ArfX2 GTPase Regulates Trafficking From the Trans-Golgi to Lysosomes and Is Necessary for Liver Abscess Formation in the Protozoan Parasite Entamoeba histolytica

Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess in humans. The parasitic lifestyle and the virulence of the protist require elaborate biological processes, including vesicular traffic and stress management against a variety of reactive oxygen and nitrogen species produced by the host immune response. Although the mechanisms for intracellular traffic of representative virulence factors have been investigated at molecular levels, it remains poorly understood whether and how intracellular traffic is involved in the defense against reactive oxygen and nitrogen species. Here, we demonstrate that EhArfX2, one of the Arf family of GTPases known to be involved in the regulation of vesicular traffic, was identified by comparative transcriptomic analysis of two isogenic strains: an animal-passaged highly virulent HM-1:IMSS Cl6 and in vitro maintained attenuated avirulent strain. EhArfX2 was identified as one of the most highly upregulated genes in the highly virulent strain. EhArfX2 was localized to small vesicle-like structures and largely colocalized with the marker for the trans-Golgi network SNARE, EhYkt6, but neither with the endoplasmic reticulum (ER)-resident chaperon, EhBip, nor the cis-Golgi SNARE, EhSed5, and Golgi-luminal galactosyl transferase, EhGalT. Expression of the dominant-active mutant form of EhArfX2 caused an increase in the number of lysosomes, while expression of the dominant-negative mutant led to a defect in lysosome formation and cysteine protease transport to lysosomes. Expression of the dominant-negative mutant in the virulent E. histolytica strain caused a reduction of the size of liver abscesses in a hamster model. This defect in liver abscess formation was likely at least partially attributed to reduced resistance to nitrosative, but not oxidative stress in vitro. These results showed that the EhArfX2-mediated traffic is necessary for the nitrosative stress response and virulence in the host.

Download Full-text

In vitro and in vivo growth inhibition of murine Melanoma K-1735 cells by a dominant negative mutant α subunit of the Gi2 protein

Cellular Signalling ◽

10.1016/0898-6568(95)02049-7 ◽

1996 ◽

Vol 8 (3) ◽

pp. 159-166 ◽

Cited By ~ 18

Author(s):

Sylvie Hermouet ◽

Sadie Aznavoorian ◽

Allen M. Spiegel

Keyword(s):

Growth Inhibition ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Α Subunit ◽

Murine Melanoma ◽

In Vivo Growth ◽

Negative Mutant

Download Full-text

Both emerin and lamin C depend on lamin A for localization at the nuclear envelope

Journal of Cell Science ◽

10.1242/jcs.114.14.2577 ◽

2001 ◽

Vol 114 (14) ◽

pp. 2577-2590 ◽

Cited By ~ 9

Author(s):

O. Anthony Vaughan ◽

Mauricio Alvarez-Reyes ◽

Joanna M. Bridger ◽

Jos L. V. Broers ◽

Frans C. S. Ramaekers ◽

...

Keyword(s):

Nuclear Envelope ◽

Cell Lines ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Lamin A ◽

Lamin B1 ◽

Physical Interactions ◽

Low Levels ◽

Negative Mutant

Physical interactions between lamins and emerin were investigated by co-immunoprecipitation of in vitro translated proteins. Emerin interacted with in vitro translated lamins A, B1 and C in co-immunprecipitation reactions. Competition reactions revealed a clear preference for interactions between emerin and lamin C. Structural associations between lamins and emerin were investigated in four human cell lines displaying abnormal expression and/or localisation of lamins A and C. In each cell line absence of lamins A and C from the nuclear envelope (NE) was correlated with mis-localisation of endogenous and exogenous emerin to the ER. In two cell lines that did not express lamin A but did express lamin C, lamin C as well as emerin was mis-localised. When GFP-lamin A was expressed in SW13 cells (which normally express only very low levels of endogenous lamin A and mis-localise endogenous emerin and lamin C), all three proteins became associated with the NE. When GFP-lamin C was expressed in SW13 cells neither the endogenous nor the exogenous lamin C was localised to the NE and emerin remained in the ER. Finally, lamins A and C were selectively eliminated from the NE of HeLa cells using a dominant negative mutant of lamin B1. Elimination of these lamins from the lamina led to the accumulation of emerin as aggregates within the ER. Our data suggest that lamin A is essential for anchorage of emerin to the inner nuclear membrane and of lamin C to the lamina.

Download Full-text

Discovering and Characterizing of Survivin Dominant Negative Mutants With Stronger Pro-apoptotic Activity on Cancer Cells and CSCs

Frontiers in Oncology ◽

10.3389/fonc.2021.635233 ◽

2021 ◽

Vol 11 ◽

Author(s):

Wei Guo ◽

Xingyuan Ma ◽

Yunhui Fu ◽

Chang Liu ◽

Qiuli Liu ◽

...

