The potassium channel protein KCNH2 is encoded by KCNH2 gene, and there are more than 300 mutations of KCNH2. Unfolded protein response (UPR) is typically initiated in response to an accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER). The present study aimed to explore the UPR process and the role of activating transcription factor 6 (ATF6) in the abnormal expression of potassium voltage-gated channel subfamily H member 2 (KCNH2)A561V. The wild-type (wt) KCNH2 and A561V mutant KCNH2 was constructed with his-tag. The 293 cells were used and divided into KCNH2wt+KCNH2A561V, KCNH2wt and KCNH2A561V groups. The expression levels of ATF6 and KCNH2 in different groups were detected by Western blotting, reverse transcription-quantitative PCR, immunofluorescence and immuno-coprecipitation assays. The protein types and abundance of immuno-coprecipitation samples were analyzed by mass spectrometry. The proteomic analysis of the mass spectrometry results was carried out by using the reactome database and GO (Gene Ontology) tool. The mRNA expression levels of KCNH2 and ATF6 in the KCNH2wt+KCNH2A561V group were higher compared with the KCNH2A561V group. However, the full-length protein expression of ATF6 was inhibited, indicating that ATF6 was highly activated and a substantial number of ATF6 was sheared in KCNH2wt+KCNH2A561V group compared with control group. Furthermore, A561V-KCNH2 mutation leading to the accumulation of the immature form of KCNH2 (135 kDa bands) in ER, resulting in the reduction of the ratio of 155 kDa/135 kDa. In addition, the abundance of UPR-related proteins in the KCNH2A561V group was higher compared with the KCNH2wt+KCNH2A561V group. The 'cysteine biosynthetic activity' of GO:0019344 process and the 'positive regulation of cytoplasmic translation activity' of GO:2000767 process in the KCNH2A561V group were higher compared with the KCNH2wt+KCNH2A561V group. Hence, co-expression of wild-type and A561V mutant KCNH2 in 293 cells activated the UPR process, which led to the inhibition of protein translation and synthesis, in turn inhibiting the expression of KCNH2. These results provided a theoretical basis for clinical treatment of Long QT syndrome.
- MeSH
- endoplazmatické retikulum metabolismus MeSH
- mutace MeSH
- proteomika * MeSH
- signální dráha UPR genetika MeSH
- transkripční faktor ATF6 * genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- ATF6 protein, human MeSH Prohlížeč
- KCNH2 protein, human MeSH Prohlížeč
- transkripční faktor ATF6 * MeSH
Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.
- Klíčová slova
- COP vesicles, ER stress, Golgi apparatus, cargo receptor, endoplasmic reticulum, epithelial cells, kidney, organoids, secretory pathway, unfolded protein response,
- MeSH
- benzamidy chemie metabolismus farmakologie MeSH
- epitelové buňky cytologie metabolismus MeSH
- heptany farmakologie terapeutické užití MeSH
- imidazolinové receptory antagonisté a inhibitory genetika metabolismus MeSH
- indukované pluripotentní kmenové buňky cytologie metabolismus MeSH
- ledviny cytologie metabolismus patologie MeSH
- lidé MeSH
- lyzozomy účinky léků metabolismus MeSH
- malá interferující RNA metabolismus MeSH
- mucin 1 chemie genetika metabolismus MeSH
- můstkové bicyklické sloučeniny farmakologie terapeutické užití MeSH
- myši transgenní MeSH
- myši MeSH
- nemoci ledvin metabolismus patologie MeSH
- posunová mutace MeSH
- RNA interference MeSH
- signální dráha UPR účinky léků MeSH
- transkripční faktor ATF6 metabolismus MeSH
- vezikulární transportní proteiny chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ATF6 protein, human MeSH Prohlížeč
- benzamidy MeSH
- BRD4780 MeSH Prohlížeč
- heptany MeSH
- imidazolinové receptory MeSH
- malá interferující RNA MeSH
- mucin 1 MeSH
- můstkové bicyklické sloučeniny MeSH
- NISCH protein, human MeSH Prohlížeč
- TMED9 protein, human MeSH Prohlížeč
- transkripční faktor ATF6 MeSH
- vezikulární transportní proteiny MeSH
BACKGROUND: Fatty acid-induced apoptosis and ER stress of pancreatic β-cells contribute to the development of type 2 diabetes, however, the molecular mechanisms involved are unclear. AIMS: In this study we have tested the role of caspase-2 and suggested ER stress mediator JNK in saturated fatty acid-induced apoptosis of the human pancreatic β-cells NES2Y. RESULTS: We found that stearic acid at apoptosis-inducing concentration activated ER stress signaling pathways, i.e. IRE1α, PERK and ATF6 pathways, in NES2Y cells. During stearic acid-induced apoptosis, JNK inhibition did not decrease the rate of apoptosis nor the activation of caspase-8, -9, -7 and -2 and PARP cleavage. In addition, inhibition of JNK activity did not affect CHOP expression although it did decrease the induction of BiP expression after stearic acid treatment. Caspase-2 silencing had no effect on PARP as well as caspase-8, -9 and -7 cleavage and the induction of CHOP expression, however, it also decreased the induction of BiP expression. Surprisingly, caspase-2 silencing was accompanied by increased phosphorylation of c-Jun. CONCLUSIONS: We have demonstrated that caspase-2 as well as JNK are not key players in apoptosis induction by saturated fatty acids in human pancreatic β-cells NES2Y. However, they appear to be involved in the modulation of saturated fatty acid-induced ER stress signaling, probably by a mechanism independent of c-Jun phosphorylation.
- MeSH
- apoptóza účinky léků MeSH
- beta-buňky cytologie metabolismus MeSH
- chaperon endoplazmatického retikula BiP MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- fosforylace MeSH
- JNK mitogenem aktivované proteinkinasy antagonisté a inhibitory metabolismus MeSH
- kaspasa 2 chemie genetika metabolismus MeSH
- kaspasa 7 metabolismus MeSH
- kaspasa 8 metabolismus MeSH
- kaspasa 9 metabolismus MeSH
- kyseliny stearové farmakologie MeSH
- lidé MeSH
- malá interferující RNA metabolismus MeSH
- poly(ADP-ribosa)-polymerasy metabolismus MeSH
- proteiny tepelného šoku metabolismus MeSH
- RNA interference MeSH
- signální transdukce účinky léků MeSH
- stres endoplazmatického retikula účinky léků MeSH
- transkripční faktor ATF6 metabolismus MeSH
- transkripční faktor CHOP metabolismus MeSH
- transkripční faktory RFX MeSH
- transkripční faktory genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ATF6 protein, human MeSH Prohlížeč
- chaperon endoplazmatického retikula BiP MeSH
- DDIT3 protein, human MeSH Prohlížeč
- DNA vazebné proteiny MeSH
- JNK mitogenem aktivované proteinkinasy MeSH
- kaspasa 2 MeSH
- kaspasa 7 MeSH
- kaspasa 8 MeSH
- kaspasa 9 MeSH
- kyseliny stearové MeSH
- malá interferující RNA MeSH
- poly(ADP-ribosa)-polymerasy MeSH
- proteiny tepelného šoku MeSH
- stearic acid MeSH Prohlížeč
- transkripční faktor ATF6 MeSH
- transkripční faktor CHOP MeSH
- transkripční faktory RFX MeSH
- transkripční faktory MeSH