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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 我们打算使用更有效的时间分辨技术来研究蛋白质：蛋白质相互作用的动力学。 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IFN-γ受体复合物上，并扩展到IL-10和干扰素-γ（IFN-g）受体复合物。 IFN-g受体复合物是特别重要的，因为已经提出了它与IFN-g受体复合物的串扰和生物协同作用。 IFN-g是一组称为I型IFNs的蛋白质的代表，当被病毒感染时，由细胞释放，并起到保护非造血细胞免受病毒感染的作用。 具体目标是： 1. 研究活细胞中受体链相互作用的动力学。 2.研究活细胞中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.分析受体复合物与配体结合时的相互作用。 以前，我们打算使用荧光共振能量转移（FRET），以确定总的结构的干扰素-γ（IFN-g）受体复合物在其天然环境中的活细胞。 我们采用这种直接的方法，因为从受体复合物的自然环境之外的研究中获得了相互矛盾的结论。 在区域激光生物技术实验室的系统设置非常适合FRET显微镜：共聚焦显微镜耦合到单色仪，允许频率分辨荧光测量生物样品上进行，而无需任何额外的操作。 频率分辨的测量是必不可少的明确建立FRET的荧光发射。 阐明存在于真实的细胞中的IFN-g信号转导级联的动力学对于鉴定IFN-g信号传导的抑制剂或激活剂将是非常重要的，所述抑制剂或激活剂可在癌症、自身免疫性疾病、病毒性疾病、组织移植排斥和其它情况中具有调节免疫系统的治疗用途。 我们跟踪了IFN-γ受体复合物激活Stat 1的动力学，发现该动力学非常不寻常：在配体结合几乎瞬间发生的条件下，除非IFN-γ R 1链或IFN-γ R 2链过量，否则Stat 1激活存在潜伏期。 先前发表的观察结果支持的对该潜伏期的一种解释是两个配体结合的IFN-γ受体复合物相互作用。 实时荧光技术的使用将在建立受体激活和运动的动力学过程中是有价值的。

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.