Cloning and Characterization of DULP, a Novel Ubiquitin-Like Molecule from Human Dendritic Cells

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1 Cellular & Molecular Immunology 27 Article Cloning and Characterization of, a Novel Ubiquitin-Like Molecule from Human Dendritic Cells Guoyan Liu 1, 2, Shuxun Liu 1, 2, Ping Li 1, Ling Tang 1, Yanmei Han 1, Huazhang An 1, Jiangyan Li 1, Xiankun Dai 1, Nan Li 1, Xuetao Cao 1 and Yizhi Yu 1, 3 We identified a novel ubiquitin-like molecule from human dendritic cells. contains a domain that shares 26% identity and 34% similarity with ubiquitin, and it possesses the corresponding Ile-44 hydrophobic patch used by mono- or poly-ubiquitin to interact with a ubiquitin-interaction motif (UIM) or ubiquitin-associated domain (UBA). Lysine residue corresponding to 6 of ubiquitin, which is involved in the formation of a multi-ubiquitin chain that can bind proteasomal subunit Rpn10/S5a, is also conserved in its ubiquitin-homology domain. However, does not possess the highly conserved C-terminus Gly-Gly required for ubiquitin conjugation or the Lys-48 required for the formation of polyubiquitin chain to target substrates for degradation, suggesting it might be a novel ubiquitin-domain protein (UDP). was found widely expressed in many cells and the ubiquitin-homology domain was not cleaved. We also confirmed that expression was enriched in the nucleus and much weaker in the cytosol. Besides, we found that overexpression of in 293T cells induced apoptosis, which might not be associated with the mitochondrial or proteasome pathway, with the specific mechanism remain unclear. Further investigations are needed to identify the precise biological functions of. Cellular & Molecular Immunology. 2009;6(1): Key Words: ubiquitin-like protein, dendritic cell, molecular cloning, apoptosis Introduction Ubiquitin-Proteasome System (UPS) is an important ATPdependent proteolytic system. Over the past twenty years most related studies have focused on its roles in regulating cellular processes, such as cell cycle progression, DNA repair, apoptosis, endocytosis of membrane receptors, and cellular location of some proteins. Over time, the proteinacious tag theme has been subsequently extended since the discovery of numerous ubiquitin-like proteins, such as NEDD8, UCRP, FAT10 and Sentrin family (1). As their names imply, these proteins contain domains that share a 15% to 60% identity with ubiquitin on the amino acid level and can be grouped in two separated classes: ubiquitin-domain proteins (UDPs) and ubiquitin-like modifiers (ULMs). UDPs bear a sequence 1 Institute of Immunology and State Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai , China; 2 Guoyan Liu and Shuxun Liu contributed equally to this study; 3 Correspondence to: Dr. Yizhi Yu, Institute of Immunology and National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai , China. Fax: , cjcb@sh163.net Received Jan 30, Accepted Feb 18, Copyright 2009 by The Chinese Society of Immunology domain similar to ubiquitin, but they are not conjugated to proteins. UBLs can be covalently conjugated to proteins by specific enzymatic cascades similar to those for ubiquitin conjugation (2, 3). Previously we identified a full-length novel gene from human dendritic cell cdna library by large-scale sequencing. Homology analysis showed that this novel molecule contained a domain similar to ubiquitin, suggesting it might be a novel ubiquitin-like protein. Therefore, we designated it as dendritic cell-derived ubiquitin-like protein () and submitted its sequence to GenBank database (accession No. AY037155). In this paper, we demonstrated that was widely found in many cells, predominately in the nucleus with weak cytosolic expression. We also found overexpression of induced apoptosis in 293T cells, which was not proteasome- or mitochondrion-dependent. Materials and Methods Cell lines and culture All cell lines were obtained from ATCC; human glioma cell line U251 was maintained in our laboratory. All cell lines were maintained in RPMI 1640 (Hyclone) or Dulbecco s modified Eagle s medium (Hyclone) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Hyclone), antibiotics, 2 mm glutamine, and 50 M 2-ME in a 5% CO 2 incubator at

