The Experts below are selected from a list of 765 Experts worldwide ranked by ideXlab platform
Gianni Colotti - One of the best experts on this subject based on the ideXlab platform.
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profiling calcium dependent interactions between Sorcin and intrinsically disordered regions of human proteome
Biochimica et Biophysica Acta, 2020Co-Authors: Gianni Colotti, Ilaria Genovese, Andrea Ilari, Annarita Fiorillo, Theo Battista, Andrea Carotti, Ylva IvarssonAbstract:Abstract Background Sorcin is a calcium sensor that exerts many calcium-related functions in the cells, e.g. it regulates calcium concentration in the cytoplasm, endoplasmic reticulum (ER) and mitochondria, by interacting with calcium pumps, exchangers and channels. Albeit Sorcin is an interesting potential cancer target, little is known about its interactors upon calcium-mediated activation. Our previous study suggested that Sorcin may recognize short linear binding motifs as the crystal structure revealed a self-interaction with a GYYPGG stretch in its N-terminus, and combinatorial peptide-phage display provided support for peptide-mediated interactions. Methods In this study we screened for motif-based interactions between Sorcin and intrinsically disordered regions of the human proteome using proteomic peptide phage display (ProP-PD). We identified a peptide belonging to protein phosphatase 1 regulatory subunit 3G (PPP1R3G) as a potential novel interactor and confirm the interaction through biophysical and cell-based approaches, and provide structural information through molecular dynamics simulations. Results Altogether, we identify a preferred motif in the enriched pool of binders and a peptide belonging to protein phosphatase 1 regulatory subunit 3G (PPP1R3G) as a preferred ligand. Conclusion Through this study we gain information on a new Sorcin binding partner and profile Sorcin's motif-based interaction. General significance The interaction between Sorcin and PPP1R3G may suggest a close dependence between glucose homeostasis and calcium concentration in the different cell compartments, opening a completely new and interesting scenery yet to be fully disclosed.
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roles of Sorcin in drug resistance in cancer one protein many mechanisms for a novel potential anticancer drug target
Cancers, 2020Co-Authors: Theo Battista, Ilaria Genovese, Andrea Ilari, Annarita Fiorillo, Valerio Chiarini, Gianni ColottiAbstract:The development of drug resistance is one of the main causes of failure in anti-cancer treatments. Tumor cells adopt many strategies to counteract the action of chemotherapeutic agents, e.g., enhanced DNA damage repair, inactivation of apoptotic pathways, alteration of drug targets, drug inactivation, and overexpression of ABC (Adenosine triphosphate-binding cassette, or ATP-binding cassette) transporters. These are broad substrate-specificity ATP-dependent efflux pumps able to export toxins or drugs out of cells; for instance, ABCB1 (MDR1, or P-glycoprotein 1), overexpressed in most cancer cells, confers them multidrug resistance (MDR). The gene coding for Sorcin (SOluble Resistance-related Calcium-binding proteIN) is highly conserved among mammals and is located in the same chromosomal locus and amplicon as the ABC transporters ABCB1 and ABCB4, both in human and rodent genomes (two variants of ABCB1, i.e., ABCB1a and ABCB1b, are in rodent amplicon). Sorcin was initially characterized as a soluble protein overexpressed in multidrug (MD) resistant cells and named “resistance-related” because of its co-amplification with ABCB1. Although for years Sorcin overexpression was thought to be only a by-product of the co-amplification with ABC transporter genes, many papers have recently demonstrated that Sorcin plays an important part in MDR, indicating a possible role of Sorcin as an oncoprotein. The present review illustrates Sorcin roles in the generation of MDR via many mechanisms and points to Sorcin as a novel potential target of different anticancer molecules.
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A22 Sorcin rescues ca (II) dysregulation and endoplasmic reticulum stress in huntington’s disease
Journal of Neurology Neurosurgery and Psychiatry, 2018Co-Authors: Gianni Colotti, Ilaria Genovese, Eris Bidollari, Jessica Rosati, Marzia Perluigi, Tito Cali, Andrea Arena, Ferdinando Squitieri, Andrea IlariAbstract:Background Sorcin is an essential penta-EF hand Ca (II) -binding protein, able to reduce Endoplasmic Reticulum (ER) stress and cell death. X-ray apo and Ca (II)-bound Sorcin structures showed that Ca (II) binding to EF1–3 hands promotes a large conformational change and the exposure of molecular surfaces through which Sorcin interacts with Ryanodine receptors (RYR), Sarco/ER Ca (II)-ATPase (SERCA), Na (I)-Ca (II) exchanger and regulates Ca (II) fluxes. Aims Sorcin is one of the most expressed Ca (II)-binding proteins in the human brain, and is hypothesized to be involved in neurodegenerative diseases. We aim at demonstrating that Sorcin in brain counteracts the increased cytosolic Ca (II) levels associated with neurodegeneration and that for this reason it is overexpressed in HD (Huntington’s Disease). Methods We identified Sorcin interactors by colocalization, coIP and SPR experiments, analysed Ca (II) currents and mitochondria-associated ER membranes (MAMs) and measured Sorcin expression in many biological samples by RT-PCR and Western Blot. Results Sorcin interacts in a Ca (II)-dependent manner with RYR; it increases both SERCA- and RYR-dependent ER Ca (II) currents and mitochondrial Ca (II) transients; Sorcin increases the number of short MAMs, essential for mitochondrial Ca (II) intake, disrupted in most neurodegenerative diseases, and is able to interact with proteins involved in the Unfolded Protein Response, i.e. ATF6, eIF2a and Sigma1R, localized in MAM. Finally, we found increased Sorcin expression levels in fibroblasts from HD patients and post-mortem cortex tissue of HD subjects. Conclusions Our preliminary results show that Sorcin, which controls Ca (II) flux across cell compartments, is overexpressed in neurons to counteract Ca (II) unbalance caused by HD. These findings indicate that Sorcin is a novel, promising HD marker and represents a defence from cellular stress dependent on neurodegeneration.
