Cofactor binding and enzymatic activity in an unevolved superfamily of de novo designed 4‐helix bundle proteins

To probe the potential for enzymatic activity in unevolved amino acid sequence space, we created a combinatorial library of de novo 4‐helix bundle proteins. This collection of novel proteins can be considered an “artificial superfamily” of helical bundles. The superfamily of 102‐residue proteins was designed using binary patterning of polar and nonpolar residues, and expressed in Escherichia coli from a library of synthetic genes. Sequences from the library were screened for a range of biological functions including heme binding and peroxidase, esterase, and lipase activities. Proteins exhibiting these functions were purified and characterized biochemically. The majority of de novo proteins from this superfamily bound the heme cofactor, and a sizable fraction of the proteins showed activity significantly above background for at least one of the tested enzymatic activities. Moreover, several of the designed 4‐helix bundles proteins showed activity in all of the assays, thereby demonstrating the functional promiscuity of unevolved proteins. These studies reveal that de novo proteins—which have neither been designed for function, nor subjected to evolutionary pressure (either in vivo or in vitro)—can provide rudimentary activities and serve as a “feedstock” for evolution.     

Inhibition of Arabidopsis growth by the allelopathic compound azetidine‐2‐carboxylate is due to the low amino acid specificity of cytosolic prolyl‐tRNA synthetase

The toxicity of azetidine‐2‐carboxylic acid (A2C), a structural analogue of L‐proline, results from its incorporation into proteins due to misrecognition by prolyl‐tRNA synthetase (ProRS). The growth of Arabidopsis thaliana seedling roots is more sensitive to inhibition by A2C than is cotyledon growth. Arabidopsis contains two ProRS isozymes. AtProRS‐Org (At5g52520) is localized in chloroplasts/mitochondria, and AtProRS‐Cyt (At3g62120) is cytosolic. AtProRS‐Cyt mRNA is more highly expressed in roots than in cotyledons. Arabidopsis ProRS isoforms were expressed as His‐tagged recombinant proteins in Escherichia coli. Both enzymes were functionally active in ATP‐PPi exchange and aminoacylation assays, and showed similar K_m for L‐proline. A major difference was observed in the substrate specificity of the two enzymes. AtProRS‐Cyt showed nearly identical substrate specificity for L‐proline and A2C, but for AtProRS‐Org the specificity constant was 77.6 times higher for L‐proline than A2C, suggesting that A2C‐sensitivity may result from lower amino acid specificity of AtProRS‐Cyt. Molecular modelling and simulation results indicate that this specificity difference between the AtProRS isoforms may result from altered modes of substrate binding. Similar kinetic results were obtained with the ProRSs from Zea mays, suggesting that the difference in substrate specificity is a conserved feature of ProRS isoforms from plants that do not accumulate A2C and are sensitive to A2C toxicity. The discovery of the mode of action of A2C toxicity could lead to development of biorational weed management strategies.     

Synthesis and application of N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde)‐protected amino acids for optimization of solid‐phase peptide synthesis using gel‐phase 19F NMR spectroscopy

N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde) protected amino acids have been prepared and applied in solid‐phase peptide synthesis monitored by gel‐phase ¹⁹F NMR spectroscopy. The Fde protective group could be cleaved with 2% hydrazine or 5% hydroxylamine solution in DMF as determined with gel‐phase ¹⁹F NMR spectroscopy. The dipeptide Ac‐L ‐Val‐L ‐Val‐NH₂ 12 was constructed using Fde‐L ‐Val‐OH and no noticeable racemization took place during the amino acid coupling with N,N′‐diisopropylcarbodiimide and 1‐hydroxy‐7‐azabenzotriazole or Fde deblocking. To extend the scope of Fde protection, the hydrophobic nonapeptide LLLLTVLTV from the signal sequence of mucin MUC1 was successfully prepared using Fde‐L ‐Leu‐OH at diagnostic positions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The relative contributions of diet and associated microbiota to the accumulation of UV‐absorbing mycosporine‐like amino acids in the freshwater copepod Boeckella antiqua

