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1.
Nucleic Acids Res ; 52(8): 4541-4555, 2024 May 08.
Article in English | MEDLINE | ID: mdl-38499490

ABSTRACT

Formation of programmed DNA double-strand breaks is essential for initiating meiotic recombination. Genetic studies on Arabidopsis thaliana and Mus musculus have revealed that assembly of a type IIB topoisomerase VI (Topo VI)-like complex, composed of SPO11 and MTOPVIB, is a prerequisite for generating DNA breaks. However, it remains enigmatic if MTOPVIB resembles its Topo VI subunit B (VIB) ortholog in possessing robust ATPase activity, ability to undergo ATP-dependent dimerization, and activation of SPO11-mediated DNA cleavage. Here, we successfully prepared highly pure A. thaliana MTOPVIB and MTOPVIB-SPO11 complex. Contrary to expectations, our findings highlight that MTOPVIB differs from orthologous Topo VIB by lacking ATP-binding activity and independently forming dimers without ATP. Most significantly, our study reveals that while MTOPVIB lacks the capability to stimulate SPO11-mediated DNA cleavage, it functions as a bona fide DNA-binding protein and plays a substantial role in facilitating the dsDNA binding capacity of the MOTOVIB-SPO11 complex. Thus, we illustrate mechanistic divergence between the MTOPVIB-SPO11 complex and classical type IIB topoisomerases.


Subject(s)
Arabidopsis Proteins , Arabidopsis , DNA Topoisomerases, Type II , Adenosine Triphosphate/metabolism , Arabidopsis/genetics , Arabidopsis/enzymology , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Archaeal Proteins , DNA Breaks, Double-Stranded , DNA Topoisomerases/metabolism , DNA Topoisomerases/genetics , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Endodeoxyribonucleases/metabolism , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/chemistry , Evolution, Molecular , Meiosis , Protein Multimerization
2.
J Virol ; 96(7): e0010722, 2022 04 13.
Article in English | MEDLINE | ID: mdl-35293767

ABSTRACT

The propagation of the hepatitis C virus (HCV) is regulated in part by the phosphorylation of its nonstructural protein NS5A that undergoes sequential phosphorylation on several highly conserved serine residues and switches from a hypo- to a hyperphosphorylated state. Previous studies have shown that NS5A sequential phosphorylation requires NS3 encoded on the same NS3-NS4A-NS4B-NS5A polyprotein. Subtle mutations in NS3 without affecting its protease activity could affect NS5A phosphorylation. Given the ATPase domain in the NS3 COOH terminus, we tested whether NS3 participates in NS5A phosphorylation similarly to the nucleoside diphosphate kinase-like activity of the rotavirus NSP2 nucleoside triphosphatase (NTPase). Mutations in the NS3 ATP-binding motifs blunted NS5A hyperphosphorylation and phosphorylation at serines 225, 232, and 235, whereas a mutation in the RNA-binding domain did not. The phosphorylation events were not rescued with wild-type NS3 provided in trans. When provided with an NS3 ATPase-compatible ATP analog, N6-benzyl-ATP-γ-S, thiophosphorylated NS5A was detected in the cells expressing the wild-type NS3-NS5B polyprotein. The thiophosphorylation level was lower in the cells expressing NS3-NS5B with a mutation in the NS3 ATP-binding domain. In vitro assays with a synthetic peptide and purified wild-type NS3 followed by dot blotting and mass spectrometry found weak NS5A phosphorylation at serines 222 and 225 that was sensitive to an inhibitor of casein kinase Iα but not helicase. When casein kinase Iα was included in the assay, much stronger phosphorylation was observed at serines 225, 232, and 235. We concluded that NS5A sequential phosphorylation requires the ATP-binding domain of the NS3 helicase and that casein kinase Iα is a potent NS5A kinase. IMPORTANCE For more than 20 years, NS3 was known to participate in NS5A sequential phosphorylation. In the present study, we show for the first time that the ATP-binding domain of NS3 is involved in NS5A phosphorylation. In vitro assays showed that casein kinase Iα is a very potent kinase responsible for NS5A phosphorylation at serines 225, 232, and 235. Our data suggest that ATP binding by NS3 probably results in conformational changes that recruit casein kinase Iα to phosphorylate NS5A, initially at S225 and subsequently at S232 and S235. Our discovery reveals intricate requirements of the structural integrity of NS3 for NS5A hyperphosphorylation and HCV replication.


