PKR: A Kinase to Remember Gal-Ben-Ari, Shunit; Barrera, Iliana; Ehrlich, Marcelo ...
Frontiers in molecular neuroscience,
01/2019, Letnik:
11
Journal Article
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Aging is a major risk factor for many diseases including metabolic syndrome, cancer, inflammation, and neurodegeneration. Identifying mechanistic common denominators underlying the impact of aging is ...essential for our fundamental understanding of age-related diseases and the possibility to propose new ways to fight them. One can define aging biochemically as prolonged metabolic stress, the innate cellular and molecular programs responding to it, and the new stable or unstable state of equilibrium between the two. A candidate to play a role in the process is protein kinase R (PKR), first identified as a cellular protector against viral infection and today known as a major regulator of central cellular processes including mRNA translation, transcriptional control, regulation of apoptosis, and cell proliferation. Prolonged imbalance in PKR activation is both affected by biochemical and metabolic parameters and affects them in turn to create a feedforward loop. Here, we portray the central role of PKR in transferring metabolic information and regulating cellular function with a focus on cancer, inflammation, and brain function. Later, we integrate information from open data sources and discuss current knowledge and gaps in the literature about the signaling cascades upstream and downstream of PKR in different cell types and function. Finally, we summarize current major points and biological means to manipulate PKR expression and/or activation and propose PKR as a therapeutic target to shift age/metabolic-dependent undesired steady states.
Cell autonomous immunity genes mediate the multiple stages of anti-viral defenses, including recognition of invading pathogens, inhibition of viral replication, reprogramming of cellular metabolism, ...programmed-cell-death, paracrine induction of antiviral state, and activation of immunostimulatory inflammation. In tumor development and/or immunotherapy settings, selective pressure applied by the immune system results in tumor immunoediting, a reduction in the immunostimulatory potential of the cancer cell. This editing process comprises the reduced expression and/or function of cell autonomous immunity genes, allowing for immune-evasion of the tumor while concomitantly attenuating anti-viral defenses. Combined with the oncogene-enhanced anabolic nature of cancer-cell metabolism, this attenuation of antiviral defenses contributes to viral replication and to the selectivity of oncolytic viruses (OVs) towards malignant cells. Here, we review the manners by which oncogene-mediated transformation and tumor immunoediting combine to alter the intracellular milieu of tumor cells, for the benefit of OV replication. We also explore the functional connection between oncogenic signaling and epigenetic silencing, and the way by which restriction of such silencing results in immune activation. Together, the picture that emerges is one in which OVs and epigenetic modifiers are part of a growing therapeutic toolbox that employs activation of anti-tumor immunity for cancer therapy.
Lipocalin-2 (LCN2) performs pleiotropic and tumor context-dependent functions in cancers of diverse etiologies. In prostate cancer (PCa) cells, LCN2 regulates distinct phenotypic features, including ...cytoskeleton organization and expression of inflammation mediators. Oncolytic virotherapy uses oncolytic viruses (OVs) to kill cancer cells and induce anti-tumor immunity. A main source of specificity of OVs towards tumor cells stems from cancer-induced defects in interferon (IFN)-based cell autonomous immune responses. However, the molecular underpinnings of such defects in PCa cells are only partially understood. Moreover, LCN2 effects on IFN responses of PCa cells and their susceptibility to OVs are unknown. To examine these issues, we queried gene expression databases for genes coexpressed with LCN2, revealing co-expression of IFN-stimulated genes (ISGs) and LCN2. Analysis of human PCa cells revealed correlated expression of LCN2 and subsets of IFNs and ISGs. CRISPR/Cas9-mediated stable knockout of LCN2 in PC3 cells or transient overexpression of LCN2 in LNCaP cells revealed LCN2-mediated regulation of IFNE (and IFNL1) expression, activation of JAK/STAT pathway, and expression of selected ISGs. Accordingly, and dependent on a functional JAK/STAT pathway, LCN2 reduced the susceptibility of PCa cells to infection with the IFN-sensitive OV, EHDV-TAU. In PC3 cells, LCN2 knockout increased phosphorylation of eukaryotic initiation factor 2α (p-eIF2α). Inhibition of PKR-like ER kinase (PERK) in PC3-LCN2-KO cells reduced p-eIF2α while increasing constitutive IFNE expression, phosphorylation of STAT1, and ISG expression; and decreasing EHDV-TAU infection. Together, these data propose that LCN2 regulates PCa susceptibility to OVs through attenuation of PERK activity and increased IFN and ISG expression.
