RESUMO
AXL, a member of the TAM (TYRO3, AXL, MER) receptor tyrosine kinase family, and its ligand, GAS6, are implicated in oncogenesis and metastasis of many cancer types. However, the exact cellular processes activated by GAS6-AXL remain largely unexplored. Here, we identified an interactome of AXL and revealed its associations with proteins regulating actin dynamics. Consistently, GAS6-mediated AXL activation triggered actin remodeling manifested by peripheral membrane ruffling and circular dorsal ruffles (CDRs). This further promoted macropinocytosis that mediated the internalization of GAS6-AXL complexes and sustained survival of glioblastoma cells grown under glutamine-deprived conditions. GAS6-induced CDRs contributed to focal adhesion turnover, cell spreading, and elongation. Consequently, AXL activation by GAS6 drove invasion of cancer cells in a spheroid model. All these processes required the kinase activity of AXL, but not TYRO3, and downstream activation of PI3K and RAC1. We propose that GAS6-AXL signaling induces multiple actin-driven cytoskeletal rearrangements that contribute to cancer-cell invasion.
Assuntos
Actinas/metabolismo , Extensões da Superfície Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Pinocitose , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Citoesqueleto de Actina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Glioblastoma/patologia , Glutamina/farmacologia , Células HEK293 , Humanos , Modelos Biológicos , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Proteínas rac1 de Ligação ao GTP/metabolismo , Receptor Tirosina Quinase AxlRESUMO
Molecular details of how endocytosis contributes to oncogenesis remain elusive. Our in silico analysis of colorectal cancer (CRC) patients revealed stage-dependent alterations in the expression of 112 endocytosis-related genes. Among them, transcription of the endosomal sorting complex required for transport (ESCRT)-I component VPS37B was decreased in the advanced stages of CRC. Expression of other ESCRT-I core subunits remained unchanged in the investigated dataset. We analyzed an independent cohort of CRC patients, which also showed reduced VPS37A mRNA and protein abundance. Transcriptomic profiling of CRC cells revealed non-redundant functions of Vps37 proteins. Knockdown of VPS37A and VPS37B triggered p21 (CDKN1A)-mediated inhibition of cell proliferation and sterile inflammatory response driven by the nuclear factor (NF)-κB transcription factor and associated with mitogen-activated protein kinase signaling. Co-silencing of VPS37C further potentiated activation of these independently induced processes. The type and magnitude of transcriptional alterations correlated with the differential ESCRT-I stability upon individual and concurrent Vps37 depletion. Our study provides novel insights into cancer cell biology by describing cellular stress responses that are associated with ESCRT-I destabilization.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Fatores de Transcrição , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , HumanosRESUMO
AXL, a TAM receptor tyrosine kinase (RTK), and its ligand growth arrest-specific 6 (GAS6) are implicated in cancer metastasis and drug resistance, and cellular entry of viruses. Given this, AXL is an attractive therapeutic target, and its inhibitors are being tested in cancer and COVID-19 clinical trials. Still, astonishingly little is known about intracellular mechanisms that control its function. Here, we characterized endocytosis of AXL, a process known to regulate intracellular functions of RTKs. Consistent with the notion that AXL is a primary receptor for GAS6, its depletion was sufficient to block GAS6 internalization. We discovered that upon receptor ligation, GAS6-AXL complexes were rapidly internalized via several endocytic pathways including both clathrin-mediated and clathrin-independent routes, among the latter the CLIC/GEEC pathway and macropinocytosis. The internalization of AXL was strictly dependent on its kinase activity. In comparison to other RTKs, AXL was endocytosed faster and the majority of the internalized receptor was not degraded but rather recycled via SNX1-positive endosomes. This trafficking pattern coincided with sustained AKT activation upon GAS6 stimulation. Specifically, reduced internalization of GAS6-AXL upon the CLIC/GEEC downregulation intensified, whereas impaired recycling due to depletion of SNX1 and SNX2 attenuated AKT signaling. Altogether, our data uncover the coupling between AXL endocytic trafficking and AKT signaling upon GAS6 stimulation. Moreover, our study provides a rationale for pharmacological inhibition of AXL in antiviral therapy as viruses utilize GAS6-AXL-triggered endocytosis to enter cells.
