Mechanistic Insights into Harmine-Mediated Inhibition of Human DNA Methyltransferases and Prostate Cancer Cell Growth.

Mammalian DNA methyltransferases (DNMTs), including DNMT1, DNMT3A, and DNMT3B, are key DNA methylation enzymes and play important roles in gene expression regulation. Dysregulation of DNMTs is linked to various diseases and carcinogenesis, and therefore except for the two approved anticancer azanucleoside drugs, various non-nucleoside DNMT inhibitors have been identified and reported. However, the underlying mechanisms for the inhibitory activity of these non-nucleoside inhibitors still remain largely unknown. Here, we systematically tested and compared the inhibition activities of five non-nucleoside inhibitors toward the three human DNMTs. We found that harmine and nanaomycin A blocked the methyltransferase activity of DNMT3A and DNMT3B more efficiently than resveratrol, EGCG, and RG108. We further determined the crystal structure of harmine in complex with the catalytic domain of the DNMT3B-DNMT3L tetramer revealing that harmine binds at the adenine cavity of the SAM-binding pocket in DNMT3B. Our kinetics assays confirm that harmine competes with SAM to competitively inhibit DNMT3B-3L activity with a Ki of 6.6 µM. Cell-based studies further show that harmine treatment inhibits castration-resistant prostate cancer cell (CRPC) proliferation with an IC50 of ∼14 µM. The CPRC cells treated with harmine resulted in reactivating silenced hypermethylated genes compared to the untreated cells, and harmine cooperated with an androgen antagonist, bicalutamide, to effectively inhibit the proliferation of CRPC cells. Our study thus reveals, for the first time, the inhibitory mechanism of harmine on DNMTs and highlights new strategies for developing novel DNMT inhibitors for cancer treatment.

A myeloid leukemia factor homolog involved in encystation-induced protein metabolism in Giardia lamblia.

BACKGROUND: Giardia lamblia differentiates into resistant cysts as an established model for dormancy. Myeloid leukemia factor (MLF) proteins are important regulators of cell differentiation. Giardia possesses a MLF homolog which was up-regulated during encystation and localized to unknown cytosolic vesicles named MLF vesicles (MLFVs). METHODS: We used double staining for visualization of potential factors with role in protein metabolism pathway and a strategy that employed a deletion mutant, CDK2m3, to test the protein degradation pathway. We also explored whether autophagy or proteasomal degradation are regulators of Giardia encystation by treatment with MG132, rapamycin, or chloroquine. RESULTS: Double staining of MLF and ISCU or CWP1 revealed no overlap between their vesicles. The aberrant CDK2m3 colocalized with MLFVs and formed complexes with MLF. MG132 increased the number of CDK2m3-localized vesicles and its protein level. We further found that MLF colocalized and interacted with a FYVE protein and an ATG8-like (ATG8L) protein, which were up-regulated during encystation and their expression induced Giardia encystation. The addition of MG132, rapamycin, or chloroquine, increased their levels and the number of their vesicles, and inhibited the cyst formation. MLF and FYVE were detected in exosomes released from culture. CONCLUSIONS: The MLFVs are not mitosomes or encystation-specific vesicles, but are related with degradative pathway for CDK2m3. MLF, FYVE, and ATG8L play a positive role in encystation and function in protein clearance pathway, which is important for encystation and coordinated with Exosomes. GENERAL SIGNIFICANCE: MLF, FYVE, and ATG8L may be involved an encystation-induced protein metabolism during Giardia differentiation.

Elucidating the tunability of binding behavior for the MERS-CoV macro domain with NAD metabolites.

