Volume 398, Issue 1

A controlled interaction of materials with their surrounding biological environment is of great interest in many fields. Multifunctional coatings aim to provide simultaneous modulation of several biological signals. They can consist of various combinations of bioactive, and bioinert components as well as of reporter molecules to improve cell-material contacts, prevent infections or to analyze biochemical events on the surface. However, specific immobilization and particular assembly of various active molecules are challenging. Herein, an overview of multifunctional coatings for biomaterials is given, focusing on synthetic strategies and the biological benefits by displaying several motifs.

The specific recognition of peptides, which we define to include unstructured regions or denatured forms of proteins, is an intrinsic part of a multitude of biochemical assays and procedures. Many cellular interactions are also based on this principle as well. While it would be highly desirable to have a stockpile of sequence-specific binders for essentially any sequence, a de novo selection of individual binders against every possible target peptide sequence would be rather difficult to reduce to practice. Modular peptide binders could overcome this problem, as preselected and/or predesigned modules could be reused for the generation of new binders and thereby revolutionize the generation of binding proteins. This minireview summarizes advances in the development of peptide binders and possible scaffolds for their design.

Nature uses the advantages of fusion proteins for multi-step reactions to facilitate the metabolism in cells as the conversion of substrates through intermediates to the final product can take place more rapidly and with less side-product formation. In a similar fashion, also for enzyme cascade reactions, the fusion of biocatalysts involved can be advantageous. In the present study, we investigated fusion of an alcohol dehydrogenase (ADH), an enoate reductase (ERED) and a Baeyer-Villiger monooxygenase (BVMO) to enable the synthesis of (chiral) lactones starting from unsaturated alcohols as substrates. The domain order and various linkers were studied to find optimal conditions with respect to expression levels and enzymatic activities. Best results were achieved for the ERED xenobiotic reductase B (XenB) from Pseudomonas putida and the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp., whereas none of the ADHs studied could be fused successfully. This fusion protein together with separately supplied ADH resulted in similar reaction rates in in vivo biocatalysis reactions. After 1.5 h we could detect 40% more dihydrocarvone lactone in in vivo reactions with the fusion protein and ADH then with the single enzymes.

Members of the vascular endothelial growth factor receptor (VEGFR) family play a central role in angiogenesis as well as lymphangiogenesis and are crucial for tumor growth and metastasis. In particular, VEGFR-3 expression is induced in endothelial cells during tumor angiogenesis. We report the design of anticalins that specifically recognize the ligand-binding domains 1 and 2 of VEGFR-3. To this end, a library of the lipocalin 2 scaffold with 20 randomized positions distributed across its binding site was subjected to phage display selection and enzyme linked immunosorbent assay (ELISA) screening using the VEGF-C binding fragment (D1-2) or the entire extracellular region (D1-7) of VEGFR-3 as target proteins. Promising anticalin candidates were produced in Escherichia coli and biochemically characterized. Three variants with different receptor binding modes were identified, and two of them were optimized with regard to target affinity as well as folding efficiency. The resulting anticalins show dissociation constants down to the single-digit picomolar range. Specific recognition of VEGFR-3 on cells was demonstrated by immunofluorescence microscopy. Competitive binding versus VEGF-C was demonstrated for two of the anticalins with K i values in the low nanomolar range. Based on these data, VEGFR-3 specific anticalins provide promising reagents for the diagnosis and/or therapeutic intervention of tumor-associated vessel growth.

Protein splicing by inteins has found diverse applications in biotechnology, protein chemistry and chemical biology. Inteins display a wide range of efficiencies and rates unpredictable from their amino acid sequences. Here, we identified positions T22S and S35 in the LacZα peptide as intein insertion sites that strictly require protein splicing, in contrast to cleavage side-reactions, to allow for complementation of β-galactosidase activity. Both the cis -variant of the M86 mutant of the Ssp DnaB intein and a split form undergoing protein trans -splicing gave rise to formation of blue colonies in the β-galactosidase read-out. Furthermore, we report the two novel, naturally split VidaL T4Lh-1 and VidaL UvsX-2 inteins whose N-terminal fragments consist of only 15 and 16 amino acids, respectively. Initial biochemical characterization with the LacZα host system of these inteins further underlines its utility. Finally, we used the LacZα host system to rapidly identify amino acid substitutions from a small randomized library at the structurally conserved intein position 2 next to the catalytic center, that are tolerated for protein splicing activity of the M86 intein. These findings demonstrate the potential of the system for initial testing and directed evolution of inteins.

