PMC:4157140 / 14682-17116
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4157140","sourcedb":"PMC","sourceid":"4157140","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4157140","text":"In order to study the effect of the protein in a cellular system, we attempted to examine APOPT1-HA in HeLa and fibroblast cell lines, by transducing a recombinant lentiviral expression construct that requires puromycin as a selectable marker.17 Although we detected high levels of recombinant APOPT1-HA transcript after selection (Figure S7A), hardly any protein was immunovisualized by immunoblot or immunofluorescence in either transduced cell line. To test whether this result was due to selective APOPT1-HA-induced cell death, we used a Tet on-off inducible vector, expressing the APOPT1-HA transcript under exposure to increasing concentrations of doxycycline. However, we were unable to detect the protein in doxycycline-treated cells expressing high levels of the APOPT1-HA transcript (Figure S7B) and failed to observe increased cell death in induced compared to control cells. Taken together, these results indicate that the APOPT1-HA cDNA is expressed transcriptionally, but the corresponding protein product is rapidly degraded by a surveillance system active in standard culturing conditions. To further explore this hypothesis, immortalized fibroblasts from either individual S2 or a control subject, stably transduced with the APOPT1-HA lentiviral vector, were treated with MG-132 (5 μM for 24 hr), a proteasome inhibitor.18 HA-immunoreactive bands corresponding to the precursor and mature APOPT1-HA species were clearly present in both MG-132-treated cell lines, in contrast with the absence of HA-immunoreactive band in the same cell lines under naive, untreated conditions (Figures 4A and 4B). These results strongly suggest that APOPT1 precursor protein is degraded by the proteasome system in standard culture conditions. Next, we tested whether the levels of the APOPT1 protein responded to oxidative19 or apoptogenic20 challenges. We exposed the same transduced cell lines to increasing concentrations of H2O2 (100 μM–1 mM) or to a standard concentration of staurosporine (1 μM), an inducer of apoptosis. Under conditions of oxidative stress (H2O2 treatment), APOPT1-HA protein increased to immunodetectable levels, with a maximum at 24 hr (Figure 4C); no protein was detected following treatment with staurosporine (data not shown). In contrast to the effect of MG-132, exposure to H2O2 determined the predominant accumulation of the mature, intramitochondrial, presumably active APOPT1-HA species (Figure 4C).","tracks":[{"project":"2_test","denotations":[{"id":"25175347-23993194-2048454","span":{"begin":243,"end":245},"obj":"23993194"},{"id":"25175347-19513526-2048455","span":{"begin":1337,"end":1339},"obj":"19513526"},{"id":"25175347-7843283-2048456","span":{"begin":1822,"end":1824},"obj":"7843283"},{"id":"25175347-18771761-2048457","span":{"begin":1839,"end":1841},"obj":"18771761"}],"attributes":[{"subj":"25175347-23993194-2048454","pred":"source","obj":"2_test"},{"subj":"25175347-19513526-2048455","pred":"source","obj":"2_test"},{"subj":"25175347-7843283-2048456","pred":"source","obj":"2_test"},{"subj":"25175347-18771761-2048457","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#d9ec93","default":true}]}]}}