Proteomic Analysis of Human Angiogenin Interactions Reveals

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Proteomic analysis of human Angiogenin interactions reveals cytoplasmic PCNA as a putative binding partner Demetra S.M. Chatzileontiadou, Martina Samiotaki, Annika N Alexopoulou, Marina Cotsiki, George Panayotou, Melina Stamatiadi, Nikolaos A. A. Balatsos, Demetres D. Leonidas, and Maria Kontou J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00335 • Publication Date (Web): 04 Aug 2017 Downloaded from http://pubs.acs.org on August 9, 2017

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Journal of Proteome Research

Proteomic analysis of human Angiogenin interactions reveals cytoplasmic PCNA as a putative binding partner

Demetra S.M. Chatzileontiadoua, Martina Samiotakib*, Annika N. Alexopouloub, Marina Cotsikib, George Panayotoub, Melina Stamatiadia, Nikolaos A.A. Balatsosa, Demetres D. Leonidasa*, Maria Kontoua*

a

Department of Biochemistry and Biotechnology, University of Thessaly, Biopolis, 41500 Larissa,

Greece b

Biomedical Sciences Research Center “Alexander Fleming”, Vari 16672, Greece.

*

Corresponding authors:

Dr Martina Samiotaki, Biomedical Sciences Research Center Alexander Fleming, Vari 16672, Greece. Phone +30 210 9656310, Fax: +30 210 9653934, e-mail: [email protected] Prof. Demetres D. Leonidas, Department of Biochemistry and Biotechnology, University of Thessaly, Biopolis, 41500 Larissa, Greece. Phone: +30 2410 565278; Fax: +30 2410 565290; e-mail: [email protected];

ACS Paragon Plus Environment


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Ass. Prof. Maria Kontou, Department of Biochemistry and Biotechnology, University of Thessaly, Biopolis, 41500 Larissa, Greece. Phone: +30 2410 565281; Fax: +30 2410 565290; e-mail: [email protected];


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Abstract Human Angiogenin (hAng) is a member of the ribonuclease A superfamily and a potent inducer of neovascularization. Herein, protein interactions of hAng in the nucleus and cytoplasm of the human umbilical vein cell line EA.hy926, have been investigated by mass spectroscopy. Data are available via ProteomeXchange with identifiers PXD006583 and PXD006584. The first gel-free analysis of hAng immunoprecipitates revealed many statistically significant potential hAng-interacting proteins involved in crucial biological pathways. Surprisingly, proliferating cell nuclear antigen (PCNA), was found to be immunoprecipitated with hAng only in the cytoplasm. The hAng-PCNA interaction and co-localization in the specific cellular compartment was validated with immunoprecipitation, immunoblotting and immunocytochemistry. The results revealed that PCNA is predominantly localized in the cytoplasm, while hAng is distributed both in the nucleus and in the cytoplasm. hAng and PCNA co-localize in the cytoplasm, suggesting that they may interact in this compartment.

Keywords: human Angiogenin (hAng); immunoprecipitation; immunocytochemistry; LC-MS/MS; proteomic analysis; mass spectrometry; cytoplasm; nucleus; statistical significance; Proliferating cell nuclear antigen (PCNA)



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Introduction Human Angiogenin (hAng) is a 14.4 kDa basic single-chain protein, a member of the pancreatic ribonuclease (RNase) superfamily,1,

2

that serves as a multiple-function protein with varied

mechanisms of action. hAng is a potent angiogenic factor by directly inducing the process of angiogenesis, and is implicated in almost every step of tumorigenesis since it promotes tumor cell survival, proliferation, adhesion, migration and invasion

3, 4

. It has also been associated with

numerous malignant and non-malignant angiogenesis-dependent diseases

3, 5, 6

. Recently hAng has

been associated with amyotrophic lateral sclerosis (ALS)7-9 and Parkinson's disease 10, revealing that it plays a role in neural development too11. The protein is also involved in many physiological processes, such as pregnancy, by promoting normal vascular development12, as well as innate immunity by presenting antibacterial and antiviral activity13-15. During human development it has been detected in many organs, such as heart, brain, lung, breast, spleen, liver, retina, colon, prostate, foreskin, and in melanocytes5, 16, 17. Angiogenin is a member of the RNase A superfamily, sharing 33 % amino acid identity and 56 % similarity with bovine pancreatic RNase A, but a 105 to 106- fold less ribonucleolytic activity. This is attributed to its unique structural characteristics, and it is an indication that hAng may have specific substrates, distinct from those of other RNases 18, 19. Albeit low, the ribonucleolytic activity of hAng is essential for its angiogenic activity

20

. Little is known about the RNA substrates targeted by

hAng21, however, there is evidence that hAng cleaves tRNA molecules to form tiRNAs (tRNAinduced small RNA molecules) in response to stress, resulting to the reprogramming of protein translation, and promotion of cell survival22. Additionally, hAng binds to a smooth muscle type αactin on endothelial cell surface 23, inducing synthesis of proteases (uPA, tPA) to generate plasmin 24, promoting thus the degradation of basement membrane and extracellular matrix, and endothelial cell migration

25

. Besides its binding to actin it has been shown that hAng interacts with fibulin-1, an

extracellular matrix and plasma glycoprotein, indicating that the complex may modulate the


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stabilization of newly formed blood vessel wall 26. Furthermore, hAng can activate several signaling pathways in different cell conditions upon its binding on endothelial cell surface. It binds to a putative 170 kDa receptor

3, 27

. Finally, hAng has a nuclear localization sequence (NLS), and it has

been found that it undergoes nuclear translocation accumulating in the nucleolus28. There, hAng contributes to the rRNA transcription and regulates transcription of certain mRNAs

3, 29

. Moreover

nuclear translocation of hAng is necessary for cell proliferation induced by other angiogenic factors 30

. Studies on HeLa cells have shown that angiogenin can constantly translocate into the nucleus, and

that down-regulation of angiogenin expression inhibits cell proliferation, rRNA transcription, ribosome biogenesis, and tumorigenesis 31. Although hAng seems to play key roles in a plethora of biological processes and activate several signaling pathways, the precise mechanisms of its function(s) remain unclear. Interaction with different protein partners may be an important regulatory mechanism for the diverse cellular functions

of

hAng.

Therefore,

examination

of

the

signal

networks

leading

to

the

association/dissociation of protein complexes with hAng at a specific time and compartment (nucleus, cytoplasm, cell membrane) might shed light on the process and regulation of its subcellular localization and assist in the elucidation of the hAng-involved control mechanisms. For this purpose, we have performed an immunoprecipitation coupled mass spectrometry (MS) analysis of the cytoplasmic and nuclear extracts from EA.hy926 cancer human cell line. The MS analysis of all biological replicates, identified many statistically significant potential hAng-interacting proteins in the two cellular compartments. The majority of these proteins are parts of multiprotein complexes, and some of them are common in nucleus and cytoplasm including spliceosome, proteasome, and molecular chaperone TRiC/CCT. These complexes are involved in crucial biological pathways, implicating thus the key role of hAng in many vital cellular processes.

