Showing papers by "Laboratory of Molecular Biology published in 2007"
TL;DR: A new web interface to the popular Primer3 primer design program, developed in close collaboration with molecular biologists and technicians regularly designing primers, that provides an intuitive user interface using present-day web technologies and allows easy expansion or integration of external software packages.
Abstract: Here we present Primer3Plus, a new web interface to the popular Primer3 primer design program as an enhanced alternative for the CGI- scripts that come with Primer3. Primer3 consists of a command line program and a web interface. The web interface is one large form showing all of the possible options. This makes the interface powerful, but at the same time confusing for occasional users. Primer3Plus provides an intuitive user interface using present-day web technologies and has been developed in close collaboration with molecular biologists and technicians regularly designing primers. It focuses on the task at hand, and hides detailed settings from the user until these are needed. We also added functionality to automate specific tasks like designing primers for cloning or step-wise sequencing. Settings and designed primer sequences can be stored locally for later use. Primer3Plus supports a range of common sequence formats, such as FASTA. Finally, primers selected by Primer3Plus can be sent to an order form, allowing tight integration into laboratory ordering systems. Moreover, the open architecture of Primer3Plus allows easy expansion or integration of external software packages. The Primer3Plus Perl source code is available under GPL license from SourceForge. Primer3Plus is available at http://www.bioinformatics.nl/primer3plus.
2,368 citations
TL;DR: It is shown that cell lines can be derived from the epiblast, a tissue of the post-implantation embryo that generates the embryo proper, and interrogated to understand how pluripotent cells generate distinct fates during early development.
Abstract: The application of human embryonic stem (ES) cells in medicine and biology has an inherent reliance on understanding the starting cell population. Human ES cells differ from mouse ES cells and the specific embryonic origin of both cell types is unclear. Previous work suggested that mouse ES cells could only be obtained from the embryo before implantation in the uterus. Here we show that cell lines can be derived from the epiblast, a tissue of the post-implantation embryo that generates the embryo proper. These cells, which we refer to as EpiSCs (post-implantation epiblast-derived stem cells), express transcription factors known to regulate pluripotency, maintain their genomic integrity, and robustly differentiate into the major somatic cell types as well as primordial germ cells. The EpiSC lines are distinct from mouse ES cells in their epigenetic state and the signals controlling their differentiation. Furthermore, EpiSC and human ES cells share patterns of gene expression and signalling responses that normally function in the epiblast. These results show that epiblast cells can be maintained as stable cell lines and interrogated to understand how pluripotent cells generate distinct fates during early development.
2,134 citations
TL;DR: The best understood system for the transport of macromolecules between the cytoplasm and the nucleus is the classical nuclear import pathway and a bioinformatics approach is taken to analyze the likely prevalence of this system in vivo.
1,132 citations
TL;DR: Pre-SCOP, a preview of the SCOP developmental version that enables earlier access to the information on new relationships, is introduced and the impact of worldwide Structural Genomics initiatives, which are producing new protein structures at an increasing rate, on the rates of discovery and growth of protein families and superfamilies is discussed.
Abstract: The Structural Classification of Proteins (SCOP) database is a comprehensive ordering of all proteins of known structure, according to their evolutionary and structural relationships. The SCOP hierarchy comprises the following levels: Species, Protein, Family, Superfamily, Fold and Class. While keeping the original classification scheme intact, we have changed the production of SCOP in order to cope with a rapid growth of new structural data and to facilitate the discovery of new protein relationships. We describe ongoing developments and new features implemented in SCOP. A new update protocol supports batch classification of new protein structures by their detected relationships at Family and Superfamily levels in contrast to our previous sequential handling of new structural data by release date. We introduce pre-SCOP, a preview of the SCOP developmental version that enables earlier access to the information on new relationships. We also discuss the impact of worldwide Structural Genomics initiatives, which are producing new protein structures at an increasing rate, on the rates of discovery and growth of protein families and superfamilies. SCOP can be accessed at http:// scop.mrc-lmb.cam.ac.uk/scop.
