Chemical genetics on protein acetylation, a key reaction regulating biological functions

在组蛋白去乙酰化酶（HDAC）中，研究了HDAC 4（其定位于HDAC 4/Bach 2体中）和HDAC 6（其定位于细胞质中并使微管蛋白去乙酰化）的分子功能。结果，我们发现HDAC 4和Bach 2在氧化应激反应中重新定位到细胞核，并在PML小体周围的点状结构中积累。转录标记实验表明，由HDAC 4/Bach 2包围的PML小体内和周围的核区域是转录失活的。在HDAC 6的情况下，我们发现HDAC 6表达抑制EGF受体和转铁蛋白受体的内吞作用。在稳定表达用于敲低HDAC 6的siRNA的肺癌细胞中，EGF诱导的EGF受体内吞作用大大增强，从而下调EGF受体的水平。然而，这些细胞可以通过过度表达下游ERK来补偿EGF信号传导而生长。这些结果表明HDAC 6通过控制内吞作用来调节受体酪氨酸激酶的信号传导途径。此外，我们使用特异性HDAC抑制剂寻找新的乙酰化蛋白，并鉴定了几种蛋白质，包括Hsp 90，SV 40大T抗原和poly（A）聚合酶（PAP）。我们还确定了十多个蛋白质的乙酰化的裂变酵母细胞。功能分析表明，乙酰化导致Hsp 90活性降低，使SV 40大T抗原不稳定，并抑制PAP的核输入。这些发现意味着乙酰化发生在各种各样的细胞蛋白质上，并深刻地调节蛋白质功能。

Among the histone deacetylase (HDAC) enzymes, molecular functions of HDAC4, which localizes in the HDAC4/Bach2 body, and HDAC6, which localizes in the cytoplasm and deacetylates tubulin, were studied. As a result, we found that HDAC4, as well as Bach2, relocalized to the nucleus in response to oxidative stress, and accumulated in the punctate structures that surround the PML bodies. The transcription labeling experiments revealed that the nuclear regions in and around the PML bodies surrounded by HDAC4/Bach2 were transcriptionally inactivated. In the case of HDAC6, we showed that HDAC6 expression repressed the endocytosis of both EGF receptor and transferrin receptor. In the lung cancer cells stably expressing siRNA for the knock down of HDAC6, EGF-induced EGF receptor endocytosis was greatly enhanced thereby down-regulating the level of EGF receptor. However, these cells can grow by compensating the EGF signaling by overexpression of the downstream ERK. These results indicate that HDAC6 regulates the signaling pathways of receptor tyrosine kinases by controlling endocytosis. Furthermore, we searched for novel acetylated proteins using specific HDAC inhibitors, and identified several proteins including Hsp90, SV40 large T-antigen, and poly(A) polymerase (PAP). We also identified more than ten proteins that are acetylated in the fission yeast cells. The functional analysis demonstrated that acetylation caused a decrease in the activity of Hsp90, destabilized the SV40 large T-antigen, and suppressed the nuclear import of PAP. These findings imply that acetylation occurs on a wide variety of cellular proteins and profoundly regulates the protein function.