Identification of the cellular receptor for anthrax toxin.
TL;DR: The cloning of the human PA receptor is described using a genetic complementation approach and a soluble version of this domain can protect cells from the action of the toxin.
Abstract: The tripartite toxin secreted by Bacillus anthracis, the causative agent of anthrax, helps the bacterium evade the immune system and can kill the host during a systemic infection. Two components of the toxin enzymatically modify substrates within the cytosol of mammalian cells: oedema factor (OF) is an adenylate cyclase that impairs host defences through a variety of mechanisms including inhibiting phagocytosis; lethal factor (LF) is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinase and causes lysis of macrophages. Protective antigen (PA), the third component, binds to a cellular receptor and mediates delivery of the enzymatic components to the cytosol. Here we describe the cloning of the human PA receptor using a genetic complementation approach. The receptor, termed ATR (anthrax toxin receptor), is a type I membrane protein with an extracellular von Willebrand factor A domain that binds directly to PA. In addition, a soluble version of this domain can protect cells from the action of the toxin.
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TL;DR: What is known about mammalian endocytic mechanisms is reviewed, with focus on the cellular proteins that control these events, and the functional relevance of distinctendocytic pathways is discussed.
Abstract: Endocytic mechanisms control the lipid and protein composition of the plasma membrane, thereby regulating how cells interact with their environments. Here, we review what is known about mammalian endocytic mechanisms, with focus on the cellular proteins that control these events. We discuss the well-studied clathrin-mediated endocytic mechanisms and dissect endocytic pathways that proceed independently of clathrin. These clathrin-independent pathways include the CLIC/GEEC endocytic pathway, arf6-dependent endocytosis, flotillin-dependent endocytosis, macropinocytosis, circular doral ruffles, phagocytosis, and trans-endocytosis. We also critically review the role of caveolae and caveolin1 in endocytosis. We highlight the roles of lipids, membrane curvature-modulating proteins, small G proteins, actin, and dynamin in endocytic pathways. We discuss the functional relevance of distinct endocytic pathways and emphasize the importance of studying these pathways to understand human disease processes.
2,685 citations
TL;DR: This review summarizes various features of furin — its structural and enzymatic properties, intracellular localization, trafficking, substrates, and roles in vivo.
Abstract: Furin catalyses a simple biochemical reaction--the proteolytic maturation of proprotein substrates in the secretory pathway. But the simplicity of this reaction belies furin's broad and important roles in homeostasis, as well as in diseases ranging from Alzheimer's disease and cancer to anthrax and Ebola fever. This review summarizes various features of furin--its structural and enzymatic properties, intracellular localization, trafficking, substrates, and roles in vivo.
1,109 citations
TL;DR: The development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis are reported.
Abstract: Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.
695 citations
TL;DR: The von Willebrand A (VWA) domain is a well-studied domain involved in cell adhesion, in extracellular matrix proteins, and in integrin receptors.
Abstract: The von Willebrand A (VWA) domain is a well-studied domain involved in cell adhesion, in extracellular matrix proteins, and in integrin receptors. A number of human diseases arise from mutations in...
674 citations
TL;DR: A soluble version of the CMG2 VWA/I domain inhibited intoxication of cells expressing endogenous toxin receptors when it was added to PA at a 3:1 ratio and identified a potent antitoxin that may prove useful for the treatment of anthrax.
