Skin aging is a complex process, and a lot of efforts have been made to identify new and specific targets that could help to diagnose, prevent, and treat skin aging. Several studies concerning skin aging have analyzed the changes in gene expression, and very few investigations have been performed at the protein level. Moreover, none of these proteomic studies has used a global quantitative labeled proteomic offgel approach that allows a more accurate description of aging phenotype. We applied such an approach on human primary keratinocytes obtained from sun-nonexposed skin biopsies of young and elderly women. A total of 517 unique proteins were identified, and 58 proteins were significantly differentially expressed with 40 that were downregulated and 18 upregulated with aging. Gene ontology and pathway analysis performed on these 58 putative biomarkers of skin aging evidenced that these dysregulated proteins were mostly involved in metabolism and cellular processes such as cell cycle and signaling pathways. Change of expression of tubulin beta-3 chain was confirmed by western blot on samples originated from several donors. Thus, this study suggested the tubulin beta-3 chain has a promising biomarker in skin aging.
Life expectancy in developed countries over the past two centuries has considerably increased, and if this trend continues through the 21st century, most babies born since 2000 in such countries will reach 100 years. Also, it is expected that by 2030, one in eight people worldwide will be 65 or above and the global aging of the population will lead to several societal, economical, and medical challenges [
Aging is a complex process influenced by multiple genetic and environmental factors and is characterized by a progressive decline in multiple physiological functions. Skin like other organs is affected by aging that can be accelerated by environmental factors such as UV radiation. Intrinsic skin aging is observed in sun-nonexposed skin and reflects the aging process of the entire organism [
Over the last decade, several transcriptomic studies have investigated the effect of aging on gene expression in several organism models and in humans [
Proteins are the workhorses of the cell and the main effectors of numerous cellular processes. Quantitative mass spectrometry-based proteomics has proven its utility for the description of protein dynamics in order to decipher complex processes and to describe normal and pathological states [
Skin is composed of several cell layers, and previous studies have investigated the protein expression level in whole skin or in
Our quantitative proteomic profiling, which expression was significantly dysregulated, of young and elderly primary human keratinocytes identified 58 proteins that are putative candidate biomarkers for intrinsic skin aging. Further western blot analysis on 14 donors confirms that tubulin beta-3 chain could be a biomarker of skin aging.
Human keratinocyte cultures were established by outgrowth from skin biopsies obtained after plastic mammary surgery (Centre Hospitalier Universitaire Grenoble Alpes, France) from healthy donors with their informed consent. Donors were Caucasian women aged 57–71 years (
Cultured keratinocytes obtained from young (27 and 32 years) and elderly (60 and 65 years) donors were cultivated up to passage 2 and harvested by trypsinization. After two washes with PBS (Life Technologies), cell pellets were kept at −80°C up to the extraction. Frozen cell pellets were lysed for 30 minutes at 4°C in a solution containing 40 mM HEPES pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.02% Triton, 0.02% sodium deoxycholate, 0.2 mM TCEP, and protease and phosphatase inhibitor cocktails (PhosSTOP) from Roche. Lysis was achieved by short sonication on ice, and the lysates were cleared by centrifugation at 14,000 rpm for 20 minutes at 4°C. The concentration of the protein extract was determined using BCA protein assay kit (Thermo Fisher Scientific, IL, USA).
