Nmr Studies Of The Regulation Of Cell Signaling

细胞信号传导调节的核磁共振研究

基本信息

批准号：
10929077
负责人：
NICO TJANDRA
金额：
$ 149.51万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
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：
https://reporter.nih.gov/project-details/10929077
关键词：
2019-nCoV Adopted Affinity Antiviral Agents Binding Binding Sites Biological Biological Assay Biological Process C-terminal Capsid Cell membrane Cells Cellular Assay Clinical Collaborations Complex Coronavirus Cysteine Degradation Pathway Dependence Development Dimerization Disease Environment Enzyme Activation Enzymes Event Exhibits Family Generations Goals HIV-1 Half-Life High Pressure Liquid Chromatography Hybrids Isomerism Knowledge Lanthanoid Series Elements Ligands Ligase Link Lobe Measures Metals Methylation Molecular Monitor Mutation N-terminal National Institute of Allergy and Infectious Disease Nature Nitrogen Nuclear Pathway interactions Peptide Signal Sequences Phase Physiological Population Predisposition Process Production Protein Dynamics Proteins Proton Pump Inhibitors Protons RNA Regulation Research Research Project Grants SARS-CoV-2 genome Scheme Signal Transduction Site Specificity Spin Labels Stimulus Structure Study models System Technology Temperature Testing Tyrosine UBD protein Ubiquitin Ubiquitination Viral Viral Proteins Virion Virus Virus-like particle Water alanylproline cell motility experimental study flexibility follow-up hepatocyte growth factor-regulated tyrosine kinase substrate imaging probe improved inhibitor molecular dynamics new technology novel particle protein degradation protein function protein protein interaction recruit response small molecule trafficking ubiquitin ligase ubiquitin-protein ligase

