Rezende SM, Simmonds RE, Lane DA (2004) Coagulation, inflammation, and apoptosis: different roles for protein S and the protein S-C4b binding protein complex, BLOOD 103 (4) pp. 1192-1201 AMER SOC HEMATOLOGY
Ogbechi J, Ruf MT, Hall BS, Bodman-Smith K, Vogel M, Wu HL, Stainer A, Esmon CT, Ahnström J, Pluschke G, Simmonds RE (2015) Mycolactone-Dependent Depletion of Endothelial Cell Thrombomodulin Is Strongly Associated with Fibrin Deposition in Buruli Ulcer Lesions,PLoS Pathogens 11 (7)
© 2015 Ogbechi et al.A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin?s substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells? ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone?s effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tissue ischemia could contribute to the development of the tissue necrosis seen in BU lesions.
Rance JB, Follows GA, Cockerill PN, Bonifer C, Lane DA, Simmonds RE (2003) Regulation of the human endothelial cell protein C receptor gene promoter by multiple Sp1 binding sites, BLOOD 101 (11) pp. 4393-4401 AMER SOC HEMATOLOGY
Mille-Baker B, Rezende SM, Simmonds RE, Mason PJ, Lane DA, Laffan MA (2003) Deletion or replacement of the second EGF-hke domain of protein S results in loss of APC cofactor activity, BLOOD 101 (4) pp. 1416-1418 AMER SOC HEMATOLOGY
Rezende SM, Simmonds RE, Zoller B, Dahlback B, Lane DA (1999) The molecular basis of type I/III protein S deficiency, THROMBOSIS AND HAEMOSTASIS pp. 426-427 F K SCHATTAUER VERLAG GMBH
Hall BS, Hill K, McKenna M, Ogbechi J, High S, Willis AE, Simmonds RE (2014) The pathogenic mechanism of the Mycobacterium ulcerans virulence factor, mycolactone, depends on blockade of protein translocation into the ER., PLoS Pathog 10 (4)
Infection with Mycobacterium ulcerans is characterised by tissue necrosis and immunosuppression due to mycolactone, the necessary and sufficient virulence factor for Buruli ulcer disease pathology. Many of its effects are known to involve down-regulation of specific proteins implicated in important cellular processes, such as immune responses and cell adhesion. We have previously shown mycolactone completely blocks the production of LPS-dependent proinflammatory mediators post-transcriptionally. Using polysome profiling we now demonstrate conclusively that mycolactone does not prevent translation of TNF, IL-6 and Cox-2 mRNAs in macrophages. Instead, it inhibits the production of these, along with nearly all other (induced and constitutive) proteins that transit through the ER. This is due to a blockade of protein translocation and subsequent degradation of aberrantly located protein. Several lines of evidence support this transformative explanation of mycolactone function. First, cellular TNF and Cox-2 can be once more detected if the action of the 26S proteasome is inhibited concurrently. Second, restored protein is found in the cytosol, indicating an inability to translocate. Third, in vitro translation assays show mycolactone prevents the translocation of TNF and other proteins into the ER. This is specific as the insertion of tail-anchored proteins into the ER is unaffected showing that the ER remains structurally intact. Fourth, metabolic labelling reveals a near-complete loss of glycosylated and secreted proteins from treated cells, whereas cytosolic proteins are unaffected. Notably, the profound lack of glycosylated and secreted protein production is apparent in a range of different disease-relevant cell types. These studies provide a new mechanism underlying mycolactone's observed pathological activities both in vitro and in vivo. Mycolactone-dependent inhibition of protein translocation into the ER not only explains the deficit of innate cytokines, but also the loss of membrane receptors, adhesion molecules and T-cell cytokines that drive the aetiology of Buruli ulcer.
