ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)

Biopolymers and Cell. 2018. Vol. 34. N 4. P 292–302

doi: http://dx.doi.org/10.7124/bc.000982

UDC 577.217.535 + 577.322.23

Mass-spectrometric and bioinformatic analysis of eEF1Bγ

interactome in the cytoplasmic fraction of A549 cells
L. M. Kapustian, I. L. Lysetsky, T. V. Bondarchuk, O. V. Novosylna, B. S. Negrutskii
Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143
[email protected]

Aim. To study protein networks containing the translation elongation factor eEF1B gamma
(eEF1Bγ) in lung carcinoma cells. Methods. The protein partners of eEF1Bγ in the cytoplas-
mic fraction of human lung adenocarcinoma A549 cells were identified by co-immunoprecip-
itation (co-IP) followed by subsequent liquid chromatography-tandem mass spectrometry
(LC-MS/MS). The protein interaction network for eEF1Bγ was determined by a Cytoscape
3.2.0 program using a MCODE plugin. Results. 222 high-scored proteins interacting with
eEF1Bγ in the cytoplasm of A549 cells have been identified. Possible functional networks
involving these protein-protein interactions were predicted using bioinformatic approaches.
Conclusions. Five protein networks were identified as possible targets of eEF1Bγ in lung
cancer cells. Apart from translation, eEF1Bγ was shown to be potentially involved in cell
cycle regulation, nucleosome remodeling, transcription, mRNA splicing and processing, and
oxidative stress response.
K e y w o r d s: eEF1Bγ, protein-protein interactions, A549 cells

Introduction cancer tissues [1.2] suggested their non-trans-

lational functioning as well. The nature of
eEF1Bg is a non-catalytic subunit of the these non-canonical functions remains mostly
eEF1B complex responsible for GDP/GTP unidentified.
exchange in translation elongation factor Recently, the information on cellular pro-
eEF1A. Apart from eEF1Bg, eEF1B contains cesses that can engage the oncogenic eEF1Bβ
two catalytic subunits, eEF1Bα and eEF1Bβ. subunit in cancer cells has been obtained [3, 4].
Altogether eEF1A and the eEF1B complex While a direct information about an onco-
provide efficient and accurate translation of genic role of the eEF1Bg subunit is absent,
mRNA on ribosomes in cytoplasm of eukary- there are several reports on its overexpression
otic cells. However, the data on the existence in cancer cells [5–8]. This suggests a possibil-
of a free pool of the eEF1B subunits in human ity of cancer-related functioning of this pro-

© 2018 L. M. Kapustian et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Bio-
polymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium,
provided the original work is properly cited

292
L. M. Kapustian, I. L. Lysetsky, T. V. Bondarchuk
 Mass-spectrometric and bioinformatic analysis of eEF1Bγ interactome in the cytoplasmic fraction of A549 cells

tein. Here, we used the combination of co- total protein) were added to pre-cleared lysates
immunoprecipitation, mass-spectrometry and and incubated overnight at 4 °C. After addition
bioinformatics to investigate a cytoplasmic of Protein G Sepharose slurry the incubation
interactome of the eEF1Bg protein in human persisted for 2 h at 4 °C with continuous shak-
cancer cells. In particular, we aimed to shed ing. Then the resin was washed with ten resin
light on non-translational cancer-related pro- volumes of the lysis buffer and treated further
cesses which can involve eEF1Bg in lung in accordance with the manufacturer’s proto-
cancer. col. After elution of eEF1Bγ-associated pro-
The obtained results show that eEF1Bγ may teins, the 12 % PAGE was performed [9]. The
be involved in cell cycle regulation, nucleo- protein bands of interest were cut and pro-
some remodeling, mRNA splicing and process- cessed for mass-spectrometry analysis
ing, viral mRNA transcription and oxidative (LC‑MS/MS).
stress response in lung cancer cells.
Mass-spectrometry LC-MS/MS
Materials and Methods The cytoplasmic extract incubated with plain
G-Sepharose was used as a control of nonspe-
Preparation of cytoplasmic fraction cific binding. The electrophoretic bands that
of human lung cells were not present in the control or were much
Human lung cancer cells A-549 were har- more extensive than in the control were cut
vested with Trypsin-EDTA. Cytoplasmic and processed for mass spectrometry analysis
fraction was isolated as in [3]. Briefly, the at the Mass Spectrometry Laboratory of the
cells were lysed with 1.5 volume of buffer Institute of Biochemistry and Biophysics
containing 10 mM HEPES pH7.9, 1.5 mM (Warsaw, Poland) using LTQ-Orbitrap Velos
MgCl2, 0.5 % NP-40, 0.2 mM PMSF, 0.5 mM mass spectrometer (Thermo Scientific)
DTT and kept on ice for 20 min. Then, the equipped with nanoAcquity (Waters
cells were centrifuged 10 min at 400 g. The Corporation) LC system, with ions score or
supernatant was subjected to centrifugation expect cut-off, 30 and significance threshold,
at 16000 g for 30 min. The obtained super- p < 0.05, as described before [3, 4].
natant was used as cytoplasmic extract. The
absence of nuclear fraction admixture was Bioinformatics analysis
verified by Western blotting with anti- Cytoscape 3.2.0 Program [10] interaction da-
Poly(ADP-ribose) polymerase and anti-His- tabase BIOGRID was supplemented with
tone 3.3 antibodies. newly identified protein partners of eEF1Bγ
and analyzed by MCODE plugin which finds
Co-immunoprecipitation highly interconnected regions (clusters) in any
The cytoplasmic extract was pre-cleared with network loaded into Cytoscape. These clusters
Protein G Sepharose (Sigma, USA) for 1 h at have been shown to represent protein com-
4 °C. Anti-eEF1Bγ antibodies (Abnova, plexes and/or parts of pathways [11]. For the
Taiwan) (1.5 μg of antibodies per 1 mg of sake of clarity, such known protein partners of

