PMC:4786408 / 28250-38024 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4786408","sourcedb":"PMC","sourceid":"4786408","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4786408","text":"Materials and methods\n\nSubjects\nStudy protocols including written informed consents have been approved by the University of Michigan Institutional Review Board and the Boğaziçi University Institutional Review Board for Research with Human Participants. Two Turkish brothers, ages 5 and 7 in 2004, presented with ataxia and developmental delay, as previously described (Yapici and Eraksoy, 2005). Parents were initially reported to be unrelated, but recently suggested they might be remotely related. Both patients were delayed in walking, had truncal ataxia and dysmetria, nystagmus, and lower IQ (68 and 70). MRI revealed cerebellar hypoplasia. Follow-up examinations showed no progression of symptoms.\n\nGenetic analysis\nDNA was isolated from peripheral whole blood using the Qiagen (Germantown, MD) Gentra Puregene isolation kit. Linkage analysis was performed using the genotype data generated with Illumina HumanOmniExpress-24 chip for the mother and the four sibs. The Allegro module (Gudbjartsson et al., 2000) of easyLINKAGE software was used, assuming autosomal recessive inheritance and parents as third cousins. No deletion or duplications common to just the two affected brothers were detected using cnvPartition plug-in in Illumina Genome Studio v.1.02 software.\nExome sequencing was performed independently twice on one subject. Capture for whole exome sequencing was performed with NimbleGen SeqCap EZ Exome Library v1.0 kit (Roche, Indianapolis, IN). Captured regions were sequenced with Illumina HiSeq2000 instruments. Variants were filtered to remove common variants based on 1000 Genomes, Exome Sequencing Project, and Exome Aggregation Consortium databases, variants outside of identified linkage regions, variants not expected to change protein coding, and variants not following a recessive model of inheritance (Exome Aggregation Consortium (ExAC), 2015; Genomes Project Consortium et al., 2012; NHLBI Go Exome Sequencing Project, 2015).\nPCR followed by Sanger sequencing was performed to validate the variant identified through exome sequencing and test for segregation within the family. The variant of interest was further examined in two separate collections of a total of 500 Turkish samples, and found absent.\n\nLymphoblast cell culture\nLymphoblastoid cell lines (LCL) of both subjects were generated from heparinized whole blood samples and cultured as described (Doyle, 1990). As they are made in house and cultured briefly, mycoplasma contamination risk is minimized.\n\nProtein co-expression and affinity purification from insect cells\nWe used a baculovirus/insect cell expression system to examine formation of the human ATG12–ATG5 conjugate in a heterologous system described previously (Qiu et al., 2013). Hi5 insect cells (Invitrogen, Carlsbad, CA) were infected with baculoviruses expressing human ATG7, ATG10, a GST-tagged version of ATG12 (residues 53–140, corresponding to the ubiquitin-like domain) and a His-tagged version of either ATG5WT or ATG5E122D. Three days post infection, lysates were subjected to glutathione affinity chromatography, and the GST-ATG12–His-ATG5 conjugate was detected by SDS-PAGE followed by Coomassie Blue staining. To confirm that the baculoviruses produce protein, Hi5 cells were coinfected with baculoviruses expressing the His-tagged WT and mutant versions of ATG5 and the N-terminal domain of ATG16L1 (residues 1–69, as an MBP fusion). At three days post infection, lysates were subjected to nickel affinity purification. The ATG5-ATG16L1 complex formation was detected by SDS-PAGE and Coomassie Blue staining.\n\nCrystallization and structure determination\nThe complex containing ATG5E122D and the N-terminal domain of ATG16L1 (residues 1–69) was expressed in Hi5 insect cells, and purified by nickel affinity, ion exchange, and size exclusion chromatography into a final buffer of 20 mM Tris, pH 8.5, 50 mM NaCl, 10 mM DTT. The complex was concentrated to 18.5 mg/ml, aliquoted, flash-frozen and stored at -80°C until further use. Crystals were grown by the hanging drop vapor diffusion method by mixing purified protein 1:1 with reservoir solutions of 37.5 mM MES, pH 5.2–5.8, 0.2 M sodium tartrate, and 11–13% polyethylene glycol 3350. Final crystals were obtained by micro-seeding with reservoir solution of 40 mM MES, pH 5.5, 0.2 M sodium tartrate, 8.5% PEG3350, 10 mM DTT. Crystals were cryoprotected in reservoir solution supplemented with 25% xylitol, and flash frozen in liquid nitrogen prior to data collection. Diffraction data were processed with XDS. The structure was determined by molecular replacement using Phaser (McCoy et al., 2007) with the structure of the WT ATG5-ATG16L1 (1–69) (PDB: 4TQ0) complex as a search model (Kim et al., 2015a). Model construction and rebuilding were performed using Coot (Emsley et al., 2010). The structure was refined using Phenix (Adams et al., 2010). Diffraction data and refinement statistics are provided in Table 1.