Enhancement of N-Methyl Amino Acid Incorporation into Proteins and Peptides Using Modified Bacterial Ribosomes and Elongation Factor P.

N-Methylated amino acids are constituents of natural bioactive peptides and proteins. Nα-methylated amino acids appear abundantly in natural cyclic peptides, likely due to their constraint of peptide conformation and contribution to peptide stability. Peptides containing Nα-methylated amino acids have long been prepared by chemical synthesis. While such natural peptides are not produced ribosomally, recent ribosomal strategies have afforded Nα-methylated peptides. Presently, we define new strategies for the ribosomal incorporation of Nα-methylated amino acids into peptides and proteins. First, we identify modified ribosomes capable of facilitating the incorporation of six N-methylated amino acids into antibacterial scorpion peptide IsCT. Also synthesized analogously was a protein domain (RRM1) from hnRNP LL; improved yields were observed for nearly all tested N-methylated amino acids. Computational modeling of the ribosomal assembly illustrated how the distortion imposed by N-methylation could be compensated by altering the nucleotides in key 23S rRNA positions. Finally, it is known that incorporation of multiple prolines (an N-alkylated amino acid) ribosomally can be facilitated by bacterial elongation factor P. We report that supplementing endogenous EF-P during IsCT peptide and RRM1 protein synthesis gave improved yields for most of the N-methylated amino acids studied.

Diagnosis, Characterization and Treatment of Emerging Pathogens.

Emerging infectious diseases are perhaps the most rapidly spreading diseases [...].

Activation of d-Asparagine and d-Glutamine Derivatives Using the Mitsunobu Reaction.

Seven d-amino acid derivatives having reactive side chains have been activated to afford their respective 3,5-dinitrobenzyl esters using the Mitsunobu reaction. This esterification was found to be difficult using traditional methods involving 3,5-dinitrobenzyl chloride under alkaline conditions. The conversion of a tRNA to the respective d-glutaminyl-tRNA using d-glutamine 3,5-dinitrobenzyl ester was catalyzed by a flexizyme, followed by purification to remove all the unacylated tRNAs and other byproducts. Both d- and l-glutamine were incorporated from their aminoacyl-tRNAs into a model peptide structurally related to IFN-ß.

Site-Selective Tyrosine Phosphorylation in the Activation of the p50 Subunit of NF-κB for DNA Binding and Transcription.

The family of NF-κB transcriptional activators controls the expression of many genes, including those involved in cell survival and development. The family consists of homo- and heterodimers constituted by combinations of five subunits. Subunit p50 includes 13 tyrosine residues, but the relationship between specific tyrosine phosphorylations and p50 function is not well understood. Subunits of p50 and p65 prepared in vitro formed a heterodimer, but this NF-κB would not bind to the interleukin-2 (IL-2) promoter DNA. Treatment of p50 with guanosine triphosphate (GTP) and a lysate from activated Jurkat cells, effected rapid p50 phosphorylation, and, in the presence of wild-type subunit p65, was accompanied on the same time scale by IL-2 promoter DNA binding. Modified p50s containing one of seven stoichiometrically phosphorylated tyrosines in NF-κB p50/p65 heterodimers, included three that facilitated binding to the IL-2 DNA promoter region to a greater extent than the wild type. One of these three stoichiometrically phosphorylated p50/p65 heterodimers of NF-κB, containing pTyr60 in the p50 subunit, was treated with a lysate from activated Jurkat cells + GTP and shown to be phosphorylated on the same time scale as wild-type p50. This modified NF-κB also developed IL-2 promoter DNA binding activity on the same time scale as the wild type but exhibited greater binding to the IL-2 DNA promoters than the wild type. The nature of this enhanced binding was studied in greater detail using a metabolically stable pTyr derivative at position 60 of p50 and cellular phosphatases. We suggest that enhanced DNA binding of modified NF-κB containing pTyr60 in the p50 subunit may reflect stoichiometric NF-κB phosphorylation at a site that is not normally fully phosphorylated, or not phosphorylated at all, and is relatively resistant to the effects of Jurkat cell tyrosine phosphatase activity. This conclusion was reinforced by demonstrating that modification of Tyr60 of p50 with a metabolically stable methylenephosphonate moiety further increased the stability of the formed NF-κB p50/p65 heterodimer against the action of activated Jurkat cell phosphatases.

