Schizosaccharomyces pombe phenocopies the DNA-damage sensitivity of strains deleted for mus81. Our results indicate an important role for the WH domain in both yeast and human MUS81 complexes.The MUS81-EME1 endonuclease maintains metazoan genomic integrity by cleaving branched DNA structures that arise during the resolution of recombination intermediates. In humans, MUS81 also forms a poorly characterized complex with EME2. Here, we identify and determine the structure of a winged helix (WH) domain from human MUS81, which binds DNA. WH domain mutations greatly reduce binding of the isolated domain to DNA and impact on incision activity of MUS81-EME1/EME2 complexes. Deletion of the WH domain reduces the endonuclease activity of both MUS81-EME1 and MUS81-EME2 complexes, and incisions made by MUS81-EME2 are made closer to the junction on substrates containing a downstream duplex, such as fork structures and nicked Holliday junctions. WH domain mutation or deletion in  These s binding . This st binding . The N-t binding .Schizosaccharomyces pombe render a similar sensitivity profile to DNA damaging agents as a mus81 deleted strain, implying that this domain has a critical role in DNA repair that has been retained in human MUS81.Here, we identify for the first time a winged helix domain (WH domain) within the N-terminal region of human MUS81 that binds DNA, increases the activity of MUS81-EME1/EME2 complexes and influences the incision position of MUS81-EME2 but not MUS81-EME1 complexes on synthetic forks, 3′ flaps and nicked Holliday junctions. Additionally, in MUS81-EME2 complexes, it stimulates the cleavage of splayed arms. Mutations in the WH domain in Escherichia coli BL21 (DE3) Rosetta pLysS (Stratagene) using the dicistronic expression plasmid derived from pGEX-KG. MUS81-EME1/EME2 wild-type and mutant complexes were purified as follows: Cultures were grown in Luria-Bertani at 37°C and induced with 25 µM Isopropyl-β-D-thiogalactoside (IPTG) at 18°C overnight. Bacteria were harvested by centrifugation at 4000 × g for 15 min at 4°C, and the pellets resuspended in 50 mM sodium phosphate (pH 8), 300 mM NaCl, 2 mM β-mercaptoethanol, 10% glycerol, 0.2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) (buffer A) supplemented with 10 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitors (Roche). Bacteria were lysed by sonication. Cell debris was cleared by centrifugation at 29 220 × g for 60 min at 4°C, and the clarified lysate was mixed with glutathione-Sepharose 4B  for 90 min at 4°C. Unbound proteins were collected, and the affinity resin was washed extensively with buffer A. Human MUS81 (hMUS81) complexes were eluted with elution buffer [buffer A containing 20 mM reduced glutathione (pH 8)]. The eluted proteins were analysed by SDS–PAGE. hMUS81 complexes were concentrated by ultrafiltration and loaded onto a 1 ml of HiTrap Heparin-Sepharose column pre-equilibrated with buffer A. The hMUS81 complexes were eluted using a NaCl gradient (up to 1 M NaCl in buffer A) over 10 column volumes on an AKTA fast performance liquid chromatography system . Peak fractions from the Heparin-Sepharose column were pooled and concentrated by ultrafiltration. The concentrated sample was loaded onto a Superose 12 HR 10/300 column  pre-equilibrated with buffer A. After analysis by SDS–PAGE, aliquots of complex-containing fractions were flash-frozen in liquid nitrogen for assaying.MUS81-EME1/EME2 complexes   were prog for 15 min at 4°C. Cells were resuspended in 50 mM sodium phosphate (pH 7), 500 mM NaCl, 5 mM dithiothreitol (DTT) (buffer A) supplemented with 10 mM benzamidine, 1 mM PMSF and 0.1 mg/ml DNase, and lysed by sonication. Cell debris was cleared by centrifugation at 29 220 × g for 30 min at 4°C, and the clarified lysate was mixed with glutathione-Sepharose 4B  for 90 min at 4°C. Unbound proteins were collected and the affinity resin was washed extensively with buffer A. hMUS81 WH domain protein was eluted from the affinity resin by addition of GST-tagged 3C protease . hMUS81 WH domain protein was concentrated by ultrafiltration and loaded onto a pre-equilibrated Superdex 75 HR 10/300 column  pre-equilibrated with 25 mM sodium phosphate (E. coli strain FB810 (BL21 (DE3) recA-) from a pET41a plasmid (Novagen). Uniformly 15N labelled WH domain protein was produced in minimal medium M9 containing 1 g/l (15NH4)2SO4 (Sigma Aldrich) as the nitrogen source. The 13C/15N labelled WH domain was produced in a similar fashion with 13C6-D-glucose (Sigma Aldrich) as the only carbon source.Cultures were grown at 37°C and induced by addition of isopropyl-β-D-thiogalactoside  and further incubation at 37°C for 120 min. Bacteria were harvested by centrifugation at 4000 × 1H frequency of 500 MHz) equipped with a triple resonance probe including a Z-axis pulse field gradient coil. Data acquisition and processing leading to structure calculations are described in Supplementary Information. Atomic coordinates of the final 20 simulated annealing MUS81 conformers and the list of experimental restraints (accession code RCSB 103460) have been deposited at the RCSB Protein Data Bank. Chemical shifts for resonance assignments for the MUS81 have been deposited at the BioMagResBank (accession code 17324).Nuclear magnetic resonance (NMR) spectra were acquired at 298 K (except where indicated) on a Varian Unity PLUS spectrometer , 5 mM MgCl2, 1 mM DTT and 100 µg/ml bovine serum albumin (BSA) in a total volume of 20 µl. Incision products were separated on a sequencing gel  for 2 h. The gel was removed and dried, and products were visualized by auroradiography, or a STORM phosphorimager (Molecular Dynamics). Quantification of data was carried out using ImageQuant TL software .Oligonucleotides used to make the structures were described previously . XO1 andFluorescence polarization anisotropy experiments were carried out as described previously .mus81 base strain was constructed in S.pombe, which is compatible for recombination-mediated cassette exchange , camptothecin , 4-nitroquinoline 1-oxide (Fluka 73265), Hydroxyurea (98% Sigma H8627) at the indicated concentrations were used. Plates were incubated for 4 days at 30°C.For DNA damage-sensitivity assays, cells were grown at 30°C overnight in yeast extract (YE) media, serially diluted and spotted onto agar plates starting with 10Whole cell protein extracts were prepared by TCA precipitation. For the chromatin fractionation assay, chromatin was separated by centrifugation through a sucrose cushion as in Liang and Stillman 1997  except t). Analysis of this region by the Phyre recognition server . Size exclusion chromatography and CD revealed the protein migrated as an 11.5 kDa protein and exhibited CD spectra consistent with a predominantly helical secondary structure (data not shown).The major portion of the human MUS81 (Accession code: NP_079404.3) amino-terminus has few characterized domains A. Our sen server  gave a bSupplementary Table S1). The structure revealed a helical domain that adopts a WH tertiary fold . In hMUS81, T206 acts as an N-cap to helix α4, hydrogen bonding to mainchain amides and also across to a carbonyl of the helix α3-ß1 loop, similar to the metal-dependent transcriptional regulator WH domains. The WH motif also includes L205 and G209 from hMUS81 that play important structural roles at the amino-end of helix α4 and contact conserved buried residues from helix α1. A213 is also frequently a small sidechain and packs against Y146 sidechain of helix α1.Structural comparisons with the hMUS81 WH domain indicate the closest similarity is to the manganese-binding MnTR , a metal-dependent bacterial repressor protein belonging to a DtxR-like family of transcriptional regulators ,21 (Supp15-labelled MUS81 WH domain and duplex DNA shifted residues within regions 131–142 (amino-terminus and helix a1) and 186–191 , thus implicating these regions in DNA binding .Residues 125–244 of MUS81 were previously reported to bind to the RecQ helicase homologue, BLM . HoweverThe contribution of different regions of the WH domain to DNA binding was measured by fluorescence polarization B. CarboxTo establish whether MUS81 WH domain is a major groove binding canonical WH domain, individual mutations within helix α3  were prepared. These mutations markedly reduced DNA binding B. CombinSupplementary Table S2). In general, MUS81-EME2 complexes converted more of its substrates to products than the same concentration of MUS81-EME1, suggesting that MUS81-EME2 is intrinsically more active (Supplementary Table S2). This is in agreement with another study (Supplementary Table S2), whereas the same amount of MUS81-EME2 converted nearly half of it into incision products (Supplementary Table S2). Deletion of residues 128–230 corresponding to the WH domain from MUS81-EME2 reduced total cleavage of the splayed arm substrate, and the incisions occurred at the same position as wild-type (Supplementary Table S2).To study the effect of the WH domain of MUS81 on incision activity, we prepared recombinant MUS81-EME1 and MUS81-EME2 complexes and compared the endonuclease activity of 25 fmol of each on splayed arm, 3′-flap, fork and Holliday junctions, A. The caer study . FurtherSupplementary Table S2). This contrasts with previous studies where it was shown that removal of 246 amino acids from the N-terminus of MUS81 did not impact on the incision activity of recombinant MUS81-EME1 complexes , an, anSupplomplexes B, also somplexes C. These in vivo migratable Holliday junction and was shown previously to be weakly cleaved by purified MUS81-EME1 (The substrate preferences of MUS81-EME2 have not been previously characterized so we tested the incision activity of MUS81-EME2 and the MUS81ΔWH domain deletion mutant on a range of substrates that included a mobile Holliday junction (X26) containing 26 centrally placed complementary base pairs allowing the junction to migrate. This structure is thought to be more representative of an S. pombe Eme1, although human EME2 is much shorter  . Combining the K176A/K181A double mutation with R165A/R168A (KER) results in a moderate increase in sensitivity to HU and 4-NQO and a similar phenotype to mus81-ΔWH. These results show that the WH domain of Mus81 is crucial for resistance to DNA-damaging agents in S. pombe and implies that this in vivo function has been retained in mammals.The structural similarities to repressors, the WH domain DNA-binding properties and effects on the endonucleolytic incision combined provide compelling evidence that in human MUS81, the WH domain of MUS81 binds duplex DNA. Human EME1 and EME2 are both distant homologues of f mus81)  reduced s81 gene . N-termiin human ] with alSaccharomyces cerevisiae . This is probably a result of enhanced bending or opening of the duplex at the junction and given that the incision pattern is exactly the same on all three of these substrates, we conclude that the WH domain must bind duplex DNA or interact with subdomains in the MUS81-EME2 complex or both. Alternatively, it is possible that it may contribute to substrate recognition by stable binding of the downstream duplex as well as the MUS81 (HhH)2 domain, as modelled by Chang et al. ; ;  thereforA hypothetical model for the interaction of MUS81-EME2 with a fork is shown in S. pombe, and we found that the WH domain is essential for both meiosis and DNA damage tolerance in this organism. The role of the Mus81WH domain in meiosis in S. pombe suggests that its DNA binding activity is necessary for the processing of intermediates such as D loops or Holliday junctions in the absence of a Yen1/GEN1 resolvase homologue ; MRC grant [G1100074 to A.M.C.].Funding for open access charge: Conflict of interest statement. None declared.


