A PDLP-NHL3 complex integrates plasmodesmal immune signaling cascades.

The plant immune system relies on the perception of molecules that signal the presence of a microbe threat. This triggers signal transduction that mediates a range of cellular responses via a collection of molecular machinery including receptors, small molecules, and enzymes. One response to pathogen perception is the restriction of cell-to-cell communication by plasmodesmal closure. We previously found that while chitin and flg22 trigger specialized immune signaling cascades in the plasmodesmal plasma membrane, both execute plasmodesmal closure via callose synthesis at the plasmodesmata. Therefore, the signaling pathways ultimately converge at or upstream of callose synthesis. To establish the hierarchy of signaling at plasmodesmata and characterize points of convergence in microbe elicitor-triggered signaling, we profiled the dependence of plasmodesmal responses triggered by different elicitors on a range of plasmodesmal signaling machinery. We identified that, like chitin, flg22 signals via RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD) to induce plasmodesmal closure. Further, we found that PLASMODESMATA-LOCATED PROTEIN 1 (PDLP1), PDLP5, and CALLOSE SYNTHASE 1 (CALS1) are common to microbe- and salicylic acid (SA)-triggered responses, identifying PDLPs as a candidate signaling nexus. To understand how PDLPs relay a signal to CALS1, we screened for PDLP5 interactors and found NON-RACE SPECIFIC DISEASE RESISTANCE/HIN1 HAIRPIN-INDUCED-LIKE protein 3 (NHL3), which is also required for chitin-, flg22- and SA-triggered plasmodesmal responses and PDLP-mediated activation of callose synthesis. We conclude that a PDLP-NHL3 complex acts as an integrating node of plasmodesmal signaling cascades, transmitting multiple immune signals to activate CALS1 and plasmodesmata closure.

Comparative phyloproteomics identifies conserved plasmodesmal proteins.

Plasmodesmata are cytosolic bridges, lined by the plasma membrane and traversed by endoplasmic reticulum; plasmodesmata connect cells and tissues, and are critical for many aspects of plant biology. While plasmodesmata are notoriously difficult to extract, tissue fractionation and proteomic analyses can yield valuable knowledge of their composition. Here we have generated two novel proteomes to expand tissue and taxonomic representation of plasmodesmata: one from mature Arabidopsis leaves and one from the moss Physcomitrium patens, and leveraged these and existing data to perform a comparative analysis to identify evolutionarily conserved protein families that are associated with plasmodesmata. Thus, we identified ß-1,3-glucanases, C2 lipid-binding proteins, and tetraspanins as core plasmodesmal components that probably serve as essential structural or functional components. Our approach has not only identified elements of a conserved plasmodesmal proteome, but also demonstrated the added power offered by comparative analysis for recalcitrant samples. Conserved plasmodesmal proteins establish a basis upon which ancient plasmodesmal function can be further investigated to determine the essential roles these structures play in multicellular organism physiology in the green lineages.

Diffusion and bulk flow of amino acids mediate calcium waves in plants.

In plants, a variety of stimuli trigger long-range calcium signals that travel rapidly along the vasculature to distal tissues via poorly understood mechanisms. Here, we use quantitative imaging and analysis to demonstrate that traveling calcium waves are mediated by diffusion and bulk flow of amino acid chemical messengers. We propose that wounding triggers release of amino acids that diffuse locally through the apoplast, activating the calcium-permeable channel GLUTAMATE RECEPTOR-LIKE 3.3 as they pass. Over long distances through the vasculature, the wound-triggered dynamics of a fluorescent tracer show that calcium waves are likely driven by bulk flow of a channel-activating chemical. We observed that multiple stimuli trigger calcium waves with similar dynamics, but calcium waves alone cannot initiate all systemic defense responses, suggesting that mobile chemical messengers are a core component of complex systemic signaling in plants.

The primary function of Six5 of Fusarium oxysporum is to facilitate Avr2 activity by together manipulating the size exclusion limit of plasmodesmata.

