Both tasks will benefit from an understanding of which structural and dynamic features of networks can emerge from evolutionary processes, through which intermediary steps these arise, and whether they embody general design principles. As natural evolution at the level of network dynamics is difficult to study, in silico evolution of network models can provide important insights. However, current tools used for in silico evolution of network dynamics are limited to ad hoc computer simulations and models. Here we introduce BioJazz, an extendable, user-friendly tool for simulating the evolution of dynamic biochemical networks. Unlike previous tools for in silico evolution, BioJazz allows for the evolution of cellular networks with unbounded complexity by combining rule-based modeling with an encoding of networks that is akin to a genome. We show that BioJazz can be used to implement biologically realistic selective pressures and allows exploration of the space of network architectures and dynamics that implement prescribed physiological functions. BioJazz is provided as an open-source tool to facilitate its further development and use. Source code and user manuals are available at: http://oss-lab.github.io/biojazz and http://osslab.lifesci.warwick.ac.uk/BioJazz.aspx.Systems biologists aim to decipher the structure and dynamics of signaling and regulatory networks underpinning cellular responses; synthetic biologists can use this insight to alter existing networks or engineer  Cellular networks allow organisms to sense and process environmental information and thereby implement phenotypic behaviors that enable survival. Hence, it is of fundamental interest to understand their structure and dynamics either by experimental and modeling studies on specific examples –3 or by in silico. This task requires computational tools that are intuitive to use, yet are sufficiently complex to capture the system dynamics of known cellular networks. Modeling of the evolution of cellular networks has so far been attempted either for exploiting evolution as a design tool  or f or fin sol e.g. –20). It . It in sol (e.g. –28 or exol e.g. –34) with within sol  is not bounded in any way . Rule-based modeling is perfectly suited for this evolutionary approach, as it is developed in the first place to overcome the combinatorial complexity arising from accounting for all possible interactions in a given biological system . Her systems ,41 and,  systems ,39,42–44 systems , becauseANC has been described previously . In brieANC implements allosteric regulation by modeling the effect of any binding event or post-translational modification on a given domain through modifying the R-T transition dynamics of that domain. Thus, other molecules binding to a given protein can be seen as ‘modifiers’, which alter the distribution of the R and T states of the domain that they bind. The transition between the R and T states is governed by the free energies of these states as well as any transition state between them (see Supplementary Data). ANC can thereby model a cellular network as a given set of proteins that comprise domains and that interact through binding and through covalent modifications of reactive sites on those domains. Any domain can be allosteric, in which case, it would have distinct R and T states with associated allosteric rate constants. Any modifications would result in altering the dynamics of the R-T transitions. As explained further below, in BioJazz's application of ANC, the in silico, one needs a method to store and mutate the corresponding protein interaction rules and parameters. In BioJazz, we encode the information in an ANC model as a binary string . We can also start evolutionary simulations from entirely random initial points (i.e. a randomly generated binary string). The coding sections of the binary string encode the structure, dynamics and interactions of proteins as explained in detail below and in Table The coding sections of the binary string contain information about the domain structure of proteins Figure . Each prIntra-action fields are binary strings located at the beginning of each domain. They encode the parameters controlling the internal allosteric properties of the domain, namely the basal kinetic rates for the transitions between the R and T states  will affect the R-T transition of that domain, i.e. they encode the parameters mentclass2pt{minimbinding profiles to determine the site-specific interactions among protein domains. In each iteration, BioJazz performs an exclusive-OR (XOR) operation on the binding profiles of two given sites. The number of ‘1's in the string resulting from this operation determines whether or not binding occurs based on a user-defined threshold , the XOR operation is repeated, this time using the forward and backward rate profiles, to determine the binding coefficients  can be altered by the structural changes that the protein undergoes. To include such changes, the model should incorporate the possibility of alterations in the kinetic rates of each reactive site with the R-T state transition of the domain. We do so by implementing a on Table . There aes Table . Note thetc. This flexibility is useful for example to ‘freeze’ all or parts of a network and use BioJazz as a design tool rather than mimicking biological evolution.The use of rule-based modeling and the encoding of such a model in a genome-like binary string allows us to implement most biologically feasible mutations in a natural way. Currently, the possible mutations included in BioJazz are point mutations, protein duplication, protein deletion, domain duplication, domain deletion, domain joining, domain splitting and domain shuffling. Of these, mutations involving domains were to our knowledge not considered before ,31,54, bPoint mutations are implemented as the flipping of specific bits in the binary string. Thus a point mutation can alter any of the qualitative flags (explained above) or reaction parameters. Of particular note are mutations on hard and soft linkers, which can result in domain splitting and fusion respectively. The mutation algorithm parses the binary string and attempts a point mutation at each location: a bit is flipped if a randomly generated number in the interval  is smaller than a user-set probability (corresponding to a genome-wide point mutation rate).In nature, the rate of gene duplication is suggested to be a function of the size of genome . Based oBioinformatics analysis of the genomes of existing organisms reveals duplication patterns of domains in proteins, where the duplication patterns show no dependence on the size of the domains involved . Thus, BBioJazz implements rearrangements between two protein-encoding sections of the binary string. The mutation rate leading to rearrangements is defined per protein. For each protein coding section of the binary string, a random number drawn from a uniform distribution in the interval  is compared to a user-defined probability. If the random number is smaller, a fragment containing a certain number of reactive sites is randomly chosen. Then, another subsection of a protein coding section of the binary string is randomly selected, copied and fused with the first selected fragment. Note that this approach combines sets of intact reactive sites, which can correspond to an entire domain, part of a domain or a sequence that covers multiple domains. Besides mimicking biological domain shuffling, shuffling is expected to create novel material for subsequent evolution.In biological systems, rearrangement of large genome chunks containing multiple genes also happens in certain probability. We also implemented this mutation operator in BioJazz. At each step of mutation, comparison between a random number and the rearrangement rate will determine occurrence of genome rearrangement. With rearrangement occurring, a continuous segment containing multiple reactive sites that possible cross several genes is randomly selected. Then either deletion of segment or duplication of segment is randomly chosen and executed.BioJazz also has implementation of horizontal gene transfer (HGT). Since HGT occurs between different genomes in nature, this mutational operator is only implemented when using population-based selection (see below). At the mutation step of each individual, the occurrence of HGT is determined by comparing a random number to a pre-defined probability (set by the user). If the random number is smaller than this probability, a continuous segment of string containing multiple reactive sites is randomly chosen and copied from the mutating individual. Then, another genome/individual is randomly selected and the copied segment is inserted into its genome at a randomly chosen site that is between any two reactive sites.in silico, we need to model selective pressures experienced by the evolving cellular networks and so link the contribution of a network's function to the overall fitness of the organism. Fitness is an abstract concept, representing the reproductive success of an organism and might be most tractable for microbes where it could be approximated by growth rate  mutant can be fixed in the population before any other mutants can arise or as occurring in a population where multiple mutants can co-exist. The former is an appropriate model for evolutionary dynamics at low mutation rate and large population size limit ,65; the w) and mutant (w’) genotypes by the following equation:Under very low mutation rates and in large populations, evolutionary dynamics can be approximated by a random walk in the genotype space. Then, a single genotype dominates the population and new mutants either get fixed or are lost rapidly under natural selection and/or genetic drift . The prokaryotes ). The coutations . A newlyHere we consider evolution dynamics in discrete generations of an asexual population of a fixed-size ,72. In aThe configuration file.BioJazz contains three key parts that are interlinked to each other: an encoding of an ANC model in the form of a binary string, evolutionary simulation of that binary string through mutations and dynamic simulation of the ANC model and derivation of a fitness score. Many of the parameters governing the structure of these three parts and their inter linkage can be defined by the user, allowing for high customizability. These parameters are stored in a single Besides the parameters already mentioned above, the configuration file also allows the setting of parameters relating to computational performance , string encoding (e.g. fields’ width and binding profiles of input and output), the evolutionary algorithm , the dynamical simulation of the ANC model , the scoring function (discussed below) and the output structure (e.g. frequency of output generation).in silico evolution studies, . ThuTo illustrate the workings of BioJazz and how it can be used to address biological questions, we consider here the evolution of signaling networks under two example selective pressures  Figure . Note thUltrasensitivity is observed in many biological networks and in particular in signaling networks implementing phosphorylation cycles ,11,12,75The fitness function used to score the ability of a given signaling network to generate an ultrasensitive signal-response relationship evaluated the response to a three-step ramp-up and three-step ramp-down signal profile as shown in Figure With this fitness function, we used BioJazz to evolve ultrasensitive signaling networks. We started evolutionary simulations from a minimal seed network composed of a receptor, a kinase, a phosphatase and an output protein Figure . The outSelecting for ultrasensitivity in the signaling network using the random-walk approach , we ran evolutionary simulations by assuming a high population size and low mutation rate regime  and by using different complexity weightings In all simulations, the fitness score increases over generations Figure  and we es Figure , i.e. wh} Figure . On the Equation . Consequ} Figure . A similOur implementation of evolution under a low mutation rate and high population size regime through Equation [5] still allows for a degree of neutral evolution. Thus, we find significant diversity in the set of ultrasensitive networks emerging at the end points of different evolutionary simulations Figure . This diin silico evolution has been successfully used to understand gene network architectures that can achieve adaptive responses , to elucidate the biological origins of ultrasensitivity. Alternatively, one can implement selection for different dynamics such as adaptive response dynamics or oscillatory dynamics. The evolved network structures could then provide insights into which biochemical networks can implement the required dynamics and inform both systems and synthetic biology studies . Networks resulting from specific simulations that implemented different selective pressures displayed specific architectures, suggesting that BioJazz can be used to study the possible repertoire of functional networks. In the case of ultrasensitivity, we found that these networks and their evolutionary dynamics highlighted known biochemical mechanisms. In particular, we found that kinetic parameters controlling binding of the enzymes and output protein evolve to favor low saturation initially for increased response amplitude and then high saturation later on for increased ultrasensitivity. BioJazz can be used to further elucidate such trends under different evolutionary scenarios. For example, the simulations we used started from high substrate and low enzyme concentrations. It would be interesting to reverse this situation and explore how ultrasensitivity can emerge under regimes where high enzyme saturation would not be possible . Similare.g. see ,24,31,32in silico evolution of gene regulatory networks to understand the emergence of different dynamics (in silico evolution model that explicitly considers systems dynamics and protein allostery and domain structure. The incorporation of protein allostery and domain structure is a unique addition to the evolutionary modeling of networks, but whose effects on system dynamics have been demonstrated (in silico evolution is a novel approach to modeling evolution and naturally includes emerging system complexity in evolutionary simulations. In particular, the rule-based modeling approach theoretically allows for simulation of arbitrarily large reaction networks as well as protein complexes.There are notable previous works on evolutionary simulation of the structure and dynamics of cellular networks. In particular, previous studies analyzed the dynamics ,35,73,85dynamics . The latdynamics  and signdynamics ,74,88 ordynamics ,27. As anstrated ,37. In aAlthough by using rule-based modeling BioJazz theoretically allows the evolution of cellular networks without restricting their complexity, there are still computational challenges when simulating large reaction networks and multi-protein complexes that give rise to the ‘curse of dimensionality’ ,40. In pSuch development of the rule-based modeling component of BioJazz would extend its focus from encoding signaling networks to include metabolic and transcriptional networks. In particular, rule-based models like Kappa and BioNetGen are able to model degradation and synthesis reactions. This ability can be combined with the binary string patterns of a BioJazz model to encode binding between proteins and genes, and thereby simulate transcription factors binding to DNA. For metabolic networks, the extension would require encoding of metabolites in a form that captures the basics of chemical conversion ,91. ThisSupplementary Data are available at NAR Online.


