Blue Native Gel Electrophoresis Research Articles

Phosphoproteomic Analysis of Isolated Mitochondria in Yeast.

Mitochondria play a central role in cellular energy metabolism and cell death. Deregulation of mitochondrial functions is associated with several human pathologies (neurodegenerative diseases, neuromuscular diseases, type II diabetes, obesity, cancer). The steadily increasing number of identified mitochondrial phosphoproteins, kinases, and phosphatases in recent years suggests that reversible protein phosphorylation plays an important part in the control of mitochondrial processes. In addition, many mitochondrial phosphoproteins probably still remain to be identified, considering that 30% of proteins are expected to be phosphorylated in eukaryotes. In this chapter, we describe two procedures for the analysis of the mitochondrial phosphoproteome. The first one is a qualitative method that combines blue native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D-BN/SDS-PAGE) and specific phosphoprotein staining. The second one is a quantitative approach that associates mitochondrial peptide labeling, phosphopeptide enrichment, and mass spectrometry.

Cyclophilin D over-expression increases mitochondrial complex III activity and accelerates supercomplex formation

Cyclophilin D (CyPD), a mitochondrial matrix protein, has been widely studied for its role in mitochondrial-mediated cell death. Unexpectedly, we previously discovered that overexpression of CyPD in a stable cell line, increased mitochondrial membrane potentials and enhanced cell survival under conditions of oxidative stress. Here, we investigated the underlying mechanisms responsible for these findings. Spectrophotometric measurements in isolated mitochondria revealed that overexpression of CyPD in HEK293 cells increased respiratory chain activity, but only for Complex III (CIII). Acute treatment of mitochondria with the immumosupressant cyclosporine A did not affect CIII activity. Expression levels of the CIII subunits cytochrome b and Rieske-FeS were elevated in HEK293 cells overexpressing CyPD. However, CIII activity was still significantly higher compared to control mitochondria, even when normalized by protein expression. Blue native gel electrophoresis and Western blot assays revealed a molecular interaction of CyPD with CIII and increased levels of supercomplexes in mitochondrial protein extracts. Radiolabeled protein synthesis in mitochondria showed that CIII assembly and formation of supercomplexes containing CIII were significantly faster when CyPD was overexpressed. Taken together, these data indicate that CyPD regulates mitochondrial metabolism, and likely cell survival, by promoting more efficient electrons flow through the respiratory chain via increased supercomplex formation.

Characterization of α-Synuclein Multimer Stoichiometry in Complex Biological Samples by Electrophoresis.

The aberrant aggregation of α-synuclein in the brain is a hallmark of Parkinson’s disease (PD). In vivo soluble α-synuclein occurs as a monomer and several multimers, the latter of which may be important for the biological function of α-synuclein. Currently, there is a lack of reproducible methods to compare α-synuclein multimer abundance between complex biological samples. Here we developed a method, termed “multimer-PAGE,” that combines in-gel chemical cross-linking with several common electrophoretic techniques to measure the stoichiometry of soluble α-synuclein multimers in brain tissue lysates. Results show that soluble α-synuclein from the rat brain exists as several high molecular weight species of approximately 56 kDa (αS56), 80 kDa (αS80), and 100 kDa (αS100) that comigrate with endogenous lipids, detergents, and/or micelles during blue native gel electrophoresis (BN-PAGE). Co-extraction of endogenous lipids with α-synuclein was essential for the detection of soluble α-synuclein multimers. Homogenization of brain tissue in small buffer volumes (>50 mg tissue per 1 mL buffer) increased relative lipid extraction and subsequently resulted in abundant soluble multimer detection via multimer-PAGE. α-Synuclein multimers captured by directly cross-linking soluble lysates resembled those observed following multimer-PAGE. The ratio of multimer (αS80) to monomer (αS17) increased linearly with protein input into multimer-PAGE, suggesting to some extent, multimers were also formed during electrophoresis. Overall, soluble α-synuclein maintains lipid interactions following tissue disruption and readily forms multimers when this lipid–protein complex is preserved. Once the multimer-PAGE technique was validated, relative stoichiometric comparisons could be conducted simultaneously between 14 biological samples. Multimer-PAGE provides a simple inexpensive biochemical technique to study the molecular factors influencing α-synuclein multimerization.

