Principals in Programming Languages: Technical Results

This is the companion technical report for ``Principals in Programming Languages'' [20]. See that document for a more readable version of these results. In this paper, we describe two variants of the simply typed $\lambda$-calculus extended with a notion of {\em principal}. The results are languages in which intuitive statements like ``the client must call $\mathtt{open}$ to obtain a file handle'' can be phrased and proven formally. The first language is a two-agent calculus with references and recursive types, while the second language explores the possibility of multiple agents with varying amounts of type information. We use these calculi to give syntactic proofs of some type abstraction results that traditionally require semantic arguments

Structure-Based Prediction of G‑Protein-Coupled Receptor Ligand Function: A β‑Adrenoceptor Case Study

The spectacular advances in G-protein-coupled receptor (GPCR) structure determination have opened up new possibilities for structure-based GPCR ligand discovery. The structure-based prediction of whether a ligand stimulates (full/partial agonist), blocks (antagonist), or reduces (inverse agonist) GPCR signaling activity is, however, still challenging. A total of 31 β₁ (β₁R) and β₂ (β₂R) adrenoceptor crystal structures, including antagonist, inverse agonist, and partial/full agonist-bound structures, allowed us to explore the possibilities and limitations of structure-based prediction of GPCR ligand function. We used all unique protein–ligand interaction fingerprints (IFPs) derived from all ligand-bound β-adrenergic crystal structure monomers to post-process the docking poses of known β₁R/β₂R partial/full agonists, antagonists/inverse agonists, and physicochemically similar decoys in each of the β₁R/β₂R structures. The systematic analysis of these 1920 unique IFP–structure combinations offered new insights into the relative impact of protein conformation and IFP scoring on selective virtual screening (VS) for ligands with a specific functional effect. Our studies show that ligands with the same function can be efficiently classified on the basis of their protein–ligand interaction profile. Small differences between the receptor conformation (used for docking) and reference IFP (used for scoring of the docking poses) determine, however, the enrichment of specific ligand types in VS hit lists. Interestingly, the selective enrichment of partial/full agonists can be achieved by using agonist IFPs to post-process docking poses in agonist-bound as well as antagonist-bound structures. We have identified optimal structure–IFP combinations for the identification and discrimination of antagonists/inverse agonist and partial/full agonists, and defined a predicted IFP for the small full agonist norepinephrine that gave the highest retrieval rate of agonists over antagonists for <i>all</i> structures (with an enrichment factor of 46 for agonists and 8 for antagonists on average at a 1% false-positive rate). This β-adrenoceptor case study provides new insights into the opportunities for selective structure-based discovery of GPCR ligands with a desired function and emphasizes the importance of IFPs in scoring docking poses

Proposed model for regulation of CXCR7 trafficking.

<p>CXCR7 requires ubiquitination of the Lys residues of its C-tail in order to reach the cell surface. Receptor activation by CXCL12 and subsequent phosphorylation of the C-terminal Ser/Thr residues results in β-arrestin recruitment by CXCR7 and receptor internalization in CCPs. In addition, β-arrestin scaffolds the interaction of CXCR7 with an unknown de-ubiquitinating enzyme (DUB) responsible for receptor deubiquitination. After chemokine degradation in early endosomes and due to the transient interaction of CXCR7 with β-arrestin, release of β-arrestin (and DUB) from the endocytosed receptor results in a CXCR7 able to undergo ubiquitination by a specific E3 ligase (E3) and subsequent delivery of the recycled receptor to the cell surface.</p

CXCR7/CXCR3 tail switch alters ubiquitination properties of the receptors.

<p>(<b>A</b>) Immunoprecipitation experiments were performed in cells expressing chimeric receptors consisting on CXCR7 with CXCR3 C-terminus (CXCR7-X3) or the reciprocal CXCR3 with CXCR7 C-terminus (CXCR3-X7). Detection of the immunoprecipitated CXCR7 and CXCR3 was done with the 11G8 and mAB160 antibodies, respectively. HA-Ub expression was confirmed blotting lysates using an anti-HA antibody and equal loading was controlled by detection of actin on the same blot. Molecular weight markers (kDa) are indicated on the sides of the blots. (<b>B</b>) Detection of total CXCR7 protein expression by ELISA in the same cells.</p

The C-terminus of CXCR7 is constitutively ubiquitinated.

