cell free system

Looking Back: Cell-Free Expression Systems Helped to Characterize Proteins Involved in Hypoxia Response

Posted on by Gary Kobs
Structur of a HIF-1a-pVHL-ElonginB-ElonginC complex
Structure of a HIF-1a-pVHL-ElonginB-ElonginC complex

William G. Kaelin Jr., Sir Peter J. Ratcliffe and Gregg L. Semenza were awarded the 2019 Nobel Prize in Physiology or Medicine for their discoveries of how cells sense and adapt to oxygen availability.

Kaelin and Ratcliffe’s labs focused their efforts on the transcription factor HIF (hypoxia-inducible factor). This transcription factor is critical in the cellular adaptation of to changes in oxygen availability.

When oxygen levels are elevated cells contain very little HIF. Ubiquitin is added to the HIF protein via the VHL complex and it is degraded in the proteasome.  When oxygen levels are low (hypoxia) the amount of HIF increases.

In 2001 both groups published articles characterizing the interaction between VHL and HIF, and these articles were referenced by the Nobel Prize Organization in their press release about this year’s award. (1,2). Both studies demonstrated that under the normal oxygen conditions hydroxylation of proline residue P564 enabled VHL to recognize and bind to HIF.

The use of cell free expression (i.e., TNT Coupled Transcription/Translation System) by both labs was key in the characterization of the VHL:HIF interaction The labs utilized HIF and VHL 35-S labeled proteins generated via the TNT system under both normal or in a hypoxic work station to:

  • Determine the affect of ferrous chloride and cobaltous chloride on the interaction
  • Map the specific region of HIF required for the interaction to occur (556-574)
  • Determine the effect of HIF point mutations on the interaction
  • Use synthetic peptides to block the interaction
  • Conclude that a factor in mammalian cells was necessary for the interaction to occur.

Literature Cited

  1. Ivan, M et al. (2001) HIF Targeted for VHL-Mediated Destruction by Proline Hydroxylation: Implications for O2 Sensing. Science 292: 464–67.
  2. Jaakkola, P. et al. (2001) Targeting of HIF-α to the von Hippel-Lindau Ubiquitylation of Complex by O2– Regulated Prolyl Hydroxylation. Science 202, 468–72 .

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Characterizing Compound Binding in Cell-Free Systems

Posted on by Gary Kobs

Dioxins (e.g., 2,3,7,8-Tetrachlorodibenzo-p-dioxin, TCDD) and related compounds (DRCs) are persistent environmental pollutants that gradually accumulate through the food chain, mainly in the fatty tissues of animals. Dioxins are highly toxic and can cause reproductive and developmental problems, damage the immune system, interfere with hormones and also cause cancer. This broad range of toxic and biological effects of DRCs is mostly mediated by the aryl hydrocarbon receptor (AHR).

In animal cells, DRCs bind to AHR in the cytoplasm and then translocate into the nucleus, where they affect the transcription of multiple target genes, including xenobiotic-metabolizing enzymes, such as CYP1A isozymes. AHR is also involved in immune system maintenance, protein degradation and cell proliferation.

The jungle crow (Corvus macrorhynchos) has been considered a suitable indicator for monitoring environmental chemicals such as DRCs. While mammals only have one AHR form, avian species have multiple AHR isoforms such as AHR1 and AHR2. To unveil the functional diversity of multiple avian AHR isoforms in terms of their contribution to responses to DRCs a recent study by Kim et al. investigated the molecular and functional characteristics of jungle crow AHR isoforms, cAHR1 and jcAHR2 (1).

cAHR1 and jcAHR2 proteins were synthesized using AHR proteins were synthesized using the TnT Quick-Coupled Reticulocyte Lysate System  to examine whether these jcAHRs have the potential to bind to TCDD. TCDD-binding affinity of the in vitro-expressed jcAHR protein was analyzed using the velocity sedimentation assay with a sucrose gradient.

The results demonstrate that both jcAHR1and jcAHR2 are capable of binding to TCDD.

Reference
Kim, E-Y (2019) The aryl hydrocarbon receptor 2 potentially mediates cytochrome P450 1A induction in the jungle crow (Corvus macrorhynchos). Ecotoxicology and Environmental Safety 171. 99–111