RESUMO
Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.
Assuntos
Autoanticorpos/genética , Doenças Autoimunes/genética , Linfócitos B/imunologia , Linfoma/genética , Animais , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos B/patologia , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas de Transporte/genética , Evolução Clonal/genética , Evolução Clonal/imunologia , Ciclina D3/genética , Guanilato Ciclase/genética , Humanos , Proteínas Imediatamente Precoces/genética , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Proteínas Inibidoras de Diferenciação/genética , Linfoma/imunologia , Linfoma/patologia , Camundongos , Mutação/genética , Mutação/imunologia , Proteínas de Neoplasias/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteínas Supressoras de Tumor/genética , Recombinação V(D)J/genéticaRESUMO
Neurons receive input from the outside world or from other neurons through neuronal receptive endings (NREs). Glia envelop NREs to create specialized microenvironments; however, glial functions at these sites are poorly understood. Here, we report a molecular mechanism by which glia control NRE shape and associated animal behavior. The C. elegans AMsh glial cell ensheathes the NREs of 12 neurons, including the thermosensory neuron AFD. KCC-3, a K/Cl transporter, localizes specifically to a glial microdomain surrounding AFD receptive ending microvilli, where it regulates K(+) and Cl(-) levels. We find that Cl(-) ions function as direct inhibitors of an NRE-localized receptor-guanylyl-cyclase, GCY-8, which synthesizes cyclic guanosine monophosphate (cGMP). High cGMP mediates the effects of glial KCC-3 on AFD shape by antagonizing the actin regulator WSP-1/NWASP. Components of this pathway are broadly expressed throughout the nervous system, suggesting that ionic regulation of the NRE microenvironment may be a conserved mechanism by which glia control neuron shape and function.
Assuntos
Caenorhabditis elegans/metabolismo , Neuroglia/metabolismo , Células Receptoras Sensoriais/metabolismo , Simportadores/metabolismo , Animais , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , GMP Cíclico/metabolismo , Guanilato Ciclase/química , Guanilato Ciclase/metabolismo , Potássio/metabolismo , Domínios Proteicos , Simportadores/química , Simportadores/genética , Sensação Térmica , Cotransportadores de K e Cl-RESUMO
Adoptive T cell therapies have produced exceptional responses in a subset of patients with cancer. However, therapeutic efficacy can be hindered by poor T cell persistence and function1. In human T cell cancers, evolution of the disease positively selects for mutations that improve fitness of T cells in challenging situations analogous to those faced by therapeutic T cells. Therefore, we reasoned that these mutations could be co-opted to improve T cell therapies. Here we systematically screened the effects of 71 mutations from T cell neoplasms on T cell signalling, cytokine production and in vivo persistence in tumours. We identify a gene fusion, CARD11-PIK3R3, found in a CD4+ cutaneous T cell lymphoma2, that augments CARD11-BCL10-MALT1 complex signalling and anti-tumour efficacy of therapeutic T cells in several immunotherapy-refractory models in an antigen-dependent manner. Underscoring its potential to be deployed safely, CARD11-PIK3R3-expressing cells were followed up to 418 days after T cell transfer in vivo without evidence of malignant transformation. Collectively, our results indicate that exploiting naturally occurring mutations represents a promising approach to explore the extremes of T cell biology and discover how solutions derived from evolution of malignant T cells can improve a broad range of T cell therapies.
Assuntos
Evolução Molecular , Imunoterapia Adotiva , Linfoma Cutâneo de Células T , Mutação , Linfócitos T , Humanos , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/biossíntese , Citocinas/imunologia , Citocinas/metabolismo , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Imunoterapia Adotiva/métodos , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/imunologia , Linfoma Cutâneo de Células T/patologia , Linfoma Cutâneo de Células T/terapia , Fosfatidilinositol 3-Quinases , Transdução de Sinais/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplanteRESUMO
Pericytes and endothelial cells (ECs) constitute the fundamental components of blood vessels. While the role of ECs in tumor angiogenesis and the tumor microenvironment is well appreciated, pericyte function in tumors remains underexplored. In this study, we used pericyte-specific deletion of the nitric oxide (NO) receptor, soluble guanylate cyclase (sGC), to investigate via single-cell RNA sequencing how pericytes influence the vascular niche and the tumor microenvironment. Our findings demonstrate that pericyte sGC deletion disrupts EC-pericyte interactions, impairing Notch-mediated intercellular communication and triggering extensive transcriptomic reprogramming in both pericytes and ECs. These changes further extended their influence to neighboring cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) through paracrine signaling, collectively suppressing tumor growth. Inhibition of pericyte sGC has minimal impact on quiescent vessels but significantly increases the vulnerability of angiogenic tumor vessels to conventional anti-angiogenic therapy. In conclusion, our findings elucidate the role of pericytes in shaping the tumor vascular niche and tumor microenvironment and support pericyte sGC targeting as a promising strategy for improving anti-angiogenic therapy for cancer treatment.
