J Michael Lord

The University of Warwick, Coventry, England, United Kingdom

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Publications (82)386.22 Total impact

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    Robert A Spooner · J Michael Lord
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    ABSTRACT: The heterodimeric plant toxin ricin binds exposed galactosyls at the cell surface of target mammalian cells, and, following endocytosis, is transported in vesicular carriers to the endoplasmic reticulum (ER). Subsequently, the cell-binding B chain (RTB) and the catalytic A chain (RTA) are separated reductively, RTA embeds in the ER membrane and then retrotranslocates (or dislocates) across this membrane. The protein conducting channels used by RTA are usually regarded as part of the ER-associated protein degradation system (ERAD) that removes misfolded proteins from the ER for destruction by the cytosolic proteasomes. However, unlike ERAD substrates, cytosolic RTA avoids destruction and folds into a catalytic conformation that inactivates its target ribosomes. Protein synthesis ceases, and subsequently the cells die apoptotically. This raises questions about how this protein avoids the pathways that are normally sanctioned for ER-dislocating substrates. In this review we focus on the molecular events that occur with non-tagged ricin and its isolated subunits at the ER-cytosol interface. This focus reveals that intra-membrane interactions of RTA may control its fate, an area that warrants further investigation.
    Full-text · Article · Jan 2015 · Toxins
  • Robert A. Spooner · J. Michael Lord
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    ABSTRACT: Type II RIPs from plants display a wide range of cytotoxicities to mammalian cells, from the highly toxic volkensin through to the essentially non-toxic ebulins and nigrins. To date, there have been few point-by-point comparisons made between these proteins. Most knowledge has accumulated for ricin, the type II RIP from Ricinus communis, providing some details of cell surface events, endosome sorting decisions, retrograde arrival in the ER, ER events including the retrotranslocation or dislocation of the RIP from the ER into the cytosol, and recovery of catalytic activity in the cytosol. Here we use ricin as an exemplar, and compare and contrast its intracellular journey with that of the bacterial type II RIP, Shiga toxin, examining points of similarity and difference, and highlighting some of the differences noted for other type II RIPs.
    No preview · Chapter · Apr 2014
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    ABSTRACT: The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the endoplasmic reticulum (ER) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show here that the proteasome has a more complex role in ricin intoxication than previously recognised, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. Our results implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated.
    Full-text · Article · Apr 2013 · Biochemical Journal
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    ABSTRACT: Escherichia coli Shiga-like toxin 1 normally traffics to the endoplasmic reticulum (ER) in sensitive mammalian cells from where the catalytic A chain (SLTxA1) dislocates to the cytosol to inactivate ribosomes. Currently, no molecular details of the dislocation process are available. To investigate the mechanism of the dislocation step we expressed SLTxA1 in the ER of Saccharomyces cerevisiae. Using a combination of growth studies and biochemical tracking in yeast knock-out strains we show that SLTxA1 follows an ER-associated degradation (ERAD) pathway to enter the cytosol in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex. ER-to-cytosol dislocation of the bulk population of SLTxA1 requires Cdc48p and its ubiquitin-handling co-factor Npl4p, and this population of toxin is terminally dispatched by proteasomal degradation. A small sub-population of SLTxA1 uncouples from this classical ERAD pathway and recovers catalytic activity in the cytosol. The pathway that leads to toxicity is also Hrd1p-dependent but, unlike that for the related ricin A chain toxin, SLTxA1 dislocation does require the catalytic cysteine of Hrd1p. However it does not depend on canonical ubiquitylation since toxin variants lacking endogenous lysyl residues also utilize this pathway, and furthermore there is no requirement for a number of Cdc48p co-factors. The fraction of SLTxA1 that disengages from the ERAD pathway thus does so upstream of Cdc48p interactions and downstream of Hrd1p interactions, in a step that possibly involves de-ubiquitylation. Mechanistically therefore, the dislocation of this toxin is quite distinct from that of conventional ERAD substrates that are normally degraded, and the toxins partially characterised to date that do not require the catalytic cysteine of the major Hrd1p component of the dislocation apparatus.
