Báo cáo khoa học: Structural and functional analysis of ataxin-2 and ataxin-3 potx

Eur J Biochem 271, 3155–3170 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04245.x Structural and functional analysis of ataxin-2 and ataxin-3 Mario Albrecht1,*, Michael Golatta2,*, Ullrich Wullner3 and Thomas Lengauer1 ă Max-Planck-Institute for Informatics, Saarbruăcken, Germany; 2Institute for Medical Biometry, Informatics, and Epidemiology, University of Bonn, Germany; 3Department of Neurology, University of Bonn, Germany Spinocerebellar ataxia types (SCA2) and (SCA3) are autosomal-dominantly inherited, neurodegenerative diseases caused by CAG repeat expansions in the coding regions of the genes encoding ataxin-2 and ataxin-3, respectively To provide a rationale for further functional experiments, we explored the protein architectures of ataxin2 and ataxin-3 Using structure-based multiple sequence alignments of homologous proteins, we investigated domains, sequence motifs, and interaction partners Our analyses focused on presumably functional amino acids and the construction of tertiary structure models of the RNAbinding Lsm domain of ataxin-2 and the deubiquitinating Josephin domain of ataxin-3 We also speculate about distant evolutionary relationships of ubiquitin-binding UIM, GAT, UBA and CUE domains and helical ANTH and UBX domain extensions Spinocerebellar ataxia types (SCA2) and (SCA3) are autosomal-dominantly inherited, neurodegenerative disorders [1,2] SCA3 has also been known as Machado– Joseph disease (MJD), and SCA2 and SCA3 belong to a heterogeneous group of trinucleotide repeat disorders This group includes Huntington disease (HD), dentatorubralpallidoluysian atrophy (DRPLA), and other spinocerebellar ataxia types such as SCA1, SCA7 and SCA17 [3–7] The age of onset of SCA2 and SCA3 is in the third to fourth decade [8] The disorders share common phenotypic features such as the degeneration of specific vulnerable neuron populations and the presence of intracellular aggregations of the mutant proteins in affected neurons In contrast, the expression of the disease-associated genes occurs in a great variety of tissues and is not restricted to neuronal cells The SCA2 and SCA3/MJD genes have been mapped to chromosomes 12q24.1 and 14q32.1 [1,2] The common underlying genetic basis of SCA2 and SCA3 is the expansion of a CAG repeat region beyond a certain threshold These CAG repeats encode a polyglutamine (polyQ) tract in the respective proteins ataxin-2 and ataxin-3 The polyQ stretch in ataxin-2 lies near the N-terminus at the 5¢end of the coding region of exon [9], but the polyQ region of ataxin-3 is contained in exon 10 close to the C-terminus [10] While ataxin-2 is located predominantly in the Golgi apparatus [11], ataxin-3 is found in both the nucleus and the cytoplasm of cells [12] To provide a rationale for further experiments, we characterized the protein architectures of ataxin-2 and ataxin-3 and investigated domains, sequence motifs, and interaction partners To explore the functional implications, we assembled a multiple sequence alignment for the Lsm domain of ataxin-2 homologues including the yeast homologue Pbp1 We also constructed a 3D structural model for the RNA-binding Lsm domain of ataxin-2 Similarly, we used a structure-based multiple sequence alignment of the Josephin domain of ataxin-3 homologues to derive a 3D model of this domain and to analyse specific residues involved in deubiquitination Correspondence to M Albrecht, Max-Planck-Institute for Informatics, Stuhlsatzenhausweg 85, 66123 Saarbrucken, Germany ă E-mail: mario.albrecht@mpi-sb.mpg.de Abbreviations: A2BP1, ataxin-2 binding protein 1; DRPLA, dentatorubral pallidoluysian atrophy; DUB, deubiquitinating enzymes; HD, Huntington disease; MJD, Machado–Joseph disease; NLS, nuclear localization signal; OTU, otubains; PABP, poly(A)-binding protein; RMSD, root mean square deviation; SCA, spinocerebellar ataxia; SnRNPs, small nuclear ribonucleoproteins; UBP, ubiquitinspecific protease; UCH, ubiquitin C-terminal hydrolase; UIM, ubiquitin-interacting motif; VCP, valosin-containing protein *Note: M Albrecht and M Golatta contributed equally to this work (Received April 2004, accepted June 2004) Keywords: spinocerebellar ataxia; Machado–Joseph disease; polyglutamine disorder; ubiquitin; valosin-containing protein Materials and methods Protein sequences were retrieved from the NCBI [13], Ensembl [14], and SWISS-PROT/TrEMBL (SPTrEMBL) [15] databases and protein domain architectures from the Pfam [16] and SCOP [17] databases Sequence accession numbers are given in the respective figure legends and Tables S1 and S2 Species names are abbreviated by first letters (Table S3) Protein structures were obtained from the PDB database [18] The secondary structure assignments of PDB structures were taken from the DSSP database [19] A single capital letter appended to the actual PDB identifier denotes the chosen structure chain We used the PSI-BLAST suite of programs [20] to search for homologues (E-value cut-off 0.005) and the web servers PSIPRED [21], SAM-T99 [22], and SSpro2 [23] to predict the secondary structure of proteins and to form a consensus prediction by majority voting [24] To predict intrinsically unstructured and disordered regions in proteins, we explored the consensus of the results returned by the DisEMBL [25], DISOPRED [26], GlobPlot [27], NORSp [28] and PONDR [10] online 3156 M Albrecht et al (Eur J Biochem 271) servers The nuclear localization signals in ataxin-3 homologues were discovered with help of the prediction server PSORT II [29] Multiple sequence alignments were assembled by means of T-COFFEE [30] and improved manually by minor adjustments based on structure prediction results and pairwise structure superpositions computed by the program CE [31] The root mean square deviations (RMSDs) were taken from the CE superpositions We investigated the results of all state-of-the-art fold recognition methods available via the online meta-server BioInfo.PL [32], which contacts a dozen other state-of-the-art prediction servers (the names of which are listed on the web site http://Bioinfo.PL/Meta/) The associated 3D-Jury system allows for the comparison and evaluation of the predicted 3D models in a consensus view [33] To model the protein structure of ataxin-2 and ataxin3, we submitted the constructed sequence–structure alignments to the 3D modelling server WHAT IF [34] The sequence alignments depicted in the figures were prepared in the SEAVIEW editor [35] and illustrated by the web service ESPript [36] The protein structure images were drawn in the Accelrys Discovery Studio ViewerLight The online version of this manuscript contains supplementary material, and our web site will provide additional pictures Results and discussion Protein architecture of ataxin-2 Ataxin-2 has 1312 residues (including 22 glutamines of the polyQ stretch) and a molecular mass of  140 kDa Ataxin2 is a highly basic protein except for one acidic region (amino acid 254–475) containing 46 acidic amino acids (Fig 1) This region covers roughly exons 2–7 and is predicted to consist of two globular domains named Lsm (Like Sm, amino acid 254–345) [37] and LsmAD (Lsmassociated domain, amino acid 353–475) The LsmAD Ó FEBS 2004 domain of ataxin-2 contains both a clathrin-mediated trans-Golgi signal (YDS, amino acid 414–416) and an endoplasmic reticulum (ER) exit signal (ERD, amino acid 426–428) [11,38] It is composed mainly of a-helices according to the results from secondary structure prediction servers The rest of ataxin-2 outside of the Lsm and LsmAD domains is only weakly conserved in eukaryotic ataxin-2 homologues and is predicted to be intrinsically unstructured according to the consensus result from the DisEMBL, DISOPRED, GlobPlot, NORSp and PONDR online servers These nonglobular, flexible N- and C-terminal tails (amino acid 1–253 and 476–1312) contain the polyQ region (amino acid 166–187), several highly conserved short sequence motifs as possible protein interaction sites, and conspicuous (R)RG peptides at the C-terminus of the LsmAD domain One of the sequence motifs constitutes a putative PABP [poly(A)-binding protein] interacting motif PAM2 (amino acid 908–925) [39], and (R)RG peptides are well-known to bind RNA in other proteins [40] The N- and C-terminal tails of ataxin-2 also have a high content of proline (179 prolines out of 1090 amino acids, 16.4%) This property and the low complexity of unstructured sequence regions may lead to several significant, but probably false-positive, hits during a PSI-BLAST search for homologues of ataxin-2 For instance, despite the use of the standard low complexity filter, our PSI-BLAST search with human ataxin-2 homologues found several questionable hits outside globular domains to homologues of the polyglutamine DRPLA gene product atrophin For instance, starting the PSI-BLAST search with an Arabidopsis thaliana ataxin-2 homologue (SPTrEMBL: Q94AM9), human atrophin is retrieved in the third iteration with an E-value of · 10)11 Conversely, using the rat atrophin homologue (SPTrEMBL: Q62901) as the start sequence, human ataxin2 was detected in the second iteration with an E-value of · 10)04 Fig Protein architectures of human ataxin-2, its yeast homologue Pbp1, and the P falciparum homologue PF13_0048 of the decapping