Protein samples were labeled with iTRAQ reagents according to the manufacturer’s instructions (iTRAQ Reagents 8 plex Applications kit; Sciex, Framingham, MA, USA). Briefly, equal amount of protein extract obtained from cells originated from young donors was pooled in order to achieve a total of 100
Peptide fractionation according to their pI was performed with 3100 OFFGEL Fractionator and the OFFGEL Kit linear pH 3–10 (Agilent Technology, Les Ulis, France) in a 24-well setup following the manufacturer’s instructions. The device was set up for the 24-fraction separation by using a 24-cm-long IPG gel strip with a linear pH gradient ranging at 3–10. iTRAQ-labeled peptide mix was dried by vacuum centrifugation and resuspended in focusing OFFGEL buffer prior to loading in each of the 24 wells. Peptides were focused with a constant current of 50
Further peptide separation was performed on an Ultimate 3000 C18 reversed phase nanoliquid chromatography (RP-nanoLC) system (Ultimate 3000, Dionex/Thermo Scientific) controlled by Chromeleon v. 6.80 software (Dionex/Thermo Scientific/LC Packings, Amsterdam, The Netherlands) and coupled with a PROBOT MALDI spotting device controlled by the
MS and MS/MS analysis of nanoLC-off-line spotted peptide samples was performed using the 4800 MALDI-TOF/TOF mass spectrometer (Sciex, Les Ulis, France) controlled by the 4000 Series Explorer software v. 3.5. The mass spectrometer was operated in a positive reflector mode. Each spectrum was externally calibrated using the Peptide Calibration Standard II (Bruker Daltonics, Bremen, Germany), and the peptide mass tolerance was set to 50 ppm
MS and MS/MS spectra were used for identification and relative quantitation by using ProteinPilot™ software v 4.0 with the Paragon™ (Sciex, Les Ulis, France) and Mascot (Matrix Science, London, UK) search engines. The analysis was performed with the UniProtKB database released on June 2015, and the taxonomy was limited to
Gene ontology and protein function analysis were performed using PANTHER (
Human primary keratinocytes were harvested and cultivated as described previously from skin biopsies of 8 young (age: 18, 21, 24, 26, 27 (2 donors), 30, and 32) and 10 elderly donors (age: 57, 59, 60, 62 (2), 65 (2), 66, 68, and 71). At early passages (2 or 3, when cells are still proliferating), cells were lysed by vortexing in RIPA Buffer (Sigma-Aldrich) containing protease inhibitors (Complete Mini protease inhibitor cocktail, Roche, Switzerland), 1 mM DTT and 100
Western blot results of tubulin beta-3 chain were illustrated by box plots, and receiver operating characteristic curve (ROC curve) was created by using GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla California USA,
In order to obtain a quantitative proteomic map of elderly and young donor-derived keratinocyte cells, we used an iTRAQ labeling coupled with OFFGEL fractionation and off-line nanoLC/MS/MS as previously described [
List of proteins significantly downregulated in elderly versus young cells (iTRAQ ratio 117/113). Statistically significant iTRAQ ratios (
Accession/variants | ID | Description | Gene | Peptide count | Spectral count | Sequence coverage (%) | Ratio [elderly/young] |
|
|
Log10 ratio |
---|---|---|---|---|---|---|---|---|---|---|
O60814 | H2B1K_HUMAN | Histone H2B type 1-K | HIST1H2BK | 9 | 131 | 4.76 | 0.170 | 6.27 |
4.36 |
−0.769 |
O75334-[2-6] | LIPA2_HUMAN | Liprin-alpha-2 | PPFIA2 | 2 | 2 | 0.64 | 0.295 | 3.83 |
