scholarly journals The mosquito protein AEG12 displays both cytolytic and antiviral properties via a common lipid transfer mechanism

2021 ◽  
Vol 118 (11) ◽  
pp. e2019251118
Author(s):  
Alexander C. Y. Foo ◽  
Peter M. Thompson ◽  
Shih-Heng Chen ◽  
Ramesh Jadi ◽  
Brianna Lupo ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Acyl Chain ◽  
Three Dimensional ◽  
Cytolytic Activity ◽  
Dimensional Structure ◽  
Lipid Transfer ◽  
Adeno Associated Virus ◽  
Nonenveloped Virus ◽  
Flavivirus Infection ◽  
Blood Meals

The mosquito protein AEG12 is up-regulated in response to blood meals and flavivirus infection though its function remained elusive. Here, we determine the three-dimensional structure of AEG12 and describe the binding specificity of acyl-chain ligands within its large central hydrophobic cavity. We show that AEG12 displays hemolytic and cytolytic activity by selectively delivering unsaturated fatty acid cargoes into phosphatidylcholine-rich lipid bilayers. This property of AEG12 also enables it to inhibit replication of enveloped viruses such as Dengue and Zika viruses at low micromolar concentrations. Weaker inhibition was observed against more distantly related coronaviruses and lentivirus, while no inhibition was observed against the nonenveloped virus adeno-associated virus. Together, our results uncover the mechanistic understanding of AEG12 function and provide the necessary implications for its use as a broad-spectrum therapeutic against cellular and viral targets.

scholarly journals Unraveling the Role of Drug-Lipid Interactions in NSAIDs-Induced Cardiotoxicity

Membranes ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 24
Author(s):  
Catarina Pereira-Leite ◽  
Marina Figueiredo ◽  
Kinga Burdach ◽  
Cláudia Nunes ◽  
Salette Reis
Keyword(s):  
Lipid Bilayers ◽  
Three Dimensional ◽  
Model Systems ◽  
Dimensional Structure ◽  
Molar Ratio ◽  
Similar Action ◽  
Lipid Interactions ◽  
Action Mechanisms ◽  
X Ray Scattering

Cardiovascular (CV) toxicity is nowadays recognized as a class effect of non-aspirin nonsteroidal anti-inflammatory drugs (NSAIDs). However, their mechanisms of cardiotoxicity are not yet well understood, since different compounds with similar action mechanisms exhibit distinct cardiotoxicity. For instance, diclofenac (DIC) is among the most cardiotoxic compounds, while naproxen (NAP) is associated with low CV risk. In this sense, this study aimed to unravel the role of drug-lipid interactions in NSAIDs-induced cardiotoxicity. For that, DIC and NAP interactions with lipid bilayers as model systems of cell and mitochondrial membranes were characterized by derivative spectrophotometry, fluorometric leakage assays, and synchrotron X-ray scattering. Both DIC and NAP were found to have the ability to permeabilize the membrane models, as well as to alter the bilayers’ structure. The NSAIDs-induced modifications were dependent on the lipid composition of the membrane model, the three-dimensional structure of the drug, as well as the drug:lipid molar ratio tested. Altogether, this work supports the hypothesis that NSAIDs-lipid interactions, in particular at the mitochondrial level, may be another key step among the mechanisms underlying NSAIDs-induced cardiotoxicity.

scholarly journals LUMENAL PLASMA MEMBRANE OF THE URINARY BLADDER

1972 ◽  
Vol 53 (1) ◽  
pp. 73-91 ◽  
Author(s):  
L. Andrew Staehelin ◽  
Francis J. Chlapowski ◽  
Mary A. Bonneville
Keyword(s):  
Urinary Bladder ◽  
Lipid Bilayers ◽  
Membrane Surface ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Transitional Epithelium ◽  
Permeability Barrier ◽  
Outer Leaflet ◽  
Cytoplasmic Filaments ◽  
Membrane Components