Assuntos
Neoplasias , Pericitos , Humanos , Pericitos/patologia , Pericitos/fisiologia , Guanilil Ciclase Solúvel , Células Endoteliais/fisiologia , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Neoplasias/genética , Neoplasias/patologia , Guanilato Ciclase , Microambiente TumoralRESUMO
Most eukaryotic cells express small regulatory RNAs. The purpose of one class, the somatic endogenous siRNAs (endo-siRNAs), remains unclear. Here, we show that the endo-siRNA pathway promotes odor adaptation in C. elegans AWC olfactory neurons. In adaptation, the nuclear Argonaute NRDE-3, which acts in AWC, is loaded with siRNAs targeting odr-1, a gene whose downregulation is required for adaptation. Concomitant with increased odr-1 siRNA in AWC, we observe increased binding of the HP1 homolog HPL-2 at the odr-1 locus in AWC and reduced odr-1 mRNA in adapted animals. Phosphorylation of HPL-2, an in vitro substrate of the EGL-4 kinase that promotes adaption, is necessary and sufficient for behavioral adaptation. Thus, environmental stimulation amplifies an endo-siRNA negative feedback loop to dynamically repress cognate gene expression and shape behavior. This class of siRNA may act broadly as a rheostat allowing prolonged stimulation to dampen gene expression and promote cellular memory formation. PAPERFLICK:
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Regulação para Baixo , Guanilato Ciclase/genética , Interferência de RNA , Células Receptoras Sensoriais/metabolismo , Adaptação Fisiológica , Animais , Butanonas/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Odorantes , Fosforilação , RNA de Helmintos/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Nitric oxide (NO) is an essential signaling molecule in biological systems. In mammals, the diatomic gas is critical to the cyclic guanosine monophosphate (cGMP) pathway as it functions as the primary activator of soluble guanylate cyclase (sGC). NO is synthesized from l-arginine and oxygen (O(2)) by the enzyme nitric oxide synthase (NOS). Once produced, NO rapidly diffuses across cell membranes and binds to the heme cofactor of sGC. sGC forms a stable complex with NO and carbon monoxide (CO), but not with O(2). The binding of NO to sGC leads to significant increases in cGMP levels. The second messenger then directly modulates phosphodiesterases (PDEs), ion-gated channels, or cGMP-dependent protein kinases to regulate physiological functions, including vasodilation, platelet aggregation, and neurotransmission. Many studies are focused on elucidating the molecular mechanism of sGC activation and deactivation with a goal of therapeutic intervention in diseases involving the NO/cGMP-signaling pathway. This review summarizes the current understanding of sGC structure and regulation as well as recent developments in NO signaling.
