The Elephant Evolved p53 Isoforms that Escape MDM2-Mediated Repression and Cancer Monikaben Padariya,1 Mia-Lyn Jooste,2 Ted Hupp,2 Robin Fåhraeus,1,3,4,5 Borek Vojtesek,4 Fritz Vollrath,6,7 Umesh Kalathiya,* ,†,1 and Konstantinos Karakostis * ,†,3,8 1 International Centre for Cancer Vaccine Science, University of Gdansk, ul. Kładki 24, Gdansk, Poland 2 Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK 3 Inserm UMRS1131, Institut de Génétique Moléculaire, Université Paris 7, Hôpital St. Louis, Paris, France 4 Research Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Brno, Czech Republic 5 Department of Medical Biosciences, Umeå University, Umeå, Sweden 6 Department of Zoology, Zoology Research and Administration Building, University of Oxford, Oxford, UK Downloaded from https://academic.oup.com/mbe/article/39/7/msac149/6632613 by guest on 16 January 2023 7 Save the Elephants Marula Manor, Marula Lane, Karen P.O. Box 54667, Nairobi, Kenya 8 Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra (Barcelona), Spain *Corresponding authors: E-mails: umesh.kalathiya@ug.edu.pl; konstantinos.karakostis@gmail.com. † These authors contributed equally to this work. Associate Editor: Harmit Malik Abstract The p53 tumor suppressor is a transcription factor with roles in cell development, apoptosis, oncogenesis, aging, and homeostasis in response to stresses and infections. p53 is tightly regulated by the MDM2 E3 ubiquitin ligase. The p53–MDM2 pathway has coevolved, with MDM2 remaining largely conserved, whereas the TP53 gene morphed into various isoforms. Studies on prevertebrate ancestral homologs revealed the transition from an environmentally induced mechanism activating p53 to a tightly regulated system involving cell signaling. The evolution of this mech- anism depends on structural changes in the interacting protein motifs. Elephants such as Loxodonta africana consti- tute ideal models to investigate this coevolution as they are large and long-living as well as having 20 copies of TP53 isoformic sequences expressing a variety of BOX-I MDM2-binding motifs. Collectively, these isoforms would enhance sensitivity to cellular stresses, such as DNA damage, presumably accounting for strong cancer defenses and other adaptations favoring healthy aging. Here we investigate the molecular evolution of the p53–MDM2 system by com- bining in silico modeling and in vitro assays to explore structural and functional aspects of p53 isoforms retaining the MDM2 interaction, whereas forming distinct pools of cell signaling. The methodology used demonstrates, for the first time that in silico docking simulations can be used to explore functional aspects of elephant p53 isoforms. Our ob- Article servations elucidate structural and mechanistic aspects of p53 regulation, facilitate understanding of complex cell signaling, and suggest testable hypotheses of p53 evolution referencing Peto’s Paradox. Key words: structural variations, model, p53 retrogenes, molecular evolution, Loxodonta africana, lifespan, Peto’s Paradox, intrinsic specificity. Introduction promoting cell cycle arrest or cellular apoptosis (Lane The p53 family, including p53, p63, and p73, takes up roles and Levine 2010), thus coordinating cellular responses in cancer, neurodegeneration, development, inflammation (Haronikova et al. 2019). The question arises as of how and tissue regeneration, cellular senescence, aging (Chusyd p53 activity is differentiated in response to the require- et al. 2021), and stress-induced mechanisms (Anbarasan ments of a cell and, of special interest to humans, how and Bourdon 2019; Fujita 2019; Beck et al. 2020). P53 family this prevents the development of cancers (Fahraeus and members are suggested to modulate gene expression in Olivares-Illana 2014; Haronikova et al. 2019; Karakostis both a full-length p53-dependent and -independent man- and Fahraeus 2019). This important topic can be addressed ner. Importantly, p53 isoforms with specific activities have by tracking the molecular evolution of p53 structures and been shown to form homo- or hetero-oligomers, providing interaction interfaces. an additional level of differentiation (Fujita 2019). The p53 The p53 gene has evolved from an ancestral p53/p63/ transcription factor targets DNA sequences through p73 gene, which gave rise to three p53-related genes, p53-responsive elements (p53Res) (Bourdon et al. 1997) with distinct roles in mammals (Belyi et al. 2010; and coregulates the transactivation of several genes, Rutkowski et al. 2010; Karakostis et al. 2016; Siau et al. © The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Open Access Mol. Biol. Evol. 39(7):msac149 https://doi.org/10.1093/molbev/msac149 Advance Access publication July 6, 2022 1 Padariya et al. · https://doi.org/10.1093/molbev/msac149 MBE 2016; Hendler et al. 2021). The tp53 transcription factor is a with the typical number of one copy found (so far) in all tumor suppressor with key roles in oncogenesis other mammals (Sulak et al. 2016). According to the (Mantovani et al. 2019; Salomao et al. 2021). It involves Peto’s Paradox referring to why larger animals with a high- two N-terminal transactivation domains (TAD I and TAD er number of cells and cell divisions do not also have a II), a DNA-binding domain (DBD), and a C-terminal oligo- higher cancer incidence (Sulak et al. 2016; Tollis et al. merization domain (Wallace et al. 2006; Logotheti et al. 2017, 2020; Callier 2019), the elephant’s multiple copies 2010). Thirteen isoforms have been shown to be produced are proposed to have evolved in order to defuse enhanced through alternative mRNA translation initiation (p53/47) DNA damage response as a way to promote cancer sup- or splicing (full-length p53 or FLp53, p53β, p53γ, pression. Despite the large body size and long-life span, ele- Δ40p53α, Δ40p53β, Δ40p53γ, Δ133p53α, Δ133p53β, phants exhibit a high resistance to cancer as cancer Δ133p53γ, Δ160p53α, Δ160p53β, Δ160p53γ) (Bourdon mortality is estimated to be <5%, as compared with hu- et al. 1997, 2005; Marcel et al. 2010, 2011). The full-length mans reaching up to 25% (Abegglen et al. 2015; p53 includes the TAD I, which is required for the induction Gaughran et al. 2016). During evolution, elephant retro- Downloaded from https://academic.oup.com/mbe/article/39/7/msac149/6632613 by guest on 16 January 2023 of p53 target genes and proapoptotic factors (p21CDKN1A, gene p53 sequences have evolved from an ancestral se- Bax, Puma, Noxa of the Bcl-2 family), leading to cell cycle quence, whereas the initial TP53 retrogene amplification arrest (G1) or apoptosis. The p53/47 isoform at the second may predate extant elephant species. The closest relative in frame AUG (+120), lacking the TAD I but retaining TAD (with sequenced genome) of elephants, the hyrax II, is activated during Unfolded Protein Response, following (Procavia capensis), only contains one copy of TP53 stress to the endoplasmic reticulum, to lead to G2/M arrest (Abegglen et al. 2015), whereas gene amplification appar- (Haronikova et al. 2019). This isoform lacks the BOX-I ently rapidly expanded in Proboscideans in parallel with in- MDM2-binding epitope (found in the TAD I). Another iso- creased body size and life span (Sulak et al. 2016). It form, the Δ133p53, lacking the first 132 amino acids, corre- appears that this event may result to an induction of cell sponding to the TAD