Screening a Molecular Fragment Library to Modulate the PED/PEA15-Phospholipase D1 Interaction in Cellular Lysate Environments.

The overexpression of PED/PEA15, the phosphoprotein enriched in diabetes/phosphoprotein enriched in the astrocytes 15 protein (here referred simply to as PED), observed in some forms of type II diabetes, reduces the transport of insulin-stimulated glucose by binding to the phospholipase D1 (PLD1). The inhibition of the PED/PLD1 interaction was shown to restore basal glucose transport, indicating PED as a pharmacological target for the development of drugs capable of improving insulin sensitivity and glucose tolerance. We here report the identification and selection of PED ligands by means of NMR screening of a library of small organic molecules, NMR characterization of the PED/PLD1 interaction in lysates of cells expressing PLD1, and modulation of such interactions using BPH03, the best selected ligand. Overall, we complement the available literature data by providing detailed information on the structural determinants of the PED/PLD1 interaction in a cellular lysate environment and indicate BPH03 as a precious scaffold for the development of novel compounds that are able to modulate such interactions with possible therapeutic applications in type II diabetes.

Structural Insight of the Full-Length Ros Protein: A Prototype of the Prokaryotic Zinc-Finger Family.

Ros/MucR is a widespread family of bacterial zinc-finger (ZF) containing proteins that integrate multiple functions such as virulence, symbiosis and/or cell cycle transcription. NMR solution structure of Ros DNA-binding domain (region 56-142, i.e. Ros87) has been solved by our group and shows that the prokaryotic ZF domain shows interesting structural and functional features that differentiate it from its eukaryotic counterpart as it folds in a significantly larger zinc-binding globular domain. We have recently proposed a novel functional model for this family of proteins suggesting that they may act as H-NS-'like' gene silencers. Indeed, the N-terminal region of this family of proteins appears to be responsible for the formation of functional oligomers. No structural characterization of the Ros N-terminal domain (region 1-55) is available to date, mainly because of serious solubility problems of the full-length protein. Here we report the first structural characterization of the N-terminal domain of the prokaryotic ZF family examining by means of MD and NMR the structural preferences of the full-length Ros protein from Agrobacterium tumefaciens.

Regulation of leukocyte recruitment by the long pentraxin PTX3.

Pentraxins are a superfamily of conserved proteins involved in the acute-phase response and innate immunity. Pentraxin 3 (PTX3), a prototypical member of the long pentraxin subfamily, is a key component of the humoral arm of innate immunity that is essential for resistance to certain pathogens. A regulatory role for pentraxins in inflammation has long been recognized, but the underlying mechanisms remain unclear. Here we report that PTX3 bound P-selectin and attenuated neutrophil recruitment at sites of inflammation. PTX3 released from activated leukocytes functioned locally to dampen neutrophil recruitment and regulate inflammation. Antibodies have glycosylation-dependent regulatory effect on inflammation. Therefore, PTX3, which is an essential component of humoral innate immunity, and immunoglobulins share functional outputs, including complement activation, opsonization and, as shown here, glycosylation-dependent regulation of inflammation.

Design, synthesis, and in vitro activity of peptidomimetic inhibitors of myeloid differentiation factor 88.

We describe the design and synthesis of a peptidomimetic library derived from the heptapeptide Ac-RDVLPGT-NH 2, belonging to the Toll/IL-1 receptor (TIR) domain of the adaptor protein MyD88 and effective in inhibiting its homodimerization. The ability of the peptidomimetics to inhibit protein-protein interaction was assessed by yeast 2-hybrid assay and further validated in a mammalian cell system by evaluating the inhibition of NF-kappaB activation, a transcription factor downstream of MyD88 signaling pathway that allows production of essential effector molecules for immune and inflammatory responses.

High-level expression and efficient purification of recombinant human long pentraxin PTX3 in Chinese hamster ovary cells.

PTX3 is a secreted multimeric glycoprotein which plays a key role in innate immunity by activating the classical complement pathway through specific recognition of the C1q subunit. A method is described for the high level expression of the recombinant human PTX3 in Chinese hamster ovary cells (CHO), adapted to a suspension growth in spinner flasks containing a serum-free chemically defined medium and producing about 50 mg of PTX3/L of culture. A purification procedure to produce a homogeneous protein preparation from the supernatant, by means of anion exchange, hydroxyapatite and size exclusion chromatography, is also reported. This three-step protocol allows us to obtain PTX3 with a recovery yield close to 70%, a purity degree exceeding 95%, and a final host cell protein (HCP) content lower than 150 ppm. The recombinant purified PTX3 retains its biological activity, as demonstrated by C1q binding ELISA assay, and displays a complex quaternary structure characterized by a high secondary structure content quite different from human short pentraxin C-reactive protein (CRP) and serum amyloid P component (SAP), as determined by circular dichroism, fluorescence analysis, and native and SDS-PAGE experiments.