The macro domain is an ADP-ribose (ADPR) binding module, which is considered to act as a sensor to recognize nicotinamide adenine dinucleotide (NAD) metabolites, including poly ADPR (PAR) and other small molecules. The recognition of macro domains with various ligands is important for a variety of biological functions involved in NAD metabolism, including DNA repair, chromatin remodeling, maintenance of genomic stability, and response to viral infection. Nevertheless, how the macro domain binds to moieties with such structural obstacles using a simple cleft remains a puzzle. We systematically investigated the Middle East respiratory syndrome-coronavirus (MERS-CoV) macro domain for its ligand selectivity and binding properties by structural and biophysical approaches. Of interest, NAD, which is considered not to interact with macro domains, was co-crystallized with the MERS-CoV macro domain. Further studies at physiological temperature revealed that NAD has similar binding ability with ADPR because of the accommodation of the thermal-tunable binding pocket. This study provides the biochemical and structural bases of the detailed ligand-binding mode of the MERS-CoV macro domain. In addition, our observation of enhanced binding affinity of the MERS-CoV macro domain to NAD at physiological temperature highlights the need for further study to reveal the biological functions.

C-terminal Redox Domain of Arabidopsis APR1 is a Non-Canonical Thioredoxin Domain with Glutaredoxin Function.

Sulfur is an essential nutrient that can be converted into utilizable metabolic forms to produce sulfur-containing metabolites in plant. Adenosine 5'-phosphosulfate (APS) reductase (APR) plays a vital role in catalyzing the reduction of activated sulfate to sulfite, which requires glutathione. Previous studies have shown that the C-terminal domain of APR acts as a glutathione-dependent reductase. The crystal structure of the C-terminal redox domain of Arabidopsis APR1 (AtAPR1) shows a conserved α/ß thioredoxin fold, but not a glutaredoxin fold. Further biochemical studies of the redox domain from AtAPR1 provided evidence to support the structural observation. Collectively, our results provide structural and biochemical information to explain how the thioredoxin fold exerts the glutaredoxin function in APR.

Structural and biochemical evidence supporting poly ADP-ribosylation in the bacterium Deinococcus radiodurans.

Poly-ADP-ribosylation, a post-translational modification involved in various cellular processes, is well characterized in eukaryotes but thought to be devoid in bacteria. Here, we solve crystal structures of ADP-ribose-bound poly(ADP-ribose)glycohydrolase from the radioresistant bacterium Deinococcus radiodurans (DrPARG), revealing a solvent-accessible 2'-hydroxy group of ADP-ribose, which suggests that DrPARG may possess endo-glycohydrolase activity toward poly-ADP-ribose (PAR). We confirm the existence of PAR in D. radiodurans and show that disruption of DrPARG expression causes accumulation of endogenous PAR and compromises recovery from UV radiation damage. Moreover, endogenous PAR levels in D. radiodurans are elevated after UV irradiation, indicating that PARylation may be involved in resistance to genotoxic stresses. These findings provide structural insights into a bacterial-type PARG and suggest the existence of a prokaryotic PARylation machinery that may be involved in stress responses.

Structural basis of interaction between dimeric cyclophilin 1 and Myb1 transcription factor in Trichomonas vaginalis.

Cyclophilin 1 (TvCyP1), a cyclophilin type peptidyl-prolyl isomerase present in the human parasite Trichomonas vaginalis, interacts with Myb1 and assists in its nuclear translocation. Myb1 regulates the expression of ap65-1 gene that encodes for a disease causing cytoadherence enzyme. Here, we determined the crystal structures of TvCyP1 and its complex with the minimum TvCyP1-binding sequence of Myb1 (Myb1104-111), where TvCyP1 formed a homodimer, unlike other single domain cyclophilins. In the complex structure, one Myb1104-111 peptide was bound to each TvCyP1 protomer, with G106-P107 and Y105 fitting well into the active site and auxiliary S2 pocket, respectively. NMR data further showed that TvCyP1 can catalyze the cis/trans isomerization of P107 in Myb1104-111. Interestingly, in the well-folded Myb1 protein (Myb135-141), the minimum binding sequence adopted a different conformation from that of unstructured Myb1104-111 peptide, that could make P107 binding to the active site of TvCyP1 difficult. However, NMR studies showed that similar to Myb1104-111 peptide, Myb135-141 also interacted with the active site of TvCyP1 and the dynamics of the Myb135-141 residues near P107 was reduced upon interaction. Together, the structure of TvCyP1 and detailed structural insights on TvCyP1-Myb1 interaction provided here could pave the way for newer drugs to treat drug-resistant strains.

