse primers, and 2.5 units of PfuTurbo. The annealing temperature was set to 58°C. The PCR products generated were digested with 10 units of Dpn I restriction enzyme at 37°C for 3 h to digest the parental DNA template. Digested PCR products were transformed into XL1 blue competent cells and plated onto agar plates containing 100 μg/mL Ampicillin. Positive clones were confirmed via DNA sequencing analysis.

Transformation of plasmid DNA

Chemically competent E. coli cells (100 μl) were thawed on ice and transferred into a chilled 14 mL polypropylene round-bottom tube (BD Biosciences). Then 50ng of the desired plasmid DNA was added to the E. coli and the E. coli/DNA mixture was incubated for 20–30 min on ice. Subsequently, this E. coli/DNA mix was heat-shocked for 40 seconds in a 42°C water bath and cooled on ice for 2 min. S.O.C medium (250 μl, Life Technologies) was added and tubes shaken at 200 rpm for 1 h at 37°C. The transformed E. coli were plated onto Lysogeny Broth (LB) agar plates with the appropriate antibiotics and incubated overnight at 37°C. For subsequent analysis of clones obtained, single colonies were picked and inoculated in LB medium containing ampicyline, incubated overnight at 37°C and plasmid DNAs were purified using the Promega Wizard mini-prep kit.

Transient transfection

The polyethylenimine (PEI) method of transfection was used to deliver plasmid DNA into U87MG cells in a 10 cm dish as previously described [19] with minor modifications. Briefly, cells were plated at the density of 0.5 x 106 per 10 cm dish and allowed to become adherent. The next day, when cells reached 60–80% confluency, the media was changed 30 min before transfection. Then, the PEI-DNA mixture was prepared using 3:1 ratio of PEI to DNA (w/w). Briefly, 30 μg of PEI (1 g PEI dissolved in 1000 mL distilled water pH7) was diluted into a total volume of 750 μl of 0.9% NaCl. Then, 10 μg of DNA was diluted into a total volume of 750 μl of 0.9% NaCl. Subsequently, the diluted DNA was added to the diluted PEI and incubated at ambient temperature for 15 min. The PEI-DNA mixture was then added dropwise to the 10 cm dish and left for 48 h, after which the cells were ready to be harvested in NP-40 lysis buffer with protease inhibitor.

Mass-spectrometry and data analysis

Briefly, IP samples were desalted, and buffer exchanged using 3-kDa Amicon filter units (EMD Millipore) using 50 mM sodium bicarbonate. Protein sample (400 μg) was reduced by adding 2 μl of Tris(2-carboxyethyl)phosphine (Sigma) and incubating samples at 60°C for 1 h. Samples were then alkylated by adding 1 μl of iodoacetamide (37 mg/mL) to block cysteine side chains (10 min, ambient temperature). Protein samples were digested using 4 μg of trypsin (sequencing grade, low autolysis trypsin; Promega), at 37°C for 16 h. Digested samples were briefly spun in a microcentrifuge and pH checked and, if necessary, adjusted to pH 9–10 with a few μL of sodium carbonate (500 mM Na2CO3). Samples were analyzed in biological triplicates (6 μg injected per run) by LC-MS/MS (HCD on the QExactive Plus: Thermo Electron, Bremen, Germany), followed by Mascot searching to identify peptides and proteins. Adaptations of previously published protocols were used [20, 21].

Only proteins with ≥2 annotated peptides in Mascot were used for further analysis. The Mascot Score is a statistical score for how well the experimental data match the database sequence [22], and Mascot parameters included using 15-ppm parent mass tolerance, 0.5 Da fragment mass tolerance, and Protein False Discovery Rate equal or lower than 1%. Searches were conducted against several databases such as Uniprot, Swissprot, and NCBI. Mascot results are statistically interpreted using a Java-based re-implementation of the Peptide Prophet Algorithm [23], which converts search engine scores into probabilities of peptide identification. Data compilation and quantification across 3 IP biological replicates for each of the experimental groups were performed using Scaffold 2.4.0 and a 95% protein probability threshold was applied. Scaffold computes protein grouping at two levels: first to determine the identified protein groups within a single MS/MS sample, and again across all MS/MS samples in the experiment to determine an experiment wide protein identification list [24]. Scaffold uses a reimplementation of the Peptide Prophet algorithm to combine peptides into protein identifications using a statistical model that considers a distribution of the number of peptides assigned to each protein. Quantitative analysis from ScaffoldQ was utilized by averaging over the 3 biological replicates and comparisons applying a value (Student’s test) criterion of < 0.05 to determine the statistically significant differentially bound proteins. Protein ontology was analyzed using DAVID gene ontology database (v6.8) using gene ontology annotations for the entire human genome as the background list. Interactomes and network properties were visually represented using CytoScape (v3.7.1). Existing evidence for protein–protein interactions of TSPO was derived from BioGRID (v3.1.180) and STRING (v10.5) databases and through literature searching.

Results

Identification of the hTSPOWT and hTSPOA147T interacting proteins in human glioma cells

Interactions of TSPO with other proteins have been characterized by conventional methods, such as in-gel digestion followed by mass-spectrometry (MS) analysis [25] or pull-down methods [26] which are limited by antibody affinity, lysis, and solubility conditions. Moreover, identification of interaction partners of the cholesterol-binding domain of TSPO (the CRAC domain) have been identified by coimmunoprecipitation [27]. However, identification of TSPO interaction partners in the context of the frequent A147T polymorphism have not been studied. To gain insight into the effects of the A147T polymorphism on mitochondrial TSPO interactions, we employed immunoprecipitation coupled to mass-spectrometry (IP-MS). The use of IP-MS is considered the gold standard in regards to sensitivity and specificity for the identification of protein interactions (complexes) [28]. We tagged full-length human TSPO and TSPO with a C-terminal V5 tag for expression in U87MG cells, a human astro-/microglioma cell line of CNS origin [29]. V5-tagged TSPO protein levels were equally transfected for both TSPO and TSPO (). Isolated mitochondria from these cells showed enrichment of the mitochondrial protein voltage-dependent anion channel 1 (VDAC1) and absence of the nuclear protein heterogenous nuclear ribonucleoproteins (hnRNP), indicating successful purification of mitochondria ( and Supporting information for uncropped blots for the referenced western blots figures throughout this study with areas displayed in the main figures highlighted as red broken boxes). Furthermore, equal expression levels of V5-tagged TSPOWT and TSPOA147T was confirmed in isolated mitochondria by the immunoblotting (). Optimization of the V5 antibody for IP showed that 2 μl of antibody per 400 μg total protein was the optimum condition to pull down both V5-tagged TSPOWT and TSPOA147T (). To assess the specificity of V5 antibody and to control for non-specific binding, both isolated mitochondria of TSPOWT and TSPOA147T expressing cells as well as non-transfected cells were immunoprecipitated using IgG antibody that served as negative control (). Immunoblot of input (10% of the total lysates) further confirmed comparable expression levels between TSPOWT and TSPOA147T whereas immunoblot of unbound V5-tagged proteins (FT = flow through) showed a relatively small amount of non-precipitated TSPO (). Next, one third of the IP samples were run by SDS PAGE, silver stained, and the result indicated that TSPOA147T had fewer bands and/or weaker intensity compared to TSPOWT samples (). Overall, these results show that V5-tagged TSPO expression in TSPOWT and TSPOA147T yields similar expression levels in isolated mitochondria of U87MG cells, which supported efficient and comparable immunoprecipitation for subsequent MS.

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 TSPOWT and TSPOA147T 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 TSPOWT and TSPOA147T. This confirms equal expression levels of V5 in both TSPOWT and TSPOA147T extracts. D) Immunoprecipitation of TSPOWT and TSPOA147T 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 TSPOWT and TSPOA147T. Immunoblot of unbound V5 tag shows the amount of non-precipitated TSPO. E) Immunoprecipitation of TSPOWT and TSPOA147T from transfected U87MG cells. Precipitated proteins were visualized by silver staining of SDS-PAGE.

To identify the interaction partners of V5-tagged TSPOWT and TSPOA147T, we performed MS of enriched mitochondrial fractions from U87MG cells at sub-confluency and transfected with constructs expressing either V5-tagged TSPO, V5-tagged TSPO, or non-transfected cells. A schematic outline of the IP-MS analysis procedure and results is shown in . For protein identification, immunoprecipitated proteins were trypsin digested in-solution and the resulting peptides were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). The mass spectrometric data contain a list of the accurate peptide masses and peptide fragment masses. These were searched against sequence databases containing known protein amino acid sequences using the Mascot software package. Based on Mascot LC-MS/MS search engine (Swissprot Database, human taxonomy), 647 unique proteins were identified, with a protein False Discovery Rate ≤1% and were identified in TSPOWT, TSPOA147T, and non-transfected (WT) mitochondria that were immunoprecipitated by either V5 antibodies or control IgG (). Further manual analysis using Scaffold software was carried out to exclude peptide sequences that were present in both the wild-type TSPO samples as well as the IgG controls, yielding a final list of 307 proteins expressed specifically in TSPOWT, TSPOA147T, samples, but not expressed (or minimally) in the controls. Of these, 44 proteins were known mitochondrial proteins, 51 proteins were of cytoplasmic localization, and the rest were nuclear GO annotated proteins. Subsequently, further manual stringent measures were taken to remove peptide sequences that were only present in less than 2 out of 3 IP TSPOWT and TSPOA147T replicates. This further limited the number of candidate proteins to 56 for TSPOWT and 47 for TSPOA147T. A significant difference (p<0.01-p<0.1) in total spectral counts between three biological replicates of each of TSPOWT and TSPOA147T was a further selection criterion applied to identify TSPO binding partners. Twenty-three candidate proteins interacted with both TSPOWT and TSPOA147T, and 7 candidate proteins interacted only with TSPOWT (). A functional protein association network analysis of the identified TSPO interaction partners was generated using STRING and Cytoscape bioinformatics software and are shown in . All of these candidate proteins were in the top 50% of the highest interaction probability and abundance (spectral counts). Of note are known interacting partners of TSPO, including VDAC1, VDAC2, VDAC3, and ATAD3A that were also identified in our IP-MS experiments, increasing confidence in our approach ().

Identification of binding partners of hTSPOWT and hTSPOA147T by label free semiquantitative proteome analysis.

A) Diagram of the immunoprecipitation-mass spectrometry approach to identify the TSPOWT and TSPOA147T interactomes. We identified 56 proteins for V5-tagged human TSPOWT and 47 proteins for human TSPOA147T, of which two thirds comprised of mitochondrial proteins and the remainder were cytoplasmic and nuclear proteins. Of these, 23 selected candidates interacted with both TSPOWT and TSPOA147T and 7 candidates interacted only with TSPOWT, 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 TSPOWT are indicated on nodes with red outlines. Nodes with no network edges represent proteins that have never previously been reported as TSPO interaction partners.

Analysis of the TSPOWT and TSPOA147T interactomes by protein domain ontology identified an enriched set of mitochondrial outer membrane proteins (. DAVID INTERPRO domain enrichment cluster 1, p = 3.7 x 10–9) and ion channel binding proteins (. DAVID INTERPRO domain enrichment cluster 2, p = 1.5 x 10–6). Mitochondrial outer membrane proteins constituting cluster 1 were VDAC1, VDAC2, VDAC3, HK2, MGST1, TOMM20, and TOMM40 (), whereas ion channel binding proteins constituting cluster 2 were HSP90AA1, HSP90AB1, YWHAE, YWHAQ, and VDAC1 ().

Our IP-MS results identify interactions of TSPOWT and TSPOA147T in cultured glial cells with proteins of heterogeneous functions, including protein transmembrane transport into intracellular organelle, ion channel binding and regulation of protein targeting, possibly explaining the multiple functions of TSPO in cells. Importantly, analysis of the TSPOA147T interactome revealed the loss of interactions with 7 proteins as compared to TSPOWT, namely mitochondrial import receptor subunit TOM40 homolog (TOMM40), 14-3-3 protein subunit epsilon (YWHAE), 14-3-3 protein subunit tau/theta (YWHAQ), hexokinase 2 (HK2), sequestosome 1 (SQSTM1), phosphatidylserine synthase 1 (PTDSS1), and trans-2,3 enoyl-CoA reductase (TECR) (). Taken together, the A147T point mutation of TSPO might result in a loss of interacting partners important for mitochondrial and cell function.

