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Thomas J Buckley - One of the best experts on this subject based on the ideXlab platform.

  • Resistance of Paroxysmal Nocturnal Hemoglobinuria Cells to the
    2016
    Co-Authors: Glycosylphosphatidylinositol-binding Toxin Aerolysin, Kim L Nelson, Tracy S Lawrence, Galina L Mukhina, Richard J Jones, A. Brodsky, Thomas J Buckley
    Abstract:

    Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder caused by a somatic mutation of the PIGA gene. The product of this gene is required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; therefore, the phenotypic hallmark of PNH cells is an absence or marked deficiency of all GPI-anchored proteins. Aerolysin is a toxin secreted by the bacterial pathogen Aeromonas hydrophila and is capable of killing target cells by forming channels in their membranes after binding to GPI-anchored receptors. We found that PNH blood cells (erythrocytes, lymphocytes, and granulocytes), but not blood cells from normals or other hematologic disorders, are resistant to the cytotoxic effects of Aerolysin. The percentage of lysis of PNH cells after aero-lysin exposure paralleled the percentage of CD591 cells in the samples measured by flow cytometry. The kinetics of re

  • multilineage glycosylphosphatidylinositol anchor deficient haematopoiesis in untreated aplastic anaemia
    British Journal of Haematology, 2001
    Co-Authors: Galina L Mukhina, Thomas J Buckley, James Barber, Richard J Jones, Robert A Brodsky
    Abstract:

    Aplastic anaemia and paroxysmal nocturnal haemoglobinuria (PNH) are closely related disorders. In PNH, haematopoietic stem cells that harbour PIGA mutations give rise to blood elements that are unable to synthesize glycosylphosphatidylinositol (GPI) anchors. Because the GPI anchor is the receptor for the channel-forming protein Aerolysin, PNH cells do not bind the toxin and are unaffected by concentrations that lyse normal cells. Exploiting these biological differences, we have developed two novel Aerolysin-based assays to detect small populations of PNH cells. CD59 populations as small as 0.004% of total red cells could be detected when cells were pretreated with Aerolysin to enrich the PNH population. All PNH patients displayed CD59-deficient erythrocytes, but no myelodysplastic syndrome (MDS) patient or control had detectable PNH cells before or after enrichment in Aerolysin. Only one aplastic anaemia patient had detectable PNH red cells before exposure to Aerolysin. However, 14 (61%) had detectable PNH cells after enrichment in Aerolysin. The inactive fluorescent proAerolysin variant (FLAER) that binds the GPI anchors of a number of proteins on normal cells was used to detect a global GPI anchor deficit on granulocytes. Flow cytometry with FLAER showed that 12 out of 18 (67%) aplastic anaemia patients had FLAER-negative granulocytes, but none of the MDS patients or normal control subjects had GPI anchor-deficient cells. These studies demonstrate that Aerolysin-based assays can reveal previously undetectable multilineage PNH cells in patients with untreated aplastic anaemia. Thus, clonality appears to be an early feature of aplastic anaemia.

  • improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent Aerolysin
    American Journal of Clinical Pathology, 2000
    Co-Authors: Robert A Brodsky, Kim L Nelson, Thomas J Buckley, Galina L Mukhina, Patricia L Chiurazzi, Michael J Borowitz
    Abstract:

    Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation in the gene PIGA, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIGA mutation results in absence or marked deficiency of more than a dozen proteins on PNH blood cells. Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of specific GPI anchor proteins, such as CD59. However, because no single GPI anchor protein is always expressed in all cell lineages, no one monoclonal antibody can be used with confidence to diagnose PNH. We describe a new diagnostic test for PNH, based on the ability of a fluorescently labeled inactive variant of the protein Aerolysin (FLAER) to bind selectively to GPI anchors. We compared GPI anchor protein expression in 8 patients with PNH using FLAER and anti-CD59. In all cases, FLAER detected similar or higher proportions of PNH monocytes and granulocytes compared with anti-CD59. Because of the increased sensitivity of detection, FLAER could detect small abnormal granulocyte populations in patients to a level of about 0.5%; samples from healthy control subjects contained substantially fewer FLAER-negative cells. FLAER gives a more accurate assessment of the GPI anchor deficit in PNH.

