Tocotrienol-Rich Fraction and Levodopa Regulate Proteins Involved in Parkinson’s Disease-Associated Pathways in Differentiated Neuroblastoma Cells: Insights from Quantitative Proteomic Analysis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents
2.2. Cell Culture and Differentiation of SH-SY5Y Human Neuroblastoma Cells
2.3. Treatment Protocol
2.4. Protein Extraction, Reduction and Alkylation
2.5. Protein Digestion and Peptide Clean-up
2.6. Liquid Chromatography Electrospray-Ionization Coupled with Tandem Mass Spectrometry Analysis
2.7. Data Computation and Protein Identification
2.8. Differential Protein Expression Analysis
2.9. Determination of Common and Unique Proteins between TRF and Levodopa Treatment
2.10. Gene Ontology (GO) Term and Pathway Enrichment Analysis
2.11. Validation of mRNA by One Step Quantitative PCR (qPCR) Assay
2.12. Statistical Analysis
3. Results
3.1. The Effects of TRF and Levodopa Treatments on Total Protein Extract from Diff-Neural Cells
3.2. Venn Diagram Analysis
3.3. Functional Gene Ontology (GO) Enrichment Analysis
3.4. Common Protein Expression between TRF and Levodopa Treatment in Diff-Neural Cells
3.5. Pathway Enrichment Analysis of the Differentially Expressed Proteins
3.6. TRF and Levodopa Implicated Differential Protein Expression in PD Pathways
3.7. Validation of Protein Expression Using qPCR Assay
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AD | Alzheimer’s disease |
ATP5F1A | ATP synthase F1 subunit α |
CALM | calmodulin |
CAMKII | calmodulin-dependent kinase-IIα |
diff-neural cells | differentiated human SH-SY5Y neuroblastoma cells |
DMSO | dimethyl sulfoxide |
ER | endoplasmic reticulum |
HSPA5 | heat shock protein family A member 5 |
HSPD1 | heat shock protein family D member 1 |
KEGG | Kyoto Encyclopedia of Genes and Genomes |
PD | Parkinson’s disease |
PPIA | peptidylprolyl isomerase A |
RA | retinoic acid |
SLC25 | ADP/ATP translocase 3 |
SOD | superoxide dismutase |
T3 | tocotrienol |
Toc | tocopherol |
TUBA | tubulin α |
TUBB | tubulin β |
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Upregulated Proteins | |||||
---|---|---|---|---|---|
Uniprot Accession | Description | Symbol | Average Mass | p Value | * Log2 (Fold Change) |
Q86VP6 | Cullin-associated NEDD8-dissociated protein 1 | CAND1 | 136,375 | 0.021 | 1.287 |
Q9UL46 | Proteasome activator complex subunit 2 | PSME2 | 27,402 | 0.005 | 0.967 |
P35637 | RNA-binding protein FUS | FUS | 53,426 | 0.003 | 0.885 |
O43143 | Pre-mRNA-splicing factor ATP-dependent RNA helicase | DHX15 | 90,933 | 0.010 | 0.666 |
P49321 | Nuclear autoantigenic sperm protein | NASP | 85,238 | 0.026 | 0.618 |
P05386 | 60S acidic ribosomal protein P1 | RPLP1 | 11,514 | 0.006 | 0.484 |
