BAG6/BAT3 modulates autophagy by affecting EP300/p300 intracellular localization

We recently reported that BAG6/BAT3 (BCL2-associated athanogene 6) is essential for basal and starvation-induced autophagy in E18.5 bag6^−/− mouse embryos and in mouse embryonic fibroblasts (MEFs) through the modulation of the EP300/p300-dependent acetylation of TRP53 and autophagy-related (ATG) proteins. We observed that BAG6 increases TRP53 acetylation during starvation and pro-autophagic TRP53-target gene expression. BAG6 also decreases the EP300 dependent-acetylation of ATG5, ATG7, and LC3-I, posttranslational modifications that inhibit autophagy. In addition, in the absence of BAG6 or when using a mutant of BAG6 exclusively located in the cytoplasm, autophagy is inhibited, ATG7 is hyperacetylated, TRP53 acetylation is abrogated, and EP300 accumulates in the cytoplasm indicating that BAG6 is involved in the regulation of the nuclear localization of EP300. We also reported that the interaction between BAG6 and EP300 occurs in the cytoplasm rather than the nucleus. Moreover, during starvation, EP300 is transported to the nucleus in a BAG6-dependent manner. We concluded that BAG6 regulates autophagy by controlling the localization of EP300 and its accessibility to nuclear (TRP53) and cytoplasmic (ATGs) substrates.     

ATG7 contributes to plant basal immunity towards fungal infection

Autophagy has an important function in cellular homeostasis. In recent years autophagy has been implicated in plant basal immunity and assigned negative (“anti-death”) and positive (“pro-death”) regulatory functions in controlling cell death programs that establish sufficient immunity to microbial infection. We recently showed that Arabidopsis mutants lacking the autophagy-associated (ATG) genes ATG5, ATG10 and ATG18a are compromised in their resistance towards infection with necrotrophic fungal pathogens but display an enhanced resistance towards biotrophic bacterial invaders. Thus, the function of autophagy as either being pro-death or anti-death depends critically on the lifestyle and infection strategy of invading microbes. Here we show that ATG7 contributes to resistance to fungal pathogens. Genetic inactivation of ATG7 results in elevated susceptibility towards the necrotrophic fungal pathogen, Alternaria brassicicola, with atg7 mutants developing spreading necrosis accompanied by production of reactive oxygen intermediates. Likewise, treatment with the fungal toxin fumonisin B1 causes spreading lesion formation in the atg7 mutant. We conclude that ATG7-dependent autophagy constitutes an “anti-death” (“pro-survival”) plant mechanism to control the containment of cell death and immunity to necrophic fungal infection.Key words: autophagy, ATG7, basal immunity, fungal resistance, arabidopsisPlants have evolved a bipartite plant immune system to cope with microbial infections. The first layer of defense relies on the recognition of pathogen-associated molecular patterns (PAMP) by pattern-recognition receptors (PAMP-triggered immunity, PTI).^1,2 To overcome this defense strategy, successful pathogens deliver so-called effector proteins into plant cells to modify host cellular processes and to suppress immune responses to enhance virulence. The presence or activities of these microbial effectors is sensed by plant resistance proteins and triggers the second layer of defense, the effector-triggered immunity (ETI).^1,2 In contrast to PTI, ETI is most often accompanied by programmed host cell death (PCD) at the site of attempted microbial invasion; however the molecular basis of this apoptosis-like hypersensitive response (HR) is largely unknown.In recent years evidence accumulated that a non-apoptotic form of cell death called autophagy is not only involved in animal PCD and innate immunity³ but is also an important component in the plant basal immune response.⁴ Generally, autophagy (auto, meaning “self” and phagy, “to eat”) is a cytoplasmic bulk degradation process in which cellular components are targeted to lysosomal or vacuolar degradation. This process is ubiquitous in eukaryotic organisms and is considered to aid cellular survival, differentiation, development and homeostasis by nutrient recycling or removal of damaged or toxic materials.^5–7     

