- Research article
- Open Access
Evolution of the highly networked deubiquitinating enzymes USP4, USP15, and USP11
© Vlasschaert et al. 2015
Received: 5 June 2015
Accepted: 17 October 2015
Published: 26 October 2015
USP4, USP15 and USP11 are paralogous deubiquitinating enzymes as evidenced by structural organization and sequence similarity. Based on known interactions and substrates it would appear that they have partially redundant roles in pathways vital to cell proliferation, development and innate immunity, and elevated expression of all three has been reported in various human malignancies. The nature and order of duplication events that gave rise to these extant genes has not been determined, nor has their functional redundancy been established experimentally at the organismal level.
We have employed phylogenetic and syntenic reconstruction methods to determine the chronology of the duplication events that generated the three paralogs and have performed genetic crosses to evaluate redundancy in mice.
Our analyses indicate that USP4 and USP15 arose from whole genome duplication prior to the emergence of jawed vertebrates. Despite having lower sequence identity USP11 was generated later in vertebrate evolution by small-scale duplication of the USP4-encoding region. While USP11 was subsequently lost in many vertebrate species, all available genomes retain a functional copy of either USP4 or USP15, and through genetic crosses of mice with inactivating mutations we have confirmed that viability is contingent on a functional copy of USP4 or USP15. Loss of ubiquitin-exchange regulation, constitutive skipping of the seventh exon and neural-specific expression patterns are derived states of USP11. Post-translational modification sites differ between USP4, USP15 and USP11 throughout evolution.
In isolation sequence alignments can generate erroneous USP gene phylogenies. Through a combination of methodologies the gene duplication events that gave rise to USP4, USP15, and USP11 have been established. Although it operates in the same molecular pathways as the other USPs, the rapid divergence of the more recently generated USP11 enzyme precludes its functional interchangeability with USP4 and USP15. Given their multiplicity of substrates the emergence (and in some cases subsequent loss) of these USP paralogs would be expected to alter the dynamics of the networks in which they are embedded.
Overexpression of these DUBs has been noted in various human cancers, which may be attributable to their collective regulation of oncogenic proteins. For instance, all three paralogs regulate the type I TGF-β receptor while USP15 and USP11 also regulate several of its downstream effectors [4, 12, 13]. Conversely, whereas USP4 and USP15 target p53-inhibiting ligases ARF-BP1  and MDM2 , respectively, USP11 stabilizes p53  as well as several other tumor suppressors including PML , BRCA2  and Mre11 complex members MRE11 & RAD50 . In sum, though these paralogs are functionally redundant in some capacities, each appears to have undergone substantial subfunctionalization and neofunctionalization. A summary of their known protein interactions is presented in Table 2.
To understand the evolutionary changes in sequence, structure, and function among these paralogs, it is very important to know the temporal sequence of duplication. This enables us to determine which are the ancestral states and which are the derived states that potentially represent adaptation in response to an ancient environment. This motivated us to do phylogenetic studies to characterize the branching pattern and the timing of duplication events. An integrative in silico approach probing these systematic changes in a comparative genomic framework was employed to trace the duplication and subsequent radiation of USP4, USP15 and USP11. We first quantified and characterized USP paralogs in a set of representative metazoan genomes and delineated their divergence times in reference to known whole genome duplication events. We then evaluated ortholog variability to gain insight into the evolutionary processes that gave rise to the three paralogs observable in humans.
Phylogenetics based on aligned nucleotide and amino acid sequence
List of coding sequences analyzed with corresponding accession numbers
Little brown bat
Green sea turtle
The branching pattern of Figs. 3 and 4 enables us to infer an approximate time for gene duplication events. The USP15 lineage splits from (USP4,USP11) during the period between the divergence of vertebrates from primitive chordates (from 581 to 460.6 millions of years ago, or MYA ) and the branching of shark from teleost (462.5 to 421.75 MYA ), corresponding to the timing of a known whole genome duplication event . A second gene duplication leads to the USP4/USP11 split which occurred in the common ancestor of bony fishes represented by gar, fugu, zebrafish and coelacanth (421.75 to 416 MYA ). USP11 is absent in shark. Given that the shark genome has evolved little , we may infer that the absence is ancestral instead of secondary loss, i.e., the USP4/USP11 split occurs after the divergence of shark from the ancestor of teleosts.
Synteny of USP11 and USP4 loci supports duplication in a Euteleostome ancestor
Summary of USP4, USP15 and USP11 interaction partners
Signature features of USP paralogs
First let us consider the molecular signatures of structured regions in USP4, USP15, and USP11. The “domain in USP” (DUSP) and “ubiquitin-like” (UBL) structured regions form the N-terminal domain that distinguishes this subgroup of USPs. DUSP-UBL domains mediate some enzyme-substrate interactions [9, 33–35] and confer intrinsic regulatory capacities that have been structurally modeled for mammalian USP4, USP15 and USP11 [20, 32, 35, 36]. For instance, USP4 dimerization occurs in equilibrium through this domain, while neither USP15 nor USP11 are expected to dimerize in vivo . The DUSP-UBL domain of USP4 also regulates ubiquitin active- site binding dynamics through its association with the unstructured insert region , though the absence of key residues impedes this regulatory function in human USP11 [32, 35]. The enzyme kinetics of USP15 are more similar to that of USP4 . Given our derivation of their duplication chronology, it seems likely that the loss of ubiquitin-exchange regulation in USP11 is a derived and not ancestral state, though structural information is available only for mammalian proteins. Fig. 7a presents an alignment of the DUSP-UBL domains of an ancestral USP with the signature sequences of USP4, USP15 and USP11. Lancelet was selected as the ancestral species because it is the closest single-copy relative (Fig. 2). In addition, the domain sequence is identical in Branchiostoma floridae and the newly sequenced B. belcheri, two lancelets that have experienced a high degree of protein evolution , suggesting that it is an accurate depiction of a pre-duplication USP. USP signature sequences indicate residues that are conserved in a majority of members from each phylogenetic clade (elimination of species-specific substitutions). While the key residues are largely conserved in USP4, USP15, and the ancestral USP, disruption of the hydrophobic pocket and shortening of DUSP-UBL linker [32, 35] are signatures of USP11. This derived state implies that USP11 has had a different mode of action throughout time.
