scholarly journals Leukemic HRX Fusion Proteins Inhibit GADD34-Induced Apoptosis and Associate with the GADD34 and hSNF5/INI1 Proteins

1999 ◽  
Vol 19 (10) ◽  
pp. 7050-7060 ◽  
Author(s):  
Haskell T. Adler ◽  
Rebecca Chinery ◽  
Daniel Y. Wu ◽  
Steven J. Kussick ◽  
John M. Payne ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Chromosomal Abnormalities ◽  
Protein A ◽  
Wild Type ◽  
Chromosomal Locus ◽  
Amino Terminal ◽  
Apoptotic Effect ◽  
The Difference ◽  
Induced Apoptosis ◽  
First Time

ABSTRACT One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene (also called MLL, ALL-1, or HTRX) at chromosomal locus 11q23, resulting in the formation of HRX fusion proteins. Using the yeast two-hybrid system and human cell culture coimmunoprecipitation experiments, we show here that HRX proteins interact directly with the GADD34 protein. We have found that transfected cells overexpressing GADD34 display a significant increase in apoptosis after treatment with ionizing radiation, indicating that GADD34 expression not only correlates with apoptosis but also can enhance apoptosis. The amino-terminal third of the GADD34 protein was necessary for this observed increase in apoptosis. Furthermore, coexpression of three different HRX fusion proteins (HRX-ENL, HRX-AF9, and HRX-ELL) had an anti-apoptotic effect, abrogating GADD34-induced apoptosis. In contrast, expression of wild-type HRX gave rise to an increase in apoptosis. The difference observed here between wild-type HRX and the leukemic HRX fusion proteins suggests that inhibition of GADD34-mediated apoptosis may be important to leukemogenesis. We also show here that GADD34 binds the human SNF5/INI1 protein, a member of the SNF/SWI complex that can remodel chromatin and activate transcription. These studies demonstrate, for the first time, a gain of function for leukemic HRX fusion proteins compared to wild-type protein. We propose that the role of HRX fusion proteins as negative regulators of post-DNA-damage-induced apoptosis is important to leukemia progression.

scholarly journals Sequence Analysis and Expression of the Attachment and Fusion Proteins of Canine Distemper Virus Wild-Type Strain A75/17

1999 ◽  
Vol 73 (3) ◽  
pp. 2263-2269 ◽  
Author(s):  
Pascal Cherpillod ◽  
Karin Beck ◽  
Andreas Zurbriggen ◽  
Riccardo Wittek
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Translation Initiation ◽  
Fusion Proteins ◽  
Canine Distemper Virus ◽  
Protein A ◽  
Biological Properties ◽  
Canine Distemper ◽  
Wild Type ◽  
Attachment Protein

ABSTRACT The biological properties of wild-type A75/17 and cell culture-adapted Onderstepoort canine distemper virus differ markedly. To learn more about the molecular basis for these differences, we have isolated and sequenced the protein-coding regions of the attachment and fusion proteins of wild-type canine distemper virus strain A75/17. In the attachment protein, a total of 57 amino acid differences were observed between the Onderstepoort strain and strain A75/17, and these were distributed evenly over the entire protein. Interestingly, the attachment protein of strain A75/17 contained an extension of three amino acids at the C terminus. Expression studies showed that the attachment protein of strain A75/17 had a higher apparent molecular mass than the attachment protein of the Onderstepoort strain, in both the presence and absence of tunicamycin. In the fusion protein, 60 amino acid differences were observed between the two strains, of which 44 were clustered in the much smaller F2 portion of the molecule. Significantly, the AUG that has been proposed as a translation initiation codon in the Onderstepoort strain is an AUA codon in strain A75/17. Detailed mutation analyses showed that both the first and second AUGs of strain A75/17 are the major translation initiation sites of the fusion protein. Similar analyses demonstrated that, also in the Onderstepoort strain, the first two AUGs are the translation initiation codons which contribute most to the generation of precursor molecules yielding the mature form of the fusion protein.

scholarly journals Specific Ligand Binding Attributable to Individual Epitopes of Gonococcal Transferrin Binding Protein A

2002 ◽  
Vol 70 (2) ◽  
pp. 732-740 ◽  
Author(s):  
Heather P. Masri ◽  
Cynthia Nau Cornelissen
Keyword(s):  
Ligand Binding ◽  
Fusion Proteins ◽  
Iron Uptake ◽  
Solid Phase ◽  
Protein A ◽  
Dot Blot ◽  
Affinity Tags ◽  
Amino Terminal ◽  
Dot Blot Assay ◽  
Specific Ligand

