scholarly journals Attenuated diphtheria toxin mediates siRNA delivery

2020 ◽  
Vol 6 (18) ◽  
pp. eaaz4848 ◽  
Author(s):  
Amy E. Arnold ◽  
Laura J. Smith ◽  
Greg Beilhartz ◽  
Laura C. Bahlmann ◽  
Emma Jameson ◽  
...  
Keyword(s):  
Diphtheria Toxin ◽  
Sirna Delivery ◽  
Significant Inhibition ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Survival Gene ◽  
First Time ◽  
Translation Initiation Factor 3

Toxins efficiently deliver cargo to cells by binding to cell surface ligands, initiating endocytosis, and escaping the endolysosomal pathway into the cytoplasm. We took advantage of this delivery pathway by conjugating an attenuated diphtheria toxin to siRNA, thereby achieving gene downregulation in patient-derived glioblastoma cells. We delivered siRNA against integrin-β1 (ITGB1)—a gene that promotes invasion and metastasis—and siRNA against eukaryotic translation initiation factor 3 subunit b (eIF-3b)—a survival gene. We demonstrated mRNA downregulation of both genes and the corresponding functional outcomes: knockdown of ITGB1 led to a significant inhibition of invasion, shown with an innovative 3D hydrogel model; and knockdown of eIF-3b resulted in significant cell death. This is the first example of diphtheria toxin being used to deliver siRNAs, and the first time a toxin-based siRNA delivery strategy has been shown to induce relevant genotypic and phenotypic effects in cancer cells.

scholarly journals Activation of AP-1-Dependent Transcription by a Truncated Translation Initiation Factor

2005 ◽  
Vol 4 (11) ◽  
pp. 1840-1850 ◽  
Author(s):  
Caroline C. L. Jenkins ◽  
Juan Mata ◽  
Richard F. Crane ◽  
Benjamin Thomas ◽  
Alexandre Akoulitchev ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Regulatory Sequences ◽  
Eukaryotic Translation ◽  
Hybridization Data ◽  
Eukaryotic Translation Initiation ◽  
First Time ◽  
Translation Initiation Factor 3

ABSTRACT Int6/eIF3e is a highly conserved subunit of eukaryotic translation initiation factor 3 (eIF3) that has also been reported to interact with subunits of the proteasome and the COP9 signalosome. Overexpression of full-length Int6 or a 13-kDa C-terminal fragment, Int6CT, in the fission yeast Schizosaccharomyces pombe causes multidrug resistance that requires the otherwise inessential AP-1 transcription factor Pap1. Here we show for the first time that Int6CT acts to increase the transcriptional activity of Pap1. Microarray hybridization data indicate that Int6CT overexpression resulted in the up-regulation of 67 genes; this expression profile closely matched that of cells overexpressing Pap1. Analysis of the upstream regulatory sequences of these genes showed that the majority contained AP-1 consensus binding sites. Partial defects in ubiquitin-dependent proteolysis have been suggested to confer Pap1-dependent multidrug resistance, but no such defect was seen on Int6CT overexpression. Indeed, none of the previously identified interactions of endogenous Int6 was required for the activation of Pap1 transcription described here. Moreover, Int6CT-induced activation of Pap1-responsive gene expression was independent of the ability of Pap1 to undergo a redox-regulated conformational change which mediates its relocalization to the nucleus and expression of oxidative stress response genes. Int6CT therefore activates Pap1-dependent transcription by a novel mechanism.

scholarly journals Dual function of Rpn5 in two PCI complexes, the 26S proteasome and COP9 signalosome

2011 ◽  
Vol 22 (7) ◽  
pp. 911-920 ◽  
Author(s):  
Zanlin Yu ◽  
Oded Kleifeld ◽  
Avigail Lande-Atir ◽  
Maisa Bsoul ◽  
Maya Kleiman ◽  
...  
Keyword(s):  
Translation Initiation Factor ◽  
26S Proteasome ◽  
Initiation Factor ◽  
Cop9 Signalosome ◽  
Dual Function ◽  
Dual Roles ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Bona Fide ◽  
Translation Initiation Factor 3

Subunit composition and architectural structure of the 26S proteasome lid is strictly conserved between all eukaryotes. This eight-subunit complex bears high similarity to the eukaryotic translation initiation factor 3 and to the COP9 signalosome (CSN), which together define the proteasome CSN/COP9/initiation factor (PCI) troika. In some unicellular eukaryotes, the latter two complexes lack key subunits, encouraging questions about the conservation of their structural design. Here we demonstrate that, in Saccharomyces cerevisiae, Rpn5 plays dual roles by stabilizing proteasome and CSN structures independently. Proteasome and CSN complexes are easily dissected, with Rpn5 the only subunit in common. Together with Rpn5, we identified a total of six bona fide subunits at roughly stoichiometric ratios in isolated, affinity-purified CSN. Moreover, the copy of Rpn5 associated with the CSN is required for enzymatic hydrolysis of Rub1/Nedd8 conjugated to cullins. We propose that multitasking by a single subunit, Rpn5 in this case, allows it to function in different complexes simultaneously. These observations demonstrate that functional substitution of subunits by paralogues is feasible, implying that the canonical composition of the three PCI complexes in S. cerevisiae is more robust than hitherto appreciated.

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