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

• 批准号: 9906434
• 负责人: Maria Gaczynska
• 金额: $ 38.93万
• 依托单位: University of Texas Health Science Center San Antonio
• 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: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9906434&HistoricalAwards=false
• 关键词: Multicorn; Example; Regulation; Proteolytic; Activities

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.

Gaczynska9906434该项目的目的是扩大我们对调节大型酶复合物活性的分子机制的了解，并为理解新大型蛋白酶的生物学作用提供基础。 Multicorn是这项研究的主题，这是真核生物中新发现的蛋白酶无处不在。 它与细胞克服蛋白酶体的部分抑制作用的能力有关，并且也可能在细胞周期进程中起作用。受控蛋白水解是在分子水平上协调细胞生理的关键过程之一。 蛋白水解的不可逆性标志着其在其他信号中的独特位置，例如翻译后修饰或寡聚化。 大型的多生蛋白水解复合物特别适合用作调节剂，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状态的这种酶促复合物的最著名例子。 蛋白酶体是由许多可交换亚基建造的，可以通过附加其他蛋白质复合物来调整其活性。尽管进行了许多研究，但我们对这种对酶活性的调节的理解远非完整。 到目前为止，真核蛋白酶体的非常复杂的结构仅允许对控制蛋白酶体作用的机制的相对有限的见解。 在约束中，Multicorn似乎非常适合作为大型酶复合物的模型，用于研究其活性的分子调节。 P.I.实验室的初步数据强烈表明，至少部分地，多层活动可以通过其寡聚化调节。 这项研究的具体目的是：I。克隆裂变酵母多型亚基的基因，并在同型和异源系统中表达它。 ii。 研究多角色的寡聚形式之间的功能差异的结构基础。 iii。 确定催化中心并探索多层寡聚形式之间的功能差异。为了实现这些目标，裂变酵母的多角色，将纯化和生化表征的精神分裂症。 结果表明，酵母多型以两种稳定的寡聚形式存在：大约900 kDa和4,000 kDa。 两种形式的催化能力和底物特异性不同。 两种形式都表现出肽酶活性，但只有大形式是蛋白酶。 已经发现两种形式都是由单个150 kDa多肽组成的。 多型活动和低聚状态的变化，具体取决于细胞的生理状态。鉴于蛋白水解在细胞生理学中的突出性，在分子水平上建立对多角色活性的控制模式将非常重要。 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物在复杂的细胞过程中的调节和调节。这将对我们对如何管理复杂生物催化剂的运行以及大型“蛋白质机器”（如Multicorn和proteasemome）的相互作用如何影响彼此的性能产生广泛的影响。蛋白水解或分解蛋白质以创建具有新特性或回收蛋白质构建块的蛋白质，现在被认为是调节细胞寿命的最重要过程之一。 蛋白水解由称为蛋白酶或蛋白水解酶进行蛋白质。 许多这样的酶充当了大型多生育络合物，因为这样他们的作用可以受到严格的调节，并与细胞的其他脆弱成分进行物理分离。 蛋白水解等事件除其他事件外，决定了细胞是否增殖，是否克服了环境应激或内部异常的影响。 解释蛋白水解的分子机制对于理解细胞生理的调节极为重要。 该项目的目的是为剖析行动的基础，并最终为名为Multicorn的新大蛋白酶的生物学作用。 为了实现目标，P.I.她的同事们从裂变酵母中纯化和生物化学表征了蛋白酶，并发现多型人以两种稳定形式存在不同尺寸的稳定形式。 Multicorn的独特特征是，尽管这两种形式显然是从相同的亚基组成的，但它们的能力分解蛋白质的能力有所不同。 该属性产生了对新蛋白酶作用的精确调节的潜力。 这尤其重要，因为多角的数量和活性在很大程度上取决于酵母的生理状态，这表明该酶在细胞中起着专业作用。 在这项工作中，将建立两种形式的多层形式之间功能差异的分子基础。 收集的知识将有助于更好地理解大型蛋白酶在细胞中的作用和作用。