293
L. M. Kapustian, I. L. Lysetsky, T. V. Bondarchuk et al.

eEF1Bγ as eEF1A1, eEF1A2 and UBC (poly- Our study was aimed to examine whether
ubiquitin-C) were excluded from the database these and other cellular processes implicate
as they interact with a huge number of cell eEF1Bγ in lung cancer cells. We identified 222
proteins and create a very complicated network proteins as the interacting partners of eEF1Bγ
of protein-protein interactions that is not as- in the cytoplasm of human lung adenocarci-
sociated with eEF1Bγ directly [3]. Also, we noma A549 cells by using co-immunoprecip-
simplified the task by taking for analysis only itation and subsequent LC-MS/MS identifica-
the first (direct) partners of eEF1Bγ partners tion of the interacting proteins. These protein
determined by the Program algorithm. MCODE partners were used for further analysis by the
analysis was performed on the hybrid super- Cytoscape program to predict functional clus-
computer “SCIT-4” at the Glushkov Institute ters, which may involve these proteins.
of Cybernetics (GIC) of National Academy of First, human BioGRID database was em-
Sciences of Ukraine (http://icybcluster.org.ua). ployed to generate by the MCODE the protein
interaction network of eEF1Bγ based on exist-
Results and Discussion ing (published) data. The resulting network
Literature data indicate that eEF1Bg may have contained 11 proteins (including eEF1Bγ).
connections with different types of cytoske­ Three of them showed direct interactions with
leton [12, 13] and interact with viral compo- eEF1Bγ (Fig. 1). Those are eEF1Bβ (Gene
nents [14, 15], it may be involved in transcrip- ID  1936), FLNC (filamin C, gamma, Gene
tion process [16–18] and oxidative stress re- ID 2318) and NUDT21 (Cleavage and poly-
sponse [19]. adenylation specificity factor subunit 5, Gene

Fig. 1. Protein cluster containing

eEF1Bγ, generated by MCODE in
the Cytoscape 3.2.0 Program from
Human BioGRID database solely on
the basis of literature data.

294
 Mass-spectrometric and bioinformatic analysis of eEF1Bγ interactome in the cytoplasmic fraction of A549 cells

ID 11051). The other members of this protein database with newly identified 222 partners of
network are: SKIV2L2 (Ski2 Like RNA eEF1Bγ. Resulting protein cluster contained
Helicase 2, Gene ID 23517), TERF1 (Telomeric 55 proteins (including eEF1Bγ) (Fig.  2).
repeat-binding factor 1, Gene ID 7014), TAF2 Several functional protein sub-clusters were
(TATA-box binding protein associated factor 2, identified among the main cluster. Sub-
Gene ID 6873), TBP (TATA-box binding pro- cluster A included the proteins associated with
tein, Gene ID 6908), ELF3 (ETS-related tran- mRNA splicing and processing. Sub-cluster B
scription factor Elf-3, Gene ID 1999), JAK1 contained the proteins participating in nucleo-
(Janus kinase 1, Gene ID 3716) and ATIC some remodeling via changes of post-transla-
(5-aminoimidazole-4-carboxamide ribonucle- tional modifications of histones and DNA bind-
otide formyltransferase/IMP cyclohydrolase, ing. The proteins from sub-cluster C were
Gene ID 471). The majority of these proteins involved in cell cycle events. Sub-cluster D
participate in synthesis and degradation of mostly comprised the members of translation
mRNA and its regulation (20–24). apparatus, and sub-cluster E contained the
Second, a protein network was generated transcription factors associated with oxidative
by MCODE after complementing the BioGrid stress response. The detailed analysis of the

C B

Fig. 2. Protein cluster containing

eEF1Bγ, generated by MCODE in
the Cytoscape 3.2.0 Program from
Human BioGRID database supple-
mented with co-IP/MS-MS experi-
D mental data. Sub-clusters: A — the
proteins associated with mRNA
splicing and processing, B — nucleo-
some remodeling which proceeds
via changes in the post-translational
modifications of histones, C — the
proteins involved in cell cycle
E events, D — translation and viral
RNA replication, E – oxidative stress
response.

