Structural basis for phosphatidylinositol phosphate kinase type Igamma binding to talin at focal adhesions

The cytoskeletal protein talin binds to a short C-terminal sequence in phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma), activating the enzyme and promoting the local production of phosphatidylinositol 4,5 bisphosphate, which regulates focal adhesion dynamics as well as clathrin-mediated endocytosis in neuronal cells. Here we show by crystallographic, NMR, and calorimetric analysis that the phosphotyrosine binding (PTB)-like domain of talin engages the PIPKIgamma C terminus in a mode very similar to that of integrin binding. However, PIPKIgamma binds in the canonical PTB-peptide mode with an SPLH motif replacing the classic NPXY motif. The tighter packing of the SPLH motif against the hydrophobic core of talin may explain the stronger binding of PIPKIgamma. Two tyrosine residues flanking the SPLH motif (Tyr-644 and Tyr-649) have been implicated in the regulation of talin binding. We show that phosphorylation at Tyr-644, a Src phosphorylation site in vivo, has little effect on the binding mode or strength, which is consistent with modeling studies in which the phosphotyrosine makes surface-exposed salt bridges, and we suggest that its strong activating effect arises from the release of autoinhibitory restraints in the full-length PIPKIgamma. Modeling studies suggest that phosphorylation of Tyr-649 will likewise have little effect on talin binding, whereas phosphorylation of the SPLH serine is predicted to be strongly disruptive. Our data are consistent with the proposal that Src activity promotes a switch from integrin binding to PIPKIgamma binding that regulates focal adhesion turnover.     

Structural analysis of Siah1-Siah-interacting protein interactions and insights into the assembly of an E3 ligase multiprotein complex

Siah1 is the central component of a multiprotein E3 ubiquitin ligase complex that targets beta-catenin for destruction in response to p53 activation. The E3 complex comprises, in addition to Siah1, Siah-interacting protein (SIP), the adaptor protein Skp1, and the F-box protein Ebi. Here we show that SIP engages Siah1 by means of two elements, both of which are required for mediating beta-catenin destruction in cells. An N-terminal dimerization domain of SIP sits across the saddle-shaped upper surface of Siah1, with two extended legs packing against the sides of Siah1 by means of a consensus PXAXVXP motif that is common to a family of Siah-binding proteins. The C-terminal domain of SIP, which binds to Skp1, protrudes from the lower surface of Siah1, and we propose that this surface provides the scaffold for bringing substrate and the E2 enzyme into apposition in the functional complex.     

Properties of recombinant human cytosolic sialidase HsNEU2. The enzyme hydrolyzes monomerically dispersed GM1 ganglioside molecules

Recombinant human cytosolic sialidase (HsNEU2), expressed in Escherichia coli, was purified to homogeneity, and its substrate specificity was studied. HsNEU2 hydrolyzed 4-methylumbelliferyl alpha-NeuAc, alpha 2-->3 sialyllactose, glycoproteins (fetuin, alpha-acid glycoprotein, transferrin, and bovine submaxillary gland mucin), micellar gangliosides GD1a, GD1b, GT1b, and alpha 2-->3 paragloboside, and vesicular GM3. alpha 2-->6 sialyllactose, colominic acid, GM1 oligosaccharide, whereas micellar GM2 and GM1 were resistant. The optimal pH was 5.6, kinetics Michaelis-Menten type, V(max) varying from 250 IU/mg protein (GD1a) to 0.7 IU/mg protein (alpha(1)-acid glycoprotein), and K(m) in the millimolar range. HsNEU2 was activated by detergents (Triton X-100) only with gangliosidic substrates; the change of GM3 from vesicular to mixed micellar aggregation led to a 8.5-fold V(max) increase. HsNEU2 acted on gangliosides (GD1a, GM1, and GM2) at nanomolar concentrations. With these dispersions (studied in detailed on GM1), where monomers are bound to the tube wall or dilutedly associated (1:2000, mol/mol) to Triton X-100 micelles, the V(max) values were 25 and 72 microIU/mg protein, and K(m) was 10 and 15 x 10(-9) m, respectively. Remarkably, GM1 and GM2 were recognized only as monomers. HsNEU2 worked at pH 7.0 with an efficiency (compared with that at pH 5.6) ranging from 4% (on GD1a) to 64% (on alpha(1)-acid glycoprotein), from 7% (on GD1a) to 45% (on GM3) in the presence of Triton X-100, and from 30 to 40% on GM1 monomeric dispersion. These results show that HsNEU2 differentially recognizes the type of sialosyl linkage, the aglycone part of the substrate, and the supramolecular organization (monomer/micelle/vesicle) of gangliosides. The last ability might be relevant in sialidase interactions with gangliosides under physiological conditions.     

Rapid in vivo transport of proteins from digested allergen across pre-sensitized gut

Although the route of sensitization to food allergens is still the subject of debate, it is generally accepted the gut immune system plays a pivotal role. However, hitherto the transport of allergens across the normal, pre-sensitized gut epithelium remained largely unknown. Our aim was to identify the route through which protein bodies and soluble proteins from digested peanuts penetrated the pre-sensitized gut epithelium in vivo and the specific cell types involved in the transport. Digestion of peanuts released a large number of protein bodies that are exclusively transported across the epithelium by specialized antigen-sampling M cells and delivered to the lymphoid tissue of Peyer's patch. Intracellular transport of soluble protein also occurred almost exclusively via M cells and it was negligible across absorptive enterocytes. We hypothesize that these conditions which are known to favour strongly the induction of immune responses rather than oral tolerance may play a significant role in the genesis of allergic reactions.     

Some factors affecting the stability of interferon alpha 2b in solution.

In this paper we evaluated the influence of the protein concentration and a formulation vehicle on the stability of recombinant human Interferon alpha 2b (rhIFN-alpha2b) in solution. The effect of the protein content (from 1 to 100 MIU/ml) at 37 degrees C, showed that higher concentration of this cytokine protected against the loss of bioactivity (antiviral titration) better than the lower concentrations. In contrast, rhIFN-alpha2b at 50 and 100 MIU/ml decreased the SDS/PAGE- and RP-HPLC-determined purity faster than samples at 1 or 10 MIU/ml. According to these results, 10 MIU/ml rhIFN-alpha2b was the best choice to evaluate the influence of a formulation on the stability of this cytokine. Taking this into consideration, we studied the stability of a liquid and albumin-free formulation of this protein at the recommended storage temperature (5+/-3 degrees C) and under accelerated conditions (28+/-2 degrees C). Accelerated storage results showed an acceptable biochemical stability of the active ingredient throughout 2 months. Real-time storage data confirmed the good biochemical stability of this formulation for 30 months.     

The role of residue Thr249 in modulating the catalytic efficiency and substrate specificity of catechol-2,3-dioxygenase from Pseudomonas stutzeri OX1

Bioremediation strategies use microorganisms to remove hazardous substances, such as aromatic molecules, from polluted sites. The applicability of these techniques would greatly benefit from the expansion of the catabolic ability of these bacteria in transforming a variety of aromatic compounds. Catechol-2,3-dioxygenase (C2,3O) from Pseudomonas stutzeri OX1 is a key enzyme in the catabolic pathway for aromatic molecules. Its specificity and regioselectivity control the range of molecules degraded through the catabolic pathway of the microorganism that is able to use aromatic hydrocarbons as growth substrates. We have used in silico substrate docking procedures to investigate the molecular determinants that direct the enzyme substrate specificity. In particular, we looked for a possible molecular explanation of the inability of catechol-2,3-dioxygenase to cleave 3,5-dimethylcatechol and 3,6-dimethylcatechol and of the efficient cleavage of 3,4-dimethylcatechol. The docking study suggested that reduction in the volume of the side chain of residue 249 could allow the binding of 3,5-dimethylcatechol and 3,6-dimethylcatechol. This information was used to prepare and characterize mutants at position 249. The kinetic and regiospecificity parameters of the mutants confirm the docking predictions, and indicate that this position controls the substrate specificity of catechol-2,3-dioxygenase. Moreover, our results suggest that Thr249 also plays a previously unsuspected role in the catalytic mechanism of substrate cleavage. The hypothesis is advanced that a water molecule bound between one of the hydroxyl groups of the substrate and the side chain of Thr249 favors the deprotonation/protonation of this hydroxyl group, thus assisting the final steps of the cleavage reaction.     

Effect of protein backbone folding on the stability of protein-ligand complexes

The role played by the degree of folding of protein backbones in explaining the binding energetics of protein-ligand interactions has been studied. We analyzed the protein/peptide interactions in the RNase-S system in which amino acids at two positions of the peptide S have been mutated. The global degree of folding of the protein S correlates in a significant way with the free energy and enthalpy of the protein-peptide interactions. A much better correlation is found with the local contribution to the degree of folding of one amino acid residue: Thr36. This residue is shown to have a destabilizing interaction with Lys41, which interacts directly with peptide S. Another system, consisting of the interactions of small organic molecules with HIV-1 protease was also studied. In this case, the global change in the degree of folding of the protease backbone does not explain the binding energetics of protein-ligand interactions. However, a significant correlation is observed between the free energy of binding and the contribution of two amino acid residues in the HVI-1 protease: Gly49 and Ile66. In general, it was observed that the changes in the degree of folding are not restricted to the binding site of the protein chain but are distributed along the whole protein backbone. This study provides a basis for further consideration of the degree of folding as a parameter for empirical structural parametrizations of the binding energetics of protein folding and binding.     

Optimisation of the purification process of a tumour-targeting antibody produced in <Emphasis Type="Italic">N. benthamiana</Emphasis> using vacuum-agroinfiltration

It was previously demonstrated that the tumour-targeting antibody mAb H10 can be transiently expressed and purified at high levels in Nicotiana benthamiana by using a vacuum-agroinfiltration system boosted by the use of a virus silencing suppressor protein. Scope of this work was to analyse different steps of protein extraction from agroinfiltrated leaves to optimise the purification process of the secretory mAb H10 providing new insights in the field of large-scale plant production. Two different extraction procedures (mechanical shearing/homogenisation and recovery of intercellular fluids -IFs-) were evaluated and compared in terms of purified antibody yields, antibody degradation and total phenolic compounds content. Mechanical grinding from fresh leaf tissues gave the highest purification yield (75 mg/kg Fresh Weight -75% intact tetrameric IgG-) and total phenolics concentration in the range of 420 μg/g FW. The second extraction procedure, based on the recovery of IFs, gave purification yields of 15–20 mg/kg FW (corresponding to 27% of total soluble protein) in which about 40% of purified protein is constituted by fully assembled IgG with a total phenolic compounds content reduced by one order of magnitude (21 μg/g FW). Despite a higher antibody degradation, purification from intercellular fluids demonstrated to be very promising since extraction procedures resulted extremely fast and amenable to scaling-up. Overall data highlight that different extraction procedures can dramatically affect the proteolytic degradation and quality of antibody purified from agroinfiltrated N. benthamiana leaves. Based on these results, we optimised a pilot-scale purification protocol using a two-step purification procedure from batches of fresh agroinfiltrated leaves (250 g) allowing purification of milligram quantities (average yield 40 mg/kg FW) of fully assembled and functional IgG with a 99.4% purity, free of phenolic and alkaloid compounds with low endotoxin levels (<1 EU/ml).