    Full-text · Article · Jul 2012 · PLoS ONE
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    ABSTRACT: We screened a panel of compounds derived from Exo2 - a drug that perturbs post-Golgi compartments and trafficking in mammalian cells - for their effect on the secretory pathway in Arabidopsis root epidermal cells. While Exo2 and most related compounds had no significant effect, one Exo2 derivative, named LG8, induced severe morphological alterations in both the Golgi (at high concentrations) and the endoplasmic reticulum (ER). LG8 causes the ER to form foci of interconnecting tubules, which at the ultrastructural level appear similar to those previously reported in Arabidopsis roots after treatment with the herbicide oryzalin. In cotyledonary leaves, LG8 causes redistribution of a trans Golgi network (TGN) marker to the vacuole. LG8 affects the anterograde secretory pathway by inducing secretion of vacuolar cargo and preventing the brassinosteroid receptor BRI1 from reaching the plasma membrane. Uptake and arrival at the TGN of the endocytic marker FM4-64 is not affected. Unlike the ADP ribosylation factor-GTP exchange factor (ARF-GEF) inhibitor brefeldin A (BFA), LG8 affects these post-Golgi events without causing the formation of BFA bodies. Up to concentrations of 50 µm, the effects of LG8 are reversible.
    Full-text · Article · Jul 2011 · Traffic
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    ABSTRACT: The small molecule Eeyarestatin I (ESI) inhibits the endoplasmic reticulum (ER)-cytosol dislocation and subsequent degradation of ERAD (ER associated protein degradation) substrates. Toxins such as ricin and Shiga/Shiga-like toxins (SLTx) are endocytosed and trafficked to the ER. Their catalytic subunits are thought to utilise ERAD-like mechanisms to dislocate from the ER into the cytosol, where a proportion uncouples from the ERAD process, recovers a catalytic conformation and destroys their cellular targets. We therefore investigated ESI as a potential inhibitor of toxin dislocation. Using cytotoxicity measurements, we found no role for ES(I) as an inhibitor of toxin dislocation from the ER, but instead found that for SLTx, ESI treatment of cells was protective by reducing the rate of toxin delivery to the ER. Microscopy of the trafficking of labelled SLTx and its B chain (lacking the toxic A chain) showed a delay in its accumulation at a peri-nuclear location, confirmed to be the Golgi by examination of SLTx B chain metabolically labelled in the trans-Golgi cisternae. The drug also reduced the rate of endosomal trafficking of diphtheria toxin, which enters the cytosol from acidified endosomes, and delayed the Golgi-specific glycan modifications and eventual plasma membrane appearance of tsO45 VSV-G protein, a classical marker for anterograde trafficking. ESI acts on one or more components that function during vesicular transport, whilst at least one retrograde trafficking pathway, that of ricin, remains unperturbed.
    Full-text · Article · Jul 2011 · PLoS ONE
  • Robert A Spooner · J Michael Lord
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    ABSTRACT: A number of protein toxins bind at the surface of mammalian cells and after endocytosis traffic to the endoplasmic reticulum, where the toxic A chains are liberated from the holotoxin. The free A chains are then dislocated, or retrotranslocated, across the ER membrane into the cytosol. Here, in contrast to ER substrates destined for proteasomal destruction, they undergo folding to a catalytic conformation and subsequently inactivate their cytosolic targets. These toxins therefore provide toxic probes for testing the molecular requirements for retrograde trafficking, the ER processes that prepare the toxic A chains for transmembrane transport, the dislocation step itself and for the post-dislocation folding that results in catalytic activity. We describe here the dislocation of ricin A chain and Shiga toxin A chain, but also consider cholera toxin which bears a superficial structural resemblance to Shiga toxin. Recent studies not only describe how these proteins breach the ER membrane, but also reveal aspects of a fundamental cell biological process, that of ER-cytosol dislocation.
    No preview · Article · Jul 2011 · Current topics in microbiology and immunology
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    J Michael Lord · Robert A Spooner
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    ABSTRACT: Ricin is a heterodimeric plant protein that is potently toxic to mammalian and many other eukaryotic cells. It is synthesized and stored in the endosperm cells of maturing Ricinus communis seeds (castor beans). The ricin family has two major members, both, lectins, collectively known as Ricinus communis agglutinin ll (ricin) and Ricinus communis agglutinin l (RCA). These proteins are stored in vacuoles within the endosperm cells of mature Ricinus seeds and they are rapidly broken down by hydrolysis during the early stages of post-germinative growth. Both ricin and RCA traffic within the plant cell from their site of synthesis to the storage vacuoles, and when they intoxicate mammalian cells they traffic from outside the cell to their site of action. In this review we will consider both of these trafficking routes.
    Full-text · Article · Jul 2011 · Toxins
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    ABSTRACT: The ribosome-inhibiting toxin ricin binds exposed β1→4 linked galactosyls on multiple glycolipids and glycoproteins on the cell surface of most eukaryotic cells. After endocytosis, internal cell trafficking is promiscuous, with only a small proportion of ricin proceeding down a productive (cytotoxic) trafficking route to the endoplasmic reticulum (ER). Here, the catalytic ricin A chain traverses the membrane to inactivate the cytosolic ribosomes, which can be monitored by measuring reduction in protein biosynthetic capacity or cell viability. Although some markers have been discovered for the productive pathway, many molecular details are lacking. To identify a more comprehensive set of requirements for ricin intoxication, the authors have developed an RNAi screen in Drosophila S2 cells, screening in parallel the effects of individual RNAi treatments alone and when combined with a ricin challenge. Initial screening of 806 gene knockdowns has revealed a number of candidates for both productive and nonproductive ricin trafficking, including proteins required for transport to the Golgi, plus potential toxin interactors within the ER and cytosol.