Materials and Methods



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Cell culture The human umbilical vein cell line, EA.hy926, was established by the fusion of primary human umbilical vein cells with a thioguanine-resistant clone of A54932, and obtained from ATCC. The cells were cultured in DMEM (Gibco) supplemented with 10 % fetal bovine serum (Gibco) and antibioticantimycotic (pen/strep, Biosera). Cells were maintained at 37 oC with 5 % humidified CO2. Antibodies Mouse monoclonal hAng (ANG I, C-1), mouse monoclonal PCNA (PCNA - PC10: sc-56) antibodies and nonspecific mouse IgG (negative control for immunoprecipitation) were used for immunoprecipitation and immunoblotting experiments. Rabbit polyclonal c-Jun and RhoGDI antibodies were used for immunoblotting experiments. Rabbit polyclonal hAng (ANG I, Η-123) and mouse monoclonal PCNA antibodies were used for immunocytochemistry. All the previous antibodies were purchased from Santa Cruz Biotechnology. The secondary antibodies used were: goat anti-mouse IgG-HRP (Santa Cruz Biotechnology), goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology), donkey anti-mouse IgG Alexa Fluor® 546 (Invitrogen), and donkey anti-rabbit (H+L) IgG Alexa Fluor® 488 (Invitrogen). Overexpression and purification of hAng Construction of the recombinant vector containing the gene of hAng and the purification procedure of the protein have been described previously33. The conversion of the purified Met(-1)-hAng form to the authentic Pyr1 human form (Pyr1-hAng), which contains a pyroglutamic acid residue at position 1, was conducted as described previously34. Nuclear and cytoplasmic extract preparation To obtain the cytoplasmic lysate, EA.hy926 cells were resuspended in NP-40 lysis buffer [10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5 % (v/v) NP-40, and protease inhibitor cocktail (Roche)] and maintained on ice for 15 minutes. Centrifugation at 600 g for 5 min at 4 °C, separated the cytoplasmic material from the nuclei. The nuclear pellet was resuspended in NP-40 lysis buffer


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and cleared by centrifugation at 600 g for 5 min at 4 °C. Subsequently, the nuclear pellet was resuspended in nuclear lysis buffer (NLB) (10 mM HEPES, pH 7.9, 100 mM KCl, 3 mM MgCl2, 0.2 mM EDTA, 20 % (v/v) glycerol supplemented with protease inhibitor cocktail (Roche)) and proteins were extracted by the dropwise addition of a 4 M KCl solution with gentle agitation on ice, up to 380 mM KCl. Nuclear proteins were allowed to extract for 1 h at 4 °C on rotator, and then centrifuged at 18,000 g for 35 min at 4 °C. The supernatant contains the nuclear lysate35. Protein concentrations were determined using the Bradford protocol. In order to ensure complete degradation of DNA and RNA molecules, the nuclear extract was diluted 3-fold using HENG buffer (10 mM HEPES-KOH, pH 9.0, 1.5 mM MgCl2, 0,25 mM EDTA, 20 % (v/v) glycerol and protease inhibitor cocktail (Roche)), and incubated with 250 U benzonase and 0,1 mg/ mL RNase A, for 1h at 4 °C on rotator, while the cytoplasmic extract with 0,1 mg/ mL RNase A. The complete degradation of DNA and RNA molecules was verified by agarose electrophoresis. Immunoprecipitation and Protein Identification i) hAng immunoprecipitation Both cell extracts were precleared by adding control IgG antibody on ice for 1 h, followed by incubation with protein A/G-conjugated agarose beads (Santa Cruz) for 30 min on rotator. The samples were then centrifuged at 10,000 g for 10 min at 4 °C. The supernatants were used for the immunoprecipitation procedure, while the pellets served as control samples. Recombinant human angiogenin was added in both cell lysates, in complex with the hAng antibody/protein beads. For the formation of the complexes, the hAng antibody was pre-incubated with the A/G protein beads in NP-40 lysis buffer (for the cytoplasmic extract) or in NLB buffer (for the nuclear extract), for 6 h at 4 oC on a rotator. After centrifugation (3,500 g, 5 min, 4 oC) and washing, the antibody/beads complexes were resuspended in NP-40 or NLB buffer, and recombinant hAng was then added in excess (molar ratio hAng/anti-hAng antibody 10/1). The samples were incubated for 12 h at 4 oC on a rotator, centrifuged at 3,500 g for 5 min at 4 oC, and washed two



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times with the same buffers. Finally, the cytoplasmic and nuclear extract, were incubated with the hAng/antibody/beads complexes for 12 h at 4 oC on a rotator, and then the immunocomplexes were pelleted by centrifugation at 3,500 g for 5 min at 4 oC. The immunocomplexes (control-IgG samples and specific antibody samples) were then washed 2 times with NP-40 or HENG buffer, for the cytoplasmic and the nuclear material, respectively. A last wash with PBS 1x containing 1 mM PMSF was performed and the samples were resuspended in SDS-PAGE loading buffer. ii) PCNA immunoprecipitation For each extract (cytoplasmic and nuclear), two complexes of antibody/protein G agarose beads (Santa Cruz) were formed; one for the specific PCNA antibody and another for the control IgG. The complexes were formed in the appropriate buffers (NP-40 or NLB), for 3 h at 4 oC on a rotator, followed by centrifugation at 3,500 g for 5 min at 4 oC, and two washes with the same buffers. Equal amounts of each extract (cytoplasmic and nuclear) were incubated for 1 h at 4 oC with recombinant hAng (molar ratio hAng/antibody = 1/1) prior to the addition of the antibody/beads complexes. The four samples were incubated for 3 h at 4 oC on a rotator. The immunocomplexes were then pelleted and washed as described for the hAng immunoprecipitation. iii) Proteomic analysis The immunocomplexes were resuspended in 8 M Urea/100 mM Tris-HCl pH 8.5, the beads were pelleted by centrifugation, and the extracted proteins were processed according to the Filter Aided Sample Preparation (FASP) protocol36 using spin filter devices with a 10 kDa cut-off (Sartorius, VN01H02). Filters were extensively washed with the urea solution, treated with 10 mg/mL iodoacetamide in the urea solution and incubated for 30 min in the dark for cysteine alkylation. Proteins on the top of the filters were washed three times with 50 mM ammonium bicarbonate and finally digested by adding 1 µg trypsin/LysC mix in 80 µL of 50 mM ammonium bicarbonate solution (Mass spec grade, Promega) and incubated overnight at 37 °C. Peptides were eluted by centrifugation and upon speed-vac-assisted solvent remova,l eluted peptides were reconstituted in 0.1


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% formic acid, 2 % acetonitrile in water. Peptide concentration was determined by nanodrop absorbance measurement at 280 nm. 2.5 µg peptides were pre-concentrated with a flow of 3 µL/ min for 10 min using a C18 trap column (Acclaim PepMap100, 100 µm×2 cm, Thermo Scientific) and then loaded onto a 50 cm C18 column (75 µm ID, particle size 2 µm, 100 Å, Acclaim PepMap RSLC, Thermo Scientific). The binary pumps of the HPLC (RSLCnano, Thermo Scientific) consisted of solution A (2 % (v/v) acetonitrile in 0.1 % (v/v) formic acid) and solution B (80 % acetonitrile in 0.1 % formic acid). The peptides were separated using a linear gradient of 4 % solution B up to 40 % in 450 min for an 8 h gradient run with a flow rate of 300 nL/ min. The column was placed in an oven operating at 35 °C. For LC-MS/MS, purified peptides were analysed by HPLC MS/MS coupled to an LTQ Orbitrap XL Mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a nanospray source. Full scan MS spectra were acquired in the orbitrap (m/z 300–1,600) in profile mode and data-dependent acquisition, with the resolution set to 60,000 at m/z 400 and automatic gain control target at 106 ions. The six most intense ions were sequentially isolated for collision-induced (CID) MS/MS fragmentation and detection in the linear ion trap. Dynamic exclusion was set to 1 min and activated for 90 s. Ions with single charge states were excluded. Lock mass of m/z 445, 120025 was used for internal calibration. Xcalibur (Thermo Scientific) was used to control the system and acquire the raw files. Raw files were analyzed using MaxQuant (version 1.5.5.1)37, using the complete human proteome database. Search parameters were precursor mass tolerance of 20 ppm, an MS/MS fragment tolerance of 0.5 Da, a maximum of two missed cleavages by trypsin, methionine oxidation, and acetylation of the N-terminus as variable modifications. The protein and peptide false discovery rate (FDR) was set to 1 %. Label-free quantification was carried out in MaxQuant and protein abundance was calculated on the basis of the normalized spectral protein intensity (Label Free Quantitation LFQ intensity).



