BioMed Research International

BioMed Research International / 2015 / Article

Research Article | Open Access

Volume 2015 |Article ID 872983 | 17 pages | https://doi.org/10.1155/2015/872983

Peripheral Blood Mononuclear Cell Proteome Changes in Patients with Myelodysplastic Syndrome

Academic Editor: Shivani Soni
Received23 Sep 2014
Accepted31 Mar 2015
Published16 Apr 2015

Abstract

Our aim was to search for proteome changes in peripheral blood mononuclear cells (PBMCs) of MDS patients with refractory cytopenia with multilineage dysplasia. PBMCs were isolated from a total of 12 blood samples using a Histopaque-1077 solution. The proteins were fractioned, separated by 2D SDS-PAGE (pI 4–7), and double-stained. The proteomes were compared and statistically processed with Progenesis SameSpots; then proteins were identified by nano-LC-MS/MS. Protein functional association and expression profiles were analyzed using the EnrichNet application and Progenesis SameSpots hierarchical clustering software, respectively. By comparing the cytosolic, membrane, and nuclear fractions of the two groups, 178 significantly (, ANOVA) differing spots were found, corresponding to 139 unique proteins. Data mining of the Reactome and KEGG databases using EnrichNet highlighted the possible involvement of the identified protein alterations in apoptosis, proteasome protein degradation, heat shock protein action, and signal transduction. Western blot analysis revealed underexpression of vinculin and advanced fragmentation of fermitin-3 in MDS patients. To the best of our knowledge, this is the first time that proteome changes have been identified in the mononuclear cells of MDS patients. Vinculin and fermitin-3, the proteins involved in cell adhesion and integrin signaling, have been shown to be dysregulated in MDS.

1. Introduction

MDS encompasses a diverse range of oncohematological diseases affecting hematopoietic stem cells and their hematopoietic microenvironment [1]. MDS is characterized by dysplastic ineffective hematopoiesis with the apoptosis of hematopoietic cells in the bone marrow and by subsequent cytopenias in the blood. It occurs in particularly elder people with incidence of 20–50 patients in 100,000 inhabitants [2]. There are several groups of MDS patients according to the WHO classification based on bone marrow and peripheral blood findings, cytogenetics, and other factors [3]. Prognostically MDS subgroups can be also stratified into low-risk and high-risk subgroups; high-risk subgroups are characterized by poor survival outcome and higher rate of progression toward acute myeloid leukemia. Refractory cytopenia with multilineage dysplasia (RCMD) is a subgroup of myelodysplastic syndrome (MDS). According to the revised WHO (World Health Organization) classification of MDS, RCMD is defined by the presence of cytopenias in peripheral blood and dysplastic changes present in 10% or more of the cells in two or more myeloid lineages in the bone marrow (with approximately 15% ringed sideroblasts) [3].

In recent years, fundamental knowledge in MDS pathophysiology based on DNA alterations has been and is still being complemented by other “omics” disciplines, in particular by proteomics, with the proteomes of plasma (of different MDS subgroups such as refractory anemia and refractory anemia with ringed sideroblasts (RA and RARS) [4], RCMD [5], refractory anemia with excess of blasts subtype 1 (RAEB-1) [6], and refractory anemia with excess of blasts subtype 2 (RAEB-2) [7]), serum [8, 9], platelets [10], and neutrophils [11] of MDS patients having been investigated. Despite research efforts the pathogenesis and exact mechanisms of MDS development still remain unclear, as there are many factors involved. For example, factors involved in the development of MDS (cytogenetic abnormalities [12], gene mutations [1315], epigenetic alterations [16], etc.) can contribute to the dysregulation of various processes in the immune system [17], a portion of which are by mononuclear blood cells (B-lymphocytes, T-lymphocytes, dendritic cells, and monocytes). Mononuclear cells represent a heterogeneous population; nevertheless, due to the rapid and simple methods of their isolation, they are believed to be a promising and interesting material to search for biomarkers [1820]. As very little is known about the role of mononuclear cells and their protein alterations in MDS the goal of this work, our aim, has been to describe the changes in the proteome of mononuclear blood cells of MDS patients and to discuss the involvement of these changes in MDS pathophysiology.

2. Methods

In this pilot study, a total of 12 samples (RCMD , control ) have been investigated. The diagnosis of RCMD was established according to the WHO classification criteria [21]. The median age of RCMD patients was 67; the patient group included 4 females (67%). The median age of sex-matched healthy control donors was 28. Patient characteristics are summarized in Table 1. All of the individuals tested agreed to participate in the study on the basis of an informed consent. All samples were obtained and analyzed in accordance with the Ethical Committee regulations of the Institute of Hematology and Blood Transfusion.


Patient123456

SexFFFFMF
Age703369597365
Hb [g/L]119839192117104
RBC [1012/L]3.152.472.332.674.292.87
WBC [109/L]2.202.815.504.155.102.89
PLT [109/L]18429126221150284
NS1.11.913.082.412.30.72
Blasts in PB [%]000000
Blasts in BM [%]0.40.93.24.80.83.4
Karyotype46, XX, inv(9)46, XX46, XX, del(5)(q13.2q34)46, XX, del(5)(q31.q31)46, XY46, XX, 1qh+, 46, XX, 1qh+, del(5)(q31)
IPSS0.500000.5
IPSS-RLowLowLowLowVery lowlow
WPSSLowLowLowLowIntermediateintermediate
Transfusion [avg./month]0.504.503.423.251.472.19

Hb: hemoglobin, RBC: red blood cells, WBC: white blood cells, PLT: platelets, NS: neutrophil segments, PB: peripheral blood, BM: bone marrow, IPSS (-R): International Prognostic Scoring System (-Revised), and WPSS: WHO Classification-Based Prognostic Scoring System.

