ATF4 Signaling in HIV-1 Infection: Viral Subversion of a Stress Response Transcription Factor.

Cellular integrated stress response (ISR), the mitochondrial unfolded protein response (UPRmt), and IFN signaling are associated with viral infections. Activating transcription factor 4 (ATF4) plays a pivotal role in these pathways and controls the expression of many genes involved in redox processes, amino acid metabolism, protein misfolding, autophagy, and apoptosis. The precise role of ATF4 during viral infection is unclear and depends on cell hosts, viral agents, and models. Furthermore, ATF4 signaling can be hijacked by pathogens to favor viral infection and replication. In this review, we summarize the ATF4-mediated signaling pathways in response to viral infections, focusing on human immunodeficiency virus 1 (HIV-1). We examine the consequences of ATF4 activation for HIV-1 replication and reactivation. The role of ATF4 in autophagy and apoptosis is explored as in the context of HIV-1 infection programmed cell deaths contribute to the depletion of CD4 T cells. Furthermore, ATF4 can also participate in the establishment of innate and adaptive immunity that is essential for the host to control viral infections. We finally discuss the putative role of the ATF4 paralogue, named ATF5, in HIV-1 infection. This review underlines the role of ATF4 at the crossroads of multiple processes reflecting host-pathogen interactions.

DREAM a little dREAM of DRM: Model organisms and conservation of DREAM-like complexes: Model organisms uncover the mechanisms of DREAM-mediated transcription regulation.

DREAM complexes are transcriptional regulators that control the expression of hundreds to thousands of target genes involved in the cell cycle, quiescence, differentiation, and apoptosis. These complexes contain many subunits that can vary according to the considered target genes. Depending on their composition and the nature of the partners they recruit, DREAM complexes control gene expression through diverse mechanisms, including chromatin remodeling, transcription cofactor and factor recruitment at various genomic binding sites. This complexity is particularly high in mammals. Since the discovery of the first dREAM complex (drosophila Rb, E2F, and Myb) in Drosophila melanogaster, model organisms such as Caenorhabditis elegans, and plants allowed a deeper understanding of the processes regulated by DREAM-like complexes. Here, we review the conservation of these complexes. We discuss the contribution of model organisms to the study of DREAM-mediated transcriptional regulatory mechanisms and their relevance in characterizing novel activities of DREAM complexes.

Lessons on SpA pathogenesis from animal models.

Understanding the complex mechanisms underlying a disorder such as spondyloarthritis (SpA) may benefit from studying animal models. Several suitable models have been developed, in particular to investigate the role of genetic factors predisposing to SpA, including HLA-B27, ERAP1, and genes related to the interleukin (IL)-23/IL-17 axis. One of the best examples of such research is the HLA-B27 transgenic rat model that fostered the emergence of original theories regarding HLA-B27 pathogenicity, including dysregulation of innate immunity, contribution of the adaptive immune system to chronic inflammation, and influence of the microbiota on disease development. Very recently, a new model of HLA-B27 transgenic Drosophila helped to expand further some of those theories in an unexpected direction involving the TGFß/BMP family of mediators. On the other hand, several spontaneous, inducible, and/or genetically modified mouse models-including SKG mouse, TNFΔARE mouse and IL-23-inducible mouse model of SpA-have highlighted the importance of TNFα and IL-23/IL-17 axis in the development of SpA manifestations. Altogether, those animal models afford not only to study disease mechanism but also to investigate putative therapeutic targets.

HLA-B27 alters BMP/TGFß signalling in Drosophila, revealing putative pathogenic mechanism for spondyloarthritis.