Proteins convey the majority of biochemical and cellular activities in organisms. Over the course of evolution, proteins undergo normal sequence mutations as well as large scale mutations involving domain duplication and/or domain shuffling. These events result in the generation of new proteins and protein families. Processes that affect proteome evolution drive species diversity and adaptation. Herein, change over the course of metazoan evolution, as defined by birth/death and duplication/deletion events within protein families and domains, was examined using the proteomes of 9 metazoan and two outgroup species.Trichinella spiralis and Gallus gallus, we propose a link between protein redundancy and evolutionary changes controlled by domain shuffling; however, the speed of adaptation among the different lineages was relatively invariant. Evaluating the functions of protein families that appeared or disappeared at the last common ancestors (LCAs) of the three metazoan clades supports a correlation with organism adaptation. Furthermore, bursts of new protein families and domains in the LCAs of metazoans and vertebrates are consistent with whole genome duplications.In studying members of the three major metazoan groups, the vertebrates, arthropods, and nematodes, we found that the number of protein families increased at the majority of lineages over the course of metazoan evolution where the magnitude of these increases was greatest at the lineages leading to mammals. In contrast, the number of protein domains decreased at most lineages and at all terminal lineages. This resulted in a weak correlation between protein family birth and domain birth; however, the correlation between domain birth and domain member duplication was quite strong. These data suggest that domain birth and protein family birth occur via different mechanisms, and that domain shuffling plays a role in the formation of protein families. The ratio of protein family birth to protein domain birth (domain shuffling index) suggests that shuffling had a more demonstrable effect on protein families in nematodes and arthropods than in vertebrates. Through the contrast of high and low domain shuffling indices at the lineages of Metazoan speciation and adaptation were explored by birth/death and duplication/deletion events among protein families and domains. Our results provide insights into protein evolution and its bearing on metazoan evolution. Proteins convey the majority of biochemical and cellular activities in organisms. Their structural and functional units are defined as domains ,2 where http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome), and their corresponding proteomes have been annotated. The culmination of these efforts is the emergence of databases consisting of well-defined protein domains such as Pfam  i.D. melanogaster and S. cerevisiae proteins identified by INPARANOID [Protein sequences from the 11 species were searched (BlastP) against each other. Based on these results, we used MCL  to clustPARANOID . ProteinEach protein sequence was searched against the PFam domain profiles ,68, usinDuplications and deletions of protein sequences and domain sequences were identified using Urec . First, Using an approach similar to that of Hughes and Friedman , proteinProtein family deaths and births were inferred by checking the states of these characters on each lineage of the 11 species tree. In a like manner, the death/birth events of each domain over its evolution were also inferred as were the unique domain losses of each species. Unique domain losses were defined as deaths of domains present in all other 10 species except the one indicated. The association of family member duplications/deletions, domain duplications/deletions, protein family deaths/births, and domain deaths/births were investigated using Pearson’s correlation coefficients. The significance of these correlation coefficients was tested using Student’s t-distribution.Organism complexity is closely related to the number of protein families and domains. In order to illustrate the changes of organism complexity over the course of evolution, we computed the log ratio of birth and death events of protein families and domains at different lineages, which we define as protein/domain change index. For example, if the number of birth events equals that of death events over a lineage, the index is 0, suggesting no complexity change over the evolution of this lineage. If the number of birth events is larger than death events, the index is larger than 0, suggesting the organism has gained complexity over the lineage.The protein family and domain change indices reflect the changes of organism complexity, but does not reflect how quickly these changes occur. To assess the spread of these changes, we used the following equation:where AI: Adaption Index; B: inferred birth events at the corresponding lineage; D: inferred death events at the corresponding lineage; l: the branch length of the corresponding lineage. If the branch length of a lineage is short and possesses a large number of birth/death events, then the adaptation index of that lineage is large, suggesting dramatic adaptation along that lineage. Only adaptation index of protein family was explored because of the limited number of domain birth events.Protein families can be generated from new domains and/or domain shuffling. To illustrate the effect of domain shuffling in protein family evolution, we examined the log ratio of protein family birth to protein domain birth at different lineages which we defined as the domain shuffling index. Similar to the adaptation, index, the birth events were first normalized by the lineage branch lengths.H. sapiens, D. melanogaster or C. elegans proteomes. Significant enrichments of GO terms were computed based on hypergeometric distributions using FUNC [C. elegans proteins were included and GO terms identified multiple times by different proteins from the same family were only counted once. When testing the probability of these data, refinement was performed by removing the GO terms identified as significant due to their derived terms. The false discovery rate (FDR) computed by FUNC was used to reduce false discovery. Therefore, unless specified otherwise, GO term enrichment was selected based on both p-value <0.001 (after refinement) and FDR <0.1.GO term functional annotation of protein families was examined using Interproscan  based oning FUNC  by compaFDR: False discovery rate; GO: Gene Ontology; LCA: Last common ancestor.The authors declare no competing financial interests.ZW, DZ and MM designed the study and wrote the manuscript. ZW, SA, and JM did the analysis. All authors read and approved the final manuscriptData summary of the species included in the analysis. The data provided represent summary of the number of proteins and domains per species and proteins within protein families.Click here for fileIDs of protein families and their members. The data provided represent the accession numbers of the proteins grouped in protein families.Click here for fileGroups of protein families with different taxonomic compositions. The provided table summarizes the numbers of protein families and their taxonomic distribution.Click here for fileTrichinella spiralis  specific domains, their putative origin and function. List of the identified domains in T. spiralis having highest sequence similarity with non-metazoan species, their putative function and species of origin. (XLS 25 kb)Click here for file


The first deformation (D1) episode is defined by a main axial-plane of parallel folds observable on a micro- to kilometer-scale, while the second episode of deformation (D2) is defined by micro-scale metamorphic folds, associated with E–W oriented stretching lineation. These processes are the result of Indosinian tectonism (Late Triassic to Early Jurassic) characterized by nearly coaxial N-S compression and deformation. This is indicated by E–W trending, sub-parallel to parallel foliation  and lineation . Most of the porphyroblasts and minerals  show two growth phases with localized growth in the third phase (muscovite). The progressive D1–D2 structure is widespread in the south of the Jiaochang area, but only D1 structure crops out at the north. The strain intensity (γ), compression ratios (c%) and octahedral strain intensity (εs) are similar across the Jiaochang structure , showing a broad range of Flinn values (K = 0.77 to 7.57). The long-axis orientations are roughly symmetric between two limbs of the structure. Therefore, we suggest that the architecture of the Jiaochang structure has been controlled by coaxial N-S shortening and deformation (D1–D2) during the Indosinian tectonic epoch, with insignificant post-D2 deformation.Integrated field data, microstructural and three-dimensional strain analyses are used to document coaxial N-S shortening and southward increase in deformation intensity and metamorphism at the Jiaochang structure. Two episodes of deformation (D The oroclinal bending, which is one of the most striking features in the Songpan-Ganzi fold belt, shows an obvious change in the orientation of structure from northwest direction in the west, to E-W direction and northeast direction farther to the east near the border with Longmenshan Fig. 1. Wang et al. 1,D2) at the Jiaochang structure, which mostly account for the architecture of the Jiaochang area.In this study, we present new observations based on field data, microstructure and three-dimensional strain analyses from the Jiaochang structure at the north Songpan-Ganzi fold-belt. We have recognized two principal episodes of N-S oriented deformation (DThe Songpan-Ganzi Fold-Belt (SGFB) is a triangular area with widespread Triassic flysch located at the eastern margin of the Tibetan Plateau . It is bMw 7.3 Diexi and 1976 Mw 6.9 Songpan earthquakes).The widespread Songpan-Ganzi flysch is comprised by 5∼15 km of mudstone and shale, exposed to low- to medium-grade greenschist-facies metamorphism Our study area in the Jiaochang structure is located at a transition region between the northern SGFB and the Maowen-Wenchuan ductile shear zone in the Longmenshan. It is characteristic by the occurrence of symmetric, parallel and parabolic structures Fig. 2. 