Blood samples were collected by venipuncture into EDTA-coated tubes. Peripheral blood mononuclear cells were isolated from 9 mL of whole blood using Histopaque-1077 (Sigma-Aldrich, Prague, Czech Republic) according to manufacturer instructions.

Protein fractionation was performed using a ProteoExtract Subcellular Proteome Extraction Kit (Merck Millipore, Darmstadt, Germany) according to manufacturer instructions to enrich the proteins according to their subcellular localization; four different subproteomes were obtained (cytosolic, membrane and membrane organelle, nuclear, and cytoskeletal). Enriched proteins were precipitated with the addition of four volumes of acetone, incubated for 60 min at −20°C, and then centrifuged at 15,000 ×g for 10 min. Protein concentration in all samples was determined using a Micro-BCA Protein Assay Kit (Thermo Fischer Scientific, Waltham, MA, USA). Protein sample concentrations of each subproteome were adjusted to the same level.

Isoelectric focusing was performed (IPG strips pI 4–7, 7.7 cm) followed by SDS-PAGE (8 × 10 cm, 10% resolving gel, 3.75% stacking gel, and 30 mA/gel), as described in detail in previous publications [46, 22]. Briefly, 40 μg of cytosolic, 50 μg of membrane and membrane organelle, and 40 μg of nuclear proteins were used for an IPG strip. The proteins of the cytoskeletal subproteome were not analyzed due to insufficient protein yield.

The gels were double-stained according to the improved fast-staining protocol [23], combining imidazole-zinc reverse and Coomassie dye-based staining. Imidazole-zinc reverse staining was used to detect as many spots as possible, followed by Coomassie dye-based staining to enable maximal spot detection and quantification. After each staining step, the gels were digitized and processed with Progenesis SameSpots software (Nonlinear Dynamics, Newcastle upon Tyne, UK) that computed the fold and values of all spots using one-way ANOVA analysis. Protein spots that differed significantly () were submitted for protein identification by tandem mass spectrometry (HCT ultra ion-trap mass spectrometer with nanoelectrospray ionization; Bruker Daltonics, Bremen, Germany) coupled to a nano-LC system (UltiMate 3000; Dionex, Sunnyvale, CA, USA); this procedure has been described in detail previously [46, 22]. Mascot (Matrix Science, London, UK) was used for database searching (Swiss-Prot). Two unique peptides (with higher Mascot scores than the minimum for identification, ) were necessary to successfully identify a protein. Exceptions were given to proteins with a molecular weight of 15 kDa or less and to proteins with more than 3 additional unique peptides identified by error tolerant search.

To analyze the functional associations between identified proteins and cellular pathways, the protein list was processed with the on-line EnrichNet application [24] using KEGG [25, 26] and Reactome [27, 28] databases. The significance of overlap between protein sets was evaluated using a combination of one-side Fisher’s exact test () and network similarity scores (XD-scores). The threshold values were estimated via EnrichNet with a regression fit equivalent to a Fisher value of 0.05 and an upper boundary of 95% confidence for linear fitting.

Dendrogram analysis was performed using Progenesis SameSpots software to reveal closely related proteins. The dendrogram is a visual representation of spot correlation data (with correlation analysis performed on log-normalized spot expression levels). Spots were clustered according to their closest correlation.

Western blot analysis was performed for vinculin and fermitin-3 proteins. Equal protein amounts of all (patient or donor) samples of appropriate protein fractions were pooled and 1 μg or 0.75 μg of total protein amounts were used for 1D western blot analyses for vinculin or fermitin-3, respectively. Briefly, following SDS-PAGE (10% resolving gel) proteins were transferred to a PVDF membrane (10 V constant voltage for 60 min) using an Owl HEP-1 Semi Dry Electroblotting System (Thermo Scientific, Waltham, MA, USA). Membranes were then incubated with a blocking buffer (3% BSA in PBS) at 30°C for 60 min and incubated with primary antibodies, anti-vinculin antibody (V9131; Sigma-Aldrich, Praque, Czech Republic) (1 : 200 dilution) or anti-kindlin-3 antibody (SAB4200013; Sigma-Aldrich, Prague, Czech Republic) (1 : 340 dilution). Then the membranes were incubated with secondary antibodies, rabbit anti-mouse IgG antibody conjugated with peroxidase (for vinculin detection, 1 : 80,000 dilution) (A9044; Sigma-Aldrich, Prague, Czech Republic) or goat anti-rabbit IgG antibody conjugated with peroxidase (for kindlin-3 detection, 1 : 120,000 dilution) (A0545; Sigma-Aldrich, Prague, Czech Republic). Visualization was performed using a chemiluminescent substrate (SuperSignal West Pico; Thermo Scientific, Waltham, MA, USA) and CL-XPosure Film (Thermo Scientific, Waltham, MA, USA).

3. Results and Discussion

Comparing the PBMC subproteomes of RCMD patients () with healthy volunteer control group subproteomes (), we found 178 spots that differed significantly in their normalized volumes (). Figures 13 indicate the positions of significantly differing spots of the cytosolic, membrane and membrane organelle, and nuclear subproteomes, respectively. The spots are marked considering the gel staining.

