Brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) is predicted to interact with its partner through an ARM-type α-helical structure
© Chen et al.; licensee BioMed Central Ltd. 2014
Received: 23 April 2014
Accepted: 30 June 2014
Published: 6 July 2014
Brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) has been identified recently as a novel regulator of estrogen signalling in breast cancer cells. Despite being a potential target for new breast cancer treatment, its amino acid sequence suggests no association with any well-characterized protein family and provides little clues as to its molecular function. In this paper, we predicted the structure, function and interactions of BIG3 using a range of bioinformatic tools.
Homology search results showed that BIG3 had distinct features from its paralogues, BIG1 and BIG2, with a unique region between the two shared domains, Sec7 and DUF1981. Although BIG3 contains Sec7 domain, the lack of the conserved motif and the critical glutamate residue suggested no potential guaninyl-exchange factor (GEF) activity. Fold recognition tools predicted BIG3 to adopt an α-helical repeat structure similar to that of the armadillo (ARM) family. Using state-of-the-art methods, we predicted interaction sites between BIG3 and its partner PHB2.
The combined results of the structure and interaction prediction led to a novel hypothesis that one of the predicted helices of BIG3 might play an important role in binding to PHB2 and thereby preventing its translocation to the nucleus. This hypothesis has been subsequently verified experimentally.
KeywordsBreast cancer Estrogen receptor-alpha BIG3 PHB2 Protein-protein interaction Bioinformatics
Breast cancer is the most common cancer among women worldwide . The majorities of breast cancers are estrogen receptor-alpha (ERα) positive and depend on the hormone estrogen for growth. Estradiol (E2) is known to induce cell proliferation by binding to ERα, resulting in the transcriptional activation of its downstream genes [2, 3]. Antagonists to ERα such as tamoxifen can block the effects of E2 on breast cancer cells and thereby interfere with estrogen-induced cell proliferation. Although tamoxifen has been a great success and improves breast cancer survival rates considerably [4–6], a significant proportion of ERα-positive breast cancer is tamoxifen-unresponsive, and tamoxifen-resistant cases have been also reported [7, 8]. The mechanism of E2/ERα signalling is not fully understood and a better understanding of the E2/ERα pathway will be essential for more effective and alternate treatments for breast cancer.
Recently, genome-wide profiling of gene expression in breast cancer cells has identified a novel regulator of E2/ERα signalling, brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3). BIG3 has been shown to be over-expressed in breast cancer cells but hardly detectable in normal human tissues . Small-interfering RNA (siRNA)-mediated knockdown of BIG3 was shown to suppress the growth of breast cancer cells significantly . Co-immunoprecipitation and immuno-blotting assays have shown that BIG3 interacts with prohibitin 2 (PHB2), a protein that can repress the activity of ER. PHB2 was shown to be localized mainly in the cytoplasm . When BIG3 is absent, E2 stimulation causes the translocation of PHB2 to the nucleus and results in the suppression of the ERα transcriptional activity. On the other hand, when BIG3 is over-expressed, PHB2 remains in the cytoplasm even with estrogen treatment and it has been shown that the intracellular localization of PHB2 is dependent on its interaction with BIG3 in the cytoplasm. Therefore, the current hypothesis is that BIG3 interacts with PHB2 and traps it in the cytoplasm and thereby prevents its nuclear translocation, resulting in increases in the transcriptional activities of ERα.
This novel mechanism of ERα regulation by BIG3 has the potential to offer molecular details of signalling events in ERα-positive breast cancer cells and can lead to new ways of therapeutic intervention. The progress has been hampered, however, by the lack of information about molecular functions of BIG3. The BIG3 protein consists of 2177 amino acid residues and its sequence suggests no association with any well-characterized protein family and provides little clues as to its molecular function. Although a series of co-immunoprecipitation assays identified residues 86-434 to be responsible for the binding of BIG3 to PHB2, further attempts at narrowing down the binding region or any other biochemical characterization had been unsuccessful until computational predictions, described in this paper, were made and subsequently verified experimentally .
In this paper, we describe details of our predictions for the structure, function and interactions of BIG3 using state-of-the-art bioinformatic tools. The prediction of protein interaction sites, supported by consistent fold recognition results, led to a specific hypothesis about the nature of the molecular interactions between BIG3 and PHB2, which was a key to the successful experimental verification studies.