5.16 |
−0.531 |
P20674 | COX5A_HUMAN | Cytochrome c oxidase subunit 5A, mitochondrial | COX5A | 2 | 3 | 9.33 | 0.309 | 1.56 |
2.23 |
−0.510 |
P04732 | MT1E_HUMAN | Metallothionein-1E | MT1E | 1 | 9 | 16.39 | 0.310 | 7.22 |
2.40 |
−0.509 |
P05204 | HMGN2_HUMAN | Nonhistone chromosomal protein HMG-17 | HMGN2 | 4 | 11 | 8.89 | 0.330 | 1.89 |
1.50 |
−0.482 |
Q15075 | EEA1_HUMAN | Early endosome antigen 1 | EEA1 | 2 | 3 | 0.64 | 0.331 | 1.71 |
1.61 |
−0.481 |
Q9UKY7-[2] | CDV3_HUMAN | Protein CDV3 homolog | CDV3 | 3 | 16 | 11.63 | 0.342 | 1.18 |
4.11 |
−0.466 |
O75152 | ZC11A_HUMAN | Zinc finger CCCH domain-containing protein 11A | ZC3H11A | 1 | 1 | 1.48 | 0.349 | 2.87 |
7.18 |
−0.457 |
P17096 | HMGA1_HUMAN | High mobility group protein |
HMGA1 | 1 | 19 | 7.48 | 0.352 | 1.56 |
8.83 |
−0.454 |
P06454-[2] | PTMA_HUMAN | Prothymosin alpha [cleaved into: |
PTMA | 5 | 24 | 12.61 | 0.357 | 4.01 |
1.34 |
−0.447 |
Q8NC51-[3] | PAIRB_HUMAN | Plasminogen activator inhibitor 1 |
SERBP1 | 11 | 90 | 4.66 | 0.361 | 1.56 |
1.82 |
−0.442 |
Q13442 | HAP28_HUMAN | 28 kDa heat- and acid-stable phosphoprotein | PDAP1 | 1 | 1 | 7.18 | 0.363 | 1.23 |
2.09 |
−0.440 |
P63313 | TYB10_HUMAN | Thymosin beta-10 | TMSB10 | 2 | 35 | 13.64 | 0.364 | 1.30 |
2.21 |
−0.439 |
O00233 | PSMD9_HUMAN | 26S proteasome non-ATPase |
PSMD9 | 1 | 6 | 5.38 | 0.383 | 2.34 |
8.33 |
−0.416 |
P05114 | HMGN1_HUMAN | Nonhistone chromosomal protein HMG-14 | HMGN1 | 3 | 13 | 8.00 | 0.384 | 1.20 |
8.80 |
−0.415 |
P62158 | CALM_HUMAN | Calmodulin | CALM1 | 10 | 147 | 8.72 | 0.385 | 2.89 |
9.13 |
−0.415 |
P02795, P13640-[2], P80297 | MT1G_HUMAN, MT1X_HUMAN, MT2_HUMAN | Metallothionein-1G, Metallothionein-1X, Metallothionein-2 | MT1G, MT1X, MT2A | 1 | 20 | 16.29 | 0.392 | 3.61 |
1.47 |
−0.406 |
P67936 | TPM4_HUMAN | Tropomyosin alpha-4 chain | TPM4 | 5 | 48 | 3.23 | 0.401 | 3.64 |
2.51 |
−0.397 |
P22528 | SPR1B_HUMAN | Cornifin-B | SPRR1B | 4 | 56 | 8.99 | 0.405 | 2.54 |
3.18 |
−0.392 |
Q92538-[2,3] | GBF1_HUMAN | Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 | GBF1 | 1 | 1 | 0.32 | 0.406 | 4.48 |
3.34 |
−0.391 |
P51858 | HDGF_HUMAN | Hepatoma-derived growth factor | HDGF | 4 | 12 | 3.75 | 0.429 | 2.37 |
1.17 |
−0.368 |
P61604 | CH10_HUMAN | 10 kDa heat shock protein, mitochondrial | HSPE1 | 12 | 121 | 7.84 | 0.455 | 2.20 |
4.16 |
−0.342 |
P07108-[2-5] | ACBP_HUMAN | Acyl-CoA-binding protein | DBI | 4 | 50 | 9.20 | 0.460 | 1.86 |
5.21 |
−0.337 |
Q9C030-[2] | TRIM6_HUMAN | Tripartite motif-containing protein 6 | TRIM6 | 2 | 8 | 1.23 | 0.463 | 1.10 |
6.03 |
−0.334 |
P20962 | PTMS_HUMAN | Parathymosin | PTMS | 4 | 11 | 8.82 | 0.468 | 1.44 |
7.30 |
−0.330 |
Q9GZP8 | IMUP_HUMAN | Immortalization upregulated protein | IMUP | 3 | 5 | 9.43 | 0.476 | 8.05 |
1.05 |
−0.322 |
Q9H299 | SH3L3_HUMAN | SH3 domain-binding glutamic acid-rich-like protein 3 | SH3BGRL3 | 3 | 36 | 10.75 | 0.490 | 1.62 |
1.82 |
−0.310 |
P62857 | RS28_HUMAN | 40S ribosomal protein S28 | RPS28 | 3 | 20 | 17.39 | 0.491 | 9.21 |
1.93 |
−0.309 |
P16949-[2] | STMN1_HUMAN | Stathmin | STMN1 | 3 | 24 | 8.72 | 0.492 | 2.51 |
2.00 |
−0.308 |
P02765 | FETUA_HUMAN | Alpha-2-HS-glycoprotein | AHSG | 4 | 61 | 3.27 | 0.507 | 4.77 |
3.38 |
−0.295 |
O15212 | PFD6_HUMAN | Prefoldin subunit 6 | PFDN6 | 1 | 9 | 9.30 | 0.519 | 1.72 |
5.20 |
−0.285 |
P52926 | HMGA2_HUMAN | High mobility group protein HMGI-C | HMGA2 | 3 | 8 | 11.93 | 0.528 | 1.02 |
7.07 |
−0.277 |
P61956 | SUMO2_HUMAN | Small ubiquitin-related modifier 2 | SUMO2 | 1 | 7 | 12.63 | 0.534 | 1.89 |
