Intramembrane-Cleaving metalloproteases of Bacillus subtilis

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（MIPs）在细菌中的作用。MIPs是膜内嵌入酶，在膜内或膜表面附近切割底物。已知细菌MIPs在产孢、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内质网对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。关于细菌MIPs的知识将促进真核生物MIPs的研究，这可能导致新的治疗方法的发展。关于MIP如何识别它们的底物或如何调节MIP活性，人们知之甚少。为了填补这一知识空白，大部分项目都集中在SpoIVFB上，它将Pro-？枯草芽孢杆菌产孢过程中的K。在体外重建了裂解反应，需要ATP。ATP和Pro-？K绑定到SpoIVFB的CBS域。CBS结构域被认为可以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-？K到酶的活性位点。为了测试这个模型并更好地理解SpoIVFB如何识别Pro-？K，生物化学，结构和遗传方法的结合提出。同样，提出了多种方法的组合，以实现对SpoIVFB天然抑制剂BofA蛋白抑制机制的分子理解。从SpoIVFB抑制、底物识别和ATP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。除SpoIVFB外，枯草芽孢杆菌还编码另外三种MIPs。其中研究最多的是RasP，它是MIPs亚家族的代表，比SpoIVFB亚家族保守得更广泛，但尚未有关于RasP的生化研究报道。RasP子家族成员包含PDZ域，而不包含CBS域。与已研究的其他含有pdz结构域的mip一样，RasP在应激反应中起作用，似乎可以裂解抗-？抗-?初始裂解后的跨膜段extracytoplasmic域。然而，有证据表明RasP在没有事先切割的情况下切割细胞分裂蛋白。遗传学和生物化学的方法被提出来测试这种潜在的新范式。两种已知的RasP底物都不能解释RasP突变体的某些缺陷或RasP耗尽的影响。提出了一种识别RasP未知底物的新方法。除了扩展RasP的知识外，该方法还可用于识别其他MIPs的底物，克服了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.