Critical steps in the assembly process of the bacterial 50S ribosomal subunit.

During assembly, ribosomal particles in bacteria fold according to energy landscapes comprised of multiple parallel pathways. Cryo-electron microscopy studies have identified a critical maturation step that occurs during the late assembly stages of the 50S subunit in Bacillus subtilis. This step acts as a point of convergency for all the parallel assembly pathways of the subunit, where an assembly intermediate accumulates in a 'locked' state, causing maturation to pause. Assembly factors then act on this critical step to 'unlock' the last maturation steps involving the functional sites. Without these factors, the 50S subunit fails to complete its assembly, causing cells to die due to a lack of functional ribosomes to synthesize proteins. In this review, we analyze these findings in B. subtilis and examine other cryo-EM studies that have visualized assembly intermediates in different bacterial species, to determine if convergency points in the ribosome assembly process are a common theme among bacteria. There are still gaps in our knowledge, as these methodologies have not yet been applied to diverse species. However, identifying and characterizing these convergency points can reveal how different bacterial species implement unique mechanisms to regulate critical steps in the ribosome assembly process.

Respiratory Vaccination with Hemagglutinin Nanoliposomes Protects Mice from Homologous and Heterologous Strains of Influenza Virus.

Intranasal vaccination offers the potential advantage of needle-free prevention of respiratory pathogens such as influenza viruses with induction of mucosal immune responses. Optimal design of adjuvants and antigen delivery vehicles for intranasal delivery has not yet been well established. Here, we report that an adjuvant-containing nanoliposome antigen display system that converts soluble influenza hemagglutinin antigens into nanoparticles is effective for intranasal immunization. Intranasal delivery of nanoliposomes in mice delivers the particles to resident immune cells in the respiratory tract, inducing a mucosal response in the respiratory system as evidenced by nasal and lung localized IgA antibody production, while also producing systemic IgG antibodies. Intranasal vaccination with nanoliposome particles decorated with nanogram doses of hemagglutinin protected mice from homologous and heterologous H3N2 and H1N1 influenza virus challenge. IMPORTANCE A self-assembling influenza virus vaccine platform that seamlessly converts soluble antigens into nanoparticles is demonstrated with various H1N1 and H3N2 influenza antigens to protect mice against influenza virus challenge following intranasal vaccination. Mucosal immune responses following liposome delivery to lung antigen-presenting cells are demonstrated.

Extracellular endosulfatase Sulf-2 harbors a chondroitin/dermatan sulfate chain that modulates its enzyme activity.

Sulfs represent a class of unconventional sulfatases which provide an original post-synthetic regulatory mechanism for heparan sulfate polysaccharides and are involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivo functions. Here we show that human Sulf-2 (HSulf-2) harbors a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate-binding domain. We demonstrate that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitro and in vivo. In addition, we show that mammalian hyaluronidase acts as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a proteoglycan-related enzyme and its GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute to clarifying the conflicting data on the activities of the Sulfs.

RbgA ensures the correct timing in the maturation of the 50S subunits functional sites.

RbgA is an essential protein for the assembly of the 50S subunit in Bacillus subtilis. Depletion of RbgA leads to the accumulation of the 45S intermediate. A strain expressing a RbgA variant with reduced GTPase activity generates spontaneous suppressor mutations in uL6. Each suppressor strain accumulates a unique 44S intermediate. We reasoned that characterizing the structure of these mutant 44S intermediates may explain why RbgA is required to catalyze the folding of the 50S functional sites. We found that in the 44S particles, rRNA helices H42 and H97, near the binding site of uL6, adopt a flexible conformation and allow the central protuberance and functional sites in the mutant 44S particles to mature in any order. Instead, the wild-type 45S particles exhibit a stable H42-H97 interaction and their functional sites always mature last. The dependence on RbgA was also less pronounced in the 44S particles. We concluded that the binding of uL6 pauses the maturation of the functional sites, but the central protuberance continues to fold. RbgA exclusively binds intermediates with a formed central protuberance and licenses the folding of the functional sites. Through this mechanism, RbgA ensures that the functional sites of the 50S mature last.

Ribosomal subunits in bacteria assemble according to energy landscapes comprised of multiple parallel pathways. In this study, the authors identified a critical maturation step in the late assembly stages of the large 50S ribosomal subunit in bacteria. This step represents a merging point where all parallel assembly pathways of the ribosomal particles converge. At this critical step, the convergent assembly intermediate that accumulates in cells exists in a 'locked' state, and its maturation is paused. The RbgA protein acts on this critical step to 'unlock' the last maturation steps involving folding of the functional sites. Through this mechanism, RbgA ensures that the functional sites of the 50S mature last.