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a22 Sorcin rescues ca ii dysregulation and endoplasmic reticulum stress in huntington s disease
Journal of Neurology Neurosurgery and Psychiatry, 2018Co-Authors: Gianni Colotti, Ilaria Genovese, Eris Bidollari, Jessica Rosati, Marzia Perluigi, Tito Cali, Andrea Arena, Ferdinando Squitieri, Andrea IlariAbstract:Background Sorcin is an essential penta-EF hand Ca (II) -binding protein, able to reduce Endoplasmic Reticulum (ER) stress and cell death. X-ray apo and Ca (II)-bound Sorcin structures showed that Ca (II) binding to EF1–3 hands promotes a large conformational change and the exposure of molecular surfaces through which Sorcin interacts with Ryanodine receptors (RYR), Sarco/ER Ca (II)-ATPase (SERCA), Na (I)-Ca (II) exchanger and regulates Ca (II) fluxes. Aims Sorcin is one of the most expressed Ca (II)-binding proteins in the human brain, and is hypothesized to be involved in neurodegenerative diseases. We aim at demonstrating that Sorcin in brain counteracts the increased cytosolic Ca (II) levels associated with neurodegeneration and that for this reason it is overexpressed in HD (Huntington’s Disease). Methods We identified Sorcin interactors by colocalization, coIP and SPR experiments, analysed Ca (II) currents and mitochondria-associated ER membranes (MAMs) and measured Sorcin expression in many biological samples by RT-PCR and Western Blot. Results Sorcin interacts in a Ca (II)-dependent manner with RYR; it increases both SERCA- and RYR-dependent ER Ca (II) currents and mitochondrial Ca (II) transients; Sorcin increases the number of short MAMs, essential for mitochondrial Ca (II) intake, disrupted in most neurodegenerative diseases, and is able to interact with proteins involved in the Unfolded Protein Response, i.e. ATF6, eIF2a and Sigma1R, localized in MAM. Finally, we found increased Sorcin expression levels in fibroblasts from HD patients and post-mortem cortex tissue of HD subjects. Conclusions Our preliminary results show that Sorcin, which controls Ca (II) flux across cell compartments, is overexpressed in neurons to counteract Ca (II) unbalance caused by HD. These findings indicate that Sorcin is a novel, promising HD marker and represents a defence from cellular stress dependent on neurodegeneration.
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binding of doxorubicin to Sorcin impairs cell death and increases drug resistance in cancer cells
Cell Death and Disease, 2017Co-Authors: Ilaria Genovese, Andrea Ilari, Annarita Fiorillo, Silvia Masciarelli, Francesco Fazi, Gianni ColottiAbstract:Sorcin is a calcium binding protein that plays an important role in multidrug resistance (MDR) in tumors, since its expression confers resistance to doxorubicin and to other chemotherapeutic drugs. In this study, we show that Sorcin is able to bind doxorubicin, vincristine, paclitaxel and cisplatin directly and with high affinity. The high affinity binding of doxorubicin to Sorcin has been demonstrated with different techniques, that is, surface plasmon resonance, fluorescence titration and X-ray diffraction. Although the X-ray structure of Sorcin in complex with doxorubicin has been solved at low resolution, it allows the identification of one of the two doxorubicin binding sites, placed at the interface between the EF5 loop the G helix and the EF4 loop. We show that Sorcin cellular localization changes upon doxorubicin treatment, an indication that the protein responds to doxorubicin and it presumably binds the drug also inside the cell, soon after drug entrance. We also demonstrate that Sorcin is able to limit the toxic effects of the chemotherapeutic agent in the cell. In addition, Sorcin silencing increases cell death upon treatment with doxorubicin, increases the accumulation of doxorubicin in cell nucleus, decreases the expression of MDR1 and doxorubicin efflux via MDR1.
Andrea Ilari - One of the best experts on this subject based on the ideXlab platform.
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profiling calcium dependent interactions between Sorcin and intrinsically disordered regions of human proteome
Biochimica et Biophysica Acta, 2020Co-Authors: Gianni Colotti, Ilaria Genovese, Andrea Ilari, Annarita Fiorillo, Theo Battista, Andrea Carotti, Ylva IvarssonAbstract:Abstract Background Sorcin is a calcium sensor that exerts many calcium-related functions in the cells, e.g. it regulates calcium concentration in the cytoplasm, endoplasmic reticulum (ER) and mitochondria, by interacting with calcium pumps, exchangers and channels. Albeit Sorcin is an interesting potential cancer target, little is known about its interactors upon calcium-mediated activation. Our previous study suggested that Sorcin may recognize short linear binding motifs as the crystal structure revealed a self-interaction with a GYYPGG stretch in its N-terminus, and combinatorial peptide-phage display provided support for peptide-mediated interactions. Methods In this study we screened for motif-based interactions between Sorcin and intrinsically disordered regions of the human proteome using proteomic peptide phage display (ProP-PD). We identified a peptide belonging to protein phosphatase 1 regulatory subunit 3G (PPP1R3G) as a potential novel interactor and confirm the interaction through biophysical and cell-based approaches, and provide structural information through molecular dynamics simulations. Results Altogether, we identify a preferred motif in the enriched pool of binders and a peptide belonging to protein phosphatase 1 regulatory subunit 3G (PPP1R3G) as a preferred ligand. Conclusion Through this study we gain information on a new Sorcin binding partner and profile Sorcin's motif-based interaction. General significance The interaction between Sorcin and PPP1R3G may suggest a close dependence between glucose homeostasis and calcium concentration in the different cell compartments, opening a completely new and interesting scenery yet to be fully disclosed.