I, TAD II, and the first 30 residues of death at lower levels of DNA damage, compared with the DBD, is abundant in early passage normal human fibro- phylogenetically close relatives, like the African rock hyrax, blasts and decreases in late passage and senescent cells the East African aardvark, and the American southern (Fujita 2019). Indeed, these isoforms have various three-banded armadillo (Callier 2019). However, other MDM2-binding capacities and are expressed in a stress- long-living large-sized mammals, like the bowhead whale dependent fashion, contributing to responses by transacti- (Balaena mysticetus), have not evolved extra copies of vating downstream pathways (Rozan and El-Deiry 2007; any tumor suppressor gene (Yim et al. 2014; Keane et al. Allen et al. 2014; Chen 2016; Haronikova et al. 2019; 2015). The variability or truncation of the elephant p53 Carlsen and El-Deiry 2021). It has been suggested that a isoformic sequences (i.e., p53 isoforms of partial length, quantitatively regulated coexpression of these distinct iso- corresponding to the N′ domains of the canonical p53 full- forms is required during cell proliferation (Khoury and length sequence), indicates an enhanced functional diver- Bourdon 2011) and that the expression of different p53 iso- sity and the formation of diverse pools of functional p53 forms is induced in response to DNA damage agents or proteins. This would increase distinct structural character- stress to the endoplasmic reticulum (Chen et al. 2009). istics conferring specialized interacting interfaces for key In addition, numerous cellular and animal model stud- functional proteins such as MDM2 and downstream acti- ies indicate that an unbalance in p53 isoform expression vating proteins. In addition to p53 variations, it has been causes cancer, premature aging, and degenerative diseases hypothesized that an accumulation of differentially ex- (Fujita 2019). An abnormal expression of p53 isoforms, or pressed, modified, and activated (by PTMs) p53 pools, col- mis-regulation of posttranslational modifications (PTMs) lectively or synergistically regulate the response to specific mediating the interactions of p53 pathway, may be in- stresses in the cell (Khoury and Bourdon 2011). These duced by mutations leading to carcinogenesis (Khoury PTMs are suggested to have evolved as a result of co- and Bourdon 2011; Karakostis et al. 2019; Salomao et al. evolutionary cues of the p53–MDM2 interactions during 2021). Importantly, experiments on mice have shown the transition from (1) an ancestral early branching inver- that extra copies of the p53 gene can be introduced to tebrate system (prevertebrate) (Barreira et al. 2021) to (2) generate “super p53” animals carrying three or four func- a derived mammalian vertebrate system (Karakostis et al. tional copies of p53, accurately reproducing the behavior 2016; Karakostis and Fahraeus 2019). of the endogenous gene but with an enhanced response The elephant’s genetic set-up thus innately confers to DNA damage (Garcia-Cao et al. 2002; Moding et al. functional diversity to p53-dependant mechanisms con- 2016). As p53 appears to regulate aging and longevity in tributing significantly to valuable protein fine tuning. a context-dependent manner (Feng et al. 2011; Wu and This is in stark contrast to the human set-up where one Prives 2018; Fujita 2019), mice with accelerated aging gene with alternatively activated isoforms is expressed in show chronic p53 activation. This suggests that the p53– response to a stress. The existence of multiple p53 forms MDM2 axis leads to elevated p53 activity caused by either in elephants, with potentially different activities, provides early aging or delayed-onset aging (Wu and Prives 2018). an ideal model for understanding how p53 activities are An exciting study discovered that elephants have 20 executed and regulated. Indeed, substitutions made by ret- copies, that is, 40 alleles, of the TP53 genes, compared rogene isoforms altering the p53 DBD and its functions, 2 The Elephant MDM2–TP53 Structures · https://doi.org/10.1093/molbev/msac149 MBE have been investigated and associated with longevity in upstream of the TAD1 that could have emerged via spli- species lifespan (Bartas et al. 2021). Important for an un- cing. This sequence is: derstanding of P53 diversity in elephants, Sulak et al. N′MFSINSTLAALVCRTSPPQNPGSLRSLLFHSLSASPLPTGK (2016) and Haupt and Haupt (2017) investigated the roles LLALTCHGDCPALCQKPRGGCWDWEFPFPCAHTGAKSF- of retrogene isoforms (TP53RTGs) forming a “pool of pro- QLFKSPKPPSWLQLAAGLWRYLVSGLGPCFQGRLHARLR- tected p53” thus acting as contributors to an enhanced FGGQIALPGAA-C′. Furthermore, a BLAST search has sensitivity of elephant cells to DNA damage. The two mod- shown a similarity of ∼50% for two other sequences, en- els (the “guardian” or the “decoy” model) explored the po- coding homologous sequences to p63 (XP_023407389.1 tential roles of the TP53RTGs in facilitating the and XP_023407390.1) and p73 (XP_010591460.1) pro- stabilization of the canonical p53, by either participating teins (Lane and Levine 2010). on the dimerization with canonical p53, or by acting as an- Nineteen sequences were identified and used for the tagonists, competing for the MDM2 interaction (Sulak analysis. The retrogene sequences were identified et al. 2016; Haupt and Haupt 2017). (supplementary tables S1 and S2, Supplementary Downloaded from https://academic.oup.com/mbe/article/39/7/msac149/6632613 by guest on 16 January 2023 Here, we explore the elephants apparently unique sys- Material online) and their sequence alignment and phylo- tem further by focusing on the p53–MDM2 interaction. genetic relation to the canonical elephant p53 were ex- We consider this interaction important as p53 is primarily posed (supplementary figs. S1 and S2, Supplementary regulated by MDM2 and that their interaction depends on Material online). These alignments were used for the prep- the BOX I motif (residing in the TAD I) (Gajjar et al. 2012; aration of a graphical illustration representing the puta- Karakostis et al. 2019), which is intrinsically disordered in tive corresponding topology of each retrogene on the turn implying greater likelihood of evolutionary changes full-length elephant TP53 (fig. 1A). In order to reveal (Brown et al. 2010). This suggests that the variations iden- which p53 isoforms might potentially bind MDM2, we tified on the elephant BOX I sequences potentially modify first performed a bioinformatic analysis to identify se- the binding epitope of MDM2 and thus alter the expres- quence variations on the encoded BOX-I peptides of sion levels and activation of p53 (Wasylyk et al. 1999; each isoform. Comparative analysis of the BOX-I se- Naski et al. 2009; Buyukpinarbasili et al. 2016). For our quences from L. africana and the human p53, showed investigation, we focus on the BOX-I sequences of the that the BOX-I is conserved in all isoforms (fig. 1B, p53 isoforms in order to test the hypothesis that p53 supplementary figs. S1 and S2 and table S1, isoforms induce distinct pools of p53 proteins with varia- Supplementary Material online). However, within these tions on the epitopes interacting with MDM2. This has isoforms, the analysis indicated six types of identical se- significant implications for functional diversity and the quences, corresponding to BOX-I motifs (supplementary