  • channel formation by the glycosylphosphatidylinositol anchored protein binding toxin Aerolysin is not promoted by lipid rafts
    Journal of Biological Chemistry, 2000
    Co-Authors: Kim L Nelson, Thomas J Buckley
    Abstract:

    Glycosylphosphatidylinositol-anchored proteins may be concentrated in membrane microdomains (lipid rafts) that are also enriched in cholesterol and sphingolipids. The glycosyl anchor of these proteins is a specific, high affinity receptor for the channel-forming protein Aerolysin. We wished to determine if the presence of rafts promotes the activity of Aerolysin. Treatment of T lymphocytes with methyl-beta-cyclodextrin, which destroys lipid rafts by sequestering cholesterol, had no measurable effect on the sensitivity of the cells to Aerolysin; nor did similar treatment of erythrocytes decrease the rate at which they were lysed by the toxin. We also studied the rate of Aerolysin-induced channel formation in liposomes containing glycosylphosphatidylinositol-anchored placental alkaline phosphatase, which we show is a receptor for Aerolysin. In liposomes containing sphingolipids as well as glycerophospholipids and cholesterol, most of the enzyme was Triton X-100-insoluble, indicating that it was localized in rafts, whereas in liposomes prepared without sphingolipids, all of the enzyme was soluble. Aerolysin was no more active against liposomes containing rafts than against those that did not. We conclude that lipid rafts do not promote channel formation by Aerolysin.

  • clostridium septicum alpha toxin uses glycosylphosphatidylinositol anchored protein receptors
    Journal of Biological Chemistry, 1999
    Co-Authors: Valery M Gordon, Rodney K Tweten, Kim L Nelson, Thomas J Buckley, Victoria L Stevens, Patrick C Elwood, Stephen H Leppla
    Abstract:

    Abstract The alpha toxin produced by Clostridium septicum is a channel-forming protein that is an important contributor to the virulence of the organism. Chinese hamster ovary (CHO) cells are sensitive to low concentrations of the toxin, indicating that they contain toxin receptors. Using retroviral mutagenesis, a mutant CHO line (BAG15) was generated that is resistant to alpha toxin. FACS analysis showed that the mutant cells have lost the ability to bind the toxin, indicating that they lack an alpha toxin receptor. The mutant cells are also resistant to Aerolysin, a channel-forming protein secreted by Aeromonas spp., which is structurally and functionally related to alpha toxin and which is known to bind to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1. We obtained evidence that the BAG15 cells lackN-acetylglucosaminyl-phosphatidylinositol deacetylase-L, needed for the second step in GPI anchor biosynthesis. Several lymphocyte cell lines lacking GPI-anchored proteins were also shown to be less sensitive to alpha toxin. On the other hand, the sensitivity of CHO cells to alpha toxin was increased when the cells were transfected with the GPI-anchored folate receptor. We conclude that alpha toxin, like Aerolysin, binds to GPI-anchored protein receptors. Evidence is also presented that the two toxins bind to different subsets of GPI-anchored proteins.

Kim L Nelson - One of the best experts on this subject based on the ideXlab platform.

  • Resistance of Paroxysmal Nocturnal Hemoglobinuria Cells to the
    2016
    Co-Authors: Glycosylphosphatidylinositol-binding Toxin Aerolysin, Kim L Nelson, Tracy S Lawrence, Galina L Mukhina, Richard J Jones, A. Brodsky, Thomas J Buckley
    Abstract:

    Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder caused by a somatic mutation of the PIGA gene. The product of this gene is required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; therefore, the phenotypic hallmark of PNH cells is an absence or marked deficiency of all GPI-anchored proteins. Aerolysin is a toxin secreted by the bacterial pathogen Aeromonas hydrophila and is capable of killing target cells by forming channels in their membranes after binding to GPI-anchored receptors. We found that PNH blood cells (erythrocytes, lymphocytes, and granulocytes), but not blood cells from normals or other hematologic disorders, are resistant to the cytotoxic effects of Aerolysin. The percentage of lysis of PNH cells after aero-lysin exposure paralleled the percentage of CD591 cells in the samples measured by flow cytometry. The kinetics of re

  • improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent Aerolysin
    American Journal of Clinical Pathology, 2000
    Co-Authors: Robert A Brodsky, Kim L Nelson, Thomas J Buckley, Galina L Mukhina, Patricia L Chiurazzi, Michael J Borowitz
    Abstract:

    Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation in the gene PIGA, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIGA mutation results in absence or marked deficiency of more than a dozen proteins on PNH blood cells. Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of specific GPI anchor proteins, such as CD59. However, because no single GPI anchor protein is always expressed in all cell lineages, no one monoclonal antibody can be used with confidence to diagnose PNH. We describe a new diagnostic test for PNH, based on the ability of a fluorescently labeled inactive variant of the protein Aerolysin (FLAER) to bind selectively to GPI anchors. We compared GPI anchor protein expression in 8 patients with PNH using FLAER and anti-CD59. In all cases, FLAER detected similar or higher proportions of PNH monocytes and granulocytes compared with anti-CD59. Because of the increased sensitivity of detection, FLAER could detect small abnormal granulocyte populations in patients to a level of about 0.5%; samples from healthy control subjects contained substantially fewer FLAER-negative cells. FLAER gives a more accurate assessment of the GPI anchor deficit in PNH.

  • channel formation by the glycosylphosphatidylinositol anchored protein binding toxin Aerolysin is not promoted by lipid rafts
    Journal of Biological Chemistry, 2000
    Co-Authors: Kim L Nelson, Thomas J Buckley
    Abstract:

    Glycosylphosphatidylinositol-anchored proteins may be concentrated in membrane microdomains (lipid rafts) that are also enriched in cholesterol and sphingolipids. The glycosyl anchor of these proteins is a specific, high affinity receptor for the channel-forming protein Aerolysin. We wished to determine if the presence of rafts promotes the activity of Aerolysin. Treatment of T lymphocytes with methyl-beta-cyclodextrin, which destroys lipid rafts by sequestering cholesterol, had no measurable effect on the sensitivity of the cells to Aerolysin; nor did similar treatment of erythrocytes decrease the rate at which they were lysed by the toxin. We also studied the rate of Aerolysin-induced channel formation in liposomes containing glycosylphosphatidylinositol-anchored placental alkaline phosphatase, which we show is a receptor for Aerolysin. In liposomes containing sphingolipids as well as glycerophospholipids and cholesterol, most of the enzyme was Triton X-100-insoluble, indicating that it was localized in rafts, whereas in liposomes prepared without sphingolipids, all of the enzyme was soluble. Aerolysin was no more active against liposomes containing rafts than against those that did not. We conclude that lipid rafts do not promote channel formation by Aerolysin.

  • clostridium septicum alpha toxin uses glycosylphosphatidylinositol anchored protein receptors
    Journal of Biological Chemistry, 1999
    Co-Authors: Valery M Gordon, Rodney K Tweten, Kim L Nelson, Thomas J Buckley, Victoria L Stevens, Patrick C Elwood, Stephen H Leppla
    Abstract:

    Abstract The alpha toxin produced by Clostridium septicum is a channel-forming protein that is an important contributor to the virulence of the organism. Chinese hamster ovary (CHO) cells are sensitive to low concentrations of the toxin, indicating that they contain toxin receptors. Using retroviral mutagenesis, a mutant CHO line (BAG15) was generated that is resistant to alpha toxin. FACS analysis showed that the mutant cells have lost the ability to bind the toxin, indicating that they lack an alpha toxin receptor. The mutant cells are also resistant to Aerolysin, a channel-forming protein secreted by Aeromonas spp., which is structurally and functionally related to alpha toxin and which is known to bind to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1. We obtained evidence that the BAG15 cells lackN-acetylglucosaminyl-phosphatidylinositol deacetylase-L, needed for the second step in GPI anchor biosynthesis. Several lymphocyte cell lines lacking GPI-anchored proteins were also shown to be less sensitive to alpha toxin. On the other hand, the sensitivity of CHO cells to alpha toxin was increased when the cells were transfected with the GPI-anchored folate receptor. We conclude that alpha toxin, like Aerolysin, binds to GPI-anchored protein receptors. Evidence is also presented that the two toxins bind to different subsets of GPI-anchored proteins.