The two parts of the structured catalytic domain of these USPs, D1 and D2, are the most highly conserved regions among paralogs and orthologs (Fig. 1). Both are required for catalytic activity, and their conservation extends beyond the USPs under current consideration to the entire USP subfamily of deubiquitinating enzymes.
Distinctive spliced isoforms
Whereas the seventh exon (E7) is alternatively spliced in USP4 and USP15, a corresponding exon is absent in USP11. The flexible linker region separating the DUSP-UBL and catalytic domains is roughly 20 residues long in USP11, its length in USP4 and USP15 short isoforms and the minimal length required for the aforementioned domain interaction . Shark USP4 and the lancelet single-copy USP, ancestral to USP11, contain E7; what is more, both long and short isoforms have been reported in chondrichthyes. Thus, the “permanent skipping” of E7 in all USP11 represents a derived state. Alterations in the stoichiometry of USP4 isoforms have been reported for a rare bone disease , though the functional consequences of E7 alternative splicing have not been studied. In all species, the polypeptides encoded by USP4 and USP15 E7 are serine-rich, and many serve as putative post-translational modification (PTM) sites as identified in large-scale studies on human proteins . In sum, the loss of E7 is a signature derived state of USP11 with potential functional or regulatory implications.
Altered cellular regulation
Post-translational modification (PTM) regulation can differ among gene duplicates. Some PTM sites are well conserved while others stably differentiate the USP paralogs in question. For one, Ser445 (a known Akt phosphorylation site ), there is conservation in all USP4, USP11, USP15 and ancestral homologs. There are, on the other hand, multiple cases wherein a putative phosphorylation site has been lost or gained in USP11 relative to its ancestor, USP4. Within the insert region, USP4 Ser675 and Ser680 (identified phosphorylation substrates in multiple studies [40–48]) are conserved in USP15 but absent from USP11. Similarly, in an alignment of all USPs, putative phosphorylation site USP4 Tyr539  is conserved in USP4 and USP15 while it is substituted by Phe in USP11. Slightly downstream, USP11 has Tyr551 (a reported phosphorylation site ) and Tyr554 whereas His and Phe, respectively, are universally present in USP4 and USP15. Still within the insert region, at positions 607 and 608, there exists in USP11 a pair of tyrosines that have been identified as phosphorylation sites in several large-scale studies . The region in question aligns poorly with other paralogs, though there are two reported, albeit low confidence, serine phosphorylation sites in USP4 and none in USP15. As previously mentioned, the alternatively spliced exon, lost in USP11, contains several reported phosphorylated serines in USP4 and USP15 .
The N and C-termini are remarkably different among USP4, USP11 and USP15. The N-terminus of USP11 is longer, more disordered and more hydrophobic (rich in alanine). In addition, the C-termini of all gnathostome USP15 present a segment rich in aspartic acid, glutamatic acid and asparagine (e.g. human: 962-DEDSNDNDNDIENEN-976; shark: 978-DEDCNENDVENEN-990), except those of teleost fish, which instead have C-terminal segment(s) exceptionally rich in glutamate (e.g. zebrafish: 775-EKEEEEEDEDEEDVNDSEQEED-795; tongue sole: 966-DEEDEEEEEEEEGEVEEEDEEEEEGRE-981, 1015-NEREDEEEEEEEEEEEEEEEQE-1035). A poly-E repetitive sequence is also found in USP11 of various organisms, including teleost fish, some reptiles, the opossum and the Chinese hamster. These regions are schematically highlighted in Fig. 2. Aspartic acid and asparagine residues can be hydroxylated , though it remains to be seen whether any hydroxylation of such residues occurs within the acidic domains of the USPs. In addition, the D- & N- rich C-terminus of non-teleost fish USP15 presents two validated serine phosphorylation sites [39, 50–55], absent from USP4, whereas human USP11 has seven of these sites [39, 50, 55–57] within its final 20 residues that are conserved among mammals. While many of these conserved and differential phosphorylation sites remain to be functionally characterized, most are all located within unstructured regions, namely the insert, linker, and C-terminal regions. This is consistent with reports that disordered region often serve as PTM substrates [58–61] and changes in PTM regulation contributes to the functional divergence of paralogs . In addition to phosphorylation and hydroxylation the disordered regions of the USPs may be subject to a number of other modifications including acetylation, methylation, and/or the addition of peptide moieties such as ubiquitin or SUMO. The contribution of this growing repertoire of PTMs to USP4, USP15, and USP11 regulation has yet to be established.
In sum, each paralog has distinctive signature features that represent common evolutionary categories namely structure-function innovations, distinctive spliced isoforms and altered cellular regulation. The fourth common differentiating trait, different spatiotemporal expression patterns, will be discussed in a later section.
