Structural Biochemistry/Protein function/HIF

Hypoxia-inducible factor (HIF)

Hypoxia-inducible factor (HIF) is the transcription factor responsible for regulating the body’s response to hypoxia. Hypoxia is a state of reduced oxygen in the blood. Without oxygen, the body’s normal pathways and cycles are disrupted, thus resulting in cellular and inevitably human death. HIF translation and degradation are studied because of its link to tumor growth. Tumor hypoxia shows resistance to cancer treatment, such as radiation and chemotherapy and an increased expression for HIF-1 enables tumor growth. Thus the regulation of HIF is a target for advances in cancer research.

HIF is a heterodimer with α and β subunits. Of the three known HIF-α isoforms (HIF-1α, HIF-2α, HIF-3α), HIF-1α is most common.

Role of HIF-1α during hypoxia

In a state of hypoxia, HIF-1α responds by promoting anaerobic metabolism through glycolysis. HIF-1α increases the transportation of glucose by upregulating the expression of glucose transporters and glycolytic enzymes. HIF-1α also upregulates pyruvate dehydrogenase kinase 1, which increases the conversion from pyruvate to lactate. This in turn steers activity away from the oxygen-dependent metabolic pathways of the Kreb’s cycle and oxidative phsophorylation by increasing lactic acid production. In addition, HIF-1α also balances the potentially toxic buildup of lactic acid and carbon dioxide by increasing the monocarboxylate transporter 4 and membrane-bound carbonic anhydrase IX which react with the lactic acid and carbon dioxide to prevent the levels from becoming toxic.

HIF-1α is also responsible for activating angiogenesis. Angiogenesis restores the supplies of oxygen and nutrients by forming new blood vessels. Although this may sound like something positive, increased angiogenesis enables tumor growth.

Degradation of HIF-1α

Studies have shown that HIF degradation occurs both in the presence and in the absence of oxygen. Oxygen-dependent degradation (ODD) includes pVHL and SUMOylation. Oxygen-independent degradation includes HAF and RACK1.

The pVHL-HIF-1α degradation pathway

Under aerobic condition, HIF-1α is hydroxylated by prolyl hydroxylases( PHD) at two conserved proline residues located within its oxygen-dependent degradation(ODD) domain. Under hypoxic condition, PHD activity is inhibited by enzyme therefore stabilizing HIF-1α. In addition, hypoxic condition also causes perturbation in the electron transport chain in mitochondria and increases the level of cytoplasmic ROS (reactive- oxygen species) which alters the oxidation state of Fe2+, a cofactor for PHD activity. This effect also inhibits PHD and promotes HIF-1α stabilization. HIF-1α hydroxylation helps pVHL bind to the HIF-1α ODD. Then, pVHL will form the substrate-recognition module of an E3 ubiquitin ligase complex which will direct HIF-1α proteasomal degradation.

pVHL (von Hippel Lindau)

In the pVHL (von Hippel Lindau) pathway, the presence of oxygen hydroxylates the HIF-1α. The hydroxylation binds HIF-1α to the pVHL which then forms an E3 ubiquitin ligase complex which essentially tags the HIF-1α for degradation. The SSAT2 regulator also binds and stabilizes the interaction between the HIF-1α and pVHL.

SUMOylation

SUMOylation is used to regulate protein properties. This modification in polypeptides have been used to study the amyloid-beta peptide levels which is linked to the human condition, Alzheimer's Disease. Because of sumoylation, it was discovered that familial dilated cardiomyopathy was caused by a decrease in lamin A sumoylation which leads to increased cell death.

SUMOylation leads HIF-1α to bind to the same E3 ligase as in the pVHL pathway but does so under hypoxic conditions. The SUMO protein binds to the HIF-1α, tagging it to attach to the E3 ligase to eventually degrade.

