RESUMO
Planar cell polarity (PCP) signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of protein phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one serine/threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling.
Assuntos
Proteínas de Drosophila , Proteínas de Membrana , Animais , Polaridade Celular/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
The core components of the planar cell polarity (PCP) signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. We find that the E3 ubiquitin ligase complex Cullin1(Cul1)/SkpA/Supernumerary limbs(Slimb) regulates the stability of one of the peripheral membrane components, Prickle (Pk). Excess Pk disrupts PCP feedback and prevents asymmetry. We show that Pk participates in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang) and Flamingo (Fmi), and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. Our results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling.
Assuntos
Polaridade Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Endocitose , Proteínas com Domínio LIM/metabolismo , Ubiquitinação , Animais , Análise por Conglomerados , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas com Domínio LIM/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Asas de Animais/metabolismoRESUMO
Microtubules (MTs) are substrates upon which plus- and minus-end directed motors control the directional movement of cargos that are essential for generating cell polarity. Although centrosomal MTs are organized with plus-ends away from the MT organizing center, the regulation of non-centrosomal MT polarity is poorly understood. Increasing evidence supports the model that directional information for planar polarization is derived from the alignment of a parallel apical network of MTs and the directional MT-dependent trafficking of downstream signaling components. The Fat/Dachsous/Four-jointed (Ft/Ds/Fj) signaling system contributes to orienting those MTs. In addition to previously defined functions in promoting asymmetric subcellular localization of 'core' planar cell polarity (PCP) proteins, we find that alternative Prickle (Pk-Sple) protein isoforms control the polarity of this MT network. This function allows the isoforms of Pk-Sple to differentially determine the direction in which asymmetry is established and therefore, ultimately, the direction of tissue polarity. Oppositely oriented signals that are encoded by oppositely oriented Fj and Ds gradients produce the same polarity outcome in different tissues or compartments, and the tissue-specific activity of alternative Pk-Sple protein isoforms has been observed to rectify the interpretation of opposite upstream directional signals. The control of MT polarity, and thus the directionality of apical vesicle traffic, by Pk-Sple provides a mechanism for this rectification.
Assuntos
Polaridade Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Proteínas com Domínio LIM/metabolismo , Microtúbulos/metabolismo , Animais , Vesículas Citoplasmáticas/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Cabelo/citologia , Cabelo/crescimento & desenvolvimento , Cabelo/metabolismo , Mutação/genética , Isoformas de Proteínas/metabolismo , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismoRESUMO
Ral is a small Ras-like GTPase that regulates membrane trafficking and signaling. Here, we show that in response to planar cell polarity (PCP) signals, Ral modulates asymmetric Notch signaling in the Drosophila eye. Specification of the initially equivalent R3/R4 photoreceptor precursor cells in each developing ommatidium occurs in response to a gradient of Frizzled (Fz) signaling. The cell with the most Fz signal (R3) activates the Notch receptor in the adjacent cell (R4) via the ligand Delta, resulting in R3/R4 cell determination and their asymmetric positions within the ommatidium. Two mechanisms have been proposed for ensuring that the cell with the most Fz activation sends the Delta signal: Fz-dependent transcriptional upregulation in R3 of genes that promote Delta signaling, and direct blockage of Notch receptor activation in R3 by localization of an activated Fz/Disheveled protein complex to the side of the plasma membrane adjacent to R4. Here, we discover a distinct mechanism for biasing the direction of Notch signaling that depends on Ral. Using genetic experiments in vivo, we show that, in direct response to Fz signaling, Ral transcription is upregulated in R3, and Ral represses ligand-independent activation of Notch in R3. Thus, prevention of ligand-independent Notch activation is not simply a constitutive process, but is a target for regulation by Ral during cell fate specification and pattern formation.