Sacre SM, Lo A, Gregory B, Simmonds RE, Williams L, Feldmann M, Brennan FM, Foxwell BM (2008) Inhibitors of TLR8 Reduce TNF Production from Human Rheumatoid Synovial Membrane Cultures, JOURNAL OF IMMUNOLOGY 181 (11) pp. 8002-8009 AMER ASSOC IMMUNOLOGISTS
Simmonds RE, Lali FV, Smallie T, Small PLC, Foxwell BM (2009) Mycolactone Inhibits Monocyte Cytokine Production by a Posttranscriptional Mechanism, JOURNAL OF IMMUNOLOGY 182 (4) pp. 2194-2202 AMER ASSOC IMMUNOLOGISTS
Simmonds RE, Ireland H, Kunz G, Lane DA, Bhavnani M, Castaman G, Hambley H, Laffan M, OConnor N, Sas G, Tew CJ, Walker ID (1996) Identification of 19 protein S gene mutations in patients with phenotypic protein S deficiency and thrombosis, BLOOD 88 (11) pp. 4195-4204 W B SAUNDERS CO
Rezende SM, Lane DA, Mille-Baker B, Samama MM, Conard J, Simmonds RE (2002) Protein S Gla-domain mutations causing impaired Ca2+-induced phospholipid binding and severe functional protein S deficiency, BLOOD 100 (8) pp. 2812-2819 AMER SOC HEMATOLOGY
Gandrille S, Borgel D, Ireland H, Lane DA, Simmonds R, Reitsma PH, Mannhalter C, Pabinger I, Saito H, Suzuki K, Formstone C, Cooper DN, Espinosa Y, Sala N, Bernardi F, Aiach M (1997) Protein S deficiency: A database of mutations, Thrombosis and Haemostasis 77 (6) pp. 1201-1214
Rezende SM, Lane DA, Zoller B, Mille-Baker B, Laffan M, Dahlback B, Simmonds RE (2002) Genetic and phenotypic variability between families with hereditary protein S deficiency, THROMBOSIS AND HAEMOSTASIS 87 (2) pp. 258-265 SCHATTAUER GMBH-VERLAG MEDIZIN NATURWISSENSCHAFTEN
Simmonds RE, Lane DA (2001) The endothelial cell protein C receptor: A candidate genetic risk factor for thrombosis, THROMBOSIS AND HAEMOSTASIS 86 (4) pp. 939-941 F K SCHATTAUER VERLAG GMBH
The virulence factor mycolactone is responsible for the immunosuppression and tissue necrosis that characterise Buruli ulcer, a disease caused by infection with Mycobacterium ulcerans. In this study, we confirm that Sec61, the protein-conducting channel that coordinates entry of secretory proteins into the endoplasmic reticulum, is a primary target of mycolactone, and characterise the nature of its inhibitory effect. We conclude that mycolactone constrains the ribosome-nascent chain-Sec61 complex, consistent with its broad-ranging perturbation of the co-translational translocation of classical secretory proteins. In contrast, the effect of mycolactone on the post-translational, ribosome-independent translocation of short secretory proteins through the Sec61 complex is dependent on both signal sequence hydrophobicity and the translocation competence of the mature domain. Changes to protease sensitivity strongly suggest that mycolactone acts by inducing a conformational change in the pore-forming Sec61± subunit. These findings establish that mycolactone inhibits Sec61-mediated protein translocation and highlight differences between the co- and post-translational routes that the Sec61 complex mediates. We propose that mycolactone also provides a useful tool for further delineating the molecular mechanisms of Sec61-dependent protein translocation.
Brenner C, Simmonds RE, Wood S, Rose V, Feldmann M, Turner J (2012) TLR signalling and adapter utilization in primary human in vitro differentiated adipocytes., Scand J Immunol 76 (4) pp. 359-370
Toll-like receptors (TLRs) are central to innate immunity and yet their expression is widespread and not restricted to professional inflammatory cells. TLRs have been reported on adipocytes and have been implicated in obesity-associated pathologies such as diabetes. Why TLRs are found on adipocytes is not clear although one hypothesis is that they may coordinate energy utilization for the energy intensive process of an immune response. We have explored TLR signalling in primary human in vitro differentiated adipocytes and investigated the specific adapter molecules that are involved. Only lipopolysaccharide (LPS), poly(I:C), Pam3CSK4 and MALP-2 could induce the production of IL-6, IL-8 and MCP-1 by adipocytes. Poly(I:C) alone caused a strong induction of type I interferons, as assessed by IP-10 production. Using siRNA, it was confirmed that LPS-dependent signalling in adipocytes occurs via TLR4 utilizing the adapter molecules MyD88, Mal and TRIF and caused rapid degradation of IºB±. Pam3CSK4 signalling utilized TLR2, MyD88 and Mal (but not TRIF). However, the response to poly(I:C) observed in these cells appeared not to require TRIF, but MyD88 was required for induction of NFºB-dependent cytokines by Poly(I:C). Despite this, IºB± degradation could not be detected in poly(I:C) stimulated adipocytes at any time-point up to 4 h. Indeed, IL-6 transcription was not induced until 8-16 h after exposure. These data suggest that Pam3CSK4 and LPS signal via the expected routes in human adipocytes, whereas poly(I:C)/TLR3 signalling may act via a TRIF-independent, MyD88-dependent route.
