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Nature Oct 2017Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin...
Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.
Topics: Animals; Apoenzymes; Cell Line, Tumor; Cell Proliferation; Crystallography, X-Ray; Female; Humans; Mice; Models, Molecular; Neoplasms; Piperidines; Proto-Oncogene Proteins c-mdm2; Pyrazoles; Pyrimidines; Substrate Specificity; Transcription, Genetic; Tumor Suppressor Protein p53; Ubiquitin-Specific Peptidase 7; Ubiquitination; Xenograft Model Antitumor Assays
PubMed: 29045389
DOI: 10.1038/nature24451 -
Nature Communications Sep 2022Schlafen 11 (SLFN11) is an interferon-inducible antiviral restriction factor with tRNA endoribonuclease and DNA binding functions. It is recruited to stalled replication...
Schlafen 11 (SLFN11) is an interferon-inducible antiviral restriction factor with tRNA endoribonuclease and DNA binding functions. It is recruited to stalled replication forks in response to replication stress and inhibits replication of certain viruses such as the human immunodeficiency virus 1 (HIV-1) by modulating the tRNA pool. SLFN11 has been identified as a predictive biomarker in cancer, as its expression correlates with a beneficial response to DNA damage inducing anticancer drugs. However, the mechanism and interdependence of these two functions are largely unknown. Here, we present cryo-electron microscopy (cryo-EM) structures of human SLFN11 in its dimeric apoenzyme state, bound to tRNA and in complex with single-strand DNA. Full-length SLFN11 neither hydrolyses nor binds ATP and the helicase domain appears in an autoinhibited state. Together with biochemical and structure guided mutagenesis studies, our data give detailed insights into the mechanism of endoribonuclease activity as well as suggestions on how SLFN11 may block stressed replication forks.
Topics: Adenosine Triphosphate; Antineoplastic Agents; Antiviral Agents; Apoenzymes; Cryoelectron Microscopy; DNA; Endoribonucleases; Humans; Interferons; Nuclear Proteins; RNA, Transfer
PubMed: 36115853
DOI: 10.1038/s41467-022-33123-0 -
Nature Aug 2021Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution. However, cryo-EM studies...
Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution. However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure-function relationships, but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1 Å. Newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure-function relationships in ribozymes.
Topics: Apoenzymes; Cryoelectron Microscopy; Holoenzymes; Models, Molecular; Nucleic Acid Conformation; RNA, Catalytic; Tetrahymena thermophila
PubMed: 34381213
DOI: 10.1038/s41586-021-03803-w -
International Journal of Molecular... Jan 2021Ferroptosis has been described recently as an iron-dependent cell death driven by peroxidation of membrane lipids. It is involved in the pathogenesis of a number of... (Review)
Review
Ferroptosis has been described recently as an iron-dependent cell death driven by peroxidation of membrane lipids. It is involved in the pathogenesis of a number of diverse diseases. From the other side, the induction of ferroptosis can be used to kill tumor cells as a novel therapeutic approach. Because of the broad clinical relevance, a comprehensive understanding of the ferroptosis-controlling protein network is necessary. Noteworthy, several proteins from this network are flavoenzymes. This review is an attempt to present the ferroptosis-related flavoproteins in light of their involvement in anti-ferroptotic and pro-ferroptotic roles. When available, the data on the structural stability of mutants and cofactor-free apoenzymes are discussed. The stability of the flavoproteins could be an important component of the cellular death processes.
