Intellectual disability in Duchenne muscular dystrophy has been associated with the loss of dystrophin‐protein 71, Dp71, the main dystrophin‐gene product in the adult brain. Dp71 shows major ...expression in perivascular macroglial endfeet, suggesting that dysfunctional glial mechanisms contribute to cognitive impairments. In the present study, we investigated the molecular alterations induced by a selective loss of Dp71 in mice, using semi‐quantitative immunogold analyses in electron microscopy and immunofluorescence confocal analyses in brain sections and purified gliovascular units. In macroglial pericapillary endfeet of the cerebellum and hippocampus, we found a drastic reduction (70%) of the polarized distribution of aquaporin‐4 (AQP4) channels, a 50% reduction of β‐dystroglycan, and a complete loss of α1‐syntrophin. Interestingly, in the hippocampus and cortex, these effects were not homogeneous: AQP4 and AQP4ex isoforms were mostly lost around capillaries but preserved in large vessels corresponding to pial arteries, penetrating cortical arterioles, and arterioles of the hippocampal fissure, indicating the presence of Dp71‐independent pools of AQP4 in these vascular structures. In conclusion, the depletion of Dp71 strongly alters the distribution of AQP4 selectively in macroglial perivascular endfeet surrounding capillaries. This effect likely affects water homeostasis and blood–brain barrier functions and may thus contribute to the synaptic and cognitive defects associated with Dp71 deficiency.
Main Points
Loss of dystrophin‐Dp71 impairs expression of AQP4 isoforms in perivascular glial endfeet of capillary, not in larger vessels.
Expression of other dystrophin proteins or utrophin paralogues may compensate for Dp71 loss in large vessels.
The cell wall is an important compartment in grain cells that fulfills both structural and functional roles. It has a dynamic structure that is constantly modified during development and in response ...to biotic and abiotic stresses. Non-structural cell wall proteins (CWPs) are key players in the remodeling of the cell wall during events that punctuate the plant life. Here, a subcellular and quantitative proteomic approach was carried out to identify CWPs possibly involved in changes in cell wall metabolism at two key stages of wheat grain development: the end of the cellularization step and the beginning of storage accumulation. Endosperm and outer layers of wheat grain were analyzed separately as they have different origins (maternal and seed) and functions in grains. Altogether, 734 proteins with predicted signal peptides were identified (CWPs). Functional annotation of CWPs pointed out a large number of proteins potentially involved in cell wall polysaccharide remodeling. In the grain outer layers, numerous proteins involved in cutin formation or lignin polymerization were found, while an unexpected abundance of proteins annotated as plant invertase/pectin methyl esterase inhibitors were identified in the endosperm. In addition, numerous CWPs were accumulating in the endosperm at the grain filling stage, thus revealing strong metabolic activities in the cell wall during endosperm cell differentiation, while protein accumulation was more intense at the earlier stage of development in outer layers. Altogether, our work gives important information on cell wall metabolism during early grain development in both parts of the grain, namely the endosperm and outer layers. The wheat cell wall proteome is the largest cell wall proteome of a monocot species found so far.
