Blue Native Polyacrylamide Gel Electrophoresis Research Articles

MecE, MecB, and MecC proteins orchestrate methyl group transfer during dichloromethane fermentation.

Dichloromethane (DCM), a common hazardous industrial chemical, is anaerobically metabolized by four bacterial genera: Dehalobacter, Dehalobacterium, Ca. Dichloromethanomonas, and Ca. Formimonas. However, the pivotal methyltransferases responsible for DCM transformation have remained elusive. In this study, we investigated the DCM catabolism of Dehalobacterium formicoaceticum strain EZ94, contained in an enriched culture, using a combination of biochemical approaches. Initially, enzymatic assays were conducted with cell-free protein extracts, after protein separation by blue native polyacrylamide gel electrophoresis. In the slices with the highest DCM transformation activity, a high absolute abundance of the methyltransferase MecC was revealed by mass spectrometry. Enzymatic activity assays with heterologously expressed MecB, MecC, and MecE from strain EZ94 showed complete DCM transformation only when all three enzymes were present. Our experimental results, coupled with the computational analysis of MecB, MecC, and MecE sequences, enabled us to assign specific roles in DCM transformation to each of the proteins. Our findings reveal that both MecE and MecC are zinc-dependent methyltransferases responsible for DCM demethylation and re-methylation of a product, respectively. MecB functions as a cobalamin-dependent shuttle protein transferring the methyl group between MecE and MecC. This study provides the first biochemical evidence of the enzymes involved in the anaerobic metabolism of DCM.IMPORTANCEDichloromethane (DCM) is a priority regulated pollutant frequently detected in groundwater. In this work, we identify the proteins responsible for the transformation of DCM fermentation in Dehalobacterium formicoaceticum strain EZ94 using a combination of biochemical approaches, heterologous expression of proteins, and computational analysis. These findings provide the basis to apply these proteins as biological markers to monitor bioremediation processes in the field.

Physioxia rewires mitochondrial complex composition to protect stem cell viability

Human induced pluripotent stem cells (hiPSCs) are an invaluable tool to study molecular mechanisms on a human background. Culturing stem cells at an oxygen level different from their microenvironmental niche impacts their viability. To understand this mechanistically, dermal skin fibroblasts of 52 probands were reprogrammed into hiPSCs, followed by either hyperoxic (20 % O2) or physioxic (5 % O2) culture and proteomic profiling. Analysis of chromosomal stability by Giemsa-banding revealed that physioxic -cultured hiPSC clones exhibited less pathological karyotypes than hyperoxic (e.g. 6 % vs. 32 % mosaicism), higher pluripotency as evidenced by higher Stage-Specific Embryonic Antigen 3 positivity, higher glucose consumption and lactate production. Global proteomic analysis demonstrated lower abundance of several subunits of NADH:ubiquinone oxidoreductase (complex I) and an underrepresentation of pathways linked to oxidative phosphorylation and cellular senescence. Accordingly, release of the pro-senescent factor IGFBP3 and β-galactosidase staining were lower in physioxic hiPSCs. RNA- and ATAC-seq profiling revealed a distinct hypoxic transcription factor-binding footprint, amongst others higher expression of the HIF1α-regulated target NDUFA4L2 along with increased chromatin accessibility of the NDUFA4L2 gene locus. While mitochondrial DNA content did not differ between groups, physioxic hiPSCs revealed lower polarized mitochondrial membrane potential, altered mitochondrial network appearance and reduced basal respiration and electron transfer capacity. Blue-native polyacrylamide gel electrophoresis coupled to mass spectrometry of the mitochondrial complexes detected higher abundance of NDUFA4L2 and ATP5IF1 and loss of incorporation into complex IV or V, respectively. Taken together, physioxic culture of hiPSCs improved chromosomal stability, which was associated with downregulation of oxidative phosphorylation and senescence and extensive re-wiring of mitochondrial complex composition.

Evidence of γ-secretase complex involved in the regulation of intramembrane proteolysis in Entamoeba histolytica

Presenilins (PSNs) are multifunctional membrane proteins involved in signal transduction, lysosomal acidification, and certain physiological processes related to mitochondria. The aspartic protease activity of PSN and the formation of a γ-secretase complex with other subunits such as nicastrin (NCT) are required for the biological functions. Although PSN is widely conserved in eukaryotes, most studies on PSN were conducted in metazoans. Homologous genes for PSN and NCT (EhPSN and EhNCT, respectively) are encoded in the genome of Entamoeba histolytica, however, their functions remain unknown. In this study, we showed that EhPSN and EhNCT form a complex on the cell membrane, demonstrating that the parasite possesses γ-secretase. The predicted structure of EhPSN was similar to the human homolog, demonstrated by the crystal structure, and phylogenetic analysis indicated good conservation between EhPSN and human PSN, supporting the premise that EhPSN functions as a subunit of γ-secretase. By contrast, EhNCT appears to have undergone remarkable structural changes during its evolution. Blue native-polyacrylamide gel electrophoresis combined with western blotting indicated that a 150-kDa single band contains both EhPSN (estimated molecular size: 47-kDa) and EhNCT (64-kDa), suggesting that the complex also contains other unknown components or post-translational modifications. Coimmunoprecipitation from amebic lysates also confirmed that EhPSN and EhNCT formed a complex. Indirect immunofluorescence analysis revealed that the complex localized to the plasma membrane. Moreover, EhPSN exhibited protease activity, which was suppressed by a γ-secretase inhibitor. This is the first report of a γ-secretase complex in protozoan parasites.

