1,720,977 research outputs found
A Genome-Wide CRISPR-Cas9 Loss-of-Function Screening to Identify Host Restriction Factors Modulating Oncolytic Virotherapy
No full textOncolytic virotherapy represents an efficient immunotherapeutic approach for cancer treatment. Oncolytic viruses (OVs) promote antitumor responses through tumor-selective cell lysis and immune system activation. However, some tumor cell lines and primary tumors display resistance to therapy. Here we describe a protocol to identify novel host factors responsible for tumor resistance to oncolysis using an unbiased genome-wide CRISPR-Cas9 loss-of-function screening. Cas9-expressing tumor cells are transduced with a library of pooled single-guide RNA (sgRNA)-expressing lentiviruses that target all human genes to obtain a cell population where each cell is knocked out for a single gene. Upon OV infection, resistant cells survive, while sensitive cells die. The relative abundance of each genome-integrated sgRNA is measured by next-generation sequencing (NGS) in resistant and control cells. This protocol is amenable to uncover host factors involved in the resistance to different OVs in multiple tumor models
A novel association between filamin A and NF-κB inducing kinase couples CD28 to inhibitor of NF-κB kinase α and NF-κB activation
No full textCD28 costimulatory molecule plays a critical role in the activation of NF-κB. Indeed, while stimulation of T cells with either professional APCs or anti-TCR plus anti-CD28 antibodies efficiently activates NF-κB, TCR alone fails to do that. Moreover, CD28 stimulation by B7 in the absence of TCR may activate IκB kinase α (IKKα) and a non-canonical NF-κB2-like pathway, in human primary CD4+ T cells. Despite its functional relevance in NF-κB activation, the molecules connecting autonomous CD28-mediated signals to IKKα and NF-κB activation remain still unknown. In searching for specific upstream activators linking CD28 to the IKKα/NF-κB cascade, we identify a novel constitutive association between filamin A (FLNa) and the NF-κB inducing kinase (NIK), in both Jurkat and human primary T cells. Following CD28 engagement by B7, in the absence of TCR, FLNa-associated NIK is activated and induces IKKα kinase activity. Both proline (P208YAP211P212) and tyrosine residues (Y206QPY209APP) within the C-terminal proline-rich motif of CD28 are involved in the recruitment of FLNa/NIK complexes to the membrane as well as in the activation of NIK and IKKα. © 2011 Elsevier B.V
The multifaceted role of PIP2 in leukocyte biology
No full textPhosphatidylinositol 4,5-bisphosphate (PIP2) represents about 1 % of plasma membrane phospholipids and behaves as a pleiotropic regulator of a striking number of fundamental cellular processes. In recent years, an increasing body of literature has highlighted an essential role of PIP2 in multiple aspects of leukocyte biology. In this emerging picture, PIP2 is envisaged as a signalling intermediate itself and as a membrane-bound regulator and a scaffold of proteins with specific PIP2 binding domains. Indeed PIP2 plays a key role in several functions. These include directional migration in neutrophils, integrin-dependent adhesion in T lymphocytes, phagocytosis in macrophages, lysosomes secretion and trafficking at immune synapse in cytolytic effectors and secretory cells, calcium signals and gene transcription in B lymphocytes, natural killer cells and mast cells. The coordination of these different aspects relies on the spatio-temporal organisation of distinct PIP2 pools, generated by the main PIP2 generating enzyme, phosphatidylinositol 4-phosphate 5-kinase (PIP5K). Three different isoforms of PIP5K, named α, β and γ, and different splice variants have been described in leukocyte populations. The isoform-specific coupling of specific isoforms of PIP5K to different families of activating receptors, including integrins, Fc receptors, toll-like receptors and chemokine receptors, is starting to be reported. Furthermore, PIP2 is turned over by multiple metabolising enzymes including phospholipase C (PLC) γ and phosphatidylinositol 3-kinase (PI3K) which, along with Rho family small G proteins, is widely involved in strategic functions within the immune system. The interplay between PIP2, lipid-modifying enzymes and small G protein-regulated signals is also discussed
The recruitment and activation of phosphatidylinositol 4-phosphate 5-kinases α critically regulate CD28-dependent signaling responses
Full text linkCD28 costimulatory receptor is a crucial determinant of the outcome of T lymphocyte activation. The engagement of CD28 by its natural ligands, B7.1/CD80 or B7.2/CD86, expressed on the surface of professional APC, lowers T cell receptor (TCR) activation threshold, thus leading to the enhancement of early signalling events necessary for efficient cytokine production, cell cycle progression, survival and regulation of T cells effector responses. CD28 is also able to act as a unique signalling receptor and to deliver TCR-independent autonomous signals, which account for its critical role in the regulation of pro-inflammatory cytokine/chemokine production and T cell survival. Most of the CD28-dependent signalling functions are initiated by the recruitment and activation of class IA phosphatidylinositol 3-kinase (PI3K), The intracytoplasmic domain of CD28 contains a N-terminal YMNM motif that following phosphorylation binds the p85 subunit of phosphatidylinositol 3-kinase (PI3K). Once activated, PI3K catalyzes the conversion of phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) and generates the docking sites for key signalling proteins. PIP2 plays a critical role in the regulation of both cytoskeleton dynamics and second messenger generation. Indeed, PIP2 is the common source for two major distinct signalling cascades involving PI3K and PLCγ1 that often colocalize in the same signalling complexes competing for the common pool of substrate. Consequently, PIP2 levels decrease following receptor activation, thus suggesting that stimulation of PIP2 synthesis may be an essential regulatory step to sustain the activation of both PI3K and PLCγ1 following CD28 engagement. The main biosynthetic pathway of PIP2 involves phosphorylation of phosphatidylinositol 4-monophosphate (PI4P) at the D5 position of the inositol ring by PIP5K. Three PIP5K isoforms (α, β and γ) have been identified. Several data obtained in different cell systems evidenced differential subcellular localizations of each isoform. PIP5Kα, for instance, is localized at the plasma membrane, where it guarantees the local availability of PIP2.
Here we show that CD28 stimulation by both B7.1/CD80 or agonistic Abs induces the recruitment and activation of PIP5Kα in human primary CD4+ T lymphocytes. This event leads to the neo-synthesis of PIP2 that is consumed by CD28-activated PI3K. By either small interference RNA (siRNA)-driven cell silencing or overexpressing a kinase dead mutant, we evidenced that PIP5Kα activation is required for both CD28 autonomous signals regulating IL-8 gene expression as well as for CD28/TCR-induced Ca2+ mobilization, NF-AT nuclear translocation and IL-2 gene transcription. Our findings identify PIP5Kα as a critical mediator of CD28-dependent responses
p53 death signal is mainly mediated by Nuc1(EndoG) in the yeast Saccharomyces cerevisiae
No full textThe tumor suppressor p53 plays a central role in the regulation of cellular growth and apoptosis. In the yeast Saccharomyces cerevisiae, the overexpression of the human p53 leads to growth inhibition and apoptotic cell death on minimal medium. In the present work, we show that p53-expressing cells are more susceptible to cell death after an apoptotic stimulus such as H2O2. The analysis of mutants involved in yeast apoptosis-like death suggests that the observed cell death is Yca1 independent and mainly mediated through Nuc1p. © 2013 Federation of European Microbiological Societies
Phosphatidylinositol 4-phosphate 5-kinase β controls recruitment of lipid rafts into the immunological synapse
No full textPhosphatidylinositol 4,5-biphosphate (PIP2) is critical for T lymphocyte activation serving as a substrate for the generation of
second messengers and the remodeling of actin cytoskeleton necessary for the clustering of lipid rafts, TCR, and costimulatory
receptors toward the T:APC interface. Spatiotemporal analysis of PIP2 synthesis in T lymphocytes suggested that distinct isoforms
of the main PIP2-generating enzyme, phosphatidylinositol 4-phosphate 5-kinase (PIP5K), play a differential role on the basis of their
distinct localization. In this study, we analyze the contribution of PIP5Kb to T cell activation and show that CD28 induces the
recruitment of PIP5Kb to the immunological synapse, where it regulates filamin A and lipid raft accumulation, as well as T cell
activation, in a nonredundant manner. Finally, we found that Vav1 and the C-terminal 83 aa of PIP5Kb are pivotal for the
PIP5Kb regulatory functions in response to CD28 stimulation
Trichostatin A up-regulates p73 and induces Bax-dependent apoptosis in cisplatin-resistant ovarian cancer cells
No full textSeveral studies in the last years evidenced that deregulation of proapoptotic and antiapoptotic pathways are key players in the onset and maintenance of chemoresistance in advanced ovarian cancers. To characterize the signaling events and molecules involved in the acquisition of cisplatin resistance, we used the human ovarian cancer cell line A2780 and its derivative cisplatin-resistant subline A2780 CIS. We found that the mitochondrial intrinsic apoptotic pathway, induced by cis-dichlorodiammineplatinum (CDDP) in A2780 wild-type cells, was compromised in the resistant subline CIS. The analysis of expression of proteins involved in mitochondria-dependent apoptosis revealed a role of Bax and p73 but not p53. Indeed, we found that CDDP treatment induced the up-regulation of p53 in both sensitive and resistant A2780 cell lines. By contrast, p73 and Bax expressions were compromised in resistant cells. Pretreatment of resistant A2780 CIS cells with the histone deacetylase inhibitor trichostatin A overcomes apoptosis resistance to CDDP by restoring both p73 and Bax but not p53 expression. Altogether, these data indicate that p73, but not p53, is involved in the regulation of apoptosis susceptibility to cisplatin in A2780 ovarian cancer cells and evidence a key contribution of histone deacetylase activation in the acquisition of chemotherapy resistance in human ovarian cancer cells
Phosphatidylinositol 4-phosphate 5-kinase α: a key modulator of CD28 costimulatory signals
No full textCD28 individual signals amplify pro-inflammatory cytokine production in relapse-remitting multiple sclerosis T lymphocytes.