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•LCN2 regulates IFNE and IFNL1 expression.•LCN2 regulates ISG expression and activation of the JAK/STAT pathway.•LCN2 restricts infection of prostate cancer cells and promotes antiviral signaling.•Restriction of EHDV-TAU infection by LCN2 is JAK dependent.•LCN2-mediated attenuation of PERK enhances antiviral signaling.
The intracellular pathogen Legionella pneumophila translocates a large number of effector proteins into host cells via the Icm/Dot type-IVB secretion system. Some of these effectors were shown to ...cause lethal effect on yeast growth. Here we characterized one such effector (LecE) and identified yeast suppressors that reduced its lethal effect. The LecE lethal effect was found to be suppressed by the over expression of the yeast protein Dgk1 a diacylglycerol (DAG) kinase enzyme and by a deletion of the gene encoding for Pah1 a phosphatidic acid (PA) phosphatase that counteracts the activity of Dgk1. Genetic analysis using yeast deletion mutants, strains expressing relevant yeast genes and point mutations constructed in the Dgk1 and Pah1 conserved domains indicated that LecE functions similarly to the Nem1-Spo7 phosphatase complex that activates Pah1 in yeast. In addition, by using relevant yeast genetic backgrounds we examined several L. pneumophila effectors expected to be involved in phospholipids biosynthesis and identified an effector (LpdA) that contains a phospholipase-D (PLD) domain which caused lethal effect only in a dgk1 deletion mutant of yeast. Additionally, LpdA was found to enhance the lethal effect of LecE in yeast cells, a phenomenon which was found to be dependent on its PLD activity. Furthermore, to determine whether LecE and LpdA affect the levels or distribution of DAG and PA in-vivo in mammalian cells, we utilized fluorescent DAG and PA biosensors and validated the notion that LecE and LpdA affect the in-vivo levels and distribution of DAG and PA, respectively. Finally, we examined the intracellular localization of both LecE and LpdA in human macrophages during L. pneumophila infection and found that both effectors are localized to the bacterial phagosome. Our results suggest that L. pneumophila utilize at least two effectors to manipulate important steps in phospholipids biosynthesis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The glucosinolate breakdown product indole‐3‐carbinol functions in cruciferous vegetables as a protective agent against foraging insects. While the toxic and deterrent effects of glucosinolate ...breakdown on herbivores and pathogens have been studied extensively, the secondary responses that are induced in the plant by indole‐3‐carbinol remain relatively uninvestigated. Here we examined the hypothesis that indole‐3‐carbinol plays a role in influencing plant growth and development by manipulating auxin signaling. We show that indole‐3‐carbinol rapidly and reversibly inhibits root elongation in a dose‐dependent manner, and that this inhibition is accompanied by a loss of auxin activity in the root meristem. A direct interaction between indole‐3‐carbinol and the auxin perception machinery was suggested, as application of indole‐3‐carbinol rescues auxin‐induced root phenotypes. In vitro and yeast‐based protein interaction studies showed that indole‐3‐carbinol perturbs the auxin‐dependent interaction of Transport Inhibitor Response (TIR1) with auxin/3‐indoleacetic acid (Aux/IAAs) proteins, further supporting the possibility that indole‐3‐carbinol acts as an auxin antagonist. The results indicate that chemicals whose production is induced by herbivory, such as indole‐3‐carbinol, function not only to repel herbivores, but also as signaling molecules that directly compete with auxin to fine tune plant growth and development.