1 fold (1) across the Songpan-Ganzi Fold-Belt, as described by Dirk et al. The Jiaochang structure is dominated by nearly E-W trending, upright-inclined F1 fold Fig. 2. 2) deformation accompanied by metamorphism in the southern region. It indicates medium to high intensity of deformation and metamorphism. Immediately south of Shuigouzhi along the Minjiang River, deformation of the upper Paleozoic strata, particularly of the marble rock, is marked by a distinct metamorphic fold (S2 and F2) .Polyharmonic fold Fig. 3c i) and matrix mineral (Se) is marked by opaque-poor bands (i.e. quartz with elongated inclusions) continuous inside and outside of the porphyroblast. It is result of progressive deformation (D1 and D2). Thus we suggest that formation of D1 and D2 took place at approximately same time, or that the D2 was formed slightly later.Few of the pervasive foliation  is preserved. The original sedimentary bedding is only preserved in metamorphic porphyroblasts Fig. 3e.2 deformation (D3) has been identified in the north of Shuigouzhi, as crenulation cleavage (S3) of pervasive metamorphic foliation (S2)  Fig. 3f.1 fold is still E-W striking with a near-horizontal plunge , defined by an alignment of biotite and elongation of quartz grains. Furthermore, the L1 (intersection lineation and bounding axis) is found to be parallel to F1 axis with NW to EW strike  Fig. 5e.1 ductile deformation has been observed, the post-D1 brittle deformation is prominent in the northern region. Some of fold limbs display different-scale transporting structure, most of which generally indicates N-oriented thrust, to accommodate fault-related fold  from the north to the south. The values range from γ = 1.6 to γ = 2.4, from c% = 18% to c% = 41%, and from εs = 0.82 to εs = 1.16, respectively. However, the Flinn values, with a range of ∼4–7, are larger than those in the northern part. The long-axis of three samples is NE striking, and roughly parallel to the fold axis. However, there are two samples (S109-01 and S125-01) with nearly N-S striking long-axis; both of them sampled from locations within meters from the fault. Thus, we suggest that thrusting deformation of the faults could account for significant change of the long-axis of our oriented samples.Five samples from the central and southern region of the Jiaochang were analyzed. No distinct change has been observed in strain intensity (γ), compression ratios (c%) and octahedral strain intensity .In the Flinn diagram most samples cluster around K = 5 within a range of 0.77∼7.57, as a triaxial-prolate ellipsoid Fig. 7. 1–D2 tectonic deformation, with insignificant post-D2 deformation occurring in the Jiaochang area. The deformation is characterized by nearly coaxial N-S compression, indicated by sub-parallel to parallel foliations  and lineations .Our observation supports progressive D2 . The early porphyroblasts show undulose extinction and are wrapped by deflected matrix minerals, in contrast to the later porphyroblasts without deflection of wrapping minerals  are always without strain shadow and deflection of wrapping minerals, overprinting the early undulose extinction minerals (i) to the matrix minerals (Se) ; only part of muscovite grew during the third episode of deformation (D3). In outcrops, the localized post-D2 mineral growth indicates that post-D2 deformation had insignificant impact on tectonic architecture of the Jiaochang area. It suggests that the progressive D1–D2 deformation predominantly accounts for the Jiaochang structure. Furthermore, there are widespread syntectonic structures and recrystallization with grain boundary migration, that makes it difficult to identify which episodes of deformation is more important. Therefore, we argue that the three-dimensional strain should be interpreted with the D1–D2 progressive process and insignificant post-D2 deformation.Therefore, we suggest that the multiphase growth of minerals and porphyroblasts is correlated with the multiphase deformation in the Jiaochang area Fig. 9. 1–D2 deformation in Jiaochang area is roughly equivalent to the pervasive D1 deformation with the superposed D2 deformation during Indosinian epoch, as described by Dirks et al. In the region, the crosscutting relationship between the folded Late Triassic sequence and the Late Triassic to Early Jurassic (Indosinian) pluton 2 deformation (D3 and/or D4) described by Dirk et al. 2 deformation is localized in the outcrops in the southern part of the SGFB. In the Jiaochang area, the progressive D1–D2 structure with ductile deformation is widespread in the south, and the D1 structure crops out in the north. These differences show clear heterogeneity in multiphase deformation and metamorphism between the SGFB and Longmenshan, as well as in the SGFB interior. Why such heterogeneous deformation (post-D2) is lacking in the Jiaochang structure remains unknown.However, the post-DThere are many factors affecting the deformation and displacement-path trajectories in curved fold-thrust belt. As discussed by Ries and Shackleton The three-dimensional strain data of our samples show three principal axis of strain sub-parallel to the fold axis with roughly E-W strike, being sub-perpendicular to the transport direction (N-S trending), except for samples S109-01 and S125-01 Fig. 6. The first hypothesis is that the juxtaposition of coaxial pure shear and simple shear results in successive increment of strain, and similar lineation switching between the principal and other axes 1–D2 deformation with insignificant post-D2 deformation; (b) a southward increase in deformation intensity and metamorphic condition; (c) although finite strain data shows similar magnitude in the Jiaochang , there is much larger strain intensity values at the western limb than at the eastern limb; (d) the long-axis orientations are subparrellel to the fold axis, and roughly symmetric between two limbs; (e) no high-angle plunge and various lineation were observed in this area, which is typical feature related to transpressional movement Given possible explanation for the strain in the Jiaochang, several features lead us to favor the hypothesis of progressive N-S shortening as predominantly accounting for the formation of the Jiaochang structure. These are: (a) a progressive DAs suggested by Dirk et al. During Late Triassic time, as result of the closure of the Paleo-Tethys Ocean, significant south-oriented compression and shortening occurred in the SGFB. The subduction occurred to the west under the Qiangtang block along western Jinshajiang-Litang suture 1 phase has accommodated the widespread upright and isoclinal F1 folds. Such structure is also widespread in the SGFB, as the first episode deformation structure. The strata are intruded by undeformed, crosscutting granitic plutons emplaced during Late Triassic to Early Jurassic time Much of the SGFB was involved in such intensive shortening and deformation during Late Triassic time. In the Jiaochang structure, the N-S shortening and deformation during D2 phase, intensive deformation and metamorphism further took place to accommodate the second foliation, lineation and minerals growth in the central and southern parts of the Jiaochang structure. Harrowfield and Wilson By progressive N-S compression during DDue to intensive deformation and metamorphism, granitic plutons being result of deeper curst melting are widespread in the SGFB 2). It was followed by post-D2 deformation during Yanshanian and Himalayan epochs, with insignificant impact on the Jiaochang architecture.The intensive deformation and metamorphism could accommodate decollement and diapiric rise of the migmatized base core in the Jiaochang, resulting in a northward thrusting structure formed in the hinterland. A curvature structure of the Jiaochang was finally formed after the second episode of deformation  and lineation . The progressive D1–D2 structure is widespread in the southern and central region of the Jiaochang area; only D1 structure crops out at the north. Thus most of the structures support a southward increase in deformation intensity and metamorphism grade.Our study of the Jiaochang structure indicates a progressive D1–D2 deformation and metamorphism in the evolution of the Jiaochang structure, in which the pervasive foliation and multiphase mineral growth predominantly took place during the first to second episodes of the deformation (D1–D2). According to three-dimensional strain analyses, the strain intensity (γ), compression ratios (c%) and octahedral strain intensity (εs), all indicate a similar feature across the Jiaochang structure . The long-axis orientations are subparrellel to the fold axis, and are roughly symmetric between two limbs in the northern region. Moreover, the Flinn values increase from ∼2 in the north to ∼6 in south. Thus, we propose that the Jiaochang structure could be interpreted by progressive N-S trending, D1–D2 coaxial shortening and deformation during the Indosinian epoch, followed by insignificant post-D2 deformation under N-S compression.Based on the micro deformation and metamorphic structures, most of the porphyroblasts and minerals (e.g. pyrite and biotite) have two growth phases with localized growth in the third phase (muscovite). It highlights the importance of the D


