The Experts below are selected from a list of 5217 Experts worldwide ranked by ideXlab platform

Doryen Bubeck - One of the best experts on this subject based on the ideXlab platform.

  • structural basis of soluble Membrane Attack Complex packaging for clearance
    Nature Communications, 2021
    Co-Authors: Anais Menny, Marie V Lukassen, Emma C Couves, Vojtech Franc, Albert J R Heck, Doryen Bubeck
    Abstract:

    Unregulated complement activation causes inflammatory and immunological pathologies with consequences for human disease. To prevent bystander damage during an immune response, extracellular chaperones (clusterin and vitronectin) capture and clear soluble precursors to the Membrane Attack Complex (sMAC). However, how these chaperones block further polymerization of MAC and prevent the Complex from binding target Membranes remains unclear. Here, we address that question by combining cryo electron microscopy (cryoEM) and cross-linking mass spectrometry (XL-MS) to solve the structure of sMAC. Together our data reveal how clusterin recognizes and inhibits polymerizing complement proteins by binding a negatively charged surface of sMAC. Furthermore, we show that the pore-forming C9 protein is trapped in an intermediate conformation whereby only one of its two transMembrane β-hairpins has unfurled. This structure provides molecular details for immune pore formation and helps explain a complement control mechanism that has potential implications for how cell clearance pathways mediate immune homeostasis. To prevent unregulated complement activation, extracellular chaperones capture soluble precursors to the Membrane Attack Complex (sMAC). Here, structural analysis of sMAC reveals how clusterin recognizes heterogeneous sMAC Complexes and inhibits polymerization of complement protein C9.

  • soluble Membrane Attack Complex biochemistry and immunobiology
    Frontiers in Immunology, 2020
    Co-Authors: Scott R. Barnum, Doryen Bubeck, Theresa N Schein
    Abstract:

    The soluble Membrane Attack Complex (sMAC, a.k.a., sC5b-9 or TCC) is generated on activation of complement and contains the complement proteins C5b, C6, C7, C8, C9 together with the regulatory proteins clusterin and/or vitronectin. sMAC is a member of the MACPF/cholesterol-dependent-cytolysin superfamily of pore-forming molecules that insert into lipid bilayers and disrupt cellular integrity and function. sMAC is a unique complement activation macromolecule as it is comprised of several different subunits. To date no complement-mediated function has been identified for sMAC. sMAC is present in blood and other body fluids under homeostatic conditions and there is abundant evidence documenting changes in sMAC levels during infection, autoimmune disease and trauma. Despite decades of scientific interest in sMAC, the mechanisms regulating its formation in healthy individuals and its biological functions in both health and disease remain poorly understood. Here, we review the structural differences between sMAC and its Membrane counterpart, MAC, and examine sMAC immunobiology with respect to its presence in body fluids in health and disease. Finally, we discuss the diagnostic potential of sMAC for diagnostic and prognostic applications and potential utility as a companion diagnostic.

  • cryoem reveals how the complement Membrane Attack Complex ruptures lipid bilayers
    Nature Communications, 2018
    Co-Authors: Anais Menny, Marina Serna, B P Morgan, Courtney M Boyd, Scott Gardner, A P Joseph, Maya Topf, Nicholas J Brooks, Doryen Bubeck
    Abstract:

    The Membrane Attack Complex (MAC) is one of the immune system’s first responders. Complement proteins assemble on target Membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the Membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for Membrane bending, C8 and C9 traverse the bilayer increasing Membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant β-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how β-pore forming proteins breach the Membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions.

  • the mystery behind Membrane insertion a review of the complement Membrane Attack Complex
    Philosophical Transactions of the Royal Society B, 2017
    Co-Authors: Charles Baylyjones, Doryen Bubeck, Michelle A Dunstone
    Abstract:

    The Membrane Attack Complex (MAC) is an important innate immune effector of the complement terminal pathway that forms cytotoxic pores on the surface of microbes. Despite many years of research, MAC structure and mechanism of action have remained elusive, relying heavily on modelling and inference from biochemical experiments. Recent advances in structural biology, specifically cryo-electron microscopy, have provided new insights into the molecular mechanism of MAC assembly. Its unique 'split-washer' shape, coupled with an irregular giant β-barrel architecture, enable an atypical mechanism of hole punching and represent a novel system for which to study pore formation. This review will introduce the complement terminal pathway that leads to formation of the MAC. Moreover, it will discuss how structures of the pore and component proteins underpin a mechanism for MAC function, modulation and inhibition.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'.

