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

反刍硒单胞菌是一种严格厌氧的革兰氏阴性细菌，它既没有游离形式也没有结合形式的脂蛋白，而脂蛋白在维持一般革兰氏阴性细菌的细胞表面结构完整性方面发挥着重要作用，相反，它含有尸胺，其与作为细胞分裂的关键成分的消化聚糖共价连接，尸胺由组成型合成。 L-赖氨酸被赖氨酸脱羧酶（LDC）转移到肽聚糖上，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)生化前景与真核鸟氨酸相似。这里，我们发现了以下证据，即(1)S. rtm inanluon LDC 在早期稳定期被 ATP 依赖性丝氨酸蛋白酶显着降解，这种降解蛋白水解作用是由腐胺在 S 中的积累引起的，被命名为 P22。 raminanlium 细胞，并且它与 LDC 紧密结合，K_D) 为 8.5X10^<-11> M。P22 似乎具有类似的生化作用与仅在哺乳动物细胞中报道过的抗酶体 (AZ) 相比，P22 被鉴定为 S. rimliuunlimn 的核糖体蛋白 LI0。L10 与 LDC 结合形成 LDC-L10 复合物可能会导致非对称构象。 LDC亚基发生变化，使其受到ATP依赖性丝氨酸蛋白酶的攻击，LDC会被分解成小肽。我们还鉴定了 L-10 分子中与 LDC 结合所必需的两个区域：A 区域 (K^<101>NKLD^<105>) 和 B 区域 (G^<106>VIRNAVYVLD^<170>)。(2 ) 我们鉴定了 ATP 依赖性丝氨酸蛋白酶，该蛋白酶参与反刍动物 S. ruminantiian LDC 的降解。 ATP 依赖性蛋白酶染色体有 3 个基因。在 S. ruminantium 中，我们重点关注其中一种，它属于 C IpXP 蛋白酶，因为它们需要由 CIpX ATP 酶和 CIpP 肽酶组成的 CIpXP 蛋白酶。我们将来自 S. ruminantiuni 的 C1pP 基因克隆到 pET15b 中，并将其携带到 CIpXP 蛋白酶上。在大肠杆菌 BL21 (DE3) 中表达并纯化。(3) 肽聚糖与主要外膜蛋白 Mep45 的 S 层同源 (SLH) 结构域之间的相互作用需要与肽聚糖共价连接的尸胺。，使用一系列通用结构为 NH_3^+(CH_2)_nNH_3^+, n=3-6) 的二胺，我们发现尸胺 (n=5) 是肽聚糖中最有效的成分，可以使细胞获得对外部损伤剂的高抵抗力，并且是 Mep45 的 SLH 结构域和肽聚糖之间有效相互作用所必需的，促进正确的锚定外膜与肽聚糖的结合较少。