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L M Roberts

Publications and source records attributed to L M Roberts.

At least 37 records · Page 2Linked to original sources
PubMed1999

Restoration of lectin activity to an inactive abrin B chain by substitution and mutation of the 2 gamma subdomain.

Abrin is a heterodimeric plant protein that occurs in several isoforms (abrin-a, abrin-b, abrin-c and abrin-d), whose B chains are believed to either have (abrin-a and abrin-d) or lack (abrin-b and abrin-c) the ability to bind galactose. The 5' signal sequence and toxin B chain (ATB)-coding region were excised from a preproabrin cDNA [K. A. Wood, J. M. Lord, E. J. Wawrzynczak, and M. Piatak (1991) Eur. J. Biochem. 198, 723-732], tentatively identified as abrin-c, which was predicted to lack lectin activity, and fused in-frame to generate pre-ATB cDNA. Transcripts, synthesized in vitro from pre-ATB cloned into the transcription vector pSP64T, were expressed after microinjection into Xenopus oocytes. The recombinant ATB was shown, using a qualitative sugar-binding assay, to be devoid of lectin activity. Lectin activity could not be restored to this nonbinding ATB by replacing the 2 gamma subdomain with the corresponding galactose-binding 2 gamma subdomain from ricin B chain, but it was restored by replacement with the active galactose-binding 2 gamma subdomain from a different abrin isoform (abrin-a). The putative galactose-binding pocket of the nonbinding ATB 2 gamma subdomain contained a His residue at the position occupied by a residue with an aromatic side chain (Tyr or Trp) in functional 2 gamma subdomains. Mutationally converting this His to either Tyr or Trp restored lectin activity to the nonbinding ATB, emphasizing the contribution of an aromatic side chain in a functional 2 gamma subdomain galactose-binding site for members of this lectin family.

Abrin↗
PubMed1999

Toxin entry: how bacterial proteins get into mammalian cells.

Certain bacteria secrete protein toxins that catalytically modify and disrupt essential processes in mammalian cells, often leading to cell death. As the substrates modified by these toxins are located in the mammalian cell cytosol, a catalytically active toxin polypeptide must reach this compartment in order to act. The toxins bind to receptors on the surface of susceptible cells and enter them by endocytic uptake. Endocytosed toxins initially accumulate in endosomes, where some of these proteins take advantage of the acidic environment within these organelles to form, or contribute to the formation of, protein-conducting channels through which the catalytic polypeptide is able to translocate into the cytosol. Other toxins are unable to respond to low pH in this way and must undergo intracellular vesicular transport to reach a compartment where pre-existing protein-conducting channels occur and can be exploited for membrane translocation--the endoplasmic reticulum. In this way, cell entry by this second group of toxins demonstrates that the secretory pathway of mammalian cells is completely reversible.

Animals↗
PubMed1999

The KDEL retrieval system is exploited by Pseudomonas exotoxin A, but not by Shiga-like toxin-1, during retrograde transport from the Golgi complex to the endoplasmic reticulum.

To investigate the role of the KDEL receptor in the retrieval of protein toxins to the mammalian cell endoplasmic reticulum (ER), lysozyme variants containing AARL or KDEL C-terminal tags, or the human KDEL receptor, have been expressed in toxin-treated COS 7 and HeLa cells. Expression of the lysozyme variants and the KDEL receptor was confirmed by immunofluorescence. When such cells were challenged with diphtheria toxin (DT) or Escherichia coli Shiga-like toxin 1 (SLT-1), there was no observable difference in their sensitivities as compared to cells which did not express these exogenous proteins. By contrast, the cytotoxicity of Pseudomonas exotoxin A (PE) is reduced by expressing lysozyme-KDEL, which causes a redistribution of the KDEL receptor from the Golgi complex to the ER, and cells are sensitised to this toxin when they express additional KDEL receptors. These data suggest that, in contrast to SLT-1, PE can exploit the KDEL receptor in order to reach the ER lumen where it is believed that membrane transfer to the cytosol occurs. This contention was confirmed by microinjecting into Vero cells antibodies raised against the cytoplasmically exposed tail of the KDEL receptor. Immunofluorescence confirmed that these antibodies prevented the retrograde transport of the KDEL receptor from the Golgi complex to the ER, and this in turn reduced the cytotoxicity of PE, but not that of SLT-1, to these cells.

