Biochemistry of Energy-Dependent (Intracellular) Protein
能量依赖性(细胞内)蛋白质的生物化学
基本信息
- 批准号:7038580
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:Escherichia coliRNA interferenceactive sitesadenosine triphosphatebacterial proteinsbioenergeticscell lineconformationcrosslinkelectron microscopyendopeptidasesenzyme activityenzyme complexenzyme structureenzyme substrategene expressionintermolecular interactionintracellularmitochondriamolecular chaperonesmutantpolymersprotein bindingprotein degradationprotein structure function
项目摘要
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. These data combined with earlier crystal structure data lead to a hypothesis that the chaperone subunits, though identical, cannot assemble into tightly bonded symmetrical complexes and must undergo some asymmetric conformation change to allow formation of a closed planar ring. Induced asymmetry may be necessary to allow the forces exerted on the unfolded substrates to be of unequal magnitude or to be applied at separate times providing a means of vectorial translocation of the extended polypeptide through the chaperone. Analysis of ClpA bound to substrates is being carried out in two ways. We have created mutants of ClpA into which we can introduce structural probes to obtain information about conformational changes and internal distances in the complexes and distances between bound ligands and sites in the protein. Cross-linking experiments are also contributing to identification of residues in the vicinity of substrate interaction sites. Preliminary cross-linking data indicates that the initial peptide binding site is in the large AAA subdomain of ClpX and ClpA-NBD1. Also, in collaboration with Dr. Di Xia, LCB, NCI, crystals of ClpA with peptide substrate bound or in a complex with the 70 kDa protein, RepA, have been obtained. Optimization of methods to obtain high resolution diffraction quality crystals is underway. We have obtained a crystal structure of ClpP with a peptide covalently linked at the active site. This structure provides the first view of the interaction of substrates in the peptide binding groove within the ClpP chamber. This work is still in progress, and we are extending these studies by the use of mutants that have low catalytic turnover rates in order to co-crystallize longer peptide substrates with ClpP to map the interaction sites on both sides of the scissile bond. Studies with Lon protease have provided the first ever structural data on the N-terminal domain. We have found that the first 120 residues of Lon adopt a unique fold and are currently extending the structural analysis by crystallizing a more complete N-domain containing the predicted coiled-coil region. Lon protease is activated in vivo by interaction with several polymers, including polyphosphate and some nucleic acids. We are currently mapping the domain of Lon responsible for interaction with these polymers and will try to visualize the complexes of Lon with them by cryo electron microscopy in collaboration with Dr. Alasdair Steven, NIAMS.Budget for 2003-2004 634,594
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。这些数据与早期的晶体结构数据相结合,导致了一个假设,即伴侣亚基虽然相同,但不能组装成紧密结合的对称复合物,必须经历一些不对称的构象变化,才能形成一个封闭的平面环。诱导不对称可能是必要的,以允许施加在未折叠底物上的力的大小不等或在不同的时间施加,从而提供通过伴侣的延伸多肽的矢量易位的手段。对与底物结合的ClpA的分析有两种方法。我们已经创建了ClpA突变体,我们可以在其中引入结构探针来获得有关构象变化和复合物内部距离以及结合配体与蛋白质中位点之间距离的信息。交联实验也有助于鉴定底物相互作用位点附近的残基。初步交联数据表明,初始肽结合位点位于ClpX和ClpA-NBD1的大AAA亚结构域。此外,在与Dr. Di Xia, LCB, NCI的合作下,已经获得了与肽底物结合或与70 kDa蛋白RepA复合物的ClpA晶体。获得高分辨率衍射质量晶体的方法正在优化中。我们得到了ClpP的晶体结构,在活性位点有一个共价连接的肽。这种结构提供了ClpP腔内肽结合槽中底物相互作用的第一个视图。这项工作仍在进行中,我们正在通过使用具有低催化周转率的突变体来扩展这些研究,以便将较长的肽底物与ClpP共结晶,以绘制可剪键两侧的相互作用位点。对Lon蛋白酶的研究首次提供了n端结构域的结构数据。我们发现Lon的前120个残基采用独特的褶皱,目前通过结晶一个更完整的n域来扩展结构分析,该n域包含预测的线圈-线圈区域。Lon蛋白酶在体内通过与几种聚合物(包括多磷酸盐和一些核酸)相互作用而被激活。我们目前正在绘制负责与这些聚合物相互作用的Lon域,并将与NIAMS的Alasdair Steven博士合作,尝试通过低温电子显微镜将Lon与它们的复合物可视化。2003-2004年预算634,594
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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MICHAEL MAURIZI其他文献
MICHAEL MAURIZI的其他文献
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{{ truncateString('MICHAEL MAURIZI', 18)}}的其他基金
The ClpP protease as a therapeutic target in bacterial and mammalian cells
ClpP 蛋白酶作为细菌和哺乳动物细胞的治疗靶点
- 批准号:
8938126 - 财政年份:
- 资助金额:
-- - 项目类别:
Biochemistry of Energy-Dependent (Intracellular) Protein Degradation
能量依赖性(细胞内)蛋白质降解的生物化学
- 批准号:
7592538 - 财政年份:
- 资助金额:
-- - 项目类别:
Biochemistry of Energy-Dependent (Intracellular) Protein
能量依赖性(细胞内)蛋白质的生物化学
- 批准号:
7337911 - 财政年份:
- 资助金额:
-- - 项目类别:
Biochemistry of Energy-Dependent (Intracellular) Protein Degradation
能量依赖性(细胞内)蛋白质降解的生物化学
- 批准号:
6433041 - 财政年份:
- 资助金额:
-- - 项目类别:
Biochemistry of Energy-Dependent (Intracellular) Protein Degradation
能量依赖性(细胞内)蛋白质降解的生物化学
- 批准号:
8937640 - 财政年份:
- 资助金额:
-- - 项目类别:
Biochemistry of Energy-Dependent (Intracellular) Protein Degradation
能量依赖性(细胞内)蛋白质降解的生物化学
- 批准号:
8762996 - 财政年份:
- 资助金额:
-- - 项目类别:
The ClpP protease as a therapeutic target in bacterial and mammalian cells
ClpP 蛋白酶作为细菌和哺乳动物细胞的治疗靶点
- 批准号:
8763529 - 财政年份:
- 资助金额:
-- - 项目类别:
Biochemistry of Energy-Dependent (Intracellular) Protein Degradation
能量依赖性(细胞内)蛋白质降解的生物化学
- 批准号:
8157185 - 财政年份:
- 资助金额:
-- - 项目类别:
The ClpP protease as a therapeutic target in bacterial and mammalian cells
ClpP 蛋白酶作为细菌和哺乳动物细胞的治疗靶点
- 批准号:
8553191 - 财政年份:
- 资助金额:
-- - 项目类别:
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