Towards the application of Tc toxins as a universal protein translocation system

AbstractTc toxins are large bacterial protein complexes that inject cytotoxic enzymes into target cells using a sophisticated syringe-like mechanism. Tc toxins are composed of a membrane translocator and a cocoon that encapsulates a toxic enzyme. The toxic enzyme varies between Tc toxins from different species and is not conserved. Here, we investigated whether the toxic enzyme can be replaced by other small proteins of different origin and properties, namely human Cdc42, herpes simplex virus ICP47, Arabidopsis thaliana iLOV, Escherichia coli DHFR, human Ras-binding domain of CRAF kinase, and tobacco etch virus protease. Using a combination of electron microscopy, X-ray crystallography and in vitro translocation assays, we demonstrate that it is possible to turn Tc toxins into customizable molecular syringes for delivering proteins of interest across membranes. We also infer the guidelines that protein cargos must obey in terms of size, charge, and fold in order to successfully take advantage of this new universal protein translocation system.

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Towards the application of Tc toxins as a universal protein translocation system

Nature Communications ◽

10.1038/s41467-019-13253-8 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 5

Author(s):

Daniel Roderer ◽

Evelyn Schubert ◽

Oleg Sitsel ◽

Stefan Raunser

Keyword(s):

Protein Translocation ◽

Protein Complexes ◽

Target Cells ◽

X Ray Crystallography ◽

Tev Protease ◽

Small Proteins ◽

Simplex Virus ◽

In Vitro Translocation ◽

Universal Protein

AbstractTc toxins are bacterial protein complexes that inject cytotoxic enzymes into target cells using a syringe-like mechanism. Tc toxins are composed of a membrane translocator and a cocoon that encapsulates a toxic enzyme. The toxic enzyme varies between Tc toxins from different species and is not conserved. Here, we investigate whether the toxic enzyme can be replaced by other small proteins of different origin and properties, namely Cdc42, herpes simplex virus ICP47, Arabidopsis thaliana iLOV, Escherichia coli DHFR, Ras-binding domain of CRAF kinase, and TEV protease. Using a combination of electron microscopy, X-ray crystallography and in vitro translocation assays, we demonstrate that it is possible to turn Tc toxins into customizable molecular syringes for delivering proteins of interest across membranes. We also infer the guidelines that protein cargos must obey in terms of size, charge, and fold in order to apply Tc toxins as a universal protein translocation system.

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Human neutrophil – mediated destruction of antibody sensitized herpes simplex virus type I infected cells

Canadian Journal of Microbiology ◽

10.1139/m78-031 ◽

1978 ◽

Vol 24 (2) ◽

pp. 182-186 ◽

Cited By ~ 10

Author(s):

Yoshaiki Fujimiya ◽

Barry T. Rouse ◽

Lorne A. Babiuk

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Human Peripheral Blood ◽

Polymorphonuclear Neutrophils ◽

Target Cells ◽

Type I ◽

Effector Cells ◽

Ability To Act ◽

Simplex Virus

Human peripheral blood polymorphonuclear neutrophils (PMN) were tested for their ability to act as effector cells in antibody-dependent cell cytotoxicity (ADCC) against Herpes simplex virus (HSV) infected target cells sensitized with anti-HSV serum. The PMN from all 29 individuals tested could mediate ADCC in the presence of a standard human anti-HSV serum. Since PMN are prominent cells early in herpes lesions, it was hypothesized that because ADCC could represent an in vitro model for antiviral recovery, perhaps the efficacy of PMN at mediating ADCC might be impaired in those subject to frequent recrudescent herpes. However, evidence for the hypothesis was not obtained since the PMN from individuals with frequent, infrequent, or unrecorded herpes labialis all showed approximately the same activity at mediating ADCC. Alternative ways in which PMN could be involved in antiviral recovery were discussed.

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Identification of a ubiquitin-binding interface using Rosetta and DEER

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1716861115 ◽

2018 ◽

Vol 115 (3) ◽

pp. 525-530 ◽

Cited By ~ 9

Author(s):

Maxx H. Tessmer ◽

David M. Anderson ◽

Adam M. Pickrum ◽

Molly O. Riegert ◽

Rocco Moretti ◽

...

