Biochemistry of Energy-Dependent (Intracellular) Protein Degradation

我们的研究重点是选择性蛋白质降解的机制和负责细胞内蛋白质降解的atp依赖性蛋白酶的结构/功能关系。Lon和Clp蛋白酶存在于所有生物体中，它们有助于调节重要蛋白质的水平，并有助于蛋白质质量控制途径。atp依赖性蛋白酶是与蛋白酶紧密相关的分子伴侣的高分子量复合物。电子显微镜下的ClpAP和ClpXP提供了一个结构模型，作为其他atp依赖性蛋白酶的范例。Clp蛋白酶有两个七元环的ClpP，两侧各有一个六元环的ClpA或ClpX。蛋白水解活性位点位于一个由ClpP环包围的大水腔中。ClpA亚基包裹着另一个水腔，这可能是未折叠的蛋白质在转运到ClpP的蛋白水解腔之前被隔离的位置。底物配合物在易位过程中的电子显微镜图像证实了动力学研究得出的模型。底物从atp酶顶端表面的结合位点迁移到轴向通道上的位置，然后转移到复合物的内部。对于ClpAP，可以在ClpA的内腔内看到一些底物，其余的在ClpP内积累。对于ClpX，底物要么在顶端表面，要么在ClpP内部，这意味着易位是一个快速而协调的过程。通过减缓易位，我们能够证明蛋白质一次只从ClpXP复合体的一侧易位，这表明复合体的两端处于通信中，并调节了一个往复的易位机制。有限的蛋白质水解表明，ClpX折叠成三个结构域，一个n端结构域，可以从蛋白质中移除而不破坏其余的全酶复合物，以及两个类似于所有AAA家族成员的atp酶结构域亚段。n端可能在促进atp依赖性的展开或底物易位中起作用，但不是激活小蛋白质或肽降解所必需的。有限的蛋白水解还发现了Lon蛋白酶中可移动的n端结构域和截断后类似的非atp依赖性活性。特异的Lon n端片段的过度表达会干扰体内依赖于Lon的降解，这意味着n端可能在底物的结合中起作用。生化研究表明，只要蛋白质含有可被ClpX识别的可接近基序，ClpX就可以展开一个稳定的蛋白质。被ClpX识别的蛋白质在展开时结合更紧密，这意味着ClpX也可以与蛋白质的未折叠区域相互作用，但通常ClpX对没有识别基序的未折叠蛋白质没有高亲和力。当存在不可水解的ATP类似物时，未折叠蛋白与ClpX和ClpA结合，但ATP水解促进结合蛋白的释放（与S. Wickner， NCI合作进行的研究）。人ClpP已得到表达和纯化。蛋白质的晶体结构表明，hClpP的折叠方式与大肠杆菌ClpP几乎相同。人酶对肽底物表现出不同的特异性。有趣的是，人类ClpP可以被大肠杆菌ClpX识别的特定底物激活并靶向，这清楚地证明了atp依赖性蛋白酶的降解特异性存在于相关的atp酶中。目前正在进行分离人类ClpX蛋白的研究，并获得可用于抑制体内内源性活性的ClpP突变体。

Our research is focused on the mechanisms of selective protein degradation and the structure/function relationships of the ATP-dependent proteases responsible for intracellular protein degradation. The Lon and Clp proteases are found in all organisms, where they help regulate the levels of important proteins and contribute to protein quality control pathways. ATP-dependent proteases are high molecular weight complexes of a molecular chaperone tightly associated with a protease. Electron microscopy of ClpAP and ClpXP has provided a structural model that serves as a paradigm for other ATP-dependent proteases. Clp proteases have 2 seven-membered rings of ClpP flanked on each side by a six-membered ring of either ClpA or ClpX. The proteolytic active sites are located in a large aqueous chamber enclosed by the rings of ClpP. The ClpA subunits enclose another aqueous chamber which may be the site where unfolded proteins are sequestered prior to translocation to the proteolytic chamber of ClpP. Electron microscopic images of substrate complexes during translocation confirm the model derived from kinetic studies. Substrates migrate from a binding site on the apical surface of the ATPase to a position over an axial channel, and thereafter are transferred to the interior of the complex. For ClpAP, some substrate can be seen within the interior chamber of ClpA, and the remainder accumulates within ClpP. For ClpX, substrate is seen either at the apical surface or within ClpP, implying that translocation is a rapid and concerted process. By slowing translocation, we were able to show that proteins are translocated from only one side of the ClpXP complex at a time, indicating that the ends of the complex are in communication and regulate a reciprocating mechanism of translocation. Limited proteolysis has shown that ClpX folds into three domains, an N-terminal domain that , which can be removed from the protein without disrupting the remainder of the holoenzyme complex, and two sub-sections of the ATPase domain analogous to those found in all AAA family members. The N-terminus may have a role in promoting ATP-dependent unfolding or translocation substrate but is not required for activating degradation of small proteins or peptides. Limited proteolysis also identified a removable N-terminal domain in Lon protease and similar residual non-ATP-dependent activity after truncation. Over-expression of specific N-terminal fragments of Lon interferes with Lon-dependent degradation in vivo, implying that the N-terminus may have a role in binding of substrates. Biochemical studies have shown that ClpX can unfold a stable protein as long as the protein contains an accessible motif recognized by ClpX. Proteins recognized by ClpX bind more tightly when they are unfolded, implying that ClpX can also interact with unfolded regions of proteins, but in general ClpX does not have high affinity for unfolded proteins without some recognition motif. Binding of unfolded proteins to both ClpX and ClpA occurs when a non-hydrolyzable analog of ATP is present, but ATP hydrolysis promotes the release of bound proteins (studies conducted in collaboration with S. Wickner, NCI). Human ClpP has been expressed and purified. The crystal structure of the protein shows that hClpP folds in a virtually identical manner as E. coli ClpP. The human enzyme shows different specificity towards peptide substrates. Interesting, the human ClpP can be activated by and can target specific substrates recognized by E. coli ClpX, providing a clear demonstration that the specificity of degradation by ATP-dependent proteases resides in the associated ATPase. Studies are underway to isolate the human ClpX protein and to obtain mutants of ClpP that can be used to inhibit endogenous activity in vivo.