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Lynne M. Roberts - One of the best experts on this subject based on the ideXlab platform.
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folding competent And folding defective forms of Ricin A chAin hAve different fAtes After retrotrAnslocAtion from the endoplAsmic reticulum
Molecular Biology of the Cell, 2010Co-Authors: Robert A. Spooner, Michael J Lord, Stuart C H Allen, Christopher P Guise, Graham Ladds, Tina Schnoder, Manfred J Schmitt, Lynne M. RobertsAbstract: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 RTAΔ), 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 RTAΔ, 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.
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Ricin A chAin insertion into endoplAsmic reticulum membrAnes is triggered by A temperAture increAse to 37 c
Journal of Biological Chemistry, 2009Co-Authors: Peter U Mayerhofer, Michael J Lord, Jonathan Cook, Judit Wahlman, Teresa T J Pinheiro, Katherine A H Moore, Arthur E Johnson, Lynne M. RobertsAbstract: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.
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protein disulphide isomerAse reduces Ricin to its A And b chAins in the endoplAsmic reticulum
Biochemical Journal, 2004Co-Authors: Robert A. Spooner, Michael J Lord, Katherine A H Moore, Peter Duncan Watson, Catherine J Marsden, Daniel C Smith, Jonathon P Cook, Lynne M. RobertsAbstract: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.
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The position of the proRicin vAcuolAr tArgeting signAl is functionAlly importAnt
Plant Molecular Biology, 2003Co-Authors: Nicholas A. Jolliffe, Aldo Ceriotti, Lorenzo Frigerio, Lynne M. RobertsAbstract: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.
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Ricin A chAin without its pArtner b chAin is degrAded After retrotrAnslocAtion from the endoplAsmic reticulum to the cytosol in plAnt cells
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Alessandra Di Cola, Michael J Lord, Lorenzo Frigerio, Aldo Ceriotti, Lynne M. RobertsAbstract:When expressed in tobAcco cells, the cAtAlytic subunit of the dimeric ribosome inActivAting protein, Ricin, is first inserted into the endoplAsmic reticulum (ER) And then degrAded in A mAnner thAt cAn be pArtiAlly inhibited by the proteAsome inhibitor clAsto-lActAcystin β-lActone. Consistent with the implicAtion of cytosolic proteAsomes, degrAdAtion of Ricin A chAin is brefeldin A-insensitive And the polypeptides thAt AccumulAte in the presence of the proteAsome inhibitor Are not processed in A vAcuole-specific fAshion. RAther, these stAbilized polypeptides Are in pArt deglycosylAted by A peptide:N-glycAnAse-like Activity. TAken together, these results indicAte thAt Ricin A chAin, Albeit A structurAlly nAtive protein, cAn behAve As A substrAte for ER to cytosol export, deglycosylAtion in the cytosol, And proteAsomAl degrAdAtion. Furthermore, retrotrAnslocAtion of this protein is not tightly coupled to proteAsomAl Activity. These dAtA Are consistent with the hypothesis thAt Ricin A chAin cAn exploit the ER-AssociAted protein degrAdAtion pAthwAy to reAch the cytosol. Although well chArActerized in mAmmAliAn And yeAst cells, the operAtion of A similAr pAthwAy to the cytosol of plAnt cells hAs not previously been demonstrAted.
Sherry Ogg - One of the best experts on this subject based on the ideXlab platform.
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novel binding mechAnisms of fusion broAd rAnge Anti infective protein Ricin A chAin mutAnt pokeweed AntivirAl protein 1 rtAm pAp1 AgAinst sArs cov 2 key proteins in silico
Toxins, 2020Co-Authors: Yasser Hassan, Sherry OggAbstract:The deAdly pAndemic nAmed COVID-19, cAused by A new coronAvirus (SARS-CoV-2), emerged in 2019 And is still spreAding globAlly At A dAngerous pAce. As of todAy, there Are no proven vAccines, therApies, or even strAtegies to fight off this virus. Here, we describe the in silico docking results of A novel broAd rAnge Anti-infective fusion protein RTAM-PAP1 AgAinst the vArious key proteins of SARS-CoV-2 using the lAtest protein-ligAnd docking softwAre. RTAM-PAP1 wAs compAred AgAinst the SARS-CoV-2 B38 Antibody, Ricin A chAin, A pokeweed AntivirAl protein from leAves, And the lectin griffithsin using the speciAl CoDockPP COVID-19 version. These experiments reveAled novel binding mechAnisms of RTAM-PAP1 with A high Affinity to numerous SARS-CoV-2 key proteins. RTAM-PAP1 wAs further chArActerized in A preliminAry toxicity study in mice And wAs found to be A potentiAl therApeutic cAndidAte. These findings might leAd to the discovery of novel SARS-CoV-2 tArgets And therApeutic protein structures with outstAnding functions.
