scholarly journals Interaction of the Amino-Terminal Domain of the ISAV Fusion Protein with a Cognate Cell Receptor

Pathogens ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 416
Author(s):  
Nicolás Ojeda ◽  
Constanza Cárdenas ◽  
Sergio Marshall
Keyword(s):  
Fusion Protein ◽  
Tertiary Structure ◽  
Structural Characteristics ◽  
Cell Receptor ◽  
Type I ◽  
Amino Terminal ◽  
Infectious Salmon Anemia ◽  
Anemia Virus ◽  
Carboxy Terminal

The infectious salmon anemia virus (ISAV), etiological agent of the disease by the same name, causes major losses to the salmon industry. Classified as a member of the Orthomyxoviridae family, ISAV is characterized by the presence of two surface glycoproteins termed hemagglutinin esterase (HE) and fusion protein (F), both of them directly involved in the initial interaction of the virus with the target cell. HE mediates receptor binding and destruction, while F promotes the fusion process of the viral and cell membranes. The carboxy-terminal end of F (F2) possesses canonical structural characteristics of a type I fusion protein, while no functional properties have been proposed for the amino-terminal (F1) region. In this report, based on in silico modeling, we propose a tertiary structure for the F1 region, which resembles a sialic acid binding domain. Furthermore, using recombinant forms of both HE and F proteins and an in vitro model system, we demonstrate the interaction of F with a cell receptor, the hydrolysis of this receptor by the HE esterase, and a crucial role for F1 in the fusion mechanism. Our interpretation is that binding of F to its cell receptor is fundamental for membrane fusion and that the esterase in HE modulates this interaction.

scholarly journals Chitosan-Based Nanoparticles for Intracellular Delivery of ISAV Fusion Protein cDNA into Melanoma Cells: A Path to Develop Oncolytic Anticancer Therapies

2020 ◽  
Vol 2020 ◽  
pp. 1-13 ◽  
Author(s):  
Claudia Robles-Planells ◽  
Giselle Sánchez-Guerrero ◽  
Carlos Barrera-Avalos ◽  
Silvia Matiacevich ◽  
Leonel E. Rojo ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Chitosan Nanoparticles ◽  
Syncytium Formation ◽  
Melanoma Cells ◽  
Current Evidence ◽  
Infectious Salmon Anemia ◽  
Melanoma Model ◽  
Anemia Virus

Oncolytic virus therapy has been tested against cancer in preclinical models and clinical assays. Current evidence shows that viruses induce cytopathic effects associated with fusogenic protein-mediated syncytium formation and immunogenic cell death of eukaryotic cells. We have previously demonstrated that tumor cell bodies generated from cells expressing the fusogenic protein of the infectious salmon anemia virus (ISAV-F) enhance crosspriming and display prophylactic antitumor activity against melanoma tumors. In this work, we evaluated the effects of the expression of ISAV-F on the B16 melanoma model, both in vitro and in vivo, using chitosan nanoparticles as transfection vehicle. We confirmed that the transfection of B16 tumor cells with chitosan nanoparticles (NP-ISAV) allows the expression of a fusogenically active ISAV-F protein and decreases cell viability because of syncytium formation in vitro. However, the in vivo transfection induces a delay in tumor growth, without inducing changes on the lymphoid populations in the tumor and the spleen. Altogether, our observations show that expression of ISAV fusion protein using chitosan nanoparticles induces cell fusion in melanoma cells and slight antitumor response.

scholarly journals Effects of point mutations in the cytidine deaminase domains of APOBEC3B on replication and hypermutation of hepatitis B virus in vitro

2007 ◽  
Vol 88 (12) ◽  
pp. 3270-3274 ◽  
Author(s):  
Marianne Bonvin ◽  
Jobst Greeve
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Point Mutations ◽  
Alternative Mechanism ◽  
Amino Terminal ◽  
Hbv Replication ◽  
B Virus ◽  
Carboxy Terminal ◽  
Deaminase Domain

APOBEC3 cytidine deaminases hypermutate hepatitis B virus (HBV) and inhibit its replication in vitro. Whether this inhibition is due to the generation of hypermutations or to an alternative mechanism is controversial. A series of APOBEC3B (A3B) point mutants was analysed in vitro for hypermutational activity on HBV DNA and for inhibitory effects on HBV replication. Point mutations inactivating the carboxy-terminal deaminase domain abolished the hypermutational activity and reduced the inhibitory activity on HBV replication to approximately 40 %. In contrast, the point mutation H66R, inactivating the amino-terminal deaminase domain, did not affect hypermutations, but reduced the inhibition activity to 63 %, whilst the mutant C97S had no effect in either assay. Thus, only the carboxy-terminal deaminase domain of A3B catalyses cytidine deaminations leading to HBV hypermutations, but induction of hypermutations is not sufficient for full inhibition of HBV replication, for which both domains of A3B must be intact.

