Agonist-induced phosphorylation of sst₂ and sst₅ tail-swap mutants.

<p><i>Top</i>, Schematic representation of the human wild-type sst₂ (depicted in grey) and human wild-type sst₅ receptors (depicted in black) and their corresponding tail-swap mutants. Phosphate acceptor sites targeted for the generation of phosphosite-specific antibodies are depicted as circles. <i>Bottom</i>, stably transfected HEK 293 cells were exposed to 1 µM SS-14 at room temperature for the indicated time periods. Cells were lysed and immunoblotted with the indicated phosphosite-specific antibodies. Blots were then stripped and reprobed with the phosphorylation-independent anti-sst₅ antibody {UMB-4} or anti-sst₂ antibody {UMB-1} to confirm equal loading of the gels. Shown are representative results from one of three independent experiments. The position of molecular mass markers is indicated on the left (in kDa).</p

Agonist-induced β-arrestin mobilization of sst₂ and sst₅ tail-swap mutants.

<p>(<i>A</i>) HEK293 cells were transiently transfected with sst₂, sst₅, sst_2-5 or sst_5-2 and β-arrestin-2-EGFP. The distribution of β-arrestin-2 was visualized sequentially in the same live cells before (0 min) and after the addition of 1 µM SS-14 to the culture medium. Shown are representative images from one of three independent experiments. (<i>B</i>) HEK293 cells were transiently cotransfected with sst₂, sst₅, sst_2-5 or sst_5-2, β-arrestin-2-EGFP and GRK2. The distribution of β-arrestin-2 was visualized sequentially in the same live cells before (0 min) and after (1 to 30 min) the addition of 1 µM SS-14 to the culture medium. Shown are representative images from one of four independent experiments performed in duplicate. <i>Scale bar</i>, 20 µm.</p

Dephosphorylation of sst₂ and sst₅ tail-swap mutants.

<p>Stably transfected HEK 293 cells were exposed to 1 µM SS-14 for 5 min, washed and incubated at room temperature in the absence of agonist for the indicated time periods. Cells were lysed and immunoblotted with the indicated phosphosite-specific antibodies. Blots were then stripped and reprobed with the phosphorylation-independent anti-sst₅ antibody {UMB-4} or anti-sst₂ antibody {UMB-1} to confirm equal loading of the gels. Shown are representative results from one of three independent experiments. The position of molecular mass markers is indicated on the left (in kDa).</p

A Prodomain Fragment from the Proteolytic Activation of Growth Differentiation Factor 11 Remains Associated with the Mature Growth Factor and Keeps It Soluble

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor β (TGF-β) family, plays diverse roles in mammalian development. It is synthesized as a large, inactive precursor protein containing a prodomain, pro-GDF11, and exists as a homodimer. Activation requires two proteolytic processing steps that release the prodomains and transform latent pro-GDF11 into active mature GDF11. In studying proteolytic activation in vitro, we discovered that a 6-kDa prodomain peptide containing residues 60–114, PDP_60–114, remained associated with the mature growth factor. Whereas the full-length prodomain of GDF11 is a functional antagonist, PDP_60–114 had no impact on activity. The specific activity of the GDF11/PDP_60–114 complex (EC₅₀ = 1 nM) in a SMAD2/3 reporter assay was identical to that of mature GDF11 alone. PDP_60–114 improved the solubility of mature GDF11 at neutral pH. As the growth factor normally aggregates/precipitates at neutral pH, PDP_60–114 can be used as a solubility-enhancing formulation. Expression of two engineered constructs with PDP_60–114 genetically fused to the mature domain of GDF11 through a 2x or 3x G4S linker produced soluble monomeric products that could be dimerized through redox reactions. The construct with a 3x G4S linker retained 10% activity (EC₅₀ = 10 nM), whereas the construct connected with a 2x G4S linker could only be activated (EC₅₀ = 2 nM) by protease treatment. Complex formation with PDP_60–114 represents a new strategy for stabilizing GDF11 in an active state that may translate to other members of the TGF-β family that form latent pro/mature domain complexes

