Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus  Sordaria macrospora has a long history as an ideal model system for cell differentiation, and we show here that the proposed cell wall integrity (CWI) MAPK module of this fungus controls differentiation of sexual fruiting bodies, cell fusion, polar growth and cell wall stress response. We further discovered that developmental protein PRO40 binds the MAPK kinase kinase (MAPKKK), the MAPK kinase (MAPKK) and upstream activator protein kinase C (PKC1) of the CWI pathway and is required for MAK1 activity, thereby providing evidence that PRO40 is a scaffold protein. Collectively, our findings reveal a molecular role for a protein implicated in development, cell fusion, symbiosis, and pathogenicity in different fungi.The specific response to environmental cues is crucial for cell differentiation and is often mediated by highly conserved eukaryotic MAP kinase (MAPK) pathways. How these pathways react specifically to huge numbers of different cues is still unclear, and current literature about adapter and scaffolding proteins remains scarce. However, gaining fundamental insight into molecular signaling determinants is pivotal for combating diseases with impaired signal transduction processes, such as Alzheimer's disease or cancer. Importantly, signal transduction can easily be studied in lower eukaryotes like filamentous fungi that are readily genetically tractable. The fungus  Saccharomyces cerevisiae, in which five MAPKs have been reported , pheromone signaling (PS), and osmosensing cascade. Notably, CWI pathway components have been studied in various fungi and are known to be not only responsible for cell wall stress response. For example, in A. fumigatus, the CWI pathway is involved in pyomelanin and gliotoxin formation, response to reactive oxygen species and siderophore biosynthesis N. crassa is necessary for polar growth, conidiation, fusion of conidial germling protrusions , and fruiting body formation Cochliobolus heterostrophus, Coniothyrium minitans, Fusarium graminearum, and Magnaporthe oryzae have further been shown to be involved in female fertility, heterokaryon formation, mycoparasitism and pathogenicity Mitogen-activated protein kinase (MAPK) cascades are central components of signaling networks in all eukaryotic organisms S. cerevisiae, the polarisome component Spa2p is known to act as a scaffold-like protein for the CWI MAPKK and MAPK during polar growth Scaffold and adapter proteins are vital for the spatiotemporally correct assembly and signaling output of MAPK pathways and are involved in decision making, thereby enabling highly specific adaptation of signaling pathways S. macrospora. This filamentous ascomycete has four major advantages over other fungal systems for the study of sexual development  and 21.3% identity to S. cerevisiae CWI MAPKKK Bck1p  S. cerevisiae genome sequence (http://www.yeastgenome.org/) N. crassa MEK-1/S. cerevisiae Mkk1p and Mkk2p . It should be noted, however, that we rarely found protoperithecia in Δmak1 .We compared sexual development of the three different kinase deletion strains to wildtype. A general observation made by ourselves and others is that a defect in sexual development is often linked to a defect in hyphal fusion mik1, mek1, and mak1 into the respective deletion strains partially complemented the growth defect. This result may be explained by mis-expression of the kinase genes from the constitutive A. nidulans gpd promoter (mik1 and mek1) and the inducible S. macrospora Smxyl promoter (mak1) To evaluate the role of MIK1, MEK1, and MAK1 in cell wall stress response, we performed growth tests on medium containing Calcofluor White (CFW). CFW is a common agent used to test fungal mutants for cell wall stress-related defects Schizosaccharomyces pombe, where MAPKKK Mkh1 and MAPKK Pek1 localize to the cytoplasm, whereas MAPK Pmk1 shuttles between the cytoplasm and the nucleus Signal transduction via a MAPK module requires several subsequent phosphorylation events, eventually leading to phosphorylation of the MAPK. Western blot analysis with deletion mutants and complemented strains showed that MIK1 and MEK1 are required for MAK1 phosphorylation, confirming the composition of the three-tiered MAPK module . We furtmek1 transcript among the most abundant transcripts in protoperithecia As mentioned above, little is known about the regulation of the fungal CWI pathway through scaffolds or adapters. We surmised that these regulators might be identified by searching for interaction partners of the kinases. In a recent transcriptomics analysis, we found the S. macrospora proteins from complex mixtures, and yields a huge number of peptides even after tandem purification MEK1 was fused to an N-terminal tandem affinity purification (TAP) tag consisting of protein A, a TEV protease cleavage site and the calmodulin-binding peptide Functionality of NTAP-MEK1 was shown by complementation analysis , and sinpro40-3xFLAG) yielding detectable amounts of PRO40-FLAG in the eluate  followed by MudPIT. The PRO40-FLAG fusion construct has already been shown to complement pro40 e eluate .A. nidulans, and PRO4/LEU1  is an enzyme that is involved in the leucine biosynthesis pathway, which is essential for fruiting body formation in S. macrosporaFLAG-AP in combination with MudPIT was performed three times with T184.2NS11 and twice with wildtype (control). A full list of proteins detected with at least two different peptides in at least two out of three (PRO40-FLAG) or two out of two (wildtype) replicate experiments is given in In addition to TAP-MudPIT with NTAP-MEK1 in a Δmek1 background, we also performed TAP-MudPIT in a Δpro40 background  and inactive (RHO1_CI) versions of RHO1 to determine interactions dependent on RHO1 activity. Protein structures of all tested proteins are given in pro40 , at septal pores in response to various stresses or hyphal injury , as well as associated with Woronin bodies (S. macrospora) S. macrospora PRO40 with Woronin bodies was already found by co-localizing PRO40 with GFP-tagged HEX1 A. oryzae. There, PKC1 is required for HEX1 phosphorylation and subsequent HEX1 self-assembly M. griseaF. graminearum, and hex1 has been found to be regulated by PS MAPK MAK2 and downstream transcription factor PP-1, homologous to yeast Ste12p, in N. crassaPRO40/SOFT homologs have been found in stress granules  of S. macrospora cDNA libraries SmI and SmII were generated using the Matchmaker Library Construction & Screening Kit . RNA was extracted from S. macrospora wildtype according to Pöggeler et al. 7/ml and 1.5×107/ml for SmI and SmII, respectively.S. macrospora cDNA and PCR fragments cloned into pGBKT7 and pGADT7 as follows:All plasmids and oligonucleotides used in this study are listed in S. macrospora cDNA using primers rho1_CA-for/rho1_CA-rev and ligated EcoRI/BamHI into pGADT7 and pGBKT7 to generate pA-RHO1_CA and pB-RHO1_CA, respectively. For pA-RHO1_CI, two PCR fragments generated with primer pairs rho1_CI-for x rho1_CIint-rev and rho1_CI-rev x rho1_CIint-for, were co-transformed into yeast with SmaI-digested pGADT7. pB-RHO1_CI was generated by ligating a 0.6 kb EcoRI/BamHI fragment from pA-RHO1_CI into EcoRI/BamHI-digested pGBKT7.For RHO1, constitutively active (RHO1_CA) and constitutively inactive (RHO1_CI) versions were generated by inserting mutations G15V/C191S and E41I/C191S, respectively SmaI-digested pGADT7 and three cDNA fragments amplified by PCR using primer pairs 4666-01-AD/4666-02, 4666-03/4666-04, and 4666-05/4666-06-AD. For pB-PKC1, a similar strategy was employed with SmaI-digested pGBKT7 and three PCR fragments produced with primer pairs 4666-01-BD/4666-02, 4666-03/4666-04, and 4666-05/4666-06-BD.To generate pA-PKC1, yeast recombination was performed with EcoRI/BamHI-digested pGADT7 and five PCR fragments produced with primer pairs 3673-1-AD/3673-2, 3673-3/3673-4, 3673-5/3673-6, 3673-7/3673-8, and 3673-9/3673-10-AD. For cloning of pB-MIK1, yeast recombination was again employed with SmaI-digested pGBKT7, two PCR fragments produced with primer pairs 3673-1-BD/3673-2 and 3673-9/3673-10-BD, and a 5254 bp NdeI/BamHI fragment from pA-MIK1.To generate pA-MIK1, yeast recombination was performed with mek1 vectors, a 1458 bp mek1 cDNA fragment was amplified with primers 6419-9/6419-10 and ligated EcoRI/BamHI into pGBKT7 and pGADT7 to generate pB-MEK1_v01 and pA-MEK1_v01, respectively. For full-length cDNA vector pA-MEK1, pA-MEK1_v01 was digested with EcoRI and transformed into yeast together with a 425 bp BglII/BamHI fragment of pA-MEK1a (see below). Likewise, pB-MEK1 was generated by yeast recombination using EcoRI-linearized pB-MEK1_v01and a 653 bp XhoI/BamHI fragment of pB-MEK1a.For mak1 vectors, a PCR-fragment produced with primers HR-mak1-for/HR-mak1-rev as well as BamHI-digested pGADT7 were transformed into yeast, generating pA-MAK1. pB-MAK1 was generated by ligation of a 1426 bp EcoRI/PstI fragment from pA-MAK1 into EcoRI/PstI-digested pGBKT7.For EcoRI/PstI in pGBKT7. pA-PRO40 was generated by yeast recombination of two PCR fragments produced with primer pairs AD-40-for1/AD-40-rev1 and AD-40-for2/AD-40-rev2, and a 3961 bp EcoRI/PstI fragment from pB-PRO40 into pGADT7/BamHI.For pB-PRO40, full-length cDNA was amplified using primers Y2H-05/Y2H-06neu and ligated S. macrospora cDNA using primer pair mek1_F1-fw/mek1_F1-rv, the PCR fragment subcloned into pDrive, cut EcoRI/BamHI and ligated into EcoRI/BamHI digested pGADT7 and pGBKT7, respectively. pA-MEK1b/pB-MEK1b, pA-MEK1c/pB-MEK1c and pA-MEK1d/pB-MEK1d were generated accordingly, using primer pairs mek1_F2-fw/mek1_F2-rv, mek1_F1-fw/mek1_F2-rv and mek1_F4-fw/6419-10, respectively.To generate pA-MEK1a and pB-MEK1a, PCR was performed on pro40 fragments were amplified from cDNA and subsequently ligated into EcoRI and BamHI sites of pGADT7 and pGBKT7. Primers used were Y2H-13/Y2H-07 for PRO40a, Y2H-08/Y2H-09 for PRO40b, Y2H-01/Y2H-12 for PRO40c, Y2H-10/Y2H-11 for PRO40d, and Y2H-03/Y2H-04 for PRO40e. pA-PRO40e was generated by ligating a 1007 bp EcoRI fragment from pB-PRO40e into the pGADT7 EcoRI site.To generate yeast two-hybrid vectors encoding PRO40 derivatives, five ScaI-digested pB-PRO40. To generate pA-PRO40AAA, a 3961 bp EcoRI/PstI-fragment from pB-PRO40AAA and a 10322 bp SalI fragment from pA-40 were recombined in yeast.Vector pB-PRO40AAA encoding PRO40 with a mutated WW domain  was generated by yeast recombination of a PCR fragment (primers 40-6/40-7) into S. macrospora pro40 cDNA was used as bait to screen both S. macrospora cDNA libraries for interacting proteins using the Matchmaker System . Yeast Y187 cells were transformed with pB-PRO40, mated with 1 ml cDNA library and plated on selective media (SD-trp-leu-ade and SD-trp-leu-his-ade). Colonies were re-inoculated on selective media lacking histidine or adenine and histidine. Growing yeast cells were subjected to two subsequent lacZ filter tests . 