Proteome analysis of human amniotic mesenchymal stem cells (h A-MSCs) reveals impaired
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1 Supplementary Information for Proteome analysis of human amniotic mesenchymal stem cells (h A-MSCs) reveals impaired antioxidant ability, cytoskeleton and metabolic functionality in maternal obesity Valentina Capobianco 1*, Marianna Caterino 1,2*, Laura Iaffaldano 1, Carmela Nardelli 1,2, Angelo Sirico 3, Luigi Del Vecchio 1,2, Pasquale Martinelli 3, Lucio Pastore 1,2, Pietro Pucci 1,4, Lucia Sacchetti 1 1 CEINGE-Biotecnologie Avanzate S.C.a R.L., Via Gaetano Salvatore 486, Naples, Italy 2 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Via S. Pansini 5, Naples, Italy 3 Dipartimento di Neuroscienze e Scienze Riproduttive ed Odontostomatologiche, Via S. Pansini 5, Naples, Italy. 4 Dipartimento di Scienze chimiche, Via Cintia, Complesso Monte Sant Angelo 21, Naples, Italy *These authors contributed equally to the paper Corresponding author: Lucia Sacchetti, sacchett@unina.it This file include: 1. Supplementary Table S1 2. Supplementary Table S2 3. Supplementary Figure S1 4. Supplementary Figure S2
2 (a) Pregnant women features Obese Non-obese Subjects (n) 12 6 Age (years) 34.0 ± ± 6.8 Weight (kg) a ± ± 5.1 Height (m) 1.60 ± ± 2.2 BMI pre-pregnancy (kg/m 2 ) a 42.7 ± ± 3.3 Weight gain in pregnancy 9.5 ± ± 4.2 Systolic blood pressure (mmhg) ± ± 0.5 Diastolic blood pressure (mmhg) 79.0 ± ± 5.7 Cardiac frequency 80.5 ± ± 3.5 Gestational age 39.0 ± ± 0.5 Glucose (mmol/l) 4.3 ± ± 0.4 Cholesterol (mmol/l) 7.1 ± ± 0.5 Triglycerides (mmol/l) 2.8 ± ± 1.3 AST (U/L) 15.1 ± ± 8.1 ALT (U/L) 13.3 ± ± 13.8 ALP (U/L) ± ± 39.0 GGT (U/L) 10.7 ± ± 5.7 PCR (mg/l) 6.4 ± ± 2.6 Albumin (g/dl ) 3.2 ± ± 0.3 Total proteins (g/dl ) 6.1 ± ± 0.4 Leptin (L) (ng/ml) b 35.0 ± ± 7.9 Adiponectin (A) (µg/ml) 7.2 ± ± 2.8 L/A c 7.4 ± ± 0.7 (b) Newborn features Obese newborns Non-obese newborns Birth weight (g) ± ± Length (cm) 50.3 ± ± 2.2 Head circumference (cm) 34.8 ± ± 0.5 Apgar ± ± 0.8 Apgar ± ± 0.5 Supplementary Table S1. Clinical and biochemical characteristics of obese and non-obese pregnant women (a) and of their newborns (b) Data are expressed as mean ± Standard Deviation. Statistically significant difference at the Student s t test. a P < ; b P = 0.002; c P = 0.01