A liposome-displayed hemagglutinin vaccine platform protects mice and ferrets from heterologous influenza virus challenge.

Recombinant influenza virus vaccines based on hemagglutinin (HA) hold the potential to accelerate production timelines and improve efficacy relative to traditional egg-based platforms. Here, we assess a vaccine adjuvant system comprised of immunogenic liposomes that spontaneously convert soluble antigens into a particle format, displayed on the bilayer surface. When trimeric H3 HA was presented on liposomes, antigen delivery to macrophages was improved in vitro, and strong functional antibody responses were induced following intramuscular immunization of mice. Protection was conferred against challenge with a heterologous strain of H3N2 virus, and naive mice were also protected following passive serum transfer. When admixed with the particle-forming liposomes, immunization reduced viral infection severity at vaccine doses as low as 2 ng HA, highlighting dose-sparing potential. In ferrets, immunization induced neutralizing antibodies that reduced the upper respiratory viral load upon challenge with a more modern, heterologous H3N2 viral strain. To demonstrate the flexibility and modular nature of the liposome system, 10 recombinant surface antigens representing distinct influenza virus strains were bound simultaneously to generate a highly multivalent protein particle that with 5 ng individual antigen dosing induced antibodies in mice that specifically recognized the constituent immunogens and conferred protection against heterologous H5N1 influenza virus challenge. Taken together, these results show that stable presentation of recombinant HA on immunogenic liposome surfaces in an arrayed fashion enhances functional immune responses and warrants further attention for the development of broadly protective influenza virus vaccines.

A Potent Cancer Vaccine Adjuvant System for Particleization of Short, Synthetic CD8+ T Cell Epitopes.

Short major histocompatibility complex (MHC) class I (MHC-I)-restricted peptides contain the minimal biochemical information to induce antigen (Ag)-specific CD8+ cytotoxic T cell responses but are generally ineffective in doing so. To address this, we developed a cobalt-porphyrin (CoPoP) liposome vaccine adjuvant system that induces rapid particleization of conventional, short synthetic MHC-I epitopes, leading to strong cellular immune responses at nanogram dosing. Along with CoPoP (to induce particle formation of peptides), synthetic monophosphoryl lipid A (PHAD) and QS-21 immunostimulatory molecules were included in the liposome bilayer to generate the "CPQ" adjuvant system. In mice, immunization with a short MHC-I-restricted peptide, derived from glycoprotein 70 (gp70), admixed with CPQ safely generated functional, Ag-specific CD8+ T cells, resulting in the rejection of multiple tumor cell lines, with durable immunity. When cobalt was omitted, the otherwise identical peptide and adjuvant components did not result in peptide binding and were incapable of inducing immune responses, demonstrating the importance of stable particle formation. Immunization with the liposomal vaccine was well-tolerated and could control local and metastatic disease in a therapeutic setting. Mechanistic studies showed that particle-based peptides were better taken up by antigen-presenting cells, where they were putatively released within endosomes and phagosomes for display on MHC-I surfaces. On the basis of the potency of the approach, the platform was demonstrated as a tool for in vivo epitope screening of peptide microlibraries comprising a hundred peptides.

Lyophilized, antigen-bound liposomes with reduced MPLA and enhanced thermostability.

Thermostability and decreased component costs are desirable features for adjuvanted, recombinant vaccines. We previously showed that a model malaria transmission-blocking vaccine candidate antigen, Pfs25, can be rendered more immunogenic when mixed with liposomes containing cobalt porphyrin-phospholipid (CoPoP) and a synthetic monophosphoryl lipid A (MPLA) variant. CoPoP can induce stable particle formation of recombinant antigens based on interaction with their polyhistidine tag. In the present work, different synthetic MPLA variants and concentrations were assessed in CoPoP liposomes. Long-term biophysical stability and immunogenicity were not adversely impacted by a 60% reduction in MPLA content. When admixed with Pfs25, the adjuvant formulations effectively induced functional antibodies in immunized mice and rabbits. Lyophilized, antigen-bound liposomes were formed using sucrose and trehalose cryoprotectants, which improved vaccine reconstitution for a variety of model antigens. Compared to liquid storage, the lyophilized Pfs25 and CoPoP liposomes exhibited thermostability with respect to size, biochemical integrity, binding capacity, protein folding and immunogenicity. Following 6 weeks of storage at 60 °C, the most extended storage period assessed, the lyophilized formulation induced functional antibodies in mice with immunization.

