By its creation in 1989, IMGT® marked the advent of immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics. IMGT® is specialized in the immunoglobulins (IG) or antibodies, T cell receptors (TR), major histocompatibility (MH), and IgSF and MhSF superfamilies. IMGT® has been built on the IMGT-ONTOLOGY axioms and concepts, which bridged the gap between genes, sequences and three-dimensional (3D) structures. The concepts include the IMGT® standardized keywords (identification), IMGT® standardized labels (description), IMGT® standardized nomenclature (classification), IMGT unique numbering and IMGT Colliers de Perles (numerotation). IMGT® comprises seven databases, 15,000 pages of web resources and 17 tools. IMGT® tools and databases provide a high-quality analysis of the IG from fish to humans, for basic, veterinary and medical research, and for antibody engineering and humanization. They include, as examples: IMGT/V-QUEST and IMGT/JunctionAnalysis for nucleotide sequence analysis and their high-throughput version IMGT/HighV-QUEST for next generation sequencing, IMGT/DomainGapAlign for amino acid sequence analysis of IG domains, IMGT/3Dstructure-DB for 3D structures, contact analysis and paratope/epitope interactions of IG/antigen complexes, and the IMGT/mAb-DB interface for therapeutic antibodies and fusion proteins for immunological applications (FPIA).IMGT For the first time, immunoglobulin (IG) or antibody and T cell receptor (TR) variable (V), diversity (D), joining (J) and constant (C) genes were officially recognized as “genes” as well as the conventional genes [IMGT system® ,2, was cal genes ,4,5,6. Tgnathostomata) more than 450 million years ago and is found in all extant jawed vertebrate species from fishes to humans. It is characterized by a remarkable immune specificity and memory, which are properties of the B and T cells owing to an extreme diversity of their antigen receptors. The specific antigen receptors comprise the IG or antibodies of the B cells and plasmacytes [The adaptive immune response was acquired by jawed vertebrates  and MH superfamily (MhSF) and related proteins of the immune system (RPI) of vertebrates and invertebrates [® provides a common access to standardized data from genome, proteome, genetics, and two-dimensional (2D) and three-dimensional (3D) structures. IMGT® is the acknowledged high-quality integrated knowledge resource in immunogenetics for exploring immune functional genomics. IMGT® comprises seven databases  [® is the global reference in immunogenetics and immunoinformatics [® online access at the 9th International Congress of Immunology, San Francisco, CA, USA) [IMGTtebrates . IMGT® puctures) ,10,11,12uctures) ,26,27,28uctures) , The IMGormatics ,43,44,45CA, USA) ,47 and tCA, USA) ,49.® data are based on IMGT-ONTOLOGY [® standards that constitute the IMGT Scientific chart: e.g., IMGT® standardized keywords (IDENTIFICATION) [® standardized labels (DESCRIPTION) [® standardized gene and allele nomenclature (CLASSIFICATION) [The accuracy and the consistency of the IMGTONTOLOGY ,51,52, tONTOLOGY ,67,68,69ONTOLOGY . The conICATION) , IMGT® sRIPTION) , IMGT® sICATION) , IMGT unICATION) ,63,64,65ICATION) ,67,68,69® tools and databases used for IG repertoire analysis, antibody engineering and humanization, and IG/Ag structures are then briefly presented: IMGT/V-QUEST [® tools and databases run against IMGT reference directories built from sequences annotated in IMGT/LIGM-DB [® nucleotide database , and from IMGT/GENE-DB [® gene database .With a focus on IG, we first review the fundamental information generated from these IMGT-ONTOLOGY concepts which led to the IMGT Scientific chart rules. The major IMGT/V-QUEST ,16,17,18/V-QUEST ,20; IMGT/V-QUEST ,22 and I/V-QUEST ; IMGT/Hi/V-QUEST ,24; IMGT/V-QUEST ,25,26 fo/V-QUEST ,10,11; aAn interface, IMGT/mAb-DB , has bee® approach is of major interest for bridging knowledge from IG repertoire in normal and pathological situations [The unified IMGTtuations ,73,74,75tuations ,77,78, Ntuations ,24, antituations ,84,85,86® standardized keywords (189 for sequences and 137 for 3D structures) were precisely defined [® databases and for the extraction of sequences and 3D structures. They have been entered in BioPortal [More than 325 IMGT defined . They reioPortal  at the NStandardized keywords are assigned at each step of the molecular synthesis of an IG. Those assigned to a nucleotide sequence are found in the “DE” (definition) and “KW” (keyword) lines of the IMGT/LIGM-DB files . They ch® [®: very similar (+ + +), similar , dissimilar , and very dissimilar (− − −).The 20 usual amino acids (AA) have been classified in 11 IMGT physicochemical classes (IMGT® . For exa® . Four ty® standardized labels (277 for sequences and 285 for 3D structures) were precisely defined [More than 560 IMGT defined . They ar defined .Highly conserved amino acids at a given position in a domain have IMGT labels . Thus th® concepts of classification allowed us, for the first time, to classify the antigen receptor genes (IG and TR) for any locus , T cell receptor alpha (TRA)), for any gene configuration  and for any species (from fishes to humans). In higher vertebrates, there are three IG major loci . The IG major loci include the immunoglobulin heavy (IGH), and for the light chains, the immunoglobulin kappa (IGK) and the immunoglobulin lambda (IGL) in higher vertebrates and the immunoglobulin iota (IGI) in fishes , the standardized classification and nomenclature of the IG and TR of humans and other vertebrate species have been under the responsibility of the IMGT Nomenclature Committee (IMGT-NC).Since the creation of IMGT® gene and allele names are based on the concepts of classification of “Group”, “Subgroup”, “Gene” and “Allele” [Homo sapiens IGHV1 subgroup. Subgroups, genes, and alleles are always associated to a species name. An allele is a polymorphic variant of a gene, which is characterized by the mutations of its sequence at the nucleotide level, identified in its core sequence, and compared to the gene allele reference sequence, designated as allele *01. For example, Homo sapiens IGHV1-2*01 is the allele *01 of the Homo sapiens IGHV1-2 gene that belongs to the Homo sapiens IGHV1 subgroup which itself belongs to the IGHV group. For the IGH locus, the constant genes are designated by the letter  corresponding to the encoded isotypes , instead of using the letter C. IG and TR genes and alleles are not italicized in publications. IMGT-ONTOLOGY concepts of classification have been entered in the NCBO BioPortal.IMGT“Allele” . “Group”® IG and TR gene names [® IG and TR gene names are the official international reference and, as such, have been entered in IMGT/GENE-DB [® human IG and TR genes were also integrated in IMGT-ONTOLOGY on the NCBO BioPortal and, on the same site, in the HUGO ontology and in the National Cancer Institute (NCI) Metathesaurus. Amino acid sequences of human IG and TR constant genes  were provided to UniProt in 2008. Since 2007, IMGT® IG gene and allele names have been used for the description of the therapeutic mAb and FPIA of the WHO-INN program [The IMGTne names ,4,5,6 wene names ,89 and wne names ,47. The /GENE-DB , in Locu/GENE-DB , in Entr/GENE-DB , in NCBI/GENE-DB , and in /GENE-DB  at the W program ,49.® Rosetta stone” that has bridged the biological and computational spheres in bioinformatics [® concepts of numerotation comprise the IMGT unique numbering [The IMGT-ONTOLOGY NUMEROTATION axiom is acknowledged as the “IMGTormatics . The IMGumbering ,63,64,65umbering ,67,68,69umbering ,61,62, tumbering .The V-DOMAIN of the IG and TR are encoded by V-(D)-J rearrangements ,4. Thus,A V-DOMAIN  comprise® definitive system [® standards [® [The V-DOMAIN strands and loops and their delimitations and lengths, based on the IMGT unique numbering for V domain ,61,62,65e system , the CDRe system  or Chothe system , are avatandards ,69,70,84For a V-DOMAIN, the CDR1-IMGT or BC loop encompasses positions 27 to 38, the CDR2-IMGT or C'C'' loop, positions 56 to 65, and the CDR3-IMGT or FG loop, positions 105 to 117. The CDR3-IMGT encompasses the V-(D)-J junction that results from a V-J or V-D-J rearrangement  and is m® that definitively solved the ambiguous situation of different CDR lengths and delimitations found in the literature. The six anchors of a V-DOMAIN are positions 26 and 39 (anchors of the BC loop or CDR1-IMGT), 55 and 66 (anchors of the C'-C'' loop or CDR2-IMGT), 104 and 118 (anchors of the FG loop or CDR3-IMGT). The CDR3-IMGT anchors are highly conserved; they are C104  and F118 or W118 . The JUNCTION of an IG or TR V-DOMAIN includes the anchors 104 and 118 and is therefore two amino acids longer than the corresponding CDR3-IMGT (positions 105–117).The V-DOMAIN strands and loops (FR-IMGT and CDR-IMGT) are visualized in the IMGT Colliers de Perles ,67,68,69In biological data, the lengths of the loops and strands are given by the number of occupied positions (unoccupied positions or “IMGT gaps” are represented with hatches in the IMGT Colliers de Perles  or by doA V-DOMAIN has five characteristic amino acids at given positions (positions with bold (online red) letters in the IMGT Colliers de Perles). Four of them are highly conserved and hydrophobic  and are ® definitive system for the characterization of a V domain is its delimitation taking into account the exon delimitations, whenever appropriate [® genomic approach integrates the strands A and G, in contrast to structural alignments that usually lack these strands due to their poor structural conservation, and thus bridges the gap between genomic data (exon) and 3D structure (domain) [A criterion used in the IMGTropriate . The exoropriate . This IM(domain) .A C-DOMAIN  comprise® standards [® [The C-DOMAIN strands, turns and loops and their delimitations and lengths, based on the IMGT unique numbering for C domain ,65, are tandards ,69,70,84dards [® , IMGT ScThe C-DOMAIN lengths of the strands and loops are visualized in the IMGT Colliers de Perles ,68,69, oA C-DOMAIN has five characteristic amino acids at given positions (positions with bold (online red) letters in the IMGT Colliers de Perles). Four of them are highly conserved and hydrophobic  and are ® genomic approach integrates the strands A and G, which are absent of structural alignments [The IG C-DOMAIN are delimited taking into account the exon delimitation, whenever appropriate . As for ignments .® Web site [The IMGT/Collier-de-Perles tool , on the Web site , is a geWeb site ,67,68,69Web site ,25,26 ; alignments and identification of the genes and alleles with the closest germline V, D, and J genes; analysis of the junctions; analysis of somatic hypermutations and amino acid changes; and, if the option “Search for insertions and deletions” was selected, identification of insertions and deletions (indels) and their correction. Customized parameters and results provided by IMGT/V-QUEST and IMGT/JunctionAnalysis have been described elsewhere ,17,18,70The IMGT/V-QUEST reference directory  compriseBy definition, the IMGT/V-QUEST reference directory contains one reference sequence for each allele. By default, the user sequences are compared with all alleles of the genes of the selected set. However, the option “With allele *01 only” is useful for: (1) “Detailed view”, if the user sequences need to be compared with different genes; and (2) “Synthesis view”, if the user sequences that use the same gene need to be aligned together  ,17,18.® Web site). Links to the IMGT/V-QUEST reference directory sets are available from the IMGT/V-QUEST Welcome page [The IMGT/V-QUEST reference directories have been set up for species which have been extensively studied, such as human and mouse. This also holds for the other species or taxons with incomplete IMGT reference directory sets. In those cases, results should be interpreted considering the status of the IMGT reference directory  or available data. Thus, a clonotype may denote either a complete antigen receptor , or only one of the two chains of the receptor , or one domain , or the CDR3 sequence of a domain. Moreover the sequence can be at the amino acid (AA) or nucleotide (nt) level, and this is rarely specified. Therefore, the goal of IMGTiguously .®, the clonotype, designated as “IMGT clonotype (AA)”, is defined by a unique V-(D)-J rearrangement  level) and a unique CDR3-IMGT AA (in-frame) junction sequence [In IMGTsequence . For idesequence .By essence, an “IMGT clonotype (AA)” is “unique” for a given dataset. For that reason, each “IMGT clonotype (AA)”, in a given dataset, has a unique set identifier and, importantly, has a unique representative sequence selected by IMGT/HighV-QUEST among the “1 copy” “single allele” (for V and J), based on the highest percent of identity of the V-REGION (“V %”) compared to that of the closest germline, and/or on the sequence length (thus the most complete V-REGION) ,70.Clonotype expression is the number of sequences that can be assigned to each IMGT clonotype (AA). The total number of sequences assigned to each given “IMGT clonotype (AA)” is calculated by a stepwise procedure that aggregates sequences to the “IMGT clonotype (AA)”, and insures that high-quality and specific characterization of the “IMGT clonotype (AA)” remains unaltered) ,70.