Keyword(s):

Stem Cells ◽

Cancer Cells ◽

A549 Cells ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Anti Cancer ◽

Apoptotic Activity ◽

Reversal Index ◽

Negative Mutant

Survivin as a member of the inhibitor of apoptosis proteins (IAPs) family is undetectable in normal cells, but highly expressed in cancer cells and cancer stem cells (CSCs) which makes it an attractive target in cancer therapy. Survivin dominant negative mutants have been reported as competitive inhibitors of endogenous survivin protein in cancer cells. However, there is a lack of systematic comparative studies on which mutants have stronger effect on promoting apoptosis in cancer cells, which will hinder the development of novel anti-cancer drugs. Here, based on the previous study of survivin and its analysis of the relationship between structure and function, we designed and constructed a series of different amino acid mutants from survivin (TmSm34, TmSm48, TmSm84, TmSm34/48, TmSm34/84, and TmSm34/48/84) fused cell-permeable peptide TATm at the N-terminus, and a dominant negative mutant TmSm34/84 with stronger pro-apoptotic activity was selected and evaluated systematically in vitro. The double-site mutant of survivin (TmSm34/84) showed more robust pro-apoptotic activity against A549 cells than others, and could reverse the resistance of A549 CSCs to adriamycin (ADM) (reversal index up to 7.01) by decreasing the expression levels of survivin, P-gp, and Bcl-2 while increasing cleaved caspase-3 in CSCs. This study indicated the selected survivin dominant negative mutant TmSm34/84 is promising to be an excellent candidate for recombinant anti-cancer protein by promoting apoptosis of cancer cells and their stem cells and sensitizing chemotherapeutic drugs.

Download Full-text

Conditional Cytomegalovirus Replication In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.79.1.486-494.2005 ◽

2005 ◽

Vol 79 (1) ◽

pp. 486-494 ◽

Cited By ~ 24

Author(s):

Brigitte Rupp ◽

Zsolt Ruzsics ◽

Torsten Sacher ◽

Ulrich H. Koszinowski

Keyword(s):

Capsid Protein ◽

Viral Genome ◽

Expression System ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Conditional Expression ◽

Regulated Expression ◽

Negative Mutant

ABSTRACT We have established a conditional gene expression system for cytomegalovirus which allows regulation of genes independently from the viral replication program. Due to the combination of all elements required for regulated expression in the same viral genome, conditional viruses can be studied in different cell lines in vitro and in the natural host in vivo. The combination of a self-sufficient tetracycline-regulated expression cassette and Flp recombinase-mediated insertion into the viral genome allowed fast construction of recombinant murine cytomegaloviruses carrying different conditional genes. The regulation of two reporter genes, the essential viral M50 gene and a dominant-negative mutant gene (m48.2) encoding the small capsid protein, was analyzed in more detail. In vitro, viral growth was regulated by the conditional expression of M50 by 3 orders of magnitude and up to a millionfold when the dominant-negative small capsid protein mutant was used. In vivo, viral growth of the dominant-negative mutant was reduced to detection limits in response to the presence of doxycycline in the organs of mice. We believe that this conditional expression system is applicable to genetic studies of large DNA viruses in general.

Download Full-text

Chlamydia infection of epithelial cells expressing dynamin and Eps15 mutants: clathrin-independent entry into cells and dynamin-dependent productive growth

Journal of Cell Science ◽

10.1242/jcs.112.10.1487 ◽

1999 ◽

Vol 112 (10) ◽

pp. 1487-1496 ◽

Cited By ~ 2

Author(s):

H. Boleti ◽

A. Benmerah ◽

D.M. Ojcius ◽

N. Cerf-Bensussan ◽

A. Dautry-Varsat

Keyword(s):

Epithelial Cells ◽

Mutant Cell ◽

Dominant Negative ◽

Chlamydia Infection ◽

Coated Vesicle ◽

Dominant Negative Mutant ◽

Vesicular Traffic ◽

Membrane Compartments ◽

Dynamin I ◽

Negative Mutant

Chlamydiae enter epithelial cells via a mechanism that still remains to be fully elucidated. In this study we investigated the pathway of entry of C. psittaci GPIC and C. trachomatis LGV/L2 into HeLa cells and demonstrated that it does not depend on clathrin coated vesicle formation. We used mutant cell lines defective in clathrin-mediated endocytosis due to overexpression of dominant negative mutants of either dynamin I or Eps15 proteins. When clathrin-dependent endocytosis was inhibited by overexpression of the dynK44A mutant of dynamin I (defective in GTPase activity), Chlamydia entry was not affected. However, in these cells there was a dramatic inhibition in the proliferation of Chlamydia and the growth of the chlamydia vacuole (inclusion). When clathrin-dependent endocytosis was inhibited by overexpression of an Eps15 dominant negative mutant, the entry and growth of Chlamydia was unaltered. These results indicate that the effect on the growth of Chlamydia in the dynK44A cells was not simply due to a deprivation of nutrients taken up by endocytosis. Instead, the dominant-negative mutant of dynamin most likely affects the vesicular traffic between the Chlamydia inclusion and intracellular membrane compartments. In addition, cytochalasin D inhibited Chlamydia entry by more than 90%, indicating that chlamydiae enter epithelial cells by an actin-dependent mechanism resembling phagocytosis. Finally, dynamin is apparently not involved in the formation of phagocytic vesicles containing Chlamydia.

Download Full-text