scholarly journals The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex

2003 ◽  
Vol 160 (7) ◽  
pp. 1139-1150 ◽  
Author(s):  
Ryan Ratts ◽  
Huiyan Zeng ◽  
Eric A. Berg ◽  
Clare Blue ◽  
Mark E. McComb ◽  
...  
Keyword(s):  
T Cell ◽  
Diphtheria Toxin ◽  
Essential Role ◽  
Reductase Activity ◽  
Translocation Factor ◽  
Thioredoxin Reductase ◽  
Cell Extracts ◽  
Human T Cell ◽  
Early Endosomes

In vitro delivery of the diphtheria toxin catalytic (C) domain from the lumen of purified early endosomes to the external milieu requires the addition of both ATP and a cytosolic translocation factor (CTF) complex. Using the translocation of C-domain ADP-ribosyltransferase activity across the endosomal membrane as an assay, the CTF complex activity was 650–800-fold purified from human T cell and yeast extracts, respectively. The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast. Further analysis of the role played by these two proteins with specific inhibitors, both in the in vitro translocation assay and in intact cell toxicity assays, has demonstrated their essential role in the productive delivery of the C-domain from the lumen of early endosomes to the external milieu. These results confirm and extend earlier observations of diphtheria toxin C-domain unfolding and refolding that must occur before and after vesicle membrane translocation. In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain. Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.

scholarly journals Interaction of the human T-cell lymphotropic virus type 1 tax transactivator with transcription factor IIA.

1996 ◽  
Vol 16 (9) ◽  
pp. 4656-4664 ◽  
Author(s):  
K E Clemens ◽  
G Piras ◽  
M F Radonovich ◽  
K S Choi ◽  
J F Duvall ◽  
...  
Keyword(s):  
Transcription Factor ◽  
T Cell ◽  
T Lymphocytes ◽  
Virus Type ◽  
Expression Vectors ◽  
Human T Lymphocytes ◽  
Cell Extracts ◽  
Human T Cell

The Tax protein of human T-cell lymphotropic virus type 1 (HTLV-1) is a 40-kDa transcriptional activator which is critical for HTLV-1 gene regulation and virus-induced cellular transformation. Tax is localized to the DNA through its interaction with the site-specific activators cyclic AMP-responsive element-binding protein, NF-kappaB, and serum response factor. It has been suggested that the recruitment of Tax to the DNA positions Tax for interaction with the basal transcriptional machinery. On the basis of several independent assays, we now report a physical and functional interaction between Tax and the transcription factor, TFIIA. First, Tax was found to interact with the 35-kDa (alpha) subunit of TFIIA in the yeast two-hybrid interaction system. Importantly, two previously characterized mutants with point mutations in Tax, M32 (Y196A, K197S) and M41 (H287A, P288S), which were shown to be defective in Tax-activated transcription were unable to interact with TFIIA in this assay. Second, a glutathione-S-transferase (GST) affinity-binding assay showed that the interaction of holo-TFIIA with GST-Tax was 20-fold higher than that observed with either the GST-Tax M32 activation mutant or the GST control. Third, a coimmunoprecipitation assay showed that in HTLV-1-infected human T lymphocytes, Tax and TFIIA were associated. Finally, TFIIA facilitates Tax transactivation in vitro and in vivo. In vitro transcription studies showed reduced levels of Tax-activated transcription in cell extracts depleted of TFIIA. In addition, transfection of human T lymphocytes with TFIIA expression vectors enhanced Tax-activated transcription of an HTLV-1 long terminal repeat-chloramphenicol acetyltransferase reporter construct. Our study suggests that the interaction of Tax with the transcription factor TFIIA may play a role in Tax-mediated transcriptional activation.

scholarly journals To be helped or not helped, that is the question

2003 ◽  
Vol 160 (7) ◽  
pp. 991-992 ◽  
Author(s):  
Emmanuel Lemichez ◽  
Patrice Boquet
Keyword(s):  
Diphtheria Toxin ◽  
Catalytic Domain ◽  
Elongation Factor ◽  
Translocation Factor ◽  
Thioredoxin Reductase ◽  
Endosomal Membrane ◽  
Elongation Factor 2 ◽  
Early Endosomes ◽  
Cellular Machinery ◽  
Catalytic Fragment