enzyme DCP2 (DCP2_Pf) Ó FEBS 2004 RNA binding of ataxin-2 The Lsm domain of ataxin-2 is typical of RNA-binding Sm and Sm-like proteins, which often form cyclic 6-, 7- or even 14-oligomers [41–43] Generally, Lsm domain proteins are involved in a variety of essential RNA processing events including RNA modification, pre-mRNA splicing, and mRNA decapping and degradation Some of them are also important components of spliceosomal small nuclear ribonucleoproteins (snRNPs) The LsmAD domain is contained in the Pfam database with the name Ataxin-2_N and also occurs in another, as yet uncharacterized Plasmodium falciparum/yoelii yoelii gene products PF13_0048/PY07327 without an Lsm domain (Fig 1) Both Plasmodium gene products have an additional N-terminal DCP2 domain (also termed box A), which is always followed by a NUDIX domain [44] in all known DCP2 homologues This NUDIX domain constitutes the catalytic subunit of the mRNA decapping holoenzyme DCP1–DCP2 [45,46] The physiological function of ataxin-2 and closely related eukaryotic homologues in RNA processing is as yet quite unexplored [47–50] Interestingly, ataxin-2 has been observed to interact with A2BP1 (ataxin-2 binding protein 1) [38], whose RNA-binding Caenorhabditis elegans homologue, fox-1, regulates tissue-specific alternative splicing [51] Disruption of the human A2BP1 gene may cause epilepsy or mental retardation [52] In addition, ataxin-2 shows significant homology to the yeast protein Pbp1 (Pab1/PABPbinding protein 1), which also contains the Lsm and LsmAD domains; regions outside of these two globular domains are predicted to be mainly unstructured in Pbp1 as in ataxin-2 Although the C-terminal tail of Pbp1 does not contain a PAM2 motif [39], this yeast protein regulates polyadenylation after pre-mRNA splicing and interacts with the C-terminal part of the yeast homologue PAB1 of the human PABP [53] A2BP1 and PABP are also evolutionarily related and possess RNA recognition motifs [38] These observations strongly suggest that ataxin-2 is involved in similar mRNA processing tasks Structural modelling of ataxin-2 First, we compiled a list of ataxin-2 homologues including the yeast homologue Pbp1 and several Lsm domains of snRNPs and other Sm and Sm-like proteins from various species Then, we assembled a structure-based multiple sequence alignment of the Lsm domains, crystallographically determined structures of which reveal a close structural homology between archaeal and eukaryotic proteins (Fig 2) [42,43,54–65] This suggests that the function and the RNA-binding mode of the Lsm domain have been preserved during evolution The RNA-binding Lsm domain is characterized by a conserved sequence motif consisting of two short segments known as Sm1 and Sm2, which are separated by a variable linker [66,67] The very strong conservation of certain glycine residues is especially striking and also demonstrates the evolutionary relationship of ataxin-2 to Lsm domain proteins The amide groups of the glycines are known to stabilize the protein structure when forming hydrogen Analysis of ataxins and (Eur J Biochem 271) 3157 bonds to adjacent b-strands [55] The secondary structure predictions of ataxin-2 and its yeast homologue Pbp1 are also in good agreement with the known structure of the Lsm domain as open b-barrel, consisting of an N-terminal a-helix followed by a strongly bent five-stranded antiparallel b-sheet with a 310 helical turn in some cases before the fifth b-strand The top two alignment rows in Fig show human ataxin-2 aligned with the Pyrococcus abyssi Sm1 protein (PDB identifier 1m8v, chain A), the crystal structure of which consists of a heptameric ring with a central cavity like other Lsm domain oligomers [65] This Sm1 protein provides the only Lsm domain structure, which is bound to RNA inside and outside of the doughnut-shaped ring at an internal and an external binding site Therefore, we used this alignment of ataxin-2 to Sm1 to model the 3D structure of the Lsm domain of ataxin-2 in complex with RNA and Lsm domains of ataxin-2 protomers (Fig 3) Functional analysis of the Lsm domain We applied the same colour scheme to functionally relevant residues shown in the multiple sequence alignment and the 3D model of ataxin-2 (Figs and 3) Based on the crystal structure of Sm1 from P abyssi bound to uridine heptamers (U7), we marked several amino acids in Sm1, which are involved in RNA binding [65] and are mostly physicochemically conserved in ataxin-2 (Sm1/ataxin-2 residue numbers) The residues forming the internal U7 binding site are H37/K299, N39/L302 and R63/K330, while ionic interactions between K22/K284, R63/K330 and D65/S332 stabilize the RNA-binding area The residues involved in the external U7 binding site are R4/R266, H10/T272 and Y34/ Y296, stabilized by a hydrogen bond between H10/T272 and Y34/Y296 It is interesting to note that Sm1 from P abyssi and from Archaeoglobus fulgidus (PDB identifier 1i4k, chain A) share identical RNA-binding residues except for H10, which is replaced by an asparagine [59,65] Furthermore, we investigated whether ataxin-2 may also form oligomers through the Lsm domain To this end, we used the detailed crystal structure analyses of the very similar snRNP heterodimers D1–D2 and D3–B [55] Because of analogous intermolecular interactions in both dimers, we focused on the complex of D3 with B This complex is stabilized mainly by the pairing of the fifth b-strand (b5) from D3 with the fourth b-strand (b4) from B (D3/ataxin-2– B/ataxin-2): R69/V335–R73/K330, L71/V337–L71/L328, and L73/F339–L69/S326 In addition, two hydrophobic clusters formed by residues of D3 and B contribute to the stability of the dimer The first cluster includes F70/V336 and I72/Q338 (both in b5 strand) of D3 and F27/Y289 (b2 strand), L67/M324, V70/I327 and L72/L328 (all in b4 strand) of B The second cluster consists of P6/M267, L10/ L271 (both in a-helix), V18/C279 (b1 strand), L32/F293 (b2 strand), I33/K294 (loop after b2 strand), I68/F334, L71/ V337 and L73/F339 (all in b5 strand) of D3 and I41/L304, C43/A306 (both in b3), L69/S326 and L71/L328 (both in b4) of B Stacking interactions between guanidinium groups of arginines R69/V335 of D3 and R25/G287 and R49/T312 of B as well as an ionic interaction between E21/Q282 of D3 and R65/S322 of B stabilize the dimer further However, the latter salt bridge is not observed in the D1–D2 complex Fig Structure-based multiple sequence alignment of the Lsm domains of ataxin-2 homologues including the yeast homologue Pbp1 (upper part) with Sm and Sm-like proteins (lower part) The known DSSP secondary structure assignment of the Sm1 protein from P abyssi is shown at the top of the alignment (cylinder for a-helix, arrow for b-strand), and the amino acid sequences of crystallographically determined PDB structures of Lsm domains are underlined accordingly (curled line for a-helix, straight line for b-strand) The corresponding secondary structure predictions for the Lsm domains of ataxin2 and Pbp1 are also given Physico-chemically similar amino acids are coloured identically The highly conserved glycines characteristic of Lsm domains are indicated In the upper part, blue text boxes point to functionally relevant amino acids forming an internal binding site for uridine heptamers bound to Sm1 from P abyssi, and green text boxes mark amino acids of the external RNA binding site In the lower part, orange text labels annotate how the dimerization of the snRNPs D3 and B is stabilized by intermolecular interactions PDB identifiers and corresponding SPTrEMBL accession numbers for Lsm proteins are given in Table S2 3158 M Albrecht et al (Eur J Biochem 271) Ó FEBS 2004 Ó FEBS 2004 Analysis of ataxins and (Eur J Biochem 271) 3159 Fig 3D model of the Lsm domain of ataxin-2 using three adjacent protomers of the Sm1 protein from P abyssi as template (PDB identifier 1m8v, chain A, B and G) The model illustrates predicted internal (blue) and external (green) binding sites of ataxin-2 to RNA (grey) a-Helices are in shown in red, b-strands are shown in cyan Only functionally relevant residues of the central ataxin-2 protomer are annotated as follows: dark blue boxes point to residues forming the internal site, and light blue boxes mark amino acids stabilizing the RNA binding area; dark green boxes highlight residues involved in the external site, and light green ones indicate stabilizing hydrogen bonds despite identical amino acids Altogether, the degree of conservation of amino acids relevant for heterodimerization is only moderate, but may still suggest that ataxin-2 may form Lsm domain oligomers Protein architecture of ataxin-3 The longest splice variant of ataxin-3 possesses 376 amino acids (including 22 glutamines of the polyQ stretch, amino acid 296–317) and an approximate molecular weight of 42 kDa Ataxin-3 consists of a globular deubiquitinating N-terminal Josephin domain (amino acid 1–170) [68,69] and a flexible C-terminal tail containing two ubiquitin-interacting motifs (UIMs) [70] (also termed LALAL motifs and PUBs [71], amino acid 223–240 and 243–260) and the polyQ region (amino acid 296–317) (Fig 4) [72] A slightly shorter alternative splice variant of ataxin-3 with 373 amino acids has a third UIM (amino acid 334–351) at the C-terminus An as yet uncharacterized ataxin-3 paralogue on the X chromosome (sequence identity 