96 randomly chosen colonies showing reporter gene activity were chosen for PCR amplification of cDNA inserts using primer pair pAD-2/pAD-FPneu and PCR products were directly sequenced with primer pADfor96er. Quantitative measurements of β-galactosidase activity were carried out as described previously The To test interactions between full-length proteins as well as derivatives of MEK1 and PRO40, strains carrying single plasmids were generated by electroporation mik1 and mak1 were generated by yeast recombination as described mik1 were PCR-amplified using S. macrospora genomic DNA and primer pairs 3673-5fw/3673-5rv and 3673-3fw/3673-3rv, respectively. Flanking regions were transformed into yeast together with an hph cassette cut EcoRI from plasmid pDrivehph EcoRI/XhoI-linearized vector pRS426 mak1 5′ (1000 bp) and 3′ (1039 bp) flanking regions amplified with primer pairs 5504-5fw/5504-5rv and 5504-3fwIT/5504-3rvIT, respectively.Deletion vectors for mek1 deletion, 5′ (832 bp) and 3′ (913 bp) flanking regions of mek1 were PCR-amplified using primer pairs KO-mek-1/KO-mek-2 and KO-mek-3/KO-mek-4, respectively, and subcloned into pDrive. Due to annotation changes concerning mek1 in genome version 02 of S. macrosporamek1_v01, nt280–1858, encoding amino acids 35–519) was used as basis for generating mek1 deletion and TAP vectors. The 5′ and 3′ regions were cut SnaBI/BamHI and XbaI/ApaI and successively ligated into the corresponding sites of vector pDrive-Hyg . Linearized pKO-MIK1, pKO-MEK1 and pKO-MAK1 were transformed into S. macrospora Δku70 mik1, mek1 or mak1 had been replaced by the hph cassette and which had the wildtype genetic background were obtained as described previously through crosses against spore color mutant fus or mutant pro40 To generate a BamHI-digested pDS21 S. macrospora genomic DNA and primer pairs NTAP-mik-fw/3673-2, 3673-3/3673-4, 3673-5/3673-6, 3673-7/3673-8, and 3673-9/NTAP-mik-rv. pRSnat-gfp-mik1 was generated by amplification of egfp from pDS23  using primers Pgpd_egfp_for/mik1_egfp_rev and subsequent recombination in yeast with HindIII-digested pNTAP-mik1. For pGFP-MIK1_NA, 5′ and 3′ mik1 sequences were amplified from S. macrospora genomic DNA with primer pairs 3673-5fw/3673-5rv-gfp and 3673-11/3673-3rv, respectively, and subsequently recombined into pRSnat-gfp-mik1, replacing the gpd promoter and trpC terminator.For pNTAP-mik1, yeast recombination was employed. Fragments used for transformation were mek1 was amplified from genomic DNA with primers mek1-BamHI-fw/NTAP-mek-BamHI-rv, subcloned into pDrive, cut with BamHI and cloned into BamHI-digested pDS21 mek1 from S. macrospora genomic DNA with primer pair Pgpd-mek1_V3/gfp-mek-rev and recombination into linearized pDS23 in yeast. For pMEK1-GFP_NA, 5′ and 3′ mek1 sequences were amplified from S. macrospora genomic DNA with primer pairs 2183-5fw_IT/mek1_F1-rv and 2183-3fw-gfp/2183-3rv_IT, respectively, and transformed into yeast together with a 2.8 kb PvuII-SpeI fragment from pRSnat-mek1-gfp_V3 and linearized pRSnat For complementation and TAP, mak1 complementation vector was constructed by amplifying mak1 using primers Pxyl-mak-for/NTAP-MAK-rv, and recombining the PCR fragment into NotI/BamHI-digested pNpX-GFP mak1 from genomic DNA with primer pair CTAP-mak1-fw/GFP-mak1-rv and transforming the PCR fragment in yeast together with HindIII-linearized pDS23. For pMAK1-GFP_NA, 5′ (5504-5fw/GFP-mak1-rv) and 3′ (5504-3fw-gfp/5504-3rv_IT) mak1 sequences were amplified and transformed in yeast together with a 0.8 kb BamHI fragment from pRSnat-mak1-gfp and linearized pRSnat.A To search for PRO40 interaction partners, pC-FLAG-PRO40 S. macrospora Δmek1 and Δpro40, and transformants were selected on media with nourseothricin. Primary transformants expressing NTAP-MEK1 were used for single spore isolation. For protein extraction, S. macrospora strains E292 (Δmek1::NTAP-MEK1) and E2544 (Δpro40::NTAP-MEK1) were grown in P-flasks with BMM liquid medium for 3 d at 27°C. TAP analysis was performed as described previously For TAP analysis, pNTAP-mek1 was transformed into S. macrospora database (smacrosporapep_v4_110909) with tryptic peptides, mass accuracy of 10 ppm, fragment ion tolerance of 0.8 Da, and with oxidation of methionine as variable modification allowing 4 missed cleavage sites. All accepted results had a high peptide confidence with a score of 10. Proteins identifies with at least two different peptides in at least two of three to four independent experiments were considered for further analysis.Tryptic digestion of proteins and MudPIT analysis To identify contaminants in TAP-MudPIT data, we used an extended background list from Bloemendal et al. Immunodetection of TAP-tagged proteins was performed as described using a polyclonal anti-calmodulin binding peptide antibody  and an anti-rabbit HRP-linked secondary antibody  2O2 for 15, 30, or 45 minutes. Mycelia were harvested by filtration, ground in liquid nitrogen, and resuspended in FLAG extraction buffer with phosphatase inhibitors . After centrifugation at 15000 rpm for 30 min, equal amounts of total protein were subjected to SDS PAGE and Western Blotting according to standard protocols For analysis of MAK1 phosphorylation status, strains were pre-cultured in liquid BMM for 2 days at 27°C. Three standardized inoculates were transferred into liquid HEPES (50 mM) -buffered BMM and cultivated for an additional three to six days at 27°C and 30 rpm. For induction of cell wall stress, cultures were subjected to 0.01% H2HPO4, 17 mM NaH2PO4, 68 mM NaCl; pH 7.4). Fluorescence was observed using filter sets (Chroma Technology) 41017  or 49002  for EGFP and filter set 49008  for tdTomato.Microscopy was performed with an AxioImager microscope . For characterization of sexual development by DIC microscopy, strains were grown on BMM-coated glass slides for 2–7 days as described previously S. macrospora reference genome Mutant pro30 from our laboratory collection was back-crossed several times to wildtype or brown-spored fus Figure S1mik1 gene is mutated in the pro30 mutant. (A) Structure of the S. macrospora mik1 gene with introns (white boxes). The deduced MIK1 protein structure is displayed above the gene structure. The C904T mutation is indicated. Domains are displayed as in mik1 gene in 20 ascospore lines from a pro30 to fus cross. Sterile strains display the C to T transition present in pro30, while fertile strains display the wildtype CAG codon. The transition leads to an exchange of the CAG codon to a TAG stop codon (boxed).The (TIF)Click here for additional data file.Figure S2mik1 genomic locus in wildtype and deletion strains. ORFs are displayed as grey arrows, with introns marked as white boxes. Flanking sequences used for homologous integration of knockout constructs are shown as thick black lines. Black arrows represent primers used for PCR; size of PCR products is given on the thin black lines depicting the PCR products. Probes used for Southern hybridization are shown as grey bars, with grey lines indicating the size of expected signals. Restriction enzymes used for digestion of genomic DNA are depicted in grey. Not drawn to scale. (B) PCR analysis of Δmik1 strains S113578 and S113494, as well as wildtype (WT). NK, negative control. (C) Southern analysis of strains from (B) with hph and mik1 probes as illustrated in (A).Generation of Δmik1 deletion strains. (A) Schematic representation of the (TIF)Click here for additional data file.Figure S3mek1 genomic locus in wildtype and deletion strains. ORFs are displayed as grey arrows, with introns marked as white boxes. Flanking sequences used for homologous integration of knockout constructs are shown as thick black lines. Black arrows represent primers used for PCR; size of PCR products is given on the thin black lines depicting the PCR products. Probes used for Southern hybridization are shown as grey bars, with grey lines indicating the size of expected signals. Restriction enzymes used for digestion of genomic DNA are depicted in grey. Not drawn to scale. (B) PCR analysis of Δmek1 strains S101796 and S101767, as well as wildtype (WT). NK, negative control. (C) Southern analysis of strains from (B) with hph and mek1 probes as illustrated in (A).Generation of Δmek1 deletion strains. (A) Schematic representation of the (TIF)Click here for additional data file.Figure S4mak1 genomic locus in wildtype and deletion strains. ORFs are displayed as grey arrows, with introns marked as white boxes. Flanking sequences used for homologous integration of knockout constructs are shown as thick black lines. Black arrows represent primers used for PCR; size of PCR products is given on the thin black lines depicting the PCR products. Probes used for Southern hybridization are shown as grey bars, with grey lines indicating the size of expected signals. Restriction enzymes used for digestion of genomic DNA are depicted in grey. Not drawn to scale. (B) PCR analysis of Δmak1 strains R8501 and R8533, as well as wildtype (WT). NK, negative control. (C) Southern analysis of strains from (B) with hph and mak1 probes as illustrated in (A).Generation of Δmak1 deletion strains. (A) Schematic representation of the (TIF)Click here for additional data file.Figure S5Affinity purification of PRO40 and MEK1. (A) SDS-PAGE and immunodetection of PRO40-FLAG. Aliquots of crude extract, washing steps, FLAG beads and elution were analyzed by immunodetection with an anti-FLAG antibody. PRO40 is strongly enriched in the elution. (B), (C), SDS-PAGE and immunodetection of NTAP-MEK1. Crude extracts of a strain E292 (B) and E2544 (C) expressing NTAP-MEK1 show the total soluble protein before purification. Aliquots of flowthrough 1 and washing step 1 (IgG beads), flowthrough 2 and washing step 2 (CBP beads), and the elution were analyzed. Immunodetection with an anti-CBP antibody shows NTAP-MEK1 with a size of 76 kDa in the crude extracts and washing steps 1 and CBP-MEK1 with a size of 57 kDa in the elution.(TIF)Click here for additional data file.Figure S6Growth control of yeast colonies from yeast two-hybrid assays. This figure is related to (TIF)Click here for additional data file.Figure S7mek1 deletion. WT, wildtype; NK, negative control. Primers are depicted in Generation of Δmek1/pro40 double mutants. (A) Sequence analysis of strains from a Δku70/Δmek1 to pro40 cross. Strains S101796 and S101767 show the wildtype (WT) sequence, whereas strains S100793 and S101820 show the pro40 mutation. (B) PCR analysis of Δmek1/pro40 strains S101793 and S101820 to verify the (TIF)Click here for additional data file.Figure S8Crossing history of strains for genome sequencing. (A) Crossing history of mutant pro30. Strains were backcrossed to wildtype (wt) or spore color mutant fus (TIF)Click here for additional data file.Figure S9S. macrospora, N. crassa, and S. cerevisiae. Alignments were generated with ClustalW  and edited in GeneDoc. Identity to the S. macrospora proteins is given in percent. Sm_MIK1, CCC09641.1; Nc_MIK-1, XP_959647.2; Sc_Bck1p, EWG95039.1.Alignment of MIK1 homologs from (TIF)Click here for additional data file.Figure S10S. macrospora, N. crassa, and S. cerevisiae. Alignments were generated with ClustalW  and edited in GeneDoc. Identity to the S. macrospora proteins is given in percent. Sm_MEK1, CCC11961.1; Nc_MEK-1, XP_957310.2; Sc_Mkk1p, EWG93186.1; Sc_Mkk2p, EWG93010.1.Alignment of MEK1 homologs from (TIF)Click here for additional data file.Figure S11S. macrospora, N. crassa, and S. cerevisiae. Alignments were generated with ClustalW  and edited in GeneDoc. Identity to the S. macrospora proteins is given in percent. Sm_MAK1, CCC12327.1; Nc_MAK-1, EAA28804.2; Sc_Mpk1p, AAB26249.1.Alignment of MAK1 homologs from (TIF)Click here for additional data file.Table S1Summary of sequence reads and small variants from genome sequencing of pro30 and wildtype.(PDF)Click here for additional data file.Table S2List of MEK1 interaction partners identified by tandem affinity purification and mass spectrometry.(XLSX)Click here for additional data file.Table S3List of PRO40 interaction partners identified by FLAG affinity purification and mass spectrometry.(XLSX)Click here for additional data file.Table S4List of proteins identified in control experiments by FLAG affinity purification and mass spectrometry.(XLSX)Click here for additional data file.Table S5Evaluation of significance of PRO40 interaction partners.(XLSX)Click here for additional data file.Table S6List of known contaminants from affinity purification experiments.(XLSX)Click here for additional data file.Table S7List of putative PRO40 interaction partners identified by yeast two-hybrid screens.(PDF)Click here for additional data file.Table S8Strains used in this study.(PDF)Click here for additional data file.Table S9Plasmids used in this study.(PDF)Click here for additional data file.Table S10Oligonucleotides used in this study.(PDF)Click here for additional data file.Text S1Supplementary methods.(PDF)Click here for additional data file.