Sphingosine-1-phosphate in mitochondrial function and metabolic diseases.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite. The past decade has witnessed exponential growth in the field of S1P research, partly attributed to drugs targeting its receptors or kinases. Accumulating evidence indicates that changes in the S1P axis (i.e., S1P production, transport, and receptors) may modify metabolism and eventually mediate metabolic diseases. Dysfunction of the mitochondria on a master monitor of cellular metabolism is considered the leading cause of metabolic diseases, with aberrations typically induced by abnormal biogenesis, respiratory chain complex disorders, reactive oxygen species overproduction, calcium deposition, and mitophagy impairment. Accordingly, we discuss decades of investigation into changes in the S1P axis and how it controls mitochondrial function. Furthermore, we summarize recent scientific advances in disorders associated with the S1P axis and their involvement in the pathogenesis of metabolic diseases in humans, including type 2 diabetes mellitus and cardiovascular disease, from the perspective of mitochondrial function. Finally, we review potential challenges and prospects for S1P axis application to the regulation of mitochondrial function and metabolic diseases; these data may provide theoretical guidance for the treatment of metabolic diseases.

Local Conformational Constraint of Firefly Luciferase Can Affect the Energy of Bioluminescence and Enzyme Stability.

Conformational dynamics contribute importantly to enzyme catalysis, such that targeted conformational constraint may affect catalysis. Firefly luciferases undergo extensive structural change during catalysis; key residues form a hydrophobic pocket, excluding water and enabling maximally energetic light production. Point mutants almost always luminesce at longer wavelengths (lower energy) than the wild type. Conformational constraint, using dipeptide analogue 3 at a position critical for optimized excited state structure, produced luciferase emission at a shorter wavelength by ~10 nm. In comparison, introduction of conformationally constrained analogues 4, 5, or 7 afforded luciferases emitting at longer wavelengths, while a related unconstrained luciferase (analogue 6) exhibited wild-type emission. The constrained luciferases tested were more stable than the wild type. Protein modeling demonstrated that the "inside" or "outside" orientation of the conformationally constrained dipeptide led to the shorter or longer emission wavelength, respectively. More broadly, these results suggest that local conformational constraint can control specific elements of enzyme behavior, both in vitro and in vivo. This represents the first example of studying enzyme function by introducing conformationally constrained dipeptides at a specific protein position. The principles discovered here in luciferase modification will enable studies to control the wavelength emission and photophysical properties of modified luciferases.

Site-selective incorporation of phosphorylated tyrosine into the p50 subunit of NF-κB and activation of its downstream gene CD40.

The NF-κB family of transcriptional activators is responsible for the expression of numerous genes that control key functions such as cell development and survival. Subunit p50 has been studied extensively and is known to include 13 tyrosines, but the extent and pattern of tyrosine phosphorylation that accompanies p50 function has not been defined in the literature, especially at the level of selectivity of gene expression. In this study, phosphorylated tyrosine (pTyr) was site-selectively incorporated into the p50 subunit using an E. coli in vitro expression system containing a modified ribosome. In human T cells, the NF-κBs containing a pTyr at position 60 or 82 of p50 strongly increased the expression of CD40, which is a potential target for cancer or viral immunotherapy. Promoter DNA binding was studied for CD40 promoters, and verified two pTyr residues in NF-κB p50/p65 heterodimers that facilitated this process, and that support the possible importance of phosphorylation stoichiometry. This study defines a new approach for studying tyrosine residues whose phosphorylation alters protein binding to DNA promoters, and contributes to the facility of DNA expression.

An assay for DNA polymerase ß lyase inhibitors that engage the catalytic nucleophile for binding.

DNA polymerase ß (Pol ß) repairs cellular DNA damage. When such damage is inflicted upon the DNA in tumor cells treated with DNA targeted antitumor agents, Pol ß thus diminishes their efficacy. Accordingly, this enzyme has long been a target for antitumor therapy. Although numerous inhibitors of the lyase activity of the enzyme have been reported, none has yet proven adequate for development as a therapeutic agent. In the present study, we developed a new strategy to identify lyase inhibitors that critically engage the lyase active site primary nucleophile Lys72 as part of the binding interface. This involves a parallel evaluation of the effect of the inhibitors on the wild-type DNA polymerase ß (Pol ß) and Pol ß modified with a lysine analogue at position 72. A model panel of five structurally diverse lyase inhibitors identified in our previous studies (only one of which has been published) with unknown modes of binding were used for testing, and one compound, cis-9,10-epoxyoctadecanoic acid, was found to have the desired characteristics. This finding was further corroborated by in silico docking, demonstrating that the predominant mode of binding of the inhibitor involves an important electrostatic interaction between the oxygen atom of the epoxy group and Nε of the main catalytic nucleophile, Lys72. The strategy, which is designed to identify compounds that engage certain structural elements of the target enzyme, could find broader application for identification of ligands with predetermined sites of binding.

