Dominant-Negative FADD Rescues the In Vivo Fitness of a Cytomegalovirus Lacking an Antiapoptotic Viral Gene

ABSTRACT Genes that inhibit apoptosis have been described for many DNA viruses. Herpesviruses often contain even more than one gene to control cell death. Apoptosis inhibition by viral genes is postulated to contribute to viral fitness, although a formal proof is pending. To address this question, we studied the mouse cytomegalovirus (MCMV) protein M36, which binds to caspase-8 and blocks death receptor-induced apoptosis. The growth of MCMV recombinants lacking M36 (ΔM36) was attenuated in vitro and in vivo. In vitro, caspase inhibition by zVAD-fmk blocked apoptosis in ΔM36-infected macrophages and rescued the growth of the mutant. In vivo, ΔM36 infection foci in liver tissue contained significantly more apoptotic hepatocytes and Kupffer cells than did revertant virus foci, and apoptosis occurred during the early phase of virus replication prior to virion assembly. To further delineate the mode of M36 function, we replaced the M36 gene with a dominant-negative FADD (FADDDN) in an MCMV recombinant. FADDDN was expressed in cells infected with the recombinant and blocked the death-receptor pathway, replacing the antiapoptotic function of M36. Most importantly, FADDDN rescued ΔM36 virus replication, both in vitro and in vivo. These findings have identified the biological role of M36 and define apoptosis inhibition as a key determinant of viral fitness.

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Ezrin Is an Effector of Hepatocyte Growth Factor–mediated Migration and Morphogenesis in Epithelial Cells

The Journal of Cell Biology ◽

10.1083/jcb.138.2.423 ◽

1997 ◽

Vol 138 (2) ◽

pp. 423-434 ◽

Cited By ~ 225

Author(s):

Tiziana Crepaldi ◽

Alexis Gautreau ◽

Paolo M. Comoglio ◽

Daniel Louvard ◽

Monique Arpin

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Cell Shape ◽

Control Cell ◽

Dominant Negative ◽

Site Directed Mutagenesis ◽

Epithelial Cell Line ◽

Hepatocyte Growth

The dissociation, migration, and remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) entail modifications in cell adhesion and in the actin cytoskeleton through unknown mechanisms. Here we report that ezrin, a membrane–cytoskeleton linker, is crucial to HGF-mediated morphogenesis in a polarized kidney-derived epithelial cell line, LLC-PK1. Ezrin is a substrate for the tyrosine kinase HGF receptor both in vitro and in vivo. HGF stimulation causes enrichment of ezrin recovered in the detergent-insoluble cytoskeleton fraction. Overproduction of wild-type ezrin, by stable transfection in LLC-PK1 cells, enhances cell migration and tubulogenesis induced by HGF stimulation. Overproduction of a truncated variant of ezrin causes mislocalization of endogenous ezrin from microvilli into lateral surfaces. This is concomitant with altered cell shape, characterized by loss of microvilli and cell flattening. Moreover, the truncated variant of ezrin impairs the morphogenic and motogenic response to HGF, thus suggesting a dominant-negative mechanism of action. Site-directed mutagenesis of ezrin codons Y145 and Y353 to phenylalanine does not affect the localization of ezrin at microvilli, but perturbs the motogenic and morphogenic responses to HGF. These results provide evidence that ezrin displays activities that can control cell shape and signaling.

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iTSP-PseAAC: Identify Tumor Suppressor Proteins by Using Fully Connected Neural Network and PseAAC

Current Bioinformatics ◽

10.2174/1574893615666210108094431 ◽

2021 ◽

Vol 15 ◽

Author(s):

Muhammad Awais ◽

Waqar Hussain ◽

Nouman Rasool ◽

Yaser Daanial Khan

Keyword(s):

