The Experts below are selected from a list of 1932 Experts worldwide ranked by ideXlab platform
Peter B. Stathopulos - One of the best experts on this subject based on the ideXlab platform.
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structural elements of Stromal Interaction Molecule mediated store operated calcium entry regulation
Journal of Biomedical Science, 2021Co-Authors: Peter B. StathopulosAbstract:Calcium (Ca2+) is a universal signaling entity in eukaryotic cells mediating diverse processes such as the immune response, hypertrophy, apoptosis, platelet aggregation and memory, to name a few. These processes require a sustained elevation of cytosolic Ca2+ levels which is facilitated by store operated Ca2+ entry (SOCE). SOCE is the process whereby endoplasmic reticulum (ER) luminal Ca2+ depletion signals the opening of ion channels on the plasma membrane (PM) which facilitate the movement of Ca2+ down the concentration gradient from the extracellular space into the cytosol. Th e principal Molecules that mediate SOCE include the ER resident Stromal Interaction Molecule-1 (STIM1) and PM ORAI1 protein subunits which assemble into a channel pore. Upon ER luminal Ca2+ depletion, STIM1 undergoes a destabilization coupled oligomerization which leads to translocation of this Ca2+ sensor to ER-PM junctions where it couples to ORAI1 subunits and opens these PM Ca2+ channels. Since the identification of STIM1 and ORAI1 as the principal Molecules driving SOCE, considerable progress has been made elucidating their high-resolution structural mechanisms of action. Author will present available structural data on the STIM1 Ca2+ sensing mechanism and how this regulator may complex to ORAI1 subunits. Th e coupling mechanism revealed using soluble human STIM1 and ORAI1 fragments are congruent with the hexameric assembly elucidated in the D. melanogaster crystal structure. Finally, author will present unpublished work showing how post translational modifications within the luminal domain of STIM1 aff ects the structural mechanisms of Ca2+ sensing. Ultimately, the post-translation modifi cation driven STIM1 structural and biophysical changes have implications in the agonist induced hypertrophic response and pinpoint a new therapeutic target for heart disease.
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Synergistic stabilization by nitrosoglutathione-induced thiol modifications in the Stromal Interaction Molecule-2 luminal domain suppresses basal and store operated calcium entry
Scientific Reports, 2020Co-Authors: Matthew J. Novello, Qingping Feng, Mengqi Zhang, Peter B. StathopulosAbstract:Stromal Interaction Molecule−1 and −2 (STIM1/2) are endoplasmic reticulum (ER) membrane-inserted calcium (Ca^2+) sensing proteins that, together with Orai1-composed Ca^2+ channels on the plasma membrane (PM), regulate intracellular Ca^2+ levels. Recent evidence suggests that S -nitrosylation of the luminal STIM1 Cys residues inhibits store operated Ca^2+ entry (SOCE). However, the effects of thiol modifications on STIM2 during nitrosative stress and their role in regulating basal Ca^2+ levels remain unknown. Here, we demonstrate that the nitric oxide (NO) donor nitrosoglutathione (GSNO) thermodynamically stabilizes the STIM2 Ca^2+ sensing region in a Cys-specific manner. We uncovered a remarkable synergism in this stabilization involving the three luminal Cys of STIM2, which is unique to this paralog. S -Nitrosylation causes structural perturbations that converge on the face of the EF-hand and sterile α motif (EF-SAM) domain, implicated in unfolding-coupled activation. In HEK293T cells, enhanced free basal cytosolic Ca^2+ and SOCE mediated by STIM2 overexpression could be attenuated by GSNO or mutation of the modifiable Cys located in the luminal domain. Collectively, we identify the Cys residues within the N-terminal region of STIM2 as modifiable targets during nitrosative stress that can profoundly and cooperatively affect basal Ca^2+ and SOCE regulation.