项目摘要

As a progression in developing new technology to characterize dynamic molecular events which regulate important biological function, we chose to look at retroviral capsid assembly. This protein is a part of the Gag-poly protein which is processed as part of the maturation of the virus. The assembly and disassembly of the capsid particle is crucial for viral budding from and entry into the host cell, respectively. We showed that capsid assembly occurs due to two types of distinct molecular interactions. The N-terminal beta hairpin promotes the elongation of helix 1 which forms the oligomerization interface of the capsid particle. This event occurs at a slower timescale than the dimerization that involves the C-terminal domain of the capsid. We could only established the above observations by using a barrage of NMR experiments. This is largely due to the dynamic nature of the molecular interactions. We also synthesized a compound (methylated-DOTA) that can coordinate lanthanide ligand with reduced flexibility. This was done in collaborating with the Imaging Probe Development Group. The goal was to achieve a substantial increase in observable Pseudo Contact Shift (PCS) and use the information for structure determination. In addition we also showed that the methylated-DOTA-lanthanide adopts two isomers. The populations of these isomers depend on the size of the lanthanide metal being coordinated. The population ratios that we measured by observing PCS on a protein matched those obtained from HPLC on the methylated-DOTA-lanthanide. We carried out temperature dependence study on the DOTA lanthanide to show that the size of the susceptibility tensor depends highly on temperature and this is due to bound water exchange rate. The slower the rate the larger the tensor. We showed that the methylated-DOTA-lanthanide is also very practical in studying intrinsically disordered proteins by introducing PCS which results in better dispersion of the typically overlapping NMR resonances. Moreover, in the case of dynamic protein-proton complexes, such as those that exhibit encounter complexes, using spin label nitroxide to get structural information can be complicated by the encounter complexes. On the other hand, we showed that by using the PCS we can determine the major form of the complex between Enzyme I and NPr of the nitrogen transfer system. Using the above unique approach we have been able to establish that encounter complexes between two paralogous systems can compete against each other. We monitored changes in Enzyme I and NPr specific and encounter complexes in the presence of HPr. We previously established that HPr doesn't interact specifically with Enzyme I. With increasing HPr concentration we showed that NPr encounter can be modulated such that the specific Enzyme I and NPr complex population is increased, effectively increasing the affinity of the complex. This is a surprising finding, therefore we decided to follow up this study with a modeling study which we could recapitulate the encounter profile between NPr and enzyme I in the presence of HPr. This study reveals the lack of understanding beyond competition of specific substrate to regulate biological function. We looked at another weak protein-protein interaction that has biological relevance. In the case of Tsg101, its interaction with ubiquitin (Ub) is rather weak. We were able to detect this interaction with the new paramagnetic technology that we developed above. We established an inhibitor to this Ub-Tsg101 interaction that has allowed us to decipher the Ub signaling in viral trafficking in the host sell. In addition, using NMR we identified another Ub binding site on Tsg101. We confirmed that Tsg101 recognized Di-Ubiquitin (Di-Ub), specifically linked at K63. We also have been able to determine using NMR that Di-Ub binds Tsg101 in two distinct sites. These two sites have different physiological consequences. One site, the so called vestigial Ub binding site controls recruitment of Tsg101 by Hiv-1 Gag to the plasma membrane, while the N-termninal Ub site seems to be correlated to the nuclear capsid determinant of trafficking Hiv-1 Gag. Interestingly, tri-Ub doesn't bind as well as Di-Ub to Tsg101. This finding is novel and allows us to distinguish multiple facets to Ub signaling in the Tsg101 (ESCRTI) pathway. We have continue to use our finding of Tsg101 and Ub interaction to develop small molecules that can inhibit their interaction. We found a family of prazoles, which have been clinically used as proton pump inhibitors, can inhibit Tsg101 and Ub interaction. We showed that this inhibition can interfere with HIV-1 virus particle release from host cells. Moreover, this inhibition also seems to reduce viral protein production, such as Gag, in the host cells. We showed that the same effect could be observed for other viruses, therefore signaling the potential for the prazole family of compounds as broad antiviral agents. Since we know exactly the molecular mechanism for Tsg101-Ub inhibition, we can use this knowledge to push for new generation of compounds that can provide more specificity and potency. This effort has expanded to test our compounds against other family of viruses, including Coronavirus. This was done through the NIAID cores for anti-viral testing. A couple of our compounds showed efficacy to block these viruses, including SARS-CoV2. We are generating new derivatives of our compounds to try to improve their selectivity. In parallel we are also investigating the reason why cells would create a pseudo-E2-ubiquitin Ligase, which is what Tsg101 is. It has the same structure as Ub E2 ligase with the catalytic cysteine replaced by a tyrosine, thus Tsg101 has no enzymatic activity. Furthermore, we also recently showed that this protein can recognize cellular RNA. This ability somehow is linked to Ub recognition in the cell. Th next phase of our research is directed towards combining all of our findings to draw a general scheme of how all of these processes are tied together to benefit cell trafficking and how viruses can modify them for their replication. We expanded our study on Tsg101 by looking at its potential interaction with Nedd4, and E3 ubiquitin ligase shown to be important for HIV-1 replication. We showed that Tsg101 UEV domain does interact with the HECT domain of the Nedd4. This interaction is quite weak. We used NMR PRE experiment to identify regions in HECT that contact Tsg101. Those residues involved in this interaction belong to the hinge region between the N- and C-lobe of HECT. We validated this finding using a rescue assay in the HIV-1 virus-like-particle release and Nedd4 mutations in the sites identified by our NMR experiment. In one of our assays to validate that the a1-helix of Nedd4 was important for its enzyme activation, we noticed that the western band in the pull down assay to identify Tsg101 and Nedd4 complex was significantly higher than what we usually observed. After further testing we showed that the level of Nedd4 is higher when more Tsg101 is present. We were able to also showed that Nedd4 gets poly-ubiquinated and degraded when Tsg101 is not present. Thus Tsg101 serves as a chaperon for Nedd4 in the cell. More over the reverse is also true, when Nedd4 is present, Tsg101 half life is also longer. There is definitely a direct interplay between these two proteins that involves ubiquitin signaling that at the end regulates their level. In addition to the above studies, we also tested the unique signaling peptide for Tsg101 interaction PTAP (Pro-The-Ala-Pro) which is all found in the protein Hrs, part of ESCRT-0, as well as in CoV2 genome (PTEP, PTQP, PNQP). We could identify a second recognition site which is specific to PTEP in Tsg101. We are carrying out hybrid (CoV2-HIV-1) cell assay to validate our findings.

