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PROGRESS IN LIPID RESEARCH (0163-7827 ): 74 pp 18-30 (2019)

Hsp70 interactions with membrane lipids regulate cellular functions in health and disease

Balogi Zsolt, Multhoff Gabrielle, Jensen Thomas Kirkegaard, Lloyd-Evans Emyr, Yamashima Tetsumori, Jäättelä Marja, Harwood John L, Vígh László

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Beyond guarding the cellular proteome the major stress inducible heat shock protein Hsp70 has been shown to interact with lipids. Non-cytosolic Hsp70 stabilizes membranes during stress challenges and, in pathophysiological states, facilitates endocytosis, counteracts apoptotic mechanisms, sustains survival pathways or represents a signal that can be recognized by the immune system. Disease-coupled lipid-associated functions of Hsp70 may be targeted via distinct subcellular localizations of Hsp70 itself or its specific interacting lipids. With a special focus on interacting lipids, here we discuss localization-dependent roles of the membrane-bound Hsp70 in the context of its therapeutic potential, particularly in cancer and neurodegenerative diseases.

AGING CELL (1474-9718 1474-9726): 18 3 Paper e12849. (2019)

JNK modifies neuronal metabolism to promote proteostasis and longevity

Wang Lifen, Davis Sonnet S, Borch Jensen Martin, Rodriguez-Fernandez Imilce A, Apaydin Cagsar, Juhasz Gabor, Gibson Bradford W, Schilling Birgit, Ramanathan Arvind, Ghaemmaghami Sina, Jasper Heinrich

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Aging is associated with a progressive loss of tissue and metabolic homeostasis. This loss can be delayed by single-gene perturbations, increasing lifespan. How such perturbations affect metabolic and proteostatic networks to extend lifespan remains unclear. Here, we address this question by comprehensively characterizing age-related changes in protein turnover rates in the Drosophila brain, as well as changes in the neuronal metabolome, transcriptome, and carbon flux in long-lived animals with elevated Jun-N-terminal Kinase signaling. We find that these animals exhibit a delayed age-related decline in protein turnover rates, as well as decreased steady-state neuronal glucose-6-phosphate levels and elevated carbon flux into the pentose phosphate pathway due to the induction of glucose-6-phosphate dehydrogenase (G6PD). Over-expressing G6PD in neurons is sufficient to phenocopy these metabolic and proteostatic changes, as well as extend lifespan. Our study identifies a link between metabolic changes and improved proteostasis in neurons that contributes to the lifespan extension in long-lived mutants.

MOLECULAR SYSTEMS BIOLOGY (1744-4292 ): 15 4 Paper e8462. 18 p. (2019)

Enzyme promiscuity shapes adaptation to novel growth substrates

Guzmán Gabriela I, Sandberg Troy E, LaCroix Ryan A, Nyerges Ákos, Papp Henrietta, de Raad Markus, King Zachary A, Hefner Ying, Northen Trent R, Notebaart Richard A, Pál Csaba, Palsson Bernhard O, Papp Balázs, Feist Adam M

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Evidence suggests that novel enzyme functions evolved from low-level promiscuous activities in ancestral enzymes. Yet, the evolutionary dynamics and physiological mechanisms of how such side activities contribute to systems-level adaptations are not well characterized. Furthermore, it remains untested whether knowledge of an organism's promiscuous reaction set, or underground metabolism, can aid in forecasting the genetic basis of metabolic adaptations. Here, we employ a computational model of underground metabolism and laboratory evolution experiments to examine the role of enzyme promiscuity in the acquisition and optimization of growth on predicted non-native substrates in Escherichia coli K-12 MG1655. After as few as approximately 20 generations, evolved populations repeatedly acquired the capacity to grow on five predicted non-native substrates-D-lyxose, D-2-deoxyribose, D-arabinose, m-tartrate, and monomethyl succinate. Altered promiscuous activities were shown to be directly involved in establishing high-efficiency pathways. Structural mutations shifted enzyme substrate turnover rates toward the new substrate while retaining a preference for the primary substrate. Finally, genes underlying the phenotypic innovations were accurately predicted by genome-scale model simulations of metabolism with enzyme promiscuity.

BRITISH JOURNAL OF PHARMACOLOGY (0007-1188 1476-5381): 176 9 pp 1222-1223 (2019)

The impact of dihydropyridine derivatives on the cerebral blood flow response to somatosensory stimulation and spreading depolarization

Szabó Írisz, Tóth Orsolya M., Török Zsolt, Varga Dániel Péter, Menyhárt Ákos, Frank Rita, Hantosi Dóra, Hunya Ákos, Bari Ferenc, Horváth Ibolya, Vigh László, Farkas Eszter

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Background and Purpose A new class of dihydropyridine derivatives, which act as co-inducers of heat shock protein but are devoid of calcium channel antagonist and vasodilator effects, has recently been developed with the purpose of selectively targeting neurodegeneration. Here, we evaluated the action of one of these novel compounds LA1011 on neurovascular coupling in the ischaemic rat cerebral cortex. As a reference, we applied nimodipine, a vasodilator dihydropyridine and well-known calcium channel antagonist. Experimental Approach Rats were treated with LA1011 or nimodipine, either by chronic, systemic (LA1011), or acute, local administration (LA1011 and nimodipine). In the latter treatment group, global forebrain ischaemia was induced in half of the animals by bilateral common carotid artery occlusion under isoflurane anaesthesia. Functional hyperaemia in the somatosensory cortex was created by mechanical stimulation of the contralateral whisker pad under alpha-chloralose anaesthesia. Spreading depolarization (SD) events were elicited subsequently by 1 M KCl. Local field potential and cerebral blood flow (CBF) in the parietal somatosensory cortex were monitored by electrophysiology and laser Doppler flowmetry. Key Results LA1011 did not alter CBF, but intensified SD, presumably indicating the co-induction of heat shock proteins, and, perhaps an anti-inflammatory effect. Nimodipine attenuated evoked potentials and SD. In addition to the elevation of baseline CBF, nimodipine augmented hyperaemia in response to both somatosensory stimulation and SD, particularly under ischaemia. Conclusions and implications In contrast to the CBF improvement achieved with nimodipine, LA1011 seems not to have discernible cerebrovascular effects but may up-regulate the stress response.

AUTOPHAGY (1554-8627 ): 14 p. (2019)

Targeted interplay between bacterial pathogens and host autophagy

Sudhakar Padhmanand, Jacomin Anne-Claire, Hautefort Isabelle, Samavedam Siva, Fatemian Koorosh, Ari Eszter, Gul Leila, Demeter Amanda, Jones Emily, Korcsmaros Tamas, Nezis Ioannis P

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Due to the critical role played by autophagy in pathogen clearance, pathogens have developed diverse strategies to subvert it. Despite previous key findings of bacteria-autophagy interplay, asystems-level insight into selective targeting by the host and autophagy modulation by the pathogens is lacking. We predicted potential interactions between human autophagy proteins and effector proteins from 56 pathogenic bacterial species by identifying bacterial proteins predicted to have recognition motifs for selective autophagy receptors SQSTM1/p62, CALCOCO2/NDP52 and MAP1LC3/LC3. Using structure-based interaction prediction, we identified bacterial proteins capable to modify core autophagy components. Our analysis revealed that autophagy receptors in general potentially target mostly genus-specific proteins, and not those present in multiple genera. The complementarity between the predicted SQSTM1/p62 and CALCOCO2/NDP52 targets, which has been shown for Salmonella, Listeria and Shigella, could be observed across other pathogens. This complementarity potentially leaves the host more susceptible to chronic infections upon the mutation of autophagy receptors. Proteins derived from enterotoxigenic and non-toxigenic Bacillus outer membrane vesicles indicated that autophagy targets pathogenic proteins rather than non-pathogenic ones. We also observed apathogen-specific pattern as to which autophagy phase could be modulated by specific genera. We found intriguing examples of bacterial proteins that could modulate autophagy, and in turn being targeted by autophagy as ahost defense mechanism. We confirmed experimentally an interplay between a Salmonella protease, YhjJ and autophagy. Our comparative meta-analysis points out key commonalities and differences in how pathogens could affect autophagy and how autophagy potentially recognizes these pathogenic effectors.


Transcriptomic atlas of mushroom development reveals conserved genes behind complex multicellularity in fungi

Krizsán Krisztina, Almási Éva, Merényi Zsolt, Sahu Neha, Virágh Máté, Kószó Tamás, Mondo Stephen, Kiss Brigitta, Bálint Balázs, Kües Ursula, Barry Kerrie, Cseklye Judit, Hegedüs Botond, Henrissat Bernard, Johnson Jenifer, Lipzen Anna, Ohm Robin A, Nagy István, Pangilinan Jasmyn, Yan Juying, Xiong Yi, Grigoriev Igor V, Hibbett David S, Nagy László G

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The evolution of complex multicellularity has been one of the major transitions in the history of life. In contrast to simple multicellular aggregates of cells, it has evolved only in a handful of lineages, including animals, embryophytes, red and brown algae, and fungi. Despite being a key step toward the evolution of complex organisms, the evolutionary origins and the genetic underpinnings of complex multicellularity are incompletely known. The development of fungal fruiting bodies from a hyphal thallus represents a transition from simple to complex multicellularity that is inducible under laboratory conditions. We constructed a reference atlas of mushroom formation based on developmental transcriptome data of six species and comparisons of >200 whole genomes, to elucidate the core genetic program of complex multicellularity and fruiting body development in mushroom-forming fungi (Agaricomycetes). Nearly 300 conserved gene families and >70 functional groups contained developmentally regulated genes from five to six species, covering functions related to fungal cell wall remodeling, targeted protein degradation, signal transduction, adhesion, and small secreted proteins (including effector-like orphan genes). Several of these families, including F-box proteins, expansin-like proteins, protein kinases, and transcription factors, showed expansions in Agaricomycetes, many of which convergently expanded in multicellular plants and/or animals too, reflecting convergent solutions to genetic hurdles imposed by complex multicellularity among independently evolved lineages. This study provides an entry point to studying mushroom development and complex multicellularity in one of the largest clades of complex eukaryotic organisms.

AUTOPHAGY (1554-8627 ): 17 p. (2019)

Proteasome dysfunction induces excessive proteome instability and loss of mitostasis that can be mitigated by enhancing mitochondrial fusion or autophagy

Tsakiri Eleni N., Gumeni Sentiljana, Vougas Konstantinos, Pendin Diana, Papassideri Issidora, Daga Andrea, Gorgoulis Vassilis, Juhász Gábor, Scorrano Luca, Trougakos Ioannis P.

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The ubiquitin-proteasome pathway (UPP) is central to proteostasis network (PN) functionality and proteome quality control. Yet, the functional implication of the UPP in tissue homeodynamics at the whole organism level and its potential cross-talk with other proteostatic or mitostatic modules are not well understood. We show here that knock down (KD) of proteasome subunits in Drosophila flies, induced, for most subunits, developmental lethality. Ubiquitous or tissue specific proteasome dysfunction triggered systemic proteome instability and activation of PN modules, including macroautophagy/autophagy, molecular chaperones and the antioxidant cncC (the fly ortholog of NFE2L2/Nrf2) pathway. Also, proteasome KD increased genomic instability, altered metabolic pathways and severely disrupted mitochondrial functionality, triggering a cncC-dependent upregulation of mitostatic genes and enhanced rates of mitophagy. Whereas, overexpression of key regulators of antioxidant responses (e.g., cncC or foxo) could not suppress the deleterious effects of proteasome dysfunction; these were alleviated in both larvae and adult flies by modulating mitochondrial dynamics towards increased fusion or by enhancing autophagy. Our findings reveal the extensive functional wiring of genomic, proteostatic and mitostatic modules in higher metazoans. Also, they support the notion that age-related increase of proteotoxic stress due to decreased UPP activity deregulates all aspects of cellular functionality being thus a driving force for most age-related diseases.

PLOS BIOLOGY (1544-9173 1545-7885): 17 3 Paper e3000182. 17 p. (2019)

Evolthon: A community endeavor to evolve lab evolution

Strauss Sivan Kaminski, Bálint Csörgő, Csaba Pál, et al.

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In experimental evolution, scientists evolve organisms in the lab, typically by challenging them to new environmental conditions. How best to evolve a desired trait? Should the challenge be applied abruptly, gradually, periodically, sporadically? Should one apply chemical mutagenesis, and do strains with high innate mutation rate evolve faster? What are ideal population sizes of evolving populations? There are endless strategies, beyond those that can be exposed by individual labs. We therefore arranged a community challenge, Evolthon, in which students and scientists from different labs were asked to evolve Escherichia coli or Saccharomyces cerevisiae for an abiotic stresslow temperature. About 30 participants from around the world explored diverse environmental and genetic regimes of evolution. After a period of evolution in each lab, all strains of each species were competed with one another. In yeast, the most successful strategies were those that used mating, underscoring the importance of sex in evolution. In bacteria, the fittest strain used a strategy based on exploration of different mutation rates. Different strategies displayed variable levels of performance and stability across additional challenges and conditions. This study therefore uncovers principles of effective experimental evolutionary regimens and might prove useful also for biotechnological developments of new strains and for understanding natural strategies in evolutionary arms races between species. Evolthon constitutes a model for community-based scientific exploration that encourages creativity and cooperation.