P14174 | Macrophage migration inhibitory factor | MIF | 12,476 | 0.018 | 0.432 |
O15240 | Neurosecretory protein VGF | VGF | 67,258 | 0.047 | 0.392 |
P06748 | Nucleophosmin | NPM1 | 32,575 | 0.003 | 0.387 |
P12236 | ADP/ATP translocase 3 | SLC25A6 | 32,866 | 0.020 | 0.369 |
P50395 | Rab GDP dissociation inhibitor beta | GDI2 | 50,663 | 0.002 | 0.342 |
P16401 | Histone H1.5 | HIST1H1B | 22,580 | 0.030 | 0.331 |
P0DP23 | Calmodulin-1 | CALM1 | 16,838 | 0.025 | 0.326 |
P0DP24 | Calmodulin-2 | CALM2 | 16,838 | 0.025 | 0.326 |
P0DP25 | Calmodulin-3 | CALM3 | 16,838 | 0.025 | 0.326 |
Q00610 | Clathrin heavy chain 1 | CLTC | 191,613 | 0.012 | 0.319 |
P52209 | 6-phosphogluconate dehydrogenase decarboxylating | PGD | 53,140 | 0.032 | 0.312 |
P25705 | ATP synthase subunit alpha mitochondrial | ATP5F1A | 59,751 | 0.005 | 0.308 |
P62841 | 40S ribosomal protein S15 | RPS15 | 17,040 | 0.005 | 0.308 |
P16104 | Histone H2AX | H2AFX | 15,145 | 0.031 | 0.303 |
Q71UI9 | Histone H2A.V | H2AFV | 13,163 | 0.007 | 0.296 |
P0C0S5 | Histone H2A.Z | H2AFZ | 13,553 | 0.007 | 0.296 |
Q9BUF5 | Tubulin beta-6 chain | TUBB6 | 49,857 | 0.022 | 0.277 |
Q13509 | Tubulin beta-3 chain | TUBB3 | 50,433 | 0.024 | 0.276 |
P16403 | Histone H1.2 | HIST1H1C | 21,365 | 0.044 | 0.264 |
P31150 | Rab GDP dissociation inhibitor alpha | GDI1 | 50,583 | 0.013 | 0.250 |
P68032 | Actin alpha cardiac muscle 1 | ACTC1 | 42,019 | 0.022 | 0.248 |
P08238 | Heat shock protein HSP 90-beta | HSP90AB1 | 83,264 | 0.025 | 0.244 |
P63261 | Actin cytoplasmic 2 | ACTG1 | 41,793 | 0.024 | 0.240 |
P00558 | Phosphoglycerate kinase 1 | PGK1 | 44,615 | 0.016 | 0.239 |
Q9BVA1 | Tubulin beta-2B chain | TUBB2B | 49,953 | 0.018 | 0.236 |
P60709 | Actin cytoplasmic 1 | ACTB | 41,737 | 0.025 | 0.234 |
P46782 | 40S ribosomal protein S5 | RPS5 | 22,876 | 0.022 | 0.232 |
P07602 | Prosaposin | PSAP | 58,113 | 0.014 | 0.224 |
P29401 | Transketolase | TKT | 67,878 | 0.043 | 0.211 |
P10599 | Thioredoxin | TXN | 11,737 | 0.012 | 0.201 |
O00571 | ATP-dependent RNA helicase DDX3X | DDX3X | 73,244 | 0.007 | 0.199 |
P37802 | Transgelin-2 | TAGLN2 | 22,391 | 0.031 | 0.197 |
P27348 | 14-3-3 protein theta | YWHAQ | 27,764 | 0.023 | 0.185 |
P62937 | Peptidyl-prolyl cis-trans isomerase A | PPIA | 18,012 | 0.024 | 0.17 |
P68371 | Tubulin beta-4B chain | TUBB4B | 49,831 | 0.035 | 0.157 |
P07437 | Tubulin beta chain | TUBB | 49,671 | 0.013 | 0.151 |
P09936 | Ubiquitin carboxyl-terminal hydrolase isozyme L1 | UCHL1 | 24,824 | 0.043 | 0.145 |
P33778 | Histone H2B type 1-B | HIST1H2BB | 13,950 | 0.015 | 0.130 |
P06899 | Histone H2B type 1-J | HIST1H2BJ | 13,904 | 0.015 | 0.130 |
P23527 | Histone H2B type 1-O | HIST1H2BO | 13,906 | 0.015 | 0.130 |
P68363 | Tubulin alpha-1B chain | TUBA1B | 50,152 | 0.048 | 0.124 |
