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Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress.

Vallelian F, Deuel JW, Opitz L, Schaer CA, Puglia M, Lönn M, Engelsberger W, Schauer S, Karnaukhova E, Spahn DR, Stocker R, Buehler PW, Schaer DJ - Cell Death Differ. (2014)

Bottom Line: Dual control of cellular heme levels by extracellular scavenger proteins and degradation by heme oxygenases is essential in diseases associated with increased heme release.These observations were explained mechanistically by demonstrating binding of heme to the proteasome that was linked to impaired proteasome function.Oxidative heme reactions and proteasome inhibition could be differentiated as synergistic activities of the porphyrin.

View Article: PubMed Central - PubMed

Affiliation: Division of Internal Medicine, University of Zurich, CH-8091 Zurich, Switzerland.

ABSTRACT
Dual control of cellular heme levels by extracellular scavenger proteins and degradation by heme oxygenases is essential in diseases associated with increased heme release. During severe hemolysis or rhabdomyolysis, uncontrolled heme exposure can cause acute kidney injury and endothelial cell damage. The toxicity of heme was primarily attributed to its pro-oxidant effects; however additional mechanisms of heme toxicity have not been studied systematically. In addition to redox reactivity, heme may adversely alter cellular functions by binding to essential proteins and impairing their function. We studied inducible heme oxygenase (Hmox1)-deficient mouse embryo fibroblast cell lines as a model to systematically explore adaptive and disruptive responses that were triggered by intracellular heme levels exceeding the homeostatic range. We extensively characterized the proteome phenotype of the cellular heme stress responses by quantitative mass spectrometry of stable isotope-labeled cells that covered more than 2000 individual proteins. The most significant signals specific to heme toxicity were consistent with oxidative stress and impaired protein degradation by the proteasome. This ultimately led to an activation of the response to unfolded proteins. These observations were explained mechanistically by demonstrating binding of heme to the proteasome that was linked to impaired proteasome function. Oxidative heme reactions and proteasome inhibition could be differentiated as synergistic activities of the porphyrin. Based on the present data a novel model of cellular heme toxicity is proposed, whereby proteasome inhibition by heme sustains a cycle of oxidative stress, protein modification, accumulation of damaged proteins and cell death.

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Heme binding to proteasome: UV/Vis spectrophotometry. (a) UV/Vis spectra of heme in DMSO or diluted in Hepes buffer (pH 7.4). The inset shows the time-dependent absorbance decay (red arrow) of heme in HEPES buffer. The spectra of heme in DMSO remained stable over days. (b) Time-dependent changes in the UV/Vis spectra of the heme in HEPES buffer sample shown in (a) after addition of proteasome-enriched cytoplasmic protein fraction. The inset shows enlarged selected spectra that reflect the major changes over time. The protein concentration in the proteasome fraction was 1.4 mg/ml. Identical results were observed with two proteasome fractions from rabbit reticulocyte (F360) and from HeLa cells (F370), respectively. (c) Changes in maximum absorbance after heme binding to the two proteasome fractions F360 and F370 across a range of heme concentrations
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fig5: Heme binding to proteasome: UV/Vis spectrophotometry. (a) UV/Vis spectra of heme in DMSO or diluted in Hepes buffer (pH 7.4). The inset shows the time-dependent absorbance decay (red arrow) of heme in HEPES buffer. The spectra of heme in DMSO remained stable over days. (b) Time-dependent changes in the UV/Vis spectra of the heme in HEPES buffer sample shown in (a) after addition of proteasome-enriched cytoplasmic protein fraction. The inset shows enlarged selected spectra that reflect the major changes over time. The protein concentration in the proteasome fraction was 1.4 mg/ml. Identical results were observed with two proteasome fractions from rabbit reticulocyte (F360) and from HeLa cells (F370), respectively. (c) Changes in maximum absorbance after heme binding to the two proteasome fractions F360 and F370 across a range of heme concentrations

Mentions: The addition of proteasome-enriched cytoplasmic protein fraction to an aqueous solution of heme induced two specific spectral changes within the Soret band region of the heme UV/Vis spectrum (Figure 5): (1) a decrease of the initial broad intensity of the Soret band at 405 nm and (2) a bathochromic shift of the Soret band with an increased intensity at 413 nm (Figure 5b). In contrast to the broad band over 370–390 nm that gradually decreases in aqueous heme solutions (Figure 5a), this newly formed λmax at 413 nm remained stable over days. This suggested that heme had transferred to one or multiple binding sites and stabilized within a coordination of amino acid residues (e.g., histidine and tyrosine). The 20–23 nm bathochromic shift of heme complexes within the protein was significantly greater than the 9–10 nm red shift known for heme complexes with albumin. Heme binding with formation of the λmax 413 nm proceeded slowly, suggesting conformational changes accompanying heme accommodation in the protein. The heme titration shown in Figure 5c was conducted until no further increase in the absorption intensity at 413 nm over the intensity at 370 nm was observed. Heme concentrations within the range of our cell culture studies resulted in a linear increase of the Soret band intensity.


Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress.