Anabaena, contains two GAF domains in tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both the GAFa and the GAFb domains are present in tandem (GAFab domain). In silico docking and mutational analysis identified distinct residues important for interaction with either cAMP or cGMP in the GAFb domain. Structural changes associated with ligand binding to the GAF domains could not be detected by bioluminescence resonance energy transfer (BRET) experiments. However, amide hydrogen-deuterium exchange mass spectrometry (HDXMS) experiments provided insights into the structural basis for cAMP-induced allosteric regulation of the GAF domains, and differences in the changes induced by cAMP and cGMP binding to the GAF domain. Thus, our findings could allow the development of molecules that modulate the allosteric regulation by GAF domains present in pharmacologically relevant proteins.GAF domains are a large family of regulatory domains, and a subset are found associated with enzymes involved in cyclic nucleotide (cNMP) metabolism such as adenylyl cyclases and phosphodiesterases. CyaB2, an adenylyl cyclase from  GMP-specific and -regulated cyclic nucleotide phosphodiesterase, Adenylyl cyclase, and E. coli transcription factor FhlA) are a family of protein domains that regulate the function of a variety of domains with which they are associated  and pPRO-Ex-B plasmid vector , respectively, using BamHI and XhoI sites, resulting in the pGEX-6p-1-GAFb and pPRO-Ex-GAFb plasmid. To generate a BRET-based sensor, the GAFb gene fragment was released and subcloned into the pGFP2-MCS-Rluc plasmid vector  using KpnI and EcoRV sites, resulting in the pGFP2-GAFb-Rluc plasmid. The I308A and T293A mutations in the GAFb domain was introduced using a single primer (The nucleotide sequence of the GAFb domain of CyaB2 from 30-CyaB2 ) using pe primer  and primBamHI and XhoI sites, resulting in the pGEX-6p-1-GAFab plasmid. To generate a BRET-based sensor construct, the tandem GAFab domain gene fragment was subcloned into the pGFP2-MCS-Rluc plasmid vector  using EcoRV and XhoI sites, resulting in the generation of pGFP2-GAFab-Rluc plasmid.The nucleotide sequence of the tandem GAFab domains of CyaB2 spanning residues S77 to L431 was amplified by PCR from the full-length CyaB2 gene cloned into pQE30 plasmid , gene fragment encoding NterGAFab domain was subcloned into the pGFP2-MCS-Rluc plasmid vector  using EcoRV and XhoI sites, resulting in the generation of pGFP2-NterGAFab-Rluc plasmid.The nucleotide sequence encoding residues M1 to L431 was PCR amplified from the full-length CyaB2 gene cloned into pQE30 plasmid (pQE30-CyaB2 E. coli BL21 (DE3) cells were transformed with specific plasmid and cells were induced using 100 µM IPTG at 37 °C for 3 h. Cells were collected by centrifugation and cell pellet was resuspended in lysis buffer containing 50 mM Tris (pH 8.2 at 4 °C), 100 mM NaCl, 10% glycerol, 2 mM PMSF, 1 mM benzamidine. Cells were lysed by sonication and lysate was centrifuged at 30,000 g for 30 min at 4 °C. Supernatant was collected and interacted with pre-equilibrated Glutathione Sepharose 4B beads  at 4 °C for 1 h. Post interaction, beads were washed with buffer containing 50 mM Tris (pH 8.2 at 4 °C), 100 mM NaCl, 0.1% TritonX-100 followed by three washes with buffer containing 50 mM Tris (pH 8.2 at 4 °C), 100 mM NaCl, 10% glycerol. The protein-bound GSH beads were resuspended in buffer containing 25 mM HEPES, 100 mM NaCl and 10% glycerol and stored at 4 °C till further use.To express and purify GST fusion proteins, 6-GAFb protein, E. coli BL21DE3 cyc− cells were transformed with pPRO-Ex-B-GAFb plasmid DNA and induced with 500 µM IPTG for 3 h at 37 °C ]. Cells were lysed by sonication and centrifuged at 17,000 g at 4 °C for 40 min. The supernatant was collected and incubated with Talon resin  at 4 °C for 1 h. The resin was then transferred into columns, and washed with lysis buffer and wash buffer (lysis buffer with 10 mM imidazole) followed by elution buffer containing lysis buffer with 150 mM imidazole. Further purification was achieved by size-exclusion chromatography on a Superdex 200 column using the AKTA FPLC System .To express the Hisat 37 °C . Cells w3H]-cAMP  either in the absence or presence of unlabeled cAMP or cGMP, in a total reaction volume of 50 µL. Reactions were incubated at 37 °C for 1 h and then filtered through GF/C filters , which were then washed with 6 mL of ice-cold washing buffer . The filters were then dried and radioactivity was measured in a liquid scintillation counter.Cyclic nucleotide binding assays were carried out essentially as described earlier  using 1–http://cactus.nci.nih.gov/services/translate/). Charges were added to the atoms and the final structure file for AutoDock was prepared using the Dundee PRODRG2 Server (http://davapc1.bioch.dundee.ac.uk/programs/prodrg/) (Docking was performed using AutoDock (Version 3.0.5)  implemenprodrg/) . Atomic 2 humidified incubator. Transfection was performed with polyethyleneimine lipid according to manufacturers’ protocols. Expression of proteins was monitored by Western Blot analysis using an antibody raised in rabbit against the Rluc protein and described below.Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco’s modified Eagle’s media (DMEM) with 10% fetal calf serum, 120 mg/L penicillin and 270 mg/L streptomycin at 37 °C in a 5% CO6-Rluc protein essentially as described previously  was in Freund’s complete adjuvant and booster dose of immunogen (∼250 µg) was in Freund’s incomplete adjuvant. The presence of antibody was detected by ELISA and Western Blot analysis.Polyclonal antibodies against Rluc protein was raised in rabbits using Hiseviously . Rluc ge2 assay components i.e., acceptor—GFP2, donor—Rluc and Rluc substrate—Coelenterazine 400a. In vitro BRET assays were performed as described previously  was added to a final concentration of 5 µM and emissions were collected for 0.8 s in a Victor3 microplate reader . Emission filters used for Rluc and GFP2 emission were 410 nm (bandpass 80 nm) and 515 nm (bandpass 30 nm), respectively. BRET was calculated as the ratio of GFP emission per second to Rluc emission per second, and the average of three such measurements is reported.All BRET assays were performed using the BRETeviously . HEK 2932-GAFb-Rluc plasmid in 10 cm tissue culture dishes. Forty eight hours post transfection, medium was removed, and monolayers treated with Dulbecco’s phosphate buffered saline containing 5 mM EDTA for 5 min at 37 °C in the incubator following which the EDTA solution was removed, and cells resuspended in phenol-red free DMEM, containing 10% fetal calf serum. Cells (∼105) were then treated with 100 µM of either forskolin (for 5 min) or sodium nitroprusside (for 2 min). BRET was determined as described above.HEK 293T cells were transfected with pGFPIntracellular levels of cyclic nucleotide monophosphates (cNMP) were measured from the cells used for BRET measurements, or parallely transfected and treated cells. Cells were lysed in 0.1 N HCl and cNMP was measured by radioimmunoassay as described earlier .2O (99.90%)  resulting in a final deuterium concentration of 90%. Hydrogen-deuterium exchange was carried out at 20 °C for various time points . The exchange reaction was quenched by adding 40 µl of prechilled quench buffer  to get a final pH read of 2.5. An aliquot (50 µl) of the quenched sample was then injected on to a chilled nanoUPLC sample manager  as previously described . Samples were prepared by adding either 1 mM cAMP or cGMP to apo GAFb domain protein. 2 µL each of apo, cAMP-, or cGMP-bound GAFb domain in 20 mM Tris–HCl (pH 7.5), 100 mM NaCl and 5 mM beta-mercaptoethanol buffer were diluted and incubated with 18 µL of Described . PeptideD method .E data from undeuterated samples using ProteinLynx Global Server 2.4  . Data on each individual peptide at all periods were extracted using this software, and exported to HX-Express   and searts, USA) . The def-Express  for analAll experimental data were analyzed using GraphPad Prism and represent the mean ± S.E.M.DK of 0.8 ± 0.2 µM  reported previously from assays monitoring cAMP-mediated activation of a related adenylyl cyclase domain  and a much reduced affinity for cGMP   . Therefoin silico docking of cGMP on the structure of the GAFb domain. We removed the bound cAMP molecule from the crystal structure of the GAFb domain and used it as the receptor  (We generated a fusion protein containing GFP sensor) . The GAF sensor) . Lysates sensor) . Basal B sensor) , indicat sensor) . To rule sensor) , and int sensor) . Althoug sensor)  and 3E.2 and Rluc in the sensor constructs. However, incubation of lysates prepared from cells expressing either the GAFab or the NterGAFab sensor with cAMP or cGMP (1 mM) did not result in a significant alteration in the BRET  resulted in the generation of multiple peptide fragments across the domain with >90% sequence coverage . Hiscoverage , thus prβ1 and β2 sheets, and β1–β2 loop showed lower dynamics in the presence of cAMP. The peptide (299–309) contains two residues that interact with cAMP, namely Ile 308 and Thr 309. As discussed previously, Ile 308 provides hydrophobic stacking interaction to cAMP while Thr 309 interacts with the N6 of cAMP, forming H-bond through the peptide backbone carbonyl oxygen.A comparison of amide exchange of various peptides in the absence and presence of cAMP showed a decrease in exchange at the central core region comprising the ligand-binding pocket , suggestα2 helix connects the GAFb domain to the GAFa domain, and the C-terminal α5 helix connects the GAFb domain to the catalytic adenylyl cyclase domain of CyaB2. This suggests that the structural changes observed here are signatures of allosteric signal transduction from the GAFb domain to both the GAFa and the adenylyl cyclase domain. The absence of any change in the BRET signal observed with the GAFb sensor construct indicates that the increase in amide exchange following cAMP binding arose from an increase in the entropy of these parts of the GAFb domain, and not as a consequence of a gross change in the relative structure of the protein.Interestingly, cAMP binding resulted in an increased solvent accessibility of peptides arising from the N- and C-terminal helices. The N-terminal Binding of cGMP, on the other hand, resulted in remarkably less changes in the amide exchange of the GAFb domain, and only some regions in the ligand-binding pocket showed an increase in exchange compared to the unliganded protein . A compaMost cyclic nucleotide binding GAF domains are specific in terms of ligand binding. Efforts have been directed towards understanding the mechanism by which these domains achieve specificity. Mutational and biochemical analysis of GAF domains from other proteins have provided some understanding of the mechanism by which these structurally similar domains discriminate nucleotides . We propα4, helix α3 and sheet β3) in the presence of cAMP confirms that the domain ‘closes’ on ligand binding. A similar mechanism has been proposed for the GAFa domain of PDE5 on binding cGMP (The crystal structure of the cAMP-bound GAFb domain showed that cAMP is largely buried, leading to the speculation that the ligand binding pocket is initially present in an open conformation ready to receive the ligand . Reductiing cGMP . InteresIn addition to the mechanism of ligand specificity by a specific residue in the ligand-binding site, comparison of ligand binding to the isolated GAFb domain and the tandem GAF domains revealed a much higher degree of ligand selectivity. This indicates that the isolated GAFb domain and the tandem GAF domains are structurally and biochemically different. Proteins exist in an ensemble of conformations at steady state. The presence of the second GAF domain could influence ligand-binding behavior of the associated GAFb domain by establishing new steady state conformations, allowing concomitant ligand binding specificity, coupled with precise ligand-induced allosteric regulation of these proteins.Our results provide insights on the basis of nucleotide selectivity and proximal conformational changes that occur following cAMP binding to the GAFb domain of CyA2. They may also allow a molecular understanding of the ‘regulated unfolding’ that needs to occur during activation of the C-terminal catalytic domains associated with the GAF domains , and als