<p><b>(A) CXCR7 gets deubiquitinated by CXCL12-stimulation.</b> HEK293T cells were transfected as indicated and processed for immunoprecipitation of the HA-Ub (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034192#s4" target="_blank">Materials and Methods</a>). (<b>A</b>) CXCR7 was stimulated with 10⁻⁸ M CXCL12 for 30 min, and removal of CXCL12 was performed by two washes of the cells and additional 30 min incubation with fresh chemokine-free media. Detection of the immunoprecipitated CXCR7 was done with the 11G8 antibody. HA-Ub expression was confirmed by blotting lysates using an anti-HA antibody and equal loading was controlled by detection of actin on the same blot. Molecular weight markers (kDa) are indicated on the right of the blot. (<b>B</b>) Detection of total CXCR7 protein expression by ELISA in the same cells.</p

Real-time monitoring of receptor ubiquitination using BRET².

<p>HEK293T cells were transfected with Ub-GFP² (white bars) or (G75A,G76A)-Ub-GFP² (filled bars) and (<b>A</b>) CXCR7-RLuc, CXCR7 ΔC-RLuc, or CXCR7 ST/A-Rluc, (<b>B</b>) CXCR4-RLuc, or (<b>C</b>) CXCR3-RLuc. BRET² was measured 30 min after stimulation with 10⁻⁸ M of CXCL12 (CXCL11 for CXCR3) by addition of coelenterazine 400a and immediate read out. Results are expressed in Net BRET normalized to basal as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034192#s4" target="_blank">Materials and Methods</a>. Data represent the mean ± SEM of 3 experiments each performed in triplicate. **, p<0.01, and ***, p<0.001, by Student t test.</p

(A) CXCR7 internalization depends on CCPs and is G protein-independent.

<p>HEK293T cells were transfected with wt CXCR7 (and β-arrestin (319–418) were indicated) and cell surface levels of the receptor after CXCL12 stimulation was detected by ELISA using the CXCR7-specific antibody 11G8. Incubation with 0.4 M Sucrose was done 30 min prior and during stimulation. PTX was incubated overnight at 25 ng/ml final concentration. <b>(B) β-arrestin1/2 knock-down prevents CXCR7 internalization.</b> HEK293/CXCR7 cells transfected with control siRNAs (white bars) or pools targeting β-arrestin1/2 (filled bars), were stimulated with CXCL12 (10⁻⁸ M) or vehicle for 45 min and receptor surface expression was determined. Knockdown of β-arrestin1 and -2, 48 hrs after transfection, was assessed in western blot using an anti-β–arrestin1/2 antibody (inset). Anti-STAT3 (mAb 79D7, Cell Signaling Technologies) was used as loading control. <b>(C) CXCR7 C-terminus is essential for receptor internalization.</b> HEK293T cells were transfected with wt CXCR7 (filled bars), CXCR7 ΔC (grey bars) or CXCR7 ST/A (white bars) and cell surface receptor levels were assessed as above. Data represent the mean ± SEM of at least 3 experiments each performed in triplicate. ***, p<0.001 by one-way ANOVA and Bonferroni post test.</p

CXCR7 recycles to the cell surface after internalization.

