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The role of the A1 subunit in the activity of shiga toxins
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Basu, Debaleena. The role of the A1 subunit in the activity of shiga toxins. Retrieved from https://doi.org/doi:10.7282/T3VH5R4T

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TitleThe role of the A1 subunit in the activity of shiga toxins
NameBasu, Debaleena (author); Tumer, Nilgun (chair); White, James (internal member); Belanger, Faith (internal member); Woychik, Nancy (outside member); Rutgers University; Graduate School - New Brunswick
Date Created2016
Other Date2016-10 (degree)
SubjectPlant Biology, Escherichia coli, Toxins
Extent1 online resource (xiii, 179 p. : ill.)
DescriptionShiga toxin (Stx) producing E.coli infections can lead to life-threatening complications, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit and a pentamer of receptor binding B subunits. Stx2-producing E.coli strains are more frequently associated with HUS than Stx1-producing strains. The role of the A subunits in the increased potency of Stx2 has not been fully investigated. This study using purified A1 subunits, provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity towards the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation and exhibit cytotoxicity at a significantly higher level than Stx1A1 in yeast and human cells. To determine if conserved arginines at the distal face of the active site contribute to the higher affinity of Stx2A1 for the ribosome, Arg172, Arg176 and Arg179 in Stx1A1 and Stx2A1 are mutated. It is seen that Arg172 and Arg176 are more important than Arg179 for depurination activity and toxicity of Stx1A1 and Stx2A1. Mutation of at least two of the three arginines is required to significantly reduce depurination by Stx2A1 in vitro and in cells in yeast and mammalian cells. Conserved arginines at the distal face of the active site are critical for interactions of Stx1A1 and Stx2A1 with the stalk, while a conserved arginine at the active site is critical for non-stalk specific interactions with the ribosome. Mutations at conserved arginines at either site reduced ribosome interactions of Stx1A1 and Stx2A1 similarly, indicating that they do not contribute to the higher affinity of Stx2A1 for the ribosome. Interchanging residues E60 and 61 in Stx1A1 and Y60 and Q61 in Stx2A1 that are located away from the active site and ribosome stalk binding site, resulted in small but significant increase in depurination level of E60Y/E61Q for Stx1A1 and a decrease in depurination level of Y60E/Q61E of Stx2A1 in vivo in yeast. A larger difference may be observed if more than two residues are simultaneously altered to change the electrostatic charge distribution of Stx1A1 and Stx2A1 sufficiently.
NotePh.D.
NoteIncludes bibliographical references
Noteby Debaleena Basu
Genretheses, ETD doctoral
Persistent URLhttps://doi.org/doi:10.7282/T3VH5R4T
Languageeng
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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