Article Figures & Data
Figures
- Figure 1.
Effects of nicotine use and mood disorder on total high-affinity [125I]-epibatidine binding in cell membranes from human temporal cortex samples. A, Nicotine use, determined by tissue cotinine content, has a significant effect on the number of total [125I]-epibatidine binding sites (two-way ANOVA with Holm–Sidak multiple comparisons: F(1,17) = 9.794, p = 0.007), while mood disorder does not have an appreciable effect on the expression of high-affinity nAChRs in the cortex (F(1,17) = 0.224, p = 0.673). B, β2*-nAChR capture was evaluated by [125I]-epibatidine binding following immunoprecipitation from human temporal cortex using mAb295-M270 Dynabeads. Error bars represent standard error of the mean.
- Figure 2.
[125I]-epibatidine binding to α4β2*-nAChRs in human temporal cortex homogenates correlates well with β2-nAChR (A) and α4-nAChR (B) subunit abundance estimated by normalized unique peptide abundance determined by peptide LC-MS/MS.
- Figure 3.
Effects of long-term treatment with saline or 4 mg/kg/h nicotine treatment on captured [125I]-epibatidine binding sites from pooled cortex from wild-type (β2+/+, W), heterozygous (β2+/−, H), double-heterozygous (α4+/−β2+/−, DH), and knock-out (β2−/−, K) mice. Gene dose-dependent changes in receptor capture are apparent (two-way ANOVA; main effect, genotype: F(3,15) = 59.314, p < 0.0009), as is the upregulation produced by long-term nicotine exposure in all but the β2−/− genotype groups (main effect, nicotine: F(1,15) = 29.05, p < 0.0009). Error bars represent standard error of the mean.
- Figure 4.
A, Representation of the log2-normalized fold change in β2*-nAChR-associated proteins in human temporal cortex relative to the non-nicotine, non-MD group average. The graph shows nAChR-associated proteins whose expression is significantly impacted by nicotine, MD, or an interaction, according to GLM. Group means above zero are upregulated by nicotine exposure, indicating that as nAChR levels increase, so do the levels of those proteins. Increases in excess of the log2 fold change for the α4- and β2-nAChR subunits indicate a superstoichiometric shift. B, Log2 protein abundance normalized to within-sample β2-nAChR protein content for five proteins that showed a significant shift with nicotine use. Normalization demonstrates the change in protein expression per molecule of the nAChR β2-subunit in human cortical samples. For these samples, LFQ proteomics allows the comparison of protein levels, even when the variation in total abundance across treatments is small. Normalization to an internal standard in each condition (β2-subunit levels) allows the measurement of more discrete changes than are apparent from fold change estimates.
Tables
- Table 1.
Tabulation of all proteins identified from mAb295-immunoisolated β2*-nAChRs from human temporal cortex, indicating which proteins have been identified in mouse cortex or whole mouse brain (Iso, isoform of protein identified; GN, gene name)