Particle-based, Pfs230 and Pfs25 immunization is effective, but not improved by duplexing at fixed total antigen dose.

BACKGROUND: The Plasmodium falciparum sexual-stage surface proteins Pfs25 and Pfs230 are antigen candidates for a malaria transmission-blocking vaccine (TBV), and have been widely investigated as such. It is not clear whether simultaneously presenting these two antigens in a particulate vaccine would enhance the transmission reducing activity (TRA) of induced antibodies. To assess this, immunization was carried out with liposomes containing synthetic lipid adjuvant monophosphoryl lipid A (MPLA), and cobalt-porphyrin-phospholipid (CoPoP), which rapidly converts recombinant, his-tagged antigens into particles. METHODS: His-tagged, recombinant Pfs25 and Pfs230C1 were mixed with CoPoP liposomes to form a bivalent vaccine. Antigens were fluorescently labelled to infer duplex particleization serum-stability and binding kinetics using fluorescence resonance energy transfer. Mice and rabbits were immunized with individual or duplexed particleized Pfs25 and Pfs230C1, at fixed total antigen doses. The resulting antibody responses were assessed for magnitude and TRA. RESULTS: Pfs230C1 and Pfs25 rapidly bound CoPoP liposomes to form a serum-stable, bivalent particle vaccine. In mice, immunization with 5 ng of total antigen (individual antigen or duplexed) elicited functional antibodies against Pfs25 and Pfs230. Compared to immunization with the individual antigen, Pfs25 antibody production was moderately lower for the bivalent CoPoP vaccine, whereas Pfs230C1 antibody production was not impacted. All antibodies demonstrated at least 92% inhibition in oocyst density at 750 µg/mL purified mouse IgG in the standard membrane feeding assay (SMFA). At lower IgG concentrations, the bivalent vaccine did not improve TRA; antibodies induced by particleized Pfs25 alone showed stronger function in these conditions. In rabbits, immunization with a 20 µg total antigen dose with the duplexed antigens yielded similar antibody production against Pfs25 and Pfs230 compared to immunization with a 20 µg dose of individual antigens. However, no enhanced TRA was observed with duplexing. CONCLUSIONS: Pfs25, Pfs230 or the duplexed combination can readily be prepared as particulate vaccines by mixing CoPoP liposomes with soluble, recombinant antigens. This approach induces potent transmission-reducing antibodies following immunization in mice and rabbits. Immunization with bivalent, particleized, Pfs230 and Pfs25 did not yield antibodies with superior TRA compared to immunization with particleized Pfs25 as a single antigen. Altogether, duplexing antigens is straightforward and effective using CoPoP liposomes, but is likely to be more useful for targeting distinct parasite life stages.

Antibody response of a particle-inducing, liposome vaccine adjuvant admixed with a Pfs230 fragment.

Pfs230 is a malaria transmission-blocking antigen candidate, expressed on the surface of Plasmodium falciparum gametocytes. A recombinant, his-tagged Pfs230 fragment (Pfs230C1; amino acids 443-731) formed serum-stable particles upon incubation with liposomes containing cobalt-porphyrin-phospholipid (CoPoP). In mice, immunization with Pfs230C1, admixed with the adjuvants Alum, Montanide ISA720 or CoPoP liposomes (also containing synthetic monophosphoryl lipid A; PHAD), resulted in elicitation of IgG antibodies, but only those induced with CoPoP/PHAD or ISA720 strongly reduced parasite transmission. Immunization with micrograms of Pfs230C1 adjuvanted with identical liposomes lacking cobalt (that did not induce particle formation) or Alum was less effective than immunization with nanograms of Pfs230C1 with CoPoP/PHAD. CoPoP/PHAD and ISA720 adjuvants induced antibodies with similar Pfs230C1 avidity but higher IgG2-to-IgG1 ratios than Alum, which likely contributed to enhanced functional activity. Unlike prior work with another transmission-blocking antigen (Pfs25), Pfs230C1 was found to be effectively taken up by antigen-presenting cells without particle formation. The anti-Pfs230C1 IgG response was durable in mice for 250 days following immunization with CoPoP/PHAD, as were antibody avidity and elevated IgG2-to-IgG1 ratios. Immunization of rabbits with 20 µg Pfs230C1 admixed with CoPoP/PHAD elicited antibodies that inhibited parasite transmission. Taken together, these results show that liposomes containing CoPoP and PHAD are an effective vaccine adjuvant platform for recombinant malaria transmission blocking antigens.

