Recently, a new class of broadly neutralizing anti-influenza virus antibodies that target the stalk domain of the viral hemagglutinin was discovered. As such, induction, isolation, characterization, and quantification of these novel antibodies has become an area of intense research and great interest. Since most of these antibodies bind to conformational epitopes, the structural integrity of hemagglutinin substrates for the detection and quantification of these antibodies is of high importance. Here we evaluate the binding of these antibodies to soluble, secreted hemagglutinins with or without a carboxy-terminal trimerization domain based on the natural trimerization domain of T4 phage fibritin. The lack of such a domain completely abolishes binding to group 1 hemagglutinins and also affects binding to group 2 hemagglutinins. Additionally, the presence of a trimerization domain positively influences soluble hemagglutinin stability during expression and purification. Our findings suggest that a carboxy-terminal trimerization domain is a necessary requirement for the structural integrity of stalk epitopes on recombinant soluble influenza virus hemagglutinin. Hemagglutinin (HA) exists as a glycoprotein trimer on the surface of the influenza virion. Each monomer is initially expressed as HA0, and is subsequently cleaved by host proteases into HA1 and HA2 subunits, which are linked via a disulfide bond. So far, 17 antigenically distinct influenza A HA subtypes have been described and they are further categorized as either group 1 or group 2 hemagglutinins  HA can be functionally divided into two domains, the globular head and the stalk. The head region contains the receptor-binding site that modulates the ability of the virus to bind to host substrates. Antibodies directed towards this region can block receptor binding and are known to be neutralizing. The stalk domain, which makes up the majority of the amino acid sequence of the HA molecule, mediates virus fusion and uncoating by virtue of the fusion peptide that is located in this domain. Following binding to sialylated host receptors, the virus is internalized by endocytosis. The endosome is then acidified, inducing a conformational change in the HA that facilitates the union of host and viral membranes so that the viral genome can be released into the cytoplasm for subsequent replication, transcription and translation Because of the importance of HA-directed antibodies in preventing influenza virus infection, recombinant HA is a valuable reagent for influenza virus research and the vaccine industry. Recombinant HA is used to assess sero-conversion of vaccinees and experimental animals, to measure binding kinetics of monoclonal antibodies, or as standard for the quantification of the HA content of vaccines. There are also attempts to use baculoviral-, mammalian- or bacterial-expressed HAs as human or veterinarian vaccines Much excitement has surrounded the discovery of antibodies that bind the stalk domain of the HA molecule. Most globular head antibodies are strain-specific because of antigenic drift in the hypervariable loops of this domain. In contrast, the stalk domain is highly conserved, and antibodies directed against the stalk are more likely to be cross-reactive, even between subtypes Sf9 insect cells (ATCC # CRL-1711) were grown in TMN-FH medium (Gemini Bio-Products) supplemented with 10% FBS , 0.1% Pluronic F68 (Sigma) and a Penicillin-Streptomycin antibiotic (Gibco) mixture. BTI-TN-5B1-4 cells (High Five - Vienna Institute of Biotechnology subclone BamHI or StuI and NotI restriction endonucleases (NEB). Primer sequences are available upon request. Two sets of constructs, HA without and with trimerization domain, were cloned: HA constructs without trimerization domain were designed so that the C-terminal transmembrane- and endodomain of the HA were replaced with a hexahistidine-tag ; the other set of constructs, HA with a trimerization domain, also lack the C-terminal transmembrane- and endodomains (HA sequence ends with V503 - H3 numbering) but include a thrombin cleavage site and a T4 foldon trimerization domain Sequences coding for HAs of H1 strains A/Puerto Rico/8/34 (PR8), A/California/04/09 , H2 strain A/Japan/305/57 (JAP57), H3 strains A/Hong Kong/1/68 (HK68), A/Wisconsin/67/05 (Wisc05) and H5 strain A/Viet Nam/1203/04 (VN04 - with removed polybasic cleavage site 6 cells/ml in HyClone SFX serum free media (Fisher Scientific) at a multiplicity of infection of 10. Expression was carried out in 1000 ml shaker flasks for 96 hours at 28°C. After 96 hours, supernatants were cleared by low speed centrifugation  and incubated with Ni-NTA (Qiagen) resin (3 ml slurry for 250 ml of culture supernatant) for two hours at room temperature (RT). The resin-supernatant mixture was then passed over 10 ml polypropylene columns (Qiagen). The retained resin was washed four times with 15 ml of washing buffer  and protein was eluted with elution buffer . The eluate was concentrated using Amicon Ultracell (Millipore) centrifugation units with a cut-off of 30 kDa and buffer was changed to phosphate buffered saline (PBS) of pH 7.4. Protein concentration was quantified using Quickstart Bradford Dye Reagent (Bio-Rad) with a bovine serum albumin standard curve. Protein purity, integrity and identity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) , Coomassie staining and Western blot or enzyme linked immunosorbent assay (ELISA). Extent of trimerization and/or multimerization was tested by crosslinking of HA with bis-[sulfosuccinimidyl]suberate (BS3 - Fisher Scientific) according to the manufacturer's recommendations. Briefly, 3 µg of HA were incubated in 30 µl of PBS in the presence of a 25 fold molar excess of BS3 crosslinker. The mixture was incubated at RT for 30 minutes and then BS3 was quenched by adding 1 M Tris-HCl buffer (pH 8) to a final concentration of 50 mM. Subsequently SDS-PAGE and/or Western blot analysis with a mouse anti-his primary antibody (Sigma) and anti-mouse horseradish peroxidase (Santa Cruz Biotechnology) or alkaline phosphatase (Santa Cruz Biotechnology) conjugated secondary antibody was performed.Baculovirus was amplified in Sf9 cells to a passage 3 stock and then used to infect BTI-TN-5B1-4 (High Five) cells at 1×102CO3/NaHCO3, pH 9.2, 50 µl/well) overnight at 4°C. The plates were then blocked for one hour at RT with PBS (pH 7.4) containing 1% Tween 20 (TBPS) and 3% non-fat dry milk powder. After blocking, plates were washed once with TPBS and then incubated with three fold dilutions of monoclonal antibody or sera  for one hour at RT. Plates were then washed trice with 100 µl of TPBS and incubated for another hour at RT with horse radish peroxidase conjugated anti-mouse IgG (Santa Cruz Biotechnology) or anti-human Fab secondary antibody (Sigma) at a dilution of 1∶3000 (50 µl per well). After three more washes, plates were developed using SigmaFAST OPD substrate (Sigma) (100 µl/well), stopped with 3 M HCl (50 µl/well) and read at an absorption of 490 nm on a Synergy 4 (BioTek) plate reader. The obtained read-out was background subtracted with values from secondary antibody-only incubated wells.Immunolon 4HBX (Fisher Scientific) plates were coated with recombinant HA with and without trimerization domain at a concentration of 5 µg/ml in coating buffer , two, three or four freeze-thaw cycles. Stability of head versus stalk binding antibodies was compared using PY102 and C179 monoclonal antibodies. Antibody-HA combinations in ELISA were done in triplicates except for stability studies where duplicates were used.We expressed the extracellular domain of various group 1 and group 2 HAs in soluble form with or without a C-terminal T4 phage trimerization domain  in the b3, a hydrophilic 11 Ångstrom chemical crosslinker that was recently used to show trimerization for HAs We were also interested in assessing whether HAs without trimerization domain would form trimers or would stay in monomeric conformation as recently reported Several broadly neutralizing antibodies that react with the stalk domain of influenza virus HA have been reported recently. We tested the reactivity of a panel of such broadly reactive, neutralizing antibodies to our HA constructs in order to determine differential binding of these antibodies to HA substrates with and without trimerization domain. We used well-defined stalk antibodies such as mouse mAb C179, human mAb CR6261 (both group 1 specific), and human mAb CR8020 (group 2 specific) along with antibodies generated by our laboratory  and VN04 (H5) HAs with and without a trimerization domain, a similar phenotype was observed, where these antibodies only reacted with trimerized forms of the protein . The samFor group 2 HA-binding antibodies, however, a different pattern emerged. In order to test the effects of a trimerization domain on reactivity of stalk antibodies with group 2 HAs, we used broadly reactive antibodies CR8020 and 12D1. CR8020 is known to bind a conformational epitope in group 2 HAs Stability during storage is an important issue for reagents as well as vaccines. In order to assess stability of stalk epitopes on HA proteins with trimerization domains during storage, we chose the PR8 HA as a model. We stored purified protein at 4°C in a laboratory refrigerator for a period of 60 days or kept it at −80°C in a freezer, and subjected the protein to either one, two, or three freeze-thaw cycles. We then coated ELISA plates with the differentially treated proteins and assessed the integrity of relevant epitopes of the recombinant HA either with the stalk-reactive antibody C179 or with the head-reactive antibody PY102. As expected, the reactivity of C179 was inversely related to the number of freeze-thaw cycles the protein was subjected to . It is oSecreted, soluble forms of HA can easily be produced in research laboratories using baculovirus- or mammalian cell-based expression systems. In this report, we investigated the influence of a C-terminal T4 trimerization domain on the binding of stalk-reactive broadly neutralizing antibodies to a secreted soluble form of HA. We expressed a variety of different group 1 and group 2 HAs and found that the addition of a C-terminal trimerization motif seems to increase the stability of these glycoproteins as trimers during expression and prevents both site-dependent as well as non-specific cleavage. Of note, we demonstrate that specific stalk-reactive antibody binding is largely dependent on trimerization of soluble HAs. Binding of stalk-reactive broadly neutralizing antibodies only occurs in the presence of a C-terminal trimerization domain for group 1 HAs. All tested mAbs bound exclusively to the HAs that had a trimerization domain; binding was completely abolished when a trimerization domain was not expressed. For group 2 HAs, a trimerization domain was not required for stalk-specific antibody binding. It is of note that while 12D1 binding was completely unaffected by trimerization domain expression, CR8020 binding was greatly enhanced when a trimerization domain was included in the construct. In contrast, antibodies directed against the globular head domain of both group 1 and group 2 HAs were not affected by the presence or absence of the trimerization motif. Our data therefore reflects a fundamental difference in terms of stalk- and head- specific antibody binding to soluble HA proteins. The above observances are also reflective of the intrinsic differences in the nature of the epitopes of group 1 and group 2 stalk-reactive antibodies. In the former, it was shown in crystal structure analysis that group 1 mAbs such as CR6261 and F10 bound to the hydrophobic groove of the stalk region formed between two monomers of the HA Our work also highlights differences between group 1 and group 2 soluble HAs. Group 1 HAs that have a C-terminal trimerization domain form uniform trimers whereas group 1 HAs lacking a trimerization domain form high molecular weight multimers. This multimerization of group 1 HAs that do not express a trimerization domain is most likely mediated by a multimerization motif present on the stalk domain of the HA1 subunit which is also functional when the HA1 subunit is expressed without the HA2 subunit Trimerization domains have previously been described for use in influenza virus HA expression. The trimerization domain (foldon) of T4 phage fibritin, a trimeric beta hairpin propeller, was first used in crystallization studies of the 1918 H1N1 HA It is of interest that stalk-reactive antibodies were crystallized with trimeric, recombinant HA proteins after the trimerization domain was removed