To determine the three-dimensional structure of the lumenal membrane of transitional epithelium, a study was made of sectioned, negatively stained, and freeze-etched specimens from intact epithelium and membrane fractions from rabbit urinary bladder. Particulate membrane components are confined to plaque regions within which the unit membrane is asymmetric, having a thicker outer leaflet. Transversely fractured freeze-etched plaques display a thick (∼80 A), particulate lumenal leaflet and a thin (∼40 A) cytoplasmic one. Four different faces of the two leaflets can be distinguished: two complementary, split, inner membrane faces exposed by freeze-cleaving the bilayer and two external (lumenal and cytoplasmic) membrane surfaces revealed by deep-etching. On the split, inner face of the lumenal leaflet appear polygonal plaques of hexagonally arranged particles. These fit into holes observed on the complementary, split, innerface of the cytoplasmic leaflet. The particles, which have a center-to-center spacing of ∼160 A, also seem to protrude from the external surface of the lumenal leaflet, where their subunits (∼50 A in diameter) are revealed by freeze-etching and negative staining. The plaques are separated from each other by smooth-surfaced regions, which cleave like simple lipid bilayers. Since the array of plaque particles covers only ∼73% of the membrane surface area, whereas 27% is taken up by particle-free interplaque regions, the presence of particles cannot in itself entirely account for the permeability barrier of the lumenal membrane. Although no particles are observed protruding from the cytoplasmic surface of the membrane, cytoplasmic filaments are attached to it by short, cross-bridge-like filaments that seem to contact the particles within the membrane. These long cytoplasmic filaments cross-link adjacent plaques. Therefore, we suggest that at least one function of the particles is to serve as anchoring sites for cytoplasmic filaments, which limit the expansion of the lumenal membrane during distention of the bladder, thereby preventing it from rupturing. The particle-free interplaque regions probably function as hinge areas between the stiff plaques, allowing the membrane to fold up when the bladder is contracted.

scholarly journals Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails

2019 ◽  
Author(s):  
M.L. Giglio ◽  
S. Ituarte ◽  
V. Milesi ◽  
M.S. Dreon ◽  
T.R. Brola ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Solution Structure ◽  
Three Dimensional ◽  
Membrane Attack Complex ◽  
Membrane Conductance ◽  
Dimensional Structure ◽  
Apple Snail ◽  
Negative Stain ◽  
Negative Stain Electron Microscopy ◽  
Macpf Domain

AbstractThe Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles but MACPF but pore-forming toxic function are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 15 Å resolution determined by negative stain electron microscopy (NS-EM) and its solution structure by small angle X-ray scattering (SAXS). We found that PV2s differ from nearly all MACPFs in two respects: it is a dimer in solution and protomers combine two immune proteins into an AB toxin. MACPF chain is linked by a single disulfide bond to a tachylectin chain, and two heterodimers are arranged head-to-tail by non-covalent forces in the native protein. MACPF domain is fused with a putative new Ct-accessory domain exclusive to invertebrates. Tachylectin is a six-bladed β-propeller, similar to animal tectonins. We experimentally validated the predicted functions of both subunits and demonstrated for the first time that PV2s are true pore-forming toxins. The tachylectin ..B.. delivery subunit would bind to target membranes, and then its MACPF ..A.. toxic subunit disrupt lipid bilayers forming large pores altering the plasma membrane conductance. These results indicate that PV2s toxicity evolved by linking two immune proteins where their combined preexisting functions give rise to a new toxic entity with a novel role in defense against predation. This structure is an unparalleled example of protein exaptation.

scholarly journals Isolation and Characterization ofEscherichia coli tolC Mutants Defective in Secreting Enzymatically Active Alpha-Hemolysin

2001 ◽  
Vol 183 (23) ◽  
pp. 6908-6916 ◽  
Author(s):  
Hema Vakharia ◽  
Greg J. German ◽  
Rajeev Misra
Keyword(s):  
Mutant Strain ◽  
Parental Strain ◽  
Time Course ◽  
Guanidine Hydrochloride ◽  
Three Dimensional ◽  
Growth Conditions ◽  
Cytolytic Activity ◽  
Dimensional Structure ◽  
Alpha Hemolysin ◽  
Isolation And Characterization