Pathogens produce effector proteins to manipulate their hosts. While most effectors act autonomously, some fungal effectors act in pairs and rely on each other for function. During the colonization of the plant vasculature, the root-infecting fungus Fusarium oxysporum (Fo) produces 14 so-called Secreted in Xylem (SIX) effectors. Two of these effector genes, Avr2 (Six3) and Six5, form a gene pair on the pathogenicity chromosome of the tomato-infecting Fo strain. Avr2 has been shown to suppress plant defense responses and is required for full pathogenicity. Although Six5 and Avr2 together manipulate the size exclusion limit of plasmodesmata to facilitate cell-to-cell movement of Avr2, it is unclear whether Six5 has additional functions as well. To investigate the role of Six5, we generated transgenic Arabidopsis lines expressing Six5. Notably, increased susceptibility during the early stages of infection was observed in these Six5 lines, but only to Fo strains expressing Avr2 and not to wild-type Arabidopsis-infecting Fo strains lacking this effector gene. Furthermore, neither PAMP-triggered defense responses, such as ROS accumulation and callose deposition upon treatment with Flg22, necrosis and ethylene-inducing peptide 1-like protein (NLP), or chitosan, nor susceptibility to other plant pathogens, such as the bacterium Pseudomonas syringae or the fungus Verticilium dahlia, were affected by Six5 expression. Further investigation of the ability of the Avr2/Six5 effector pair to manipulate plasmodesmata (PD) revealed that it not only permits cell-to-cell movement of Avr2, but also facilitates the movement of two additional effectors, Six6 and Six8. Moreover, although Avr2/Six5 expands the size exclusion limit of plasmodesmata (i.e., gating) to permit the movement of a 2xFP fusion protein (53 kDa), a larger variant, 3xFP protein (80 kDa), did not move to the neighboring cells. The PD manipulation mechanism employed by Avr2/Six5 did not involve alteration of callose homeostasis in these structures. In conclusion, the primary function of Six5 appears to function together with Avr2 to increase the size exclusion limit of plasmodesmata by an unknown mechanism to facilitate cell-to-cell movement of Fo effectors.

Everything You Always Wanted to Know About Organoid-Based Models (and Never Dared to Ask).

Homeostatic functions of a living tissue, such as the gastrointestinal tract, rely on highly sophisticated and finely tuned cell-to-cell interactions. These crosstalks evolve and continuously are refined as the tissue develops and give rise to specialized cells performing general and tissue-specific functions. To study these systems, stem cell-based in vitro models, often called organoids, and non-stem cell-based primary cell aggregates (called spheroids) appeared just over a decade ago. These models still are evolving and gaining complexity, making them the state-of-the-art models for studying cellular crosstalk in the gastrointestinal tract, and to investigate digestive pathologies, such as inflammatory bowel disease, colorectal cancer, and liver diseases. However, the use of organoid- or spheroid-based models to recapitulate in vitro the highly complex structure of in vivo tissue remains challenging, and mainly restricted to expert developmental cell biologists. Here, we condense the founding knowledge and key literature information that scientists adopting the organoid technology for the first time need to consider when using these models for novel biological questions. We also include information that current organoid/spheroid users could use to add to increase the complexity to their existing models. We highlight the current and prospective evolution of these models through bridging stem cell biology with biomaterial and scaffold engineering research areas. Linking these complementary fields will increase the in vitro mimicry of in vivo tissue, and potentially lead to more successful translational biomedical applications. Deepening our understanding of the nature and dynamic fine-tuning of intercellular crosstalks will enable identifying novel signaling targets for new or repurposed therapeutics used in many multifactorial diseases.

Morphogen Signals Shaping the Gastric Glands in Health and Disease.

