Trypanosoma brucei TBC-B. An efficient recombinant expression system was prepared, material-purified, and characterized by CD. Extensive crystallization screening, allied with the use of limited proteolysis, led to structures of the N-terminal ubiquitin-like and C-terminal cytoskeleton-associated protein with glycine-rich segment domains at 2.35-Å and 1.6-Å resolution, respectively. These are compact globular domains that appear to be linked by a flexible segment. The ubiquitin-like domain contains two lysines that are spatially conserved with residues known to participate in ubiquitinylation, and so may represent a module that, through covalent attachment, regulates the signalling and/or protein degradation associated with the control of microtubule assembly, catastrophe, or function. The TBC-B C-terminal cytoskeleton-associated protein with glycine-rich segment domain, a known tubulin-binding structure, is the only such domain encoded by the T. brucei genome. Interestingly, in the crystal structure, the peptide-binding groove of this domain forms intermolecular contacts with the C-terminus of a symmetry-related molecule, an association that may mimic interactions with the C-terminus of α-tubulin or other physiologically relevant partners. The interaction of TBC-B with the α-tubulin C-terminus may, in particular, protect from post-translational modifications, or simply assist in the shepherding of the protein into polymerization.Tubulin-binding cofactor (TBC)-B is implicated in the presentation of α-tubulin ready to polymerize, and at the correct levels to form microtubules. Bioinformatics analyses, including secondary structure prediction, CD, and crystallography, were combined to characterize the molecular architecture of  Key to the regulation of many biological processes is the tight control exercised over protein biosynthesis, folding, and degradation. With respect to tubulin, a central component of the cytoskeleton, the correct folding and polymerization involves distinct stages that are influenced by several chaperones or cofactors Caenorhabditis elegansDrosophila melanogaster [Protein Data bank (PDB) code 2KJR], Arabidopsis thaliana (2KJ6), and Mus musculus (1V6E). The mouse TBC-B CAP-Gly domain NMR structure has also been determined (1WHG), but the only crystal structure known is that of the C. elegans TBC-B CAP-Gly domain Trypanosoma brucei, an organism that is considered to be a useful model for the study of MT biology T. brucei genome (http://www.genedb.org/) with mouse, human and C. elegans TBC-B sequences identified a single protein, Tb10.61.2930, with ∼ 40% amino acid sequence identity. This protein, T. brucei TBC-B (TbTBC-B), consists of 232 amino acids organized into the N-terminal Ubl domain (∼ 90 residues) and a CAP-Gly domain  domain and a C-terminal cytoskeleton-associated protein with glycine-rich segment (CAP-Gly) domain. There are NMR structures of the TBC-B Ubl domains from We now report the construction of an efficient bacterial recombinant expression system, protein purification and the use of CD and bioinformatics approaches to investigate secondary structure content and predicted flexibility. Crystallization of the full-length protein was achieved, but the samples were poorly ordered. Consequently, structural analyses of the individual domains were carried out. The fortuitous observation in the crystal structure of the CAP-Gly domain of intermolecular contacts with the C-terminus of a symmetry-related molecule suggests how interactions with partners, including α-tubulin, might occur. Comparisons with ubiquitin suggest a functional link between the structure of TBC-B and the regulation of distinct populations of proteins associated with tubulin biology by proteasome-dependent degradation.TbTBC-B was produced in Escherichia coli and purified. The final step in purification was size exclusion gel chromatography, and this indicated that the full-length protein is monomeric in solution. The protein crystallized readily; however, despite displaying a good appearance (data not shown), X-ray diffraction from the crystals did not extend beyond 7 Å of resolution. Limited proteolysis produced two polypeptides, one of which, the Ubl domain, gave highly ordered crystals, and the structure was solved with anomalous dispersion methods Recombinant full-length TbTBC-B crystallized in the tetragonal space group P41212 with a single polypeptide in the asymmetric unit. Size exclusion chromatography employed during purification indicated that the protein was monomeric in solution.The N-terminal Ubl domain of The Ubl domain is a small globular entity consisting of a mixed four-strand β-sheet that forms a concave groove in which a single α-helix is placed . This isT. brucei and mouse TBC-Bs share ∼ 30% and 55% sequence identity, respectively. Although remarkably similar in structure to ubiquitin (rmsd of 0.83 Å over 41 Cα atoms with PDB code 1UBQ), the TbTBC-B Ubl domain shares only 10% sequence identity. Ubiquitin contains seven lysines  that are targets for covalent modification TbTBC-B contains two lysines (Lys34 and Lys62), which correspond to Lys27 and Lys48 of ubiquitin, and a structural overlay reveals strong structural similarity in terms of the positioning of these residues  encodes a glutamate in this position.Although the sequence derived from the genome assigns residue 223 as aspartate, the cloned gene encodes a glutamate at this position, an observation confirmed by the structural analysis. This conservative difference could be an artefact of the cloning process, or, more likely, could result from a natural variation in the strain that from which genomic DNA was obtained. We note that the TbTBC-B CAP-Gly domain is a small globular structure with a similar fold to other CAP-Gly domains, and is an important module for the recognition and binding of the C-terminal tail of α-tubulin TbTBC-B matches closely to other CAP-Gly domains  The  domains B. The C.On the other side of the domain from the peptide-binding groove, there are two salt bridges formed by Arg159 with Asp191, and Arg172 with Glu189, which link β5 with β7, and β6 with β7, respectively. In addition, hydrogen bonds between Arg159 and the backbone oxygen of Phe227 link β5 to the C-terminal segment of β9 .TbTBC-B CAP-Gly domain has a different sequence in this tubulin-binding motif at residues 195–199, GKGDG. Glycine rather than asparagine occupies the third position, and this nonconservative change precludes the formation of a side chain hydrogen bond with the terminal residue of an EE[Y/F] peptide.CAP-Gly domains, including those of TBC-B, possess a highly conserved pentapeptide tubulin-binding motif with a sequence that is almost always GKNDG TbTBC-B CAP-Gly domain with peptides either. However, and fortuitously, in our structure the C-terminus of chain A forms intermolecular contacts with the basic groove of a symmetry-related chain B . The loss of these intermolecular contacts may help to explain why constructs in which the C-terminus had been truncated did not crystallize. The C-terminus of TbTBC-B has the sequence EVF, which is similar to the α-tubulin tail EE[Y/F] motif, and so these intermolecular interactions can be taken to mimic the association with a relevant binding partner. The terminal residue of chain A, Phe232, anchors the peptide in the groove with a combination of hydrophobic and hydrophilic interactions. The aromatic side chain interacts with a hydrophobic patch consisting of the chain B residues Leu180, Val201, and Phe216. The carboxylate of this terminal residue forms a hydrogen bond with the main chain amide of Phe216, and participates in a water-mediated hydrogen-bonding network with Asp198 and Thr200. The C-terminal peptide then arches away from β8, trailing back to Glu230, which forms a salt bridge with Arg167. Another residue, Gln221, interacts with Glu230, as well as contributing to a water-mediated interaction with the backbone of Pro228. Gln221 is conserved in the trypanosomatid TBC-B sequences, but is highly variable in other species. The final interaction that chain A undergoes as it exits the groove is a salt bridge between Asp226 and Arg218.No crystal structures have been solved with an α-tubulin tail-like ligand bound to TBC-B, and, despite our efforts, it was not possible to cocrystallize the  chain B A. The syThe NMR structure of the CAP-Gly domain of human CLIP-170 complexed with an α-tubulin tail peptide provides an example for comparative purposes T. brucei α-tubulin. A range of concentrations of the protein domain and the peptide were tested, but no heat changes were observed (data not shown).The Gly/Asp difference at position 4 in the tubulin-binding loop, discussed earlier, allows the C-terminus of chain A to bind further along the groove than the α-tubulin tail peptide in the CLIP-170 structure. This causes a relative shift in the position of the Val231 side chain, which is directed out of the groove, and undergoes no interactions with the CAP-Gly domain. The third residue from the end of chain A, Glu230 A3 in , is in tThe peptide-binding groove on chain A is occupied by two formate molecules, derived from the crystallization mixture, which bind in similar fashion to the negatively charged moieties of the C-terminal peptide just discussed (data not shown). One formate interacts with Arg167 and the other with Gln221.T. cruzi and Trypanosoma vivax α-tubulin, this sequence is matched exactly. In T. brucei gambiense and T. brucei 427, the sequence is EMF, and, as just described, we present structural data to show that the sequence EVF can also bind to a CAP-Gly domain. Our structure of TbTBC-B indicates that the penultimate residue, whether glutamate or methionine, is probably directed out of the peptide-binding groove, and, with no direct interactions involving the side chain, the identity would appear to be less important for binding.CAP-Gly domains recognize the highly conserved α-tubulin C-terminal tail with the sequence EE[Y/F] T. brucei is Tb927.3.2680, a 530-residue protein with ∼ 25% sequence identity. The protozoan protein contains a leucine-rich repeat segment and a Ubl domain but, surprisingly, lacks a CAP-Gly domain. In fission yeast, the TBC-E homologue Alp21 also lacks a CAP-Gly domain, but is indispensible for maintaining α-tubulin levels, MT integrity, and cell survival Both TBC-B and TBC-E retain highly conserved CAP-Gly domains across numerous different species, and this probably reflects important roles in tubulin biology T. brucei genome, even in proteins that usually contain such modules, e.g. kinesins. Perhaps, in trypanosomatids, TBC-B is sufficient to provide this interaction with the α-tubulin tail, or other modules, yet to be discovered, may compensate for this function.We could not identify any other CAP-Gly domains encoded by the As it was not possible to obtain crystallographic data on the linker region between the globular domains (residues 89–153), the CD spectrum of full-length TBC-B was analysed. The spectra indicated a low α-helical content of ∼ 10% and a β-strand content of ∼ 35%. The α-helical content of the Ubl domain itself is ∼ 8% of the overall structure, and, together with the prediction of a short helical segment between residues 116 and 123, ∼ 4% of the sequence, there is excellent agreement with the CD data. Residues with a β-strand conformation constitute nearly 30% of the full-length structure, which is also in good agreement with the spectroscopic data. Both the CD results and the structural analyses also agreed well with the predicted secondary structure, and overall indicate a two-domain structure with a flexible linker.TbTBC-B, the first such protist structures, were determined to 2.3-Å and 1.6-Å resolution, respectively. It was not possible to solve the full-length structure, as the crystals did not diffract