Non-specific lipid-transfer protein 1

Lipid transfer proteins (LTP) are members of the family of pathogenesis-related proteins (PR-14) that play a key role in plant defense mechanisms. In this study, a novel gene TdLTP4 encoding an antifungal protein from wheat (cv. Om Rabiaa) was cloned, overexpressed in Escherichia coli BL-21 (DE3) and enriched using ammonium sulfate fractionation. The TdLTP4 fusion protein was then tested against a panel of pathogens, food-borne and spoilage bacteria and fungi in order to evaluate the antimicrobial properties. TdLTP4 was applied to 0.5 μg/mL LPS-induced RAW 264.7 macrophages in vitro at different concentrations (5, 10, 20, 50 and 100 μg/mL). Levels of nitric oxide (NO), pro-inflammatory cytokines interleukin (IL)-1β (IL-1 β), interleukin (IL)-6 (IL-6), tumor necrosis factor (TNF-α) and anti-inflammatory cytokine IL-10 in the supernatant fraction were measured using enzyme-linked immunosorbent assay (ELISA). Expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were detected via Western blot. The inhibition zones and minimal inhibitory concentration (MIC) values of bacterial strains were in the range of 14-26 mm and 62.5-250 μg/mL, respectively. Moreover, a remarkable activity against several fungal strains was revealed. TdLTP4 (5-100 μg/mL) decreased the production of NO (IC50 = 4.32 μg/mL), IL-6 (IC50 = 11.52 μg/mL), IL-1β (IC50 = 7.87 μg/mL) and TNF-α (IC50 = 8.66 μg/mL) by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. TdLTP4 could modulate the macrophages inflammatory mode by causing reduction in iNOS and COX-2. According to these findings, TdLTP4 fusion protein could be used as natural anti-inflammatory and antimicrobial agent in food preservation and human health.

Background: Trachyspermum ammi, commonly known as Ajwain, is a member of the Apiaceae family. It is a therapeutic herbal spice with diverse pharmacological properties, used in traditional medicine for various ailments. However, all previous studies were conducted using small molecule extracts, leaving the protein's bioactivity undiscovered. Aim: The current study aimed to demonstrate the cytotoxic activity of Ajwain non-specific lipid transfer protein (nsLTP1) in normal breast (MCF10A), breast cancer (MCF-7), and pancreatic cancer (AsPC-1) cell lines. Also, to evaluate its structural stability in human serum as well as at high temperature conditions. Methods: The cytotoxic activity of Ajwain nsLTP1 was evaluated in MCF-7 and AsPC-1 cell lines using MTT assay. Annexin V-FITC and PI staining were used to detect the early apoptotic and late apoptotic cells. The role of nsLTP1 in inducing apoptosis was further studied by quantifying Bcl-2, Bax, Caspase-3, Survivin, EGFR, and VEGF genes expression using RT-PCR. CD spectroscopy analyzed the nsLTP1 conformational changes after thermal treatment for structure stability determination. The RP-HPLC was used to analyze the nsLTP1 degradation rate in human serum at different time intervals incubated at 37 °C. Results: Ajwain nsLTP1 showed a potent cytotoxic effect in MCF-7 and AsPC-1. The IC50 value obtained in MCF-7 was 8.21 μM, while for AsPC-1 4.17 μM. The effect of nsLTP1 on stimulating apoptosis revealed that the proportions of apoptotic cells in both cell lines were relatively increased depending on the concentration. The apoptotic cells percentage at 20 μM was in MCF-7 71% (***P < 0.001) and AsPC-1 88% (***P < 0.001). These results indicate that nsLTP1 might efficaciously induce apoptosis in multiple types of cancerous cells. Genes expression in MCF-7 and AsPC-1 showed significant upregulation in Bax and Caspase-3 and downregulation in Bcl-2, Survivin, EGFR, and VEGF protein. The CD analysis of nsLTP1 showed a significant thermostable property. In serum, nsLTP1 showed a slow degradation rate, indicating high stability with a half-life of ~ 8.4 h. Conclusion: Our results revealed the potential anticancer activity of Ajwain nsLTP1 and its mechanism in inducing apoptosis. It further exhibited thermostable properties at high temperatures and in human serum, which suggested this protein as a promising anticancer agent.

Background: Brassica napus is an important agricultural species, improving stress resistance was one of the main breeding goals at present. Non-specific lipid transfer proteins (nsLTPs) are small, basic proteins which are involved in some biotic or abiotic stress responses. B. napus is susceptible to a variety of fungal diseases, so identify the BnLTPs and their expression in disease responses is very important. The common reference genome of B. napus does not contain all B. napus genes because of gene presence/absence variations between individuals. Therefore, it was necessary to search for candidate BnLTP genes in the B. napus pangenome. Results: In the present study, the BnLTP genes were identified throughout the pangenome, and different BnLTP genes were presented among varieties. Totally, 246 BnLTP genes were identified and could be divided into five types (1, 2, C, D, and G). The classification, phylogenetic reconstruction, chromosome distribution, functional annotation, and gene expression were analyzed. We also identified potential cis-elements that respond to biotic and abiotic stresses in the 2 kb upstream regions of all BnLTP genes. RNA sequencing analysis showed that the BnLTP genes were involved in the response to Sclerotinia sclerotiorum infection. We identified 32 BnLTPs linked to blackleg resistance quantitative trait locus (QTL). Conclusion: The identification and analysis of LTP genes in the B. napus pangenome could help to elucidate the function of BnLTP family members and provide new information for future molecular breeding in B. napus.