Jasmonate regulates the FAMA/mediator complex subunit 8-THIOGLUCOSIDE GLUCOHYDROLASE 1 cascade and myrosinase activity.

Myrosinases are ß-thioglucoside glucosidases that are unique to the Brassicales order. These enzymes hydrolyze glucosinolates to produce compounds that have direct antibiotic effects or that function as signaling molecules in the plant immune system, protecting plants from pathogens and insect pests. However, the effects of jasmonic acid (JA), a plant hormone that is crucial for plant disease resistance, on myrosinase activity remain unclear. Here, we systematically studied the effects of JA on myrosinase activity and explored the associated internal transcriptional regulation mechanisms. Exogenous application of JA significantly increased myrosinase activity, while the inhibition of endogenous JA biosynthesis and signaling reduced myrosinase activity. In addition, some myrosinase genes in Arabidopsis (Arabidopsis thaliana) were upregulated by JA. Further genetic and biochemical evidence showed that transcription factor FAMA interacted with a series of JASMONATE ZIM-DOMAIN proteins and affected JA-mediated myrosinase activity. However, among the JA-upregulated myrosinase genes, only THIOGLUCOSIDE GLUCOHYDROLASE 1 (TGG1) was positively regulated by FAMA. Further biochemical analysis showed that FAMA bound to the TGG1 promoter to directly mediate TGG1 expression in conjunction with Mediator complex subunit 8 (MED8). Together, our results provide evidence that JA acts as an important signal upstream of the FAMA/MED8-TGG1 pathway to positively regulate myrosinase activity in Arabidopsis.

The Role of Glycoside Hydrolases in Phytopathogenic Fungi and Oomycetes Virulence.

Phytopathogenic fungi need to secrete different hydrolytic enzymes to break down complex polysaccharides in the plant cell wall in order to enter the host and develop the disease. Fungi produce various types of cell wall degrading enzymes (CWDEs) during infection. Most of the characterized CWDEs belong to glycoside hydrolases (GHs). These enzymes hydrolyze glycosidic bonds and have been identified in many fungal species sequenced to date. Many studies have shown that CWDEs belong to several GH families and play significant roles in the invasion and pathogenicity of fungi and oomycetes during infection on the plant host, but their mode of function in virulence is not yet fully understood. Moreover, some of the CWDEs that belong to different GH families act as pathogen-associated molecular patterns (PAMPs), which trigger plant immune responses. In this review, we summarize the most important GHs that have been described in eukaryotic phytopathogens and are involved in the establishment of a successful infection.

Activity of CcpA-Regulated GH18 Family Glycosyl Hydrolases That Contributes to Nutrient Acquisition and Fitness in Enterococcus faecalis.

The ability of Enterococcus faecalis to colonize host anatomical sites is dependent on its adaptive response to host conditions. Three glycosyl hydrolase gene clusters, each belonging to glycosyl hydrolase family 18 (GH18) (ef0114, ef0361, and ef2863), in E. faecalis were previously found to be upregulated under glucose-limiting conditions. The GH18 catalytic domain is present in proteins that are classified as either chitinases or ß-1,4 endo-ß-N-acetylglucosaminidases (ENGases) based on their ß-1,4 endo-N-acetyl-ß-d-glucosaminidase activity, and ENGase activity is commonly associated with cleaving N-linked glycoprotein, an abundant glycan structure on host epithelial surfaces. Here, we show that all three hydrolases are negatively regulated by the transcriptional regulator carbon catabolite protein A (CcpA). Additionally, we demonstrate that a constitutively active CcpA variant represses the expression of CcpA-regulated genes irrespective of glucose availability. Previous studies showed that the GH18 catalytic domains of EndoE (EF0114) and EfEndo18A (EF2863) were capable of deglycosylating RNase B, a model high-mannose-type glycoprotein. However, it remained uncertain which glycosidase is primarily responsible for the deglycosylation of high-mannose-type glycoproteins. In this study, we show by mutation analysis as well as a dose-dependent analysis of recombinant protein expression that EfEndo18A is primarily responsible for deglycosylating high-mannose glycoproteins and that the glycans removed by EfEndo18A support growth under nutrient-limiting conditions in vitro. In contrast, IgG is representative of a complex-type glycoprotein, and we demonstrate that the GH18 domain of EndoE is primarily responsible for the removal of this glycan decoration. Finally, our data highlight the combined contribution of glycosidases to the virulence of E. faecalis in vivo.

Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease.

ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.

