Intramembrane-Cleaving metalloproteases of Bacillus subtilis

枯草芽孢杆菌的膜内切割金属蛋白酶

• 批准号: 8308390
• 负责人: LEE R KROOS
• 金额: $ 31.22万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8308390
• 关键词: Accounting; Active Sites; Animal Model; Antibiotics; Bacillus (bacterium); Bacillus subtilis; Bacteria; Binding; Biochemical; Cell division; Cells; Cleaved cell; Clostridium; Code; Complex; Critical Pathways; Defect; Development; Disease; Endoplasmic Reticulum; Enterococcus faecalis; Enzymes; Escherichia coli; Eukaryota; Failure; Gene Expression; Gene Expression Regulation; Genetic; Goals; Grant; Health; Human; In Vitro; Infection; Knowledge; Lead; Length; Life; Mass Spectrum Analysis; Measures; Membrane; Metalloproteases; Modeling; Molecular; Morphogenesis; Mothers; Mutation; Organism; Outcome; Partner in relationship; Peptide Hydrolases; Play; Process; Proteins; Reaction; Reporting; Research; Role; Site; Staphylococcus aureus; Streptococcus pneumoniae; Structure; Surface; Testing; Work; biological adaptation to stress; experience; in vivo; inhibitor/antagonist; innovation; insight; interest; lipid metabolism; member; mutant; novel; novel therapeutics; protease So; reconstitution; response; stem; transcription factor

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand how metallo intramembrane-cleaving proteases (MIPs) function in bacteria. MIPs are membrane-embedded enzymes that cleave their substrates within a membrane or near the membrane surface. Bacterial MIPs are known to play important roles during sporulation, stress responses, mating, polar morphogenesis, cell division, and infection. Understanding how MIPs function in bacteria could lead to the development of new antibiotics. In eukaryotes, MIPs cleave transcription factors that regulate lipid metabolism and the response to unfolded proteins in the endoplasmic reticulum. These pathways are critical for human health. Knowledge about bacterial MIPs will facilitate studies of eukaryotic MIPs, which could lead to the development of novel therapeutics. Little is known about how MIPs recognize their substrates or how MIP activity can be modulated. To fill this knowledge gap, most of the project focuses on SpoIVFB, which cleaves Pro-?K during Bacillus subtilis sporulation. The cleavage reaction has been reconstituted in vitro and requires ATP. Both ATP and Pro-?K bind to the CBS domain of SpoIVFB. CBS domains have been proposed to sense cellular energy levels and regulate the activity of a variety of proteins. The CBS domain of SpoIVFB may sense the energy level in the mother cell compartment of the sporangium and regulate access of Pro-?K to the active site of the enzyme. To test this model and to better understand how SpoIVFB recognizes Pro-?K, a combination of biochemical, structural, and genetic approaches is proposed. Likewise, a combination of approaches is proposed to achieve a molecular understanding of the mechanism of SpoIVFB inhibition by its natural inhibitor, the protein BofA. Knowledge from studies of SpoIVFB inhibition, substrate recognition, and the role of ATP could guide efforts to develop MIP modulators that benefit human health. B. subtilis codes for three other MIPs in addition to SpoIVFB. The most-studied of these, RasP, is representative of a subfamily of MIPs that is even more broadly conserved than the SpoIVFB subfamily, yet no biochemical studies on RasP have been reported. RasP subfamily members contain a PDZ domain and do not contain a CBS domain. Like certain other PDZ-domain-containing MIPs that have been studied, RasP functions in stress response and appears to cleave an anti-? transmembrane segment after initial cleavage of the anti-? extracytoplasmic domain. However, evidence suggests that RasP cleaves a cell division protein without a prior cleavage. Genetic and biochemical approaches are proposed to test this potential new paradigm. Neither of the known substrates of RasP accounts for certain defects of a rasP mutant or for the effects of RasP depletion. An innovative approach is proposed to identify the unknown substrate(s) of RasP. In addition to expanding knowledge of RasP, the approach could be used to identify substrates of other MIPs, overcoming a critical barrier to progress in the field.

描述（由申请人提供）：该项目的长期目标是了解细菌中金属内膜内切割蛋白酶（MIPS）的功能。 MIPS是膜上的酶，将其底物裂解在膜或膜表面附近。已知细菌MIP在孢子形成，压力反应，交配，极性形态发生，细胞分裂和感染过程中起重要作用。了解MIP在细菌中的功能如何导致新抗生素的发展。在真核生物中，MIPS裂解转录因子，这些转录因子调节脂质代谢以及对内质网中展开的蛋白质的反应。这些途径对于人类健康至关重要。关于细菌MIP的知识将促进对真核MIP的研究，这可能导致新型治疗剂的发展。 关于MIP如何识别其底物或如何调节MIP活动的知之甚少。为了填补这一知识差距，大多数项目都集中在SpoiVFB上，在枯草芽孢杆菌孢子芽孢杆菌期间，哪些裂解。切割反应已在体外重构，需要ATP。 ATP和Pro- k均与SpoIVFB的CBS结构域结合。已经提出了CBS结构域来感知细胞能级并调节多种蛋白质的活性。 SPOIVFB的CBS结构域可能会感觉到孢子囊的母细胞室中的能级，并调节对酶的活性位点的访问。为了测试该模型并更好地了解SpoiVFB如何识别pro- K，提出了生化，结构和遗传方法的结合。同样，提出了一种方法的组合来实现其天然抑制剂蛋白质BOFA对SPOIVFB抑制的机理的分子理解。来自SPOIVFB抑制作用，底物识别以及ATP的作用的知识可以指导开发有益于人类健康的MIP调节剂的努力。 B.枯草厂除SPOIVFB外还代码其他三个MIP。其中最受研究的RASP代表了MIP的亚科，比SPOIVFB亚科更广泛地保守，但尚无关于RASP的生化研究。 RASP亚家族成员包含一个PDZ域，不包含CBS域。像某些已研究的其他含PDZ域的MIP一样，RASP在压力反应中起作用，并且似乎可以裂开抗？抗 - 抗裂解后的跨膜段？外质域。但是，有证据表明，RASP在没有先前裂解的情况下切开细胞分裂蛋白。提出了遗传和生化方法来测试这种潜在的新范式。 RASP的已知底物均未说明RASP突变体的某些缺陷或RASP耗竭的作用。提出了一种创新的方法来识别RASP的未知底物。除了扩大RASP知识外，该方法还可以用于识别其他MIP的底物，从而克服了该领域进步的关键障碍。