880 citations
TL;DR: It is proposed that Wnts induce coclustering of receptors and Dvl in LRP6-signalosomes, which in turn triggers LRP 6 phosphorylation to promote Axin recruitment and β-catenin stabilization.
Abstract: Multiple signaling pathways, including Wnt signaling, participate in animal development, stem cell biology, and human cancer. Although many components of the Wnt pathway have been identified, unresolved questions remain as to the mechanism by which Wnt binding to its receptors Frizzled and Low-density lipoprotein receptor-related protein 6 (LRP6) triggers downstream signaling events. With live imaging of vertebrate cells, we show that Wnt treatment quickly induces plasma membrane-associated LRP6 aggregates. LRP6 aggregates are phosphorylated and can be detergent-solubilized as ribosome-sized multiprotein complexes. Phospho-LRP6 aggregates contain Wnt-pathway components but no common vesicular traffic markers except caveolin. The scaffold protein Dishevelled (Dvl) is required for LRP6 phosphorylation and aggregation. We propose that Wnts induce coclustering of receptors and Dvl in LRP6-signalosomes, which in turn triggers LRP6 phosphorylation to promote Axin recruitment and beta-catenin stabilization.
838 citations
TL;DR: The nuclear import of proteins through nuclear pore complexes (NPCs) illustrates how a complex biological function can be generated by a spatially and temporally organized cycle of interactions between cargoes, carriers and the Ran GTPase.
Abstract: The nuclear import of proteins through nuclear pore complexes (NPCs) illustrates how a complex biological function can be generated by a spatially and temporally organized cycle of interactions between cargoes, carriers and the Ran GTPase. Recent work has given considerable insight into this process, especially about how interactions are coordinated and the basis for the molecular recognition that underlies the process. Although considerable progress has been made in identifying and characterizing the molecular interactions in the soluble phase that drive the nuclear protein import cycle, understanding the precise mechanism of translocation through NPCs remains a major challenge.
834 citations
TL;DR: The intrinsic requirement for miR-155 is shown in B cell responses to thymus-dependent and -independent antigens and implicate post-transcriptional regulation of gene expression for establishing the terminal differentiation program of B cells.
789 citations
TL;DR: Several previously unsuspected key links between multiple ESCRT subunits, phospholipids and ubiquitin have now been elucidated, which, together with the detailed morphological analyses of ESCRT-depletion phenotypes, provide new insights into the mechanism of MVB biogenesis.
Abstract: The past two years have seen an explosion in the structural understanding of the endosomal sorting complex required for transport (ESCRT) machinery that facilitates the trafficking of ubiquitylated proteins from endosomes to lysosomes via multivesicular bodies (MVBs). A common organization of all ESCRTs is a rigid core attached to flexibly connected modules that recognize other components of the MVB pathway. Several previously unsuspected key links between multiple ESCRT subunits, phospholipids and ubiquitin have now been elucidated, which, together with the detailed morphological analyses of ESCRT-depletion phenotypes, provide new insights into the mechanism of MVB biogenesis.
710 citations
01 Jan 2007
595 citations
TL;DR: What is known about all the members of the Arf family, along with the known regulatory molecules that convert them between GDP- and GTP-bound states are reviewed.
Abstract: Small G proteins play a central role in the organization of the secre- tory and endocytic pathways. The majority of such small G proteins are members of the Rab family, which are anchored to the bilayer by C-terminal prenyl groups. However, the recruitment of some effectors, including vesicle coat proteins, is mediated by a second class of small G proteins that is unique in having an N-terminal amphipathic helix that becomes available for membrane insertion upon GTP binding. Sar1, Arf1, and Arf6 are the best-characterized members of this ADP-ribosylation factor (Arf ) family. In addition, all eukaryotes contain additional distantly related G proteins, often called Arf like, or Arls. The complete Arf family in humans has 29 members. The roles of these related G proteins are poorly under- stood, but recent work has shown that some are involved in mem- brane traffic or organizing the cytoskeleton. Here we review what is known about all the members of the Arf family, along with the known regulatory molecules that convert them between GDP- and GTP-bound states.
