Sequence Analysis and Phylogenetic Studies of Hypoxia-Inducible Factor-1α

Abstract

Hypoxia-inducible factors (HIF) belong to the basic helix loop helix-PER ARNT SIM (bHLH-PAS) family of transcription factors that induce metabolic reprogramming under hypoxic condition. The phylogenetic studies of hypoxia-inducible factor-1α (HIF-1α) sequences across different organisms/species may leave a clue on the evolutionary relationships and its probable correlation to tumorigenesis and adaptation to low oxygen environments. In this study, we have aimed at the evolutionary investigation of the protein HIF-1α across different species to decipher their sequence variations/mutations and look into the probable causes and abnormal behaviour of this molecule under exotic conditions. In total, 16 homologous sequences for HIF-1α were retrieved from the National Center for Biotechnology Information. Sequence identity was performed using the Needle program. Multiple aligned sequences were used to construct the phylogeny using the neighbour-joining method. Most of the changes were observed in oxygen-dependent degradation domain and inhibitory domain. Sixteen sequences were clustered into 5 groups. The phylogenetic analysis clearly highlighted the variations that were observed at the sequence level. Comparisons of the HIF-1α sequence among cancer-prone and cancer-resistant animals enable us to find out the probable clues towards potential risk factors in the development of cancer.

Keywords: Hypoxia-inducible factor-1α (HIF-1α); cancer resistance; hypoxia; oxygen-dependent degradation domain; phylogeny.

Figure 1

Multiple sequence alignment. (A) bHLH domain. The DNA-interacting basic amino acids K21, R29, K19, R30, R27, D55, and K56 are highlighted in red. (B) PAS-A, (C) PAS-B, and (D) PAS-C. PAS-B is involved in interaction with aryl hydrocarbon receptor nuclear transporter (ARNT) and HSP90. Residues interacting with PAS-B and ARNT were highlighted in red. PAS-C also includes residues interacting with ARNT as mentioned in crystal structure PDB ID (4H6J), and it is highlighted in red.

Figure 2

Multiple sequence alignment of oxygen-dependent degradation domain. Hydroxylation sites P402 and P564 are highlighted in yellow. The acetylation site (K532) is shown in orange red. The N-terminal transactivation domain is shown in green, and the C-terminal VHL recognition site is shown in red box.

Figure 3

Multiple sequence analysis of (A) inhibitory domain and (B) C-terminal transactivation domain (C-TAD). There are 2 binding sites in C-TAD. Site 1 in human hypoxia-inducible factor-1α carboxy-terminal activation domain encompasses residues 795 to 806 and contains the hydroxylated asparagine (N803), and site 2 includes residues 812 to 823 and shows only weak binding independent of site 1. These sites are highlighted in green.

Figure 4

Evolutionary relationships of taxa for hypoxia-inducible factor-1α of selected organisms.

Figure 5

Phylogeny of the oxygen-dependent degradation domain. The tree shows similar trend in group formation like the tree for entire hypoxia-inducible factor-1α. Shuffle is only observed in the group of even-toed ungulates.

Figure 6

Correlation of changes in human hypoxia-inducible factor-1α oxygen-dependent degradation domain with respect to blind-mole rat. The critical residues such as P402, P564, and K532 are well conserved.