Accession_HUMAN Description Mouse Ctx Mouse Brain 1433E 14-3-3 protein ε GN = YWHAE Iso Iso 1433G;1433F 14-3-3 protein γ GN = YWHAG Iso Iso 1433S 14-3-3 protein σ GN = SFN Iso Iso 1433Z 14-3-3 protein ζ/δ GN = YWHAZ Iso Yes ACHA4;ACHA2;ACHA6 Neuronal acetylcholine receptor subunit α4 GN = CHRNA4 Yes Yes ACHB2 Neuronal acetylcholine receptor subunit β2 GN = CHRNB2 Yes Yes ACTB;ACTBM;POTEE Actin, cytoplasmic 1 GN = ACTB Yes Iso ACTC;ACTA Actin, α cardiac muscle 1 GN = ACTC1 Iso Yes ACTN1;ACTN3 α-Actinin-1 GN = ACTN1 No Yes ACTN2 α-Actinin-2 GN = ACTN2 No Yes ACTN4 α-Actinin-4 GN = ACTN4 No Iso ADDA α-Adducin GN = ADD1 No No ADT1;ADT2;ADT4 ADP/ATP translocase 1 GN = SLC25A4 Yes Yes ALBU Serum albumin GN = ALB No No ANK2 Ankyrin-2 GN = ANK2 No Yes AP1B1 AP-1 complex subunit β1 GN = AP1B1 No No ARP2 Actin-related protein 2 GN = ACTR2 No Iso ARP3 Actin-related protein 3 GN = ACTR3 No Yes ARPC3 Actin-related protein 2/3 complex subunit 3 GN = ARPC3 No Iso ARPC4 Actin-related protein 2/3 complex subunit 4 GN = ARPC4 No Iso AT1A1;AT12A Sodium/potassium-transporting ATPase subunit α1 GN = ATP1A1 Iso Iso AT1A2 Sodium/potassium-transporting ATPase subunit α2 GN = ATP1A2 Iso Yes AT1A3;AT1A4;ATP4A Sodium/potassium-transporting ATPase subunit α3 GN = ATP1A3 Yes Yes AT1B1 Sodium/potassium-transporting ATPase subunit β1 GN = ATP1B1 Yes Yes AT2B2;AT2B1 Plasma membrane calcium-transporting ATPase 2 GN = ATP2B2 No No ATPA ATP synthase subunit α, mito GN = ATP5A1 Yes Yes ATPB ATP synthase subunit β, mito GN = ATP5B Yes Yes BASP1 Brain acid-soluble protein 1 GN = BASP1 No No CALM Calmodulin GN = CALM1 Yes Yes CANB1 Calcineurin subunit B type 1 GN = PPP3R1 No No CLH1 Clathrin heavy chain 1 GN = CLTC SV = 5 Yes No CNTN1 Contactin-1 GN = CNTN1 No Yes COX2 Cytochrome c oxidase subunit 2 GN = MT-CO2 No Yes COX41 Cytochrome c oxidase subunit 4 isoform 1, mito GN = COX4I1 No Yes COX6C Cytochrome c oxidase subunit 6C GN = COX6C No Yes CX6B1 Cytochrome c oxidase subunit 6B1 GN = COX6B1 No Yes CX7A2 Cytochrome c oxidase subunit 7A2, mito GN = COX7A2 No Yes DREB Drebrin GN = DBN1 No Yes DYN1 Dynamin-1 GN = DNM1 No No E41L3 Band 4.1-like protein 3 GN = EPB41L3 No No EAA1 Excitatory amino acid transporter 1 GN = SLC1A3 Iso Iso EAA2 Excitatory amino acid transporter 2 GN = SLC1A2 Yes Yes EF1A1;EF1A2 Elongation factor 1-α1 GN = EEF1A1 No No FCGBP IgGFc-binding protein GN = FCGBP No No FRIH Ferritin heavy chain GN = FTH1 No No FRIL Ferritin light chain GN = FTL No No G3P Glyceraldehyde-3-phosphate dehydrogenase GN = GAPDH Yes Yes GBB1;GBB2;GBB3;GBB4 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit β1 GN = GNB1 Yes Yes GELS Gelsolin GN = GSN No Yes GFAP Glial fibrillary acidic protein GN = GFAP Yes Yes GNAO;GNA12 Guanine nucleotide-binding protein G(o) subunit α GN = GNAO1 Yes Yes GPM6A Neuronal membrane glycoprotein M6-a GN = GPM6A No No H13 Histone H1.3 GN = HIST1H1D No Iso H2A1B Histone H2A type 1-B/E GN = HIST1H2AB No Yes H2B1M;H2B1A Histone H2B type 1-M GN = HIST1H2BM No Yes H4 Histone H4 GN = HIST1H4A No Yes HBA Hemoglobin subunit α GN = HBA1 No Iso HBB;HBD Hemoglobin subunit β GN = HBB No Yes HORN Hornerin GN = HRNR No No HS12A Heat shock 70 kDa protein 12A GN = HSPA12A No Iso HS71L Heat shock 70 kDa protein 1-like GN = HSPA1L No Iso HSPB1 Heat shock protein β1 GN = HSPB1 No Iso IGHG4;IGHG2 Ig γ-4 