N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG’ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG’ases , the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans  were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG’ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody  was performed using SKBR-3 and BT-474 as target cells, and revealed that the glycoform influenced ADCC activity.Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain  N-glycans attached at a single conserved site within the Fc region are critical for the antibody’s effector functions. In addition, X-ray crystallographic . Ma. MaN-glys report  and idens report    Figs. Th7.56 Hz) . Althoug , had the same affinity as homogeneous G0-mAb. These relationships between glycan structure and affinity for FcγRIIIa-V158 were clearer than those in a previous report using humanized IgG in glycoengineered Pichia pastoris -, respectively. The decomposed ratio was calculated based on ion intensities.A2-Oxa  was dissolved in 100 mM sodium phosphate buffer (pH 6.2–8.0). The decomposed ratio (A2OH/A2OH and A2Oxa) was monitored by MALDI-TOF MS in negative mode using α-cyano-4-hydroxycinnamic acid diethylammonium salt as the matrix at 0, 15, 30, 60, 120, 180, 300, and 480 min. A2-OH and A2-OXa were observed as (TIF)Click here for additional data file.S16 FigN-glycans are shown as 54.1 kDa proteins, and unreacted aglycosylated heavy chains are shown as 52.5 kDa proteins. Light chains are shown as 29.8 kDa proteins.Translycosylated heavy chains with G2 (TIF)Click here for additional data file.S17 FigThe transglycosylated anti-Her2 mAbs were a mixture of fully glycosylated mAb, which consisted of two reacted glycosylated heavy chains and two light chains, and hemi-glycosylated mAb, which consisted of a single reacted glycosylated heavy chain and a single unreacted aglycosylated heavy chain and two light chains. The transglycosylated anti-Her2 mAbs migrates two bands of reacted glycosylated and unreacted aglycosylated heavy chains.(TIF)Click here for additional data file.S18 FigMono S column chromatogram of transglycosylated anti-Her2 mAbs with A2-OXa (A). Cation-exchange HPLC analyses of the transglycosylated anti-Her2 mAbs with A2-OXa (B) and fraction 1 isolated from Mono S column chromatograph in S18A Fig (C).(TIF)Click here for additional data file.S19 FigMono S column chromatogram of transglycosylated anti-Her2 mAbs with G2-OXa (A). Cation-exchange HPLC analyses of the transglycosylated anti-Her2 mAbs with G2-OXa (B) and fraction 1 isolated from Mono S column chromatograph in S19A Fig (C).(TIF)Click here for additional data file.S20 FigMono S column chromatogram of the transglycosylated anti-Her2 mAbs with G0-OXa (A). Cation-exchange HPLC analyses of the transglycosylated anti-Her2 mAbs with G0-OXa (B) and fraction 1 isolated from Mono S column chromatograph in S20A Fig (C).(TIF)Click here for additional data file.S21 FigMono S column chromatogram of the transglycosylated anti-Her2 mAbs with M3-OXa (A). Cation-exchange HPLC analyses of the transglycosylated anti-Her2 mAbs with G0-OXa (B) and fraction 1 isolated from Mono S column chromatograph in S21A Fig(C).(TIF)Click here for additional data file.S22 Fig(TIF)Click here for additional data file.S1 Text(TXT)Click here for additional data file.


Anxa7-deficient mice and validated in human cancers. In the androgen-resistant prostate cancer cells, ANXA7 and p53 showed similar cytotoxicity levels. However, in the androgen-sensitive LNCaP, ANXA7 greatly exceeded the p53-induced cytotoxicity. We hypothesized that the p53 underperformance in LNCaP could be due to the involvement of p53-responsive SGK1 and FOXO3A. In this study, we show that p53 failed to match programmed cell death (PCD) and G1-arrest that were induced by ANXA7 in LNCaP. WT-ANXA7 preserved total FOXO3A expression with no hyperphosphorylation that could enable FOXO3A nuclear translocation and proapoptotic transcription. In contrast, in the p53-transfected LNCaP cells with maintained cell proliferation, the phosphorylated  FOXO3A fraction was increased implying a predominantly cytoplasmic localization and, subsequently, a lack of FOXO3A proapoptotic transcription. In addition, p53 reduced the expression of aberrant SGK1 protein form in LNCaP. Using Ingenuity Pathway Analysis and p53-signature genes, we elucidated the role of distinct SGK1/FOXO3A-associated regulation in p53 versus ANXA7 responses and proposed that aberrant SGK1 could affect reciprocal SGK1-FOXO3A-Akt regulation. Thus, the failure of the cell growth regulator p53 versus the phospholipid-binding ANXA7 could be potentially attributed to its diverse effects on SGK1-FOXO3A-Akt pathway in the PTEN-deficient LNCaP.Tumor suppressor function of the calcium/phospholipid-binding Annexin-A7 (ANXA7) has been shown in  Anxa7(+/−) murine model and ANXA7 tissue microarray profiling in normal versus tumor tissues [ in vitro [The tumor suppressor gene (TSG) function of the Ca/phospholipid-binding Annexin-A7  has been demonstrated in our previous studies, including tissues . Loss of tissues  and a paThe PTEN-mutant LNCaP possess a constitutively active Akt which negatively regulates the forkhead transcription factor FOXO3A/FKHRL1. A FOXO3A decrease followed by the p53-downstream p27 promoter transactivation was reported in the LNCaP progression to androgen independence , 7, the Hence, we studied the SGK1/FOXO3A-associated effects of p53 versus ANXA7 that were anticipated to reveal the alterations in canonical p53 cell survival control as well as beneficial ANXA7 tumor suppressor effects in the PTEN-deficient LNCaP.Normal prostate (PrEC) and androgen-sensitive LNCaP prostate cancer cells (ATCC) were cultured as suggested by the manufacturer and transfected with ANXA7 and p53 constructs. Adenoviral vector-based plasmids for infection  are “empty” vector, wild-type (WT) or dominant-negative (DN) ANXA7, and p53. WT-ANXA7 corresponded to a more abundant short ANXA7-isoform . DN-ANXA7 (which is known to inhibit WT-ANXA7-induced phosphatidylserine liposome aggregation) contained triple mutations against calcium-binding site which was intended to affect COOH-terminal residues in annexin repeats 2, 3, and 4 .t-test at P < 0.05 level of significance. Cell cycle analysis was performed in parental and transfected  LNCaP cells (18 h), using propidium iodide staining (Sigma-Aldrich) and flow cytometry .Programmed cell death (PCD) detection was performed in single GFP-positive cells. Early (phosphatidylserine exposure) and late (membrane permeabilization) PCD stages were studied by Annexin V-PE Apoptosis Detection Kit I (BD Pharmingen) and flow cytometry . DNA fragmentation was studied in the end-stage PCD by TUNEL-  based APO-BRDU Kit (BD Pharmingen) and flow cytometry . To avoid the overlap with GFP-marker, FITC-labeled anti-BRDU mAb was substituted with the PE-conjugated anti-BRDU mAb (BD Pharmingen). Results were analyzed as the mean ± SE, and compared using an independent two-sample β-actin were used as control.Western blotting was performed using standard procedures, and equal amounts of total protein were electrophoresed on 4–20% Tris-Glycine gels and MagicMark . Antibodies used are FOXO3A and phospho-FOXO3A-Ser318/321 ; SGK1  and Confluent parental and vector-, WT/DN-ANXA7-, and p53-transfected LNCaP and DU145 cells were harvested, and total RNA was isolated with RNAqueous-4 PCR Kit  and used for reverse transcription . SGK1 C-terminal fragments were amplified in duplex PCR using the following primers: SGK1  and beta-actin . Full-length SGK1 was amplified using the following primers: 5′-TTTGAGCGCTAACGTCTTTCTGT-3′ and 5′-TTGCTAAGCTTCCAGAGATGTGC-3′. SGK1 cDNA was purified from agarose gels and sequenced .P < 0.01. The two identified canonical pathways, PI3K/Akt and PTEN signaling, were first enriched with p53, ANXA7, FOXO3A, and SGK1 relations from the IPA database. Next, the PI3K/Akt and PTEN pathways were modified using Path Designer, IPA, and were further customized by adding ANXA7 versus p53 connections (genes as nodes and relationships as edges) from the current report.Ingenuity Pathways Analysis (IPA)  was used for the identification of SGK1/FOXO3A-associated molecular paths of p53 versus ANXA7 in LNCaP. The p53-signature genes (210) from Comparative Marker Selection (GenePattern) (data not shown) were mapped to corresponding genes/proteins in IPA's database. IPA yielded the p53-centered interactome network that was analyzed for significant alterations in major cancer-related cell survival pathways with a threshold of P < 0.001 for both comparisons) in LNCaP cells. In APO-BRDU assay (P < 0.001) inducing DNA fragmentation almost in half of LNCaP cells. In the meantime, p53 caused only a slight increase with the phosphatidylserine exposure causing a potentially reversible early apoptosis. In contrast, WT-ANXA7 and p53 induced comparable levels of PCD and cell growth inhibition in androgen-resistant DU145 and PC3 [P < 0.001), whereas p53 failed to reach the cell growth inhibition effects of WT- as well as DN-ANXA7. These results along with DNA fragmentation (APO-BRDU) as well as by phosphatidylserine exposure and membrane permeabilization (AnnexinV-PE) suggest that wild-type- (WT-) ANXA7 surpassed the p53-induced PCD and cell cycle inhibition in androgen-sensitive LNCaP.Programmed cell death (PCD) responses to WT/DN-ANXA7 and p53 (18 hr) were assessed by Annexin V-PE and APO-BRDU. In AnnexinV-PE assay , WT-ANXADU assay , WT-ANXA E2F induction by p53 compared to WT-ANXA7 contributed to the p53 insufficiency in androgen-sensitive LNCaP [While similar tumor suppressor effects of WT-ANXA7 and p53 in DU145 were accompanied by similar RB-E2F profiles, the lack of RB1 dephosphorylation andve LNCaP , thus pr SGK1 cDNA, thereby validating a slightly smaller size of the extra-SGK1-transcript (not shown). cDNA sequence analysis verified that the SGK1 sequences from LNCaP lacked the entire exon11 (90nt). In addition, SGK1 from DU145 had a G/C-change (∗) in the same exon as compared to wild-type SGK1 reference , almost all predicted SGK1 splice isoforms lack the C-terminal exon11 . Compared to the longer splice variants overexpressed in tumors , the exo Anxa7-deficient mice that have defective nutrient response [The opposing effects of WT- and DN-ANXA7  specifically underlined the role of annexin properties in the cell survival regulation by ANXA7. The lipid phosphatase PTEN and inositol-1,4,5-triphosphate receptor ITPR3 were reduced in our tumorigenesis-proneresponse . On the  SGK1 transcript that lacks exon11. Consistent with that, SGK1 protein profile included the extra-low-molecular-weight-SGK1-form that was abolished by p53. The initially high SGK1-specific S318/321 phosphorylation of FOXO3A was further enhanced by p53 in LNCaP, but not in DU145, implied a cytoplasmic retention. In contrast, WT-ANXA7 dephosphorylated FOXO3A in the PTEN-deficient LNCaP. The SGK1-antagonists that block androgen effects on LNCaP growth are considered prostate cancer therapeutics [In conclusion, unlike p53 that was incapacitated in LNCaP, the Ca/phospholipid-binding WT-ANXA7 revived the PTEN/FOXO3A-associated cell survival control in these PTEN-deficient and androgen-sensitive prostate cancer cells. For the first time, we demonstrated that LNCaP possess aberrantapeutics . While papeutics . We prop


