National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin, 09 W6Y4, Ireland.
Alltech, Dunboyne, Meath, Ireland.
In Vitro Cell Dev Biol Anim. 2021 Mar;57(3):359-371. doi: 10.1007/s11626-020-00540-8. Epub 2021 Feb 8.
We previously described a non-monotonic dose response curve at low copper concentrations where 3.125 μM CuSO (the early inflection point) was more toxic than 25 μM CuSO in Caco-2 cells. We employed global proteomics to investigate this observation. The altered expression levels of a small number of proteins displaying a temporal response may provide the best indication of the underlying mechanism; more well-known copper response proteins including the metal binding metallothioneins (MT1X, MT1F, MT2A) and antioxidant response proteins including Heme oxygenase were upregulated to a similar level in both copper concentrations and so are less likely to underpin this phenomenon.The temporal response proteins include Granulins, AN1-type zinc finger protein 2A (ZFAND2A), and the heat shock proteins (HSPA6 and HSPA1B). Granulins were decreased after 4 h only in 25 μM CuSO but from 24 h, were decreased in both copper concentrations to a similar level. Induction of ZFAND2A and increases in HSPA6 and HSPA1B were observed at 24 h only in 25 μM CuSO but were present at 48 h in both copper conditions. The early expression of ZFAND2A, HSPs, and higher levels of α-crystallin B (CRYAB) correlated with lower levels of misfolded proteins in 25 μM CuSO compared to 3.125 μM CuSO at 48 h. These results suggest that 3.125 μM CuSO at early time points was unable to activate the plethora of stress responses invoked by the higher copper concentration, paradoxically exposing the Caco-2 cells to higher levels of misfolded proteins and greater proteotoxic stress.
我们之前描述了在低铜浓度下呈非单调剂量反应曲线的现象，其中 3.125μM 的 CuSO（早期拐点）比 25μM 的 CuSO 在 Caco-2 细胞中更为毒性。我们采用了全局蛋白质组学来研究这一观察结果。少数具有时间响应的蛋白质表达水平的改变可能是潜在机制的最佳指示；在两种铜浓度下，包括金属结合金属硫蛋白（MT1X、MT1F、MT2A）和抗氧化反应蛋白在内的更为知名的铜反应蛋白以及血红素加氧酶都被上调到相似的水平，因此不太可能是这一现象的基础。时间响应蛋白包括颗粒蛋白、AN1 型锌指蛋白 2A（ZFAND2A）和热休克蛋白（HSPA6 和 HSPA1B）。仅在 25μM 的 CuSO 中，颗粒蛋白在 4 小时后才减少，但从 24 小时开始，在两种铜浓度下都减少到相似的水平。仅在 25μM 的 CuSO 中观察到 ZFAND2A 的诱导和 HSPA6 和 HSPA1B 的增加，但在两种铜条件下都在 48 小时时出现。在 25μM 的 CuSO 中，ZFAND2A、HSPs 和较高水平的α-晶体蛋白 B（CRYAB）的早期表达与 48 小时时较低水平的错误折叠蛋白相关，与 3.125μM 的 CuSO 相比，在 25μM 的 CuSO 中，早期表达水平较低。这些结果表明，在早期时间点，3.125μM 的 CuSO 无法激活高浓度铜所引发的大量应激反应，这反而使 Caco-2 细胞暴露于更高水平的错误折叠蛋白和更大的蛋白毒性应激下。
