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内膜裂解酶(MIP)如何在细菌中发挥作用。MIPS是一种膜嵌入酶，它在膜内或膜表面附近裂解底物。细菌分子印迹蛋白在孢子形成、胁迫反应、交配、极地形态发生、细胞分裂和感染等过程中发挥重要作用。了解MIP在细菌中的功能可以导致新抗生素的开发。在真核生物中，MIP切割转录因子，这些转录因子调节脂类代谢和对内质网中未折叠蛋白质的反应。这些途径对人类健康至关重要。有关细菌分子印迹聚合物的知识将有助于真核生物分子印迹聚合物的研究，这可能会导致新疗法的发展。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，该项目的大部分重点是SpoIVFB，它在枯草芽孢杆菌产孢期裂解Pro-？K。切割反应已经在体外重组，需要ATP。ATP和Pro-？K都与SpoIVFB的CBS结构域结合。CBS结构域被认为可以感知细胞的能量水平，并调节各种蛋白质的活性。SpoIVFB的CBS结构域可能感知孢子囊母细胞室的能量水平，调节Pro-K对酶活性部位的访问。为了测试这一模型，并更好地理解SpoIVFB如何识别Pro-K，提出了一种生化、结构和遗传方法的组合。同样，我们提出了一系列方法的组合，以实现对SpoIVFB被其天然抑制剂BofA抑制的机制的分子理解。来自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。除了SpoIVFB外，枯草杆菌还编码另外三个MIP。其中研究最多的是rasp，它代表了一个比SpoIVFB亚家族更保守的MIP亚家族，但目前还没有关于rasp的生物化学研究报道。RASP亚家族成员包含PDZ结构域，但不包含CBS结构域。与已研究的其他某些含有PDZ结构域的MIP一样，RASP在应激反应中发挥作用，似乎切割了一种抗？初步切割后的跨膜片段抗？胞外结构域。然而，有证据表明，RASP在没有事先切割的情况下就能切割细胞分裂蛋白。提出了遗传和生化方法来测试这一潜在的新范式。已知的rasp底物都不能解释rasp突变体的某些缺陷，也不能解释rasp耗尽的影响。提出了一种识别RASP未知底物(S)的新方法。除了扩大关于RASP的知识外，该方法还可用于确定其他MIP的底物，从而克服在该领域取得进展的关键障碍。

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.