Effects of Nucleobase Amino Acids on the Binding of Rob to Its Promoter DNA: Differential Alteration of DNA Affinity and Phenotype.

Nucleic acid binding proteins have been studied extensively, but the nature of the interactions that control their affinity, selectivity, and DNA and RNA functions is still not well understood. To understand the nature and functional consequences of such interactions, we introduced nucleobase amino acids at specific positions of the transcriptional regulator Rob protein in vivo and succeeded in demonstrating that an alteration of the protein-DNA affinity can affect specific phenotypes associated with Rob protein-DNA interactions. Previously, we inserted different nucleobase amino acids in lieu of Arg40; this residue is known (via X-ray crystallography) to interact with the micF DNA promoter A-box residue Gua6. The interactions predominantly involved Watson-Crick-like H bonding. The present study focused primarily on the micF DNA promoter B-box; the crystallographically determined interaction involves H bonding between the agmatine moiety of Arg90 within an HTH motif of Rob and a phosphate oxygen anion to the 5'-side of Thy14. We had two main goals, the first of which was to demonstrate enhanced Rob-binding to the micF promoter DNA and the functional consequences resulting from the interaction of micF DNA with Rob analogues containing Arg90 nucleobase mimics. The second was to explore the possible functional consequences of enhancing the protein-DNA affinity with nucleobase replacements, which mechanistically mediate interactions differently than those reported to be operative for specific protein-DNA interactions. Nucleobase replacement at position 90 with Arg isosteres enhanced the Rob protein-micF DNA affinity in parallel with increasing antibiotic and Hg2+ resistance, while aromatic amino acid replacements increased the affinity but not the antibiotic or Hg2+ resistance. The demonstration of an increased affinity through strong base stacking interactions was notable.

Preparation of Poloxamer188-b-PCL and Study on in vitro Radioprotection Activity of Curcumin-Loaded Nanoparticles.

A novel polymer of poloxamer188-b-PCL was synthesized via a ring-opening polymerization. Fourier transform infrared spectroscopy (FTIR), Raman, and 1H nuclear magnetic resonance (1H NMR) spectra were used to study the structures of obtained poloxamer188-b-PCL. The thermo-stability of poloxamer188 -b-PCL was carried out with a thermal gravimetric analyzer (TGA), and cytotoxicity was obtained using the CCK8 method. Cargo-free and curcumin (CUR)-loaded poloxamer188-b-PCL NPs were fabricated via the solvent evaporation method. The morphology, particle size distribution, and stability of cargo-free NPs were studied with a scanning electron microscope (SEM) and laser particle analyzer. The in vitro radioprotection activity of CUR-loaded NPs was performed. FTIR, Raman, and 1H NMR spectra confirmed that poloxamer188-b-PCL was obtained. TGA curves suggested poloxamer188-b-PCL had better thermo-stability than original poloxamer188. Cell tests suggested that the cargo-free NPs had no cytotoxicity. SEM image showed that the cargo-free NPs were spherical with a diameter of 100 nm. Free radical scavenging experiments proved that CUR-loaded NPs had better antioxidant activity than CUR solutions. CUR-loaded NPs could be detected in all tissues, including liver, kidneys and lung. In summary, this work demonstrated a feasibility of developing an injective formulation of CUR and provided a protection agent in caner radiotherapy.

Alteration of Transcriptional Regulator Rob In Vivo: Enhancement of Promoter DNA Binding and Antibiotic Resistance in the Presence of Nucleobase Amino Acids.

