Plasma membrane proteins synthesised at the endoplasmic reticulum are delivered to the cell surface via sorting pathways. Hydrophobic mismatch theory based on the length of the transmembrane domain (TMD) dominates discussion about determinants required for protein sorting to the plasma membrane. Transmembrane adaptor proteins (TRAP) are involved in signalling events which take place at the plasma membrane. Members of this protein family have TMDs of varying length. We were interested in whether palmitoylation or other motifs contribute to the effective sorting of TRAP proteins. We found that palmitoylation is essential for some, but not all, TRAP proteins independent of their TMD length. We also provide evidence that palmitoylation and proximal sequences can modulate sorting of artificial proteins with TMDs of suboptimal length. Our observations point to a unique character of each TMD defined by its primary amino acid sequence and its impact on membrane protein localisation. We conclude that, in addition to the TMD length, secondary sorting determinants such as palmitoylation or flanking sequences have evolved for the localisation of membrane proteins.

• MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• extracelulární prostor chemie   MeSH
• glykosylace MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• Jurkat buňky MeSH
• lidé MeSH
• lipoylace * MeSH
• membránové proteiny chemie   metabolismus   MeSH
• terciární struktura proteinů MeSH
• transport proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Linker for activation in T cells (LAT) is a critical regulator of T-cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane, is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using in silico analysis, confocal and super-resolution imaging and flow cytometry, we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics are further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of nonpalmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.

• MeSH
• adaptorové proteiny signální transdukční chemie   genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• glycin genetika   metabolismus   MeSH
• interferenční mikroskopie MeSH
• Jurkat buňky MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• mutace MeSH
• prolin genetika   metabolismus   MeSH
• proteinové domény MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• T-lymfocyty metabolismus   MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The functional relevance of aromatic residues in the upper part of the transmembrane domain-1 of purinergic P2X receptors (P2XRs) was examined. Replacement of the conserved Tyr residue with Ala had a receptor-specific effect: the P2X1R was non-functional, the P2X2R, P2X4R, and P2X3R exhibited enhanced sensitivity to ATP and alphabeta-meATP accompanied by prolonged decay of current after washout of agonists, and the P2X7R sensitivity for agonists was not affected, though decay of current was delayed. The replacement of the P2X4R-Tyr42 with other amino acids revealed the relevance of an aromatic residue at this position. Mutation of the neighboring Phe and ipsilateral Tyr/Trp residues, but not the contralateral Phe residue, also affected the P2X2R, P2X3R, and P2X4R function. Double mutation of ipsilateral Tyr42 and Trp46 P2X4R residues restored receptor function, whereas the corresponding P2X2R double mutant was not functional. In contrast, mutation of the contralateral Phe48 residue in the P2X4R-Y42A mutant had no effect. These results indicate that aromatic residues in the upper part of TM1 play important roles in the three-dimensional structure of the P2XRs and that they are required not only for ion conductivity but also for specificity of agonist binding and/or channel gating.

• MeSH
• adenosintrifosfát analogy a deriváty   farmakologie   MeSH
• aminokyseliny aromatické genetika   metabolismus   MeSH
• biofyzika MeSH
• elektrická stimulace MeSH
• financování organizované MeSH
• konformace proteinů MeSH
• lidé MeSH
• membránové potenciály genetika   účinky léků   MeSH
• metoda terčíkového zámku metody   MeSH
• mutageneze genetika   MeSH
• purinergní receptory P2 genetika   klasifikace   metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální transdukce fyziologie   účinky léků   MeSH
• terciární struktura proteinů genetika   fyziologie   MeSH
• transfekce metody   MeSH
• transformované buněčné linie MeSH
• vazba proteinů fyziologie   genetika   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zelené fluorescenční proteiny MeSH
• Check Tag
• lidé MeSH

