Multicorn as an Example of Regulation of Proteolytic Activities of Large Complexes on a Molecular Level
以多角蛋白为例在分子水平上调节大型复合物的蛋白水解活性
基本信息
- 批准号:9906434
- 负责人:
- 金额:$ 38.93万
- 依托单位:
- 依托单位国家:美国
- 项目类别:Continuing Grant
- 财政年份:1999
- 资助国家:美国
- 起止时间:1999-07-01 至 2003-06-30
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
Gaczynska9906434The aim of this project is to broaden our knowledge about molecular mechanisms regulating activities of large enzymatic complexes and to provide the basis for understanding the biological role of a new large protease. Multicorn is the subject of this study, which is a newly discovered protease ubiquitous among Eukaryotes. It is associated with the ability of cells to overcome the effects of partial inhibition of the proteasome, and may also play a role in the cell cycle progression. Controlled proteolysis is one of the key processes coordinating cellular physiology on the molecular level. Irreversibility of proteolysis marks its unique position among other signals, like posttranslational modifications or oligomerization. Large, multisubunit proteolytic complexes are especially well suited to serve as regulators because their actions can be precisely controlled by intracellular signals. The proteasome is the best-known example of such enzymatic complexes of organelle-like status in the cell. The proteasome is built from numerous exchangeable subunits, and its activities can be adjusted by attaching additional protein complexes. Despite numerous studies our understanding of such regulation of enzymatic activities is far from complete. The very complicated structure of the eukaryotic proteasome has thus far allowed only a relatively limited insight into mechanisms which govern proteasomal actions. In constrast, multicorn seems to be very well suited to the role as a model of a large enzymatic complex for studies on molecular regulation of its activities. Preliminary data from the P.I.'s lab strongly suggest that, at least partially, the multicorn activities may be modulated by its oligomerization. The specific aims of this study are to: I. Clone the gene of the fission yeast multicorn subunit and express it in homo- and heterologous systems. II. Study the structural basis for functional differences between the oligomeric forms of the multicorn. III. Identify catalytic centers and explore functional differences between oligomeric forms of the multicorn. To accomplish these goals, the multicorn from fission yeast, Schizosaccharomyces pombe will be purified and biochemically characterized. It was shown that the yeast multicorn exists in two stable oligomeric forms: about 900 kDa and more than 4,000 kDa. The two forms differ in their catalytic abilities and substrate specificities. Both forms display peptidase activity, but only the large form is a proteinase. Both forms have been found to be composed from a single 150 kDa polypeptide. Multicorn activities and oligomerization status change depending on the physiological status of the cell. In the light of the prominence of proteolysis in cellular physiology it will be very important to establish the modes of control of the multicorn activities on the molecular level. Results to be obtained from this study will contribute to better understanding of how large enzymatic complexes are regulated in and by the intricate web of cellular processes. It will have a broad impact on our understanding of how to manage the operation of complex biological catalysts, and how interplay of large "protein machines", like the multicorn and the proteasome, may affect each others' performance. Proteolysis, or breaking down proteins to create proteins with new properties or to recycle the protein building blocks, is now considered one of the most important processes regulating the life of the cell. Proteolysis is performed by proteins called proteases, or proteolytic enzymes. Many such enzymes act as large multisubunit complexes because this way their actions can be tightly regulated and physically separated from other vulnerable components of the cell. Proteolysis, among other events, decides if the cell proliferates, if it overcomes the effects of environmental stress or internal abnormalities. Explaining molecular mechanisms of proteolysis is extremely important for understanding the regulation of cellular physiology. The aim of this project is to provide the basis for dissecting the actions and, ultimately, the biological role of a new large protease named the multicorn. To reach the goal, the P.I. and her colleagues have purified and biochemically characterized the protease from fission yeast, and have found that the multicorn exists in two stable forms of different size. The unique feature of the multicorn is that, although the two forms are composed from apparently the same subunit, they differ in their abilities to break down proteins. This property creates a potential for precise regulation of the actions of the new protease. This is especially important because the amount and activity of the multicorn forms strongly depend on the physiological status of the yeast, which suggest that the enzyme plays a specialized role in the cell. In this work, the molecular basis for the functional differences between the two forms of the multicorn will be established. The knowledge gathered will contribute to better understanding of the role and actions of large proteases in the cell.
Gaczynska 9906434这个项目的目的是扩大我们对大型酶复合体活性调节分子机制的了解,并为理解一种新的大型蛋白酶的生物学作用提供基础。多角蛋白是一种在真核生物中普遍存在的新发现的蛋白水解酶。它与细胞克服部分抑制蛋白酶体的影响的能力有关,也可能在细胞周期进程中发挥作用。受控蛋白降解是在分子水平上协调细胞生理的关键过程之一。蛋白质降解的不可逆性标志着它在其他信号中的独特位置,如翻译后修饰或寡聚。大的、多亚单位的蛋白分解复合体特别适合作为调节剂,因为它们的作用可以由细胞内信号精确控制。蛋白酶体是细胞中细胞器样状态的这种酶复合体的最著名的例子。蛋白酶体是由许多可交换的亚基组成的,其活性可以通过附加蛋白复合体来调节。尽管有大量的研究,但我们对酶活性的这种调控的了解还远远不完整。到目前为止,真核蛋白酶体的非常复杂的结构使得人们对控制蛋白酶体活动的机制只有相对有限的了解。相比之下,多角兽似乎非常适合作为大型酶复合体的模型来研究其活性的分子调控。来自P.I.S实验室的初步数据强烈表明,至少在一定程度上,多角兽的活动可能受到其寡聚的调节。本研究的具体目的是:1.克隆裂解酵母多角亚单位基因,并在同源和异源系统中表达。II.研究多角体寡聚形式之间功能差异的结构基础。确定催化中心并探索多角体寡聚形式之间的功能差异。为了实现这些目标,将从裂殖酵母中分离出多角裂殖酵母,并对其进行生化特征分析。结果表明,酵母多角体以约900 kDa和4000 kDa以上两种稳定的寡聚体形式存在。这两种形式的催化能力和底物特性不同。这两种形式都显示了多肽酶活性,但只有大的形式是一种蛋白酶。这两种形式都被发现由一个150 kDa的多肽组成。多角体活性和寡聚状态取决于细胞的生理状态。鉴于蛋白质分解在细胞生理学中的重要性,在分子水平上建立多角体活性的调控模式将是非常重要的。从这项研究中获得的结果将有助于更好地理解大型酶复合体是如何在细胞过程的复杂网络中调节的,并受其调控。这将对我们理解如何管理复杂生物催化剂的运行,以及大型蛋白质机器的相互作用,如多角体和蛋白酶体,可能如何影响彼此的性能产生广泛的影响。蛋白质分解,即分解蛋白质以产生具有新性质的蛋白质或回收蛋白质构建块,现在被认为是调节细胞生命的最重要的过程之一。蛋白水解酶是由一种叫做蛋白水解酶的蛋白质来完成的。许多这样的酶充当大的多亚单位复合体,因为这样它们的活动可以受到严格的调控,并在物理上与细胞的其他脆弱成分分开。在其他事件中,蛋白质分解决定了细胞是否增殖,是否克服了环境压力或内部异常的影响。解释蛋白质分解的分子机制对于理解细胞生理的调节是极其重要的。这个项目的目的是为剖析这些作用并最终了解一种名为多角体的新的大型蛋白酶的生物学作用提供基础。为了达到这一目标,P.I.和她的同事们提纯了从分裂酵母中提取的蛋白酶,并对其进行了生化特征分析,发现这种多角体以两种不同大小的稳定形式存在。多角兽的独特之处在于,尽管这两种形式显然由相同的亚基组成,但它们分解蛋白质的能力不同。这一特性为精确调控新的蛋白水解酶的行为创造了可能性。这一点尤其重要,因为多角体的数量和活性强烈依赖于酵母的生理状态,这表明该酶在细胞中发挥着特殊的作用。在这项工作中,将建立两种形式的多角兽之间功能差异的分子基础。收集到的知识将有助于更好地了解大型蛋白酶在细胞中的作用和行动。
项目成果
期刊论文数量(0)
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科研奖励数量(0)
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Maria Gaczynska其他文献
Scanning Probe Microscopy of Serpin Polymers
- DOI:
10.1016/j.bpj.2011.11.3208 - 发表时间:
2012-01-31 - 期刊:
- 影响因子:
- 作者:
Pawel A. Osmulski;Maria Gaczynska;Przemek Karpowicz;Srividya Madabhushi;Monika Tokmina-Lukaszewska;Christine Stuart;Wesley Jong;Malgosia Mikolajczyk;Martha C. Anderson;Ewa Marszal - 通讯作者:
Ewa Marszal
γ-Interferon and expression of MHC genes regulate peptide hydrolysis by proteasomes
γ-干扰素和 MHC 基因的表达调节蛋白酶体对肽的水解
- DOI:
10.1038/365264a0 - 发表时间:
1993-09-16 - 期刊:
- 影响因子:48.500
- 作者:
Maria Gaczynska;Kenneth L. Rock;Alfred L. Goldberg - 通讯作者:
Alfred L. Goldberg
The binding of a polyclonal antibody against human band 3to <em>in vitro</em> aged erythrocytes
- DOI:
10.1016/s0065-1281(11)80249-7 - 发表时间:
1990-01-01 - 期刊:
- 影响因子:
- 作者:
Matthias H.F. Klinger;Karl-Jurgen Halbhuber;Werner Linss;Herwart Feuerstein;Maria Gaczynska - 通讯作者:
Maria Gaczynska
Macrophages Support the Aggressive Mechanical Phenotype of Circulating Tumor Cells in Prostate Cancer
- DOI:
10.1016/j.bpj.2020.11.619 - 发表时间:
2021-02-12 - 期刊:
- 影响因子:
- 作者:
Pawel A. Osmulski;Alessandra Cunsolo;Yusheng Qian;Meizhen Chen;Chun-Lin Lin;Chia-Nung Hung;Devalingam Mahalingam;Nameer Kirma;Chun-Liang Chen;Josephine Taverna;Michael Liss;Ian M. Thompson;Tim H. Huang;Maria Gaczynska - 通讯作者:
Maria Gaczynska
Cells in motion: model of circulating tumor cells in prostate cancer
- DOI:
10.1016/j.bpj.2021.11.673 - 发表时间:
2022-02-11 - 期刊:
- 影响因子:
- 作者:
Yusheng Qian;Pawel A. Osmulski;Maria Gaczynska - 通讯作者:
Maria Gaczynska
Maria Gaczynska的其他文献
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