Variable mechanisms of USP11 loss
In vivo demonstration of a minimal requirement for USP4 or USP15
Pooled progeny of USP4- and USP15-null mouse crosses
In the present work we have established the duplication chronology of a subgroup of highly networked ubiquitin-specific proteases, USP4, USP15 and USP11, and have characterized their subsequent radiation. According to the widely accepted 2R theory , vertebrate genomes have undergone two rounds of whole genome duplication (WGD). While it was conventionally assumed that these WGD events predated the divergence of jawless and jawed vertebrates , recent analysis of the elephant shark genome  placed at least one WGD event median to cyclostome and gnathostome divergence. In fact, subsequent studies have suggested that the 2R events occurred independently in cyclostomes and gnathostomes [63, 64], and that the former expansion was further shaped by an additional, lamprey-specific WGD. Thus, the USP15-like duplicate in lampreys is likely not orthologous to gnathostome USP4; rather, USP4 and USP15 appear to be ohnologs derived from WGD in a jawed vertebrate ancestor. In addition, the USP15-likeness of the lamprey version suggests that this paralog is the ancestral sequence, though there is no consensus among invertebrates as to whether their single copy most resembles USP4 or USP15 (Fig. 2). The issue cannot be settled by genomic synteny reconstruction, which is considerably more difficult in earlier species due to increased divergence time and the present lack of chromosomal assembly data for many species. In contrast, well-conserved intragenomic synteny points to the emergence of USP11 as the result of a more recent duplication event that does not coincide with any reported WGD event; it is likely the product of a small-scale duplication (SSD). The characterization of USP4/15 and USP4/11 duplications as WGD and SSD, respectively, corroborates well with reported trends for these phenomena: SSD-derived paralog sequences tend to evolve faster and are more functionally divergent .
Several notable differences in USP composition exist between and within clades. Primate species appear to have inconsistent USP repertoires: USP15 was inactivated by insertion of a LINE1 element in the gibbon, while erasure of USP11 and reduction of unstructured USP4 domains can both be observed in the gorilla. USP11 was also lost in the avian ancestor, inferred by the consistent absence of its genomic locus in all bird genomes (Fig. 8). Curiously, avian USP4 presents notable deviations from the signatures of this paralog: of the six bird genomes surveyed, all bear mutations in crucial residues for the ubiquitin-exchange mechanism , i.e. Arg40 and/or Met24 mutated in all, disruption of DUSP-UBL linker residues (a.a. 88–92) in chicken, QQD box region deleted in duck, and so on. In fact, USP4 is also more divergent in other species where USP11 was lost, which lead for example to the consistently incorrect branching patterns for frog and gorilla USP4 (Figs. 3 and 4; Additional file 1: Figure S1 and Additional file 2: Figure S2). What is more, avian USP4 adopts some USP11 signatures: these have collectively lost the Ser675 and Ser680 phosphorylation sites, while USP4 of the pigeon and zebra finch have also lost E7. The loss of these features that define all other USP4 (and USP15) is a derived state of USP11 and may thus represent a homoplastic convergence of avian USP4 toward the USP11 sequence.
Most of the signature features distinguish USP11 from USP4 and USP15, though the divergence of these last two is of practical interest due to their high protein sequence identity (Fig. 1), functional overlap (Table 2), and capacity for reciprocal rescue at the organismal level (Table 3). USP4 and USP15 differ in their codon usage: USP15, located in GC-poor isochores, employs more AT-ending codons than USP4. Low GC content is common in germ-line specific genes . USP15 is in fact expressed at notably elevated levels in mature oocytes  (oocytes being the cell type for which its expression is the highest in mice ) while USP4 is at low abundance throughout oocyte maturation . USP4 is predominantly expressed in somatic cells, particularly those of the immune system . The distinct spatiotemporal expression patterns of USP4 and USP15 could explain why these redundant proteins have been maintained: vertebrate genomes could optimally encode two versions of an ancestral protein to accommodate its important roles in germ and somatic cells. While we show that one functional copy of USP4 or USP15 is a minimum requirement for viability (Table 3), the observed departure from Mendelian ratios may arise from a functional deficiency in oocytes haploinsufficient for USP15. Planned experiments (including in vitro culture of early embryos) should be informative in this regard. The expansion of TGF-β pathway substrates in USP15 may reflect an enhanced role in the regulation of oocyte development [69–71], while USP4 may have become the USP of greater importance in innate immunity pathways, as reflected by an increased number of substrates (Table 2). Further, an inserted in-frame zebrafish-specific repetitive element has modified the USP15 catalytic domain coding sequence of this species. While it remains to be seen whether the enzymatic activity of USP15 has been altered or inactivated by this insertion, we anticipate that perturbation of USP15 will provide insights into DUB network rewiring in the zebrafish. As a model system that is amenable to the testing of hypotheses through genome manipulation, the zebrafish should be ideal for future investigations of the respective roles and expression patterns of USP4 and USP15. The expression pattern of USP11 is notably distinct: without exception in human, mouse, rat, and pig its expression is predominantly neuronal . In contrast to its paralogs , USP11 exerts a protective effect in glioma  as it stabilizes many tumor suppressors (Table 2).
Identification and proper annotation of homologs in an array of species is a first essential step in studying the evolution of duplicated genes. Coding sequences were retrieved from GenBank  and from genome project databases [62, 73, 74] using the well-annotated human sequences for USP4, USP15 and USP11 as tBLASTn queries . Reciprocal Best BLAST Hit (RBBH) annotation transfer was applied to unannotated genomes. Accession IDs for all sequences are below.
GC content analysis
The seqinr package in R was employed to generate plots for the GC content of the third codon positions (GC3) of the Homo sapiens USP4, USP15 and USP11 coding sequences using a sliding window of width 10. A heatmap of GC3 content was generated using Circos .
