Fr Francium

Alzheimer's disease (AD) and type 2 diabetes (T2D) are linked to the self-association of β-amyloid peptide (Aβ) and islet amyloid polypeptide (IAPP), respectively. We have shown that IAPP-GI, a soluble IAPP analogue and mimic of nonamyloidogenic and nontoxic IAPP, binds Aβ with high affinity and blocks its cytotoxic self-assembly and fibrillogenesis. We have also shown that IAPP and Aβ interact with each other into nonfibrillar and nontoxic heterocomplexes that suppress cytotoxic self-association by both polypeptides. The Aβ–IAPP interaction might thus be a molecular link between AD and T2D. We studied the role of individual IAPP-GI and IAPP regions in their inhibitory function on Aβ40 self-association and cytotoxicity. We found that the presence of the two hot-spot regions of the Aβ–IAPP interaction interface in IAPP(8–28) is not sufficient for inhibitory function and that, in addition to IAPP(8–28), the presence of the N-terminal region IAPP(1–7) is absolutely required. By contrast, the C-terminal region, IAPP(30–37), is not required although its presence together with IAPP(1–7) in IAPP-GI results in a marked enhancement of the inhibitory effect as compared to IAPP(1–28)-GI. We suggest that the inhibitory effect of IAPP-GI and IAPP on Aβ40 fibrillogenesis and cell toxicity is mediated primarily by interactions involving the hot regions of the Aβ–IAPP interaction interface and the N terminus of IAPP while a concerted and likely structure-stabilizing action of the N- and C-terminal IAPP regions potentiates this effect. These results identify important molecular determinants of the amyloid suppressing function of the Aβ40–IAPP interaction and could contribute to the design of novel inhibitors of Aβ40 aggregation and cell degeneration.

Block buster: We have previously found that the interaction of the type 2 diabetes islet amyloid polypeptide (IAPP) and its nonamyloidogenic mimic IAPP-GI with the Alzheimer's disease β-amyloid peptide (Aβ) blocks Aβ aggregation and cytotoxicity. Here we uncover the role of individual IAPP-GI and IAPP regions in the inhibitory function on Aβ40 amyloidogenesis and cytotoxicity and suggest a molecular basis for this effect.

Althiomycin biosynthetic gene cluster: The potent broad-spectrum antibiotic althiomycin is a product of a nonribosomal peptide/polyketide assembly line. The biosynthetic gene cluster from the natural producer Myxococcus xanthus DK897 was identified and characterized.

Cubane revolution: We have constructed various mutants to probe the role of [Fe₄S₄] cubane. The results show that [Fe₄S₄] cubane, which is bridged to a [Ni_pNi_d] center via Cys509 cysteinate in the A-cluster of acetyl-coenzyme A synthase (see figure), modulates the redox properties of the catalytic metal Ni_p and stabilizes the low valence of Ni_p through the {[Fe₄S₄](SR)Ni_p} “conjugation system”.

Quadruple alliance: By performing a screen for binders of the human telomeric repeat sequence with small molecule libraries, the principle of which is outlined in the scheme, we have identified novel compounds that stabilise the G quadruplex structure. Some of these could be suitable leads for further optimisation as potent and selective quadruplex ligands.

Aspergillus oryzae is a fungus widely used in traditional Japanese fermentation industries. Its inability to produce mycotoxins, due to mutation or transcriptional repression of the genes responsible for their biosynthesis, is consistent with the hypothesis that A. oryzae is a domesticated species derived from A. flavus, a wild species that is a well-known producer of aflatoxin. In contrast, the cyclopiazonic acid (CPA) biosynthetic gene (cpa) cluster in A. oryzae contains genes that have been lost in A. flavus. Through targeted gene inactivation, isolation of the corresponding metabolite, and evaluation of biological activity of the metabolite, we demonstrated that an A. oryzae-specific gene—cpaH—mediates the conversion of CPA into the less toxic 2-oxocyclopiazonic acid, a new analogue of CPA. The detoxifying properties of cpaH, which have been lost in the A. flavus pathway, reflect the relationship of the two species.

The end product of a more ancient four-step cyclopiazonic acid pathway existing in the domesticated fungus A. oryzae is less toxic than that of the prevalent three-step pathway in the wild fungus A. flavus. The detoxifying properties of cpaH, which have been lost in the A. flavus pathway, reflect the relationship of the two species.

Biofilm, friend not foe: Single species biofilms can be engineered to form robust biocatalysts with greater catalytic activity and significantly improved catalytic longevity than purified and immobilised enzymes. We report the engineering, structural analysis and biocatalytic capability of a biofilm that can mediate the conversion of serine and haloindoles to halotryptophans (see scheme).

