Biochemistry of Energy-Dependent (Intracellular) Protein Degradation

在蛋白质生物化学部门进行的研究主要集中在细菌和人类细胞中蛋白质降解的功能和控制。细胞内蛋白质降解在控制细胞调节蛋白水平方面起着关键作用，是蛋白质质量控制系统的重要组成部分。细胞质内的蛋白质降解是由atp依赖的蛋白酶进行的。机器的核心是atp驱动的蛋白质展开酶，它结合特定的蛋白质靶标，破坏其结构，并将未折叠的蛋白质易位到紧密相关的自区隔内肽酶的蛋白质水解室中。我们的研究包括对细菌和人类线粒体中atp依赖性Clp和Lon蛋白酶的结构和生化分析，以及它们的生物活性测定。我们专注于三个主要领域：ClpAP选择衬底和接合的结构基础；ClpP的双环结构与其允许未折叠蛋白进入降解室的能力之间的关系；以及应激条件下人类ClpXP在线粒体功能和信号传导中的作用。我们还与合作者一起获得了Lon蛋白酶的高分辨率结构信息。ClpAP负责细菌细胞中n端规则蛋白的降解。这种活性是由一个接头蛋白ClpS介导的，ClpS结合到ClpA的n结构域，并结合到带有N-degrons（标记蛋白质降解的疏水氨基酸）的蛋白质的n端。当蛋白质除了N-degron没有其他降解信号时，ClpS是绝对需要的。含有N-degrons的肽抑制clps介导的降解，但对ClpA本身没有影响。我们发现，尽管存在6个可用于与ClpS相互作用的n结构域，ClpS的单个分子仍优先与ClpA六聚体结合。这一发现可以用两种替代模型来解释：一种是ClpS第一个分子的结合重组了ClpA的n结构域，从而限制了其他ClpS结合位点的可及性；或者，第二种是ClpS第一个分子与n结构域结合，而ClpS的一部分占据了轴向通道，提供了更高的亲和力，并阻断了其他ClpS分子的结合。我们已经证明ClpS的n端20个氨基酸对于增强ClpS的结合和活性是必需的，未来的研究将解决ClpS的n端与ClpA结合的相互作用位点。为了了解含有N-degrons的蛋白质是如何在细胞中产生的，我们将首先测量不同生长条件下的细胞和受到各种应激的细胞中N-degrons的总量。为了寻找带有N-degron的特异性蛋白，我们构建了缺乏其他蛋白酶的突变体，这些蛋白酶将保留特异性靶向N-degron蛋白的能力。我们表达了无活性的ClpP来捕获N-degron蛋白，并通过串联质谱法对其进行鉴定。在ClpA的其他研究中，我们已经生成了D1和D2结构域Walker B共识（部分催化位点）发生改变的ClpA突变体，并表明它们与ATP结合并组装成稳定的复合物。D2突变体尤其缺乏atp酶和其他活性。通过组装野生型ClpA和D2突变体的混合六聚体，我们发现ClpA D2结构域之间的变构相互作用是由底物结合调节的。此外，D2和D1位点之间的负相互作用表明，在atp酶周期中，两个结构域之间的通信需要协调底物加工。D1中ATP水解缺陷的突变体完全无法展开稳定的蛋白质，这表明D1结构域为D2的有效易位准备了底物。在合作研究中，高分辨率低温电子显微镜已被用于检测高移动轴环在ClpA的D2域。为了使通道内的蛋白质相互作用位点在易位过程中参与和释放底物，可能需要移动性。我们还可视化了ClpA结合时ClpP轴向孔的打开，并检测了ClpA与ClpP对接位点接合的环。这些研究为分析ClpAP在底物结合和易位过程中的动力学奠定了基础。在对ClpP的研究中，我们发现ClpP的n端肽影响了四聚体的稳定性。ClpP是两个七聚体的复合体，封闭的四聚体是活性所必需的。ClpP的n端沿轴向通道排列，并从根尖表面突出。n端与表面或轴向通道的相互作用会影响四聚体的活性和稳定性，肽结合也会影响两种七聚体之间的相互作用。这些影响影响了底物通过轴向通道的方式，可以影响ClpP的构象和催化位点的活性，也影响了进入底物的方式，可能影响肽降解产物的排放，从而为新的蛋白质底物清除腔室。在合作研究中，我们发现一种酰基沉积肽抗生素（ADEP）与ClpP结合并影响其十四聚体的稳定性。ADEP激活蛋白质降解的水平与ClpA和ClpX复合物观察到的atp依赖性降解相当。然而，在ADEP存在的情况下，只有未折叠的蛋白才能被ClpP摄取。交联的ClpP，七聚体不能分离，也会被ADEP激活以降解蛋白质，这意味着蛋白质必须通过轴向通道进入，而不是从环之间进入，进一步表明ADEP结合改变了n末端的位置，从而扩大了轴向通道以允许蛋白质进入。对人类ClpXP的研究证实，在DNA损伤、死亡受体结合和激酶抑制的作用下，人类ClpP的过表达会影响凋亡细胞死亡的时间和程度。对三种不同胁迫信号通路的相似反应表明，hClpP改变了线粒体的基本结构或生理。我们发现，在缺乏营养的细胞和siRNA阻断hClpP表达后应激的细胞中，hClpP蛋白丢失。缺乏hClpP的细胞失去线粒体膜的完整性。这些数据表明，hClpP在线粒体生长过程中是必需的，但在非生长条件下被消除。siRNA处理后hClpX的缺失也会导致细胞死亡。hClpX下调引发的线粒体特异性未折叠蛋白反应通过JNK1和JNK2途径起作用。我们在线粒体中表达了一种新的hClpXP底物GFP-SsrA，并表明当hClpP或hClpX从线粒体中消失时，其水平会增加。然而，纯化hClpXP的研究表明，GFP-SsrA是野生型和突变型hClpXP的一个非常差的底物，这表明需要线粒体中的其他因素来帮助介导降解。目前的研究方向是鉴定一种能够激活hClpXP特异性蛋白质降解的衔接蛋白。在合作研究中，我们获得了第一个完整的Lon蛋白酶六聚体的x射线晶体结构。这种结构揭示了lon蛋白酶的几个新方面，并为lon蛋白酶的区隔提供了一个清晰的模型[摘要截断为7800个字符]。

Research conducted in the Biochemistry of Proteins Section is focused on the function and control of protein degradation in bacterial and human cells. Intracellular protein degradation plays a critical part in controlling the levels of cellular regulatory proteins and is an essential part of the protein quality control system. Protein degradation within the cytosol is carried out by ATP-dependent proteases. The core of the machine is an ATP-driven protein unfoldase that binds a specific protein target, disrupts its structure, and translocates the unfolded protein into the proteolytic chamber of a tightly associated self-compartmentalized endopeptidase. Our studies encompass structural and biochemical analysis of the ATP-dependent Clp and Lon proteases from bacteria and from human mitochondria and assays of their biological activities. We have focused on three major areas: the structural basis for substrate selection