Proteins of the spots detected were submitted to protein identification by mass spectrometry, and 139 unique proteins were identified. The list of all spots, including ANOVA values, their multiplication (fold value), protein identification with the number of identified peptides (unique peptides above the identity threshold score), and protein accession number (Swiss-Prot), is summarized in Table 2.


SpotProteinUniprot ACPeptidesFold

1Vinculin P1820650.00172.7

2Tubulin alpha-1C chain; tubulin alpha-1B chain Q9BQE3; P6836340.004782.6
Annexin A6 P08133 2

3Alpha-enolase P06733 80.026−2.5

4Alpha-enolase P06733 60.0345−2.4

5Cytosolic nonspecific dipeptidase Q96KP4 140.00179−2.3
Tubulin beta chain P07437 5

6Annexin A1 P04083 100.00387−2.4

7Leukotriene A-4 hydrolase P09960 120.0000401−2.1

8T-complex protein 1 subunit theta P50990 60.00287−1.7

9Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 30.01332.1

10Plastin-2P13796170.000467−3.3

11Protein S100-A9 P0670210.0259−2.0

12Neutral alpha-glucosidase ABQ1469730.012−2.0

13Leukocyte elastase inhibitor P3074080.0114−2.0

14Alpha-enolase P0673340.000782−2.0
Adenylosuccinate synthetase isozyme 2P305205
Rab GDP dissociation inhibitor beta P503953
C-terminal-binding protein 1Q133632

15Annexin A7 P2007330.00019−2.0
AdenosylhomocysteinaseP235263
Proliferation-associated protein 2G4 Q9UQ801

16Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 30.02471.9
T-complex protein 1 subunit beta P783712

17Vinculin P1820650.0002671.9

18Triosephosphate isomerase P6017450.000113−1.7

19Eukaryotic initiation factor 4A-IP6084260.000589−1.9
Heterogeneous nuclear ribonucleoprotein FP525974
Heterogeneous nuclear ribonucleoprotein QO605064
T-complex protein 1 subunit thetaP509903

20Plastin-2P13796140.00156−1.9

21Alpha-enolase P06733 70.00129−1.9

22Plastin-2P13796160.0363−1.9

23Proteasome subunit alpha type-6P6090040.000319−1.8
UPF0568 protein C14orf166 Q9Y2244
Hypoxanthine-guanine phosphoribosyltransferaseP004922

24Alpha-enolase P06733 60.01721.8

25Ubiquitin carboxyl-terminal hydrolase 5P4597450.00228−1.7

26Proteasome activator complex subunit 1Q0632320.00171.7
Coagulation factor XIII A chain P004882

27Heat shock cognate 71 kDa protein P1114240.00034−1.8
Rho-related GTP-binding protein RhoC P081342

2840S ribosomal protein SA P0886570.00126−1.8

29Triosephosphate isomerase P6017470.0217−1.8

30T-complex protein 1 subunit zeta P4022740.00972−1.8

31WD repeat-containing protein 1O7508390.0183−1.8

32Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 20.001521.7
Heterogeneous nuclear ribonucleoprotein KP619782
ADP-sugar pyrophosphatase Q9UKK92

33Coronin-1A P3114620.00433−1.7

3426S proteasome non-ATPase regulatory subunit 14O0048720.000458−1.7

35m7GpppX diphosphatase Q96C8640.00316−1.5
Transaldolase P378373

36Proliferation-associated protein 2G4 Q9UQ8030.000667−1.7
Annexin A1 P04083 2

37Macrophage-capping protein P4012130.0279−1.7

38Talin-1Q9Y49050.004971.7

39Heat shock 70 kDa protein 4P3493250.0282−1.7

40Plastin-2P13796170.0131−1.6

41Glutathione S-transferase PP0921160.000628−1.8

42Protein S100-A7 P3115120.00218−1.6

43Talin-1Q9Y49040.008041.6

44Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform P3015370.00245−1.6

45Ribonuclease inhibitor P1348930.0247−1.6

46Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 30.001811.6

47Tubulin beta-1 chain Q9H4B720.01441.6

48Vinculin P1820630.01041.6

49Annexin A5 P08758120.00129−1.8

50Proteasome activator complex subunit 2Q9UL4630.000338−1.6

51Annexin A6 P08133200.000508−2.6
Heat shock 70 kDa protein 1A/1BP081073
Heat shock 70 kDa protein 1-like P349312
Heat shock protein HSP 90-alpha P079002

52Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 20.000127−1.6
Glutathione S-transferase PP092113

53Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 20.03521.5

54Triosephosphate isomerase P6017420.00181−1.5

55Ras GTPase-activating-like protein IQGAP1 P46940130.0219−1.7

56Actin, cytoplasmic 1; Actin, cytoplasmic 2P60709; P63261 20.0191.5

57Rab GDP dissociation inhibitor beta P5039530.0336−1.5
26S proteasome non-ATPase regulatory subunit 11 O002312

58Rab GDP dissociation inhibitor beta P50395130.0000838−1.5
Alpha-enolase P06733 6
Adenylosuccinate synthetase isozyme 2P305205
Beta-centractin P420253

59Tubulin alpha-1C chain Q9BQE350.03931.5
Tubulin alpha-1A chain Q71U365
Tubulin alpha-4A chain P683664