NMR assignments of the macro domain from Middle East respiratory syndrome coronavirus (MERS-CoV).

The newly emerging human pathogen, Middle East respiratory syndrome coronavirus (MERS-CoV), contains a macro domain in the highly conserved N-terminal region of non-structural protein 3. Intense research has shown that macro domains bind ADP-ribose and other derivatives, but it still remains intangible about their exact function. In this study we report the preliminary structural analysis through solution NMR spectroscopy of the MERS-CoV macro domain. The near complete NMR assignments of MERS-CoV macro domain provide the basis for subsequent structural and biochemical investigation in the context of protein function.

Macro Domain from Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Is an Efficient ADP-ribose Binding Module: CRYSTAL STRUCTURE AND BIOCHEMICAL STUDIES.

The newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) encodes the conserved macro domain within non-structural protein 3. However, the precise biochemical function and structure of the macro domain is unclear. Using differential scanning fluorimetry and isothermal titration calorimetry, we characterized the MERS-CoV macro domain as a more efficient adenosine diphosphate (ADP)-ribose binding module than macro domains from other CoVs. Furthermore, the crystal structure of the MERS-CoV macro domain was determined at 1.43-Å resolution in complex with ADP-ribose. Comparison of macro domains from MERS-CoV and other human CoVs revealed structural differences in the α1 helix alters how the conserved Asp-20 interacts with ADP-ribose and may explain the efficient binding of the MERS-CoV macro domain to ADP-ribose. This study provides structural and biophysical bases to further evaluate the role of the MERS-CoV macro domain in the host response via ADP-ribose binding but also as a potential target for drug design.

Crystallization of the C-terminal redox domain of the sulfur-assimilatory enzyme APR1 from Arabidopsis thaliana.

Plant-type APS reductase (APR), which catalyzes the reduction of activated sulfate to sulfite in plants, consists of a reductase domain and a C-terminal redox domain showing sequence homology to thioredoxin but possessing the activity of glutaredoxin. In order to understand the structural and biochemical properties of the redox domain of plant-type APS reductase, the C-terminal domain of APR1 (APR1C) from Arabidopsis thaliana was crystallized using the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to a resolution of 2.70 Šon the SPXF beamline BL13B1 at the NSRRC, Taiwan. The crystals belonged to space group P43212 or P41212, with unit-cell parameters a = b = 58.2, c = 86.7 Å. With one molecule per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.64 Å(3) Da(-1), which corresponds to a solvent content of approximately 53.49%. Further structure-based functional studies of APR1C would extend knowledge of the molecular mechanism and regulation of APR.

Functional redundancy of two Pax-like proteins in transcriptional activation of cyst wall protein genes in Giardia lamblia.

The protozoan Giardia lamblia differentiates from a pathogenic trophozoite into an infectious cyst to survive outside of the host. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately induced. Pax family transcription factors are involved in a variety of developmental processes in animals. Nine Pax proteins have been found to play an important role in tissue and organ development in humans. To understand the progression from primitive to more complex eukaryotic cells, we tried to identify putative pax genes in the G. lamblia genome and found two genes, pax1 and pax2, with limited similarity. We found that Pax1 may transactivate the encystation-induced cwp genes and interact with AT-rich initiatior elements that are essential for promoter activity and transcription start site selection. In this study, we further characterized Pax2 and found that, like Pax1, Pax2 was present in Giardia nuclei and it may specifically bind to the AT-rich initiator elements of the encystation-induced cwp1-3 and myb2 genes. Interestingly, overexpression of Pax2 increased the cwp1-3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of nuclear localization, DNA-binding activity, and transactivation activity of Pax2. These results are similar to those found in the previous Pax1 study. In addition, the profiles of gene expression in the Pax2 and Pax1 overexpressing cells significantly overlap in the same direction and ERK1 associated complexes may phosphorylate Pax2 and Pax1, suggesting that Pax2 and Pax1 may be downstream components of a MAPK/ERK1 signaling pathway. Our results reveal functional redundancy between Pax2 and Pax1 in up-regulation of the key encystation-induced genes. These results illustrate functional redundancy of a gene family can occur in order to increase maintenance of important gene function in the protozoan organism G. lamblia.