560 citations
TL;DR: It is found that, in response to Ca2+ binding, synaptotagmin-1 could promote SNARE-mediated fusion by lowering this activation barrier by inducing high positive curvature in target membranes on C2-domain membrane insertion.
Abstract: Synaptic vesicles loaded with neurotransmitters are exocytosed in a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent manner after presynaptic depolarization induces calcium ion (Ca2+) influx. The Ca2+ sensor required for fast fusion is synaptotagmin-1. The activation energy of bilayer-bilayer fusion is very high (approximately 40 k(B)T). We found that, in response to Ca2+ binding, synaptotagmin-1 could promote SNARE-mediated fusion by lowering this activation barrier by inducing high positive curvature in target membranes on C2-domain membrane insertion. Thus, synaptotagmin-1 triggers the fusion of docked vesicles by local Ca2+-dependent buckling of the plasma membrane together with the zippering of SNAREs. This mechanism may be widely used in membrane fusion.
European Bioinformatics Institute1, University of Manchester2, Wellcome Trust Sanger Institute3, Wellcome Trust Centre for Human Genetics4, Centre national de la recherche scientifique5, University College London6, French Institute for Research in Computer Science and Automation7, Laboratory of Molecular Biology8, Georgetown University9
TL;DR: The latest InterPro release (13.0) contains more than 13 000 entries, covering over 78% of all proteins in UniProtKB, and there have been several new developments in InterPro, including an additional reading field, new database links, extensions to the web interface and additional match XML files.
Abstract: InterPro is an integrated resource for protein families, domains and functional sites, which integrates the following protein signature databases: PROSITE, PRINTS, ProDom, Pfam, SMART, TIGRFAMs, PIRSF, SUPERFAMILY, Gene3D and PANTHER. The latter two new member databases have been integrated since the last publication in this journal. There have been several new developments in InterPro, including an additional reading field, new database links, extensions to the web interface and additional match XML files. InterPro has always provided matches to UniProtKB proteins on the website and in the match XML file on the FTP site. Additional matches to proteins in UniParc (UniProt archive) are now available for download in the new match XML files only. The latest InterPro release (13.0) contains more than 13 000 entries, covering over 78% of all proteins in UniProtKB. The database is available for text- and sequence-based searches via a webserver (http://www.ebi.ac.uk/interpro), and for download by anonymous FTP (ftp://ftp.ebi.ac.uk/pub/databases/interpro). The InterProScan search tool is now also available via a web service at http://www.ebi.ac.uk/Tools/webservices/WSInterProScan.html.
TL;DR: In this paper, the authors used an oligonucleotide microarray to monitor rice (Oryza sativa) microRNA expression profile under drought stress and identified two drought-induced microRNAs.
TL;DR: In vivo and in vitro studies reveal a central role for Fbxl3 in mammalian circadian timekeeping and identify a mouse mutation, after hours (Afh), which results in long free-running rhythms of about 27 hours in homozygotes.
Abstract: By screening N-ethyl-N-nitrosourea–mutagenized animals for alterations in rhythms of wheel-running activity, we identified a mouse mutation, after hours (Afh). The mutation, a Cys358Ser substitution in Fbxl3, an F-box protein with leucine-rich repeats, results in long free-running rhythms of about 27 hours in homozygotes. Circadian transcriptional and translational oscillations are attenuated in Afh mice. The Afh allele significantly affected Per2 expression and delayed the rate of Cry protein degradation in Per2::Luciferase tissue slices. Our in vivo and in vitro studies reveal a central role for Fbxl3 in mammalian circadian timekeeping.
TL;DR: It is shown that Snail activation and consequent repression of E-cadherin may depend on AKT-mediated nuclear factor-κB activation, and that NF-κBs induces Snail expression, which is a potential target for antimetastatic therapeutics.
