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Small extracellular vesicles (sEVs) are an important part of intercellular communication. They are phospholipid bilayer particles that carry active biomolecules such as proteins, various nucleic acids, and lipids. In recipient cells, sEVs can alter cellular functions, including cancer development and premetastatic niche formation in distant organs. Moreover, sEVs can carry cancer-specific features, which makes them promising biomarker candidates. However, the interactions of sEVs with biological barriers and consequences thereof, are not clarified yet. The blood-saliva barrier is crucial for preventing the entry of pathogens and (in)organic substances into the bloodstream, as well as molecule filtration from blood to saliva. The effects of brain derived DU145 prostate cancer (PCa) sEVs on a human submandibular salivary gland barrier (SSGB) in vitro were investigated. Small EVs were harvested from normoxic (N, atmospheric O2) or hypoxic (H, 1% O2) conditions, fluorescently labeled with CellTrackerTM Orange and thoroughly characterized. HTB-41 B2 cells were used as SSGB model cultured on 24-well ThinCert® inserts. After model optimization indicating effects of serum and serum-sEVs on barrier properties, PCa sEVs were applied to the basolateral (blood) side in either 10% serum, or serum-free conditions, and barrier integrity was continuously monitored for 40 hours. This study found that H and N PCa sEVs were uptaken by the SSGB in vitro model in similar quantities regardless of the media composition in the basolateral compartment. Permeation of fluorescent PCa sEVs into the apical compartment was not detectable with the applied methods. However, treatment with H and N sEVs under different serum conditions revealed distinct molecular clusters after hierarchical analysis of mRNA data measured by high-throughput qPCR, which were partly reflected at the protein level. For example, serum-reduction dependent decrease of barrier properties was accompanied with the decrease of CDH1 or Claudin-7 expression. Interestingly, the presence of H sEVs significantly increased the number of sEV-sized particles in the apical compartment of the SSGB model compared to basolaterally added N sEVs. This functional effect on the number of particles in the saliva (apical) compartment induced by different sEVs applied in the blood (basolateral) compartment might be a new approach to understand one possible mechanism how differences of salivary EVs might occur which then could be used as biomarker.
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BACKGROUND: Blood-brain barrier (BBB) models based on primary murine, bovine, and porcine brain capillary endothelial cell cultures have long been regarded as robust models with appropriate properties to examine the functional transport of small molecules. However, species differences sometimes complicate translating results from these models to human settings. During the last decade, brain capillary endothelial-like cells (BCECs) have been generated from stem cell sources to model the human BBB in vitro. The aim of the present study was to establish and characterize a human BBB model using human induced pluripotent stem cell (hiPSC)-derived BCECs from the hIPSC line SBAD0201. METHODS: The model was evaluated using transcriptomics, proteomics, immunocytochemistry, transendothelial electrical resistance (TEER) measurements, and, finally, transport assays to assess the functionality of selected transporters and receptor (GLUT-1, LAT-1, P-gp and LRP-1). RESULTS: The resulting BBB model displayed an average TEER of 5474 ± 167 Ω·cm2 and cell monolayer formation with claudin-5, ZO-1, and occludin expression in the tight junction zones. The cell monolayers expressed the typical BBB markers VE-cadherin, VWF, and PECAM-1. Transcriptomics and quantitative targeted absolute proteomics analyses revealed that solute carrier (SLC) transporters were found in high abundance, while the expression of efflux transporters was relatively low. Transport assays using GLUT-1, LAT-1, and LRP-1 substrates and inhibitors confirmed the functional activities of these transporters and receptors in the model. A transport assay suggested that P-gp was not functionally expressed in the model, albeit antibody staining revealed that P-gp was localized at the luminal membrane. CONCLUSIONS: In conclusion, the novel SBAD0201-derived BBB model formed tight monolayers and was proven useful for studies investigating GLUT-1, LAT-1, and LRP-1 mediated transport across the BBB. However, the model did not express functional P-gp and thus is not suitable for the performance of drug efflux P-gp reletated studies.