P63104 | 14-3-3 protein zeta/delta | YWHAZ | 27,745 | 0.013 | 0.039 |
P62136 | Serine/threonine-protein phosphatase PP1-alpha catalytic subunit | PPP1CA | 37,512 | 0.040 | 0.037 |
P62140 | Serine/threonine-protein phosphatase PP1-beta catalytic subunit | PPP1CB | 37,187 | 0.040 | 0.037 |
P36873 | Serine/threonine-protein phosphatase PP1-gamma catalytic subunit | PPP1CC | 36,984 | 0.040 | 0.037 |
P07737 | Profilin-1 | PFN1 | 15,054 | 0.004 | 0.037 |
P10809 | 60 kDa heat shock protein mitochondrial | HSPD1 | 61,055 | 0.034 | 0.025 |
Down-Regulated Proteins | |||||
Uniprot Accession | Description | Symbol | Average Mass | p value | * Log2 (Fold Change) |
P24534 | Elongation factor 1-beta | EEF1B2 | 24,764 | 0.011 | −0.055 |
P60660 | Myosin light polypeptide 6 | MYL6 | 16,930 | 0.012 | −0.088 |
Q16555 | Dihydropyrimidinase-related protein 2 | DPYSL2 | 73,503 | 0.012 | −0.121 |
Q05639 | Elongation factor 1-alpha 2 | EEF1A2 | 50,470 | 0.035 | −0.125 |
P14625 | Endoplasmin | HSP90B1 | 92,469 | 0.023 | −0.132 |
P00338 | L-lactate dehydrogenase A chain | LDHA | 36,689 | 0.041 | −0.178 |
Q00839 | Heterogeneous nuclear ribonucleoprotein U | HNRNPU | 90,585 | 0.017 | −0.195 |
P30041 | Peroxiredoxin-6 | PRDX6 | 25,035 | 0.035 | −0.200 |
P39687 | Acidic leucine-rich nuclear phosphoprotein 32 family member A | ANP32A | 19,997 | 0.013 | −0.248 |
P32119 | Peroxiredoxin-2 | PRDX2 | 21,892 | 0.001 | −0.312 |
Q08211 | ATP-dependent RNA helicase A | DHX9 | 140,958 | 0.041 | −0.324 |
P62249 | 40S ribosomal protein S16 | RPS16 | 16,445 | 0.014 | −0.336 |
P19338 | Nucleolin | NCL | 76,615 | 0.000 | −0.364 |
Q02878 | 60S ribosomal protein L6 | RPL6 | 32,728 | 0.000 | −0.366 |
Q06830 | Peroxiredoxin-1 | PRDX1 | 10,676 | 0.033 | −0.394 |
P11021 | Endoplasmic reticulum chaperone BiP | HSPA5 | 72,333 | 0.005 | −0.405 |
P09429 | High mobility group protein B1 | HMGB1 | 24,894 | 0.002 | −0.414 |
P07237 | Protein disulfide-isomerase | P4HB | 57,116 | 0.033 | −0.415 |
P22626 | Heterogeneous nuclear ribonucleoproteins A2/B1 | HNRNPA2B1 | 37,430 | 0.027 | −0.451 |
P60842 | Eukaryotic initiation factor 4A-I | EIF4A1 | 46,154 | 0.015 | −0.459 |
P35232 | Prohibitin | PHB | 22,271 | 0.002 | −0.462 |
P29373 | Cellular retinoic acid-binding protein 2 | CRABP2 | 15,693 | 0.001 | −0.508 |
P40939 | Trifunctional enzyme subunit alpha mitochondrial | HADHA | 86,372 | 0.032 | −0.510 |
P05387 | 60S acidic ribosomal protein P2 | RPLP2 | 11,665 | 0.031 | −0.521 |
P23396 | 40S ribosomal protein S3 | RPS3 | 26,688 | 0.045 | −0.580 |
P46781 | 40S ribosomal protein S9 | RPS9 | 22,591 | 0.036 | −0.586 |
P35579 | Myosin-9 | MYH9 | 226,530 | 0.045 | −0.727 |
P27824 | Calnexin | CANX | 67,568 | 0.001 | −1.187 |
