Related Experiment Video

Updated: Jul 10, 2026

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
09:17

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Published on: August 2, 2018

The interaction of superoxide with nitric oxide destabilizes hypoxia-inducible factor-1alpha

B Herr1, J Zhou, S Dröse

  • 1Institute of Biochemistry I, Pathobiochemistry, Faculty of Medicine, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590, Frankfurt, Germany.

Cellular and Molecular Life Sciences : CMLS
|November 9, 2007
PubMed

Insights

Hypoxia-inducible factor-1 (HIF-1) degradation in renal carcinoma cells is mediated by calpain activation. This process requires mitochondria-derived reactive oxygen species and calcium signaling, independent of the proteasome.

Area of Science:

  • Cell Biology
  • Biochemistry
  • Oncology

Background:

  • Hypoxia-inducible factor-1 (HIF-1) is constitutively expressed in renal carcinoma cells (RCC4) due to von Hippel Lindau protein deficiency.
  • Calpain can degrade HIF-1 independently of the 26S proteasome under hypoxia/nitric oxide (NO) conditions.

Purpose of the Study:

  • To investigate the molecular mechanisms underlying calpain activation for HIF-1 degradation.
  • To elucidate the role of mitochondria and reactive oxygen species in this pathway.

Main Methods:

  • Utilized respiratory-deficient RCC4-rho0 cells to assess the requirement for mitochondria.
  • Employed a superoxide-generating redox cycler (2,3-dimethoxy-1,4-naphthoquinone) to study reactive oxygen species (ROS) involvement.
  • Investigated the role of intracellular calcium transients and calpain activation.
  • Used uric acid to interfere with NO/ROS signaling.

Main Results:

  • Hypoxia/NO failed to degrade HIF-1alpha in respiratory-deficient RCC4-rho0 cells, indicating a requirement for mitochondria-derived ROS.
  • Superoxide (O(2)(-)) in combination with NO was necessary for HIF-1alpha destabilization.
  • HIF-1alpha degradation was dependent on intracellular calcium transients and calpain activation.
  • Interference with NO/ROS signaling using uric acid blocked HIF-1alpha degradation and attenuated calcium increase.

Conclusions:

  • An oxidative signal from co-formed NO and O(2)(-) triggers a calcium increase.
  • This calcium increase activates calpain, leading to HIF-1alpha degradation.
  • The degradation pathway is independent of the 26S proteasome.

More Related Videos

Induction and Testing of Hypoxia in Cell Culture07:01

Induction and Testing of Hypoxia in Cell Culture

Published on: August 12, 2011

Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds08:23

Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds

Published on: February 16, 2022

Related Concept Videos

Regulation of Angiogenesis and Blood Supply01:24

Regulation of Angiogenesis and Blood Supply

Rapidly dividing tumors, embryos, and wounded tissues require more oxygen than usual, lowering the oxygen concentration in the blood. At low oxygen or hypoxic conditions, an oxygen-sensitive transcription factor called the hypoxia-inducible factor 1 or HIF1 is activated. HIF1 is a dimeric protein of alpha (ɑ) and beta (β) subunits.  Under optimal oxygen conditions, HIF1β is present in the nucleus while HIF1ɑ remains in the cytosol. HIF1ɑ is hydroxylated by prolyl hydroxylase and factor...
Oxygen Transport in the Blood01:27

Oxygen Transport in the Blood

Hemoglobin (Hb) is a crucial molecule in the human body, consisting of four polypeptide chains, each bound to an iron-containing heme group. This unique structure enables hemoglobin to bind to oxygen, with each molecule capable of combining with four molecules of oxygen, leading to rapid and reversible oxygen loading. When fully loaded with oxygen, it is called oxyhemoglobin, while hemoglobin that has released oxygen is called reduced hemoglobin or deoxyhemoglobin. As hemoglobin binds oxygen,...
Hypoxia01:23

Hypoxia

Hypoxia is a medical condition characterized by an inadequate oxygen supply to body tissues. It typically manifests as a bluish discoloration of the skin and mucosae, especially in fair-skinned individuals, when hemoglobin (Hb) saturation drops below 75%.
Types of Hypoxia
There are four primary types of hypoxia, each resulting from a different cause:
1. Anemic hypoxia: This type occurs due to insufficient oxygen delivery caused by a lack of red blood cells (RBCs) or RBCs with abnormal or...
Electron Transport Chain: Complex III and IV01:43

Electron Transport Chain: Complex III and IV

During the electron transport chain, electrons from NADH and FADH2 are first transferred to complexes I and II, respectively. These two complexes then transfer the electrons to ubiquinol, which carries them further to complex III. Complex III passes the electrons across the intermembrane space to Cyt c, which carries them further to complex IV. Complex IV donates electrons to oxygen and reduces it to water. As electrons pass through complexes I, III, and IV, the energy released aids the pumping...
Nitric Oxide Signaling Pathway01:28

Nitric Oxide Signaling Pathway

Nitric oxide (NO), an inorganic gas, acts as a potent second messenger in most animal and plant tissues. NO diffuses out of the cells that produce it and enters the neighboring cells to generate a downstream response. NO synthase (NOS) catalyzes NO production by the deamination of the amino acid arginine. There are three isoforms of NOS. Endothelial cells have endothelial NOS (eNOS), nerve and muscle cells have neuronal NOS (nNOS), and macrophages produce inducible NOS (iNOS) upon exposure to...
The Electron Transport Chain01:30

The Electron Transport Chain

The electron transport chain or oxidative phosphorylation is an exothermic process in which free energy released during electron transfer reactions is coupled to ATP synthesis. This process is a significant source of energy in aerobic cells, and therefore inhibitors of the electron transport chain can be detrimental to the cell's metabolic processes.
Inhibitors of the electron transport chain
Rotenone, a widely used pesticide, prevents electron transfer from Fe-S cluster to ubiquinone or Q in...