Biomedical subjects
Publications and source records attributed to J Michael Lord.
Misfolded proteins in the ER require the p97 AAA ATPase for dislocation across the membrane prior to degradation by the cytosolic proteasome. The mechanism by which dislocated proteins are delivered to the proteasome from p97 is unclear, but recent studies suggest an important regulatory role for the protein ataxin-3.
The plant toxin ricin is synthesized in castor bean seeds as an endoplasmic reticulum (ER)-targeted precursor. Removal of the signal peptide generates proricin in which the mature A- and B-chains are joined by an intervening propeptide and a 9-residue propeptide persists at the N terminus. The two propeptides are ultimately removed in protein storage vacuoles, where ricin accumulates. Here we have demonstrated that the N-terminal propeptide of proricin acts as a nonspecific spacer to ensure efficient ER import and glycosylation. Indeed, when absent from the N terminus of ricin A-chain, the non-imported material remained tethered to the cytosolic face of the ER membrane, presumably by the signal peptide. This species appeared toxic to ribosomes. The propeptide does not, however, influence catalytic activity per se or the vacuolar targeting of proricin or the rate of retrotranslocation/degradation of A-chain in the cytosol. The likely implications of these findings to the survival of the toxin-producing tissue are discussed.
Internalisation of the plant toxin ricin occurs by retrograde transport which delivers the toxin to the ER where it intersects with the MHC class I system for peptide antigen display. Here, we describe the generation of an inactivated, non-toxic, ricin molecule fused to a peptide which elicits a CD8+ T-cell response in mice directed against pneumonia virus of mice, a pneumovirus related to human respiratory syncytial virus. The ricin fusion elicited a significant T-cell response when delivered by intraperitoneal inoculation in the absence of adjuvent. Challenge experiments showed that the T-cell response resulting from inoculation with the ricin-peptide fusion molecule delayed the onset of virus-induced disease.
A model has been presented for retrograde transport of certain toxins and viruses from the cell surface to the ER that suggests an obligatory interaction with a glycolipid receptor at the cell surface. Here we review studies on the ER trafficking cholera toxin, Shiga and Shiga-like toxins, Pseudomonas exotoxin A and ricin, and compare the retrograde routes followed by these protein toxins to those of the ER trafficking SV40 and polyoma viruses. We conclude that there is in fact no obligatory requirement for a glycolipid receptor, nor even with a protein receptor in a lipid-rich environment. Emerging data suggests instead that there is no common pathway utilised for retrograde transport by all of these pathogens, the choice of route being determined by the particular receptor utilised.
Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the intoxication of mammalian cells by Pseudomonas exotoxin A (PEx). The toxin binds the alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein. Here, we show that in HeLa cells, PEx recruits a proportion of this receptor to detergent-resistant microdomains (DRMs). Uptake of receptor-bound PEx involves transport steps both directly from early endosomes to the trans-Golgi network (TGN) independently of Rab9 function and from late endosomes to the TGN in a Rab9-dependent manner. Furthermore, treatments that simultaneously perturb both Arf1-dependent and Rab6-dependent retrograde pathways show that PEx can use multiple routes to reach the ER. The Rab6-dependent route has only been described previously for cargo with lipid-sorting signals. These findings suggest that partial localization of PEx within DRM permits a choice of trafficking routes consistent with a model that DRM-associated toxins reach the ER on a lipid-dependent sorting pathway whilst non-DRM-associated PEx exploits the previously characterized KDEL receptor-mediated uptake pathway. Thus, unexpectedly, an ER-directed toxin with a proteinaceous receptor shows promiscuity in its intracellular trafficking pathways, exploiting routes controlled by both lipid- and protein-sorting signals.
Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the productive intoxication of susceptible mammalian cells by Shiga-like toxin-1 (SLTx). Recently, it has been proposed that the observed association of certain ER-directed toxins and viruses with detergent-resistant membranes (DRM) may provide a general mechanism for their retrograde transport to endoplasmic reticulum (ER). Here, we show that DRM recruitment of SLTx bound to its globotriosylceramide (Gb(3)) receptor is mediated by the availability of other glycosphingolipids. Reduction in glucosylceramide (GlcCer) levels led to complete protection against SLTx and a reduced cell surface association of bound toxin with DRM. This reduction still allowed efficient binding and transport of the toxin to the ER. However, toxin sequestration within DRM of the ER was abolished under reduced GlcCer conditions, suggesting that an association of toxin with lipid microdomains or rafts in the ER (where these are defined by detergent insolubility) is essential for a later step leading to or involving retro-translocation of SLTx across the ER membrane. In support of this, we show that a number of ER residents, proteins intimately involved in the process of ER dislocation of misfolded proteins, are present in DRM.
The quality control system known as ERAD removes misfolded proteins from the ER to the cytosol for degradation. The AAA ATPase Cdc48p and ubiquitin ligases play crucial roles; their relationship has been unclear, but recent work has shown that the membrane protein Ubx2p links their functions in yeast.