Identification of a multi-protein reductive dehalogenase complex in Dehalococcoides mccartyi strain CBDB1 suggests a protein-dependent respiratory electron transport chain obviating quinone involvement.

Dehalococcoides mccartyi strain CBDB1 is an obligate organohalide-respiring bacterium using only hydrogen as electron donor and halogenated organics as electron acceptor. Here, we studied proteins involved in the respiratory chain under non-denaturing conditions. Using blue native gel electrophoresis (BN-PAGE), gel filtration and ultrafiltration an active dehalogenating protein complex with a molecular mass of 250-270 kDa was identified. The active subunit of reductive dehalogenase (RdhA) colocalised with a complex iron-sulfur molybdoenzyme (CISM) subunit (CbdbA195) and an iron-sulfur cluster containing subunit (CbdbA131) of the hydrogen uptake hydrogenase (Hup). No colocalisation between the catalytically active subunits of hydrogenase and reductive dehalogenase was found. By two-dimensional BN/SDS-PAGE the stability of the complex towards detergents was assessed, demonstrating stepwise disintegration with increasing detergent concentrations. Chemical cross-linking confirmed the presence of a higher molecular mass reductive dehalogenase protein complex composed of RdhA, CISM I and Hup hydrogenase and proved to be a potential tool for stabilising protein-protein interactions of the dehalogenating complex prior to membrane solubilisation. Taken together, the identification of the respiratory dehalogenase protein complex and the absence of indications for quinone participation in the respiration suggest a quinone-independent protein-based respiratory electron transfer chain in D. mccartyi.

Fos-Zippered GH Receptor Cytosolic Tails Act as Jak2 Substrates and Signal Transducers.

Members of the Janus kinase (Jak) family initiate the majority of downstream signaling events of the cytokine receptor family. The prevailing principle is that the receptors act in dimers: 2 Jak2 molecules bind to the cytosolic tails of a cytokine receptor family member and initiate Jak-signal transducer and activator of transcription signaling upon a conformational change in the receptor complex, induced by the cognate cytokine. Due to the complexity of signaling complexes, there is a strong need for in vitro model systems. To investigate the molecular details of the Jak2 interaction with the GH receptor (GHR), we used cytosolic tails provided with leucine zippers derived from c-Fos to mimic the dimerized state of GHR. Expressed together with Jak2, fos-zippered tails, but not unzippered tails, were stabilized. In addition, the Jak-signal transducer and activator of transcription signaling pathway was activated by the fos-zippered tails. The stabilization depended also on α-helix rotation of the zippers. Fos-zippered GHR tails and Jak2, both purified from baculovirus-infected insect cells, interacted via box1 with a binding affinity of approximately 40nM. As expected, the Jak kinase inhibitor Ruxolitinib inhibited the stabilization but did not affect the c-Fos-zippered GHR tail-Jak2 interaction. Analysis by blue-native gel electrophoresis revealed high molecular-weight complexes containing both Jak2 and nonphosphorylated GHR tails, whereas Jak2-dissociated tails were highly phosphorylated and monomeric, implying that Jak2 detaches from its substrate upon phosphorylation.

Renal Mitochondrial Lipid Peroxidation during Sepsis.

Sepsis can provoke kidney injury, which increases mortality. Human and animal studies have documented increased renal oxidative injury and mitochondrial damage during sepsis. However, few studies have attempted to dissect specific renal targets and/or types of oxidative injury using the cecal ligation and puncture (CLP) murine model of sepsis. The purpose of this short communication is to examine the extent of lipid peroxidation within renal mitochondria using CLP and blue native gel electrophoresis which separates intact mitochondrial respiratory complexes. Our results show that CLP induced increased 4-hydroxy-nonenal protein adduction (marker of lipid peroxidation) in renal homogenates and mitochondrial fractions. Blue native gel electrophoresis revealed that respiratory complex III was selectively targeted within mitochondrial fractions. This supports our prior report showing renal complex III inactivation following CLP. Future studies will identify specific renal proteins within complex III that are modified during sepsis to provide mechanistic insight on how mitochondrial respiration is inhibited during sepsis.