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roles of Sorcin in drug resistance in cancer one protein many mechanisms for a novel potential anticancer drug target
Cancers, 2020Co-Authors: Theo Battista, Ilaria Genovese, Andrea Ilari, Annarita Fiorillo, Valerio Chiarini, Gianni ColottiAbstract:The development of drug resistance is one of the main causes of failure in anti-cancer treatments. Tumor cells adopt many strategies to counteract the action of chemotherapeutic agents, e.g., enhanced DNA damage repair, inactivation of apoptotic pathways, alteration of drug targets, drug inactivation, and overexpression of ABC (Adenosine triphosphate-binding cassette, or ATP-binding cassette) transporters. These are broad substrate-specificity ATP-dependent efflux pumps able to export toxins or drugs out of cells; for instance, ABCB1 (MDR1, or P-glycoprotein 1), overexpressed in most cancer cells, confers them multidrug resistance (MDR). The gene coding for Sorcin (SOluble Resistance-related Calcium-binding proteIN) is highly conserved among mammals and is located in the same chromosomal locus and amplicon as the ABC transporters ABCB1 and ABCB4, both in human and rodent genomes (two variants of ABCB1, i.e., ABCB1a and ABCB1b, are in rodent amplicon). Sorcin was initially characterized as a soluble protein overexpressed in multidrug (MD) resistant cells and named “resistance-related” because of its co-amplification with ABCB1. Although for years Sorcin overexpression was thought to be only a by-product of the co-amplification with ABC transporter genes, many papers have recently demonstrated that Sorcin plays an important part in MDR, indicating a possible role of Sorcin as an oncoprotein. The present review illustrates Sorcin roles in the generation of MDR via many mechanisms and points to Sorcin as a novel potential target of different anticancer molecules.
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a22 Sorcin rescues ca ii dysregulation and endoplasmic reticulum stress in huntington s disease
Journal of Neurology Neurosurgery and Psychiatry, 2018Co-Authors: Gianni Colotti, Ilaria Genovese, Eris Bidollari, Jessica Rosati, Marzia Perluigi, Tito Cali, Andrea Arena, Ferdinando Squitieri, Andrea IlariAbstract:Background Sorcin is an essential penta-EF hand Ca (II) -binding protein, able to reduce Endoplasmic Reticulum (ER) stress and cell death. X-ray apo and Ca (II)-bound Sorcin structures showed that Ca (II) binding to EF1–3 hands promotes a large conformational change and the exposure of molecular surfaces through which Sorcin interacts with Ryanodine receptors (RYR), Sarco/ER Ca (II)-ATPase (SERCA), Na (I)-Ca (II) exchanger and regulates Ca (II) fluxes. Aims Sorcin is one of the most expressed Ca (II)-binding proteins in the human brain, and is hypothesized to be involved in neurodegenerative diseases. We aim at demonstrating that Sorcin in brain counteracts the increased cytosolic Ca (II) levels associated with neurodegeneration and that for this reason it is overexpressed in HD (Huntington’s Disease). Methods We identified Sorcin interactors by colocalization, coIP and SPR experiments, analysed Ca (II) currents and mitochondria-associated ER membranes (MAMs) and measured Sorcin expression in many biological samples by RT-PCR and Western Blot. Results Sorcin interacts in a Ca (II)-dependent manner with RYR; it increases both SERCA- and RYR-dependent ER Ca (II) currents and mitochondrial Ca (II) transients; Sorcin increases the number of short MAMs, essential for mitochondrial Ca (II) intake, disrupted in most neurodegenerative diseases, and is able to interact with proteins involved in the Unfolded Protein Response, i.e. ATF6, eIF2a and Sigma1R, localized in MAM. Finally, we found increased Sorcin expression levels in fibroblasts from HD patients and post-mortem cortex tissue of HD subjects. Conclusions Our preliminary results show that Sorcin, which controls Ca (II) flux across cell compartments, is overexpressed in neurons to counteract Ca (II) unbalance caused by HD. These findings indicate that Sorcin is a novel, promising HD marker and represents a defence from cellular stress dependent on neurodegeneration.
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A22 Sorcin rescues ca (II) dysregulation and endoplasmic reticulum stress in huntington’s disease
Journal of Neurology Neurosurgery and Psychiatry, 2018Co-Authors: Gianni Colotti, Ilaria Genovese, Eris Bidollari, Jessica Rosati, Marzia Perluigi, Tito Cali, Andrea Arena, Ferdinando Squitieri, Andrea IlariAbstract:Background Sorcin is an essential penta-EF hand Ca (II) -binding protein, able to reduce Endoplasmic Reticulum (ER) stress and cell death. X-ray apo and Ca (II)-bound Sorcin structures showed that Ca (II) binding to EF1–3 hands promotes a large conformational change and the exposure of molecular surfaces through which Sorcin interacts with Ryanodine receptors (RYR), Sarco/ER Ca (II)-ATPase (SERCA), Na (I)-Ca (II) exchanger and regulates Ca (II) fluxes. Aims Sorcin is one of the most expressed Ca (II)-binding proteins in the human brain, and is hypothesized to be involved in neurodegenerative diseases. We aim at demonstrating that Sorcin in brain counteracts the increased cytosolic Ca (II) levels associated with neurodegeneration and that for this reason it is overexpressed in HD (Huntington’s Disease). Methods We identified Sorcin interactors by colocalization, coIP and SPR experiments, analysed Ca (II) currents and mitochondria-associated ER membranes (MAMs) and measured Sorcin expression in many biological samples by RT-PCR and Western Blot. Results Sorcin interacts in a Ca (II)-dependent manner with RYR; it increases both SERCA- and RYR-dependent ER Ca (II) currents and mitochondrial Ca (II) transients; Sorcin increases the number of short MAMs, essential for mitochondrial Ca (II) intake, disrupted in most neurodegenerative diseases, and is able to interact with proteins involved in the Unfolded Protein Response, i.e. ATF6, eIF2a and Sigma1R, localized in MAM. Finally, we found increased Sorcin expression levels in fibroblasts from HD patients and post-mortem cortex tissue of HD subjects. Conclusions Our preliminary results show that Sorcin, which controls Ca (II) flux across cell compartments, is overexpressed in neurons to counteract Ca (II) unbalance caused by HD. These findings indicate that Sorcin is a novel, promising HD marker and represents a defence from cellular stress dependent on neurodegeneration.