Structural consequences of the interaction of RbgA with a 50S ribosomal subunit assembly intermediate.

Bacteria harbor a number GTPases that function in the assembly of the ribosome and are essential for growth. RbgA is one of these GTPases and is required for the assembly of the 50S subunit in most bacteria. Homologs of this protein are also implicated in the assembly of the large subunit of the mitochondrial and eukaryotic ribosome. We present here the cryo-electron microscopy structure of RbgA bound to a Bacillus subtilis 50S subunit assembly intermediate (45SRbgA particle) that accumulates in cells upon RbgA depletion. Binding of RbgA at the P site of the immature particle stabilizes functionally important rRNA helices in the A and P-sites, prior to the completion of the maturation process of the subunit. The structure also reveals the location of the highly conserved N-terminal end of RbgA containing the catalytic residue Histidine 9. The derived model supports a mechanism of GTP hydrolysis, and it shows that upon interaction of RbgA with the 45SRbgA particle, Histidine 9 positions itself near the nucleotide potentially acting as the catalytic residue with minimal rearrangements. This structure represents the first visualization of the conformational changes induced by an assembly factor in a bacterial subunit intermediate.

Mass spectrometry analysis of the human endosulfatase Hsulf-2.

The human 6-O-endosulfatases HSulf-1 and -2 catalyze the region-selective hydrolysis of the 6-O-sulfate group of the glucosamine residues within sulfated domains of heparan sulfate, thereby ensuring a unique and original post-biosynthetic modification of the cell surface proteoglycans. While numerous studies point out the role of HSulf-2 in crucial physiological processes as well as in pathological conditions particularly in cancer, its structural organization in two chains and its functional properties remain poorly understood. In this study, we report the first characterization by mass spectrometry (MS) of HSulf-2. An average molecular weight of 133,115 Da was determined for the whole enzyme by MALDI-TOF MS, i.e. higher than the naked amino acid backbone (98,170 Da), highlighting a significant contribution of post-translational modifications. The HSulf-2 protein sequence was determined by Nano-LC-MS/MS, leading to 63% coverage and indicating at least four N-glycosylation sites at Asn 108, 147, 174 and 217. These results provide a platform for further structural investigations of the HSulf enzymes, aiming at deciphering the role of each chain in the substrate binding and specificities and in the catalytic activities.

Expression and purification of recombinant extracellular sulfatase HSulf-2 allows deciphering of enzyme sub-domain coordinated role for the binding and 6-O-desulfation of heparan sulfate.

Through their ability to edit 6-O-sulfation pattern of Heparan sulfate (HS) polysaccharides, Sulf extracellular endosulfatases have emerged as critical regulators of many biological processes, including tumor progression. However, study of Sulfs remains extremely intricate and progress in characterizing their functional and structural features has been hampered by limited access to recombinant enzyme. In this study, we unlock this critical bottleneck, by reporting an efficient expression and purification system of recombinant HSulf-2 in mammalian HEK293 cells. This novel source of enzyme enabled us to investigate the way the enzyme domain organization dictates its functional properties. By generating mutants, we confirmed previous studies that HSulf-2 catalytic (CAT) domain was sufficient to elicit arylsulfatase activity and that its hydrophilic (HD) domain was necessary for the enzyme 6-O-endosulfatase activity. However, we demonstrated for the first time that high-affinity binding of HS substrates occurred through the coordinated action of both domains, and we identified and characterized 2 novel HS binding sites within the CAT domain. Altogether, our findings contribute to better understand the molecular mechanism governing HSulf-2 substrate recognition and processing. Furthermore, access to purified recombinant protein opens new perspectives for the resolution of HSulf structure and molecular features, as well as for the development of Sulf-specific inhibitors.

Binding of an amphiphilic phthalocyanine to pre-formed liposomes confers light-triggered cargo release.

Liposomes are able to load a range of cargos and have been used for drug delivery applications, including for stimuli-triggered drug release. Here, we describe an approach for imparting near infrared (NIR) light-triggered release to pre-formed liposomes, using a newly-synthesized cationic, amphiphilic phthalocyanine. When simply mixed in aqueous solution with cargo-loaded liposomes, the cationic amphiphilic phthalocyanine, but not a cationic hydrophilic azaphthalocyanine, spontaneously incorporates into the liposome bilayer. This enables subsequent release of loaded cargo (doxorubcin or basic orange) upon irradiation with NIR light. The rate of release could be altered by varying the amount of photosensitizer added to the liposomes. In the absence of NIR light exposure, stable cargo loading of the liposomes was maintained. Introduction.