The adult gastric mucosa is characterised by deep invaginations of the epithelium called glands. These tissue architectural elements are maintained with the contribution of morphogen signals. Morphogens are expressed in specific areas of the tissue, and their diffusion generates gradients in the microenvironment. Cells at different positions in the gland sense a specific combination of signals that instruct them to differentiate, proliferate, regenerate, or migrate. Differentiated cells perform specific functions involved in digestion, such as the production of protective mucus and the secretion of digestive enzymes or gastric acid. Biopsies from gastric precancerous conditions usually display tissue aberrations and change the shape of the glands. Alteration of the morphogen signalling microenvironment is likely to underlie those conditions. Furthermore, genes involved in morphogen signalling pathways are found to be frequently mutated in gastric cancer. We summarise the most recent findings regarding alterations of morphogen signalling during gastric carcinogenesis, and we highlight the new stem cell technologies that are improving our understanding of the regulation of human tissue shape.

Suppression of AMPK/aak-2 by NRF2/SKN-1 down-regulates autophagy during prolonged oxidative stress.

NF-E2-related factor 2 (NRF2) transcription factor has a fundamental role in cell homeostasis maintenance as one of the master regulators of oxidative and electrophilic stress responses. Previous studies have shown that a regulatory connection exists between NRF2 and autophagy during reactive oxygen species-generated oxidative stress. The aim of the present study was to investigate how autophagy is turned off during prolonged oxidative stress, to avoid overeating and destruction of essential cellular components. AMPK is a key cellular energy sensor highly conserved in eukaryotic organisms, and it has an essential role in autophagy activation at various stress events. Here the role of human AMPK and its Caenorhabditis elegans counterpart AAK-2 was explored upon oxidative stress. We investigated the regulatory connection between NRF2 and AMPK during oxidative stress induced by tert-butyl hydroperoxide (TBHP) in HEK293T cells and C. elegans. Putative conserved NRF2/protein skinhead-1 binding sites were found in AMPK/aak-2 genes by in silico analysis and were later confirmed experimentally by using EMSA. After addition of TBHP, NRF2 and AMPK showed a quick activation; AMPK was later down-regulated, however, while NRF2 level remained high. Autophagosome formation and Unc-51-like autophagy activating kinase 1 phosphorylation were initially stimulated, but they returned to basal values after 4 h of TBHP treatment. The silencing of NRF2 resulted in a constant activation of AMPK leading to hyperactivation of autophagy during oxidative stress. We observed the same effects in C. elegans demonstrating the conservation of this self-defense mechanism to save cells from hyperactivated autophagy upon prolonged oxidative stress. We conclude that NRF2 negatively regulates autophagy through delayed down-regulation of the expression of AMPK upon prolonged oxidative stress. This regulatory connection between NRF2 and AMPK may have an important role in understanding how autophagy is regulated in chronic human morbidities characterized by oxidative stress, such as neurodegenerative diseases, certain cancer types, and in metabolic diseases.-Kosztelnik, M., Kurucz, A., Papp, D., Jones, E., Sigmond, T., Barna, J., Traka, M. H., Lorincz, T., Szarka, A., Banhegyi, G., Vellai, T., Korcsmaros, T., Kapuy, O. Suppression of AMPK/aak-2 by NRF2/SKN-1 down-regulates autophagy during prolonged oxidative stress.

Systems-Level Feedbacks of NRF2 Controlling Autophagy upon Oxidative Stress Response.