Species tree reconstruction
A taxonomic phylogeny was generated using PhyloT . Paralog affiliations were attributed as per their RBBH (described in Sequence retrieval). Putative non-processed pseudogene loci were confirmed using GenScan . The SynMap function in CoGe  enabled comparison of the synteny of USP neighbouring regions in Anolis carolinensis, Homo sapiens, and Gallus gallus, which was visualized using Circos.
USP4, USP15 and USP11 codon sequences were aligned using MUSCLE . The maximum-likelihood method using estimated transition/transversion ratio and F84 model, as implemented in DAMBE , was used to derive a phylogeny rooted on Amphimedon queenslandica. Molecular clock analyses were also conducted using DAMBE .
Codon alignments were produced by the MUSCLE algorithm using default GBlocks parameters as implemented in TranslatorX . AIC and LRT nucleotide substitution model tests in DAMBE  designated the Generalized Time Reversible (GTR) as most appropriate for all alignments. BEAST v.1.8.2 , a Bayesian Markov chain Monte Carlo (MCMC)-based phylogenetic dating program, was employed to quantify USP age-calibrated divergence times. All analyses used a log normal relaxed molecular clock. The USP4/USP11 analysis used six calibration points obtained from TimeTree  (in millions of years): Dog-Cow[USP4,USP11]: 60, Human-Opossum[USP4]: 112, Human-Anole [USP4, USP11]: 320 & Gar-Human [USP4]: 418. The RBM5/RBM10 analysis employed four pairs of calibration points: Human-Gorilla [RBM5,RBM10]: 8, Mouse-Rat[RBM5,RBM10]: 10.4, Human-Anole[RBM5,RBM10]: 320 & Human-Zebrafish [RBM5,RBM10]: 425. Tracer was used to verify similar convergence after 20 million steps for 3 runs in each case.
Mouse genetic crosses
TF2497 and TF2834 strains were purchased from Taconic Laboratories (Germantown, New York, USA), and were housed in a barrier facility at the University of Ottawa under protocol ME-305, approved by the Animal Care Committee, University of Ottawa. The strains were crossed to obtain mice heterozygous for proviral insertions in both the Usp4 and Usp15 genes. Eight pairs of compound heterozygous mice were mated under standard conditions, and progeny were obtained for genotyping. Genotyping was performed at 3–4 weeks of age, using tissue from ear punches. DNA was prepared using the REDExtract-N-Amp™ Tissue PCR Kit (Sigma-Aldrich Canada, Oakville, Ontario) and polymerase chain reaction was performed using the kit reagents. For genotyping of USP4 the forward primer used was derived from the third exon (upstream of the proviral insertion site): 5′- CCAGCAGCCTATTGTCAGAA -3′, where reverse primers were derived from the third intron (downstream of the proviral insertion site): 5′- TCAGTACTTAGGGATCTCTGA -3′ or from the neomycin phosphotransferase gene within the provirus: 5′- AACCTGCGTGCAATCCATCT -3′. Amplification conditions for USP4 were as follows: initial denaturation at 95C for 3 min followed by 30 cycles of 95C for 30 s, 57C for 30 s and 72C for 60 s and a final cycle at 72C for 5 min. A PCR product of approximately 250 base pairs was generated from the wild type gene, whereas the disrupted gene generated a product of approximately 1000 bp as detected by ethidium bromide staining of 1 % agarose gels. For USP15 a similar strategy was adopted using the forward primer: 5′ – GGTTTGAAGGATAACGTAGGC -3′, and reverse primers 5′ – ATAAACCCTCTTGCAGTTGCATC -3′ and 5′- GAGTACCTAACAGGCACTTGAGACG -3′. USP15 PCR conditions were similar except that annealing was done at 55C for 30 s and elongation at 72C was reduced to 45 s.
Availability of supporting data
This work was supported by the Natural Sciences and Engineering Research Council of Canada through grant RGPIN-2015-05879 (awarded to DAG). Caitlyn Vlasschaert was the recipient of a Frederick Banting and Charles Best Scholarship from the Canadian Institutes of Health Research. We thank Dr. Stéphane Aris-Brosou for his time and guidance in troubleshooting the BEAST phylogenetic analyses. We are grateful to David Cook for his help with Circos, and acknowledge the kind assistance of Tim Ramsay in the statistical analysis of mouse crosses.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Grabbe C, Husnjak K, Dikic I. The spatial and temporal organization of ubiquitin networks. Nat Rev Mol Cell Biol. 2011;12:295–307.PubMed CentralView ArticlePubMedGoogle Scholar
- Sowa ME, Bennett EJ, Gygi SP, Harper JW. Defining the human deubiquitinating enzyme interaction landscape. Cell. 2009;138:389–403.PubMed CentralView ArticlePubMedGoogle Scholar
- Aggarwal K, Massagué J. Ubiquitin removal in the TGF-β pathway. Nat Cell Biol. 2012;14:656–7.View ArticlePubMedGoogle Scholar