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Statistical analysis was performed using Perseus (version 1.2.7.4)38. Proteins identified as contaminants, “reverse” and “only identified by site” were filtered out. The LFQ intensities were transformed to logarithmic. Zero intensities were imputed — replaced by normal distribution, assuming that the corresponding protein is present in low amounts in the sample. The replicas were grouped for each set of conditions, ie “hAng Ab” and “IgG Ab” (three biological replicas for the cytoplasmic fraction and four biological replicas for the nuclear fraction), and a twosided Student's t-test of the grouped proteins was performed using p values for truncation. Western blotting Protein samples were resolved by 12 % SDS-PAGE and then transferred to PVDF membranes (Macherey-Nagel) by semi-dry blotting (Wealtec Corp.) in transfer buffer (10 % methanol, 25 mM Tris, 192 mM glycine, 0.1 % SDS). Membranes were blocked with 5 % skimmed milk powder in phosphate buffered saline with 0.1 % Tween-20 (PBST) and incubated with primary antibodies at 4 o

C overnight. After 3 washes with PBST, membranes were incubated with HRP-conjugated

secondary antibodies, and proteins were visualized using the ECL chemiluminescence substrate (GenScript) and the FluorChem E system (Protein Simple). All primary and secondary antibodies were diluted in 5 % skimmed milk powder in PBST. Immunocytochemistry To visualize the cytoplasmic localization of hAng and/or PCNA, EA.hy926 cells were grown on glass coverslips and fixed with pre-chilled methanol for 10 min at -20 °C. After blocking with 1 % BSA for 30 min, cells were incubated overnight at 4 oC with hAng and/or PCNA antibodies, diluted 1:100 (final concentration 2 µg/ mL) and 1:200 (final concentration 1 µg/ mL), respectively. Incubation with the secondary antibodies was carried out at room temperature for 1 h in the dark. Slides were mounted in ProLong® Gold antifade mountant with DAPI (Life Technologies) and observed under a Zeiss AxioVert 200 inverted microscope.


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Results and Discussion Identification and functional classification of putative hAng interacting proteins Immunoprecipitation combined with a label-free LC-MS/MS was used for the qualitative and quantitative analysis of the potential hAng-interacting partners. As expected, hAng was immunoprecipitated with the hAng specific antibody in the nuclear and the cytoplasmic extract, while no hAng amounts were detected in the control (control IgG) samples (Figure 1A). Nuclear proteins were efficiently separated from the cytoplasmic ones, as confirmed by no detectible crosscontamination of the c-Jun nuclear marker or the Rho GDP-dissociation inhibitor (Rho-GDI) cytoplasmic marker (Figure 1B).

Figure 1. Immunoprecipitation of hAng in the nuclear and cytoplasmic fraction of EA.hy926 cells. A. Immunoprecipitation of hAng with a hAng antibody or control IgG, followed by immunoblotting with hAng antibody. B. Equivalent nuclear and cytoplasmic extracts, were subjected to immunoblotting with c-Jun and RhoGDI antibodies.

The activity and the integrity of the recombinant human angiogenin34 used in the immunoprecipitation experiments, have been verified by kinetic33, X-ray crystallography33, NMR39 and in vivo studies (manuscript submitted for publication). The mass-spectrometric data from all biological replicates of each cellular compartment were analyzed with the quantitative proteomics software package MaxQuant37 enabling its LFQ



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function40, and thus the first gel-free analysis of hAng immunoprecipitates was performed. The results were statistically processed and visualized within the Perseus38 software. Proteins were categorized as specific or nonspecific based on the relative enrichment of the protein in the specific (hAng antibody) versus control samples (IgG) of each cellular compartment. Volcano plots were constructed, by plotting the t-test logarithmic difference between the two groups (“hAng Ab” and “IgG Ab”) versus the negative logarithm of the p values for each protein (Figure 2).

Figure 2A


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Figure 2B Figure 2. Volcano plots. Comparison of hAng Ab group vs control group (IgG Ab) fractions from the (A) nuclear and (B) cytoplasmic cellular compartments, respectively. Grey nodes are non significant/non specific proteins, orange nodes are proteins with p-value < 0.2 to > 0.05, and red nodes are proteins with p-value ≤ 0.05. Proliferating cell nuclear antigen is shown in black (A).

Putative specific interactors were identified based on p-value ≤ 0.05 and log2 Fold Difference ≥ 1 and selected for subsequent data analysis (Tables 1, 2). The peptides found for these proteins (Tables 1, 2) are shown in the supplementary Tables S3 and S4. The list of the putative hAng-interacting proteins was subjected to functional annotation analysis using the enrichment analysis tool FunRich41. The majority of these proteins (Tables 1, 2) were classified according to their primary biological processes. The wide spectrum of activity of hAng-binding proteins indicates a possible involvement of hAng in unexpected functions.



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Table 1: List of the statistically significant nuclear hAng-co-precipitated proteins identified by Quantitative (LFQ) LC-MS/MS analysis. Log2 Fold UniProt Protein name / synonyms

Gene name

Change

p-value

accession Difference Angiogenin

ANG

P03950

12.40805

1.27E-06

CCT4

P50991

1.804967

0.000141

ORC2

Q13416

1.420966

0.000878

CTBP2

P56545

3.884568

0.001497

SFPQ

P23246

1.342132

0.002283

NELFCD

Q8IXH7

2.537101

0.002915

PUF60

Q9UHX1

1.575066

0.002998

CCAR2

Q8N163

2.629205

0.003968

SNTB2

Q13425

2.51676

0.00761

RBBP4;

Q09028; 1.323516

0.008397

RBBP7

Q16576

NONO

Q15233

1.210911

0.011032

MRE11A

P49959

1.562704

0.01214

PICALM

Q13492

3.553107

0.012571

EEF1A1;

P68104;

1.488594

0.016911

T-complex protein 1 subunit delta Origin recognition complex subunit 2 C-terminal-binding protein 2 Splicing factor, proline- and glutamine-rich Negative elongation factor C/D Poly(U)-binding-splicing factor PUF60 Cell cycle and apoptosis regulator protein 2 Beta-2-syntrophin Histone-binding protein RBBP4; Histone-binding protein RBBP7 Non-POU domain-containing octamer-binding protein Double-strand break repair protein MRE11A Phosphatidylinositol-binding clathrin assembly protein Elongation factor 1-alpha 1;


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Putative elongation factor 1-

EEF1A1P5

Q5VTE0

RUVBL2

Q9Y230

2.676107

0.019184

ALDH18A1

P54886

4.649745

0.021542

MCM3

P25205

1.531037

0.024602

Poly(rC)-binding protein 1

PCBP1

Q15365

1.62869

0.025114

Apoptosis inhibitor 5

API5

Q9BZZ5

3.149391

0.025181

GMPS

P49915

1.26363

0.026242

NCAPD2

Q15021

0.992823

0.026245

HNRNPH1

P31943

0.919016

0.0269

PCBP2

Q15366

1.996495

0.028486

KHSRP

Q92945

3.932938

0.028664

MTA1;

Q13330; 2.543787

0.030188

MTA3

Q9BTC8

Citron Rho-interacting kinase

CIT

O14578

1.313169

0.031563

Galectin-1

LGALS1

P09382

2.881368

0.032307

CCT5

P48643

3.251119

0.033781

Intron-binding protein aquarius

AQR

O60306

0.906875

0.034928

AP-2 complex subunit beta

AP2B1

P63010

2.155357

0.036566

Elongation factor 1-gamma

EEF1G

P26641

1.462585

0.039503

CCT3

P49368

2.641495

0.039729

alpha-like 3 RuvB-like 2 Delta-1-pyrroline-5-carboxylate synthase DNA replication licensing factor MCM3

GMP synthase [glutaminehydrolyzing] Condensin complex subunit 1 Heterogeneous nuclear ribonucleoprotein H Poly(rC)-binding protein 2 Far upstream element-binding protein 2 Metastasis-associated protein MTA1; Metastasis-associated protein MTA3

T-complex protein 1 subunit epsilon

T-complex protein 1 subunit gamma



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Nucleolysin TIA-1 isoform p40

Page 16 of 54

TIA1

P31483

2.908225

0.043578

CYFIP2

Q96F07

1.486047

0.045253

EIF4A1

P60842

1.138265

0.047069

DDX49

Q9Y6V7

1.710062

0.048236

PSMC3

P17980

2.119186

0.050884

Cyclin-dependent kinase 1;

CDK1;

P06493;

Cyclin-dependent kinase 2;

CDK2;

P24941;

2.766289

0.050927

Cyclin-dependent kinase 3

CDK3

Q00526

PDZ and LIM domain protein 7

PDLIM7

Q9NR12

1.403693

0.051272

Protein unc-45 homolog A

UNC45A

Q9H3U1

3.815187

0.053289

DDX39A

O00148

2.249216

0.053999

Cytoplasmic FMR1-interacting protein 2 Eukaryotic initiation factor 4A-I Probable ATP-dependent RNA helicase DDX49 26S protease regulatory subunit 6A

