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Human elongation factor 2 is the translocase that is responsible for the movement of tRNA from the A- to P- and P- to E-site on the ribosome during the elongation phase of translation. Being a vital factor of protein biosynthesis, its function is highly controlled and regulated. It has been implicated in numerous diseases and pathologies, and as such it is important to have a source for isolated pure and active protein for biomedical and biochemical studies. Here we report development of a purification protocol for native human elongation factor 2 from HEK-293S cells. The resulting protein is active, pure, has an intact diphtamide and is obtainable in yields suitable for functional and structural studies.
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Factor 2 de Elongación Peptídica/química , Factor 2 de Elongación Peptídica/aislamiento & purificación , Células HEK293 , HumanosRESUMEN
Signalling proteins are intrinsic to all biological processes and interact with each other in tightly regulated and orchestrated signalling complexes and pathways. Characterization of protein binding can help to elucidate protein function within signalling pathways. This information is vital for researchers to gain a more comprehensive knowledge of cellular networks which can then be used to develop new therapeutic strategies for disease. However, studying protein-protein interactions (PPIs) can be challenging as the interactions can be extremely transient downstream of specific environmental cues. There are many powerful techniques currently available to identify and confirm PPIs. Choosing the most appropriate range of techniques merits serious consideration. The aim of this review is to provide a starting point for researchers embarking on a PPI study. We provide an overview and point of reference for some of the many methods available to identify interactions from in silico analysis and large scale screening tools through to the methods used to validate potential PPIs. We discuss the advantages and disadvantages of each method and we also provide a workflow chart to highlight the main experimental questions to consider when planning cell lysis to maximize experimental success.
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Biología Computacional/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas/metabolismo , Proteómica/métodos , Animales , Simulación por Computador , Humanos , Unión Proteica , Reproducibilidad de los Resultados , Investigación/tendenciasRESUMEN
In vitro preclinical testing of chimeric antigen receptor (CAR) T cells is mostly carried out in monolayer cell cultures. However, alternative strategies are needed to take into account the complexity and the effects of the tumor microenvironment. Here, we describe the modulation of CAR T-cell activity by malignant cells and fibroblasts in human three-dimensional (3D) in vitro cell models of increasing complexity. In models combining mucin-1 (MUC1) and TnMUC1 CAR T cells with human high-grade serous ovarian cancer cell spheroids, malignant cell-intrinsic resistance to CAR T-cell killing was due to defective death receptor signaling involving TNFα. Adding primary human fibroblasts to spheroids unexpectedly increased the ability of CAR T cells to kill resistant malignant cells as CCL2 produced by fibroblasts activated CCR2/4+ CAR T cells. However, culturing malignant cells and fibroblasts in collagen gels engendered production of a dense extracellular matrix that impeded CAR T-cell activity in a TGFß-dependent manner. A vascularized microfluidic device was developed that allowed CAR T cells to flow through the vessels and penetrate the gels in a more physiological way, killing malignant cells in a TNFα-dependent manner. Complex 3D human cell models may provide an efficient way of screening multiple cytotoxic human immune cell constructs while also enabling evaluation of mechanisms of resistance involving cell-cell and cell-matrix interactions, thus accelerating preclinical research on cytotoxic immune cell therapies in solid tumors. Significance: Three-dimensional in vitro models of increasing complexity uncover mechanisms of resistance to CAR T cells in solid tumors, which could help accelerate development of improved CAR T-cell constructs.
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Inmunoterapia Adoptiva , Neoplasias Ováricas , Receptores Quiméricos de Antígenos , Esferoides Celulares , Linfocitos T , Microambiente Tumoral , Humanos , Femenino , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Inmunoterapia Adoptiva/métodos , Microambiente Tumoral/inmunología , Esferoides Celulares/inmunología , Linfocitos T/inmunología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Fibroblastos/patología , Línea Celular TumoralRESUMEN
Most biologists know of the interferons IFNα, IFNß, and IFNγ and their roles in immunity and infection, but they may not have heard of IFNε. A recent study in Nature suggests that IFNε can act as a tumor suppressor in serous ovarian cancers.
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Interferones , Neoplasias Ováricas , Femenino , Humanos , Neoplasias Ováricas/inmunología , Interferones/metabolismoRESUMEN
Studies of the high-grade serous ovarian cancer (HGSOC) tumor microenvironment, the most lethal gynecological cancer, aim to enhance the efficiency of established therapies. Cell motility is an important process of anti-tumor response. Using ex vivo human and mouse HGSOC tumor slices combined with time-lapse imaging, we assessed the motility of CD8+ T and myeloid cells. We developed a semi-supervised analysis of cell movements, identifying four cell behaviors: migrating, long migrating, static, and wobbling. Tumor slices were maintained 24h ex vivo, retaining viability and cell movements. Ex vivo treatments with lipopolysaccharide altered CD8+ T and myeloid cell behavior. In vivo chemotherapy reduced ex vivo cell movements in human and mouse tumors and differentially affected CD8+ T and myeloid cells in chemo-sensitive and chemo-resistant mouse models. Ex vivo tumor slices can extend in vivo mouse studies to human, providing a stepping stone to translate mouse cancer studies to clinical trials.