Abscisic Acid-Triggered Persulfidation of the Cys Protease ATG4 Mediates Regulation of Autophagy by Sulfide

Hydrogen sulfide is a signaling molecule that regulates essential processes in plants, such as autophagy. In Arabidopsis (Arabidopsis thaliana), hydrogen sulfide negatively regulates autophagy independently of reactive oxygen species via an unknown mechanism. Comparative and quantitative proteomic analysis was used to detect abscisic acid-triggered persulfidation that reveals a main role in the control of autophagy mediated by the autophagy-related (ATG) Cys protease AtATG4a. This protease undergoes specific persulfidation of Cys170 that is a part of the characteristic catalytic Cys-His-Asp triad of Cys proteases. Regulation of the ATG4 activity by persulfidation was tested in a heterologous assay using the Chlamydomonas reinhardtii CrATG8 protein as a substrate. Sulfide significantly and reversibly inactivates AtATG4a. The biological significance of the reversible inhibition of the ATG4 by sulfide is supported by the results obtained in Arabidopsis leaves under basal and autophagy-activating conditions. A significant increase in the overall ATG4 proteolytic activity in Arabidopsis was detected under nitrogen starvation and osmotic stress and can be inhibited by sulfide. Therefore, the data strongly suggest that the negative regulation of autophagy by sulfide is mediated by specific persulfidation of the ATG4 protease.     

Autophagy differentially controls plant basal immunity to biotrophic and necrotrophic pathogens

In plants, autophagy has been assigned 'pro-death' and 'pro-survival' roles in controlling programmed cell death associated with microbial effector-triggered immunity. The role of autophagy in basal immunity to virulent pathogens has not been addressed systematically, however. Using several autophagy-deficient (atg) genotypes, we determined the function of autophagy in basal plant immunity. Arabidopsis mutants lacking ATG5, ATG10 and ATG18a develop spreading necrosis upon infection with the necrotrophic fungal pathogen, Alternaria brassicicola, which is accompanied by the production of reactive oxygen intermediates and by enhanced hyphal growth. Likewise, treatment with the fungal toxin fumonisin B1 causes spreading lesion formation in atg mutant genotypes. We suggest that autophagy constitutes a 'pro-survival' mechanism that controls the containment of host tissue-destructive microbial infections. In contrast, atg plants do not show spreading necrosis, but exhibit marked resistance against the virulent biotrophic phytopathogen, Pseudomonas syringae pv. tomato. Inducible defenses associated with basal plant immunity, such as callose production or mitogen-activated protein kinase activation, were unaltered in atg genotypes. However, phytohormone analysis revealed that salicylic acid (SA) levels in non-infected and bacteria-infected atg plants were slightly higher than those in Col-0 plants, and were accompanied by elevated SA-dependent gene expression and camalexin production. This suggests that previously undetected moderate infection-induced rises in SA result in measurably enhanced bacterial resistance, and that autophagy negatively controls SA-dependent defenses and basal immunity to bacterial infection. We infer that the way in which autophagy contributes to plant immunity to different pathogens is mechanistically diverse, and thus resembles the complex role of this process in animal innate immunity.     

Functional characterization of the cleavage specificity of the sapovirus chymotrypsin-like protease

Sapovirus is a positive-stranded RNA virus with a translational strategy based on processing of a polyprotein precursor by a chymotrypsin-like protease. So far, the molecular mechanisms regulating cleavage specificity of the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the predicted forms of the viral protease, the 3C-like protease (NS6) and the 3CD-like protease-polymerase (NS6-7), were examined in vitro. The purified NS6 and NS6-7 were able to cleave synthetic peptides (15 to 17 residues) displaying the cleavage sites of the sapovirus polyprotein, both NS6 and NS6-7 proteins being active forms of the viral protease. High-performance liquid chromatography and subsequent mass spectrometry analysis of digested products showed a specific trans cleavage of peptides bearing Gln-Gly, Gln-Ala, Glu-Gly, Glu-Pro, or Glu-Lys at the scissile bond. In contrast, peptides bearing Glu-Ala or Gln-Asp at the scissile bond (NS4-NS5 and NS5-NS6, or NS6-NS7 junctions, respectively) were resistant to trans cleavage by NS6 or NS6-7 proteins, whereas cis cleavage of the Glu-Ala scissile bond of the NS5-NS6 junction was evidenced. Interestingly, the presence of a Phe at position P4 overruled the resistance to trans cleavage of the Glu-Ala junction (NS5-NS6), whereas substitutions at the P1 and P2′ positions altered the cleavage efficiency. The differential cleavage observed is supported by a model of the substrate-binding site of the sapovirus protease, indicating that the P4, P1, and P2′ positions in the substrate modulate the cleavage specificity and efficiency of the sapovirus chymotrypsin-like protease.     