Subject(s)
Hepacivirus , Hepatitis C , RNA-Dependent RNA Polymerase , Viral Nonstructural Proteins , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Casein Kinase Ialpha/metabolism , Hepacivirus/enzymology , Hepacivirus/genetics , Hepatitis C/virology , Humans , Phosphorylation , Polyproteins/metabolism , Protein Domains/genetics , RNA-Dependent RNA Polymerase/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
3.
Proc Natl Acad Sci U S A ; 119(1)2022 01 04.
Article in English | MEDLINE | ID: mdl-34969844

ABSTRACT

Deoxypodophyllotoxin contains a core of four fused rings (A to D) with three consecutive chiral centers, the last being created by the attachment of a peripheral trimethoxyphenyl ring (E) to ring C. Previous studies have suggested that the iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, deoxypodophyllotoxin synthase (DPS), catalyzes the oxidative coupling of ring B and ring E to form ring C and complete the tetracyclic core. Despite recent efforts to deploy DPS in the preparation of deoxypodophyllotoxin analogs, the mechanism underlying the regio- and stereoselectivity of this cyclization event has not been elucidated. Herein, we report 1) two structures of DPS in complex with 2OG and (±)-yatein, 2) in vitro analysis of enzymatic reactivity with substrate analogs, and 3) model reactions addressing DPS's catalytic mechanism. The results disfavor a prior proposal of on-pathway benzylic hydroxylation. Rather, the DPS-catalyzed cyclization likely proceeds by hydrogen atom abstraction from C7', oxidation of the benzylic radical to a carbocation, Friedel-Crafts-like ring closure, and rearomatization of ring B by C6 deprotonation. This mechanism adds to the known pathways for transformation of the carbon-centered radical in Fe/2OG enzymes and suggests what types of substrate modification are likely tolerable in DPS-catalyzed production of deoxypodophyllotoxin analogs.


Subject(s)
Berberidaceae/enzymology , Drugs, Chinese Herbal/chemistry , Ligases/chemistry , Plant Proteins/chemistry , Podophyllotoxin/analogs & derivatives , Oxidation-Reduction , Podophyllotoxin/chemistry
4.
ACS Catal ; 11(12): 7186-7192, 2021 Jun 18.
Article in English | MEDLINE | ID: mdl-35721870

ABSTRACT

Nature has developed complexity-generating reactions within natural product biosynthetic pathways. However, direct utilization of these pathways to prepare compound libraries remains challenging due to limited substrate scopes, involvement of multiple-step reactions, and moderate robustness of these sophisticated enzymatic transformations. Synthetic chemistry, on the other hand, offers an alternative approach to prepare natural product analogs. However, owing to complex and diverse functional groups appended on the targeted molecules, dedicated design and development of synthetic strategies are typically required. Herein, by leveraging the power of chemo-enzymatic synthesis, we report an approach to bridge the gap between biological and synthetic strategies in the preparation of quinolone alkaloid analogs. Leading by in silico analysis, the predicted substrate analogs were chemically synthesized. The AsqJ-catalyzed asymmetric epoxidation of these substrate analogues was followed by an Lewis Acid-triggered ring contraction to complete the viridicatin formation. We evaluated the robustness of this method in gram-scale reactions. Lastly, through chemoenzymatic cascades, a library of quinolone alkaloids is effectively prepared.

5.
J Med Chem ; 63(15): 8485-8494, 2020 08 13.
Article in English | MEDLINE | ID: mdl-32663396

ABSTRACT

Phosphodiesterase 5A1 (PDE5) is a key target for treating cardiovascular diseases and erectile dysfunction. Here, we report the crystal structure of PDE5 complexed with the sole second generation drug avanafil. Analysis of protein-drug interactions revealed the structural basis of avanafil's superior isoform selectivity. Moreover, a halogen bonding was observed between avanafil and a backbone carbonyl oxygen of an adjacent α-helix, whose contribution to inhibitory potency illustrates the feasibility of exploiting α-helix backbone in structure-based drug design.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 5/chemistry , Phosphodiesterase 5 Inhibitors/pharmacology , Pyrimidines/pharmacology , Crystallography, X-Ray , Cyclic Nucleotide Phosphodiesterases, Type 5/metabolism , Drug Design , Humans , Molecular Docking Simulation , Protein Conformation/drug effects , Protein Conformation, alpha-Helical/drug effects , Protein Isoforms/chemistry , Protein Isoforms/metabolism
6.
J Am Chem Soc ; 142(13): 6268-6284, 2020 04 01.
Article in English | MEDLINE | ID: mdl-32131594

