Biomedical subjects

Lynne M Roberts

Publications and source records attributed to Lynne M Roberts.

27 records · Page 2Linked to original sources

PubMed2003

The position of the proricin vacuolar targeting signal is functionally important.

Ricin is synthesised as an ER-targeted precursor containing an enzymatic A chain and a galactose-binding B chain separated by a 12-amino acid linker propeptide. This internal propeptide is known to contain a sequence-specific vacuolar sorting signal whose functionality depends on the presence of an isoleucine residue. Conversion of this isoleucine to glycine completely abolished vacuolar targeting of proricin and led to its secretion. However, when this mutated signal was positioned at the C-terminus of a normally secreted reporter, vacuolar targeting of a significant fraction still occurred. Likewise, when the corrupted linker was C-terminally exposed within its natural context following the mature ricin A chain, and then co-expressed with ricin B chain, toxin heterodimers were still partially transported to tobacco cell vacuoles. By contrast, when placed at the N-terminus of the secreted reporter, or at the N-terminus of ricin B chain for co-expression with ricin A chain, the propeptide behaved most strikingly as a sequence-specific vacuolar targeting signal that, when mutated, resulted in complete secretion of the proteins. It would appear that the position of the linker peptide influences the specificity of its vacuolar targeting function.

Amino Acid Sequence↗

PubMed2003

Sequence-specific, Golgi-dependent vacuolar targeting of castor bean 2S albumin.

The targeting of the castor bean (Ricinus communis) 2S albumin precursor has been investigated by expressing cDNA in transformed tobacco (Nicotiana tabacum) leaf cells and by following biosynthesis in the native tissue. Correct targeting in both tissues was accompanied by processing of the precursor. Delivery to vacuoles was sensitive to brefeldin A (BFA) treatment in both tissues and to perturbation of COPII function in tobacco, supporting the view that transport through the Golgi is required. The targeting signal for this Golgi-dependent routing lies within the propeptide of the first heterodimer of proalbumin. This propeptide directed a normally secreted reporter protein to the vacuoles of tobacco cells in a Golgi-dependent manner in vivo, unless a critical Leu residue was mutated, supporting the view that a sequence-specific signal was needed to target a seed storage protein to the vacuoles of a vegetative cell.

2S Albumins, Plant↗

PubMed2003

Ricin. Mechanisms of cytotoxicity.

Ricin is a heterodimeric protein produced in the seeds of the castor oil plant (Ricinus communis). It is exquisitely potent to mammalian cells, being able to fatally disrupt protein synthesis by attacking the Achilles heel of the ribosome. For this enzyme to reach its substrate, it must not only negotiate the endomembrane system but it must also cross an internal membrane and avoid complete degradation without compromising its activity in any way. Cell entry by ricin involves a series of steps: (i) binding, via the ricin B chain (RTB), to a range of cell surface glycolipids or glycoproteins having beta-1,4-linked galactose residues; (ii) uptake into the cell by endocytosis; (iii) entry of the toxin into early endosomes; (iv) transfer, by vesicular transport, of ricin from early endosomes to the trans-Golgi network; (v) retrograde vesicular transport through the Golgi complex to reach the endoplasmic reticulum; (vi) reduction of the disulphide bond connecting the ricin A chain (RTA) and the RTB; (vii) partial unfolding of the RTA to render it translocationally-competent to cross the endoplasmic reticulum (ER) membrane via the Sec61p translocon in a manner similar to that followed by misfolded ER proteins that, once recognised, are targeted to the ER-associated protein degradation (ERAD) machinery; (viii) avoiding, at least in part, ubiquitination that would lead to rapid degradation by cytosolic proteasomes immediately after membrane translocation when it is still partially unfolded; (ix) refolding into its protease-resistant, biologically active conformation; and (x) interaction with the ribosome to catalyse the depurination reaction. It is clear that ricin can take advantage of many target cell molecules, pathways and processes. It has been reported that a single molecule of ricin reaching the cytosol can kill that cell as a consequence of protein synthesis inhibition. The ready availability of ricin, coupled to its extreme potency when administered intravenously or if inhaled, has identified this protein toxin as a potential biological warfare agent. Therapeutically, its cytotoxicity has encouraged the use of ricin in 'magic bullets' to specifically target and destroy cancer cells, and the unusual intracellular trafficking properties of ricin potentially permit its development as a vaccine vector. Combining our understanding of the ricin structure with ways to cripple its unwanted properties (its enzymatic activity and promotion of vascular leak whilst retaining protein stability and important immunodominant epitopes), will also be crucial in the development of a long awaited protective vaccine against this toxin.

Animals↗

PubMed2002

Exogenous peptides delivered by ricin require processing by signal peptidase for transporter associated with antigen processing-independent MHC class I-restricted presentation.

In this study we demonstrate that a disarmed version of the cytotoxin ricin can deliver exogenous CD8(+) T cell epitopes into the MHC class I-restricted pathway by a TAP-independent, signal peptidase-dependent pathway. Defined viral peptide epitopes genetically fused to the N terminus of an attenuated ricin A subunit (RTA) that was reassociated with its partner B subunit were able to reach the early secretory pathway of sensitive cells, including TAP-deficient cells. Successful processing and presentation by MHC class I proteins was not dependent on proteasome activity or on recycling of MHC class I proteins, but rather on a functional secretory pathway. Our results demonstrated a role for signal peptidase in the generation of peptide epitopes associated at the amino terminus of RTA. We showed, first, that potential signal peptide cleavage sites located toward the N terminus of RTA can be posttranslationally cleaved by signal peptidase and, second, that mutation of one of these sites led to a loss of peptide presentation. These results identify a novel MHC class I presentation pathway that exploits the ability of toxins to reach the lumen of the endoplasmic reticulum by retrograde transport, and suggest a role for endoplasmic reticulum signal peptidase in the processing and presentation of MHC class I peptides. Because TAP-negative cells can be sensitized for CTL killing following retrograde transport of toxin-linked peptides, application of these results has direct implications for the development of novel vaccination strategies.