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binding of doxorubicin to Sorcin impairs cell death and increases drug resistance in cancer cells
Cell Death and Disease, 2017Co-Authors: Ilaria Genovese, Andrea Ilari, Annarita Fiorillo, Silvia Masciarelli, Francesco Fazi, Gianni ColottiAbstract:Sorcin is a calcium binding protein that plays an important role in multidrug resistance (MDR) in tumors, since its expression confers resistance to doxorubicin and to other chemotherapeutic drugs. In this study, we show that Sorcin is able to bind doxorubicin, vincristine, paclitaxel and cisplatin directly and with high affinity. The high affinity binding of doxorubicin to Sorcin has been demonstrated with different techniques, that is, surface plasmon resonance, fluorescence titration and X-ray diffraction. Although the X-ray structure of Sorcin in complex with doxorubicin has been solved at low resolution, it allows the identification of one of the two doxorubicin binding sites, placed at the interface between the EF5 loop the G helix and the EF4 loop. We show that Sorcin cellular localization changes upon doxorubicin treatment, an indication that the protein responds to doxorubicin and it presumably binds the drug also inside the cell, soon after drug entrance. We also demonstrate that Sorcin is able to limit the toxic effects of the chemotherapeutic agent in the cell. In addition, Sorcin silencing increases cell death upon treatment with doxorubicin, increases the accumulation of doxorubicin in cell nucleus, decreases the expression of MDR1 and doxorubicin efflux via MDR1.
Emilia Chiancone - One of the best experts on this subject based on the ideXlab platform.
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activation of the cardiac na ca2 exchanger by Sorcin via the interaction of the respective ca2 binding domains
Journal of Molecular and Cellular Cardiology, 2010Co-Authors: Carlotta Zamparelli, Daniela Verzili, Niall Macquaide, Gianni Colotti, Tim Seidler, Godfrey L Smith, Emilia ChianconeAbstract:Sorcin is a penta-EF-hand protein that interacts with intracellular target proteins after Ca2+ binding. The sarcolemmal Na+/Ca2+ exchanger (NCX1) may be an important Sorcin target in cardiac muscle. In this study, RNAi knockdown of Sorcin, purified Sorcin or Sorcin variants was employed in parallel measurements of: (i) NCX activity in isolated rabbit cardiomyocytes using electrophysiological techniques and (ii) Sorcin binding to the NCX1 calcium binding domains (CBD1 and (iii) using surface plasmon resonance and gel overlay techniques. Sorcin is activated by Ca2+ binding to the EF3 and EF2 regions, which are connected by the D helix. To investigate the importance of this region in the interaction with NCX1, three variants were examined: W105G and W99G, mutated respectively near EF3 and EF2, and E124A that does not bind Ca2+ due to a mutation at EF3. Downregulation of Sorcin decreased and supplementation with wt Sorcin (3 μM) increased NCX activity in isolated cardiomyocytes. The relative stimulatory effects of the Sorcin variants were: W105G > wt Sorcin > Sorcin Calcium Binding Domain (SCBD) > W99G > E124A. Sorcin binding to both CBD1 and 2 was observed. In the presence of 50 µM Ca2+, the interaction with CBD1 followed the order W105G > SCBD > wt Sorcin > W99G > E124A. In Sorcin, the interacting surface can be mapped on the C-terminal Ca2+-binding domain in the D helix region comprising W99. The fast association/dissociation rates that characterize the interaction of Sorcin with CBD1 and 2 may permit complex formation/dissociation during an excitation/contraction cycle.
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complex modulation of l type ca2 current inactivation by Sorcin in isolated rabbit cardiomyocytes
Pflügers Archiv: European Journal of Physiology, 2009Co-Authors: Mark R Fowler, Emilia Chiancone, Gianni Colotti, Tim Seidler, Yoshiharu Higuchi, Godfrey L SmithAbstract:Modulation of the L-type Ca2+ channel (LTCC) by Sorcin was investigated by measuring the L-type Ca2+ current (I Ca,L) in isolated rabbit ventricular myocytes using ruptured patch, single electrode voltage clamp in the absence of extracellular Na+. Fifty millimolars EGTA (170 nM Ca2+) in the pipette solution buffered bulk cytoplasmic [Ca2+], but retained rapid Ca2+-dependant inactivation of I Ca,L,. Recombinant Sorcin (3 μM) in the pipette significantly slowed time-dependant inactivation (τ fast: 8.8 ± 0.9 vs. 15.1 ± 1.7 ms). Sorcin had no significant effect on I Ca,L, after inhibition of the sarcoplasmic reticulum (SR). Using 10 mM 1,2-bis(o-N,N,N′,N′-tetraacetic acid (170 nM Ca2+), I Ca,L inactivation was then determined by a Ca2+ -independent, voltage-dependant process. Under these conditions, 3 μM Sorcin speeded up inactivation. A similar effect was observed by substitution of Ca2+ with Ba2+. Down-regulation of endogenous Sorcin to 27 ± 7% using an RNAi adenoviral vector slowed inactivation of I Ca,L by ∼42%. The effects of Sorcin on voltage-dependant inactivation were mimicked by a truncated form of the protein containing only the Ca2+-binding domain. This data is consistent with two independent actions of Sorcin on the LTCC: (1) slowing Ca2+-dependant inactivation and (2) stimulating voltage-dependant inactivation. The net effect of Sorcin on the time-dependent inactivation of I Ca,L was a balance between these two effects. Under normal conditions, Sorcin slows I Ca,L inactivation because the effects of Ca2+-dependant inactivation out-weigh the effects on voltage-dependant inactivation.