The identification of proteins that bind selectively to nucleic acid sequences is an ongoing challenge. We previously synthesized nucleobase amino acids designed to replace proteinogenic amino acids; these were incorporated into proteins to bind specific nucleic acids predictably. An early example involved selective cell free binding of the hnRNP LL RRM1 domain to its i-motif DNA target via Watson-Crick-like H-bonding interactions. In this study, we employ the X-ray crystal structure of transcriptional regulator Rob bound to its micF promoter, which occurred without DNA distortion. Rob proteins modified in vivo with nucleobase amino acids at position 40 exhibited altered DNA promoter binding, as predicted on the basis of their Watson-Crick-like H-bonding interactions with promoter DNA A-box residue Gua-6. Rob protein expression ultimately controls phenotypic changes, including resistance to antibiotics. Although Rob proteins with nucleobase amino acids were expressed in Escherichia coli at levels estimated to be only a fraction of that of the wild-type Rob protein, those modified proteins that bound to the micF promoter more avidly than the wild type in vitro also produced greater resistance to macrolide antibiotics roxithromycin and clarithromycin in vivo, as well as the ß-lactam antibiotic ampicillin. Also demonstrated is the statistical significance of altered DNA binding and antibiotic resistance for key Rob analogues. These preliminary findings suggest the ultimate utility of nucleobase amino acids in altering and controlling preferred nucleic acid target sequences by proteins, for probing molecular interactions critical to protein function, and for enhancing phenotypic changes in vivo by regulatory protein analogues.

Double-component diazeniumdiolate derivatives as anti-cancer agents.

In this study, we synthesized a series of double-component O2-aryl diazeniumdiolate (DDNO) derivatives, of which each molecule can release up to four nitric oxide molecules. These compounds showed cytotoxic activities to cancer cells, such as human leukemia, breast cancer and lung cancer. Among them, compound 1 (DDNO-1) showed the highest specific activity to human leukemia cells. It induced cell apopotosis and arrest cell cycle of G2/M phase. The JNK and p38 protein kinases were activated by compound 1 to induce cancer cell apoptosis. Compound 1 also increased pro-apoptotic Bax level, which is a same function compared to a reported NO donor, JS-K. More interestingly, it decreased the level of an anti-apoptotic member Bcl-2, which is an opposite effect compared to JS-K. Compound 1 could be developed as a new anti-cancer agent since it increases the Bax/Bcl-2 ratio to overcome the drug resistance.

Enhancing Antitumor Efficacy of Nucleoside Analog 5-Fluorodeoxyuridine on HER2-Overexpressing Breast Cancer by Affibody-Engineered DNA Nanoparticle.

BACKGROUND: Chemotherapy, as an adjuvant treatment strategy for HER2-positive breast cancer, can effectively improve clinical symptoms and overcome the drug resistance of therapeutic monoclonal antibodies. Nucleoside analogues are a class of traditional chemotherapeutic drugs that are widely applied in adjuvant therapy. However, there are many critical issues that limit their clinical efficiency, including poor selectivity and stability, severe side effects and suboptimal therapeutic efficacy. Hence, this work aims to develop a new DNA nanocarrier for targeted drug delivery to solve the above problems. METHODS: Four 41-mer DNA strands were synthesized and 10 FUdR molecules were attached to 5' end of each DNA strand by DNA solid-phase synthesis. An affibody molecule was connected to the end of polymeric FUdR through a linker in one of the four strands. The affibody-FUdR-tetrahedral DNA nanostructures (affi-F/TDNs) were self-assembled through four DNA strands, in which one vertex was connected to an affibody at the end of a polymeric FUdR tail and three vertices were only polymeric FUdR tails. In vitro cellular uptake of affi-F/TDNs was examined visually with confocal fluorescence microscopy and flow cytometry, and the cytotoxicity of affi-F/TDNs against cancer cells was investigated with MTT assay. Cell apoptosis was detected by Annexin V-FITC/PI double staining method. Using NOD/SCID (Mus Musculus) mice model, the targeted killing efficacy of affi-F/TDNs was also evaluated. RESULTS: The drug-loading of FUdR in affi-TDNs was 19.6% in mole ratio. The in vitro results showed that affi-F/TDNs had high selectivity and inhibition (81.2%) for breast cancer BT474 cells overexpressing HER2 and low toxicity in MCF-7 cells with low HER2 expression. During the in vivo application, affi-F/TDNs displayed good stability in the blood circulation, achieved specific accumulation in tumor region and the best antitumor efficacy (inhibition ratio of 58.1%), and showed excellent biocompatibility. CONCLUSIONS: The affibody-DNA tetrahedrons, as a simple and effective active targeting delivery nanocarrier, provided a new avenue for the transport of nucleoside antitumor drugs.

