RESUMO
The topology of most membrane proteins is defined by the successive integration of α-helical transmembrane domains at the Sec61 translocon. The translocon provides a pore for the transfer of polypeptide segments across the membrane while giving them lateral access to the lipid. For each polypeptide segment of â¼20 residues, the combined hydrophobicities of its constituent amino acids were previously shown to define the extent of membrane integration. Here, we discovered that different sequences preceding a potential transmembrane domain substantially affect its hydrophobicity requirement for integration. Rapidly folding domains, sequences that are intrinsically disordered or very short or capable of binding chaperones with high affinity, allow for efficient transmembrane integration with low-hydrophobicity thresholds for both orientations in the membrane. In contrast, long protein fragments, folding-deficient mutant domains, and artificial sequences not binding chaperones interfered with membrane integration, requiring higher hydrophobicity. We propose that the latter sequences, as they compact on their hydrophobic residues, partially folded but unable to reach a native state, expose hydrophobic surfaces that compete with the translocon for the emerging transmembrane segment, reducing integration efficiency. The results suggest that rapid folding or strong chaperone binding is required for efficient transmembrane integration.
Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Citoplasma , Regulação Fúngica da Expressão Gênica , Imunoprecipitação , Chaperonas Moleculares , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Since the start of the COVID-19 outbreak, pharmaceutical companies and research groups have focused on the development of vaccines and antiviral drugs against SARS-CoV-2. Here, we apply a drug repurposing strategy to identify drug candidates that are able to block the entrance of the virus into human cells. By combining virtual screening with in vitro pseudovirus assays and antiviral assays in Human Lung Tissue (HLT) cells, we identify entrectinib as a potential antiviral drug.
Assuntos
Benzamidas/farmacologia , Tratamento Farmacológico da COVID-19 , Indazóis/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/farmacologia , Benzamidas/metabolismo , COVID-19/metabolismo , Linhagem Celular , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos/métodos , Humanos , Indazóis/metabolismo , Pulmão/patologia , Pulmão/virologia , Simulação de Acoplamento Molecular , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Células Vero , Ligação Viral/efeitos dos fármacosRESUMO
Molecular processes within cells have traditionally been studied with biochemical methods due to their high degree of specificity and ease of use. In recent years, cell-based assays have gained more and more popularity since they facilitate the extraction of mode of action, phenotypic, and toxicity information. However, to provide specificity, cellular assays rely heavily on biomolecular labels and tags while label-free cell-based assays only offer holistic information about a bulk property of the investigated cells. Here, we introduce a cell-based assay for protein-protein interaction analysis. We achieve specificity by spatially ordering a membrane protein of interest into a coherent pattern of fully functional membrane proteins on the surface of an optical sensor. Thereby, molecular interactions with the coherently ordered membrane proteins become visible in real time, while nonspecific interactions and holistic changes within the living cell remain invisible. Due to its unbiased nature, this new cell-based detection method presents itself as an invaluable tool for cell signaling research and drug discovery.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Membrana/metabolismo , Arrestina/química , Arrestina/genética , Arrestina/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mapas de Interação de Proteínas , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismoRESUMO
Cell fate choices during metazoan development are driven by the highly conserved Notch signalling pathway. Notch receptor activation results in release of the Notch intracellular domain (NICD) that acts as transcriptional co-activator of the DNA-binding protein CSL. In the absence of signal, a repressor complex consisting of CSL bound to co-repressors silences Notch target genes. The Drosophila repressor complex contains the fly CSL orthologue Suppressor of Hairless [Su(H)] and Hairless (H). The Su(H)-H crystal structure revealed a large conformational change within Su(H) upon H binding, precluding interactions with NICD. Based on the structure, several sites in Su(H) and H were determined to specifically engage in complex formation. In particular, three mutations in Su(H) were identified that affect interactions with the repressor H but not the activator NICD. To analyse the effects these mutants have on normal fly development, we introduced these mutations into the native Su(H) locus by genome engineering. We show that the three H-binding deficient Su(H) alleles behave similarly. As these mutants lack the ability to form the repressor complex, Notch signalling activity is strongly increased in homozygotes, comparable to a complete loss of H activity. Unexpectedly, we find that the abundance of the three mutant Su(H) protein variants is altered, as is that of wild type Su(H) protein in the absence of H protein. In the presence of NICD, however, Su(H) mutant protein persists. Apparently, Su(H) protein levels depend on the interactions with H as well as with NICD. Based on these results, we propose that in vivo levels of Su(H) protein are stabilised by interactions with transcription-regulator complexes.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/genética , Mutação , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Alelos , Animais , Sítios de Ligação , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Ligação Proteica , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
