TIME-RESOLVED STUDY OF PROTEIN:PROTEIN INTERACTIONS IN SINGLE CELLS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们打算使用更有效的时间分辨技术来研究蛋白质的动力学：蛋白质相互作用。此外，我们希望使用先进的成像技术来详细说明细胞中FRET的变化发生在哪里。我们的研究将继续主要集中在干扰素-伽马受体复合体上，并延伸到IL-10和干扰素-伽马(干扰素-g)受体复合体。干扰素-g受体复合体特别重要，因为它的串扰和与干扰素-g受体复合体的生物协同作用已被提出。干扰素-g是一组被称为I型干扰素的蛋白质的代表，当细胞感染病毒时会释放这种蛋白质，并发挥保护非造血细胞免受病毒感染的作用。具体目标是： 1.研究活细胞内受体链相互作用的动力学。 2.研究活体细胞中Jak激酶与受体链相互作用的动力学。 3.研究受体运动、Jak激酶运动和Stat运动的动力学。 4.分析与配体结合时受体复合体之间的相互作用。 以前，我们打算使用荧光共振能量转移(FRET)来确定干扰素-γ(IFN-g)受体复合体在活细胞中天然环境中的大体结构。我们采用了这种直接的方法，因为对自然环境以外的受体复合体的研究得出了相互矛盾的结论。区域激光生物技术实验室的系统设置非常适合FRET显微镜：共焦显微镜与单色仪耦合，无需任何额外操作即可在生物样品上进行频率分辨荧光测量。频率分辨测量对于明确建立荧光发射中的FRET是必不可少的。 阐明在真实细胞中存在的干扰素-g信号转导级联的动力学对于表征干扰素-g信号的抑制剂或激活剂具有重要意义，这些信号抑制剂或激活剂可能在癌症、自身免疫性疾病、病毒疾病、组织移植排斥反应和其他情况下具有治疗作用。我们跟踪了干扰素-g受体复合体激活STAT1的动力学，发现这种动力学非常不寻常：在配体结合几乎瞬间发生的条件下，除非干扰素-GR1链或干扰素-GR2链过多，否则STAT1的激活存在潜伏期。对这种潜伏期的一种解释是，两个配体结合的干扰素-g受体复合体相互作用，这一解释得到了先前发表的观察结果的支持。使用实时荧光技术在建立受体激活和运动的动力学过程中将是有价值的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We intend to use techniques that are more efficiently time-resolved to study the dynamics of protein:protein interactions. Additionally, we want to use advanced imaging techniques to detail where in the cell changes in FRET are occurring. Our studies will continue to focus primarily on the IFN-gamma receptor complex, with extensions into the IL-10 and interferon-gamma (IFN-g) receptor complexes. The IFN-g receptor complex is especially important as its crosstalk and biological synergy with the IFN-g receptor complex has been suggested. IFN-g is representative of a group of proteins called Type I IFNs that are released by cells when infected by virus, and function to protect nonhematopoietic cells from virus infection. The specific aims are: 1. To investigate the dynamics of receptor chain interactions in live cells. 2. To investigate the dynamics of interactions of Jak kinases with receptor chains in live cells. 3. To investigate the kinetics of receptor movements, Jak kinase movements and Stat movements. 4. To analyze the interactions among receptor complexes when bound by ligand. Previously we intended to use Fluorescence Resonance Energy Transfer (FRET) to ascertain the gross structure of the interferon-gamma (IFN-g) receptor complex in its native environment in live cells. We employed this direct approach because conflicting conclusions were obtained from studies of the receptor complex outside its natural environment. The system setup at the Regional Laser Biotechnology Laboratory is ideally suited for FRET microscopy: a confocal microscope is coupled to a monochromator, allowing frequency-resolved fluorescence measurements to be done on biological samples without any additional manipulations. Frequency-resolved measurements are essential in the unequivocal establishment of FRET in fluorescence emission. The elucidation of the kinetics of the IFN-g signal transduction cascade as it exists in real cells will be of great importance in the characterization of inhibitors or activators of IFN-g signaling that could have therapeutic use in modulating the immune system in cancer, autoimmune disease, viral disease, tissue transplant rejection, and other cases. We followed the kinetics of Stat1 activation by the IFN-g receptor complex and found that the kinetics were very unusual: under conditions where ligand-binding occurs almost instantaneously, a latency period in the activation of Stat1 exists unless either the IFN-gR1 chain or the IFN-gR2 chain is in excess. One explanation of this latency period that is supported by previously published observations is that two ligand-bound IFN-g receptor complexes are interacting. The use of real-time fluorescent techniques will be valuable in establishing the kinetic process of receptor activation and movement.