Assuntos
Barreira Hematoencefálica , Células-Tronco Pluripotentes Induzidas , Humanos , Animais , Bovinos , Camundongos , Suínos , Barreira Hematoencefálica/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Linhagem Celular , Transporte Biológico , Encéfalo/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Células CultivadasRESUMO
BACKGROUND: The function of the blood-brain barrier (BBB) is impaired in late-onset Alzheimer disease (LOAD), but the associated molecular mechanisms, particularly with respect to the high-risk APOE4/4 genotype, are not well understood. For this purpose, we developed a multicellular isogenic model of the neurovascular unit (NVU) based on human induced pluripotent stem cells. METHODS: The human NVU was modeled in vitro using isogenic co-cultures of astrocytes, brain capillary endothelial-like cells (BCECs), microglia-like cells, neural stem cells (NSCs), and pericytes. Physiological and pathophysiological properties were investigated as well as the influence of each single cell type on the characteristics and function of BCECs. The barriers established by BCECs were analyzed for specific gene transcription using high-throughput quantitative PCR. RESULTS: Co-cultures were found to tighten the barrier of BCECs and alter its transcriptomic profile under both healthy and disease conditions. In vitro differentiation of brain cell types that constitute the NVU was not affected by the LOAD background. The supportive effect of NSCs on the barrier established by BCECs was diminished under LOAD conditions. Transcriptomes of LOAD BCECs were modulated by different brain cell types. NSCs were found to have the strongest effect on BCEC gene regulation and maintenance of the BBB. Co-cultures showed cell type-specific functional contributions to BBB integrity under healthy and LOAD conditions. CONCLUSIONS: Cell type-dependent transcriptional effects on LOAD BCECs were identified. Our study suggests that different brain cell types of the NVU have unique roles in maintaining barrier integrity that vary under healthy and LOAD conditions. .
Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Humanos , Barreira Hematoencefálica/metabolismo , Transcriptoma , Doença de Alzheimer/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Encéfalo , Astrócitos/metabolismoRESUMO
Here, we report an experimental setup to benchmark different receptors for targeted therapeutic antibody delivery at the blood-brain barrier. We used brain capillary endothelial-like cells derived from induced pluripotent stem cells (hiPSC-BECs) as a model system and compared them to colon epithelial Caco-2 cells. This approach helped to identify favourable receptors for transport into the cell layer itself or for directing transport for transcytosis across the cell layer. The sorting receptors transferrin receptor and sortilin were shown to be efficient as antibody cargo receptors for intracellular delivery to the cell layer. In contrast, the cell surface receptors CD133 and podocalyxin were identified as static and inefficient receptors for delivering cargo antibodies. Similar to in vivo studies, the hiPSC-BECs maintained detectable transcytotic transport via transferrin receptor, while transcytosis was restricted using sortilin as a cargo receptor. Based on these findings, we propose the application of sortilin as a cargo receptor for delivering therapeutic antibodies into the brain microvascular endothelium.
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Barreira Hematoencefálica , Transcitose , Humanos , Barreira Hematoencefálica/metabolismo , Células CACO-2 , Transporte Biológico , Encéfalo/metabolismo , Receptores da Transferrina/metabolismoRESUMO
An in vitro model of the human blood-brain barrier was developed, based on a collagen hydrogel containing astrocytes, overlaid with a monolayer of endothelium, differentiated from human induced pluripotent stem cells (hiPSCs). The model was set up in transwell filters allowing sampling from apical and basal compartments. The endothelial monolayer had transendothelial electrical resistance (TEER) values >700Ω.cm2 and expressed tight-junction markers, including claudin-5. After differentiation of hiPSCs the endothelial-like cells expressed VE-cadherin (CDH5) and von-Willebrand factor (VWF) as determined by immunofluorescence. However, electron microscopy indicated that at set-up (day 8 of differentiation), the endothelial-like cells still retained some features of the stem cells, and appeared immature, in comparison with primary brain endothelium or brain endothelium in vivo. Monitoring showed that the TEER declined gradually over 10 days, and transport studies were best carried out in a time window 24-72hrs after establishment of the model. Transport studies indicated low permeability to paracellular tracers and functional activity of P-glycoprotein (ABCB1) and active transcytosis of polypeptides via the transferrin receptor (TFR1).