Abstract: Carcinoma progression is associated with the loss of epithelial features, and the acquisition of mesenchymal characteristics and invasive properties by tumour cells. The loss of cell-cell contacts may be the first step of the epithelium mesenchyme transition (EMT) and involves the functional inactivation of the cell-cell adhesion molecule E-cadherin. Repression of E-cadherin expression by the transcription factor Snail is a central event during the loss of epithelial phenotype. Akt kinase activation is frequent in human carcinomas, and Akt regulates various cellular mechanisms including EMT. Here, we show that Snail activation and consequent repression of E-cadherin may depend on AKT-mediated nuclear factor-kappaB (NF-kappaB) activation, and that NF-kappaB induces Snail expression. Expression of the NF-kappaB subunit p65 is sufficient for EMT induction, validating this signalling module during EMT. NF-kappaB pathway activation is associated with tumour progression and metastasis of several human tumour types; E-cadherin acts as a metastasis suppressor protein. Thus, this signalling and transcriptional network linking AKT, NF-kappaB, Snail and E-cadherin during EMT is a potential target for antimetastatic therapeutics.
TL;DR: The properties of CTCF, and its newly described genomic distribution, suggest that it may play an important role in large-scale nuclear architecture, perhaps mediated by the co-factors with which it interacts in vivo.
TL;DR: AM-hMSCs may emerge as a remarkable tool for the cell therapy of multiple diseased tissues due to their angiogenic potential and may also pave the way for novel approaches in the development of tissue-engineered vascular grafts which are useful when vascularization of ischemic tissues is required.
Abstract: Background
Term Amniotic membrane (AM) is a very attractive source of Mesenchymal Stem Cells (MSCs) due to the fact that this fetal tissue is usually discarded without ethical conflicts, leading to high efficiency in MSC recovery with no intrusive procedures. Here we confirmed that term AM, as previously reported in the literature, is an abundant source of hMSCs; in particular we further investigated the AM differentiation potential by assessing whether these cells may also be committed to the angiogenic fate. In agreement with the recommendation of the International Society for Cellular Therapy, the mesenchymal cells herein investigated were named Amniotic Membrane-human Mesenchymal Stromal Cells (AM-hMSC).
TL;DR: The role of local SCN-synchronised clocks in controlling vital processes, including xenobiotic detoxification, cell division and nutrient metabolism, is essential to health, and disturbances to circadian timing arising from modern working schedules are becoming recognised as an increasingly relevant factor in major systemic illness.
Abstract: Daily and seasonal rhythms in the endocrine system are co-ordinated by a hypothalamic pacemaker, the suprachiasmatic nuclei (SCN) that is synchronised to solar time by direct retinal afferents. Individual SCN neurons are circadian clocks, their intrinsic oscillator consisting of a series of interlinked autoregulatory transcriptional/post-translational feedback loops incorporating Period (Per) and Cryptochrome (Cry) genes. Mutations that alter the rate of transcription of Per and Cry genes or the stability of Per and Cry proteins affect clock speed. Molecular timekeeping in SCN neurons is synchronised and sustained by interneuronal neuropeptidergic signals. A molecular clock mechanism comparable to that of the SCN is present in most major organ systems. These tissue clocks are synchronised by endocrine, autonomic and behavioural cues that are dependent on the SCN, and in turn they drive the circadian expression of local transcriptomes, thereby co-ordinating circadian metabolism and physiology. Rhythmic glucocorticoid signalling is a prominent mediator of SCN output and internal synchroniser. The role of local SCN-synchronised clocks in controlling vital processes, including xenobiotic detoxification, cell division and nutrient metabolism, is essential to health, and disturbances to circadian timing arising from modern working schedules are becoming recognised as an increasingly relevant factor in major systemic illness. Moreover, the newly identified molecular components of circadian control systems provide novel avenues for therapeutic intervention.