In the case of the Cullin3 (Cul3), it forms a BTB-Cul3-Rbx1 (BCR) ubiquitin ligase complex where it is believed that a BTB domain-containing protein performs dual functions where it serves as both the substrate adaptor and the substrate recognition protein.Cullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, such as Skp1 or Elongin B, and a substrate-binding protein with a conserved protein-protein interacting domain, such as Cul3-bound proteins that contain either the LRR or WD40 domain (CLWs). Further biochemical analysis revealed that the LRR domain-containing CLWs could bind both Cul3 and BTB domain-containing proteins. The dual binding role for the LRR domain-containing CLWs results in causing the BTB-domain protein to become a substrate instead of an adaptor.Tandem affinity purification and LC/MS-MS analysis of the BCR complex led to the identification of 10,225 peptides. After the SEQUEST algorithm and CDART program were used for protein identification and domain prediction, we discovered a group of To further distinguish potential substrates from other components that are part of the BCR ubiquitin ligase complex, we altered the parameters in the SEQUEST algorithm to select for peptide fragments with a modified lysine residue. This method not only identifies the potential substrates of the BCR ubiquitin ligase complex, but it also pinpoints the lysine residue in which the post-translational modification occurs. Interestingly, none of the CLWs were identified by this method, supporting our hypothesis that CLWs were not potential substrates but rather additional components of the BCR ubiquitin ligase complex.Our study identified a new set of Cul3-binding proteins known as CLWs via tandem affinity purification and LC/MS-MS analysis. Subsequently, our biochemical analysis revealed that some CLWs modify binding of BTB domain-containing proteins to the complex, causing degradation of the BTB domain-containing protein. As these CLWs were excluded from our list of substrates, we propose that CLWs serve as unique Cul3 binding proteins that provide an alternative regulatory mechanism for the complex. Skp1-Cullin-F-box (SCF) complex that acquires substrate specificity via two proteins: a Skp1 linker protein and a substrate adaptor protein containing an F-box domain[Elongin-Cul2-SOCS-box (ECS) ubiquitin ligase complex with a ubiquitin-like protein Elongin B, a Skp1-like linker protein Elongin C and a substrate adaptor SOCS-box containing protein[BTB-Cul3-Rbx1 (BCR) ubiquitin ligase complex, in which a BTB domain-containing protein serves as both a linker and a substrate adaptor module[Seven cullins have been identified in mammalian cells ; each cullin assembles a unique set of ubiquitin ligase complexes that catalyze the formation of polyubiquitin chains to signal the degradation of target proteins by the ubiquitin-dependent proteolytic pathway[ protein. Conversr module. It is er module.Previous studies have uncovered new substrates and unique components of cullin ubiquitin ligase complexes that suggest new mechanisms for substrate recruitment,10-13. FMultidimensional Protein Identification Technology (MudPIT) mass spectrometry to identify new components of the BCR complex. Several known Cul3-binding proteins and substrates of the BCR ubiquitin ligase complex were identified by this method. In an attempt to rapidly analyze the potential of the BCR ubiquitin ligase complex from this list of proteins, we modified the parameters in the SEQUEST algorithm so that it would recognize the peptide sequence with a lysine residue that is attached to two glycine residues on its side chain, which is indicative of a protein that has been conjugated to ubiquitin or a ubiquitin-like molecule prior to the tryptic digest.Here we performed tandem affinity purification (TAP) followed by Cul3-bound proteins with either Leucine-rich repeats (LRR) or a WD40 domain, which will be referred to as CLWs in this study.In addition to many known Cul3 binding proteins and new substrates, identified by remnants of conjugated ubiquitin, we have identified two classes of In this study, a combination of tandem affinity purification (TAP) and MudPIT mass spectrometry were used to identify new members of the BCR ubiquitin ligase complex. To obtain highly purified Cul3 complexes, an expression cassette encoding a FLAG-TEV-CBP-Cul3 fusion protein was constructed so that the Cul3 complex could be purified using anti-FLAG Sepharose beads and calmodulin-conjugated beads Figure A. The siFive independent MudPIT experiments were performed for the Cul3 fusion protein as well as one actin control. The SEQUEST algorithm was set to match the experimentally obtained mass spectra with theoretical peptides from the human NCBI nr database, revealing a total of 5,964 peptides from the purified actin control Table , top andProteins that corresponded to the identified peptide spectra were subjected to the domain prediction program CDART and categorized based on conserved domains. When coCul3-bound proteins with LRR or WD40 domains (CLWs) to verify their binding to Cul3. The cDNAs were fully sequenced and cloned into mammalian expression vectors in frame with the HA-tag. The proteins were co-expressed with FLAG-tagged Cul3 (FLAG-Cul3). Immunoprecipitation assays revealed that FLAG-Cul3 was able to specifically bind the HA-tagged CLWs with either the LRR ) and two WD40 domain-containing proteins (W14 and W16) were cloned from the list of 32 MudPIT-identified To clarify how other Cul3 binding proteins affect or regulate the binding of LRRs to Cul3, we tested a variety of Cul3 mutants that were unable to bind various Cul3 binding partners. Mutants that could not be neddylated K712R) or could not bind RBX1 (F665D) were able to bind the LRR proteins in an identical fashion to wild type Cul3 (not shown). Surprisingly, mutants that could not bind BTB domain-containing proteins  was co-expressed with MYC-Ctb73 and HA-LRR5. FLAG-beads were used to purify the Cul3 complex, which were subsequently treated with TEV protease to release the protein complex from the beads. The resulting soluble proteins were subjected to a second round of immunoprecipitation with MYC antibody to pull down MYC-Ctb73. The resulting protein complexes were analyzed by Western blot analysis to detect the presence of the third protein, HA-LRR5. Two sequential affinity purifications of FLAG-Cul3 and MYC-Ctb73 pulled down significant amounts HA-LRR5 only in the presence of all three proteins   based on our MudPIT data indicating that the K414 residue on Cul3 contains a ubiquitination site based on the extra mass that is equivalent to two glycine residues Table . Biochem4R based Cul3-bound proteins containing either LRR domains or WD40 domains (CLWs).Since the discovery of Cul3, the characterization of the BCR ubiquitin ligase has progressed rapidly and has resulted in the identification of protein substrates and novel subunits of the BCR ubiquitin ligase complex,26-30. IBased on our chemical analysis, we propose that the LRR domain-containing proteins (LRRs) function to modify the binding of the BTB domain containing protein to Cul3 such that the BTB domain containing protein becomes a substrate. LRRs bind to Cul3 not only via the BTB domain-containing protein, but also through other regions of the Cul3 complex, as the Cul3 mutant that cannot bind BTB domain-containing proteins (Cul3Δ51-67) can still bind LRRs. In addition, these Cul3-bound LRRs were not ubiquitinated, suggesting they are unlikely to be substrates of the BCR ubiquitin ligase complex. This conclusion is further supported by the fact that substrates are unlikely to share a domain (such as an LRR domain) because the mechanism of substrate recognition is usually based on short sequences,32.We discovered that the LRR domain-containing proteins modulated the binding of the BTB domain-containing proteins binding to Cul3 such that they became substrates instead of substrate adaptors. We found that a region on the BTB domain-containing protein just N-terminal to the BTB domain appears to be mediating the interaction with LRR domain-containing proteins. This region on Ctb73 is extremely proline-rich; it is interesting to note that other BTB domain proteins that have been shown to bind Cul3 also have similar proline rich regions in their N-terminus, like actinfilin, BTBD2, and FAZF (PLZF does not have this region and does not bind LRR5). Thus, this region may play a role in a number of other BTB-domain protein/LRR-domain protein interactions. In addition the LRR domain-containing proteins contain a sequence that resembles a B/C box that mediates binding to the BTB domain-containing proteins.Based on the identification of the WD40 domain in substrate adaptor subunits of the Cul4 complex, we first proposed that our MudPIT-identified WD40 domain-containing proteins also behave similarly in the Cul3 complex. However, mutational analysis revealed that the interaction site for the WD40 domain-containing proteins lies within the conserved cullin domain in C-terminal region of Cul3, suggesting that their binding interactions did not resemble those found on the LRRs (not shown). Therefore, the WD40 domain-containing proteins may function in a completely different way than the LRRs.To distinguish novel Cul3-binding proteins from potential substrates of the BCR ubiquitin ligase complex, we adjusted the parameters in the SEQUEST algorithm to recognize the ubiquitin-modified lysine residue in the peptide spectra. Manual validation of the mass spectra revealed that the altered SEQUEST parameters could be used to identify ubiquitinated precursor peptides that are derived from the ubiquitin-conjugated proteins. This technique for the identification of ubiquitin-conjugated proteins using MudPIT mass spectrometry is an approach that can be applied to the analysis of ubiquitinated proteins bound to other ubiquitin ligase complexes. Using this technique, our list of MudPIT-identified Cul3-binding proteins also includes GluR-1, Mei1 and p60/katanin , Skp1 and an F-box containing protein. The F-box containing protein recognizes substrates and recruits them to the E3 complex Figure A. PrevioIn this report, we have identified an additional group of Cul3-binding proteins that contain either LRR domains or WD40 domains. We propose that the B/C-like box domain on the LRR domain-containing proteins and a unique, proline rich domain on the BTB domain containing protein Ctb73 is responsible for the binding between the BTB domain-containing protein, the LRRs and Cul3. However, the binding interaction may be more complex because deletion of the BTB domain resulted in enhanced LRR binding to Cul3. These observations led us to further investigate these interactions. We found that one of these LRR domain-containing