P2X receptors (P2X1-7) are trimeric ion channels activated by extracellular ATP. Each P2X subunit contains two transmembrane helices (TM1 and TM2). We substituted all residues in TM1 of rat P2X7 with alanine or leucine one by one, expressed mutants in HEK293T cells, and examined the pore permeability by recording both membrane currents and fluorescent dye uptake in response to agonist application. Alanine substitution of G27, K30, H34, Y40, F43, L45, M46, and D48 inhibited agonist-stimulated membrane current and dye uptake, and all but one substitution, D48A, prevented surface expression. Mutation V41A partially reduced both membrane current and dye uptake, while W31A and A44L showed reduced dye uptake not accompanied by reduced membrane current. Mutations T28A, I29A, and L33A showed small changes in agonist sensitivity, but they had no or small impact on dye uptake function. Replacing charged residues with residues of the same charge (K30R, H34K, and D48E) rescued receptor function, while replacement with residues of opposite charge inhibited (K30E and H34E) or potentiated (D48K) receptor function. Prolonged stimulation with agonist-induced current facilitation and a leftward shift in the dose-response curve in the P2X7 wild-type and most functional mutants, but sensitization was absent in the W31A, L33A, and A44L. Detailed analysis of the decay of responses revealed two kinetically distinct mechanisms of P2X7 deactivation: fast represents agonist unbinding, and slow might represent resetting of the receptor to the resting closed state. These results indicate that conserved and receptor-specific TM1 residues control surface expression of the P2X7 protein, non-polar residues control receptor sensitization, and D48 regulates intrinsic channel properties.

• MeSH
• adenosintrifosfát farmakologie   metabolismus   MeSH
• biologický transport MeSH
• HEK293 buňky MeSH
• iontové kanály * metabolismus   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• mutace genetika   MeSH
• proteinové domény MeSH
• purinergní receptory P2X7 * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Extracellular and cytosolic leaflets in cellular membranes are distinctly different in lipid composition, yet they contribute together to signaling across the membranes. Here we consider a mechanism based on long-chain gangliosides for coupling the extracellular and cytosolic membrane leaflets together. Based on atomistic molecular dynamics simulations, we find that long-chain GM1 in the extracellular leaflet exhibits a strong tendency to protrude into the opposing bilayer leaflet. This interdigitation modulates the order in the cytosolic monolayer and thereby strengthens the interaction and coupling across a membrane. Coarse-grained simulations probing longer time scales in large membrane systems indicate that GM1 in the extracellular leaflet modulates the phase behavior in the cytosolic monolayer. While short-chain GM1 maintains phase-symmetric bilayers with a strong membrane registration effect, the situation is altered with long-chain GM1. Here, the significant interdigitation induced by long-chain GM1 modulates the behavior in the cytosolic GM1-free leaflet, weakening and slowing down the membrane registration process. The observed physical interaction mechanism provides a possible means to mediate or foster transmembrane communication associated with signal transduction.

• MeSH
• G(M1) gangliosid chemie   MeSH
• lipidové dvojvrstvy chemie   MeSH
• simulace molekulární dynamiky MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Bcl-2 protein family comprises both pro- and antiapoptotic members that control the permeabilization of the mitochondrial outer membrane, a crucial step in the modulation of apoptosis. Recent research has demonstrated that the carboxyl-terminal transmembrane domain (TMD) of some Bcl-2 protein family members can modulate apoptosis; however, the transmembrane interactome of the antiapoptotic protein Mcl-1 remains largely unexplored. Here, we demonstrate that the Mcl-1 TMD forms homooligomers in the mitochondrial membrane, competes with full-length Mcl-1 protein with regards to its antiapoptotic function, and induces cell death in a Bok-dependent manner. While the Bok TMD oligomers locate preferentially to the endoplasmic reticulum (ER), heterooligomerization between the TMDs of Mcl-1 and Bok predominantly takes place at the mitochondrial membrane. Strikingly, the coexpression of Mcl-1 and Bok TMDs produces an increase in ER mitochondrial-associated membranes, suggesting an active role of Mcl-1 in the induced mitochondrial targeting of Bok. Finally, the introduction of Mcl-1 TMD somatic mutations detected in cancer patients alters the TMD interaction pattern to provide the Mcl-1 protein with enhanced antiapoptotic activity, thereby highlighting the clinical relevance of Mcl-1 TMD interactions.

