Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants

Cold storage of tubers of potato (Solanum tuberosum L.) compromises tuber quality in many cultivars by the accumulation of hexose sugars in a process called cold-induced sweetening. This is caused by the breakdown of starch to sucrose, which is cleaved to glucose and fructose by vacuolar acid invertase. During processing of affected tubers, the high temperatures involved in baking and frying cause the Maillard reaction between reducing sugars and free amino acids, resulting in the accumulation of acrylamide. cDNA clones with deduced proteins homologous to known invertase inhibitors were isolated and the two most abundant forms, termed INH1 and INH2, were shown to possess apoplastic and vacuolar localization, respectively. The INH2 gene showed developmentally regulated alternative splicing, so, in addition to the INH2α transcript encoding the full-length protein, two hybrid mRNAs (INH2β*A and INH2β*B) that encoded deduced vacuolar invertase inhibitors with divergent C-termini were detected, the result of mRNA splicing of an upstream region of INH2 to a downstream region of INH1. Hybrid RNAs are common in animals, where they may add to the diversity of the proteome, but are rarely described in plants. During cold storage, INH2α and the hybrid INH2β mRNAs accumulated to higher abundance in cultivars resistant to cold-induced sweetening than in susceptible cultivars. Increased amounts of invertase inhibitor may contribute to the suppression of acid invertase activity and prevent cleavage of sucrose. Evidence for increased RNA splicing activity was detected in several resistant lines, a mechanism that in some circumstances may generate a range of proteins with additional functional capacity to aid adaptability

CRISTALLOGENESE DU BPTI A PH ACIDE (ETUDE DES RELATIONS ENTRE L'ETAT D'ASSOCIATION DES MOLECULES EN SOLUTION ET A L'ETAT CRISTALLIN PAR DIFFUSION ET DIFFRACTION DES RAYONS X)

ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Crystal structure of Epiphyas postvittana pheromone binding protein 3

The insect olfactory system operates as a well-choreographed ensemble of molecules which functions to selectively translate volatile chemical messages present in the environment into neuronal impulses that guide insect behaviour. Of these molecules, binding proteins are believed to transport hydrophobic odorant molecules across the aqueous lymph present in antennal sensilla to receptors present in olfactory sensory neurons. Though the exact mechanism through which these proteins operate is still under investigation, these carriers clearly play a critical role in determining what an insect can smell. Binding proteins that transport important sex pheromones are colloquially named pheromone binding proteins (PBPs). Here, we have produced a functional recombinant PBP from the horticultural pest, Epiphyas postvittana (EposPBP3), and experimentally solved its apo-structure through X-ray crystallography to a resolution of 2.60 Å. Structural comparisons with related lepidopteran PBPs further allowed us to propose models for the binding of pheromone components to EposPBP3. The data presented here represent the first structure of an olfactory-related protein from the tortricid family of moths, whose members cause billions of dollars in losses to agricultural producers each year. Knowledge of the structure of these important proteins will allow for subsequent studies in which novel, olfactory molecule-specific insecticides can be developed

Structure of an antennally-expressed carboxylesterase suggests lepidopteran odorant degrading enzymes are broadly tuned

Insects rely on the detection of chemical cues present in the environment to guide their foraging and reproductive behaviour. As such, insects have evolved a sophisticated chemical processing system in their antennae comprised of several types of olfactory proteins. Of these proteins, odorant degrading enzymes are responsible for metabolising the chemical cues within the antennae, thereby maintaining olfactory system function. Members of the carboxyl/cholinesterase gene family are known to degrade odorant molecules with acetate-ester moieties that function as host recognition cues or sex pheromones, however, their specificity for these compounds remains unclear. Here, we evaluate expression levels of this gene family in the light-brown apple moth, Epiphyas postvittana, via RNAseq and identify putative odorant degrading enzymes. We then solve the apo-structure for EposCCE24 by X-ray crystallography to a resolution of 2.43 Å and infer substrate specificity based on structural characteristics of the enzyme's binding pocket. The specificity of EposCCE24 was validated by testing its ability to degrade biologically relevant and non-relevant sex pheromone components and plant volatiles using GC–MS. We found that EposCCE24 is neither capable of discriminating between linear acetate-ester odorant molecules of varying chain length, nor between molecules with varying double bond positions. EposCCE24 efficiently degraded both plant volatiles and sex pheromone components containing acetate-ester functional groups, confirming its role as a broadly-tuned odorant degrading enzyme in the moth olfactory organ

Structure of Spa15, a type III secretion chaperone from Shigella flexneri with broad specificity

Type III secretion (TTS) systems are used by many Gram-negative pathogens to inject virulence proteins into the cells of their hosts. Several of these virulence effectors require TTS chaperones that maintain them in a secretion-competent state. Whereas most chaperones bind only one effector, Spa15 from the human pathogen Shigella flexneri and homologous chaperones bind several seemingly unrelated effectors, and were proposed to form a special subgroup. Its 1.8 Å crystal structure confirms this specific classification, showing that Spa15 has the same fold as other TTS effector chaperones, but forms a different dimer. The presence of hydrophobic sites on the Spa15 surface suggests that the different Spa15 effectors all possess similar structural elements that can bind these sites. Furthermore, the Spa15 structure reveals larger structural differences between class I chaperones than previously anticipated, which does not support the hypothesis that chaperone–effector complexes are structurally conserved and function as three-dimensional secretion signals