Assuntos
Olho Composto de Artrópodes/metabolismo , Proteínas de Drosophila/metabolismo , Receptores Frizzled/metabolismo , Proteínas de Membrana/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Proteínas ral de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Linhagem da Célula/fisiologia , Polaridade Celular/fisiologia , Drosophila , Proteínas de Drosophila/genética , Receptores Frizzled/genética , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Receptores Acoplados a Proteínas G/genética , Receptores Notch/genética , Proteínas ral de Ligação ao GTP/genéticaRESUMO
The growth and survival of cells with different fitness, such as those with a proliferative advantage or a deleterious mutation, is controlled through cell competition. During development, cell competition enables healthy cells to eliminate less fit cells that could jeopardize tissue integrity, and facilitates the elimination of pre-malignant cells by healthy cells as a surveillance mechanism to prevent oncogenesis. Malignant cells also benefit from cell competition to promote their expansion. Despite its ubiquitous presence, the mechanisms governing cell competition, particularly those common to developmental competition and tumorigenesis, are poorly understood. Here, we show that in Drosophila, the planar cell polarity (PCP) protein Flamingo (Fmi) is required by winners to maintain their status during cell competition in malignant tumors to overtake healthy tissue, in early pre-malignant cells when they overproliferate among wildtype cells, in healthy cells when they later eliminate pre-malignant cells, and by supercompetitors as they compete to occupy excessive territory within wildtype tissues. "Would-be" winners that lack Fmi are unable to over-proliferate, and instead become losers. We demonstrate that the role of Fmi in cell competition is independent of PCP, and that it uses a distinct mechanism that may more closely resemble one used in other less well-defined functions of Fmi.
RESUMO
Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. Epsin's mechanism of function is complex and context-dependent. Consequently, how Epsin promotes ligand endocytosis and thus Notch signaling is unclear, as is why Notch signaling is uniquely dependent on Epsin. Here, by generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, we tested each of the five protein or lipid interaction modules for a role in Notch activation by each of the two ligands, Serrate and Delta. There are five main results of this work that impact present thinking about the role of Epsin in ligand cells. First, we discovered that deletion or mutation of both UIMs destroyed Epsin's function in Notch signaling and had a greater negative impact on Epsin activity than removal of any other module type. Second, only one of Epsin's two UIMs was essential. Third, the lipid-binding function of the ENTH domain was required only for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins were necessary collectively for Epsin activity, their functions were highly redundant; most unexpected was the finding that Epsin's Clathrin binding motifs were dispensable. Finally, we found that signaling from either ligand, Serrate or Delta, required the same Epsin modules. All of these observations are consistent with a model where Epsin's essential function in ligand cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins. We propose that Epsin's specificity for Notch signaling simply reflects its unique ability to interact with the plasma membrane, Ubiquitin, and proteins that bind Clathrin.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Receptores Notch/metabolismo , Ubiquitina/metabolismo , Complexo 2 de Proteínas Adaptadoras/química , Complexo 2 de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Clatrina/química , Clatrina/metabolismo , Drosophila melanogaster/genética , Feminino , Deleção de Genes , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Jagged-1 , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serrate-Jagged , Transdução de Sinais , Ubiquitina/químicaRESUMO
Planar Cell Polarity (PCP) signaling polarizes epithelial cells in a plane orthogonal to their apical-basal axis. A core PCP signaling module both generates molecular asymmetry within cells and coordinates the direction of polarization between neighboring cells. Two subcomplexes of core proteins segregate to opposite sides of the cell, defining a polarity axis. Homodimers of the atypical cadherin Flamingo are thought to be the scaffold upon which these subcomplexes assemble and are required for intercellular polarity signaling. The central role for Flamingo homodimers in scaffolding and intercellular communication suggests that cells in which intercellular signaling via Flamingo is disabled should fail to polarize. We show that cells lacking Flamingo, or bearing a truncated Flamingo that cannot homodimerize do in fact polarize. Cell polarization requires both positive and negative feedback, and in a multicellular tissue, feedback might involve both intracellular and intercellular pathways. We identify positive and negative feedback pathways that operate cell autonomously to drive polarization.
RESUMO
PCP signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of Protein Phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one Serine/Threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling.
RESUMO
The capacity of SARS-CoV-2 to evolve poses challenges to conventional prevention and treatment options such as vaccination and monoclonal antibodies, as they rely on viral receptor binding domain (RBD) sequences from previous strains. Additionally, animal CoVs, especially those of the SARS family, are now appreciated as a constant pandemic threat. We present here a new antiviral approach featuring inhalation delivery of a recombinant viral trap composed of ten copies of angiotensin-converting enzyme 2 (ACE2) fused to the IgM Fc. This ACE2 decamer viral trap is designed to inhibit SARS-CoV-2 entry function, regardless of viral RBD sequence variations as shown by its high neutralization potency against all known SARS-CoV-2 variants, including Omicron BQ.1, BQ.1.1, XBB.1 and XBB.1.5. In addition, it demonstrates potency against SARS-CoV-1, human NL63, as well as bat and pangolin CoVs. The multivalent trap is effective in both prophylactic and therapeutic settings since a single intranasal dosing confers protection in human ACE2 transgenic mice against viral challenges. Lastly, this molecule is stable at ambient temperature for more than twelve weeks and can sustain physical stress from aerosolization. These results demonstrate the potential of a decameric ACE2 viral trap as an inhalation solution for ACE2-dependent coronaviruses of current and future pandemic concerns.