作为开发新技术来表征调节重要生物功能的动态分子事件的进展，我们选择研究逆转录病毒衣壳组装。该蛋白是 Gag-poly 蛋白的一部分，Gag-poly 蛋白是病毒成熟过程中进行加工的一部分。衣壳颗粒的组装和分解分别对于病毒从宿主细胞出芽和进入宿主细胞至关重要。我们发现衣壳组装是由于两种不同的分子相互作用而发生的。 N 端 β 发夹促进螺旋 1 的延长，螺旋 1 形成衣壳颗粒的寡聚化界面。该事件发生的时间尺度比涉及衣壳 C 端结构域的二聚化要慢。我们只能通过一系列核磁共振实验来证实上述观察结果。这主要是由于分子相互作用的动态性质。我们还合成了一种可以配位镧系配体但灵活性降低的化合物（甲基化-DOTA）。这是与成像探针开发小组合作完成的。目标是实现可观测的伪接触位移（PCS）的大幅增加，并利用该信息进行结构测定。此外我们还发现甲基化-DOTA-镧系元素有两种异构体。这些异构体的数量取决于所配位的镧系金属的大小。我们通过观察蛋白质上的 PCS 测量的群体比率与通过甲基化 DOTA 镧系元素的 HPLC 获得的群体比率相匹配。我们对 DOTA 镧系元素进行了温度依赖性研究，结果表明磁化率张量的大小高度依赖于温度，这是由于束缚水交换率所致。速率越慢，张量越大。我们表明，通过引入 PCS，甲基化-DOTA-镧系元素在研究本质无序蛋白质方面也非常实用，PCS 可以更好地分散典型重叠的 NMR 共振。此外，在动态蛋白质-质子复合物的情况下，例如那些表现出相遇复合物的情况，使用自旋标记硝基氧来获取结构信息可能会因相遇复合物而变得复杂。另一方面，我们表明，通过使用 PCS，我们可以确定氮转移系统的酶 I 和 Npr 之间的复合物的主要形式。使用上述独特的方法，我们已经能够确定两个旁系同源系统之间的相遇复合体可以相互竞争。我们监测了酶 I 和 Npr 特异性的变化，并在 HPr 存在的情况下遇到复合物。我们之前确定 HPr 不会与酶 I 特异性相互作用。随着 HPr 浓度的增加，我们表明可以调节 NPr 相遇，从而增加特定酶 I 和 NPr 复合物的数量，从而有效增加复合物的亲和力。这是一个令人惊讶的发现，因此我们决定通过建模研究来跟进这项研究，我们可以在 HPr 存在的情况下重现 NPr 和酶 I 之间的相遇情况。这项研究揭示了人们对特定底物竞争之外的生物功能调节缺乏了解。我们研究了另一种具有生物学相关性的弱蛋白质-蛋白质相互作用。就 Tsg101 而言，它与泛素 (Ub) 的相互作用相当弱。我们能够利用我们上面开发的新顺磁技术检测到这种相互作用。我们建立了一种针对 Ub-Tsg101 相互作用的抑制剂，使我们能够破译宿主病毒贩运中的 Ub 信号。此外，使用 NMR，我们鉴定了 Tsg101 上的另一个 Ub 结合位点。我们确认 Tsg101 识别 Di-Ubiquitin (Di-Ub)，特别连接于 K63。我们还能够使用 NMR 确定 Di-Ub 在两个不同的位点结合 Tsg101。这两个位点具有不同的生理后果。其中一个位点，即所谓的残留 Ub 结合位点，控制 Hiv-1 Gag 将 Tsg101 募集至质膜，而 N 末端 Ub 位点似乎与运输 HIV-1 Gag 的核衣壳决定因素相关。有趣的是，tri-Ub 与 Tsg101 的结合不如 Di-Ub 好。这一发现是新颖的，使我们能够区分 Tsg101 (ESCRTI) 通路中 Ub 信号传导的多个方面。我们继续利用 Tsg101 和 Ub 相互作用的发现来开发可以抑制它们相互作用的小分子。我们发现临床上已用作质子泵抑制剂的普拉唑家族可以抑制Tsg101和Ub相互作用。我们发现这种抑制作用可以干扰宿主细胞中 HIV-1 病毒颗粒的释放。此外，这种抑制似乎还会减少宿主细胞中病毒蛋白的产生，例如 Gag。我们表明，对于其他病毒也可以观察到相同的效果，因此表明普拉唑家族化合物作为广泛的抗病毒药物的潜力。由于我们确切地了解 Tsg101-Ub 抑制的分子机制，因此我们可以利用这些知识来推动新一代化合物的开发，以提供更高的特异性和效力。这项工作已扩大到测试我们的化合物对抗其他病毒家族，包括冠状病毒。这是通过 NIAID 抗病毒测试核心完成的。我们的几种化合物显示出能够有效阻止这些病毒，包括 SARS-CoV2。我们正在生成化合物的新衍生物，以尝试提高其选择性。与此同时，我们还在研究细胞会产生伪 E2 泛素连接酶（Tsg101）的原因。它与Ub E2连接酶结构相同，只是催化的半胱氨酸被酪氨酸取代，因此Tsg101没有酶活性。此外，我们最近还证明这种蛋白质可以识别细胞RNA。这种能力在某种程度上与细胞中的 Ub 识别有关。我们研究的下一阶段旨在结合我们的所有发现，制定一个总体方案，说明所有这些过程如何结合在一起以有利于细胞运输，以及病毒如何修改它们以进行复制。我们通过观察 Tsg101 与 Nedd4 和 E3 泛素连接酶的潜在相互作用来扩展对 Tsg101 的研究，而 E3 泛素连接酶对 HIV-1 复制很重要。我们证明 Tsg101 UEV 结构域确实与 Nedd4 的 HECT 结构域相互作用。这种相互作用是相当微弱的。我们使用 NMR PRE 实验来识别 HECT 中与 Tsg101 接触的区域。参与这种相互作用的那些残基属于 HECT N 和 C 叶之间的铰链区。我们使用 HIV-1 病毒样颗粒释放的救援测定和 NMR 实验确定的位点中的 Nedd4 突变验证了这一发现。在我们验证 Nedd4 的 a1 螺旋对其酶激活很重要的一项测定中，我们注意到用于鉴定 Tsg101 和 Nedd4 复合物的 Pull Down 测定中的 Western 条带明显高于我们通常观察到的条带。经过进一步测试，我们发现当存在更多 Tsg101 时，Nedd4 的水平更高。我们还能够证明，当 Tsg101 不存在时，Nedd4 会被多泛素化并降解。因此，Tsg101 在细胞中充当 Nedd4 的伴侣。反之亦然，当 Nedd4 存在时，Tsg101 半衰期也更长。这两种蛋白质之间肯定存在直接相互作用，涉及泛素信号传导，最终调节它们的水平。除了上述研究之外，我们还测试了 Tsg101 相互作用的独特信号肽 PTAP (Pro-The-Ala-Pro)，该信号肽均存在于 ESCRT-0 的 Hrs 蛋白以及 CoV2 基因组 (PTEP) 中。、PTQP、PNQP）。我们可以识别 Tsg101 中 PTEP 特有的第二个识别位点。我们正在进行混合 (CoV2-HIV-1) 细胞测定以验证我们的研究结果。