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expression of novel fusion AntivirAl proteins Ricin A chAin pokeweed AntivirAl proteins rtA pAps in escherichiA coli And their inhibition of protein synthesis And of hepAtitis b virus in vitro
BMC Biotechnology, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:Ricin A chAin (RTA) And Pokeweed AntivirAl proteins (PAPs) Are plAnt-derived N-glycosidAse ribosomAl-inActivAting proteins (RIPs) isolAted from Ricinus communis And PhytolAccA AmericAnA respectively. This study wAs to investigAte the potentiAl production AmenAbility And sub-toxic AntivirAl vAlue of novel fusion proteins between RTA And PAPs (RTA-PAPs). In brief, RTA-Pokeweed AntivirAl protein isoform 1 from seeds (RTA-PAPS1) wAs produced in An E. coli in vivo expression system, purified from inclusion bodies using gel filtrAtion chromAtogrAphy And protein synthesis inhibitory Activity AssAyed by compArison to the production of A control protein LuciferAse. The AntivirAl Activity of the RTA-PAPS1 AgAinst HepAtitis B virus (HBV) in HepAD38 cells wAs then determined using A dose response AssAy by quAntifying supernAtAnt HBV DNA compAred to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells wAs determined by meAsuring cell viAbility using A tetrAzolium dye uptAke AssAy. The fusion protein wAs further optimized using in silico tools, produced in An E. coli in vivo expression system, purified by A three-step process from soluble lysAte And confirmed in A protein synthesis inhibition Activity AssAy. Results showed thAt RTA-PAPS1 could effectively be recovered And purified from inclusion bodies. The refolded protein wAs bioActive with A 50% protein synthesis inhibitory concentrAtion (IC50) of 0.06 nM (3.63 ng/ml). The results Also showed thAt RTA-PAPS1 hAd A synergetic Activity AgAinst HBV with A hAlf-mAximAl response concentrAtion vAlue (EC50) of 0.03 nM (1.82 ng/ml) And A therApeutic index of > 21,818 with noticeAble steric hindrAnce. Results Also showed thAt the optimized protein Ricin A chAin mutAnt-Pokeweed AntivirAl protein isoform 1 from leAves (RTAM-PAP1) could be recovered And purified from soluble lysAtes with gAin of function on protein synthesis inhibition Activity, with An IC50 of 0.03 nM (1.82 ng/ml), And with minimAl, if Any, steric hindrAnce. Collectively, our results demonstrAte thAt RTA-PAPs Are AmenAble to effective production And purificAtion in nAtive form, possess significAnt gAin of function on protein synthesis inhibition And Anti-HBV Activities in vitro with A high therApeutic index And, thus, merit further development As potentiAl potent AntivirAl Agents AgAinst chronic HBV infection to be used As A stAndAlone or in combinAtion with existent therApies.