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Sorcin modulates cardiac l type ca2 current by functional interaction with the α1c subunit in rabbits
Experimental Physiology, 2008Co-Authors: Mark R Fowler, Emilia Chiancone, Gianni Colotti, Godfrey L Smith, Ian M FearonAbstract:We examined the modulation of the cardiac L-type Ca2+ channel (LTCC) by the regulatory protein Sorcin and tested the hypothesis that modulation occurred by direct interaction. Whole-cell patch-clamp recordings were made on native rabbit ventricular myocytes and HEK 293 cells expressing cardiac α1C subunits. In ventricular cells, Sorcin increased peak current when using either Ca2+ or Ba2+ as charge carriers. In HEK 293 cells, Sorcin increased peak current density when using Ba2+ as a charge carrier but not when using Ca2+. In ventricular myocytes, current inactivation (τfast, in ms) was slowed by Sorcin with Ca2+ as the charge carrier, whilst in the presence of Ba2+ it was enhanced. In HEK 293 cells, Sorcin significantly enhanced τfast, but no significant change was observed with Ba2+. This trend was mimicked by the truncated peptide, Sorcin Ca2+-binding domain, which lacks the N-terminal domain. These data suggest that Sorcin interacts with LTCC via its C-terminal domain, which alters current magnitude and τfast. These effects appear to be influenced by the prevailing experimental conditions.
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Sorcin modulates cardiac L‐type Ca2+ current by functional interaction with the α1C subunit in rabbits
Experimental Physiology, 2008Co-Authors: Mark R Fowler, Emilia Chiancone, Gianni Colotti, Godfrey L Smith, Ian M FearonAbstract:We examined the modulation of the cardiac L-type Ca2+ channel (LTCC) by the regulatory protein Sorcin and tested the hypothesis that modulation occurred by direct interaction. Whole-cell patch-clamp recordings were made on native rabbit ventricular myocytes and HEK 293 cells expressing cardiac α1C subunits. In ventricular cells, Sorcin increased peak current when using either Ca2+ or Ba2+ as charge carriers. In HEK 293 cells, Sorcin increased peak current density when using Ba2+ as a charge carrier but not when using Ca2+. In ventricular myocytes, current inactivation (τfast, in ms) was slowed by Sorcin with Ca2+ as the charge carrier, whilst in the presence of Ba2+ it was enhanced. In HEK 293 cells, Sorcin significantly enhanced τfast, but no significant change was observed with Ba2+. This trend was mimicked by the truncated peptide, Sorcin Ca2+-binding domain, which lacks the N-terminal domain. These data suggest that Sorcin interacts with LTCC via its C-terminal domain, which alters current magnitude and τfast. These effects appear to be influenced by the prevailing experimental conditions.
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Complex modulation of L-type Ca^2+ current inactivation by Sorcin in isolated rabbit cardiomyocytes
Pflügers Archiv - European Journal of Physiology, 2008Co-Authors: Mark R Fowler, Emilia Chiancone, Gianni Colotti, Tim Seidler, Yoshiharu Higuchi, Godfrey L SmithAbstract:Modulation of the L-type Ca^2+ channel (LTCC) by Sorcin was investigated by measuring the L-type Ca^2+ current ( I _Ca,L) in isolated rabbit ventricular myocytes using ruptured patch, single electrode voltage clamp in the absence of extracellular Na^+. Fifty millimolars EGTA (170 nM Ca^2+) in the pipette solution buffered bulk cytoplasmic [Ca^2+], but retained rapid Ca^2+-dependant inactivation of I _Ca,L,. Recombinant Sorcin (3 μM) in the pipette significantly slowed time-dependant inactivation ( τ _fast: 8.8 ± 0.9 vs. 15.1 ± 1.7 ms). Sorcin had no significant effect on I _Ca,L, after inhibition of the sarcoplasmic reticulum (SR). Using 10 mM 1,2-bis( o - N , N , N ′, N ′-tetraacetic acid (170 nM Ca^2+), I _Ca,L inactivation was then determined by a Ca^2+ -independent, voltage-dependant process. Under these conditions, 3 μM Sorcin speeded up inactivation. A similar effect was observed by substitution of Ca^2+ with Ba^2+. Down-regulation of endogenous Sorcin to 27 ± 7% using an RNAi adenoviral vector slowed inactivation of I _Ca,L by ∼42%. The effects of Sorcin on voltage-dependant inactivation were mimicked by a truncated form of the protein containing only the Ca^2+-binding domain. This data is consistent with two independent actions of Sorcin on the LTCC: (1) slowing Ca^2+-dependant inactivation and (2) stimulating voltage-dependant inactivation. The net effect of Sorcin on the time-dependent inactivation of I _Ca,L was a balance between these two effects. Under normal conditions, Sorcin slows I _Ca,L inactivation because the effects of Ca^2+-dependant inactivation out-weigh the effects on voltage-dependant inactivation.
Ilaria Genovese - One of the best experts on this subject based on the ideXlab platform.
