A Manually Curated Network of the PML Nuclear Body Interactome
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Int. J. Biol. Sci. 2010, 6 51 International Journal of Biological Sciences 2010; 6(1):51-67 © Ivyspring International Publisher. All rights reserved Review A manually curated network of the PML nuclear body interactome reveals an important role for PML-NBs in SUMOylation dynamics Ellen Van Damme1, Kris Laukens2, Thanh Hai Dang2, Xaveer Van Ostade1 1. Laboratory of Protein Chemistry, Proteomics and Signal Transduction, Department of Biomedical Sciences, University of Antwerp (Campus Drie Eiken), Universiteitsplein 1 – Building T, 2610 Wilrijk, Belgium. 2. Intelligent Systems Laboratory (ISLab) Department of Mathematics and Computer Science, University of Antwerp (Campus Middelheim), Middelheimlaan 1 – Building G, 2020 Antwerp, Belgium Correspondence to: Prof. Dr. Xaveer Van Ostade, [email protected], tel: 0032 (0)3 365 23 19 Received: 2009.10.20; Accepted: 2010.01.09; Published: 2010.01.12 Abstract Promyelocytic Leukaemia Protein nuclear bodies (PML-NBs) are dynamic nuclear protein aggregates. To gain insight in PML-NB function, reductionist and high throughput techniques have been employed to identify PML-NB proteins. Here we present a manually curated network of the PML-NB interactome based on extensive literature review including data- base information. By compiling ‘the PML-ome’, we highlighted the presence of interactors in the Small Ubiquitin Like Modifier (SUMO) conjugation pathway. Additionally, we show an enrichment of SUMOylatable proteins in the PML-NBs through an in-house prediction algo- rithm. Therefore, based on the PML network, we hypothesize that PML-NBs may function as a nuclear SUMOylation hotspot. Key words: PML-NB, SUMOylation, Cytoscape, protein-protein interaction, network Introduction Nuclear domain 10 (also called Kremer bodies cross-brace conformation. This structure is ideal for or PODs) was first observed almost 50 years ago by the formation of larger protein aggregates and there- electron microscopy as nuclear dense granular bod- fore crucial in processes such as cell growth, onco- ies[1, 2]. These punctuate structures are now dubbed genesis, apoptosis, and RNA trafficking during viral “PML nuclear bodies” (PML-NBs) after their scaffold infections [5, 6]. The B-box provides the correct ori- protein Promyelocytic Leukaemia Protein (PML, also entation and alignment of the coiled-coil domain [7]. known as TRIM19 or MYL). The PML gene consists of When correctly positioned, the coiled-coil domain is nine exons and alternative splicing yields six nuclear responsible for homo and hetero-dimer interactions PML isoforms (PML I-VI) and one smaller cytoplas- between TRIM proteins[3]. The RBCC motif of the matic isoform (PMLVII). However, of these seven PML protein makes it possible to sustain a highly isoforms there are several alternative spliced variants dynamic turnover of partner proteins during the cell yielding at least 18 different forms of PML[3]. At the cycle [8, 9] which could explain the functionally pro- conserved N-terminus, PML contains a motif that miscuous nature of the PML-NBs. PML is not only defines the TRIM (Tripartite Motif) family of proteins. responsible for maintaining the integrity of the com- It is characterized by an RBCC motif composed of a plex but also for the recruitment and correct localiza- conserved RING domain, one or two B-boxes, and an tion of all PML-NB proteins, e.g. p53, Sp100, Daxx α-helical coiled-coil domain [4, 5]. The RING domain and CBP [4]. SUMOylation plays an important role in is a cysteine rich domain with zinc bound in a the scaffold function of PML. SUMO-3 attachment http://www.biolsci.org Int. J. Biol. Sci. 2010, 6 52 has been reported to be responsible for the correct way[14] (Figure 1). However, unlike ubiquitination, nuclear localization of PML [10], and without SUMO1 which targets proteins for degradation, SUMOylation modification several protein partners are not re- modifies proteins to interact in nuclear transport[10, cruited to the PML-NB [10-12]. However, unSU- 15], transcriptional regulation[14, 16], apoptosis[17, MOylatable mutants of PML (3KR) still show the 18] and protein trafficking[19]. typical nuclear speckle pattern [11] indicating that SUMO is synthesized as a precursor and its also other factors play a role in PML-NB formation. conjugation requires exposure of two C-terminal gly- Recently, PML was identified as the first protein de- cine residues. The processing of immature SUMO is graded by SUMO-dependent polyubiquitination after one of the three functions of SUMO specific proteases treatment with arsenic [12]. (SENPs). In addition, SENPs are also capable of dis- SUMOylation is a post-translational modifica- mantling SUMO chains and reversing SUMOylation tion where one of the four isoforms of the Small by cleaving the isopeptide bonds between SUMO and Ubiquitin-like Modifier (SUMO) protein is conjugated its protein targets or between the several SUMOs[20]. to a target protein as a single SUMO