® standardized approach allows a clear distinction and accurate evaluation between the clonal diversity (nb of “IMGT clonotypes (AA)”), and the clonal expression ”) [® users.For the first time for NGS antigen receptor data analysis, the IMGTe (AA)”) ,70. These (AA)”) ,70. The Clonal diversity is also visualized in the online results with histograms which represent the number of IMGT clonotypes (AA) per V, D (for IGH), and J genes (in pink) . Clonal ® online tool for the analysis of amino acid sequences and 2D structures of domains   . It anal® “gene” and “allele” names. Data are comprehensive for human and mouse IG  whereas for other species and other IgSF and MhSF they are added progressively. The IMGT/DomainSeq reference directory comprises AA sequences of domains, or of core regions for V and J, of functional (F), ORF (open reading frame) and in frame pseudogene (P) genes. As IMGT® alleles are characterized at the nucleotide level, identical sequences at the amino acid level may therefore correspond to different alleles, in the IMGT/DomainSeq reference directory. The sequences can be displayed by querying IMGT/DomainDisplay [The IMGT/DomainSeq reference directory  comprisenDisplay .® structure database; provides IMGT® annotation and contact analysis of IG 3D structures; and paratope/epitope description of IG/antigen complexes  are identified with the integrated IMGT/DomainGapAlign [The “Chain details” section comprises information first on the chain itself, then per domain ,10,11. CGapAlign ,26, whic“Contact analysis” gives access to a table with the different “Domain pair contacts” of the 3D structure ’). “Domain pair contacts” refer to contacts between a pair of domains or between a domain and a ligand. Clicking on “DomPair” gives access to the contacts between amino acids for a given “Domain pair contacts”. Contacts between VH and the Ligand  and the V-KAPPA and the Ligand (Ag) of an IG/Ag complex are shown in In IMGT/3Dstructure-DB, all contacts are described as atom pair contacts. Atom pair contacts are obtained by a local program in which atoms are considered to be in contact when no water molecule can take place between them ,10. AtomClicking on “R@P” gives access to the IMGT identity card of a given residue  at a given position or Residue@Position. The IMGT R@P card can also be accessed from the amino acid sequences of the IMGT/3Dstructure-DB card or from the IMGT Collier de Perles, by clicking on one amino acid. In an IMGT R@P card, the Residue@Position is defined by the IMGT position numbering in a domain, or if not characterized, in the chain, the amino acid name , the IMGT domain description, and the IMGT chain ID, e.g., “103—TYR (Y)—VH—1hzh_H” ,10,11. TIn an IG/Ag complex, the amino acids in contact at the interface between the IG and the Ag constitute the paratope on the IG V-DOMAIN surface and the epitope on the Ag surface. For IG/Ag, the paratope and epitope are displayed in Contact analysis B,C, but “Renumbered IMGT file” allows viewing (or downloading) of an IMGT coordinate file renumbered according to the IMGT unique numbering, and with added IMGT specific information on chains and domains (added in the “REMARK 410” lines (blue online) and identical to the “Chain details” annotation).“IMGT numbering comparison” provides, per domain, the IMGT DOMAIN numbering by comparison with the PDB numbering and the residue (3-letter and 1-letter names), which allows standardized IMGT representations using generic tools.Tools associated with IMGT/3Dstructure-DB include IMGT/StructuralQuery  and IMGT® criteria of standardized identification [IMGT/2Dstructure-DB was created as an extension of IMGT/3Dstructure-DB ,10,11 tofication , descripfication , nomenclfication  and numefication ,63,64,65The current IMGT/2Dstructure-DB entries include amino acid sequences of antibodies from Kabat   is only available in the corresponding IMGT/3Dstructure-DB cards if the antibodies have been crystallized.The objective of antibody humanization is to graft at the DNA level the CDR of an antibody V domain, from mouse (or other species) and of a given specificity, onto a human V domain framework, thus preserving the specificity of the original (murine or other species) antibody while decreasing its immunogenicity . IMGT/Do® [Analyses performed on humanized therapeutic antibodies underline the importance of a correct delimitation of the CDR and FR. As an example, two amino acid changes were required in the first version of the humanized VH of alemtuzumab, in order to restore the specificity and affinity of the original rat antibody. The positions of these amino acid changes (S28 > F and S35 > F) are now known to be located in the CDR1-IMGT and should have been directly grafted, but at the time of this mAb humanization they were considered as belonging to the FR according to the Kabat numbering . In contHomo sapiens” IG V genes at the top of the results, in the case of a successfully “humanized” V. In contrast, the query will display V genes of species other than Homo sapiens for an unsuccessful humanization: in that case the V gene is “non-human” and the IG chain to which it belongs is “chimeric”.IMGT/DomaingapAlign is used to evaluate the degree of “humanization” of an IG V sequence, either obtained from a species other than human , or obtained from engineered human sequences . IMGT/DomaingapAlign provides an objective assessment of the degree of humanization of the user sequence, based on sequence alignments, independently on the source of the starting sequence  and independently on the experimental methodology that was used with the objective of humanizing it. A query of the user sequence against “V” of “any” species will display “Allotypes are polymorphic markers of an IG subclass that correspond to amino acid changes and are detected serologically by antibody reagents . In therThe allotypes G1m, G2m and G3m are carried by the constant region of the gamma1, gamma2 and gamma3 chains, encoded by the IGHG1, IGHG2 and IGHG3 genes, respectively . The gamIn the IGHG1 CH1, the lysine at position 120 (K120) in strand G corresponds to the G1m17 allotype  Figure D. The isIn the IGHG1 CH3, the aspartate D12 and leucine L14 (strand A) correspond to G1m1, whereas glutamate E12 and methionine M14 correspond to the nG1m1 isoallotype  Table 6Table 6. A N-linked glycosylation site is present in the CH2 domain of the constant region of the human IG heavy chains of the four IgG isotypes. The N-linked glycosylation site belongs to the classical N-glycosylation motif N-X-S/T  and is defined as CH2 N84.4. As shown in the IMGT Collier de Perles, this asparagine is localized at the DE turn. The IMGT unique numbering has the advantage of identifying the C domain  and, in the domain, the amino acid and its localization  which can be visualized in the IMGT Collier de Perles and correlated with the 3D structure ,84,85.® information system, which are at the origin of immunoinformatics [IMGT-ONTOLOGY and the IMGTormatics , have pr® standards for IG are used in clinical applications. Thus, IMGT/V-QUEST is frequently used by clinicians for the analysis of IG somatic hypermutations in leukemia, lymphoma and myeloma, and more particularly in chronic lymphocytic leukemia (CLL) [® standards use more needed as ever. More generally, the IMGT/HighV-QUEST web portal is a paradigm for identification of IMGT clonotype diversity and expression in NGS immune repertoire analysis of the adaptive immune response in infectious diseases, in vaccination, and for next generation repertoire immunoprofiling [The IMGTia (CLL) ,73,74,75ia (CLL) . The seqia (CLL) ,20 are arofiling .® standardization allows repertoire analysis and antibody humanization studies to move to novel high-throughput methodologies with the same high-quality criteria. The CDR-IMGT lengths are now required for mAb INN applications and are included in the WHO-INN definitions [The therapeutic monoclonal antibody engineering field represents the most promising potential in medicine. A standardized analysis of IG genomic and expressed sequences, structures and interactions is crucial for a better molecular understanding and comparison of the mAb specificity, affinity, half-life, Fc effector properties and potential immunogenicity. IMGT-ONTOLOGY concepts have become a necessity for IG loci description of newly sequenced genomes, antibody structure/function characterization, antibody engineering (single chain Fragment variable (scFv), phage displays, combinatorial libraries) and antibody humanization  ,84,85,86initions , bringin


We devise a scoring metric to measure the plasticity potential for these domain sets, and evaluate how function has changed over time for different species. Applying this metric to a phylogenetic tree of eukaryotic genomes, we find that the involvement of each function is not random but highly selective. For certain lineages there is strong bias for evolution to involve domains related to certain functions. In general eukaryotic genomes, particularly animals, expand complex functional activities such as signalling and regulation, but at the cost of reducing metabolic processes. We also observe differential evolution of transcriptional regulation and a unique evolutionary role of channel regulators; crucially this is only observable in terms of the architecture plasticity potential. Our findings provide a new layer of information to understand the significance of function in eukaryotic genome evolution. A web search tool, available at   •A new concept to measure domain architecture plasticity potential in a genome.•We reveal the function-selective role in eukaryotic genome evolution.•Eukaryotic genomes expand signalling and regulations but reduce metabolism.•We observe differential evolution between trans- and cis-acting regulations.•We observe a unique role of channel regulators in separating eukaryotic kingdoms. We present a first attempt towards a systematic description and comparison of protein function over the evolution of eukaryotic proteomes. A proteome is an entire protein repertoire (encoded by a genome), and is composed of proteins comprised of structural units or domains 1.2The Structural Classification of Proteins (SCOP) database is a gold standard for classifying protein domains of known structure 1.3‘architecture plasticity potential’, the capacity of a domain superfamily to occur in different architectural contexts (i.e. the number of different architectures) within a genome. From this concept, architecture plasticity potential differs from one superfamily to another. The upper panel of To better describe the relationship between genomes, superfamilies and architectures, we introduce a new concept of 1.4Based on preliminary data present in 22.1Domain assignments for sequenced genomes were obtained from the SUPERFAMILY database 2.2Recently, we have published the sTOL 2.3GO annotations for domain superfamilies were obtained from the dcGO database 2.4MED) as an averaging metric to quantify the plasticity potential PPds(g) of a domain set (ds) for a given genome (g):d∈ds denotes a list of individual domains belonging to the domain set ds and Nd for the number of architectures associated with the domain d. To allow for comparison across genomes and across terms (i.e. domain sets), we next consider the null distribution of the PPds(g). A null distribution was estimated using a randomization procedure, which simultaneously respects the size of the domain set, the domain repertoires present in the genome, and the collection of annotatable domains (e.g. only those annotated by one GO term or more). Specifically, we generated a random instance of domain set dsb, which contained the same number of domains as in ds but being randomly sampled from the annotatable domain repertoires present in the genome g. In a similar way to PPds(g), we calculated the randomized PP for this instance, denoted as B times (2000 or higher), we obtained a sampling of the null distribution: b = 1 … B. This null distribution was then used to estimate the sample mean μds(g), sample standard deviation σds(g), and the plasticity potential score (PP-score). They are formulated as:The tendency for an individual domain to occur in distinct/different architectures (i.e. its architecture diversity) can be described by a ‘plasticity potential’ (PP). High PP for a domain means it is frequently found in diverse architectures, while low PP indicates a domain occurring in few distinct architectures. The number of different architectures is counted over every protein sequence present in a genome (i.e. the proteome) without using any filtering criteria. With this definition, the PP is genome-specific since the same domain may have different PP across different genomes. In this way, the PP can be used for studying the dynamic changes of architecture diversity (attached to a given domain), e.g. during eukaryotic genome evolution. In a similar way the PP for a domain set can also be estimated by calculating an average number of architectures per domain in the set. To ensure the estimate is insensitive to extreme values, we used the median . Following this, there are four smaller circles below, each denoting the architecture repertories/landscape of the associated superfamily. The number of architectures (colour-coded hexagons) defines the PP for an individual domain. This definition can be naturally extended to a collection of related domains annotated by an ontology term (i.e. a domain set sharing certain common characteristics). Without loss of generality, hereinafter we view an ontology term as equivalent to a domain set. As exemplified in A, B and C it annotates, and ‘Term 2’ (the right panel) to the two superfamilies A and D. The PP for a domain set is quantified by taking the median number of architectures per domain . To allow for the PP to be comparable across genomes and across terms, we further devise a new scoring metric called ‘PP-score’. The PP-score takes into account the mean and standard deviation of the PP that would be expected when randomly sampling any domain set of the same size within this genome. Using this randomisation, the negative PP-score for the ‘Term 1’ is