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Barreira Hematoencefálica , Células-Tronco Pluripotentes Induzidas , Humanos , Células Cultivadas , Hidrogéis , Técnicas de Cocultura , Diferenciação CelularRESUMO
Significant advancements in the field of preclinical in vitro blood-brain barrier (BBB) models have been achieved in recent years, by developing monolayer-based culture systems towards complex multi-cellular assays. The coupling of those models with other relevant organoid systems to integrate the investigation of blood-brain barrier permeation in the larger picture of drug distribution and metabolization is still missing. Here, we report for the first time the combination of a human induced pluripotent stem cell (hiPSC)-derived blood-brain barrier model with a cortical brain and a liver spheroid model from the same donor in a closed microfluidic system (MPS). The two model compounds atenolol and propranolol were used to measure permeation at the blood-brain barrier and to assess metabolization. Both substances showed an in vivo-like permeation behavior and were metabolized in vitro. Therefore, the novel multi-organ system enabled not only the measurement of parent compound concentrations but also of metabolite distribution at the blood-brain barrier.
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Barreira Hematoencefálica , Células-Tronco Pluripotentes Induzidas , Preparações Farmacêuticas , Humanos , Atenolol/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo , Células-Tronco Pluripotentes Induzidas/metabolismo , Fígado , Preparações Farmacêuticas/metabolismo , Propranolol/metabolismoRESUMO
Neurological complications are common in COVID-19. Although SARS-CoV-2 has been detected in patients' brain tissues, its entry routes and resulting consequences are not well understood. Here, we show a pronounced upregulation of interferon signaling pathways of the neurovascular unit in fatal COVID-19. By investigating the susceptibility of human induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (BCECs) to SARS-CoV-2 infection, we found that BCECs were infected and recapitulated transcriptional changes detected in vivo. While BCECs were not compromised in their paracellular tightness, we found SARS-CoV-2 in the basolateral compartment in transwell assays after apical infection, suggesting active replication and transcellular transport of virus across the blood-brain barrier (BBB) in vitro. Moreover, entry of SARS-CoV-2 into BCECs could be reduced by anti-spike-, anti-angiotensin-converting enzyme 2 (ACE2)-, and anti-neuropilin-1 (NRP1)-specific antibodies or the transmembrane protease serine subtype 2 (TMPRSS2) inhibitor nafamostat. Together, our data provide strong support for SARS-CoV-2 brain entry across the BBB resulting in increased interferon signaling.
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Barreira Hematoencefálica/virologia , Sistema Nervoso Central/virologia , SARS-CoV-2/fisiologia , Internalização do Vírus , Anticorpos/farmacologia , Benzamidinas/farmacologia , COVID-19/patologia , COVID-19/virologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Guanidinas/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Internalização do Vírus/efeitos dos fármacosRESUMO
Early detection of cancer in order to facilitate timely therapeutic interventions is an unsolved problem in today's clinical diagnostics. Tumors are detected so far mostly after pathological symptoms have emerged (usually already in progressed disease states), within preventive screenings, or occasionally as incidental finding. The emergence of extracellular vesicle (EV) analytics in combination with liquid biopsy sampling opened a plethora of new possibilities for the detection of tumors (and other diseases). This review gives an overview of the diversity of currently known EV species and the relevant cargo molecules representing potential biomarkers to detect, identify and characterize tumor cells. A number of molecules reported in recent years to be valuable targets for different aspects of cancer diagnostics, are presented. Furthermore, we discuss (technical) challenges and pitfalls related to the various potential applications (screening, diagnosis, prognosis, monitoring) of liquid biopsy based EV analytics, and give an outlook to possible future directions of this emerging field in oncology.