Assuntos
Endocitose , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/metabolismo , COVID-19/terapia , Clatrina/metabolismo , Clatrina/fisiologia , Endocitose/efeitos dos fármacos , Endocitose/genética , Endocitose/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Neoplasias/metabolismo , Neoplasias/terapia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/fisiologia , Receptor Tirosina Quinase AxlRESUMO
BACKGROUND: Overexpression of FGFR1 is observed in numerous tumors and therefore this receptor constitutes an attractive molecular target for selective cancer treatment with cytotoxic conjugates. The success of cancer therapy with cytotoxic conjugates largely relies on the precise recognition of a cancer-specific marker by a targeting molecule within the conjugate and its subsequent cellular internalization by receptor mediated endocytosis. We have recently demonstrated that efficiency and mechanism of FGFR1 internalization are governed by spatial distribution of the receptor in the plasma membrane, where clustering of FGFR1 into larger oligomers stimulated fast and highly efficient uptake of the receptor by simultaneous engagement of multiple endocytic routes. Based on these findings we aimed to develop a modular, self-assembly system for generation of oligomeric cytotoxic conjugates, capable of FGFR1 clustering, for targeting FGFR1-overproducing cancer cells. METHODS: Engineered FGF1 was used as FGFR1-recognition molecule and tailored for enhanced stability and site-specific attachment of the cytotoxic drug. Modified streptavidin, allowing for controlled oligomerization of FGF1 variant was used for self-assembly of well-defined FGF1 oligomers of different valency and oligomeric cytotoxic conjugate. Protein biochemistry methods were applied to obtain highly pure FGF1 oligomers and the oligomeric cytotoxic conjugate. Diverse biophysical, biochemical and cell biology tests were used to evaluate FGFR1 binding, internalization and the cytotoxicity of obtained oligomers. RESULTS: Developed multivalent FGF1 complexes are characterized by well-defined architecture, enhanced FGFR1 binding and improved cellular uptake. This successful strategy was applied to construct tetrameric cytotoxic conjugate targeting FGFR1-producing cancer cells. We have shown that enhanced affinity for the receptor and improved internalization result in a superior cytotoxicity of the tetrameric conjugate compared to the monomeric one. CONCLUSIONS: Our data implicate that oligomerization of the targeting molecules constitutes an attractive strategy for improvement of the cytotoxicity of conjugates recognizing cancer-specific biomarkers. Importantly, the presented approach can be easily adapted for other tumor markers.
Assuntos
Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais , Linhagem Celular Tumoral , Humanos , Ligação Proteica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
The endosomal sorting complexes required for transport (ESCRTs) machinery consists of four protein assemblies (ESCRT-0 to -III subcomplexes) which mediate various processes of membrane remodeling in the cell. In the endocytic pathway, ESCRTs sort cargo destined for degradation into intraluminal vesicles (ILVs) of endosomes. Cargos targeted by ESCRTs include various signaling molecules, mainly internalized cell-surface receptors but also some cytosolic proteins. It is therefore expected that aberrant trafficking caused by ESCRT dysfunction affects different signaling pathways. Here we review how perturbation of ESCRT activity alters intracellular transport of membrane receptors, causing their accumulation on endocytic compartments, decreased degradation and/or altered recycling to the plasma membrane. We further describe how perturbed trafficking of receptors impacts the activity of their downstream signaling pathways, with or without changes in transcriptional responses. Finally, we present evidence that ESCRT components can also control activity and intracellular distribution of cytosolic signaling proteins (kinases, other effectors and soluble receptors). The underlying mechanisms involve sequestration of such proteins in ILVs, their sorting for degradation or towards non-lysosomal destinations, and regulating their availability in various cellular compartments. All these ESCRT-mediated processes can modulate final outputs of multiple signaling pathways.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Transporte Biológico , HumanosRESUMO
Cytokine receptors, such as tumor necrosis factor receptor I (TNFRI, also known as TNFRSF1A) and lymphotoxin ß receptor (LTßR), activate inflammatory nuclear factor (NF)-κB signaling upon stimulation. We have previously demonstrated that depletion of ESCRT components leads to endosomal accumulation of TNFRI and LTßR, and their ligand-independent signaling to NF-κB. Here, we studied whether other perturbations of the endolysosomal system could trigger intracellular accumulation and signaling of ligand-free LTßR. While depletion of the CORVET components had no effect, knockdown of Rab7a or HOPS components, or pharmacological inhibition of lysosomal degradation, caused endosomal accumulation of LTßR and increased its interaction with the TRAF2 and TRAF3 signaling adaptors. However, the NF-κB pathway was not activated under these conditions. We found that knockdown of Rab7a or HOPS components led to sequestration of LTßR in intraluminal vesicles of endosomes, thus precluding NF-κB signaling. This was in contrast to the LTßR localization on the outer endosomal membrane that was seen after ESCRT depletion and was permissive for signaling. We propose that the inflammatory response induced by intracellular accumulation of endocytosed cytokine receptors critically depends on the precise receptor topology within endosomal compartments.