Assuntos
Guanilato Ciclase/química , Óxido Nítrico/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Animais , GMP Cíclico/metabolismo , Guanilato Ciclase/isolamento & purificação , Guanilato Ciclase/metabolismo , Humanos , Isoenzimas/metabolismo , Receptores Citoplasmáticos e Nucleares/isolamento & purificação , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Guanilil Ciclase SolúvelRESUMO
It is unclear how quiescence is enforced in naive T cells, but activation by foreign antigens and self-antigens is allowed, despite the presence of inhibitory signals. We showed that active transforming growth factor ß (TGF-ß) signaling was present in naive T cells, and T cell receptor (TCR) engagement reduced TGF-ß signaling during T cell activation by downregulating TGF-ß type 1 receptor (TßRI) through activation of caspase recruitment domain-containing protein 11 (CARD11) and nuclear factor κB (NF-κB). TGF-ß prevented TCR-mediated TßRI downregulation, but this was abrogated by interleukin-6 (IL-6). Mitigation of TCR-mediated TßRI downregulation through overexpression of TßRI in naive and activated T cells rendered T cells less responsive and suppressed autoimmunity. Naive T cells in autoimmune patients exhibited reduced TßRI expression and increased TCR-driven proliferation compared to healthy subjects. Thus, TCR-mediated regulation of TßRI-TGF-ß signaling acts as a crucial criterion to determine T cell quiescence and activation.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linfócitos T CD4-Positivos/imunologia , Guanilato Ciclase/metabolismo , Ativação Linfocitária/imunologia , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Autoimunidade/imunologia , Proteínas Adaptadoras de Sinalização CARD/genética , Linhagem Celular , Proliferação de Células , Colite/imunologia , Colite/patologia , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Guanilato Ciclase/genética , Células HEK293 , Humanos , Interleucina-6/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/biossíntese , Transdução de Sinais/imunologia , Fator de Crescimento Transformador beta1/biossínteseRESUMO
Light perception for orientation in zoospore-forming fungi is linked to homo- or heterodimeric rhodopsin-guanylyl cyclases (RGCs). Heterodimeric RGCs, first identified in the chytrid Rhizoclosmatium globosum, consist of an unusual near-infrared absorbing highly fluorescent sensitizer neorhodopsin (NeoR) that is paired with a visual light-absorbing rhodopsin responsible for enzyme activation. Here, we present a comprehensive analysis of the distribution of RGC genes in early-branching fungi using currently available genetic data. Among the characterized RGCs, we identified red-sensitive homodimeric RGC variants with maximal light activation close to 600 nm, which allow for red-light control of GTP to cGMP conversion in mammalian cells. Heterodimeric RGC complexes have evolved due to a single gene duplication within the branching of Chytridiales and show a spectral range for maximal light activation between 480 to 600 nm. In contrast, the spectral sensitivity of NeoRs is reaching into the near-infrared range with maximal absorption between 641 and 721 nm, setting the low energy spectral edge of rhodopsins so far. Based on natural NeoR variants and mutational studies, we reevaluated the role of the counterion-triad proposed to cause the extreme redshift. With the help of chimera constructs, we disclose that the cyclase domain is crucial for functioning as homo- or heterodimers, which enables the adaptation of the spectral sensitivity by modular exchange of the photosensor. The extreme spectral plasticity of retinal chromophores in native photoreceptors provides broad perspectives on the achievable spectral adaptation for rhodopsin-based molecular tools ranging from UVB into the near-infrared.
Assuntos
Retina , Rodopsina , Animais , Rodopsina/genética , Células Fotorreceptoras , Luz , Guanilato Ciclase/genética , MamíferosRESUMO
The cardiac natriuretic peptides (NPs) control pivotal physiological actions such as fluid and electrolyte balance, cardiovascular homeostasis, and adipose tissue metabolism by activating their receptor enzymes [natriuretic peptide receptor-A (NPRA) and natriuretic peptide receptor-B (NPRB)]. These receptors are homodimers that generate intracellular cyclic guanosine monophosphate (cGMP). The natriuretic peptide receptor-C (NPRC), nicknamed the clearance receptor, lacks a guanylyl cyclase domain; instead, it can bind the NPs to internalize and degrade them. The conventional paradigm is that by competing for and internalizing NPs, NPRC blunts the ability of NPs to signal through NPRA and NPRB. Here we show another previously unknown mechanism by which NPRC can interfere with the cGMP signaling function of the NP receptors. By forming a heterodimer with monomeric NPRA or NPRB, NPRC can prevent the formation of a functional guanylyl cyclase domain and thereby suppress cGMP production in a cell-autonomous manner.