8.65 |
−0.272 |
Q9UHV9 | PFD2_HUMAN | Prefoldin subunit 2 | PFDN2 | 1 | 5 | 9.09 | 0.544 | 1.81 |
1.16 |
−0.265 |
P20929-[2,3] | NEBU_HUMAN | Nebulin | NEB | 4 | 4 | 0.13 | 0.563 | 5.23 |
2.03 |
−0.250 |
P09429 | HMGB1_HUMAN | High mobility group protein B1 | HMGB1 | 5 | 33 | 5.58 | 0.563 | 9.96 |
2.07 |
−0.249 |
P62328 | TYB4_HUMAN | Thymosin beta-4 | TMSB4X | 1 | 16 | 15.91 | 0.582 | 4.60 |
3.41 |
−0.235 |
P09497-[2] | CLCB_HUMAN | Clathrin light chain B | CLTB | 7 | 24 | 3.49 | 0.593 | 1.87 |
4.46 |
−0.227 |
P35749-[2-4] | MYH11_HUMAN | Myosin-11 | MYH11 | 1 | 5 | 0.56 | 0.649 | 4.13 |
1.53 |
−0.188 |
Q16629-[2-4] | SRSF7_HUMAN | Serine/arginine-rich splicing factor 7 | SRSF7 | 2 | 13 | 3.78 | 0.651 | 3.09 |
1.59 |
−0.187 |
List of proteins significantly upregulated in elderly versus young cells (iTRAQ ratio 117/113). Statistically significant iTRAQ ratios (
Accession/variants | ID | Description | Gene | Peptide count | Spectral count | Sequence |
Ratio [elderly/young] |
|
|
Log10 ratio |
---|---|---|---|---|---|---|---|---|---|---|
P26373 | RL13_HUMAN | 60S ribosomal protein L13 | RPL13 | 4 | 15 | 4.27 | 1.455 | 2.54 |
3.04 |
0.163 |
P01861 | IGHG4_HUMAN | Ig gamma-4 chain C region | IGHG4 | 1 | 4 | 4.89 | 1.482 | 2.00 |
2.46 |
0.171 |
Q13200 | PSMD2_HUMAN | 26S proteasome non-ATPase regulatory |
PSMD2 | 1 | 5 | 1.65 | 1.511 | 4.13 |
1.96 |
0.179 |
P13797 | PLST_HUMAN | Plastin-3 | PLS3 | 5 | 13 | 1.90 | 1.584 | 2.13 |
1.08 |
0.200 |
P49721 | PSB2_HUMAN | Proteasome subunit beta type-2 | PSMB2 | 1 | 22 | 5.47 | 1.635 | 1.82 |
7.01 |
0.214 |
P48643 | TCPE_HUMAN | T-complex protein 1 subunit epsilon | CCT5 | 6 | 35 | 1.29 | 1.696 | 2.17 |
4.15 |
0.229 |
P11166 | GTR1_HUMAN | Solute carrier family 2, facilitated glucose transporter member 1 | SLC2A1 | 4 | 46 | 2.03 | 1.891 | 2.60 |
7.28 |
0.277 |
P61158 | ARP3_HUMAN | Actin-related protein 3 | ACTR3 | 6 | 26 | 2.63 | 2.040 | 3.21 |
1.84 |
0.310 |
P09211 | GSTP1_HUMAN | Glutathione S-transferase P | GSTP1 | 10 | 117 | 7.62 | 2.308 | 3.65 |
1.47 |
0.363 |
P78417 | GSTO1_HUMAN | Glutathione S-transferase omega-1 | GSTO1 | 4 | 25 | 5.81 | 2.482 | 2.89 |
2.77 |
0.395 |
P13667 | PDIA4_HUMAN | Protein disulfide-isomerase A4 | PDIA4 | 3 | 4 | 1.09 | 2.633 | 2.99 |
6.60 |
0.420 |
P62277 | RS13_HUMAN | 40S ribosomal protein S13 | RPS13 | 1 | 1 | 7.95 | 2.638 | 2.88 |
6.27 |
0.421 |
Q13509 | TBB3_HUMAN | Tubulin beta-3 chain | TUBB3 | 4 | 19 | 4.00 | 2.664 | 4.21 |
4.87 |
0.426 |
P30048 | PRDX3_HUMAN | Thioredoxin-dependent peroxide reductase, mitochondrial | PRDX3 | 1 | 1 | 5.47 | 2.769 | 3.86 |
1.80 |
0.442 |
Q01813-[2] | PFKAP_HUMAN | ATP-dependent 6-phosphofructokinase, |
PFKP | 2 | 3 | 2.68 | 3.116 | 3.23 |
6.75 |
0.494 |
P50213 | IDH3A_HUMAN | Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial | IDH3A | 3 | 3 | 2.19 | 3.239 | 6.67 |
2.14 |
0.510 |
Q9Y6N5 | SQRD_HUMAN | Sulfide:quinone oxidoreductase, mitochondrial | SQRDL | 1 | 2 | 2.22 | 3.487 | 8.97 |
2.17 |
0.543 |
P38606-[2] | VATA_HUMAN | V-type proton ATPase catalytic subunit A | ATP6V1A | 2 | 2 | 2.43 | 4.833 | 1.38 |
1.75 |
0.684 |
The 58 proteins previously identified were analyzed using PANTHER [
List of proteins identified as differently expressed with age status and their functional classification relating to protein family, protein class, molecular function, biological process, cellular component, and pathway according to PANTHER and the GO database.