Animals↗

PubMed2002

The low lysine content of ricin A chain reduces the risk of proteolytic degradation after translocation from the endoplasmic reticulum to the cytosol.

Several protein toxins, including the A chain of ricin (RTA), enter mammalian cells by endocytosis and subsequently reach their cytosolic substrates by translocation across the endoplasmic reticulum (ER) membrane. To achieve this export, such toxins exploit the ER-associated protein degradation (ERAD) pathway but must escape, at least in part, the normal degradative fate of ERAD substrates. Toxins that translocate from the ER have an unusually low lysine content. Since lysyl residues are potential ubiquitination sites, it has been proposed that this paucity of lysines reduces the chance of ubiquitination and subsequent ubiquitin-mediated proteasomal degradation [Hazes, B., and Read, R. J. (1997) Biochemistry 36, 11051-11054]. Here we provide experimental support for this hypothesis. The two lysyl residues within RTA were changed to arginyl residues. Their replacement in RTA did not have a significant stabilizing effect, suggesting that the endogenous lysyl residues are not the usual sites for ubiquitin attachment. However, when four additional lysines were introduced into RTA in a way that did not compromise the activity, structure, or stability of the toxin, degradation was significantly enhanced. Enhanced degradation resulted from ubiquitination that predisposed the toxin to proteasomal degradation. Treatment with the proteasome inhibitor clasto-lactacystin beta-lactone increased the cytotoxicity of the lysine-rich RTA to a level approaching that of wild-type ricin. The introduction of four additional lysyl residues into a second ribosome-inactivating protein, abrin A chain, also dramatically decreased the cytotoxicity of the holotoxin compared to wild-type abrin. This effect could also be reversed by proteasomal inhibition. Our data support the hypothesis that the evolution of a low lysine content is a degradation-avoidance strategy for toxins that retrotranslocate from the ER.

Animals↗

PubMed2002

ER dislocation: Cdc48p/p97 gets into the AAAct.

Misfolded or unassembled proteins present in the lumen of the endoplasmic reticulum are exported to the cytosol and degraded. Recent studies have implicated a complex containing the AAA ATPase Cdc48p/p97 in the export process.

Adenosine Triphosphatases↗

PubMed2002

Binding of ricin A-chain to negatively charged phospholipid vesicles leads to protein structural changes and destabilizes the lipid bilayer.

Ricin is a heterodimeric protein toxin in which a catalytic polypeptide (the A-chain or RTA) is linked by a disulfide bond to a cell-binding polypeptide (the B-chain or RTB). During cell entry, ricin undergoes retrograde vesicular transport to reach the endoplasmic reticulum (ER) lumen, from where RTA translocates into the cytosol, probably by masquerading as a substrate for the ER-associated protein degradation (ERAD) pathway. In partitioning studies in Triton X-114 solution, RTA is predominantly found in the detergent phase, whereas ricin holotoxin, native RTB, and several single-chain ribosome-inactivating proteins (RIPs) are in the aqueous phase. Fluorescence spectroscopy and far-UV circular dichroism (CD) demonstrated significant structural changes in RTA as a result of its interaction with liposomes containing negatively charged phospholipid (POPG). These lipid-induced structural changes markedly increased the trypsin sensitivity of RTA and, on the basis of the protein fluorescence determinations, abolished its ability to bind to adenine, the product resulting from RTA-catalyzed depurination of 28S ribosomal RNA. RTA also released trapped calcein from POPG vesicles, indicating that it destabilized the lipid bilayer. We speculate that membrane-induced partial unfolding of RTA during cell entry may facilitate its recognition as an ERAD substrate.

Fluoresceins↗

PubMed2002

1st class ticket to class I: protein toxins as pathfinders for antigen presentation.

A number of bacterial toxins have evolved diverse strategies for crossing membrane barriers in order to reach their substrates in the mammalian cytosol. Recent studies show that this property can be exploited for the delivery of fused antigens into the major histocompatibility complex class I-restricted presentation pathway, with the goal of eliciting a specific immune response. Here we discuss the peculiarities of the trafficking pathways of a variety of toxins, and how these may allow the toxins to be used as delivery vehicles for therapeutic and diagnostic purposes.

Cytosol↗

PubMed1999

Proteolytic cleavage of the A subunit is essential for maximal cytotoxicity of Escherichia coli O157:H7 Shiga-like toxin-1.

Members of the bacterial Shiga toxin family consist of a single A subunit that is non-covalently associated with a pentamer of B subunits. These toxins bind to receptors on susceptible mammalian cells and enter the cells by endocytic uptake. During cell entry, the 32 kDa A subunit is cleaved by the membrane-anchored protease furin to generate a catalytically active, 27.5 kDa A1 fragment and a 4.5 kDa A2 fragment. Previous studies have shown that mutating the furin site to prevent cleavage did not significantly affect toxin potency, suggesting that cleavage is not required for toxin activity. Here it is confirmed that preventing cleavage at the usual processing site does not prevent proteolytic processing of the Escherichia coli Shiga-like toxin-1 A subunit. However, simultaneous mutation of both the primary furin-recognition site and a nearby putative furin cleavage site did prevent intracellular processing of the A subunit. Comparison of the cytotoxicities of purified recombinant toxins to cultured mammalian cells demonstrated that even on prolonged incubation with toxin, the unprocessed mutant was 60-fold less toxic than the wild-type protein or other mutants still capable of being proteolytically processed during cell entry.

Animals↗