Fr Francium

Graft order: The putative binding epitopes of peptides of the NPY family at their cognate GPCR, the Y1 receptor, were grafted onto a stable β-barrel scaffold. We demonstrate the synthetic feasibility of such an approach and discuss its potential benefits. The integrity of the scaffold as well as its interaction with NPY was studied by using solution NMR spectroscopy.

Ansamitocins are potent antitumor agents produced by Actinosynnema pretiosum. As deduced from their structures, an N-methylation on the amide bond is required among the various modifications. The protein encoded by asm10 belongs to the SAM-dependent methyltransferase family. Through gene inactivation and complementation, asm10 was proved to be responsible for the N-methylation of ansamitocins. Asm10 is a 33.0 kDa monomer, as determined by gel filtration. By using N-desmethyl-ansamitocin P-3 as substrate, the optimal temperature and pH for Asm10 catalysis were determined to be 32 °C and 10.0, respectively. Asm10 also showed broad substrate flexibility toward other N-desmethyl-ansamycins and synthetic indolin-2-ones. Through site-directed mutagenesis, Asp154 and Leu155 of Asm10 were confirmed to be essential for its catalysis, possibly through the binding of SAM. The characterization of this unique N-methyltransferase has enriched the toolbox for engineering N-methylated derivatives from both natural and synthetic compounds; this will allow known potential drugs to be modified.

N-methylation in ansamitocins: The protein encoded by asm10 is a unique N-methyltransferase responsible for the N-methylation in the biosynthesis of ansamitocins.

Fourth amendment: RNA quadruplexes located in the 5′-untranslated regions (5′-UTR in the figure) of mRNAs inhibit gene expression. This effect can be amplified by targeting RNA quadruplexes with specific bisquinolinium compounds.

Triptolide is a potent natural product, with documented antiproliferative, immunosuppressive, anti-inflammatory, antifertility, and antipolycystic kidney disease effects. Despite a wealth of knowledge about the biology of this compound, direct intracellular target proteins have remained elusive. We synthesized a biotinylated photoaffinity derivative of triptolide, and used it to identify dCTP pyrophosphatase 1 (DCTPP1) as a triptolide-interacting protein. Free triptolide interacts directly with recombinant DCTPP1, and inhibits the enzymatic activity of this protein. Triptolide is thus the first dCTP pyrophosphatase inhibitor identified, and DCTPP1 is a biophysically validated target of triptolide.

Nucleotide denied by triptolide: Using a photoaffinity pull-down approach, we found that the natural product triptolide binds to and inhibits the function of dCTP pyrophosphatase (DCTPP1; see scheme). Triptolide will be a useful tool for probing the function of this newly characterized metabolic enzyme.

Aurachins are quinoline alkaloids isolated from the myxobacterium Stigmatella aurantiaca. They are substituted with an isoprenoid side chain and act as potent inhibitors in the electron transport chain. A biosynthetic gene cluster that contains at least five genes (auaA–auaE) has been identified for aurachin biosynthesis. In this study, auaA, the gene encoding a putative prenyltransferase of 326 amino acids, was cloned and overexpressed in Escherichia coli. Biochemical investigations showed that AuaA catalyzes the prenylation of 2-methyl-4-hydroxyquinoline in the presence of farnesyl diphosphate (FPP), thereby resulting in the formation of aurachin D. The hydroxyl group at position C4 of the quinoline ring is essential for an acceptance by AuaA; this was concluded by testing 18 quinoline derivatives or analogues with AuaA and FPP. ¹H NMR and HR-EI-MS analyses of six isolated enzyme products revealed the presence of a farnesyl moiety at position C3 of the quinoline ring. K_M values of 43 and 270 μM were determined for FPP and 2-methyl-4-hydroxyquinoline, respectively. Like other known membrane-bound prenyltransferases, the reaction catalyzed by AuaA is dependent on the presence of metal ions such as Mg²⁺, Mn²⁺ and Co²⁺, although no typical (N/D)DXXD binding motif was found in the sequence.

To strut and prenylate: In the biosynthesis of aurachins in the myxobacterium Stigmatella aurantiaca, membrane-bound prenyltransferase AuaA catalyzes the formation of aurachin D by transfering a farnesyl moiety from FPP to position C3 of 2-methyl-4-hydroxyquinoline. With improved catalysis rates, this unusual enzyme could have useful biotechnological applications.