G protein-coupled receptors (GPCRs) are cellular master regulators that translate extracellular stimuli such as light, small molecules or peptides into a cellular response. Upon ligand binding, they bind intracellular proteins such as G proteins or arrestins, modulating intracellular signaling cascades. Here, we use a protein-fragment complementation approach based on nanoluciferase (split luciferase assay) to assess interaction of all four known human arrestins with four different GPCRs (two class A and two class B receptors) in live cells. Besides directly tagging the 11S split-luciferase subunit to the receptor, we also could demonstrate that membrane localization of the 11S subunit with a CAAX-tag allowed us to probe arrestin recruitment by endogenously expressed GPCRs. Varying the expression levels of our reporter constructs changed the dynamic behavior of our assay, which we addressed with an advanced baculovirus-based multigene expression system. Our detection assay allowed us to probe the relevance of each of the two arrestin binding sites in the different GPCRs for arrestin binding. We observed remarkable differences between the roles of each arresting binding site in the tested GPCRs and propose that the distinct advantages of our system for probing receptor interaction with effector proteins will help elucidate the molecular basis of GPCR signaling efficacy and specificity in different cell types.
Assuntos
Arrestinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Domínio Catalítico , Dosagem de Genes , Genes Reporter , Células HEK293 , Humanos , Cinética , Luciferases/genética , Nucleopoliedrovírus/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Receptores de Vasopressinas/metabolismo , Proteínas Recombinantes/metabolismo , Transdução Genética , beta-Arrestina 2/metabolismoRESUMO
This review focusses on the energetics of protein translocation via the Sec translocation machinery. First we complement structural data about SecYEG's conformational rearrangements by insight obtained from functional assays. These include measurements of SecYEG permeability that allow assessment of channel gating by ligand binding and membrane voltage. Second we will discuss the power stroke and Brownian ratcheting models of substrate translocation and the role that the two models assign to the putative driving forces: (i) ATP (SecA) and GTP (ribosome) hydrolysis, (ii) interaction with accessory proteins, (iii) membrane partitioning and folding, (iv) proton motive force (PMF), and (v) entropic contributions. Our analysis underlines how important energized membranes are for unravelling the translocation mechanism in future experiments.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Canais de Translocação SEC/química , Canais de Translocação SEC/metabolismo , Trifosfato de Adenosina/metabolismo , Eletrofisiologia , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Methanocaldococcus/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Transporte Proteico/fisiologia , Força Próton-Motriz/fisiologiaRESUMO
BACKGROUND: DNA damage generally results in the activation of ATM/ATR kinases and the downstream checkpoint kinases Chk1/Chk2. In Drosophila melanogaster, the ATR homologue meiotic 41 (mei-41) is pivotal to DNA damage repair and cell cycle checkpoint signalling. Although various mei-41 mutant alleles have been analyzed in the past, no gain-of-function allele is yet available. To fill this gap, we have generated transgenic flies allowing temporal and tissue-specific induction of mei-41. RESULTS: Overexpression of mei-41 in wing and eye anlagen affects proliferation and a G2/M checkpoint even in the absence of genomic stress. Similar consequences were observed following the overexpression of the downstream kinase Grapes (Grp) but not of Loki (Lok), encoding the respective Drosophila Chk1 and Chk2 homologues, in agreement with their previously reported activities. Moreover, we show that irradiation induced cell cycle arrest was prolonged in the presence of ectopic mei-41 expression. Similar to irradiation stress, mei-41 triggered the occurrence of a slower migrating form of Grp, implying specific phosphorylation of Grp in response to either signal. Using a p53R-GFP biosensor, we further show that overexpression of mei-41 was sufficient to elicit a robust p53 activation in vivo. CONCLUSION: We conclude that overexpression of the Drosophila ATR homologue mei-41 elicits an effectual DNA damage response irrespective of irradiation.
