BACKGROUND: Carbonic anhydrase IX (CA IX) is a tumor-associated, highly active, transmembrane carbonic anhydrase isoform regulated by hypoxia and implicated in pH control and adhesion-migration-invasion. CA IX ectodomain (ECD) is shed from the tumor cell surface to serum/plasma of patients, where it can signify cancer prognosis. We previously showed that the CA IX ECD release is mediated by disintegrin and metalloproteinase ADAM17. Here we investigated the CA IX ECD shedding in tumor cells undergoing apoptosis in response to cytotoxic drugs, including cycloheximide and doxorubicin. METHODS: Presence of cell surface CA IX was correlated to the extent of apoptosis by flow cytometry in cell lines with natural or ectopic CA IX expression. CA IX ECD level was assessed by ELISA using CA IX-specific monoclonal antibodies. Effect of recombinant CA IX ECD on the activation of molecular pathways was evaluated using the cell-based dual-luciferase reporter assay. RESULTS: We found a significantly lower occurrence of apoptosis in the CA IX-positive cell subpopulation than in the CA IX-negative one. We also demonstrated that the cell-surface CA IX level dropped during the death progress due to an increased ECD shedding, which required a functional ADAM17. Inhibitors of metalloproteinases reduced CA IX ECD shedding, but not apoptosis. The CA IX ECD release induced by cytotoxic drugs was connected to elevated expression of CA IX in the surviving fraction of cells. Moreover, an externally added recombinant CA IX ECD activated a pathway driven by the Nanog transcription factor implicated in epithelial-mesenchymal transition and stemness. CONCLUSIONS: These findings imply that the increased level of the circulating CA IX ECD might be useful as an indicator of an effective antitumor chemotherapy. Conversely, elevated CA IX ECD might generate unwanted effects through autocrine/paracrine signaling potentially contributing to resistance and tumor progression.

• Klíčová slova
• Apoptosis, Carbonic anhydrase IX, Chemotherapy, Ectodomain, Hypoxia, Metalloproteinase, Shedding,
• MeSH
• apoptóza účinky léků   genetika   MeSH
• cykloheximid aplikace a dávkování   MeSH
• epitelo-mezenchymální tranzice genetika   MeSH
• HeLa buňky MeSH
• hypoxie buňky genetika   MeSH
• karboanhydrasa IX aplikace a dávkování   genetika   metabolismus   MeSH
• lidé MeSH
• monoklonální protilátky aplikace a dávkování   MeSH
• nádory genetika   patologie   MeSH
• protein ADAM17 genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ADAM17 protein, human MeSH Prohlížeč
• cykloheximid MeSH
• karboanhydrasa IX MeSH
• monoklonální protilátky MeSH
• protein ADAM17 MeSH

The P2X7 receptor (P2X7R) is a member of the ATP-gated ion channel family that exhibits distinct electrophysiological and pharmacological properties. This includes low sensitivity to ATP, lack of desensitization, a sustained current growth during prolonged receptor stimulation accompanied with development of permeability to large organic cations, and the coupling of receptor activation to cell blebbing and death. The uniquely long C-terminus of P2X7R accounts for many of these receptor-specific functions. The aim of this study was to understand the role of conserved ectodomain cysteine residues in P2X7R function. Single- and double-point threonine mutants of C119-C168, C129-C152, C135-C162, C216-C226, and C260-C269 cysteine pairs were expressed in HEK293 cells and studied using whole-cell current recording. All mutants other than C119T-P2X7R responded to initial and subsequent application of 300-μM BzATP and ATP with small amplitude monophasic currents or were practically nonfunctional. The mutagenesis-induced loss of function was due to decreased cell-surface receptor expression, as revealed by assessing levels of biotinylated mutants. Coexpression of all double mutants with the wild-type receptor had a transient or, in the case of C119T/C168T double mutant, sustained inhibitory effect on receptor trafficking. The C119T-P2X7R mutant was expressed on the plasma membrane and was fully functional with a slight decrease in the sensitivity for BzATP, indicating that interaction of liberated Cys168 with another residue rescues the trafficking of receptor. Thus, in contrast to other P2XRs, all disulfide bonds of P2X7R are individually essential for the proper receptor trafficking.