Validation of selected human TSPO protein-protein interactions

Specific interactions of several identified candidate binding partners of human TSPOWT and TSPOA147T were confirmed by co-immunoprecipitation (co-IP) from transiently transfected cells followed by immunoblotting. The chosen candidate binding partners were voltage-dependent anion-selective channel protein 1 (VDAC1), heat shock protein HSP90-alpha (HSP90AA1), 14-3-3 protein subunit theta (YWHAQ) and sequestosome-1 (SQSTM1). Two approaches of co-IP were employed. The first approach involved pull-down of cells overexpressing either V5-tagged TSPOWT or TSPOA147T, with antibodies against their respective interacting proteins and then immunoblotted for TSPO with V5 antibodies. The candidate proteins validated with this approach were VDAC1 and HSP90AA1. Cells overexpressing V5-tagged pcDNA was used as a negative control. As expected from our MS results, there were no differences in VDAC1 () and HSP90AA1 interaction () with either TSPOWT or TSPOA147T, respectively.

Validated interactions of hTSPOWT and hTSPOA147T.

A) Co-immunoprecipitation (IP) of voltage-dependent anion channel 1 (VDAC1) and hTSPOWT or hTSPOA147T 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 TSPOWT and TSPOA147T. B) Co-IP of heat shock protein HSP90-alpha (Hsp90AA1) and hTSPOWT or hTSPOA147T 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 TSPOWT and TSPOA147T. C-D) 14-3-3 θ and sequestosome 1 (SQSTM1) were expressed in U87MG cells together with V5-tagged human TSPOWT or TSPOA147T. C) Co-IP of 14-3-3 θ and hTSPOWT or hTSPOA147T 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 TSPOA147T compare with TSPOWT. D) Co-IP of SQSTM1 and hTSPOWT or hTSPOA147T with V5 antibody and detected by immunoblotting for myc and V5. Comparable amount of SQSTM1 was co-purified with TSPOWT and TSPOA147T. Results from 3 independent experiments were quantified by densitometry using Image J and represented as mean ± S.D. (Student t test) * p <0.05.

The second approach involved co-IP with V5 antibody, of cells overexpressing either V5-tagged TSPOWT, TSPOA147T or pcDNA together with their respective interacting proteins tagged with Myc. The candidate proteins validated with this approach were 14-3-3 protein subunit theta (YWHAQ) and sequestosome-1 (SQSTM1). TSPOA147T had significantly lower interaction of YWHAQ compared to TSPOWT, indicating weaker YWHAQ interaction due to the A147T mutation (). Whereas there was no detectable difference in SQSTM1 interaction with TSPO between TSPOWT and TSPOA147T (). 14-3-3 protein subunit eta (YWHAH) and 14-3-3 subunit beta (YWHAB) were not on the interacting proteins list, and were therefore used as negative controls. Both YWHAH and YWHAB did not co-purified with TSPO, as expected, indicating the robustness and specificity of the mass-spectrometry list of interacting partners ().

Discussion

Protein interactions of human full-length TSPO and its domain involving cholesterol recognition/interaction amino acid consensus (CRAC) motif have been documented in previous studies, which used blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to MS [25] and computational modelling with in-silico screening [30], respectively. However, the effect of the common TSPO polymorphism A147T on protein interactions remains unknown. To date, only the effect of A147T TSPO on protein structure and stability have been reported [31, 32]. Our current study identified putative binding partners of human full-length TSPO and its A147T polymorphism variant using IP-MS biased towards mitochondrial proteins. Employing IP-MS of mitochondrial proteins isolated from cells overexpressing either TSPOWT and TSPOA147T and downstream rigorous peptide analysis using Scaffold software [24], we detected 30 protein-protein interactions (PPIs) of hTSPOWT and 23 PPIs of hTSPOA147T with high confidence, suggesting that the A147T variant of human TSPO results in an alteration of the TSPO protein interactome. The A147T mutation not only significantly alters the protein flexibility and stability, but also decreases the half-life of the mutant protein by about 25 percent [31]. Such structural changes would increase TSPOA147T turnover, and may, at least in part, explain its effect on reducing the number of protein interactions.