Assuntos
Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/genética , Dano ao DNA , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Animais Geneticamente Modificados , Divisão Celular , Drosophila melanogaster/efeitos da radiação , Fase G2RESUMO
BACKGROUND: Gold nanoparticles (AuNPs) are a popular choice for use in medical and biomedical research applications. With suitable functionalisation AuNPs can be applied in drug delivery systems, or can aid in disease diagnosis. One such functionalisation is with chitosan, which enables efficient interaction and permeation of cellular membranes, providing an effective adjuvant. As both AuNPs and chitosan have been shown to have low toxicity and high biocompatibility their proposed use in nanomedicine, either individually or combined, is expanding. However, further toxicological and immunological assessments of AuNP-chitosan conjugates are still needed. Therefore, we have evaluated how AuNP functionalisation with chitosan can affect uptake, cytotoxicity, and immunological responses within mononuclear cells, and influence the interaction of AuNPs with biomolecules within a complex biofluid. The AuNPs used were negatively charged through citrate-coating, or presented either low or high positive charge through chitosan-functionalisation. Uptake by THP-1 cells was assessed via transmission electron microscopy and electron energy loss spectroscopy, pro-inflammatory responses by ELISA and qRT-PCR, and cell death and viability via lactate dehydrogenase release and mitochondrial activity, respectively. Interactions of AuNPs with protein components of a frequently used in vitro cell culture medium supplement, foetal calf serum, were investigated using mass spectrometry. RESULTS: Although cells internalised all AuNPs, uptake rates and specific routes of intracellular trafficking were dependent upon chitosan-functionalisation. Accordingly, an enhanced immune response was found to be chitosan-functionalisation-dependent, in the form of CCL2, IL-1ß, TNF-α and IL-6 secretion, and expression of IL-1ß and NLRP3 mRNA. A corresponding increase in cytotoxicity was found in response to chitosan-coated AuNPs. Furthermore, chitosan-functionalisation was shown to induce an increase in unique proteins associating with these highly charged AuNPs. CONCLUSIONS: It can be concluded that functionalisation of AuNPs with the perceived non-toxic biocompatible molecule chitosan at a high density can elicit functionalisation-dependent intracellular trafficking mechanisms and provoke strong pro-inflammatory conditions, and that a high affinity of these NP-conjugates for biomolecules may be implicit in these cellular responses.
Assuntos
Quitosana/química , Endocitose , Ouro/química , Nanopartículas Metálicas/química , Fagócitos/metabolismo , Proteínas de Transporte/metabolismo , Morte Celular , Linhagem Celular , Meios de Cultura/química , Humanos , Inflamassomos/metabolismo , Inflamação/patologia , Nanopartículas Metálicas/ultraestrutura , Proteína 3 que Contém Domínio de Pirina da Família NLR , Fagócitos/patologiaRESUMO
Chemical inducers of dimerization (CIDs) have been developed to orchestrate protein dimerization and translocation. Here we present a novel photocleavable HaloTag- and SNAP-tag-reactive CID (MeNV-HaXS) with excellent selectivity and intracellular reactivity. Excitation at 360â nm cleaves the methyl-6-nitroveratryl core of MeNV-HaXS. MeNV-HaXS covalently links HaloTag- and SNAP-tag fusion proteins, and enables targeting of selected membranes and intracellular organelles. MeNV-HaXS-mediated translocation has been validated for plasma membrane, late endosomes, lysosomes, Golgi, mitochondria, and the actin cytoskeleton. Photocleavage of MeNV-HaXS liberates target proteins and provides access to optical manipulation of protein relocation with high spatiotemporal and subcellular precision. MeNV-HaXS supports kinetic studies of protein dynamics and the manipulation of subcellular enzyme activities, which is exemplified for Golgi-targeted cargo and the assessment of nuclear import kinetics.
Assuntos
Permeabilidade da Membrana Celular , Proteínas de Fluorescência Verde/metabolismo , Luz , Fármacos Fotossensibilizantes/farmacologia , Multimerização Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Endocitose/fisiologia , Endocitose/efeitos da radiação , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/genética , Guanina/análogos & derivados , Guanina/química , Guanina/farmacologia , Células HeLa , Humanos , Cinética , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Fármacos Fotossensibilizantes/química , Transporte Proteico , Proteínas Recombinantes de Fusão/genéticaRESUMO
Tandem affinity purification (TAP) is an efficient method for the purification and characterization of large macromolecular complexes. To elucidate the role of specific components of such complexes, it is important to address the question of how loss of a specific factor affects complex composition. Here, we introduce a method that combines TAP of large macromolecular assemblies with inducible shRNA-mediated protein depletion in human somatic cells. As a proof of principle, we have applied this method to the purification of human pre-ribosomal particles. Using inducible expression of ribosome assembly factors as bait proteins, different pre-40S particles could be isolated and characterized, revealing high conservation of the ribosome biogenesis pathway from yeast to human cells. Besides known ribosome maturation factors, C21orf70 was identified as a novel pre-40S component. By combining TAP of pre-40S particles with shRNA-mediated depletion of the pre-40S-associated protein kinase Rio2, we observed that increased levels of the nuclear HEAT-repeat protein Rrp12 are associated with 40S precursors in absence of Rio2. Further analyses revealed that Rrp12 is partially mislocalized to the cytoplasm and trapped on late 40S precursors upon loss of Rio2, and therefore fails to efficiently recycle to the nucleus. Thus, the combination of tandem affinity purification and shRNA induction provided further insights into late cytoplasmic 40S maturation steps, demonstrating the high potential of this method.
Assuntos
Cromatografia de Afinidade , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/farmacologia , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Northern Blotting , Células Cultivadas , Imunofluorescência , Células HeLa , Humanos , Imunoprecipitação , Rim/citologia , Rim/metabolismo , Complexos Multiproteicos/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Carvedilol is among the most effective ß-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of ß1-adrenoceptors, arrestin-biased signalling via ß2-adrenoceptors is a molecular mechanism proposed to explain the survival benefits. Here, we offer an alternative mechanism to rationalize carvedilol's cellular signalling. Using primary and immortalized cells genome-edited by CRISPR/Cas9 to lack either G proteins or arrestins; and combining biological, biochemical, and signalling assays with molecular dynamics simulations, we demonstrate that G proteins drive all detectable carvedilol signalling through ß2ARs. Because a clear understanding of how drugs act is imperative to data interpretation in basic and clinical research, to the stratification of clinical trials or to the monitoring of drug effects on the target pathway, the mechanistic insight gained here provides a foundation for the rational development of signalling prototypes that target the ß-adrenoceptor system.
Assuntos
Antagonistas Adrenérgicos beta , Infarto do Miocárdio , Humanos , Carvedilol/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Receptores Adrenérgicos beta 2/genética , Infarto do Miocárdio/tratamento farmacológicoRESUMO
The Notch signaling pathway is pivotal to cellular differentiation. Activation of this pathway involves proteolysis of the Notch receptor and the release of the biologically active Notch intracellular domain, acting as a transcriptional co-activator of Notch target genes. While the regulation of Notch signaling dynamics at the level of ligand-receptor interaction, endocytosis, and transcriptional regulation has been well studied, little is known about factors influencing Notch cleavage. We identified EP555 as a suppressor of the Notch antagonist Hairless (H). EP555 drives expression of CG32521 encoding membrane-bound proteins, which we accordingly rename membrane-bound Notch regulator (mnr). Within the signal-receiving cell, upregulation of Mnr stimulates Notch receptor activation, whereas a knockdown reduces it, without apparent influence on ligand-receptor interaction. We provide evidence that Mnr plays a role in γ-secretase-mediated intramembrane cleavage of the Notch receptor. As revealed by a fly-eye-based reporter system, γ-secretase activity is stimulated by the overexpression of Mnr, and is inhibited by its knockdown. We conclude that Mnr proteins support Notch signaling activity by fostering the cleavage of the Notch receptor. With Mnr, we identified a membrane-bound factor directly augmenting Notch intra-membrane processing, thereby acting as a positive regulator of Notch signaling activity.
Assuntos
Drosophila melanogaster , Receptores Notch , Secretases da Proteína Precursora do Amiloide , Animais , Transdução de SinaisRESUMO
The highly conserved Notch signaling pathway controls a multitude of developmental processes including hematopoiesis. Here, we provide evidence for a novel mechanism of tissue-specific Notch regulation involving phosphorylation of CSL transcription factors within the DNA-binding domain. Earlier we found that a phospho-mimetic mutation of the Drosophila CSL ortholog Suppressor of Hairless [Su(H)] at Ser269 impedes DNA-binding. By genome-engineering, we now introduced phospho-specific Su(H) mutants at the endogenous Su(H) locus, encoding either a phospho-deficient [Su(H) S269A ] or a phospho-mimetic [Su(H) S269D ] isoform. Su(H) S269D mutants were defective of Notch activity in all analyzed tissues, consistent with impaired DNA-binding. In contrast, the phospho-deficient Su(H) S269A mutant did not generally augment Notch activity, but rather specifically in several aspects of blood cell development. Unexpectedly, this process was independent of the corepressor Hairless acting otherwise as a general Notch antagonist in Drosophila. This finding is in agreement with a novel mode of Notch regulation by posttranslational modification of Su(H) in the context of hematopoiesis. Importantly, our studies of the mammalian CSL ortholog (RBPJ/CBF1) emphasize a potential conservation of this regulatory mechanism: phospho-mimetic RBPJ S221D was dysfunctional in both the fly as well as two human cell culture models, whereas phospho-deficient RBPJ S221A rather gained activity during fly hematopoiesis. Thus, dynamic phosphorylation of CSL-proteins within the DNA-binding domain provides a novel means to fine-tune Notch signal transduction in a context-dependent manner.
RESUMO
The binding pockets of aminergic G protein-coupled receptors are often targeted by drugs and virtual screening campaigns. In order to find ligands with unprecedented scaffolds for one of the best-investigated receptors of this subfamily, the ß2 -adrenergic receptor, we conducted a docking-based screen insisting that molecules would address previously untargeted residues in extracellular loop 2. We here report the discovery of ligands with a previously undescribed coumaran-based scaffold. Furthermore, we provide an analysis of the added value that X-ray structures in different conformations deliver for such docking screens.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 2/farmacologia , Receptores Adrenérgicos beta 2/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2/química , Relação Dose-Resposta a Droga , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Translocation of many secretory proteins through the bacterial plasma membrane is facilitated by a complex of the SecYEG channel with the motor protein SecA. The ATP-free complex is unstable in detergent, raising the question how SecA may perform several rounds of ATP hydrolysis without being released from the membrane embedded SecYEG. Here we show that dual recognition of (i) SecYEG and (ii) vicinal acidic lipids confers an apparent nanomolar affinity. High-speed atomic force microscopy visualizes the complexes between monomeric SecA and SecYEG as being stable for tens of seconds. These long-lasting events and complementary shorter ones both give rise to single ion channel openings of equal duration. Furthermore, luminescence resonance energy transfer reveals two conformations of the SecYEG-SecA complex that differ in the protrusion depth of SecA's two-helix finger into SecYEG's aqueous channel. Such movement of the finger is in line with the power stroke mechanism of protein translocation.
RESUMO
Notch signaling activity governs widespread cellular differentiation in higher animals, including humans, and is involved in several congenital diseases and different forms of cancer. Notch signals are mediated by the transcriptional regulator RBPJ in a complex with activated Notch (NICD). Analysis of Notch pathway regulation in humans is hampered by a partial redundancy of the four Notch receptor copies, yet RBPJ is solitary, allowing its study in model systems. In Drosophila melanogaster, the RBPJ orthologue is encoded by Suppressor of Hairless [Su(H)]. Using genome engineering, we replaced Su(H) by murine RBPJ in order to study its function in the fly. In fact, RBPJ largely substitutes for Su(H)'s function, yet subtle phenotypes reflect increased Notch signaling activity. Accordingly, the binding of RBPJ to Hairless (H) protein, the general Notch antagonist in Drosophila, was considerably reduced compared to that of Su(H). An H-binding defective RBPJLLL mutant matched the respective Su(H)LLL allele: homozygotes were lethal due to extensive Notch hyperactivity. Moreover, RBPJLLL protein accumulated at lower levels than wild type RBPJ, except in the presence of NICD. Apparently, RBPJ protein stability depends on protein complex formation with either H or NICD, similar to Su(H), demonstrating that the murine homologue underlies the same regulatory mechanisms as Su(H) in Drosophila. These results underscore the importance of regulating the availability of RBPJ protein to correctly mediate Notch signaling activity in the fly.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alelos , Animais , Animais Geneticamente Modificados , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Homozigoto , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Camundongos , Fenótipo , Ligação Proteica , Estabilidade Proteica , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Germline stem cell development and differentiation is tightly controlled by the surrounding somatic cells of the stem cell niche. In Drosophila females, cells of the niche emit various signals including Dpp and Wg to balance stem cell renewal and differentiation. Here, we show that the gene pzg is autonomously required in cells of the germline to sustain the interplay between niche and stem cells. Loss of pzg impairs stem cell differentiation and provokes the death of cells in the germarium. As a consequence of pzg loss, increased growth signalling activity predominantly of Dpp and Wg/Wnt, was observed, eventually disrupting the balance of germ cell self-renewal and differentiation. Whereas in the soma, apoptosis-induced compensatory growth is well established, the induction of self-renewal signals during oogenesis cannot compensate for dying germ cells, albeit inducing a new niche-like microenvironment. Instead, they impair the further development of germ cells and cause in addition a forward and feedback loop of cell death.
Assuntos
Proteínas de Ciclo Celular/deficiência , Morte Celular , Microambiente Celular , Proteínas de Drosophila/deficiência , Óvulo/citologia , Nicho de Células-Tronco , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica , Mães , Transdução de SinaisRESUMO
Activation and repression of Notch target genes is mediated by transcription factor CSL, known as Suppressor of Hairless (Su(H)) in Drosophila and CBF1 or RBPJ in human. CSL associates either with co-activator Notch or with co-repressors such as Drosophila Hairless. The nuclear translocation of transcription factor CSL relies on co-factor association, both in mammals and in Drosophila. The Drosophila CSL orthologue Su(H) requires Hairless for repressor complex formation. Based on its role in transcriptional silencing, H protein would be expected to be strictly nuclear. However, H protein is also cytosolic, which may relate to its role in the stabilization and nuclear translocation of Su(H) protein. Here, we investigate the function of the predicted nuclear localization signals (NLS 1-3) and single nuclear export signal (NES) of co-repressor Hairless using GFP-fusion proteins, reporter assays and in vivo analyses using Hairless wild type and shuttling-defective Hairless mutants. We identify NLS3 and NES to be critical for Hairless function. In fact, HâNLS3 mutant flies match H null mutants, whereas HâNLS3âNES double mutants display weaker phenotypes in agreement with a crucial role for NES in H export. As expected for a transcriptional repressor, Notch target genes are deregulated in HâNLS3 mutant cells, demonstrating nuclear requirement for its activity. Importantly, we reveal that Su(H) protein strictly follows Hairless protein localization. Together, we propose that shuttling between the nucleo-cytoplasmic compartments provides the possibility to fine tune the regulation of Notch target gene expression by balancing of Su(H) protein availability for Notch activation.