It has been proposed that the aggregation of nascent transmembrane segments of polytopic proteins is prevented by chaperones present in the endoplasmic reticulum membrane; now the first experimental support for this proposal has been reported.
Knowledge of the uptake, membrane translocation, refolding and ribosome interaction of the ribosome-inactivating toxin ricin is incomplete at the present time. Ricin A chain (RTA) is the catalytic subunit of holotoxin and is also of particular interest as a vaccine candidate. For many studies into the uptake and immunological applications of ricin, it is essential to have inactive variants. Here, following error-prone polymerase chain reaction of the RTA open reading frame, we have used a modified gap-repair protocol in Saccharomyces cerevisiae to show that it is possible to rapidly generate a panel of inactive RTA mutants. Since yeast cells have ribosomes that are highly sensitive to RTA, we utilized a genetic selection based on the viability of transformants. This enabled the recovery of a number of mutations, some not previously identified, which permitted production of full-length but non-toxic RTA proteins. Such disarmed toxins may have utility as tools to study the cytosolic entry and action of RTA, and as potential vaccine candidates.
Several protein toxins, such as the potent plant toxin ricin, enter mammalian cells by endocytosis and undergo retrograde transport via the Golgi complex to reach the endoplasmic reticulum (ER). In this compartment the catalytic moieties exploit the ER-associated degradation (ERAD) pathway to reach their cytosolic targets. Bacterial toxins such as cholera toxin or Pseudomonas exotoxin A carry KDEL or KDEL-like C-terminal tetrapeptides for efficient delivery to the ER. Chimeric toxins containing monomeric plant ribosome-inactivating proteins linked to various targeting moieties are highly cytotoxic, but it remains unclear how these molecules travel within the target cell to reach cytosolic ribosomes. We investigated the intracellular pathways of saporin, a monomeric plant ribosome-inactivating protein that can enter cells by receptor-mediated endocytosis. Saporin toxicity was not affected by treatment with Brefeldin A or chloroquine, indicating that this toxin follows a Golgi-independent pathway to the cytosol and does not require a low pH for membrane translocation. In intoxicated Vero or HeLa cells, ricin but not saporin could be clearly visualized in the Golgi complex using immunofluorescence. The saporin signal was not evident in the Golgi, but was found to partially overlap with that of a late endosome/lysosome marker. Consistently, the toxicities of saporin or saporin-based targeted chimeric polypeptides were not enhanced by the addition of ER retrieval sequences. Thus, the intracellular movement of saporin differs from that followed by ricin and other protein toxins that rely on Golgi-mediated retrograde transport to reach their retrotranslocation site.
Ricin is a potent cytotoxin that can be rapidly internalized into mammalian cells leading to cell death. The ease in obtaining the toxin and its deadly nature combine to implicate ricin as a convenient agent for bioterrorism. Research into the mechanism of toxicity, as well as strategies for treatment and protection from the toxin has been widely undertaken for a number of years. This article reviews the current understanding of the mechanism of action of the toxin, the clinical effects of ricin intoxication and how these relate to current and continuing prospects for vaccine development.
Proteins that fail to fold in the endoplasmic reticulum (ER) or cannot find a pattern for assembly are often disposed of by a process named ER-associated degradation (ERAD), which involves transport of the substrate protein across the ER membrane (dislocation) followed by rapid proteasome-mediated proteolysis. Different ERAD substrates have been shown to be ubiquitinated during or soon after dislocation, and an active ubiquitination machinery has been found to be required for the dislocation of certain defective proteins. We have previously shown that, when expressed in tobacco (Nicotiana tabacum) protoplasts, the A chain of the heterodimeric toxin ricin is degraded by a pathway that closely resembles ERAD but is characterized by an unusual uncoupling between the dislocation and the degradation steps. Since lysine (Lys) residues are a major target for ubiquitination, we have investigated the effects of changing the Lys content on the retrotranslocation and degradation of ricin A chain in tobacco protoplasts. Here we show that modulating the number of Lys residues does not affect recognition events within the ER lumen nor the transport of the protein from this compartment to the cytosol. Rather, the introduced modifications have a clear impact on the degradation of the dislocated protein. While the substitution of the two Lys residues present in ricin A chain with arginine slowed down degradation, the introduction of four extra lysyl residues had an opposite effect and converted the ricin A chain to a standard ERAD substrate that is disposed via a process in which dislocation and degradation steps are tightly coupled.