chain C region GN = IGHG4 No Iso IGHM Ig μ chain C region GN = IGHM No Yes IGKC Ig κ chain C region GN = IGKC No Iso KCC2A;KCC2D Calcium/calmodulin-dependent protein kinase type II subunit α GN = CAMK2A Yes Yes KCC2B;KCC2G Calcium/calmodulin-dependent protein kinase type II subunit β GN = CAMKIIB Yes Yes MBP Myelin basic protein GN = MBP Yes Yes ML12A;MYL9 Myosin regulatory light chain 12A GN = MYL12A No Iso MOG Myelin-oligodendrocyte glycoprotein GN = MOG No No MPCP Phosphate carrier protein, mito GN = SLC25A3 Yes Yes MYH10 Myosin-10 GN = MYH10 Yes Yes MYH9;MYH11;MYH14 Myosin-9 GN = MYH9 Iso Yes MYL6;MYL6B Myosin light polypeptide 6 GN = MYL6 Yes Yes MYO5A;MYO5B Unconventional myosin-Va GN = MYO5A No Yes MYPR Myelin proteolipid protein GN = PLP1 No Yes NDUA4 Cytochrome c oxidase subunit NDUFA4 GN = NDUFA4 Yes Iso NDUS3 NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mito GN = NDUFS3 Yes Yes NDUV2 NADH dehydrogenase [ubiquinone] flavoprotein 2, mito GN = NDUFV2 Yes Yes NFL Neurofilament light polypeptide GN = NEFL Yes Yes NFM Neurofilament medium polypeptide GN = NEFM Yes Yes NSF Vesicle-fusing ATPase GN = NSF No Yes ODB2 Lipoamide acyltransferase of branched-chain α-keto acid dehydrogenase mito GN = DBT No Yes ODO2 Dihydrolipoyllysine-residue succinyltransferase 2-oxoglutarate dehydrogenase mito GN = DLST Yes Yes QCR2 Cytochrome b-c1 complex subunit 2, mito GN = UQCRC2 No Yes RAB1A Ras-related protein Rab-1A GN = RAB1A Iso Yes RL40 Ubiquitin-60S ribosomal protein L40 GN = UBA52 No No RO52 E3 ubiquitin-protein ligase TRIM21 GN = TRIM21 No No S10A7;S1A7A Protein S100-A7 GN = S100A7 No No S10A8 Protein S100-A8 GN = S100A8 No No S10A9 Protein S100-A9 GN = S100A9 No No SIR2 NAD-dependent protein deacetylase sirtuin-2 GN = SIRT2 No No SNP25 Synaptosomal-associated protein 25 GN = SNAP25 Yes Yes SPTB2;SPTB1 Spectrin β chain, nonerythrocytic 1 GN = SPTBN1 Yes Yes SPTN1 Spectrin α chain, nonerythrocytic 1 GN = SPTAN1 Yes Yes SPTN2 Spectrin β chain, nonerythrocytic 2 GN = SPTBN2 Iso Iso SSBP Single-stranded DNA-binding protein, mito GN = SSBP1 No No STX1A Syntaxin-1A GN = STX1A Iso Iso STX1B;STX2 Syntaxin-1B GN = STX1B Yes Yes STXB1 Syntaxin-binding protein 1 GN = STXBP1 No Yes SV2A Synaptic vesicle glycoprotein 2A GN = SV2A No No SYT1 Synaptotagmin-1 GN = SYT1 Yes Yes TBA1A,C;TBA3C,E;TBA8 Tubulin α1A chain GN = TUBA1A Iso Yes TBA1B Tubulin α1B chain GN = TUBA1B Iso Iso TBA4A;TBA4B;TBAL3 Tubulin α4A chain GN = TUBA4A Yes Yes TBB2A;TBB1;TBB2B Tubulin β2A chain GN = TUBB2A Yes Yes TBB3 Tubulin β3 chain GN = TUBB3 Yes Yes TBB4A;TBB6,8;TBB8L Tubulin β4A chain GN = TUBB4A Yes Yes TBB4B Tubulin β4B chain GN = TUBB4B Yes Yes TBB5 Tubulin β chain GN = TUBB Yes Yes THY1 Thy-1 membrane glycoprotein GN = THY1 Yes No TMOD2 Tropomodulin-2 GN = TMOD2 No Yes TPM4;TPM1 Tropomyosin α4 chain GN = TPM4 Iso No VA0D1;VA0D2 V-type proton ATPase subunit d 1 GN = ATP6V0D1 Yes Iso VAMP3 Vesicle-associated membrane protein 3 GN = VAMP3 No Iso VATA V-type proton ATPase catalytic subunit A GN = ATP6V1A Yes Yes VISL1 Visinin-like protein 1 GN = VSNL1 No No VPP1 V-type proton ATPase 116 kDa subunit a isoform 1 GN = ATP6V0A1 PE = 2 Yes Iso - Table 2.
