However, mutation of this region of Core typically disrupts nucleocapsid formation in the cytoplasm, making it difficult to ascertain if the Core PPAY motif constitutes a functional L-domain that mediates HBV release in the context of replicating virus. Since many viral L-domains are functionally interchangeable between different virus families, and such swapping experiments have been used as a tool to identify other viral sequences with L-domain activity, we generated chimeric constructs between murine leukemia virus (MLV) Gag and HBV Core to determine if the potential HBV L-domain motif is sufficient to stimulate virus release. We found that the HBV Core PPAY motif, but not the PNAP motif, demonstrates L-domain activity in the context of MLV replication to direct virus release and infectious virion production. Additionally, we found that overexpression of the cellular Nedd4 or WWP1 ubiquitin ligases stimulates release of a partially defective PPAY domain mutant, providing further evidence supporting a role for the Nedd4 ubiquitin ligase in promoting HBV release. These studies lend further insight into the mechanisms used by HBV to mediate its release from infected cells.The hepatitis B virus (HBV) Core protein encodes a late (L)-domain like motif ( Current therapies for chronic hepatitis B virus (HBV) infection include antiviral drugs that block HBV genome replication, such as adefovir and entecavir, or interferon-α (IFN-α), which stimulates the immune response for clearance of the virus Many viral structural proteins encode highly conserved late domain (L-domain) sequence motifs that mediate virus release 129PPAYRPPNAP138. The Nedd4 Ub ligase interacts with the Core L-domain-like PPAY motif and mutation of the tyrosine residue to alanine (Y132A) abolishes the Nedd4 interaction The HBV Core protein contains two L-domain-like sequences located within a single motif: The observation that some viral L-domains are functionally interchangeable between heterologous viruses has led to the identification of new viral L-domain sequences and motif binding partners Since viral L-domains are functionally interchangeable between different virus families, and complementation can be used as a tool to identify sequences with L-domain activity, we generated chimeric constructs between MLV Gag and HBV Core to determine if the two potential PPAY and PNAP L-domain motifs are sufficient to stimulate viral release as well as recruit cellular ubiquitin-dependent proteins of the endocytic pathway. We found that the Core PPAY motif, but not the PNAP motif, demonstrates L-domain activity in the context of MLV replication to direct infectious virion production. Furthermore, we provide additional evidence that the Nedd4 Ub ligase can promote virus release in an HBV L-domain dependent manner.Core-YFP L-domain chimeras, pUC57 expression plasmids were synthesized (Genscript) to encode the MLV Gag gene with the endogenous L-domain sequence (nucleotides 382 to 393) replaced by the HBV Core L-domain sequence motif PPAY/PNAP (WIRTPPAYRPPNAPILST), PPAY (WIRTPPAYRP), or PNAP (RPPNAPILST). An XhoI fragment containing the MLV GagCore chimera gene sequence was subcloned into MLV Gag-YFP to generate MLV GagPPAY/PNAP-YFP, MLV GagPPAY-YFP, or MLV GagPNAP-YFP expression constructs and a AflIII/XhoI DNA fragment containing the Core L-domain was subsequently subcloned into MLV GagPol to generate MLV GagPolPPAY/PNAP, MLV GagPolPPAY, and MLV GagPolPNAP expression constructs, respectively. The MLV GagPPAY/PNAP-YFP construct was used as a template for mutagenesis of the Core PPAY motif and an AflIII/XhoI DNA fragment was subsequently subcloned in MLV GagPol to generate MLV GagPolLPAY/PNAP, MLV GagPolPPAF/PNAP, or MLV GagPolPPAA/PNAP. HECT Ub ligase expression constructs used in this study that contained the full length  or the truncated  versions of the proteins with YFP fused to the N-terminus were a gift from Paul Bieniasz (The Rockefeller University) and were previously described in Martin-Serrano et al. MLV GagPol plasmid, MLV GagPol4A plasmid, MLV Gag-YFP plasmid, Friend MLV Env plasmid, and MLV long terminal repeat-lacZ reporter construct (MLV LTR-LacZ) were previously described in Sherer et al. LPAY/PNAP), 5-GGA TTC GCA CTC TTC CAG CTT ATA GAC CAC CAA ATG-3; for the PPAF/PNAP mutation (MLV GagPolPPAF/PNAP), 5-GAT TCG CAC TCC TCC AGC TTT TAG ACC ACC AAA TG-3; for the PPAA/PNAP mutation (MLV GagPolPPAA/PNAP), 5-GAT TCG CAC TCC TCC AGC TGC TAG ACC ACC AAA TG-3.All mutations in the expression plasmids were introduced using the QuikChange II XL site-directed mutagenesis kit  with the following primers and verified by sequencing: for the LPAY/PNAP mutation  as recommended by the manufacturer.The avian DFJ8 cell line that stably expresses the murine ecotropic receptor MCAT-1 293T cells were seeded in a 6-well plate  and transfected the following day with wild type, chimeric, or mutant MLV GagPol plasmids, a Friend MLV Env expression plasmid, and a MLV LTR-LacZ reporter plasmid. In experiments where the ability of HECT Ub ligases or Ub ligase fragments were used to enhance or inhibit viral release, various amounts of YFP-fusion protein expression plasmid was cotransfected with the viral constructs as indicated. At 24 h after transfection, the cell culture medium was clarified by low speed centrifugation and subsequently filtered through a 0.45-µm-pore-size syringe filter before isolation of virus particles by centrifugation at 13,000 rpm for 60 min. After centrifugation, the supernatant was removed and the virus pellet was suspended in 50 µl NP-40 lysis buffer  then boiled in 1X SDS sample buffer for 10 min at 95°C. Cells were washed with 1X PBS and lysed in 500 µl of NP-40 lysis buffer for 20 min on ice. Cell lysates were cleared by centrifugation at 13,000 rpm for 10 min, and the supernatant was collected and boiled in 1X SDS sample buffer for 10 min at 95°C. Proteins in cell or virus lysates were separated on a 10% SDS acrylamide gel and transferred to a nitrocellulose membrane. The membranes were probed with a rat monoclonal anti-MLV CA antibody (1∶1000) or a rabbit polyclonal anti-GFP antibody , followed by detection with a peroxidase-conjugated secondary antibody using chemiluminescence. Membranes that were stripped for loading controls were reprobed with goat polyclonal anti-β-actin antibody . Proteins were quantified by using ImageGauge software version 4.22 (Fuji Film).As described above, virus particles were produced using transient transfection of 293T cells with viral expression plasmids. In experiments where the ability of HECT Ub ligases or Ub ligase fragments was used to enhance or inhibit viral release, various amounts of YFP-fusion protein expression plasmid were cotransfected with the viral constructs as indicated. At 24 h post-transfection the cell culture medium was clarified by low speed centrifugation and passed through a 0.45-µm-pore-size syringe filter. Subconfluent cultures of DFJ8 target cells were infected with dilutions of clarified media containing virus. At 24 h after infection, β-galactosidase activity in infected target cells was determined with a β-galactosidase assay kit (Stratagene) and amounts of infectious MLV produced were quantified by counting blue stained target cells.129PPAYRPPNAP138 sequence constitutes a genuine L-domain motif that directs release of a heterologous virus, expression constructs were generated to encode an MLV GagPolCore chimera protein where the endogenous PPPY L-domain of Gag was replaced with the putative Core L-domain motif  and with tyrosine for phenylalanine at Core position 132 (GagPolPPAF/PNAP). An additional construct was made with tyrosine 132 mutated to alanine (GagPolPPAA/PNAP) as this mutation abolishes HBV capsid assembly and therefore cannot be analyzed in the context of replicating HBV PPAY/PNAP, GagLPAY/PNAP, GagPPAF/PNAP, or GagPPAA/PNAP. Media and cellular lysates were harvested from virus-replicating cells 1 day post transfection and resolved by SDS-PAGE to detect virion-associated Gag or cellular-associated Gag protein by western blot  of full-length expression constructs for the Nedd4 Ub ligase family members Nedd4, Nedd4L, WWP1, WWP2, Smurf1, Smurf2, and Itch in cells co-expressing wild-type GagPol, GagPol4A, GagPolPPAY/PNAP, or the GagPolPPAF/PNAP partial release mutant. The effects on infectious virion release and virion-associated Gag proteins were then examined. Three criteria were applied to identify the specific HECT ligases that are important for L-domain dependent release of the MLV GagPolPPAF/PNAP mutant chimera  of HBV Core, we found that the PPAY motif at positions 129–132 was able to promote viral release and thus exhibits L-domain activity. Mutation of key motif residues within the L-domain chimeras  reduced viral release, further supporting the argument for L-domain activity by the HBV Core sequence. While it is intriguing that an additional L-domain-like motif is found adjacent to the PPAY motif, a PNAP sequence at position 