indicative of the tendency to form less diversified architectures than by chance. Conversely, the positive PP-score for the ‘Term 2’ suggests the tendency to form more different architectures than by chance. A key point of this strategy is to use term-specific architecture plasticity potentials for correlating functions (carried by GO terms) with genome evolution. Our recent progress in building relevant databases makes this strategy feasible. The SUPERFAMILY database We use a hypothetical genome , and the second occurring during the shift from the animal ancestor (Metazoa) to its subkingdom (Eumetazoa). In contrast, we only observed a dramatic increase when shifting to Opisthokonta for activities related to channel/enzyme regulation, i.e. expansion of kinase and peptidase families. In animal evolution, the rise of multicellularity  is a key transition, for which signalling systems are a prerequisite Bilateria. This observation clarifies the previous view that signalling systems were likely to have arisen before the split with bilaterians (and even the metazoans) cis-acting (binding to nucleic acids) and trans-acting (binding to proteins). We found that the ‘trans-acting’ mode has a dramatic increase in architectural plasticity potentials at the rise of the animal-fungi common ancestor; thereafter, such potentials are maintained. In sharp contrast, the ‘cis-acting’ mode experiences a steady increase along with increasing organism complexity. This observation is not just limited to human-specific evolution . It offers a wide spectrum of ontology terms, representing over 5000 research topics not just on functions, but also on phenotypes and diseases, in the hope of exploring their evolutionary importance over the eukaryotic tree of life. The users can pick up any terms of their interest, and similarly in All findings described above are drawn from a functional perspective, but in principle the same analysis can be applied to any subject, such as the evolution of diseases and phenotypes. To facilitate attempts in these promising directions, we provide a web search tool ‘Pevo’ (4vs. metabolism). Because of this robustness, we are also able to uncover the evolutionary importance of signalling systems for multicellularity, differential evolution between trans-acting and cis-acting transcriptional regulation, and the uniqueness of channel regulators in the formation of the three kingdoms. Although some of our findings are as expected, great care needs to be taken when making interpretations. Take these two terms as examples: one is the metabolism-related term ‘electron carrier activity’, and the other the signalling-related term ‘molecular transducer activity’. Both terms have almost the same positive PP-scores in the eukaryotic common ancestor. Along the evolutionary lineage to human, there is a steady decrease in the PP-score (even becoming negative) for the metabolism term, whereas the opposite trend is observed for the signalling term. This strong function-selective pattern cannot be simply explained by saying that this metabolism is no longer needed by human. Instead, as a basic activity it is still essential but just gives way to the need for increased signalling, seen as a reduction in the architectural repertoire. It is a matter of balance: in human we see much more architecture diversity being formed for signalling in place of metabolism; but in the eukaryotic common ancestor we see a relative equilibrium. The plasticity potential is a layer of information revealing the functional selection, which can be imagined as ‘invisible software’, operating on the architectural design of the proteins, which can be imagined as the ‘visible hardware’.We introduce and implement the concept of architecture plasticity potentials to better understand the role of function in eukaryotic genome evolution. We observe that the way nature shapes architecture diversity is not random. It is more economical from an evolutionary viewpoint to re-use existing protein domains by diversifying the repertoire of architectures (in a function-selective manner), than it is to diversify by creating new domains and deleting existing ones. Probably under the pressure of increasing organism complexity, the diversity of architectures harbouring superfamilies that are involved in complex functions increases at the cost of the diversity of architectures containing metabolism-related superfamilies. This is particularly prominent in animal evolution, suggesting that there is a robust signal underlying our observations (i.e. of complex functions The authors declare that no competing interests exist.


To provide Dutch normal values for rehabilitation outpatients with chronic pain or musculoskeletal diseases utilizing the World Health Organization Quality of Life questionnaire abbreviated version (WHOQOL-BREF) and analyse influence of diagnosis and patient characteristics on normal values and increase understanding in those values.Five hundred and forty-two outpatients were referred to a rehabilitation psychologist. Referral diagnoses were “musculoskeletal”, “chronic pain”, “neurological” and “miscellaneous”. Comparisons between groups were made for each of the four domains of the WHOQOL-BREF (scoring range 4–20).Domain scores of rehabilitation outpatients were physical domain 11.0 (±2.7), psychological domain 13.6 (±2.4), social domain 14.8 (±3.4) and environmental domain 14.2 (±2.2). Outpatients with chronic pain reported the lowest scores on the WHOQOL-BREF when compared to the “musculoskeletal”, “neurological” and “miscellaneous” groups. Increased age, lower education, living alone and unemployment had a negative impact on WHOQOL-BREF scores. Compared to the general Dutch population, rehabilitation outpatients scored, unadjusted for age, significantly lower difference for the physical domain 4.5 , the environment domain 1.7 , the psychological domain 1.1  and the social domain 0.4 .WHOQOL-BREF scores of rehabilitation outpatients are lower and differed significantly from normal values of a Dutch population in all four domains. Therefore, the WHOQOL-BREF can be used to measure the subjective impact of their disease or injury. The subjective impact of chronic pain was found to be particularly high. Due to modern health care, more and more patients with potentially lethal diseases are cured or disease progression is reduced . TherefoIt should be noted that apart from disease and injury, QOL is also influenced by social functioning , 5, educThe negative influence on the QOL by somatic and psychiatric diseases is found in all domains. This influence is well understood since Engel introduced the biopsychosocial model . This moThe aims of this study were to provide normal values for Dutch rehabilitation outpatients with chronic pain or musculoskeletal diseases utilizing the WHOQOL-BREF, to analyse the influence of diagnosis and patient characteristics of rehabilitation outpatients on normal values and to compare normal values with those of the general Dutch population and psychiatric outpatients.Between January 2008 and January 2013, 607 outpatients from the Department of Rehabilitation Medicine of the University Medical Centre Groningen (UMCG) were referred to a psychologist. They were referred by a rehabilitation specialist for a psychological assessment and/or treatment. Prior to this assessment, a set of questionnaires and a consent form were sent by mail to the patients with a request to fill out all forms. During the assessment, a semi-structured interview was conducted to determine a treatment plan. During the intake procedure, patient’s gender, age, educational level, employment and marital status were collected. The rehabilitation specialist’s referral medical diagnosis was retrieved from the medical records.The general Dutch population reference group was based on the Dutch manual WHOQOL-100 and WHOQOL-BREF. This group of 626 persons had a mean age of 53.9 (SD 16.2), and 67.5 % of the group were women .The psychiatric reference group consisted of 410 psychiatric outpatients with a mean age of 33.5 (SD 8.3), and 58.8 % of the group were women. It was a mixed diagnostic group: 54 persons who did not obtain a DSM-IV diagnosis, 224 with a single axis diagnosis and 132 with a diagnosis on axis 1 as well as axis 2 .r ranges between 0.88 and 0.96) , marital status , education  and employment  did not differ significantly between the different diagnosis groups. Employment was almost a significant difference between groups, and most deviant were the neurological group and the miscellaneous group. The four domains of the QOL were normally distributed. Cronbach’s alpha for the WHOQOL-BREF was 0.90. Removing items from the questionnaire resulted in lower values of alpha.Gender [Compared to the total group rehabilitation outpatients, the chronic pain group scored significantly lower in every domain except the environment, and the musculoskeletal group scored significantly higher in all four domains. There is a significant difference between the musculoskeletal group and the chronic pain group in all four domains Table .Table 3CThe results of the regression analyses are summarized in Table R square of the regression models for physical domain was 0.082, for the psychological domain 0.070, for the social domain 0.073 and for the environment domain 0.091.After correcting for patient characteristics, the diagnosis chronic pain contributed significantly to the regression equation in all domains.Compared to the general Dutch population, rehabilitation outpatient’s scores were significantly lower in all domains. Compared to psychiatric outpatients, rehabilitation outpatients’ scores were significantly higher except the physical domain Fig. ; Table 5The current study aimed to provide normal values of the WHOQOL-BREF for outpatients in rehabilitation and to gain insight into the influence of diagnosis and patient characteristics on QOL. Compared to the general Dutch population, rehabilitation outpatients scored lower on all domains of WHOQOL-BREF, the physical domain most strongly. A higher age had a negative impact on QOL in all domains except the psychological domain. Unemployment had a negative impact on all domains except the physical domain. Living alone influenced the psychological and environmental domains negatively. Lower education influenced the physical and environmental domains negatively. Finally, gender had no significant influence on any domain.In all four domains, the patients suffering from chronic pain were found to have a lower QOL than the musculoskeletal group. This influence was also significant after correcting for patient characteristics in all domains of WHOQOL-BREF. This finding corresponds with the concept that the emotional component plays an important role in chronic pain , 25.n = 4628), including healthy people and people suffering from different health conditions, effects of age on WHOQOL-BREF scores were small [Both psychiatric outpatients and rehabilitation outpatients scored lower on the physical domain than the general Dutch population, with the rehabilitation patients scoring the lowest. The psychiatric patients scored lower in the other three domains compared to the general Dutch population and to the rehabilitation outpatients. Further analyses revealed that the chronic pain patients had a lower score on the psychological domain but not as low as the psychiatric patients. The comparison with the psychiatric patients was not adjusted for age and gender. The comparison with the general Dutch population was not adjusted for age because data to do so were not available. Some age differences were present in our study. The mean age of the general Dutch population was 53.9 (SD 16.2), of the rehabilitation outpatients 41.0 (SD 14.0) and of the psychiatric outpatients 33.5 (SD 8.3). In a large WHOQOL-BREF study in the UK (re small . There wThe ability of the WHOQOL-BREF to evaluate change over time was investigated in a study within an outpatient rehabilitation setting. That study concluded that the questionnaire was a useful instrument for outcome measurement . Also, sIn the current study, QOL was measured once. The largest difference between the general Dutch population and the rehabilitation outpatients was in the physical domain, approximately 4 points on a 4–20 scale. The difference between the general Dutch population and rehabilitation outpatients was 1.1 point on the psychological domain and only 0.4 on the social domain. In our opinion, the differences in the psychological and social domain are small. This finding upholds one of the conclusions of the aforementioned study of a limited responsiveness .The strength of the current study is the number of consecutive participants over a 5-year period. All referred patients were asked to participate. Of these participants, only 11 % were excluded. Limitation of the current study is a missing baseline measurement of QOL before the trauma or disease. However, these data cannot be obtained.In rehabilitation outpatients, scores on all WHOQOL-BREF domains were significantly lower than those of the general Dutch population. Therefore, the WHOQOL-BREF can be used to measure the subjective impact of their disease or injury in rehabilitation outpatients. A small but significant negative effect of increased age and unemployment was found on three domains, of living alone on two domains and of lower education also on two domains of QOL.Patients with chronic pain were found to exhibit a significant lower QOL in all four domains when compared to the group of patients with musculoskeletal problems. The differences between the rehabilitation outpatients and the general Dutch population on the psychological and social domain are small.


Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase, which plays critical roles in a wide spectrum of neuronal functions including neuronal survival, neurite outgrowth, and synapse development and plasticity. Cdk5 activity is controlled by its specific activators: p35 or p39. While knockout studies reveal that Cdk5/p35 is critical for neuronal migration during early brain development, functions of Cdk5/p35 have been unraveled through the identification of the interacting proteins of p35, most of which are Cdk5/p35 substrates. However, it remains unclear whether p35 can regulate neuronal functions independent of Cdk5 activity. Here, we report that a nuclear protein, nuclear hormone receptor coregulator (NRC)-interacting factor 1 (NIF-1), is a new interacting partner of p35. Interestingly, p35 regulates the functions of NIF-1 independent of Cdk5 activity. NIF-1 was initially discovered as a transcriptional regulator that enhances the transcriptional activity of nuclear hormone receptors. Our results show that p35 interacts with NIF-1 and regulates its nucleocytoplasmic trafficking via the nuclear export pathway. Furthermore, we identified a nuclear export signal on p35; mutation of this site or blockade of the CRM1/exportin-dependent nuclear export pathway resulted in the nuclear accumulation of p35. Intriguingly, blocking the nuclear export of p35 attenuated the nuclear accumulation of NIF-1. These findings reveal a new p35-dependent mechanism in transcriptional regulation that involves the nucleocytoplasmic shuttling of transcription regulators. Neural development in response to various stimuli such as neural activity, neurotrophic factors, and nuclear hormones is a tightly coordinated process that involves the concerted regulation of gene expression Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase. It has two neuronal-specific Cdk5 activators, p35 and p39 The p35-associated Cdk5 activity is mainly localized to membrane fractions. Nonetheless, Cdk5 and p35 are also expressed in the nuclei of neurons The present study aimed to discover novel roles of p35 in neural development through identification of its new interacting partner(s). We identified a new p35-interacting protein, nuclear hormone receptor coregulator (NRC)-interacting factor 1 (NIF-1). These findings reveal an unexpected role of p35 in mediating the nucleocytoplasmic shuttling of NIF-1. NIF-1 protein, which was originally identified to associate with NRC 5′-TCCAGGCGGTCGCAGCAACCTGCGCATACCTCTCCTACT, 3′-AGTAGGAGAGGTATGCGCAGGTTGCTGCGACCGCCTGGA), and the cDNA fragment was subcloned into pEGFP-N3. All constructs were confirmed by sequencing.Complementary DNAs (cDNAs) encoding p35, p25, and p10 were subcloned into the yeast GAL4 DNA-binding vector, pAS2-1 . Full-length p35 was used as bait in the yeast two-hybrid screen Antibodies specific for Cdk5 , p35 , and HA were purchased from Santa Cruz Biotechnology . A polyclonal antibody against p39 was raised against a synthetic peptide . A custom antibody against NIF-1 was raised against the GST fusion protein of NIF-1 C-terminus . The specificity of the antibody was confirmed by pre-absorption with the His fusion protein of the NIF-1 C-terminus .A yeast two-hybrid screen was performed following the Matchmaker two-hybrid screen protocol (Clontech). p35 was used as bait to screen for a mouse muscle cDNA library in a GAL4 transcriptional activation vector, pACT2. The bait and library plasmid were co-transformed into yeast strain Y190. The transformants were selected on SD-Trp-Leu-His plates, and the selected clones were subjected to a β-galactosidase filter assay. The C-terminal region of NIF-1 encoding amino acids 772–1,291 was isolated from the two-hybrid screen. To map the interacting domains between p35 and NIF-1, cDNA fragments corresponding to different domains of p35 and NIF-1 were subcloned into the yeast DNA binding-domain vector, pAS2-1, and transcriptional activation-domain vector, pACT2, respectively. Subsequent two-hybrid interaction analyses were carried out by co-transformation of plasmids containing the GAL4 DNA-binding  and activation  domains into yeast.2. Primary cortical neuron cultures were prepared from embryonic day 18 (E18) SD rats. E18 timed pregnant rats were euthanized by carbon dioxide inhalation and rapid decapitation. The E18 embryos subsequently removed and sacrificed by decapitation. Cortices were then dissociated in trypsin and plated on poly-d-lysine–coated culture plates. Cells were cultured in Neurobasal medium supplemented with 2% B27 and antibiotics. All animal experiments were conducted in accordance with the protocol number 2009056 approved by the Animal Care Committee of the Hong Kong University of Science and Technology. To examine the effects of blocking the nuclear export and inhibition of Cdk5 activity, the cells were treated with leptomycin B  or roscovitine , respectively, for 4 or 8 h before analysis.COS-7 and Neuro-2a cells  were cultured in DMEM (Invitrogen) supplemented with 10% heat-inactivated FBS plus antibiotics in an incubator at 37°C with 5% COCOS-7 and Neuro-2a cells were transfected with Lipofectamine Plus reagents (Invitrogen). Meanwhile, cortical neurons were transfected with the nucleofector transfection kit (Amaxa) or a calcium phosphate transfection assay.Escherichia coli (BL21 strain) and purified using a glutathione-Sepharose 4B column according to the manufacturer's instructions . Purified protein (1–2 µg) was bound to the glutathione-Sepharose beads and subsequently incubated with (1–2 µg) recombinant p35 protein (Abcam) or 500 µg lysates from p35-expressing COS-7 cells. The beads were washed with lysis buffer, and the bound proteins were resuspended in SDS loading buffer, resolved by SDS-PAGE, and analyzed by western blot analysis.For the pull-down analysis, recombinant proteins encoding GST-NIF-1-C, GST-NIF-1-N, and GST-NIF-1A were expressed in in vitro phosphorylation assay. The kinase assay was performed in kinase buffer  containing 1 µCi [γ-32P] ATP for 30 min at 30°C. GST protein and histone H1 served as negative and positive controls, respectively. The phosphorylated proteins were separated by SDS-PAGE and visualized by autoradiography.To determine if Cdk5 can phosphorylate the C-terminal of NIF-1, 0.5 µg GST-NIF-1-C recombinant protein was used as substrate for reconstituted Cdk5/p25 in the 2+ and 500 µM Mg2+. The cells were fixed by 4% paraformaldehyde and 5% sucrose in PBS with Ca2+ and Mg2+ for 30 min and subsequently washed 3 times with 1× PBS. After blocking with 1% bovine serum albumin, 4% goat serum, and 0.1% Triton X-100 (Sigma) for 20 min, the cells were incubated with the corresponding primary antibody overnight at 4°C and subsequently washed 3 times with 1× PBS. Alexa Fluor secondary antibody was added with 1% bovine serum albumin for 1 h at room temperature in the dark. The cells were washed 5 times with 1× PBS, stained with DAPI, and mounted with MOWIOL for fluorescent microscopy.To determine the subcellular localizations of NIF-1, p35, p25, and p39, COS-7 cells were washed with 1× PBS with 900 µM Cat-test and one-way ANOVA.All data are expressed as the arithmetic mean ± standard error of the mean (SEM). Comparisons between groups were made using Student's th zinc finger and leucine zipper-like motif  were sufficient for binding to p35 (As a first step to identify p35-interacting protein(s), full-length p35 was used as a bait to screen a postnatal day 14 mouse muscle cDNA library using a yeast two-hybrid screen g to p35 . On the g to p35 .To confirm that NIF-1 directly binds to p35, lysate of p35-expressing COS-7 cells or recombinant p35 protein was incubated with GST fusion proteins encoding different regions of NIF-1. Intriguingly, p35 was pulled down efficiently by the C-terminal fragments of NIF-1 . Although active Cdk5/p25 phosphorylated NIF-1 , the inhNext, we examined whether p35 indeed shuttles between the nucleus and cytoplasm in neuronal cells. While p35WT was prominently detected in the cell bodies and along the neurites of Neuro-2a cells, its expression was barely detected in the nucleus. In contrast, p35NES was prominently expressed in the nuclei of Neuro-2a cells . More imThe neuronal activator of Cdk5, p35, is believed to exert its functions through its interaction with Cdk5, resulting in the phosphorylation of various cellular substrates Deficiency of p35 results in aberrant brain development and adult lethality, suggesting that the protein is critical for neural development Transcriptional activation induced by neuronal activity and growth factors is a pivotal control mechanism of neuronal development Many transcription factors that were initially found to be localized in the nucleus were subsequently shown to be expressed in the cytoplasmic regions and exert specific functions. For example, the localization of the transcription factors Stat3 and neurogenin-3 to cytoplasmic compartments is important for their functions in tumorigenesis and synaptogenesis, respectively Taken together, the present findings reveal a newly identified NES on p35 that regulates the nucleocytoplasmic shuttling of p35. This protein trafficking mechanism may result in the redistribution of p35 and its interacting partners  between the nucleus and cytoplasm, thus demonstrating a new molecular mechanism by which p35 modulates gene transcription.


