Schizosaccharomyces pombe. We found that deletion of ubp8, encoding the Spt-Ada-Gcn5-Acetyl transferase (SAGA) complex associated DUB, suppressed temperature-sensitive phenotypes of APC/C mutants cut9-665, lid1-6, cut4-533, and slp1-362. Our analysis revealed that Ubp8 antagonizes APC/C function in a mechanism independent of the spindle assembly checkpoint and proteasome activity. Notably, suppression of APC/C mutants was linked to loss of Ubp8 catalytic activity and required histone H2B ubiquitination. On the basis of these data, we conclude that Ubp8 antagonizes APC/C function indirectly by modulating H2B ubiquitination status.Ubiquitination and deubiquitination of proteins are reciprocal events involved in many cellular processes, including the cell cycle. During mitosis, the metaphase to anaphase transition is regulated by the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C). Although the E3 ubiquitin ligase function of the APC/C has been well characterized, it is not clear whether deubiquitinating enzymes (DUBs) play a role in reversing APC/C substrate ubiquitination. Here we performed a genetic screen to determine what DUB, if any, antagonizes the function of the APC/C in the fission yeast  The purpose of cell division is to accurately replicate the genetic material of a dividing cell and evenly distribute it between mother and daughter cells. Precise degradation of critical cell-cycle regulators by the ubiquitin proteasome system (UPS) ensures unidirectionality of cell-cycle progression  E3 ubiquitin ligase. The APC/C carries out its mitotic function by promoting the degradation of securin and cyclin B through the UPS (reviewed in In Ubiquitin is removed from proteins by deubiquitinating enzymes (DUBs)   were grown in yeast extract (YE) medium -tagged ubiquitin was achieved by growth in the absence of thiamine for 18-22 hr, whereas repression was achieved by growth in the presence of 5 μg/mL of thiamine. htb1-Flag and htb1-K119R-Flag strains were a gift from Dr. Jason Tanny (McGill University).ubp8 was tagged after the stop codon of its endogenous open reading frame (ORF) with sequences encoding the Kanamycin resistance gene by a polymerase chain reaction (PCR)-mediated strategy (strategy . Proper ubp8 gene including 500 bp upstream and downstream of the open reading frame was amplified by PCR and ligated into a PCR-Blunt vector (Life Technologies) and then subcloned into a pIRT2 vector (ubp8-C154S H387A was created by mutagenizing a pIRT2-plasmid containing +ubp8 using a QuikChange site-directed mutagenesis kit (Agilent Technologies). For ubp8 gene replacements, a haploid ubp8::ura4+ strain was transformed with a linear ubp8 gene fragment (digested from pIRT2- ubp8-C154S H387A plasmid) using standard lithium acetate transformations. Integrants were selected based on resistance to 5-FOA and validated by colony PCR using primers homologous to endogenous sequences that flank the genomic clone within pIRT2 in combination with those within the ORF. All constructs were sequenced to ensure their accuracy.All plasmids were generated by standard molecular biology techniques. The Cell pellets were frozen in a dry ice/ethanol bath and lysed by bead disruption in NP-40 lysis buffer under denaturing sodium dodecyl sulfate (SDS) lysis conditions as previously described (wildtype and ubp8∆ strains utilizing the thiamine repressible nmt1 promoter in pREP1 (2+-NTA agarose beads (QIAGEN) for 3–4 hr at room temperature. After incubation, beads were washed 4 times with buffer 3  and eluted in buffer 4 . The pH of the eluate was adjusted to 8 and streptavidin ultra-link resin (Pierce) was added and incubated overnight at room temperature. After the overnight incubation, streptavidin beads were washed 4 times with buffer 6  and once with buffer 7 . Purifications were performed in duplicate and purified proteins were subjected to mass spectrometric (MS) analysis.HBH-tagged ubiquitin was overexpressed in in pREP1 . Ubiquit2 were subjected to MS3 fragmentation. MS data analysis was done as previously described . Proteins were reduced with 3mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride), alkylated with 10mM iodoacetamide, and digested with trypsin . Two-dimensional liquid chromatography–mass spectrometry 2D-LC-MS/MS analysis was performed in the following manner. Peptides were loaded onto 26-cm columns with a bomb pressure cell and then separated and analyzed by three-phase multidimensional protein identification technology on a Velos LTQ mass spectrometer  coupled to a nanoHPLC . The NanoAcquity autosampler was used for the 12 salt elution steps, each with 2 μL of ammonium acetate. Each injection was followed by elution of peptides with a 0–40% acetonitrile gradient (60 min) except the first and last injections, in which a 0–90% acetonitrile gradient was used. One full precursor MS scan (400–2000 mass-to-charge ratio) and five tandem MS scans of the most abundant ions detected in the precursor MS scan under dynamic exclusion was performed. Ions with a neutral loss of 98 Da (singly charged), 49 Da (doubly charged), or 32.7 Da (triply charged) from the parent ions during MSS. pombe, the deletion of that DUB should suppress a mutant defective in APC/C activity. Thus, we crossed each of the 19 nonessential DUB deletion mutants and a temperature-sensitive mutant of rpn11  to cut9-665, an APC/C mutant (cut9-665 (ubp8∆ suppressed cut9-665 at its semipermissive temperature (32°)  (We surmised that if a DUB antagonizes APC/C function in C mutant . Only thcut9-665 . In a sere (32°) . cut9-66B rpn11) . These Dlid1+ and cut4+ encode other components of the APC/C whereas slp1+ encodes the APC/C coactivator termed Cdc20p in other organisms , indicating that suppression of APC/C mutants is likely not mediated by enhanced proteasome function.The APC/C is responsible for ubiquitinating critical cell-cycle regulatory proteins, targeting them for their subsequent degradation by the 26S proteasome  led to increased levels of ubiquitinated H2B in vivo . Histone H2B, an important substrate of Ubp8 .To better understand how Ubp8 inhibits APC/C function, we identified putative substrates of the SAGA DUB module by semiquantitative comparison of all ubiquitinated proteins (the ubiquitinome) in wild-type cells relative to that of ubp8∆ to suppress APC/C mutants required histone H2B ubiquitination. BRE1-like Brl1 and small histone ubiquitination factor Shf1, components of the HULC ubiquitin ligase complex, are required in S. pombe for H2B ubiquitination (brl1 and shf1 (brl1∆ and shf1∆) abolished ubiquitination of histone H2B as did a lysine to arginine mutant of histone H2B in which the ubiquitin acceptor site is lost (htb1-K119R)  . ubp8∆ nbolished . Furthercut9-665 . Togetheubp8∆, reduced the requirement for APC/C function in S. pombe. Our data support a model wherein Ubp8 antagonizes APC/C indirectly via modulating the ubiquitination status of H2B.In this work we found that of all DUB single deletions, only one, ubp14∆ and pad1-1, mutations in the conserved DUBs Ubp14 and Rpn11, respectively, showed negative genetic interactions with APC/C mutants, indicating that these DUBs are important for APC/C function. Ubp14 cleaves free ubiquitin chains to liberate monomeric ubiquitin, thereby replenishing the ubiquitin pool  mutants have compromised ubiquitin-dependent proteasome function and arrest at metaphase at the restrictive temperature (ubp14∆ and pad1-1 (rpn11-ts) mutants, we predict that the degradation of APC/C substrates is compromised, further exacerbating the cut9-665 temperature sensitive phenotype.In our genetic approach to identify DUB(s) that antagonize APC/C function, we found that S. cerevisiae, each subunit of the Ubp8-Sgf11-Sus1-Sgf73 complex makes extensive contact with all other subunits and all four components are required for DUB activity in vitro and in vivo . In contrast, in Drosophila melanogaster and in human cells, the presence of Ataxin-7 (Sgf73) is not necessary for DUB activity, and, instead, loss of Ataxin-7 results in increased deubiquitination and reduced levels of H2B ubiquitination  . However, more complicated mechanisms of influencing the requirement for APC/C function through an effect of H2B ubiquitination on centromeric chromatin structure cannot be ruled out.A second possibility for the reduced requirement of APC/C function when H2B ubiquitination levels are raised in S. cerevisiae (A third possibility is that H2B ubiquitination regulates a downstream signaling event that modulates APC/C activity. For example, H2B ubiquitination is required for methylation of the Dam1 kinetochore component (revisiae . Thus, iBecause of its essential roles in chromosome segregation and mitotic progression, the APC/C has become a therapeutic target for the treatment of multiple neoplastic diseases (


Arabidopsis, along with HPLC/MS analysis, the present study shows that two novel cytochrome P450 (CYP450) enzymes, CYP76AD6 and CYP76AD5, and the previously described CYP76AD1 can perform this initial step. Co-expressing these CYP450s with DOPA 4,5-dioxygenase in yeast, and overexpression of these CYP450s in yellow beets show that CYP76AD1 efficiently uses L-DOPA leading to red betacyanins while CYP76AD6 and CYP76AD5 lack this activity. Furthermore, CYP76AD1 can complement yellow beetroots to red while CYP76AD6 and CYP76AD5 cannot. Therefore CYP76AD1 uniquely performs the beet R locus function and beets appear to be genetically redundant for tyrosine hydroxylation. These new functional data and ancestral character state reconstructions indicate that tyrosine hydroxylation alone was the most likely ancestral function of the CYP76AD alpha and beta groups and the ability to convert L-DOPA to cyclo-DOPA evolved later in the alpha group.Yellow and red-violet betalain plant pigments are restricted to several families in the order Caryophyllales, where betacyanins play analogous biological roles to anthocyanins. The initial step in betalain biosynthesis is the hydroxylation of tyrosine to form L-DOPA. Using gene expression experiments in beets, yeast, and  Betalains are water-soluble nitrogenous pigments classified as the red-violet betacyanins and yellow betaxanthins. These pigments variously accumulate in flowers, fruits, and vegetative tissues of plants in most core families of the Caryophyllales order, and where they exist, betalains exclusively replace anthocyanins. The betacyanins play important and analogous roles to anthocyanins in attracting pollinators and seed dispersers. While the structural and regulatory genes of anthocyanin biosynthesis are well known, betalain biosynthesis has been poorly understood at the molecular level creating a phylogenetic mystery surrounding their origin(s).Beta vulgaris (beet) as BvCYP76AD1 [Much of the biosynthetic pathway involved in betalain biosynthesis has recently been described , yet theCYP76AD1 , which iPhytolacca americana; Phytolaccaceae [Portulaca grandiflora (Portulacaceae), forming L-DOPA from tyrosine and cyclo-DOPA from L-DOPA in vitro. Mushroom tyrosinases have been shown to perform the tyrosine hydroxylation in vitro and in transgenic plant tissues [in vivo [Investigators have proposed the hydroxylation of L-tyrosine to L-DOPA was performed by the monophenolase activity of a tyrosinase (or a polyphenoloxidase-PPO) ,8. Thereaccaceae , and PPOaccaceae  and red accaceae . A tyrosaccaceae  activitiaccaceae  have bee tissues . Even th[in vivo –15. Thus[in vivo . Poltura[in vivo  subsequeCYP76AD5 and CYP76AD6) were identified with mRNA levels positively correlated with high betalain levels. These two genes are in the beta clade of CYP76AD genes described by Brockington et al. [CYP76AD1 is in the alpha clade. The newly identified CYP76AD5 and CYP76AD6 and the previously characterized CYP76AD1 [Mirabilis jalapa (Nyctaginaceae), have the same activities as alpha and beta clade members from beets (Amaranthaceae). The maintenance of both alpha and beta CYP76AD proteins would allow plants to differentially regulate these genes to produce different ratios of yellow and red pigment, thus allowing yellow, orange, red, or patterned pigmentation.Here, the search for the genes/enzymes responsible for tyrosine hydroxylation in the beet betalain biosynthetic pathway is described. Two novel beet cytochrome CYP450 genes , CYP76AD6 . These clones were recombined into the plant TDNA vector pB7WG2 [CYP76AD5, and CYP76AD6 coding regions in plants. These plasmids were transformed into Agrobacterium rhizogenes ARqua1 [Agrobacterium tumefaciens GV3101 [Previous work reported the identification and cloning of  R locus ,18. CYP7r pB7WG2 . This crs ARqua1  for beets GV3101  for ArabPortulaca grandiflora DODA cDNA (PgDODA) into pDONRZeo and subsequently into the yeast vectors, pVV214 or pVV200 [A. tumefaciens GV3101 [Cloning of the r pVV200  has beenr pVV200  to make CYP76AD3 and CYP76AD15 were amplified from 7-day-old red 4 o’clock seedling cDNA using the following primers: CYP76AD3StartF 5’ATGGATTTCTTAACCCTTGTCATG3’; CYP76AD3StopR 5’TCAATATTTAATAAGAGGAATAATCTC3’; CYP76AD15StartF 5’ACGTTCAAGCATAGACTAACC3’; CYP76AD15StopR 5’TGATTATCTCCCAACCATCG3’. Gateway recombination sequences were included in all primers but are not shown. The products were recombined into pDONR222 (Invitrogen) and sequenced: CYP76AD3  CYP76AD15 . These were recombined into pVV214 for yeast expression.The cDNA sequences for A. rhizogenes strains were placed into a 1 ml tuberculin syringe. A 30-gauge needle was used to make wounds on the hypocotyls of 10-day-old seedlings, injecting the A. rhizogenes culture into the wounds. Inoculated seedlings were maintained on MS media with 3% sucrose for 2 days and then transferred to the same media with Timentin 200mg/L to kill the bacteria. Plates were incubated at 22°C in continuous light. “Hairy roots” emerged from wound sites on the hypocotyl in about 2–3 weeks. Roots were excised from the wound site and maintained on MS with 3% sucrose and 200 mg/L Timentin. At least ten independent transgenic lines with consistent phenotype were produced for each construct.Seeds of yellow beet cultivar Golden Globe (from W. Atlee Burpee & Co.) were sterilized and germinated on MS media with 3% sucrose in petri plates. Overnight liquid LB cultures of the Arabidopsis thaliana mutant ttg1-1 in the Ler ecotype  was used for all experiments because it does not produce anthocyanin pigments. Arabidopsis plants were transformed by vacuum infiltration with the described constructs in A. tumefaciens GV3101 [s GV3101 .CYP76AD1, CYP76AD5, or CYP76AD6. Total RNA was extracted using Qiagen plant RNeasy mini kit and used in 20 μL reverse transcriptase reactions using 1 μg RNA and 1 μg oligo-dT. qRT-PCR was performed as described [For gene expression analysis, tissue was collected from 7-day-old untransformed hypocotyls of the beet cultivars Bull’s Blood  and Albina Vereduna  or of transformed hairy roots generated on Golden Globe by overexpressing escribed . Four teCYP76AD1, CYP76AD6, CYP76AD5, CYP76AD3, and CYP76AD15 in yeast expression vector pVV200 [CYP76AD1; pVV200/CYP76AD5; pVV200CYP76AD6; and pVV200/empty) were grown overnight in minimal medium with galactose and supplemented with 100 mg/L leucine, 20 mg/L histidine, and 40 mg/L adenine. The cultures were pelleted by centrifugation. The yeast were resuspended to an OD600 of 1.1 in 3 mL of above media with 10 mM tyrosine and 25 mM ascorbic acid.The cDNAs of r pVV200 , and pVVr pVV200 . The WATPgDODA cloned into pVV214 [CYP76AD1 + pVV214/PgDODA; pVV200/CYP76AD6 + pVV214/PgDODA; pVV200/CYP76AD5 + pVV214/PgDODA; pVV200/CYP76AD3 + pVV214/PgDODA; pVV200/CYP76AD15 + pVV214/PgDODA; and pVV200/empty vector + pVV214/PgDODA. These yeast co-expression cultures were also grown overnight in minimal medium  and resuspended in 3 mL as above with three separate substrate feedings: 10 mM L-DOPA + 25 mM ascorbic acid; 10 mM tyrosine + 25 mM ascorbic acid; and no enzyme substrate + 25 mM ascorbic acid, for each independent strain. Cultures were grown for 24 hrs after feeding and pelleted by centrifugation and 500 μL of the supernatant was retained for pigment analysis as described below in the pigment chemical analysis section.The WAT11 yeast strain was also co-transformed with the above vector constructs and o pVV214  to make The preparation of pigment standards for High Performance Liquid Chromatography / Mass Spectrometry (HPLC/MS) analysis was performed by preparative Thin Layer Chromatography (TLC) from red and yellow beet extracts as described .CYP76AD1, CYP76AD5, or CYP76AD6 in beet cultivar Golden Globe, were filtered through a 0.2 μm membrane filter. The filter residue was rinsed with 100% methanol to elute the pigment completely. Eluted pigment was concentrated by vacuum at 30°C and resuspended in 100 μL of 0.1% ascorbic acid. Resuspended pigments were analyzed by HPLC/MS.The supernatants from the yeast feeding assays, or plant pigment extracts from transgenic hairy roots overexpressing 0 C, drying gas flow was 12 L/min, nebulizer pressure was 50 psig, VCap was 5000 V, fragmentor voltage was 200 V, the skimmer was set to 65 V. The mass spec data was acquired scanning the m/z range from 100 m/z to 3200 m/z.Five microliter aliquots of extracts were injected into an Agilent 1260 Infinity HPLC system interfaced with an Agilent 6530 Accurate-Mass QTOF mass spectrometer. The samples were separated on a ZORBAX Eclipse XDB-C18 column  using a two-stage LC program. The first stage of the program was a 2 minute isocratic run with a mobile phase of 1% formic acid in water at a flow rate of 0.1 mL/min. The second stage of the program, lasting 6 minutes, included a flow rate gradient from 0.1 mL/min to 0.5 mL/min and a mobile phase gradient of 0–33% B, where mobile phase A is 1% formic acid in water and mobile phase B is 0.1% formic acid in acetonitrile. Mass spec detection of the eluent was performed using positive mode electrospray ionization. Gas temp was 350The exact mass of L-DOPA is 197.188 g/mol. The observed mass for L-DOPA in http://www.ebi.ac.uk/Tools/msa/clustalo/). The alignment was analyzed for residues that appeared to diverge according to the phylogenetic clades (highlighted in http://weblogo.berkeley.edu/logo.cgi). The number for each residue corresponds to its position in CYP76AD1 and the height of the residue is proportional to its appearance within the clade members.The sequences used to create the phylogeny  were aliBvCYP76AD1, BvCYP76AD5, and BvCYP76AD6 were each submitted to BLAST searches at NCBI and OneKP to identify orthologs from across the Caryophyllales and beyond. The resulting set of sequences and selected outgroup sequences (members of two other CYP76 subfamilies) were aligned using the MUSCLE alignment tool in GENEIOUS [2 [The cDNA sequences of GENEIOUS  and editEIOUS [2  to deterEIOUS [2  under GT1.3.1 [R9 [Ancestral amino acid sequences were generated for all internal nodes of the CYP76 gene tree using CODEML as implemented in PAMLX version 1.3.1 . Input f.3.1 [R9 . New ancTo identify betalain pathway gene/s responsible for tyrosine hydroxylation, RNAseq was performed on three white beet cultivars—Blankoma, Albina Vereduna, and sugar beet cv. C869. Red beets contain a dominant Y betalain regulatory locus allele ,30 whileCYP76AD1: BvCYP76AD5 and BvCYP76AD6  and CYP76AD1, are down-regulated many fold in all three white beet varieties: BvDODA1 is down 71, 207, 57 fold while CYP76AD1 is down 19, 22, 26 fold in Albina Vereduna, Blankoma, and C869, respectively.The reads from the RNAseq data of the white beet varieties were mapped onto a red beet reference sequence database  and geneCYP76AD6 , S1 Fig.CYP76AD1, CYP76AD5 and CYP76AD6 showed that these genes were expressed at 7%, 2%, and 8% respectively, in Albina Vereduna white beets vs. red beet cultivar Bull’s Blood  to confirm the expression differences observed in the RNAseq data. qRT-PCR of ’s Blood .PPO  was also identified in the RNAseq database and in the sugar beet genome [BvPPO was 2.1 fold higher in Albina Vereduna and 3.8 fold higher in sugar beet C869 than in the dark red cultivar Bull’s Blood. This lack of correlation between BvPPO expression and betalain production does not support a role for this gene in betalain biosynthesis.A single t genome  and its CYP76AD5 and CYP76AD6 were amplified from red beet seedlings. CYP76AD5 and CYP76AD6 have open reading frames of 1506 bp and 1500 bp respectively; the nucleotide identity is 81% while the amino acid identity is 85%. The amino acid identity between CYP76AD1 and CYP76AD6 is 72% while the identity between CYP76AD1 and CYP76AD5 is 69%.cDNAs for CYP76AD1, which lies at the R locus. Ectopic expression of CYP76AD1 in Golden Globe complements it from yellow to red. To test whether CYP76AD5 or CYP76AD6 can also perform step 3, these genes were overexpressed in Golden Globe tissue via Agrobacterium rhizogenes and pigment phenotypes were observed in transformed hairy roots. Overexpression of CYP76AD1 in yellow beetroots complemented them to red (CYP76AD5 (CYP76AD6 (not shown) in yellow beets resulted in yellow, non-complemented roots.Previous work showed that CYP76AD1 performs step 3  of the bm to red . OverexpCYP76AD5  and CYP7qRT-PCR was performed to verify that the respective genes were overexpressed in all hairy root cultures . IncreasCYP76AD1, CYP76AD6, and CYP76AD5 were also expressed in yeast to test for tyrosine hydroxylation activity by detecting L-DOPA formation after growing in media containing 10 mM tyrosine ; PgDODA + CYP76AD6; PgDODA + CYP76AD5 produced visible yellow to orange pigmentation; while PgDODA + CYP76AD1 produced red pigmentation. Of the cultures fed tyrosine, PgDODA + empty vector produced no color; PgDODA + CYP76AD6; PgDODA + CYP76AD5 produced yellow to orange pigmentation; while PgDODA + CYP76AD1 produced red pigmentation. In the absence of added substrate feeding all four cultures produced colors similar to tyrosine feeding but with lower intensity. Pigments produced without supplemental feeding were undoubtedly through the conversion of native yeast-produced tyrosine to betalain pigments.The CYP450s were also co-expressed in yeast with the DODA gene (step 2) from ormation . These wormation ; or no sPgDODA + CYP76AD1 as previously reported [PgDODA + CYP76AD6 and PgDODA + CYP76AD5 at about 27 and 100 fold less intensity, respectively  was detected at high intensities in both PgDODA + CYP76AD6 and PgDODA + CYP76AD5 samples. The tyrosine-bx level was very low in PgDODA + CYP76AD1 and absent in PgDODA + empty vector  as a representative of yellow betaxanthins . Betanidreported  and inteectively . These dectively . Betanidy vector .Arabidopsis thaliana. Expression of 35S::PgDODA alone results in yellow betaxanthin production only when the plants are fed L-DOPA (data not shown), the same as seen in yeast, and the same that has been previously demonstrated in Arabidopsis [While expression in yeast clearly indicates the activities of the divergent beet CYP76ADs, these activities were verified by expression in bidopsis .35S::CYP76AD5 expression alone in Arabidopsis results in no pigmentation in most of the tissues of the living plant . When PgDODA and CYP76AD1 are co-expressed in ypocotyl , and thaypocotyl .B. vulgaris (Amaranthaceae family). The sequence of Mirabilis jalapa  MjCYP76AD3 is BvCYP76AD1-like  while MjCYP76AD15 is BvCYP76AD5/6-like (beta clade). Co-expression of MjCYP76AD3 and PgDODA in yeast produced red pigment  and the CYP76AD1 enzyme responsible for conversion of L-DOPA to cyclo-DOPA in beet (step 3)  and thesCYP76AD6 . It has  R locus . While tA. thaliana [Antirrhinum majus (snapdragon) cells expressing