Search PubMed⌕ Search

Biomedical subjects

L M Roberts

Publications and source records attributed to L M Roberts.

At least 55 records · Page 3Linked to original sources
PubMed1996

Free ricin A chain reaches an early compartment of the secretory pathway before it enters the cytosol.

During the intoxication of mammalian cells by ricin, the catalytically active A chain must cross the membrane of an intracellular compartment in order to reach its ribosomal substrates in the cytosol. The actual site of ricin A chain translocation is unclear, and conflicting views hold that it enters the cytosol from endosomes or from an early compartment of the secretory pathway, possibly the lumen of the endoplasmic reticulum. Here we show that treating cells with brefeldin A, or transiently overexpressing mutant GTPases known to inhibit biochemical complexes mediating anterograde and retrograde transport between the endoplasmic reticulum and the Golgi complex, protected cells from intoxication by free ricin A chain. These data indicate that ricin A chain, either free or as part of intact ricin, reaches an early compartment of the secretory pathway before translocation into the cytosol occurs.

Amino Acid Sequence↗
PubMed1996

Ricin A chain fused to a chloroplast-targeting signal is unfolded on the chloroplast surface prior to import across the envelope membranes.

The initial stages of chloroplast protein import involve the binding of precursor proteins to surface-bound receptors prior to translocation across the envelope membranes in a partially folded conformation. We have analyzed the unfolding process by examining the conformation of a construct, comprising the presequence of a chloroplast protein linked to ricin A chain, before and after binding to the chloroplast surface. We show that the presequence is highly susceptible to proteolysis in solution, probably reflecting a lack of tertiary structure, whereas the A chain passenger protein is resistant to extremely high concentrations of protease, unless deliberately unfolded using denaturant. The A chain moiety is furthermore active, indicating that the presence of the presequence does not prevent formation of a tightly folded, native state. In contrast, receptor-bound p33KRA (fusion protein comprising the 33-kDa presequence plus 22 residues of mature protein, linked to the A chain of ricin) is quantitatively digested by protease concentrations that have little effect on the A chain in solution. We conclude that protein unfolding can take place on the chloroplast surface in the absence of translocation and without the aid of soluble factors.

Chloroplasts↗
PubMed1996

Major structural differences between pokeweed antiviral protein and ricin A-chain do not account for their differing ribosome specificity.

Pokeweed antiviral protein (PAP) and the A-chain of ricin (RTA) are two members of a family of ribosome-inactivating proteins (RIPS) that are characterised by their ability to catalytically depurinate eukaryotic ribosomes, a modification that makes the ribosomes incapable of protein synthesis. In contrast to RTA, PAP can also inactivate prokaryotic ribosomes. In order to investigate the reason for this differing ribosome specificity, a series of PAP/RTA hybrid proteins was prepared to test for their ability to depurinate prokaryotic and eukaryotic ribosomes. Information from the X-ray structures of RTA and PAP was used to design gross polypeptide switches and specific peptide insertions. Initial gross polypeptide swaps created hybrids that had altered ribosome inactivation properties. Preliminary results suggest that the carboxy-terminus of the RIPs (PAP 219-262) does not contribute to ribosome recognition, whereas polypeptide swaps in the amino-terminal half of the proteins did affect ribosome inactivation. Structural examination identified three loop regions that were different in both structure and composition within the amino-terminal region. Directed substitution of RTA sequences into PAP at these sites, however, had little effect on the ribosome inactivation characteristics of the mutant PAPs, suggesting that the loops were not crucial for prokaryotic ribosome recognition. On the basis of these results we have identified regions of RIP primary sequence that may be important in ribosome recognition. The implications of this work are discussed.

Antiviral Agents↗
PubMed1995

A hydrophobic region of ricin A chain which may have a role in membrane translocation can function as an efficient noncleaved signal peptide.