Assuntos
Guanilato Ciclase , Receptores do Fator Natriurético Atrial , Guanilato Ciclase/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Receptores de Peptídeos/metabolismo , Peptídeos Natriuréticos , Transdução de Sinais , Fator Natriurético Atrial/metabolismo , GMP Cíclico/metabolismoRESUMO
Mutations in receptor guanylyl cyclase C (GC-C) cause severe gastrointestinal disease, including meconium ileus, early onset acute diarrhea, and pediatric inflammatory bowel disease that continues into adulthood. Agonists of GC-C are US Food and Drug Administration-approved drugs for the treatment of constipation and irritable bowel syndrome. Therapeutic strategies targeting GC-C are tested in preclinical mouse models, assuming that murine GC-C mimics human GC-C in its biochemical properties and downstream signaling events. Here, we reveal important differences in ligand-binding affinity and GC activity between mouse GC-C and human GC-C. We generated a series of chimeric constructs of various domains of human and mouse GC-C to show that the extracellular domain of mouse GC-C contributed to log-orders lower affinity of mouse GC-C for ligands than human GC-C. Further, the Vmax of the murine GC domain was lower than that of human GC-C, and allosteric regulation of the receptor by ATP binding to the intracellular kinase-homology domain also differed. These altered properties are reflected in the high concentrations of ligands required to elicit signaling responses in the mouse gut in preclinical models and the specificity of a GC inhibitor towards human GC-C. Therefore, our studies identify considerations in using the murine model to test molecules for therapeutic purposes that work as either agonists or antagonists of GC-C, and vaccines for the bacterial heat-stable enterotoxin that causes watery diarrhea in humans.
Assuntos
Receptores Acoplados a Guanilato Ciclase , Animais , Criança , Humanos , Camundongos , Diarreia , Enterotoxinas , Guanilato Ciclase/metabolismo , Ligantes , Receptores de Enterotoxina/genética , Receptores Acoplados a Guanilato Ciclase/antagonistas & inibidores , Receptores Acoplados a Guanilato Ciclase/genética , Receptores Acoplados a Guanilato Ciclase/metabolismo , Gastroenteropatias/tratamento farmacológico , Gastroenteropatias/metabolismo , Gastroenteropatias/patologiaRESUMO
Although T cell activation can result from signaling via T cell antigen receptor (TCR) alone, physiological T cell responses require costimulation via the coreceptor CD28. Through the use of an N-ethyl-N-nitrosourea-mutagenesis screen, we identified a mutation in Rltpr. We found that Rltpr was a lymphoid cell-specific, actin-uncapping protein essential for costimulation via CD28 and the development of regulatory T cells. Engagement of TCR-CD28 at the immunological synapse resulted in the colocalization of CD28 with both wild-type and mutant Rltpr proteins. However, the connection between CD28 and protein kinase C-θ and Carma1, two key effectors of CD28 costimulation, was abrogated in T cells expressing mutant Rltpr, and CD28 costimulation did not occur in those cells. Our findings provide a more complete model of CD28 costimulation in which Rltpr has a key role.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/imunologia , Antígenos CD28/imunologia , Proteínas de Transporte/imunologia , Guanilato Ciclase/imunologia , Proteína Quinase C/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/genética , Citometria de Fluxo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos , Microscopia Confocal , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Alinhamento de Sequência , Análise de Sequência de DNA , Organismos Livres de Patógenos EspecíficosRESUMO
A strategy combining targeted therapies is effective in B-cell lymphomas (BCL), such as mantle cell lymphoma (MCL), but acquired resistances remain a recurrent issue. In this study, we performed integrative longitudinal genomic and single-cell RNA-sequencing analyses of patients with MCL who were treated with targeted therapies against CD20, BCL2, and Bruton tyrosine kinase (OAsIs trial). We revealed the emergence of subclones with a selective advantage against OAsIs combination in vivo and showed that resistant cells were characterized by B-cell receptor (BCR)-independent overexpression of NF-κB1 target genes, especially owing to CARD11 mutations. Functional studies demonstrated that CARD11 gain of function not only resulted in BCR independence but also directly increased the transcription of the antiapoptotic BCL2A1, leading to resistance against venetoclax and OAsIs combination. Based on the transcriptional profile of OAsIs-resistant subclones, we designed a 16-gene resistance signature that was also predictive for patients with MCL who were treated with conventional chemotherapy, underlying a common escape mechanism. Among druggable strategies to inhibit CARD11-dependent NF-κB1 transduction, we evaluated the selective inhibition of its essential partner MALT1. We demonstrated that MALT1 protease inhibition led to a reduction in the expression of genes involved in OAsIs resistance, including BCL2A1. Consequently, MALT1 inhibition induced synergistic cell death in combination with BCL2 inhibition, irrespective of CARD11 mutational status, both in vitro and in vivo. Taken together, our study identified mechanisms of resistance to targeted therapies and provided a novel strategy to overcome resistance in aggressive BCL. The OAsIs trial was registered at www.clinicaltrials.gov #NCT02558816.