Although the primary role of autophagy-dependent cellular self-eating is cytoprotective upon various stress events (such as starvation, oxidative stress, and high temperatures), sustained autophagy might lead to cell death. A transcription factor called NRF2 (nuclear factor erythroid-related factor 2) seems to be essential in maintaining cellular homeostasis in the presence of either reactive oxygen or nitrogen species generated by internal metabolism or external exposure. Accumulating experimental evidence reveals that oxidative stress also influences the balance of the 5' AMP-activated protein kinase (AMPK)/rapamycin (mammalian kinase target of rapamycin or mTOR) signaling pathway, thereby inducing autophagy. Based on computational modeling here we propose that the regulatory triangle of AMPK, NRF2 and mTOR guaranties a precise oxidative stress response mechanism comprising of autophagy. We suggest that under conditions of oxidative stress, AMPK is crucial for autophagy induction via mTOR down-regulation, while NRF2 fine-tunes the process of autophagy according to the level of oxidative stress. We claim that the cellular oxidative stress response mechanism achieves an incoherently amplified negative feedback loop involving NRF2, mTOR and AMPK. The mTOR-NRF2 double negative feedback generates bistability, supporting the proper separation of two alternative steady states, called autophagy-dependent survival (at low stress) and cell death (at high stress). In addition, an AMPK-mTOR-NRF2 negative feedback loop suggests an oscillatory characteristic of autophagy upon prolonged intermediate levels of oxidative stress, resulting in new rounds of autophagy stimulation until the stress events cannot be dissolved. Our results indicate that AMPK-, NRF2- and mTOR-controlled autophagy induction provides a dynamic adaptation to altering environmental conditions, assuming their new frontier in biomedicine.

The small molecule AUTEN-99 (autophagy enhancer-99) prevents the progression of neurodegenerative symptoms.

Autophagy functions as a main route for the degradation of superfluous and damaged constituents of the cytoplasm. Defects in autophagy are implicated in the development of various age-dependent degenerative disorders such as cancer, neurodegeneration and tissue atrophy, and in accelerated aging. To promote basal levels of the process in pathological settings, we previously screened a small molecule library for novel autophagy-enhancing factors that inhibit the myotubularin-related phosphatase MTMR14/Jumpy, a negative regulator of autophagic membrane formation. Here we identify AUTEN-99 (autophagy enhancer-99), which activates autophagy in cell cultures and animal models. AUTEN-99 appears to effectively penetrate through the blood-brain barrier, and impedes the progression of neurodegenerative symptoms in Drosophila models of Parkinson's and Huntington's diseases. Furthermore, the molecule increases the survival of isolated neurons under normal and oxidative stress-induced conditions. Thus, AUTEN-99 serves as a potent neuroprotective drug candidate for preventing and treating diverse neurodegenerative pathologies, and may promote healthy aging.

AUTEN-67, an autophagy-enhancing drug candidate with potent antiaging and neuroprotective effects.

Autophagy is a major molecular mechanism that eliminates cellular damage in eukaryotic organisms. Basal levels of autophagy are required for maintaining cellular homeostasis and functioning. Defects in the autophagic process are implicated in the development of various age-dependent pathologies including cancer and neurodegenerative diseases, as well as in accelerated aging. Genetic activation of autophagy has been shown to retard the accumulation of damaged cytoplasmic constituents, delay the incidence of age-dependent diseases, and extend life span in genetic models. This implies that autophagy serves as a therapeutic target in treating such pathologies. Although several autophagy-inducing chemical agents have been identified, the majority of them operate upstream of the core autophagic process, thereby exerting undesired side effects. Here, we screened a small-molecule library for specific inhibitors of MTMR14, a myotubularin-related phosphatase antagonizing the formation of autophagic membrane structures, and isolated AUTEN-67 (autophagy enhancer-67) that significantly increases autophagic flux in cell lines and in vivo models. AUTEN-67 promotes longevity and protects neurons from undergoing stress-induced cell death. It also restores nesting behavior in a murine model of Alzheimer disease, without apparent side effects. Thus, AUTEN-67 is a potent drug candidate for treating autophagy-related diseases.

NRF2-ome: an integrated web resource to discover protein interaction and regulatory networks of NRF2.