135–138, this sequence did not exhibit L-domain activity. Although not the canonical P(T/S)AP sequence, the PNAP motif has previously been described in 5-lipoxygenase as a potential interaction site for cytoskeletal proteins and proline-rich SH3 binding domain in Grb2, an adaptor protein for tyrosine kinase-mediated cell signaling These studies show that an HBV Core amino acid sequence containing the PPAY motif can functionally substitute for the MLV Gag L-domain to promote viral release by recruitment of Nedd4 family Ub ligases. Given that some L-domains from different viruses are functionally interchangeable, the use of L-domain swapping experiments can serve as a tool to identify viral sequences that exhibit L-domain activity LPAY/PNAP, GagPolPPAF/PNAP, and GagPolPPAA/PNAP L-domain mutant chimeras, while less than wild-type MLV, was not as strongly reduced as that observed for the GagPol4A mutant that lacks L-domain activity. This indicates that the L-domain mutants demonstrate a partial release phenotype that may be attributed to residual L-domain binding of host factors that direct viral release. Such L-domain mutants have been previously used to identify HECT ligases that can restore release and are therefore identified as potential binding partners for the viral L-domain motif PPAF/PNAP as demonstrated by the increased levels of virion-associated p30 CA, which is indicative of mature virion release. Interestingly, while the total level of virion-associated Gag increased with Nedd4 and WWP1 overexpression, the ratio of p30 CA to Pr65 Gag also increased with a correlative increase in the infectivity of target cells. Although maximum target cell infectivity by virions produced in cells expressing the L-domain chimera mutant corresponded to the peak production of virion-associated p30 CA, infectivity did not reach levels comparable to the GagPolPPAY/PNAP chimera that contains a functional L-domain (data not shown). This was unexpected considering that maximum p30 CA levels with Nedd4 or WWP1 ligase overexpression were comparable to the p30 CA levels released by GagPolPPAY/PNAP.The levels of virus release by the GagPolPPAF/PNAP in a PPxY-dependent manner by overexpressing ligase WW-domain fragments in GagPolPPAY/PNAP to determine if release would be inhibited. Surprisingly, the Nedd4 WW-domain did not inhibit release of the L-domain chimera. While it may be that Nedd4 binds to the Core L-domain but is not required for promoting viral release similar to what is observed in HIV-1 and Ebola L-domain chimera studies We attempted to confirm that Nedd4 and WWP1 enhanced release of GagPolPPAF/PNAP but instead was comparable to the total level of Gag produced by GagPolPPAY/PNAP. Additionally, low levels of Itch overexpression significantly reduced the amount of Pr65 Gag released by GagPolPPAF/PNAP and significantly increased p30 CA levels. The maximum level of p30 CA produced by GagPolPPAF/PNAP due to Itch overexpression was comparable to p30 CA levels released by the functional L-domain chimera, but unlike Nedd4 and WWP1 overexpression, Itch enhanced virion infectivity to significantly higher levels and decreased virion infectivity of wild-type MLV and the GagPolPPAY/PNAP L-domain chimera. Surprisingly, Itch overexpression also significantly enhanced production of infectious virions released by GagPol4A expressing cells indicating this effect could act in an L-domain independent manner. The observation that low levels of Itch overexpression significantly increases infectivity for both GagPolPPAF/PNAP and GagPol4A suggests that Itch may correct Gag processing defects of the MLV L-domain mutants to enhance release of infectious virions. However, in contrast to the WW-domain of Nedd4L ligase that also acts in an L-domain independent manner to stimulate release of GagPol4A, overexpression of the Itch WW-domain in GagPolPPAY/PNAP replicating cells significantly inhibited release of the functional L-domain chimera demonstrating that Itch can act in an L-domain dependent manner as well. These results suggest that Itch can rescue the release defects of L-domain mutants by associating with Gag either directly through the PPxY motif or through another as yet unidentified motif within Gag, and/or indirectly through Gag-associated proteins.In contrast to Nedd4 and WWP1 overexpression, Itch overexpression did not increase the total level of virion-associated Gag produced by GagPolDespite recent evidence supporting the involvement of the cellular ubiquitin pathway, the exact mechanisms and host factors involved in HBV assembly and release are still not fully understood. Microscopy studies have demonstrated that HBV proteins traffic to intracellular vesicles reminiscent of MVBs or late endosomes Although an interaction between Nedd4 and HBV Core had previously been demonstrated Retrovirus assembly and release is traditionally thought to occur primarily at the plasma membrane and is therefore a distinct pathway from the intracellular assembly and envelopment of HBV virions. However, recent studies have shown that retroviral release can also occur through the MVB pathway


The S-domain serine/threonine receptor-like kinases (SRLKs) comprise one of the largest and most rapidly expanding subfamilies in the plant receptor-like/Pelle kinase (RLKs) family. The founding member of this subfamily, the S-locus receptor kinase (SRK), functions as the female determinant of specificity in the self-incompatibility (SI) responses of crucifers. Two classes of proteins resembling the extracellular S domain  or the intracellular kinase domain  of SRK are also ubiquitous in land plants, indicating that the SRLKs are composite molecules that originated by domain fusion of the two component proteins. Here, we explored the origin and diversification of SRLKs by phylogenomic methods.Based on the distribution patterns of SRLKs and SRLCKs in a reconciled species-domain tree, a maximum parsimony model was then established for simultaneously inferring and dating gene duplication/loss and fusion /fission events in SRLK evolution. Various SRK alleles from crucifer species were then included in our phylogenetic analyses to infer the origination of SRKs by identifying the proper outgroups.Two gene fusion events were inferred and the major gene fusion event occurred in the common ancestor of land plants generated almost all of extant SRLKs. The functional diversification of duplicated SRLKs was illustrated by molecular evolution analyses of SRKs. Our findings support that SRKs originated as two ancient haplotypes derived from a pair of tandem duplicate genes through random regulatory neo-/sub- functionalization in the common ancestor of the Brassicaceae. S locus. SLG, which was the first S locus-derived gene identified, encodes a secreted glycoprotein, whereas SRK encodes a transmembrane receptor kinase with an extracellular domain that shares extensive sequence similarity with SLG [S haplotype-specific binding of SRK to its cognate pollen-borne ligand S locus cystein rich protein/S locus protein 11 (SCR/SP11) activates the SRK and triggers a signaling cascade that culminates in “self-pollen” rejection [Arabidopsis thaliana also suggested that SRLKs are not monophyletic and probably arose via independent recruitment of S domains [The “S domain” (SD) was initially defined by the S-locus glycoprotein (SLG) and the S-locus receptor kinase (SRK), which are encoded by two closely-linked genes in the Brassica self-incompatibility (SI) determining locus, the with SLG . SRK is ejection ,3. The eejection ,5, whereejection ,7. With ejection -11. In aejection ,12. Inteejection -15, sugg domains .Gene fusion is considered to be an important evolutionary path to create novelty in protein architectures (the linear arrangement of protein domains) and functions by forming composite proteins and linking components of extant signaling/biochemical pathways . In agreS haplotypes took place after the separation of the two genera [S-haplotype system causing incomplete suppression of selfing, with further differentiation among S haplotypes and enhancement of SI expression having evolved subsequently [As the only members of the SRLK subfamily whose function is known, SRKs are well suited to investigate the functional diversification of SRLKs. In Brassica species but not Arabidopsis species, SRKs may be clearly divided into two classes, class I and class II . Moreoveo genera . Furtherequently ,31. ThisIn this study, we first retrieved SRLKs from five sequenced genomes representing the major lineages of land plants. SRLCKs were then delineated by their close phylogenetic relationship to SRLKs based on a maximum likelihood (ML) kinase domain tree. On the basis of a reconciled species-domain tree including both SRLKs and SRLCKs, gene duplication/loss and fusion events in SRLK evolution were inferred and dated by integrating the maximum parsimony ancestral architecture inference algorithm ,26 into SRLKs are composed principally of three modular