Protein-ligand interface engineering for allele-specific regulation of histone demethylases and epigenome editing

用于组蛋白去甲基化酶和表观基因组编辑等位基因特异性调控的蛋白质-配体界面工程

• 批准号: 1817692
• 负责人: Kabirul Islam
• 金额: $ 64.91万
• 依托单位: University of Pittsburgh
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1817692&HistoricalAwards=false
• 关键词: Protein; ligand; interface; engineering; allele

In eukaryotes, genes can be turned on or off by changing the way the genome is packaged in the nucleus. Normally, the genome is compacted by winding the DNA around barrel-like cores of proteins. Tight packaging turns genes off, but chemical modification of the protein cores can relax the DNA and allow genes to be turned on. The modifications are carried out by a large group of enzymes, but how and where they function remains unknown. This project will take an interdisciplinary approach, including organic chemistry, protein engineering, and cell biology--to uncover the specificity of these modifiers and to design enzymes capable of turning on specific genes at will. The research setting will provide a unique training ground for graduate and undergraduate students. Furthermore, a chemical biology laboratory course will engage undergraduate students in inquiry-based exercises where students will learn to design and carry out real-world experiments aimed at developing their critical thinking and independent learning skills. Early implementation of such a research-based course is expected to bridge a gap existing in the current educational curriculum by motivating STEM students in science education and research with the long-term benefit of generating a skilled workforce.Changes in gene expression in eukaryotic organisms like humans can be achieved by reversible chemical modifications on the histone protein components of chromatin. The focus of this project is to study a particular type of modification, called lysine methylation, which is removed by a class of enzymes known as lysine demethylases. How specific demethylases contribute to gene expression has remained largely unexplored, due in part to the lack of tools capable of rapidly interrogating a given demethylase in intact cells under carefully controlled conditions. Using one particular lysine demethylase as paradigm, this research will focus on developing a novel chemical-genetic platform that combines pharmacological and genetic engineering to perturb specific isoforms with rationally designed small molecules and precise temporal control. Furthermore, the engineered demethylation apparatus will be combined with the spatial selectivity of programmable CRISPR-Cas9 to develop a new type of conditional epigenome editing tool for regulating gene transcription in space and time. The approach will be applied to reprogram expression of genes that underlie faithful cell division, cellular differentiation, lineage commitment, and ultimately, organismal development. These unique tools will be made broadly available to researchers interested in addressing how reversible histone methylation regulates eukaryotic biology.This project is funded jointly by the Genetic Mechanisms Cluster, Division of Molecular and Cellular Biosciences in the Directorate of Biological Sciences and the Chemistry of Life Processes Program, Division of Chemistry in the Directorate of Mathematical and Physical Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在真核生物中，可以通过更改基因组包装在细胞核中的方式来打开或关闭基因。 通常，通过将DNA缠绕在蛋白质的桶状核周围来使基因组紧凑。紧密包装关闭基因，但是蛋白质核的化学修饰可以放松DNA并允许打开基因。 修饰是由一大批酶进行的，但是它们的作用方式和何处仍然未知。该项目将采用跨学科的方法，包括有机化学，蛋白质工程和细胞生物学 - 揭示这些修饰符的特异性，并设计能够随意开启特定基因的酶。研究环境将为研究生和本科生提供独特的培训场。此外，化学生物学实验室课程将吸引本科生进行基于询问的练习，学生将学习设计和进行旨在发展其批判性思维和独立学习技能的现实世界实验。 预计这种基于研究的课程的早期实施将弥合当前教育课程中存在的差距，通过激励STEM学生从事科学教育和研究，具有产生熟练劳动力的长期益处。在像人类（例如人类）基因表达的基因表达中，可以通过对Chratemin的组蛋白蛋白质成分的可逆化学修饰来实现人类（如人类）的基因表达。 该项目的重点是研究一种称为赖氨酸甲基化的特定类型的修饰，该修饰被称为赖氨酸脱甲基酶的一类酶去除。特定的脱甲基酶如何对基因表达有何贡献，这在很大程度上尚未开发，部分原因是缺乏能够在精心控制的条件下能够快速审问完整细胞中给定脱甲基酶的工具。这项研究将使用一种特定的赖氨酸去甲基酶作为范式，将重点放在开发一种新型的化学遗传平台上，该平台将药理学和遗传工程结合在一起，以扰动特异性同工型与合理设计的小分子和精确的时间控制。此外，工程化的脱甲基化设备将与可编程CRISPR-CAS9的空间选择性相结合，以开发一种新型的条件表格编辑工具，用于调节时空中的基因转录。该方法将应用于基于忠实细胞分裂，细胞分化，谱系承诺以及最终是生物体发育的基因的重编程表达。这些独特的工具将在有兴趣解决可逆组蛋白甲基化如何调节真核生物生物学的研究人员中广泛使用。该项目由遗传机制集群，分子和细胞生物科学局在生物学科学局和生命统计过程中的化学和物理统治和物理学的化学和物理学的化学和物理学的化学委员会和物理学的化学措施。使用基金会的知识分子优点和更广泛的审查标准，被认为值得通过评估来支持。

项目成果

Catalytic Space Engineering as a Strategy to Activate C-H Oxidation on 5-Methylcytosine in Mammalian Genome.

• DOI: 10.1021/jacs.1c03815
• 发表时间: 2021-08-11
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Sappa, Sushma;Dey, Debasis;Sudhamalla, Babu;Islam, Kabirul
• 通讯作者: Islam, Kabirul

Allele-Specific Chemical Rescue of Histone Demethylases Using Abiotic Cofactors.

• DOI: 10.1021/acschembio.1c00335
• 发表时间: 2022-12-16
• 期刊: ACS CHEMICAL BIOLOGY
• 影响因子: 4
• 作者: Scott, Valerie;Dey, Debasis;Kuwik, Jordan;Hinkelman, Kathryn;Waldman, Megan;Islam, Kabirul
• 通讯作者: Islam, Kabirul

Complementary Steric Engineering at the Protein-Ligand Interface for Analogue-Sensitive TET Oxygenases.

• DOI: 10.1021/jacs.8b05283
• 发表时间: 2018-08-15
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Sudhamalla B;Wang S;Snyder V;Kavoosi S;Arora S;Islam K
• 通讯作者: Islam K

Engineering a methyllysine reader with photoactive amino acid in mammalian cells.

• DOI: 10.1039/d0cc03814h
• 发表时间: 2020-10-18
• 期刊: Chemical communications (Cambridge, England)
• 作者: Arora S ;Sappa S ;Hinkelman K ;Islam K
• 通讯作者: Islam K

Allele-Specific Inhibition of Histone Demethylases.

组蛋白去甲基酶的等位基因特异性抑制。

• DOI: 10.1002/cbic.201800756
• 发表时间: 2019
• 期刊: Chembiochem : a European journal of chemical biology
• 作者: Wagner,Shana;Waldman,Megan;Arora,Simran;Wang,Sinan;Scott,Valerie;Islam,Kabirul
• 通讯作者: Islam,Kabirul