NRF2 is the master transcriptional regulator of oxidative and xenobiotic stress responses. NRF2 has important roles in carcinogenesis, inflammation, and neurodegenerative diseases. We developed an online resource, NRF2-ome, to provide an integrated and systems-level database for NRF2. The database contains manually curated and predicted interactions of NRF2 as well as data from external interaction databases. We integrated NRF2 interactome with NRF2 target genes, NRF2 regulating TFs, and miRNAs. We connected NRF2-ome to signaling pathways to allow mapping upstream NRF2 regulatory components that could directly or indirectly influence NRF2 activity totaling 35,967 protein-protein and signaling interactions. The user-friendly website allows researchers without computational background to search, browse, and download the database. The database can be downloaded in SQL, CSV, BioPAX, SBML, PSI-MI, and in a Cytoscape CYS file formats. We illustrated the applicability of the website by suggesting a posttranscriptional negative feedback of NRF2 by MAFG protein and raised the possibility of a connection between NRF2 and the JAK/STAT pathway through STAT1 and STAT3. NRF2-ome can also be used as an evaluation tool to help researchers and drug developers to understand the hidden regulatory mechanisms in the complex network of NRF2.

The NRF2-related interactome and regulome contain multifunctional proteins and fine-tuned autoregulatory loops.

NRF2 is a well-known, master transcription factor (TF) of oxidative and xenobiotic stress responses. Recent studies uncovered an even wider regulatory role of NRF2 influencing carcinogenesis, inflammation and neurodegeneration. Prompted by these advances here we present a systems-level resource for NRF2 interactome and regulome that includes 289 protein-protein, 7469 TF-DNA and 85 miRNA interactions. As systems-level examples of NRF2-related signaling we identified regulatory loops of NRF2 interacting proteins (e.g., JNK1 and CBP) and a fine-tuned regulatory system, where 35 TFs regulated by NRF2 influence 63 miRNAs that down-regulate NRF2. The presented network and the uncovered regulatory loops may facilitate the development of efficient, NRF2-based therapeutic agents.

A role for SKN-1/Nrf in pathogen resistance and immunosenescence in Caenorhabditis elegans.

A proper immune response ensures survival in a hostile environment and promotes longevity. Recent evidence indicates that innate immunity, beyond antimicrobial effectors, also relies on host-defensive mechanisms. The Caenorhabditis elegans transcription factor SKN-1 regulates xenobiotic and oxidative stress responses and contributes to longevity, however, its role in immune defense is unknown. Here we show that SKN-1 is required for C. elegans pathogen resistance against both Gram-negative Pseudomonas aeruginosa and Gram-positive Enterococcus faecalis bacteria. Exposure to P. aeruginosa leads to SKN-1 accumulation in intestinal nuclei and transcriptional activation of two SKN-1 target genes, gcs-1 and gst-4. Both the Toll/IL-1 Receptor domain protein TIR-1 and the p38 MAPK PMK-1 are required for SKN-1 activation by PA14 exposure. We demonstrate an early onset of immunosenescence with a concomitant age-dependent decline in SKN-1-dependent target gene activation, and a requirement of SKN-1 to enhance pathogen resistance in response to longevity-promoting interventions, such as reduced insulin/IGF-like signaling and preconditioning H(2)O(2) treatment. Finally, we find that wdr-23(RNAi)-mediated constitutive SKN-1 activation results in excessive transcription of target genes, confers oxidative stress tolerance, but impairs pathogen resistance. Our findings identify SKN-1 as a novel regulator of innate immunity, suggests its involvement in immunosenescence and provide an important crosstalk between pathogenic stress signaling and the xenobiotic/oxidative stress response.

Increased life span from overexpression of superoxide dismutase in Caenorhabditis elegans is not caused by decreased oxidative damage.

