Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)
2015; Elsevier BV; Volume: 15; Issue: 2 Linguagem: Inglês
10.1074/mcp.m115.055079
ISSN1535-9484
AutoresFarr Niere, Sanjeev V. Namjoshi, Ehwang Song, Geoffrey A. Dilly, Grant L. Schoenhard, Boris V. Zemelman, Yehia Mechref, Kimberly F. Raab‐Graham,
Tópico(s)Mast cells and histamine
ResumoMany biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer's disease, and autism spectrum disorder—neurological disorders that exhibit elevated mTORC1 activity. Through a protein–protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson's disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states. Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer's disease, and autism spectrum disorder—neurological disorders that exhibit elevated mTORC1 activity. Through a protein–protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson's disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states. The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) 1The abbreviations used are:mTORC1mechanistic/mammalian target of rapamycin complex 1ADAlzheimer's diseaseAHAazidohomoalanineAPPamyloid precursor proteinASDautism spectrum disorderBONCATbioorthogonal noncanonical amino acid taggingCIconfidence intervalcKOconditional knockoutCreCre-recombinaseDAVIDdatabase for annotation, visualization and integrated discoveryDIVday in vitroDlg4discs large homolog 4DMSOdimethyl sulfoxideEASEexpression analysis systematic explorerFDAFood and Drug AdministrationGAP-43growth-associated protein-43GOgene ontologyGRINglutamate receptor, ionotropic NMDA subtypeICCimmunocytochemistryGluNglutamate receptor, NMDA subtypeKARkainic acid receptorKcna1potassium voltage-gated channel subfamily A member 1 geneKEGGKyoto Encyclopedia of Genes and GenomeKOknockoutKv1.1voltage-gated potassium channel subfamily A member 1 type 1 proteinLlysatesLTPlong-term potentiationLTDlong-term depressionMapk3mitogen-activated protein kinase 3mGlumetabotropic glutamate receptormRNAmessenger ribonucleic acidmTORmechanistic/mammalian target of rapamycinNCBINational Center for Biotechnology InformationNMDAN-methyl-D-aspartatePpostsynaptic density/pelletP-bodiesprocessing bodiesPark7/DJ-1Parkinson protein 7PLAproximity ligation assayPPIprotein–protein interactionPSDpostsynaptic densityPSD-95PSD 95kDa proteinRaparapamycinS/SOLsoluble/supernatantSEMstandard error of the meanSFARISimons Foundation Autism Research InitiativeSNAP-25synaptosomal-associated 25 kDaSNCAα-synucleinSUMO3small ubiquitin-like modifier 3SYN1synapsin 1TSCtuberous sclerosis complexTX-100triton X-100UTUniversity of TexasWTwildtype. is a serine/threonine protein kinase that is highly expressed in many cell types (1.Kim D.H. 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Optogenetic and potassium channel gene therapy in a rodent model of focal neocortical epilepsy.Sci. Transl. Med. 2012; 4: 161ra152Crossref PubMed Scopus (184) Google Scholar). Because both active and inactive mTORC1 permit protein synthesis, we sought to determine the proteins whose expression is altered when mTORC1 phosphorylation is reduced in vivo. Rapamycin is an FDA-approved, immunosuppressive drug that inhibits mTORC1 activity (38.Seto B. Rapamycin and mTOR: a serendipitous discovery and implications for breast cancer.Clin. Transl. Med. 2012; 1: 29Crossref PubMed Google Scholar). We capitalized on the ability of rapamycin to reduce mTORC1 activity in vivo and the unbiased approach of mass spectrometry to identify changes in protein expression. Herein, we provide evidence that mTORC1 activation bidirectionally regulates protein expression, especially in the PSD where roughly an equal distribution of proteins dynamically appear and disappear. Remarkably, using protein–protein interaction networks facilitated the novel discovery that PARK7, a protein thus far only implicated in Parkinson's disease, (1) is up-regulated by increased mTORC1 activity, (2) resides in the PSD only when mTORC1 is active, and (3) is aberrantly expressed in a rodent model of TSC, an mTORC1-related disease that has symptoms of epilepsy and autism. Collectively, these data provide the first comprehensive