OBJECTIVES: The human leucocyte antigen (HLA)-B27 confers an increased risk of spondyloarthritis (SpA) by unknown mechanism. The objective of this work was to uncover HLA-B27 non-canonical properties that could explain its pathogenicity, using a new Drosophila model. METHODS: We produced transgenic Drosophila expressing the SpA-associated HLA-B*27:04 or HLA-B*27:05 subtypes, or the non-associated HLA-B*07:02 allele, alone or in combination with human ß2-microglobulin (hß2m), under tissue-specific drivers. Consequences of transgenes expression in Drosophila were examined and affected pathways were investigated by the genetic interaction experiments. Predictions of the model were further tested in immune cells from patients with SpA. RESULTS: Loss of crossveins in the wings and a reduced eye phenotype were observed after expression of HLA-B*27:04 or HLA-B*27:05 in Drosophila but not in fruit flies expressing the non-associated HLA-B*07:02 allele. These HLA-B27-induced phenotypes required the presence of hß2m that allowed expression of well-folded HLA-B conformers at the cell surface. Loss of crossveins resulted from a dominant negative effect of HLA-B27 on the type I bone morphogenetic protein (BMP) receptor saxophone (Sax) with which it interacted, resulting in elevated mothers against decapentaplegic (Mad, a Drosophila receptor-mediated Smad) phosphorylation. Likewise, in immune cells from patients with SpA, HLA-B27 specifically interacted with activin receptor-like kinase-2 (ALK2), the mammalian Sax ortholog, at the cell surface and elevated Smad phosphorylation was observed in response to activin A and transforming growth factor ß (TGFß). CONCLUSIONS: Antagonistic interaction of HLA-B27 with ALK2, which exerts inhibitory functions on the TGFß/BMP signalling pathway at the cross-road between inflammation and ossification, could adequately explain SpA development.

The endoplasmic reticulum unfolded protein response varies depending on the affected region of the tissue but independently from the source of stress.

Accumulation of unfolded proteins and calcium dyshomeostasis induces endoplasmic reticulum (ER) stress, which can be resolved by the unfolded protein response (UPR). We have previously reported that activation of the PERK/ATF4 branch of the UPR, by overexpressing Presenilin in part of the vestigial domain of Drosophila wing imaginal discs, induces both a caspase-dependent apoptosis and a Slpr/JNK/Dilp8-dependent developmental delay that allows compensation of cell death in the tissue. Recently, dDad1 depletion in Drosophila in engrailed-expressing cells of wing imaginal discs was also reported to activate the PERK/ATF4 branch but induced Mekk1/JNK-dependent apoptosis. Here, we assessed whether the stressed cell location in the wing imaginal disc could explain these differences in response to chronic ER stress or whether the stress source could be responsible for the signaling discrepancy. To address this question, we overexpressed a Rhodopsin-1 mutant prone to aggregate either in vestigial- or engrailed-expressing cells. We observed similar responses to the Presenilin overexpression in the vestigial domain and to the dDad1 depletion in the engrailed domain. Therefore, the consequences of a PERK/ATF4 branch activation depend on the position of the cell in the Drosophila wing imaginal disc, suggesting interactions of PERK signaling with developmental pathways involved in the determination or maintenance of wing domains.

Screening of suppressors of bax-induced cell death identifies glycerophosphate oxidase-1 as a mediator of debcl-induced apoptosis in Drosophila.

Members of the Bcl-2 family are key elements of the apoptotic machinery. In mammals, this multigenic family contains about twenty members, which either promote or inhibit apoptosis. We have previously shown that the mammalian pro-apoptotic Bcl-2 family member Bax is very efficient in inducing apoptosis in Drosophila, allowing the study of bax-induced cell death in a genetic animal model. We report here the results of the screening of a P[UAS]-element insertion library performed to identify gene products that modify the phenotypes induced by the expression of bax in Drosophila melanogaster. We isolated 17 putative modifiers involved in various function or process: the ubiquitin/proteasome pathway; cell growth, proliferation and death; pathfinding and cell adhesion; secretion and extracellular signaling; metabolism and oxidative stress. Most of these suppressors also inhibit debcl-induced phenotypes, suggesting that the activities of both proteins can be modulated in part by common signaling or metabolic pathways. Among these suppressors, Glycerophosphate oxidase-1 is found to participate in debcl-induced apoptosis by increasing mitochondrial reactive oxygen species accumulation.

