HIGH RESOLUTION SOLUTION STUDIES OF LIN 12 DOMAIN OF HUMAN NOTCH I: LEUKEMIA

人类 NOTCH I 的 LIN 12 域的高分辨率解决方案研究：白血病

• 批准号: 6118651
• 负责人: Stephen C. Blacklow
• 金额: $ 0.21万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-15 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6118651
• 关键词: biological products; biomedical resource; enzymes

Notchl is a member of a conserved family of large modular type I transmembrane receptors that control differentiation in multicellular animals. Notch function is mediated through a novel signal transduction pathway involving successive ligand-induced proteolytic cleavages that serve to release the intracellular domain of Notch, which then translocates to the nucleus and activates downstream transcription factors. The extracellular domain of all Notch receptors have three iterated LIN 12 modules that appear to act as negative regulatory domains, possibly by limiting proteolysis. Each LIN 12 module contains three disulfide bonds and three conserved aspartate (D) or asparagine (N) residues. To begin to understand the structural basis for LIN12 function, the first LIN12 module of human Notchl (rLIN12.1) has been expressed recombinantly in E. coli and purified in reduced form. In redox buffers, rLIN12.1 forms only one disulfide isomer in the presence of millimolar Ca++, whereas multiple disulfide isomers are observed in the presence of Mg ,-, and EDTA. One-dimensional proton nuclear magnetic resonance shows that Ca + induces a dramatic increase in chemical shift dispersion of the native rLIN12.1 amide protons, as seen for Ca ++ -binding LDL-A modules. We have identified conditions suitable for determining the solution structure of rLIN 12. 1 by NMR. Using recombinant '5N-labelled protein, we have completed backbone assignments of rLIN12.1 and side-chain assignments are in progress. In addition, we have successfully refolded a recombinant fragment of Notchl that spans all three LIN12 modules and intend to pursue solution structural studies of the entire LIN12 domain.

Notchl是一个保守的大模I型家族的成员 控制多细胞分化的跨膜受体 动物。缺口函数是通过一种新的信号来调节的 连续配基诱导蛋白水解酶的信号转导途径 用来释放Notch细胞内结构域的裂解， 然后移位到细胞核并在下游激活 转录因子。All Notch的胞外结构域 受体有三个重复的LIN 12模块，看起来像是 负调控结构域，可能通过限制蛋白分解。每个人 LIN 12模中含有三个二硫键和三个保守的键 天冬氨酸(D)或天冬酰胺(N)残留物。要开始理解 人类第一个LIN12模块--LIN12功能的结构基础 Notch1(rLIN12.1)已在大肠杆菌中获得重组表达 以简化形式提纯的。在氧化还原缓冲区中，rLIN12.1仅形成一个 在毫摩尔钙离子存在的情况下，二硫键异构体，而多个 在镁、-和EDTA存在的情况下，观察到二硫键异构体。 一维质子核磁共振显示，Ca+ 导致原生分子的化学位移分散度急剧增加 RLIN12.1酰胺质子，如钙结合低密度脂蛋白-A模块所见。我们 已经确定了适合确定溶液的条件 用核磁共振技术研究Rlin12.1的结构。利用重组‘5N标记 蛋白质，我们已经完成了rLIN12.1和rLIN12.1的骨架指定 侧链分配正在进行中。此外，我们还有 成功地重新折叠了Notchl的一个跨越所有 三个LIN12模块，并打算进行解决方案结构研究 整个LIN12结构域。