R)-3-hydroxybutyrate (PHB), a linear polymer of R-3-hydroxybutyrate (R-3HB), is a fundamental constituent of biological cells. Certain prokaryotes accumulate PHB of very high molecular weight , which is segregated within granular deposits in the cytoplasm; however, all prokaryotes and all eukaryotes synthesize PHB of medium-chain length (~100–200 residues) which resides within lipid bilayers or lipid vesicles, and PHB of short-chain length (<12 residues) which is conjugated to proteins (cPHB), primarily proteins in membranes and organelles. The physical properties of cPHB indicate it plays important roles in the targeting and folding of cPHB-proteins. Here we review the occurrence, physical properties and molecular characteristics of cPHB, and discuss its influence on the folding and structure of outer membrane protein A (OmpA) of Escherichia coli.Poly-( R)-3-hydroxybutyrate (PHB), a linear polymer of R-3-hydroxybutyrate (R-3HB) . The polymers in these complex subcellular organelles, known as carbanosomes, are very long-chain , and are covered by a layer of lipids and proteins, which include enzymes involved in PHB synthesis and degradation . Wh. Wh44]. et al. [Although the cPHB backbone is highly flexible at physiological temperatures, it becomes increasingly more rigid as temperatures approach the glass temperature. As a consequence, cPHB-modified proteins would fold very slowly or be unable to fold into their native conformations when maintained below physiological temperatures. The presence of cPHB may also render protein function(s) highly sensitive to temperature in this range. For example, Zakharian et al. ,43 found1H-NMR resonances of PHB are distinctive  and fatty-acid metabolism—participate in the synthesis of cPHB in n system –50. Rych [et al.  used strd Reusch  found thd Reusch . The outproteins , which iproteins .E. coli. OmpA is a major protein of the cell envelope with many functions [The physical properties and distribution of cPHB imply that it plays important roles in protein structure and physiology. The best example to-date of the influence of cPHB on protein targeting and protein folding is the outer membrane protein A (OmpA) of unctions . It stabunctions , acts asunctions ,58, partunctions ,60, is aunctions , mediateunctions –65 and punctions . In addiunctions .N-terminal residues form a narrow eight β-barrel pore in the outer membrane and 154 C-terminal residues form a globular structure that interacts with peptidoglycan in the periplasm has vied with a large-pore, single-domain structure in which all or most of the 325 residues create a large pore in the outer membrane.The mature conformation of OmpA has been a matter of contention for several decades. A narrow-pore, two-domain structure in which 171 N-terminal domain—residues 1–171—has been described by fluorescence, circular dichroism, Raman and NMR spectroscopy [N-terminal domain forms an antiparallel eight-stranded-β-barrel connected by long, unstructured and mobile loops at the extracellular side and short, tight loops at the periplasmic side. The interior of the β-barrel, lined with charged and polar residues, encloses several unconnected aqueous cavities. The C-terminal domain has been crystallized [A two-domain conformation of OmpA was proposed based on bacteriophage mapping ,58, prottroscopy –75, by Xtroscopy ,77, and troscopy ,79. The tallized ,81 and ctallized .C-terminal domain in formation of a large pore is further indicated by studies of OmpA homologues, Pseudomonas aeruginosa OprF by Rawling et al. [Salmonella enterica OmpA  by Singh et al. [C-terminal epitopes of the proteins were exposed on the cell surface of intact bacteria.The structure of the single-domain large pore is yet unknown; however, a large pore structure is consistent with the known functions of OmpA as a receptor for bacteriophages ,58 and pg et al.  and Salmh et al. , in whicC-terminal domain in pore formation. Jeanteur et al. [C-terminal domain, and additional C-terminal transmembrane segments were predicted by algorithms of Schirmer and Cowan [C-terminal domain that is consistent with the varied biochemical, immunological, and genetic topological data concerning OmpA  made to the protein in the cytoplasm but only OmpA extracted from the outer membranes will have acquired any additional modification(s) that may be made in the periplasm.OmpA must be transported from the ribosome to the cytoplasmic membrane, conveyed across the cytoplasmic membrane into the periplasm, conducted across the periplasm, inserted into the outer membrane and folded therein in its mature conformation. During this journey, modifications may be made to the protein by cytoplasmic and/or periplasmic enzymes. Studies of OmpA structure have been performed with OmpA obtained by protein overexpression into cytoplasmic inclusion granules or with OmpA directly extracted from the outer membranes of N-terminal domain of OmpA, known as the sorting signal, are essential for the integration of OmpA into the outer membrane. When the sorting signal is removed, OmpA remains in the periplasm. Xian et al. [The OmpA precursor (ProOmpA) is synthesized in the cytoplasm as a 325-residue protein with a 21-residue signal sequence at the amino terminal. Studies by the Henning group –99 have n et al.  establis1H-NMR spectra [N-terminal domains. Due to its low concentrations and highly flexible backbone, cPHB would resemble detergent or lipid molecules in these spectra. However, the modification of OmpA peptide 162–174 by up to 10 R-3HB residues is visible in the MALDI-MS spectra [cPHB is not discernible in the Raman or  spectra –75 or X- spectra ,77 of th spectra  (Figure  spectra . Continu spectra .et al. [Negoda et al.  demonstrC-terminal domain must insert into the outer membrane bilayer, and this requires an increase in their hydrophobicity. This may be accomplished by the attachment of cPHB to specific amino acids.After translocation across the cytoplasmic membrane, the signal sequence is cleaved and OmpA, assisted by periplasmic chaperones and lipopolysaccharides, is inserted into the outer membrane and folded into its native form –105. In C-terminal domain by cPHB is a decisive event in the translocation and folding of OmpA into the outer membrane in its mature large-pore conformation. Studies by Negoda et al [C-terminal domain of OmpA 264–325 is modified by cPHB and that this modification takes place in the periplasm, i.e., Western Blot and chemical assays indicated that segment 264–325 contains cPHB when OmpA is isolated from outer membranes, but not when OmpA is isolated from cytoplasmic inclusion bodies. The identity of cPHB on segment 264–325 of OmpA was confirmed by 1H-NMR. The increased hydrophobicity conferred to this segment by addition of cPHB is indicated by the solubility of segment 264–325 in chloroform when it is obtained from outer membranes, in contrast to its insolubility in chloroform when it is obtained from inclusion granules. The specific residues that are modified by cPHB within this segment have not been identified. It is also unknown whether other segments of the C-terminal domain are modified by cPHB.Modification of the da et al . demonstIn vivo, the folding process may be assisted by chaperones that lower the activation energy.Studies by Zakharian and Reusch ,93 illusOnce the conformational rearrangement has been completed, the large pore is very stable. The transformation is irreversible. Conversion of large pores to narrow pores with decreasing temperature was not observed. Even extended periods at low temperatures did not reverse the transition. Large pores are converted to narrow pores only when they are subjected to denaturing conditions. The sum of the data indicates strongly that the mature large pore structure is the end-product of the folding cascade .C-terminal domain that must traverse the bilayer to reach the extracellular aqueous medium. Another periplasmic modification that is also critical to the proper folding of OmpA is the formation of a disulfide bond between Cys290 and Cys302 of the C-terminal domain [N-terminal domain and thus it was considered to be irrelevant to the proper folding of the protein. In the Stathopoulos model of OmpA [et al. [Although cPHB-modification is an effective process for increasing the hydrophobicity of polypeptide segments destined to remain within the bilayer, it may not be suitable for polar segments of the l domain  by the ol domain ,107. Thi [et al.  suggest  [et al. , which sE. coli is that of a single-domain large membrane pore, and that the two-domain structure is a folding intermediate that is unusually stable at lower temperatures. It is clear that cPHB-modification is essential to the formation of both the narrow pore and large pore conformations. Without cPHB modification of the N-terminal domain of OmpA in the cytoplasm, the narrow pore either does not form or it cannot fully insert into the bilayer; without cPHB modification of the C-terminal domain and creation of the Cys290–Cys302 disulfide bond in the periplasm, the large pore does not form. Studies of OmpA, which concluded that the native structure is a two-domain narrow pore, obtained the protein by overexpression [From these studies, one may infer that the native structure of OmpA in pression ,102,103,pression ,90 or itpression . In summcPHB constitutes yet another class of protein conjugates. This polyester is a ubiquitous constituent of both prokaryotic and eukaryotic cells in which it is primarily found covalently attached to proteins located within membranes and organelles. cPHB possesses physical properties that enable it to play important roles in the targeting, folding and function of these proteins. Clearly, the prospect of cPHB-modification should always be considered when designing protocols for isolating and refolding proteins for structural studies. Protein overexpression is a convenient method of obtaining ample amounts of protein for spectroscopic and crystallographic studies, but a functional protein is more than a chain of amino acids. Posttranslational modifiers of the protein should be identified and their potential roles in attaining the native structure carefully evaluated. In addition, it is important to realize that although the polypeptide structure of the protein is not strongly temperature-dependent, modifiers such as lipids and cPHB may make it so. Unless all temperature-dependent modifiers are ruled out, protein folding is best observed within the temperature range at which it occurs in nature.