Lytic polysaccharide monooxygenases and other histidine-brace copper proteins: structure, oxygen activation and biotechnological applications.

Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper enzymes that catalyse the oxidative cleavage of glycosidic bonds. They are characterised by two histidine residues that coordinate copper in a configuration termed the Cu-histidine brace. Although first identified in bacteria and fungi, LPMOs have since been found in all biological kingdoms. LPMOs are now included in commercial enzyme cocktails used in industrial biorefineries. This has led to increased process yield due to the synergistic action of LPMOs with glycoside hydrolases. However, the introduction of LPMOs makes control of the enzymatic step in industrial stirred-tank reactors more challenging, and the operational stability of the enzymes is reduced. It is clear that much is still to be learned about the interaction between LPMOs and their complex natural and industrial environments, and fundamental scientific studies are required towards this end. Several atomic-resolution structures have been solved providing detailed information on the Cu-coordination sphere and the interaction with the polysaccharide substrate. However, the molecular mechanisms of LPMOs are still the subject of intense investigation; the key question being how the proteinaceous environment controls the copper cofactor towards the activation of the O-O bond in O2 and cleavage of the glycosidic bonds in polysaccharides. The need for biochemical characterisation of each putative LPMO is discussed based on recent reports showing that not all proteins with a Cu-histidine brace are enzymes.

PhiA, a Peptidoglycan Hydrolase Inhibitor of Brucella Involved in the Virulence Process.

The peptidoglycan in Gram-negative bacteria is a dynamic structure in constant remodeling. This dynamism, achieved through synthesis and degradation, is essential because the peptidoglycan is necessary to maintain the structure of the cell but has to have enough plasticity to allow the transport and assembly of macromolecular complexes in the periplasm and outer membrane. In addition, this remodeling has to be coordinated with the division process. Among the multiple mechanisms bacteria have to degrade the peptidoglycan are the lytic transglycosidases, enzymes of the lysozyme family that cleave the glycan chains generating gaps in the mesh structure increasing its permeability. Because these enzymes can act as autolysins, their activity has to be tightly regulated, and one of the mechanisms bacteria have evolved is the synthesis of membrane bound or periplasmic inhibitors. In the present study, we identify a periplasmic lytic transglycosidase inhibitor (PhiA) in Brucella abortus and demonstrate that it inhibits the activity of SagA, a lytic transglycosidase we have previously shown is involved in the assembly of the type IV secretion system. A phiA deletion mutant results in a strain with the incapacity to synthesize a complete lipopolysaccharide but with a higher replication rate than the wild-type parental strain, suggesting a link between peptidoglycan remodeling and speed of multiplication.

Molecular mechanism of Aspergillus fumigatus biofilm disruption by fungal and bacterial glycoside hydrolases.

During infection, the fungal pathogen Aspergillus fumigatus forms biofilms that enhance its resistance to antimicrobials and host defenses. An integral component of the biofilm matrix is galactosaminogalactan (GAG), a cationic polymer of α-1,4-linked galactose and partially deacetylated N-acetylgalactosamine (GalNAc). Recent studies have shown that recombinant hydrolase domains from Sph3, an A. fumigatus glycoside hydrolase involved in GAG synthesis, and PelA, a multifunctional protein from Pseudomonas aeruginosa involved in Pel polysaccharide biosynthesis, can degrade GAG, disrupt A. fumigatus biofilms, and attenuate fungal virulence in a mouse model of invasive aspergillosis. The molecular mechanisms by which these enzymes disrupt biofilms have not been defined. We hypothesized that the hydrolase domains of Sph3 and PelA (Sph3h and PelAh, respectively) share structural and functional similarities given their ability to degrade GAG and disrupt A. fumigatus biofilms. MALDI-TOF enzymatic fingerprinting and NMR experiments revealed that both proteins are retaining endo-α-1,4-N-acetylgalactosaminidases with a minimal substrate size of seven residues. The crystal structure of PelAh was solved to 1.54 Å and structure alignment to Sph3h revealed that the enzymes share similar catalytic site residues. However, differences in the substrate-binding clefts result in distinct enzyme-substrate interactions. PelAh hydrolyzed partially deacetylated substrates better than Sph3h, a finding that agrees well with PelAh's highly electronegative binding cleft versus the neutral surface present in Sph3h Our insight into PelAh's structure and function necessitate the creation of a new glycoside hydrolase family, GH166, whose structural and mechanistic features, along with those of GH135 (Sph3), are reported here.

