Caraway proteimics
Background: Carum carvi (caraway) belongs to the Apiaceae family. In many cultures, it is a condiment cooking spice and integrated into their folk medicine. While comprehensive documentation of the biological activity of caraway whole extract and essential oil has been achieved, proteomic analyses are limited, necessitating further research. The current study aimed to characterize nonspecific lipid transfer protein (nsLTP1) from caraway seed and to study the three-dimensional structure of the protein using an in silico approach. Additionally, we also evaluated nsLTP1 in vitro cytotoxic effect and investigated the antioxidant activity.
Methods: Caraway nsLTP1 was purified using two-dimensional chromatography. The complete amino acid sequence of nsLTP1 was achieved by intact protein sequence for the first 20 residues and the overlapping digested peptides. The three-dimensional structure was predicted using MODELLER. Autodock Vina software was employed for docking fatty acids against caraway nsLTP1. MEGA 11 was utilized to construct the phylogenetic tree employing nsLTP1 sequences from different genera against caraway nsLTP1. Thermal and pH stability of the nsLTP1 was examined by circular dichroism (CD) spectroscopy. Assessment of nsLTP1 cytotoxic activity was achieved by MTS assay, and the total antioxidant capacity (TAC) was investigated.
Results: Caraway nsLTP1 is composed of 91 residues and weighs 9652 Da. The three-dimensional structure of caraway nsLTP1 sequence was constructed based on searching known structures available in the PDB. We chose nsLTP of Solanum melongena (PDB ID: 5TVI) as the modeling template with the highest identity among all other homologous proteins. Docking linolenic acid with caraway protein showed a maximum binding score of -3.6 Kcal/mol. In the Apiaceae family, C. carvi shares the most common ancestor with Trachyspermum ammi and Daucus carota. Assessment of nsLTP1 demonstrated high thermal stability. A preliminary screening of caraway nsLTP1 suppressed the proliferation of human breast cancer cell lines MDA-MB-231 and MCF-7 in a dose‑dependent manner with an IC50 value of 52.93 and 44.76 μM, respectively. Also, nsLTP1 (0.4 mg/mL) showed TAC up to 750.4 μM Trolox equivalent.
Conclusion: To the best of our knowledge, this is the first study carried out on nsLTP1 from caraway seeds. Our study on caraway nsLTP1 revealed its amino acid sequence, tertiary structure model (in silico), and lipid binding affinity (in silico). The experiments suggest thermal stability besides potential anticancer and antioxidant bioactivity.
Background
Traditional health systems continue to progress, but plant rituals are mainly the shared practice in between them. Various parts of plants have been frequently used as conventional remedies worldwide. Besides, numerous extracted small molecule phytochemicals, the plant bioactive compounds, continually contribute to drug discovery. According to Newman & Cragg (2020), “Natural products still hold out the best options for finding novel agents/active templates” to produce new lead compounds for drug discovery if combined with emerging techniques. Nonetheless, the plant’s biologically active proteins and peptides are underestimated areas in the nutraceutical industry that could promise drug discoveries.
Nonspecific lipid transfer proteins (nsLTPs) are present in various plant species and expressed extensively in most tissues. The first reported LTP was isolated by Kader (1975) from potato tuber (Solanum tuberosum). Though new classifications emerged, conventional classification is still in use and classifies them based on the molecular mass as 10 kDa for nsLTP1 and 7 kDa for nsLTP2 [3]. NsLTPs are cysteine-rich proteins that contain eight cysteine motifs that form four disulfide bonds. These bonds stabilize the protein structure against high temperature and denaturing agents. Moreover, their tunnel-like structure forms an internal hydrophobic cavity that can bind to different lipids and transfer them [4]. Therefore, its name comes from its general role, mediating nonspecific lipids transfer between membranes in the cytoplasm. They also exhibit lipid sensing, lipid presenting, and lipid-modifying functions [5].