  • structural basis of complement Membrane Attack Complex formation
    Nature Communications, 2016
    Co-Authors: Marina Serna, Paul B Morgan, Joanna L Giles, Doryen Bubeck
    Abstract:

    In response to complement activation, the Membrane Attack Complex (MAC) assembles from fluid-phase proteins to form pores in lipid bilayers. MAC directly lyses pathogens by a 'multi-hit' mechanism; however, sublytic MAC pores on host cells activate signalling pathways. Previous studies have described the structures of individual MAC components and subComplexes; however, the molecular details of its assembly and mechanism of action remain unresolved. Here we report the electron cryo-microscopy structure of human MAC at subnanometre resolution. Structural analyses define the stoichiometry of the complete pore and identify a network of interaction interfaces that determine its assembly mechanism. MAC adopts a 'split-washer' configuration, in contrast to the predicted closed ring observed for perforin and cholesterol-dependent cytolysins. Assembly precursors partially penetrate the lipid bilayer, resulting in an irregular β-barrel pore. Our results demonstrate how differences in symmetric and asymmetric components of the MAC underpin a molecular basis for pore formation and suggest a mechanism of action that extends beyond Membrane penetration.

Michelle A Dunstone - One of the best experts on this subject based on the ideXlab platform.

  • the mystery behind Membrane insertion a review of the complement Membrane Attack Complex
    Philosophical Transactions of the Royal Society B, 2017
    Co-Authors: Charles Baylyjones, Doryen Bubeck, Michelle A Dunstone
    Abstract:

    The Membrane Attack Complex (MAC) is an important innate immune effector of the complement terminal pathway that forms cytotoxic pores on the surface of microbes. Despite many years of research, MAC structure and mechanism of action have remained elusive, relying heavily on modelling and inference from biochemical experiments. Recent advances in structural biology, specifically cryo-electron microscopy, have provided new insights into the molecular mechanism of MAC assembly. Its unique 'split-washer' shape, coupled with an irregular giant β-barrel architecture, enable an atypical mechanism of hole punching and represent a novel system for which to study pore formation. This review will introduce the complement terminal pathway that leads to formation of the MAC. Moreover, it will discuss how structures of the pore and component proteins underpin a mechanism for MAC function, modulation and inhibition.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'.

  • packing a punch the mechanism of pore formation by cholesterol dependent cytolysins and Membrane Attack Complex perforin like proteins
    Current Opinion in Structural Biology, 2012
    Co-Authors: Michelle A Dunstone, Rodney K Tweten
    Abstract:

    The bacterial cholesterol dependent cytolysins (CDCs) and Membrane Attack Complex/perforin-like proteins (MACPF) represent two major branches of a large, exceptionally diverged superfamily. Most characterized CDC/MACPF proteins form large pores that function in immunity, venoms, and pathogenesis. Extensive structural, biochemical and biophysical studies have started to address some of the questions surrounding how the soluble, monomeric form of these remarkable molecules recognize diverse targets and assemble into oligomeric Membrane embedded pores. This review explores mechanistic similarities and differences in how CDCs and MACPF proteins form pores.