An understanding of the activities of enzymes could help to elucidate the metabolic pathways of thousands of chemical reactions that are catalyzed by enzymes in living systems. Sophisticated applications such as drug design and metabolic reconstruction could be developed using accurate enzyme reaction annotation. Because accurate enzyme reaction annotation methods create potential for enhanced production capacity in these applications, they have received greater attention in the global market. We propose the enzyme reaction prediction (ERP) method as a novel tool to deduce enzyme reactions from domain architecture. We used several frequency relationships between architectures and reactions to enhance the annotation rates for single and multiple catalyzed reactions. The deluge of information which arose from high-throughput techniques in the postgenomic era has improved our understanding of biological data, although it presents obstacles in the data-processing stage. The high computational capacity provided by cloud computing has resulted in an exponential growth in the volume of incoming data. Cloud services also relieve the requirement for large-scale memory space required by this approach to analyze enzyme kinetic data. Our tool is designed as a single execution file; thus, it could be applied to any cloud platform in which multiple queries are supported. Enzymes are biochemical agents that efficiently catalyze the conversion of substrates into products in organisms. Enzymes are essential to the metabolic activity of living systems, and they share 3 features: catalytic power, specificity, and regulation . CatalytThe catalytic power and specificity of enzymes can enhance productivity in industrial applications. A recent study published by the BBC research group estimated that the global market for industrial enzymes was at $3.3 billion in 2010 and was expected to reach $4.4 billion by 2015 [There are 3 main types of enzyme reaction annotations: sequence similarity, chemical structure comparison, and domain architecture fingerprint. Certain annotation methods, such as profils pour l'identification automatique du métabolisme (PRIAM)  and CataThe second type of enzyme reaction annotation is based on chemical structure comparison because the conversion of a particular reactant into a product with a specific molecular structure in an uncatalyzed chemical reaction can often be achieved by enzyme catalysis in an organism. Problems are frequently encountered when an enzyme catalyzes several reactions and when the same reaction is catalyzed by different enzymes. Several reported computational methods exist for assigning EC numbers that use the physicochemical and topological properties of reactants, products, and bonds involved in the reaction –12. Domain architecture fingerprint is the third type for enzyme reaction annotation. Substrates bind to an enzyme at its active site, where they undergo reaction. An enzyme reaction is intimately linked to the compact protein structure of a domain. As a general rule, enzymes of similar domain architectures catalyze similar reactions; this creates a difficult mapping problem from the architecture space into the reaction space. Various machine-learning methods have been applied to the mapping problem, including the association rule algorithm , the decThe advent of genomics technologies, including next-generation sequencing and mass spectrometry-based flow cytometry , 21, creProteins comprise polypeptide chains that form several compact, occasionally loosely connected, global units called structural domains. Regarding the protein structure, structural domains are considered fundamental units of protein function, folding, and evolution . It is rFrom the viewpoint of protein function, enzymes are agents of metabolic function, which control the rate of biochemical activities in living organisms . The firUniProtKB  is a comRelying on the rationale that structural domains are related to protein functions, we integrated enzymes sharing the same domain architecture as a single entry. For example, Q5VT25, Q9BZL6, and E0W264 share the {SSF50729, SSF56112, SSF57889} architecture with EC numbers 2.7.11.1, 2.7.11.13, and 1.3.1.74 and are considered a single entry . There aOur proposed method accounts for the frequency of each potential type of domain architecture from a set, and a rank is assigned according to several criteria. After determining the domain architecture that has the greatest score, we obtain the corresponding enzyme reactions.Because domains are fundamental structural units that can fold into a compact block, we considered the appearance of a domain in an enzyme and omitted the repetition of domains. As a result, the number of domain architectures is nearly 5 times the number of types of SUPERFAMILY domains but does not grow exponentially. This shows a tendency for one domain to accompany others to form one type of domain architecture for a protein. The ERP method is used to predict enzyme reactions from components of domain architectures. In the model-building process, there are 2 main phases: “domain architecture enumeration” and “enzyme reaction ranking.”Before building the prediction model, we divided 5,203 entries into 2 sets, the training set and the testing set. The training set is used to establish the prediction model and the testing set is adopted for verification. The details of the model simulation are described in the 5-fold cross-validation section.The first phase of model building is based on the rationale that one domain has a tendency to accompany others to form one type of domain architecture. We enumerated all possible subsets from domain architectures in the training set and estimated each subset according to 4 measurements: comprising existence, succinctness, consistency, and simplicity. The domain architecture candidate with the highest priority was thus obtained. In the second phase, we ranked enzyme reactions in a list according to their intensity values associated with one specific type of domain architecture.Domain Architecture Enumeration. After inspecting the set of domain architectures, we learned that the number of types of adjacent domains was considerably lower than the numbers encountered when enumerating every possible combination. Thousands of architectures are enumerated exhaustively among all the possible subsets associated with the domain architectures of proteins. A possibility of tandem domains appearing with the expression of enzyme-catalyzed reactions also exists. Thus, we propose the following 4 measurements to sequentially estimate each domain's architecture during the domain architecture enumeration phase. For example, if the domain architecture {SSF50729, SSF56112, SSF57889} could not be found, 6 subarchitectures comprising {SSF50729, SSF56112}, {SSF50729, SSF57889}, {SSF56112, SSF57889}, {SSF50729}, {SSF56112}, and {SSF57889} are considered.1) Existence of the Protein Consisting of a Given Domain Subset. In the process of enumerating all possible subsets of domain architectures, many putative subsets may be produced. If one subset matches one type of domain architecture of an enzyme, it is reasonable that this domain subset contributes directly to its catalyzed reactions and is awarded higher priority than other subsets are.(2) Succinctness Measurement of the Domain Architecture of Enzymes. One reaction can be catalyzed by various enzymes that can comprise a variety of domain architectures. Among them, each subset of one type of domain architecture could also include another type of an enzyme. The Succinctnessdomain_arch equation  in {SSF56112, SSF57889} is estimated as 0.6. We assign priority to the candidate with the greatest succinctness value because the corresponding chemical reactions proceed without requiring auxiliary domains as follows:(equation  is desig3) Multiplicity of Enzyme Reactions from One Type of Domain Architecture. An enzyme can catalyze different reactions; alternatively, different enzymes may share the same domain architecture. Considering a domain subset domain_arch, we collected all entries that have domain architectures containing domains of domain_arch. Among these entries, the number of involved reactions is defined similarly to the definition of |ECsincluded| in the previous paragraph, but we denoted it as k for simplicity. To clearly observe the expression of one specific reaction among various architectures, we separated an entry with multiple reactions into several entries with a single reaction, and the number of entries with a single reaction is counted as N , such that N = ∑i=1kni. The mean value i. Because k and Entriesdomain_arch are variables dependent on the set of domains in domain_arch, we provided Consistencydomain_arch (N and weighted with (ni/N) for each reaction for comparison with other architecture candidates. Simplicity of Domain Architecture. In the case that no protein matching the query architecture domain_arch is found, the fewest number of domains in an architecture candidate is preferred. the union of item sets X and Y must appear frequently in T, and (2) the relationship between item sets X and Y is close. A frequent set satisfies the condition that the number of transactions containing that set is higher than the support threshold. If set Y accompanies set X in various transactions, a close relationship exists between sets X and Y. The confidence value can be estimated as the ratio of the number of transactions containing both item sets (X and Y) to that containing item set X alone. If the confidence value of the item sets in a rule is higher than the given confidence threshold, it is placed into the rule set.In the field of data mining, the association rule (AR) method is an established method for detecting the relationship between items, particularly for a large database, In a classification model, the parameters of the model are optimized to fit the training set as much as possible during the fitting process. An overfitting problem results when another independent validation data set (from the same population) is used to test the model and does not fit as well as the training set did. Cross-validation is a technique used to infer the goodness of fit of a model to a validation set. We used 5-fold cross-validation, in which the sample is randomly divided into 5 subsets: one subset is retained as the testing set, and the other subsets are assigned to the training set. The numbers of entries in each fold for the 4-numerical-block EC number set are shown in A chemical reaction may be catalyzed by more than one enzyme, and an enzyme may catalyze more than one reaction. By considering the relationship between enzymes and chemical reactions as a mapping problem, we create a many-to-many mapping problem. Although there are various methods available that approach this type of problem from different viewpoints, we present this intuitive method, which is based on the frequency of domain architecture and, in an enzyme, the associated catalyzed reactions. To examine the feasibility of our method, we compiled data from the UniProtKB and SUPERFAMILY domains of the InterPro database. A total of 1,664,839 proteins are associated with 1,218 SUPERFAMILY domains and 3,306 4-numerical-block EC numbers. The population of the 6 NC-IUBMB classes is shown in To avoid the bias caused by the selection of the training data set, we used 20 runs of 5-fold cross-validation. From 5,203 entries, approximately 4,160 entries were used for model building, and the remaining 1,040 entries were used for verification. According to the complexity of classification problems, it is difficult to predict multiple reactions of entries from domain architectures. We separated 1,040 entries into 2 sets: 624 entries for the single-EC set and 416 entries for the multiple-EC set; hence, there are 2 main rows in For comparison with our ERP method, we used the established Apriori algorithm  to mine The accuracy is provided by the ratio of the number of entries predicted correctly to the number of entry-matching rules of each method in In the model-building phase, we implemented the AR method in a server equipped with 12 CPUs  and 128 GB of memory, and the server used for the ERP method was equipped with 2 CPUs  and 8 GB of memory. The average model-building time was over 1 hour for the AR method and 15 minutes for the ERP method. The reasons may be that the AR method needed to produce frequent item sets and many redundant rules was generated. Furthermore, estimates of the prediction time for a batch of query domain architectures are shown in A substantial demand exists for enzymes for industrial and medical applications in the global market; thus, enzyme function annotation is receiving considerable attention because it offers reductions in the cost of chemical processes. In this study, we proposed the ERP tool for annotating enzyme reactions based on the query domain architecture . After pTo implement the deduction of enzyme reactions from the domain architectures of enzymes, we designed a tool by using the Perl script language as follows. The set of domains in a protein must be listed before applying the ERP method. In the “Domain set” dialog, the domain set may be comma-or space-delimited. When the domain set is ready, pressing the “Predict” button starts processing according to the flowchart in If the ERP tool cannot deduce the corresponding enzyme reactions from the ERP integrated universe set, a failure message, such as the domain architecture failure notice {SSF54211, SSF54236} shown in In deducible cases, the existence of enzymes sharing the same architecture is considered. If the corresponding protein exists, succinctness and consistency values expressing the strength of the domain architecture are listed. If no enzyme sharing the same architecture is located, subsets of the domain architecture are evaluated, and the domain subset with the highest priority is selected. Two main situations in which analysis of the entered domain set could fail are described as follows.3.4.24.21 is calculated as 1. The protein consisting of the architecture {SSF51110, SSF55486} is shown in In the event that the same architecture protein  is found2.7.7.8 is calculated as 1. The protein with the accession number D9PMT6 and UniProt ID D9PMT6_9ZZZZ consisting of this type of domain architecture {SSF54814} is listed in In the absence of a protein consisting of the same architecture , the subIn this study, we investigated the intimate relationship between domain architecture and enzyme-catalyzed reactions by applying various criteria to the compiled universe data set of domains and EC numbers. The advent of high-throughput techniques has produced numerous gene sequences, and annotating each enzyme reaction based on experimental results is difficult. However, we can consider domains as segments of sequences that fold into compact structural units; thus, we can model protein sequences and structures as these folded domains. We can identify and retrieve domains by integrating established sequence alignment tools with the proposed ERP tool.