1. Mycosporine‐like amino acids (MAAs) are ubiquitous compounds in aquatic organisms that are usually considered sunscreens that protect them from harmful ultraviolet radiation. Given that virtually all animals lack the metabolic pathways to synthesise MAAs de novo, they must acquire them either from their diet or from microorganisms living in close association. In freshwater copepods, accumulation of MAAs is stimulated by exposure to ultraviolet and/or visible radiation. 2. A 2 × 2 factorial experiment was performed to assess the contributions of dietary and microbial sources of MAAs in the freshwater copepod Boeckella antiqua. The treatments consisted of two different diets: an MAA‐free diet, including only Chlamydomonas reinhardtii, and an MAA‐rich diet, including both C. reinhardtii and Peridinium inconspicuum, crossed with two antibiotic treatments, with and without chloramphenicol. Treatment with chloramphenicol was intended to inhibit the development of bacteria associated with the copepods. 3. MAA concentration in B. antiqua was affected by the experimental conditions: (i) exposure to artificial PAR + UVR stimulated the accumulation of several MAAs (up to 62% increase in total MAA concentration with respect to the initial concentration); (ii) the presence of chloramphenicol in the culture media reduced the MAA concentration in copepods fed an MAA‐free diet; (iii) in the absence of chloramphenicol, copepods fed the MAA‐rich diet had significantly higher total MAA concentration than those fed the MAA‐deficient diet; but (iv) dietary supplementation with an MAA‐rich algae in the presence of chloramphenicol failed to significantly increase total MAA concentration. 4. Analysis of profiles from denaturing gradient gel electrophoresis (DGGE) showed that the prokaryotic community associated with the copepods was affected by chloramphenicol. Dendograms constructed from digitalised DGGE images consistently grouped the antibiotics treatments separately from the initial samples and the treatments without antibiotics. Two band positions were exclusive to treatments without antibiotics. 5. We conclude that when offered an MAA‐rich diet, B. antiqua may accumulate a proportion of MAAs from diet. However, we suspect that in the absence of an MAA‐rich dietary source (as in its natural habitat), virtually all MAAs present in B. antiqua are produced by copepod‐associated prokaryotes.     

Use of high resolution melting analysis to genotype the avirulence AvrLm4‐7 gene of Leptosphaeria maculans,a fungal pathogen of Brassica napus

The oilseed rape (Brassica napus) stem canker disease, due to the fungal pathogen Leptosphaeria maculans, is mainly controlled by host genetic resistance. Since 2004, the specific resistance gene Rlm7 is widely used in France. Specific resistance is effective when fungal populations are mainly composed of avirulent isolates. The development of molecular tools for the identification of virulent isolates towards Rlm7 was needed to undertake large‐scale surveys and to monitor the emergence of virulent populations in fields. Previous studies have described a large diversity of molecular events leading to virulence towards Rlm7, rendering conventional polymerase chain reaction (PCR) methods inapplicable to identify virulent isolates. Interestingly, a very limited nucleotide polymorphism was observed for avirulent, AvrLm7, alleles. Such characteristics were exploited here to develop a diagnostic method based on high resolution melting (HRM) analysis of the AvrLm4‐7 gene. High resolution melting analysis of a collection of 206 reference isolates revealed only four different profiles within 100 avirulent isolates and 87% of virulent isolates showed either no amplification or HRM curves distinct from those of avirulent isolates. The reliability of the method was confirmed using a second set of 119 unknown isolates, for which biological phenotyping and HRM genotyping were in agreement for 93% of the isolates. HRM combined with the PCR amplification of a larger fragment encompassing AvrLm4‐7 led to a correct diagnostic for 97.5% of the isolates.     

The aspartimide problem persists: Fluorenylmethyloxycarbonyl‐solid‐phase peptide synthesis (Fmoc‐SPPS) chain termination due to formation of N‐terminal piperazine‐2,5‐diones

Aspartimide (Asi) formation is a notorious side reaction in peptide synthesis that is well characterized and described in literature. In this context, we observed significant amounts of chain termination in Fmoc‐SPPS while synthesizing the N‐terminal Xaa‐Asp‐Yaa motif. This termination was caused by the formation of piperazine‐2,5‐diones. We investigated this side reaction using a linear model peptide and independently synthesizing its piperazine‐2,5‐dione derivative. Nuclear magnetic resonance (NMR) data of the side product present in the crude linear peptide proves that exclusively the six‐membered ring is formed whereas the theoretically conceivable seven‐membered 1,4‐diazepine‐2,5‐dione is not found. We propose a mechanism where nucleophilic attack of the N‐terminal amino function takes place at the α‐carbon of the carbonyl group of the corresponding Asi intermediate. In addition, we systematically investigated the impact of (a) different adjacent amino acid residues, (b) backbone protection, and (c) side chain protection of flanking amino acids. The side reaction is directly related to the Asi intermediate. Hence, hindering or avoiding Asi formation reduces or completely suppresses this side reaction.     

A 19‐Mb de novo deletion on BTA 22 including MITF leads to microphthalmia and the absence of pigmentation in a Holstein calf

Mutations in MITF lead to a large variety of phenotypes in human, mice and other species. They mostly affect pigmentation and hearing, whereas in mice, they may additionally cause microphthalmia and osteopetrosis. In this study, we report a single case of a Holstein calf with lack of pigmentation and microphthalmia born to healthy parents. Mendelian analysis of high‐density SNP genotypes reveals a large number of parentage errors showing missing paternal alleles in the offspring, indicating a deletion encompassing 19 Mb on BTA 22. The genomic deletion affects the paternal allele and includes MITF and 131 other annotated genes. As the calf shows only one copy of the BTA 22 segment, the observed phenotype is probably caused by haploinsufficiency of the genes in that genomic region. Both the observed lack of skin pigmentation and reduced eye size can most likely be explained by a lack of MITF function.     

Combined computational design of a zinc‐binding site and a protein–protein interaction: One open zinc coordination site was not a robust hotspot for de novo ubiquitin binding

We computationally designed a de novo protein–protein interaction between wild‐type ubiquitin and a redesigned scaffold. Our strategy was to incorporate zinc at the designed interface to promote affinity and orientation specificity. A large set of monomeric scaffold surfaces were computationally engineered with three‐residue zinc coordination sites, and the ubiquitin residue H68 was docked to the open coordination site to complete a tetrahedral zinc site. This single coordination bond was intended as a hotspot and polar interaction for ubiquitin binding, and surrounding residues on the scaffold were optimized primarily as hydrophobic residues using a rotamer‐based sequence design protocol in Rosetta. From thousands of independent design simulations, four sequences were selected for experimental characterization. The best performing design, called Spelter, binds tightly to zinc (K_d < 10 nM) and binds ubiquitin with a K_d of 20 µM in the presence of zinc and 68 µM in the absence of zinc. Mutagenesis studies and nuclear magnetic resonance chemical shift perturbation experiments indicate that Spelter interacts with H68 and the target surface on ubiquitin; however, H68 does not form a hotspot as intended. Instead, mutation of H68 to alanine results in tighter binding. Although a 3/1 zinc coordination arrangement at an interface cannot be ruled out as a means to improve affinity, our study led us to conclude that 2/2 coordination arrangements or multiple‐zinc designs are more likely to promote high‐affinity protein interactions. Proteins 2013; 81:1245–1255. © 2013 Wiley Periodicals, Inc.     