integration of diverse signaling outcomes. Our observa- table S1, Supplementary Material online). The generally tions are discussed and compared with previous models, highly conserved MDM2-interacting BOX-I motif exploring the diverse roles of elephant p53 isoforms FxxxWxxL is well conserved in only one of the sequences (Sulak et al. 2016). We also explore potential translational (elephant X1, Type A). The retro gene sequences 2, 5, 6, 7, insights derived from the molecular evolution (interspe- 8, 9, 11, 13, and 17 (Type B) carry an FxxxGxxL domain. In cies) of the p53–MDM2 axis and the evolved structural addition, more variations are present in retrogene 3 (Type modules. C), retrogene 4 (Type D), retrogene 10 (Type E), and retro- genes 12, 14, 15, 16, and 18 (Type F), potentially altering the structures and the binding affinity to MDM2 (fig. 1B, Results supplementary table S1, Supplementary Material online). Modeling and experimental results show that elephant Each BOX-I Type was linked to corresponding ENSEMBL re- p53 isoformic putative proteins exhibit a spectrum ferences for each p53 sequence, to facilitate a comparative of reduced docking capacity to MDM2, strongly study with previous findings (Sulak et al. 2016) indicating a range of an isoform-specific downstream (supplementary table S3, Supplementary Material online). p53 functionality. It is noted that ENSLAFG00000025553 (TP53RTG1) and ENSLAFG00000032258 (TP53RTG19) are annotated by ENSEMBL as pseudogenes. For assessing the interaction cap- Elephant p53 Isoforms Exhibit Diverse Docking acity of each putative protein, the PDB model 1YCR, deriv- Association to MDM2 ing crystallographic data, was used (Kussie et al. 1996). The In this work, we identified p53 sequences in Loxodonta af- 1YCR structure illustrates human MDM2 bound to the p53 ricana, and grouped them based on their MDM2-binding TAD. According to this structure, the amino acid sites F, W, motifs (p53 BOX-I), found at the N′ terminal and within and L are positioned to the interphase of MDM2, mediating the TAD I. The “XP_010594888.1” (cellular tumor antigen the interaction. Based on this structure, each type of BOX-I p53 isoform X2 [L. africana]) was assigned as “Type A.” In sequence was modeled and the interaction interphases addition, our findings identified the “XP_003416950” (cel- were calculated (fig. 1C). Type A exhibits the highest dock- lular tumor antigen p53 isoform X1 [L. africana]), long as ing capacity, whereas B, C, and E exhibit a comparatively the canonical p53 sequence and another sequence of decreased docking capacity. Types D and F exhibit a 519 aa. It contains an additional 5′ coding sequence poor docking capacity (fig. 1C, supplementary table S1, 3 Padariya et al. · https://doi.org/10.1093/molbev/msac149 MBE A FIG. 1. Docking models of the MDM2–p53 N′ terminal (BOX-I) interaction, with calcu- lated GBVI/WSA dG (–kcal/ mol) values. (A) Graphical mapping of the topography of each elephant p53 isoformic Downloaded from https://academic.oup.com/mbe/article/39/7/msac149/6632613 by guest on 16 January 2023 sequence on the canonical p53. (B) Alignment of the hom- ologous BOX-I sequences from Loxodonta africana and human p53. (C) Docking model of the interaction of MDM2 with dif- B ferent elephant p53 sequences: Type A: X1, Type B: 2, 5, 6, 7, 8, 9, 11, 13, and 17; Type C: retro- gene 3; Type D: retrogene 4; Type E: retrogene 10; and Type F: retrogenes 12, 14, 15, 16, and 18. (C) Docking model of the interaction of MDM2 with mutated Type A elephant p53: humanized Y > D or E > K. The humanized elephant p53 sequences exhibit an increased docking capacity, compared with the wt elephant p53 X1 (Type A). To comparatively illustrate the interaction C D capacity among the Types (C) and the effect of intro- duced mutations on the full- length (D), the binding affinity values were normalized, setting the highest value at 100. In pa- nel (C), Type A has the highest value and all the measure- ments were normalized after setting it at 100. In addition, for panel (D), the humanized E > K mutation had the highest value and was set at 100. These results show that variations found in the isoforms, as well as single variants, like the Y > D, strongly effect on the inter- action of p53 with MDM2, by modifying the docking interfaces. Supplementary Material online). All the compared se- and the positioning of the BOX-I motif of p53 at the inter- quences carry the FxxxxxxL motif; however, the W to G vari- phase with MDM2. ation is only present in the sequences exhibiting a decreased In order to explore additional resides modifying the docking capacity. These results indicate that the FxxxWxxL docking capacity, we introduced two mutations, simulat- motif on the Box-I of p53, increases the binding capacity to ing the human BOX-I orthologue, Y to D and E to MDM2, and variations induce changes on the conformation K. These mutations lead to an increased docking capacity 4 The Elephant MDM2–TP53 Structures · https://doi.org/10.1093/molbev/msac149 MBE Downloaded from https://academic.oup.com/mbe/article/39/7/msac149/6632613 by guest on 16 January 2023 FIG. 2. Illustration of the MDM2–p53 peptides docking models, explaining the association of each p53 peptide to MDM2, in human (i) and elephant (iii–ix). The hydrophobicity is visualized in blue for hydrophilic and brown for hydrophobic. Yellow represents the binding pattern FxxxWxxL. Blue letters represent residues that may stabilize the peptide when binding MDM2. compared with the elephant Type A p53 (fig. 1D, crucial residues F, W, and L, play a crucial role in the dy- supplementary table S1, Supplementary Material online), namics or the positioning of the BOX-I motif on the indicating that these residues, positioned in between the MDM2 cleft. 5 Padariya et al. · https://doi.org/10.1093/molbev/msac149 MBE A Modeling Six Elephant p53 BOX-I Sequences Interacting with MDM2, Reveals Variable Stereochemistry In line with the calculated binding affinity (kcal/mol; Generalized Born/Volume Integral [GBVI]/WSA dG) va- lues of the docking models, the visualization of each model revealed the crucial role of the positioning of the F, W/G, L residues at the MDM2 cleft (fig. 2). Indeed, the human se- B quence (fig. 2i), as well as the earlier mentioned cross- species mutations of humanized elephant p53 Type A (fig. 2ii and iii), leading to an increased docking capacity. This demonstrates the crucial role of the positioning of Downloaded from https://academic.oup.com/mbe/article/39/7/msac149/6632613 by guest on 16 January 2023 the F, W, and L residues extended toward separate direc- tions within the elephant MDM2 cleft. Similarly, the ele- phant sequence Type A and the above mentioned cross-species mutations Y > D and E > K, show an in- creased docking efficiency (fig. 2ii and iii). According to the models, sequences of decreased dock- ing capacity due to variations on the BOX-I sequence, ex- hibit a limited exposure of these residues within the hydrophobic cleft of MDM2. Interestingly, the W > G vari- C ation, decreased docking in the isoforms Types B–F (fig. 2v–ix). In addition, the positioning of the Types D and F approaching the MDM2 cleft, was comparatively poor. These results together point toward a model whereby ele- phant p53 putative proteins may play a fine-tuning regu- lative role by exhibiting a spectrum of docking capacity on