Upregulated Proteins | |||||
---|---|---|---|---|---|
Uniprot Accession | Description | Symbol | Average Mass | p Value | * Log2 (Fold Change) |
P15121 | Aldose reductase | AKR1B1 | 35,853 | 0.002 | 0.928 |
Q09666 | Neuroblast differentiation-associated protein | AHNAK | 629,114 | 0.025 | 0.870 |
P35637 | RNA-binding protein FUS | FUS | 53,426 | 0.002 | 0.707 |
P49321 | Nuclear autoantigenic sperm protein | NASP | 85,238 | 0.027 | 0.707 |
P30153 | Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform | PPP2R1A | 65,309 | 0.027 | 0.689 |
P05386 | 60S acidic ribosomal protein P1 | RPLP1 | 11,514 | 0.005 | 0.611 |
P06748 | Nucleophosmin | NPM1 | 32,575 | 0.010 | 0.422 |
P04792 | Heat shock protein beta-1 | HSPB1 | 22,783 | 0.007 | 0.372 |
P08238 | Heat shock protein 90kDa alpha (Cytosolic) class B member 1 isoform CRA_a | HSP90AB1 | 83,264 | 0.007 | 0.22 |
P62937 | Peptidyl-prolyl cis-trans isomerase A | PPIA | 18,012 | 0.015 | 0.209 |
P16401 | Histone H1.5 | HIST1H1B | 22,580 | 0.000 | 0.203 |
Q9BVA1 | Tubulin beta-2B chain | TUBB2B | 49,953 | 0.038 | 0.190 |
P60709 | Actin cytoplasmic 1 | ACTB | 41,737 | 0.011 | 0.169 |
P63261 | Actin cytoplasmic 2 | ACTG1 | 41,793 | 0.010 | 0.168 |
P07437 | Tubulin beta chain | TUBB | 49,671 | 0.000 | 0.156 |
P07195 | L-lactate dehydrogenase B chain | LDHB | 36,639 | 0.005 | 0.150 |
Q9BUF5 | Tubulin beta-6 chain | TUBB6 | 49,857 | 0.003 | 0.148 |
P09936 | Ubiquitin carboxyl-terminal hydrolase isozyme L1 | UCHL1 | 24,824 | 0.017 | 0.145 |
P68032 | Actin alpha cardiac muscle 1 | ACTC1 | 42,019 | 0.023 | 0.137 |
Q71U36 | Tubulin alpha-1A chain | TUBA1A | 50,136 | 0.038 | 0.135 |
P16104 | Histone H2AX | H2AFX | 15,145 | 0.038 | 0.134 |
Q13509 | Tubulin beta-3 chain | TUBB3 | 50,433 | 0.004 | 0.132 |
P68371 | Tubulin beta-4B chain | TUBB4B | 49,831 | 0.005 | 0.124 |
P68363 | Tubulin alpha-1B chain | TUBA1B | 50,152 | 0.046 | 0.122 |
P04406 | Glyceraldehyde-3-phosphate dehydrogenase | GAPDH | 36,053 | 0.007 | 0.122 |
P11142 | Heat shock cognate 71 kDa protein | HSPA8 | 70,898 | 0.008 | 0.122 |
P09455 | Retinol-binding protein 1 | RBP1 | 15,850 | 0.004 | 0.115 |
P16403 | Histone H1.2 | HIST1H1C | 21,365 | 0.025 | 0.098 |
P68104 | Elongation factor 1-alpha 1 | EEF1A1 | 50,141 | 0.006 | 0.080 |
P25705 | ATP synthase subunit alpha mitochondrial | ATP5F1A | 59,751 | 0.017 | 0.066 |
P60660 | Myosin light polypeptide 6 | MYL6 | 16,930 | 0.046 | 0.055 |
P10809 | 60 kDa heat shock protein mitochondrial | HSPD1 | 61,055 | 0.013 | 0.038 |
Down-regulated proteins | |||||
Uniprot Accession | Description | Symbol | Average Mass | p Value | * Log2 (Fold Change) |
P27824 | Calnexin | CANX | 67,568 | 0.010 | −1.331 |
Q01105 | Protein SET | SET | 33,489 | 0.001 | −1.292 |