ABSTRACT The gonococcal transferrin receptor complex comprises two iron-regulated proteins, TbpA and TbpB. TbpA is essential for transferrin-iron uptake and is a TonB-dependent integral outer membrane protein. TbpB is thought to increase the efficiency of iron uptake from transferrin and is lipid modified and surface exposed. To evaluate the structure-function relationships in one of the components of the receptor, TbpA, we created constructs that fused individual putative loops of TbpA with amino-terminal affinity tags. The recombinant proteins were then overexpressed in Escherichia coli, and the fusions were recovered predominately from inclusion bodies. Inclusion body proteins were solubilized, and the epitope fusions were renatured by slow dialysis. To assess transferrin binding capabilities, the constructs were tested in a solid-phase dot blot assay followed by confirmatory quantitative chemiluminescent enzyme-linked immunosorbent assays. The constructs with only loop 5 and with loops 4 and 5 demonstrated dose-dependent specific ligand binding in spite of being out of the context of the intact receptor. The immunogenicities of individual TbpA-specific epitopes were investigated by generating rabbit polyclonal antisera against the fusion proteins. Most of the fusion proteins were immunogenic under these conditions, and the resulting sera recognized full-length TbpA in immunoblots. These results suggest that individual epitopes of TbpA are both immunogenic and functional with respect to ligand binding capabilities, and the vaccine implications of these findings are discussed.

Punicic Acid Inhibits Glioblastoma Migration and Proliferation via the PI3K/AKT1/mTOR Signaling Pathway

2019 ◽  
Vol 19 (9) ◽  
pp. 1120-1131 ◽  
Author(s):  
Mesut Mete ◽  
Ulkun U. Unsal ◽  
Işıl Aydemir ◽  
Pınar K. Sönmez ◽  
Mehmet I. Tuglu
Keyword(s):  
Signaling Pathway ◽  
Caspase 3 ◽  
Treated Group ◽  
Punicic Acid ◽  
Glioblastoma Cells ◽  
Electron Microscopy Analysis ◽  
Apoptotic Effect ◽  
The Difference ◽  
And Migration ◽  
Induced Apoptosis

Background:Punicic Acid (PA) is a polyunsaturated fatty acid that accounts for approximately 70%- 80% of Pomegranate Seed Oil (PSO). PA possesses strong antioxidant, anti-inflammatory, anti-atherogenic effects, and anti-tumorigenic properties. Pomegranate extracts have been shown to have anticancer activity in many studies. However, there is no evidence for the effect of PSO on T98 glioblastoma cells. Therefore, the present study was the first to investigate the mechanisms induced by PA on T98 cells, which is one of the major compounds extracted from PSO.Methods:The effects of PA on cell viability; oxidative stress; and migration, proliferation, and apoptosis at the IC50 dose were studied.Results:The proliferation and migration were inhibited in the treated group compared to the non-treated group by 9.85µl/ml PA. The difference was statistically significant (***p<0.001). Furthermore, PA-induced apoptosis in the T98 glioblastoma cells compared to non-treated group and the difference was statistically significant (***p<0.001). Apoptosis was determined via immunocytochemistry staining of caspase-3, caspase-9 and TUNEL methods. Apoptosis was checked by flow cytometry (using caspase 3 methods) and Scanning Electron Microscopy Analysis. We also investigated the potential signaling pathway underlying this apoptotic effect. The immunocytochemical stainings of PI3K/ Akt-1/ mTOR-1 demonstrated that Akt-1 staining was increased with PA treatment similar to mTOR-1 and PI3K staining (***p<0.001). These increases were statistically significant compared to the non-treated group.Conclusion:PA exhibited exceptional abilities as an anticancer agent against GBM cells. The use of punicic acid in combination with other drugs used in the treatment of glioblastoma may increase the efficacy of the treatment. This study provided a basis for future investigation of its use in preclinical and clinical studies.

scholarly journals Amino-Terminal Protein Fusions to the TraR Quorum-Sensing Transcription Factor Enhance Protein Stability and Autoinducer-Independent Activity

2005 ◽  
Vol 187 (4) ◽  
pp. 1219-1226 ◽  
Author(s):  
Yunrong Chai ◽  
Stephen C. Winans
Keyword(s):  
Quorum Sensing ◽  
Fusion Proteins ◽  
Antibiotic Resistance Gene ◽  
Homoserine Lactone ◽  
Amino Terminus ◽  
Original Strain ◽  
Wild Type ◽  
Terminal Protein ◽  
Amino Terminal ◽  
Strain Data