Aβ1–42 oligomer‐induced leakage in an in vitro blood–brain barrier model is associated with up‐regulation of RAGE and metalloproteinases,and down‐regulation of tight junction scaffold proteins

Accumulating evidence indicates that abnormal deposition of amyloid‐β (Aβ) peptide in the brain is responsible for endothelial cell damage and consequently leads to blood–brain barrier (BBB) leakage. However, the mechanisms underlying BBB disruption are not well described. We employed an monolayer BBB model comprising bEnd.3 cell and found that BBB leakage was induced by treatment with Aβ_1–42, and the levels of tight junction (TJ) scaffold proteins (ZO‐1, Claudin‐5, and Occludin) were decreased. Through comparisons of the effects of the different components of Aβ_1–42, including monomer (Aβ_1–42‐Mono), oligomer (Aβ_1–42‐Oligo), and fibril (Aβ_1–42‐Fibril), our data confirmed that Aβ_1–42‐Oligo is likely to be the most important damage factor that results in TJ damage and BBB leakage in Alzheimer's disease. We found that the incubation of bEnd.3 cells with Aβ_1–42 significantly up‐regulated the level of receptor for advanced glycation end‐products (RAGE). Co‐incubation of a polyclonal antibody to RAGE and Aβ_1–42‐Oligo in bEnd.3 cells blocked RAGE suppression of Aβ_1–42‐Oligo‐induced alterations in TJ scaffold proteins and reversed Aβ_1–42‐Oligo‐induced up‐regulation of RAGE, matrix metalloproteinase (MMP)‐2, and MMP‐9. Furthermore, we found that these effects induced by Aβ_1–42‐Oligo treatment were effectively suppressed by knockdown of RAGE using small interfering RNA (siRNA) transfection. We also found that GM 6001, a broad‐spectrum MMP inhibitor, partially reversed the Aβ_1–42‐Oligo‐induced inhibitor effects in bEnd.3 cells. Thus, these results suggested that RAGE played an important role in Aβ‐induced BBB leakage and alterations of TJ scaffold proteins, through a mechanism that involved up‐regulation of MMP‐2 and MMP‐9.

     

The natural killer cell dysfunction of aged mice is due to the bone marrow stroma and is not restored by IL‐15/IL‐15Rα treatment

Immune dysfunctions in the elderly result in increased susceptibility to infectious diseases, cancer, and autoimmune diseases. Natural killer (NK) cells are bone marrow‐derived lymphocytes crucial for host defense against several infections and cancer. We have previously shown that compared to young, aged C57BL/6 mice have decreased numbers of mature NK cells in the blood, spleen, and bone marrow, resulting in susceptibility to mousepox, a lethal disease caused by ectromelia virus. Here, we describe further age‐related defects in NK cells including reduced proliferation in vivo, additional signs of immaturity, and dysregulated expression of activating and inhibitory receptors. Aging also alters the expression of collagen‐binding integrins in conventional NK cells and the frequency and phenotype of liver tissue‐resident NK cells. We additionally show that the defect in NK maturation is the consequence of deficient maturational cues provided by bone marrow stromal cells. Moreover, we demonstrate that in aged mice, treatment with complexes of the cytokine IL‐15 and IL‐15Rα induce massive expansion of the NK cells, but most of these NK cells remain immature and are unable to restore resistance to mousepox. The use of rodent model to understand immunosenescence may help the development of treatments to improve the immune fitness of the aged. Our work with NK cells should contribute toward this goal.     

The two‐phase partitioning system – a powerful technique to purify integral membrane proteins of Corynebacterium glutamicum for quantitative shotgun analysis

We established a single consecutive strategy which assigned the most comprehensive number of integral membrane proteins from Gram‐positive bacteria to date. For this purpose, we adapted a biphasic partitioning system for the biotechnologically intensively used Corynebacterium glutamicum and proved for the first time that such a system is well suited for quantitative comparison. 297 integral membrane proteins were identified by our integrated approach, which depletes stringently cytosolic proteins. In combination with our previously developed SIMPLE strategy, our data comprise 61% (374 integral membrane proteins) of the entire membrane proteome, which aims towards an almost comprehensive coverage. Wild type and a production strain of C. glutamicum were compared by ¹⁵N metabolic labelling and quantitation was obtained by spectral counting and peak areas. Both quantification strategies display a consistent trend in up or downregulation of proteins. Nevertheless, spectral counting often provides results indicating a much stronger regulation compared to ProRata values. Either spectral counting seems to exaggerate protein regulation or ProRata tends to attenuate the information about the regulation level. We highlight some of the biologically relevant candidates, which prove that our approach helps to give a deeper quantitative insight towards the understanding of transport and other membrane associated processes, important for strain development of C. glutamicum.     