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Detecção Precoce de Câncer/métodos , Vesículas Extracelulares/patologia , Neoplasias/diagnóstico , Animais , Biomarcadores Tumorais/análise , Vesículas Extracelulares/química , Humanos , Biópsia Líquida/métodos , Neoplasias/patologiaRESUMO
The exposure of humans to nano-and microplastic particles (NMPs) is an issue recognized as a potential health hazard by scientists, authorities, politics, non-governmental organizations and the general public. The concentration of NMPs in the environment is increasing concomitantly with global plastic production and the usage of plastic materials. NMPs are detectable in numerous aquatic organisms and also in human samples, therefore necessitating a risk assessment of NMPs for human health. So far, a comprehensive risk assessment of NMPs is hampered by limited availability of appropriate reference materials, analytical obstacles and a lack of definitions and standardized study designs. Most studies conducted so far used polystyrene (PS) spheres as a matter of availability, although this polymer type accounts for only about 7% of total plastic production. Differently sized particles, different concentration and incubation times, and various biological models have been used, yielding hardly comparable data sets. Crucial physico-chemical properties of NMPs such as surface (charge, polarity, chemical reactivity), supplemented additives and adsorbed chemicals have been widely excluded from studies, although in particular the surface of NMPs determines the interaction with cellular membranes. In this manuscript we give an overview about the critical parameters which should be considered when performing risk assessments of NMPs, including novel reference materials, taking into account surface modifications (e.g., reflecting weathering processes), and the possible role of NMPs as a substrate and/or carrier for (pathogenic) microbes. Moreover, we make suggestions for biological model systems to evaluate immediate toxicity, long-term effects and the potential of NMPs to cross biological barriers. We are convinced that standardized reference materials and experimental parameters along with technical innovations in (nano)-particle sampling and analytics are a prerequisite for the successful realization of conclusive human health risk assessments of NMPs.
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Plásticos , Poluentes Químicos da Água , Organismos Aquáticos , Humanos , Microplásticos , Nanopartículas/análise , Plásticos/toxicidade , Poliestirenos , Poluentes Químicos da Água/análiseRESUMO
LAP2-Emerin-MAN1 (LEM) domain-containing proteins represent an abundant group of inner nuclear membrane proteins involved in diverse nuclear functions, but their functional redundancies remain unclear. Here, using the biotinylation-dependent proximity approach, we report proteome-wide comparative interactome analysis of the two structurally related LEM proteins MAN1 (LEMD3) and LEM2 (LEMD2), and the more distantly related emerin (EMD). While over 60% of the relatively small group of MAN1 and emerin interactors were also found in the LEM2 interactome, the latter included a large number of candidates (>85%) unique for LEM2. The interacting partners unique for emerin support and provide further insight into the previously reported role of emerin in centrosome positioning, and the MAN1-specific interactors suggest a role of MAN1 in ribonucleoprotein complex assembly. Interestingly, the LEM2-specific interactome contained several proteins of the nucleotide excision repair pathway. Accordingly, LEM2-depleted cells, but not MAN1- and emerin-depleted cells, showed impaired proliferation following ultraviolet-C (UV-C) irradiation and prolonged accumulation of γH2AX, similar to cells deficient in the nucleotide excision repair protein DNA damage-binding protein 1 (DDB1). These findings indicate impaired DNA damage repair in LEM2-depleted cells. Overall, this interactome study identifies new potential interaction partners of emerin, MAN1 and particularly LEM2, and describes a novel potential involvement of LEM2 in nucleotide excision repair at the nuclear periphery.