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profiling calcium dependent interactions between Sorcin and intrinsically disordered regions of human proteome
Biochimica et Biophysica Acta, 2020Co-Authors: Gianni Colotti, Ilaria Genovese, Andrea Ilari, Annarita Fiorillo, Theo Battista, Andrea Carotti, Ylva IvarssonAbstract:Abstract Background Sorcin is a calcium sensor that exerts many calcium-related functions in the cells, e.g. it regulates calcium concentration in the cytoplasm, endoplasmic reticulum (ER) and mitochondria, by interacting with calcium pumps, exchangers and channels. Albeit Sorcin is an interesting potential cancer target, little is known about its interactors upon calcium-mediated activation. Our previous study suggested that Sorcin may recognize short linear binding motifs as the crystal structure revealed a self-interaction with a GYYPGG stretch in its N-terminus, and combinatorial peptide-phage display provided support for peptide-mediated interactions. Methods In this study we screened for motif-based interactions between Sorcin and intrinsically disordered regions of the human proteome using proteomic peptide phage display (ProP-PD). We identified a peptide belonging to protein phosphatase 1 regulatory subunit 3G (PPP1R3G) as a potential novel interactor and confirm the interaction through biophysical and cell-based approaches, and provide structural information through molecular dynamics simulations. Results Altogether, we identify a preferred motif in the enriched pool of binders and a peptide belonging to protein phosphatase 1 regulatory subunit 3G (PPP1R3G) as a preferred ligand. Conclusion Through this study we gain information on a new Sorcin binding partner and profile Sorcin's motif-based interaction. General significance The interaction between Sorcin and PPP1R3G may suggest a close dependence between glucose homeostasis and calcium concentration in the different cell compartments, opening a completely new and interesting scenery yet to be fully disclosed.
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roles of Sorcin in drug resistance in cancer one protein many mechanisms for a novel potential anticancer drug target
Cancers, 2020Co-Authors: Theo Battista, Ilaria Genovese, Andrea Ilari, Annarita Fiorillo, Valerio Chiarini, Gianni ColottiAbstract:The development of drug resistance is one of the main causes of failure in anti-cancer treatments. Tumor cells adopt many strategies to counteract the action of chemotherapeutic agents, e.g., enhanced DNA damage repair, inactivation of apoptotic pathways, alteration of drug targets, drug inactivation, and overexpression of ABC (Adenosine triphosphate-binding cassette, or ATP-binding cassette) transporters. These are broad substrate-specificity ATP-dependent efflux pumps able to export toxins or drugs out of cells; for instance, ABCB1 (MDR1, or P-glycoprotein 1), overexpressed in most cancer cells, confers them multidrug resistance (MDR). The gene coding for Sorcin (SOluble Resistance-related Calcium-binding proteIN) is highly conserved among mammals and is located in the same chromosomal locus and amplicon as the ABC transporters ABCB1 and ABCB4, both in human and rodent genomes (two variants of ABCB1, i.e., ABCB1a and ABCB1b, are in rodent amplicon). Sorcin was initially characterized as a soluble protein overexpressed in multidrug (MD) resistant cells and named “resistance-related” because of its co-amplification with ABCB1. Although for years Sorcin overexpression was thought to be only a by-product of the co-amplification with ABC transporter genes, many papers have recently demonstrated that Sorcin plays an important part in MDR, indicating a possible role of Sorcin as an oncoprotein. The present review illustrates Sorcin roles in the generation of MDR via many mechanisms and points to Sorcin as a novel potential target of different anticancer molecules.
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a22 Sorcin rescues ca ii dysregulation and endoplasmic reticulum stress in huntington s disease
Journal of Neurology Neurosurgery and Psychiatry, 2018Co-Authors: Gianni Colotti, Ilaria Genovese, Eris Bidollari, Jessica Rosati, Marzia Perluigi, Tito Cali, Andrea Arena, Ferdinando Squitieri, Andrea IlariAbstract:Background Sorcin is an essential penta-EF hand Ca (II) -binding protein, able to reduce Endoplasmic Reticulum (ER) stress and cell death. X-ray apo and Ca (II)-bound Sorcin structures showed that Ca (II) binding to EF1–3 hands promotes a large conformational change and the exposure of molecular surfaces through which Sorcin interacts with Ryanodine receptors (RYR), Sarco/ER Ca (II)-ATPase (SERCA), Na (I)-Ca (II) exchanger and regulates Ca (II) fluxes. Aims Sorcin is one of the most expressed Ca (II)-binding proteins in the human brain, and is hypothesized to be involved in neurodegenerative diseases. We aim at demonstrating that Sorcin in brain counteracts the increased cytosolic Ca (II) levels associated with neurodegeneration and that for this reason it is overexpressed in HD (Huntington’s Disease). Methods We identified Sorcin interactors by colocalization, coIP and SPR experiments, analysed Ca (II) currents and mitochondria-associated ER membranes (MAMs) and measured Sorcin expression in many biological samples by RT-PCR and Western Blot. Results Sorcin interacts in a Ca (II)-dependent manner with RYR; it increases both SERCA- and RYR-dependent ER Ca (II) currents and mitochondrial Ca (II) transients; Sorcin increases the number of short MAMs, essential for mitochondrial Ca (II) intake, disrupted in most neurodegenerative diseases, and is able to interact with proteins involved in the Unfolded Protein Response, i.e. ATF6, eIF2a and Sigma1R, localized in MAM. Finally, we found increased Sorcin expression levels in fibroblasts from HD patients and post-mortem cortex tissue of HD subjects. Conclusions Our preliminary results show that Sorcin, which controls Ca (II) flux across cell compartments, is overexpressed in neurons to counteract Ca (II) unbalance caused by HD. These findings indicate that Sorcin is a novel, promising HD marker and represents a defence from cellular stress dependent on neurodegeneration.