Structure-guided mutational evidence and postulates explaining how a glycohydrolase from Pyrococcus furiosus functions simultaneously as an amylase and as a 4-α-glucanotransferase.

Pyrococcus furiosus exoamylase-cum-4-α-glucanotransferase (4-α-GTase; PF0272; PfuAmyGT) is reported to both (i) act upon starch, and (ii) catalyze 'disproportionation' of maltooligosaccharides (with glucose as the smallest product). PfuAmyGT shares ∼65% sequence identity with a homo-dimeric Thermococcus litoralis 4-α-GTase, for which structures are available in complex with a non-hydrolysable analog of maltotetraose (acarbose) bound to one subunit and maltose (of unknown origin) bound to the other subunit. We structurally transposed the maltose onto the acarbose-bound subunit and discovered that the two molecules lie juxtaposed in what could be perfect 'acceptor' and 'donor' substrate-binding sites, respectively. We also discovered that there is a loop between the two sites which could use an available aspartate to excise a glucose from the donor, and an available tryptophan to transfer the glucose to the non-reducing end of the acceptor glucan. We derived a structure for PfuAmyGT through homology-based modeling, identified the potential donor site, acceptor site, glucan-transferring loop, and catalytically important residues, and mutated these to alanine to examine effect(s) upon activity. Mutation D362A abolished creation of shorter, or longer, maltooligosaccharides. Mutation W365A abolished creation of longer oligosaccharides. Mutation H366A had no effect on activity. We propose that D362 facilitates glucose excision, and that W365 facilitates its transfer, either (a) directly into solution (allowing PfuAmyGT to act as an exoamylase), or (b) by glycoside bond formation with an acceptor (allowing PfuAmyGT to act as a 4-α-glucanotransferase), depending upon whether the acceptor site is vacant or occupied in a reaction cycle.

Enzymatic and molecular characterization of α-1,3-glucanase (AglST2) from Streptomyces thermodiastaticus HF3-3 and its relation with α-1,3-glucanase HF65 (AglST1).

Extracellular α-1,3-glucanase HF90 (AglST2), with a sodium dodecyl sulfate (SDS)-PAGE-estimated molecular mass of approximately 91 kDa, was homogenously purified from the culture filtrate of Streptomyces thermodiastaticus HF3-3. AglST2 showed a high homology with mycodextranase in an amino acid sequence and demonstrated specificity with an α-1,3-glycosidic linkage of homo α-1,3-glucan. It has been suggested that AglST2 may be a new type of α-1,3-glucanase. The optimum pH and temperature of AglST2 were pH 5.5 and 60°C, respectively. AglST2 action was significantly stimulated in the presence of 5-20% (w/v) NaCl, and 1 mM metal ions Mn2+ and Co2+. On the other hand, it was inhibited by 1 mM of Ag+, Cu2+, Fe2+ and Ni2+. Regarding the stability properties, AglST2 retained more than 80% of its maximum activity over a pH range of 5.0-7.0 at up to 60°C and in the presence of 0-20% (w/v) NaCl. Based on these results, the properties of AglST2 were comparable with those of AglST1, which had been previously purified and characterized from S. thermodiastaticus HF3-3 previously. The N-terminal amino acid sequence of AglST2 showed a good agreement with that of AglST1, suggesting that AglST1 was generated from AglST2 by proteolysis during cultivation. MALDI-TOF mass analysis suggested that AglST1 might be generated from AglST2 by the proteolytic removal of C-terminus polypeptide (approximately 20 kDa). Our investigation thus revealed the properties of AglST2, such as tolerance against high temperature, salts, and surfactants, which have promising industrial applications.

Structural and functional characterization of a family GH53 ß-1,4-galactanase from Bacteroides thetaiotaomicron that facilitates degradation of prebiotic galactooligosaccharides.

Galactooligosaccharides (GOS) are prebiotic compounds synthesized from lactose using bacterial enzymes and are known to stimulate growth of beneficial bifidobacteria in the human colon. Bacteroides thetaiotaomicron is a prominent human colon commensal bacterial species that hydrolyzes GOS using an extracellular Glycosyl Hydrolase (GH) family GH53 endo-galactanase enzyme (BTGH53), releasing galactose-based products for growth. Here we dissect the molecular basis for GOS activity of this B. thetaiotaomicron GH53 endo-galactanase. Elucidation of its X-ray crystal structure revealed that BTGH53 has a relatively open active site cleft which was not observed with the bacterial enzyme from Bacillus licheniformis (BLGAL). BTGH53 acted on GOS with degree of polymerization ≤3 and therefore more closely resembles activity of fungal GH53 enzymes (e.g. Aspergillus aculeatus AAGAL and Meripileus giganteus MGGAL). Probiotic lactobacilli that lack galactan utilization systems constitute a group of bacteria with relevance for a healthy (infant) gut. The strains tested were unable to use GOS ≥ DP3. However, they completely consumed GOS in the presence of BTGH53, resulting in clear stimulation of their extent of growth. The extracellular BTGH53 enzyme thus may play an important role in carbohydrate metabolism in complex microbial environments such as the human colon. It also may find application for the development of synergistic synbiotics.

