The Participation of the Intrinsically Disordered Regions of the bHLH-PAS Transcription Factors in Disease Development

Abstract

The basic helix-loop-helix/Per-ARNT-SIM (bHLH-PAS) proteins are a family of transcription factors regulating expression of a wide range of genes involved in different functions, ranging from differentiation and development control by oxygen and toxins sensing to circadian clock setting. In addition to the well-preserved DNA-binding bHLH and PAS domains, bHLH-PAS proteins contain long intrinsically disordered C-terminal regions, responsible for regulation of their activity. Our aim was to analyze the potential connection between disordered regions of the bHLH-PAS transcription factors, post-transcriptional modifications and liquid-liquid phase separation, in the context of disease-associated missense mutations. Highly flexible disordered regions, enriched in short motives which are more ordered, are responsible for a wide spectrum of interactions with transcriptional co-regulators. Based on our in silico analysis and taking into account the fact that the functions of transcription factors can be modulated by posttranslational modifications and spontaneous phase separation, we assume that the locations of missense mutations inducing disease states are clearly related to sequences directly undergoing these processes or to sequences responsible for their regulation.

Keywords: ARNT2; AhR; AhRR; BMAL1; Catalogue of Somatic Mutations in Cancer (COSMIC); D2P2; Hif-2α; HuVarBase; LLPS prediction; NPAS4; PScore; SIM2; STRING; Single-Minded Protein 1 (SIM1); Waltz; cancer; catGranule; disease-associated mutation; disorder prediction; intrinsically disordered region (IDR); liquid-liquid phase separation (LLPS); post-translational modifications (PTM).

Figure 2

Schematic presentation of results for (A) AhR (P35869) and (B) AhRR (A9YTQ3) analysis. (a) Post-translational modifications based on PhosphoSitePlus server [99]; (b) the domain structure of protein: green indicates the bHLH domain (27–80aa AhR; 28–81aa AhRR), purple represents PAS domains (111–181aa PAS1, 275–342aa PAS2 AhR; 112–182aa PAS AhRR), whereas blue indicates PAC (348–386aa PAC AhR). Predicted MoRFs [100] are indicated as orange rectangles, (c) D²P² database disorder regions predictions based on the protein amino acids sequence (find the legend in the plot for description). Grey shadow presents the averaged disorder profile, and a score over 0.5 indicates a high probability of disorder. Positions of disease-linked mutations are marked as black vertical lines (listed in HuVarBase database [101], Supplementary Materials), (d) liquid–liquid phase separation (LLPS) propensity predictions based on catGranules (blue line) [102] and PScore (purple line) [103] servers; positions of disease-linked mutations are marked as black vertical lines (listed in HuVarBase database [101], Supplementary Materials).

Figure 3

Schematic presentation of results for (A) SIM1 (P81133) and (B) SIM2 (Q14190) analysis. (a) Post-translational modifications based on PhosphoSitePlus server [99], (b) the domain structure of protein, green indicates the bHLH domain (1–63aa SIM1; 1–53aa SIM2), purple represents PAS domains (77–147aa PAS1 SIM1, 77–149aa PAS1 SIM2, 218–288aa PAS2 SIM1/2), whereas blue indicates PAC (292–335aa PAC SIM1/2). Predicted MoRFs [100] are indicated as orange rectangles, (c) D²P² database disorder regions predictions based on the protein amino acids sequence (find the legend in the plot for description). Grey shadow presents the averaged disorder profile, and a score over 0.5 indicates a high probability of disorder. Positions of disease-linked mutations are marked as black vertical lines (listed in HuVarBase database [101], Supplementary Materials), (d) LLPS propensity predictions based on catGranules (blue line) [102] and PScore (purple line) [103] servers; positions of disease-linked mutations are marked as black vertical lines (listed in HuVarBase database [101], Supplementary Materials).

Figure 5

Schematic presentation of results for (A) ARNT2 (Q9HBZ2) and (B) BMAL1 (O00327) analysis. (a) Post-translation modifications based on PhosphoSitePlus server [99]; (b) the domain structure of protein, green indicates the bHLH domain (63–116aa ARNT2;72–125aa BMAL1); purple represents PAS domains (134–209aa PAS1, 323–393aa PAS2, ARNT2; 143–215aa PAS1, 326–396aa PAS2 BMAL1), whereas blue indicates PAC (398–441aa PAC ARNT2; 401–444aa PAC BMAL1). Predicted MoRFs [100] are indicated as orange rectangles, (c) D²P² database disorder regions predictions based on the protein amino acids sequence (find the legend in the plot for description). Grey shadow presents the averaged disorder profile, and a score over 0.5 indicates a high probability of disorder. Positions of disease-linked mutations are marked as black vertical lines (listed in HuVarBase database [101], Supplementary Materials), (d) LLPS propensity predictions based on catGranules (blue line) [102] and PScore (purple line) [103] servers; positions of disease-linked mutations are marked as black vertical lines (listed in HuVarBase database [101], Supplementary Materials).

Figure 1

Structure organization of basic helix–loop–helix/Per-ARNT-SIM (bHLH-PAS) proteins. (A) The domain structure of bHLH-PAS proteins [12]; green indicates the bHLH domain, purple indicates PAS domains, and blue indicates PAS-associated C-terminal (PAC), respectively, (B) crystal structure of the heterodimeric NPAS3-ARNT complex with Hypoxia Response Element (HRE) DNA (PDB: 5SY7) [13]. The bHLH domain, responsible for DNA binding, is colored in green, whereas PAS-Domain Containing Protein 1 (PAS1) and PAS2 domains are colored in purple.

Figure 4

Schematic presentation of results for (A) Hif-2α (Q99814) and (B) NPAS4 (Q8IUM7) (B) analysis. (a) Post-translational modifications based on PhosphoSitePlus server [99]; (b) the domain structure of protein, green indicates the bHLH domain (14–47aa Hif-2α; 1–53aa NPAS4), purple represents PAS domains (84–154aa PAS1, 230–300aa PAS2 Hif-2α; 70–144aa PAS1, 203–273aa PAS2 NPAS4), whereas blue indicates PAC (304–347aa PAC Hif-2α; 278–317aa PAC NPAS4). Predicted MoRFs [100] are indicated as orange rectangles, (c) D²P² database disorder regions predictions based on the protein amino acids sequence (find the legend in the plot for description). Grey shadow presents the averaged disorder profile, and a score over 0.5 indicates a high probability of disorder. Positions of disease-linked mutations are marked as black vertical lines (listed in HuVarBase database [101], Supplementary Materials), (d) LLPS propensity predictions based on catGranules (blue line) [102] and PScore (purple line) [103] servers; positions of disease-linked mutations are marked as black vertical lines (listed in HuVarBase database [101], Supplementary Materials).

Figure 6

In silico prediction of amylogenic regions for AhR, AhRR, SIM1, SIM2, Hif-2α, NPAS4, ARNT2, and BMAL1 using Waltz predictor [109].

Figure 7

STRING-based interactome between selected representatives of bHLH-PAS transcription factor (TF) proteins (an internal protein-protein interaction network (PPI)). In the corresponding STRING-generated network, the nodes correspond to proteins, whereas the edges show predicted or known functional associations. Seven types of evidence are used to build the corresponding network, where they are indicated by the differently colored lines: a green line represents neighborhood evidence; a red line—the presence of fusion evidence; a purple line—experimental evidence; a blue line—co-occurrence evidence; a light blue line—database evidence; a yellow line—text mining evidence; and a black line—co-expression evidence.

Figure 8

STRING-based external interactome of selected bHLH-PAS TFs with the “first shell” interactors. A confidence level of 0.5 was used in this analysis.