Assuntos
Receptor beta de Linfotoxina/metabolismo , NF-kappa B/metabolismo , Endossomos/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Transporte Proteico , Transdução de Sinais , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
BACKGROUND: Lymphotoxin ß receptor (LTßR) is a member of tumor necrosis factor receptor (TNFR) superfamily which regulates the immune response. At the cellular level, upon ligand binding, the receptor activates the pro-inflammatory NF-κB and AP-1 pathways. Yet, the intracellular distribution of LTßR, the routes of its endocytosis and their connection to the signaling activation are not characterized. Here, we investigated the contribution of LTßR internalization to its signaling potential. METHODS: Intracellular localization of LTßR in unstimulated and stimulated cells was analyzed by confocal microscopy. Endocytosis impairment was achieved through siRNA- or CRISPR/Cas9-mediated depletion, or chemical inhibition of proteins regulating endocytic routes. The activation of LTßR-induced signaling was examined. The levels of effector proteins of the canonical and non-canonical branches of the NF-κB pathway, and the phosphorylation of JNK, Akt, ERK1/2, STAT1 and STAT3 involved in diverse signaling cascades, were measured by Western blotting. A transcriptional response to LTßR stimulation was assessed by qRT-PCR analysis. RESULTS: We demonstrated that LTßR was predominantly present on endocytic vesicles and the Golgi apparatus. The ligand-bound pool of the receptor localized to endosomes and was trafficked towards lysosomes for degradation. Depletion of regulators of different endocytic routes (clathrin-mediated, dynamin-dependent or clathrin-independent) resulted in the impairment of LTßR internalization, indicating that this receptor uses multiple entry pathways. Cells deprived of clathrin and dynamins exhibited enhanced activation of canonical NF-κB signaling represented by increased degradation of IκBα inhibitor and elevated expression of LTßR target genes. We also demonstrated that clathrin and dynamin deficiency reduced to some extent LTßR-triggered activation of the non-canonical branch of the NF-κB pathway. CONCLUSIONS: Our work shows that the impairment of clathrin- and dynamin-dependent internalization amplifies a cellular response to LTßR stimulation. We postulate that receptor internalization restricts responsiveness of the cell to subthreshold stimuli. Video Abstract.
Assuntos
Clatrina/metabolismo , Dinaminas/metabolismo , Endocitose , Receptor beta de Linfotoxina/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Células A549 , Endossomos/metabolismo , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Ligantes , Lisossomos/metabolismo , ProteóliseRESUMO
Platelet-derived growth factor receptor ß (PDGFRß) is a receptor tyrosine kinase which upon activation by PDGF-BB stimulates cell proliferation, migration and angiogenesis. Ligand binding induces intracellular signaling cascades but also internalization of the receptor, eventually resulting in its lysosomal degradation. However, endocytic trafficking of receptors often modulates their downstream signaling. We previously reported that internalization of PDGFRß occurs via dynamin-dependent and -independent pathways but their further molecular determinants remained unknown. Here we show that, in human fibroblasts expressing endogenous PDGFRß and stimulated with 50â ng/ml PDGF-BB, ligand-receptor uptake proceeds via the parallel routes of clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE). CME involves the canonical AP2 complex as a clathrin adaptor, while CIE requires RhoA-ROCK, Cdc42 and galectin-3, the latter indicating lectin-mediated internalization via clathrin-independent carriers (CLICs). Although different uptake routes appear to be partly interdependent, they cannot fully substitute for each other. Strikingly, inhibition of any internalization mechanism impaired activation of STAT3 but not of other downstream effectors of PDGFRß. Our data indicate that multiple routes of internalization of PDGFRß contribute to a transcriptional and mitogenic response of cells to PDGF.
Assuntos
Endocitose/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Complexo 2 de Proteínas Adaptadoras/metabolismo , Clatrina/metabolismo , DNA/biossíntese , Dinaminas/metabolismo , Endocitose/genética , Galectina 3/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/metabolismo , Masculino , Transdução de Sinais/genética , Transcrição Gênica/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Lymphotoxin ß receptor (LTßR) plays important roles in the development of the immune system and immune response. At the cellular level, ligand-bound LTßR activates the pro-inflammatory NF-κB pathway but the detailed mechanisms regulating its signaling remain unknown. Understanding them is of high importance since LTßR and its ligands are promising therapeutic targets. Here, we studied the consequences of perturbed cellular cholesterol content on LTßR-induced NF-κB signaling. METHODS: To modulate cholesterol availability and/or level in lung carcinoma A549 and H2228, and endothelial HUVEC cells different treatment regimens with filipin, methyl-ß-cyclodextrin and simvastatin were applied. LTßR localization was studied by confocal microscopy. The activity of LTßR-induced NF-κB pathway was assessed by measuring the levels of NF-κB pathway inhibitor IκBα and phosphorylation of RelA transcription factor by Western blotting. The NF-κB transcriptional response, production of chemokines and adhesion molecules were examined by qRT-PCR, ELISA, and Western blotting, respectively. Adherence of different types of primary immune cells to epithelial A549 cells and endothelial HUVECs was measured fluorometrically. Interactions of LTßR with its protein partners were investigated by immunoprecipitation. RESULTS: We showed that filipin-mediated sequestration of cholesterol or its depletion from the plasma membrane with methyl-ß-cyclodextrin impaired LTßR internalization and potentiated LTßR-dependent activation of the canonical branch of the NF-κB pathway. The latter was manifested by enhanced degradation of IκBα inhibitor, elevated RelA phosphorylation, substantial increase in the expression of NF-κB target genes encoding, among others, cytokines and adhesion molecules known to play important roles in immune response. It was followed by robust secretion of CXCL8 and upregulation of ICAM1, that favored the adhesion of immune cells (NK and T cells, neutrophils) to A549 cells and HUVECs. Mechanistically, we showed that cholesterol depletion stabilized interactions of ligand-stimulated LTßR with modified forms of TRAF2 and NEMO proteins. CONCLUSIONS: Our results showed that the reduction of the plasma membrane content of cholesterol or its sequestration strongly potentiated signaling outcome initiated by LTßR. Thus, drugs modulating cholesterol levels could potentially improve efficacy of LTßR-based therapies. Video abstract.
Assuntos
Colesterol/farmacologia , Receptor beta de Linfotoxina/antagonistas & inibidores , Receptor beta de Linfotoxina/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células A549 , Células Cultivadas , Humanos , Células Jurkat , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismoRESUMO
Maintenance of physiologic cellular functions and homeostasis requires highly coordinated interactions between different cellular compartments. In this regard, the endocytic system, which plays a key role in cargo internalization and trafficking within the cell, participates in upkeep of intracellular dynamics, while communicating with multiple organelles. This chapter will discuss the function of endosomes from a standpoint of cellular integration. We will present examples of different types of interactions between endosomes and other cellular compartments, such as the endoplasmic reticulum (ER), mitochondria, the plasma membrane (PM), and the nuclear envelope. In addition, we will describe the incorporation of endocytic components, such as endosomal sorting complexes required for transport (ESCRT) proteins and Rab small GTPases, into cellular processes that operate outside of the endolysosomal pathway. The significance of endosomal interactions for processes such as signaling regulation, intracellular trafficking, organelle dynamics, metabolic control, and homeostatic responses will be reviewed. Accumulating data indicate that beyond its involvement in cargo transport, the endocytic pathway is comprehensively integrated into other systems of the cell and plays multiple roles in the complex net of cellular functions.
