Citrullination at the Nuclear Localization Signal of the Inhibitor of Growth 4 (ING4) Interferes With its Binding to Importin a3

Abstract

The inhibitor of growth 4 (ING4) acts as a tumor suppressor regulating chromatin structure. Due to this nuclear function, ING4 has a nuclear localization signal (NLS), which is recognized by the cellular translocation machinery ‒ mainly formed by proteins named importins, which include the isoform importin α3 (Impα3) ‒ to allow movement through the nuclear membrane. Peptidyl arginine iminohydrolases (PADIs) are enzymes involved in the posttranslational modification of arginine to citrulline. PADI4, one of the five isoforms of PADI in humans, citrullinates ING4 at the NLS region. We studied in vitro and in silico how the different degrees of citrullination affected binding of the NLS of ING4 to PADI4, Impα3 and its truncated species (ΔImpα3), lacking the importin binding domain, by using several biophysical techniques and molecular simulations. To that end, we synthesized eight peptides encompassing the NLS of ING4 (residues 130–152), with single, double and triple citrulline replacements at Arg132, Arg142 and Arg144. The peptides were monomeric and disordered, as tested by DOSY, 1D- and 2D-1H NMR experiments. All the peptides were capable of binding to PADI4 with low micromolar affinities, but their affinity decreased as the fraction of citrullination increased. Moreover, all peptides could bind to both importin species with affinities in the low micromolar range, and their affinities were also dependent on the citrullination degree. The peptides targeted the canonical NLS binding site for cargo proteins of both importin species. These findings suggest that: (i) citrullination at the NLS might interfere with ING4 nuclear translocation; and (ii) successive citrullination at the NLS affected binding to PADI4.