DDX49 is an RNA helicase that affects translation by regulating mRNA export and the levels of pre-ribosomal RNA.

Among the proteins predicted to be a part of the DExD box RNA helicase family, the functions of DDX49 are unknown. Here, we characterize the enzymatic activities and functions of DDX49 by comparing its properties with the well-studied RNA helicase, DDX39B. We find that DDX49 exhibits a robust ATPase and RNA helicase activity, significantly higher than that of DDX39B. DDX49 is required for the efficient export of poly (A)+ RNA from nucleus in a splicing-independent manner. Furthermore, DDX49 is a resident protein of nucleolus and regulates the steady state levels of pre-ribosomal RNA by regulating its transcription and stability. These dual functions of regulating mRNA export and pre-ribosomal RNA levels enable DDX49 to modulate global translation. Phenotypically, DDX49 promotes proliferation and colony forming potential of cells. Strikingly, DDX49 is significantly elevated in diverse cancer types suggesting that the increased abundance of DDX49 has a role in oncogenic transformation of cells. Taken together, this study shows the physiological role of DDX49 in regulating distinct steps of mRNA and pre-ribosomal RNA metabolism and hence translation and potential pathological role of its dysregulation, especially in cancers.

Structural, Mechanical, Imaging and in Vitro Evaluation of the Combined Effect of Gd3+ and Dy3+ in the ZrO2-SiO2 Binary System.

Mechanical strength and biocompatibility are considered the main prerequisites for materials in total hip replacement or joint prosthesis. Noninvasive surgical procedures are necessary to monitor the performance of a medical device in vivo after implantation. To this aim, simultaneous Gd3+ and Dy3+ additions to the ZrO2-SiO2 binary system were investigated. The results demonstrate the effective role of Gd3+ and Dy3+ to maintain the structural and mechanical stability of cubic zirconia ( c-ZrO2) up to 1400 °C, through their occupancy of ZrO2 lattice sites. A gradual tetragonal to cubic zirconia ( t-ZrO2 → c-ZrO2) phase transition is also observed that is dependent on the Gd3+ and Dy3+ content in the ZrO2-SiO2. The crystallization of either ZrSiO4 or SiO2 at elevated temperatures is delayed by the enhanced thermal energy consumed by the excess inclusion of Gd3+ and Dy3+ at c-ZrO2 lattice. The addition of Gd3+ and Dy3+ leads to an increase in the density, elastic modulus, hardness, and toughness above that of unmodified ZrO2-SiO2. The multimodal imaging contrast enhancement of the Gd3+ and Dy3+ combinations were revealed through magnetic resonance imaging and computed tomography contrast imaging tests. Biocompatibility of the Gd3+ and Dy3+ dual-doped ZrO2-SiO2 systems was verified through in vitro biological studies.

DDX39B promotes translation through regulation of pre-ribosomal RNA levels.

DDX39B, a DExD RNA helicase, is known to be involved in various cellular processes such as mRNA export, splicing and translation. Previous studies showed that the overexpression of DDX39B promotes the global translation but inhibits the mRNA export in a dominant negative manner. This presents a conundrum as to how DDX39B overexpression would increase the global translation if it inhibits the nuclear export of mRNAs. We resolve this by showing that DDX39B affects the levels of pre-ribosomal RNA by regulating its stability as well as synthesis. Furthermore, DDX39B promotes proliferation and colony forming potential of cells and its levels are significantly elevated in diverse cancer types. Thus, increase in DDX39B enhances global translation and cell proliferation through upregulation of pre-ribosomal RNA. This highlights a possible mechanism by which dysregulation of DDX39B expression could lead to oncogenesis.

Synthesis, characterization, mechanical and magnetic characteristics of Gd3+ /PO4 3 - substituted zircon for application in hard tissue replacements.