N-acetyl galactosamine , a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense oligonucleotides (ASOs) 6–10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S-cEt  gapmer ASOs, ∼60-fold enhancement in potency relative to the parent MOE (2′-O-methoxyethyl RNA) ASO was observed. GN3-conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3-ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3-ASO conjugate was detected in plasma suggesting that GN3 acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver. GalNAc conjugation also enhanced potency and duration of the effect of two ASOs targeting human apolipoprotein C-III and human transthyretin (TTR) in transgenic mice. The unconjugated ASOs are currently in late stage clinical trials for the treatment of familial chylomicronemia and TTR-mediated polyneuropathy. The ability to translate these observations in humans offers the potential to improve therapeutic index, reduce cost of therapy and support a monthly dosing schedule for therapeutic suppression of gene expression in the liver using ASOs.Triantennary


Cdk5 has been implicated in a multitude of processes in neuronal development, cell biology and physiology. These influence many neurological disorders, but the very breadth of Cdk5 effects has made it difficult to synthesize a coherent picture of the part played by this protein in health and disease. In this review, we focus on the roles of Cdk5 in neuronal function, particularly synaptic homeostasis, plasticity, neurotransmission, subcellular organization, and trafficking. We then discuss how disruption of these Cdk5 activities may initiate or exacerbate neural disorders. A recurring theme will be the sensitivity of Cdk5 sequelae to the precise biological context under consideration. Cyclin-dependent kinase 5 (Cdk5) is a protein serine, threonine kinase that regulates neuronal migration, neurite extension and compartmentalization –3, both In this review, we summarize the roles that Cdk5 plays in synaptic homeostasis, plasticity, neurotransmission, synaptic position, and intracellular trafficking, with an eye to gaining insight into how disruption of these activities of Cdk5 contributes to neural disease. A common theme throughout this review will be the seemingly contradictory effects of Cdk5 on various aspects of neuronal activity and structure in different experimental systems, reflecting the remarkable dependence of Cdk5 action upon the cell type, age, homeostatic set point and other aspects of the biological context of the neuron under study.in vivo, though they appear to be interchangeable in most experimental paradigms [Cdk5 is a member of the cyclin-dependent kinase family of protein kinases, closely related to the Cdk regulators of the cell division cycle. Unlike other members of the Cdk family, however, Cdk5 is not activated by canonical cyclins , and doearadigms . Cdk5 isaradigms . This pharadigms . The staaradigms .Cdk5 is a major regulator of synaptic function through its control of vesicle exocytosis and endocytosis . Indeed,Cdk5 uses multiple routes to modulate vesicle endocytosis, and although the targets are largely agreed upon, the outcomes of these interactions are often disputed. For example, Cdk5 phosphorylates synapsin-I, although whether this interaction increases or decreases synaptic transmission is unknown . FurtherCdk5, and more specifically its balance with calcineurin, also directly determines the rate of vesicle recycling, with acute inhibition of Cdk5 serving to free resting vesicles into the recyclable vesicle pool. The dynamic between Cdk5 and calcineurin is further influenced by pre-synaptic activity, since chronic silencing of neuronal activity by tetrodotoxin (TTX) suppresses presynaptic Cdk5 activity .Cdk5 has profound effects on nervous system activity through altering the synthesis or receptor function of acetylcholinergic –35, cateMr 32 kDa), a signal transduction molecule that regulates the amount of dopamine signaling in neostriatal neurons [2+, inducing calpain proteolysis of p35 to p25, and leading to increased Cdk5 activation. This in turn induces hyperphosphorylation of tau producing signs of neurodegeneration, including cell death [Cdk5 regulates dopamine (DA) synthesis via the rate-limiting enzyme in catecholamine synthesis, Tyrosine Hydroxylase (TH). Through phosphorylation of serine 31 of TH, Cdk5 increases the stability and activity of the protein in the substantia nigra of the midbrain ,38. The  neurons ,39. The ll death .Regulation of glutamatergic transmission by Cdk5 is largely postsynaptic, and again involves a variety of molecular pathways. NMDA-type glutamate receptors are direct targets of Cdk5, with the kinase phosphorylating the NR2A subunit of NMDA receptors on Ser- 1232 to increase channel activity . In thisBeyond modification of the receptor itself, Cdk5 also regulates glutamatergic transmission at the level of structural proteins of the postsynaptic density. The post-synaptic scaffolding protein PSD-95 regulates clustering and density of ionotropic glutamate receptors, and is a substrate of Cdk5 ,42. PSD-Many of the synaptic effects of Cdk5 modulate neural plasticity and synaptic homeostasis. To protect against excessive excitation, pre-and post-synaptic neurons engage in a homeostatic process known as synaptic scaling, which is a form of plasticity that allows individual neurons to regulate their overall rate of firing action potentials . During Another mechanism of PSD-linked, Cdk5-dependent synapse loss, in frontal cortical neurons, stems from the interaction of Cdk5 with the postsynaptic density scaffolding protein GKAP from the MAGUK family of proteins . SolubleThe net effect of Cdk5 phosphorylation of a multitude of synaptic targets is regulation of global organismal processes such as cognition and behavior. Hebbian Long-Term Potentiation (LTP) strengthens synapses and is presumed to be a central mechanism of learning and memory. Cdk5 activity clearly modulates LTP, but in just what way remains controversial. For example, inhibition of Cdk5 with roscovitine has been reported to block induction of LTP . Moreoveenhanced associative and spatial memory for hippocampus-dependent tasks, though no differences were observed in hippocampus-independent tasks. Indeed, Ris et al. [Cdk5 function has profound effects on various forms of hippocampal learning and memory ,7,58,59.s et al.  hypothess et al. . Inactivs et al. , but is s et al. . OverallBeyond regulating the composition and transmission properties of individual synapses, Cdk5 also modulates the overall structure and sub cellular organization of neurons. This includes the polarization of the neuron, compartmentalization of the axon and trafficking of its components, the structure of dendrites and synapse density . A growiAn early step in organizing a mammalian neuron is the selection of one of its neurites to be the nascent axon while the others differentiate into dendrites. Axon formation is based on tightly regulated events of cytoskeletal reorganization  in respoin vivo. Separate studies, however, showed that Sema3A function is mediated by Cdk5 [The possibility of a very different and indeed opposite, effect on axon formation for Cdk5 is suggested by its interaction with the repulsive guidance molecule Semaphorin3A (Sema3A) . Sema3A  by Cdk5 . Cdk5 ph by Cdk5 ,69. ThisDrosophila show that Cdk5 is a dose-dependent regulator of AIS size [Axons are not just featureless shafts. They have a complex internal organization that is fundamental to neuronal function and maintenance. The Axon Initial Segment (AIS) is the site of action potential initiation, and it acts as a gatekeeper in intra-axonal traffic, segregating the somatodendritic vs axonal compartments . MoreoveAIS size . Reductivs other regions of the cell. For another potassium channel Kv2.1, Cdk5-mediated phosphorylation regulates the steady state level of channel clustering in somatic and axonal compartments [The unique electrophysiological properties of the AIS, particularly initiation of action potentials, rely on selective enrichment of specific subtypes of sodium, potassium and calcium channels, and exclusion of others . In matuartments . This phartments ,75.Propagation of action potentials in myelinated neurons depends on the function of ion channels clustered at nodes of Ranvier ,77. ClusDendrites are dynamic structures. Their morphology and organization control how neurons process information , and strCdk5 controls dendritic spine density using a variety of molecular mechanisms. Cdk5 phosphorylation of the WAVE1 protein inhibits its interaction with the Arp2/3 actin polymerization complex, and consequently inhibits spine morphogenesis –87. ThisNeuronal excitability and neurotransmission are profoundly sensitive to long-range intracellular trafficking. Cdk5 regulates this process by modifying motors, their cargo, the scaffolding proteins that connect them, and the cytoskeletal proteins themselves, as we now discuss.Outward transport from the soma to the distal tips of the axon and dendrites is largely powered by the diverse family of kinesins (KIFs) ,92. KineCdk5 has a well-established role in regulating retrograde trafficking in the axon by controlling the activity of dynein as well as its interaction with different cargos. Cdk5 phosphorylates NUDEL (Ndel1), a protein that binds both dynein and the dynein interacting protein, LIS1 ,96. PhosC. elegans have offered particularly rich opportunities for dissecting some of the many ways that this kinase regulates axonal trafficking. For example, in cholinergic DA9 motoneurons Cdk5 cooperates with the PCTAIRE kinase (PCT-1) to suppress retrograde transport by negatively regulating dynein function [Cdk5 mutants and the number of retrogradely-moving vesicles was significantly increased in PCT-1, Cdk-5 double mutants. That this reflects a shift in a dynamic balance of anterograde vs retrograde motility is demonstrated by the observation that mutations in the genes encoding dynein heavy chain or Ndel can suppress the mislocalization phenotype of Cdk5 mutants.Studies of Cdk5 function in function . In thisC. elegans neurons, Cdk5 interacts with PCT-1 in different ways to control the balance of anterograde and retrograde transport. In a subset of cholinergic neurons PCT-1 was shown to be a downstream target of Cdk5 rather than acting redundantly [In other undantly . In yet undantly . These nActin-based mechanisms of polarized transport also exist in neurons, particularly for short-range trafficking, and are also modulated by Cdk5 ,101. TheC. elegans, Cdk5 promotes trafficking of glutamate receptors (GLR-1), probably acting at an early stage in the secretory process, in the soma [The specificity of neuronal trafficking relies heavily on adaptor and scaffolding proteins that link cargos to their appropriate motors, and Cdk5 regulation is often directed at these interactions. In the soma . It is tthe soma . By thisC. elegans [vs dynein and thus promoting polarized trafficking in axons and preventing dynein dependent trafficking into dendrites.Cargo selective effects of Cdk5 on transport are also apparent in the specific trafficking of neuropeptide filled Dense Core Vesicles (DCVs) in cholinergic DB motoneurons of  elegans . Cdk5 ac elegans  suggesteIn several cases, Cdk5-regulated adaptor proteins are implicated in neurological disorders making them of particular interest. Huntingtin (Htt), the protein mutated in Huntington’s disease, acts as a scaffolding protein that facilitates interaction between dynein and kinesin motors and their cargo, and phosphorylation of Htt by Akt kinase regulates the directionality of Htt-dependent transport ,106. CdkAnother Cdk5-associated adaptor protein is Disrupted in schizophrenia 1 (DISC1), which has multiple functions including microtubule-mediated transport and regulation of the structure and composition of dendritic spines . MutatioPost translation modifications of MTs control their stability and availability for interactions with other molecules, including motors. Cdk5/p35 binds tubulin, promoting