The IMD pathway regulates lysozyme-like proteins (LLPs) in the silkmoth Antheraea mylitta.

Lysozyme-like proteins (LLPs) are members of the glycoside hydrolase family 22 (CAZY GH22). Unlike conventional c-type lysozymes (EC 3.2.1.17), LLPs lack specific catalytic amino acid residues essential for muramidase activity. Previous reports indicated upregulation of LLPs upon bacterial infection in the wild silkworm, Antheraea mylitta as well as in the domesticated silkworm, Bombyx mori. In the present work, we studied the signaling pathways mediating the production of LLPs using RNA interference-mediated knockdown of Spätzle, Relish and STAT, the key regulators of Toll, IMD (Immune deficiency) and JAK/STAT pathways, respectively. We observed that knockdown of the Relish variant RD1 resulted in reduced expression levels of the ALLP1. We also showed that recombinant LLP has antiviral activity. We infer that LLPs showing both antibacterial and antiviral activity are regulated by the conventional IMD pathway in the silkmoths.

Cloning, expression, and characterization of thermal-stable and pH-stable agarase from mangrove sediments.

AgaM1, a ß-agarase belonging to glycoside hydrolases family 16 (GH16), was cloned from the environmental DNA of mangrove sediments. The gene agaM1 is 2136 bp in length and encodes a protein of 712 amino acids. The properties of recombinant AgaM1 (rAgaM1) were studied using prokaryotic expression. The optimum temperature and pH were 50 °C and 7.0, respectively, and rAgaM1 exhibited a high adaptability to wide ranges of temperature and pH. A relatively high activity was retained at from 30 to 60 °C and from pH 6.0 to 9.0. Thermal stability was showed more than 70% relative activity after pre-incubation at 40 °C for 60 h. Outstanding pH stability were observed for rAgaM1 from pH 5.0 to 10.0 after pre-incubation for 60 h. Thin-layer chromatography revealed neoagarotetraose (NA4) and neoagarohexaose (NA6) were the end-products of rAgaM1-degraded agarose. Besides, rAgaM1 were found with a Km of 1.82 mg ml-1 and a Vm of 357.14 U mg-1 for agarose. The Km was smaller than those of most agarases reported previously. This discrepancy revealed the high affinity of rAgaM1 to agarose. Overall, the results indicated the potential of rAgaM1 in future industrial application.

Proteomic analyses identify ARH3 as a serine mono-ADP-ribosylhydrolase.

ADP-ribosylation is a posttranslational modification that exists in monomeric and polymeric forms. Whereas the writers (e.g. ARTD1/PARP1) and erasers (e.g. PARG, ARH3) of poly-ADP-ribosylation (PARylation) are relatively well described, the enzymes involved in mono-ADP-ribosylation (MARylation) have been less well investigated. While erasers for the MARylation of glutamate/aspartate and arginine have been identified, the respective enzymes with specificity for serine were missing. Here we report that, in vitro, ARH3 specifically binds and demodifies proteins and peptides that are MARylated. Molecular modeling and site-directed mutagenesis of ARH3 revealed that numerous residues are critical for both the mono- and the poly-ADP-ribosylhydrolase activity of ARH3. Notably, a mass spectrometric approach showed that ARH3-deficient mouse embryonic fibroblasts are characterized by a specific increase in serine-ADP-ribosylation in vivo under untreated conditions as well as following hydrogen peroxide stress. Together, our results establish ARH3 as a serine mono-ADP-ribosylhydrolase and as an important regulator of the basal and stress-induced ADP-ribosylome.

Arabidopsis choline transporter-like 1 (CTL1) regulates secretory trafficking of auxin transporters to control seedling growth.

Auxin controls a myriad of plant developmental processes and plant response to environmental conditions. Precise trafficking of auxin transporters is essential for auxin homeostasis in plants. Here, we report characterization of Arabidopsis CTL1, which controls seedling growth and apical hook development by regulating intracellular trafficking of PIN-type auxin transporters. The CTL1 gene encodes a choline transporter-like protein with an expression pattern highly correlated with auxin distribution and is enriched in shoot and root apical meristems, lateral root primordia, the vascular system, and the concave side of the apical hook. The choline transporter-like 1 (CTL1) protein is localized to the trans-Golgi network (TGN), prevacuolar compartment (PVC), and plasma membrane (PM). Disruption of CTL1 gene expression alters the trafficking of 2 auxin efflux transporters-Arabidopsis PM-located auxin efflux transporter PIN-formed 1 (PIN1) and Arabidopsis PM-located auxin efflux transporter PIN-formed 3 (PIN3)-to the PM, thereby affecting auxin distribution and plant growth and development. We further found that phospholipids, sphingolipids, and other membrane lipids were significantly altered in the ctl1 mutant, linking CTL1 function to lipid homeostasis. We propose that CTL1 regulates protein sorting from the TGN to the PM through its function in lipid homeostasis.

A new vesicle trafficking regulator CTL1 plays a crucial role in ion homeostasis.

Ion homeostasis is essential for plant growth and environmental adaptation, and maintaining ion homeostasis requires the precise regulation of various ion transporters, as well as correct root patterning. However, the mechanisms underlying these processes remain largely elusive. Here, we reported that a choline transporter gene, CTL1, controls ionome homeostasis by regulating the secretory trafficking of proteins required for plasmodesmata (PD) development, as well as the transport of some ion transporters. Map-based cloning studies revealed that CTL1 mutations alter the ion profile of Arabidopsis thaliana. We found that the phenotypes associated with these mutations are caused by a combination of PD defects and ion transporter misregulation. We also established that CTL1 is involved in regulating vesicle trafficking and is thus required for the trafficking of proteins essential for ion transport and PD development. Characterizing choline transporter-like 1 (CTL1) as a new regulator of protein sorting may enable researchers to understand not only ion homeostasis in plants but also vesicle trafficking in general.

The molecular structure of the glycoside hydrolase domain of Cwp19 from Clostridium difficile.

Clostridium difficile is a burden to healthcare systems around the world, causing tens of thousands of deaths annually. The S-layer of the bacterium, a layer of protein found of the surface of cells, has received a significant amount of attention over the past two decades as a potential target to combat the growing threat presented by C. difficile infections. The S-layer contains a wide range of proteins, each of which possesses three cell wall-binding domains, while many also possess a "functional" region. Here, we present the high resolution structure of the functional region of one such protein, Cwp19 along with preliminary functional characterisation of the predicted glycoside hydrolase. Cwp19 has a TIM barrel fold and appears to possess a high degree of substrate selectivity. The protein also exhibits peptidoglycan hydrolase activity, an order of magnitude slower than that of lysozyme and is the first member of glycoside hydrolase-like family 10 to be characterised. This research goes some way to understanding the role of Cwp19 in the S-layer of C. difficile. DATABASE: Structural data are available in the PDB under the accession numbers 5OQ2 and 5OQ3.

Structural and functional characterization of a highly secreted α-l-arabinofuranosidase (GH62) from Aspergillus nidulans grown on sugarcane bagasse.

Carbohydrate-Active Enzymes are key enzymes for biomass-to-bioproducts conversion. α-l-Arabinofuranosidases that belong to the Glycoside Hydrolase family 62 (GH62) have important applications in biofuel production from plant biomass by hydrolyzing arabinoxylans, found in both the primary and secondary cell walls of plants. In this work, we identified a GH62 α-l-arabinofuranosidase (AnAbf62Awt) that was highly secreted when Aspergillus nidulans was cultivated on sugarcane bagasse. The gene AN7908 was cloned and transformed in A. nidulans for homologous production of AnAbf62Awt, and we confirmed that the enzyme is N-glycosylated at asparagine 83 by mass spectrometry analysis. The enzyme was also expressed in Escherichia coli and the studies of circular dichroism showed that the melting temperature and structural profile of AnAbf62Awt and the non-glycosylated enzyme from E. coli (AnAbf62Adeglyc) were highly similar. In addition, the designed glycomutant AnAbf62AN83Q presented similar patterns of secretion and activity to the AnAbf62Awt, indicating that the N-glycan does not influence the properties of this enzyme. The crystallographic structure of AnAbf62Adeglyc was obtained and the 1.7Å resolution model showed a five-bladed ß-propeller fold, which is conserved in family GH62. Mutants AnAbf62AY312F and AnAbf62AY312S showed that Y312 was an important substrate-binding residue. Molecular dynamics simulations indicated that the loop containing Y312 could access different conformations separated by moderately low energy barriers. One of these conformations, comprising a local minimum, is responsible for placing Y312 in the vicinity of the arabinose glycosidic bond, and thus, may be important for catalytic efficiency.

