Intramembrane-Cleaving metalloproteases of Bacillus subtilis

描述（由申请人提供）：本项目的长期目标是了解金属膜内裂解蛋白酶（MIP）在细菌中的功能。MIP是膜包埋的酶，其在膜内或膜表面附近切割其底物。已知细菌MIPs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MIPs在细菌中的作用方式可能会导致新抗生素的开发。在真核生物中，MIP切割调节脂质代谢和对内质网中未折叠蛋白的反应的转录因子。这些途径对人类健康至关重要。对细菌MIP的了解将促进真核MIP的研究，这可能导致新疗法的开发。 关于MIP如何识别它们的底物或MIP活性如何被调节的知之甚少。为了填补这一知识空白，该项目的大部分重点是SpoIVFB，它分裂Pro-？枯草芽孢杆菌产孢过程中的K。裂解反应已在体外重建，需要ATP。ATP和Pro？K与SpoIVFB的CBS结构域结合。CBS结构域已被提出用于感知细胞能量水平并调节多种蛋白质的活性。SpoIVFB的CBS结构域可能感知孢子囊母细胞室的能量水平，并调节Pro-？酶的活性位点。为了测试这个模型，并更好地了解如何SpoIVFB识别亲？K，生物化学，结构和遗传方法的组合提出。同样，提出了一种方法的组合，以实现SpoIVFB抑制其天然抑制剂，蛋白质BofA的机制的分子理解。从SpoIVFB抑制，底物识别和ATP的作用的研究中获得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。除了SpoIVFB之外，还使用了其他三种MIP的subtilis代码。其中研究最多的是RasP，它代表了一个比SpoIVFB亚家族更为保守的MIP亚家族，但还没有关于RasP的生物化学研究报道。RasP亚家族成员含有PDZ结构域，不含CBS结构域。像某些其他PDZ结构域已被研究的MIP，RasP的功能在应激反应，并出现切割抗？跨膜段后，初始切割的抗？胞质外结构域然而，有证据表明，RasP切割细胞分裂蛋白，而无需预先切割。遗传和生物化学的方法，提出了测试这种潜在的新范式。RasP的已知底物都不能解释rasP突变体的某些缺陷或rasP缺失的影响。提出了一种新的方法来识别RasP的未知底物。除了扩展RasP的知识外，该方法还可用于识别其他MIP的底物，克服该领域进展的关键障碍。

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.