Quantification of Ataxin-3 and Ataxin-7 aggregates formed in vivo in Drosophila reveals a threshold of aggregated polyglutamine proteins associated with cellular toxicity.

Polyglutamine diseases are nine dominantly inherited neurodegenerative pathologies caused by the expansion of a polyglutamine domain in a protein responsible for the disease. This expansion leads to protein aggregation, inclusion formation and toxicity. Despite numerous studies focusing on the subject, whether soluble polyglutamine proteins are responsible for toxicity or not remains debated. To focus on this matter, we evaluated the level of soluble and insoluble truncated pathological Ataxin-3 in vivo in Drosophila, in presence or absence of two suppressors (i.e. Hsp70 and non-pathological Ataxin-3) and along aging. Suppressing truncated Ataxin-3-induced toxicity resulted in a lowered level of aggregated polyglutamine protein. Interestingly, aggregates accumulated as flies aged and reached a maximum level when cell death was detected. Our results were similar with two other pathological polyglutamine proteins, namely truncated Ataxin-7 and full-length Ataxin-3. Our data suggest that accumulation of insoluble aggregates beyond a critical threshold could be responsible for toxicity.

p62 plays a protective role in the autophagic degradation of polyglutamine protein oligomers in polyglutamine disease model flies.

Oligomer formation and accumulation of pathogenic proteins are key events in the pathomechanisms of many neurodegenerative diseases, such as Alzheimer disease, ALS, and the polyglutamine (polyQ) diseases. The autophagy-lysosome degradation system may have therapeutic potential against these diseases because it can degrade even large oligomers. Although p62/sequestosome 1 plays a physiological role in selective autophagy of ubiquitinated proteins, whether p62 recognizes and degrades pathogenic proteins in neurodegenerative diseases has remained unclear. In this study, to elucidate the role of p62 in such pathogenic conditions in vivo, we used Drosophila models of neurodegenerative diseases. We found that p62 predominantly co-localizes with cytoplasmic polyQ protein aggregates in the MJDtr-Q78 polyQ disease model flies. Loss of p62 function resulted in significant exacerbation of eye degeneration in these flies. Immunohistochemical analyses revealed enhanced accumulation of cytoplasmic aggregates by p62 knockdown in the MJDtr-Q78 flies, similarly to knockdown of autophagy-related genes (Atgs). Knockdown of both p62 and Atgs did not show any additive effects in the MJDtr-Q78 flies, implying that p62 function is mediated by autophagy. Biochemical analyses showed that loss of p62 function delays the degradation of the MJDtr-Q78 protein, especially its oligomeric species. We also found that loss of p62 function exacerbates eye degeneration in another polyQ disease fly model as well as in ALS model flies. We therefore conclude that p62 plays a protective role against polyQ-induced neurodegeneration, by the autophagic degradation of polyQ protein oligomers in vivo, indicating its therapeutic potential for the polyQ diseases and possibly for other neurodegenerative diseases.

The drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the ß-TrCP homologue slimb.

The ubiquitin-proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, we identified the ubiquitin activating enzyme Uba1 as a suppressor of bax-induced cell death. We then demonstrated that Uba1 also regulates apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, we show that these apoptotic processes involve the same multimeric E3 ligase-an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of ß-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family.

zVAD-fmk upregulates caspase-9 cleavage and activity in etoposide-induced cell death of mouse embryonic fibroblasts.

