Essential role of hyperacetylated microtubules in innate immunity escape orchestrated by the EBV-encoded BHRF1 protein.

Innate immunity constitutes the first line of defense against viruses, in which mitochondria play an important role in the induction of the interferon (IFN) response. BHRF1, a multifunctional viral protein expressed during Epstein-Barr virus reactivation, modulates mitochondrial dynamics and disrupts the IFN signaling pathway. Mitochondria are mobile organelles that move through the cytoplasm thanks to the cytoskeleton and in particular the microtubule (MT) network. MTs undergo various post-translational modifications, among them tubulin acetylation. In this study, we demonstrated that BHRF1 induces MT hyperacetylation to escape innate immunity. Indeed, the expression of BHRF1 induces the clustering of shortened mitochondria next to the nucleus. This "mito-aggresome" is organized around the centrosome and its formation is MT-dependent. We also observed that the α-tubulin acetyltransferase ATAT1 interacts with BHRF1. Using ATAT1 knockdown or a non-acetylatable α-tubulin mutant, we demonstrated that this hyperacetylation is necessary for the mito-aggresome formation. Similar results were observed during EBV reactivation. We investigated the mechanism leading to the clustering of mitochondria, and we identified dyneins as motors that are required for mitochondrial clustering. Finally, we demonstrated that BHRF1 needs MT hyperacetylation to block the induction of the IFN response. Moreover, the loss of MT hyperacetylation blocks the localization of autophagosomes close to the mito-aggresome, impeding BHRF1 to initiate mitophagy, which is essential to inhibiting the signaling pathway. Therefore, our results reveal the role of the MT network, and its acetylation level, in the induction of a pro-viral mitophagy.

Reactive oxygen species, AMP-activated protein kinase, and the transcription cofactor p300 regulate α-tubulin acetyltransferase-1 (αTAT-1/MEC-17)-dependent microtubule hyperacetylation during cell stress.

Beyond its presence in stable microtubules, tubulin acetylation can be boosted after UV exposure or after nutrient deprivation, but the mechanisms of microtubule hyperacetylation are still unknown. In this study, we show that this hyperacetylation is a common response to several cellular stresses that involves the stimulation of the major tubulin acetyltransferase MEC-17. We also demonstrate that the acetyltransferase p300 negatively regulates MEC-17 expression and is sequestered on microtubules upon stress. We further show that reactive oxygen species of mitochondrial origin are required for microtubule hyperacetylation by activating the AMP kinase, which in turn mediates MEC-17 phosphorylation upon stress. Finally, we show that preventing microtubule hyperacetylation by knocking down MEC-17 affects cell survival under stress conditions and starvation-induced autophagy, thereby pointing out the importance of this rapid modification as a broad cell response to stress.

Autophagy and microtubules - new story, old players.

Both at a basal level and after induction (especially in response to nutrient starvation), the function of autophagy is to allow cells to degrade and recycle damaged organelles, proteins and other biological constituents. Here, we focus on the role microtubules have in autophagosome formation, autophagosome transport across the cytoplasm and in the formation of autolysosomes. Recent insights into the exact relationship between autophagy and microtubules now point to the importance of microtubule dynamics, tubulin post-translational modifications and microtubule motors in the autophagy process. Such factors regulate signaling pathways that converge to stimulate autophagosome formation. They also orchestrate the movements of pre-autophagosomal structures and autophagosomes or more globally organize and localize immature and mature autophagosomes and lysosomes. Most of the factors that now appear to link microtubules to autophagosome formation or to autophagosome dynamics and fate were identified initially without the notion that sequestration, recruitment and/or interaction with microtubules contribute to their function. Spatial and temporal coordination of many stages in the life of autophagosomes thus underlines the integrative role of microtubules and progressively reveals hidden parts of the autophagy machinery.

Starvation-induced hyperacetylation of tubulin is required for the stimulation of autophagy by nutrient deprivation.