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does Stromal Interaction Molecule 1 have five senses
Cell Calcium, 2019Co-Authors: Peter B. Stathopulos, Mitsuhiko IkuraAbstract:Abstract A single calcium (Ca2+) binding site within the canonical EF-hand loop was thought to govern the Stromal Interaction Molecule-1 (STIM1) structural changes that lead to activation of Orai1 Ca2+ channels. Recent work by Gudlur et al., published in Nat Commun [9(1):4536], suggests that the STIM1 endoplasmic reticulum (ER) luminal domain has ∼5 additional Ca2+ binding sites, which underlie a surprising new proposal for Ca2+ sensing.
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the 2β splice variation alters the structure and function of the Stromal Interaction Molecule coiled coil domains
International Journal of Molecular Sciences, 2018Co-Authors: Steve Chung, Mengqi Zhang, Peter B. StathopulosAbstract:Stromal Interaction Molecule (STIM)-1 and -2 regulate agonist-induced and basal cytosolic calcium (Ca2+) levels after oligomerization and translocation to endoplasmic reticulum (ER)-plasma membrane (PM) junctions. At these junctions, the STIM cytosolic coiled-coil (CC) domains couple to PM Orai1 proteins and gate these Ca2+ release-activated Ca2+ (CRAC) channels, which facilitate store-operated Ca2+ entry (SOCE). Unlike STIM1 and STIM2, which are SOCE activators, the STIM2β splice variant contains an 8-residue insert located within the conserved CCs which inhibits SOCE. It remains unclear if the 2β insert further depotentiates weak STIM2 coupling to Orai1 or independently causes structural perturbations which prevent SOCE. Here, we use far-UV circular dichroism, light scattering, exposed hydrophobicity analysis, solution small angle X-ray scattering, and a chimeric STIM1/STIM2β functional assessment to provide insights into the molecular mechanism by which the 2β insert precludes SOCE activation. We find that the 2β insert reduces the overall α-helicity and enhances the exposed hydrophobicity of the STIM2 CC domains in the absence of a global conformational change. Remarkably, incorporation of the 2β insert into the STIM1 context not only affects the secondary structure and hydrophobicity as observed for STIM2, but also eliminates the more robust SOCE response mediated by STIM1. Collectively, our data show that the 2β insert directly precludes Orai1 channel activation by inducing structural perturbations in the STIM CC region.
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a charge sensing region in the Stromal Interaction Molecule 1 luminal domain confers stabilization mediated inhibition of soce in response to s nitrosylation
Journal of Biological Chemistry, 2018Co-Authors: Jinhui Zhu, Qingping Feng, Peter B. StathopulosAbstract:Store-operated Ca2+ entry (SOCE) is a major Ca2+ signaling pathway facilitating extracellular Ca2+ influx in response to the initial release of intracellular endo/sarcoplasmic reticulum (ER/SR) Ca2+ stores. Stromal Interaction Molecule 1 (STIM1) is the Ca2+ sensor that activates SOCE following ER/SR Ca2+ depletion. The EF-hand and the adjacent sterile α-motif (EFSAM) domains of STIM1 are essential for detecting changes in luminal Ca2+ concentrations. Low ER Ca2+ levels trigger STIM1 destabilization and oligomerization, culminating in the opening of Orai1-composed Ca2+ channels on the plasma membrane. NO-mediated S-nitrosylation of cysteine thiols regulates myriad protein functions, but its effects on the structural mechanisms that regulate SOCE are unclear. Here, we demonstrate that S-nitrosylation of Cys49 and Cys56 in STIM1 enhances the thermodynamic stability of its luminal domain, resulting in suppressed hydrophobic exposure and diminished Ca2+ depletion–dependent oligomerization. Using solution NMR spectroscopy, we pinpointed a structural mechanism for STIM1 stabilization driven by complementary charge Interactions between an electropositive patch on the core EFSAM domain and the S-nitrosylated nonconserved region of STIM1. Finally, using live cells, we found that the enhanced luminal domain stability conferred by either Cys49 and Cys56 S-nitrosylation or incorporation of negatively charged residues into the EFSAM electropositive patch in the full-length STIM1 context significantly suppresses SOCE. Collectively, our results suggest that S-nitrosylation of STIM1 inhibits SOCE by interacting with an electropositive patch on the EFSAM core, which modulates the thermodynamic stability of the STIM1 luminal domain.