The "in and out" of glucosamine 6-O-sulfation: the 6th sense of heparan sulfate.

The biological properties of Heparan sulfate (HS) polysaccharides essentially rely on their ability to bind and modulate a multitude of protein ligands. These interactions involve internal oligosaccharide sequences defined by their sulfation patterns. Amongst these, the 6-O-sulfation of HS contributes significantly to the polysaccharide structural diversity and is critically involved in the binding of many proteins. HS 6-O-sulfation is catalyzed by 6-O-sulfotransferases (6OSTs) during biosynthesis, and it is further modified by the post-synthetic action of 6-O-endosulfatases (Sulfs), two enzyme families that remain poorly characterized. The aim of the present review is to summarize the contribution of 6-O-sulfates in HS structure/function relationships and to discuss the present knowledge on the complex mechanisms regulating HS 6-O-sulfation.

Syndecan-1 alters heparan sulfate composition and signaling pathways in malignant mesothelioma.

Syndecan-1 is a proteoglycan that acts as co-receptor through its heparan sulfate (HS) chains and plays important roles in cancer. HS chains are highly variable in length and sulfation pattern. This variability is enhanced by the SULF1/2 enzymes, which remove 6-O-sulfates from HS. We used malignant mesothelioma, an aggressive tumor with poor prognosis, as a model and demonstrated that syndecan-1 over-expression down-regulates SULF1 and alters the HS biosynthetic machinery. Biochemical characterization revealed a 2.7-fold reduction in HS content upon syndecan-1 over-expression, but an overall increase in sulfation. Consistent with low SULF1 levels, trisulfated disaccharides increased 2.5-fold. ERK1/2 activity was enhanced 6-fold. Counteracting ERK activation, Akt, WNK1, and c-Jun were inhibited. The net effect of these changes manifested in G1 cell cycle arrest. Studies of pleural effusions showed that SULF1 levels are lower in pleural malignancies compared to benign conditions and inversely correlate with the amounts of syndecan-1, suggesting important roles for syndecan-1 and SULF1 in malignant mesothelioma.

Post-Synthetic Regulation of HS Structure: The Yin and Yang of the Sulfs in Cancer.

Heparan sulfate (HS) is a complex polysaccharide that takes part in most major cellular processes, through its ability to bind and modulate a very large array of proteins. These interactions involve saccharide domains of specific sulfation pattern (S-domains), the assembly of which is tightly orchestrated by a highly regulated biosynthesis machinery. Another level of structural control does also take place at the cell surface, where degrading enzymes further modify HS post-synthetically. Amongst them are the Sulfs, a family of extracellular sulfatases (two isoforms in human) that catalyze the specific 6-O-desulfation of HS. By targeting HS functional sulfated domains, Sulfs dramatically alter its ligand binding properties, thereby modulating a broad range of signaling pathways. Consequently, Sulfs play major roles during development, as well as in tissue homeostasis and repair. Sulfs have also been associated with many pathologies including cancer, but despite increasing interest, the role of Sulfs in tumor development still remains unclear. Studies have been hindered by a poor understanding of the Sulf enzymatic activities and conflicting data have shown either anti-oncogenic or tumor-promoting effects of these enzymes, depending on the tumor models analyzed. These opposite effects clearly illustrate the fine tuning of HS functions by the Sulfs, and the need to clarify the mechanisms involved. In this review, we will detail the present knowledge on the structural and functional properties of the Sulfs, with a special focus on their implication during tumor progression. Finally, we will discuss attempts and perspectives of using the Sulfs as a biomarker of cancer prognosis and diagnostic and as a target for anti-cancer therapies.

HSulf sulfatases catalyze processive and oriented 6-O-desulfation of heparan sulfate that differentially regulates fibroblast growth factor activity.