The molecular mechanisms underlying microtubule participation in autophagy are not known. In this study, we show that starvation-induced autophagosome formation requires the most dynamic microtubule subset. Upon nutrient deprivation, labile microtubules specifically recruit markers of autophagosome formation like class III-phosphatidylinositol kinase, WIPI-1, the Atg12-Atg5 conjugate, and LC3-I, whereas mature autophagosomes may bind to stable microtubules. We further found that upon nutrient deprivation, tubulin acetylation increases both in labile and stable microtubules and is required to allow autophagy stimulation. Tubulin hyperacetylation on lysine 40 enhances kinesin-1 and JIP-1 recruitment on microtubules and allows JNK phosphorylation and activation. JNK, in turn, triggers the release of Beclin 1 from Bcl-2-Beclin 1 complexes and its recruitment on microtubules where it may initiate autophagosome formation. Finally, although kinesin-1 functions to carry autophagosomes in basal conditions, it is not involved in motoring autophagosomes after nutrient deprivation. Our results show that the dynamics of microtubules and tubulin post-translational modifications play a major role in the regulation of starvation-induced autophagy.

BHRF1, a BCL2 viral homolog, disturbs mitochondrial dynamics and stimulates mitophagy to dampen type I IFN induction.

Mitochondria respond to many cellular functions and act as central hubs in innate immunity against viruses. This response is notably due to their role in the activation of interferon (IFN) signaling pathways through the activity of MAVS (mitochondrial antiviral signaling protein) present at the mitochondrial surface. Here, we report that the BHRF1 protein, a BCL2 homolog encoded by Epstein-Barr virus (EBV), inhibits IFNB/IFN-ß induction by targeting the mitochondria. Indeed, we have demonstrated that BHRF1 expression modifies mitochondrial dynamics and stimulates DNM1L/Drp1-mediated mitochondrial fission. Concomitantly, we have shown that BHRF1 is pro-autophagic because it stimulates the autophagic flux by interacting with BECN1/Beclin 1. In response to the BHRF1-induced mitochondrial fission and macroautophagy/autophagy stimulation, BHRF1 drives mitochondrial network reorganization to form juxtanuclear mitochondrial aggregates known as mito-aggresomes. Mitophagy is a cellular process, which can specifically sequester and degrade mitochondria. Our confocal studies uncovered that numerous mitochondria are present in autophagosomes and acidic compartments using BHRF1-expressing cells. Moreover, mito-aggresome formation allows the induction of mitophagy and the accumulation of PINK1 at the mitochondria. As BHRF1 modulates the mitochondrial fate, we explored the effect of BHRF1 on innate immunity and showed that BHRF1 expression could prevent IFNB induction. Indeed, BHRF1 inhibits the IFNB promoter activation and blocks the nuclear translocation of IRF3 (interferon regulatory factor 3). Thus, we concluded that BHRF1 can counteract innate immunity activation by inducing fission of the mitochondria to facilitate their sequestration in mitophagosomes for degradation.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; BCL2: BCL2 apoptosis regulator; CARD: caspase recruitment domain; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CI: compaction index; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole, dihydrochloride; DDX58/RIG-I: DExD/H-box helicase 58; DNM1L/Drp1: dynamin 1 like; EBSS: Earle's balanced salt solution; EBV: Epstein-Barr virus; ER: endoplasmic reticulum; EV: empty vector; GFP: green fluorescent protein; HEK: human embryonic kidney; IFN: interferon; IgG: immunoglobulin G; IRF3: interferon regulatory factor 3; LDHA: lactate dehydrogenase A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MMP: mitochondrial membrane potential; MOM: mitochondrial outer membrane; PINK1: PTEN induced kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.

Early mitochondrial fragmentation is a potential in vitro biomarker of environmental stress.

Mitochondria are essential dynamic organelles that ordinarily balance between fragmentation and fusion. Under stress conditions, a shift toward fragmentation or hyper-fusion is observed as a pro-survival reaction. Fragmentation of mitochondria occurs within minutes or hours after the beginning of the stress and occurs in response to a large number of stress stimuli, including those triggered by environmental contaminants. In this study, we tested whether the change in the mitochondrial phenotype, from tubular to fragmented, could be used as a potential environmental stress biomarker in cells and compared this response with the standard MTT-based viability assay. Firstly, we show that mitochondrial fragmentation induced by selected stressors not only increases with concentrations, but also correlates positively with the cytotoxicity. Secondly, we found that the mitochondrial fragmentation that occurs in the first hour of stress correlated with the viability measured after a 24-h stress, allowing the establishment of a linear relation between mitochondrial fragmentation at 1 h and the predictable associated cytotoxicity of environmental contaminants alone or in mixture. In conclusion, we have succeeded in developing a model of predictable 24 h-cytotoxicity given mitochondrial fragmentation at 1 h with a set of chemicals. This model has been successful applied to three environmental toxicants and to a set of two chemical mixtures. We thus propose that mitochondrial fragmentation is a response that could be used as an early in vitro biomarker of environmental stress for toxicants alone or in mixture.