Diphtheria toxin (DT)**Abbreviations used in this paper: DT, diphtheria toxin; DT-C, DT catalytic fragment; DT-T, DT transmembrane; EF-2, elongation factor 2. is the paradigm of the powerful A-B toxins. These bacterial poisons bind to cells, are endocytosed, and inject their catalytic domain into the cytosol causing the irreversible modification of a key component of the the host cellular machinery. The mechanism by which the hydrophilic enzymatic fragment of DT crosses the endosomal membrane and is released into the cytosol remains controversial. In this issue, Ratts et al. (2003) demonstrate that delivery of the DT catalytic domain from the lumen of purified early endosomes to the external medium requires the addition of a cytosolic translocation factor complex composed in part of Hsp90 and thioredoxin reductase.

scholarly journals DL4-μbeads induce T cell lineage differentiation from stem cells in a stromal cell-free system

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ashton C. Trotman-Grant ◽  
Mahmood Mohtashami ◽  
Joshua De Sousa Casal ◽  
Elisa C. Martinez ◽  
Dylan Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Stromal Cell ◽  
Hematopoietic Stem ◽  
Free System ◽  
Cell Therapies ◽  
Immunodeficient Mice ◽  
Human T Cell

AbstractT cells are pivotal effectors of the immune system and can be harnessed as therapeutics for regenerative medicine and cancer immunotherapy. An unmet challenge in the field is the development of a clinically relevant system that is readily scalable to generate large numbers of T-lineage cells from hematopoietic stem/progenitor cells (HSPCs). Here, we report a stromal cell-free, microbead-based approach that supports the efficient in vitro development of both human progenitor T (proT) cells and T-lineage cells from CD34+cells sourced from cord blood, GCSF-mobilized peripheral blood, and pluripotent stem cells (PSCs). DL4-μbeads, along with lymphopoietic cytokines, induce an ordered sequence of differentiation from CD34+ cells to CD34+CD7+CD5+ proT cells to CD3+αβ T cells. Single-cell RNA sequencing of human PSC-derived proT cells reveals a transcriptional profile similar to the earliest thymocytes found in the embryonic and fetal thymus. Furthermore, the adoptive transfer of CD34+CD7+ proT cells into immunodeficient mice demonstrates efficient thymic engraftment and functional maturation of peripheral T cells. DL4-μbeads provide a simple and robust platform to both study human T cell development and facilitate the development of engineered T cell therapies from renewable sources.

scholarly journals Biochemical Characterization, Specificity and Inhibition Studies of HTLV-1, HTLV-2, and HTLV-3 Proteases

Life ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 127
Author(s):  
Norbert Kassay ◽  
János András Mótyán ◽  
Krisztina Matúz ◽  
Mária Golda ◽  
József Tőzsér
Keyword(s):  
T Cell ◽  
Biochemical Characterization ◽  
Leukemia Virus ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Cell Leukemia Virus Type ◽  
Adult T Cell Leukemia ◽  
Human T Cell ◽  
T Cell Leukemia Virus

The human T-lymphotropic viruses (HTLVs) are causative agents of severe diseases including adult T-cell leukemia. Similar to human immunodeficiency viruses (HIVs), the viral protease (PR) plays a crucial role in the viral life-cycle via the processing of the viral polyproteins. Thus, it is a potential target of anti-retroviral therapies. In this study, we performed in vitro comparative analysis of human T-cell leukemia virus type 1, 2, and 3 (HTLV-1, -2, and -3) proteases. Amino acid preferences of S4 to S1′ subsites were studied by using a series of synthetic oligopeptide substrates representing the natural and modified cleavage site sequences of the proteases. Biochemical characteristics of the different PRs were also determined, including catalytic efficiencies and dependence of activity on pH, temperature, and ionic strength. We investigated the effects of different HIV-1 PR inhibitors (atazanavir, darunavir, DMP-323, indinavir, ritonavir, and saquinavir) on enzyme activities, and inhibitory potentials of IB-268 and IB-269 inhibitors that were previously designed against HTLV-1 PR. Comparative biochemical analysis of HTLV-1, -2, and -3 PRs may help understand the characteristic similarities and differences between these enzymes in order to estimate the potential of the appearance of drug-resistance against specific HTLV-1 PR inhibitors.