3 (a) Pathways predicted by DAVID Annotation Cluster 1 Enrichment Score: 4.62 Proteins P_Value GOTERM_BP_FAT response to unfolded protein HSPH1, VCP, HSPB1, CLIC1, HSPD1, SERPINH1, HSPA8 3.2E-7 SP_PIR_KEYWORDS stress response HSPH1, HSPB1, HSPD1, SERPINH1, HSPA8, HSPA9 1.8E-6 GOTERM_BP_FAT response to protein stimulus HSPH1, VCP, HSPB1, CLIC1, HSPD1, SERPINH1, HSPA8 3.6E-6 SP_PIR_KEYWORDS heat shock HSPB1, HSPD1, SERPINH1 5.5E-5 SP_PIR_KEYWORDS stress-induced protein HSPB1, HSPD1, SERPINH1 2.6E-4 GOTERM_BP_FAT response to organic substance HSPH1, VCP, ALDOC, HSPB1, CLIC1, HSPD1, ANXA5, SERPINH1, HSPA8 6.6E-3 Annotation Cluster 2 Enrichment Score: 4.46 Proteins P_Value GOTERM_CC_FAT pigment granule ANXA6, HSP90B1, SND1, PDCD6IP, PDIA4, HSPA5, HSPA8, ANXA2 5.6E-8 GOTERM_CC_FAT melanosome ANXA6, HSP90B1, SND1, PDCD6IP, PDIA4, HSPA5, HSPA8, ANXA2 5.6E-8 GOTERM_CC_FAT cytoplasmic membrane-bounded vesicle ANXA6, SEC23A, HSP90B1, SND1, ACTN1, PDCD6IP, PDIA4, HSPA5, HSPD1, HSPA8, ANXA2 5.2E-5 GOTERM_CC_FAT membrane-bounded vesicle ANXA6, SEC23A, HSP90B1, SND1, ACTN1, PDCD6IP, PDIA4, HSPA5, HSPD1, HSPA8, ANXA2 6.8E-5 GOTERM_CC_FAT cytoplasmic vesicle ANXA6, SEC23A, HSP90B1, SND1, ACTN1, PDCD6IP, PDIA4, HSPA5, HSPD1, HSPA8, ANXA2 1.9E-4 GOTERM_CC_FAT vesicle ANXA6, SEC23A, HSP90B1, SND1, ACTN1, PDCD6IP, PDIA4, HSPA5, HSPD1, HSPA8, ANXA2 2.7E-4 Annotation Cluster 3 Enrichment Score: 4.33 Proteins P_Value SP_PIR_KEYWORDS molecular chaperone LONP1, CCT6A, HSPD1, SERPINH1, HSPA8 5.6E-8 SP_PIR_KEYWORDS stress response HSPH1, HSPB1, HSPD1, SERPINH1, HSPA8, HSPA9 1.8E-6 INTERPRO Heat shock protein Hsp70 HSPH1, HSPA5, HSPA8, HSPA9 9.1E-6 INTERPRO Heat shock protein 70 HSPH1, HSPA5, HSPA8, HSPA9 9.1E-6 unfolded protein binding HSP90B1, CCT6A, HSPA5, HSPD1, SERPINH1, HSPA8, HSPA9 1.1E-5 INTERPRO Heat shock protein 70, conserved site HSPH1, HSPA5, HSPA8, HSPA9 1.9E-5 SP_PIR_KEYWORDS Chaperone HSP90B1, CCT6A, HSPD1, SERPINH1, HSPA8, HSPA9 1.2E-4 PIR_SUPERFAMILY PIRSF002581:chaperone HSP70 HSPA5, HSPA8, HSPA9 9.9E-4 GOTERM_BP_FAT protein folding HSP90B1, CCT6A, HSPD1, HSPA8, HSPA9 5.0E-3 GOTERM_CC_FAT cell surface HSPB1, HSPA5, HSPD1, HSPA8, HSPA9 5.0E-2 Annotation Cluster 4 Enrichment Score: 3.76 Proteins P_Value SP_PIR_KEYWORDS molecular chaperone LONP1, CCT6A, HSPD1, SERPINH1, HSPA8 5.6E-8 SP_PIR_KEYWORDS ATP LONP1, PFKP, HK1, HSPD1, HSPA8 5.9E-3