The study reports on the use of sol-gel technique to yield zircon type [Zr(1-0.1-x) GdxTi0.1 ] [(SiO4 )1-x (PO4 )x ] solid solution. Titanium has been used as a mineralizer to trigger zircon formation while equimolar concentrations of Gd3+ and PO4 3- were added to determine their accommodation limits in the zircon structure. The crystallization of t-ZrO2 as a dominant phase alongside the crystallization of m-ZrO2 and zircon were detected at 1200°C while their further annealing revealed the formation of zircon as a major phase at 1300°C. Heat treatment at 1400°C revealed the formation of zircon-type solid solution [Zr(1-0.1-x) Gdx Ti0.1 ][(SiO4 )1-x (PO4 )x ] comprising the accommodation of 10 mol.% of Gd3+ /PO4 3- at the zircon lattice. Beyond 10 mol.% of Gd3+ /PO4 3- , the crystallization of GdPO4 as a secondary phase is noticed. Structural analysis revealed the expansion of zircon lattice due to the simultaneous occupancy of Gd3+ /PO4 3- for the corresponding Zr4+ /SiO4 4- sites. The mechanical strength of single-phase zircon solid solution was higher in comparison to that of multiphase materials, namely in the presence of GdPO4 formed as a secondary phase in samples with added equimolar Gd3+ /PO4 3- contents beyond 10 mol.%. Nevertheless, the paramagnetic behavior of the samples demonstrated a steady surge as a function of enhanced Gd3+ content.

The ATRX-ADD domain binds to H3 tail peptides and reads the combined methylation state of K4 and K9.

Mutations in the ATRX protein are associated with the alpha-thalassemia and mental retardation X-linked syndrome (ATR-X). Almost half of the disease-causing mutations occur in its ATRX-Dnmt3-Dnmt3L (ADD) domain. By employing peptide arrays, chromatin pull-down and peptide binding assays, we show specific binding of the ADD domain to H3 histone tail peptides containing H3K9me3. Peptide binding was disrupted by the presence of the H3K4me3 and H3K4me2 modification marks indicating that the ATRX-ADD domain has a combined readout of these two important marks (absence of H3K4me2 and H3K4me3 and presence of H3K9me3). Disease-causing mutations reduced ATRX-ADD binding to H3 tail peptides. ATRX variants, which fail in the H3K9me3 interaction, show a loss of heterochromatic localization in cells, which indicates the chromatin targeting function of the ADD domain of ATRX. Disruption of H3K9me3 binding may be a general pathogenicity pathway of ATRX mutations in the ADD domain which may explain the clustering of disease mutations in this part of the ATRX protein.

Enhancing the zircon yield through the addition of calcium phosphates into ZrO2-SiO2 binary systems: synthesis and structural, morphological, mechanical and in vitro analysis.

The crystallization of ZrSiO4 is generally accomplished by the addition of mineralizers into ZrO2-SiO2 binary oxides. The current investigation aimed to investigate the effect of adding calcium phosphates into ZrO2-SiO2 binary oxides on the yield of ZrSiO4. The concentration of calcium phosphate additions were varied to obtain ZrSiO4 that fetches improved mechanical and biological properties for application in hard tissue replacements. The findings highlight the significant role of Ca2+ and P5+ in triggering the ZrSiO4 formation via their accommodation at the Zr4+ and Si4+ sites. Especially, calcium phosphate additions trigger the t- → m-ZrO2 transition beyond 1000 °C, which consequently reacts with SiO2 to promote ZrSiO4 formation. Calcium phosphates are accommodated at the lattice sites of ZrSiO4 with a maximum limit of 20 mol%, beyond which the crystallization of ß-Ca3(PO4)2 is noticed. The optimum amount of 20 mol% of calcium phosphates displayed a better strength than that of all the investigated specimens. More than 80% of cell viability in MG-63 cells was invariably determined in all the calcium phosphate-added ZrSiO4 systems.

Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail.