the PgDODA gene and fed L-DOPA produce not only yellow betaxanthin but also the red betacyanin pigments [If CYP76AD1, CYP76AD5 and CYP76AD6 are the only enzymes responsible for tyrosine hydroxylation in beet, then knocking them all out should prevent any betalain pigment production. It has been shown that VIGS against CYP76AD1 and CYP76AD6 leads to loss of betalain pigmentation . Based othaliana . Howeverpigments . The argpigments  so snapdRecent phylogenetic analyses indicate that both the CYP76AD and DODA alpha and beta groups have arisen via gene duplication and subsequent diversification and that these events may underlie the origin of betalains . At thisr mutant, AD5/6 cannot replace CYP76AD1’s step 3 activity.It will be interesting to determine whether the CYP76AD1 (step 1 and 3 performing enzymes), or CYP76AD5/6 (step 1 only enzymes) CYP450s better represent the ancestral type. If the CYP76AD5/6 type predates the CYP76AD1 type, it would indicate that those first betalain-producing plants only had yellow betalains, which would be unlikely to supplant anthocyanins through analogous biological function. The latter could not have occurred until the new, step 3 activity evolved, providing betacyanins with hues similar to anthocyanins. If the CYP76AD1 type evolved first, this would indicate that red betalains predominated, allowing anthocyanins to be functionally replaced in many or all contexts. It would also mean that evolving CYP450s that limit the production of red while leaving yellow intact was an important subsequent evolutionary event providing a selective advantage. Of the polymorphisms that differentiate the CYP76AD1 and CYP76AD5/6 groups, the next closest orthologs (gamma group) are more similar to the CYP76AD5/6 genes, suggesting that tyrosine hydroxylation (step 1) was the ancestral function and cyclo-DOPA formation (step 3) evolved later in the CYP76AD1 lineage. Further, ancestral sequence reconstructions indicate that the ancestral copy of the alpha and beta groups would have had a leucine at the pivotal amino acid position 309, which likely would have limited its activity to step 1, thus producing only yellow pigments. This is confounded by the finding here that CYP76AD5/6 appear to have weak step 3 activity when overexpressed in yeast; however, in the beet The current taxonomic distribution of betalains has been a perplexing feature of angiosperm evolution. The most recent phylogenetic analyses suggest that betalains have had a single origin in the Caryophyllales, with subsequent betalain losses and reversions to anthocyanin production. What remains unclear is why betalains may have arisen in the first place and how they supplanted the well-entrenched and successful anthocyanins. As an alternative to outright anthocyanin replacement by betalains, the betalain pathway may have evolved in a lineage that lost the ability to synthesize anthocyanins. Indeed, some extant species in the Caryophyllales reportedly make neither betalains nor anthocyanins . In thisS1 FigThe tree presented here is very similar to that presented by Brockington et al. , except (TIF)Click here for additional data file.S2 FigAmino acids that appear to differentiate the three primary groups resolved in the gene tree are highlighted. The top  group consists of the CYP76AD1-like homologs ; center (beta) group, CYP76AD5/6-like ; bottom (gamma) group, unknown function. Red: conserved among CYP76AD1-like. Yellow: conserved among CYP76AD5/6-like. Blue: conserved among CYP76AD1, 5, and 6. Green: conserved among the unknown CYP76AD members. The numbers below the alignment are the amino acid positions in CYP76AD1 and correspond to the amino acids used in the LOGO analysis.(DOC)Click here for additional data file.S1 TableShown are the species name, family, gene name, and accession number for all genes referenced in the manuscript.(DOCX)Click here for additional data file.


The adaptation of H5N1 avian influenza viruses to human poses a great threat to public health. Previous studies indicate the adaptive mutations in viral polymerase of avian influenza viruses are major contributors in overcoming the host species barrier, with the majority of mammalian adaptive mutations occurring in the PB2 protein. However, the adaptive mutations in the PA protein of the H5N1 avian influenza virus are less defined and poorly understood. In this study, we identified the synergistic effect of the PA/224P + 383D of H5N1 avian influenza viruses and its ability to enhance the pathogenicity and viral replication in a mammalian mouse model. Interestingly, the signature of PA/224P + 383D mainly exists in mammalian isolates of the H5N1 influenza virus and pdmH1N1 influenza virus, providing a potential pathway for the natural adaptation to mammals which imply the effects of natural adaptation to mammals. Notably, the mutation of PA/383D, which is highly conserved in avian influenza viruses, increases the polymerase activity in both avian and human cells, and may have roles in maintaining the avian influenza virus in their avian reservoirs, and jumping species to infect humans. The adaptive mutations3467The highly pathogenic avian influenza (HPAI) H5N1 viruses continue circulation in avian populations results in sporadic infections in humans with a high mortality rate. Human H5N1 infections have a potential for human to human transmissibility1et al. demonstrated that two amino acid residues in the matrix protein M1 contribute to differences in virulence of the H5N1 viruses in micePrevious studies have demonstrated that multiple viral components play a role in pathogenicity and adaptation of the avian influenza virus in mammals. Strains of the human influenza virus preferentially bind α-2,6 sialic acid (SA) while strains of the avian influenza virus bind α-2,3 SA910141314et al. reported that a threonine-to-isoleucine substitution at position 97 in the PA protein enhanced the pathogenicity of the H5N2 strain in mice and viral replication in mammalian cellset al. reported the substitution of human-origin PA subunits into the avian influenza virus polymerase alleviated restriction in human cellset al. reported the PA and NS genes from pdmH1N1 made the H5N1 virus transmissible through respiratory droplets in guinea pigset al. found the amino acid residue at position 185 of PA could affect the pathogenicity in mice for avian H5N1 virusesThe viral polymerase, the nucleoprotein (NP), and the viral RNA genome form the ribonucleoprotein (RNP) complex, which is required for both transcription and viral genome replication345192021222324253451927293031323350), <0.8 versus 4.6 log10 50% egg infectious dose (EID50)]. We investigated the effects of mutations of S224P and N383D in the PA protein of H5N1 influenza viruses on the pathogenicity and viral replication in mice. In addition, most mammalian adaptive mutations in the polymerase of avian influenza virus are correlated with enhanced polymerase activity in mammalian cells1920232430We previously reported two genetically similar H5N1 AIVs, A/duck/Hubei/49/05 (R-DK/49) and A/goose/Hubei/65/05 (R-GS/65), that showed significant difference in their virulence in ducks3550 of 2.8log10EID50 in contrast to MLD50 of 4.6log10EID50 in R-GS/65, indicating the double substitution significantly increases the virulence of the parent virus R-GS/65 in mice (50 increase compared with parent virus R-DK/49 in mice (50 in both the backbone of R-DK/49 and R-GS/65 (6 EID50 then euthanized on day 3 post-infection (p.i.). The lungs, kidneys, spleens, and brains were collected for viral titration. The R-GS/65 replicated only in the lungs, while its derived mutant GS/65-PA224P + 383D replicated more efficiently in multiple organs . In 293T cells, the polymerase activity of mutant DK/49-PA383N was reduced to 16%  . SimilarTo determine the residue changes at position 383 in the PA protein, we aligned a total of 10524 PA sequences of influenza viruses from avian isolates and 7252 PA sequences from human isolates . We found PA/383D is dominant (99.7%) with only 0.09% (9/10542) of strains harboring the mutation PA/383N in avian influenza viruses. Surprisingly, PA/383D only took up 26.4% and the residue PA/383N took up 73.3% in human influenza viruses . This reOur previous study demonstrated that mutations at the residue 224 and 383 in the PA protein greatly contributed to the virulence of H5N1 avian influenza in domestic ducks3839Intriguingly, PA/383D is highly conserved in avian isolates (99.7%) but not in human isolates (26.4%) of the influenza viruses. The PA/383D mutation is associated with an increased polymerase activity in both avian and human cells. The PA/383D mutation may play an important role in maintaining the avian influenza viruses in avian reservoirs and be a key component for adaptation to humans. The mutation of residue PA/383 is not sufficient for altering viral pathogenicity in mice or ducks and requires the additional mutation of residue PA/224. The synergistic effect of S224P and N383D substitutions in the PA of H5N1 avian influenza on viral pathogenicity and replication was successfully demonstrated in a mouse model, but the underlying mechanisms remain unknown and further investigation is needed. In summary, we identified that the mutations at position of 224 and 383 in the PA protein significantly altered the pathogenicity and viral replication of H5N1 viruses in mice, and the single mutation at residue PA/383 can greatly affect viral polymerase activity in both avian and mammalian cells.Studies with highly pathogenic H5N1 avian influenza viruses were conducted in a biosecurity level 3 laboratory approved by the Chinese Ministry of Agriculture. All animal studies were approved by the Review Board of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The study was carried out in strict accordance with the recommendation in the Guide for the Care and Use of Laboratory Animals.Human embryonic kidney cells (293T) and chicken embryo fibroblasts cells (DF1) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum plus Penicillin-Streptomycin (100 U/mL) The construction of plasmids for virus rescue and polymerase activity was performed as described previouslyThe parental viruses and their derived mutant viruses were rescued as previously described6 EID50) were euthanized on day 3 p.i. Organs were collected and titrated for virus infectivity in eggs as described previously50 was determined by inoculating mice (n = 5) with 10-fold serial dilutions of the virus (50 μl) and calculated by the Reed and Muench methodGroups of 6-week-old female BALB/c mice  were anesthetized with CO2 and inoculated intranasally with the indicated dose of H5N1 influenza virus (50 μl). Mice n = 3)  and pcDNA3.1 + plasmid constructs expressing the polymerase PB2, PB1, PA, and NP genes (0.5 μg each) plus Lipofectamine 2000 (Invitrogen) as recommended by the manufacturer. Renilla luciferase expressed by pTK-RL was used as an internal control to normalize transfection efficiency. Cell extracts were harvested 30 h post-transfection, and luciferase activity was assayed by using the luciferase assay system (Promega).The assay was standardized against the Renilla luciferase activity. All experiments were performed in triplicate.p < 0.01 and indicated with an asterisk (*).Data are expressed as mean ± standard deviation (SD). Statistical significance was determined using unpaired Student’s t-test with two-tailed analysis . Significance is defined as H.C. and J.S. designed the study and wrote the manuscript. J.S., J.X., JZ.S. and Y.L. performed the experiments. All authors reviewed the manuscript.How to cite this article: Song, J. et al. Synergistic Effect of S224P and N383D Substitutions in the PA of H5N1 Avian Influenza Virus Contributes to Mammalian Adaptation. Sci. Rep.5, 10510; doi: 10.1038/srep10510 (2015).