  • expression and properties of an Aerolysin clostridium septicum alpha toxin hybrid protein
    Molecular Microbiology, 1999
    Co-Authors: Dzung B Diep, Bret R Sellman, Kim L Nelson, Tracy S Lawrence, Thomas J Buckley
    Abstract:

    Summary Aerolysin is a bilobal channel-forming toxin secreted by Aeromonas hydrophila. The alpha toxin produced by Clostridium septicum is homologous to the large lobe of Aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of Aerolysin to alpha toxin, producing a hybrid protein that should structurally resemble Aerolysin. Unlike Aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the Aerolysin heptamer. Like Aerolysin, the hybrid was far more active than alpha toxin against human erythrocytes and mouse T lymphocytes. Both Aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas alpha toxin was unaffected. We conclude that Aerolysin contains two receptor binding sites, one for glycosylphosphatidylinositol-anchored proteins that is located in the large lobe and is also found in alpha toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.

Rodney K Tweten - One of the best experts on this subject based on the ideXlab platform.

  • identification of functional domains of clostridium septicum alpha toxin
    Biochemistry, 2006
    Co-Authors: Jody Meltonwitt, Lori Bentsen, Rodney K Tweten
    Abstract:

    Alpha toxin (AT) is the major virulence factor of Clostridium septicum that is a proteolytically activated pore-forming toxin that belongs to the Aerolysin-like family of toxins. AT is predicted to be a three-domain molecule on the basis of its functional and sequence similarity with Aerolysin, for which the crystal structure has been determined. In this study, we have substituted the entire primary structure of AT with alanine or cysteine to identify those amino acids that comprise functional domains involved in receptor binding, oligomerization, and pore formation. These studies revealed that receptor binding is restricted to domain 1 of the AT structure, whereas domains 1 and 3 are involved in oligomerization. These studies also revealed the presence of a putative functional region of AT proximal to the receptor-binding domain but distal from the pore-forming domain that is proposed to regulate the insertion of the transmembrane β-hairpin of the prepore oligomer.

  • clostridium septicum alpha toxin uses glycosylphosphatidylinositol anchored protein receptors
    Journal of Biological Chemistry, 1999
    Co-Authors: Valery M Gordon, Rodney K Tweten, Kim L Nelson, Thomas J Buckley, Victoria L Stevens, Patrick C Elwood, Stephen H Leppla
    Abstract:

    Abstract The alpha toxin produced by Clostridium septicum is a channel-forming protein that is an important contributor to the virulence of the organism. Chinese hamster ovary (CHO) cells are sensitive to low concentrations of the toxin, indicating that they contain toxin receptors. Using retroviral mutagenesis, a mutant CHO line (BAG15) was generated that is resistant to alpha toxin. FACS analysis showed that the mutant cells have lost the ability to bind the toxin, indicating that they lack an alpha toxin receptor. The mutant cells are also resistant to Aerolysin, a channel-forming protein secreted by Aeromonas spp., which is structurally and functionally related to alpha toxin and which is known to bind to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1. We obtained evidence that the BAG15 cells lackN-acetylglucosaminyl-phosphatidylinositol deacetylase-L, needed for the second step in GPI anchor biosynthesis. Several lymphocyte cell lines lacking GPI-anchored proteins were also shown to be less sensitive to alpha toxin. On the other hand, the sensitivity of CHO cells to alpha toxin was increased when the cells were transfected with the GPI-anchored folate receptor. We conclude that alpha toxin, like Aerolysin, binds to GPI-anchored protein receptors. Evidence is also presented that the two toxins bind to different subsets of GPI-anchored proteins.