The Arabidopsis LECTIN RECEPTOR KINASE-VI.2 is a functional protein kinase and is dispensable for basal resistance to Botrytis cinerea

Sensing of microbial pathogens by pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs) elicits a defense program known as PAMP-triggered immunity (PTI). Recently, we have shown that the Arabidopsis thaliana L-TYPE LECTIN RECEPTOR KINASE-VI.2 (LecRK-VI.2) positively regulates bacterial PTI. In this report, we suggest by in silico analysis that the kinase domain of LecRK-VI.2 is functional. LecRK-VI.2 also demonstrated auto-phosphorylation activity in vitro in the presence of divalent metal cations indicating that LecRK-VI.2 has the ability to auto-phosphorylate. We further investigate the role of LecRK-VI.2 in Arabidopsis resistance to the necrotrophic fungal pathogen Botrytis cinerea. Disruption of LecRK-VI.2 did not affect Arabidopsis resistance to B. cinerea. Accordingly, wild-type upregulation levels of PTI-responsive WRKY53, FRK1, NHL10, CYP81F2 and CBP60 g after treatment with the fungal PAMP chitin were observed in lecrk-VI.2-1. These data provide evidences that the kinase domain of LecRK-VI.2 is active and show that LecRK-VI.2 is not critical for resistance to the fungal pathogen B. cinerea.     

ENHANCED DISEASE RESISTANCE4 Associates with CLATHRIN HEAVY CHAIN2 and Modulates Plant Immunity by Regulating Relocation of EDR1 in Arabidopsis

Obligate biotrophs, such as the powdery mildew pathogens, deliver effectors to the host cell and obtain nutrients from the infection site. The interface between the plant host and the biotrophic pathogen thus represents a major battleground for plant-pathogen interactions. Increasing evidence shows that cellular trafficking plays an important role in plant immunity. Here, we report that Arabidopsis thaliana ENHANCED DISEASE RESISTANCE4 (EDR4) plays a negative role in resistance to powdery mildew and that the enhanced disease resistance in edr4 mutants requires salicylic acid signaling. EDR4 mainly localizes to the plasma membrane and endosomal compartments. Genetic analyses show that EDR4 and EDR1 function in the same genetic pathway. EDR1 and EDR4 accumulate at the penetration site of powdery mildew infection, and EDR4 physically interacts with EDR1, recruiting EDR1 to the fungal penetration site. In addition, EDR4 interacts with CLATHRIN HEAVY CHAIN2 (CHC2), and edr4 mutants show reduced endocytosis rates. Taken together, our data indicate that EDR4 associates with CHC2 and modulates plant immunity by regulating the relocation of EDR1 in Arabidopsis.     