P < 0.01) and D + Vit B6 (P < 0.05) groups. Plasma and tissue malondialdehyde levels were reduced in the C + Vit B6 and D + Vit B6 groups. Whole blood glutathione peroxidase (GSH-Px) and erythrocyte superoxide dismutase (SOD) activities were higher in the D group (P < 0.05). GSH-Px and SOD activities were increased in C + Vit B6 group while these parameters decreased in the D + Vit B6 group. Paraoxonase and arylesterase activities were decreased in the D group while they were increased in C + Vit B6 and D + Vit B6 groups. The results of present study suggest that vitamin B6 supplementation might be a promising adjunctive agent for improving oxidative stress and metabolic disturbances and for preventing diabetic complications including atherogenesis.The purpose of this study was to investigate the effects of vitamin B6 (Vit B6) on oxidant and antioxidant status in streptozotocin-induced diabetic rats. Thirty-two Wistar rats were divided into four groups: control (C), control + Vit B6 group (C + Vit B6), diabetes (D), and diabetes + Vit B6 group (D + Vit B6). Vit B6 (4 mg/kg body weight) was administered in drinking water for 4 weeks after the induction of diabetes. Vitamin B6 reduced serum total cholesterol level in the C + Vit B6 ( Diabetes mellitus is a chronic metabolic disorder characterized by chronic hyperglycemia and disturbances of carbohydrate, lipid, and protein metabolism  those whVitamin B6, also called pyridoxine, is one of water-soluble B vitamins that assist in the metabolism of proteins, fats, and carbohydrates . PyridoxThe number of studies about the effect of vitamin B6 on metabolic changes and oxidative stress in diabetes mellitus is limited –27. In tThe experiments were performed with 32 male Wistar strain rats (age: 5 months) weighing approximately 300–350 g. The rats were housed at room temperature (25 ± 2°C) under a 12 h light-dark cycle. Rats were given free access to standard laboratory chow  and tap water for one week before the experiment. Four rats were housed per cage. Experimental protocol was approved by the accordance with ethical procedures and policies approved by the Animal Care and Use Committee of Uludağ University, Bursa. Group I: normal control rats (C), Group II: control rats with orally administered vitamin B6 (C + Vit B6), Group III: STZ induced diabetic rats (D), Group IV: diabetic rats with orally administered vitamin B6 (D + Vit B6).The rats were divided into four groups of 8 rats each:Diabetes was induced with a single intraperitoneal injection of 65 mg/kg STZ  freshly dissolved in sodium citrate buffer (pH 4.5). Control rats received an injection of citrate buffer. Blood glucose level was measured 48 h after STZ injection. Rats with blood glucose levels >11 mmol/L were considered diabetic and were used for the study. Blood glucose concentration was measured with a Glucostix strip test in a glucometer . Streptozotocin injection may result in fatal hypoglycemia related to massive insulin release. To prevent hypoglycemia, rats were kept on a 5% glucose solution diet for 24 h after the injection.Vitamin B6  was prepared daily and administrated in drinking water at concentration (4 mg/kg body weight) to C + Vit B6 and D + Vit B6 groups for four weeks after STZ-injection.At the end of the experimental period, blood samples were obtained by cardiac puncture under light ether anesthesia following 10–12 h of fasting. Liver, kidney, heart, and skeletal muscle (musculus gastrocnemius) tissues were removed immediately after blood collection, rinsed with cold saline, blotted with gauze, and stored at −20°C until analysis. Blood samples were drawn in heparin-coated, EDTA-containing, and nonadditive tubes. A part of whole blood was frozen for GSH-Px determination. Erythrocytes for SOD determination were washed by saline and frozen after hemolysis. Blood samples were stored at −20°C until analysis process.Paraoxonase activity was determined as described by Eckerson et al. . The ratSera TC, TG, and HDL-C levels were determined by standard laboratory methods using an autoanalyzer . Plasma vitamin B6  concentration was measured by high-performance liquid chromatography .U test. A level of P < 0.05 was accepted as statistically significant. Statistical analyses were carried out by SPSS 13.0 program for Windows.Data are presented as mean ± SEM. Kruskal Wallis test was used, followed by the Mann-Whitney P < 0.05), and TG levels (P < 0.01) were significantly increased, and final body weight (P < 0.05) significantly decreased compared with the C group. Serum TC levels were significantly decreased in C + Vit B6 (P < 0.01) and D + Vit B6 (P < 0.05) groups compared with the C and D groups, respectively. HDL-C levels were significantly increased in the C + Vit B6 (P < 0.01) and D + Vit B6 (P < 0.05) groups compared with those of the C and D groups, respectively. There was no difference in the final body weight, food, fluid consumption, and blood glucose levels of C + Vit B6 and D + Vit B6 groups compared with those of the C and D groups, respectively.P < 0.05); however, supplementation of vitamin B6 for 4 weeks increased serum vitamin B6 levels in C + Vit B6 and D + Vit B6 compared with the C and D groups, respectively (P < 0.05).Concentrations of serum vitamin B6 values are shown in P < 0.01); however, these parameters were significantly increased in the C + Vit B6 and D + Vit B6 group compared with the C and D groups, respectively (P < 0.05) (P < 0.05). Whole blood GSH-Px (P < 0.05) and erythrocyte SOD (P < 0.01) activities were increased in the C + Vit B6 but decreased in D + Vit B6 compared with the C and D groups, respectively (P < 0.05) (Serum paraoxonase and arylesterase activities in the D group were significantly decreased compared with those of the C group ( < 0.05) . Compare < 0.05) .P < 0.05), heart, skeletal muscle, and liver tissue MDA levels were significantly higher in the D group compared with the C group (P < 0.01). However, plasma and tissues MDA levels were decreased in the C + Vit B6 and D + Vit B6 groups compared with the C and D groups, respectively.As shown in In the present study, we demonstrated that STZ administrated rats were obviously diabetic and under oxidative stress as reflected by changes in body weight, food and fluid intake, and laboratory parameters such as, plasma glucose, plasma, and tissue MDA levels. MDA, one of the best indicators of oxidative stress , is a pr2O2) is neutralized to H2O by Gpx or CAT. There is currently no consensus regarding response of antioxidant enzymes in the diabetes mellitus; some studies reported increased [P < 0.05). Increase in the activity of these antioxidant enzymes might be a compensatory defensive response to overproduction of ROS since oxidative damage related to hyperglycemia is predominantly caused by mitochondrial superoxide over production [Antioxidant enzymes SOD, CAT, and GSH-Px exist in all oxygen-metabolizing cells to prevent cells from damage exerted by free radicals and provide a repair mechanism for oxidized components . SOD disncreased , 36 and ncreased , 38 SOD oduction –23.In the present study, serum paraoxonase and arylesterase activities were reduced in the STZ-diabetic rats, which were consistent with previous diabetic human and rat studies –43. DecrP < 0.05). There are contradictory reports regarding vitamin B6 levels in the type 1 diabetes mellitus. Leklem and Hollenbeck reported that [P < 0.05).In the present study, in line with previous reports –50 we obted that  hyperglyted that , 51 suggted that . Vitaminted that , 48, 52.The results of present study suggest that, besides oxidative stress and other metabolic changes, diabetes is accompanied with reduced levels of vitamin B6 and it seems likely that supplementation of vitamin B6 improves oxidative stress and lipid profile in diabetes. Furthermore, vitamin B6 supplementation enhanced serum paraoxonase and arylesterase activities which might be related to a possible direct effect of this vitamin on the enzyme and/or related to its ability to reduce oxidative stress. Taking the data of this study into consideration vitamin B6 supplementation might be a promising adjunctive agent for improving oxidative stress and metabolic disturbances and for preventing diabetic complications including atherogenesis.


The DNA damage response (DDR) is a conglomerate of pathways designed to detect DNA damage and signal its presence to cell cycle checkpoints and to the repair machinery, allowing the cell to pause and mend the damage, or if the damage is too severe, to trigger apoptosis or senescence. Various DDR branches are regulated by kinases of the phosphatidylinositol 3-kinase-like protein kinase family, including ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). Replication intermediates and linear double-stranded genomes of DNA viruses are perceived by the cell as DNA damage and activate the DDR. If allowed to operate, the DDR will stimulate ligation of viral genomes and will inhibit virus replication. To prevent this outcome, many DNA viruses evolved ways to limit the DDR. As part of its attack on the DDR, adenovirus utilizes various viral proteins to cause degradation of DDR proteins and to sequester the MRN damage sensor outside virus replication centers. Here we show that adenovirus evolved yet another novel mechanism to inhibit the DDR. The E4orf4 protein, together with its cellular partner PP2A, reduces phosphorylation of ATM and ATR substrates in virus-infected cells and in cells treated with DNA damaging drugs, and causes accumulation of damaged DNA in the drug-treated cells. ATM and ATR are not mutually required for inhibition of their signaling pathways by E4orf4. ATM and ATR deficiency as well as E4orf4 expression enhance infection efficiency. Furthermore, E4orf4, previously reported to induce cancer-specific cell death when expressed alone, sensitizes cells to killing by sub-lethal concentrations of DNA damaging drugs, likely because it inhibits DNA damage repair. These findings provide one explanation for the cancer-specificity of E4orf4-induced cell death as many cancers have DDR deficiencies leading to increased reliance on the remaining intact DDR pathways and to enhanced susceptibility to DDR inhibitors such as E4orf4. Thus DDR inhibition by E4orf4 contributes both to the efficiency of adenovirus replication and to the ability of E4orf4 to kill cancer cells. The cellular DNA damage response (DDR) network interprets the presence of replicating viral DNA genomes as DNA damage and strives to repair it, leading to inhibition of virus replication. Many DNA viruses, including adenovirus, evolved mechanisms to inhibit the DDR, thus increasing the efficiency of virus replication. In this study we identify a novel mechanism used by adenovirus to inhibit the DDR. The viral E4orf4 protein, together with its cellular partner, the PP2A phosphatase, inhibits damage signaling by reducing phosphorylation of proteins belonging to different DDR branches. As a result, E4orf4 causes accumulation of DNA damage in the cells. Inhibition of the DDR regulators ATM and ATR, as well as expression of E4orf4, enhances infection efficiency. Moreover, E4orf4 sensitizes cells to killing by sub-lethal concentrations of DNA damaging drugs, likely because it inhibits DNA repair. These findings could provide one explanation for the previously reported ability of E4orf4 to induce cancer-specific cell death, as many cancers have DDR deficiencies leading to their increased reliance on the remaining intact DDR pathways and to enhanced susceptibility