Lipolysis-Stimulated Lipoprotein Receptor Acts as Sensor to Regulate ApoE Release in Astrocytes.

Astroglia play an important role, providing de novo synthesized cholesterol to neurons in the form of ApoE-lipidated particles; disruption of this process can increase the risk of Alzheimer's disease. We recently reported that glia-specific suppression of the lipolysis-stimulated lipoprotein receptor (LSR) gene leads to Alzheimer's disease-like memory deficits. Since LSR is an Apo-E lipoprotein receptor, our objective of this study was to determine the effect of LSR expression modulation on cholesterol and ApoE output in mouse astrocytes expressing human ApoE3. qPCR analysis showed that siRNA-mediated lsr knockdown significantly increased expression of the genes involved in cholesterol synthesis, secretion, and metabolism. Analysis of media and lipoprotein fractions showed increased cholesterol and lipidated ApoE output in HDL-like particles. Further, lsr expression could be upregulated when astrocytes were incubated 5 days in media containing high levels (two-fold) of lipoprotein, or after 8 h treatment with 1 µM LXR agonist T0901317 in lipoprotein-deficient media. In both conditions of increased lsr expression, the ApoE output was repressed or unchanged despite increased abca1 mRNA levels and cholesterol production. We conclude that LSR acts as a sensor of lipoprotein content in the medium and repressor of ApoE release, while ABCA1 drives cholesterol efflux, thereby potentially affecting cholesterol load, ApoE lipidation, and limiting cholesterol trafficking towards the neuron.

Functional domain analysis of LmSAP protein reveals the crucial role of the zinc-finger A20 domain in abiotic stress tolerance.

Stress-associated proteins (SAPs), such as A20/AN1 zinc-finger domain-containing proteins, have emerged as a novel class of proteins involved in abiotic stress signaling, and they are important candidates for preventing the loss of yield caused by exposure to environmental stresses. In a previous report, it was found that the ectopic-expression of Lobularia maritima stress-associated protein, LmSAP, conferred tolerance to abiotic and heavy metal stresses in transgenic tobacco plants. This study aimed to investigate the functions of the A20 and AN1 domains of LmSAP in salt and osmotic stress tolerance. To this end, in addition to the full-length LmSAP gene, we have generated three LmSAP-truncated forms (LmSAPΔA20, LmSAPΔAN1, and LmSAPΔA20-ΔAN1). Heterologous expression in Saccharomyces cerevisiae of different truncated forms of LmSAP revealed that the A20 domain is essential to increase cell tolerance to salt, ionic, and osmotic stresses. Transgenic tobacco plants overexpressing LmSAP and LmSAPΔAN1 constructs exhibited higher tolerance to salt and osmotic stresses in comparison to the non-transgenic plants (NT) and lines transformed with LmSAPΔA20 and LmSAPΔA20-ΔAN1 constructs. Similarly, transgenic plants overexpressing the full-length LmSAP gene and LmSAPΔAN1 truncated domain maintained higher superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) enzymatic activities due to the high expression levels of the genes encoding these key antioxidant enzymes, MnSOD, POD, and CAT1, as well as accumulated lower levels of malondialdehyde (MDA) under salt and osmotic stresses compared to NT and LmSAPΔA20 and LmSAPΔA20-ΔAN1 forms. These findings provide insights into the pivotal role of A20 and AN1 domains of LmSAP protein in salt and osmotic stress tolerance.

Identification and molecular characterization of allergenic non-specific lipid-transfer protein from durum wheat (Triticum turgidum).