Selective Detection of Dengue Virus Serotypes Using Tandem Toehold-Mediated Displacement Reactions.

Dengue virus (DENV) is the most common human arboviral infection worldwide and can present with severe clinical manifestations. Timely DENV detection improves clinical outcomes, and identification of the DENV serotype (DENV-1-4) may provide beneficial epidemiologic data to inform the initiation of control measures. Here, DENV RNA-triggered, enzyme-free tandem toehold-mediated displacement reactions were developed to identify and serotype DENV in RNA controls and contrived samples through the amplification of a fluorescent signal detected by the use of a fluorescent scanner and a confocal microscope. Each DENV serotype was detected selectively using both imaging methods. In addition, a 384-well plate was used to prepare an array for diagnosis of the four DENV RNA serotypes from contrived clinical samples. The four serotypes of dengue virus were detected using novel enzyme-free amplification reactions, which are more facile than amplification using reverse transcriptase PCR.

In Cellulo Synthesis of Proteins Containing a Fluorescent Oxazole Amino Acid.

Genetic code expansion has enabled many noncanonical amino acids to be incorporated into proteins in vitro and in cellulo. These have largely involved α-l-amino acids, reflecting the substrate specificity of natural aminoacyl-tRNA synthetases and ribosomes. Recently, modified E. coli ribosomes, selected using a dipeptidylpuromycin analogue, were employed to incorporate dipeptides and dipeptidomimetics. Presently, we report the in cellulo incorporation of a strongly fluorescent oxazole amino acid (lacking an asymmetric center or α-amino group) by using modified ribosomes and pyrrolysyl-tRNA synthetase (PylRS). Initially, a plasmid encoding the RRM1 domain of putative transcription factor hnRNP LL was cotransformed with plasmid pTECH-Pyl-OP in E. coli cells, having modified ribosomes able to incorporate dipeptides. Cell incubation in a medium containing oxazole 2 resulted in the elaboration of RRM1 containing the oxazole. Green fluorescent protein, previously expressed in vitro with several different oxazole amino acids at position 66, was also expressed in cellulo containing oxazole 2; the incorporation was verified by mass spectrometry. Finally, oxazole 2 was incorporated into position 13 of MreB, a bacterial homologue of eukaryotic cytoskeletal protein actin F. Modified MreB expressed in vitro and in cellulo comigrated with wild type. E. coli cells expressing the modified MreB were strongly fluorescent and retained the E. coli cell rod-like phenotype. For each protein studied, the incorporation of oxazole 2 strongly increased oxazole fluorescence, suggesting its potential utility as a protein tag. These findings also suggest the feasibility of dramatically increasing the repertoire of amino acids that can be genetically encoded for protein incorporation in cellulo.

DNA-affibody nanoparticle delivery system for cisplatin-based breast cancer chemotherapy.

Cisplatin is the most widely used anticancer drug, but its side effects limit the maximum systemic dose. To circumvent the side effects, a DNA tetrahedron-affibody nanoparticle was prepared by combination of a DNA chain with cisplatin via interstrand crosslinks or adducts. Each nanocarrier can bind ∼68 molecules of cisplatin. This cisplatin nanoparticle exhibited high selectivity and inhibition for breast cancer HER2 overexpressing cells BT474 and lower toxicity in MCF-7 cells with low HER2 expression. The nano-drug inhibited the growth of BT474 cells by 94.57% at 512 nM (containing 33.3 µM cisplatin), which was higher than that of cisplatin (82.9%, 33.3 µM).

Rapid detection of a dengue virus RNA sequence with single molecule sensitivity using tandem toehold-mediated displacement reactions.

A DNA tetrahedron having a single-strand DNA edge was used to detect a dengue virus RNA sequence. Novel tandem toehold-mediated displacement reactions (tTMDR) were developed to amplify the fluorescence signal from the DNA tetrahedron. Using an excess of the DNA tetrahedron each target RNA was recycled about 103 times during the tTMDR process. This amplification process was used for the sensitive detection of dengue virus RNA in this study. As few as 6 copies of RNA per sample could be detected using a photon count technique with single molecule sensitivity.