Inhibition of ER stress-mediated apoptosis in macrophages by nuclear-cytoplasmic relocalization of eEF1A by the HIV-1 Nef protein

HIV-1 Nef protein has key roles at almost all stages of the viral life cycle. We assessed the role of the Nef/eEF1A (eukaryotic translation elongation factor 1-alpha) complex in nucleocytoplasmic shuttling in primary human macrophages. Nuclear retention experiments and inhibition of the exportin-t (Exp-t) pathway suggested that cytoplasmic relocalization of eEF1A, mediated by Exp-t, occurs in Nef-treated monocyte-derived macrophages (MDMs). We observed the presence of tRNA in the Nef/eEF1A complexes. Nucleocytoplasmic relocalization of the Nef/eEF1A complexes prevented stress-induced apoptosis of MDMs treated with brefeldin-A. Blockade of stress-induced apoptosis of MDMs treated with HIV-1 Nef resulted from enhanced nucleocytoplasmic transport of eEF1A with decreased release of mitochondrial cytochrome c, and from increased tRNA binding to cytochrome c, ultimately leading to an inhibition of caspase activation. Our results indicate that HIV-1 Nef, through the nucleocytoplasmic relocalization of eEF1A and tRNAs, enhances resistance to stress-induced apoptosis in primary human macrophages.     

Human immunodeficiency virus type 1 inhibits DNA damage-triggered apoptosis by a Nef-independent mechanism

It is controversial whether the accessory human immunodeficiency virus type 1 (HIV-1) Nef protein inhibits or enhances apoptosis. To address this issue, we investigated the effect of Nef on programmed cell death with vectors or proviral HIV-1 constructs coexpressing Nef and green fluorescent protein from single bicistronic RNAs. This approach allows us to readily identify transfected or infected cells and to correlate cell death directly with Nef expression levels. We demonstrate that Nef does not significantly affect apoptosis in transfected or HIV-1-infected Jurkat T cells or primary human peripheral blood mononuclear cells. Unexpectedly, however, both nef⁺ and nef-defective HIV-1 infection blocked apoptosis in cells treated with UV light or etoposide but not cell death induced by CD95 antibody, TRAIL, Ly294002, or serum starvation. Our results show that HIV-1 infection inhibits DNA damage-induced but not death receptor-dependent cell death by a Nef-independent mechanism.     

Yeast translation elongation factor eEF3 promotes late stages of tRNA translocation

In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the ribosomal A and E sites, its exact mechanism of action is unclear. Here, we show that eEF3 acts at the mRNA–tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl‐tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slowdown in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo‐EM analysis of native eEF3‐ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non‐rotated ribosomal states, as well as by opening the L1 stalk to release the E‐site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.     

In vitro treatment of human monocytes/macrophages with myristoylated recombinant Nef of human immunodeficiency virus type 1 leads to the activation of mitogen-activated protein kinases, IkappaB kinases, and interferon regulatory factor 3 and to the release of beta interferon

The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-kappaB and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT3. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the alpha and beta subunits of the IkappaB kinase complex and of JNK, ERK1/2, and p38 mitogen-activated protein kinase family members. In addition, we have observed the activation of interferon regulatory factor 3, leading to the synthesis of beta interferon mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation.     

Exogenous Nef Is an Inhibitor of Mycobacterium tuberculosis-induced Tumor Necrosis Factor-�� Production and Macrophage Apoptosis

Human immunodeficiency virus-1 (HIV-1) impairs tumor necrosis factor-α (TNF-α)-mediated macrophage apoptosis induced by Mycobacterium tuberculosis (Mtb). HIV Nef protein plays an important role in the pathogenesis of AIDS. We have tested the hypothesis that exogenous Nef is a factor that inhibits TNF-α production/apoptosis in macrophages infected with Mtb. We demonstrate that Mtb and Nef individually trigger TNF-α production in macrophages. However, TNF-α production is dampened when the two are present simultaneously, probably through cross-regulation of the individual signaling pathways leading to activation of the TNF-α promoter. Mtb-induced TNF-α production is abrogated upon mutation of the Ets, Egr, Sp1, CRE, or AP1 binding sites on the TNF-α promoter, whereas Nef-mediated promoter activation depends only on the CRE and AP1 binding sites, pointing to differences in the mechanisms of activation of the promoter. Mtb-dependent promoter activation depends on the mitogen-activated kinase (MAPK) kinase kinase ASK1 and on MEK/ERK signaling. Nef inhibits ASK1/p38 MAPK-dependent Mtb-induced TNF-α production probably by inhibiting binding of ATF2 to the TNF-α promoter. It also inhibits MEK/ERK-dependent Mtb-induced binding of FosB to the promoter. Nef-driven TNF-α production occurs in an ASK1-independent, Rac1/PAK1/p38 MAPK-dependent, and MEK/ERK-independent manner. The signaling pathways used by Mtb and Nef to trigger TNF-α production are therefore distinctly different. In addition to attenuating Mtb-dependent TNF-α promoter activation, Nef also reduces Mtb-dependent TNF-α mRNA stability probably through its ability to inhibit ASK1/p38 MAPK signaling. These results provide new insight into how HIV Nef probably exacerbates tuberculosis infection by virtue of its ability to dampen Mtb-induced TNF-α production.     

Importance of cytochrome c redox state for ceramide-induced apoptosis of human mammary adenocarcinoma cells

Background

Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides.

Methods

The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry.

Results

We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c.

Conclusions

Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione.

General significance

Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides.     

tRNA and cytochrome c in cell death and beyond

Both transfer RNA (tRNA) and cytochrome c are essential to cellular function: tRNA mediates protein synthesis while cytochrome c is required for oxidative phosphorylation and apoptosis induction. tRNA has recently been implicated as a direct regulator of the well-conserved apoptotic role of cytochrome c. Interaction between these molecules could potentially coordinate biosynthesis, energy production and apoptosis. Here we review the diversity and dynamics of tRNA and how this class of non-coding RNAs may regulate the role of cytochrome c in apoptosis. We comment on unanswered questions in the cell biology of this interaction and how answers may influence our understanding of disease.Key words: tRNA, cytochrome c, apoptosis, caspase, mitochondria, protein synthesis, cancer     

Cordycepin induces cell cycle arrest and apoptosis by inducing DNA damage and up-regulation of p53 in Leukemia cells

Cordycepin, an adenosine analog derived from Cordyceps militaris has been shown to exert anti-tumor activity in many ways. However, the mechanisms by which cordycepin contributes to the anti-tumor still obscure. Here our present work showed that cordycepin inhibits cell growth in NB-4 and U937 cells by inducing apoptosis. Further study showed that cordycepin increases the expression of p53 which promotes the release of cytochrome c from mitochondria to the cytosol. The released cytochrome c can then activate caspase-9 and trigger intrinsic apoptosis. Cordycepin also blocks MAPK pathway by inhibiting the phosphorylation of ERK1/2, and thus sensitizes the apoptosis. In addition, our results showed that cordycepin inhibits the expression of cyclin A2, cyclin E, and CDK2, which leads to the accumulation of cells in S-phase. Moreover, our study showed that cordycepin induces DNA damage and causes degradation of Cdc25A, suggesting that cordycepin-induced S-phase arrest involves activation of Chk2-Cdc25A pathway. In conclusion, cordycepin-induced DNA damage initiates cell cycle arrest and apoptosis which leads to the growth inhibition of NB-4 and U937 cells.     

Perturbation of apoptosis upon binding of tRNA to the heme domain of cytochrome c

In response to apoptotic stimuli, cytochrome c, an inter-membrane space protein is released from mitochondria to activate the cascade of caspases that leads to apoptosis. Recent evidence suggests that cytochrome c interacts with tRNA in the cytoplasm and this interaction was shown to inhibit the caspase mediated apoptotic process. Interestingly, cytochrome c does not contain any putative RNA binding domain. In this report, we sought to define the structural component of cytochrome c that is involved in binding of tRNA. By using gel mobility shift assays, we show that holocytochrome c can interact with tRNA but not apocytochrome c that lacks the heme domain suggesting that heme is essential for the interaction of cytochrome c to tRNA. In addition, using in vitro cross linking and circular dichroism spectroscopic studies, we show that cytochrome c can undergo heme mediated oligomerization. Prevention of heme mediated oligomerization of cytochrome c by potassium ferricyanide treatment prevents the binding of tRNA and promotes caspase activation. Our studies provide a novel regulation of apoptosis by heme dependent tRNA interaction to cytochrome c.     

Morphine and galectin-1 modulate HIV-1 infection of human monocyte-derived macrophages

Morphine is a widely abused, addictive drug that modulates immune function. Macrophages are a primary reservoir of HIV-1; therefore, they play a role in the development of this disease, as well as impact the overall course of disease progression. Galectin-1 is a member of a family of β-galactoside-binding lectins that are soluble adhesion molecules and that mediate direct cell-pathogen interactions during HIV-1 viral adhesion. Because the drug abuse epidemic and the HIV-1 epidemic are closely interrelated, we propose that increased expression of galectin-1 induced by morphine may modulate HIV-1 infection of human monocyte-derived macrophages (MDMs). In this article, we show that galectin-1 gene and protein expression are potentiated by incubation with morphine. Confirming previous studies, morphine alone or galectin-1 alone enhance HIV-1 infection of MDMs. Concomitant incubation with exogenous galectin-1 and morphine potentiated HIV-1 infection of MDMs. We used a nanotechnology approach that uses gold nanorod-galectin-1 small interfering RNA complexes (nanoplexes) to inhibit gene expression for galectin-1. We found that nanoplexes silenced gene expression for galectin-1, and they reversed the effects of morphine on galectin-1 expression. Furthermore, the effects of morphine on HIV-1 infection were reduced in the presence of the nanoplex.     

Human Immunodeficiency virus type 1 Nef potently induces apoptosis in primary human brain microvascular endothelial cells via the activation of caspases

The lentiviral protein Nef plays a major role in the pathogenesis of human immunodeficiency virus type I (HIV-1) infection. Although the exact mechanisms of its actions are not fully understood, Nef has been shown to be essential for the maintenance of high-titer viral replication and disease pathogenesis in in vivo models of simian immunodeficiency virus infection of monkeys. Nef has also been suggested to play a pivotal role in the depletion of T cells by promoting apoptosis in bystander cells. In this context, we investigated the ability of extracellular and endogenously expressed HIV-1 Nef to induce apoptosis in primary human brain microvascular endothelial cells (MVECs). Human brain MVECs were exposed to baculovirus-expressed HIV-1 Nef protein, an HIV-1-based vector expressing Nef, spleen necrosis virus (SNV)-Nef virus (i.e., SNV vector expressing HIV-1 Nef as a transgene), and the HIV-1 strain ADA and its Nef deletion mutant, ADADeltaNef. We observed that ADA Nef, the HIV-1 vector expressing Nef, and SNV-Nef were able to induce apoptosis in a dose-dependent manner. The mutant virus with a deletion in Nef was able to induce apoptosis in MVECs to modest levels, but the effects were not as pronounced as with the wild-type HIV-1 strain, ADA, the HIV-1-based vector expressing Nef, or SNV-Nef viruses. We also demonstrated that relatively high concentrations of exogenous HIV-1 Nef protein were able to induce apoptosis in MVECs. Gene microarray analyses showed increases in the expression of several specific proapoptotic genes. Western blot analyses revealed that the various caspases involved with Nef-induced apoptosis are processed into cleavage products, which occur only during programmed cell death. The results of this study demonstrate that Nef likely contributes to the neuroinvasion and neuropathogenesis of HIV-1, through its effects on select cellular processes, including various apoptotic cascades.     

Neuroglobin upregulation induced by 17β-estradiol sequesters cytocrome c in the mitochondria preventing H2O2-induced apoptosis of neuroblastoma cells

The sex steroid hormone 17β-estradiol (E2) upregulates the levels of neuroglobin (NGB), a new neuroprotectant globin, to elicit its neuroprotective effect against H₂O₂-induced apoptosis. Several mechanisms could be proposed to justify the NGB involvement in E2 prevention of stress-induced apoptotic cell death. Here, we evaluate the ability of E2 to modulate the intracellular NGB localization and the NGB interaction with mitochondrial cytochrome c following the H₂O₂-induced toxicity. Present results demonstrate that NGB is expressed in the nuclei, mitochondria, and cytosol of human neuroblastoma SK-N-BE cells. E2, but not H₂O₂ treatment of SK-N-BE cells, reallocates NGB mainly at the mitochondria and contemporarily reduces the number of apoptotic nuclei and the levels of cleaved caspase-3. Remarkably, the E2 treatment strongly increases NGB–cytochrome c association into mitochondria and reduces the levels of cytochrome c into the cytosol of SK-N-BE cells. Although both estrogen receptors (ERα and ERβ) are expressed in the nucleus, mitochondria, and cytosol of SK-N-BE cells, this E2 effect specifically requires the mitochondrial ERβ activity. As a whole, these data demonstrate that the interception of the intrinsic apoptotic pathway into mitochondria (i.e., the prevention of cytochrome c release) is one of the pivotal mechanisms underlying E2-dependent NGB neuroprotection against H₂O₂ toxicity.     

HIV-1 Nef protects human-monocyte-derived macrophages from HIV-1-induced apoptosis

HIV-1 Nef is the regulatory protein expressed earliest and most abundantly in the infection cycle. Its expression has been correlated with a plethora of effects detectable either in producer, target, and bystander cells, as well as in the viral particles. Even if the relationship between Nef expression and apoptosis has been already matter of investigation in infected lymphocytes, whose resistance to HIV infection is however limited to few days, this remains to be investigated in cells that in vivo well resist the HIV cytopathic effect. In such an instance, we were interested in establishing whether Nef influences the apoptotic processes in primary human-monocyte-derived macrophages (MDM). High efficiency HIV-1 infection of MDM allowed us to establish that virus-expressed Nef strongly counteracts the HIV-1-induced apoptosis. The Nef mutant analysis suggested that this effect relies on the interaction with different protein partners and cell compartments. We also observed that the Nef protection to the HIV-1-induced apoptosis correlated with the hyper-phosphorylation and consequent inactivation of the pro-apoptotic Bad protein. On the basis of these results, we propose the Nef anti-apoptotic effect as a relevant part of the mechanism of the in vivo establishment of the HIV macrophage reservoirs.     

Cyclophilin A as negative regulator of apoptosis by sequestering cytochrome c

The release of cytochrome c from the mitochondrial intermembrane space is a decisive event in programed cell death. Once in the cytoplasm, cytochrome c is involved in the formation of the macromolecular complex termed apoptosome, which activates procaspase-9 which in turn activates downstream procaspase-3. There are increasing evidence indicating that cyclophilin A is highly expressed in many tumors and cell lines where it exerts an anti-apoptotic function. In brain tissue, which over-expresses constitutively cyclophilin A, we found mixed dimers composed of cyclophilin A and cytochrome c. In a cell-free system we observed that pure cyclophilin A inhibited cytochrome c-dependent procaspase-3 activation. Moreover, we detected cyclophilin A-cytochrome c complexes within the cytoplasm of HCT116 cells following staurosporine-induced apoptosis. Our results strongly support that, in tumor cells, cyclophilin A is able to inhibit procaspase-3 activation by sequestering cytochrome c.     

HIV-1 Nef Dimerization Is Required for Nef-Mediated Receptor Downregulation and Viral Replication

Nef, a human immunodeficiency virus type 1 (HIV-1) accessory factor capable of interaction with a diverse array of host cell signaling molecules, is essential for high-titer HIV replication and AIDS progression. Previous biochemical and structural studies have suggested that Nef may form homodimers and higher-order oligomers in HIV-infected cells, which may be required for both immune and viral receptor downregulation as well as viral replication. Using bimolecular fluorescence complementation, we provide the first direct evidence for Nef dimers within HIV host cells and identify the structural requirements for dimerization in vivo. Bimolecular fluorescence complementation analysis shows that the multiple hydrophobic and electrostatic interactions found within the dimerization interface of the Nef X-ray crystal structure are essential for dimerization in cells. Nef dimers localized to the plasma membrane as well as the trans-Golgi network, two subcellular localizations essential for Nef function. Mutations in the Nef dimerization interface dramatically reduced both Nef-induced CD4 downregulation and HIV replication. Viruses expressing dimerization-defective Nef mutants were disabled to the same extent as HIV that fails to express Nef in terms of replication. These results identify the Nef dimerization region as a potential molecular target for antiretroviral drug discovery.