Simmonds RE, Hermida J, Rezende SM, Lane DA (2001) Haemostatic genetic risk factors in arterial thrombosis, THROMBOSIS AND HAEMOSTASIS 86 (1) pp. 374-385 F K SCHATTAUER VERLAG GMBH
Hall BS, Simmonds R, Benbow ME, Mosi L, Roberts S, Williamso Jordan H (2015) Mycobacterium ulcerans and Buruli Ulcer, In: Singh SK (eds.), Human Emerging and Re-emerging Infections: Viral and Parasitic Infections, Volume I 1 44 John Wiley and Sons
Buruli Ulcer (BU) is the third most common mycobacterium disease following only tuberculosis and leprosy. Though BU is thought to be associated with large-and small-scale disturbances to the landscape and bodies of water frequented by human populations, primary prevention of BU is difficult because the mode of transmission is not known. This chapter reviews the most common environmental risk factors for BU and recent research into understanding its transmission. It is predicted that the proteins affected by mycolactone may share an underlying mechanism of production that could explain their co-regulation. Early work on the mechanism of suppression by mycolac-tone was carried out in Jurkat T cells using ASLs and focused on the suppression of IL-2 production. A multidisciplinary approach to treatment and patient care is essential for optimizing treatment outcomes. Physiotherapy is paramount minimizing and/or preventing disabilities.
Simmonds RE, Ireland H, Lane DA, Zoller B, de Frutos PG, Dahlback B (1998) Clarification of the risk for venous thrombosis associated with hereditary protein S deficiency by investigation of a large kindred with a characterized gene defect, ANNALS OF INTERNAL MEDICINE 128 (1) pp. 8-+ AMER COLL PHYSICIANS
Gandrille S, Borgel D, Ireland H, Lane DA, Simmonds R, Reitsma PH, Mannhalter C, Pabinger I, Saito H, Suzuki K, Formstone C, Cooper DN, Espinosa Y, Sala N, Bernardi F, Alach M (1997) Protein S deficiency: A database of mutations - For the Plasma Coagulation Inhibitors Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis, THROMBOSIS AND HAEMOSTASIS 77 (6) pp. 1201-1214 F K SCHATTAUER VERLAG GMBH
Biguzzi E, Razzari C, Lane DA, Castaman G, Cappellari A, Bucciarelli P, Fontana G, Margaglione M, D'Andrea G, Simmonds RE, Rezende SM, Preston R, Prisco D, Faioni EM (2005) Molecular diversity and thrombotic risk in Protein S deficiency: The PROSIT study, HUMAN MUTATION 25 (3) pp. 259-269 WILEY-LISS
White SJ, Simmonds RE, Lane DA, Baker AH (2005) Efficient isolation of peptide ligands for the endothelial cell protein C receptor (EPCR) using candidate receptor phage display biopanning, PEPTIDES 26 (7) pp. 1264-1269 ELSEVIER SCIENCE INC
Simmonds RE, Philippou H, Rance J, Lane DA (1999) Structural and functional implications of the intron/exon organisation of the human endothelial cell protein C/activated protein C receptor (EPCR) gene. Comparison with the structure of CD1/major histocompatibility complex alpha 1 and alpha 2 domains, THROMBOSIS AND HAEMOSTASIS pp. 14-14 F K SCHATTAUER VERLAG GMBH
Simmonds RE, Lane DA (1999) Structural and functional implications of the intron exon organization of the human endothelial cell protein C activated protein C receptor (EPCR) gene: Comparison with the structure of CD1/major histocompatibility complex alpha 1 and alpha 2 domains, BLOOD 94 (2) pp. 632-641 AMER SOC HEMATOLOGY