Using peptide arrays and binding to native histone proteins, we show that the ADD domain of Dnmt3a specifically interacts with the H3 histone 1-19 tail. Binding is disrupted by di- and trimethylation of K4, phosphorylation of T3, S10 or T11 and acetylation of K4. We did not observe binding to the H4 1-19 tail. The ADD domain of Dnmt3b shows the same binding specificity, suggesting that the distinct biological functions of both enzymes are not related to their ADD domains. To establish a functional role of the ADD domain binding to unmodified H3 tails, we analyzed the DNA methylation of in vitro reconstituted chromatin with Dnmt3a2, the Dnmt3a2/Dnmt3L complex, and the catalytic domain of Dnmt3a. All Dnmt3a complexes preferentially methylated linker DNA regions. Chromatin substrates with unmodified H3 tail or with H3K9me3 modification were methylated more efficiently by full-length Dnmt3a and full-length Dnmt3a/3L complexes than chromatin trimethylated at H3K4. In contrast, the catalytic domain of Dnmt3a was not affected by the H3K4me3 modification. These results demonstrate that the binding of the ADD domain to H3 tails unmethylated at K4 leads to the preferential methylation of DNA bound to chromatin with this modification state. Our in vitro results recapitulate DNA methylation patterns observed in genome-wide DNA methylation studies.

Special Issue "Structure, Activity, and Function of Protein Methyltransferases".

Post-translational modifications (PTMs) largely expand the functional diversity of the proteome [...].

Acetylene containing 2-(2-hydrazinyl)thiazole derivatives: design, synthesis, and in vitro and in silico evaluation of antimycobacterial activity against Mycobacterium tuberculosis.

Mycobacterium tuberculosis resistance to commercially available drugs is increasing day by day. To address this issue, various strategies were planned and are being implemented. However, there is a need for new drugs and rapid diagnostic methods. For this endeavour, in this paper, we present the synthesis of acetylene containing 2-(2-hydrazinyl) thiazole derivatives and in vitro evaluation against the H37Rv strain of Mycobacterium tuberculosis. Among the developed 26 acetylene containing 2-(2-hydrazinyl) thiazole derivatives, eight compounds inhibited the growth of Mycobacterium tuberculosis with MIC values ranging from 100 µg ml-1 to 50 µg ml-1. The parent acetylene containing thiosemicarbazones showed promising antimycobacterial activity by inhibiting up to 75% of the Mycobacterium at 50 µg ml-1. In addition, in silico studies were employed to understand the binding mode of all the novel acetylene-containing derivatives against the KasA protein of the Mycobacterium. Interestingly, the KasA protein interactions with the compounds were similar to the interactions of KasA protein with thiolactomycin and rifampicin. Cytotoxicity study results indicate that the compounds tested are non-toxic to human embryonic kidney cells.

The Dnmt3a PWWP domain reads histone 3 lysine 36 trimethylation and guides DNA methylation.

The Dnmt3a DNA methyltransferase contains in its N-terminal part a PWWP domain that is involved in chromatin targeting. Here, we have investigated the interaction of the PWWP domain with modified histone tails using peptide arrays and show that it specifically recognizes the histone 3 lysine 36 trimethylation mark. H3K36me3 is known to be a repressive modification correlated with DNA methylation in mammals and heterochromatin in Schizosaccharomyces pombe. These results were confirmed by equilibrium peptide binding studies and pulldown experiments with native histones and purified native nucleosomes. The PWWP-H3K36me3 interaction is important for the subnuclear localization of enhanced yellow fluorescent protein-fused Dnmt3a. Furthermore, the PWWP-H3K36me3 interaction increases the activity of Dnmt3a for methylation of nucleosomal DNA as observed using native nucleosomes isolated from human cells after demethylation of the DNA with 5-aza-2'-deoxycytidine as substrate for methylation with Dnmt3a. These data suggest that the interaction of the PWWP domain with H3K36me3 is involved in targeting of Dnmt3a to chromatin carrying that mark, a model that is in agreement with several studies on the genome-wide distribution of DNA methylation and H3K36me3.

