Pex11 proteins are involved in membrane elongation and division processes associated with the multiplication of peroxisomes. Human Pex11pβ has recently been linked to a new disorder affecting peroxisome morphology and dynamics. Here, we have analyzed the exact membrane topology of Pex11pβ. Studies with an epitope-specific antibody and protease protection assays show that Pex11pβ is an integral membrane protein with two transmembrane domains flanking an internal region exposed to the peroxisomal matrix and N- and C-termini facing the cytosol. A glycine-rich internal region within Pex11pβ is dispensable for peroxisome membrane elongation and division. However, we demonstrate that an amphipathic helix (Helix 2) within the first N-terminal 40 amino acids is crucial for membrane elongation and self-interaction of Pex11pβ. Interestingly, we find that Pex11pβ self-interaction strongly depends on the detergent used for solubilization. We also show that N-terminal cysteines are not essential for membrane elongation, and that putative N-terminal phosphorylation sites are dispensable for Pex11pβ function. We propose that self-interaction of Pex11pβ regulates its membrane deforming activity in conjunction with membrane lipids. Mutations in many PEX genes have been identified as the cause of severe and often lethal peroxisome biogenesis disorders (e.g. Zellweger syndrome) Peroxisomes are single-membrane bound, multifunctional and highly dynamic organelles of most eukaryotic cells, which fulfil important metabolic functions in hydrogen peroxide and lipid metabolism. Their function has also been linked to developmental processes, stress response, age-related disorders, and antiviral innate immunity de novo biogenesis from the ER, growth and division of peroxisomes follows a multistep maturation pathway, which results in the formation of new daughter peroxisomes Peroxisome formation by growth and division involves the deformation and elongation of the peroxisomal membrane, its constriction and final scission It has recently been reported that Pex11 proteins feature amphipathic helices that can insert into the peroxisomal membrane, thus influencing membrane bending PEX11β gene was identified Pex11 proteins are conserved amongst species; however, many organisms contain various “isoforms” which are poorly characterized on a functional level, and may differ in their biochemical properties. Furthermore, their membrane topology is not entirely clear and may vary amongst different species. The mammalian genome encodes for three Pex11 proteins, Pex11pα, Pex11pβ, and Pex11pγ, which are thought to be integral membrane proteins with their N- and C-termini facing the cytosol HsPex11pβ, and demonstrated that HsPex11pβ is an integral transmembrane protein with two transmembrane domains flanking an internal region exposed to the peroxisomal matrix. Based on the topology data, we characterized functional motifs including potential phosphorylation sites and cysteine residues in its N-terminal domain. We demonstrate that a previously uncharacterized amphipathic helix (Helix 2) is essential for peroxisome membrane elongation and self-interaction of HsPex11pβ. Furthermore, we show that complex formation of HsPex11pβ strongly depends on the detergent used for solubilization. We propose that self-interaction of HsPex11pβ regulates its membrane deforming activity.Here, we addressed the exact membrane topology of HsPex11pα-Myc, HsPex11pβ-Myc, Myc-HsPex11pα, Myc-HsPex11pβ, Myc-HsPex11pγ, YFP-HsPex11pβ and HsPex11pβ-YFP were described before HsPex11pβ deletion constructs were generated by subcloning: N-terminal deletions Pex11pβΔN40-Myc, Pex11pβΔN60-Myc, and Pex11pβΔN70-Myc; Myc-Pex11pβΔGly is missing a glycine-rich region  (Table S1). Mutations were introduced through site-directed mutagenesis via PCR with oligonucleotide pairs harbouring the respective mutation (Table S1). The following plasmids were generated: phospho-mimicking mutants Pex11pβ-MycS11A, Pex11pβ-MycS11D, Pex11pβ-MycS38A and Pex11pβ-MycS38D; cysteine mutants Pex11pβ-MycC18S, Pex11pβ-MycC25S, Pex11pβ-MycC85S, Pex11pβ-MycC18S-C25S and Pex11pβ-MycC18S-C25S-C85S (Table S1). Plasmid Pex11pβ-MycA21P was obtained by cloning the coding sequence of Pex11pβ-MycA21P  into pcDNA3  (Table S1). In-frame insertion and mutations of all constructs were verified by sequencing . For a schematic overview of all plasmids used, see Fig. S1.Plasmids encoding for http://bp.nuap.Nagoya-u.ac.jp/sosui/); Toppred tool, Mobyle@Pasteur ; HMMTOP (http://www.enzim.hu/hmmtop/); TMPred (http://www.ch.embnet.org/software/TMPRED_form.html); TMHMM Server, v 2.0 (http://www.cbs.dtu.dk/services/TMHMM/); PredictProtein (http://www.predictprotein.org/); Split 4.0 server (http://split.pmfst.hr/split/4/) . Protein fragment size after proteinase K digest was calculated using PeptideMass (http://web.expasy.org/peptide_mass/).Potential transmembrane domains were predicted using SOSUI (http://kinasephos2.mbc.nctu.edu.tw/index.html); NetPhos 2.0 (http://www. cbs.dtu.dk/services/NetPhos/); DISPHOS (http://core.ist.temple.edu/pred/pred.html); NetPhosK (http://www.cbs.dtu.dk/services/NetPhosK/), Scansite MotifScan (http://scansite.mit.edu/motifscan_seq.phtml); ScanProsite (http://expasy.org/tools/scanprosite/) and ELM (http://elm.eu. org/). Alignment of Pex11β protein sequences from different species was performed using the ClustalW2 tool  .Potential phosphorylation sites or potential binding sites for kinases were predicted by KinasePhos 2.0  (see our Abcam Abreview) and anti-Pex14p . The following mouse monoclonal antibodies were used: anti-Pex19p,  and anti-Myc epitope 9E10 . Species-specific anti-IgG antibodies conjugated to HRP or to the fluorophores TRITC and Alexa 488 were obtained from BioRad , Dianova , Molecular Probes Europe  and Invitrogen .Rabbit polyclonal antibodies were used as follows: anti-GFP , anti-acyl-CoA oxidase (AOX), anti-PMP70 2-humidified incubator. For some experiments, cells were transferred to lipid-free Panserin™ 401 medium, a serum-free nutrient mixture . Cells were transfected with polyethylenimine  or by electroporation using the ECM 630 Electro Cell Manipulator  t test used to determine statistical differences. p values <0.05 were considered as significant, and p values <0.01 were considered as highly significant.COS-7 cells (ATCC CRL-1651) were maintained in DMEM supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin and 10% FCS  at 37°C in a 5% COCOS-7 cells transfected with Pex11pβ constructs were fixed with 4% para-formaldehyde, washed with PBS and treated with Triton X-100 (see above). The resulting supernatants were collected and cleared by centrifugation . The remaining cells (and non-treated controls) were rinsed with PBS and centrifuged . Cell pellets were then lysed . The samples were passed ten times through a 26-gauge syringe needle, incubated by mixing at 4°C for 30 min and cleared by centrifugation . Protein concentrations were determined using the Bradford assay  and proteins were precipitated with TCA. Equal amounts of protein were separated by SDS-PAGE (10–15% PAA gels) under reducing and non-reducing conditions, transferred to nitrocellulose  by semi-dry transfer  and analysed by immunoblotting. Immunoblots were processed using HRP-conjugated secondary antibodies and enhanced chemiluminescence reagents .Peroxisome-enriched fractions were prepared from COS-7 cells transfected with the indicated constructs and from controls (four 100 mm cell culture dishes) by differential centrifugation as described 2CO3, pH 11.5). Samples and controls were incubated for 30 minutes on ice with gentle shaking every 5 minutes and centrifuged at 223,000×g  in an Optima LE-80K Ultracentrifuge . The supernatant was collected and the final membrane pellet was resuspended in peroxisome homogenization buffer. Protein concentrations of all fractions were determined before SDS-PAGE and immunoblotting.For carbonate extraction, peroxisome-enriched fractions were resuspended in ice-cold carbonate buffer . The gradient was eluted into 12 fractions of 1 ml each. For gradient calibration a “Kit for Molecular Weights 14,000–500,000 - Non-denaturing PAGE2” from Sigma-Aldrich  was used. Samples were analyzed by immunoblotting.Twenty four hours after transfection, COS-7 cells expressing Pex11pβ-Myc were treated for 30 min at room temperature with 1 mM dithiobis[succinimidyl propionate] . After quenching with 50 mM Tris-HCl, pH 7.4 for 15 min at room temperature, cells were harvested, solubilized with Triton X-100 (or digitonin) and lysed as described above. Alternatively, lysates were prepared without previous cross-linking substituting Triton X-100 by 1% digitonin in the lysis buffer. Lysates were applied on top of a 10–47% sucrose step gradient in 50 mM Tris acetate buffer, pH 7.2 with 1 mM EDTA, 0.1% digitonin (Triton X-100) and were centrifuged for 3 h at 125,000×gScPex11p AtPex11a HsPex11pγ was recently reported to dock on the cytosolic site of the peroxisomal membrane in silico analysis of Pex11pβ does not always result in the prediction of two transmembrane domains as initially proposed Fig. S2B). Determination of the exact topology of Pex11pβ was so far limited by the lack of specific antibodies; however, a newly available antibody directed against an internal site (aa 110–140) corresponding to a region roughly behind the predicted first transmembrane domain enabled further characterization. First, COS-7 cells were transfected with Pex11pβ-Myc and processed for immunofluorescence microscopy by applying antibodies to the Myc-epitope at the very C-terminus and to Pex11pβ itself , proteinase K digest would result in an almost complete degradation of the protein (and hence the epitope); therefore no signal would be detected. Similarly, the antibody epitope would be digested if a first transmembrane domain at aa 170–200 is assumed . If a first transmembrane domain would be present approximately in the middle of the protein (aa 85–105), the antibody’s epitope would be rendered protease protected, resulting in the formation of a protein fragment of approximately 17 kDa . For protease-protection assays, COS-7 cells were transfected with YFP-Pex11pβ. Peroxisome-enriched fractions from non-transfected controls (UT) and transfected cells were mock treated, or incubated with proteinase K in the absence or presence of Triton X-100 . Proteinase K digest resulted in a band shift yielding a fragment of approx. 