International Journal of Genomics

International Journal of Genomics / 2014 / Article
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Drug-Related Genomics in Cancer and Immunological Diseases

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Research Article | Open Access

Volume 2014 |Article ID 402603 | 9 pages | https://doi.org/10.1155/2014/402603

Molecular Evolution of the Vertebrate FK506 Binding Protein 25

Academic Editor: Huai-Rong Luo
Received25 Nov 2013
Accepted16 Jan 2014
Published02 Mar 2014

Abstract

FK506 binding proteins (FKBPs) belong to immunophilins with peptidyl-prolyl isomerases (PPIases) activity. FKBP25 (also known as FKBP3) is one of the nuclear DNA-binding proteins in the FKBPs family, which plays an important role in regulating transcription and chromatin structure. The calculation of nonsynonymous and synonymous substitution rates suggested that FKBP25 undergoes purifying selection throughout the whole vertebrate evolution. Moreover, the result of site-specific tests showed that no sites were detected under positive selection. Only one PPIase domain was detected by searching FKBP25 sequences at Pfam and SMART domain databases. Mammalian FKBP25 possess exon-intron conservation, although conservation in the whole vertebrate lineage is incomplete. The result of this study suggests that the purifying selection triggers FKBP25 evolutionary history, which allows us to discover the complete role of the PPIase domain in the interaction between FKBP25 and nuclear proteins. Moreover, intron alterations during FKBP25 evolution that regulate gene splicing may be involved in the purifying selection.

1. Introduction

Immunophilins include three families with peptidyl-prolyl isomerases (PPIases) activity, FK506 binding proteins (FKBPs), cyclophilins, and parvulins. FKBPs are named for binding to the immunosuppressive drug FK506, characterized by one or more PPIase domains. The 15 identified members of human FKBPs are divided into 4 groups: cytoplasmic, TPR domain, endoplasmic reticulum (ER), and nucleus. FKBP25 and FKBP133 locate in the nucleus, containing a single PPIase domain [1].

FKBP25 (also known as FKBP3) is the first mammalian FKBP with a calculated molecular mass of 25 kDa found in the nucleus, which plays a role in regulating transcription and chromatin structure. The FKBP25 comprises a conserved PPIase domain at its C-terminus with a 43% sequence identity to FKBP12 and a helix-loop-helix (HLH) motif at its unique hydrophilic N-terminal [2, 3]. This conserved PPIase domain functions in binding to the immunosuppressive agent FK506 or rapamycin. Unlike another FKBPs, FKBP25 shows a strong affinity for binding rapamycin (Ki = 0.9 nM) over FK506 (Ki = 200 nM) [4]. The FKBP25 was reported to be associated with nuclear proteins including transcription factor Yin-Yang1 (YY1), mouse double minute 2 (MDM2), and histone deacetylases (HDACs) [5]. FKBP25 binds to YY1 at N-terminal and increases its DNA-binding activity without the involvement of the FK506/rapamycin binding domain [6]. In addition, the level and activity of the tumor suppressor protein p53 are negatively regulated by MDM2. The HLH motif of FKBP25 mediates protein-protein interaction to enhance ubiquitination and degradation of oncogene MDM2, increasing the expression of tumor suppressor p53 and its downstream effector p21 [7]. Moreover, the protein-protein interaction contributes to form HDAC complexes, which is critical for the chromatin structure [2].

In 1992, Jin et al. reported the molecule cloning of human FKBP25 and performed a homology comparison between FKBP25 and FKBP12/FKBP13 [8]. Furthermore, Mas et al. showed the molecule cloning of mouse FKBP25 and expression pattern of FKBP25 gene during cerebral cortical neurogenesis [9]. However, the relationships between nuclear functions and evolution in FKBP25 are seldom reported. In this study, we exhibit an evolutional analysis not only on selective pressure but also on intron-exon conversion among vertebrate FKBP25 genes.

