Reductive Activation of Type 2 Ribosome-inactivating Proteins Is Promoted by Transmembrane Thioredoxin-related Protein

Pasetto, Matteo; Barison, Erika; Castagna, Monica; Cristina, Pietro Della; Anselmi, Cristina; Colombatti, Marco

doi:10.1074/jbc.m111.316828

Cited by 20 publications

(21 citation statements)

References 19 publications

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“…In the case of ricin, PDI is sufficient to reduce the holotoxin into RTA and RTB, as well as promote the reassociation of the toxin subunits (Spooner et al, 2004). Another PDI family member known as thioredoxin-like transmembrane protein (TMX) has just recently been shown to also play a role in the reductive activation of ricin (Pasetto et al, 2012). …”

Section: Discussionmentioning

confidence: 99%

Neutralizing monoclonal antibodies against ricin's enzymatic subunit interfere with protein disulfide isomerase-mediated reduction of ricin holotoxin in vitro

O’Hara

Mantis

2013

Journal of Immunological Methods

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The penultimate event in the intoxication of mammalian cells by ricin toxin is the reduction, in the endoplasmic reticulum (ER), of the intermolecular disulfide bond that links ricin’s enzymatic (RTA) and binding (RTB) subunits. In this report we adapted an in vitro protein disulfide isomerase (PDI)-mediated reduction assay to test the hypothesis that the RTA-specific neutralizing monoclonal antibody (mAb) IB2 interferes with the liberation of RTA from RTB. IB2 recognizes an epitope located near the interface between RTA and RTB and, like a number of other RTA-specific neutralizing mAbs, is proposed to neutralize ricin intracellularly. In this study, we found that IB2 virtually eliminated the reduction of ricin holotoxin into RTA and RTB in vitro. Surprisingly, three other neutralizing mAbs (GD12, R70 and SyH7) that bind epitopes at considerable distance from ricin’s disulfide bond were as effective (or nearly as effective) as IB2 in interfering with PDI-mediated liberation of RTA from RTB. By contrast, two non-neutralizing RTA-specific mAbs, FGA12 and SB1, did not affect PDI-mediated reduction of ricin. These data reveal a possible mechanism by which RTA-specific antibodies may neutralize ricin intracellularly, provided they are capable of trafficking in association with ricin from the cell surface to the ER.

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Section: Discussionmentioning

confidence: 99%

Neutralizing monoclonal antibodies against ricin's enzymatic subunit interfere with protein disulfide isomerase-mediated reduction of ricin holotoxin in vitro

O’Hara

Mantis

2013

Journal of Immunological Methods

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“…Interaction of ricin with the ER-luminal chaperone protein disulfide isomerase (PDI) is thought to remodel the holotoxin structure to open the interface between RTA and RTB, thus permitting reductive cleavage [ 2 , 32 , 33 ]. Since PDI selects its substrates by binding permanently or transiently unfolded regions [ 34 ], and ricin holotoxin purified from seeds is thermally stable in vitro [ 35 ], then this suggests that ricin is either transiently unstable under ER conditions, or is destabilized in transit.…”

Section: Er Luminal Eventsmentioning

confidence: 99%

Ricin Trafficking in Cells

Spooner

Lord

2015

Toxins

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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.

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“…For example, intra‐ and intermolecular disulfide bonds in ERAD substrates as well as interchain disulfides linking the regulatory and catalytic subunits of bacterial toxins are reduced with the intervention of PDI and/or PDI family members (e.g. ERdj5 , ERp57 , ERp72, ERp29 , TMX1 , ERFAD‐ERp90 and ERO1 ).…”

Section: Specificity Of the Erad Machinerymentioning

confidence: 99%

Specificity and Regulation of the Endoplasmic Reticulum‐Associated Degradation Machinery

Merulla

Fasana

Soldà

et al. 2013

Traffic

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The endoplasmic reticulum-associated degradation (ERAD) machinery selects native and misfolded polypeptides for dislocation across the ER membrane and proteasomal degradation. Regulated degradation of native proteins is an important aspect of cell physiology. For example, it contributes to the control of lipid biosynthesis, calcium homeostasis and ERAD capacity by setting the turnover rate of crucial regulators of these pathways. In contrast, degradation of native proteins has pathologic relevance when caused by viral or bacterial infections, or when it occurs as a consequence of dysregulated ERAD activity. The efficient disposal of misfolded proteins prevents toxic depositions and persistent sequestration of molecular chaperones that could induce cellular stress and perturb maintenance of cellular proteostasis. In the first section of this review, we survey the available literature on mechanisms of selection of native and non-native proteins for degradation from the ER and on how pathogens hijack them. In the second section, we highlight the mechanisms of ERAD activity adaptation to changes in the ER environment with a particular emphasis on the post-translational regulatory mechanisms collectively defined as ERAD tuning. The cellular proteome is mostly synthesized by cytosolic ribosomes to operate in the cytosol and, upon appropriate targeting, in various intracellular organelles or in the extracellular space. Cellular compartments where protein folding occurs [e.g. the cytosol, mitochondria, the endoplasmic reticulum (ER)] contain two classes of non-native polypeptides: (i) newly synthesized polypeptide chains that must be assisted by folding chaperones and enzymes to attain the native mono-or oligomeric structure; (ii) terminally misfolded conformers that must efficiently be degraded to prevent the formation of toxic deposits and the persistent sequestration of chaperones that could eventually inhibit the cellular protein folding capacity and elicit stress (1-3) ( Figure 1A). The distinction between the two classes of non-native chains, one to be preserved, the second to be cleared from the folding compartment, is not an easy task for the cellular quality control machineries. Selection for disposal might be a stochastic process: the longer the persistency of structural defects in the ER, the greater is the probability to be selected for destruction. Therefore, mutations that delay folding may channel the polypeptide into destructive pathways, even if they do not compromise the function of the mutated protein.In the ER, non-native, but also native proteins might be selected for degradation by components of the endoplasmic reticulum-associated degradation (ERAD) machinery that deliver them at dislocation sites embedded in the ER membrane. Dislocation sites consist of a multitude of luminal and membrane-bound specialized ER-resident proteins as well as a number of cytosolic factors insuring dislocation across the ER membrane, poly-ubiquitylation and disposal of ERAD substrates by 26S proteasomes (Specificity of...

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Reductive Activation of Type 2 Ribosome-inactivating Proteins Is Promoted by Transmembrane Thioredoxin-related Protein

Cited by 20 publications

References 19 publications

Neutralizing monoclonal antibodies against ricin's enzymatic subunit interfere with protein disulfide isomerase-mediated reduction of ricin holotoxin in vitro

Neutralizing monoclonal antibodies against ricin's enzymatic subunit interfere with protein disulfide isomerase-mediated reduction of ricin holotoxin in vitro

Ricin Trafficking in Cells

Specificity and Regulation of the Endoplasmic Reticulum‐Associated Degradation Machinery

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