295
L. M. Kapustian, I. L. Lysetsky, T. V. Bondarchuk et al.

protein components of the sub-clusters is Association of eEF1Bγ with sub-cluster A

shown below. suggests its participation in the mRNA splicing
Sub-cluster A contained 3 experimentally and processing events. Peculiarly, the splicing
identified in this paper protein partners of complexes are normally localized in the nu-
eEF1Bγ — DDX5 (Probable ATP-dependent cleus, whereas the interaction of eEF1Bγ with
RNA helicase DDX5, Gene ID 1655), the members of sub-cluster A is observed in
SYNCRIP (Heterogeneous nuclear ribonu- the cytoplasmic fraction of the cells. One of
cleoprotein Q, Gene ID 10492) and U2AF2 the possible explanation is that eEF1Bγ par-
(Splicing factor U2AF 65 kDa subunit, Gene ticipates in cyto-nuclear transport of the com-
ID 11338) . The proteins CDC5L (Cell divi- ponents of the sub-cluster A. As indicated in
sion cycle 5-like, Gene ID 988), SRRM1 the Materials and Methods section, the absence
(Serine/arginine repetitive matrix 1, Gene of admixture of the nuclear proteins in the
ID 10250), SNRPD1 (Small nuclear ribonu- cytoplasmic fraction was routinely controlled
cleoproteinD1polypeptide 16 RDa, Gene by Western blotting with anti-Poly(ADP-ri-
ID 6632), PRPF19 (Pre-mRNA processing bose) polymerase and anti-Histone 3.3 anti-
factor 19, Gene ID 27339), rev P19 (Protein bodies. This significantly diminishes the pos-
Rev p19, Gene ID 155908) and SNPD2 (Small sibility of artefact presence of the components
nuclear ribonucleoprotein D2 polypeptide of the splicing machinery in cytoplasm.
16.5 kDa, Gene ID 6633) formed the central According to bioinformatics analysis, sub-
core of sub-cluster A. The outer layer of this cluster B is linked to sub-cluster A via several
group contained the proteins HNRNPR proteins: СHD4 (Chromodomain helicase
(Heterogeneous nuclear ribonucleoprotein R, DNA binding protein 4, Gene ID 1108), MTA2
Gene ID 10236), SF3A (Splicing factor 3A, (Metastasis associated 1 family, member 2,
subunit 1, Gene ID 10291), EFTUD2 Gene ID 9219) and MBD3 (Methyl-ChG bind-
(Elongation factor Tu GTP binding domain ing domain protein 3, Gene ID 53615). There
containing 2, Gene ID 9343), SSBP1 (Single are six more proteins in this sub-cluster:
stranded DNA binding protein 1, Gene NR2C1 (Nuclear receptor subfamily 2,
ID 6742), PRPF8 (Pre-mRNA processing fac- group C, member 1, Gene ID 7181), RBBP7
tor 8, Gene ID 10594), PRPF6 (Pre-mRNA (Retinoblastoma Binding Protein 7, Gene
processing factor 6, Gene ID 24148), YBX3 ID  5931), KDM5B (Lysine-specific demeth-
(Y box binding protein 3, Gene ID 8531), ylase 5B, Gene ID 10765), MTA3 (metastasis
TADA2A (Transcriptional adaptor 2A, Gene associated 1 family, member 3, Gene
ID 6871), SYNCRIP (Heterogeneous nuclear ID 57504), GATAD2A (GATA zinc finger do-
ribonucleoprotein Q, Gene ID 10492), U2AF2 main containing 2A, Gene ID 54815), and
(U2 small nuclear RNA auxiliary factor 2, SOX2 (SRY (Sex determining region Y)-box
Gene ID 11338), DDX5 (Probable ATP- 2, Gene ID 6657). The main function of sub-
dependent RNA helicase DDX5, Gene cluster B is suggested to be a nucleosome re-
ID 1655), and C14orf166 (Chromosome 14 modeling which proceeds via changes in the
Open Reading Frame 166, Gene ID 51637). post-translational modifications of histones.