ABSTRACT TraR of Agrobacterium tumefaciens is a member of the LuxR family of quorum-sensing transcription factors and regulates genes required for conjugation and vegetative replication of the tumor-inducing (Ti) plasmid in the presence of the autoinducer 3-oxooctanoyl-homoserine lactone (OOHL). In the absence of OOHL, TraR is rapidly destroyed by proteolysis, suggesting that this ligand is required for TraR folding. To date, no TraR variant has been found that is active in the absence of OOHL. In this study, we conducted whole-cell and plasmid mutagenesis experiments to search for constitutive mutations of traR and identified two constitutive alleles. Surprisingly, neither contained a point mutation within the traR gene, but rather, both encoded fusion proteins between TraR and the N-terminal domain of an aminoglycoside N-acetyltransferase, encoded by a plasmid-borne antibiotic resistance gene present in the original strain. Data from Western immunoblot assays, pulse-chase assays, and immunoprecipitation assays show that these fusion proteins are far more stable to proteolysis than native apo-TraR. We also constructed a library of traR alleles encoding random amino-terminal fusions and selected for constitutive TraR activity. Five independent fusion proteins were identified by this approach. These fusion proteins accumulated to far higher levels than wild-type TraR in the absence of OOHL. One of these fusions was overexpressed in Escherichia coli and showed detectable tra box binding in the absence of OOHL. These data suggest that the native amino terminus of TraR may signal proteolysis and that fusing it to other proteins might sequester it from intracellular proteases.

Wild-type, mitochondrial and ER-restricted Bcl-2 inhibit DNA damage-induced apoptosis but do not affect death receptor-induced apoptosis

2001 ◽  
Vol 114 (23) ◽  
pp. 4161-4172
Author(s):  
Justine Rudner ◽  
Albrecht Lepple-Wienhues ◽  
Wilfried Budach ◽  
Johannes Berschauer ◽  
Björn Friedrich ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Transmembrane Domain ◽  
Cytochrome B5 ◽  
Death Receptor ◽  
Jurkat Cells ◽  
Wild Type ◽  
Specific Sequence ◽  
Apoptotic Effect ◽  
Radiation Induced ◽  
Induced Apoptosis

The proto-oncogene Bcl-2 is expressed in membranes of mitochondria and endoplasmic reticulum and mediates resistance against a broad range of apoptotic stimuli. Although several mechanisms of Bcl-2 action have been proposed, its role in different cellular organelles remains elusive. Here, we analyzed the function of Bcl-2 targeted specifically to certain subcellular compartments in Jurkat cells. Bcl-2 expression was restricted to the outer mitochondrial membrane by replacing its membrane anchor with the mitochondrial insertion sequence of ActA (Bcl-2/MT) or the ER-specific sequence of cytochrome b5 (Bcl-2/ER). Additionally, cells expressing wild-type Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/ΔTM) were employed. Apoptosis induced by ionizing radiation or by the death receptors for CD95L or TRAIL was analyzed by determination of the mitochondrial membrane potential (ΔΨm) and activation of different caspases. Bcl-2/WT and Bcl-2/MT strongly inhibited radiation-induced apoptosis and caspase activation, whereas Bcl-2/ΔTM had completely lost its anti-apoptotic effect. Interestingly, Bcl-2/ER conferred protection against radiation-induced mitochondrial damage and apoptosis similarly to Bcl-2/MT. The finding that ER-targeted Bcl-2 interfered with mitochondrial ΔΨm breakdown and caspase-9 activation indicates the presence of a crosstalk between both organelles in radiation-induced apoptosis. By contrast, Bcl-2 in either subcellular position did not influence CD95- or TRAIL-mediated apoptosis.

scholarly journals The Baculovirus PE38 Protein Augments Apoptosis Induced by Transactivator IE1

1999 ◽  
Vol 73 (8) ◽  
pp. 6691-6699 ◽  
Author(s):  
Elena A. Prikhod’ko ◽  
Lois K. Miller
Keyword(s):  
Leucine Zipper ◽  
Nuclear Protein ◽  
Ring Finger ◽  
Leucine Zipper Motif ◽  
Wild Type ◽  
Amino Terminal ◽  
Baculovirus Infection ◽  
Apoptotic Activity ◽  
Induced Apoptosis ◽  
Ring Finger Motif

