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: 6355149
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6355149
• 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是一个保守的大模块I型家族的成员 控制多细胞分化的跨膜受体 动物 Notch功能通过一种新的信号介导 涉及连续配体诱导的蛋白水解的转导途径 用于释放Notch细胞内结构域的裂解， 然后转移到细胞核并在下游激活 转录因子 所有Notch的细胞外结构域 受体有三个重复的LIN 12模块， 负调控域，可能通过限制蛋白水解。 每个 LIN 12模块包含三个二硫键和三个保守的 天冬氨酸（D）或天冬酰胺（N）残基。 为了开始理解 LIN 12功能的结构基础，人类的第一个LIN 12模块 Notchl（rLIN12.1）已在大肠杆菌中重组表达。杆菌和 以还原形式纯化。 在氧化还原缓冲液中，rLIN12.1仅形成一个 二硫化物异构体在存在毫摩尔Ca++，而多个 在Mg 2+和EDTA存在下观察到二硫化物异构体。 一维质子核磁共振显示Ca + 诱导本征的化学位移分散的急剧增加， rLIN 12.1酰胺质子，如Ca ++结合LDL-A模块所见。 我们 确定了适合于确定溶液的条件 rLIN 12的结构。 1 NMR 使用重组'5 N-标记 蛋白质，我们已经完成了rLIN12.1和 侧链分配正在进行中。 另外我们有 成功地重折叠了Notchl的重组片段， 三个LIN 12模块，并打算进行解决方案结构研究 整个LIN 12域。