microtubule polymerization and stability ,102,112.per se, or to the effects of the modification on wild type functions of tau in processes such as trafficking. In this context, it is important to remember that neurotoxic effects of altered Cdk5 activity have been associated with changes in trafficking and clustering of various cellular organelles, including ER and mitochondria [Cdk5 also regulates the microtubule cytoskeleton through its effects on Microtubule Associated Proteins (MAPs) and intermediate filaments (IFs). Two of the most prominent MAPs, MAP2 and tau, are well-established targets of CDK5 activity ,115,116.chondria ,126.Neurofilaments (NFs) also have multiple potential Cdk5 phosphorylation sites on their C-terminal tails . The extThus, Cdk5 regulates neuronal trafficking at a variety of levels, and in ways that are highly specialized depending on cell type, cellular compartment and physiological state. Many of these activities of Cdk5 are undoubtedly connected to its roles in neuropathology, as we will discuss below.In the final section of this review we will consider how disruptions in the function of Cdk5, and particularly those functions related to neuronal excitability, neurotransmission, protein trafficking and the compartmentalization of the neuron, contribute to neurological disease.Given the many ways in which Cdk5 modulates neural structure and activity, it is not surprising that alterations of Cdk5 activity are associated with an equally broad range of neurological disorders. The neurotransmission effects of Cdk5 have many impacts on neural function, sometimes in complex and unexpected ways. For example, Cdk5 can alter dopaminergic transduction, and, in turn, DA and NMDA receptor activity can cause over activation of Cdk5 that leads to toxicity and cell death. Paoletti et al.  propose Emotional disorders may also be linked to Cdk5, both through its generalized effects on neuronal excitability and more specifically those on DA metabolism and function. Dopamine and the dopaminergic nuclei  are important in reward signaling. Reward or reinforcement cause a behavior to increase in intensity or frequency and is critical to the development of habit. Almost all addictive drugs cause increased dopamine release and this signaling is an important neural basis of substance abuse. Cdk5 is implicated in regulating the neural processes underlying cocaine addiction through its effects on dopaminergic neurotransmission . ChronicIn addition to drug-based models of Cdk5-mediated emotional disorders, p35 loss-of-function has been proposed as a model of hyperactivity , and thrDisruption of ion channel trafficking is often associated with neurological disorders including epilepsy, ataxia, pain and autism . ConsistAmong axonopathies, amyotrophic lateral sclerosis (ALS) has probably the clearest association with axon transport as the motor neurons affected by the disease have unique characteristics such as very long axons and high energy metabolism . ALS patSOD-1 gene caused Cdk5 mislocalization in the cell and elevated activity of Cdk5 due to a changed p35/p25 ratio, and it caused hyperphosphorylation of tau and NFs. Previous studies had shown reduction in both fast and slow anterograde axonal transport of NFs and other cytoskeleton proteins in SOD1-ALS neurons. This was accompanied by accumulation of NFs and NF inclusions in the perikarya and proximal axon of motor neurons and reduced levels of NFs in the distal axon [Alteration of Cdk5 function was shown in motor neurons in an SOD1-ALS mouse model . ALS-typtal axon ,144. Givtal axon .Morphologically, transport defects often are manifested as accumulation of materials in the soma and neurites accompanied by swollen axon segments or spheroids –146. AnaDrosophila central brain at a level exceeding that in physiologically matched controls [alpha-synuclein mutation [Recent results implicate Cdk5 activity in regulation of autophagy, though without addressing whether subcellular localization plays a role in Cdk5-dependent autophagy activation or inhibition. Loss of Cdk5 activity causes age-dependent accumulation of autophagosomes in the controls . In a mocontrols . VPS34 icontrols . Cdk5-mecontrols . In contmutation . In thesvia its regulation of the autophagy pathway stem in part from this dual nature of autophagy itself, and will depend on the details of a specific pathological context. It may also be relevant that autophagy has quite different properties in the axon vs the soma. The abundance of autophagy core complexes at the synapse and the trafficking dynamics of the process depend strongly on their interaction with both kinesin and dynein motors in primary neuronal culture [The question of whether autophagy enhances or suppresses neurodegeneration has been contentious. Autophagy can clearly be part of the mechanism of pathological degeneration, but just as clearly, lack of autophagy can trigger degeneration by the failure to remove dysfunctional mitochondria and toxic aggregates. It seems likely that the contradictory effects of Cdk5 on degeneration  culture . It is pvs inhibition, stability vs turnover, transmitter release vs recycling, transport towards vs away from the soma and many others. Many neural disorders are linked to the failure of one or more of these delicate equilibria, and in a remarkable number of cases, Cdk5 plays a central role in the establishment and maintenance of the necessary physiological balance. Here we have reviewed a few illustrative cases, particularly those associated with neurotransmission and the affiliated processes of neuronal subcellular organization. Exploiting Cdk5 in the treatment of neural disorders and disease will require a nuanced understanding of the subtle physiology to which it contributes.Neuronal physiology depends on a series of balancing acts: excitation


The bacterial ferrous iron acquisition protein FeoB assembles as a homotrimer that is predicted to form a central pore lined by conserved cysteine residues. Structure-function analysis of FeoB indicates a putative mechanism more akin to a GTP-gated channel than a transporter. Pseudomonas aeruginosa further reveals a putative iron sensor domain that could connect GTP binding/hydrolysis to the opening of the pore. These results indicate that FeoB might not act as a transporter, but rather as a GTP-gated channel.Iron is essential for the survival and virulence of pathogenic bacteria. The FeoB transporter allows the bacterial cell to acquire ferrous iron from its environment, making it an excellent drug target in intractable pathogens. The protein consists of an N-terminal GTP-binding domain and a C-terminal membrane domain. Despite the availability of X-ray crystal structures of the N-terminal domain, many aspects of the structure and function of FeoB remain unclear, such as the structure of the membrane domain, the oligomeric state of the protein, the molecular mechanism of iron transport, and how this is coupled to GTP hydrolysis at the N-terminal domain. In the present study, we describe the first homology model of FeoB. Due to the lack of sequence homology between FeoB and other transporters, the structures of four different proteins were used as templates to generate the homology model of full-length FeoB, which predicts a trimeric structure. We confirmed this trimeric structure by both blue-native-PAGE (BN-PAGE) and AFM. According to our model, the membrane domain of the trimeric protein forms a central pore lined by highly conserved cysteine residues. This pore aligns with a central pore in the N-terminal GTPase domain (G-domain) lined by aspartate residues. Biochemical analysis of FeoB from  Pseudomonas aeruginosa is a ubiquitous Gram-negative opportunistic pathogen that is intrinsically resistant to multiple classes of antimicrobials  and 50 μl of the diluted protein was adsorbed on to freshly cleaved mica for 10 min. The sample was then washed with 10x 1 ml of BPC water and dried gently in a stream of nitrogen gas. After tuning and engaging of the AFM tip, 2×2 μm2 areas were scanned at 3–4 Hz. Further processing (flattening of images) and analysis of the observed particles  were done using the SPIP software (Image Metrology). The detection threshold was 0.25 nm and adjacent particles were split when gaps were below 3 nm. Particles with a molecular volume (Z Net Volume) below 50 nm3 were neglected. Molecular volume based on molecular mass was calculated using the equation: Vc=(M0/N0)(V1 + dV2), where M0 is the molecular mass, N0 is Avogadro's number, V1 and V2 are the partial specific volumes of particle (0.74 cm3/g) and water (1 cm3/g) respectively, and d is the extent of protein hydration (taken as 0.4 g water/g protein).AFM imaging was carried out using a Bruker Multimode 2 instrument and a Nanoscope controller IIIa equipped with a 120 μm J-scanner and a dry imaging cell. Silicon cantilevers had a typical drive frequency of ∼300 kHz and a specified spring constant of ∼42 N/m . The NV10 polymer used for the BN-PAGE was not compatible with AFM as it formed a sheet on the mica substrate. For this reason, Cfor FeoB . IsolateIt has been shown previously  that forThe C429S and C675S mutants of FeoB were generated by PCR with pFeoBH as a template, and using primers 5′-CGTTCATGTCCAGCGGCGCGCGCCTGGCGATC-3′ and 5′-GGCGCGCGCCGCTGGACATGAACGGCGCCATC-3′ for the C429S substitution and 5′-CTACGTGCCCAGCG-TGACCGCCATGGGCGC-3′ and 5′-GGCGGTCACGCT-GGGCACGTAGAGCAGGACG-3′ for the C675S substitution. PCR was performed using the high-fidelity Phusion DNA polymerase kit. The wild-type plasmid DNA template was removed by digestion of the PCR product with DpnI (New England Biolabs). The PCR products were digested with NdeI/HindIII (Fermentas) and ligated into digested pET41a(+) to yield plasmids pC429S-FeoBH and pC675S-FeoBH. The cloned PCR products were sequenced to ensure that only the intended changes were introduced.i released during hydrolysis of GTP. Reactions were prepared to give a final volume of 150 μl, containing the assay buffer (BioAssay Systems), 10 mM MgSO4, 1 mM GTP and 20–50 μg/ml of purified protein in 20 mM K-HEPES pH 7.0 buffer. The GTPase assay was performed at 37°C with continuous shaking after initiation of the reaction by addition of GTP (1 mM). Samples were taken at different time intervals over a total of 4 h. Reactions were terminated by addition of the colouring reagent (100 μl) and the A630 values were read after 30 min using a BioTek EL800 plate reader. A standard curve was prepared simultaneously using phosphate dilutions (0–5 nM).GTPase activity was determined with a malachite green assay ,53 usingSo far, all structural and most functional studies on FeoB have focused on the soluble domain, NFeoB. However, in order to understand the molecular mechanism of iron transport and how this is linked to GTP hydrolysis, it is imperative to study the full-length protein. To this end, it would be extremely valuable to have a structure on which to base experimental design. However, progress in this field is hampered by the general difficulty in handling membrane proteins. Moreover, FeoB is such a unique type of transporter that it is difficult to find models that could act as a suitable blueprint.E. coli and P. aeruginosa, our model of NFeoB was based on the template structure of E. coli NFeoB (PDB code: 3HYT), which was shown previously to crystallize as a homotrimer [2+ binding in all G-proteins, as well as the effector-binding regions recognized as Switch I (residues 25–40) and Switch II (residues 70–85), were identified based on the alignment shown in z-DOPE score for our model was −0.26 whereas the RMSD value that compares the template and model was ∼3.6. The GA341 score (an indication for fold assessment) was 1.Modelling of the cytoplasmic domain of FeoB  was relatively straightforward owing to the existence of several crystal structures of this domain from various organisms. Due to the high level of identity (59%) between the FeoB proteins from P. horikoshii [In contrast with the cytosolic domain, it was difficult to find modelling templates for the TM domain