Programmed death-ligand 1 (PD-L1) is a key immune regulatory protein that interacts with programmed cell death protein 1 (PD-1), leading to T-cell suppression. Whilst this interaction is key in self-tolerance, cancer cells evade the immune system by overexpressing PD-L1. Inhibition of the PD-1/ PD-L1 pathway with standard monoclonal antibodies has proven a highly effective cancer treatment; however, single domain antibodies (VHH) may offer numerous potential benefits. Here, we report the identification and characterization of a diverse panel of 16 novel VHHs specific to PD-L1. The panel of VHHs demonstrate affinities of 0.7 nM to 5.1 mu M and were able to completely inhibit PD-1 binding to PD-L1. The binding site for each VHH on PD-L1 was determined using NMR chemical shift perturbation mapping and revealed a common binding surface encompassing the PD-1-binding site. Additionally, we solved crystal structures of two representative VHHs in complex with PD-L1, which revealed unique binding modes. Similar NMR experiments were used to identify the binding site of CD80 on PD-L1, which is another immune response regulatory element and interacts with PD-L1 localized on the same cell surface. CD80 and PD-1 were revealed to share a highly overlapping binding site on PD-L1, with the panel of VHHs identified expected to inhibit CD80 binding. Comparison of the CD80 and PD-1 binding sites on PD-L1 enabled the identification of a potential antibody binding region able to confer specificity for the inhibition of PD-1 binding only, evasion of the immune system.

Interleukin-13 (IL-13) is a cytokine involved in T-cell immune responses and is a well validated therapeutic target for the treatment of asthma, along with other allergic and inflammatory diseases. IL-13 signals through a ternary signalling complex formed with the receptors IL-13Rα1 and IL-4Rα. This complex is assembled by IL-13 initially binding IL-13Rα1, followed by association of the binary IL-13:IL-13Rα1 complex with IL-4Rα. The receptors are shared with IL-4, but IL-4 initially binds IL-4Rα. Here we report the identification and characterisation of a diverse panel of single-domain antibodies (VHHs) that bind to IL-13 (KD 40 nM-5.5 μM) and inhibit downstream IL-13 signalling (IC50 0.2-53.8 μM). NMR mapping showed that the VHHs recognise a number of epitopes on IL-13, including previously unknown allosteric sites. Further NMR investigation of VHH204 bound to IL-13 revealed a novel allosteric mechanism of inhibition, with the antibody stabilising IL-13 in a conformation incompatible with receptor binding. This also led to the identification of a conformational equilibrium for free IL-13, providing insights into differing receptor signalling complex assembly seen for IL-13 compared to IL-4, with formation of the IL-13:IL-13Rα1 complex required to stabilise IL-13 in a conformation with high affinity for IL-4Rα. These findings highlight new opportunities for therapeutic targeting of IL-13 and we report a successful 19F fragment screen of the IL-13:VHH204 complex, including binding sites identified for several hits. To our knowledge, these 19F containing fragments represent the first small-molecules shown to bind to IL-13 and could provide starting points for a small-molecule drug discovery programme.

Functionally important proteins at the interface of cell and soil are of potentially low abundance when compared with commonly recovered intracellular proteins. A novel approach was developed and used to extract the metaexoproteome, the subset of proteins found outside the cell, in the context of a soil enriched with the nitrogen-containing recalcitrant polymer chitin. The majority of proteins recovered was of bacterial origin and localized to the outer membrane or extracellular milieu. A wide variety of transporter proteins were identified, particularly those associated with amino-acid and phosphate uptake. The metaexoproteome extract retained chitinolytic activity and we were successful in detecting Nocardiopsis-like chitinases that correlated with the glycoside hydrolase family 18 (GH18) chi gene data and metataxonomic analysis. Nocardiopsis-like chitinases appeared to be solely responsible for chitinolytic activity in soil. This is the first study to detect and sequence bacterial exoenzymes with proven activity in the soil enzyme pool.

A good model to experimentally explore evolutionary hypothesis related to enzyme function is the ancient-like dual-substrate (beta alpha)(8) phosphoribosyl isomerase A (PriA), which takes part in both histidine and tryptophan biosynthesis in Streptomyces coelicolor and related organisms. In this study, we determined the Michaelis-Menten enzyme kinetics for both isomerase activities in wild-type PriA from S. coelicolor and in selected single-residue monofunctional mutants, identified after Escherichia coli in vivo complementation experiments. Structural and functional analyses of a hitherto unnoticed residue contained on the functionally important beta -> alpha loop 5, namely, Arg(139), which was postulated on structural grounds to be important for the dual-substrate specificity of PriA, is presented for the first time. Indeed, enzyme kinetics analyses done on the mutant variants PriA_Ser(81)Thr and PriA_Arg(139)Asn showed that these residues, which are contained on beta -> alpha loops and in close proximity to the N-terminal phosphate-binding site, are essential solely for the phosphoribosyl anthranilate isomerase activity of PriA. Moreover, analysis of the X-ray crystallographic structure of PriA_Arg(139)Asn elucidated at 1.95 angstrom herein strongly implicates the occurrence of conformational changes in this beta -> alpha a loop as a major structural feature related to the evolution of the dual-substrate specificity of PriA. It is suggested that PriA has evolved by tuning a fine energetic balance that allows the sufficient degree of structural flexibility needed for accommodating two topologically dissimilar substrates-within a bifunctional and thus highly constrained active site-without compromising its structural stability.