The complete genome sequences of three foot-and-mouth disease viruses  were directly sequenced from RNA extracted from clinical bovine samples, demonstrating the feasibility of full-genome sequencing from strong positive samples taken from symptomatic animals. Picornaviridae, Aphthovirus), exists in seven distinct serotypes , with multiple subtypes  is a primary livestock disease of economic importance, affecting wild and domestic cloven-hoofed animals . The etisubtypes . To evaluate the feasibility of complete genome sequencing from clinical samples using unbiased RNA sequencing (RNA-seq) methods, three FMDV strongly positive epithelial samples from symptomatic cattle were selected using real-time reverse transcription-PCR (RT-PCR) . From thhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Stretches containing unidentified nucleotides (“N”) were trimmed using Cutadapt version 1.3  (De novo assembly was performed using Newbler version 2.9 (Roche). The protein-coding genes were predicted relative to sequences with accession numbers AF540910, HM191257, and AY593842 using GATU  . De novoThe complete genome sequences of SAT2/ZAM18/2009 and O/ZAM14/2010 were obtained, with average coverages of 2,151× and 732×, respectively. These FMDV genomes contain a single open reading frame (ORF) of 7,008 (SAT2/ZAM18/2009) and 6,999 nucleotides (nt) (O/ZAM14/2010) encoding a polypeptide precursor protein, and they share high nucleotide homology with AF540910 and HM191257, respectively. The contig representing SAT1/NAM01/2010 contains a single 7,020-nt ORF (polypeptide precursor protein) and shares a high nucleotide homology with AY593842. As only a limited number of FMDV reads were available for the latter sample, two gaps (a 67-nt gap around the poly(C) tract and a 110-nt gap centered around contig position 6000) were closed using PCR amplification and Sanger sequencing, while the average coverage was <10×.TC] range of 14.63 to 16.18 for the samples used in this study) using an unbiased cDNA sequencing approach. However, targeted approaches using FMDV-specific cDNA synthesis primers (These data demonstrate the feasibility of direct sequencing of complete FMDV coding sequences from samples from symptomatic animals (real-time RT-PCR threshold cycle [ primers  or PCR aKU821590 to KU821592.The complete coding sequences for SAT2/ZAM18/2009, O/ZAM14/2010, and SAT1/NAM01/2010 were assigned DDBJ/EMBL/GenBank accession numbers


Myocardial first-pass perfusion (MPI) requires single-shot imaging of multiple slices per cycle. Breath-holding supports advanced high-resolution MPI methods, but tolerance to respiratory motion is desirable. Respiratory misregistration can induce aliasing artefacts by inaccurate coil sensitivity calibration, particularly at higher acceleration factors and during stress hyperpnea. Coil calibration can be adapted for respiratory motion, but autocalibration ("integrated") methods limit acceleration, and temporal coil-calibration methods may reduce SNR or cause temporal smoothing. We evaluated a motion-tracking modification of "prescan" parallel imaging specifically for MPI.To compare the conventional and new methods, rest MPI was acquired in 20 consented patients referred for late-enhancement imaging (0.1 mmol/kg GBCA). Patients were asked to breathe "slowly and deeply" during MPI to mimic potentially increased motion due to stress . The new method repeats coil prescans over the range of free-breathing motion  before acquiring MPI. For each reconstructed image, MFP aimed to use a prescan at the closest respiratory position. For this respiratory position-matched prescan selection, the MPI image was initially reconstructed using any of the prescans. The anterior chest wall location in this potentially artifactual image was compared with its location in every prescan, using a semi-automatic edge-detection algorithm. This enabled selection of the optimal coil prescan for each image of a final GRAPPA reconstruction (labeled MFP). The initial MPI image using a Single Conventional Prescan for its GRAPPA reconstruction (labeled SCP) was compared with MFP by randomised blinded scoring of parallel artefact (0 = none to 4 = non-diagnostic). In 5 further patients, MFP was compared with other coil calibrations  at increasing accelerations (R 2-6) by retrospective subsampling of full k-space MPI scans. The root-mean-square difference error (RMSE) over the FOV compared accelerated vs fully-sampled images.Accelerated patient scans showed significantly p < 0.02; n = 20) reduced artefact scores (MFP 1.15 ± 0.88 vs SCP 2.40 ± 1.31). Retrospective subsampling at R = 4 resulted in the lowest  mean RMSE by MFP 11.0 ± 1.3 vs Integrated 18.8 ± 3.8, SCP 13.6 ± 0.8, Temporal 11.8 ± 1.0 (Figure ; n = 20 The MFP method by semi-automated image-based selection of a position-matched prescan reduces parallel imaging aliasing artefact in free-breathing MPI.Biomedical Research Unit, National Institute for Health Research.


L. Moreover, none of the classical Puma transcription factors such as p53, p73 or p65 NFκB were involved in HSV-1-induced apoptosis. Our data suggest that viruses use a Puma protein-dependent mechanism to trigger MOMP and apoptosis in host cells.Viruses can trigger apoptosis of infected host cells if not counteracted by cellular or viral anti-apoptotic proteins. These protective proteins either inhibit the activation of caspases or they act as Bcl-2 homologs to prevent Bax/Bak-mediated outer mitochondrial membrane permeabilization (MOMP). The exact mechanism by which viruses trigger MOMP has however remained enigmatic. Here we use two distinct types of viruses, a double stranded DNA virus, herpes simplex virus-1 (HSV-1) and a positive sense, single stranded RNA virus, Semliki Forest virus (SFV) to show that the BH3-only protein Puma is the major mediator of virus-induced Bax/Bak activation and MOMP induction. Indeed, when Puma was genetically deleted or downregulated by shRNA, mouse embryonic fibroblasts and IL-3-dependent monocytes as well as human colon carcinoma cells were as resistant to virus-induced apoptosis as their Bax/Bak double deficient counterparts (Bax/Bak-/-). Puma protein expression started to augment after 2 h postinfection with both viruses. Puma mRNA levels increased as well, but this occurred after apoptosis initiation (MOMP) because it was blocked in cells lacking Bax/Bak or overexpressing Bcl-x The role of apoptotic, programmed cell death as an ancestral form of host cellular response to inhibit viral replication and limit viral spread and the co-evolutionary capacity of viruses to counteract apoptosis have been extensively investigated in the past years –4. PartiAlphaherpesvirinae subfamily of herpesviruses. Particularly, herpes simplex virus type 1 (HSV-1) is a human common pathogen that rapidly and efficiently replicates at a portal entry of the host before retrograde transportation to nuclei of sensory neurons. In these cells, HSV-1 remains latent for the lifetime of its host and can be reactivated to cause lesions at or near the initial site of infection. This complex cycle of infection is tightly controlled by an ordered sequence of molecular events, involving a regulated expression of both viral and cellular genes [Herpes simplex viruses (HSV) are double-stranded DNA viruses belonging to the ar genes . As a coar genes ,10. Sevear genes , ICP27 [ar genes  and ICP2ar genes , the latar genes –16, the ar genes –20, and ar genes . On the ar genes ,22,23 anar genes  seem to ar genes ,23. NFκBar genes . Thus, dar genes . HoweverIn fact, depending on a variety of both viral and cellular factors, host cells can also die after HSV-1 infection. For example, whilst apoptosis is usually prevented by wild type HSV-1 in fully permissive epithelial cells, the same virus and the closely related herpes simplex virus type-2 (HSV-2) can induce apoptosis as an exclusive cytopathic effect in human monocytic cells . InductiAlphaviridae do not carry any survival factors in their genome and induce apoptosis of many different mammalian host cell types [Other viruses such as the positive sense, single stranded RNA virus Semliki Forest (SFV) of the genus ll types . The maill types . Recentlll types . While tActivation of Bax/Bak leads to mitochondrial outer membrane permeabilization (MOMP), which is an essential step for the release of apoptogenic factors such as cytochrome c to activate effectors caspase-3 and -7 along the intrinsic, mitochondrial signalling pathway of apoptosis . This acIn addition to the intrinsic mitochondrial pathways some viruses also induce apoptosis via the extrinsic death receptor pathway. Here caspase-8 is the major target as viruses produce factors, which inhibit this caspase  . CaspaseHere we show that both HSV-1 and SFV primarily use Bax/Bak-dependent, death receptor-independent signalling to induce apoptosis in human monocytes and colon carcinoma cells as well as in mouse embryo fibroblasts and monocytes. The BH3 sensor was uncovered to be Puma for both viruses. Although Puma mRNA was consistently upregulated in response to both HSV-1 and SFV, this effect was dependent on Bax/Bak indicating that it occurred after apoptosis induction. However, Puma protein levels were increased early after HSV-1 and SFV infection by a so far unknown mechanism indicating that this posttranslational regulation is most likely the way by which both viruses trigger Bax/Bak-mediated MOMP and apoptosis if not counteracted by Bcl-2-like survival factors.HSV-1 only minorly induces apoptosis of permissive human cells, probably because the viral proteins ICP4 and US3 block caspase-dependent and-independent cell death ,13. HoweTo further characterize HSV-1-induced apoptosis in a genetically more amenable system, we used mouse embryo fibroblasts (MEFs), either transformed by SV40 T antigen (TAg) or spontaneously immortalized (3T9). Following infection with 10 moi of HSV-1, a high percentage of SV40 TAg WT MEFs stained positive for the env protein gD  indicatiIndeed, when we infected SV40 TAg Bax/Bak-/- MEFs with HSV-1, these cells did not show any cytochrome c release , active We confirmed the Bax/Bak requirement for HSV-1-induced apoptosis in other mouse cells, IL-3 (factor)-dependent monocytes (FDMs) as well as in human HCT116 colon carcinoma cells. As shown in Since Bax/Bak-/- MEFs still died in a protracted manner by both caspase-dependent and-independent mechanisms, we envisaged the possibility that HSV-1 could also either engage the extrinsic death receptor and/or the necroptotic signalling pathway(s). Necroptosis can be induced by cellular treatment with TNFα + ZVAD and is mediated by RIP1 and RIP3 kinases . To studThe activation of Bax/Bak depends on the transcriptional induction or posttranslational modification of upstream BH3-only proteins. To uncover which BH3-only protein was engaged by HSV-1 we infected various 3T9-immortalized or SV40 TAg-transformed MEFs deficient for particular BH3-only proteins with 10 moi of HSV-1 and tested their apoptosis sensitivity. SV40 TAg MEFs lacking Bid, Bad or Bik or 3T9 MEFs lacking Bim or Noxa died in a similar fashion in response to HSV-1 infection as the respective WT cell lines . SV40 TATo assure that the protection from HSV-1-induced apoptosis in Puma-/- cells was not due to another cell death resistance mechanism that had been acquired through the generation of Puma-/- mice or the culturing and/or immortalization of isolated Puma-/- MEFs, we knocked-down Puma expression in both 3T9 and SV40 TAg MEFs by shRNA. As shown in L (SV40 TAg Bcl-xL MEFs)  . MoreovexL MEFs) .We finally wanted to know if the requirement of Puma was unique to apoptosis induction by HSV-1 or if other unrelated viruses used the same BH3-only protein to kill their target cells. We recently reported that the positive sense, single stranded RNA virus Semliki Forest (SFV) induced Bax/Bak-dependent and-independent apoptosis of mammalian cells ,33. The Considering the crucial role of Puma for SFV-induced apoptosis, we wanted to know if Puma is similarly regulated as in HSV-1 infections. We first performed a qRT-PCR of Puma from WT and Bax/Bak-/- SV40 TAg MEFs infected with 10 moi of SFV for up to 24 h. As shown in While mechanisms utilized by viruses to prevent premature apoptotic death of their host cells are well known, relatively few data are available on molecular events that initiate and drive an apoptotic innate response to virus infection. In this study we show for the first time that two phylogenetically distant viruses, such as the double stranded DNA virus HSV-1 and the single stranded RNA virus SFV, use the same BH3-only protein, Puma, to trigger Bax/Bak-mediated MOMP, effector caspase-3/-7 activation and apoptosis of different mammalian cell types. In both