  • the primary structure of clostridium septicum alpha toxin exhibits similarity with that of aeromonas hydrophila Aerolysin
    Infection and Immunity, 1995
    Co-Authors: Jimmy D Ballard, Jean E Crabtree, Rodney K Tweten
    Abstract:

    The gene for Clostridium septicum alpha-toxin was cloned and expressed in Escherichia coli from C. septicum BX96. The toxin was determined to be 443 amino acids in length, with a 31-residue signal peptide that was removed from the toxin during secretion. No extended hydrophobic regions were observed in the mature toxin sequence. Expression of alpha-toxin in E. coli BL21 resulted in the production of ATpro, which was identical to native toxin from C. septicum with respect to activity and activation. The proteolytic activation site for alpha-toxin was determined to be on the carboxy-terminal side of arginine 398, which lies within the sequence KKRRGKR-398SVD. Previous work showing similarities in activation and mechanism between alpha-toxin and Aeromonas hydrophila Aerolysin was extended to the primary structures of both toxins. The DNA-derived primary sequence of alpha-toxin exhibited 27% identity and 72% similarity over a 387-residue region with the primary structure of the A. hydrophila Aerolysin toxin, a level of similarity heretofore unobserved between toxins produced by a gram-positive organism and a gram-negative organism.

Juan Pelta - One of the best experts on this subject based on the ideXlab platform.

  • Electrical recognition of the twenty proteinogenic amino acids using an Aerolysin nanopore.
    Nature biotechnology, 2019
    Co-Authors: Hadjer Ouldali, Kumar Sarthak, Tobias Ensslen, Fabien Piguet, Philippe Manivet, Juan Pelta, Jan C. Behrends, Aleksei Aksimentiev, Abdelghani Oukhaled
    Abstract:

    Efforts to sequence single protein molecules in nanopores1-5 have been hampered by the lack of techniques with sufficient sensitivity to discern the subtle molecular differences among all twenty amino acids. Here we report ionic current detection of all twenty proteinogenic amino acids in an Aerolysin nanopore with the help of a short polycationic carrier. Application of molecular dynamics simulations revealed that the Aerolysin nanopore has a built-in single-molecule trap that fully confines a polycationic carrier-bound amino acid inside the sensing region of the Aerolysin. This structural feature means that each amino acid spends sufficient time in the pore for sensitive measurement of the excluded volume of the amino acid. We show that distinct current blockades in wild-type Aerolysin can be used to identify 13 of the 20 natural amino acids. Furthermore, we show that chemical modifications, instrumentation advances and nanopore engineering offer a route toward identification of the remaining seven amino acids. These findings may pave the way to nanopore protein sequencing.

  • Dynamics of a polyelectrolyte through Aerolysin channel as a function of applied voltage and concentration
    European Physical Journal E: Soft matter and biological physics, 2018
    Co-Authors: Manuela Pastoriza-gallego, Laurent Bacri, Benedicte Thiebot, Loic Auvray, Juan Pelta
    Abstract:

    We describe the behaviour of a polyelectrolyte in confined geometry. The transport of a polyelectrolyte, dextran sulfate, through a recombinant protein channel, Aerolysin, inserted into a planar lipid bilayer is studied as a function of applied voltage and polyelectrolyte concentration and chain length. The Aerolysin pore has a weak geometry asymmetry, a high number of charged residues and the polyelectrolyte is strongly negatively charged. The resulting current blockades were characterized by short and long dwelling times. Their frequency varies exponentially as a function of applied voltage and linearly as a function of polyelectrolyte concentration. The long blockade duration decreases exponentially when the electrical force increases. The ratio of the population of short events to the one of long events decreases when the applied voltage increases and displays an exponential variation. The long residence time increases with the polyelectrolyte chain length. We measure a reduction of the effective charge of the polyelectrolyte at the pore entry and inside the channel. For a fixed applied voltage, + / - 100 mV, at both sides of the protein pore entrance, the events frequency is similar as a function of dextran sulfate concentration. The mean blockade durations are independent of polyelectrolyte concentration and are similar for both entrances of the pore and remain constant as a function of the electrical force.

