RESUMO
Co-translational transport of polypeptides into the endoplasmic reticulum (ER) involves the Sec61 channel and additional components such as the ER lumenal Hsp70 BiP and its membrane-resident co-chaperone Sec63p in yeast. We investigated whether silencing the SEC61A1 gene in human cells affects co- and post-translational transport of presecretory proteins into the ER and post-translational membrane integration of tail-anchored proteins. Although silencing the SEC61A1 gene in HeLa cells inhibited co- and post-translational transport of signal-peptide-containing precursor proteins into the ER of semi-permeabilized cells, silencing the SEC61A1 gene did not affect transport of various types of tail-anchored protein. Furthermore, we demonstrated, with a similar knockdown approach, a precursor-specific involvement of mammalian Sec63 in the initial phase of co-translational protein transport into the ER. By contrast, silencing the SEC62 gene inhibited only post-translational transport of a signal-peptide-containing precursor protein.
Assuntos
DNA Helicases/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos/metabolismo , Animais , DNA Helicases/genética , Retículo Endoplasmático/genética , Inativação Gênica , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Camundongos , Chaperonas Moleculares , Células NIH 3T3 , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas de Ligação a RNA , Canais de Translocação SECRESUMO
BACKGROUND: We previously reported that over-expression of the SEC62 gene is a widespread phenomenon in prostate cancer. Since the use of endoplasmic reticulum (ER) stress-inducing substances such as thapsigargin in prostate cancer therapy is widely discussed in the literature, we investigated the influence of Sec62 protein content on the cellular response to these drugs. METHODS: Growth effects were analyzed by real-time cell analysis and viability tests in DU145-cells representing an increased SEC62 expression or PC3- and LNCaP-cells representing a similar SEC62 expression compared to non-tumor cells. Ca(2+) -imaging in an established HeLa-system with fluorescent dye was used to study molecular effects of Sec62 depletion. RESULTS: We found a lower propensity toward apoptotic cell death after thapsigargin treatment for DU145 cells compared to PC3 or LNCaP and siRNA-mediated silencing of SEC62 resulted in a reduced viability of thapsigargin-treated PC3 cells, indicating that Sec62 functions in cellular stress response. Measurement of cytosolic [Ca(2+) ] demonstrated the influence of Sec62 on the cellular response to thapsigargin on a molecular level. Using real-time cell analysis, we observed the loss of androgen stimulation of LNCaP cells in the presence of thapsigargin, and an additional negative effect on cell growth of Sec62 depletion. Also, for PC3- and DU145-cells Sec62 depletion inhibited growth after thapsigargin treatment. CONCLUSIONS: Our data indicate a crucial function of Sec62 in the response to thapsigargin-induced ER stress. This will be of great significance on the background of elevated Sec62 protein levels in prostate cancer cells when treatment with thapsigargin analogs is considered.
Assuntos
Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Humanos , Proteínas de Membrana Transportadoras/genética , RNA Interferente Pequeno , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Tapsigargina/farmacologia , Células Tumorais CultivadasRESUMO
Sec62 is part of the protein translocation apparatus in the membrane of the endoplasmic reticulum (ER). In yeast, Sec62 participates in the post-translational translocation of proteins into the ER, but its function in mammals remains elusive. Previously we described the amplification and over-expression of the SEC62 gene in prostate cancer cell lines and the protein has been described as a potential target gene in prostate cancer. In the current study we show that in the tumor tissue of prostate cancer patients Sec62 protein levels are elevated compared with tumor-free tissue derived from the same patients or from prostates of control group patients and that the higher Sec62 protein content correlates with an increasing de-differentiation of the cells. Therefore, up-regulation of Sec62 protein content indeed is a phenomenon associated with prostate cancer progression. Analysis of a multi-tissue tumor array showed that in addition to prostate cancer, overproduction of Sec62 is observed in various other tumors, most significantly in tumors of the lung and the thyroid. To examine the tumor-related functions of Sec62, we silenced the SEC62 gene in the prostate cancer cell-line PC3 as well as in a set of other tumor cell-lines with two different siRNAs. In general, after silencing of SEC62 the cell migration and the invasive potential of the cells was blocked or at least dramatically reduced while cell viability was hardly affected. Thus, the SEC62 gene may indeed be considered as a target gene in the therapy of various tumors.