Application of Celluspots peptide arrays for the analysis of the binding specificity of epigenetic reading domains to modified histone tails.

BACKGROUND: Epigenetic reading domains are involved in the regulation of gene expression and chromatin state by interacting with histones in a post-translational modification specific manner. A detailed knowledge of the target modifications of reading domains, including enhancing and inhibiting secondary modifications, will lead to a better understanding of the biological signaling processes mediated by reading domains. RESULTS: We describe the application of Celluspots peptide arrays which contain 384 histone peptides carrying 59 post translational modifications in different combinations as an inexpensive, reliable and fast method for initial screening for specific interactions of reading domains with modified histone peptides. To validate the method, we tested the binding specificities of seven known epigenetic reading domains on Celluspots peptide arrays, viz. the HP1ß and MPP8 Chromo domains, JMJD2A and 53BP1 Tudor domains, Dnmt3a PWWP domain, Rag2 PHD domain and BRD2 Bromo domain. In general, the binding results agreed with literature data with respect to the primary specificity of the reading domains, but in almost all cases we obtained additional new information concerning the influence of secondary modifications surrounding the target modification. CONCLUSIONS: We conclude that Celluspots peptide arrays are powerful screening tools for studying the specificity of putative reading domains binding to modified histone peptides.

Structure, Activity and Function of the Protein Arginine Methyltransferase 6.

Members of the protein arginine methyltransferase (PRMT) family methylate the arginine residue(s) of several proteins and regulate a broad spectrum of cellular functions. Protein arginine methyltransferase 6 (PRMT6) is a type I PRMT that asymmetrically dimethylates the arginine residues of numerous substrate proteins. PRMT6 introduces asymmetric dimethylation modification in the histone 3 at arginine 2 (H3R2me2a) and facilitates epigenetic regulation of global gene expression. In addition to histones, PRMT6 methylates a wide range of cellular proteins and regulates their functions. Here, we discuss (i) the biochemical aspects of enzyme kinetics, (ii) the structural features of PRMT6 and (iii) the diverse functional outcomes of PRMT6 mediated arginine methylation. Finally, we highlight how dysregulation of PRMT6 is implicated in various types of cancers and response to viral infections.

The ribosomal protein eL21 interacts with the protein lysine methyltransferase SMYD2 and regulates its steady state levels.

The protein lysine methyltransferase, SMYD2 is involved in diverse cellular events by regulating protein functions through lysine methylation. Though several substrate proteins of SMYD2 are well-studied, only a limited number of its interaction partners have been identified and characterized. Here, we performed a yeast two-hybrid screening of SMYD2 and found that the ribosomal protein, eL21 could interact with SMYD2. SMYD2-eL21 interaction in the human cells was confirmed by immunoprecipitation methods. In vitro pull-down assays revealed that SMYD2 interacts with eL21 directly through its SET and MYND domain. Computational mapping, followed by experimental studies identified that Lys81 and Lys83 residues of eL21 are important for the SMYD2-eL21 interaction. Evolutionary analysis showed that these residues might have co-evolved with the emergence of SMYD2. We found that eL21 regulates the steady state levels of SMYD2 by promoting its transcription and inhibiting its proteasomal degradation. Importantly, SMYD2-eL21 interaction plays an important role in regulating cell proliferation and its dysregulation might lead to tumorigenesis. Our findings highlight a novel extra-ribosomal function of eL21 on regulating SMYD2 levels and imply that ribosomal proteins might regulate wide range of cellular functions through protein-protein interactions in addition to their core function in translation.

PRMT3 interacts with ALDH1A1 and regulates gene-expression by inhibiting retinoic acid signaling.