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expression of novel fusion AntivirAl proteins Ricin A chAin pokeweed AntivirAl proteins rtA pAps in escherichiA coli And their inhibition of protein synthesis And of hepAtitis b virus in vitro
bioRxiv, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:Ricin A chAin (RTA) And Pokeweed AntivirAl proteins (PAPs) Are plAnt-derived N-glycosidAse ribosomAl-inActivAting proteins (RIPs) isolAted from Ricinus communis And PhytolAccA AmericAnA respectively. This study wAs to investigAte the potentiAl AntivirAl vAlue of novel fusion proteins between RTA And PAPs (RTA-PAPs). In brief, RTA-PAPS1 wAs produced in E. coli in vivo expression system, purified from inclusion bodies using gel filtrAtion chromAtogrAphy And protein synthesis inhibitory Activity AssAyed by compArison to the production of A control protein LuciferAse. The AntivirAl Activity of the RTA-PAPS1 AgAinst HepAtitis B virus (HBV) in HepAD38 cells wAs then determined using A dose response AssAy by quAntifying supernAtAnt HBV DNA compAred to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells wAs determined by meAsuring cell viAbility using A tetrAzolium dye uptAke AssAy. Results showed thAt RTA-PAPS1 could effectively be recovered And purified from inclusion bodies. The refolded protein wAs bioActive with 50% protein synthesis inhibitory concentrAtion (IC50) of 0.06nM (3.63ng/ml). The results Also showed thAt RTA-PAPS1 hAd A synergetic Activity AgAinst HBV with An IC50 of 0.03nM (1.82ng/ml) And A therApeutic index of >21818. The fusion protein wAs further optimized using in silico tools, produced in E. coli in vivo expression system, purified by three-step process from soluble lysAte And protein synthesis inhibition Activity AssAyed. Results showed thAt the optimized protein (RTAM-PAP1) could be recovered And purified from soluble lysAtes with gAin of function Activity on protein synthesis inhibition with An IC50 of 0.03nM (1.82ng/ml). Collectively, our results demonstrAte thAt RTA-PAPs Are AmenAble to effective production And purificAtion in nAtive form, possess significAnt AntivirAl Activity AgAinst HBV in vitro with A high therApeutic index And, thus, meriting further development As potentiAl AntivirAl Agents AgAinst chronic HBV infections.
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Expression of novel fusion AntivirAl proteins Ricin A chAin-pokeweed AntivirAl proteins (RTA-PAPs) in EscherichiA coli And their inhibition of protein synthesis And of hepAtitis B virus in vitro
BMC, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:AbstrAct BAckground Ricin A chAin (RTA) And Pokeweed AntivirAl proteins (PAPs) Are plAnt-derived N-glycosidAse ribosomAl-inActivAting proteins (RIPs) isolAted from Ricinus communis And PhytolAccA AmericAnA respectively. This study wAs to investigAte the potentiAl production AmenAbility And sub-toxic AntivirAl vAlue of novel fusion proteins between RTA And PAPs (RTA-PAPs). In brief, RTA-Pokeweed AntivirAl protein isoform 1 from seeds (RTA-PAPS1) wAs produced in An E. coli in vivo expression system, purified from inclusion bodies using gel filtrAtion chromAtogrAphy And protein synthesis inhibitory Activity AssAyed by compArison to the production of A control protein LuciferAse. The AntivirAl Activity of the RTA-PAPS1 AgAinst HepAtitis B virus (HBV) in HepAD38 cells wAs then determined using A dose response AssAy by quAntifying supernAtAnt HBV DNA compAred to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells wAs determined by meAsuring cell viAbility using A tetrAzolium dye uptAke AssAy. The fusion protein wAs further optimized using in silico tools, produced in An E. coli in vivo expression system, purified by A three-step process from soluble lysAte And confirmed in A protein synthesis inhibition Activity AssAy. Results Results showed thAt RTA-PAPS1 could effectively be recovered And purified from inclusion bodies. The refolded protein wAs bioActive with A 50% protein synthesis inhibitory concentrAtion (IC50) of 0.06 nM (3.63 ng/ml). The results Also showed thAt RTA-PAPS1 hAd A synergetic Activity AgAinst HBV with A hAlf-mAximAl response concentrAtion vAlue (EC50) of 0.03 nM (1.82 ng/ml) And A therApeutic index of > 21,818 with noticeAble steric hindrAnce. Results Also showed thAt the optimized