Mitsuhiko Ikura - One of the best experts on this subject based on the ideXlab platform.
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does Stromal Interaction Molecule 1 have five senses
Cell Calcium, 2019Co-Authors: Peter B. Stathopulos, Mitsuhiko IkuraAbstract:Abstract A single calcium (Ca2+) binding site within the canonical EF-hand loop was thought to govern the Stromal Interaction Molecule-1 (STIM1) structural changes that lead to activation of Orai1 Ca2+ channels. Recent work by Gudlur et al., published in Nat Commun [9(1):4536], suggests that the STIM1 endoplasmic reticulum (ER) luminal domain has ∼5 additional Ca2+ binding sites, which underlie a surprising new proposal for Ca2+ sensing.
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Structural elements of Stromal Interaction Molecule function.
Cell Calcium, 2018Co-Authors: Matthew J. Novello, Qingping Feng, Mitsuhiko Ikura, Peter B. StathopulosAbstract:Abstract Stromal Interaction Molecule (STIM)-1 and -2 are multi-domain, single-pass transmembrane proteins involved in sensing changes in compartmentalized calcium (Ca2+) levels and transducing this cellular signal to Orai1 channel proteins. Our understanding of the molecular mechanisms underlying STIM signaling has been dramatically improved through available X-ray crystal and solution NMR structures. This high-resolution structural data has revealed that intricate intramolecular and intermolecular protein-protein Interactions are involved in converting STIMs from the quiescent to activation-competent states. This review article summarizes the current high resolution structural data on specific EF-hand, sterile α motif and coiled-coil Interactions which drive STIM function in the activation of Orai1 channels. Further, the work discusses the effects of post-translational modifications on the structure and function of STIMs. Future structural studies on larger STIM:Orai complexes will be critical to fully defining the molecular bases for STIM function and how post-translational modifications influence these mechanisms.
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a coiled coil clamp controls both conformation and clustering of Stromal Interaction Molecule 1 stim1
Journal of Biological Chemistry, 2014Co-Authors: Marc Fahrner, Peter B. Stathopulos, Mitsuhiko Ikura, Martin Muik, Rainer Schindl, Carmen Butorac, Le Zheng, Isaac Jardin, Christoph RomaninAbstract:Abstract Store-operated Ca2+ entry, essential for the adaptive immunity, is initiated by the endoplasmic reticulum (ER) Ca2+ sensor STIM1. Ca2+ entry occurs through the plasma membrane resident Ca2+ channel Orai1 that directly interacts with the C-terminal STIM1 domain, named SOAR/CAD. Depletion of the ER Ca2+ store controls this STIM1/Orai1 Interaction via transition to an extended STIM1 C-terminal conformation, exposure of the SOAR/CAD domain, and STIM1/Orai1 co-clustering. Here we developed a novel approach termed FRET-derived Interaction in a Restricted Environment (FIRE) in an attempt to dissect the interplay of coiled-coil (CC) Interactions in controlling STIM1 quiescent as well as active conformation and cluster formation. We present evidence of a sequential activation mechanism in the STIM1 cytosolic domains where the Interaction between CC1 and CC3 segment regulates both SOAR/CAD exposure and CC3-mediated higher-order oligomerization as well as cluster formation. These dual levels of STIM1 auto-inhibition provide efficient control over the coupling to and activation of Orai1 channels.