Stress-induced hyperacetylation of microtubule enhances mitochondrial fission and modulates the phosphorylation of Drp1 at 616Ser.

Mitochondria dynamics results from fission and fusion events that may be unbalanced in favor of mitochondrial fragmentation upon cell stress. During oxidative stress, microtubules are hyperacetylated in a mitochondria-dependent manner. In this study, we show that under stress conditions, most of the mitochondria form foci with microtubule domains that carry Drp1. We also demonstrate that stress-induced hyperacetylation of microtubules is required for the effective induction of Drp1 phosphorylation at 616Ser, in a kinesin-1- and c-Jun N-terminal kinase-dependent manner. Furthermore, hyperacetylation of microtubules contributes to the recruitment of total Drp1 to mitochondria to enhance fission. These results highlight a new way of interaction between microtubules and mitochondria dynamics.

UVA-induced cyclobutane pyrimidine dimers form predominantly at thymine-thymine dipyrimidines and correlate with the mutation spectrum in rodent cells.

Ligation-mediated PCR was employed to quantify cyclobutane pyrimidine dimer (CPD) formation at nucleotide resolution along exon 2 of the adenine phosphoribosyltransferase (aprt) locus in Chinese hamster ovary (CHO) cells following irradiation with either UVA (340-400 nm), UVB (295-320 nm), UVC (254 nm) or simulated sunlight (SSL; lambda > 295 nm). The resulting DNA damage spectrum for each wavelength region was then aligned with the corresponding mutational spectrum generated previously in the same genetic target. The DNA sequence specificities of CPD formation induced by UVC, UVB or SSL were very similar, i.e., in each case the overall relative proportion of this photoproduct forming at TT, TC, CT and CC sites was approximately 28, approximately 26, approximately 16 and approximately 30%, respectively. Furthermore, a clear correspondence was noted between the precise locations of CPD damage hotspots, and of 'UV signature' mutational hotspots consisting primarily of C-->T and CC-->TT transitions within pyrimidine runs. However, following UVA exposure, in strong contrast to the above situation for UVC, UVB or SSL, CPDs were generated much more frequently at TT sites than at TC, CT or CC sites (57% versus 18, 11 and 14%, respectively). This CPD deposition pattern correlates well with the strikingly high proportion of mutations recovered opposite TT dipyrimidines in UVA- irradiated CHO cells. Our results directly implicate the CPD as a major promutagenic DNA photoproduct induced specifically by UVA in rodent cells.

Xenoestrogens modulate genotoxic (UVB)-induced cellular responses in estrogen receptors positive human breast cancer cells.

Human populations and wildlife are exposed to mixtures of both anthropogenic and natural chemicals. Some of these compounds are known to interact principally with the endocrine function, whereas others act mainly on genomic DNA. Given this evidence, we wanted to address the question of whether concomitant exposure of such chemicals was able to interact at the cellular level. We have previously shown that 17ß-Estradiol (E(2)) modulates the DNA repair capacity of cells. In this work, we wanted to examine if other xenoestrogens (i.e. industrial compounds, pesticides or pharmaceuticals) were able to interact with the UVB-induced cellular response as E(2) does. Here, we show that xenoestrogens modulate the capacity of cells to repair their DNA damage according to the type of compounds. For example, the oral contraceptive 17α-Ethinylestradiol down-regulated the repair of UVB-induced DNA damage whereas the UV filter Eusolex 6007 up-regulated this pathway. The notion that xenoestrogens could interact with a genotoxic stress is reinforced by the modulation of the estrogens-dependent luciferase reporter gene expression when cells are UVB-irradiated. Finally, these observations suggested the potential role of xenoestrogens in carcinogenesis by their capacity to modulate cells responses to genotoxic stress.

Assessment of river contamination by estrogenic compounds in Paris area (France).