- Eichhorn PJA, Rodón L, Gonzàlez-Juncà A, Dirac A, Gili M, Martínez-Sáez E, et al. USP15 stabilizes TGF-β receptor I and promotes oncogenesis through the activation of TGF-β signaling in glioblastoma. Nat Med. 2012;18:429–35.View ArticlePubMedGoogle Scholar
- Zhao B, Schlesiger C, Masucci MG, Lindsten K. The ubiquitin specific protease 4 (USP4) is a new player in the Wnt signalling pathway. J Cell Mol Med. 2009;13:1886–95.View ArticlePubMedGoogle Scholar
- Fan Y-H, Yu Y, Mao R-F, Tan X-J, Xu G-F, Zhang H, et al. USP4 targets TAK1 to downregulate TNFα-induced NF-κB activation. Cell Death Differ. 2011;18:1547–60.PubMed CentralView ArticlePubMedGoogle Scholar
- Xiao N, Li H, Luo J, Wang R, Chen H, Chen J, et al. Ubiquitin-specific protease 4 (USP4) targets TRAF2 and TRAF6 for deubiquitination and inhibits TNFα-induced cancer cell migration. Biochem J. 2012;441:979–86.View ArticlePubMedGoogle Scholar
- Schweitzer K, Bozko PM, Dubiel W, Naumann M. CSN controls NF‐κB by deubiquitinylation of IκBα. EMBO J. 2007;26:1532–41.PubMed CentralView ArticlePubMedGoogle Scholar
- Song EJ, Werner SL, Neubauer J, Stegmeier F, Aspden J, Rio D, et al. The Prp19 complex and the Usp4Sart3 deubiquitinating enzyme control reversible ubiquitination at the spliceosome. Genes Dev. 2010;24:1434–47.PubMed CentralView ArticlePubMedGoogle Scholar
- Long L, Thelen JP, Furgason M, Haj-Yahya M, Brik A, Cheng D, et al. The U4/U6 recycling factor SART3 has histone chaperone activity and associates with USP15 to regulate H2B deubiquitination. J Biol Chem. 2014;289:8916–30.PubMed CentralView ArticlePubMedGoogle Scholar
- Breuer K, Foroushani AK, Laird MR, Chen C, Sribnaia A, Lo R, et al. InnateDB: systems biology of innate immunity and beyond—recent updates and continuing curation. Nucleic Acids Res. 2013;41:D1228–33.PubMed CentralView ArticlePubMedGoogle Scholar
- Al-Salihi MA, Herhaus L, Macartney T, Sapkota GP. USP11 augments TGFβ signalling by deubiquitylating ALK5. Open Biol. 2012;2:120063.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang L, Zhou F, Drabsch Y, Snaar-Jagalska E, Mickanin C, Huang H, et al. USP4 is regulated by Akt phosphorylation and deubiquitylates TGF-beta type I receptor. Nat Cell Biol. 2012;14:717–26.View ArticlePubMedGoogle Scholar
- Zhang X, Berger FG, Yang J, Lu X. USP4 inhibits p53 through deubiquitinating and stabilizing ARF‐BP1. EMBO J. 2011;30:2177–89.PubMed CentralView ArticlePubMedGoogle Scholar
- Zou Q, Jin J, Hu H, Li HS, Romano S, Xiao Y, et al. USP15 stabilizes MDM2 to mediate cancer-cell survival and inhibit antitumor T cell responses. Nat Immunol. 2014;15:562–70.PubMed CentralView ArticlePubMedGoogle Scholar
- Ke J, Dai C, Wu W, Gao J, Xia A, Liu G, et al. USP11 regulates p53 stability by deubiquitinating p53. J Zhejiang Univ Sci B. 2014;15:1032–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Wu H-C, Lin Y-C, Liu C-H, Chung H-C, Wang Y-T, Lin Y-W, et al. USP11 regulates PML stability to control Notch-induced malignancy in brain tumours. Nat Commun. 2014;5:3214.PubMedGoogle Scholar
- Schoenfeld AR, Apgar S, Dolios G, Wang R, Aaronson SA. BRCA2 is ubiquitinated in vivo and interacts with USP11, a deubiquitinating enzyme that exhibits prosurvival function in the cellular response to DNA damage. Mol Cell Biol. 2004;24:7444–55.PubMed CentralView ArticlePubMedGoogle Scholar
- Clague MJ, Barsukov I, Coulson JM, Liu H, Rigden DJ, Urbé S. Deubiquitylases from genes to organism. Physiol Rev. 2013;93:1289–315.View ArticlePubMedGoogle Scholar
- Elliott PR, Liu H, Pastok MW, Grossmann GJ, Rigden DJ, Clague MJ, et al. Structural variability of the ubiquitin specific protease DUSP-UBL double domains. FEBS Lett. 2011;585:3385–90.View ArticlePubMedGoogle Scholar
- Jacq X, Kemp M, Martin NMB, Jackson SP. Deubiquitylating enzymes and DNA damage response pathways. Cell Biochem Biophys. 2013;67:25–43.PubMed CentralView ArticlePubMedGoogle Scholar
- Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792–7.PubMed CentralView ArticlePubMedGoogle Scholar
- Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol. 2000;17:540–52.View ArticlePubMedGoogle Scholar
- Xia X. DAMBE5: a comprehensive software package for data analysis in. Mol Biol Evol. 2013;30:1720–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Vinogradov AE. Isochores and tissue-specificity. Nucleic Acids Res. 2003;31:5212–20.PubMed CentralView ArticlePubMedGoogle Scholar
- Rodin SN, Parkhomchuk DV. Position-associated GC asymmetry of gene duplicates. J Mol Evol. 2004;59:372–84.View ArticlePubMedGoogle Scholar
- Rodríguez-Ezpeleta N, Brinkmann H, Roure B, Lartillot N, Lang BF, Philippe H. Detecting and overcoming systematic errors in genome-scale phylogenies. Syst Biol. 2007;56:389–99.View ArticlePubMedGoogle Scholar