TL;DR: It is demonstrated that the DIX domain of DVL2 mediates dynamic polymerization, which is essential for the signaling activity of Dvl2, the purified domain of which polymerizes gradually, reversibly and in a concentration dependent manner, ultimately forming fibrils.
Abstract: The Wnt signaling pathway controls numerous cell fates in animal development and is also a major cancer pathway. Dishevelled (Dvl) transduces the Wnt signal by interacting with the cytoplasmic Axin complex. Dvl and Axin each contain a DIX domain whose molecular properties and structure are unknown. Here, we demonstrate that the DIX domain of Dvl2 mediates dynamic polymerization, which is essential for the signaling activity of Dvl2. The purified domain polymerizes gradually, reversibly and in a concentration dependent manner, ultimately forming fibrils. The Axin DIX domain has a novel structural fold largely composed of β-strands that engage in head-to-tail self-interaction to form filaments in the crystal. The DIX domain thus seems to mediate the formation of a dynamic interaction platform with a high local concentration of binding sites for transient Wnt signaling partners; this represents a previously uncharacterized mechanistic principle, signaling by reversible polymerization.
TL;DR: A novel pathway for the induction of AHR is defined and it is suggested that IL-25 represents an important therapeutic target for the treatment of asthma and blocks the binding of human IL- 25 to its receptor.
Abstract: Background IL-25 (IL-17E), a member of the IL-17 family of immunoregulatory cytokines, has been implicated in the regulation of type 2 immunity. Its roles in antigen-driven airway inflammation and airway hyperresponsiveness (AHR) remain to be fully established. Objective We sought to determine whether a neutralizing antibody against IL-25 represents a novel therapeutic for airway inflammation and hyperresponsiveness. Methods We generated a neutralizing mAb against IL-25 and used this to inhibit IL-25 in a mouse model of allergic airway disease. Results Blocking IL-25 in an experimental model of allergic asthma prevented AHR, a critical feature of clinical asthma. Administration of anti–IL-25 mAb during the sensitization phase resulted in significantly reduced levels of IL-5 and IL-13 production, eosinophil infiltration, goblet cell hyperplasia, and serum IgE secretion, and prevented AHR. Even more striking was the ability of anti–IL-25 mAb, administered only during the challenge phase of the response, specifically to prevent AHR even during an ongoing type 2 inflammatory response in the lungs. Conclusion IL-25 is critical for development of AHR. Clinical implications We define a novel pathway for the induction of AHR and suggest that IL-25 represents an important therapeutic target for the treatment of asthma. Significantly, our antibody also blocks the binding of human IL-25 to its receptor.
TL;DR: The crystallographic and biochemical approaches used to gain insight into activating mutations in two noncatalytic p110α domains—the adaptor-binding and the helical domains provide insight into how two classes of mutations that cause a gain in function can lead to cancer.
Abstract: Many human cancers involve up-regulation of the phosphoinositide 3-kinase PI3Kalpha, with oncogenic mutations identified in both the p110alpha catalytic and the p85alpha regulatory subunits. We used crystallographic and biochemical approaches to gain insight into activating mutations in two noncatalytic p110alpha domains-the adaptor-binding and the helical domains. A structure of the adaptor-binding domain of p110alpha in a complex with the p85alpha inter-Src homology 2 (inter-SH2) domain shows that oncogenic mutations in the adaptor-binding domain are not at the inter-SH2 interface but in a polar surface patch that is a plausible docking site for other domains in the holo p110/p85 complex. We also examined helical domain mutations and found that the Glu545 to Lys545 (E545K) oncogenic mutant disrupts an inhibitory charge-charge interaction with the p85 N-terminal SH2 domain. These studies extend our understanding of the architecture of PI3Ks and provide insight into how two classes of mutations that cause a gain in function can lead to cancer.
TL;DR: The most potent immunotoxins are made from bacterial and plant toxins as discussed by the authors, which are composed of an antibody fragment linked to a toxin, and the immunotoxin binds to a surface antigen on a cancer cell, enters the cell by endocytosis, and kills it.