Assuntos
Endocitose/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/genética , Transporte Proteico/genética , Endossomos/metabolismo , Humanos , Mitocôndrias/genética , Transdução de Sinais/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Platelet-derived growth factor (PDGF) isoforms regulate cell proliferation, migration and differentiation both in embryonic development and adult tissue remodeling. At the cellular level, growth-factor signaling is often modulated by endocytosis. Despite important functions of PDGF, its endocytosis remains poorly studied, mainly for lack of tools to track internalized ligand by microscopy. Here, we developed such a tool and quantitatively analyzed internalization and endosomal trafficking of PDGF-BB in human fibroblasts. We further show that PDGF can be internalized in the presence of dynamin inhibitors, arguing that both dynamin-dependent and dynamin-independent pathways can mediate PDGF uptake. Although these routes operate with somewhat different kinetics, they both ultimately lead to lysosomal degradation of PDGF. Although acute inhibition of dynamin activity only moderately affects PDGF endocytosis, it specifically decreases downstream signaling of PDGF via signal transducer and activator of transcription 3 (STAT3). This correlates with reduced expression of MYC and impaired cell entry into S-phase, indicating that dynamin activity is required for PDGF-induced mitogenesis. Our data support a general view that the components governing endocytic trafficking may selectively regulate certain signaling effectors activated by a growth factor.
Assuntos
Dinaminas/antagonistas & inibidores , Endocitose , Sistema de Sinalização das MAP Quinases , Fator de Crescimento Derivado de Plaquetas/metabolismo , Linhagem Celular , Fibroblastos/metabolismo , Humanos , Lisossomos/metabolismo , Transporte Proteico , Proteólise , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fase S/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Transcrição GênicaRESUMO
Rab GTPases and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are evolutionarily conserved essential components of the eukaryotic intracellular transport system. Although pairing of cognate SNAREs is sufficient to fuse membranes in vitro, a complete reconstitution of the Rab-SNARE machinery has never been achieved. Here we report the reconstitution of the early endosomal canine Rab5 GTPase, its key regulators and effectors together with SNAREs into proteoliposomes using a set of 17 recombinant human proteins. These vesicles behave like minimal 'synthetic' endosomes, fusing with purified early endosomes or with each other in vitro. Membrane fusion measured by content-mixing and morphological assays requires the cooperativity between Rab5 effectors and cognate SNAREs which, together, form a more efficient 'core machinery' than SNAREs alone. In reconstituting a fusion mechanism dependent on both a Rab GTPase and SNAREs, our work shows that the two machineries act coordinately to increase the specificity and efficiency of the membrane tethering and fusion process.
Assuntos
Endossomos/fisiologia , Fusão de Membrana/fisiologia , Proteínas SNARE/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Cricetinae , Citosol/metabolismo , Cães , Endossomos/metabolismo , Humanos , Microscopia Eletrônica , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas Recombinantes/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
It is becoming clear that intracellular signaling events are intimately linked with the membrane transport processes. In addition to the long known role of endocytosis in downregulating plasma membrane receptors, more recent data uncover several sophisticated modes by which endocytosis affects the type and duration of signals. Particularly striking are various roles of endocytic compartments as membrane platforms for compartmentalized assembly or sequestration of specific signaling complexes. Here we review some recent examples illustrating how endosomes may mediate ligand-stimulated apoptotic signaling and how multivesicular bodies affect Wnt signaling by regulated sequestration of signaling molecules or their secretion in exosomes. We also discuss evidence documenting the involvement of endocytic proteins in the regulation of p53 activity and stability, which suggests a possible cross-talk between endocytic processes and transcriptional responses.
Assuntos
Endocitose/fisiologia , Transdução de Sinais/fisiologia , Animais , Exossomos/genética , Exossomos/metabolismo , Exossomos/fisiologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Corpos Multivesiculares/genética , Corpos Multivesiculares/metabolismo , Corpos Multivesiculares/fisiologia , Estabilidade Proteica , Transporte Proteico , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
APPL1 is a multifunctional adaptor protein that binds membrane receptors, signaling proteins and nuclear factors, thereby acting in endosomal trafficking and in different signaling pathways. Here, we uncover a novel role of APPL1 as a positive regulator of transcriptional activity of NF-κB under basal but not TNFα-stimulated conditions. APPL1 was found to directly interact with TRAF2, an adaptor protein known to activate canonical NF-κB signaling. APPL1 synergized with TRAF2 to induce NF-κB activation, and both proteins were necessary for this process and function upstream of the IKK complex. Although TRAF2 was not detectable on APPL endosomes, endosomal recruitment of APPL1 was required for its function in the NF-κB pathway. Importantly, in the canonical pathway, APPL1 appeared to regulate the proper spatial distribution of the p65 subunit of NF-κB in the absence of cytokine stimulation, since its overexpression enhanced and its depletion reduced the nuclear accumulation of p65. By analyzing the patterns of gene transcription upon APPL1 overproduction or depletion we found altered expression of NF-κB target genes that encode cytokines. At the molecular level, overexpressed APPL1 markedly increased the level of NIK, the key component of the noncanonical NF-κB pathway, by reducing its association with the degradative complex containing TRAF2, TRAF3 and cIAP1. In turn, high levels of NIK triggered nuclear translocation of p65. Collectively, we propose that APPL1 regulates basal NF-κB activity by modulating the stability of NIK, which affects the activation of p65. This places APPL1 as a novel link between the canonical and noncanonical machineries of NF-κB activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Núcleo Celular/metabolismo , Endossomos/metabolismo , Estabilidade Enzimática , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Ligação Proteica , Transporte Proteico , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/metabolismo , Fator de Transcrição RelA/metabolismo , Quinase Induzida por NF-kappaBRESUMO
The diverse range of organizations contributing to the global research ecosystem is believed to enhance the overall quality and resilience of its output. Mid-sized autonomous research institutes, distinct from universities, play a crucial role in this landscape. They often lead the way in new research fields and experimental methods, including those in social and organizational domains, which are vital for driving innovation. The EU-LIFE alliance was established with the goal of fostering excellence by developing and disseminating best practices among European biomedical research institutes. As directors of the 15 EU-LIFE institutes, we have spent a decade comparing and refining our processes. Now, we are eager to share the insights we've gained. To this end, we have crafted this Charter, outlining 10 principles we deem essential for research institutes to flourish and achieve ground-breaking discoveries. These principles, detailed in the Charter, encompass excellence, independence, training, internationality and inclusivity, mission focus, technological advancement, administrative innovation, cooperation, societal impact, and public engagement. Our aim is to inspire the establishment of new institutes that adhere to these principles and to raise awareness about their significance. We are convinced that they should be viewed a crucial component of any national and international innovation strategies.
Assuntos
Disciplinas das Ciências Biológicas , Pesquisa Biomédica , Academias e InstitutosRESUMO
APPL endosomes are a recently identified subpopulation of early endosomes characterized by the presence of two homologous Rab5 effector proteins APPL1 and APPL2. They exhibit only limited colocalization with EEA1, another Rab5 effector and a marker of the canonical early endosomes. Although APPL endosomes appear to play important roles in cargo trafficking and signal transduction, their protein composition and biochemical properties remain largely unknown. Here we employed membrane fractionation methods to characterize APPL endosomes biochemically. We demonstrate that they represent heterogeneous membrane structures which can be discriminated from the canonical EEA1-positive early endosomes by their partly different physical properties and a distinct migration pattern in the continuous density gradients. In search for other potential markers of APPL endosomes we identified Annexin A2 as an interacting partner of both APPL1 and APPL2. Annexin A2 is a Ca(2+) and phosphatidylinositol 4,5-bisphosphate binding protein, previously implicated in several endocytic steps. We show that Annexin A2 co-fractionates and colocalizes with APPL endosomes. Moreover, silencing of its expression causes solubilization of APPL2 from endosomes. Although Annexin A2 is not an exclusive marker of APPL endosomes, our data suggest that it has an important function in membrane recruitment of APPL proteins, acting in parallel to Rab5.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anexina A2/metabolismo , Membrana Celular/metabolismo , Endossomos/química , Endossomos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Anexina A2/genética , Cálcio/metabolismo , Fracionamento Celular/métodos , Membrana Celular/ultraestrutura , Células HEK293 , Células HeLa , Humanos , Fosfatidilinositóis/metabolismo , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Anti-CD20 monoclonal antibodies (mAbs) are successfully used in the management of non-Hodgkin lymphomas and chronic lymphocytic leukemia. We have reported previously that statins induce conformational changes in CD20 molecules and impair rituximab-mediated complement-dependent cytotoxicity. Here we investigated in more detail the influence of farnesyltransferase inhibitors (FTIs) on CD20 expression and antitumor activity of anti-CD20 mAbs. Among all FTIs studied, L-744,832 had the most significant influence on CD20 levels. It significantly increased rituximab-mediated complement-dependent cytotoxicity against primary tumor cells isolated from patients with non-Hodgkin lymphomas or chronic lymphocytic leukemia and increased CD20 expression in the majority of primary lymphoma/leukemia cells. Incubation of Raji cells with L-744,832 led to up-regulation of CD20 at mRNA and protein levels. Chromatin immunoprecipitation assay revealed that inhibition of farnesyltransferase activity was associated with increased binding of PU.1 and Oct-2 to the CD20 promoter sequences. These studies indicate that CD20 expression can be modulated by FTIs. The combination of FTIs with anti-CD20 mAbs is a promising therapeutic approach, and its efficacy should be examined in patients with B-cell tumors.
