ABSTRACT
Transcription factors are generally considered challenging, if not "undruggable", targets but they promise new therapeutic options due to their fundamental involvement in many diseases. In this study, we aim to assess the ligandability of the C-terminal Rel-homology domain of nuclear factor of activated T cells 1 (NFAT1), a TF implicated in T-cell regulation. Using a combination of experimental and computational approaches, we demonstrate that small molecule fragments can indeed bind to this protein domain. The newly identified binder is the first small molecule binder to NFAT1 validated with biophysical methods and an elucidated binding mode by X-ray crystallography. The reported eutomer/distomer pair provides a strong basis for potential exploration of higher potency binders on the path toward degrader or glue modalities.
Subject(s)
NFATC Transcription Factors , Binding Sites , Crystallography, X-Ray , Ligands , NFATC Transcription Factors/metabolism , NFATC Transcription Factors/chemistry , Protein Binding , Protein Domains , Structure-Activity RelationshipABSTRACT
BACKGROUND: Cardiac valvulogenesis is a highly conserved process among vertebrates and cause unidirectional flow of blood in the heart. It was precisely regulated by signal pathways such as VEGF, NOTCH, and WNT and transcriptional factors such as TWIST1, TBX20, NFATC1, and SOX9. Tricuspid atresia refers to morphological deficiency of the valve and confined right atrioventricular traffic due to tricuspid maldevelopment, and is one of the most common types of congenital valve defects. METHODS: We recruited a healthy couple with two fetuses aborted due to tricuspid atresia and identified related gene mutations using whole-exome sequencing. We then discussed the pathogenic significance of this mutation by bioinformatic and functional analyses. RESULTS: PROVEAN, PolyPhen, MutationTaster, and HOPE indicated the mutation could change the protein function and cause disease; Western blotting showed the expression of NFATC1 c.964G>A mutation was lower than the wild type. What's more, dual-luciferase reporter assay showed the transcriptional activity of NFATC1 was impact by mutation and the expression of downstream DEGS1 was influenced. CONCLUSION: Taken together, the c.964G>A mutation might be pathological and related to the occurrence of disease. Our research tended to deepen the understanding of etiology of tricuspid atresia and gene function of NFATC1, and provide some references or suggestions for genetic diagnosis of tricuspid atresia.
Subject(s)
NFATC Transcription Factors/genetics , Tricuspid Atresia/genetics , Aborted Fetus/abnormalities , Adult , Animals , Cell Line , Cells, Cultured , Fatty Acid Desaturases/genetics , Fatty Acid Desaturases/metabolism , Female , Humans , Male , Mice , Mutation , NFATC Transcription Factors/chemistry , NFATC Transcription Factors/metabolism , Pedigree , Protein Domains , Tricuspid Atresia/pathologyABSTRACT
The activation of NFAT (nuclear factor of activated T cells) transcription factors by calcium-dependent phosphatase calcineurin is a key step in controlling T cell activation and plays a vital role during carcinogenesis. NFATs are overexpressed in many cancers, including the most common primary brain tumor, gliomas. In the present study, we demonstrate the expression of NFATs and NFAT-driven transcription in several human glioma cells. We used a VIVIT peptide for interference in calcineurin binding to NFAT via a conserved PxIxIT motif. VIVIT was expressed as a fusion protein with a green fluorescent protein (VIVIT-GFP) or conjugated to cell-penetrating peptides (CPP), Sim-2 or 11R. We analyzed the NFAT expression, phosphorylation, subcellular localization and their transcriptional activity in cells treated with peptides. Overexpression of VIVIT-GFP decreased the NFAT-driven activity and inhibited the transcription of endogenous NFAT-target genes. These effects were not reproduced with synthetic peptides: Sim2-VIVIT did not show any activity, and 11R-VIVIT did not inhibit NFAT signaling in glioma cells. The presence of two calcineurin docking sites in NFATc3 might require dual-specificity blocking peptides. The cell-penetrating peptides Sim-2 or 11R linked to VIVIT did not improve its action making it unsuitable for evaluating NFAT dependent events in glioma cells with high expression of NFATc3.
