The chromatin nuclear protein NUPR1L is intrinsically disordered and binds to the same proteins as its paralogue

Abstract

NUPR1 is a protumoral multifunctional intrinsically disordered protein (IDP), which is activated during the acute phases of pancreatitis. It interacts with other IDPs such as prothymosin α, as well as with folded proteins such as the C-terminal region of RING1-B (C-RING1B) of the Polycomb complex; in all those interactions, residues around Ala33 and Thr68 (the 'hot-spot' region) of NUPR1 intervene. Its paralogue, NUPR1L, is also expressed in response to DNA damage, it is p53-regulated, and its expression down-regulates that of the gene. In this work, we characterized the conformational preferences of isolated NUPR1L and its possible interactions with the same molecular partners of NUPR1. Our results show that NUPR1L was an oligomeric IDP from pH 2.0 to 12.0, as judged by steady-state fluorescence, circular dichroism (CD), dynamic light scattering, 1DH-NMR (nuclear magnetic resonance), and as indicated by structural modelling. However, in contrast with NUPR1, there was evidence of local helical- or turn-like structures; these structures were not rigid, as judged by the lack of sigmoidal behaviour in the chemical and thermal denaturation curves obtained by CD and fluorescence. Interestingly enough, NUPR1L interacted with prothymosin α and C-RING1B, and with a similar affinity to that of NUPR1 (in the low micromolar range). Moreover, NUPR1L hetero-associated with NUPR1 with an affinity of 0.4 µM and interacted with the 'hot-spot' region of NUPR1. Thus, we suggest that the regulation of gene by NUPR1L does not only happen at the DNA level, but it could also involve direct interactions with NUPR1 natural partners.

The Paralogue of the Intrinsically Disordered Nuclear Protein 1 Has a Nuclear Localization Sequence that Binds to Human Importin α3

“…We first determined whether intact NUPR1L could bind to Impα3 and ∆Impα3, keeping in mind that this IDP has a high tendency to aggregate [ 14 ]. We first mapped, by fluorescence and CD, whether there was binding between NUPR1L and each importin species, by comparing changes in the fluorescence and far-UV CD spectra of the complex with those obtained from the sum of the corresponding spectra of each molecule.…”

“…The results of CD and fluorescence for ∆Impα3 indicate the following: (i) there were changes in the environment around tryptophan residues of at least one of the two proteins (i.e., NUPR1L or ∆Impα3) upon binding (fluorescence spectra, Figure 1 A), and (ii) there were changes in the secondary structure of at least one of the proteins upon binding (CD spectra; Figure 1 B). As NUPR1L has no well-defined structure [ 14 ], and ∆Impα3 is a large protein with a rigid, well-formed helical fold [ 25 ], we suggest that the changes in CD spectra were due to the acquisition of structure by NUPR1L. We did not attempt to deconvolute the spectrum of the complex because of the presence of two polypeptide chains, and the fact that we do not know the exact conformation of NUPR1L.…”

“…In this aspect, NLS-NUPR1L behaves not differently from any other NLS region of a well-folded protein [ 15 , 20 , 22 , 40 ]; that is, it is disordered both in isolation and when participating in forming the complex with importins (in our studies, the latter conclusion was obtained from our docking simulations). Finally, it is important to pinpoint that Trp62 is also involved in the binding of NUPR1L to prothymosin α [ 14 ]; therefore, it seems that this residue can be classified as a hot spot of NUPR1L in the association with other molecular partners.…”

“…Comparison of the values of Table 1 for NLS-NUPR1L with those of the NLS region of NUPR1 (1.7 μM for Impα3 and 0.95 μM for ∆Impα3 [ 41 ]) indicate that the binding is stronger in the case of NUPR1. Therefore, although both paralogues bind to the same molecules ([ 14 ] and this work), their affinity for the different partners is dissimilar. This could provide a mechanism to explain the regulation between these proteins, not only at a DNA level, but also at a post-translational stage.…”

“…Expression of the NUPR1 gene is down-regulated by the presence of NUPR1L, a 97-residue-long paralogue of NUPR1; in turn, the expression of NUPR1L is p53-regulated [ 13 ]. We have recently shown that NUPR1L is also an IDP, but it has a higher tendency to self-associate than NUPR1 [ 14 ], and it shows regions with conformations including turn- or helix-like structures.…”