Multicorn as an Example of Regulation of Proteolytic Activities of Large Complexes on a Molecular Level

该项目的目的是拓宽我们对调节大型酶复合物活性的分子机制的认识，并为理解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酶。它与细胞克服蛋白酶体部分抑制作用的能力有关，也可能在细胞周期进程中发挥作用。受控蛋白水解是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（如翻译后修饰或寡聚化）中的独特地位。大的、多亚基的蛋白水解复合物特别适合作为调节因子，因为它们的作用可以被细胞内信号精确地控制。蛋白酶体是细胞中具有类似细胞器状态的酶复合物的最著名的例子。蛋白酶体由许多可交换的亚基组成，其活性可以通过附加额外的蛋白质复合物来调节。尽管进行了大量的研究，但我们对酶活性的这种调节的理解还远远不够完整。真核蛋白酶体非常复杂的结构迄今为止只允许对控制蛋白酶体作用的机制有相对有限的了解。相比之下，多角玉米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。来自私家侦探的初步数据他的实验室强烈建议，至少部分地，多玉米的活性可能是由其寡聚化调节的。本研究的具体目的是：1 .克隆分裂酵母多玉米亚基基因，并在同源和异源系统中表达。二世。研究多角玉米低聚形态功能差异的结构基础。三世。鉴定催化中心和探索多角星的低聚形式之间的功能差异。为了实现这些目标，我们将对分裂酵母，裂糖酵母的多粒进行纯化和生化表征。结果表明，酵母多粒存在两种稳定的低聚物形式：约900 kDa和超过4000 kDa。这两种形式在催化能力和底物特异性上有所不同。两种形式都显示肽酶活性，但只有大形式是蛋白酶。发现这两种形式都是由一个150 kDa的多肽组成的。多粒活性和寡聚化状态的变化取决于细胞的生理状态。鉴于蛋白质水解在细胞生理学中的重要地位，在分子水平上建立多角酶活性的控制模式具有重要意义。从这项研究中获得的结果将有助于更好地理解在复杂的细胞过程网络中如何调节大型酶复合物。它将对我们理解如何管理复杂生物催化剂的操作，以及大型“蛋白质机器”（如多角星和蛋白酶体）如何相互作用可能影响彼此的性能产生广泛的影响。蛋白质水解，或分解蛋白质以产生具有新特性的蛋白质或回收蛋白质结构块，现在被认为是调节细胞生命的最重要的过程之一。蛋白质水解是由一种叫做蛋白酶或蛋白水解酶的蛋白质进行的。许多这样的酶作为大的多亚基复合物，因为这样它们的行为可以被严格调节，并与细胞的其他易损成分物理分离。在其他事件中，蛋白质水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。解释蛋白质水解的分子机制对于理解细胞生理调控具有极其重要的意义。该项目的目的是为剖析一种名为多角蛋白的新型大型蛋白酶的作用和最终的生物学作用提供基础。为了实现这一目标，P.I.和她的同事们从裂变酵母中纯化并对蛋白酶进行了生化鉴定，并发现多角玉米粒以两种不同大小的稳定形式存在。多角玉米的独特之处在于，尽管这两种形式显然是由相同的亚基组成的，但它们分解蛋白质的能力不同。这种特性为精确调节新蛋白酶的作用创造了可能。这一点尤其重要，因为多角形式的数量和活性强烈依赖于酵母的生理状态，这表明这种酶在细胞中起着特殊的作用。在这项工作中，将建立两种形式的多角玉米之间功能差异的分子基础。所收集的知识将有助于更好地理解细胞中大型蛋白酶的作用和作用。

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.