proteins, LRR5/FMOD, causes a BTB domain-containing protein, FAZF, to become a substrate for the BCR complex. Possibly, this mechanism is a way for BTB domain proteins to be removed from substrates. There are several know examples of cullin based E3 ligases degrading their substrate adaptors, thus weFull-length cDNA sequences of Cul3, BTBs, and CLWs were cloned from a human testis cDNA library and inserted in-frame into the 3XFLAG-24 (Sigma-Aldrich), CS2 + MT, and CS2 + HA vectors, respectively. All clones were fully sequenced. Mutations in the Cul3 protein were made using the QuikChange site-directed mutagenesis kit (Stratagene). All plasmids were sequenced after mutagenesis. Expression plasmids for MudPIT analysis were constructed by cloning the full-length Cul3 sequence into the 3XFLAGNEO-26 plasmid (Sigma-Aldrich) in-frame with the FLAG tag. A TEV recognition sequence and a calmodulin-binding protein sequence were inserted in-frame between the 3XFLAG epitope and the Cul3 sequence, creating the 3XFLAG-TEV-CBP-Cul3 plasmid that was used for tandem affinity purification. As a control, a 3XFLAG-TEV-CBP-actin plasmid was created by cloning the full-length actin sequence from a human testis cDNA library and inserting it in-frame into the 3XFLAG-TEV-CBP plasmid.HEK293 cell lines were maintained in DMEM media supplemented with 10% fetal bovine serum (FBS). For transient protein expression, HEK293 cells were transfected with either 3XFLAG-TEV-CBP-Cul3 or 3XFLAG-TEV-CBP-actin plasmids as described previously.pGildaLRR5 (FMOD) was screened using the Matchmaker LexA two hybrid system (Clontech). A human testis library was used and 2.7 million clones were screened. The only interacting clone identified was FAZF.Transfected HEK293 cells were harvested in 2.7 mM EDTA in phosphate-buffered saline and cell lysates were prepared by sonication for 10 seconds in radioimmunoprecipitation assay (RIPA) buffer . For immunoprecipitation assays, the clarified lysates were incubated with primary antibody, followed by protein A-Sepharose beads for 1 hr each at 4°C. Beads were spun down and washed twice with RIPA buffer. Protein samples were separated by electrophoresis on 10% polyacrylamide gels, transferred to PVDF membranes, and incubated overnight in primary antibodies. Proteins were visualized using enhanced chemiluminescence followed by digital acquisition using an AlphaInnotech Fluorochem SP system. The following antibodies were used in these experiments: anti-FLAG M2 monoclonal antibody (Sigma-Aldrich), anti-actin polyclonal antibody (Sigma-Aldrich), anti-HA monoclonal antibody (Covance), anti-MYC sc-789 polyclonal antibody (Santa Cruz Biotechnology), and anti-Cul3 antibody which has been previously described. The ant2) to recover the Cul3 complexes that were cleaved but remained bound to the beads. A sample of FLAG beads prior to TEV cleavage and protein samples from each eluted fraction were immunoblotted and probed with the appropriate antibodies to determine the efficiency of TEV cleavage and to estimate the protein yield for the next purification step.Clarified cell lysates were incubated with anti-FLAG M2 agarose slurry (Sigma-Aldrich) at 4°C for 1 hour. The protein-bound beads were washed twice in RIPA buffer and three times in tobacco etch virus (TEV) buffer . The FLAG beads were then incubated with the AcTEV protease enzyme (Life Technologies) in TEV buffer overnight at 4°C with constant agitation. After TEV cleavage was complete, the supernatant containing Cul3 complexes was collected and the FLAG beads were washed three times with TEV protease buffer and twice with calmodulin-binding buffer  for 90 minutes at 4°C. The beads were spun down at 750 × g and washed three times with the calmodulin-binding buffer and twice with the calmodulin-rinsing buffer . The bound protein complexes were then eluted from the calmodulin beads using calmodulin-eluting buffer  and collected in separate fractions. A sample of protein-bound calmodulin beads and protein samples of each eluted fraction were immunoblotted and probed with the appropriate antibodies to estimate the relative protein yield of each eluted fraction. The fractions with the most amount of protein were selected for further analysis using MudPIT techonology.Collected protein samples from the FLAG immunoprecipitation and the TEV protease cleavage were supplemented with 1 M CaClAn equal volume of 8 M urea, pH 8.3, was added to the eluants from the calmodulin bead purification and the mixture was denatured at 95°C for 5 minutes. After the mixture had cooled to room temperature, trypsin (Promega) was added to purified proteins in a 1:40 w/w ratio and the mixture was incubated overnight at 37°C. Once the digestion was complete, the peptide mixture was diluted with 0.1% acetic acid, adjusted to pH 3, and then loaded onto a C18 reverse phase peptide macrotrap cartridge (Michrom Bioresources) that had been equilibrated with 0.1% acetic acid. The cartridge with the bound protein was washed twice with 0.1% acetic acid and the peptides were eluted with 70% acetonitrile in 0.1% acetic acid. The peptide mixture was dried using a SAVANT SC110A SpeedVac centrifuge with an RVT400 refrigerated vapor trap (Thermo Electron Corp.) and stored at -80°C.The dried peptides were reconstituted in 0.1% acetic acid to a concentration of 0.5 mg/ml. They were subsequently loaded onto a strong cation capillary column (home-packed on a pressure bomb) with an inner diameter of 247.0 μm (Polymicro technology), 20 cm in length, and fritted by M-520 (Upchurch) containing polysulfoethyl A™ bulk material of 5 μm diameter and 300 Å pore size . The samples were eluted with a 12-step salt gradient between buffer C  and buffer D  with 0.01 ml/min flow rate for 10 minutes in each gradient step. Each solution step consists of a different ratio of buffer C to buffer D (C/D): step 1 = 100/0, step 2 = 98/2, step 3 = 96/4, step 4 = 94/6, step 5 = 92/8, step 6 = 90/10, step 7 = 88/12, step 8 = 86/14, step 9 = 84/16, step 10 = 80/20, step 11 = 50/50, and step 12 = 0/100. The eluted peptides from each salt gradient step were collected separately, dried and stored at -80°C until the next step.In preparation for analysis by mass spectrometry, the dried peptide samples from each salt step fraction were reconstituted in 0.1% acetic acid. The peptides were loaded onto a pre-column (360 μm outer diameter × 75 μm inner diameter) containing 2 cm of 5 μm Monitor C18 resin (Column Engineering). They were then eluted into the mass spectrometer  through an analytical column  by using a linear HPLC gradient ranging from 0-70% acetonitrile over 30 minutes using buffer A (0.1 M acetic acid) and buffer B (0.1 M acetic acid in acetonitrile) with peak parting. The eluted peptides were then processed by an LTQ-FTICR mass spectrometer (data-dependent scanning with 1 MS scan followed by 5 MS/MS scans). MS/MS spectra were generated and automatically searched against the human NCBI non-redundant protein database using the SEQUEST algorithm provided with Bioworks 3.2 SR (Thermo Electron Corp.). The list of predicted proteins was imported into a custom-made FileMaker Pro relational database for data analysis.MS/MS spectra with mass error of less than 20 ppm and high Xcorr value  were chosen for further analysis. The exact predicted peptide sequences were searched against sequences in the NCBI protein-protein database to uncover potential Cul3-binding proteins. All corThe data sets supporting the results of this article are included within the article .BACK: BTB and C-terminal Kelch; BCR: BTB-Cul3-Rbx1; BPB: β-propeller domain; BTB: Broad-Complex, Tramtrack and Bric a brac; CAND1: Cullin-associated and neddylation-dissociated 1; CBP: Calmodulin-binding protein; CLW: Cul3-bound proteins that contain either the LRR or WD40 domain; Ctb: Cul3-binding protein; DDB1: DNA damage-binding protein1; DWD: DDB1 binding WD40; ECS: Elongin-Cul2-SOCS-box; Gly: Glycine; KLHL5: Kelch-like family member 5; LC/MS-MS: Liquid chromatography-tandem mass spectrometry; LRR: Leucine-rich repeat; Mei1: Meiosis inhibitor 1; MudPIT: Multidimensional Protein Identification Technology; Nedd8: Neural precursor cell expressed; Rbx1: RING-box protein 1; Rpn: Ribophorin; SCF: Skp1-Cullin-F-box; Skp1: S-phase kinase-associated protein 1; Skp2: S-phase kinase-associated protein 2, SOCS, suppressors of cytokine signaling; STIP1: Stress-induced-phosphoprotein 1; TAP: Tandem affinity purification, TEV, Tobacco etch virus.The authors declare that they have no competing interests.WW did the cloning, expressed the Cul3 complexes, performed tandem affinity purification and immunoprecipitation  or with MYC-tagged Ctb62 (first lane) or with MYC-tagged Ctb62 deleted for its BTB domain. Lower gel shows an immunoblot of levels of expression of LRR3 and the upper blot shows binding of LRR3 to Ctb62 by immunoprecipitating Ctb62 followed by an immunoblot for LRR3. Figure S2. Cul3 is ubiquitinated at the lysine 414 residue. HEK293 cells were transfected with vectors expressing wild-type Cul3, Cul3K414R mutant, and HA-tagged ubiquitin. Lysates were prepared, checked for protein expression (bottom), and immunoprecipitated with anti-HA antibody. The precipitates were separated by SDS-PAGE and analyzed by immunoblot for Cul3.Summary of conserved domains of potential Cul3 and actin-binding proteins. Click here for fileSummary of proteins that contain conserved domains of interest from MudPIT-identified Cul3-binding proteins.Click here for file


Low complexity protein sequences are often intrinsically unstructured and many have the potential to polymerize into amyloid aggregates including filaments and hydrogels. RNA-binding proteins are unusually enriched in such sequences raising the question as to what function these domains serve in RNA metabolism. One such yeast protein, Nab3, is an 802 amino acid termination factor that contains an RNA recognition motif and a glutamine/proline rich domain adjacent to a region with structural similarity to a human hnRNP. A portion of the C-terminal glutamine/proline-rich domain assembles into filaments that organize into a hydrogel. Here we analyze the determinants of filament formation of the isolated low complexity domain as well as examine the polymerization properties of full-length Nab3. We found that the C-terminal region with structural homology to hnRNP-C is not required for assembly, nor is an adjacent stretch of 16 glutamines. However, reducing the overall glutamine composition of this 134-amino acid segment from 32% to 14% destroys its polymerization ability. Importantly, full-length wildtype Nab3 also formed filaments with a characteristic cross-β structure which was dependent upon the glutamine/proline-rich region. When full length Nab3 with reduced glutamine content in its low complexity domain was exchanged for wildtype Nab3, cells were not viable. This suggests that polymerization of Nab3 is normally required for its function. In an extension of this idea, we show that the low complexity domain of another yeast termination factor, Pcf11, polymerizes into amyloid fibers and a hydrogel. These findings suggest that, like many other RNA binding proteins, termination factors share a common biophysical trait that may be important for their function. This initial survey has been seminal in the study of LCDs. We recently showed that LCDs from the transcription termination factors Nrd1 and Nab3 which were predicted to be prion-like, indeed form amyloid-like filamentous polymers in vitro [The study of low complexity protein domains (LCDs), particularly those that are considered intrinsically