Statistical analyses indicating significance of variance with levels of the β2-nAChR subunit
Accession_HUMAN ANOVA (p) Peptides ACTB;ACTBM;POTEE;POTEF;POTEI;POTEJ 1.10E-11 30 S10A9 1.67E-11 5 ODO2 2.50E-11 2 ACTC;ACTA 1.51E-10 1 BASP1 2.15E-10 2 ACTN2 1.03E-09 5 KCC2A;KCC2D 2.14E-09 11 TBA4A;TBA4B;TBAL3 2.40E-09 3 TBA1A;TBA1C;TBA3C;TBA3E;TBA8 2.92E-09 2 MYH9;MYH11;MYH14 3.28E-09 6 S10A7;S1A7A 3.97E-09 2 AT1A1;AT12A 6.35E-09 7 AT1B1 9.37E-09 3 TBA1B 6.14E-08 2 VAMP3 2.62E-07 1 TBB4A;TBB6;TBB8;TBB8L 3.00E-07 10 H2B1M;H2B1A 3.68E-07 4 RL40 8.67E-07 2 MYH10 1.11E-06 18 FCGBP 1.34E-06 19 ODB2 2.48E-06 5 COX41 2.57E-06 2 GELS 3.76E-06 10 HS12A 3.82E-06 4 AT1A3;AT1A4;ATP4A 4.09E-06 19 TBB2A;TBB1;TBB2B 4.12E-06 25 ACTN1;ACTN3 1.03E-05 19 TBB5 1.04E-05 2 ADT1;ADT2;ADT4 1.09E-05 3 IGHG4;IGHG2 1.34E-05 3 EF1A1;EF1A2 1.53E-05 2 DREB 2.67E-05 5 COX2 3.01E-05 2 CLH1 4.69E-05 2 CNTN1 5.48E-05 1 IGKC 5.53E-05 1 STX1B;STX2 6.30E-05 6 H13 8.79E-05 1 EAA2 1.11E-04 2 CX7A2 1.19E-04 1 ANK2 1.43E-04 3 VISL1 1.84E-04 2 MYO5A;MYO5B 2.45E-04 8 ML12A;MYL9 4.10E-04 4 S10A8 4.70E-04 2 ATPA 5.39E-04 3 ACTN4 5.80E-04 5 GFAP 6.13E-04 16 ARPC4 6.76E-04 2 ACHB2 7.24E-04 10 ARPC3 1.22E-03 1 CANB1 1.35E-03 1 STXB1 1.55E-03 3 TBB4B 1.58E-03 1 TBB3 1.73E-03 2 MPCP 2.04E-03 1 1433Z 2.60E-03 2 H4 2.92E-03 3 MYPR 2.96E-03 5 NDUS3 3.08E-03 1 GPM6A 3.13E-03 2 E41L3 3.53E-03 1 CALM 3.66E-03 2 H2A1B 4.17E-03 1 FRIH 4.43E-03 3 SPTN1 4.48E-03 57 GNAO;GNA12 4.51E-03 4 ACHA4;ACHA2;ACHA6 5.52E-03 23 DYN1 5.67E-03 4 ARP3 5.99E-03 2 SIR2 6.01E-03 1 HS71L 7.68E-03 1 ARP2 8.71E-03 4 SPTB2;SPTB1 0.013000 48 QCR2 0.019305 1 SPTN2 0.019379 3 AP1B1 0.023924 1 VA0D1;VA0D2 0.026761 4 1433E 0.028173 1 HBB;HBD 0.031713 4 AT2B2;AT2B1 0.033005 2 HORN 0.037459 2 VPP1 0.047673 2 MBP 0.053508 6 1433G;1433F 0.066781 2 TMOD2 0.066906 1 IGHM 0.067934 1 SV2A 0.069290 1 KCC2B;KCC2G 0.070110 2 ATPB 0.074512 3 RAB1A 0.076581 1 FRIL 0.083836 1 VATA 0.089288 1 ADDA 0.093382 2 AT1A2 0.126331 3 RO52 0.126544 12 EAA1 0.131182 1 1433S 0.149390 1 STX1A 0.155190 4 SNP25 0.193139 7 ALBU 0.249829 2 HBA 0.254867 2 SYT1 0.257254 2 G3P 0.263379 3 MYL6;MYL6B 0.365416 5 GBB1;GBB2;GBB3;GBB4 0.378267 7 TPM4;TPM1 0.409469 3 COX6C 0.456303 1 NDUA4 0.511653 2 CX6B1 0.580761 1 SSBP 0.640567 2 NSF 0.666753 8 MOG 0.744318 2 NFM 0.769224 2 THY1 0.778619 2 NDUV2 0.802323 1 NFL 0.842694 5 HSPB1 0.941399 1 - Table 3.
