RESUMEN
Post-translational modifications (PTMs) are crucial covalent processes that alter protein properties, achieved through proteolytic cleavage or addition of modifying groups like acetyl, phosphoryl, glycosyl, or methyl to amino acids. ADP-ribosylation is a reversible post-translational modification, where ADP-ribose units are covalently attached to target protein side chains. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that plays a key role in physiological and pathological conditions. Studies have reported that ADP-ribosylation affects VEGF's ability to bind to VEGF receptors, impacting angiogenesis signalling. However, the specific amino acid undergoing ADP-ribosylation on VEGF remained unknown. To understand the mechanism of ADP-ribose addition to VEGF, an in silico study was designed. The study initially checked for the presence of any conserved motif where ADP-ribosylation could potentially occur and identified the presence of the EIE motif in VEGF, a probable site for ADP-ribosylation for many proteins. Subsequently, the amino acids near this motif were selected and their structural properties were analyzed. Surface-exposed amino acids were chosen, and ADP-ribose was then added to their side chains. The results revealed that the amino acids ASP (67) and GLU (70) underwent glycosidic linkage with ADP-ribose, indicating that they are the most probable modification sites. Subsequently, Molecular dynamic simulation analysis such as RMSD, RMSF, Rg, PCA, and FEL, along with MM-PBSA binding free energy calculations were performed to understand the stability of the VEGF-ADP-ribose complexes. The analysis revealed that amino acid at position 67 (ASP67) is the most probable site for ADP-ribosylation in VEGF.Communicated by Ramaswamy H. Sarma.
RESUMEN
The biological activity of vascular endothelial growth factor (VEGF), the major cytokine regulating the process of angiogenesis is tightly controlled at multiple levels including processes involving post-translational modification such as ADP-ribosylation and glycosylation. ADP-ribosylation is a reversible NAD+-dependent modification, catalyzed by poly ADP-ribose polymerase (PARP) or ADP-ribosyl transferase (ADPRTs) and has been reported by us and others as a modification that reduces the biological activity of VEGF. The factors responsible for any such modification should occur in the secretory pathway, i.e., in the endoplasmic reticulum and Golgi. Our investigation carried out in this direction revealed that ADP-ribosylation of VEGF requires the interplay between members of poly ADP-ribose polymerase (PARP) family in the secretory pathway, viz., ER associated PARP-16 and Golgi associated Tankyrase-2 (TNKS-2). The data presented in this manuscript suggest that PARP-16 catalysis the priming mono ADP-ribosylation of VEGF which is a prerequisite for poly ADP-ribosylation of VEGF by TNKS-2.
Asunto(s)
Poli ADP Ribosilación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Procesamiento Proteico-Postraduccional , Tanquirasas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Humanos , Poli(ADP-Ribosa) Polimerasas/genética , Tanquirasas/genéticaRESUMEN
Adulteration of medicinally valuable curcumin (CU) with harmful chemicals as metanil yellow (MY) in recent years have demanded for quick detection techniques of the adulterants. The voltammetric behavior of CU and MY on bare glassy carbon electrode (BGCE) and carbon quantum dots modified glassy carbon electrode (CQDs/GCE) was studied by both cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in phosphate buffer solution of pHâ¯5.4. The MY responded to the CQDs/GCE with two anodic peaks at -0.004â¯V and 0.136â¯V and two cathodic peaks at -0.112 and -0.048â¯V. Under similar conditions CU exhibited two anodic peaks at 0.28â¯V and 0.55â¯V and one cathodic peak at 0.25â¯V. The overlapped voltammogram obtained for CU and MY on BGCE was well separated on the CQDs/GCE. The interference studies revealed that the compounds, demethoxycurcumin and bisdemethoxycurcumin, which are commonly associated with CU, did not interfere with the detection of MY. Real sample was analyzed with fabricated electrode and the recovery values >98% were obtained.