4 ATPase activity LONP1, VCP, RUVBL1, HSPD1, MYH9, HSPA8 1.5E-2 Annotation Cluster 5 Enrichment Score: 3.68 Proteins P_Value SP_PIR_KEYWORDS SP_PIR_KEYWORDS atp-binding nucleotide-binding ATP binding adenyl ribonucleotide binding adenyl nucleotide binding purine nucleoside binding nucleoside binding purine ribonucleotide binding ribonucleotide binding nucleotide binding purine nucleotide binding PGD, PFKP, HK1, ACLY, CCT6A, MYH9, HNRNPA1, ACTG1, HSPH1, LONP1, HSP90B1, VCP, UBA1, 2.8E-7 7.7E-6 1.3E-4 1.6E-4 2.9E-4 3.5E-4 3.8E-4 1.7E-3 1.7E-3 2.4E-3 2.8E-3 Annotation Cluster 6 Enrichment Score: 3.66 Proteins P_Value SP_PIR_KEYWORDS GOTERM_CC_FAT GOTERM_CC_FAT GOTERM_CC_FAT cytoskeleton intracellular non-membrane-bounded organelle non-membrane-bounded organelle cytoskeleton ACTG1, LIMA1, LASP1, CALD1, HSPB1, ACTN1, RANGAP1, RUVBL1, PDCD6IP, FLNB, FLNA RPSA, LIMA1, RRBP1, CALD1, ALDOC, VIM, UCHL1, LMNA, ANXA1, ACTN1, RANGAP1, DPYSL3, MYH9, FLNB, HNRNPA1, FLNA, ACTG1, LONP1, VCP, LASP1, HSPB1, PDCD6IP, RUVBL1, HSPA9 RPSA, LIMA1, RRBP1, CALD1, ALDOC, VIM, UCHL1, LMNA, ANXA1, ACTN1, RANGAP1, DPYSL3, MYH9, FLNB, HNRNPA1, FLNA, ACTG1, LONP1, VCP, LASP1, HSPB1, PDCD6IP, RUVBL1, HSPA9 LIMA1, ALDOC, CALD1, VIM, LMNA, ANXA1, ACTN1, DPYSL3, MYH9, FLNB, FLNA, ACTG1, LASP1, HSPB1, PDCD6IP, RUVBL1 2.1E-5 5.5E-5 5.5E-5 2.2E-4
5 GOTERM_CC_FAT cytoskeletal part LIMA1, LASP1, CALD1, VIM, LMNA, HSPB1, RUVBL1, PDCD6IP, MYH9 3.4E-2 Annotation Cluster 7 Enrichment Score: 3.64 Proteins P_Value SP_PIR_KEYWORDS acetylated amino end ANXA6, ACTG1, APEH, PGD, ANXA1, ANXA5, HNRNPA1, ANXA2 2.3E-8 SP_PIR_KEYWORDS duplication ANXA6, ANXA1, PFKP, HK1, ACTN1, PDIA4, ANXA5, FLNA, ANXA2 4.5E-7 SMART SM00335:ANX ANXA6, ANXA1, ANXA5, ANXA2 4.4E-6 SP_PIR_KEYWORDS endonexin fold ANXA6, ANXA1, ANXA5, ANXA2 4.4E-6 SP_PIR_KEYWORDS annexin ANXA6, ANXA1, ANXA5, ANXA2 9.7E-6 UP_SEQ_FEATURE repeat:annexin 1 ANXA6, ANXA1, ANXA5, ANXA2 9.9E-6 UP_SEQ_FEATURE repeat:annexin 2 ANXA6, ANXA1, ANXA5, ANXA2 9.9E-6 UP_SEQ_FEATURE repeat:annexin 3 ANXA6, ANXA1, ANXA5, ANXA2 9.9E-6 UP_SEQ_FEATURE repeat:annexin 4 ANXA6, ANXA1, ANXA5, ANXA2 9.9E-6 SP_PIR_KEYWORDS calcium/phospholipid-binding ANXA6, ANXA1, ANXA5, ANXA2 1.2E-5 INTERPRO IPR001464:Annexin ANXA6, ANXA1, ANXA5, ANXA2 1.5E-5 INTERPRO IPR018252:Annexin repeat, conserved site ANXA6, ANXA1, ANXA5, ANXA2 1.5E-5 INTERPRO IPR018502:Annexin repeat ANXA6, ANXA1, ANXA5, ANXA2 1.5E-5 SP_PIR_KEYWORDS calcium binding ANXA6, HSP90B1, ANXA1, ACTN1, ANXA5, ANXA2 1.6E-5 SP_PIR_KEYWORDS phospholipid binding ANXA6, ANXA1, ANXA5, ANXA2 4.0E-5 PIR_SUPERFAMILY PIRSF002359:annexin I ANXA6, ANXA1, ANXA5, ANXA2 4.5E-5 GO: ~calcium-dependent phospholipid binding ANXA6, ANXA1, ANXA5, ANXA2 1.9E-4 GO: ~phospholipase inhibitor activity ANXA1, ANXA5, ANXA2 1.2E-3 GO: ~lipase inhibitor activity ANXA1, ANXA5, ANXA2 1.9E-3 GO: ~enzyme inhibitor activity