Topics: Animals; Ferroptosis; Flavoproteins; Humans; Iron; Protein Stability
PubMed: 33406703
DOI: 10.3390/ijms22010430 -
International Journal of Tryptophan... 2022Major species differences in tryptophan (Trp) metabolism and disposition exist with important physiological, functional and toxicity implications. Unlike mammalian and... (Review)
Review
Major species differences in tryptophan (Trp) metabolism and disposition exist with important physiological, functional and toxicity implications. Unlike mammalian and other species in which plasma Trp exists largely bound to albumin, teleosts and other aquatic species possess little or no albumin, such that Trp entry into their tissues is not hampered, neither is that of environmental chemicals and toxins, hence the need for strict measures to safeguard their aquatic environments. In species sensitive to toxicity of excess Trp, hepatic Trp 2,3-dioxygenase (TDO) lacks the free apoenzyme and its glucocorticoid induction mechanism. These species, which are largely herbivorous, however, dispose of Trp more rapidly and their TDO is activated by smaller doses of Trp than Trp-tolerant species. In general, sensitive species may possess a higher indoleamine 2,3-dioxygenase (IDO) activity which equips them to resist immune insults up to a point. Of the enzymes of the kynurenine pathway beyond TDO and IDO, 2-amino-3-carboxymuconic acid-6-semialdehyde decarboxylase (ACMSD) determines the extent of progress of the pathway towards NAD synthesis and its activity varies across species, with the domestic cat () being the leading species possessing the highest activity, hence its inability to utilise Trp for NAD synthesis. The paucity of current knowledge of Trp metabolism and disposition in wild carnivores, invertebrates and many other animal species described here underscores the need for further studies of the physiology of these species and its interaction with Trp metabolism.
PubMed: 36325027
DOI: 10.1177/11786469221122511 -
Protein Science : a Publication of the... May 2020Nickel enzymes, present in archaea, bacteria, plants, and primitive eukaryotes are divided into redox and nonredox enzymes and play key functions in diverse metabolic... (Review)
Review
Nickel enzymes, present in archaea, bacteria, plants, and primitive eukaryotes are divided into redox and nonredox enzymes and play key functions in diverse metabolic processes, such as energy metabolism and virulence. They catalyze various reactions by using active sites of diverse complexities, such as mononuclear nickel in Ni-superoxide dismutase, glyoxylase I and acireductone dioxygenase, dinuclear nickel in urease, heteronuclear metalloclusters in [NiFe]-carbon monoxide dehydrogenase, acetyl-CoA decarbonylase/synthase and [NiFe]-hydrogenase, and even more complex cofactors in methyl-CoM reductase and lactate racemase. The presence of metalloenzymes in a cell necessitates a tight regulation of metal homeostasis, in order to maintain the appropriate intracellular concentration of nickel while avoiding its toxicity. As well, the biosynthesis and insertion of nickel active sites often require specific and elaborated maturation pathways, allowing the correct metal to be delivered and incorporated into the target enzyme. In this review, the phylogenetic distribution of nickel enzymes will be briefly described. Their tridimensional structures as well as the complexity of their active sites will be discussed. In view of the latest findings on these enzymes, a special focus will be put on the biosynthesis of their active sites and nickel activation of apo-enzymes.
Topics: Biocatalysis; Catalytic Domain; Dioxygenases; Enzymes; Hydrogenase; Lactoylglutathione Lyase; Nickel; Protein Conformation; Superoxide Dismutase; Urease
PubMed: 32022353
DOI: 10.1002/pro.3836 -
The Journal of Biological Chemistry Aug 2017The biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is a multistage, multicompartment process that is essential for a broad range of cellular functions,... (Review)
Review
The biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is a multistage, multicompartment process that is essential for a broad range of cellular functions, including genome maintenance, protein translation, energy conversion, and the antiviral response. Genetic and cell biological studies over almost 2 decades have revealed some 30 proteins involved in the synthesis of cellular [2Fe-2S] and [4Fe-4S] clusters and their incorporation into numerous apoproteins. Mechanistic aspects of Fe/S protein biogenesis continue to be elucidated by biochemical and ultrastructural investigations. Here, we review recent developments in the pursuit of constructing a comprehensive model of Fe/S protein assembly in the mitochondrion.
