scholarly journals Activation of contact-dependent antibacterial tRNase toxins by translation elongation factors

2017 ◽  
Vol 114 (10) ◽  
pp. E1951-E1957 ◽  
Author(s):  
Allison M. Jones ◽  
Fernando Garza-Sánchez ◽  
Jaime So ◽  
Christopher S. Hayes ◽  
David A. Low
Keyword(s):  
Coiled Coil ◽  
Elongation Factor ◽  
Nuclease Activity ◽  
Ternary Complexes ◽  
Cell Contact ◽  
Translation Elongation ◽  
Direct Cell ◽  
Dependent Growth

Contact-dependent growth inhibition (CDI) is a mechanism by which bacteria exchange toxins via direct cell-to-cell contact. CDI systems are distributed widely among Gram-negative pathogens and are thought to mediate interstrain competition. Here, we describetsfmutations that alter the coiled-coil domain of elongation factor Ts (EF-Ts) and confer resistance to the CdiA-CTEC869tRNase toxin from enterohemorrhagicEscherichia coliEC869. Although EF-Ts is required for toxicity in vivo, our results indicate that it is dispensable for tRNase activity in vitro. We find that CdiA-CTEC869binds to elongation factor Tu (EF-Tu) with high affinity and this interaction is critical for nuclease activity. Moreover, in vitro tRNase activity is GTP-dependent, suggesting that CdiA-CTEC869only cleaves tRNA in the context of translationally active GTP·EF-Tu·tRNA ternary complexes. We propose that EF-Ts promotes the formation of GTP·EF-Tu·tRNA ternary complexes, thereby accelerating substrate turnover for rapid depletion of target-cell tRNA.

scholarly journals Overexpression of Translation Elongation Factor 1A Affects the Organization and Function of the Actin Cytoskeleton in Yeast

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1425-1436
Author(s):  
Raj Munshi ◽  
Kimberly A Kandl ◽  
Anne Carr-Schmid ◽  
Johanna L Whitacre ◽  
Alison E M Adams ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Elongation Factor ◽  
Nucleotide Exchange ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
And Function

Abstract The translation elongation factor 1 complex (eEF1) plays a central role in protein synthesis, delivering aminoacyl-tRNAs to the elongating ribosome. The eEF1A subunit, a classic G-protein, also performs roles aside from protein synthesis. The overexpression of either eEF1A or eEF1Bα, the catalytic subunit of the guanine nucleotide exchange factor, in Saccharomyces cerevisiae results in effects on cell growth. Here we demonstrate that overexpression of either factor does not affect the levels of the other subunit or the rate or accuracy of protein synthesis. Instead, the major effects in vivo appear to be at the level of cell morphology and budding. eEF1A overexpression results in dosage-dependent reduced budding and altered actin distribution and cellular morphology. In addition, the effects of excess eEF1A in actin mutant strains show synthetic growth defects, establishing a genetic connection between the two proteins. As the ability of eEF1A to bind and bundle actin is conserved in yeast, these results link the established ability of eEF1A to bind and bundle actin in vitro with nontranslational roles for the protein in vivo.

scholarly journals Structural basis for EarP-mediated arginine glycosylation of translation elongation factor EF-P

2017 ◽  
Author(s):  
Ralph Krafczyk ◽  
Jakub Macošek ◽  
Daniel Gast ◽  
Swetlana Wunder ◽  
Pravin Kumar Ankush Jagtap ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Structural Data ◽  
Enzyme Characterization ◽  
Structural Basis ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Enzymatic Mechanism ◽  
Bacterial Translation

ABSTRACTGlycosylation is a universal strategy to post-translationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida. The enzyme is composed of two opposing domains with Rossmann-folds, thus constituting a GT-B glycosyltransferase. While TDP-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the EF-P via its KOW-like N-domain. Based on our structural data combined with an in vitro /in vivo enzyme characterization, we propose a mechanism of inverting arginine glycosylation. As EarP is essential for pathogenicity in P. aeruginosa our study provides the basis for targeted inhibitor design.

scholarly journals IL-15Rα chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation

2008 ◽  
Vol 205 (5) ◽  
pp. 1213-1225 ◽  
Author(s):  
Erwan Mortier ◽  
Tammy Woo ◽  
Rommel Advincula ◽  
Sara Gozalo ◽  
Averil Ma
Keyword(s):  
Nk Cells ◽  
Biosynthetic Pathway ◽  
Cell Activation ◽  
Nk Cell ◽  
Cell Contact ◽  
Rapid Responses ◽  
Direct Cell ◽  
Membrane Bound