BACKGROUND: Common wheat (Triticum aestivum) and durum wheat (T. turgidum) are both involved in Baker's asthma (BA) and food allergy (FA) including wheat-dependent exercise-induced asthma (WDEIA). However, allergens in durum wheat have not been described, and the over-expression of T. turgidum non-specific lipid-transfer protein (nsLTPs) is considered to increase resistance to phytopathogens. OBJECTIVE: To identify and assess the allergenicity of nsLTP from T. turgidum. METHODS: Recombinant T. turgidum nsLTP Tri tu 14 was generated and tested for structural integrity (circular dichroism-spectroscopy) and purity (SDS-PAGE). Thirty-two wheat allergic patients were enrolled: 20 Spanish patients (BA) with positive bronchial challenge to wheat flour, and 12 Italian patients (wheat FA/WDEIA) with positive double-blind placebo-controlled food challenge/open food challenge (OFC) to pasta. IgE values to wheat, Tri tu 14, Tri a 14 (T. aestivum) and Pru p 3 (P. persica) were determined by ImmunoCAP testing. Allergenic potency (in vitro mediator release) and IgE cross-reactivity were investigated. RESULTS: Tri tu 14 was found to share 49% and 52% amino acid identity with Tri a 14 and Pru p 3, respectively. Among 25 Tri a 14 CAP positive sera, 23 (92%) were reactive to wheat extract, 22 (88%) to Tri tu 14 and 20 (80%) to Pru p 3. The correlation between Tri a 14 and Tri tu 14 specific IgE levels was r = 0.97 (BA) and r = 0.93 (FA/WDEIA), respectively. FA/WDEIA patients showed higher specific IgE values to Tri tu 14 and Pru p 3 than BA patients. Tri tu 14 displayed allergenic activity by mediator release from effector cells and IgE cross-reactivity with Pru p 3. The degree of IgE cross-reactivity between the two wheat nsLTPs varied between individual patients. CONCLUSIONS AND CLINICAL RELEVANCE: Sensitization to Tri tu 14 likely appears to be more important in wheat FA/WDEIA than in BA. Over-expression of Tri tu 14 in wheat would represent a risk for patients with nsLTP-mediated FA.

Promoter of the wheat lipid transfer protein, TdLTP4, drives leaf-preferential expression in transgenic Arabidopsis plants.

In a previous report, a gene encoding a durum wheat lipid transfer protein, TdLTP4, was characterised as induced by abiotic and biotic stresses. In the present work, we investigated the regulation of the gene TdLTP4. A TdLTP4 promoter (PrTdLTP4) region of around 868-bp was isolated and sequenced. Its analysis revealed the presence of several DNA boxes known to be important mainly in the regulation of genes expressed under abiotic stress (salt and dehydration), abscisic acid (ABA) and pathogen responsiveness. The whole PrTdLTP4 fragment was fused to the reporter gene ß-glucuronidase (gusA) and analysed in transgenic Arabidopsis plants. Histochemical assays of transgenic Arabidopsis plants showed that the 868-bp fragment of TdLTP4 gene promoter was found to be sufficient for both spatial and temporal patterns of its expression. Under control conditions, GUS histochemical staining was observed significantly only in young leaves of 8- and 12-day-old plants. Whereas after stress challenge especially with NaCl and mannitol, GUS transcripts expression increased substantially in leaves of 30-day-old transgenic seedlings. Real-time qPCR expression analysis of the gusA gene, confirmed the results of histochemical assays. Taken together these data provide evidence that PrTdLTP4 functions as abiotic-stress-inducible promoter in a heterologous dicot system and could be an excellent tool for future crop improvement.

A wheat lipid transfer protein (TdLTP4) promotes tolerance to abiotic and biotic stress in Arabidopsis thaliana.

Lipid transfer proteins (LTPs) are members of the family of pathogenesis-related proteins (PR-14) that are believed to be involved in plant defense responses. In this study, we report the isolation and characterization of a novel gene TdLTP4 encoding an LTP protein from durum wheat [Triticum turgidum L. subsp. Durum Desf.]. Molecular Phylogeny analyses of wheat TdLTP4 gene showed a high identity to other plant LTPs. Predicted three-dimensional structural model revealed the presence of six helices and nine loop turns. Expression analysis in two local durum wheat varieties with marked differences in salt and drought tolerance, revealed a higher transcript accumulation of TdLTP4 under different stress conditions in the tolerant variety, compared to the sensitive one. The overexpression of TdLTP4 in Arabidopsis resulted in a promoted plant growth under various stress conditions including NaCl, ABA, JA and H2O2 treatments. Moreover, the LTP-overexpressing lines exhibit less sensitivity to jasmonate than wild-type plants. Furthermore, detached leaves from transgenic Arabidopsis expressing TdLTP4 gene showed enhanced fungal resistance against Alternaria solani and Botrytis cinerea. Together, these data provide the evidence for the involvement of TdLTP4 gene in the tolerance to both abiotic and biotic stresses in crop plants.