17 kDa, which was properly recognized by anti-Pex11pβ. Addition of Triton X-100 rendered the fragment sensitive to proteinase K digest . The protein fragment was also digested after membrane rupture by sonication . In the absence of proteinase K, the 56 kDa band of YFP-Pex11pβ was detected using an anti-GFP antibody. In the presence of proteinase K, the YFP fusion tag was removed from Pex11pβ and remained unaffected by the action of the protease . However, similar band intensities were detected in the absence or presence of Triton X-100, verifying equal loading of lanes. Further incubation of the blotting membranes with anti-AOX antibodies routinely served to ensure integrity of the peroxisomal membrane before addition of Triton X-100  and a second one between aa 230–255. Thus, Pex11pβ has a major part of its N-terminus exposed to the cytosol and possesses two hydrophobic transmembrane domains flanking an internal region which extends into the peroxisomal matrix or is buried within the lipid bilayer.The N-terminal YFP-tag of YFP-Pex11pβ appeared to be quite resilient to protease action, most probably due to the compact β-barrel structure of GFP and its analogues, and thus served as an excellent loading control after membrane stripping . To examine if the glycine-rich region  is required for Pex11pβ function, we deleted this region resulting in construct Myc-Pex11pβΔGly . Expression in COS-7 cells showed proper targeting to peroxisomes as revealed by immunofluorescence microscopy (We observed that human Pex11pβ contains a glycine-rich region at aa positions 159–182 (gsggvpggsetgglggpgtpggg), which is absent in Pex11pα or Pex11pγ. This region is located between the two transmembrane domains and based on our topology studies, is exposed to the peroxisomal matrix . The results were combined with a homology screen of various Pex11pβ protein sequences examined for conservation of putative phosphorylation sites . Several conserved sites were identified at positions S11, S38, S70, S154, S160, S168 and T178 within the human protein, which showed high probability for possible phosphorylation . Here, we focused on the putative phosphorylation sites S11 and S38 in the 40 aa N-terminal portion of Pex11pβ, because deletion of the 40 aa was sufficient to inhibit membrane elongation and homo-dimerization (see below). Furthermore, our topology results indicate that the residues S11 and S38 are present in the cytosolic portion of Pex11pβ and are thus potentially accessible to cytosolic kinases. Individual point mutations were generated by site-directed mutagenesis. We converted the respective serines to alanine to block putative phosphorylation resulting in constructs Pex11pβ-MycS11A and Pex11pβ-MycS38A. Furthermore, to generate phospho-mimicking (constitutively phosphorylated) versions we mutated the sequences encoding S11 or S38 to aspartate resulting in constructs Pex11pβ-MycS11D and Pex11pβ-MycS38D. The constructs were expressed in COS-7 cells and alterations of peroxisome morphology were analyzed at different time points by immunofluorescence microscopy using anti-Myc and Pex14p antibodies . Wild type Pex11pβ induces a prominent elongation of peroxisomes which is followed by division into spherical organelles over time ; Fig. S4). A similar pattern of morphological alterations was observed in all mutants generated. No enlarged or otherwise altered morphologies were detected and division proceeded normally over time when Pex11pβ-MycS11A or Pex11pβ-MycS38A were expressed. Similarly, expression of Pex11pβ-MycS11D or Pex11pβ-MycS38D did not result in division at a faster rate. These findings indicate that modifications of S11 and S38 have no impact on peroxisome elongation or division, but do not exclude that other putative phosphorylation sites within Pex11pβ may modulate its activity. So far, we have not obtained any indication that Pex11pβ is phosphorylated under our experimental conditions (e.g. by phospho-labelling). These findings may indicate that yeast and mammalian Pex11 proteins rely on different regulatory mechanisms.To identify potential phosphorylation sites in Pex11pβ, we performed an S2A) in vitro studies using peptides matching Helix 3 of ScPex11p, HpPex11p, and HsPex11pα and HsPex11pβ showed the ability of the peptides to elongate negatively charged small unilamellar vesicles in situ, we generated N-terminally truncated versions  and analyzed their effect on peroxisome morphology . This is in contrast to the expression of full-length Pex11pβ-Myc, which typically induced a significant membrane elongation S2A). This indicates that although peptides matching Helix 3 are capable of elongating liposomal structures in vitro, also Helix 2 (and region H1) is required for peroxisome elongation in living cells.Pex11 proteins possess amphipathic regions which are supposed to play important roles in membrane remodelling and peroxisome proliferation rphology Fig. 5. A21P), and analyzed the effect of this version on peroxisome morphology . Interestingly, expression of this mutant construct did not result in prominent peroxisome elongation, thus confirming the importance of Helix 2 for proper Pex11pβ function.To verify this assumption, we introduced a proline at position 21, which breaks the helical structure . We now discovered that co-expression of YFP-Pex11pβ and Pex11pβ-Myc induced the formation of TPAs  can be extracted from peroxisomal membranes after postfixation Triton X-100 treatment Our postfixation Triton X-100 assay revealed the formation of Pex11pβ dimers, but higher ordered oligomeric structures were not detected. However, high molecular mass complexes ranging from 230–430 kDa were recently reported by induction of peroxisome proliferation with docosahexaenoic acid To investigate the requirement of lipids in Pex11pβ-mediated membrane elongation and division, we cultured COS-7 cells stably expressing a GFP-fusion protein carrying a peroxisomal targeting signal (GFP-PTS1) under lipid- and serum-free conditions. At different time points, cells transfected with Myc-Pex11pβ were analyzed by immunofluorescence microscopy using anti-Myc antibodies. Interestingly, cells cultured under lipid-free conditions revealed alterations in peroxisome morphology exhibiting enlarged, spherical organelles Fig. 8. S2A). To analyze if these cysteines contribute to Pex11pβ self-interaction or conformation, e.g. by the formation of intra-molecular disulfide bridges, we replaced the cysteines by serine  and generated triple (Pex11pβ-MycC18S-C25S-C85S) as well as double (Pex11pβ-MycC18S-C25S) and single mutants . After expression in COS-7 cells, all versions were properly targeted to peroxisomes as demonstrated by immunofluorescence microscopy using anti-Myc and anti-Pex14p antibodies Click here for additional data file.Figure S2(A) Overview of the location of putative amphipathic helices, transmembrane domains, potential phosphorylation sites and cysteine residues within the N-terminal portion of HsPex11pβ (aa sequence). (B) Predicted positions of the transmembrane domains of human Pex11pβ. A variety of in silico screening tools were applied to determine the position of the transmembrane domains in HsPex11pβ. Based on these results, the expected size of the protein fragment between the two transmembrane domains was calculated using PeptideMass counter.(TIF)Click here for additional data file.Figure S3Determination of potential phosphorylation sites within HsPex11pβ. (A) Overview of multiple hits for different amino acid positions. Several online screening tools were used to determine potential phosphorylation sites in the sequence of human Pex11pβ. The various tools are plotted against the positions given. (B) Scheme depicting phosphorylation-sites chosen for subsequent studies. Based on the screening, several putative phosphorylation sites were selected whose location is indicated in the upper scheme . Based on our findings regarding the topology of Pex11pβ, intra-peroxisomal sites were excluded . Furthermore, based on studies regarding deletions of the N-terminus, the phosphorylation sites listed on the bottom were chosen. (C) Overview of conserved amino acids within Pex11pβ protein sequences across species. The putative phosphorylation-sites are depicted in red brackets. Note that the one at position S11 is highly conserved.(TIF)Click here for additional data file.Figure S4 COS-7 cells were transfected with Pex11pβ-Myc , Pex11pβ-MycS11A , Pex11pβ-MycS11D , Pex11pβ-MycS38A  and Pex11pβ-MycS38D . Cells were fixed after 24 and 72 h, processed for immunofluorescence and labeled with antibodies directed to the Myc-epitope (A1-J1) and the peroxisomal marker protein Pex14p (A2-J2). Bars, 20 µm.(TIF)Click here for additional data file.Table S1Plasmids and oligonucleotides used in this study.(TIF)Click here for additional data file.


The molecular mechanism of its function, including ion selectivity and gating,has not been fully characterized. Recently we reported a new structure of a CorA homologue fromMethanocaldococcus jannaschii, which provided novel structural details that offeredthe conception of a unique gating mechanism involving conversion of an open hydrophilic gate into aclosed hydrophobic one. In the present study we report functional evidence for this novel gatingmechanism in the Thermotoga maritima CorA together with an improved crystalstructure of this CorA to 2.7 Å (1 Å=0.1 nm) resolution. The latter reveals theorganization of the selectivity filter to be similar to that of M. jannaschii CorAand also the previously unknown organization of the second signature motif of the CorA family. Theproposed gating is achieved by a helical rotation upon the binding of a metal ion substrate to theregulatory binding sites. Additionally, our data suggest that the preference of this CorA forCo2+ over Mg2+ is controlled by the presence of threonine side chains in thechannel. Finally, the roles of the intracellular metal-binding sites have been assigned to increasedthermostability and regulation of the gating. These mechanisms most likely apply to the entire CorAfamily as they are regulated by the highly conserved amino acids.The CorA family of divalent cation transporters utilizes Mg Mg2+ is themost abundant divalent cation among all living organisms and is involved in a myriad of biologicalactivities. Co2+ is an essential trace element and, as the cofactor for cobalamin, it isinvolved in several cellular metabolic pathways, especially the synthesis of DNA and fatty acids. Inaddition, Co2+ is the essential cofactor for many enzymes in several micro-organisms. Theintracellular concentrations of these ions must be kept under tight control, as imbalances may leadto major consequences that could ultimately result in cell death. The CorA family of divalent cationtransporters is ubiquitous among prokaryotes, with functional homologues in eukaryotes  and 0.05%DDM} to remove the cleaved His6 tag from the solution. The eluate was then incubated with5 mM EDTA to remove any residual divalent metals and desalted with a PD10 column equilibratedwith GF buffer. The desalted eluate underwent a final purification step with Superdex 200 16/60  equilibrated with GF buffer.Plasmids carrying the mutant eviously , with thD600 of 1.0 and thensubjected to SDS/PAGE using 12% Bis-Tris gels (Invitrogen) according to the manufacturer'srecommendations. Protein bands were then transferred from the gel on to nitrocellulose membranesusing the I-Blot system (Invitrogen), according to the manufacturer's recommendations. The membraneswere then incubated in TBST , blocked with 5% BSA (Sigma–Aldrich) and probed with an horseradishperoxidase-conjugated His6 probe. West-Pico (Pierce) was used to visualize the bandsaccording to the manufacturer's recommendations.Whole bacterial cells were adjusted to a E. coli MG1655 with a knockout corA was transformed with the mutatedcorA genes. The knock-out corA strains transformed with either theempty pBAD vector or T. maritima corA were used as negative and positive controlsrespectively. Cultures were grown overnight at 37°C in LB (Luria–Bertani) medium(Formedium) supplemented with 100 μg/ml each of ampicillin and kanamycin. Theovernight culture was diluted 12-fold into 10 ml of fresh LB supplemented with100 μg/ml each of ampicillin and kanamycin. Cells were left to grow at 37°C for0.5–2 h depending on the proteins’ particular expression requirements. Proteinexpression was then induced with 0.02% l-arabinose (Sigma–Aldrich) for 2 h.Subsequently, the cells were harvested at 3000 g for10 min at 25°C and washed twice by resuspension in 10 ml of N-buffer. The cells were then normalized to a D600 of 0.2 bydilution with N-buffer containing various concentrations of Co2+. The mixture was thenincubated for 10 min at 37°C. Three equivalents of LB containing the sameconcentrations of Co2+ were added into each mixture, and the solution was incubated at37°C for an additional 3 h. The final D600 value wasrecorded and analysed in comparison with the starting D600. TheMg2+ competition assays were conducted