Natural killer (NK) cells are innate immune effectors that mediate rapid responses to viral antigens. Interleukin (IL)-15 and its high affinity IL-15 receptor, IL-15Rα, support NK cell homeostasis in resting animals via a novel trans presentation mechanism. To better understand how IL-15 and IL-15Rα support NK cell activation during immune responses, we have used sensitive assays for detecting native IL-15 and IL-15Rα proteins and developed an assay for detecting complexes of these proteins. We find that IL-15 and IL-15Rα are preassembled in complexes within the endoplasmic reticulum/Golgi of stimulated dendritic cells (DCs) before being released from cells. IL-15Rα is required for IL-15 production by DCs, and IL-15 that emerges onto the cell surface of matured DCs does not bind to neighboring cells expressing IL-15Rα. We also find that soluble IL-15–IL-15Rα complexes are induced during inflammation, but membrane-bound IL-15–IL-15Rα complexes, rather than soluble complexes, support NK cell activation in vitro and in vivo. Finally, we provide in vivo evidence that expression of IL-15Rα specifically on DCs is critical for trans presenting IL-15 and activating NK cells. These studies define an unprecedented cytokine–receptor biosynthetic pathway in which IL-15Rα serves as a chaperone for IL-15, after which membrane-bound IL-15Rα–IL-15 complexes activate NK cells via direct cell–cell contact.

scholarly journals The cytotoxic effect of diphtheria toxin on the actin cytoskeleton

2012 ◽  
Vol 17 (1) ◽  
Author(s):  
Başak Varol ◽  
Muhammet Bektaş ◽  
Rüstem Nurten ◽  
Engin Bermek
Keyword(s):  
Actin Cytoskeleton ◽  
Dna Cleavage ◽  
Diphtheria Toxin ◽  
Gel Filtration ◽  
Elongation Factor ◽  
Nuclease Activity ◽  
Entry Rate ◽  
Adp Ribosyltransferase

AbstractDiphtheria toxin (DT) and its N-terminal fragment A (FA) catalyse the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) into a covalent linkage with eukaryotic elongation factor 2 (eEF2). DT-induced cytotoxicity is versatile, and it includes DNA cleavage and the depolymerisation of actin filaments. The inhibition of the ADP-ribosyltransferase (ADPrT) activity of FA did not affect the deoxyribonuclease activity of FA or its interaction with actin. The toxin entry rate into cells (HUVEC) was determined by measuring the ADP-ribosyltransferase activity. DT uptake was nearly 80% after 30 min. The efficiency was determined as Km = 2.2 nM; Vmax = 0.25 pmol.min−1. The nuclease activity was tested with hyperchromicity experiments, and it was concluded that G-actin has an inhibitory effect on DT nuclease activity. In thepresence of DT and mutant of diphtheria toxin (CRM197), F-actin depolymerisation was determined with gel filtration, WB and fluorescence techniques. In the presence of DT and CRM197, 60–65% F-actin depolymerisation was observed. An in vitro FA-actin interaction and F-actin depolymerisation were reported in our previous paper. The present study thus confirms the depolymerisation of actin cytoskeleton in vivo.

scholarly journals Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A

Science ◽  
2021 ◽  
Vol 371 (6532) ◽  
pp. 926-931 ◽  
Author(s):  
Kris M. White ◽  
Romel Rosales ◽  
Soner Yildiz ◽  
Thomas Kehrer ◽  
Lisa Miorin ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Elongation Factor ◽  
Resistant Mutant ◽  
Host Protein ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Eukaryotic Translation ◽  
Drug Resistant Mutant

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.

Bovine oocyte maturation: acquisition of developmental competence

2020 ◽  
Vol 32 (2) ◽  
pp. 98
Author(s):  
Bernard A. J. Roelen
Keyword(s):  
Oocyte Maturation ◽  
Developmental Competence ◽  
Cell Contact ◽  
Intimate Contact ◽  
Homologous Chromosomes ◽  
Paracrine Signalling ◽  
Sister Chromatids ◽  
Direct Cell

Although millions of oocytes are formed during embryo and fetal development in the cow, only a small fraction of these will form a developmentally competent oocyte and be fertilised. Development to competence relies on an intimate contact between the oocyte and the surrounding somatic cells in ovarian follicles, via both direct cell–cell contact and paracrine signalling. An important aspect of oocyte maturation is the segregation of homologous chromosomes and subsequently sister chromatids to form a haploid oocyte. Furthermore, the cytoplasm needs to be prepared for the formation of pronuclei and nuclear reprogramming to form a totipotent zygote. Conditions such as high levels of fatty acids or oxidative stress constrain the developmental competence of oocytes, and a better insight into these processes may help improve in vitro and in vivo oocyte maturation success. In addition, identification of the developmentally competent oocyte is useful for the efficiency of (artificial) reproduction.