2 28 Characterization of a Novel Ubiquitin-Like Molecule () 1 tgc ttc agg tgc ctg cga cct ccg cgc ctc ccg ccc gga agt gcc cga ggg ggg ccg cga tgg agc tgg ggg agc cgg gcg ctc ggt agc gcg gcg ggc aag gca ggc gcc ATG ACC CTG ATT GAA GGG GTG GGT GAT GAG GTG ACC GTC CTT TTC TCG GTG CTT GCC TGC 171 M T L I E G V G D E V T V L F S V L A C 172 CTT CTG GTG CTG GCC CTT GCC TGG GTC TCA ACG CAC ACC GCT GAG GGC GGG GAC CCA CTG 231 L L V L A L A W V S T H T A E G G D P L 232 CCC CAG CCG TCA GGG ACC CCA ACG CCA TCC CAG CCC AGC GCA GCC ATG GCA GCT ACC GAC 291 P Q P S G T P T P S Q P S A A M A A T D 292 AGC ATG AGA GGG GAG GCC CCA GGG GCA GAG ACC CCC AGC CTG AGA CAC AGA GGT CAA GCT 351 S M R G E A P G A E T P S L R H R G Q A 352 GCA CAG CCA GAG CCC AGC ACG GGG TTC ACA GCA ACA CCG CCA GCC CCG GAC TCC CCG CAG 411 A Q P E P S T G F T A T P P A P D S P Q 412 GAG CCC CTC GTG CTA CGG CTG AAA TTC CTC AAT GAT TCA GAG CAG GTG GCC AGG GCC TGG 471 E P L V L R L K F L N D S E Q V A R A W 472 CCC CAC GAC ACC ATT GGC TCC TTG AAA AGG ACC CAG TTT CCC GGC CGG GAA CAG CAG GTG 531 P H D T I G S L K R T Q F P G R E Q Q V 532 CGA CTC ATC TAC CAA GGG CAG CTG CTA GGC GAC GAC ACC CAG ACC CTG GGC AGC CTT CAC 591 R L I Y Q G Q L L G D D T Q T L G S L H 592 CTC CCT CCC AAC TGC GTT CTC CAC TGC CAC GTG TCC ACG AGA GTC GGT CCC CCA AAT CCC 651 L P P N C V L H C H V S T R V G P P N P 652 CCC TGC CCG CCG GGG TCC GAG CCC GGC CCC TCC GGG CTG GAA ATC GGC AGC CTG CTG CTG 711 P C P P G S E P G P S G L E I G S L L L 712 CCC CTG CTG CTC CTG CTG TTG CTG CTG CTC TGG TAC TGC CAG ATC CAG TAC CGG CCC TTC 771 P L L L L L L L L L W Y C Q I Q Y R P F 772 TTT CCC CTG ACC GCC ACT CTG GGC CTG GCC GGC TTC ACC CTG CTC CTC AGT CTC CTG GCC 831 F P L T A T L G L A G F T L L L S L L A 832 TTT GCC ATG TAC CGC CCG TAG 852 F A M Y R P 853 tgc ctc cgc ggg cgc ttg gca gcg tcg ccg gcc cct ccg gac ctt gct ccc cgc gcc gcg gcg gga gct gct gcc tgc cca ggc ccg cct ctc cgg cct gcc tct tcc cgc tgc cct gga gcc cag ccc tgc gcc gca gag gac tcc cgg gac tgg cgg agg ccc cgc cct gcg acc gcc ggg gct cgg ggc cac ctc ccg ggg ctg ctg acc ctc agc ccg cac tgg gag tgg gct cct cgg ggt cgg gca tct gct gtc gct gcc tcg gcc ccg ggc aga gcc ggg ccg ccc cgg ggg ccc gtc tta gtg ttc tgc cgg agg acc cag ccg cct cca atc cct gac agc tcc ttg ggc tga gtt ggg gac gcc agg tcg gtg gga ggc tgg tga agg gga gcg ggg agg ggc aga gga gtt ccc cgg aac ccg tgc aga tta aag taa ctg tga agt ttt caa aaa aaa aaa aaa aaa 1332 Figure 1. Nucleotide and amino acid sequence of full-length cdna encoding. The 5 and 3 UTR sequences are indicated by lowercases. The nucleotide and predicted amino acid sequences of were shown. Underlined nucleotide were start and stop codons. The upstream inframe stop codon and the poly(a) + tail are shadowed. The grey box denotes the ubiquitin domain. The data are available from GenBank under accession number AY C according to ATCC instructions. Isolation of full-length cdna The full-length cdna was isolated from human dendritic cell cdna library by large-scale random sequencing. A full-length cdna clone was found to potentially encode a protein containing a domain sharing great homology with ubiquitin. So we designated it as. The full-length sequence of human is available in GenBank database under accession NO. AY RT-PCR assay mrna expression profiles in various cell lines were analyzed by RT-PCR. Total RNA was extracted using the Trizol reagent (Invitrogen), and first strand cdna was synthesized from 1 g of the total RNA with an oligo dt 15 primer (Invitrogen). cdna synthesis was checked by PCR, with human -actin primers serving as a positive control. The amplification of fragment with the primers 5 -ATA AGA TCT CTC GTG CTA CGG CTG AAA-3 (sence) and 5 -GGA ATT CGC ACT ACG GGC GGT ACA TG-3 (antisence) was performed for 37 cycles (30 s at 94 C, 30 s at 50 C, 1.5 min at 72 C). Plasmid construction and cell transfection Expression plasmids containing were constructed by PCR-based amplification and subsequent insertion of the corresponding fragment into pcmv/myc vector (Invitrogen)