Caspases are key effectors of programmed cell death. Down- and up-regulation of their activity are involved in different pathologies. In most cells, zVAD-fmk prevents apoptosis. However, unexpected effects of zVAD-fmk have been characterized in different laboratories, cell models and cell death processes. We have previously shown that zVAD-fmk accelerates p53-dependent apoptosis in rat embryonic fibroblasts. In this study, we pursued our investigations on zVAD-fmk effects and focused our study at the mitochondrial level in mouse embryonic fibroblasts (MEFs). In both primary and immortalized (by AgT or 3T9 protocol) MEFs, zVAD-fmk increased etoposide-induced loss of ΔΨm. This increase correlated with an increase of the number of apoptotic cells in primary and 3T9 MEFs, but did not in AgT MEFs. In both types of immortalized MEFs, zVAD-fmk regulated neither p53 levels nor transcriptional activities, suggesting that zVAD-fmk acts downstream of p53. In MEFs, zVAD-fmk increased p53-dependent loss of ΔΨm, cytochrome c release and caspase-9 activity. Indeed, zVAD-fmk inhibited effector caspases (caspases-3, -6, -7) as expected but increased caspase-9 cleavage and activity in etoposide-treated MEFs. Q-VD-OPh, another caspase inhibitor, also increased both loss of ΔΨm and caspase-9 cleavage in etoposide-treated MEFs. Invalidation of bax and bak suppressed p53-dependent cell death and zVAD-fmk regulation of this process. Invalidation of caspase-9 did not inhibit mitochondrial membrane depolarization but suppressed zVAD-fmk amplification of this process. Altogether, our data suggest that caspase-9 activity is up-regulated by zVAD-fmk and is involved in an amplification loop of etoposide-induced cell death at the mitochondrial level in MEFs.

Mitochondria, Bcl-2 family proteins and apoptosomes: of worms, flies and men.

Initiator caspases are activated within specialized complexes, one of which is the apoptosome. The apoptosome is always constituted by at least an initiator caspase and a caspase activator. Apoptosome activation enables maturation of the associated caspase and constitutes a key step for cell fate. This activating complex is found throughout metazoans but its composition and regulation seem slightly different from one species to another. This review focuses on the composition and activation of the apoptosome in different species and details the role of mitochondrial factors and Bcl-2 family members in this activation.

Life span extension by dietary restriction is reduced but not abolished by loss of both SIR2 and HST2 in Podospora anserina.

Dietary restriction (DR) extends life span of many organisms, from yeast to mammals. The question of whether or not the SIR2 protein functions to mediate life span extension in response to DR remains debated. In this paper, we studied the relationship between SIR2 and DR in the filamentous fungus Podospora anserina. We show that the loss of PaSir2, PaHst2 or PaPnc1 does not alter life span under standard conditions. PaHst2 is the closest paralog of PaSir2 and the ortholog of yeast HST2 and PaPnc1 is the ortholog of the yeast PNC1 which encodes a nicotinamidase that deaminates nicotinamide, a natural inhibitor of SIR2. As observed for other organisms, overexpression of PaSir2 weakly increases life span under standard condition. Under DR conditions, deletion of the PaSir2 or PaHst2 genes induce a significant reduction in life span extension, while the double mutant strongly reduces life span extension. However, a clear response to DR subsists in the double mutant, demonstrating that DR acts through a SIR2/HST2 independent pathway.

Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain.

P62 has been proposed to mark ubiquitinated protein bodies for autophagic degradation. We report that the Drosophila melanogaster p62 orthologue, Ref(2)P, is a regulator of protein aggregation in the adult brain. We demonstrate that Ref(2)P localizes to age-induced protein aggregates as well as to aggregates caused by reduced autophagic or proteasomal activity. A similar localization to protein aggregates is also observed in D. melanogaster models of human neurodegenerative diseases. Although atg8a autophagy mutant flies show accumulation of ubiquitin- and Ref(2)P-positive protein aggregates, this is abrogated in atg8a/ref(2)P double mutants. Both the multimerization and ubiquitin binding domains of Ref(2)P are required for aggregate formation in vivo. Our findings reveal a major role for Ref(2)P in the formation of ubiquitin-positive protein aggregates both under physiological conditions and when normal protein turnover is inhibited.

The PI 3-kinase regulator Vps15 is required for autophagic clearance of protein aggregates.

