Styrene-maleic acid (SMA) and similar amphiphilic copolymers are known to cut biological membranes into lipid nanoparticles/nanodiscs containing membrane proteins apparently in their relatively native membrane lipid environment. Our previous work demonstrated that membrane raft microdomains resist such disintegration by SMA. The use of SMA in studying membrane proteins is limited by its heterogeneity and the inability to prepare defined derivatives. In the present paper, we demonstrate that some amphiphilic peptides structurally mimicking SMA also similarly disintegrate cell membranes. In contrast to the previously used copolymers, the simple peptides are structurally homogeneous. We found that their membrane-disintegrating activity increases with their length (reaching optimum at 24 amino acids) and requires a basic primary structure, that is, (XXD)n, where X represents a hydrophobic amino acid (optimally phenylalanine), D aspartic acid, and n is the number of repeats of these triplets. These peptides may provide opportunities for various well-defined potentially useful modifications in the study of membrane protein biochemistry. Our present results confirm a specific character of membrane raft microdomains.

• Klíčová slova
• leukocyte, lipid raft, lymphocyte, membrane, membrane proteins, peptides,
• MeSH
• buněčná membrána metabolismus   chemie   MeSH
• buněčné linie MeSH
• lidé MeSH
• maleáty chemie   MeSH
• membránové mikrodomény metabolismus   chemie   MeSH
• membránové proteiny * chemie   metabolismus   MeSH
• peptidy * chemie   MeSH
• polystyreny chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The RLTPR cytosolic protein, also known as CARMIL2, is essential for CD28 co-stimulation in mice, but its importance in human T cells and mode of action remain elusive. Here, using affinity purification followed by mass spectrometry analysis, we showed that RLTPR acts as a scaffold, bridging CD28 to the CARD11/CARMA1 cytosolic adaptor and to the NF-κB signaling pathway, and identified proteins not found before within the CD28 signaling pathway. We further demonstrated that RLTPR is essential for CD28 co-stimulation in human T cells and that its noncanonical pleckstrin-homology domain, leucine-rich repeat domain, and proline-rich region were mandatory for that task. Although RLTPR is thought to function as an actin-uncapping protein, this property was dispensable for CD28 co-stimulation in both mouse and human. Our findings suggest that the scaffolding role of RLTPR predominates during CD28 co-stimulation and underpins the similar function of RLTPR in human and mouse T cells. Along that line, the lack of functional RLTPR molecules impeded the differentiation toward Th1 and Th17 fates of both human and mouse CD4+ T cells. RLTPR was also expressed in both human and mouse B cells. In the mouse, RLTPR did not play, however, any detectable role in BCR-mediated signaling and T cell-independent B cell responses.

• MeSH
• aminokyselinové motivy MeSH
• antigeny CD28 metabolismus   MeSH
• biologické modely MeSH
• buňky NK metabolismus   MeSH
• dendritické buňky metabolismus   MeSH
• endocytóza MeSH
• genový targeting MeSH
• HEK293 buňky MeSH
• Jurkat buňky MeSH
• lidé MeSH
• lymfocyty metabolismus   MeSH
• mapování interakce mezi proteiny MeSH
• mikrofilamentové proteiny chemie   metabolismus   MeSH
• multimerizace proteinu MeSH
• mutace genetika   MeSH
• myeloidní buňky metabolismus   MeSH
• myši MeSH
• proteinové domény MeSH
• proteomika MeSH
• regulační T-lymfocyty metabolismus   MeSH
• signální transdukce MeSH
• T-lymfocyty metabolismus   MeSH
• thymocyty metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD28 MeSH
• CARMIL1 protein, human MeSH Prohlížeč
• Carmil1 protein, mouse MeSH Prohlížeč
• mikrofilamentové proteiny MeSH

Transmembrane adaptor proteins (TRAPs) are important organisers for the transduction of immunoreceptor-mediated signals. Prr7 is a TRAP that regulates T cell receptor (TCR) signalling and potently induces cell death when overexpressed in human Jurkat T cells. Whether endogenous Prr7 has a similar functional role is currently unknown. To address this issue, we analysed the development and function of the immune system in Prr7 knockout mice. We found that loss of Prr7 partially impairs development of single positive CD4+ T cells in the thymus but has no effect on the development of other T cell subpopulations, B cells, NK cells, or NKT cells. Moreover, Prr7 does not affect the TCR signalling pathway as T cells derived from Prr7 knockout and wild-type animals and stimulated in vitro express the same levels of the activation marker CD69, and retain their ability to proliferate and activate induced cell death programs. Importantly, Prr7 knockout mice retained the capacity to mount a protective immune response when challenged with Listeria monocytogenes infection in vivo. In addition, T cell effector functions (activation, migration, and reactivation) were normal following induction of experimental autoimmune encephalomyelitis (EAE) in Prr7 knockout mice. Collectively, our work shows that loss of Prr7 does not result in a major immune system phenotype and suggests that Prr7 has a dispensable function for TCR signalling.