Vallelian F, Deuel JW, Opitz L, Schaer CA, Puglia M, Lönn M, Engelsberger W, Schauer S, Karnaukhova E, Spahn DR, Stocker R, Buehler PW, Schaer DJ - Cell Death Differ. (2014)

Heme binding to proteasome: UV/Vis spectrophotometry. (a) UV/Vis spectra of heme in DMSO or diluted in Hepes buffer (pH 7.4). The inset shows the time-dependent absorbance decay (red arrow) of heme in HEPES buffer. The spectra of heme in DMSO remained stable over days. (b) Time-dependent changes in the UV/Vis spectra of the heme in HEPES buffer sample shown in (a) after addition of proteasome-enriched cytoplasmic protein fraction. The inset shows enlarged selected spectra that reflect the major changes over time. The protein concentration in the proteasome fraction was 1.4 mg/ml. Identical results were observed with two proteasome fractions from rabbit reticulocyte (F360) and from HeLa cells (F370), respectively. (c) Changes in maximum absorbance after heme binding to the two proteasome fractions F360 and F370 across a range of heme concentrations
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4356336&req=5

fig5: Heme binding to proteasome: UV/Vis spectrophotometry. (a) UV/Vis spectra of heme in DMSO or diluted in Hepes buffer (pH 7.4). The inset shows the time-dependent absorbance decay (red arrow) of heme in HEPES buffer. The spectra of heme in DMSO remained stable over days. (b) Time-dependent changes in the UV/Vis spectra of the heme in HEPES buffer sample shown in (a) after addition of proteasome-enriched cytoplasmic protein fraction. The inset shows enlarged selected spectra that reflect the major changes over time. The protein concentration in the proteasome fraction was 1.4 mg/ml. Identical results were observed with two proteasome fractions from rabbit reticulocyte (F360) and from HeLa cells (F370), respectively. (c) Changes in maximum absorbance after heme binding to the two proteasome fractions F360 and F370 across a range of heme concentrations
Mentions: The addition of proteasome-enriched cytoplasmic protein fraction to an aqueous solution of heme induced two specific spectral changes within the Soret band region of the heme UV/Vis spectrum (Figure 5): (1) a decrease of the initial broad intensity of the Soret band at 405 nm and (2) a bathochromic shift of the Soret band with an increased intensity at 413 nm (Figure 5b). In contrast to the broad band over 370–390 nm that gradually decreases in aqueous heme solutions (Figure 5a), this newly formed λmax at 413 nm remained stable over days. This suggested that heme had transferred to one or multiple binding sites and stabilized within a coordination of amino acid residues (e.g., histidine and tyrosine). The 20–23 nm bathochromic shift of heme complexes within the protein was significantly greater than the 9–10 nm red shift known for heme complexes with albumin. Heme binding with formation of the λmax 413 nm proceeded slowly, suggesting conformational changes accompanying heme accommodation in the protein. The heme titration shown in Figure 5c was conducted until no further increase in the absorption intensity at 413 nm over the intensity at 370 nm was observed. Heme concentrations within the range of our cell culture studies resulted in a linear increase of the Soret band intensity.

Bottom Line: Dual control of cellular heme levels by extracellular scavenger proteins and degradation by heme oxygenases is essential in diseases associated with increased heme release.These observations were explained mechanistically by demonstrating binding of heme to the proteasome that was linked to impaired proteasome function.Oxidative heme reactions and proteasome inhibition could be differentiated as synergistic activities of the porphyrin.

View Article: PubMed Central - PubMed

Affiliation: Division of Internal Medicine, University of Zurich, CH-8091 Zurich, Switzerland.

ABSTRACT
Dual control of cellular heme levels by extracellular scavenger proteins and degradation by heme oxygenases is essential in diseases associated with increased heme release. During severe hemolysis or rhabdomyolysis, uncontrolled heme exposure can cause acute kidney injury and endothelial cell damage. The toxicity of heme was primarily attributed to its pro-oxidant effects; however additional mechanisms of heme toxicity have not been studied systematically. In addition to redox reactivity, heme may adversely alter cellular functions by binding to essential proteins and impairing their function. We studied inducible heme oxygenase (Hmox1)-deficient mouse embryo fibroblast cell lines as a model to systematically explore adaptive and disruptive responses that were triggered by intracellular heme levels exceeding the homeostatic range. We extensively characterized the proteome phenotype of the cellular heme stress responses by quantitative mass spectrometry of stable isotope-labeled cells that covered more than 2000 individual proteins. The most significant signals specific to heme toxicity were consistent with oxidative stress and impaired protein degradation by the proteasome. This ultimately led to an activation of the response to unfolded proteins. These observations were explained mechanistically by demonstrating binding of heme to the proteasome that was linked to impaired proteasome function. Oxidative heme reactions and proteasome inhibition could be differentiated as synergistic activities of the porphyrin. Based on the present data a novel model of cellular heme toxicity is proposed, whereby proteasome inhibition by heme sustains a cycle of oxidative stress, protein modification, accumulation of damaged proteins and cell death.

Show MeSH
Related in: MedlinePlus