Background. Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies worldwide. It is characterized by its high invasive and metastatic potential. Leprecan-like 1 (LEPREL1) has been demonstrated to be downregulated in the HCC tissues in previous proteomics studies. The present study is aimed at a new understanding of LEPREL1 function in HCC. Methods. Quantitative RT-PCR, immunohistochemical analysis, and western blot analysis were used to evaluate the expression of LEPREL1 between the paired HCC tumor and nontumorous tissues. The biology function of LEPREL1 was investigated by Cell Counting Kit-8 (CCK8) assay and colony formation assay in HepG2 and Bel-7402 cells. Results. The levels of LEPREL1 mRNA and protein were significantly lower in the HCC tissues as compared to those of the nontumorous tissues. Reduced LEPREL1 expression was not associated with conventional clinical parameters of HCC. Overexpression of LEPREL1 in HepG2 and Bel-7402 cells inhibited cell proliferation (P < 0.01) and colony formation (P < 0.05). LEPREL1 suppressed tumor cell proliferation through regulation of the cell cycle by downregulation of cyclins. Conclusions. Clinical parameters analysis suggested that LEPREL1 was an independent factor in the development of HCC. The biology function experiments showed that LEPREL1 might serve as a potential tumor suppressor gene by inhibiting the HCC cell proliferation. HCC is one of the most prevalent tumors worldwide and the third leading cause of cancer-related deaths around the world , 2. HCC β catenin (about 25%) [Hepatocarcinogenesis is a complex multistep process in which many signaling cascades are altered. The most common mutations include the tumor suppressor gene TP53  and CTNNB1 gene for out 25%) . Epigeneout 25%) . It has out 25%) , 8, 9. Iout 25%) .Leprecan-like 1 (LEPREL1) is a protein with an extensive similarity to the Gros1/Leprecan. This protein mainly concentrates in the endoplasmic apparatus and Golgi complex in the cell and is abundant in the basement membranes , 12. OurA total of 80 HCC patients who were treated with hepatectomy in our hospital  in the second half of 2011 were enrolled in this study. The HCC was preoperatively diagnosed by appropriate imaging characteristics and was verified by histological examination after the operation. The cancer samples and matched noncancerous samples were obtained during the surgery. None of the patients received presurgical chemo- or radiation therapy. Clinicopathologic data were available for each of the 80 patients. Approval for these studies was obtained from the Institutional Review Board of the Faculty of our hospital in accordance with the ethical standards of the responsible committee on human experimentation as well as the declaration of Helsinki.2.Human HCC cell lines (HepG2 and Bel-7402) were obtained from the cell bank of the Chinese Academy of Sciences . The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium (Gibco-BRL), supplemented with 10% fetal bovine serum, in a 37°C incubator with 5% COμg of total RNA was used for the first-strand cDNA synthesis with PrimeScript reverse transcriptase (TaKaRa) according to the manufacturer's instructions. Real-time PCR was performed using SYBR Premix PCR kit (TaKaRa) and an ABI PRISM 7500 Sequence Detector. Real-time PCR programs were as follows: one cycle of 95°C for 5 min, 95°C for 5 s, 50°C annealing for 30 s, and 72°C for 34 s, followed by 40 cycles. Three independent experiments were performed for each sample. The relative gene expression levels were determined using the 2-ΔΔCt method. The threshold cycle (CT) was measured during the exponential amplification phase and the amplification plots were analyzed by SDS 1.9.1 software . Specific primer pairs were used for LEPREL1  and GAPDH .Total RNA was isolated using TRIzol reagent (invitrogen) according to the protocols recommended by the manufacturer. The total RNA concentration and quantity were assessed by absorbance at 260 nm using Nano Drop ND-2000 spectrophotometer. A total of 1 μg) were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) gels (Invitrogen). After electrophoresis, the separated proteins were transferred into polyvinylidene fluoride (PVDF) membranes (Millipore) and were blocked with Tris-buffered saline (TBS) containing 5% nonfat milk for 1 h. The membranes were then incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal LEPREL1(Abcam), rabbit polyclonal GAPDH, rabbit polyclonal Cyclin A2, rabbit polyclonal Cyclin B1, rabbit polyclonal Cyclin D1, rabbit polyclonal Cyclin E2, rabbit polyclonal CDK2, and rabbit polyclonal CDK4 (Epitomics). Primary antibodies were diluted in TBST with 5% nonFAT milk at 1 : 1000. They were then incubated with a secondary goat anti-rabbit IgG monoclonal antibody (Epitomics) conjugated with horseradish peroxidase at 1 : 1000 dilution for 1 h at room temperature. Finally, the protein bands were detected by chemiluminescence using EZ-ECL chemiluminescent detection kit (BIOIND).12 pairs of HCC samples (100 mg) or cells were lysed in the cold RIPA lysis buffer (Beyotime) supplemented with protease inhibitor phenylmethanesulfonyl fluoride (PMSF). The protein concentration was measured by bicinchoninic acid assay. Protein extracts  under microscope was calculated and was compared in different specimens by two separate observers in a double-blind fashion. It was described as a score of 1 (<10% positive cells), 2 (10–20% positive cells), and 3 (>20% positive cells) .After transfection with pcDNA3.1-LEPREL1 or control vectors for 24 h, the HepG2 and Bel-7402 cells were collected and placed onto the six-well plate (1000 cells per well). After a 14-day growth, the surviving colonies were fixed in methanol, washed two times with phosphate-buffered saline, dried, stained with liquid crystal violet , and counted. The experiments were repeated in triplicate.μL of complete culture medium. After adhesion for 24 hours, the cells were transfected with pcDNA3.1-LEPREL1 or control vectors and were cultured for another 24, 48, 72, and 96 h. The wells where only the culture medium was added in them served as blanks. At each time point, the supernatant was removed and 100 μL of DMEM medium containing 10 μL of CCK8 (Dojindo) was added to each well for another 2 h at 37°C. The absorbance was recorded at 450 nm. All the experiments were independently repeated at least seven times.HepG2 and Bel-7402 cells were plated in 96-well plates at the density of 5,000 cells per well with 100 t-test. The clinicopathological features of the patients were compared using chi-square test (Fisher's exact test) for categorical variables. A P value of less than 0.05 indicated that the differences were statistically significant.The SPSS 10.0 statistical analysis software was used to statistically process the experimental data. The CT values of LEPREL1 in tumor and nontumor samples and the effects of LEPREL1 on cell viability were compared using two-tailed Student's P < 0.001, n = 80, In this study, we first detected the expression level of LEPREL1 using qRT-PCR in 80 pairs of HCC and the matched nontumor tissues. It was noticed that the downregulation of LEPREL1 was detected in 61/80 (76.3%) of the HCC tissues. The CT values of the LEPREL1 in tumor and nontumor tissues were then subjected to the appropriate statistical analysis. The results showed that the expression level of LEPREL1 was obviously lower in the tumor tissues as compared to that of the adjacent nontumor tissues  of the 86 HCC specimens as compared with that of the adjacent noncancerous livers . The exaP > 0.05).The expression of LEPREL1 was analyzed in HCC with respect to several standard clinic-pathological features . HoweverP < 0.05, To investigate the tumor suppression ability of LEPREL1 in the HCC cells, we observed the effect of LEPREL1 expression on cell proliferation and colony formation. Cell growth assay showed that the growth of HepG2 and Bel-7402 cells was significantly reduced after the transfection with pcDNA3.1-LEPREL1 as compared to that of the cells transfected with an empty pcDNA3.1 vector  in the CpG islands in intragenic, promoter, and intergenic regions is a common epigenetic mechanism in the eukaryotic DNA, which plays an important role during differentiation and in response to some types of physiological changes. Increasing evidence has shown that DNA methylation could be involved in genomic instability and silencing of the tumor suppressor genes in many cancers, including HCC –21. ReceThe antiproliferation mechanism of LEPREL1 was further studied by detecting cell cycle regulatory proteins. Overexpression of these cyclins and CDKs altered the cell cycle progression which is closely associated with malignancy. Many studies suggested that overexpression of Cyclins D1, E, and CDK4 protein levels resulting in uncontrolled cell proliferation is closely associated with HCC , 25. CycWith a cohort of 86 randomly selected HCC patients, we investigated the potential downregulation of the LEPREL1 with numerous clinical parameters, including age, gender, tumor size, Edmondson grade, vascular invasion, and AFP. However, the expression of the LEPREL1 was not associated with any of the above clinical parameters. This study is the first to examine the role of LEPREL1 in HCC and reveals that the LEPREL1 played a key role in proliferation inhibition of the HCC cell lines. Our results suggested that the LEPREL1 might be a tumor suppressor gene.