  • Electrophoresis and Electroosmosis in Aerolysin and Hemolysin Nanopores
    Biophysical Journal, 2016
    Co-Authors: Mordjane Boukhet, Juan Pelta, Jan C. Behrends, Abdelghani Oukhaled
    Abstract:

    Voltage-dependent interaction of macromolecules with biological nanopores can arise from electroosmotic (EOF) or electrophoretic (EPF) forces. Both poly(ethylene-glycol) (PEG), and cyclodextrins (β- and α-CD, respectively) block the ionic current through α-hemolysin (αHL) and Aerolysin (AeL) pore-forming proteins with well-documented voltage-dependence. Here, we attempt to relate differential effects of the electrolytes KCl and LiCl on the voltage-dependence of frequency and dwell-time of blocks to the relative contributions of EOF and EPF.For AeL, irrespective of electrolyte, blocks by α-CD only occurred from the pore's cis-side and with trans-positive voltages. Blocks in LiCl were much longer than those in KCl (11ms vs. 760μs at 90mV) but dwell times showed a positive, linear voltage dependence in both cases (LiCl:6fold/100mV; KCl:3fold/100mV). In KCl, cis-side PEG blocked AeL with trans-negative voltages only.1 Surprisingly, in LiCl, blocks occurred only at trans-positive voltages and at more than 1000 fold PEG-concentration but we still are in a very diluted regime. Blocks were shorter than in KCl by a factor of 5 (60 vs. 320μs at 100mV), but frequency (3fold/100mV) and dwell time (2fold/100mV) depended linearly on voltage, reminiscent of the behavior of α-CD in both electrolytes.Linear voltage dependence of block frequency might indicate dominance of EOF for pore entry of PEG in LiCl and for α-CD generally, while an exponential voltage-dependence would be expected for an electrophoretic force. Indeed, for PEG in KCl, which may be positively charged due to K+-chelation (see Ref.1 for discussion), we found an exponential increase in frequency for blocks of AeL and αHL with increasing trans-negative and trans-positive voltages, respectively. We cannot, however, exclude voltage and ion-dependent effects on the binding reaction, which, rather than partitioning, may be rate-limiting for the electrophysiologically detectable blockages.

  • Dynamics and Energy Contributions for Transport of Unfolded Pertactin through a Protein Nanopore
    2015
    Co-Authors: Benjamin Cressiot, Juan Pelta, Abdelghani Oukhaled, Esther Braselmann, Adrian H. Elcock, Patricia L. Clark
    Abstract:

    To evaluate the physical parameters governing translocation of an unfolded protein across a lipid bilayer, we studied protein transport through Aerolysin, a passive protein channel, at the single-molecule level. The protein model used was the passenger domain of pertactin, an autotransporter virulence protein. Transport of pertactin through the Aerolysin nanopore was detected as transient partial current blockades as the unfolded protein partially occluded the Aerolysin channel. We compared the dynamics of entry and transport for unfolded pertactin and a covalent end-to-end dimer of the same protein. For both the monomer and the dimer, the event frequency of current blockades increased exponentially with the applied voltage, while the duration of each event decreased exponentially as a function of the electrical potential. The blockade time was twice as long for the dimer as for the monomer. The calculated activation free energy includes a main enthalpic component that we attribute to electrostatic interactions between pertactin and the Aerolysin nanopore (despite the low Debye length), plus an entropic component due to confinement of the unfolded chain within the narrow pore. Comparing our experimental results to previous studies and theory suggests that unfolded proteins cross the membrane by passing through the nanopore in a somewhat compact conformation according to the “blob” model of Daoud and de Gennes

  • wild type mutant protein unfolding and phase transition detected by single nanopore recording
    ACS Chemical Biology, 2012
    Co-Authors: Celine Merstorf, Abdelghani Oukhaled, Manuela Pastorizagallego, Loic Auvray, Jeanmichel Betton, Benjamin Cressiot, Juan Pelta
    Abstract:

    Understanding protein folding remains a challenge. A difficulty is to investigate experimentally all the conformations in the energy landscape. Only single molecule methods, fluorescence and force spectroscopy, allow observing individual molecules along their folding pathway. Here we observe that single-nanopore recording can be used as a new single molecule method to explore the unfolding transition and to examine the conformational space of native or variant proteins. We show that we can distinguish unfolded states from partially folded ones with the Aerolysin pore. The unfolding transition curves of the destabilized variant are shifted toward the lower values of the denaturant agent compared to the wild type protein. The dynamics of the partially unfolded wild type protein follows a first-order transition. The denaturation curve obtained with the Aerolysin pore is similar to that obtained with the α-hemolysin pore. The nanopore geometry or net charge does not influence the folding transition but changes the dynamics.

