PubMed · 7803547
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J García Tamayo. 1994. [Cell death].. https://pubmed.ncbi.nlm.nih.gov/7803547/
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Transcription activator-like effectors (TALEs) in plant-pathogenic Xanthomonas bacteria activate expression of plant genes and support infection or cause a resistance response. PthA4AT is a TALE with a particularly short DNA-binding domain harboring only 7.5 repeats which triggers cell death in Nicotiana benthamiana; however, the genetic basis for this remains unknown. To identify possible target genes of PthA4AT that mediate cell death in N. benthamiana, we exploited the modularity of TALEs to stepwise enhance their specificity and reduce potential target sites. Substitutions of individual repeats suggested that PthA4AT-dependent cell death is sequence specific. Stepwise addition of repeats to the C-terminal or N-terminal end of the repeat region narrowed the sequence requirements in promoters of target genes. Transcriptome profiling and in silico target prediction allowed the isolation of two cell death inducer genes, which encode a patatin-like protein and a bifunctional monodehydroascorbate reductase/carbonic anhydrase protein. These two proteins are not linked to known TALE-dependent resistance genes. Our results show that the aberrant expression of different endogenous plant genes can cause a cell death reaction, which supports the hypothesis that TALE-dependent executor resistance genes can originate from various plant processes. Our strategy further demonstrates the use of TALEs to scan genomes for genes triggering cell death and other relevant phenotypes.
It has been proposed that unconjugated type I ribosome-inactivating proteins (RIP) enter cells through passive mechanisms such as fluid-phase pinocytosis. However, some observations, such as the difference in sensitivity to type I RIP among different cell types, and the organ-specific toxicity of type I RIP, indicate a specific mechanism for the entry of these proteins into target cells. The alpha 2-macroglobulin receptor (alpha 2MR) is responsible for the binding and endocytosis of several ligands, including alpha 2-macroglobulin/proteinase complexes, plasminogen-activator-inhibitor complexes, apoE-enriched beta-very low density lipoproteins, and lipoprotein lipase. Here we demonstrate that saporin, a potent type I RIP, binds specifically to purified alpha 2MR and the binding is prevented by some alpha 2MR ligands. Moreover, the occupancy of specific ligand-binding sites on cell surface alpha 2MR decreases the cytotoxicity of saporin. The A chain of ricin, a type II RIP, also interacts with alpha 2MR. This, and the fact that saporin and ricin A chain both interact also with alpha 2-macroglobulin, indicates a general mechanism of complex interactions between RIP and cellular membranes that is mediated by alpha 2-macroglobulin and the alpha 2MR system.
One of the mechanisms in which anticancer therapies function is to induce cell death by apoptosis. In this regard, the biological activity of p53 may be critical since the presence of p53 appears to play a role in apoptosis following genotoxic stress. In experimental systems using in vitro transformed primary cells, there is a direct correlation between the presence of p53 and apoptosis. For example, E1A/ras-transformed primary mouse fibroblasts are less viable and undergo apoptosis following genotoxic stress if these cells contain a wild type p53. In comparison, similarly transformed cells which are p53-deficient are more viable and will not undergo apoptosis under these conditions. Although these are important observations, it remains to be established whether there exists a similar relationship between the presence of wild type p53 and loss of cell viability following therapy in human tumour cells. One way to address this question is to target wild type p53 in human tumour cells using human papillomavirus E6 which mediates the degradation of wild type p53 through the ubiquitin pathway. In this manner, E6 engineered p53-deficient and parental p53-containing human tumour cells provides an appropriate experimental system in which to determine whether wild type p53 in tumour cells has influence on cell viability following genotoxic anticancer treatments. In the present study, the wild type p53 protein in human fibrosarcoma HT1080 cells were targeted with HPV-18 E6 and the viability of these cells in response to treatment with adriamycin, u.v.-irradiation and gamma-irradiation was examined. Data is presented which shows that p53-containing and p53-deficient cells were equally sensitive to these treatments. These data argue that the wild type p53 in these tumour cells does not cause these cells to be less viable when treated with anticancer agents or u.v.-irradiation. Therefore, the status of p53 alone in tumour cells may not be an indicator of response to anticancer treatments.