Abstract: Immunotoxins are proteins used to treat cancer that are composed of an antibody fragment linked to a toxin. The immunotoxin binds to a surface antigen on a cancer cell, enters the cell by endocytosis, and kills it. The most potent immunotoxins are made from bacterial and plant toxins. Refinements over many years have produced recombinant immunotoxins; these therapeutic proteins are made using protein engineering. Individual immunotoxins are designed to treat specific cancers. To date, most success has been achieved treating hematologic tumors. Obstacles to successful treatment of solid tumors include poor penetration into tumor masses and the immune response to the toxin component of the immunotoxin, which limits the number of cycles that can be given. Strategies to overcome these limitations are being pursued.
TL;DR: SS1P is well tolerated with pleuritis as the DLT at the highest dose level and Phase II clinical trials of SS1P are being planned for malignant mesothelioma and other mesothelin-expressing malignancies.
Abstract: Purpose: To determine the toxicities, maximum tolerated dose (MTD) and pharmacokinetics of the recombinant immunotoxin SS1P (anti-mesothelin dsFv-PE38) in patients with mesothelin-expressing cancers. Experimental Design: SS1P given as a 30-min i.v. infusion every other day (QOD) for six or three doses was administered to 34 patients with advanced mesothelioma ( n = 20), ovarian ( n = 12), and pancreatic ( n = 2) cancer. Results: The initial cohort of 17 patients received SS1P QOD × 6 doses and the MTD was 18 μg/kg/dose. Dose-limiting toxicities (DLT) included grade 3 uticaria (one patient) and grade 3 vascular leak syndrome (two patients). To allow further SS1P dose escalation, 17 patients were treated on the QOD × 3 schedule and the MTD was 45 μg/kg/dose. The DLT was grade 3 pleuritis and was seen in two of two patients treated at a dose of 60 μg/kg and in one of nine patients treated at a dose of 45 μg/kg. At the MTD of 45 μg/kg, the mean C max of SS1P was 483 ng/mL and half-life was 466 min. Of the 33 evaluable patients treated, 4 had minor responses, 19 had stable disease (including 2 with resolution of ascites), and 10 had progressive disease. Conclusions: SS1P is well tolerated with pleuritis as the DLT at the highest dose level. Evidence of clinical activity was noted in a group of heavily pretreated patients. Phase II clinical trials of SS1P are being planned for malignant mesothelioma and other mesothelin-expressing malignancies.
TL;DR: JARID1B is identified as a demethylase capable of removing three methyl groups from histone H3 lysine 4 and up-regulated in prostate cancer and associates with androgen receptor and regulates its transcriptional activity.
Abstract: Histone methylation is a dynamic process that participates in a diverse array of cellular processes and has been found to associate with cancer. Recently, several histone demethylases have been identified that catalyze the removal of methylation from histone H3 lysine residues. Through bioinformatic and biochemical analysis, we identified JARID1B as a H3K4 demethylase. Overexpression of JARID1B resulted in loss of tri-, di-, and monomethyl H3K4 but did not affect other histone lysine methylations. In vitro biochemical experiments demonstrated that JARID1B directly catalyzes the demethylation. The enzymatic activity requires the JmjC domain and uses Fe(II) and α-ketoglutarate as cofactors. Furthermore, we found that JARID1B is up-regulated in prostate cancer tissues, compared with benign prostate samples. We also demonstrated that JARID1B associates with androgen receptor and regulates its transcriptional activity. Thus, we identified JARID1B as a demethylase capable of removing three methyl groups from histone H3 lysine 4 and up-regulated in prostate cancer.
TL;DR: The results suggest that eIF1 and eif1A promote an open, scanning-competent preinitiation complex that closes upon start codon recognition and eIF 1 release to stabilize ternary complex binding and clamp down on mRNA.