Proteins coimmunopurified with β2*-nAChRs from postmortem human temporal cortex with abundances that correlate significantly with the abundance of the β2 nAChR subunit
Accession Description Correlation (r) p Value n ACHB2_HUMAN Neuronal acetylcholine receptor subunit β2 1 0 18 ACHA4_HUMAN Neuronal acetylcholine receptor subunit α4 0.972 <0.0009 18 AT1B1_HUMAN Sodium/potassium-transporting ATPase subunit β1 0.889 <0.0009 18 AT1A3_HUMAN Sodium/potassium-transporting ATPase subunit α3 0.888 <0.0009 18 SNP25_HUMAN Synaptosomal-associated protein 25 0.88 <0.0009 18 STX1B_HUMAN Syntaxin-1B 0.874 <0.0009 18 AT1A1_HUMAN Sodium/potassium-transporting ATPase subunit α1 0.871 <0.0009 18 ANK2_HUMAN Ankyrin-2 0.856 <0.0009 18 STX1A_HUMAN Syntaxin-1A 0.844 <0.0009 18 GPM6A_HUMAN Neuronal membrane glycoprotein M6-a 0.823 <0.0009 18 AT2B2_HUMAN Plasma membrane calcium-transporting ATPase 2 0.781 <0.0009 18 GNAO_HUMAN Guanine nucleotide-binding protein G(o) subunit α 0.781 <0.0009 18 VPP1_HUMAN V-type proton ATPase 116 kDa subunit a isoform 1 0.777 <0.0009 18 MYPR_HUMAN Myelin proteolipid protein 0.762 <0.0009 18 GBB1_HUMAN Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit β1 0.702 0.001 18 EAA2_HUMAN Excitatory amino acid transporter 2 0.701 0.001 18 VA0D1_HUMAN V-type proton ATPase subunit d 1 0.644 0.004 18 MYH9_HUMAN Myosin-9 0.631 0.005 18 AT1A2_HUMAN Sodium/potassium-transporting ATPase subunit α2 0.609 0.007 18 TBB5_HUMAN Tubulin β chain 0.576 0.012 18 TBA1A_HUMAN Tubulin α1A chain 0.56 0.016 18 1433Z_HUMAN 14-3-3 protein zeta/delta 0.548 0.019 18 STXB1_HUMAN Syntaxin-binding protein 1 0.526 0.025 18 SYT1_HUMAN Synaptotagmin-1 0.523 0.026 18 CLH1_HUMAN Clathrin heavy chain 1 0.515 0.041 18 TBA4A_HUMAN Tubulin α4A chain 0.514 0.029 18 S10A7_HUMAN Protein S100-A7 0.511 0.03 18 TBA1B_HUMAN Tubulin α1B chain 0.506 0.032 18 RO52_HUMAN E3 ubiquitin-protein ligase TRIM21 0.492 0.038 18 ODO2_HUMAN Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex 0.483 0.042 18 TBB4A_HUMAN Tubulin β4A chain 0.476 0.046 18 MYO5A_HUMAN Unconventional myosin-Va 0.475 0.046 18 TPM4_HUMAN Tropomyosin α4 chain 0.473 0.047 18 High correlation indicates the identified protein is either specifically interacting with the receptor at the time of tissue lysis or is part of a complex that stays intact following extraction in Triton X-100. The cluster of proteins with correlation coefficients <0.7 is evidence of significant enrichment of presynaptic components critical for neurotransmitter release and neuronal excitability.
- Table 4.
Tabulation of all proteins identified in common between two biological replicates of eight-plex iTRAQ-labeled β2*-nAChRs immunoisolated with mAb295 from mouse cortex, indicating which proteins have been identified in human temporal cortex or whole mouse brain (Iso, isoform of protein identified; GN, gene name)
Accession_MOUSE Description Human Ctx Mouse brain 1433T 14-3-3 protein theta GN = Ywhaq Iso Iso ACHA4 Neuronal acetylcholine receptor subunit α4 GN = Chrna4 Yes Yes ACHA5 Neuronal acetylcholine receptor subunit α5 GN = Chrna5 Iso Iso ACHB2 Neuronal acetylcholine receptor subunit β2 GN = Chrnb2 Yes Yes ACPM Acyl carrier protein, mito GN = Ndufab1 No Yes ACTG; ACTB Actin, cytoplasmic 2 GN = Actg1; actin, cytoplasmic 1 GN = Actb Iso Yes ADT1 ADP/ATP translocase 1 GN = Slc25a4 SV = 4 Yes Yes ADT2 ADP/ATP translocase 2 GN = Slc25a5 Yes Yes AINX α-Internexin GN = Ina No Yes AT1A3 Sodium/potassium-transporting ATPase subunit α3 GN = Atp1a3 Yes Yes AT1B1 Sodium/potassium-transporting ATPase subunit β1 GN = Atp1b1 Yes Yes AT5F1 ATP synthase subunit b, mito GN = Atp5f1 Iso Yes ATP5H ATP synthase subunit d, mito GN = Atp5h Iso Yes ATP5I ATP synthase subunit e, mito GN = Atp5i Iso Yes ATPA ATP synthase subunit α, mito GN = Atp5a1 Yes Yes ATPB ATP synthase subunit β, mito GN = Atp5b Yes Yes ATPD ATP synthase subunit delta, mito GN = Atp5d Iso Yes C1QA Complement C1q subcomponent subunit A GN = C1qa No No CALM Calmodulin GN = Calm1 Yes Yes CLH1 Clathrin heavy chain 1 GN = Cltc Yes No CMC1 Calcium-binding mito carrier protein Aralar1 GN = Slc25a12 No Yes CN37 2',3'-cyclic-nucleotide 3'-phosphodiesterase GN = Cnp No Yes EAA2 Excitatory amino acid transporter 2 GN = Slc1a2 Yes Yes G3P Glyceraldehyde-3-phosphate dehydrogenase GN = Gapdh Yes Yes GBB1 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit β1 GN = Gnb1 Yes Yes GFAP Glial fibrillary acidic protein GN = Gfap SV = 4 Yes Yes GLNA Glutamine synthetase GN = Glul SV = 6 No Yes GNAO