HAF (Hypoxia-associated factor)

The HAF (Hypoxia-associated factor) is a multi-functional protein. At the C-terminus it has an E3 ligase which binds to the ODD domain of HIF-1α and tags it for degradation. This occurs regardless of the presence or absence of oxygen. At the N-terminus it promotes the translation for some of the HIF-1α targets, thus contradicting the degradation work of the C-terminus. However, HAF selectively activates some of the targets, but not all. This dual functionality provides potential opportunities for therapeutic regulation.

RACK1

RACK1 binds with HIF-1α and tags it with the E3 ligase for degradation. In contrast to the SSAT2 regulator which promotes the ODD pathway of pVHL, the SSAT1 regulator stabilizes the bond between RACK1 and HIF-1α .Calcium affects this pathway. HIF-1α degradation is inhibited when calcineurin A dephosphorylates RACK1, thus preventing it from binding with HIF-1α.

Translation of HIF-1α

When in a state of hypoxia, general protein translation is inhibited to in order to decrease the amount of energy consumption. However, the translation of HIF-1α is not disrupted. The exact mechanism and reasoning behind this phenomenon is not completely understood. One proposed pathway is through RNA sequences that do not need a cap-binding complex to form secondary and tertiary structures and to bind directly to the ribosome.

Factors increasing HIF-1α

• Modulator of Degradation:
  • Oxygen Dependent:
    • EPF UCP (degrades pHVL)
    • VDU2 (de-ubiquitinates HIF-1α)
    • SUMOylation (via RSUME)
    • DeSUMOylation ( via SENP1)
  • Oxygen independent:
    • Calcineurin A ( Ca2+ dependent via RACK1)
• Modulators of translation:
  • RNA- binding proteins, PTB and HuR
  • PtdIns3K and MAPK pathways
  • IRES-mediated translation
  • calcium signaling
  • miRNAs

Factors decreasing HIF-1α

• Modulator of Degradation:
  • Oxygen Dependent:
    • PHD, pVHL, OS-9 and SSAT2
    • SUMOylation
  • Oxygen independent
    • RACK1 and SSAT1
    • GSK3β
    • FOXO4
• Modulators of translation:
  • Calcium signaling
  • miRNAs

HIF Switch

Modern research have disclosed the presence of HIF switch, which are mechanisms that are qualified of directly altering the HIF-α isoform. Examples of HIF switches include Hsp70/CHIP axis, which encourages the particular deterioration of HIF-1α in diabetes-associated hypoxia and hyperglycemia. As a consequence, this gives diabetic complications affiliated with impaired hypoxic response and cell destruction. Histone deacetylase SIRT1, another HIF switch, which has a tendency to deacetylate HIF-2α, and increases HIF-2 activity during hypoxia. Recent evidences have shown that SIRT1 has left traces in regulating HIF-1. HAF, a crucial HIF-α isoform target regulator, specifically attached to and degrades HIF-1α in an oxygen-independent case. However, it also enhances HIF-2α transactivation and constancy. HAF encounters a decrease when exposed to chronic hypoxia, but develops with extended hypoxic exposure. Regardless, the switch from HIF-1α towards HIF-2α is a necessity for cells.

Even though there is much research needed to aid the understanding of HIF-1α to HIF-2α switch, current knowledge holds value in the growth of cancer. Identified HAD is a crucial component has proven to enhance tumor initiation and progression.

References

1. Yee Koh M. & Powis G. “HAF: the new player in oxygen-independent HIF-1α degradation.” Cell Cycle. 2009 May 1; 8(9): 1359–1366.
2. Yee Koh M, Spivak-Kroizman TR, Powis G. “HIF-1 regulation: not so easy come, easy go.” Trends Biochem Sci. 2008 Nov;33(11):526-34. Epub 2008 Sep 21.

Mei Yee Koh, Garth Powis, Passing the baton: the HIF switch, Trends in Biochemical Sciences, Volume 37, Issue 9, September 2012. <http://www.sciencedirect.com/science/article/pii/S0968000412000795>