3 Cellular & Molecular Immunology 29 Figure 2. Multiple alignments of, human ubiquitin and other ubiquitin-like proteins sentrin-1 (SUMO-1), human NEDD8 and UCRP. Identical residues were boxed in black, and similar residues were in grey. Asterisks indicate conserved positions of Lys-6, Ile-44, Lys-48, Lys-63 and C-terminus Gly-Gly site in ubiquitin, respectively. to generate myc-tagged expression vector, with pcmv/myc serving as a mock control. The full-length coding region of cdna was also cloned into the VNG/GFP vector for GFP-tagged fusion protein expression. The recombinant plasmid was transiently transfected into 293T cells or MCF7 cells using Lipofectamine Reagent (Invitrogen) according to the protocol provided by the manufacturer. The transfected cells were harvested for analysis 48 h after transfection. Western blotting analysis Cells transiently transfected with myc-tagged expression vector or pcmv/myc mock vector were harvested and extracted with protein extraction reagent (Pierce) supplemented with proteinase inhibitors (Sigma) on ice. A BCA protein assay reagent kit (Pierce) was used to measure protein levels. Equal amount of total cell extracts were separated by 12% SDS-PAGE, and were transferred to nitrocellulose membranes (Schleicher-schnell Inc.). Membranes were probed with primary antibodies anti-myc (Santa Cruz Biotechnology, Santa Cruz, CA) and incubated with appropriate horseradish peroxidase (HRP)-coupled secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. The relevant protein bands were Volume 6 Number 1 visualized using enhanced chemiluminescence (Pierce) according to the manufacturer s introduction. Fluorescence confocal microscopic analysis MCF7 cells growing on glass coverslips placed in 6-well plates were transiently transfected with -GFP expression vectors. Forty-eight hours after transfection, cells were stained with Mitotrackor Orange CMTMRos (M-7510) specific for mitochondria, Brefeldin A BODIPY 558/568 conjugate isomer1 (B-7449) for ER and Glogi apparatus, and Lysotracker Red DND99 (L-7528) (Molecular Probes) for lysosome at 1 M for 20 min at 37 C in the dark. Cells were washed with PBS, fixed in 4% polyformaldehyde for 15 min at room temperature and observed by fluorescence confocal microscopy (LSM 510, Carl Zeiss). Apoptosis assay Apoptotic cells were analyzed by either R123-PI double staining or Annexin V-PI double staining according to the manufacturer s instructions. Cells were stained with 1 nm Rhodamine 123 (Molecular Probes) for 30 min at 37 C, protected from light. Then cells were harvested, washed and stained in 5 g/ml Propidium iodide for flow cytometric analysis. For Annexin V-PI staining, cells were harvested and February 2009

4 30 Characterization of a Novel Ubiquitin-Like Molecule () A SW620 MCF-7 A549 U251 A172 LS174T HT-29 Hela LoVo MCF7- MCF7- mock MCF7 28 kda B KG-1 THP-1 Molt-4 Hut78 NB4 K562 Jurkat -actin Figure 4. Western blotting analysis for pcmv- expression in transfected MCF7 cells. MCF7 cells were transiently transfected with pcmv- recombinant vector. After 48 h, cells were harvested for Western blotting using anti-myc tag antibody and HRP-secondary antibody. washed in PBS, then resuspended in pre-diluted binding buffer and stained with fluorescein isothiocyanate-conjugated Annexin V (Bender) for 10 min at room temperature, protected from