2. Materials and Methods

2.1. Sequence Data Collection

All the FKBP25 gene and amino acid sequences were obtained from the ENSEMBL (http://www.ensembl.org/index.html) [10], based on orthologous and paralogous relationships. The gained FKBP25 sequences were applied as queries to search known FKBP25 genes using BLAST at the National Center for Biotechnology Information (NCBI), in order to confirm whether their best hit was an FKBP25 gene [11].

Incomplete sequences of FKBP25 genes in four species (tree shrew, horse, platypus, and turkey) were retrieved from both ENSEMBL and NCBI. After eliminating these incomplete sequences, 28 sequences were applied for this study. The 28 sequences from 23 species comprised human (ENSG00000100442), chimpanzee (ENSPTRG00000006305), gorilla (ENSGGOG00000013322), orangutan (ENSPPYG00000005778), macaque (ENSMMUG00000016512), marmoset (ENSCJAG00000015972), mouse (ENSMUSG00000020949), rat (ENSRNOG00000004629), guinea pig (ENSCPOG00000001444), rabbit1 (ENSOCUG00000007535), rabbit2 (ENSOCUG00000026892), dog1 (ENSCAFG00000014018), dog2 (ENSCAFG00000014093), dog3 (ENSCAFG00000024192), dog4 (ENSCAFG00000000578), cow (ENSBTAG00000002610), elephant1 (ENSLAFG00000003572), elephant2 (ENSLAFG00000027553), opossum (ENSMODG00000007352), chicken (ENSGALG00000012466), zebra finch (ENSTGUG00000013231), anole lizard (ENSACAG00000004080), xenopus (ENSXETG00000003052), fugu (ENSTRUG00000011887), medaka (ENSORLG00000015070), stickleback (ENSGACG00000012834), tetraodon (ENSTNIG00000010980), and zebrafish (ENSDARG00000079018).

2.2. Molecular Phylogenetic Analyses

The protein coding sequences of FKBP25 were aligned using CLUSTAL W program in MEGA 5.05. We constructed a maximum likelihood (ML) tree of FKBP25 amino acid sequences by MEGA 5.05 with the optimal model (Kimura 2-parameter model). The relative support of internal node was performed by bootstrap analyses with 1000 replications for ML reconstructions [12].

2.3. Selection Pressure Analyses

The numbers of nonsynonymous substitutions per nonsynonymous site (dN) and the numbers of synonymous substitutions per synonymous site (dS) were computed by MEGA 5.05 with the modified Nei-Gojobori method. The dN/dS <1, =1 and >1 demonstrate purifying selection, neutral selection, and positive selection, respectively [13]. The dN is the numbers of nonsynonymous substitutions per nonsynonymous site, and the dS is the numbers of synonymous substitutions per synonymous site. The transition/transversion ratio was 1.55 estimated using the ML method by MEGA 5.05 [14].

The FASTA format of FKBP25 sequences was converted to the PAML format using DAMBE software for subsequent site analyses [13]. The CODEML program implemented in the PAML 4.7 package was used to detect positive selection of individual sites. The site-specific model was exerted using likelihood ratio tests (LRT) to compare M7 (null model) with M8 model. M7 is a null model that does not allow for any codons with , whereas M8 model allows for positively selective sites (). When the M8 model fitted the data significantly (-value < 0.05) better than the null model (M7), the presence of sites with is suggested. On the contrary, the results of value > 0.05 proved the absence of sites with . The twice log likelihood difference between the two compared models () is compared against with critical values 5.99 and 9.21 at 0.05 and 0.01 significance levels, respectively [15].

2.4. Protein Domain and Motif Analyses

Protein domain analyses of FKBP25 were shown at Pfam domains database (http://pfam.sanger.ac.uk) [16]. SMART (http://smart.embl-heidelberg.de/) was used to make sure the presence of FKBP25 domains [17]. The motifs of FKBP25 were analyzed by the MEME software (http://meme.sdsc.edu/meme/website/intro.html) with a maximum of 10 motifs to find [18].

2.5. Exon-Intron Conservation Analyses

We collected elaborate information about FKBP25 exon and intron from ENSEMBL (http://www.ensembl.org/index.html) [19]. The number and length of FKBP25 exon and intron in 28 sequences were investigated for exon-intron conservation analyses.