The body of disease mutations with known phenotypic relevance continues to increase and is expected to do so even faster with the advent of new experimental techniques such as whole-genome sequencing coupled with disease association studies. However, genomic association studies are limited by the molecular complexity of the phenotype being studied and the population size needed to have adequate statistical power. One way to circumvent this problem, which is critical for the study of rare diseases, is to study the molecular patterns emerging from functional studies of existing disease mutations. Current gene-centric analyses to study mutations in coding regions are limited by their inability to account for the functional modularity of the protein. Previous studies of the functional patterns of known human disease mutations have shown a significant tendency to cluster at protein domain positions, namely position-based domain hotspots of disease mutations. However, the limited number of known disease mutations remains the main factor hindering the advancement of mutation studies at a functional level. In this paper, we address this problem by incorporating mutations known to be disruptive of phenotypes in other species. Focusing on two evolutionarily distant organisms, human and yeast, we describe the first inter-species analysis of mutations of phenotypic relevance at the protein domain level.The results of this analysis reveal that phenotypic mutations from yeast cluster at specific positions on protein domains, a characteristic previously revealed to be displayed by human disease mutations. We found over one hundred domain hotspots in yeast with approximately 50% in the exact same domain position as known human disease mutations.We describe an analysis using protein domains as a framework for transferring functional information by studying domain hotspots in human and yeast and relating phenotypic changes in yeast to diseases in human. This first-of-a-kind study of phenotypically relevant yeast mutations in relation to human disease mutations demonstrates the utility of a multi-species analysis for advancing the understanding of the relationship between genetic mutations and phenotypic changes at the organismal level. The aet al. . The autet al. [et al. conclude that these edgetic perturbations can cause clinically distinct phenotypes when disrupting different protein domain regions of the same protein. Thus, a domain-centric study of disease mutations has the potential to differentiate among genomic variations by accounting for protein modularity that would have otherwise been grouped together by whole-protein studies.The vast majority of studies related to human disease mutations are performed by comparison of whole proteins, which here will be denoted by the genes that encode them. However, these whole-protein approaches are of limited applicability to the study of disease mutations due to the fact that they mostly fail to account for protein modularity. Most proteins contain multiple domains that can be recombined in different arrangements to create proteins with different functions -15. As aet al.  in theirhttp://bioinf.umbc.edu/dmdm/) [et al. [et al. [et al. [To capture the disruption of domains by genetic mutations, we have previously created a database to visualize the aggregation patterns of disease mutations at the protein and domain levels for human genomics data (Domain Mapping of Disease Mutations database (DMDM), freely available at u/dmdm/) . More reu/dmdm/) . We demou/dmdm/) . In addi [et al. , Nehrt e [et al. , and Pet [et al.  have furIn this paper, we describe the first inter-species analysis of mutations of phenotypic relevance at the protein domain level for human and yeast genomes. We perform the comparison between these species by mapping human and yeast mutations into the corresponding domain sites. Protein domains, such as those defined by CDD  and PfamIn summary, the work described in this paper demonstrates that domain-centric, inter-species mutation analyses lead to the identification of new domain sites of relevance to human diseases even when performed among species separated by long evolutionary distances. The patterns of evolutionarily conserved and functional mutations associated with phenotypic changes emerging from this study represent a step towards a new paradigm for the analysis of large-scale genomic studies of human diseases.th, 2012 (http://downloads.yeastgenome.org/sequence/S288C_reference/orf_protein/). The Homologene database [th, 2011. A protein domain set was obtained from the Conserved Domain Database (CDD version 2.25) [ftp://ftp.ncbi.nih.gov/pub/mmdb/cdd), totaling 1,727 unique functional features. The non-overlapping set of human, non-synonymous disease mutations was created from the OMIM [http://www.uniprot.org/docs/humsavar) respectively. The resulting human mutation dataset consists of 32,653 mutations related to human diseases. The set of phenotypic yeast mutations was downloaded from SGD (the phenotype_data.tab database obtained from http://www.yeastgenome.org/download-data/curation/) and was filtered to exclude records without allelic information and records listing the phenotypic change as "normal," as these records refer to mutations with no phenotypic change. Finally, the yeast mutation database was manually curated to extract single point mutations and to ensure that each mutation record referred to a single point mutation. Mutation records referring to multiple mutations for a single phenotype were separated into multiple records. The final yeast mutational database is comprised of 1,490 unique mutations associated with phenotypic changes and is available upon request.A human protein database containing 54,372 proteins was created with 33,963 proteins from RefSeq  and 20,4database  was downon 2.25) , which ion 2.25) , COG [28on 2.25)  databasethe OMIM  and SwisHidden Markov models for protein domains from SMART, COG, CDD, and Pfam were built using multiple sequence alignments from CDD with the hmmerbuild tool (HMMer version 2.3.2) . HMMer'sj in a protein domain multiple sequence alignment, we used the AL2CO [For each column he AL2CO  method tp is the frequency of amino acid ai at position j. We then estimated a threshold for identifying highly conserved positions by adding one standard deviation to the average of all AL2CO scores on all domain positions. As a result, a threshold of entropy less than or equal to 0.533 was used to determine conservation. The average entropy of each domain model was determined by estimating the mean of the entropy scores for all positions in the domain model.where n be the total number of mutations in the domain and let L be the number of possible positions in the domain. The random sample, X1, ... , XL consists of the numbers of mutations aggregated to the domain level from a single organism  be the smallest of these Xi , and X(2) be the next order of magnitude, ... , and X(L) is the largest Xi. We used the probability mass function, P(X = x), to test whether the n mutations are randomly distributed into L positions. Finally, we defined our position-based DS-Score by the negative logarithm transformation of the binomial probability of observing a cluster of mutations of a particular size, given the total available positions in a domain and the total number of mutations observed. For consistency, when the number of mutations is equal in m positions, we assigned the same score to all m positions. Where k is the kth order of the number of mutations at domain positions, we define the DS-Score asWe used a method previously developed by our group  to estimm Figure  or from m Figure  and let et al. [http://www.r-project.org) were used to determine and distribute the DS-Scores for each domain position.We defined domain hotspots using three levels of significance from the Fisher's scale of evidence for interpreting p-values as described by Yue et al. , namely et al. . Perl anDue to large domain superfamily hierarchies and duplicate domains from different sources, a single protein mutation can be mapped to many redundant domains. As a consequence, multiple domain hotspots can be identified on different domains that have originated from the same cluster of mutations. On one hand, to ensure that the DS-Scores and domain hotspots are estimated and identified for all protein domain models, all mutations in our analysis were distributed to all domains that map to the mutated protein position. The results of our analysis for each individual domain model, even for those domains from superfamily hierarchies and disparate sources for which we expect high redundancy, are available upon request. On the other hand, to prevent overestimating the number of hotspots shared between yeast and human, we designed a procedure for the non-redundification of domain hotspots. As a result, the numbers reported for all domain hotspots and multi-species domain hotspots originated from a unique set of mutation clusters. We excluded hotspots that originated from identical sets of mutations using the following method to select a unique representative domain for cluster of mutations. The representative domain is only used for visualization and internal calculations and does not affect the reported results. To select the representative domain for a mutation cluster, all domains were ordered alphanumerically from lowest to highest accession identifier. Preference is given to domains that are listed first within the list and were defined as root in the domain hierarchy. If none of the domains in the list is the root domain of a hierarchy, the representative domain is the first domain in the list that contain known functional annotated sites. In addition, when comparing hotspots using the multi-species DS-Score, the representative domain model is selected only among those that are shared among the species.The significance of overlapping human diseases and yeast phenotypes was calculated using a right-sided Fisher's exact test. The Fisher's test for each possible pair of human diseases and yeast phenotypic changes was estimated using the following values: the number of times the human disease and yeast phenotypic change (H and Y respectively) overlap, the number of times H overlaps with a yeast phenotype that is not Y, the number of times Y overlaps with a human disease that is not H, and the total number of overlaps between yeast and human. Human diseases and yeast phenotypes were considered to overlap if the associated mutations were found to localize at the same position of an identical domain. To avoid overestimation due to domain model redundancy, no protein mutation was counted more than once as overlapping with a single human disease or yeast phenotype.To study the distribution of phenotypically relevant mutations at the protein domain level, all proteins from the considered species, i.e., human and yeast, were aligned to one or multiple domains and their mutations mapped into these domains. First, we mapped all phenotypically relevant mutations from yeast and disease mutations from human to protein domains and analyzed the distribution of the location of these mutations with respect to their functional annotation and conservation over species as measured by the entropy of the domain site. Due to the redundancy and large hierarchies within the protein domain database, we observed that the 1,490 yeast and 32,653 human protein mutations were propagated to 11,016 and 323,840 domain mutations respectively , glucose-6-phosphate dehydrogenase (G6PD), neural cell adhesion molecule L1 (L1CAM), phenylalanine hydroxylase (PAH), paired box 6 (PAX6), the X-linked retinoschisis gene, and a gene associated with tuberous sclerosis (TSC2). From the study of mutations in these seven genes and their conservation across 20 organisms, including human, the authors concluded that these mutations are in highly conserved protein positions. Here, we reach similar conclusions, but we estimated conservation based on the protein domain models and not at the gene level. Additionally, our findings at the domain level are consistent with Mooney et al. [Our study of phenotypically