scenarios, Puma transcription is increased, but as a consequence rather than a cause of Bax/Bak-induced MOMP. Moreover, p53, p73 and p65 NFκB, the major transcriptional inducers of Puma expression, are not involved in HSV-1-induced apoptosis. Instead Puma protein levels increased rapidly after infection of both viruses in a Bax/Bak-independent fashion suggesting that a yet unknown molecular mechanism to stabilize the Puma protein is crucial for virus-induced apoptosis.We firmly believe that our finding uncovers a more general mechanism by which viruses induce apoptosis of their host cells. Cruz et al.  reportedL. This indicates that Puma transcription in response to HSV-1 and SFV infection is not an early event of apoptosis but occurs later under the control of MOMP and caspase-3/-7 activation. Perhaps a substrate that is cleaved by caspase-3/-7 directly or indirectly triggers Puma transcription therefore stimulating a feed-forward loop to amplify virus-induced apoptosis. Consistent with this notion, the genetic deletion of p53, p73 or p65 NFκB in MEFs did not protect the cells from HSV-1 or SFV-induced apoptosis although cell death was slightly delayed in both cases , Hoechst 33334, propidium iodide (PI), actinomycin D (Act D), bovine serum albumin (BSA), Triton X-100 (TX) and NP40 were bought from Sigma-Aldrich, the caspase inhibitors Q-VD-OPH and ZVAD-fmk from Biomedicals, lipofectamine from Invitrogen and the fluorogenic caspase-3 substrate DEVD-AMC (acetyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-7-amino-4-methylcoumarin) from Alexis Biochemicals. Acrylamide and dithiothreitol (DTT) were from AppliChem, Necrostatin-1 (Nec-1) from Calbiochem and PageRuler Prestained Protein Ladder from Thermo Scientific. Recombinant mouse TNFα and neutralizing monoclonal anti-mouse TNF-R1 antibodies were purchased from R&D Systems. Lentiviral shRNAs to knock-down mouse Puma, mouse RIP3 and the respective scrambled shRNAs (sh-Ctrl) were obtained from Sigma-Aldrich. Recombinant CD95/FasL was kindly provided by P. Schneider, Lausanne, Switzerland, mouse CD95/Fas-Fc by T. Brunner, Konstanz, Germany, and leucine zipper-tagged TRAIL (lz-TRAIL), TRAIL-R2-Fc and the F(ab)’2 fragment of a neutralizing anti-TRAIL-R1 antibody by H. Walczak, London College, UK). The monoclonal anti-gD antibody was kindly provided by G. Cohen and R. Eisenberg, University of Pennsylvania, Philadelphia, PA, the polyclonal anti-SFV-capsid (C) antibody by J. Pavlovic, Zürich, Switzerland.Rabbit polyclonal anti-caspase-3 antibodies recognizing the 32 kD proform (#9661) and the cleaved active 17 kD form (#9662) were purchased from Cell Signaling, rabbit polyclonal anti-Puma from ProSci, mouse monoclonal anti-actin (clone C4) from BD Biosciences, mouse monoclonal anti-COX-VIc from Life Technologies, mouse monoclonal anti-cytochrome c (clone 6H2.B4), horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies from Jackson Immunoresearch Laboratories, rabbit polyclonal anti-Bax (NT), rabbit polyclonal anti-Bak (06–536), neutralizing anti-human CD95/Fas antibody (clone ZB4) and FITC-conjugated goat anti-mouse IgG from Millipore and PE-conjugated goat anti-rabbit IgG, F(ab’)L were generated as described [2 incubator. The insect cell line Aedes albopictus was maintained at 28°C in L15 medium (Gibco) supplemented with 10% fetal calf serum (FCS) and 4% Difco Bacto phosphate tryptose broth.SV40 large T antigen (TAg)-transformed WT, Bid-/- , Bad-/- escribed . Jurkat escribed . U937 stescribed . The Pumescribed . Similarescribed . All theA. albopictus cells (SFV) [9 PFU/ml.The F-strain of HSV-1  and the virulent L10 strain of SFV  were usels (SFV) . Virus tls (SFV) . Both viSV40 TAg-transformed or 3T9-immortalized MEFs, mouse monocytic FDMs, human monocytic U937 cells and human colon carcinoma HCT116 cells were infected with 10 moi of SFV or 50 moi of HSV-1 while shaking in DMEM plus 0.5% FCS. After 1 h at 37°C, viral infection was stopped and the cells incubated in DMEM plus 10% FCS until processed for further experiments. Alternatively, the cells were treated with 50 ng/ml FasL , 10 ng/ml TNFα (R&D Systems) or 500 ng/ml leucine zipper (lz) TRAIL . Apoptosis was quantified by annexin-V-GFP/propidium iodide (PI) FACS analysis using a FACS Calibur equipment from Becton Dickinson, and caspase-3/-7 activity was measured by the DEVDase assay as described . FluoresTo study the implication of CD95/Fas, TRAIL-R1, TNF-R1 or their respective ligands CD95/FasL, TRAIL or TNFα, we incubated human Jurkat cells with neutralizing antibodies against human Fas (anti-hCD95/Fas) or MEFs with neutralizing antibodies against mouse TRAIL-R1 (anti-mTRAIL-R1 Fab2) or mouse TNF-R1 (anti-mTNF-R1) or recombinant receptor Fc proteins directed against mouse FasL (mCD95/Fas-Fc) or mouse TRAIL (mTRAIL-R2-Fc) at 37°C for 1 h before either adding the respective ligands or infecting the cells with 10 moi of HSV-1 for up to 24 h, exactly as described for SFV .2, 2 mM EGTA) containing 1% of Triton-X-100 or in buffer B  containing 1% NP40 and complete protease inhibitors (Roche). After 20 min incubation on ice, the suspension was centrifuged at 4°C and 13000 rpm for 5 min. The supernatant contained the cytosolic and TX- or NP40-solubilized membrane proteins. Protein concentration was determined using Bradford assay. An amount of 80 μg of proteins was boiled in SDS sample buffer , and then subjected to electrophoresis on 12.5–15% denaturing polyacrylamide gel followed by transferring to nitrocellulose membrane. The membranes were blocked in 1 x PBS supplemented with 5% non-fat dried milk and 0.05% Tween-20 for 1 h and incubated with primary antibodies at 4°C overnight. After three washings in PBS, secondary horseradish peroxidase-conjugated antibodies were added and the membranes incubated at RT for 90 min. Proteins were visualized with the enhanced chemiluminescence SuperSignal West Pico Chemiluminescent.For total extracts, cells were collected on ice by scraping. The cells were washed with ice-cold 1 x PBS and lysed in buffer A . The cells were then incubated with mouse monoclonal anti-gD or mouse monoclonal anti-cytochrome c and rabbit polyclonal anti-caspase-3 antibodies followed by FITC-conjugated goat anti-mouse IgG, (Millipore) and PE-conjugated goat anti-rabbit IgG, F(ab’)2 fragment (Santa Cruz Biotechnology) secondary antibodies (1:200) for 90 min. Nuclei were stained with Hoechst 33334 . The samples were directly viewed under a Leica DMRE fluorescence microscope.5 x 106 mock- or SFV- or HSV-1-infected WT or knock-out MEFs using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The amount of total RNA isolation was quantified by optical density at 260 nm. 1–2 μg of total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Transcription kit  according to the manufacturer’s instructions. To digest the leftover RNA, 1 μl RNase H was added and incubated at 37°C for 20 min. Quantitative polymerase chain reaction (qPCR) amplification of the various cDNAs was carried out in a 25 μl solution containing 11.25 μl SYBR Green PCR Master Mix (Bio-Rad laboratories), 150 nM forward and reverse primers, 750 ng cDNA template, 10.25 μl H2O and the Hotstar meteor Taq polymerase. The sequences of the primer used for the qPCR were: Puma forward murine primer 5’-GCCCAGCAGCACTTAGAGTC- 3'; Puma reverse murine primer 5'- GGTGTCGATGCTGCTCTTCT -3’; 18S forward primer 5’-GTAACCCGTTGAACCCCATT-3’; 18S reverse primer 5’CCATCCAATCGGTAGTAGCG-3’. Samples were heated for 10 min at 95°C and then subjected to 45 cycles of PCR amplification, each cycle consisting of 15 s at 95°C and 60 s at 60°C. Within each experiment, a no-template control and the housekeeping 18S gene were run in parallel. Each run was completed with a melting curve analysis to confirm the specificity of amplification and lack of unspecific products and primer dimers. Quantification was performed using the Ct comparative method. The relative gene mRNA levels were calculated as follows: ΔCt values (Ct (gene of interest)—Ct ) represent the difference, in threshold cycle number, between genes of interest and the housekeeping gene 18S. To compare the expression of different genes, ΔCt values were normalized to the mean value of ΔCt from the least expressed gene (2-ΔΔCt = 2-(ΔCt (gene of interest)—mean value of the less expressed gene ΔCt )).Total RNA was extracted from 1 x 10p values) was analyzed by a two-tailed Student’s t test. Data are the means of at least three experiments using two to three independent cell clones ± SEM.Statistical significance , SV40 TAg WT or Bax/Bak-/- MEFs (B), WT, Bax/Bak-/- or Puma-/- FDMs (C), or WT, Bax/Bak-/- or Puma-/- HCT116 cells (D), infected with 50 (A) or 10 (B-D) moi of HSV-1 in the presence or absence of 25 μM of the general caspase inhibitor QVD for 0 (mock), 12, 24, 36 or 48 h (hpi). The x-axis (FL1-H) shows annexin-V-GFP, the y-axis (FL2-H) shows PI staining. The lower left quadrant depicts the percentage of double negative, surviving cells, the lower right quadrant the percentage of single positive (annexin-V) apoptotic cells and the upper right quadrant the percentage of double positive (annexin-V/PI) secondary necrotic cells.A representative set of original annexin-V/PI FACS dot plot data of U937 cells carrying the pcDNA3 vector or expressing a dominant-negative version of IκBα (mIκBα) (TIF)Click here for additional data file.S2 Fig(A) Anti-env gD immunofluorescence analysis of SV40 TAg WT and Bax/Bak-/- MEFs infected with 10 moi of HSV-1 for 16 h (hpi). gD positivity represents viral infection, Hoechst 33334 stains nuclear DNA. (B) The number of gD positive cells in (A) were determined by counting 10 different fields under the fluorescent microscope. The data represent the means of 3 independent stainings (counting 10 fields each) ± SEM. The p values are the following: HSV-1 versus mock, p < 0.001 for 24 and 48 h; HSV-1-infected Bax/Bak-/- versus HSV-1-infected WT cells: p = 0.01 for 24 h, p = 0.05 for 48 h, p < 0.001 for 72 h, n = 5. (C) Viral titers determined by the plaque assay and depicted as Log10 Plaque Forming Units (PFU)/ml after infecting U937 vector control (pMEP) and Bcl-2-overexpressing (Bcl-2) monocytes with 50 moi or infecting SV40 TAg WT and Bax/Bak-/- MEFs with 10 moi of HSV-1 for up to 72 h. Data are the means of at least three independent experiments ± SEM. The p values are < 0.001 for U937 Bcl-2 versus pMEP and SV40 TAg Bax/Bak-/- versus WT at 48 and 72 hpi, n = 4.(TIF)Click here for additional data file.S3 FigAnti-RIP3 western blot analysis of total extracts from mixed populations of puromycin-selected, SV40 TAg WT and Bax/Bak-/- MEFs infected with lentiviruses carrying a scrambled shRNA (sh-Ctrl) or an shRNAs for mouse RIP3 (sh-Rip3). Anti-actin as loading control.(TIF)Click here for additional data file.S4 FigAnti-Puma western blot analysis of total extracts from mixed populations of puromycin-selected, SV40 TAg-transformed and 3T9-immortalized MEFs infected with lentiviruses carrying a scrambled shRNA (sh-Ctrl) or shRNAs for mouse Puma (Sigma Open Labs). For comparison, an extract from 3T9 Puma-/- MEFs is shown. Anti-actin as loading control.(TIF)Click here for additional data file.S5 Fig-∆∆Ct relative to mock cells  of Puma mRNA isolated from 3T9-immortalized WT and Bax/Bak-/- MEFs infected with 10 moi of HSV-1 for 0, 0.5, 1, 2, 3, 6, 12, 18 or 24 h. The mRNA values were normalized to the ribosomal housekeeping S18 gene and depicted as 2(TIF)Click here for additional data file.S6 Fig(A) 3T9-immortalized WT, p53-/+ and p53-/- MEFs, (B) SV40 TAg-transformed WT and p73-/- MEFs or (C) SV40 TAg-transformed WT and p65 NFκB-/- MEFs, infected with 10 moi of HSV-1 for 0, 14, 24 or 36 h (hpi). In (A) and (B) the cells were also exposed to UV light (100 J/m2) for 24 h as a positive control. Data are the means of at least three independent experiments using two different clones of WT and knock-out cells ± SEM. The p values are < 0.001 for UV-treated p53-/- versus WT and UV-treated p73-/- versus WT MEFs, n = 3.Annexin-V/PI FACS analysis of (TIF)Click here for additional data file.


