A High-Throughput Search for SFXN1 Physical Partners Led to the Identification of ATAD3, HSD10 and TIM50.

Sideroflexins (SFXN, SLC56) are a family of evolutionarily conserved mitochondrial carriers potentially involved in iron homeostasis. One member of the SFXN family is SFXN1, recently identified as a human mitochondrial serine transporter. However, little is known about the SFXN1 interactome, necessitating a high-throughput search to better characterize SFXN1 mitochondrial functions. Via co-immunoprecipitation followed by shotgun mass spectrometry (coIP-MS), we identified 96 putative SFXN1 interactors in the MCF7 human cell line. Our in silico analysis of the SFXN1 interactome highlights biological processes linked to mitochondrial organization, electron transport chains and transmembrane transport. Among the potential physical partners, ATAD3A and 17ß-HSD10, two proteins associated with neurological disorders, were confirmed using different human cell lines. Nevertheless, further work will be needed to investigate the significance of these interactions.

Keeping Cell Death Alive: An Introduction into the French Cell Death Research Network.

Since the Nobel Prize award more than twenty years ago for discovering the core apoptotic pathway in C. elegans, apoptosis and various other forms of regulated cell death have been thoroughly characterized by researchers around the world. Although many aspects of regulated cell death still remain to be elucidated in specific cell subtypes and disease conditions, many predicted that research into cell death was inexorably reaching a plateau. However, this was not the case since the last decade saw a multitude of cell death modalities being described, while harnessing their therapeutic potential reached clinical use in certain cases. In line with keeping research into cell death alive, francophone researchers from several institutions in France and Belgium established the French Cell Death Research Network (FCDRN). The research conducted by FCDRN is at the leading edge of emerging topics such as non-apoptotic functions of apoptotic effectors, paracrine effects of cell death, novel canonical and non-canonical mechanisms to induce apoptosis in cell death-resistant cancer cells or regulated forms of necrosis and the associated immunogenic response. Collectively, these various lines of research all emerged from the study of apoptosis and in the next few years will increase the mechanistic knowledge into regulated cell death and how to harness it for therapy.

Insights into the Roles of the Sideroflexins/SLC56 Family in Iron Homeostasis and Iron-Sulfur Biogenesis.

Sideroflexins (SLC56 family) are highly conserved multi-spanning transmembrane proteins inserted in the inner mitochondrial membrane in eukaryotes. Few data are available on their molecular function, but since their first description, they were thought to be metabolite transporters probably required for iron utilization inside the mitochondrion. Such as numerous mitochondrial transporters, sideroflexins remain poorly characterized. The prototypic member SFXN1 has been recently identified as the previously unknown mitochondrial transporter of serine. Nevertheless, pending questions on the molecular function of sideroflexins remain unsolved, especially their link with iron metabolism. Here, we review the current knowledge on sideroflexins, their presumed mitochondrial functions and the sparse-but growing-evidence linking sideroflexins to iron homeostasis and iron-sulfur cluster biogenesis. Since an imbalance in iron homeostasis can be detrimental at the cellular and organismal levels, we also investigate the relationship between sideroflexins, iron and physiological disorders. Investigating Sideroflexins' functions constitutes an emerging research field of great interest and will certainly lead to the main discoveries of mitochondrial physio-pathology.

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.

FGF1 induces resistance to chemotherapy in ovarian granulosa tumor cells through regulation of p53 mitochondrial localization.

Ovarian cancer remains associated with a high mortality rate and relapse is too frequently seen after chemotherapeutic treatment of granulosa cell tumors (GCTs) or epithelial ovarian cancers (EOCs). It is thus of major importance to progress in the knowledge of the molecular mechanisms underlying chemoresistance of ovarian tumors. Overexpression of Fibroblast Growth Factor 1 (FGF1) is observed in various cancers, correlates with poor survival and could be responsible for resistance to platinum-based chemotherapy of serous ovarian cancers. How FGF1 promotes escape to chemotherapy remains unknown. In previous studies, we showed that FGF1 inhibits p53 transcriptional activities, leading to increased cell survival of neuronal or fibroblast cell lines. In this study, we show that FGF1 favors survival of COV434 cells upon treatment with etoposide and cisplatin, two common chemotherapeutic molecules used for ovarian cancer. Etoposide and cisplatin induced mitochondrial depolarization, cytochrome c release and caspase activation in COV434 cells. Overexpression of FGF1 counteracts these events and thus allows increased survival of ovarian cells. In this study, FGF1 had little effect on p53 stability and transcriptional activities. Etoposide induced p21 expression as expected, but p21 protein levels were even increased in the presence of FGF1. Using RNA interference, we showed that p21 exerts an anti-apoptotic activity in COV434 cells. However abrogating this activity was not sufficient to restore cell death of FGF1-overexpressing cells. We also show for the first time that p53 mitochondrial pathway is involved in the cell death of COV434 cells. Indeed, p53 accumulates at mitochondria upon etoposide treatment and inhibition of p53 mitochondrial localization using pifithrin-µ inhibits apoptosis of COV434 cells. FGF1 also decreases mitochondrial accumulation of p53 induced by etoposide. This constitutes a novel mechanism of action for FGF1 to promote cell survival in response to chemotherapy.

FGF1 protects neuroblastoma SH-SY5Y cells from p53-dependent apoptosis through an intracrine pathway regulated by FGF1 phosphorylation.

Neuroblastoma, a sympathetic nervous system tumor, accounts for 15% of cancer deaths in children. In contrast to most human tumors, p53 is rarely mutated in human primary neuroblastoma, suggesting impaired p53 activation in neuroblastoma. Various studies have shown correlations between fgf1 expression levels and both prognosis severity and tumor chemoresistance. As we previously showed that fibroblast growth factor 1 (FGF1) inhibited p53-dependent apoptosis in neuron-like PC12 cells, we initiated the study of the interaction between the FGF1 and p53 pathways in neuroblastoma. We focused on the activity of either extracellular FGF1 by adding recombinant rFGF1 in media, or of intracellular FGF1 by overexpression in human SH-SY5Y and mouse N2a neuroblastoma cell lines. In both cell lines, the genotoxic drug etoposide induced a classical mitochondrial p53-dependent apoptosis. FGF1 was able to inhibit p53-dependent apoptosis upstream of mitochondrial events in SH-SY5Y cells by both extracellular and intracellular pathways. Both rFGF1 addition and etoposide treatment increased fgf1 expression in SH-SY5Y cells. Conversely, rFGF1 or overexpressed FGF1 had no effect on p53-dependent apoptosis and fgf1 expression in neuroblastoma N2a cells. Using different FGF1 mutants (that is, FGF1K132E, FGF1S130A and FGF1S130D), we further showed that the C-terminal domain and phosphorylation of FGF1 regulate its intracrine anti-apoptotic activity in neuroblastoma SH-SY5Y cells. This study provides the first evidence for a role of an intracrine growth factor pathway on p53-dependent apoptosis in neuroblastoma, and could lead to the identification of key regulators involved in neuroblastoma tumor progression and chemoresistance.

Evolutionary conservation of Notch signaling inhibition by TMEM131L overexpression.

Human KIAA0922/TMEM131L encodes a transmembrane protein, TMEM131L, that regulates the canonical Wnt/ß-catenin signaling pathway by eliciting the lysosome-dependent degradation of phosphorylated LRP6 co-receptor. Here, we use a heterospecific Drosophila transgenic model to examine the potential evolutionary conservation of TMEM131L function. Analysis of TMEM131L transgenic flies shows that TMEM131L interference with the Wnt pathway results primarily from a Notch-dependent decrease in Wingless production. Consistently, lentivirus-mediated overexpression of TMEM131L in human CD34+ hematopoietic progenitor cells leads to decreased susceptibility to Notch1 ligation and defective commitment toward the T lineage. These results show that TMEM131L corresponds to an evolutionary conserved regulator of the Notch signaling pathway.

[The comeback of mitochondria in Drosophila apoptosis]. / Le come-back de la mitochondrie dans l'apoptose chez la drosophile.

The role of the mitochondrion in mammalian cell apoptosis has been established since the mid-1990s. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, notably because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and apoptosis in Drosophila cell death occurs at the mitochondrial level. Numerous proteins that appear key for Drosophila apoptosis regulation constitutively or transiently bind to mitochondria. They participate in the cell death process at different levels such as degradation of an IAP caspase inhibitor, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. The aim of this review is to take stock of these events that might have their counterpart in humans.

Two different specific JNK activators are required to trigger apoptosis or compensatory proliferation in response to Rbf1 in Drosophila.

The Jun Kinase (JNK) signaling pathway responds to diverse stimuli by appropriate and specific cellular responses such as apoptosis, differentiation or proliferation. The mechanisms that mediate this specificity remain largely unknown. The core of this signaling pathway, composed of a JNK protein and a JNK kinase (JNKK), can be activated by various putative JNKK kinases (JNKKK) which are themselves downstream of different adaptor proteins. A proposed hypothesis is that the JNK pathway specific response lies in the combination of a JNKKK and an adaptor protein upstream of the JNKK. We previously showed that the Drosophila homolog of pRb (Rbf1) and a mutant form of Rbf1 (Rbf1(D253A)) have JNK-dependent pro-apoptotic properties. Rbf1(D253A) is also able to induce a JNK-dependent abnormal proliferation. Here, we show that Rbf1-induced apoptosis triggers proliferation which depends on the JNK pathway activation. Taking advantage of these phenotypes, we investigated the JNK signaling involved in either Rbf1-induced apoptosis or in proliferation in response to Rbf1-induced apoptosis. We demonstrated that 2 different JNK pathways involving different adaptor proteins and kinases are involved in Rbf1-apoptosis (i.e. Rac1-dTak1-dMekk1-JNK pathway) and in proliferation in response to Rbf1-induced apoptosis (i.e., dTRAF1-Slipper-JNK pathway). Using a transient induction of rbf1, we show that Rbf1-induced apoptosis activates a compensatory proliferation mechanism which also depends on Slipper and dTRAF1. Thus, these 2 proteins seem to be key players of compensatory proliferation in Drosophila.

