Nmr Studies Of The Regulation Of Cell Signaling

细胞信号传导调节的核磁共振研究

• 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
• 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

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.

作为开发新技术以表征调节重要生物学功能的动态分子事件的进展，我们选择查看逆转录病毒的衣壳组件。该蛋白是Gag-Poly蛋白的一部分，该蛋白是作为病毒成熟的一部分加工的。衣壳颗粒的组装和拆卸对于分别从进入宿主细胞的病毒萌芽至关重要。我们表明，由于两种类型的不同分子相互作用，衣壳组件发生。 N末端β发夹促进了螺旋1的伸长率，该螺旋形成形成衣壳颗粒的寡聚界面。该事件发生在较慢的时间表上，而不是涉及capsid的C末端域的二聚化。我们只能通过使用NMR实验的弹跳来确定上述观察结果。这主要是由于分子相互作用的动态性质。 我们还合成了一个化合物（甲基化dota），该化合物可以与柔韧性降低，该化合物（甲基化dota）可以协调灯笼配体。这是与成像探针开发小组合作完成的。目的是实现可观察到的伪接触转移（PC）的大幅增加，并使用该信息进行结构确定。此外，我们还表明，甲基化的氟乙烷采用了两个异构体。这些异构体的种群取决于与之协调的灯笼金属的大小。我们通过在蛋白质上观察PC与从HPLC上获得的甲基化 - 甲基苯胺的PC相匹配来测量的种群比率。我们对DOTA灯笼进行了温度依赖研究，以表明易感性张量的大小在很大程度上取决于温度，这是由于绑定的水汇率所致。速率越慢，张量越大。我们表明，通过引入PC来研究本质上无序的蛋白质，甲基化的甲基苯胺也非常实用，从而可以更好地分散通常重叠的NMR共振。此外，在动态蛋白质 - 普罗替型复合物（例如表现出遇到复合物的蛋白质复合物）的情况下，使用自旋标记氮氧化物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PC，我们可以确定氮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寄生系统之间会遇到复合物可以相互竞争。我们监视了HPR存在的酶I和NPR特异性的变化，并遇到了复合物。我们先前确定HPR与酶I的相互作用并未特别相互作用。随着HPR浓度的增加，我们表明可以调节NPR遭遇，以使特定的酶I和NPR复合物种群增加，从而有效地增加了复合物的亲和力。这是一个令人惊讶的发现，因此我们决定通过建模研究跟进这项研究，在HPR存在的情况下，我们可以概括NPR和酶I之间的相遇概况。这项研究表明，除了特定底物调节生物学功能的竞争之外，缺乏理解。 我们研究了具有生物学相关性的另一种弱蛋白质蛋白质相互作用。在TSG101的情况下，它与泛素（UB）的相互作用相当薄弱。我们能够检测与上面开发的新的顺磁技术的这种相互作用。我们建立了这种UB-TSG101相互作用的抑制剂，使我们能够在宿主出售的病毒贩运中破译UB信号传导。另外，使用NMR我们确定了TSG101上的另一个UB结合位点。我们证实TSG101识别di-upiquitin（Di-UB），该二氨基酸素（DI-UB）是在K63处链接的。我们还能够使用DI-UB在两个不同位点结合TSG101的NMR确定。这两个部位具有不同的生理后果。一个所谓的遗迹UB结合位点控制了HIV-1 GAG对质膜募集TSG101的位置，而N-Termninal UB位点似乎与运输HIV-1 GAG的核帽决定因素相关。有趣的是，TRI-UB与TSG101的绑定不如DI-UB。这一发现是新颖的，使我们能够在TSG101（ESCRTI）途径中区分多个方面。 我们已经继续使用我们的TSG101和UB相互作用的发现来发展可以抑制它们相互作用的小分子。我们发现，在临床上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。我们表明，这种抑制作用会干扰从宿主细胞中释放HIV-1病毒颗粒。此外，这种抑制作用似乎还会减少宿主细胞中的病毒蛋白产生，例如GAG。我们表明，对于其他病毒，可以观察到相同的效果，因此可以向普拉唑家族作为广泛的抗病毒剂的潜力表明潜力。由于我们确切地知道TSG101-UB抑制的分子机制，因此我们可以使用这些知识来推动新一代的化合物，这些化合物可以提供更具体和效力。这项工作已扩大，以测试我们针对其他病毒（包括冠状病毒）的化合物。这是通过NIAID核心进行抗病毒测试完成的。我们的几个化合物显示出阻断这些病毒的功效，包括SARS-COV2。我们正在生成新化合物的新衍生物，以提高其选择性。 同时，我们还研究了细胞会产生伪-E2-泛素连接酶的原因，这就是TSG101的意思。它具有与UB E2连接酶相同的结构，其催化性半胱氨酸被酪氨酸取代，因此TSG101没有酶促活性。此外，我们最近还表明该蛋白可以识别细胞RNA。这种功能以某种方式与细胞中的UB识别有关。我们研究的下一阶段是针对所有发现的结合，以制定一般方案，即如何将所有这些过程捆绑在一起以使细胞运输受益以及病毒如何修改它们的复制。 我们通过查看其与NEDD4的潜在相互作用来扩展了对TSG101的研究，而E3泛素连接酶对HIV-1复制很重要。我们表明TSG101 UEV结构域确实与NEDD4的Hect域相互作用。这种相互作用非常薄弱。我们使用NMR PRE实验来识别接触TSG101的区域。涉及这种相互作用的那些残基属于hect的n-和c-lobe之间的铰链区域。我们使用NMR实验确定的位点中的HIV-1病毒样片段释放中的救援测定法和NEDD4突变验证了这一发现。在我们的一种测定中证明NEDD4的A1螺旋对于其酶激活很重要，我们注意到下拉测定中的西部带以鉴定TSG101和NEDD4复合物的明显高于我们通常观察到的。经过进一步测试，我们表明当存在更多TSG101时，NEDD4的水平较高。我们还能够表明，当不存在TSG101时，NEDD4会被多泛纤维化和降解。因此，TSG101充当细胞中NEDD4的伴侣。相反的情况也是如此，当存在NEDD4时，TSG101半寿命也更长。这两种蛋白质之间肯定有一个直接的相互作用，涉及泛素信号，最终调节其水平。 除上述研究外，我们还测试了TSG101相互作用PTAP（Pro-the-ala-Pro）的独特信号肽，该肽都在蛋白质HRS（ESCRT-0的一部分）以及COV2基因组（PTEP，PTQP，PTQP，PNQP）中找到。我们可以确定一个针对TSG101中PTEP的第二个识别站点。我们正在进行混合（COV2-HIV-1）细胞测定法，以验证我们的发现。

项目成果

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.

• DOI: 10.1021/bi902045p
• 发表时间: 2010-03-09
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Bermejo, Guillermo A.;Strub, Marie-Paule;Ho, Chien;Tjandra, Nico
• 通讯作者: Tjandra, Nico