list of proteins whose abundance or subcellular distributions are altered with acute changes in mTORC1 activity in vivo. We used three sets of paired sibling male Sprague-Dawley rats (seven to 9-weeks old) that were housed together. Within each pair, one received rapamycin (10 mg/kg) and the other received an equal volume of DMSO (carrier, control) via intraperitoneal (intraperitoneal) injection. After one hour, the animals were sacrificed. Cortices were homogenized, nuclei were pelleted by low speed centrifugation (100 × g), and the resulting supernatant was analyzed as cell lysates (L). To obtain synaptoneurosomes, homogenized cortices were processed as described (39.Workman E.R. Niere F Raab-Graham K.F. mTORC1-dependent protein synthesis underlying rapid antidepressant effect requires GABABR signaling.Neuropharmacology. 2013; 73: 192-203Crossref PubMed Scopus (49) Google Scholar). An aliquot of the synaptoneurosome fraction from each animal was solubilized in 1% triton X-100 (10min) and centrifuged (12,000 × g) to yield a triton X-100-soluble fraction (soluble, S) and a triton X-100-insoluble fraction (pellet, PSD). To prepare protein for LC-MS/MS, lysates, soluble, and PSD fractions were further solubilized in SDS-sample buffer. SDS-solubilized fractions were run on a 10% SDS-polyacrylamide gel (4min). The migration was stopped as the ladder began to separate. Gel plugs containing the sample were sectioned and sent for mass spectrometry analysis. Animal experiments were performed according to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and approved by the UT-Austin Institutional Care and Usage Committee. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE). To visualize the proteins, we used the following antibodies: rabbit anti-phospho-mTOR Ser2448 (1:2000; Cell Signaling, Danvers, MA), mouse anti-mTOR (1:5000; Life Technologies, Grand Island, NY), mouse anti-Park7 (1:2000; Novus Biologicals, Littleton, CO) mouse anti-PSD-95 (1:10,000; NeuroMab, Davis, CA), mouse antisynapsin 1 (1:10,000; Synaptic Systems, Goettingen, Germany), rabbit polyclonal anti-α tubulin (1:50,000; Abcam, Cambridge, MA), mouse ribosomal S6 (1:1000, Cell Signaling) and rabbit anti-phospho-S6 (1:1000, Cell Signaling). Membranes were subsequently incubated in fluorescence-conjugated secondary antibodies (AF680, Life Technologies; AF800, LiCor, Lincoln, NE; 1:5000). Using the Odyssey CLx infrared imaging system, we obtained fluorescent images of the membranes. ImageJ (National Institutes of Health) software was used for densitometry analyses of proteins. The gels were cut into 1 mm cubes after washing gels with water for 15min. Gel pieces were transferred to a clean Eppendorf tube followed by adding 100 μl of water and incubating for 15min. All the water was removed. This washing step was repeated with 50% acetonitrile (ACN) and 100% ACN two times. A 100 μl volume of 100 mm ammonium bicarbonate buffer was added followed by a 5min incubation. All the liquid was removed and the samples were subjected to drying in speed-vacuum until they were completely dried. For reduction/alkylation, 100 μl of 10 mm dithiothreitol (DTT) suspended in 100 mm ammonium bicarbonate buffer was added to the dried samples. This was followed by incubation in a water bath at 55 °C for 45min. After removing the alkylation solution, 100 μl of 55 mm iodoacetamide (IAA) suspended in 100 mm ammonium bicarbonate buffer was added into the reduced samples followed by incubating at 37.5 °C for 30 min. The alkylated samples were then washed with 100 μl of 100 mm ammonium bicarbonate buffer and incubated for 5min. A 100 μl of acetonitrile was added to make one to one ratio of solutions with incubation for 15min. All the solutions were removed. The samples were then dried before digestion. Trypsin digestion solution was prepared with 4 μl trypsin in 96 μl of 100 mm ammonium bicarbonate buffer. The amount of trypsin was determined using enzyme/protein ratio of 1:50 w/w because 200 μg of proteins were loaded into the gel. A 100 μl of trypsin digestion solution was added to the alkylated samples and then incubated for 45 min on ice. This allows the gels to absorb the