domains in a configuration of S domain (SD)-transmembrane domain (TM)-kinase domain (KD). SDs are further divided into three subdomains in a configuration of B_lectin-SLG-PAN_APPLE Figure . The B_lChlamydomonas reinhardtii and Ostreococcus tauri, whereas sequences of stand-alone SLG domains or any combination of B_lectin, PAN_APPLE, SLG, and KD domains were not detected. In view of the fact that plant RLKs were likely generated after the divergence of land plants from green algae [Physcomitrella patens (moss), Selaginella moellendorffii (spikemoss), Oryza sativa (rice), Arabidopsis thaliana (Arabidopsis), and Populus trichocarpa (poplar) respectively  or SLGs (B_lectin-SLG-PAN_APPLE) were retrieved and are hereafter referred to as typical SRLKs and SRLPs respectively  phylogenetic tree , because its function in SI has been demonstrated [Capsella rubella is also included [The s Figure . We thuss Figure A. A funcnstrated . In addiincluded .A. thalina non-SRK SRLKs, Arabidopsis Receptor Kinase (ARK1/ARK2/ARK3), do not fall within the outgroups in the kinase domain tree  and most SRKs from A. lyrata, A. halleri, and Capsella grandiflora also form a distinct orthologous group in the Arabidopsis/Capsella lineage. In addition, Brassica class-I SRKs and Arabidopsis/Capsella SRKs (except for AlSRK20) appear to form a large orthologous group in Brassicaceae, albeit with a relatively low bootstrap value  and ARK3 (At4g21380) are located in different orthologous groups and are arranged in tandem  of the most recent common ancestors (MRCAs) at the leaf nodes with lost genes can only be inferred by the parsimony principle  and ancestor of SRKII (A_SRKII). Subsequently, random neo-fuctionalzation and/or sub-funtionalization of A_SRKI and A_SRKII produced two ancient S haplotypes, from which SRKs might have been derived by further diversification , ARC1 homologs of A. thaliana[Functional diversification following the origination of multiple-domain proteins is a common theme in studies of protein family evolution -43. Althe Figure B. Since n Figure . This mosicaceae ,31. Howes Figure  and the and PLENA as well rawberry . Paralogications . Howevers Figure , suggest thaliana. In view thaliana-53, the Physcomitrella patens (moss), Selaginella moellendorffii (spikemoss), Oryza sativa (rice), Arabidopsis thaliana (Arabidopsis), and Populus trichocarpa (poplar), which represent the major lineages in land plant evolution, were used in this study. The annotated protein sequences of the 5 sequenced genomes were downloaded (http://www.phytozome.net). To identify SRLK and SRLP sequences, an HMMer search was performed by the standard profiles of the modular domains of S-domains [http://bioinformatics.psb.ugent.be/plaza/), we thus retrieved RLCK sequences from the homologous group , and then updated each RLCK sequence with the best hit from Phytozome v9.0 using BLASTP. At last, these hits filtered for only one kinase domain using Pfam, were used as candidates for SRLCKs , ARK1 (At1g65790), ARK2 (At1g65800), and ARK3 (At4g21380) sequences as well as 7 SRLKs from rice and poplar form a well-supported subgroup. Kinase domain sequences from these 11 proteins and Brassicaceae 47 SRK sequences , gene duplicate- on/loss and fusion events were inferred and dated. Based on the reconciled tree, the ancestral protein architectures were inferred by a modified maximum parsimonious protein ancestral architecture inference algorithm [A reconciled species-domain tree was generated using the Notung program, which offers a unified framework for incorporating duplication-loss parsimony into phylogenetic analysis . After rlgorithm ,26. The AlSRK and AlARKs in A. lyrata organs. Total RNA from root, stem, leaf, stamen, style, and stigma tissues of A. lyrata was isolated using the Trizol reagent  according to the manufacturer’s instructions. The residual genomic DNA in the total RNA was removed by treatment with RNase-free DNaseI and the total RNA was further purified with phenol chloroform-isoamyl alcohol. RT-PCR was performed using SuperScript™II RNase HI Reverse Transcriptase  using the following primer pairs: 5′-GACAACGCGTGTGAGACCTAT-3′ and 5′-CATTAGG AGCTGCAGTTGCTC-3′ for AlSRKa, and 5′-GACCAATGCGATGATTACAAAG -3′ and 5′-CAGTAGCTGTTTCAATTAGT-3′ for AlARK3. The PCR conditions of AlSRKa/AlARK3 were 94°C for 3 min followed by 40 cycles of the following: 94°C for 30 s, 58°C/52°C for 30 s, and 72°C for 40 s. The amplification products were then analyzed with agarose gel electrophoresis.RT-PCR was used to examine the spatial expression of The data sets supporting the results of this article are included within the article S-locus receptor kinase; SI: Self-incompatibility; SRLP: S-domain receptor-like protein; SRLCK: S-domain receptor-like cytoplasmic kinases; SD: S domain; SLG: S-locus glycoprotein; SCR/SP11: S locus cystein rich protein/S locus protein 11; KD: Kinase domain; ARC1: Arm Repeat Containing; TM: Transmembrane domain; ARK1/ARK2/ARK3: Arabidopsis Receptor Kinase; MRCA: Most recent common ancestor; A_SRK: Common ancestor of Brassicaceae SRKs; A_SRKI: Ancestor of SRKI; A_SRKII: Ancestor of SRKII; AtPUB-ARM: Arabidopsis plant U-box/ARM-repeat; RIO1: Right Open Reading 1.SRLK: S-domain serine/threonine receptor-like kinase; RLK: plant receptor-like/Pelle kinase; SRK: The authors declare that they have no competing interests.SX and PL participated in the design of study, and conducted the phylogenetic analysis. PL and SX prepared the manuscript. ML carried out lab work. All authors participated in reading and approving the manuscript.List of SRLK and SRLP sequences retrieved from Phytozome v9.0 and homologous RLCK sequences retrieved from Plaza v2.5 databases.Click here for fileA maximum likelihood (ML) phylogenetic tree of KD sequences from 392 SRLKs and 96 homologous RLCKs. The distribution patterns of SRLKs (filled triangles) and SRLCKs (open triangles) from five plants are shown in the same color scheme as in Figure 2. The domain fusion events are also shown in the same color scheme as in Figure 2.Click here for fileInference of gene duplication/loss and fusion events in SRLK evolution. The architectures of leaves are indicated by the minus sign for SRLCKs. Gene fusion events are indicated by filled circles at internal nodes. Gene duplication events are indicated by filled rectangles at internal nodes and gene loss events are indicated by “LOST” at the leaves.Click here for fileList of Brassicaceae SRKs sequences retrieved from UniProt.Click here for fileBrassica class-I, Brassica class-II, and Arabidopsis/Capsella as well as out groups are shown.A ML S-domain tree constructed by including 7 SRLK members in SD-1 g group and 47 full length SRKs from Brassicaceae species as in Figure 6. The aLRT bootstrap values of the major internal nodes are indicated by numbers. Three main clusters of Click here for fileSupporting dataset 1. Alignment of KD sequences from 392 SRLKs and 96 homologous RLCKs.Click here for fileSupporting dataset 2. Alignment of KD sequences from 47 Brassicaceae SRK sequences.Click here for file


These are typical motifs of extracellular proteins that mediate protein–protein interactions. We have reconstructed the phylogeny of this new family of oxidoreductases and show the presence of four invertebrate clades as well as one vertebrate clade that includes also two different human representatives. The variability of domain assembly in the various clades was analyzed, as was the occurrence of relevant catalytic residues in the peroxidase domain based on the knowledge of catalysis of the mammalian homologues. Finally, the few reports on expression, localization, enzymatic activity, and physiological roles in the model organisms Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens are critically reviewed. Roles attributed to peroxidasins include antimicrobial defense, extracellular matrix formation, and consolidation at various developmental stages. Many research questions need to be solved in future, including detailed biochemical/physical studies and elucidation of the three dimensional structure of a model peroxidasin as well as the relation and interplay of the domains and the in vivo functions in various organisms including man.Peroxidasins represent the subfamily 2 of the peroxidase-cyclooxygenase superfamily and are closely related to chordata peroxidases (subfamily 1) and peroxinectins (subfamily 3). They are multidomain proteins containing a heme peroxidase domain with high homology to human lactoperoxidase that mediates one- and two-electron oxidation reactions. Additional domains of the secreted and glycosylated metalloproteins are type C-like immunoglobulin domains, typical leucine-rich repeats, as well as a  Based oDrosophila , imp, impDrosdysozoan . Finallydysozoan . In the dysozoan , localizdysozoan , enzymatdysozoan –18, but i.e., invertebrate and vertebrate peroxidasins, and discuss the function of the enzymatic domain based on the knowledge about catalysis of human peroxidases , al, al14], in vivo function of peroxidasins. Focusing on the enzymatic domain only, one could speculate about similar biological roles as reported for the closely related and homologous chordata peroxidases including MPO, EPO, and LPO. The latter are involved in innate immune-defense reactions against invading pathogens and are stored in high concentration in granules of neutrophilic (MPO) or eosinophilic leukocytes (EPO) that are recruited to sites of pathogen invasion and inflammation , wh, whin viammation . These pe.g., MPO) can have both damaging and protective effects on the ECM, including inactivation of metalloproteinases [Another physiological role of chordata peroxidases is cell adhesion and formation of extracellular matrix (ECM). It has been demonstrated that the extracellular oxidants generated by cationic mammalian peroxidases  might give some indications about the functionality of peroxidasins, although from this subfamily (that has no vertebrate representative) only very few reports can be found in the literature. These metalloenzymes seem to be involved in the promotion of cell–ECM adhesion via their integrin-binding motifs . InteresClade 5). The motifs typical of extracellular proteins  might contribute to association with ECM. Both the roles in innate immune defense and extracellular matrix consolidation are often addressed in various contexts in the literature.Regarding the overall structure and size as well as domain assembly, peroxidasins are the most complex representatives of peroxidase-domain-containing enzymes of the peroxidase-cyclooxygenase superfamily. Sequence analysis clearly suggests an enzymatic functionality similar to LPO and EPO  vertebrate peroxidasin was performed on group 1 peroxidasin ( in 2005 . This re2O2 and do not release O2•− at reasonable rates.Investigations on human peroxidasins started in 1999 . It was via two ester bonds very similar to LPO or EPO [−). However, the reported activities were very low compared to mammalian peroxidases. The finding of chlorination activity at neutral pH, which so far was only attributed to MPO, is peculiar, since hsPxd01 cannot form the MPO-typical (e− withdrawing) sulfonium-ion linkage and thus this reactivity must be addressed in future biochemical studies.Finally, the cloning of human and mice peroxidasins and initial characterization of hsPxd01 were reported . Heme-coO or EPO . The pro2•− that dismutates to H2O2 necessary to initiate both peroxidase and halogenation activity.Based on its halogenation activity and localization, the role of hsPxd01 in low-density lipoprotein oxidation and endothelial cell apoptosis was investigated . It was Peterfi et al. [i et al.  demonstrIn another study , homozygin vivo activity and might contribute to extracellular matrix consolidation as well as antimicrobial defense. The additional domains  are typical for extracellular proteins and may help in targeting the peroxidase to its site of action. However, a distinct function of the individual domains and their relation to the reported in vivo roles has not been demonstrated so far. Clearly, more detailed biochemical and biophysical studies as well as the elucidation of the three-dimensional structure of peroxidasins are necessary. These studies must include invertebrate and both groups of vertebrate peroxidasins, since – as sequence analysis suggests – they most probably differ in catalysis and, in consequence, in function.Summing up, peroxidasins are multidomain heme peroxidases with a substrate spectrum similar to mammalian peroxidases. Based on sequence analysis and reported biochemical data, they perform one-electron oxidation reactions including tyrosine oxidation as well as two-electron oxidation reactions of (pseudo-) halides. Hypochlorous acid formation is questionable and must be addressed in future investigations. In any case, physiological studies have shown that the enzymatic domain is essential for the Austrian Science Fund and the doctoral program BioToP – Biomolecular Technology of Proteins (FWF W1224).This project was supported by the Uniprot, NCBI, PeroxiBase, Ensembl). Selected representatives of non-mammalian vertebrate peroxidase , HsTPO Human thyroid peroxidase, and Aedes aegypti peroxinectin (AaePxt01) sequences were retrieved from the PeroxiBase database [Table.All currently (December 2011) available 106 peroxidasin sequences were collected from public databases  method of the MEGA5 package [Jones–Taylor–Thornton (JTT) model of amino acid substitution, pairwise deletion of gaps, and homogenous pattern among lineages. The test of phylogeny was conducted with the bootstrap method, by setting the bootstrap replications to 1000. The optimized γ parameter for this set was determined as γ = 1.75. package  with theThe phylogeny was also reconstructed using the ME method of the MEGA5 package  with folAdditionally, the ML trees were inferred with the MEGA5 package  by usingProtein secondary and tertiary structure prediction of the various domains of peroxidasin was performed with PSIPRED  and EsyP


Short C-terminal domain). This domain is widespread in bacteria with over a thousand examples. But we found it is missing from the most commonly studied model organisms, despite being present in closely related species. It's predominantly C-terminal location, co-occurrence with numerous other domains and short size is reminiscent of the Gram-positive anchor motif, however it is present in a much wider range of species. We suggest several hypotheses about the function of SHOCT, including oligomerisation and nucleic acid binding. Our initial experiments do not support its role as an oligomerisation domain.We have identified a new protein domain, which we have named the SHOCT domain ( Protein domains are regarded as the structural and functional components of proteins. These domains are globular, independently folding units. Many proteins also contain motifs, which are shorter structural elements One of our goals in the Pfam database of protein domains and families is to increase our coverage of known protein sequences Anaerotruncus colihominis DSM 17241, a bacterium that was isolated from Human faeces and may be of clinical significance Clostridium and Ruminococcus.In this paper we describe the identification and preliminary analysis of a domain that is prevalent in bacteria. This domain was discovered in a protein from a Firmicute, http://hmmer.janelia.org/) Anaerotruncus colihominis DSM 17241 (UniProtKB accession B0PET9.1) using the default parameters against the UniProtKB database et al. 2012We initially identified the SHOCT domain using the Jackhmmer program, which forms a part of the HMMER3 package written by Sean Eddy . Proteins were blotted onto PVDF membranes (Amersham) and blocked in 2% BSA. Membranes were incubated with peroxidase-conjugated streptavidin (Jackson Immuno Research), washed in 1×PBS 0.1% tween 20 (Sigma) and proteins detected using SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific).Purified proteins were resolved by SDS-PAGE under reducing conditions using NuPAGE 4–12% Bis Tris precast gels (Invitrogen) and visualised by Coomassie Brilliant Blue R-250 (Sigma) staining.Proteins were purified from 300 ml of spent tissue culture supernatant by Nickel affinity chromatography on 1 ml His-Trap columns  following manufacturer's instructions using an ÄKTA Xpress . Purified proteins were further analysed by size exclusion chromatography on a Superdex 2000 Tricorn 10/600 column in HBS-EP buffer . Molecular masses were calculated based on the elution volumes compared to known protein standards .A short conserved domain was found near the N-terminus of our query protein (UniProtKB accession B0PET9.1), this domain is found in 1,403 sequence regions on 1,381 different proteins from 882 distinct species in UniProt release 2011_06 SHOrt C-Terminal domain). The entire domain is found within 50 amino acids of the C-terminus of the sequence in 89.2% of proteins in which it is found (in Pfam release 26.0).The SHOCT domain was so named because it is a short domain present at the C-terminus of many proteins (http://www.compbio.dundee.ac.uk/www-jpred/) The domain is around 30 amino acids in length and contains a strongly conserved glycine, a strongly conserved glutamic acid, two additional reasonably well-conserved charged amino acids, and five reasonably well conserved hydrophobic amino acids . The secWe have termed this a domain, but we cannot rule out the possibility that SHOCT is in fact a motif. There are few structural domains that are as short as SHOCT, and those that do exist tend to be stabilised by disulphide bonds and/or interactions with metal ions as found in zinc finger domains. The lack of conserved cysteine and histidine residues in SHOCT indicates this this is not the case here. There are exceptions to this, one such short domain is the WW domain, which binds to proline-rich proteinsAs a control computational experiment to ensure that our HMM model was not identifying