项目成果

期刊论文数量（31）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Characterization of the N-terminal tail domain of histone H3 in condensed nucleosome arrays by hydrogen exchange and NMR.

DOI：
10.1021/ja9070078
发表时间：
2009-10-28
期刊：
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
影响因子：
15
作者：
Kato, Hidenori;Gruschus, James;Ghirlando, Rodolfo;Tjandra, Nico;Bai, Yawen
通讯作者：
Bai, Yawen

Author Correction: Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymes.

作者更正：TTLL 家族酶引发和延长聚谷氨酸链的结构基础。

DOI：
10.1038/s41594-020-0498-1
发表时间：
2020
期刊：
Nature structural & molecular biology
影响因子：
16.8
作者：
Mahalingan,KishoreK;KeithKeenan,E;Strickland,Madeleine;Li,Yan;Liu,Yanjie;Ball,HaydnL;Tanner,MartinE;Tjandra,Nico;Roll-Mecak,Antonina
通讯作者：
Roll-Mecak,Antonina

Temperature dependence of molecular interactions involved in defining stability of glutamine binding protein and its complex with L-glutamine.

DOI：
10.1021/bi201494h
发表时间：
2012-01-17
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Pistolesi, Sara;Tjandra, Nico
通讯作者：
Tjandra, Nico

Increasing the Chemical-Shift Dispersion of Unstructured Proteins with a Covalent Lanthanide Shift Reagent.

DOI：
10.1002/anie.201607261
发表时间：
2016-11-14
期刊：
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
影响因子：
16.6
作者：
Goebl, Christoph;Resch, Moritz;Strickland, Madeleine;Hartlmueller, Christoph;Viertler, Martin;Tjandra, Nico;Madl, Tobias
通讯作者：
Madl, Tobias

Ligand-free open-closed transitions of periplasmic binding proteins: the case of glutamine-binding protein.