70%) is expressed in testis (ataxin-3t) [10] The Josephin domain is also found without a C-terminal tail in other, as yet uncharacterized, proteins named josephins (Fig 5) [73] A highly conserved, putative nuclear localization signal (NLS) is found upstream of the polyQ stretch (RKRR, amino acid 282–285), which may be bipartite in the Caenorhabditis elegans homologue of ataxin-3, consisting of 17 residues (RRDRQKFLERFEKKKEE, amino acid 296–312) This NLS follows a potential casein kinase II (CK-II) phosphorylation site (TSEE, amino acid 277–280), which may determine the rate of the observed ataxin-3 transport into the nucleus [74] Ataxin-3 may also contain a nuclear export signal (NES) following the Josephin domain (ADQLLQMIRV, amino acid 174–183) based on our comparison with a published sequence profile of nuclear export signals [75] Furthermore, ataxin-3 contains several conserved sequence motifs similar to NR- and CoRNRboxes L-x-x-L-L/[IL]-x-x-[IV]-I of transcriptional coactivators and corepressors, respectively [73] Indeed, ataxin-3 interacts with histones and the histone acetyltransferases CBP, p300, and PCAF, which work as transcriptional coactivators In particular, dependent on these cofactors, ataxin-3 represses histone acetylation and transcription [76], and altered protein acetylation has already been implicated in polyglutamine disease processes [77] Generally, the (de-)ubiquitination of histones has been linked to transcriptional regulation [78], which may also explain the observed interactions of ataxin-3 Ataxin-3 is evolutionarily conserved in eukaryotes including P falciparum and plants, but not yeast The P falciparum homologue PFL1295w of ataxin-3 (ataxin-3_Pf), whose gene expression is upregulated similarly to the P falciparum josephin homologue PF11_0125 in gametocytes [79–81], constitutes an exception because it has only the second UIM conserved (amino acid 250–267) and has an additional ubiquitin-like UBX domain [82–85] at the Ó FEBS 2004 3160 M Albrecht et al (Eur J Biochem 271) Fig Protein architectures of human ataxin-3, its P falciparum homologue PFL1295w (ataxin-3_Pf), and human josephin C-terminus (amino acid 271–381) instead of the polyQcontaining region [69] Like human ataxin-3, this ataxin-3 homologue PFL1295w also has a potential casein kinase II phosphorylation site (TSDE, amino acid 278–281) close to basic amino acids, which can be indicative of an NLS (KKIH, amino acid 293–296) near the N-terminus of the UBX domain In contrast, the prediction server PSORT II returns another region inside the UBX domain as a possible NLS (PRRK, amino acid 339–342) It is unclear which NLS motif may be functionally more relevant because both NLS motifs correspond to amino acids at solvent exposed N-termini of the second and fourth b-strand in the crystal structure of the UBX domain of the cofactor p47 (PDB identifier 1s3s) [86] Similar to the P falciparum homologue, the Cryptosporidium parvum homologue of ataxin-3 also possesses only one UIM motif (amino acid 266–283) and a C-terminal UBX domain (amino acid 288–397) instead of a polyQ region Ubiquitin binding of ataxin-3 Ubiquitination fulfills many cellular functions in cytoplasmic trafficking, guiding specific proteins through the endocytic pathways, and targeting proteins to the protea- some [84,87–93] Above all, the ubiquitin–proteasomal pathway is involved in processing mutant or damaged proteins that cause neurodegenerative diseases The small ubiquitin protein can be covalently linked to other proteins as single molecule or polyubiquitin chain Recently, the two UIMs between the Josephin domain and the polyQ stretch of ataxin-3 have been shown to be capable of binding tetraubiquitin and polyubiquitinated proteins [68,94–97] In our previous study, we used the C-terminal ANTH domain extension, which consists of an antiparallel three-helix bundle, to model the structure of the UIMs in the C-terminal tail of ataxin-3 [73] In fact, novel structure determinations have shown that UIM peptides are a-helices and can form helix bundles in the crystal structure [98] In contrast, the NMR solution structures of UIM peptides reveal that they are single amphiphatic a-helices connected by unstructured linkers [99,100] The latter observation is in agreement with the observed flexibility of the C-terminal tail of ataxin-3 [72] Furthermore, the ANTH domain itself is evolutionarily, structurally, and functionally related to a VHS domain [101] Lately, the structure of the GAT (GGAs and Tom1) domain directly following the VHS domain of Tom1 and GGAs (Golgi-associate, c-adaptin ear-containing, Arfbinding proteins) was determined crystallographically [102–105] The GAT domain contains a three-helix bundle, which we found to superimpose very well with the helical bundle of the C-terminal ANTH domain extension (RMSD ˚ 3.1 A, PDB identifiers 1o3x and 1hx8, A chains) Interestingly, the GAT domains of GGAs and Tom1 have been reported to interact with ubiquitin [106–108] The corresponding ubiquitin binding site was located to the third a-helix of the GAT three-helix bundle, and hydrophobic amino acids like leucines are important for the interaction (Fig 5) The same residue type also plays an essential role in binding ubiquitin to the UIM a-helix [98– 100] and the third a-helix of the helical bundle in the homologous CUE and UBA domains [109] However, the sequence similarity is quite low, and thus it is difficult to deduce an evolutionary relationship, although the ubiquitin binding sequence of GGAs and Tom1 resembles a noncanonical UIM whose, otherwise strictly conserved, serine residue is replaced by an asparagine except in case of human GGA3 (Fig 5) Further interaction partners of ataxin-3 It has been shown that ataxin-3 interacts with the ubiquitinlike (UBL) domain of the homologous ubiquitin- and proteasome-binding factors hHR23A and hHR23B, whose yeast orthologue is Rad23 [96,110–112] The latter factors are also involved in the nucleotide excision repair pathway by targeting the ubiquitinated nucleotide excision repair factor XPC/Rad4 to the proteasome [113] Their UBL domain binds to a UIM helix of the 26S proteasome subunit S5a, and this interaction disrupts the interdomain contacts between the N-terminal ubiquitin-mimicking UBL domain and the two C-terminal ubiquitin-binding UBA domains, thereby inducing the change from a closed to an open protein conformation [109,111,114,115] Rad23 and the yeast orthologue Rpn10 of S5a serve as alternative ubiquitin receptors for the proteasome [116], and the UBA domains Ó FEBS 2004 Analysis of ataxins and (Eur J Biochem 271) 3161 Fig Multiple sequence alignment of UIM peptides, divided into groups by horizontal lines from top to bottom: UIM sequences of the Pfam seed alignment including first, second, and third UIMs of ataxin-3 homologues, UIM-like peptides from GGAs and Tom1, and related AP180 sequences The latter are derived from the 3D structure superposition of the GAT domain of human GGA1 with the AP180 extensions from Rattus norvegicus and D melanogaster (PDB identifiers 1hf8 and 1hx8, respectively) The second group of UIMs in ataxin-3 homologues also includes the similar N-terminal a-helix of the UBX domain extension of p47 (PDB identifier 1s3s) For each group, amino acids in alignment columns with a majority of identical residues are printed on a black background, and similar amino acids are highlighted in grey of Rad23 inhibit proteasome-catalysed proteolysis by sequestering Lys48-linked polyubiquitin chains [117,118] In particular, the NMR solution structures of the UBL domain of hHR23A/B bound to a UIM peptide of S5a [99,119] could be used to model the complex of hHR23A/B and ataxin-3 Similarly, the complex of a UIM of ataxin-3 with ubiquitin could be modelled based on the NMR solution structure of the UIM of the Vps27 protein bound to ubiquitin [100] The C-terminal region of ataxin-3 including the polyQ region interacts with the N-terminal cofactor/substratebinding adaptor domain of the valosin-containing protein VCP/p97/Cdc48/VAT/ter94 [96,120–123] VCP is an important multifunctional AAA+ ATPase with two Cterminal ATPase domains after the adaptor domain, which provide the energy for major conformational changes [124] VCP forms hexamers and works as molecular chaperone involved in a variety of intracellular functions including cell cycle progression, membrane fusion, vesicle-mediated transport, transcription activation, apoptosis prevention, and ubiquitin-proteasome degradation, modulating polyglutamine-induced neurodegeneration [96,120–123,125–127] VCP binds the ubiquitin E3 ligase and the chain assembly factor UFD2a/E4B, which is a U box homologue of yeast Ufd2 [128], and interacts with and regulates the degradation of the proteasome-associated ataxin-3, forming a trimeric complex of ataxin-3, VCP, and UFD2a [96,127,129–131] Interestingly, Ufd2 binds the UBL domain of Rad23 and competes with Rad23 for binding to the Rpn1 proteasome subunit, while the N-terminal UBL domain of the ubiquitin C-terminal hydrolase Ubp6 interacts with Rpn1 without competition with Rad23 [116,132] Furthermore, VCP also binds the C-terminal UBX domain of the membrane fusion adaptor p47/SHP1/EYC/ Ubx3 [85,86,133], which consists of three domains UBASEP-UBX [134] The crystallographically determined complex of the N-terminal