• Publikační typ
• časopisecké články MeSH

Non-T cell activation linker (NTAL; also called LAB or LAT2) is a transmembrane adaptor protein that is expressed in a subset of hematopoietic cells, including mast cells. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FcεRI) signaling. Studies carried out on mast cells derived from mice with NTAL knock out (KO) and wild type mice suggested that NTAL is a negative regulator of FcεRI signaling, while experiments with RNAi-mediated NTAL knockdown (KD) in human mast cells and rat basophilic leukemia cells suggested its positive regulatory role. To determine whether different methodologies of NTAL ablation (KO vs KD) have different physiological consequences, we compared under well defined conditions FcεRI-mediated signaling events in mouse bone marrow-derived mast cells (BMMCs) with NTAL KO or KD. BMMCs with both NTAL KO and KD exhibited enhanced degranulation, calcium mobilization, chemotaxis, tyrosine phosphorylation of LAT and ERK, and depolymerization of filamentous actin. These data provide clear evidence that NTAL is a negative regulator of FcεRI activation events in murine BMMCs, independently of possible compensatory developmental alterations. To gain further insight into the role of NTAL in mast cells, we examined the transcriptome profiles of resting and antigen-activated NTAL KO, NTAL KD, and corresponding control BMMCs. Through this analysis we identified several genes that were differentially regulated in nonactivated and antigen-activated NTAL-deficient cells, when compared to the corresponding control cells. Some of the genes seem to be involved in regulation of cholesterol-dependent events in antigen-mediated chemotaxis. The combined data indicate multiple regulatory roles of NTAL in gene expression and mast cell physiology.

• MeSH
• adaptorové proteiny vezikulární transportní genetika   metabolismus   MeSH
• genetická transkripce fyziologie   MeSH
• mastocyty metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• receptory IgE metabolismus   MeSH
• signální transdukce imunologie   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny vezikulární transportní MeSH
• LAB protein, mouse MeSH Prohlížeč
• receptory IgE MeSH
• vápník MeSH

Transmembrane adaptor proteins are membrane-anchored proteins consisting of a short extracellular part, a transmembrane domain, and a cytoplasmic part with various protein-protein interaction motifs but lacking any enzymatic activity. They participate in the regulation of various signaling pathways by recruiting other proteins to the proximity of cellular membranes where the signaling is often initiated and propagated. In this work, we show that LST1/A, an incompletely characterized protein encoded by MHCIII locus, is a palmitoylated transmembrane adaptor protein. It is expressed specifically in leukocytes of the myeloid lineage, where it localizes to the tetraspanin-enriched microdomains. In addition, it binds SHP-1 and SHP-2 phosphatases in a phosphotyrosine-dependent manner, facilitating their recruitment to the plasma membrane. These data suggest a role for LST1/A in negative regulation of signal propagation.

• MeSH
• buněčná membrána metabolismus   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• hlavní histokompatibilní komplex fyziologie   MeSH
• intracelulární signální peptidy a proteiny MeSH
• Jurkat buňky MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• myeloidní buňky cytologie   metabolismus   MeSH
• plakiny metabolismus   MeSH
• primární buněčná kultura MeSH
• pseudopodia metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální transdukce fyziologie   MeSH
• terciární struktura proteinů fyziologie   MeSH
• transport proteinů fyziologie   MeSH
• tyrosinfosfatasa nereceptorového typu 11 metabolismus   MeSH
• tyrosinfosfatasa nereceptorového typu 6 metabolismus   MeSH
• U937 buňky MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• intracelulární signální peptidy a proteiny MeSH
• LST1 protein, human MeSH Prohlížeč
• membránové proteiny MeSH
• plakiny MeSH
• PTPN11 protein, human MeSH Prohlížeč
• PTPN6 protein, human MeSH Prohlížeč
• tyrosinfosfatasa nereceptorového typu 11 MeSH
• tyrosinfosfatasa nereceptorového typu 6 MeSH

Aggregation of the high-affinity IgE receptor (FcεRI) initiates a cascade of signaling events leading to release of preformed inflammatory and allergy mediators and de novo synthesis and secretion of cytokines and other compounds. The first biochemically well defined step of this signaling cascade is tyrosine phosphorylation of the FcεRI subunits by Src family kinase Lyn, followed by recruitment and activation of spleen tyrosine kinase (Syk). Activity of Syk is decisive for the formation of multicomponent signaling assemblies, the signalosomes, in the vicinity of the receptors. Formation of the signalosomes is dependent on the presence of transmembrane adaptor proteins (TRAPs). These proteins are characterized by a short extracellular domain, a single transmembrane domain, and a cytoplasmic tail with various motifs serving as anchors for cytoplasmic signaling molecules. In mast cells five TRAPs have been identified [linker for activation of T cells (LAT), non-T cell activation linker (NTAL), linker for activation of X cells (LAX), phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG), and growth factor receptor-bound protein 2 (Grb2)-binding adaptor protein, transmembrane (GAPT)]; engagement of four of them (LAT, NTAL, LAX, and PAG) in FcεRI signaling has been documented. Here we discuss recent progress in the understanding of how TRAPs affect FcεRI-mediated mast cell signaling. The combined data indicate that individual TRAPs have irreplaceable roles in important signaling events such as calcium response, degranulation, cytokines production, and chemotaxis.

• Klíčová slova
• IgE receptor, LAT/LAT1, LAX, NTAL/Lab/LAT2, PAG/Cbp, mast cells, plasma membrane, transmembrane adaptor proteins,
• Publikační typ
• časopisecké články MeSH