Lipid transfer proteins are expressed in various plants’ seeds, leaves, stems, roots, flowers, and fruits. Inhibition of LTP gene expression results in a number of hypotheses regarding their possible role in the development of plants during their vegetative and reproductive phases, and a decrease in their resistance to infection [4]. Nonetheless, their capacity to bind and transfer lipids are considered accountable for many of their functions, and their abundance would imply their critical roles in plant survival and reproduction. Numerous studies discovered nsLTPs involvement in plant vital roles, e.g., signaling, cuticular wax accumulation, liquid secretion, seed germination, cell expansion, nodule formation, and root suberin synthesis [6]. In addition, nsLTPs are critical in pollen, seed, and fruit development. Their variability and transferability might be promising for drug discovery and drug delivery studies. Variously reported nsLTPs exhibited biological activities, for example, antiproliferative activity against MCF-7, AsPC-1, HL-60, and HepG2 human cancer cells; antifungal activity against C. tropicalis and C. albicans; antibacterial activity against S. aureus and P. aeruginosa; antiviral activity against RSV, H1N1, and HIV-1 reverse transcriptase; and enzyme inhibition activity against human salivary α-amylase [4,7–10].
The cultivation and consumption of caraway in Europe are believed to exist longer than any other spice. Carum carvi is a plant that belongs to the family Apiaceae. C. carvi fruit is dry and indehiscent; therefore, known as a caraway seed [11,12]. In many cultures, caraway seed was a common aromatic spice in cuisines and baked goods. It is also integrated into their folk medicine for indigestion, flatulence, appetite loss, galactagogue, pneumonia, and eczema [11]. The seeds of caraway are still used in traditional medicine. Many studies have recently investigated caraway seeds’ bioactivities through in vitro, in vivo, and clinical studies. In vitro studies showed antimicrobial, antioxidant, anti-diabetic, anti-inflammatory, and chemopreventive effects [13–15]. Besides, in vivo experiments displayed anti-colitic activity, anti-convulsant, hepatoprotective, and wound healing properties [13–15]. In clinical trials, caraway exhibited an anti-obesity effect, functional dyspepsia management, and irritable bowel syndrome soothing [13–15]. The terpenes present in caraway essential oil, predominantly carvone, and limonene, are the active components accountable for caraway bioactivity [13].
While it is commonly used in folk medicine, its scientific studies are limited. To date, whole caraway extract and essential oil bioactivity have been studied, but no proteomics studies are available. This study focuses on nsLTP1 primary structure characterization from the seeds of caraway by N-terminal amino acid sequencing. In addition, the 3D structural modeling, lipid binding potential, and a phylogenetic tree will be achieved using several bioinformatics tools. The study also evaluated its secondary conformations at different temperatures and pH using circular dichroism (CD) spectroscopy. Furthermore, the prospective biological anticancer and antioxidant activity studies have also been done.
Conclusion
In the present study, we isolated, purified, and characterized the amino acid sequence of a 9.6 kDa protein member of nsLTP1 from caraway (C. carvi) seed. We also predicted the 3D structure modeling using eggplant (S. melongena) 5TVI as a template. Moreover, in silico studies for lipid affinity and evolutionary connections were conducted. NsLTPs appeal promising for drug discovery and development applications due to their revealed biological activities. Hence, this study also reports preliminary studies for the in vitro bioactivity of caraway nsLTP1. The protein displayed antiproliferative activity against MDA-MB-231 and MCF-7 human breast cancer cell lines. An in-depth molecular study is required to investigate the involvement of tumor suppressor proteins and signal transduction pathways in the process. Further confirmation of antiproliferative effects will require in vivo studies. Caraway seed also exhibited valued Trolox equivalent antioxidant capacity. Besides, caraway nsLTP1 exhibited a high degree of stability in temperatures ranging from 20-90 °C and stability in different pH conditions. Establishing further stability studies is essential to ensure its properties.