296
 Mass-spectrometric and bioinformatic analysis of eEF1Bγ interactome in the cytoplasmic fraction of A549 cells

Thus, eEF1Bγ can be possibly involved in The protein MAPK14 (mitogen-activated

controlling the pre-translational gene expres- protein kinase 14, Gene ID1432) links sub-
sion events. clusters C and D. This kinase belongs to the
The proteins SOX2 and MTA3 function- family of MAP-kinases, which integrate ubiq-
ally link sub-clusters B and C. The core of uitous cellular signals and participate in regu-
sub-cluster C contains five proteins: CCNB1 lation of transcription, differentiation, prolif-
(cyclin B1, Gene ID 891), CDC16 (Cell eration and cell development. MAPK14 is
Division Cycle 16, Gene ID 8881), ANAPC1 implicated in the control of the genotoxic
(Anaphase Promoting Complex Subunit 1, stress response and in the stress-induced tran-
Gene ID 64682), ANAPC4 (Anaphase scription and cell cycle regulation [26].
Promoting Complex Subunit 4, Gene According to the scheme, in sub-cluster D this
ID 29945), and CDC27 (Cell Division protein interacts with SKP1 (S-Phase Kinase-
Cycle 27, Gene ID 996). All these proteins, Associated Protein1, Gene ID 6500), which
except cyclin B1, are the components of the participates in regulation of ubiquitination, and
Anaphase Promoting Complex (APC), which KARS (lysyl-tRNA synthetase, Gene ID 3735).
maintains the metaphase–anaphase transition KARS is associated with protein biosynthesis
due to the degradation of cyclin B1. Cyclin and also interacts with gag Pr55 (Gag poly-
B1 is associated with G2/M transition of mi- protein (Human immunodeficiency virus 1),
totic cell cycle. Other two proteins of sub- Gene ID 155030). KARS interaction with gag
cluster C are TP53BP1 (Tumor Protein P53 Pr55 assists effective packaging of tRNA3Lys,
Binding Protein 1, Gene ID 7158) and MDC1 which works as a primer for initiation of the
(Mediator of DNA-Damage Checkpoint 1, reverse transcription, to viral particles [27].
Gene ID 9656). The last one is connected with The protein AIMP2 (Aminoacyl tRNA
sub-cluster A by two links. Importantly, pos- Synthetase Complex-Interacting
sible association of eEF1Bγ with the cell Multifunctional Protein 2, Gene ID 7965) from
cycle regulation components has been re- sub-cluster D interacts with KARS and LARS
ported [25]. (Leucyl-TRNA Synthetase, Gene ID 51520)
The protein FBXO5 (F- box protein 5, Gene in the macromolecular aminoacyl-tRNA syn-
ID 26271), which can inhibit APC complex thetase complex. Interestingly, another com-
due to its ubiquitin ligase activity, connects ponent of HIV-1 virus, the protein rev p19, is
sub-cluster C with sub-clusters A and D also found in the interactome of eEF1Bγ (sub-
through SKP1 (S-phase kinase-associated pro- cluster A). Thus, on the non-translational side,
tein 1, Gene ID 6500). The SKP1 protein takes the data on sub-clusters D and A provide in-
part in ubiquitination of FBXW4 (F-Box and dependent support to the notion that eEF1Bγ
WD Repeat Domain Containing 4, Gene can take part in functioning of HIV-1 virus
ID 6468), which in turn interacts with two [14]. In particular, it may play a role of struc-
proteins of sub-cluster A, one of them (DDX5) tural platform for spatial immobilization of
was experimentally identified as a partner of some viral and host-cell proteins leading to
eEF1Bγ in this paper. more effective viral replication.

297
L. M. Kapustian, I. L. Lysetsky, T. V. Bondarchuk et al.

The protein PCK1 (Phosphoenolpyruvate Inducible Factor 1, Alpha Subunit (Basic

Carboxykinase 1, Gene ID 5105) connects Helix-Loop-Helix Transcription Factor, Gene
sub-clusters A, B, C, and E. This protein is at ID 3091), a subunit of transcription factors
the main checkpoint in control of gluconeo- complex HIF-1, which is a main regulator of
genesis. PCK1 is linked to sub-cluster A via anti-hypoxic homeostatic cell response. This
the protein HSPA1L (Heat Shock 70kDa factor is responsible for the transcription acti-
Protein 1-Like, Gene ID 5105). PCK1 is con- vation of many genes involved in energetic
nected to sub-claster B through the protein metabolism, angiogenesis, apoptosis as well
C1QBP (Complement Component 1, Q Sub­ as of the genes that product an enhanced oxy-
com­ponent Binding Protein, Gene ID 708), a gen transition or raise anti-hypoxic metabolic
multifunctional protein, associated with in- adaptation level [30]. The protein EPAS1
flammation and infection processes, apoptosis, (Endothelial PAS Domain Protein 1, Gene
transcription and pre-mRNA splicing regula- ID 2034) is a link between HIF1A and ELOB.
tion, and ribosome biogenesis [28]. C1QBP This protein is a transcription factor associ-
interacts with the protein SOX2 from sub- ated with induction of the genes regulated by
cluster B that links sub-clusters C and B. It oxygen. The protein FEM1B (Fem-1
also interacts with the protein YBX3 (Y Box Homolog B, Gene ID 10116) belongs to a
Binding Protein 3, Gene ID 8531) from sub- family of death receptor-associated proteins
cluster A, which can act as a transcription essential for apoptosis. Thus, sub-cluster E
factor specifically binding certain DNA se- mainly contains the transcription factors par-
quences, in particular ds-DNA. PCK1 is linked ticipating in the induction of the oxidative
to sub-cluster E through interaction with the stress response, which can play adaptive or
proteins ELOC (Elongin-C, Gene ID 6921) apoptotic role.
and ELOB (Elongin-B, Gene ID 6923). ELOC Several papers suggested possible participa-
and ELOB are the regulatory subunits of the tion of eEF1Bγ in oxidative stress response
transcription factor B (SIII) complex, which [19, 31, 32], however the details of such con-
activates RNA-polymerase II–mediated tran- nection were not presented. Our data predict
scription elongation. Interestingly, eEF1Bγ existence of the direct links between eEF1Bγ
was reported to interact with RNA-poly­ and transcription factors involved in induction
merase II [16]. of this response. This connection can be used
Sub-cluster E also includes LRR1 (Leucine- as a new target for drugs with a capacity of
rich repeat protein 1, Gene ID 122769), a jugulating oxidative stress response in human
negative regulator of 4-1BB-mediated signal- cells.
ing, which leads to the NK-kappaB and JNK1 Translation initiation factor eIF1B (sub-
activation [29]. An independent study showed cluster D) interacts with transcription factor
that eEF1Bγ is a positive regulator of NF- ELOC (sub-cluster E) and with translation
кappaB signaling pathway, with unknown initiation factor eIF3L (Eukaryotic Translation
mechanism of action [17]. Another component Initiation Factor 3, Subunit L, Gene ID 51386).
of this sub-cluster E is HIF1A (Hypoxia Factor eIF3L interacts with eEF1Bγ closing