    Full-text · Article · Mar 2011 · Journal of Biomolecular Screening
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    No preview · Article · Dec 2010 · ChemInform
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    ABSTRACT: Brefeldin A-mediated inhibition of ADP ribosylation factor (Arf) GTPases and their guanine nucleotide exchange factors, Arf-GEFs, has been a cornerstone of membrane trafficking research for many years. Brefeldin A (BFA) is relatively non-selective inhibiting at least three targets in human cells, Golgi brefeldin A resistance factor 1 (GBF1), brefeldin A inhibited guanine nucleotide exchange factor 1 (BIG1) and brefeldin A inhibited guanine nucleotide exchange factor 2 (BIG2). Here, we show that the previously described compound Exo2 acts through inhibition of Arf-GEF function, but causes other phenotypic changes that are not GBF1 related. We describe the engineering of Exo2 to produce LG186, a more selective, reversible inhibitor of Arf-GEF function. Using multiple-cell-based assays and GBF1 mutants, our data are most consistent with LG186 acting by selective inhibition of GBF1. Unlike other Arf-GEF and reported GBF1 inhibitors including BFA, Exo2 and Golgicide A, LG186 induces disassembly of the Golgi stack in both human and canine cells.
    Full-text · Article · Dec 2010 · Traffic
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    ABSTRACT: The small molecule 4-hydroxy-3-methoxybenzaldehyde (5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-yl)hydrazone (Exo2) stimulates morphological changes at the mammalian Golgi and trans-Golgi network that are virtually indistinguishable from those induced by brefeldin A. Both brefeldin A and Exo2 protect cells from intoxication by Shiga(-like) toxins by acting on other targets that operate at the early endosome, but do so at the cost of high toxicity to target cells. The advantage of Exo2 is that it is much more amenable to chemical modification and here we report a range of Exo2 analogues produced by modifying the tetrahydrobenzothienopyrimidine core, the vanillin moiety and the hydrazone bond that links these two. These compounds were examined for the morphological changes they stimulated at the Golgi stack, the trans-Golgi network and the transferrin receptor-positive early endosomes and this activity correlated with their inherent toxicity towards the protein manufacturing ability of the cell and their protective effect against toxin challenge. We have developed derivatives that can separate organelle morphology, target specificity, innate toxicity and toxin protection. Our results provide unique compounds with low toxicity and enhanced specificity to unpick the complexity of membrane trafficking networks.
    Full-text · Article · Oct 2010 · Molecular BioSystems
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    ABSTRACT: We report that a toxic polypeptide retaining the potential to refold upon dislocation from the endoplasmic reticulum (ER) to the cytosol (ricin A chain; RTA) and a misfolded version that cannot (termed RTA(Delta)), follow ER-associated degradation (ERAD) pathways in Saccharomyces cerevisiae that substantially diverge in the cytosol. Both polypeptides are dislocated in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex and subsequently degraded. Canonical polyubiquitylation is not a prerequisite for this interaction because a catalytically inactive Hrd1p E3 ubiquitin ligase retains the ability to retrotranslocate RTA, and variants lacking one or both endogenous lysyl residues also require the Hrd1p complex. In the case of native RTA, we established that dislocation also depends on other components of the classical ERAD-L pathway as well as an ongoing ER-Golgi transport. However, the dislocation pathways deviate strikingly upon entry into the cytosol. Here, the CDC48 complex is required only for RTA(Delta), although the involvement of individual ATPases (Rpt proteins) in the 19S regulatory particle (RP) of the proteasome, and the 20S catalytic chamber itself, is very different for the two RTA variants. We conclude that cytosolic ERAD components, particularly the proteasome RP, can discriminate between structural features of the same substrate.
    Full-text · Article · Aug 2010 · Molecular biology of the cell
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    ABSTRACT: The cytotoxic plant protein ricin comprises a lectin B chain that binds promiscuously to glycolipids and glycoproteins at the surface of mammalian cells, disulphide-coupled to a toxic A chain which depurinates target ribosomes. To find these cytosolic targets, the A chain has to cross a biological membrane, which is not a simple task for a folded protein. The secretory pathway of eukaryotic cells is reversible and ricin can take advantage of this to move from the plasma membrane, via the Golgi, to the ER whose membrane is crossed to gain access to the cytosol. Since membrane traversal is preceded by an unfolding step, there is a clear requirement for cytosolic re-folding of ricin to gain a catalytic conformation. This final step for ricin is accomplished after triage by cytosolic chaperones, underlining the central role of these in cellular protein folding.
