The RNA-N-glycosidase activity of Shiga-like toxin I: Kinetic parameters of the native and activated toxin

Brigotti, Maurizio; Carnicelli, Domenica; Alvergna, Paola; Mazzaracchio, R; Sperti, S; Montanaro, Lucio

doi:10.1016/s0041-0101(96)00225-5

Cited by 27 publications

(21 citation statements)

References 20 publications

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“…However, after trypsin and TCEP treatment to release the A1 subunits, Stx1A1 and Stx2A1 were able to depurinate the yeast ribosome at 374-fold and 92-fold higher levels than Stx1 and Stx2 holotoxins, respectively, at 0.5 µM ribosome concentration (Figure 6a). A previous study examined the k cat and K m of Stx1 holotoxin on Artemia salina ribosomes before and after treatment with trypsin, urea and DTT [33]. Our results are consistent with this study in the requirement for activation, although this study used a different assay, different source of ribosomes, different method of activation, and much higher toxin concentrations [33].…”

Section: Resultssupporting

confidence: 89%

“…A previous study examined the k cat and K m of Stx1 holotoxin on Artemia salina ribosomes before and after treatment with trypsin, urea and DTT [33]. Our results are consistent with this study in the requirement for activation, although this study used a different assay, different source of ribosomes, different method of activation, and much higher toxin concentrations [33]. …”

Section: Resultssupporting

confidence: 89%

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Differences in Ribosome Binding and Sarcin/Ricin Loop Depurination by Shiga and Ricin Holotoxins

Tumer

2017

Toxins

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Both ricin and Shiga holotoxins display no ribosomal activity in their native forms and need to be activated to inhibit translation in a cell-free translation inhibition assay. This is because the ribosome binding site of the ricin A chain (RTA) is blocked by the B subunit in ricin holotoxin. However, it is not clear why Shiga toxin 1 (Stx1) or Shiga toxin 2 (Stx2) holotoxin is not active in a cell-free system. Here, we compare the ribosome binding and depurination activity of Stx1 and Stx2 holotoxins with the A1 subunits of Stx1 and Stx2 using either the ribosome or a 10-mer RNA mimic of the sarcin/ricin loop as substrates. Our results demonstrate that the active sites of Stx1 and Stx2 holotoxins are blocked by the A2 chain and the B subunit, while the ribosome binding sites are exposed to the solvent. Unlike ricin, which is enzymatically active, but cannot interact with the ribosome, Stx1 and Stx2 holotoxins are enzymatically inactive but can interact with the ribosome.

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Section: Resultssupporting

confidence: 89%

Section: Resultssupporting

confidence: 89%

Differences in Ribosome Binding and Sarcin/Ricin Loop Depurination by Shiga and Ricin Holotoxins

Tumer

2017

Toxins

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“…In previous studies, differences were not observed for the protein synthesis inhibition abilities of Stx1 and Stx2 by using cell-free translation assays (23,24,52). While in some of these studies, the holotoxin was used (24), in others, the holotoxin was activated by digestion with trypsin to release the A1 chain from the A2-B5 complex or by chemical treatment with urea or DTT to break the disulfide bond between the A1 and A2 chains (23,24,53,54).…”

Section: Discussionmentioning

confidence: 88%

The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1

Basu

Kahn

et al. 2016

Infect Immun

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Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae. Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1. Shiga toxin-producing Escherichia coli (STEC) is an emerging foodborne and waterborne pathogen. STEC infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), with potentially lethal consequences (1). Due to a very low infectious dose and ease of person-to-person spread, STEC infection is the leading cause of death from foodborne bacterial infection in children (2). Presently, there are no postexposure therapeutics or vaccines available for STEC infection. Due to recent outbreaks of E. coli O157:H7 in the United States and the emergence of highly virulent new strains, such as E. coli O104:H4, which caused the deadliest HUS outbreak in Germany in 2011, STEC remains a major challenge for food safety and public health (3-6).The primary virulence factors of STEC, Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), are AB 5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits and are known as type II ribosome-inactivating proteins (RIPs). The A subunits of Stx1 and Stx2 consist of the catalytically active A1 (residues 1 to 251 in Stx1 and 1 to 250 in Stx2) and A2 (residues 252 to 293 in Stx1 and 251 to 297 in Stx2) chains, which are cleaved by the protease furin and kept together by a disulfide bond (7). The B subunits bind ...

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“…Both A chain fragments remain linked by a single disulfide bond which is thought to be reduced in the ER lumen [8], [9], [10]. The A 1 domain is then retrotranslocated to the cytosol by virtue of its newly exposed hydrophobic C-terminus, where it eventually docks onto ribosomes and subsequently depurinates a single adenine base (A 4324 ) in the sarcin-ricin loop (SRL) of 28S rRNA [11], [12], [13], [14], [15]. This depurination event creates an apurinic site that prevents elongation factor 1 (EF-1)-dependent amino-acyl tRNA from binding to the ribosome and EF-2-catalysed translocation during elongation, leading to an inhibition of protein synthesis [16], [17], [18].…”

Section: Introductionmentioning

confidence: 99%

Charged and Hydrophobic Surfaces on the A Chain of Shiga-Like Toxin 1 Recognize the C-Terminal Domain of Ribosomal Stalk Proteins

et al. 2012

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Shiga-like toxins are ribosome-inactivating proteins (RIP) produced by pathogenic E. coli strains that are responsible for hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A1 chain of Shiga-like toxin 1 (SLT-1), a representative RIP, first docks onto a conserved peptide SD[D/E]DMGFGLFD located at the C-terminus of all three eukaryotic ribosomal stalk proteins and halts protein synthesis through the depurination of an adenine base in the sarcin-ricin loop of 28S rRNA. Here, we report that the A1 chain of SLT-1 rapidly binds to and dissociates from the C-terminal peptide with a monomeric dissociation constant of 13 µM. An alanine scan performed on the conserved peptide revealed that the SLT-1 A1 chain interacts with the anionic tripeptide DDD and the hydrophobic tetrapeptide motif FGLF within its sequence. Based on these 2 peptide motifs, SLT-1 A1 variants were generated that displayed decreased affinities for the stalk protein C-terminus and also correlated with reduced ribosome-inactivating activities in relation to the wild-type A1 chain. The toxin-peptide interaction and subsequent toxicity were shown to be mediated by cationic and hydrophobic docking surfaces on the SLT-1 catalytic domain. These docking surfaces are located on the opposite face of the catalytic cleft and suggest that the docking of the A1 chain to SDDDMGFGLFD may reorient its catalytic domain to face its RNA substrate. More importantly, both the delineated A1 chain ribosomal docking surfaces and the ribosomal peptide itself represent a target and a scaffold, respectively, for the design of generic inhibitors to block the action of RIPs.

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The RNA-N-glycosidase activity of Shiga-like toxin I: Kinetic parameters of the native and activated toxin

Cited by 27 publications

References 20 publications

Differences in Ribosome Binding and Sarcin/Ricin Loop Depurination by Shiga and Ricin Holotoxins

Differences in Ribosome Binding and Sarcin/Ricin Loop Depurination by Shiga and Ricin Holotoxins

The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1

Charged and Hydrophobic Surfaces on the A Chain of Shiga-Like Toxin 1 Recognize the C-Terminal Domain of Ribosomal Stalk Proteins

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