• MeSH
• cystein biosyntéza   genetika   fyziologie   MeSH
• HEK293 buňky MeSH
• konzervovaná sekvence * MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• mutace fyziologie   MeSH
• purinergní receptory P2X7 genetika   metabolismus   MeSH
• transport proteinů fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• cystein MeSH
• purinergní receptory P2X7 MeSH

BACKGROUND: Carbonic anhydrase IX (CA IX) is a hypoxia-induced enzyme regulating tumour pH and facilitating cell migration/invasion. It is primarily expressed as a transmembrane cell-surface protein, but its ectodomain can be shed by ADAM17 to extracellular space. This study aims to elucidate the impact of CA IX shedding on cancer cells. METHODS: We generated a non-shed CA IX mutant by deletion of amino acids 393-402 from the stalk region and studied its phenotypic effects compared to full-length, shedding-competent CA IX using a range of assays based on immunodetection, confocal microscopy, in vitro real-time cell monitoring and in vivo tumour cell inoculation using xenografted NMRI and C57BL/6J female mice. RESULTS: We demonstrated that the impairment of shedding does not alter the ability of CA IX to bind ADAM17, internalise, form oligomers and regulate pH, but induces cancer-promoting changes in extracellular proteome. Moreover, it affects intrinsic properties of cells expressing the non-shed variant, in terms of their increased ability to migrate, generate primary tumours and form metastatic lesions in lungs. CONCLUSIONS: Our results show that the ectodomain shedding controls pro-tumorigenic and pro-metastatic roles of the cell-associated CA IX and suggest that this phenomenon should be considered when developing CA IX-targeted therapeutic strategies.

• MeSH
• fenotyp MeSH
• invazivní růst nádoru patologie   MeSH
• karboanhydrasa IX metabolismus   MeSH
• karcinogeneze metabolismus   patologie   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory metabolismus   patologie   MeSH
• protein ADAM17 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• karboanhydrasa IX MeSH
• protein ADAM17 MeSH

Mammalian P2X receptors contain 10 conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). Here, we analyzed the relevance of these disulfide pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin, an allostenic modulator of these channels. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in decreased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor function was not affected when the 217 cysteine was replaced with threonine or arginine or when the 261 cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating.

• MeSH
• cystein chemie   genetika   MeSH
• gating iontového kanálu fyziologie   MeSH
• HEK293 buňky MeSH
• konzervovaná sekvence MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• purinergní receptory P2X4 chemie   genetika   metabolismus   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• cystein MeSH
• purinergní receptory P2X4 MeSH

Lectin-like transcript 1 (LLT1, gene clec2d) was identified to be a ligand for the single human NKR-P1 receptor present on NK and NK-T lymphocytes. Naturally, LLT1 is expressed on the surface of NK cells, stimulating IFN-γ production, and is up-regulated upon activation of other immune cells, e.g. TLR-stimulated dendritic cells and B cells or T cell receptor-activated T cells. While in normal tissues LLT1:NKR-P1 interaction (representing an alternative "missing-self" recognition system) play an immunomodulatory role in regulation of crosstalk between NK and antigen presenting cells, LLT1 is upregulated in glioblastoma cells, one of the most lethal tumors, where it acts as a mediator of immune escape of glioma cells. Here we report transient expression and characterization of soluble His176Cys mutant of LLT1 ectodomain in an eukaryotic expression system of human suspension-adapted HEK293S GnTI(-) cell line with uniform N-glycans. The His176Cys mutation is critical for C-type lectin-like domain stability, leading to the reconstruction of third canonical disulfide bridge in LLT1, as shown by mass spectrometry. Purified soluble LLT1 is homogeneous, deglycosylatable and forms a non-covalent homodimer whose dimerization is not dependent on presence of its N-glycans. As a part of production of soluble LLT1, we have adapted HEK293S GnTI(-) cell line to growth in suspension in media facilitating transient transfection and optimized novel high cell density transfection protocol, greatly enhancing protein yields. This transfection protocol is generally applicable for protein production within this cell line, especially for protein crystallography.

