Hugo L. Monaco

Pleurotus ostreatus Lectin (POL) is a 353 amino acid chain lectin that can be purified from the fruiting bodies of the very well-known and widely diffused edible oyster mushrooms (Pleurotus ostreatus). The lectin has been partially characterized by different groups and, although it was crystallized about 20 years ago, its three-dimensional structure and the details of its interactions with carbohydrates are still unknown. This paper reports the three-dimensional structure and ligand-binding properties of POL. We have determined the X-ray structure of the apo-protein purified from the fruiting bodies of the mushroom and that of the recombinant protein in complex with melibiose to a resolution of about 2 Å. The lectin is a homodimer in which the two polypeptide chains are linked by a disulfide bridge. A POL monomer is composed of two highly homologous β-jellyroll domains each of which containing a calcium-dependent carbohydrate binding site. A high degree of sequence similarity is observed between the two carbohydrate binding modules present in each monomer. The structure of the lectin in complex with melibiose reveals that a POL dimer has four calcium dependent carbohydrate-binding sites. The interaction with sugars in solution has been characterized by ITC and Saturation transfer difference (STD) NMR and it sheds new light on the molecular determinants of POL specificity. The lectin exhibits in vitro antiproliferative effects against human cancer cell lines and presents structural similarity with the prototype member of the CBM67 family, the non-catalytic domain of Streptomyces avermitilis α-Rhamnosidase.

For targeted brain delivery, nanoparticles (NPs) should bypass the blood‐brain barrier (BBB). Novel functionalization strategies, based on low‐density lipoprotein receptor (LDLR) binding domain, have been here tested to increase the brain targeting efficacy of polyd,l‐lactic‐co‐glycolic acid (PLGA) NPs, biodegradable and suited for biomedical applications. Custom‐made PLGA NPs were functionalized with an apolipoprotein E modified peptide (pep‐apoE) responsible for LDLR binding, or with lipocalin‐type prostaglandin‐d‐synthase (L‐PGDS), highly expressed in the brain. At the comparison of pep‐apoE and L‐PGDS sequences, a highly homologs region was here identified, indicating that also L‐PGDS could bind LDLR. Non‐functionalized and functionalized NPs did not affect the viability of cultured human dendritic cells, protagonists of the immune response, and did not activate them to a proinflammatory profile. At 2 h after intravenous injection in mice, functionalized, but not the non‐functionalized ones, fluorescent‐tagged NPs were observed in the cerebral cortex parenchyma. The NPs were mostly internalized by neurons and microglia; glial cells showed a weak activation. The findings indicate that the tested functionalization strategies do not elicit adverse immune responses and that the peptidic moieties enable BBB traversal of the NPs, thus providing potential brain drug carriers. These could be especially effective for brain diseases in which LDLR is involved. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 847–858, 2017.

The combined use of in vitro (¹⁹F-NMR) and in silico (molecular docking) procedures demonstrates the affinity of a number of human calycins (lipid-binding proteins from ileum, liver, heart, adipose tissue and epidermis, and retinol-binding protein from intestine) for different drugs (mainly steroids and vastatins). Comparative evaluations on the complexes outline some of the features relevant for interaction (non-polar character of the drugs; amino acids and water molecules in the protein calyx most often involved in binding). Dissociation constants (K_i) for drugs typically lie in the same range as K_i for natural ligands; in most instances (different proteins and docking conditions), vastatins are the strongest interactors, with atorvastatin ranking top in half of the cases. The affinity of some calycins for some of the vastatins is in the order of magnitude of the drug C_max after systemic administration in humans. The possible biological implications of this feature are discussed in connection with drug delivery parameters (route of administration, binding to carrier proteins, distribution to, and accumulation in, human tissues).

Carp FEL (fishelectin or fish-egg lectin) is a 238-amino-acid lectin that can be purified from fish eggs by exploiting its selective binding to Sepharose followed by elution withN-acetylglucosamine. Its amino-acid sequence and other biochemical properties have previously been reported. The glycoprotein has four disulfide bridges and the structure of the oligosaccharides linked to Asn27 has been described. Here, the three-dimensional structures of apo carp FEL (cFEL) and of its complex withN-acetylglucosamine determined by X-ray crystallography at resolutions of 1.35 and 1.70 Å, respectively, are reported. The molecule folds as a six-bladed β-propeller and internal short consensus amino-acid sequences have been identified in all of the blades. A calcium atom binds at the bottom of the funnel-shaped tunnel located in the centre of the propeller. Two ligand-binding sites, α and β, are present in each of the two protomers in the dimer. The first site, α, is closer to the N-terminus of the chain and is located in the crevice between the second and the third blades, while the second site, β, is located between the fourth and the fifth blades. The amino acids that participate in the contacts have been identified, as well as the conserved water molecules in all of the sites. Both sites can bind the two anomers, α and β, ofN-acetylglucosamine, as is clearly recognizable in the electron-density maps. The lectin presents sequence homology to members of the tachylectin family, which are known to have a function in the innate immune system of arthropods, and homologous genes are present in the genomes of other fish and amphibians. This structure is the first of a protein of this group and, given the degree of homology with other members of the family, it is expected that it will be useful to experimentally determine other crystal structures using the coordinates of cFEL as a search probe in molecular replacement.

Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the isomerization of the 9,11-endoperoxide group of PGH₂(prostaglandin H₂) to produce PGD₂(prostaglandin D₂) with 9-hydroxy and 11-keto groups. The product of the reaction, PGD₂, is the precursor of several metabolites involved in many regulatory events. L-PGDS, the first member of the important lipocalin family to be recognized as an enzyme, is also able to bind and transport small hydrophobic molecules and was formerly known as β-trace protein, the second most abundant protein in human cerebrospinal fluid. Previous structural work on the mouse and human proteins has focused on the identification of the amino acids responsible and the proposal of a mechanism for catalysis. In this paper, the X-ray structures of the apo and holo forms (bound to PEG) of the C65A mutant of human L-PGDS at 1.40 Å resolution and of the double mutant C65A/K59A at 1.60 Å resolution are reported. The apo forms of the double mutants C65A/W54F and C65A/W112F and the triple mutant C65A/W54F/W112F have also been studied. Mutation of the lysine residue does not seem to affect the binding of PEG to the ligand-binding cavity, and mutation of a single or both tryptophans appears to have the same effect on the position of these two aromatic residues at the entrance to the cavity. A solvent molecule has also been identified in an invariant position in the cavity of virtually all of the molecules present in the nine asymmetric units of the crystals that have been examined. Taken together, these observations indicate that the residues that have been mutated indeed appear to play a role in the entrance–exit process of the substrate and/or other ligands into/out of the binding cavity of the lipocalin.

Familial and idiopathic Parkinson's disease (PD) is associated with the abnormal neuronal accumulation of α-synuclein (aS) leading to β-sheet-rich aggregates called Lewy Bodies (LBs). Moreover, single point mutation in aS gene and gene multiplication lead to autosomal dominant forms of PD. A connection between PD and the 14-3-3 chaperone-like proteins was recently proposed, based on the fact that some of the 14-3-3 isoforms can interact with genetic PD-associated proteins such as parkin, LRRK2 and aS and were found as components of LBs in human PD. In particular, a direct interaction between 14-3-3η and aS was reported when probed by co-immunoprecipitation from cell models, from parkinsonian brains and by surface plasmon resonance in vitro. However, the mechanisms through which 14-3-3η and aS interact in PD brains remain unclear. Herein, we show that while 14-3-3η is unable to bind monomeric aS, it interacts with aS oligomers which occur during the early stages of aS aggregation. This interaction diverts the aggregation process even when 14-3-3η is present in sub-stoichiometric amounts relative to aS. When aS level is overwhelmingly higher than that of 14-3-3η, the fibrillation process becomes a sequestration mechanism for 14-3-3η, undermining all processes governed by this protein. Using a panel of complementary techniques, we single out the stage of aggregation at which the aS/14-3-3η interaction occurs, characterize the products of the resulting processes, and show how the processes elucidated in vitro are relevant in cell models. Our findings constitute a first step in elucidating the molecular mechanism of aS/14-3-3η interaction and in understanding the critical aggregation step at which 14-3-3η has the potential to rescue aS-induced cellular toxicity.