Subject(s)
Brain Neoplasms/pathology , Calcineurin/metabolism , Glioma/pathology , NFATC Transcription Factors/metabolism , Oligopeptides/pharmacology , Signal Transduction , Amino Acid Sequence , Basic Helix-Loop-Helix Transcription Factors/metabolism , Brain Neoplasms/genetics , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Proliferation/drug effects , Cell-Penetrating Peptides/pharmacology , Glioma/genetics , Green Fluorescent Proteins/metabolism , Humans , NFATC Transcription Factors/chemistry , Oligopeptides/chemistry , Peptides/pharmacology , Protein Transport/drug effects , Transcription, Genetic/drug effectsABSTRACT
Acute respiratory distress syndrome (ARDS) is an inflammatory lung disease with a high morbidity and mortality rate, for which no pharmacologic treatment is currently available. Our previous studies discovered that a pivotal step in the disease process is the activation of the nuclear factor of activated T cells (NFAT) c3 in lung macrophages, suggesting that inhibitors against the upstream protein phosphatase calcineurin should be effective for prevention/treatment of ARDS. Herein, we report the development of a highly potent, cell-permeable, and metabolically stable peptidyl inhibitor, CNI103, which selectively blocks the interaction between calcineurin and NFATc3, through computational and medicinal chemistry. CNI103 specifically inhibited calcineurin signaling in vitro and in vivo and exhibited a favorable pharmacokinetic profile, broad tissue distribution following different routes of administration, and minimal toxicity. Our data indicate that CNI103 is a promising novel treatment for ARDS and other inflammatory diseases.
Subject(s)
Calcineurin/metabolism , NFATC Transcription Factors/metabolism , Acute Lung Injury/pathology , Acute Lung Injury/prevention & control , Amino Acid Sequence , Animals , Binding Sites , Calcineurin/chemistry , Calcineurin Inhibitors/chemistry , Calcineurin Inhibitors/metabolism , Calcineurin Inhibitors/pharmacology , Calcineurin Inhibitors/therapeutic use , Half-Life , Humans , Lipopolysaccharides/toxicity , Lung/diagnostic imaging , Lung/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Dynamics Simulation , NFATC Transcription Factors/chemistry , Peptides/chemistry , Peptides/pharmacokinetics , Peptides/pharmacology , Peptides/therapeutic use , Protein Interaction Domains and Motifs/drug effects , Signal Transduction/drug effects , Tissue DistributionABSTRACT
Nuclear factor of activated T cells (NFAT) leads to the transcription of diverse inducible genes involved in many biological processes; therefore, aberrant NFAT expression is responsible for the development and exacerbation of various disorders. Since five isoforms of NFAT (NFATc1-c4, NFAT5) exhibit distinct and overlapping functions, selective control of a part, but not all, of NFAT family members is desirable. By comparing the binding activity of each NFATc1-c4 with its regulatory enzyme, calcineurin (CN), using a quantitative immunoprecipitation assay, we found a new CN-binding region (CNBR) selectively functioning in NFATc1 and NFATc4. This region, termed CNBR3, is located between two preexisting CNBR1 and CNBR2, within the Ca2+ regulatory domain. The nuclear translocation of NFATc1 but not NFATc2 in T cells was suppressed by ectopic expression of CNBR3 and, accordingly, NFATc1-dependent cytokine expression was downregulated. Through competition assays using NFATc1-derived partial peptides and mass spectrometry with photoaffinity technology, we identified 18 amino acids in NFATc1 (Arg258 to Pro275 ) and 13 amino acids in CN catalytic subunit (CNA) (Asn77 to Gly89 ) responsible for CNA/CNBR3 binding in which Cys263 and Asp82 , respectively, played crucial roles. The possible selective regulation of NFAT-mediated biological processes by targeting this new CN/NFAT-binding region is suggested.