and engagement by ClpAP; the relationship between the double-ringed structure of ClpP and its ability to allow unfolded proteins to enter the degradation chamber; and the role of human ClpXP in mitochondrial function and signaling under conditions of stress. We have also worked with collaborators to obtain high resolution structural information on Lon protease. ClpAP is responsible for degradation of N-end rule proteins in bacterial cells. This activity is mediated by an adaptor protein, ClpS, which binds to the N-domains of ClpA and binds to the N-terminus of proteins bearing N-degrons (hydrophobic amino acids that mark proteins for degradation). ClpS is absolutely required when proteins have no other degradation signals other than the N-degron. Peptides with N-degrons inhibit ClpS-mediated degradation but have no effect on ClpA itself. We have found that a single molecule of ClpS is preferentially bound to ClpA hexamers, despite the presence of six N-domains that should be available for interaction with ClpS. This finding can be explained by two alternative models: one, binding of the first molecule of ClpS reorganizes the N-domains of ClpA in a way that limits the accessibility of other sites for binding ClpS, or, two, the first ClpS molecule binds to an N-domain and a portion of the ClpS occupies the axial channel, providing increased affinity and occluding the binding of other ClpS molecules. We have shown that the N-terminal 20 amino acids of ClpS is needed for enhanced binding and activity of ClpS and future studies will address the interaction site where the N-terminus of ClpS binds to ClpA. To learn how proteins with N-degrons arise in cells, we will first measure the total amount of N-degrons in cells under different growth conditions and in cells subjected to various kinds of stress. To look for specific proteins with N-degrons, we have constructed mutants lacking other proteases that will retain the ability to specifically target N-degron proteins to ClpAP. We have expressed inactive ClpP to trap N-degron proteins, identify them by tandem mass spectrometry. In other studies of ClpA, we have generated ClpA mutants altered in the Walker B consensus (part of the catalytic site) of the D1 and D2 domains and showed that they bind ATP and assemble into stable complexes. D2 mutants in particular are deficient in ATPase and other activities. By assembling mixed hexamers of wild type ClpA and D2 mutants, we have found that allosteric interactions between the D2 domains of ClpA are modulated by substrate binding. Also, negative interactions between the D2 and D1 sites suggest that communication between the two domains during the ATPase cycle is needed to coordinate substrate processing. Mutants defective in ATP hydrolysis in D1 are completely unable to unfold stable proteins, demonstrating that the D1 domain prepares substrates for efficient translocation by D2. In collaborative studies, high resolution cryo electron microscopy has been used to detect the highly mobile axial loops in the D2 domain of ClpA. Mobility is likely required to enable the protein interaction sites within the channel to engage and release substrates during translocation. We have also visualized the opening of the axial pore of ClpP when ClpA binds and detected the loop of ClpA that engages the