Myocardial T1 mapping is increasingly used to diagnose and quantify disease burden in patients with known or suspected cardiac amyloid. Optimal timing for diagnostic application of T1 mapping is not established.Myocardial T1 mapping was performed in two cohorts - (1) "amyloid +" subjects, defined by biopsy-proven systemic amyloid with associated remodeling suggestive of cardiac involvement ; (2) normative controls without risk factors for amyloid or cardiovascular disease. CMR (1.5T) included 2 components - cine-CMR (SSFP) for cardiac structure/function, and T1 mapping for myocardial tissue characterization. T1 mapping was performed using a conventional modified look locker inversion recovery (MOLLI) sequence , with T1 calculated using an established formula (T1 = T1* (B/A-1), T1*, A, and B obtained via three-parameter exponential fit). To test time dependent differences in myocardial T1, MOLLI was acquired at sequential time points  following intravenous administration of gadolinium (0.2 mmol/kg).10 subjects  were studied ; all amyloid affected subjects had biopsy-confirmed disease . Amyloid subjects had higher LV mass , lower myocardial contraction fraction , and larger left atrial area  than controls, but similar LV end-diastolic volume , stroke volume , and LVEF . MOLLI was successfully acquired in all subjects at each time point: T1 differed significantly  between amyloid and control groups at all times (Figure MOLLI-quantified myocardial T1 yields maximal difference between amyloid-affected subjects and normative controls within 3 minutes following gadolinium administration. Increased magnitude of T1 difference early post contrast may relate to altered gadolinium contrast kinetics due to amyloid-associated increases in cardiac extracellular volume, and/or pulse-sequence aspects of the MOLLI approach. Current findings support use of early post-contrast MOLLI T1 mapping for identification of cardiac amyloid.M. Cooper was supported by NSF GFRP DGE-0707428. J. Weinsaft was supported by NIH K23HL102249-01.


Despite this, little evidence of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) was found in CHIP−/−-mice over the first 8–9-months of life. We herein document that this lack of tissue injury is largely due to the concurrent up-regulation and/or activation of the adiponectin-5′-AMP-activated protein kinase (AMPK)-forkhead box O (FOXO)-signaling axis stemming from at the least three synergistic features: Up-regulated expression of adipose tissue adiponectin and its hepatic adipoR1/adipoR2 receptors, stabilization of hepatic AMPKα1-isoform, identified herein for the first time as a CHIP-ubiquitination substrate (unlike its AMPKα2-isoform), as well as nuclear stabilization of FOXOs, well-known CHIP-ubiquitination targets. Such beneficial predominance of the adiponectin-AMPK-FOXO-signaling axis over the sustained JNK-elevation and injurious insulin resistance in CHIP−/−-livers apparently counteracts/delays rapid progression of the hepatic microvesicular steatosis to the characteristic macrovesicular steatosis observed in clinical NASH and/or rodent NASH-models.Genetic ablation of C-terminus of Hsc70-interacting protein (CHIP) E3 ubiquitin-ligase impairs hepatic cytochrome P450 CYP2E1 degradation. Consequent CYP2E1 gain of function accelerates reactive O CHIP Ub-ligase is physiologically relevant, as its genetic ablation in mice results in premature aging, shortened lifespan, and various pathologies including widespread oxidative damage due to disrupted protein quality control2O2 and other ROS 16−/−-livers. Additionally, we document that such chronic CYP2E1-elicited oxidative stress in CHIP−/−-hepatocytes is associated with the sustained activation of stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK)-signaling cascades, nuclear factor κB (NF-κB) and inflammatory cytokines and chemokines and the Nod-like receptor P3 (NLRP3)-inflammasome, which may significantly contribute to the age-dependent cellular ballooning and microvesicular steatosis observed in CHIP−/−-livers. However, in spite of this concatenation of nonalcoholic fatty liver disease (NAFLD)/NASH-like events, little evidence of NAFLD/NASH, as signaled by its hallmark macrovesicular steatosis, was actually found in CHIP−/−-livers over the first 8–9 months of life. The significant activation of the hepatic energy- and ROS-sensor 5′AMP-activated protein kinase (AMPK) coupled with the significantly up-regulated expression of epididymal white adipose tissue (EWAT) adiponectin and its hepatic adipoR1/adipoR2-receptors, observed early in 2-month-old CHIP−/−-mice relative to that in age-matched CHIP+/+-controls, indeed presaged the concurrent up-regulation and activation of the adiponectin-AMPK-forkhead box O (FOXO)-signaling axis. In this, FOXO/FKHR transcription factors, critical nodes at the intersection of the JNK- and AMPK-signaling networks1819202122−/−-livers. Our findings reinforce the growing awareness of the beneficial role of adiponectin-AMPK-FOXO-signaling pathway in the pathogenesis of NAFLD/NASH24CYPs 3A and CYP2E1 undergo futile oxidative cycling that generates HIB) analyses, appreciably higher CYP3A and CYP2E1 protein stabilization was observed in cultured CHIP−/−-hepatocyte lysates than in CHIP+/+-hepatocyte lysates, irrespective of mouse age , thereby verifying that CHIP-knockout stabilized both hepatic CYP3A and CYP2E1 content. This increased P450 content in CHIP−/−-hepatocytes was functional, as documented by the CYP3A and CYP2E1 functional probes .Upon Western-immunoblotting (2t-isoprostane (15-F2t-IP) and malondialdehyde (MDA) basal levels (+/+- and CHIP−/−-hepatocytes were concomitantly pretreated with both dexamethasone (DEX) and isoniazid (INH) to restore basal CYP3A and CYP2E1 content and then cotreated with KTZ (5 μM) plus 4-MP (2.5 mM), basal 15-F2t-IP levels were decreased by 56% and 80%, respectively -protein conjugation and oxidized protein side-chain carbonyls provided additional evidence for the relatively enhanced oxidative stress in cultured CHIPatocytes . In situatocytes  revealed7 months . Togethe−/−- and CHIP+/+-mouse livers histologically following staining with hematoxylin and eosin (H&E), Oil red O for detection of lipid accumulation, and Masson’s trichrome for detection of fibrosis  was quite marked in 9-month-old CHIP−/−-mice relative to age-matched CHIP+/+-controls  found striking evidence of “fibrosis due to mild cardiovascular congestion in the central vein stemming from the onset of heart failure”, a plausible cause of death in these prematurely aging CHIP−/−-mice manifesting cardiac hypertrophy and compromised cardiac functionWe characterized the age-related morphological changes in 2-, 4- and 9-month-old CHIPfibrosis . No greacontrols . No evidcontrols . Trichro−/−-livers, we screened cultured hepatocytes from 2-, 4- and 9-month-old mice using the PathScan Intracellular signaling array kit. This slide-based antibody array provides a broad snapshot of age-dependent activation of signaling modules and/or proapoptotic processes . Several key signaling transducers  activated early (≈2 months) and in a CYP2E1-dependent manner in CHIP−/−-livers were thus identified: (i) Mitogen-activated protein kinase (MAPK) JNK, activated by pro-inflammatory cytokines and cellular stresses8112930313233−/−-hepatocytes . By contrast to JNK, p38MAPK, another similarly activated kinase and the extracellular-signal-regulated kinase (ERK1/2) were markedly activated in CHIP−/−-hepatocytes but only at 9 months .To identify any plausible signaling cascades affected by CYP2E1-mediated persistent oxidative stress in CHIP−/−-hepatocytes relative to age-matched CHIP+/+-controls was documented by the activation of the pro-apoptotic signal transducer and activator of transcription 1 (STAT1)-signaling and marked activation of caspase 3  via endoproteolytic cleavage at Asp214 and subsequent cleavage of its principal target polyADP-ribose polymerase 1 (PARP1), involved in DNA repair . Such enhanced caspase 3-activation was not 4-MP-sensitive and thus CYP2E1-independent. Most likely, consistent with the pyknotic nuclei in 9-month-old CHIP−/−-livers -JNK-MAPK-protooncogene c-Jun/activator protein 1 (AP1)-signaling cascade in their native and activated/phosphorylated forms81011293031−/−-hepatocytes at 2 months over age-matched WT-controls. Neither increase was CYP2E1-dependent. MAPK-kinase MKK4 levels were comparable in CHIP−/−-hepatocytes and WT-controls; but activated MKK4 (pMKK4) levels were dramatically increased in CHIP−/−-hepatocytes relative to WT-controls, in a 4-MP-sensitive manner -proteolytic processing and consequent transcriptional activation of SREBP target genessrebp-1 c and srebp-2a gene expression was also concurrently upregulated, thus synergistically upregulating lipogenic target genes responsible for FA-synthesis  and lipid-uptake (ATP-binding cassette transporter abc-a1), consistent with the observed hepatic microvesicular steatosisThe concomitant age-dependent microvesicular steatosis and hepatocyte ballooning observed in CHIPcontrols  led us t−/−-livers as early as 2 months. Their expression along with that of IL-1β remained upregulated at 9 months. In CHIP−/−-livers, this TNFα-up-regulation is paralleled by their NF-κB-activation profile−/−- and CHIP+/+-livers revealed the very early relative activation of p65/p50 NF-κB-heterodimers in CHIP−/−-livers at 2 and 4 months, that peaked at 8 months but reverted to WT-levels at 12 months (see below). Similarly, IL-6 up-regulation was also consistent with CYP2E1-dependent pro-inflammatory activation of STAT3-signaling . Additionally, a decisive elevation of NLRP3 (cryopyrin), an NLRP3-inflammasome component involved in pro-IL-1β-activation to IL-1β−/−-hepatocytes but not earlier, or in age-matched controls , interleukin 6 (IL-6) and macrophagic chemokine monocyte chemotactic protein 1 (MCP-1) was also significantly detected in CHIP2 months , possiblcontrols . Such up−/−-hepatocytes, we monitored cytosolic alanine aminotransferase (ALT)-leakage into the medium as a hepatocellular-injury marker over a 24 h-period. Although this was slightly, albeit significantly higher in CHIP−/−-hepatocytes from 2-month-old mice relative to those from age-matched WT, little basal cytotoxicity was evident under conditions of routine culture (Williams medium E (WME)/5 days) . Because all these mice were fed a standard chow-diet, relative predisposition to cell injury conceivably could be elicited upon hepatocyte culture in a reportedly steatogenic methionine-choline deficient (MCD)-WME medium−/−-hepatocytes, this was only nominally higher than that of