P27797 | Calreticulin | CALR | 48,142 | 0.026 | −1.006 |
P62851 | 40S ribosomal protein S25 | RPS25 | 13,742 | 0.003 | −0.925 |
P07237 | Protein disulfide-isomerase | P4HB | 57,116 | 0.042 | −0.777 |
O75531 | Barrier-to-autointegration factor | BANF1 | 10,059 | 0.000 | −0.775 |
P35232 | Prohibitin | PHB | 29,804 | 0.002 | −0.745 |
Q92928 | Putative Ras-related protein Rab-1C | RAB1C | 22,017 | 0.032 | −0.538 |
P29373 | Cellular retinoic acid-binding protein 2 | CRABP2 | 15,693 | 0.005 | −0.474 |
Q15084 | Protein disulfide-isomerase A6 | PDIA6 | 48,121 | 0.022 | −0.406 |
P49327 | Fatty acid synthase | FASN | 273,424 | 0.007 | −0.398 |
P62249 | 40S ribosomal protein S16 | RPS16 | 16,445 | 0.001 | −0.390 |
P62753 | 40S ribosomal protein S6 | RPS6 | 28,681 | 0.023 | −0.271 |
P40227 | T-complex protein 1 subunit zeta | CCT6A | 58,024 | 0.020 | −0.246 |
P32119 | Peroxiredoxin-2 | PRDX2 | 21,892 | 0.026 | −0.230 |
P14618 | Pyruvate kinase PKM | PKM | 57,937 | 0.039 | −0.189 |
P62424 | 60S ribosomal protein L7a | RPL7A | 29,996 | 0.043 | −0.184 |
P21796 | Voltage-dependent anion-selective channel protein 1 | VDAC1 | 30,773 | 0.042 | −0.165 |
Q9Y277 | Voltage-dependent anion-selective channel protein 3 | VDAC3 | 30,659 | 0.042 | −0.165 |
P23396 | 40S ribosomal protein S3 | RPS3 | 26,688 | 0.027 | −0.164 |
Q00839 | Heterogeneous nuclear ribonucleoprotein U | HNRNPU | 90,585 | 0.002 | −0.162 |
P13639 | Elongation factor 2 | EEF2 | 95,338 | 0.018 | −0.154 |
P00338 | L-lactate dehydrogenase A chain | LDHA | 36,689 | 0.001 | −0.141 |
P23284 | Peptidyl-prolyl cis-trans isomerase B | PPIB | 23,743 | 0.038 | −0.080 |
O15240 | Neurosecretory protein VGF | VGF | 67,258 | 0.002 | −0.014 |
Compartment | Total Proteins | Elements |
---|---|---|
TRF and levodopa | 32 | HSPD1, HSP90AB1, RPS16, CANX, LDHA, TUBB, NASP, TUBB6, RPLP1, PPIA, ACTB, TUBB4B, MYL6, HIST1H1B, UCHL1, NPM1, FUS, TUBB3, HIST1H1C, TUBA1B, RPS3, CRABP2, VGF, P4HB, H2AFX, PRDX2, ACTC1, ACTG1, TUBB2B, ATP5F1A, HNRNPU, PPP2R1A |
Unique to TRF | 49 | PRDX6, PPP1CB, PGD, SLC25A6, CALM3, PPP1CC, TXN, RPS9, CALM2, HSP90B1, NCL, HSPA5, CAND1, TKT, YWHAQ, HIST1H2BJ, HNRNPA2B1, DHX15, CLTC, MYH9, HADHA, EEF1B2, TAGLN2, PSME2, MIF, PFN1, RPL6, GDI2, HIST1H2BO, DPYSL2, EIF4A1, H2AFZ, RPS5, GDI1, PPP1CA, HMGB1, HIST1H2BB, YWHAZ, PGK1, DHX9, ANP32A, CALM1, RPLP2, DDX3X, H2AFV, EEF1A2, PSAP, PRDX1, RPS15 |
Unique to levodopa | 25 | SET, RPS25, RPS6, HSPB1, PPIB, VDAC3, VDAC1, RAB1C, CCT6A, HSPA8, PHB, EEF2, GAPDH, EEF1A1, PDIA6, AHNAK, BANF1, LDHB, RBP1, TUBA1A, PKM, RPL7A, CALR, AKR1B1, FASN |
KEGG-Pathways | Pathway Accession | Differentially Regulated Proteins by TRF | Differentially Regulated Proteins by Levodopa |
---|---|---|---|