Topics: Acyl Carrier Protein; Adrenodoxin; Animals; Apoenzymes; Gene Expression Regulation, Enzymologic; Humans; Iron-Binding Proteins; Iron-Sulfur Proteins; Mitochondria; Mitochondrial Proteins; Models, Biological; Models, Molecular; Protein Conformation; Protein Folding; Protein Multimerization; Protein Transport; Saccharomyces cerevisiae Proteins; Species Specificity; Sulfurtransferases; Frataxin
PubMed: 28615445
DOI: 10.1074/jbc.R117.787101 -
International Journal of Molecular... Aug 2020Hydrogenases are complex metalloenzymes, showing tremendous potential as H-converting redox catalysts for application in light-driven H production, enzymatic fuel cells... (Review)
Review
Hydrogenases are complex metalloenzymes, showing tremendous potential as H-converting redox catalysts for application in light-driven H production, enzymatic fuel cells and H-driven cofactor regeneration. They catalyze the reversible oxidation of hydrogen into protons and electrons. The apo-enzymes are not active unless they are modified by a complicated post-translational maturation process that is responsible for the assembly and incorporation of the complex metal center. The catalytic center is usually easily inactivated by oxidation, and the separation and purification of the active protein is challenging. The understanding of the catalytic mechanisms progresses slowly, since the purification of the enzymes from their native hosts is often difficult, and in some case impossible. Over the past decades, only a limited number of studies report the homologous or heterologous production of high yields of hydrogenase. In this review, we emphasize recent discoveries that have greatly improved our understanding of microbial hydrogenases. We compare various heterologous hydrogenase production systems as well as in vitro hydrogenase maturation systems and discuss their perspectives for enhanced biohydrogen production. Additionally, activities of hydrogenases isolated from either recombinant organisms or in vivo/in vitro maturation approaches were systematically compared, and future perspectives for this research area are discussed.
Topics: Bacterial Proteins; Hydrogenase; Industrial Microbiology; Iron-Sulfur Proteins; Protein Engineering
PubMed: 32824336
DOI: 10.3390/ijms21165890 -
EcoSal Plus Apr 2021Vitamin B is an ensemble of six interconvertible vitamers: pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and their 5'-phosphate derivatives, PNP, PMP, and PLP.... (Review)
Review
Vitamin B is an ensemble of six interconvertible vitamers: pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and their 5'-phosphate derivatives, PNP, PMP, and PLP. Pyridoxal 5'-phosphate is a coenzyme in a variety of enzyme reactions concerning transformations of amino and amino acid compounds. This review summarizes all known and putative PLP-binding proteins found in the MG1655 proteome. PLP can have toxic effects since it contains a very reactive aldehyde group at its 4' position that easily forms aldimines with primary and secondary amines and reacts with thiols. Most PLP is bound either to the enzymes that use it as a cofactor or to PLP carrier proteins, protected from the cellular environment but at the same time readily transferable to PLP-dependent apoenzymes. and its relatives synthesize PLP through the seven-step deoxyxylulose-5-phosphate (DXP)-dependent pathway. Other bacteria synthesize PLP in a single step, through a so-called DXP-independent pathway. Although the DXP-dependent pathway was the first to be revealed, the discovery of the widespread DXP-independent pathway determined a decline of interest in vitamin B metabolism. In , as in most organisms, PLP can also be obtained from PL, PN, and PM, imported from the environment or recycled from protein turnover, via a salvage pathway. Our review deals with all aspects of vitamin B metabolism in , from transcriptional to posttranslational regulation. A critical interpretation of results is presented, in particular, concerning the most obscure aspects of PLP homeostasis and delivery to PLP-dependent enzymes.
Topics: Escherichia coli; Pyridoxal Phosphate; Pyridoxine; Vitamin B 6; Vitamins
PubMed: 33787481
DOI: 10.1128/ecosalplus.ESP-0004-2021 -
Nucleic Acids Research Apr 2020Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5' exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL)....
Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO-POL enzyme.
Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5' exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3'-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain-especially the O helix-to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3' exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO-POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.
Topics: Binding Sites; Crystallography, X-Ray; DNA Polymerase I; DNA Replication; DNA-Directed DNA Polymerase; Flap Endonucleases; Magnesium; Mycobacterium; Nucleic Acid Conformation; Nucleotides; Phosphodiesterase I
PubMed: 32034423
DOI: 10.1093/nar/gkaa075