Peptidases Involved in Signal Peptide Generation and Degradation

该项目的长期目标是了解细菌中参与信号肽产生和降解的蛋白酶之间的结构和功能关系。通常，靶向特定亚细胞区室如内质网、线粒体膜间空间或叶绿体类囊体腔的蛋白质含有提供输出信息的可裂解信号序列。信号肽酶是通过从输出的蛋白质中去除信号序列而产生信号肽的蛋白酶。然后信号肽水解酶降解可能对细胞有毒或干扰蛋白质输出的信号肽。本研究的目的是了解信号肽酶催化结构域的哪个区域与膜相互作用，以了解酶如何在膜表面切割前分泌蛋白。该项目还研究了信号肽肽酶A的作用，这是另一种新的肽酶，通过非常规机制进行催化。该项目将1）定义信号肽酶内的结构元件，使其能够精确地切割前蛋白，2）确定信号肽酶活性位点下方的疏水反平行β链在膜结合中的作用，3）阐明信号肽肽酶A的催化机制，膜拓扑结构和底物特异性。定点诱变、化学修饰、遗传学和生物物理学将用于实现这些目标。这项研究将确定有多少信号肽酶的催化结构域是在双层的脂相。该项目的更广泛的影响是，它将有助于理解非常规丝氨酸蛋白酶的机制，并有助于阐明催化如何在膜表面或膜内发生。一个高度优先事项将是本科生和研究生的培训。

The long-term objective of this project is to understand the structural and functional relationships among the proteases involved in signal peptide generation and degradation in bacteria. Typically, proteins that are targeted to specific subcellular compartments such as the endoplasmic reticulum, mitochondria intermembrane space, or chloroplast thylakoid lumen contain cleavable signal sequences that provide information for export. Signal peptidases are proteases that generate the signal peptide by removing the signal sequence from exported proteins. The signal peptide hydrolases then degrade the signal peptides that can be toxic to the cell or interfere with protein export. The objective of this research is to understand which region of the signal peptidase catalytic domain interacts with the membrane in order to understand how the enzyme cleaves presecretory proteins at the membrane surface. This project also examines the role of signal peptide peptidase A, another novel peptidase, that carries out catalysis by an unconventional mechanism. The project will 1) define the structural elements within signal peptidase that allow it to cleave preproteins with exquisite accuracy, 2) determine the role of the signal peptidase hydrophobic antiparallel Beta-strand below the active site in membrane association, and 3) elucidate the catalytic mechanism, membrane topology, and substrate specificity of signal peptide peptidase A. Site-directed mutagenesis, chemical modification, genetics and biophysics will be used to achieve these aims. The research will determine how much of the catalytic domain of signal peptidase is within the lipid phase of the bilayer. The broader impact of the project is that it will contribute to the understanding of the mechanism of the unconventional serine proteases and help elucidate how catalysis occurs within or at the membrane surface. A high priority will be the training of both undergraduate and graduate students.