Subject(s)
Calcineurin/chemistry , Molecular Docking Simulation , NFATC Transcription Factors/chemistry , Animals , Binding Sites , Calcineurin/genetics , Calcineurin/metabolism , Cell Line , Cricetinae , Cricetulus , Humans , Jurkat Cells , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Protein BindingABSTRACT
The NFAT family of transcription factors plays an important role in immune system development and function. NFATc1 and NFATc2 are highly expressed in peripheral T cells, and several isoforms are produced via the use of different promoters and polyadenylation sites. The specific isoforms with relatively long C-termini, NFATc1/C and NFATc2/A, have been shown to be modified by SUMO within their specific C-terminal regions, which regulates NFAT protein localization and transactivation activity. Here, we demonstrate that an isoform NFATc1/A, which has a short C-terminus and does not contain the sumoylation sites found in the long isoforms, is also modified by SUMO. NFATc1/A sumoylation increased with low level expression of SUMO E3 ligases, specifically PIAS1, PIAS3, and PIASy, in co-transfected cells. PIAS3 interacted with NFATc1/A and an active site mutant failed to promote NFATc1/A sumoylation, indicating a role for PIAS3 as a SUMO E3 ligase. A lysine residue at 351 within the central regulatory domain was identified as the major SUMO attachment site in both co-transfection and in vitro assays. Sumoylation of NFATc1/A did not affect nuclear translocation upon ionomycin and phorbol 12-myristate 13-acetate treatment. However, although sumoylation of NFATc1/A slightly increased protein stability, it inhibited transactivation activity for reporter genes driven by promoters containing NFAT sites. Our results indicate that the transactivation activity of NFATc1/A is negatively regulated by PIAS protein-mediated sumoylation, and that SUMO is a general regulator of NFAT family members with either long or short C-termini.
Subject(s)
NFATC Transcription Factors/metabolism , Protein Inhibitors of Activated STAT/metabolism , Sumoylation , Transcriptional Activation , Amino Acid Sequence , Cell Line , Humans , NFATC Transcription Factors/chemistry , Protein StabilityABSTRACT
Intrinsically disordered proteins and regions (IDPs and IDRs) lack well-defined tertiary structures, yet carry out various important cellular functions, especially those associated with cell signaling and regulation. In eukaryotes, IDPs and IDRs contain the preferred loci for both alternative splicing (AS) and many post-translational modifications (PTMs). Furthermore, AS and/or PTMs at these loci generally alter the signaling outcomes associated with these IDPs or IDRs, where the functional cooperation of these three features is named the IDP-AS-PTM toolkit. However, the prevalence of such functional modulations remains unknown. Also, the signal-altering mechanisms by which AS, and PTMs modulate function and the extent to which AS and PTMs collaborate in their signaling modulations have not been well defined for particular protein examples. Here we focus on three important signaling and regulatory IDR-containing protein families in humans, namely, G protein-coupled receptors (GPCRs), which are transmembrane proteins; the nuclear factors of activated T cells (NFATs), which are transcription factors; and the Src family kinases (SFKs), which are signaling enzymes. The goals here are to determine how AS and PTMs individually alter the outcomes of the signaling carried out by the various IDRs and to determine whether AS and PTMs work together to bring about differential cellular responses. We also present data indicating that a wide range of other signaling IDPs or IDRs undergo both AS- and PTM-based modifications, suggesting that they, too, likely take advantage of signal outcome modulations that result from collaboration between these two events. Hence, we propose that the widespread cooperation of IDPs, AS and/or PTMs provides an IDP-AS-PTM toolkit and substantially contributes to the vast complexity of eukaryotic cell signaling systems.
Subject(s)
Alternative Splicing , Intrinsically Disordered Proteins/metabolism , Protein Processing, Post-Translational , Biochemical Phenomena , Humans , NFATC Transcription Factors/chemistry , NFATC Transcription Factors/metabolism , Protein Binding , Protein Conformation , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , src-Family Kinases/chemistry , src-Family Kinases/metabolismABSTRACT
Calcineurin is a phosphatase that targets the transcription factor, nuclear factor of activated T-cells (NFAT) dephosphorylates multiple sites along NFAT's regulatory domain. The calcineurin-NFAT complex interaction is mediated through two conserved binding motifs known as the PxIxIT and LxVP, which are located at the N- and C- terminus to the phosphorylation sites. The vast range of cellular processes regulated by the calcineurin-NFAT interaction has aroused great interest in the investigation of the structural aspects that govern their complex formation and in the discovery of protein-protein interaction inhibitors; the latter interfere with calcineurin-NFAT complex formation while keeping calcineurin's catalytic site free. To assist additional biophysical study of the calcineurin-NFAT structure-function relation and to screen for new inhibitors, we present a robust and cost-effective Enzyme Linked Immuno Sorbent Assay (ELISA) that is based on the interaction of calcineurin with the NFAT homology region. The latter includes the two calcineurin's binding sites, in addition to the phosphorylation sites. The ELISA experiment shown here can thus be applied towards the study of important structural aspects of the complex and for the discovery of new inhibitors. This will allow for a better understanding of T-cell activation switch.