Tumor Suppressor ◽

Cross Validation ◽

Control Cell ◽

Normal Function ◽

In Vivo Studies ◽

Tumor Suppressor Proteins ◽

Proposed Model ◽

Self Consistency

Background: The uncontrolled growth due to accumulation of genetic and epigenetic changes as a result of loss or reduction in the normal function of Tumor Suppressor Genes (TSGs) and Pro-oncogenes is known as cancer. TSGs control cell division and growth by repairing of DNA mistakes during replication and restrict the unwanted proliferation of a cell or activities, those are the part of tumor production. Objectives: This study aims to propose a novel, accurate, user-friendly model to predict tumor suppressor proteins, which would be freely available to experimental molecular biologists to assist them using in vitro and in vivo studies. Methods: The predictor model has used the input feature vector (IFV) calculated from the physicochemical properties of proteins based on FCNN to compute the accuracy, sensitivity, specificity, and MCC. The proposed model was validated against different exhaustive validation techniques i.e. self-consistency and cross-validation. Results: Using self-consistency, the accuracy is 99%, for cross-validation and independent testing has 99.80% and 100% accuracy respectively. The overall accuracy of the proposed model is 99%, sensitivity value 98% and specificity 99% and F1-score was 0.99. Conclusion: It concludes, the proposed model for prediction of the tumor suppressor proteins can predict the tumor suppressor proteins efficiently, but it still has space for improvements in computational ways as the protein sequences may rapidly increase, day by day.

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Towards plant resistance to viruses using protein-only RNase P

Nature Communications ◽

10.1038/s41467-021-21338-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Anthony Gobert ◽

Yifat Quan ◽

Mathilde Arrivé ◽

Florent Waltz ◽

Nathalie Da Silva ◽

...

Keyword(s):

Mosaic Virus ◽

Virus Replication ◽

Yield Loss ◽

Rnase P ◽

Plant Viruses ◽

Yellow Mosaic Virus ◽

Resistance To Viruses ◽

Target Plant

AbstractPlant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5′ maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases.

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The Diagnostic Journey of a Patient with Prader–Willi-Like Syndrome and a Unique Homozygous SNURF-SNRPN Variant; Bio-Molecular Analysis and Review of the Literature

Genes ◽

10.3390/genes12060875 ◽

2021 ◽

Vol 12 (6) ◽

pp. 875

Author(s):

Karlijn Pellikaan ◽

Geeske M. van Woerden ◽

Lotte Kleinendorst ◽

Anna G. W. Rosenberg ◽

Bernhard Horsthemke ◽

...

Keyword(s):

Intellectual Disability ◽

Single Nucleotide Polymorphism Array ◽

Genetic Defect ◽

Missense Variant ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Pituitary Hormone ◽

Negative Effect

Prader–Willi syndrome (PWS) is a rare genetic condition characterized by hypotonia, intellectual disability, and hypothalamic dysfunction, causing pituitary hormone deficiencies and hyperphagia, ultimately leading to obesity. PWS is most often caused by the loss of expression of a cluster of genes on chromosome 15q11.2-13. Patients with Prader–Willi-like syndrome (PWLS) display features of the PWS phenotype without a classical PWS genetic defect. We describe a 46-year-old patient with PWLS, including hypotonia, intellectual disability, hyperphagia, and pituitary hormone deficiencies. Routine genetic tests for PWS were normal, but a homozygous missense variant NM_003097.3(SNRPN):c.193C>T, p.(Arg65Trp) was identified. Single nucleotide polymorphism array showed several large regions of homozygosity, caused by high-grade consanguinity between the parents. Our functional analysis, the ‘Pipeline for Rapid in silico, in vivo, in vitro Screening of Mutations’ (PRiSM) screen, showed that overexpression of SNRPN-p.Arg65Trp had a dominant negative effect, strongly suggesting pathogenicity. However, it could not be confirmed that the variant was responsible for the phenotype of the patient. In conclusion, we present a unique homozygous missense variant in SNURF-SNRPN in a patient with PWLS. We describe the diagnostic trajectory of this patient and the possible contributors to her phenotype in light of the current literature on the genotype–phenotype relationship in PWS.

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A Novel Fully Human Agonistic Single Chain Fragment Variable Antibody Targeting Death Receptor 5 with Potent Antitumor Activity In Vitro and In Vivo

International Journal of Molecular Sciences ◽

10.3390/ijms18102064 ◽

2017 ◽

Vol 18 (10) ◽

pp. 2064 ◽

Cited By ~ 3

Author(s):

Gaoxin Lei ◽

Menglong Xu ◽

Zhipan Xu ◽

Lili Gu ◽

Chenchen Lu ◽

...