Acinus integrates AKT1 and subapoptotic caspase activities to regulate basal autophagy

How cellular stresses up-regulate autophagy is not fully understood. One potential regulator is the Drosophila melanogaster protein Acinus (Acn), which is necessary for autophagy induction and triggers excess autophagy when overexpressed. We show that cell type–specific regulation of Acn depends on proteolysis by the caspase Dcp-1. Basal Dcp-1 activity in developing photoreceptors is sufficient for this cleavage without a need for apoptosis to elevate caspase activity. On the other hand, Acn was stabilized by loss of Dcp-1 function or by the presence of a mutation in Acn that eliminates its conserved caspase cleavage site. Acn stability also was regulated by AKT1-mediated phosphorylation. Flies that expressed stabilized forms of Acn, either the phosphomimetic Acn^S641,731D or the caspase-resistant Acn^D527A, exhibited enhanced basal autophagy. Physiologically, these flies showed improvements in processes known to be autophagy dependent, including increased starvation resistance, reduced Huntingtin-induced neurodegeneration, and prolonged life span. These data indicate that AKT1 and caspase-dependent regulation of Acn stability adjusts basal autophagy levels.     

Digestive proteases in bodies and faeces of the two-spotted spider mite,Tetranychus urticae

Digestive proteases of the phytophagous mite Tetranychus urticae have been characterised by comparing their activity in body and faecal extracts. Aspartyl, cathepsin B- and L-like and legumain activities were detected in both mite bodies and faeces, with a specific activity of aspartyl and cathepsin L-like proteases about 5- and 2-fold higher, respectively, in mite faeces than in bodies. In general, all these activities were maintained independently of the host plant where the mites were reared (bean, tomato or maize). Remarkably, this is the first report in a phytophagous mite of legumain-like activity, which was characterised for its ability to hydrolyse the specific substrate Z-VAN-AMC, its activation by DTT and inhibition by IAA but not by E-64. Gel free nanoLC–nanoESI-QTOF MS/MS proteomic analysis of mite faeces resulted in the identification of four cathepsins L and one aspartyl protease (from a total of the 29 cathepsins L, 27 cathepsins B, 19 legumains and two aspartyl protease genes identified the genome of this species). Gene expression analysis reveals that four cathepsins L and the aspartyl protease identified in the mite faeces, but also two cathepsins B and two legumains that were not detected in the faeces, were expressed at high levels in the spider mite feeding stages (larvae, nymphs and adults) relative to embryos. Taken together, these results indicate a digestive role for cysteine and aspartyl proteases in T. urticae. The expression of the cathepsins B and L, legumains and aspartyl protease genes analysed in our study increased in female adults after feeding on Arabidopsis plants over-expressing the HvCPI-6 cystatin, that specifically targets cathepsins B and L, or the CMe trypsin inhibitor that targets serine proteases. This unspecific response suggests that in addition to compensation for inhibitor-targeted enzymes, the increase in the expression of digestive proteases in T. urticae may act as a first barrier against ingested plant defensive proteins.     

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism

Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells.     

Cell surface presenilin-1 participates in the gamma-secretase-like proteolysis of Notch

Presenilin-1 (PS1), a polytopic membrane protein primarily localized to the endoplasmic reticulum, is required for efficient proteolysis of both Notch and beta-amyloid precursor protein (APP) within their trans- membrane domains. The activity that cleaves APP (called gamma-secretase) has properties of an aspartyl protease, and mutation of either of the two aspartate residues located in adjacent transmembrane domains of PS1 inhibits gamma-secretase processing of APP. We show here that these aspartates are required for Notch processing, since mutation of these residues prevents PS1 from inducing the gamma-secretase-like proteolysis of a Notch1 derivative. Thus PS1 might function in Notch cleavage as an aspartyl protease or di-aspartyl protease cofactor. However, the ER localization of PS1 is inconsistent with that hypothesis, since Notch cleavage occurs near the cell surface. Using pulse-chase and biotinylation assays, we provide evidence that PS1 binds Notch in the ER/Golgi and is then co-transported to the plasma membrane as a complex. PS1 aspartate mutants were indistinguishable from wild-type PS1 in their ability to bind Notch or traffic with it to the cell surface, and did not alter the secretion of Notch. Thus, PS1 appears to function specifically in Notch proteolysis near the plasma membrane as an aspartyl protease or cofactor.     

