Upon Ca2+ influx, synaptic vesicles fuse with the presynaptic plasma membrane (PM) to release neu... more Upon Ca2+ influx, synaptic vesicles fuse with the presynaptic plasma membrane (PM) to release neurotransmitters. Membrane fusion is triggered by synaptotagmin-1, a transmembrane protein in the vesicle membrane (VM), but the mechanism is under debate. Synaptotagmin-1 contains a single transmembrane helix (TM) and two tandem C2-domains (C2A and C2B). The present study aimed to use molecular dynamics simulations to elucidate how Ca2+-bound synaptotagmin-1, by simultaneously associating with VM and PM, brings them together for fusion. While C2A stably associates with VM via two Ca2+-binding loops, C2B has a propensity to partially dissociate. Importantly, an acidic motif in the TM-C2A linker competes with VM for interacting with C2B, thereby flipping its orientation to face PM. Subsequently C2B can readily associate with PM via a polybasic cluster and a Ca2+-binding loop. These results delineate the functional process of fusion triggered by synaptotagmin-1.
Tissue factor (TF) mediated blood clot is the key event in cellular physiology. During vascular i... more Tissue factor (TF) mediated blood clot is the key event in cellular physiology. During vascular injury or endothelial cell rupture, factor VII (FVII) which is present normally in flowing blood as zymogenic form, upon binding with its cofactor TF (present beneath endothelial cell), get converted into its activated form, FVIIa; makes TF-FVIIa protease complex for the conversion of downstream protease members into activated form. Upon successful transformation of FX to FXa, prothrombin to thrombin, soluble fibrinogen get converted into its insoluble form fibrin, which accumulates at the site of vascular injury in the presence of platelets, resulting into the blood clot. Recent studies have shown that TF is solely responsible for the structural modulation of FVIIa. However, due to lack of well-resolved crystal structure of the zymogenic form of FVII and full-length TF-FVIIa binary complex, the actual allosteric mechanism induced by TF and structural changes observed in FVIIa is not full...
Metastasis, the hallmark of cancer propagation is attributed by the modification of phenotypic/fu... more Metastasis, the hallmark of cancer propagation is attributed by the modification of phenotypic/functional behavior of cells to break attachment and migrate to distant body parts. Cancer cell-secreted microvesicles (MVs) contribute immensely in disease propagation. These nano-vesicles, generated from plasma membrane outward budding are taken up by nearby healthy cells thereby inducing phenotypic alterations in those recipient cells. Protease activated receptor 2 (PAR2), activated by trypsin, also contributes to cancer progression by increasing metastasis, angiogenesis etc. Here, we report that PAR2 activation promotes pro-metastatic MVs generation from human breast cancer cell line, MDA-MB-231. Rab5a, located at the plasma membrane plays vital roles in MVs biogenesis. We show that PAR2 stimulation promotes AKT phosphorylation which activates Rab5a by converting inactive Rab5a-GDP to active Rab5a-GTP. Active Rab5a polymerizes actin which critically regulates MVs shedding. Not only MVs...
Cell invasion is attributed to the synthesis and secretion of proteolytically active matrix-metal... more Cell invasion is attributed to the synthesis and secretion of proteolytically active matrix-metalloproteinases (MMPs) by tumor cells to degrade extracellular matrix (ECM) and promote metastasis. The role of protease-activated receptor 2 (PAR2) in human breast cancer migration/invasion via MMP-2 up-regulation remains ill-defined; hence we investigated whether TF-FVIIa/trypsin-mediated PAR2 activation induces MMP-2 expression in human breast cancer. MMP-2 expression and the signaling mechanisms were analyzed by western blotting and RT-PCR. MMP-2 activity was measured by gelatin zymography. Cell invasion was analyzed by transwell invasion assay whereas; wound healing assay was performed to understand the cell migratory potential. Here, we highlight that TF-FVIIa/trypsin-mediated PAR2 activation leads to enhanced MMP-2 expression in human breast cancer cells contributing to tumor progression. Knock-down of PAR2 abrogated TF-FVIIa/trypsin-induced up-regulation of MMP-2. Again, genetic ma...
Apart from blood coagulation, coagulation proteases are involved inextricably in cancer progressi... more Apart from blood coagulation, coagulation proteases are involved inextricably in cancer progression/propagation via intra/inter‐cellular signaling, mediated predominantly by protease‐activated receptors (PARs). Microvesicles (MVs), a plasma membrane shredded component, has recently been identified as an important contributor to human breast cancer metastasis. However, the role of PAR2 in promoting MVs generation from breast cancer cells remains largely unexplored. The objective of this study is to investigate whether coagulation protease‐mediated human breast cancer propagation commences via MVs and also to decipher the underlying signaling mechanism. Here, we elicited that coagulation factor‐FVIIa and Trypsin activates PAR2, which governs MVs shedding from MDAMB231 cells by altering actomyosin dynamics. Treatment of cells with PAR2 activators facilitate MVs generation by activating three independent (MAPK, P38, and Rho) signaling cascades. MAPK, signals through activating MLCK foll...