What are the main proteins in the hemolymph of Haemaphysalis flava ticks?

Haemaphysalis flava is a notorious parasite for humans and animals worldwide. The organs of H. flava are bathed in hemolymph, which is a freely circulating fluid. Nutrients, immune factors, and waste can be transported to any part of the body via hemolymph. The main soluble components in hemolymph are proteins. However, knowledge of the H. flava proteome is limited. The hemolymph was collected from fully engorged H. flava ticks by leg amputation. Hemolymph proteins were examined by both blue native polyacrylamide gel electrophoresis (BN-PAGE) and sodium dodecyl sulfate PAGE (SDS-PAGE). Proteins extracted from the gels were further identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two bands (380 and 520 kDa) were separated from tick hemolymph by BN-PAGE and were further separated into four bands (105, 120, 130, and 360 kDa) by SDS-PAGE. LC-MS/MS revealed that seven tick proteins and 13 host proteins were present in the four bands. These tick proteins mainly belonged to the vitellogenin (Vg) family and the α-macroglobulin family members. In silico structural analysis showed that these Vg family members all had common conserved domains, including the N-terminus lipid binding domain (LPD-N), the C-terminus von Willebrand type D domain (vWD), and the domain of unknown function (DUF). Additionally, two of the Vg family proteins were determined to belong to the carrier protein (CP) by analyzing the unique N-terminal amino acid sequences and the cleaving sites. These findings suggest that the Vg family proteins and α-macroglobulin are the primary constituents of the hemolymph in the form of protein complexes. Our results provide a valuable resource for further functional investigations of H. flava hemolymph effectors and may be useful in tick management.

Shikonin blocks CAF-induced TNBC metastasis by suppressing mitochondrial biogenesis through GSK-3β/NEDD4-1 mediated phosphorylation-dependent degradation of PGC-1α

BackgroundTriple-negative breast cancer (TNBC) is characterized by its high metastatic potential, which results in poor patient survival. Cancer-associated fibroblasts (CAFs) are crucial in facilitating TNBC metastasis via induction of mitochondrial biogenesis. However, how to inhibit CAF-conferred mitochondrial biogenesis is still needed to explore.MethodsWe investigated metastasis using wound healing and cell invasion assays, 3D-culture, anoikis detection, and NOD/SCID mice. Mitochondrial biogenesis was detected by MitoTracker green FM staining, quantification of mitochondrial DNA levels, and blue-native polyacrylamide gel electrophoresis. The expression, transcription, and phosphorylation of peroxisome-proliferator activated receptor coactivator 1α (PGC-1α) were detected by western blotting, chromatin immunoprecipitation, dual-luciferase reporter assay, quantitative polymerase chain reaction, immunoprecipitation, and liquid chromatography-tandem mass spectrometry. The prognostic role of PGC-1α in TNBC was evaluated using the Kaplan–Meier plotter database and clinical breast cancer tissue samples.ResultsWe demonstrated that PGC-1α indicated lymph node metastasis, tumor thrombus formation, and poor survival in TNBC patients, and it was induced by CAFs, which functioned as an inducer of mitochondrial biogenesis and metastasis in TNBC. Shikonin impeded the CAF-induced PGC-1α expression, nuclear localization, and interaction with estrogen-related receptor alpha (ERRα), thereby inhibiting PGC-1α/ERRα-targeted mitochondrial genes. Mechanistically, the downregulation of PGC-1α was mediated by synthase kinase 3β-induced phosphorylation of PGC-1α at Thr295, which associated with neural precursor cell expressed developmentally downregulated 4e1 recognition and subsequent degradation by ubiquitin proteolysis. Mutation of PGC-1α at Thr295 negated the suppressive effects of shikonin on CAF-stimulated TNBC mitochondrial biogenesis and metastasis in vitro and in vivo.ConclusionsOur findings indicate that PGC-1α is a viable target for blocking TNBC metastasis by disrupting mitochondrial biogenesis, and that shikonin merits potential for treatment of TNBC metastasis as an inhibitor of mitochondrial biogenesis through targeting PGC-1α.