MDM2. Biochemical Data Confirm the Structural Interface Models Our docking model simulations were further tested in vi- tro by Sandwich ELISA, using recombinant human MDM2 protein (fig. 3A). The experimental results are in line with the bioinformatic predictive models, showing an increased binding of the elephant p53, Type A, followed by the Types B and E, whereas the Types D and F showed decreased binding (fig. 3B). These results indicate that MDM2 structure is well conserved in human and elephant, FIG. 3. Interaction of MDM2–p53 (protein–peptide) carried out by and that the cross-species proteins retain their binding re- Sandwich ELISA. These results are in line with the docking models. lationships. However, the elephant p53 Type C did not The statistical significance was calculated by t-test (two-tailed) interact with recombinant human MDM2. In order to un- and the P-values are indicated as follows: P ≤ 0.05 (*), P ≤ 0.01 (**), cover why this result is not in line with the bioinformatic P ≤ 0.001 (***), and P ≤ 0.0001 (****). Unpaired test results are indi- docking model, we performed another docking calculation cated on the graph. Nonsignificant measurements are indicated by “ns” and negative controls as: “n/c.” The y-axis is the normalized in order to compare the docking of this isoform to human (%) values derived from the ELISA measurements, which indicate and elephant MDM2 proteins (supplementary fig. 3, the interaction of MDM2 with each of the p53 peptides. The Supplementary Material online). It emerged that the y-axis represents the “p53 (BOX-I)–MDM2 interaction” and the Type C isoform (Y > C in the elephant and D > C com- bars show the standard deviation of the measurements. The y-axis pared with the human) (fig. 1B) shows a high docking ef- in (B) is divided in two segments, for illustrative purposes. (A) Scheme illustrating the set-up of the sandwich ELISA. Biotinylated ficiency to elephant MDM2, but a decreased one to p53 peptides are fixed on streptavidin-coated plates. An human MDM2. anti-MDM2 antibody tagged with HRP and a secondary anti-IgG This observation is in line with figure 1C (docking model were used. (B) Elephant (Loxodonta africana) p53 peptides Types: using elephant MDM2) and figure 3B (ELISA using human A–F exhibit a variable capacity to bind MDM2. (C ) The interaction MDM2). In addition, the mutations Y to D and E to K, of MDM2 with humanized Type A elephant p53: Y > D or E > K, is increased compared with the wt Type A. Experiments were tested simulating the human orthologues, exhibited an increased in three technical replicates (see supplementary table S4, binding capacity, in line with the models (figs. 1D and 3C). Supplementary Material online). These results collectively demonstrate that peptides 6 The Elephant MDM2–TP53 Structures · https://doi.org/10.1093/molbev/msac149 MBE Downloaded from https://academic.oup.com/mbe/article/39/7/msac149/6632613 by guest on 16 January 2023 FIG. 4. Summary of human and elephant p53 sequences, grouping the different BOX-I domains into six types (A–F ) illustrating their capacity to interact/dock with MDM2. *1: The Y > D increases binding; *2: W > G and P > L/- prevent binding; *3: The E > K increases binding, depending on additional variations. encoded by human p53 and elephant isoforms carrying of p53 (Gajjar et al. 2012; Nguyen et al. 2018; Haronikova the conserved residue corresponding W23 in human p53 et al. 2019). have an increased MDM2-binding affinity, whereas pep- Nineteen TP53RTGs have been accumulated primarily tides carrying the W > G variation show decreased binding by segmental duplication and drift, following an initial ret- activity with MDM2 (fig. 4). Indeed, the W > G and P > rotransposition of the p53 gene. Studies by Sulak and col- L/-variations prevent binding, whereas the Y > D or the laborators indicated that most of these RTGs are under the E > K variations increase binding. control of the RTE-type non-LTR retrotransposon We conclude that a group of the p53 isoforms putatively (RTE1_LA) promoter. Using RNA-Seq data, the authors expressed in the elephant L. africana retain the capacity to showed that TP53 and TP53RTG12 genes were transcribed find to MDM2, thus forming distinct pools of variable (grad- in African and Asian elephant samples (Sulak et al. 2016). ually decreasing) binding affinity. Isoforms with the W resi- This work largely supports previous studies (Cortez et al. due are expected to be regulated by MDM2 via 2014; Reddy et al. 2015); whereas the TP53RTG3 and well-described pathways, signaling p53 for degradation un- TP53RTG18 transcripts were considerably less abundant der normal conditions. However, an underlying mechanism, (Sulak et al. 2016). Though TP53RTG12 is annotated as a combining activities synergistically deriving from these iso- pseudogene at the Ensemble database, an elephant- forms, may have evolved to orchestrate and fine-tune a se- specific protein band of the expected size for lective activation of specific tumor suppressors in response TP53RTG19 (22.3 kDa) was identified by Sulak et al., as to comorbiditing stress signals (Abegglen et al. 2015) (fig. 4). well as a band at the size of TP53RTG12 protein (19.6 kDa). Further, these authors employed treatment with low doses of different genotoxic agents (mitomycin Discussion c, doxorubicin) or with UV-C in order to induce an up- The p53–MDM2 axis has been thoroughly studied in hu- regulation of TP53 signaling and apoptosis (Cas3/7 activ- mans and mice, as well as in a range of other species. It ity) aiming to study whether elephant cells exhibit an is well-established that during normal conditions, the enhanced TP53 response in response. However, these ex- MDM2 E3 ubiquitin ligase binds the p53 protein via the periments did not indicate the activation of an expanded BOX-I motif and catalyses the poly-ubiquitination of p53, TP53 gene repertoire (Sulak et al. 2016). Correlating with targeting it for degradation via the 26S proteasomal path- previous findings (Karakostis et al. 2016), the abrogation way. Several studies have shown the interaction interfaces of the MDM2–p53 PPI by nutlin 3a lead to the stabilization of p53–MDM2 (Chene 2003; Moll and Petrenko 2003; Pant of p53, with cells were resistant to apoptosis. Indeed, nutlin et al. 2013) and have directly linked the prevention of this 3a prevents p53 from MDM2-mediated degradation. interaction to the stabilization or/and activation of p53. In However, the activation of p53 further requires PTMs addition, it is worth-noting that the abrogation of the (e.g., phosphorylation by ATM, acetylation by p300), p53–MDM2 (protein–protein) interaction constitutes a mediated in response to stress signals (Meek and druggable target in anticancer therapies (e.g., Nutlin 3a), Anderson 2009; Haronikova et al. 2019). aiming to stabilize p53. Moreover, preventing the capacity Previously, Karakostis et al. (2019) described a mechan- of p53 isoforms to bind MDM2, directly promotes the sta- ism activating p53 (S15 phosphorylation) in response to bilization, activation, and downstream signaling functions DNA damage (doxorubicin) that requires the MDM2– 7 Padariya et al. · https://doi.org/10.1093/molbev/msac149 MBE p53 mRNA interaction. Indeed, it appears that MDM2 and MDM2 and each p53 BOX-I motif constituting the p53 have coevolved since the early Metazoan with the MDM2-binding