Journal of biomolecular structure & dynamics, Jan 17, 2017
Tissue factor (TF)-mediated factor VII (FVII) activation and a subsequent proteolytic TF-FVIIa bi... more Tissue factor (TF)-mediated factor VII (FVII) activation and a subsequent proteolytic TF-FVIIa binary complex formation is the key step initiating the coagulation cascade, with implications in various homeostatic and pathologic scenarios. TF binding allosterically modifies zymogen-like free FVIIa to its highly catalytically active form. As a result of unresolved crystal structure of the full-length TF1-263-FVIIa binary complex and free FVIIa, allosteric alterations in FVIIa following its binding to full-length TF and the consequences of these on function are not entirely clear. The present study aims to map and identify structural alterations in FVIIa and TF resulting from full-length TF binding to FVIIa and the key events responsible for enhanced FVIIa activity in coagulation. We constructed the full-length TF1-263-FVIIa membrane bound complex using computational modeling and subjected it to molecular dynamics (MD) simulations. MD simulations showed that TF alters the structure of ...
Journal of biomolecular structure & dynamics, Jan 21, 2018
Two distinct populations: active and cryptic form of tissue factor (TF) resides on the cell surfa... more Two distinct populations: active and cryptic form of tissue factor (TF) resides on the cell surface. Apart from phospholipid contribution, various models have been introduced to explain decryption/encryption of TF. The proposed model, the switching of Cys186-Cys209 bond of TF has become the matter of controversy. However, it is well accepted that this disulfide has an immense influence upon ligand factor VIIa (FVIIa) for its binding. However, molecular level understanding for this remains unveiled due to lack of detailed structural information. In this regard, we have done the molecular dynamic study of membrane bound TF/TF-FVIIa in both the forms (±Cys186-Cys209 allosteric disulfide bond), individually. Dynamic study depicts that disulfide bond provides structural rigidity of TF in both free and ligand-bound forms. This disulfide bond also governs the conformation of FVIIa structure as well as the binding affinity of FVIIa towards TF. Significant differences in lipid-protein intera...
Upon Ca2+ influx, synaptic vesicles fuse with the presynaptic plasma membrane (PM) to release neu... more Upon Ca2+ influx, synaptic vesicles fuse with the presynaptic plasma membrane (PM) to release neurotransmitters. Membrane fusion is triggered by synaptotagmin-1, a transmembrane protein in the vesicle membrane (VM), but the mechanism is under debate. Synaptotagmin-1 contains a single transmembrane helix (TM) and two tandem C2-domains (C2A and C2B). The present study aimed to use molecular dynamics simulations to elucidate how Ca2+-bound synaptotagmin-1, by simultaneously associating with VM and PM, brings them together for fusion. While C2A stably associates with VM via two Ca2+-binding loops, C2B has a propensity to partially dissociate. Importantly, an acidic motif in the TM-C2A linker competes with VM for interacting with C2B, thereby flipping its orientation to face PM. Subsequently C2B can readily associate with PM via a polybasic cluster and a Ca2+-binding loop. These results delineate the functional process of fusion triggered by synaptotagmin-1.
Tissue factor (TF) mediated blood clot is the key event in cellular physiology. During vascular i... more Tissue factor (TF) mediated blood clot is the key event in cellular physiology. During vascular injury or endothelial cell rupture, factor VII (FVII) which is present normally in flowing blood as zymogenic form, upon binding with its cofactor TF (present beneath endothelial cell), get converted into its activated form, FVIIa; makes TF-FVIIa protease complex for the conversion of downstream protease members into activated form. Upon successful transformation of FX to FXa, prothrombin to thrombin, soluble fibrinogen get converted into its insoluble form fibrin, which accumulates at the site of vascular injury in the presence of platelets, resulting into the blood clot. Recent studies have shown that TF is solely responsible for the structural modulation of FVIIa. However, due to lack of well-resolved crystal structure of the zymogenic form of FVII and full-length TF-FVIIa binary complex, the actual allosteric mechanism induced by TF and structural changes observed in FVIIa is not full...