60Alpha-enolase P06733 70.04591.5
Phosphoglycerate kinase 1P005588

61Lymphocyte-specific protein 1P3324120.00972−1.5
Secernin-1Q127653

62Eukaryotic translation initiation factor 3 subunit HO1537230.00335−1.5
T-complex protein 1 subunit eta Q998324

63L-Lactate dehydrogenase B chain P0719580.00156−1.5
Pyruvate kinase isozymes M1/M2P146185

646-PhosphogluconolactonaseO9533650.0029−1.5
Endoplasmic reticulum resident protein 29 P300404

65Pyruvate kinase isozymes M1/M2 P1461880.000801−1.7
Sorting nexin-6Q9UNH72

66Proteasome subunit beta type-3P4972030.00454−1.5
Triosephosphate isomerase P601745

67Pyruvate kinase isozymes M1/M2 P14618160.0219−1.5

6814-3-3 protein zeta/delta P63104 60.0053−1.6

694-trimethylaminobutyraldehyde dehydrogenase P4918920.0183−1.4
Caspase-1P294662

70Proteasome subunit beta type-4P2807020.000337−1.4

71Chloride intracellular channel protein 1O0029960.00012−1.7
Tumor protein D5O43399 2

72Major vault protein Q1476480.0122−1.7
Ubiquitin-like modifier-activating enzyme 1P223148

73Transitional endoplasmic reticulum ATPase P55072130.0132−1.8
Ras GTPase-activating-like protein IQGAP1 P469403

74Serum albumin P02768100.00454−1.7

75Heat shock protein beta-1P0479220.001871.5

76Vinculin P18206 60.00886−1.5

77Gelsolin P0639670.0459−2.1

78Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 50.02211.9
Annexin A1 P04083 3

79Tryptophan-tRNA ligase, cytoplasmic P2338170.00185−1.6

80Vinculin P18206 70.0369−1.7

81Proteasome activator complex subunit 1Q0632370.0328−1.5

82Cofilin-1P2352830.000586−1.7

83Endoplasmin P1462550.00491−2.6

84Alpha-enolase P0673340.0001662.4
Monoglyceride lipase Q996851

85Alpha-actinin-1P1281420.007772.4

86Heterogeneous nuclear ribonucleoprotein H2 P5579540.000792−2.3
Heterogeneous nuclear ribonucleoprotein HP319433

87Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 30.007522.3
Alpha-actinin-1P128142

88Delta(3,5)-delta(2,4)-dienoyl-CoA isomerase, mitochondrialQ1301130.000218−2.2
Estradiol 17-beta-dehydrogenase 8Q925062

89Heterogeneous nuclear ribonucleoproteins A2/B1P2262630.000986−2.2

90Transitional endoplasmic reticulum ATPaseP5507250.00163−2.1

91Aconitate hydratase, mitochondrialQ9979870.000362−2.1
EndoplasminP146252

92EndoplasminP1462570.000319−2.0

93Heat shock 70 kDa protein 1A/1B P0810740.00155−2.0
Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrialP105156
Stress-70 protein, mitochondrialP386463
Annexin A6P081333

94Heat shock protein 75 kDa, mitochondrialQ1293130.00144−2.0

9578 kDa glucose-regulated protein P11021170.0018−2.5
Protein disulfide-isomerase A4P136674
Endoplasmin P146252

96Endoplasmic reticulum resident protein 29 P3004060.00318−1.9
ATP synthase subunit gamma, mitochondrial P365422

97EndoplasminP1462570.0109−1.9

98Tropomyosin alpha-4 chain P6793640.007552.1

9978 kDa glucose-regulated proteinP1102170.00377−1.8

100EndoplasminP1462550.0108−1.8

10140S ribosomal protein SA P0886530.00000707−1.8
VimentinP086704

102F-actin-capping protein subunit beta P4775620.000949−1.8

10378 kDa glucose-regulated proteinP11021150.00946−2.1

104Superoxide dismutase [Cu-Zn]P0044110.001391.7
ATP synthase subunit alpha, mitochondrial P257051

105Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 40.001381.7

106F-actin-capping protein subunit alpha-1P5290720.002471.7

107Purine nucleoside phosphorylaseP0049120.00353−1.7
Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1P048432

108Stress-70 protein, mitochondrialP38646100.00224−1.6
Heat shock cognate 71 kDa protein P111424

109Stress-70 protein, mitochondrialP3864620.01371.7

110Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 20.002011.7

111Delta(3,5)-delta(2,4)-dienoyl-CoA isomerase, mitochondrialQ1301130.00338−1.7

11260 kDa heat shock protein, mitochondrial P1080960.003851.7
Integrin beta-3P051064
ATP synthase subunit beta, mitochondrial P065762

113Aldehyde dehydrogenase, mitochondrial P0509190.000949−1.7

114Heat shock cognate 71 kDa protein P1114280.0122−1.7
Stress-70 protein, mitochondrialP386464
EndoplasminP146254

115T-complex protein 1 subunit beta P7837150.0215−1.7

116MoesinP2603840.000902−1.6
T-complex protein 1 subunit zeta P783712

117Macrophage-capping proteinP4012120.0114−1.6

118Stress-70 protein, mitochondrialP38646130.0106−1.7
Annexin A6P081333

11960 kDa heat shock protein, mitochondrial P10809110.000211−1.6
Heterogeneous nuclear ribonucleoprotein KP619782

120Elongation factor Tu, mitochondrial P4941140.00392−1.5
Isocitrate dehydrogenase [NADP] cytoplasmicO758742