Among the 30 TSPOWT PPIs and 23 TSPOA147T PPIs were a number of known mitochondrial TSPO interaction partners, increasing our confidence in our proteomic data sets. These included the voltage-dependent anion-selective channel protein 1 (VDAC1) [27], voltage-dependent anion-selective channel protein 2 (VDAC2), voltage-dependent anion-selective channel protein 3 (VDAC3) [33], and ATPase family AAA domain-containing protein 3A (ATAD3A) [25]. Since we performed the IP-MS using isolated mitochondria, it is not surprising that, mitochondrial proteins were the most prevalent amongst the 30 TSPOWT PPIs and 23 TSPOA147T PPIs identified. However, we also identified a number of nuclear and cytoplasmic proteins as TSPO interaction partners. Although this may reflect impurities during mitochondrial preparations, an alternative explanation is that this reflects interactions at the surface of mitochondria, where TSPO resides [34]. We found that the A147T polymorphism in TSPO is associated with loss of a small subset of interaction partners implicated in mitochondrial outer membrane organization, ion channel binding, and protein targeting functions. Of the confirmed interaction partners, only 14-3-3 protein subunit theta (YWHAQ) showed weaker interaction with TSPOA147T, suggesting this point mutation modulates specifically protein interactions. Our results suggest that the A147T polymorphism of human TSPO partially changes its interactome, and thus its molecular function, as compared with non-mutant full-length human TSPO. Our IP-MS data suggests differential interaction of TSPOWT and TSPOA147T with outer mitochondrial membrane proteins HK2 and TOMM40 with hTSPOA147T, as well as ion channel binding proteins and targeting proteins YWHAE and YWHAQ. Whether these normal interactions are directly with TSPOWT or indirectly remains to be shown. Indirect interactions of TSPO with nuclear proteins have been reported for YWHAE through VDAC1 protein [35]. Indirect interaction via VDAC1 or VDAC2 [27, 36] are unlikely, since their interaction with TSPO was not compromised by the A147T polymorphism. TSPO interaction with YWHAG is mediated through Ser-194 of the steroidogenic acute regulatory protein (STAR) [37]. However, YWHAG was not identified in our study, possibly due to the different cell types used by Aghazadeh and colleagues (MA10 mouse Leydig cells), that have a very different protein expression pattern compared to the human glioblastoma cells (U87MG) used in our study [38].

To our knowledge, this is the first report of YWHAQ as an interactor of TSPOWT and with reduced affinity to hTSPOA147T. More than 200 proteins have been found to interact with 14-3-3 family members (there are seven isoforms in mammals: β, γ, ε, η, ζ, σ, and τ/θ), a group of proteins which are not only steroidogenesis-related proteins but also include protein kinases, enzymes, cell cycle control-related proteins, and apoptosis-related proteins [39]. In addition, 14-3-3 proteins localize to disease-specific injury sites and toxic protein aggregates in the brain, which may contribute to the regulation of disease pathogenesis [40]. As a result, 14-3-3 proteins may have either a protective role by facilitating sequestration of pathogenic proteins, or a detrimental role by promoting protein aggregate formation, neurotoxicity, and/or loss of stabilization of binding partners [41]. For example, YWHAQ and YWHAE have been found to inhibit apoptosis induced by rotenone in a cellular model of PD [42]. Based on DAVID Functional Clustering enrichment analysis, a possible role of the TSPO’s interaction with YWHAQ could be the regulation of TSPO’s targeting into mitochondrial membranes involved in apoptotic signaling pathways. But future studies are required to provide direct evidence. Since YWHAQ is known to inhibit apoptosis [42], and TSPO has been shown to be involved in apoptosis via its interaction with VDAC by generating ROS production which leads to activation of mitophagy [43], carriers of the A147T TSPO may be more prone to cellular apoptosis through its weaker interaction with YWHAQ. Understanding the role(s) of this interaction with TSPO warrants further investigation.

