在上世纪九十年代中期，美国西南医学中心的两位科学家同时也是 1985年诺贝尔生理学奖或医学奖得主 Michael S. Brown 和Joseph L. Goldstein 发现一种在生物膜中工作的蛋白水解酶（intramembrane protease）。膜内水解酶切割底物的穿膜区，释放其在细胞质内的信号蛋白，其进入细胞核内调控基因的转录。这种调控的机制，被称做调节性膜内蛋白酶解（regulated intramembrane proteolysis, RIP)，它影响着从细菌到人类几乎所有的生物，对未折叠蛋白的处理，细胞分化，脂类合成，老年痴呆症等等都与其相关。 膜内蛋白酶因为活性位点和工作机理分为四大类，Rhomboid, Site-2 Protease (S2P), Signal Peptide Peptidase (SPP)和Presenilin。S2P 是一种膜内金属蛋白酶，其活性中心是一个Zn离子，S2P 不仅存在于原核还存在于真核生物中，它在人类的胆固醇代谢，微生物相关疾病中都发挥着至关重要的作用。很多实验表明 S2P 在切割底物必须在另外一种蛋白酶 S1P 切割底物后发生，但是机理尚不明。本文通过体外对大肠杆菌中 S2P 通路的重组，结合生物化学和结构生物学研究，发现底物在被 S1P 切割后，C 端所暴露出来的氨基酸 Val-148 对 S2P 的工作起着至关重要的作用。如果对 C 端所暴露出来的氨基酸进行突变，则会造成 S2P 不能正常酶切底物。通过解析底物末端肽链与 S2P 第二个 PDZ 结构域复合体 1.67Å 的结构，阐释了底物和 S2P 的相互作用。因为 S2P 在生物进化上的保守性，我们推测在高等哺乳动物的S2P 也存在着同样的调控机制。 本文的另一部分内容是关于果蝇细胞凋亡通路的结构生物学研究。细胞凋亡即细胞程序性死亡，广泛存在于各种真核生物中。果蝇的凋亡抑制蛋白DIAP1 存在着一个自抑制的构象，它不能抑制细胞中的 effector caspase drICE 的活性。但是当DIAP1的前20个氨基酸被成熟的drICE所切除后，它便可以抑制drICE的活性，从而促使drICE 泛素化降解。本文通过解析2.4Å的DIAP1－BIR1 结构域结构，阐述了DIAP1 的自抑制机制；通过一系列的生物化学研究并且解析了3.5Å的DIAP1－BIR1 结构域与 drICE 的复合体结构，揭示了 DIAP1 对drICE 的抑制机理。
In the mid-1990s, two scientists Michael S. Brown and Joseph L. Goldstein, the Nobel Prize in Physiology or Medicine Laureates in 1985, found a kind of enzymes that worked in the biological membrane. The substract can be cleaved within the plane of the membrane to liberate cytosolic fragments that enter the nucleus to control gene transcription. This mechanism, called regulated intramembrane proteolysis (RIP), from bacteria to human, influences processes as diverse as the response to unfolded proteins, cellular differentiation, lipid metabolism, and Alzheimer's disease. There are four kinds of intramembrane proteases, Rhomboid, Site-2 Protease (S2P), Signal Peptide Peptidase (SPP) and Presenilin. S2P is a metalloprotease, containing a Zn as the active center. S2P plays a very important role in the metabolism of sterol in human. A central unanswered question is: Why must the action of S2P be preceded by S1P cleavage? We reconstituted sequential, in vitro Escherichia coli S2P pathway. After S1P cleavage, the newly exposed carboxyl-terminal residue Val-148 of substract plays an essential role for S2P cleavage, and its mutation to charged or dissimilar amino acids crippled the Site-2 cleavage. Structural analysis reveals that the putative peptide-binding groove in the second PDZ domain of S2P is poised for binding to a single hydrophobic amino acid. These observations suggest that after S1P cleavage, the newly exposed carboxyl terminus of RseA may facilitate Site-2 cleavage. Because of conserved evolution, we supposed this mechanism also presents in eukaryotic animals. Another part is concerning apoptosis. The Drosophila inhibitor of apoptosis protein DIAP1 exists in an auto-inhibited conformation, unable to suppress the effector caspase drICE. Auto-inhibition is disabled by caspase-mediated cleavage of DIAP1. The cleaved DIAP1 binds to mature drICE, inhibits its protease activity and targets drICE for ubiquitylation. We report a 2.4Å crystal structure of uncleaved DIAP1–BIR1, which reveals the mechanism of auto-inhibition, and a 3.5Å crystal structure of active drICE bound to cleaved DIAP1–BIR1, which provides a structural explanation to DIAP1-mediated inhibition of drICE. These structures and associated biochemical analyses shows how DIAP1 inhibits the activity of drICE.