121Pre-mRNA-processing factor 19 Q9UMS420.00523−1.6

122Prohibitin P35232 90.0121−1.6
Annexin A2 P073556

12378 kDa glucose-regulated proteinP1102160.0216−1.6
ATP synthase subunit beta, mitochondrial P065764
Integrin alpha-IIb P085142

124DnaJ homolog subfamily B member 11 Q9UBS420.00315−1.6
Moesin P26038 2

125Protein disulfide-isomeraseP0723770.0422−1.6

126GelsolinP0639630.02941.5

12714-3-3 protein zeta/delta P6310450.03371.5
14-3-3 protein eta Q049172

12860 kDa heat shock protein, mitochondrial P10809180.00252−1.5

129Endoplasmin P1462540.0251−1.6

130Synaptosomal-associated protein 23 O0016130.02541.5

131Stress-induced-phosphoprotein 1P3194810.0329−1.5

132CalreticulinP2779720.00435−1.5

133UPF0568 protein C14orf166 Q9Y22440.0208−1.5

13460 kDa heat shock protein, mitochondrial P1080970.0004121.5
ATP synthase subunit beta, mitochondrial P065762
Integrin beta-3P051062

135Alpha-enolase P0673340.0357−1.5

136Myosin-9P3557950.01061.5

137Myosin regulatory light polypeptide 9P2484430.03251.7
Protein disulfide-isomerase A6 Q150842

138Endoplasmic reticulum resident protein 29P3004060.0104−1.5

139Peroxiredoxin-4Q1316220.0362−1.5

140Tyrosine-protein phosphatase nonreceptor type 6 P2935030.0319−1.5
Protein disulfide-isomerase A3 P301012

141Protein disulfide-isomerase A3 P3010120.0125−1.5

142PDZ and LIM domain protein 1O0015120.003061.5

143ATP synthase subunit beta, mitochondrial P06576180.00266−1.4
Protein disulfide-isomerase A6 Q150846
78 kDa glucose-regulated proteinP110214
Protein disulfide-isomerase P072372
VimentinP086702

144Plastin-2P13796150.000372−1.6

145Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 30.009051.5

146Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 40.01561.8

14760 kDa heat shock protein, mitochondrial P1080990.0181.5

148Filamin-AP2133340.0001232.6

149Filamin-AP2133330.001162.6

150Alpha-actinin-1P1281470.001042.4

151Ficolin-1O0060220.000967−2.4

152EzrinP1531130.0136−2.4

153Coronin-1A P3114610.00915−2.2

154Alpha-actinin-1P1281440.001092.2

155Fermitin family homolog 3Q86UX720.0002612.1

156Filamin-AP2133320.0005682.1

157Alpha-actinin-1P1281450.00172.0

158Filamin-AP2133320.01231.9

159Ficolin-1 O0060220.0221−1.8

160Alpha-actinin-1P1281450.0005161.6
Filamin-AP213332

16114-3-3 protein zeta/delta P6310430.0221.8

162Prohibitin P3523230.0414−1.8

163Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 20.01331.7

164Nucleoprotein TPR P1227010.009521.7

165Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 50.0371.7
Filamin-AP213333
Beta-parvin Q9HBI12

166Alpha-actinin-1P1281430.009691.7

167Filamin-AP2133320.01091.7

168Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 20.005292.8

169EzrinP1531120.0174−1.7

170Beta-actin-like protein 2 Q562R1 20.02261.6

171Filamin-AP2133320.009751.6

172Filamin-AP2133330.03191.6

173Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 20.02021.6

174Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 30.02071.6

175Myosin-9P3557920.02251.6

176Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 40.0009912.2

177Filamin-AP2133330.01311.5

178Filamin-AP2133350.0312.1
Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261 3

We compared these summarized results with our previous research that investigated the plasma proteomes of patients with different MDS subgroups [47]. It is known that PBMCs can secrete proteins into the plasma [29, 30]; therefore, we searched for changes in plasma proteomes that could be related to PBMCs. Nevertheless, none of the proteins identified in this study corresponded to any proteins identified in our previous proteomic studies of the plasma. This observation strongly indicates that the alterations observed in plasma proteins (possibly secreted by PBMCs) are caused by posttranslational modifications of such proteins instead of the changes in their level. For example, we have shown in our previous plasma proteome MDS studies [47] that inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) was extensively fragmented and the changes observed (for ITIH4) were related to these fragments; similar results were observed for complement proteins. However, it should be taken into account that PBMCs are not the only source of these plasma proteins.

In order to estimate the possible involvement of the identified proteins in the PBMC cellular and metabolic pathways and thus reveal the processes influenced by RCMD pathogenesis, we processed the protein dataset in EnrichNet. Enrichment analysis using KEGG and Reactome databases revealed implications of the identified proteins in several cellular pathways (Tables 3 and 4). XD-scores were considered to be more than 0.78 and 1.0 threshold values, as estimated by the application for the KEGG and Reactome databases, respectively.