The BPTI decamer observed in acidic pH crystal forms pre-exists as a stable species in solution

International audienceBovine pancreatic trypsin inhibitor (BPTI) crystallizes under acidic pH conditions in the presence of thiocyanate, chloride and sulfate ions, yielding three different polymorphs in P2$_1$, P6$_4$22 and P6$_3$22 space groups, respectively. In all three crystal forms, the same decamer is found in the packing (ten BPTI molecules organized through two perpendicular 2-fold and 5-fold axes as a well-defined and compact object) in contrast to the monomeric crystal forms observed at basic pH conditions. The crystallization of BPTI under acidic conditions (pH 4.5) was investigated by small angle X-ray scattering with both under- and supersaturated BPTI solutions. Data showed the oligomerization of BPTI molecules under all investigated conditions. Accordingly, various mixtures of discrete oligomers ($n$=1 to 10) were considered. Calculated scattering curves were obtained using models based on the crystallographic structures, and the experimental patterns were analyzed as a linear combination of the model curves using a non-linear curve fitting procedure. The results, confirmed by gel filtration experiments, unambiguously demonstrate the co-existence of two different BPTI particles in solution: a monomer and a decamer, with no evidence of any other intermediates. Moreover, using both approaches, the fraction of decamers was found to increase with increasing salt concentration, even beyond the solubility curve. We therefore propose that at acidic pH, BPTI crystallizes following a two step process: decamers are first built in under- and supersaturated solutions, upon which crystal growth proceeds by decamer stacking. Indeed, those BPTI crystals should best be described as “BPTI decamer” crystals

The BPTI decamer observed in acidic pH crystal forms pre-exists as a stable species in solution

International audienceBovine pancreatic trypsin inhibitor (BPTI) crystallizes under acidic pH conditions in the presence of thiocyanate, chloride and sulfate ions, yielding three different polymorphs in P2$_1$, P6$_4$22 and P6$_3$22 space groups, respectively. In all three crystal forms, the same decamer is found in the packing (ten BPTI molecules organized through two perpendicular 2-fold and 5-fold axes as a well-defined and compact object) in contrast to the monomeric crystal forms observed at basic pH conditions. The crystallization of BPTI under acidic conditions (pH 4.5) was investigated by small angle X-ray scattering with both under- and supersaturated BPTI solutions. Data showed the oligomerization of BPTI molecules under all investigated conditions. Accordingly, various mixtures of discrete oligomers ($n$=1 to 10) were considered. Calculated scattering curves were obtained using models based on the crystallographic structures, and the experimental patterns were analyzed as a linear combination of the model curves using a non-linear curve fitting procedure. The results, confirmed by gel filtration experiments, unambiguously demonstrate the co-existence of two different BPTI particles in solution: a monomer and a decamer, with no evidence of any other intermediates. Moreover, using both approaches, the fraction of decamers was found to increase with increasing salt concentration, even beyond the solubility curve. We therefore propose that at acidic pH, BPTI crystallizes following a two step process: decamers are first built in under- and supersaturated solutions, upon which crystal growth proceeds by decamer stacking. Indeed, those BPTI crystals should best be described as “BPTI decamer” crystals

Structural mimicry for vinculin activation by IpaA, a virulence factor of Shigella flexneri

Invasion of epithelial cells by Shigella flexneri is characterized by cytoskeletal rearrangements of the host cell membrane, promoting internalization of the bacterium. The bacterial effector IpaA is injected into the epithelial cell by a type III secretion apparatus and recruits vinculin to regulate actin polymerization at the site of entry. We analysed the complex formed between a carboxy-terminal fragment of IpaA (IpaA(560−633)) and the vinculin D1 domain (VD1), both in crystals and in solution. We present evidence that IpaA(560−633) has two α-helical vinculin-binding sites that simultaneously bind two VD1 molecules. The interaction of IpaA(560−633) with VD1 is highly similar to the interaction of the endogenous, eukaryotic proteins talin and α-actinin with VD1, showing that Shigella uses a structural mimicry strategy to activate vinculin

The decameric structure of bovine pancreatic trypsin inhibitor (BPTI) crystallized from thiocyanate at 2.7 Å resolution

International audienceThe structure of a monoclinic form of bovine pancreatic trypsin inhibitor (BPTI) crystallized from a thiocyanate solution has been determined and refined at 2.7 Å resolution. The space group is P2$_1$ with $a$ = 71.56, $b$ = 73.83, $c$ = 64.47 Å, $\beta$ = 93.9° and Z = 20. The ten independent molecules were located by a multi-body molecular-replacement search as developed in the AMoRe program, starting from a single monomer model (PDB code: 6PTI). The molecular arrangement of the subunits is a decamer resulting from the combination of two orthogonal fivefold and twofold non-crystallographic axes. This builds a globular micelle-like particle which minimizes hydrophobic interactions with the solvent. The refinement was conducted with non-crystallographic symmetry constraints up to a final residual of R = 0.20 (R$_{free}$ = 0.26). The root-mean-square deviations from ideal geometry were 0.015 Å and 1.6° on bond distances and bond angles, respectively. Several sites for thiocyanate ions were analyze