Assuntos
Infecções por Coronavirus , Coronavirus , Animais , Camundongos , Humanos , Enzima de Conversão de Angiotensina 2/metabolismo , Ligação Proteica , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/metabolismo , Glicoproteína da Espícula de CoronavírusRESUMO
Planar cell polarity (PCP) signaling regulates several polarization events during development of ommatidia in the Drosophila eye, including directing chirality by polarizing a cell fate choice and determining the direction and extent of ommatidial rotation. The pksple isoform of the PCP protein Prickle is known to participate in the R3/R4 cell fate decision, but the control of other polarization events and the potential contributions of the three Pk isoforms have not been clarified. Here, by characterizing expression and subcellular localization of individual isoforms together with re-analyzing isoform specific phenotypes, we show that the R3/R4 fate decision, its coordination with rotation direction, and completion of rotation to a final ±90° rotation angle are separable polarization decisions with distinct Pk isoform requirements and contributions. Both pksple and pkpk can enforce robust R3/R4 fate decisions, but only pksple can correctly orient them along the dorsal-ventral axis. In contrast, pksple and pkpk can fully and interchangeably sustain coordination of rotation direction and rotation to completion. We propose that expression dynamics and competitive interactions determine isoform participation in these processes. We propose that the selective requirement for pksple to orient the R3/R4 decision and their interchangeability for coordination and completion of rotation reflects their previously described differential interaction with the Fat/Dachsous system which is known to be required for orientation of R3/R4 decisions but not for coordination or completion of rotation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Olho/metabolismo , Proteínas com Domínio LIM/metabolismo , Animais , Encéfalo/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Edição de Genes , Genótipo , Proteínas com Domínio LIM/antagonistas & inibidores , Proteínas com Domínio LIM/genética , Proteínas de Membrana/metabolismo , Fenótipo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/genéticaRESUMO
Subcellular asymmetry directed by the planar cell polarity (PCP) signaling pathway orients numerous morphogenetic events in both invertebrates and vertebrates. Here, we describe a morphogenetic movement in which the intertwined socket and shaft cells of the Drosophila anterior wing margin mechanosensory bristles undergo PCP-directed apical rotation, inducing twisting that results in a helical structure of defined chirality. We show that the Frizzled/Vang PCP signaling module coordinates polarity among and between bristles and surrounding cells to direct this rotation. Furthermore, we show that dynamic interplay between two isoforms of the Prickle protein determines right- or left-handed bristle morphogenesis. We provide evidence that, Frizzled/Vang signaling couples to the Fat/Dachsous PCP directional signal in opposite directions depending on whether Pkpk or Pksple predominates. Dynamic interplay between Pk isoforms is likely to be an important determinant of PCP outcomes in diverse contexts. Similar mechanisms may orient other lateralizing morphogenetic processes.
Our right and left hands are mirror images of each other and cannot be precisely superimposed. This property, known as chirality, is vital for many tissues and organs to form correctly in humans and other animals. For example, fruit flies have hair-like sensory organs on the edges of their wings known as bristles. One of the cells in each bristle forms a shaft that generally tilts away from the main body of the fly and is anchored in place by another cell known as the socket.A signaling pathway known as PCP signaling controls the directions in which many chiral tissues and organs in animals form. The pathway contains two signaling modules: the global module collects "directional" information about the orientation of the body and sends it to the core module, which interprets this information to control how the tissue or organ grows.Fruit flies have two different versions of one of the core module components known as Prickle and Spiny legs that are thought to alter the direction the core module responds to the information it receives. Mutant flies known as pkpk mutants are unable to make Prickle and their wing bristles tilt in the opposite direction compared to those in normal flies, but it was not clear exactly why this happens.To address this question, Cho et al. studied PCP signaling in the wings of normal and pkpk mutant flies. The experiments showed that Prickle directed the bristles on the right wing of a normal fly to grow in left-handed corkscrew-like patterns in which the emerging shaft and socket of each bristle twisted around each other. As a result, the bristles tilted away from the bodies of the flies. In the pkpk mutants, however, Spiny legs substituted for Prickle, causing the equivalent bristles to grow in a right-handed corkscrew pattern and tilt towards the body.The findings of Cho et al. show that PCP signaling controls the direction fly bristles grow by selectively using Prickle and Spiny legs. In the future, this work may also aid efforts to develop effective screening and treatments for birth defects that result from the failure of chiral tissues and organs to form properly.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Domínio LIM/fisiologia , Asas de Animais/embriologia , Alelos , Animais , Padronização Corporal , Polaridade Celular , Proteínas de Ligação a DNA/química , Proteínas de Drosophila/química , Genótipo , Proteínas com Domínio LIM/química , Morfogênese , Mutação , Isoformas de Proteínas , Transdução de Sinais , Asas de Animais/metabolismoRESUMO
Serological analysis of cDNA expression library (SEREX) was employed to identify cancer-associated genes. By screening cDNA expression libraries with sera of patients with lung cancers, we identified a total of 49 genes that specifically reacted with the sera of patients with lung cancers. Among these, we characterized a novel gene with expression pattern similar to that of cancer/testis antigens. Its open reading frame is 1920 bp in size and encodes for putative protein composed of 639 amino acids. Southern blot analysis reveals that this gene exists as single copy. In vitro transcription/translation and Western blot analysis confirm that this gene encodes a protein of 73 kDa in size. The comparison of cDNA and genomic sequences reveals that it is composed of 11 exons and 10 introns. This gene displays testis-specific expression among normal tissues, and wide expression among various cancer tissues and cancer cell lines. A study using GFP fusion construct reveals mainly nuclear localization of CAGE-1 protein. The expression of this clone is relatively higher in cancer tissues compared with their surrounding non-cancerous tissues. This suggests that overexpression of CAGE-1 may be associated with the progression of tumor. Because of its association with cancer, this gene was named cancer-associated gene-1 (CAGE-1). Given the fact that several cancer/testis antigens reportedly induce cytolytic T lymphocyte (CTL) reactions, it is reasonable that this gene will be a valuable target for cancer immunotherapy. The exact functional role of CAGE-1 in tumorigenesis remains to be seen.
Assuntos
Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Sequência de Aminoácidos , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Sequência de Bases , DNA Complementar/isolamento & purificação , Escherichia coli/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/imunologia , Masculino , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Proteínas Nucleares/química , Testículo/imunologia , Testículo/metabolismo , TransfecçãoRESUMO
We discovered recently that the Drosophila Ral GTPase regulates Notch signaling and thereby affects cell patterning in the eye. Although Ral functions in the ligand signaling cells, Ral does not stimulate ligand signaling directly. Rather, in cells that express both Notch receptor and ligand, Ral activity promotes a cell to become the signaler by inhibiting Notch receptor activation in that cell. Moreover, Ral inhibits a particular pathway of Notch activation-receptor activation that occurs independent of ligand binding. In this Commentary, we discuss the phenomenon of ligand-independent Notch receptor activation and how this event might be regulated by Ral.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Olho/crescimento & desenvolvimento , Receptores Notch/metabolismo , Transdução de Sinais , Proteínas ral de Ligação ao GTP/metabolismo , Animais , Padronização Corporal , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/análise , Proteínas de Drosophila/genética , Endossomos/metabolismo , Olho/citologia , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Receptores Notch/análise , Receptores Notch/genética , Proteínas ral de Ligação ao GTP/análise , Proteínas ral de Ligação ao GTP/genéticaRESUMO
Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. Here, we use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, we find that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, we find that Drosophila female germline cells, the one cell type known to signal without clathrin, also do not require auxilin to signal. Third, we find that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling.
Assuntos
Auxilinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Endocitose , Receptores Notch/metabolismo , Transdução de Sinais , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Auxilinas/genética , Clatrina/metabolismo , Proteínas de Drosophila/genética , Olho/metabolismo , Olho/patologia , Feminino , Ligantes , Mutação/genética , Ovário/citologia , Ovário/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
We applied serological analysis of cDNA expression library technique to identify cancer-associated genes. We screened cDNA expression libraries of human testis and gastric cancer cell lines with sera of patients with gastric cancers. We identified a gene whose expression is testis-specific among normal tissues. We cloned and characterized this novel gene. It contains D-E-A-D box domain and encodes a putative protein of 630 amino acids with possible helicase activity. It showed wide expression in various cancer tissues and cancer cell lines. The corresponding gene was named cancer-associated gene (CAGE). PCR of human x hamster Radiation Hybrids showed localization of CAGE on the human chromosome Xp22. Transient transfection of CAGE showed predominantly nuclear localization. Both Western blot and plaque assay indicated seroreactivity of CAGE protein. We found that demethylation played a role in the activation of CAGE in some cancer cell lines that do not express it. Cell synchronization experiments showed that the expression of CAGE was related with cell cycle. This suggests that CAGE might play a role in cellular proliferation. Because CAGE is expressed in a variety of cancers but not in normal tissues except testis, this gene can be a target of antitumor immunotherapy.