Cell transformation by avian defective leukaemia viruses

1980 ◽  
Vol 210 (1180) ◽  
pp. 397-409 ◽  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Cell Transformation ◽  
Nucleic Acid Hybridization ◽  
Phenotypic Analysis ◽  
Amino Terminal ◽  
Cell Specificity ◽  
Core Proteins ◽  
Carboxy Terminal

A comparative study of seven independently isolated defective leukaemia viruses has been carried out. Phenotypic analysis of the chicken bone marrow cells transformed in vitro allowed the separation of these seven viruses into three groups based on the differentiation phenotype of the transformed cell. Nucleic acid hybridization studies revealed that these seven viruses had acquired cellular sequences. Interestingly, these studies also showed that the viruses within the same biological grouping had acquired related sequences. This indicates that viruses that have acquired the same or similar cellular sequences have very similar oncogenic capabilities. Analysis of proteins expressed in cells transformed by these viruses demonstrated that the cellular sequences were usually inserted within the gene for the viral core proteins, gag . Therefore the cellular sequences are expressed as a gag -related fusion protein which has an amino-terminal region derived from the gag gene and a carboxy-terminal half derived from the cellular sequences. Two exceptions to this are discussed. The general conclusion from these studies is that defective leukaemia viruses transform cells by virtue of acquired host cellular sequences. The ability of these viruses to transform cells and the target cell specificity of the transformation depends on these cellular sequences.

Structural requirements for enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck

1992 ◽  
Vol 12 (6) ◽  
pp. 2720-2729
Author(s):  
L Caron ◽  
N Abraham ◽  
T Pawson ◽  
A Veillette
Keyword(s):  
T Cell ◽  
Protein Phosphorylation ◽  
Phospholipase C ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Amino Terminal ◽  
Src Homology ◽  
Phospholipase C Gamma ◽  
Domain 3

To understand the mechanism(s) by which p56lck participates in T-cell receptor (TCR) signalling, we have examined the effects of mutations in known regulatory domains of p56lck on the ability of F505 p56lck to enhance the responsiveness of an antigen-specific murine T-cell hybridoma. A mutation of the amino-terminal site of myristylation (glycine 2), which prevents stable association of p56lck with the plasma membrane, completely abolished the ability of F505 p56lck to enhance TCR-induced tyrosine protein phosphorylation. Alteration of the major site of in vitro autophosphorylation, tyrosine 394, to phenylalanine diminished the enhancement of TCR-induced tyrosine protein phosphorylation by F505 p56lck. Such a finding is consistent with the previous demonstration that this site is required for full activation of p56lck by mutation of tyrosine 505. Strikingly, deletion of the noncatalytic Src homology domain 2, but not of the Src homology domain 3, markedly reduced the improvement of TCR-induced tyrosine protein phosphorylation by F505 Lck. Additional studies revealed that all the mutations tested, including deletion of the Src homology 3 region, abrogated the enhancement of antigen-triggered interleukin-2 production by F505 p56lck, thus implying more stringent requirements for augmentation of antigen responsiveness by F505 Lck. Finally, it was also observed that expression of F505 p56lck greatly increased TCR-induced tyrosine phosphorylation of phospholipase C-gamma 1, raising the possibility that phospholipase C-gamma 1 may be a substrate for p56lck in T lymphocytes. Our results indicate that p56lck regulates T-cell antigen receptor signalling through a complex process requiring multiple distinct structural domains of the protein.

scholarly journals 173 Expression of a membrane-tethered IL-15/IL-15 receptor fusion protein enhances the persistence of MSLN-targeted TRuC-T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A185-A185
Author(s):  
Michelle Fleury ◽  
Derrick McCarthy ◽  
Holly Horton ◽  
Courtney Anderson ◽  
Amy Watt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Receptor ◽  
Cytokine Production ◽  
Cell Receptor ◽  
Great Promise ◽  
And Function

