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: 8185465
• 负责人: LEE R KROOS
• 金额: $ 31.27万
• 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/8185465
• 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. PUBLIC HEALTH RELEVANCE: Intramembrane-cleaving metalloproteases play important roles in bacteria when they infect humans, so these proteases are potential targets for the development of new antibiotics, and a closely related protease in humans functions in pathways critical for human health, making it a potential target for the development of novel therapeutics. The project aims to understand how intramembrane-cleaving metalloproteases function in bacteria, using the model organism Bacillus subtilis, which is closely related to several Bacillus species and Clostridia that cause disease, as well as to disease-causing bacteria such as Enterococcus faecalis, Staphylococcus aureus, and Streptococcus pneumoniae. Knowledge about how intramembrane-cleaving metalloproteases of Bacillus subtilis function is expected to facilitate studies of these proteases in other bacteria, as well as studies of the human protease.

描述（由申请人提供）：该项目的长期目标是了解金属膜内切割蛋白酶（MIP）如何在细菌中发挥作用。 MIP 是膜嵌入酶，可在膜内或膜表面附近裂解其底物。已知细菌 MIP 在孢子形成、应激反应、交配、极性形态发生、细胞分裂和感染过程中发挥重要作用。了解 MIP 如何在细菌中发挥作用可能有助于新抗生素的开发。在真核生物中，MIP 裂解调节脂质代谢和内质网中未折叠蛋白反应的转录因子。这些途径对人类健康至关重要。有关细菌 MIP 的知识将有助于对真核 MIP 的研究，从而促进新疗法的开发。 关于 MIP 如何识别其底物或如何调节 MIP 活性，人们知之甚少。为了填补这一知识空白，该项目的大部分重点是 SpoIVFB，它在枯草芽孢杆菌孢子形成过程中裂解 Pro-?K。裂解反应已在体外重建并需要 ATP。 ATP 和 Pro-?K 均与 SpoIVFB 的 CBS 结构域结合。 CBS 结构域已被提议用于感知细胞能量水平并调节多种蛋白质的活性。 SpoIVFB 的 CBS 结构域可以感知孢子囊母细胞区室中的能量水平并调节 Pro-αK 进入酶的活性位点。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、结构和遗传方法的组合。同样，提出了多种方法的组合，以实现对 SpoIVFB 天然抑制剂蛋白 BofA 抑制的机制的分子理解。 SpoIVFB 抑制、底物识别和 ATP 作用的研究知识可以指导开发有益于人类健康的 MIP 调节剂。 除 SpoIVFB 外，枯草芽孢杆菌还编码其他三种 MIP。其中研究最多的 RasP 是 MIP 亚家族的代表，该亚家族比 SpoIVFB 亚家族更广泛保守，但尚未报道 RasP 的生化研究。 RasP 亚家族成员包含 PDZ 域，但不包含 CBS 域。与已研究的某些其他含有 PDZ 结构域的 MIP 一样，RasP 在应激反应中发挥作用，并且似乎会裂解抗？抗-初始裂解后的跨膜片段？胞质外结构域。然而，有证据表明 RasP 无需事先裂解即可裂解细胞分裂蛋白。提出了遗传和生化方法来测试这种潜在的新范式。 RasP 的已知底物均不能解释 rasP 突变体的某些缺陷或 RasP 耗竭的影响。提出了一种创新方法来识别 RasP 的未知底物。除了扩展 RasP 的知识之外，该方法还可用于识别其他 MIP 的底物，从而克服该领域进展的关键障碍。 公共健康相关性：膜内切割金属蛋白酶在细菌感染人类时发挥着重要作用，因此这些蛋白酶是开发新抗生素的潜在目标，而人类中的一种密切相关的蛋白酶在对人类健康至关重要的途径中发挥作用，使其成为开发新型疗法的潜在目标。该项目旨在利用模式生物枯草芽孢杆菌了解膜内切割金属蛋白酶如何在细菌中发挥作用，枯草芽孢杆菌与几种引起疾病的芽孢杆菌和梭状芽胞杆菌以及粪肠球菌、金黄色葡萄球菌和肺炎链球菌等致病细菌密切相关。有关枯草芽孢杆菌膜内切割金属蛋白酶如何发挥作用的知识有望促进其他细菌中这些蛋白酶的研究以及人类蛋白酶的研究。