DIABETOLOGIA 62 : 4 pp. 717-725. , 9 p. (2019)

Expression of GLP-1 receptors in insulin-containing interneurons of rat cerebral cortex

Csajbók ÉA, Kocsis ÁK, Faragó N, Furdan S, Kovács B, Lovas S, Molnár G, Likó I, Zvara Á, Puskás LG, Patócs A, Tamás G

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Aims/hypothesisGlucagon-like peptide 1 (GLP-1) receptors are expressed by pancreatic beta cells and GLP-1 receptor signalling promotes insulin secretion. GLP-1 receptor agonists have neural effects and are therapeutically promising for mild cognitive impairment and Alzheimer's disease. Our previous results showed that insulin is released by neurogliaform neurons in the cerebral cortex, but the expression of GLP-1 receptors on insulin-producing neocortical neurons has not been tested. In this study, we aimed to determine whether GLP-1 receptors are present in insulin-containing neurons.MethodsWe harvested the cytoplasm of electrophysiologically and anatomically identified neurogliaform interneurons during patch-clamp recordings performed in slices of rat neocortex. Using single-cell digital PCR, we determined copy numbers of Glp1r mRNA and other key genes in neurogliaform cells harvested in conditions corresponding to hypoglycaemia (0.5mmol/l glucose) and hyperglycaemia (10mmol/l glucose). In addition, we performed whole-cell patch-clamp recordings on neurogliaform cells to test the effects of GLP-1 receptor agonists for functional validation of single-cell digital PCR results.ResultsSingle-cell digital PCR revealed GLP-1 receptor expression in neurogliaform cells and showed that copy numbers of mRNA of the Glp1r gene in hyperglycaemia exceeded those in hypoglycaemia by 9.6 times (p<0.008). Moreover, single-cell digital PCR confirmed co-expression of Glp1r and Ins2 mRNA in neurogliaform cells. Functional expression of GLP-1 receptors was confirmed with whole-cell patch-clamp electrophysiology, showing a reversible effect of GLP-1 on neurogliaform cells. This effect was prevented by pre-treatment with the GLP-1 receptor-specific antagonist exendin-3(9-39) and was absent in hypoglycaemia. In addition, single-cell digital PCR of neurogliaform cells revealed that the expression of transcription factors (Pdx1, Isl1, Mafb) are important in beta cell development.Conclusions/interpretationOur results provide evidence for the functional expression of GLP-1 receptors in neurons known to release insulin in the cerebral cortex. Hyperglycaemia increases the expression of GLP-1 receptors in neurogliaform cells, suggesting that endogenous incretins and therapeutic GLP-1 receptor agonists might have effects on these neurons, similar to those in pancreatic beta cells

CELLULAR AND MOLECULAR LIFE SCIENCES (1420-682X 1420-9071): 76 5 pp 865-871 (2019)

Heparan sulfate proteoglycan (HSPG) can take part in cell division: inside and outside

Ughy Bettina, Schmidthoffer Ildiko, Szilak Laszlo

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Prior to the cytokinesis, the cell-matrix interactions should be disrupted, and the mitotic cells round up. Prerequisite of mitosis, the centrosomes duplicate, spindle fibers are generated and move away from each other to opposite sides of the cells marking the cell poles. Later, an invagination in the plasma membrane is formed a few minutes after anaphase. This furrow ingression is driven by a contractile actomyosin ring, whose assembly is regulated by RhoA GTPase. At the completion of cytokinesis, the two daughter cells are still connected by a thin intercellular bridge, which is subjected to abscission, as the terminal step of cytokinesis. Here, it is overviewed, how syndecan-4, a transmembrane, heparan sulfate proteoglycan, can contribute to these processes in a phosphorylation-dependent manner.

NATURE MICROBIOLOGY (2058-5276 ): 4 3 pp 447-458 (2019)

Phylogenetic barriers to horizontal transfer of antimicrobial peptide resistance genes in the human gut microbiota

Kintses Bálint, Méhi Orsolya, Ari Eszter, Számel Mónika, Györkei Ádám, Jangir Pramod K, Nagy István, Pál Ferenc, Fekete Gergely, Tengölics Roland, Nyerges Ákos, Likó István, Bálint Anita, Molnár Tamás, Bálint Balázs, Vásárhelyi Bálint Márk, Bustamante Misshelle, Papp Balázs, Pál Csaba

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The human gut microbiota has adapted to the presence of antimicrobial peptides (AMPs), which are ancient components of immune defence. Despite its medical importance, it has remained unclear whether AMP resistance genes in the gut microbiome are available for genetic exchange between bacterial species. Here, we show that AMP resistance and antibiotic resistance genes differ in their mobilization patterns and functional compatibilities with new bacterial hosts. First, whereas AMP resistance genes are widespread in the gut microbiome, their rate of horizontal transfer is lower than that of antibiotic resistance genes. Second, gut microbiota culturing and functional metagenomics have revealed that AMP resistance genes originating from phylogenetically distant bacteria have only a limited potential to confer resistance in Escherichia coli, an intrinsically susceptible species. Taken together, functional compatibility with the new bacterial host emerges as a key factor limiting the genetic exchange of AMP resistance genes. Finally, our results suggest that AMPs induce highly specific changes in the composition of the human microbiota, with implications for disease risks.

PLOS BIOLOGY (1544-9173 1545-7885): 17 1 Paper e3000131. (2019)

Pathogen diversity drives the evolution of generalist MHC-II alleles in human populations

Manczinger Máté, Boross Gábor, Kemény Lajos, Müller Viktor, Lenz Tobias L., Papp Balázs, Pál Csaba

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Central players of the adaptive immune system are the groups of proteins encoded in the major histocompatibility complex (MHC), which shape the immune response against pathogens and tolerance to self-peptides. The corresponding genomic region is of particular interest, as it harbors more disease associations than any other region in the human genome, including associations with infectious diseases, autoimmune disorders, cancers, and neuropsychiatric diseases. Certain MHC molecules can bind to a much wider range of epitopes than others, but the functional implication of such an elevated epitope-binding repertoire has remained largely unclear. It has been suggested that by recognizing more peptide segments, such promiscuous MHC molecules promote immune response against a broader range of pathogens. If so, the geographical distribution of MHC promiscuity level should be shaped by pathogen diversity. Three lines of evidence support the hypothesis. First, we found that in pathogen-rich geographical regions, humans are more likely to carry highly promiscuous MHC class II DRB1 alleles. Second, the switch between specialist and generalist antigen presentation has occurred repeatedly and in a rapid manner during human evolution. Third, molecular positions that define promiscuity level of MHC class II molecules are especially diverse and are under positive selection in human populations. Taken together, our work indicates that pathogen load maintains generalist adaptive immune recognition, with implications for medical genetics and epidemiology.

JOURNAL OF MEDICINAL CHEMISTRY (0022-2623 1520-4804): (2019)

Antioxidant-inspired drug discovery: antitumor metabolite is formed in situ from a hydroxycinnamic acid derivative upon free radical scavenging

Fási Laura, Di Meo Florent, Kuo Ching-Ying, Stojkovic Buric Sonja, Martins Ana, Kúsz Norbert, Béni Zoltán, Dékány Miklós, Balogh György T., Pesic Milica, Wang Hui-Chun, Trouillas Patrick, Hunyadi Attila

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Cancer cells generally possess higher levels of reactive oxygen species than normal cells, and this can serve as a possible therapeutic target. In this proof-of-concept study, an antioxidant-inspired drug discovery strategy was evaluated using a hydroxycinnamic acid derivative. The processing of oxidized mixtures of p-coumaric acid methyl ester (pcm) revealed a new antitumor lead, graviquinone. Graviquinone bypassed ABCB1-mediated resistance, induced DNA damage in lung carcinoma cells but exerted DNA protective activity in normal keratinocytes, and modulated DNA damage response in MCF-7 cells. The cytotoxic effect of pcm in MCF-7 cells was potentiated under H2O2-induced oxidative stress, and the formation of graviquinone was confirmed by Fenton's reaction on pcm. In silico density functional theory calculations suggested graviquinone as a kinetic product of pcm-scavenging (OH)-O-center dot radicals. Our results demonstrate the pharmacological value of an in situ-formed, oxidative stress-related metabolite of an antioxidant. This might be of particular importance for designing new strategies for antioxidant-based drug discovery.


Cytotoxic CD8(+) T lymphocytes expressing ALS-causing SOD1 mutant selectively trigger death of spinal motoneurons

Coque Emmanuelle, Salsac Celine, Espinosa-Carrasco Gabriel, Varga Bela, Degauque Nicolas, Cadoux Marion, Crabe Roxane, Virenque Anais, Soulard Claire, Fierle Julie K., Brodovitch Alexandre, Libralato Margot, Vegh Attila G., Venteo Stephanie, Scamps Frederique, Boucraut Jose, Laplaud David, Hernandez Javier, Gergely Csilla, Vincent Thierry, Raoul Cedric

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Adaptive immune response is part of the dynamic changes that accompany motoneuron loss in amyotrophic lateral sclerosis (ALS). CD4(+) T cells that regulate a protective immunity during the neurodegenerative process have received the most attention. CD8(+) T cells are also observed in the spinal cord of patients and ALS mice although their contribution to the disease still remains elusive. Here, we found that activated CD8(+) T lymphocytes infiltrate the central nervous system (CNS) of a mouse model of ALS at the symptomatic stage. Selective ablation of CD8(+) T cells in mice expressing the ALS-associated superoxide dismutase-1 (SOD1)(G93A) mutant decreased spinal motoneuron loss. Using motoneuron-CD8(+) T cell coculture systems, we found that mutant SOD1-expressing CD8(+) T lymphocytes selectively kill motoneurons. This cytotoxicity activity requires the recognition of the peptide-MHC-I complex (where MHC-I represents major histocompatibility complex class I). Measurement of interaction strength by atomic force microscopy-based single-cell force spectroscopy demonstrated a specific MHC-I-dependent interaction between motoneuron and SOD1(G93A) CD8(+) T cells. Activated mutant SOD1 CD8(+) T cells produce interferon-gamma, which elicits the expression of the MHC-I complex in motoneurons and exerts their cytotoxic function through Fas and granzyme pathways. In addition, analysis of the clonal diversity of CD8(+) T cells in the periphery and CNS of ALS mice identified an antigen-restricted repertoire of their T cell receptor in the CNS. Our results suggest that self-directed immune response takes place during the course of the disease, contributing to the selective elimination of a subset of motoneurons in ALS.

BRIEFINGS IN BIOINFORMATICS (1467-5463 1477-4054): 20 1 pp 89-101 (2019)

Discovering cooperative biomarkers for heterogeneous complex disease diagnoses

Sun Duanchen, Xianwen Ren, Ari Eszter, Korcsmáros Tamás, Csermely Péter, Ling-Yun Wu

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Biomarkers with high reproducibility and accurate prediction performance can contribute to comprehending the underlying pathogenesis of related complex diseases and further facilitate disease diagnosis and therapy. Techniques integrating gene expression profiles and biological networks for the identification of network-based disease biomarkers are receiving increasing interest. The biomarkers for heterogeneous diseases often exhibit strong cooperative effects, which implies that a set of genes may achieve more accurate outcome prediction than any single gene. In this study, we evaluated various biomarker identification methods that consider gene cooperative effects implicitly or explicitly, and proposed the gene cooperation network to explicitly model the cooperative effects of gene combinations. The gene cooperation network-enhanced method, named as MarkRank, achieves superior performance compared with traditional biomarker identification methods in both simulation studies and real data sets. The biomarkers identified by MarkRank not only have a better prediction accuracy but also have stronger topological relationships in the biological network and exhibit high specificity associated with the related diseases. Furthermore, the top genes identified by MarkRank involve crucial biological processes of related diseases and give a good prioritization for known disease genes. In conclusion, MarkRank suggests that explicit modeling of gene cooperative effects can greatly improve biomarker identification for complex diseases, especially for diseases with high heterogeneity.


Crystal and solution structures of calcium complexes relevant to problematic waste disposal: calcium gluconate and calcium isosaccharinate

Bugris V, Dudás C, Kutus B, Harmat V, Csankó K, Brockhauser S, Palinko I, Turner P, Sipos P

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The single-crystal structures of calcium D-gluconate and calcium α-D-isosaccharinate have been determined using X-ray diffraction at 100 K. Surprisingly, given its significance in industrial and medical applications, the structure of calcium D-gluconate has not previously been reported. Unexpectedly, the gluconate crystal structure comprises coordination polymers. Unusually, the calcium coordination number is nine. Adjacent metal centres are linked by three μ-oxo bridges, with a metal–metal separation of 3.7312(2)Å. One of the gluconate ligands contradicts a suggestion from 1974 that a straight chain conformation is associated with an intramolecular hydrogen bond. This ligand binds to three adjacent metal centres. The use of synchrotron radiation provided an improved crystal structure with respect to that previously reported for the isosaccharinate complex, allowing the location of the hy­droxy hydrogen sites to be elucidated. In contrast to the gluconate structure, there are no μ-oxo bridges in the isosaccharinate coordination polymer and the isosaccharinate bridging coordination is such that the distance between adjacent metal centres, each of which is eight-coordinate, is 6.7573(4)Å. Complementing the crystal structure determinations, modelling studies of the geometries and coordination modes for the aqueous [CaGluc]+ and [CaIsa]+ complexes are presented and discussed.