298
 Mass-spectrometric and bioinformatic analysis of eEF1Bγ interactome in the cytoplasmic fraction of A549 cells

the loop of interactions in sub-cluster D. On Conclusions

the other hand, eIF3L interacts with TADA2A 222 proteins were identified by co-immuno-
(sub-cluster A) which is a member of the his- precipitation, with subsequent LC-MS-MS, as
tone-acetylase complex PCAF. It is one more interacting with eEF1Bγ in the cytoplasm of
link that connects eEF1Bγ with sub-cluster A, human lung adenocarcinoma A549 cells. The
the function of which is associated with the eEF1Bγ protein partners were used to construct
nucleosome structure modification. possible functional networks by the Cytoscape
Several identified protein partners of 3.2.0 program. We identified five protein net-
eEF1Bγ are associated with human diseases. works (sub-clusters), which can involve
The proteins CCNB1 and FBXO5 from sub- eEF1Bγ in human cancer cells. They are linked
cluster C are associated with tetraploidy [33]. to mRNA splicing and processing; nucleosome
The proteins CCNB1, MDC1 and MTA3 (sub- remodeling; cell cycle regulation; viral RNA
cluster C) are linked to breast cancer [34–36]. transcription; oxidative stress response. Thus,
The protein PRPF8 (sub-cluster A) is related our data support and detail the previously re-
to myeloid neoplasms [37]. The proteins ported cases of eEF1Bγ linkage to the pro-
CDC5L and PRPF19 from sub-cluster A par- cesses of cell cycle, transcription of viral
ticipate in the development of poikiloderma RNAs and oxidative stress response. Moreover,
with neutropenia [38]. The proteins CDC27 they indicate, for the first time, a possible
(sub-cluster C) and SSBP1 (sub-cluster A) are participation of eEF1Bγ in the mRNA matura-
associated with herpes infection [39,40]. The tion and nucleosome remodeling in human
proteins MAPK14 (sub-cluster D) and HIF1A cancer cells.
(sub-cluster E) are involved in vascular disease
[41, 42]. DDX5 (sub-cluster A) participates in Acknowledgments
the necrosis development [43]. This work was partially financed by the
Surprisingly, seven proteins from sub-clus- Interdisciplinary Program of Scientific re-
ters A, B, C and protein PCK1, which connects search of NAS of Ukraine “Molecular and
these sub-clusters, are involved in corneal cell biotechnologies for medicine, industry
diseases, particularly retinoblastoma. It opens and agriculture” and Collaborative Program
an interesting possibility of the eEF1Bγ in- of NAS of Ukraine and PAN 2018–2020. The
volvement in the development of human cor- financing by Ministry of Science and
neal diseases. Education of Ukraine and Ministry of Science
As eEF1Bγ has no evident catalytic activ- and High Education of Poland is appreciated.
ity, we suggest that the potential molecular We thank Prof. M. Dadlez for an expert help
mechanisms of its participation in non-trans- in performing MS analysis. We appreciate
lational events could involve its ability to form the contribution of V. Zakon and Dr. I.
multimeric structures. In such way, eEF1Bγ Groisman to the categorization of MS data.
can serve as a scaffold for various protein We are grateful to M. M. Ilchenko for valu-
partners participating in a variety of cellular able advices.
processes.