disordered, has gained focus recently because of the unusually large number of proteins that contain them, and their association with prion-like structural properties , 2. RNA in vitro . In vivoin vitro .One mechanism for terminating transcription by RNA polymerase II (pol II) employs the hnRNP-like proteins Nab3 and Nrd1 and the helicase-like protein, Sen1 . The Nabin vivo to form higher order structural assemblies. Nab3 contains a lengthy prion-like domain that is glutamine and proline rich over 235 amino acids, including a tract of 16 glutamines. The domain ends with a C-terminal region with structural homology to an α-helix found in human hnRNP-C that is known to self-associate into bundles [Recently, it has been shown that Nab3 can employ its LCD to detoxify a derivative of the polyglutamine-containing huntingtin protein in living yeast , 21. Thi bundles . Mutatio bundles . Genetic bundles , 12. Rec bundles –27. Henc bundles . We now E. coli and expressed by autoinduction as described [Full-length Nab3 expressed from a modified pET41a plasmid  was transformed into BL21(DE3) escribed . Cells wThe sequence of Nab3 encoding its C-terminal domain was deleted from pDL469 using Phusion DNA polymerase (NEB) and the oligonucleotides 5’-aggtggttgaggaggcggacc-3’ and 5’-gaaaacctgtattttcagggacaccacc-3’. This plasmid (pET41-Nab3Δ134) was transformed into BL21(DE3) cells and this truncated version of Nab3 was expressed and purified as described above for full-length Nab3.134) protein were expressed from plasmids pET32a-Nab3-134, pET32-Nab3134ΔQ16, pET32-Nab3134Δα, and pET32-134QE. pET32a-Nab3-134 has been described [BglII and XhoI sites of pET32a. pET32-Nab3134ΔQ16, was made by deleting the DNA encoding the stretch of sixteen Qs from pET32a-Nab3-134 using Phusion DNA polymerase and the oligonucleotides 5’-cctgctggcaataatgttcaaagtctatta-3’ and 5’-aggtggcggaggttggtgtgac-3’ in mutagenic PCR. pET32-Nab3134Δα was similarly made by deleting the last 18 amino acids of Nab3 from pET32a-Nab3-134 using the oligonucleotides 5’-aggttgctgttgctgttgctgttgctg-3’ and 5’-tgactcgagcaccaccaccaccac-3’. pET32-134QE was created by synthesis  of the portion of Nab3 shown in EcoRV site of pUC57. Synthesis included BglII and XhoI sites at the 5’ and 3’ ends respectively which were used to excise the fragment for insertion into similarly digested pET32a. All plasmids were transformed into E. coli, which were induced with IPTG, lysed with lysozyme, sonicated, and the lysate was chromatographed over HisTrap media as described above. Pooled fractions were digested with thrombin, washed back into lysis buffer, and re-chromatographed on HisTrap columns as described above. The resulting proteins were concentrated by centrifugal filtration and left undisturbed at 4°C or 22°C.Derivatives of the Nab3 low complexity region  and inserted in place of the corresponding wildtype fragment in pRS315-Nab3 to create pRS315-Nab324Q>E. Control plasmids containing 11 or 14 glutamine to glutamate substitutions in this region were made through multiple rounds of site-directed mutagenesis using mutagenic oligonucleotides to generate the plasmids pRS315-Nab3-11QE and pRS315-Nab3-14QE. The plasmid pRS315-PCF11 [et al., [PCF11-ΔPrLD.Plasmids for introduction into yeast started with pRS315-Nab3 . DNA encitutions  encompas15-PCF11  was usedNAB3 and a wildtype copy of NAB3 on a URA3-marked plasmid, was used as a starting strain for plasmid shuffling. It was transformed with the LEU2-marked plasmids pRS315-Nab3-11QE, pRS315-Nab3-14QE, or pRS315-Nab3 to yield strains with two plasmids. Counterselection against the URA3 plasmid on 5-fluoroortic acid (FOA)-containing medium yielded the strains DY368, DY384, and DY357, respectively. DY382 was made by transforming DY3111 with pRS315-Nab324Q>E. For PCF11 shuffles, H-314 (courtesy of Dr. M. Hampsey) was used as a starting strain. pRS315-PCF11 or pRS315-PCF11-ΔPrLD were transformed into H-314 to generate DY361 and DY362, respectively. Counterselection against the URA3-marked plasmid following growth in the presence of FOA led to strains DY363 and DY364, respectively.Strains used in this study are listed in Purified protein samples were adjusted to 2% SDS, 140 mM β-mercaptoethanol, 10% glycerol, 0.002% bromphenol blue, 80 mM Tris, pH 6.8 and separated by electrophoresis in agarose gels (1.5% w/v) in 40 mM Tris-acetate, pH 7.8; 1 mM EDTA, 0.1% SDS run at 4°C. Bio-Rad Precision Plus Protein Kaleidoscope (Cat. No. 161–0375) molecular weight markers were run as standards. Proteins were blotted to Protran nitrocellulose transfer membrane (Whatman) by capillary action for 18 hrs. Filters were blocked in 5% (w/v) nonfat dry milk in Tris-buffered saline with 0.1% Tween-20  and probFive μl of sample suspension were placed on a 400-mesh carbon coated copper grid that had been made hydrophilic by glow discharge. After 5 minutes, the grid was rinsed by briefly touching the sample side to a drop of distilled water. The residual water was then removed by blotting to filter paper. For negative staining, 5μl 1% aqueous phosphotungstic acid (pH 6.5) was applied to the grid immediately after water removal, and excess liquid was removed by blotting after 30 seconds. The grid was air dried before viewing on a JEOL  IEM-1400 transmission electron microscope equipped with a Gatan (Pleasanton CA) 2k x 2k US1000 CCD camera. While filaments and less well-defined amorphous material were both apparent, the bulk of the negatively stained material resided in filaments. Representative images were captured digitally as.tif files.Assays were carried out as described by LeVine . ThioflaSaccharomyces cerevisiae Nab3 LCD  formed amyloid polymers and an ordered hydrogel [134 with a specific set of mutations. Because we identified mutations in Nab3 that impact its termination activity by deleting all or part of 16 consecutive glutamines in the LCD [We previously showed that a recombinant version of the hydrogel , 12. Thi the LCD , 23, we  the LCD .134 is 32% glutamine, and it resembles prion-like domains of yeast that polymerize into filaments by stacking their β-sheet-forming, low complexity domains perpendicular to the direction of filament polymerization [E. coli. The introduction of charged glutamic acid residues resulted in an anomalously slow mobility of the polypeptide on SDS-PAGE relative to the wildtype LCD  and analyzed by Western blotting with an anti-Nab3 antibody. We found that the pelletable fibers were indeed formed from full-length protein  to separate monomeric protein from high molecular weight polymers . As show24Q>E was ineffective in supporting cell survival. In fact, when cells with both plasmids (one containing wildtype NAB3 marked with URA3 and another with Nab324Q>E marked with LEU2) were grown on media selective only for the latter (leucine-dropout media), both were retained because, even though there was no selection for URA3, wildtype NAB3 had to be retained to cover for the inactive nab324Q>E allele.If assembly of Nab3 through its LCD is important for its function, the 24 glutamine to glutamate substitutions in the LCD that impair filament and hydrogel formation should impact cell physiology. To test this, we introduced this low-glutamine version of the LCD into full length Nab3 expressed from its own promoter and tested if it could support cell viability by plasmid shuffling. This test revealed that cells are not viable with only the mutant form of Nab3 . To insuE. coli formed filaments that could be observed by electron microscopy (134 (LEU2-marked plasmid was transformed into yeast cells harboring a URA3-marked plasmid encoding wildtype Pcf11. Cells that lost the latter plasmid were selected on FOA-containing medium and were able to grow well when the only copy of PCF11 was the one lacking its LCD (A number of yeast transcription termination factors have a low complexity domain that appears prion-like when analyzed by a Hidden Markov Model algorithm trained on well-characterized prions . Since, croscopy . Polymercroscopy . The filcroscopy  and theyopy (134 . Deletio its LCD . Thus, PS. cerevisiae Nab3 is aligned with that from five other Saccharomyces species (in vivo [A growing body of evidence indicates that RNA binding proteins often possess low complexity, unstructured regions that can form amyloid polymers , 3, 33.  species . While t species , is amon species . This po(in vivo .in vitro. This domain is contained in the termination factor Pcf11, thereby extending the trend for termination complexes to contain subunits with this ability. Pcf11 co-exists in the CFI polyadenylation complex with Hrp1, whose prion-like domain scores very strongly in the study of Alberti et al., including the ability to form filaments in vitro [in vitro assembly had not been detected [E. coli. In any case, our findings suggest that under certain conditions there may be additional members of the computationally-derived set of predicted yeast prion-like domains identified by Alberti et al. that indeed polymerize.We also show that the Nab3 LCD, which independently forms amyloid polymers, is responsible for filament formation of the full-length protein with its RNA-binding domain. The finding that full length Nab3 forms filaments is an important extension of the initial computational analysis identifying LCDs in the yeast proteome that have assembly potential . In thisin vitro . Combinein vitro , and thein vitro , LCD poldetected . One pos


This paper presents the crystal structure of the MID domain of a Piwi Argonaute protein. Docking experiments show that this domain specifies recognition of the 5′ uridine of piRNAs. Domain-swapping experiments reveal an unexpected role for the MID domain indicating strand orientation of its bound piRNA. Piwi-interacting RNAs (piRNAs) guide Piwi Argonautes to suppress transposon activity in animal gonads. Known piRNA populations are extremely complex, with millions of individual sequences present in a single organism. Despite this complexity, specific Piwi proteins incorporate piRNAs with distinct nucleotide- and transposon strand-biases (antisense or sense) of unknown origin. Here, we examined the contribution of structural domains in Piwi proteins toward defining these biases. We report the first crystal structure of the MID domain from a Piwi Argonaute and use docking experiments to show its ability to specify recognition of 5′ uridine (1U-bias) of piRNAs. Mutational analyses reveal the importance of 5′ end-recognition within the MID domain for piRNA biogenesis in vivo. Finally, domain-swapping experiments uncover an unexpected role for the MID-PIWI module of a Piwi protein in dictating the transposon strand-orientation of its bound piRNAs. Our work identifies structural features that allow distinguishing individual Piwi members during piRNA biogenesis. Argonaute proteins are expressed in most organisms ranging from bacteria to animals and plants . They arBiogenesis of piRNAs is only beginning to be understood, but analysis of factor requirements and precursor RNA features points to the process being mechanistically distinct from that of other small RNA classes. Single-stranded, often spliced, kilobases-long RNAs transcribed from discrete genomic loci called piRNA clusters are major sources for piRNAs . These aDrosophila proteins Aub and Piwi (Drosophila Ago3 do not show 1U-bias. One source of this bias could be the specificity of nuclease(s) generating the 5′ end of primary piRNAs. In this context, the Zucchini endonuclease acting in an unknown step during primary processing lacks any such specificity in vitro (Bombyx mori (silkworm) ovary-derived BmN4 