epitope. Our research provides novel ex- BOX-I encoding sequence evolving two MDM2 interac- perimental and in silico evidence that reveal how elephant tions, one RNA for controlling synthesis that takes place p53 isoforms may have modified BOX-I motifs to exhibit in prevertebrates and one protein for regulating degrad- reduced binding capacity to MDM2 (Kubbutat et al. ation that occurs in vertebrates. The MDM2–p53 inter- 1997; Klein and Vassilev 2004; Coffill et al. 2016). action also evolved to impose allosteric regulation of Alternations in the coding sequences of p53 BOX-I iso- both proteins as they gained complex interactomes to in- forms induce structural variations in the elephant p53, as crease and their functional repertoires. Hence, the p53– shown by modeling and experimental (ELISA) studies, con- MDM2 interactions not only serve to control p53 expres- stituting a range of putative functional p53 proteins with sion but also as ligands to control their respective confor- different activities toward various stress inducers. Our mations and functions. In mammals that carry a single findings support previous co-IP evidence, which show p53 gene, the MDM2–p53 interaction is controlled by alter- that the Type B BOX-I isoforms (corresponding to the Downloaded from https://academic.oup.com/mbe/article/39/7/msac149/6632613 by guest on 16 January 2023 natively spliced or translated isoforms. In elephants, in con- TP53RTG12) prevent the interaction of p53 with MDM2 trast, the retrogenes serve to differentiate the function and (Sulak et al. 2016) (figs. 1 and 3). Additionally, these find- regulation of p53 into specific isoforms that contribute to ings also show how other BOX-I Types (corresponding to an enhanced sensitivity to cellular stresses, such as DNA TP53RTGs) also limit the interaction with MDM2, consti- damage. Indeed, isoforms derived by gene duplication fol- tutively contributing in a range of MDM2-binding p53 mo- lowed by subfunctionalization, have been hypothesized to lecules with variable binding efficiencies (decreased), thus allow an intrinsic specificity, by partitioning ancestral inter- preventing MDM2-dependant control. acting partners along each lineage (Wheeler and Harms Our observations do not exclude the “guardian model” 2021). Elephant species show variations in the TP53 se- proposed by Sulak et al (2016). Indeed, our results add by quences and copy numbers, with the smallest species ex- suggesting a range of district and/or contributing activities pressing a higher number (Tollis et al. 2021). Such of these isoforms, potentially dynamically regulating the variations may be epigenetically introduced (mutations) activation of p53 toward various stresses. We note that or be endogenously expressed via alternative splicing me- even though these p53 isoforms are truncated compared chanisms and synthesis of several retrogenes. Hints con- with the canonical full-length p53, they retain the TAD cerning the coevolution of the p53–MDM2 interaction and parts of the DBD regulating downstream targets reflected by variations on the sequences expressing the (fig. 1A). After all, the canonical p53 regulates the expres- binding interphases, are crucial for the appropriate regula- sion of more than 3,000 genes and the interactions be- tion of the P53–MDM2 pathway, constituting the basis tween p53 isoforms are considered to be direct via for the evolution of specific activation mechanisms of p53 oligomerization or indirect, via promoter-dependent regulation in response to diver stresses. Conceptually, this oligomerization (Anbarasan and Bourdon 2019). interpretation is embedded in the hypothesis that an accu- mulation of differentially expressed and structurally modi- fied p53 pools (either isoformic or modified by PTMs), Structural Variations on the p53–MDM2 Interface in collectively and synergistically regulates the response to spe- Different Species cific stresses in the cell (Khoury and Bourdon 2011). To ad- Despite intensive academic or commercial (biomedical) dress this paradigm, Sulak et al. (2016) developed a model studies over the last 40 years (Marcel et al. 2018; Duffy for TP53RTG function to explore whether the retrogenes et al. 2020; Levine 2020; Yang et al. 2021), we lack a clear act as “decoys” or “guardians” of the canonical p53 with picture of how p53 may be regulated, how it functions the view to understand how several TP53RTGs contribute and how cells differentiate p53 activity to best suit intra- to an enhanced sensitivity of elephant cells to DNA damage. cellular and extracellular changes to their environment Indeed, the proposed “guardian” model, is supported by the (Salomao et al. 2021). It is still very much an open question interaction between TP53 and the retrogress TP53RTG12, how the human p53, which is expressed from a single gene but not between TP53RTG12 and MDM2. According to can have so many different activities and how these are all this model, Sulak et al. suggested that TP53RTG proteins di- regulated. Insights into the molecular evolution of the an- merize with canonical TP53 to block the formation of TP53 cestral p53/p63/p73 gene to the mammalian p53 family of tetramers, thus preventing the MDM2-dependant negative genes and comparative studies on the MDM2–p53 path- regulation of the canonical TP53 protein. The alternative way, are beginning to unravel the biological roles of p53 “decoy” model, describing TP53RTGs acting as decoys for homologs in different species. Mechanistic and structural the MDM2 interaction, was also tested and discussed by studies on interacting partners can be approached by em- the authors (Sulak et al. 2016). Although confirming the dy- ploying different model systems. In our current study, we namics of the oligomerization states will be challenging, introduced the p53 isoforms from elephant as an interest- both hypotheses (models) provide a testable approach to ing model system for structural studies of the p53–MDM2 address TP53RTGs functions. interplay. From the analysis so far, as reflected by the high Here, we aim to advance previous findings and add to number of p53 isoformic sequences (both the high copy the discussions, by focusing on the interaction between number and the existence of several diverse or/and partial 8 The Elephant MDM2–TP53 Structures · https://doi.org/10.1093/molbev/msac149 MBE p53 sequences), it emerges that the elephant may employ Since many of the encoded proteins (i.e., the p53 retrogene a sequence-specific strategy to respond to p53-mediated 12) retain the MDM2 interaction motif at the TAD and di- regulation, depending on a stress. It remains unclear merization sites at the DBD, they might act in favor of p53 whether the p53 PTM palette (including phosphorylation, activation via binding competition to the MDM2 complex acetylation, methylation, ubiquitination, sumoylation, or by dimerization with the canonical p53 (Kubbutat et al. etc.), is conserved in the elephant as it is for example in 1998; Sulak et al. 2016). In correlation with this, our results the human, although the documented functional diversity show that retrogene 12 along with retrogenes 14, 15, 16, of elephant p53 sequences escaping MDM2-binding and and 18, form the group “Type F,” which shows poor bind- protein degradation, points toward a mechanism whereby ing to MDM2 (fig. 4) strongly indicating that these puta- p53 