Substantial evidence suggests that soluble prefibrillar oligomers of the Aβ42 peptide associated with Alzheimer's disease are the most cytotoxic aggregated Aβ isoform. Limited previous work has revealed that aromatic compounds capable of remodeling Aβ oligomers into nontoxic conformers typically do so by converting them into off-pathway aggregates instead of dissociating them into monomers. Towards identifying small-molecule antagonists capable of selectively dissociating toxic Aβ oligomers into soluble peptide at substoichiometric concentrations, we have investigated the pathways used by polyphenol aglycones and their glycosides to remodel Aβ soluble oligomers. We find that eleven polyphenol aglycones of variable size and structure utilize the same remodeling pathway whereby Aβ oligomers are rapidly converted into large, off-pathway aggregates. Strikingly, we find that glycosides of these polyphenols all utilize a distinct remodeling pathway in which Aβ oligomers are rapidly dissociated into soluble, disaggregated peptide. This disaggregation activity is a synergistic combination of the aglycone and glycone moieties because combinations of polyphenols and sugars fail to disaggregate Aβ oligomers. We also find that polyphenolic glycosides and aglycones use the same opposing pathways to remodel Aβ fibrils. Importantly, both classes of polyphenols fail to remodel nontoxic Aβ oligomers (which are indistinguishable in size and morphology to Aβ soluble oligomers) or promote aggregation of freshly disaggregated Aβ peptide; thus revealing that they are specific for remodeling toxic Aβ conformers. We expect that these and related small molecules will be powerful chemical probes for investigating the conformational and cellular underpinnings of Aβ-mediated toxicity.

Correcting the fold: We have investigated the pathways used by polyphenol aglycones and their glycosides to selectively remodel Aβ soluble oligomers into nontoxic conformers. We find that polyphenol aglycones rapidly remodel Aβ oligomers into off-pathway aggregates, while their glycosides rapidly dissociate Aβ oligomers into monomers.

Despite their flexible natures, glycine residues adopt only a subset of their sterically available conformations in protein structures. With the aid of quantum calculations we have found that hyperconjugation plays a significant role in influencing glycine conformations by stabilizing planar backbone orientations.

Have you Met this? Methionine prototrophic E. coli harboring a single genomic copy of an engineered methionyl-tRNA synthetase (MetRS) gene, metG*, are more efficient at incorporating azidonorleucine (ANL) into recombinant proteins than prototrophic E. coli containing a multicopy plasmid expressing metG* and a genomic copy of the native MetRS (metG).

Twinkle, twinkle little bone! A short peptide derived from a natural bone binding protein, osteocalcin, shows excellent selectivity and affinity to hydroxyapatite, the main component of bone. With a fluorescent label, the intravenously injected peptide revealed detailed bone structures in mice, like an X-ray image (see figure).

The effect of exposure of guanine in double-stranded oligonucleotides to aqueous solvent during oxidation by one-electron oxidants was investigated by introducing single synthetic tetrahydrofuran-type abasic sites (Ab) either adjacent to or opposite tandem GG sequences. The selective oxidation of guanine was initiated by photoexcitation of the aromatic sensitizers riboflavin and a pyrene derivative, and by the relatively small negatively charged carbonate radical anion. The relative rates of oxidation of the 5′- and 3′ side G in runs of 5′⋅⋅⋅GG⋅⋅⋅ (evaluated by standard hot alkali treatment of the damaged DNA strand followed by high resolution gel electrophoresis of the cleavage fragments) are markedly affected by adjacent abasic sites either on the same or opposite strand. For example, in fully double-stranded DNA or one with an Ab adjacent to the 5′-G, the 5′-G/3′-G damage ratio is ≥4, but is inverted (<1.0) with the Ab adjacent to the 3′-G. These striking effects of Ab are attributed to the preferential localization of the “hole” on the most solvent-exposed guanine regardless of the size, charge, or reduction potential of the oxidizing species.

Damage assessment: We have investigated the effects of solvent exposure on the oxidation of guanine in GG sequences in oligonucleotide duplexes. The results show that a single abasic site (Ab) inverts the extent of damage in GG sequences induced by one-electron oxidation when it flanks the 3′ side guanine in double-stranded DNA (see histograms showing relative oxidation at G sites).