Annotation (pathway/process)XD-scoreFisher-test, value

Pathogenic Escherichia coli infection1.3719
Proteasome1.2793
Glycolysis/gluconeogenesis0.9755
Pyruvate metabolism0.9392
Antigen processing and presentation0.9051


Annotation (pathway/process)XD-scoreFisher-test, value

Formation of tubulin folding intermediates by CCT Tric3.7875
Further platelet releasate3.1660
Prefoldin mediated transfer of substrate to CCT Tric2.8529
Activation of chaperones by IRE1 alpha2.4375
Postchaperonin tubulin folding pathway2.3845
Chaperonin mediated protein folding1.6524
Formation of ATP by chemiosmotic coupling1.5375
Smooth muscle contraction1.4518
Cell-extracellular matrix interactions1.4250
Glycolysis1.3738
p53 independent DNA damage response1.2026
Stabilization of p531.1070
Regulation of ornithine decarboxylase1.0779
VIF mediated degradation of APOBEC3G1.0779
Platelet degranulation1.0545
SCF beta TRCP mediated degradation of EMI11.0500
Association of Tric CCT with target proteins during biosynthesis1.0232

Dendrogram analysis was performed using Progenesis SameSpots, which grouped spots by their expression profiles using an automatic correlation analysis and hierarchical clustering. We chose the top ten spot expression profile groups (with distance parameters less than 0.5) with almost identical expression profiles. Thus, each group contained spots with similar expression profiles, suggesting that these spots may be coregulated, colocalized, or by another way coaffected. The list of the expression profile groups (denoted as A, B, etc.), including the spot number, protein identification, and its accession number, is presented in Tables 57. Proteins in the F1 groups (cytosolic subproteome) were associated with the cytoskeleton, microtubule metabolism, cellular homeostasis maintained by heat shock proteins (HSPs), and proteasome. Proteins in the F2 groups (membrane and membrane organelle subproteome) were associated with the mitochondria and apoptosis. The protein identified in most cases in the F3 groups (nuclear subproteome) was Filamin-A, which is released from the apoptotic nucleus [31]. Other proteins in these groups were mainly actin and actin-binding proteins. Thus, the actual associations corresponded to the subproteomes as anticipated. Due to thematic similarity of the protein groups obtained via EnrichNet and dendrogram analysis, we discuss the results in parallel. Further in relation to MDS or other hematological malignancies, we summarize the most interesting protein groups that could be affected in connection with pathophysiological processes.


Group F1A
SpotProteinUniprot AC

1Vinculin P18206
17Vinculin P18206
43Talin-1Q9Y490
48Vinculin P18206
59Tubulin alpha-1C chain Q9BQE3
Tubulin alpha-1A chain Q71U36
Tubulin alpha-4A chain P68366
26Proteasome activator complex subunit 1Q06323
Coagulation factor XIII A chain P00488
47Tubulin beta-1 chain Q9H4B7

Group F1B
SpotProteinUniprot AC

56Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261
46Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261
2Tubulin alpha-1C chain; tubulin alpha-1B chain Q9BQE3; P68363
Annexin A6 P08133
9Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261
16Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261
T-complex protein 1 subunit beta P78371
38Talin-1Q9Y490

Group F1C
SpotProteinUniprot AC

51Annexin A6 P08133
Heat shock 70 kDa protein 1A/1BP08107
Heat shock 70 kDa protein 1-like P34931
Heat shock protein HSP 90-alpha P07900
7Leukotriene A-4 hydrolase P09960
15Annexin A7 P20073
AdenosylhomocysteinaseP23526
Proliferation-associated protein 2G4 Q9UQ80
58Rab GDP dissociation inhibitor beta P50395
Alpha-enolase P06733
Adenylosuccinate synthetase isozyme 2P30520
Beta-centractin P42025
36Proliferation-associated protein 2G4 Q9UQ80
Annexin A1 P04083
14Alpha-enolase P06733
Adenylosuccinate synthetase isozyme 2P30520
Rab GDP dissociation inhibitor beta P50395
C-terminal-binding protein 1Q13363
21Alpha-enolase P06733
3Alpha-enolase P06733
35m7GpppX diphosphatase Q96C86
Transaldolase P37837
3426S proteasome non-ATPase regulatory subunit 14O00487
23Proteasome subunit alpha type-6P60900
UPF0568 protein C14orf166 Q9Y224
Hypoxanthine-guanine phosphoribosyltransferaseP00492
18Triosephosphate isomerase P60174
66Proteasome subunit beta type-3P49720
Triosephosphate isomerase P60174
54Triosephosphate isomerase P60174
70Proteasome subunit beta type-4P28070
27Heat shock cognate 71 kDa protein P11142
Rho-related GTP-binding protein RhoC P08134
50Proteasome activator complex subunit 2Q9UL46


Group F2A
SpotProteinUniprot AC

85Alpha-actinin-1P12814
11260 kDa heat shock protein, mitochondrial P10809
Integrin beta-3P05106
ATP synthase subunit beta, mitochondrial P06576
130Synaptosomal-associated protein 23 O00161
107Purine nucleoside phosphorylaseP00491
Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1P04843
142PDZ and LIM domain protein 1O00151
136Myosin-9P35579

Group F2B
SpotProteinUniprot AC

91Aconitate hydratase, mitochondrialQ99798
EndoplasminP14625
94Heat shock protein 75 kDa, mitochondrialQ12931
116MoesinP26038
T-complex protein 1 subunit zeta P78371
132CalreticulinP27797
86Heterogeneous nuclear ribonucleoprotein H2 P55795
Heterogeneous nuclear ribonucleoprotein HP31943
83Endoplasmin P14625
10140S ribosomal protein SA P08865
VimentinP08670