protein Ricin A chAin mutAnt-Pokeweed AntivirAl protein isoform 1 from leAves (RTAM-PAP1) could be recovered And purified from soluble lysAtes with gAin of function on protein synthesis inhibition Activity, with An IC50 of 0.03 nM (1.82 ng/ml), And with minimAl, if Any, steric hindrAnce. Conclusions Collectively, our results demonstrAte thAt RTA-PAPs Are AmenAble to effective production And purificAtion in nAtive form, possess significAnt gAin of function on protein synthesis inhibition And Anti-HBV Activities in vitro with A high therApeutic index And, thus, merit further development As potentiAl potent AntivirAl Agents AgAinst chronic HBV infection to be used As A stAndAlone or in combinAtion with existent therApies
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gene cloning And construction of prokAryotic And plAnt expression vectors of Ricin A chAin pAp s1 fusion protein And its inhibition of protein synthesis
bioRxiv, 2016Co-Authors: Yasser Hassan, Sherry OggAbstract:Pokeweed AntivirAl protein (PAP) is A single-chAin ribosome-inActivAting protein thAt exists in severAl forms isolAted from vArious orgAns And At different stAges of development of PhytolAccA AmericAnA (pokeweed). In this study, PAP-S1, one of the two known isoforms found in seeds, wAs isolAted And PCR Amplified using primers bAsed on the known mRNA of PAP-S2, the other known form found in seeds. The complete cDNA encoding PAP-S1 wAs determined here for the first time. PAP-S1 is A potent AntivirAl protein with mAny potentiAl clinicAl ApplicAtions. However, it wAs found to be dosAge dependent with observed side effects At high dosAge. In this study, we report the production of A recombinAnt AntivirAl peptide-fusion protein between Ricin A-chAin And PAP-S1. The peptide-fusion recombinAnt proteins Ricin-A-ChAin/PAP-S1 And PAP-S1/Ricin-A-ChAin were generAted by joining the N-terminus of PAP-S1 to the C-terminus of Ricin A-chAin And the C-terminus of PAP-S1 to the N-terminus of Ricin A-chAin respectively, And were expressed in An EscherichiA coli cell free expression systems. The peptide-fusion recombinAnt protein Ricin-A-ChAin/PAP-S1 (F2) wAs found to be more Active thAn the PAP-S1/Ricin-A-chAin (F1) And similAr to PAP-S1 in A cell free prokAryotic environment, And both showed much stronger Activity in A cell free eukAryotic environment. The DNA sequence of the complete cDNA of PAP-S1 And of the peptide-fusion protein Ricin-A-ChAin/PAP-S1 with the PAP-S1 signAl peptide At the N-terminus of Ricin A-chAin were inserted in plAnt destinAtion binAry vectors for A. tumefAciens mediAted trAnsformAtion. It is the opinion of the Authors thAt AdditionAl reseArch should be done in order to determine both cytotoxicity And selectivity of fusion protein F2 compAred to PAP-S1, As it could be A viAble, more potent And less cytotoxic AlternAtive to PAP-S1 Alone At high dosAge, for both AgriculturAl And therApeutic ApplicAtions.
Yasser Hassan - One of the best experts on this subject based on the ideXlab platform.
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novel binding mechAnisms of fusion broAd rAnge Anti infective protein Ricin A chAin mutAnt pokeweed AntivirAl protein 1 rtAm pAp1 AgAinst sArs cov 2 key proteins in silico
Toxins, 2020Co-Authors: Yasser Hassan, Sherry OggAbstract:The deAdly pAndemic nAmed COVID-19, cAused by A new coronAvirus (SARS-CoV-2), emerged in 2019 And is still spreAding globAlly At A dAngerous pAce. As of todAy, there Are no proven vAccines, therApies, or even strAtegies to fight off this virus. Here, we describe the in silico docking results of A novel broAd rAnge Anti-infective fusion protein RTAM-PAP1 AgAinst the vArious key proteins of SARS-CoV-2 using the lAtest protein-ligAnd docking softwAre. RTAM-PAP1 wAs compAred AgAinst the SARS-CoV-2 B38 Antibody, Ricin A chAin, A pokeweed AntivirAl protein from leAves, And the lectin griffithsin using the speciAl CoDockPP COVID-19 version. These experiments reveAled novel binding mechAnisms of RTAM-PAP1 with A high Affinity to numerous SARS-CoV-2 key proteins. RTAM-PAP1 wAs further chArActerized in A preliminAry toxicity study in mice And wAs found to be A potentiAl therApeutic cAndidAte. These findings might leAd to the discovery of novel SARS-CoV-2 tArgets And therApeutic protein structures with outstAnding functions.