Assuntos
Citoplasma/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Receptores Notch/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Drosophila/genética , Feminino , Sinais de Exportação Nuclear/genética , Sinais de Localização Nuclear/genética , Fenótipo , Receptores Notch/genética , Fatores de Transcrição/genética , Asas de Animais/crescimento & desenvolvimentoRESUMO
Label-free biosensors are ideally suited for the quantitative analysis of specific interactions among biomolecules or of biomolecules with drugs, as well as for quantitation of diagnostic markers in biofluids. In contrast to the label-dependent methods, a new assay for a particular prey molecule can be set up within few minutes by immobilizing the corresponding bait molecule on the sensor surface, using one of the common immobilization procedures. Unfortunately, the extensive application of label-free biosensors is still hampered by the fact that the immobilization of the bait molecule is usually irreversible; for that reason, a new chip (which is expensive) is required for every successful or futile attempt. Here, we present a general method for the switchable immobilization of biotinylated bait molecules on a new desthiobiotin surface, using wild-type streptavidin as a robust bridge between the chip and the biotinylated bait. The immobilization of the bait is very stable, so that many cycles of prey injection and subsequent prey removal can be carried out. For the latter, common reagents like HCl, Na2CO3, glycine buffer, or SDS are employed. When desired, however, streptavidin plus the biotinylated bait can be completely removed by 3min injections of biotin, guanidinium thiocyanate, pepsin, and SDS, which makes it possible to immobilize new biotinylated bait. The number of in situ regeneration cycles is unlimited during the lifetime of the chip (2-3 weeks). One chip can easily be shared by many users with unrelated tasks (as is typical in academics), or used for the fully automated screening of many different interactions (for example in pharmaceutical research). In comparison to other regenerative chips, the new chip surface has much wider applicability and all of its structural and functional parameters have been disclosed.
Assuntos
Técnicas Biossensoriais , Biotina/análogos & derivados , Biotinilação/métodos , Técnicas Analíticas Microfluídicas , Biotina/química , Guanidinas/química , Indicadores e Reagentes/química , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Estreptavidina/química , Ressonância de Plasmônio de Superfície , Tiocianatos/químicaRESUMO
One of the key players in genome surveillance is the tumour suppressor p53 mediating the adaptive response to a multitude of stress signals. Here we identify Cyclin G (CycG) as co-factor of p53-mediated genome stability. CycG has been shown before to be involved in double-strand break repair during meiosis. Moreover, it is also important for mediating DNA damage response in somatic tissue. Here we find it in protein complexes together with p53, and show that the two proteins interact physically in vitro and in vivo in response to ionizing irradiation. In contrast to mammals, Drosophila Cyclin G is no transcriptional target of p53. Genetic interaction data reveal that p53 activity during DNA damage response requires the presence of CycG. Morphological defects caused by overexpression of p53 are ameliorated in cycG null mutants. Moreover, using a p53 biosensor we show that p53 activity is impeded in cycG mutants. As both p53 and CycG are likewise required for DNA damage repair and longevity we propose that CycG plays a positive role in mediating p53 function in genome surveillance of Drosophila.