ABSTRACT

Mechanisms of enzymatic epoxidation via oxygen atom transfer (OAT) to an olefin moiety is mainly derived from the studies on thiolate-heme containing epoxidases, such as cytochrome P450 epoxidases. The molecular basis of epoxidation catalyzed by nonheme-iron enzymes is much less explored. Herein, we present a detailed study on epoxidation catalyzed by the nonheme iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, AsqJ. The native substrate and analogues with different para substituents ranging from electron-donating groups (e.g., methoxy) to electron-withdrawing groups (e.g., trifluoromethyl) were used to probe the mechanism. The results derived from transient-state enzyme kinetics, Mössbauer spectroscopy, reaction product analysis, X-ray crystallography, density functional theory calculations, and molecular dynamic simulations collectively revealed the following mechanistic insights: (1) The rapid O2 addition to the AsqJ Fe(II) center occurs with the iron-bound 2OG adopting an online-binding mode in which the C1 carboxylate group of 2OG is trans to the proximal histidine (His134) of the 2-His-1-carboxylate facial triad, instead of assuming the offline-binding mode with the C1 carboxylate group trans to the distal histidine (His211); (2) The decay rate constant of the ferryl intermediate is not strongly affected by the nature of the para substituents of the substrate during the OAT step, a reactivity behavior that is drastically different from nonheme Fe(IV)-oxo synthetic model complexes; (3) The OAT step most likely proceeds through a stepwise process with the initial formation of a C(benzylic)-O bond to generate an Fe-alkoxide species, which is observed in the AsqJ crystal structure. The subsequent C3-O bond formation completes the epoxide installation.


Subject(s)
Aspergillus nidulans/metabolism , Epoxy Compounds/metabolism , Fungal Proteins/metabolism , Ketoglutaric Acids/metabolism , Oxygen/metabolism , Oxygenases/metabolism , Aspergillus nidulans/chemistry , Aspergillus nidulans/enzymology , Crystallography, X-Ray , Epoxy Compounds/chemistry , Fungal Proteins/chemistry , Iron/chemistry , Iron/metabolism , Models, Molecular , Oxygen/chemistry , Oxygenases/chemistry
7.
J Exp Clin Cancer Res ; 38(1): 5, 2019 Jan 06.
Article in English | MEDLINE | ID: mdl-30612578

ABSTRACT

BACKGROUND: The Slug-E-cadherin axis plays a critical role in non-small-cell lung cancers (NSCLCs) where aberrant upregulation of Slug promotes cancer metastasis. Now, the post-translational modifications of Slug and their regulation mechanisms still remain unclear in lung cancer. Hence, exploring the protein linkage map of Slug is of great interest for investigating the scenario of how Slug protein is regulated in lung cancer metastasis. METHODS: The Slug associated proteins, Ubc9 and SUMO-1, were identified using yeast two-hybrid screening; and in vitro SUMOylation assays combined with immunoprecipitation and immunoblotting were performed to explore the detail events and regulations of Slug SUMOylation. The functional effects of SUMOylation on Slug proteins were examined by EMSA, reporter assay, ChIP assay, RT-PCR, migration and invasion assays in vitro, tail vein metastatic analysis in vivo, and also evaluated the association with clinical outcome of NSCLC patients. RESULTS: Slug protein could interact with Ubc9 and SUMO-1 and be SUMOylated in cells. Amino acids 130-212 and 33-129 of Slug are responsible for its binding to Ubc9 and protein inhibitor of activated STAT (PIAS)y, respectively. SUMOylation could enhance the transcriptional repression activity of Slug via recruiting more HDAC1, resulting in reduced expression of downstream Slug target genes and enhanced lung cancer metastasis. In addition, hypoxia could increase Slug SUMOylation through attenuating the interactions of Slug with SENP1 and SENP2. Finally, high expression Slug and Ubc9 levels were associated with poor overall survival among NSCLC patients. CONCLUSIONS: Ubc9/PIASy-mediated Slug SUMOylation and subsequent HDAC1 recruitment may play a crucial role in hypoxia-induced lung cancer progression, and these processes may serve as therapeutic targets for NSCLC.


