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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 -
Critical Reviews in Biochemistry and... Feb 2021The focus of this review is the human de novo purine biosynthetic pathway. The pathway enzymes are enumerated, as well as the reactions they catalyze and their physical... (Review)
Review
The focus of this review is the human de novo purine biosynthetic pathway. The pathway enzymes are enumerated, as well as the reactions they catalyze and their physical properties. Early literature evidence suggested that they might assemble into a multi-enzyme complex called a metabolon. The finding that fluorescently-tagged chimeras of the pathway enzymes form discrete puncta, now called purinosomes, is further elaborated in this review to include: a discussion of their assembly; the role of ancillary proteins; their locus at the microtubule/mitochondria interface; the elucidation that at endogenous levels, purinosomes function to channel intermediates from phosphoribosyl pyrophosphate to AMP and GMP; and the evidence for the purinosomes to exist as a protein condensate. The review concludes with a consideration of probable signaling pathways that might promote the assembly and disassembly of the purinosome, in particular the identification of candidate kinases given the extensive phosphorylation of the enzymes. These collective findings substantiate our current view of the de novo purine biosynthetic metabolon whose properties will be representative of how other metabolic pathways might be organized for their function.
Topics: Adenosine Monophosphate; Biosynthetic Pathways; Cyclic AMP; Cyclic GMP; Guanosine Monophosphate; Humans; Microtubules; Mitochondria; Multienzyme Complexes; Phosphoribosyl Pyrophosphate; Phosphorylation; Proteins; Purines; Signal Transduction
PubMed: 33179964
DOI: 10.1080/10409238.2020.1832438 -
Science (New York, N.Y.) Nov 2020The 14-subunit metazoan-specific Integrator contains an endonuclease that cleaves nascent RNA transcripts. Here, we identified a complex containing Integrator and...
The 14-subunit metazoan-specific Integrator contains an endonuclease that cleaves nascent RNA transcripts. Here, we identified a complex containing Integrator and protein phosphatase 2A core enzyme (PP2A-AC), termed INTAC. The 3.5-angstrom-resolution structure reveals that nine human Integrator subunits and PP2A-AC assemble into a cruciform-shaped central scaffold formed by the backbone and shoulder modules, with the phosphatase and endonuclease modules flanking the opposite sides. As a noncanonical PP2A holoenzyme, the INTAC complex dephosphorylates the carboxy-terminal repeat domain of RNA polymerase II at serine-2, -5, and -7 and thus regulates transcription. Our study extends the function of PP2A to transcriptional regulation and reveals how dual enzymatic activities-RNA cleavage and RNA polymerase II dephosphorylation-are structurally and functionally integrated into the INTAC complex.
Topics: Chromatin; Cryoelectron Microscopy; Holoenzymes; Humans; Multienzyme Complexes; Protein Domains; Protein Phosphatase 2; RNA Polymerase II
PubMed: 33243860
DOI: 10.1126/science.abb5872 -
Nature Sep 2020The mitochondrial electron transport chain (ETC) is necessary for tumour growth and its inhibition has demonstrated anti-tumour efficacy in combination with targeted...
The mitochondrial electron transport chain (ETC) is necessary for tumour growth and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies. Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria. However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP-that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)-an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX), which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX) targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity.
Topics: Animals; Cell Line, Tumor; Cell Proliferation; Ciona intestinalis; Citric Acid Cycle; Cytosol; Dihydroorotate Dehydrogenase; Electron Transport; Electron Transport Complex I; Electron Transport Complex II; Electron Transport Complex III; Humans; Levilactobacillus brevis; Male; Mice; Mitochondria; Mitochondrial Proteins; Multienzyme Complexes; NAD; NADH, NADPH Oxidoreductases; Neoplasms; Oxidative Phosphorylation; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Plant Proteins; Ubiquinone
PubMed: 32641834
DOI: 10.1038/s41586-020-2475-6 -
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 -
Biomacromolecules Jun 2020Liquid-liquid phase separation forms condensates that feature a highly concentrated liquid phase, a defined yet dynamic boundary, and dynamic exchange at and across the...
Liquid-liquid phase separation forms condensates that feature a highly concentrated liquid phase, a defined yet dynamic boundary, and dynamic exchange at and across the boundary. Phase transition drives the formation of dynamic multienzyme complexes in cells, for example, the purinosome, which forms subcellular macrobodies responsible for purine biosynthesis. Here, we construct synthetic versions of multienzyme biosynthetic systems by assembling enzymes in protein condensates. A synthetic protein phase separation system using component proteins from postsynaptic density in neuronal synapses, GKAP, Shank, and Homer provides the scaffold for assembly. Three sets of guest proteins: a pair of fluorescent proteins (CFP and YFP), three sequential enzymes in menaquinone biosynthesis pathway (MenF, MenD, and MenH), and two enzymes in terpene biosynthesis pathway (Idi and IspA) are assembled via peptide-peptide interactions in the condensate. First, we discover that coassembly of CFP and YFP exhibited a broad distribution of the FRET signal within the condensate. Second, a spontaneous enrichment of the rate-limiting enzyme MenD in the condensate is sufficient to increase the 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate production rate by 70%. Third, coassembly of both Idi and IspA in the protein condensate increases the farnesyl pyrophosphate production rate by more than 50%. Altogether, we show here that phase separation significantly accelerates the efficiency of multienzyme biocatalysis.
Topics: Biocatalysis; Multienzyme Complexes; Phase Transition; Proteins
PubMed: 32343131
DOI: 10.1021/acs.biomac.0c00321 -
The Journal of Clinical Investigation Apr 2023After androgen deprivation, prostate cancer frequently becomes castration resistant (CRPC), with intratumoral androgen production from extragonadal precursors that...
After androgen deprivation, prostate cancer frequently becomes castration resistant (CRPC), with intratumoral androgen production from extragonadal precursors that activate the androgen receptor pathway. 3β-Hydroxysteroid dehydrogenase-1 (3βHSD1) is the rate-limiting enzyme for extragonadal androgen synthesis, which together lead to CRPC. Here, we show that cancer-associated fibroblasts (CAFs) increased epithelial 3βHSD1 expression, induced androgen synthesis, activated the androgen receptor, and induced CRPC. Unbiased metabolomics revealed that CAF-secreted glucosamine specifically induced 3βHSD1. CAFs induced higher GlcNAcylation in cancer cells and elevated expression of the transcription factor Elk1, which induced higher 3βHSD1 expression and activity. Elk1 genetic ablation in cancer epithelial cells suppressed CAF-induced androgen biosynthesis in vivo. In patient samples, multiplex fluorescent imaging showed that tumor cells expressed more 3βHSD1 and Elk1 in CAF-enriched areas compared with CAF-deficient areas. Our findings suggest that CAF-secreted glucosamine increases GlcNAcylation in prostate cancer cells, promoting Elk1-induced HSD3B1 transcription, which upregulates de novo intratumoral androgen synthesis to overcome castration.
Topics: Male; Humans; Prostatic Neoplasms; Androgens; Receptors, Androgen; Prostatic Neoplasms, Castration-Resistant; Androgen Antagonists; Up-Regulation; Glucosamine; Cancer-Associated Fibroblasts; Multienzyme Complexes; Cell Line, Tumor
PubMed: 37009898
DOI: 10.1172/JCI161913 -
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 -
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