ISGylation directly modifies hypoxia-inducible factor-2α and enhances its polysome association

Area of Science:

• Molecular Biology
• Cellular Biology
• Biochemistry

Background:

• Hypoxia-inducible factors (HIFs) are crucial for cellular adaptation to low oxygen, regulating gene expression in development and cancer.
• HIF-2α also functions as a translation factor, influencing protein synthesis.
• The ubiquitin-like modifier ISG15 plays roles in cellular responses, but its interplay with HIFs is not well understood.

Purpose of the Study:

• To investigate the regulatory relationship between ISG15 and HIF-1α/HIF-2α under hypoxia and interferon stimulation.
• To elucidate the mechanisms by which ISG15 influences HIF activity and function.
• To determine the impact of ISG15 modification on the HIF transcriptional program and mRNA translation.

Main Methods:

• Cell culture under hypoxic and interferon-induced conditions.
• Western blotting and immunoprecipitation to detect ISG15 conjugation (ISGylation) and HIF-α stabilization.
• Quantitative PCR and RNA sequencing to assess transcriptional changes.
• Analysis of protein fractions and polysome profiling to evaluate translation activity.

Main Results:

• ISG15 induction and HIF-α stabilization occur concurrently.
• Both HIF-1α and HIF-2α promote ISGylation of proteins, including the first evidence of HIF-2α ISGylation.
• ISGylation of HIFs leads to systemic alterations in the HIF transcriptional program.
• ISGylation enhances the cytoplasmic/nuclear localization and translation activity of HIF-2α.

Conclusions:

• ISG15 acts as a regulator of HIF activity, influencing both transcription and translation under hypoxic conditions.
• The ISG15-HIF interaction modulates cellular responses to hypoxia, impacting gene expression and protein synthesis.
• This crosstalk has significant implications for understanding immune responses and disease progression, particularly in cancer.