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Nature Jul 2020The cellular NADH/NAD ratio is fundamental to biochemistry, but the extent to which it reflects versus drives metabolic physiology in vivo is poorly understood. Here we...
The cellular NADH/NAD ratio is fundamental to biochemistry, but the extent to which it reflects versus drives metabolic physiology in vivo is poorly understood. Here we report the in vivo application of Lactobacillus brevis (Lb)NOX, a bacterial water-forming NADH oxidase, to assess the metabolic consequences of directly lowering the hepatic cytosolic NADH/NAD ratio in mice. By combining this genetic tool with metabolomics, we identify circulating α-hydroxybutyrate levels as a robust marker of an elevated hepatic cytosolic NADH/NAD ratio, also known as reductive stress. In humans, elevations in circulating α-hydroxybutyrate levels have previously been associated with impaired glucose tolerance, insulin resistance and mitochondrial disease, and are associated with a common genetic variant in GCKR, which has previously been associated with many seemingly disparate metabolic traits. Using LbNOX, we demonstrate that NADH reductive stress mediates the effects of GCKR variation on many metabolic traits, including circulating triglyceride levels, glucose tolerance and FGF21 levels. Our work identifies an elevated hepatic NADH/NAD ratio as a latent metabolic parameter that is shaped by human genetic variation and contributes causally to key metabolic traits and diseases. Moreover, it underscores the utility of genetic tools such as LbNOX to empower studies of 'causal metabolism'.
Topics: Adaptor Proteins, Signal Transducing; Animals; Cytosol; Disease Models, Animal; Fibroblast Growth Factors; Genetic Variation; Glucose Tolerance Test; Humans; Insulin Resistance; Levilactobacillus brevis; Liver; Male; Mice; Multienzyme Complexes; NAD; NADH, NADPH Oxidoreductases; Oxidation-Reduction; Stress, Physiological; Triglycerides
PubMed: 32461692
DOI: 10.1038/s41586-020-2337-2 -
Chemical Reviews Jul 2022Gases like H, N, CO, and CO are increasingly recognized as critical feedstock in "green" energy conversion and as sources of nitrogen and carbon for the agricultural and... (Review)
Review
Gases like H, N, CO, and CO are increasingly recognized as critical feedstock in "green" energy conversion and as sources of nitrogen and carbon for the agricultural and chemical sectors. However, the industrial transformation of N, CO, and CO and the production of H require significant energy input, which renders processes like steam reforming and the Haber-Bosch reaction economically and environmentally unviable. Nature, on the other hand, performs similar tasks efficiently at ambient temperature and pressure, exploiting gas-processing metalloenzymes (GPMs) that bind low-valent metal cofactors based on iron, nickel, molybdenum, tungsten, and sulfur. Such systems are studied to understand the biocatalytic principles of gas conversion including N fixation by nitrogenase and H production by hydrogenase as well as CO and CO conversion by formate dehydrogenase, carbon monoxide dehydrogenase, and nitrogenase. In this review, we emphasize the importance of the cofactor/protein interface, discussing how second and outer coordination sphere effects determine, modulate, and optimize the catalytic activity of GPMs. These may comprise ionic interactions in the second coordination sphere that shape the electron density distribution across the cofactor, hydrogen bonding changes, and allosteric effects. In the outer coordination sphere, proton transfer and electron transfer are discussed, alongside the role of hydrophobic substrate channels and protein structural changes. Combining the information gained from structural biology, enzyme kinetics, and various spectroscopic techniques, we aim toward a comprehensive understanding of catalysis beyond the first coordination sphere.
Topics: Aldehyde Oxidoreductases; Carbon Dioxide; Formate Dehydrogenases; Hydrogenase; Multienzyme Complexes; Nitrogenase; Oxidation-Reduction
PubMed: 35849738
DOI: 10.1021/acs.chemrev.1c00914 -
Nature Communications Oct 2023Pervasive transcription of the human genome generates an abundance of RNAs that must be processed and degraded. The nuclear RNA exosome is the main RNA degradation...