of FeoB with traditional methods as there are no structures of membrane proteins with a high sequence similarity to FeoB. We therefore decided to use a protein with a related function, such as a membrane transport protein, which also contains motifs present in FeoB. As a result, we selected the glutamate transporter GltPh from rikoshii  which isrikoshii ,55. UnliThree algorithms were used to characterize the topology of the crystallized GltPh protein . Of these, only MPEx was able to predict TM helix (TMH) topology successfully, since MPEx was the only algorithm that identified the TMH4 and the TMH8 of the glutamate transporter . None of429) and TMH7 (Cys675). Cysteine residues are good ligands for soft Lewis acids, such as Fe2+, suggesting that the conserved cysteine residues in FeoB could be involved in metal binding during the transport process [429 in the TMH4, suggesting that this helix may form the pore of the transporter. In the homotrimer, the three cysteine residues line a ring in the middle of the membrane domain with a diameter of 4.9 Å (1 Å=0.1 nm), which would represent an ‘open’ state and could allow Fe2+ to pass  in the Pfam database , certain process . The pre to pass . The othInterestingly, the linker (residues 264–286) that connects the soluble domain to the membrane domain of FeoB could form an α-helix according to the sequence identity with the EHD ATPase protein . The space constraints derived from the modelling forced this helix into a semicircle; however, it is more likely to be a straight helix in the full-length protein once the GTP domain is active.2+-binding cysteine/histidine-rich region, presumed to be located in the cytosol. This feature is somewhat similar to the metal-binding motifs (CXXC), which may have a chaperone function, found in the metal-transporting P-type ATPases [2+ translocation [The C-terminal region of FeoB (residues 740–766) was modelled using as a template the C-terminal domain of the dihydrodipicolinate reductase from the glyceraldehyde-3-phosphate dehydrogenase-like superfamily  . The assembly state of FeoB was further investigated at single-molecule resolution using AFM. AFM images of FeoB in 0.5% Cus sizes A. More sus sizes B that pr 844 nm3 C. As sho2+ transport. In order to investigate the roles of these residues, we first established a method for measuring the direct involvement of FeoB in Fe2+ transport. ATPases and GTPases normally have a low basal activity which is stimulated by their substrates. Given the predicted function of FeoB as a Fe2+ transporting protein, the GTPase activity of the protein was tested in the presence of Fe2+. To ensure that the reduced state of iron was maintained throughout the reaction, FeSO4 was prepared in ascorbic acid, and this reducing agent was also included in the reaction to prevent the oxidation of iron. The GTPase activity of FeoB was monitored in the presence of varying Fe2+ concentrations. As predicted, an increase in Fe2+ concentration caused a progressive increase in GTPase activity, up to a maximum at 1–1.5 mM Fe2+  are involved in Fe2+ transport, mutants of FeoB were prepared in which the cysteine residues were replaced with serine residues. The GTPase activities of C426S FeoB and C675S FeoB were determined in the absence or presence of Fe2+. The basal GTPase activity of FeoB was not affected by either mutation (results not shown). As Cys429 lines a central pore in the derived structure, it was expected to play a central role in Fe2+ transport. Unexpectedly, the Fe2+-stimulated GTPase activity of C429S FeoB was not different from that of the wild-type protein . The pore in NFeoB is aligned with the pore in the TM domain, so that it could potentially form a continuous pathway for Fe2+. In NFeoB from E. coli, the nucleotide-free and nucleotide-bound structures correspond to the closed and open states of a central cytoplasmic pore respectively [2+ [The NFeoB is conserved, and the modelled structure was consistent with our expectations  and 5. Ites flow –71. The tes flow ,72. In sn Gate 1 . The por(E. coli . Our finectively . The 4.5vely [2+ .675) was positioned on TMH7 at the membrane-periplasm interface in Gate 2  would hamper Fe2+-stimulated GTPase activity, whereas a mutation in the ‘pore’ motif (Cys429) would only affect substrate transport. 2+-stimulated GTPase activity.The second conserved cysteine residue . It has been suggested that FeoA, the small cytosolic protein from the Feo operon, could act as a GAP to activate FeoB [Corynebacterium diphtheria. Further, FeoA contains an SH3 domain, which would typically mediate protein–protein interactions [E. coli could be stimulated in part by K+ ions [P. aeruginosa (results not shown).FeoB has always presented a conundrum. The G-domain is needed for Feate FeoB . FeoA shractions ,74. Thisractions . Howeverractions . The GTP K+ ions ,77; howe−/H+ exchange transporter, indicating that the boundary between channels and transporters is not clear cut [2+/H+ antiporter still needs to be established. The placement of the residues in our model also still has to be verified experimentally. However, the notion of sensor and pore domains in a GTPase is completely novel. Our model not only provides the first structure of full-length FeoB, but also predicts many interesting new features of the protein. We suggest that use of this model could inform many future experiments into the structure and molecular mechanism of FeoB proteins.At present the most plausible mechanism of FeoB action, which is compatible with the biochemical measurements, is that FeoB does not act as a transporter, but rather as a GTP-gated channel, just as ATP hydrolysis drives gating in the cystic fibrosis transmembrane conductance regulator (CFTR). Similar to FeoB, CFTR also has a very low nucleotide hydrolysis rate that is only loosely connected with channel activity . In factlear cut . Whether


N-linked glycan intermediates displayed on proteins serve as quality tags. The ER folding-sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) operates as the gatekeeper for ER quality control by specifically transferring monoglucose residues to incompletely folded glycoproteins, thereby allowing them to interact with lectin chaperone complexes to facilitate their folding. Despite its functional importance, no structural information is available for this key enzyme to date. To elucidate the folding-sensor mechanism in the ER, we performed a structural study of UGGT. Based on bioinformatics analyses, the folding-sensor region of UGGT was predicted to harbour three tandem thioredoxin (Trx)-like domains, which are often found in proteins involved in ER quality control. Furthermore, we determined the three-dimensional structure of the third Trx-like domain, which exhibits an extensive hydrophobic patch concealed by its flexible C-terminal helix. Our structural data suggest that this hydrophobic patch is involved in intermolecular interactions, thereby contributing to the folding-sensor mechanism of UGGT.The endoplasmic reticulum (ER) possesses a protein quality control system that supports the efficient folding of newly synthesized glycoproteins. In this system, a series of  N-linked oligosaccharides displayed on polypeptide chains function as quality tags for the determination of glycoprotein fates, i.e. folding, transport or degradation, that are selectively recognized by certain intracellular lectins245In eukaryotic cells, proteins destined for the secretory pathway are translocated to the endoplasmic reticulum (ER) for folding, assembly and post-translational modification, including asparagine-linked glycosylation. To guarantee that only correctly folded glycoproteins are transported to the Golgi apparatus, the ER possesses a sophisticated protein quality control system1234563Man9GlcNAc2), which contains three non-reducing terminal branches 797121314151617In the ER, newly synthesized proteins are cotranslationally modified with high mannose-type tetradecasaccharide Glc3ManGlcNAc2, UGGT acts as the gatekeeper in this system because this enzyme is capable of sensing the folding states of glycoproteins as potential substrates. UGGT only transfers monoglucose residues to incompletely folded glycoproteins7121319Chaetomium thermophilum, a thermophilic fungus, which survives at temperatures of up to 60°CIn this study, to elucidate the working mechanism of UGGT, we attempted to characterize the three-dimensional (3D) structure of its N-terminal folding-sensor region. We selected To investigate the structure of the N-terminal folding-sensor region of UGGT, we subjected its amino acid sequence (residues 28–1198) to bioinformatics analysis using the programs PSIPREDversus Trx2, 22.1%; Trx1 versus Trx3, 23.3%; Trx2 versus Trx3, 16.2% in C. thermophilum), suggesting variability in their three-dimensional structures.Next, we attempted to identify structural domain(s) within the N-terminal folding-sensor region using InterProEscherichia coli cells. Therefore, we made tandem constructs for their expression. Consequently, we were able to express Trx1-Trx2, Trx2-Trx3 and Trx1-Trx2-Trx3 proteins in their soluble form. Of these constructs, we successfully crystallized the Trx3 domain with the optimization of its N- and C-terminal sequences (residues 671–831), based on the identification of proteolytically stable fragments. However, despite extensive trials, we were unable to obtain crystals of the tandem constructs Trx1-Trx2, Trx2-Trx3 or Trx1-Trx2-Trx3.Based on the bioinformatic prediction that folding-sensor region of UGGT possesses three tandem Trx-like domains, we performed bacterial expression, purification and crystallization of a series of Trx domains. First, we expressed each of the three Trx domains. Although we were able to express the Trx3 domain as a soluble protein, the Trx1 and Trx2 domains formed inclusion bodies in Rwork of 23.5% and Rfree of 29.2%  than the average value . As representative of the DsbA/C structure, the well-characterized crystal structure of E. coli DsbC (PDB code: 1EEJ)E. coli thioredoxin trxAE. coli trxAAs expected from the bioinformatics analysis, the crystal structure displayed a typical Trx-like fold, i.e. a five-stranded β-sheet with a β1–β3–β2–β4–β5 arrangement surrounded by six α-helices . In the 79.7 Å2; . CompariThe C-terminal α6 helix, which is followed by a putatively flexible linker region in UGGT, was completely disordered in the crystal structure of Form 2, suggesting the instability of this helix , left. Ba, b, b′ and a′), of which a and a′ domains have a CXXC catalytic motif, whereas b and b′ domains do not31cis-Pro loop adjacent to the CXXC motif, a hallmark of redox-active Trx-fold proteins3334In this study, we proposed that the folding-sensor region of UGGT contains three tandem Trx-like domains and, solved the first 3D structure of a structural domain, i.e. the third Trx-like domain, of this functional region . Trx-lik29Growing evidence implies that UGGT exhibits glucosyltransferase activity only against incompletely folded glycoproteins, suggesting that the folding-sensor region has exposed the hydrophobic patch as a principal substrate-binding site7121335In summary, our bioinformatic analyses predicted that the folding-sensor region of UGGT harbours three tandem Trx-like domains. Moreover, we provided snapshots of the 3D structure of the third Trx-like domain, in which a putative substrate-binding hydrophobic patch is intramolecularly masked or involved in an intermolecular interaction, offering a key breakthrough toward understanding of the functional mechanisms of this ER folding-sensor enzyme.C. thermophilum var. thermophilum La Touche (DSM 1495) was obtained from DSMZ, Braunschweig, Germany. Total RNA was isolated using TRIzol® reagent (Life Technologies). The cDNA was synthesized using SuperScript® III Reverse Transcriptase (Life Technologies) with oligo d(T) primers according to the manufacturer's instructions. Full-length UGGT cDNA was cloned by PCR using a C. thermophilum genomic DNA databaseS-transferase (GST)-fused proteins. The Trx1 (residues 168–379), Trx2 (residues 467–624), Trx3 (residues 671–831), Trx1-Trx2 (residues 168–624), Trx2-Trx3 (residues 467–831) and Trx1-Trx2-Trx3 (residues 168–831) domains were amplified by PCR and subcloned into the BamHI and XbaI sites of a modified pCold-GST vector (Takara Bio Inc.)E. coli BL21 Star™ cells (Life Technologies) according to the manufacturer's protocols (Takara Bio Inc.). GST-fused proteins were purified using glutathione-Sepharose™ columns . Subsequently, the GST tag was removed by adding TEV protease to the resin for 12 h at 277 K, leaving two additional residues Gly-Ser at the N-terminus. The resultant proteins were further purified by size-exclusion chromatography  using a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.1 mM EDTA. The selenomethione (SeMet)-labelled Trx3 domain was expressed in E. coli B834 (DE3) using M9 minimal medium with SeMet. Expression and purification were performed following the same protocol as that for the native protein. Purified proteins were dialyzed against a buffer containing 10 mM Tris-HCl (pH 7.5) and 100 mM NaCl. The integrity of the protein samples was validated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis using an AXIMA-CFR™ spectrometer (Shimadzu) and N-terminal Edman sequencing with a Procise 494HT protein sequenator (ABI/Life Technologies).· 3,6,9,12,15,18,21,24-octaoxahexatriacontan-1-ol) mixed with an equal volume of precipitant solution containing 23% PEG3350, 0.1 M Tris-HCl (pH 7.0) and 0.2 M ammonium acetate for 6 days at 289 K. The crystals were transferred into the reservoir solution and flash-cooled in liquid nitrogen. Data sets for Forms 1 and 2 were collected using synchrotron radiation at 13B1 of the National Synchrotron Radiation Research Center  and AR-NW12A of the Photon Factory , respectively. All diffraction data were processed using HKL2000The crystals of the Trx3 domain of UGGT  were grown in a buffer containing 60% Tacsimate (pH 7.0) for 2 weeks at 289 K. The crystals of the Trx3 domain of UGGT (Form 2) were obtained by equilibrating a solution of 8 mg/ml protein with 1.2 mM ANAPOE C12E8 (polyoxyethylene[8]dodecyl ether http://www.pymol.org/). Homology modeling of the Trx1 and Trx2 domains were performed using Phyre2Neisseria gonorrhoeae DsbC-like protein (PDB code: 3GV1) and Neisseria meningitidis DsbA1 (PDB code: 3DVW) as templates, respectively.The 1.70 Å-resolution crystal structure of the Trx3 domain of UGGT (Form 2) was solved using the SAD method. The initial phase was determined using the SHELX C/D/E programT.S. and K.K. conceived and designed the experiments; T.Z. and T.S. performed the bioinformatics analyses and crystallographic experiments; all authors wrote and reviewed the manuscript.Accession codes The coordinates and structural factors of the crystal structures of the Trx3 domain of C. thermophilum UGGT (Forms 1 and 2) have been deposited in the Protein Data Bank under the accession numbers 3WZT and 3WZS, respectively.Supplementary information


Miniaturization of magnonic devices for wave-like computing requires emission of short-wavelength spin waves, a key feature that cannot be achieved with microwave antennas. In this paper, we propose a tunable source of short-wavelength spin waves based on highly localized and strongly pinned magnetic domain walls in ferroelectric-ferromagnetic bilayers. When driven into oscillation by a microwave spin-polarized current, the magnetic domain walls emit spin waves with the same frequency as the excitation current. The amplitude of the emitted spin waves and the range of attainable excitation frequencies depend on the availability of domain wall resonance modes. In this respect, pinned domain walls in magnetic nanowires are particularly attractive. In this geometry, spin wave confinement perpendicular to the nanowire axis produces a multitude of domain wall resonances enabling efficient spin wave emission at frequencies up to 100 GHz and wavelengths down to 20 nm. At high frequency, the emission of spin waves in magnetic nanowires becomes monochromatic. Moreover, pinning of magnetic domain wall oscillators onto the same ferroelectric domain boundary in parallel nanowires guarantees good coherency between spin wave sources, which opens perspectives towards the realization of Mach-Zehnder type logic devices and sensors. Collective spin wave excitations in ferromagnetic materials provide a promising platform for low power wave-like computing in nanoscale devices up to THz frequencies124671516173 layer, but the concept can be extended to other ferroelectric materials and magnetoelectric coupling mechanisms. Strain-coupling between the ferroelectric and ferromagnetic layer originates in the transfer of in-plane lattice tetragonality from the ferroelectric domains to the ferromagnetic material, leading to modulations of magnetic anisotropy via inverse magnetostriction. While other domain patterns are also suitable, the ferroelectric layer considered in this paper consist of a regular repetition of a1- and a2 stripe domains. This domain pattern is characterized by abrupt 90 degree rotations of the ferroelectric polarization and lattice tetragonality in the film plane. Because of strain coupling, uniaxial magnetic anisotropy is induced in the ferromagnetic film that covers the alternating a1- and a2 stripe domains. The orientation of the uniaxial magnetic anisotropy follows the lattice tetragonality in the ferroelectric layer, i.e. it rotates by 90 degrees at domain boundaries. The regular modulation of magnetic anisotropy in the ferromagnetic film leads to full domain pattern transfer from the ferroelectric layer, an effect that has been experimentally demonstrated for various magnetic materials on BaTiO320212224In this work, we propose a highly tunable source of short-wavelength spin waves based on strongly pinned oscillating domain walls in a ferromagnetic-ferroelectric bilayer. In particular, we consider ferromagnetic films that are strain-coupled to a ferroelectric BaTiOTo analyze spin wave excitation from a pinned magnetic domain wall and the propagation of these waves in the ferromagnetic domains, micromagnetic simulations are performed. The domain wall is excited by an ac spin-polarized current26μm2 region with an a1 and a2 domain, is shown in x- and y-direction to mimic stripe domains with infinite stripe length  and a width of 3.2 μm. Strain coupling to the ferroelectric layer is implemented by a magnetic anisotropy (Ku) and abrupt 90 degree rotation of the uniaxial anisotropy axis at the a1 − a2 domain boundary. Simulations are performed for zero applied magnetic field after initialization of a head-to-tail magnetization configuration in the ferromagnetic domains  wave-vector spectra  and perpendicular (surface spin waves) to the magnetization. In the ferromagnetic domains of the hybrid bilayer that we consider, the remanent magnetization direction is set by the uniaxial magnetic anisotropy. The oscillating domain wall emits spin waves at an angle of 45 degrees with respect to this magnetization direction. To obtain further insights in the propagation of spin waves inside the ferromagnetic domains, we interpret the domain wall emitter as a line source that consists of an infinite amount of point sources. Each point along the oscillating domain wall emits spin waves which interfere to an overall spin wave pattern following the Huygens-Fresnel principle. In the following simulations, we analyze the anisotropic propagation properties of spin waves that are emitted from a point-like source by exciting the magnetic domain wall over a length of only 50 nm. Magnetization profiles after 20 excitation periods are shown in The simulations reveal that spin waves propagate along the domain wall and into the ferromagnetic domains. At  GHz , the magquencies , nodal lal bands , one at n an integer)) suggests that modes with Spin wave propagation into the ferromagnetic domains is highly anisotropic . It is mϕ between the wave vector and local magnetization. In this model, the 0th order approximation for ω , which originate from the congruent isofrequency contours in reciprocal space. The dispersion diagram for spin wave propagation along the x-direction (irection  contains3), which makes the application of a bias field superfluous. The dispersion diagram of 2 to increase the spin wave signal. Similar to the extended film geometry, a broad non-dispersive band is obtained around nth-DE mode and the nanowire width (w) is given by The thin-film domain wall oscillator that we described thus far generates spin waves with wavelengths down to  GHz see . At highwall see . The resf = 50 GHz in λ = 25 nm. The reason for these attractive emission properties is twofold: (i) Contrary to the extended film geometry, higher order domain wall resonances exist at high frequency because of spin wave confinement perpendicular to the nanowire axis. As a result, large-amplitude spin waves are emitted into the nanowire over a wide range of excitation frequencies. The spin wave profiles of f = 50 GHz, for instance, the 12th-DE resonance mode generates short-wavelength spin waves that propagate into the nanowire over several micrometers. (ii) The second key feature of the magnetic domain wall oscillator is its straight and narrow excitation area, which is provided by strong pinning onto the few nanometers wide a1 − a2 domain boundary in the ferroelectric layer. The uniformity of the pinning potential is thus not limited by the resolution of a lithography process. These important attributes and the absence of long-range magnetic bias fields open up perspectives towards the development of miniaturized magnonic devices. In such sub-micrometer magnonic elements, damping of spin waves is no longer a limiting factor, while large group velocities guarantee fast signal throughput in a broad GHz frequency range.Most interestingly, the dispersion diagram evidences that the implementation of a domain wall oscillator in a magnetic nanowire allows for the emission of short-wavelength spin waves at high frequencies. For example, the wave vector of propagating spin waves at an excitation frequency of f = 50 GHz is δk ≈ 0.01 rad/nm, which corresponds to a wavelength distribution of only δλ ≈ 1 nm. Moreover, domain wall oscillators in parallel nanowires that are pinned onto the same ferroelectric domain wall  and short, typically 10-100 nm, opening new avenues for the realization of nanoscale magnonic devices.m in the ferromagnetic layer is governed by the Landau-Lifshitz-Gilbert equationj36The time evolution of the magnetization γ is the gyromagnetic ratio, α is the damping constant and 3 and 3 which, in practice, depends on the magnetoelastic properties of the ferromagnetic layer and the efficiency of strain transfer. Furthermore, in expression (4) e is the electron charge and 3. While the induced anisotropy strength x- and y-directions. For the magnetic nanowire, this is only done in the x-direction. In all simulations, a large damping constant of α is adjusted to give the best possible analysis of different spin wave properties: to determine Here, cale see ; to ensun an integer. This is when the domain wall displacement is maximal and the domain wall velocity is zero. This way, dynamic energy contributions due to domain wall tilting at non-zero domain wall velocities do not affect the data. The obtained energy versus displacement relation  it is In addition to micromagnetic simulations, we developed a 1-dimensional model to describe the driven oscillations of a 90 degree magnetic domain wall. Again, we consider an anisotropy boundary at We express the local magnetization in spherical coordinates, where the domain wall width is given by By linearizing the LLG equation, we then obtain linear equations of motion for the collective coordinates ε is the areal energy density ν being the volume energy density. Moreover, where From the definition of potential stiffness one obtains ulations .How to cite this article: Wiele, B. V. et al. Tunable short-wavelength spin wave excitation from pinned magnetic domain walls. Sci. Rep.6, 21330; doi: 10.1038/srep21330 (2016).