  • the structure and function of mammalian Membrane Attack Complex perforin like proteins
    Tissue Antigens, 2010
    Co-Authors: S C Kondos, Ruby H P Law, Tamas Zsolt Hatfaludi, Ilia Voskoboinik, Joseph A Trapani, James C Whisstock, Michelle A Dunstone
    Abstract:

    The Membrane-Attack Complex (MAC) of complement pathway and perforin (PF) are important tools deployed by the immune system to target pathogens. Both perforin and the C9 component of the MAC contain a common 'MACPF' domain and form pores in the cell Membrane as part of their function. The MAC targets gram-negative bacteria and certain pathogenic parasites, while perforin, released by natural killer cells or cytotoxic T lymphocytes (CTLs), targets virus-infected and transformed host cells (1). Remarkably, recent structural studies show that the MACPF domain is homologous to the pore-forming portion of bacterial cholesterol-dependent cytolysins; these data have provided important insight into the mechanism of pore-forming MACPF proteins. In addition to their role in immunity, MACPF family members have been identified as animal venoms, factors required for pathogen migration across host cell Membranes and factors that govern developmental processes such as embryonic patterning and neuronal guidance (2). While most MACPF proteins characterized to date either form pores or span lipid Membranes, some do not (e.g. the C6 component of the MAC). A current challenge is thus to understand the role, pore forming or otherwise, of MACPF proteins in developmental biology. This review discusses structural and functional diversity of the mammalian MACPF proteins.

Nalini S Bora - One of the best experts on this subject based on the ideXlab platform.

  • role of complement and complement Membrane Attack Complex in laser induced choroidal neovascularization
    American Journal of Ophthalmology, 2005
    Co-Authors: Puran S Bora, J H Sohn, Jose M C Cruz, H Nishihori, Yali Wang, S Kaliappan, Henry J Kaplan, Nalini S Bora
    Abstract:

    Choroidal neovascularization (CNV) is the hallmark of exudative age-related macular degeneration and a leading cause of visual loss after age 55. The pathogenesis of new choroidal vessel formation is poorly understood. Although inflammation has been implicated in the development of CNV, the role of complement in CNV has not been experimentally explored. A reliable way to produce CNV in animals is to rupture Bruch’s Membrane with laser photocoagulation. A murine model of laser-induced CNV in C57BL/6 mice revealed the deposition of C3 and Membrane Attack Complex (MAC) in the neovascular Complex. CNV was inhibited by complement depletion using cobra venom factor and did not develop in C3(−/−) mice. Anti-murine C6 Abs in C57BL/6 mice inhibited MAC formation and also resulted in the inhibition of CNV. Vascular endothelial growth factor, TGF-beta2, and beta-fibroblast growth factor were elevated in C57BL/6 mice after laser-induced CNV; complement depletion resulted in a marked reduction in the level of these angiogenic factors. Thus, activation of complement, specifically the formation of MAC, is essential for the development of laser-induced choroidal angiogenesis in mice. —Hans E. Grossniklaus

  • role of complement and complement Membrane Attack Complex in laser induced choroidal neovascularization
    Journal of Immunology, 2005
    Co-Authors: Puran S Bora, J H Sohn, Jose M C Cruz, H Nishihori, Yali Wang, S Kaliappan, Henry J Kaplan, Nalini S Bora
    Abstract:

    Choroidal neovascularization (CNV), or choroidal angiogenesis, is the hallmark of age-related macular degeneration and a leading cause of visual loss after age 55. The pathogenesis of new choroidal vessel formation is poorly understood. Although inflammation has been implicated in the development of CNV, the role of complement in CNV has not been explored experimentally. A reliable way to produce CNV in animals is to rupture Bruch’s Membrane with laser photocoagulation. A murine model of laser-induced CNV in C57BL/6 mice revealed the deposition of C3 and Membrane Attack Complex (MAC) in the neovascular Complex. CNV was inhibited by complement depletion using cobra venom factor and did not develop in C3−/− mice. Anti-murine C6 Abs in C57BL/6 mice inhibited MAC formation and also resulted in the inhibition of CNV. Vascular endothelial growth factor, TGF-β2, and β-fibroblast growth factor were elevated in C57BL/6 mice after laser-induced CNV; complement depletion resulted in a marked reduction in the level of these angiogenic factors. Thus, activation of complement, specifically the formation of MAC, is essential for the development of laser- induced choroidal angiogenesis in mice. It is possible that a similar mechanism may be involved in the pathophysiology of other angiogenesis essential diseases.