Two structures of phosphoribosyl isomerase A (PriA) from Streptomyces coelicolor, involved in both histidine and tryptophan biosynthesis, were solved at 1.8Å resolution. A closed conformer was obtained, which represents the first complete structure of PriA, revealing hitherto unnoticed molecular interactions and the occurrence of conformational changes. Inspection of these conformers, including ligand-docking simulations, allowed identification of residues involved in substrate recognition, chemical catalysis and conformational changes. These predictions were validated by mutagenesis and functional analysis. Arg19 and Ser81 were shown to play critical roles within the carboxyl and amino phosphate-binding sites, respectively; the catalytic residues Asp11 and Asp130 are responsible for both activities; and Thr166 and Asp171, which make an unusual contact, are likely to elicit the conformational changes needed for adopting the active site architectures. This represents the first report of the structure/function relationship of this (βα)8-isomerase.

24 European Crystallographic Meeting, ECM24, Marrakech, 2007 Page s126 Acta Cryst. (2007). A63, s126 Escherichia coli beta-galactosidase is known to be active only in the form of tetramers, while the cold-active Arthrobacter sp. C2-2 beta-galactosidase forms compact hexamers with active sites oriented into an internal cavity, connected by three types of channels with exterior solvent. Additionally, sequence differences between both enzymes exist in the active site. Acknowledgement: The research was supported by the Grant Agency of the Czech Republic (project 204/02/0843/A) and by the Grant Agency of the Academy of Sciences of the Czech Republic (project B500500512).

Escherichia coli beta-galactosidase is known to be active only in the form of tetramers, while the cold-active Arthrobacter sp.C2-2 beta-galactosidase forms compact hexamers with active sites oriented into an internal cavity, connected by three types of channels with exterior solvent.Additionally, sequence differences between both enzymes exist in the active site.

Acylaminoacyl peptidase (also known as acylamino-acid-releasing enzyme or acylpeptide hydrolase; EC 3.4.19.1) is an unusual member of the prolyl oligopeptidase family catalysing the hydrolysis of an N-acylated peptide to an acylamino acid and a peptide with a free N-terminus. Acylaminoacyl peptidase purified from porcine liver has been crystallized in mother liquor containing 0.1 M Tris-HCl pH 7.0, 10%(w/v) polyethylene glycol 8000, 50 mM MgCl2 and 1%(w/v) CHAPS using the hanging-drop vapour-diffusion technique. A full data set to 3.4 angstrom resolution was collected at ESRF beamline ID14-4 and space group C222 was assigned, with unit-cell parameters a = 84.8, b = 421.1, c = 212.0 angstrom and four molecules in the asymmetric unit.

The priA gene encoding the enzyme phosphoribosyl isomerase from Streptomyces coelicolor, a novel bifunctional enzyme involved in both histidine and tryptophan biosynthesis, was heterologously expressed and purified in Escherichia coli as an N-terminal His-tag fusion. The purified recombinant enzyme was crystallized using the hanging-drop method in 1.50 M ammonium sulfate and 100 mM sodium citrate pH 4.8. Crystals were obtained of up to 0.05 x 0.05 x 0.3 mm in size. A full data set to 2 A resolution was collected at the ESRF beamline ID14-1 and space group P3(1,2)21 was assigned, with unit-cell parameters a = 65.1, c = 104.7 A.

The acute histological effects of a commercially available copper vapour laser (CVL) on normal and atherosclerotic arterial wall were studied. The multi-cyclic CVL produces a quasi continuous wave output at 511 nm (green) and 578 nm (yellow) [green/yellow ratio 2∶1]. Tissue craters were produced in segments of normal and atherosclerotic human femoral artery using a maximum of 8 J of laser energy delivered with a bare 1 mm quartz fibre in contact and perpendicular to the tissue. Crater dimensions and ablation volumes were determined histologically using an optical graticule. Ablation of atheroma was almost three times more efficient than ablation of normal arterial wall (p<0.001). A narrow zone of vacuolization and coagulative thermal damage lined the crater margins suggesting a predominantly photothermal ablative mechanism. In conclusion, selective ablation of atheroma using low power copper vapour laser light is possible. The CVL is an attractive alternative to the argon ion laser because of its low running costs and applicability to other medical disciplines. Therefore, its potential application in laser angioplasty merits further study, preferably using a modified optical fibretip delivery system.

A histological technique for the measurement of laser-induced thermal tissue damage is described using the stain picrosirius red F3BA. This stain enhances the birefringence of normal collagen when viewed in polarized light. Areas of (thermally) denatured collagen, however, have no optical activity and can be measured directly by optical micrometry. The technique has been applied to experimental studies on both laser angioplasty and laser vascular anastomosis.

Previous articleNext article No AccessBook ReviewsAmerican Portrait Prints. Proceedings of the Tenth Annual American Print Conference. Wendy Wick Reaves Helena WrightHelena Wright Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by Winterthur Portfolio Volume 20, Number 2/3Summer - Autumn, 1985 Published for the Henry Francis du Pont Winterthur Museum, Inc. Article DOIhttps://doi.org/10.1086/496228 Views: 1Total views on this site Copyright 1985 The Henry Francis du Pont Winterthur MuseumPDF download Crossref reports no articles citing this article.