Asunto(s)
Compuestos Azo/análisis , Curcumina/análisis , Técnicas Electroquímicas/métodos , Puntos Cuánticos/química , Carbono/química , ElectrodosRESUMEN
Curcumin is a polyphenol derived from the herb Curcuma longa, which has been extensively studied in terms of its antitumour, antioxidant, and chemopreventive activity as well as various other effects. In the present work we compared curcumin with its synthetic analogue dimethoxycurcumin (dimc) in terms of its antioxidant enzyme-modulating effects in human peripheral blood mononuclear cells (PBMC). We found that these compounds modulate antioxidant enzymes differentially. Both curcumin and dimethoxycurcumin effected a decrease in lipid peroxidation status in PBMC, however, curcumin had better activity in this regard. An increase in the activity of catalase was seen in the case of curcumin-treated PBMC, whereas dimc increased catalase activity significantly to almost twofold level. Real time-polymerase chain reaction (RT-PCR) analysis revealed significant up-regulation of catalase at mRNA level post treatment with curcumin as well as dimc, however, dimc had better activity in this regard. Glutathione reductase (GR) activity and reduced glutathione levels increased in the case of peripheral blood mononuclear cells (PBMC) treated with curcumin, however, the trend was reversed with dimethoxycurcumin where, both glutathione reductase activity and reduced glutathione levels were significantly reduced. RT-PCR analysis of glutathione reductase mRNA levels showed decrease in mRNA levels post treatment with dimethoxycurcumin (dimc) further corroborating GR enzyme assay results, however, we could not obtain significant result post curcumin treatment. NFkB reporter assay and western blot analysis of nuclear as well as cytosolic fractions of NFkB revealed that curcumin inhibits NFkB activation whereas inhibition was much less with dimc. It has been reported that curcumin and dimc exerts differential cytotoxicity in normal and tumour cells and the reason for this had been attributed to the differential uptake of these compounds by normal cells and tumour cells. Based on our results we propose that differential modulation of antioxidant enzymes via NFkB pathway could be the reason behind differential cytotoxicity of dimc as well as curcumin in normal cells and tumour cells in addition to differential uptake of these compounds as reported previously.
Asunto(s)
Antioxidantes/farmacología , Curcumina/análogos & derivados , Curcumina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Catalasa/genética , Catalasa/metabolismo , Glutatión/genética , Glutatión/metabolismo , Glutatión Reductasa/genética , Glutatión Reductasa/metabolismo , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Peroxidación de Lípido/efectos de los fármacos , FN-kappa B/genética , FN-kappa B/metabolismo , Cultivo Primario de Células , Transducción de SeñalRESUMEN
Recent studies indicate that horizontal transfer of genetic material can act as a communication tool between heterogenous populations of tumour cells, thus altering the chemosensitivity of tumour cells. The present study was designed to check whether the horizontal transfer of miRNAs released by cisplatin resistant (Cp-r) Hepatocarcinoma cells can alter the sensitivity of cervical cancer cells. For this exosomes secreted by cisplatin resistant and cisplatin sensitive HepG2 cells (EXres and EXsen) were isolated and characterised. Cytotoxicity analysis showed that EXres can make Hela cells resistant to cisplatin. Analysis of miR-106a/b levels in EXres and EXsen showed that their levels vary. Mechanistic studies showed that miR-106a/b play an important role in EXsen and EXres mediated change in chemosensitivity of Hela cells to cisplatin. Further SIRT1 was identified as a major target of miR-106a/b using in silico tools and this was proved by experimentation. Also the effect of miR-106a/b in chemosensitivity was seen to be dependent on regulation of SIRT1 by miR-106a/b. In brief, this study brings into light, the SIRT1 dependent mechanism of miR-106a/b mediated regulation of chemosensitivity upon the horizontal transfer from one cell type to another.