In proteomics, the interpretation of mass spectra representing peptides carrying multiple complex modifications remains challenging, as it is difficult to strike a balance between reasonable ...execution time, a limited number of false positives, and a huge search space allowing any number of modifications without a priori. The scientific community needs new developments in this area to aid in the discovery of novel post-translational modifications that may play important roles in disease. To make progress on this issue, we implemented SpecGlobX (SpecGlob eXTended to eXperimental spectra), a standalone Java application that quickly determines the best spectral alignments of a (possibly very large) list of Peptide-to-Spectrum Matches (PSMs) provided by any open modification search method, or generated by the user. As input, SpecGlobX reads a file containing spectra in MGF or mzML format and a semicolon-delimited spreadsheet describing the PSMs. SpecGlobX returns the best alignment for each PSM as output, splitting the mass difference between the spectrum and the peptide into one or more shifts while considering the possibility of non-aligned masses (a phenomenon resulting from many situations including neutral losses). SpecGlobX is fast, able to align one million PSMs in about 1.5 min on a standard desktop. Firstly, we remind the foundations of the algorithm and detail how we adapted SpecGlob (the method we previously developed following the same aim, but limited to the interpretation of perfect simulated spectra) to the interpretation of imperfect experimental spectra. Then, we highlight the interest of SpecGlobX as a complementary tool downstream to three open modification search methods on a large simulated spectra dataset. Finally, we ran SpecGlobX on a proteome-wide dataset downloaded from PRIDE to demonstrate that SpecGlobX functions just as well on simulated and experimental spectra. We then carefully analyzed a limited set of interpretations. SpecGlobX is helpful as a decision support tool, providing keys to interpret peptides carrying complex modifications still poorly considered by current open modification search software. Better alignment of PSMs enhances confidence in the identification of spectra provided by open modification search methods and should improve the interpretation rate of spectra.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A deficiency in the shortest dystrophin-gene product, Dp71, is a pivotal aggravating factor for intellectual disabilities in Duchenne muscular dystrophy (DMD). Recent advances in preclinical research ...have achieved some success in compensating both muscle and brain dysfunctions associated with DMD, notably using exon skipping strategies. However, this has not been studied for distal mutations in the
gene leading to Dp71 loss. In this study, we aimed to restore brain Dp71 expression in the Dp71-null transgenic mouse using an adeno-associated virus (AAV) administrated either by intracardiac injections at P4 (ICP4) or by bilateral intracerebroventricular (ICV) injections in adults. ICP4 delivery of the AAV9-Dp71 vector enabled the expression of 2 to 14% of brain Dp71, while ICV delivery enabled the overexpression of Dp71 in the hippocampus and cortex of adult mice, with anecdotal expression in the cerebellum. The restoration of Dp71 was mostly located in the glial endfeet that surround capillaries, and it was associated with partial localization of Dp71-associated proteins, α1-syntrophin and AQP4 water channels, suggesting proper restoration of a scaffold of proteins involved in blood-brain barrier function and water homeostasis. However, this did not result in significant improvements in behavioral disturbances displayed by Dp71-null mice. The potential and limitations of this AAV-mediated strategy are discussed. This proof-of-concept study identifies key molecular markers to estimate the efficiencies of Dp71 rescue strategies and opens new avenues for enhancing gene therapy targeting cognitive disorders associated with a subgroup of severely affected DMD patients.
Recent emphasis has been placed on the role that cerebellar dysfunctions could have in the genesis of cognitive deficits in Duchenne muscular dystrophy (DMD). However, relevant genotype-phenotype ...analyses are missing to define whether cerebellar defects underlie the severe cases of intellectual deficiency that have been associated with genetic loss of the smallest product of the
gene, the Dp71 dystrophin. To determine for the first time whether Dp71 loss could affect cerebellar physiology and functions, we have used patch-clamp electrophysiological recordings in acute cerebellar slices and a cerebellum-dependent behavioral test battery addressing cerebellum-dependent motor and non-motor functions in
null transgenic mice. We found that Dp71 deficiency selectively enhances excitatory transmission at glutamatergic synapses formed by climbing fibers (CFs) on Purkinje neurons, but not at those formed by parallel fibers. Altered basal neurotransmission at CFs was associated with impairments in synaptic plasticity and clustering of the scaffolding postsynaptic density protein PSD-95. At the behavioral level,
-null mice showed some improvements in motor coordination and were unimpaired for muscle force, static and dynamic equilibrium, motivation in high-motor demand and synchronization learning.
-null mice displayed altered strategies in goal-oriented navigation tasks, however, suggesting a deficit in the cerebellum-dependent processing of the procedural components of spatial learning, which could contribute to the visuospatial deficits identified in this model. In all, the observed deficits suggest that Dp71 loss alters cerebellar synapse function and cerebellum-dependent navigation strategies without being detrimental for motor functions.
Ovalbumin (OVA) is a food allergen whose allergenicity is modulated by heating. We aimed to establisha molecular connection between heat-induced modifications and the modulation of the IgE ...bindingcapacity of OVA. For this, we used model samples of heat-modified OVA with increasing complexity;glycated, aggregated or glycated and aggregated. Using sera from egg-allergic individuals, we showthat both aggregation and glycation strongly impacted IgE binding capacity, despite limited structuralchanges for glycated OVA. A molecular exploration at the amino acid level using high-resolution massspectrometry = revealed extensive cross-linking, mostly through disulfide and dehydroprotein bridges,and moderate but significant glycation. Structural modifications affected residues located within or ata few amino acids distance of known human linear IgE epitopes, such as C121, K123, S169, K190, K207,H332 and C368. We thus unveil key amino residues implicated in the changes in IgE binding of OVAinduced by heating
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•The allergenic risk of exposure to hemp seed was assessed.•7S vicilin-like proteins and edestins are potential hemp seed allergens.•A cross-reactivity exists between hazelnut and ...hemp seed proteins.