Discovery of the Showdomycin Gene Cluster from Streptomyces showdoensis ATCC 15227 Yields Insight into the Biosynthetic Logic of C-Nucleoside Antibiotics.

Nucleoside antibiotics are a large class of pharmaceutically relevant chemical entities, which exhibit a broad spectrum of biological activities. Most nucleosides belong to the canonical N-nucleoside family, where the heterocyclic unit is connected to the carbohydrate through a carbon-nitrogen bond. However, atypical C-nucleosides were isolated from Streptomyces bacteria over 50 years ago, but the molecular basis for formation of these metabolites has been unknown. Here, we have sequenced the genome of S. showdoensis ATCC 15227 and identified the gene cluster responsible for showdomycin production. Key to the detection was the presence of sdmA, encoding an enzyme of the pseudouridine monophosphate glycosidase family, which could catalyze formation of the C-glycosidic bond. Sequence analysis revealed an unusual combination of biosynthetic genes, while inactivation and subsequent complementation of sdmA confirmed the involvement of the locus in showdomycin formation. The study provides the first steps toward generation of novel C-nucleosides by pathway engineering.

Specific killing of DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) glycohydrolase.

Poly(ADP-ribosylation) of proteins following DNA damage is well studied and the use of poly(ADP-ribose) polymerase (PARP) inhibitors as therapeutic agents is an exciting prospect for the treatment of many cancers. Poly(ADP-ribose) glycohydrolase (PARG) has endo- and exoglycosidase activities which can cleave glycosidic bonds, rapidly reversing the action of PARP enzymes. Like addition of poly(ADP-ribose) (PAR) by PARP, removal of PAR by PARG is also thought to be required for repair of DNA strand breaks and for continued replication at perturbed forks. Here we use siRNA to show a synthetic lethal relationship between PARG and BRCA1, BRCA2, PALB2, FAM175A (ABRAXAS) and BARD1. In addition, we demonstrate that MCF7 cells depleted of these proteins are sensitive to Gallotannin and a novel and specific PARG inhibitor PDD00017273. We confirm that PARG inhibition increases endogenous DNA damage, stalls replication forks and increases homologous recombination, and propose that it is the lack of homologous recombination (HR) proteins at PARG inhibitor-induced stalled replication forks that induces cell death. Interestingly not all genes that are synthetically lethal with PARP result in sensitivity to PARG inhibitors, suggesting that although there is overlap, the functions of PARP and PARG may not be completely identical. These data together add further evidence to the possibility that single treatment therapy with PARG inhibitors could be used for treatment of certain HR deficient tumours and provide insight into the relationship between PARP, PARG and the processes of DNA repair.

GH16 and GH81 family ß-(1,3)-glucanases in Aspergillus fumigatus are essential for conidial cell wall morphogenesis.

The fungal cell wall is a rigid structure because of fibrillar and branched ß-(1,3)-glucan linked to chitin. Softening of the cell wall is an essential phenomenon during fungal morphogenesis, wherein rigid cell wall structures are cleaved by glycosylhydrolases. During the search for glycosylhydrolases acting on ß-(1,3)-glucan, we identified seven genes in the Aspergillus fumigatus genome coding for potential endo-ß-(1,3)-glucanase. ENG1 (previously characterized and named ENGL1, Mouyna et al., ), belongs to the Glycoside-Hydrolase 81 (GH81) family, while ENG2 to ENG7, to GH16 family. ENG1 and four GH16 genes (ENG2-5) were expressed in the resting conidia as well as during germination, suggesting an essential role during A. fumigatus morphogenesis. Here, we report the effect of sequential deletion of AfENG2-5 (GH16) followed by AfENG1 (GH81) deletion in the Δeng2,3,4,5 mutant. The Δeng1,2,3,4,5 mutant showed conidial defects, with linear chains of conidia unable to separate while the germination rate was not affected. These results show, for the first time in a filamentous fungus, that endo ß-(1,3)-glucanases are essential for proper conidial cell wall assembly and thus segregation of conidia during conidiation.