Keyword(s):

Antitumor Activity ◽

Death Receptor ◽

Death Receptor 5 ◽

Single Chain ◽

Chain Fragment ◽

Single Chain Fragment Variable ◽

Antibody Targeting ◽

Single Chain Fragment

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420 Small interfering RNA(siRNA)-mediated inhibition of hepatitis C virus replication using bicistronic vector in vivo (cell line Huh-7) and in vitro

Journal of Hepatology ◽

10.1016/s0168-8278(05)81832-x ◽

2005 ◽

Vol 42 ◽

pp. 154 ◽

Cited By ~ 1

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Line ◽

Virus Replication ◽

Small Interfering Rna ◽

Bicistronic Vector ◽

Interfering Rna

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Extracellular Signal-Regulated Kinase Binds to TFII-I and Regulates Its Activation of the c-fosPromoter

Molecular and Cellular Biology ◽

10.1128/mcb.20.4.1140-1148.2000 ◽

2000 ◽

Vol 20 (4) ◽

pp. 1140-1148 ◽

Cited By ~ 37

Author(s):

Dae-Won Kim ◽

Brent H. Cochran

Keyword(s):

Map Kinase ◽

Transcriptional Activation ◽

Dominant Negative ◽

Binding Motif ◽

Dependent Manner ◽

Consensus Map ◽

Interaction Domain ◽

Extracellular Signal

ABSTRACT We have previously shown that TFII-I enhances transcriptional activation of the c-fos promoter through interactions with upstream elements in a signal-dependent manner. Here we demonstrate that activated Ras and RhoA synergize with TFII-I for c-fospromoter activation, whereas dominant-negative Ras and RhoA inhibit these effects of TFII-I. The Mek1 inhibitor, PD98059 abrogates the enhancement of the c-fos promoter by TFII-I, indicating that TFII-I function is dependent on an active mitogen-activated protein (MAP) kinase pathway. Analysis of the TFII-I protein sequence revealed that TFII-I contains a consensus MAP kinase interaction domain (D box). Consistent with this, we have found that TFII-I forms an in vivo complex with extracellular signal-related kinase (ERK). Point mutations within the consensus MAP kinase binding motif of TFII-I inhibit its ability to bind ERK and its ability to enhance the c-fos promoter. Therefore, the D box of TFII-I is required for its activity on the c-fos promoter. Moreover, the interaction between TFII-I and ERK can be regulated. Serum stimulation enhances complex formation between TFII-I and ERK, and dominant-negative Ras abrogates this interaction. In addition, TFII-I can be phosphorylated in vitro by ERK and mutation of consensus MAP kinase substrate sites at serines 627 and 633 impairs the phosphorylation of TFII-I by ERK and its activity on the c-fos promoter. These results suggest that ERK regulates the activity of TFII-I by direct phosphorylation.

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Cleavage and Inactivation of ATM during Apoptosis

Molecular and Cellular Biology ◽

10.1128/mcb.19.9.6076 ◽

1999 ◽

Vol 19 (9) ◽

pp. 6076-6084 ◽

Cited By ~ 67

Author(s):

Graeme C. M. Smith ◽

Fabrizio d’adda di Fagagna ◽

Nicholas D. Lakin ◽

Stephen P. Jackson

Keyword(s):

Dna Damage ◽

Dna Binding ◽

Caspase 3 ◽

Cysteine Proteases ◽

Dominant Negative ◽

Protein Kinase Activity ◽

Dna Damage Signalling ◽

In Cells

ABSTRACT The activation of the cysteine proteases with aspartate specificity, termed caspases, is of fundamental importance for the execution of programmed cell death. These proteases are highly specific in their action and activate or inhibit a variety of key protein molecules in the cell. Here, we study the effect of apoptosis on the integrity of two proteins that have critical roles in DNA damage signalling, cell cycle checkpoint controls, and genome maintenance—the product of the gene defective in ataxia telangiectasia, ATM, and the related protein ATR. We find that ATM but not ATR is specifically cleaved in cells induced to undergo apoptosis by a variety of stimuli. We establish that ATM cleavage in vivo is dependent on caspases, reveal that ATM is an efficient substrate for caspase 3 but not caspase 6 in vitro, and show that the in vitro caspase 3 cleavage pattern mirrors that in cells undergoing apoptosis. Strikingly, apoptotic cleavage of ATM in vivo abrogates its protein kinase activity against p53 but has no apparent effect on the DNA binding properties of ATM. These data suggest that the cleavage of ATM during apoptosis generates a kinase-inactive protein that acts, through its DNA binding ability, in a trans-dominant-negative fashion to prevent DNA repair and DNA damage signalling.

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109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0109 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A121-A121

Author(s):

Nina Chu ◽

Michael Overstreet ◽

Ryan Gilbreth ◽

Lori Clarke ◽

Christina Gesse ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Dominant Negative ◽

Car T Cells ◽

Car T Cell ◽

Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

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