Amyotrophic lateral sclerosis (ALS) is a heterogeneous group of incurable motor neuron diseases (MNDs) characterized by a selective loss of upper and lower motor neurons in the brain and spinal cord. Most cases of ALS are sporadic, while approximately 5–10% cases are familial. More than 16 causative genes for ALS/MNDs have been identified and their underlying pathogenesis, including oxidative stress, endoplasmic reticulum stress, excitotoxicity, mitochondrial dysfunction, neural inflammation, protein misfolding and accumulation, dysfunctional intracellular trafficking, abnormal RNA processing, and noncell-autonomous damage, has begun to emerge. It is currently believed that a complex interplay of multiple toxicity pathways is implicated in disease onset and progression. Among such mechanisms, ones that are associated with disturbances of protein homeostasis, the ubiquitin-proteasome system and autophagy, have recently been highlighted. Although it remains to be determined whether disease-associated protein aggregates have a toxic or protective role in the pathogenesis, the formation of them results from the imbalance between generation and degradation of misfolded proteins within neuronal cells. In this paper, we focus on the autophagy-lysosomal and endocytic degradation systems and implication of their dysfunction to the pathogenesis of ALS/MNDs. The autophagy-endolysosomal pathway could be a major target for the development of therapeutic agents for ALS/MNDs. Amyotrophic lateral sclerosis (ALS) is a heterogeneous group of inexorable neurodegenerative disorders characterized by a selective loss of upper and lower motor neurons in the brain and spinal cord , 2. MostSOD1, ALS2, SETX, SPG11, FUS, VAPB, ANG, TARDBP, FIG4, OPTN, ATXN2, VCP, C9orf72, UBQLN2, SIGMAR1, and CHMP2B have been identified [http://neuromuscular.wustl.edu/index.html)  . The fol Among these pathogenic mechanisms, ones that are associated with disturbances of protein homeostasis have been highlighted, as the accumulation of insoluble protein aggregates is the cardinal pathological feature for ALS and other neurodegenerative diseases . AlthougIn this paper, we aim to give a comprehensive view on the autophagy-endolysosomal system and implication of its dysfunction to the pathogenesis of ALS/MNDs. Excellent review articles specialized on the role of the UPS in ALS/MNDs can be found elsewhere , 19.Autophagy is an evolutionally conserved lysosomal degradation system that is tightly linked to a wide variety of physiological processes such as protein homeostasis, removal of pathogens, and antigen presentation. There are at least three forms of autophagic pathways; macroautophagy, microautophagy, and chaperon-mediated autophagy, among which macroautophagy, hereafter referred to as “autophagy”, plays a crucial role in the removal of cytoplasmic long-lived as well as misfolded proteins , 20–23. These multiple steps of autophagy are highly orchestrated by a common group of proteins called ATG (autophagy-related), such as Atg5 and Atg7 , and RabWhichever autophagic mechanisms except Atg5/Atg7-independent alternative  are invoEndocytosis is an evolutionally conserved cellular process involving the internalization of a wide variety of molecules from the surface of cells. There are at least four distinct well-recognized endocytic pathways in eukaryotes: phagocytosis, macropinocytosis, clathrin-mediated endocytosis (CME), and caveola-mediated endocytosis –38 Figu. In addiNascent autophagosomes undergo a stepwise maturation, resulting in the creation of amphisomes and autolysosomes by fusion with multiple endocytic compartments, such as early endosomes, multivesicular bodies (MVBs), late endosomes, and lysosomes , 42. AmpElaborate molecular mechanisms regulating the maturation of intracellular vesicular/vacuolar compartments have recently begun to emerge. Those include Rab-switching, phosphatidylinositol (PI) conversion, endosomal sorting complex required for transport (ESCRT) machinery, as well as lumenal acidification, as reviewed in detail elsewhere , 42, 44.Since autophagosomes as well as endosomes are motile within cells , 52, it The final step of the autophagy-endolysosomal system is degradation of cargo molecules within lysosomes. Two classes of proteins; lysosomal acid hydrolases and lysosomal membrane proteins (LMPs), play essential roles in degradation of cargos in lysosomes. Lysosomal acid hydrolases such as cathepsins are involved not only in bulk degradation of substrates (cargos) but also in other physiological processes such as antigen processing. On the other hand, LMPs have a wide variety of functions including lumenal acidification, import of cytosolic proteins, and transport of degraded materials to cytosol. Excellent review article specialized on lysosome biogenesis is available elsewhere .Lysosomal positioning is dynamically regulated by nutritional conditions, in which a starvation induces preferential relocalization of lysosomes from cell peripheries to the juxtanuclear regions close to the microtubule-organizing center (MTOC), thereby regulating the autophagic flux in cells . In neurSOD1 [FIG4 [VCP [CHMP2B [SQSTM1 [DCTN1 [DYNC1H1 [RAB7A [ALS2 [TARDBP [OPTN [UBQLN2 [Growing evidence supports a role of the autophagy-endolysosomal pathway in the pathogenesis of ALS/MNDs. Indeed, the accumulation of autophagosomes were observed in the spinal cord of sporadic ALS patients , indicatSOD1 , FIG4 [6D1 [FIG4 , VCP [62 [CHMP2B , SQSTM1  [SQSTM1 , DCTN1 [1 [DCTN1 , DYNC1H1[DYNC1H1 , and RAB1 [RAB7A   account for an approximately 20% of familial ALS cases [G93A)-expressing ALS mouse model [Mutations in LS cases , 77. RatLS cases . RecentlLS cases , 79. SODLS cases , 81. Impse model –84. Mostse model . It is ase model –88  [FIG4 [FIG4 is implicated in the pathogenesis of both peripheral neuropathy and ALS/MNDs. FIG4 encodes a phosphoinositide 5-phosphatase, Fig4, that regulates the intracellular level of phosphatidylinositol-3,5,-bisphosphate P2). It has been shown that mutation in FIG4 results in a significant reduction of the PIP2 level in cultured cells [FIG4-linked ALS/MNDs (Mutations in  (CMT4J) . InteresJ) [FIG4 , indicated cells . Furthered cells . Thus, dALS/MNDs .VCP have previously been identified in patients with inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) [VCP mutations as a cause of familial ALS, accounting for 1-2% of familial ALS [VCP encodes valosin-containing protein (VCP/p97) that belongs to the AAA+ (ATPases associated with various activities) protein family, being implicated in multiple cellular processes including the UPS [Mutations in (IBMPFD) . Recentllial ALS . VCP enc the UPS , 93. A r the UPS . Further the UPS , and exp the UPS , 95–97. CHMP2B have been identified in patients with FTD and ALS-FTD [CHMP2B encodes charged multivesicular body protein 2B (CHMP2B), a component of the ESCRT-III complex. The ESCRT complexes are known to play important roles in MVB biogenesis and autophagosomal-endolysosomal maturation [CHMP2B-linked FTD and ALS-FTD  [κB activation through the binding with aPKCs but also the selective-autophagy via association with ubiquitinated misfolded proteins [Sqstm1 in mice results in the accumulation of hyperphosphorylated tau and neurodegeneration [SQSTM1 have originally been identified in patients with Paget disease of bone (PDB) [SQSTM1 in familial as well as sporadic ALS [C9orf72 [VCP for IBMPFD and SQSTM1 for PDB, are also associated with ALS and/or ALS-FTD, and that both VCP and p62 are key regulators for the autophagy-endolysosomal system, dysregulation of such VCP/p62-associated common pathological pathways might account for these seemingly different diseases. (aPKCs) . It has proteins –104. Furproteins . Conversneration . Althougne (PDB) , a recenadic ALS . It is n[C9orf72 , 109. Co DCTN1 encoding the p150 subunit of the transporter protein dynactin has been identified in autosomal dominant form of lower MNDs [DYNC1H1 encoding cytoplasmic dynein heavy chain in patients with dominant form of axonal CMT [Dync1h1 have also been identified, resulting in progressive motor neuron degeneration in mice [Mutation inwer MNDs . Dynactiwer MNDs . It has wer MNDs . Moreovewer MNDs . On the onal CMT . Mutatio in mice . It has  in mice , 87. Fur in mice . Interes in mice . Collect in mice   is an autosomal-dominant peripheral neuropathy caused by the missense mutations in RAB7A . These mRAB7A , a regulRAB7A –116 , juvenile primary lateral sclerosis (JPLS), and infantile-onset ascending hereditary spastic paralysis (IAHSP) [ALS2 gene encodes a 184 kDa protein of 1657 amino acids, ALS2 or alsin, comprising three predicted guanine nucleotide exchange factor (GEF) domains: the N-terminal RCC1-like domain (RLD), the central Dbl homology and pleckstrin homology (DH/PH) domain, and the C-terminal vacuolar protein sorting 9 (VPS9) domain [Loss of function mutations in the  (IAHSP) , 120. Th) domain . Indeed,) domain , and reg) domain , 123 (Fi) domain , 125. In) domain . Further) domain , and in ) domain –130.H46R-expressing ALS mouse model exhibits the aberrant accumulation of autophagosomes and vesicular compartments in axons, delayed protein degradation by the autophagy-endolysosomal system, accelerated neurodegeneration, and earlier death [ Recently, we have demonstrated that activated Rac1 interacts with ALS2 and induces the relocalization of ALS2 from cytoplasm to membranous compartments; for example, membrane ruffle, macropinosome, and endosome . This Raer death . AlthougG93A-expressing ALS mouse model [in vivo. As such, our understanding of the intricate autophagy-endolysosomal system and its functional linking to other physiological systems in the CNS is still incomplete. Future studies, which could uncover the molecular mechanisms of a selective neurodegeneration in greater detail, will be required for the development of proper and effective therapeutic agents for the treatment of ALS/MNDs and other neurodegenerative diseases.Thus far, a large number of successful therapeutic interventions in preclinical animal studies have failed to translate into human clinical applications in ALS/MNDs. Even in such a discouraging situation, enormous efforts have continuously been made towards defining the molecular pathogenesis of these devastating diseases. The autophagy-endolysosomal system is among the underlying mechanisms, whose dysfunction is tightly associated with a variety of neurodegenerative diseases. It plays a pivotal role not only in ALS/MNDs as discussed in this paper, but also in other neurodegenerative diseases including Alzheimer's disease , Parkinsse model . Howeverse model . These c