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Stromal Interaction Molecule stim 1 and stim2 calcium sensing regions exhibit distinct unfolding and oligomerization
2009Co-Authors: Peter B. Stathopulos, Le Zheng, Mitsuhiko IkuraAbstract:Stromal InteractionMolecules (STIM) 1 and STIM2 are regulators of store-operated calcium (Ca2 ) entry as well as basal cytoplasmic Ca2 levels in human cells. Despite a high sequence similarity (>65%) and analogous sequence-based domain architectures, STIM1 and STIM2 differentially influence these phenomena. Among all eukaryotes, the endoplasmic reticulum luminal portion of STIM proteins minimally encode EF-hand and sterile -motif (SAM) domains (EF-SAM), which are responsible for sensing changes in Ca2 levels and initiating oligomerization. STIM oligomerization is a key induction step in the activation of Ca2 -permeable channels on the plasma membrane. Here, we show that the kinetic half-timeof conversion fromamonomeric to a steadyoligomeric state is>70 shorter for STIM1EF-SAMthan STIM2 under similar conditions. Urea-induced rates of unfolding forSTIM1EF-SAMare>3 quickerwhencomparedwithSTIM2, coherent with partial unfolding-coupled aggregation. Additionally, we demonstrate that the isoform-specificN-terminal residues beyond EF-SAMcan influence the stability of this region.We postulate that distinct oligomerization dynamics of STIM isoforms have evolved to adapt to differential roles inCa2 homeostasis and signaling.
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Stromal Interaction Molecule (STIM) 1 and STIM2 calcium sensing regions exhibit distinct unfolding and oligomerization kinetics.
The Journal of biological chemistry, 2008Co-Authors: Peter B. Stathopulos, Le Zheng, Mitsuhiko IkuraAbstract:Abstract Stromal Interaction Molecules (STIM) 1 and STIM2 are regulators of store-operated calcium (Ca2+) entry as well as basal cytoplasmic Ca2+ levels in human cells. Despite a high sequence similarity (>65%) and analogous sequence-based domain architectures, STIM1 and STIM2 differentially influence these phenomena. Among all eukaryotes, the endoplasmic reticulum luminal portion of STIM proteins minimally encode EF-hand and sterile α-motif (SAM) domains (EF-SAM), which are responsible for sensing changes in Ca2+ levels and initiating oligomerization. STIM oligomerization is a key induction step in the activation of Ca2+-permeable channels on the plasma membrane. Here, we show that the kinetic half-time of conversion from a monomeric to a steady oligomeric state is >70× shorter for STIM1 EF-SAM than STIM2 under similar conditions. Urea-induced rates of unfolding for STIM1 EF-SAM are >3× quicker when compared with STIM2, coherent with partial unfolding-coupled aggregation. Additionally, we demonstrate that the isoform-specific N-terminal residues beyond EF-SAM can influence the stability of this region. We postulate that distinct oligomerization dynamics of STIM isoforms have evolved to adapt to differential roles in Ca2+ homeostasis and signaling.
Lan Huang - One of the best experts on this subject based on the ideXlab platform.
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knockdown of Stromal Interaction Molecule 1 down regulates the differentiation of endothelial progenitor cells and reendothelialization after vascular injury
International Journal of Cardiology, 2011Co-Authors: Chunyan Kuang, Lan HuangAbstract:Objectives The study was to investigate the effect of Stromal Interaction Molecule 1(STIM1) silencing on endothelial progenitor cells (EPCs) differentiation and reendothelialization. Methods Following balloon injury, EPCs, which were transfected with Ad-si/rSTIM1, Ad-hSTIM1 and Ad–non silencing control (NSC), were transplanted to the rat. Evans Blue dye was performed to measured reendothelialization at 7 and 14 day after injury, the neointimal formation was evaluated by staining with hematoxylin and eosin at 14 day after injury. EPCs differentiation was examined by western blot, and rat gene expression array was detected. Results At 7 and 14 day, the reendothelialized area in the Ad-si/rSTIM1-EPCs infected arteries was obviously less than that in NSC-infected groups and Ad-si/rSTIM1+ Ad-hSTIM1-EPCs group(p Conclusions Silencing of STIM1 inhibited EPCs differentiation and reendothelialization, indicating a possible new mechanism through EPCs underlying the process of vascular repair.