Subject(s)
Lung Neoplasms/complications , Sumoylation/genetics , Cell Hypoxia , Cell Line, Tumor , Humans , Lung Neoplasms/pathology , Neoplasm Metastasis , Transfection
8.
Nat Commun ; 9(1): 3085, 2018 08 06.
Article in English | MEDLINE | ID: mdl-30082834

ABSTRACT

Type IIA topoisomerases (Top2s) manipulate the handedness of DNA crossovers by introducing a transient and protein-linked double-strand break in one DNA duplex, termed the DNA-gate, whose opening allows another DNA segment to be transported through to change the DNA topology. Despite the central importance of this gate-opening event to Top2 function, the DNA-gate in all reported structures of Top2-DNA complexes is in the closed state. Here we present the crystal structure of a human Top2 DNA-gate in an open conformation, which not only reveals structural characteristics of its DNA-conducting path, but also uncovers unexpected yet functionally significant conformational changes associated with gate-opening. This structure further implicates Top2's preference for a left-handed DNA braid and allows the construction of a model representing the initial entry of another DNA duplex into the DNA-gate. Steered molecular dynamics calculations suggests the Top2-catalyzed DNA passage may be achieved by a rocker-switch-type movement of the DNA-gate.


Subject(s)
DNA Topoisomerases, Type II/chemistry , DNA/chemistry , Nucleic Acid Conformation , Poly-ADP-Ribose Binding Proteins/chemistry , Allosteric Site , Catalysis , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Conformation , Molecular Dynamics Simulation , Protein Conformation
9.
Angew Chem Int Ed Engl ; 57(7): 1831-1835, 2018 02 12.
Article in English | MEDLINE | ID: mdl-29314482

ABSTRACT

AsqJ, an iron(II)- and 2-oxoglutarate-dependent enzyme found in viridicatin-type alkaloid biosynthetic pathways, catalyzes sequential desaturation and epoxidation to produce cyclopenins. Crystal structures of AsqJ bound to cyclopeptin and its C3 epimer are reported. Meanwhile, a detailed mechanistic study was carried out to decipher the desaturation mechanism. These findings suggest that a pathway involving hydrogen atom abstraction at the C10 position of the substrate by a short-lived FeIV -oxo species and the subsequent formation of a carbocation or a hydroxylated intermediate is preferred during AsqJ-catalyzed desaturation.


Subject(s)
Epoxy Compounds/metabolism , Fungal Proteins/metabolism , Peptides/metabolism , Aspergillus nidulans/enzymology , Biocatalysis , Catalytic Domain , Cytochrome P-450 Enzyme System/metabolism , Epoxy Compounds/chemistry , Ferric Compounds/chemistry , Fungal Proteins/chemistry , Ketoglutaric Acids/chemistry , Ketoglutaric Acids/metabolism , Molecular Dynamics Simulation , Peptides/chemistry , Quantum Theory , Stereoisomerism
10.
Biochem J ; 475(2): 373-398, 2018 01 23.
Article in English | MEDLINE | ID: mdl-29363591

ABSTRACT

Many cancer type-specific anticancer agents have been developed and significant advances have been made toward precision medicine in cancer treatment. However, traditional or nonspecific anticancer drugs are still important for the treatment of many cancer patients whose cancers either do not respond to or have developed resistance to cancer-specific anticancer agents. DNA topoisomerases, especially type IIA topoisomerases, are proved therapeutic targets of anticancer and antibacterial drugs. Clinically successful topoisomerase-targeting anticancer drugs act through topoisomerase poisoning, which leads to replication fork arrest and double-strand break formation. Unfortunately, this unique mode of action is associated with the development of secondary cancers and cardiotoxicity. Structures of topoisomerase-drug-DNA ternary complexes have revealed the exact binding sites and mechanisms of topoisomerase poisons. Recent advances in the field have suggested a possibility of designing isoform-specific human topoisomerase II poisons, which may be developed as safer anticancer drugs. It may also be possible to design catalytic inhibitors of topoisomerases by targeting certain inactive conformations of these enzymes. Furthermore, identification of various new bacterial topoisomerase inhibitors and regulatory proteins may inspire the discovery of novel human topoisomerase inhibitors. Thus, topoisomerases remain as important therapeutic targets of anticancer agents.