spurious matches, we searched the UniProtKB database with a reversed version of the SHOCT seed alignment using the same parameters. None of the hits to this reversed alignment had a bit score of greater than or equal to our selected sequence inclusion threshold of 24.0 bits . There iNaegleria gruberi, and the slime mold Dictyostelium discoideum. The SHOCT domain is only found in one species of higher plant  and one insect species (Culicoides sonorensis). The R. communis protein SHOCT is found in, UniProtKB accession B9TKH1.1, only has similarity to bacterial proteins and not to other eukaryotic proteins. It is likely that this is not a true plant protein and that it arises from bacterial contamination. The insect protein is a fragment arising from a translation of an expressed sequence tag (EST), and has low similarity to any other proteins in UniProtKB.In Pfam release 26.0, the SHOCT domain is found in proteins from 882 distinct species. These are mainly bacteria (790 species) and archaea (53 species). It is widely distributed amongst both Gram-positive and Gram-negative bacteria, with 29.6% of the bacterial species being Firmicutes, 25.3% Proteobacteria, 19.1% Actinobacteria, 5.2% Bacteroidetes and 4.1% Cyanobacteria. Besides the bacteria and archaea, it is also found in proteins from 15 different bacteriophage species, 19 eukaryotic species and a small number of unclassified species from metagenomic studies. The eukaryotic species include trypanosomes, the amoeba Bacillus including one strain of B. subtilis, but is not found in Bacillus subtilis strain 168Pseudomonas species, but absent from Pseudomonas aeruginosa. Of the 23 bacterial model organisms analysed by Hedges (2002), the SHOCT domain is only present in 6 . The band 7 proteins are physiologically important proteins found in both prokaryotes and eukaryotesThe SHOCT domain is associated with many domains found in oligomeric proteins . It is mPleckstrin homology (PH) domains are found in a large number of eukaryotic proteins involved in intracellular signalling and cytoskeletal regulation There are nine Trypanosome sequences in which SHOCT domain is present with a CorA domain (PF01544), in these sequences the SHOCT domain occurs at the N-terminus. CorA proteins are divalent metal ion transporters. In bacteria, CorA forms a funnel-shaped pentameric structure with two transmembrane helices towards the C-terminus of the protein Rhizobium loti reveals that it is a homotetramer comprised of two dimers. Each subunit contains transmembrane helices in the N-terminal half of the protein and a cyclic nucleotide-binding domain (PF00027) at the C-terminus. The cyclic nucleotide-binding domain is often associated with ion channels, in the R. loti protein this domain appears likely to mediate dimerisation Rattus norvegicus small-conductance Ca2+-activated K+ channel (SK2) is an ion channel which includes a central ion channel domain, this protein contains a calmodulin-binding domain  at the C-terminus. CaMBD consists of two long alpha-helices, which form dimers The SHOCT domain is present at the C-terminus of three proteins with an ion channel domain (PF07885). The structure of a protein containing an ion channel domain from The SHOCT domain is also present at the C-terminus of one protein in an ion transport family (PF00520). This family is in the same clan as the ion channel domain (PF07885). The human voltage-gated hydrogen channel 1 (HV1) is a member of the ion transport family. This protein dimerises through interactions between coiled coils at its C-terminus In addition to ion transport channels, SHOCT domains are also associated with protein transport channels. Tic20 (PF09685) is a channel protein that forms a part of the Tic complex, which is responsible for protein precursor import into chloroplasts The above examples include proteins where short alpha helical domains or motifs at the C-termini are responsible for oligomer formation, including stomatin, major vault protein and the CorA ion transport familyChlamydia trachomatis protein kinase pkn1 contains both an N-terminal protein kinase and a C-terminal formylglycine generating sulfatase domain. This enzyme has been shown to interact with, and phosphorylate, the membrane protein IncG and may play a role in host-pathogen interactions The SHOCT domain is found in several different enzymes . One proMagnetospirillum magneticum contains a protein kinase domain (PF00069) and a C-terminal SHOCT domain. The protein kinase domain is found in association with numerous different domains. Amongst the most commonly associated domains are the PASTA domain (PF03793), which binds to the beta-lactam rings of antibiotics Several other kinases contain a SHOCT domain. A predicted serine/threonine kinase from the proteobacterium The SHOCT domain is often found associated with the phospholipase D-nuclease N-terminal domain (PF13396), which is frequently found at the C-terminus of cardiolipin synthetase enzymes. The probable active site of these enzymes is not in the phospholipase D-nuclease N-terminal domain but is likely to be in a duplicated PLD-like domain (PF00614) The SHOCT domain is found in association with several different domains that are involved in binding to proteins and binding to DNA .It is found in a number of putative DNA-binding proteins. In eight cases it occurs at the C-terminus of proteins containing the double zinc ribbon domain (PF12773). This domain is a member of the zinc-beta ribbon superfamily Thermotoga maritima. DisA functions as a diadenylate cyclase. It consists of three domains: a globular N-terminal nucleotide binding domain (PF02457), which binds a cyclic diadenosine phosphate, a central alpha-helical domain  (PF10635) and a C-terminal helix-hairpin-helix motif (PF00633). DisA forms octamers, the crystal structure of T. maritima DisA reveals that the formation of octamers is largely mediated by interactions between two of the alpha helices in the helical spine Two proteins containing the SHOCT domain have a TM2 domain (PF05154) at the N-terminus, a SHOCT domain in the centre of the protein and a helix-hairpin-helix motif (PF12836) at the C-terminus. The TM2 domain consists of a short pair of transmembrane alpha-helices connected by a short linker, the biological function of this domain is unknown. The helix-hairpin-helix motif is a short DNA-binding motif In addition to DNA-binding domains, the SHOCT domain is also found in several proteins that contain protein-binding domains. Amongst these are three proteins that contain an N-terminal SHOCT domain followed by a duplicated PUB domain (PF09409), and one protein containing a PUB domain and a C-terminal SHOCT domain. PUB domains, which are also known as PUG domains, are found in peptide-N-glycanases where they bind to the AAA ATPase p97 Naegleria gruberi, in the majority of these proteins one SHOCT domain is present, usually at the C-terminus. Two proteins from Naegleria gruberi contain three copies of the SHOCT domain, and one protein (UniProtKB accession D2VS68.1) contains eight repeats of the SHOCT domain. In these proteins containing multiple SHOCT domains the repeats are not identical.Although most SHOCT domain-containing proteins only include one copy of SHOCT there are several proteins that contain multiple copies. The SHOCT domain is found in 14 proteins from the amoeba We have identified a new protein domain that is present in proteins with various functions from a wide range of predominantly bacterial species. We have named this the SHOCT domain. Its widespread distribution suggests that it may have an important function. It is surprising that it has not been discovered previously, but this may be because it is missing from well-studied species. One possible hypothesis for the function of this domain is as an oligomerisation domain, we tested this hypothesis experimentally and found that it did not appear to function as an oligomerisation domains.Another possible hypothesis for the function of the SHOCT domain is that it may bind to something other than itself. It may bind to other protein domains/motifs or to nucleic acid. It's secondary structure is reminiscent of the helix-hairpin-helix motif, with both consisting of a pair of alpha helices separated by a small number of amino acids. It is therefore possible that SHOCT is a distant evolutionary relative of other helix-hairpin-helix proteins and may therefore bind to DNA.Alternatively, SHOCT may function as a localisation domain. Its predominantly C-terminal location is similar to that of the Gram-positive cell wall sorting LPXTG motif, which is required for the anchoring of surface proteins to the bacterial cell wall Table S1Sequences of expressed peptides.(DOCX)Click here for additional data file.Table S2Primer sequences. The underlined nucleotides represent the AscI and NotI restriction sites.(DOCX)Click here for additional data file.Table S3Domain architectures of SHOCT domain-containing proteins.(DOCX)Click here for additional data file.