similarly by adding Mg2+ at variousconcentrations. All the data and statistics were analysed by Prism 6 (GraphPad).The assay was performed as described previously , withso2+ concentrations added to thealiquot. Samples were incubated for 20 min at room temperature and then further incubated for10 min at every increment of 10°C up to 85°C in a thermocycler (AgilentTechnologies). The samples were transferred to a 96-well filter plate (0.65 μm)(Millipore) to remove precipitated proteins. The yield of the filtered protein was analysed bySDS/PAGE stained with Coomassie Brilliant Blue (Merck). Each experiment was carried out at leastthree times to obtain the S.D.Purified protein samples suspended in GF buffer were diluted to a concentration of0.5 mg/ml. The solution was aliquoted into 30 μl volumes, with1 μl of a stock solution of different Cohttp://www.pymol.org). The final structure andstructural factors were deposited to the PDB under the accession code 4I0U.TmCorA was crystallized essentially as described previously . Data we2+was identified as being co-ordinated by the carbonyl groups of glycine and the hydroxy groups ofasparagine of the GMN motif [http://www.biochemj.org/bj/451/bj4510365add.htm). The universally conserved GMN motif isarranged in an almost exact way as that of MjCorA, creating a polar entry made of theAsn314 side chain with the underlying ring of the carbonyl group from Gly312, similar to what was seen in MjCorA [311 as well as Tyr309 and Phe315, the other two highly conservedresidues (the YGMNF motif). Hence the conservation of the structure of the extracellular entry ofCorA is now confirmed by comparing the new improved structure of TmCorA with the recently reportedstructure of MjCorA.The previously resolved crystal structures of TmCorA –11 were MN motif . FollowiMN motif , we werem Gly312C. A stron MjCorA  and in an MjCorA . The hyd316 faces towards theconcavity, and most probably facilitates the initial trawling of Mg2+ ions, as predictedpreviously [316 to be the main binding and selection site fora fully hydrated Mg2+ [320) of the conserved MPEL motif. Itis facing away from the concavity and is trapped in a hydrophobic patch surrounded by highlyconserved tyrosine and proline residues  , whereasTmCorA contains the conserved region EYMPEL (where MPEL is the second signature motif of the CorAfamily). The latter is thus more charged compared with the more hydrophobic motif in MjCorA. Thestructure of the TmCorA loop reveals that the negatively charged Glueviously . Biochemted Mg2+ ,22. Howemonomer) D as well288, Thr295 andThr299) was identified on the first transmembrane helix (TM1), close to the hydrophobicconstriction site . To study the Co2+ transportactivity, we used our previously reported Co2+ transport assay, where acorA-deficient E. coli strain that is resistant to Co2+becomes highly Co2+-sensitive upon expressing recombinant TmCorA [2+ sensitivity, indicating that theleucine residue mutation(s) had completely blocked Co2+ transport . These data suggest that these threeresidues are essential to the function of the transporter. It is likely that they face the channeland create a polar environment suitable for transport.To validate our hypothesis, we performed site-directed mutagenesis on Asnransport A. The siectively A. The do299 site, itcould indicate that the ion passage through the pore is narrowest at the cytoplasm/membraneinterface (at Asn288) and becomes wider towards Thr299. This interface is inturn comparable with the size and shape of the pore in its closed conformation. To further explorethis idea, we performed the Co2+ transport assay on TmCorA bearing either the T295M orthe T299M mutations. The inhibitory effect of the T295M mutation on the TmCorA Co2+transport was almost identical with that of the T295L mutation  and aidstheir transport [As the leucine-mediated blockage of the channel decreases towards the Thrmutation B. Howevemutation B. Methioransport .305 is the lone polar residue inside thechannel. A corresponding threonine residue in the MjCorA structure, Thr264, was observedto mediate Mg2+ binding in the closed conformation, thus it is possible thatThr305 of TmCorA performs the similar role. To verify whether its hydroxy group ispositioned in the channel in the open conformation of TmCorA, we mutated this threonine residue to aleucine. If TmCorA with the T305L mutation displays resistance to Co2+, it would indicatethat Thr305 faces the channel during ion transport and thus is indeed involved in the ionco-ordination. In fact, the Co2+ transport assay revealed that the T305L mutationinhibited the transport ability of TmCorA , E.coli CorA and Haemophilus influenzae CorA, whereas the TmCorA of subgroupA is a Co2+-selective transporter. The presence of threonine in subgroup A and serine insubgroup B appears to be the only main difference in the polar side chains that are active in iontransport. We hypothesized that the differences in substrate specificity between the two subgroupscould be due to the presence of threonine or serine residues. To test this hypothesis, we performedsite-directed mutagenesis to create the T305S, T299S and T295S mutations in TmCorA. These mutantswere then subjected to the Co2+-transport assay. As shown in 2+ transport activity of TmCorA carrying the T295Smutation did not dramatically affect Co2+ sensitivity. However, both the T305S and T299Smutations completely impaired Co2+ transport. This finding clearly indicates theimportance of threonine residues in Co2+ selection. Thr305 andThr299 are situated closer to the periplasmic entrance, hence they are apparently moreeffective in ion selection. Thr295 is situated much farther from the channel entrance,which may prevent it from affecting Co2+ selection. Nevertheless, to further explorewhether T295S is less Co2+-selective, and also whether the presence of serine in TM1 canincrease the Mg2+-selectivity of CorA, we included Mg2+ as a competitor toCo2+ at various concentrations while assaying the Co2+ transport activity ofthe T295S mutant. Already in the presence of 5 μM Mg2+, the T295S channelwas unable to select for Co2+, whereas wild-type TmCorA was highly sensitive to lowconcentrations of Co2+, even in the presence of 250 μM Mg2+(2+selection. The data also suggest that Mg2+ is better selected by serine residues,explaining the presence of serine in the transmembrane region of Mg2+-selectivetransporters.Asn μM Mg2+, in the 2+ bindingat the M1 site that is tightly co-ordinated by the carboxyl groups of Asp89 andAsp253. The distance between Co2+ and these carboxyl groups is approximately 2Å, thus excluding any bridging water molecules. If the binding of Co2+ to the M1and M2 sites is required to close the channel, then a disruption in metal co-ordination would resultin a permanently open channel. Hence a Co2+ sensitivity that is similar to or worse thanthat of wild-type TmCorA will be detected. The D89K mutation resulted in the reverse effect, withsignificantly reduced Co2+ sensitivity, indicating impaired Co2+ transport(253 and thus triggered channel closure, as suggested earlier by Payandeh et al. [2+ transportability of TmCorA carrying the D89N mutation. Similar to the D89K mutation, the D89N mutation didnot trigger Co2+ sensitivity to the same degree as the wild-type TmCorA (89 for the stability of TmCorA. We recently showed that the stability of TmCorA atphysiological temperatures was dependent on the presence of its Co2+ substrate, withoutwhich it stays stable only up to 75°C; the addition of micromolar Co2+ ensuredTmCorA stability up to 95°C [89 is essential for both the functionality and stability of TmCorA, in which thelatter is also dependent on the presence of Co2+. As Asp89 is clearly involvedin the co-ordination of the Co2+ bound to the M1 site, its importance in maintainingstability and functionality must be directly linked with the role of the M1-site in the stability ofTmCorA. To test this further, we performed additional site-directed mutagenesis to specificallytarget the M1-site. His257 appears to co-ordinate the Co2+ in the M1-site(2+, in contrast with Mg2+, where such a type of co-ordinationis common only in chlorophylls [257 coulddefine the specificity of the M1-site for Co2+. Hence the H257A TmCorA mutant was createdand its Co2+ transport ability was analysed. The experiment showed that mutation at thisposition affects the transport ability of TmCorA in a more pronounced manner than theAsp89 mutations (253 for the functionality andstability of TmCorA. The D253K mutation did not cause any significant change in the Co2+transport activity of TmCorA (256, is located adjacent to the M1-site, but notto the M2-site. This residue could be involved in Co2+ co-ordination at the M1-site, inthe absence of Asp253. Therefore we created the double mutation D253K/D256A. TheCo2+ transport assay revealed that this double mutation made TmCorA more resistant toCo2+ in a similar manner to the H257A mutation (253 andAsp256, as the single mutations failed to significantly affect the transport activity ofTmCorA. Collectively, the M1-binding site was identified as the stabilizing metal-binding site,which needs to be intact, and perhaps constantly occupied by Co2+, to maintain thefunctionality of TmCorA.An anti-clockwise rotation along helix 7 of TmCorA would also cause the disruption of metalco-ordination in the metal-binding sites, M1 and M2, as observed in the closed conformation A. Thus, ransportB. This mh et al. . To verie TmCorA B. These  to 95°C . Thermos M1-siteA. Owing utations B. Additiutations C. Finallf TmCorA B. This rmutation B. It was2+passes through a polar channel co-ordinated by hydroxy groups of threonine and asparagine; threonineis also instrumental to select for Co2+. The metal-binding site M1 is constantly andtightly occupied to maintain the stability and functionality of TmCorA. When the intracellularconcentration of Co2+ is elevated, Co2+ also binds to the M2 site. Thisbinding causes helix 7 to turn clockwise, which sequentially will result in the removal of the polarresidues from the channel and replace them with hydrophobic leucine and methionine residues, andthus this will close the gate.Altogether, the mechanism of ion transport and gating can be summarized as illustrated in 305. Most likely, this polarity begins at the entry with the conservedAsn314 from the GMN motif. From Met302 towards the cytoplasmic side, thediameter of the pore decreases and the interior residues become completely hydrophobic. Ahydrophobic pore is completely closed to ions with radii of 4.5 Å or smaller [299,Thr295 and Asn288, along with that of Thr305, are aligned on helix7 and face the channel. This is consistent with our recent model based on the crystal structure ofMjCorA, where we also identified partially hydrated Mg2+ within the channel co-ordinatedby polar residues [2+ transport in StCorA [2+ and Mg2+ transport and not the negatively charged carboxyl or carbonylgroups. Accordingly, a clockwise turn along helix 7 will remove Thr299, Thr295and Asn288 from the pore and replace them with mainly Met302,Leu294 and Met291 to close the pore. Presumably, such a rotation would be moreenergetically favourable than the sideway movements of helices, or combination of co-ordinatedmovement in three directions assured by breaking and forming pairs of salt bridges. However, theposition of Thr305 is not changed and the helical turn appears to be stopped at theThr299/Met302 site. This stop appears to be mediated by the hydrophobicinteractions between the side chains of Phe301 of one monomer and Pro303 of anadjacent monomer, which creates a kink in the TM1 (As seen in the crystal structure, the pore is polar near the entrance at residueThr smaller . Such a  smaller .Chakrab smaller  have pos