Biochemistry of Energy-Dependent (Intracellular) Protein

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基本机制和调节。控制细胞内蛋白质降解是至关重要的，服务于调节和蛋白质质量控制功能。大多数细胞内蛋白质降解是由多组分、自区隔的atp依赖蛋白酶进行的，它选择性地筛选潜在的靶标，控制对隔离蛋白水解位点的访问，并可以产生离散的降解产物，这些降解产物被循环利用为氨基酸，或者有时作为各种细胞反应的信号信使和激活剂。我们的研究主要针对atp依赖性Clp和Lon蛋白酶的结构和生化特性的表征。去年，在几个领域取得了进展。我们（与NIAMS的Bijan Ahvazi博士合作）改进并发表了人类线粒体ClpP的晶体结构，这为ClpP的n端肽在调节或促进底物通过易位通道进入活性位点室中的作用提供了独特模型的基础。诱变研究表明，从n端删除一个以上的氨基酸会导致ClpP活性急剧下降或完全丧失。在与Ann Ginsburg博士（NHLBI）合作完成的其他研究中，我们已经表明，人类ClpP形成稳定的七聚体，需要伴侣成分ClpX组装成具有隔离活性位点的双层结构。这些数据表明，ClpP的组装可能在人类细胞中作为控制ClpP活性的量或特异性的一种手段而受到调节。我们已经获得了稳定的人类癌细胞系，过表达人类ClpP的活性或非活性突变形式。初步数据表明，过量的人ClpP对顺铂诱导的细胞凋亡的时间或程度有影响。我们将检测线粒体中促凋亡和抗凋亡蛋白水平的变化，以响应ClpP表达的改变。目前正在努力利用siRNA技术操纵人类ClpP的细胞含量，并确定迄今为止观察到的人类ClpX在细胞对ClpP的反应中的作用。ClpAP和ClpXP的生化研究提供了一些关于伴侣成分结合底物的有趣见解。含有ClpA或ClpX识别的序列基序的短肽已被证明与每六聚体一个肽的化学计量结合。有趣的是，ClpA识别的具有不同序列基序的肽会竞争结合，这表明肽相互作用位点可能是可变形的，可以适应不同的基序，或者这些位点结构复杂，包含多个结合不同基序的对接位点。由于ClpA是一个六重对称复合体，限制与一个肽的结合需要一种机制，在与第一个配体结合后，将肽排除在剩余的等效位点之外。我们正在研究Clp伴侣是否在肽结合上经历构象变化以解释负合作性的结合，或者肽结合位点是否靠近环的中心并相互重叠或空间干扰。对可以改变ClpA底物偏好的接头蛋白ClpS的研究表明，ClpS对ClpA的影响也以每六聚体一个ClpS的化学计量量产生。

Research conducted in the Biochemistry of Proteins Section is focused on basic mechanisms and regulation of protein degradation in bacterial and human cells. Controlled intracellular protein degradation is vitally important, serving both regulatory and protein quality control functions. Most intracellular protein degradation is carried out by multi-component, self-compartmentalized ATP-dependent proteases, which selectively screen potential targets, control access to sequestered proteolytic sites, and can generate discrete degradation products that are recycled to amino acids or sometimes serve as signaling messengers and activators of various cellular responses. Our efforts have been directed toward characterization of the structural and biochemical properties of the ATP-dependent Clp and Lon proteases. In the last year, progress has been made in several areas. We have (in collaboration with Dr. Bijan Ahvazi, NIAMS) refined and published the crystal structure of human mitochondrial ClpP, which has provided the basis for a unique model for the role of the N-terminal peptide of ClpP in regulating or facilitating the passage of substrates through the translocation channel into the active site chamber. Mutagenesis studies have shown that deletion of more than one amino acid from the N-terminus leads to a dramatic decrease or total loss of ClpP activity. In other studies done in collaboration with Dr. Ann Ginsburg, NHLBI, we have shown that human ClpP forms stable heptamers that require the chaperone component, ClpX, to assemble into the bilayered structure with sequestered active sites. These data suggest that assembly of ClpP may be regulated in human cells as a means of controlling the amount or the specificity of ClpP activity. We have obtained stable human cancer cell lines over-expressing either active or inactive mutant forms of human ClpP. Preliminary data suggests that excess human ClpP has effect on the timing or extent of cisplatin-induced apoptosis. We will examine changes in the level of pro- and anti-apoptotic proteins in mitochondria in response to altered expression of ClpP. Efforts are underway to manipulate the cellular content of human ClpP using siRNA techniques and to determine the role of human ClpX on the cellular responses to ClpP so far observed. Biochemical studies of ClpAP and ClpXP have provided several intriguing insights regarding substrate binding by the chaperone component. Short peptides containing sequence motifs recognized by ClpA or ClpX have been shown to bind with a stoichiometry of one peptide per hexamer. Interesting, peptides with different sequence motifs recognized by ClpA compete for binding, indicating that peptide interaction sites may be deformable and adaptable to different motifs or that the sites are structurally complex and contain multiple docking sites that bind different motifs. Because ClpA is a six-fold symmetric complex, limiting binding to one peptide requires a mechanism to exclude peptides from the remaining equivalent sites following binding of the first ligand. We are investigating whether Clp chaperones undergo a conformational change on peptide binding to explain negative cooperativity of binding or whether the peptide binding sites lie close together near the center of the ring and either overlap or sterically interfere with each other. Studies with the adaptor protein, ClpS, which can alter the substrate preference of ClpA, show that ClpS exerts its effect on ClpA also at a stoichiometry of one ClpS per hexamer.