Mammalian 20S proteasome isotypes: from structure and function to selective inhibition

20S蛋白酶体核心颗粒（CP）是非溶酶体蛋白降解途径的关键参与者，控制着大量必需的细胞内过程，是一个经过验证的药物靶点。虽然简单的真核生物只表达一种CP，但哺乳动物的进化及其复杂性的增加导致了CP的多样化和分化，最终产生了四种同型。存在于所有细胞中，组成性蛋白酶体（cCP）完成了蛋白质稳态和生存所需的大部分多肽降解。相比之下，免疫蛋白酶体（iCP）和胸腺蛋白酶体（tCP）参与形成功能性适应性免疫系统，而精子蛋白酶体（sCP）的作用尚未阐明。虽然CP同型的整体结构是相同的，但它们的亚基组成不同。不区分CP同型的抑制剂是治疗血癌的重磅药物，而CP选择性化合物的其他医学应用，例如慢性炎症或自身免疫性疾病，正在临床试验中探索。在目前的资助期内，对cCP和iCP进行了广泛的基于结构的抑制剂和诱变研究，我们已在11篇出版物中报道了其结果。迄今为止，人们对tCP和sCP都知之甚少，对它们的结构检查将分别提供对独特亚基ß5t和α 4s的更彻底的了解。因此，在应用后续项目中，我们的目标是对tCP进行结构和生化表征。特别是，tCP与cCP和iCP的裂解模式分析将用于开发tCP特异性配体。这些化合物可能有助于未来探索tCP的生物学功能及其选择性失活的任何医学相关性。与我们在iCP上的工作类似，酵母诱变的工作被设想为确定ß5t的关键元素，用于tCP的组装和功能。此外，还计划对sCP进行详细的实验。主要目的是表达、纯化和结晶真正的人类sCP或嵌合CP，即含有人类sCP特异性α4s实体的酵母CP。本提案设想的跨学科概念必将丰富基础蛋白酶体研究，并可能开辟CP药物开发的未来方向。我的实验室拥有良好的基础设施和长期的专业知识，可以期待对20S蛋白酶体同型的新见解。

The 20S proteasome core particle (CP) is the key player of the non-lysosomal protein degradation pathway, controls a plethora of essential intracellular processes and is a validated drug target. While simple eukaryotes express only one type of CP, the evolution of mammals and their increased complexity led to diversification and differentiation of the CP, ultimately resulting in four isotypes. Present in all cells, the constitutive proteasome (cCP) accomplishes the bulk of polypeptide degradation required for protein homoeostasis and survival. In contrast, the immunoproteasome (iCP) and the thymoproteasome (tCP) are engaged in shaping a functional adaptive immune system, whereas the role of the spermatoproteasome (sCP) is yet to be elucidated. Although the overall architecture of CP isotypes is identical, their subunit composition varies. Inhibitors that do not discriminate between CP isotypes are blockbuster drugs for the treatment of blood cancers and additional medicinal applications of iCP-selective compounds, e.g. chronic inflammatory or autoimmune diseases, are explored in clinical trials. During the current funding period extensive structure-based inhibitor and mutagenesis studies were performed on cCP and iCP, the results of which we have reported in 11 publications. To date, both tCP and sCP are poorly understood and their structural examination would provide a more thorough understanding of the unique subunits ß5t and Alpha 4s, respectively. Therefore, in the applied follow-up project we aim to structurally and biochemically characterize the tCP. In particular, the analysis of cleavage patterns of tCP versus cCP and iCP will be used to develop tCP-specific ligands. Such compounds may serve future efforts to probe the biological function of the tCP and any medicinal relevance of its selective inactivation. Similar to our work on the iCP, yeast mutagenesis efforts are envisioned to determine crucial elements of ß5t for assembly and function of the tCP. Besides, elaborate experiments on the sCP are planned. The primary goal is expression, purification and crystallization of the genuine human sCP or of a chimeric CP, i.e. a yeast CP that contains the human sCP-specific α4s entity. The envisioned interdisciplinary concept of this proposal certainly will enrich basic proteasome research and may open up future directions in CP drug development. The excellent infrastructure and the long lasting expertise of my lab allow to expect also promising new insights into 20S proteasome isotypes.