Malignancy-induced alterations to cytokine signaling in tumor cells differentially regulate their interactions with the immune system and oncolytic viruses. The abundance of inflammatory cytokines in ...the tumor microenvironment suggests that such signaling plays key roles in tumor development and therapy efficacy. The JAK-STAT axis transduces signals of interleukin-6 (IL-6) and interferons (IFNs), mediates antiviral responses, and is frequently altered in prostate cancer (PCa) cells. However, how activation of JAK-STAT signaling with different cytokines regulates interactions between oncolytic viruses and PCa cells is not known. Here, we employ LNCaP PCa cells, expressing (or not) JAK1, activated (or not) with IFNs (α or γ) or IL-6, and infected with RNA viruses of different oncolytic potential (EHDV-TAU, hMPV-GFP, or HIV-GFP) to address this matter. We show that in JAK1-expressing cells, IL-6 sensitized PCa cells to viral cell death in the presence or absence of productive infection, with dependence on virus employed. Contrastingly, IFNα induced a cytoprotective antiviral state. Biochemical and genetic (knockout) analyses revealed dependency of antiviral state or cytoprotection on STAT1 or STAT2 activation, respectively. In IL-6-treated cells, STAT3 expression was required for anti-proliferative signaling. Quantitative proteomics (SILAC) revealed a core repertoire of antiviral IFN-stimulated genes, induced by IL-6 or IFNs. Oncolysis in the absence of productive infection, induced by IL-6, correlated with reduction in regulators of cell cycle and metabolism. These results call for matching the viral features of the oncolytic agent, the malignancy-induced genetic-epigenetic alterations to JAK/STAT signaling and the cytokine composition of the tumor microenvironment for efficient oncolytic virotherapy.
Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We ...have detected the formation of coats by monitoring incorporation of fluorescently tagged clathrin or its adaptor AP-2; we have also followed clathrin-mediated uptake of transferrin and of single LDL or reovirus particles. The intensity of a cargo-loaded clathrin cluster grows steadily during its lifetime, and the time required to complete assembly is proportional to the size of the cargo particle. These results are consistent with a nucleation-growth mechanism and an approximately constant growth rate. There are no strongly preferred nucleation sites. A proportion of the nucleation events are weak and short lived. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit. Our data lead to a model in which coated pits initiate randomly but collapse unless stabilized, perhaps by cargo capture.
Stromal cells and pro-inflammatory cytokines play key roles in promoting the aggressiveness of triple-negative breast cancers (TNBC; Basal/Basal-like). In our previous study we demonstrated that ...stimulation of TNBC and mesenchymal stem cells (MSCs) co-cultures by the pro-inflammatory cytokine tumor necrosis factor α (TNFα) has led to increased metastasis-related properties
and
. In this context, elevated release of the pro-metastatic chemokines CXCL8 (IL-8) and CCL5 (RANTES) was noted in TNFα- and interleukin-1β (IL-1β)-stimulated TNBC:MSC co-cultures; the process was partly (CXCL8) and entirely (CCL5) dependent on physical contacts between the two cell types. Here, we demonstrate that DAPT, inhibitor of γ-secretase that participates in activation of Notch receptors, inhibited the migration and invasion of TNBC cells that were grown in "Contact" co-cultures with MSCs or with patient-derived cancer-associated fibroblasts (CAFs), in the presence of TNFα. DAPT also inhibited the contact-dependent induction of CXCL8, but not of CCL5, in TNFα- and IL-1β-stimulated TNBC:MSC/CAF co-cultures; some level of heterogeneity between the responses of different TNBC cell lines was noted, with MDA-MB-231:MSC/CAF co-cultures being the most sensitive to DAPT. Patient dataset studies comparing basal tumors to luminal-A tumors, and mRNA analyses of Notch receptors in TNBC and luminal-A cells pointed at Notch1 as possible mediator of CXCL8 increase in TNFα-stimulated TNBC:stroma "Contact" co-cultures. Accordingly, down-regulation of Notch1 in TNBC cells by siRNA has substantially reduced the contact-dependent elevation in CXCL8 in TNFα- and also in IL-1β-stimulated TNBC:MSC "Contact" co-cultures. Then, studies in which CXCL8 or p65 (NF-κB pathway) were down-regulated (siRNAs; CRISPR/Cas9) in TNBC cells and/or MSCs, indicated that upon TNFα stimulation of "Contact" co-cultures, p65 was activated and led to CXCL8 production mainly in TNBC cells. Moreover, our findings indicated that when tumor cells interacted with stromal cells in the presence of pro-inflammatory stimuli, TNFα-induced p65 activation has led to elevated Notch1 expression and activation, which then gave rise to elevated production of CXCL8. Overall, tumor:stroma interactions set the stage for Notch1 activation by pro-inflammatory signals, leading to CXCL8 induction and consequently to pro-metastatic activities. These observations may have important clinical implications in designing novel therapy combinations in TNBC.
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