BIOCHEMISTRY OF ENERGY DEPENDENT (INTRACELLULAR) PROTEIN DEGRADATION

我们的研究主要集中在结构/功能关系上。 依赖于ATP的CLP和Lon蛋白酶，它们能降解重要的调节作用 大肠杆菌和人的蛋白质以及损伤和变性蛋白质 细胞。野生型Lon可以用牛痘表达 系统，蛋白质被定位到线粒体并进行加工。 人类Lon的加工似乎是自我催化的，因为一个突变体 活性部位丝氨酸残基已被改变的Lon为靶标 到线粒体但未加工和共表达的突变体和 野生型Lon蛋白水解酶可用于突变体的加工。 野生型Lon在HeLa细胞中的表达导致细胞四舍五入 并失去活力，而丝氨酸突变体的表达没有 有害的影响。该系统将被用来定义函数 不同Lon突变体的变化及其生理靶点的确定 人类Lon蛋白水解酶。稳定的人Lon基因转染体已经建立 使用以mdr1为主要可选标记的构造获取。 在这个系统中，失活突变体的高水平表达 不是野生型的Lon已经成为可能。我们克隆并表达了E. Coli ClpX，是CLP ATPase家族的成员。ClpX依赖于ATP 伴侣活性，是某些特定的ATP依赖所必需的 蛋白水解性依赖于ClpP。凝胶过滤和电子 显微镜观察显示，ClpX亚基(Mr 46000)结合形成六个- 通过结合三磷酸腺苷而稳定的成员环(Mr 280,000)。在 在ATP存在下，六聚体ClpX与十四聚体ClpP相互作用 由叠加的七元环组成，形成稳定的络合物 可以通过凝胶过滤将其分离出来。在建筑群中， ClpP的两侧各有一个ClpX环。一种对称 因此，在ClpP的七元环和 ClpXP和ClpAP的六元ATPase环。竞争研究 显示ClpX和ClpA具有几乎相同的结合亲和力 ClpP，但没有证据表明ClpA、ClpX和ClpP的混合络合物 表明特定的ATPase与一个面结合 有利于将类似的ATPase结合到ClpP的相反面上。 寡肽FAPHMALVPV在存在的情况下被ClpXP切割 三磷酸腺苷的非水解性类似物，其周转次数为10000min-1 (根据ClpP的十四聚体)，表明ClpX和ClpA一样， 变构激活影响ClpP使活性部位更多 并增强蛋白质水解物的催化效率 活动站点。对ClpP亚基相互作用的研究表明 环之间的接触可以在不破坏亚基的情况下被破坏 七聚体环内的相互作用。高盐处理 浓度和低温导致可逆分离 ClpP环，可与酶的完全修复重新结合 活动。

Our research is focused on the structure/function relationships of the ATP-dependent Clp and Lon proteases, which degrade important regulatory proteins as well as damaged and denatured proteins in E. coli and human cells. Wild-type Lon can be expressed using a Vaccinia expression system, and the protein is targeted to mitochondria and processed. Processing of human Lon appears to be autocatalytic, because a mutant Lon in which the active site serine residue has been altered is targeted to mitochondria but is not processed and co-expression of the mutant and the wild-type Lon proteases leads to processing of the mutant. Expression of wild-type Lon in HeLa cells causes the cells to round up and lose viability, whereas expression of the serine mutant has no deleterious effects. This system will be exploited to define functional changes in different Lon mutants and to identify physiological targets of human Lon protease. Stable transfectants of human Lon have been obtained using a construct with MDR1 as the dominant selectable marker. In this system also, high level expression of the inactive mutant but not of wild-type Lon has been possible. We have cloned and expressed E. coli ClpX, a member of the Clp family of ATPases. ClpX has ATP-dependent chaperone activity and is required for some specific ATP-dependent proteolytic activities dependent on ClpP. Gel filtration and electron microscopy show that ClpX subunits (Mr 46,000) associate to form a six- membered ring (Mr 280,000) that is stabilized by binding of ATP. In the presence of ATP, hexameric ClpX interacts with ClpP, a tetradecamer composed of superimposed seven-membered rings, to form a stable complex that can be isolated by gel filtration. In the complex, the rings of ClpP are flanked on each side by a single ring of ClpX. A symmetry mismatch thus exists between the seven-membered rings of ClpP and the six-membered ATPase rings for both ClpXP and ClpAP. Competition studies showed that ClpX and ClpA have nearly equal affinity for binding to ClpP, however no evidence for mixed complexes of ClpA, ClpX, and ClpP were observed suggesting that binding of a specific ATPase to one face of ClpP favors binding of a like ATPase to the opposite face of ClpP. The oligopeptide, FAPHMALVPV, is cleaved by ClpXP in the presence of non- hydrolyzable analogs of ATP with a turnover number of 10,000 min-1 (per tetradecamer of ClpP), indicating that ClpX, as does ClpA, allosterically activates affects ClpP to make the active site more accessible and to potentiate the catalytic efficiency of the proteolytic active site. Studies of subunit interactions in ClpP indicate that contacts between rings can be disrupted without breaking the subunit interactions within the heptameric rings. Treatment with high salt concentrations and low temperature lead to reversible separation of the ClpP rings, which can reassociate with complete restoration of enzymatic activity.