sufficiently well, and attempts to extend structural information into the region linking the two domains were also unsuccessful. It was determined that the missing linker region is mostly unstructured, with only a short segment of α-helix being noted, and potentially flexible.Structures of the Ubl and CAP-Gly domains of Saccharomyces cerevisiae TBC-E interacts with the ubiquitin receptor Rpn10 via the Ubl domain, and subsequently with a ubiquitin ligase complex, providing a route to protein degradation TbTBC-B is conserved in the Ubl domains of TBC-E (data not shown).The fold of the Ubl domain is highly conserved, despite low sequence identity within TBC-B proteins and, indeed, ubiquitin itself. A striking similarity is observed in the spatial location of two functionally important lysines in ubiquitin, and strongly supports a biological function for the Ubl domain, and by implication for TBC-B. Through reversible post-translational modification, TBC-B can influence aspects of tubulin biology in a proteasome-dependent manner. Such a conclusion is consistent with other studies. Yeast two-hybrid studies suggest that proteasome-dependent degradation of human TBC-B is driven by gigaxonin binding to the Ubl domain In the final stages of MT assembly, α-tubulin and β-tubulin form a heterodimer with the encouragement of the TBC proteins. A balance has to be struck between the availability of free tubulin, heterodimer assembly, and release of MT structures. This process involves a number of highly abundant proteins, and the presence of Ubl domains offers a means whereby protein folding and the population of complex assemblies can be regulated by protein degradation or recycling. The use of a Ubl protein rather than ubiquitin itself may provide specificity with regard to this aspect of tubulin biology. Cognate activating enzymes might then contribute to the regulation of levels of free tubulin heterodimers, tubulin polymerization, and MT catastrophe, or to avoid miscommunication during a stress response. It will therefore be of great interest to now identify, from a plethora of candidates, the specific ligases and proteases that might participate in the control of MT disassembly/assembly as opposed to other biological functions.TbTBC-B. A glycine replaces asparagine at position 3 of the normally conserved GKNDG sequence, and allows the C-terminus of chain A to bind further down in the peptide-binding groove. This appears to be a trypanosomatid-specific feature.The molecular packing in the crystal structure of the CAP-Gly domain places the C-terminal tail of one CAP-Gly domain in the peptide-binding groove of a symmetry-related molecule, and allows examination of interactions in this binding site. The binding differs slightly from that observed in a CLIP-170 CAP-Gly domain bound to an α-tubulin tail peptide, owing to a difference in the β7–β8 tubulin-binding loop of Tubulin and MT assembly are subject to C-terminal post-translational modifications, which provide important tracking positions for a range of MT-binding partners T. brucei α-tubulin by using ITC. According to previous work, the presence of TBC-E might be required for TBC-B to form a stable complex with α-tubulin We were unable to observe any binding of the CAP-Gly domain with a hexapeptide representing the terminal residues of TbTBC-B was amplified from genomic DNA (strain 927) with 5′-CATATGTCCGTTGTTAAAGTATCGC-3′ and 5′-CTCGAGTTAAAACACCTCCGGGGGAAAGTC-3′ as forward and reverse primers, respectively . The restriction enzyme sites for NdeI and XhoI are in bold. The PCR product was ligated into pCR2.1-TOPO with the TOPO TA Cloning Kit , and then cloned into a modified pET15b  expression vector, which adds an N-terminal His6 tag and a tobacco etch virus (TEV) protease cleavage site to the product. Sequencing confirmed the identity of the construct, and the vector was heat-shock-transformed into E. coli Rosetta (DE3) pLysS cells for expression. DNA encoding the CAP-Gly domain, residues 157–222, was amplified from the vector containing the TbTBC-B gene with 5′-CATATGGCAGAGACAATACATGTGGGGG-3′ and 5′-CTCGAGTTAAAACACCTCCGGGGGAAAGTC-3′ as forward and reverse primers, respectively (ThermoFisher Scientific). The gene fragment was cloned into the modified pET15b vector as described above, and transformed into E. coli Rosetta (DE3) pLysS cells for protein production.The gene encoding full-length TbTBC-B and the CAP-Gly domain. Typically, cells were grown at 37 °C in 1 L of LB medium supplemented with carbenicillin (50 μg·L−1) and chloramphenicol (25 μg·L−1). Gene expression was induced, at a D600 nm of 0.6, by addition of 1 mm isopropyl thio-β-d-galactoside. The culture was incubated for a further 16 h at 22 °C, and cells were then harvested by centrifugation at 3500 g for 30 min at 4 °C. The cells were resuspended in a lysis buffer  containing DNase I and EDTA-free protease inhibitors , and then lysed with a French press at 16000 p.s.i. The resultant lysate was centrifuged at 35 000 g for 30 min at 4 °C, and the supernatant was loaded onto a pre-equilibrated HisTrap HP 5-mL column  precharged with Ni2+. A linear gradient of 25 mm to 1 m imidazole was applied to elute the proteins, and the derived fractions were analysed by SDS/PAGE. Samples were pooled and incubated with His-tagged TEV protease at 30 °C for 3 h, and then dialyzed into 50 mm Tris/HCl (pH 7.5) and 250 mm NaCl. The sample was loaded onto a HisTrap HP 5-mL column to remove the His-tagged TEV protease, uncleaved product, and histidine-rich contaminants. A Superdex 20 026/60 size exclusion column  equilibrated with 50 mm Tris/HCl (pH 7.5) and 250 mm NaCl was used to further purify the protein. This column had been calibrated with BioRad Gel Filtration standards. Fractions containing the proteins were pooled and concentrated to 10 mg·L−1 with Amicon Ultra devices (Millipore) for subsequent use. The purity and identity of the proteins were confirmed by MALDI-TOF MS. Yields of ∼ 20 mg·L−1 cell culture for full-length TBC-B and ∼ 8 mg·L−1 cell culture for the CAP-Gly domain were obtained.Similar protocols were applied to purify full-length A series of constructs encoding five polypeptide fragments, covering residues 91–232, 101–232, 113–232, 128–226, and 143–232, were also produced in an attempt to extend structural knowledge between the domains. These polypeptides proved to be either insoluble or failed to crystallize, and so no further details are provided.−1 protein solutions and a 0.02-cm-pathlength quartz cuvette. Five scans were accumulated and averaged with the following parameters: scan rate, 50 nm·min−1; response, 0.5 s; and bandwidth, 1 nm. Protein CD spectra were corrected by subtracting the appropriate buffer spectrum and correcting for protein concentration. Protein secondary structure estimates were obtained with the contin procedure dichroweb server CD spectra were recorded on a Jasco J-810 spectropolarimeter. Far-UV CD spectra were obtained with 0.5 mg·mL2O, and then submitted for MALDI-TOF MS analysis. This gave a molecular mass of 12 069 Da for the polypeptide. The fragment was isolated by SDS/PAGE and then transferred onto a poly(vinylidene difluoride) membrane. The band was then submitted for N-terminal Edman sequencing, and the sequence GHMSVVKV was identified. This corresponds to the N-terminus of TbTBC-B, with the first two residues being remnants of the TEV cleavage site after treatment to remove the His6 tag. These data indicated that the Ubl domain had crystallized.Crystals of the full-length protein did not diffract beyond ∼ 7-Å resolution (data not shown). Limited proteolysis by addition of chymotrypsin to the crystallization drops TbTBC-B (50 mg) was incubated with chymotrypsin (0.02 mg) overnight, and then dialyzed into 50 mm Tris/HCl (pH 7.5) and 50 mm NaCl. The mixture was loaded onto a HiTrap Q HP 5-mL column, and eluted with a linear gradient of 50 mm to 1 m NaCl. The products of the cleavage eluted as two peaks, and were analysed by SDS/PAGE. Sample 1 eluted at 50 mm NaCl, and sample 2 at 220 mm NaCl. MALDI-TOF MS of sample 1, the Ubl domain, gave a mass of 12 002 Da, and MALDI-TOF MS of sample 2 gave a mass of 12 116 Da. Both protein fragment samples were concentrated to 5 mg·mL−1.Full-length −1 of cell culture. Analysis by MALDI-TOF MS indicated full incorporation of four SeMet residues.A SeMet-substituted Ubl domain was obtained from material generated as described above, and with incorporation achieved following metabolic inhibition Initial crystallization conditions were established by sitting drop vapour diffusion with a Phoenix Liquid Handling System  and commercially available screens. Several conditions were optimized for hanging drop vapour diffusion, with drops of 1 μL of protein solution and 1 μL of reservoir.m Hepes (pH 7.5), 1% poly(ethylene glycol) 400, and 2 m ammonium sulfate, and a protein concentration of 2.5 mg·mL−1. Crystals of the CAP-Gly domain, derived from expression of a gene fragment, were rod-like, with dimensions of 1 × 0.05 × 0.05 mm, and were obtained with reservoir conditions of 0.2 m potassium formate, 30% poly(ethylene glycol) 3350 and a 7.5 mg·mL−1 protein solution in 50 mm Tris/HCl (pH 7.5) and 250 mm NaBr.Rectangular crystals (dimensions 0.3 × 0.1 × 0.1 mm) of the Ubl domain, which had been isolated after proteolysis of the full-length protein, were obtained with a reservoir of 0.1 ++ dual image plate detector. Subsequently, data were collected with ADSC charge-coupled device detectors at the Diamond light source beamline I03 for the Ubl domain and at beamline I02 for the CAP-Gly domain.Crystals were transferred to a solution containing a mixture of their reservoir solution and 40% poly(ethylene glycol) 400 for ∼ 15 s before being cooled to −173 °C in a stream of nitrogen. The crystals were characterized in-house with a Micromax HF007 copper-rotating anode X-ray generator equipped with an R-axis IVxdsscalaf′ maximum wavelength (λ = 0.98 Å) in the ccp4 pipeline crankbp3 solomonbuccaneerData from the Ubl domain crystal were processed with imosflmscalaphyre server M. musculus CAP-Gly domain of TBC-B (PDB code 1WHG), sharing a sequence identity of ∼ 55%, as a suitable model for molecular replacement. A poly-Ala model was prepared with chainsawphaserR-factor of 49% to 1.6-Å resolution.Data from the CAP-Gly domain crystal were scaled and processed with cootrefmacmolprobitypymolareaimolBoth structures were refined with rounds of map inspection and model manipulation with phyrepsipreddaliconsurfmafftpsiblastconsurf, with identity > 50% were used for comparisons. However, there were not enough homologues with identities of > 30% of the Ubl domain for consurf to be used, so 31 sequences with identities in the range 31–38%, were aligned by the use of muscleSecondary structure and disorder predictions were obtained from m Tris/HCl (pH 7.5) and 250 mm NaCl. Typically, 8 μL of peptide was injected from the stirred syringe (305 r.p.m.) 30 times into the sample cell. The peptides were dissolved in the minimum amount of the buffer, and then diluted to the appropriate concentration just before use. Four experiments were carried out with CAP-Gly/hexapeptide concentrations of 1 : 10, 10 : 100, 7 : 500 and 45 : 500 μL. No binding events were observed.ITC experiments were performed at 25 °C with a VP-ITC calorimeter . The 1.4-mL sample cell and 300-μL syringe were filled with TBC-B CAP-Gly domain and peptide EDVEEY in a buffer of 50 m