Moyo N, Westcott D, Simmonds R, Steinbach F (2012) Equine arteritis virus replication in monocytic cells suppresses differentiation and function of dendritic cells, IMMUNOLOGY 137 pp. 625-625 WILEY-BLACKWELL
Gandrille S, Borgel D, Sala N, Espinosa-Parrilla Y, Simmonds R, Rezende S, Lind B, Mannhalter C, Pabinger I, Reitsma PH, Formstone C, Cooper DN, Saito H, Suzuki K, Bernardi F, Aiach M (2000) Protein S deficiency: A database of mutations - Summary of the first update, THROMBOSIS AND HAEMOSTASIS 84 (5) pp. 918-918 F K SCHATTAUER VERLAG GMBH
Mycolactone is a polyketide macrolide lipid-like secondary metabolite synthesized by Mycobacterium ulcerans, the causative agent of BU (Buruli ulcer), and is the only virulence factor for this pathogen identified to date. Prolonged exposure to high concentrations of mycolactone is cytotoxic to diverse mammalian cells (albeit with varying efficiency), whereas at lower doses it has a spectrum of immunosuppressive activities. Combined, these pleiotropic properties have a powerful influence on local and systemic cellular function that should explain the pathophysiology of BU disease. The last decade has seen significant advances in our understanding of the molecular mechanisms underlying these effects in a range of different cell types. The present review focuses on the current state of our knowledge of mycolactone function, and its molecular and cellular targets, and seeks to identify commonalities between the different functional and cellular systems. Since mycolactone influences fundamental cellular processes (cell division, cell death and inflammation), getting to the root of how mycolactone achieves this could have a profound impact on our understanding of eukaryotic cell biology.
Mycolactone is the exotoxin virulence factor produced by Mycobacterium ulcerans, the pathogen responsible for Buruli ulcer. The skin lesions and immunosuppression characteristic of this disease result from the action of mycolactone, which targets the Sec61 complex and inhibits the co-translational translocation of secretory proteins into the endoplasmic reticulum. In this study, we investigate the effect of mycolactone on the Sec61-dependent biogenesis of different classes of transmembrane protein (TMP). Our data suggest that the effect of mycolactone on TMP biogenesis depends on how the nascent chain initially engages the Sec61 complex. For example, translocation of TMP lumenal domains driven by an N-terminal, cleavable signal sequence is efficiently inhibited by mycolactone. In contrast, the effect of mycolactone on protein translocation driven solely by a non-cleavable signal anchor/transmembrane domain depends on which flanking region is translocated. For example, while translocation of the region N-terminal to a signal anchor/transmembrane domain is refractive to mycolactone, C-terminal translocation is efficiently inhibited. Our findings highlight the diversity of Sec61-dependent translocation and provide a molecular basis for understanding the effect of mycolactone on the biogenesis of different TMPs.