Bacillus subtilis). It has been proposed that the MtnBD fusion enzyme in Tetrahymena thermophila catalyzes four sequential reactions from the dehydratase to dioxygenase steps, based on the results of molecular biological analyses of mutant yeast strains with knocked-out MSP genes, suggesting that new catalytic function can be acquired by fusion of enzymes. This result raises the question of how the MtnBD fusion enzyme can catalyze four very different reactions, especially since there are no homologous domains for enolase and phosphatase  in the peptide. Here, we tried to identify the domains responsible for catalyzing the four reactions using recombinant proteins of full-length MtnBD and each domain alone. UV-visible and 1H-NMR spectral analyses of reaction products revealed that the MtnB domain catalyzes dehydration and enolization and the MtnD domain catalyzes dioxygenation. Contrary to a previous report, conversion of 5-methylthioribulose-1-phosphate to 2-keto-4-methylthiobutyrate was dependent on addition of an exogenous phosphatase from B. subtilis. This was observed for both the MtnB domain and full-length MtnBD, suggesting that MtnBD does not catalyze the phosphatase reaction. Our results suggest that the MtnB domain of T. thermophila MtnBD acquired the new function to catalyze both the dehydratase and enolase reactions through evolutionary gene mutations, rather than fusion of MSP genes.To recycle reduced sulfur to methionine in the methionine salvage pathway (MSP), 5-methylthioribulose-1-phosphate is converted to 2-keto-4-methylthiobutyrate, the methionine precursor, by four steps; dehydratase, enolase, phosphatase, and dioxygenase reactions (catalyzed by MtnB, MtnW, MtnX and MtnD, respectively, in  Duplication, fusion, mutation, and homologous recombination of genes encoding enzymes have contributed to the emergence, diversification, and development of metabolic pathways during evolution Tetrahymena thermophila has 52 genes for fusion enzymes mtnA and mtnK, and mtnB and mtnD, which encode enzymes in the methionine salvage pathway (MSP), are present in the genome of T. thermophilaBacillus subtilis  and adenine by MTA nucleosidase (MtnN) (step 1) Bacillus-specific enzyme (step 6). DHK-MTPene is converted into 2-keto-4-methylthiobutyrate (KMTB) and formate by a DHK-MTPene dioxygenase (MtnD) (step 7) It has been predicted that subtilis  [18]–, [1rmophila  [37]–, motif II [(S/T)XX], and motif III [K-(X)n-(G/S)(D/S)XXX(D/N)] essential for catalysis in dephosphorylation and metal coordination. The multiple alignment was created manually and highlighted with Boxshade.(PDF)Click here for additional data file.


Protein structures are comprised of modular elements known as domains. These units are used and re-used over and over in nature, and usually serve some particular function in the structure. Thus it is useful to be able to break up a protein of interest into its component domains, prior to similarity searching for example. Numerous computational methods exist for doing so, but most operate only on a single protein chain and many are limited to making a series of cuts to the sequence, while domains can and do span multiple chains.This study presents a novel clustering-based approach to domain identification, which works equally well on individual chains or entire complexes. The method is simple and fast, taking only a few milliseconds to run, and works by clustering either vectors representing secondary structure elements, or buried alpha-carbon positions, using average-linkage clustering. Each resulting cluster corresponds to a domain of the structure. The method is competitive with others, achieving 70% agreement with SCOP on a large non-redundant data set, and 80% on a set more heavily weighted in multi-domain proteins on which both SCOP and CATH agree.It is encouraging that a basic method such as this performs nearly as well or better than some far more complex approaches. This suggests that protein domains are indeed for the most part simply compact regions of structure with a higher density of buried contacts within themselves than between each other. By representing the structure as a set of points or vectors in space, it allows us to break free of any artificial limitations that other approaches may depend upon. It is well understood that proteins are made up of structural and functional subunits or 'domains'. Ever since domains were first described, numerouAs a result of these different paradigms, there still does not exist a precise definition for a protein domain, nor do experts always agree on the number or location of domains within a given structure. This makes it extremely difficult to come up with a fully automated algorithm, then, to assign domain boundaries. That said, the SCOP and CATHSequence-based domain identification usually involves comparing the sequence in question to a database of protein sequences where the domains have already been defined (such as SCOP) using an alignment tool such as BLAST. More adStructure-based algorithms should in theory be simple and straightforward, and often to the human eye it is obvious where domain boundaries should be drawn when viewing a 3D structure. Nevertheless, it has proved a difficult computational problem and no automated algorithm agrees more than about 80% of the time with SCOP or CATH assignments. A wide variety of methods exist, some based on graph theory and contact maps, some based on secondary structure layout. Some allow only single cuts to be made resulting in domains made of contiguous segments only and a maximum of 3 or 4 domains per chain, others do not have this restriction. PUU builds aDespite the number of algorithms that have been described, most of comparable performance, it seems each has certain disadvantages. As mentioned some methods cannot deal with domains comprised of multiple contiguous segments, and most cannot deal with genetic domains (those with pieces from multiple chains). Some methods are very slow, and some cannot place boundaries midway through secondary structure elements. This study investigates a novel, intuitive algorithm for domain identification by simply clustering α-carbon positions or secondary structure vectors in space. It is very fast, taking under one second for all but the largest proteins, and intuitively obvious. By its nature it has no maximum number of domains it can define, nor limitation on where domain boundaries can occur. Even domains comprised of pieces from multiple chains, such as when domain swapping occurs,20, are Two distinct but related algorithms were studied, as described in Methods: the α-carbon based algorithm (CA) and the secondary structure element based algorithm (SS). Both make use of average-linkage clustering to produce and then cut a dendrogram; they differ only in the objects that they cluster. The main data set used to optimize the algorithms was the ASTRAL30 set, consisting of 8792 domains in 7178 non-redundant protein chains. Only 7076 of these chains actually still existed in the current Protein Data Bank (PDB) however and so comprised the training set used in this study for the CA algorithm. Only 6841 of these had sufficient secondary structure to use the SS algorithm, so this slightly smaller training set was used in that case. Both algorithms have only two adjustable parameters: the minimum value for cutting the cluster dendrogram, m, and the step size to determine whether to make a cut, s.For the CA algorithm, a range of these values were tested, and the performance on the training set recorded for each of 1-, 2-, 3-, and 4-domain chains, summarized in TableSimilar runs for the SS algorithm are shown in TableFor each algorithm we also tested removing the adjacency constraint - i.e. enforcing a distance of 4Å for Cαs in the same secondary structure element, for the CA algorithm, or a distance of 4Å between consecutive secondary structure elements in the SS algorithm. In both cases removing this had a slight detrimental effect on the success rate , so the constraints were left in.For the CA algorithm, initial tests were done clustering all α-carbons. Using the buried α-carbons only (see Methods) resulted in marked improvement however, increasing single, 2-, 3- and 4-domain proteins from 70%, 55%, 41% and 24%  to 75%, 58%, 45% and 32%, respectively . Focusing only on the more buried residues helps make the domain boundaries more clear to the clustering algorithm and so became a permanent part of the algorithm.The effect of the ‘gold standard’ chosen was also investigated. As mentioned, the success rates in TablesDoing the same for the CA algorithm Table, again iTableWith optimization complete, the algorithms were then run on the Benchmark_2 test set. This set (see Methods) is significant in that the distribution of number of domains is intended to match that of the genome, and not the over-weighting of single domain proteins found in the PDB. Additionally, SCOP and CATH, as well as the structure authors, agree on the number of domains for all structures in this data set making the correct result less ambiguous. Note that this test set only contains 4 proteins with 4 domains, so reporting success rates for these is not statistically meaningful.TableLastly we tested the present methods on the Benchmark_3 set requiring 90% or better overlap. Benchmark_3 is a subset of the Benchmark_2 structures in which both SCOP and CATH also agree upon the exact boundaries of the domains, within a small tolerance, suggesting that the domain boundaries are sharply defined in this set. As seen in TableIt is instructive to look at the types of mistakes made by the CA algorithm, which performed best overall on the test data sets, of the two methods developed in this work. There have already been detailed comparisons of SCOP and CATH published so we wi2PCD chain M is a single domain assigned as