Robert A Brodsky - One of the best experts on this subject based on the ideXlab platform.

  • HEMATOPOIESIS PIG-A mutations in normal hematopoiesis
    2016
    Co-Authors: Galina L Mukhina, Robert A Brodsky, Richard J Jones, Steven Piantadosi, Jamie P. Barber, Paroxysmal Nocturnal Hemoglobinuria
    Abstract:

    (PNH) is caused by phosphatidylinositol glycan–class A (PIG-A) mutations in he-matopoietic stem cells (HSCs). PIG-A mu-tations have been found in granulocytes from most healthy individuals, suggest-ing that these spontaneous PIG-A muta-tions are important in the pathogenesis of PNH. It remains unclear if these PIG-A mutations have relevance to those found in PNH. We isolated CD34 progenitors from 4 patients with PNH and 27 controls. The frequency of PIG-A mutant progeni-tors was determined by assaying for colony-forming cells (CFCs) in methylcel-lulose containing toxic doses of Aerolysin (1 109 M). Glycosylphosphatidylinositol (GPI)–anchored proteins serve as recep-tors for Aerolysin; thus, PNH cells are resis-tant to Aerolysin. The frequency of Aerolysin resistant CFC was 14.7 4.0 106 in the bone marrow of healthy donors and was 57.0 6.7 106 from mobilized pe-ripheral blood. DNA was extracted from individual day-14 Aerolysin-resistant CFCs and the PIG-A gene was sequenced to determine clonality. Aerolysin-resistant CFCs from patients with PNH exhibited clonal PIG-A mutations. In contrast, PIG-A mutations in the CFCs from controls were polyclonal, and did not involve T cells. Our data confirm the finding that PIG-A mutations are relatively common in nor-mal hematopoiesis; however, the finding suggests that these mutations occur in differentiated progenitors rather tha

  • paroxysmal nocturnal hemoglobinuria from bench to bedside
    Clinical and Translational Science, 2011
    Co-Authors: Robert A Brodsky
    Abstract:

    Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disease that presents with protean manifestations. Clinical and laboratory investigation over the past 25 years have uncovered most of the basic science underpinnings of PNH and have led to the development of a highly effective targeted therapy. PNH originates from a multipotent hematopoietic stem cell (HSC) that acquires a somatic mutation in a gene called phosphatidylinositol glycan anchor biosynthesis, class A (PIG-A). The PIG-A gene is required for the first step in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Failure to synthesize GPI anchors leads to an absence of all proteins that utilize GPI to attach to the plasma membrane. Two GPI-anchor proteins, CD55 and CD59, are complement regulatory proteins; their absence on the surface of PNH cells leads to complement-mediated hemolysis. The release of free hemoglobin leads to scavenging of nitric oxide and contributes to many clinical manifestations, including esophageal spasm, fatigue, and possibly thrombosis. Aerolysin, a pore-forming toxin, binds GPI anchored proteins and kills normal cells, but not PNH cells. A fluorescinated Aerolysin variant (FLAER) binds GPI-anchor and serves as a novel reagent diagnosing PNH. Eculizumab, a humanized monoclonal antibody against C5, is the first effective drug therapy for PNH.