ABSTRACT This study describes the isolation and characterization of a unique class of TolC mutants that, under steady-state growth conditions, secreted normal levels of largely inactive alpha-hemolysin. Unlike the reduced activity in the culture supernatants, the cell-associated hemolytic activity in these mutants was identical to that in the parental strain, thus reflecting a normal intracellular toxin activation event. Treatment of the secreted toxin with guanidine hydrochloride significantly restored cytolytic activity, suggesting that the diminished activity may have been due to the aggregation or misfolding of the toxin molecules. Consistent with this notion, sedimentation and filtration analyses showed that alpha-hemolysin secreted from the mutant strain has a mass greater than that secreted from the parental strain. Experiments designed to monitor the time course of alpha-hemolysin release showed delayed appearance of toxin in the culture supernatant of the mutant strain, thus indicating a possible defect in alpha-hemolysin translocation or release. Eight different TolC substitutions displaying this toxin secretion defect were scattered throughout the protein, of which six localized in the periplasmically exposed α-helical domain, while the remaining two mapped within the outer membrane-embedded β-barrel domain of TolC. A plausible model for the secretion of inactive alpha-hemolysin in these TolC mutants is discussed in the context of the recently determined three-dimensional structure of TolC.

scholarly journals Aβ(1-42) tetramer and octamer structures reveal edge pores as a mechanism for membrane damage

2019 ◽  
Author(s):  
Sonia Ciudad ◽  
Eduard Puig ◽  
Thomas Botzanowski ◽  
Moeen Meigooni ◽  
Andres S. Arango ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Membrane Damage ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Ion Permeation ◽  
The Core ◽  
Hydrophilic Residues ◽  
Dynamics Simulations ◽  
Β Sheet ◽  
Aβ Oligomer

AbstractThe formation of amyloid-beta (Aβ) oligomer pores in the membrane of neurons has been proposed as the means to explain neurotoxicity in Alzheimer’s disease (AD). It is therefore critical to characterize Aβ oligomer samples in membrane-mimicking environments. Here we present the first three-dimensional structure of an Aβ oligomer formed in dodecyl phosphocholine (DPC) micelles, namely an Aβ(1-42) tetramer. It comprises a β-sheet core made of six β-strands, connected by only two β-turns. The two faces of the β-sheet core are hydrophobic and surrounded by the membrane-mimicking environment. In contrast, the edges of the core are hydrophilic and are solvent-exposed. By increasing the concentration of Aβ(1-42), we prepared a sample enriched in Aβ(1-42) octamers, formed by two Aβ(1-42) tetramers facing each other forming a β-sandwich structure. Notably, samples enriched in Aβ(1-42) tetramers and octamers are both active in lipid bilayers and exhibit the same types of pore-like behaviour, but they show different occurrence rates. Remarkably, molecular dynamics simulations showed a new mechanism of membrane disruption in which water and ion permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core β-sheets edges of the Aβ(1-42) tetramers and octamers.

scholarly journals Structural Characterization of the Dual Glycan Binding Adeno-Associated Virus Serotype 6

2010 ◽  
Vol 84 (24) ◽  
pp. 12945-12957 ◽  
Author(s):  
Robert Ng ◽  
Lakshmanan Govindasamy ◽  
Brittney L. Gurda ◽  
Robert McKenna ◽  
Olga G. Kozyreva ◽  
...  
Keyword(s):  
Amino Acids ◽  
Sialic Acid ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Transduction Efficiency ◽  
Fold Axis ◽  
Adeno Associated Virus ◽  
Virus Serotype ◽  
Loop Regions

ABSTRACT The three-dimensional structure of adeno-associated virus (AAV) serotype 6 (AAV6) was determined using cryo-electron microscopy and image reconstruction and using X-ray crystallography to 9.7- and 3.0-Å resolution, respectively. The AAV6 capsid contains a highly conserved, eight-stranded (βB to βI) β-barrel core and large loop regions between the strands which form the capsid surface, as observed in other AAV structures. The loops show conformational variation compared to other AAVs, consistent with previous reports that amino acids in these loop regions are involved in differentiating AAV receptor binding, transduction efficiency, and antigenicity properties. Toward structure-function annotation of AAV6 with respect to its unique dual glycan receptor (heparan sulfate and sialic acid) utilization for cellular recognition, and its enhanced lung epithelial transduction compared to other AAVs, the capsid structure was compared to that of AAV1, which binds sialic acid and differs from AAV6 in only 6 out of 736 amino acids. Five of these residues are located at or close to the icosahedral 3-fold axis of the capsid, thereby identifying this region as imparting important functions, such as receptor attachment and transduction phenotype. Two of the five observed amino acids are located in the capsid interior, suggesting that differential AAV infection properties are also controlled by postentry intracellular events. Density ordered inside the capsid, under the 3-fold axis in a previously reported, conserved AAV DNA binding pocket, was modeled as a nucleotide and a base, further implicating this capsid region in AAV genome recognition and/or stabilization.