Ricin A chain is a polypeptide of 267 amino acids containing a hydrophobic region near its carboxyl-terminus (residues 245-256) which has been implicated in the membrane translocation step necessary for this catalytically active toxin to reach its intracellular substrate. DNA fusions were constructed that encoded hybrid proteins consisting of carboxyl-terminal residues 233-267 or residues 238-267 of ricin A chain preceding mouse dihydrofolate reductase. When in vitro transcripts prepared from these constructs were translated in cell-free systems, the ricin A chain-derived sequences functioned as efficient signal peptides which directed dihydrofolate reductase into microsomes or into proteoliposomes containing microsomal membrane components.

Amino Acid Sequence↗
PubMed1995

Mutational analysis of the Ricinus lectin B-chains. Galactose-binding ability of the 2 gamma subdomain of Ricinus communis agglutinin B-chain.

Ricin B-chain (RTB) is a galactose-specific lectin that folds into two globular domains, each of which binds a single galactoside. The two binding sites are structurally similar and both contain a conserved tripeptide kink and an aromatic residue that comprises a sugar-binding platform. Whereas the critical RTB residues implicated in lectin activity are conserved in domain 1 of Ricinus communis agglutinin (RCA) B-chain, the sugar platform aromatic residue Tyr-248 present in domain 2 of RTB is replaced by His in RCA B-chain. In this study, key residues in the vicinity of the binding sites of the Ricinus lectin B-chains were altered by site-directed mutagenesis. The recombinant B-chains were produced in Xenopus oocytes in soluble, stable, and core-glycosylated forms. Both sites of RCA B-chain must be simultaneously modified in order to abolish lectin activity, indicating the presence of two independent, functional binding sites/molecule. Activity associated with the domain 2 site of RCA B-chain is abrogated by the conversion of Trp-258 to Ser. Moreover, the domain 2 site appears responsible for a weak binding interaction recombinant RCA B-chain with GalNAc, not observed with native tetrameric RCA. Finally, the introduction of His at position 248 of RTB severely disrupts but does not abolish GalNAc binding.

Acetylgalactosamine↗
PubMed1995

Point mutations in the hydrophobic C-terminal region of ricin A chain indicate that Pro250 plays a key role in membrane translocation.

A series of mutations have been made in the carboxyl terminus of ricin A chain, centred on the hydrophobic region between amino acid residues Val245 and Val256. The mutant ricin A chains were expressed to a high level in an Escherichia coli system and the proteins purified to homogeneity. The enzymic activity of each of these A chain molecules was tested on rabbit reticulocyte ribosomes; in all cases, the activities were found to be comparable to wild-type recombinant ricin A chain. Following reassociation of these A chains to ricin B chain, Vero cells were challenged with these holotoxins and the cytotoxicities determined. Mutant ricin A chain with Ile247-->Ala was unable to reassociate and form holotoxin, indicating the importance of this residue in the interaction with ricin B chain. Mutant ricin A chain with Pro250-->Ala readily reassociated with ricin B chain, forming holotoxin with a 170-fold reduction in cytotoxicity to Vero cells. Other mutations in this region also produced A chain proteins which gave marked reductions in holotoxin cytotoxicity. We propose therefore that the C-terminal hydrophobic region of ricin A chain may be involved in membrane interactions prior to the translocation of this subunit into the cytosol, and that Pro250 plays a key role in one or both of these steps.

Amino Acid Sequence↗
PubMed1995

Ricin cytotoxicity is sensitive to recycling between the endoplasmic reticulum and the Golgi complex.

Cytotoxic proteins that kill mammalian cells by catalytically inhibiting protein synthesis must enter the cytosol in order to reach their substrates. With the exception of diphtheria toxin, which enters the cytosol from acidified endosomes, the intracellular site of translocation of other toxins including ricin, Escherichia coli Shiga-like toxin-1, and Pseudomonas exotoxin A is likely to involve early compartments of the secretory pathway. We have used a molecular approach to identify the site and mechanism of toxin delivery to the cytosol by transiently expressing mutant GTPases that inhibit the assembly of biochemical complexes mediating anterograde and retrograde transport in the exocytic and endocytic pathways. The results provide evidence to suggest that receptors actively recycling between the endoplasmic reticulum and terminal Golgi compartments are essential for toxin translocation to the cytosol from the endoplasmic reticulum. The rapid kinetics of intoxication demonstrate a substantial level of bidirectional membrane flow and sorting through the early secretory pathway.