Subject(s)
Calcineurin/chemistry , NFATC Transcription Factors/chemistry , Calcineurin/metabolism , Enzyme-Linked Immunosorbent Assay/methods , HumansABSTRACT
Studies over the past decade have highlighted the functional significance of intrinsically disordered proteins (IDPs). Due to conformational heterogeneity and inherent dynamics, structural studies of IDPs have relied mostly on NMR spectroscopy, despite IDPs having characteristics that make them challenging to study using traditional 1H-detected biomolecular NMR techniques. Here, we develop a suite of 3D 15N-detected experiments that take advantage of the slower transverse relaxation property of 15N nuclei, the associated narrower linewidth, and the greater chemical shift dispersion compared with those of 1H and 13C resonances. The six 3D experiments described here start with aliphatic 1H magnetization to take advantage of its higher initial polarization, and are broadly applicable for backbone assignment of proteins that are disordered, dynamic, or have unfavorable amide proton exchange rates. Using these experiments, backbone resonance assignments were completed for the unstructured regulatory domain (residues 131-294) of the human transcription factor nuclear factor of activated T cells (NFATC2), which includes 28 proline residues located in functionally important serine-proline (SP) repeats. The complete assignment of the NFATC2 regulatory domain enabled us to study phosphorylation of NFAT by kinase PKA and phosphorylation-dependent binding of chaperone protein 14-3-3 to NFAT, providing mechanistic insight on how 14-3-3 regulates NFAT nuclear translocation.
Subject(s)
Magnetic Resonance Spectroscopy , NFATC Transcription Factors/chemistry , Nitrogen Isotopes/chemistry , Protein ConformationABSTRACT
Calcineurin (CN) is involved in many physiological processes and interacts with multiple substrates. Most of the substrates contain similar motifs recognized by CN. Recent studies revealed a new CN substrate, transcription factor EB (TFEB), which is involved in autophagy. We showed that a 15-mer QSYLENPTSYHLQQS peptide from TFEB (TFEB-YLENP) bound to CN. When the TFEB-YLENP peptide was changed to YLAVP, its affinity for CN increased and it had stronger CN inhibitory activity. Molecular dynamics simulations revealed that the TFEB-YLENP peptide has the same docking sites in CN as the 15-mer DQYLAVPQHPYQWAK motif of the nuclear factor of activated T cells, cytoplasmic 1 (NFATc1-YLAVP). Moreover expression of the NFATc1-YLAVP peptide suppressed the TFEB activation in starved Hela cells. Our studies first identified a CN binding site in TFEB and compared the inhibitory capability of various peptides derived from CN substrates. The data uncovered a diversity in recognition sequences that underlies the CN signaling within the cell. Studies of CN-substrate interactions should lay the groundwork for developing selective CN peptide inhibitors that target CN-substrate interaction in vitro experiments.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/chemistry , Calcineurin/metabolism , NFATC Transcription Factors/chemistry , Peptide Fragments/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Calcineurin/chemistry , HeLa Cells , Humans , Mice , Peptide Fragments/chemistry , Protein BindingABSTRACT
BACKGROUND: The tumor acidic microenvironment, a common biochemical event in solid tumors, offers evolutional advantage for tumors cells and even enhances their aggressive phenotype. However, little is known about the molecular mechanism underlying the acidic microenvironment-induced invasion and metastasis. METHODS: We examined the expression of the acid-sending ion channel (ASIC) family members after acidic exposure using RT-PCR and immunofluoresence. Gene manipulation was applied to reveal the potential of ASIC2 on invasion, proliferation, colony formation of colorectal cancer (CRC). We assessed the in vivo tumor growth by subcutaneous transplantation and metastasis by spleen xenografts. Chromatin immunoprecipitation-sequencing was used to uncover the binding sites of NFAT1. Finally, we examined the expression of ASIC2 in CRC tissues using immunohistochemistry. RESULTS: Acidic exposure led to up-regulation of the acid-sensing ion channel, ASIC2, in colorectal cancer (CRC) cells. ASIC2 overexpression in CRC cell lines, SW480 and HCT116, significantly enhanced cell proliferation in vitro and in vivo, while ASIC2 knockdown had the reverse effect. Importantly, ASIC2 promoted CRC cell invasion under acidosis in vitro and liver metastasis in vivo. Mechanistically, ASIC2 activated the calcineurin/NFAT1 signaling pathway under acidosis. Inhibition of the calcineurin/NFAT pathway by cyclosporine A (CsA) profoundly attenuated ASIC2-induced invasion under acidosis. ChIP-seq assay revealed that the nuclear factor, NFAT1, binds to genes clustered in pathways involved in Rho GTPase signaling and calcium signaling. Furthermore, immunohistochemistry showed that ASIC2 expression is increased in CRC samples compared to that in adjacent tissues, and ASIC2 expression correlates with T-stage, distant metastasis, recurrence, and poor prognosis. CONCLUSION: ASIC2 promotes metastasis of CRC cells by activating the calcineurin/NFAT1 pathway under acidosis and high expression of ASIC2 predicts poor outcomes of patients with CRC.