light, washed again and resuspended in binding buffer. Propidium iodide (1 g/ml) was added and the cell suspension was immediately subjected to flow cytometric analysis. All of the data shown in this article are representative of at least three independent experiments. Results -actin Figure 3. RT-PCR analysis of expression of mrna in tumor cell lines. (A) Solid tumor cell lines SW620, MCF-7, A549, U251, A172, LS174T, HT-29, Hela and LoVo, respectively; (B) Leukemia and lymphoma cell lines KG-1, THP-1, Molt-4, Hut78, NB4, K562 and Jurkat, respectively. -actin was used as positive control (lower panel). Identification and sequence analysis of The cdna clone was directly isolated from a human dendritic cell (DC) cdna library by large-scale random sequencing. As shown in Figure 1, the full-length 1,332 bp cdna contained a single open reading frame (ORF) of 738 bp, potentially encoding a 246-amino-acid peptide with a theoretical molecular mass of 27 kda. Homology analysis by NCBI database showed that this novel molecule contained a domain spanning from 103 aa to 176 aa, which was 26% identical and 34% homologous to human ubiquitin (Figure 2). According to its primary structure and the homologous domain, we presumed that this novel molecule might be a member of the ubiquitin-like protein family. Therefore, we designated it as. With specific primer, we obtained the full-length cdna by PCR from human monocytederived dendritic cells. Amino acid alignments of, human ubiquitin and other ubiquitin-like proteins showed that contained the amino acid residues equivalent to the essential Ile-44 hydrophobic patch on the surface of ubiquitin. Ile-44 and the nearby residues of ubiquitin are important for interaction with ubiquitin-associated domain (UBA) and ubiquitininteraction motif (UIM) (4). Lysine residue corresponding to 6 of ubiquitin, which is involved in formation of a multi-ubiquitin chain that can bind proteasome subunit Rpn10/S5a (5), was also conserved in. As shown, ubiquitin, FAT10, NEDD8, UCRP and Sentrin-1 (SUMO-1) all had a diglycine motif which is required for conjugation formation (6-10). However, this highly conserved motif was substituted by Val-Gly in the ubiquitin-homology domain of. Furthermore, amino acid corresponding to the conserved Lys48 residue in ubiquitin is Gln in, which is identical to that in Sentrin-1 (11) (Figure 2). By BlastN-htgs of NCBI database, the gene was mapped in Chromosome 7q36.1 and close to FASTK (Fas-activated serine/threonine kinase) gene (data not shown). The genomic structure contained two exons and three introns. Expression pattern of the human The result of RT-PCR showed that mrna was widely expressed in many cells. Expression of mrna was found in solid tumor cells, such as glioblastoma A172,lung carcinoma A549, breast adenocarcinoma MCF-7 and colon adeno- carcinoma SW620 cells, but no expression was detected in LS174T, LoVo, HT-29, U251 and Hela cell lines (Figure 3A). mrna was also widely expressed in myeloid leukemia cells, including all cell lines we detected. Hut78 and K562 cells expressed mrna at a relative high level (Figure 3B). To observe the protein expression, we cloned full-length code region of into mammalian expression vector pcmv/myc and transfected it into MCF7 cells. After 48 h, we detected a single band of about 28 kda, which corresponded to the calculated molecule weight of myctagged protein in pcmv- transfected MCF7 cell lysate (Figure 4). Subcellular location of To identify the location of protein in cells, we constructed VNG/ expression vector, which expressed green fluorescent protein (GFP)-tagged protein, and transiently transfected it into MCF7 cells. As shown in Figure 5, GFP-tagged protein expression was highly enriched in the nucleus and weaker in the cytosol. Moreover, distribution of lysosome, mitochondria, ER or Golgi apparatus was not similar to distribution of.