ADP Ribose Transferases↗
PubMed1998

The deoxyribonuclease activity attributed to ribosome-inactivating proteins is due to contamination.

The mode of action of ribosome-inactivating proteins (RIPs) has, for many years, been considered to be depurination of a specific adenyl residue of ribosomal RNA, resulting in inhibition of protein synthesis. Recently, this view has been challenged by the observation that many RIP preparations have significant DNase activity in addition to their N-glycosidase activity. In this study, we have investigated the putative DNase activity of two RIPs, ricin and pokeweed antiviral protein (PAP), and show that, in both cases, the DNase activity is due to the presence of contaminating nucleases. The N-glycosidase and DNase activities of PAP were separately and specifically inactivated by chemical modification and heat. Gel filtration of ricin allowed physical separation of the two activities. Furthermore, neither recombinant PAP nor recombinant ricin A-chain purified from Escherichia coli displayed DNase activity.

Chromatography, Gel↗
PubMed1998

The lipid-free structure of apolipoprotein A-I: effects of amino-terminal deletions.

Deletion mutants of human apolipoprotein A-I (apo hA-I) have been produced from a bacterial expression system to explore the function of the specific domains comprising residues 1-43, 1-65, 88-98, and 187-243, respectively, in the lipid-free conformation and in the lipid-binding mechanism of apo hA-I. Initial studies on apo Delta(1-43)A-I and apo Delta(187-243)A-I have already been reported. To aid purification of these mutants, a histidine-containing N-terminal extension was incorporated (+his); in cases where comparison with the (-his) construct was possible, little effect on the physical properties due to the (+his) extension was found. All mutants have folded structures in their lipid-free state, however these structures differ widely in their relative thermodynamic stability and extent of secondary structure. The mutant with the fewest residues deleted, apo Delta(88-98)A-I(+his), has the least secondary structure (only 34% helix) and is also the least stable (DeltaG = 2.9 kcal/mol). Determined from sedimentation velocity measurements on the lipid-free proteins, all but apo Delta(1-65)A-I(+his) exhibited a range of conformers in solution, which fluctuated around a highly elongated species (dimensions equal to approximately (14-16) x approximately 2.3 nm). Apo Delta(1-65)A-I(+his) exhibited a discrete species which was less asymmetric (dimensions equal to 9 x 2.9 nm). Apo Delta(88-98)A-I(+his) showed extreme heterogeneity with no predominating conformer. Spectroscopic studies (ANS binding and circular dichroism) indicate that there is little difference in the lipid-free structure of the carboxy-terminal deletion mutant, apo Delta(187-243)A-I(+/-his) compared to wild-type (wt) apo wtA-I(+/-his), but substantial differences are observed between wt and the amino-terminal deletion mutants, apo Delta(1-43)A-I, apo Delta(1-65)A-I(+his), and apo Delta(88-98)A-I(+his). In contrast, the lipid-binding properties are impaired for apo Delta(187-243)A-I(+/-his), as measured by dimyristoyl phosphatidylcholine (DMPC) liposome turbidity clearance kinetics and palmitoyloleoyl phosphatidylcholine (POPC) equilibrium binding. Apo Delta(1-43)A-I, apo Delta(1-65)A-I(+his), and apo Delta(88-98)A-I(+his) show lipid affinities statistically similar to apo wtA-I(+his), but significantly defective DMPC clearance kinetics. Interestingly, lecithin:cholesterol acyltransferase (LCAT) activation results correlate qualitatively with the lipid-binding affinity for all mutants but apo Delta(88-98)A-I(+his), suggesting that this mutant has an altered and possibly noncooperative lipid-bound structure as well as an altered lipid-free structure. These results suggest helix 1 (residues 44-65) and helix 10 (residues 220-240) are both required for native lipid-binding properties, while the presence of internal residues, at least helix 3 (residues 88-98), is essential for proper folding of both the lipid-free and lipid-bound conformations. Importantly, studies on apo Delta(88-98)A-I(+his) provide the first experimental evidence that a native-like structure is not necessary for native-like lipid affinity, but apparently is necessary for both DMPC solubilization and LCAT activation. These results provide support for a hypothetical, multistep structure-based mechanism for apo hA-I lipid binding.

