We previously described a non-monotonic dose response curve at low copper concentrations where 3.125 μM CuSO₄ (the early inflection point) was more toxic than 25 μM CuSO₄ 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 CuSO₄ 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 CuSO₄ 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 CuSO₄ compared to 3.125 μM CuSO₄ at 48 h. These results suggest that 3.125 μM CuSO₄ 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.

我们之前描述了低铜浓度下的非单调剂量反应曲线，其中在 Caco-2 细胞中 3.125 μM C​​uSO ₄（早期拐点）比 25 μM C​​uSO ₄毒性更大。我们采用全局蛋白质组学来研究这一观察结果。显示时间响应的少量蛋白质的表达水平改变可能提供潜在机制的最佳指示；更知名的铜反应蛋白，包括金属结合金属硫蛋白（MT1X、MT1F、MT2A）和抗氧化反应蛋白，包括血红素加氧酶，在两种铜浓度下均上调至相似水平，因此不太可能支持这种现象。

时间反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 25 μM C​​uSO _{4 中，}颗粒蛋白在 4 小时后降低，但从 24 小时开始，两种铜浓度均降低至相似水平。仅在 25 μM C​​uSO _{4 中}在 24 小时观察到 ZFAND2A 的诱导以及 HSPA6 和 HSPA1B 的增加，但在两种铜条件下均在 48 小时时出现。与48 小时时的3.125 μM C​​uSO ₄相比，ZFAND2A、HSP 和较高水平的 α-晶状体蛋白 B (CRYAB) 的早期表达与 25 μM C​​uSO _{4 中}较低水平的错误折叠蛋白相关。这些结果表明 3.125 μM C​​uSO ₄ 在早期时间点无法激活由较高铜浓度引起的过多应激反应，自相矛盾地将 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。