Cryo-slicing Blue Native-Mass Spectrometry (csBN-MS), a Novel Technology for High Resolution Complexome Profiling

Blue native (BN) gel electrophoresis is a powerful method for protein separation. Combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), it enables large scale identification of protein complexes and their subunits. Current BN-MS approaches, however, are limited in size resolution, comprehensiveness, and quantification. Here, we present a new methodology combining defined sub-millimeter slicing of BN gels by a cryo-microtome with high performance LC-MS/MS and label-free quantification of protein amounts. Application of this cryo-slicing BN-MS approach to mitochondria from rat brain demonstrated a high degree of comprehensiveness, accuracy, and size resolution. The technique provided abundance-mass profiles for 774 mitochondrial proteins, including all canonical subunits of the oxidative respiratory chain assembled into 13 distinct (super-)complexes. Moreover, the data revealed COX7R as a constitutive subunit of distinct super-complexes and identified novel assemblies of voltage-dependent anion channels/porins and TOM proteins. Together, cryo-slicing BN-MS enables quantitative profiling of complexomes with resolution close to the limits of native gel electrophoresis.

Impaired mitochondrial oxidative phosphorylation and supercomplex assembly in rectus abdominis muscle of diabetic obese individuals.

Skeletal muscle mitochondrial dysfunction has been documented in patients with type 2 diabetes mellitus; however, specific respiratory defects and their mechanisms are poorly understood. The aim of the current study was to examine oxidative phosphorylation and electron transport chain (ETC) supercomplex assembly in rectus abdominis muscles of 10 obese diabetic and 10 obese non-diabetic individuals. Twenty obese women undergoing Roux-en-Y gastric bypass surgery were recruited for this study. Muscle samples were obtained intraoperatively and subdivided for multiple analyses, including high-resolution respirometry and assessment of supercomplex assembly. Clinical data obtained from referring physicians were correlated with laboratory findings. Participants in both groups were of a similar age, weight and BMI. Mitochondrial respiration rates were markedly reduced in diabetic vs non-diabetic patients. This defect was observed during maximal ADP-stimulated respiration in the presence of complex I-linked substrates and complex I- and II-linked substrates, and during maximal uncoupled respiration. There were no differences in fatty acid (octanoyl carnitine) supported respiration, leak respiration or isolated activity of cytochrome c oxidase. Intriguingly, significant correlations were found between glycated haemoglobin (HbA1c) levels and maximal respiration or respiration supported by complex I, complex I and II or fatty acid. In the muscle of diabetic patients, blue native gel electrophoresis revealed a striking decrease in complex I, III and IV containing ETC supercomplexes. These findings support the hypothesis that ETC supercomplex assembly may be an important underlying mechanism of muscle mitochondrial dysfunction in type 2 diabetes mellitus.

Tim62, a Novel Mitochondrial Protein in Trypanosoma brucei, Is Essential for Assembly and Stability of the TbTim17 Protein Complex

Trypanosoma brucei, the causative agent of human African trypanosomiasis, possesses non-canonical mitochondrial protein import machinery. Previously, we characterized the essential translocase of the mitochondrial inner membrane (TIM) consisting of Tim17 in T. brucei. TbTim17 is associated with TbTim62. Here we show that TbTim62, a novel protein, is localized in the mitochondrial inner membrane, and its import into mitochondria depends on TbTim17. Knockdown (KD) of TbTim62 decreased the steady-state levels of TbTim17 post-transcriptionally. Further analysis showed that import of TbTim17 into mitochondria was not inhibited, but its half-life was reduced >4-fold due to TbTim62 KD. Blue-native gel electrophoresis revealed that TbTim62 is present primarily in ∼150-kDa and also in ∼1100-kDa protein complexes, whereas TbTim17 is present in multiple complexes within the range of ∼300 to ∼1100 kDa. TbTim62 KD reduced the levels of both TbTim62 as well as TbTim17 protein complexes. Interestingly, TbTim17 was accumulated as lower molecular mass complexes in TbTim62 KD mitochondria. Furthermore, depletion of TbTim62 hampered the assembly of the ectopically expressed TbTim17-2X-myc into TbTim17 protein complex. Co-immunoprecipitation analysis revealed that association of TbTim17 with mHSP70 was markedly reduced in TbTim62 KD mitochondria. All together our results demonstrate that TbTim62, a unique mitochondrial protein in T. brucei, is required for the formation of a stable TbTim17 protein complex. TbTim62 KD destabilizes this complex, and unassembled TbTim17 degrades. Therefore, TbTim62 acts as a novel regulatory factor to maintain the levels of TIM in T. brucei mitochondria.