Results and discussion
BIG3 has features distinct from BIG1 and BIG2
BIG3 is likely to adopt α-helical repeat structures similar to that of the armadillo (ARM) family
The ARM repeat, first discovered in armadillo gene of Drosophila, is an approximately 40 amino acid long tandem repeat, forming a super-helix of helices. Proteins in the ARM family are known to function in various processes, including cytoskeletal regulation, signalling, tumor suppression and nuclear translocation. It has been proposed that ARM may mediate protein-protein interactions but currently, no typical feature of target proteins is known. Of particular note is that the nuclear transport protein importin, known to recognize nuclear localization signals (NLSs), is a member of the ARM family. Given its predicted structure, BIG3 might also bind to its partners in a similar manner (see below).
Prediction of protein binding sites suggested how BIG3 could possibly inhibit the nuclear translocation of PHB2
To pursue this possibility further, we attempted to predict protein-binding sites on BIG3 using PSIVER  and examined the results within the predicted ARM repeats, as these repeats fell within residues 1-250, a region that had been shown experimentally to be responsible for the binding of BIG3 to PHB2 .
PHB2 is known to be involved in several biological processes and found in different cellular compartments, including the nucleus, mitochondria and cell membrane [37–40]. Although the mechanism of translocation of PHB2 is still unclear, one possibility is that it is mediated by importin (or importin-like proteins), and BIG3 could possibly dislocate importin and interact with PHB2, preventing it from being transported to the nucleus. In light of this hypothesis, it is highly suggestive that BIG3 is predicted to adopt the same fold as that of importin.
Based on the differences in sequence and the lack of conserved motif in the Sec7 domain, BIG3 was shown to have distinct features from its paralogues BIG1 and BIG2. Structural analysis showed that BIG3 would adopt α-helical repeat structures similar to that of the ARM family. Prediction of interaction sites between BIG3 and PHB2 provided a new insight into how BIG3 would interfere the translocation of PHB2 and suggested a specific, testable hypothesis.
Protein sequences of BIG3 [Swiss-Prot:Q5TH69] and PHB2 [Swiss-Prot:Q99623] were retrieved from Uniprot. Pfam (http://pfam.xfam.org/) and SMART (http://smart.embl-heidelberg.de/) searches were performed using their web servers. BLAST was run on the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST/) using default parameters. A multiple sequence alignment of the Sec7 domains of BIG1 [Swiss-Prot:Q9Y6D6], BIG2 [Swiss-Prot:Q9Y6D5], ARNO [Swiss-Prot:Q99418], GBF1 [Swiss-Prot:Q92538], GRP1 [Swiss-Prot:O43739], GNOM [Swiss-Prot:Q42510], GEP100 [Swiss-Prot:Q6ND90] and BIG3 was generated using CLUSTALW and formatted by Jalview . A multiple sequence alignment of the N-terminal portions of BIG3 and its homologues was generated by MAFFT version 7 (http://mafft.cbrc.jp/alignment/server/). The sequences included were human BIG1 [Swiss-Prot:Q9Y6D6] and BIG2 [Swiss-Prot:Q9Y6D5], mouse BIG1 [Swiss-Prot:G3X9K3], BIG2 [Swiss-Prot:A2A5R2] and BIG3 [Swiss-Prot:Q3UGY8], rat BIG1 [Swiss-Prot:D4A631] and BIG2 [Swiss-Prot:Q7TSU1] and bovine BIG1 [Swiss-Prot:O46382].
Secondary structure was predicted by using a local installation of PSIPRED  with the default script. Disordered regions were predicted using both POODLE-L and POODLE-W on the POODLE server (http://mbs.cbrc.jp/poodle/index.html) and PrDOS (http://prdos.hgc.jp/index.html). Coiled-coil was predicted using Paircoil2 (http://groups.csail.mit.edu/cb/paircoil2/)  and COILS (http://www.ch.embnet.org/software/COILS_form.html) . Sequence repeats were predicted using REP (http://www.bork.embl.de/~andrade/papers/rep/search.html) , HHrep (http://toolkit.tuebingen.mpg.de/hhrep)  and REPRO (http://www.ibi.vu.nl/programs/reprowww/) . Fold recognition was performed using FUGUE (http://tardis.nibio.go.jp/fugue/) and HHpred (http://toolkit.tuebingen.mpg.de/hhpred/, with the HMM database of pdb70_18Dec10)  using the three segments defined in Figure 1 as queries.
Interaction site prediction
Interaction sites on BIG3 and PHB2 were predicted using PSIVER . The default threshold of 0.390 was used in this study. Interacting pair positions between the two proteins were predicted using PPiPP  with default parameters.
Helical wheel projection
The helical wheel projection was generated by a custom script derived from the original code by Zidovetzki and Armstrong .
This study was in part supported by the Industrial Technology Research Grant Program in 2007 (Grant Number 07C46056a) from New Energy and Industrial Technology Development Organization (NEDO) of Japan, and also by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (Grant Numbers 25430186 and 25293079).
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