BackgroundAdoptive cell therapies have shown great promise in hematological malignancies but have yielded little progress in the context of solid tumors. We have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes the full complement of TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. In clinical trials, mesothelin (MSLN)-targeting TRuC-T cells (TC-210 or gavo-cel) have shown unprecedented results in patients suffering from advanced mesothelioma and ovarian cancer. To potentially increase the depth of response, we evaluated strategies that can promote intra-tumoral T cell persistence and function. Among the common ??-chain cytokines, IL-15 uniquely supports the differentiation and maintenance of memory T cell subsets by limiting terminal differentiation and conferring resistance to IL-2 mediated activation-induced cell death (AICD). In the studies described here, we evaluated the potential of IL-15 as an enhancement to TRuC-T cell phenotype, persistence and function against MSLN+ targets.MethodsPrimary human T cells were activated and transduced with a lentiviral vector encoding an anti-MSLN binder fused to CD3ε alone or co-expressed with a membrane-tethered IL-15rα/IL-15 fusion protein (IL-15fu). Transduced T cells were expanded for 9 days and characterized for expression of the TRuC, IL-15rα and memory phenotype before subjecting them to in vitro functional assays to evaluate cytotoxicity, cytokine production, and persistence. In vivo efficacy was evaluated in MHC class I/II deficient NSG mice bearing human mesothelioma xenografts.ResultsIn vitro, co-expression of the IL-15fu led to similar cytotoxicity and cytokine production as TC-210, but notably enhanced T-cell expansion and persistence upon repeated stimulation with MSLN+ cell lines. Furthermore, the IL-15fu-enhanced TRuC-T cells sustained a significantly higher TCF-1+ population and retained a stem-like phenotype following activation. Moreover, the IL-15fu-enhanced TRuCs demonstrated robust in vivo expansion and intra-tumoral accumulation as measured by ex vivo analysis of TRuC+ cells in the tumor and blood, with a preferential expansion of CD8+ T cells. Finally, IL-15fu-enhanced TRuC-T cells could be observed in the blood long after the tumors were cleared.ConclusionsThese pre-clinical studies suggest that the IL-15fu can synergize with TC-210 to increase the potency and durability of response in patients with MSLN+ tumors.Ethics ApprovalAll animal studies were approved by the respective Institutional Animal Care and Use Committees.

Distinct domains of a nucleolar protein mediate protein kinase binding, interaction with nucleic acids and nucleolar localization

1998 ◽  
Vol 111 (17) ◽  
pp. 2615-2623 ◽  
Author(s):  
A. Das ◽  
J.H. Park ◽  
C.B. Hagen ◽  
M. Parsons
Keyword(s):  
Protein Kinase ◽  
Nucleic Acids ◽  
Protozoan Parasite ◽  
In Vitro Transcription ◽  
Nucleic Acid Binding ◽  
Binding Interaction ◽  
Amino Terminal ◽  
Amino Acid Region ◽  
Carboxy Terminal

Nopp44/46 is a phosphoprotein of the protozoan parasite Trypanosoma brucei that is localized to the nucleolus. Based on the primary sequence, Nopp44/46 appears to be a protein composed of distinct domains. This communication describes the relationship of these domains to the known functional interactions of the molecule and suggests that the amino-terminal region defines a novel homology region that functions in nucleolar targeting. We have previously shown that Nopp44/46 is capable of interacting with nucleic acids and associating with a protein kinase. Using in vitro transcription and translation, we now demonstrate that the nucleic acid binding function maps to the carboxy-terminal domain of the molecule, a region rich in arginine-glycine-glycine motifs. Our experiments reveal that a central region containing a high proportion of acidic residues is required for association with the protein kinase. Analysis of transfectants expressing epitope-tagged Nopp44/46 deletion constructs showed that the amino-terminal 96 amino acids allowed nuclear and nucleolar accumulation of the protein. This region of the molecule shows homology to several recently described nucleolar proteins. Deletion of a 27-amino-acid region within this domain abrogated nucleolar, but not nuclear, localization. These studies show that Nopp44/46 is composed of distinct modules, each of which plays a different role in molecular interactions. We suggest that this protein could facilitate interactions between sets of nucleolar molecules.

Collagen fibrillogenesis in vitro: interaction of types I and V collagen regulates fibril diameter

1990 ◽  
Vol 95 (4) ◽  
pp. 649-657 ◽  
Author(s):  
D.E. Birk ◽  
J.M. Fitch ◽  
J.P. Babiarz ◽  
K.J. Doane ◽  
T.F. Linsenmayer
Keyword(s):  
Type I Collagen ◽  
Small Diameter ◽  
Collagen Molecule ◽  
Type I ◽  
Collagen Types ◽  
Type V Collagen ◽  
Amino Terminal ◽  
Type V ◽  
Fibril Diameter