smaller . Their n smaller . Thereforesidues . A partin StCorA . Most li the TM1 A.A simi320 of the conserved MPEL motif located inthe periplasmic loop is puzzling. This motif is highly conserved and preferred among bacteria. Theside chain of Glu320 is positioned towards the hydrophobic environment. Such anon-favoured location of Glu320 might make the loop less rigid and more flexible than thecorresponding loop in MjCorA. This might explain why it was tremendously difficult to get thestructure of the periplasmic loop in the case of TmCorA, compared with MjCorA where MPEL issubstituted with the LPLA motif, which provides the extreme rigidness of the loop. Perhaps suchflexibility will in turn allow for better movements, either during the ion uptake, or during thehelical rotation and gating.The position of the negatively charged Glu2+ and Co2+. However, TmCorA from subgroup A has shown high selectivity forCo2+ over Mg2+, whereas members from subgroup B, such as StCorA, have shown astronger preference for Mg2+. The entrance of all CorAs is made of highly conservedresidues and therefore the selection between Mg2+ and Co2+ is most likely notat the entrance. Our data show that the Co2+ selectivity of TmCorA is highly dependent onthe presence of threonine residues in the channel. The fact that removal of the methyl group fromthe threonine residues in the channel turns TmCorA into being more selective for Mg2+ iscertainly remarkable and intriguing. In line with that, the pore-forming TM1 contains more serinethan threonine residues in the Mg2+-selective subgroup B. Both Mg2+ andCo2+ have six water molecules in their first hydration shell [2+ has beenconsistently reported to contain 12 water molecules in its second hydration shell, whereas forCo2+ this number has varied from six to 14 (see [2+, butnot for Co2+. Thus it is reasonable to assume that perhaps Co2+ has a moreflexible second hydration shell as compared with Mg2+. The rigidity in the secondhydration shell of Mg2+ may prevent it from readily interacting with threonine, whichcontains a bulky methyl group, as compared with that of serine.All members of the CorA family characterized so far have shown the ability to transport bothMgon shell ,28. Howe 14 (see ,28 and t2+ is not limited to the threonine residues in thechannel. The regulatory cytoplasmic metal-binding site is also Co2+-specific, as shown byvarious competition studies as well as thermostability studies, in which Co2+consistently outcompeted Mg2+ [2+ to these sites. Hence, during ion transport, these sites would beunoccupied; upon an increase in intracellular Co2+, the ions would bind to these sites,for which the co-ordinating residues on helix 7 would need to turn clockwise. Nevertheless, someconsiderations could render such a sequence of events rather debatable. For example, the metal ionin M1 binds directly to Asp89 and Asp253, as their carboxyl groups replace thewater molecules of the first hydration shell. Such dehydration requires a significant energy input;likewise, the rehydration would also cost additional energy. The latter could lead to the questionof whether the M1 site is always occupied and holds a different role other than regulating thegating process. As has been shown, TmCorA requires Co2+ to remain stable at physiologicaltemperatures [89 and His257. Thelatter is highly conserved in subgroup A of the CorA family, to which the Co2+transporter TmCorA belongs; the residue is missing in subgroup B of the Mg2+transporters, as well as in the archaean CorA from MjCorA, which is also a selective Mg2+transporter. This organism is also hyperthermophilic and prefers optimal surrounding temperatures ofup to 94°C [2+[2+ requirement for thethermostability of TmCorA. Therefore we believe that the M1 site is indeed always occupied withCo2+ and its main role is to maintain the stability and thus the functionality of TmCorA.When the intracellular Co2+ concentration is increased, it will bind to the M2 site. TheM2 site, in contrast with the M1 site, is occupied by a partially hydrated ion, which binds to theco-ordinating amino acids via transient hydrogen bonds. The occupation of the M2 site will theninduce a pulling force, which may be enhanced by the occupancy of the M1 site. The latter reasoningis based on the involvement of Asp253 in co-ordinating the ions in both sites. Thispulling force is supported by the carbonyls from the backbone of Leu12 andPro13 (2+ transporters of subgroup B and MjCorA.The specificity of TmCorA for Coted Mg2+ ,9. The geratures . Disrupt to 94°C . Our the94°C [2+, contrarndPro13 A. These The structure of the ion pathway and the gating mechanism of the TmCorA all involve highlyconserved amino acids within the CorA family. Therefore the functional mechanisms suggested in thepresent study most likely represent those of the entire family. In addition, the differences withinthe CorA subgroups reflect their unique differences to suit their physiological roles.2+ or Mg2+.In conclusion, CorA transports its substrate in a partially hydrated form. A novel gatingmechanism, in which substrate binding to the cytoplasmic binding sites converts a narrow openhydrophilic pore into a narrow closed hydrophobic pore, regulates this transport. The presence ofthreonine or serine residues in the CorA channel determines the preference of the channel foreither Co


Ascosphaera in wild solitary bees to investigate the diversity of the genus in nature and the ecology of these fungi with their bee hosts. A new morphologically distinctive species was discovered which also has a unique nrITS sequence. This new species, here named Ascosphaera callicarpa, is common on the larval feces of the solitary bee Chelostoma florisomne which nests in the Phragmites reeds of thatched roofs in Europe. Because collections of Ascosphaera from wild bees are scarce and because little is known about the ecology and distribution of the majority of the species in the genus, a key to the species thus far reported for Europe is included.We studied the bee specialist fungus  Ascosphaera is a genus of 28 species of bee specialist fungi with a worldwide distribution in the temperate to tropical regions. The genus is remarkable for its host and habitat specificity with all species completing their entire life cycle within the nests of bees (Apoidea: Anthophila). Ascosphaera was first discovered in the early 20th century in Europe after A. apis, the type species, was identified as the causative agent of a brood disease affecting honeybees Ascosphaera is widely known as the chalkbrood fungus, although at least half of the species lead a saprotrophic rather than pathogenic lifestyle Ascosphaera species flourish on diverse substrates within the bee nest, for example on pollen provisions, on materials used by the bees to construct the nest and on larval feces Ascosphaera is placed in Ascosphaeraceae (Pezizomycotina: Eurotiomycetidae), a small family of ascomycetes primarily characterized by a unique fruiting body type called a spore cyst. Spore cysts are unicellular, cyst-like fruiting bodies that form from the expansion of a single cell called a nutriocyte Ascosphaera and its relatives remained uncertain until ontological studies led C.F. Spiltoir and L.S. Olive Ascosphaera is the presence of spore balls A distinguishing feature of Ascosphaera species afflict only the larval stage of bees. Typically diseased larvae die in the larval stage; however, in rare occurrences, larvae have been observed to enter pupation before being overcome by the fungus (Wynns pers. obs.). Pathogenic species of Ascosphaera appear to be highly specialized fungi with ascospores typically germinating only when within the midgut of their host. Spore germination is followed by rapid hyphal growth, with the fungus consuming the larva from the inside out Ascosphaera aggregata and A. apis, are of economic interest because of their potential to negatively impact populations of commercial pollinators, namely Apis mellifera L. and Megachile rotundata (Fabricius) Pathogenic Ascosphaera lives in association with both solitary and social bees the majority of species (25 out of 28) were originally described from solitary bees. Within the nests of solitary bees Ascosphaera grows on pollen provisions where an egg has failed to develop, on larval feces, on the surface of cocoons, within larvae, and on the diverse materials used by different bee species for brood cell construction Although AscosphaeraMegachile rotundata. While limited in scope, this monograph, which included the first key to the genus, remains the most useful and comprehensive reference for the identification of Ascosphaera species. Given the importance of wild pollinators and their increasing role in buffering the loss of honeybee pollination services The only monographic work on Ascosphaera are currently known from Europe. Here we describe a new species from Denmark occurring in the nests of the wild solitary bee Chelostoma florisomne L. To stimulate interest and to facilitate the identification of Ascosphaera species so far known from Europe, we provide a key and descriptions for these species. Cumulative host reports and species distributions are also included with the hope that this information will result in additional records for these under-collected fungi.Seven of the 28 described species of Descriptions of spore cysts and ascospores were made from observations of spore cysts mounted in water on a glass slide. Measurements and light photomicrographs were made on an Olympus AX70 Provis light microscope and Olympus SZX16 dissecting microscope. Herbarium acronyms follow those of Index Herbariorum 2 as described in Wynns et al. Attempts to isolate and culture the fungus were made by placing spore cysts and hyphae on three different solid agar media: malt agar with 20% dextrose (MY20), V8® agar with 2% yeast extract (V8YE), and malt extract agar (MEA). To induce spore germination spore suspensions were prepared from spore cysts placed in a modified V8 spore germination broth Genomic DNA was obtained by plucking 5–10 spore cysts and grinding them inside a 1.5 ml Eppendorf tube. DNA was isolated using the Qiagen DNeasy Plant Mini Kit  using the standard protocol and eluted in two separate 50–100 µl fractions to avoid over-dilution.A. callicarpa sp. nov. Genomic DNA was amplified using ITS1F and ITS4 primers Taq polymerase reaction buffer (Sigma®), 1.0 µl 10 mM dNTPs, 3.0 µl 25 mM MgCl2, 0.2 µl Taq DNA polymerase (Sigma®), 5.0 µl each 10 µM primer and 1 µl of genomic DNA template. PCR was performed on a Biometra® thermocycler (Whatman) under the following conditions: step 1) 1 min at 95 C, 2) 45 sec at 95 C, 3) 40 sec at 52 C, 4) 1 min 30 sec at 72 C, 5) return to step 2 30 times, 6) final step of 10 min at 72 C. Samples were kept at 4 C until electrophoresis was performed on a 1% agarose TAE gel and visualized with EZvision One® (Amresco). PCR reactions were cleaned using Qiaquick® PCR purification kit (Qiagen) and sent to Eurofins MWG Operon AG  for sequencing. The nucleotide sequence was assembled using BioEdit We sequenced the entire nuclear ribosomal ITS region (ITS1-5.8S-ITS2) for http://www.mycobank.org/MB/. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS.The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies. In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix Ascosphaera callicarpa.Despite repeated attempts, we were unable to obtain in vitro mycelial growth or induce ascospore germination of A. callicarpa (GenBank accession: JX070046). A BLASTn search of the ITS sequence revealed a highest sequence-similarity to other Ascosphaera species.An ITS sequence was obtained for A. callicarpa A.A. Wynns  4.0×1.6(−2.0) µm; saprotrophA. Wynns ig. 11. Wall of spore cyst with minute or conspicuous dark spots; ascospores not cylindrical; saprotroph or pathogen2A. tenax Skou  L.S. Olive & Spiltoir  Skou (7. Ascospores (2.4–) 2.8–4.0 (−5.0)×1.0–1.8 (−2.0) µmbl) Skou igs. 2D–EA. proliperda Skou .T.Mating system unknown but possibly homothallic Ascosphaera aggregata is an obligate pathogen with a preference for bees belonging to the family Megachilidae. This species has a broad distribution, with reports from both North America and Europe. Like A. apis, the distribution of A. aggregata is probably closely tied to the exchange and transport of bees (e.g. M. rotundata) for the pollination of commercial crops. In Europe A. aggregata is known from Denmark A. aggregata in culture often results in the co-isolation of another pathogenic species, A. proliperda. Although A. proliperda and A. aggregata can be difficult to separate based on microscopic morphological features their growth in culture is strikingly different (A. proliperda for further discussion on its co-occurrence with A. aggregata).lberta: Brooks, on larvae of M. rotundata, 1988, J Jakobsen s.n. (c). DENMARK. Thurø: Svendborg Kommune, on Osmia rufa ( = O. bicornis L.), 1974, J.P. Skou s.n., paratype (c); On O. rufa, 1974, J.P. Skou s.n., holotype, (c); Zealand: Frederikssund Kommune, Slangerup, organic apple orchard, on larva of O. rufa, 2010, A.A. Wynns 5152; Roskilde Kommune, Roskilde, on larva of O. rufa, 2010, A.A. Wynns 5144; Taastrup Kommune, Taastrup, Højbakkegård, on larvae of O. rufa, 2010, A.A. Wynns 5145 (c). USA. Nevada: On Megachile pacifica (Panzer) ( = M. rotundata), 1975, K. Hackett s.n. paratype (c). SPAIN: On M. rotundata, 1972, J.P. Skou s.n., paratype (c); on M. rotundata, 1973, J.P. Skou s.n. paratype (c).CANADA. AAscosphaera apis (Maasen ex Claussen) L.S. Olive & Spiltoir in Spiltoir & Olive, Mycologia 47: 242, 1955.≡Pericystis apis Maasen ex Claussen, Mitt. biol. BundAnst. Ld-u. Forstw.10: 470. 1921.Mating system heterothallic. Pathogenic. Infected larvae shrunken, pale buff, covered by a weft of hyphae, with or without the production of ascomata. Ascomata greenish (immature) to black (mature) spore cysts produced on aerial hyphae above the larval cuticle, 40–119 µm in diameter; wall pale greenish to yellowish-brown, nearly smooth with minute punctae at high magnification. Spore balls hyaline to pale yellowish, without granules, 7–20 µm in diameter, mostly persistent. Ascospores ellipsoid to sub-allantoid, 2.1–3.9×1.1–1.7 µm. Culture on SDA with rapid growth after 2–6 days, white with abundant production of spore cysts when both mating strains are present.See Skou Ascosphaera apis is an opportunistic pathogen of honeybees. Experimental trials showed A. apis is able to induce chalkbrood in the solitary bee M. rotundataA. apis is not known to live in association with solitary bees in nature. Reports of chalkbrood caused by A. apis in solitary bees before 1972 are most likely attributable to pathogenic species described after this time; e.g., A. major, A. aggregata or A. proliperda (the later two species are pathogens specific to solitary bees). Originally described from Germany, A. apis, is now known from all continents where honeybees are kept.exas: Weslaco, 26 Jun 2003, K.D. Murray s.n., ARSEF 7405 (+), 7406 (−).Additional specimens examined. USA. TAscosphaera atra Skou & K. Hackett, Friesia 11: 279, 1979.ype: U.S.A. Nevada, isolated from larva of Megachile pacifica with ragged-brood disease, 36836 (c), CBS 524.75 .TMating system homothallic. Ascomata black, globose spore cysts, 30–140 µm; wall dark brown, punctate, punctae appearing as uniform dark circles often of variable size. Spore balls hyaline to pale yellowish brown, 8–17 µm diameter, evanescent. Ascospores ellipsoid to broadly ellipsoid, 4–7.9×2.3–6.5 µm, with or without small granules attached to the surface of the spore wall. Culture on SDA with moderate growth after 7 days, white to greyish-buff with abundant production of black spore cysts on aerial hyphae and on hyphae growing beneath the surface of the agar.Ascosphaera atra is a fast-growing saprotroph associated primarily with solitary bees. This species is typically found growing on pollen provisions. Less common substrates from which A. atra has been isolated include the surface of a diseased M. rotundata larva with chalkbrood caused by A. aggregataM. rotundata larva A. melliferaAscosphaera atra is the only species of the genus that has been found growing on plant material (grass silage) outside of the bee habitat A. atra is not a pathogen of solitary bees; however, Vojvodic et al. A. atra is comparable to some of its bee- pathogen congeners e.g. A. aggregata and A. apis. The perceived pathogenicity of this species in honey bee larvae may be more closely tied to its rapid growth on suitable substrates. Ascosphaera atra is the most extensively studied saprotrophic species of Ascosphaera. This is reflected in the multiple reports from N. America Ascosphaera atra. AUSTRALIA. Peel: Waroona, A. mellifera honey, Nov 1994, D.L. Anderson 198, ARSEF 5147. CANADA. Alberta: Beaverlodge, Peace River region, from pollen in M. rotundata cells, Jan 1985, D. Farney s.n., DAOM 188981. USA. Oregon: Ontario. M. rotundata, Jun 1979, J.D. Vandenberg 6, ARSEF 693.Additional specimens examined. Ascosphaera callicarpa A.A. Wynns, sp. nov. [urn:lsid:indexfungorum.org:names:518624]ype. DENMARK. Zealand: Lejre Kommune, Sagnlandet (“Land of Legends”) Lejre, Landbohusene, on fecal pellets of Chelostoma florisomne nesting in the Phragmites reeds of thatched roof of shed behind 19th century cottage, 55°37′11″N; 11°22′13″, 2010, A.A.Wynns 5165 .TMating system unknown. Ascomata pale brown, semi-transparent and somewhat iridescent spore cysts ig. 1B, gC. florisomne where it grows on the fecal pellets of this bee. Although not definitely known, the distribution of A. callicarpa is probably closely tied to that of C. florisomne. This fungus was not found in association with other bees, e.g. Osmia and Megachile, although these bees were observed nesting in the same Phragmites reeds as C. florisomne. Ascosphaera callicarpa appears to be solely saprotrophic; it was not found in association with diseased bees or where a larva had failed to develop. Ascosphaera callicarpa is so far known only from the island of Zealand, Denmark.Common in the nest reeds of the solitary bee callicarpa means with beautiful fruits, here referring to the spore cysts.The epithet Ascosphaera callicarpa should be sought in other aggregations of C. florisomne in thatched roofs throughout Europe in order to assess its conservation status. As a possible obligate associate of the bee C. florisomne, the conservation of this fungus should be considered dependent on the conservation of its host.ealand: Lejre Kommune, Sagnlandet Lejre, Landbohusene, shed behind 19th century houses. All specimens on fecal pellets of C. florisomne nesting in the Phragmites reeds of the thatched roof, 2008, A.A.Wynns 5011, 5012, 5013, 5014, 5015, 5018, 5025, 5026, 5027, 5072, 5074, 5136, all specimens in c; 2011, A.A.Wynns 5166, 5168 (c); Sorø Kommune, Kristiansminde, University of Copenhagen field station, east facing side of classroom building, growing on the fecal pellets of C. florisomne nesting in Phragmites reeds of the thatched roof, 2012, A.A. Wynns 5169, 5170 (c).DENMARK. ZAscosphaera callicarpa most closely resembles A. fimicola Skou which also grows on the fecal pellets of bees. This new species is distinguishable from A. fimicola by a pale brown, highly transparent fragile spore cyst .TMating system not known. Ascomata light to dark brown, somewhat iridescent, glistening spore cysts, (25–) 64–125 µm in diameter; wall brown, punctate, punctae minute and of uniform in size. Spore balls yellowish, with small granules on the surface, (5–)10–15(−20) µm in diameter, mostly persistent. Ascospores ellipsoid to sub-allantoid, 3.0–5.0×1.3–1.8 µm with or without small granules attached to the spore wall. Mycelium on natural substrate noticeable, stringy, white and opaque. No growth in culture.Ascosphaera fimicola grows saprotrophically on the larval feces and cocoons of the solitary bee Osmia bicornis (syn. O. rufa) and was recently collected on the larval feces of Cacoxenus indagator (Diptera: Drosophilidae) a cleptoparasite of this bee. Despite extensive collecting, A. fimicola was not found on the larval feces of the solitary bee C. florisomne. The composition of the pollen provisions of these bees may play a role in the absence or presence of A. fimicola in their nests. Chelostoma florisomne feeds exclusively on pollen from the plant genus Ranunculus (Ranunculaceae) O. bicornis often collects pollen from the plant family Rosaceae A. fimicola prior to our study was in 1975 A. fimicola is restricted to Denmark but, like other species in the genus, this narrow distribution is most likely an artifact of under-collecting because of a more focused interest in the pathogens rather than the saprotrophs.ealand: Taastrup Kommune, Højbakkegård Allé 3, on feces of Cacoxenus indagator in O. rufa brood cell, 2008, A.A. Wynns 5123 (c); on feces and cocoon of O. rufa, 2010, A.A. Wynns 5147, 5167 (c). Frederikssund Kommune, Slangerup, residence and farm of Verner Andersen, on pollen and feces in nest cell of O. rufa, 2010, A.A. Wynns 5149, 5151 (c). langeland: Langeland Kommune, Rudkøbing, Skovsgaard, Kågårdsvej 12, on cocoon of O. rufa and C. indagator, 2008, A.A. Wynns 5130 (c); on cocoon and feces of O. rufa, 2008, A.A. Wynns 5131 (c). THURØ: Svendborg Kommune, on fecal pellets of O. rufa, 1972, J.P. Skou s.n., paratype (c).DENMARK. ZAscosphaera major (Prökschl & Zobl) Skou, Friesia 10:15, 1972.type: DENMARK. Zealand: Glostrup, isolated from chalkbrood cells of Megachile centuncularis, cbs 686.71 .Pericystis apis Maasen ex Claussen var. major Prökschl & Zobl in Prökschl, Arch. Microbiol. 18: 200. 1953.≡Ascosphaera apis (Maasen ex Claussen) L.S. Olive & Spiltoir var. major (Prökschl & Zobl) L.S. Olive & Spiltoir in Spiltoir & Olive, Mycologia 47: 243. 1955.≡Mating system heterothallic. Ascomata dark brown to black, spore cysts, 60–150(−380) µm in diameter; wall greenish brown, with indistinct puntcae or small granules attached to the inner surface, occasionally with larger crystalliferous brown precipitations with age. Spore balls hyaline to greyish-brown, (6–)14–18(−24) µm in diameter, usually with granules attached to the surface. Ascospores suballantoid or bacilliform. (2.4–)2.8–4.0(−5.0)×1.0–1.8(−2.0) µm, at least some with small granules attached to the spore wall. Mycelium white to greyish-white. Culture on V8YE with moderate growth after 10 days, with abundant production of spore cysts when both strains are present.Ascosphaera major causes chalkbrood in Apis melliferaMegachile centuncularisM. centuncularisA. major was found growing on the larval feces and leaf material lining the brood cell of a species of Megachile and on the larval feces and pollen provisions of O. bicornis. The frequency of A. major as a cause of chalkbrood in honeybees is not known. Outwardly A. apis and A. major induce the same disease symptoms; therefore, the etiology of chalkbrood in honeybees should be carefully verified by morphological study of the fungus to distinguish infections by A. apis or A. major or to identify co-infection with both species. Ascosphaera major is known from N. America ealand: Frederikssund Kommune, Slangerup, organic apple orchard belonging to Verner Andersen, growing on pollen and feces of O. bicornis, 2010, A.A. Wynns 5150 (c); Lejre Kommune, Lejre Forsøgscenter, growing on cocoon and between walls of leaf lining of healthy Megachile sp., 2008, A.A. Wynns 5038 (c); Roskilde Kommune, Roskilde, Gøderupvej 5, on leaf-lining of brood cell belonging to Megachile sp., 2010, A.A. Wynns 5173, on larval feces of Megachile sp. without disease, 2010, A.A. Wynns 5175.DENMARK. ZAscosphaera proliperda Skou, Friesia 10: 15. 