Asunto(s)
Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Técnicas de Transferencia de Gen , Neoplasias Hepáticas/genética , MicroARNs/genética , Neoplasias del Cuello Uterino/genética , Secuencia de Bases , Exosomas/efectos de los fármacos , Exosomas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , MicroARNs/metabolismo , Modelos Biológicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
MMP9 is a member of the family of zinc-containing endopeptidases which degrade various components of the extracellular matrix, thereby regulating matrix remodeling. Since matrix remodeling plays an important role during growth and progression of cancer and considering the fact that, tumor cells switch to aerobic glycolysis as its major energy source, this study was designed to analyze if partial inhibition of glycolysis (the major energy pathway during hypoxia) can be used as a means to control matrix remodeling in terms of MMP9 activity and expression. For this, human epithelial carcinoma cells were treated with glycolytic inhibitor, 2-deoxy glucose (2DG) at sub-lethal concentrations followed by analysis of the expression and activity of MMP2 and MMP9. The experimental findings demonstrate that exposure of cancer cells to glycolytic inhibitor at concentration that does not induce ER stress, downregulates the activity and expression of MMP9 without affecting the expression levels and activity of MMP2. Further mechanistic analysis revealed that the regulation of MMP9 was mediated in a SIRT-1 dependent mechanism and did not alter the NFkB signaling pathway. The overall results presented here, therefore suggest that the use of glycolytic inhibitor, 2DG at concentration that do not affect cell viability or induce ER stress can be an effective strategy to control matrix remodeling.
Asunto(s)
Desoxiglucosa/farmacología , Metaloproteinasa 9 de la Matriz/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Sirtuina 1/metabolismo , Células HeLa , Humanos , Metaloproteinasa 2 de la Matriz/metabolismoRESUMEN
One of the possible mechanisms of the angiogenic effect of laminin (Ln) involves modulation of the biological activity of VEGF by regulating poly ADP ribosylation (PAR). PAR modification of VEGF was found to be related with the changes in NAD(+) associated with a shift in LDH isoenzymes. Further investigations on LDH gene expression in HUVECs suggested that the effect of Ln was mediated through alpha(6)beta(4) integrin-FAK-src-p38 MAPK pathway. This was evidenced by (a) co-immunoprecipitation of beta(4) integrin with alpha(6) subunit, (b) activation by tyrosine phosphorylation of beta(4) integrin and FAK, (c) co-immunoprecipitation of FAK with beta(4) and with adapter protein, src, (d) increased phosphorylation of p38 MAPK in cells maintained on Ln and (e) blocking of effect of Ln on LDH-B gene expression by inhibition of p38 MAPK. Increase in serine phosphorylation of c-fos and c-jun and higher levels of heterodimers of AP-1 in the nucleus in cells maintained on Ln suggested activation of AP-1 transcription factor. These results provide evidence for modulation of endothelial cell function relevant to angiogenesis by Ln through alpha(6)beta(4) integrin.
Asunto(s)
Células Endoteliales/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Regulación Enzimológica de la Expresión Génica , Integrina alfa6beta4/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Laminina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Western Blotting , Células Cultivadas , Humanos , Inmunoprecipitación , Isoenzimas/metabolismo , Unión Proteica , Transducción de Señal/fisiologíaRESUMEN
Investigations were carried out to understand the molecular basis of the effect of ursolic acid on angiogenesis by analysing its effects on the expression of modulators of angiogenesis by HUVECs in culture. Treatment with ursolic acid increased the expression of adhesion molecules such as E-selectin, CD-31 and I-CAM, upregulated angiogenic growth factors such as VEGF and FGF-2 and their receptors and caused increase in the ratio of PGE(2) to PGD(2). Reversal of the effect of ursolic acid by inhibition of PI3K-Akt pathway and increase in the level of phospho Akt suggest that the ursolic acid effect is mediated through PI3K-Akt pathway.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Triterpenos/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Células Cultivadas , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Selectina E/metabolismo , Células Endoteliales/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Ácido UrsólicoRESUMEN
Alterations in both cell-cell and cell-matrix interactions are associated with the activation of endothelial cells that initiate angiogenesis. Cell-matrix interactions are affected by changes in both cell surface receptors for matrix proteins and the composition of ECM. One of the molecular mechanisms involved in changes in these components is the action of neutral proteinases, particularly matrix metalloproteinases. To understand the involvement of MMPs in angiogenic processes, the in vitro model of human umbilical vein endothelial cells in culture was used. Zymography and ELISA showed that, as cell-cell contact and network-like structures were formed, there was down regulation of MMP-2 and MMP-9 associated with high levels of their endogenous inhibitors TIMP-1 and TIMP-2. On treatment with aspirin, which inhibited the cell-cell contact and network-like structure formation, there was no down regulation of MMPs and cells continued to produce MMP-2 and MMP-9. These results indicate a temporal relationship between MMP-2 and MMP-9 production by endothelial cells and the onset of angiogenic event.