Accession | ID | Description | Gene | PANTHER |
PANTHER protein class | PANTHER GO-slim |
PANTHER GO-slim biological process | PANTHER GO-slim |
Pathway |
---|---|---|---|---|---|---|---|---|---|
P61158 | ARP3_ |
Actin-related |
ACTR3 | Actin-related protein 3 |
Actin and actin-related |
Structural constituent |
Cytokinesis(GO:0009987); |
Actin cytoskeleton(GO:0043226); |
|
|
|||||||||
P02765 | FETUA_ |
Alpha-2-HS-glycoprotein | AHSG | Alpha-2-HS-glycoprotein |
Extracellular matrix |
Cysteine-type peptidase |
Immune system |
Extracellular region |
|
|
|||||||||
P38606 | VATA_ |
V-type proton |
ATP6V1A | V-type proton atpase |
ATP synthase(PC00227); anion channel (PC00068); ligand-gated ion channel (PC00002); ligand-gated ion channel (PC00133); DNA-binding protein (PC00049); hydrolase |
Hydrolase activity |
Respiratory electron |
Proton-transporting |
|
|
|||||||||
P62158 | CALM_ |
Calmodulin | CALM1 | Calmodulin |
Calmodulin (PC00060) | Calcium ion binding (GO:0005488); |
Cellular component |
CCKR signaling | |
P48643 | TCPE_ |
T-complex |
CCT5 | T-complex protein |
Chaperonin (PC00072) | Protein folding |
|||
|
|||||||||
Q9UKY7 | CDV3_ |
Protein CDV3 |
CDV3 | Protein CDV3 homolog (PTHR16284:SF13) | |||||
|
|||||||||
P09497 | CLCB_ |
Clathrin light |
CLTB | Clathrin light |
Vesicle coat |
Intracellular protein |
Vesicle coat (GO:0032991); |
Heterotrimeric | |
|
|||||||||
P20674 | COX5A_ |
Cytochrome C |
COX5A | Cytochrome C |
Oxidase(PC00176) | Oxidoreductase |
Oxidative phosphorylation |
||
|
|||||||||
P07108 | ACBP_ |
Acyl-CoA-binding |
DBI | Acyl-CoA-binding |
Transfer/carrier |
Catalytic activity |
Lipid metabolic |
||
|
|||||||||
Q15075 | EEA1_ |
Early endosome |
EEA1 | Early endosome |
|||||
|
|||||||||
Q92538 | GBF1_ |
Golgi-specific |
GBF1 | Golgi-specific brefeldin |
Signaling molecule |
GTPase activity |
Phosphate-containing |
Organelle (GO:0043226); |
|
|
|||||||||
P78417 | GSTO1_ |
Glutathione |
GSTO1 | Glutathione |
Transferase (PC00220); |
Oxidoreductase |
Immune system |
Cytoskeleton (GO:0043226); |
|
|
|||||||||
P09211 | GSTP1_ |
Glutathione |
GSTP1 | Glutathione S-transferase P |
|||||
|
|||||||||
P51858 | HDGF_ |
Hepatoma-derived |
HDGF | Hepatoma-derived |
Transcription |
Transcription |
Immune system |
||
|
|||||||||
O60814 | H2B1K_ |
Histone H2B |
HIST1H2BK | Histone H2B type 1-K |
Histone (PC00171) | Nucleic acid |
Nitrogen compound |
Protein DNA complex |
|
|
|||||||||
P17096 | HMGA1_ |
High mobility |
HMGA1 | High mobility group |
DNA-binding |
Nucleic acid-binding |
Nitrogen compound |
Organelle (GO:0043226); |
|
|
|||||||||
P52926 | HMGA2_ |
High mobility |
HMGA2 | High mobility group |
DNA-binding |
Nucleic acid-binding |
Nitrogen compound |
Organelle (GO:0043226); |
|
|
|||||||||
P09429 | HMGB1_ |
High mobility |
HMGB1 | High mobility group |
HMG box transcription |
p53 pathway→high | |||
|
|||||||||
P05114 | HMGN1_ |
Nonhistone |
HMGN1 | Nonhistone |
Chromatin/ |
Nucleic acid |
DNA replication |
||
|
|||||||||
P05204 | HMGN2_ |
Nonhistone |
HMGN2 | Nonhistone |
Chromatin/chromatin- |
Nucleic acid binding (GO:0005488); |
DNA replication |
||
|
|||||||||
P61604 | CH10_ |
10 kDa heat |
HSPE1 | 10 kDa heat shock |
Chaperonin (PC00072) | Protein metabolic |
|||
|
|||||||||
P50213 | IDH3A_ |
Isocitrate |
IDH3A | Isocitrate dehydrogenase |
Dehydrogenase |
Oxidoreductase |
Generation of precursor |
Ascorbate | |
|
|||||||||
P01861 | IGHG4_ |
Ig gamma-4 |
IGHG4 | IG gamma-1 chain c |
|||||
|
|||||||||
Q9GZP8 | IMUP_ |
Immortalization |
IMUP | Immortalization |
|||||
|
|||||||||
P04732 | MT1E_ |