Enantioselectivity of bunitrolol 4‐hydroxylation is reversed by the change of an amino acid residue from valine to methionine at position 374 of cytochrome P450‐2D6

The enantioselectivity of 4‐hydroxylation of bunitrolol (BTL), a β‐adrenoceptor blocking drug, was studied in microsomes from human liver, human hepatoma (Hep G2) cells expressing CYP2D6, and lymphoblastoid cells expressing CYP2D6. Kinetics in human liver microsomes showed that the Vmax value for (+)‐BTL was 2.1‐fold that of (−)‐BTL, and that the Km value for (+)‐BTL was lower than that for the (−)‐antipode, resulting in the intrinsic clearance (Vmax/Km) of (+)‐BTL being 2.1‐fold over its (−)‐antipode. CYP2D6 (CYP2D6‐met) expressed in Hep G2 cells had a methionine residue at position 373 of the amino acid sequence and a rat‐type N‐terminal peptide (MELLNGTGLWSM) instead of the human‐type (MGLEALVPLAVIV), and showed enantioselectivity of [(+)‐BTL < (−)‐BTL] for the rate of BTL 4‐hydroxylation. In contrast, enantioselectivity [(+)‐BTL > (−)‐BTL] for Hep G2‐CYP2D6 (CYP2D6‐val) with a human‐type N‐terminal peptide that had a valine residue at 374, which corresponds to the methionine of the CYP2D6‐met variant, was the same as that for human liver microsomes. We further confirmed that CYP2D6‐met and CYP2D6‐val expressed in human lymphoblastoid cells, both of which have methionine and valine, respectively, at position 374 and a human‐type N‐terminal peptide, exhibited the same enantioselectivities as those obtained from CYP2D6‐met and CYP2D6‐val expressed in the Hep G2 cell system. These results indicate that the amino acid at 374 of CYP2D6 is one of the key factors influencing the enantioselectivity of BTL 4‐hydroxylation. Chirality 11:1–9, 1999. © 1999 Wiley‐Liss, Inc.     

The evolutionary appearance of non‐cyanogenic hydroxynitrile glucosides in the Lotus genus is accompanied by the substrate specialization of paralogous β–glucosidases resulting from a crucial amino acid substitution

Lotus japonicus, like several other legumes, biosynthesizes the cyanogenic α–hydroxynitrile glucosides lotaustralin and linamarin. Upon tissue disruption these compounds are hydrolysed by a specific β–glucosidase, resulting in the release of hydrogen cyanide. Lotus japonicus also produces the non‐cyanogenic γ‐ and β–hydroxynitrile glucosides rhodiocyanoside A and D using a biosynthetic pathway that branches off from lotaustralin biosynthesis. We previously established that BGD2 is the only β–glucosidase responsible for cyanogenesis in leaves. Here we show that the paralogous BGD4 has the dominant physiological role in rhodiocyanoside degradation. Structural modelling, site‐directed mutagenesis and activity assays establish that a glycine residue (G211) in the aglycone binding site of BGD2 is essential for its ability to hydrolyse the endogenous cyanogenic glucosides. The corresponding valine (V211) in BGD4 narrows the active site pocket, resulting in the exclusion of non‐flat substrates such as lotaustralin and linamarin, but not of the more planar rhodiocyanosides. Rhodiocyanosides and the BGD4 gene only occur in L. japonicus and a few closely related species associated with the Lotus corniculatus clade within the Lotus genus. This suggests the evolutionary scenario that substrate specialization for rhodiocyanosides evolved from a promiscuous activity of a progenitor cyanogenic β–glucosidase, resembling BGD2, and required no more than a single amino acid substitution.