The small-diameter fibrils of the chick corneal stroma are heterotypic, composed of both collagen types I and V. This tissue has a high concentration of type V collagen relative to other type I-containing tissues with larger-diameter fibrils, suggesting that heterotypic interactions may have a regulatory role in the control of fibril diameter. The interactions of collagen types I and V were studied using an in vitro self-assembly system. Collagens were purified from lathyritic chick embryos in the presence of protease inhibitors. The type V collagen preparations contained higher molecular weight forms of the alpha 1(V) and alpha 2(V) chains constituting 60–70% of the total. Rotary-shadow electron micrographs showed a persistence of a small, pepsin-sensitive terminal region in an amount consistent with that seen by electrophoresis. In vitro, this purified type V collagen formed thin fibrils with no apparent periodicity, while type I collagen fibrils had a broad distribution of large diameters. However, when type I collagen was mixed with increasing amounts of type V collagen a progressive and significant decrease in both the mean fibril diameter and the variance was observed for D periodic fibrils. The amino-terminal domain of the type V collagen molecule was required for this regulatory effect and in its absence little diameter reducing activity was observed. Electron microscopy using collagen type-specific monoclonal antibodies demonstrated that the fibrils formed were heterotypic, containing both collagen types I and V. These data indicate that the interaction of type V with type I collagen is one mechanism modulating fibril diameter and is at least partially responsible for the regulation of collagen fibril formation.

Radioimmunoassay for the pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen: a new serum marker of bone collagen degradation

1993 ◽  
Vol 39 (4) ◽  
pp. 635-640 ◽  
Author(s):  
J Risteli ◽  
I Elomaa ◽  
S Niemi ◽  
A Novamo ◽  
L Risteli
Keyword(s):  
Type I Collagen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bone Collagen ◽  
Collagen Molecule ◽  
Type I ◽  
Serum Samples ◽  
Amino Terminal ◽  
Wide Range ◽  
Carboxy Terminal

Abstract We developed a radioimmunoassay (RIA) for the carboxy-terminal telopeptides of type I collagen (ICTP), cross-linked with the helical domain of another type I collagen molecule, after isolation from human femoral bone. The cross-linked peptide was liberated by digesting insoluble, denatured bone collagen either with bacterial collagenase or with trypsin, and purified by two successive reversed-phase separations on HPLC, with monitoring of pyridinoline-specific fluorescence. The purity of the peptide was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its origin in the type I collagen fibers was determined by amino-terminal amino acid sequencing. Polyclonal antibodies and a separation reagent containing second antibody and polyethylene glycol are used in the RIA. An immunologically identical, somewhat larger antigen is present in human serum; its concentration increases in multiple myeloma and in rheumatoid arthritis. The ICTP antigen seems to be cleared from the circulation by the kidneys, because glomerular filtration rates that are two-thirds of normal or less are associated with increased circulating ICTP concentrations. The CVs of the method are between 3% and 8% for a wide range of concentrations. The analysis of 40 serum samples can be completed in 4 h.

scholarly journals LeuRS can leucylate type I and type II tRNALeus in Streptomyces coelicolor

2019 ◽  
Vol 47 (12) ◽  
pp. 6369-6385
Author(s):  
Jia-Yi Fan ◽  
Qian Huang ◽  
Quan-Quan Ji ◽  
En-Duo Wang
Keyword(s):  
Tertiary Structure ◽  
Streptomyces Coelicolor ◽  
Catalytic Efficiency ◽  
Type I ◽  
Type Ii ◽  
Specific Domain ◽  
Recognition Mechanism ◽  
Variable Loop

Abstract Transfer RNAs (tRNAs) are divided into two types, type I with a short variable loop and type II with a long variable loop. Aminoacylation of type I or type II tRNALeu is catalyzed by their cognate leucyl-tRNA synthetases (LeuRSs). However, in Streptomyces coelicolor, there are two types of tRNALeu and only one LeuRS (ScoLeuRS). We found that the enzyme could leucylate both types of ScotRNALeu, and had a higher catalytic efficiency for type II ScotRNALeu(UAA) than for type I ScotRNALeu(CAA). The results from tRNA and enzyme mutagenesis showed that ScoLeuRS did not interact with the canonical discriminator A73. The number of nucleotides, rather than the type of base of the variable loop in the two types of ScotRNALeus, was determined as important for aminoacylation. In vitro and in vivo assays showed that the tertiary structure formed by the D-loop and TψC-loop is more important for ScotRNALeu(UAA). We showed that the leucine-specific domain (LSD) of ScoLeuRS could help LeuRS, which originally only leucylates type II tRNALeu, to aminoacylate type I ScotRNALeu(CAA) and identified the crucial amino acid residues at the C-terminus of the LSD to recognize type I ScotRNALeu(CAA). Overall, our findings identified a rare recognition mechanism of LeuRS to tRNALeu.

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