<p>(<b>A</b>) HEK293T stably expressing CXCR7 were stimulated with 10⁻⁸ M CXCL11, CXCL12 or vehicle for 45 min or 3 h and fixed immediately. CXCR7 was detected using the specific 11G8 antibody and an Alexa-488-conjugated secondary antibody. Scale bar represents 10 µm. (<b>B</b>) HEK293T cells expressing CXCR7 (filled symbols) or CXCR3 (open symbols) were incubated with CXCL11 (10⁻⁸ M, squares), CXCL12 (10⁻⁸ M, triangles) or vehicle (circles) for the indicated times. Cell surface receptor levels were detected by ELISA using CXCR7- or CXCR3-specific antibodies (11G8 and mAB160, respectively). Results were normalized to basal surface protein levels, and data represent the mean ± SEM of 4 experiments each performed in triplicate. (<b>C</b>) ELISA was performed as in B in cells pre-incubated for 2 h with the <i>de novo</i> protein synthesis inhibitor cycloheximide (10 µg/ml). (<b>D</b>) ELISA performed as in C on intact HEK293/CXCR7 cells treated with vehicle or 1 µM of bafilomycin A1 (Baf A1), 30 min prior to incubation with CXCL12. <b>(E) C-terminal Ser/Thr clusters determine receptor fate after internalization.</b> HEK293T cells were transiently transfected with CXCR7 wt (white bars) or with a chimeric receptor consisting of CXCR7 harboring the C-terminal sequence of CXCR3 (CXCR7-X3, filled bars). To assess the cell surface expression of the receptor, ELISA experiments were performed after 30 min or 3 hours of incubation with 10⁻⁸ M CXCL12. Data represent the mean ± SEM of 3 experiments each performed in triplicate. ***, p<0.001, **, p<0.01, and *, p<0.05 by one-way ANOVA and Bonferroni post test.</p

β-arrestin2 recruitment to CXCR7 is dependent on C-terminal Ser/Thr residues.

<p>(<b>A</b>) CXCL11 or CXCL12-mediated β-arrestin2 recruitment to CXCR7. HEK293T co-expressing RLuc-tagged CXCR7 and YFP-tagged β-arrestin2 were stimulated with increasing concentrations of CXCL11 (open circles) or CXCL12 (filled circles) (<b>B</b>) HEK293T co-expressing RLuc-tagged CXCR7 and YFP-tagged β-arrestin2 were incubated overnight with 25 ng/ml of PTX or for 30 min with the CXCR7-specific antibody 8F11 prior to the BRET measurement. (<b>C</b>) CXCL12-induced β-arrestin2 recruitment to CXCR7 wt (filled circles), a truncated CXCR7 lacking the C-terminus (CXCR7 ΔC, filled triangles) or a mutant CXCR7 for which all the Ser and Thr residues were mutated to Ala (CXCR7 ST/A, open squares). HEK293T cells coexpressing <i>RLuc</i>-tagged CXCR7 mutants and YFP-tagged β-arrestin2 were stimulated with increasing concentrations of CXCL12 prior to BRET measurements. Data represent the mean ± SEM of 4 experiments each performed in triplicate. Results are expressed in Net BRET as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034192#s4" target="_blank">Materials and Methods</a>. ***, p<0.001 by one-way ANOVA and Bonferroni post test.</p

Domain structure of GlPDE and alignment of its catalytic domain sequence with other PDEs.

<p><b>(A)</b> Schematic representation of the domain structure. The predicted transmembrane helices (TMHs) between amino acids 1 and 573 are shown as red boxes. One segment is predicted with less confidence (predicted by 4 of 14 algorithms) and is depicted as a hatched box. The C-terminal catalytic domain is shown in blue and the GlPDE-specific 35aa-insert between helix 6 and 7 is indicated by a light-blue box. The N-terminal 71 amino acids that were recognized by the program MitoProtII as mitochondrial-targeting signal are indicated with an orange bar. GlPDE regions 588–1371 and 983–1371 corresponding to the recombinant proteins expressed in yeast are marked with grey arrows. <b>(B)</b> Sequence alignment of the catalytic domains (α-helices 3–16) of GlPDE, human PDE4B, human PDE3B and <i>T</i>. <i>brucei</i> TbrPDEB1. Secondary structure features are shown in dark blue (α-helices) and light blue (3₁₀-helices). α-helices are additionally numbered above the sequences (H3-H16). Residues of the substrate-binding subpocket are indicated with dots above the alignment. Thereof, the invariant glutamine is additionally marked with an @-sign (highlighted in yellow) and the hydrophobic P-clamp residues are tagged with green labels. Amino acids that are conserved in all 11 human PDE families and in GlPDE are shown in red letters and indicated with an asterisk below the alignment. Asterisks are underlined at the eight strictly conserved residues of the metal-binding pocket at the site of catalysis. The single histidine residue that is conserved among all hPDEs, but is substituted by an alanine in GlPDE (Ala-1175) is marked with a black triangle.</p