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International Journal of Molecular... Nov 2022Mitochondrial oxidative phospho rylation, the center of cellular metabolism, is pivotal for the energy production in eukaryotes. Mitochondrial oxidative phosphorylation... (Review)
Review
Mitochondrial oxidative phospho rylation, the center of cellular metabolism, is pivotal for the energy production in eukaryotes. Mitochondrial oxidative phosphorylation relies on the mitochondrial respiratory chain, which consists of four main enzyme complexes and two mobile electron carriers. Mitochondrial enzyme complexes also assemble into respiratory chain supercomplexes (SCs) through specific interactions. The SCs not only have respiratory functions but also improve the efficiency of electron transfer and reduce the production of reactive oxygen species (ROS). Impaired assembly of SCs is closely related to various diseases, especially neurodegenerative diseases. Therefore, SCs play important roles in improving the efficiency of the mitochondrial respiratory chain, as well as maintaining the homeostasis of cellular metabolism. Here, we review the structure, assembly, and functions of SCs, as well as the relationship between mitochondrial SCs and diseases.
Topics: Electron Transport; Mitochondrial Membranes; Mitochondria; Reactive Oxygen Species; Oxidative Phosphorylation; Multienzyme Complexes
PubMed: 36430359
DOI: 10.3390/ijms232213880 -
Nature Jun 2022Chromosome replication is performed by a complex and intricate ensemble of proteins termed the replisome, where the DNA polymerases Polδ and Polε, DNA polymerase...
Chromosome replication is performed by a complex and intricate ensemble of proteins termed the replisome, where the DNA polymerases Polδ and Polε, DNA polymerase α-primase (Polα) and accessory proteins including AND-1, CLASPIN and TIMELESS-TIPIN (respectively known as Ctf4, Mrc1 and Tof1-Csm3 in Saccharomyces cerevisiae) are organized around the CDC45-MCM-GINS (CMG) replicative helicase. Because a functional human replisome has not been reconstituted from purified proteins, how these factors contribute to human DNA replication and whether additional proteins are required for optimal DNA synthesis are poorly understood. Here we report the biochemical reconstitution of human replisomes that perform fast and efficient DNA replication using 11 purified human replication factors made from 43 polypeptides. Polε, but not Polδ, is crucial for optimal leading-strand synthesis. Unexpectedly, Polε-mediated leading-strand replication is highly dependent on the sliding-clamp processivity factor PCNA and the alternative clamp loader complex CTF18-RFC. We show how CLASPIN and TIMELESS-TIPIN contribute to replisome progression and demonstrate that, in contrast to the budding yeast replisome, AND-1 directly augments leading-strand replication. Moreover, although AND-1 binds to Polα, the interaction is dispensable for lagging-strand replication, indicating that Polα is functionally recruited via an AND-1-independent mechanism for priming in the human replisome. Collectively, our work reveals how the human replisome achieves fast and efficient leading-strand and lagging-strand DNA replication, and provides a powerful system for future studies of the human replisome and its interactions with other DNA metabolic processes.
Topics: DNA; DNA Helicases; DNA Replication; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Humans; Multienzyme Complexes; Time Factors
PubMed: 35585232
DOI: 10.1038/s41586-022-04759-1 -
Biochimica Et Biophysica Acta. Gene... Feb 2021Histone post-translational modifications are essential for the regulation of gene expression in eukaryotes. Gcn5 (KAT2A) is a histone acetyltransferase that catalyzes... (Review)
Review
Histone post-translational modifications are essential for the regulation of gene expression in eukaryotes. Gcn5 (KAT2A) is a histone acetyltransferase that catalyzes the post-translational modification at multiple positions of histone H3 through the transfer of acetyl groups to the free amino group of lysine residues. Gcn5 catalyzes histone acetylation in the context of a HAT module containing the Ada2, Ada3 and Sgf29 subunits of the parent megadalton SAGA transcriptional coactivator complex. Biochemical and structural studies have elucidated mechanisms for Gcn5's acetyl- and other acyltransferase activities on histone substrates, for histone H3 phosphorylation and histone H3 methylation crosstalks with histone H3 acetylation, and for how Ada2 increases Gcn5's histone acetyltransferase activity. Other studies have identified Ada2 isoforms in SAGA-related complexes and characterized variant Gcn5 HAT modules containing these Ada2 isoforms. In this review, we highlight biochemical and structural studies of Gcn5 and its functional interactions with Ada2, Ada3 and Sgf29.
Topics: Acetylation; Cryoelectron Microscopy; Histone Acetyltransferases; Histones; Isoenzymes; Methylation; Multienzyme Complexes; Phosphorylation; Protein Processing, Post-Translational; Saccharomyces cerevisiae Proteins; Transcription Factors; p300-CBP Transcription Factors
PubMed: 32890768
DOI: 10.1016/j.bbagrm.2020.194629 -
Protein Science : a Publication of the... Apr 2017The SET1 family of proteins, and in particular MLL1, are essential regulators of transcription and key mediators of normal development and disease. Here, we summarize... (Review)
Review
The SET1 family of proteins, and in particular MLL1, are essential regulators of transcription and key mediators of normal development and disease. Here, we summarize the detailed characterization of the methyltransferase activity of SET1 complexes and the role of the key subunits, WDR5, RbBP5, ASH2L, and DPY30. We present new data on full kinetic characterization of human MLL1, MLL3, SET1A, and SET1B trimeric, tetrameric, and pentameric complexes to elaborate on substrate specificities and compare our findings with what has been reported before. We also review exciting recent work identifying potent inhibitors of oncogenic MLL1 function through disruption of protein-protein interactions within the MLL1 complex.
Topics: Enzyme Inhibitors; Histone-Lysine N-Methyltransferase; Humans; Multienzyme Complexes; Myeloid-Lymphoid Leukemia Protein
PubMed: 28160335
DOI: 10.1002/pro.3129 -
Proceedings of the National Academy of... Mar 2022SignificanceThe use of biological enzyme catalysts could have huge ramifications for chemical industries. However, these enzymes are often inactive in nonbiological...
SignificanceThe use of biological enzyme catalysts could have huge ramifications for chemical industries. However, these enzymes are often inactive in nonbiological conditions, such as high temperatures, present in industrial settings. Here, we show that the enzyme PETase (polyethylene terephthalate [PET]), with potential application in plastic recycling, is stabilized at elevated temperature through complexation with random copolymers. We demonstrate this through simulations and experiments on different types of substrates. Our simulations also provide strategies for designing more enzymatically active complexes by altering polymer composition and enzyme charge distribution.
Topics: Hydrolases; Multienzyme Complexes; Plastics; Polyethylene Terephthalates; Polymers; Recycling
PubMed: 35312375
DOI: 10.1073/pnas.2119509119 -
PLoS Biology Dec 2019Peptide-based intercellular communication is a ubiquitous and ancient process that predates evolution of the nervous system. Cilia are essential signaling centers that...
Peptide-based intercellular communication is a ubiquitous and ancient process that predates evolution of the nervous system. Cilia are essential signaling centers that both receive information from the environment and secrete bioactive extracellular vesicles (ectosomes). However, the nature of these secreted signals and their biological functions remain poorly understood. Here, we report the developmentally regulated release of the peptide amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), and the presence of peptidergic signaling machinery (including propeptide precursors, subtilisin-like prohormone convertases, amidated products, and receptors) in ciliary ectosomes from the green alga Chlamydomonas. One identified amidated PAM product serves as a chemoattractant for mating-type minus gametes but repels plus gametes. Thus, cilia provide a previously unappreciated route for the secretion of amidated signaling peptides. Our study in Chlamydomonas and the presence of PAM in mammalian cilia suggest that ciliary ectosome-mediated peptidergic signaling dates to the early eukaryotes and plays key roles in metazoan physiology.
Topics: Cell Communication; Cell-Derived Microparticles; Chlamydomonas; Chlorophyta; Cilia; Mixed Function Oxygenases; Multienzyme Complexes; Peptides; Signal Transduction
PubMed: 31809498
DOI: 10.1371/journal.pbio.3000566 -
Science (New York, N.Y.) Apr 2016A decline in electron transport chain (ETC) activity is associated with many human diseases. Although diminished mitochondrial adenosine triphosphate production is...
A decline in electron transport chain (ETC) activity is associated with many human diseases. Although diminished mitochondrial adenosine triphosphate production is recognized as a source of pathology, the contribution of the associated reduction in the ratio of the amount of oxidized nicotinamide adenine dinucleotide (NAD(+)) to that of its reduced form (NADH) is less clear. We used a water-forming NADH oxidase from Lactobacillus brevis (LbNOX) as a genetic tool for inducing a compartment-specific increase of the NAD(+)/NADH ratio in human cells. We used LbNOX to demonstrate the dependence of key metabolic fluxes, gluconeogenesis, and signaling on the cytosolic or mitochondrial NAD(+)/NADH ratios. Expression of LbNOX in the cytosol or mitochondria ameliorated proliferative and metabolic defects caused by an impaired ETC. The results underscore the role of reductive stress in mitochondrial pathogenesis and demonstrate the utility of targeted LbNOX for direct, compartment-specific manipulation of redox state.
Topics: Catalysis; Cytosol; Electron Transport; Electron Transport Chain Complex Proteins; Genetic Complementation Test; Gluconeogenesis; HeLa Cells; Humans; Levilactobacillus brevis; Mitochondria; Mitochondrial Diseases; Multienzyme Complexes; NAD; NADH, NADPH Oxidoreductases; Oxidation-Reduction
PubMed: 27124460
DOI: 10.1126/science.aad4017 -
Cell Apr 2017A complex interplay of environmental factors impacts the metabolism of human cells, but neither traditional culture media nor mouse plasma mimic the metabolite...
A complex interplay of environmental factors impacts the metabolism of human cells, but neither traditional culture media nor mouse plasma mimic the metabolite composition of human plasma. Here, we developed a culture medium with polar metabolite concentrations comparable to those of human plasma (human plasma-like medium [HPLM]). Culture in HPLM, relative to that in traditional media, had widespread effects on cellular metabolism, including on the metabolome, redox state, and glucose utilization. Among the most prominent was an inhibition of de novo pyrimidine synthesis-an effect traced to uric acid, which is 10-fold higher in the blood of humans than of mice and other non-primates. We find that uric acid directly inhibits uridine monophosphate synthase (UMPS) and consequently reduces the sensitivity of cancer cells to the chemotherapeutic agent 5-fluorouracil. Thus, media that better recapitulates the composition of human plasma reveals unforeseen metabolic wiring and regulation, suggesting that HPLM should be of broad utility.
Topics: Aged; Animals; Cell Culture Techniques; Cell Line, Tumor; Culture Media; Fluorouracil; Glucose; Humans; Leukemia, Myeloid, Acute; Male; Mice; Middle Aged; Multienzyme Complexes; Orotate Phosphoribosyltransferase; Orotidine-5'-Phosphate Decarboxylase; Protein Domains; Pyrimidines; Uric Acid
PubMed: 28388410
DOI: 10.1016/j.cell.2017.03.023 -
The EMBO Journal Aug 2016Cells contain numerous, molecularly distinct cellular compartments that are not enclosed by lipid bilayers. These compartments are implicated in a wide range of cellular... (Review)
Review
Cells contain numerous, molecularly distinct cellular compartments that are not enclosed by lipid bilayers. These compartments are implicated in a wide range of cellular activities, and they have been variously described as bodies, granules, or organelles. Recent evidence suggests that a liquid-liquid phase separation (LLPS) process may drive their formation, possibly justifying the unifying term "droplet organelle". A veritable deluge of recent publications points to the importance of low-complexity proteins and RNA in determining the physical properties of phase-separated structures. Many of the proteins linked to such structures are implicated in human diseases, such as amyotrophic lateral sclerosis (ALS). We provide an overview of the organizational principles that characterize putative "droplet organelles" in healthy and diseased cells, connecting protein biochemistry with cell physiology.
Topics: Cell Physiological Phenomena; Cytosol; Macromolecular Substances; Multienzyme Complexes
PubMed: 27357569
DOI: 10.15252/embj.201593517 -
Nature Nov 2022The SEA complex (SEAC) is a growth regulator that acts as a GTPase-activating protein (GAP) towards Gtr1, a Rag GTPase that relays nutrient status to the Target of...
The SEA complex (SEAC) is a growth regulator that acts as a GTPase-activating protein (GAP) towards Gtr1, a Rag GTPase that relays nutrient status to the Target of Rapamycin Complex 1 (TORC1) in yeast. Functionally, the SEAC has been divided into two subcomplexes: SEACIT, which has GAP activity and inhibits TORC1, and SEACAT, which regulates SEACIT. This system is conserved in mammals: the GATOR complex, consisting of GATOR1 (SEACIT) and GATOR2 (SEACAT), transmits amino acid and glucose signals to mTORC1. Despite its importance, the structure of SEAC/GATOR, and thus molecular understanding of its function, is lacking. Here, we solve the cryo-EM structure of the native eight-subunit SEAC. The SEAC has a modular structure in which a COPII-like cage corresponding to SEACAT binds two flexible wings, which correspond to SEACIT. The wings are tethered to the core via Sea3, which forms part of both modules. The GAP mechanism of GATOR1 is conserved in SEACIT, and GAP activity is unaffected by SEACAT in vitro. In vivo, the wings are essential for recruitment of the SEAC to the vacuole, primarily via the EGO complex. Our results indicate that rather than being a direct inhibitor of SEACIT, SEACAT acts as a scaffold for the binding of TORC1 regulators.
Topics: Animals; Cryoelectron Microscopy; GTP Phosphohydrolases; GTPase-Activating Proteins; Mammals; Mechanistic Target of Rapamycin Complex 1; Multienzyme Complexes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Protein Subunits; Amino Acids; Glucose; COP-Coated Vesicles
PubMed: 36289347
DOI: 10.1038/s41586-022-05370-0