Assuntos
Linfoma Difuso de Grandes Células B , Linfoma de Célula do Manto , Adulto , Humanos , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linhagem Celular Tumoral , Mutação com Ganho de Função , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma de Célula do Manto/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Nuclear receptors recruit multiple coactivators sequentially to activate transcription. This "ordered" recruitment allows different coactivator activities to engage the nuclear receptor complex at different steps of transcription. Estrogen receptor (ER) recruits steroid receptor coactivator-3 (SRC-3) primary coactivator and secondary coactivators, p300/CBP and CARM1. CARM1 recruitment lags behind the binding of SRC-3 and p300 to ER. Combining cryo-electron microscopy (cryo-EM) structure analysis and biochemical approaches, we demonstrate that there is a close crosstalk between early- and late-recruited coactivators. The sequential recruitment of CARM1 not only adds a protein arginine methyltransferase activity to the ER-coactivator complex, it also alters the structural organization of the pre-existing ERE/ERα/SRC-3/p300 complex. It induces a p300 conformational change and significantly increases p300 HAT activity on histone H3K18 residues, which, in turn, promotes CARM1 methylation activity on H3R17 residues to enhance transcriptional activity. This study reveals a structural role for a coactivator sequential recruitment and biochemical process in ER-mediated transcription.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteína p300 Associada a E1A/metabolismo , Receptor alfa de Estrogênio/metabolismo , Guanilato Ciclase/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Transcrição Gênica , Acetilação , Sítios de Ligação , Proteínas Adaptadoras de Sinalização CARD/química , Proteínas Adaptadoras de Sinalização CARD/genética , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína p300 Associada a E1A/química , Proteína p300 Associada a E1A/genética , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Guanilato Ciclase/química , Guanilato Ciclase/genética , Células HEK293 , Células HeLa , Histonas/química , Histonas/metabolismo , Humanos , Células MCF-7 , Metilação , Modelos Moleculares , Complexos Multiproteicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Coativador 3 de Receptor Nuclear/química , Coativador 3 de Receptor Nuclear/genética , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Fatores de Tempo , Fatores de Transcrição , Ativação Transcricional , TransfecçãoRESUMO
Nitric oxide (NO) signaling in biology relies on its activating cyclic guanosine monophosphate (cGMP) production by the NO receptor soluble guanylyl cyclase (sGC). sGC must obtain heme and form a heterodimer to become functional, but paradoxically often exists as an immature heme-free form in cells and tissues. Based on our previous finding that NO can drive sGC maturation, we investigated its basis by utilizing a fluorescent sGC construct whose heme level can be monitored in living cells. We found that NO generated at physiologic levels quickly triggered cells to mobilize heme to immature sGC. This occurred when NO was generated within cells or by neighboring cells, began within seconds of NO exposure, and led cells to construct sGC heterodimers and thus increase their active sGC level by several-fold. The NO-triggered heme deployment involved cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-heme complexes and required the chaperone hsp90, and the newly formed sGC heterodimers remained functional long after NO generation had ceased. We conclude that NO at physiologic levels triggers assembly of its own receptor by causing a rapid deployment of cellular heme. Redirecting cellular heme in response to NO is a way for cells and tissues to modulate their cGMP signaling and to more generally tune their hemeprotein activities wherever NO biosynthesis takes place.