Dysfunction of these enzymes may cause, for instance, life-threatening diseases in humans, the loss of sex determination in nematodes and embryo lethality in plants. Although the calpain family is well characterized in animal and plant model organisms, there is a great lack of knowledge about these genes in unicellular eukaryote species (i.e. protists). Here, we study the distribution and evolution of calpain genes in a wide range of eukaryote genomes from major branches in the tree of life.Calpains are CaOur investigations reveal 24 types of protein domains that are combined with the calpain-specific catalytic domain CysPc. In total we identify 41 different calpain domain architectures, 28 of these domain combinations have not been previously described. Based on our phylogenetic inferences, we propose that at least four calpain variants were established in the early evolution of eukaryotes, most likely before the radiation of all the major supergroups of eukaryotes. Many domains associated with eukaryotic calpain genes can be found among eubacteria or archaebacteria but never in combination with the CysPc domain.de novo eukaryote domains, have been assembled into many novel domain combinations along the evolutionary history of eukaryotes. Some of the new calpain genes show a narrow distribution in a few branches in the tree of life, likely representing lineage-specific innovations. Hence, the functionally important classical calpain genes found among humans and vertebrates make up only a tiny fraction of the calpain family. In fact, a massive expansion of the calpain family occurred by domain shuffling among unicellular eukaryotes and contributed to a wealth of functionally different genes.The analyses presented here show that ancient modules present in prokaryotes, and a few  Defecbstrates -7. Furthin fungi ,9. Plantr plants -12.2+ required for their activation [In vivo, calpain activity depends on the presence of three key catalytic amino acid residues  in the CysPc domain [The calpain family belongs to protease clan CA, and is classified into classical and non-classical forms based on domain architecture . The clativation . In vivoc domain ,15, althc domain . Comparac domain ,14,17,18c domain . For insc domain ,21. Usinc domain . The phyPlasmodium falciparum to 14 genes in Trypanosoma brucei) indicate a large variation of calpain diversity in single-celled eukaryotes. In fact, many unknown calpain genes may exist among hitherto unexplored unicellular organisms, some of which diverged early after eukaryotes arose and that display ancient morphological features and cellular structures [Entamoeba and slime molds Dictyostelium), Excavata , Plantae , SAR , Alveolata , and Rhizaria ) and a loose assemblage of Hacrobia . Despite several attempts, the evolutionary relationships between these supergroups have not been completely resolved, but recent reports suggest that Plantae, SAR and Hacrobia are constituting a mega group [Thecamonas and Collodictyon, that may occupy distinct positions in the tree of life [Until now, studies on the diversity and evolutionary history of the calpain gene family have been focused on multicellular eukaryotes, including animals, land plants and fungi. Although calpain-like genes were also reported in several protozoan genomes such as those of the apicomplexan and kinetoplastid parasites ,23, the ructures ,25. Currructures -28. Thesga group , while Oga group ,31. In a of life ,33. TheyIn this study, we have searched for calpain genes in genomes representing a broad taxonomic sampling from all eukaryote supergroups with emphasis on unicellular organisms. We present a massively expanded calpain gene family, in which a large number of the new calpain genes are composed of many other domains than previously reported. Evolutionary inferences suggest that most of the calpain variants arose from the combinations of ancient domains through domain shuffling mechanisms. Domains derived more recently have contributed to the innovation of the calpain family by multiple independent insertion events. The majority of the new calpain variants are considered as non-classical types, implying that the classical calpains typical for animal and human genomes comprise only a small subset of the total gene family. The vast diversity of calpains described here provides a new framework for addressing the function of calpain genes in unicellular eukaryotes, and for elucidating the various levels by which these important proteins are regulated to prevent diseases or developmental defects in higher eukaryotes.Our survey of calpain diversity identified a total of 41 different domain arrangements, of which 28 have not been previously reported  the amoeboa-flagella Naegleria gruberi (Excavata)and the ciliate Paramecium tetraurelia (SAR) also contain a large number of genes encoding seven, five and four calpain variants, respectively. In contrast, one single calpain was found in all land plants (named Dek1), Entamoeba histolytica, Spizellomyces punctatus, Plasmodium falciparum, Cryptosporidium parvum and Thalassiosira pseudonana. No CysPc domain was detected in Giardia intestinalis, Dictyostelium discoideum, Chlorella NC64A or red algae (Cyanidioschyzon merolae), even if we loosened the threshold  in the BLAST searches. Thus, we found a large variation in calpain gene numbers in the 44 representative taxa. Even closed related species, such as species in genera Trichomonas and Giardia, have very different calpain gene content.Among the investigated species, Of all 41 calpain types, we identified that 13 variants are present in more than one supergroup. For instance, types 1 (CysPc), 3 CysPc-C2L) and 4 (CysPc-C2L-C2L) show a scattered taxonomic distribution  probably arose soon after the early duplication of domain CysPc based on its presence in nearly all supergroups . In addition, it was found in L probablEctocarpus, Thalassiosira, Fragilariopsis and Leishmania  have one duplicated C2L at the C-terminus, forming MIT-CysPc-C2L-C2L. In contrast, the presence of one duplicated MIT domain at the N-terminus of CysPc is observed in vertebrates  outside the Metazoa and hence strengthens the notion that these genes are animal-specific [Paramecium (type 32: EF-CysPc-C2L) and Tetrahymena (type 33: EF-CysPc), respectively , the basal fungi , the pathological parasites , green algae (i.e. Chlamydomonas reinhardtii and Volvox carteri), brown algae , phagotrophic protists  and marine phytoplankton . In addition, three early diverged multicellular species  in Metazoa together with seven representative land animals and plants were involved in taxonomic sampling as well. For a full overview of the species in our analyses and how they are related to the corresponding supergroup, see Additional file We sampled 34 unicellular eukaryotic organisms for surveying and characterizing calpain diversity. These taxa represented a wide selection from the proposed supergroups including the Choanozoa (i.e. -10). We used a relatively strict criterion to collect calpain-like genes with a high-quality sequence. Specifically, we included sequences that showed more than 40% overlap with the CysPc query. Here, one gene encoding a ‘calpain-like cysteine protease’ was found in Dictyostelium discoideum AX4 [A primary BlastP or tBlastN  search wdeum AX4 , but thideum AX4 -49. We odeum AX4 . The dethttp://www.mn.uio.no/bio/english/people/aca/kamran/data/Calpain_final_247_accession.dat . ProtTest 3.0 was used for amino acid substitution model selection using the Akaike Information Criterion (AIC) to choose the best-fitting tested model (LG + GAMMA) for phylogenetic analyses [The CysPc sequences were then aligned by MAFFT using the L-INS-i algorithm , followianalyses .Reconstruction of maximum likelihood (ML) phylogenies from the calpain sequence alignment was performed using RAxML v7.2.6 . For mulThe authors declare that they have no competing interests.OAO and KST and conceived, designed and supervised this study. ZL and SZ collected data and performed analyses. SZ, ZL, RW, OAO and KST interrepted the data. SZ, ZL, KST, OAO, RW contributed to drafting the manuscript. VD and WJ read and revised the mansucript. All authors read and approved the final manuscript.Table S1. Accession numbers, domain combination of calpains.Click here for fileFigure S1. Eukaryotic calpain phylogeny inferred from the CysPc domain alignment . The phylogeny is obtained from the consensus between two independent Bayesian inferences. For each node, support values are marked (numbers from left to right: Bayesian posterior probabilities (PP) inferred under LG /CAT models and maximum-likelihood bootstraps (% BP) inferred using PROTGAMMALG model) if all are more than 80% BP and 0.8 PP (filled circle) or more than 50% BP and 0.5 PP (open circle). Dashes ‘-’ show the support values are marked< 50% BP or 0.5 PP. For multiple CysPc domains found in the calpain sequence,1st, 2nd and 3rd indicate their orders from N-terminus to C-terminus of the calpain sequence. The domains marked by ‘*’ indicate their identities only have marginal significance of e-value in domain database research due to high sequence divergence. The branches and clades are assigned to the numbers that represent specified domain combinations shown in Figure Click here for fileFigure S2. Eukaryotic calpain phylogeny rooted by the bacterial outgroup . The phylogeny is obtained from the consensus between two independent Bayesian inferences. For each node, support values are marked (numbers from left to right: Bayesian posterior probabilities (PP) inferred under LG (left) /CAT (middle) models and maximum-likelihood bootstraps (% BP) inferred using PROTGAMMALG (right) model) if all are more than 80% BP and 0.8 PP (filled circle) or more than 50% BP and 0.5 PP (open circle). Dashes ‘-’ show the support values < 50% BP or 0.5 PP. For multiple CysPc domains found in the calpain sequence,1st, 2nd and 3rd indicate their orders from N-terminus to C-terminus of the calpain sequence. The domains marked by ‘*’ indicate their identities only have marginal significance of e-value in domain database research due to high sequence divergence. The branches and clades are assigned to the numbers that represent specified domain combinations shown in Figure http://www.mn.uio.no/bio/english/people/aca/kamran/data/Calpain_final_261_accession_outgroup.dat.Click here for fileFigure S3. Phylogeny of the calpain genes with MIT modules. The tree is obtained from the consensus between two independent Bayesian inferences. Support values are marked at the nodes, and numbers from left to right represent Bayesian posterior probabilities (PP) inferred under CAT /LG models and the maximum-likelihood bootstraps (% BP) inferred using PROTGAMMALG model in RAxML v7.2.5. All support values more than 80% BP and 0.8 PP or more than 50% BP and 0.5 PP are shown as full or open circles. Dashes ‘-’ show the support values < 50% or 0.5 PP. The MIT calpain genes found in Tetraodon nigroviridis, Takifugu rubripes, Gasterosteus aculeatus, Gadus morhua, Salmo salar, Danio rerio, Oryzia latipes, Gallus gallus, Dasypus novemcinctus, Ailuropoda melanoleuca, Sus scrofa, Canis familiaris, Bos taurus and Rattus norvegicus are added to this analysis. The domains marked by ‘*’ indicate their identities only have marginal significance of e-value in domain database research due to high sequence divergence. The branches and clades are assigned to the numbers that correspond to specified domain combinations shown in Figure http://www.mn.uio.no/bio/english/people/aca/kamran/data/Calpain_MIT_accession.dat.Click here for fileFigure S4. Proposed origin of calpain superfamily domain combinations shown on the global eukaryote phylogeny. The tree is rooted according to Cavalier-Smith T 2010. Biol Lett 6(3):342-345. Red bars on the branches indicate the hypothetical origin of specified domain combinations. Calpain variants only found in one taxon or among closely related species are marked in red within the supergroup rectangles, which likely constitute lineage-specific domain combinations.Click here for fileFigure S5. Phylogeny of calpains in Metazoa. The topology is obtained from the consensus between two independent Bayesian inferences. The representative taxa sampled here include Homo species, Mus musculus, Gallus gallus, Xenopus laevis, Danio rerio, Nematostella vectensis, Drosophila melanogaster, Caenorhabditis elegans, Trichoplas adhaerens and Amphimedon queenslandica. Support values are marked at the nodes, and number from left to right represent Bayesian posterior probabilities (PP) inferred under CAT /LG models and the maximum-likelihood bootstraps (% BP) inferred using PROTGAMMALG model. All support values more than 80% BP and 0.8 PP or more than 50% BP and 0.5 PP are marked by full or open circles. Dashes ‘-’ indicate the support values < 50% BP or 0.5 PP. The branches and clades are assigned to the numbers that represent specified domain combinations listed in Figure http://www.mn.uio.no/bio/english/people/aca/kamran/data/Calpain_Metazoa_accession.dat.Click here for file