Protein arginine methyltransferase 3 (PRMT3) regulates protein functions by introducing asymmetric dimethylation marks at the arginine residues in proteins. However, very little is known about the interaction partners of PRMT3 and their functional outcomes. Using yeast-two hybrid screening, we identified Retinal dehydrogenase 1 (ALDH1A1) as a potential interaction partner of PRMT3 and confirmed this interaction using different methods. ALDH1A1 regulates variety of cellular processes by catalyzing the conversion of retinaldehyde to retinoic acid. By molecular docking and site-directed mutagenesis, we identified the specific residues in the catalytic domain of PRMT3 that facilitate interaction with the C-terminal region of ALDH1A1. PRMT3 inhibits the enzymatic activity of ALDH1A1 and negatively regulates the expression of retinoic acid responsive genes in a methyltransferase activity independent manner. Our findings show that in addition to regulating protein functions by introducing methylation modifications, PRMT3 could also regulate global gene expression through protein-protein interactions.

The uncharacterized protein FAM47E interacts with PRMT5 and regulates its functions.

Protein arginine methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues in various proteins affecting diverse cellular processes such as transcriptional regulation, splicing, DNA repair, differentiation, and cell cycle. Elevated levels of PRMT5 are observed in several types of cancers and are associated with poor clinical outcomes, making PRMT5 an important diagnostic marker and/or therapeutic target for cancers. Here, using yeast two-hybrid screening, followed by immunoprecipitation and pull-down assays, we identify a previously uncharacterized protein, FAM47E, as an interaction partner of PRMT5. We report that FAM47E regulates steady-state levels of PRMT5 by affecting its stability through inhibition of its proteasomal degradation. Importantly, FAM47E enhances the chromatin association and histone methylation activity of PRMT5. The PRMT5-FAM47E interaction affects the regulation of PRMT5 target genes expression and colony-forming capacity of the cells. Taken together, we identify FAM47E as a protein regulator of PRMT5, which promotes the functions of this versatile enzyme. These findings imply that disruption of PRMT5-FAM47E interaction by small molecules might be an alternative strategy to attenuate the oncogenic function(s) of PRMT5.

Protein lysine methyltransferase G9a acts on non-histone targets.

By methylation of peptide arrays, we determined the specificity profile of the protein methyltransferase G9a. We show that it mostly recognizes an Arg-Lys sequence and that its activity is inhibited by methylation of the arginine residue. Using the specificity profile, we identified new non-histone protein targets of G9a, including CDYL1, WIZ, ACINUS and G9a (automethylation), as well as peptides derived from CSB. We demonstrate potential downstream signaling pathways for methylation of non-histone proteins.

In situ formation, structural, mechanical and in vitro analysis of ZrO2/ZnFe2O4 composite with assorted composition ratios.

The investigation underline the in situ formation of ZrO2/ZnFe2O4 composites and the resultant structural, morphological, mechanical and magnetic properties. The characterization results ensured the crystallization of tetragonal ZrO2 (t-ZrO2) and ZnFe2O4 phases at 900 °C. Depending on Zn2+/Fe3+ content, the composite system revealed a gradual increment in the phase yield of ZnFe2O4. The significance of monoclinic ZrO2 (m-ZrO2) is also evident in all the systems at 900 °C; however, the incremental heat treatment to 1300 °C indicated its corresponding loss, thus indicating the reverse m- â†’ t-ZrO2 transition. The crystallization of ZnFe2O4 as a secondary phase in the t-ZrO2 matrix is also affirmed from the morphological analysis. Mechanical studies accomplished good uniformity in all the investigated compositions despite the variation in the phase content of ZnFe2O4 in composite system. All the t-ZrO2/ZnFe2O4 composites ensured strong ferrimagnetic features and moreover better biocompatibility and non-toxicity characteristics were displayed from in vitro tests.

Investigation on the structural, mechanical and in vitro biocompatibility features of CaZr4 (PO4 )6 influenced by Zn2+ substitutions.