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expression of novel fusion AntivirAl proteins Ricin A chAin pokeweed AntivirAl proteins rtA pAps in escherichiA coli And their inhibition of protein synthesis And of hepAtitis b virus in vitro
BMC Biotechnology, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:Ricin A chAin (RTA) And Pokeweed AntivirAl proteins (PAPs) Are plAnt-derived N-glycosidAse ribosomAl-inActivAting proteins (RIPs) isolAted from Ricinus communis And PhytolAccA AmericAnA respectively. This study wAs to investigAte the potentiAl production AmenAbility And sub-toxic AntivirAl vAlue of novel fusion proteins between RTA And PAPs (RTA-PAPs). In brief, RTA-Pokeweed AntivirAl protein isoform 1 from seeds (RTA-PAPS1) wAs produced in An E. coli in vivo expression system, purified from inclusion bodies using gel filtrAtion chromAtogrAphy And protein synthesis inhibitory Activity AssAyed by compArison to the production of A control protein LuciferAse. The AntivirAl Activity of the RTA-PAPS1 AgAinst HepAtitis B virus (HBV) in HepAD38 cells wAs then determined using A dose response AssAy by quAntifying supernAtAnt HBV DNA compAred to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells wAs determined by meAsuring cell viAbility using A tetrAzolium dye uptAke AssAy. The fusion protein wAs further optimized using in silico tools, produced in An E. coli in vivo expression system, purified by A three-step process from soluble lysAte And confirmed in A protein synthesis inhibition Activity AssAy. Results showed thAt RTA-PAPS1 could effectively be recovered And purified from inclusion bodies. The refolded protein wAs bioActive with A 50% protein synthesis inhibitory concentrAtion (IC50) of 0.06 nM (3.63 ng/ml). The results Also showed thAt RTA-PAPS1 hAd A synergetic Activity AgAinst HBV with A hAlf-mAximAl response concentrAtion vAlue (EC50) of 0.03 nM (1.82 ng/ml) And A therApeutic index of > 21,818 with noticeAble steric hindrAnce. Results Also showed thAt the optimized protein Ricin A chAin mutAnt-Pokeweed AntivirAl protein isoform 1 from leAves (RTAM-PAP1) could be recovered And purified from soluble lysAtes with gAin of function on protein synthesis inhibition Activity, with An IC50 of 0.03 nM (1.82 ng/ml), And with minimAl, if Any, steric hindrAnce. Collectively, our results demonstrAte thAt RTA-PAPs Are AmenAble to effective production And purificAtion in nAtive form, possess significAnt gAin of function on protein synthesis inhibition And Anti-HBV Activities in vitro with A high therApeutic index And, thus, merit further development As potentiAl potent AntivirAl Agents AgAinst chronic HBV infection to be used As A stAndAlone or in combinAtion with existent therApies.
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expression of novel fusion AntivirAl proteins Ricin A chAin pokeweed AntivirAl proteins rtA pAps in escherichiA coli And their inhibition of protein synthesis And of hepAtitis b virus in vitro
bioRxiv, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:Ricin A chAin (RTA) And Pokeweed AntivirAl proteins (PAPs) Are plAnt-derived N-glycosidAse ribosomAl-inActivAting proteins (RIPs) isolAted from Ricinus communis And PhytolAccA AmericAnA respectively. This study wAs to investigAte the potentiAl AntivirAl vAlue of novel fusion proteins between RTA And PAPs (RTA-PAPs). In brief, RTA-PAPS1 wAs produced in E. coli in vivo expression system, purified from inclusion bodies using gel filtrAtion chromAtogrAphy And protein synthesis inhibitory Activity AssAyed by compArison to the production of A control protein LuciferAse. The AntivirAl Activity of the RTA-PAPS1 AgAinst HepAtitis B virus (HBV) in HepAD38 cells wAs then determined using A dose response AssAy by quAntifying supernAtAnt HBV DNA compAred to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells wAs determined by meAsuring cell viAbility using A tetrAzolium dye uptAke AssAy. Results showed thAt RTA-PAPS1 could effectively be recovered And purified from inclusion bodies. The refolded protein wAs bioActive with 50% protein synthesis inhibitory concentrAtion (IC50) of 0.06nM (3.63ng/ml). The results Also showed thAt RTA-PAPS1 hAd A synergetic Activity AgAinst HBV with An IC50 of 0.03nM (1.82ng/ml) And A therApeutic index of >21818. The fusion protein wAs further optimized using in silico tools, produced in E. coli in vivo expression system, purified by three-step process from soluble lysAte And protein synthesis inhibition Activity AssAyed. Results showed thAt the optimized protein (RTAM-PAP1) could be recovered And purified from soluble lysAtes with gAin of function Activity on protein synthesis inhibition with An IC50 of 0.03nM (1.82ng/ml). Collectively, our results demonstrAte thAt RTA-PAPs Are AmenAble to effective production And purificAtion in nAtive form, possess significAnt AntivirAl Activity AgAinst HBV in vitro with A high therApeutic index And, thus, meriting further development As potentiAl AntivirAl Agents AgAinst chronic HBV infections.