Biotin protein ligase (BPL) mediates the covalent attachment of biotin to a specific lysine residue of biotin carboxyl carrier protein (BCCP). This biotinylation in Sulfolobus tokodaii is unique in that BPL forms a tight complex with the product, biotinylated BCCP, and this property was exploited for fluorescent labeling of a membrane protein. Thus, the truncated form of BCCP (BCCPΔ100, 69 residues) was fused to either the N or C terminus of the bradykinin B2 receptor (B2R). The resulting fusion proteins, BCCPΔ100–B2R and B2R–BCCPΔ100, respectively, were separately expressed in mammalian HEK293 cells, and labeled with BPL conjugated with a fluorophore: either fluorescein, DyLight549 or green fluorescent protein. The fusion proteins were biotinylated and bound to BPL, thereby giving rise to strong fluorescence along the periphery of the cell. Some were capable of binding bradykinin and an antagonist. When stimulated with the former, the receptor translocated to the cytosol; this suggests that the labeled receptor retains its integrity in terms of ligand-binding and translocation.

Protein labeling in living cells: We describe a fluorescent protein-labeling method that uses a unique biotinylation mechanism from the archaeon Sulfolobus tokodaii. A membrane protein fused to the biotin acceptor sequence (BCCP) was labeled by using biotin protein ligase (BPL) conjugated with fluorophores, both outside and inside the cell.

The family-B DNA polymerases obtained from the order Thermococcales, for example, Pyrococcus furiosus (Pfu-Pol) are commonly used in the polymerase chain reaction (PCR) because of their high thermostability and low error rates. Most of these polymerases contain four cysteines, arranged as two disulfide bridges. With Pfu-Pol C429–C443 forms one of the disulfides (DB1) and C507–C510 (DB2) the other. Although the disulfides are well conserved in the enzymes from the hyperthermophilic Thermococcales, they are less prevalent in euryarchaeal polymerases from other orders, and tend to be only found in other hyperthermophiles. Here, we report on the effects of deleting the disulfide bridges by mutating the relevant cysteines to serines. A variety of techniques, including differential scanning calorimetry and differential scanning fluorimetry, have shown that both disulfides make a contribution to thermostability, with DB1 being more important than DB2. However, even when both disulfides are removed, sufficient thermostability remains for normal (identical to the wild type) performance in PCR and quantitative (real-time) PCR. Therefore, polymerases totally lacking cysteine are fully compatible with most PCR-based applications. This observation opens the way to further engineering of polymerases by introduction of a single cysteine followed by appropriate chemical modification.

Some like it hot: The family-B DNA polymerases from the Thermococcales are extensively used in PCR. They usually contain two disulfide bridges (see figure), the role of which in thermostability has been probed with site directed mutagenesis. The disulfides play a role in stability but even deletion of both does not compromise PCR performance. This opens the way to single cysteine variants, amenable to useful chemical modification.

Making it traceless: A traceless Staudinger reaction between a specifically functionalized sugar phosphine and an azide-tagged protein allows the construction of well-defined reverse N-linked glycoproteins (see scheme). This reaction was applied to different azidoprotein substrates and its potentials and limitations are outlined.

Perillyl alcohol is the terminal hydroxylation product of the cheap and readily available terpene, limonene. It has high potential as an anti-tumor substance, but is of limited availability. In principle, cytochrome P450 monooxygenases, such as the self-sufficient CYP102A1, are promising catalysts for the oxidation of limonene or other inert hydrocarbons. The wild-type enzyme converts (4R)-limonene to four different oxidation products; however, terminal hydroxylation at the allylic C7 is not observed. Here we describe a generic strategy to engineer this widely used enzyme to hydroxylate exclusively the exposed, but chemically less reactive, primary C7 in the presence of other reactive positions. The approach presented here turns CYP102A1 into a highly selective catalyst with a shifted product spectra by successive rounds of modeling, the design of small focused libraries, and screening. In the first round a minimal CYP102A1 mutant library was rationally designed. It contained variants with improved or strongly shifted regio-, stereo- and chemoselectivity, compared to wild-type. From this library the variant with the highest perillyl alcohol ratio was fine-tuned by two additional rounds of molecular modeling, diversification, and screening. In total only 29 variants needed to be screened to identify the triple mutant A264V/A238V/L437F that converts (4R)-limonene to perillyl alcohol with a selectivity of 97 %. Focusing mutagenesis on a small number of relevant positions identified by computational approaches is the key for efficient screening for enzyme selectivity.

Successive rounds of modeling (MD simulations of enzyme–substrate complexes to identify hotspots for selectivity), diversification (design of minimal library) and screening of a minimal library is shown to be an efficient approach to shift and maximize regioselectivity of CYP102A1, and thus to generate the valuable oxidation product perillyl alcohol from cheap and readily available limonene.