3. Results

3.1. Phylogenetic Analyses of FKBP25

All the FKBP25 gene and protein sequences were collected from the ENSEMBL and checked by BLAST at NCBI. The sequence and structural alignment of FKBP25 was shown in Figure 1. The phylogenetic tree was constructed according to the protein coding sequences of FKBP25 using the maximum likelihood method (Figure 2, left panel). The FKBP25 genes from the primate lineage and teleost lineage form a species-specific cluster, respectively. Four FKBP25 isoforms of dog exhibited a close relationship and clustered together, according to the phylogenetic tree. There were similar phenomena in rabbit and elephant.

3.2. Selection Pressure Analyses

The nonsynonymous to synonymous rate ratio (dN/dS) may demonstrate the selective pressures of involved protein. We calculated the pairwise distance of FKBP25 sequences using MEGA 5.05. There was a significantly lower dN than dS in the pairwise comparisons of these sequences. Most values of dN/dS in these sequences were distributed blow the diagonal, showing that the presence of a purifying selection existed in the FKBP25 (Figure 3). The comparisons of average dN and dS in various vertebrate groups were shown in Figure 4, respectively. Furthermore, site-specific tests were performed for searching the positive selection sites in vertebrate, mammalian, primate, and mammalian excluding primate, rodent and teleost lineages. Although some positive selection sites were computed, each of M7 and M8 <5.99 indicated that the M8 model was not significantly better than the M7 model to fit the data. Consequently, we concluded that the site-specific analyses also compute no positive selection sites acting on FKBP25 using PAML4.7 (Table 1).


SpeciesModelsEstimates of parameterslnL Positively selected sites

VertebrateM7    −5463.9384650.003806NA
M8    −5463.940368None
( )  

MammalianM7 −2182.2447890.000258NA
M8 −2182.244918None
  ( )  

PrimateM7 −997.0773890.000102NA
M8    −997.077440None
  ( )  

Mammalian excluding primateM7 −2242.3062220.000160NA
M8 −2242.306302NS
  ( )  

RodentM7 −1372.9021640.000058NA
M8 −1372.902193NS
  ( )  

TeleostM7 −2354.9231810.000408NA
M8 −2354.923385NS
( )  

lnL: the log-likelihood difference between the two models; : twice the log-likelihood difference between the two models (In all the species, , the -value is more than the significance level 0.05, indicating that M8 model is not better than M7 model); NA: not allowed; NS: not shown (it means the sites under positive selection but not reaching the significance level of 0.9).
3.3. Protein Domain and Motif Analyses

Early studies reported that mammalian FKBP25 have two portions: one is a putative helix-loop-helix motif within N-terminal unique sequence (Figure 5(a)) and the other is the PPIase domain at its C-terminus (Figure 5(b)) [20].

The domain distribution of FKBP25 was investigated using FKBP25 to search amino acid sequences at the Pfam database firstly. Only one domain (PPIase domain) was found in the Pfam database. The PPIase domain within FKBP25 sequences generally started at position 122 and ended at position 221. Similarly, we further make sure that the FKBP25 domain is at SMART, resulting in the single PPIase domain at position 119 to 221.

We then performed a detailed domain and motif analyses using the MEME software. Except two dog isoforms, dog2 and dog3, the FKBP25 sequences used in this study contain a conversed PPIase domain within motif 1 (shown in Figure 2) at its C-terminus. In addition, the result implied that motif 2 located in the N-terminal contained an HLH motif [6], which was associated with DNA binding and dimerization [21]. However, HLH motif was not found in dog3, anole lizard, and teleost lineage, implying that these FKBP25 proteins may function on gene expression in another pathway.

3.4. Exon-Intron Conservation Analyses

The exon-intron information collected from the ENSEMBL database was shown in Table 2 and Figure 6. Most of the FKBP25 genes have 7 exons with similar length in different species (Table 2). Mammalian FKBP25 shows exon-intron conservation with 6 introns and similar sizes of each intron. Intron deletions existed in several isoforms of species. The rabbit2 isoform had 2 exons, and elephant2 isoform had only one exon. The exon numbers of dog2, dog3, and dog4 isoforms were less than seven. Except mammalian FKBP25 genes, anole lizard reduced one exon compared with mammalian and birds, but the xenopus and teleost maintained 7 exons. The intron deletions of FKBP25 genes may happen in the evolutionary process from amphibian to reptile. Then, a subsequent intron insertion occurred in the evolution from reptile to more advanced animals. The FKBP25 genes also had intron insertion in zebra finch and zebra fish.