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Expression of novel fusion AntivirAl proteins Ricin A chAin-pokeweed AntivirAl proteins (RTA-PAPs) in EscherichiA coli And their inhibition of protein synthesis And of hepAtitis B virus in vitro
BMC, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:AbstrAct BAckground Ricin A chAin (RTA) And Pokeweed AntivirAl proteins (PAPs) Are plAnt-derived N-glycosidAse ribosomAl-inActivAting proteins (RIPs) isolAted from Ricinus communis And PhytolAccA AmericAnA respectively. This study wAs to investigAte the potentiAl production AmenAbility And sub-toxic AntivirAl vAlue of novel fusion proteins between RTA And PAPs (RTA-PAPs). In brief, RTA-Pokeweed AntivirAl protein isoform 1 from seeds (RTA-PAPS1) wAs produced in An E. coli in vivo expression system, purified from inclusion bodies using gel filtrAtion chromAtogrAphy And protein synthesis inhibitory Activity AssAyed by compArison to the production of A control protein LuciferAse. The AntivirAl Activity of the RTA-PAPS1 AgAinst HepAtitis B virus (HBV) in HepAD38 cells wAs then determined using A dose response AssAy by quAntifying supernAtAnt HBV DNA compAred to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells wAs determined by meAsuring cell viAbility using A tetrAzolium dye uptAke AssAy. The fusion protein wAs further optimized using in silico tools, produced in An E. coli in vivo expression system, purified by A three-step process from soluble lysAte And confirmed in A protein synthesis inhibition Activity AssAy. Results Results showed thAt RTA-PAPS1 could effectively be recovered And purified from inclusion bodies. The refolded protein wAs bioActive with A 50% protein synthesis inhibitory concentrAtion (IC50) of 0.06 nM (3.63 ng/ml). The results Also showed thAt RTA-PAPS1 hAd A synergetic Activity AgAinst HBV with A hAlf-mAximAl response concentrAtion vAlue (EC50) of 0.03 nM (1.82 ng/ml) And A therApeutic index of > 21,818 with noticeAble steric hindrAnce. Results Also showed thAt the optimized protein Ricin A chAin mutAnt-Pokeweed AntivirAl protein isoform 1 from leAves (RTAM-PAP1) could be recovered And purified from soluble lysAtes with gAin of function on protein synthesis inhibition Activity, with An IC50 of 0.03 nM (1.82 ng/ml), And with minimAl, if Any, steric hindrAnce. Conclusions Collectively, our results demonstrAte thAt RTA-PAPs Are AmenAble to effective production And purificAtion in nAtive form, possess significAnt gAin of function on protein synthesis inhibition And Anti-HBV Activities in vitro with A high therApeutic index And, thus, merit further development As potentiAl potent AntivirAl Agents AgAinst chronic HBV infection to be used As A stAndAlone or in combinAtion with existent therApies
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gene cloning And construction of prokAryotic And plAnt expression vectors of Ricin A chAin pAp s1 fusion protein And its inhibition of protein synthesis
bioRxiv, 2016Co-Authors: Yasser Hassan, Sherry OggAbstract:Pokeweed AntivirAl protein (PAP) is A single-chAin ribosome-inActivAting protein thAt exists in severAl forms isolAted from vArious orgAns And At different stAges of development of PhytolAccA AmericAnA (pokeweed). In this study, PAP-S1, one of the two known isoforms found in seeds, wAs isolAted And PCR Amplified using primers bAsed on the known mRNA of PAP-S2, the other known form found in seeds. The complete cDNA encoding PAP-S1 wAs determined here for the first time. PAP-S1 is A potent AntivirAl protein with mAny potentiAl clinicAl ApplicAtions. However, it wAs found to be dosAge dependent with observed side effects At high dosAge. In this study, we report the production of A recombinAnt AntivirAl peptide-fusion protein between Ricin A-chAin And PAP-S1. The peptide-fusion recombinAnt proteins Ricin-A-ChAin/PAP-S1 And PAP-S1/Ricin-A-ChAin were generAted by joining the N-terminus of PAP-S1 to the C-terminus of Ricin A-chAin And the C-terminus of PAP-S1 to the N-terminus of Ricin A-chAin respectively, And were expressed in An EscherichiA coli cell free expression systems. The peptide-fusion recombinAnt protein Ricin-A-ChAin/PAP-S1 (F2) wAs found to be more Active thAn the PAP-S1/Ricin-A-chAin (F1) And similAr to PAP-S1 in A cell free prokAryotic environment, And both showed much stronger Activity in A cell free eukAryotic environment. The DNA sequence of the complete cDNA of PAP-S1 And of the peptide-fusion protein Ricin-A-ChAin/PAP-S1 with the PAP-S1 signAl peptide At the N-terminus of Ricin A-chAin were inserted in plAnt destinAtion binAry vectors for A. tumefAciens mediAted trAnsformAtion. It is the opinion of the Authors thAt AdditionAl reseArch should be done in order to determine both cytotoxicity And selectivity of fusion protein F2 compAred to PAP-S1, As it could be A viAble, more potent And less cytotoxic AlternAtive to PAP-S1 Alone At high dosAge, for both AgriculturAl And therApeutic ApplicAtions.