ATP-dependent RNA helicase DDX39A

Table 2: List of the statistically significant cytoplasmic hAng-co-precipitated proteins identified by Quantitative (LFQ) LC-MS/MS analysis. Log2 Fold UniProt Protein name / synonyms

Gene name

Change

p-value

accession Difference Tripeptidyl-peptidase 1

TPP1

O14773

4.355183

2.09E-05

PSMD5

Q16401

4.170012

5.79E-05

Proliferating cell nuclear antigen

PCNA

P12004

8.343543

0.000132

Tubulin beta-8 chain

TUBB8

Q3ZCM7

8.016228

0.000162

Heat shock 70 kDa protein 1B;

HSPA1B;

P0DMV9;

5.256535

0.000562

26S proteasome non-ATPase regulatory subunit 5


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Heat shock 70 kDa protein 1A

HSPA1A

P0DMV8

RuvB-like 1

RUVBL1

Q9Y265

4.717649

0.001409

Transmembrane protein 33

TMEM33

P57088

3.675543

0.001467

14-3-3 protein eta

YWHAH

Q04917

3.666375

0.001839

DDX39A;

O00148; 6.647326

0.002041

DDX39B

Q13838

Actin-related protein 2

ACTR2

P61160

4.562078

0.002134

Protein unc-45 homolog A

UNC45A

Q9H3U1

7.20316

0.002257


TUBB3

Q13509

2.973211

0.002309

ATP5B

P06576

6.383986

0.002447

ANP32B

Q92688

5.19521

0.00254

Importin subunit beta-1

KPNB1

Q14974

3.621418

0.002797

Melanoma-associated antigen D2

MAGED2

Q9UNF1

3.262938

0.003087

EFEMP1

Q12805

6.138875

0.003421

PCMT1

P22061

5.97185

0.00392

MAT2A

P31153

4.474179

0.003931

DDB1

Q16531

4.895379

0.004556

ACAT1

P24752

5.859104

0.004596

Tubulin alpha-1C chain

TUBA1C

Q9BQE3

3.048147

0.004932

Endoglin

ENG

P17813

4.181399

0.005783

Catenin alpha-1

CTNNA1

P35221

3.686558

0.006245

ATP-dependent RNA helicase DDX39A; Spliceosome RNA helicase DDX39B

ATP synthase subunit beta, mitochondrial Acidic leucine-rich nuclear phosphoprotein 32 family member B

EGF-containing fibulin-like extracellular matrix protein 1 Protein-L-isoaspartate Omethyltransferase S-adenosylmethionine synthase isoform type-2 DNA damage-binding protein 1 Acetyl-CoA acetyltransferase, mitochondrial



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Angiogenin

ANG

P03950

10.62103

0.007614

RuvB-like 2

RUVBL2

Q9Y230

6.055868

0.007177

MSH2

P43246

4.729833

0.007307

ACADM

P11310

4.632466

0.007327

Tubulin beta chain

TUBB

P07437

2.810893

0.009049

Myc target protein 1

MYCT1

Q8N699

4.169182

0.009375

Synembryn-A

RIC8A

Q9NPQ8

3.801528

0.009423

T-complex protein 1 subunit theta

CCT8

P50990

5.311

0.00988


TUBB6

Q9BUF5

2.734499

0.010534

Tubulin beta-4B chain

TUBB4B

P68371

3.111849

0.011957

45 kDa calcium-binding protein

SDF4

Q9BRK5

4.45485

0.01281

14-3-3 protein theta

YWHAQ

P27348

2.495167

0.013194

Tubulin alpha-1B chain;

TUBA1B;

P68363; 2.332371

0.013893

Tubulin alpha-4A chain

TUBA4A

P68366

MSH6

P52701

4.119052

0.014618

HSPD1

P10809

5.377247

0.015314

MAP1B

P46821

1.968246

0.017049

IPO7

O95373

5.278625

0.017081

PRKAA1

Q13131

3.631046

0.018248

CAND1

Q86VP6

3.294813

0.02054

MCM3

P25205

4.080209

0.02065

DNA mismatch repair protein Msh2 Medium-chain specific acyl-CoA dehydrogenase, mitochondrial

DNA mismatch repair protein Msh6 60 kDa heat shock protein, mitochondrial Microtubule-associated protein 1B Importin-7 5-AMP-activated protein kinase catalytic subunit alpha-1 Cullin-associated NEDD8dissociated protein 1 DNA replication licensing factor MCM3


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Heat shock protein beta-1

HSPB1

P04792

3.4584

0.021024

EEF1A1;

P68104;

EEF1A1P5

Q5VTE0

1.570248

0.02111

PFKP

Q01813

2.72386

0.023578

ACTG1

P63261

2.816229

0.024109

ARHGEF1

Q92888

3.229576

0.024564

CCT3

P49368

4.251968

0.0259

HSPA8

P11142

1.318996

0.028545

MTHFD1

P11586

2.152489

0.030237

SSBP1

Q04837

2.003373

0.030327

CCT7

Q99832

5.922038

0.0333

PFKL

P17858

4.616782

0.035179

Maleylacetoacetate isomerase

GSTZ1

O43708

2.671729

0.036978

Elongator complex protein 1

IKBKAP

O95163

3.473028

0.039637

PSMD2

Q13200

4.986183

0.041326

TCP1

P17987

5.337725

0.041681

Elongation factor 1-alpha 1; Putative elongation factor 1alpha-like 3 ATP-dependent 6phosphofructokinase, platelet type Actin, cytoplasmic 2 Rho guanine nucleotide exchange factor 1 T-complex protein 1 subunit gamma Heat shock cognate 71 kDa protein Methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1 Single-stranded DNA-binding protein T-complex protein 1 subunit eta ATP-dependent 6phosphofructokinase, liver type

26S proteasome non-ATPase regulatory subunit 2 T-complex protein 1 subunit



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alpha Nucleosome assembly protein 1NAP1L1

P55209

1.883533

0.041799

AKAP13

Q12802

1.113161

0.04442

DNAJA2

O60884

2.384314

0.046035

RAB3A

P20336

1.73407

0.046533

UMPS

P11172

3.606234

0.046853

DNAJB1

P25685

2.569375

0.047529

CCT5

P48643

3.235831

0.050126

SLC25A3

Q00325

1.544589

0.050227

ATP5A1

P25705

4.842325

0.051212

PPP2CA;

P67775; 3.127401

0.052937

PPP2CB

P62714

SUCLG1

P53597

2.925508

0.053501

MCM7

P33993

3.200167

0.05397

like 1 A-kinase anchor protein 13 DnaJ homolog subfamily A member 2 Ras-related protein Rab-3A Uridine 5-monophosphate synthase DnaJ homolog subfamily B member 1 T-complex protein 1 subunit epsilon Phosphate carrier protein, mitochondrial ATP synthase subunit alpha, mitochondrial Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform; Serine/threonineprotein phosphatase 2A catalytic subunit beta isoform Succinyl-CoA ligase [ADP/GDPforming] subunit alpha, mitochondrial DNA helicase;DNA replication licensing factor MCM7


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The annotation revealed that the proteins immunoprecipitated with hAng in the nuclear extract (Table 3, Figure 3A) are mainly involved in transcription (13 proteins), RNA splicing via spliceosome and subsequent mRNA processing (10), DNA replication (7), apoptotic process (6), protein stabilization (4), DNA repair (5), cell proliferation (5), and proteasome (4). The proteins from the cytoplasmic extract (Table 4, Figure 3B) are mainly involved in protein stabilization (12 proteins), microtubule-based process (8), regulation of transcription (8), cell proliferation (6), negative regulation of apoptotic process (5), movement of cell or subcellular component (5), DNA repair (5), regulation of mRNA stability (5), mRNA splicing including the mRNA export from the nucleus and mRNA 3’-end processing (4), proteasome (4), intracellular signal transduction (3), regulation of cell adhesion (2), and ATP biosynthetic process (2).