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Three-dimensional (3D), multicellular invitro models provide a useful platform for studying human cancer biology, particularly through deconvolution of the tumor microenvironment, or where animal models do not recapitulate the human condition. Here, we detail a protocol for building human multicellular models made of patient-derived primary cells and malignant cell lines, which recapitulate features of the tumor microenvironment. This protocol is optimized for building 3D models of high-grade serous ovarian cancer omental metastasis but can be adapted for modeling other cancers. For complete details on the use and execution of this profile, please refer to Delaine-Smith et al. (2021) and Malacrida et al. (2021).
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Neoplasias Ováricas , Neoplasias Peritoneales , Animales , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Ováricas/metabolismo , Microambiente TumoralRESUMEN
Interleukin-8 (CXCL8) produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. In human cancer, IL8 mRNA levels correlate with IL1B and TNF transcripts. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids, and fresh human tumor explants. Although IL8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1ß upregulate CXCL1 and CXCL2 in tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1ß induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα blockers infliximab and etanercept or the IL-1ß inhibitor anakinra was able to interfere with this pathogenic cytokine loop. Finally, in paired plasma samples of patients with cancer undergoing TNFα blockade with infliximab in a clinical trial, reductions of circulating IL-8 were substantiated. SIGNIFICANCE: IL-8 attracts immunosuppressive protumor myeloid cells to the tumor microenvironment, and IL-8 levels correlate with poor response to checkpoint inhibitors. TNFα and IL-1ß are identified as major inducers of IL-8 expression on malignant cells across cancer types and models in a manner that is druggable with clinically available neutralizing agents. This article is highlighted in the In This Issue feature, p. 2007.
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Citocinas , Factor de Necrosis Tumoral alfa , Animales , Citocinas/metabolismo , Humanos , Infliximab/farmacología , Infliximab/uso terapéutico , Interleucina-1beta/metabolismo , Interleucina-8/genética , Ratones , Microambiente Tumoral , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
In a multi-level "deconstruction" of omental metastases, we previously identified a prognostic matrisome gene expression signature in high-grade serous ovarian cancer (HGSOC) and twelve other malignancies. Here, our aim was to understand how six of these extracellular matrix (ECM) molecules, COL11A1, cartilage oligomeric matrix protein, FN1, versican, cathepsin B, and COL1A1, are upregulated in cancer. Using biopsies, we identified significant associations between TGFßR activity, Hedgehog (Hh) signaling, and these ECM molecules and studied the associations in mono-, co-, and tri-culture. Activated omental fibroblasts (OFs) produced more matrix than malignant cells, directed by TGFßR and Hh signaling cross talk. We "reconstructed" omental metastases in tri-cultures of HGSOC cells, OFs, and adipocytes. This combination was sufficient to generate all six ECM proteins and the matrisome expression signature. TGFßR and Hh inhibitor combinations attenuated fibroblast activation and gel and ECM remodeling in these models. The tri-culture model reproduces key features of omental metastases and allows study of diseased-associated ECM.
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Guided by a multi-level "deconstruction" of omental metastases, we developed a tetra (four cell)-culture model of primary human mesothelial cells, fibroblasts, adipocytes, and high-grade serous ovarian cancer (HGSOC) cell lines. This multi-cellular model replicated key elements of human metastases and allowed malignant cell invasion into the artificial omental structure. Prompted by findings in patient biopsies, we used the model to investigate the role of platelets in malignant cell invasion and extracellular matrix, ECM, production. RNA (sequencing and quantitative polymerase-chain reaction), protein (proteomics and immunohistochemistry) and image analysis revealed that platelets stimulated malignant cell invasion and production of ECM molecules associated with poor prognosis. Moreover, we found that platelet activation of mesothelial cells was critical in stimulating malignant cell invasion. Whilst platelets likely activate both malignant cells and mesothelial cells, the tetra-culture model allowed us to dissect the role of both cell types and model the early stages of HGSOC metastases.
