Site-directed cross-linking with KLK proteases from prostate

与前列腺 KLK 蛋白酶进行定点交联

• 批准号: 409661645
• 负责人: Professor Dr. Nediljko Budisa
• 金额: --
• 依托单位: Arbeitsgruppe Struktur und Mechanismus von Proteinen in der Immunologie und Blutgerinnung
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/409661645?language=en
• 关键词: Site; directed; cross; linking; KLK

Many biological processes depend on proteins that bind other molecules, which are often proteins as well. Some of these complexes are very stable and can be analyzed in structural biology. However, there are also short-lived complexes, which are much more difficult to investigate. The current procedures for cross-linking molecular components of unstable complexes are rather unspecific. Thus, new approaches for site-directed linking are desirable. So-called click chemistry reactions result in the formation of covalent bonds at defined positions in proteins, among them the well-established 1,3-dipolar cycloaddition, also known as azide-alkyne click reaction, and the newly developed thiol-ene coupling for proteins. Both approaches are based on non-natural or non-canonical amino acids (nnAA), which can be incorporated into selected proteins by manipulating the genetic code and the protein synthesis in cell cultures. A suitable model system for protein-ligand interactions are the human kallikrein-related peptidases (KLKs) from prostate, in particular KLKs 2, 3, 4, 5, and 11. These serine proteases have important physiological functions in fertilization and altered roles under pathological conditions as prostate cancer.Knowledge of several KLK crystal structures is a good basis for placing nnAAs at selected positions of the proteases for linking them to other molecules. To this end, mainly inactive variants of these KLKs will be employed, which do not cleave the linked molecules after the click reactions. The two approaches of the click reactions will be applied in two steps: 1. Inactive KLK variants containing reactive nnAAs are linked with peptides containing an azide and an alkyne as nnAA reaction partner, or a cysteine and an olefinic side-chain, respectively. These peptides are derived from natural substrates, for which the prostatic KLKs are specific. 2. The nnAA-KLKs will be linked to natural substrates, with corresponding modifications by recombinant production. After purification of the stabilized complexes, they are biochemically characterized and crystallized. If this procedure yields suitable crystals for X-ray diffraction, most likely the structures of the protein complexes can be determined.Site-directed cross-linking of proteins with nnAAs could become a general method, which is applicable to protein complexes with other biomolecules, such as nucleic acids, lipids, and sugars. Especially in this project information can be gained on the three-dimensional structural elements of the KLKs, which determine their substrate specificity, which is highly valuable for elucidating biological and disease-related processes, e.g. in prostate cancer. Knowledge of this “tertiary specificity” of the KLK proteases will enable the preparation of novel drugs with unprecedented efficacy.

许多生物过程依赖于结合其他分子的蛋白质，这些分子通常也是蛋白质。其中一些复合物非常稳定，可以在结构生物学中进行分析。然而，也有一些短暂的复合体，这些复合体的调查难度要大得多。目前用于交联不稳定复合物的分子组分的方法相当不特异。因此，需要用于定点连接的新方法。所谓的点击化学反应导致在蛋白质中的限定位置处形成共价键，其中包括公认的1，3-偶极环加成，也称为叠氮化物-炔点击反应，以及新开发的蛋白质硫醇-烯偶联。这两种方法都是基于非天然或非规范氨基酸（nnAA），可以通过操纵遗传密码和细胞培养物中的蛋白质合成将其掺入选定的蛋白质中。用于蛋白质-配体相互作用的合适的模型系统是来自前列腺的人激肽释放酶相关肽酶（KLK），特别是KLK 2、3、4、5和11。这些丝氨酸蛋白酶在受精过程中具有重要的生理功能，在前列腺癌等病理条件下具有改变的作用，几种KLK晶体结构的知识是将nnAA置于蛋白酶的选定位置以将其连接到其他分子的良好基础。为此，将使用这些KLK的主要无活性变体，其在点击反应后不切割连接的分子。点击反应的两种方法将分两步应用：1.含有反应性nnAA的非活性KLK变体分别与含有叠氮化物和炔作为nnAA反应伴侣或半胱氨酸和烯属侧链的肽连接。这些肽衍生自天然底物，前列腺KLK对天然底物具有特异性。2. nnAA-KLK将与天然底物连接，并通过重组生产进行相应的修饰。在纯化稳定的复合物后，对其进行生物化学表征和结晶。如果这一过程产生合适的晶体的X射线衍射，最有可能的蛋白质复合物的结构可以determined.Site-directed交联的蛋白质与nnAA可能成为一种通用的方法，这是适用于蛋白质复合物与其他生物分子，如核酸，脂质和糖。特别是在该项目中，可以获得关于KLK的三维结构元件的信息，这些结构元件决定其底物特异性，这对于阐明生物学和疾病相关过程（例如前列腺癌）非常有价值。了解KLK蛋白酶的这种“三级特异性”将能够制备具有前所未有功效的新药。