Wogonin preferentially kills malignant lymphocytes and suppresses T-cell tumor growth by inducing PLCγ1- and Ca2+-dependent apoptosis

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2354-2363 ◽  
Author(s):  
Sven Baumann ◽  
Stefanie C. Fas ◽  
Marco Giaisi ◽  
Wolfgang W. Müller ◽  
Anette Merling ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chinese Medicine ◽  
Traditional Chinese Medicine ◽  
Therapeutic Potential ◽  
Human T Cell ◽  
Voltage Dependent ◽  
Ca2 Channels ◽  
Malignant T Cells

Herbs have successfully been used in traditional Chinese medicine for centuries. However, their curative mechanisms remain largely unknown. In this study, we show that Wogonin, derived from the traditional Chinese medicine Huang-Qin (Scutellaria baicalensis Georgi), induces apoptosis in malignant T cells in vitro and suppresses growth of human T-cell leukemia xenografts in vivo. Importantly, Wogonin shows almost no toxicity on T lymphocytes from healthy donors. Wogonin induces prolonged activation of PLCγ1 via H2O2 signaling in malignant T cells, which leads to sustained elevation of cytosolic Ca2+ in malignant but not normal T cells. Subsequently, a Ca2+ overload leads to disruption of the mitochondrial membrane. The selective effect of Wogonin is due to its differential regulation of the redox status of malignant versus normal T cells. In addition, we show that the L-type voltage-dependent Ca2+ channels are involved in the intracellular Ca2+ mobilization in T cells. Furthermore, we show that malignant T cells possess elevated amounts of voltage-dependent Ca2+ channels compared with normal T cells, which further enhance the cytotoxicity of Wogonin for malignant T cells. Taken together, our data show a therapeutic potential of Wogonin for the treatment of hematologic malignancies.

scholarly journals Interleukin 2 receptor-targeted cytotoxicity. Interleukin 2 receptor-mediated action of a diphtheria toxin-related interleukin 2 fusion protein.

1988 ◽  
Vol 167 (2) ◽  
pp. 612-622 ◽  
Author(s):  
P Bacha ◽  
D P Williams ◽  
C Waters ◽  
J M Williams ◽  
J R Murphy ◽  
...  
Keyword(s):  
T Cells ◽  
Protein Synthesis ◽  
T Cell ◽  
Diphtheria Toxin ◽  
Interleukin 2 ◽  
Elongation Factor ◽  
High Affinity ◽  
Interleukin 2 Receptor ◽  
Human T Cell ◽  
Inhibition Of Protein Synthesis

The IL-2 toxin-mediated inhibition of protein synthesis in high affinity IL-2-R-positive murine and human T cell lines has been examined. Both excess free IL-2 and mAb to the Tac epitope of the p55 subunit of IL-2-R are shown to block the action of IL-2 toxin; whereas, agents that interact with other receptors or antigens on the T cell surface have no effect. We show that IL-2 toxin, like diphtheria toxin, must pass through an acidic vesicle in order to intoxicate target T cells. Finally, we demonstrate that the IL-2 toxin-mediated inhibition of protein synthesis in both human and murine T cells that bear the high affinity IL-2-R is due to the classic diphtheria toxin fragment A-catalyzed ADP ribosylation of elongation factor 2.

Thioredoxin reductase gene expression and activity among human T‐cell lymphotropic virus type 1–infected patients

2018 ◽  
Author(s):  
Neda Yaghoubi ◽  
Masoud Youssefi ◽  
Seyed Isaac Hashemy ◽  
Houshang Rafat Panah ◽  
Barat Ali Mashkani ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Virus Type ◽  
Thioredoxin Reductase ◽  
Human T Cell ◽  
Reductase Gene ◽  
Expression And Activity
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