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A22 Sorcin rescues ca (II) dysregulation and endoplasmic reticulum stress in huntington’s disease
Journal of Neurology Neurosurgery and Psychiatry, 2018Co-Authors: Gianni Colotti, Ilaria Genovese, Eris Bidollari, Jessica Rosati, Marzia Perluigi, Tito Cali, Andrea Arena, Ferdinando Squitieri, Andrea IlariAbstract:Background Sorcin is an essential penta-EF hand Ca (II) -binding protein, able to reduce Endoplasmic Reticulum (ER) stress and cell death. X-ray apo and Ca (II)-bound Sorcin structures showed that Ca (II) binding to EF1–3 hands promotes a large conformational change and the exposure of molecular surfaces through which Sorcin interacts with Ryanodine receptors (RYR), Sarco/ER Ca (II)-ATPase (SERCA), Na (I)-Ca (II) exchanger and regulates Ca (II) fluxes. Aims Sorcin is one of the most expressed Ca (II)-binding proteins in the human brain, and is hypothesized to be involved in neurodegenerative diseases. We aim at demonstrating that Sorcin in brain counteracts the increased cytosolic Ca (II) levels associated with neurodegeneration and that for this reason it is overexpressed in HD (Huntington’s Disease). Methods We identified Sorcin interactors by colocalization, coIP and SPR experiments, analysed Ca (II) currents and mitochondria-associated ER membranes (MAMs) and measured Sorcin expression in many biological samples by RT-PCR and Western Blot. Results Sorcin interacts in a Ca (II)-dependent manner with RYR; it increases both SERCA- and RYR-dependent ER Ca (II) currents and mitochondrial Ca (II) transients; Sorcin increases the number of short MAMs, essential for mitochondrial Ca (II) intake, disrupted in most neurodegenerative diseases, and is able to interact with proteins involved in the Unfolded Protein Response, i.e. ATF6, eIF2a and Sigma1R, localized in MAM. Finally, we found increased Sorcin expression levels in fibroblasts from HD patients and post-mortem cortex tissue of HD subjects. Conclusions Our preliminary results show that Sorcin, which controls Ca (II) flux across cell compartments, is overexpressed in neurons to counteract Ca (II) unbalance caused by HD. These findings indicate that Sorcin is a novel, promising HD marker and represents a defence from cellular stress dependent on neurodegeneration.
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binding of doxorubicin to Sorcin impairs cell death and increases drug resistance in cancer cells
Cell Death and Disease, 2017Co-Authors: Ilaria Genovese, Andrea Ilari, Annarita Fiorillo, Silvia Masciarelli, Francesco Fazi, Gianni ColottiAbstract:Sorcin is a calcium binding protein that plays an important role in multidrug resistance (MDR) in tumors, since its expression confers resistance to doxorubicin and to other chemotherapeutic drugs. In this study, we show that Sorcin is able to bind doxorubicin, vincristine, paclitaxel and cisplatin directly and with high affinity. The high affinity binding of doxorubicin to Sorcin has been demonstrated with different techniques, that is, surface plasmon resonance, fluorescence titration and X-ray diffraction. Although the X-ray structure of Sorcin in complex with doxorubicin has been solved at low resolution, it allows the identification of one of the two doxorubicin binding sites, placed at the interface between the EF5 loop the G helix and the EF4 loop. We show that Sorcin cellular localization changes upon doxorubicin treatment, an indication that the protein responds to doxorubicin and it presumably binds the drug also inside the cell, soon after drug entrance. We also demonstrate that Sorcin is able to limit the toxic effects of the chemotherapeutic agent in the cell. In addition, Sorcin silencing increases cell death upon treatment with doxorubicin, increases the accumulation of doxorubicin in cell nucleus, decreases the expression of MDR1 and doxorubicin efflux via MDR1.
Marian B. Meyers - One of the best experts on this subject based on the ideXlab platform.
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expression of a Sorcin missense mutation in the heart modulates excitation contraction coupling
The FASEB Journal, 2007Co-Authors: Leon Collis, Marian B. Meyers, William A Coetzee, Jie Zhang, Colin K L Phoon, Eric A Sobie, Glenn I. FishmanAbstract:Sorcin is a Ca2+ binding protein implicated in the regulation of intracellular Ca2+ cycling and cardiac excitation-contraction coupling. Structural and human genetic studies suggest that a naturally occurring sequence variant encoding L112-Sorcin disrupts an E-F hand Ca2+ binding domain and may be responsible for a heritable form of hypertension and hypertrophic heart disease. We generated transgenic mice overexpressing L112-Sorcin in the heart and characterized the effects on Ca2+ regulation and cardiac function both in vivo and in dissociated cardiomyocytes. Hearts of SorcinF112L transgenic mice were mildly dilated but ventricular function was preserved and systemic blood pressure was normal. SorcinF112L myocytes were smaller than control cells and displayed complex alterations in Ca2+ regulation and contractility, including a slowed inactivation of L-type Ca2+ current, enhanced Ca2+ spark width, duration, and frequency, and increased Na+-Ca2+ exchange activity. In contrast, mice with cardiac-specific o...
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Sorcin regulates excitation contraction coupling in the heart
Journal of Biological Chemistry, 2003Co-Authors: Marian B. Meyers, Avi Fischer, Coeli M Lopes, Tibor Rohacs, Tomoe Y Nakamura, Yingying Zhou, Ruth A Altschuld, Sylvia A Mccune, William A Coetzee, Glenn I. FishmanAbstract:Abstract Sorcin is a penta-EF hand Ca2+-binding protein that associates with both cardiac ryanodine receptors and L-type Ca2+ channels and has been implicated in the regulation of intracellular Ca2+ cycling. To better define the function of Sorcin, we characterized transgenic mice in which Sorcin was overexpressed in the heart. Transgenic mice developed normally with no evidence of cardiac hypertrophy and no change in expression of other calcium regulatory proteins. In vivo hemodynamics revealed significant reductions in global indices of contraction and relaxation. Contractile abnormalities were also observed in isolated adult transgenic myocytes, along with significant depression of Ca2+ transient amplitudes. Whole cell ICa density and the time course of activation were normal in transgenic myocytes, but the rate of inactivation was significantly accelerated. These effects of Sorcin on L-type Ca2+ currents were confirmed in Xenopus oocyte expression studies. Finally, we examined the expression of Sorcin in normal and failing hearts from spontaneous hypertensive heart failure rats. In normal myocardium, Sorcin extensively co-localized with ryanodine receptors at the Z-lines, whereas in myopathic hearts the degree of co-localization was markedly disrupted. Together, these data indicate that Sorcin modulates intracellular Ca2+ cycling and Ca2+ influx pathways in the heart.