  • role of complement and complement Membrane Attack Complex in laser induced choroidal neovascularization
    Journal of Immunology, 2005
    Co-Authors: Puran S Bora, J H Sohn, Jose M C Cruz, H Nishihori, Yali Wang, S Kaliappan, Henry J Kaplan, P Jha, Nalini S Bora
    Abstract:

    Choroidal neovascularization (CNV), or choroidal angiogenesis, is the hallmark of age-related macular degeneration and a leading cause of visual loss after age 55. The pathogenesis of new choroidal vessel formation is poorly understood. Although inflammation has been implicated in the development of CNV, the role of complement in CNV has not been explored experimentally. A reliable way to produce CNV in animals is to rupture Bruch's Membrane with laser photocoagulation. A murine model of laser-induced CNV in C57BL/6 mice revealed the deposition of C3 and Membrane Attack Complex (MAC) in the neovascular Complex. CNV was inhibited by complement depletion using cobra venom factor and did not develop in C3(-/-) mice. Anti-murine C6 Abs in C57BL/6 mice inhibited MAC formation and also resulted in the inhibition of CNV. Vascular endothelial growth factor, TGF-beta2, and beta-fibroblast growth factor were elevated in C57BL/6 mice after laser-induced CNV; complement depletion resulted in a marked reduction in the level of these angiogenic factors. Thus, activation of complement, specifically the formation of MAC, is essential for the development of laser- induced choroidal angiogenesis in mice. It is possible that a similar mechanism may be involved in the pathophysiology of other angiogenesis essential diseases.

Michel Fardeau - One of the best experts on this subject based on the ideXlab platform.

  • x linked vacuolated myopathy Membrane Attack Complex deposition on muscle fiber Membranes with calcium accumulation on sarcolemma
    Annals of Neurology, 1997
    Co-Authors: Jean-pierre Louboutin, Brigitte Lucasherson, Marcello Villanova, Michel Fardeau
    Abstract:

    : Cytochemical localization of calcium on muscle fibers from patients with X-linked vacuolated myopathy demonstrated strong sarcolemmal or vacuolar calcium deposits in histologically abnormal muscle fibers. All Membrane Attack Complex-positive fibers demonstrated such calcium accumulation. Total content of calcium was significantly elevated in muscles from patients with X-linked vacuolated myopathy compared to control muscles. Taken together, these results suggest that calcium accumulation on sarcolemma could be probably secondary to Membrane Attack Complex deposition on the cell surface.

  • x linked vacuolated myopathy complement Membrane Attack Complex on surface Membrane of injured muscle fibers
    Annals of Neurology, 1995
    Co-Authors: Marcello Villanova, Jean-pierre Louboutin, Danielle Chateau, B Eymard, M Sagniez, F M S Tome, Michel Fardeau
    Abstract:

    We describe a probable recessive X-linked myopathy characterized by the presence of vacuolated muscle fibers. Four males and their shared maternal grandfather were affected. Clinical characteristics include juvenile onset, very slow progression, and predominant proximal muscle involvement. The clinical picture and the morphological findings are compared with those previously described in a family. By immunofluorescence, all histologically abnormal muscle fibers, in particular those vacuolated, showed a strong deposition of the complement C5b-9 Membrane Attack Complex over the whole muscle fiber surface. Weak immunostaining for Membrane Attack Complex was also found in endomysial capillaries and perimysial vessel walls. Muscle fibers showed sarcolemmal immunolabeling with anti-major histocompatibility Complex I, which was also present on the margins of many vacuoles. All vacuoles were stained by antidystrophin antibody, which colocalized in most of them with antilaminin immunostaining. Taken together, these results suggest that the deposition of Membrane Attack Complex on the damaged cell surface Membrane could be important in the pathogenesis of this muscle disorder, and that the Membrane-bounded vacuoles could be a consequence of sarcolemmal invagination.

Bart W Hoogenboom - One of the best experts on this subject based on the ideXlab platform.