trypsin digestion solution. The solution was removed if it was excessive. The samples were incubated at 37.5 °C overnight. The addition of 2% trifluoroacetic acid (TFA) acidified the digestion. The tryptic digests were extracted from the gel by adding 0.1%TFA and incubating in ice water bath with sonication. The solutions were then collected in a separate clean Eppendorf tube. This extraction step was repeated with 30% ACN/0.1% TFA and 60% ACN/0.1% TFA three times. The collected peptides were then dried in a speed-vacuum and resuspended in 0.1% formic acid prior to LC-MS/MS analysis. Trypsin digested samples were subjected to LC-MS/MS analysis using Dionex 3000 Ultimate nano-LC system (Dionex, Sunnyvale, CA) interfaced to LTQ Orbitrap Velos mass spectrometer (Thermo Scientific, San Jose, CA) equipped with a nano-ESI source. The samples were initially online-purified using a PepMap 100 C18 cartridge (3 μm, 100Å, Dionex). The purified peptides were then separated using a PepMap 100 C18 capillary column (75 μm id × 150 mm, 2 μm, 100Å, Dionex). The separation of peptides was achieved at 350 nl/min flow rate, using the following gradient: 0–10 min 5% solvent B (98% ACN with 0.1% formic acid), 10–65 min ramping solvent B 5–20%, 55–90 min ramping solvent B 20–30%, 90–105 min ramping solvent B 30–50%, 105–110min maintaining solvent B at 80%, 110–111 min decreasing solvent B 80–5%, and 111–120 min sustaining solvent B at 5%. Solvent A was a 2% ACN aqueous solution containing 0.1% formic acid. The separation and scan time was set to 120 min. The LTQ Orbitrap Velos mass spectrometer was operated with three scan events. The first scan event was a full MS scan of 380–2000 m/z at a mass resolution of 15,000. The second scan event was CID MS/MS of parent ions selected from the first scan event with an isolation width of 3.0 m/z, a normalized collision energy (CE) of 35%, and an activation Q value of 0.250. The third scan event was set to acquire HCD MS/MS of the parent ions selected from the first scan event. The isolation width of HCD experiment was set to 3.0 m/z while the normalized CE was set to 45% with an activation time of 0.1 ms. The CID and HCD MS/MS were performed on the five most intense ions observed in the MS scan event. Quantitation was attained employing normalized spectral counts that were calculated by Scaffold Q+ (Proteome Software, Inc., Portland, OR). The identification of proteins/peptides was achieved using MASCOT database (40.Brosch M. Yu L. Hubbard T. Choudhary J. Accurate and sensitive peptide identification with Mascot Percolator.J. Proteome Res. 2009; 8: 3176-3181Crossref PubMed Scopus (332) Google Scholar). Proteome Discoverer version 1.2 software (Thermo Scientific, San Jose, CA) was used to generate a mascot generic format file (*.mgf) that was subsequently employed for database searching using MASCOT version 2.3.2 (Matrix Science Inc., Boston, MA). Parent ions were selected from a mass range of 350–10000Da with a minimum peak count of one. The parameters from Mascot Daemon were set to search against the UniProt Rattus database (UniProt release 2013_11). Oxidation of methionine was set as a variable modification while carbamidomethylation of cysteine was as a fixed modification. The formation of propionamide adducts on cysteine, N terminus and C terminus of peptides was added as variable modification because of the use of polyacrylamide gels. Trypsin was selected with missed cleavages up to two. Peptides were searched with a precursor ion mass tolerance of 6ppm or better and fragment ion mass tolerance of 1.5Da. The MASCOT results were imported to Scaffold version 3.6.3 (41.Searle B.C. Scaffold: a bioinformatic tool for validating MS/MS-based proteomic studies.Proteomics. 2010; 10: 1265-1269Crossref PubMed Scopus (395) Google Scholar) (Proteome Software, Inc.). Scaffold probabilistically validates the identification of peptides and proteins assigned by MASCOT using PeptideProphet (42.Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3886) Google Scholar) and ProteinProphet (43.Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectromet
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