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

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
蛋白质生物化学部分进行的研究重点是细菌和人类细胞中蛋白质降解的基本机制和调节。受控的细胞内蛋白质降解至关重要,具有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、自区室化的 ATP 依赖性蛋白酶进行的,这些蛋白酶选择性地筛选潜在靶标,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,这些产物被回收为氨基酸或有时充当各种细胞反应的信号信使和激活剂。我们的努力主要针对 ATP 依赖性 Clp 和 Lon 蛋白酶的结构和生化特性的表征。去年,在多个领域取得了进展。我们(与 NIAMS 的 Bijan Ahvazi 博士合作)完善并发表了人类线粒体 ClpP 的晶体结构,这为 ClpP 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的模型基础。诱变研究表明,从 N 末端删除一个以上氨基酸会导致 ClpP 活性急剧下降或完全丧失。在与 NHLBI 的 Ann Ginsburg 博士合作进行的其他研究中,我们表明人类 ClpP 形成稳定的七聚体,需要伴侣成分 ClpX 才能组装成具有隔离活性位点的双层结构。这些数据表明,人类细胞中的 ClpP 组装可能受到调节,作为控制 ClpP 活性的量或特异性的一种手段。我们已经获得了过度表达活性或非活性突变形式的人类 ClpP 的稳定人类癌细胞系。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。我们将检查线粒体中促凋亡蛋白和抗凋亡蛋白水平的变化,以响应 ClpP 表达的改变。目前正在努力使用 siRNA 技术操纵人 ClpP 的细胞含量,并确定人 ClpX 在迄今为止观察到的 ClpP 细胞反应中的作用。 ClpAP 和 ClpXP 的生化研究提供了有关分子伴侣成分与底物结合的一些有趣的见解。含有 ClpA 或 ClpX 识别的序列基序的短肽已被证明可以与每个六聚体一个肽的化学计量结合。有趣的是,具有被 ClpA 识别的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并适应不同的基序,或者这些位点结构复杂并包含多个结合不同基序的对接位点。由于 ClpA 是六重对称复合物,因此限制与一个肽的结合需要一种机制,以在第一个配体结合后将肽从剩余的等效位点中排除。我们正在研究 Clp 分子伴侣是否会在肽结合上发生构象变化以解释结合的负协同性,或者肽结合位点是否靠近环中心并相互重叠或空间干扰。对接头蛋白 ClpS 的研究表明,ClpS 也以每个六聚体一个 ClpS 的化学计量对 ClpA 发挥作用,该蛋白可以改变 ClpA 的底物偏好。这些数据与早期的晶体结构数据相结合,得出这样的假设:伴侣亚基虽然相同,但不能组装成紧密结合的对称复合物,并且必须经历一些不对称构象变化才能形成闭合的平面环。诱导的不对称可能是必要的,以允许施加在未折叠底物上的力具有不相等的大小或在不同的时间施加,从而提供延伸的多肽通过伴侣的矢量易位的手段。与底物结合的 ClpA 的分析以两种方式进行。我们已经创建了 ClpA 突变体,我们可以在其中引入结构探针,以获得有关构象变化和复合物内部距离以及结合配体和蛋白质位点之间距离的信息。交联实验也有助于识别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 子结构域中。此外,与 LCB、NCI 的 Di Xia 博士合作,获得了与肽底物结合或与 70 kDa 蛋白 RepA 形成复合物的 ClpA 晶体。获得高分辨率衍射质量晶体的方法正在优化。我们获得了 ClpP 的晶体结构,其中肽在活性位点共价连接。该结构提供了 ClpP 室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,以便与 ClpP 共结晶更长的肽底物,以绘制可剪键两侧的相互作用位点。 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 个残基采用独特的折叠,目前正在通过结晶包含预测的卷曲螺旋区域的更完整的 N 结构域来扩展结构分析。 Lon 蛋白酶在体内通过与多种聚合物(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 Alasdair Steven 博士合作,尝试通过冷冻电子显微镜可视化 Lon 与它们的复合物,NIAMS.Budget for 2003-2004 634,594

项目成果

期刊论文数量(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 Protein Degradation
能量依赖性蛋白质降解的生物化学
  • 批准号:
    6558935
  • 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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通过 RNA 干扰改善骨骼肌功能预防衰弱的研究
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CAREER: Investigating the Role of an RNA Interference Pathway in Safeguarding the Tetrahymena Thermophila Somatic Genome
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