Sulfs are extracellular sulfatases that have emerged recently as critical regulators of heparan sulfate (HS) activities through their ability to catalyze specific 6-O-desulfation of the polysaccharide. Consequently, Sulfs have been involved in many physiological and pathological processes, and notably for Sulf-2, in the development of cancers with poor prognosis. Despite growing interest, little is known about the structure and activity of these enzymes and the way they induce dynamic remodeling of HS 6-O-sulfation status. Here, we have combined an array of analytical approaches, including mass spectrometry, NMR, HS oligosaccharide sequencing, and FACS, to dissect HSulf-2 sulfatase activity, either on a purified octasaccharide used as a mimic of HS functional domains, or on intact cell-surface HS chains. In parallel, we have studied the functional consequences of HSulf-2 activity on fibroblast growth factor (FGF)-induced mitogenesis and found that the enzyme could differentially regulate FGF1 and FGF2 activities. Notably, these data supported the existence of precise 6-O-sulfation patterns for FGF activation and provided new insights into the saccharide structures involved. Altogether, our data bring to light an original processive enzymatic mechanism, by which HSulfs catalyze oriented alteration of HS 6-O-desulfation patterns and direct fine and differential regulation of HS functions.

Dual function of mitochondrial Nm23-H4 protein in phosphotransfer and intermembrane lipid transfer: a cardiolipin-dependent switch.

The nucleoside diphosphate kinase Nm23-H4/NDPK-D forms symmetrical hexameric complexes in the mitochondrial intermembrane space with phosphotransfer activity using mitochondrial ATP to regenerate nucleoside triphosphates. We demonstrate the complex formation between Nm23-H4 and mitochondrial GTPase OPA1 in rat liver, suggesting its involvement in local and direct GTP delivery. Similar to OPA1, Nm23-H4 is further known to strongly bind in vitro to anionic phospholipids, mainly cardiolipin, and in vivo to the inner mitochondrial membrane. We show here that such protein-lipid complexes inhibit nucleoside diphosphate kinase activity but are necessary for another function of Nm23-H4, selective intermembrane lipid transfer. Mitochondrial lipid distribution was analyzed by liquid chromatography-mass spectrometry using HeLa cells expressing either wild-type Nm23-H4 or a membrane binding-deficient mutant at a site predicted based on molecular modeling to be crucial for cardiolipin binding and transfer mechanism. We found that wild type, but not the mutant enzyme, selectively increased the content of cardiolipin in the outer mitochondrial membrane, but the distribution of other more abundant phospholipids (e.g. phosphatidylcholine) remained unchanged. HeLa cells expressing the wild-type enzyme showed increased accumulation of Bax in mitochondria and were sensitized to rotenone-induced apoptosis as revealed by stimulated release of cytochrome c into the cytosol, elevated caspase 3/7 activity, and increased annexin V binding. Based on these data and molecular modeling, we propose that Nm23-H4 acts as a lipid-dependent mitochondrial switch with dual function in phosphotransfer serving local GTP supply and cardiolipin transfer for apoptotic signaling and putative other functions.

Inhibition of AMPK signalling by doxorubicin: at the crossroads of the cardiac responses to energetic, oxidative, and genotoxic stress.

AIMS: Cardiotoxic side effects of anthracyclines, the most widely used anticancer drugs, are well documented, while mechanisms involved are not fully elucidated. The cellular energy sensor and regulator AMP-activated protein kinase (AMPK) was suggested as a putative mediator of cardiotoxicity of doxorubicin, the leading anthracycline drug, by our earlier work. Here, we study the interference of doxorubicin with AMPK signalling and potentially involved mechanisms. METHODS AND RESULTS: Effects of doxorubicin on cell signalling are studied in isolated Langendorff-perfused Wistar rat hearts and in hearts from doxorubicin-treated Wistar rats. In both models, doxorubicin induces energetic, oxidative, and genotoxic stress. Despite energy depletion and unaffected AMPK upstream signalling, doxorubicin does not activate the AMPK pathway and even reduces basal phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase. In contrast, oxidative and genotoxic stress do activate pro-survival mitogen-activated protein kinase (MAPK) and Akt pathways, the latter via DNA-dependent protein kinase activation triggered by DNA damage. Combined inhibition of AMPK and activation of Akt and MAPK lead to activation of growth-stimulating mammalian target of rapamycin (mTOR) signalling. CONCLUSION: Our results suggest that in the doxorubicin-challenged heart, a combined energetic, oxidative, and genotoxic stress elicits a specific, hierarchical response where AMPK is inhibited at least partially by the known negative cross-talk with Akt and MAPK pathways, largely triggered by DNA damage signalling. Although such signalling can be protective, e.g. by limiting apoptosis, it primarily induces a negative feedback that increases cellular energy deficits, and via activation of mTOR signalling, it also contributes to the pathological cardiac phenotype in chronic doxorubicin toxicity.