relevant mutations using a protein domain framework confirms that both yeast and human mutations show a significant tendency to fall within conserved and annotated functional protein domain sites. This is in agreement with the conclusions by Miller et al.. In theiy et al. . The autIRE1 gene was propagated to over a hundred domains within the catalytic protein kinase domain family , resultdatabase , was conIn this first-of-a-kind study of yeast mutations at the domain level, we demonstrate that phenotypically relevant mutations in yeast cluster at the domain level just as human disease mutations do, forming yeast and human domain hotspots that are the focus of this study. The hotspots in yeast present the same patterns as human domain hotspots in terms of enrichment at protein domain sites that are conserved and also in sites with known functional annotation. Neither the yeast nor human DS-Scores were found to correlate with conservation (as measured by entropy of the domain site), making the DS-Score method a complement to other methods for prioritization of mutations with putative phenotypic relevance such as SIFT , that usWe compared the newly found position-based and feature-based domain hotspots in yeast against those arising from human mutations. Given the limited number of mutations with known phenotypic association for each species, we expect the overlap between hotspots in both species to be low or non-existent. Indeed, the only position-based domain hotspot shared between yeast and human is at position 246 on PKc (cd00180), a kinase domain. Surprisingly, the number of hotspots in yeast and human sharing a common domain and functional annotation is 108, 114 and 177 when estimated using feature-based DS-score greater or equal to 1.6, 1.3, and 1.0 respectively. From this analysis, we concluded that the lower threshold provides a significant increase in the number of common feature-based domain hotspots found at the cost of including hotspots with lower significance. On the other hand, the similarity of mutation clusters between species increases when using feature-based DS-Score, a method designed to exploit the manually curated functional annotation provided by CDD . The feaARF2, SEC4, GTR1, and NOG1 that may not have been identified without analyzing mutations at the domain level due to the low sequence similarity of NOG1 . The yeast mutations in position ten of cd00882 in genes ARF2, SEC4, GTR1, and NOG1 were associated with increased sensitivity to cold [ARL6 gene contains a mutation associated with Bardet-Biedl syndrome type 3 [HRAS and KRAS genes, from which many mutations have been implicated in diseases such as Costello syndrome [HRAS and KRAS belong to the same protein family and are thus often implicated in the same studies, domain position five also aligns to position 38 of a gene from a different family, GNAT1, which is not similar in sequence to HRAS . The GNAT1 mutation has been associated with congenital stationary night blindness [An example of a domain with several domain hotspots in human and yeast that highlights the advantages of using feature-based domain hotspots is the Ras-like GTPase domain  or KRASlindness . Additiolindness , somaticlindness , Noonan lindness , and chylindness . Thus, bet al. [Furthermore, the domain-centric approach across species introduced here could also be extended to compare the particular phenotypes across organisms that can be related through mutations clustered with this approach, analogous to the phenotype similarities described by McGary et al.  using geet al. . While tThis first-of-a-kind study demonstrates the aggregation of mutations from species spanning large evolutionary distances such as yeast and human. Using the DS-Score method as the framework for the integration of molecular characteristics, such as domain location and functional annotation of phenotypically relevant mutations, we were able to identify common mutation patterns from two distantly related species. The domain-centric approach introduced in this paper provides an ideal framework for the analysis of mutational data across species since the number of mutations that can be related from one species to another is much higher than what could be related through gene orthology. The feature-based method to compare mutations across species introduced here represents a unique way to integrate functional annotation of domains into the statistical analysis of mutations, shown here to be extremely advantageous for capturing similarities between mutations across distantly related species. This analysis also suggests that the approach is useful in relating phenotypes from yeast and human resulting from a particular pattern of mutations, such as being localized at the same domain position or functional site. We plan to perform a detailed analysis of the molecular basis of these related phenotypes. Hypotheses derived from this analysis have great potential for discovering new relationships between pathways and networks in both species. In addition, we plan to extend this study to other species to identify patterns across more closely related species including mouse, and to increase the number of known phenotypically relevant domain hotspots by including all mutational data available for a wide range of organisms.The authors declare that they have no competing interests.MGK designed the study, TAP performed the bioinformatics analysis, MGK and TAP prepared and analysed the data and drafted the manuscript. DP developed the algorithm for computing the mutation frequency significance thresholds. All authors read and approved the final manuscript.This file contains information related to the mapping of hotspots in human to mutations in yeast. This file is in PDF format, and can be viewed using Adobe Reader or similar applications.Click here for fileThis file contains the human disease and yeast phenotypic changes the co-occur at domain sites. This file is in Microsoft Excel format, and can be viewed using Microsoft Excel or similar applications.Click here for file


Pseudomonas putida, a representative example of CheR-TPR. The affinities of WspC-Pp for S-adenosylmethionine and S-adenosylhomocysteine were comparable to those of prototypal CheR, indicating that WspC-Pp activity is in analogy to prototypal CheRs controlled by product feed-back inhibition. The removal of the TPR domain did not impact significantly on the binding constants and consequently not on the product feed-back inhibition. WspC-Pp was found to be monomeric, which rules out a role of the TPR domain in self-association.Chemosensory pathways correspond to major signal transduction mechanisms and can be classified into the functional families flagellum-mediated taxis, type four pili-mediated taxis or pathways with alternative cellular functions (ACF). CheR methyltransferases are core enzymes in all of these families. CheR proteins fused to tetratricopeptide repeat (TPR) domains have been reported and we present an analysis of this uncharacterized family. We show that CheR-TPRs are widely distributed in GRAM-negative but almost absent from GRAM-positive bacteria. Most strains contain a single CheR-TPR and its abundance does not correlate with the number of chemoreceptors. The TPR domain fused to CheR is comparatively short and frequently composed of 2 repeats. The majority of CheR-TPR genes were found in gene clusters that harbor multidomain response regulators in which the REC domain is fused to different output domains like HK, GGDEF, EAL, HPT, AAA, PAS, GAF, additional REC, HTH, phosphatase or combinations thereof. The response regulator architectures coincide with those reported for the ACF family of pathways. Since the presence of multidomain response regulators is a distinctive feature of this pathway family, we conclude that CheR-TPR proteins form part of ACF type pathways. The diversity of response regulator output domains suggests that the ACF pathways form a superfamily which regroups many different regulatory mechanisms, in which all CheR-TPR proteins appear to participate. In the second part we characterize WspC of  Protein-protein interactions are essential for life. In many cases single-domain proteins have evolved with the capacity to recognize other proteins or to oligomerize. An alternative evolutionary strategy consists in fusion proteins in which one domain is dedicated to binding other proteins. An example for such domains is the tetratricopeptide repeat (TPR) containing domain The primary function of TPR domains consists in the binding to other proteins which is reviewed in Allan and Ratajczak Bacterial signal transduction is primarily mediated by one- and two-component systems as well as by chemosensory pathways Chemoreceptor mediated signaling is based on the concerted action of the excitatory pathway and adaptational mechanism(s). The initial step of the excitatory pathway consists in the recognition of signal molecules by chemoreceptors, which produces a molecular stimulus that is transduced across the membrane to the signaling domain that forms a ternary complex with CheA and CheW. This molecular stimulus alters CheA autophosphorylation and in turn transphosphorylation activity towards the response regulator CheY Based on a bioinformatic analysis Wuichet and Zhulin Myxococcus xanthusPseudomonas aeruginosa has 4 chemosensory clusters of which two are involved in flagellum-mediated taxis Chemosensory pathways have been first described in the context of flagellum-mediated taxis, where it was shown that the phosphorylation of CheY induces a conformational change that permits an interaction with the flagellar motor cheR genes abolished or impaired chemotactic behavior in many species M. xanthus. FrzF is composed of a methyltransferase domain that is fused to a TPR domain. The authors show that full-length FrzF methylates a single amino acid, whereas a truncated version lacking the TPR domain methylated the protein at 3 amino acids. This implies that the presence of the TPR domain inhibits methylation activity. The mechanism of action of this CheR-TPR fusion protein is unknown, but Scott et al. The classification of CheR as a core protein is supported by experimental data which showed that mutation of the There appear to be two families of bacterial methyltransferases: the prototypal CheR and the family of CheR-TPR fusions. Prototypal CheRs have been studied in depth which contrasts the scarce information available on the CheR-TPR family. We present here a first analysis of the family of CheR-TPR proteins. In the initial part of this article we have screened completed bacterial genomes sequences to retrieve CheR-TPR sequences, which were then submitted to bioinformatic analyses. In the second part we report the analysis of a representative example of a CheR-TPR protein as well as truncated versions thereof lacking the TPR domain.Heliobacterium modesticaldum) contained a CheR-TPR sequence. As shown in The analysis of translated open reading frames of completed genome sequences resulted in the detection of 160 CheR-TPR sequences which were then manually curated to exclude false positive hits. The final list contained 132 CheR-TPR proteins that were encoded in 96 genomes. Detailed information on these strains and proteins is compiled in the Stigmatella aurantiaca is an outlier, since this species has 19 CheRs of which 8 are CheR-TPR fusions . These data are consistent with the observation that most species contain a single CheR-TPR independent of the number of chemoreceptors  family is the presence of multidomain response regulators, whereas the response regulators that participate in flagellum-mediated taxis (Fla) pathways or type four pili-mediated taxis (Tfp) are with high frequency single domain response regulators egulator . Only 20egulator . To evalcheR-TPR containing gene clusters are multidomain proteins  is devoid of linker and TPR domain. WspC-Ppshort can thus be considered as prototypal CheR. The three proteins were expressed in E. coli and purified from the soluble fraction of the cell lysate.Following the bioinformatic studies we proceeded with the functional characterization of a representative member of the