Computational/manual annotations of protein functions are one of the first routes to making sense of a newly sequenced genome. Protein domain predictions form an essential part of this annotation process. This is due to the natural modularity of proteins with domains as structural, evolutionary and functional units. Sometimes two, three, or more adjacent domains  are the operational unit responsible for a function, e.g. via a binding site at the interface. These supra-domains have contributed to functional diversification in higher organisms. Traditionally functional ontologies have been applied to individual proteins, rather than families of related domains and supra-domains. We expect, however, to some extent functional signals can be carried by protein domains and supra-domains, and consequently used in function prediction and functional genomics.http://supfam.org/SUPERFAMILY/dcGO including an enrichment analysis tool.Here we present a domain-centric Gene Ontology (dcGO) perspective. We generalize a framework for automatically inferring ontological terms associated with domains and supra-domains from full-length sequence annotations. This general framework has been applied specifically to primary protein-level annotations from UniProtKB-GOA, generating GO term associations with SCOP domains and supra-domains. The resulting 'dcGO Predictor', can be used to provide functional annotation to protein sequences. The functional annotation of sequences in the Critical Assessment of Function Annotation (CAFA) has been used as a valuable opportunity to validate our method and to be assessed by the community. The functional annotation of all completely sequenced genomes has demonstrated the potential for domain-centric GO enrichment analysis to yield functional insights into newly sequenced or yet-to-be-annotated genomes. This generalized framework we have presented has also been applied to other domain classifications such as InterPro and Pfam, and other ontologies such as mammalian phenotype and disease ontology. The dcGO and its predictor are available at As functional units, domains offer a unique perspective on function prediction regardless of whether proteins are multi-domain or single-domain. The 'dcGO Predictor' holds great promise for contributing to a domain-centric functional understanding of genomes in the next generation sequencing era. Although these massive amounts of data offer an unprecedented opportunity for addressing many fundamental questions in the field of biomedical science ,2, yet mructures ,12. Hencsuperfamily and/or family levels are decent choices regarding the above-mentioned three aspects  of protein modularity [superfamily (or evolutionary) level, domains are distantly related with evidence for common ancestry; within the same superfamily, domains are further divided into the family level wherein domains are often related by sequence similarity [superfamily and family levels [SCOP domains defined at dularity . At the milarity . Based oy levels . Consequy levels . To betty levels . Althougy levels , we findy levels . The comy levels . In otheIn an attempt to address the questions mentioned above, we first generalized our previous framework for capturing GO terms suitable for annotating both individual domains and supra-domains. Using the concept of reverse engineering, at the core of this domain-centric approach is: if a GO term tends to annotate a set of proteins containing a certain domain (or a set of proteins containing a supra-domain), then this term should also carry out functional signals for that domain (or supra-domain). Biologically, the resulting domain-centric GO (dcGO) annotations have carried on hidden functional signals buried under existing annotated proteins. Methodologically, this resource has taken into account the structural organization of GO by performing two types of statistical inference. Because of these considerations, a domain (supra-domain) can be associated with multiple GO terms (if any) that are informative to annotate it, and thus allowing multiple associations between domains and GO terms (quite similar to those between proteins and GO terms). Since the inferred dcGO can preserve the input information at the protein level, to some extent our approach addresses the challenges like one-domain-many-functions and one-function-many-domains (if there exists any evidence to support one2many associations). With the dcGO annotations at hand, we then developed 'dcGO Predictor' to predict functions of the target sequences in the CAFA experiment, an international competition for automatic protein function and critical assessment . FinallyThe structural domain information of a protein is closely relevant to biological functions it has. To reveal the extent of functional signals carried by protein domains (and supra-domains in the multi-domain proteins), we developed a domain-centric Gene Ontology (dcGO) approach . When it comes to annotation density, however, these triple domain types share the same range for each of three GO ontologies, including Biological Process , Molecular Function  and Cellular Component . This un-skewed annotation density partially implies that dcGO approach has no biases towards processing individual domains and supra-domains. Although many more annotations will be inferred in the future as primary source databases improve, we do not expect the annotation density to change dramatically from one fortnight update to the next.Table To maximize performance, protein function prediction should integrate different kinds of predictive features -33, inclWe first examined the PR-RC curves of our prediction using both domains and supra-domains for eukaryotic sets Figure . Conside'dcGO Predictor' is not just computationally efficient, but brings additional understanding to the annotation at a sub-protein resolution. Using modular domains is not just conceptually more intuitive, but easier to implement than other more complex methods. For example, we have extended the generalized domain-centric approach to other InterPro signatures . The addThe 'dcGO Predictor' for function prediction in the CAFA experiment validates the quality of this dcGO annotations resource. To extend the usefulness of the resource, we generated meta-GO terms Figure . SimilarOikopleura dioica [Capsaspora owczarzaki, Monosiga brevicollis, and Proterospongia). These genomes are phylogetically located at the animal-fungal boundary and thus afford an important look at the origin of the multicellular animals [Since the derived meta-GO terms provide a grain-specific overview, restricting their related dcGO annotations  is particularly useful for GO term enrichment analysis. This kind of enrichment analysis is commonly employed for interpreting transcriptome data  with a GO annotations for proteins over a wide spectrum of species  were downloaded from the UniProtKB on 19th, November 2011 . Only thsuperfamily (SF) level and 3,246 distinct domains at SCOP family (FA) level. A vast number of analyzable UniProt protein space  secures the adequate power of statistical inference carried out in this study.In the SUPERFAMILY database , proteinsuperfamily level) or gaps (estimated to be one or more unknown domains). The SUPERFAMILY database contains a total of 14,531 fully-annotated supra-domains (i.e. containing no unknown domains) that are present in analyzable UniProt protein space.In multidomain proteins, a certain domain tends to co-occur/co-evolve with other domains. Considering such promiscuous nature  and follThe brief summary is illustrated in Figure We took UniProt proteins with experimentally or manually curated GO annotations and high-coverage domain/supra-domain assignments as two training inputs. The correspondence matrix between domains/supra-domains and GO terms are tabulated with each entry as observed number of Uniprot proteins which contain that domain/supra-domain (given in column) and can be annotated by that GO term (given in row).We used the hypergeometric distribution as a null-hypothesis and performed statistical test  to infer the possible associations between a GO term and a domain/supra-domain. Terms in GO are not isolated; rather they are organized as a directed acyclic graph (DAG) by viewing individual terms as a node and its relations to parental terms  as directed edges. Moreover, GO follows 'true-path rule', that is, a protein/domain annotated to a term should also be annotated by its all parent terms. To respect DAG structure and true-path rule, we conducted two types of statistical inferences. First, we calculated an overall p-value  using all analyzable UniProt proteins  as the background. We also calculated a relative p-value (and the corresponding relative hypergeometric score) using the background of only those UniProt proteins annotated to all direct parental GO terms.-3) was accepted to statistically infer GO annotations of individual domains and supra-domains. In addition to FDR as significant measure, we also took the smaller of the overall and relative hypergeometric scores from (2) to indicate the strength of associations, denoted as h-score.We first took the larger one of the overall and relative p-values from (2) to indicate the likelihood of associations between that GO term and that domain/supra-domain. To account for the multiple hypothesis testing, the Benjamini-Hochberg derived FDR  rather t-3) and the h-score . The latter was used for the evaluation of protein function prediction.According to the true-path rule, the inferred GO terms for a domain/supra-domain were propagated to all ancestor terms, along with the FDR and h-score . The inferred originally were called as direct annotations, the propagated as inherited annotations; both of them constituted a GO annotation profile in DAG. Notably, each annotation was associated with the FDR . The higher value of the p-score indicates the more evident the prediction is. Each target (if predictable) was accompanied by a list of GO terms along with the corresponding predictive scores. The intuition behind this simple calculation is to rank the predictive p-scores for precision-recall analysis below.For the prediction part , we firSUM is the sum of all h-scores to support a GO term transferred to the target, MIN and MAX are respectively the minimum and maximum of SUM over a whole list of predicted GO terms for the target.where t) using Eq. 2 and Eq. 3. All these calculations were done separately for GO terms in BP and GO terms in MF. From individual target-specific PR vs. RC values, the precision  of each of eukaryotic sets (and the whole sets) was further calculated as an average over all targets contained in each set (and the whole sets).For the evaluation part, we first obtained a total of about 246,000 annotations newly added until 19th, November 2011. Based on these gold standards, then we estimated precision (PR) and recall (RC) for each sequence target under a given p-score  overlapped with gold standards, FP for false positives - the number of the