Keyword(s):

Enzymatic Activity ◽

Tissue Injury ◽

Target Cells ◽

Biological Assays ◽

X Ray Crystallography ◽

Protein Protein Interaction ◽

Binding Interface ◽

Ubiquitin Binding ◽

Double Electron Electron Resonance

ExoU is a type III-secreted cytotoxin expressing A2 phospholipase activity when injected into eukaryotic target cells by the bacterium Pseudomonas aeruginosa. The enzymatic activity of ExoU is undetectable in vitro unless ubiquitin, a required cofactor, is added to the reaction. The role of ubiquitin in facilitating ExoU enzymatic activity is poorly understood but of significance for designing inhibitors to prevent tissue injury during infections with strains of P. aeruginosa producing this toxin. Most ubiquitin-binding proteins, including ExoU, demonstrate a low (micromolar) affinity for monoubiquitin (monoUb). Additionally, ExoU is a large and dynamic protein, limiting the applicability of traditional structural techniques such as NMR and X-ray crystallography to define this protein–protein interaction. Recent advancements in computational methods, however, have allowed high-resolution protein modeling using sparse data. In this study, we combine double electron–electron resonance (DEER) spectroscopy and Rosetta modeling to identify potential binding interfaces of ExoU and monoUb. The lowest-energy scoring model was tested using biochemical, biophysical, and biological techniques. To verify the binding interface, Rosetta was used to design a panel of mutations to modulate binding, including one variant with enhanced binding affinity. Our analyses show the utility of computational modeling when combined with sensitive biological assays and biophysical approaches that are exquisitely suited for large dynamic proteins.

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RNA and RNA–Protein Complex Crystallography and its Challenges

Australian Journal of Chemistry ◽

10.1071/ch14319 ◽

2014 ◽

Vol 67 (12) ◽

pp. 1741 ◽

Cited By ~ 3

Author(s):

Janine K. Flores ◽

James L. Walshe ◽

Sandro F. Ataide

Keyword(s):

Protein Complex ◽

Protein Complexes ◽

Future Directions ◽

Vast Number ◽

X Ray Crystallography ◽

Rna Biology ◽

Non Coding Rnas ◽

Rna Protein Complexes

RNA biology has changed completely in the past decade with the discovery of non-coding RNAs. Unfortunately, obtaining mechanistic information about these RNAs alone or in cellular complexes with proteins has been a major problem. X-ray crystallography of RNA and RNA–protein complexes has suffered from the major problems encountered in preparing and purifying them in large quantity. Here, we review the available techniques and methods in vitro and in vivo used to prepare and purify RNA and RNA–protein complex for crystallographic studies. We also discuss the future directions necessary to explore the vast number of RNA species waiting for their atomic-resolution structure to be determined.

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Inhibition of Herpes Simplex Virus Type 1 and Type 2 Infections by Peptide-Derivatized Dendrimers

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00149-11 ◽

2011 ◽

Vol 55 (7) ◽

pp. 3231-3239 ◽

Cited By ~ 54

Author(s):

Anna Luganini ◽

Silvia Fabiole Nicoletto ◽

Lorena Pizzuto ◽

Giovanna Pirri ◽

Andrea Giuliani ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Serum Proteins ◽

Antiviral Agents ◽

Target Cells ◽

Synergistic Activity ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

Hsv 1

ABSTRACTIn response to the need for new antiviral agents, dendrimer-based molecules have been recognized as having a large number of potential therapeutic applications. They include peptide-derivatized dendrimers, which are hyperbranched synthetic well-defined molecules which consist of a peptidyl branching core and covalently attached surface functional peptides. However, few studies have addressed their applications as direct-acting antiviral agents. Here, we report on the ability of the peptide dendrimer SB105 and its derivative, SB105_A10, to directly inhibit herpes simplex virus 1 (HSV-1) and HSV-2in vitroreplication, with favorable selective indexes discerned for both compounds. An analysis of their mode of action revealed that SB105 and SB105_A10 prevent HSV-1 and HSV-2 attachment to target cells, whereas SB104, a dendrimer with a different amino acid sequence within the functional group and minimal antiviral activity, was ineffective in blocking HSV attachment. Moreover, both SB105 and SB105_A10 retained their ability to inhibit HSV adsorption at pH 3.0 and 4.0 and in the presence of 10% human serum proteins, conditions mimicking the physiological properties of the vagina, a potential therapeutic location for such compounds. The inhibition of HSV adsorption is likely to stem from the ability of SB105_A10 to bind to the glycosaminoglycan moiety of cell surface heparan sulfate proteoglycans, thereby blocking virion attachment to target cells. Finally, when combined with acyclovir in checkerboard experiments SB105_A10 exhibited highly synergistic activity. Taken together, these findings suggest that SB105 and SB105_A10 are promising candidates for the development of novel topical microbicides for the prevention of HSV infections.