Amino Acid Sequence↗
PubMed1995

Catalytic and cytotoxic activities of recombinant ricin A chain mutants with charged residues added at the carboxyl terminus.

Ricin A chain (RTA) mutants which had been modified by the addition of three lysine residues, three lysines and an alanine, or six histidine residues at the carboxyl terminus were expressed in Escherichia coli. The recombinant proteins were purified to homogeneity by ion-exchange chromatography on CM-Sepharose CL-6B. The 28S ribosomal RNA N-glycosidase activities of the three RTA mutants were indistinguishable from each other and from the activity of wild-type recombinant RTA. The RTA mutants were not impaired, compared with wild-type RTA, in their ability to reassociate with ricin B chain to form ricin holotoxin. Holotoxins containing mutant RTAs were as readily dissociated into subunits under reducing conditions as native holotoxin, and the RTA mutants were indistinguishable from wild-type RTA in the extent of their interaction with biological membranes. Ricin holotoxins containing the RTA mutants were, however, less cytotoxic to Vero cells than ricin containing wild-type RTA. At equivalent concentrations, a time course assay showed that holotoxin containing the mutant RTAs took longer to kill target cells than that containing wild-type recombinant RTA, suggesting that the mutant forms of RTA are less efficiently processed or translocated across an intracellular membrane than is wild-type RTA.

Amino Acids↗
PubMed1995

Preliminary investigation of a Bayesian network for mammographic diagnosis of breast cancer.

Bayesian networks use the techniques of probability theory to reason under conditions of uncertainty. We investigated the use of Bayesian networks for radiological decision support. A Bayesian network for the interpretation of mammograms (MammoNet) was developed based on five patient-history features, two physical findings, and 15 mammographic features extracted by experienced radiologists. Conditional-probability data, such as sensitivity and specificity, were derived from peer-reviewed journal articles and from expert opinion. In testing with a set of 77 cases from a mammography atlas and a clinical teaching file, MammoNet performed well in distinguishing between benign and malignant lesions, and yielded a value of 0.881 (+/- 0.045) for the area under the receiver operating characteristic curve. We conclude that Bayesian networks provide a potentially useful tool for mammographic decision support.

Bayes Theorem↗
PubMed1994

Analysis of oxidative titrations of Desulfovibrio gigas hydrogenase; implications for the catalytic mechanism.

The oxidative titrations of hydrogenase (Hase) from Desulfovibrio gigas [Barondeau, D. P., Roberts, L. M., & Lindahl, P. A. (1994) J. Am. Chem. Soc. 116, 3442] were simulated using model descriptions of the redox reactions in the enzyme. The data fit best to a model that assumed Hase contains one [Fe3S4]1+/0 cluster, two [Fe4S4]2+/1+ clusters, and a Ni center stable in four redox states (Ni-B, Ni-SI, Ni-C, and Ni-R), each separated by one electron. A model in which Ni-SI, Ni-C, and Ni-R correspond to Nickel(2+) dithiolate, nickel(1+) dithiol, and nickel(2+) dithiol hydride, respectively, is compatible with all established relevant properties of the Ni center. This model and the concept of redox microstates were employed to define electronic states of the enzyme and to reformulate the catalytic mechanism initially proposed by Cammack et al. [Cammack, R., Patil, D. S., Hatchikian, E. C., & Fernandez, V. M. (1987) Biochim. Biophys. Acta 912, 98] into three interconnected catalytic cycles. These cycles differ in the average oxidation level of the Fe4S4 clusters. The cycle with the most reduced clusters appears to operate reversibly (catalyzing both H2 oxidation and H+ reduction), while those with more oxidized clusters function only to oxidize H2. The difference in reversibility is explained by assuming that Ni-R prefers to reduce an [Fe4S4]2+ cluster instead of H+ and that H+ is reduced only when that Fe4S4 cluster is in its reduced state.