Group F2C
SpotProteinUniprot AC

122Prohibitin P35232
Annexin A2 P07355
96Endoplasmic reticulum resident protein 29 P30040
ATP synthase subunit gamma, mitochondrial P36542
111Delta(3,5)-delta(2,4)-dienoyl-CoA isomerase, mitochondrialQ13011
89Heterogeneous nuclear ribonucleoproteins A2/B1P22626
124DnaJ homolog subfamily B member 11 Q9UBS4
Moesin P26038
113Aldehyde dehydrogenase, mitochondrial P05091
11960 kDa heat shock protein, mitochondrial P10809
Heterogeneous nuclear ribonucleoprotein KP61978

Group F2D
SpotProteinUniprot AC

9978 kDa glucose-regulated proteinP11021
10378 kDa glucose-regulated proteinP11021
9578 kDa glucose-regulated protein P11021
Protein disulfide-isomerase A4P13667
Endoplasmin P14625
143ATP synthase subunit beta, mitochondrial P06576
Protein disulfide-isomerase A6 Q15084
78 kDa glucose-regulated proteinP11021
Protein disulfide-isomerase P07237
VimentinP08670
144Plastin-2P13796
12860 kDa heat shock protein, mitochondrial P10809
114Heat shock cognate 71 kDa protein P11142
Stress-70 protein, mitochondrialP38646
EndoplasminP14625
118Stress-70 protein, mitochondrialP38646
Annexin A6P08133
140Tyrosine-protein phosphatase nonreceptor type 6 P29350
Protein disulfide-isomerase A3 P30101
88Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrialQ13011
Estradiol 17-beta-dehydrogenase 8Q92506

Group F2E
SpotProteinUniprot AC

97EndoplasminP14625
92EndoplasminP14625
90Transitional endoplasmic reticulum ATPaseP55072
115T-complex protein 1 subunit beta P78371
117Macrophage-capping proteinP40121
102F-actin-capping protein subunit beta P47756


Group F3A
SpotProteinUniprot AC

167Filamin-AP21333
156Filamin-AP21333
176Actin, cytoplasmic 1; actin, cytoplasmic 2P60709; P63261
172Filamin-AP21333
155Fermitin family homolog 3Q86UX7

Group F3B
SpotProteinUniprot AC

16114-3-3 protein zeta/delta P63104
148Filamin-AP21333
154Alpha-actinin-1P12814
157Alpha-actinin-1P12814
150Alpha-actinin-1P12814

PBMCs are metabolically active cells (carbohydrate metabolism, cellular respiration) and as cells of the immune system are involved in antigen processing and presentation. Our KEGG results analysis (Table 3) highlighted the relationships of the identified proteins to infectious agents (E. coli). This observation is not surprising, as it is known that the immune system in MDS is dysregulated and MDS patients tend to be especially vulnerable to infections [17, 32]. Therefore, this observation is most likely related to the manifestation of secondary complications, rather than MDS itself.

T-complex protein 1 (TCP-1) subunits (four of eight identified, Table 2) with a degree of functional autonomy [33] are a part of the TRiC cytosolic chaperone (TCP-1 ring complex), which acts in tubulin biosynthesis (Table 4). This chaperone was originally thought to fold only cytoskeletal proteins but now is known to have a more general role in protein folding in eukaryotic cytosol [34]. TRiC also assists in the formation of BBSome, a part of the primary cilium, nonmotile microtubule-based sensory organelle transporting signals within the cell [35]. Primary cilium has not been under closer analysis until in the last decade, and many questions surrounding it are still unanswered. For example, it is not entirely known, whether the primary cilium is present in PBMCs [3639]. There is evidence that components which contribute to the assembly of the primary cilium are expressed by PBMCs [36]. Therefore, there is a possibility that TCP subunits are part of the machinery of primary cilium formation or function, as a chaperone not only of cytoskeletal proteins (see Table 5, Groups F1A, F1B). Changes in TCP subunits could cause changes or even errors in BBSome formation [40], thus an effect on signaling within the cell. In last decade, the function of primary cilium in several cancers has been described, but its role in hematological malignancies has not yet been unraveled. There is also the possibility that a change in TCP subunits could affect the proper folding of proteins involved in hematopoiesis and its regulation and therefore may contribute to MDS pathogenesis.