cells that participate in secondary processing via the Ping-pong cycle , and PIWI domains. Numerous crystal structures of archaeal, bacterial, and eukaryotic AGO proteins in complex with nucleic acids have indicated recognition of the 5′ monophosphorylated end within a conserved basic pocket formed by the MID domain, while the 3′ hydroxyl end is anchored in the PAZ domain . The cry A, or C . This prStructural analysis of Piwi proteins is still in its infancy, with the PAZ domain being the only one characterized to date. The solution structure of mouse MIWI  and crysNeurospora crassa QDE-2 MID-PIWI and full-length eukaryotic Argonaute structures  in the MIWI MID structure by docking analysis. In hAgo2, a proline-flanked rigid specificity loop A is showBombyx mori BmN4 cell culture model. BmN4 expresses two Piwi proteins, Siwi (with 1U-bias) and Ago3 (without the bias), which we used for mutational analyses to probe the importance of the specificity loop and 5′ phosphate recognition within the conserved MID domain . Unfortunately, these were not loaded with piRNAs in vivo. So, to avoid drastic changes to the protein structure, we focused on a single asparagine (N) in the specificity loop of Siwi for mutagenesis (Arabidopsis AGO clade member AGO1 (Bombyx Siwi was mutated by converting the asparagine to a glutamine (N602Q). After expression in BmN4 cells, immunoprecipitation and 5′-end labeling revealed unaffected levels of bound small RNAs  to abolish stacking or with phenylalanine (F) to maintain it, although both mutations are predicted to destroy hydrogen bonding with the 5′ phosphate of the bound small RNA  (Bombyx Siwi with glutamate (Y607E) or extension of the C-terminal end by a single alanine (A) residue (C-term +A), which presumably affects the C terminus's contribution to 5′ phosphate recognition within the MID domain  C and traor Ago3) D of piRND domain , were foD domain . MutatioD domain . Taken tBombyx Siwi and Ago3 expressed in BmN4 cells , compared to that of Siwi (85%) and Ago3 (40%) F. This iBombyx Siwi and Ago3). Furthermore, the N termini of Piwi proteins are decorated with a variable number of arginine residues that are targets for symmetrical dimethylation by PRMT5  was cloned into pProExHtb vector (Invitrogen). HA-tagged escribed . Point mescribed A that weescribed .6-tagged MIWI MID domain was expressed in Escherichia coli BL21 Rosetta cells. Soluble protein was purified by a Ni2+-affinity chromatography using chelating sepharose beads . Tag removal was performed by proteolytic digestion using Tobacco Etch Virus (TEV) protease and a subsequent Ni2+-affinity step. The protein was further purified by size-exclusion chromatography in 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM dithiothreitol (DTT), performed on a Superdex 75 10/300 GL column . The selenomethionine derivative (SeMet) of the MIWI MID domain (S480–E616) was expressed in E. coli B834 (DE3) cells grown in minimal M9 medium supplemented with 50 mg/mL L-SeMet (Sigma) and induced with 1 mM IPTG (EMBL Protein Expression and Purification Core facility). Purification was the same as the wild-type protein.The His2, and 26%–32% polyethylene glycol 4000. Crystals appeared after 3 d and were flash-frozen at 100K after transferring them to identical crystallization conditions containing 10% glycerol. The crystals were orthorhombic, space group P212121, contained four molecules in the asymmetric unit, and the best one diffracted to 2.3 Å. A highly redundant 2.8-Å SeMet anomalous data set was collected at the peak of the SeMet signal, as measured by X-ray fluorescence for experimental phasing. All X-ray data were collected on beamline ID14-4  centered on Y569. Surface conservation representation was based on sequence alignment of MID domains from the following: mouse Piwi proteins  and Bombyx Piwi proteins (Siwi and Ago3), and human Ago2.AutoDock Tools  was usedBombyx mori ovarian cell line BmN4 for piRNA studies is described  0.5% Triton X-100, 50 μg mL−1 tRNA, and 1× vanadyl ribonucleoside complex [Sigma]). Immunoprecipitation of HA-tagged wild-type and mutant Piwi proteins was performed with HA-affinity beads (Roche). After multiple washes in IP buffer , immunoprecipitated samples were processed for Western blotting and extraction of associated RNAs. Bound RNAs were revealed by 5′-end labeling and 15% urea-PAGE. Western analyses were performed with mouse anti-HA antibody (kind gift of Marc Bühler) and anti-actin (Santa Cruz), both at a 1:200 dilution.BmN4 cells were lysed in buffer  according to the manufacturer's instructions. Libraries were sequenced on an Illumina HiSeq platform (EMBL Heidelberg Gene Core facility). Small RNA analyses were as previously reported .BmN4 cells were grown on cover glasses and fixed with a 4% paraforamaldehyde solution (Sigma). Endogenous and HA-tagged Piwi proteins were detected using primary antibodies at a 1:200 dilution, while secondary antibodies coupled to Alexa 488, 594, and 647 (Invitrogen) were used for visualization at a 1:250 dilution. Antibodies used were the following: anti-Ago3 and anti-Siwi polyclonal antibodies , and mouThe crystallographic coordinates for the MIWI MID domain are deposited with the Protein Data Bank (PDB) under the accession code 4P1Z. Deep-sequencing data sets used in this study are deposited with Gene Expression Omnibus (GEO) under the accession number GSE55451.


Plasmodium falciparum are not well studied. Identification of PfCENP-C, an inner kinetochore protein (the homologue of human CENP-C) and its co-localization with PfCENH3 was recently reported. This study aims to decipher the functions of inner kinetochore protein, PfCENP-C as a centromere protein in P. falciparum.The conserved centromere-associated proteins, CENH3 (or CENP-A) and CENP-C are indispensable for the functional centromere-kinetochore assembly, chromosome segregation, cell cycle progression, and viability. The presence and functions of centromere proteins in PfCENP-C, and the functions of PfCENP-C domains were demonstrated by functional complementation assays in the temperature sensitive (TS) mutant strains (mif2-3 and mif2-2) of Saccharomyces cerevisiae with MIF2p (the yeast homologue of CENP-C) loss-of-function. By site-directed mutagenesis, the key residues essential for PfCENP-C functions were determined. The chromatin immunoprecipitation was carried out to determine the in vivo binding of PfCENP-C to the Plasmodium centromeres and the in vivo interactions of PfCENP-C with PfCENH3, and mitotic spindles were shown by co-immunopreciptation experiments.Bio-informatic tools were employed to demarcate the two conserved domains of PfCENP-C is able to functionally complement MIF2p functions. The essential role of some of the key residues: F1993, F1996 and Y2069 within the PfCENP-C dimerization domain in mediating its functions and maintenance of mitotic spindle integrity is evident from this study. The pull-down assays show the association of PfCENP-C with PfCENH3 and mitotic spindles. The ChIP-PCR experiments confirm PfCENP-C-enriched Plasmodium centromeres. These studies thus provide an insight into the roles of this inner kinetochore protein and establish that the centromere proteins are evolutionary conserved in the parasite.The studies demonstrate that the motif and the dimerization domain of PfCENP-C is a true CENP-C homologue in P. falciparum which binds to the centromeric DNA and its dimerization domain is essential for its in vivo functions as a centromere protein. The identification and functional characterization of the P. falciparum centromeric proteins will provide mechanistic insights into some of the mitotic events that occur during the chromosome segregation in human malaria parasite, P. falciparum.The online version of this article (doi:10.1186/1475-2875-13-475) contains supplementary material, which is available to authorized users. A functional kinetochore assembly depends on the recruitment of the CENP-C to the CENP-A-containing centromeric nucleosomes and the subsequent CENP-C-dependent recruitment of a subset of inner and outer kinetochore proteins on the centromere  in the Plasmodium database [PfCENP-C, MIF2p and CENP-C shows the maximum sequence homology in the conserved regions of the CENP-C motif and the C-terminal dimerization domain  displays 30 and 63% amino acid sequence similarities with S. cerevisiae and human CENP-C motif, respectively  shares 50 and 46% amino acid sequence similarities with S. cerevisiae and human dimerization domains, respectively  stages of the parasite, Western blot analysis was performed using anti-PfCENP-C antibodies on the parasite lysates prepared from the tightly synchronized ring, early trophozoite, late trophozoite and schizont stages  is detected at all the stages but the expression is down regulated during the early trophozoite stage as compared to ring, late trophozoite and schizont stages, suggesting its differential constitutive expression at all the developmental stages of the IE cycle. The equal protein loading was confirmed by anti-GAPDH antibodies , PfCENP-C motif, PfCENP-C dimerization domain (DD), MIF2p, and an empty vector alone were performed at permissive (23°C) and non-permissive (37°C) temperatures  are expressed in the TS mutants at both the permissive as well as non-permissive temperatures  and the vector control show aberrant large bud morphology with dispersed and/or fragmented nuclei along with unviable cells. Similarly, at 37°C, both the TS mutants expressing the N-terminal region (NTD) of PfCENP-C show predominantly large bud morphology with fragmented nuclear mass along the mother-bud neck and a subset of unviable cells lacking detectable nuclear staining. These abnormal phenotypes reflect impaired chromosome segregation on MIF2p loss-of-function. At 37°C, the mif2-3 TS mutants expressing the PfCENP-C motif show normal bud morphology but with chromosome mis-segregation  show normal bud morphology with proper chromosome segregation apparent by the intracellular location of the nucleus that is equally segregated between the bud and the mother cell. The TS mutants expressing budding yeast MIF2p also show restoration of the normal phenotype at 37°C. The ability of PfCENP-C motif in mif2-2 TS mutants and the PfCENP-C dimerization domain in mif2-2 and mif2-3 TS mutants to rescue the mitotically arrested cells and overcome the chromosome segregation defects demonstrates their role in mediating accurate chromosome segregation. Collectively, these results suggest that the putative PfCENP-C is a true CENP-C homologue.In regation . The larregation . To dete Figures C, i-iii.in vivo kinetochore assembly [PfCENP-C dimerization domain, point mutations in some of the conserved amino acid residues of this domain were incorporated. In budding yeast MIF2p, the residues Y451, F452 and F523 within the dimerization domain are shown to be essential for its dimerization ability upon which mutation results in arrested growth while the mutation of another conserved residue T488 which is present outside the dimer interface is dispensable for growth [P. falciparum and the budding yeast shows that the amino acid residues F1993, F1996 and Y2069 of PfCENP-C dimerization domain corresponds to the residues Y451, F452 and F523 of MIF2p dimerization domain, respectively . At the non-permissive temperature , the mif2-3 TS mutant is very well characterized. The mif2-3 TS mutant has a point mutation, P505L in the MIF2p dimerization domain where proline at position 505 is substituted by leucine [P505L mutation confers the aberrant TS phenotypes to the mif2-3 TS mutants and renders