5 Cellular & Molecular Immunology A BFA T Merge B Lysol 293Tmock 293T 28 kda Figure 6. Western blotting analysis for expression in transfected 293T cells. 293T cells were transiently transfected with pcmv-, the protein was detected by anti-myc antibody. Merge (data not shown). Mitochondria were reportedly involved in some cases of apoptosis; in this study, the colocalization of with mitochondria was not determined by confocal microscope analysis in 293T cells transfected with pcmv- (data not shown). C Mito Discussion Merge Figure 5. Subcellular location of protein in transfected MCF7 cells. MCF7 cells were transiently transfected with VNG, which expressed GFP-tagged protein. After transfection for 48 h, cells were respectively stained with red-fluorescence probes specific for ER and Golgi apparatus (A), lysosome (B), or mitochondrion (C). Overexpression of in 293T cells induced cell apoptosis To investigate the biological property of, we transfected several human normal and tumor cell lines with pcmv- recombinant expression vector transiently. Western blotting analysis detected a single band of about 28 kda, which corresponded to the calculated molecule weight of myc-tagged protein in pcmv- transfected 293T cells (Figure 6). Compared with the pcmv transfected cells, pcmv- transfected 293T cells underwent apoptosis; meanwhile, apoptosis was not detected in other cell lines transfected with pcmv-, including MCF7, A549 and Lovo (data not shown). Thirty-six hours after transfection with pcmv-, some 293T cells were weakly adherent and floated in the medium, losing their normal fusiform appearance (Figure 7). The results of flow cytometry showed that 293T cells underwent apoptosis after transfection with pcmv- (Figure 8). To uncover the underlying mechanism of the apoptosis, we used proteasome inhibitor (MG-132) to detect whether the apoptosis still occurs after the proteasome was blocked. We found that proteasome inhibitor did not block the apoptosis of 293T cells induced by overexpression of Volume 6 Number 1 The ubiquitin-proteasome system plays a fundamental regulatory role in eukaryotic organisms (12). It is not surprising that ubiquitin system also has an important role in the immune system, especially for transcriptional activation (13, 14). Most studies have focused on the following two aspects of the ubiquitin system: (1) ubiquitin system degrades receptors on the surface of immunological cells, ensuring a moderate immune response; (2) ubiquitin system controls the activation of NF-κB. Therefore, it seems that the immune system is the best to demonstrate the multiple roles of ubiquitin system, and the discovery of novel ubiquitin-like proteins in immune cells can help us to know more about the immune system. In this study, we identified a full-length novel ubiquitinlike gene by large-scale sequencing of human dendritic cell pcmv pcmv Figure 7. Apoptosis of 293T cells after transiently transfected with pcmv-. Cells were transiently transfected with pcmv or pcmv-. Cells were observed by microscope 36 h after transfection (magnification: left, 50 ; right, 100 ). February 2009

6 32 Characterization of a Novel Ubiquitin-Like Molecule () A PI B PI 293T 293T-mock 293T R T 293T-mock 293T Annexin V Figure 8. Apoptosis of 293T cells after transiently transfected with pcmv- detected by flow cytometry. (A) Apoptosis of 293T cells detected by R123/PI double staining. Cells were transiently transfected with pcmv (mock) or pcmv-. After 36 h, cells were harvested and the percentage of apoptotic cells was quantified by staining with Rhodamine123 and Propidium iodide (PI). The PI-single positive staining cells were considered to be apoptotic cells on the late stage or already being necrotic cells. (B) Apoptosis of 293T cells detected by Annexin V/PI double staining. Cells were transiently transfected with pcmv (mock) or pcmv-. After 36 h, cells were harvested and the Annexin V-single positive staining cells, Annexin V- and PI-double positive staining cells were considered to be apoptotic. cdna library. Dendritic cells