• Klíčová slova
• C-type lectin-like, Glycosylation, HEK293S GnTI(−), LLT1, NK cell, Transfection,
• MeSH
• buňky NK metabolismus   MeSH
• disulfidy metabolismus   MeSH
• DNA metabolismus   MeSH
• glykosylace MeSH
• HEK293 buňky MeSH
• krystalizace MeSH
• lektiny typu C chemie   izolace a purifikace   metabolismus   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• N-acetylglukosaminyltransferasy metabolismus   MeSH
• polyethylenimin chemie   MeSH
• polysacharidy metabolismus   MeSH
• rozpustnost MeSH
• roztoky MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• stabilita proteinů MeSH
• terciární struktura proteinů MeSH
• transfekce metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• disulfidy MeSH
• DNA MeSH
• lektiny typu C MeSH
• N-acetylglukosaminyltransferasy MeSH
• polyethylenimin MeSH
• polysacharidy MeSH
• roztoky MeSH

Basic fibroblast growth factor (FGF-2) functions as a natural inducer of mesoderm, regulator of cell differentiation and autocrine modulator of cell growth and transformation. The FGF-2 signals are transduced through receptors with intrinsic protein tyrosine kinase activity. However, receptor binding and activation is governed by extracellular matrix, cell surface or soluble proteoglycans. This paper focuses on the role of proteoglycans synthesized by embryonic cells, embryoglycans, in FGF-2 signaling via FGF receptor-1 (FGFR-1). We found that embryoglycan ectodomain Lewis X, analog of developmentally regulated embryonic cell surface epitope TEC 1, promotes oligomerization of FGF-2 in the cell free chemical crosslinking. In vitro assays show that a large molar excess of extracellular Lewis X does not inhibit binding of FGF-2 to embryonic stem (ES) cells, but prevents the mitogenic effect of FGF-2. Western blot analysis of ES cells revealed the presence of abundant 52 kDa and trace amounts of 67 and 125 kDa isoforms of FGFR-1. However, none of these isoforms undergo any detectable changes in tyrosine phosphorylation under the conditions that modulate the mitogenic effect of FGF-2. Rather, a primary substrate of all receptor tyrosine kinases, phospholipase C gamma (PLC gamma), is activated by both FGF-2 and Lewis X. The combination, FGF-2 plus Lewis X, leads to weak inhibition, when compared with the effects of FGF-2 and Lewis X, respectively. In accordance, the level of phosphorylation of non-receptor tyrosine kinase c-Src is reduced in a reversed pattern to PLC(gamma). Furthermore, in this particular cell type we show the presence of activated forms of extracellular signal-related kinase (ERK) in all nontreated and treated cells. These findings demonstrate that embryoglycan ectodomains may act as negative regulators of FGF-2-induced ES cell proliferation, most likely through the FGFR-1-independent signaling pathway.

• MeSH
• antigen Lewis X metabolismus   farmakologie   MeSH
• buněčné dělení účinky léků   MeSH
• buněčné linie MeSH
• fibroblastový růstový faktor 2 chemie   metabolismus   farmakologie   MeSH
• fosfolipasa C gama MeSH
• fosfolipasy typu C metabolismus   MeSH
• fosforylace MeSH
• izoenzymy metabolismus   MeSH
• kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• myši MeSH
• polysacharidy metabolismus   MeSH
• proteoglykany metabolismus   MeSH
• reagencia zkříženě vázaná MeSH
• receptor fibroblastových růstových faktorů, typ 1 MeSH
• receptory fibroblastových růstových faktorů metabolismus   MeSH
• signální transdukce MeSH
• skupina kinas odvozených od src-genu metabolismus   MeSH
• tyrosinkinasové receptory * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigen Lewis X MeSH
• embryoglycan MeSH Prohlížeč
• Fgfr1 protein, mouse MeSH Prohlížeč
• fibroblastový růstový faktor 2 MeSH
• fosfolipasa C gama MeSH
• fosfolipasy typu C MeSH
• izoenzymy MeSH
• polysacharidy MeSH
• proteoglykany MeSH
• reagencia zkříženě vázaná MeSH
• receptor fibroblastových růstových faktorů, typ 1 MeSH
• receptory fibroblastových růstových faktorů MeSH
• skupina kinas odvozených od src-genu MeSH
• tyrosinkinasové receptory * MeSH