Guanine nucleotide-binding protein G(o) subunit α GN = Gnao1 Yes Yes KCC2A Calcium/calmodulin-dependent protein kinase type II subunit α GN = CaMKIIa Yes Yes KCC2B Calcium/calmodulin-dependent protein kinase type II subunit β GN = CaMKIIb Yes Yes M2OM Mito 2-oxoglutarate/malate carrier protein GN = Slc25a11 No Yes MBP Myelin basic protein GN = Mbp Yes Yes MPCP Phosphate carrier protein, mito GN = Slc25a3 Yes Yes MYH10 Myosin-10 GN = Myh10 Yes Yes MYL6 Myosin light polypeptide 6 GN = Myl6 Yes Yes NDUA2 NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 2 GN = Ndufa2 Iso Yes NDUA4 NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 4 GN = Ndufa4 Yes Iso NDUA5 NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 5 GN = Ndufa5 Iso Yes NDUA6 NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 6 GN = Ndufa6 Iso Yes NDUA7 NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 7 GN = Ndufa7 Iso Yes NDUA9 NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 9, mito GN = Ndufa9 Iso Yes NDUAA NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 10, mito GN = Ndufa10 Iso Yes NDUAC NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 12 GN = Ndufa12 Iso Yes NDUAD NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 13 GN = Ndufa13 Iso Yes NDUB3 NADH dehydrogenase [ubiquinone] 1 β subcomplex subunit 3 GN = Ndufb3 Iso Yes NDUB4 NADH dehydrogenase [ubiquinone] 1 β subcomplex subunit 4 GN = Ndufb4 Iso Yes NDUB5 NADH dehydrogenase [ubiquinone] 1 β subcomplex subunit 5, mito GN = Ndufb5 Iso Yes NDUB6 NADH dehydrogenase [ubiquinone] 1 β subcomplex subunit 6 GN = Ndufb6 Iso Yes NDUB7 NADH dehydrogenase [ubiquinone] 1 β subcomplex subunit 7 GN = Ndufb7 Iso Yes NDUB8 NADH dehydrogenase [ubiquinone] 1 β subcomplex subunit 8, mito GN = Ndufb8 Iso Yes NDUB9 NADH dehydrogenase [ubiquinone] 1 β subcomplex subunit 9 GN = Ndufb9 Iso Yes NDUC2 NADH dehydrogenase [ubiquinone] 1 subunit C2 GN = Ndufc2 Iso Yes NDUS1 NADH-ubiquinone oxidoreductase 75 kDa subunit, mito GN = Ndufs1 Iso Yes NDUS2 NADH dehydrogenase [ubiquinone] iron-sulfur protein 2, mito GN = Ndufs2 Iso Yes NDUS3 NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mito GN = Ndufs3 Yes Yes NDUS4 NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mito GN = Ndufs4 Iso Yes NDUS5 NADH dehydrogenase [ubiquinone] iron-sulfur protein 5 GN = Ndufs5 Iso Yes NDUS6 NADH dehydrogenase [ubiquinone] iron-sulfur protein 6, mito GN = Ndufs6 Iso Yes NDUS7 NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mito GN = Ndufs7 Iso Yes NDUS8 NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, mito GN = Ndufs8 Iso Yes NDUV1 NADH dehydrogenase [ubiquinone] flavoprotein 1, mito GN = Ndufv1 Iso Yes NDUV2 NADH dehydrogenase [ubiquinone] flavoprotein 2, mito GN = Ndufv2 Yes Yes NEST Nestin GN = Nes No No NFH Neurofilament heavy polypeptide GN = Nefh Iso Iso NFL Neurofilament light polypeptide GN = Nefl SV = 5 Yes Yes NFM Neurofilament medium polypeptide GN = Nefm SV = 4 Yes Yes OCAD2 OCIA domain-containing protein 2 GN = Ociad2 No Yes ODO2 Dihydrolipoyl-lysine residue succinyltransferas 2-oxoglutarate dehydrogenase complex, mito GN = Dlst Yes Yes ODPA Pyruvate dehydrogenase E1 component subunit α, somatic form, mito GN = Pdha1 No Yes ODPB Pyruvate dehydrogenase E1 component subunit β, mito GN = Pdhb No Yes PHB Prohibitin GN = Phb No Yes PHB2 Prohibitin-2 GN = Phb2 No Yes RAB3A Ras-related protein Rab-3A GN = Rab3a Iso Yes SFXN3 Sideroflexin-3 GN = Sfxn3 No Yes SNP25 Synaptosomal-associated protein 25 GN = Snap25 Yes Yes SPTB2 Spectrin β chain, nonerythrocytic 1 GN = Sptbn1 Yes Yes SPTN1 Spectrin α chain, nonerythrocytic 1 GN = Sptan1 SV = 4 Yes Yes STX1B Syntaxin-1B GN = Stx1b Yes Yes SYPH Synaptophysin GN = Syp No No SYT1 Synaptotagmin-1 GN = Syt1 Yes Yes TBA4A Tubulin α4A chain GN = Tuba4a Yes Yes TBB2A Tubulin β2A chain GN = Tubb2a Yes Yes TBB3 Tubulin β3 chain GN = Tubb3 Yes Yes TBB4A Tubulin β4A chain GN = Tubb4a Yes Yes TBB4B Tubulin β4B chain GN = Tubb4b Yes Yes TBB5 Tubulin β5 chain GN = Tubb5 Yes Yes THY1 Thy-1 membrane glycoprotein GN = Thy1 Yes No TPM3 Tropomyosin α3 chain GN = Tpm3 Iso No VA0D1 V-type proton ATPase subunit d 1 GN = Atp6v0d1 Yes Iso VATA V-type proton ATPase catalytic subunit A GN = Atp6v1a Yes Yes VDAC1 Voltage-dependent anion-selective channel protein 1 GN = Vdac1 No Yes VDAC2 Voltage-dependent anion-selective channel protein 2 GN = Vdac2 No Yes VIME Vimentin GN = Vim No Yes VPP1 V-type proton ATPase 116 kDa subunit a isoform 1 GN = Atp6v0a1 Yes Iso - Table 5.