Apolipoprotein A-I↗
PubMed1998

Free ricin A chain, proricin, and native toxin have different cellular fates when expressed in tobacco protoplasts.

The catalytic A subunit of ricin can inactivate eukaryotic ribosomes, including those of Ricinus communis where the toxin is naturally produced. How such plant cells avoid intoxication has remained an open question. Here we report the transient expression of a number of ricin A chain-encoding cDNA constructs in tobacco protoplasts. Ricin A chain entered the endoplasmic reticulum lumen, where it was efficiently glycosylated, but it was toxic to the cells and disappeared with time in a brefeldin A-insensitive manner, suggesting reverse translocation to the cytosol and eventual degradation. Proricin (the natural precursor form containing A and B chains joined together by a linker sequence) was glycosylated, transported to the vacuole, and processed to its mature form, but was not toxic. Free ricin A chain and proricin were not secreted, whereas free ricin B chain was found entirely in the extracellular medium. The coexpression of ricin A and B chains resulted in the formation of disulfide-linked, transport-competent heterodimers, which were secreted, with a concomitant reduction in the observed cytotoxicity. These results suggest that the production of ricin as a precursor is essential for its routing to the vacuole and for protection of ricin-producing cells.

Ricinus communis↗
PubMed1998

Expression of mutant dynamin protects cells against diphtheria toxin but not against ricin.

Diphtheria toxin is believed to enter sensitive mammalian cells via receptor-mediated endocytosis from clathrin-coated pits, while ricin can enter via both clathrin-dependent and clathrin-independent endocytosis. The present study has confirmed this by determining the toxin sensitivity of COS-7y cells which were transiently overexpressing a trans dominant negative mutant of dynamin, a GTPase required for the budding of clathrin-coated vesicles from the plasma membrane. Cells overexpressing wild-type dynamin showed normal receptor-mediated endocytosis of transferrin and remained sensitive to both diphtheria toxin and ricin. Cells overexpressing a mutant dynamin defective in GTP binding and hydrolysis were unable to endocytose transferrin and were protected against diphtheria toxin, but they remained completely sensitive to ricin intoxication. Treating non-transfected cells or cells overexpressing mutant dynamin with nystatin caused a redistribution of the caveolae membrane marker protein VIP21-caveolin from the cell surface to intracellular locations, but did not affect their sensitivity to ricin. The redistribution of caveolin seen after nystatin treatment may reflect the disappearance of caveolae. If this is the case, caveolae are not responsible for the endocytosis of ricin. An alternative clathrin-independent route may operate for ricin, since cellular uptake, intracellular transport, and translocation into the cytosol remain unaffected when clathrin-dependent endocytosis is effectively blocked.

Animals↗
PubMed1998

Structural analysis of apolipoprotein A-I: effects of amino- and carboxy-terminal deletions on the lipid-free structure.

An amino-terminal deletion mutant (residues 1-43) and a carboxy-terminal deletion mutant (residues 187-243) of human apoliprotein A-I (apo hA-I) have been produced from a bacterial expression system to explore the importance of the missing residues for the conformation of apo hA-I. Our focus has been to study the lipid-free structure of apo hA-I to understand how discrete domains influence the conformational plasticity of the protein and, by inference, the mechanism of lipid binding. All spectral and physical measurements indicate that both apo delta(1-43)A-I and apo delta(187-243)A-I have folded, tertiary structures. These structures differ in the specific arrangement of helical domains based, in part, on their relative thermodynamic stability, near- and far-UV CD, limited proteolysis, and the accessibility of tryptophans to fluorescence quenchers. In addition, all data indicate that the folded domains of apo hA-I and apo delta(187-243)A-I are very similar. Results from analytical ultracentrifugation suggest that lipid-free apo hA-I and the deletion mutants each exist in a dynamic equilibrium between a loosely folded, helical bundle and an elongated monomeric helical hairpin. The conformational heterogeneity is consistent with significant ANS binding exhibited by all three proteins and could help to explain the facile lipid binding properties of apo hA-I.