Apoptosis in Drosophila: which role for mitochondria?

It is now well established that the mitochondrion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human.

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.

The Drosophila retinoblastoma protein, Rbf1, induces a Debcl- and Drp1-dependent mitochondrial apoptosis.

In accordance with its tumor suppressor role, the retinoblastoma protein pRb can ensure pro-apoptotic functions. Rbf1, the Drosophila homolog of Rb, also displays a pro-apoptotic activity in proliferative cells. We have previously shown that the Rbf1 pro-apoptotic activity depends on its ability to decrease the level of anti-apoptotic proteins such as the Bcl-2 family protein Buffy. Buffy often acts in an opposite manner to Debcl, the other Drosophila Bcl-2-family protein. Both proteins can localize at the mitochondrion, but the way they control apoptosis still remains unclear. Here, we demonstrate that Debcl and the pro-fission gene Drp1 are necessary downstream of Buffy to trigger a mitochondrial fragmentation during Rbf1-induced apoptosis. Interestingly, Rbf1-induced apoptosis leads to a Debcl- and Drp1-dependent reactive oxygen species production, which in turn activates the Jun Kinase pathway to trigger cell death. Moreover, we show that Debcl and Drp1 can interact and that Buffy inhibits this interaction. Notably, Debcl modulates Drp1 mitochondrial localization during apoptosis. These results provide a mechanism by which Drosophila Bcl-2 family proteins can control apoptosis, and shed light on a link between Rbf1 and mitochondrial dynamics in vivo.

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.

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.

Mutating RBF can enhance its pro-apoptotic activity and uncovers a new role in tissue homeostasis.

The tumor suppressor retinoblastoma protein (pRb) is inactivated in a wide variety of cancers. While its role during cell cycle is well characterized, little is known about its properties on apoptosis regulation and apoptosis-induced cell responses. pRb shorter forms that can modulate pRB apoptotic properties, resulting from cleavages at caspase specific sites are observed in several cellular contexts. A bioinformatics analysis showed that a putative caspase cleavage site (TELD) is found in the Drosophila homologue of pRb(RBF) at a position similar to the site generating the p76Rb form in mammals. Thus, we generated a punctual mutant form of RBF in which the aspartate of the TELD site is replaced by an alanine. This mutant form, RBFD253A, conserved the JNK-dependent pro-apoptotic properties of RBF but gained the ability of inducing overgrowth phenotypes in adult wings. We show that this overgrowth is a consequence of an abnormal proliferation in wing imaginal discs, which depends on the JNK pathway activation but not on wingless (wg) ectopic expression. These results show for the first time that the TELD site of RBF could be important to control the function of RBF in tissue homeostasis in vivo.

Mitochondrial p53 mediates a transcription-independent regulation of cell respiration and interacts with the mitochondrial F1F0-ATP synthase.

We and others previously reported that endogenous p53 can be located at mitochondria in the absence of stress, suggesting that p53 has a role in the normal physiology of this organelle. The aim of this study was to characterize in unstressed cells the intramitochondrial localization of p53 and identify new partners and functions of p53 in mitochondria. We find that the intramitochondrial pool of p53 is located in the intermembrane space and the matrix. Of note, unstressed HCT116 p53(+/+) cells simultaneously show increased O2 consumption and decreased mitochondrial superoxide production compared with their p53-null counterpart. This data was confirmed by stable H1299 cell lines expressing low levels of p53 specifically targeted to the matrix. Using immunoprecipitation and mass spectrometry, we identified the oligomycin sensitivity-conferring protein (OSCP), a subunit of the F1F0-ATP synthase complex, as a new partner of endogenous p53, specifically interacting with p53 localized in the matrix. Interestingly, this interaction seems implicated in mitochondrial p53 localization. Moreover, p53 localized in the matrix promotes the assembly of F1F0-ATP synthase. Taking into account that deregulations of mitochondrial respiration and reactive oxygen species production are tightly linked to cancer development, we suggest that mitochondrial p53 may be an important regulator of normal mitochondrial and cellular physiology, potentially exerting tumor suppression activity inside mitochondria.

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.