TL;DR: Three-dimensional structures of multidomain proteins that have been characterized experimentally are analyzed and it is observed that where the interface is small and loosely packed, or unstructured, the folding of the domains is independent.
Abstract: Analyses of genomes show that more than 70% of eukaryotic proteins are composed of multiple domains. However, most studies of protein folding focus on individual domains and do not consider how interactions between domains might affect folding. Here, we address this by analysing the three-dimensional structures of multidomain proteins that have been characterized experimentally and observe that where the interface is small and loosely packed, or unstructured, the folding of the domains is independent. Furthermore, recent studies indicate that multidomain proteins have evolved mechanisms to minimize the problems of interdomain misfolding.
TL;DR: It is demonstrated that IL‐33 is a selective Th2 chemoattractant associated with the pro‐inflammatory property of this novel cytokine.
Abstract: IL-33 is a novel cytokine of the IL-1 family and mediates its biological effect via the receptor ST2, which is selectively expressed on Th2 cells but not Th1 cells. IL-33 drives production of Th2-associated cytokines including IL-5 and IL-13 and thereby promotes defense and pathology in mucosal organs. Cell locomotion is crucial to the induction of an effective immune response. We report here the chemoattraction of Th2 cells by IL-33. Recombinant IL-33 increased the proportion of human Th2 cells, but not Th1 cells, in polarized morphology in vitro and stimulated their subsequent invasion into collagen gels in an IL-33 concentration-dependent manner. Injection of recombinant IL-33 into the footpad of ST2–/– mice which had been adoptively transferred with polarized Th2 cells, led to local accumulation of the transferred Th2 cells. These data therefore demonstrate that IL-33 is a selective Th2 chemoattractant associated with the pro-inflammatory property of this novel cytokine.
TL;DR: The function of the yeast SBDS ortholog Sdo1 is identified, showing that it is critical for the release and recycling of the nucleolar shuttling factor Tif6 from pre-60S ribosome, a key step in 60S maturation and translational activation of ribosomes.
Abstract: The Shwachman-Bodian-Diamond syndrome protein mediates translational activation of ribosomes in yeast
TL;DR: Characterization of the IgG binding site for TRIM 21 PRYSPRY reveals TRIM21 as a superantigen analogous to bacterial protein A and suggests that an antibody bipolar bridging mechanism may contribute to the pathogenic accumulation of anti-TRIM21 autoantibody immune complex in autoimmune disease.
Abstract: The human tripartite motif (TRIM) family comprises 70 members, including HIV restriction factor TRIM5α and disease-associated proteins TRIM20 (pyrin) and TRIM21. TRIM proteins have conserved domain architecture but diverse cellular roles. Here, we describe how the C-terminal PRYSPRY domain mediates diverse TRIM functions. The crystal structure of TRIM21 PRYSPRY in complex with its target IgG Fc reveals a canonical binding interface comprised of two discrete pockets formed by antibody-like variable loops. Alanine scanning of this interface has identified the hot-spot residues that control TRIM21 binding to Fc; the same hot-spots control HIV/murine leukemia virus restriction by TRIM5α and mediate severe familial Mediterranean fever in TRIM20/pyrin. Characterization of the IgG binding site for TRIM21 PRYSPRY reveals TRIM21 as a superantigen analogous to bacterial protein A and suggests that an antibody bipolar bridging mechanism may contribute to the pathogenic accumulation of anti-TRIM21 autoantibody immune complex in autoimmune disease.
TL;DR: It is shown here how a careful understanding of a given biological pathway can refine an interactome approach, which permits the elucidation of additional design principles and of spatio-temporal dynamics behind pathways, and aids in experimental design and interpretation.
Abstract: The strength of network biology lies in its ability to derive cell biological information without a priori mechanistic or molecular knowledge. It is shown here how a careful understanding of a given biological pathway can refine an interactome approach. This permits the elucidation of additional design principles and of spatio-temporal dynamics behind pathways, and aids in experimental design and interpretation.