In the field of cell signaling research, the signal transducer and activator of transcription (STAT) family plays a critical role, with abnormal activation closely related to various diseases such as cancer and autoimmune diseases, making it an important drug target. However, the long-standing lack of effective tools for real-time monitoring of STAT activation has hindered related biological research and drug development. Recently, a study published in Nature Chemical Biology developed a class of highly sensitive gene-encoded biosensors, STATeLights, providing a breakthrough to address this issue.
The STAT family consists of seven members, among which STAT5 is crucial in immune cell signaling. Under normal circumstances, STAT5 undergoes tyrosine phosphorylation and conformational changes to form active dimers that enter the nucleus to initiate target gene transcription in response to stimuli such as cytokines. The precise regulation of this process is significant for cell proliferation and differentiation. Previous methods for detecting STAT5 activation, such as immunostaining, require cell fixation and cannot achieve real-time monitoring; commercial reporter cell lines also have limitations. Although methods based on fluorescence resonance energy transfer (FRET) have potential, they have previously struggled to continuously monitor STAT5 activation in real-time.
The design of the STATeLights biosensor is both clever and efficient. The research team first focused on STAT5A, using AlphaFold polymer simulations to predict its full-length dimer structure, discovering that STAT5A exists in both inactive antiparallel dimers and active parallel dimers. Based on this, they selected mNeonGreen (mNG) and mScarlet-I (mSC-I) as the FRET donor and acceptor fluorescent protein pair, monitoring conformational changes by fusing these two fluorescent proteins at appropriate positions in STAT5A. After screening eight different fusion strategies, they identified the optimal STAT5A biosensor, STATeLight5A, which produces significant FRET signal changes under IL-2 stimulation, with fluorescence lifetime changes primarily observed in the cytoplasm, consistent with the cellular localization characteristics of STAT5 activation.
STATeLight5A demonstrates excellent performance and broad applicability. In terms of specificity, IL-2 and IL-15 significantly reduce its mNG fluorescence lifetime, while IL-4, which does not activate STAT5, shows no such effect. The changes in fluorescence lifetime are highly correlated with the dimerization ratio of STAT5A. Dose-response experiments show that it can detect STAT5A dimerization induced by IL-2 as low as 0.01 ng/ml, with a half-maximal effective concentration of 2.369 ng/ml, and can track the dynamic activation of STAT5A in real-time, detecting dimer formation within five minutes and reaching a peak at seventy minutes. Additionally, STATeLight5A can form heterodimers with endogenous STAT5B without interfering with the endogenous STAT5 signaling pathway, and after transfecting PTPN1, it can also monitor the dephosphorylation process of STAT5, demonstrating reversibility.
The application scenarios for this biosensor are extensive. In studies of other members of the STAT family, STATeLight5B, STATeLight1-4, and STATeLight6, developed based on similar design strategies, can effectively detect corresponding STAT homodimerization and heterodimerization. In mutation analysis, using STATeLight5A to study loss-of-function (LOF) and gain-of-function (GOF) mutations of STAT5A clearly reveals the impact of different mutations on STAT5A dimerization, such as LOF mutations failing to form dimers, while GOF mutations exhibit higher dimerization efficiency and stronger resistance to dephosphorylation. For cancer-related STAT5B mutations, it can also accurately assess their effects on STAT5B activation, transcriptional activity, and resistance to dephosphorylation.
In the field of drug screening, STATeLight5A also performs excellently. The research team used it to test various JAK inhibitors and STAT5 inhibitors, accurately quantifying the inhibitory effects of the inhibitors on STAT5A dimerization, such as determining the half-maximal inhibitory concentrations of upadacitinib and filgotinib, and observing the time-resolved inhibition patterns of different JAK inhibitors.
Moreover, STATeLight5A has also been successfully applied to human primary CD4+ T cells, achieving real-time monitoring of STAT5 activation in primary immune cells, and can detect the inhibitory effects of JAK inhibitors on STAT5 activation in these cells, providing a powerful tool for studying STAT signaling under physiologically relevant conditions.
The emergence of STATeLights biosensors has brought revolutionary changes to STAT biology research and drug development. In the future, by further optimizing their dynamic range, such as trying different combinations of fluorescent proteins or integrating techniques like single-molecule localization microscopy, it is expected to reveal the regulatory mechanisms of the JAK-STAT signaling pathway in greater depth, promoting the development of diagnostic tools and drug screening strategies for related diseases, contributing more to human health.