No full textCD28 may be considered one of the most important costimulatory receptor that by binding its cognate ligands B7.1/CD80 or B7.2/CD86 on the surface of professional antigen presenting cells (APCs), lowers TCR activation threshold and leads to the augmentation of early signalling events necessary for efficient cytokine production, cell cycle progression and survival. The role of CD28 in multiple sclerosis (MS) has been often evaluated as the source of costimulatory signals integrating those delivered by TCR and blockade of CD28/B7 interaction by recombinant CTLA4-Ig, which binds B7 molecules with high affinity, is being evaluated for the use in MS patients. However, CD28 is also able to act as a unique signalling receptor and to arise TCR-independent autonomous signals. We have previously demonstrated that CD28 stimulation by agonistic antibodies or B7 expressed on APCs, in the absence of TCR engagement, is able to activate a specific NF-κB pathway in peripheral CD4+ T cells, leading to both the production of pro-inflammatory cytokine/chemokines and to the activation of survival genes.. NF-κB has been defined as a central mediator of the immune responses, by regulating the expression of over 150 genes including chemokines, cytokines and adhesion molecules involved in inflammation. Thus, CD28 stimulation may provide TCR-independent signals, which sustain the inflammatory response in MS.
By comparing the cytokine and chemokine patterns of peripheral blood CD4+ T cells isolated from relapsing-remitting MS (RRMS) patients and healthy donors (HD), we found that in RRMS but not in HD, CD28 stimulation selectively induces the expression of IL-6, IL-21 and IL-17A, all cytokines related to the Th17 cell profile phenotype. We also demonstrate that the up-regulation of all pro-inflammatory cytokines tested was dependent on CD28-mediated phosphatydilinositol 3 kinase (PI3K) activation. Therefore,CD28 could be considered as a novel receptor molecule that may contribute to amplify the inflammatory response in RRMS by favouring pro-inflammatory cytokine production and Th17 amplification
Virus-like particle – mediated delivery of the RIG-I agonist M8 induces a type I interferon response and protects cells against viral infection
Full text linkVirus-Like Particles (VLPs) are nanostructures that share conformation and self-assembly properties with viruses, but lack a viral genome and therefore the infectious capacity. In this study, we produced VLPs by co-expression of VSV glycoprotein (VSV-G) and HIV structural proteins (Gag, Pol) that incorporated a strong sequence-optimized 5’ppp-RNA RIG-I agonist, termed M8. Treatment of target cells with VLPs-M8 generated an antiviral state that conferred resistance against multiple viruses. Interestingly, treatment with VLPs-M8 also elicited a therapeutic effect by inhibiting ongoing viral replication in previously infected cells. Finally, the expression of SARS-CoV-2 Spike glycoprotein on the VLP surface retargeted VLPs to ACE2 expressing cells, thus selectively blocking viral infection in permissive cells. These results highlight the potential of VLPs-M8 as a therapeutic and prophylactic vaccine platform. Overall, these observations indicate that the modification of VLP surface glycoproteins and the incorporation of nucleic acids or therapeutic drugs, will permit modulation of particle tropism, direct specific innate and adaptive immune responses in target tissues, and boost immunogenicity while minimizing off-target effects
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