two domains Figure. HoweverA more clear failure of the algorithm is 1YUA chain A, which is a two domain protein assigned to be a single domain. Visually the protein is clearly two distinct domains, and the problem here is that they are just very small. Lowering our minimum cut value, m, to 19Å and running the assignment again Figure gets it 1GDD chain A is a two-domain protein assigned as three domains - the smaller domain location is assigned correctly, but the larger one is split in two Figure. The SS 1PKY chain A is an example of a three-domain protein we assign as two-domain Figure. This PyFinally, 5EAU chain A was correctly assigned as 2-domain but had an overlap of only 73% ; and for very small domains it will tend to undercut. DDomain, domainparser2 and PDP also fail mostly due to incorrect number of domains rather than overlap under 75%, and in each case roughly half the incorrect assignments overlap with the CA method’s failures. Thus the CA algorithm correctly assigns about half the failures of each of the other ones. In total 10 failed assignments are unique to the CA method including 2PCD, 1GDD and 5EAU above. Interestingly there are 5 structures that none of the algorithms assign correctly .An example where the CA algorithm assigned two domains correctly while the others all assigned three domains is 1FMT chain A Figure. This isand the Benchmark data sets. The algorithms in this study also have the advantage that they can be run on arbitrary numbers of chains, and have no artificial limitations on how many domains or segments they may assign. The CA algorithm is not limited to assigning cuts only at secondary structure boundaries, either.This work presents two novel, related, domain assignment algorithms, one based on clustering buried α-carbons and one clustering secondary structure elements. They are appealing due to their intuitiveness, speed and extreme simplicity – having only two adjustable parameters – and are able to perform competitively with the best algorithms available. The CA algorithm is several times faster than other methods, and comes within a few percent of the top performer on all the data sets investigated, making its use appealing. It is worth noting that no one algorithm performed best on the ASTRAL30 The examples studied indicate that the CA algorithm should not be used when very small domains are expected. Also when multiple copies of a chain exist in the asymmetric unit, it should be run on each separately and perhaps the consensus assignment taken due to its sensitivity to small perturbations in coordinates. Keeping these limitations in mind, it is encouraging that such simple, fast methods can perform as well as they do.et al.[Domain assignment within 3D protein structures is a difficult subject to tackle, due to it being an ill-defined problem to begin with. Different people have different definitions of what a domain is, and this definition might change depending on the intended application. Thus measuring the performance of a particular method, and comparing it to others, is difficult at best. That said, in some cases there is a clear and unambiguous split and the data sets from Holland et al. go a lonEven the best methods are still far from perfect, and this is in part due to the subjective nature of the problem. With a problem like domain assignment, rather than focusing on which method is a few percent closer to SCOP or CATH, for example, it is perhaps more prudent to simply look at the cases where assignments differ from the ‘correct’ answer and ask ‘is this reasonable’?Two distinct though related algorithms were developed for this study, one using α-carbons and one secondary structure elements. Both methods attempt to cluster pieces of the structure using hierarchical agglomerative techniques.All α-carbons within the structure were identified, and all other atoms were ignored for the remainder of the process. The atoms were then divided into two sets: 'buried' and 'exposed'. Buried α-carbons were defined as those with 9 or more α-carbons within 7Å. These values were found empirically to correspond well to an intuitive definition of buriedness.Next, an NxN pairwise distance matrix is constructed for all N buried α-carbons and these are clustered using average linkage clustering to produBy cutting the dendrogram at a specific level, clusters of the original N objects are formed. Thus cutting this dendrogram at a specific point produces a number of clusters of α-carbons, which can then be defined as the domains. Obviously choosing where, and when, to cut the dendrogram is the key problem as this determines the number (and location) of domains Figure. We defi1. Start at D = max d, the root of the dendrogram2. If D < m then stop without making a cut3. If no branch node of the dendrogram is traversed between D and D - s, stop and make the cut4. Set D = d’, the value at the branch node traversed in the previous step5. Return to step 2 and repeatThus the algorithm seeks to cut at a region of clear separation in the dendrogram, but not making the domains too small. These parameters were optimized on a number of test sets as described in Results, and values of m = 22Å and s = 5Å produced the best results.Residues which were initially classified as exposed are at this point added to the cluster of the nearest buried atom. Clustering was also tested on all α-carbons, but using just the buried ones both tended to produce better results, and was also faster, there being less points to cluster.Lastly, a bit of 'clean-up' is performed. This clustering technique can sometimes result in some small clusters of just a few residues being created, and these were eliminated by simply deleting any clusters less than 10% of the size of the largest cluster. Also, because no heed is paid to chain or residue sequence, the algorithm would frequently produce small stretches of a few amino acids from one domain, within the sequence of another when adding back the exposed α-carbons to the clusters. In order to try to minimize the number of small segments like this, the sequence is scanned linearly for segments less than 20 residues in length. Any such short segments which are enclosed on both sides by residues of the same cluster, or which appear at the ends of a chain, are consumed by the adjacent cluster and become a part of it.A minor variation on the algorithm was tested which helped prevent placing domain boundaries midway through secondary structure elements. When building the pairwise distance matrix, all residues pairs which were within the same secondary structure element, as defined by DSSP, were giWe also experimented with a routine that looked only at secondary structure elements. Its performance was comparable to the α-carbon approach and was faster, there being less objects to cluster. Ultimately as shown in Results, the α-carbon method was found to be superior, and preferable, being independent of any particular secondary structure definition.First secondary structure elements are identified, using DSSP. Elements are represented by vectors, with direction computed as the largest eigenvector of the covariance matrix of the Cα coordinates comprising the helix or strand Figure. We denoA special check is made for elements spanning chain breaks - these are broken into two elements, one on either side of the break. Although helices and β-strands often curve, the curve is usually gentle and we found that they are represented sufficiently well by a single vector.Next, as in the previous algorithm, an NxN distance matrix is constructed. Here the distance between two secondary structure elements was defined either as the distance of closest approach of the two corresponding secondary structure vectors, or as the distance between the centers of the vectors. In practice the latter was found to work better. Again average linkage clustering was employed to produce a dendrogram, and the same procedure as in the previous algorithm was used to determine where and if to cut the dendrogram to produce clusters of the secondary structure elements. In this case m = 22Å and s = 5Å were found to be the optimal values, interestingly the same values used for the α-carbon algorithm despite the fact that much larger objects were now being clustered.As before, very small domains are undesirable so all clusters of one or two secondary structure elements were discarded. Lastly, domain cut points were defined midway along the sequence between consecutive secondary structure elements that belonged to different clusters. This choice is somewhat arbitrary but usually produces satisfactory results.A variation to this algorithm which obtained slightly improved results, was to mark secondary structure elements that were adjacent in sequence space as having a distance of 4Å when constructing the distance matrix before clustering. This was analogous to the variation in the α-carbon algorithm where those atoms in the same helix or sheet were set to have a distance of 4Å in that distance matrix. This modification tended to keep consecutive elements within the same cluster unless there was a good reason not to, and thus resulted overall in fewer disjoint segments among the assignments.Both algorithms have been implemented within MOE using thet al.[As mentioned earlier, there is no ideal test set for domain assignment, which makes it difficult to evaluate the performance in an unbiased manner. Holland et al. have pubAdditionally, a second much larger data set, ASTRAL30, was used. This is a non-redundant set of SCOP domains with no more than 30% sequence identity between any two domains. The entire chain, for all chains with at least one domain in the ASTRAL30 set, was included for the purposes of this work. For this data set, when SCOP and CATH disagreed on domain assignment, SCOP was chosen as the ‘correct’ one except where otherwise noted. This set is heavily biased, with 75% of the 7076 chains having a single domain.et al.[In this work, if an assignment had a different number of domains than the value in the test set, it was considered incorrect. When the number of domains matched, a procedure similar to that described in Holland et al. was usedThe author declares that they have no competing interests.HF performed all tasks.