Metastasis, the hallmark of cancer propagation is attributed by the modification of phenotypic/fu... more Metastasis, the hallmark of cancer propagation is attributed by the modification of phenotypic/functional behavior of cells to break attachment and migrate to distant body parts. Cancer cell-secreted microvesicles (MVs) contribute immensely in disease propagation. These nano-vesicles, generated from plasma membrane outward budding are taken up by nearby healthy cells thereby inducing phenotypic alterations in those recipient cells. Protease activated receptor 2 (PAR2), activated by trypsin, also contributes to cancer progression by increasing metastasis, angiogenesis etc. Here, we report that PAR2 activation promotes pro-metastatic MVs generation from human breast cancer cell line, MDA-MB-231. Rab5a, located at the plasma membrane plays vital roles in MVs biogenesis. We show that PAR2 stimulation promotes AKT phosphorylation which activates Rab5a by converting inactive Rab5a-GDP to active Rab5a-GTP. Active Rab5a polymerizes actin which critically regulates MVs shedding. Not only MVs...
Cell invasion is attributed to the synthesis and secretion of proteolytically active matrix-metal... more Cell invasion is attributed to the synthesis and secretion of proteolytically active matrix-metalloproteinases (MMPs) by tumor cells to degrade extracellular matrix (ECM) and promote metastasis. The role of protease-activated receptor 2 (PAR2) in human breast cancer migration/invasion via MMP-2 up-regulation remains ill-defined; hence we investigated whether TF-FVIIa/trypsin-mediated PAR2 activation induces MMP-2 expression in human breast cancer. MMP-2 expression and the signaling mechanisms were analyzed by western blotting and RT-PCR. MMP-2 activity was measured by gelatin zymography. Cell invasion was analyzed by transwell invasion assay whereas; wound healing assay was performed to understand the cell migratory potential. Here, we highlight that TF-FVIIa/trypsin-mediated PAR2 activation leads to enhanced MMP-2 expression in human breast cancer cells contributing to tumor progression. Knock-down of PAR2 abrogated TF-FVIIa/trypsin-induced up-regulation of MMP-2. Again, genetic ma...
Apart from blood coagulation, coagulation proteases are involved inextricably in cancer progressi... more Apart from blood coagulation, coagulation proteases are involved inextricably in cancer progression/propagation via intra/inter‐cellular signaling, mediated predominantly by protease‐activated receptors (PARs). Microvesicles (MVs), a plasma membrane shredded component, has recently been identified as an important contributor to human breast cancer metastasis. However, the role of PAR2 in promoting MVs generation from breast cancer cells remains largely unexplored. The objective of this study is to investigate whether coagulation protease‐mediated human breast cancer propagation commences via MVs and also to decipher the underlying signaling mechanism. Here, we elicited that coagulation factor‐FVIIa and Trypsin activates PAR2, which governs MVs shedding from MDAMB231 cells by altering actomyosin dynamics. Treatment of cells with PAR2 activators facilitate MVs generation by activating three independent (MAPK, P38, and Rho) signaling cascades. MAPK, signals through activating MLCK foll...
Journal of biomolecular structure & dynamics, Jan 17, 2017
Tissue factor (TF)-mediated factor VII (FVII) activation and a subsequent proteolytic TF-FVIIa bi... more Tissue factor (TF)-mediated factor VII (FVII) activation and a subsequent proteolytic TF-FVIIa binary complex formation is the key step initiating the coagulation cascade, with implications in various homeostatic and pathologic scenarios. TF binding allosterically modifies zymogen-like free FVIIa to its highly catalytically active form. As a result of unresolved crystal structure of the full-length TF1-263-FVIIa binary complex and free FVIIa, allosteric alterations in FVIIa following its binding to full-length TF and the consequences of these on function are not entirely clear. The present study aims to map and identify structural alterations in FVIIa and TF resulting from full-length TF binding to FVIIa and the key events responsible for enhanced FVIIa activity in coagulation. We constructed the full-length TF1-263-FVIIa membrane bound complex using computational modeling and subjected it to molecular dynamics (MD) simulations. MD simulations showed that TF alters the structure of ...
Journal of biomolecular structure & dynamics, Jan 21, 2018
Two distinct populations: active and cryptic form of tissue factor (TF) resides on the cell surfa... more Two distinct populations: active and cryptic form of tissue factor (TF) resides on the cell surface. Apart from phospholipid contribution, various models have been introduced to explain decryption/encryption of TF. The proposed model, the switching of Cys186-Cys209 bond of TF has become the matter of controversy. However, it is well accepted that this disulfide has an immense influence upon ligand factor VIIa (FVIIa) for its binding. However, molecular level understanding for this remains unveiled due to lack of detailed structural information. In this regard, we have done the molecular dynamic study of membrane bound TF/TF-FVIIa in both the forms (±Cys186-Cys209 allosteric disulfide bond), individually. Dynamic study depicts that disulfide bond provides structural rigidity of TF in both free and ligand-bound forms. This disulfide bond also governs the conformation of FVIIa structure as well as the binding affinity of FVIIa towards TF. Significant differences in lipid-protein intera...
Uploads
Papers by Ramesh Prasad