the dimerization domain non-functional [P. falciparum CENP-C at the analogous position  could rescue the abnormal spindle morphology which is evident by the presence of disintegrated spindles in the mif2-3 and mif2-2 TS cells expressing all the above mentioned PfCENP-C dimerization domain mutants. The mif2-3 and mif2-2 TS mutants expressing PfCENP-CN2060A show intact normal spindles confirming its dispensable role in chromosome segregation. The loss of spindle integrity in the absence of dimerization domain is consistent with a defective centromere protein. These results confirm the essential role of the PfCENP-C dimerization domain in maintaining the mitotic spindles integrity during chromosome segregation.In spindles , 21. To PfCENP-C on centromeric regions in vivo, ChIP assays were performed as previously described [PfCENP-C and PfCENH3 antibodies  [PfCENH3, PfCENP-C is also enriched on all the six P. falciparum centromeric regions analysed. However, at the non-centromeric sites, upstream as well as downstream of each centromere, very weak or no enrichment of PfCENH3 and PfCENP-C is observed. The in vivo enrichment of PfCENP-C on the centromeric regions confirms its functions as a centromere protein in P. falciparum. Like PfCENH3, the results also establish PfCENP-C as the centromere binding protein in the parasite.To determine the enrichment of escribed , 50 withs Figure A. As thetromeres , ChIP wi and 12)  were carPfCENH3 and PfCENP-C on the centromeres and their co-localization throughout the cell cycle [in vivo interactions of PfCENH3with PfCENP-C by co-immunoprecipitation (co-IP) assay. The pull-down of PfCENH3 using anti-PfCENP-C antibodies and vice-versa were performed. The presence of PfCENH3 and PfCENP-C in the pull-down immunocomplexes was confirmed by the Western blot analyses  may be due to the failure of PfCENP-C to recruit and interact with the proteins that generate the stable spindle tension during the sister chromatids separation. This imbalance in the tension thus results in the abrupt breakage of the mitotic spindles. Alternatively, the spindles may fall apart due to the impaired spindle elongation during anaphase, resulting in structurally defective spindles. However, the spindle defects in the TS mutants are overcome by the PfCENP-C dimerization domain demonstrating its role in the maintenance of the structure of mitotic spindles. This also supports the idea that the PfCENP-C dimerization domain plays an important role in the kinetochore assembly at the centromeres and establishes connection with the mitotic spindles to ensure accurate chromosome segregation. The enrichment of PfCENP-C on the centromeric regions of P. falciparum as compared to the non-centromeric regions and its interaction with PfCENH3 and microtubules further strengthens its centromeric function in the malaria parasite.It has been proposed that the loss of spindle integrity in the MIF2p-deficient cells is due to the defective spindle elongation during the anaphase but not the failure in spindle formation . CENP-C PfCENP-C domains especially the dimerization domain and its role in formation of a functional centromere-kinetochore structure.These findings thus offer insights into the structural-functional details of various PfCENP-C, the PfCENP-C motif and the PfCENP-C dimerization domain have been mapped by bio-informatic analysis. The functional complementation assays establish the putative PfCENP-C as a true CENP-C homologue involved in chromosome segregation, stable mitotic spindle morphology and cell viability. The key residues within the PfCENP-C dimerization domain essential for its in vivo functions as a centromere protein have also been identified. The binding of PfCENP-C to the P. falciparum centromeres and its in vivo interactions with PfCENH3 and mitotic spindles further strengthens its centromere-associated functions.The two conserved domains of Functional complementation ofSaccharomyces cerevisiaeMIF2p (A and B). Complementation and ten-fold serial dilution assays with mif2-2 TS mutant expressing various PfCENP-C domains: -NTD, -motif and -DD, MIF2p and vector control at 23°C. (C) (i) The confocal images show the normal phenotypes of mif2-2 TS mutants carrying various PfCENP-C domains, MIF2p, vector control and the parent strain alone at 23°C. The nucleus (blue) is stained with Hoechst. (ii and iii) The graphs show the percentage of normal phenotype (y-axis) exhibited by mif2-3 and mif2-2 TS mutant cells expressing various PfCENP-C domains, MIF2p, vector control and the parent TS mutant strain (x-axis) at 37°C. The normal phenotypes were scored for the cells showing both the normal bud morphology as well as proper chromosome segregation. Number of cells counted = 100. NTD- N-terminal region, DD-dimerization domain. (PDF 140 KB)Additional file 1: Functionally essential residues within thePfCENP-C dimerization domain. (A and B) Growth and ten-fold serial dilution assays with the mif2-2 TS mutants expressing the PfCENP-C-DD mutants, PfCENP-CF1993A, PfCENP-CF1996A, PfCENP-CY2069A and PfCENP-CN2060A at 23°C. All the constructs show normal growth at permissive temperature. (C) The phenotypes of mif2-2 TS mutants expressing PfCENP-CF1993A, PfCENP-CF1996A, PfCENP-CY2069A and PfCENP-CN2060A at 23°C. The Hoechst stained nucleus (blue) shows proper chromosome segregation in all the constructs. (PDF 86 KB)Additional file 2: The mitotic spindle integrity is maintained by the dimerization domain. (A) Confocal images showing the mitotic spindle (green) and the Hoechst stained nucleus (blue) of the mif2-2 TS mutant cells expressing the PfCENP-C domains: -NTD, -motif and -DD, MIF2p and empty vector alone at 23°C. (B) The confocal images represent the mitotic spindles of mif2-2 TS mutants expressing PfCENP-C-DD mutants, PfCENP-CF1993A, PfCENP-CF1996A, PfCENP-CY2069A and PfCENP-CN2060A at 23°C. At permissive temperature, normal spindle morphology is observed by all the constructs expressed by mif2-2 TS mutants. The insets show the mitotic spindle structures. (PDF 218 KB)Additional file 3:


Biosecurity measures are important tools to maintain animal health in pig herds. Within the MINAPIG project, whose overall aim is to evaluate strategies to raise pigs with less antimicrobial use, biosecurity was evaluated in medium to large farrow-to-finish pig herds in Sweden. In 60 farrow-to-finish herds with more than 100 sows, the biosecurity level was evaluated using a previously developed protocol (BioCheck). In a detailed questionnaire, internal and external biosecurity was scored in six subcategories each. An overall score for biosecurity was also provided. Information regarding production parameters as well as gender and educational level of personnel working with the pigs was also collected. Descriptive statistics were used to examine the recorded data.The median scores for external and internal biosecurity were 68 and 59, respectively, where 0 indicates total absence of biosecurity and 100 means maximal possible biosecurity. The subcategories for external and internal biosecurity that had the highest scores were “Purchase of animals”  and “Nursery unit”/“Fattening unit” , while “Feed, water and equipment supplies”  and “Measures between compartments and equipment”/“Cleaning and disinfection”  received the lowest scores. A female caretaker in the farrowing unit, a farmer with fewer years of experience and more educated personnel were positively associated with higher scores for some of the external and internal subcategories. In herds with <190 sows, fattening pigs were mixed between batches significantly more often than in larger herds.The herds in this study had a high level of external biosecurity, as well as good internal biosecurity. Strong biosecurity related to the purchase of animals, protocols for visitors, the use of all-in, all-out systems, and sanitary period between batches. Still, there is room for improvement in preventing both the introduction of disease to herds  and the spread of infections within herds . Systems for animal transport can be improved and with respect to internal biosecurity, there is especially room for improvement regarding hygiene measures in and between compartments, as well as the staff’s working procedures between different groups of pigs.The online version of this article (doi:10.1186/s13028-015-0103-5) contains supplementary material, which is available to authorized users. In pig herds, biosecurity is an important aspect of preventing the transmission of diseases, thus improving health and reducing the need for antimicrobials (AMs) . ExternaSweden, situated in Scandinavia in northern Europe, produces around 2.5 million slaughter pigs yearly, corresponding to 1% of EU production . Most ofwww.minapig.eu), an EU project with the overall aim to evaluate strategies to raise pigs with less AM use, a cross-sectional study was conducted to assess the level of biosecurity in farrow-to-finish herds in four European countries with different levels of AM use. The aim of this study was to describe biosecurity and health management practices in Swedish farrow-to-finish herds using an established scoring system, based on interviews with the farmers.Within MINAPIG , i.e. herds in which veterinarians from the SvDHV served as herd veterinarians, or herds with previous contact with researchers at the National Veterinary Institute, in Uppsala. In total, a convenience sample of 60 herds was recruited from across Sweden. Recruited herds were visited once during the period April to September 2013, either by the herd veterinarian (48 herds) or by the first or second author (12 herds). Herd veterinarians (n = 15) were instructed on how to perform the visit/interview before the start of the study. The farmer was interviewed about biosecurity measures and a tour around the farm was conducted. An amount of approximately EUR330 was paid in compensation to each participating farm at the end of the study.www.biocheck.ugent.be), was used. BioCheck was originally developed by Laanen et al. [To evaluate the biosecurity in the herds, a pre-established protocol, BioCheck  and larger herds (190–1200 sows), and farms where farmers had <23 (range 5–22) versus ≥23 (23–41) years of experience, and differences in overall scores and subcategory scores were compared between the groups. Also, differences in overall scores and subcategory scores between farms with female versus male workers responsible for piglet and sow care were studied, between farms with basic – lower versus higher – university education level of responsible person, and between conventional farms and outdoor herds, satellite herds and SPF herds respectively. The significance of the differences between groups was assessed by Mann–Whitney test. Microsoft Excel  and Minitab 16  were used for data handling and statistical analyses.www.svenskapig.se) and are based on reports on 59,000 sows . The average herd size of herds participating in this study was considerably lower than the mean figures from PigWin data, but production numbers, mortality figures and age at weaning were comparable. Our herd size (see Table Nineteen (32%) of the 60 herds in the study were satellites in a sow pool, a system whereby pregnant sows are leased from a central herd unit to several other herds (“satellite” herds). The sows are transported to the satellite herds before farrowing, and return to the central unit after weaning. Three (5%) were specific pathogen-free (SPF) herds and three (5%) had outdoor production. For Swedish herds altogether, the corresponding figures are 25% sow pool herds, 4% SPF and 1% organic