Biophysics of Macromolecular Complexes
大分子复合物的生物物理学
基本信息
- 批准号:10697735
- 负责人:
- 金额:$ 39.64万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:2019-nCoVAffinityAnalytical CentrifugationArchitectureBindingBiologicalBiological AssayBiophysicsC-terminalCCCTC-binding factorCOVID-19 treatmentCell NucleusChromatinChromatin StructureCollaborationsComplexCoronavirus InfectionsDNADNA Binding DomainDNA-Binding ProteinsDataDimerizationDiseaseEnzymesEventEvolutionFDA Emergency Use AuthorizationFeline CoronavirusGenerationsGenetic TranscriptionGenomeGenomic DNAGenomicsGrantHistonesHumanMacromolecular ComplexesMeasuresMediatingMethodsN-terminalNational Institute of Diabetes and Digestive and Kidney DiseasesNonstructural ProteinNuclearOralOrthologous GenePeptide HydrolasesPlayPolyproteinsPropertyProtease InhibitorProteinsRNARoleShapesSpermatogenesisTailVirus ReplicationWorkZinc Fingersanalogcancer typecohesindesigndimerinhibitorinterestmacromolecular assemblymonomersedimentation equilibriumsedimentation velocitystoichiometrytool
项目摘要
Chromatin structure and architecture.
Histone proteins package and condense genomic DNA into chromatin within the cell nucleus. Proteins such as the CCCTC binding factor (CTCF) help direct chromatin higher-order organization through passive and active mechanisms by imposing topological constraints, mediating long-range genomic interactions, and participating in transcriptional events. CTCF is a highly conserved DNA binding protein found exclusively in bilaterians. In humans, the protein consists of an eleven zinc-finger DNA binding domain, flanked by conserved N-terminal and C-terminal tails constituting about 57 percent of the protein. While the zinc-fingers specifically recognize DNA motifs and interact with RNA, the roles of the N- and C-terminal domains remain primarily unknown. These termini interact with cohesin and help stabilize the complexes delineating topologically associated chromatin domains. However, it is unlikely that this is their sole role. We have shown that these N- and C-terminal domains are intrinsically disordered in solution, a property shared by many nuclear and DNA binding proteins. Current work focuses on identifying protein partners that bind to the N- and C-termini of CTCF and a study of the complexes formed to understand how CTCF regulates higher-order genome organization within the eukaryotic nucleus.
Human CTCF has an ortholog CTCFL, primarily associated with spermatogenesis and some cancer types. While CTCF and CTCFL have a highly conserved eleven zinc-finger DNA binding domain and recognize identical DNA motifs, they differ significantly in their N- and C-termini suggesting that the diverse roles for these proteins arise from their termini. Similarly, while conserved among bilaterians and across evolution with a core zinc-finger DNA binding domain, CTCF may have divergent termini across phyla. We are interested in characterizing protein partners for CTCF from select species to dissect further the multiple roles that the protein plays in organizing the genome.
Macromolecular assemblies of biological interest.
We utilize hydrodynamic methods, particularly sedimentation velocity and equilibrium analytical centrifugation, to characterize critical biological assemblies, determine their shape and stoichiometry, and measure their interaction affinity. In collaboration with John Louis (LCP-NIDDK), we are studying the maturation of the dimeric SARS-CoV-2 main protease (MPro) and the effects of transition-state analog inhibitors. MPro functions as a dimer and is indispensable for viral replication and propagation. MPro is formed as a monomer, as part of a polyprotein chain. MPro dimerization is required for its release and the generation of non-structural proteins necessary for viral replication and transcription. Oral COVID-19 therapies recently granted emergency use authorization by the FDA target the main protease MPro, highlighting its importance. Using a carefully designed monomeric form of MPro(M) and comparing its properties to that of the wild-type dimeric MPro(WT), we show that protease activity requires the dimeric form of the enzyme. GC376, a transition-state analog protease inhibitor used in veterinary applications to treat feline coronavirus infections, interacts with MPro(M) and MPRo(WT). Interestingly, MPro(M) binds to GC376 with a higher affinity than MPro(WT). Furthermore, using activity assays and hydrodynamic methods, we show that GC376 promotes MPro(M) dimerization in a concentration-dependent manner. GC376 does not restore the catalytic activity observed for MPro(WT), reflecting structural differences in the monomeric MPro(M) design. However, data demonstrate that the binding of the transition state analog favors the dimer form of the enzyme, enhancing catalytic activity. These observations may provide a valuable tool for designing and identifying more effective protease inhibitors.