Catalysis↗
PubMed1994

Introduction of a disulfide bond into ricin A chain decreases the cytotoxicity of the ricin holotoxin.

Wild type ricin A chain (RTA) contains two cysteine residues (Cys171 and Cys259). Cys259 forms the interchain disulfide bond of ricin holotoxin with Cys4 of ricin B chain (RTB). We have used site-directed mutagenesis of RTA cDNA to convert Cys171 to Ser and to introduce a disulfide bond into RTA by converting Ser215 and Met255 to Cys residues. Mutant RTA was expressed in Escherichia coli and directed to the oxidizing environment of the periplasmic space where the Cys215-Cys255 disulfide bond was formed. The disulfide-containing RTA mutant had an in vitro catalytic activity similar to that of an identical form of recombinant RTA that lacked the S215C and M255C mutations. In the presence of glutathione and protein disulfide isomerase, this RTA variant reassociated with RTB to form ricin holotoxin. Incubation of this holotoxin with increasing concentrations of dithiothreitol showed that the interchain disulfide bond joining RTA and RTB was more readily reduced than the intrachain disulfide bond in RTA. Ricin in which the RTA moiety contained the disulfide bond was 15-18-fold less cytotoxic to HeLa or Vero cells than ricin in which the RTA did not contain the stabilizing disulfide cross-link. Since these ricin molecules had identical RTB cell binding and RTA catalytic activities, we suggest that the observed reduction in cytotoxicity caused by the introduced disulfide bond resulted from a constraint on the unfolding of RTA, indicating that such unfolding is necessary for the membrane translocation of RTA during its entry into the cytosol.

Animals↗
PubMed1994

Pokeweed antiviral protein (PAP) mutations which permit E.coli growth do not eliminate catalytic activity towards prokaryotic ribosomes.

Pokeweed antiviral protein (PAP) has N-glycosidase activity towards both eukaryotic and prokaryotic ribosomes. This is in marked contrast with the A chains of type 2 ribosome inactivating proteins (RIPs) such as ricin and abrin, which inactivate only eukaryotic ribosomes. A recent report described spontaneous mutations in PAP that implicated specific amino acids to be involved in determining the activity of PAP towards prokaryotic ribosomes. As part of an ongoing study into RIP--ribosome interactions these mutations were specifically recreated in a PAP clone encoding the mature 262 amino acid PAP sequence. Mutants were tested for their N-glycosidase activity by analysing the integrity of eukaryotic and prokaryotic ribosomes after mutant protein expression. Mutations of F196Y and K211R, either individually or within the same clone, were active toward both classes of ribosome, indicating that these amino acid positions are not involved in differentiating ribosomal substrates. Mutation R68G led to a protein that appeared to be inactive towards prokaryotic ribosomes, but also very poorly active towards eukaryotic ribosomes. This mutation is currently under further investigation.

Base Sequence↗
PubMed1994

Ricin B chain fragments expressed in Escherichia coli are able to bind free galactose in contrast to the full length polypeptide.

Deleted forms of ricin B chain (RTB) containing only one of the two galactose binding sites were produced in E. coli and targeted to the periplasm by fusion to the ompA or ompF signal sequences. The proteins were then isolated from the periplasm and their sugar binding properties assessed. Previous studies investigating the properties of such proteins produced in Xenopus laevis oocytes suggested that deleted forms of RTB, when not glycosylated, retain their ability to bind simple sugars, unlike the full-length unglycosylated proteins. When produced in E. coli however we found that only one, EB733, of a number of deleted forms of RTB closely related to those previously produced in Xenopus laevis oocytes, bound to simple sugars. All of the deletion forms of RTB were found to bind in the asialofetuin binding assay; an assay which has been previously utilized to measure binding of lectins to the terminal galactose residues of glycoprotein oligosaccharides. However, in contrast to glycosylated RTB, binding of the deletion mutants could be competed to only a small degree or not at all with galactose. The only deletion mutant observed to bind to free galactose when produced in E. coli corresponded closely to the complete domain 2 of RTB. It is assumed that this mutant forms a stable structure similar to that of the C-terminal domain in the full-length protein. The structural integrity of EB733 was not only suggested by its sugar binding properties and solubility but also by its consistently higher level of expression and the absence of any apparent susceptibility to E. coli proteases.