Apoptosis, an important phenomenon in MDS, in a highly regulated manner removes the excess or potentially dangerous cells from the organism. The apoptotic process relies heavily on the cleavage of proteins/proteolysis. Any proteolytic pathway involved in cell death regulation must be precise; therefore, a highly regulated proteasome pathway is a good candidate for the regulation of protein composition during apoptosis [41]. Evidence of cross talk between the apoptotic pathways, HSPs, and proteasome system exists [42]; the relation of these processes is also suggested in our dendrogram analysis results (see Table 5, Group F1C). It is difficult to define a clear role or the involvement of the proteasome system in apoptosis, because in some systems proteasome activity induces apoptosis while in others it does not [42]. It is possible that increased proteasome activity causes the suppression of apoptosis, and this could be one of the reasons for the transformation from MDS to AML [43]. In relation to programmed cell death, researchers have identified changes in the regulatory subunits of the 19S cap complex [44, 45]. We observed changes in several proteasomal proteins and in the non-ATPase regulatory subunits of the 26S complex (Table 5, Group F1C), a part of which is the 19S cap complex [46]. We also observed changes in several HSPs, including HSP90α (identified in spot 51), whose overexpression has already been linked to apoptosis and MDS [47]. HSPs assist in proper protein folding, as molecular chaperones. They are fundamental for cell life and death decisions, and their abnormal expression is linked to oncogenesis [48]. When a protein is misfolded, HSPs are induced and associate with the misfolded protein, trying to refold it. When this process fails, the protein is ubiquitinated and determined to be processed by the proteasome. In case there are not enough HSPs or the proteasome function is impaired, proteins tend to aggregate with HSPs, ubiquitin, and proteasome to an insoluble compartment and trigger apoptosis [42, 49, 50]. HSPs can act in apoptosis at three levels: (i) in upstream mitochondria by regulating signaling molecules [51] (see Table 5, Group F1C); (ii) at the mitochondrial level by controlling mitochondrial membrane permeabilization and thus the release of cytochrome c [52] (see Table 6); and (iii) downstream of the mitochondria by affecting apoptosome formation [53] (see Table 5, Group F1C). Their role in apoptosis is also controversial; HSP function in apoptosis may be impacted by posttranslational modifications and the interaction with cochaperones (e.g., the DnaJ-family proteins identified in spot 124) [54]. The overexpression of HSPs has been shown to block apoptosis; and on the other hand, the depletion of HSPs increases sensitivity to apoptotic stimuli [55]. We observed both a decrease and an increase in normalized volumes in the spots containing HSPs (Table 2). However, from 2D SDS-PAGE data it is not possible to claim whether a change is caused by protein expression alteration or by protein posttranslational modification. Therefore, there is the possibility that the HSPs identified are posttranslationally modified, differently expressed, or a combination of both. Changes in proteasomal proteins and HSPs could be involved in cells’ decisions regarding the triggering of apoptosis. Because of the inconsistency in the roles of both the proteasome and HSPs in apoptosis, we can only speculate whether the changes are the cause of the apoptosis in MDS.

In order to provide further insight into the possible PMBCs alterations in MDS patients additional to those suggested by the 2D electrophoresis data, western blot analysis was performed for the two of identified proteins: fermitin-3 and vinculin. Both the proteins were uniquely identified in the corresponding spots (without coidentified proteins) and they are both involved in particular in cell adhesion and integrin signaling [56, 57]. While vinculin was identified in five different spots with both increasing and decreasing spot normalized volumes (which strongly indicates the presence of posttranslational modifications and the alterations of individual proteoforms), fermitin-3 was observed in one spot only. The results of western blot analysis are shown in Figure 4.

It is apparent that vinculin is underexpressed in PBMCs of MDS patients when compared to the healthy donor group. Therefore, while the prevalent vinculin form is decreased in MDS there is also a minority of posttranslationally modified forms altered as estimated from 2D electrophoresis and LC-MS/MS data. Vinculin is an actin-binding protein that is involved especially in cell adhesion dynamics and cell migration [56, 58]. Downregulation of vinculin expression could be possibly related to the immune system dysregulation in MDS as vinculin underexpression was described in the study by Kim et al. [56] investigating proteome changes in PBMCs of patients with atopic dermatitis, a chronic inflammatory skin disease. The presence of vinculin posttranslational modifications (as indicated in this work) was also observed in the proteomic study of PBMCs collected from amyotrophic lateral sclerosis (ALS) patients; significantly higher level of vinculin nitration was observed for sporadic ALS patients compared to healthy donors [59].

Western blot analysis of fermitin-3 showed the presence of several protein forms; in addition to the uncleaved protein, various fragments of fermitin-3 were observed. The uncleaved fermitin-3 band was observed with a lower intensity in MDS patients compared to the healthy controls; this observation may suggest fermitin-3 underexpression in MDS patients. However, the total intensity of the unaltered fermitin-3 together with its fragments estimated with ImageJ software [60] showed that there is no difference between MDS patients and the healthy controls. Therefore, while fermitin-3 expression did not seem to differ between the studied groups, it was shown that there was advanced fermitin-3 fragmentation in MDS patients. Fermitin-3 is a member of the family of focal adhesion proteins exclusively expressed in hematopoietic cells [61]. Its role in adhesion is essential for the function of blood cells including leukocytes [57, 62, 63]. Thus, higher rate of fermitin-3 fragmentation observed in this work could substantially influence PMBCs function in MDS. Moreover, fermitin-3 and vinculin are known to be colocalized to hematopoietic cell adhesion structure called podosome [64]. Therefore, since vinculin and fermitin-3 are both involved in cell adhesion and integrin signaling (and thus could play an important role in clinical applications) their changes observed in this work could initiate future research efforts.

4. Conclusion

In conclusion, we have compared the peripheral blood mononuclear cell proteome of myelodysplastic syndrome patients with refractory cytopenia with multilineage dysplasia against the proteome of healthy donors using two-dimensional electrophoresis combined with mass spectrometry. Through data mining of the Reactome and KEGG databases using EnrichNet, we highlighted the possible involvement of the identified protein alterations in apoptosis, protein degradation by proteasome, heat shock protein action, and signal transduction. Western blot analysis showed substantial changes in vinculin and fermitin-3, proteins involved in cell adhesion and integrin signaling. Vinculin was found to be underexpressed in MDS patients; advanced fragmentation of fermitin-3 was shown to take place in PBMCs of MDS patients.

To the best of our knowledge, our pilot study represents the first report on the proteome changes of peripheral blood mononuclear cells in myelodysplastic syndrome.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This study was supported by the Czech Science Foundation P205/12/G118 and by the state project (Ministry of Health, Czech Republic) for the conceptual development of the research organization (Institute of Hematology and Blood Transfusion).

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Copyright © 2015 Klara Pecankova et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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