NUCLEIC ACIDS RESEARCH (0305-1048 1362-4962): 46 19 pp 10272-10285 (2018)

Mb- and FnCpf1 nucleases are active in mammalian cells

Tóth Eszter, Czene Bernadett C, Kulcsár Péter I, Krausz Sarah L, Tálas András, Nyeste Antal, Varga Éva, Huszár Krisztina, Weinhardt Nóra, Ligeti Zoltán, Borsy Adrienn É, Fodor Elfrieda, Welker Ervin

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Cpf1s, the RNA-guided nucleases of the class II clustered regularly interspaced short palindromic repeats system require a short motive called protospacer adjacent motif (PAM) to be present next to the targeted sequence for their activity. The TTTV PAM sequence of As- and LbCpf1 nucleases is relatively rare in the genome of higher eukaryotic organisms. Here, we show that two other Cpf1 nucleases, Fn- and MbCpf1, which have been reported to utilize a shorter, more frequently occurring PAM sequence (TTN) when tested in vitro, carry out efficient genome modification in mammalian cells. We found that all four Cpf1 nucleases showed similar activities and TTTV PAM preferences. Our approach also revealed that besides their activities their PAM preferences are also target dependent. To increase the number of the available targets for Fn- and MbCpf1 we generated their RVR and RR mutants with altered PAM specificity and compared them to the wild-type and analogous As- and LbCpf1 variants. The mutants gained new PAM specificities but retained their activity on targets with TTTV PAMs, redefining RR-Cpf1's PAM-specificities as TTYV/TCCV, respectively. These variants may become versatile substitutes for wild-type Cpf1s by providing an expanded range of targets for genome engineering applications.

NATURE MICROBIOLOGY (2058-5276 ): 3 12 pp 1451-1460 (2018)

Collapse of genetic division of labour and evolution of autonomy in pellicle biofilms

Dragos Anna, Martin Marivic, Garcia Carolina Falcon, Kricks Lara, Pausch Patrick, Heimerl Thomas, Balint Balazs, Maroti Gergely, Bange Gert, Lopez Daniel, Lieleg Oliver, Kovacs Akos T.

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Closely related microorganisms often cooperate, but the prevalence and stability of cooperation between different genotypes remain debatable. Here, we track the evolution of pellicle biofilms formed through genetic division of labour and ask whether partially deficient partners can evolve autonomy. Pellicles of Bacillus subtilis rely on an extracellular matrix composed of exo-polysaccharide (EPS) and the fibre protein TasA. In monocultures, Delta eps and Delta tasA mutants fail to form pellicles, but, facilitated by cooperation, they succeed in co-culture. Interestingly, cooperation collapses on an evolutionary timescale and Delta tasA gradually outcompetes its partner Delta eps. Pellicle formation can evolve independently from division of labour in Delta eps and Delta tasA monocultures, by selection acting on the residual matrix component, TasA or EPS, respectively. Using a set of interdisciplinary tools, we unravel that the TasA producer (Delta eps) evolves via an unconventional but reproducible substitution in TasA that modulates the biochemical properties of the protein. Conversely, the EPS producer (Delta tasA) undergoes genetically variable adaptations, all leading to enhanced EPS secretion and biofilms with different biomechanical properties. Finally, we revisit the collapse of division of labour between Delta eps and Delta tasA in light of a strong frequency versus exploitability trade-off that manifested in the solitarily evolving partners. We propose that such trade-off differences may represent an additional barrier to evolution of division of labour between genetically distinct microorganisms.

CELL REPORTS (2211-1247 ): 24 12 pp 3251-3261 (2018)

Opposing Roles of FANCJ and HLTF Protect Forks and Restrain Replication during Stress

Peng M, Cong K, Panzarino NJ, Nayak S, Calvo J, Deng B, Zhu LJ, Morocz M, Hegedus L, Haracska L, Cantor SB

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The DNA helicase FANCJ is mutated in hereditary breast and ovarian cancer and Fanconi anemia (FA). Nevertheless, how loss of FANCJ translates to disease pathogenesis remains unclear. We addressed this question by analyzing proteins associated with replication forks in cells with or without FANCJ. We demonstrate that FANCJ-knockout (FANCJ-KO) cells have alterations in the replisome that are consistent with enhanced replication stress, including an aberrant accumulation of the fork remodeling factor helicase-like transcription factor (HLTF). Correspondingly, HLTF contributes to fork degradation in FANCJ-KO cells. Unexpectedly, the unrestrained DNA synthesis that characterizes HLTF-deficient cells is FANCJ dependent and correlates with S1 nuclease sensitivity and fork degradation. These results suggest that FANCJ and HLTF promote replication fork integrity, in part by counteracting each other to keep fork remodeling and elongation in check. Indicating one protein compensates for loss of the other, loss of both HLTF and FANCJ causes a more severe replication stress response.

NATURE COMMUNICATIONS (2041-1723 ): 9 Paper 4476. 6 p. (2018)

An optical reaction micro-turbine

Bianchi Silvio, Vizsnyiczai Gaszton, Ferretti Stefano, Maggi Claudio, Di Leonardo Roberto

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To any energy flow there is an associated flow of momentum, so that recoil forces arise every time an object absorbs or deflects incoming energy. This same principle governs the operation of macroscopic turbines as well as that of microscopic turbines that use light as the working fluid. However, a controlled and precise redistribution of optical energy is not easy to achieve at the micron scale resulting in a low efficiency of power to torque conversion. Here we use direct laser writing to fabricate 3D light guiding structures, shaped as a garden sprinkler, that can precisely reroute input optical power into multiple output channels. The shape parameters are derived from a detailed theoretical analysis of losses in curved microfibers. These optical reaction micro-turbines can maximally exploit light's momentum to generate a strong, uniform and controllable torque.

AUTOPHAGY 14:(9) pp. 1499-1519. (2018)

Developmentally regulated autophagy is required for eye formation in Drosophila.

Billes V, Kovacs T, Manzeger A, Lorincz P, Szincsak S, Regos A, Kulcsar PI, Korcsmaros T, Lukacsovich T, Hoffmann G, Erdelyi M, Mihaly J, Takacs-Vellai K, Sass M, Vellai T

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The compound eye of the fruit fly Drosophila melanogaster is one of the most intensively studied and best understood model organs in the field of developmental genetics. Herein we demonstrate that autophagy, an evolutionarily conserved selfdegradation process of eukaryotic cells, is essential for eye development in this organism. Autophagic structures accumulate in a specific pattern in the developing eye disc, predominantly in the morphogenetic furrow (MF) and differentiation zone. Silencing of several autophagy genes (Atg) in the eye primordium severely affects the morphology of the adult eye through triggering ectopic cell death. In Atg mutant genetic backgrounds however genetic compensatory mechanisms largely rescue autophagic activity in, and thereby normal morphogenesis of, this organ. We also show that in the eye disc the expression of a key autophagy gene, Atg8a, is controlled in a complex manner by the anterior Hox paralog lab (labial), a master regulator of early development. Atg8a transcription is repressed in front of, while activated along, the MF by lab. The amount of autophagic structures then remains elevated behind the moving MF. These results indicate that eye development in Drosophila depends on the cell death-suppressing and differentiating effects of the autophagic process. This novel, developmentally regulated function of autophagy in the morphogenesis of the compound eye may shed light on a more fundamental role for cellular self-digestion in differentiation and organ formation than previously thought.

NATURE NEUROSCIENCE 21:(9) pp. 1185-1195. (2018)

Transcriptomic and morphophysiological evidence for a specialized human cortical GABAergic cell type

Boldog E, Bakken TE, Baka J, Borde S, Farago N, Kocsis AK, Kovacs B, Molnar G, Olah G, Ozsvar A, Rozsa M, Puskas LG, Barzo P, Tamas G

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We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large 'rosehip'-like axonal boutons and compact arborization. These rosehip cells show an immunohistochemical profile (GAD1(+)CCK(+), CNR1(-)SST(-)CALB2(-)PVALB(-)) matching a single transcriptomically defined cell type whose specific molecular marker signature is not seen in mouse cortex. Rosehip cells in layer 1 make homotypic gap junctions, predominantly target apical dendritic shafts of layer 3 pyramidal neurons, and inhibit backpropagating pyramidal action potentials in microdomains of the dendritic tuft. These cells are therefore positioned for potent local control of distal dendritic computation in cortical pyramidal neurons.

NATURE COMMUNICATIONS 9: Paper 3487. 9 p. (2018)

Megahertz data collection from protein microcrystals at an X-ray free-electron laser

Grunbein ML, Bielecki J, Brockhauser S, Schlichting I

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X-ray free-electron lasers (XFELs) enable novel experiments because of their high peak brilliance and femtosecond pulse duration. However, non-superconducting XFELs offer repetition rates of only 10-120 Hz, placing significant demands on beam time and sample consumption. We describe serial femtosecond crystallography experiments performed at the European XFEL, the first MHz repetition rate XFEL, delivering 1.128 MHz X-ray pulse trains at 10 Hz. Given the short spacing between pulses, damage caused by shock waves launched by one XFEL pulse on sample probed by subsequent pulses is a concern. To investigate this issue, we collected data from lysozyme microcrystals, exposed to a similar to 15 mu m XFEL beam. Under these conditions, data quality is independent of whether the first or subsequent pulses of the train were used for data collection. We also analyzed a mixture of microcrystals of jack bean proteins, from which the structure of native, magnesium-containing concanavalin A was determined.


Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset

Miton CM, Jonas S, Fischer G, Duarte F, Mohamed MF, van Loo B, Kintses B, Kamerlin SCL, Tokuriki N, Hyvonen M, Hollfelder F

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The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono-and diester hydrolyses were only marginally affected (<= 50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E.S, enzyme-substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate (beta(leaving) (group) from -1.08 to -0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes.

NUCLEIC ACIDS RESEARCH 46:(12) pp. 6087-6098. (2018)

CHD3 and CHD4 recruitment and chromatin remodeling activity at DNA breaks is promoted by early poly(ADP-ribose)-dependent chromatin relaxation

Smith R, Sellou H, Chapuis C, Huet S, Timinszky G

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One of the first events to occur upon DNA damage is the local opening of the compact chromatin architecture, facilitating access of repair proteins to DNA lesions. This early relaxation is triggered by poly(ADP-ribosyl)ation by PARP1 in addition to ATP-dependent chromatin remodeling. CHD4 recruits to DNA breaks in a PAR-dependent manner, although it lacks any recognizable PAR-binding domain, and has the ability to relax chromatin structure. However, its role in chromatin relaxation at the site of DNA damage has not been explored. Using a live cell fluorescence three-hybrid assay, we demonstrate that the recruitment of CHD4 to DNA damage, while being poly(ADP-ribosyl) ation-dependent, is not through binding poly(ADP-ribose). Additionally, we show that CHD3 is recruited to DNA breaks in the same manner as CHD4 and that both CHD3 and CHD4 play active roles in chromatin remodeling at DNA breaks. Together, our findings reveal a two-step mechanism for DNA damage induced chromatin relaxation in which PARP1 and the PAR-binding re-modeler activities of Alc1/CHD1L induce an initial chromatin relaxation phase that promotes the subsequent recruitment of CHD3 and CHD4 via binding to DNA for further chromatin remodeling at DNA breaks.


Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance.

Nyerges A, Csorgo B, Draskovits G, Kintses B, Szili P, Ferenc G, Revesz T, Ari E, Nagy I, Balint B, Vasarhelyi BM, Bihari P, Szamel M, Balogh D, Papp H, Kalapis D, Papp B, Pal C

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Antibiotic development is frequently plagued by the rapid emergence of drug resistance. However, assessing the risk of resistance development in the preclinical stage is difficult. Standard laboratory evolution approaches explore only a small fraction of the sequence space and fail to identify exceedingly rare resistance mutations and combinations thereof. Therefore, new rapid and exhaustive methods are needed to accurately assess the potential of resistance evolution and uncover the underlying mutational mechanisms. Here, we introduce directed evolution with random genomic mutations (DIvERGE), a method that allows an up to million-fold increase in mutation rate along the full lengths of multiple predefined loci in a range of bacterial species. In a single day, DIvERGE generated specific mutation combinations, yielding clinically significant resistance against trimethoprim and ciprofloxacin. Many of these mutations have remained previously undetected or provide resistance in a species-specific manner. These results indicate pathogen-specific resistance mechanisms and the necessity of future narrow-spectrum antibacterial treatments. In contrast to prior claims, we detected the rapid emergence of resistance against gepotidacin, a novel antibiotic currently in clinical trials. Based on these properties, DIvERGE could be applicable to identify less resistance-prone antibiotics at an early stage of drug development. Finally, we discuss potential future applications of DIvERGE in synthetic and evolutionary biology.