  • bacterial killing by complement requires direct anchoring of Membrane Attack Complex precursor c5b 7
    PLOS Pathogens, 2020
    Co-Authors: Dennis J Doorduijn, Bart W Hoogenboom, Edward S Parsons, Dani A C Heesterbeek, Maartje Ruyken, Bart W Bardoel, Georgina Benn
    Abstract:

    An important effector function of the human complement system is to directly kill Gram-negative bacteria via Membrane Attack Complex (MAC) pores. MAC pores are assembled when surface-bound convertase enzymes convert C5 into C5b, which together with C6, C7, C8 and multiple copies of C9 forms a transMembrane pore that damages the bacterial cell envelope. Recently, we found that bacterial killing by MAC pores requires local conversion of C5 by surface-bound convertases. In this study we aimed to understand why local assembly of MAC pores is essential for bacterial killing. Here, we show that rapid interaction of C7 with C5b6 is required to form bactericidal MAC pores on Escherichia coli. Binding experiments with fluorescently labelled C6 show that C7 prevents release of C5b6 from the bacterial surface. Moreover, trypsin shaving experiments and atomic force microscopy revealed that this rapid interaction between C7 and C5b6 is crucial to efficiently anchor C5b-7 to the bacterial cell envelope and form complete MAC pores. Using complement-resistant clinical E. coli strains, we show that bacterial pathogens can prevent complement-dependent killing by interfering with the anchoring of C5b-7. While C5 convertase assembly was unaffected, these resistant strains blocked efficient anchoring of C5b-7 and thus prevented stable insertion of MAC pores into the bacterial cell envelope. Altogether, these findings provide basic molecular insights into how bactericidal MAC pores are assembled and how bacteria evade MAC-dependent killing.

  • bacterial killing by complement requires direct anchoring of Membrane Attack Complex precursor c5b 7
    bioRxiv, 2019
    Co-Authors: Dennis J Doorduijn, Bart W Hoogenboom, Edward S Parsons, Dani A C Heesterbeek, Maartje Ruyken, Bart W Bardoel, Georgina Benn
    Abstract:

    Abstract An important effector function of the human complement system is to directly kill Gram-negative bacteria via Membrane Attack Complex (MAC) pores. MAC pores are assembled when surface-bound convertase enzymes convert C5 into C5b, which together with C6, C7, C8 and multiple copies of C9 forms a transMembrane pore that damages the bacterial cell envelope. Recently, we found that bacterial killing by MAC pores requires local conversion of C5 by surface-bound convertases. In this study we aimed to understand why local assembly of MAC pores is essential for bacterial killing. Here, we show that rapid interaction of C7 with C5b6 is required to form bactericidal MAC pores. Binding experiments with fluorescently labelled C6 show that C7 prevents release of C5b6 from the bacterial surface. Moreover, trypsin shaving experiments and atomic force microscopy revealed that this rapid interaction between C7 and C5b6 is crucial to efficiently anchor C5b-7 to the bacterial cell envelope and form complete MAC pores. Using complement-resistant clinical E. coli strains, we show that bacterial pathogens can prevent complement-dependent killing by interfering with the anchoring of C5b-7. While C5 convertase assembly was unaffected, these resistant strains blocked efficient anchoring of C5b-7 and thus prevented stable insertion of MAC pores into the bacterial cell envelope. Altogether, these findings provide basic molecular insights into how bactericidal MAC pores are assembled and how bacteria evade MAC-dependent killing.

  • the Membrane Attack Complex perforin and cholesterol dependent cytolysin superfamily of pore forming proteins
    Journal of Cell Science, 2016
    Co-Authors: Natalya Lukoyanova, Bart W Hoogenboom, Helen R Saibil
    Abstract:

    The Membrane Attack Complex and perforin proteins (MACPFs) and bacterial cholesterol-dependent cytolysins (CDCs) are two branches of a large and diverse superfamily of pore-forming proteins that function in immunity and pathogenesis. During pore formation, soluble monomers assemble into large transMembrane pores through conformational transitions that involve extrusion and refolding of two α-helical regions into transMembrane β-hairpins. These transitions entail a dramatic refolding of the protein structure, and the resulting assemblies create large holes in cellular Membranes, but they do not use any external source of energy. Structures of the Membrane-bound assemblies are required to mechanistically understand and modulate these processes. In this Commentary, we discuss recent advances in the understanding of assembly mechanisms and molecular details of the conformational changes that occur during MACPF and CDC pore formation.