Subject(s)
Acid Sensing Ion Channels/metabolism , Acidosis/metabolism , Calcineurin/metabolism , Colorectal Neoplasms/pathology , Liver Neoplasms/secondary , NFATC Transcription Factors/metabolism , Aged , Animals , Binding Sites , Calcium/metabolism , Cell Line, Tumor , Cell Proliferation , Colorectal Neoplasms/metabolism , Female , Gene Expression Regulation, Neoplastic , HCT116 Cells , HT29 Cells , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Mice , Middle Aged , NFATC Transcription Factors/chemistry , Neoplasm Invasiveness , Neoplasm Staging , Neoplasm Transplantation , Signal Transduction , Tumor Microenvironment , rho GTP-Binding Proteins/metabolismABSTRACT
Nuclear factor of activated T cells (NFAT) was first identified as a transcription factor about 3 decades ago and was not well studied until the development of immunosuppressant. Numerous studies confirm that calcineurin/NFAT signaling is very important in the development of vasculature and cardiovascular system during embryogenesis and is involved in the development of vascular diseases such as hypertension, atherosclerosis and restenosis. Recent studies demonstrated that NFAT proteins also regulate immune response and vascular cells in the pulmonary microenvironment. In this review, we will discuss how different NFAT isoforms contribute to pulmonary vascular remodeling and potential new therapeutic targets for treating pulmonary arterial hypertension.
Subject(s)
Hypertension, Pulmonary/metabolism , NFATC Transcription Factors/metabolism , Animals , Humans , Hypertension, Pulmonary/physiopathology , Models, Biological , Molecular Targeted Therapy , NFATC Transcription Factors/chemistry , Signal Transduction , Vascular RemodelingABSTRACT
NFAT5 is a transcription factor originally identified because it is activated by hypertonicity and that activation increases expression of genes that protect against the adverse effects of the hypertonicity. However, its targets also include genes not obviously related to tonicity. The transactivating domain of NFAT5 is contained in its COOH-terminal region, which is predicted to be unstructured. Unstructured regions are common in transcription factors particularly in transactivating domains where they can bind co-regulatory proteins essential to their function. To identify potential binding partners of NFAT5 from either cytoplasmic or nuclear HEK293 cell extracts, we used peptide affinity chromatography followed by mass spectrometry. Peptide aptamer-baits consisted of overlapping 20 amino acid peptides within the predicted COOH-terminal unstructured region of NFAT5. We identify a total of 351 unique protein preys that associate with at least one COOH-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from cells incubated at various tonicities (NaCl varied). In addition to finding many proteins already known to associate with NFAT5, we found many new ones whose function suggest novel aspects of NFAT5 regulation, interaction, and function. Relatively few of the proteins pulled down by peptide baits from NFAT5 are generally involved in transcription, and most, therefore, are likely to be specifically related to the regulation of NFAT5 or its function. The novel associated proteins are involved with cancer, effects of hypertonicity on chromatin, development, splicing of mRNA, transcription, and vesicle trafficking.