predicted GO terms that are not in gold standards, FN for false negatives - the number of gold standards that are not in the predicted GO terms.where We used information content (IC) of a GO term to measure its specificity in meaning of individual domain and supra-domain annotations (directed and inherited). For a given GO term, we defined IC as negative 10-based log-transformed frequency of domains/supra-domains annotated to that term. The reason behind using IC rather than the GO tree-like structure is that the GO was originally designed for annotating proteins, and some parts of GO structure might be irrelevant to annotate domains/supra-domains. Similar to our previous report , a searcIn additional to two SCOP-orientated and GO-orientated options for the navigations (the most-left 'BROWSE'), we also provided flat files and MySQL tables for the download on the dcGO website .BP: Biological Process; CC: Cellular Component; dcGO: domain-centric Gene Ontology; DAG: directed acyclic graph; FDR: false discovery rate; FN: false negatives; FP: false positives; IC: information content; MF: Molecular Function; PR: precision; RC: recall; FA: SCOP family; SF: SCOP superfamily; TP: true positives; UniProtKB: UniProt Knowledgebase.The authors declare that they have no competing interests.HF conceived and designed the study, performed the data analysis and interpretation, and wrote manuscript. JG conceived and coordinated the study, contributed to data interpretations and manuscript preparation. Both authors read and approved the final manuscript.CAFA function prediction using SCOP individual domains and supra-domains plus InterPro domains. (A) Precision-recall curves based on GO annotations of both domains and supra-domains. The left panel is for the Molecular Function (MF), and the right panel for Biological Process (BP). (B) The same as in (A) but using additional InterPro domains .Click here for filePerformance comparisons between the currently revised version of 'dcGO Predictor' and the old version. The revised version uses h-scores to calculate p-score while the old version  uses the FDR to calculate p-scores.Click here for fileOikopleura dioicaEnriched GO terms for domain repertoire present at a rapidly evolving metazoan, .Click here for fileEnriched GO terms for domain repertoire present at each of three genomes of animal-fungal boundary.Click here for file


Neuroblastoma are pediatric tumors of the sympathetic nervous system with a poor prognosis. Apoptosis is often deregulated in cancer cells, but only a few defects in apoptotic routes have been identified in neuroblastoma.http://r2.amc.nl). BIRC6 was selected for further analysis as a tumor driving gene. Knockdown experiments were performed using BIRC6 lentiviral shRNA and phenotype responses were analyzed by Western blot and MTT-assays. In addition, DIABLO levels and interactions were investigated with immunofluorescence and co-immunoprecipitation.Here we investigated genomic aberrations affecting genes of the intrinsic apoptotic pathway in neuroblastoma. We analyzed DNA profiling data (CGH and SNP arrays) and mRNA expression data of 31 genes of the intrinsic apoptotic pathway in a dataset of 88 neuroblastoma tumors using the R2 bioinformatic platform , that can bind and degrade the cytoplasmic fraction of the pro-apoptotic protein DIABLO. DIABLO mRNA expression was exceptionally high in neuroblastoma but the protein was only detected in the mitochondria. Upon silencing of BIRC6 by shRNA, DIABLO protein levels increased and cells went into apoptosis. Co-immunoprecipitation confirmed direct interaction between DIABLO and BIRC6 in neuroblastoma cell lines.We observed frequent gain of the Our findings indicate that BIRC6 may have a potential oncogenic role in neuroblastoma by inactivating cytoplasmic DIABLO. BIRC6 inhibition may therefore provide a means for therapeutic intervention in neuroblastoma. BIRC6  is a cytoplasmic protein with a dual role. Firstly, BIRC6 has an anti-apoptotic function in the intrinsic apoptotic pathway by antagonizing the pro-apoptotic DIABLO protein. BIRC6 can bind the cytoplasmic DIABLO fraction and induce ubiquitination and proteasomal degradation of this protein,2. BIRC6BIRC6 is highly expressed in several types of cancer. BIRC6 over-expression in acute myeloid leukemia is correlated with a poor outcome[BIRC6 gene was gained in a few samples[BIRC6 expression in colon cancer stem cells is associated with drug resistance[ outcome. A genom samples. Additiosistance.BIRC6 is located on chromosome 2p in the region which shows frequent gain in neuroblastoma[MYCN and ALK, two well characterized oncogenes in neuroblastoma. Amplification of MYCN occurs in 20–30% of neuroblastoma and strongly correlates with a poor prognosis[ALK was recently found to be mutated in 6-10% of primary neuroblastoma[MYCN amplification and ALK mutations seem independent of the gain of chromosome 2p[Neuroblastoma are pediatric tumors that originate from the embryonal precursor cells of the sympathetic nervous system. High stage tumors have a poor prognosis with 20 to 40% overall survival-14. BIRCblastoma. This rerognosis-15. ALK blastoma-20. MYCNosome 2p and therTP53 is mostly intact in primary neuroblastoma although functional defects in the p53 pathway have been described[Caspase 8 (CASP8) is hypermethylated and thereby inactivated in some neuroblastoma resulting in an inactive extrinsic apoptotic pathway[BIRC5 (Survivin) is located on the chromosome 17q region which is frequently gained in neuroblastoma and high BIRC5 expression correlates with a poor prognosis[BCL2 protein is highly expressed in neuroblastoma. Targeted inhibitors against BIRC5[The apoptotic pathway has been widely investigated in neuroblastoma and only a few tumor driving events have been described. escribed. Caspase pathway. The IAPrognosis-26. Alsost BIRC5-32 and Bst BIRC5 are currBIRC5 and BIRC6 to be frequently gained and CASP9 often lost. Since BIRC6 was not previously evaluated in a neuroblastoma model, we studied the potency of BIRC6 as a potential new target for neuroblastoma therapy. Silencing of BIRC6 induced apoptosis and up-regulation of DIABLO. We established BIRC6 to physically interact with DIABLO, indicating that BIRC6 can degrade DIABLO very effectively.To identify patterns in aberrations of genes involved in intrinsic apoptotic signaling we combined high throughput analysis of DNA copy number and mRNA expression of these genes in a dataset of 88 neuroblastoma tumors. We found http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE16476[We used a neuroblastoma tumor panel for Affymetrix Microarray analysis containing 88 primary neuroblastoma tumor samples of untreated patients of which 87 neuroblastoma tumor samples were also used for CGH analysis and SNP array. All neuGSE16476. Public GSE16476 and LastTotal RNA of neuroblastoma tumors was extracted using Trizol reagent  according to the manufacturer’s protocol. RNA concentration and quality were determined using the RNA 6000 nano assay on the Agilent 2100 Bioanalyzer (Agilent Technologies). Fragmentation of cRNA, hybridization to hg-u133 plus 2.0 microarrays and scanning were performed according to the manufacturer’s protocol (Affymetrix inc).High-molecular-weight DNA was isolated from tumor tissue by a standard salt-chloroform extraction method. For refTumor DNA was extracted as previously described, quantified with NanoDrop and the quality was determined by the Abs 260/280 and 230/260 ratio. SNP arrays were processed for analysis of copy number variations with the Infinium II assay on Human370/660-quad arrays containing > 370 000/> 660 000 markers and run on the Illumina Beadstation  according to the manufacturer’s recommendations. Raw data were processed using Illumina’s BeadStudio software suite (Genotyping module 3.0), producing report files containing normalized intensity data and SNP genotypes. Subsequently, log 2 Ratio and B-allele frequency data were imported into the R2 web application for detailed analysis and comparison with the CGH and expression data.http://r2.amc.nl. The expression data were normalized with the MAS5.0 algorithm within the GCOS program of Affymetrix Inc. Target intensity was set to 100. For scoring genomic aberrations of the 31 included genes , supplemented with 10% fetal calf serum, 10 mM L-glutamine, 10 U/ml penicillin/streptomycin, Non Essential Amino Acids (1x) and 10 μg/ml streptomycin. Cells were maintained at 37°C under 5% COr et al..Lentiviral particles were produced in HEK293T cells by co-transfection of lentiviral vector containing the short hairpin RNA (shRNA) with lentiviral packaging plasmids pMD2G, pRRE and pRSV/REV using FuGene HD. Supernatant of the HEK293T cells was harvested at 48 and 72 h after transfection, which was purified by filtration and ultracentrifugation. The concentration was determined by a p24 ELISA. Cells were plated at a 10% confluence. After 24 h cells were transduced with lentiviral DIABLO shRNA  or BIRC6 shRNA  in various concentrations (Multiplicity of infection (MOI): 1–3). SHC-002 shRNA (non-targeting shRNA: CAACAAGATGAAGAGCACCAA) was used as a negative control. Medium was refreshed 24 h after transduction and puromycin was added to select for transduced cells. Protein was harvested 72 h after transduction and analyzed by Western blot.ABT263, a small molecule BCL2 inhibitor, was dissolved in DMSO to a concentration of 20 mM as stock solution. A final concentration of 200 nM ABT263 was used. For the experiment using this compound we chose SJNB12 instead of IMR32 or SKNSH for its high expression of BCL2 and its subsequent high sensitivity to this compound.Z-Val-Asp(OMe)-Val-Ala-Asp(OMe)-FMK  was added to the cells following manufacturer’s protocol in a concentration of 20 μM.Attached and floating cells were harvested on ice 72 h after transduction with shRNA. Cells were lyzed with Laemmlibuffer . Protein concentrations were quantified with RC-DC protein assay . Lysates were separated on a 10% SDS-Page gel and transferred onto a blotting membrane . Blocking and incubation were performed in OBB according to manufacturer’s protocol (LI-COR). Primary antibodies used were anti-BIRC6 , anti-DIABLO , anti-PARP  and anti-BCL2 . Protein loading was checked by anti-β-actin  or anti-α-tubulin . The secondary antibodies used were provided by LI-COR. Proteins were visualized with the Odyssey bioanalyzer (LI-COR).Cells were fixed with 4% paraformaldehyde for 20 min 48 h after transduction with BIRC6 shRNA. Blocking and incubation were performed in OBB according to manufacturer’s protocol (LI-COR). Primary antibodies used were anti-BIRC6  and anti-β-actin mouse monoclonal . Proteins were visualized with the Odyssey bioanalyzer (LI-COR) and quantified and corrected for β-actin using the Odyssey software.Cells were grown on glass slides in 6-well plates and were fixed with 4% paraformaldehyde in PBS 48 h after transduction. We used anti-DIABLO  as a primary antibody, and anti-rabbit  as a secondary antibody. Mitochondria were stained using Mitotracker . Antibodies were dissolved in 5% ELK in PBS/0.2% tween-20. Slides were stained with DAPI (1:1000) in vectashield (Vector Laboratories).Protein was harvested and fractionated using the Subcellular Proteome Extraction Kit according to manufacturer’s protocol . Fraction I (cytosol) and II (membrane/organelle) were used for Western blot.Cells were lyzed in a buffer containing 150 mM NaCl, 50 mM Hepes, 5 mM EDTA, 0.3% NP-40, 10 mM β-glycerophosphate, 6% glycerol, protease inhibitors  and Phosphatase inhibitors . The antibody used for IP was anti-BIRC6 ; negative controls were anti-flag  and protein without antibody. Protein-G agarose beads (Roche) and antibody were incubated for pre-coupling overnight after which lysates were added and incubated overnight. Immunocomplexes were washed, heated at 95°C for 10 min and loaded on a gel for Western blot. Primary antibodies used were anti-BIRC6 and anti-DIABLO . Blots were incubated overnight with primary antibodies, after which a one hour incubation step with anti-rabbit IgG  was performed followed by incubation with the secondary antibody provided by LI-COR.Cells were seeded at 30% confluence in a 48-well plate, transduced with BIRC6 shRNA and after 24 h treated with ZVDVAD. Seventy-two hours after treatment, 25 μl of Thiazolyl blue tetrazolium bromide  was added. After 4–6 h of incubation, 250 μl of 10% SDS, 0.01 M HCl was added to stop the reaction. The absorbance was measured at 570 nm and 720 nm using a plate-reader (Biotek).BIRC5 gene, which is located in the smallest region of overlap (SRO) of gain of chromosomal band 17q25[CASP9 is located at the SRO of deletions of 1p36[BIRC6, which is located on 2p22, is gained in 24% of the neuroblastoma tumors. Distal chromosome 2p is a known region of gain in neuroblastoma. BIRC6 is not located in the SRO of this gained region, but is gained in 84% of the tumors with 2p gain  (FigureBIRC6 is also highly expressed compared to several adult tumors and various normal tissues (FigureBIRC6 is at least partially caused by genomic aberrations often occurring in neuroblastoma tumors.To identify patterns in the aberrations of genes involved in intrinsic apoptotic signaling, we combined high throughput analysis of DNA copy number and mRNA expression of these genes. We included all 31 genes that are directly involved in the mitochondrial apoptotic pathway and their downstream target genes (Tablend 17q25, is gain of 1p36 and it iBIRC6 levels indeed counteract apoptosis in neuroblastoma. We used 2 lentiviral shRNAs targeting different parts of the coding sequence of BIRC6. SKNSH, one of the neuroblastoma cell lines with the highest BIRC6 expression, was transduced with these vectors (FigureWe investigated whether the high The BIRC6 protein functions both by silencing of DIABLO and in the formation of the midbody ring during cell division. Inhibition of each of these functions can result in cell death. We therefore investigated which process causes apoptosis after BIRC6 silencing in neuroblastoma. Apoptosis induced after inhibiting the midbody-related function of BIRC6 has been found to be mediated by CASP2,8,38. WeAlternatively, BIRC6 functions as an IAP that binds DIABLO in the cytoplasm thereby inducing ubiquitination and degradation of DIABLO,2. DIABLUntreated cells have low cytoplasmic DIABLO levels Figured. These These experiments suggest that BIRC6 may effectively inactivate cytoplasmic DIABLO in neuroblastoma cells and can thereby prevent an apoptotic response.In this paper we analyzed aberrations in gene copy number and mRNA expression of genes directly involved in the intrinsic apoptotic pathway in neuroblastoma. BIRC6, known as an inhibitor of the pro-apoptotic protein DIABLO, showed gene copy number gains and increased expression. Silencing of BIRC6 with two shRNAs targeting different parts of the coding sequence resulted in increased cytoplasmic DIABLO levels and triggered apoptosis. As expected, BIRC6 directly interacted with DIABLO proteins.The apoptotic response in neuroblastoma cells upon BIRC6 silencing occurs in a background of surprisingly high DIABLO expression levels. High cytoplasmic levels of DIABLO can be tumor inhibitory through binding of IAPs in the cytoplasm. We suggest two mechanisms why high DIABLO expression does not induce apoptosis in neuroblastoma. Firstly, we show that the major fraction of DIABLO protein has a mitochondrial localization. This mitochondrial sequestration is probably mediated by the exceptionally high levels of BCL2, which occurs in most neuroblastoma tumors. TargeteThe interaction of BIRC6 with CASP9, which has been reported previously,2,45, haRelease of the strongly increased mitochondrial DIABLO levels would offer a therapeutic potential in neuroblastoma. This suggests that the proteins that impair the pro-apoptotic function of DIABLO could be effective drug targets. The previously shown efficacy of targeted BCL2 inhibition could relate to high DIABLO levels. MoreoveThe authors declare that they have no competing interests.All authors have made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. All authors have been involved in drafting the manuscript or revising it critically for important intellectual content and have given final approval of the version to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/285/prepub


Proteolytic shedding of cell surface proteins generates paracrine signals involved in numerous signaling pathways. Neuregulin 1 (NRG1) type III is involved in myelination of the peripheral nervous system, for which it requires proteolytic activation by proteases of the ADAM family and BACE1. These proteases are major therapeutic targets for the prevention of Alzheimer’s disease because they are also involved in the proteolytic generation of the neurotoxic amyloid β-peptide. Identification and functional investigation of their physiological substrates is therefore of greatest importance in preventing unwanted side effects. Here we investigated proteolytic processing of NRG1 type III and demonstrate that the ectodomain can be cleaved by three different sheddases, namely ADAM10, ADAM17, and BACE1. Surprisingly, we not only found cleavage by ADAM10, ADAM17, and BACE1 C-terminal to the epidermal growth factor (EGF)-like domain, which is believed to play a pivotal role in signaling, but also additional cleavage sites for ADAM17 and BACE1 N-terminal to that domain. Proteolytic processing at N- and C-terminal sites of the EGF-like domain results in the secretion of this domain from NRG1 type III. The soluble EGF-like domain is functionally active and stimulates ErbB3 signaling in tissue culture assays. Moreover, the soluble EGF-like domain is capable of rescuing hypomyelination in a zebrafish mutant lacking BACE1. Our data suggest that NRG1 type III independent myelination is not only controlled by membrane-retained NRG1 type III, but also in a paracrine manner via proteolytic liberation of the EGF-like domain.


LNX proteins function as E3 ubiquitin ligases and their domain organisation suggests that their ubiquitin ligase activity may be targeted to specific substrates or subcellular locations by PDZ domain-mediated interactions. Indeed, numerous interaction partners for LNX proteins have been identified, but the LNX gene family. We find that a LNX1/LNX2-like gene arose in an early metazoan lineage by gene duplication and fusion events that combined a RING domain with four PDZ domains. These PDZ domains are closely related to the four carboxy-terminal domains from multiple PDZ domain containing protein-1 (MUPP1). Duplication of the LNX1/LNX2-like gene and subsequent loss of PDZ domains appears to have generated a gene encoding a LNX3/LNX4-like protein, with just two PDZ domains. This protein has novel carboxy-terminal sequences that include a potential modular LNX3 homology domain. The two ancestral LNX genes are present in some, but not all, invertebrate lineages. They were, however, maintained in the vertebrate lineage, with further duplication events giving rise to five LNX family members in most mammals. In addition, we identify novel interactions of LNX1 and LNX2 with three known MUPP1 ligands using yeast two-hybrid asssays. This demonstrates conservation of binding specificity between LNX and MUPP1 PDZ domains.To gain insights into their function we examined the phylogenetic origins and evolution of the LNX gene family has an early metazoan origin with a LNX1/LNX2-like protein likely giving rise to a LNX3/LNX4-like protein through the loss of PDZ domains. The absence of LNX orthologs in some lineages indicates that LNX proteins are not essential in invertebrates. In contrast, the maintenance of both ancestral LNX genes in the vertebrate lineage suggests the acquisition of essential vertebrate specific functions. The revelation that the LNX PDZ domains are phylogenetically related to domains in MUPP1, and have common binding specificities, suggests that LNX and MUPP1 may have similarities in their cellular functions.The  Ligand of Numb Protein-X) or PDZRN (PDZ and RING) family of proteins consists of five members that, in the interest of clarity, we shall refer to hereafter as LNX1 - LNX5. These proteins are characterized by the presence of a RING domain  followed by between one and four PDZ domains . Within the LNX family, LNX1 and LNX2 are closely related and share an identical domain structure consisting of one RING and four PDZ domains -19. Of td in vivo. Despited in vivo,22. Howeet al (2006) who provided the first detailed description of LNX3 function [in vivo[LNX3  was first identified based on its interaction in a yeast two-hybrid screen with a Semaphorin (Sema4C). A similfunction . They refunction . In thisfunction . Here LN [in vivo. Overallin silico based on homology to LNX3 [LNX4 (PDZRN4/PDZRN4S) and LNX5 (PDZRN4L/PDZK4) were identified  to LNX3 ,3. LNX4  to LNX3 . LNX5 waLNX gene family for the first time. In particular, we focus on the phylogenetic origins of the family and the evolutionary relationship between LNX1/2-like and LNX3/4-like proteins. Our analysis traces the ancestral LNX gene to the earliest metazoan lineage. We characterize what may be a novel, modular protein domain present in LNX3, -4 and -5. We also reveal a close phylogenetic relationship between the LNX and MUPP1 protein families. Based on this relationship, we identify novel LNX1- and LNX2-interacting proteins.In the present study, we take advantage of the availability of complete genome sequences for diverse metazoan species to examine the evolution of the LNX1 and LNX2 are found in virtually all vertebrates for which complete genome sequences are available. In addition, fish, marsupial, amphibian and bird genomes have a third LNX1/2-like gene that has been termed LNX-like or LNX2b in zebra fish [LNX3 in chicken and marsupials [LNX2b to reflect the fact that it encodes a protein that is most similar to LNX2. Eutherian mammals have only two LNX1/2-like genes with the loss of LNX2b occuring when it underwent pseudogenization and contributed several exons to the non-coding Xist RNA that controls X-chromosome inactivation [LNX1 and LNX2 share exactly the same domain architecture . LNX1/2-related proteins that lack the RING domain were identified in the hemichordate Saccoglossus kowalevskii and the echinoderm Strongylocentrotus pupuratus. Examining more basal metazoan lineages, we did not find LNX1/2 orthologs in cnidaria or placozoa, but a LNX1/2 ortholog is found in the poriferan Amphimedon queenslandica[LNX1/2 ortholog was found in choanoflagellates or any other non-metazoan species. Thus the ancestral LNX1/2-like gene appears to have an early metazoan origin prior to the divergence of porifera (sponges) from other metazoans, but has subsequently been lost from several lineages. This gene is likely to have been duplicated in the vertebrate ancestor, giving rise to vertebrate LNX1, LNX2 and LNX2b.Database searches for invertebrate nslandica,33. No LT. adhaerens MUPP1 proteins as well as those from A. queenslandica LNX2 . Specifically, Canis familiaris (dog) and Ornithorhynchus anatinus (platypus) appear to lack LNX3, while no LNX4 homolog is found in Monodelphis domestica (opossum). LNX5 is found in most amphibian, fish and mammalian species but seems to be absent from birds. Invertebrate orthologs of LNX3/LNX4 containing RING and PDZ domains are found in some metazoan lineages including hemichordates  and echinoderms(S. pupuratus), cnidarians (Hydra magnipapillata) and placozoans (Trichoplax adhaerens)  Figure . All of a genome , nor wasT. adhaerens. Within this region, the last 150 amino acids are particularly similar and the degree of cross-species sequence identity is as high as that for the PDZ and RING domains. In fact, many invertebrate lineages have LNX3/4-like proteins that contain this highly conserved carboxy-terminal region, but lack the characteristic RING domain and may also be missing one or both of the PDZ domains , nematodes (C. elegans) and cephalochordates (B. floridae). Katoh and Katoh (2004) had previously identified conserved sequences shared by LNX3/4 and D. melanogaster Slip1 (CG1783). With the availability of sequences from other species, we can better define this conserved region that we term the LNX3 homology (LNX3H) domain. Figure D. melanogaster Slip1 is shorter than all other sequences examined including several other arthropods, thus the consensus LNX3 homology region is more extensive than that previously identified [The carboxy-terminal half of LNX3 lacks any identifiable domains annotated in the Pfam, SMART or InterPro databases -36, yet s Figure . Lineagend Katoh 004 had pWe used the multiple sequence alignment to generate a protein secondary structure prediction for the LNX3H domain on the Jpred 3 server . The Jprn) appears to be unique to the LNX family. In principle, however, the common domain architecture of LNX1/2-like and LNX3/4-like proteins may have arisen independently. PDZ and RING domains are abundant in metazoan genomes and so the independent evolution of a RING-PDZn architecture more than once is plausible. Indeed, in arthropods this domain combination does seem to have arisen independently, but in the reverse order (PDZ-RING). Since LNX1/2 and LNX3/4 have only limited sequence homology in non-modular regions we examined the relationships between the RING and PDZ domains of LNX1-4. Using a BLASTP search, we find that the most similar protein sequences to the RING domain of human LNX1 are the RING domains of LNX2, LNX4 and LNX3 respectively . To examine relationships between the PDZ domains, we have generated a phylogenetic tree from an alignment of all human PDZ domains  a LNX1/2-like protein with four PDZ domains might have arisen from a LNX3/4 like ancestor through acquisition/duplication of PDZ domains; 2) a LNX3/4-like protein with two PDZ domain may have arisen from a LNX1/2-like ancestor protein through loss of two PDZ domains. The poriferan s Figure . The higrd and 4th PDZ domains (RING-PDZ2) as prey. These truncated constructs were used because, in contrast to LNX2, full-length LNX1 protein was not functional in the yeast two-hybrid system. HTR2C did not show any evidence of an interaction with LNX1 constructs and showed only moderate expression of one reporter gene when binding to LNX2 was assessed , were identified by searching the primary literature and protein interaction databases. Among these proteins, Claudin-1 and CAR, have been independently reported to interact with LNX1 and/or LNX2 ,16,40,41Our strategy of screening known MUPP1 interacting proteins was thus successful in identifying three novel ligands for the LNX1 and LNX2 PDZ domains. We also confirmed the common interaction of LNX1, LNX2 and MUPP1 with EphA7 and show that EphA7 can bind, not only to an isolated LNX1 PDZ domain , but alsA. queenslandica has a LNX1/2 ortholog. Thus the ancestral LNX1/2 gene would have evolved in very primitive metazoans, that likely consisted of layers of cells but lacked distinct tissues. This suggests that the original LNX1/2 protein probably had a general cellular function, one that perhaps related to the development of multicellular metazoans but was not specific to a particular tissue type or organ system. It remains to be seen whether the function of the LNX1/2 ortholog in present day poriferans is closely related to its ancestral function and whether studying LNX1/2 in poriferans could provide clues as to the roles of LNX1/2 orthologs in other lineages.We have undertaken the current phylogenetic analysis with a view to gleaning some insights into the functions of the poorly characterized LNX protein family. We find that the sponge LNX1/2 gene was maintained in the lineage leading to vertebrates and duplicated to give rise to the paralogous LNX1, LNX2 and LNX2b genes in vertebrates. The duplication of the ancestral LNX1/2 gene was probably a result of the two rounds (2R hypothesis) of large-scale gene or whole-genome duplication that are thought to have occurred early in the vertebrate lineage [LNX-2b through pseudogenization in eutherian mammals is indicative of some redundancy of function between the LNX1/2 paralogs, but the maintenance of at least two paralogs in all vertebrates is suggestive of an essential function for LNX1/2-like proteins in vertebrates [in vivo significance of this interaction is clarified by loss of function studies. During zebra fish embryogenesis, LNX-2b functions in the establishment of the dorso-ventral axis by modulating the activity of the transcriptional repressor Bozozok [Whatever the role of the ancestral LNX1/2 protein was, it is apparent that this function is not essential in invertebrates since many lineages, such as arthropods and nematodes, lack a LNX1/2-like protein. Nevertheless the  lineage ,51. The tebrates . LNX1 watebrates ,11. LNX2 Bozozok ,30. WhetPhylogenetic analysis reveals that the original LNX1/2 protein shared a common ancestor with MUPP1 and INADL/PATJ - proteins that are found at tight junctions in epithelial cells ,52. LNX1In addition to common interactions with claudin-1 and CAR, we have identified two known MUPP1 ligands, TAPP1 and SynGAP1 as novel interaction partners for LNX1and/or LNX2. Furthermore, we have confirmed a common interaction of MUPP1 and LNX1/LNX2 with EphA7. Full length LNX2 shows convincing interactions with EphA7 and SynGAP, while full length LNX1 interacts with EphA7. By contrast strong evidence for interactions of TAPP1 and SynGAP with LNX1 is only observed using the carboxy-terminal truncated RING-PDZ2 construct. This may be because PDZ1 of LNX1 can bind its own carboxy-terminus, thereby preventing other proteins from binding efficiently to this domain [TAPP1 is a phosphatidylinositol  bisphosphate binding protein that appears to function as a negative regulator of phosphoinositide 3-kinase signaling . SynGAP1T. adhaerens. This protein, which has a RING and one PDZ domain, may represent an intermediate step in the evolution of LNX3/4. If this is the case, then the ancestral LNX1/2 would, in fact, have lost three PDZ domains and the single remaining PDZ domain might have duplicated to generate the two PDZ domains found in other LNX3/4 orthologs. Supporting this idea, an analysis of splicing junctions shows that both PDZ domains in LNX3 and in LNX4 and the single domain in LNX5 have a splice site at the same position as PDZ1 of LNX1/2  ancestor gave rise to a LNX3/4-like (RING-PDZ2) protein. We cannot be certain of the evolutionary mechanisms underlying the loss of the two C-terminal PDZ domains and appearance of the LNX3H domain and other conserved region in LNX3/4/5. A duplication-degeneration mechanism [de novo evolution of the putative LNX3H domain is one possibility, but a fusion event that inserted the LNX3 homology-encoding region from an existing gene into the ancestral LNX gene is also plausible. Sequencing of LNX homologs from additional basal metazoan lineages may resolve these possibilities.During evolution rearrangements of existing protein domains give rise to novel domain combinations and architectures that facilitate functional diversification of proteins without the need to invent new protein modules . Increasy origin ,62-65. Hy origin ,67, and echanism  followedT. adhaerens, all invertebrate and vertebrate LNX3/4 orthologs contain the highly conserved carboxy-terminal region of 150 amino acids that we define here as the LNX3H domain. A previous comparison of LNX3 and the D. melanogaster LNX3/4-like protein Slip1 had identified two conserved regions apart from the PDZ domains [D. melanogaster sequence is thus anomalous and the region of highly conserved sequence in LNX3/4-like proteins is about 60 amino acids longer than previously described. Secondary structure prediction for the LNX3H region indicates a high helical content, while an examination of amino acid composition reveals a very high proportion of charged amino acids, especially positively charged lysines and arginines (25%). These observations are consistent with this region folding as a globular domain, but biophysical and structural approaches must now be applied to determine this definitively.Apart from  domains . By analC. elegans have a protein that contains the LNX3H domain in the absence of any other recognizable domains. Together, these observations suggest that the LNX3H domain has functions that are independent of the RING and PDZ domains and that may be more important than the functions of the other domains, at least in invertebrates. To date, however, the function of the LNX3H domain in any species is completely unclear. Since the LNX3H domain is conserved in LNX5, its presence in a protein may be a better way of defining the LNX3/4/5 subfamily than the presence of both a RING and PDZ domain.Cnidarians make up the most basal lineage that have the LNX3H domain, and representative organisms in all bilaterian lineages examined encode a protein in which this domain is present. The basic LNX3/4 domain architecture seen in cnidaria has been maintained in vertebrates, yet in many invertebrate lineages, the RING and in some cases the PDZ domains have been lost. The LNX3H domain is thus more conserved across diverse invertebrate lineages than the RING and PDZ domains. Some species such as This study represents the first, thorough examination of the molecular evolution of the LNX gene family. Based on our findings an exploration of common interactions and cellular functions of LNX1/2 and MUPP1 is merited. Structural and functional characterization of the LNX3H domain should also provide insights into the LNX3/4/5 subfamily. Our analysis thus provides a useful framework for the further characterization of the enigmatic LNX proteins.Several approaches were used to retrieve a comprehensive set of both vertebrate and invertebrate LNX protein sequences. Initially, a search was carried out within the NCBI Protein Database using the following keywords as a query; "Ligand of numb protein X 1", "Ligand of numb protein X 2", "Ligand of numb protein X 3", "Ligand of numb protein X 4", "LNX1", "LNX2", "LNX3", "LNX4", "PDZRN3", "PDZRN4", "PDZD4", "SEMACAP3" and "Semaphorin cytoplasmic domain-associated protein 3". Sequences were retrieved and each of these sequences was used to query the NCBI Protein Database using BLASTp  to identA. queenslandica was initially obtained from Sakarya et al., who assembled sequences of proteins containing PDZ domains from EST and genomic trace data [A. queenslandica genome the LNX1/2 sequence is identified as transcript Aqu1.226425 http://www.metazome.net/amphimedon. tBLASTN searches of this draft sequence did not reveal any LNX3/4 ortholog.To complement the above approach the Conserved Domain Architecture Retrieval Tool (CDART) was also used . A list ace data . The proAlignments of all retrieved seqeuences were generated using ClustalX and visually inspected to verify that sequences were indeed LNX homologs and to distinguish which LNX protein each sequence represented, as many automatically generated annotations were incorrect. Domain architectures for all identified LNX homologs were determined using SMART . The numSequences of all human PDZ (PF00595) domains were retrieved from the Pfam database . DuplicaHomo sapiens, Mus musculus, Gallus gallus, Xenopus tropicalis and Danio rerio LNX2 sequences was generated using MUSCLE [A. queenslandica LNX2 and H. sapiens and T. adhaerens MUPP1. Domain boundries were identified by searching against the PFAM database. An alignment of the extracted PDZ domains was first generated in MUSCLE [A phylogenetic tree for LNX2 and MUPP1 PDZ domains from vertebrate and early metazoan was generated as follows. An alignment of the g MUSCLE ,74. A cog MUSCLE ). Sequenn MUSCLE ,74. Phyln MUSCLE ,79. 1000n MUSCLE . The evon MUSCLE .M. musculus LNX3, LNX4, LNX5 and various invertebrate LNX3 and LNX3-like orthologs were aligned using ClustalX [M. musculus LNX3 was used as input sequence. For ProDom searches the blastp and multiple alignment options and an expect value of 0.01 were selected. The ADDA database was searched using default parameters.ClustalX . To idenClustalX -36. SearM. musculus LNX3 was performed using the Jpred3 secondary structure prediction server [Secondary structure analysis for the LNX3H region of n server . SecondaSaccharomyces cerevisiae L40 reporter strain and grown on selection agar plates lacking leucine and tryptophan to select for plasmids. His3-reporter gene activation was monitored by restreaking multiple colonies onto plates that also lacked histidine. Filter assays for β galactosidase activity were performed as described in the Clontech manual, using X-gal  as color substrate. Briefly, yeast cells were transferred to nylon membranes  and frozen twice in liquid nitrogen to lyse cells. The membrane was then placed on a filter paper soaked in Z buffer  and incubated for 6-12 hours at 37°C.Oligonucleotides coding for the carboxy-terminal ten amino acids of murine HTR2C, TAPP1, SynGAP1 and EphA7 were cloned into the pLex bait vector . cDNAs sMF identified and characterised LNX orthologs and generated alignments and phylogenetic trees for individual PDZ domains. OS carried out searches for MUPP1 ligands and performed yeast-two-hybrid analysis. PY conceived the project and analysed the LNX3 homology domain. MF, OS and PY wrote the paper and all authors read and approved the final manuscript.Analysis of splicing junctions in LNX1, LNX2, LNX3, LNX4 and MUPP1 PDZ domains. An alignment of PDZ domain sequences with splicing junctions highlighted.Click here for fileMultiple sequence alignment of LNX3, LNX4 and LNX5 and their invertebrate orthologs. An alignment of full length murine LNX3, LNX4 and LNX5, as well as LNX3/4 orthologs from diverse invertebrate species.Click here for filePhylogenetic tree of all human PDZ domains. Clustering of related LNX PDZ domains is highlighted in a phylogenetic tree of all human PDZ domains.Click here for fileConservation of ligand-binding residues in LNX1, LNX2 and MUPP1. Table showing the amino acids identified as determinants of PDZ domain specificity for LNX1, LNX2 and MUPP1.Click here for filePredicted interactions of LNX1, LNX2, MUPP1 & INADl PDZ domains. Table showing predicted PDZ domain interactions for LNX1 PDZ1-4, LNX2 PDZ1-4, MUPP1 PDZ10-13 and INADl PDZ 8-10.Click here for fileGenbank accession numbers. Table showing Genbank accession numbers for LNX and MUPP1 homologs referred to in this study.Click here for file


UTPB is a large evolutionarily conserved complex in the 90S small subunit processome that mediates early processing of 18S rRNA. UTPB consists of six proteins Utp1/Pwp1, Utp6, Utp12/Dip2, Utp13, Utp18 and Utp21 and has abundant WD domains. Here, we determined the crystal structure of the tandem WD domain of yeast Utp21 at 2.1 Å resolution, revealing two open-clamshell-shaped β-propellers. The bottom faces of both WD domains harbor several conserved patches that potentially function as molecular binding sites. We show that residues 100–190 of Utp18 bind to the tandem WD domain of Utp21. Structural mapping of previous crosslinking data shows that the WD domains of Utp18 and Utp1 are organized on two opposite sides of the Utp21 WD domains. This study reports the first structure of a UTPB component and provides insight into the structural organization of the UTPB complex.Assembly of the eukaryotic ribosome requires a large number of  Ribosomes are large molecular machines that consist of four rRNAs and ∼80 ribosomal proteins in eukaryotes. These components are assembled in a highly complicated and dynamic process Saccharomyces cerevisiae. Most of them are conserved throughout eukaryotes, although humans have additional ribosome synthesis factors that are absent in yeast Genetic and biochemical studies have found over two hundred proteins involved in ribosome synthesis in The SSU processome is an enormous particle that contains 35S pre-rRNA, U3 snoRNA, ∼50 assembly factors and a subgroup of r-proteins UTPB is a large complex composed of six proteins Utp1/Pwp2, Utp6, Utp12/Dip2, Utp13, Utp18 and Utp21 The WD domain (aka WD40 domain) is composed of 40–60 residue long WD repeats arranged in a β-propeller structure and commonly mediates protein-protein interactions in diverse cellular processes Interactions between the six UTPB proteins have been mapped by yeast two-hybrid assay, but the precise domain of each protein participating in binding is mostly unclear As part of an effort to understand the structure of UTPB, we have determined the crystal structure of the tandem WD domain of Utp21, the first structure of a UTPB component. We further identified a region of Utp18 that mediates the interaction with Utp21. The structure allows us to integrate available crosslinking and interaction data to obtain a better understanding of the organization of UTPB.Utp21 has been shown to interact with Utp18 in a two-hybrid assay 6-tag. The full-length Utp21, the full-length Utp18 and the CTD of Utp21 could not be examined due to protein expression problems. The Utp21 and Utp18 proteins in different combinations were mixed and pulled down using Ni beads. SDS-PAGE analyses show that Utp18 100–190, but not Utp18 1–100 and Utp18 183–594, efficiently copurified with Utp21 1–684  and screened these complexes for crystal formation. The complex that copurified with Utp18 100–139 was the only one that crystallized. The structure was determined by single isomorphous replacement with anomalous signal (SIRAS) phasing using a native and Se-derivative dataset. The current model was refined to 2.1 Å resolution with ectively , Table 1The tandem WD domains of Utp21 fold into two 7-blade β-propellers, resembling an open clamshell . Each blOnly a few structures have been determined for tandem WD domain proteins, including Aip1 Utp21 has been shown to interact with multiple proteins, including Utp18, Utp1, Utp12 and Utp6 in the UTPB complex and SSU biogenesis factors Sas10/Utp3 and Utp25 Traditionally, the surface of WD propellers composed by the B-C and D-A loops is termed the top surface, whereas the opposite surface covered by the A-B and C-D loops is termed the bottom surface. The bottom surface of the WD1 domain displays an extensive conserved region that spans blades 4–7 . The A-BThe WD2 domain contains two separated conserved patches also on the bottom surface . One is In contrast to the conserved bottom surfaces, other surface areas are variable in either of the WD domains . This suThe interaction and spatial proximity of the six UTPB proteins have been mapped to some extent In addition, Utp21 was found to form eleven intermolecular crosslinks with Utp18, Utp1 and Utp12 Utp6 has been shown to bind Utp18 via its N-terminal region and to bind Utp21 via its HAT domain Four Utp1 residues  mainly from the WD1 domain were previously shown to make six crosslinks with four residues  in the WD domains of Utp21 . In the Understanding the structure of the entire UTPB complex is going to be a challenging task. A practical way to do so is to determine the structure of the individual domains and subcomplexes and to then assemble them with low-resolution constraints, such as crosslinking data, obtained from the entire complex. Toward this goal, we have analyzed the structure and interactions of the Utp21 tandem WD domains. Utp21 occupies a central position in the UTPB complex as it interacts with all of the other proteins except Utp13 The crystal structure of the Utp21 tandem WD domain reveals an overall topology similar to those found in Aip1 and 2YMU, indicating that this topology is the prevalent, although not exclusive (Apaf1 is an exception), way to arrange tandem WD domains. In UTPB, Utp1, Utp12 and Utp13 are predicted to also contain tandem WD domains, which the Utp21 structure could provide a good template to model. We have found several highly conserved patches on the bottom face of each of the Utp21 WD domains that are potential binding sites for other molecules. We identified residues 100–190 of Utp18 as interacting with the tandem WD domain of Utp21, but the structural details of the interaction await further investigation. Finally, the atomic structure of the Utp21 WD domains serves as a 3-dimensional model to interpret the previous crosslinking data. By determining additional structures of the individual components and subcomplexes of UTPB, we hope to assemble a complete UTPB structure in the future.6-Smt3-tag.The Utp21 and Utp18 genes were PCR-amplified from yeast genomic DNA. Gene cloning was conducted with the ligation-based or In-fusion (TaKaRa) method. The DNA of Utp21 1–684 was cloned into pGEX-6P-1 and expressed as a GST-fusion protein. The DNA of various Utp18 fragments was cloned into pET28a and expressed with an N-terminal His6-Smt3-Utp18 fragment were co-transformed into E. coli strain BL21(DE3). The cells were cultured in LB medium, and protein expression was induced with 0.2 mM isopropyl β-D-1-thiogalactopyranoside for 12 h at 22°C. The cells were collected by centrifugation and resuspended in lysis buffer containing 25 mM Tris-HCl pH 7.5 and 500 mM NaCl. The cells were broken using a high-pressure JN-3000 cell disruptor (JNBIO) and clarified by centrifugation at 4°C. After incubation with Ulp1 for 4 h at 4°C to cleave the His6-Smt3 tag from Utp18, the supernatant was loaded onto a Glutathione Sepharose column followed by a wash with lysis buffer. The GST-fusion protein was incubated on-bead with PreScission protease for 8 h at 4°C. The released protein was eluted with lysis buffer, concentrated and further purified in a HiLoad 16/60 Superdex 200 PG column with buffer (10 mM Tris-HCl pH 7.5 and 200 mM NaCl). The pooled fractions of the target protein were concentrated to 5–10 mg/ml and stored at −80°C.To purify the Utp21/Utp18 complex, two plasmids encoding GST-Utp21 1–684 and one HisThe selenomethionine(SeMet)-labeled protein was expressed in M9 minimal medium with inhibition of the methionine biosynthesis pathway The copurified Utp21 1–684/Utp18 100–139 complex was crystallized using the vapor diffusion sitting drop method at 20°C. One microliter of the protein sample  was mixed with an equal volume of mother solution (0.15 M sodium citrate pH 8.5 and 15% PEG 3350). SeMet-labeled crystals were crystallized under the same conditions using native microcrystals as seed. The crystal was cryoprotected in 25% glycerol made from the mother solution.All diffraction data were collected at the BL17U beamline of the Shanghai Synchrotron Radiation Facility (SSRF) and processed with HKL2000 The yeast two-hybrid assay was performed using the MATCHMAKER GAL4 two-hybrid system (Clontech) as described 6-Smt3-tagged Utp18 proteins were purified by HisTrap and Q chromatography. Concentrations of each protein were determined using the measured absorbance at 280 nm and the theoretic molar extinction coefficient. The Utp18 proteins (0.95 nmol) were incubated with Utp21 1–684 (1.9 nmol) for 2 h at 4°C and then bound to Ni Sepharose Fast Flow beads . The beads were washed with buffer  three times, followed by boiling in SDS loading buffer at 95°C for 5 min. The proteins were separated on an SDS-PAGE gel and stained with Coomassie blue.The Utp21 1–684 protein was purified as described above. The HisThe coordinates and structural factors have been deposited into Protein Data Bank with the accession code 4NSX.