Foe or friend? Janus-faces of the neurovascular unit in the formation of brain metastases.

Wilhelm I, Fazakas C, Molnar K, Vegh AG, Hasko J, Krizbai IA

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Despite the potential obstacle represented by the blood-brain barrier for extravasating malignant cells, metastases are more frequent than primary tumors in the central nervous system. Not only tightly interconnected endothelial cells can hinder metastasis formation, other cells of the brain microenvironment (like astrocytes and microglia) can also be very hostile, destroying the large majority of metastatic cells. However, malignant cells that are able to overcome these harmful mechanisms may benefit from the shielding and even support provided by cerebral endothelial cells, astrocytes and microglia, rendering the brain a sanctuary site against anti-tumor strategies. Thus, cells of the neurovascular unit have a Janus-faced attitude towards brain metastatic cells, being both destructive and protective. In this review, we present the main mechanisms of brain metastasis formation, including those involved in extravasation through the brain vasculature and survival in the cerebral environment.

BRAIN BEHAVIOR AND IMMUNITY 64: pp. 220-231. (2017)

Expression of pattern recognition receptors and activation of the non-canonical inflammasome pathway in brain pericytes.

Nyul-Toth A, Kozma M, Nagyoszi P, Nagy K, Fazakas C, Hasko J, Molnar K, Farkas AE, Vegh AG, Varo G, Galajda P, Wilhelm I, Krizbai IA

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Cerebral pericytes are mural cells embedded in the basement membrane of capillaries. Increasing evidence suggests that they play important role in controlling neurovascular functions, i.e. cerebral blood flow, angiogenesis and permeability of the blood-brain barrier. These cells can also influence neuroinflammation which is highly regulated by the innate immune system. Therefore, we systematically tested the pattern recognition receptor expression of brain pericytes. We detected expression of NOD1, NOD2, NLRC5, NLRP1-3, NLRP5, NLRP9, NLRP10 and NLRX mRNA in non-treated cells. Among the ten known human TLRs, TLR2, TLR4, TLR5, TLR6 and TLR10 were found to be expressed. Inflammatory mediators induced the expression of NLRA, NLRC4 and TLR9 and increased the levels of NOD2, TLR2, inflammasome-forming caspases and inflammasome-cleaved interleukins. Oxidative stress, on the other hand, upregulated expression of TLR10 and NLRP9. Activation of selected pattern recognition receptors can lead to inflammasome assembly and caspase-dependent secretion of IL-1beta. TNF-alpha and IFN-gamma increased the levels of pro-IL-1beta and pro-caspase-1 proteins; however, no canonical activation of NLRP1, NLRP2, NLRP3 or NLRC4 inflammasomes could be observed in human brain vascular pericytes. On the other hand, we could demonstrate secretion of active IL-1beta in response to non-canonical inflammasome activation, i.e. intracellular LPS or infection with E. coli bacteria. Our in vitro results indicate that pericytes might have an important regulatory role in neuroinflammation.

ELIFE 7: Paper e31700. 20 p. (2018)

Coordination of robust single cell rhythms in the Arabidopsis circadian clock via spatial waves of gene expression

Gould PD, Domijian M, Greenwood M, Tokuda IT, Rees H, Kozma-Bognar L, Hall AJW, Locke JCW

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The Arabidopsis circadian clock orchestrates gene regulation across the day/night cycle. Although a multiple feedback loop circuit has been shown to generate the 24-hr rhythm, it remains unclear how robust the clock is in individual cells, or how clock timing is coordinated across the plant. Here we examine clock activity at the single cell level across Arabidopsis seedlings over several days under constant environmental conditions. Our data reveal robust single cell oscillations, albeit desynchronised. In particular, we observe two waves of clock activity; one going down, and one up the root. We also find evidence of cell-to-cell coupling of the clock, especially in the root tip. A simple model shows that cell-to-cell coupling and our measured period differences between cells can generate the observed waves. Our results reveal the spatial structure of the plant clock and suggest that unlike the centralised mammalian clock, the Arabidopsis clock has multiple coordination points.

PLOS GENETICS 14:(4) Paper e1007359. 23 p. (2018)

Non-canonical role of the SNARE protein Ykt6 in autophagosome-lysosome fusion.

Takats S, Glatz G, Szenci G, Boda A, Horvath GV, Hegedus K, Kovacs AL, Juhasz G

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The autophagosomal SNARE Syntaxin17 (Syx17) forms a complex with Snap29 and Vamp7/8 to promote autophagosome-lysosome fusion via multiple interactions with the tethering complex HOPS. Here we demonstrate that, unexpectedly, one more SNARE (Ykt6) is also required for autophagosome clearance in Drosophila. We find that loss of Ykt6 leads to large-scale accumulation of autophagosomes that are unable to fuse with lysosomes to form autolysosomes. Of note, loss of Syx5, the partner of Ykt6 in ER-Golgi trafficking does not prevent autolysosome formation, pointing to a more direct role of Ykt6 in fusion. Indeed, Ykt6 localizes to lysosomes and autolysosomes, and forms a SNARE complex with Syx17 and Snap29. Interestingly, Ykt6 can be outcompeted from this SNARE complex by Vamp7, and we demonstrate that overexpression of Vamp7 rescues the fusion defect of ykt6 loss of function cells. Finally, a point mutant form with an RQ amino acid change in the zero ionic layer of Ykt6 protein that is thought to be important for fusion-competent SNARE complex assembly retains normal autophagic activity and restores full viability in mutant animals, unlike palmitoylation or farnesylation site mutant Ykt6 forms. As Ykt6 and Vamp7 are both required for autophagosome-lysosome fusion and are mutually exclusive subunits in a Syx17-Snap29 complex, these data suggest that Vamp7 is directly involved in membrane fusion and Ykt6 acts as a non-conventional, regulatory SNARE in this process.

CELL REPORTS 23:(8) pp. 2273-2282. (2018)

Ubiquitylation Dynamics of the Clock Cell Proteome and TIMELESS during a Circadian Cycle

Áron Szabó, Christian Papin, David Cornu, Elisabeth Chélot, Zoltán Lipinszki, Andor Udvardy, Virginie Redeker, Ugo Mayor, François Rouyer

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Circadian clocks have evolved as time-measuring molecular devices to help organisms adapt their physiology to daily changes in light and temperature. Transcriptional oscillations account for a large fraction of rhythmic protein abundance. However, cycling of various posttranslational modifications, such as ubiquitylation, also contributes to shape the rhythmic protein landscape. In this study, we used an in vivo ubiquitin labeling assay to investigate the circadian ubiquitylated proteome of Drosophila melanogaster. We find that cyclic ubiquitylation affects MEGATOR (MTOR), a chromatin-associated nucleoporin that, in turn, feeds back to regulate the core molecular oscillator. Furthermore, we show that the ubiquitin ligase subunits CULLIN-3 (CUL-3) and SUPERNUMERARY LIMBS (SLMB) cooperate for ubiquitylating the TIMELESS protein. These findings stress the importance of ubiquitylation pathways in the Drosophila circadian clock and reveal a key component of this system.

CELL REPORTS 22:(10) pp. 2541-2549. (2018)

PI3K/Akt Cooperates with Oncogenic Notch by Inducing Nitric Oxide-Dependent Inflammation

Villegas SN, Gombos R, Garcia-Lopez L, Gutierrez-Perez I, Garcia-Castillo J, Vallejo DM, Da Ros VG, Ballesta-Illan E, Mihaly J, Dominguez M

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The PI3K/Akt signaling pathway, Notch, and other oncogenes cooperate in the induction of aggressive cancers. Elucidating how the PI3K/Akt pathway facilitates tumorigenesis by other oncogenes may offer opportunities to develop drugs with fewer side effects than those currently available. Here, using an unbiased in vivo chemical genetic screen in Drosophila, we identified compounds that inhibit the activity of proinflammatory enzymes nitric oxide synthase (NOS) and lipoxygenase (LOX) as selective suppressors of Notch-PI3K/Akt cooperative oncogenesis. Tumor silencing of NOS and LOX signaling mirrored the antitumor effect of the hit compounds, demonstrating their participation in Notch-PI3K/Akt- induced tumorigenesis. Oncogenic PI3K/Akt signaling triggered inflammation and immunosuppression via aberrant NOS expression. Accordingly, activated Notch tumorigenesis was fueled by hampering the immune response or by NOS overexpression to mimic a protumorigenic environment. Our lead compound, the LOX inhibitor BW B70C, also selectively killed human leukemic cells by dampening the NOTCH1-PI3K/AKT-eNOS axis.


Cotranslational protein assembly imposes evolutionary constraints on homomeric proteins.

Natan E , Endoh T , Haim-Vilmovsky L , Flock T , Chalancon G , Hopper JTS , Kintses B , Horvath P , Daruka L , Fekete G , Pal C , Papp B , Oszi E , Magyar Z , Marsh JA , Elcock AH , Babu MM , Robinson CV , Sugimoto N , Teichmann SA

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Cotranslational protein folding can facilitate rapid formation of functional structures. However, it can also cause premature assembly of protein complexes, if two interacting nascent chains are in close proximity. By analyzing known protein structures, we show that homomeric protein contacts are enriched toward the C termini of polypeptide chains across diverse proteomes. We hypothesize that this is the result of evolutionary constraints for folding to occur before assembly. Using high-throughput imaging of protein homomers in Escherichia coli and engineered protein constructs with N- and C-terminal oligomerization domains, we show that, indeed, proteins with C-terminal homomeric interface residues consistently assemble more efficiently than those with N-terminal interface residues. Using in vivo, in vitro and in silico experiments, we identify features that govern successful assembly of homomers, which have implications for protein design and expression optimization.


Underground metabolism: network-level perspective and biotechnological potential.

Notebaart RA , Kintses B , Feist AM , Papp B

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A key challenge in molecular systems biology is understanding how new pathways arise during evolution and how to exploit them for biotechnological applications. New pathways in metabolic networks often evolve by recruiting weak promiscuous activities of pre-existing enzymes. Here we describe recent systems biology advances to map such 'underground' activities and to predict and analyze their contribution to new metabolic functions. Underground activities are prevalent in cellular metabolism and can form novel pathways that either enable evolutionary adaptation to new environments or provide bypass to genetic lesions. We also illustrate the potential of integrating computational models of underground metabolism and experimental approaches to study the evolution of novel metabolic phenotypes and advance the field of biotechnology.

SCIENCE TRANSLATIONAL MEDICINE 10:(429) Paper eaal3973. (2018)

High-throughput metabolomic analysis predicts mode of action of uncharacterized antimicrobial compounds.

Zampieri M, Szappanos B, Buchieri MV, Trauner A, Piazza I, Picotti P, Gagneux S, Borrell S, Gicquel B, Lelievre J, Papp B, Sauer U

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Rapidly spreading antibiotic resistance and the low discovery rate of new antimicrobial compounds demand more effective strategies for early drug discovery. One bottleneck in the drug discovery pipeline is the identification of the modes of action (MoAs) of new compounds. We have developed a rapid systematic metabolome profiling strategy to classify the MoAs of bioactive compounds. The method predicted MoA-specific metabolic responses in the nonpathogenic bacterium Mycobacterium smegmatis after treatment with 62 reference compounds with known MoAs and different metabolic and nonmetabolic targets. We then analyzed a library of 212 new antimycobacterial compounds with unknown MoAs from a drug discovery effort by the pharmaceutical company GlaxoSmithKline (GSK). More than 70% of these new compounds induced metabolic responses in M. smegmatis indicative of known MoAs, seven of which were experimentally validated. Only 8% (16) of the compounds appeared to target unconventional cellular processes, illustrating the difficulty in discovering new antibiotics with different MoAs among compounds used as monotherapies. For six of the GSK compounds with potentially new MoAs, the metabolome profiles suggested their ability to interfere with trehalose and lipid metabolism. This was supported by whole-genome sequencing of spontaneous drug-resistant mutants of the pathogen Mycobacterium tuberculosis and in vitro compound-proteome interaction analysis for one of these compounds. Our compendium of drug-metabolome profiles can be used to rapidly query the MoAs of uncharacterized antimicrobial compounds and should be a useful resource for the drug discovery community.

ELIFE 7: Paper e29845. 23 p. (2018)

Hsp70-associated chaperones have a critical role in buffering protein production costs.