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When breast cancer progresses to a metastatic stage, survival rates decline rapidly and it is considered incurable. Thus, deciphering the critical mechanisms of metastasis is of vital importance to develop new treatment options. We hypothesize that studying the proteins that are newly synthesized during the metastatic processes of migration and invasion will greatly enhance our understanding of breast cancer progression. We conducted a mass spectrometry screen following bioorthogonal noncanonical amino acid tagging to elucidate changes in the nascent proteome that occur during epidermal growth factor stimulation in migrating and invading cells. Annexin A2 was identified in this screen and subsequent examination of breast cancer cell lines revealed that Annexin A2 is specifically upregulated in estrogen receptor negative (ER-) cell lines. Furthermore, siRNA knockdown showed that Annexin A2 expression promotes the proliferation, wound healing and directional migration of breast cancer cells. In patients, Annexin A2 expression is increased in ER- breast cancer subtypes. Additionally, high Annexin A2 expression confers a higher probability of distant metastasis specifically for ER- patients. This work establishes a pivotal role of Annexin A2 in breast cancer progression and identifies Annexin A2 as a potential therapeutic target for the more aggressive and harder to treat ER- subtype.
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Anexina A2/metabolismo , Neoplasias de la Mama/metabolismo , Anexina A2/genética , Western Blotting , Neoplasias de la Mama/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Inmunoprecipitación , Células MCF-7 , Espectrometría de Masas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismoRESUMEN
The histone H3 variant CENP-A epigenetically defines the centromere and is critical for chromosome segregation. Here we report an interaction between CENP-A and subunits of the mitochondrial ATP synthase complex in the germline of male Drosophila. Furthermore, we report that knockdown of CENP-A, as well as subunits ATPsyn-α, -ßlike (a testis-specific paralogue of ATPsyn-ß) and -γ disrupts sister centromere cohesion in meiotic prophase I. We find that this disruption is likely independent of reduced ATP levels. We identify that ATPsyn-α and -ßlike localise to meiotic centromeres and that this localisation is dependent on the presence of CENP-A. We show that ATPsyn-α directly interacts with the N-terminus of CENP-A in vitro and that truncation of its N terminus perturbs sister centromere cohesion in prophase I. We propose that the CENP-A N-terminus recruits ATPsyn-α and -ßlike to centromeres to promote sister centromere cohesion in a nuclear function that is independent of oxidative phosphorylation.
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Proteína A Centromérica/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Meiosis , ATPasas de Translocación de Protón Mitocondriales/genética , Subunidades de Proteína/genética , Espermatocitos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Centrómero/metabolismo , Centrómero/ultraestructura , Proteína A Centromérica/metabolismo , Segregación Cromosómica , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Epigénesis Genética , Fertilidad/genética , Histonas/genética , Histonas/metabolismo , Masculino , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Subunidades de Proteína/metabolismo , Espermatocitos/citologíaRESUMEN
AIM: RACK1 is a multifunctional scaffolding protein that is expressed in many cellular compartments, orchestrating a number of signaling processes. RACK1 acts as a signaling hub to localize active enzymes to discrete locations; therefore tight control of RACK1 is vital to cellular homeostasis. Our aim was to identify the mechanisms responsible for RACK1 turnover and show that degradation is directed by the ubiquitin proteasome system. RESULTS: Using siRNA screening, we identified RAB40C as the ubiquitin E3 ligase responsible for ubiquitination of RACK1, and that the action of RAB40C in controlling RACK1 levels is crucial to both cancer cell growth and migration of T cells. CONCLUSION: Our data suggest that manipulation of RACK1 levels in this way may provide a novel strategy to explore RACK1 function.
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BACKGROUND AND PURPOSE: Dehydroepiandrosterone (DHEA) is thought to be an anti-glucocorticoid hormone known to be fully functional in young people but deficient in aged humans. Our previous data suggest that DHEA not only counteracts the effect of cortisol on RACK1 expression, a protein required both for the correct functioning of immune cells and for PKC-dependent pathway activation, but also modulates the inhibitory effect of cortisol on LPS-induced cytokine production. The purpose of this study was to investigate the effect of DHEA on the splicing mechanism of the human glucocorticoid receptor (GR). EXPERIMENTAL APPROACH: The THP1 monocytic cell line was used as a cellular model. Cytokine production was measured by specific elisa. Western blot and real-time RT-PCR were used, where appropriate, to determine the effect of DHEA on GRs, serine/arginine-rich proteins (SRp), and RACK1 protein and mRNA. Small-interfering RNA was used to down-regulate GRß. KEY RESULTS: DHEA induced a dose-related up-regulation of GRß and GRß knockdown completely prevented DHEA-induced RACK1 expression and modulation of cytokine release. Moreover, we showed that DHEA influenced the expression of some components of the SRps found within the spliceosome, the main regulators of the alternative splicing of the GR gene. CONCLUSIONS AND IMPLICATIONS: These data contribute to our understanding of the mechanism of action of DHEA and its effect on the immune system and as an anti-glucocorticoid agent.