Amino Acid Sequence↗
PubMed1998

Retrograde transport: going against the flow.

Certain protein toxins act by catalytically modifying substrates in the cytosol of mammalian cells. To reach this compartment, these proteins undergo retrograde transport from the cell surface, via the Golgi complex, to the endoplasmic reticulum.

Animals↗
PubMed1997

Proteolytic processing of ricin A chain is not required for cytotoxicity.

[125I]-labeled ricin A chain was endocytosed by macrophages and Vero cells either on its own or as part of the ricin holotoxin. Subsequently [125I]-ricin A chain was recovered from intoxicated cells by immunoprecipitation or acetone precipitation. The recovered protein was shown to have the same electrophoretic mobility as the material supplied to the cells. These data challenge an earlier suggestion that limited intracellular processing of ricin A chain by proteolytic enzymes is required for maximal toxicity.

Animals↗
PubMed1997

Ricin A chain can transport unfolded dihydrofolate reductase into the cytosol.

Ricin is a heterodimeric protein toxin. The ricin A chain is able to cross the membrane of intracellular compartments to reach the cytosol where it catalytically inactivates protein synthesis. It is linked via a disulfide bond to the B chain, a galactose-specific lectin, which allows ricin binding at the cell surface and endocytosis. To examine the potential of ricin A to carry proteins into the cytosol and the requirement for unfolding of the passenger protein, we connected mouse dihydrofolate reductase (DHFR) to ricin A by gene fusion via a spacer peptide. DHFR-ricin A expressed in Escherichia coli displayed the biological activities of the parent proteins and associated quantitatively with ricin B to form DHFR-ricin. The resulting toxin was highly cytotoxic to cells (4-8-fold less than recombinant ricin). DHFR-ricin cytotoxicity was inhibited by methotrexate, a DHFR inhibitor stabilizing DHFR-ricin A in a folded conformation. The DHFR moiety of DHFR ricin bound to the plasma membrane. Although methotrexate prevented this binding, it did not significantly affect DHFR-ricin endocytosis, which proceeded via ricin B chain. Intoxication kinetics data and a cell-free translocation assay demonstrated that protection of cells from DHFR-ricin cytotoxicity resulted from a selective inhibition by methotrexate of DHFR-ricin A translocation. We conclude that ricin A is a potential carrier of proteins to the cytosol, provided that the passenger protein is able to unfold for transmembrane transport.

Animals↗
PubMed1997

Characterization of prokaryotic recombinant Aspergillus ribotoxin alpha-sarcin.

The Aspergillus ribonuclease alpha-sarcin is toxic to intact mammalian cells but the mechanism by which it enters the cells to reach its ribosomal RNA substrate is unclear. Here we have compared the cytotoxicity of alpha-sarcin to that of ricin, another catalytic toxin that targets the same rRNA sequence but whose mechanism of cell entry is better understood. Intact ricin binds to cell surface components and enters the cells by receptor-mediated endocytosis, whereas the catalytic polypeptide of ricin (the A chain or RTA) which, like alpha-sarcin, is unable to bind to surface components directly and enters cells by fluid phase uptake. Recombinant alpha-sarcin was produced in Escherichia coli and purified to homogeneity. The protein was soluble, stable and its ability to inhibit in vitro protein synthesis was indistinguishable from that of native alpha-sarcin. Further, recombinant alpha-sarcin had the same in vitro protein synthesis inhibition activity as ricin A chain. The cytotoxicity of alpha-sarcin and ricin A chain to HeLa cells was also the same. The cytotoxicity of alpha-sarcin was due to its RNAase activity rather than to specific membrane effects at the cell surface, since a mutant containing a single substitution at a putative key catalytic residue had reduced ribonuclease activity and an equivalent reduction in cytotoxicity. One interpretation of the data is that a-sarcin enters mammalian cells in the same way as free ricin A chain.