- Hedges SB, Dudley J, Kumar S. TimeTree: a public knowledge-base of divergence times among organisms. Bioinformatics. 2006;22:2971–2.View ArticlePubMedGoogle Scholar
- Dehal P, Boore JL. Two rounds of whole genome duplication in the ancestral vertebrate. PLoS Biol. 2005;3:e314.PubMed CentralView ArticlePubMedGoogle Scholar
- Venkatesh B, Lee AP, Ravi V, Maurya AK, Lian MM, Swann JB, et al. Elephant shark genome provides unique insights into gnathostome evolution. Nature. 2014;505:174–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Nguyen Ba AN, Strome B, Hua JJ, Desmond J, Gagnon-Arsenault I, Weiss EL, et al. Detecting functional divergence after gene duplication through evolutionary changes in posttranslational regulatory sequences. PLoS Comput Biol. 2014;10:e1003977.PubMed CentralView ArticlePubMedGoogle Scholar
- Clerici M, Luna-Vargas MPA, Faesen AC, Sixma TK. The DUSP-Ubl domain of USP4 enhances its catalytic efficiency by promoting ubiquitin exchange. Nat Commun. 2014;5:5399.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhao B, Velasco K, Sompallae R, Pfirrmann T, Masucci MG, Lindsten K. The ubiquitin specific protease-4 (USP4) interacts with the S9/Rpn6 subunit of the proteasome. Biochem Biophys Res Commun. 2012;427:490–6.View ArticlePubMedGoogle Scholar
- Hayes SD, Liu H, MacDonald E, Sanderson CM, Coulson JM, Clague MJ, et al. Direct and indirect control of mitogen-activated protein kinase pathway-associated components, BRAP/IMP E3 ubiquitin ligase and CRAF/RAF1 kinase, by the deubiquitylating enzyme USP15. J Biol Chem. 2012;287:43007–18.PubMed CentralView ArticlePubMedGoogle Scholar
- Harper S, Gratton HE, Cornaciu I, Oberer M, Scott DJ, Emsley J, et al. Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains. Biochemistry (Mosc). 2014;53:2966–78.View ArticleGoogle Scholar
- Harper S, Besong TMD, Emsley J, Scott DJ, Dreveny I. Structure of the USP15 N-terminal domains: a β-hairpin mediates close association between the DUSP and UBL domains. Biochemistry (Mosc). 2011;50:7995–8004.View ArticleGoogle Scholar
- Huang S, Chen Z, Yan X, Yu T, Huang G, Yan Q, et al. Decelerated genome evolution in modern vertebrates revealed by analysis of multiple lancelet genomes. Nat Commun. 2014;5:5896.PubMed CentralView ArticlePubMedGoogle Scholar
- Klinck R, Laberge G, Bisson M, McManus S, Michou L, Brown JP, et al. Alternative splicing in osteoclasts and Paget’s disease of bone. BMC Med Genet. 2014;15:98.PubMed CentralView ArticlePubMedGoogle Scholar
- Hornbeck PV, Kornhauser JM, Tkachev S, Zhang B, Skrzypek E, Murray B, et al. PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse. Nucleic Acids Res. 2012;40 (Database issue):D261–70.PubMed CentralView ArticlePubMedGoogle Scholar
- Zanivan S, Gnad F, Wickström SA, Geiger T, Macek B, Cox J, et al. Solid tumor proteome and phosphoproteome analysis by high resolution mass spectrometry. J Proteome Res. 2008;7:5314–26.View ArticlePubMedGoogle Scholar
- Huttlin EL, Jedrychowski MP, Elias JE, Goswami T, Rad R, Beausoleil SA, et al. A tissue-specific atlas of mouse protein phosphorylation and expression. Cell. 2010;143:1174–89.PubMed CentralView ArticlePubMedGoogle Scholar
- Yu Y, Yoon S-O, Poulogiannis G, Yang Q, Ma XM, Villén J, et al. Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling. Science. 2011;332:1322–6.PubMed CentralView ArticlePubMedGoogle Scholar
- Gnad F, Young A, Zhou W, Lyle K, Ong CC, Stokes MP, et al. Systems-wide analysis of K-Ras, Cdc42, and PAK4 signaling by quantitative phosphoproteomics. Mol Cell Proteomics. 2013;12:2070–80.PubMed CentralView ArticlePubMedGoogle Scholar
- Lundby A, Secher A, Lage K, Nordsborg NB, Dmytriyev A, Lundby C, et al. Quantitative maps of protein phosphorylation sites across 14 different rat organs and tissues. Nat Commun. 2012;3:876.PubMed CentralView ArticlePubMedGoogle Scholar
- Nishi H, Fong JH, Chang C, Teichmann SA, Panchenko AR. Regulation of protein-protein binding by coupling between phosphorylation and intrinsic disorder: analysis of human protein complexes. Mol Biosyst. 2013;9:1620–6.PubMed CentralView ArticlePubMedGoogle Scholar
- Trost M, Sauvageau M, Hérault O, Deleris P, Pomiès C, Chagraoui J, et al. Posttranslational regulation of self-renewal capacity: insights from proteome and phosphoproteome analyses of stem cell leukemia. Blood. 2012;120:e17–27.View ArticlePubMedGoogle Scholar
- Zhou H, Di Palma S, Preisinger C, Peng M, Polat AN, Heck AJR, et al. Toward a comprehensive characterization of a human cancer cell phosphoproteome. J Proteome Res. 2013;12:260–71.View ArticlePubMedGoogle Scholar
- Sharma K, D’Souza RCJ, Tyanova S, Schaab C, Wiśniewski JR, Cox J, et al. Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling. Cell Rep. 2014;8:1583–94.View ArticlePubMedGoogle Scholar