299
L. M. Kapustian, I. L. Lysetsky, T. V. Bondarchuk et al.

10. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT,

References Ramage D, Amin N, Schwikowski B, Ideker T. Cy-
1. Veremieva M, Khoruzhenko A, Zaicev S, Ne- toscape: a software environment for integrated mod-
grutskii B, El’skaya A. Unbalanced expression of els of biomolecular interaction networks. Genome
the translation complex eEF1 subunits in human Res. 2003;13(11):2498–504.
cardioesophageal carcinoma. Eur J Clin Invest. 11. Bader GD, Hogue CW. An automated method for
2011;41(3):269–76. finding molecular complexes in large protein inter-
2. Veremieva M, Kapustian L, Khoruzhenko A, action networks. BMC Bioinformatics. 2003;4:2.
Zakharychev V, Negrutskii B, El’skaya A. Indepen- 12. Serpinskaya AS, Tuphile K, Rabinow L, Gelfand VI.
dent overexpression of the subunits of translation Protein kinase Darkener of apricot and its substrate
elongation factor complex eEF1H in human lung EF1γ regulate organelle transport along microtu-
cancer. BMC Cancer. 2014;14:913. bules. J Cell Sci. 2014;127(Pt 1):33–9.
3. Kapustian LM, Dadlez M, Negrutskii BS. Non-ca- 13. Kim S, Kellner J, Lee CH, Coulombe PA. Interaction
nonical interactions of the β subunit of the transla- between the keratin cytoskeleton and eEF1Bgamma
tion elongation complex eEF1B and analysis of their affects protein synthesis in epithelial cells. Nat
possible functional role. Biopolym Cell. 2016; Struct Mol Biol. 2007;14(10):982–3.
32(5):347–58. 14. Warren K, Wei T, Li D, Qin F, Warrilow D, Lin MH,
4. Kapustian LM, Dadlez M, Negrutskii BS. Protein Sivakumaran H, Apolloni A, Abbott CM, Jones A,
partners of the eEF1Bβ subunit of the translation Anderson JL, Harrich D. Eukaryotic elongation
elongation complex eEF1B in the nuclear fraction factor 1 complex subunits are critical HIV-1 reverse
of human lung carcinoma cells. Biopolym. Cell. transcription cofactors. Proc Natl Acad Sci U S A.
2017; 33(4):243–55. 2012;109(24):9587–92.
5. Shuda M, Kondoh N, Tanaka K, Ryo A, Wakatsuki T, 15. Zhang Z, Pan L, Ding Y, Lv J, Zhou P, Fang Y, Liu X,
Hada A, Goseki N, Igari T, Hatsuse K, Aihara T, Zhang Y, Wang Y. eEF1G interaction with foot-and-
Horiuchi S, Shichita M, Yamamoto N, Yamamoto M. mouth disease virus nonstructural protein 2B: Iden-
Enhanced expression of translation factor mRNAs tification by yeast two-hybrid system. Microb Pat-
in hepatocellular carcinoma. Anticancer Res. hog. 2017;112:111–116.
2000;20(4):2489–94. 16. Corbi N, Batassa EM, Pisani C, Onori A, Di Cer-
6. Lew Y, Jones DV, Mars WM, Evans D, Byrd D, to MG, Strimpakos G, Fanciulli M, Mattei E, Pas-
Frazier ML. Expression of elongation factor-1 gam- sananti C. The eEF1γ subunit contacts RNA poly-
ma-related sequence in human pancreatic cancer. merase II and binds vimentin promoter region. PLoS
Pancreas. 1992;7(2):144–52. One. 2010;5(12):e14481.
7. Mimori K, Mori M, Tanaka S, Akiyoshi T, Sugima- 17. Liu D, Sheng C, Gao S, Jiang W, Li J, Yao C,
chi K. The overexpression of elongation factor Chen H, Wu J, Chen S, Huang W. eEF1Bγ is a
1 gamma mRNA in gastric carcinoma. Cancer. positive regulator of NF-кB signaling pathway.
1995;75(6 Suppl):1446–9. Biochem Biophys Res Commun. 2014;446(2):523–8.
8. Chi K, Jones DV, Frazier ML. Expression of an 18. Pisani C, Onori A, Gabanella F, Delle Monache F,
elongation factor 1 gamma-related sequence in ad- Borreca A, Ammassari-Teule M, Fanciulli M, Gra-
enocarcinomas of the colon. Gastroenterology. zia Di Certo M, Passananti C, Corbi N. eEF1Bγ
1992;103(1):98–102. binds the Che-1 and TP53 gene promoters and their
9. Kang D-H, Gho Y-S, Suh M-K, Kang C-H. Highly transcripts. J Exp Clin Cancer Res. 2016; 35(1):146.
sensitive and fast protein detection with coomassie 19. Olarewaju O, Ortiz PA, Chowdhury WQ, Chatter-
brilliant blue in sodium dodecyl sulfate-polyacryl- jee I, Kinzy TG. The translation elongation factor
amide gel electrophoresis. Bull Korean Chem Soc. eEF1B plays a role in the oxidative stress response
2002; 23(11):1511–12. pathway. RNA Biol. 2004;1(2):89–94.