herds, which in Sweden stipulates outdoor production ,15. SeveOn the farms included, the person who made biosecurity decisions and who was mainly responsible for the pigs  had a median of 23 years (range 5–41 years) of relevant experience in pig farming. The educational level of the pig farm managers was lower agricultural education 38%, university education 27%, higher agricultural education 17%, not specified 13% and basic school education 5%. The gender distribution was 48% female and 52% male in the farrowing and nursing units, and 30% female, 67% male and 3% not stated in the finishing units. The results suggest that the educational level of the farming personnel is appropriate for the task on the majority of farms and that there is a gender balance, at least among persons in charge of pigs, on Swedish pig farms. A median number of three persons (range two to 15) worked regularly in the pig barns.The average score for external biosecurity was 68 (median 68) and for internal 59 median 61), while the average total score was 64 (median 65). Distributions of the scores for each subcategory are shown in Figure , while tet al. [et al. [The average score for internal biosecurity in the present study was slightly higher compared with Belgian herds  described in a recent study by Laanen et al.  using th [et al. . This isP < 0.01; data not shown). In Table There were no significant correlations between total or subcategory scores and herd size, number of weaned piglets per sow per year, or years of pig farming experience Figure . Larger There were no significant differences in internal, external or total biosecurity scores when comparing conventional farms with farms with outdoor production, but only a few outdoor herds participated. Satellite herds had borderline significantly higher internal biosecurity scores than conventional farms. Not surprisingly, SPF herds had significantly higher score for external biosecurity than conventional farms but not for internal biosecurity. A higher education level for the person responsible for the animals was associated with a significantly higher internal biosecurity score, and the subcategories that were significantly different was the “Nursery period” and “Cleaning and disinfection”.Of 19 herds that purchased breeding pigs, 17 used a quarantine stable where a strict all-in, all-out routine was applied in all cases. The mean minimal length of the quarantine period was 23 days. It should be noted that the satellite herds (n = 19) receive sows that could have spent the previous farrowing period in another satellite herd, without quarantine. However, sows from different satellites will spend the dry period together in the central unit. Therefore, sow pools are considered to be one single epidemiological unit. Only five herds purchased piglets, so these were not investigated further. Forty-two of the herds purchased semen , but of these, 100% acquired semen from boar stations with a higher or equal health status. For details see Additional file A weak point with the farms included in this study was that 95% of farms answered that feed transports used the clean road, which can be interpreted as a general lack of distinction between clean and dirty roads on Swedish farms. Also, 95% of herds did not have specific measures for pass-through of material supplies Figure . MoreoveThe subcategory “Personnel and visitors” received a median score of 65. About 90% of herds provided visitors with boots and clothing, and about 85% obliged visitors to check in before entering the farm. A hygiene lock was used, washing and disinfection of hands was done and a quarantine period of more than 12 hours after visiting other farms was applied for visitors in more than half of the farms. However, these measures were carried out by farmers and staff themselves on only 32% of farms, which reduced the score for this subcategory. Further details are presented in Additional file 2, and in the fattening unit 80% of herds had >0.6 m2 per pig, which is larger than the space requirement stipulated by the EU (Council Directive 2008/120/EC) [An all-in, all-out system in the fattening unit was practised in 90% of herds, for all compartments. In the nursery unit, a strict all-in, all-out protocol was practised at a pen level in 95% of the herds, but the situation at the compartment level was not covered by the questionnaire. In the nursery, 77% of herds achieved the maximum score for a pig density of three or fewer pigs per m/120/EC) . See AddScores for “Disease management” varied the most between herds, but were based on four questions only. Strengths within the subcategory were that 97% of the herds had a plan for vaccination and treatments that was complied with, and that the disease status of the herd was regularly checked Figure . WeaknesTo the authors’ knowledge, only one previous study, conducted in 2006, describes biosecurity measures in Swedish pig herds, although that also included specialized fattening farms . The stuThe source population were mainly herds affiliated to the SvDHV’s full service programme, which excludes herds with herd veterinarians outside this organization. To be able to make cross-country comparisons within the MINAPIG project, the inclusion criterion for herd size was set to >100 sows. In Sweden, more than half of farrow-to-finish herds are smaller , and theIt can be concluded that middle-sized to large Swedish farrow-to-finish systems have a high level of external biosecurity, and a good internal biosecurity. Good routines for purchase of animals and protocols for visitors are applied in the majority of herds. Furthermore, there are low stocking densities and an all-in, all-out protocol is applied in most herds. The external biosecurity can be improved regarding transports for animals and feed. For internal biosecurity, there is room for improvement especially regarding hygiene measures such as washing of hands and changing of clothes and boots in and between compartments and the order in which work is carried out between different groups of pigs. A female caretaker in the nursery unit or a farmer with <23 years of experience was positively associated with higher scores for certain external and internal biosecurity scores, and a higher level of education was likewise associated with higher scores for internal biosecurity.


Judging from the large number of C2 domains present in nearly all ciliary transition zone proteins identified, the structure presented here seems to constitute a cilia-specific module present in multi-protein transition zone complexes.RPGRIP1 (RPGR interacting protein 1) is mutated in the severe eye disease Leber´s Congenital Amaurosis (LCA), while the structural homologue RPGRIP1L (RPGRIP1-like) is mutated in many different ciliopathies. Both are large multi-domain proteins predicted to interact with RPGR, the Retinitis Pigmentosa G-Protein Regulator. RPGR is mutated in X-linked Retinitis Pigmentosa and located in photoreceptors and in primary cilia. We solved the crystal structure between the RPGR-interacting domain (RID) of RPGRIP1 and RPGR and demonstrate that RPGRIP1L binds to RPGR in a similar mode. RPGRIP1 binding to RPGR affects the interaction with PDEð, the cargo shuttling factor for prenylated ciliary proteins. The RPGRIP1-RID is a type II C2 domain with a canonical ß-sandwich structure. However, it does not bind Ca


To probe the mechanism and selectivity of diolide formation, we sought to reconstitute ring formation in vitro by using a non-natural substrate. Incubation of recombinant elaiophylin thioesterase/cyclase with a synthetic pentaketide analogue of the presumed monomeric polyketide precursor of elaiophylin, specifically its N-acetylcysteamine thioester, produced a novel 16-membered C2-symmetric macrodiolide. A linear dimeric thioester is an intermediate in ring formation, which indicates iterative use of the thioesterase active site in ligation and subsequent cyclization. Furthermore, the elaiophylin thioesterase acts on a mixture of pentaketide and tetraketide thioesters to give both the symmetric decaketide diolide and the novel asymmetric hybrid nonaketide diolide. Such thioesterases have potential as tools for the in vitro construction of novel diolides.Elaiophylin is an unusual C Modular type I polyketide synthases (PKSs) are giant multifunctional enzymes, principally from actinomycete bacteria, that use a remarkable assembly-line logic for the biosynthesis of a diverse array of bioactive natural products, includinThe directness of the link between the PKS gene sequence and the chemical structure of the end product has revolutionized our view of the evolution of antibiotic biosynthesis, and has C2-symmetric macrocyclic dilactones, or diolides.[C2-symmetric diolides of diverse ring size have now been characterized from numerous sources including bacteria,[1 b; Scheme An intriguing and relatively rare variation in the mode of polyketide release from modular PKS assembly lines leads to diolides. C2-symmebacteria, fungi,[1bacteria, and maribacteria, A better1 b on the polyketide synthase, as illustrated in Scheme N-acetylcysteaminyl thioesters (SNAC thioesters) of tetraketide and pentaketide analogues of the natural octaketide monomers, which in vivo are acted upon by the TE while tethered to an adjacent ACP domain in the multienzyme assembly line, we show here that the TE can catalyze homodimerization of the synthetic pentaketide 3 b , while pentaketide SNAC thioester 3 b was obtained in 13 steps  with Ela-TE (40 μm) in 0.1 m potassium phosphate buffer (pH 8.2) containing 10 % DMSO produced, in a time- and enzyme-dependent manner, the symmetric decaketide diolide 5, the structure of which was confirmed by HRMS and 1D- and 2D-NMR techniques ; as well as the hydrolysis product 3 a. These were accompanied by a further species eluted after 20.5 min . Notably, dimerization of 3 b gave only the symmetrical 16-membered macrodiolide, as found in natural 1 b, with no evidence of regioisomers with a different ring size being formed. We also tested the chain-terminating cyclase/thioesterase from the erythromycin pathway (DEBS-TE)[3 b, but this enzyme exclusively catalyzed hydrolysis to 3 a . These observations show that the TE is competent to catalyze both the ligation of two monomeric polyketide chains and subsequent diolide formation and they support the mechanism of route 2) . Interestingly, when an equimolar mixture of 2 b and 3 b was incubated with Ela-TE under the same conditions, the asymmetric 16-membered macrodiolide nonaketide 6, the structure of which was confirmed by HRMS and 1D- and 2D-NMR , was produced in addition to 5 and in comparable amounts (Figure In contrast to pentaketide analogue 4 (To the best of our knowledge, this is the first example of a “hybrid” macrodiolide polyketide produced enzymatically in vitro. In this experiment, LC–MS revealed the presence of a species with the mass predicted for a linear nonaketide thioester with the same retention time as 4 . HoweverPrevious structural studies on PKS TE domains catalyzing either hydrolysis or macroThe iterative Ela-TE must catalyze a total of two acylation and two deacylation reactions to form the diolide, and further structural and functional studies will be needed to understand the respective selectivity of these steps. Nevertheless, our present results open the perspective of using such diolide TE domains preparatively to synthesize not only novel macrodiolides but also linear dimeric or even trimeric polyketide esters. Such studies with the purified TE domain of the oxazole diolide conglobatin are unde