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folding competent And folding defective forms of Ricin A chAin hAve different fAtes After retrotrAnslocAtion from the endoplAsmic reticulum
Molecular Biology of the Cell, 2010Co-Authors: Robert A. Spooner, Michael J Lord, Stuart C H Allen, Christopher P Guise, Graham Ladds, Tina Schnoder, Manfred J Schmitt, Lynne M. RobertsAbstract: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 RTAΔ), 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 RTAΔ, 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.
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Ricin A chAin insertion into endoplAsmic reticulum membrAnes is triggered by A temperAture increAse to 37 c
Journal of Biological Chemistry, 2009Co-Authors: Peter U Mayerhofer, Michael J Lord, Jonathan Cook, Judit Wahlman, Teresa T J Pinheiro, Katherine A H Moore, Arthur E Johnson, Lynne M. RobertsAbstract: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.
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protein disulphide isomerAse reduces Ricin to its A And b chAins in the endoplAsmic reticulum
Biochemical Journal, 2004Co-Authors: Robert A. Spooner, Michael J Lord, Katherine A H Moore, Peter Duncan Watson, Catherine J Marsden, Daniel C Smith, Jonathon P Cook, Lynne M. RobertsAbstract: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.
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Ricin A chAin without its pArtner b chAin is degrAded After retrotrAnslocAtion from the endoplAsmic reticulum to the cytosol in plAnt cells
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Alessandra Di Cola, Michael J Lord, Lorenzo Frigerio, Aldo Ceriotti, Lynne M. RobertsAbstract:When expressed in tobAcco cells, the cAtAlytic subunit of the dimeric ribosome inActivAting protein, Ricin, is first inserted into the endoplAsmic reticulum (ER) And then degrAded in A mAnner thAt cAn be pArtiAlly inhibited by the proteAsome inhibitor clAsto-lActAcystin β-lActone. Consistent with the implicAtion of cytosolic proteAsomes, degrAdAtion of Ricin A chAin is brefeldin A-insensitive And the polypeptides thAt AccumulAte in the presence of the proteAsome inhibitor Are not processed in A vAcuole-specific fAshion. RAther, these stAbilized polypeptides Are in pArt deglycosylAted by A peptide:N-glycAnAse-like Activity. TAken together, these results indicAte thAt Ricin A chAin, Albeit A structurAlly nAtive protein, cAn behAve As A substrAte for ER to cytosol export, deglycosylAtion in the cytosol, And proteAsomAl degrAdAtion. Furthermore, retrotrAnslocAtion of this protein is not tightly coupled to proteAsomAl Activity. These dAtA Are consistent with the hypothesis thAt Ricin A chAin cAn exploit the ER-AssociAted protein degrAdAtion pAthwAy to reAch the cytosol. Although well chArActerized in mAmmAliAn And yeAst cells, the operAtion of A similAr pAthwAy to the cytosol of plAnt cells hAs not previously been demonstrAted.
