stromal interaction molecule 1
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SDF-1, nový cytokin z rodiny a-chemokinů, hraje klíčovou roli v regulaci hematopoézy. Vyskytuje se ve dvou formách (alfa a beta), které vznikají cestou alternativního splicingu. Jeho vysoká exprese v mikroprostředí kostní dřeně je zodpovědná za uvolňování progenitorových buněk do cirkulace a představuje prevenci nekontrolovaného úniku CD34+ buněk. Zvláště významná je jeho stimulace proliferace progenitorů B-řady, u ostatních krevních řad se projevuje jako senzibilizují faktor vůči jiným cytokinům. Schopnost indukovat agregaci trombocytů odhaluje roli SDF-1 v trombogenezi a obliteraci lumina cév postižených aterosklerózou. Výlučným receptorem pro SDF-1 je CXCR4, jehož přítomnost byla prokázána v řadě tkání a orgánů. Jejich přítomnost byla potvrzena i v mozkových tumorech, přičemž míra jejich exprese stoupá s gradingem, angiogenezí a výskytem nekrotických změn v tumoru. Díky této vlastnosti bude pravděpodobně možné určovat i prognózu pacientů. SDF-1 je také supresorem imunitní odpovědi skrze jeho facilitující účinek na interakci makrofágů s CD8+ T-lymfocyty. Afinita T-lymfocytotropního HIV k CXCR4 dává naději na možné ovlivnění infekce pomocí SDF-1. Význam SDF-1 a jeho receptoru CXCR4 potvrzují morfologické a funkční odchylky novorozených myší při jejich absenci, zvl. poruchy hematopoézy, angiogeneze a vývoje srdeční a nervové tkáně.
SDF-1, a novel cytokine from a-chemokine family, plays a key role in regulation of haematopoiesis. It exists in two forms (alpha and beta) that originate from alternative splicing. Its high expression in the bone marrow microenvironment accounts for the release of progenitor cells in the circulation and represents a prevention of uncontrolled leak of CD34+ cells. Notably significant is its stimulation of proliferation of B-lineage progenitors, in other haematopoietic lineages it functions as a facilitating factor of other cytokines. Ability of induction of platelet aggregation reveales the role of SDF-1 in thrombogenesis and vascular lumen obliteration in vessels affected by atherosclerosis. The only receptor for SDF-1 in thrombogenesis and vascular lumen obliteration in vessels affected by atherosclerosis. The only receptor for SDF-1 is CXCR, whose presence was proved in great numbers of tissues and organs. Their presence was also verified in brain tumours, whereas degree of their expression raises with grading, angiogenesis and occurrence of necrotic changes in tumour. Thanks to this feature it will probably be possible to estimate the prognosis of the patients. SDF-1 is also a suppressor of immune response via its facilitating activity on the interaction of the macrophages and CD8+ T lymphocytes. Affinity of the T-lymphocytotropic HIV to CXCR4 holds out hopes for a possible modulation of the infection with SDF-1. The significance of SDF-1 and its receptor CXCR4 is supported by morphological and functional abnormalities of new-born mice in their absence, especially disorders in haematopoiesis, angiogenesis and development of cardiac and nervous tissues.
The family of stromal interaction molecules (STIM) includes two widely expressed single-pass endoplasmic reticulum (ER) transmembrane proteins and additional splice variants that act as precise ER-luminal Ca2+ sensors. STIM proteins mainly function as one of the two essential components of the so-called Ca2+ release-activated Ca2+ (CRAC) channel. The second CRAC channel component is constituted by pore-forming Orai proteins in the plasma membrane. STIM and Orai physically interact with each other to enable CRAC channel opening, which is a critical prerequisite for various downstream signalling pathways such as gene transcription or proliferation. Their activation commonly requires the emptying of the intracellular ER Ca2+ store. Using their Ca2+ sensing capabilities, STIM proteins confer this Ca2+ content-dependent signal to Orai, thereby linking Ca2+ store depletion to CRAC channel opening. Here we review the conformational dynamics occurring along the entire STIM protein upon store depletion, involving the transition from the quiescent, compactly folded structure into an active, extended state, modulation by a variety of accessory components in the cell as well as the impairment of individual steps of the STIM activation cascade associated with disease.
Rituximab improves the outcome of patients with non-Hodgkin lymphoma, but does not completely eradicate residual B-cell populations in the microenvironment of the bone marrow and lymph nodes. Adhesion to stromal cells can protect B-cells from apoptosis induced by chemotherapy drugs [(cell adhesion-mediated drug resistance (CAM-DR)]. A similar mechanism of resistance to rituximab has not, to our knowledge, been described. We tested the hypothesis that the microenvironment protects malignant B-cells from rituximab-induced apoptosis, and that blocking these interactions with natalizumab, an antibody targeting VLA-4 (integrin alfa-4-beta-1/CD49d), can overcome this protection. VLA-4 is an adhesion molecule constitutively expressed on malignant B-cells and is important for pro-survival signalling in the bone marrow and lymph node microenvironment. The human bone marrow stromal cell line HS-5 was shown to strongly protect B-cell lymphoma cells from rituximab cytotoxicity, suggesting the existence of a stromal cell adhesion-mediated antibody resistance (CAM-AR) mechanism analogous to CAM-DR. Natalizumab decreased B-lymphocyte adherence to fibronectin by 75-95% and partially overcame stromal protection against rituximab and cytotoxic drugs. These pre-clinical findings suggest that the addition of stromal adhesion-disruptive drugs to rituximab-containing therapy could improve treatment efficacy.