Table 3: Function annotations of the nuclear putative hAng interactors by FunRich. Biological process

Proteins (UniProt ID) P56545, P23246, Q9UHX1, Q8N163, Q09028,

Transcription

Q16576, Q15233, P68104, Q9Y230, Q92945, Q13330, Q8IXH7, Q9BTC8

RNA splicing via spliceosome – mRNA

P23246, Q9UHX1, Q8N163, Q15233, Q92945,

processing

Q15365, P31943, Q15366, O60306, O00148 Q13416, Q09028, Q16576, P49959, P25205,

DNA replication P06493, P24941 Q9UHX1, Q9BZZ5, P09382, P31483, Q96F07, Apoptotic process P06493 Protein stabilization – protein folding

P50991, P48643, P49368, Q9Y230

DNA repair

Q15233, P49959, Q9Y230, P06493, P24941

Cell proliferation

Q16576, P49959, Q13492, P06493, Q00526

Proteasome-mediated ubiquitin-

Q15366, Q13330, P17980, P06493



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dependent protein catabolic process

Table 4: Function annotations of the cytoplasmic putative hAng interactors by FunRich. Biological process

Proteins (UniProt ID) Q9Y230, P50990, P49368, P11142, Q99832,

Protein stabilization: protein folding/ P17987, P48643, P0DMV9, P0DMV8, P10809, protein refolding P11142, O60884 Q3ZCM7, Q13509, Q9BQE3, P07437, Q9BUF5, Microtubule-based process P68371, P68363, P68366 Q12805, P17813, Q13131, P68104, P67775, Regulation of transcription Q9Y265, Q9Y230, P11142 P12004, Q92888, P33993, Q13131, P55209, Cell proliferation O60884 Negative regulation of apoptotic process

P50990, P49368, Q99832, P17987, P48643

Movement of cell or subcellular P61160, P07437, P68371, P04792, P63261 component DNA repair

Q9Y265, Q16531, Q9Y230, P43246, P52701

Regulation of mRNA stability

Q16401, P0DMV8, P04792, P11142, Q13200

RNA splicing - RNA export from nucleus – P11142, P67775, O00148, Q13838 mRNA 3’-end processing Proteasome-mediated ubiquitinQ16401, Q16531, Q13200, P62714 dependent protein catabolic process Intracellular signal transduction

Q13131, P04792, Q12802

Regulation of cell adhesion

P17813, P67775

ATP biosynthetic process

P25705, P06576


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Until now, a total of 19 hAng’s partners have been reported based on experimental evidence3. Most of these partners have been also found in our study (supplementary Tables S1, S2), thus validating our results. In the cytoplasmic extract of our study, apart from members of the actin family, members of the tubulin family have been also identified (Table S2). Both actins and tubulins being the two major components of the cytoskeleton42 play crucial roles in microfilaments and microtubules assembly, respectively.

Figure 3. Pie-chart representation of gene ontology (GO) annotation results from the nuclear (A) and the cytoplasmic (B) cellular compartment. Proteins were grouped into sub-categories of biological processes using GeneCodis annotation tool43.



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Clustering of putative hAng partners that constitute parts of large protein complexes Taking into consideration that slight differences on the experimental conditions and on the state of cells (phase of the cell cycle) might affect the accurate reproducibility among the independent immunoprecipitation experiments, especially concerning the indirect (secondary) interactions of hAng, we performed a less stringent statistical analysis. Therein, we mainly focused on the quantitative difference between specific and control samples, rather than on the statistical significance among the three replicates. The proteins which were more abundant in the specific than in the control samples generating an abundance ratio greater than 2 and presented p-value ≤ 0.2 are shown in Table S1 for the nucleus and Table S2 for the cytoplasm. The majority of these proteins were classified according to their cellular role as part of stable complexes. To achieve this annotation the CORUM database44 was used. The multiprotein complexes formed by the proteins identified, were analysed based on the hypothesis that if hAng directly interacts with a member of a protein complex, it is possible that other members of the specific complex will be co-precipitated as well. The complexes identified are presented in Tables 5 and 6 for the nuclear and the cytoplasmic extract, respectively. The most statistically significant proteins (p-value ≤ 0.05) are highlighted (Tables 5 and 6) since they might be the direct hAng interactors. This classification revealed that hAng is possibly implicated in many crucial biological processes in both the nucleus and the cytoplasm. Reviewing the corresponding literature, we discuss below the potential biological roles of the putative interactions that hAng forms with some of these proteins (Tables 5 and 6).

Table 5: The multiprotein complexes formed by the putative hAng interactors identified in the nuclear fraction, annotated by the CORUM database. The proteins in bold presented threshold values log2 Fold Difference ≥ 0.9 and p-value ≤ 0.05.


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Nuclear proteins Complex name

Protein name (Gene name) Serine/arginine-rich splicing factor 7 (SRSF7) Splicing factor U2AF 65 kDa subunit (U2AF) Poly(U)-binding-splicing factor PUF60 (PUF60) Heterogeneous nuclear ribonucleoprotein A/B (HNRNPΑΒ) Luc7-like protein 3 (LUC7L3) Galectin-1 (LGALS1) Serine-threonine kinase receptor-associated protein (STRAP) Regulator of nonsense transcripts 2 (UPF2)

Spliceosome

Nuclear RNA export factor 1 (NXF1) Spliceosome RNA helicase DDX39B (DDX39B) ATP-dependent RNA helicase DDX39A (DDX39A) Cleavage and polyadenylation specificity factor subunit 6 (CPSF6) Splicing factor, proline- and glutamine-rich (SFPQ) Heterogeneous nuclear ribonucleoprotein H (HNRNPH1) Eukaryotic initiation factor 4A-III (EIF4A3) Intron-binding protein aquarius (AQR) Cactin (CACTIN) Structural maintenance of chromosomes protein 4 (SMC4)

Condensin I Condensin complex subunit 1 (NCAPD2) Proteasome subunit alpha type-2 (PSMA2) 26S protease regulatory subunit 4 (PSMC1, Rpt2)

Proteasome

26S protease regulatory subunit 6A (PSMC3, Rpt5) 26S protease regulatory subunit 8 (PSMC5 Rpt6) 26S protease regulatory subunit 10B (PSMC6, Rpt4)

Mi-2/NuRD

Histone deacetylase 1 (HDAC1) Histone deacetylase 2 (HDAC2)

(Nucleosome Histone-binding protein RBBP4 (RBBP4, RbAp48)

Remodeling Histone-binding protein RBBP7 (RBBP7, RbAp46)

Deacetylase) BASC (BRCA1associated genome

Metastasis-associated protein MTA2 (MTA2,MTA1-L1) DNA mismatch repair protein Msh6 (MSH6) DNA repair protein RAD50 (RAD50)



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surveillance)

Double-strand break repair protein MRE11A (MRE11A) T-complex protein 1 subunit beta (CCT2)

TRiC/CCT T-complex protein 1 subunit gamma (CCT3)

(Molecular chaperone T-complex protein 1 subunit delta (CCT4)

TCP1 ring T-complex protein 1 subunit epsilon (CCT5)

complex/chaperonin T-complex protein 1 subunit zeta (CCT6A)

containing TCP1) T-complex protein 1 subunit theta (CCT8)

Table 6: The multiprotein complexes formed by the putative hAng interactors identified in the cytoplasmic fraction, annotated by the CORUM database. The proteins in bold presented threshold values log2 Fold Difference ≥ 0.9 and p-value ≤ 0.05. Cytoplasmic proteins Complex name

Protein name (Gene name) 26S protease regulatory subunit 6A (PSMC3, Rpt5) 26S protease regulatory subunit 7 (PSMC2, Rpt1) 26S protease regulatory subunit 4 (PSMC1, Rpt2) 26S protease regulatory subunit 8 (PSMC5, Rpt6) 26S protease non-ATPase regulatory subunit 13 (PSMD13, Rpn9)

Proteasome 26S proteasome non-ATPase regulatory subunit 2 (PSMD2, Rpn1) 26S proteasome non-ATPase regulatory subunit 5 (PSMD5) 26S proteasome non-ATPase regulatory subunit 8 (PSMD8, Rpn12) 26S proteasome non-ATPase regulatory subunit 10 (PSMD10) 26S proteasome non-ATPase regulatory subunit 11 (PSMD11, Rpn6) DNA mismatch repair protein Msh6 (MSH6)

MutSα DNA mismatch repair protein Msh2 (MSH2) 14-3-3 protein epsilon (YWHAE) 14-3-3 protein theta (YWHAQ)

14-3-3 proteins 14-3-3 protein eta (YWHAH) 14-3-3 protein gamma (YWHAG)