Farkas Z, Kalapis D, Bodi Z, Szamecz B, Daraba A, Almasi K, Kovacs K, Boross G, Pal F, Horvath P, Balassa T, Molnar C, Pettko-Szandtner A, Klement E, Rutkai E, Szvetnik A, Papp B, Pal C

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Proteins are necessary for cellular growth. Concurrently, however, protein production has high energetic demands associated with transcription and translation. Here, we propose that activity of molecular chaperones shape protein burden, that is the fitness costs associated with expression of unneeded proteins. To test this hypothesis, we performed a genome-wide genetic interaction screen in baker's yeast. Impairment of transcription, translation, and protein folding rendered cells hypersensitive to protein burden. Specifically, deletion of specific regulators of the Hsp70-associated chaperone network increased protein burden. In agreement with expectation, temperature stress, increased mistranslation and a chemical misfolding agent all substantially enhanced protein burden. Finally, unneeded protein perturbed interactions between key components of the Hsp70-Hsp90 network involved in folding of native proteins. We conclude that specific chaperones contribute to protein burden. Our work indicates that by minimizing the damaging impact of gratuitous protein overproduction, chaperones enable tolerance to massive changes in genomic expression.

NATURE COMMUNICATIONS 9:(1) p. 226. (2018)

Intelligent image-based in situ single-cell isolation.

Brasko C, Smith K, Molnar C, Farago N, Hegedus L, Balind A, Balassa T, Szkalisity A, Sukosd F, Kocsis K, Balint B, Paavolainen L, Enyedi MZ,Nagy I, Puskas LG, Haracska L, Tamas G, Horvath P

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Quantifying heterogeneities within cell populations is important for many fields including cancer research and neurobiology; however, techniques to isolate individual cells are limited. Here, we describe a high-throughput, non-disruptive, and cost-effective isolation method that is capable of capturing individually targeted cells using widely available techniques. Using high-resolution microscopy, laser microcapture microscopy, image analysis, and machine learning, our technology enables scalable molecular genetic analysis of single cells, targetable by morphology or location within the sample.

REDOX BIOLOGY 14: pp. 439-449. (2018)

Menthol evokes Ca2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells

Nazıroğlu M, Blum W, Jósvay K, Çiğ B, Henzi T, Oláh Z, Vizler C, Schwaller B, Pecze L

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Menthol is a naturally occurring monoterpene alcohol possessing remarkable biological properties including antipruritic, analgesic, antiseptic, anti-inflammatory and cooling effects. Here, we examined the menthol-evoked Ca2+ signals in breast and prostate cancer cell lines. The effect of menthol (50–500 µM) was predicted to be mediated by the transient receptor potential ion channel melastatin subtype 8 (TRPM8). However, the intensity of menthol-evoked Ca2+ signals did not correlate with the expression levels of TRPM8 in breast and prostate cancer cells indicating a TRPM8-independent signaling pathway. Menthol-evoked Ca2+ signals were analyzed in detail in Du 145 prostate cancer cells, as well as in CRISPR/Cas9 TRPM8-knockout Du 145 cells. Menthol (500 µM) induced Ca2+ oscillations in both cell lines, thus independent of TRPM8, which were however dependent on the production of inositol trisphosphate. Results based on pharmacological tools point to an involvement of the purinergic pathway in menthol-evoked Ca2+ responses. Finally, menthol (50–500 µM) decreased cell viability and induced oxidative stress independently of the presence of TRPM8 channels, despite that temperature-evoked TRPM8-mediated inward currents were significantly decreased in TRPM8-knockout Du 145 cells compared to wild type Du 145 cells. © 2017 The Authors

MOLECULAR & CELLULAR PROTEOMICS 17:(1) pp. 2-17. (2018)

Analysis of Mammalian O-Glycopeptides-We Have Made a Good Start, but There is a Long Way to Go.

Darula Z, Medzihradszky KF

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Glycosylation is perhaps the most common post-translational modification. Recently there has been growing interest in cataloging the glycan structures, glycoproteins, and specific sites modified and deciphering the biological functions of glycosylation. Although the results are piling up for N-glycosylation, O-glycosylation is seriously trailing behind. In our review we reiterate the difficulties researchers have to overcome in order to characterize O-glycosylation. We describe how an ingenious cell engineering method delivered exciting results, and what could we gain from "wild-type" samples. Although we refer to the biological role(s) of O-glycosylation, we do not provide a complete inventory on this topic.

JOURNAL OF CELL BIOLOGY 217:(1) pp. 361-374. (2018)

Molecular mechanisms of developmentally programmed crinophagy in Drosophila.

Csizmadia T, Lorincz P, Hegedus K, Szeplaki S, Low P, Juhasz G

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At the onset of metamorphosis, Drosophila salivary gland cells undergo a burst of glue granule secretion to attach the forming pupa to a solid surface. Here, we show that excess granules evading exocytosis are degraded via direct fusion with lysosomes, a secretory granule-specific autophagic process known as crinophagy. We find that the tethering complex HOPS (homotypic fusion and protein sorting); the small GTPases Rab2, Rab7, and its effector, PLEKHM1; and a SNAP receptor complex consisting of Syntaxin 13, Snap29, and Vamp7 are all required for the fusion of secretory granules with lysosomes. Proper glue degradation within lysosomes also requires the Uvrag-containing Vps34 lipid kinase complex and the v-ATPase proton pump, whereas Atg genes involved in macroautophagy are dispensable for crinophagy. Our work establishes the molecular mechanism of developmentally programmed crinophagy in Drosophila and paves the way for analyzing this process in metazoans.

ANTIOXIDANTS & REDOX SIGNALING Paper ahead of print. 53 p. (2017)

Concentration does matter: The beneficial and potentially harmful effects of ascorbate in humans and plants

Tóth SZ, Lőrincz T, Szarka A

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Significance: Ascorbate (Asc) is an essential compound both in animals and plants, mostly due to its reducing properties, thereby playing a role in scavenging reactive oxygen species (ROS) and acting as a cofactor in various enzymatic reactions. Recent Advances: Growing number of evidence shows that excessive Asc accumulation may have negative effects on cellular functions both in humans and plants; inter alia it may negatively affect signaling mechanisms, cellular redox status, and contribute to the production of ROS via the Fenton reaction. Critical Issues: Both plants and humans tightly control cellular Asc levels, possibly via biosynthesis, transport, and degradation, to maintain them in an optimum concentration range, which, among other factors, is essential to minimize the potentially harmful effects of Asc. On the contrary, the Fenton reaction induced by a high-dose Asc treatment in humans enables a potential cancer-selective cell death pathway. Future Directions: The elucidation of Asc induced cancer selective cell death mechanisms may give us a tool to apply Asc in cancer therapy. On the contrary, the regulatory mechanisms controlling cellular Asc levels are also to be considered, for example, when aiming at generating crops with elevated Asc levels. Antioxid. Redox Signal. 00, 000-000.

PLANT CELL AND ENVIRONMENT 40:(11) pp. 2457-2468. (2017)

New insights of red light-induced development

Viczian A, Klose C, Adam E, Nagy F

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The red/far-red light absorbing photoreceptors phytochromes regulate development and growth and thus play an essential role in optimizing adaptation of the sessile plants to the ever-changing environment. Our understanding of how absorption of a red/far-red photon by phytochromes initiates/modifies diverse physiological responses has been steadily improving. Research performed in the last 5 years has been especially productive and led to significant conceptual changes about the mode of action of these photoreceptors. In this review, we focus on the phytochrome B photoreceptor, the major phytochrome species active in light-grown plants. We discuss how its light-independent inactivation (termed dark/thermal reversion), post-translational modification, including ubiquitination, phosphorylation and sumoylation, as well as heterodimerization with other phytochrome species modify red light-controlled physiological responses. Finally, we discuss how photobiological properties of phytochrome B enable this photoreceptor to function also as a thermosensor.

NATURE CELL BIOLOGY 19:(11) pp. 1326-1335. (2017)

Synthetic hydrogels for human intestinal organoid generation and colonic wound repair

Cruz-Acuna R, Quiros M, Farkas AE, Dedhia PH, Huang S, Siuda D, Garcia-Hernandez V, Miller AJ, Spence JR, Nusrat A, Garcia AJ

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In vitro differentiation of human intestinal organoids (HIOs) from pluripotent stem cells is an unparalleled system for creating complex, multicellular three-dimensional structures capable of giving rise to tissue analogous to native human tissue. Current methods for generating HIOs rely on growth in an undefined tumour-derived extracellular matrix (ECM), which severely limits the use of organoid technologies for regenerative and translational medicine. Here, we developed a fully defined, synthetic hydrogel based on a four-armed, maleimide-terminated poly(ethylene glycol) macromer that supports robust and highly reproducible in vitro growth and expansion of HIOs, such that three-dimensional structures are never embedded in tumour-derived ECM. We also demonstrate that the hydrogel serves as an injection vehicle that can be delivered into injured intestinal mucosa resulting in HIO engraftment and improved colonic wound repair. Together, these studies show proof-of-concept that HIOs may be used therapeutically to treat intestinal injury.

ACS PHOTONICS 4:(9) pp. 2171-2178. (2017)

Large band-edge Photocurrent Peak in thick methylammonium lead iodide photosensors with symmetric metal electrodes

Hantz P, Sarlós F, Jalsovszky I, Plesz B

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This study examines the photocurrent (photoconductivity) spectra of thick methylammonium lead iodide perovskite layers coupled to symmetric metal electrodes. A large photocurrent peak has been observed in the near-band-gap wavelength region. The origin of this current peak is related to an interplay between the penetration depths of light having a certain wavelength and its ability to raise the concentration of the charge carriers. A model including the generation, position-specific or uniform recombination, as well as diffusion of charge carrier pairs, made it possible to reproduce the experimental findings. Besides the microscopic approach, we also present a phenomenological model and a simulation providing the conductance for an arbitrary relative orientation of the illuminating beam and the field imposed by the electrodes. The results enabled us to outline the functioning principle of a sensor for determining the angle of incidence of electromagnetic radiation. Furthermore, a method for estimating optical absorption spectra from photocurrent spectra is delineated.

GENOME BIOLOGY 18: Paper 190. 17 p. (2017)

Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage.

Kulcsar PI, Talas A, Huszar K, Ligeti Z, Toth E, Weinhardt N, Fodor E, Welker E

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BACKGROUND: The propensity for off-target activity of Streptococcus pyogenes Cas9 (SpCas9) has been considerably decreased by rationally engineered variants with increased fidelity (eSpCas9; SpCas9-HF1). However, a subset of targets still generate considerable off-target effects. To deal specifically with these targets, we generated new "Highly enhanced Fidelity" nuclease variants (HeFSpCas9s) containing mutations from both eSpCas9 and SpCas9-HF1 and examined these improved nuclease variants side by side to decipher the factors that affect their specificities and to determine the optimal nuclease for applications sensitive to off-target effects. RESULTS: These three increased-fidelity nucleases can routinely be used only with perfectly matching 20-nucleotide-long spacers, a matching 5' G extension being more detrimental to their activities than a mismatching one. HeFSpCas9 exhibit substantially improved specificity for those targets for which eSpCas9 and SpCas9-HF1 have higher off-target propensity. The targets can also be ranked by their cleavability and off-target effects manifested by the increased fidelity nucleases. Furthermore, we show that the mutations in these variants may diminish the cleavage, but not the DNA-binding, of SpCas9s. CONCLUSIONS: No single nuclease variant shows generally superior fidelity; instead, for highest specificity cleavage, each target needs to be matched with an appropriate high-fidelity nuclease. We provide here a framework for generating new nuclease variants for targets that currently have no matching optimal nuclease, and offer a simple means for identifying the optimal nuclease for targets in the absence of accurate target-ranking prediction tools.

FRONTIERS IN IMMUNOLOGY 8: Paper 1369. 9 p. (2017)

Interaction of THP-1 monocytes with conidia and hyphae of different Curvularia strains

Tóth EJ, Boros É, Hoffmann A, Szebenyi C, Homa M, Nagy G, Vágvölgyi C, Nagy I, Papp T

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Interaction of the human monocytic cell line, THP-1 with clinical isolates of three Curvularia species were examined. Members of this filamentous fungal genus can cause deep mycoses emerging in both immunocompromised and immunocompetent patients. It was found that monocytes reacted only to the hyphal form of Curvularia lunata. Cells attached to the germ tubes and hyphae and production of elevated levels of interleukin (IL)-8 and IL-10 and a low level of TNF-alpha were measured. At the same time, monocytes failed to produce IL-6. This monocytic response, especially with the induction of the anti-inflammatory IL-10, correlates well to the observation that C. lunata frequently cause chronic infections even in immunocompetent persons. Despite the attachment to the hyphae, monocytes could not reduce the viability of the fungus and the significant decrease in the relative transcript level of HLA-DRA assumes the lack of antigen presentation of the fungus by this cell type. C. spicifera and C. hawaiiensis failed to induce the gathering of the cells or the production of any analyzed cytokines. Monocytes did not recognize conidia of Curvularia species, even when melanin was lacking in their cell wall.


