Mechanisms and Regulation of Nf-κB Nuclear Import Through Importin α Isoforms

Tyler J Florio, Thomas Jefferson University

Abstract

Nucleocytoplasmic transport is a complex, highly regulated process vital to cell physiology in all eukaryotes. In the ‘classical’ nuclear import pathway, cargos bearing an exposed nuclear localization sequence (NLS) are recognized by a heterodimer of the importin β receptor and importin α adapter and are shuttled into the nucleus. The importin β receptor is necessary to translocate through the NPC by binding favorably with the flexible FG-Nups lining the NPC. Nuclear Import requires a gradient of RanGTP across the nuclear envelope, which dictates nucleocytoplasmic directionality and triggers the nuclear import complex to dissociate from high-affinity binding sites inside the NPC. Chapter one of this thesis provides an introduction to nuclear import mechanisms and key players, emphasizing the role of β-karyopherins, importin α isoforms diversity, NLS-cargo specificity, and the regulation of protein import by phosphorylation in the NLS. In chapter two, we interrogate the molecular mechanisms for importin α3 specificity towards the canonical NF-κB dimers. We used X-ray crystallography to obtain high-resolution crystal structures of NF-κB NLSs bound to importin α isoforms. This structural work led to the discovery of a novel in trans bipartite NLS in the p65:p50 and p65:p65 dimers bound with importin α, explaining the specificity for the isoform 3. Through structural and biochemical analysis, we have discovered that the p50-NLS is dominant compared to the p65-NLS and essential for association with importin α. Coupling high-resolution crystal structures with SEC-SAXS modeling, we determined the overall molecular architecture of the canonical NF-κB:importin α3 nuclear import complexes. These quaternary structures revealed that importin α3 intrinsic flexibility mediates differential recognition of the NF-κB dimers. In chapter three, we investigate the poorly understood second NLS (NLS2) of Influenza A virus NP and its nuclear import mechanism by importin α isoforms. We used site-directed mutagenesis and transfection studies to assess the functional role of NLS2 in Influenza A viral infection. This work revealed that the NLS2 is a functional NLS essential for Influenza A viral infection. Using X-ray crystallography, ITC, and quantitative pull-downs, we probed the molecular mechanisms governing NLS2 association with importin α isoforms. We determined NP binding to importin α7 is enhanced by the increased avidity from two NLSs and propose NP binds to importin α7 as an in trans bipartite NLS. In chapter four, we dissect the molecular architecture of the yeast inositol phosphatase Siw14 that we used as a model system to understand substrate specificity for phospho-inositols. Using a ‘divide and conquer’ approach that employs biochemical and biophysical techniques (X-ray crystallography, size-exclusion chromatography, small-angle X-ray scattering, analytical ultracentrifugation, circular dichroism), we discovered a surprisingly deep catalytic cleft in Siw14 that mediates specificity for di-phosphorylated inositol pyrophosphates. Overall, the studies described in this thesis examine the mechanisms and regulation of nuclear import and the role this vital pathway plays in cellular signaling.

Subject Area

Biochemistry|Cellular biology

Recommended Citation

Florio, Tyler J, "Mechanisms and Regulation of Nf-κB Nuclear Import Through Importin α Isoforms" (2021). ETD Collection for Thomas Jefferson University. AAI28720871.
https://jdc.jefferson.edu/dissertations/AAI28720871

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