300
 Mass-spectrometric and bioinformatic analysis of eEF1Bγ interactome in the cytoplasmic fraction of A549 cells

20. Brumbaugh J, Di Stefano B, Wang X, Borkent M, 30. Ziello JE, Jovin IS, Huang Y. Hypoxia-Inducible
Forouzmand E, Clowers KJ, Ji F, Schwarz BA, Factor (HIF)-1 regulatory pathway and its potential
Kalocsay M, Elledge SJ, Chen Y, Sadreyev RI, for therapeutic intervention in malignancy and isch-
Gygi SP, Hu G, Shi Y, Hochedlinger K. Nudt21 emia. Yale J Biol Med. 2007;80(2):51–60.
Controls Cell Fate by Connecting Alternative Poly- 31. Esposito AM, Kinzy TG. The eukaryotic translation
adenylation to Chromatin Signaling. Cell. elongation Factor 1Bgamma has a non-guanine
2018;172(1–2):106-120.e21. nucleotide exchange factor role in protein metabo-
21. Fan J, Kuai B, Wu G, Wu X, Chi B, Wang L, Wang K, lism. J Biol Chem. 2010;285(49):37995–8004.
Shi Z, Zhang H, Chen S, He Z, Wang S, Zhou Z, 32. O’Keeffe G, Jöchl C, Kavanagh K, Doyle S. Exten-
Li G, Cheng H. Exosome cofactor hMTR4 competes sive proteomic remodeling is induced by eukaryotic
with export adaptor ALYREF to ensure balanced translation elongation factor 1Bγ deletion in Asper-
nuclear RNA pools for degradation and export. gillus fumigatus. Protein Sci. 2013;22(11):1612–22.
EMBO J. 2017;36(19):2870–2886. 33. Lehman NL, Verschuren EW, Hsu JY, Cherry AM,
22. Feigerle JT, Weil PA. The C Terminus of the RNA Jackson PK. Overexpression of the anaphase pro-
Polymerase II Transcription Factor IID (TFIID) moting complex/cyclosome inhibitor Emi1 leads to
Subunit Taf2 Mediates Stable Association of Subunit tetraploidy and genomic instability of p53-deficient
Taf14 into the Yeast TFIID Complex. J Biol Chem. cells. Cell Cycle. 2006;5(14):1569–73.
2016;291(43):22721–22740. 34. Ding K, Li W, Zou Z, Zou X, Wang C. CCNB1 is a
23. Teves SS, An L, Bhargava-Shah A, Xie L, Darzacq X, prognostic biomarker for ER+ breast cancer. Med
Tjian R. A stable mode of bookmarking by TBP Hypotheses. 2014;83(3):359–64.
recruits RNA polymerase II to mitotic chromo- 35. Zou R, Zhong X, Wang C, Sun H, Wang S, Lin L,
somes. Elife. 2018;7. pii: e35621. Sun S, Tong C, Luo H, Gao P, Li Y, Zhou T, Li D,
24. Hubbard SR. Mechanistic Insights into Regulation Cao L, Zhao Y. MDC1 Enhances Estrogen Receptor-
of JAK2 Tyrosine Kinase. Front Endocrinol (Lau- mediated Transactivation and Contributes to Breast
sanne). 2018;8:361. Cancer Suppression. Int J Biol Sci. 2015;11(9):992–
25. Yu L, Peña Castillo L, Mnaimneh S, Hughes TR, 1005.
Brown GW. A survey of essential gene function in 36. Zhang H, Stephens LC, Kumar R. Metastasis tumor
the yeast cell division cycle. Mol Biol Cell. antigen family proteins during breast cancer progres-
2006;17(11):4736–47. sion and metastasis in a reliable mouse model for
26. Bulavin DV, Higashimoto Y, Popoff IJ, Gaarde WA, human breast cancer. Clin Cancer Res.
Basrur V, Potapova O, Appella E, Fornace AJ Jr. 2006;12(5):1479–86.
Initiation of a G2/M checkpoint after ultraviolet 37. Kurtovic-Kozaric A, Przychodzen B, Singh J, Konar-
radiation requires p38 kinase. Nature. ska MM, Clemente MJ, Otrock ZK, Nakashima M,
2001;411(6833):102–7. Hsi ED, Yoshida K, Shiraishi Y, Chiba K, Tanaka H,
27. Kleiman L, Jones CP, Musier-Forsyth K. Formation Miyano S, Ogawa S, Boultwood J, Makishima H,
of the tRNALys packaging complex in HIV-1. FEBS Maciejewski JP, Padgett RA. PRPF8 defects cause
Lett. 2010;584(2):359–65. missplicing in myeloid malignancies. Leukemia.
28. McGee AM, Douglas DL, Liang Y, Hyder SM, Ba- 2015;29(1):126–36.
ines CP. The mitochondrial protein C1qbp promotes 38. Shchepachev V, Wischnewski H, Missiaglia E, Sone-
cell proliferation, migration and resistance to cell son C, Azzalin CM. Mpn1, mutated in poikiloderma
death. Cell Cycle. 2011;10(23):4119–27. with neutropenia protein 1, is a conserved 3’-to-5’
29. Jang LK, Lee ZH, Kim HH, Hill JM, Kim JD, RNA exonuclease processing U6 small nuclear
Kwon BS. A novel leucine-rich repeat protein RNA. Cell Rep. 2012;2(4):855–65.
(LRR‑1): potential involvement in 4-1BB-mediated 39. Sun Z, Xiao B, Jha HC, Lu J, Banerjee S, Robert-
signal transduction. Mol Cells. 2001;12(3):304–12. son ES. Kaposi’s sarcoma-associated herpesvirus-