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Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Centrossomo/metabolismo , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Domínios Proteicos , Transfecção , Raios UltravioletaRESUMO
Sirtuin 2 (SIRT2) is an NAD-dependent sirtuin deacetylase that regulates microtubule and chromatin dynamics, gene expression and cell cycle progression, as well as nuclear envelope reassembly. Recent proteomic analyses have identified Golgi proteins as SIRT2 interactors, indicating that SIRT2 may also play a role in Golgi structure formation. Here, we show that SIRT2 depletion causes Golgi fragmentation and impairs Golgi reassembly at the end of mitosis. SIRT2 interacts with the Golgi reassembly stacking protein GRASP55 (also known as GORASP2) in mitosis when GRASP55 is highly acetylated on K50. Expression of wild-type and the K50R acetylation-deficient mutant of GRASP55, but not the K50Q acetylation-mimetic mutant, in GRASP55 and GRASP65 (also known as GORASP1) double-knockout cells, rescued the Golgi structure and post-mitotic Golgi reassembly. Acetylation-deficient GRASP55 exhibited a higher self-interaction efficiency, a property required for Golgi structure formation. These results demonstrate that SIRT2 regulates Golgi structure by modulating GRASP55 acetylation levels.
Assuntos
Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Microtúbulos/metabolismo , Sirtuína 2/metabolismo , Humanos , Mitose/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
Sirtuin 2 (SIRT2) is an NAD-dependent deacetylase known to regulate microtubule dynamics and cell cycle progression. SIRT2 has also been implicated in the pathology of cancer, neurodegenerative diseases and progeria. Here, we show that SIRT2 depletion or overexpression causes nuclear envelope reassembly defects. We link this phenotype to the recently identified regulator of nuclear envelope reassembly ANKLE2. ANKLE2 acetylation at K302 and phosphorylation at S662 are dynamically regulated throughout the cell cycle by SIRT2 and are essential for normal nuclear envelope reassembly. The function of SIRT2 therefore extends beyond the regulation of microtubules to include the regulation of nuclear envelope dynamics.
Assuntos
Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Sirtuína 2/metabolismo , Acetilação , Biotinilação , Ciclo Celular , Forma do Núcleo Celular , Cromatografia de Afinidade , Células HEK293 , Humanos , Modelos Biológicos , Fosforilação , Ligação Proteica , ProteômicaRESUMO
BACKGROUND: Ankyrin repeats and LEM domain containing protein 1 (Ankle1) belongs to the LEM protein family, whose members share a chromatin-interacting LEM motif. Unlike most other LEM proteins, Ankle1 is not an integral protein of the inner nuclear membrane but shuttles between the nucleus and the cytoplasm. It contains a GIY-YIG-type nuclease domain, but its function is unknown. The mammalian genome encodes only one other GIY-YIG domain protein, termed Slx1. Slx1 has been described as a resolvase that processes Holliday junctions during homologous recombination-mediated DNA double strand break repair. Resolvase activity is regulated in a spatial and temporal manner during the cell cycle. We hypothesized that Ankle1 may have a similar function and its nucleo-cytoplasmic shuttling may contribute to the regulation of Ankle1 activity. Hence, we aimed at identifying the domains mediating Ankle1 shuttling and investigating whether cellular localization is affected during DNA damage response. RESULTS: Sequence analysis predicts the presence of two canonical nuclear import and export signals in Ankle1. Immunofluorescence microscopy of cells expressing wild-type and various mutated Ankle1-fusion proteins revealed a C-terminally located classical monopartite nuclear localization signal and a centrally located CRM1-dependent nuclear export signal that mediate nucleo-cytoplasmic shuttling of Ankle1. These sequences are also functional in heterologous proteins. The predominant localization of Ankle1 in the cytoplasm, however, does not change upon induction of several DNA damage response pathways throughout the cell cycle. CONCLUSIONS: We identified the domains mediating nuclear import and export of Ankle1. Ankle1's cellular localization was not affected following DNA damage.