scholarly journals Jagged1-Notch1-deployed tumor perivascular niche promotes breast cancer stem cell phenotype through Zeb1

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Huimin Jiang ◽  
Chen Zhou ◽  
Zhen Zhang ◽  
Qiong Wang ◽  
Huimin Wei ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Antiangiogenic Therapy ◽  
Cell Phenotype ◽  
Cell Contact ◽  
Perivascular Niche ◽  
Cancer Aggressiveness ◽  
E Box ◽  
Direct Cell

Abstract Zinc finger E-box binding homeobox 1 (Zeb1) has been demonstrated to participate in the acquisition of the properties of cancer stem cells (CSCs). However, it is largely unknown how signals from the tumor microenvironment (TME) contribute to aberrant Zeb1 expression. Here, we show that Zeb1 depletion suppresses stemness, colonization and the phenotypic plasticity of breast cancer. Moreover, we demonstrate that, with direct cell-cell contact, TME-derived endothelial cells provide the Notch ligand Jagged1 (Jag1) to neighboring breast CSCs, leading to Notch1-dependent upregulation of Zeb1. In turn, ectopic Zeb1 in tumor cells increases VEGFA production and reciprocally induces endothelial Jag1 in a paracrine manner. Depletion of Zeb1 disrupts this positive feedback loop in the tumor perivascular niche, which eventually lessens tumor initiation and progression in vivo and in vitro. In this work, we highlight that targeting the angiocrine Jag1-Notch1-Zeb1-VEGFA loop decreases breast cancer aggressiveness and thus enhances the efficacy of antiangiogenic therapy.

scholarly journals Requirement of direct contact between chondrocytes and macrophages for the maturation of regenerative cartilage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kengo Kanda ◽  
Yukiyo Asawa ◽  
Ryoko Inaki ◽  
Yuko Fujihara ◽  
Kazuto Hoshi ◽  
...  
Keyword(s):  
Cartilage Regeneration ◽  
Host Cells ◽  
Raw264.7 Cells ◽  
Cell Contact ◽  
Humoral Factors ◽  
Donor Cells ◽  
Direct Cell ◽  
Cell Cell

AbstractRegenerative cartilage prepared from cultured chondrocytes is generally immature in vitro and matures after transplantation. Although many factors, including host cells and humoral factors, have been shown to affect cartilage maturation in vivo, the requirement of direct cell–cell contact between host and donor cells remains to be verified. In this study, we examined the host cells that promote cartilage maturation via cell–cell contact. Based on analysis of the transplanted chondrocytes, we examined the contribution of endothelial cells and macrophages. Using a semiclosed device that is permeable to tissue fluids while blocking host cells, we selectively transplanted chondrocytes and HUVECs or untreated/M1-polarized/M2-polarized RAW264.7 cells. As a result, untreated RAW264.7 cells induced cartilage regeneration. Furthermore, an in vitro coculture assay indicated communication between chondrocytes and RAW264.7 cells mediated by RNA, suggesting the involvement of extracellular vesicles in this process. These findings provide insights for establishing a method of in vitro cartilage regeneration.

scholarly journals Extracellular vesicles mediate the intercellular exchange of nanoparticles

2021 ◽  
Author(s):  
Xian Wu ◽  
Tang Tang ◽  
Yushuang Wei ◽  
Katherine A. Cummins ◽  
David K. Wood ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Cell Contact ◽  
Therapeutic Effects ◽  
Cellular Receptors ◽  
Transcellular Transport ◽  
Direct Cell ◽  
Intercellular Exchange

AbstractIn order to exert their therapeutic effects, nanoparticles (NPs) often need to travel into the tissues composed of multilayered cells. Accumulative evidence has revealed the central role of transcellular transport route (entry into one cell, exocytosis, and re-entry into another) in this process. While NP endocytosis and subcellular transport have been intensively characterized, the exocytosis and re-entry steps are poorly understood, which becomes a barrier to improve NP delivery into complex tissues. Here, we termed the exocytosis and re-entry steps together as intercellular exchange. We developed a novel collagen-based 3D cellular assay to specifically monitor and quantify the intercellular exchange events of NPs and distinguish the contributions of several potential mechanisms. Our results showed that NPs can be exocytosed freely or enclosed inside extracellular vesicles (EVs) for re-entry, while direct cell-cell contact is hardly involved. EVs account for a significant fraction of NP intercellular exchange, and its importance in NP delivery was demonstrated in vitro and in vivo. Intriguingly, while freely released NPs engage with the same cellular receptors for re-entry, EV-enclosed ones bypass this dependence. These studies provide an easy and precise system to investigate the intercellular exchange stage of NP delivery, and shed the first light in the importance of EVs in NP transport between cells and across complex tissues.

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