Hippocampal receptor complexes paralleling LTP reinforcement in the spatial memory holeboard test in the rat

The current study was designed to examine learning-induced transformation of early-LTP into late-LTP. Recording electrodes were implanted into the dentate gyrus of the hippocampus in male rats and early-LTP was induced by weak tetanic stimulation of the medial perforant path. Dorsal right hippocampi were removed, membrane proteins were extracted, separated by blue-native gel electrophoresis with subsequent immunoblotting using brain receptor antibodies. Spatial training resulted into reinforcement of LTP and the reinforced LTP was persistent for 6h. Receptor complex levels containing GluN1 and GluN2A of NMDARs, GluA1 and GluA2 of AMPARs, nAchα7R and the D(1A) dopamine receptor were significantly-elevated in rat hippocampi of animals underwent spatial learning, whilst levels of GluA3 and 5-HT1A receptor containing complexes were significantly reduced. Evidence for complex formation between GluN1 and D(1A) dopamine receptor was provided by antibody shift assay, co-immunoprecipitation and mass spectrometric analysis.Thus our results propose that behavioural stimuli like spatial learning reinforce early LTP into late LTP and this reinforced LTP is accompanied by changes in certain receptor levels in the membrane fraction of the rat hippocampus.

Interaction between phage BFK20 helicase gp41 and its host Brevibacterium flavum primase DnaG

Protein–protein interactions have been identified between the replication proteins of corynephage BFK20 and its host Brevibacterium flavum CCM 251. We tested the interactions between phage proteins gp40, gp41, gp42, gp43 and gp44 and between these phage proteins and host replication proteins DnaZX, DnaN, Dnaδ, DnaG, DnaA, RecF, TrxC, TrxB1 and SSB using a bacterial two-hybrid system. Phage proteins gp41, gp42 and gp43 self-associated, demonstrating that these proteins are oligomers in vivo. Interactions were also detected between phage protein gp41 and host proteins DnaZX, DnaN, Dnaδ, DnaG and SSB. β-galactosidase activity measurements showed that the strongest interaction was between gp41 and DnaG. The interaction was studied further using 2-dimensional blue native SDS-polyacrylamide gel electrophoresis and Western blot analysis.

A novel mutation in TTC19 associated with isolated complex III deficiency, cerebellar hypoplasia, and bilateral basal ganglia lesions.

Isolated complex III (cIII) deficiency is a rare biochemical finding in mitochondrial disorders, mainly associated with mutations in mitochondrial DNA MTCYB gene, encoding cytochrome b, or in assembly factor genes (BCS1L, TTC19, UQCC2, and LYRM7), whereas mutations in nuclear genes encoding cIII structural subunits are extremely infrequent. We report here a patient, a 9 year old female born from first cousin related parents, with normal development till 18 months when she showed unsteady gait with frequent falling down, cognitive, and speech worsening. Her course deteriorated progressively. Brain MRI showed cerebellar vermis hypoplasia and bilateral lentiform nucleus high signal lesions. Now she is bed ridden with tetraparesis and severely impaired cognitive and language functions. Biochemical analysis revealed isolated cIII deficiency in muscle, and impaired respiration in fibroblasts. We identified a novel homozygous rearrangement in TTC19 (c.213_229dup), resulting in frameshift with creation of a premature termination codon (p.Gln77Argfs*30). Western blot analysis demonstrated the absence of TTC19 protein in patient’s fibroblasts, while Blue-Native Gel Electrophoresis analysis revealed the presence of cIII-specific assembly intermediates. Mutations in TTC19 have been rarely associated with mitochondrial disease to date, being described in about ten patients with heterogeneous clinical presentations, ranging from early onset encephalomyopathy to adult forms with cerebellar ataxia. Contrariwise, the biochemical defect was a common hallmark in TTC19 mutant patients, confirming the importance of TTC19 in cIII assembly/stability. Therefore, we suggest extending the TTC19 mutational screening to all patients with cIII deficiency, independently from their phenotypes.

Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane Reveals Roles of Acrosomal Proteins in ZP3 Binding.

The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (∼750-1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with anti-zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation.

Na+/K+-ATPase β1-subunit is recruited in Na-K-2Cl co-transporter isoform 2 multiprotein complexes in rat kidneys: possible role in blood pressure regulation.

The progression from prehypertensive to hypertensive state in spontaneous hypertensive rats (SHRs) is accompanied by a significant increase in membrane expression of Na-K-2Cl co-transporter isoform 2 (NKCC2), suggesting that the altered NKCC2 trafficking and activity are directly related with the development of hypertension in this strain. The aim of this work is to gain insights on the molecular mechanism that underlies this phenomenon. We performed a comparative analysis of NKCC2 multiprotein complexes (MPCs) in the kidney of SHRs versus Wistar Kyoto rats by Blue Native difference gel electrophoresis combined with mass spectrometry. We found that the recruitment of the β-subunit isoform 1 of the Na(+)-K(+)-ATPase (β1NK) in NKCC2 MPCs was significantly increased in the kidneys of SHR compared with Wistar Kyoto rat control strain. Co-immunoprecipitation experiments showed that β1NK actually interacts with NKCC2 in the native tissue. The analysis of the physiological role of β1NK-NKCC2 interaction in human embryonic kidney cells showed that β1NK increased the steady-state membrane expression and activity of NKCC2 enhancing NKCC2 trafficking toward the plasma membrane. We identify a new NKCC2-interacting partner involved in the modulation of NKCC2 intracellular trafficking and possibly involved in the regulation of blood pressure.

Evidence of functional trimeric chlorophyll a/c2-peridinin proteins in the dinoflagellate Symbiodinium

The chlorophyll a-chlorophyll c2-peridinin-protein (apcPC), a major light harvesting component in peridinin-containing dinoflagellates, is an integral membrane protein complex. We isolated functional acpPC from the dinoflagellate Symbiodinium. Both SDS-PAGE and electrospray ionization mass spectrometry (ESI-MS) analysis quantified the denatured subunit polypeptide molecular weight (MW) as 18kDa. Size-exclusion chromatography (SEC) and blue native gel electrophoresis (BN-PAGE) were employed to estimate the size of native acpPC complex to be 64–66kDa. We also performed native ESI-MS, which can volatilize and ionize active biological samples in their native states. Our result demonstrated that the native acpPC complex carried 14 to 16 positive charges, and the MW of acpPC with all the associated pigments was found to be 66.5kDa. Based on these data and the pigment stoichiometry, we propose that the functional light harvesting state of acpPC is a trimer. Our bioinformatic analysis indicated that Symbiodinium acpPC shares high similarity to diatom fucoxanthin Chl a/c binding protein (FCP), which tends to form a trimer. Additionally, acpPC protein sequence variation was confirmed by de novo protein sequencing. Its sequence heterogeneity is also discussed in the context of Symbiodinium eco-physiological adaptations.

Analysis of mutant SOD1 electrophoretic mobility by Blue Native gel electrophoresis; evidence for soluble multimeric assemblies.