TSPO interaction with VDAC has been well-documented for more than two decades [36] and is consistently observed in the transduceosome that bridges the inner and outer mitochondrial membrane. This interaction is best characterized for its role in hormone-induced steroidogenesis [44]. The direct interaction of TSPO with VDAC has been demonstrated with various methods including blue native PAGE [25], immunoprecipitation and microscopy [44] and copurification [45]. However, the mode of this interaction at the molecular level is still not well characterized, despite the advent of high-resolution crystal structures of both TSPO [46] and VDAC [47, 48]. Heat shock proteins (HSPs), including HSP90AA1, are known to play an important role in delivering proteins to the outer mitochondrial membrane (OMM) for import and facilitating translocation in an ATP dependent manner [49].

Validated non-interactors of hTSPOWT and hTSPOA147T.

A-B) 14-3-3 η protein (YWHAH) and 14-3-3 β (YWHAB) were expressed in U87MG cells together with V5-tagged human TSPOWT or TSPOA147T. A) Co-IP of YWHAH and hTSPOWT or hTSPOA147T with V5 antibody and detected by immunoblotting for myc. YWHAH was not co-purified with V5. B) Co-IP of YWHAB and hTSPOWT or hTSPOA147T with V5 antibody and detected by immunoblotting for myc. YWHAB was not co-purified with V5.

(PDF)

Click here for additional data file.

Mascot LC-MS/MS results from immunoprecipitations of hTSPOWT, hTSPOA147T and non-transfected (WT) mitochondria that were immunoprecipitated by either V5 antibodies or control IgG.

647 unique proteins were identified, based on 1 or more unique peptide(s) with total spectrum count >2 and with a protein False Discovery Rate ≤1%.

(XLSX)

Click here for additional data file.

27 Jul 2021

PONE-D-21-18913

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

PLOS ONE

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Additional Editor Comments:

This is very good attempt to see the interactions, but results are not very promising. Further supporting data are required . Author may give insights of additional studies if possible and should add a detailed future studies required in this directions. It will help to those who are trying to establish new insights for this protein in different pathological conditions.

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The manuscript of the paper entitled "Differential mitochondrial protein interaction profile between human translocator protein and its A147T polymorphism variant' is presented in a proper manner. However, author should consider following corrections before resubmission:

1.Page 10, L16 replace ‘cOmplete with ‘complete’

2.Page 10, L17; Page 10, L 22; Page 11, L 6, replace ‘))’ with ‘)’

3.Page 11, L 13 replace ‘inhibitor .’ with ‘inhibitor.’

4.Maintain consistency in units such as 50 mM instead of 50mM, 4oC instead of 4o C, min instead of Minutes, h instead of hour, either mL or ml, g instead of gram, 10 cm instead of 10cm, μl instead of microlitres etc

5.Page 12, L 13-15, L16-18 correct the sentences

6.Page 17, L12-14, reframe the sentence

7.Page 20, line 8, Replace ‘experiments .’ with ’experiments.’

I found the experiments were executed in a planned way. Technically manuscript is good.

Reviewer #2: An interesting research work by Lars M Ittner and team.

This is first such study to see interaction profile between human translocator protein and its A147T polymorphism variant.

In summary, the results identified the loss of a subset of interaction partners with the 6 A147T polymorphism variant of human TSPO but one of the positive aspects seems

"TSPO’s interaction with YWHAQ 7 might contribute to the regulation of TSPO’s targeting into mitochondrial membranes involved 8 in apoptotic signaling pathways"

Does author further support it by additional evidence.

**********

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Reviewer #1: Yes: Dr Pooja Srivastava

Reviewer #2: No

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7 Feb 2022

Point-to-point response to reviewer comments

We thank both reviewers for their comments that have been addressed as indicated below. We hope you find the revised version of the manuscript ready for publication in PLOS ONE.

Reviewer #1: The manuscript of the paper entitled "Differential mitochondrial protein interaction profile between human translocator protein and its A147T polymorphism variant' is presented in a proper manner. However, author should consider following corrections before resubmission:

1.Page 10, L16 replace ‘cOmplete with ‘complete’

2.Page 10, L17; Page 10, L 22; Page 11, L 6, replace ‘))’ with ‘)’

3.Page 11, L 13 replace ‘inhibitor .’ with ‘inhibitor.’