Abstract: Bacillus anthracis secretes two bipartite toxins thought to be involved in anthrax pathogenesis and resulting death of the host. The current model for intoxication is that protective antigen (PA) toxin subunits bind a single group of cell-surface anthrax toxin receptors (ATRs), encoded by the tumor endothelial marker 8 (TEM8) gene. The ATR/TEM8-PA interaction is mediated by the receptor's extracellular domain related to von Willebrand factor type A or integrin inserted domains (VWA/I domains). A metal ion-dependent adhesion site (MIDAS) located within this domain of the ATR/TEM8 protein chelates a divalent cation critical for PA binding. In this report, we identify a second PA receptor encoded by capillary morphogenesis gene 2 (CMG2), which has 60% amino acid identity to ATR/TEM8 within the VWA/I domain, as well as a conserved MIDAS motif. A recombinant CMG2 protein bound PA and mediated toxin internalization when expressed on receptor-deficient cells. Binding between the CMG2 VWA/I domain and PA was shown to be direct and metal-dependent, although the cation specificity of this interaction is different than that observed with ATR/TEM8. Northern blot analysis revealed that CMG2 is widely expressed in human tissues, indicating that this receptor is likely to be relevant for disease pathogenesis. Finally, a soluble version of the CMG2 VWA/I domain inhibited intoxication of cells expressing endogenous toxin receptors when it was added to PA at a 3:1 ratio. These studies distinguish CMG2 as a second anthrax toxin receptor and identify a potent antitoxin that may prove useful for the treatment of anthrax.
628 citations
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4,950 citations
TL;DR: It is demonstrated that tumor and normal endothelium are distinct at the molecular level, a finding that may have significant implications for the development of anti-angiogenic therapies.
Abstract: To gain a molecular understanding of tumor angiogenesis, we compared gene expression patterns of endothelial cells derived from blood vessels of normal and malignant colorectal tissues. Of over 170 transcripts predominantly expressed in the endothelium, 79 were differentially expressed, including 46 that were specifically elevated in tumor-associated endothelium. Several of these genes encode extracellular matrix proteins, but most are of unknown function. Most of these tumor endothelial markers were expressed in a wide range of tumor types, as well as in normal vessels associated with wound healing and corpus luteum formation. These studies demonstrate that tumor and normal endothelium are distinct at the molecular level, a finding that may have significant implications for the development of anti-angiogenic therapies.
1,912 citations
TL;DR: A second selection process occurs during immune responses in which a new antibody repertoire is generated through somatic hypermutation, where only mutants binding antigen with high affinity survive to become memory cells.
Abstract: Each antibody-producing B cell makes antibodies of unique specificity, reflecting a series of ordered gene rearrangements which must be successfully performed if the cell is to survive. A second selection process occurs during immune responses in which a new antibody repertoire is generated through somatic hypermutation. Here only mutants binding antigen with high affinity survive to become memory cells. Cells expressing autoreactive receptors are counter-selected at both stages. This stringent positive and negative selection allows the generation and diversification of cells while rigorously controlling their specificity.
1,705 citations
TL;DR: The ability to concentrate vesicular stomatitis virus G glycoprotein pseudotyped vectors will facilitate gene therapy model studies and other gene transfer experiments that require direct delivery of vectors in vivo, and facilitate genetic studies in nonmammalian species, including the important zebrafish developmental system.
Abstract: The restricted host-cell range and low titer of retroviral vectors limit their use for stable gene transfer in eukaryotic cells. To overcome these limitations, we have produced murine leukemia virus-derived vectors in which the retroviral envelope glycoprotein has been completely replaced by the G glycoprotein of vesicular stomatitis virus. Such vectors can be concentrated by ultracentrifugation to titers > 10(9) colony-forming units/ml and can infect cells, such as hamster and fish cell lines, that are ordinarily resistant to infection with vectors containing the retroviral envelope protein. The ability to concentrate vesicular stomatitis virus G glycoprotein pseudotyped vectors will facilitate gene therapy model studies and other gene transfer experiments that require direct delivery of vectors in vivo. The availability of these pseudotyped vectors will also facilitate genetic studies in nonmammalian species, including the important zebrafish developmental system, through the efficient introduction and expression of foreign genes.
1,503 citations
TL;DR: The successful isolation of these prepuberal cell types was accomplished by defining distinctive morphological characteristics of the cells and assessing the identity and purity of the isolated cell types by microscopy.
Abstract: A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).
1,342 citations