1972.type: DENMARK. Zealand: Frederiksberg Kommune, Frederiksberg, in Megachile centuncularis larvae collected from the greenhouse of the Royal Veterinary and Agricultural University, J.P. Skou s.n., Jun 1967, CBS 687.71 .Mating system homothallic Ascosphaera proliperda causes chalkbrood in Megachile centuncularisM. rotundataO. bicornis [present study]. Ascosphaera proliperda has repeatedly been isolated from surface sterilized chalkbrood cadavers of M. rotundata and O. bicornis infected with A. aggregataA. aggregata infection i.e. the cuticle of the host is intact with spore cysts just below the cuticle rather than above the cuticle as is typical for A. proliperda. The co-occurrence of A. proliperda and A. aggregata in chalkbrood larvae adds to the difficulty of isolating A. aggregata since the former species is much faster growing. Unless isolation or PCR diagnostics .GERMANY. Isolated from the surface of a sterilized Ascosphaera tenax Skou & S.N. Holm, Mycotaxon 35: 212, 1989.ype: DENMARK, Nekselø: Kalundborg Kommune, inside cocoons of Megachile willughbiella, 1985, J.P. Skou s.n., .TMating system unknown. Ascomata lustrous black, less often dark brown, spore cysts, (33–)40–90(−105) µm diameter; wall dark brown,1.5 µm thick, tough and leathery, smooth or minutely punctate. Spore balls hyaline, (7.7–)9–14(−15.4) µm diameter. Ascospores sub-falcate, with a tendency to be trigonal when viewed on-end, 1.9–3.5×0.6–0.9 µm.Ascosphaera tenax grows saprotrophically on pollen provisions, larval feces and the inner side of cocoons of Megachile willughbiella and M. rotundata. Spore cysts are common beneath the inside of the leaf cap of Megachile cells. The last collections of A. tenax date from 1988, when the species was found growing in nearly half (18 out of 44) M. willughbiella cocoons examined Ascosphaera tenax is known only from Denmark on the islands of Nekselø and Zealand. More focused collecting is needed to determine its real geographical range.Ascosphaera. With the addition of A. callicarpa sp. nov., eight Ascosphaera species are now known from Europe (A. fimicola (see discussion under A. fimicola) from a dipteran cleptoparasite of Osmia bicornis add to the mounting evidence that, although undoubtedly a bee specialist, Ascosphaera is not restricted to bees; further evidence includes an isolated report of the saprotroph A. atra growing on grass Ascosphaera DNA from Eristalis (Diptera: Syrphidae) and Vespula (Hymenoptera: Vespidae) species Ascosphaera should be sought outside the bee habitat in association with other pollenivorous insects and where high-sugar substrates are available. Reports of Ascosphaera in non-apoidean insects are quite possibly relevant for the control of chalkbrood in commercial bee pollinators since these insects may act as pathogen reservoirs or vectors of Ascosphaera. More frequent collections of Ascosphaera are needed to begin to grasp the diversity and ecology of these fungi in nature and to elucidate their potentially significant role within the bee habitat. Additional regional keys, such as the one provided here, may ease identification for the non-specialist and bring attention to the lesser-known species of both saprotrophs and pathogens.Our study is the first to provide a regional key to m Europe . Our col


However, the effectiveness of inhibitors is difficult to assess, as nophysiological substrates or downstream effectors have been identified that could be exploited todevelop a robust cell-based assay. We recently established that LRRK2 bound 14-3-3 protein isoformsvia its phosphorylation of Ser910 and Ser935. In the present study we showthat treatment of Swiss 3T3 cells or lymphoblastoid cells derived from control or a Parkinson'sdisease patient harbouring a homozygous LRRK2(G2019S) mutation with two structurally unrelatedinhibitors of LRRK2 (H-1152 or sunitinib) induced dephosphorylation of endogenous LRRK2 atSer910 and Ser935, thereby disrupting 14-3-3 interaction. Our results suggestthat H-1152 and sunitinib induce dephosphorylation of Ser910 and Ser935 byinhibiting LRRK2 kinase activity, as these compounds failed to induce significant dephosphorylationof a drug-resistant LRRK2(A2016T) mutant. Moreover, consistent with the finding thatnon-14-3-3-binding mutants of LRRK2 accumulated within discrete cytoplasmic pools resemblinginclusion bodies, we observed that H-1152 causes LRRK2 to accumulate within inclusion bodies. Thesefindings indicate that dephosphorylation of Ser910/Ser935, disruption of14-3-3 binding and/or monitoring LRRK2 cytoplasmic localization can be used as an assay to assessthe relative activity of LRRK2 inhibitors in vivo. These results will aid theelaboration and evaluation of LRRK2 inhibitors. They will also stimulate further research tounderstand how phosphorylation of Ser910 and Ser935 is controlled by LRRK2,and establish any relationship to development of Parkinson's disease.LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson'sdisease patients. Since a common mutation that replaces Gly P81 phosphocellulose paper was from Whatman. Pepceuticals synthesized Nictide. TheFlp-in T-REx system was from Invitrogen and stable cell lines, generated according to themanufacturer's instructions by selection with hygromycin, have been described previously ATP(~500–1000 c.p.m./pmol) in the presence of 20 μM Nictide peptidesubstrate. Reactions were terminated by applying 40 μl of the reaction mixture on toP81 phosphocellulose paper and immersion in 50 mM phosphoric acid. After extensive washing,reaction products were quantified by Cerenkov counting.Peptide kinase assays were set up in a total volume of 50 μl withimmunoprecipitated LRRK2 as a source of kinase, in 50 mM Tris/HCl, pH 7.5,0.1 mM EGTA, 10 mM MgCl2™-coated chamber slides (Nunc). At 1 day afterplating, cells were induced with 0.1 μg/ml doxycycline and 24 h later, cellswere fixed in 4% (w/v) PFA  in PBS. For staining of microtubules, cells, inducedas above, were washed twice in 37 °C medium, and fixed with 4% (w/v) PFA for15 min. Cells were washed with PBS, permeabilized with 1% (v/v) NP-40 in PBS for10 min then blocked with 10% (v/v) normal goat serum after washing. Microtubules were stainedwith rat monoclonal clone YL1/2 . Cells werestained with goat anti-rat antibody conjugated to Alexa Fluor®594 (Molecular Probes). Cellswere mounted in ProLong Gold (Invitrogen) and imaged under the same settings for each mutant, on aZeiss LSM 700 confocal microscope using an αPlan-Apochromat ×100 objective.HEK-293 Flp-in T-REx were purchased from Invitrogen and stable cells harbouring GFP-taggedwild-type and mutant forms of LRRK2 were generated using standard protocols. Cells were plated infour-well glass-bottomed CC910/Ser935 phosphorylation, we initially treated Swiss 3T3 cells withincreasing amounts of the LRRK2 inhibitors H-1152 .To investigate how inhibition of LRRK2 protein kinase activity affects onSers H-1152 Aor sunis H-1152 C. Strikif H-1152 B andsunf H-1152 D on endo did not, suggest910/Ser935phosphorylation and 14-3-3 binding resulted from inhibition of LRRK2 protein kinase activity, wetreated HEK-293 cells overexpressing LRRK2(G2019S) or the H-1152/sunitinib-resistantLRRK2(A2016T/G2019S) double-mutant with LRRK2 inhibitors. As observed with the endogenous LRRK2, wefound that H-1152 and sunitinib induced a dose-dependent dephosphorylation of the Parkinson'sdisease LRRK2(G2019S) mutant at Ser910 and Ser935, as well as disruptingbinding to 14-3-3 (910 or Ser935phosphorylation or 14-3-3 binding to the drug-resistant LRRK2(A2016T/G2019S) mutant  2–4-fold more potently than wild-type LRRK2 , we foun910 and Ser935. H-1152 and sunitinib inducedmarked dephosphorylation of Ser910 and Ser935 in both wild-type andLRRK2(G2019S) human lymphoblastoid cell lines. However, consistent with the increased sensitivity ofLRRK2(G2019S) for these drugs we observed that H-1152 and sunitinib inhibited phosphorylation ofSer910 and Ser935 in the LRRK2(G2019S) cells more potently than in thewild-type cells  mutation, incomparison with those derived from a second individual without an LRRK2 mutation, who presented withno disease . This reMutants of LRRK2 that do not interact with 14-3-3, rather than being diffusely localizedthroughout the cytoplasm, accumulate within cytoplasmic aggregates . This pr910 andSer935, we treated Swiss 3T3 cells with no drug, 30 μM H-1152 or10 μM sunitinib to induce dephosphorylation of Ser910 and Ser935, mTOR , ERK, p38, JNK  and innate immunesignalling pathways . All inhibitors were utilized at the higher limits ofconcentrations known to suppress signalling pathways. As expected, 10 μM sunitinibinduced marked dephosphorylation of Ser910 and Ser935, whereas 32 of theinhibitors tested did not significantly affect dephosphorylation ofSer910/Ser935. Some dephosphorylation of Ser910 andSer935 was observed with the relatively non-specific JNK inhibitor SP600125, which wasused at a concentration of 15 μM and is known to inhibit many protein kinases morepotently than JNK  protein kinase [Our finding that all of the structurally diverse LRRK2 inhibitors tested to date inducedephosphorylation of Sern kinase , whereasn kinase . In the n kinase  and Rictn kinase  to regul910 and Ser935 is dependent upon LRRK2 activity and hence mediates bindingto 14-3-3 isoforms. We propose that monitoring LRRK2 phosphorylation atSer910/Ser935 and/or 14-3-3 binding, and/or cytoplasmic localization of LRRK2,can be utilized as a cell-based read-out to evaluate the relative potency of LRRK2 inhibitors. Theseassays could be deployed in cell lines, tissues of animals or humans treated with LRRK2 inhibitors.Our results show that LRRK2 inhibitors induced dephosphorylation and disrupted 14-3-3 binding toendogenous LRRK2 from patient-derived lymphoblastoid cell lines  or phosphatase(s) and the role that LRRK2 plays in influencing feedback phosphorylation ofthese residues. It would be fascinating to explore whether the Ser910/Ser935protein kinase and/or phosphatase enzymes might also comprise a drug target for the treatment ofParkinson's disease. Finally, it will be vital to work out how 14-3-3 affects LRRK2 function andwhether this is relevant to understanding the role of LRRK2 in Parkinson's disease.In ll lines . This in