adaptor domain of VCP with this UBX domain (PDB identifier 1s3s) indicates the interacting residues [86] and could be used to model the putative complex of VCP with the C-terminal UBX domain of the ataxin-3 homologue from P falciparum (ataxin-3_Pf) Like the UBX domain of p47, ataxin-3_Pf contains the conserved loop that is essential for an interaction with VCP because it inserts into a hydrophobic pocket of VCP [86] The UBX domain structure of p47 is extended at its N-terminus by a disordered peptide structure and an additional a-helix of as yet unknown functional relevance [86] The length of this a-helix is similar to a UIM a-helix (Fig 5), and such a UIM also precedes the UBX domain of ataxin-3_Pf Therefore, this a-helix of p47 might be related to the second UIM in ataxin-3 homologues (recall that the first UIM is missing in ataxin-3_Pf) In addition, the arrangement of one UIM helix followed by a C-terminal UBX domain is also found in the cofactor Ubx2 with domain architecture UBA-UAS- 3162 M Albrecht et al (Eur J Biochem 271) UIM-UBX [133] The UIM of Ubx2 binds ubiquitin chains, and the UBX domain interacts with VCP Thus the same interactions can be expected for ataxin-3_Pf The C-terminal, presumably VCP-binding, UBX domain of ataxin-3_Pf appears to correspond to the VCP-binding C-terminal part of human ataxin-3, which follows the second UIM and includes the polyQ region [120,123,131] In addition, the polyQ tract of ataxin-3 has been shown to be indispensable for the interaction with VCP, and its length correlates with the strength of the interaction These observations raise the question how human ataxin-3 binds VCP in contrast to its P falciparum homologue This is particularly interesting because VCP may suppress polyQ induced neurodegeneration, and mutations in VCP have been observed to cause cytoplasmic vacuoles followed by cell death because of a dysfunctional second ATPase domain and inclusion body formation [120–123,127,135,136] We also observed that all VCP sequence variations associated with Paget disease of bone and frontotemporal dementia (IBMPFD) [135] are not located in the binding interface of a UBX domain with the N-terminal adaptor domain of VCP, but are involved in interactions between protein regions (for details see the online supplement) Therefore, motions of the adaptor domain, which are essential for proper VCP function [124,127], may be impaired by IBMPFD-associated mutations According to a recent yeast-2-hybrid screen [137], a josephin homologue from Drosophila melanogaster (CG3781) on the X chromosome interacts with the heat shock protein HSP60b (CG2830), which is involved in spermatogenesis [138,139], suppresses ubiquitination [140] and associates with 38 further proteins including a ubiquitin E3 ligase, but no other deubiquitinating enzyme except josephin (CG8184) Interestingly, HSP40 and HSP70 chaperones have already been observed to associate with VCP, and they also colocalize with intranuclear ataxin-3 aggregates and may play an important role in the disease process and the impairment of the ubiquitin-proteasome system [121,141–149] Structural modelling of the Josephin domain Recently, it has been observed that the Josephin domain contains highly conserved amino acids reminiscent of the catalytic residues of a deubiquitinating cysteine protease [69], and first experimental results support this function hypothesis [68]: decrease of polyubiquitination of 125Ilabelled lysozyme by removal of ubiquitin, cleavage of the ubiquitin protease substrate ubiquitin-AMC, and binding of the specific ubiquitin protease inhibitor ubiquitin-aldehyde (Ubal) Mutating the catalytic cysteine in ataxin-3 inhibits these functions [68] Previously, we modeled the 3D structure of ataxin-3 based on the ANTH domain [150] of the adaptin AP180 as structural template [73] However, this prediction has to be revised with regard to the N-terminal Josephin domain because of the identified cysteine protease signature [69] In contrast to our previous prediction [73], which relied on the secondary structure prediction from a single server, we now formed the consensus result of the three state-ofthe-art secondary structure prediction servers PSIPRED, SAM-T99, and SSpro2 All three online servers basically Ó FEBS 2004 returned the same secondary structure for human ataxin-3 and josephin 1, resulting in a much more reliable secondary structure prediction of b-strands besides a-helices We propose that the increased accuracy of this prediction is due, at least in part, to a substantial growth of protein sequence and structure databases The predicted b-strands in the Josephin domain corroborate a cysteine protease fold of deubiquitinating enzymes (DUBs) and not support the ANTH domain structure consisting solely of a-helices In hindsight, the fold recognition methods applied in the past to predict the structure of ataxin-3 may have been misguided by the pronounced prediction of a-helices only DUBs process ubiquitin proteolytically at the C-terminus and can be divided into at least two evolutionarily related families of cysteine proteases, UBPs (ubiquitin-specific proteases) and UCHs (ubiquitin C-terminal hydrolases) [151,152] However, new ubiquitin-specific families such as otubains (OTU) and JAMMs with low sequence similarity to known DUBs are still being discovered [151] A consensus of fold recognition servers now selects both available UCH domain structures of human UCH-L3 [153] and yeast YUH1 [154], which superimpose with a low ˚ RMSD of 2.0 A (PDB identifiers 1uch and 1cmxA, respectively), as best modelling templates with a moderate confidence score for human josephin 1, but still with only a weak score for ataxin-3 The pairwise sequence–structure alignments returned by the structure prediction servers for 3D modelling differ mainly in the central part of the Josephin domain (amino acid 47–117 in ataxin-3) aligned to DUBs This finding underpins the distant relationship of the Josephin domain to known DUBs The central part does not contain catalytic residues and is thus less conserved, containing insertions of variable length and structure in other cysteine proteases [155] Based on a multiple sequence alignment of Josephin domain homologues (Fig 6), we used the crystallographically determined structure of YUH1 bound to the ubiquitinlike inhibitor Ubal (PDB identifier 1cmx, chains A and B, respectively) to model the tertiary structure of the Josephin domain of ataxin-3 in complex with Ubal (Fig 7) Thus, the structure of ataxin-3 is predicted to be distinct from the finger–palm–thumb architecture of UBPs such as USP7/ HAUSP [156] Because of the low degree of conservation in the central part, we believe that ataxin-3 and josephin adopt slightly different structures in this part, which are not very similar to YUH1 In addition, we observed that the Josephin domain also resembles the OTU domain because both have a highly conserved histidine three residues downstream of the catalytic cysteine Interestingly, like ataxin-3, the deubiquitinating OTU domain protein VCIP135 interacts with the N-terminal adaptor domain of VCP through the C-terminal tail including a UBL domain and dissociates p47 from the complex with VCP during ATP hydrolysis of VCP [157,158] This observation also indicates a close functional relationship of the homologous ubiquitin-like UBL and UBX domains Functional analysis of the Josephin domain The active site of UCHs is divided into two parts as follows (YUH1/ataxin-3 residue numbers) [153,154]: The Ó FEBS 2004 Analysis of ataxins and (Eur J Biochem 271) 3163 Fig Structure-based multiple sequence alignment of the Josephin domains of ataxin-3 homologues with the crystallographically determined UCH domains of human UCH-L3 and yeast YUH1 The known DSSP secondary structure assignments of UCH-L3 and YUH1 are shown at the top of the alignment (curled lines for a-helix, arrows for b-strands) The corresponding consensus secondary structure predictions for human ataxin-3 and josephin are also depicted Alignment columns with identical residues are highlighted in purple-coloured boxes, those with more than 50% physico-chemically similar amino acids in yellow boxes (bold-printed letters) Text labels (including UCH-L3/YUH1 and ataxin-3/josephin residue numbers) point to catalytic residues (four grey-shaded boxes) and to other highly conserved amino acids in the Josephin domain The PDB/ SPTrEMBL identifiers of UCH-L3 and YUH1 are 1uch/P15374 and 1cmxA/P35127, respectively NCBI or Ensembl accession numbers for Josephin domain homologues are given in Table S3 N-terminal part consists of a glutamine (Q84/Q9) upstream of a cysteine (C90/C14), both of which form an oxyanion hole to accommodate the negative charge on the substrate carbonyl oxygen during catalysis The C-terminal part contains a histidine (H166/H119), which is thought to be deprotonated, and an asparagine or aspartate (D181/N134), both of which activate the side chain of the cysteine to unleash a nucleophilic attack on the carbonyl carbon atom of the scissile peptide bond The cysteine, histidine, and asparagine/aspartate constitute the catalytic triad characteristic of cysteine proteases such as papain While all four discussed catalytic residues are strictly conserved in the Josephin domain (Fig 6), a functionally relevant disordered loop (E144–N164/V79–Q100) 3164 M Albrecht et al (Eur J Biochem 271) Ó FEBS 2004 Fig (Continued) positioned over the catalytic cleft is aligned in the less conserved central part This loop maintains an inaccessible active site, but becomes ordered upon binding of Ubal [154] Therefore, it may control substrate specificity together with further strongly conserved amino acids such as N88/L13, which forms hydrogen bonds with main chain groups of the loop, and Y167/W120 next to the catalytic histidine [154] Unfortunately, the structure of the central part and the loop function remains unclear for the Josephin domain because of insufficient sequence similarity to UCHs The Josephin domain is also missing the N-terminal extensions of UCHs, which are involved in substrate recognition [154] In addition, a functional relevance of a second strictly conserved histidine H17, two highly conserved asparagines N20 and N21, and another identical glutamine Q24 downstream of the catalytic cysteine C14 cannot be derived either from the structural model of the Josephin domain (Figs and 7) However, considering their distance from the active site and location inside the protein, they may be solely important for the stability of the domain fold This may also hold true for the strictly conserved S135 and P140 after the catalytically active N134 In contrast, it is easy to interpret an alternative splice variant of ataxin-3 [10], which consists of a deletion of the residues from E10 to Q64 including the catalytic cysteine and thus cannot possess proteolytic activity Ó FEBS 2004 Analysis of ataxins and (Eur J Biochem 271) 3165 Fig 3D model of the deubiquitinating Josephin domain of ataxin-3 using the structure of yeast YUH1 bound to the ubiquitin-like inhibitor Ubal (in CPK view mode) as template (PDB identifier 1cmx, chains A and B, respectively) Grey-shaded text labels indicate the four catalytic residues (balland-stick view) forming the active site of the ubiquitin hydrolase The remaining text boxes point to other residues, which are highly conserved in the Josephin domain Residues are coloured in agreement with the alignment columns in Fig The N-terminal extension of YUH1, which is missing in ataxin-3 homologues, is depicted in the background as thin dark brown protein backbone only The less conserved central part of ataxin-3 is shown in green; it could not be modelled reliably using YUH1 as template because of low sequence similarity Comparison to other polyQ proteins Conclusions The polyQ stretch of both ataxin-2 and ataxin-3 lies in sequence regions whose degree of conservation is very low in contrast to the globular domains and which are predicted to be intrinsically unstructured This prediction has been confirmed experimentally for ataxin-3 [72], and polyQ tracts themselves also adopt a random coil conformation [159] So we decided to investigate other polyglutamine disease proteins such as ataxin-1 (SCA1), ataxin-7 (SCA7), atrophin (DRPLA) and huntingtin (HD) as to whether their polyQ regions are also predicted to be surrounded by disordered structure For this purpose, we used several online prediction servers (DisEMBL, DISOPRED, GlobPlot, NORSp, PONDR), which consensus basically indicates that the polyQ tracts are generally located in unstructured regions (Table S4) This is also in agreement with secondary structure prediction results, which not indicate globular domains consisting of a-helices or b-strands (data not shown), and other computational predictions of locally unfolded regions [160] Therefore, it is not surprising that mutant polyglutamine proteins can readily form aggregates via the solvent-exposed polyQ region We presented a detailed analysis of ataxin-2 homologues including the yeast homologue Pbp1, using a structurebased multiple sequence alignment of Sm and Sm-like proteins and a 3D model of the Lsm domain of ataxin-2 Our comparison revealed a high degree of conservation of chemical properties for RNA-binding residues in the aligned Lsm domains in general and between Sm1 from P abyssi and human ataxin-2 in particular Based on this observation, we propose that ataxin-2 is capable of binding RNA by the identified residues Therefore, an essential function of ataxin-2 homologues in RNA processing should be explored experimentally and could implicate the regulation of polyadenylation of mRNA as it is known for Pbp1 In addition, the similarity of amino acids involved in the formation of Lsm domain oligomers as derived from the D1–D2 and D3–B heterodimers may suggest that ataxin-2 may also form such complexes Our structural model of the Josephin domain of ataxin-3 confirms the evolutionary relationship with deubiquitinating cysteine proteases of the UCH family Interestingly, this relates ataxin-3 to another ubiquitin hydrolase termed USP14, which is involved in synaptic dysfunction in ataxic Ó FEBS 2004 3166 M Albrecht et al (Eur J Biochem 271) mice [161,162] Moreover, the polyglutamine disease protein ataxin-1 interacts with the ubiquitin-specific protease USP7/ HAUSP, and the length of the polyQ region influences the strength of the interaction [163] Unfortunately, the central part of the Josephin domain is difficult to model because of low sequence similarity Therefore, it cannot be deduced whether the ataxin-3 mechanism of ubiquitin recognition works similarly to UCHs It is striking that both human ataxin-3 and its P falciparum homologue ataxin-3_Pf can bind the N-terminal adaptor domain of the molecular chaperone VCP at their C-termini, although they differ considerably and an evolutionary relationship is not apparent: ataxin-3 contains the polyQ region, but the P falciparum homologue has a ubiquitin-like UBX domain Another open question is whether a deubiquitinating analogue of ataxin-3 exists in yeast, since the Josephin domain is not found in any yeast protein, but complexes of the ataxin-3 and proteasome binding proteins hHR23A/B, VCP and UFD2a are conserved in yeast Generally, it remains to be seen how the normal functions of ataxin-2 and ataxin-3 are affected in mutant proteins with an expanded polyglutamine tract 11 12 13 14 15 16 Acknowledgements Part of this research was funded by the German Research Foundation (DFG) under contract no LE 491/14–1, by the Federal Ministry of Education and Research (BMBF) under contract no 01gs0115-NVS02T12, and by the European Commission through the EUROSCA project under contract no LSHM-CT-2004-503304 17 18 References Kawaguchi, Y., Okamoto, T., Taniwaki, M., Aizawa, M., Inoue, M., Katayama, S., Kawakami, H., Nakamura, S., Nishimura, M., Akiguchi, I et al (1994) CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1 Nat Genet 8, 221–228 Pulst, S.M., Nechiporuk, A., Nechiporuk, T., Gispert, S., Chen, X.N., Lopes-Cendes, I., Pearlman, S., Starkman, S., OrozcoDiaz, G., Lunkes, A., DeJong, P., Rouleau, G.A., Auburger, G., Korenberg, J.R., Figueroa, C & Sahba, S (1996) Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type Nat Genet 14, 269–276 Evert, B.O., Wullner, U & Klockgether, T (2000) Cell death in ă polyglutamine diseases Cell Tissue Res 301, 189–204 Zoghbi, H.Y & Orr, H.T (2000) Glutamine repeats and neurodegeneration Annu Rev Neurosci 23, 217–247 Margolis, R.L (2002) The spinocerebellar ataxias: order emerges from chaos Curr Neurol Neurosci Report 2, 447–456 Stevanin, G., Durr, A & Brice, A (2002) Spinocerebellar ataxias ¨ caused by polyglutamine expansions Adv Exp Med Biol 516, 47–77 Wullner, U (2003) Genes implicated in the pathogenesis of ă spinocerebellar ataxias Drugs Today (Barc) 39, 927937 Gusella, J.F & MacDonald, M.E (2000) Molecular genetics: unmasking polyglutamine triggers in neurodegenerative disease Nat Rev Neurosci 1, 109–115 Sahba, S., Nechiporuk, A., Figueroa, K.P., Nechiporuk, T & Pulst, S.M (1998) Genomic structure of the human gene for spinocerebellar ataxia type (SCA2) on chromosome 12q24.1 Genomics 47, 359–364 10 Ichikawa, Y., Goto, J., Hattori, M., Toyoda, A., Ishii, K., Jeong, S.Y., Hashida, H., Masuda, N., Ogata, K., Kasai, F., Hirai, M., 19 20 21 22 23 24 25 26 27 Maciel, P., Rouleau, G.A., Sakaki, Y & Kanazawa, I (2001) The genomic structure and expression of MJD, the MachadoJoseph disease gene J Hum Genet 46, 413–422 Huynh, D.P., Yang, H.T., Vakharia, H., Nguyen, D & Pulst, S.M (2003) Expansion of the polyQ repeat in ataxin-2 alters its Golgi localization, disrupts the Golgi complex and causes cell death Hum Mol Genet 12, 1485–1496 Trottier, Y., Cancel, G., An-Gourfinkel, I., Lutz, Y., Weber, C., Brice, A., Hirsch, E & Mandel, J.L (1998) Heterogeneous intracellular localization and expression of ataxin-3 Neurobiol Dis 5, 335–347 Wheeler, D.L., Church, D.M., Edgar, R., Federhen, S., Helmberg, W., Madden, T.L., Pontius, J.U., Schuler, G.D., Schriml, L.M., Sequeira, E et al (2004) Database resources of the National Center for Biotechnology – information update Nucleic Acids Res 32 (Database issue), D35–40 Birney, E., Andrews, D., Bevan, P., Caccamo, M., Cameron, G., Chen, Y., Clarke, L., Coates, G., Cox, T., Cuff, J et al (2004) Ensembl Nuceic Acids Res 32 (Database issue), D468–470 Apweiler, R., Bairoch, A., Wu, C.H., Barker, W.C., Boeckmann, B., Ferro, S., Gasteiger, E., Huang, H., Lopez, R., Magrane, M et al (2004) UniProt: the Universal Protein knowledgebase Nucleic Acids Res 32 (Database issue), D115– 119 