Regulation of a Myb transcription factor by cyclin-dependent kinase 2 in Giardia lamblia.

The protozoan Giardia lamblia parasitizes the human small intestine to cause diseases. It undergoes differentiation into infectious cysts by responding to intestinal stimulation. How the activated signal transduction pathways relate to encystation stimulation remain largely unknown. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately up-regulated by a Myb2 transcription factor. Because cell differentiation is linked to cell cycle regulation, we tried to understand the role of cell cycle regulators, cyclin-dependent kinases (Cdks), in encystation. We found that the recombinant Myb2 was phosphorylated by Cdk-associated complexes and the levels of phosphorylation increased significantly during encystation. We have identified a putative cdk gene (cdk2) by searching the Giardia genome database. Cdk2 was found to localize in the cytoplasm with higher expression during encystation. Interestingly, overexpression of Cdk2 resulted in a significant increase of the levels of cwp gene expression and cyst formation. In addition, the Cdk2-associated complexes can phosphorylate Myb2 and the levels of phosphorylation increased significantly during encystation. Mutations of important catalytic residues of Cdk2 resulted in a significant decrease of kinase activity and ability of inducing cyst formation. Addition of a Cdk inhibitor, purvalanol A, significantly decreased the Cdk2 kinase activity and the levels of cwp gene expression and cyst formation. Our results suggest that the Cdk2 pathway may be involved in phosphorylation of Myb2, leading to activation of the Myb2 function and up-regulation of cwp genes during encystation. The results provide insights into the use of Cdk inhibitory drugs in disruption of Giardia differentiation into cysts.

A novel E2F-like protein involved in transcriptional activation of cyst wall protein genes in Giardia lamblia.

Giardia lamblia differentiates into resistant walled cysts for survival outside the host and transmission. During encystation, synthesis of cyst wall proteins is coordinately induced. The E2F family of transcription factors in higher eukaryotes is involved in cell cycle progression and cell differentiation. We asked whether Giardia has E2F-like genes and whether they influence gene expression during Giardia encystation. Blast searches of the Giardia genome database identified one gene (e2f1) encoding a putative E2F protein with two putative DNA-binding domains. We found that the e2f1 gene expression levels increased significantly during encystation. Epitope-tagged E2F1 was found to localize to nuclei. Recombinant E2F1 specifically bound to the thymidine kinase and cwp1-3 gene promoters. E2F1 contains several key residues for DNA binding, and mutation analysis revealed that its binding sequence is similar to those of the known E2F family proteins. The E2F1-binding sequences were positive cis-acting elements of the thymidine kinase and cwp1 promoters. We also found that E2F1 transactivated the thymidine kinase and cwp1 promoters through its binding sequences in vivo. Interestingly, E2F1 overexpression resulted in a significant increase of the levels of CWP1 protein, cwp1-3 gene mRNA, and cyst formation. We also found E2F1 can interact with Myb2, a transcription factor that coordinate up-regulates the cwp1-3 genes during encystation. Our results suggest that E2F family has been conserved during evolution and that E2F1 is an important transcription factor in regulation of the Giardia cwp genes, which are key to Giardia differentiation into cysts.

A novel pax-like protein involved in transcriptional activation of cyst wall protein genes in Giardia lamblia.

