A Novel Anchoring Mechanism for Prokaryotic Surface Proteins

原核表面蛋白的新型锚定机制

• 批准号: 1413158
• 负责人: Mechthild Pohlschroder
• 金额: $ 74.23万
• 依托单位: University of Pennsylvania
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2018-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1413158&HistoricalAwards=false
• 关键词: Novel; Anchoring; Mechanism; Prokaryotic; Surface

Archaea are prokaryotic organisms that are distinct from bacteria and although they are well known for the ability of many species to thrive in extreme environments, they are ubiquitous and can be found even in our gut and on our skin. Many cell surface proteins are essential to cellular functions (processes such as nutrient uptake, surface adhesion, and mating) and they must be effectively anchored to the cell surface in order to carry out their functions. In archaea, these attachment mechanisms are poorly understood and this project will characterize a novel surface protein anchoring mechanism that is also predicted to be used by many bacteria. The research project team will engage in science education to inspire an interest in science in young children, particularly those in underserved schools in West Philadelphia. The PI will develop microbiology experiments that can be utilized by schools with limited financial resources, and the researchers will participate in both in-class instruction and teacher-training workshops hosted by the University of Pennsylvania. In silico data suggest that many prokaryotes use a previously unknown mechanism to covalently link the C-terminus of a substrate to a lipid in the cell membrane. In archaea, this anchoring mechanism is likely catalyzed by an archaeosortase (ArtA) in a process that is reminiscent of the anchoring of surface proteins by the evolutionarily distinct bacterial sortase, an enzyme that processes and covalently links the C-termini of substrates to the cell wall. In vivo evidence supports the hypothesis that the Haloferax volcanii S-layer glycoprotein is processed and covalently linked to the lipid bilayer in an ArtA-dependent manner and has also revealed that other ArtA substrates play vital roles in important biological processes. The goal of the proposed research is to determine the specific roles that this ArtA-dependent anchoring mechanism plays in H. volcanii cellular processes, using biochemical, mass spectrometric, genetic and microscopic approaches. The proposed research will: 1) characterize post-translational modifications involving ArtA in greater detail; 2) determine structure-function relationships for ArtA, including the importance of a putative catalytic amino acid triad, as well as identifying other proteins that interact with ArtA; and 3) characterize the functional roles played by ArtA substrates in cellular processes such as mating, motility and adhesion.

古细菌是与细菌不同的原核生物，虽然众所周知，许多物种能够在极端环境中繁衍生息，但它们无处不在，甚至可以在我们的肠道和皮肤中找到。许多细胞表面蛋白对于细胞功能（营养吸收、表面粘附和交配等过程）至关重要，它们必须有效地锚定在细胞表面才能发挥其功能。在古细菌中，人们对这些附着机制知之甚少，该项目将描述一种新颖的表面蛋白锚定机制，预计该机制也将被许多细菌使用。研究项目团队将从事科学教育，以激发幼儿对科学的兴趣，特别是那些西费城服务不足的学校的孩子。首席研究员将开发可供财力有限的学校使用的微生物学实验，研究人员将参加宾夕法尼亚大学主办的课堂教学和教师培训研讨会。 计算机数据表明，许多原核生物使用以前未知的机制将底物的 C 末端与细胞膜中的脂质共价连接。在古细菌中，这种锚定机制很可能是由古菌分选酶 (ArtA) 催化的，这一过程让人想起进化上独特的细菌分选酶对表面蛋白的锚定，细菌分选酶是一种处理底物 C 末端并将其共价连接到细胞壁的酶。体内证据支持这样的假设：Haloferax volcanii S 层糖蛋白以 ArtA 依赖性方式被加工并与脂质双层共价连接，并且还揭示了其他 ArtA 底物在重要的生物过程中发挥着至关重要的作用。本研究的目标是利用生化、质谱、遗传和微观方法确定这种依赖 ArtA 的锚定机制在 H. volcanii 细胞过程中发挥的具体作用。拟议的研究将：1）更详细地描述涉及 ArtA 的翻译后修饰； 2) 确定 ArtA 的结构-功能关系，包括假定的催化氨基酸三联体的重要性，以及鉴定与 ArtA 相互作用的其他蛋白质； 3) 表征 ArtA 底物在细胞过程（如交配、运动和粘附）中所发挥的功能作用。