molecule Thus far, there are six known SENP isoforms (SUMO1) or in polymeric chains (SUMO2/3)[13]. The (SENP1-3 and SENP5-7)[20] with different assign- enzymatic cascade is similar to the ubiquitin path- ments in the cascade (Table 1). Immature SUMO ATP + UBA2 Maturation Activation AOS1 SENP SUMO GG SUMO GG UBA2 AOS1 K UBC9 Target De-SUMOylation UBA2 UBC9 SENP Target AOS1 SUMO GG K SUMO GG UBC9 Target Ligation of SUMO to protein target E3 Ligase Transfer to conjugation enzyme UBC9 Figure 1. Schematic representation of the SUMOylation pathway. Before conjugation, immature SUMO is processed by SUMO specific proteases (SENPs) to expose two C-terminal glycine residues required for conjugation. After processing, mature SUMO is activated in an ATP dependent manner by E1 enzymes AOS1 and UBA2. Subsequently, SUMO is trans- ferred to E2 conjugation enzyme UBC9 and finally conjugated to a target protein by an E3 ligase. SUMO conjugation can be reversed by protease activity of SENPs thereby releasing free SUMO and target. K= Lysine residue. Table 1. The SENP isoforms show different affinity for the several SUMO isoforms in maturation processing and de-conjugation. SENP isoform Maturation De-conjugation SENP1 SUMO1>SUMO2>>SUMO3[21] SUMO1, SUMO2[22, 23] SENP2 SUMO2>SUMO1, poor activity towards SUMO3[24, 25] SUMO2>SUMO1, poor activity towards SUMO3[24, 25] SENP3 No processing activity SUMO2, SUMO3[26, 27] SENP5 SUMO3[26;27] SUMO1[24, 25] SENP6 No processing activity[20, 28] SUMO2, SUMO3[29] SENP7 No processing activity[20, 28] SUMO2, SUMO3>>>SUMO1[28] http://www.biolsci.org Int. J. Biol. Sci. 2010, 6 53 After maturation, SUMO is activated in an ATP methyltransferases, histone deacetylases or DNA dependent manner by thio-ester bond formation with methyltransferases, suggesting that PML-NBs may the E1 activation proteins UBA2/AOS1[14]. The acti- play an important role as epigenetic regulator of vating E1 enzyme Uba2p was first identified in yeast transcription and replication (for a review see [67] where it is, together with Aos1, necessary and suffi- and references therein). The third hypothesis is that cient for Smt3p (yeast homologue of SUMO) activa- PML-NBs provide a nuclear platform for tion in vitro [30]. The human homologue hUBA2 was post-translational modification (PTM) such as phos- shown to act in the same way[31]. Subsequently phorylation [68, 69], acetylation[70] and SUMOyla- SUMO is transferred from UBA2 to the SUMO con- tion[71]. jugating enzyme UBC9 [14, 32]. UBC9 recognizes the These three hypotheses support the concept of consensus sequence ΨKXE where Ψ is a large hy- dynamic interactions with PML and a high turnover drophobic amino acid and K is the lysine for SUMO of protein associates, instead of a static complex with conjugation[33]. UBC9 is believed to be the only a fixed number of partners. The involvement of SUMO conjugating enzyme[32, 34] and SUMOylation PML-NBs in such a variety of processes has led to an of its binding partners is enhanced by interaction enormous research interest in this enigmatic protein with p14ARF[35]. Finally, an isopropyl bond between which has culminated in an overwhelming number of the terminal glycine of SUMO and the ε-aminogroup publications. In recent years, several techniques such of lysine of the target protein is formed by an E3 li- as affinity capture, yeast two hybrid, co-localization gase. Several unrelated proteins have been attributed and mass spectrometry have been employed to iden- E3 SUMO ligase activity: nucleoporin protein tify proteins of the PML complex which could lead to RANBP2[36], TOPORS[37, 38], several PIAS pro- a better understanding of the PML-NB function. This teins[39-41], polycomb group protein Pc2[42], and reductionist approach has successfully identified RNF4[43]. Recently an anti-SUMO ubiquitin-protein several of the components of the PML-NBs but added isopeptide ligase (E3) has been described : mel-18 in- more pieces to the puzzle instead of narrowing the teracts both with HSF2 and the SUMO E2 UBC9 and spectrum of possible functions. Therefore an integra- inhibits UBC9’s activity which decreases HSF2 SU- tive “Systems Biology” approach could be essential to MOylation[44]. grasp the true complexity of the PML-NBs and to al- Because of the many associated proteins, low us to generate hypotheses based upon the vast PML-NBs are involved in a wide variety of cellular amount of information available in distributed processes such as DNA damage and repair[45, 46], sources. apoptosis[47-49], senescence[50, 51], cell cycle[52], Not all PML partners described in literature are transcription[53], carcinogenesis[54] and virus infec- well represented in protein databases such as tion[55, 56]. Despite the involvement in such vital IntAct[72], Molecular Interactions Database processes, PML deficient mice are normal, fertile and (MINT)[73], BIOGRID[74], Human Protein Reference do not have a higher occurrence of tumors [57]. Al- Database (HPRD)[75] and Nuclear Protein Database though the actual function of the bodies still has to be (NPD)[76].