Base Sequence↗
PubMed1994

Correlation between the activities of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection.

The rRNA depurination activities of five ribosome-inactivating proteins (RIPs) were compared in vitro using yeast and tobacco leaf ribosomes as substrates. All of the RIPs (pokeweed antiviral protein (PAP), dianthin 32, tritin, barley RIP and ricin A-chain) were active on yeast ribosomes. PAP and dianthin 32 were highly active and ricin A-chain weakly active on tobacco ribosomes, whereas tritin and barley RIP were inactive. PAP and dianthin 32 were highly effective in inhibiting the formation of local lesions caused by tobacco mosaic virus (TMV) on tobacco leaves, whereas tritin, barley RIP and ricin A-chain were ineffective. The apparent anomaly between the in vitro rRNA depurination activity, but lack of antiviral activity of ricin A-chain was further investigated by assaying for rRNA depurination in situ following the topical application of the RIP to leaves. No activity was detected, a finding consistent with the apparent lack of antiviral activity of this RIP. Thus, it is concluded that there is a positive correlation between RIP-catalysed depurination of tobacco ribosomes and antiviral activity which gives strong support to the hypothesis that the antiviral activity of RIPs works through ribosome inactivation.

Aniline Compounds↗
PubMed1994

Ricin: structure, mode of action, and some current applications.

Ricin is an abundant protein component of Ricinus communis seeds (castor beans) that is exquisitely toxic to mammalian cells. It consists of an enzymic polypeptide that catalyzes the N-glycosidic cleavage of a specific adenine residue from 28S ribosomal RNA, joined by a single disulfide bond to a galactose (cell)-binding lectin. The enzymatic activity renders ribosomes containing depurinated 28S RNA incapable of protein synthesis. The bipartite molecular structure of ricin allows it to bind to the mammalian cell surface, enter via endocytic uptake, and deliver the catalytically active polypeptide into the cell cytosol where it irreversibly inhibits protein synthesis causing cell death. Because of its cytotoxic potency, modified ricin is being used for the selective killing of unwanted cells and for the toxigenic ablation of cell lineages in transgenic organisms.

Animals↗
PubMed1993

Addition of an endoplasmic reticulum retrieval sequence to ricin A chain significantly increases its cytotoxicity to mammalian cells.

An Escherichia coli expression system was used to produce recombinant ricin A chain (RTA) and RTA modified either by the addition of a carboxyl-terminal endoplasmic reticulum retrieval sequence Lys-Asp-Glu-Leu (RTAKDEL) or a nonfunctional analogue Lys-Asp-Glu-Ala (RTAKDEA). These RTA molecules can enter mammalian cells by fluid phase endocytosis. RTAKDEL was significantly more cytotoxic than either RTA or RTAKDEA to both Vero cells and HeLa cells (250- and 10-fold, respectively), despite the fact that all these RTA molecules had comparable enzymatic activities. This difference did not result from KDEL-mediated binding of RTAKDEL to the cell surface. Enhanced cytotoxicity could be correlated with an increased level of ribosome inactivation, measured as the RTA-catalyzed depurination of 28 S ribosomal RNA. These results indicate that the added KDEL sequence facilitated RTA entry into the cytosol. We propose that interaction with the intracellular KDEL receptor promotes retrograde transport of the toxin to the endoplasmic reticulum, where translocation of RTA into the cytosol occurs.

Amino Acid Sequence↗