- MeSH
- apoptóza * fyziologie účinky léků MeSH
- B-buněčný lymfom farmakoterapie patologie MeSH
- B-lymfocyty fyziologie účinky léků MeSH
- buněčná adheze účinky léků MeSH
- buňky kostní dřeně fyziologie MeSH
- buňky stromatu * fyziologie MeSH
- chemorezistence účinky léků MeSH
- humanizované monoklonální protilátky farmakologie MeSH
- integrin alfa4beta1 antagonisté a inhibitory fyziologie MeSH
- kokultivační techniky MeSH
- lidé MeSH
- myší monoklonální protilátky * farmakologie MeSH
- nádorové buňky kultivované MeSH
- nádorové mikroprostředí účinky léků MeSH
- natalizumab MeSH
- protinádorové látky * farmakologie MeSH
- rituximab MeSH
- Check Tag
- lidé MeSH
Cardiac fibrotization is a well-known process characteristic of many cardiac pathological conditions. The key element is excessive activation of cardiac fibroblasts, their transdifferentiation into myofibroblasts, increased production, and accumulation of extracellular matrix proteins, resulting in cardiac stiffness. The exact cellular mechanisms and molecular components involved in the process are not fully elucidated, but the SOCE mechanism could play an important role. Its key molecules are the molecular sensor of calcium in ER/SR - STIM and the highly selective calcium channels Orai located in the plasma membrane. This study aims to evaluate selected SOCE-associated genes in the activation of HCF cell culture by several known substances (phenylephrine, isoprenaline) that represent cardiovascular overload. After cell cultivation, cell medium was collected to measure the soluble collagen content. From the harvested cells, qRT-PCR was performed to determine the mRNA levels of the corresponding genes. The activation of cells was based on changes in the relative expression of collagen genes as well as the collagen content in the medium of the cell culture. We detected an increase in the expression of the Orai2 isoform, a change in the Orai1/Orai3 ratio and also an increase in the expression of the STIM2 isoform. These results suggest an increased activation of the SOCE mechanism under stress conditions of fibroblasts, which supports the hypothesis of fibroblast activation in pathological processes by altering calcium homeostasis through the SOCE mechanism.
The stromal interaction molecule 1 (STIM1) has two important functions, Ca2+ sensing within the endoplasmic reticulum and activation of the store-operated Ca2+ channel Orai1, enabling plasma-membrane Ca2+ influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca2+-dependent conformations of the luminal STIM1 domain upon activation. Furthermore, we identified the residues within the canonical and noncanonical EF-hand domains that can bind to multiple Ca2+ ions. In MD simulations, a single Ca2+ ion was sufficient to stabilize the luminal STIM1 complex. Ca2+ store depletion destabilized the two EF hands, triggering disassembly of the hydrophobic cleft that they form together with the stable SAM domain. Point mutations associated with tubular aggregate myopathy or cancer that targeted the canonical EF hand, and the hydrophobic cleft yielded constitutively clustered STIM1, which was associated with activation of Ca2+ entry through Orai1 channels. On the basis of our results, we present a model of STIM1 Ca2+ binding and refine the currently known initial steps of STIM1 activation on a molecular level.
- MeSH
- algoritmy MeSH
- buněčná membrána metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- HEK293 buňky MeSH
- hydrofobní a hydrofilní interakce MeSH
- konfokální mikroskopie MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- motivy EF-ruky MeSH
- mutace MeSH
- nádorové buněčné linie MeSH
- nádorové proteiny chemie genetika metabolismus MeSH
- protein ORAI1 chemie metabolismus MeSH
- protein STIM1 chemie genetika metabolismus MeSH
- proteinové domény * MeSH
- rozbalení proteinů * MeSH
- simulace molekulární dynamiky * MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Highly Ca2+ selective channels trigger a large variety of cellular signaling processes in both excitable and non-excitable cells. Among these channels, the Orai channel is unique in its activation mechanism and its structure. It mediates Ca2+ influx into the cytosol with an extremely small unitary conductance over longer time-scales, ranging from minutes up to several hours. Its activation is regulated by the Ca2+ content of the endoplasmic reticulum (ER). Depletion of luminal [Ca2+]ER is sensed by the STIM1 single transmembrane protein that directly binds and gates the Orai1 channel. Orai mediated Ca2+ influx increases cytosolic Ca2+ from 100 nM up to low micromolar range close to the pore and thereby forms Ca2+ microdomains. Hence, these features of the Orai channel can trigger long-term signaling processes without affecting the overall Ca2+ content of a single living cell. Here we focus on the architecture and dynamic conformational changes within the Orai channel. This review summarizes current achievements of molecular dynamics simulations in combination with live cell recordings to address gating and permeation of the Orai channel with molecular precision.
The store-operated calcium channels Orai1-3 form extraordinary long and funnel like pores, in stark contrast to a classical pore loop architecture. A hydrophobic segment centrally located in the Orai pore controls gating. Here, we comment on a recent work that describes decisive binding between three residues that controls the open and closed conformation of Orai channels.
Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca2+ sensor protein that induces permeation of Orai Ca2+ channels upon endoplasmic reticulum Ca2+-store depletion. A drop in luminal Ca2+ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca2+ depletion from our molecular dynamics (MD) simulations with a recent 2D NMR structure. Simulation- and structure-based results showed unfolding of two α-helices in the canonical and in the non-canonical EF-hand. Further, we structurally and functionally evaluated mutations in the non-canonical EF-hand that have been shown to cause tubular aggregate myopathy. We found these mutations to cause full constitutive activation of Ca2+-release-activated Ca2+ currents (ICRAC) and to promote autophagic processes. Specifically, heterologously expressed STIM1 mutations in the non-canonical EF-hand promoted translocation of the autophagy transcription factors microphthalmia-associated transcription factor (MITF) and transcription factor EB (TFEB) into the nucleus. These STIM1 mutations additionally stimulated an enhanced production of autophagosomes. In summary, mutations in STIM1 that cause structural unfolding promoted Ca2+ down-stream activation of autophagic processes.
- MeSH
- autofagie * MeSH
- kationty dvojmocné metabolismus MeSH
- konformace proteinů, alfa-helix MeSH
- lidé MeSH
- motivy EF-ruky MeSH
- mutace MeSH
- myopatie strukturální vrozené genetika metabolismus MeSH
- nádorové proteiny chemie genetika metabolismus MeSH
- protein STIM1 chemie genetika metabolismus MeSH
- rozbalení proteinů MeSH
- simulace molekulární dynamiky MeSH
- vápník metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Galectin-3 (Gal-3) is a β-galactoside-binding protein that influences various cell functions, including cell adhesion. We focused on the role of Gal-3 as an extracellular ligand mediating cell-matrix adhesion. We used human adipose tissue-derived stem cells and human umbilical vein endothelial cells that are promising for vascular tissue engineering. We found that these cells naturally contained Gal-3 on their surface and inside the cells. Moreover, they were able to associate with exogenous Gal-3 added to the culture medium. This association was reduced with a β-galactoside LacdiNAc (GalNAcβ1,4GlcNAc), a selective ligand of Gal-3, which binds to the carbohydrate recognition domain (CRD) in the Gal-3 molecule. This ligand was also able to detach Gal-3 newly associated with cells but not Gal-3 naturally present on cells. In addition, Gal-3 preadsorbed on plastic surfaces acted as an adhesion ligand for both cell types, and the cell adhesion was resistant to blocking with LacdiNAc. This result suggests that the adhesion was mediated by a binding site different from the CRD. The blocking of integrin adhesion receptors on cells with specific antibodies revealed that the cell adhesion to the preadsorbed Gal-3 was mediated, at least partially, by β1 and αV integrins-namely α5β1, αVβ3, and αVβ1 integrins.
- MeSH
- buněčná adheze * MeSH
- endoteliální buňky pupečníkové žíly (lidské) cytologie fyziologie MeSH
- galektiny metabolismus MeSH
- integriny metabolismus MeSH
- krevní proteiny metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- mezenchymální kmenové buňky cytologie fyziologie MeSH
- spoje buňka-matrix metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
We report a new NMR-scale purification procedure for two recombinant wild type fragments of the stromal interaction molecule 1 (STIM1). This protein acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol accumulating at ER - plasma membrane (PM) junctions upon calcium store depletion ultimately leading to activation of the Orai/CRAC channel. The functionally relevant cytosolic part of STIM1 consists of three coiled coil domains, which are mainly involved in intra- and inter-molecular homomeric interactions as well as coupling to and gating of CRAC channels. The optimized one-step rapid purification procedure for two 15N,13C isotope-labeled cytosolic coiled coil fragments, which avoids the problems of previous approaches. The high yields of soluble well folded 15N,13C isotope-labeled cytosolic coiled coil fragments followed by detergent screening provide for initial NMR characterization of these domains. The longer 30.5 kDa fragment represents the largest STIM1 wild type fragment that has been recombinantly prepared and characterized in solution without need for mutation or refolding.
- MeSH
- chromatografie afinitní MeSH
- dynamický rozptyl světla MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- izotopové značení MeSH
- izotopy dusíku chemie izolace a purifikace MeSH
- izotopy uhlíku chemie izolace a purifikace MeSH
- lidé MeSH
- nádorové proteiny chemie izolace a purifikace MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- protein STIM1 chemie izolace a purifikace MeSH
- proteinové domény MeSH
- rekombinantní proteiny chemie izolace a purifikace MeSH
- rozpustnost MeSH
- sbalování proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