Metallothionein-1E | MT1E | Metallothionein-1E (PTHR23299:SF2) | |||||
|
|||||||||
P13640 | MT1G_ |
Metallothionein-1G, |
MT1G | Metallothionein-1G (PTHR23299:SF3) | |||||
|
|||||||||
P35749 | MYH11_ |
Myosin-11 | MYH11 | Myosin-11 |
G-protein modulator |
Motor activity |
Metabolic process |
Plasma membrane |
Inflammation mediated |
|
|||||||||
P20929 | NEBU_ |
Nebulin | NEB | Nebulin (PTHR11039:SF37) | |||||
|
|||||||||
Q13442 | HAP28_ |
28 kDa heat- |
PDAP1 | 28 kDa heat- and |
|||||
|
|||||||||
P13667 | PDIA4_ |
Protein disulfide- |
PDIA4 | Protein |
Isomerase activity |
Protein folding (GO:0008152); |
Organelle (GO:0043226); |
||
|
|||||||||
Q9UHV9 | PFD2_ |
Prefoldin subunit 2 | PFDN2 | PREFOLDIN |
Chaperone (PC00072) | protein folding |
|||
|
|||||||||
O15212 | PFD6_ |
Prefoldin subunit 6 | PFDN6 | Prefoldin subunit 6 (PTHR21431:SF0) | Protein binding |
Protein complex |
Protein complex |
||
|
|||||||||
Q01813 | PFKAP_ |
ATP-dependent |
PFKP | 6-Phosphofructokinase |
Carbohydrate kinase |
Carbohydrate kinase |
Glycolysis (GO:0008152); |
||
|
|||||||||
P13797 | PLST_ |
Plastin-3 | PLS3 | Plastin-3 (PTHR19961:SF32) | Nonmotor actin-binding protein (PC00085) | Structural constituent of |
Cellular process |
Actin cytoskeleton |
|
|
|||||||||
O75334 | LIPA2_ |
Liprin-alpha-2 | PPFIA2 | Liprin-alpha-2 (PTHR12587:SF6) | Cellular process |
||||
|
|||||||||
P30048 | PRDX3_ |
Thioredoxin- |
PRDX3 | Thioredoxin-dependent |
Peroxidase (PC00176) | Oxidoreductase activity |
Metabolic process |
||
|
|||||||||
P49721 | PSB2_ |
Proteasome |
PSMB2 | Proteasome subunit |
|||||
Q13200 | PSMD2_ |
26S proteasome |
PSMD2 | 26S proteasome |
Enzyme modulator (PC00095) | Catalytic activity |
Proteolysis (GO:0008152); |
Ubiquitin proteasome | |
|
|||||||||
O00233 | PSMD9_ |
26S proteasome |
PSMD9 | 26S proteasome |
Enzyme modulator (PC00095) | Protein complex |
Protein complex |
Ubiquitin proteasome | |
|
|||||||||
P06454 | PTMA_ |
Prothymosin |
PTMA | Prothymosin alpha (PTHR22745:SF0) | Nucleobase-containing |
||||
|
|||||||||
P20962 | PTMS_ |
Parathymosin | PTMS | Parathymosin |
Nucleobase-containing |
||||
|
|||||||||
P26373 | RL13_ |
60S ribosomal |
RPL13 | 60S ribosomal protein |
Ribosomal protein (PC00171) | Structural constituent |
Translation (GO:0008152) | ||
|
|||||||||
P62277 | RS13_ |
40S ribosomal |
RPS13 | 40S ribosomal protein S13 |
Ribosomal protein (PC00171) | Structural constituent of ribosome (GO:0005198); |
Protein metabolic |
||
|
|||||||||
P62857 | RS28_ |
40S ribosomal |
RPS28 | 40S ribosomal |
Ribosomal protein (PC00171) | Structural molecule |
Cellular process |
Ribosome (GO:0032991); |
|
|
|||||||||
Q8NC51 | PAIRB_ |
Plasminogen |
SERBP1 | Plasminogen |
RNA-binding protein (PC00171) | RNA binding |
Primary metabolic |
||
|
|||||||||
Q9H299 | SH3L3_ |
SH3 domain- |
SH3BGRL3 | SH3 domain-binding |
|||||
|
|||||||||
P11166 | GTR1_ |
Solute carrier |
SLC2A1 | Solute carrier family 2, |
Gonadotropin releasing | ||||
|
|||||||||
P22528 | SPR1B_ |
Cornifin-B | SPRR1B | Cornifin-B |
|||||
|
|||||||||
Q9Y6N5 | SQRD_ |
Sulfide:quinone |
SQRDL | Sulfide:quinone |
|||||
|
|||||||||
Q16629 | SRSF7_ |
Serine/arginine- |
SRSF7 | ||||||
|
|||||||||
P16949 | STMN1_ |
Stathmin | STMN1 | Stathmin |
Cytoskeletal protein |
Cellular process |
Intracellular (GO:0044464); |
Cytoskeletal regulation | |
|
|||||||||
P61956 | SUMO2_ |
Small |
SUMO2 | Small ubiquitin-related |
Cellular protein |
||||
|
|||||||||
P63313 | TYB10_ |
Thymosin beta-10 | TMSB10 | Thymosin beta-10 |
|||||
|
|||||||||
P62328 | TYB4_ |
Thymosin beta-4 | TMSB4X | Thymosin beta-4 |
|||||
|
|||||||||
P67936 | TPM4_ |