SpeciesLength (bp)
Exon1Intron1Exon2Intron2Exon3Intron3Exon4Intron4Exon5Intron5Exon6Intron6Exon7Intron7Exon8Total exons

Human 1083548102797108817313653068276198177555675
Chimpanzee 1083524102797108889813653068272598178955675
Gorilla 1083538102796108821413653068275398177855675
Orangutan 1083498102793108839513653368245798143255675
Macaque 1083496102786108827313653168284598181855675
Marmoset 1083592102780108564413650768253798210055675
Mouse 108376210284110822241368376819619893755675
Rat 1083528102816108203013694268166798111855675
Guinea pig 10832321027721083600136141668134698134055675
Rabbit1 108218910210821084634136111568182698126655675
Rabbit2 6204055675
Dog1 10825731021076108208813646868182398121655675
Dog2 296132294129654
Dog3 302195425223323312102645
Dog4 427190248675
Cow 1082332102603108283513648468170698130955675
Elephant1 10831761021089108475613648368158098172555675
Elephant2 675675
Opossum 10825601021484108280713610516812619855455675
Chicken 111761027511440813610406810119882955684
Zebra finch 111112102761084941368929720541669534955678
Anole lizard 186169910813331361078688249861055651
Xenopus 1112319102403108418136129681869878755678
Fugu 10537510278105651368268689810655669
Medaka 10510910271997381367568709880455663
Stickleback 10529410276102931361356881989655666
Tetraodon 1053051028010275136916870987555666
Zebra fish 10525271024471611172024416359199042810811666

Intron8Exon9Intron9Exon10Intron10Exon11Intron11Exon12Intron12Exon13Intron13Exon14Intron14Exon15Intron15

1042243162670814991098311071526761784

Exon16Intron16Exon17

9811855

4. Discussion

FKBP25 is a nuclear member of the FKBPs family that is associated with transcription and chromatin structure [2]. The interactions of FKBP25 with nuclear proteins are closely associated with HLH motif at the N-terminal of FKBP25. However, whether the PPIase domain at C-terminus is important for these interactions remains uncertain. The selection pressure analyses revealed that the purifying selection triggered a whole evolutionary history of FKBP25 in vertebrates, even in each lineage of vertebrates. Purifying selection is one of the natural selections that resist deleterious mutations with negative selective coefficients [22]. The mutations that disrupt the correct folding of the FKBP25 domain can weaken PPIase activity and may be the deleterious mutations [5]. It was hypothesized that the mutations of PPIase domain were one of explanations behind the purifying selection throughout FKBP25 evolution. Therefore, although the PPIase domain of FKBP25 was not found to be involved in the protein interactions previously, the PPIase domain might have some associations with the YY1 DNA-binding, MDM2 autoubiquitination and degradation, and HDACs complex formation. These inferences will become a potent direction for exploring the relationship between nuclear proteins and PPIase domain in the future.

The protein-coding sequence length of vertebrate FKBP25 is highly conversed that almost all the taxa are 224 bp; nevertheless the original gene length and exon-intron status are tremendously various among vertebrate species. However, mammalian FKBP25 exhibit exon-intron conservation with 6 introns and similar sizes of each intron. Chicken FKBP25 maintains 6 introns, but zebra finch has one more intron that inserts in the gene. Similarly, a large variability of intron number and sizes among all the taxa shown in Figure 6 revealed that intron insertion and deletion events happened frequently during the FKBP25 evolutionary history from teleost to birds. In particular, zebrafish demonstrated the maximum number of introns in this study, and the size of exon is much smaller than other teleost species (Figure 6(g)). The intron loss of FKBP25 gene from species more advanced than zebrafish is likely to induce alterations of gene expression due to the absence of specific intron splicing. Under the purifying selection, the FKBP25 gene expression event continuously removes the pernicious mutations that may associate with intron splicing regulation [23].