Wastewater treatment plants (WWTPs) receive a large spectrum of endocrine disrupting compounds (EDCs) that are partially eliminated during treatment processes and discharged into rivers. Given the lack of information in France about river contamination by EDCs, we chose to examine estrogenic potential of WWTP influents, effluents and receiving waters in Paris and its suburbs. Water samples were analyzed using gas chromatography coupled with mass spectrometry for quantifying natural and synthetic estrogens combined with an in vitro estrogenicity bioassay associated to a high pressure liquid chromatography fractionation. The four estrogens investigated, Estrone (E1), 17beta-Estradiol (E2), Estriol (E3) and 17alpha-Ethinylestradiol (EE2) were found in all WWTP and river samples at concentrations ranging from 2.7 to 17.6 ng/l and 1.0 to 3.2 ng/l, respectively. The synthetic estrogen EE2 seems more resistant to biodegradation in WWTPs and thus accounted for 35-50% of the estimated estrogenic activity in rivers. However, fractionation of samples and differences between concentrations of E1, E2, E3 and EE2 and the estrogenic activity measured by the in vitro bioassay suggested a complexity of mechanisms underlying the biological response that could not be attributed only to the investigated molecules.

The ins and outs of tubulin acetylation: more than just a post-translational modification?

Microtubules are highly dynamic polymers of α/ß tubulin heterodimers that play key roles in cell division and in organizing cell cytoplasm. Although they have been discovered more than two decades ago, tubulin post-translational modifications recently gained a new interest as their role was increasingly highlighted in neuron differentiation and neurodegenerative disorders. Here, we specifically focus on tubulin acetylation from its discovery to recent studies that provide new insights into how it is regulated in health and disease and how it impacts microtubule functions. Even though new mechanisms involving tubulin acetylation are regularly being uncovered, the molecular links between its location inside the microtubule lumen and its regulators and effectors is still poorly understood. This review highlights the emerging roles of tubulin acetylation in multiple cellular functions, ranging from cell motility, cell cycle progression or cell differentiation to intracellular trafficking and signalling. It also points out that tubulin acetylation should no longer be seen as a passive marker of microtubule stability, but as a broad regulator of microtubule functions.

Tubulin acetylation favors Hsp90 recruitment to microtubules and stimulates the signaling function of the Hsp90 clients Akt/PKB and p53.

Involved in a wide range of cellular processes such as signal transduction, microtubules are highly dynamic polymers that accumulate various post-translational modifications including polyglutamylation, polyglycylation, carboxyterminal cleavage and acetylation, the functions of which just begin to be uncovered. The molecular chaperone Hsp90, which is essential for the folding and activity of numerous client proteins involved in cell proliferation and apoptosis, associates with the microtubule network but the effects of tubulin post-translational modifications on its microtubule binding has not yet been investigated. Herein, we show that both the constitutive (beta) and the inducible (alpha) Hsp90 isoforms bind to microtubules in a way that depends on the level of tubulin acetylation. Tubulin acetylation also stimulates the binding and the signaling function of at least two of its client proteins, the kinase Akt/PKB and the transcription factor p53. This study highlights the role of tubulin acetylation in modulating microtubule-based transport of Hsp90-chaperoned proteins and thus in regulating signaling dynamics in the cytoplasm.

Evaluation of the estrogenic potential of river and treated waters in the Paris area (France) using in vivo and in vitro assays.

For many years, surface waters have been shown to be contaminated by endocrine-disrupting compounds (EDCs), which can cause adverse effects on human and wildlife growth, development, and reproduction. It is therefore of primary importance to determine if drinking water could be contaminated by EDCs when produced from polluted surface waters. It is also essential to determine if disinfection by-products can account for estrogenic activity in treated waters. The estrogenic potential of river and treated waters was investigated using an in vivo assay. Adult male zebrafish were placed in three drinking water treatment plants (DWTPs) in the Paris area and exposed for 1 month to the two types of waters. After exposure, vitellogenin (VTG) was measured in the plasma of fish using a competitive ELISA. In addition, an in vitro assay (MELN cells) was used to assess the estrogenic potential of 10 major chlorination by-products. No significant induction of VTG was observed in fish exposed to river or treated waters. Among the 10 chlorination by-products tested, only 2-chlorophenol was found to be weakly estrogenic at concentrations up to 1mg/L. Therefore, the risk for the three DWTPs studied to produce drinking water with significant level of estrogenic substances appears to be low.