4.Maintain consistency in units such as 50 mM instead of 50mM, 4oC instead of 4o C, min instead of Min, h instead of h, either mL or ml, g instead of gram, 10 cm instead of 10cm, μl instead of microlitres etc

5.Page 12, L 13-15, L16-18 correct the sentences

6.Page 17, L12-14, reframe the sentence

7.Page 20, line 8, Replace ‘experiments .’ with ’experiments.’

Response: We thank this reviewer for the detailed comments and have made the requested changes. All changes have been highlighted in the marked version of the revised manuscript for your convenience.

I found the experiments were executed in a planned way. Technically manuscript is good.

Response: This is very much appreciated.

Reviewer #2: An interesting research work by Lars M Ittner and team.

This is first such study to see interaction profile between human translocator protein and its A147T polymorphism variant.

In summary, the results identified the loss of a subset of interaction partners with the 6 A147T polymorphism variant of human TSPO but one of the positive aspects seems

"TSPO’s interaction with YWHAQ 7 might contribute to the regulation of TSPO’s targeting into mitochondrial membranes involved 8 in apoptotic signaling pathways"

Does author further support it by additional evidence.

Response: We have carefully reworded this sentence to indicate that future investigation is needed to provide direct evidence. It reads now: “Based on DAVID Functional Clustering enrichment analysis, a possible role of the TSPO’s interaction with YWHAQ could be the regulation of TSPO’s targeting into mitochondrial membranes involved in apoptotic signaling pathways. But future studies are required to provide direct evidence.”

Submitted filename: PtP letter revision.pdf

Click here for additional data file.

6 Apr 2022

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

PONE-D-21-18913R1

Dear Author

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Anjani Kumar Tiwari, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

This form of manuscript is ok for publication.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: N/A

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: No

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: As i can see that nearly all the questions asked by experts have been replied in revised manuscript therefore in my opinion Academic Editor may permit for publication in PLOS ONE

Reviewer #3: All the queries raised by previous reviewers have been replied, therefore it can be considered for publication in PLOS One.

**********

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Reviewer #2: No

Reviewer #3: No

28 Apr 2022

PONE-D-21-18913R1

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

Dear Dr. Ittner:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

Dr. Anjani Kumar Tiwari

Academic Editor

PLOS ONE

Table 1

Functional annotation cluster analysis of TSPO binding partners extracted from from V5-tagged TSPOWT and TSPOA147T-transfected samples and detected by MS.

Top 3 DAVID Bioinformatics Resources annotation clusters (n = 31 proteins from three biological replicates). Fold change refers to enrichment in comparison to the whole human proteome (background list).

Biological process	Gene count	p-Value	Genes	Fold change	FDR
Annotation cluster 1. Enrichment score 4.41
Mitochondrial outer membrane	8	3.7 x 10−9	HK2, MGST1, TOMM20, TOMM40, TSPO, VDAC1, VDAC2, VDAC3	32	4 x 10−6
Mitochondria	12	2.5 x 10−7	ATAD3A, HK2, MGST1, PRKCA, SLC25A1, TFB1M, TOMM20, TOMM40, TSPO, VDAC1, VDAC2, VDAC3	7.1	2.7 x 10−4
Anion transport	4	1.7 x 10−6	TSPO, VDAC1, VDAC2, VDAC3	160	2.2 x 10−3
HLTV-I infection	5	6.6 x 10−3	CREB1, TSPO, VDAC1, VDAC2, VDAC3	6.2	7.2
Transport	8	2.1 x 10−2	SCAMP3, SLC25A1, TOMM20, TOMM40, TSPO, VDAC1, VDAC2, VDAC3	2.7	2.1
Annotation cluster 2. Enrichment score 3.3
Ion channel binding	6	1.5 x 10−6	HSP90AA1, HSP90AB1, TSPO, YWHAE, YWHAQ, VDAC1	29	1.8 x 10−3
Annotation cluster 3. Enrichment score 3.14
Protein targeting	4	4.1 x 10−5	TOMM20, YWHAE, YWHAQ, YWHAZ	57	5.5 x 10−2

Abbreviations: FDR, False Discovery Rate; MS, mass spectrometry; HLTV-I, Human T-cell leukemia virus type 1.