FKBP25 gene knockdown declined the expression levels of p53 and p21, which emphasized the significance of FKBP25 in regulating p53 and subsequently p21 expression through controlling the ubiquitination of MDM2. Both the FKBP25 PPIase domain and its N-terminal portion were critical for the ubiquitination and degradation of MDM2 [2]. Moreover, Jin et al. reported that FKBP25 prefers to bind to rapamycin rather than FK506, implying that FKBP25 may be an important target molecule for immunosuppression by rapamycin [8]. All the evolution analyses indicated the conservation of FKBP25 gene in vertebrates. Therefore, FKBP25 possesses some basic functions in vertebrate species, like regulating p53 and p21 expression and binding to rapamycin for immunosuppression, reinforcing the suggestion that the purifying selection triggered the evolution of vertebrate FKBP25.

In conclusion, FKBP25 as a nuclear FKBP subjects to the purifying selection throughout the whole evolution, which implied the complete role of the PPIase domain involved in the interaction between FKBP25 and the nuclear proteins that are needed to be discovered continually. Additionally, incomplete exon-intron conservation of FKBP25 meets the vertebrate lineage. The intron gain or loss among the taxa is likely to be involved in the purifying selection.

Conflict of Interests

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

Authors’ Contribution

Fei Liu and Xiao-Long Wei contributed to this paper equally.

Acknowledgments

This project was sponsored by the Grants from the National Natural Science Foundation of China (81273593, 81273274, and 81302331), the Priority Academic Program Development of Jiangsu Higher Education Institutions, National Major Scientific, Technological Special Project for “Significant New Drugs Development” (2011ZX09302-003-02), Jiangsu Province Major Scientific and Technological Special Project (BM2011017), Jiangsu Province’s Key Provincial Talents Program (RC201170 and H201108), and the Foundation of the Nanjing Pharmaceutical Association, China (Nanjing, China) (Grant no. H2011YX001).