• MeSH
• apoptóza fyziologie   MeSH
• buněčná smrt fyziologie   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• HeLa buňky MeSH
• lidé MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• protein MCL-1 metabolismus   MeSH
• proteinové domény MeSH
• protoonkogenní proteiny c-bcl-2 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Transient receptor potential ankyrin 1 (TRPA1) is a temperature-sensitive ion channel activated by various pungent and irritant compounds that can produce pain in humans. Its activation involves an allosteric mechanism whereby electrophilic agonists evoke interactions within cytosolic domains and open the channel pore through an integrated nexus formed by intracellular membrane proximal regions that are densely packed beneath the lower segment of the S1-S4 sensor domain. Studies indicate that this part of the channel may contain residues that form a water-accessible cavity that undergoes changes in solvation during channel gating. We identified conserved polar residues facing the putative lower crevice of the sensor domain that were crucial determinants of the electrophilic, voltage, and calcium sensitivity of the TRPA1 channel. This part of the sensor may also comprise a domain capable of binding to membrane phosphoinositides through which gating of the channel is regulated in a state-dependent manner.

• MeSH
• alosterická regulace MeSH
• gating iontového kanálu * MeSH
• HEK293 buňky MeSH
• kationtový kanál TRPA1 chemie   fyziologie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• membránové potenciály * MeSH
• molekulární modely MeSH
• mutace MeSH
• mutageneze cílená MeSH
• proteinové domény MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakty MeSH

WW domain binding protein 1-like (WBP1L), also known as outcome predictor of acute leukaemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which correlates with ETV6-RUNX1 (t(12;21)(p13;q22)) translocation and favourable prognosis in childhood leukaemia. It has a broad expression pattern in haematopoietic and in non-haematopoietic cells. However, its physiological function has been unknown. Here, we show that WBP1L negatively regulates signalling through a critical chemokine receptor CXCR4 in multiple leucocyte subsets and cell lines. We also show that WBP1L interacts with NEDD4-family ubiquitin ligases and regulates CXCR4 ubiquitination and expression. Moreover, analysis of Wbp1l-deficient mice revealed alterations in B cell development and enhanced efficiency of bone marrow cell transplantation. Collectively, our data show that WBP1L is a novel regulator of CXCR4 signalling and haematopoiesis.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• glykoproteiny metabolismus   MeSH
• HEK293 buňky MeSH
• hematopoetické kmenové buňky metabolismus   MeSH
• hematopoéza * MeSH
• homeostáza MeSH
• lidé MeSH
• lipoylace MeSH
• malá interferující RNA metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• receptory CXCR4 metabolismus   MeSH
• signální transdukce * MeSH
• ubikvitinace MeSH
• ubikvitinligasy metabolismus   MeSH
• vazba proteinů MeSH
• zárodečné buňky metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

DCL-1 (CD302) is a single-pass type one transmembrane protein which is predominantly expressed on myeloid cell lines. It possess the ability of endocytosis and is assumed to play a role in cell adhesion and migration. It has been also connected to several illnesses but more on the level of mRNA than on the protein expression level. More interestingly it is alternatively expressed in the form of a fusion protein with another single-pass type one transmembrane protein DEC205 (CD205) which is normally involved in antigen-uptake and endocytosis. The fusion protein has been assigned to have altered function compared to the wild type proteins. We have performed NMR structural analysis of the 16.2 kDa extracellular domain of DCL-1 to get a better insight onto this molecule. We have been able to assign nearly 97 % of resonance frequencies for the (15)N and (13)C labeled recombinant protein. The assignments have been deposited into Biological Magnetic Resonance Data Bank under the accession number 25802.

• MeSH
• extracelulární prostor * MeSH
• lektiny typu C chemie   MeSH
• lidé MeSH
• nukleární magnetická rezonance biomolekulární * MeSH
• proteinové domény MeSH
• receptory buněčného povrchu chemie   MeSH
• sekundární struktura proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH