scholarly journals The mitochondrial protein Bak is pivotal for gliotoxin-induced apoptosis and a critical host factor of Aspergillus fumigatus virulence in mice

2006 ◽  
Vol 174 (4) ◽  
pp. 509-519 ◽  
Author(s):  
Julian Pardo ◽  
Christin Urban ◽  
Eva M. Galvez ◽  
Paul G. Ekert ◽  
Uwe Müller ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Molecular Basis ◽  
Mammalian Cells ◽  
Fungal Infections ◽  
Apoptotic Cell ◽  
Mitochondrial Protein ◽  
Wild Type ◽  
Apoptotic Pathway

Aspergillus fumigatus infections cause high levels of morbidity and mortality in immunocompromised patients. Gliotoxin (GT), a secondary metabolite, is cytotoxic for mammalian cells, but the molecular basis and biological relevance of this toxicity remain speculative. We show that GT induces apoptotic cell death by activating the proapoptotic Bcl-2 family member Bak, but not Bax, to elicit the generation of reactive oxygen species, the mitochondrial release of apoptogenic factors, and caspase-3 activation. Activation of Bak by GT is direct, as GT triggers in vitro a dose-dependent release of cytochrome c from purified mitochondria isolated from wild-type and Bax- but not Bak-deficient cells. Resistance to A. fumigatus of mice lacking Bak compared to wild-type mice demonstrates the in vivo relevance of this GT-induced apoptotic pathway involving Bak and suggests a correlation between GT production and virulence. The elucidation of the molecular basis opens new strategies for the development of therapeutic regimens to combat A. fumigatus and related fungal infections.

scholarly journals Ficolins Promote Fungal Clearance in vivo and Modulate the Inflammatory Cytokine Response in Host Defense against Aspergillus fumigatus

2016 ◽  
Vol 8 (6) ◽  
pp. 579-588 ◽  
Author(s):  
Ninette Genster ◽  
Elisabeth Præstekjær Cramer ◽  
Anne Rosbjerg ◽  
Katrine Pilely ◽  
Jack Bernard Cowland ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Host Defense ◽  
Knockout Mice ◽  
Fungal Infections ◽  
Inflammatory Cells ◽  
Lectin Pathway ◽  
Wild Type ◽  
Opportunistic Fungal Pathogen

Aspergillus fumigatus is an opportunistic fungal pathogen that causes severe invasive infections in immunocompromised patients. Innate immunity plays a major role in protection against A. fumigatus. The ficolins are a family of soluble pattern recognition receptors that are capable of activating the lectin pathway of complement. Previous in vitro studies reported that ficolins bind to A. fumigatus, but their part in host defense against fungal infections in vivo is unknown. In this study, we used ficolin-deficient mice to investigate the role of ficolins during lung infection with A. fumigatus. Ficolin knockout mice showed significantly higher fungal loads in the lungs 24 h postinfection compared to wild-type mice. The delayed clearance of A. fumigatus in ficolin knockout mice could not be attributed to a compromised recruitment of inflammatory cells. However, it was revealed that ficolin knockout mice exhibited a decreased production of proinflammatory cytokines in the lungs compared to wild-type mice following A. fumigatus infection. The impaired clearance and cytokine production in ficolin knockout mice was independent of complement, as shown by equivalent levels of A. fumigatus-mediated complement activation in ficolin knockout mice and wild-type mice. In conclusion, this study demonstrates that ficolins are important in initial innate host defense against A. fumigatus infections in vivo.

scholarly journals Desferrioxamine B-Mediated Pre-Clinical In Vivo Imaging of Infection by the Mold Fungus Aspergillus fumigatus

2021 ◽  
Vol 7 (9) ◽  
pp. 734 ◽  
Author(s):  
Matthias Misslinger ◽  
Milos Petrik ◽  
Joachim Pfister ◽  
Isabella Hubmann ◽  
Katerina Bendova ◽  
...  
Keyword(s):  
Molecular Imaging ◽  
Aspergillus Fumigatus ◽  
Mammalian Cells ◽  
Bacterial Infections ◽  
Fungal Infections ◽  
Cellular Transport ◽  
Desferrioxamine B ◽  
Mold Fungus

Fungal infections are a serious threat, especially for immunocompromised patients. Early and reliable diagnosis is crucial to treat such infections. The bacterially produced siderophore desferrioxamine B (DFO-B) is utilized by a variety of microorganisms for iron acquisition, while mammalian cells lack the uptake of DFO-B chelates. DFO-B is clinically approved for a variety of long-term chelation therapies. Recently, DFO-B-complexed gallium-68 ([68Ga]Ga-DFO-B) was shown to enable molecular imaging of bacterial infections by positron emission tomography (PET). Here, we demonstrate that [68Ga]Ga-DFO-B can also be used for the preclinical molecular imaging of pulmonary infection caused by the fungal pathogen Aspergillus fumigatus in a rat aspergillosis model. Moreover, by combining in vitro uptake studies and the chemical modification of DFO-B, we show that the cellular transport efficacy of ferrioxamine-type siderophores is impacted by the charge of the molecule and, consequently, the environmental pH. The chemical derivatization has potential implications for its diagnostic use and characterizes transport features of ferrioxamine-type siderophores.

scholarly journals Review of T-2307, an Investigational Agent That Causes Collapse of Fungal Mitochondrial Membrane Potential

2021 ◽  
Vol 7 (2) ◽  
pp. 130
Author(s):  
Nathan P. Wiederhold
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Candida Species ◽  
Mammalian Cells ◽  
Mitochondrial Membrane ◽  
Fungal Infections ◽  
Published Data ◽  
Candida Auris

Invasive infections caused by Candida that are resistant to clinically available antifungals are of increasing concern. Increasing rates of fluconazole resistance in non-albicans Candida species have been documented in multiple countries on several continents. This situation has been further exacerbated over the last several years by Candida auris, as isolates of this emerging pathogen that are often resistant to multiple antifungals. T-2307 is an aromatic diamidine currently in development for the treatment of invasive fungal infections. This agent has been shown to selectively cause the collapse of the mitochondrial membrane potential in yeasts when compared to mammalian cells. In vitro activity has been demonstrated against Candida species, including C. albicans, C. glabrata, and C. auris strains, which are resistant to azole and echinocandin antifungals. Activity has also been reported against Cryptococcus species, and this has translated into in vivo efficacy in experimental models of invasive candidiasis and cryptococcosis. However, little is known regarding the clinical efficacy and safety of this agent, as published data from studies involving humans are not currently available.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

Rhogtpase RAC2 Is Required for In Vivo Transformation Activity by P210 Bcr-Abl Fusion Oncogene.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 717-717
Author(s):  
Nithya Krishnan ◽  
Jeff R. Bailey ◽  
Victoria Summey-Harner ◽  
Claudio Brunstein ◽  
Catherine M. Verfaillie ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Philadelphia Chromosome ◽  
Protein Domain ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Functions ◽  
Empty Vector

Abstract Bcr-Abl, the translocation product of the Philadelphia chromosome implicated in human chronic myelogenous leukemia (CML), is a kinase affecting hematopoietic stem cell (HSC) behavior with respect to proliferation, apoptosis, adhesion and migration. Rho GTPases, particularly the Rac subfamily, have been shown to regulate these same cell functions in normal HSC and also regulate gene expression in many mammalian cells. BCR contains a “GTPase-activating protein” domain and a guanine nucleotide exchange domain, the latter or which is preserved in p210 Bcr-Abl. Since HSC functions regulated by Bcr-Abl and Rac are similar, we studied the potential involvement of Rac activation in Bcr-Abl signaling cascade. Human CML samples demonstrate baseline activation of Rac proteins that is reversed by in vitro treatment with STI571. To study the specific involvement of Rac2, we used a gene targeted mouse model with Rac2 null bone marrow. Using retovirus-mediated gene transfer, we introduced p210 Bcr-Abl in the MSCV vector into wild-type or Rac2−/− HSC/P and studied the behavior of these cells in vitro and in vivo. Irradiated recipient mice injected with LDBM cells transduced with p210 developed a uniformly fatal myeloproliferative syndrome (Median survival: 45 days, N=12), while mice injected with p210 transduced Rac2−/− LDBM cells (N=12, 2 independent exp.) had 100% survival and no development of leukocytosis, splenomegaly or organ infiltration of hematopoietic cells. These data suggest that Rac GTPases are critical for the transformation of HSC by Bcr-Abl and provide an additional therapeutic target for intervention in CML. WILD TYPE Rac 2 −/− Empty Vector MSCV-p210 Empty vector MSCV-p210 *p < 0.01 vs WT-MIEG3, **p< 0.01 vs WT-p210 bcr-abl. Proliferation (CPM) Medium 562 ± 278 16,207± 1605* 819.7 ± 363 3,135.5 ± 498** SCF (100ng/ml) 856 ± 187 23,226 ± 2203* 853.7 ± 524 3,756.8 ± 207** Cytokines (SCF, GCSF, MGDF) 8011± 1412 42,711± 13393* 4833 ±1019 3,614.5 ± 1982** Migration (%) Fibronectin 7 ± 0.4 38 ± 1.9* 0.4 ± 0.0 0.8 ± 0.1** SDF-1α 30 ±2.8 13 ±1.1* 0.5 ± 0.0 0.6 ± 0.0** Adhesion (% ) Fibronectin 76± 2.9 40 ±3* 4 ±0.4 10 ±0.1 **

scholarly journals Human Monocyte Recognition of Adenosine-Based Cyclic Dinucleotides Unveils the A2a GαsProtein-Coupled Receptor Tonic Inhibition of Mitochondrially Induced Cell Death

2014 ◽  
Vol 35 (2) ◽  
pp. 479-495 ◽  
Author(s):  
Marie Tosolini ◽  
Frédéric Pont ◽  
Delphine Bétous ◽  
Emmanuel Ravet ◽  
Laetitia Ligat ◽  
...  
Keyword(s):  
Cell Death ◽  
Mammalian Cells ◽  
Mitochondrial Permeability Transition ◽  
Human Monocyte ◽  
Inverse Agonist ◽  
Apoptotic Pathway ◽  
Cyclic Dinucleotides ◽  
Agonist Ligands

Cyclic dinucleotides are important messengers for bacteria and protozoa and are well-characterized immunity alarmins for infected mammalian cells through intracellular binding to STING receptors. We sought to investigate their unknown extracellular effects by adding cyclic dinucleotides to the culture medium of freshly isolated human blood cellsin vitro. Here we report that adenosine-containing cyclic dinucleotides induce the selective apoptosis of monocytes through a novel apoptotic pathway. We demonstrate that these compounds are inverse agonist ligands of A2a, a Gαs-coupled adenosine receptor selectively expressed by monocytes. Inhibition of monocyte A2a by these ligands induces apoptosis through a mechanism independent of that of the STING receptors. The blockade of basal (adenosine-free) signaling from A2a inhibits protein kinase A (PKA) activity, thereby recruiting cytosolic p53, which opens the mitochondrial permeability transition pore and impairs mitochondrial respiration, resulting in apoptosis. A2a antagonists and inverse agonist ligands induce apoptosis of human monocytes, while A2a agonists are antiapoptotic.In vivo, we used a mock developing human hematopoietic system through NSG mice transplanted with human CD34+cells. Treatment with cyclic di-AMP selectively depleted A2a-expressing monocytes and their precursors via apoptosis. Thus, monocyte recognition of cyclic dinucleotides unravels a novel proapoptotic pathway: the A2a Gαsprotein-coupled receptor (GPCR)-driven tonic inhibitory signaling of mitochondrion-induced cell death.

scholarly journals Loss of Heterochromatin Protein 1 (HP1) chromodomain function in mammalian cells by intracellular antibodies causes cell death

2002 ◽  
Vol 115 (9) ◽  
pp. 1803-1813 ◽  
Author(s):  
Ilaria Filesi ◽  
Alessio Cardinale ◽  
Sjaak van der Sar ◽  
Ian G. Cowell ◽  
Prim B. Singh ◽  
...  
Keyword(s):  
Cell Death ◽  
Mammalian Cells ◽  
Wide Spectrum ◽  
Human Fibroblasts ◽  
Protein S ◽  
Single Chain ◽  
Heterochromatin Protein 1 ◽  
Apoptotic Pathway

The chromodomain (CD) is a highly conserved motif present in a variety of animal and plant proteins, and its probable role is to assemble a variety of macromolecular complexes in chromatin. The importance of the CD to the survival of mammalian cells has been tested. Accordingly, we have ablated CD function using two single-chain intracellular Fv (scFv) fragments directed against non-overlapping epitopes within the HP1 CD motif. The scFv fragments can recognize both CD motifs of HP1 and Polycomb (Pc) in vitro and, when expressed intracellularly, interact with and dislodge the HP1 protein(s) from their heterochromatin localization in vivo. Mouse and human fibroblasts expressing anti-chromodomain scFv fragments show a cell-lethal phenotype and an apoptotic morphology becomes apparent soon after transfection. The mechanism of cell death appears to be p53 independent, and the cells are only partly rescued by incubation with the wide spectrum caspase inhibitor Z-VAD fmk. We conclude that expression of anti-chromodomain intracellular antibodies is sufficient to trigger a p53-independent apoptotic pathway that is only partly dependent on the known Z-VAD-inhibitable caspases, suggesting that CD function is essential for cell survival.