Vacuolar proteases from Candida glabrata: Acid aspartic protease PrA,neutral serine protease PrB and serine carboxypeptidase CpY. The nitrogen source influences their level of expression

BackgroundThe Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata.AimsThe present paper is the first report on proteolytic activity in the C. glabrata vacuole.MethodsBiochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA.ResultsOur results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source.ConclusionsThe proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen.     

Linkage of autophagy to fungal development,lipid storage and virulence in Metarhizium robertsii

Autophagy is a highly conserved process that maintains intracellular homeostasis by degrading proteins or organelles in all eukaryotes. The effect of autophagy on fungal biology and infection of insect pathogens is unknown. Here, we report the function of MrATG8, an ortholog of yeast ATG8, in the entomopathogenic fungus Metarhizium robertsii. MrATG8 can complement an ATG8-defective yeast strain and deletion of MrATG8 impaired autophagy, conidiation and fungal infection biology in M. robertsii. Compared with the wild-type and gene-rescued mutant, Mratg8Δ is not inductive to form the infection-structure appressorium and is impaired in defense response against insect immunity. In addition, accumulation of lipid droplets (LDs) is significantly reduced in the conidia of Mratg8Δ and the pathogenicity of the mutant is drastically impaired. We also found that the cellular level of a LD-specific perilipin-like protein is significantly lowered by deletion of MrATG8 and that the carboxyl terminus beyond the predicted protease cleavage site is dispensable for MrAtg8 function. To corroborate the role of autophagy in fungal physiology, the homologous genes of yeast ATG1, ATG4 and ATG15, designated as MrATG1, MrATG4 and MrATG15, were also deleted in M. robertsii. In contrast to Mratg8Δ, these mutants could form appressoria, however, the LD accumulation and virulence were also considerably impaired in the mutant strains. Our data showed that autophagy is required in M. robertsii for fungal differentiation, lipid biogenesis and insect infection. The results advance our understanding of autophagic process in fungi and provide evidence to connect autophagy with lipid metabolism.     

A region within a lumenal loop of Saccharomyces cerevisiae Ycf1p directs proteolytic processing and substrate specificity

Ycf1p, a member of the yeast multidrug resistance-associated protein (MRP) subfamily of ATP-binding cassette proteins, is a vacuolar membrane transporter that confers resistance to a variety of toxic substances such as cadmium and arsenite. Ycf1p undergoes a PEP4-dependent processing event to yield N- and C-terminal cleavage products that remain associated with one another. In the present study, we sought to determine whether proteolytic cleavage is required for Ycf1p activity. We have identified a unique region within lumenal loop 6 of Ycf1p, designated the loop 6 insertion (L6_ins), which appears to be necessary and sufficient for proteolytic cleavage, since L6_ins can promote processing when moved to new locations in Ycf1p or into a related transporter, Bpt1p. Surprisingly, mutational results indicate that proteolytic processing is not essential for Ycf1p transport activity. Instead, the L6_ins appears to regulate substrate specificity of Ycf1p, since certain mutations in this region lower cellular cadmium resistance with a concomitant gain in arsenite resistance. Although some of these L6_ins mutations block processing, there is no correlation between processing and substrate specificity. The activity profiles of the Ycf1p L6_ins mutants are dramatically affected by the strain background in which they are expressed, raising the possibility that another cellular component may functionally impact Ycf1p activity. A candidate component may be a new full-length MRP-type transporter (NFT1), reported in the Saccharomyces Genome Database as two adjacent open reading frames, YKR103w and YKR104w, but which we show here is present in most Saccharomyces strains as a single open reading frame.     

Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila

Phagophore-derived autophagosomes deliver cytoplasmic material to lysosomes for degradation and reuse. Autophagy mediated by the incompletely characterized actions of Atg proteins is involved in numerous physiological and pathological settings including stress resistance, immunity, aging, cancer, and neurodegenerative diseases. Here we characterized Atg17/FIP200, the Drosophila ortholog of mammalian RB1CC1/FIP200, a proposed functional equivalent of yeast Atg17. Atg17 disruption inhibits basal, starvation-induced and developmental autophagy, and interferes with the programmed elimination of larval salivary glands and midgut during metamorphosis. Upon starvation, Atg17-positive structures appear at aggregates of the selective cargo Ref(2)P/p62 near lysosomes. This location may be similar to the perivacuolar PAS (phagophore assembly site) described in yeast. Drosophila Atg17 is a member of the Atg1 kinase complex as in mammals, and we showed that it binds to the other subunits including Atg1, Atg13, and Atg101 (C12orf44 in humans, 9430023L20Rik in mice and RGD1359310 in rats). Atg17 is required for the kinase activity of endogenous Atg1 in vivo, as loss of Atg17 prevents the Atg1-dependent shift of endogenous Atg13 to hyperphosphorylated forms, and also blocks punctate Atg1 localization during starvation. Finally, we found that Atg1 overexpression induces autophagy and reduces cell size in Atg17-null mutant fat body cells, and that overexpression of Atg17 promotes endogenous Atg13 phosphorylation and enhances autophagy in an Atg1-dependent manner in the fat body. We propose a model according to which the relative activity of Atg1, estimated by the ratio of hyper- to hypophosphorylated Atg13, contributes to setting low (basal) vs. high (starvation-induced) autophagy levels in Drosophila.     

Disrupting Autophagy Restores Peroxisome Function to an Arabidopsis lon2 Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation

Peroxisomes house critical metabolic reactions that are essential for seedling development. As seedlings mature, metabolic requirements change, and peroxisomal contents are remodeled. The resident peroxisomal protease LON2 is positioned to degrade obsolete or damaged peroxisomal proteins, but data supporting such a role in plants have remained elusive. Arabidopsis thaliana lon2 mutants display defects in peroxisomal metabolism and matrix protein import but appear to degrade matrix proteins normally. To elucidate LON2 functions, we executed a forward-genetic screen for lon2 suppressors, which revealed multiple mutations in key autophagy genes. Disabling core autophagy-related gene (ATG) products prevents autophagy, a process through which cytosolic constituents, including organelles, can be targeted for vacuolar degradation. We found that atg2, atg3, and atg7 mutations suppressed lon2 defects in auxin metabolism and matrix protein processing and rescued the abnormally large size and small number of lon2 peroxisomes. Moreover, analysis of lon2 atg mutants uncovered an apparent role for LON2 in matrix protein turnover. Our data suggest that LON2 facilitates matrix protein degradation during peroxisome content remodeling, provide evidence for the existence of pexophagy in plants, and indicate that peroxisome destruction via autophagy is enhanced when LON2 is absent.     

BAG3: a multifaceted protein that regulates major cell pathways

Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110–124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is instead expressed in several tumor types. In different tumor contexts, BAG3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. In some tumor types, down-modulation of BAG3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. This review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression.     

The LysM receptor kinase CERK1 mediates bacterial perception in Arabidopsis

Plants use pattern recognition receptors (PRRs) to perceive pathogen-associated molecular pattern (PAMPs) and initiate defence responses. PAMP-triggered immunity (PTI) plays an important role in general resistance, and constrains the growth of most microbes on plants. Despite the importance of PRRs in plant immunity, the vast majority of them remain to be identified. We recently showed that the Arabidopsis LysM receptor kinase CERK1 is required not only for chitin signalling and fungal resistance, but plays an essential role in restricting bacterial growth on plants. We proposed that CERK1 may mediate the perception of a bacterial PAMP, or an endogenous plant cell wall component released during infection, through its extracellular carbohydrate-binding LysM-motifs. Here we report reduced activation of a PAMP-induced defence response on plants lacking the CERK1 gene after treatment with crude bacterial extracts. This demonstrates that CERK1 mediates perception of an unknown bacterial PAMP in Arabidopsis.Key words: PAMP, PRR, PTI, LysM, chitin, bacteria, carbohydrate     

Fundamental Roles of the Golgi-Associated Toxoplasma Aspartyl Protease,ASP5, at the Host-Parasite Interface

Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite’s ability to modulate host signalling pathways and immune responses.