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Structural and Functional Overview

Hypoxia-inducible factor 1 beta (HIF-1β), also known as aryl hydrocarbon receptor nuclear translocator (ARNT), is a pivotal component of the HIF transcription factor complex, which orchestrates cellular responses to hypoxic conditions [4]. Structurally, HIF-1β is characterised by its role as a constitutively expressed protein that forms a heterodimer with the oxygen-regulated HIF-1α subunit [5]. Both proteins belong to the basic helix-loop-helix (bHLH) family of transcription factors, sharing several critical structural domains that are essential for their functionality [7].

The primary structure of HIF-1β consists of several key regions, each contributing significantly to its overall functionality. At the forefront is the N-terminal bHLH domain, which is essential for DNA binding [6]. This domain enables the HIF complex to interact with hypoxia response elements (HREs) found within the promoters of target genes. Its unique structural configuration allows for precise recognition and binding to specific DNA sequences, thereby initiating the process of transcriptional activation [1][3]. Moving inward, the central Per-ARNT-Sim (PAS) domain plays a crucial role in facilitating heterodimerization with HIF-1α, ensuring the stability and functionality of the entire HIF complex. Notably, this PAS domain also serves as a sensor for environmental / oxygenic signals, allowing it to modulate the activity of the dimer in response to various stimuli [4][5]. Completing this intricate structure is the C-terminal domain, which is instrumental in recruiting a variety of transcriptional co-regulatory proteins necessary for activating target genes under hypoxic conditions. The interactions orchestrated by this domain are vital for amplifying the transcriptional output of the HIF complex, thereby ensuring a robust and coordinated response to low oxygen levels [1][2].

Functionally, HIF-1β does not directly respond to hypoxia; rather, as mentioned before, it serves as an essential partner for HIF-1α in forming a transcriptionally active complex [6]. Under normoxic conditions, HIF-1α undergoes hydroxylation at specific proline residues mediated by prolyl-hydroxylases, leading to its ubiquitination and subsequent degradation via proteasomes. In stark contrast, HIF-1β remains stable and is persistently present within the nucleus, poised to engage with HIF-1α once oxygen levels diminish [3][5].

In hypoxic environments, the activity of prolyl-hydroxylases decreases due to limited oxygen availability, resulting in the stabilisation and accumulation of HIF-1α [6]. This stabilised form then dimerizes with HIF-1β, allowing the complex to bind to specific DNA sequences and activate transcription of target genes that are crucial for adaptive responses to fluctuations in oxygen availability such as increased erythropoietin production and enhanced vascularization, consequently contributing to the maintenance of cell homeostasis [3][4][5][6].

Recent studies have illuminated additional layers of complexity within this regulatory framework. For instance, mutations in genes associated with succinate dehydrogenase can induce a phenomenon known as "pseudohypoxia," wherein HIF pathways become activated even under normoxic conditions due to disruptions in metabolic processes [2]. This highlights the intricate regulatory mechanisms governing HIF activity and its implications in various pathophysiological contexts, including cancer progression, where HIF-1β has been shown to contribute to tumour angiogenesis and metabolic reprogramming [1][2].

References:

[1] Guo, H., Huang, J., Liang, Y. et al. (2022) Focusing on the hypoxia-inducible factor pathway: role, regulation, and therapy for osteoarthritis. Eur J Med Res 27, 288. https://doi.org/10.1186/s40001-022-00926-2

[2] Infantino, V., Santarsiero, A., Convertini, P., Todisco, S., Iacobazzi, V. (2021) Cancer Cell Metabolism in Hypoxia: Role of HIF-1 as Key Regulator and Therapeutic Target. International Journal of Molecular Sciences.; 22(11):5703. https://doi.org/10.3390/ijms22115703

[3] Lisy, K., Peet, D. (2008) Turn me on: regulating HIF transcriptional activity. Cell Death Differ 15, 642–649. https://doi.org/10.1038/sj.cdd.4402315

[4] Mandl, M., Depping, R. (2014) Hypoxia-Inducible Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) (HIF-1β): Is It a Rare Exception?. Mol Med 20, 215–220. https://doi.org/10.2119/molmed.2014.00032

[5] Mole, D. R., Blancher, C., Copley, R. R., Pollard, P. J., Gleadle, J. M., Ragoussis, J., & Ratcliffe, P. J. (2009). Genome-wide association of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha DNA binding with expression profiling of hypoxia-inducible transcripts. The Journal of biological chemistry, 284(25), 16767–16775. https://www.jbc.org/article/S0021-9258(18)66494-4/fulltext

[6] Pugh, C. W., O'Rourke, J. F., Nagao, M., Gleadle, J. M., & Ratcliffe, P. J. (1997). Activation of hypoxia-inducible factor-1; definition of regulatory domains within the alpha subunit. The Journal of biological chemistry, 272(17), 11205–11214. https://www.jbc.org/article/S0021-9258(18)40624-2/fulltext

[7] Sadeghi, F., Kardar, G.A., Bolouri, M.R. et al. (2020) Overexpression of bHLH domain of HIF-1 failed to inhibit the HIF-1 transcriptional activity in hypoxia. Biol Res 53, 25. https://doi.org/10.1186/s40659-020-00293-4

HIF–1β expression in yeast

To test the functionality of the HIF system in the humanised yeast strain, we aimed to test the expression of HIF–1β protein. The humanised yeast strain contained the bidirectional GAL1,10 promoter, which is activated in the presence of galactose and the absence of glucose. Thus, we cultivated overnight cultures of our yeast strains in selective SD media containing 2% glucose, then washed the cells and cultivated them for 6 hours in SD media containing 0.5% glucose. After 6 hours we added galactose to the media to reach the final concentration of 2%. The yeast strains were then cultivated in these conditions for 20 hours. After 20 hours, cell samples were washed and fixed for immunofluorescence assay of HIF–1β protein.

After incubation with primary (HIF–1 alpha Monoclonal Antibody or HIF–1 beta Monoclonal Antibody) antibodies, we used Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody conjugated with Alexa Fluor™ 488 to visualise the results. The visualisation of results using epifluorescence microscopy (Alexa Fluor 488 filters, 1000x magnification) revealed interesting growth morphology of the yeast strains. In all samples, a varied count of elongated yeast cells were observed. The elongated cells were emitting green light, not enough to be considered fluorescent, however, green colour, expected from Alexa Fluor, was present. In comparison to immunofluorescence microscopy for HIF-1α, HIF–1β revealed more green light emission from the elongated cells.

Figure 1: Immunofluorescence microscopy for HIF-1β of humanised yeast strain HG21 after 20 hours of galactose induction, elongated cells in red rectangles. Accessible at: https://static.igem.wiki/teams/5415/results/alise2.webp

The phenomenon of elongated cell morphology might be due to galactose induction, when no glucose and limited nitrogen were present in the media. In theory, this can lead to cell cycle arrest or abnormal cell morphology in yeast cells. Moreover, the mechanism of HIF–1β degradation in normal oxygen conditions, however, is not known due to the fact that it is usually expressed constitutively in human cellssynthesised in hypoxic conditions only. To eliminate the rapid degradation of HIF system proteins to some extent, stabilisation techniques could be used and have to be tested.