The increasing exposure of the population to Cannabis sativa has revealed allergies to different parts of the plant, among which hemp seed. Nonetheless, the major hemp seed allergens remain to be identified. Several known families of allergens are present in hemp seed, including notably seed storage proteins. We therefore aimed to investigate the potential allergenicity of the hemp seed storage proteins and their potential cross-reactivity to different seeds and nuts. For this, we extracted hemp seed proteins sequentially using buffers with increasing levels of salinity (H2O, T2 and T3) to yield extracts differentially enriched in storage proteins. We used these extracts to perform immunoblots and ELISAs using sera of patients either sensitized to hemp seeds or sensitized/allergic to other seeds and nuts. Immunoblots and proteomics analyses identified vicilins and edestins as potential hemp seed allergens. Moreover, ELISA analyses revealed a correlation between sensitization to hazelnut and the hemp seed T3 extract (enriched in storage proteins). The possible cross-reactivity between hazelnut and hemp seed proteins was further strengthened by the results from inhibition ELISAs: the incubation of sera from hazelnut-sensitized individuals with increasing concentrations of the T3 extract inhibited serum IgE binding to the hazelnut extract by about 25–30%. Our study thus identifies vicilins and edestins as potential hemp seed allergens and highlights a possible cross-reactivity with hazelnut. The clinical relevance of this cross-reactivity between hemp seed and hazelnut needs to be further investigated in hazelnut-allergic individuals.
Pectin, the major non-cellulosic component of primary cell wall can be degraded by polygalacturonases (PGs) and pectin methylesterases (PMEs) during pathogen attack on plants. We characterized two ...novel enzymes, VdPG2 and VdPME1, from the fungal plant pathogen Verticillium dahliae. VdPME1 was most active on citrus methylesterified pectin (55–70%) at pH 6 and a temperature of 40 °C, while VdPG2 was most active on polygalacturonic acid at pH 5 and a temperature of 50 °C. Using LC-MS/MS oligoprofiling, and various pectins, the mode of action of VdPME1 and VdPG2 were determined. VdPME1 was shown to be processive, in accordance with the electrostatic potential of the enzyme. VdPG2 was identified as endo-PG releasing both methylesterified and non-methylesterified oligogalacturonides (OGs). Additionally, when flax roots were used as substrate, acetylated OGs were detected. The comparisons of OGs released from Verticillium-susceptible and partially resistant flax cultivars identified new possible elicitor of plant defence responses.
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•Heterologous expression of two novel pectin degrading enzymes from Verticillium dahliae.•Mode of action determined by LC-MS/MS oligogalacturonides (OGs) profiling.•Computational approaches reveal PME processivity•OGs produced from Verticillium-susceptible or partially resistant flax cultivars differ
The plant cuticle is deposited on the surface of primary plant organs, such as leaves, fruits, and floral organs,forming a diffusion barrier and protecting the plant against various abiotic and ...biotic stresses. Cutin, thestructural polyester of the plant cuticle, is synthesized in the apoplast. Plasma-membrane-localized ATP-binding cassette (ABC) transporters of the G family have been hypothesized to export cutin precursors.Here, we characterizeSlABCG42of tomato representing an ortholog ofAtABCG32inArabidopsis.SlABCG42expression inArabidopsiscomplements the cuticular deficiencies of theArabidopsis pec1/abcg32mutant.RNAi-dependent downregulation of both tomato genes encoding proteins highly homologous to AtABCG32(SlABCG36 and SlABCG42) leads to reduced cutin deposition and formation of a thinner cuticle in tomatofruits. By using a tobacco (Nicotiana benthamiana) protoplast system, we show that AtABCG32 andSlABCG42 have an export activity for 10,16-dihydroxy hexadecanoyl-2-glycerol, a cutin precursorin vivo.Interestingly, also freeu-hydroxy hexadecanoic acid as well as hexadecanedioic acid were exported,furthering the research on the identification of cutin precursorsin vivoand the respective mechanisms of their integration into the cutin polymer.
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