ABSTRACT While studying apoptosis induced by baculovirus transactivator IE1 in SF-21 cells, we found that the levels of IE1-induced apoptosis were increased approximately twofold upon cotransfection with the baculovirus early pe38 gene. However, no apoptotic activity was observed in cells transfected with pe38 alone, even when placed under the control of a constitutive promoter. Thus,pe38 was able to augment IE1-induced apoptosis but was unable to induce apoptosis when expressed in SF-21 cells alone. PE38, the full-length product of pe38, is a nuclear protein with RING finger and leucine zipper motifs. Deletion of the amino-terminal region, which contains a putative nuclear localization motif, resulted in cytoplasmic localization of the PE38 mutants. These N-terminal deletion mutants were unable to enhance IE1-induced apoptosis. Mutation of a single conserved leucine (L242) of the leucine zipper motif also eliminated the ability of PE38 to augment apoptosis induced by IE1. In contrast, PE38 mutants with alanine substitutions for conserved cysteine residues (C109 or C138) of the RING finger motif were able to increase IE1-induced apoptosis to levels equivalent to those of wild-type PE38. We propose that PE38 is one of at least two viral factors which collectively evoke a cellular apoptotic response during baculovirus infection.

scholarly journals Caspase-8 mutations associated with head and neck cancer differentially retain functional properties related to TRAIL-induced apoptosis and cytokine induction

2021 ◽  
Vol 12 (8) ◽  
Author(s):  
Zhibin Cui ◽  
Hadas Dabas ◽  
Brandon C. Leonard ◽  
Jamie V. Shiah ◽  
Jennifer R. Grandis ◽  
...  
Keyword(s):  
Head And Neck ◽  
Functional Properties ◽  
Human Head ◽  
Caspase 8 ◽  
Loss Of Function ◽  
Wild Type ◽  
Apoptosis Pathway ◽  
Amino Terminal ◽  
Cytokine Induction ◽  
Induced Apoptosis

AbstractThe cysteine protease, caspase-8, undergoes dimerization, processing, and activation following stimulation of cells with death ligands such as TRAIL, and mediates TRAIL induction of the extrinsic apoptosis pathway. In addition, caspase-8 mediates TRAIL-induced activation of NF-κB and upregulation of immunosuppressive chemokines/cytokines, via a mechanism independent of caspase-8 catalytic activity. The gene encoding procaspase-8 is mutated in 10% of human head and neck squamous cell carcinomas (HNSCCs). Despite a paucity of experimental evidence, HNSCC-associated caspase-8 mutations are commonly assumed to be loss of function. To investigate their functional properties and phenotypic effects, 18 HNSCC-associated caspase-8 mutants were expressed in doxycycline-inducible fashion in cell line models wherein the endogenous wild-type caspase-8 was deleted. We observed that 5/8 mutants in the amino-terminal prodomain, but 0/10 mutants in the carboxyl-terminal catalytic region, retained an ability to mediate TRAIL-induced apoptosis. Caspase-8 proteins with mutations in the prodomain were defective in dimerization, whereas all ten of the catalytic region mutants efficiently dimerized, revealing an inverse relationship between dimerization and apoptosis induction for the mutant proteins. Roughly half (3/8) of the prodomain mutants and 9/10 of the catalytic region mutants retained the ability to mediate TRAIL induction of immunosuppressive CXCL1, IL-6, or IL-8. Doxycycline-induced expression of wild-type caspase-8 or a representative mutant led to an increased percentage of T and NKT cells in syngeneic HNSCC xenograft tumors. These findings demonstrate that HNSCC-associated caspase-8 mutants retain properties that may influence TRAIL-mediated apoptosis and cytokine induction, as well as the composition of the tumor microenvironment.

Strategies for Oral Delivery of Metal-Saturated Lactoferrin

2019 ◽  
Vol 20 (11) ◽  
pp. 1046-1051 ◽  
Author(s):  
Przemysław Gajda-Morszewski ◽  
Klaudyna Śpiewak-Wojtyła ◽  
Maria Oszajca ◽  
Małgorzata Brindell
Keyword(s):  
Biological Activity ◽  
Oral Delivery ◽  
Therapeutic Potential ◽  
Structure And Function ◽  
The Difference ◽  
And Function ◽  
First Time

Lactoferrin was isolated and purified for the first time over 50-years ago. Since then, extensive studies on the structure and function of this protein have been performed and the research is still being continued. In this mini-review we focus on presenting recent scientific efforts towards the elucidation of the role and therapeutic potential of lactoferrin saturated with iron(III) or manganese(III) ions. The difference in biological activity of metal-saturated lactoferrin vs. the unmetalated one is emphasized. The strategies for oral delivery of lactoferrin, are also reviewed, with particular attention to the metalated protein.

Sign in / Sign up

Export Citation Format

Share Document