similarly cultured CHIP+/+-hepatocytes. Comparable findings were observed when cell injury was incited with hepatotoxic acetaminophen concentrations−/−-hepatocytes were no more predisposed to cell injury than their WT-counterparts.Given this remarkable collective pathogenic profile of CHIP−/−-livers . Thus, on the one hand, Akt-activation critical for insulin signaling was apparently impaired in CHIP−/−-livers, as inferred from the somewhat feeble activation of glycogen synthase kinase (GSK) 3β, an Akt-targetsee below). On the other, the down-regulation of insulin-regulated hepatic insig-1 and insig-2 genes signaled adequate insulin-availability in CHIP−/−-livers . These conflicting insulin-dependent responses obfuscated the real status of insulin signaling in CHIP−/−-livers and its plausible impact on their relative NAFLD/NASH-susceptibility. Because of this and the significant basal pancreatic CHIP-expression−/−-livers  and protein content  was significantly lower than in age-matched controls at 2 months, and further declined over 4–8 months  and the adipocyte adipokine adiponectin (adipoQ) was significantly up-regulated at 2 months in CHIP−/−-livers relative to age-matched WT, but the latter began to decline at 9 months  levels were relatively increased over 2–8 months in CHIP−/−-hepatocytes  was associated with concurrent hepatic activation of the adiponectin-AMPK-FOXO-signaling, an event that could potentially delay NAFLD/NASH-onset. Indeed, the expression of hepatic adiponectin receptors . Furthermore, such AMPKα1- but not AMPKα2-ubiquitination could be enhanced upon treatment of the cotransfected cells with the proteasomal inhibitor MG132  (1819202122Atg14 and lpl autophagic/lipophagic genes2351−/−-livers (see below) -transcription factors181823ionation . Such enionation . As expe (acox1) , consiste below) .−/−-mice revealed that rather than the deep red exhibited by age-matched WT-controls, their livers were typically light brown in color , which rely on CHIP for their ERAD. Studies with a specific quencher of mitochondrial ROS (MitoTEMPO) coupled with CYP2E1/CYP3A functional inhibitors (4-MP/KTZ) as probes  increased content of ASK1, the priming member of this signaling cascade and a known CHIP-ubiquitination target−/−-livers  would also magnify this JNK1-activation response, thereby transcriptionally up-regulating pro-inflammatory/inflammatory cytokines/chemokines such as TNFα, IL-6, and MCP-1 detected early (2 months), with subsequent IL-1β up-regulation at >9 months. Remarkably, TNFα coupled with elevated ROS can synergistically foster vicious, upward-spiraling pathogenic cycles entailing activation of JNK1-/p38- and IKK-NF-κB-signaling via Complex I as well as proapoptotic caspases 8 and 3 via Complex II841538−/−-livers relative to age-matched controls.Similar stabilization of the ubiquitously expressed CYP2E1 in nonhepatic CHIP−/−-livers most closely mirrored their temporal JNK1-activation profile. Similarly, the concurrent temporal activation of ASK1-JNK1-ATF2- and ASK1-JNK1-c-Jun-signaling cascades suggests that pATF2 preferentially interacts with the AP-1-component pc-Jun for its nuclear transcriptional activation of pro-inflammatory cytokines/chemokines and apoptotic effectors i.e. acetylcholinesterase (ache)−/−-livers. Such severe sustained stress is expected to eventually lead to ATF2-mediated disruption of the outer mitochondrial membrane permeability38−/−-livers to acute cell injury  is indeed remarkable.Although JNK, p38MAPK and ERK1/2 all activate ATF2, the marked ATF2-activation in 2-month-old CHIP−/−-livers relative to age-matched WT-controls was relatively defective as judged by two telltale indices (i) the relatively increased IRS1-S307-phosphorylation secondary to the sustained JNK1-activation5845−/−-livers became insulin-resistant around 4 months of age. Conditions such as Type 2 diabetes and obesity that promote cellular insulin resistance and contribute to the “metabolic syndrome” are generally known to aggravate the clinical severity of NAFLD/NASH, worsening its prognosis243031−/−-livers, little evidence exists of their simple hepatic steatosis progressing rapidly into NAFLD/NASH steatohepatitis. Apparently, CHIP−/−-mice only succumb to NAFLD/NASH much later in life ≈9–12 months. We posit that the early activation of the antisteatogenic adiponectin-LKB1-AMPK-FOXO-signaling axis effectively protects the CHIP−/−-liver from aggravated NAFLD/NASH-manifestations.This is all the more remarkable, given that insulin-signaling in CHIP−/−-mice relative to age-matched WT-controls  the canonical AMPK energy-sensing mechanism stemming from ROS-elicited oxidative inactivation of mitochondrial ATP-synthesis and potential ATF2-mediated mitochondrial disruption, with consequently increased cellular AMP/ATP ratiosnon-canonical” activation, wherein ROS trigger the oxidation and subsequent glutathionylation of two conserved AMPKα-subunit Cys-residues21−/−-livers would be its significant anti-steatogenic action via pSer79ACC2-mediated attenuation of malonyl-CoA production, thereby derepressing carnitine palmitoyltransferase 1 activity, and enhancing mitochondrial FA uptake and β-oxidationA significant additional contributor to AMPK-activation in CHIPpgc-1α, as well as hepatic cell surface adiponectin receptors (adipoR1/adipoR2)2021−/−-livers coupled with the increased EWAT adiponectin production would enhance hepatic adiponectin-sensitivity, thereby further stimulating the LKB1-AMPK-FOXO-signaling, and establishing a feed-forward mechanism to counteract the inherent oxidative stress through transcriptional activation of oxidative stress resistance genes196465−/−-livers: First, FOXO1, itself being a CHIP substrate2120−/−-livers . Thus, only after this concurrent salutary activation of the adiponectin-LKB1-AMPK-FOXO-signaling axis wanes  detection assay from Cayman , Oxyblot detection materials and EMSA gel shift assay from Millipore , Pathscan Intracellular Signaling array kit from Cell Signaling Technology , BSA protein assay, Pico ECL reagents, Nuclear and Cytoplasmic extraction reagents from Thermo Fisher . pcDNA3-CHIP, pcDNA3-CHIP-∆U-box, pcDNA3-CHIP-∆TPR were provided by the Patterson lab. pEBG-AMPKα1 was provided by Dr. Rob Onyenwoke . pCW45-AMPKα2 was obtained from DNA Resource Core of Harvard Medical School .Common cell culture media, supplements, culture plasticware, and commercial sources of protease inhibitors and DEX have been reported previously+/−- and female CHIP−/−-mice generated as described14IB) analyses of hepatocyte lysates . Mice were fed a standard laboratory chow-diet, given water ad libitum and maintained under a normal diurnal light cycle. All animal experiments were carried out strictly by protocols specifically approved by the UCSF/Institutional Animal Care and Use Committee (IACUC) and its care and use of laboratory animal guidelines. CHIP−/−-mice were maintained from birth to ≈12 months, as their median lifespan is <1 yearMale CHIP+/+-, CHIP−/−-, or CHIP+/−-livers were cultured as describedCells from CHIP2t-IP-products were assayed in the culture medium by a fluorescent immunoassay as per the manufacturer’s instructions. Levels of MDA, a byproduct of unsaturated FA-oxidation, were monitored in cell lysates as thiobarbituric acid-reactive adductsin situ with a HNE-specific antibodyFour commonly employed oxidative stress indicesCYP3A and CYP2E1 function was assessed by diagnostic assays of BFC 7-O-debenzylation and MFC 7-O-demethylation, respectively, as describedIB analysis against anti-phospho-serine/threonine antibody.Nuclear and cytoplasmic subfractions were prepared from cultured hepatocytes washed with chilled phosphate-buffered saline (PBS) for 10 min with nuclear/cytosolic extraction reagents (NER/CER)  according to the manufacturer’s instructions. Briefly, cells were mixed with ice-cold CER-I and then incubated on ice for 10 min. CER-II was then added and the cell mixture further incubated on ice for 1 min. Nuclei were harvested by centrifugation (100 × g), and the resulting supernatant was collected as the cytoplasmic extract. Nuclear pellet was suspended with ice-cold NER buffer, and nuclear protein was extracted at 4 °C for 1 h, sedimented at 16,000 × g for 10 min at 4 °C to obtain the supernatant (nuclear extract). Total protein (200 μg) from each subfraction was precleared with protein A/G-Sepharose  for 1 h and then immunoprecipitated with anti-FOXO1 or FOXO3 antibody (2 μg) on a rocking platform at 4 °C for 2 h, followed by the addition of protein A/G-Sepharose beads (20 μl) at 4 °C for 16 h as describedadipoQ was similarly analyzed with total RNA extracted using a combined TRIZOL  and RNeasy mini-kit protocol. Relative gene expression level was determined by normalizing its threshold cycle (Ct) to that of Gapdh Ct. Primers used are listed , treated with DNase , and reverse-transcribed with Accupower RT-PCR kit (Bioneer) for cDNA synthesis, in Power SYBR Green PCR Master Mix  with Agilent Mx3005P System. Adipose tissue e listed .http://rsbweb.nih.gov/ij/), using corresponding actin-loading controls for normalization.Upon harvesting, cultured hepatocytes were washed once with ice-cold PBS for 10 min and lysed in a cell-lysis buffer . The whole-cell lysates were clarified by centrifugation at 12,000 × g for 10 min. Protein concentrations were measured using the bicinchoninic acid (BCA) protein assay reagent (Pierce), 10 μg of proteins were resolved by 4–15% gradient SDS-PAGE, transferred to nitrocellulose membrane , blocked in 5% skim-milk for 1 h, and probed with primary antibodies for 16 h at 4 °C, washed with 1X Tris-buffered saline containing 0.1% Tween 20 for >5 times. Following incubation with anti-mouse IgG HRP-linked antibody or anti-rabbit IgG HRP-linked antibody, bound immunoglobulins were detected using enhanced chemiluminescence (Pierce). Antibodies used for this study and their commercial sources are provided . ImmunobMouse liver sections were fixed, stained, and subjected to light-microscopy and imaging by the UCSF Liver Center Pathology and Gladstone Institute, Histology and Light Microscopy Cores.Hepatic nuclear extracts were probed with a biotin-labeled oligonucleotide specific for the NF-κB-consensus sequence, as detailedp values <0.05 were considered statistically significant.Experiments were generally performed in triplicate. Data were compared by analysis of variance, and How to cite this article: Kim, S.-M. et al. Chip−/−-Mouse Liver: Adiponectin-AMPK-FOXO-Activation Overrides CYP2E1-Elicited JNK1-Activation, Delaying Onset of NASH: Therapeutic Implications. Sci. Rep.6, 29423; doi: 10.1038/srep29423 (2016).


Moreover, G-MDSC exo promoted the expansion of Tregs in vitro. Taken together, these results suggest that G-MDSC exo attenuate DSS-induced colitis through inhibiting Th1 cells proliferation and promoting Tregs expansion.Myeloid-derived suppressor cells (MDSC) have been described in inflammatory bowel disease (IBD), but their role in the disease remains controversial. We sought to define the effect of granulocytic MDSC-derived exosomes (G-MDSC exo) in dextran sulphate sodium (DSS)-induced murine colitis. G-MDSC exo-treated mice showed greater resistance to colitis, as reflected by lower disease activity index, decreased inflammatory cell infiltration damage. There was a decrease in the proportion of Th1 cells and an increase in the proportion of regulatory T cells (Tregs) in mesenteric lymph nodes (MLNs) from G-MDSC exo-treated colitis mice. Moreover, lower serum levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α were detected in G-MDSC exo-treated colitis mice. Interestingly, inhibition of arginase (Arg)-1 activity in G-MDSC exo partially abrogated the spontaneous improvement of colitis. In addition, G-MDSC exo could suppress CD4 The counts per minute (CPM) values of various wells were detected with an LS6500 multi-purpose scintillation counter .For escribed . Briefly+ T cells isolated from C57BL/6 mice splenocytes were cultured with anti-CD3 and anti-CD28 mAbs in the presence or absence of 5 ng/ml TGF-β in a 24-well plate for 72 h in complete RPMI medium (1.5 × 106 cells/well) with or without exo isolated from G-MDSC or neutrophils. The percentages of CD25+Foxp3+ cells in CD4+ T cells were determined by FCM after 72 h.CD4DTH was induced in mice by challenging the footpad of previously sensitized mice with OVA as described previously . In brieQ test using the SPSS version 16.0 for Windows . The data are presented as the mean ± SEM from at least three independent experiments. A p-value < 0.05 was considered statistically as significant.The statistical significance of differences between groups was analyzed by ANOVA and


Under ER stress, PKR-like ER-resident kinase (PERK) phosphorylates translation initiation factor eIF2α, resulting in repression of global protein synthesis and concomitant upregulation of the translation of specific mRNAs such as activating transcription factor 4 (ATF4). This PERK function is important for cell survival under ER stress and poor nutrient conditions. However, mechanisms of the PERK signaling pathway are not thoroughly understood. Here we identify transducin (beta)-like 2 (TBL2) as a novel PERK-binding protein. We found that TBL2 is an ER-localized type-I transmembrane protein and preferentially binds to the phosphorylated form of PERK, but not another eIF2α kinase GCN2 or ER-resident kinase IRE1, under ER stress. Immunoprecipitation analysis using various deletion mutants revealed that TBL2 interacts with PERK via the N-terminus proximal region and also associates with eIF2α via the WD40 domain. In addition, TBL2 knockdown can lead to impaired ATF4 induction under ER stress or poor nutrient conditions such as glucose and oxygen deprivation. Consistently, TBL2 knockdown rendered cells vulnerable to stresses similarly to PERK knockdown. Thus, TBL2 serves as a potential regulator of the PERK pathway. The unfolded protein response (UPR) is a survival stress response enabling the cell to cope with the accumulation of unfolded proteins in the endoplasmic reticulum (ER) causing ER stress. Three ER-membrane sensor proteins, PERK, activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1), play important roles in the UPR signaling PERK has been known to induce a response that represses general mRNA translation and promotes translation of a subset of mRNAs TBL2 gene is typically deleted. Patients with WBS suffer a developmental disorder caused by deletion of 26–28 genes at chromosome 7q11.23 TBL2 gene, are much less clear. The Tbl2 knockout mouse exhibited increased mean body weight, length, and change in bone metabolism TBL2 gene has been reported to associate with increased blood triglycerides, a lipidemia marker, although the effect of SNP on TBL2 function is unknown TBL2 is a ubiquitously expressed protein with a predicted transmembrane region, WD40 repeats, and a coiled coil domain Using mass spectrometry, we identified TBL2 as a novel PERK-interacting protein. Our experiments revealed that TBL2 is a type I ER transmembrane protein and preferentially associates with phospho-PERK. Importantly, TBL2 was involved in induction of ATF4 expression under stress conditions such as glucose/oxygen deprivation and the cell growth. Thus, our results indicate that TBL2 is a new player on the PERK signaling pathway.2-Deoxyglucose , histidinol (Sigma) and DTT  was dissolved in distilled, sterilized water. Tunicamycin  and thapsigargin  were dissolved in dimethyl sulfoxide. Hydrogen peroxide was purchased from WAKO. These compounds were added to culture medium, with the solvent being less than 0.5% of the medium’s volume. The following commercially available antibodies were used: rabbit anti-TBL2 and anti-ATF4 , anti-PERK, anti-eIF2 alpha , anti-phospho-PERK , anti-phospho-eIF2 alpha (Ser51), anti-calnexin , anti-KDEL for GRP78  anti-FLAG M2 (Sigma), and HRP- or FITC-conjugated anti-V5 (Invitrogen), HRP-conjugated anti-rabbit or mouse IgG .2. Glucose-free RPMI 1640 medium was obtained from Invitrogen  and supplemented with 10% heat-inactivated fetal bovine serum for experimental use. To create hypoxic conditions, cells were placed in a plastic box with Anaero Pack Kenki for Cell .We used following cell lines: Human fibrosarcoma HT1080 cells pFLAG-PERK plasmids were constructed by inserting full-length PERK into the p3XFLAG-CMV-14 or pFLAG-cmv 5c vector (Sigma) at the KpnI site. pFLAG-PERK-DN plasmid was constructed by inserting PERK lacking kinase domain into pFLAG-cmv 5c vector (Sigma) at the KpnI site. pFLAG-TBL2 WT or each mutant and pFLAG-IRE-1 were constructed by ligating each cDNA amplified by RT-PCR at the HindIII/NotI site into the pFlag-CMV-5c vector (Sigma). V5-tagged TBL2 was constructed by ligating cDNA amplified by RT-PCR into pcDNA3.1 TOPO V5/His (Invitrogen). pShooter pCMV/Myc/ER/GFP (Invitrogen), which produces GFP with the N-terminal ER signal peptide and the C-terminal ER retention signal sequence, was used as an ER marker. Transient transfections were performed using Lipofectamine 2000 (Invitrogen) or lipofectamine RNAi MAX (Invitrogen), according to the manufacturer’s protocol.5/well in a type I, collagen-coated, 6-well plate and were cultured overnight. The cells were transfected for 6 h with siRNA (10 nM), using the Lipofectamine RNAi MAX reagent according to the manufacturer’s protocol. Then, the cells were reseeded from 1 well to 3 wells. Two days after transfection, the cells were used for experimentation. Sequences are TBL2 #1∶5′- UCU UCU UGU AUU CCA CAU CUG UGU C −3′, 5′- GAC ACA GAU GUG GAA UAC AAG AAG A-3′; TBL2 #2∶5′- UCA UCU UGA AGA CAC GGA GGG UGU C −3′, 5′- GAC ACC CUC CGU GUC UUC AAG AUG A −3′; PERK #1∶5′- UUU ACU GUG AAG AAA CUC CAC UGC C-3′, 5′- GGC AGU GGA GUU UCU UCA CAG UAA A-3′; PERK #2∶5′- AAU ACC UCU GGU UUG CUA AGG CUG G-3′, 5′- CCA GCC UUA GCA AAC CAG AGG UAU U-3′.Stealth siRNAs against TBL2  or PERK  were purchased from Invitrogen. For transient transfection of siRNA, cells were seeded at a density of 4×10HEK293T cells were transfected with a pFLAG-PERK plasmid and the cell lysate was immunoprecipitated with a FLAG antibody. PERK-binding proteins were analyzed by direct nano-flow liquid chromatography/electrospray tandem mass spectrometry, as described earlier Immunoblot analysis was performed as described previously Immunoprecipitation was performed as described previously Cells on a poly-lysine-coated cover slip were fixed and permeabilized for 10 min in PBS-containing 4% paraformaldehyde and 0.1% Triton X-100. After blocking for 1 h in PBS with 10% BSA, the cells were incubated with primary ) and subsequent secondary antibodies (Alexa-fluor 488–conjugated anti-mouse Ig (1∶1000), or Alexa-fluor 568–conjugated anti-rabbit Ig (1∶1000) (Invitrogen)) in PBS with 1.5% BSA for 1 h at room temperature. The cover slips were mounted on microscope slides. Fluorescence images were obtained with an Olympus Fluoview 500 confocal microscope using 488-nm laser excitation for Alexa-488 or 543-nm for Alexa-568.Subcellular fractionation was modified according to a previously reported procedure Trypsin digestion assay was modified according to a previously reported procedure 5/well in a 6-well plate). After 48 h, cells were incubated for 5 min in Met-Cys-free DMEM supplemented with 2 mM glutamine, 10% dialyzed FBS. After pretreatment with thapsigargin for 20 min, the cells were labeled for 20 min using the 100 µCi/ml easy tag EXPRES 35S protein labeling mix  in the presence of thapsigargin. Then, each sample was boiled for 5 min in SDS buffer and subjected to SDS-PAGE. After gel drying, the incorporated [35S]Met/Cys was visualized with the Typhoon9410 .293 cells were transfected transiently with stealth siRNA  were purchased from Sigma-Aldrich. Stable shRNA-expressing 786-O cells were established according to the manufacturer’s protocol and were selected using puromycin.2-deprived conditions or under both. Immediately thereafter, cells were reseeded onto 12-well plates and incubated in normal culture medium for 3 days. Relative cell numbers after 3 days were measured using an MTT assay. To analyze viability of cells treated with thapsigargin, intracellular ATP level was measured by using a commercial kit (promega: Cell Titer-Glo Luminescent Cell Viability Assay). For cell proliferation assay, cells were reseeded to 6-well plate after incubation for 12 h under glucose- and/or O2-deprived conditions. The cell numbers at each time point (1–4 days) were measured automatically using a Beckman Coulter Counter .Control-, TBL2- and PERK-shRNA-expressing 786-O cells were incubated for 12 h under glucose- or OStatistical analysis was performed using student's t-test. We considered a P-value of <0.05 statistically significant.http://smart.embl.de/) suggested that TBL2 contained the N-terminal proximal transmembrane region (TM: 9-31aa), the WD40, and the C-terminal coiled coil domains (http://www.cbs.dtu.dk/services/TMHMM/) predicted one transmembrane domain corresponding to 9-31aa  for 1 h. The cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with the indicated antibody.(TIFF)Click here for additional data file.Figure S2TBL2 knockdown has little effects on XBP1 splicing and GRP78 induction. (A) Analysis of XBP1 transcript in TBL2 knockdown cells. The cells were transiently transfected with non-silencing siRNA, TBL2 siRNA or PERK siRNA. After 48 h, the cells were treated with 300 nM thapsigargin for the indicated times. To detect XBP1 mRNA splicing valiant, we amplified each cDNA using a specific primer pair that produces amplicon sizes of 441 bp (unspliced form) and 415 bp (spliced form). (B) The cells were transiently transfected with non-silencing siRNA, TBL2 siRNA or PERK siRNA. After 48 h, the cells were treated with 300 nM thapsigargin for the indicated times. Each lysate sample was subjected to immunoblot with the indicated antibody.(TIFF)Click here for additional data file.


Immunotherapy for allergic rhinitis with/without conjunctivitis (AR/C) may exhibit different efficacy characteristics in patients with multiple allergen sensitizations than monosensitized patients. It has been considered that monosensitized patients may benefit more from immunotherapy than polysensitized patients. Evidence from randomized, blinded, placebo-controlled trials of Timothy grass sublingual immunotherapy tablet (SLIT-T) MK-7243 (Merck/ALK-Abelló) indicates that treatment in mono- and polysensitized subjects is equally effective.Ambrosia artemisiifolia; Merck/ALK-Abelló). Pooled data from 2 randomized placebo-controlled trials investigating MK-3641 (6 and 12 Amb a 1-U doses) were used. The primary efficacy outcome was the total combined score (TCS=symptom+medication scores) during the 15-day peak season.A prospective efficacy analysis was performed between monosensitized and polysensitized subjects treated with the short-ragweed SLIT-T MK-3641 (P<0.001 for both). Differences versus placebo in the monosensitized MK-3641 pool (n = 175) were 15%  and 19%  for 6 and 12 Amb a 1-U, respectively. In the polysensitized MK-3641 pool (n = 784) difference versus placebo were 21%  and 27%  for 6 and 12 Amb a 1-U, respectively.Differences versus placebo for the MK-3641 6 and 12 Amb a 1-U pooled groups (mono- and polysensitized subjects combined) for the peak season TCS were 20%  and 23% , respectively (In the whole study population, treatment with MK-3641 6 and 12 Amb a 1-U for ragweed-induced AR/C was superior to placebo. Although the sample size for the 2 subpopulations was not balanced and data must be interpreted cautiously, it appears that the treatment effect is similar in the mono- and polysensitized subpopulations, with a numerical trend of a greater treatment effect in polysensitized subjects.ClinicalTrials.gov Identifiers: NCT00783198; NCT00770315