Autophagy is involved in cellular clearance of aggregate-prone proteins, thereby having a cytoprotective function. Studies in yeast have shown that the PI 3-kinase Vps34 and its regulatory protein kinase Vps15 are important for autophagy, but the possible involvement of these proteins in autophagy in a multicellular animal has not been addressed genetically. Here, we have created a Drosophila deletion mutant of vps15 and studied its role in autophagy and aggregate clearance. Homozygous Deltavps15 Drosophila died at the early L3 larval stage. Using GFP-Atg8a as an autophagic marker, we employed fluorescence microscopy to demonstrate that fat bodies of wild type Drosophila larvae accumulated autophagic structures upon starvation whereas vps15 fat bodies showed no such response. Likewise, electron microscopy revealed starvation-induced autophagy in gut cells from wild type but not Deltavps15 larvae. Fluorescence microscopy showed that Deltavps15 mutant tissues accumulated profiles that were positive for ubiquitin and Ref(2)P, the Drosophila homolog of the sequestosome marker SQSTM1/p62. Biochemical fractionation and Western blotting showed that these structures were partially detergent insoluble, and immuno-electron microscopy further demonstrated the presence of Ref(2)P positive membrane free protein aggregates. These results provide the first genetic evidence for a function of Vps15 in autophagy in multicellular organisms and suggest that the Vps15-containing PI 3-kinase complex may play an important role in clearance of protein aggregates.

An arginine/lysine-rich motif is crucial for VCP/p97-mediated modulation of ataxin-3 fibrillogenesis.

Arginine/lysine-rich motifs typically function as targeting signals for the translocation of proteins to the nucleus. Here, we demonstrate that such a motif consisting of four basic amino acids in the polyglutamine protein ataxin-3 (Atx-3) serves as a recognition site for the interaction with the molecular chaperone VCP. Through this interaction, VCP modulates the fibrillogenesis of pathogenic forms of Atx-3 in a concentration-dependent manner, with low concentrations of VCP stimulating fibrillogenesis and excess concentrations suppressing it. No such effect was observed with a mutant Atx-3 variant, which does not contain a functional VCP interaction motif. Strikingly, a stretch of four basic amino acids in the ubiquitin chain assembly factor E4B was also discovered to be critical for VCP binding, indicating that arginine/lysine-rich motifs might be generally utilized by VCP for the targeting of proteins. In vivo studies with Drosophila models confirmed that VCP selectively modulates aggregation and neurotoxicity induced by pathogenic Atx-3. Together, these results define the VCP-Atx-3 association as a potential target for therapeutic intervention and suggest that it might influence the progression of spinocerebellar ataxia type 3.

reaper and bax initiate two different apoptotic pathways affecting mitochondria and antagonized by bcl-2 in Drosophila.

bcl-2 was the first regulator of apoptosis shown to be involved in oncogenesis. Subsequent studies in mammals, in the nematode and in Drosophila revealed wide evolutionary conservation of the regulation of apoptosis. Although dbok/debcl, a member of the bcl-2 gene family described in Drosophila, shows pro-apoptotic activities, no anti-apoptotic bcl-2 family gene has been studied in Drosophila. We have previously reported that the human anti-apoptotic gene bcl-2 is functional in Drosophila, suggesting that the fruit fly shares regulatory mechanisms with vertebrates and the nematode, involving anti-apoptotic members of the bcl-2 family. We now report that bcl-2 suppresses rpr-induced apoptosis in Drosophila. Additionally, we have compared features of bax- and rpr-induced apoptosis. Flow cytometry analysis of wing disc cells demonstrate that both killers trigger mitochondrial defects. Interestingly, bcl-2 suppresses both bax- and rpr-induced mitochondrial defects while the caspase-inhibitor p35 is specific to the rpr pathway. Finally, we show that the inhibition of apoptosis by bcl-2 is associated with the down-regulation of rpr expression.