- Iyuke FO, Green JR, Willmore WG. Active Learning for the Prediction of Asparagine/Aspartate Hydroxylation Sites on Proteins. Calgary: ACTAPRESS; 2011.View ArticleGoogle Scholar
- Dephoure N, Zhou C, Villén J, Beausoleil SA, Bakalarski CE, Elledge SJ, et al. A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci U S A. 2008;105:10762–7.PubMed CentralView ArticlePubMedGoogle Scholar
- Gauci S, Helbig AO, Slijper M, Krijgsveld J, Heck AJR, Mohammed S. Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach. Anal Chem. 2009;81:4493–501.View ArticlePubMedGoogle Scholar
- Olsen JV, Vermeulen M, Santamaria A, Kumar C, Miller ML, Jensen LJ, et al. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci Signal. 2010;3:ra3.View ArticlePubMedGoogle Scholar
- Christensen GL, Kelstrup CD, Lyngsø C, Sarwar U, Bøgebo R, Sheikh SP, et al. Quantitative phosphoproteomics dissection of seven-transmembrane receptor signaling using full and biased agonists. Mol Cell Proteomics. 2010;9:1540–53.PubMed CentralView ArticlePubMedGoogle Scholar
- Phanstiel DH, Brumbaugh J, Wenger CD, Tian S, Probasco MD, Bailey DJ, et al. Proteomic and phosphoproteomic comparison of human ES and iPS cells. Nat Methods. 2011;8:821–7.PubMed CentralView ArticlePubMedGoogle Scholar
- Bian Y, Song C, Cheng K, Dong M, Wang F, Huang J, et al. An enzyme assisted RP-RPLC approach for in-depth analysis of human liver phosphoproteome. J Proteomics. 2014;96:253–62.View ArticlePubMedGoogle Scholar
- Van Hoof D, Muñoz J, Braam SR, Pinkse MWH, Linding R, Heck AJR, et al. Phosphorylation dynamics during early differentiation of human embryonic stem cells. Cell Stem Cell. 2009;5:214–26.View ArticlePubMedGoogle Scholar
- Kettenbach AN, Schweppe DK, Faherty BK, Pechenick D, Pletnev AA, Gerber SA. Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells. Sci Signal. 2011;4:rs5.View ArticlePubMedGoogle Scholar
- Kurotani A, Tokmakov AA, Kuroda Y, Fukami Y, Shinozaki K, Sakurai T. Correlations between predicted protein disorder and post-translational modifications in plants. Bioinformatics. 2014;30:1095–103.PubMed CentralView ArticleGoogle Scholar
- Vuzman D, Hoffman Y, Levy Y: Modulating protein-DNA interactions by post-translational modifications at disordered regions. Pac Symp Biocomput 2012:188–199.Google Scholar
- Dyson HJ, Wright PE. Intrinsically unstructured proteins and their functions. Nat Rev Mol Cell Biol. 2005;6:197–208.View ArticlePubMedGoogle Scholar
- Ma B, Nussinov R. Regulating highly dynamic unstructured proteins and their coding mRNAs. Genome Biol. 2009;10:204.PubMed CentralView ArticlePubMedGoogle Scholar
- Smith JJ, Kuraku S, Holt C, Sauka-Spengler T, Jiang N, Campbell MS, et al. Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution. Nat Genet. 2013;45:415–21. 421e1–2.PubMed CentralView ArticlePubMedGoogle Scholar
- Mehta TK, Ravi V, Yamasaki S, Lee AP, Lian MM, Tay B-H, et al. Evidence for at least six Hox clusters in the Japanese lamprey (Lethenteron japonicum). Proc Natl Acad Sci. 2013;110:16044–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Nah GSS, Tay B-H, Brenner S, Osato M, Venkatesh B. Characterization of the Runx gene family in a jawless vertebrate, the Japanese lamprey (lethenteron japonicum). PLoS One. 2014;9, e113445.PubMed CentralView ArticlePubMedGoogle Scholar
- Fares MA, Keane OM, Toft C, Carretero-Paulet L, Jones GW. The roles of whole-genome and small-scale duplications in the functional specialization of saccharomyces cerevisiae genes. PLoS Genet. 2013;9, e1003176.PubMed CentralView ArticlePubMedGoogle Scholar
- Kocabas AM, Crosby J, Ross PJ, Otu HH, Beyhan Z, Can H, et al. The transcriptome of human oocytes. Proc Natl Acad Sci. 2006;103:14027–32.PubMed CentralView ArticlePubMedGoogle Scholar
- Zimmermann P, Hennig L, Gruissem W. Gene-expression analysis and network discovery using genevestigator. Trends Plant Sci. 2005;10:407–9.View ArticlePubMedGoogle Scholar
- Chen J, Torcia S, Xie F, Lin C-J, Cakmak H, Franciosi F, et al. Somatic cells regulate maternal mRNA translation and developmental competence of mouse oocytes. Nat Cell Biol. 2013;15:1415–23.PubMed CentralView ArticlePubMedGoogle Scholar
- Dragovic RA, Ritter LJ, Schulz SJ, Amato F, Thompson JG, Armstrong DT, et al. Oocyte-secreted factor activation of SMAD 2/3 signaling enables initiation of mouse cumulus cell expansion. Biol Reprod. 2007;76:848–57.View ArticlePubMedGoogle Scholar
- Elvin JA, Yan C, Matzuk MM. Oocyte-expressed TGF-beta superfamily members in female fertility. Mol Cell Endocrinol. 2000;159:1–5.View ArticlePubMedGoogle Scholar