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Núcleo Celular/metabolismo , Endonucleases/metabolismo , Sinais de Exportação Nuclear , Sinais de Localização Nuclear/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Dano ao DNA , Análise Mutacional de DNA , Endonucleases/química , Genes Reporter , Humanos , Hidroxiureia/farmacologia , Mitose/efeitos dos fármacos , Domínios ProteicosRESUMO
Ankyrin repeat and LEM-domain containing protein 1 (ANKLE1) is a GIY-YIG endonuclease with unknown functions, mainly expressed in mouse hematopoietic tissues. To test its potential role in hematopoiesis we generated Ankle1-deficient mice. Ankle1Δ/Δ mice are viable without any detectable phenotype in hematopoiesis. Neither hematopoietic progenitor cells, myeloid and lymphoid progenitors, nor B and T cell development in bone marrow, spleen and thymus, are affected in Ankle1Δ/Δ-mice. Similarly embryonic stress erythropoiesis in liver and adult erythropoiesis in bone marrow and spleen appear normal. To test whether ANKLE1, like the only other known GIY-YIG endonuclease in mammals, SLX1, may contribute to Holliday junction resolution during DNA repair, Ankle1-deficient cells were exposed to various DNA-damage inducing agents. However, lack of Ankle1 did not affect cell viability and, unlike depletion of Slx1, Ankle1-deficiency did not increase sister chromatid exchange in Bloom helicase-depleted cells. Altogether, we show that lack of Ankle1 does neither affect mouse hematopoiesis nor DNA damage repair in mouse embryonic fibroblasts, indicating a redundant or non-essential function of ANKLE1 in mouse.
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Endonucleases/fisiologia , Hematopoese/fisiologia , Animais , Endonucleases/genética , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genéticaRESUMO
The LEM proteins comprise a heterogeneous family of chromatin-associated proteins that share the LEM domain, a structural motif mediating interaction with the DNA associated protein, Barrier-to-Autointegration Factor (BAF). Most of the LEM proteins are integral proteins of the inner nuclear membrane and associate with the nuclear lamina, a structural scaffold of lamin intermediate filament proteins at the nuclear periphery, which is involved in nuclear mechanical functions and (hetero-)chromatin organization. A few LEM proteins, such as Lamina-associated polypeptide (LAP)2α and Ankyrin and LEM domain-containing protein (Ankle)1 lack transmembrane domains and localize throughout the nucleoplasm and cytoplasm, respectively. LAP2α has been reported to regulate cell proliferation by affecting the activity of retinoblastoma protein in tissue progenitor cells and numerous studies showed upregulation of LAP2α in cancer. Ankle1 is a nuclease likely involved in DNA damage repair pathways and single nucleotide polymorphisms in the Ankle1 gene have been linked to increased breast and ovarian cancer risk. In this review we describe potential mechanisms of the involvement of LEM proteins, particularly of LAP2α and Ankle1 in tumorigenesis and we provide evidence that LAP2α expression may be a valuable diagnostic and prognostic marker for tumor analyses.
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Proteínas de Ligação a DNA/fisiologia , Proteínas de Membrana/fisiologia , Neoplasias/fisiopatologia , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/química , Humanos , Proteínas de Membrana/química , Neoplasias/genética , Neoplasias/patologiaRESUMO
The LEM domain (for lamina-associated polypeptide, emerin, MAN1 domain) defines a group of nuclear proteins that bind chromatin through interaction of the LEM motif with the conserved DNA crosslinking protein, barrier-to-autointegration factor (BAF). Here, we describe a LEM protein annotated in databases as 'Ankyrin repeat and LEM domain-containing protein 1' (Ankle1). We show that Ankle1 is conserved in metazoans and contains a unique C-terminal GIY-YIG motif that confers endonuclease activity in vitro and in vivo. In mammals, Ankle1 is predominantly expressed in hematopoietic tissues. Although most characterized LEM proteins are components of the inner nuclear membrane, ectopic Ankle1 shuttles between cytoplasm and nucleus. Ankle1 enriched in the nucleoplasm induces DNA cleavage and DNA damage response. This activity requires both the catalytic C-terminal GIY-YIG domain and the LEM motif, which binds chromatin via BAF. Hence, Ankle1 is an unusual LEM protein with a GIY-YIG-type endonuclease activity in higher eukaryotes.