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T-complex protein 1 subunit alpha (TCP1) T-complex protein 1 subunit beta (CCT2)

TRiC/CCT T-complex protein 1 subunit gamma (CCT3)

(Molecular chaperone T-complex protein 1 subunit delta (CCT4)

TCP1 ring T-complex protein 1 subunit epsilon (CCT5)

complex/chaperonin T-complex protein 1 subunit zeta (CCT6A)

containing TCP1) T-complex protein 1 subunit eta (CCT7) T-complex protein 1 subunit theta (CCT8) ATP-dependent RNA helicase DDX39A (DDX39A)

Splicing

Spliceosome RNA helicase DDX39B (DDX39B) Protein arginine N-methyltransferase 5 (PRMT5) RuvB-like 1 (RUVBL1)

RuvBL1/RuvBL2 RuvB-like 2 (RUVBL2) ATP synthase subunit beta, mitochondrial (ATP5B)

F1FO ΑΤΡ synthase ATP synthase subunit alpha, mitochondrial (ATP5A1)

Complexes common in cytoplasm/ nucleus Spliceosome/ splicing A significant number of proteins immunoprecipitated with hAng in the nucleus are components of the spliceosome45, and they are implicated in many different steps of the splicing machinery. More specifically, LUC7L3, U2AF and PUF60 (Table 5) are involved in the recruitment of snRNPs onto pre-mRNA molecules46-48, while STRAP and LGALS1 (Table 5), are components of the Survival of Motor Neurons (SMN) complex49,

50

that functions in the cytoplasmic assembly of snRNP core

particles and in their subsequent transport to the nucleus. Additionally, SMN binds directly to the modified domains of Sm proteins51, a process carried out by the methylosome, a complex containing the methyltransferase PRMT551, a protein that participates in splicing52 and was immunoprecipitated with hAng in the cytoplasmic fraction (Table 6). These putative interactions of hAng indicate that hAng may participate in the cytoplasmic assembly of snRNPs through its recruitment to the SMN



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complex, and that it is subsequently translocated to the nucleus for the pre-mRNA processing. However, the majority of the proteins identified (Table 5) participate in alternative splicing (SRSF753, HNRNPH154) or in the exon junction complex (EJC) (AQR55, UPF256, NXF157, DDX39B58), while EIF4A3 is implicated in both processes59,

60

. The EJC consists of an

heterotetramer core that includes EIF4A361 (Table 5), and serves as a binding platform for other factors necessary for the mRNA pathway62. Many of the EJC factors play a certain role in the nuclear mRNA export, including SRSF763, NXF157, DDX39A64 and DDX39B58 (Table 5). As shown in Table 6 hAng may interact with DDX39A and DDX39B in the cytoplasm as well, indicating that hAng is possibly recruited to the EJC in the nucleus and remains bound to the complex after the mRNA export to the cytoplasm. Our hypothesis for the implication of hAng in such a crucial biological process is further supported by recent studies which suggested that the mRNA export is regulated by the PI3 kinase/AKT pathway65 that in turn is activated by hAng66. Furthermore, the EJCs, which play a major role in mRNA surveillance, are found in the nonsense mediated decay pathway (NMD)67 and three factors involved in this process, EIF4A368, AQR55, and UPF269, were immunoprecipitated with hAng (Table 5). Therefore, it is likely hAng to be involved in ΝΜD, maybe by degrading mRNA molecules. We could speculate that when hAng exists in its free form, it cannot recognize and degrade the mRNA molecules in the cytoplasm. This presumption relies on the poor catalytic activity of hAng that may have evolved to maximize specificity for the target substrate and that the target may have a specific secondary structure, such as a hairpin or a pseudo-knot, or may be part of a protein-nucleic acid complex70. Thus, in its bound form with the EJC factors that are found in the nonsense mediated decay pathway, hAng may be recruited to the target mRNA molecules promoting their degradation. Moreover, three putative hAng-interacting factors of the splicing machinery, U2AF2, SFPQ, and CPSF6 (Table 5), are also members of the nuclear cleavage factor IIAm complex (CF IIAm) that catalyzes the 3′ end formation of mammalian pre-mRNAs by


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endonucleolytic cleavage and polyadenylation and might be involved in the coupling of transcription and splicing with 3′ end processing71. Proteasome A significant number of proteasome subunits were immunoprecipitated with hAng in both the nucleus and the cytoplasm (Tables 5 and 6). In addition to directing ubiquitin-dependent proteolysis, the proteasome and especially the 19S regulatory particle has been shown to have nonproteolytic roles, being implicated in the regulation of gene expression by recruiting activators and basal transcriptional machinery to gene promoters, playing certain roles in transcription initiation and elongation as well as in DNA repair

72-74

. As shown in Tables 5 and 6, hAng seems to interact

especially with the 19S regulatory particle in both cellular compartments, indicating that hAng may regulate proteasome activity. This assumption is associated with a previous study on ΕΑ.hy926 cells which showed that plasminogen activator inhibitor type 2 (PAI-2) interacts with proteasome in both nucleus and cytoplasm, prevents the degradation of p53 and inhibits proteasome, favoring proapoptotic signaling75. Thus, we could assume that while PAI-2 acts as a proteasome inhibitor promoting apoptosis, the interaction of hAng with proteasome could possibly, vice versa, promote cell survival and cell proliferation. Furthermore, it would be worth studying the possibility that hAng selectively “drives” proteins for degradation. One such paradigm would be the tumor suppressor protein p53 which interacts with hAng resulting in the inhibition of p53 phosphorylation, increased p53-Mdm2 interaction, and consequently increased p53 degradation through an ubiquitin-dependent pathway on nuclear and cytoplasmic 26S proteasomes76, 77. Molecular chaperone TCP1 ring complex/chaperonin containing TCP1 (TRiC/CCT) TRiC/CCT is a cytoplasmic chaperonin that facilitates protein folding and consists of two stacked rings of eight paralogous subunits each (CCT1-CCT8)78. It is known to fold a plethora of substrates, among them cytoskeletal proteins, cell cycle regulatory proteins and proteins involved in oncogenesis79, 80. TRiC/CCT plays a crucial role in the development and progression of cancer80, 81,



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however it is not clear whether this is due to other functions besides its role in protein folding80. TRiC also interacts with proteasomal subunits and ribosomal proteins suggesting a possible crosstalk among protein-synthesis, folding and degradation process82. Furthermore, a large group of protein complexes impacted by TRiC are involved in modifying or remodelling histones, implying that TRiC could perform protein folding/assembly functions inside the nucleus, consistent with reports of nuclear localisation for CCT subunits83. It is noteworthy that all subunits of the complex were immunoprecipitated with hAng in the cytoplasmic fraction (Table 6) and six of them in the nuclear (Table 5). There are many studies in progress for the identification of new TRiC/CCT substrates and co-factors that would reveal its functions in each cellular compartment. The putative interaction of hAng with TRiC/CCT in both the cytoplasm and the nucleus is of great interest. We could suggest that (1) hAng may consist a novel substrate of the complex, (2) TRiC/CCT may assist the assembly of macromolecular complexes that hAng forms with other proteins, and (3) the interaction is maybe due to other functions of TRiC/CCT besides its role as a chaperone. BRCA1-associated genome surveillance complex (BASC)/ MutSα complex BASC complex is a group of proteins that associate with breast cancer type 1 susceptibility protein (BRCA1) and includes tumor suppressors and DNA damage repair proteins (MSH2, MSH6, MLH1, ATM, BLM) as well as the MRN complex (RAD50–MRE11–NBS1)84. The members of the complex that were immunoprecipitated with hAng in the nuclear fraction are: MSH6 (DNA mismatch repair protein Msh6), RAD50 (DNA repair protein RAD50) and MRE11A (Double-strand break repair protein MRE11A) (Table 5). BASC probably serves as a sensor for DNA damage, since all members of this complex have roles in the recognition of abnormal DNA structures or damaged DNA84, implicating that hAng is maybe involved in the maintenance of the DNA integrity. Moreover, MSH6 was immunoprecipitated with hAng in the cytoplasm as well, along with MSH2 (Table 6). While this complex, MutSα (MSH6-MSH2), is formed in the cytoplasm, it is subjected to nuclear translocation in response to DNA damage85, 86. We can assume that hAng could somehow be involved in the


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heterodimerization of MSH6 and MSH2 in the cytoplasm and during the nuclear translocation to be recruited with these factors in the BASC complex. Cytoplasmic only complexes F1FO ΑΤΡ synthase Two subunits of ATP synthase (ATP5A1 and ATP5B) were immunoprecipitated with hAng in the cytoplasmic extract, presenting statistically significant threshold values (Table 6), suggesting thus a strong interaction. Besides the inner membrane of mitochondria, F1FO-ATP synthase is also located on the outside of the plasma membrane of tumor cells as well as some types of normal cells (such as endothelial cells), and the catalytic activity of ectopic ATP synthase is up-regulated in tumor-like microenvironments87. Studies on HUVEC cells have shown that angiostatin, a potent inhibitor of angiogenesis and endothelial cell proliferation, binds the α/β-subunits of ATP synthase on the surface of human endothelial cells88. Endothelial cells are more resistant to hypoxic challenge than other cell types, since they are able to maintain a high level of intracellular ATP89. Binding of angiostatin to the ATP synthase on the cell surface may mediate its antiangiogenic effects, probably by disrupting the production of additional ATP, and down-regulates endothelial cell proliferation and migration88. In our study where an endothelial cell line was used, the putative interaction of hAng with the α/β-subunits of ATP synthase on the cell surface, may result in the synthesis of additional ATP amounts, up-regulating thus endothelial cell proliferation and angiogenesis. RuvBL1/RuvBL2 complex Another complex that forms a large net of protein-protein interactions and was immunoprecipitated with hAng is RuvB-like 1/RuvB-like 2 (RuvBL1/RuvBL2), two highly conserved AAA+ ATPases (Table 6). RuvBL1 and RuvBL2 are involved in a plethora of biological processes90, 91. RuvBL1 and RuvBL2 activities may not always involve co-operation between them and they may also act independently and in opposing fashion, e.g. RuvBL2 shows distinct localization during mitosis91. This is an important observation, since hAng immunoprecipitated RuvBL1/RuvBL2 complex in the



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cytoplasmic extract (Table 6), but only RuvBL2 in the nuclear extract (Table S1). The putative interaction of hAng with RuvBL1/RuvBL2 in the cytoplasm may be related to cell adhesion and cytoskeletal reorganization, two cellular processes that are associated with RuvBL1/RuvBL292, and/or with mRNA decay (nonsense-mediated decay, NMD)93. The putative interaction of hAng with RuvBL1 and RuvBL2 in both the nucleus and the cytoplasm enhances the possibility that hAng is involved in NMD. Moreover, concerning the putative nuclear interaction hAng-RuvBL2, proteins RuvBL1/2 have been found to be part of histone acetyltransferase complex and chromatin remodelling complexes94. On the other hand, hAng, can promote 47S pre-rRNA transcription, by binding the promoter of ribosomal DNA (rDNA) and inducing the assembly of the initiation complex by epigenetic activation through promoter methylation and histone modification. There is also evidence that hAng acts as a chromatin remodeling activator to regulate mRNA transcription3. Since there is no evidence that hAng possesses methyl- or acetyltransferase activities, it is possible that its putative interaction with RuvBL2 may be involved in the recruitment of other modifying enzymes to the DNA. Nuclear only complexes Nucleosome Remodeling Deacetylase (Mi-2/NuRD) The fact that five out of the seven subunits of Mi-2/NuRD (Nucleosome Remodeling Deacetylase) complex (HDAC1, HDAC2, RbAp48, RbAp46 και MTA2) were immunoprecipitated with hAng in the nuclear extract (Table 5) further supports our hypothesis for the implication of hAng in gene expression regulation as a transcription factor. Mi-2/NuRD complex is unique since it couples both histone deacetylation (HDAC1/HDAC2) and chromatin remodeling ATPase activities, playing crucial role in the regulation of gene expression95. Condensin I Two members of the five-membered condensin I complex, SMC4 and NCAPD2 were immunoprecipated with hAng in the nuclear extract (Table 5). The putative interaction of hAng with


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condensin I, suggests potential roles of angiogenin since condensin I plays a central role in mitotic chromosome condensation, a crucial cellular process that ensures the faithful segregation of chromosomes in both meiosis and mitosis96. hAng interacts with PCNA in the cytoplasm of EA.hy926 cells Among the statistically significant proteins, proliferating cell nuclear antigen (PCNA), a factor with critical role in DNA replication in the nucleus, was immunoprecipitated with hAng in the cytoplasmic fraction (Table 2), presenting among the highest confidence threshold values [log2 Fold Difference (difference hAng Ab - Control IgG Ab) ≥ 8 and p-value ≤ 0.05] (Fig.2A). The observation that PCNA showed the highest difference, apart from the bait (hAng), between the “hAng Ab” and the “IgG” groups, together with the unexpected finding that the interaction was observed in the cytoplasmic fraction only, prompted us to further investigate this interaction. To

confirm

the

interaction

between

hAng

and

PCNA,

we

performed

independent

immunoprecipitation experiments with hAng and PCNA antibodies in the two cellular compartments followed by western blot analysis. The results confirmed that endogenous PCNA was immunoprecipitated with the hAng specific antibody in the cytoplasmic fraction of EA.hy926 cells but not in the nuclear one (Fig. 4). However, the lack of interaction in the nuclear fraction could be due to the low levels of PCNA in this compartment (Fig. 5). Moreover, the interaction of hAng with endogenous PCNA in the cytoplasm is specific since there was low nonspecific background with the control IgG (Fig. 4).



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Figure 4. Immunoprecipitation of hAng in the nuclear and cytoplasmic fraction of EA.hy926 cells, with a hAng antibody or control IgG, followed by immunoblotting with hAng and PCNA antibodies.

The converse experiment showed that the PCNA antibody binds PCNA in the cytoplasm of EA.hy926 cells, while no band corresponding to PCNA was observed in the nuclear fragment (Fig. 5). The immunoblot analysis revealed that PCNA was predominantly found in the cytoplasmic fraction, whereas in the nuclear one, a faded band was evident, using equal total protein amounts (20 µg) of each extract (Fig. 5). hAng, was immunoprecipitated with PCNA in the cytoplasmic fraction, while there is a faded band of hAng in the control samples (IgG), probably due to the weak nonspecific binding of PCNA to the control IgG, in the cytoplasm (Fig. 5). The immunoprecipitation experiments supported the MS results, indicating that endogenous PCNA interacts with hAng in the cytoplasm of EA.hy926 cells.

Figure 5. Immunoprecipitation of endogenous PCNA in the nuclear and cytoplasmic fraction of EA.hy926 cells, with a PCNA antibody or control IgG, followed by immunoblotting with hAng and PCNA antibodies. Input: equal amounts of nuclear and cytoplasmic proteins (20 µg each sample) prior to the addition of exogenous hAng, were subjected to immunoblotting with PCNA and hAng antibodies.

We then examined the interaction between endogenous hAng and PCNA in EA.hy926 cells. Immunofluorescence results revealed that significant amounts of PCNA were localized to the cytoplasm of this cell line (Fig. 6), which is in agreement with the in vitro observations. hAng was


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distributed both in nucleus and cytoplasm (Fig. 6). Both proteins exist in the cytoplasm of EA.hy926 cells, and hence this can suggest that they can interact on the level of endogenous proteins in this compartment.

Figure 6. hAng interacts with PCNA in the cytoplasm of EA.hy926 cells. Subcellular localization of endogenous hAng and PCNA. EA.hy926 cells were subjected to immunofluorescence staining with hAng and PCNA antibodies. DAPI was used to stain the nucleus. Bar scale: 20 µm.

Eukaryotic PCNA is a homotrimeric complex with an established role in DNA replication as the processivity factor for DNA polymerases delta and epsilon97. It also serves as a molecular platform that facilitates a plethora of protein–DNA and protein–protein interactions at the replication fork97, most of them mediated through specific binding motifs such as the PIP-box (PCNA-interaction protein box) and the KA-box98, while it is further involved in nucleic acid metabolism, in DNA repair, cell cycle progression, apoptosis, in chromatin assembly and gene transcription99-101. Apart from the certain roles of PCNA in the nucleus which have been established through its interaction partners and are controlled by its specific post-translational modifications, there is a



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significant cytosolic fraction as well, but very little is known about the biological function of PCNA in this compartment98.