Bateman, A., Coin, L., Durbin, R., Finn, R.D., Hollich, V., Griffiths-Jones, S., Khanna, A., Marshall, M., Moxon, S., Sonnhammer, E.L, et al (2004) The Pfam protein families database Nucleic Acids Res 32 (Database issue), D138–141 Andreeva, A., Howorth, D., Brenner, S.E., Hubbard, T.J., Chothia, C & Murzin, A.G (2004) (2004) SCOP database in: refinements integrate structure and sequence family data Nucleic Acids Res 32 (Database issue), D226–229 Bourne, P.E., Addess, K.J., Bluhm, W.F., Chen, L., Deshpande, N., Feng, Z., Fleri, W., Green, R., Merino-Ott, J.C., Townsend-Merino, W., et al (2004) The distribution and query systems of the RCSB Protein Data Bank Nucleic Acids Res 32 (Database issue), D223–225 Kabsch, W & Sander, C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features Biopolymers 22, 2577–2637 Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W & Lipman, D.J (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Nuceic Acids Res 25, 3389–402 McGuffin, L.J., Bryson, K & Jones, D.T (2000) The PSIPRED protein structure prediction server Bioinformatics 16, 404– 405 Karplus, K., Barrett, C & Hughey, R (1998) Hidden Markov models for detecting remote protein homologies Bioinformatics 14, 846–856 Pollastri, G., Przybylski, D., Rost, B & Baldi, P (2002) Improving the prediction of protein secondary structure in three and eight classes using recurrent neural networks and profiles Proteins 47, 228–235 Albrecht, M., Tosatto, S.C., Lengauer, T & Valle, G (2003) Simple consensus procedures are effective and sufficient in secondary structure prediction Protein Eng 16, 459–462 Linding, R., Jensen, L.J., Diella, F., Bork, P., Gibson, T.J & Russell, R.B (2003) Protein disorder prediction: implications for structural proteomics Structure (Camb) 11, 1453–1459 Ward, J.J.,.McGuffin, L.J., Bryson, K., Buxton, B.F & Jones, D.T (2004) The DISOPRED server for the prediction of protein disorder Bioinformatics (in press) Linding, R., Russell, R.B., Neduva, V & Gibson, T.J (2003) GlobPlot: Exploring protein sequences for globularity and disorder Nucleic Acids Res 31, 3701–3708 Ó FEBS 2004 28 Liu, J & Rost, B (2003) NORSp: Predictions of long regions without regular secondary structure Nucl Acids Res 31, 3833– 3835 29 Nakai, K & Horton, P (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization Trends Biochem Sci 24, 34–36 30 Poirot, O., O’Toole, E & Notredame, C (2003) Tcoffee@igs: a web server for computing, evaluating and combining multiple sequence alignments Nucleic Acids Res 31, 3503–3506 31 Shindyalov, I.N & Bourne, P.E (1998) Protein structure alignment by incremental combinatorial extension (CE) of the optimal path Protein Eng 11, 739–747 32 Bujnicki, J.M., Elofsson, A., Fischer, D & Rychlewski, L (2001) Structure prediction meta server Bioinformatics 17, 750–1 33 Ginalski, K & Rychlewski, L (2003) Detection of reliable and unexpected protein fold predictions using 3D-Jury Nucleic Acids Res 31, 3291–3292 34 Rodriguez, R., Chinea, G., Lopez, N., Pons, T & Vriend, G (1998) Homology modeling, model and software evaluation: three related resources Bioinformatics 14, 523–528 35 Galtier, N., Gouy, M & Gautier, C (1996) SEAVIEW and PHYLO_WIN two graphic tools for sequence alignment and molecular phylogeny Comput Appl Biosci 12, 543–548 36 Gouet, P., Robert, X & Courcelle, E (2003) ESPript/ENDscript extracting and rendering sequence and 3D information from atomic structures of proteins Nucleic Acids Res 31, 3320–3323 37 Neuwald, A.F & Koonin, E.V (1998) Ataxin-2, global regulators of bacterial gene expression, and spliceosomal snRNP proteins share a conserved domain J Mol Med 76, 3–5 38 Shibata, H., Huynh, D.P & Pulst, S.M (2000) A novel protein with RNA-binding motifs interacts with ataxin-2 Hum Mol Genet 9, 1303–1313 39 Albrecht, M & Lengauer, T (2004) Survey on the PABC recognition motif PAM2 Biochem Biophys Res Commun 316, 129–138 40 Dreyfuss, G., Matunis, M.J., Pinol-Roma, S & Burd, C.G (1993) hnRNP proteins and the biogenesis of mRNA Annu Rev Biochem 62, 289–321 41 He, W & Parker, R (2000) Functions of Lsm proteins in mRNA degradation and splicing Curr Opin Cell Biol 12, 346– 350 42 Mura, C., Phillips, M., Kozhukhovsky, A & Eisenberg, D (2003) Structure and assembly of an augmented Sm-like archaeal protein 14-mer Proc Natl Acad Sci USA 100, 4539– 4544 43 Sauter, C., Basquin, J & Suck, D (2003) Sm-like proteins in Eubacteria: the crystal structure of the Hfq protein from Escherichia coli Nucleic Acids Res 31, 4091–4098 44 Bessman, M.J., Frick, D.N & O’Handley, S.F (1996) The MutT proteins or ÔNudixÕ hydrolases, a family of versatile, widely distributed, ÔhousecleaningÕ enzymes J Biol Chem 271, 25059– 25062 45 Piccirillo, C., Khanna, R & Kiledjian, M (2003) Functional characterization of the mammalian mRNA decapping enzyme hDcp2 RNA 9, 1138–1147 46 She, M., Decker, C.J., Sundramurthy, K., Liu, Y., Chen, N., Parker, R & Song, H (2004) Crystal structure of Dcp1p and its functional implications in mRNA decapping Nat Struct Mol Biol 11, 249–256 47 Kiehl, T.R., Shibata, H & Pulst, S.M (2000) The ortholog of human ataxin-2 is essential for early embryonic patterning in C elegans J Mol Neurosci 15, 231–241 48 Meunier, C., Bordereaux, D., Porteu, F., Gisselbrecht, S., Chretien, S & Courtois, G (2002) Cloning and characterization of a family of proteins associated with Mpl J Biol Chem 277, 9139–9147 Analysis of ataxins and (Eur J Biochem 271) 3167 49 Satterfield, T.F., Jackson, S.M & Pallanck, L.J (2002) A Drosophila homolog of the polyglutamine disease gene SCA2 is a dosage-sensitive regulator of actin filament formation Genetics 162, 1687–1702 50 Figueroa, K.P & Pulst, S.M (2003) Identification and expression of the gene for human ataxin-2-related protein on chromosome 16 Exp Neurol 184, 669–678 51 Jin, Y., Suzuki, H., Maegawa, S., Endo, H., Sugano, S., Hashimoto, K., Yasuda, K & Inoue, K (2003) A vertebrate RNAbinding protein Fox-1 regulates tissue-specific splicing via the pentanucleotide GCAUG EMBO J 22, 905–912 52 Bhalla, K., Phillips, H.A., Crawford, J., McKenzie, O.L., Mulley, J.C., Eyre, H., Gardner, A.E., Kremmidiotis, G & Callen, D.F (2004) The de novo chromosome 16 translocations of two patients with abnormal phenotypes (mental retardation and epilepsy) disrupt the A2BP1 gene, J Hum Genet 49, 308–311 53 Mangus, D.A., Amrani, N & Jacobson, A (1998) Pbp1p, a factor interacting with Saccharomyces cerevisiae poly(A)-binding protein, regulates polyadenylation Mol Cell Biol 18, 7383– 7396 54 Achsel, T., Brahms, H., Kastner, B., Bachi, A., Wilm, M & Luhrmann, R (1999) A doughnut-shaped heteromer of human ă Sm-like proteins binds to the 3Â-end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro EMBO J 18, 5789– 5802 55 Kambach, C., Walke, S., Young, R., Avis, J.M., de la Fortelle, E., Raker, V.A., Luhrmann, R., Li, J & Nagai, K (1999) Crystal ă structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs Cell 96, 375–387 56 Achsel, T., Stark, H & Luhrmann, R (2001) The Sm domain is ă an ancient RNA-binding motif with oligo(U) specicity Proc Natl Acad Sci USA 98, 3685–3689 57 Collins, B.M., Harrop, S.J., Kornfeld, G.D., Dawes, I.W., Curmi, P.M & Mabbutt, B.C (2001) Crystal structure of a heptameric Sm-like protein complex from archaea: implications for the structure and evolution of snRNPs J Mol Biol 309, 915–923 58 Mura, C., Cascio, D., Sawaya, M.R & Eisenberg, D.S (2001) The crystal structure of a heptameric archaeal Sm protein: Implications for the eukaryotic snRNP core Proc Natl Acad Sci USA 98, 5532–5537 ´ 59 Toro, I., Thore, S., Mayer, C., Basquin, J., Seraphin, B & ¨ ¨ Suck, D (2001) RNA binding in an Sm core domain: X-ray structure and functional analysis of an archaeal Sm protein complex EMBO J 20, 2293–2303 60 Urlaub, H., Raker, V.A., Kostka, S & Luhrmann, R (2001) Sm ă protein–Sm site RNA interactions within the inner ring of the spliceosomal snRNP core structure EMBO J 20, 187–196 61 Schumacher, M.A., Pearson, R.F., Møller, T., ValentinHansen, P & Brennan, R.G (2002) Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein EMBO J 21, 3546–3556 62 Toro, I., Basquin, J., Teo-Dreher, H & Suck, D (2002) Archaeal ă ă Sm proteins form heptameric and hexameric complexes: crystal structures of the Sm1 and Sm2 proteins from the hyperthermophile Archaeoglobus fulgidus J Mol Biol 320, 129–142 63 Collins, B.M., Cubeddu, L., Naidoo, N., Harrop, S.J., Kornfeld, G.D., Dawes, I.W., Curmi, P.M & Mabbutt, B.C (2003) Homomeric ring assemblies of eukaryotic Sm proteins have affinity for both RNA and DNA Crystal structure of an oligomeric complex of yeast SmF J Biol Chem 278, 17291– 17298 64 Mura, C., Kozhukhovsky, A., Gingery, M., Phillips, M & Eisenberg, D (2003) The oligomerization and ligand-binding properties of Sm-like archaeal proteins (SmAPs) Protein Sci 12, 832–847 Ó FEBS 2004 3168 M Albrecht et al (Eur J Biochem 271) 65 Thore, S., Mayer, C., Sauter, C., Weeks, S & Suck, D (2003) Crystal structures of the Pyrococcus abyssi Sm core and its complex with RNA Common features of RNA binding in archaea and eukarya J Biol Chem 278, 1239–1247 66 Hermann, H., Fabrizio, P., Raker, V.A., Foulaki, K., Hornig, H., Brahms, H & Luhrmann, R (1995) snRNP Sm proteins share ă two evolutionarily conserved sequence motifs which are involved in Sm protein–protein interactions EMBO J 14, 2076–2088 67 Seraphin, B (1995) Sm and Sm-like proteins belong