The superoxide free radical (O(2)(•-)) has been viewed as a likely major contributor to aging. If this is correct, then superoxide dismutase (SOD), which removes O(2)(•-), should contribute to longevity assurance. In Caenorhabditis elegans, overexpression (OE) of the major cytosolic Cu/Zn-SOD, sod-1, increases life span. But is this increase caused by enhanced antioxidant defense? sod-1 OE did not reduce measures of lipid oxidation or glycation and actually increased levels of protein oxidation. The effect of sod-1 OE on life span was dependent on the DAF-16/FoxO transcription factor (TF) and, partially, on the heat shock TF HSF-1. Similarly, overexpression of sod-2 (major mitochondrial Mn-SOD) resulted in life-span extension that was daf-16 dependent. sod-1 OE increased steady-state hydrogen peroxide (H(2)O(2)) levels in vivo. However, co-overexpression of catalase did not suppress the life-span extension, arguing against H(2)O(2) as a cause of longevity. sod-1 OE increased hsp-4 expression, suggesting increased endoplasmic reticulum (ER) stress. Moreover, longevity was partially suppressed by inactivation of ire-1 and xbp-1, mediators of the ER stress response. This suggests that high levels of SOD-1 protein may challenge protein-folding homeostasis, triggering a daf-16- and hsf-1-dependent stress response that extends life span. These findings imply that SOD overexpression increases C. elegans life span, not by removal of O(2)(•-), but instead by activating longevity-promoting transcription factors.

The heat shock connection of metabolic stress and dietary restriction.

Molecular chaperones and the heat shock response guard and modulate protein conformation, protect proteins from misfolding and aggregation, and maintain signalling and organellar networks. Overnutrition and the metabolic syndrome represent a pro-aging condition, and dietary restriction is the most robust environmental intervention that induces longevity from yeast to mammals. In recent years a considerable effort has been made to elucidate the signaling pathways involved in metabolic signaling. Here we review the current understanding on the connection between metabolic stress, dietary restriction and the heat shock response and highlight results showing chaperone induction as a promising therapeutic strategy to promote healthy aging and to prevent metabolic disorders.

Studies on the interactions between C-reactive protein and complement proteins.

Several studies have investigated the interactions between C-reactive protein (CRP) and various complement proteins but none of them took into consideration the different structural forms of CRP. The aim of our study was to investigate whether the different antigenic forms of CRP are able to bind C1q, to trigger activation of the C1 complex and to study the ability of the various CRP forms to bind complement factor H (FH) and C4b-binding protein (C4BP). Interactions between various CRP forms and complement proteins were analysed in enzyme-linked immunosorbent assay and surface plasmon resonance tests and activation of the C1 complex was followed in a reconstituted system using purified C1q, C1r and C1s in the presence of C1-INH. Native, ligand-unbound CRP activated the classical pathway weakly. After binding to phosphocholine, native CRP bound C1q and significantly activated C1. Native CRP complexed to phosphocholine did not bind the complement regulatory proteins FH and C4BP. After disruption of the pentameric structure of CRP, as achieved by urea-treatment or by site-directed mutagenesis, C1q binding and C1 activation further increased and the ability of CRP to bind complement regulatory proteins was revealed. C1q binds to CRP through its globular head domain. The binding sites on CRP for FH and C4BP seemed to be different from that of C1q. In conclusion, in parallel with the increase in the C1-activating ability of different CRP structural variants, the affinity for complement regulatory proteins also increased, providing the biological basis for limitation of excess complement activation.

Development of a sensitive assay for the measurement of antibodies against heat shock protein binding protein 1 (HspBP1): increased levels of anti-HspBP1 IgG are prevalent in HIV infected subjects.

The 70 kDa heat shock protein (Hsp70) is generally considered to be an intracellular protein, however, there is evidence that Hsp70 can be found in the extracellular environment. Hsp70 and antibodies against Hsp70 have been reported in human serum. Recent evidence has shown that Hsp70 antibodies are elevated in HIV infected individuals. This study reports on the antibody levels against a co-chaperone, HspBP1, that regulates Hsp70 activity. We have developed a solid-phase enzyme linked assay for the determination of anti-HspBP1 IgG antibodies. We report here that HspBP1 antibodies are present in human serum and the levels are elevated approximately twofold in HIV infected patients. There was no correlation between HspBP1 antibody levels and clinical parameters nor was there a relation between anti-Hsp70 levels and anti-HspBP1 levels. The presence of HspBP1 antibodies in human serum suggests that the protein may also be present in the serum. The increased level of HspBP1 antibodies in HIV infected individuals suggests a relationship directly to the virus or indirectly to secondary consequences of HIV infection.