The overexpression of MYC genes is frequently found in many human cancers, including adult and pediatric malignant brain tumors. Targeting MYC genes continues to be challenging due to their undruggable nature. Using our prediction algorithm, the nine-amino-acid activation domain (9aaTAD) has been identified in all four Yamanaka factors, including c-Myc. The predicted activation function was experimentally demonstrated for all these short peptides in transactivation assay. We generated a set of c-Myc constructs (1-108, 69-108 and 98-108) in the N-terminal regions and tested their ability to initiate transcription in one hybrid assay. The presence and absence of 9aaTAD (region 100-108) in the constructs strongly correlated with their activation functions (5-, 3- and 67-times respectively). Surprisingly, we observed co-activation function of the myc region 69-103, called here acetyl-TAD, previously described by Faiola et al. (Mol Cell Biol 25:10220-10234, 2005) and characterized in this study as a new domain collaborating with the 9aaTAD. We discovered strong interactions on a nanomolar scale between the Myc-9aaTAD activation domains and the KIX domain of CBP coactivator. We showed conservation of the 9aaTADs in the MYC family. In summary for the c-Myc oncogene, the acetyl-TAD and the 9aaTAD domains jointly mediated activation function. The c-Myc protein is largely intrinsically disordered and therefore difficult to target with small-molecule inhibitors. For the c-Myc driven tumors, the strong c-Myc interaction with the KIX domain represents a promising druggable target.

• MeSH
• aktivace transkripce MeSH
• lidé MeSH
• proteinové domény MeSH
• protoonkogenní proteiny c-myc * metabolismus   genetika   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Adar null mutant mouse embryos die with aberrant double-stranded RNA (dsRNA)-driven interferon induction, and Adar Mavs double mutants, in which interferon induction is prevented, die soon after birth. Protein kinase R (Pkr) is aberrantly activated in Adar Mavs mouse pup intestines before death, intestinal crypt cells die, and intestinal villi are lost. Adar Mavs Eifak2 (Pkr) triple mutant mice rescue all defects and have long-term survival. Adenosine deaminase acting on RNA 1 (ADAR1) and PKR co-immunoprecipitate from cells, suggesting PKR inhibition by direct interaction. AlphaFold studies on an inhibitory PKR dsRNA binding domain (dsRBD)-kinase domain interaction before dsRNA binding and on an inhibitory ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA provide a testable model of the inhibition. Wild-type or editing-inactive human ADAR1 expressed in A549 cells inhibits activation of endogenous PKR. ADAR1 dsRNA binding is required for, but is not sufficient for, PKR inhibition. Mutating the ADAR1 dsRBD3-PKR contact prevents co-immunoprecipitation, ADAR1 inhibition of PKR activity, and co-localization of ADAR1 and PKR in cells.

• MeSH
• adenosindeaminasa * metabolismus   genetika   MeSH
• aktivace enzymů MeSH
• buňky A549 MeSH
• dvouvláknová RNA * metabolismus   MeSH
• kinasa eIF-2 * metabolismus   MeSH
• lidé MeSH
• myši MeSH
• proteinové domény MeSH
• proteiny vázající RNA * metabolismus   genetika   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

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

The Gal4 protein is a well-known prototypic acidic activator that has multiple activation domains. We have previously identified a new activation domain called the nine amino acid transactivation domain (9aaTAD) in Gal4 protein. The family of the 9aaTAD activators currently comprises over 40 members including p53, MLL, E2A and other members of the Gal4 family; Oaf1, Pip2, Pdr1 and Pdr3. In this study, we revised function of all reported Gal4 activation domains. Surprisingly, we found that beside of the activation domain 9aaTAD none of the previously reported activation domains had considerable transactivation potential and were not involved in the activation of transcription. Our results demonstrated that the 9aaTAD domain is the only decisive activation domain in the Gal4 protein. We found that the artificial peptides included in the original Gal4 constructs were results of an unintended consequence of cloning that were responsible for the artificial transcriptional activity. Importantly, the activation domain 9aaTAD, which is the exclusive activation domain in Gal4, is also the central part of a conserved sequence recognized by the inhibitory protein Gal80. We propose a revision of the Gal4 regulation, in which the activation domain 9aaTAD is directly linked to both activation function and Gal80 mediated inhibition.

• MeSH
• aktivace transkripce genetika   fyziologie   MeSH
• DNA vazebné proteiny chemie   genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• proteinové domény MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• transkripční faktory chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

The nine-amino-acid transactivation domains (9aaTAD) was identified in numerous transcription factors including Gal4, p53, E2A, MLL, c-Myc, N-Myc, and also in SP, KLF, and SOX families. Most of the 9aaTAD domains interact with the KIX domain of transcription mediators MED15 and CBP to activate transcription. The NFkB activation domain occupied the same position on the KIX domain as the 9aaTADs of MLL, E2A, and p53. Binding of the KIX domain is established by the two-point interaction involving 9aaTAD positions p3-4 and p6-7. The NFkB primary binding region (positions p3-4) is almost identical with MLL and E2A, but secondary NFkB binding region differs by the position and engages the distal NFkB region p10-11. Thus, the NFkB activation domain is five amino acids longer than the other 9aaTADs. The NFkB activation domain includes an additional region, which we called the Omichinski Insert extending activation domain length to 14 amino acids. By deletion, we demonstrated that Omichinski Insert is an entirely non-essential part of NFkB activation domain. In summary, we recognized the NFkB activation domain as prolonged 9aaTAD conserved in evolution from humans to amphibians.