Sequence-specific and programmable binding of proteins to RNA bears the potential to detect and manipulate target RNAs. Applications include analysis of subcellular RNA localization or post-transcriptional regulation but require sequence-specificity to be readily adjustable to any target RNA. The Pumilio homology domain binds an eight nucleotide target sequence in a predictable manner allowing for rational design of variants with new specificities. We describe a high-throughput system for screening Pumilio variants based on fluorescence-activated cell sorting of E. coli . Our approach should help optimizing variants obtained from rational design regarding folding and stability or identifying new variants with alternative binding modes.

Alpha-synuclein (α-syn) is an abundant neuronal protein whose physiological function, even if still not completely understood, has been consistently related to synaptic function and vesicle trafficking. A group of disorders known as synucleinopathies, among which Parkinson’s disease (PD), is deeply associated with the misfolding and aggregation of α-syn, which can give rise to proteinaceous inclusion known as Lewy bodies (LB). Proteostasis stress is a relevant aspect in these diseases and, currently, the presence of oligomeric α-syn species rather than insoluble aggregated forms, appeared to be associated with cytotoxicity. Many observations suggest that α-syn is responsible for neurodegeneration by interfering with multiple signaling pathways. α-syn protein can directly form plasma membrane channels or modify with their activity, thus altering membrane permeability to ions, abnormally associate with mitochondria and cause mitochondrial dysfunction (i.e. mitochondrial depolarization, Ca 2+ dys-homeostasis, cytochrome c release) and interfere with autophagy regulation. The picture is further complicated by the fact that single point mutations, duplications and triplication in α-syn gene are linked to autosomal dominant forms of PD. In this review we discuss the multi-faced aspect of α-syn biology and address the main hypothesis at the basis of its involvement in neuronal degeneration.

In a search for plant homologues of dipeptidyl peptidase III (DPP III) family, we found a predicted protein from the moss Physcomitrella patens (UniProt entry: A9TLP4), which shared 61% sequence identity with the Arabidopsis thaliana uncharacterized protein, designated Nudix hydrolase 3. Both proteins contained all conserved regions of the DPP III family, but instead of the characteristic hexapeptide HEXXGH zinc-binding motif, they possessed a pentapeptide HEXXH, and at the N-terminus, a Nudix box, a hallmark of Nudix hydrolases, known to act upon a variety of nucleoside diphosphate derivatives. To investigate their biochemical properties, we expressed heterologously and purified Physcomitrella (PpND) and Arabidopsis (AtND) protein. Both hydrolyzed, with comparable catalytic efficiency, the isopentenyl diphosphate (IPP), a universal precursor for the biosynthesis of isoprenoid compounds. In addition, PpND dephosphorylated four purine nucleotides (ADP, dGDP, dGTP, and 8-oxo-dATP) with strong preference for oxidized dATP. Furthermore, PpND and AtND showed DPP III activity against dipeptidyl-2-arylamide substrates, which they cleaved with different specificity. This is the first report of a dual activity enzyme, highly conserved in land plants, which catalyzes the hydrolysis of a peptide bond and of a phosphate bond, acting both as a dipeptidyl peptidase III and an atypical Nudix hydrolase.

The question as to why a protein exerts oncogenic properties is answered mainly by well-established ideas that these proteins interfere with cellular signaling pathways. However, the knowledge about structural and functional peculiarities of the oncoproteins causing these effects is far from comprehensive. The 97.5% homologous tissue-specific A1 and A2 isoforms of mammalian translation elongation factor eEF1A represent an interesting model to study a difference between protein variants of a family that differ in oncogenic potential. We propose that the different oncogenic impact of A1 and A2 might be explained by differences in their ability to communicate with their respective cellular partners. Here we probed this hypothesis by studying the interaction of eEF1A with two known partners – calmodulin and actin. Indeed, an inability of the A2 isoform to interact with calmodulin is shown, while calmodulin is capable of binding A1 and interferes with its tRNA-binding and actin-bundling activities in vitro . Both A1 and A2 variants revealed actin-bundling activity; however, the form of bundles formed in the presence of A1 or A2 was distinctly different. Thus, a potential inability of A2 to be controlled by Ca 2+ -mediated regulatory systems is revealed.

Acyl-carrier-protein (acpP) is an essential protein in fatty acid biosynthesis of Staphylococcus aureus [Cronan, J.E. and Thomas, J. (2009). Complex enzymes in microbial natural product biosynthesis, part B: polyketides, aminocoumarins and carbohydrates. Method. Enzymol. 459 , 395–433; Halavaty, A.S., Kim, Y., Minasov, G., Shuvalova, L., Dubrovska, I., Winsor, J., Zhou, M., Onopriyenko, O., Skarina, T., Papazisi, L., et al. (2012). Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. Acta Crystallogr. Sect. D Biol. Crystallogr. 68 , 1359–1370]. The inactive apo-form is converted to the active holo-enzyme by acyl-carrier protein synthase (acpS) through addition of a 4′-phosphopantetheine group from coenzyme A to a conserved serine residue of acpP [Flugel, R.S., Hwangbo, Y., Lambalot, R.H., Cronan, J.E., and Walsh, C.T. (2000). Holo-(acyl-carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli . J. Biol. Chem. 275 , 959–968; Lambalot, R.H. and Walsh, C.T. (1995). Cloning, overproduction, and characterization of the Escherichia coli holo-acyl-carrier protein synthase. J. Biol. Chem. 270 , 24658–24661]. Once activated, acpP acts as an anchor for the growing fatty acid chain. Structural data from X-ray crystallographic analysis reveals that, despite its small size (8 kDa), acpP adopts a distinct, mostly α-helical structure when complexed with acpS [Halavaty, A.S., Kim, Y., Minasov, G., Shuvalova, L., Dubrovska, I., Winsor, J., Zhou, M., Onopriyenko, O., Skarina, T., Papazisi, L., et al. (2012). Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. Acta Crystallogr. Sect. D Biol. Crystallogr. 68 , 1359–1370; Byers, D.M. and Gong, H. (2007). Acyl carrier protein: structure–function relationships in a conserved multifunctional protein family. Biochem. Cell Biol. 85 , 649–662]. We expressed and purified recombinant, active S. aureus acpP from Escherichia coli and mimicked the beginning of fatty acid biosynthesis by employing an [ 14 C]-acp loading assay. Surprisingly, acpP remained functional even after heat treatment at 95°C for up to 10 min. NMR data from 2D-HSQC experiments as well as interaction studies with acpS confirmed that acpP is structured and active both before and after heat treatment, with no significant differences between the two. Thus, our data suggest that S. aureus acpP is a highly stable protein capable of maintaining its structure at high temperatures.

Metarhizium anisopliae is an entomopathogenic fungus relevant in biotechnology with applications like malaria vector control. Studies of its virulence factors are therefore of great interest. Fungal ribotoxins are toxic ribonucleases with extraordinary efficiency against ribosomes and suggested as potential insecticides. Here we describe this ribotoxin characteristic activity in M. anisopliae cultures. Anisoplin has been obtained as a recombinant protein and further characterized. It is structurally similar to hirsutellin A, the ribotoxin from the entomopathogen Hirsutella thompsonii . Moreover, anisoplin shows the ribonucleolytic activity typical of ribotoxins and cytotoxicity against insect cells. How Metarhizium uses this toxin and possible applications are of interest.