Computational de Novo Design and Characterization of a Protein That Selectively Binds a Highly Hyperpolarizable Abiological Chromophore

This work reports the first example of a single-chain protein computationally designed to contain four α-helical segments and fold to form a four-helix bundle encapsulating a supramolecular abiological chromophore that possesses exceptional nonlinear optical properties. The 109-residue protein, designated <i><b>SCRPZ-1</b></i>, binds and disperses an insoluble hyperpolarizable chromophore, ruthenium­(II) [5-(4′-ethynyl-(2,2′;6′,2″-terpyridinyl))-10,20-bis­(phenyl)­porphinato]­zinc­(II)-(2,2′;6′,2″-terpyridine)²⁺ (<i><b>RuPZn</b></i>) in aqueous buffer solution at a 1:1 stoichiometry. A 1:1 binding stoichiometry of the holoprotein is supported by electronic absorption and circular dichroism spectra, as well as equilibrium analytical ultracentrifugation and size exclusion chromatography. <i><b>SCRPZ-1</b></i> readily dimerizes at micromolar concentrations, and an empirical redesign of the protein exterior produced a stable monomeric protein, <i><b>SCRPZ-2</b></i>, that also displayed a 1:1 protein:cofactor stoichiometry. For both proteins in aqueous buffer, the encapsulated cofactor displays photophysical properties resembling those exhibited by the dilute <i><b>RuPZn</b></i> cofactor in organic solvent: femtosecond, nanosecond, and microsecond time scale pump–probe transient absorption spectroscopic data evince intensely absorbing holoprotein excited states having large spectral bandwidth that penetrate deep in the near-infrared energy regime; the holoprotein electronically excited triplet state exhibits a microsecond time scale lifetime characteristic of the <i><b>RuPZn</b></i> chromophore. Hyper-Rayleigh light scattering measurements carried out at an incident irradiation wavelength of 1340 nm for these holoproteins demonstrate an exceptional dynamic hyperpolarizabilty (β₁₃₄₀ = 3100 × 10^–30 esu). X-ray reflectivity measurements establish that this de novo-designed hyperpolarizable protein can be covalently attached with high surface density to a silicon surface without loss of the cofactor, indicating that these assemblies provide a new approach to bioinspired materials that have unique electro-optic functionality

Stunted growth. Proceedings of the 23rd Aschauer Soiree, held at Aschauhof, Germany, November 7th 2015

Twenty-four scientists met at Aschauhof, Altenhof, Germany, to discuss the associations between child growth and development, and nutrition, health, environment and psychology. Meta-analyses of body height, height variability and household inequality, in historic and modern growth studies published since 1794, highlighting the enormously flexible patterns of child and adolescent height and weight increments throughout history which do not only depend on genetics, prenatal development, nutrition, health, and economic circumstances, but reflect social interactions. A Quality of Life in Short Stature Youth Questionnaire was presented to cross-culturally assess health-related quality of life in children. Changes of child body proportions in recent history, the relation between height and longevity in historic Dutch samples and also measures of body height in skeletal remains belonged to the topics of this meeting. Bayesian approaches and Monte Carlo simulations offer new statistical tools for the study of human growth

Stunted growth. Proceedings of the 23rd Aschauer Soiree, held at Aschauhof, Germany, November 7th 2015

Twenty-four scientists met at Aschauhof, Altenhof, Germany, to discuss the associations between child growth and development, and nutrition, health, environment and psychology. Meta-analyses of body height, height variability and household inequality, in historic and modern growth studies published since 1794, highlighting the enormously flexible patterns of child and adolescent height and weight increments throughout history which do not only depend on genetics, prenatal development, nutrition, health, and economic circumstances, but reflect social interactions. A Quality of Life in Short Stature Youth Questionnaire was presented to cross-culturally assess health-related quality of life in children. Changes of child body proportions in recent history, the relation between height and longevity in historic Dutch samples and also measures of body height in skeletal remains belonged to the topics of this meeting. Bayesian approaches and Monte Carlo simulations offer new statistical tools for the study of human growth