RIG: Regulated Intramembrane Proteolytic Activation of Membrane-anchored Transcription Factors in Anabaena

智力优势 基因转录调控是细胞对内外环境变化做出反应的最重要机制之一。 为了确保快速反应，转录调节因子通常以休眠形式合成和储存，仅在响应特定信号时才变得活跃。 转录反应的生物学兴趣主要集中在转录因子的活性是如何调节的。 通过暂时附着在细胞膜上，将转录因子从它们的DNA靶点上隔离，正成为一种调节它们活性的优雅机制。 几个膜锚定转录因子（MTF）已被实验确定，其进入转录机制的调节蛋白水解裂解从膜。 这一过程被广泛地称为调节性膜内蛋白水解（RIP）。 RIP是一种新发现的机制，它控制着从细菌到人类的许多重要信号通路。 RIP在生物学相关过程中的重要作用的发现使得理解RIP功能的全部范围变得重要。 然而，RIP的了解很少，到目前为止，还没有任何生物体RIP的系统研究。 该项目是对可变鱼腥藻29413中RIP的全基因组研究，鱼腥藻29413是一种多细胞蓝细菌，能够同时进行农业相关的O2生产光合作用和O2不稳定的N2固定，以及生物太阳能H2气体生产。 此外，它的营养细胞可以分化成其他三种类型的细胞，以响应环境的变化，这提供了一个难得的机会，研究基因调控的几个分化过程中的一个单一的细菌。 鉴于其独特的能力，A。variabilis为研究基础生物学问题，特别是农业和生物能源相关问题提供了一个非常有吸引力的实验系统。 对A. variabilis鉴定了19个推定的RIP基因，编码5种RIP蛋白酶和14种MTF。 文献调查表明，一些直系同源的RIP基因可能参与调节胁迫反应，叶绿体发育，细胞分化，细胞间通讯，光合作用，以及有氧固氮。 因此，在A. variabilis可能发挥重要的生物学作用。 本研究的目的是：1）通过对19个RIP基因的失活，获得RIP基因的细胞功能线索; 2）确定RIP基因的表达模式，进一步阐明RIP基因在A. 3）测定重组E. variabilis中5种RIP蛋白酶的蛋白水解活性。coli系统; 4）鉴定特异性MTF/RIP蛋白酶对。更广泛的影响 PI有指导学生的历史，在这个项目中，将通过在密歇根州立大学与一位参加查尔斯·德鲁方案的少数民族女教授建立合作的外联活动来扩大参与。 查尔斯·德鲁计划是一个保留和学术提高计划，为高成就的本科生和毕业生从代表性不足的少数群体。 PI将在该计划中指导学生，为参与者提供职业发展指导，并担任感兴趣的学生的研究顾问。 在这个项目中，PI将为四名本科生提供实践研究经验。

Intellectual Merit The regulation of gene transcription is one of the most important mechanisms used by cells to respond to alterations in their internal or external environment. To ensure a rapid response, transcriptional regulators are often synthesized and stored in a dormant form, becoming active only in response to a specific signal. Much of the biological interest in transcriptional responses focuses on how the activity of transcription factors is regulated. Sequestration of transcription factors away from their DNA targets, by temporary attachment to cellular membranes, is emerging as an elegant mechanism to regulate their activities. Several membrane-anchored transcription factors (MTFs) have been experimentally identified whose access to the transcriptional machinery is regulated by proteolytic cleavage from the membrane. This process is broadly referred to as regulated intramembrane proteolysis (RIP). RIP is a newly discovered mechanism that controls many important signaling pathways conserved from bacteria to humans. The discovery of an essential role for RIP in biologically relevant processes makes it important to understand the full gamut of RIP function. However, RIP is little understood and so far there has been no systematic study of RIP in any organism. This project is for a genome-wide study of RIP in Anabaena variabilis 29413, a multicellular cyanobacterium, capable of simultaneously carrying out agriculturally relevant O2-producing photosynthesis and O2-labile N2-fixation, as well as bio-solar H2 gas production. In addition, its vegetative cells can differentiate into three other types of cells in response to environmental changes, which presents a rare opportunity to investigate gene regulation of several differentiation processes in a single bacterium. Given its unique capabilities, A. variabilis provides a very attractive experimental system to study fundamental biological problems, especially for agriculture and bio-energy related problems. An analysis of the genomic sequence of A. variabilis identified 19 putative RIP genes that encode 5 RIP proteases and 14 MTFs. Literature surveys imply that some orthologs of the putative RIP genes may be involved in regulation of stress responses, chloroplast development, cellular differentiation, cell-cell communication, and photosynthesis, as well as aerobic N2-fixation. Therefore, the 19 putative RIP genes in A. variabilis may play important biological roles. The aims of the project are 1) to obtain clues for the cellular functions of 19 putative RIP genes by inactivating these genes; 2) to determine the expression patterns of these genes for further definition of their functions during A. variabilis growth and development, and in response to various stress conditions; 3) to determine the proteolytic activities of 5 putative RIP proteases in a reconstituted E. coli system; and 4) to identify specific MTF/RIP protease pairs. Broader Impact The PI has a history of mentoring students and in this project will broaden participation through outreach activities in an established collaboration at Michigan State University with a minority female professor who participates in the Charles Drew program. The Charles Drew Program is a retention and academic enhancement program for high-achieving undergraduate students and graduates from underrepresented minority groups. The PI will mentor students in the program, provide career development guidance to the participants, and serve as a research advisor to interested students. In this project the PI will provide hands-on research experiences to four undergraduates.