• MeSH
• aktivace transkripce MeSH
• aminokyseliny * metabolismus   MeSH
• lidé MeSH
• nádorový supresorový protein p53 * metabolismus   MeSH
• NF-kappa B metabolismus   MeSH
• sekvence aminokyselin MeSH
• transkripční faktory metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α1 and CC1α2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.

• MeSH
• abnormální erytrocyty MeSH
• dyslexie genetika   MeSH
• HEK293 buňky MeSH
• ichtyóza genetika   MeSH
• kanály aktivované uvolněním vápníku metabolismus   MeSH
• klonování DNA MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• metoda terčíkového zámku MeSH
• migréna genetika   MeSH
• mióza genetika   MeSH
• molekulární modely MeSH
• mutace genetika   MeSH
• nádorové proteiny genetika   MeSH
• protein ORAI1 genetika   MeSH
• protein STIM1 genetika   MeSH
• slezina abnormality   MeSH
• svalová únava genetika   MeSH
• trombocytopatie genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• imunoglobuliny - fragmenty MeSH
• vztahy mezi strukturou a aktivitou MeSH

CD200/CD200R are highly conserved type I paired membrane glycoproteins that belong to the Ig superfamily containing a two immunoglobulin‑like domain (V, C). CD200 is broadly distributed in a variety of cell types, whereas CD200R is primarily expressed in myeloid and lymphoid cells. They fulfill multiple functions in regulating inflammation. The interaction between CD200/CD200R results in activation of the intracellular inhibitory pathway with RasGAP recruitment and thus contributes to effector cell inhibition. It was confirmed that the CD200R activation stimulates the differentiation of T cells to the Treg subset, upregulates indoleamine 2,3‑dioxygenase activity, modulates cytokine environment from a Th1 to a Th2 pattern, and facilitates an antiinflammatory IL‑10 and TGF‑β synthesis. CD200/CD200R are required for maintaining self‑tolerance. Many studies have demonstrated the importance of CD200 in controlling autoimmunity, inflammation, the development and spread of cancer, hypersensitivity, and spontaneous fetal loss.

• MeSH
• antigeny povrchové fyziologie   MeSH
• CD antigeny fyziologie   MeSH
• imunita fyziologie   MeSH
• lidé MeSH
• receptory buněčného povrchu fyziologie   MeSH
• signální transdukce MeSH
• zánět patofyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH

N-Methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for synaptic plasticity and memory. Receptor activation involves glycine- and glutamate-stabilized closure of the GluN1 and GluN2 subunit ligand binding domains that is allosterically regulated by the amino-terminal domain (ATD). Using single molecule fluorescence resonance energy transfer (smFRET) to monitor subunit rearrangements in real-time, we observe a stable ATD inter-dimer distance in the Apo state and test the effects of agonists and antagonists. We find that GluN1 and GluN2 have distinct gating functions. Glutamate binding to GluN2 subunits elicits two identical, sequential steps of ATD dimer separation. Glycine binding to GluN1 has no detectable effect, but unlocks the receptor for activation so that glycine and glutamate together drive an altered activation trajectory that is consistent with ATD dimer separation and rotation. We find that protons exert allosteric inhibition by suppressing the glutamate-driven ATD separation steps, and that greater ATD separation translates into greater rotation and higher open probability.

• MeSH
• alosterická regulace MeSH
• glycin chemie   metabolismus   MeSH
• HEK293 buňky MeSH
• kinetika MeSH
• konfokální mikroskopie MeSH
• konformace proteinů * MeSH
• kyselina glutamová chemie   metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• multimerizace proteinu * MeSH
• receptory N-methyl-D-aspartátu chemie   genetika   metabolismus   MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Trf4/5p-Air1/2p-Mtr4p polyadenylation complex (TRAMP) is an essential component of nuclear RNA surveillance in yeast. It recognizes a variety of nuclear transcripts produced by all three RNA polymerases, adds short poly(A) tails to aberrant or unstable RNAs and activates the exosome for their degradation. Despite the advances in understanding the structural features of the isolated complex subunits or their fragments, the details of complex assembly, RNA recognition and exosome activation remain poorly understood. Here we provide the first understanding of the RNA binding mode of the complex. We show that Air2p is an RNA-binding subunit of TRAMP. We identify the zinc knuckles (ZnK) 2, 3 and 4 as the RNA-binding domains, and reveal the essentiality of ZnK4 for TRAMP4 polyadenylation activity. Furthermore, we identify Air2p as the key component of TRAMP4 assembly providing bridging between Mtr4p and Trf4p. The former is bound via the N-terminus of Air2p, while the latter is bound via ZnK5, the linker between ZnK4 and 5 and the C-terminus of the protein. Finally, we uncover the RNA binding part of the Mtr4p arch, the KOW domain, as the essential component for TRAMP-mediated exosome activation.

• MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• DEAD-box RNA-helikasy chemie   metabolismus   MeSH
• DNA-dependentní DNA-polymerasy chemie   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• proteiny vázající RNA chemie   metabolismus   MeSH
• ribonukleasy metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH