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New regulation mechanism of polyamine biosynthesis mediated by ribosomal Protein, L10 as an antizyme

核糖体蛋白L10作为抗酶介导多胺生物合成的新调控机制

基本信息

批准号：
20380054
负责人：
KAMIO Yoshiyuki
金额：
$ 12.73万
依托单位：
Shokei Gakuin College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2010
项目状态：
已结题

项目摘要

Selenomonas ruminantium, which is a strictly anaerobic, Gram-negative bacterium has neither free nor bound forms of lipoprotein which plays an important role of the maintenance of the structural integrity of the cell surface in general Gram-negative bacteria. Instead, it has cadaverine, which links covalently to the pepticloglycan as a pivotal constituent for the cell devision. In S. rmuinantimn cadaverine is synthesized constitutively from L-lysine by lysine decarboxylase (LDC) and transferred to the peptidoglycan as an essential constituent for normal cell growth. Furthermore, S. rwninantuan LDC has two unique characteristics ; (1) it has decarboxylase activity towards both L-lysine and L-ornithine. (2) biochemical future is similar to that of eukaryotic ornithine. Here, we found the following evidences, i.e.(1) S. rtm inanluon LDC was dramatically degraded at the early stationary phase by ATP -dependent serine protease(s). This degradation proteolysis was required a degradation fact … More or, which was named as P22. P22 was induced by the accumulation of putrescine in S. raminanlium cells, and it bound tightly to LDC with a K_D) of 8.5X10^<-11> M. P22 seems to have similar biochemical and biophysical characteristics to those of an antizynte (AZ), which have been reported only in mammalian cells. P22 was identified as a ribosomal protein LI0 of S. rimliuunlimn. The binding of L10 to LDC for forming LDC-L10 complex may cause a conformational change(s) of LDC subunit, which allow it to be attacked by ATP-dependent serine protease(s), and the LDC would be broken down to small peptides. We also identified the two regions, A region (K^<101>NKLD^<105>) and B region (G^<106>VIRNAVYVLD^<170>) in L-10 molecule essential for its binding to LDC.(2) We identified the ATP-dependent serine protease(s), which is involved in the degradation of S. ruminantiian LDC. There were three genes for ATP-dependent protease chromosomal in S. ruminantium and we focused on one of them, which belongs to C IpXP protease(s) since they require an adapter protein. CIpXP protease consists of the CIpX ATPase and the CIpP peptidase. In S. rmninantitan, L10 may work as an adapter protein and carry the LDC to CIpXP protease. We cloned C1pP gene from S. ruminantiuni into pET15b and expressed it in E, coli BL21 (DE3) and purified.(3) Cadaverine covalently linked to the peptidoglycan is required for the interaction between the peptidoglycan and the S-layer homologous (SLH) domain of the major outer membrane protein, Mep45. Here, using a series of diamines with a general structure of NH_3^+(CH_2)_nNH_3^+, n=3-6), we found that cadaverine (n=5) specifically serves as the most efficient constituent of the peptidoglycan to acquire the high resistance of the cell to the external damage agents, and is required for the effective interaction between the SLH domain of Mep45 and the peptidoglycan facilitating the correct anchoring of the outer membrane to the peptidoglycan. Less

反刍月形单胞菌（Selenomonasruminantium）是一种严格厌氧的革兰氏阴性菌，其脂蛋白既不含游离形式，也不含结合形式，在维持一般革兰氏阴性菌细胞表面结构完整性方面起着重要作用。相反，它有尸胺，它共价连接到作为细胞分裂的关键成分的肽聚糖。In S.通过赖氨酸脱羧酶（LDC）从L-赖氨酸组成型合成rmuinantin尸胺，并将其转移到肽聚糖中作为正常细胞生长的必需成分。此外，S. rwninantuan LDC具有两个独特的特征：（1）它对L-赖氨酸和L-鸟氨酸都具有脱羧酶活性。(2)生物化学的未来类似于真核生物的鸟氨酸。在这里，我们发现了以下证据，即。(1)S. rtm inanluon LDC在稳定期早期被ATP依赖性丝氨酸蛋白酶显著降解。这种降解蛋白水解是降解事实所必需的 ...更多信息或者，它被命名为P22。P22是由腐胺在S.与树突状细胞结合紧密，结合常数K_D为8.5 × 10 ~（-1）μ <-11>M。P22似乎具有类似的生物化学和生物物理特性的antizynte（AZ），这已被报道只在哺乳动物细胞。P22为S.边缘L10与LDC结合形成LDC-L10复合物可能引起LDC亚基构象变化，这使得其能够被ATP依赖性丝氨酸蛋白酶攻击，并且LDC将被分解成小肽。我们还鉴定了L-10分子中与LDC结合所必需的两个区域，A区（K^<101>NKLD^<105>）和B区（G^<106>VIRNAVYVLD^<170>）。(2)我们鉴定了参与S.反刍动物LDC。S.反刍动物，我们关注其中之一，其属于ClpXP蛋白酶，因为它们需要衔接蛋白。ClpXP蛋白酶由ClpX ATP酶和ClpP肽酶组成。In S. rminantantan，L10可能作为衔接蛋白，并携带LDC到CIpXP蛋白酶。我们克隆了S.重组质粒pET 15 b在大肠杆菌BL 21（DE 3）中表达并纯化。(3)与肽聚糖共价连接的尸胺是肽聚糖与主要外膜蛋白Mep 45的S层同源（SLH）结构域之间相互作用所必需的。在这里，我们使用一系列具有一般结构NH_3^+（CH_2）_nNH_3^+，n=3-6）的二胺，发现尸胺（n=5）特异性地作为肽聚糖的最有效组分，以获得细胞对外部损伤剂的高抗性，并且是Mep 45的SLH结构域和肽聚糖之间有效相互作用所必需的，从而促进外膜正确锚定到肽聚糖上。少