SERPINB6, ANXA1, HSPA5, ANXA5, SERPINH1, ANXA2 6.5E-3 GO: ~phospholipid binding ANXA6, ANXA1, ANXA5, ANXA2 4.3E-2 (b) Pathways predicted by KEGG Protein processing in endoplasmic reticulum P=7.27e-11; adjp=2.33e-09 SEC23A Sec23 homolog A (S. cerevisiae) HSPH1 heat shock 105kDa/110kDa protein 1 HSP90B1 heat shock protein 90kDa beta (Grp94), member 1 HSPA5 heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) HSPA8 heat shock 70kDa protein 8 RRBP1 ribosome binding protein 1 homolog 180kDa (dog) VCP valosin containing protein
6 PDIA4 protein disulfide isomerase family A, member 4 Glycolysis / Gluconeogenesis P=3.91e-10; adjp=6.26e-09 ALDH7A1 aldehyde dehydrogenase 7 family, member A1 HK1 hexokinase 1 PKM pyruvate kinase, muscle ENO1 enolase 1, (alpha) ALDOC aldolase C, fructose-bisphosphate PFKP phosphofructokinase, platelet Metabolic pathways P=2.87e-06; adjp=3.06e-05 ALDOC aldolase C, fructose-bisphosphate ACLY ATP citrate lyase ALDH7A1 aldehyde dehydrogenase 7 family, member A1 HK1 hexokinase 1 PKM pyruvate kinase, muscle ENO1 enolase 1, (alpha) DLST dihydrolipoamide S-succinyltransferase PFKP phosphofructokinase, platelet MTAP methylthioadenosine phosphorylase PGD phosphogluconate dehydrogenase Focal adhesion P=8.33e-06; adjp=5.33e-05 ACTN1 actinin, alpha 1 FLNA filamin A, alpha PPP1R12A protein phosphatase 1, regulatory subunit 12A ACTG1 actin, gamma 1 FLNB filamin B, beta ALDOC Pentose phosphate pathway P=6.95e-06; adjp=5.33e-05 aldolase C, fructose-bisphosphate
7 PFKP PGD phosphofructokinase, platelet phosphogluconate dehydrogenase Fructose and mannose P=1.68e-05; adjp=8.96e-05 HK1 hexokinase 1 ALDOC aldolase C, fructose-bisphosphate PFKP phosphofructokinase, platelet Arrhythmogenic right ventricular cardiomyopathy (ARVC) ACTN1 actinin, alpha 1 ACTG1 actin, gamma 1 LMNA lamin A/C P=0.0001; adjp= HSPA9 HSPD1 ENO1 RNA degradation P=0.0001; adjp= heat shock 70kDa protein 9 (mortalin) heat shock 60kDa protein 1 (chaperonin) enolase 1, (alpha) Regulation of actin cytoskeleton P=0.0002; adjp= MYH9 myosin, heavy chain 9, non-muscle ACTN1 actinin, alpha 1 PPP1R12A protein phosphatase 1, regulatory subunit 12A ANXA2 annexin 2 ACTG1 actin, gamma 1 Supplementary Table S2. List of the significantly impaired pathways in Ob-hA-MSCs respect to Co-hA-MSCs, and proteins included in each of them as predicted by DAVID (a) and KEGG (b).
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Hs LOC Hs.7100 T Hs YARS Tyrosyl-tRNA synthetase 0.24
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