Subject(s)
Antineoplastic Agents/chemistry , DNA Topoisomerases, Type II/chemistry , DNA, Neoplasm/chemistry , Molecular Targeted Therapy/methods , Neoplasms/drug therapy , Topoisomerase Inhibitors/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/pharmacology , Catalytic Domain , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Breaks, Double-Stranded , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Drug Design , Gene Expression , Humans , Molecular Docking Simulation , Neoplasms/enzymology , Neoplasms/genetics , Neoplasms/pathology , Protein Structure, Secondary , Structure-Activity Relationship , Topoisomerase Inhibitors/pharmacology
12.
Nucleic Acids Res ; 45(18): 10861-10871, 2017 10 13.
Article in English | MEDLINE | ID: mdl-28977631

ABSTRACT

Human type II topoisomerase (Top2) isoforms, hTop2α and hTop2ß, are targeted by some of the most successful anticancer drugs. These drugs induce Top2-mediated DNA cleavage to trigger cell-death pathways. The potency of these drugs correlates positively with their efficacy in stabilizing the enzyme-mediated DNA breaks. Structural analysis of hTop2α and hTop2ß revealed the presence of methionine residues in the drug-binding pocket, we therefore tested whether a tighter Top2-drug association may be accomplished by introducing a methionine-reactive Pt2+ into a drug to further stabilize the DNA break. Herein, we synthesized an organoplatinum compound, etoplatin-N2ß, by replacing the methionine-juxtaposing group of the drug etoposide with a cis-dichlorodiammineplatinum(II) moiety. Compared to etoposide, etoplatin-N2ß more potently inhibits both human Top2s. While the DNA breaks arrested by etoposide can be rejoined, those captured by etoplatin-N2ß are practically irreversible. Crystallographic analyses of hTop2ß complexed with DNA and etoplatin-N2ß demonstrate coordinate bond formation between Pt2+ and a flanking methionine. Notably, this stable coordinate tether can be loosened by disrupting the structural integrity of drug-binding pocket, suggesting that Pt2+ coordination chemistry may allow for the development of potent inhibitors with protein conformation-dependent reversibility. This approach may be exploited to achieve isoform-specific targeting of human Top2s.


Subject(s)
Antineoplastic Agents/chemistry , DNA Breaks , DNA-Binding Proteins/antagonists & inhibitors , Organoplatinum Compounds/chemistry , Podophyllotoxin/analogs & derivatives , Topoisomerase II Inhibitors/chemistry , Antigens, Neoplasm/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , DNA/chemistry , DNA Topoisomerases, Type II/chemistry , DNA-Binding Proteins/chemistry , HL-60 Cells , Humans , Methionine/chemistry , Organoplatinum Compounds/pharmacology , Podophyllotoxin/chemistry , Podophyllotoxin/pharmacology , Poly-ADP-Ribose Binding Proteins , Protein Conformation , Topoisomerase II Inhibitors/pharmacology
13.
Sci Rep ; 7(1): 2370, 2017 05 24.
Article in English | MEDLINE | ID: mdl-28539630

ABSTRACT

The programmed induction of meiotic DNA double-strand breaks (DSBs) by the evolutionarily conserved SPO-11 protein, which is structurally related to archaeal Topo VIA topoisomerases, triggers meiotic recombination. Identification of several meiosis-specific factors that are required for SPO-11-mediated DSB formation raises the question whether SPO-11 alone can cleave DNA. Here, we have developed procedures to express and purify C. elegans SPO-11 in a soluble, untagged, and monodispersed form. Our biochemical and biophysical analyses demonstrate that SPO-11 is monomeric and binds DNA, double-stranded DNA in particular. Importantly, SPO-11 does not exhibit DNA cleavage activity under a wide range of reaction conditions, suggesting that co-factors are needed for DSB induction activity. Our SPO-11 purification system and the findings reported herein should facilitate future mechanistic studies directed at delineating the mechanism of action of the SPO-11 ensemble in meiotic DSB formation.


Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , DNA Breaks, Double-Stranded , Endodeoxyribonucleases/genetics , Meiosis/genetics , Amino Acid Sequence , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , DNA/genetics , DNA/metabolism , DNA Repair , Endodeoxyribonucleases/metabolism , Protein Binding , Sequence Homology, Amino Acid
14.
15.
Sci Rep ; 6: 38071, 2016 12 09.
Article in English | MEDLINE | ID: mdl-27934872

ABSTRACT

Methyltransferases play crucial roles in many cellular processes, and various regulatory mechanisms have evolved to control their activities. For methyltransferases involved in biosynthetic pathways, regulation via feedback inhibition is a commonly employed strategy to prevent excessive accumulation of the pathways' end products. To date, no biosynthetic methyltransferases have been characterized by X-ray crystallography in complex with their corresponding end product. Here, we report the crystal structures of the glycine sarcosine N-methyltransferase from the halophilic archaeon Methanohalophilus portucalensis (MpGSMT), which represents the first structural elucidation of the GSMT methyltransferase family. As the first enzyme in the biosynthetic pathway of the osmoprotectant betaine, MpGSMT catalyzes N-methylation of glycine and sarcosine, and its activity is feedback-inhibited by the end product betaine. A structural analysis revealed that, despite the simultaneous presence of both substrate (sarcosine) and cofactor (S-adenosyl-L-homocysteine; SAH), the enzyme was likely crystallized in an inactive conformation, as additional structural changes are required to complete the active site assembly. Consistent with this interpretation, the bound SAH can be replaced by the methyl donor S-adenosyl-L-methionine without triggering the methylation reaction. Furthermore, the observed conformational state was found to harbor a betaine-binding site, suggesting that betaine may inhibit MpGSMT activity by trapping the enzyme in an inactive form. This work implicates a structural basis by which feedback inhibition of biosynthetic methyltransferases may be achieved.


Subject(s)
Glycine N-Methyltransferase/chemistry , Glycine N-Methyltransferase/metabolism , Methanosarcinaceae/enzymology , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Betaine/metabolism , Catalytic Domain , Crystallography, X-Ray , Feedback, Physiological , Gene Expression Regulation, Archaeal , Gene Expression Regulation, Enzymologic , Glycine/metabolism , Methanosarcinaceae/chemistry , Methylation , Models, Molecular , Protein Structure, Secondary , Sarcosine/metabolism
16.
J Med Chem ; 59(21): 9906-9918, 2016 11 10.
Article in English | MEDLINE | ID: mdl-27748121

ABSTRACT

Targeting thymidylate kinase (TMPK) that catalyzes the phosphotransfer reaction for formation of dTDP from dTMP is a new strategy for anticancer treatment. This study is to understand the inhibitory mechanism of a previously identified human TMPK (hTMPK) inhibitor YMU1 (1a) by molecular docking, isothermal titration calorimetry, and photoaffinity labeling. The molecular dynamics simulation suggests that 1a prefers binding at the catalytic site of hTMPK, whereas the hTMPK inhibitors that bear pyridino[d]isothiazolone or benzo[d]isothiazolone core structure in lieu of the dimethylpyridine-fused isothiazolone moiety in 1a can have access to both the ATP-binding and catalytic sites. The binding sites of hTMPK inhibitors were validated by photoaffinity labeling and mass spectrometric studies. Taking together, 1a and its analogues stabilize the conformation of ligand-induced degradation (LID) region of hTMPK and block the catalytic site or ATP-binding site, thus attenuating the ATP binding-induced closed conformation that is required for phosphorylation of dTMP.


Subject(s)
Nucleoside-Phosphate Kinase/antagonists & inhibitors , Phosphates/metabolism , Protein Kinase Inhibitors/pharmacology , Proteolysis/drug effects , Animals , Binding Sites/drug effects , Calorimetry , Cell Line , Cell Survival/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Humans , Mice , Models, Molecular , Molecular Structure , Nucleoside-Phosphate Kinase/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity Relationship
17.
Cancer Res ; 76(20): 6043-6053, 2016 10 15.
Article in English | MEDLINE | ID: mdl-27569210

ABSTRACT

SMYD3 methyltransferase is nearly undetectable in normal human tissues but highly expressed in several cancers, including breast cancer, although its contributions to pathogenesis in this setting are unclear. Here we report that histone H2A.Z.1 is a substrate of SMYD3 that supports malignancy. SMYD3-mediated dimethylation of H2A.Z.1 at lysine 101 (H2A.Z.1K101me2) increased stability by preventing binding to the removal chaperone ANP32E and facilitating its interaction with histone H3. Moreover, a microarray analysis identified cyclin A1 as a target coregulated by SMYD3 and H2A.Z.1K101me2. The colocalization of SMYD3 and H2A.Z.1K101me2 at the promoter of cyclin A1 activated its expression and G1-S progression. Enforced expression of cyclin A1 in cells containing mutant H2A.Z.1 rescued tumor formation in a mouse model. Our findings suggest that SMYD3-mediated H2A.Z.1K101 dimethylation activates cyclin A1 expression and contributes to driving the proliferation of breast cancer cells. Cancer Res; 76(20); 6043-53. ©2016 AACR.