Biological role of PCNA in the cytoplasm and potential biological role of the interaction hAngPCNA Studies on neutrophils (non-proliferative cells), provided the initial indications for the biological role of PCNA in the cytoplasm. In neutrophils, PCNA is localized exclusively in the cytoplasm regulating their survival. PCNA exerts antiapoptotic activity by associating to procaspases thus preventing their activation, while when it interacts with the cyclin-dependent kinase inhibitor 1 (p21WAF1/Cip1) it sensitizes cells to apoptosis. Its localization to the cytoplasm involves a nuclear export signal (NES) and only monomeric PCNA can expose this signal during granulocyte differentiation102. Whereas nuclear PCNA functions are tightly linked to its ring-shaped structure (trimer), the neutrophil cytosol contains both trimeric and monomeric PCNA that participate in the regulation of the survival/apoptosis balance102,

103

. Since our results suggest that hAng interacts with PCNA in the

cytoplasm but not in the nucleus, this could be attributed to the recognition of monomeric PCNA by hAng but not of the trimeric form. The study by Naryzhny and Lee104 on cancer cells showed that PCNA interacts in the cytoplasm with 14 proteins. None of them contained the canonical PIP-box, while only five of them contained sequences similar to the KA-box. This is an interesting finding which may indicate that PCNA interactions in the cytoplasm could be slightly different from those in the nucleus. However, it should be noted that many of the known PCNA-binding proteins do not contain either PIP or KA box. Furthermore, it is not known whether PCNA is subjected to different post-translational modifications in the cytoplasm. The study revealed that six out of 14 proteins are involved in the regulation of steps 4–9 in the glycolysis pathway, while PCNA is also associated with cytoskeletal proteins including annexin A2, and oncoproteins including malate dehydrogenase and elongation


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factor EF1A. Thus, cytoplasmic PCNA may be involved in the regulation of the glycolysis pathway, oncogenesis, and cytoskeleton integrity104. Rosental and coworkers, showed that PCNA is highly expressed in cancer cells and is recruited to the NK (natural killer) immunological synapse105. NK cells, one of the main players of the immune system, eliminate rapidly infected or malignantly transformed cells. The natural cytotoxicity receptors (NCRs) NKp30, NKp44, and NKp46 trigger the immune response since they are important mediators of NK cell cytotoxicity106. The study revealed that cytoplasmic PCNA is recruited to the NK immunological synapse upon interaction of target cancer cells with NKp44-expressing NK cells and after uptake into the (NK) cell, binds to the cytoplasmic tail of NKp44 inhibiting NKp44 signaling. Consequently, PCNA promotes cancer survival through immune evasion, by inhibiting NKp44-mediated NK cell attack105. It is noteworthy that hAng might be involved in the suppression of the immune system in tumor microenvironment as well107. The membrane localization of PCNA was reported in the study of Rosental et al. where it has been speculated that the recruitment of PCNA to the cell membrane is facilitated by annexin A2, a membrane-associated protein involved in membrane trafficking and recruitment of proteins to lipid rafts105. Annexin A2 is a Ca2+-dependent phospholipid-binding protein, which associates with the plasma membrane and the endosomal system. It promotes plasmin formation as it exists in a heterotetrameric complex with S100A10/p11 proteins on the plasma membrane that acts as a receptor for tissue plasminogen activator (t-PA)108,

109

. PCNA interacts with annexin A2104,

110

, and protein

S100A6 (S100 Calcium Binding Protein A6)110. Both annexin A2 and S100A6 are expressed in response to tubular injury, and are involved in the recovery of tubular cells in acute renal failure110. In addition, other studies on cancer cells showed that the interaction between S100A6 and annexin A2 promotes or facilitates the translocation of annexin A2 to the cell membrane, and may enhance cell motility probably due to increased tPA (tissue plasminogen activator) activity111.



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Interestingly, apart from PCNA, annexin A2 also interacts with hAng as shown in previous studies109, 112 and was identified as a putative hAng interactor in our study as well (Table S1). hAng interacts with the plasminogen activation system (uPAR, uPA, Annexin A2, and S100-A10) at the cell membranes, promoting cell migration through the proteolytic cleavage of plasminogen to generate plasmin109,

112

. Therefore, the interaction between hAng and PCNA could be involved in

cell migration (Fig. 7). One more common interaction between hAng and PCNA is the tumor suppressor protein p53. PCNA interacts both with p53, promoting its polyubiquitination, and E3 ubiquitin-protein ligase MDM2, regulating thus their stability113. Similarly to PCNA, hAng induces p53 polyubiquitination, promoting p53-MDM2 interaction76. Therefore, an alternative scenario for the interaction between hAng and PCNA could be to get implicated in the inhibition of p53 resulting in apoptosis inhibition and cell survival (Fig. 7). Further studies will elucidate the hAng-PCNA interaction in terms of strength and structure and will place it in the context of the cellular functions induced and regulated by hAng (i.e. angiogenesis).

Figure 7. Schematic representation of the potential biological role of the interaction between hAng and PCNA.


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There is a plethora of proteins, like PCNA, that are distributed both in the nucleus and in the cytoplasm although they have been characterized as nuclear proteins due to their primary role in this compartment. Some of them have been identified as putative hAng interactors from the MS data in the present study (common proteins in cytoplasm/nucleus). At the present work we focused on the hAng-PCNA interaction. Additional experiments could indicate other specific interactions with a nuclear component much like PCNA in the cytoplasm, revealing many possible hAng-involved control pathways.

Conclusions To elucidate the biological function(s) of hAng we focused on its protein interaction network since the identification and validation of new partners at a specific compartment can shed light on the process and regulation of hAng’s subcellular localization, revealing hAng-involved control mechanisms. Thus, in the present study, the first gel-free MS analysis of hAng immunoprecipitates identified many statistically significant potential hAng-interacting proteins in two cellular compartments. The majority of these proteins are parts of multiprotein complexes, involved in crucial biological pathways, and reveal potential novel key roles for hAng. Apart from the protein complexes, the most intriguing finding is that hAng interacts with PCNA in the cytoplasm as revealed by immunoprecipitation, immunoblotting and immunocytochemistry. It is very interesting that in the endothelial cell line EA.hy926, PCNA, a critical nuclear factor, is predominantly localized in the cytoplasm where very little is known about its biological function.

ASSOCIATED CONTENT The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE114 partner repository with the dataset identifier PXD006583 (Project Name: Human



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Angiogenin Cytoplasmic Interactomics) and PXD006584 (Project Name: Human Angiogenin Nuclear Interactions).

Reviewer account details: Human Angiogenin Cytoplasmic Interactomics (PXD006583) Username: [email protected] Password: OFgKG7TZ

Human Angiogenin Nuclear Interactions (PXD006584) Username: [email protected] Password: URhR5pKi

Supporting Information. Experimental details and results. Quantitative difference between specific (hAng IP) and control samples (IgG); Table S1: List of the statistically significant nuclear hAng-co-precipitated proteins identified by LFQ-MS analysis presenting threshold values: log2 Fold Difference ≥ 0.88 and p-value ≤ 0.2; Table S2: List of the statistically significant cytoplasmic hAng-co-precipitated proteins identified by LFQ-MS analysis presenting threshold values: log2 Fold Difference ≥ 1 and p-value ≤ 0.2. Excel file with the peptide lists; Table S3: List of peptides found for the nuclear hAng-co-precipitated proteins listed in Table 1; Table S4: List of peptides found for the cytoplasmic hAng-co-precipitated proteins listed in Table 2. Figure S1: Heat maps of the hierarchical clustering of the hAng-co-precipitated proteins in the two cellular compartments (A: Nuclear and B: Cytoplasmic);


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Acknowledgements This work was supported in part by the Postgraduate Programs ‘‘Biotechnology-Quality assessment in Nutrition and the Environment”, ‘‘Application of Molecular Biology-Molecular GeneticsMolecular Markers”, Department of Biochemistry and Biotechnology, University of Thessaly. D.S.M.C. would also like to acknowledge financial support from the Hellenic State Scholarships Foundation funded by the SIEMENS-Program.

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Proteomic Analysis of Human Angiogenin Interactions Reveals

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