    No preview · Chapter · Jul 2010
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    No preview · Article · May 2010 · ChemInform
  • J. Michael Lord · Gareth D. Griffiths

    No preview · Chapter · Dec 2009
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    ABSTRACT: The oxidative cyclisation of a range of benzothieno[2,3-d]pyrimidine hydrazones (7a-j) to the 1,2,4-triazolo[4,3-c]pyrimidines (8a-j) catalysed by lithium iodide or to the 1,2,4-triazolo[1,5-c]pyrimidines (10a-j) with sodium carbonate is presented. A complementary synthesis of the 1,2,4-triazolo[1,5-c]pyrimidines starting from the amino imine 11 is also reported. The effect of these compounds on Shiga toxin (STx) trafficking in HeLa cells and comparison to the previously reported Exo2 is also detailed.
    Full-text · Article · Oct 2009 · European Journal of Medicinal Chemistry
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    ABSTRACT: Disruption of Golgi alpha-mannosidase II activity can result in type II congenital dyserythropoietic anemia and induce lupus-like autoimmunity in mice. Here, we isolated a mutant human embryonic kidney (HEK) 293T cell line called Lec36, which displays sensitivity to ricin that lies between the parental HEK 293T cells, in which the secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which produce only oligomannose-type N-linked glycans. Stem cell marker 19A was transiently expressed in the HEK 293T Lec36 cells and in parental HEK 293T cells with and without the potent Golgi alpha-mannosidase II inhibitor, swainsonine. Negative ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, were dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi alpha-mannosidase II: a point mutation that mapped to the active site was found in one allele, and an in-frame deletion of 12 nucleotides was found in the other allele. Expression of the wild type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and as a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia.
    Full-text · Article · Jun 2009 · Journal of Biological Chemistry
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    ABSTRACT: After endocytic uptake by mammalian cells, the heterodimeric plant toxin ricin is transported to the endoplasmic reticulum (ER), where the ricin A chain (RTA) must cross the ER membrane to reach its ribosomal substrates. Here, using gel filtration chromatography, sedimentation, fluorescence, fluorescence resonance energy transfer, and circular dichroism, we show that both fluorescently labeled and unlabeled RTA bind both to ER microsomal membranes and to negatively charged liposomes. The binding of RTA to the membrane at 0-30 degrees C exposes certain RTA residues to the nonpolar lipid core of the bilayer with little change in the secondary structure of the protein. However, major structural rearrangements in RTA occur when the temperature is increased. At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer. RTA is then stably bound to the membrane because it is nonextractable with carbonate. The sharp temperature dependence of the structural changes does not coincide with a lipid phase change because little change in fluorescence-detected membrane mobility occurred between 30 and 37 degrees C. Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol. The sharp temperature dependence of these changes in RTA further suggests that they occur optimally in mammalian targets of the plant toxin.
    Preview · Article · Mar 2009 · Journal of Biological Chemistry
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    ABSTRACT: The plant cytotoxin ricin enters target mammalian cells by receptor-mediated endocytosis and undergoes retrograde transport to the endoplasmic reticulum (ER). Here, its catalytic A chain (RTA) is reductively separated from the cell-binding B chain, and free RTA enters the cytosol where it inactivates ribosomes. Cytosolic entry requires unfolding of RTA and dislocation across the ER membrane such that it arrives in the cytosol in a vulnerable, nonnative conformation. Clearly, for such a dislocated toxin to become active, it must avoid degradation and fold to a catalytic conformation. Here, we show that, in vitro, Hsc70 prevents aggregation of heat-treated RTA, and that RTA catalytic activity is recovered after chaperone treatment. A combination of pharmacological inhibition and cochaperone expression reveals that, in vivo, cytosolic RTA is scrutinized sequentially by the Hsc70 and Hsp90 cytosolic chaperone machineries, and that its eventual fate is determined by the balance of activities of cochaperones that regulate Hsc70 and Hsp90 functions. Cytotoxic activity follows Hsc70-mediated escape of RTA from an otherwise destructive pathway facilitated by Hsp90. We demonstrate a role for cytosolic chaperones, proteins typically associated with folding nascent proteins, assembling multimolecular protein complexes and degrading cytosolic and stalled, cotranslocational clients, in a toxin triage, in which both toxin folding and degradation are initiated from chaperone-bound states.
    Full-text · Article · Dec 2008 · Proceedings of the National Academy of Sciences