are the key antigen-presenting cells which process the antigen and initiate immune response; we expected that the study of this novel ubiquitin-like molecule could contribute to our knowledge of the mechanism of immune response. Multiple alignment analysis of, ubiquitin and other ubiquitin-like proteins showed that ubiquitin, NEDD8, UCRP and Sentrin-1 (SUMO-1) all had a diglycine motif situated either directly at the carboxyl terminus of the UBL or within the sequence of a UBL precursor, which is required for conjugation formation (15-18), but the ubiquitin domain in did not possess the highly conserved C-terminus Gly-Gly for attachment to -amino group of substrate protein s Lys residues. Moreover, the sequence analysis also showed three conserved Lys residues within ubiquitin: Lys29, Lys48 and Lys63. These positions can potentially serve as sites for conjugate. It is believed that the way by which ubiquitin is linked in polyubiquitin chains decides the fate of the modified proteins. The targeting signal for the proteasome pathway is a polyubiquitin chain which uses Lys48 residue of the proximal ubiquitin for further rounds of ubiquitination. The formation of Lys63-linked ubiquitin polymers, in contrast, does not lead to degradation but seems to play a role in DNA repair and endocytosis (19). Among these ubiquitin-like proteins, Lys48 was conserved in the ubiquitin domains of NEDD8 and UCRP, while the ubiquitin domains in Sentrin-1 and possess Q48 instead of K48. Therefore, we speculated that this conserved Q48 residue might be essential for the biological function of. In the present study, we found that mrna was widely expressed in many cells. Ubiquitin is evenly distributed in both cytosol and nucleus. protein expression was shown in both the nucleus and cytosol, but was highly enriched in the nucleus, which was very similar to those of other two ubiquitin-like proteins: NEDD8 and SUMO-1 (20), suggesting that may mainly function in the nucleus. Ubiquitin system plays an important regulatory role in many cellular biological processes. The recent discovery of ubiquitin-like proteins suggests that post-translational modification of proteins is more common than previously expected (21-23). In contrast to ubiquitin, these proteins seem to play non-proteolytic roles. Conjugation of UCRP appears to target conjugates to the cytoskeleton. SUMO-1 was found covalently linked to RanGAP1, which is involved in the regulation of nucleocytoplasmic trafficking (24, 25). was widely expressed and it contained a ubquitin domain. We transfected several human normal and tumor cell lines with pcmv- recombinant expression vector and found that pcmv- transfected 293T cells undergoing apoptosis. In our further study we found that the proteasome inhibitor could not block overexpression-induced 293T apoptosis, so we speculated that proteasome might not be involved in this process. Confocal microscope analysis showed that overexpression-induced apoptosis of 293T cells was independent of mitochondrion. Over the past twenty years, the complexity of ubiquitination system is further compounded by the identification of some ubiquitin-like molecules. In this paper, we report a novel molecule cloned from human dendritic cells. contains a domain homology with ubiquitin and is widely expressed in many cells. expression is mainly restricted to cell nuclear, with minor cytosolic expression, but is not similar to the distribution of any organelles. Although we found that overexpression in 293T cells can induce cell apoptosis, the underlying mechanism and the precise biological property of need to be further studied. Acknowledgements We appreciate the technical assistance of Ms. Xianwei Ma. This work was supported by grants from the National Key Basic Research Program of China (2004CB518807, 2007CB512403), the National Natural Science Foundation of China ( , , ) and the Natural Science Foundation of Shanghai (06QA14069). References 1. Yeh ET, Gong L, Kamitani T. Ubiquitin-like proteins: new

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