- Jackowska M, Kempisty B, Woźna M, Piotrowska H, Antosik P, Zawierucha P, et al. Differential expression of GDF9, TGFB1, TGFB2 and TGFB3 in porcine oocytes isolated from follicles of different size before and after culture in vitro. Acta Vet Hung. 2013;61:99–115.View ArticlePubMedGoogle Scholar
- Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Lipman DJ, Ostell J, et al. GenBank. Nucleic Acids Res. 2013;41(Database issue):D36–42.PubMed CentralView ArticlePubMedGoogle Scholar
- Simakov O, Marletaz F, Cho S-J, Edsinger-Gonzales E, Havlak P, Hellsten U, et al. Insights into bilaterian evolution from three spiralian genomes. Nature. 2013;493:526–31.PubMed CentralView ArticlePubMedGoogle Scholar
- Wyffels JL, King B, Vincent J, Chen C, Wu CH, Polson SW. SkateBase, an elasmobranch genome project and collection of molecular resources for chondrichthyan fishes. F1000Res. 2014;3:191.PubMed CentralPubMedGoogle Scholar
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.View ArticlePubMedGoogle Scholar
- Krzywinski MI, Schein JE, Birol I, Connors J, Gascoyne R, Horsman D, et al. Circos: an information aesthetic for comparative genomics. Genome Res. 2009;19:1639–45.PubMed CentralView ArticlePubMedGoogle Scholar
- Letunic I, Bork P. Interactive Tree Of Life v2: online annotation and display of phylogenetic trees made easy. Nucleic Acids Res. 2011;39 suppl 2:W475–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Burge C, Karlin S. Prediction of complete gene structures in human genomic DNA. J Mol Biol. 1997;268:78–94.View ArticlePubMedGoogle Scholar
- Lyons E, Pedersen B, Kane J, Freeling M. The value of nonmodel genomes and an example using SynMap within CoGe to dissect the hexaploidy that predates the Rosids. Trop Plant Biol. 2008;1:181–90.View ArticleGoogle Scholar
- Drummond AJ, Suchard MA, Xie D, Rambaut A. Bayesian Phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol. 2012;29:1969–73.PubMed CentralView ArticlePubMedGoogle Scholar
- Herhaus L, Al-Salihi MA, Dingwell KS, Cummins TD, Wasmus L, Vogt J, et al. USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling. Open Biol. 2014;4:140065.PubMed CentralView ArticlePubMedGoogle Scholar
- Blanchette P, Gilchrist CA, Baker RT, Gray DA. Association of UNP, a ubiquitin-specific protease, with the pocket proteins pRb, p107 and p130. Oncogene. 2001;20:5533–7.View ArticlePubMedGoogle Scholar
- Villeneuve NF, Tian W, Wu T, Sun Z, Lau A, Chapman E, et al. USP15 negatively regulates Nrf2 through deubiquitination of Keap1. Mol Cell. 2013;51:68–79.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang L, Zhao W, Zhang M, Wang P, Zhao K, Zhao X, et al. USP4 positively regulates RIG-I-mediated antiviral response through deubiquitination and stabilization of RIG-I. J Virol. 2013;87:4507–15.PubMed CentralView ArticlePubMedGoogle Scholar
- Pauli E-K, Chan YK, Davis ME, Gableske S, Wang MK, Feister KF, et al. The ubiquitin-specific protease USP15 promotes RIG-I-mediated antiviral signaling by deubiquitylating TRIM25. Sci Signal. 2014;7:ra3.PubMed CentralView ArticlePubMedGoogle Scholar
- Di Donato F, Chan EK, Askanase AD, Miranda-Carus M, Buyon JP. Interaction between 52 kDa SSA/Ro and deubiquitinating enzyme UnpEL: a clue to function. Int J Biochem Cell Biol. 2001;33:924–34.View ArticlePubMedGoogle Scholar
- Hou X, Wang L, Zhang L, Pan X, Zhao W. Ubiquitin-specific protease 4 promotes TNF-α-induced apoptosis by deubiquitination of RIP1 in head and neck squamous cell carcinoma. FEBS Lett. 2013;587:311–6.View ArticlePubMedGoogle Scholar
- Yamaguchi T, Kimura J, Miki Y, Yoshida K. The deubiquitinating enzyme USP11 controls an IkappaB kinase alpha (IKKalpha)-p53 signaling pathway in response to tumor necrosis factor alpha (TNFalpha). J Biol Chem. 2007;282:33943–8.View ArticlePubMedGoogle Scholar
- Sun W, Tan X, Shi Y, Xu G, Mao R, Gu X, et al. USP11 negatively regulates TNFalpha-induced NF-kappaB activation by targeting on IkappaBalpha. Cell Signal. 2010;22:386–94.PubMed CentralView ArticlePubMedGoogle Scholar
- Huang X, Langelotz C, Hetfeld-Pechoc BKJ, Schwenk W, Dubiel W. The COP9 signalosome mediates beta-catenin degradation by deneddylation and blocks adenomatous polyposis coli destruction via USP15. J Mol Biol. 2009;391:691–702.View ArticlePubMedGoogle Scholar
- Milojevic T, Reiterer V, Stefan E, Korkhov VM, Dorostkar MM, Ducza E, et al. The ubiquitin-specific protease Usp4 regulates the cell surface level of the A2A receptor. Mol Pharmacol. 2006;69:1083–94.View ArticlePubMedGoogle Scholar
- Maertens GN, El Messaoudi-Aubert S, Elderkin S, Hiom K, Peters G. Ubiquitin-specific proteases 7 and 11 modulate Polycomb regulation of the INK4a tumour suppressor. EMBO J. 2010;29:2553–65.PubMed CentralView ArticlePubMedGoogle Scholar