DNA binding dynamics and oligomerization of sensors of the pyhin family in pattern recognition

The National Institute of Allergy and Infectious Diseases (NIAID) awarded the University of California, Merced a Project Grant of $971,109 under the Allergy and Infectious Diseases Research program (CFDA 93.855) on June 10, 2025, with a completion date of May 31, 2028. This award supports research on DNA binding dynamics and oligomerization of sensors in the PYHIN (Pyrin and HIN-domain-containing) family of pattern recognition receptors. The research focuses on Absent in Melanoma 2 (AIM2) and Interferon-Inducible Protein 16 (IFI16), two intracellular proteins that detect pathogenic DNA and trigger inflammasome assembly and inflammatory responses. The project will advance mechanistic understanding of how these proteins recognize and bind pathogenic DNA at the single-molecule level using advanced laboratory techniques including optical traps and confocal fluorescence microscopy.

The research outcomes will contribute to elucidating fundamental mechanisms of innate immunity while generating knowledge applicable to therapeutic interventions. Because dysregulation of inflammation contributes to multiple diseases including cancer, diabetes, and cardiovascular disorders, and because AIM2 and IFI16 dysfunction is implicated in autoimmune disorders and cancer drug resistance, this research has direct relevance to developing anti-inflammatory and anticancer therapies. The work will be conducted at UC Merced's research facilities in California and aligns with NIAID's mission to support investigator-initiated biomedical research advancing scientific knowledge of infectious and immunologic diseases.

Abstract

Pattern recognition receptors are membrane and cytosolic proteins that alert the organism's defense mechanisms by detecting pathogen-associated and danger-associated molecular patterns. The recognition of pathogenic DNA from bacteria and viruses by DNA sensors triggers an inflammatory response indicating the danger of infection. Absent in melanoma 2 (AIM2) and interferon-inducible protein 16 (IFI16) are intracellular pattern recognition receptors that detect pathogenic DNA. Upon DNA binding, AIM2 and IFI16 oligomerize and recruit other proteins leading to inflammasome assembly. Inflammasomes are multiprotein complexes that trigger inflammation by activating cytokines in response to danger signals from pathogens or cell dysfunction. Although inflammation is a fundamental defense mechanism, its dysregulation leads to the onset of several diseases, including cancer, diabetes, and cardiovascular disorders. The functions of AIM2 and IFI16 and the signaling pathways they elicit are implicated in autoimmune disorders and drug resistance of certain types of cancer. Thus, AIM2 and IFI16 are desired targets in anti-inflammatory and anticancer therapies. AIM2 and IFI16 belong to the pyhin family of multidomain proteins bearing HIN domains for DNA binding and PYD domains for oligomerization. Evidence suggests that the HIN domains participate in DNA recognition to different extents by contributing to receptor oligomerization at various levels and engaging in heterotypic HIN-HIN interactions that could be involved in receptor inhibition. Our laboratory has reported key mechanistic information on the assembly of AIM2 on DNA at the single-molecule level using optical traps combined with confocal fluorescence microscopy. We showed that the binding of AIM2 to DNA promotes AIM2 self-association into oligomers of different sizes. Our real-time data on AIM2-DNA binding allowed US to establish that single AIM2 molecules and oligomers do not significantly diffuse along the DNA. We are extending these studies to IFI16 HIN domains, revealing they also oligomerize on DNA. In contrast to AIM2, IFI16 HIN domains demonstrate significant mobility. We have corroborated these results with biochemical methods showing that IFI16-HIN oligomerization on double- and single-stranded DNA depends on DNA length. In this project, we will demonstrate that individual HIN domains play different roles in molecular pattern recognition based on their different diffusivity and oligomerization on DNA and their capability to engage in heterotypic HIN-HIN interactions. We will use optical traps and confocal fluorescence microscopy to establish relationships between mobility, oligomerization, and HIN-DNA complex survival time. In addition, we will determine the stoichiometry of HIN-DNA complexes as a function of DNA length and identify heterotypic HIN-HIN interactions by biochemical methods. The successful outcome of this project will define the impact of oligomerization and binding dynamics on the survival of HIN- DNA complexes, which is key for subsequent signaling steps of innate immunity and inflammatory processes.