STRUCTURE/FUNCTION ANALYSIS OF MITOTIC REGULATORS
STRUCTURE/FUNCTION ANALYSIS OF MITOTIC REGULATORS
批准号:
6180560
负责人:
Joseph Patrick Noel
金额:
$26.87万
依托单位国家:
美国
项目类别:
财政年份:
1998
资助国家:
美国
项目状态:
已结题
起止时间:
1998-05-01 至 2002-04-30
中文摘要
我们的主要目标是提供一个立体化学的基础,
一种新的有丝分裂调节因子Pin 1所起的生物学作用。 的
人肽基脯氨酰顺反异构酶(PPIase)Pin 1是一种保守的
对真核细胞的G2到M转变至关重要的有丝分裂调节因子
细胞周期 我们已经确定了高分辨率的x射线晶体
与含脯氨酸的底物复合的Pin 1的结构。 在
此外,我们已经开始在体外和体内对Pin 1进行功能分析,
为了将观察到的这种结构特征与
具有调节有丝分裂进入和退出的作用的新型PPIase。
晶体结构连同pH滴定研究,
活性位点半胱氨酸诱变提示催化机制
包括一般的酸碱和共价催化作用,
键异构化 Pin 1显示出对酸性的强烈偏好。
由于以下物质的相互作用,异构化脯氨酸键的N-末端残基
这种酸性侧链在入口处具有保守的碱性簇
PPIase结构域的活性位点 正在进行结构性工作,
证实实现了与该基本群集的最佳交互
与含磷酸苏氨酸或磷酸丝氨酸的底物反应。 已知
细胞周期蛋白依赖性蛋白激酶(CDKs)的特异性是针对苏氨酸
或脯氨酸氨基末端的丝氨酸残基。Pin 1,带有活动站点
理想地适合于结合磷酸化的-SP-或-TP-的结构
含有底物,可能在进展过程中调节CDK靶点
从细胞周期的间期到有丝分裂期。 特别是,
PinI直接调节有丝分裂磷酸酶CDC 25 C的活性
通过结合其高度磷酸化的N-末端区域。 这一地区
富含CDK位点,是磷酸化的,这种磷酸化是
对CDC 25 C活性至关重要。 关于这项建议,我们会
采用两层方法,包括大分子
Pin 1高分辨结构的X射线晶体学分析
和许多与底物复合的Pin 1突变体,包括
CDC 25 C,以及Pin 1底物特异性的体外功能分析
和作用机制。 除了结构性工作,我们还将
确定控制Pin 1-target的动力学和热力学参数
蛋白质相互作用 此外,我们将完成结构
Pin 1相互作用激酶的四聚化结构域的阐明
并开始结晶和结构解析
Pin 1与NIMA激酶的该结构域复合。 最后提出
设计、合成和测试了几种结构化的Pin 1
抑制剂作为新的抗有丝分裂剂。
英文摘要
Our major goal is to provide a stereochemical foundation for the
biological roles played by a novel mitotic regulator known as Pin1. The
human peptidyl-prolyl cis-trans isomerase (PPIase) Pin1 is a conserved
mitotic regulator essential for the G2 to M transition of the eukaryotic
cell cycle. We have determined the high resolution x-ray crystal
structure of Pin1 complexed with a proline containing substrate. In
addition, we have begun a functional analysis of Pin1 in vitro and in
vivo in order to correlate the observed structural features of this
novel PPIase with its role in regulating entry and exit from mitosis.
The crystallographic structure together with pH titration studies and
mutagenesis of an active site cysteine suggest a catalytic mechanism
that includes general acid-base and covalent catalysis during peptide
bond isomerization. Pin1 displays a strong preference for an acidic
residue N-terminal to the isomerized proline bond due to interaction of
this acidic side chain with a conserved basic cluster at the entrance
to the PPIase domain's active site. Structural work in progress,
confirms that optimal interaction with this basic cluster is achieved
with phosphothreonine or phosphoserine containing substrates. The known
specificity of cyclin-dependent protein kinases (CDKs) is for threonine
or serine residues amino terminal to proline. Pin1, with an active site
structure ideally suited for binding phosphorylated -SP- or -TP-
containing substrates, likely regulates CDK targets during progression
from the inter- to the mitotic phase of the cell cycle. In particular,
PinI directly regulates the activity of the mitotic phosphatase CDC25C
by binding to its hyperphosphorylated N-terminal region. This region
rich in CDK sites, is phosphorylated, and this phosphorylation is
essential for CDC25C activity. With regard to this proposal, we will
employ a two tiered approach involving a combination of macromolecular
x-ray crystallography for high resolution structural analysis of Pin1
and a number of Pin1 mutants in complex with substrates including
CDC25C, and in vitro functional analysis of Pin1's substrate specificity
and mechanism of action. In addition to the structural work, we will
determine the kinetic and thermodynamic parameters governing Pin1-target
protein interactions. In addition, we will complete the structural
elucidation of the tetramerization domain of a Pin1 interacting kinase
known as NIMA and begin crystallization and structural elucidation of
Pin1 complexed to this domain of the NIMA kinase. Finally, we propose
to design, synthesize and test several structurally based Pin1
inhibitors as new anti-mitotic agents.
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海外基金