Assuntos
Anticorpos Monoclonais/química , Antígenos CD20/biossíntese , Proteínas do Sistema Complemento/química , Dimetilaliltranstransferase/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Citometria de Fluxo/métodos , Células HEK293 , Humanos , Linfoma de Células B/metabolismo , Metionina/análogos & derivados , Metionina/farmacologia , Regiões Promotoras GenéticasRESUMO
Inhibitors of proteasomes have been shown to affect endocytosis of multiple membrane receptors, in particular at the step of cargo sorting for lysosomal degradation. Here we demonstrate that the inhibition of proteasomes causes specific redistribution of an endosomal adaptor APPL1, which undergoes initial solubilization from APPL endosomes followed by clustering in the perinuclear region. MG132 treatment decreases APPL1 labeling of endosomes while the staining of the canonical early endosomes with EEA1 remains unaffected. Upon prolonged treatment with proteasome inhibitors, endogenous APPL1 localizes to the site of aggresome formation, with perinuclear APPL1 clusters encapsulated within a vimentin cage and co-localizing with aggregates positive for ubiquitin. The clustering of APPL1 is concomitant with increased ubiquitination and decreased solubility of this protein. We determined that the ubiquitin ligase Nedd4 enhances polyubiquitination of APPL1, and the ubiquitin molecules attached to APPL1 are linked through lysine-63. Taken together, these results add APPL1 to only a handful of endogenous cellular proteins known to be recruited to aggresomes induced by proteasomal stress. Moreover, our studies suggest that the proteasome inhibitors that are already in clinical use affect the localization, ubiquitination and solubility of APPL1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Corpos de Inclusão/metabolismo , Inibidores de Proteassoma , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Inibidores de Cisteína Proteinase/metabolismo , Endossomos/metabolismo , Células HEK293 , Células HeLa , Humanos , Leupeptinas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
AXL, a receptor tyrosine kinase from the TAM (TYRO3 AXL and MER) subfamily, and its ligand growth arrest-specific 6 (GAS6) are implicated in pathogenesis of a wide array of cancers, acquisition of resistance to diverse anticancer therapies and cellular entry of viruses. The continuous development of AXL inhibitors for treatment of patients with cancer and COVID-19 underscores the need to better characterize the cellular effects of AXL targeting.In the present study, we compared the cellular phenotypes of CRISPR-Cas9-induced depletion of AXL and its pharmacological inhibition with bemcentinib, LDC1267 and gilteritinib. Specifically, we evaluated GAS6-AXL signaling, cell viability and invasion, the endo-lysosomal system and autophagy in glioblastoma cells. We showed that depletion of AXL but not of TYRO3 inhibited GAS6-induced phosphorylation of downstream signaling effectors, AKT and ERK1/2, indicating that AXL is a primary receptor for GAS6. AXL was also specifically required for GAS6-dependent increase in cell viability but was dispensable for viability of cells grown without exogenous addition of GAS6. Furthermore, we revealed that LDC1267 is the most potent and specific inhibitor of AXL activation among the tested compounds. Finally, we found that, in contrast to AXL depletion and its inhibition with LDC1267, cell treatment with bemcentinib and gilteritinib impaired the endo-lysosomal and autophagy systems in an AXL-independent manner. IMPLICATIONS: Altogether, our findings are of high clinical importance as we discovered that two clinically advanced AXL inhibitors, bemcentinib and gilteritinib, may display AXL-independent cellular effects and toxicity.