26S蛋白酶体膜定位的调控机制与功能研究

The 26S proteasome is an essential machinery for protein degradation in all eukaryotic cells. Despite its well established importance for cell viability, how this protein complex itself is regulated under various pathophysiological conditions remains largely unknown. We and others have provided initial evidence that chemical modifications (such as phosphorylation) play important roles in fine-tuning proteasome assembly, activity as well as intracellular localization. As such, proteasome regulation has gained increasing attention in recent years and is now positioned at the frontier of interdisciplinary research. In this Proposal, we focus on three types of modification that occur on a single subunit, Rpt2, of the 26S proteasome, including its N-terminal myristoylation, serine phosphorylation and tyrosine phosphorylation. Our preliminary data indicate that N-myristoylation, which is conserved from yeast to human, is critical for membrane anchoring of Rpt2 and probably of the whole proteasome. This effect is counterbalanced by phosphorylation of Ser4, a site that is conserved in vertebrates but not present in lower organisms. Membrane localization is also found to be a prerequisite for phosphorylation of Rpt2 at its penultimate residue, Tyr439, which is within the C-terminal HbYX motif critical for proteasome assembly. Phospho-proteomic studies have suggested that an oncogenic form of EGFR, a membrane protein, may be responsible for Rpt2-Y439 phosphorylation. Together, these conserved but rarely studied modifications may determine the localization of a select pool of 26S proteasomes, linking membrane proteasome assembly to cancer cell signaling. Here we propose a broad array of mechanistic and functional studies using canonical biochemical and cell biology approaches in conjunction with cutting-edge technology including gene editing, click chemistry, super-resolution microscopy, quantitative mass spectrometry and new animal models, in order to elucidate the molecular underpinnings of those proteasome modifications and the biological relevance of membrane-tethered proteasomes in health and disease and during evolution.

26S蛋白酶体是负责蛋白质降解的关键细胞器，对细胞功能至关重要，但其自身如何被调控还很不清楚。我们发现蛋白酶体的Rpt2亚基上存在三种化学修饰，即N端肉豆蔻酰化、丝氨酸磷酸化和酪氨酸磷酸化。首先，Rpt2的肉豆蔻酰化在进化中高度保守，很可能对于整个蛋白酶体在细胞膜上的定位至关重要。其次，Rpt2-Ser4的磷酸化会抑制Rpt2插膜，从而动态调控蛋白酶体的膜定位。第三，只有膜定位的Rpt2才会发生Tyr439位点的酪氨酸磷酸化。该位点对于蛋白酶体的组装非常重要，而负责该位点磷酸化的有可能是膜蛋白EGFR。综上，Rpt2亚基上这三种鲜有研究的修饰之间相互作用，决定了蛋白酶体的膜定位，并将膜上蛋白酶体的组装与细胞信号转导联系起来。我们将利用包括基因编辑、点击化学、定量质谱和动物模型在内的一系列手段，阐明上述蛋白酶体修饰的调控机制，解读蛋白酶体膜定位的生理功能及其在进化中的意义。

26S蛋白酶体负责真核细胞中绝大部分蛋白的降解，与几乎所有生命活动和多种疾病密切相关。通常人们认为蛋白酶体主要游离于细胞质和细胞核中，但过去三十多年间时有报导表明蛋白酶体可与细胞中不同的膜结合，其分子机制和生物学意义一直不清。.本项目首次对哺乳动物细胞中由肉豆蔻酰化修饰介导的蛋白酶体膜定位进行了深入系统的研究。我们发现：.1. 蛋白酶体Rpt2亚基的N-端肉豆蔻酰化在进化中严格保守，丰度较高，在多种细胞类型中广泛存在，对于蛋白酶体膜定位起到关键作用。.2. Rpt2-G2A突变阻断其肉豆蔻酰化修饰。质谱显示，与正常细胞相比，G2A细胞中有相当数量的蛋白水平发生改变，其中2/3的蛋白与多种细胞膜结构相关。膜定位蛋白酶体不仅直接参与膜相关蛋白的降解，而且可以通过调控细胞器的形态功能间接影响多种蛋白的包内运输、定位、修饰和活性，实现membrane-associated proteasome-mediated quality control (MAP-QC)。.3. Rpt2-G2A细胞在黏附、迁移、膜信号转导等方面较正常细胞均有显著缺陷。G2A纯合小鼠于E16.5之前死亡，胚胎肝脏等器官明显发育异常。此外，Rpt2肉豆蔻酰化对于癌细胞在裸鼠中成瘤至关重要。.4. 蛋白酶体膜定位同时还受到Rpt2-Ser4磷酸化的可逆调控。.5. 发生肉豆蔻酰化修饰的Rpt2可发生Tyr439位点的磷酸化。该修饰由膜定位的Src（激酶）和PTPN2（磷酸酶）共同调控。Rpt2-Y439磷酸化抑制膜定位蛋白酶体的组装和活性，对于Src抑制剂saracatinib的抑癌功能非常重要。.综上，本项目聚焦于Rpt2亚基上相互关联的三种化学修饰，以此揭示了高等生物中26S蛋白酶体膜定位的机制和生理功能，加深了我们对于蛋白质区域化降解的认识，并为器官发育、抗癌治疗等研究提供了新的视角。