Pervasive transcription of the human genome generates an abundance of RNAs that must be processed and degraded. The nuclear RNA exosome is the main RNA degradation machinery in the nucleus. However, nuclear exosome must be recruited to its substrates by targeting complexes, such as NEXT or PAXT. By proteomic analysis, we identify additional subunits of PAXT, including many orthologs of MTREC found in S. pombe. In particular, we show that polyA polymerase gamma (PAPγ) associates with PAXT. Genome-wide mapping of the binding sites of ZFC3H1, RBM27 and PAPγ shows that PAXT is recruited to the TSS of hundreds of genes. Loss of ZFC3H1 abolishes recruitment of PAXT subunits including PAPγ to TSSs and concomitantly increases the abundance of PROMPTs at the same sites. Moreover, PAPγ, as well as MTR4 and ZFC3H1, is implicated in the polyadenylation of PROMPTs. Our results thus provide key insights into the direct targeting of PROMPT ncRNAs by PAXT at their genomic sites.
Topics: Humans; Exosome Multienzyme Ribonuclease Complex; Exosomes; Proteomics; RNA; RNA Stability; RNA, Untranslated; Polynucleotide Adenylyltransferase
PubMed: 37875486
DOI: 10.1038/s41467-023-42620-9 -
International Journal of Molecular... Jan 2022Antibody-enzyme complexes (AECs) are ideal molecular recognition elements for immunosensing applications. One molecule possesses both a binding ability to specific... (Review)
Review
Antibody-enzyme complexes (AECs) are ideal molecular recognition elements for immunosensing applications. One molecule possesses both a binding ability to specific targets and catalytic activity to gain signals, particularly oxidoreductases, which can be integrated into rapid and sensitive electrochemical measurements. The development of AECs using fragment antibodies rather than intact antibodies, such as immunoglobulin G (IgG), has attracted attention for overcoming the ethical and cost issues associated with the production of intact antibodies. Conventionally, chemical conjugation has been used to fabricate AECs; however, controlling stoichiometric conjugation using this method is difficult. To prepare homogeneous AECs, methods based on direct fusion and enzymatic conjugation have been developed, and more convenient methods using Catcher/Tag systems as coupling modules have been reported. In this review, we summarize the methods for fabricating AECs using fragment antibodies developed for sensing applications and discuss the advantages and disadvantages of each method.
Topics: Animals; Antibodies; Humans; Immunoassay; Immunoglobulin G; Multienzyme Complexes
PubMed: 35163258
DOI: 10.3390/ijms23031335 -
Biochemistry. Biokhimiia Oct 2022Summarized results of investigation of regulation of electron transport and associated processes in the photosynthetic membrane using methods of mathematical and... (Review)
Review
Summarized results of investigation of regulation of electron transport and associated processes in the photosynthetic membrane using methods of mathematical and computer modeling carried out at the Department of Biophysics, Faculty of Biology, Lomonosov Moscow State University, are presented in this review. Detailed kinetic models of processes in the thylakoid membrane were developed using the apparatus of differential equations. Fitting of the model curves to the data of spectral measurements allowed us to estimate the values of parameters that were not determined directly in experiments. The probabilistic method of agent-based Monte Carlo modeling provides ample opportunities for studying dynamics of heterogeneous systems based on the rules for the behavior of individual elements of the system. Algorithms for simplified representation of Big Data make it possible to monitor changes in the photosynthetic apparatus in the course of culture growth in a photobioreactor and for the purpose of environmental monitoring. Brownian and molecular models describe movement and interaction of individual electron carrier proteins and make it possible to study electrostatic, hydrophobic, and other interactions leading to regulation of conformational changes in the reaction complexes. Direct multiparticle models explicitly simulate Brownian diffusion of the mobile protein carriers and their electrostatic interactions with multienzyme complexes both in solution and in heterogeneous interior of a biomembrane. The combined use of methods of kinetic and Brownian multiparticle and molecular modeling makes it possible to study the mechanisms of regulation of an integral system of electron transport processes in plants and algae at molecular and subcellular levels.