employs isoformic sequences of variable BOX-I motifs tive proteins escape MDM2-dependant regulation. In and partial DBDs. These complexes (1) form dimers with addition, our in silico analysis and the experimental the canonical p53 protein, preventing its degradation via ELISA findings both show that all the peptides carrying MDM2 (guardian model, suggested by Sulak et al 2016); the W > G variation exhibit decreased binding activity Downloaded from https://academic.oup.com/mbe/article/39/7/msac149/6632613 by guest on 16 January 2023 or/and (2) independently contribute to p53 mechanisms with MDM2 (figs. 1 and 3). In fact, the alignment of the ele- potentially substituting the function of PTMs-mediated phant BOX-I sequences of the canonical p53 and the regulation, described in human (e.g., during the DNA dam- TP53RTGs, illustrates at least two vital substitutions which age response). The physiological significance and the func- are conserved in all the TP53RTGs (W > G and N > K) tion (1) of extra copies of the gene and (2) of the (fig. 4). This indicates that these sequences derive from a expression of various isoforms, are highly relevant for fur- common initial duplication. This is in line with Sulak ther studies of the paradigm. For example, in connection et al. (2016), providing evidence confirming that these iso- to human or other systems expressing a single p53 gene, forms result from a single retrotransposition event fol- the acquisition of variations during molecular evolution, lowed by repeated rounds of segmental duplication of integrated on a single p53 gene, may elegantly explain chromosomal loci containing the TP53RTG genes. the employment of PTMs that can tangle differential ex- Thus collectively these variations contribute to the for- pression and signal-specific responses, thus compensating mation of altered p53 BOX-I structures, thus resulting to a or substituting the need of several isoforms or copy num- range of interaction capacities for these isoforms. These re- ber variations. Importantly, genetically engineered mouse sults are in line with previous co-IP data, which show that models indicate that p53 may also regulate longevity the TP53RTG12 isoform (BOX-I, Type B), coimmunopreci- and aging, in a context-specific manner (Donehower pitated with p53 but not with MDM2, suggests that these 2009). Additionally, single nucleotide variations such as isoforms dimerize with p53, whereas preventing the inter- the mouse P72, were shown to better retain self-renewal action with MDM2 (requiring tetramerization of p53), and functions of stem/progenitor cells compared with R72 thus act as guardians against MDM2-dependant p53 deg- mice (Zhao et al. 2018). Overexpression of an N-terminal radation. These findings, like the guardian model proposed truncated p53 that starts at the second AUG and lacks by Sulak et al. (2016), support our thesis that p53 peptides the first 40 amino acids (Δ40p53) results in premature exhibit a spectrum of reduced p53–MDM2 interaction im- aging in mice and altered stem cell pluripotency (Ota plicated in p53 activity. Moreover, considering that these et al. 2017; Levandowski et al. 2021). Surprisingly, this isoforms carry the TADs and partial sequences of the phenotype is dependent on the presence of full-length DBD (fig. 1A), it is likely that their reduced binding cap- p53. In addition, a truncated carboxy-terminal p53 frag- acity to MDM2 allows these isoforms/retrogenes to evade ment leads to activated p53 and mice expressing this trun- degradation via specific processes, that is, the activation of cated version along with p53wt (p53+/m) exhibited specific pools of downstream genes independently from enhanced resistance to spontaneous tumors compared the canonical FL p53. Additional PTMs identified in hu- with wild-type (p53+/+) mice, but an early onset of aging man, including acetylation of C-terminal lysines (K370, (Tyner et al. 2002). Recurrently in species, increased TP53 K372, K373, K381, K382, and K386) by p300/CBP, which expression comes with a cost, whereas the elephants, ex- boosts p53 binding to its target gene loci to activate down- pressing 20 copies and 19 truncated isoforms of p53, po- stream pathways, like cell cycle arrest, senescence or apop- tentially may potentially compensate it (Sulak et al. tosis, remain to be addressed in the elephant p53 isoforms 2016), even though some of these result from (Liu et al. 2019; Chen et al. 2020). Much remains to be done retrotransposition. to decipher the physiological significance of these findings; In the current study, we make use of a naturally evolved for now, Peto’s Paradox has opened Pandora’s Box of im- system, the Elephant’s genome, endogenously expressing a portant questions revolving around the all-round function variety of truncated and variable p53 sequences. As such, of p53. the Elephant p53 sequences constitute an ideal model for exploring the multifaceted functions of p53. Even though elephant p53 retrogenes have not been shown Implications in Clinical Translation Research to directly function as transcription factors, they might en- It is argued that the p53–MDM2 axis constitutes a drug- hance DNA damage sensitivity and induction of apoptosis gable target for cancer therapeutics (Hientz et al. 2017; by regulating the p53 signaling pathway (Sulak et al. 2016). Sharma et al. 2019; Karagiannakos et al. 2022). We assert 9 Padariya et al. · https://doi.org/10.1093/molbev/msac149 MBE that addressing the coevolution of the p53–MDM2 inter- indicate the structural basis of the interfaces, which can action may refine predictive studies aiming to identify and be used for the development of highly specific antibodies characterize cancer biomarkers for translation research. In to target neoantigens and cancer mutations that are diffi- respect to diagnostics, immune biomarkers from serum, cult to target in conventional ways (Hsiue et al. 2021). for example, the cytokines (tumor necrosis factor-alpha, Insights into structural modifications and activation to- interferon-gamma, and interleukins), which are known to ward signaling mechanisms, illuminate the factors that induce p53 up-regulation have also been documented to prevent or promote carcinogenesis. Studies on the p53 iso- be indicative of immune responses to various pathologies forms (Zhang et al. 2019; Levandowski et al. 2021) will help in the elephant (Edwards et al. 2020). Additionally, identi- to illuminate how their respective activities are regulated fication and characterization of the functional implica- in response to cellular damages and pathogen infections tions of such variants and of their physiological (Breton et al. 2021; Mehta et al. 2021). This will not only significance to onco-immunology and aging, constitute result in a better understanding of p53 roles and regulation an unforeseen opportunity for personalized medicine in elephants and in its role in protecting elephants from Downloaded from https://academic.oup.com/mbe/article/39/7/msac149/6632613 by guest on 16 January 2023 (Abegglen et al. 2015). Here, we show that the elephant cancers as well as probably other “inflictions”, such as para- BOX-I Type F isoform, has an E to G variation at residue sites (Lynsdale et al. 2017), but it also facilitates the devel- 17. We note that variations on E17 in human p53 are can- opment of new therapeutic strategies