CheR-TPR family. WspC of To study SAM and SAH binding, the three proteins were submitted to isothermal titration calorimetry studies Although the linker and TPR domains did not alter significantly the equilibrium constants of binding it became apparent that removal of TPR/linker or TPR domains altered significantly the thermodynamics of binding, namely the ratio of enthalpy to entropy changes. This was particularly pronounced for SAM where an unfavorable entropy change was measured for the full-length protein, whereas ligand binding to the shortened versions was characterized by favorable entropy changes which were in their magnitude comparable to the enthalpy changes. The same tendency is observed for SAH, although to a lesser extent. It can be concluded that the linker/TPR domains have a major impact on the thermodynamics of substrate/product binding, which, however, is not reflected in the affinity of binding.S. typhimurium CheR is monomeric is solution. To determine the oligomeric state of WspC-Pp, the protein was analyzed by analytical ultracentrifugation techniques. In sedimentation velocity studies a major peak centered at 2.7 S and a very minor peak at 5.0 S were observed . TPR domains are frequently involved in mediating protein self-association http://pfam.sanger.ac.uk/search#tabview=tab1) of Pfam. The criterion for this search was the presence of members of the TPR clan (CL0020) and the E-value threshold for this search was set at 1.0 e−4. From the multiple families which form the TPR clan CL002, CheR proteins were found to be fused to TPR domains that belong exclusively to 3 families, namely TPR1 (PF00515), TPR2 (PF07719) and TPR4 (PF07721). The resulting list was then manually curated to confirm the presence of TPR domains. The criteria for the confirmation of a CheR-TPR was the identification of TPR by Interpro (presence of TPR domains IPR011990 or IPR013026) and by the “Search for conserved domains” tool of NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) using the presence of cd00189 domains as criterion. The curated list contained 132 sequences from 96 different genomes. In order to establish whether these sequences belong to a che-like gene cluster, their respective genomic contexts were investigated using the “Gene” database of NCBI (http://www.ncbi.nlm.nih.gov/gene). This allowed the identification of the cheY type response regulator genes in cheR-TPR containing gene clusters. The domain organization of response regulators was extracted from the conserved domains database of NCBI. Data of the retrieved CheR-TPR sequences, information of the host bacterium as well as links to the relevant databases from which information was extracted are provided in To retrieve all bacterial CheR genes the Interpro database was searched for sequences which are identified by IPR000780 . In total 1499 genomes were used for this analysis. This search resulted in the identification of 2898 sequences. To identify those CheR sequences that contain an additional TPR domain, the retrieved CheR sequences were then screened for the presence of TPR domains. This search was done using the “batch search” tool (http://pfam.sanger.ac.uk/search#tabview=tab1) of Pfam. The criterion for this search was the presence of members of the TPR clan (CL0020) and the E-value threshold for this search was set at 1.0 e−4. The transmembrane regions of chemoreceptor-TPR fusion proteins were determined using the TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/). The sequence fragments of chemoreceptor predicted to form a TPR domain, flanked by 5 amino acids at each side, were submitted to the three-dimensional homology modeling server CPHmodels 3.2 (http://www.cbs.dtu.dk/services/CPHmodels/). Multiple sequence alignments were carried out using the CLUSTALW algorithm of the NPSA server . The GONNET matrix was used and the gap opening and gap extension penalties were set at 10 and 0.1, respectively.Initially the CheR-TPR containing genomes were analyzed for sequences that match InterPro signatures IPR011990  and IPR004089 . Subsequently, all currently available chemoreceptor sequences were retrieved by a search for hits in all currently available genome sequences using IPR004089. This search resulted in the detection of 29 425 sequences. To verify whether these sequences contain a TPR domain, a series of batch searches were conducted using the “batch search” tool (P. aeruginosa PilF (Q51526), M. xanthus AglT (Q1D2U6) and M. xanthus AgmK (Q1D2V1). The criteria for considering a protein as homologue was a sequence identity of more than 30% as well as a size corresponding to ±30% of the search protein. Links of proteins retrieved are provided in Each of the CheR-TPR containing strains  was searThe strains and plasmids used in this study are provided in Pseudomonas putida KT2440 , its shortened versions, WspC-Pp-short (amino acids 1–240 of WspC-Pp) and WspC-Pp-middle (amino acids 1–355 of WspC-Pp) were amplified by PCR using the oligonucleotides indicated in P. putida KT2440 as template. The resulting products were digested with NdeI and BamHI and cloned into pET28b(+) (Novagen) linearized with the same enzymes. The resulting plasmids pET28b-WspC-Pp, pET28b-WspC-Ppshort and pET28b-WspC-Ppmiddle were verified by sequencing the insert and flanking regions.DNA sequences encoding full-length WspC-Pp of E. coli BL21 (DE3) was transformed with each of the three plasmids constructed above. The resulting strains were grown in 2l Erlenmeyer flask containing 500 ml of LB medium supplemented with 50 µg/ml of kanamycin at 30°C. At an OD660 of 0.6 protein production was induced by the addition of 0.1 mM IPTG and growth was continued at 16 degrees C overnight prior to cell harvest by centrifugation at 10 000×g for 30 min. Cell pellets were resuspended in buffer A  and broken using French press at 1000 psi. After centrifugation at 20 000×g for 1 hour, the supernatant was loaded into 5 ml of HisTrap HP column (Amersham Bioscience) equilibrated with buffer A and eluted with an imidazole gradient of 45–500 mM in buffer A.For protein expression 2, 0.1 mM EDTA, and 1 mM DTT, pH 7.5 and placed into the sample cell of the instrument. The ligand solutions were made up in the dialysis buffer and placed into the injector syringe. Typically 10–30 µM of protein was titrated with 3.2–8 µl aliquots of SAM (1 mM) or SAH (0.5–1 mM). In all cases, heat changes resulting from the titration of buffer with the respective ligands were subtracted from the titration data. Integrated, dilution-corrected and concentration-normalized peak areas of raw data were fitted with the “One binding site” model of the MicroCal version of ORIGIN. The parameters ΔH , KA , and n (reaction stoichiometry) were determined from the curve fit. The change in free energy (ΔG) and in entropy (ΔS) was calculated from the values of KA and ΔH using the equation ΔG  = −RT ln KA  =  ΔH−TΔS, where R is the universal molar gas constant and T is the absolute temperature.Measurements were done on a VP-microcalorimeter  at 25°C. Protein was dialyzed into 20 mM Tris/HCl, 150 mM NaCl, 2 mM MgClc(s), were calculated by least squares boundary modeling of sedimentation velocity data using the program SEDFIT s values. Short column (85 µl) sedimentation equilibrium was conducted at two speeds (12000 and 15000 rpm). Following the equilibrium scans, the solutions were centrifuged at high speed (40000 rpm) to deplete the meniscus and obtain the corresponding baseline offsets. The measured equilibrium concentration  gradients of the proteins were fitted by the equation that characterizes the equilibrium gradient of an ideally sedimenting solute  was used to perform the analytical ultracentrifugation experiments of WspC-Pp (0.25–1.0 mg/ml) in the absence and presence of ligands. The detection was carried out by means of a UV-visible absorbance detection system. Experiments were conducted at 20°C using an AnTi50 eight-hole rotor and Epon-charcoal standard double sector centerpieces . Absorbance scans were taken at the appropriate wavelength (280–295 nm). Sedimentation velocity experiments were performed at 48000 rpm using 400 µl samples in buffer consisting of 50 mM Tris/HCl pH 8.0, 300 mM NaCl and 1 µM DTT. Differential sedimentation coefficient distributions, Figure S1Distribution of CheR-TPR sequences in function of protein length. The lengths of translated proteins were classified into groups comprising 10 amino acids. The relative abundance of sequences in these groups is shown.(TIF)Click here for additional data file.Figure S2Distribution of CheR-TPR sequences in function of linker length. The segment between the CheR domain and the TPR domain was determined for each CheR-TPR sequence and its length classified into groups comprising 10 amino acids. The relative abundance of sequences in these groups is shown.(TIF)Click here for additional data file.Figure S3Three dimensional structure of the CheR methyltransferase from Salmonella typhimurium. The structure is deposited in the protein data bank with accession code 1af7. A) Structure of the entire protein, bound SAH as well as catalytic residues R98 and Y235 are shown in ball-and-stick mode. B) Zoom of the active site.(TIF)Click here for additional data file.Figure S4Sequence alignment of a selection of CheR-TPR sequences with the TPR domain on the C-terminal part of the protein (A) and with the TPR domain on the N-terminal part of the protein (B). In both alignments the sequences of CheR from E. coli and S. typhimurium have been included. The residues R98 and Y235 (see Y235 see  identifi(PDF)Click here for additional data file.Figure S5Relative abundance of TPR genes compared to total number of genes per genome. Shown are the means and corresponding standard deviations for the different taxa. TPR genes were identified by a search in Pfam using clan CL0020 (Tetratrico peptide repeat superfamily) and an E-value threshold of 1.0 e−4. CheR-TPRs are characterized by a detection using InterPro signature IPR000780 .(TIF)Click here for additional data file.Table S1Presence of CheR-TPR genes in different bacterial genomes.(XLS)Click here for additional data file.Table S2Analysis of the genetic environemnt of cheR-TPR genes. Shown is the architecture of the response regulator (RR) present in CheR-TPR containing clusters.(XLS)Click here for additional data file.Table S3Analysis of total TPR proteins encoded by the CheR-TPR containing genomes.(XLS)Click here for additional data file.Analysis S1Analysis of chemoreceptor-TPR fusion proteins. Sequences were retrieved from InterPro by a search of the CheR-TPR containing genomes (http://www.cbs.dtu.dk/services/TMHMM-2.0/) are shaded in yellow. The sequence fragment predicted to be located in the cytosol is underlined. Shown are also images from the InterPro output of each sequence. The sequence fragments shaded in red containing 5 additional amino acids at each side were submitted to the three dimensional homology modeling server CPHmodels 3.2  CPHmodels-3.0 Nucleic Acids Research 38, doi:10.1093/nar/gkq535). Shown are the resulting homology models and the protein database ID of the templates used for modeling. In each case the model showed the typical structure of a TPR domain containing 2–3 TPR. For reference, the three dimensional structure of the TPR domain of pdb entry 2C2L is shown below. This template was used to generate some of the homology models. At the end of this document a sequence alignment of members of the chemoreceptor-TPR family is shown. The alignment was made using the CLUSTALW algorithm of the NPSA server . The GONNET matrix was used and the gap opening and gap extension penalties were set at 10 and 0.1, respectively. The TPR regions are shaded in yellow. Amino acids in red are fully conserved, those in green strongly similar and those in blue weakly similar. genomes  for matc(DOCX)Click here for additional data file.Analysis S2TPR proteins shown or proposed to be involved in non-flagellum mediated motility. Shown are domain annotations in Interpro and homology models created using CPHmodels 3.2  CPHmodels-3.0 Nucleic Acids Research 38, doi:10.1093/nar/gkq535).(DOCX)Click here for additional data file.