A Novel Anchoring Mechanism for Prokaryotic Surface Proteins

细菌是原核生物，与细菌不同，尽管它们以许多物种在极端环境中茁壮成长的能力而闻名，但它们无处不在，甚至可以在我们的肠道和皮肤上找到。许多细胞表面蛋白质对细胞功能（如营养摄取，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以执行其功能。在古细菌中，这些附着机制知之甚少，该项目将描述一种新的表面蛋白质锚定机制，该机制预计也将被许多细菌使用。该研究项目团队将从事科学教育，以激发幼儿对科学的兴趣，特别是那些在费城西部服务不足的学校。PI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。 计算机模拟数据表明，许多原核生物使用以前未知的机制将底物的C-末端共价连接到细胞膜中的脂质。在古细菌中，这种锚定机制可能是由古细菌分选酶（阿尔塔）催化的，这一过程让人联想到进化上不同的细菌分选酶对表面蛋白的锚定，细菌分选酶是一种将底物的C-末端加工并共价连接到细胞壁的酶。体内证据支持的假设，Haloferax volcanii S-层糖蛋白的加工和共价连接的脂质双层的依赖性的方式，也揭示了其他的阿尔塔底物在重要的生物过程中发挥重要作用。这项研究的目的是确定这种依赖于ArtA的锚定机制在H。volcanii细胞过程，使用生物化学，质谱，遗传和微观方法。拟议的研究将：1）更详细地表征涉及阿尔塔的翻译后修饰; 2）确定阿尔塔的结构-功能关系，包括推定的催化氨基酸三联体的重要性，以及鉴定与阿尔塔相互作用的其他蛋白质;和3）表征阿尔塔底物在细胞过程中发挥的功能作用，例如交配、运动和粘附。

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.