Blue Native electrophoresis to study mitochondrial and other protein complexes

Nijtmans, Leo; Henderson, Nadine S.; Holt, Ian

doi:10.1016/s1046-2023(02)00038-5

Cited by 336 publications

(245 citation statements)

References 24 publications

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“…Complex I, IV and V were assayed as described. 38 Briefly, gels were washed in distilled water before incubation at RT with the following solutions: Complex I solution -2 mM Tris-HCl, pH 7.4, 0.1 mg/ml NADH, 2.5 mg/ml nitrotetrazolium blue for 15 min at RT. Complex IV solution -5 mg 3:3′-diamidobenzidine tetrahydrochloride dissolved in 9 ml of 5 mM phosphate buffer (0.05 M, pH 7.4), 1 nM catalase (20 U/ml), 10 mg Cyt c and 750 mg sucrose overnight at RT.…”

Section: Discussionmentioning

confidence: 99%

“…GB treatment rapidly reduced both the glutamate/malate-and succinatedependent respiration of isolated mitochondria with kinetics similar to complex I cleavage (Figures 6a and b). Interestingly, when purified intact mitochondria were treated with GB and respiratory complexes were analyzed by blue native gel electrophoresis (BNGE), 38,39 GB induced a dose-dependent inhibition of complex I activity that was significant within 1 min as monitored by in-gel activity assay (Figure 6c). Unexpectedly, GB also rapidly inhibited complex III activity, whereas the activities of complexes IV and V were unaffected.…”

Section: Gb-induced Ros Requires a Functional Respiratory Chainmentioning

confidence: 99%

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Granzyme B-induced mitochondrial ROS are required for apoptosis

et al. 2014

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Caspases and the cytotoxic lymphocyte protease granzyme B (GB) induce reactive oxygen species (ROS) formation, loss of transmembrane potential and mitochondrial outer membrane permeabilization (MOMP). Whether ROS are required for GBmediated apoptosis and how GB induces ROS is unclear. Here, we found that GB induces cell death in an ROS-dependent manner, independently of caspases and MOMP. GB triggers ROS increase in target cell by directly attacking the mitochondria to cleave NDUFV1, NDUFS1 and NDUFS2 subunits of the NADH: ubiquinone oxidoreductase complex I inside mitochondria. This leads to mitocentric ROS production, loss of complex I and III activity, disorganization of the respiratory chain, impaired mitochondrial respiration and loss of the mitochondrial cristae junctions. Furthermore, we have also found that GB-induced mitocentric ROS are necessary for optimal apoptogenic factor release, rapid DNA fragmentation and lysosomal rupture. Interestingly, scavenging the ROS delays and reduces many of the features of GB-induced death. Consequently, GB-induced ROS significantly promote apoptosis. Cell Death and Differentiation (2015) 22, 862-874; doi:10.1038/cdd.2014.180; published online 31 October 2014To induce cell death, human granzyme B (GB) activates effector caspase-3 or acts directly on key caspase substrates, such as the proapoptotic BH3 only Bcl-2 family member Bid, inhibitor of caspase-activated DNase (ICAD), poly-(ADPribose) polymerase-1 (PARP-1), lamin B, nuclear mitotic apparatus protein 1 (NUMA1), catalytic subunit of the DNAdependent protein kinase (DNA-PKcs) and tubulin. [1][2][3] Consequently, caspase inhibitors have little effect on human GB-mediated cell death and DNA fragmentation. 2 GB causes reactive oxygen species (ROS) production, dissipation of the mitochondrial transmembrane potential (ΔΨm) and MOMP, which leads to the release of apoptogenic factors such as cytochrome c (Cyt c), HtrA2/Omi, endonuclease G (Endo G), Smac/Diablo and apoptosis-inducing factor, from the mitochondrial intermembrane space to the cytosol. [4][5][6][7][8][9][10][11] Interestingly, cells deficient for Bid, Bax and Bak are still sensitive to human GB-induced cell death, 5,11-13 suggesting that human GB targets the mitochondria in another way that needs to be characterized. Altogether, much attention has been focused on the importance of MOMP in the execution of GB-mediated cell death, leaving unclear whether ROS production is a bystander effect or essential to the execution of GBinduced apoptosis. The mitochondrial NADH: ubiquinone oxidoreductase complex I is a key determinant in steadystate ROS production. This 1 MDa complex, composed of 44 subunits, 14 couples the transfer of two electrons from NADH to ubiquinone with the translocation of four protons to generate the ΔΨm. The importance of ROS has been previously demonstrated for caspase-3 and granzyme A (GA) pathways through the cleavage of NDUFS1 and NDUFS3, respectively. [15][16][17][18] GA induces cell death in a Bcl-2-insensitive and caspase-and MOMP-indepen...