染色质结构和体系结构。
组蛋白将基因组 DNA 包装并浓缩成细胞核内的染色质。 CCCTC 结合因子 (CTCF) 等蛋白质通过施加拓扑约束、介导远程基因组相互作用和参与转录事件,通过被动和主动机制帮助指导染色质高阶组织。 CTCF 是一种高度保守的 DNA 结合蛋白,仅在两侧对称动物中发现。 在人类中,该蛋白质由 11 个锌指 DNA 结合域组成,两侧是保守的 N 端和 C 端尾部,约占该蛋白质的 57%。 虽然锌指特异性识别 DNA 基序并与 RNA 相互作用,但 N 端和 C 端结构域的作用仍然基本未知。 这些末端与粘连蛋白相互作用,有助于稳定描绘拓扑相关染色质结构域的复合物。 然而,这不太可能是他们唯一的角色。 我们已经证明,这些 N 端和 C 端结构域在溶液中本质上是无序的,这是许多核和 DNA 结合蛋白共有的特性。 目前的工作重点是识别与 CTCF N 端和 C 端结合的蛋白质伙伴,并对形成的复合物进行研究,以了解 CTCF 如何调节真核细胞核内的高阶基因组组织。
人类 CTCF 具有直系同源物 CTCFL,主要与精子发生和某些癌症类型相关。 虽然 CTCF 和 CTCFL 具有高度保守的 11 个锌指 DNA 结合结构域并识别相同的 DNA 基序,但它们的 N 末端和 C 末端显着不同,表明这些蛋白质的不同作用源于它们的末端。 类似地,虽然 CTCF 在两侧对称动物中以及在进化过程中具有核心锌指 DNA 结合结构域,但 CTCF 可能在不同的门中具有不同的末端。 我们感兴趣的是从选定的物种中表征 CTCF 的蛋白质伙伴,以进一步剖析该蛋白质在组织基因组中发挥的多重作用。
具有生物学意义的大分子组装体。
我们利用流体动力学方法,特别是沉降速度和平衡分析离心,来表征关键的生物组件,确定它们的形状和化学计量,并测量它们的相互作用亲和力。 我们与 John Louis (LCP-NIDDK) 合作,研究二聚体 SARS-CoV-2 主蛋白酶 (MPro) 的成熟以及过渡态类似物抑制剂的作用。 MPro 作为二聚体发挥作用,对于病毒复制和传播是不可或缺的。 MPro 作为单体形成,作为多蛋白链的一部分。 MPro 二聚化是其释放和病毒复制和转录所需的非结构蛋白生成所必需的。 口服 COVID-19 疗法最近获得 FDA 的紧急使用授权,针对主要蛋白酶 MPro,凸显了其重要性。 使用精心设计的 MPro(M) 单体形式并将其特性与野生型二聚体 MPro(WT) 进行比较,我们表明蛋白酶活性需要酶的二聚体形式。 GC376 是一种过渡态类似蛋白酶抑制剂,用于兽医应用中治疗猫冠状病毒感染,与 MPro(M) 和 MPRo(WT) 相互作用。 有趣的是,MPro(M) 与 GC376 的结合亲和力高于 MPro(WT)。 此外,使用活性测定和流体动力学方法,我们表明 GC376 以浓度依赖性方式促进 MPro(M) 二聚化。 GC376 不会恢复 MPro(WT) 观察到的催化活性,反映了单体 MPro(M) 设计的结构差异。 然而,数据表明过渡态类似物的结合有利于酶的二聚体形式,从而增强催化活性。 这些观察结果可能为设计和鉴定更有效的蛋白酶抑制剂提供有价值的工具。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Gary Felsenfeld其他文献
Gary Felsenfeld的其他文献
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{{ truncateString('Gary Felsenfeld', 18)}}的其他基金
Organization and regulation of the human insulin locus
人胰岛素基因座的组织和调节
- 批准号:
8741425 - 财政年份:
- 资助金额:
$ 39.64万 - 项目类别:
Organization and regulation of the human insulin locus
人胰岛素基因座的组织和调节
- 批准号:
10006694 - 财政年份:
- 资助金额:
$ 39.64万 - 项目类别:
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