Co-repressor proteins, such as SMRT and NCoR, mediate the repressive activity of unliganded nuclear receptors and other transcription factors. They appear to act as intrinsically disordered “hub proteins” that integrate the activities of a range of transcription factors with a number of histone modifying enzymes. Although these co-repressor proteins are challenging targets for structural studies due to their largely unstructured character, a number of structures have recently been determined of co-repressor interaction regions in complex with their interacting partners. These have yielded considerable insight into the mechanism of assembly of these complexes, the structural basis for the specificity of the interactions and also open opportunities for targeting these interactions therapeutically. The nuclear receptor family comprises 48 receptors in humans, and includes receptors for which the ligand is known, adopted orphan receptors and orphan receptors for which the ligand remains as yet unknown . NuclearTwo of the best studied of the nuclear receptor co-repressors are the homologous proteins SMRT and NCoR, that were first identified through their interaction with nuclear receptors in the absence of a ligand . SMRT an2SMRT and NCoR are large, homologous proteins (ca. 2500 aa) with an overall sequence identity of 40% b. AnalysPredictions of secondary structure and of intrinsic disorder suggest that there are only a few regions that possess an intrinsically folded structure. Two of the regions that are predicted to be structured are proposed to fold into SANT-like domains . The firThe largely intrinsically disordered nature of SMRT and NCoR, as well as other transcriptional co-regulators, seems to be a characteristic feature of these proteins and such properties are often associated with so-called hub proteins that have many interaction partners . The cha3As mentioned earlier in Section In general, nuclear receptor co-activators bind to ligand bound nuclear receptors and co-repressors to un-liganded nuclear receptors (reviewed in Within NCoR a third receptor interaction domain has been identified (ID3) . The thyStructures of NR LBDs with co-activator peptides bound  of the Estrogen Related Receptor (ERR) family are constitutively active orphan receptors showing activity in the absence of bound ligand. Structures of the apo form of ERRγ show that the receptor adopts an active conformation in the absence of ligand . CrystalAnother co-repressor that is recruited to ligand bound nuclear receptors is LCoR (Ligand Dependent Co-repressor), which is recruited to ligand bound ERα again by means of an LxxLL motif. Like RIP140, LCoR functions by binding directly to the CtBP and to HDACs .Nuclear receptors and their co-regulatory proteins are subject to various post-translational modifications including phosphorylation, acetylation, SUMOylation, ubiquitinylation and methylation. These modifications act to modulate nuclear receptor activity and their interaction with co-regulators . Indeed 5SMRT and NCoR interact with a variety of transcription factors in addition to nuclear receptors. In many cases these interactions have only been loosely mapped to broad regions of the co-repressor. However, in a few cases, detailed mapping and structural information about the peptides that mediate these interactions is available b.The transcriptional repressor BCL-6 is important for normal B cell development. In B cell lymphomas, structural alterations of the BCL-6 promoter region leads to unregulated expression of BCL-6 and lymphomagenesis . BCL6 coThe crystal structure of residues 1414–1430 of SMRT interacting with the BTB domain of BCL6 has been solved e and f . The BCLInterestingly, although they both interact with the BCL-6 BTB domain, the interaction peptides from SMRT and BCoR show no sequence similarity. The structure of the BTB domain in complex with a BCoR co-repressor peptide revealed that BCoR binds to the same lateral groove on BCL6 as SMRT thus explaining their mutually exclusive binding . The recThe AML1/ETO chimeric protein results from a t chromosome translocation which is found in M2 subtype acute myeloid leukemia (AML) . AML1/ETThe NMR solution structure of the AML1/ETO MYND domain in complex with a peptide from SMRT (residues 1101–1113) shows that the AML1/ETO MYND domain is structurally homologous to the PHD and RING finger family of proteins, adopting an interleaved zinc-chelating topology and coordinates two zinc atoms g Liu et. The pep6In addition to interacting with repressive transcription factors, SMRT and NCoR also mediate interactions with another large co-repressor protein called SHARP. SHARP (SMRT/HDAC1-associated repressor protein) was identified as a component in transcriptional repression complexes recruited by both nuclear receptors and the Notch-signaling factor RBP-Jk . SHARP iA crystal structure of the SPOC domain (3495–3664) shows that the structure consists of a seven stranded β-barrel framed by seven α-helices a and b . The str7The interactions discussed so far in this review concern fairly weak and transient, but nevertheless specific, interactions that are made between short peptide motifs in SMRT and NCoR with various transcription factors. The motifs adopt a fixed conformation in the respective complexes, with key residues determining the specificity of the various interactions. In addition to these transient partners, there are also core interaction proteins that appear to be stably associated with the co-repressors and which are essential for the core role of the repressor complexes since they effect the chromatin remodeling functions. These core components interact with a highly conserved amino-terminal region of SMRT that has a strong transcriptional repression activity and has been termed repression domain 1 (RD1). This region contains binding sites for at least three proteins: HDAC3, TBL1/TBLR1 and GPS2. Together with SMRT, or NCoR, these proteins form the core repression complex. The region in SMRT that recruits this core complex, spans amino acids 167–480 and is predicted to be largely unstructured with the exception of a C-terminal helical domain that both recruits and activates HDAC3 and has been termed the deacetylase activation domain (DAD). The amino terminal portion of RD1 recruits both GPS2 and TBL1/TBLR1. Since TBL1 and GPS2 also interact directly with each other, a stable three-way complex is formed. These interactions were originally mapped by 7.1The DAD domain recruits and activates HDAC3  and compThe NMR structure of the DAD domain  shows thMutational analysis showed that this cleft is involved in the binding and activation of HDAC3 d. In addThe site of interaction with the DAD domain on HDAC3 has been mapped to the N-terminus and C-terminus of HDAC3 and deletion of either of these domains results in the loss of both interaction with the DAD domain and HDAC3 activation . Truncat7.2As mentioned earlier, GPS2 and TBL1 interact with the N-terminal region of the SMRT RD1. In addition to their interaction with SMRT, GPS2 and TBL1 also interact directly with each other. The NMR structure of the interacting regions of SMRT (167–207) and GPS2 (53–90) shows that the interaction is mediated by an anti-parallel coiled-coil  Fig. 4ccFig. 4c.TBL1 and its closely related homologue TBLR1  have a highly conserved N-terminal domain and a C-terminal WD40 domain. The N-terminal domain of TBL1 contains a LisH domain and mediates the interaction with both GPS2 and SMRT. The crystal structure of the N-terminal domain of TBL1 (residues 1–90) shows that TBL1 is a homotetramer, and would be expected to readily form heterotetramers with TBLR1 a and b . The priMutagenesis experiments show that both GPS2 and SMRT interact with the same hydrophobic groove on TBL1. This groove is formed between the four helix bundle and the third helix. This result seems surprising but can be explained in the context of a heterodimer of GPS2–SMRT binding to a homodimer of TBL1. Since SMRT and GPS2 interact in an anti-parallel fashion this then places the potential interaction helices with TBL1 in opposite orientations meaning that SMRT and GPS2 can make similar interactions with TBL1. A short sequence motif with the consensus A-x-x-A/L-H-R/K-x-Φ (where Φ is a large non-polar residue and x any residue) was identified in both GPS2 and SMRT. Modeling of the interaction of the peptides from GPS2/SMRT with the hydrophobic groove on TBL1 using HADDOCK identified similar interactions for both the GPS2 and SMRT sequences. The common residues that define the interaction motif all make important interactions at the interface. The histidine and non-polar residues are buried at the interface and the basic residue (Arg or Lys) on the back face of the helix interacts with two glutamic acid residues (Glu61 and Glu68) in TBL1. Glu7 (TBL1) is positioned so as to “cap” the SMRT and GPS2 interaction helices, interacting favorably with the helix dipole.The amino acids involved in dimerisation and tetramerisation of TBL1 are conserved in the closely related protein TBLR1. Hence tetramerisation of TBL1 means that the core co-repressor complex will probably contain four TBL1 or TBLR1 molecules as a scaffold for two GPS2 molecules, two SMRT or NCoR molecules and two HDAC3 molecules. Recent experiments in support of this stoichiometry show that SMRT is able to form homodimers and heterodimers with NCoR . This coTBL1 contains a C-terminal WD40 domain and in this regard is similar to co-repressor proteins such as Sif2p and Tup1 in yeast, Groucho in Drosophila and TLE proteins in humans. PHYRE modeling  of the T8Transcriptional regulator hub proteins, such as the SMRT and NCoR co-repressors, mediate and integrate interactions with a large number of other factors including transcription factors and enzymes that execute the repressive function of the complex. Structure/function analyses of these proteins and their complexes are challenging due to the largely unstructured nature of these proteins in the absence of their interacting partners. What we have learned to date has relied upon a combination of NMR, crystallographic, mutagenesis and functional assays. Such structural studies are of considerable interest not only to gain insight into the mechanisms through which these enzymatic activities are targeted to chromatin, but also for the potential to develop inhibitor molecules that can specifically inhibit co-repressor interactions with individual transcription factors. A good example of this is the potentially therapeutically useful inhibition of BCL-6 activity through blocking co-repressor recruitment.