docking site on ClpP. These studies set the stage for analysis of the dynamics of ClpAP in the process of substrate binding and translocation. In studies of ClpP, we found that the N-terminal peptide of ClpP influences the stability of the tetradecamer. ClpP is a complex of two heptamers and the closed tetradecamer is needed for activity. The N-termini of ClpP line the axial channel and protrude from the apical surface. Interactions of the N-termini either with the surface or the axial channel affect activity and influence the stability of the tetradecamer, and peptide binding also affects the interaction between the two heptamers. These effects have implications for the way in which substrates passing through the axial channel can affect the conformation of ClpP and the activity of the catalytic site and also for the manner in which entering substrates might affect the discharge of peptide degradation products to clear the chamber for new protein substrates. In collaborative studies, we have found that an acyldepsipeptide antibiotic (ADEP) binds ClpP and affects stability of the tetradecamer. ADEP activates protein degradation to levels comparable to the ATP-dependent degradation observed with ClpA and ClpX complexes. However, only unfolded proteins can be taken up by ClpP in the presence of ADEP. Cross-linked ClpP, in which the heptamers cannot separate, also are activated for protein degradation by ADEP, implying that proteins must enter through the axial channel and not between the rings and further implying that ADEP binding changes the positions of the N-termini in a manner that enlarges the axial channel to permit proteins to enter. Studies of human ClpXP have confirmed that over expression of human ClpP affects the timing and extent of apoptotic cell death in response to DNA damage, death receptor binding, and kinase inhibition. The similarity in response to three divergent stress signaling pathways suggests that hClpP alters the basal structure or physiology of mitochondria. We have found that hClpP protein is lost from nutritionally deprived cells and from cells that are stressed following blockage of hClpP expression with siRNA. Cells lacking hClpP lose mitochondrial membrane integrity. These data suggest that hClpP is needed in mitochondrial during growth but is eliminated under non-growing conditions. Depletion of hClpX following treatment with siRNA also leads to cell death. A mitochondria-specific unfolded protein response elicited by down regulation of hClpX acts through the JNK1 and JNK2 pathways. We have expressed a novel substrate for hClpXP, GFP-SsrA, in mitochondria and shown that its levels are increased when either hClpP or hClpX is depleted from mitochondria. However, studies with purified hClpXP show that GFP-SsrA is a very poor substrate for wild type and mutant forms of hClpXP, suggesting that other factors in the mitochondria are needed to help mediate degradation. Studies are now directed at identifying an adaptor protein that can activate specific protein degradation by hClpXP. In collaborative studies, we have obtained the first X-ray crystal structure of an intact hexamer of Lon protease. This structure reveals several novel aspects of Lon protease and provides a clear model for the compartmentation o [summary truncated at 7800 characters]