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Effect of Complexes of Cobalt With Aminoacids on the Replication of Herpes Simplex Virus Type 1 (HSV-1)

Metal-Based Drugs ◽

10.1155/mbd.1996.149 ◽

1996 ◽

Vol 3 (3) ◽

pp. 149-154 ◽

Cited By ~ 1

Author(s):

T. Varadinova ◽

S. Shishkov ◽

M. Panteva ◽

P. Bontchev

Keyword(s):

The Body ◽

Target Cells ◽

Stability Structure ◽

Charge Stability ◽

Low Toxicity ◽

Virucidal Effect ◽

Simplex Virus ◽

Hsv 1

Cobalt, being essential metal, influences different physiological and enzymatic functions. As cobalt does not accumulate in the body, Co-compounds have relatively low toxicity. The aim of the present study is the effect of complexes of Co(II) with aminoacids - lysine, arginine, histidine and serine on HSV-1 replication. No effect of [O2Co(his)4].nH2O and [O2Co(arg)2].nH2O on HSV-1 infection in vitro was found. Both, [O2Co(lys)2].nH2O and [O2Co(ser)2].nH2O suppress the attachement of HSV-1 particles onto target cells and the viral replication as well. Moreover, the properties of the particular Co-complex (charge, stability, structure) are manifestated by their virucidal effect. Thus, [O2Co(ser)2].nH2O irreversibly inhibits the infectious activity of free HSV-1 virions, while virucidal effect of [O2Co(lys)2].nH2O is completely reversible after the 2h of contact.

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Ultrastructural Changes in Tumor Cells During Human Peripheral Blood Monocyte-Mediated Cytotoxicity

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098757 ◽

1981 ◽

Vol 39 ◽

pp. 452-453

Author(s):

K. E. Muse ◽

D. G. Fischer ◽

H. S. Koren

Keyword(s):

Target Cell ◽

Human Peripheral Blood ◽

Mononuclear Phagocytes ◽

Ultrastructural Changes ◽

Target Cells ◽

Limited Information ◽

Blood Monocytes ◽

Tumoricidal Activity ◽

Activated State

Mononuclear phagocytes, a pluripotential cell line, manifest an array of basic extracellular functions. Among these physiological regulatory functions is the expression of spontaneous cytolytic potential against tumor cell targets.The limited observations on human cells, almost exclusively blood monocytes, initially reported limited or a lack of tumoricidal activity in the absence of antibody. More recently, freshly obtained monocytes have been reported to spontaneously impair the biability of tumor target cells in vitro (Harowitz et al., 1979; Montavani et al., 1979; Hammerstrom, 1979). Although the mechanism by which effector cells express cytotoxicity is poorly understood, discrete steps can be distinguished in the process of cell mediated cytotoxicity: recognition and binding of effector to target cells,a lethal-hit stage, and subsequent lysis of the target cell. Other important parameters in monocyte-mediated cytotoxicity include, activated state of the monocyte, effector cell concentrations, and target cell suseptibility. However, limited information is available with regard to the ultrastructural changes accompanying monocyte-mediated cytotoxicity.

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STEROID HORMONE METABOLISM IN RESPONSIVE TISSUES IN VITRO

Acta Endocrinologica ◽

10.1530/acta.0.068s279 ◽

1971 ◽

Vol 68 (1_Suppl) ◽

pp. S279-S294 ◽

Cited By ~ 12

Author(s):

Paul Robel

Keyword(s):

Steroid Hormone ◽

Physiological Activity ◽

Physiological State ◽

Target Cells ◽

Target Tissue ◽

Hormone Metabolism ◽

Metabolizing Enzymes ◽

Relationship Of

ABSTRACT Of the information available on steroid hormone metabolism in responsive tissues, only that relating hormone metabolism to physiological activity is reviewed, i. e. metabolite activity in isolated in vitro systems, binding of metabolites to target tissue receptors, specific steroid hormone metabolizing enzymes and relationship of hormone metabolism to target organ physiological state. Further, evidence is presented in the androgen field, demonstrating 5α-reduced metabolites, formed inside the target cells, as active compounds. This has led to a consideration of testosterone as a »prehormone«. The possibility that similar events take place in tissues responding to progesterone is discussed. Finally, the role of hormone metabolism in the regulation of hormone availability and/or renewal in target cells is discussed. In this context, reference is made to the potential role of plasma binding proteins and cytosol receptors.

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