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impact of Stromal Interaction Molecule 1 silencing on cell cycle of endothelial progenitor cells
Chinese journal of cardiovascular diseases, 2011Co-Authors: Chunyan Kuang, Lan Huang, Mengyang Deng, Kui Wang, Dehui QianAbstract:OBJECTIVE To investigate the effect of Stromal Interaction Molecule 1 (STIM1) silencing on EPCs cell cycle. METHODS Rat bone marrow derived endothelial progenitor cells (EPCs) were isolated and cultured in L-DMEM with 20% FBS. Ad-si/rSTIM1 and Ad-hSTIM1 were then transfected into EPCs and the expression of STIM1 mRNA was detected by RT-PCR. The cell cycle was determined using flow cytometry analysis and intracellular free Ca2+ was measured using LSCM. Co-immunoprecipitation was performed to examine the Interaction between STIM1 and TRPC1. Protein levels of inositol 1, 4, 5-trisphosphate were analyzed with ELISA assay. RESULTS Forty-eight hours after transfection, the expression of STIM1 mRNA was significantly downregulated (0.37 +/- 0.02 vs. 1.00 +/- 0.02, P 0.05). CONCLUSION siRNA-mediated knockdown of STIM1 inhibited EPCs proliferation by reducing intracellular free Ca2+ through TRPC1-SOC signaling pathway.
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silencing Stromal Interaction Molecule 1 by rna interference inhibits the proliferation and migration of endothelial progenitor cells
Biochemical and Biophysical Research Communications, 2010Co-Authors: Chunyan Kuang, Kui Wang, Dehui Qian, Yankun Shi, Ruiwei Guo, Mengyang Den, Lan HuangAbstract:Research highlights: {yields} STIM1 and TRPC1 are expressed in EPCs. {yields} Knockdown of STIM1 inhibits the proliferation, migration and SOCE of EPCs. {yields} TRPC1-SOC cooperates with STIM1 to mediate the SOCE of EPCs. -- Abstract: Knockdown of Stromal Interaction Molecule 1 (STIM1) significantly suppresses neointima hyperplasia after vascular injury. Endothelial progenitor cells (EPCs) are the major source of cells that respond to endothelium repair and contribute to re-endothelialization by reducing neointima formation after vascular injury. We hypothesized that the effect of STIM1 on neointima hyperplasia inhibition is mediated through its effect on the biological properties of EPCs. In this study, we investigated the effects of STIM1 on the proliferation and migration of EPCs and examined the effect of STIM1 knockdown using cultured rat bone marrow-derived EPCs. STIM1 was expressed in EPCs, and knockdown of STIM1 by adenoviral delivery of small interfering RNA (siRNA) significantly suppressed the proliferation and migration of EPCs. Furthermore, STIM1 knockdown decreased store-operated channel entry 48 h after transfection. Replenishment with recombinant human STIM1 reversed the effects of STIM1 knockdown. Our data suggest that the store-operated transient receptor potential canonical 1 channel is involved in regulating the biological properties of EPCs through STIM1. STIM1 is a potentmore » regulator of cell proliferation and migration in rat EPCs and may play an important role in the biological properties of EPCs.« less
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knockdown of Stromal Interaction Molecule 1 attenuates hepatocyte growth factor induced endothelial progenitor cell proliferation
Experimental Biology and Medicine, 2010Co-Authors: Yankun Shi, Mingbao Song, Ruiwei Guo, Hong Wang, Pan Gao, Weibin Shi, Lan HuangAbstract:Increased Ca2+ entry through store-operated Ca2+ channels (SOCCs) plays an essential role in the regulation of hepatocyte growth factor (HGF)-induced cell proliferation. Stromal Interaction Molecule 1 (STIM1) is thought to transmit endoplasmic reticulum (ER) Ca2+ store depletion signals to the plasma membrane (PM), causing the opening of SOCCs in the PM. However, the relationship between HGF and STIM1 in endothelial progenitor cell (EPC) proliferation remains uncharacterized. The objective of this study was to evaluate the potential involvement of STIM1 in HGF-induced EPC proliferation. For this purpose, we used cultured rat bone marrow-derived EPCs and found that HGF-induced EPC proliferation at low concentrations. Store-operated Ca2+ entry (SOCE) was elevated in HGF-treated EPCs, and the SOCC inhibitors 2-aminoethoxydiphenyl borate (2-APB) and BTP-2 inhibited the HGF-induced proliferation response. Moreover, STIM1 mRNA and protein expression levels were increased in response to HGF stimulation and knock...