The present study explores the possibility of Zn2+ substituted calcium zirconium phosphate [CaZr4 (PO4 )6 ] as a potential replacement for the existing materials in load bearing orthopedic applications. Pure CaZr4 (PO4 )6 ) and wide range of Zn2+ substitutions in CaZr4 (PO4 )6 have been synthesized through citrate assisted sol-gel technique. The characterization results confirmed the extraordinary structural stability displayed by CaZr4 (PO4 )6 until 1,550°C. Further, the flexibility of CaZr4 (PO4 )6 lattice to accommodate 40 mol% of Zn2+ has been determined. The microstructures of CaZr4 (PO4 )6 and Zn2+ substituted CaZr4 (PO4 )6 demonstrated irregular sized grains and cracks alongside the negligence to obtain definite grain boundaries. This has been reflected in the moderate mechanical properties of the investigated specimen; nevertheless, Zn2+ substituted CaZr4 (PO4 )6 displayed enhanced mechanical stability. Further, in vitro tests signified the remarkable biocompatibility and alkaline phosphatase activity of Zn2+ substituted CaZr4 (PO4 )6 .

Identification of novel quinoline inhibitor for EHMT2/G9a through virtual screening.

G9a (also known as EHMT2 - Euchromatin histone methyltransferase 2) is a protein lysine methyltransferase which introduces methylation modification in variety of proteins including histones. G9a catalyzes the dimethylation of lysine 9 on histone 3 (H3K9me2) which is a repressive epigenetic modification. H3K9me2 is associated with the silencing of several genes including tumor suppressor genes in many cancers and hence G9a is a well characterized drug target for cancer therapy. Here, we report the discovery of CSV0C018875 as a novel quinoline based G9a inhibitor through virtual screening strategy from a HTS database. Sub-structure querying based on the known G9a inhibitors, followed by docking based virtual screening, led to the identification of CSV0C018875 as G9a inhibitor. We found that CSV0C018875 inhibits the activity of G9a in both enzyme and cell based assays. Importantly, the toxicity of CSV0C018875 is much lesser than that of the well-studied G9a inhibitor, BIX-01294. Molecular dynamics simulations shows that CSV0C018875 binds deeper inside the active site cavity of G9a, which facilitates the tight binding and also increases the compounds residence time, which in turn reflects better G9a inhibition. The novel quinoline CSV0C018875 could be further optimized to improve the ADME as well pharmacodynamic property.

2,4-Di-Tert-Butylphenol Isolated From an Endophytic Fungus, Daldinia eschscholtzii, Reduces Virulence and Quorum Sensing in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is among the top three gram-negative bacteria according to the WHO's critical priority list of pathogens against which newer antibiotics are urgently needed and considered a global threat due to multiple drug resistance. This situation demands unconventional antimicrobial strategies such as the inhibition of quorum sensing to alleviate the manifestation of classical resistance mechanisms. Here, we report that 2,4-di-tert-butylphenol (2,4-DBP), isolated from an endophytic fungus, Daldinia eschscholtzii, inhibits the quorum-sensing properties of P. aeruginosa. We have found that treating P. aeruginosa with 2,4-DBP substantially reduced the secretion of virulence factors as well as biofilm, and its associated factors that are controlled by quorum sensing, in a dose-dependent manner. Concomitantly, 2,4-DBP also significantly reduced the expression of quorum sensing-related genes, i.e., lasI, lasR, rhlI, and rhlR significantly. Importantly, 2,4-DBP restricted the adhesion and invasion of P. aeruginosa to the A549 lung alveolar carcinoma cells. In addition, bactericidal assay with 2,4-DBP exhibited synergism with ampicillin to kill P. aeruginosa. Furthermore, our computational studies predicted that 2,4-DBP could bind to the P. aeruginosa quorum-sensing receptors LasR and RhlR. Collectively, these data suggest that 2,4-DBP can be exploited as a standalone drug or in combination with antibiotic(s) as an anti-virulence and anti-biofilm agent to combat the multidrug resistant P. aeruginosa infection.