Neutrophil extracellular trap-induced ferroptosis promotes abdominal aortic aneurysm formation via SLC25A11-mediated depletion of mitochondrial glutathione

BackgroundNeutrophil extracellular traps (NETs) induce oxidative stress, which may initiate ferroptosis, an iron-dependent programmed cell death, during abdominal aortic aneurysm (AAA) formation. Mitochondria regulate the progression of ferroptosis, which is characterized by the depletion of mitochondrial glutathione (mitoGSH) levels. However, the mechanisms are poorly understood. This study examined the role of mitoGSH in regulating NET-induced ferroptosis of smooth muscle cells (SMCs) during AAA formation. MethodsConcentrations of NET markers were tested in plasma samples. Western blotting and immunofluorescent staining were performed to detect the expression and localization of NET and ferroptosis markers in tissue samples. The role of NETs and SMC ferroptosis during AAA formation was investigated using peptidyl arginine deiminase 4 gene (Padi4) knockout or treatment with a PAD4 inhibitor, ferroptosis inhibitor or activator in an angiotensin II-induced AAA mouse model. The regulatory effect of SLC25A11, a mitochondrial glutathione transporter, on mitoGSH and NET-induced ferroptosis of SMCs was investigated using in vitro and in vivo experiments. Transmission electron microscopy was used to detect mitochondrial damage. Blue native polyacrylamide gel electrophoresis was used to analyze the dimeric and monomeric forms of the protein. ResultsSignificantly elevated levels of NETosis and ferroptosis markers in aortic tissue samples were observed during AAA formation. Specifically, NETs promoted AAA formation by inducing ferroptosis of SMCs. Subsequently, SLC25A11 was identified as a potential biomarker for evaluating the clinical prognosis of patients with AAA. Furthermore, NETs decreased the stability and dimerization of SLC25A11, leading to the depletion of mitoGSH. This depletion induced the ferroptosis of SMCs and promoted AAA formation. ConclusionDuring AAA formation, NETs regulate the stability of the mitochondrial carrier protein SLC25A11, leading to the depletion of mitoGSH and subsequent activation of NET-induced ferroptosis of SMCs. Preventing mitoGSH depletion and ferroptosis in SMCs is a potential strategy for treating AAA.

Physical and functional interaction between NHE3 and SGLT2 in the renal proximal tubule of male and female rats

Sodium-glucose cotransporter type 2 (SGLT2) and sodium/hydrogen exchanger-3 (NHE3) serve as the primary renal proximal tubule transporters responsible for the apical uptake of filtered glucose and NaHCO3/NaCl, respectively. Our previous studies demonstrated that chronic treatment with the selective SGLT2 inhibitor empagliflozin reduces NHE3 activity in the proximal tubule. Furthermore, we observed that the non-specific SGLT1/SGLT2 inhibitor, phlorizin, acutely inhibits NHE3 in the absence of luminal glucose, suggesting that the impact of SGLT inhibition on NHE3 may not be attributed to high tubular glucose concentrations. Additionally, our investigations revealed that SGLT2, but not SGLT1, colocalizes with NHE3 in the renal proximal tubule. This study aimed to gain insights into the functional and physical association of NHE3 and SGLT2 in the renal proximal tubule. In vivo microperfusion was conducted in male and female Wistar rats using glucose-free perfusates containing vehicle or empagliflozin (1 μM). Empagliflozin treatment significantly decreased the flow rate of bicarbonate reabsorption (JHCO3−) in both male (2.109±0.091 vs. 1.119±0.096 nmol/cm2∙s) and female rats (1.750±0.037 vs. 1.128±0.062 nmol/cm2∙s), indicating that SGLT2 inhibitors inhibit NHE3 activity through glucose-independent mechanisms. By employing nondenaturant detergents, we utilized blue native polyacrylamide gel electrophoresis (BN-PAGE) to preserve native protein-protein interactions. Double staining with antibodies against specific partner candidates revealed that bands attributed to NHE3, MAP17, PDZK1, and SGLT2 migrate similarly at ~480 kDa in samples from rat renal cortex, as supported by independent experiments. Rigorous controls were implemented to address concerns related to autofluorescence, antibody cross-reactivity, and signal specificity, confirming the validity of our results. Furthermore, complexes involving SGLT2 and NHE3 at an apparent ~480 kDa molecular weight were observed in male and female rats. Unlike SGLT2, SGLT1 bands in BN-PAGE did not overlap with the NHE3 signal, which is consistent with our earlier immunofluorescence experiments. Collectively, our data suggest that NHE3 and SGLT2 are functionally and physically associated with the renal proximal tubule. Future experiments will be undertaken to further characterize the physical association between NHE3 and SGLT2 and to determine whether the inhibition of NHE3 by SGLT2 inhibitors is mediated through the disruption of the NHE3-PDZK1-MAP17-SGLT2 multiprotein complex. Funding: São Paulo Research Foundation (FAPESP). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Differential stability of bacterial photosynthetic apparatus of Rhodobacter alkalitolerans strain JA916T under alkaline and light environment.