  • multilineage glycosylphosphatidylinositol anchor deficient haematopoiesis in untreated aplastic anaemia
    British Journal of Haematology, 2001
    Co-Authors: Galina L Mukhina, Thomas J Buckley, James Barber, Richard J Jones, Robert A Brodsky
    Abstract:

    Aplastic anaemia and paroxysmal nocturnal haemoglobinuria (PNH) are closely related disorders. In PNH, haematopoietic stem cells that harbour PIGA mutations give rise to blood elements that are unable to synthesize glycosylphosphatidylinositol (GPI) anchors. Because the GPI anchor is the receptor for the channel-forming protein Aerolysin, PNH cells do not bind the toxin and are unaffected by concentrations that lyse normal cells. Exploiting these biological differences, we have developed two novel Aerolysin-based assays to detect small populations of PNH cells. CD59 populations as small as 0.004% of total red cells could be detected when cells were pretreated with Aerolysin to enrich the PNH population. All PNH patients displayed CD59-deficient erythrocytes, but no myelodysplastic syndrome (MDS) patient or control had detectable PNH cells before or after enrichment in Aerolysin. Only one aplastic anaemia patient had detectable PNH red cells before exposure to Aerolysin. However, 14 (61%) had detectable PNH cells after enrichment in Aerolysin. The inactive fluorescent proAerolysin variant (FLAER) that binds the GPI anchors of a number of proteins on normal cells was used to detect a global GPI anchor deficit on granulocytes. Flow cytometry with FLAER showed that 12 out of 18 (67%) aplastic anaemia patients had FLAER-negative granulocytes, but none of the MDS patients or normal control subjects had GPI anchor-deficient cells. These studies demonstrate that Aerolysin-based assays can reveal previously undetectable multilineage PNH cells in patients with untreated aplastic anaemia. Thus, clonality appears to be an early feature of aplastic anaemia.

  • glycophosphatidylinositol anchored protein deficiency as a marker of mutator phenotypes in cancer
    Cancer Research, 2001
    Co-Authors: Rui Chen, James R Eshleman, Robert A Brodsky, Edward M Medof
    Abstract:

    Phosphatidylinositol glycan-A ( PIGA ) is a gene that encodes an element required for the first step in glycosylphosphatidylinositol (GPI) anchor assembly. Because PIGA is X-located, a single mutation is sufficient to abolish cell surface GPI-anchored protein expression. In this study, we investigated whether mutation of the PIGA gene could be exploited to identify mutator ( Mut ) phenotypes in cancer. We examined eight Mut colon cancer lines and four non- Mut colon cancers as controls. In every case, flow cytometric analyses of cells sorted for low fluorescence after staining for GPI-linked CD59 and CD55 revealed negative peaks in the Mut lines but not in the controls. Single cell cloning of purged and sorted GPI-anchor− HCT116 cells and sequencing of the PIGA gene in each clone uniformly showed mutations. Pretreatment of the Mut lines with anti-CD55 or anti-CD59 antibodies and complement or with the GPI-anchor-reactive bacterial toxin Aerolysin enriched for the GPI-anchor− populations. Expansion of purged GPI-anchor+ cells in the Mut lines and analyses using Aerolysin in conjunction with flow cytometry yielded PIGA gene mutation frequencies of 10−5 to 10−4, values similar to the mutation frequencies of the hprt gene. This novel approach allows for the detection of as yet undescribed repair or replication defects and in addition to its considerably greater ease of use than existing techniques and in principle would not require the production of cell lines.

  • improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent Aerolysin
    American Journal of Clinical Pathology, 2000
    Co-Authors: Robert A Brodsky, Kim L Nelson, Thomas J Buckley, Galina L Mukhina, Patricia L Chiurazzi, Michael J Borowitz
    Abstract:

    Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation in the gene PIGA, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIGA mutation results in absence or marked deficiency of more than a dozen proteins on PNH blood cells. Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of specific GPI anchor proteins, such as CD59. However, because no single GPI anchor protein is always expressed in all cell lineages, no one monoclonal antibody can be used with confidence to diagnose PNH. We describe a new diagnostic test for PNH, based on the ability of a fluorescently labeled inactive variant of the protein Aerolysin (FLAER) to bind selectively to GPI anchors. We compared GPI anchor protein expression in 8 patients with PNH using FLAER and anti-CD59. In all cases, FLAER detected similar or higher proportions of PNH monocytes and granulocytes compared with anti-CD59. Because of the increased sensitivity of detection, FLAER could detect small abnormal granulocyte populations in patients to a level of about 0.5%; samples from healthy control subjects contained substantially fewer FLAER-negative cells. FLAER gives a more accurate assessment of the GPI anchor deficit in PNH.