Changes in aggregation states of light-harvesting complexes as a mechanism for modulating energy transfer in desert crust cyanobacteria

Bar Eyal L, Ranjbar Choubeh R, Cohen E, Eisenberg I, Tamburu C, Dorogi M, Unnep R, Appavou MS, Nevo R, Raviv U, Reich Z, Garab G, van Amerongen H, Paltiel Y, Keren N

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In this paper we propose an energy dissipation mechanism that is completely reliant on changes in the aggregation state of the phycobilisome light-harvesting antenna components. All photosynthetic organisms regulate the efficiency of excitation energy transfer (EET) to fit light energy supply to biochemical demands. Not many do this to the extent required of desert crust cyanobacteria. Following predawn dew deposition, they harvest light energy with maximum efficiency until desiccating in the early morning hours. In the desiccated state, absorbed energy is completely quenched. Time and spectrally resolved fluorescence emission measurements of the desiccated desert crust Leptolyngbya ohadii strain identified (i) reduced EET between phycobilisome components, (ii) shorter fluorescence lifetimes, and (iii) red shift in the emission spectra, compared with the hydrated state. These changes coincide with a loss of the ordered phycobilisome structure, evident from small-angle neutron and X-ray scattering and cryo-transmission electron microscopy data. Based on these observations we propose a model where in the hydrated state the organized rod structure of the phycobilisome supports directional EET to reaction centers with minimal losses due to thermal dissipation. In the desiccated state this structure is lost, giving way to more random aggregates. The resulting EET path will exhibit increased coupling to the environment and enhanced quenching.

PLANT PHYSIOLOGY 175:(1) pp. 555-567. (2017)

Proline accumulation is regulated by transcription factors associated with phosphate starvation

Aleksza D, Horvath GV, Sandor G, Szabados L

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Proline accumulation in plants is a well-documented physiological response to osmotic stress caused by drought or salinity. In Arabidopsis thaliana the stress and ABA-induced Delta1-PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 (P5CS1) gene was previously shown to control proline biosynthesis in such adverse conditions. To identify regulatory factors which control the transcription of P5CS1, yeast one hybrid (Y1H) screens were performed with a genomic fragment of P5CS1, containing 1.2 kB promoter and 0.8 kB transcribed regions. The transcription factors PHOSPHATE STARVATION RESPONSE 1 (PHR1) and PHR1-LIKE 1 (PHL1) were identified to bind to P5CS1 sequences in the first intron, which carry a conserved PHR1-binding site (P1BS) motif. PHR1 and PHL1 binding to P1BS was confirmed by Y1H, electrophoretic mobility assay (EMSA) and chromatin immune precipitation (ChIP). Phosphate starvation led to gradual increase in proline content in wild type Arabidopsis plants as well as transcriptional activation of P5CS1 and PROLINE DEHYDROGENASE 2 (PDH2) genes. Induction of P5CS1 transcription and proline accumulation during phosphate deficiency was considerably reduced by phr1 and phl1 mutations and was impaired in the ABA deficient aba2-3 and ABA insensitive abi4-1 mutants. Growth and viability of phr1phl1 double mutant was reduced in phosphate-depleted medium, it was slightly diminished in the aba2-3 mutant, suggesting that ABA mediates growth retardation in such stress. Our results reveal a previously unknown link between proline metabolism and phosphate nutrition, and show that proline biosynthesis is target of crosstalk between ABA signaling and regulation of phosphate homeostasis through PHR1 and PHL1-mediated transcriptional activation of the P5CS1 gene.

PLOS GENETICS 13:(8) Paper e1006968. 35 p. (2017)

Kek-6: A truncated-Trk-like receptor for Drosophila neurotrophin 2 regulates structural synaptic plasticity

Ulian-Benitez S, Bishop S, Foldi I, Wentzell J, Okenwa C, Forero MG, Zhu BF, Moreira M, Phizacklea M, McIlroy G, Li GY, Gay NJ, Hidalgo A

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Neurotrophism, structural plasticity, learning and long-term memory in mammals critically depend on neurotrophins binding Trk receptors to activate tyrosine kinase (TyrK) signaling, but Drosophila lacks full-length Trks, raising the question of how these processes occur in the fly. Paradoxically, truncated Trk isoforms lacking the TyrK predominate in the adult human brain, but whether they have neuronal functions independently of full-length Trks is unknown. Drosophila has TyrK-less Trk-family receptors, encoded by the kekkon (kek) genes, suggesting that evolutionarily conserved functions for this receptor class may exist. Here, we asked whether Keks function together with Drosophila neurotrophins (DNTs) at the larval glutamatergic neuromuscular junction (NMJ). We tested the eleven LRR and Igcontaining (LIG) proteins encoded in the Drosophila genome for expression in the central nervous system (CNS) and potential interaction with DNTs. Kek-6 is expressed in the CNS, interacts genetically with DNTs and can bind DNT2 in signaling assays and co-immunoprecipitations. Ligand binding is promiscuous, as Kek-6 can also bind DNT1, and Kek-2 and Kek-5 can also bind DNT2. In vivo, Kek-6 is found presynaptically in motoneurons, and DNT2 is produced by the muscle to function as a retrograde factor at the NMJ. Kek-6 and DNT2 regulate NMJ growth and synaptic structure. Evidence indicates that Kek-6 does not antagonise the alternative DNT2 receptor Toll-6. Instead, Kek-6 and Toll-6 interact physically, and together regulate structural synaptic plasticity and homeostasis. Using pull-down assays, we identified and validated CaMKII and VAP33A as intracellular partners of Kek-6, and show that they regulate NMJ growth and active zone formation downstream of DNT2 and Kek-6. The synaptic functions of Kek-6 could be evolutionarily conserved. This raises the intriguing possibility that a novel mechanism of structural synaptic plasticity involvingtruncated Trk-family receptors independently of TyrK signaling may also operate in the human brain.

ELIFE 6: Paper e29942. 3 p. (2017)


Moeller GK, Timinszky G

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The modification of serines by molecules of ADP-ribose plays an important role in signaling that the DNA in a cell has been damaged and needs to be repaired.

EMBO JOURNAL 36:(13) pp. 1811-1836. (2017)

Molecular definitions of autophagy and related processes.

Galluzzi L, Baehrecke EH, Juhasz G, Kroemer G

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Over the past two decades, the molecular machinery that underlies autophagic responses has been characterized with ever increasing precision in multiple model organisms. Moreover, it has become clear that autophagy and autophagy-related processes have profound implications for human pathophysiology. However, considerable confusion persists about the use of appropriate terms to indicate specific types of autophagy and some components of the autophagy machinery, which may have detrimental effects on the expansion of the field. Driven by the overt recognition of such a potential obstacle, a panel of leading experts in the field attempts here to define several autophagy-related terms based on specific biochemical features. The ultimate objective of this collaborative exchange is to formulate recommendations that facilitate the dissemination of knowledge within and outside the field of autophagy research.

JOURNAL OF CELL BIOLOGY 216:(7) pp. 1937-1947. (2017)

Rab2 promotes autophagic and endocytic lysosomal degradation.

Lorincz P, Toth S, Benko P, Lakatos Z, Boda A, Glatz G, Zobel M, Bisi S, Hegedus K, Takats S, Scita G, Juhasz G

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Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes.

CELL SYSTEMS 4:(6) pp. 651-655. (2017)

Advanced Cell Classifier: User-Friendly Machine-Learning-Based Software for Discovering Phenotypes in High-Content Imaging Data.

Piccinini F, Balassa T, Szkalisity A, Molnar C, Paavolainen L, Kujala K, Buzas K, Sarazova M, Pietiainen V, Kutay U, Smith K, Horvath P

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High-content, imaging-based screens now routinely generate data on a scale that precludes manual verification and interrogation. Software applying machine learning has become an essential tool to automate analysis, but these methods require annotated examples to learn from. Efficiently exploring large datasets to find relevant examples remains a challenging bottleneck. Here, we present Advanced Cell Classifier (ACC), a graphical software package for phenotypic analysis that addresses these difficulties. ACC applies machine-learning and image-analysis methods to high-content data generated by large-scale, cell-based experiments. It features methods to mine microscopic image data, discover new phenotypes, and improve recognition performance. We demonstrate that these features substantially expedite the training process, successfully uncover rare phenotypes, and improve the accuracy of the analysis. ACC is extensively documented, designed to be user-friendly for researchers without machine-learning expertise, and distributed as a free open-source tool at www.cellclassifier.org.


A Novel ‘Splice Site’ HCN4 Gene mutation, c.1737+1 G>T, Causes Familial Bradycardia, Reduced Heart Rate Response, Impaired Chronotropic Competence and Increased Short-Term Heart Rate Variability

Lidia Hategan, Beáta Csányi, Balázs Ördög, Kornél Kákonyi, Annamária Tringer, Orsolya Kiss, Andrea Orosz, László Sághy, István Nagy, Zoltán Hegedűs, László Rudas, Márta Széll, András Varró, Tamás Forster, Róbert Sepp

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Background: The most important molecular determinant of heart rate regulation in sino-atrial pacemaker cells includes hyperpolarization-activated, cyclic nucleotide-gated ion channels, the major isoform of which is encoded by the HCN4 gene. Mutations affecting the HCN4 gene are associated primarily with sick sinus syndrome.
Methods and results: A novel c. 1737+ 1 G>T 'splice-site' HCN4 mutation was identified in a large family with familial bradycardia which co-segregated with the disease providing a two-point LOD score of 4.87. Twelve out of the 22 investigated familymembers [4 males, 8 females average age 36 (SD 6) years] were considered as clinically affected (heart rate < 60/min on resting ECG). Minimum[36 (SD 7) vs. 47 (SD 5) bpm, p = 0.0087) and average heart rates [62 (SD 8) vs. 73 (SD 8) bpm, p = 0.0168) were significantly lower in carriers on 24-hour Holter recordings. Under maximum exercise test carriers achieved significantly lower heart rates than non-carrier family members, and percent heart rate reserve and percent corrected heart rate reserve were significantly lower in carriers. Applying rigorous criteria for chronotropic incompetence a higher number of carriers exhibited chronotropic incompetence. Parameters, characterizing short-term variability of heart rate (i.e. rMSSD and pNN50%) were increased in carrier family members, even after normalization for heart rate, in the 24-hour ECG recordings with the same relative increase in 5-minute recordings.
Conclusions: The identified novel 'splice-site' HCN4 gene mutation, c. 1737+ 1 G>T, causes familial bradycardia and leads to reduced heart rate response, impaired chronotropic competence and increased short-term heart rate variability in the mutation carriers. (C) 2017 Elsevier Ireland Ltd. All rights reserved.


Host-secreted antimicrobial peptide enforces symbiotic selectivity in Medicago truncatula.

Wang Q, Yang S, Liu J, Terecskei K, Abraham E, Gombar A, Domonkos A, Szucs A, Kormoczi P, Wang T, Fodor L, Mao L, Fei Z, Kondorosi E, Kalo P, Kereszt A, Zhu H

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Legumes engage in root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. In nodule cells, bacteria are enclosed in membrane-bound vesicles called symbiosomes and differentiate into bacteroids that are capable of converting atmospheric nitrogen into ammonia. Bacteroid differentiation and prolonged intracellular survival are essential for development of functional nodules. However, in the Medicago truncatula-Sinorhizobium meliloti symbiosis, incompatibility between symbiotic partners frequently occurs, leading to the formation of infected nodules defective in nitrogen fixation (Fix-). Here, we report the identification and cloning of the M. truncatula NFS2 gene that regulates this type of specificity pertaining to S. meliloti strain Rm41. We demonstrate that NFS2 encodes a nodule-specific cysteine-rich (NCR) peptide that acts to promote bacterial lysis after differentiation. The negative role of NFS2 in symbiosis is contingent on host genetic background and can be counteracted by other genes encoded by the host. This work extends the paradigm of NCR function to include the negative regulation of symbiotic persistence in host-strain interactions. Our data suggest that NCR peptides are host determinants of symbiotic specificity in M. truncatula and possibly in closely related legumes that form indeterminate nodules in which bacterial symbionts undergo terminal differentiation.

PLANT CELL AND ENVIRONMENT 40:(7) pp. 1104-1114. (2017)

Expression of the UVR8 photoreceptor in different tissues reveals tissue-autonomous features of UV-B signalling

Bernula P, Crocco CD, Arongaus AB, Ulm R, Nagy F, Viczian A

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The Arabidopsis UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) orchestrates the expression of hundreds of genes, many of which can be associated with UV-B tolerance. UV-B does not efficiently penetrate into tissues, yet UV-B regulates complex growth and developmental responses. To unravel to what extent and how UVR8 located in different tissues contributes to UV-B-induced responses, we expressed UVR8 fused to the YELLOW FLUORESCENT PROTEIN (YFP) under the control of tissue-specific promoters in a uvr8 null mutant background. We show that (1) UVR8 localized in the epidermis plays a major role in regulating cotyledon expansion, and (2) expression of UVR8 in the mesophyll is important to protect adult plants from the damaging effects of UV-B. We found that UV-B induces transcription of selected genes, including the key transcriptional regulator ELONGATED HYPOCOTYL 5 (HY5), only in tissues that express UVR8. Thus, we suggest that tissue-autonomous and simultaneous UVR8 signalling in different tissues mediates, at least partly, developmental and defence responses to UV-B.