Giardia lamblia differentiates into infectious cysts to survive outside of the host. It is of interest to identify factors involved in up-regulation of cyst wall proteins (CWPs) during this differentiation. Pax proteins are important regulators of development and cell differentiation in Drosophila and vertebrates. No member of this gene family has been reported to date in yeast, plants, or protozoan parasites. We have identified a pax-like gene (pax1) encoding a putative paired domain in the G. lamblia genome. Epitope-tagged Pax1 localized to nuclei during both vegetative growth and encystation. Recombinant Pax1 specifically bound to the AT-rich initiator elements of the encystation-induced cwp1 to -3 and myb2 genes. Interestingly, overexpression of Pax1 increased cwp1 to -3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of the transactivation function of Pax1. Our results indicate that the Pax family has been conserved during evolution, and Pax1 could up-regulate the key encystation-induced genes to regulate differentiation of the protozoan eukaryote, G. lamblia.

A novel WRKY-like protein involved in transcriptional activation of cyst wall protein genes in Giardia lamblia.

Synthesis of a protective cyst wall is required for survival outside of the host and for infection of Giardia lamblia. Little is known of gene regulation of the cyst wall proteins (CWPs) during differentiation into dormant cysts. WRKY homologues constitute a large family of DNA-binding proteins in plants that are involved in several key cellular functions, including disease resistance, stress response, dormancy, and development. A putative wrky gene has been identified in the G. lamblia genome. We found that wrky expression levels increased significantly during encystation. The epitope-tagged WRKY was translocated into the nuclei during encystation. Recombinant WRKY specifically bound to its own promoter and the encystation-induced cwp1 and cwp2 promoters. WRKY contains several key residues for DNA binding, and mutation analysis revealed that its binding sequences are similar to those of the known plant WRKY proteins and that two of them are positive cis-acting elements of the wrky and cwp2 promoters. Overexpression of WRKY increased the cwp1-2 and myb2 mRNA levels, and these gene promoters were bound by WRKY in vivo. Interestingly, the wrky and cwp1-2 genes were up-regulated by ERK1 (extracellular signal-related kinase 1) overexpression, suggesting that WRKY may be a downstream component of the ERK1 pathway. In addition, a WRKY mutant that cannot enter nuclei and an ERK1 mutant lacking the predicted kinase domain showed decreased cwp1-2 gene expression. Our results suggest that the WRKY family has been conserved during evolution and that WRKY is an important transactivator of the cwp1-2 genes during G. lamblia differentiation into dormant cysts.

Regulation of cyst wall protein promoters by Myb2 in Giardia lamblia.

Myb family transcription factors are important in regulating cell proliferation, differentiation, and cell cycle progression. Giardia lamblia differentiates into infectious cysts to survive outside of the host. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately induced. We have identified an encystation-induced Myb2 protein, which binds to the promoter regions of the cwp genes and myb2 itself in vitro. To elucidate the role of Myb2 in G. lamblia, we tested the hypothesis that Myb2 can activate encystation-induced genes. We found that overexpression of Myb2 resulted in an increase of expression of CWP1 at both protein and mRNA levels. Interestingly, the Myb2-overexpressing trophozoites had increased capability to differentiate into cysts. In cotransfection assays, Myb2 was able to transactivate the cwp promoters and its own promoter in vivo, suggesting that its gene can be positively autoregulated. Moreover, deletion of the N- or C-terminal domain resulted in a decrease of transactivation and autoregulation function of Myb2. We also found that the promoter of a newly identified encystation-induced gene, the giardial myeloid leukemia factor-like gene, has the Myb2 binding sites and that its mRNA levels were increased by Myb2 overexpression. Chromatin immunoprecipitation assays confirmed that Myb2 was bound to the promoters with its binding sites. Transfection of the myb2 antisense construct reduced the levels of the cwp1 transcripts and cyst formation. Our results suggest that Myb2 is a potent transactivator of the cwp genes and other endogenous genes and plays an important role in G. lamblia differentiation into cysts.