Several membrane-anchored signal mediators such as cytokines (e.g. TNFα) and growth factors are proteolytically shed from the cell surface by the metalloproteinase ADAM17, which, thus, has an essential role in inflammatory and developmental processes. The membrane proteins iRhom1 and iRhom2 are instrumental for the transport of ADAM17 to the cell surface and its regulation. However, the structure-function determinants of the iRhom-ADAM17 complex are poorly understood. We used AI-based modelling to gain insights into the structure-function relationship of this complex. We identified different regions in the iRhom homology domain (IRHD) that are differentially responsible for iRhom functions. We have supported the validity of the predicted structure-function determinants with several in vitro, ex vivo and in vivo approaches and demonstrated the regulatory role of the IRHD for iRhom-ADAM17 complex cohesion and forward trafficking. Overall, we provide mechanistic insights into the iRhom-ADAM17-mediated shedding event, which is at the centre of several important cytokine and growth factor pathways.

• Klíčová slova
• ADAM17, EGFR ligand release iRhom–ADAM17 complex structure, Ectodomain shedding, TNF signalling, iRhom, iRhom homology domain,
• MeSH
• buněčná membrána metabolismus   MeSH
• cytokiny metabolismus   MeSH
• membránové proteiny * metabolismus   MeSH
• modely strukturální MeSH
• protein ADAM17 metabolismus   MeSH
• transportní proteiny * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokiny MeSH
• membránové proteiny * MeSH
• protein ADAM17 MeSH
• transportní proteiny * MeSH

Ivermectin (IVM) applied extracellularly increases the sensitivity of P2X4 receptor (P2X4R) to ATP, enhances the maximum current amplitudes, and greatly prolongs the deactivation kinetics. In this manuscript, we focused on identification of receptor-specific residues responsible for IVM effects on channel gating using the wild-type rat homomeric P2X4R, several chimeric P2X2/P2X4 receptors, and single-point P2X4R-specific mutants in the ectodomain and two transmembrane domains. Experiments with chimeric receptors revealed that the Val49-Val61 but not the Val64-Tyr315 ectodomain sequence is important for the effects of IVM on channel deactivation. Receptor-specific mutations placed in the Gly29-Val61 and Asp338-Leu358 regions showed the importance of Trp50, Val60, and Val357 residues in IVM regulation of the rate of channel deactivation, but not on the maximum current amplitude. These results suggest that the transmembrane domains and the nearby ectodomain region contribute to the effects of IVM on channel deactivation.

• MeSH
• adenosintrifosfát chemie   MeSH
• antiparazitární látky farmakologie   MeSH
• ivermektin chemie   farmakologie   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• purinergní receptory P2 chemie   MeSH
• purinergní receptory P2X4 MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• tryptofan chemie   MeSH
• valin chemie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• adenosintrifosfát MeSH
• antiparazitární látky MeSH
• ivermektin MeSH
• P2RX4 protein, human MeSH Prohlížeč
• P2rx4 protein, rat MeSH Prohlížeč
• purinergní receptory P2 MeSH
• purinergní receptory P2X4 MeSH
• tryptofan MeSH
• valin MeSH