References

  1. E. A. Blackburn and M. D. Walkinshaw, “Targeting FKBP isoforms with small-molecule ligands,” Current Opinion in Pharmacology, vol. 11, no. 4, pp. 365–371, 2011. View at: Publisher Site | Google Scholar
  2. Y.-L. Yao, Y.-C. Liang, H.-H. Huang, and W.-M. Yang, “FKBPs in chromatin modification and cancer,” Current Opinion in Pharmacology, vol. 11, no. 4, pp. 301–307, 2011. View at: Publisher Site | Google Scholar
  3. S. Riviere, A. Menez, and A. Galat, “On the localization of FKBP25 in T-lymphocytes,” FEBS Letters, vol. 315, no. 3, pp. 247–251, 1993. View at: Publisher Site | Google Scholar
  4. J. Liang, D. T. Hung, S. L. Schreiber, and J. Clardy, “Structure of the human 25 kDa FK506 binding protein complexed with rapamycin,” Journal of the American Chemical Society, vol. 118, no. 5, pp. 1231–1232, 1996. View at: Publisher Site | Google Scholar
  5. G. Gudavicius, H. Soufari, S. Upadhyay et al., “Resolving the functions of peptidylprolyl isomerases: insights from the mutagenesis of the nuclear FKBP25 enzyme,” Biochemical Society Transactions, vol. 41, no. 3, pp. 761–768, 2013. View at: Google Scholar
  6. W.-M. Yang, Y.-L. Yao, and E. Seto, “The Fk506-binding protein 25 functionally associates with histone deacetylases and with transcription factor YY1,” EMBO Journal, vol. 20, no. 17, pp. 4814–4825, 2001. View at: Publisher Site | Google Scholar
  7. A. M. Ochocka, P. Kampanis, S. Nicol et al., “FKBP25, a novel regulator of the p53 pathway, induces the degradation of MDM2 and activation of p53,” FEBS Letters, vol. 583, no. 4, pp. 621–626, 2009. View at: Publisher Site | Google Scholar
  8. Y.-J. Jin, S. J. Burakoff, and B. E. Bierer, “Molecular cloning of a 25-kDa high affinity rapamycin binding protein, FKBP25,” Journal of Biological Chemistry, vol. 267, no. 16, pp. 10942–10945, 1992. View at: Google Scholar
  9. C. Mas, I. Guimiot-Maloum, F. Guimiot et al., “Molecular cloning and expression pattern of the Fkbp25 gene during cerebral cortical neurogenesis,” Gene Expression Patterns, vol. 5, no. 5, pp. 577–585, 2005. View at: Publisher Site | Google Scholar
  10. P. Flicek, I. Ahmed, M. R. Amode et al., “Ensembl 2013,” Nucleic Acids Research, vol. 41, no. D1, pp. D48–D55, 2013. View at: Google Scholar
  11. M. Johnson, I. Zaretskaya, Y. Raytselis, Y. Merezhuk, S. McGinnis, and T. L. Madden, “NCBI BLAST: a better web interface,” Nucleic Acids Research, vol. 36, supplement 2, pp. W5–W9, 2008. View at: Publisher Site | Google Scholar
  12. S. Kumar, M. Nei, J. Dudley, and K. Tamura, “MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences,” Briefings in Bioinformatics, vol. 9, no. 4, pp. 299–306, 2008. View at: Publisher Site | Google Scholar
  13. X. Xia and Z. Xie, “DAMBE: software package for data analysis in molecular biology and evolution,” Journal of Heredity, vol. 92, no. 4, pp. 371–373, 2001. View at: Google Scholar
  14. K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar, “MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods,” Molecular Biology and Evolution, vol. 28, no. 10, pp. 2731–2739, 2011. View at: Publisher Site | Google Scholar
  15. Z. Yang, “PAML 4: phylogenetic analysis by maximum likelihood,” Molecular Biology and Evolution, vol. 24, no. 8, pp. 1586–1591, 2007. View at: Publisher Site | Google Scholar
  16. R. D. Finn, J. Tate, J. Mistry et al., “The Pfam protein families database,” Nucleic Acids Research, vol. 36, no. 1, pp. D281–D288, 2008. View at: Publisher Site | Google Scholar
  17. I. Letunic, T. Doerks, and P. Bork, “SMART 7: recent updates to the protein domain annotation resource,” Nucleic Acids Research, vol. 40, no. D1, pp. D302–D305, 2012. View at: Google Scholar
  18. T. L. Bailey and C. Elkan, “Fitting a mixture model by expectation maximization to discover motifs in biopolymers,” in Proceedings of the International Conference on Intelligent Systems for Molecular Biology, vol. 2, pp. 28–36, 1994. View at: Google Scholar
  19. T. Hubbard, D. Barker, E. Birney et al., “The Ensembl genome database project,” Nucleic Acids Research, vol. 30, no. 1, pp. 38–41, 2002. View at: Google Scholar
  20. M. Leclercq, F. Vinci, and A. Galat, “Mammalian FKBP-25 and its associated proteins,” Archives of Biochemistry and Biophysics, vol. 380, no. 1, pp. 20–28, 2000. View at: Publisher Site | Google Scholar
  21. W. D. Kohn, C. T. Mant, and R. S. Hodges, “α-helical protein assembly motifs,” Journal of Biological Chemistry, vol. 272, no. 5, pp. 2583–2586, 1997. View at: Publisher Site | Google Scholar
  22. Z. Yang and J. R. Bielawski, “Statistical methods for detecting molecular adaptation,” Trends in Ecology and Evolution, vol. 15, no. 12, pp. 496–503, 2000. View at: Publisher Site | Google Scholar
  23. A. Resch, Y. Xing, A. Alekseyenko, B. Modrek, and C. Lee, “Evidence for a subpopulation of conserved alternative splicing events under selection pressure for protein reading frame preservation,” Nucleic Acids Research, vol. 32, no. 4, pp. 1261–1269, 2004. View at: Publisher Site | Google Scholar

Copyright © 2014 Fei Liu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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