Assuntos
Adenoma/genética , Antígenos de Neoplasias/genética , Carcinoma/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Neoplasias Gástricas/genética , Testículo/imunologia , Adenoma/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Antígenos Nucleares , Sequência de Bases , Carcinoma/imunologia , Ciclo Celular/fisiologia , Cricetinae , RNA Helicases DEAD-box , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Alinhamento de Sequência , Neoplasias Gástricas/imunologia , Testículo/química , Testículo/metabolismo , Distribuição Tecidual , Células Tumorais Cultivadas , Cromossomo XRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of antinuclear antibodies. We performed serological analysis of cDNA expression library (SEREX) to identify autoantibodies associated with SLE. The screening of three different cDNA expression libraries with pooled sera of patients with SLE yielded 11 independent clones that reacted with pooled sera of patients with SLE. In this screening, autoantibodies to poly(ADP-ribose) polymerase (PARP), U1snRNP, and galectin-3 were prevalent in the sera of patients with SLE (26/68, 25/68, 12/63, respectively). The frequency of autoantibody to PARP was significantly higher in SLE than that of healthy donors (0/76) (38.2% vs 0%, p<0.00001). The autoantibody to PARP was infrequently detected in the serum of patients with RA (1/50). However, autoantibody to PARP was not found in the sera of patients with other rheumatic diseases including Sjogren's syndrome (0/19), systemic sclerosis (0/18), and polymyositis/myositis (0/37). The frequency of autoantibody to human galectin-3 (12/63) was significantly higher in SLE than that of healthy donors (0/56) (19% vs 0%, p=0.0006). Autoantibody to galectin-3 was not found in the sera of patients with rheumatoid arthritis (0/50), Sjogren's syndrome (0/18), and systemic sclerosis (0/19). Interestingly, autoantibody to galectin-3 was also prevalent in the sera of patients with polymyositis/dermatomyositis (16/37, 43.2%). Further functional characterization of these autoantibodies would be necessary to determine their value as diagnostic markers or to define clinical subsets of patients with SLE. Statistical analysis revealed that the presence of autoantibody to PARP was inversely related with pleurisy, and the presence of autoantibody to galectin-3 related with renal disease.
Assuntos
Autoanticorpos/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Adulto , Antígenos de Diferenciação/imunologia , Autoanticorpos/sangue , Autoanticorpos/genética , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Feminino , Galectina 3 , Biblioteca Gênica , Humanos , Lúpus Eritematoso Sistêmico/diagnóstico , Masculino , Poli(ADP-Ribose) Polimerases/imunologia , RNA Mensageiro/biossíntese , Ribonucleoproteína Nuclear Pequena U1/imunologiaRESUMO
Previously, we reported the identification and characterization of a novel cancer/testis antigen gene, CAGE(4), that was expressed in various histological types of tumors, but not in normal tissues, with the exception of the testis. To date, molecular mechanisms for the expression of CAGE have never been studied. In our expression analysis, we found that some cancer cell lines did not express CAGE. The expression of CAGE could be restored in these cell lines by treatment with 5(')-aza-2(')-deoxycytidine, suggesting that the expression of CAGE is mainly suppressed by hypermethylation. Bisulfite sequencing analysis of the 16 CpG sites of the CAGE promoter in various cancer cell lines and tissues revealed a close relationship between the methylation status of the CAGE promoter and the expression of CAGE. The transient transfection experiments displayed that the methylation of CpG sites inhibited the CAGE promoter activity in luciferase reporter assays. The methylation of the CpG sites inhibited the binding of transcription factors, shown by a mobility shift assay. A methylation-specific PCR analysis revealed that hypomethylation of the CAGE promoter was present at frequencies of more than 60% in breast, gastric, and lung cancers, and hepatocellular carcinomas, and at frequencies of less than 40% in prostate, uterine cervical, and laryngeal cancers. Promoter hypomethylation was found in chronic gastritis (19/55, 34.5%) and liver cirrhosis (13/22, 59%), but not in normal prostate, normal colon, or chronic hepatitis. These results suggest that the methylation status of the CpG sites of CAGE determines its expression, that the hypomethylation of CAGE precedes the development of gastric cancer and hepatocellular carcinoma, and that the high frequencies of hypomethylation of CAGE, in various cancers would be valuable as a cancer diagnostic marker.