Subject(s)
Breast Neoplasms/pathology , Cell Cycle , Cell Proliferation , Histone-Lysine N-Methyltransferase/physiology , Histones/metabolism , Animals , Cell Line, Tumor , Cyclin A1/genetics , Female , Humans , Methylation , Mice
18.
Org Biomol Chem ; 13(41): 10324-7, 2015 Nov 07.
Article in English | MEDLINE | ID: mdl-26309036

ABSTRACT

The jadomycins are a family of secondary metabolites produced by S. venezuelae ISP5230. Specific jadomycins have been shown to possess a variety of anticancer, antifungal, and antibacterial properties, with different molecular mechanisms of action. Herein we demonstrate qualitative and quantitative direct binding between the validated anticancer target human topoisomerase IIß and jadomycin DS using WaterLOGSY NMR spectroscopy. Additionally, we report for the first time, that jadomycin DS also binds a variety of other proteins, likely in a non-specific manner. Such interactions may rationalize the potential polypharmacology of jadomycin DS.


Subject(s)
DNA Topoisomerases, Type II/chemistry , DNA-Binding Proteins/chemistry , Isoquinolines/chemistry , Binding Sites , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Humans , Magnetic Resonance Spectroscopy , Molecular Conformation , Stereoisomerism , Water/chemistry
19.
Proc Natl Acad Sci U S A ; 112(36): 11229-34, 2015 Sep 08.
Article in English | MEDLINE | ID: mdl-26305948

ABSTRACT

Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az1) and antizyme inhibitor (AzIN). Az1 suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az1 binding. The structural basis of the Az1-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az1 complexed with either ODC or AzIN. Structural analysis revealed that Az1 sterically blocks ODC homodimerization. Moreover, Az1 binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az1 for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az1-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN-Az1 structure suggests how AzIN may effectively compete with ODC for Az1 to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation.


Subject(s)
Carrier Proteins/chemistry , Homeostasis , Ornithine Decarboxylase/chemistry , Polyamines/chemistry , Proteins/chemistry , Amino Acid Sequence , Biocatalysis , Carrier Proteins/metabolism , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Ornithine Decarboxylase/metabolism , Polyamines/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Conformation , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Proteins/metabolism , Proteolysis , Sequence Homology, Amino Acid
20.
Nucleic Acids Res ; 43(15): 7612-23, 2015 Sep 03.
Article in English | MEDLINE | ID: mdl-26150423

ABSTRACT

The mer operon confers bacterial resistance to inorganic mercury (Hg(2+)) and organomercurials by encoding proteins involved in sensing, transport and detoxification of these cytotoxic agents. Expression of the mer operon is under tight control by the dual-function transcriptional regulator MerR. The metal-free, apo MerR binds to the mer operator/promoter region as a repressor to block transcription initiation, but is converted into an activator upon Hg(2+)-binding. To understand how MerR interacts with Hg(2+) and how Hg(2+)-binding modulates MerR function, we report here the crystal structures of apo and Hg(2+)-bound MerR from Bacillus megaterium, corresponding respectively to the repressor and activator conformation of MerR. To our knowledge, the apo-MerR structure represents the first visualization of a MerR family member in its intact and inducer-free form. And the Hg(2+)-MerR structure offers the first view of a triligated Hg(2+)-thiolate center in a metalloprotein, confirming that MerR binds Hg(2+) via trigonal planar coordination geometry. Structural comparison revealed the conformational transition of MerR is coupled to the assembly/disassembly of a buried Hg(2+) binding site, thereby providing a structural basis for the Hg(2+)-mediated functional switching of MerR. The pronounced Hg(2+)-induced repositioning of the MerR DNA-binding domains suggests a plausible mechanism for the transcriptional regulation of the mer operon.


Subject(s)
Bacterial Proteins/chemistry , DNA-Binding Proteins/chemistry , Mercury/chemistry , Repressor Proteins/chemistry , Trans-Activators/chemistry , Bacillus megaterium/genetics , Binding Sites , Models, Molecular , Operon , Protein Binding , Protein Conformation
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