Mycolactone is the exotoxin virulence factor of Mycobacterium ulcerans that causes the
neglected tropical disease Buruli ulcer. We recently showed it to be a broad spectrum
inhibitor of Sec61-dependent co-translational translocation of proteins into the endoplasmic
reticulum (ER). An outstanding question is the molecular pathway linking this to its known
cytotoxicity. We have now used translational profiling to better understand the
reprogramming that occurs in cells exposed to mycolactone. Gene ontology identified
enrichment in genes involved in cellular response to stress, and apoptosis signalling
amongst those showing enhanced translation. Validation of these results supports a
mechanism by which mycolactone activates an integrated stress response meditated by
phosphorylation of eIF2± via multiple kinases (PERK, GCN, PKR) without activation of the
ER stress sensors IRE1 or ATF6. The response therefore uncouples the integrated stress
response from ER stress, and features translational and transcription modes of genes
expression that feature the key regulatory transcription factor ATF4. Emphasizing the
importance of this uncoupled response in cytotoxicity, downstream activation of this pathway
is abolished in cells expressing mycolactone-resistant Sec61± variants. Using multiple
genetic and biochemical approaches, we demonstrate that eIF2± phosphorylation is
responsible for mycolactone-dependent translation attenuation, which initially protects cells
from cell death. However, chronic activation without stress remediation enhances autophagy
and apoptosis of cells by a pathway facilitated by ATF4 and CHOP. Our findings
demonstrate that priming events at the ER can result in the sensing of stress within different
Zong Guanghui, Hu Zhijian, O?Keefe Sarah, Tranter Dale, Iannotti Michael J., Baron Ludivine, Hall Belinda, Corfield Katherine, Paatero Anja O., Henderson Mark J., Roboti Peristera, Zhou Jianhong, Sun Xianwei, Govindarajan Mugunthan, Rohde Jason M., Blanchard Nicolas, Simmonds Rachel, Inglese James, Du Yuchun, Demangel Caroline, High Stephen, Paavilainen Ville O., Shi Wei Q. (2019) Ipomoeassin F Binds Sec61± to Inhibit Protein Translocation,Journal of the American Chemical Society 141 (21) pp. 8450-8461
American Chemical Society
Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit nanomolar potency. However, its biological and pharmacological properties have remained largely unexplored. Building upon our earlier achievements in total synthesis and medicinal chemistry, we used chemical proteomics to identify Sec61± (protein transport protein Sec61 subunit alpha isoform 1), the pore-forming subunit of the Sec61 protein translocon, as a direct binding partner of ipomoeassin F in living cells. The interaction is specific and strong enough to survive lysis conditions, enabling a biotin analogue of ipomoeassin F to pull down Sec61± from live cells, yet it is also reversible, as judged by several experiments including fluorescent streptavidin staining, delayed competition in affinity pulldown, and inhibition of TNF biogenesis after washout. Sec61± forms the central subunit of the ER protein translocation complex, and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of protein translocation in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the unique resistance profile demonstrated by specific amino acid single-point mutations in Sec61± provides compelling evidence that Sec61± is the primary molecular target of ipomoeassin F and strongly suggests that the binding of this natural product to Sec61± is distinctive. Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61± function and to further investigate its potential as a therapeutic target for drug discovery.
Background: The innate immune response is a tightly regulated process that reacts rapidly in response to pathogen-associated molecular patterns(PAMPs) such as lipopolysaccharide (LPS). Evidence is accumulating thatmicroRNAs contribute to this, although few studies have examined the earlyevents that constitute the ?primary? response. LPS-dependent changes to miRNA expression were studied inMethods:primary human monocyte-derived macrophages (1°MDMs). An unbiasedscreen by microarray was validated by qPCR and a method for the absolutequantitation of miRNAs was also developed, utilising 5? phosphorylatedRNA oligonucleotide templates. RNA immunoprecipitation was performedto explore incorporation of miRNAs into the RNA-induced silencingcomplex (RISC). The effect of miRNA functional inhibition on TNFexpression (mRNA and secretion) was investigated. Of the 197 miRNAs expressed in 1°MDMs, only five were inducedResults:>1.5-fold. The most strongly induced was miR-155-3p, the partner strand tomiR-155-5p, which are both derived from the MIR155HG/BIC gene(pri-miR-155). The abundance of miR-155-3p was induced transiently~250-fold at 2-4hrs and then returned towards baseline, mirroringpri-miR-155. Other PAMPs, IL-1², and TNF caused similar responses.IL-10, NF-ºB, and JNK inhibition reduced these responses,unlike cytokine-suppressing mycolactone. Absolute quantitation revealedthat miRNA abundance varies widely from donor-to-donor, and showed thatmiR-155-3p abundance is substantially less than miR-155-5p inunstimulated cells. However, at its peak there were 446-1,113 copies/cell,and miR-155-3p was incorporated into the RISC with an efficiency similar tomiR-16-5p and miR-155-5p. Inhibition of neither miRNA affected TNFsecretion after 2hrs in 1°MDMs, but technical challenges here are noted. Dynamic regulation of miRNAs during the primary responseConclusions:is rare, with the exception of miR-155-3p. Further work is required toestablish whether its low abundance, even at the transient peak, issufficient for biological activity and to determine whether there are specificmechanisms determining its biogenesis from miR-155 precursor.