Mutations in superoxide dismutase 1 (SOD1) cause familial forms of amyotrophic lateral sclerosis (fALS). Disease causing mutations have diverse consequences on the activity and half-life of the protein, ranging from complete inactivity and short half-life to full activity and long-half-life. Uniformly, disease causing mutations induce the protein to misfold and aggregate and such aggregation tendencies are readily visualized by over-expression of the proteins in cultured cells. In the present study we have investigated the potential of using immunoblotting of proteins separated by Blue-Native gel electrophoresis (BNGE) as a means to identify soluble multimeric forms of mutant protein. We find that over-expressed wild-type human SOD1 (hSOD1) is generally not prone to form soluble high molecular weight entities that can be separated by BNGE. For ALS mutant SOD1, we observe that for all mutants examined (A4V, G37R, G85R, G93A, and L126Z), immunoblots of BN-gels separating protein solubilized by digitonin demonstrated varied amounts of high molecular weight immunoreactive entities. These entities lacked reactivity to ubiquitin and were partially dissociated by reducing agents. With the exception of the G93A mutant, these entities were not reactive to the C4F6 conformational antibody. Collectively, these data demonstrate that BNGE can be used to assess the formation of soluble multimeric assemblies of mutant SOD1.

Abstract 337: The Cardiolipin-targeting Peptide Bendavia Preserves Post-ischemic Mitochondrial Energetics by Sustaining Respiratory Supercomplexes

Bendavia is a mitochondria-targeting peptide currently being tested in the EMBRACE-STEMI trial for reducing injury during acute coronary syndromes. We previously showed that the cardioprotective effects of Bendavia involved improving cardiolipin-dependent mitochondrial membrane fluidity. As membrane fluidity influences the ability of proteins to assemble, we hypothesized that a consequence of augmented membrane fluidity would be higher expression of mitochondrial respiratory supercomplexes. Rat hearts (n=42) were subjected to ischemia-reperfusion (I/R) with or without 1nM Bendavia perfusion beginning at the onset of reperfusion. Left ventricular tissue was split into one of two study arms: 1. Supercomplex expression using blue-native gel electrophoresis (BN-PAGE), or 2. High-resolution respirometry using permeabilized ventricular fibers. For BN-PAGE studies, respiratory supercomplex bands were decreased with I/R, and restored with Bendavia (see figure). High-resolution respirometry studies indicated suppressed Complex I-dependent respiration after I/R (208±19 v 42±9 pmol O2/mg*s in control v I/R, respectively; P<0.05). Complex II-dependent respiration was also lower (753±41 v 168±13 pmol/mg*s in control versus I/R; P<0.05). Perfusion with Bendavia during reperfusion significantly increased Complexes I- (100±13pmol O2/mg*s) and II-dependent (334±63 pmol O2/mg*s) respiration (P<0.05 versus untreated IR for both). Taken together, these data suggest that Bendavia’s protective mechanism of action involves preserving supercomplex-dependent mitochondrial function during cardiac reperfusion.

Native Protein Separation by Isoelectric Focusing and Blue Gel Electrophoresis-Coupled Two-Dimensional Microfluidic Chip Electrophoresis

The use of microfluidic chip-based two-dimensional separation holds great promise in the proteomics field, given its portability, simplicity, speed, efficiency, and throughput. However, inclusion of sodium dodecyl sulfate, reported to be necessary for increasing protein-resolving capability, was also accompanied by the loss of both protein conformation and biological function. Here, we describe separation of native proteins by introducing blue native gel electrophoresis into isoelectric focusing and gel electrophoresis (IEF/CGE)-coupled protein two-dimensional microfluidic chip electrophoresis. After assessing the influence of various experimental conditions, the best separation ability and reproducibility of blue native IEF/CGE (IEF/BN-CGE) chip electrophoresis achieved until now were demonstrated no matter whether with a simple simulated mixture or with a complex mixture of total Escherichia coli proteins. Finally, instead of theoretical calculations, the image analysis technique was also used for the first time to quantitatively evaluate the actual peak capacities of chip electrophoresis. According to the number of features abstracted in the electrophoresis patterns, the superiority of the IEF/BN-CGE two-dimensional microfluidic chip electrophoresis was then exhibited quantitatively. The high native protein separation performance makes this established chip electrophoresis method possible for further application in widely needed drug screening, analysis of bio-molecular function, and assays of protein–protein interactions.