for human, based on cer mutants, frequently associated with female genital can- structural molecular data. cers (Petitjean et al. 2007; Ganguly and Chen 2015). The K24N mutation found in colorectal cancers, is thought to implicate ubiquitination by TRAF6, thereby facilitating Conclusion and Perspective p53 acetylation by p300 (Lai et al. 2022). Isothermal titra- The elephant has naturally evolved several p53 isoforms of tion calorimetry measurements demonstrated that this variable lengths and sequences. It thus constitutes an ideal structural variation did not prevent the binding affinity model for studying the functions of p53 variants in this for p53TAD–MDM2 (Zhan et al. 2013). However, the natural “experiment.” With the discovery that elephants K24N human mutation was shown by computational have 20 copies of p53 and strong evidence that elephants and NMR secondary chemical shift analysis, to reduce only rarely get cancer, a whole new front of research has the helicity in residues 18–27, from an average of ∼10– opened up for studies of p53 (Sulak et al. 2016; Vazquez 5% by disrupting the Asp21–Lys24 salt bridge, which stabi- et al. 2018). In this context, the elephant constitutes an lizes the local partial helices. In parallel, it also reduced the ideal model to comprehend how molecular evolution probability of contact formation between residues 21 and may lead to the expression of multiple p53 isoforms 24, by ∼50% thus increasing the flexibility of the p53 TAD with district RNA and protein structures and functions and affecting several molecular interactions of p53 as well as opposed to human where p53 incorporates multiple as posttranslational regulation (Ganguly and Chen 2015). functions under a tightly regulated signaling mechanism Other alternations on the BOX-I motif documented (employing PTMs). here via the comparative interspecies analysis, have also In this work, we employed bioinformatic modeling ana- been suggested to be implicated in cancer cell lines with lysis to identify the homology of the BOX-I motifs of p53 inactivating TP53 point mutations (i.e., P27, Sonkin 2015 isoforms from the elephant L. africana and calculate their and N29, Thirion et al. 2002). In addition, the cross-species docking capacities (binding) to the main p53 regulator; mutations Y > D and E > K, showed an increased docking MDM2. We also used in vitro synthesized peptides and re- efficiency. Interestingly, the glutamic acid (E), asparagine combinant versions of human MDM2, to experimentally (N), tryptophan (W), and tyrosine (Y) residues are highly confirm the interactions in vitro, employing sandwich conserved in disordered proteins as they play crucial role ELISA. These results suggest that mutations altering the in forming protein–protein interfaces (Brown et al. structures of the interacting partners (MDM2–p53 inter- 2010). As such, it emerges that cross-species studies, like face) may have dramatic functional consequences for our pilot study presented here, on genetic variations of cell signaling mechanisms, constituting potentially critical p53 can lead to several novel insights into altered struc- therapeutic or diagnostic targets (Karagiannakos et al. tural and functional outcomes of p53 action (Wang et al. 2022). Our findings support previous models pointing to- 2007; Lu et al. 2009; Belyi et al. 2010; Joerger et al. 2014; ward contributing roles of p53 retrogenes in the regulation Coffill et al. 2016; Bartas et al. 2019; Biscotti et al. 2019; of p53 and increased sensitivity to DNA damage responses Fischer 2019). Derived discoveries will in turn lead to novel, (Sulak et al. 2016). Furthermore, we show that the modi- testable hypotheses concerning clinical aspects of p53 with fied BOX-I sequences of these isoforms induce structural links to the development of drug-resistance or cancer pro- variations that prevent the interaction with MDM2, thus gression (Somarelli, Gardner, et al. 2020; Salomao et al. evading MDM2-dependant degradation. Moreover, their 2021). Accumulating evidence present how comparative truncated DBDs indicate a selective transactivation of genomic studies aiming to shed light on complex human downstream targets. traits with several translational aspects (Sulak et al. 2016; We envision that our observations and conclusions Tollis et al. 2019; Somarelli, Boddy, et al. 2020; Somarelli, form a solid basis for further experiments aiming to ad- Gardner, et al. 2020; Farre et al. 2021; Stakyte et al. 2021) dress how each isoform contributes to the activation 10 The Elephant MDM2–TP53 Structures · https://doi.org/10.1093/molbev/msac149 MBE of p53, as a result of structural variability. The method- Modeling L. africana p53–MDM2 interaction ology presented here for the first time demonstrates The homology modeling approach was applied for con- how in silico docking simulations can be used to explore structing the MDM2 and p53 protein structures. The functional aspects of these p53 isoforms and sets the ba- 1YCR crystal structure, illustrates MDM2 bound to the sis for perspective studies aiming to explore the dynamics p53 TAD, modeling the sequences >1YCR_2|Chain B| of the interactions with MDM2 under stress-inducing P53| SQETFSDLWKLLPEN and >1YCR_1|Chain A| conditions. Studies using elephant cell lines or tissues MDM2|Homo sapiens (9606) (Kussie et al. 1996). The mod- to measure the expression levels of the isoforms (qPCR) eled sequence of human MDM2 is the following: and knock-down assays (siRNAs) targeting combinations ETLVRPKPLLLKLLKSVGAQKDTYTMKEVLFYLGQYI of the p53 isoforms, are highly anticipated to probe func- MTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYT- tional and physiological aspects, potentially contributing MIYRNLVV. 1YCR_1 was used as a template to build the to the dissection/resolution of the Peto’s Paradox. In a L. africana MDM2 protein structure (Kussie et al. 1996; broader view, such studies may also address the mechan- Downloaded from https://academic.oup.com/mbe/article/39/7/msac149/6632613 by guest on 16 January 2023 Berman et al. 2000). The sequence of the elephant MDM2 isms whereby tumor suppressor genes and duplications is the following: ETLVRPKPLLLKLLKSVGAQKDTYTMKE regulate cellular senescence to drive lifespan and body VIFYLGQYIMAKRLYDEKQQHMVYCSNDLLGDLFGEPSFSVK mass (Farre et al. 2021; Tejada-Martinez et al. 2022). DHRKIYTMIYRNLVV. Homology modeling was performed using the Molecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, QC, Canada) Materials and Methods package (Vilar et al. 2008) along with the CHARMM27 force- field (Brooks et al. 2009). In silico Analysis and Phylogenetic Characterization The modeled MDM2 structure was further analyzed for the Determination of p53 Sequences in L. africana with energy minimization within the MOE package Homologous sequences of human p53 and MDM2 were (Berman et al. 2000). To build the tertiary structures of identified by searches in NCBI (http://www.nlm.nih.gov/). the p53 peptides, the human p53 from pdb id.: 1YCR Nineteen isoformic sequences were identified and used (Kussie et al. 1996) was used as a template, which was fur- for the analysis. The sequences are the following: X1 ther optimized by applying CHARMM27 forcefield (Brooks (XP_010594888.1) and X2 (XP_003416950.2) sharing an et al. 2009). These different MDM2 and p53 protein struc- identical BOX-I sequence; isoform 1 (KF715855.1); tures were considered for the protein–peptide docking isoform 2 (KF715856.1); isoform 3 (KF715857.1); isoform protocol, to calculate their binding patterns. In order to 4 (KF715858.1); isoform 5 (KF715859.1); isoform 6 achieve different conformations of p53 peptides with (KF715860.1); isoform 7 (KF715861.1); isoform MDM2 protein, the rigid body docking protocol described 8 (KF715862.1); isoform 9 (KF715863.1); isoform in MOE (Berman et al. 2000) was implemented. The 10 (KF715864.1); isoform 11 (KF715865.1); isoform 12 CHARMM27 forcefield (Vilar et al. 2008) was used to (KF715866.1); isoform 13 (KF715867.1); isoform 14 rank the binding score of individual p53 peptides with (KF715868.1); isoform 15 (KF715869.1); isoform 16 MDM2 protein, using the GBVI/WSA dG (kcal/mol) scor- (KF715870.1); isoform 17 (KF715871.1); and isoform 18 ing function (Labute 2008; Padariya et al. 2021). (KF715872.1). The human p53 sequence BAC16799.1, was Furthermore, considering molecular mechanics a refine- used for the comparative analysis. Sequences were ana- ment of individual clusters protein–peptide complex was lyzed in silico with the ClustalOmega software (Sievers performed and best 100 poses were analyzed. The et al. 2011). The BOX-1 sequences (MDM2-binding motif) BIOVIA Discovery Studio visualizer (Dassault Systèmes, of the elephant p53 sequences were analyzed. Sequence BIOVIA Corp., San Diego, CA, USA) package was used to alignment, comparisons, and phylogeny were generated prepare representation of the MDM2–p53 complexes. using the ClustalOmega software for Multiple Sequence Alignment (EMBL, EBI) (Sievers et al. 2011) and the tree is a Neighbour-joining tree. Sequences were grouped based Peptide Synthesis and Protein–Protein Sandwich on their variations into six types and a graphical represen- ELISA tation is shown (fig. 1A, supplementary figs. S1 and S2 and Each of the six types elephant BOX-I peptide was synthe- table S1, Supplementary Material online). A phylogenetic sized in vitro with a conjugated Biotin-N-terminal tag, fol- tree of the BOX-I sequences indicates the phylogeny lowed by SGSG spacer, then the amino acid sequence among the elephant p53 types (supplementary fig. S2, terminating with a C-terminal amide, and obtained from Supplementary Material online). This information may be Mimotopes (Australia). Recombinant human glutathione of interest for exploring the molecular evolution of the S-transferase (GST)-tagged MDM2 protein was used for BOX-I motif, its coevolution with MDM2, and the putative the MDM2–p53, protein–peptide sandwich ELISA, follow- duplication events that lead to the formation of several ing the protocol published previously (Picksley et al. 1994). BOX-I isoforms in the elephant. The corresponding Peptides from Mimotopes were resuspended in DMSO to ENSEMBL id numbers of each BOX-I Type are mentioned 1 mg/ml and then diluted into Elisa Buffer (used through- on supplementary table S3, Supplementary Material out the entire experiment which is PBS plus 0.1% Tween-20 online. and 3% BSA). White, high protein binding 96-well ELISA 11 Padariya et al. · https://doi.org/10.1093/molbev/msac149 MBE plates (Costar) were coated with streptavidin (1 µg/100 µl International Research Agendas program of the in H2O) by incubation overnight at 37 °C. Wells were Foundation for Polish Science cofinanced by the European washed 3× with PBS + Tween-20 (0.1%) and then blocked Union under the European Regional Development Fund. with 100 µl of ELISA buffer at room temperature for Authors would also like to thank the PL-Grid 60 min. Peptides were added at 100 ng/well in 100 ul of Infrastructure, Poland for providing their hardware and soft- ELISA buffer to allow capture of the biotinylated peptide ware resources. into the well at room temperature for 60 min. After wash- ing three times with PBS + Tween-20 (0.1%), MDM2 was di- luted to 100 ng per well in 100 µl of ELISA buffer. After Author contributions incubation at room temperature for 60 min, wells were M.P., M.-L.J., T.H., U.K. conducted the experiments; T.H., washed three times with PBS + Tween-20 (0.1%). The R.F., B.V., F.V., K.K. conceptualized the study, designed MDM2 antibody was diluted 1:1000 in ELISA buffer and the experiments, and wrote the paper. 100 µl was added per well and after incubation at room Downloaded from https://academic.oup.com/mbe/article/39/7/msac149/6632613 by guest on 16 January 2023 temperature for 60 min, wells were washed three times with PBS + Tween-20 (0.1%). The secondary HRP-anti-IgG Data availability antibody (DAKO, P260 for mouse and P217 for rabbit) was diluted 1:1000 in ELISA buffer and 100 µl was added The article complies with the MBE’s policy on the availabil- per well and after incubation at room temperature for ity of data and materials. 60 min, wells were washed three times with PBS + Tween-20 (0.1%), and enhanced chemiluminescence solu- tion was added followed by reading the plate with a reader References (Perkin Elmer). GST-tagged human MDM2 (purchased Abegglen LM, Caulin AF, Chan A, Lee K, Robinson R, Campbell MS, from Dundee University protein production facilities, Kiso WK, Schmitt DL, Waddell PJ, Bhaskara S, et al. 2015. Potential mechanisms for cancer resistance in elephants and DU43570) (fig. 3A). The human p53 peptide comparative cellular response to DNA damage in humans. Biotin-SGSG-SQETFSDLWKLLPENNV was used as a posi- JAMA 314:1850–1860. tive control. Each sample was tested in triplicates and the Allen MA, Andrysik Z, Dengler VL, Mellert HS, Guarnieri A, Freeman Prism software was used for the preparation of the graphs. JA, Sullivan KD, Galbraith MD, Luo X, Kraus WL, et al. 2014. The values were normalized to the highest value (i.e., 100% Global analysis of p53-regulated transcription identifies its direct targets and unexpected regulatory mechanisms. Elife 3:e02200. was set for the largest mean in each dataset and 0% for the Anbarasan T, Bourdon JC. 2019. The emerging landscape of P53 iso- smallest mean). The statistical significance was calculated forms in physiology, cancer and degenerative diseases. Int J Mol by t-test (two-tailed) comparing the values of each pair Sci. 20:6257. of Type (BOX-I) interaction with MDM2 and the P-values Barreira SN, Nguyen AD, Fredriksen MT, Wolfsberg TG, Moreland are indicated in the graph using the asterisk symbol scoring RT, Baxevanis AD. 2021. Aniprotdb: a collection of consistently system: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), and P ≤ generated metazoan proteomes for comparative genomics stud- ies. Mol Biol Evol. 38:4628–4633. 0.0001 (****). Nonsignificant comparisons are indicated by Bartas M, Brazda V, Cerven J, Pecinka P. 2019. Characterization of P53 “ns” and negative controls as: “n/c.” Both paired and un- family homologs in evolutionary remote branches of Holozoa. Int paired t-tests were performed (supplementary table S4, J Mol Sci. 21:6. Supplementary Material online). Scores derived from the Bartas M, Brazda V, Volna A, Cerven J, Pecinka P, Zawacka-Pankau JE. unpaired t-test are indicated (fig. 3B and C). 2021. The changes in the P53 protein across the animal kingdom point to its involvement in longevity. Int J Mol Sci. 22:8512. Beck J, Turnquist C, Horikawa I, Harris C. 2020. Targeting cellular sen- escence in cancer and aging: roles of P53 and its isoforms. 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