Tropomyosin |
TPM4 | Tropomyosin |
Actin-binding motor protein (PC00085) | Motor activity |
Metabolic process |
Actin cytoskeleton |
|
Q9C030 | TRIM6_ |
Tripartite |
TRIM6 | Tripartite |
|||||
|
|||||||||
Q13509 | TBB3_ |
Tubulin beta-3 |
TUBB3 | Tubulin beta-3 chain (PTHR11588:SF43) | Tubulin (PC00085) | Structural constituent |
Cell cycle (GO:0009987); |
Protein complex |
Huntington |
|
|||||||||
O75152 | ZC11A_ |
Zinc finger |
ZC3H11A | Zinc finger CCCH |
Nucleic acid binding (PC00171) | Nucleic |
Functional distribution of the 58 proteins identified as dysregulated with aging according to biological processes (a) and PANTHER protein class (b) categories. Assignments were made with PANTHER tool. The numbers in brackets correspond to the percentage of identified proteins classified in the category. If a protein is classified into 2 ontology terms that are not parent or child to each other, it counts in the 2 classes.
Pathway enrichment analysis was performed using PathVisio [
List of top enriched pathways provided after overrepresentation analysis with PathVisio. Positive (
Pathway | Positive ( |
Measured ( |
Total | % |
|
|
---|---|---|---|---|---|---|
Zinc homeostasis | 4 | 4 | 39 | 100.00% | 3.30 | 0.000 |
Copper homeostasis | 5 | 6 | 58 | 83.33% | 3.13 | 0.000 |
Arachidonate epoxygenase/epoxide hydrolase | 2 | 2 | 17 | 100.00% | 2.33 | 0.001 |
DNA replication | 2 | 2 | 50 | 100.00% | 2.33 | 0.009 |
Retinoblastoma (RB) in cancer | 2 | 2 | 98 | 100.00% | 2.33 | 0.012 |
Histone modifications | 11 | 25 | 69 | 44.00% | 1.98 | 0.027 |
Aryl hydrocarbon receptor | 1 | 1 | 51 | 100.00% | 1.65 | 0.048 |
Cardiac hypertrophic response | 1 | 1 | 60 | 100.00% | 1.65 | 0.063 |
Constitutive androstane receptor pathway | 1 | 1 | 34 | 100.00% | 1.65 | 0.029 |
Dual hijack model of Vif in HIV infection | 1 | 1 | 9 | 100.00% | 1.65 | 0.007 |
Endochondral ossification | 1 | 1 | 69 | 100.00% | 1.65 | 0.072 |
Endothelin pathways | 1 | 1 | 47 | 100.00% | 1.65 | 0.047 |
Gastric cancer network 1 | 1 | 1 | 32 | 100.00% | 1.65 | 0.027 |
Melatonin metabolism and effects | 1 | 1 | 55 | 100.00% | 1.65 | 0.057 |
NOTCH1 regulation of human endothelial cell calcification | 1 | 1 | 18 | 100.00% | 1.65 | 0.014 |
Notch signaling pathway | 1 | 1 | 62 | 100.00% | 1.65 | 0.058 |
RalA downstream regulated genes | 1 | 1 | 13 | 100.00% | 1.65 | 0.015 |
T-cell receptor and costimulatory signaling | 1 | 1 | 45 | 100.00% | 1.65 | 0.050 |
TarBasePathway | 1 | 1 | 19 | 100.00% | 1.65 | 0.019 |
Type II interferon signaling (IFNG) | 1 | 1 | 38 | 100.00% | 1.65 | 0.043 |
Metapathway biotransformation | 2 | 3 | 189 | 66.67% | 1.55 | 0.120 |
Cytoplasmic ribosomal proteins | 13 | 34 | 89 | 38.24% | 1.54 | 0.121 |
Circadian rythm-related genes | 5 | 11 | 210 | 45.45% | 1.40 | 0.146 |
Apoptosis modulation and signaling | 3 | 6 | 97 | 50.00% | 1.28 | 0.190 |
Alzheimers disease | 4 | 9 | 163 | 44.44% | 1.19 | 0.259 |
Electron transport chain | 4 | 9 | 118 | 44.44% | 1.19 | 0.252 |
Oxidative stress | 2 | 4 | 32 | 50.00% | 1.04 | 0.232 |
Preimplantation embryo | 2 | 4 | 60 | 50.00% | 1.04 | 0.299 |
Vitamin B12 metabolism | 2 | 4 | 118 | 50.00% | 1.04 | 0.337 |
TNF alpha signaling pathway | 3 | 7 | 97 | 42.86% | 0.95 | 0.340 |
In proteomic quantitation analysis, tubulin beta-3 chain was evidenced as a promising candidate protein biomarker of aging. Indeed, tubulin beta-3 chain was upregulated in elderly donors with an iTRAQ ratio of protein expression level of 2.66 (elderly versus young) and significant