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effects of adenovirus mediated Sorcin overexpression on excitation contraction coupling in isolated rabbit cardiomyocytes
Circulation Research, 2003Co-Authors: Tim Seidler, Stewart L W Miller, Christopher M Loughrey, Astrid Kania, Annika Burow, Sarah Kettlewell, Nils Teucher, Stefan Wagner, Harald Kogler, Marian B. MeyersAbstract:To evaluate the effect of Sorcin on cardiac excitation-contraction coupling, adult rabbit ventricular myocytes were transfected with a recombinant adenovirus coding for human Sorcin (Ad-Sorcin). A β-galactosidase adenovirus (Ad-LacZ) was used as a control. Fractional shortening in response to 1-Hz field stimulation (at 37°C) was significantly reduced in Ad-Sorcin–transfected myocytes compared with control myocytes (2.10±0.05% [n=311] versus 2.42±0.06% [n=312], respectively; P P 2+ ] i was observed after Ad-Sorcin transfection. L-type Ca 2+ current amplitude and time course were unaffected. Caffeine-induced Ca 2+ release from the sarcoplasmic reticulum (SR) and the accompanying inward Na + -Ca 2+ exchanger (NCX) current revealed a significantly lower SR Ca 2+ content and faster Ca 2+ -extrusion kinetics in Ad-Sorcin–transfected cells. Higher NCX activity after Ad-Sorcin transfection was confirmed by measuring the NCX current-voltage relationship. β-Escin–permeabilized rabbit cardiomyocytes were used to study the effects of Sorcin overexpression on Ca 2+ sparks imaged with fluo 3 at 145 to 160 nmol/L [Ca 2+ ] using a confocal microscope. Under these conditions, caffeine-mediated SR Ca 2+ release was not different between the two groups. Spontaneous spark frequency, duration, width, and amplitude were lower in Sorcin-overexpressing myocytes. In summary, Sorcin overexpression in rabbit cardiomyocytes decreased Ca 2+ -transient amplitude predominately by lowering SR Ca 2+ content via increased NCX activity. The effect of Sorcin overexpression on Ca 2+ sparks indicates an effect on the ryanodine receptor that may also influence excitation-contraction coupling.
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presenilin 2 interacts with Sorcin a modulator of the ryanodine receptor
Journal of Biological Chemistry, 2000Co-Authors: Eunju Packchung, Amy M Brownawell, Marian B. Meyers, Warren P Pettingell, Robert D Moir, Isaac Cheng, Rudolph E TanziAbstract:Abstract Perturbed Ca2+ homeostasis is a common molecular consequence of familial Alzheimer's disease-linked presenilin mutations. We report here the molecular interaction of the large hydrophilic loop region of presenilin 2 (PS2) with Sorcin, a penta-EF-hand Ca2+-binding protein that serves as a modulator of the ryanodine receptor intracellular Ca2+channel. The association of endogenous Sorcin and PS2 was demonstrated in cultured cells and human brain tissues. Membrane-associated Sorcin and a subset of the functional PS2 complexes were co-localized to a novel subcellular fraction that is distinctively positive for calcineurin B. Sorcin was found to interact with PS2 endoproteolytic fragments but not full-length PS2, and the Sorcin/PS2 interaction was greatly enhanced by treatment with the Ca2+ ionophore A23187. Our findings reveal a molecular link between PS2 and intracellular Ca2+ channels (i.e. ryanodine receptor) and substantiate normal and/or pathological roles of PS2 in intracellular Ca2+ homeostasis.
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nmdar1 in the caudate putamen nucleus ultrastructural localization and co expression with Sorcin a 22 000 mol wt calcium binding protein
Neuroscience, 1999Co-Authors: K N Gracy, Marian B. Meyers, Catherine L Clarke, Virginia M. PickelAbstract:Abstract Entry of calcium through N -methyl- d -aspartate-type glutamate receptors in the caudate–putamen nucleus is essential for normal motor activity, but can produce cytotoxicity with continued stimulation and subsequent release of intracellular calcium. To determine potential functional sites for N -methyl- d -aspartate receptor activation in this region, we examined the ultrastructural localization of the R1 subunit of the N -methyl- d -aspartate receptor (NMDAR1) in rat brain. In addition, we comparatively examined the localization of NMDAR1 and Sorcin, a 22,000 mol. wt calcium binding protein present in certain striatal neurons and involved in calcium-induced calcium release. NMDAR1-like immunoreactivity was seen at synaptic and non-synaptic sites on neuronal plasma membranes. Of 1 514 NMDAR1-labeled profiles, 62% were dendrites and dendritic spines and the remainder were mainly unmyelinated axons and axon terminals. Sorcin-like immunoreactivity was present in 39% of the profiles that contained NMDAR1 labeling, most (533/595) of which were dendrites and dendritic spines. Of 1 807 Sorcin-labeled profiles, 42% were identified, however, as small processes including spine necks and unmyelinated axons or axon terminals. These profiles also occasionally contained NMDAR1 or showed synaptic or appositional contacts with other NMDAR1-immunoreactive neurons. The results of this study suggest that in the caudate–putamen nucleus, activation of NMDA receptors permits calcium influx at plasmalemmal sites mainly on dendrites where Sorcin may play a role in calcium-induced calcium release. The presence of Sorcin in some, but not all NMDA-containing neurons in the caudate–putamen nucleus has potential implications for the known differential vulnerability of certain striatal neurons to excitotoxins.