Pathways of neurodegeneration-multiple diseases | hsa05022 | ATP5F1A↑, CALM1↑, CALM2↑, CALM3↑, FUS↑, HSPA5↓, SLC25A6↑, TUBA1B↑, TUBB↑, TUBB2B↑, TUBB3↑, TUBB4B↑, TUBB6↑, UCHL1↑ | ATP5F1A↑, FUS↑, TUBA1A↑, TUBA1B↑, TUBB↑, TUBB2B↑, TUBB3↑, TUBB4B↑, TUBB6↑, UCHL1↑, VDAC1↓, VDAC3↓ |
Parkinson’s disease | hsa05012 | ATP5F1A↓, CALM1↑, CALM2↑, CALM3↑, HSPA5↓, SLC25A6↑, TUBA1B↑, TUBB↑, TXN↑, TUBB2B↑, TUBB3↑, TUBB4B↑, TUBB6↑, UCHL1↑ | ATP5F1A↑, TUBA1A↑, TUBA1B↑, TUBB↑, TUBB2B↑, TUBB3↑, TUBB4B↑, TUBB6↑, UCHL1↑, VDAC1↓, VDAC3↓ |
Amyotrophic lateral sclerosis | hsa05014 | ACTB↑, ACTG1↑, ATP5F1A↓, FUS↑, HNRNPA2B1↓, HSPA5↓, PFN1↑, TUBA1B↑, TUBB↑, TUBB2B↑, TUBB3↑, TUBB4B↑, TUBB6↑ | ACTB↑, ACTG1↑, ATP5F1A↑, FUS↑, TUBA1A↑, TUBA1B↑, TUBB↑, TUBB2B↑, TUBB3↑, TUBB4B↑, TUBB6↑, VDAC1↓ |
Alzheimer’s disease | hsa05010 | ATP5F1A↓, CALM1↑, CALM2↑, CALM3↑, SLC25A6↑, TUBA1B↑, TUBB↑, TUBB2B↑, TUBB3↑, TUBB4B↑, TUBB6↑ | ATP5F1A↑, GAPDH↑, TUBA1A↑, TUBA1B↑, TUBB↑, TUBB2B↑, TUBB3↑, TUBB4B↑, TUBB6↑, VDAC1↓, VDAC3↓ |
Huntington disease | hsa05016 | ATP5F1A↓, CLTC↑, SLC25A6↑, TUBA1B↑, TUBB↑, TUBB2B↑, TUBB3↑, TUBB4B↑, TUBB6↑ | ATP5F1A↑, TUBA1A↑, TUBA1B↑, TUBB↑, VDAC3↓ TUBB2B↑, TUBB3↑, TUBB4B↑, TUBB6↑, VDAC1↓ |
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Magalingam, K.B.; Ramdas, P.; Somanath, S.D.; Selvaduray, K.R.; Bhuvanendran, S.; Radhakrishnan, A.K. Tocotrienol-Rich Fraction and Levodopa Regulate Proteins Involved in Parkinson’s Disease-Associated Pathways in Differentiated Neuroblastoma Cells: Insights from Quantitative Proteomic Analysis. Nutrients 2022, 14, 4632. https://doi.org/10.3390/nu14214632
Magalingam KB, Ramdas P, Somanath SD, Selvaduray KR, Bhuvanendran S, Radhakrishnan AK. Tocotrienol-Rich Fraction and Levodopa Regulate Proteins Involved in Parkinson’s Disease-Associated Pathways in Differentiated Neuroblastoma Cells: Insights from Quantitative Proteomic Analysis. Nutrients. 2022; 14(21):4632. https://doi.org/10.3390/nu14214632
Chicago/Turabian StyleMagalingam, Kasthuri Bai, Premdass Ramdas, Sushela Devi Somanath, Kanga Rani Selvaduray, Saatheeyavaane Bhuvanendran, and Ammu Kutty Radhakrishnan. 2022. "Tocotrienol-Rich Fraction and Levodopa Regulate Proteins Involved in Parkinson’s Disease-Associated Pathways in Differentiated Neuroblastoma Cells: Insights from Quantitative Proteomic Analysis" Nutrients 14, no. 21: 4632. https://doi.org/10.3390/nu14214632
APA StyleMagalingam, K. B., Ramdas, P., Somanath, S. D., Selvaduray, K. R., Bhuvanendran, S., & Radhakrishnan, A. K. (2022). Tocotrienol-Rich Fraction and Levodopa Regulate Proteins Involved in Parkinson’s Disease-Associated Pathways in Differentiated Neuroblastoma Cells: Insights from Quantitative Proteomic Analysis. Nutrients, 14(21), 4632. https://doi.org/10.3390/nu14214632