to DDR inhibitors such as E4orf4. Thus, inhibition of the DDR by E4orf4 contributes both to viral replication efficiency and to E4orf4-induced cancer cell killing. Genome integrity is constantly challenged by endogenous and exogenous agents that cause different kinds of DNA lesions. The cells have evolved a DNA damage response (DDR) which includes several mechanisms to detect and signal the presence of damaged DNA or replication stress, resulting in checkpoint activation and DNA repair, or if the damage is too extensive, resulting in senescence or cell death , 2. FormProtein phosphatase 2A (PP2A) is composed of three subunits: the catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits encoded by at least four unrelated gene families: PR55/B55/B, PR61/B56/B’, B”, and B”‘, which dDuring infection with DNA viruses, stretches of single-stranded DNA in replication intermediates and the ends of the double stranded viral DNA are perceived by the cell as damaged DNA, inducing the DDR . SoDrosophila to mammalian cells [The Ad E4orf4 protein is a multifunctional viral regulator. Within the context of the virus E4orf4 contributes to temporal regulation of the progression of viral infection by down-regulating early viral genes and cellular genes affecting Ad replication, controlling alternative splicing of viral RNAs, and influencing protein translation –50. Whenan cells –59, undean cells , indicatan cells .Studies of the mechanisms underlying E4orf4 action have revealed several E4orf4 partners, including the B55 subunit of protein phosphatase 2A (PP2A) . PP2A isHere we report that E4orf4 provides a novel mechanism to inhibit DNA damage signaling during Ad infection. We show that E4orf4 expression leads to decreased phosphorylation of several proteins that participate in the DDR, in both ATM- and ATR-regulated pathways and causes accumulation of DNA damage. This novel E4orf4 function requires its interaction with the B55 subunit of PP2A. Inhibition of the DDR by E4orf4 has biological significance to Ad infection and to E4orf4-induced cell death.dl366*, lacking the E4 region, or with dl366*+E4orf4, lacking all E4 orfs except E4orf4 [dl366* led to phosphorylation of several DDR proteins, including ATM, 53BP1, Smc1, Nbs1, Chk1, and Chk2, while these proteins were not phosphorylated in mock-infected cells. In contrast, infection with a similar multiplicity of infection (MOI) of dl366*+E4orf4 mutant Ad resulted in decreased phosphorylation levels of all tested DDR proteins belonging to both ATM- and ATR-regulated pathways. The reduced phosphorylation was confirmed by quantification of the protein bands and normalization to total protein levels. The E4orf4-induced decrease in DDR protein phosphorylation did not represent a global reduction of cellular phosphorylation as E4orf4 did not reduce Akt-S473 phosphorylation, but rather enhanced it. Thus the combined results suggest that E4orf4 reduces DDR induction by an E4-deficient mutant Ad. This conclusion was further validated by similar experiments carried out in non-transformed, primary HUVEC cells  induction. Protein extracts were prepared 24 hrs post-infection and were subjected to Western blot analysis. Similarly to our previous observations in HeLa and HUVEC cells  that were barely phosphorylated in mock-infected cells. Infection with a similar MOI of dl366*+E4orf4 resulted in decreased phosphorylation of these sites without a similar reduction in Chk1 protein levels  or the R81F84A E4orf4 mutant that does not bind PP2A (clone 3) were induced with Dox for three hrs, or were left uninduced, and were then treated with hydroxyurea (HU), which induces replication stress or with neocarzinostatin (NCS), a drug that induces DSBs. Protein extracts were prepared and subjected to Western blot analysis. As seen in 2O2 for 30 min or were left untreated and average comet tail moment was determined. The results demonstrate a significant increase in the number of comets seen in H2O2-treated cells in the presence of E4orf4, indicating a defect in damage repair in cells expressing the viral protein . dl366*+E4orf4 viruses were higher than in WT cells . Additidl366* and dl366*+E4orf4 at 30 ffu/cell, in the presence or absence of an ATR inhibitor. Morphological changes in the cells and production of infectious viruses were assayed 24 hrs after infection. The cytopathic effects (CPE) that accompany Ad infection, including aggregation, rounding up, and detachment of cells were quantified in two ways. First, the ratio of area covered with adherent cells to the total area was determined (ce), indicating changes in cell number or a clustering of cells. Second, the ratio of area covered with cell clusters larger than a predetermined size to total cell area was calculated (cl), indicating the appearance of large cell clusters. As seen in dl366* infection of A-T cells reconstituted with the empty vector, which lack ATM, than in A-T cells expressing WT ATM  = 2.44) and 5.68-fold (ln(fold change) = 1.76) respectively) and the combination of both led to more than 270-fold increase in dl366* virus propagation (ln(fold change) = 6). Addition of E4orf4 to the E4-deficient virus further enhanced virus multiplication. The temporal progression of infection with dl366* and dl366*+E4orf4 mutant viruses in A-T cells versus matching WT cells was also examined and is demonstrated in dl366*+E4orf4 viruses (24 hrs), and then gradually appeared in A-T cells infected with dl366* and in WT cells infected with dl366*+E4orf4. No CPE was observed in WT cells infected with dl366* viruses up to 48 hrs p.i. (dl366* produced 11-fold more progeny virus in A-T cells than in WT cells (ln(fold change = 2.46) and dl366*+E4orf4 produced 17-fold more progeny virus (ln(fold change = 3), indicating that ATM inhibits virus replication as was recently reported [dl366*+E4orf4 produced significantly more progeny virus than dl366* both in WT and in A-T cells at late times of infection (2.44-fold and 3.73-fold change respectively (ln(fold change) = 0.83 and 1.39)). Taken together, the results indicate that deficiency in both ATM- and ATR-regulated DDR as well as expression of E4orf4, which inhibits both these DDR pathways, led to a more efficient progression of Ad replication.Since E4orf4 appears to inhibit the ATM- and ATR-regulated DDR Figs , 2 and 4sing ATM , c2. Howhrs p.i. . Virus ireported , 78. Furdl366* and dl366*+E4orf4, in the presence or absence of an ATR inhibitor (ATRi), as described above  = 6) and E4orf4 further increased replication efficiency by close to 3.3-fold (ln(fold) = 1.11). These results suggest that inhibition of both ATM and ATR by Ad proteins contributes to the efficiency of virus replication. However, besides its contribution to ATM and ATR inhibition, E4orf4 has additional functions that are beneficial for virus replication, such as down-regulation of early gene expression, regulation of splicing of viral RNAs and control of protein translation [Our results indicate that ATM and ATR inhibition, as well as E4orf4 expression, increase the efficiency of Ad replication to various extents Figs –7. In A-5.7-fold . When bonslation –49. Execdl366* [dl1004 or dl1007 [dl366* infection of HeLa cells occurred earlier than ATR activation (represented by Chk1 phosphorylation) . The sir dl1007 , indicatylation) , suggestThe results presented here indicate that E4orf4 increases the accumulation of DNA damage following treatment of cells with DNA damaging drugs , resultiIt has been reported that when E4orf4 is expressed alone, it induces a unique mode of cancer-specific cell death , 61. InhIn summary, E4orf4 employs a novel mechanism to inhibit the DDR, which improves Ad replication and may contribute to induction of cancer-specific cell death by the viral protein. Investigation of this novel mechanism may provide a better understanding of critical DDR nodes that are targeted by E4orf4 and are required for successful application of a combinatorial treatment of cancer. Moreover, our results are important for the understanding of Ad-host cell interactions. The lengths to which Ad goes to inhibit the DDR indicate that DDR inhibition is central to the virus life cycle and our findings enhance this conclusion. Ads have been identified in recent years as significant pathogens in immunocompromised patients . As therHeLa cells (American Type Culture Collection) were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). Clone 13 cells containing tetracycline-inducible E4orf4, clone 3 cells containing a tetracycline-inducible E4orf4 mutant that does not bind PP2A, and clone L11 cells containing a tetracycline-inducible PP2A-B55 shRNA were generated by introducing a tetracycline-inducible pSUPERIOR vector (OligoEngine) containing cDNAs for WT E4orf4 , the E4oA PP2A-B55alpha shRNA-expressing pSUPER plasmid was a gift from S. Strack  and the dl366*, lacking the complete E4 region, and dl366*+E4orf4, lacking all E4 open reading frames (orfs) except E4orf4, were previously described [Adenoviral mutants escribed  and wereescribed ). W162 cescribed : 1 μM. APRPH2 gene determined by a parallel quantitative PCR with the primers: TM684-fw: 5’-CTGAAGCCGTACCTGGCTATC-3’; TM685-rev: GTGTCCCGGTAGTACTTCATGC.Relative levels of viral DNA were monitored by quantitative PCR, as previously described . Total iWhole cell extracts were prepared in SDS sample buffer  and viscosity was reduced by passing the extracts several times through a 27G needle after three min incubation at 95°C. Proteins were analyzed by Western blots using the indicated antibodies. Blot images were scanned with an Epson Photo 4990 scanner and were processed using Adobe PhotoShop 5.0 or 7.0. Band intensities were quantified using the TotalLab software.Antibodies specific for the following proteins were used in this work: E4orf4 , DBP  (Sigma). Immunostaining was visualized using an Axiocam camera linked to a Zeiss Axioskop at the indicated magnification.Clone 13 cells were treated with Dox for 2 hrs to induce E4orf4 expression or were left uninduced and were then treated with hydroxyurea (HU)  or neocarzinostatin (NCS)  for three hrs or left untreated. The cells were then harvested, counted, and plated in several decimal dilutions. Medium (without Dox) was changed every 3–4 days and colonies were counted after two weeks.2O2 for 30 minutes. An alkaline comet assay was performed as described before [Clone 13 cells were induced with Dox for 2.5 hours or were left uninduced and were then treated with 100 μM Hd before . BrieflyBox plots were generated using R software (version 3.1.2) and package ‘ggplot2’ (version 1.0.0). Data for box plot analysis was subjected to natural log transformation. Statistical significance was calculated using t-test.S1 Figdl366* and dl366*+E4orf4 mutant viruses at 30 ffu/cell or were left uninfected (Mock). Protein extracts were prepared 24 hrs post-infection and E1B-55K proteins were detected by Western blot analysis. Alpha-Tubulin staining served as a loading control.W162 cells were infected with (TIF)Click here for additional data file.S2 FigExpression of ATM and ATR in A-T cells reconstituted with an empty vector or with a vector expressing ATM was detected by Western blot analysis. Alpha-Tubulin staining served as a loading control.(TIF)Click here for additional data file.S1 TableVirus titers and ln(virus titers) from two independent experiments described in (DOCX)Click here for additional data file.S2 TableVirus titers and ln(virus titers) from two independent experiments described in (DOCX)Click here for additional data file.