301
L. M. Kapustian, I. L. Lysetsky, T. V. Bondarchuk et al.

encoded LANA can induce chromosomal instability найдено п’ять білкових мереж, у яких може брати
through targeted degradation of the mitotic check- участь eEF1Bγ в клітинах аденокарциноми легені
point kinase Bub1. J Virol. 2014;88(13):7367–78. людини. Показано, що крім трансляційних компонен-
40. Nicolas A, Alazard-Dany N, Biollay C, Arata L, тів, ці мережі формуються білками, задіяними у регу-
Jolinon N, Kuhn L, Ferro M, Weller SK, Epstein AL, ляції клітинного циклу, ремоделюванні нуклеосом,
Salvetti A, Greco A. Identification of rep-associated транскрипції, сплайсингу і процесінгу мРНК та клі-
factors in herpes simplex virus type 1-induced тинної відповіді на оксидативний стрес.
adeno-associated virus type 2 replication compart- К л ю ч о в і с л о в а: eEF1Bγ, білок-білкові взаємодії,
ments. J Virol. 2010;84(17):8871–87. клітини А549.
41. Waterworth DM, Li L, Scott R, Warren L, Gillson C,
Aponte J, Sarov-Blat L, Sprecher D, Dupuis J, Rei­ Мас-спектрометрический
ner A, Psaty BM, Tracy RP, Lin H, McPherson R, и биоинформационный анализ интерактома
Chissoe S, Wareham N, Ehm MG. A low-frequency eEF1Bγ в цитоплазматической фракции
variant in MAPK14 provides mechanistic evidence клеток A549
of a link with myeloperoxidase: a prognostic car-
diovascular risk marker. J Am Heart Assoc. Л. М. Капустян, И. Л. Лисецкий, Т. В. Бондарчук,
2014;3(4). pii: e001074. А. В. Новосильная, Б. С. Негруцкий
42. Semenza GL. Hypoxia-inducible factor 1 and car- Цель. Выявить белковые сети, членом которых может
diovascular disease. Annu Rev Physiol. 2014;76: быть фактор элонгации трансляции eEF1Bγ в клетках
39–56. карциномы легкого. Методы. С помощью ко-иммуно-
43. Dai TY, Cao L, Yang ZC, Li YS, Tan L, Ran XZ, Shi преципитации с последующей жидкостной хромато-
CM. P68 RNA helicase as a molecular target for графией и тандемной масс-спектрометрией были иден-
cancer therapy. J Exp Clin Cancer Res. 2014;33:64. тифицированы белки-партнеры eEF1Bγ в цитоплазма-
тической фракции клеток аденокарциномы легкого
Мас-спектрометричний та біоінформаційний людини А549. Белковые сети, в состав которых входит
аналіз інтерактома eEF1Bγ в цитоплазматичній eEF1Bγ, определяли с помощью программы Cytoscape
фракції клітин A549 3.2.0 с плагином MCODE. Результаты. Иденти­фи­ци­
Л. М. Капустян, І. Л. Лисецький, Т. В. Бондарчук, ро­ваны 222 белка-партнера eEF1Bγ в цитоплазмати-
О. В. Новосильна, Б. С. Негруцький ческой фракции клеток А549. Функциональные сети,
которые могут формироваться этими белками, были
Мета. Виявити білкові мережі, до яких може входити
определены биоинформатически. Выводы. На осно-
фактор елонгації трансляції eEF1Bγ в клітинах карци-
вании экспериментальных данных найдено пять бел-
номи легені. Методи. Білки-партнери eEF1Bγ у цито-
кових сетей, в которых может участвовать eEF1Bγ в
плазматичній фракції клітин аденокарциноми легені
клетках аденокарциномы легкого человека. Показано,
людини А549 були ідентифіковані за допомогою ко-
что кроме трансляционных компонентов, эти сети
іммунопреципітації із наступною рідинною хромато-
формируются белками, задействованными в регуляции
графією та тандемною мас-спектрометрією (LC-MS/
клеточного цикла, ремоделировании нуклеосом, транс-
MS). Білкові мережі, до яких входить eEF1Bγ, визна-
крипции, сплайсинга и процессинга мРНК и клеточ-
чали за допомогою програми Cytoscape 3.2.0 із плагі-
ного ответа на оксидативный стресс.
ном MCODE. Результати. Ідентифіковано 222 білки-
партнери eEF1Bγ в цитоплазматичній фракції клітин К л ю ч е в ы е с л о в а: eEF1Bγ, белок-белковые взає-
А549. Функціональні мережі, які можуть формуватися модействия, клетки А549.
цими білками, були визначені біоінформатично.
Висновки. На основі експериментальних даних ви- Received 01.04.2018

302