to a large family: identification of proteins of the U6 as well as the U1, U2, U4 and U5 snRNPs EMBO J 14, 2089–2098 68 Burnett, B., Li, F & Pittman, R.N (2003) The polyglutamine neurodegenerative protein ataxin-3 binds polyubiquitylated proteins and has ubiquitin protease activity Hum Mol Genet 12, 3195–3205 69 Scheel, H., Tomiuk, S & Hofmann, K (2003) Elucidation of ataxin-3 and ataxin-7 function by integrative bioinformatics Hum Mol Genet 12, 2845–2852 70 Hofmann, K & Falquet, L (2001) A ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems Trends Biochem Sci 26, 347–350 71 Suzuki, T., Park, H., Till, E.A & Lennarz, W.J (2001) The PUB domain: a putative protein–protein interaction domain implicated in the ubiquitin-proteasome pathway Biochem Biophys Res Commun 287, 1083–1087 72 Masino, L., Musi, V., Menon, R.P., Fusi, P., Kelly, G., Frenkiel, T.A., Trottier, Y & Pastore, A (2003) Domain architecture of the polyglutamine protein ataxin-3: a globular domain followed by a flexible tail FEBS Lett 549, 21–25 73 Albrecht, M., Hoffmann, D., Evert, B.O., Schmitt, I., Wullner, ă U & Lengauer, T (2003) Structural modeling of ataxin-3 reveals distant homology to adaptins Proteins 50, 355–370 74 Tait, D., Riccio, M., Sittler, A., Scherzinger, E., Santi, S., Ognibene, A., Maraldi, N.M., Lehrach, H & Wanker, E.E (1998) Ataxin-3 is transported into the nucleus and associates with the nuclear matrix Hum Mol Genet 7, 991–997 75 la Cour, T., Gupta, R., Rapacki, K., Skriver, K., Poulsen, F.M & Brunak, S (2003) NESbase, Version 1.0 a database of nuclear export signals Nucleic Acids Res 31, 393–396 76 Li, F., Macfarlan, T., Pittman, R.N & Chakravarti, D (2002) Ataxin-3 is a histone-binding protein with two independent transcriptional corepressor activities J Biol Chem 277, 45004– 45012 77 Bodai, L., Pallos, J., Thompson, L.M & Marsh, J.L (2003) Altered protein acetylation in polyglutamine diseases Curr Med Chem 10, 2577–2587 78 Zhang, Y (2003) Transcriptional regulation by histone ubiquitination and deubiquitination Genes Dev 17, 2733–2740 79 Florens, L., Washburn, M.P., Raine, J.D., Anthony, R.M., Grainger, M., Haynes, J.D., Moch, J.K., Muster, N., Sacci, J.B., Tabb, D.L., et al (2002) A proteomic view of the Plasmodium falciparum life cycle Nature 419, 520–526 80 Bozdech, Z., Llinas, M., Pulliam, B.L., Wong, E.D., Zhu, J & DeRisi, J.L (2003) The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum PLoS Biol 1, E5 81 Le Roch, K.G., Zhou, Y., Blair, P.L., Grainger, M., Moch, J.K., Haynes, J.D., De La Vega, P., Holder, A.A., Batalov, S., Carucci, D.J & Winzeler, E.A (2003) Discovery of gene function by expression profiling of the malaria parasite life cycle Science 301, 1503–1508 82 Buchberger, A., Howard, M.J., Proctor, M & Bycroft, M (2001) The UBX domain: a widespread ubiquitin-like module J Mol Biol 307, 17–24 83 Yuan, X., Shaw, A., Zhang, X., Kondo, H., Lally, J., Freemont, P.S & Matthews, S (2001) Solution structure and interaction 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 surface of the C-terminal domain from p47: a major p97-cofactor involved in SNARE disassembly J Mol Biol 311, 255–263 Buchberger, A (2002) From UBA to UBX: new words in the ubiquitin vocabulary Trends Cell Biol 12, 216–221 Yuan, X., Simpson, P., Kondo, H., McKeown, C., Dreveny, I., Zhang, X., Freemont, P.S & Matthews, S (2004) Complete Backbone Resonance Assignments of p47: The 41kDa Adaptor Protein of the AAA ATPase p97 J Biomol NMR 28, 309–310 Dreveny, I., Kondo, H., Uchiyama, K., Shaw, A., Zhang, X & Freemont, P.S (2004) Structural basis of the interaction between the AAA ATPase p97/VCP and its adaptor protein p47 EMBO J 23, 1030–1039 Aguilar, R.C & Wendland, B (2003) Ubiquitin: not just for proteasomes anymore Curr Opin Cell Biol 15, 184–190 Bach, I & Ostendorff, H.P (2003) Orchestrating nuclear functions: ubiquitin sets the rhythm Trends Biochem Sci 28, 189–195 Baek, K.H (2003) Conjugation and deconjugation of ubiquitin regulating the destiny of proteins Exp Mol Med 35, 1–7 Berke, S.J & Paulson, H.L (2003) Protein aggregation and the ubiquitin proteasome pathway: gaining the UPPer hand on neurodegeneration Curr Opin Genet Dev 13, 253–261 Di Fiore, P.P., Polo, S & Hofmann, K (2003) When ubiquitin meets ubiquitin receptors: a signalling connection Nat Rev Mol Cell Biol 4, 491–497 Hicke, L & Dunn, R (2003) Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins Annu Rev Cell Dev Biol 19, 141–172 Passmore, L.A & Barford, D (2004) Getting into position: The catalytic mechanisms of protein ubiquitylation Biochem J 379, 513–525 Chai, Y., Berke, S.S., Cohen, R.E & Paulson, H.L (2003) Polyubiquitin binding by the polyglutamine disease protein ataxin-3 links its normal function to protein surveillance pathways J Biol Chem 279, 3605–3611 Donaldson, K.M., Li, W., Ching, K.A., Batalov, S., Tsai, C.C & Joazeiro, C.A (2003) Ubiquitin-mediated sequestration of normal cellular proteins into polyglutamine aggregates Proc Natl Acad Sci USA 100, 8892–8897 Doss-Pepe, E.W., Stenroos, E.S., Johnson, W.G & Madura, K (2003) Ataxin)3 interactions with rad23 and valosin-containing protein and its associations with ubiquitin chains and the proteasome are consistent with a role in ubiquitin-mediated proteolysis Mol Cell Biol 23, 6469–6483 Miller, S.L.,.Malotky, E & O’Bryan, J.P (2004) Analysis of the role of ubiquitin-interacting motifs (UIMs) in ubiquitin binding and ubiquitylation J Biol Chem (in press) Fisher, R.D., Wang, B., Alam, S.L., Higginson, D.S., Robinson, H., Sundquist, W.I & Hill, C.P (2003) Structure and ubiquitin binding of the ubiquitin-interacting motif J Biol Chem 278, 28976–28984 Mueller, T.D & Feigon, J (2003) Structural determinants for the binding of ubiquitin-like domains to the proteasome EMBO J 22, 4634–4645 Swanson, K.A., Kang, R.S., Stamenova, S.D., Hicke, L & Radhakrishnan, I (2003) Solution structure of Vps27 UIMubiquitin complex important for endosomal sorting and receptor downregulation EMBO J 22, 4597–4606 Evans, P.R & Owen, D.J (2002) Endocytosis and vesicle trafficking Curr Opin Struct Biol 12, 814–821 Collins, B.M., Watson, P.J & Owen, D.J (2003) The structure of the GGA1-GAT domain reveals the molecular basis for ARF binding and membrane association of GGAs Dev Cell 4, 321– 332 Shiba, T., Kawasaki, M., Takatsu, H., Nogi, T., Matsugaki, N., Igarashi, N., Suzuki, M., Kato, R., Nakayama, K & Ó FEBS 2004 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 Wakatsuki, S (2003) Molecular mechanism of membrane recruitment of GGA by ARF in lysosomal protein transport Nat Struct Biol 10, 386–393 Suer, S., Misra, S., Saidi, L.F & Hurley, J.H (2003) Structure of the GAT domain of human GGA1: a syntaxin amino-terminal domain fold in an endosomal trafficking adaptor, Proc Natl Acad Sci USA 100, 4451–4456 Zhu, G., Zhai, P., He, X., Terzyan, S., Zhang, R., Joachimiak, A., Tang, J & Zhang, X.C (2003) Crystal structure of the human GGA1 GAT domain Biochemistry 42, 6392–6399 Katoh, Y., Shiba, Y., Mitsuhashi, H., Yanagida, Y., Takatsu, H & Nakayama, K (2004) Tollip and Tom1 form a complex and recruit ubiquitin-conjugated proteins onto early endosomes J Biol Chem 279, 24435–24443 Mattera, R., Puertollano, R., Smith, W.J & Bonifacino, J.S (2004) The tri-helical bundle subdomain of the GGA proteins interacts with multiple partners through overlapping but distinct sites J Biol Chem 14, R357–359 Pelham, H.R (2004) Membrane traffic: GGAs sort ubiquitin Curr Biol 14, R357–R359 Mueller, T.D., Kamionka, M & Feigon, J (2004) Specificity of the interaction between UBA domains and ubiquitin J Biol Chem 279, 11926–11936 Wang, G., Sawai, N., Kotliarova, S., Kanazawa, I & Nukina, N (2000) Ataxin-3, the MJD1 gene product, interacts with the two human homologs of yeast DNA repair protein RAD23, HHR23A and HHR23B Hum Mol Genet 9, 1795–1803 Walters, K.J., Lech, P.J., Goh, A.M., Wang, Q & Howley, P.M (2003) DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a Proc Natl Acad Sci USA 100, 12694–12699 Upadhya, S.C & Hegde, A.N (2003) A potential proteasomeinteracting motif within the ubiquitin-like domain of parkin and other proteins Trends Biochem Sci 28, 280–283 Sweder, K & Madura, K (2002) Regulation of repair by the 26S proteasome J Biomed Biotechnol 2, 94–105 Ryu, K.S., Lee, K.J., Bae, S.H., Kim, B.K., Kim, K.A & Choi, B.S (2003) Binding surface mapping of intra– and interdomain interactions among hHR23B, ubiquitin, and polyubiquitin binding site of S5a J Biol Chem 278, 36621–36627 Wang, Q., Goh, A.M., Howley, P.M & Walters, K.J (2003) Ubiquitin recognition by the DNA repair protein hHR23a Biochemistry 42, 13529–13535 Elsasser, S., Chandler-Militello, D., Mueller, B., Hanna, J & Finley, D (2004) Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome J Biol Chem 279, 26817–26822 Lambertson, D., Chen, L & Madura, K (2003) Investigating the importance of proteasome interaction for Rad23 function Curr Genet 42, 199–208 Rassi, S & Pickart, C.M (2003) Rad23 ubiquitin-associated domains (UBA) inhibit 26 S proteasome-catalyzed proteolysis by sequestering lysine 48-linked polyubiquitin chains J Biol Chem 278, 8951–8959 Fujiwara, K., Tenno, T., Sugasawa, K., Jee, J.G., Ohki, I., Kojima, C., Tochio, H., Hiroaki, H., Hanaoka, F & Shirakawa, M (2004) Structure of the ubiquitin-interacting motif of S5a bound to the ubiquitin-like domain of HR23B J