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Section: Discussionmentioning

confidence: 99%

Section: Gb-induced Ros Requires a Functional Respiratory Chainmentioning

confidence: 99%

Granzyme B-induced mitochondrial ROS are required for apoptosis

et al. 2014

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“…1-1.5 mg of mitochondrial protein from prefrontal cortex (PD n = 11, Ctrl n = 11), striatum (PD n = 6, Ctrl n = 6), and cerebellar cortex (PD n = 8, Ctrl n = 2) were solubilized using 1.6 mg dodecyl-maltoside/mg protein to analyse complex I by blue-native gel electrophoresis and Western blot as described [26,33,44]. 50 μg of mitochondrial protein was separated by electrophoresis and complexes I and II were immuno-visualised using anti-NDUFB8 (Abcam ab110242, dilution 1:1000) and anti-SDHB (Abcam ab14714, dilution 1:1000), respectively.…”

Section: Blue-native Gel Electrophoresis and Immunoblottingmentioning

confidence: 99%

Neuronal complex I deficiency occurs throughout the Parkinson’s disease brain, but is not associated with neurodegeneration or mitochondrial DNA damage

et al. 2017

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Mitochondrial complex I deficiency occurs in the substantia nigra of individuals with Parkinson's disease. It is generally believed that this phenomenon is caused by accumulating mitochondrial DNA damage in neurons and that it contributes to the process of neurodegeneration. We hypothesized that if these theories are correct, complex I deficiency should extend beyond the substantia nigra to other affected brain regions in Parkinson's disease and correlate tightly with neuronal mitochondrial DNA damage. To test our hypothesis, we employed a combination of semiquantitative immunohistochemical analyses, Western blot and activity measurements, to assess complex I quantity and function in multiple brain regions from an extensively characterized population-based cohort of idiopathic Parkinson's disease (n = 18) and gender and age matched healthy controls (n = 11). Mitochondrial DNA was assessed in single neurons from the same areas by real-time PCR. Immunohistochemistry showed that neuronal complex I deficiency occurs throughout the Parkinson's disease brain, including areas spared by the neurodegenerative process such as the cerebellum. Activity measurements in brain homogenate confirmed a moderate decrease of complex I function, whereas Western blot was less sensitive, detecting only a mild reduction, which did not reach statistical significance at the group level. With the exception of the substantia nigra, neuronal complex I loss showed no correlation with the load of somatic mitochondrial DNA damage. Interestingly, α-synuclein aggregation was less common in complex I deficient neurons in the substantia nigra. We show that neuronal complex I deficiency is a widespread phenomenon in the Parkinson's disease brain which, contrary to mainstream theory, does not follow the anatomical distribution of neurodegeneration and is not associated with the neuronal load of mitochondrial DNA mutation. Our findings suggest that complex I deficiency in Parkinson's disease can occur independently of mitochondrial DNA damage and may not have a pathogenic role in the neurodegenerative process.

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“…Mitochondrial membranes were isolated from 2.5 Â 10 6 cells or from 200 lg of pure mitochondria (PM) as described previously (55). Cells were solubilized with 3% digitonin (wt/vol) (SigmaAldrich) and 0.4% (wt/vol) lauryl maltoside (Sigma-Aldrich) or 1% digitonin (wt/vol).…”

Section: Blue Native Electrophoresis (Bn-page) and Sds-pagementioning

confidence: 99%

“…PM were solubilized with two rounds of digitonin (6g/g and 4g/g). Twenty ll of samples were electrophoresed on a 5-13% gradient polyacrylamide gel as described previously (55). Transfer of proteins onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) was carried out overnight at 30 V at 4 C. For second-dimension gel electrophoresis, a lane excised from the first dimension native gel was first treated for 30 min with denaturing buffer containing 1% b-mercaptoethanol and 1% SDS and then washed in 1% SDS for 1 h. The gel strip was electrophoresed on a tricine-SDS-polyacrylamide gel as described previously (56).…”

Section: Blue Native Electrophoresis (Bn-page) and Sds-pagementioning

confidence: 99%

Disrupted in schizophrenia 1 (DISC1) is a constituent of the mammalian mitochondrial contact site and cristae organizing system (MICOS) complex, and is essential for oxidative phosphorylation

et al. 2016

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Disrupted in Schizophrenia-1 (DISC1) has been associated with a broad spectrum of mental disorders. DISC1 is a multicompartmentalized protein found in the cytoplasm, centrosome, nuclei and mostly enriched in mitochondria. In order to shed light on DISC1 mitochondrial function, we have studied its topology within the organelle. We show in here that in mammals DISC1 resides in the 'Mitochondrial contact site and Cristae Organizing system' (MICOS) complex, involved in cristae organization. DISC1 knockdown in SH-SY5Y cells causes MICOS disassembly and fragmentation of the mitochondrial morphology network. Moreover, DISC1 depleted cells have decreased mitochondrial DNA (mtDNA) content and steady state levels of oxidative phosphorylation (OXPHOS) subunits. As a consequence, OXPHOS complexes and supercomplexes are partially disassembled in DISC1 knockdown cells, which suffer severe bioenergetic defects, evidenced by impaired oxygen consumption, adenosine triphosphate synthesis and mitochondrial membrane potential. Transfection of recombinant full-length human DISC1 restores MICOS complex assembly and rescues OXPHOS function, meanwhile overexpression of the DISC1 truncated form D597-854, known to be pathogenic, fails to rescue the bioenergetic impairment caused by DISC1 knockdown. These results should contribute to reveal DISC1 physiological function and potential pathogenic role in severe mental illnesses.

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Blue Native electrophoresis to study mitochondrial and other protein complexes

Cited by 336 publications

References 24 publications

Granzyme B-induced mitochondrial ROS are required for apoptosis

Granzyme B-induced mitochondrial ROS are required for apoptosis

Neuronal complex I deficiency occurs throughout the Parkinson’s disease brain, but is not associated with neurodegeneration or mitochondrial DNA damage

Disrupted in schizophrenia 1 (DISC1) is a constituent of the mammalian mitochondrial contact site and cristae organizing system (MICOS) complex, and is essential for oxidative phosphorylation

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