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Clivagem do DNA , Endonucleases/química , Endonucleases/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Sequência Conservada , Citoplasma/metabolismo , Dano ao DNA , Endonucleases/análise , Endonucleases/genética , Perfilação da Expressão Gênica , Sistema Hematopoético/metabolismo , Humanos , Imunoprecipitação , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Transporte Proteico , Transdução de SinaisRESUMO
The nuclear envelope in eukaryotic cells has important roles in chromatin organization. The inner nuclear membrane contains over 60 transmembrane proteins. LEM [LAP2 (lamina-associated polypeptide 2)/emerin/MAN1] domain-containing proteins of the inner nuclear membrane are involved in tethering chromatin to the nuclear envelope and affect gene expression. They contain a common structural, bihelical motif, the so-called LEM domain, which mediates binding to a conserved chromatin protein, BAF (barrier to autointegration factor). Interestingly, this domain is highly related to other bihelical motifs, termed HeH (helix-extension-helix) and SAP {SAF (scaffold attachment factor)/acinus/PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)]} motifs, which are directly linked to DNA. In the present paper, we summarize evidence that the LEM motif evolved from the HeH and SAP domains concomitantly with BAF. In addition, we discuss the potential evolution of HeH/SAP and LEM domain-containing proteins and their role in chromatin tethering and gene regulation from unicellular eukaryotes to mammals.
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Cromatina/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Sequência de Aminoácidos , Animais , Evolução Molecular , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/química , Dados de Sequência MolecularRESUMO
Cell polarization is a fundamental process underpinning organismal development, and tissue homeostasis, which requires an orchestrated interplay of nuclear, cytoskeletal, and centrosomal structures. The underlying molecular mechanisms, however, still remain elusive. Here we report that kinesin-1/nesprin-2/SUN-domain macromolecular assemblies, spanning the entire nuclear envelope (NE), function in cell polarization by anchoring cytoskeletal structures to the nuclear lamina. Nesprin-2 forms complexes with the kinesin-1 motor protein apparatus by associating with and recruiting kinesin light chain 1 (KLC1) to the outer nuclear membrane. Similar to nesprin-2, KLC1 requires lamin A/C for proper NE localization. The depletion of nesprin-2 or KLC1, or the uncoupling of nesprin-2/SUN-domain protein associations impairs cell polarization during wounding and dislodges the centrosome from the NE. In addition nesprin-2 loss has profound effects on KLC1 levels, the cytoskeleton, and Golgi apparatus organization. Collectively these data show that NE-associated proteins are pivotal determinants of cell architecture and polarization.
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Centrossomo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Membrana Nuclear/metabolismo , Animais , Linhagem Celular , Polaridade Celular , Chlorocebus aethiops/metabolismo , Dineínas/metabolismo , Humanos , Cinesinas/metabolismo , Regiões de Interação com a Matriz , Camundongos , Proteínas do Tecido Nervoso/metabolismoRESUMO
Recently, we found epigenetic silencing of the Ras effector genes NORE1B and/or RASSF1A in 97% of the hepatocellular carcinoma (HCC) investigated. This is strong evidence that the two genes are of major significance in hepatocarcinogenesis. Although RASSF1A serves as a tumor suppressor gene, the functions of NORE1B are largely unknown. Here, we studied the role of NORE1B for growth and transformation of cells. To understand the molecular mechanisms of action of the gene, we used the wild-type form and deletion mutants without the NH(2) terminus and CENTRAL domain, the Ras association (RA) domain, or the COOH-terminal SARAH-domain. Intact RA and SARAH-domains were found to be necessary for NORE1B (a) to increase the G(0)-G(1) fraction in hepatoma cells, (b) to suppress c-Myc/Ha-Ras-induced cell transformation, and (c) to interact closely with RASSF1A, as determined with fluorescence resonance energy transfer. In further studies, cell cycle delay by NORE1B was equally effective in hepatocyte cell lines with wild-type or mutant Ras suggesting that NORE1B does not interact with either Ras. In conclusion, NORE1B suppresses replication and transformation of cells as effectively as RASSF1A and thus is a putative tumor suppressor gene. NORE1B interacts physically with RASSF1A and functional loss of one of the interacting partners may lead to uncontrolled growth and transformation of hepatocytes. This may explain the frequent epigenetic silencing of NORE1B and/or RASSF1A in HCC.