PLOS BIOLOGY 15:(5) Paper e2000644. 26 p. (2017)

Phenotypic heterogeneity promotes adaptive evolution

Bodi Z, Farkas Z, Nevozhay D, Kalapis D, Lazar V, Csorgo B, Nyerges A, Szamecz B, Fekete G, Papp B, Araujo H, Oliveira JL, Moura G, Santos MAS, Szekely T, Balazsi G, Pal C

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Genetically identical cells frequently display substantial heterogeneity in gene expression, cellular morphology and physiology. It has been suggested that by rapidly generating a subpopulation with novel phenotypic traits, phenotypic heterogeneity (or plasticity) accelerates the rate of adaptive evolution in populations facing extreme environmental challenges. This issue is important as cell-to-cell phenotypic heterogeneity may initiate key steps in microbial evolution of drug resistance and cancer progression. Here, we study how stochastic transitions between cellular states influence evolutionary adaptation to a stressful environment in yeast Saccharomyces cerevisiae. We developed inducible synthetic gene circuits that generate varying degrees of expression stochasticity of an antifungal resistance gene. We initiated laboratory evolutionary experiments with genotypes carrying different versions of the genetic circuit by exposing the corresponding populations to gradually increasing antifungal stress. Phenotypic heterogeneity altered the evolutionary dynamics by transforming the adaptive landscape that relates genotype to fitness. Specifically, it enhanced the adaptive value of beneficial mutations through synergism between cell-to-cell variability and genetic variation. Our work demonstrates that phenotypic heterogeneity is an evolving trait when populations face a chronic selection pressure. It shapes evolutionary trajectories at the genomic level and facilitates evolutionary rescue from a deteriorating environmental stress.

BIOTECHNOLOGY FOR BIOFUELS 10: Paper 116. 5 p. (2017)

On the pathways feeding the H-2 production process in nutrient-replete, hypoxic conditions. Commentary on the article "Low oxygen levels contribute to improve photohydrogen production in mixotrophic non-stressed Chlamydomonas cultures", by Jurado-Oller et al., Biotechnology for Biofuels, published September 7, 2015; 8: 149

Scoma A, Toth SZ

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Background: Under low O-2 concentration ( hypoxia) and low light, Chlamydomonas cells can produce H-2 gas in nutrient-replete conditions. This process is hindered by the presence of O-2, which inactivates the [FeFe]-hydrogenase enzyme responsible for H-2 gas production shifting algal cultures back to normal growth. The main pathways accounting for H-2 production in hypoxia are not entirely understood, as much as culture conditions setting the optimal redox state in the chloroplast supporting long-lasting H-2 production. The reducing power for H-2 production can be provided by photosystem II (PSII) and photofermentative processes during which proteins are degraded via yet unknown pathways. In hetero- or mixotrophic conditions, acetate respiration was proposed to indirectly contribute to H-2 evolution, although this pathway has not been described in detail.
Main body: Recently, Jurado-Oller et al. (Biotechnol Biofuels 8: 149, 7) proposed that acetate respiration may substantially support H-2 production in nutrient-replete hypoxic conditions. Addition of low amounts of O-2 enhanced acetate respiration rate, particularly in the light, resulting in improved H-2 production. The authors surmised that acetate oxidation through the glyoxylate pathway generates intermediates such as succinate and malate, which would be in turn oxidized in the chloroplast generating FADH(2) and NADH. The latter would enter a PSII-independent pathway at the level of the plastoquinone pool, consistent with the light dependence of H-2 production. The authors concluded that the water-splitting activity of PSII has a minor role in H-2 evolution in nutrient-replete, mixotrophic cultures under hypoxia. However, their results with the PSII inhibitor DCMU also reveal that O-2 or acetate additions promoted acetate respiration over the usually dominant PSII-dependent pathway. The more oxidized state experienced by these cultures in combination with the relatively short experimental time prevented acclimation to hypoxia, thus precluding the PSII-dependent pathway from contributing to H-2 production.
Conclusions: In Chlamydomonas, continuous H-2 gas evolution is expected once low O-2 partial pressure and optimal reducing conditions are set. Under nutrient-replete conditions, the electrogenic processes involved in H-2 photoproduction may rely on various electron transport pathways. Understanding how physiological conditions select for specific metabolic routes is key to achieve economic viability of this renewable energy source.

PLANT PHYSIOLOGY 173:(3) pp. 1750-1762. (2017)

ABA Suppresses Root Hair Growth via the OBP4 Transcriptional Regulator

Rymen B, Kawamura A, Schafer S, Breuer C, Iwase A, Shibata M, Ikeda M, Mitsuda N, Koncz C, Ohme-Takagi M, Matsui M, Sugimoto K

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Plants modify organ growth and tune morphogenesis in response to various endogenous and environmental cues. At the cellular level, organ growth is often adjusted by alterations in cell growth, but the molecular mechanisms underlying this control remain poorly understood. In this study, we identify the DNA BINDING WITH ONE FINGER (DOF)-type transcription regulator OBF BINDING PROTEIN4 (OBP4) as a repressor of cell growth. Ectopic expression of OBP4 in Arabidopsis (Arabidopsis thaliana) inhibits cell growth, resulting in severe dwarfism and the repression of genes involved in the regulation of water transport, root hair development, and stress responses. Among the basic helix-loop-helix transcription factors known to control root hair growth, OBP4 binds the ROOT HAIR DEFECTIVE6-LIKE2 (RSL2) promoter to repress its expression. The accumulation of OBP4 proteins is detected in expanding root epidermal cells, and its expression level is increased by the application of abscisic acid (ABA) at concentrations sufficient to inhibit root hair growth. ABA-dependent induction of OBP4 is associated with the reduced expression of RSL2. Furthermore, ectopic expression of OBP4 or loss of RSL2 function results in ABA-insensitive root hair growth. Taken together, our results suggest that OBP4-mediated transcriptional repression of RSL2 contributes to the ABA-dependent inhibition of root hair growth in Arabidopsis.


Morphotype of bacteroids in different legumes correlates with the number and type of symbiotic NCR peptides

Montiel J, Downie JA, Farkas A, Bihari P, Herczeg R, Balint B, Mergaert P, Kereszt A, Kondorosi E

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In legume nodules, rhizobia differentiate into nitrogen-fixing forms called bacteroids, which are enclosed by a plant membrane in an organelle-like structure called the symbiosome. In the Inverted Repeat-Lacking Clade (IRLC) of legumes, this differentiation is terminal due to irreversible loss of cell division ability and is associated with genome amplification and different morphologies of the bacteroids that can be swollen, elongated, spherical, and elongatedbranched, depending on the host plant. In Medicago truncatula, this process is orchestrated by nodule-specific cysteine-rich peptides (NCRs) delivered into developing bacteroids. Here, we identified the predicted NCR proteins in 10 legumes representing different subclades of the IRLC with distinct bacteroid morphotypes. Analysis of their expression and predicted sequences establishes correlations between the composition of the NCR family and the morphotypes of bacteroids. Although NCRs have a single origin, their evolution has followed different routes in individual lineages, and enrichment and diversification of cationic peptides has resulted in the ability to impose major morphological changes on the endosymbionts. The wide range of effects provoked by NCRs such as cell enlargement, membrane alterations and permeabilization, and biofilm and vesicle formation is dependent on the amino acid composition and charge of the peptides. These effects are strongly influenced by the rhizobial surface polysaccharides that affect NCR-induced differentiation and survival of rhizobia in nodule cells.

JOURNAL OF CELL BIOLOGY 216:(5) pp. 1421-1438. (2017)

Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila

Foldi I, Anthoney N, Harrison N, Gangloff M, Verstak B, Nallasivan MP, AlAhmed S, Zhu BF, Phizacklea M, Losada-Perez M, Moreira M, Gay NJ, Hidalgo A

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Cell number plasticity is coupled to circuitry in the nervous system, adjusting cell mass to functional requirements. In mammals, this is achieved by neurotrophin (NT) ligands, which promote cell survival via their Trk and p75NTR receptors and cell death via p75NTR and Sortilin. Drosophila NTs (DNTs) bind Toll receptors instead to promote neuronal survival, but whether they can also regulate cell death is unknown. In this study, we show that DNTs and Tolls can switch from promoting cell survival to death in the central nervous system (CNS) via a three-tier mechanism. First, DNT cleavage patterns result in alternative signaling outcomes. Second, different Tolls can preferentially promote cell survival or death. Third, distinct adaptors downstream of Tolls can drive either apoptosis or cell survival. Toll-6 promotes cell survival via MyD88-NF-kappa B and cell death via Wek-Sarm-JNK. The distribution of adaptors changes in space and time and may segregate to distinct neural circuits. This novel mechanism for CNS cell plasticity may operate in wider contexts.

EMBO JOURNAL 36:(9) pp. 1261-1278. (2017)

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control

Horvath BM, Kourova H, Nagy S, Nemeth E, Magyar Z, Papdi C, Ahmad Z, Sanchez-Perez GF, Perilli S, Blilou I, Pettkó-Szandtner A, Darula Z, Meszaros T, Binarova P, Bogre L, Scheres B

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The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta. Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.

NATURE COMMUNICATIONS 8: Paper 15127. 12 p. (2017)

De novo evolved interference competition promotes the spread of biofilm defectors

Martin M, Dragos A, Holscher T, Maroti G, Balint B, Westermann M, Kovacs AT

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Biofilms are social entities where bacteria live in tightly packed agglomerations, surrounded by self-secreted exopolymers. Since production of exopolymers is costly and potentially exploitable by non-producers, mechanisms that prevent invasion of non-producing mutants are hypothesized. Here we study long-term dynamics and evolution in Bacillus subtilis biofilm populations consisting of wild-type (WT) matrix producers and mutant non-producers. We show that non-producers initially fail to incorporate into biofilms formed by the WT cells, resulting in 100-fold lower final frequency compared to the WT. However, this is modulated in a long-term scenario, as non-producers evolve the ability to better incorporate into biofilms, thereby slightly decreasing the productivity of the whole population. Detailed molecular analysis reveals that the unexpected shift in the initially stable biofilm is coupled with newly evolved phage-mediated interference competition. Our work therefore demonstrates how collective behaviour can be disrupted as a result of rapid adaptation through mobile genetic elements.


Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression

Nagymihaly M, Veluchamy A, Gyorgypal Z, Ariel F, Jegu T, Benhamed M, Szucs A, Kereszt A, Mergaert P, Kondorosi E

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The formation of symbiotic nodule cells in Medicago truncatula is driven by successive endoreduplication cycles and transcriptional reprogramming in different temporal waves including the activation of more than 600 cysteine-rich NCR genes expressed only in nodules. We show here that the transcriptional waves correlate with growing ploidy levels and have investigated how the epigenome changes during endoreduplication cycles. Differential DNA methylation was found in only a small subset of symbiotic nodule-specific genes, including more than half of the NCR genes, whereas in most genes DNA methylation was unaffected by the ploidy levels and was independent of the genes' active or repressed state. On the other hand, expression of nodule-specific genes correlated with ploidy-dependent opening of the chromatin as well as, in a subset of tested genes, with reduced H3K27me3 levels combined with enhanced H3K9ac levels. Our results suggest that endoreduplication-dependent epigenetic changes contribute to transcriptional reprogramming in the differentiation of symbiotic cells.

NEW PHYTOLOGIST 214:(2) pp. 668-681. (2017)

Expression of the eRF1 translation termination factor is controlled by an autoregulatory circuit involving readthrough and nonsense-mediated decay in plants

Nyiko T, Auber A, Szabadkai L, Benkovics A, Auth M, Merai Z, Kerenyi Z, Dinnyes A, Nagy F, Silhavy D

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When a ribosome reaches a stop codon, the eukaryotic Release Factor 1 (eRF1) binds to the A site of the ribosome and terminates translation. In yeasts and plants, both over- and underexpression of eRF1 lead to altered phenotype indicating that eRF1 expression should be strictly controlled. However, regulation of eRF1 level is still poorly understood. Here we show that expression of plant eRF1 is controlled by a complex negative autoregulatory circuit, which is based on the unique features of the 3'untranslated region (3'UTR) of the eRF1-1 transcript. The stop codon of the eRF1-1 mRNA is in a translational readthrough promoting context, while its 3'UTR induces nonsense-mediated decay (NMD), a translation termination coupled mRNA degradation mechanism. We demonstrate that readthrough partially protects the eRF1-1 mRNA from its 3'UTR induced NMD, and that elevated eRF1 levels inhibit readthrough and stimulate NMD. Thus, high eRF1 level leads to reduced eRF1-1 expression, as weakened readthrough fails to protect the eRF1-1 mRNA from the more intense NMD. This eRF1 autoregulatory circuit might serve to finely balance general translation termination efficiency.