Protein-protein interactions (PPIs) are frequently mediated by the binding of a modular domain in one protein to a short, linear peptide motif in its partner. The advent of proteomic methods such as peptide and protein arrays has led to the accumulation of a wealth of interaction data for modular interaction domains. Although several computational programs have been developed to predict modular domain-mediated PPI events, they are often restricted to a given domain type. We describe DomPep, a method that can potentially be used to predict PPIs mediated by any modular domains. DomPep combines proteomic data with sequence information to achieve high accuracy and high coverage in PPI prediction. Proteomic binding data were employed to determine a simple yet novel parameter Ligand-Binding Similarity which, in turn, is used to calibrate Domain Sequence Identity and Position-Weighted-Matrix distance, two parameters that are used in constructing prediction models. Moreover, DomPep can be used to predict PPIs for both domains with experimental binding data and those without. Using the PDZ and SH2 domain families as test cases, we show that DomPep can predict PPIs with accuracies superior to existing methods. To evaluate DomPep as a discovery tool, we deployed DomPep to identify interactions mediated by three human PDZ domains. Subsequent in-solution binding assays validated the high accuracy of DomPep in predicting authentic PPIs at the proteome scale. Because DomPep makes use of only interaction data and the primary sequence of a domain, it can be readily expanded to include other types of modular domains. Intrinsic specificity determinants, such as the amino acid sequence of a domain and the characteristics of its ligand-binding site, are context-independent because they are retained even in the isolated domains in silico models to predict novel PPI events Cellular signal transduction is regulated by the formation and dissolution of specific protein complexes. These complexes is often mediated by modular protein-interaction domains such as the Src homology 2 (SH2) or 3 (SH3) and the Post-synaptic density protein, Disc large, Zonula occludens-1 (or PDZ) domain that are capable of binding partner proteins via short peptide motifs in silico prediction of binding partners for that domain or for other members of the same domain family. Scansite et al.et al.Intrinsic specificity information of a domain, obtained from sequence analysis and/or experimental data on domain-peptide binding, may be harnessed for While the above methods make use of specificity and sequence information for PPI prediction, other approaches exploit the binding site characteristics of the relevant domain-peptide complex structures. For instance, a position-specific domain-ligand-contact frequency matrix could be constructed from the contacting residues in the corresponding domain-peptide complex structures To overcome limitations of the existing methods for domain-based PPI prediction, we developed DomPep. Unlike previous methods that group related domains according to a single parameter , DomPep identifies specificity-similar domains by combining two parameters, namely DSI and Position-Weighted-Matrix (PWM) distance. PWM, represented by the sequence logo of the bound peptides Using this new strategy of domain classification, we have built DomPep prediction models for the SH2 and PDZ domains that differ significantly in specificity. The accuracy of prediction in each case was benchmarked against experimental data DomPep employs both the DSI and PWM distance to assess the similarity in specificity for two domains. The PWM of a domain is a matrix  are used to calculate the PWM distance (in vitro and in vivo), the specificity-similar domains to a query domain are identified. These domains are iteratively grouped together with the query and the binding data for each group of the domains are employed to construct prediction models. These models are compared, using leave-one-out cross-validation, with the one constructed using the peptides of the query itself and the model with the best accuracy is retained as the final model for the query. The same procedure is repeated for every domain for model construction. We employed the SVM algorithm to build the DomPep models DomPep comprises two modules – model construction and PPI prediction . The moddistance . Domain The utility of the DomPep was demonstrated on the PDZ and SH2 domain families that have distinct specificities and for which comprehensive binding data are available from large-scale proteomic analysis and conventional experiments d cutoff of 100 µM was applied, 731 positive interactions and 1,361 negative interactions were identified that involved 85 PDZ domains and 181 peptides d cutoff was adopted. The five C-terminal residues of the bound peptides were considered in our analysis as they were shown to be the major specificity-determinants in PDZ binding We analyzed the specificity of PDZ domains based on published data. MacBeath and colleagues recently conducted a study to characterize the specificity of 157 mouse PDZ domains using 217 peptides representing potential physiological PDZ-binding sites We first investigated the relationship between LBS and DSI. To ensure that the calculation for LBS and subsequently, for PWM distance was accurate, PDZ domains with fewer than 10 experimentally verified ligands were omitted . As showTo further explore the relationship between DSI and binding specificity, we calculated the PWM distances see . PWM disTo characterize the relationship between domain sequence identity and ligand-binding specificity, we plotted the percentile distribution of different DSI groups against the PWM distance. As shown in PDZ domains with 10 or more binding peptides identified from the arrays were analyzed in pairs to generate the corresponding DSI, PWM distance and LBS values. We used the corresponding LBS value to calibrate DSI and PWM distances. To this end, we arbitrarily set LBS = 0.7 as the cutoff to assign two PDZ domains similar (when LBS ≥ 0.7) or dissimilar (when LBS<0.7). This cutoff value was used, in turn, to determine the threshold values for DSI and PWM distance . In the We compared DomPep to two methods developed recently by MacBeath and coworkers −4, Wilcoxon signed-rank test; −4). Therefore, we concluded that the DomPep strategy leads to improved prediction accuracy.The training set was previously employed to construct both the MDSM and SPSSM models −3, Ideally a prediction method for domain-ligand interactions should be capable of identifying binding partners not only for domains included in the training set but also for family members not included in training. We therefore compared the performance of DomPep with that of SPSSM, a method that can be used for predicting binding partners of an untrained PDZ domain http://netphorest.info/and used to evaluate the accuracy of DomPep in predicting SH2-peptide ligand interactions. We selected peptide sequences corresponding to residues from positions –2 to +4 with respect to the phosphotyrosine (pY) because these residues are sufficient in conferring specificity to most SH2 domains studies The specificity of SH2 domains was systematically analyzed by probing an array of 6,200 phosphotyrosine-containing peptides respectively with 66 human SH2 domains We further investigated the relationship between PWM distance and LBS for SH2 domain pairs grouped according to DSI . A reverSimilar to PDZ domains, we considered SH2 pairs with LBS≥0.7 as having similar specificity. This threshold was used to determine the cutoffs for DSI and PWM distance, respectively. According to the above analysis , LBS≥0.7We evaluated the performance of SH2 DomPep models by comparing them to Netphorest The training set was based on the published SH2 domain-peptide array data Several differences between the two methods may have contributed to the improved performance of DomPep over NetPhorest for the eight SH2 domains tested. For example, the two methods employ different strategies for the identification of specificity-similar domains. Furthermore, while DomPep randomly selected negative peptides of the training set from the Swiss-Prot protein sequence database, NetPhorest used positive peptides of other domains with small DSIs as negative training peptides for the query domain . We found that the difference in performance originated largely from the different strategies by which the negative peptide training set was selected. DomPep showed comparable performance to NetPhorest when the same negative peptide sets were used as in the latter (data not shown). This is consistent with our observation  that twoin-vitro array binding data that may contain non-specific binding events.Because DomPep compared favorably to MDSM in identifying ligands for a PDZ domain included in the training set and to SPSSM in identifying binding peptides for an untrained PDZ domain, we next tested it whether it could reliably PPIs mediated by a PDZ domain on the genome scale. To this end, we employed three DomPep models to predict interactions between the Scrib PDZ domains and human proteins from the Swiss-Prot protein database. The three prediction models, which were generated for the mouse Scrib PDZ-1, PDZ-2 and PDZ-3 domains, were trained with 6, 5 and 30 positive peptide, respectively . Searchihttp://lilab.uwo.ca/DomPep.html) to predict binding ligands for the PDZ and SH2 domains . LBS is defined as the percentage of peptides commonly selected by a domain pair ij is the number of peptides selected by both domains i and j, BiNBj is the number of peptides that bind to domain i but not to domain j. LBS ranges from 0 to 1, where “1” indicates that both domains have the same set of binding and non-binding peptides, or identical specificity. This formula, suitable for processing the MacBeath dataset on the PDZ domain, needs to be modified to accommodate the SH2 domain-peptide array binding data used in NetPhorest Where BAccording to this formula, the LBS value for the ABL1 and ABL2 SH2 domains increases to 1  program SVMlight with the conventional linear kernel to build prediction models Peptide arrays were synthesized following established protocols Figure S1Phylogenetic tree of the human PDZ Domains. 5 pairs of domains with similar specificities selected as example are shown with different colors. They are the first PDZ domain in protein ZO2 (ZO2/1) and HtrA3 with SI = 15% (pink), DLG3/2 and MAGI3/2 with SI = 17% (grey), SHANK1 and PDZK1/1 with SI = 23% (blue), MAGI1/5 and MAGI3/4 with SI = 24% (green), LRRC7 and SCRIB/2 with SI = 36% (red). The first three pairs are identified by DomPep which have BS>0.7 based on PDZ domain arrays (TIF)Click here for additional data file.Figure S2Respective binding profiles for the SH2 domains from HSH2D (A), GRB7 (B), GADS (C) and for control GST (D) on arrays of phosphotyrosine-containing peptide ligands. These peptides are derived from the PhosphoSite database and were predicted to be highly connected to the four SH2 domains using SMALI program (TIF)Click here for additional data file.Table S1Comparison of DomPep with MSMD on predicting PDZ domain-ligand interactions.(DOC)Click here for additional data file.Table S2Mus musculus.Comparison of DomPep with SPSSM for ability to predict PDZ domain-ligand interactions for PDZ domains in other species than (DOC)Click here for additional data file.Table S3Comparison of DomPep with Netphorest with ANN models on predicting SH2 domain-ligand interactions.(DOC)Click here for additional data file.Table S4Peptides tested for binding the SH2 domains of GADS, GRB7 and HSH2D in the peptide array experiment.(DOC)Click here for additional data file.