Aspergillus↗
PubMed1997

Structural analysis of apolipoprotein A-I: limited proteolysis of methionine-reduced and -oxidized lipid-free and lipid-bound human apo A-I.

The domain structures of lipid-free and lipid-bound apolipoprotein A-I (apo A-I) containing reduced and oxidized methionines were analyzed by limited proteolysis. Lipid-free apo A-I is cleaved primarily in the extreme carboxy-terminus and, to a much lesser extent, in the central region of the protein between residues 115 and 136. Oxidation of methionines 112 and 148 to the corresponding sulfoxides in putative amphipathic helices 4 (P99-E120) and 6 (P143-A164), respectively, causes helices 1 (L44-G65), 2 (P66-S87), and 7 (P165-G186) to become susceptible to protease digestion. These results are consistent with a discrete, globular tertiary structure for the lipid-free protein minimally formed from amphipathic helices 1, 2, 4, 6, and 7. In distinct contrast to lipid-free apo A-I, lipid-bound apo A-I is most susceptible to cleavage in the extreme amino-terminus and, to a lesser extent, in both the central and carboxy-terminal regions. The observed cleavage pattern for the reduced lipid-bound protein supports the existence of many of the turns between helices predicted by sequence analysis of the lipid-bound protein. Methionine oxidation of lipid-bound protein results in a decreased protease susceptibility in the extreme amino-terminus and a concomitant increase in protease susceptibility in the central and carboxy-terminal regions. The results from methionine oxidation indicate the oxidation state of the protein is an important determinant in defining the conformation of both lipid-free and lipid-bound apo A-I.

Amino Acid Sequence↗
PubMed1997

Restoration of lectin activity to a non-glycosylated ricin B chain mutant by the introduction of a novel N-glycosylation site.

Ricin B chain (RTB) is an N-glycosylated, galactose-specific lectin. Removal of the two native N-glycosylation sites at Asn95 and Asn135 by site-directed mutagenesis generated a recombinant protein devoid of lectin activity. Two novel N-glycosylation sites were introduced into RTB at Asn42 and Asn123, either singly or in combination. Microinjection of pre-RTB transcripts into Xenopus oocytes showed that these novel sites became glycosylated in vivo. The single oligosaccharide site chain at Asn42 restored lectin activity to RTB, whereas glycosylation at Asn123 or simultaneous glycosylation at Asn42 and Asn123 failed to do so.

Animals↗
PubMed1997

Comparison of ribosome-inactivating proteins in the induction of apoptosis.

The aim of this study was to evaluate the ability of verocytotoxin-1 (VT1), VT1 B chain alone, ricin and a hybrid toxin (RASTA2) consisting of ricin A chain linked to VT1 B chain to inhibit protein synthesis and to induce apoptosis. The lethal effects of the toxins were compared using vero cells (originating from green African monkey kidney tissue). As previously described cell death occurred through apoptosis which was quantified using the diphenylamine assay. DNA fragmentation was seen with VT1 at 10 pg/ml but there was no effect with B chain alone. Fragmentation with ricin was seen at 10 ng/ml and with RASTA2 at 1 ng/ml. Protein synthesis inhibition was measured by [(35)S]methionine incorporation. VT1 had an IC50 of 0.0024 ng/ml, B chain alone was ineffective at inhibiting protein synthesis. Ricin had an IC50 of 0.39 ng/ml and RASTA2 of 1.7 ng/ml. In vero cells the B chain of these toxins does not participate in cell killing.

Animals↗
PubMed1997

Construction of a Bayesian network for mammographic diagnosis of breast cancer.

Bayesian networks use the techniques of probability theory to reason under uncertainty, and have become an important formalism for medical decision support systems. We describe the development and validation of a Bayesian network (MammoNet) to assist in mammographic diagnosis of breast cancer. MammoNet integrates five patient-history features, two physical findings, and 15 mammographic features extracted by experienced radiologists to determine the probability of malignancy. We outline the methods and issues in the system's design, implementation, and evaluation. Bayesian networks provide a potentially useful tool for mammographic decision support.

Bayes Theorem↗