In vitro and in vivo assays suggested that MdHXK1 interacted directly with and phosphorylated an anthocyanin-associated bHLH transcription factor (TF) MdbHLH3 at its Ser361 site in response to glucose. Furthermore, both the hexokinase_2 domain and signal peptide are crucial for the MdHXK1-mediated phosphorylation of MdbHLH3. Moreover, phosphorylation modification stabilized MdbHLH3 protein and enhanced its transcription of the anthocyanin biosynthesis genes, thereby increasing anthocyanin biosynthesis. Finally, a series of transgenic analyses in apple calli and fruits demonstrated that MdHXK1 controlled glucose-induced anthocyanin accumulation at least partially, if not completely, via regulating MdbHLH3. Overall, our findings provide new insights into the mechanism of the glucose sensor HXK1 modulation of anthocyanin accumulation, which occur by directly regulating the anthocyanin-related bHLH TFs in response to a glucose signal in plants.Glucose induces anthocyanin accumulation in many plant species; however, the molecular mechanism involved in this process remains largely unknown. Here, we found that apple hexokinase MdHXK1, a glucose sensor, was involved in sensing exogenous glucose and regulating anthocyanin biosynthesis.  +-ATPase B1 (VHA-B1) and the 19S regulatory particle of proteasome subunit (RPT5B), which influences the transcription of target genes. However, it is yet unknown if and how HXK1 directly targets TFs to modulate their function in the nucleus in plants. Our results reveal the important roles of MdHXK1 protein kinase in phosphorylating MdbHLH3 TF to modulate anthocyanins accumulation in response to glucose in apple.Glucose is considered as a major regulatory molecule in addition to being essential metabolic nutrients and structural components in higher plants. As is well known, hexokinase1 (HXK1) is a glucose sensor that integrates diverse signals to govern gene expression and plant growth in response to environmental cues. Previously, it is reported that the nuclear HXK1 forms a glucose signaling complex core with the vacuolar H In higher plants, sugars function as major regulatory molecules in addition to being essential metabolic nutrients and structural components. Sugars control gene expression to affect developmental and metabolic processes during the entire plant life cycle and function in response to biotic and abiotic stresses –3. ThereAmong the myriad of sugars in photosynthesis, glucose is the preferred carbon and energy source. Glucose is involved in many metabolic pathways, including the glycolytic process, in organisms ranging from unicellular microbes to plants and animals ,5. In adArabidopsis gin2 (glucose insensitive 2) mutants, both of which are mapped to the HXK1 gene  with 100 μM ATP and [λ-32P]ATP (0.2 mCi per reaction) at room temperature for 30 min. Recombinant MdbHLH3S361A-GST was served as a negative control in the in vitro kinase assay. The phosphorylated proteins were visualized using autoradiography after separation on a 12% SDS-PAGE gel.A total of 0.2 μg of recombinant His-tagged protein kinase MdHXK1 and 1 μg of MdbHLH3MdHXK1 and a conserved fragment of MdbHLH3 were amplified with RT-PCR using apple fruit cDNA as the template. The resulting products were cloned into the tobacco rattle virus (TRV) vector in the antisense orientation under the control of the dual 35S promoter. The vectors were named TRV-MdHXK1 and TRV-MdbHLH3. To generate overexpression viral vectors, full-length cDNA of MdHXK1 and MdbHLH3 were inserted into the IL-60 vector under the control of the 35S promoter. The resulting vectors were named MdHXK1-IL and MdbHLH3-IL.To construct antisense expression viral vectors, a specific fragment of Agrobacterium tumefaciens strain GV3101 for inoculations. Fruit infiltrations were performed as previously described [The antisense expression viral vectors were transformed into escribed . The inj2, 0.6 mM EDTA, 9 mM KCl, 1 mM NAD, 1 mM ATP, and 1 unit of NAD-dependent glucose-6-phosphate dehydrogenase (G6PDH). To assay glucose phosphorylation, 25 μL of the desalted extract was added to start the reaction under 25°C. Reduction of NAD within 5 min was monitored by the increase in absorption at 340 nm. Activity was calculated in terms of μmol of NAD reduced per minute.Glucose phosphorylation activity was measured using an enzyme-linked assay according to Schaffer and Petreikov . The assProtoplasts isolated apple calli cells were prepared and transformed as described by Sheen . The fluet al. [The total anthocyanins were extracted using a methanol-HCl method and detected as described by Hu et al. .t-test. A difference at P≤0.01 was considered significant, and P≤0.001 was considered extremely significant.Samples were analyzed in triplicates, and the data are expressed as the mean ± standard deviation unless noted otherwise. Statistical significance was determined using Student’s S1 Fig(A) Different concentration of glucose was tested for their ability to induce anthocyanin accumulation in in vitro shoot cultures of the ‘Gala’ apple cultivar. The shoot cultures of apple were plated on Murashige and Skoog (MS) agar containing 0.6 mg L-1 6-BA and 0.2 mg L-1 IAA plus different concentration of glucose  as indicated. Anthocyanin accumulation in apple leaves was measured after 7 days of growth under 17°C low temperature induction and continuous light. (B) Anthocyanin content of apple leaves in (A). (C) The phenotype as indicated by a red color for anthocyanin accumulation in in vitro shoot cultures of the ‘Gala’ apple cultivar treated with 6% glucose and 6% mannitol or 6% glucose plus glucosamine. (D) The anthocyanin content of apple shoot cultures in (C). The data are shown as the mean ± SE, which were analyzed based on more than 9 replicates. Statistical significance was determined using Student’s t-test in apple shoot cultures. n.s., p > 0.01; **p < 0.001.(TIF)Click here for additional data file.S2 FigThe conserved amino acid residues were labeled with black boxes. The alignment of sequences was generated using a ‘‘multiple sequence alignment” method with DNAMAN software.(TIF)Click here for additional data file.S3 Fig(A) WT, 35S::MdHXK1 (WT background), 35S::MdHXK1G104D (WT background), and 35S::MdHXK1S177A (WT background) transgenic apple calli showed anthocyanin accumulation phenotype on MS agar media containing 1% glucose. The apple calli were placed at 10°C under long-day conditions (16 h light/8 h dark) for 10 days. (B) and (C) Anthocyanin content (B) and glucose phosphorylation activity (C) in WT and transgenic apple calli in (A). In (B) and (C), data are shown as mean±SE, which were analyzed based on more than 9 replicates. Statistical significance was determined using Student’s t test in different apple calli lines. n.s., P > 0.01; *P < 0.01; **P < 0.001.(TIF)Click here for additional data file.S4 Fig(A) Western blotting assay of MdHXK1 protein abundance by using Myc antibody in wild type (WT) and 35S::MdHXK1-Myc transgenic apple calli. The ACTIN was served as a protein-loading control. (B) Co-IP assay of MdHXK1-interacting proteins in MdHXK1-Myc transgenic apple calli. Co-immunoprecipitation assay was performed by using monoclonal Anti-Myc antibody to screen the MdHXK1-binding proteins. The resultant IPed proteins was detected by coomassie blue staining. (C) Schematic diagram of the domain structures of MdHXK1. The domain prediction was performed on the website http://smart.embl-heidelberg.de/. The pink rectangle indicates the signal peptide, while the gray rectangle shows the hexokinase_1 and hexokinase_2 domains respectively. The numbers below domains indicated the predicted starting and ending numbers of amino acid.(TIF)Click here for additional data file.S5 Fig(A) Western blotting assay of MdbHLH3 protein abundance by using anti-Myc antibody in wild type (WT) and 35S::MdbHLH3-Myc transgenic apple calli. The ACTIN was served as a protein-loading control. (B) Western blotting assay of the specificity of the anti-MdbHLH3S361 antibody. The MdbHLH3-Myc transgenic apple calli was pre-incubated in MS medium plus 6% glucose for 3 hours. Subsequently, the proteins extraction was used for Western blotting assays with an antibody of MdbHLH3S361 phosphorylation site. (C) Glucose induced the phosphorylation of MdbHLH3 protein and was abolished by CIP in WT apple plants. The apple plants were pre-incubated in MS medium plus glucose (0 or 6%) and 5 U of CIP for 1 or 3 hours. Subsequently, the proteins extraction was used for Western blotting assays with an antibody of MdbHLH3S361 phosphorylation site.(TIF)Click here for additional data file.S6 Fig(A) Relative expression level of MdHXK1 in WT and 35S::antiMdHXK1 transgenic apple calli. Data are shown as mean±SE, which were analyzed based on more than 9 replicates. Statistical significance was determined using Student’s t test in different apple calli lines. *P < 0.01. (B) The protein abundance of MdHXK1 in WT and 35S::antiMdHXK1 transgenic apple calli.(TIF)Click here for additional data file.S7 FigS361A-GST). The phosphorylated MdbHLH3-GST protein were detected with anti-MdbHLH3S361 antibody. Note: MdbHLH3-GST-P represent the phosphorylated MdbHLH3-GST protein.The kinase assay was initiated by adding radiolabeled ATP to the mixture of MdHXK1-His kinase and MdbHLH3-GST (or MdbHLH3(TIF)Click here for additional data file.S8 Fig(A-C) MdHXK1 in vivo kinase activity stabilizes MdbHLH3 protein. The isolated total proteins from 35S::MdHXK1(A), WT (B) and 35S::antiMdHXK1(C) apple calli were treated with 20 μg /ml cycloheximide for 0, 1, 3 and 6 h. The degradation of MdbHLH3 protein was followed by western blotting with anti-MdbHLH3 antibody. (D) The graph shows the quantitation of the western blot data in (A), (B), and (C).(TIF)Click here for additional data file.S9 FigThe ACTIN was served as a protein-loading control.(TIF)Click here for additional data file.S10 Fig(A) The effectors and reporter constructs in the binary vectors were introduced into apple calli for GUS activity assays. pMdANR::GUS transgenic apple calli were transformation with 35S::MdHXK1 (MdHXK1-overexpressing vector), empty vector and 35S::antiMdHXK1 (MdHXK1-suppressing vector) were grown at 25°C in the dark and stained to detect GUS activity. (B) GUS activity in the transgenic apple calli as labeled in (A). The means and standard deviations were calculated from the results of three independent experiments.(TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S12 Fig(A) Glucose-mediated HXK1 promotes anthocyanin biosynthesis in Arabidopsis. The WT (Ler), AtHXK1 mutant gin2, MdHXK1 overexpressor line HXK1-OVX1 and function complementary line HXK1-R1 were grown on one-half-strength MS medium without sugar (Control) or with 6% glucose (w/v), and 6% mannitol (w/v) at 10°C under long-day conditions (16 h light/8 h dark) for 10 days. (B) Anthocyanin content of WT and transgenic arabidopsis as indicated in (A). (C) Amino acid alignment of the conserved Ser361 of HXK1 proteins in apple and other species. The conserved serine residue at position 361 were indicated with red pentagram. The alignment of all the sequences was generated using a ‘‘multiple sequence alignment” method with DNAMAN software.(TIF)Click here for additional data file.S1 Text(XLS)Click here for additional data file.S2 Text(XLS)Click here for additional data file.S3 Text(XLS)Click here for additional data file.S4 Text(PDF)Click here for additional data file.S5 Text(DOCX)Click here for additional data file.


PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.Loss-of-function mutations in  PINK1 or PARKIN genes are the most common causes of recessively inherited PD. Together the encoded proteins coordinate a protective cellular quality control pathway that allows elimination of impaired mitochondria in order to prevent further cellular damage and ultimately death. Although it is known that the kinase PINK1 operates upstream and activates the E3 Ubiquitin ligase Parkin, the molecular mechanisms remain elusive. Here, we combined state-of-the art computational and functional biological methods to demonstrate that Parkin is sequentially activated through PINK1-dependent phosphorylation and subsequent structural rearrangement. The induced motions result in release of Parkin's closed, auto-inhibited conformation to liberate its enzymatic functions. We provide for the first time a complete protein structure of Parkin at an all atom resolution and a comprehensive molecular dynamics simulation of its activation and opening conformations. The generated models will allow uncovering the exact mechanisms of regulation and enzymatic activity of Parkin and potentially the development of novel therapeutics through a structure-function-based drug design.Parkinson's disease (PD) is a devastating neurological condition caused by the selective and progressive degeneration of dopaminergic neurons in the brain. Loss-of-function mutations in the  PINK1) and PARKIN genes are the most common causes of autosomal recessive Parkinson's disease (PD) PINK1 and PARKIN, abrogate this presumably neuroprotective pathway through distinct molecular mechanisms and at different steps along the sequential process Mutations in the PTEN-induced putative kinase 1  domain Consistent with its notoriously weak enzymatic activity, several partial crystal structures of Parkin in silico predict a structural rearrangement of the UBL domain that initiates a sequential release of Parkin's intra-molecular interactions. Along these opening conformations, we have docked the E2-Ub complex, required for Parkin's activation and enzymatic functions. Importantly, the presented computational predictions are consistent with our cell biological studies. We provide the basis for a better understanding of the molecular mechanisms of auto-inhibition and liberation of Parkin's catalytic activity.Here, we set out to provide structural models for human Parkin that would allow release of its auto-inhibited conformation and consequently activation of its E3 Ub ligase functions. We have applied highly accurate molecular modeling methods to provide for the first time an all-atom resolution of human full-length Parkin. Given the suggested auto-regulatory role of the UBL domain In order to understand the activation mechanisms of the neuroprotective E3 Ub ligase Parkin , while mutations to the negatively charged Aspartic (S65D) or Glutamic (S65E) acid are used as phospho-mimic variants. For critical analysis of the intermolecular interactions induced by these mutations , we comp2) within the pocket and counted the number of water molecules at common time intervals (from the simulation). The average number of water molecules within the pocket ranges from 9 to 12 for the initial time point (t = 0), 6 to 12 for t = 5 ns, and 12 to 19 for t = 100 ns  for Parkin wild type at 2 h CCCP . All ParTo model putative opening structures, Parkin models were analyzed using both traditional MDS and enhanced sampling MDS techniques to generate a large pool of conformers. We used Targeted Molecular Dynamics or Maxwell's demon Molecular Dynamics (MdMD) We repeated this simulation using a randomizer in our molecular dynamics sprint interval that ensures non-duplicative runs, and show the superposition of replicates alongside . Using tDuring the trajectory of the UBL domain across and alongside Parkin, several structural changes were noted that are potentially relevant for E2 co-enzyme binding and Ub charging of Parkin. Following phosphorylation of Ser65 as a trigger, we found that residues within the linker region undergo repeated contacts with the RING1 and RING2 domains during movement of the UBL domain . Our datFirst, the UBL-linker region must dissociate from RING1 and IBR domains in order to loosen the entire structure . Based oSecond, the repressor element of Parkin (REP), a region between IBR and RING2, blocks access of an Ub-loaded E2 co-enzyme  in RING1 . This in2 with transient peaks of 20 Å2. This increase in solvent exposure over time is indicative of a conformational reorientation, which could allow the active site Cys431 to receive a thioester-bonded Ub moiety from the E2 enzyme.Third, the RING0 domain buries Parkin's active site Cys431, making it unavailable to receive Ub from an incoming E2 . During Taken together, our MDS and subsequent calculations revealed a sequential release of Parkin's safety belts preventing its activation. The dissociation of the inhibitory N-terminus is triggered by PINK1-dependent phosphorylation of Ser65 in the UBL domain. As a consequence, Parkin's entire structure is loosened and further perpetuates the liberation of Parkin's presumed E2 binding region and of its active center as pre-requisite for enzymatic activity.To identify the E2 enzyme binding site in RING1 and to dock an E2∼Ub complex, we scanned across an evenly spaced distribution of ∼50 structures spanning the opening of Parkin using Piper for protein-protein docking The top performing structures were further studied with unbiased (free) MDS. Following docking of the UbcH5a-Ub complex with Parkin, we completed simulations where the Ub-loaded E2 makes substantial movements toward the active site region of Parkin . The dis2+ coordinating cysteines, the catalytic residue Cys431 is not making any intra-molecular interaction  and thereby induces larger scale conformational motions over time. It should be noted that the simulation time scale is not an exact match for what is actually occurring in the cell, but is idealized. In the cell, Parkin protein will require more than the calculated time to undergo major structural rearrangements and binding of the E2 co-enzyme in order to become fully active. pSer65-dependent hydration of the surrounding cavity induces the release of the UBL domain, which appears to act as the first safety belt that keeps Parkin's activity in check under basal conditions. Upon dissociation of the UBL domain, we next detected release of the inhibitory REP region from the E2 binding site in RING1 that could facilitate association of an Ub-charged co-enzyme. As a further consequence we also observed conformational adjustments in RING0 that intervenes between RING1 and RING2 burying Parkin's active site when unmodified. In its inactive state, the E2 binding site and Parkin's catalytic center are separated by a distance of more than 50 Å, which would not allow for Ub transfer. However, during opening of Parkin, we measured significant changes in distances, RMSD and SASA values, indicating differences in positioning and solvation of the region surrounding Cys431 that would be important for charging of Parkin.These promising measurements prompted us to dock an Ub-charged E2 enzyme (UbcH5a/UBE2D1) during the opening phase of Parkin. Indeed, we found conformations that were able to accommodate the co-enzyme and allowed us to generate an energetically favorable Parkin:E2-Ub complex. Strikingly, subsequent free MDS, showed a reorientation of the E2 to better position the Ub-Gly76 residue towards Parkin's active site. This coincides with hydration around Cys431 and results in a significantly reduced distance between the catalytic centers of the E2 and Parkin. The amide linkage in the E2-Ub co-crystal prevented further insights into the transfer of the Ub moiety from the E2 enzyme onto Parkin's active center, but we noted a repositioning of the REP region back into the E2 binding site in RING1. This putative reset mechanism may allow dissociation of the discharged E2 for the next binding event during consecutive ubiquitinations rounds. During mitophagy, multiple E2 enzymes are utilized by Parkin for Ub charging, mitochondrial translocation and substrate ubiquitinations In this context, it is important to note that Parkin has been suggested to self-associate through a PINK1-dependent mechanism upstream of its mitochondrial translocation in vitroStrikingly, it has been shown that PINK1 also phosphorylates the modifier Ub itself at the conserved Ser65 residue PARK2 gene, was taken from the NCBI Reference Sequence: NP_004553.2. The following 465 amino acid residues (full-length) were used for modeling: MIVFVRFNSSHGFPVEVDSDTSIFQLKEVVAKRQGVPADQLRVIFAGKELRNDWTVQNCDLDQQSIVHIVQRPWRKGQEMNATGGDDPRNAAGGCEREPQSLTRVDLSSSVLPGDSVGLAVILHTDSRKDSPPAGSPAGRSIYNSFYVYCKGPCQRVQPGKLRVQCSTCRQATLTLTQGPSCWDDVLIPNRMSGECQSPHCPGTSAEFFFKCGAHPTSDKETSVALHLIATNSRNITCITCTDVRSPVLVFQCNSRHVICLDCFHLYCVTRLNDRQFVHDPQLGYSLPCVAGCPNSLIKELHHFRILGEEQYNRYQQYGAEECVLQMGGVLCPRPGCGAGLLPEPDQRKVTCEGGNGLGCGFAFCRECKEAYHEGECSAVFEASGTTTQAYRVDERAAEQARWEAASKETIKKTTKPCPRCHVPVEKNGGCMHMKCPQPQCRLEWCWNCGCEWNRVCMGDHWFDV.The protein sequence of the human E3 Ub ligase Parkin (isoform 1) (Parkin), encoded by the Parkin has several conserved domains to be modeled (from the N-terminus to the C-terminus): UBL (residues 1–76), undefined linker region (residues 77–140), RING0 (or UPD) domain (residues 141–216), RING1 domain (residues 217–328), IBR domain (residues 329–378), REP region (residues 379–410), and RING2 domain (residues 411–465). The Parkin sequence was aligned, with each domain modeled as a separate unit built into a composite structure see  – Part 1ab initio approach of ORCHESTRAR The modeling was built as a hybrid model from consensus between the programs PRIME  Refinement of the fragments was completed using YASARA's refinement module. These refinements started with the Secondary Structure Prediction (SSP) feature of YASARA. Both homology and fold recognition were considered and a final refinement with the entire model was completed using YASARA for 250 ps of MD using knowledge-based force fields. Additionally, YASARA supports an extensive and large loop library for modeling loops and gaps. The sequence and identity of each fragment was reasonable see  – Part 2The model conformation was verified with WHAT-IF and PROCHECK and has a valid conformation consistent with good phi-psi space MDS was completed on each model for conformational sampling, using methods previously described in the literature 5 atoms including proteins, counter-ions, solvent ions, and solvent waters. Simulations were carried out using the particle mesh Ewald technique with repeating boundary conditions with a 9 Å nonbonded cut-off, using SHAKE with a 2-fs timestep. Pre-equilibration was started with 100,000 steps of minimization followed by 10000 ps of heating under MD, with the atomic positions of protein fixed. Then, two cycles of minimization (100000 steps each) and heating (2000 ps) were carried out with restraints of 10 and 5 kcal/(mol·Å2) applied to all protein atoms. Next, 50000-steps of minimization were performed with solute restraints reduced by 1 kcal/(mol·Å2). Then, 1000 ps of unrestrained MD were carried out, and the system was slowly heated from 1 to 310 K. The production MD runs were carried out with constant pressure boundary conditions (relaxation time of 1.0 ps). A constant temperature of 300 K was maintained using the Berendsen weak-coupling algorithm with a time constant of 1.0 ps. SHAKE constraints were applied to all hydrogens to eliminate X-H vibrations, which yielded a longer simulation time step (2 fs). Our methods for equilibration and production run protocols are in the literature Charmm27, Amber, and OPLS2005 force fields were tested with the current release of NAnoscale Molecular Dynamics 2 engine. The protein with hydrogens consists of 7,083 atoms. In all cases, we neutralized with counter-ions, and then created a solvent with 150 mM Na+ Cl- to recreate physiological strength. TIP3P water molecules were added around the protein at a depth of 15–18 Å from the edge of the molecule depending upon the side The MdMD algorithm has been previously described in exhaustive detail for smaller systems Using Schrödinger's Conformational Search Suite, we employed the following scheme. Potential terms were set using the OPLS2005 force field with water solvent and charges designated from the force field. We used an extended cutoff for electrostatic calculations . We utilized constraints fixed on each zinc atom and the associated zinc-finger amino acid residues , which were determined using harmonic restraints at a force constant of 50 kcal/mol per selected atom. Additionally, torsions of the adjacent amino acid to the zinc-binding amino acid was softly constrained with a 10 kcal/mol force constant. Every other atom of the system was left to freely move under the conditions of the search algorithm, and the constrained pairs were assigned relative to each other, not to coordinate space, such that the zinc-finger could move as a single unit during conformational searches. The Powell-Reeves conjugate gradient energy minimization method was utilized on conformations achieved to return the structure to its local minimum for that particular conformation Several search schemes were applied to Parkin to look for large global conformational variation. Using the