Oral mite anaphylaxis is a new syndrome characterized by severe allergic manifestations occurring in atopic patients shortly after the intake of foods made with mite-contaminated wheat flour. This clinical entity, observed more frequently in tropical/subtropical environments, is more often triggered by pancakes and for that reason it has been designated "pancake syndrome". Because cooked foods are able to induce the symptoms, it has been proposed that thermoresistant allergens are involved in its production. A novel variety of this syndrome occurs during physical exercise and therefore has been named dust mite ingestion-associated exercise-induced anaphylaxis. To prevent mite proliferation and the production of anaphylaxis, it has been recommended that wheat flour be stored at low temperatures in the refrigerator. Domestic mites have been recognized as the most important allergen source responsible for respiratory allergic diseases, such as rhinitis and asthma, of high prevalence worldwide . Since 1The present article summarizes the observations made by our group and other investigators on this new allergic condition. The first reports on oral anaphylaxis from mite ingestion in the United States came from Detroit, Mich, and Philadelphia, Pa ,3.Tyroglyphus farinae (Acarus siro) in stools and gut [Tyroglyphus longior Gervais, a member of the family Tyroglyphidae, arthropods frequently found in dried fruits, dried seeds, grains, cereals, and cheese [Previously, other clinical manifestations associated with the intake of mite-contaminated foods had been published. In 1963 Herranz reported the case of a 56-year-old female patient who died after a massive ingestion of mites contained in a pap made with milk and wheat flour. Autopsy revealed intense intestinal irritation, disseminated granulomas in multiple organs, and the presence of many mites of the species  and gut . Hinman Various elements with pathogenic potential, including allergenic contaminants, may be present in the food. They include microorganisms , toxins, chemicals, food additives (dyes and preservatives), food allergens, allergens from other contaminating foods, cross-reactive allergens from pollens or latex, drugs , arthropods (mites), and insects (cockroaches).There are reports from various countries, including the United States, Japan, Brazil, Taiwan, Spain, and Venezuela, where OMA has been observed Table  2,3,6-1-16-13. UIn general, there is a predominance of reports from countries located in the intertropical area, probably because in those regions there are environmental conditions favorable for mite reproduction, especially higher temperature and relative humidity, for longer periods of time.Patients described in Detroit, Mich, and Philadelphia, Pa, had anaphylaxis after eating beignets made with flour delivered by mail from New Orleans,,3 whereaTable Clinical manifestations are summarized in Table Oral allergy syndrome  is characterized by an immediate hypersensitivity reaction generally localized to the lips and oral mucosa after contact with crude fruits or vegetables containing allergens that share homology with pollen proteins, the original source of sensitization. Because the responsible allergens are thermolabile proteins, cooked foods are tolerated. OAS is not associated with oral mite anaphylaxis.Incriminated foods are usually prepared with wheat flour, including pancakes, sponge cakes, pizza, pasta, bread, parmigiana steak (prepared with contaminated grated bread), and white sauce. Pancakes are the foods most frequently involved (53.3% of our patients) and for that reason we proposed in 2001 the designation of "Pancake syndrome" for this clinical entity, a nomencOther foods have also been found to induce OMA, including beignets, "okonomi-yaki" (bonito and mackerel covered with flour), cornmeal cakes made with a commercial mix containing wheat and corn flour, and polenta. Other foods that can be contaminated with mites when stored for long periods at room temperature are cheese, ham, chorizo, and salami -21.The observation that symptoms appeared after eating foods cooked by heating led us to perform skin prick tests with contaminated wheat flour unheated and after heating at 100°C for 1 hour in 13 mite-allergic volunteers. A significant reduction of wheal diameters was observed when the tests were done with heated flour; nevertheless, the tests continued to be positive. These results suggest that allergens inducing OMA are thermoresistant.Dermatophagoides pteronyssinus and Dermatophagoides farinae), and storage mites such as Suidasia spp., Aleuroglyphus ovatus, Lepidoglyphus destructor, Tyrophagus putrescentiae, Thyreophagus entomophagus, Blomia tropicalis, and Blomia freemani. The issues on allergenic cross-reactivity between different domestic and storage mite species have been recently reviewed by Fernández-Caldas et al [Different mite species are present in the flours producing OMA. Among them are house dust mites  to aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) has been observed in patients who develop OMA ,9 see T. In 19972 (PGE2) synthesis [The reasons for this association are not known because mite allergy is mediated by a specific IgE response to mite allergens, whereas most patients with urticaria and angioedema from NSAIDs react to various anti-inflammatory drugs of diverse chemical composition ("cross-reactions") that are regarded to be nonallergic hypersensitivity reactions likely mediated by the inhibition of the enzyme cyclooxy-genase-1 of arachidonic acid metabolism, which results in enhanced production of cysteinil-leukotrienes and decreased 26 prostaglandin Eleukotriene C4 synthase (LTC4S), the enzyme responsible for the synthesis of leukotrienes, in cross-reactive NSAID hypersensitive patients with urticaria and/or angioedema. In such study we confirmed our previous results on the increased prevalence of the atopic trait in those patients [In collaboration with Canadian investigators we demonstrated that allergenic mite extracts can inhibit cyclooxy-genase-1 in vitro . More repatients .LTC4S gene, the main locus that regulates cysteinyl leukotriene synthesis in humans. We previously proposed that perhaps this colocalization could explain the association between atopy  and NSAID hypersensitivity [Various lines of evidence suggest an interaction between IgE-specific immune responses and the leukotriene pathway. The so-called "atopic" genes implicated in allergic inflammation, including those determining the production of the cytokines IL-4, IL-5, and IL-13, are located in the 5q22-q35 chromosomal region of chromosome 5, close to the sitivity .B. tropicalis allergenic extract than atopic non-NSAID-sensitive patients [A-444C allele of the LTC4S gene and low levels of IgE antibodies to D. pteronyssinus, low total IgE, and monosensitization and suggested that LTC4S could be involved in the regulation of IgE response to mite allergens [More recently, we observed that patients cross-reactive to NSAIDs show larger wheals when prick-tested with patients . In thisllergens .LTC4S knockout mice have markedly reduced antigen-induced TH2 pulmonary inflammation [Cysteinyl leukotrienes can enhance IgE and IgG production by human B cells . LTC4S kammation . Also, vammation -35.There are also some recent clinical and experimental observations that support a modulating role for leukotrienes in allergy. For example, Aihara et al reported that acetylsalicylic acid facilitates food-dependent exercise-induced anaphylaxis, and aspiAdditionally, in experimental animals it has been observed that aspirin increases the permeability of the gastric mucosa to proteins and the development of anaphylaxis . Figure The following elements have been identified as factors increasing the risk for OMA: (1) History of atopic disease; (2) sensitization to mites; (3) cutaneous hypersensitivity to NSAIDs (urticaria and angioedema); (4) ingestion of pancakes or other foods prepared with mite-contaminated wheat flour; (5) ingestion of more than 1 mg of mite allergen (more than 500 mites per gram of flour) .The diagnosis is based on the following findings: (1) Compatible symptoms occurring after the intake of foods prepared with wheat flour; (2) previous history of rhinitis, asthma, atopic dermatitis, and/or food allergy; (3) in vivo or in vitro demonstration of IgE-mediated sensitization to mite allergens; (4) positive immediate-type skin test with extract of the suspected flour; (5) negative skin tests to wheat and to uncontaminated flour extract; (6) clinical tolerance of foods made with uncontaminated wheat flour; (7) microscopic identification of mites in the suspected flour; (8) presence of mite allergens in the flour; (9) hypersensitivity to NSAIDs.Recently, we observed a 16-year-old girl with previous history of asthma, rhinoconjunctivitis, atopic dermatitis, and allergy to clams, who presented generalized itching, facial angioedema, and breathlessness when playing soccer 30 minutes after eating a lunch consisting exclusively of pancakes. Treatment in the emergency room consisted of intravenous corticosteroids, chlorpheniramine, and nebulized albuterol. After discharge, oral methylprednisolone, desloratadine, inhaled budesonide, and formoterol were given.D. pteronyssinus, B. tropicalis, and the extract of the flour used to make the pancakes, whereas they were negative for wheat and other food extracts and for other inhalant allergens. Microscopic analysis of the flour showed the presence of Suidasia medanensis, quantified in 4814.8 mites per gram of flour. Der p1 and Der f1 allergens were not detected in the flour.Results of skin prick tests done to the patient 2 weeks after the episode are presented in Table S. medanensis, B. tropicalis, and D. farinae [The results of skin tests are in agreement with previous studies that showed a high degree of cross-reactivity between  farinae .This variant of pancake syndrome has been designated as dust mite ingestion-associated exercise-induced anaphylaxis . A similWe have observed mites in closed packages of wheat flour. Because the exposure to low temperatures inhibits mite proliferation and results in the transformation into protonymphal stages that are resistant to cold and do not reproduce actively, the recommendation has been made to store the flour in sealed containers in the refrigerator . Kasti e


In vitro reaction conditions, such as substrate specificity, cofactor usage, and cellular binding partners for such reactions by the three distinct domains of prototype foamy viral integrase (PFV-IN) have been described well in several reports. Recent studies on the three‑dimensional structure of the interacting complexes between PFV-IN and DNA, cofactors, binding partners, or inhibitors have explored the mechanistic details of such interactions and shown its utilization as an important target to develop anti-retroviral drugs. The presence of a potent, non-transferable nuclear localization signal in the PFV C-terminal domain extends its use as a model for investigating cellular trafficking of large molecular complexes through the nuclear pore complex and also to identify novel cellular targets for such trafficking. This review focuses on recent advancements in the structural analysis and in vitro functional aspects of PFV-IN.Successful integration of retroviral DNA into the host chromosome is an essential step for viral replication. The process is mediated by virally encoded integrase (IN) and orchestrated by 3'-end processing and the strand transfer reaction.  IN rerget DNA . However unknown .35, Glu, known as the D, D35, E motif, essential for catalysis of 3' processing, DNA strand transfer [2+ or Mg2+ [Retroviral IN proteins are comprised of the N-terminal domain (NTD), the central catalytic core domain (CCD), and the C-terminal domain (CTD) with varying evolutionary conservation . The higtransfer ,8, and dtransfer ,10. A ditransfer ,12,13. T or Mg2+ ,14.in vitro culture conditions [Foamy viruses (FVs) are classified into the subfamily Spumaretrovirinae based upon their differences from orthoretroviruses . The ininditions ,21. The ability of FVs to infect and integrate into the genome of a wide range of cells leads to the construction of a number of gene therapy vectors . The nonpol genes are expressed as Gag-Pol fusion proteins [pol genes are expressed from a spliced mRNA separately from gag [Generally, retroviral proteins . Howeverfrom gag ,29,30. Ffrom gag ,32,33. Efrom gag ,35,36.Autocatalytic cleavage of the Pol precursor protein of other retroviruses leads to the production of protease (PR), reverse transcriptase (RT) and RNase H (RN), and IN, whereas a single cleavage event in FVs causes cleavage of the 127 kDa Pol protein into two nuclear proteins, an approximately 85 kDa protein containing the PR, RT, and RN domains and a 40-kDa protein consisting of only the IN domain ,38. 2+ coordination occurs through His and Cys residues of the invariant HHCC motif [35, E amino acid sequence motif [64, D116, and E152 for HIV-1) are responsible for catalysis of all enzymatic activities such as 3' processing, DNA strand transfer [Among the three domains of retroviral IN proteins the structure of the NTD in solution reveals a compact three-helical bundle in which ZnCC motif ,40. Simice motif ,9, and ttransfer ,41 and dtransfer . The CTDtransfer ,43,44.Gammaretrovirus (murine leukemia virus and the feline leukemia virus) [Spumavirus [in vitro and is homologous to other retroviral INs [in vitro activity [Protein mixing experiments have revealed similar IN domain organizations from a virus)  and Spumumavirus . PFV IN iral INs ,48. Althiral INs ,49. The iral INs , which hactivity  however activity .in vitro experimentation and crystallographic determination of the retroviral intasome. The crystal structure of the PFV intasome provided insight into the HIV-1 drug resistance mechanism, as it has a 22% amino acid sequence similarity within the active sites of CCD domains of HIV-1 [Although numerous efforts have focused on structural analysis of HIV IN to develop new antiviral compounds and to unveil the enzymatic mechanisms, the exact three-dimensional structure of the full protein or its functional mechanism is still unknown ,51. The of HIV-1 ,56.2+, and pre-processed U5 viral DNA substrate retained concerted integration activity [2+-IN-vDNA assembly and the presence of ligands: Mn2+ or Mg2+, target DNA, or inhibitors were yielded from the PFV system [2 revealed metal bound intasomal active sites [Symmetric intasome complexes assembled with full-length PFV IN, Znactivity . IntasomV system . The intV system ,58. The V system ). HoweveV system ,60. Soakve sites  that supve sites .N2) reactions. Both steps are simultaneously accomplished by metal-dependent nucleotidyl transferase and nuclease action, like transposases and RNase H enzymes and both are catalyzed by a triad of acidic residues in a characteristic D, D35, E motif found in all retroviral INs [2+ ions to activate the attacking nucleophile  and destabilize the scissile phosphodiester bonds [The function of IN is to insert viral DNA into the host genome, and the reaction is accomplished by 3'-processing and DNA strand transfer steps, which are bimolecular nucleophilic substitution  enzymatic activities [Several detergents are generally used for improving the solubility of recombinant IN and these might have effects on the tivities . We evalin vitro integration [The isolated CCD domain from HIV-1  and ASLVegration ,73. Mixtegration ,75. HIV-egration ,76. Sizeegration .PFV IN shows efficient catalytic properties that make it a more elegant IN to study different enzymatic activities than HIV IN . A very Escherichia coli ruvC gene derived endonuclease), bacteriophage MuA transposase, and nuclease component of the RISC complex Argonaute [in vitro [. However, the PFV IN tetramer efficiently processes a pair of LTR ends in crystallo [Retroviral INs belong to the retroviral IN superfamily (RISF) which comprises nucleic acid metabolizing enzymes such as RNase H, RuvC  was the first antiretroviral agent introduced in 1986 . A totaltegravir . The higtegravir  implies tegravir ,105,106.tegravir . Crystaltegravir .Successful infection and replication of retroviruses in their host depend largely on the reverse transcription of their RNA genome to cDNA and integration into the host cell chromosome (reviewed in ). IntegrSeveral studies have indicated that FV IN contains NLS sequences ,130. A mWe investigated the karyophilic determinant of PFV IN, and our results demonstrated that PFV IN possesses a potent NLS in its C-terminal domain spanning from residues 289–371 . StudiesAlthough much effort has long been undertaken to use HIV-1 IN as a target for anti-retroviral drug development, the exact structural elucidation of full-length protein remains unknown. Rapid emergence of HIV-1 IN inhibitor resistant strains necessitate the knowledge of the structural details of active site geometry and IN domain interactions in order to develop more effective drugs. PFV IN is equally sensitive to HIV-1 IN inhibitors therefore the crystal structures of PFV intasome as the only determined retroviral intasome will help to elucidate the structural basis of HIV-1 drug resistance to clinical INSTIs. This review provides insights about the biochemical and enzymatic activities of FV IN and the reasons for using FV IN as a model system to investigate the structural basis of anti-retroviral therapy as well as to improve the safety of FV vector systems.


Yersinia, three have been shown to be virulent and pathogenic to humans and animals—Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis. In order to be so, they are armoured with various factors that help them adhere to tissues and organelles, cross the cellular barrier and escape the immune system during host invasion. The group of proteins that mediate pathogen–host interactions constitute adhesins. Invasin, Ail, YadA, YadB, YadC, Pla, and pH 6 antigen belong to the most prominent and best-known Yersinia adhesins. They act at different times and stages of infection complementing each other by their ability to bind a variety of host molecules such as collagen, fibronectin, laminin, β1 integrins, and complement regulators. All the proteins are anchored in the bacterial outer membrane (OM), often forming rod-like or fimbrial-like structures that protrude to the extracellular milieu. Structural studies have shown that the anchor region forms a β-barrel composed of 8, 10, or 12 antiparallel β-strands. Depending on the protein, the extracellular part can be composed of several domains belonging to the immunoglobulin fold superfamily, or form a coiled-coil structure with globular head domain at the end, or just constitute several loops connecting individual β-strands in the β-barrel. Those extracellular regions define the activity of each adhesin. This review focuses on the structure and function of these important molecules, and their role in pathogenesis.Among the seventeen species of the Gram-negative genus  Yersiniae belong to the Enterobacteriaceae family; they are Gram-negative, facultative anaerobes. Seventeen different species of Yersinia genus have so far been reported, of which three have been shown to be pathogenic to humans and animals. These are the enteropathogens Y. enterocolitica and Y. pseudotuberculosis, and one zoonotic pathogen Y. pestis. Pathogenicity is correlated mostly with 70-kb virulence plasmid pYV carried by all three of them and with additional chromosomally encoded proteins . Bacteria resistant to phagocytosis spread into the bloodstream causing septicaemic plague. Moreover, infection can continue and bacteria further colonize blood, liver, spleen, or even lungs, which leads to secondary pneumonic plague   species of inv gen, which is maximally expressed at 25°C, pH 8 or at 37°C, pH 5.5 but poorly at 37°C, pH 8. This indicates that invasin is expressed prior to oral uptake , which may be beneficial for rapid transcytosis through the epithelial layer, or in the intestinal tissue , and silencing mediated by negative regulator H-NS (nucleoid structuring protein), which forms higher order complexes in a concentration-dependent manner and causes gene silencing  are closely related sequentially and structurally to the intimins, OM proteins from the EPEC and enterohemorrhagic (EHEC) By having a β-barrel at the N-terminus and the extracellular domain at the C-terminus Figure , InvasinThe barrel, similarly to AT β domains, is composed of 12 β-strands arranged in an antiparallel fashion. The barrel pore is filled with a linker, which in contrast to transporter domains of AT is not α-helical but instead adopts an extended conformation. In addition, the periplasmic α-helix connecting the transporter domain with a linker to the passenger domain does not occur in ATs Figure .Y. enterocolitica. The D4–D5 domains form a functional module that is sufficient for binding to integrins with an affinity about 100 times higher than fibronectin   terminated with an α +β domain D5. D1–D4 form bacterial immunoglobulin-like (BIG) domains, from which D1 belongs to the I2 set of the immunoglobulin superfamily, D2 and D3 belong to the I1 set, and D4 to the C1 set. D5 has a folding topology related to the C-type lectin-like domains Figure  Hamburg. D2 is n) Figure  are impo) Figure . The S-S) Figure  is very ) Figure .Yersinia, invasin initiates the internalization of bacteria to the epithelial cells of the small intestine, in particular to M cells, and maintains the invasion of the Payer's patches .YadA is encoded on pYV, the virulence plasmid, and expressed as a virulence factor by EPEC Parts of the collagen-binding domain of YadA (1P9H)  domain and a C-terminal β-barrel (translocation domain) anchored in the OM. In TAAs the passenger domain is highly variable in sequence and size (200–3000 amino acid residues). The exact reason for the huge variability is not known, but Linke et al.  suggest The passenger domains contain three main regions: stalk, connector, and head Figure  Szczesn, which aY. enterocolitica was the first to show the left-handed parallel β-roll (LPBR) fold  is adhesion. It binds fibrillar collagen types I, II, III, V  in 6 M urea  preferentially binds fibronectin, but not collagen and laminin. This is because of the presence of an additional 31 amino acid residues in the head region  and YadC (61.6 kDa) are analogous to YadA but share similarity only in the C-terminal part (14% identity and 20% similarity over 264 residues) and within a short neck domain   is a chromosomally encoded 17 kDa protein that is associated with Yersinia virulence   Figure  (Yamashi) Figure . Ail forY. pestis Ail have not been determined. However, the location of the residues conferring pathogenic activity has been identified in Y. enterocolitica Ail. They are in the extracellular loops 2 and 3, of which loop 2 is absolutely required for both invasion and serum resistance  and C4 binding protein (C4bp), and so downregulates complement activation in the same way as YadA, leading to bacterial survival , it is functional both at 28 and 37°C  is a virulence factor, adhesin and protease  differ from those found in other proteases suggesting a new catalytic mechanism, involving an Asp-His catalytic dyad and an Asp-Asp couple that activates the nucleophilic water molecule Figure  Kramer . The preE. coli)  (plasmin deactivator) allows Y. pestis to invade the host rapidly and to migrate to lymphatic tissue  , but also related adhesins in other bacteria species.The first Yersinia adhesin identified was pH 6 antigen, discovered in 1961 , and BadA (from Bartonella henselae) are good examples of this. The adhesins play crucial roles in cell attachment, invasion, serum resistance and so in the maintenance and spread of the infection, by interacting with different cell types and host molecules. Even though we continually gain insight into their molecular mechanism, the exact role of adhesins during infection remains unclear. This is mainly because each bacterium expresses multiple adhesins that have overlapping functions and binding affinities and even after deletion of one adhesin another typically compensates for its function at least in part. Moreover, due to the complexity of bacteria–bacteria and bacteria–host interactions, exactly which adhesins are expressed during the various phases of infection has not been easy to establish: the in vitro experiments altering pH and temperature only crudely mimic the situation in vivo, when the bacteria experience shear stresses due to, e.g., blood flow and form biofilms. In this, the use of genuine tissue samples such as primary umbilical cord (Riess et al., ex vivo under conditions close to in vivo. Finally, while the structure of many adhesins is known, there are no structures of adhesin-ligand complexes. That makes even more difficult to establish how they function.All the bacteria possessing genes for adhesins express them on the cell surface during the first stages of host infection. Usually, they comprise the majority of proteins present in the OM facing outside of the cell; YadA, UspA1 (from ex vivo experiments. Understanding the interactions of adhesins with their ligands at the molecular level by solving structures between bacterial adhesins and host ligands, and using this information for drug and vaccine design, is the next major challenge.To resolve the contribution of each adhesin in phatogenicity will require highly sensitive protein techniques to identify the cell-surface molecules expressed in small primary populations of bacteria taken directly from host tissue as well as The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.