In purple bacteria, photosynthesis is performed by densely packed pigment-protein complexes, including the light-harvesting complexes known as RC-LH1 and LH2, with carotenoids to assist in the functioning of photosynthesis. Most photosynthetic bacteria are exposed to various abiotic stresses such as light, temperature, alkalinity-acidity, and salinity. Rhodobacter (R.) alkalitolerans was discovered from the alkaline pond; here, we report the comparative study of the photosynthetic apparatus of R. alkalitolerans in various light intensities in relation to its high pH tolerance ability. With increased light intensity, the stability of photosystem complexes decreased in normal pH (npH pH 6.80 ± 0.05) conditions, whereas in high pH (hpH pH 8.60 ± 0.05), acclimation was observed to high light. The content of bacteriochlorophyll a, absorbance spectra, and circular dichroism data shows that the integrity of photosystem complexes is less affected in hpH compared with npH conditions. Large pore blue native polyacrylamide gel electrophoresis of photosystem protein complexes and sucrose density gradient of n-dodecyl β-D-maltoside solubilized intracytoplasmic membranes show that LH2 is more affected in npH than in hpH, whereas RC-LH1 monomer or dimer has shown interplay between monomer and dimer in hpH, although the dimer and monomer both increased in npH. Increased content and expression level of ATPase protein complex and subunit-"c" of ATPase, fast relaxation kinetics of p515, and relatively higher membrane lipid content in hpH along with less photooxidative stress and subsequently lesser superoxide dismutase activity exemplify photoprotection in hpH. Furthermore, the increased expression levels of antiporter NhaD in hpH signify its role in the maintenance of homeostatic balance in hpH.

Junctional Adhesion Molecule a (JAM-A) Associates with CD36 upon Platelet Activation and Cytoskeleton in a Phosphorylation-Dependent Manner to Promote Platelet-Platelet Contacts

Background: Platelets play a fundamental role inmaintaining hemostasis. Stability of hemostatic plug is crucial for arresting blood loss during vascular breach. JAM-A is a member of the immunoglobulin superfamily that restricts α IIbβ 3 to its low affinity state by recruiting CSK to the α IIbβ 3-c-Src complex in resting platelets. Agonist-induced inside-out signaling causes JAM-A to dissociate from the α IIbβ 3-Src complex, thus facilitating activation of α IIbβ 3 and outside-in signaling. However, the function of JAM-A in the process of hemostasis is poorly understood. Aim: To evaluate the role of JAM-A in activated platelets and on hemostasis. Methods: Washed human platelets were used in these studies. Full-length cytoplasmic domain of JAM-A (cyto JAM-A) and JAM-A cytoplasmic domain lacking C-terminal PDZ-domain-binding motif (ΔPDZ cyto JAM-A) were expressed as GST-fusion proteins in bacteria using pGEX4T-1 vector. GST-fusion proteins were purified using glutathione beads. Pull-down assays were performed by incubating glutathione GST-fusion proteins beads with lysates of washed human platelets (4×10 8/mL). GST beads alone without the fusion proteins were used as a control. Immunoprecipitation studies were performed using washed human platelets (4x10 8/mL) lysed with CHAPS lysis buffer and precleared by using Protein G Sepharose beads. Specific primary antibodies and isotype specific IgGs (control) were used for immunoprecipitation followed by western blotting. To identify protein complexes two-dimensional blue-native polyacrylamide gel electrophoresis was performed in which protein complexes were first separated based on their size (1 st dimension) and individual proteins in each complex were separated using SDS PAGE (2 nd dimension). JAM-A S 284 phosphorylation was detected using anti-Phospho-S 284. Rap1 activation assay was performed by pull-down GTP-bound Rap1 following the manufacturer's instructions. Results: CD9 and α IIbβ 3 co-immunoprecipitated with JAM-A from resting platelet lysates suggesting that CD9 is a part of the JAM-A/α IIbβ 3 complex. In a pull-down assay recombinant full-length JAM-A cytoplasmic tail fused with GST, but not JAM-A tail lacking the PDZ domain motif fused with GST pull-down CD9 and α IIbβ 3 from resting platelet lysates suggesting that PDZ-domain binding motif of JAM-A is responsible for its interaction with CD9/α IIbβ 3 complex. Interestingly, platelet activation resulted in the dissociation of JAM-A from the CD9/α IIbβ 3 complex as seen by loss of interaction between JAM-A and CD9/α IIbβ 3 in co-immunoprecipitation and blue-native PAGE assays. It is reported that platelet activation results in rapid phosphorylation of JAM-A on S 284 in a PKC-dependent manner. We found that S 284 phosphorylated JAM-A associates with CD36 and this complex moves to the Triton X-100 insoluble cytoskeletal fraction upon platelet activation suggesting that phosphorylation of JAM-A may assist its localization to cytoskeleton. Interestingly, we found that induction of conformational change in α IIbβ 3 using Mn 2+ (10μM) activates JAM-A S 284 phosphorylation. Furthermore, Mn 2+ also induced a robust activation of Rap1, a key signaling molecule involved in junctional assembly. Both JAM-A phosphorylation and Rap1 activation were significantly ( P<0.05) inhibited by a pan PKC inhibitor, bisindolylmaleimide (10μM) suggesting that S 284 phosphorylated JAM-A/CD36 complex may support junctional assembly within the platelet plug. Conclusion: JAM-A, through its cytoplasmic domain, associates with α IIbβ 3 and CD9 in resting platelets. Upon platelet activation JAM-A rapidly dissociates from this complex and is phosphorylated on S 284. Phosphorylated JAM-A then associates with CD36 and cytoskeleton leading to activation of Rap1, a protein important in junctional assembly between platelets within a hemostatic plug.

Biophysical characterization of the cetacean morbillivirus haemagglutinin glycoprotein

Cetacean morbillivirus (CeMV) is an enveloped, non-segmented, negative-stranded RNA virus that infects marine mammals, spreading across species and causing lethal disease outbreaks worldwide. Among the eight proteins encoded by the CeMV genome, the haemagglutinin (H) glycoprotein is responsible for the virus attachment to host cell receptors. CeMV H represents an attractive target for antiviral and diagnostic research, yet the elucidation of the molecular mechanisms underlying its role in infection and inter-species transmission was hampered thus far due to the unavailability of recombinant versions of the protein. Here we present the cloning, expression and purification of a recombinant CeMV H ectodomain (rH-ecto), providing an initial characterization of its biophysical and structural properties. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis (PAGE) combined to Western blot analysis and periodic acid Schiff assay showed that CeMV rH-ecto is purifiable at homogeneity from insect cells as a secreted, soluble and glycosylated protein. Miniaturized differential scanning fluorimetry, Blue Native PAGE and size exclusion chromatography coupled to multiangle light scattering revealed that CeMV rH-ecto is globularly folded, thermally stable and exists in solution in the oligomeric states of dimer and multiple of dimers. Furthermore, negative stain electron microscopy single particle analysis allowed us to delineate a low-resolution molecular architecture of the CeMV rH-ecto dimer, which recapitulates native assemblies from other morbilliviral H proteins, such as those from measles virus and canine distemper virus. This set of experiments by orthogonal techniques validates the CeMV rH-ecto as an experimental model for future biochemical studies on its structure and functions.

Development of a Cell-Based Assay Using a Split-Luciferase Reporter for Compound Screening.

Recently, the finding of recurrent mutations in the spliceosome components in cancer has indicated that the spliceosome is a potential target for cancer therapy. However, the number of small molecules known to affect the cellular spliceosome is currently limited probably because of the lack of a robust cell-based approach to identify small molecules that target the spliceosome. We have previously reported the development of a genetic reporter to detect the cellular levels of small nuclear ribonucleoproteins (snRNPs), which are subunits of the spliceosome, using a split luciferase. However, the original protocol was designed for small scale experiments and was not suitable for compound screening. Here, we found that the use of cell lysis buffer used in blue native polyacrylamide gel electrophoresis (BN-PAGE) dramatically improved the sensitivity and the robustness of the assay. Improved assay conditions were used to discover a small molecule that altered the reporter activity. Our method may be used with other cellular macromolecular complexes and may assist in the discovery of small bioactive molecules.

Hippocampal versus cortical deletion of cholinergic receptor muscarinic 1 in mice differentially affects post-translational modifications and supramolecular assembly of respiratory chain-associated proteins, mitochondrial ultrastructure, and respiration: implications in Alzheimer's disease.

Introduction: In a previous retrospective study using postmortem human brain tissues, we demonstrated that loss of Cholinergic Receptor Muscarinic 1 (CHRM1) in the temporal cortex of a subset of Alzheimer's patients was associated with poor survival, whereas similar loss in the hippocampus showed no such association. Mitochondrial dysfunction underlies Alzheimer's pathogenesis. Therefore, to investigate the mechanistic basis of our findings, we evaluated cortical mitochondrial phenotypes in Chrm1 knockout (Chrm1-/-) mice. Cortical Chrm1 loss resulted in reduced respiration, reduced supramolecular assembly of respiratory protein complexes, and caused mitochondrial ultrastructural abnormalities. These mouse-based findings mechanistically linked cortical CHRM1 loss with poor survival of Alzheimer's patients. However, evaluation of the effect of Chrm1 loss on mouse hippocampal mitochondrial characteristics is necessary to fully understand our retrospective human tissue-based observations. This is the objective of this study. Methods: Enriched hippocampal and cortical mitochondrial fractions (EHMFs/ECMFs, respectively) derived from wild-type and Chrm1-/- mice were used to measure respiration by quantifying real-time oxygen consumption, supramolecular assembly of oxidative phosphorylation (OXPHOS)-associated proteins by blue native polyacrylamide gel electrophoresis, post-translational modifications (PTMs) by isoelectric focusing (IEF), and mitochondrial ultrastructure by electron microscopy. Results: In contrast to our previous observations in Chrm1-/- ECMFs, EHMFs of Chrm1-/- mice significantly increased respiration with a concomitant increase in the supramolecular assembly of OXPHOS-associated proteins, specifically Atp5a and Uqcrc2, with no mitochondrial ultrastructural alterations. IEF of ECMFs and EHMFs from Chrm1-/- mice showed a decrease and an increase, respectively in a negatively charged (pH∼3) fraction of Atp5a relative to the wild-type mice, with a corresponding decrease or increase in the supramolecular assembly of Atp5a and respiration indicating a tissue-specific signaling effect. Discussion: Our findings indicate that loss of Chrm1 in the cortex causes structural, and physiological alterations to mitochondria that compromise neuronal function, whereas Chrm1 loss in the hippocampus may benefit neuronal function by enhancing mitochondrial function. This brain region-specific differential effect of Chrm1 deletion on mitochondrial function supports our human brain region-based findings and Chrm1-/- mouse behavioral phenotypes. Furthermore, our study indicates that Chrm1-mediated brain region-specific differential PTMs of Atp5a may alter complex-V supramolecular assembly which in turn regulates mitochondrial structure-function.

Loss of cholinergic receptor muscarinic 1 impairs cortical mitochondrial structure and function: implications in Alzheimer's disease.

Introduction: Cholinergic Receptor Muscarinic 1 (CHRM1) is a G protein-coupled acetylcholine (ACh) receptor predominantly expressed in the cerebral cortex. In a retrospective postmortem brain tissues-based study, we demonstrated that severely (≥50% decrease) reduced CHRM1 proteins in the temporal cortex of Alzheimer's patients significantly correlated with poor patient outcomes. The G protein-mediated CHRM1 signal transduction cannot sufficiently explain the mechanistic link between cortical CHRM1 loss and the appearance of hallmark Alzheimer's pathophysiologies, particularly mitochondrial structural and functional abnormalities. Therefore, the objective of this study was to analyze the molecular, ultrastructural, and functional properties of cortical mitochondria using CHRM1 knockout (Chrm1-/-) and wild-type mice to identify mitochondrial abnormalities. Methods: Isolated and enriched cortical mitochondrial fractions derived from wild-type and Chrm1-/- mice were assessed for respiratory deficits (oxygen consumption) following the addition of different substrates. The supramolecular assembly of mitochondrial oxidative phosphorylation (OXPHOS)-associated protein complexes (complex I-V) and cortical mitochondrial ultrastructure were investigated by blue native polyacrylamide gel electrophoresis and transmission electron microscopy (TEM), respectively. A cocktail of antibodies, specific to Ndufb8, Sdhb, Uqcrc2, Mtco1, and Atp5a proteins representing different subunits of complexes I-V, respectively was used to characterize different OXPHOS-associated protein complexes. Results: Loss of Chrm1 led to a significant reduction in cortical mitochondrial respiration (oxygen consumption) concomitantly associated with reduced oligomerization of ATP synthase (complex V) and supramolecular assembly of complexes I-IV (Respirasome). Overexpression of Chrm1 in transformed cells (lacking native Chrm1) significantly increased complex V oligomerization and respirasome assembly leading to enhanced respiration. TEM analysis revealed that Chrm1 loss led to mitochondrial ultrastructural defects and alteration in the tinctorial properties of cortical neurons causing a significant increase in the abundance of dark cortical neurons (Chrm1-/- 85% versus wild-type 2%). Discussion: Our findings indicate a hitherto unknown effect of Chrm1 deletion in cortical neurons affecting mitochondrial function by altering multiple interdependent factors including ATP synthase oligomerization, respirasome assembly, and mitochondrial ultrastructure. The appearance of dark neurons in Chrm1-/- cortices implies potentially enhanced glutamatergic signaling in pyramidal neurons under Chrm1 loss condition. The findings provide novel mechanistic insights into Chrm1 loss with the appearance of mitochondrial pathophysiological deficits in Alzheimer's disease.

The metabolic state of the heart regulates mitochondrial supercomplex abundance in mice

Mitochondrial supercomplexes are observed in mammalian tissues with high energy demand and may influence metabolism and redox signaling. Nevertheless, the mechanisms that regulate supercomplex abundance remain unclear. In this study, we examined the composition of supercomplexes derived from murine cardiac mitochondria and determined how their abundance changes with substrate provision or by genetically induced changes to the cardiac glucose-fatty acid cycle. Protein complexes from digitonin-solubilized cardiac mitochondria were resolved by blue-native polyacrylamide gel electrophoresis and were identified by mass spectrometry and immunoblotting to contain constituents of Complexes I, III, IV, and V as well as accessory proteins involved in supercomplex assembly and stability, cristae architecture, carbohydrate and fat oxidation, and oxidant detoxification. Respiratory analysis of high molecular mass supercomplexes confirmed the presence of intact respirasomes, capable of transferring electrons from NADH to O2. Provision of respiratory substrates to isolated mitochondria augmented supercomplex abundance, with fatty acyl substrate (octanoylcarnitine) promoting higher supercomplex abundance than carbohydrate-derived substrate (pyruvate). Mitochondria isolated from transgenic hearts that express kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (GlycoLo), which decreases glucose utilization and increases reliance on fatty acid oxidation for energy, had higher mitochondrial supercomplex abundance and activity compared with mitochondria from wild-type or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-expressing hearts (GlycoHi), the latter of which encourages reliance on glucose catabolism for energy. These findings indicate that high energetic reliance on fatty acid catabolism bolsters levels of mitochondrial supercomplexes, supporting the idea that the energetic state of the heart is regulatory factor in supercomplex assembly or stability.

A FRET-based respirasome assembly screen identifies spleen tyrosine kinase as a target to improve muscle mitochondrial respiration and exercise performance in mice

Aerobic muscle activities predominantly depend on fuel energy supply by mitochondrial respiration, thus, mitochondrial activity enhancement may become a therapeutic intervention for muscle disturbances. The assembly of mitochondrial respiratory complexes into higher-order “supercomplex” structures has been proposed to be an efficient biological process for energy synthesis, although there is controversy in its physiological relevance. We here established Förster resonance energy transfer (FRET) phenomenon-based live imaging of mitochondrial respiratory complexes I and IV interactions using murine myoblastic cells, whose signals represent in vivo supercomplex assembly of complexes I, III, and IV, or respirasomes. The live FRET signals were well correlated with supercomplex assembly observed by blue native polyacrylamide gel electrophoresis (BN-PAGE) and oxygen consumption rates. FRET-based live cell screen defined that the inhibition of spleen tyrosine kinase (SYK), a non-receptor protein tyrosine kinase that belongs to the SYK/ zeta-chain-associated protein kinase 70 (ZAP-70) family, leads to an increase in supercomplex assembly in murine myoblastic cells. In parallel, SYK inhibition enhanced mitochondrial respiration in the cells. Notably, SYK inhibitor administration enhances exercise performance in mice. Overall, this study proves the feasibility of FRET-based respirasome assembly assay, which recapitulates in vivo mitochondrial respiration activities.

Assessment of NLRP3 Inflammasome Activation and NLRP3-NEK7 Complex Assembly.

The NLRP3 inflammasome is a critical component of innate immunity that activates caspase-1 to induce inflammation in response to a wide spectrum of endogenous and exogenous stimuli. NLRP3 inflammasome activation has been shown by assays for the cleavage of caspase-1 and gasdermin D, the maturation of IL-1β and IL-18, and ASC speck formation in innate immune cells such as macrophages and monocytes. Recently, NEK7 has been revealed as an essential regulator for NLRP3 inflammasome activation by forming high-molecular-weight complexes with NLRP3. Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to study multi-protein complexes in many experimental systems. Here, we provide a detailed protocol to detect NLRP3 inflammasome activation and NLRP3-NEK7 complex assembly in mouse macrophages by Western blot and BN-PAGE.

Functional Characterization of 12 Dihydropyrimidinase Allelic Variants in Japanese Individuals for the Prediction of 5-Fluorouracil Treatment-Related Toxicity.

The drug 5-fluorouracil (5-FU) is the first-choice chemotherapeutic agent against advanced-stage cancers. However, 10% to 30% of treated patients experience grade 3 to 4 toxicity. The deficiency of dihydropyrimidinase (DHPase), which catalyzes the second step of the 5-FU degradation pathway, is correlated with the risk of developing toxicity. Thus, genetic polymorphisms within DPYS, the DHPase-encoding gene, could potentially serve as predictors of severe 5-FU-related toxicity. We identified 12 novel DPYS variants in 3554 Japanese individuals, but the effects of these mutations on function remain unknown. In the current study, we performed in vitro enzymatic analyses of the 12 newly identified DHPase variants. Dihydrouracil or dihydro-5-FU hydrolytic ring-opening kinetic parameters, Km and Vmax , and intrinsic clearance (CLint = Vmax /Km ) of the wild-type DHPase and eight variants were measured. Five of these variants (R118Q, H295R, T418I, Y448H, and T513A) showed significantly reduced CLint compared with that in the wild-type. The parameters for the remaining four variants (V59F, D81H, T136M, and R490H) could not be determined as dihydrouracil and dihydro-5-FU hydrolytic ring-opening activity was undetectable. We also determined DHPase variant protein stability using cycloheximide and bortezomib. The mechanism underlying the observed changes in the kinetic parameters was clarified using blue-native polyacrylamide gel electrophoresis and three-dimensional structural modeling. The results suggested that the decrease or loss of DHPase enzymatic activity was due to reduced stability and oligomerization of DHPase variant proteins. Our findings support the use of DPYS polymorphisms as novel pharmacogenomic markers for predicting severe 5-FU-related toxicity in the Japanese population. SIGNIFICANCE STATEMENT: DHPase contributes to the degradation of 5-fluorouracil, and genetic polymorphisms that cause decreased activity of DHPase can cause severe toxicity. In this study, we performed functional analysis of 12 DHPase variants in the Japanese population and identified 9 genetic polymorphisms that cause reduced DHPase function. In addition, we found that the ability to oligomerize and the conformation of the active site are important for the enzymatic activity of DHPase.

Two-Dimensional Blue Native/SDS Polyacrylamide Gel Electrophoresis for Analysis of Brazilian Bothrops Snake Venoms.

Viperidae snakes are the most important agents of snakebites in Brazil. The protein composition of snake venoms has been frequently analyzed by means of electrophoretic techniques, but the interaction of proteins in venoms has barely been addressed. An electrophoretic technique that has gained prominence to study this type of interaction is blue native polyacrylamide gel electrophoresis (BN-PAGE), which allows for the high-resolution separation of proteins in their native form. These protein complexes can be further discriminated by a second-dimension gel electrophoresis (SDS-PAGE) from lanes cut from BN-PAGE. Once there is no study on the use of bidimensional BN/SDS-PAGE with snake venoms, this study initially standardized the BN/SDS-PAGE technique in order to evaluate protein interactions in Bothrops atrox, Bothrops erythromelas, and Bothrops jararaca snake venoms. Results of BN/SDS-PAGE showed that native protein complexes were present, and that snake venom metalloproteinases and venom serine proteinases maintained their enzymatic activity after BN/SDS-PAGE. C-type lectin-like proteins were identified by Western blotting. Therefore, bidimensional BN/SDS-PAGE proved to be an easy, practical, and efficient method for separating functional venom proteins according to their assemblage in complexes, as well as to analyze their biological activities in further details.

AQP4-A25Q Point Mutation in Mice Depolymerizes Orthogonal Arrays of Particles and Decreases Polarized Expression of AQP4 Protein in Astrocytic Endfeet at the Blood-Brain Barrier.

Aquaporin-4 (AQP4) is characterized by the formation of orthogonal arrays of particles (OAPs) comprising its M1 and M23 isoforms in the plasma membrane. However, the biological importance of OAP formation is obscure. Here, we developed an OAP depolymerization male mouse model by transgenic knock-in of an AQP4-A25Q mutation. Analyses of the mutant brain tissue using blue native polyacrylamide gel electrophoresis, super-resolution imaging, and immunogold electron microscopy revealed remarkably reduced OAP structures and glial endfeet localization of the AQP4-A25Q mutant protein without effects on its overall mRNA and protein expression. AQP4A25Q/A25Q mice showed better survival and neurologic deficit scores when cerebral edema was induced by water intoxication or middle cerebral artery occlusion/reperfusion. The brain water content and swelling of pericapillary astrocytic endfeet processes in AQP4A25Q/A25Q mice were significantly reduced, functionally supporting decreased AQP4 protein expression at the blood-brain barrier. The infarct volume and neuronal damage were also reduced in AQP4A25Q/A25Q mice in the middle cerebral artery occlusion/reperfusion model. Astrocyte activation in the brain was alleviated in AQP4A25Q/A25Q mice, which may be associated with decreased cell swelling. We conclude that the OAP structure of AQP4 plays a key role in its polarized expression in astrocytic endfeet processes at the blood-brain barrier. Therefore, our study provided new insights into intervention of cerebral cellular edema caused by stroke and traumatic brain injury through regulating AQP4 OAP formation.SIGNIFICANCE STATEMENT Aquaporin-4 (AQP4) is characterized by orthogonal arrays of particles (OAPs) comprising the M1 and M23 isoforms in the membrane. Here, an OAP depolymerization male mouse model induced by AQP4-A25Q mutation was first established, and the functions of OAP depolymerization in cerebral edema have been studied. The results revealed that AQP4 lost its OAP structure without affecting AQP4 mRNA and protein levels in AQP4-A25Q mice. AQP4-A25Q mutation mice has neuroprotective effects on cerebral edema induced by water intoxication and middle cerebral artery occlusion/reperfusion through relieving the activation of astrocytes and suppressed microglia-mediated neuroinflammation. We concluded that the OAP structure of AQP4 plays a key role in its polarized expression in astrocytic endfeet processes at the blood-brain barrier. Therefore, our study provided new insights into intervention of cerebral cellular edema caused by stroke and traumatic brain injury through regulating AQP4 OAP formation.

The photosynthesis apparatus of European mistletoe (Viscum album).

European mistletoe (Viscum album) is known for its special mode of cellular respiration. It lacks the mitochondrial NADH dehydrogenase complex (Complex I of the respiratory chain) and has restricted capacities to generate mitochondrial adenosine triphosphate (ATP). Here, we present an investigation of the V. album energy metabolism taking place in chloroplasts. Thylakoids were purified from young V. album leaves, and membrane-bound protein complexes were characterized by Blue native polyacrylamide gel electrophoresis as well as by the complexome profiling approach. Proteins were systematically identified by label-free quantitative shotgun proteomics. We identified >1,800 distinct proteins (accessible at https://complexomemap.de/va_leaves), including nearly 100 proteins forming part of the protein complexes involved in the light-dependent part of photosynthesis. The photosynthesis apparatus of V. album has distinct features: (1) comparatively low amounts of Photosystem I; (2) absence of the NDH complex (the chloroplast pendant of mitochondrial Complex I involved in cyclic electron transport (CET) around Photosystem I); (3) reduced levels of the proton gradient regulation 5 (PGR5) and proton gradient regulation 5-like 1 (PGRL1) proteins, which offer an alternative route for CET around Photosystem I; (4) comparable amounts of Photosystem II and the chloroplast ATP synthase complex to other seed plants. Our data suggest a restricted capacity for chloroplast ATP biosynthesis by the photophosphorylation process. This is in addition to the limited ATP supply by the mitochondria. We propose a view on mistletoe's mode of life, according to which its metabolism relies to a greater extent on energy-rich compounds provided by the host trees.