NEW PHYTOLOGIST 214:(2) pp. 668-681. (2017)

Regulation of ascorbate biosynthesis in green algae has evolved to enable rapid stress-induced response via the VTC2 gene encoding GDP-l-galactose phosphorylase

Vidal-Meireles A, Neupert J, Zsigmond L, Rosado-Souza L, Kovacs L, Nagy V, Galambos A, Fernie AR, Bock R, Toth SZ

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Ascorbate (vitamin C) plays essential roles in stress resistance, development, signaling, hormone biosynthesis and regulation of gene expression; however, little is known about its biosynthesis in algae. In order to provide experimental proof for the operation of the Smirnoff-Wheeler pathway described for higher plants and to gain more information on the regulation of ascorbate biosynthesis in Chlamydomonas reinhardtii, we targeted the VTC2 gene encoding GDP-l-galactose phosphorylase using artificial microRNAs. Ascorbate concentrations in VTC2 amiRNA lines were reduced to 10% showing that GDP-l-galactose phosphorylase plays a pivotal role in ascorbate biosynthesis. The VTC2 amiRNA lines also grow more slowly, have lower chlorophyll content, and are more susceptible to stress than the control strains. We also demonstrate that: expression of the VTC2 gene is rapidly induced by H2 O2 and 1 O2 resulting in a manifold increase in ascorbate content; in contrast to plants, there is no circadian regulation of ascorbate biosynthesis; photosynthesis is not required per se for ascorbate biosynthesis; and Chlamydomonas VTC2 lacks negative feedback regulation by ascorbate in the physiological concentration range. Our work demonstrates that ascorbate biosynthesis is also highly regulated in Chlamydomonas albeit via mechanisms distinct from those previously described in land plants.

MOLECULAR BIOLOGY AND EVOLUTION 34:(2) pp. 380-390. (2017)

No Evidence That Protein Noise-Induced Epigenetic Epistasis Constrains Gene Expression Evolution

Boross G, Papp B

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Changes in gene expression can affect phenotypes and therefore both its level and stochastic variability are frequently under selection. It has recently been proposed that epistatic interactions influence gene expression evolution: gene pairs where simultaneous knockout is more deleterious than expected should evolve reduced expression noise to avoid concurrent low expression of both proteins. In apparent support, yeast genes with many epistatic partners have low expression variation both among isogenic individuals and between species. However, the specific predictions and basic assumptions of this verbal model remain untested. Using bioinformatics analysis, we first demonstrate that the model's predictions are unsupported by available large-scale data. Based on quantitative biochemical modeling, we then show that epistasis between expression reductions (epigenetic epistasis) is not expected to aggravate the fitness cost of stochastic expression, which is in sharp contrast to the verbal argument. This nonintuitive result can be readily explained by the typical diminishing return of fitness on gene activity and by the fact that expression noise not only decreases but also increases the abundance of proteins. Overall, we conclude that stochastic variation in epistatic partners is unlikely to drive noise minimization or constrain gene expression divergence on a genomic scale.

PLANT CELL AND ENVIRONMENT 40:(3) pp. 378-389. (2017)

Dissecting the Photoprotective Mechanism Encoded by the flv4-2 Operon: a Distinct Contribution of Sll0218 in Photosystem II Stabilization

Bersanini L, Allahverdiyeva Y, Battchikova N, Heinz S, Lespinasse M, Ruohisto E, Mustila H, Nickelsen J, Vass I, Aro EM

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In Synechocystis sp. PCC 6803, the flv4-2 operon encodes the flavodiiron proteins Flv2 and Flv4 together with a small protein, Sll0218, providing photoprotection for Photosystem II (PSII). Here, the distinct roles of Flv2/Flv4 and Sll0218 were addressed, using a number of flv4-2 operon mutants. In the sll0218 mutant, the presence of Flv2/Flv4 rescued PSII functionality as compared with sll0218-flv2, where neither Sll0218 nor the Flv2/Flv4 heterodimer are expressed. Nevertheless, both the sll0218 and sll0218-flv2 mutants demonstrated deficiency in accumulation of PSII proteins suggesting a role for Sll0218 in PSII stabilization, which was further supported by photoinhibition experiments. Moreover, the accumulation of PSII assembly intermediates occurred in Sll0218-lacking mutants. The YFP-tagged Sll0218 protein localized in a few spots per cell at the external side of the thylakoid membrane, and biochemical membrane fractionation revealed clear enrichment of Sll0218 in the PratA-defined membranes, where the early biogenesis steps of PSII occur. Further, the characteristic antenna uncoupling feature of the flv4-2 operon mutants is shown to be related to PSII destabilization in the absence of Sll0218. It is concluded that the Flv2/Flv4 heterodimer supports PSII functionality, while the Sll0218 protein assists PSII assembly and stabilization, including optimization of light harvesting. This work clarifies and dissects the roles of the flv4-2 operon-encoded proteins, Flv2/Flv4 heterodimer and the elusive Sll0218, in photoprotection of the photosynthetic apparatus in Synechosystis. While Flv2/Flv4 heterodimer is involved in an alternative electron transfer route, the Sll0218 protein is localized to specific cell compartments where photosynthetic complexes are assembled, and it is involved in the stabilization of Photosystem II complexes.

NUCLEIC ACIDS RESEARCH 45:(1) Paper gkw1315. 17 p. (2017)

DNA-dependent protease activity of human Spartan facilitates replication of DNA-protein crosslink-containing DNA.

Morocz M, Zsigmond E, Toth R, Enyedi MZ, Pinter L, Haracska L

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Mutations in SPARTAN are associated with early onset hepatocellular carcinoma and progeroid features. A regulatory function of Spartan has been implicated in DNA damage tolerance pathways such as translesion synthesis, but the exact function of the protein remained unclear. Here, we reveal the role of human Spartan in facilitating replication of DNA-protein crosslink-containing DNA. We found that purified Spartan has a DNA-dependent protease activity degrading certain proteins bound to DNA. In concert, Spartan is required for direct DPC removal in vivo; we also show that the protease Spartan facilitates repair of formaldehyde-induced DNA-protein crosslinks in later phases of replication using the bromodeoxyuridin (BrdU) comet assay. Moreover, DNA fibre assay indicates that formaldehyde-induced replication stress dramatically decreases the speed of replication fork movement in Spartan-deficient cells, which accumulate in the G2/M cell cycle phase. Finally, epistasis analysis mapped these Spartan functions to the RAD6-RAD18 DNA damage tolerance pathway. Our results reveal that Spartan facilitates replication of DNA-protein crosslink-containing DNA enzymatically, as a protease, which may explain its role in preventing carcinogenesis and aging.

BLOOD 129:(11) pp. E26-E37. (2017)

Ex vivo drug response profiling detects recurrent sensitivity patterns in drug-resistant acute lymphoblastic leukemia

Frismantas V, Dobay MP, Horvath P, Bourquin JP

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Drug sensitivity and resistance testing on diagnostic leukemia samples should provide important functional information to guide actionable target and biomarker discovery. We provide proof of concept data by profiling 60 drugs on 68 acute lymphoblastic leukemia (ALL) samples mostly from resistant disease in cocultures of bone marrow stromal cells. Patient-derived xenografts retained the original pattern of mutations found in the matched patient material. Stromal coculture did not prevent leukemia cell cycle activity, but a specific sensitivity profile to cell cycle-related drugs identified samples with higher cell proliferation both in vitro and in vivo as leukemia xenografts. In patients with refractory relapses, individual patterns of marked drug resistance and exceptional responses to new agents of immediate clinical relevance were detected. The BCL2inhibitor venetoclax was highly active below 10 nM in B-cell precursor ALL (BCP-ALL) subsets, including MLL-AF4 and TCF3-HLF ALL, and in some T-cell ALLs (T-ALLs), predicting in vivo activity as a single agent and in combination with dexamethasone and vincristine. Unexpected sensitivity to dasatinib with half maximal inhibitory concentration values below 20 nM was detected in 2 independent T-ALL cohorts, which correlated with similar cytotoxic activity of the SRC inhibitor KX2-391 and inhibition of SRC phosphorylation. A patient with refractory T-ALL was treated with dasatinib on the basis of drug profiling information and achieved a 5-month remission. Thus, drug profiling captures disease-relevant features and unexpected sensitivity to relevant drugs, which warrants further exploration of this functional assay in the context of clinical trials to develop drug repurposing strategies for patients with urgent medical needs.

NATURE 541:(7637) pp. 417-420. (2017)

Microenvironmental autophagy promotes tumour growth.

Katheder NS, Khezri R, O'Farrell F, Schultz SW, Jain A, Rahman MM, Schink KO, Theodossiou TA, Johansen T, Juhasz G, Bilder D, Brech A, Stenmark H, Rusten TE

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As malignant tumours develop, they interact intimately with their microenvironment and can activate autophagy, a catabolic process which provides nutrients during starvation. How tumours regulate autophagy in vivo and whether autophagy affects tumour growth is controversial. Here we demonstrate, using a well characterized Drosophila melanogaster malignant tumour model, that non-cell-autonomous autophagy is induced both in the tumour microenvironment and systemically in distant tissues. Tumour growth can be pharmacologically restrained using autophagy inhibitors, and early-stage tumour growth and invasion are genetically dependent on autophagy within the local tumour microenvironment. Induction of autophagy is mediated by Drosophila tumour necrosis factor and interleukin-6-like signalling from metabolically stressed tumour cells, whereas tumour growth depends on active amino acid transport. We show that dormant growth-impaired tumours from autophagy-deficient animals reactivate tumorous growth when transplanted into autophagy-proficient hosts. We conclude that transformed cells engage surrounding normal cells as active and essential microenvironmental contributors to early tumour growth through nutrient-generating autophagy.

EUROPEAN UROLOGY 71:(3) pp. 319-327. (2017)

Comprehensive Drug Testing of Patient-derived Conditionally Reprogrammed Cells from Castration-resistant Prostate Cancer

Saeed K, Rahkama V, Eldfors S, Bychkov D, Mpindi JP, Yadav B, Paavolainen L, Aittokallio T, Heckman C, Wennerberg K, Peehl DM, Horvath P, Mirtti T, Rannikko A, Kallioniemi O, Östling P, af Hällström TM

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Background: Technology development to enable the culture of human prostate cancer (PCa) progenitor cells is required for the identification of new, potentially curative therapies for PCa. Objective: We established and characterized patient-derived conditionally reprogrammed cells (CRCs) to assess their biological properties and to apply these to test the efficacies of drugs. Design, setting, and participants: CRCs were established from seven patient samples with disease ranging from primary PCa to advanced castration-resistant PCa (CRPC). The CRCs were characterized by genomic, transcriptomic, protein expression, and drug profiling. Outcome measurements and statistical analysis: The phenotypic quantification of the CRCs was done based on immunostaining followed by image analysis with Advanced Cell Classifier using Random Forest supervised machine learning. Copy number aberrations (CNAs) were called from whole-exome sequencing and transcriptomics using in-house pipelines. Dose-response measurements were used to generate multiparameter drug sensitivity scores using R-statistical language. Results and limitations: We generated six benign CRC cultures which all had an androgen receptor-negative, basal/transit-amplifying phenotype with few CNAs. In three-dimensional cell culture, these cells could re-express the androgen receptor. The CRCs from a CRPC patient (HUB.5) displayed multiple CNAs, many of which were shared with the parental tumor. We carried out high-throughput drug-response studies with 306 emerging and clinical cancer drugs. Using the benign CRCs as controls, we identified the Bcl-2 family inhibitor navitoclax as the most potent cancer-specific drug for the CRCs from a CRPC patient. Other drug efficacies included taxanes, mepacrine, and retinoids. Conclusions: Comprehensive cancer pharmacopeia-wide drug testing of CRCs from a CRPC patient highlighted both known and novel drug sensitivities in PCa, including navitoclax, which is currently being tested in clinical trials of CRPC. Patient summary: We describe an approach to generate patient-derived cancer cells from advanced prostate cancer and apply such cells to discover drugs that could be applied in clinical trials for castration-resistant prostate cancer. Proof-of-concept study with the aim of generating patient-derived ex vivo models of prostate cancer combined with high-throughput drug testing to identify potential efficacies among 306 existing and emerging cancer drugs highlighting the effect of navitoclax in an advanced disease model. © 2016 European Association of Urology.


Two-Dimensional Spectroscopy of Chlorophyll a Excited-State Equilibration in Light-Harvesting Complex II

Akhtar P, Zhang C, Do TN, Garab G, Lambrev PH, Tan HS

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Excited-state relaxation dynamics and energy-transfer processes in the chlorophyll a (Chl a) manifold of the light-harvesting complex II (LHCII) were examined at physiological temperature using femtosecond two-dimensional electronic spectroscopy (2DES). The experiments were done under conditions free from singlet-singlet annihilation and anisotropic decay. Energy transfer between the different domains of the Chl a manifold was found to proceed on time scales from hundreds of femtoseconds to five picoseconds, before reaching equilibration. No component slower than 10 ps was observed in the spectral equilibration dynamics. We clearly observe the bidirectional (uphill and downhill) energy transfer of the equilibration process between excited states. This bidirectional energy flow, although implicit in the modeling and simulation of the EET processes, has not been observed in any prior transient absorption studies. Furthermore, we identified the spectral forms associated with the different energy transfer lifetimes in the equilibration process.