β2*-nAChR interacting proteins identified by iTRAQ analysis of receptors isolated from transgenic mouse cortex receiving long-term treatment with nicotine and not receiving nicotine treatment (GN, gene name)
Accession Description Correlation (r) p Value sp|O70174|ACHA4_MOUSE Neuronal acetylcholine receptor subunit α4 OS = Mus musculus GN = Chrna4 PE = 2 SV = 2 0.97 <0.0009 sp|Q2MKA5|ACHA5_MOUSE Neuronal acetylcholine receptor subunit α5 OS = Mus musculus GN = Chrna5 PE = 2 SV = 1 0.91 <0.0009 sp|Q9ERK7|ACHB2_MOUSE Neuronal acetylcholine receptor subunit β2 OS = Mus musculus GN = Chrnb2 PE = 2 SV = 1 1 <0.0009 sp|Q6PIC6|AT1A3_MOUSE Sodium/potassium-transporting ATPase subunit α3 OS = Mus musculus GN = Atp1a3 PE = 1 SV = 1 0.55 0.042 sp|Q62277|SYPH_MOUSE Synaptophysin OS = Mus musculus GN = Syp PE = 1 SV = 2 0.59 0.026 Similar to the results from human cortex samples, presynaptic elements related to vesicle fusion (synaptophysin) and neuronal excitability (ATP1A3) are significantly correlated with nAChR levels.
- Table 6.
List of proteins identified in three independent proteomic studies: human and mouse cortex (current study) and whole mouse brain (McClure-Begley et al., 2013) (GN, gene name)
Accession Description No. of experiments (found/total) ACHA4 Neuronal acetylcholine receptor subunit α4 GN = Chrna4 0/411 ACHB2 Neuronal acetylcholine receptor subunit β2 GN = Chrnb2 0/411 ADT1 ADP/ATP translocase 1 GN = Slc25a4 195/411 ADT2 ADP/ATP translocase 2 GN = Slc25a5 223/411 AT1A3 Sodium/potassium-transporting ATPase subunit α3 GN = Atp1a3 101/411 AT1B1 Sodium/potassium-transporting ATPase subunit β1 GN = Atp1b1 1/411 ATPA ATP synthase subunit α, mito GN = Atp5a1 210/411 ATPB ATP synthase subunit β, mito GN = Atp5b 211/411 CALM Calmodulin GN = Calm1 133/411 EAA2 Excitatory amino acid transporter 2 GN = Slc1a2 2/411 G3P Glyceraldehyde-3-phosphate dehydrogenase GN = GAPDH 248/411 GBB1 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit β1 GN = Gnb1 59/411 GFAP Glial fibrillary acidic protein GN = GFAP 73/411 GNAO Guanine nucleotide-binding protein G(o) subunit α GN = Gnao1 42/411 KCC2A Calcium/calmodulin-dependent protein kinase type II subunit α GN = CaMKIIa 7/411 KCC2B Calcium/calmodulin-dependent protein kinase type II subunit β GN = CaMKIIb 7/411 MBP Myelin basic protein GN = Mbp 0/411 MPCP Phosphate carrier protein, mito GN = Slc25a3 130/411 MYH10 Myosin-10 GN = Myh10 166/411 MYL6 Myosin light polypeptide 6 GN = Myl6 190/411 NDUS3 NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mito GN = Ndufs3 19/411 NDUV2 NADH dehydrogenase [ubiquinone] flavoprotein 2, mito GN = Ndufv2 3/411 NFL Neurofilament light polypeptide GN = Nefl Not in database NFM Neurofilament medium polypeptide GN = Nefm 85/411 ODO2 Dihydrolipoyllysine-res succinyltransferase 2-oxoglutarate dehydrogenase complex, mito GN = Dlst 57/411 SNP25 Synaptosomal-associated protein 25 GN = Snap25 0/411 SPTB2 Spectrin β chain, nonerythrocytic 1 GN = Sptbn1 143/411 SPTN1 Spectrin α chain, nonerythrocytic 1 GN = Sptan1 161/411 STX1B Syntaxin-1B GN = Stx1b 0/411 SYT1 Synaptotagmin-1 GN = Syt1 0/411 TBA4A Tubulin α4A chain GN = Tuba4a 377/411 TBB2A