Subject(s)
Chromatography, Affinity/methods , NFATC Transcription Factors/metabolism , Peptides/metabolism , Amino Acid Sequence , Cell Extracts , HEK293 Cells , Humans , NFATC Transcription Factors/chemistry , Osmosis , Protein Binding , Protein Domains , Protein Interaction Maps , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Sodium Chloride/pharmacologyABSTRACT
Plasmacytoid dendritic cells (pDCs) rapidly produce large amounts of type 1 interferon (IFN) after Toll-like receptor 7 and 9 engagements. This specialized function of type 1 IFN production is directly linked to the constitutive expression of IRF7, the master transcription factor for type 1 IFN production. However, the IRF7 regulatory network in pDCs remains largely unknown. In this study, we identify that the transcription factor NFATC3 specifically binds to IRF7 and enhances IRF7-mediated IFN production. Furthermore, knockout of NFATC3 greatly reduced the CpG DNA-induced nuclear translocation of IRF7, which resulted in impaired type 1 IFN production in vitro and in vivo. In addition, we found that NFATC3 and IRF7 both bound to type 1 IFN promoters and that the NFAT binding site in IFN promoters was required for IRF7-mediated IFN expression. Collectively, our study shows that the transcription factor NFATC3 binds to IRF7 and functions synergistically to enhance IRF7-mediated IFN expression in pDCs.
Subject(s)
Dendritic Cells/metabolism , Interferon Regulatory Factor-7/genetics , NFATC Transcription Factors/metabolism , Transcription, Genetic , Animals , Base Sequence , CRISPR-Cas Systems/drug effects , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cells, Cultured , Dendritic Cells/drug effects , Gene Knockdown Techniques , HEK293 Cells , Humans , Interferon Regulatory Factor-7/metabolism , Interferon Type I/genetics , Interferon Type I/metabolism , Mice , NFATC Transcription Factors/chemistry , Oligodeoxyribonucleotides/pharmacology , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Protein Binding/genetics , Protein Domains , Protein Transport/drug effects , Signal Transduction/drug effects , Structure-Activity Relationship , Transcription, Genetic/drug effectsABSTRACT
Calcineurin (CN) is a unique calcium/calmodulin (CaM)-activated serine/threonine phosphatase. To perform its diverse biological functions, CN communicates with many substrates and other proteins. In the physiological activation of T cells, CN acts through transcriptional factors belonging to the NFAT family and other transcriptional effectors. The classic immunosuppressive drug cyclosporin A (CsA) can bind to cyclophilin (CyP) and compete with CN for the NFAT LxVP motif. CsA has debilitating side effects, including nephrotoxicity, hypertension and tremor. It is desirable to develop alternative immunosuppressive agents. To this end, we first tested the interactions between CN and the LxVP-type substrates, including endogenous regulators of calcineurin (RCAN1) and NFAT. Interestingly, we found that quercetin, the primary dietary flavonol, can inhibit the activity of CN and significantly disrupt the associations between CN and its LxVP-type substrates. We then validated the inhibitory effects of quercetin on the CN-NFAT interactions in cell-based assays. Further, quercetin also shows dose-dependent suppression of cytokine gene expression in mouse spleen cells. These data raise the possibility that the interactions of CN with its LxVP-type substrates are potential targets for immunosuppressive agents.
Subject(s)
Calcineurin/metabolism , Immunosuppressive Agents/pharmacology , NFATC Transcription Factors/chemistry , NFATC Transcription Factors/metabolism , Quercetin/pharmacology , Active Transport, Cell Nucleus/drug effects , Amino Acid Motifs , Amino Acid Sequence , Animals , Calcineurin/chemistry , Cell Nucleus/metabolism , Cytokines/genetics , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Male , Mice , Models, Molecular , Protein Binding/drug effects , Protein ConformationABSTRACT
The NFAT (nuclear factor of activated T cells) family of transcription factors consists of four Ca(2+)-regulated members (NFAT1-NFAT4), which were first described in T lymphocytes. In addition to their well-documented role in T lymphocytes, where they control gene expression during cell activation and differentiation, NFAT proteins are also expressed in a wide range of cells and tissue types and regulate genes involved in cell cycle, apoptosis, angiogenesis and metastasis. The NFAT proteins share a highly conserved DNA-binding domain (DBD), which allows all NFAT members to bind to the same DNA sequence in enhancers or promoter regions. The same DNA-binding specificity suggests redundant roles for the NFAT proteins, which is true during the regulation of some genes such as IL-2 and p21. However, it has become increasingly clear that different NFAT proteins and even isoforms can have unique functions. In this review, we address the possible reasons for these distinct roles, particularly regarding N- and C-terminal transactivation regions (TADs) and the partner proteins that interact with these TADs. We also discuss the genes regulated by NFAT during cell cycle regulation and apoptosis and the role of NFAT during tumorigenesis.