scholarly journals Characterization of a Novel Thermostable Carboxylesterase from Geobacillus kaustophilus HTA426 Shows the Existence of a New Carboxylesterase Family

2009 ◽  
Vol 191 (9) ◽  
pp. 3076-3085 ◽  
Author(s):  
Silvia Montoro-García ◽  
Irene Martínez-Martínez ◽  
José Navarro-Fernández ◽  
Hideto Takami ◽  
Francisco García-Carmona ◽  
...  
Keyword(s):  
Acyl Chain ◽  
Three Dimensional ◽  
Catalytic Triad ◽  
Dimensional Structure ◽  
E Coli ◽  
Optimum Growth Temperature ◽  
Geobacillus Kaustophilus ◽  
Chain Lengths

ABSTRACT The gene GK3045 (741 bp) from Geobacillus kaustophilus HTA426 was cloned, sequenced, and overexpressed into Escherichia coli Rosetta (DE3). The deduced protein was a 30-kDa monomeric esterase with high homology to carboxylesterases from Geobacillus thermoleovorans NY (99% identity) and Geobacillus stearothermophilus (97% identity). This protein suffered a proteolytic cut in E. coli, and the problem was overcome by introducing a mutation in the gene (K212R) without affecting the activity. The resulting Est30 showed remarkable thermostability at 65°C, above the optimum growth temperature of G. kaustophilus HTA426. The optimum pH of the enzyme was 8.0. In addition, the purified enzyme exhibited stability against denaturing agents, like organic solvents, detergents, and urea. The protein catalyzed the hydrolysis of p-nitrophenyl esters of different acyl chain lengths, confirming the esterase activity. The sequence analysis showed that the protein contains a catalytic triad formed by Ser93, Asp192, and His222, and the Ser of the active site is located in the conserved motif Gly91-X-Ser93-X-Gly95 included in most esterases and lipases. However, this carboxylesterase showed no more than 17% sequence identity with the closest members in the eight families of microbial carboxylesterases. The three-dimensional structure was modeled by sequence alignment and compared with others carboxylesterases. The topological differences suggested the classification of this enzyme and other Geobacillus-related carboxylesterases in a new α/β hydrolase family different from IV and VI.

Conformation of Ribosomes from Escherichia Coli

1974 ◽  
Vol 32 ◽  
pp. 210-211
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Fluorescent Dyes ◽  
Protein Biosynthesis ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Dimensional Structure ◽  
Complete Understanding ◽  
And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

Three-Dimensional Reconstruction Using Stereo Pairs from Serial Thick Sections

1977 ◽  
Vol 35 ◽  
pp. 562-563
Author(s):  
Robert Glaeser ◽  
Thomas Bauer ◽  
David Grano
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Serial Sections ◽  
Thin Sections ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Dimensional Reconstruction ◽  
Stereo Imagery ◽  
Whole Mounts

In transmission electron microscopy, the 3-dimensional structure of an object is usually obtained in one of two ways. For objects which can be included in one specimen, as for example with elements included in freeze- dried whole mounts and examined with a high voltage microscope, stereo pairs can be obtained which exhibit the 3-D structure of the element. For objects which can not be included in one specimen, the 3-D shape is obtained by reconstruction from serial sections. However, without stereo imagery, only detail which remains constant within the thickness of the section can be used in the reconstruction; consequently, the choice is between a low resolution reconstruction using a few thick sections and a better resolution reconstruction using many thin sections, generally a tedious chore. This paper describes an approach to 3-D reconstruction which uses stereo images of serial thick sections to reconstruct an object including detail which changes within the depth of an individual thick section.

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