RESUMEN
Food proteins and their constituent peptides impart huge health benefits besides their nutritional attributes. Sorghum bicolor protein hydrolysates (SPH) and derived bioactive peptides generated by simulated gastrointestinal digestion were studied for DPP-4 inhibitory properties using in vitro and in situ assays. Identified peptides, LSICGEESFGTGSDHIR (PEP1), SLGESLLQEDVEAHK (PEP2) and QLRDIVDK (PEP4) displayed potent DPP-4 inhibition with IC50 values of 73.5, 82.5 and 8.55 µM respectively. DPP-4 inhibition mechanism by the peptides was investigated by DPP4-peptide inhibition kinetics, molecular docking and microscale thermophoresis binding studies. The peptides bound to DPP-4 with micromolar affinities and PEP4 showed significantly increased affinity. The mixed type enzyme inhibition by peptides suggested that the peptides either block the active site of DPP-4 or changes the enzyme conformation via a secondary binding site. Overall, the results demonstrate that sorghum seeds are an adequate source of peptides with DPP-4 inhibitory properties that could be used in functional food formulations.
Asunto(s)
Inhibidores de la Dipeptidil-Peptidasa IV , Sorghum , Dipeptidil Peptidasa 4 , Simulación del Acoplamiento Molecular , PéptidosRESUMEN
BACKGROUND: Cotton leaf curl Kokhran Virus-Dabawali (CLCuKV-Dab) is a monopartite begomovirus encoding two proteins V1 and V2 in the virion sense and four proteins C1, C2, C3 and C4 in the complementary sense. The C4 protein of monopartite begomoviruses has been implicated to play a role in symptom determination and virus movement. The present work aims at the biochemical characterization of this protein. METHODS: The C4 protein of CLCuKV-Dab was purified in fusion with GST and tested for the ability to hydrolyze ATP and other phosphate containing compounds. ATPase activity was assayed by using radiolabeled γ-[32P]-ATP and separating the product of reaction by thin layer chromatography. The hydrolysis of other compounds was monitored by the formation of a blue colored phosphomolybdate complex which was estimated by measuring the absorbance at 655nm. RESULTS: The purified GST-C4 protein exhibited metal ion dependent ATPase and inorganic pyrophosphatase activities. Deletion of a sequence resembling the catalytic motif present in phosphotyrosine phosphatases resulted in 70% reduction in both the activities. Mutational analysis suggested arginine 13 to be catalytically important for the ATPase and cysteine 8 for the pyrophosphatase activity of GST-C4. Interaction of V2 with GST-C4 resulted in an increase in both the enzymatic activities of GST-C4. CONCLUSIONS: The residues important for the enzymatic activities of GST-C4 are present in a motif different from the classical Walker motifs and the non-classical ATP binding motifs reported so far. GENERAL SIGNIFICANCE: The C4 protein of CLCuKV-Dab, a putative natively unfolded protein, exhibits enzymatic activities.
Asunto(s)
Adenosina Trifosfatasas/química , Begomovirus/enzimología , Pirofosfatasa Inorgánica/química , Proteínas Virales/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencias de Aminoácidos , Begomovirus/genética , Dominio Catalítico , Pirofosfatasa Inorgánica/genética , Pirofosfatasa Inorgánica/metabolismo , Pliegue de Proteína , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The functional attributes of coat protein (CP) and V2 of the monopartite begomovirus, Cotton leaf curl Kokhran virus- Dabawali were analyzed in vitro and in vivo by their overexpression in E. coli, insect cells and transient expression in the plant system. Purified recombinant V2 and CP proteins were shown to interact with each other using ELISA and surface plasmon resonance. Confocal microscopy of Sf21 cells expressing V2 and CP proteins revealed that V2 localized to the cell periphery and CP to the nucleus. Deletion of the N terminal nuclear localization signal of CP restricted its distribution to the cytoplasm. GFP-V2 and YFP-CP transiently expressed in N. benthamiana plants by agroinfiltration substantiated the localization of V2 to the cell periphery and CP predominantly to the nucleus. Interestingly, upon coinfiltration, CP was found both in the nucleus and in the cytoplasm along with V2. These results suggest that the interaction of V2 and CP may have important implications in the cell to cell movement.