Patients suffering from Systemic Lupus Erythematous (SLE) have elevated type I interferon (IFN) levels which correlate with disease activity and severity. TRIM21, an autoantigen associated with SLE, has been identified as an ubiquitin E3 ligase that targets the transcription factor IRF3 in order to turn off and limit type I IFN production following detection of viral and bacterial infection by Toll Like Receptors (TLRs). However, how the activity of TRIM21 is regulated downstream of TLRs is unknown. In this study we demonstrate that TRIM21 is tyrosine phosphorylated following TLR3 and TLR4 stimulation, suggesting that its activity is potentially regulated by tyrosine phosphorylation. Using Netphos, we have identified three key tyrosines that are strongly predicted to be phosphorylated, two of which are conserved between the human and murine forms of TRIM21, at residues 343, 388, and 393, all of which have been mutated from tyrosine to phenylalanine . We have observed that tyrosine phosphorylation of TRIM21 only occurs in the substrate binding PRY/SPRY domain, and that Y393, and to a lesser extent, Y388 are required for TRIM21 to function as a negative regulator of IFN-β promoter activity. Further studies revealed that mutating Y393 to phenylalanine inhibits the ability of TRIM21 to interact with its substrate, IRF3, thus providing a molecular explanation for the lack of activity of Y393 on the IFN-β promoter. Our data demonstrates a novel role for tyrosine phosphorylation in regulating the activity of TRIM21 downstream of TLR3 and TLR4. Given the pathogenic role of TRIM21 in systemic autoimmunity, these findings have important implications for the development of novel therapeutics. Mechanisms that regulate and limit protective immune responses are critical in the prevention of excessive and pathological production of type I IFNs or pro-inflammatory cytokines. The concept that ubiquitin-mediated degradation of key targets on the pathway may be important in this context is not new, the canonical example being the regulation of NF-κB activation via the activity of the E3 ubiquitin ligases A20 and CYLD. Both A20 and CYLD limit the duration of NFκB activation via a ubiquitin editing mechanism. A20 for example has dual deubiquitinating and ubiquitinating activity and replaces K63-linked ubiquitin chains on two key signalling proteins on the NFκB pathway, IRAK1 and TRAF6, for K48 linked chains and thus targets them for proteasomal degradation et al have demonstrated that lack of functional TRIM21 in mice results in enhanced levels of circulating type I IFN, IL-23, and enhanced responses to TLR7 and TLR9 ligands et al, no observable phenotype in immune cells was observed, the authors suggesting that upregulation of other TRIM family members acted to compensate for the loss of TRIM21 TRIM21 (Ro52/SSA1) is a member of the TRIM (Tripartite Motif) family of single-protein E3 ligases and was originally identified as an autoantigen prevalent in Systemic Lupus Erythematosus (SLE). We have since identified TRIM21 as an E3 ligase that negatively regulates type I IFN production downstream of the pathogen recognition receptors (PRRs) TLR-3, TLR-4, TLR-7 and TLR-9, innate immune receptors specific for recognising viral and bacterial pathogens and inducing an immune response appropriate to the pathogen detected Phosphorylation of ubiquitin E3 ligases has previously been shown to be a key mechanism for regulating their activity. For example, Pellino3, which functions as a direct negative regulator of innate immune signalling by regulating IRAK degradation via its E3 ligase activity To date however the mechanism by which TRIM21 activity is regulated has not been investigated. Our work suggests that tyrosine phosphorylation of TRIM21 positively regulates its activity and identify tyrosine 393 in its PRY/SPRY domain as the critical tyrosine in this respect. Critically, mutation of tyrosine 393 to phenylalanine results in the loss of activity of TRIM21 and reduces its ability to interact with its substrate, IRF3. Our data therefore suggests a novel mechanism by which TRIM21 activity is regulated via promoting interaction with its substrate and provides insights into TRIM21 activity that may be manipulated in order to regulate type I IFN production in the treatment of autoimmune disorders such as SLE, in which overproduction of type I IFN and IL-23 play an important role in the pathogenesis of the disease.et al has determined TRIM21 to be an IgG receptor that binds normal serum IgG through its PRY/SPRY domain et al have recently shown that mouse Abs do not bind to human TRIM21 Work by our group and others has demonstrated a critical role for TRIM21 in regulating TLR–driven cytokine production Having demonstrated that TRIM21 is tyrosine phosphorylated in murine macrophages we next assessed its ability to be similarly regulated in human peripheral blood mononuclear cells (PBMCs). Accordingly PBMCs isolated from healthy volunteers were stimulated with the TLR3 agonist Poly I:C for 0, 15, 30 and 60 minutes as indicated in Like other TRIM family members, TRIM21 is a single protein E3 ligase with an N terminal RING domain (for interaction with its cognate E2 enzyme), a central coiled-coil domain, and a C terminal substrate interaction domain, in the case of TRIM21 a PRY/SPRY domain. To determine which domain of TRIM21 was tyrosine phosphorylated we next performed overexpression studies in HEK-293T cells, employing TRIM21 constructs missing the RING domain (Δexon1) or the C terminal PRY/SPRY domain (Δexon6) . As withY393 is important for TRIM21 function– Using NetPhos (http://www.cbs.dtu.dk/services/NetPhos/), tyrosine 343, 388 and 393 were strongly predicted to be phosphorylated  from wild type mice, a kind gift from Dr. Egil Lien and Prof. Douglas Golenbock , were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum and 1% penicillin-streptomycin. Primary human peripheral mononuclear cells (PBMCs) were isolated from whole blood from healthy donors, under ethical approval from Royal College of Surgeons in Ireland research ethics committee REC269, using a Ficoll gradient and cultured in RPMI-1640 media supplemented with 10% (v/v) fetal calf serum and 1% penicillin-streptomycin. Informed consent from all participants involved in this study was obtained in a written manner. Participants involved in this study were only recruited from, and experimentation conducted at, Royal College of Surgeons in Ireland.Xpress-tagged TRIM21, Xpress-tagged TRIM21ΔExon1, Xpress-tagged TRIM21ΔExon6, and pGEX-PRY/SPRY TRIM21 were gifts from Dr. David Rhodes , and the Flag-tagged pCMV-IRF3 and the IFN-β promoter construct were gifts from Dr. Kate Fitzgerald . TRIM21 Y393F, TRIM21 Y343,388F, and TRIM21 Y343,388,393F were generated by Genscript USA Inc. . Polyclonal Ab against TRIM21 was made by Sigma Genosys. Primary antibodies used were anti-Xpress, anti-V5 (Invitrogen Life Technologies), anti-phospho-IκB , anti-phospho-TBK and anti-alpha-actinin (Santa Cruz Biotechnologies), anti-Flag (Sigma), anti-GST , and anti-β-actin (Abcam). Anti-phosphotyrosine agarose beads were obtained from Sigma.cactctggaaaacatttctgggaggtagatgtg (forward) and cacatctacctcccagaaatgttttccagagtg (reverse); Y388F, gtggaacaaacaaaaattcgaggctggcacctaccc (forward) and gggtaggtgccagcctcgaatttttgtttgttccac (reverse); Y393F, gggattttcctggacttcgaggctggcatggtc (forward) and gaccatgccagcctcgaagtccaggaaaatccc (reverse).V5-TRIM21 was generated from cDNA and inserted into pcDNA3.1 using the Gateway™ vector system (Invitrogen Life Technologies). The V5-TRIM21 and the pGEX-PRY/SPRY TRIM21 tyrosine to phenylalanine mutants were generated from the V5-TRIM21 plasmid and pGEX-PRY/SPRY TRIM21 plasmid using the QuikChange Lightning site-directed mutagenesis kit (Stratagene). The primers used for the mutagenesis are as follows: Y343F, 3VO4, 1 µg/ml pepstatin) followed by immunoprecipitation with anti-phosphotyrosine-conjugated agarose beads (Sigma). For recombinant pulldowns, lysates were incubated with 500 ng of GST-PRY/SPRY TRIM21 bound to GSH-sepharose beads (Qiagen). Immunoprecipitates were analysed by Western blot. Each blot is representative of three independent experiments.Immunoblots were performed as previously described 5 cells/ml) onto 96-well plates 24 hrs pre-transfection with 50 ng of the indicated reporter constructs and increasing amounts of the relevant TRIM21 constructs  as indicated. HEK293T cells were also co-transfected with TRIF (50 ng) in order to drive the system. Luciferase activity was standardised to Renilla luciferase plasmid activity to normalise for transfection efficiency.HEK293T cells were seeded .


Blattella germanica Linnaeus (Blatodea: Blattellidae), was investigated in the laboratory using a topical application bioassay. The results showed the 6 week-old nymphs were more tolerant to dichlorvos and propoxur than the other ages tested. LD50 values of dichlorvos and propoxur for the 6 week-old nymphs were 2.003 µµg per insect and 5.296 µµg per insect, respectively. Tolerance ratios of 18.55-fold and 4.98-fold for LD50 were obtained from 6-week-old nymphs compared to 4 week-old nymphs. The specific activity of acetylcholinesterase (AChE) from 1 week-old nymphs was the highest among all tested developmental stages of nymphs and adult males and females. The specific activity of AChE decreased significantly with increasing age. The sensitivity of AChE to dichlorvos was the highest with a ki value of 3.12××104 mol-1min-1 in the last nymphal stage of B. germanica (about 6 weeks-old). The AChE from 4 week-old nymphs was the most sensitive to propoxur, with the highest ki value being 2.63××105 mol-1min-1. These results indicated that the different developmental stages and sexes of B. germanica affected the inhibition of AChE by dichlorvos and propoxur.Stage-dependent dichlorvos and propoxur tolerance in a field population of the German cockroach,  Blattella germanica Linnaeus (Blatodea: Blattellidae), is considered one of the most serious pests in the urban environment. In Beijing, large amounts of insecticides are applied annually in an effort to control this pest. However, B. germanica has developed resistance to most of the traditional insecticides  used for its control  were significantly more tolerant to insecticides including pyrethroid, carbamate, and organophosphate insecticides than adult males in topical and residual insecticide bioassays  males and females were maintained in separate tubs and removed after anesthesia with carbon dioxide.A field population of the German cockroach, e groups  where 2 www.huayang.com), and dichlorvos was purchased from Tianjin Qianjin Pesticide Factory (www.tjqianjin.com). Acetylthiocholine iodide (ATCh), 5, 5′?-dithiobis (2-nitrobenzoic acid) (DTNB), bovine serum albumin (BSA), and Triton X-100 were purchased from Fluka Chemical Co. . All other chemicals used in the experiments were of analytical grade.Propoxur (99% pure) was purchased from Shandong Huayang Technology Co., Ltd.,  was applied to the first abdominal sternite 2 hr prior to propoxur application. Mortality (no response to probing) was recorded 24 hr after treatment. Data were analyzed by probit analysis .B. germanica were frozen at -80°° C prior to biochemical assays. The whole bodies of 10 cockroaches were homogenized in ice-cold 0.1M phosphate buffer, pH 7.5, containing 1%(v/v) Triton X-100 , and the crude homogenate was centrifuged at 12,000 g for 30 min at 4°° C using an ASTEC Microtec 1524R centrifuge . The supernatant was used as an enzyme source for measuring AChE activity.All stages of www.perkinelmer.com). The control samples contained no enzyme during the incubation. After addition of the color reagent, appropriate amounts of enzyme solutions were added to the controls.AChE activity was measured using the method described by Ellman et al.  with minProtein content was determined by the method of Bradford  using bo-5 mol L-1.Inhibitors were pre-incubated with the enzyme, and then the mixture of inhibitors and enzymes were added to the tube containing the equivalent volume of substrate at 1 min intervals. The residual enzyme activity was measured and the percentage of inhibition was calculated compared with that of the control. Each assay was represented by three replicates. The final concentration of each inhibitor was 1 ×× 10Km) and Maximal velocity (Vmax) for ATCh was determined by enzymatic activity measures at 11 substrate concentrations ranging from 0.2 mmol L-1 to 2.2 mmol L-1. Results comprise the mean values of three separate preparations with three determinations for 11 concentrations. The kinetic parameters, Km and Vmax, were calculated with Enzifit software (The apparent Michaelis-Menten constant (software .kd (dissociation constant), k2 (phosphorylation rate constant or carbamylation rate constant), and ki (bimolecular rate constant) were determined by methods described in Main (vi/vo) versus time was linear for a given inhibitor concentration. Bimolecular rate constants (ki) and phosphorylation (or carbamylationrate) rate constants (k2) were calculated by nonlinear regression using Enzifit software . The data were statistically analyzed using one-way ANOVA and two-tail P value. The Tukey's test was used among the different stages of B. germanica in multiple comparisons with p <0.05.All statistical tests were performed using the software InStat  of AChE was also less in 6 week-old nymphs than in other developmental stages.The sensitivity of AChE to dichlorvos in 6 week-old nymphs was the highest in all tested developmental stages with a tages of . There wki value of 2.635××105 mol-1 min-1 which was 2–3 times that of other stages  of AChE was also less in 4 week-old nymphs than in other developmental stages.The sensitivity of AChE to propoxur in 4 week old nymphs was the highest in all tested developmental stages with a r stages . A signiet al.  of AChE varied dependent on the ages of B. germanica, which indicates that characteristics of AChE can be different among the ages of B. germanica.Valles et al.  showed tB. germanica than in 5 week-old and 4 week-old B. germanica. This suggests that inhibition of propoxur to AChE was weaker in 6 week-old B. germanica than in 5 week-old and 4 week-old B. germanica. The inhibition of AChE corresponded with the toxicity of propoxur to different ages of B. germanica.The consistent relationship between inhibition of AChE and the tolerance to dichlorvos was not observed in this study. However, the residual activity of AChE after the treatment with propoxur was higher in 6 week-old et al. (ki) for the inhibition of AChE by propoxur in 4 week-old nymphs was statistically higher than that in other tested stages. It was also consistent with the bioassay data of propoxur, indicating that higher sensitivity of AChE may be one of factors that are responsible for the more susceptibility of 4 week-old nymph to propoxur with a lower LD50 value.Valles et al.  reportedB. germanica.The sensitivity of AChE from 6 week-old nymphs to dichlorvos was the highest in the tested nymph stages, but the bioassay data showed that the 4 week-old nymph was the most sensitive stage. It demonstrated that there was no direct relationship between the stage-and gender-dependent differential susceptibility and the inhibitory effects of dichlorvos on AChE in B. germanica. Therefore, selective application of insecticide for controlling B. germanica is very important based on developmental changes of AChE sensitivity to organophosphate and carbamate insecticides. However, it is impossible to explain the relationship between the inhibitory effects on AChE and the difference of insecticide susceptibility against developmental stages without the insecticide bioassay data. Results from the present study including biochemical and bioassay data, have demonstrated that stage-and gender-dependent differential susceptibility were consistent with the inhibitory effects of propoxur on acetylcholinesterase in B. germanica, but to dichlorvos there was no direct relationship observed.Acetylcholinesterase is the target site for carbamate and organophosphate insecticides. The sensitivity of AChE to inhibitors varied with the different developmental stages of B. germanica. Further work, including a better understanding of the molecular mechanisms that are operating, is necessary to improve control of B. germanica with organophosphate and carbamate insecticides.In the present paper some preliminary results are reported on the relationship between the stage- and gender-dependent differential susceptibility and the inhibitory effects of dichlorvos and propoxur on acetylcholinesterase in


REDD1 is known to be transcriptionally upregulated in hypoxia. During hypoxic stress, REDD1 plays an important role as a mediator of mTORC1 inhibition. REDD1 is also subject to highly dynamic transcriptional regulation in response to a variety of other stress signals. In addition, the REDD1 protein is highly unstable. However, it is currently not well understood how REDD1 protein stability is regulated. In this study, we discovered that mTORC1 regulates REDD1 protein stability in a 26S proteasome dependent manner. Inhibition of mTORC1 resulted in reduced REDD1 protein stability and a consequent decrease in REDD1 expression. Conversely, activation of the mTORC1 pathway increases REDD1 protein levels. We show that REDD1 degradation is not regulated by HUWE1, Cul4a or other Cullin E3 ubiquitin ligases. Our study shows that mTORC1 increases REDD1 protein stability and reveals a novel mTORC1-REDD1 feedback loop. This feedback mechanism may limit the inhibitory action of REDD1 on mTORC1. The mechanistic target of rapamycin complex I (mTORC1) is a key regulator of cell proliferation. Its main function is to regulate protein synthesis through phosphorylation of its downstream targets S6 kinase 1 (S6K1), eukaryotic initiation factor 4E (eIF4E)-binding protein 4E-BP1 and eukaryotic elongation factor 2 kinase (eEF2K) REDD1 gene  belongs to a family of genes that includes its paralog REDD2 (TRP801L/Dig2/DDIT4L) Scylla and CharybdisREDD1 is upregulated through transcriptional mechanisms in response to different stress stimuli such as DNA damage, ER stress, hypoxia, serum deprivation, glucocorticoid-, hydrogen peroxide-, dexamethasone-treatment REDD1 is mediated by different transcription factors including p53, p63, ATF4, Sp1 and HIF1, indicating that REDD1 is an important regulator in response to diverse stress conditions REDD1 (Regulated in Development and DNA Damage responses 1) is a negative regulator of mTORC1 in hypoxia and functions in a TSC2 dependent manner REDD1 is a highly unstable protein. The REDD1 protein half-life has been reported to be between 5–7 mins In this study, we identified a novel mTORC1-REDD1 feedback loop whereby mTORC1 regulates REDD1 protein stability. Furthermore, we observed that REDD1 stability is not regulated by GSK3β dependent phosphorylation and that REDD1 is not ubiquitinated by Cul4a or other Cullin RING E3 ubiquitin ligases.We initially observed that overexpression of REDD1-V5 in HEK293 cells led to reduced levels of endogenous REDD1 protein when compared to REDD1 protein in untransfected cells . High le+/+ and TSC2−/− mouse embryonic fibroblasts (MEFs). These cells have differential mTORC1 activities due to the presence or absence of the mTORC1 upstream negative regulator TSC2. In the TSC2−/− MEFs where mTORC1 is constitutively active, REDD1 levels are much higher compared to TSC2+/+ cells  conjugating enzyme, induced with tetracycline, in HEK293 cells It has been reported that REDD1 degradation is mediated by the Cul4a E3 ubiquitin ligase complex and that this in turn is dependent on REDD1 phosphorylation at Thr23 and Thr25 sites by GSK3β. Therefore, to confirm this, we overexpressed a tetracycline inducible dominant negative Cul4a-V5 plasmid  in HEK293 cells We also used siRNA mediated silencing of Cul4a to confirm the results obtained with the dnCul4a cell line. We used two different siRNA oligonucleotides and Cul4a siRNA-2 proved more effective . We obseet al. reported that GSK3β dependent phosphorylation of REDD1 causes a faster migration in SDS gels It has been reported that GSK3β phosphorylates REDD1 at residues Thr23 and Thr25, resulting in REDD1 recruitment to the Cullin 4a-β-Trcp E3 ligase complex. To confirm involvement of these threonine residues in the regulation of REDD1 protein stability, we mutated both Thr23 and Thr25 to alanines. These mutations would be expected to stabilize REDD1 protein. However, we did not observe any significant difference in the stability of REDD1 mutant compared to controls . AdditioREDD1 is an important negative regulator of mTORC1 in response to stress. In this study, we have shown that mTORC1 in turn also regulates REDD1. mTORC1 dependent regulation of REDD1 is at the level of the REDD1 protein stability. Inhibition of mTORC1 using the small molecules rapamycin and PP242 or by overexpressing REDD1 led to reduced REDD1 protein stability and a consequent decrease in REDD1 expression. This mTORC1-REDD1 feedback loop would limit the inhibitory action of REDD1 on mTORC1 . The phyIn further experiments, we studied the mechanism through which REDD1 stability is regulated. We found that REDD1 degradation in response to mTORC1 inhibition is proteasome dependent. However, contrary to a previous report Our results suggest that an alternative E3 ligase is responsible for both basal REDD1 ubiquitination and ubiquitination that is induced upon mTORC1 inhibition. Using pharmacological and genetic inhibitory approaches, we have ruled out any role for members of the Cullin RING E3 ligase family, which constitutes about half of all cellular E3 ubiquitin ligases. Although knockdown of the HUWE1 E3 ubiquitin ligase resulted in increased REDD1 protein steady state levels, our further studies indicated that this effect is not due to an effect of HUWE1 on REDD1 protein stability. Hence, the identity of the E3 ligase that mediates basal REDD1 ubiquitination and ubiquitination upon mTORC1 inhibition is currently unknown. Identification of this E3 ligase is important as this ligase may be a unique drug target for specific inhibition of mTORC1.+/+-p53−/− and TSC2−/−p53−/− MEFs were kindly provided by D.J. Kwiatkowski  +/+-p53−/− and TSC2−/−p53−/− MEF cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% inactivated fetal bovine serum, 2 mM L-glutamine and 1% penicillin-streptomycin (Invitrogen) and maintained in a 5% CO2 incubator at 37°C. For overexpression experiments, sub-confluent cells were transfected using Genejuice (Novagen) according to the manufacturer's instructions. Knockdown experiments using siRNAs  were performed using Lipofectamine RNAiMax (Invitrogen) according to the instructions by the manufacturer.TSC2The human REDD1-V5 pcDNA3 plasmid was constructed by PCR amplification from human brain cDNA and subsequently inserted into the pcDNA3 vector with a C terminal V5 tag using KpnI and XbaI restriction sites with a SacII restriction site inserted between REDD1 and the V5 tag. Mutagenesis of REDD1 Threonines 23 and 25 to Alanines was carried out using the Stratagene site-directed mutagenesis kit. The HA-p70S6K1 T389D pcDNA3 and HA-p70S6K1 T389A pcDNA3 plasmids were constructed from the pRK7-HA-S6K1-WT (Addgene Plasmid 8984) 2 and 2 mM DTT in the presence or absence of 1 mM ATP and/or the recombinant protein GSK3β. Following the reaction, the samples were denatured in SDS-sample buffer and subjected to SDS-PAGE and immunoblotting.FLAG-immunoprecipitates (REDD1-FLAG or FRAT-FLAG) from HEK293 cell lysates were incubated on a shaking platform for 45 minutes at room temperature in 50 mM Tris pH 7.5, 25 mM MgCl3VO4, 20 mM sodium β-glycerophosphate, 10 mM sodium pyrophosphate, and 0.5% Triton X-100 containing freshly added protease inhibitor cocktail (Roche Diagnostics) and 0.1% β-mercaptoethanol. Equal amounts of protein from each sample were separated by SDS–PAGE (10%) and transferred onto nitrocellulose membranes. The blots were probed with a primary antibody followed by a secondary antibody conjugated to horseradish peroxidase. The following primary antibodies were used: rabbit anti-REDD1 , rabbit anti-phospho-p70 S6 kinase (Thr389) , rabbit anti-p70 S6 kinase , mouse anti-HIF-1α , mouse anti-p27 , mouse anti-HECTH9 (AX8D1)/HUWE1 , mouse anti-GSK3β , mouse anti-α-tubulin , mouse anti-V5 , mouse anti-Mcl-1 . Protein levels on the blots were detected using the enhanced chemiluminescence system  according to the manufacturer’s instructions. All western blots are representative of at least two independent experiments.Whole cell lysates were prepared by rinsing the cells in ice cold 1x PBS followed by cell lysis using lysis buffer with the following composition: 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mM Na