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结构/功能关系的ATP依赖性蛋白酶负责细胞内蛋白质降解。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结合的分子伴侣的高分子量复合物。电子显微镜的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蛋白酶具有2个ClpP的七元环，每侧上侧接ClpA或ClpX的六元环。蛋白水解活性位点位于由ClpP的环包围的大的水性室中。ClpA亚基包围另一个水室，该水室可能是未折叠的蛋白质在易位到ClpP的蛋白水解室之前被隔离的位点。移位过程中底物复合物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酶顶端表面上的结合位点迁移到轴向通道上方的位置，然后转移到复合物的内部。对于ClpAP，可以在ClpA的内室内看到一些底物，其余的在ClpP内积累。对于ClpX，在顶端表面或ClpP内观察到底物，这意味着易位是一个快速和协调的过程。通过减慢易位，我们能够表明蛋白质一次仅从ClpXP复合物的一侧易位，表明复合物的末端处于通信中并调节易位的往复机制。有限的蛋白水解显示ClpX折叠成三个结构域，N-末端结构域，其可以从蛋白质中去除而不破坏全酶复合物的其余部分，以及ATP酶结构域的两个亚部分，其类似于在所有AAA家族成员中发现的那些。N-末端可能在促进ATP依赖性解折叠或易位底物中起作用，但对于激活小蛋白或肽的降解不是必需的。有限的蛋白水解也确定了一个可移动的N-末端结构域的Lon蛋白酶和类似的残留非ATP依赖性的活性截短后。过表达的特定N-末端片段的Lon干扰Lon-dependent降解在体内，这意味着N-末端可能有一个作用，在结合底物。生物化学研究表明，ClpX可以展开一个稳定的蛋白质，只要该蛋白质含有一个可接近的基序识别的ClpX。ClpX识别的蛋白质在未折叠时结合更紧密，这意味着ClpX也可以与蛋白质的未折叠区域相互作用，但一般来说，ClpX对没有一些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，未折叠蛋白质与ClpX和ClpA结合，但ATP水解促进结合蛋白质的释放（与S. Wickner，NCI）。 人ClpP已被表达和纯化。 蛋白质的晶体结构显示hClpP以与E. coli ClpP. 人酶对肽底物显示出不同的特异性。 有趣的是，人ClpP可以被E. coliClpX，提供了一个明确的证明，由ATP依赖性蛋白酶降解的特异性驻留在相关的ATP酶。正在进行研究以分离人ClpX蛋白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.