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an essential role for Stromal Interaction Molecule 1 in neointima formation following arterial injury
Cardiovascular Research, 2009Co-Authors: Ruiwei Guo, Yankun Shi, Mingbao Song, Hong Wang, Pan Gao, Chunyu Zeng, Jianfei Chen, Lan HuangAbstract:Aims There is evidence to suggest that Stromal Interaction Molecule 1 (STIM1) functions as a Ca2+ sensor on the endoplasmic reticulum, leading to transduction of signals to the plasma membrane and opening of store-operated Ca2+ channels (SOC). SOC have been detected in vascular smooth muscle cells (VSMCs) and are thought to have an essential role in the regulation of contraction and cell proliferation. We hypothesized that knockdown of STIM1 inhibits VSMC proliferation and suppresses neointimal hyperplasia. Methods and results We examined the effect of the knockdown of STIM1 using a rat balloon injury model and cultured rat aortic VSMCs. Interestingly, knockdown of rat STIM1 by adenovirus delivery of small interfering RNA (siRNA) significantly suppressed neointimal hyperplasia in a rat carotid artery balloon injury model at 14 days after injury. The re-expression of human STIM1 to smooth muscle reversed the effect of STIM1 knockdown on neointimal formation. Rat aortic VSMCs were used for the in vitro assays. Knockdown of endogenous STIM1 significantly inhibited proliferation and migration of VSMCs. Moreover, STIM1 knockdown induced cell-cycle arrest in G0/G1 and resulted in a marked decrease in SOC. Replenishment with recombinant human STIM1 reversed the effect of siRNA knockdown. These results suggest STIM1 has a critical role in neointimal formation in a rat model of vascular injury. Conclusion STIM1 may represent a novel therapeutic target in the prevention of restenosis after vascular interventions.
Le Zheng - One of the best experts on this subject based on the ideXlab platform.
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a coiled coil clamp controls both conformation and clustering of Stromal Interaction Molecule 1 stim1
Journal of Biological Chemistry, 2014Co-Authors: Marc Fahrner, Peter B. Stathopulos, Mitsuhiko Ikura, Martin Muik, Rainer Schindl, Carmen Butorac, Le Zheng, Isaac Jardin, Christoph RomaninAbstract:Abstract Store-operated Ca2+ entry, essential for the adaptive immunity, is initiated by the endoplasmic reticulum (ER) Ca2+ sensor STIM1. Ca2+ entry occurs through the plasma membrane resident Ca2+ channel Orai1 that directly interacts with the C-terminal STIM1 domain, named SOAR/CAD. Depletion of the ER Ca2+ store controls this STIM1/Orai1 Interaction via transition to an extended STIM1 C-terminal conformation, exposure of the SOAR/CAD domain, and STIM1/Orai1 co-clustering. Here we developed a novel approach termed FRET-derived Interaction in a Restricted Environment (FIRE) in an attempt to dissect the interplay of coiled-coil (CC) Interactions in controlling STIM1 quiescent as well as active conformation and cluster formation. We present evidence of a sequential activation mechanism in the STIM1 cytosolic domains where the Interaction between CC1 and CC3 segment regulates both SOAR/CAD exposure and CC3-mediated higher-order oligomerization as well as cluster formation. These dual levels of STIM1 auto-inhibition provide efficient control over the coupling to and activation of Orai1 channels.
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Stromal Interaction Molecule stim 1 and stim2 calcium sensing regions exhibit distinct unfolding and oligomerization
2009Co-Authors: Peter B. Stathopulos, Le Zheng, Mitsuhiko IkuraAbstract:Stromal InteractionMolecules (STIM) 1 and STIM2 are regulators of store-operated calcium (Ca2 ) entry as well as basal cytoplasmic Ca2 levels in human cells. Despite a high sequence similarity (>65%) and analogous sequence-based domain architectures, STIM1 and STIM2 differentially influence these phenomena. Among all eukaryotes, the endoplasmic reticulum luminal portion of STIM proteins minimally encode EF-hand and sterile -motif (SAM) domains (EF-SAM), which are responsible for sensing changes in Ca2 levels and initiating oligomerization. STIM oligomerization is a key induction step in the activation of Ca2 -permeable channels on the plasma membrane. Here, we show that the kinetic half-timeof conversion fromamonomeric to a steadyoligomeric state is>70 shorter for STIM1EF-SAMthan STIM2 under similar conditions. Urea-induced rates of unfolding forSTIM1EF-SAMare>3 quickerwhencomparedwithSTIM2, coherent with partial unfolding-coupled aggregation. Additionally, we demonstrate that the isoform-specificN-terminal residues beyond EF-SAMcan influence the stability of this region.We postulate that distinct oligomerization dynamics of STIM isoforms have evolved to adapt to differential roles inCa2 homeostasis and signaling.
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Stromal Interaction Molecule (STIM) 1 and STIM2 calcium sensing regions exhibit distinct unfolding and oligomerization kinetics.
The Journal of biological chemistry, 2008Co-Authors: Peter B. Stathopulos, Le Zheng, Mitsuhiko IkuraAbstract:Abstract Stromal Interaction Molecules (STIM) 1 and STIM2 are regulators of store-operated calcium (Ca2+) entry as well as basal cytoplasmic Ca2+ levels in human cells. Despite a high sequence similarity (>65%) and analogous sequence-based domain architectures, STIM1 and STIM2 differentially influence these phenomena. Among all eukaryotes, the endoplasmic reticulum luminal portion of STIM proteins minimally encode EF-hand and sterile α-motif (SAM) domains (EF-SAM), which are responsible for sensing changes in Ca2+ levels and initiating oligomerization. STIM oligomerization is a key induction step in the activation of Ca2+-permeable channels on the plasma membrane. Here, we show that the kinetic half-time of conversion from a monomeric to a steady oligomeric state is >70× shorter for STIM1 EF-SAM than STIM2 under similar conditions. Urea-induced rates of unfolding for STIM1 EF-SAM are >3× quicker when compared with STIM2, coherent with partial unfolding-coupled aggregation. Additionally, we demonstrate that the isoform-specific N-terminal residues beyond EF-SAM can influence the stability of this region. We postulate that distinct oligomerization dynamics of STIM isoforms have evolved to adapt to differential roles in Ca2+ homeostasis and signaling.
Joseph P Yuan - One of the best experts on this subject based on the ideXlab platform.
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molecular determinants mediating gating of transient receptor potential canonical trpc channels by Stromal Interaction Molecule 1 stim1
Journal of Biological Chemistry, 2014Co-Authors: Kyu Pil Lee, Shmuel Muallem, Seok Choi, Jeong Hee Hong, Malini Ahuja, Sarabeth Graham, Dong Min Shin, Joseph P YuanAbstract:Transient receptor potential canonical (TRPC) channels mediate a critical part of the receptor-evoked Ca(2+) influx. TRPCs are gated open by the endoplasmic reticulum Ca(2+) sensor STIM1. Here we asked which Stromal Interaction Molecule 1 (STIM1) and TRPC domains mediate the Interaction between them and how this Interaction is used to open the channels. We report that the STIM1 Orai1-activating region domain of STIM1 interacts with the TRPC channel coiled coil domains (CCDs) and that this Interaction is essential for opening the channels by STIM1. Thus, disruption of the N-terminal (NT) CCDs by triple mutations eliminated TRPC surface localization and reduced binding of STIM1 to TRPC1 and TRPC5 while increasing binding to TRPC3 and TRPC6. Single mutations in TRPC1 NT or C-terminal (CT) CCDs reduced Interaction and activation of TRPC1 by STIM1. Remarkably, single mutations in the TRPC3 NT CCD enhanced Interaction and regulation by STIM1. Disruption in the TRPC3 CT CCD eliminated regulation by STIM1 and the enhanced Interaction caused by NT CCD mutations. The NT CCD mutations converted TRPC3 from a TRPC1-dependent to a TRPC1-independent, STIM1-regulated channel. TRPC1 reduced the FRET between BFP-TRPC3 and TRPC3-YFP and between CFP-TRPC3-YFP upon stimulation. Accordingly, knockdown of TRPC1 made TRPC3 STIM1-independent. STIM1 dependence of TRPC3 was reconstituted by the TRPC1 CT CCD alone. Knockout of Trpc1 and Trpc3 similarly inhibited Ca(2+) influx, and inhibition of Trpc3 had no further effect on Ca(2+) influx in Trpc1(-/-) cells. Cell stimulation enhanced the formation of Trpc1-Stim1-Trpc3 complexes. These findings support a model in which the TRPC3 NT and CT CCDs interact to shield the CT CCD from Interaction with STIM1. The TRPC1 CT CCD dissociates this Interaction to allow the STIM1 Orai1-activating region within STIM1 access to the TRPC3 CT CCD and regulation of TRPC3 by STIM1. These studies provide evidence that the TRPC channel CCDs participate in channel gating.
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the ca2 sensor Stromal Interaction Molecule 1 stim1 is necessary and sufficient for the store operated ca2 entry function of transient receptor potential canonical trpc 1 and 4 channels in endothelial cells
Molecular Pharmacology, 2012Co-Authors: Premanand Sundivakkam, Joseph P Yuan, Marc Freichel, Vandana Singh, Stephen M Vogel, Veit Flockerzi, Asrar B Malik, Chinnaswamy TiruppathiAbstract:We addressed the requirement for Stromal Interaction Molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca2+-sensor, and Orai1, a Ca2+ selective channel, in regulating Ca2+ entry through the store-operated channels mouse transient receptor potential canonical (TRPC) 4 or human TRPC1. Studies were made using murine and human lung endothelial cells (ECs) challenged with thrombin known to induce Ca2+ entry via TRPC1/4. Deletion or knockdown of TRPC4 abolished Ca2+ entry secondary to depletion of ER Ca2+ stores, preventing the disruption of the endothelial barrier. Knockdown of STIM1 (but not of Orai1or Orai3) or expression of the dominant-negative STIM1K684E-K685E mutant in ECs also suppressed Ca2+ entry secondary to store depletion. Ectopic expression of WT-STIM1 or WT-Orai1 in TRPC4(−/−)-ECs failed to rescue Ca2+ entry; however, WT-TRPC4 expression in TRPC4(−/−)-ECs restored Ca2+ entry indicating the requirement for TRPC4 in mediating store-operated Ca2+ entry. Moreover, expression of the dominant-negative Orai1R91W mutant or Orai3E81W mutant in WT-ECs failed to prevent thrombin-induced Ca2+ entry. In contrast, expression of the dominant-negative TRPC4EE647-648KK mutant in WT-ECs markedly reduced thrombin-induced Ca2+ entry. In ECs expressing YFP-STIM1, ER-store Ca2+ depletion induced formation of fluorescent membrane puncta in WT but not in TRPC4(−/−) cells, indicating that mobilization of STIM1 and engagement of its Ca2+ sensing function required TRPC4 expression. Coimmunoprecipitation studies showed coupling of TRPC1 and TRPC4 with STIM1 on depletion of ER Ca2+ stores. Thus, TRPC1 and TRPC4 can interact with STIM1 to form functional store-operated Ca2+-entry channels, which are essential for mediating Ca2+ entry-dependent disruption of the endothelial barrier.