Assuntos
Heme/metabolismo , Óxido Nítrico/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Cálcio , GMP Cíclico , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Guanilato Ciclase/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Modelos Biológicos , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Transdução de Sinais , Guanilil Ciclase Solúvel/metabolismoRESUMO
Transcription factors (TFs) play critical roles in hematopoiesis, and their aberrant expression can lead to various types of leukemia. The t(8;21) leukemogenic fusion protein AML1-ETO (AE) is the most common fusion protein in acute myeloid leukemia and can enhance hematopoietic stem cell renewal while blocking differentiation. A key question in understanding AE-mediated leukemia is what determines the choice of AE to activate self-renewal genes or repress differentiation genes. Toward the resolution of this problem, we earlier showed that AE resides in the stable AETFC complex and that its components colocalize on up- or down-regulated target genes and are essential for leukemogenesis. In the current study, using biochemical and genomic approaches, we show that AE-containing complexes are heterogeneous, and that assembly of the larger AETFC (containing AE, CBFß, HEB, E2A, LYL1, LMO2, and LDB1) requires LYL1. Furthermore, we provide strong evidence that the LYL1-containing AETFC preferentially binds to active enhancers and promotes AE-dependent gene activation. Moreover, we show that coactivator CARM1 interacts with AETFC and facilitates gene activation by AETFC. Collectively, this study describes a role of oncoprotein LYL1 in AETFC assembly and gene activation by recruiting CARM1 to chromatin for AML cell survival.
Assuntos
Leucemia Mieloide Aguda , Proteínas de Fusão Oncogênica , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas Adaptadoras de Sinalização CARD , Cromatina , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Guanilato Ciclase , Humanos , Proteínas com Homeodomínio LIM/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína-Arginina N-Metiltransferases , Ativação TranscricionalRESUMO
Human cone outer segment (COS) length changes in response to stimuli bleaching up to 99% of L- and M-cone opsins were measured with high resolution, phase-resolved optical coherence tomography (OCT). Responses comprised a fast phase (â¼5 ms), during which COSs shrink, and two slower phases (1.5 s), during which COSs elongate. The slower components saturated in amplitude (â¼425 nm) and initial rate (â¼3 nm ms-1) and are well described over the 200-fold bleaching range as the sum of two exponentially rising functions with time constants of 80 to 90 ms (component 1) and 1,000 to 1,250 ms (component 2). Measurements with adaptive optics reflection densitometry revealed component 2 to be linearly related to cone pigment bleaching, and the hypothesis is proposed that it arises from cone opsin and disk membrane swelling triggered by isomerization and rate-limited by chromophore hydrolysis and its reduction to membrane-localized all-trans retinol. The light sensitivity and kinetics of component 1 suggested that the underlying mechanism is an osmotic response to an amplified soluble by-product of phototransduction. The hypotheses that component 1 corresponds to G-protein subunits dissociating from the membrane, metabolites of cyclic guanosine monophosphate (cGMP) hydrolysis, or by-products of activated guanylate cyclase are rejected, while the hypothesis that it corresponds to phosphate produced by regulator of G-protein signaling 9 (RGS9)-catalyzed hydrolysis of guanosine triphosphate (GTP) in G protein-phosphodiesterase complexes was found to be consistent with the results. These results provide a basis for the assessment with optoretinography of phototransduction in individual cone photoreceptors in health and during disease progression and therapeutic interventions.
Assuntos
Opsinas dos Cones , GTP Fosfo-Hidrolases , Fosfatos , Proteínas RGS , Células Fotorreceptoras Retinianas Cones , Catálise , Opsinas dos Cones/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Guanosina Monofosfato/metabolismo , Guanosina Trifosfato/metabolismo , Guanilato Ciclase/metabolismo , Humanos , Osmose , Fosfatos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Subunidades Proteicas/metabolismo , Proteínas RGS/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Vitamina A/metabolismoRESUMO
Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5R22A) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5WT). Here, we present ITC, NMR, and functional analysis of GCAP5R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5R22A binds a total of 3 Ca2+, including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5WT were not detected by ITC, suggesting that R22A may affect the binding of Ca2+ to these sites. The NMR-derived structure of GCAP5R22A is overall similar to that of GCAP5WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5R22A with â¼25% higher affinity compared to GCAP5WT, consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.