Topics: Humans; Electron Transport; Photosynthesis; Computer Simulation; Plants; Multienzyme Complexes; Carrier Proteins; Models, Biological
PubMed: 36273876
DOI: 10.1134/S0006297922100017 -
Endocrinology Dec 2021Peptides derived from proopiomelanocortin (POMC) are well-established neuropeptides and peptide hormones that perform multiple functions, including regulation of body... (Review)
Review
Peptides derived from proopiomelanocortin (POMC) are well-established neuropeptides and peptide hormones that perform multiple functions, including regulation of body weight. In humans and some animals, these peptides include α- and β-melanocyte-stimulating hormone (MSH). In certain rodent species, no β-MSH is produced from POMC because of a change in the cleavage site. Enzymes that convert POMC into MSH include prohormone convertases (PCs), carboxypeptidases (CPs), and peptidyl-α-amidating monooxygenase (PAM). Humans and mice with inactivating mutations in either PC1/3 or carboxypeptidase E (CPE) are obese, which was assumed to result from defective processing of POMC into MSH. However, recent studies have shown that selective loss of either PC1/3 or CPE in POMC-expressing cells does not cause obesity. These findings suggest that defects in POMC processing cannot alone account for the obesity observed in global PC1/3 or CPE mutants. We propose that obesity in animals lacking PC1/3 or CPE activity depends, at least in part, on deficient processing of peptides in non-POMC-expressing cells either in the brain and/or the periphery. Genetic background may also contribute to the manifestation of obesity.
Topics: Animals; Brain; Carboxypeptidases; Disease Models, Animal; Humans; Mice; Mice, Obese; Mixed Function Oxygenases; Multienzyme Complexes; Obesity; Pro-Opiomelanocortin; Proprotein Convertase 2; Proprotein Convertases
PubMed: 34333593
DOI: 10.1210/endocr/bqab155 -
The Journal of Biological Chemistry Dec 2023Pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH) are vital entry points for monosaccharides and amino acids into the Krebs cycle and thus integral... (Review)
Review
Pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH) are vital entry points for monosaccharides and amino acids into the Krebs cycle and thus integral for mitochondrial bioenergetics. Both complexes produce mitochondrial hydrogen peroxide (mHO) and are deactivated by electrophiles. Here, we provide an update on the role of PDH and KGDH in mitochondrial redox balance and their function in facilitating metabolic reprogramming for the propagation of oxidative eustress signals in hepatocytes and how defects in these pathways can cause liver diseases. PDH and KGDH are known to account for ∼45% of the total mHO formed by mitochondria and display rates of production several-fold higher than the canonical source complex I. This mHO can also be formed by reverse electron transfer (RET) in vivo, which has been linked to metabolic dysfunctions that occur in pathogenesis. However, the controlled emission of mHO from PDH and KGDH has been proposed to be fundamental for oxidative eustress signal propagation in several cellular contexts. Modification of PDH and KGDH with protein S-glutathionylation (PSSG) and S-nitrosylation (PSNO) adducts serves as a feedback inhibitor for mHO production in response to glutathione (GSH) pool oxidation. PSSG and PSNO adduct formation also reprogram the Krebs cycle to generate metabolites vital for interorganelle and intercellular signaling. Defects in the redox modification of PDH and KGDH cause the over generation of mHO, resulting in oxidative distress and metabolic dysfunction-associated fatty liver disease (MAFLD). In aggregate, PDH and KGDH are essential platforms for emitting and receiving oxidative eustress signals.
Topics: Hydrogen Peroxide; Ketoglutarate Dehydrogenase Complex; Oxidation-Reduction; Oxidative Stress; Pyruvate Dehydrogenase Complex; Humans; Hepatocytes; Mitochondria, Liver; Non-alcoholic Fatty Liver Disease; Animals; Mice
PubMed: 37898400
DOI: 10.1016/j.jbc.2023.105399 -
Nature Jun 2022Eukaryotic genomes are compacted into loops and topologically associating domains (TADs), which contribute to transcription, recombination and genomic stability. Cohesin...
Eukaryotic genomes are compacted into loops and topologically associating domains (TADs), which contribute to transcription, recombination and genomic stability. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered. Little is known about whether loop extrusion is impeded by DNA-bound machines. Here we show that the minichromosome maintenance (MCM) complex is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C (high-resolution chromosome conformation capture) of mouse zygotes reveals that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, which suggests that loop extrusion is impeded before reaching CTCF. This effect extends to HCT116 cells, in which MCMs affect the number of CTCF-anchored loops and gene expression. Simulations suggest that MCMs are abundant, randomly positioned and partially permeable barriers. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocation in vitro. Notably, chimeric yeast MCMs that contain a cohesin-interaction motif from human MCM3 induce cohesin pausing, indicating that MCMs are 'active' barriers with binding sites. These findings raise the possibility that cohesin can arrive by loop extrusion at MCMs, which determine the genomic sites at which sister chromatid cohesion is established. On the basis of in vivo, in silico and in vitro data, we conclude that distinct loop extrusion barriers shape the three-dimensional genome.
Topics: Animals; CCCTC-Binding Factor; Cell Cycle Proteins; Chromatids; Chromosomal Proteins, Non-Histone; DNA; G1 Phase; HCT116 Cells; Humans; Mice; Minichromosome Maintenance Complex Component 3; Minichromosome Maintenance Proteins; Multienzyme Complexes; Nucleic Acid Conformation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Cohesins
PubMed: 35585235
DOI: 10.1038/s41586-022-04730-0 -
Biochemical Society Transactions Dec 2021Mitochondria are one of the most exhaustively investigated organelles in the cell and most attention has been paid to the components of the mitochondrial electron... (Review)
Review
Mitochondria are one of the most exhaustively investigated organelles in the cell and most attention has been paid to the components of the mitochondrial electron transport chain (ETC) in the last 100 years. The ETC collects electrons from NADH or FADH2 and transfers them through a series of electron carriers within multiprotein respiratory complexes (complex I to IV) to oxygen, therefore generating an electrochemical gradient that can be used by the F1-F0-ATP synthase (also named complex V) in the mitochondrial inner membrane to synthesize ATP. The organization and function of the ETC is a continuous source of surprises. One of the latest is the discovery that the respiratory complexes can assemble to form a variety of larger structures called super-complexes (SCs). This opened an unexpected level of complexity in this well-known and fundamental biological process. This review will focus on the current evidence for the formation of different SCs and will explore how they modulate the ETC organization according to the metabolic state. Since the field is rapidly growing, we also comment on the experimental techniques used to describe these SC and hope that this overview may inspire new technologies that will help to advance the field.
Topics: Animals; Electron Transport Chain Complex Proteins; Mitochondria; Multienzyme Complexes; Oxidative Phosphorylation
PubMed: 34747989
DOI: 10.1042/BST20210460 -
Neurological Sciences : Official... Nov 2022GNE myopathy is a hereditary muscle disorder characterized by muscle atrophy and weakness initially involving the lower distal extremities. The treatment of GNE myopathy... (Review)
Review
GNE myopathy is a hereditary muscle disorder characterized by muscle atrophy and weakness initially involving the lower distal extremities. The treatment of GNE myopathy mainly focuses on a sialic acid deficiency caused by a mutation in the GNE gene, but it has not achieved the expected effect. The main pathological features of GNE myopathy are myofiber atrophy and rimmed vacuoles, including accumulation of amyloid β, which is mainly found in atrophic muscle fibers. Although the role of amyloid β and other misfolded proteins on the nervous system has been widely recognized, the cause and process of the formation of amyloid β in the pathological process of GNE myopathy are unclear. In addition, amyloid β has been reported to be linked to quality control mechanisms of proteins, such as molecular chaperones, the ubiquitin-proteasome system, and the autophagy-lysosome system. Herein, we summarize the possible reasons for amyloid β deposition and illustrate amyloid β-mediated events in the cells and their role in muscle atrophy in GNE myopathy. This review represents an overview of amyloid β and GNE myopathy that could help identify a potential mechanism and thereby a plausible therapeutic for the disease.
Topics: Humans; Amyloid beta-Peptides; Multienzyme Complexes; Distal Myopathies; Muscular Diseases; Mutation; Muscular Atrophy; Muscle, Skeletal
PubMed: 35904705
DOI: 10.1007/s10072-022-06301-7