Tauopathies are neurodegenerative diseases characterized by intracellular amyloid deposits of tau protein. Missense mutations in the tau gene ( MAPT ) correlate with aggregation propensity and cause dominantly inherited tauopathies, but their biophysical mechanism driving amyloid formation is poorly understood. Many disease-associated mutations localize within tau’s repeat domain at inter-repeat interfaces proximal to amyloidogenic sequences, such as 306 VQIVYK 311 . We use cross-linking mass spectrometry, recombinant protein and synthetic peptide systems, in silico modeling, and cell models to conclude that the aggregation-prone 306 VQIVYK 311 motif forms metastable compact structures with its upstream sequence that modulates aggregation propensity. We report that disease-associated mutations, isomerization of a critical proline, or alternative splicing are all sufficient to destabilize this local structure and trigger spontaneous aggregation. These findings provide a biophysical framework to explain the basis of early conformational changes that may underlie genetic and sporadic tau pathogenesis.

Gabarapl1 (gec1) was first described as an estrogen regulated gene which shares a high sequence homology with the gabarap gene. We previously demonstrated that GABARAPL1, like GABARAP, interacts with the GABAA receptor and tubulin and promotes tubulin polymerization. Previous work has demonstrated that the GABARAP family members (GABARAP, LC3, GATE-16 and Atg8) are not only involved in the transport of proteins or vesicles but are also implicated in various mechanisms such as autophagy, cell death, cell proliferation and tumor progression. We therefore asked whether GABARAPL1 might also play a role in autophagy. First, we showed that GABARAPL1 is cleaved at glycine 116, a residue which is conserved in other members of the family. We also demonstrated that GABARAPL1 is linked to phospholipids, delipidated by Atg4B, associated with intracellular membranes and accumulated in intracellular vesicles after inhibition of lysosomal activity. Finally, we showed that GABARAPL1 partially colocalizes with LC3 or Lysotracker green in intracellular vesicles. Taken together, our results demonstrate that GABARAPL1 associates with autophagic vesicles.

Odorant-binding proteins (OBPs) are small abundant extracellular proteins thought to participate in perireceptor events of odor-pheromone detection by carrying, deactivating, and/or selecting odor stimuli. The honeybee queen pheromone is known to play a crucial role in colony organization, in addition to drone sex attraction. We identified, for the first time in a social insect, a binding protein called antennal-specific protein 1 (ASP1), which binds at least one of the major queen pheromone components. ASP1 was characterized by cDNA cloning, expression in Pichia pastoris, and pheromone binding. In situ hybridization showed that it is specifically expressed in the auxiliary cell layer of the antennal olfactory sensilla. The ASP1 sequence revealed it as a divergent member of the insect OBP family. The recombinant protein presented the exact characteristics of the native protein, as shown by mass spectrometry, and N-terminal sequencing and exclusion-diffusion chromatography showed that recombinant ASP1 is dimeric. ASP1 interacts with queen pheromone major components, opposite to another putative honeybee OBP, called ASP2. ASP1 biosynthetic accumulation, followed by nondenaturing electrophoresis during development, starts at day 1 before emergence, in concomitance with the functional maturation of olfactory neurons. The isobar ASP1b isoform appears simultaneously to ASP1a in workers, but only at ϳ2 weeks after emergence in drones. Comparison of in vivo and heterologous expressions suggests that the difference between ASP1 isoforms might be because of dimerization, which might play a physiological role in relation with mate attraction.

Elicitins, produced by most of the phytopathogenic fungi of the genus Phytophthora, provoke in tobacco both remote leaf necrosis and the induction of a resistance against subsequent attack by various microorganisms. Despite the recent description of the three-dimensional crystal structure of cryptogein~CRY!, the molecular basis of the interactions between Phytophthora and plants largely remains unknown. The X-ray crystal structure, refined at 2.1 Å, of a ligand complexed, mutated CRY, K13H, is reported. Analysis of this structure reveals that CRY is able to encapsulate a ligand that induces only a minor conformational change in the protein structure. The ligand has been identified as an ergosterol by gas chromatographic analysis coupled with mass spectrometry analysis. This result is consistent with biochemical data that have shown that elicitins are a distinct class of Sterol Carrier Proteins~SCP!. Data presented here provide the first structural description of the pertinent features of the elicitin sterol interaction and permit a reassessment of the importance of both the key residue 13 and the mobility of the omega loop for the accessibility of the sterol to the cavity. The biological implications thereof are discussed. This paper reports the first structure of a SCP0sterol complex.

After characterization of a novel odorant-binding protein (OBP) variant isolated from the rat nasal mucus, the corresponding cDNA was cloned by RT-PCR. Recombinant OBP-1F, the sequence of which is close to that of previously reported rat OBP-1, has been secreted by the yeast Pichia pastoris at a concentration of 80 mg´L 21 in a form identical to the natural protein as shown by MS, N-terminal sequencing and CD. We observed that, in contrast with porcine OBP-1, purified recombinant OBP-1F is a homodimer exhibiting two disulfide bonds (C44±C48 and C63±C155), a pairing close to that of hamster aphrodisin. OBP-1F interacts with fluorescent probe 1-aminoanthracene (1-AMA) with a dissociation constant of 0.6^0.3 mm. Fluorescence experiments revealed that 1-AMA was displaced efficiently by molecules including usual solvents such as EtOH and dimethylsulfoxide. Owing to the large OBP-1F amounts expressed, we set up a novel biomimetic assay (volatileodorant binding assay) to study the uptake of airborne odorants without radiolabelling and attempted to understand the odorant capture by OBP in the nasal mucus under natural conditions. The assay permitted observations on the binding of airborne odorants of different chemical structures and odors (2-isobutyl-3-methoxypyrazine, linalool, isoamyl acetate, 1-octanal, 1-octanol, dimethyl disulfide and methyl thiobutyrate). Uptake of airborne odorants in nearly physiological conditions strengthens the role of OBP as volatile hydrophobic odorant carriers in the mucus of the olfactory epithelium through the aqueous barrier towards the chemo-sensory cells.Keywords: heterologous expression; odorant-binding protein; olfaction; Pichia pastoris; rat.In order to reach their membrane receptors embedded in the membrane of the olfactory neurons, airborne odorants, which are commonly hydrophobic molecules, have to be conveyed through the aqueous nasal mucus by carriers. The odorantbinding proteins (OBP), which are abundant low-molecular mass soluble proteins (< 20 kDa) secreted by the olfactory epithelium in the nasal mucus of vertebrates, have been thought to play such a role [1]. These proteins reversibly bind odorants with dissociation constants in the micromolar range. Although their functions are still unclear, OBP are also suspected to participate in the deactivation of odorants [2]. Most OBP are homodimers, but monomers and heterodimers have also been reported [3±6]. X-ray analysis has revealed that bovine and porcine OBP are folded in the typical b-barrel structure of lipocalins [7±9]. OBP have been identified in a variety of species, including pig, rabbit, mouse and rat [5,6,10±12] since the discovery of the first vertebrate OBP isolated from the bovine nasal mucus [3,13]. Different OBP subtypes have been reported to occur simultaneously in the same animal species, two in pig [5,14], four in mouse [15±17], three in rabbit [4] and at least eight in porcupine [18]. In rat, two OBP (called OBP-1 and OBP-2) have been cloned with quite different sequences [12,19,20]. The latter was specif...