Subject(s)
Apoptosis , NFATC Transcription Factors/metabolism , CASP8 and FADD-Like Apoptosis Regulating Protein/chemistry , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Cell Cycle Checkpoints , Cell Transformation, Neoplastic , Fas Ligand Protein/chemistry , Fas Ligand Protein/metabolism , Humans , MEF2 Transcription Factors/chemistry , MEF2 Transcription Factors/metabolism , NFATC Transcription Factors/chemistry , Nuclear Proteins/metabolism , Receptors, Estrogen/chemistry , Receptors, Estrogen/metabolism , p300-CBP Transcription Factors/chemistry , p300-CBP Transcription Factors/metabolismABSTRACT
The adhesion G protein-coupled receptors (aGPCRs) are a large yet poorly understood family of seven-transmembrane proteins. A defining characteristic of the aGPCR family is the conserved GAIN domain, which has autoproteolytic activity and can cleave the receptors near the first transmembrane domain. Several aGPCRs, including ADGRB1 (BAI1 or B1) and ADGRG1 (GPR56 or G1), have been found to exhibit significantly increased constitutive activity when truncated to mimic GAIN domain cleavage (ΔNT). Recent reports have suggested that the new N-terminal stalk, which is revealed by GAIN domain cleavage, can directly activate aGPCRs as a tethered agonist. We tested this hypothesis in studies on two distinct aGPCRs, B1 and G1, by engineering mutant receptors lacking the entire NT including the stalk (B1- and G1-SL, with "SL" indicating "stalkless"). These receptors were evaluated in a battery of signaling assays and compared with full-length wild-type and cleavage-mimicking (ΔNT) forms of the two receptors. We found that B1-SL, in multiple assays, exhibited robust signaling activity, suggesting that the membrane-proximal stalk region is not necessary for its activation. For G1, however, the results were mixed, with the SL mutant exhibiting robust activity in several signaling assays (including TGFα shedding, activation of NFAT luciferase, and ß-arrestin recruitment) but reduced activity relative to ΔNT in a distinct assay (activation of SRF luciferase). These data support a model in which the activation of certain pathways downstream of aGPCRs is stalk-dependent, whereas signaling to other pathways is stalk-independent.
Subject(s)
Angiogenic Proteins/agonists , Models, Molecular , Receptors, G-Protein-Coupled/agonists , Signal Transduction , Allosteric Regulation , Amino Acid Substitution , Angiogenic Proteins/chemistry , Angiogenic Proteins/genetics , Angiogenic Proteins/metabolism , Arrestins/chemistry , Arrestins/genetics , Arrestins/metabolism , Conserved Sequence , Genes, Reporter , HEK293 Cells , Humans , Ligands , NFATC Transcription Factors/agonists , NFATC Transcription Factors/chemistry , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Peptide Fragments/agonists , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Point Mutation , Protein Conformation , Protein Interaction Domains and Motifs , Proteolysis , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Transforming Growth Factor alpha/chemistry , Transforming Growth Factor alpha/genetics , Transforming Growth Factor alpha/metabolism , Ubiquitination , beta-ArrestinsABSTRACT
The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) undergoes remarkable adaptive changes during hibernation. Interestingly, skeletal muscle remodelling occurs during the torpor-arousal cycle of hibernation to prevent net muscle loss despite inactivity. Reversible cardiomyocyte hypertrophy occurs in cardiac muscle, allowing the heart to preserve cardiac output during hibernation, while avoiding chronic maladaptive hypertrophy post-hibernation. We propose that calcium signalling proteins [calcineurin (Cn), calmodulin (CaM), and calpain], the nuclear factor of activated T cell (NFAT) family of transcription factors, and the NFAT targets myoferlin and myomaker contribute significantly to adaptations taking place in skeletal and cardiac muscle during hibernation. Protein-level analyses were performed over several conditions: euthermic room temperature (ER), euthermic cold room (EC), entrance into (EN), early (ET), and late torpor (LT) time points, in addition to early (EA), interbout (IA), and late arousal (LA) time points using immunoblotting and DNA-protein interaction (DPI) enzyme-linked immunosorbent assay (ELISAs). In skeletal and cardiac muscle, NFATc2 protein levels were elevated during torpor. NFATc4 increased throughout the torpor-arousal cycle in both tissues, and NFATc1 showed this trend in cardiac muscle only. NFATc3 showed an elevation in DNA-binding activity but not expression during torpor. Myoferlin protein levels dramatically increased during torpor in both skeletal and cardiac muscle. Myomaker levels also increased significantly in cardiac muscle during torpor. Cardiac Cn levels remained stable, whereas CaM and calpain decreased throughout the torpor-arousal cycle. Activation and/or upregulation of NFATc2, c3, myoferlin, and myomaker at torpor could be part of a stress-response mechanism to preserve skeletal muscle mass, whereas CaM and calpain appear to initiate the rapid reversal of cardiac hypertrophy during arousal through downregulation of the NFAT-Cn pathway.
Subject(s)
Hibernation , Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Myocardium/metabolism , NFATC Transcription Factors/metabolism , Sciuridae/physiology , Amino Acid Sequence , Animals , DNA/metabolism , Muscle Proteins/chemistry , NFATC Transcription Factors/chemistry , Protein BindingABSTRACT
A limited repertoire of PPP family of serine/threonine phosphatases with a highly conserved catalytic domain acts on thousands of protein targets to orchestrate myriad central biological roles. A major structural reorganization of human calcineurin, a ubiquitous Ser/Thr PPP regulated by calcium and calmodulin and targeted by immunosuppressant drugs cyclosporin A and FK506, is unveiled here. The new conformation involves trans- to cis-isomerization of proline in the SAPNY sequence, highly conserved across PPPs, and remodels the main regulatory site where NFATc transcription factors bind. Transitions between cis- and trans-conformations may involve peptidyl prolyl isomerases such as cyclophilin A and FKBP12, which are known to physically interact with and modulate calcineurin even in the absence of immunosuppressant drugs. Alternative conformations in PPPs provide a new perspective on interactions with substrates and other protein partners and may foster development of more specific inhibitors as drug candidates.
Subject(s)
Calcineurin/metabolism , Amino Acid Sequence , Binding Sites , Calcineurin/chemistry , Calcineurin/genetics , Catalytic Domain , Crystallography, X-Ray , Cyclophilin A/metabolism , Cyclosporine/chemistry , Cyclosporine/metabolism , HEK293 Cells , Humans , Isomerism , Molecular Dynamics Simulation , Molecular Sequence Data , NFATC Transcription Factors/chemistry , NFATC Transcription Factors/metabolism , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence Alignment , Tacrolimus Binding Protein 1A/metabolismABSTRACT
Calcineurin (CN), a serine and threonine protein phosphatase that depends on Ca(2+) and calmodulin for its activity, is the target of the immunosuppressant drugs cyclosporin A (CsA) and tacrolimus (FK506). CN dephosphorylates and activates members of the NFATc (nuclear factor of activated T cells) family of transcription factors in T cells by binding to their conserved PxIxIT motif. Upon dephosphorylation, NFATc proteins translocate to the nucleus, where they stimulate the expression of genes encoding cytokines and chemokines that are required for T cell proliferation and the immune response. We performed a pharmacophore-based virtual screening of ~5.5 million commercially available, "drug-like" compounds to identify nonpeptidic compounds that inhibited the CN-dependent activation of NFATc signaling and that could serve as potential drug candidates for immunosuppressive therapy. Of 32 compounds that mimicked the PxIxIT motif, 7 competed with NFATc for binding to CN in vitro without interfering with the phosphatase activity of CN. Furthermore, in activated human CD4(+) T cells, four of the seven compounds inhibited the expression of NFATc-dependent genes, cytokine production, and cell proliferation, suggesting that these may have therapeutic potential as immunosuppressive agents.