Membrane-tethered signalling proteins such as TNFα and many EGF receptor ligands undergo shedding by the metalloproteinase ADAM17 to get released. The pseudoproteases iRhom1 and iRhom2 are important for the transport, maturation and activity of ADAM17. Yet, the structural and functional requirements to promote the transport of the iRhom-ADAM17 complex have not yet been thoroughly investigated. Utilising in silico and in vitro methods, we here map the conserved iRhom homology domain (IRHD) and provide first insights into its structure and function. By focusing on iRhom2, we identified different structural and functional factors within the IRHD. We found that the structural integrity of the IRHD is a key factor for ADAM17 binding. In addition, we identified a highly conserved motif within an unstructured region of the IRHD, that, when mutated, restricts the transport of the iRhom-ADAM17 complex through the secretory pathway in in vitro, ex vivo and in vivo systems and also increases the half-life of iRhom2 and ADAM17. Furthermore, the disruption of this IRHD motif was also reflected by changes in the yet undescribed interaction profile of iRhom2 with proteins involved in intracellular vesicle transport. Overall, we provide the first insights into the forward trafficking of iRhoms which is critical for TNFα and EGF receptor signalling.

• Klíčová slova
• ADAM17, Ectodomain shedding, Growth factors, TNF, iRhom, iRhom homology domain,
• MeSH
• aminokyselinové motivy MeSH
• buněčné linie MeSH
• epidermální růstové faktory metabolismus   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• mutageneze MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• poločas MeSH
• protein ADAM17 chemie   metabolismus   MeSH
• proteinové domény MeSH
• RNA interference MeSH
• signální transdukce MeSH
• TNF-alfa metabolismus   MeSH
• transport proteinů MeSH
• transportní proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• epidermální růstové faktory MeSH
• iRhom1 protein, mouse MeSH Prohlížeč
• iRhom2 protein, mouse MeSH Prohlížeč
• malá interferující RNA MeSH
• membránové proteiny MeSH
• protein ADAM17 MeSH
• TNF-alfa MeSH
• transportní proteiny MeSH

A disintegrin and metalloproteinase 10 (ADAM10) plays a major role in the ectodomain shedding of important surface molecules with physiological and pathological relevance including the amyloid precursor protein (APP), the cellular prion protein, and different cadherins. Despite its therapeutic potential, there is still a considerable lack of knowledge how this protease is regulated. We have previously identified tetraspanin15 (Tspan15) as a member of the TspanC8 family to specifically associate with ADAM10. Cell-based overexpression experiments revealed that this binding affected the maturation process and surface expression of the protease. Our current study shows that Tspan15 is abundantly expressed in mouse brain, where it specifically interacts with endogenous ADAM10. Tspan15 knockout mice did not reveal an overt phenotype but showed a pronounced decrease of the active and mature form of ADAM10, an effect which augmented with aging. The decreased expression of active ADAM10 correlated with an age-dependent reduced shedding of neuronal (N)-cadherin and the cellular prion protein. APP α-secretase cleavage and the expression of Notch-dependent genes were not affected by the loss of Tspan15, which is consistent with the hypothesis that different TspanC8s cause ADAM10 to preferentially cleave particular substrates. Analyzing spine morphology revealed no obvious differences between Tspan15 knockout and wild-type mice. However, Tspan15 expression was elevated in brains of an Alzheimer's disease mouse model and of patients, suggesting that upregulation of Tspan15 expression reflects a cellular response in a disease state. In conclusion, our data show that Tspan15 and most likely also other members of the TspanC8 family are central modulators of ADAM10-mediated ectodomain shedding in vivo.

• Klíčová slova
• ADAM10, N-cadherin, Prion protein, Tetraspanin,
• MeSH
• Alzheimerova nemoc genetika   metabolismus   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• mozek metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• neurony metabolismus   MeSH
• protein ADAM10 genetika   metabolismus   MeSH
• regulace genové exprese * MeSH
• stanovení celkové genové exprese metody   MeSH
• synapse metabolismus   MeSH
• tetraspaniny genetika   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein ADAM10 MeSH
• tetraspaniny MeSH