Assuntos
Proteínas Ativadoras de Guanilato Ciclase , Guanilato Ciclase , Modelos Moleculares , Animais , Cálcio/metabolismo , Ativação Enzimática/genética , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Guanilato Ciclase/química , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/química , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Multimerização Proteica , Retina/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismoRESUMO
Cilia serve as cellular antennae that translate sensory information into physiological responses. In the sperm flagellum, a single chemoattractant molecule can trigger a Ca2+ rise that controls motility. The mechanisms underlying such ultra-sensitivity are ill-defined. Here, we determine by mass spectrometry the copy number of nineteen chemosensory signaling proteins in sperm flagella from the sea urchin Arbacia punctulata. Proteins are up to 1,000-fold more abundant than the free cellular messengers cAMP, cGMP, H+ , and Ca2+ . Opto-chemical techniques show that high protein concentrations kinetically compartmentalize the flagellum: Within milliseconds, cGMP is relayed from the receptor guanylate cyclase to a cGMP-gated channel that serves as a perfect chemo-electrical transducer. cGMP is rapidly hydrolyzed, possibly via "substrate channeling" from the channel to the phosphodiesterase PDE5. The channel/PDE5 tandem encodes cGMP turnover rates rather than concentrations. The rate-detection mechanism allows continuous stimulus sampling over a wide dynamic range. The textbook notion of signal amplification-few enzyme molecules process many messenger molecules-does not hold for sperm flagella. Instead, high protein concentrations ascertain messenger detection. Similar mechanisms may occur in other small compartments like primary cilia or dendritic spines.
Assuntos
Arbacia/fisiologia , Quimiotaxia , Proteômica , Transdução de Sinais , Animais , Arbacia/ultraestrutura , Cálcio/metabolismo , Cílios/fisiologia , Cílios/ultraestrutura , GMP Cíclico/metabolismo , Tomografia com Microscopia Eletrônica , Flagelos/fisiologia , Flagelos/ultraestrutura , Guanilato Ciclase/metabolismo , Masculino , Espectrometria de Massas , Espermatozoides/fisiologia , Espermatozoides/ultraestruturaRESUMO
Revertant mosaicism, or "natural gene therapy," refers to the spontaneous in vivo reversion of an inherited mutation in a somatic cell. Only approximately 50 human genetic disorders exhibit revertant mosaicism, implicating a distinctive role played by mutant proteins in somatic correction of a pathogenic germline mutation. However, the process by which mutant proteins induce somatic genetic reversion in these diseases remains unknown. Here we show that heterozygous pathogenic CARD14 mutations causing autoinflammatory skin diseases, including psoriasis and pityriasis rubra pilaris, are repaired mainly via homologous recombination. Rather than altering the DNA damage response to exogenous stimuli, such as X-irradiation or etoposide treatment, mutant CARD14 increased DNA double-strand breaks under conditions of replication stress. Furthermore, mutant CARD14 suppressed new origin firings without promoting crossover events in the replication stress state. Together, these results suggest that mutant CARD14 alters the replication stress response and preferentially drives break-induced replication (BIR), which is generally suppressed in eukaryotes. Our results highlight the involvement of BIR in reversion events, thus revealing a previously undescribed role of BIR that could potentially be exploited to develop therapeutics for currently intractable genetic diseases.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Replicação do DNA , Guanilato Ciclase/genética , Recombinação Homóloga , Proteínas de Membrana/genética , Mosaicismo , Mutação , Pitiríase Rubra Pilar/patologia , Psoríase/patologia , Estresse Fisiológico , Ciclo Celular , Humanos , Pitiríase Rubra Pilar/genética , Psoríase/genéticaRESUMO
The CARMA1-Bcl10-MALT1 (CBM) signalosome is a crucial module of NF-κB activation in B cell receptor (BCR) signaling. Biophysical studies have shown that the E3 ubiquitin ligase TRAF6 cooperatively modifies the CBM signalosome; however, the specific details regarding how TRAF6 is involved in BCR signal-induced CBM formation remain unclear. In this study, we aimed to reveal the influences of TRAF6 on CBM formation and TAK1 and IKK activities using DT40 B cells which lack all the exons of TRAF6. In TRAF6-null cells we found: (i) attenuation of TAK1 activity and abolishment of IKK activity and (ii) sustained binding of CARMA1 to Bcl10. To account for the molecular mechanism causing these dynamics, we performed a mathematical model analysis. The mathematical model analysis showed that the regulation of IKK activation by TRAF6 can reproduce TAK1 and IKK activities in TRAF6 null cells, and that the TRAF6 related signal-dependent inhibitor suppresses CARMA1 binding to Bcl10 in wild-type cells. These results suggest that TRAF6 contributes to the positive regulation of IKK activation via TAK1, alongside the negative signal-dependent regulation of CARMA1 binding to Bcl10.