Tubulin β2A chain GN = Tubb2a 372/411 TBB3 Tubulin β3 chain GN = Tubb3 Not in database TBB4A Tubulin β4A chain GN = Tubb4a 366/411 TBB4B Tubulin β4B chain GN = Tubb4b 376/411 TBB5 Tubulin β5 chain GN = Tubb5 Not in database VATA V-type proton ATPase catalytic subunit A GN = Atp6v1a 62/411 Numbers in the right hand column represent the number of times each protein has been identified in control studies designed to evaluate background contaminants (Mellacheruvu et al., 2013).
- Table 7.
β2*-nAChR-associated proteins with abundances that are significantly affected by nicotine use and/or mood disorder status in human temporal cortex
Accession Description Significant effect F p Value 1433Z_HUMAN 14-3-3 protein zeta/delta MD x Nicotine F(1,17) = 4.943 0.043 ACHA4_HUMAN Neuronal acetylcholine receptor subunit α4 Nicotine F(1,17) = 16.596 <0.0009 ACHB2_HUMAN Neuronal acetylcholine receptor subunit β2 Nicotine F(1,17) = 12.881 <0.0009 AT1A1_HUMAN Sodium/potassium-transporting ATPase subunit α1 Nicotine F(1,17) = 7.460 0.016 AT1A2_HUMAN Sodium/potassium-transporting ATPase subunit α2 Nicotine, MD × nicotine F(1,17) = 5.749; F(1,17) = 4.740 0.031; 0.047 AT1A3_HUMAN Sodium/potassium-transporting ATPase subunit α3 Nicotine F(1,17) = 5.820 0.03 AT1B1_HUMAN Sodium/potassium-transporting ATPase subunit β1 Nicotine F(1,17) = 7.537 0.016 AT2B2_HUMAN Plasma membrane calcium-transporting ATPase 2 Nicotine F(1,17) = 13.246 0.003 GNAO_HUMAN Guanine nucleotide-binding protein G(o) subunit α Nicotine F(1,17) = 5.077 0.041 MYPR_HUMAN Myelin proteolipid protein Nicotine F(1,17) = 8.779 0.01 S10A7_HUMAN Protein S100-A7 Nicotine F(1,17) = 5.850 0.03 SNP25_HUMAN Synaptosomal-associated protein 25 Nicotine F(1,17) = 11.990 0.004 STX1A_HUMAN Syntaxin-1A Nicotine F(1,17) = 11.506 0.004 STX1B_HUMAN Syntaxin-1B Nicotine F(1,17) = 11.792 0.004 VPP1_HUMAN V-type proton ATPase 116 kDa subunit a isoform 1 Nicotine F(1,17) = 5.946 0.029 Nicotine use has the largest statistical effect observed in terms of its impact on β2*-nAChR expression and the abundance of associated proteins. Only two proteins (14-3-3ζ and ATP1A2) show a significant interaction between nicotine use and mood disorder.
- Table 8.
β2*-nAChR-associated proteins that show a significant effect of nicotine use on their “per-β2” abundance from human temporal cortex
Accession Description Significant effect F p Value GBB1_HUMAN Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit β1 Nicotine F(1,17) = 10.085 0.007 RO52_HUMAN E3 ubiquitin-protein ligase TRIM21 Nicotine F(1,17) = 5.676 0.031 STX1A_HUMAN Syntaxin-1A Nicotine F(1,17) = 5.375 0.036 TPM4_HUMAN Tropomyosin α4-chain Nicotine F(1,17) = 5.911 0.029 VAOD1_HUMAN V-type proton ATPase subunit d 1 Nicotine F(1,17) = 6.700 0.021 These proteins have abundance indices that correlate significantly with levels of β2-nAChR, but the effect of nicotine is apparent only when protein content is normalized to total β2-nAChR levels, suggesting that their expression does not track linearly with increases in nAChR due to nicotine-induced upregulation and likely represent limiting interactions.
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