Oligomerization of optineurin and its oxidative stress- or E50K mutation-driven covalent cross-linking: possible relationship with glaucoma pathology.

The optineurin gene, OPTN, is one of the causative genes of primary open-angle glaucoma. Although oligomerization of optineurin in cultured cells was previously observed by gel filtration analysis and blue native gel electrophoresis (BNE), little is known about the characteristics of optineurin oligomers. Here, we aimed to analyze the oligomeric state of optineurin and factors affecting oligomerization, such as environmental stimuli or mutations in OPTN. Using BNE or immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we demonstrated that both endogenous and transfected optineurin exist as oligomers, rather than monomers, in NIH3T3 cells. We also applied an in situ proximity ligation assay to visualize the self-interaction of optineurin in fixed HeLaS3 cells and found that the optineurin oligomers were localized diffusely in the cytoplasm. Optineurin oligomers were usually detected as a single band of a size equal to that of the optineurin monomer upon SDS-PAGE, while an additional protein band of a larger size was observed when cells were treated with H2O2. We showed that larger protein complex is optineurin oligomers by immunoprecipitation and termed it covalent optineurin oligomers. In cells expressing OPTN bearing the most common glaucoma-associated mutation, E50K, covalent oligomers were formed even without H2O2 stimulation. Antioxidants inhibited the formation of E50K-induced covalent oligomers to various degrees. A series of truncated constructs of OPTN was used to reveal that covalent oligomers may be optineurin trimers and that the ubiquitin-binding domain is essential for formation of these trimers. Our results indicated that optineurin trimers may be the basic unit of these oligomers. The oligomeric state can be affected by many factors that induce covalent bonds, such as H2O2 or E50K, as demonstrated here; this provides novel insights into the pathogenicity of E50K. Furthermore, regulation of the oligomeric state should be studied in the future.

What Goes Wrong With Mitochondria During Renal Cold Storage Plus Transplantation and Can It Be Fixed?

Purpose: The overall goal of this study is to elucidate mechanisms involved with cold storage (CS) mediated mitochondrial and renal injury, which could lead to better therapies (or preservation solutions) that are designed to minimize damage, increase the donor pool, and improve renal transplant outcomes. Our published reports showed that cold storage induces mitochondrial and renal damage, and we recently reported that a mitochondrial targeted antioxidant, MitoQ, blunted mitochondrial damage in a porcine kidney model. Mitochondrial damage can be alleviated by biogenesis of new healthy mitochondria; however, few studies have investigated the mitochondrial biogenesis pathway during renal CS + transplantation. Methods: Male rat kidneys were isolated and cold stored for 0-18 hr +/- MitoQ (100 uM) followed by transplantation. Mitochondrial function (oxygen consumption) was assessed via high resolution respirometry and ATP measurement of renal biopsies post-transplant (18 hr). Quantitative proteomics of mitochondrial extracts separated using blue native gel electrophoresis was used as a proteomic approach to identify key mitochondrial respiratory complex proteins altered during CS + transplantation. Mitochondrial biogenesis was assessed by small fragment PCR of mitochondrial DNA (mtDNA) as well as mitochondrial protein expression. Pilot studies using ‘discarded' paired human kidneys obtained from our local procurement office were also treated with MitoQ. Results: Renal CS + transplantation lead to decreased mtDNA integrity and copy numbers, reduced respiratory complex I and II/III function, and impaired mitochondrial biogenesis. Quantitative proteomics of complex I showed that 8 complex I subunits were decreased following CS + transplantation. We also showed that adding MitoQ increased ATP and mtDNA after CS alone, and improved renal function following CS + transplantation. Early results using discarded human kidneys showed reduced mtDNA integrity and biogenesis in these kidneys; addition of MitoQ restored respiratory complex activity in these kidneys. Summary: These studies suggest cold storage leads to damaged respiratory complexes as well as damage to the mtDNA, which can both lead to reduced mitochondrial function and loss of the reparative biogenesis component. Interestingly, inclusion of MitoQ during cold storage lead to protection against mitochondrial and renal damage during cold storage and transplantation. Supported by NIH DK089659 (LAMC).