Mechanisms of STAT1 and STAT3 canonical and persistent activation in physiology and disease

Summary Rapid plasma membrane-to-nucleus relocalization of Signal Transducer and Activator of Transcription (STAT) family members upon cytokines stimulation is vital for signal transduction. Countless cell processes, including immune cell’s ability to fight pathogens, depend directly on STAT signaling. Extracellular signals typically result in STAT phosphorylation, triggering a conformational change that leads to importin recruitment and nuclear translocation. In the nucleus, STATs function as transcription factors, inducing the expression of a plethora of genes involved in native immunity, survival, cell growth, proliferation, and pathogen response. Gain-of-function (GOF) mutations in STAT1 and STAT3 are associated with various immunodeficiencies characterized by over- activated immune cells, autoimmunity, and increased risk of infections. This proposal focuses on STAT1 and STAT3. Leveraging the power of cryogenic electron microscopy and biochemical, biophysical, and cellular methodologies, we aim to illuminate what constitutes a knowledge gap at the center of innate antimicrobial protection in STAT signaling. Aim 1 will decipher the mechanisms activated STAT1 and STAT3 enter the cell nucleus under physiological conditions. STATs do not have a classical Nuclear Localization Signal (NLS) yet use importin α/β and the GTPase Ran for nuclear entry. The molecular mechanisms of STAT nuclear import have not been elucidated and appear to differ in different STAT family members. Understanding STAT’s quaternary structure is crucial in deciphering how the nuclear import machinery is recruited without a classical NLS. Aim 2 will catalog GOF mutations identified in STAT1 and STAT3 and examine their impact on STAT quaternary structure, NLS exposure, and nuclear signaling, establishing a causative relationship between GOF mutations and persistent nuclear activation of STAT1 and STAT3. Our integrative approach to molecularly characterize STAT1 and STAT3 GOF mutations based on genotype- phenotype correlations will generate a comprehensive atlas of all known mutations discovered thus far.