\n\nImmunoblotting\nCells or tissues were lysed in cell lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1% Triton X-100) or RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40, 0.1% SDS) containing protease inhibitor cocktail (Roche). After being clarified with centrifugation, lysates were boiled in SDS sample buffer, separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes and probed with the indicated antibodies. ATG5 (12994), LC3 (3868) and SQSTM1/p62 (5114) antibodies were purchased from Cell Signaling Technology. Hemagglutinin (HA, 3F10) antibody was from Roche. Actin (JLA20) and tubulin (T5168) antibodies were from Developmental Studies Hybridoma Bank and Sigma, respectively. Ref(2)P antibody was previously described (Pircs et al., 2012).\n\nHEK293 cell culture\nWild-type human ATG5-coding sequence was from Addgene #24922 (deposited by Dr. Toren Finkel) (Lee et al., 2008). The E122D mutation was introduced into ATG5 by PCR-based site-directed mutagenesis. ATG5WT and ATG5E122D were cloned into the plasmid pLU-CMV-Flag. The HA-ATG12-expressing plasmid was from Addgene #22950 (deposited by Dr. Noboru Mizushima) (Mizushima et al., 1998b). HEK293 cells (the 293 A substrain from Invitrogen, tested negative for mycoplasma by PCR) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 10% fetal bovine serum (FBS) and penicillin/streptomycin at 37°C in 5% CO2. For transient expression of proteins, HEK293 cells were transfected with purified plasmid constructs and polyethylenimine (PEI, Sigma) as previously described (Horbinski et al., 2001). Cells were harvested 24 hr after transfection for immunoblot analyses.\n\nYeast model\nSaccharomyces cerevisiae strain WLY176 was used to generate an ATG5 knockout strain (atg5∆) as previously described (Gueldener et al., 2002). The MKO strain YCY137 (SEY6210 atg1∆, 2∆, 4∆, 5∆, 6∆, 7∆, 8∆, 9∆, 10∆, 11∆, 12∆, 13∆, 14∆, 16∆, 17∆, 18∆, 19∆, 20∆, 21∆, 23∆, 24∆, 27∆, 29∆) (Cao et al., 2008), was used for in vivo reconstitution of Atg8 conjugation. Site-directed mutagenesis was performed to generate ATG5 amplicons with the E141D mutation as previously described (Liu and Naismith, 2008). A pRS406 empty plasmid was digested with Spel and SalI, and then ligated with a DNA fragment encoding either wild-type or mutant Atg5-PA. atg5∆ was transformed with an empty pRS406 vector, or plasmids encoding Atg5-PA WT or Atg5-PA E141D. Wild-type WLY176 colonies were transformed with empty pRS406 vector as a control. Colonies were grown on SMD-URA medium and starved in nitrogen-deficient medium. Pho8Δ60 and western blot analyses were performed as described previously (Noda and Klionsky, 2008; Shintani and Klionsky, 2004). Quantification was performed using ImageJ software. The pATG8∆R-ATG7-ATG10(414), pATG5(WT)-HA-ATG12(416) and pATG5(WT)-HA-ATG12-ATG16(416) plasmids were described previously (Cao et al., 2008). The pATG5(E141D)-HA-ATG12(416) and pATG5(E141D)-HA-ATG12-ATG16(416) plasmids were made by site-directed mutagenesis based on the wild-type constructs.\n\nDrosophila genetics\nAtg5-null Drosophila flies (Atg55cc5) were generated by CRISPR-Cas9-mediated genome editing, using a double gRNA approach, both targeting the same gene, as described (Kondo and Ueda, 2013). The Atg55cc5 mutant was recovered by screening viable candidate lines for accumulation of the specific autophagy cargo Ref(2)P using western blots, followed by PCR and sequencing. Atg55cc5 mutants have a deletion in X:7,322,242–7,323,717 residues (Drosophila melanogaster R6.06), which deletes five out of six exons of the Atg5 gene, eliminating more than 85% of protein-coding sequences including the translation start site (Figure 6A). The PhiC31 integrase-mediated site-specific transformation method was used to express human ATG5WT and ATG5E122D from an identical genomic locus (Bateman et al., 2006; Bischof et al., 2007; Venken et al., 2006). In brief, flag-tagged ATG5WT and ATG5E122D were cloned into a pUAST-attB vector (Bischof et al., 2007) and fully sequenced. pUAST-attB-ATG5WT and pUAST-attB-ATG5E122D were microinjected into y1 M{vas-int.Dm}ZH-2A w*; M{3xP3-RFP.attP}ZH-51D flies and stable transformants were isolated by the presence of the mini-white+ marker (Figure 6B). The UAS-ATG5WT or UAS-ATG5E122D transgenes were crossed with a double balancer strain (Bl/CyO; TM2/TM6B) and then with +/CyO; Tub-Gal4/TM2 to be constructed as stable Tub\u003eATG5 lines (UAS-ATG5/UAS-ATG5; Tub-Gal4/TM6B). The Tub\u003eATG5 male flies were crossed with Atg55cc5/FM7 female flies to generate Atg5-null flies expressing human ATG5 transgenes. Climbing assays and TUNEL staining were performed as previously described (Kim et al., 2015b). 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