Biol Chem 279, 4760–4767 Hirabayashi, M., Inoue, K., Tanaka, K., Nakadate, K., Ohsawa, Y., Kamei, Y., Popiel, A.H., Sinohara, A., Iwamatsu, A., Kimura, Y., Uchiyama, Y., Hori, S & Kakizuka, A (2001) VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration Cell Death Differ 8, 977–984 Higashiyama, H., Hirose, F., Yamaguchi, M., Inoue, Y.H., Fujikake, N., Matsukage, A & Kakizuka, A (2002) Identifica- Analysis of ataxins and (Eur J Biochem 271) 3169 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 tion of ter94, Drosophila VCP, as a modulator of polyglutamineinduced neurodegeneration Cell Death Differ 9, 264–273 Kobayashi, T., Tanaka, K., Inoue, K & Kakizuka, A (2002) Functional ATPase activity of p97/valosin-containing protein (VCP) is required for the quality control of endoplasmic reticulum in neuronally differentiated mammalian PC12 cells J Biol Chem 277, 47358–47365 Kobayashi, T & Kakizuka, A (2003) Molecular analyses of Machado-Joseph disease Cytogenet Genome Res 100, 261– 275 DeLaBarre, B & Brunger, A.T (2003) Complete structure of p97/valosin-containing protein reveals communication between nucleotide domains Nat Struct Biol 10, 856–863 Kimura, Y & Kakizuka, A (2003) Polyglutamine diseases and molecular chaperones IUBMB Life 55, 337–345 Woodman, P.G (2003) p97, a protein coping with multiple identities J Cell Sci 116, 4283–4290 Wang, Q., Song, C & Li, C.C (2004) Molecular perspectives on p97-VCP: progress in understanding its structure and diverse biological functions J Struct Biol 146, 44–57 Hatakeyama, S & Nakayama, K.I (2003) Ubiquitylation as a quality control system for intracellular proteins J Biochem (Tokyo) 134, 1–8 Koegl, M., Hoppe, T., Schlenker, S., Ulrich, H.D., Mayer, T.U & Jentsch, S (1999) A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly Cell 96, 635–644 Kaneko, C., Hatakeyama, S., Matsumoto, M., Yada, M., Nakayama, K & Nakayama, K.I (2003) Characterization of the mouse gene for the U-box-type ubiquitin ligase UFD2a Biochem Biophys Res Commun 300, 297–304 Matsumoto, M., Yada, M., Hatakeyama, S., Ishimoto, H., Tanimura, T., Tsuji, S., Kakizuka, A., Kitagawa, M & Nakayama, K.I (2004) Molecular clearance of ataxin-3 is regulated by a mammalian E4 EMBO J 23, 659–669 Kim, I., Mi, K & Rao, H (2004) Multiple interactions of Rad23 suggest a mechanism for ubiquitylated substrate delivery important in proteolysis Mol Biol Cell 15, 3357–3365 Hartmann-Petersen, R., Wallace, M., Hofmann, K., Koch, G., Johnsen, A.H., Hendil, K.B & Gordon, C (2004) The Ubx2 and Ubx3 cofactors direct Cdc48 activity to proteolytic and nonproteolytic ubiquitin-dependent processes Curr Biol 14, 824– 828 Yuan, X., Simpson, P., McKeown, C., Kondo, H., Uchiyama, K., Wallis, R., Dreveny, I., Keetch, C., Zhang, X., Robinson, C., Freemont, P & Matthews, S (2004) Structure, dynamics and interactions of p47, a major adaptor of the AAA ATPase, p97 EMBO J 23, 1463–1473 Watts, G.D., Wymer, J., Kovach, M.J., Mehta, S.G., Mumm, S., Darvish, D., Pestronk, A., Whyte, M.P & Kimonis, V.E (2004) Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosincontaining protein Nat Genet 36, 377–381 Yamanaka, K., Okubo, Y., Suzaki, T & Ogura, T (2004) Analysis of the two p97/VCP/Cdc48p proteins of Caenorhabditis elegans and their suppression of polyglutamine-induced protein aggregation J Struct Biol 146, 242–250 Giot, L., Bader, J.S., Brouwer, C., Chaudhuri, A., Kuang, B., Li, Y., Hao, Y.L., Ooi, C.E., Godwin, B., Vitols, E., et al (2003) A protein interaction map of Drosophila melanogaster Science 302, 1727–1736 Castrillon, D.H., Gonczy, P., Alexander, S., Rawson, R., Eberă hart, C.G., Viswanathan, S., DiNardo, S & Wasserman, S.A (1993) Toward a molecular genetic analysis of spermatogenesis in Drosophila melanogaster: characterization of male-sterile mutants generated by single P element mutagenesis Genetics 135, 489–505 Ó FEBS 2004 3170 M Albrecht et al (Eur J Biochem 271) 139 Spradling, A.C., Stern, D., Beaton, A., Rhem, E.J., Laverty, T., Mozden, N., Misra, S & Rubin, G.M (1999) The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes Genetics 153, 135–177 140 Shan, Y.X., Yang, T.L., Mestril, R & Wang, P.H (2003) Hsp10 and Hsp60 suppress ubiquitination of insulin-like growth factor1 receptor and augment insulin-like growth factor-1 receptor signaling in cardiac muscle: implications on decreased myocardial protection in diabetic cardiomyopathy J Biol Chem 278, 45492–45498 141 Chai, Y., Koppenhafer, S.L., Bonini, N.M & Paulson, H.L (1999) Analysis of the role of heat shock protein (Hsp) molecular chaperones in polyglutamine disease J Neurosci 19, 10338– 10347 142 Chai, Y., Koppenhafer, S.L., Shoesmith, S.J., Perez, M.K & Paulson, H.L (1999) Evidence for proteasome involvement in polyglutamine disease: localization to nuclear inclusions in SCA3/MJD and suppression of polyglutamine aggregation in vitro Hum Mol Genet 8, 673–682 143 Warrick, J.M., Chan, H.Y., Gray-Board, G.L., Chai, Y., Paulson, H.L & Bonini, N.M (1999) Suppression of polyglutaminemediated neurodegeneration in Drosophila by the molecular chaperone HSP70 Nat Genet 23, 425–428 144 Bonini, N.M (2002) Chaperoning brain degeneration Proc Natl Acad Sci USA 99 16407–16411 145 Schmidt, T., Lindenberg, K.S., Krebs, A., Schols, L., Laccone, ă F., Herms, J., Rechsteiner, M., Riess, O & Landwehrmeyer, G.B (2002) Protein surveillance machinery in brains with spinocerebellar ataxia type 3: redistribution and differential recruitment of 26S proteasome subunits and chaperones to neuronal intranuclear inclusions Ann Neurol 51, 302–310 146 Thoms, S (2002) Cdc48 can distinguish between native and nonnative proteins in the absence of cofactors FEBS Lett 520, 107– 110 147 Ciechanover, A & Brundin, P (2003) The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg Neuron 40, 427–446 148 Jana, N.R & Nukina, N (2003) Recent advances in understanding the pathogenesis of polyglutamine diseases: involvement of molecular chaperones and ubiquitin-proteasome pathway J Chem Neuroanat 26, 95–101 149 Tarlac, V & Storey, E (2003) Role of proteolysis in polyglutamine disorders J Neurosci Res 74, 406–416 150 Legendre-Guillemin, V., Wasiak, S., Hussain, N.K., Angers, A & McPherson, P.S (2004) ENTH/ANTH proteins and clathrinmediated membrane budding J Cell Sci 117, 9–18 151 Kim, J.H., Park, K.C., Chung, S.S., Bang, O & Chung, C.H (2003) Deubiquitinating enzymes as cellular regulators J Biochem (Tokyo) 134, 9–18 152 Wing, S.S (2003) Deubiquitinating enzymes – the importance of driving in reverse along the ubiquitin-proteasome pathway Int J Biochem Cell Biol 35, 590–605 153 Johnston, S.C., Larsen, C.N., Cook, W.J., Wilkinson, K.D & Hill, C.P (1997) Crystal structure of a deubiquitinating enzyme ˚ (human UCH-L3) at 1.8 A resolution EMBO J 16, 3787– 3796 154 Johnston, S.C., Riddle, S.M., Cohen, R.E & Hill, C.P (1999) Structural basis for the specificity of ubiquitin C-terminal hydrolases EMBO J 18, 3877–3887 155 Barrett, A.J & Rawlings, N.D (2001) Evolutionary lines of cysteine peptidases Biol Chem 382, 727–733 156 Hu, M., Li, P., Li, M., Li, W., Yao, T., Wu, J.W., Gu, W., Cohen, R.E & Shi, Y (2002) Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde Cell 111, 1041–1054 157 Uchiyama, K., Jokitalo, E., Kano, F., Murata, M., Zhang, X., Canas, B., Newman, R., Rabouille, C., Pappin, D., Freemont, P & Kondo, H (2002) VCIP135, a novel essential factor for p97/ p47-mediated membrane fusion, is required for Golgi and ER assembly in vivo J Cell Biol 159, 855–866 158 Wang, Y., Satoh, A., Warren, G & Meyer, H.H (2004) VCIP135 acts as a deubiquitinating enzyme during p97-p47mediated reassembly of mitotic Golgi fragments J Cell Biol 164, 973–978 159 Masino, L., Kelly, G., Leonard, K., Trottier, Y & Pastore, A (2002) Solution structure of polyglutamine tracts in GST-polyglutamine fusion proteins FEBS Lett 513, 267–272 160 Chen, Y.W (2003) Local protein unfolding and pathogenesis of polyglutamine-expansion diseases Proteins 51, 68–73 161 Wilson, S.M., Bhattacharyya, B., Rachel, R.A., Coppola, V., Tessarollo, L., Householder, D.B., Fletcher, C.F., Miller, R.J., Copeland, N.G & Jenkins, N.A (2002) Synaptic defects in ataxia mice result from a mutation in Usp14, encoding a ubiquitin-specific protease Nat Genet 32, 420–425 162 Ehlers, M.D (2003) Ubiquitin and synaptic dysfunction: ataxic mice highlight new common themes in neurological disease Trends Neurosci 26, 4–7 163 Hong, S., Kim, S.J.KaS., Choi, I & Kang, S (2002) USP7, a ubiquitin-specific protease, interacts with ataxin-1, the SCA1 gene product Mol Cell Neurosci 20, 298–306 Supplementary material The following material is available from http://blackwellpublishing.com/products/journals/suppmat/ ejb/ejb4245/ejb4245sm.htm Appendix Supplementary online material  ... b5 strand) of D3 and F27/Y289 (b2 strand), L67/M324, V70/I327 and L72/L328 (all in b4 strand) of B The second cluster consists of P6/M267, L10/ L271 (both in a-helix), V18/C279 (b1 strand), L32/F293... solution structures of the UBL domain of hHR23A/B bound to a UIM peptide of S5a [99,119] could be used to model the complex of hHR23A/B and ataxin-3 Similarly, the complex of a UIM of ataxin-3 with... detailed analysis of ataxin-2 homologues including the yeast homologue Pbp1, using a structurebased multiple sequence alignment of Sm and Sm-like proteins and a 3D model of the Lsm domain of ataxin-2