(a) Western blot analysis of protein extract from young and elderly keratinocytes with tubulin beta-3 chain. (b) Box plots of relative protein expression of tubulin beta-3 chain in young and elderly donors (based on western blot data by considering the relative intensity of the specific antibodies on the membrane versus the amount of protein loaded on the gel for young and elderly donors,
Skin aging is a complex process with multifactorial origins that can decipher using new technological approach such as global quantitative proteomics. We carried out an iTRAQ-MALDI-TOF/TOF MS and MS/MS analysis to identify and quantify changes in human primary keratinocyte proteomes from young and elderly donors. 517 proteins were identified including proteins found mainly in keratinocytes such as cornifin-B and keratin-2E which are associated with keratinocyte activation, proliferation, and keratinization [
Comparison of our results with previous gene and protein expression studies of skin aging shows some similarities. We found that more proteins are downregulated (40) than upregulated (18) with aging which is consistent with the previous results from a gene expression study in women [
In this work, tubulin beta-3 chain expression is upregulated with aging in our proteomic experiment and also in western blot analyses on samples from several donors. Statistical analysis of these data has shown that tubulin beta-3 chain may discriminate age status. Tubulin beta-3 chain is a component of the microtubules that are complex polymers composed of tandem repeats of
In our study, we identified other dysregulated proteins such as peroxiredoxin 3, 6-phosphofructokinase, platelet type, and cornifin-B. Thioredoxin-dependent peroxide reductase, mitochondrial, also known as peroxiredoxin 3 (PrxIII), a mitochondrial member of the antioxidant family of thioredoxin (Trx) peroxidases, was found upregulated with aging in our study. Two other family members, peroxiredoxins 1 and 2, were also upregulated in a previous report [
Comparing our results with other studies aimed at identifying biomarkers of skin aging shows some differences that may be explained by different type/origin of skin samples, gender, difference in sample processing all along the workflow, and the variable correlation between mRNA and protein expression levels [
Defining the differential protein signature with aging even if these changes could be initiating adaptive or compensatory events is crucial to further increase our knowledge of skin aging. Our aim was to identify some biomarker candidates for skin aging from human primary keratinocyte culture. 58 proteins were dysregulated with aging, and from this, tubulin beta-3 chain was also observed dysregulated in western blot analysis of keratinocyte extracts isolated from multiple donors. Statistical analysis has confirmed especially that an increase in tubulin beta-3 chain is associated with aging. Further studies will be needed in order to evaluate the effect of this change of expression on the complex process of aging.
This study brings a new effort to reach a better understanding of the biology of skin aging and to identify new and specific targets that could help to diagnose, prevent, and treat skin aging. Indeed, emerging diagnostic tools now require a combination of multiple biomarkers to achieve a better accuracy, and we propose that tubulin beta-3 chain could be one of these.
The authors declare that there is no conflict of interest regarding the publication of this paper.
The authors would like to thank Valérie Cunin, Sylvie Michelland, and David Beal for providing their help and technical assistance and Friederike Ehrhart for the help with PathVisio.