RIP30A - Protein synthesis inhibitor II - Hordeum …
Inhibits the elongation phase of protein synthesis
To investigate if apoptosis was induced in the transfected cells, activation of caspase 3/7 was investigated in MAC-T cells 19 h after transfection (). Caspase activity was induced similarly by pre- and mature WT RTA (2.7 ± 0.2 and 2.8 ± 0.2-fold, respectively; mean ± SE of 9 experiments) relative to vector controls. The active site mutant E177K elicited negligible caspase activation which corresponded with the lack of ribosome depurination and protein synthesis inhibition. In contrast the pre- and mature forms of E145K, G212E and P95L/E145K as well as mature S215F activated caspase activity to the same degree as their respective WT RTA controls. PreS215F activated caspase 3/7 to a lesser extent than WT preRTA, which corresponded with decreases in depurination and protein synthesis inhibition. The E177Q mutant also showed a difference between the pre- and mature forms in terms of caspase activation, with mature E177Q eliciting a greater increase in caspase 3/7 activity relative to preE177Q. Interestingly, both the pre- and mature forms of P95L activated caspase to a greater extent relative to their WT counterparts.
Protein synthesis inhibitor : Wikis (The Full Wiki)
A GFP transfection assay was used to determine if protein synthesis inhibition corresponded with changes in ribosome depurination. Overall, the pattern of ribosome depurination observed with the mutated RTA proteins was reflected in the degree of protein synthesis inhibition (). Fluorescence was almost nondetectable in cells transfected with pre- or mature WT RTA relative to cells transfected with plasmid vector alone. Similar results were obtained with P95L, which depurinated ribosomes similarly to WT RTA based on qRT-PCR results. Pre- and mature E177K did not inhibit protein synthesis which corresponded with the very slight degree of depurination observed. Protein synthesis levels observed with pre- and mature E177Q were intermediate between E177K and the other mutants. Surprisingly, a greater level of inhibition of protein synthesis was observed with mature E177Q (60% inhibition of vector controls) compared to preE177Q (40% inhibition of vector controls) even though they showed similar levels of depurination at 19 h after transfection. Protein synthesis was inhibited significantly less relative to WT RTA for preP95L/E145K and for pre- and mature S215F and G212E, however, this still represented an 80 to 90% inhibition of protein synthesis relative to vector controls. Interestingly, the mature RTA mutants tended to have a greater effect on protein synthesis inhibition than their preRTA counterparts. Specifically, preE177Q, P95L/E145K, S215F and G212E inhibited protein synthesis less than their corresponding mature forms and less than WT preRTA.