scholarly journals Antifungal effect of all-trans retinoic acid against Aspergillus fumigatus in vitro and in a pulmonary aspergillosis in vivo model

2020 ◽  
Author(s):  
Elena Campione ◽  
Roberta Gaziano ◽  
Elena Doldo ◽  
Daniele Marino ◽  
Mattia Falconi ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Aspergillus Fumigatus ◽  
Rat Model ◽  
Fungal Infections ◽  
Invasive Pulmonary Aspergillosis ◽  
Antifungal Drugs ◽  
Pulmonary Aspergillosis ◽  
Fungistatic Activity

AIM: Aspergillus fumigatus is the most common opportunistic fungal pathogen and causes invasive pulmonary aspergillosis (IPA), with high mortality among immunosuppressed patients. Fungistatic activity of all-trans retinoic acid (ATRA) has been recently described in vitro. We evaluated the efficacy of ATRA in vivo and its potential synergistic interaction with other antifungal drugs. MATERIALS AND METHODS: A rat model of IPA and in vitro experiments were performed to assess the efficacy of ATRA against Aspergillus in association with classical antifungal drugs and in silico studies used to clarify its mechanism of action. RESULTS: ATRA (0.5 and 1 mM) displayed a strong fungistatic activity in Aspergillus cultures, while at lower concentrations, synergistically potentiated fungistatic efficacy of sub-inhibitory concentration of Amphotericin B (AmB) and Posaconazole (POS). ATRA also enhanced macrophagic phagocytosis of conidia. In a rat model of IPA, ATRA reduced mortality similarly to Posaconazole. CONCLUSION: Fungistatic efficacy of ATRA alone and synergistically with other antifungal drugs was documented in vitro, likely by inhibiting fungal Hsp90 expression and Hsp90-related genes. ATRA reduced mortality in a model of IPA in vivo. Those findings suggest ATRA as suitable fungistatic agent, also to reduce dosage and adverse reaction of classical antifungal drugs, and new therapeutic strategies against IPA and systemic fungal infections.

scholarly journals Identification of Novel Interaction Partners of AIF Protein on the Outer Mitochondrial Membrane

Acta Naturae ◽  
2018 ◽  
Vol 10 (4) ◽  
pp. 100-109 ◽  
Author(s):  
N. P. Fadeeva ◽  
N. V. Antipova ◽  
V. O. Shender ◽  
K. S. Anufrieva ◽  
G. A. Stepanov ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mitochondrial Membrane ◽  
Bioinformatic Analysis ◽  
Amino Acid Sequences ◽  
Outer Mitochondrial Membrane ◽  
Apoptotic Pathway ◽  
Survival Prognosis ◽  
High Level

In response to the wide variety of external and internal signals, mammalian cells undergo apoptosis, programmed cell death. Dysregulation of apoptosis is involved in multiple human diseases, including cancer, autoimmunity, and ischemic injuries. Two types of apoptosis have been described: the caspase-dependent one, leading to digestion of cellular proteins, and caspase-independent apoptosis, resulting in DNA fragmentation. The latter type of apoptosis is executed by AIF protein and is believed to have appeared first during evolution. The key step in the caspase-independent apoptosis program is the dissociation of AIF from the outer mitochondrial membrane (OMM). However, the molecular mechanism of interaction between AIF and OMM remains poorly understood. In this study, we demonstrated that AIF can bind to OMM via mortalin protein. We confirmed interaction between AIF and mortalin both in vitro and in vivo and mapped the amino acid sequences that are important for the binding of these proteins. Next, we showed that apoptosis induction by chemotherapy leads to downregulation of AIF-mortalin interaction and dissociation of AIF from the OMM. Finally, a bioinformatic analysis demonstrated that a high level of mortalin expression correlates with a worse survival prognosis for glioma patients. Altogether, our data revealed that mortalin plays an important role in the regulation of the caspase-independent apoptotic pathway and allowed us to speculate that inhibition of AIF-mortalin interaction may induce a dissociation of AIF from the OMM and subsequent apoptosis of cancer cells.

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