Cells expressing ricin B chain within the secretory pathway are significantly more resistant to intoxication by ricin holotoxin but not to other cytotoxins that exploit similar endocytic routes to the cytosol. Furthermore, cells expressing the related B chain of abrin are protected against both incoming abrin and ricin. These phenotypes can be correlated with the abilities of the respective B chains to form disulphide-linked A-B holotoxins, since abrin B chain forms heterodimers with either abrin or ricin A chains, whereas ricin B chain forms heterodimers with ricin A chain only. In the ricin B-expressing cells, this newly made lectin disappears with biphasic kinetics comprising a retention phase followed by slow turnover and disposal after disengagement from calnexin cycle components. Interference with ricin cytotoxicity occurs during the early retention phase when ricin B chain is associated with PDI (protein disulphide-isomerase). The data show that retrotranslocation of incoming toxin is impeded by PDI-catalysed formation of heterodimers between endogenous B and A chains derived from reduced holotoxin, thus proving that reduction of ricin occurs in the endoplasmic reticulum. In contrast with other toxins, ricin does not appear to require either proteolytic cleavage or unfolding for PDI-catalysed reduction.
The insertion of a specific 25-residue internal peptide into ricin toxin A chain (RTA) reduced the catalytic activity of this protein approximately 300-fold. Directed proteolytic cleavage of the peptide insert essentially restored catalytic activity of the resulting two peptide A chain to normal levels. Ricin holotoxin containing unprocessed mutant A chain was not toxic to cultured mammalian cells, due to enhanced proteasomal degradation, nor was it toxic when injected into rats at a concentration that is lethal in the case of native ricin. Rats treated in this way were completely resistant to native ricin when subsequently challenged with a potentially lethal dose of the toxin. These ricin-resistant animals had a significant anti-ricin antibody titer, indicating that this approach has potential for developing an effective vaccine against this toxin.
Several bacterial toxins either promote or inhibit the maturation of human monocyte-derived DC. Since the potent plant toxin ricin exploits the same cell entry pathway used by these bacterial toxins and shares identical catalytic activity with some of them, we have studied the capacity of ricin to induce DC maturation in vitro. Here, we show that in contrast to the bacterial proteins, ricin neither induces DC maturation nor interferes with LPS-induced DC maturation. There is no correlation between the absence of DC maturation and ricin dysfunction. Indeed, some of the ricin variants retain significant ribotoxicity and catalytic activity. We have extended these observations to ebulin-1, suggesting that this may be a general characteristic of plant-derived cytotoxic ribosome-inactivating toxins. The human immune system may therefore have evolved to recognize and rapidly respond to the bacterial proteins, whilst being less responsive to the equivalent plant cytotoxins. Understanding the effect of ricin on professional APC may provide insights into the generation of an anti-ricin vaccine and into the use of inactivated ricin A chains as delivery vectors as part of a vaccination protocol.
A number of proteins produced by certain bacteria and plants are potently toxic to mammalian cells. This toxicity results from their ability to catalytically modify macromolecules that are required for essential cellular functions such as vesicular trafficking, cytoskeletal assembly, signalling or protein synthesis. To reach their targets, these proteins bind specific surface receptors before endocytosis and translocation across an internal membrane. The surface receptors exploited by different toxins include a range of proteins and lipids. Here we focus on specific glycosphingolipid receptors and two well-characterised subsets of toxins that exploit them for surface binding, intracellular trafficking, and signalling events.
Models for the binding of the sarcin-ricin loop (SRL) of 28S ribosomal RNA to ricin A chain (RTA) suggest that several surface exposed arginine residues surrounding the active site cleft make important interactions with the RNA substrate. The data presented in this study suggest differing roles for these arginyl residues. Substitution of Arg48 or Arg213 with Ala lowered the activity of RTA 10-fold. Furthermore, substitution of Arg213 with Asp lowered the activity of RTA 100-fold. The crystal structure of this RTA variant showed it to have an unaltered tertiary structure, suggesting that the positively charged state of Arg213 is crucial for activity. Substitution of Arg258 with Ala had no effect on activity, although substitution with Asp lowered activity 10-fold. Substitution of Arg134 prevented expression of folded protein, suggesting a structural role for this residue. Several models have been proposed for the binding of the SRL to the active site of RTA in which the principal difference lies in the conformation of the second 'G' in the target GAGA motif in the 28S rRNA substrate. In one model, the sidechain of Asn122 is proposed to make interactions with this G, whereas another model proposes interactions with Asp75 and Asn78. Site-directed mutagenesis of these residues of RTA favours the first of these models, as substitution of Asn78 with Ser yielded an RTA variant whose activity was essentially wild-type, whereas substitution of Asn122 reduced activity 37.5-fold. Substitution of Asp75 failed to yield significant folded protein, suggesting a structural role for this residue.
Ribosome-inactivating proteins (RIPs) are a heterogeneous group of enzymes found mainly in plants and a few bacteria that possess N-glycosidase activity on ribosomes and a related polynucleotide adenosine glycosidase activity on naked nucleic acids. They encompass single enzymatic chains, heterodimeric toxic lectins and related agglutinins. Plants commonly produce several RIP isoforms encoded by multi-gene families. The toxic lectins possess adaptations related to their cytotoxic role.