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Ricin A chAin utilises the endoplAsmic reticulum AssociAted protein degrAdAtion pAthwAy to enter the cytosol of yeAst
FEBS Letters, 1999Co-Authors: Jeremy C Simpson, Lynne M. Roberts, Karin Romisch, John Davey, Dieter H Wolf, Michael J LordAbstract:Cytotoxic proteins such As Ricin A chAin (RTA) hAve tArget substrAtes in the cytosol And therefore hAve to reAch this cellulAr compArtment in order to Act. RTA is thought to trAnslocAte into the cytosol from the lumen of the endoplAsmic reticulum (ER), Although how it trAverses the ER membrAne hAs not been estAblished. Using yeAst mutAnts defective in vArious Aspects of the ER-AssociAted protein degrAdAtion (ERAD) pAthwAy, we show thAt RTA introduced into the yeAst ER subverts this pAthwAy to enter the cytosol viA the Sec61p trAnslocon. A significAnt proportion of the exported RTA Avoided proteAsomAl degrAdAtion. These dAtA Are consistent with the contention thAt the RTA component from Ricin endocytosed by mAmmAliAn cells mAy likewise exploit ERAD to trAnslocAte into the cytosol.
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Ricin A ChAin CAn TrAnsport Unfolded DihydrofolAte ReductAse into the Cytosol
Journal of Biological Chemistry, 1997Co-Authors: Bruno Beaumelle, J. Michael Lord, Marie-pierre Taupiac, Lynne RobertsAbstract:Ricin is A heterodimeric protein toxin. The Ricin A chAin is Able to cross the membrAne of intrAcellulAr compArtments to reAch the cytosol where it cAtAlyticAlly inActivAtes protein synthesis. It is linked viA A disulfide bond to the B chAin, A gAlActose-specific lectin, which Allows Ricin binding At the cell surfAce And endocytosis. To exAmine the potentiAl of Ricin A to cArry proteins into the cytosol And the requirement for unfolding of the pAssenger protein, we connected mouse dihydrofolAte reductAse (DHFR) to Ricin A by gene fusion viA A spAcer peptide. DHFR-Ricin A expressed in EscherichiA coli displAyed the biologicAl Activities of the pArent proteins And AssociAted quAntitAtively with Ricin B to form DHFR-Ricin. The resulting toxin wAs highly cytotoxic to cells (4-8-fold less thAn recombinAnt Ricin). DHFR-Ricin cytotoxicity wAs inhibited by methotrexAte, A DHFR inhibitor stAbilizing DHFR-Ricin A in A folded conformAtion. The DHFR moiety of DHFR Ricin bound to the plAsmA membrAne. Although methotrexAte prevented this binding, it did not significAntly Affect DHFR-Ricin endocytosis, which proceeded viA Ricin B chAin. IntoxicAtion kinetics dAtA And A cell-free trAnslocAtion AssAy demonstrAted thAt protection of cells from DHFR-Ricin cytotoxicity resulted from A selective inhibition by methotrexAte of DHFR-Ricin A trAnslocAtion. We conclude thAt Ricin A is A potentiAl cArrier of proteins to the cytosol, provided thAt the pAssenger protein is Able to unfold for trAnsmembrAne trAnsport.
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CorrelAtion between the Activities of five ribosome‐inActivAting proteins in depurinAtion of tobAcco ribosomes And inhibition of tobAcco mosAic virus infection
Plant Journal, 1994Co-Authors: Sally Taylor, Andrea J Massiah, J. Michael Lord, George P Lomonossoff, Lynne M. Roberts, Martin R HartleyAbstract:SummAry 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.
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Expression of immunoglobulin heAvy chAin-Ricin A chAin fusions in mAmmAliAn cells
Molecular Immunology, 1994Co-Authors: Robert A. Spooner, Deborah J. Allen, Agamemnon A. Epenetos, J. Michael LordAbstract:MAmmAliAn cell lines were trAnsfected with Antibody heAvy (H) chAin-Ricin A chAin gene fusions in Attempts to Assemble A recombinAnt immunotoxin. We found thAt A light chAin-secreting mouse plAsmAcytomA cell line cAn be trAnsfected stAbly with such A chimAeric gene, but only if the Ricin A chAin portion is disArmed by genetic meAns prior to trAnsfection; if not, stAble trAnsfection AppeArs to select for genetic inActivAtion of the trAnsfected gene. Co-expression of An Antibody heAvy chAin-Ricin A chAin fusion with light chAin in non-lymphoid cells results in cell deAth. We conclude thAt the Ricin A chAin moiety retAins biologicAl Activity precluding the expression of biologicAlly Active Antibody-Ricin A chAin fusion proteins in mAmmAliAn cells.