conformational search engine, we examined torsional sampling with MCMM, Mixed-torsional/Low-mode sampling, Large-scale Low-mode sampling, and Mixed torsional/Large-scale low-mode sampling (LC-MOD). When using the LLMOD search, we set the initial convergence criteria to 1.000. To customize the search we used enhanced torsion sampling options with distance and torsion checking. Maximum number of steps per attempt was 5,000 with 100 steps per rotatable bond, saving 10 structures per search. The default energy window for saving structures is 5.02 kcal/mol. Probability of a torsion rotation/molecule translation is 0.5. Minimum distance for a low-mode move is 3.0 Å and maximum is 7.5 Å. The search was continued for 100 s of conformers, retaining only lowest energy models and discarding unrealistic structures. Structures from the LCMOD search form guidepost structures for MdMD biasing of the initial model to traverse the landscape of structures generated using Schrödinger's Large-scale sampling using the initial crude guideposts and various biasing schemes. Final simulations relied on MdMD for smooth pathway transitions.Mutations of amino acids were completed using the Maestro within the Schrödinger suite with the mutate residue feature. Additionally, the build panel in Maestro conveniently allows for placing mutated residues (or growing them) automatically within an existing peptide chain. Also, MacroModel features within Maestro allow for the quick minimization of the structure for local geometry fixes to correct stereochemistry and packing of the amino acids. Modifications for amino acids, such as phosphorylation of Serine-65 (pSer65), was achieved using the 2D sketcher and importing as an new amino acid type which can be parameterized using the Schrödinger force fields. Using OPLS2005 or YASARA2, one can parameterize these modification and import into existing molecular dynamics integration engines, as the parameters for the modification are well documented for YASARA and Schrödinger MMB is an internal coordinate mechanics (ICM) ZEMu is implemented in MMB wt) with the KB potential FoldX mutant complex to obtain the respective free energy of unfolding (ΔGmutant). The difference between these two quantities is our estimate of the experimental change in binding free energy induced by the mutation wt−ΔGmutant. Using MMB and ZEMu with these equilibration and binding energy evaluation required an average of 15 minutes per protein, on a single core of a 3.00 GHz AMD Opteron 6220 processor. All other calculations in the above MD sections were completed on a Xeon-based cluster with 120 hexa-core processors with 256 GB RAM available.For each proposed mutation locus, we equilibrated the corresponding flexibility zone in the wild type complex and evaluated the free energy of unfolding  were obtained from the ATCC (American Type Culture Collection), human embryonic kidney cells (HEK293E) from Invitrogen. Cells were maintained in DMEM containing 10% FBS at 37°C under humidified conditions and 5% COThe Parkin mitochondrial translocation assay has been previously been described HeLa cells were plated onto glass coverslips coated with poly-D-lysine (Sigma), fixed with 4% paraformaldehyde and permeabilized with 1% Triton-X-100 in PBS. Cells were incubated with primary anti-TOM20  and anti-p62 antibodies  followed by incubation with secondary antibodies anti-mouse IgG Alexa Fluor-647 and anti-rabbit Alexa Fluor-568 (Molecular Probes) diluted 1∶1000. Nuclei were stained with Hoechst 33342 (1∶5000). Coverslips were mounted onto slides using fluorescent mounting medium (Dako). Confocal fluorescent images were taken with an AxioObserver microscope equipped with an ApoTome Imaging System (Zeiss).HeLa cells were transfected with FLAG-Parkin C431S variants using Lipofectamine 2000 according to manufacturer's protocol and medium was replaced 4 h later. The next day, cells were treated with 10 µM CCCP for 0, 1, 2, 4, or 16 h. Cells were harvested in preheated (95°C) SDS lysis buffer . Lysates were homogenized by 10 strokes through a 23G needle. Protein concentration was determined by use of bicinchoninic acid (Pierce Biotechnology). To verify the band shift by oxyester formation, aliquots of lysates were treated with or without NaOH  for 1 h at 37°C. NaOH was neutralized by addition of equal amounts of HCl before samples were run on 8–16% Tris-Glycine gels and transferred onto polyvinylidene fluoride membranes (Millipore). Membranes were incubated with anti-FLAG antibody  overnight at 4°C followed by HRP-conjugated anti-mouse secondary antibodies . Bands were visualized with ImmobilonWestern Chemiluminescent HRP Substrate (Millipore) using a LAS-3000 Imager (Fuji).2) before supernatant was completely removed. Phosphatase treated samples were washed 2× in IP buffer before supernatant was removed and 8 µl H2O, 2 µl 10× buffer and 10 U phosphatase  were added to the beads for 30 min at 37°C. Beads were then washed twice with 1× Ub buffer before ubiquitination reaction. Recombinant proteins were purchased from Boston Biochem. Ubiquitination reaction contained 100 nM E1 enzyme , 1 µM E2 enzyme , 2.5 mM ATP, 2 mM DTT, 5 µg untagged ubiquitin (#U-100H), 1 µg N-terminally biotinylated ubiquitin (#UB-560) in 1× Ub buffer in a 20 µl reaction. Ubiquitination reaction was carried out for 1.5 h at 37°C. Laemmli buffer was added and samples boiled for 10 min at 95°C. Samples were run on a 8–16% Tris-glycine gel, blotted and probed with horseradish peroxidase-coupled Streptavidin  and anti-FLAG antibody .HEK293E cells transfected with FLAG-Parkin wild type were incubated with or without 10 µM CCCP for 1 h prior to lysis. Cells were washed once in cold 1× HBS  and lysed in IP buffer ). PhosStop (Roche) was added to all samples that were not treated with phosphatase. 750 µg protein was immunoprecipitated for 4 h using 20 µl of anti-FLAG EZview beads (Sigma). Beads were washed twice in cold IP buffer and once in 1× Ub buffer Click here for additional data file.Figure S2Comparison between Parkin with and without pSer65. RMSD comparison between human full-length pSer65 Parkin structural model and X-ray structures  for backbone residues 141–465, which measure to the initial conformation of pSer65 the following RMS values: 3.56 Å, 3.48 Å, 2.92 Å, and 1.72 Å, respectively.(TIF)Click here for additional data file.Figure S3Zoom into Parkin crystal structure (4K95). Region surrounding Ser65 in the UBL domain is shown. Residues Trp45, Leu61, Val67 and Ser65 are highlighted. Ser65 establishes a hydrogen bond with the main chain of Asp62, while this interaction is lost in all mutants. Though S65D and S65E introduce a negative charge that should perturb the electron distribution in the adjacent hydrophobic pocket and decrease the stability of the UBL domain, we obtained ΔΔG values lower than expected from structure gazing . On the other hand, S65A should further stabilize the UBL domain by expanding the hydrophobic pocket electron distribution resulting in a ΔΔG of −0.7 kcal.mol−1. The RMS error associated with the method is 1.53 kcal/mol for all mutants  and 1.15 kcal/mol for the stabilizing mutants (TIF)Click here for additional data file.Figure S4Mutations of Ser65 delay Parkin translocation, p62 recruitment and mitochondrial clustering.A) Analyzed GFP-Parkin constructs had similar expression levels. Shown is the average signal intensity of the cytoplasmic plus the nuclear GFP signal at 0 h CCCP treatment across all analyzed wells after nontransfected, GFP-negative or low level expressing cells were excluded. GFP alone showed a higher expression levels than the GFP-Parkin fusion proteins . B–D) Shown are representative immunofluorescence images before and after 2 h or 4 h CCCP treatment. Individual and merged channels are given: GFP-Parkin in green, TOM20 (mitochondria) in red, Ub adaptor protein p62 in cyan and nuclei (Hoechst) in blue. Scale bars correspond to 10 µM.(TIF)Click here for additional data file.Figure S5Ramanchandran plot for the average structure from MDS indicating normal distribution of Phi-Psi space for protein geometry. Additional measurements using What-If within Yasara were completed for backbone measurements, 1D/2D packing, and dihedrals/rotamers (see Methods). Both α-helical and β-sheet quadrants on the left side are well populated, random coil is shown on the right.(TIF)Click here for additional data file.Figure S6Linearity of MdMD algorithm at driving Parkin from state 1 to state 5. MdMD algorithm is applied following 10 ns production (post-equilibration) MD to allow the structure(s) to relax. After MdMD algorithm starts, the RMS rapidly and linearly descends toward State 5 with final RMSDs <3 Å for all replicates. All RMS calculations are relative to the final state (State 5) from generated models described in methods.(TIF)Click here for additional data file.Figure S7Stability of the C-terminal region of Parkin (146–465) protein during MdMDS and MDS simulations. RMSD for protein backbone of the four replicates stays between 2 and 4.5 Å. RMSD was calculated relative to the final state from MdMD with the replicate in brown indicated the reference structure (RMSD goes to zero at end).(TIF)Click here for additional data file.Figure S8RMS fluctuation of Parkin amino acid residues during free MDS versus MdMD simulation. The fluctuation of amino acids from UBL versus residues 141–465 is shown with the MdMD line in black and the free MDS line in red. Residues 141–465 (black) stay within 4–6 Å of initial structural model. Apparently the zinc fingers greatly stabilize those regions of the structure. The UBL domain and residues through 140 show greatest fluctuation during MdMD, which traverses from IBR to C431. For the free MDS trajectory, the overall structure is stays under 7–8 Å for RMSF (red line). The red line is for Ser65 simulation and black line is for pSer65 simulation.(TIF)Click here for additional data file.Figure S9RMSD of UBL-linker versus residues 141–465 from four replicates. Residues 141–465 (blue) are <5 Å of initial structure for duration. Only local conformational fluctuations occur in the main body, which is rigidified by the presence of eight Zinc-fingers. The MdMD algorithm moves each replicate toward State 5 giving rise to large RMS changes that are shown in red, green and beige. For each replicate, the movement is almost similar giving closely spaced RMS changes during the common MD sprint interval.(TIF)Click here for additional data file.Figure S10Parkin crystal structure close-up of the active site region. The active site residue for Parkin (Cys431) is given with its neighboring residues that are implicated in coordinating the thioester linkage with Ub. In this inactive state, Cys431 is not making any contacts. The C431S mutation does not result in any significant structural changes.(TIF)Click here for additional data file.Movie S1The improved human Parkin model. A model for human full-length Parkin is shown with legend key for domain residues in lower left corner. Initial closed, auto-inhibited state for Parkin is shown. As animation progresses the surface of the protein is shown semi-transparent to account for the solvent-accessible surface area of Parkin. Surfaces are colored to match domains as indicated in (MP4)Click here for additional data file.Movie S2Ser65 Parkin from free MDS for closed conformation (100 ns). Representative animation for molecular dynamics of unmodified human full-length Parkin (Ser65) model is shown. Domains are colored as indicated in (MP4)Click here for additional data file.Movie S3pSer65 Parkin from free MDS for closed conformation (200 ns). Representative animation for molecular dynamics of modified human full-length Parkin (pSer65) model is shown. Domains are colored as indicated in (MP4)Click here for additional data file.Movie S4pSer65 cleft opening with MDS starting in closed conformation. Close-up view of the cleft pocket, which contains residues Met1, Ile2, Phe4, Arg6, Gln63, Gln64, Arg97, Pro113, and Ser116 in close proximity to pSer65 during the MDS run. Ribbons are colored by secondary structure and surface is shown to illustrate widening of gap between UBL and linker during simulation.(MP4)Click here for additional data file.Movie S5MdMD pathway from a representative excursion of the UBL-linker domain across Parkin towards the active site. A representative pathway via MdMD excursions between the initial MDS state 1 to the active site region in MDS state 5 is shown (see 2+ atoms (spheres) as well as the coordinating Cys/His residues are shown.hown see . The Par(MP4)Click here for additional data file.Movie S6MdMD replicates of Parkin's opening. Replicates of MdMD runs show the same trajectory as in 2+ atoms are shown as red spheres.(MP4)Click here for additional data file.Movie S7Successful docking of the E2∼Ub complex onto Parkin with unbiased MDS. The E2 enzyme is shown in dark green, the Ub moiety in brown.(MPG)Click here for additional data file.Movie S8Poor docking of the E2∼Ub complex onto Parkin with unbiased MDS. The E2 enzyme is shown in dark green, the Ub moiety in brown.(MPG)Click here for additional data file.Text S1In silico modeling of human Parkin. Part 1: The modeling of individual fragments of Parkin is provided. Part 2: The consensus model for all fragments combined into complete full-length Parkin structure is provided.(DOCX)Click here for additional data file.