However, very few biochemical data are available regarding this enzyme. This paper represents the first complete molecular characterization of a novel NMN deamidase from the halotolerant and alkaliphilic bacterium Oceanobacillus iheyensis (OiPncC). The enzyme was active over a broad pH range, with an optimum at pH 7.4, whilst maintaining 90 % activity at pH 10.0. Surprisingly, the enzyme was quite stable at such basic pH, maintaining 61 % activity after 21 days. As regard temperature, it had an optimum at 65 °C but its stability was better below 50 °C. OiPncC was a Michaelian enzyme towards its only substrate NMN, with a Km value of 0.18 mM and a cat/Kkm of 2.1 mM-1 s-1. To further our understanding of these enzymes, a complete phylogenetic and structural analysis was carried out taking into account the two Pfam domains usually associated with them (MocF and CinA). This analysis sheds light on the evolution of NMN deamidases, and enables the classification of NMN deamidases into 12 different subgroups, pointing to a novel domain architecture never before described. Using a Logo representation, conserved blocks were determined, providing new insights on the crucial residues involved in the binding and catalysis of both CinA and MocF domains. The analysis of these conserved blocks within new protein sequences could permit the more efficient data curation of incoming NMN deamidases. Nicotinamide mononucleotide (NMN) deamidase is one of the key enzymes of the bacterial pyridine nucleotide cycle (PNC). It catalyzes the conversion of NMN to nicotinic acid mononucleotide, which is later converted to NAD Salmonella typhimurium and Escherichia coli in the late 70s, as one of the reactions implicated in the Pyridine Nucleotide Cycle (PNC), which in turn, is associated with bacterial NAD+ salvage  . Un of Moco  was less binding .2+- and K+-dependent ADP-ribose (APDr) pyrophosphatase activity. In fact, ADPr docking in 3KBQ renders a similar picture . All the above described blocks are also compatible with the recently described results in which MocF domain, called COG1058 by Raffaelli’s group , is endo picture  to that  picture , but wit picture . In addiES, which in the archaea lineage 1.3 is followed with a conserved xhAP. The block i-II was conserved in all lineages with a consensus sequence EVxhR/Kh, except for lineage 1.3. No information on the structure of the interdomain segment is available since no crystals of two-domain PncCs have been described. However, two relatively conserved blocks have been identified . The firAgrobacterium tumefaciens CinA (PDB code 2A9S) (AESCTGG) was highly conserved in all lineages, including the inactive single-domain form of the enzyme   appeared to be responsible of the enzyme inactivity , was not involved in active site formation   . Block PD/P E/Q) , blue co YxNxxK; . In the activity . Howeveractivity . Residueactivity , yellow.ormation , pink. Holi YfaY . Block Poli YfaY  of the CMN [GTV) ; which, MN [GTV) , cyan, wMN [GTV) . This reMN [GTV)  and was MN [GTV) . FinallyLxxLxxxL . This se+-degrading enzymes, there are several enzymes involved in NAD+ regeneration according to the Pyridine Nucleotide Cycle and NAD+ biosynthetic pathways shown in Oceanobacillus iheyensis, where no other enzymes, such as NadV, NadM or NadR are present  and regulatory pathways (acting as substrate for numerous protein- and nucleic acid-modifying reactions). Regardless the origin of these NAD present , relyingIn addition, NMN deamidase activity in this deep-sea microorganism runs in parallel with another key enzyme in the PNC pathway, nicotinamidase or PncA . We haveE. coli YfaY, where only block P-I, P-VIII and P-IX are relatively conserved, while the rest of them strongly differed from the CinA domain, indicating a massive accumulation of mutations or, more plausibly, a insertion of a mobile genetic element in the middle of CinA motif. A milder form of gene decay seems to have occurred in Lineage 2.2.2.2  and the tyrosine of block P-III were mutated. A particular case is also Lineage 2.2.3.4  and S. oneidensis COG1058/PncC (Uniprot code: Q8EK32) were only active towards ADPr in the presence of Co2+ and K+ ions, but not with Mg2+, which also required Molybdate (MoO42-) in the MPT-AMP cleavage carried out by MoeA, gephyrin and Cnx1 to give Moco [Finally, the present work revealed some important gaps in the study of these NMN deamidases. Firstly, this work highlights the need for crystals structure of a functional two-domain PncC to be compared with that of one MocF domain enzymes (like 3KBQ) or one domain CinA enzymes (like 2A9S). In addition, the role of the interdomain sequence in the final structure of two-domain PncCs is still unknown. Moreover, further experiments using MPT-AMP as possible substrate for MocF domain are required to fully explore the catalytic role of MocF-containing enzymes. The recent work assigning a pyrophosphatase function towards ADPr to those enzymes  establisive Moco . The aboFigure S1O. iheyensis (OiPncC) and related nicotinamide mononucleotide deamidases.Multiple sequence alignment for  ESPript outputs [Oceanobacillus iheyensis PncC , Shewanella oneidensis PncC , Agrobacterium tumefaciens PncC , Escherichia coli YGAD PncC , E. coli YDEJ PncC  and E. coli YFAY PncC  were aligned with CLUSTAL-W [O. iheyensis NMN deamidase, springs represent helices and arrows represent β-strands. Conserved residues of the MocF domain are marked with a blue triangle. Conserved residues of the inter-domain segment are marked with a green triangle. Conserved residues of the CinA domain are marked with a red triangle.  outputs  obtainedLUSTAL-W . Residue(TIF)Click here for additional data file.Figure S2SDS-PAGE of the OiPncC purification. M: molecular weight standards (New England Biolabs: P7708S). Lane 1: cell extract after disruption. Lane 2: cell extract after 50 kDa tangential ultrafiltration. Lane 3: OiPncC after HisTrap column step (purified protein is about 47 kDa). Each lane contained 20 µg of protein.(TIF)Click here for additional data file.Figure S3Enzymatic activity of OiPncC. A) Assayed by HPLC. Aliquots of the reaction were removed and stopped at 0 minutes (solid line), 10 minutes (dashed-line) and 15 minutes (dotted line) of the reaction course. B) Assayed by enzyme-coupled assay using glutamate dehydrogenase. The standard reaction conditions at 37 °C were used. (TIF)Click here for additional data file.Figure S4Effect of NMN concentration on OiPncC activity. A) Measured by HPLC under the standard reaction conditions at 37 °C and increasing concentrations of NMN (0.01 mM to 1 mM). B) Measured by the enzyme-coupled spectrophotometric method under the standard reaction conditions at 37 °C and increasing concentrations of NMN (0.01 mM to 1 mM).(TIF)Click here for additional data file.Figure S5Conserved blocks in the MocF domain and interdomain segment in the different lineages. Red background indicates strictly conserved amino acids; orange background indicates conserved amino acids; “c”, a charged residue; “h”, a hydrophobic residue; “p”, a polar residue; and “x”, any residue. Alternative amino acids at a given position are shown within brackets.(TIF)Click here for additional data file.Figure S6Conserved blocks in the CinA domain in the different lineages. Red background indicates strictly conserved amino acids; orange background indicates conserved amino acids; “c”, a charged residue; “h”, a hydrophobic residue; “p”, a polar residue; and “x”, any residue. Alternative amino acids at a given position are shown within brackets. (TIF)Click here for additional data file.Figure S7ESPript outputs obtained with the sequences from the different lineages. Residues strictly conserved across NMN deamidase enzymes have a red background. Symbols above blocks of sequences represent the secondary structure of the most representative enzyme from each lineage, springs represent helices and arrows represent β-strands. Conserved blocks are marked under the corresponding sequences for each lineage.(PDF)Click here for additional data file.Figure S8WEBLOGO outputs obtained from the above alignment of the sequences from the different lineages. Conserved blocks are marked under the corresponding sequences for each lineage.(PDF)Click here for additional data file.Table S1NMN deamidases used in the phylogenetic analysis.(PDF)Click here for additional data file.


It has been demonstrated that oxidative stress has a ubiquitous role in neurodegenerative diseases. Major source of oxidative stress due to reactive oxygen species (ROS) is related to mitochondria as an endogenous source. Although there is ample evidence from tissues of patients with neurodegenerative disorders of morphological, biochemical, and molecular abnormalities in mitochondria, it is still not very clear whether the oxidative stress itself contributes to the onset of neurodegeneration or it is part of the neurodegenerative process as secondary manifestation. This paper begins with an overview of how oxidative stress occurs, discussing various oxidants and antioxidants, and role of oxidative stress in diseases in general. It highlights the role of oxidative stress in neurodegenerative diseases like Alzheimer's, Parkinson's, and Huntington's diseases and amyotrophic lateral sclerosis. The last part of the paper describes the role of oxidative stress causing deregulation of cyclin-dependent kinase 5 (Cdk5) hyperactivity associated with neurodegeneration. Oxidative stress is caused by the imbalance in the production of ROS and the biological system's inability to detoxify those species and repair the resulting damage. The effects of oxidative stress depend upon its impact on the cellular system, whether the system is able to handle well and regain its original state or not. However, high levels of oxidative stress can cause necrosis, ATP depletion, and prevention of controlled apoptotic death . In normROS are of two types: less reactive and aggressive. Most of these less reactive species are produced at a low level by normal aerobic metabolism, and the damage they cause to cells is constantly repaired. However, in some instances even the less reactive species like superoxide are readily converted by oxidoreduction reactions with transition metals or other redox cycling compounds into more aggressive radical species that cause extensive cellular damage which includes damage inflicted on DNA . Like inROS plays an important role in cell signaling, a process termed redox signaling. Thus, to maintain proper cellular homeostasis, a balance must be struck between reactive oxygen production and consumption. Excessive ROS or free radicals need to be either quenched by converting them into metabolically nondestructive molecules or be scavenged/neutralized right after their formation. This protective mechanism is called the antioxidant defense system preventing free radical mediated damage of cells leading to various diseases and aging .The most common naturally occurring anti-oxidants are Vitamin A (retinol), C (ascorbic acid), and E (tocopherol), besides polyphenol antioxidants like flavonoids. Some enzymes also play an important role in combating the oxidative stress and one of the best studied is superoxide dismutase (SOD). Catalase, glutathione peroxidase, aldehyde dehydrogenases, and sulfiredoxin all belong to the class of enzymatic antioxidants , 6. The As previously mentioned the imbalance between the reactive oxygen/nitrogen species formed and quenched leads to the cell/tissue damage ultimately becoming the cause of many diseases. In humans, oxidative stress is associated with diseases in different ways. Oxidative stress could be generated as a secondary effect of a preexisting diseased condition or could be the central cause of the disease itself. Besides being implicated in aging by the accumulation of the ROS and mutations in the mitochondrial DNA (mtDNA), oxidative stress is associated with various pathological conditions. It plays a central role in chronic lung disease (CLD) which is usually found in the preterm infants subjected to high oxygen concentration , 8. HighOne of the most metabolically active organs of the body is the brain, including the spinal cord comprising central nervous system (CNS), which, even at rest, utilizes an estimated 20% of the total oxygen uptake. During active state this percentage substantially increases and in order to carry out normal physiological actions, it requires an uninterrupted oxygen-rich blood supply. Any blockage or deprivation of this oxygen supply even for a few seconds can have severe and irreversible detrimental effect to the cells of brain (both neurons and glia). Consumption of oxygen leads to production of free radicals and the brain requirement for higher amount of oxygen leads to even higher number of reactive oxygen/nitrogen species. Though CNS has high requirement for oxygen but unexpectedly, is relatively deficient in the enzymes that metabolize a number of oxygen-based reactants to innocuous species . On the Among others the three main neurodegenerative diseases are Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) which are not only sporadic, but also have rare familial forms. These diseases occur mainly as sporadic (90%) where as the familial cause is usually limited (10%). Sporadic forms occur due to a complicated and heterogeneous mixture of hereditary, environmental factors, and lifestyle stresses. In contrast Huntington's disease (HD), another neurodegenerative disorder is completely hereditary. HD is predominantly inherited and is strictly autosomal disorder. In the following sections role of oxidative stress in all the above-mentioned neurodegenerative disorders is briefly described. β42) aggregates and the intracellular neurofilament tangles (NFT) formed by hyperphosphorylated microtubule associated protein, tau (τ) [τ could be altered via activation of different signaling pathway.Clinically, Alzheimer's disease (AD) is characterized as a late-onset, age-dependent, progressive cognitive decline which results in irreversible loss of neurons especially in the cortex and hippocampus. The major pathological hallmarks of AD are the formation of extracellular senile plaques due to amyloid beta 42 peptide (A tau (τ) . In AD, α-secretase followed by γ-secretase to yield nonamyloidogenic fragments [α-secretase by β-secretase (BACE1), followed by γ-secretase to yield amyloidogenic Aβ42 which aggregates to form insoluble plaques. Various cell culture studies have shown Aβ42 causes toxicity and induces cell death via apoptosis [Usually most of the AD cases are late onset and sporadic but there are 5–10% familial AD (FAD) occurring as an early-onset and in an autosomal-dominant manner. FAD can be caused due to mutations in three genes namely, amyloid precursor protein (APP), presenilin 1, and 2 . During normal physiological conditions proteolytic cleavage of APP is initiated by ragments . Mutatiopoptosis . τ functions to regulate microtubule (MT) assembly and transport. Where as in AD τ is hyperphosphorylated and dissociates from MT, resulting in destabilizing MTs and impairment of axonal transport. The phospho-τ aggregates form the paired helical filaments (PHFs) which further combine to form the insoluble NFTs [β42 and also exhibit tau hyperphosphorylation, neuronal degeneration, motor, and behavioral deficits [In contrast to senile plaques, NFTs are so far not associated with mutations but are due to modulations of kinase and phosphatase activities. Normally ble NFTs . Abnormable NFTs . Among vble NFTs . Conformdeficits , 26.τ is one of the major causes of AD pathology as it not only fails in its normal function of stabilizing microtubules, but also exhibits a “gain of toxic function” due to its sequestering normal τ. Absence of normal τ results in the disruption of microtubules [τ accumulates to form the insoluble intraneuronal PHF-τ which could reflect inhibitory binding of oxidatively damaged protein to the proteasome. Under physiological conditions any aggregate which could be a potential toxic to the system is cleaned up by the proteasome. Inhibition of this cleanup process by the proteasome is sufficient to induce neuronal degeneration and death [Hyperphosphorylated otubules , 28. Hypnd death . Also, vnd death , 31. Fornd death . HoweverAmyotrophic lateral sclerosis (ALS) is clinically characterized by progressive weakness, atrophy, and spasticity of muscle tissue. Pathologically, ALS is an adult-onset neurodegenerative disease reflecting degeneration of upper and lower motor neurons in the cortex, brainstem, and spinal cord . Like ADIn order to better understand the ALS pathogenesis, most of the recent studies have focused on mouse models that express the mutant human SOD1 forms. These animals experience age-dependent motor neuron degeneration with cellular and biochemical damage to nerve fibers and spinal cord tissue as well as increased protein and lipid oxidation . TransgeWith evidences from the mutSOD1 mouse model, the most accepted theory is that the different point mutations create a misfolding defect, leading to small amyloid-like aggregates that appear in late stages of the disease. These misfolded aggregated proteins could produce toxic effect towards neurons, similar to the neurotoxicity that arises in other amyloidoses , 45. MisSimilar to mutSOD-1, more evidence propose alteration in proteins related to mRNA metabolism leading to protein misfolding as a salient feature of ALS pathogenesis , 51. In in vivo studies in the generation of ROS and oxidative damage, and trials of antioxidant therapies have been disappointing [The above data suggests that both a toxic gain of function due to protein aggregation/mislocation together with a loss of normal biological function upon mutations contribute to disease pathogenesis. There have been mixed results of pointing . If protα-synuclein [Parkinson's disease (PD) is a common neurodegenerative movement disorder and is clinically characterized by progressive rigidity, bradykinesia, and tremor. Pathologically PD is characterized by loss of melanin-pigmented nigral neurons accompanied by depletion of dopamine in the striatum and the presence of Lewy bodies , 60. Lewynuclein . Althougα-synuclein and dardarin) and three are autosomal recessive (including parkin) [DJ-1, phosphatase and tensin homologue- (PTEN-) induced kinase 1 (PINK1), leucine-rich-repeat kinase 2 (LRRK2), the nuclear receptor NURR1, HTRA2, and τ [α-synuclein is to maintain synapse and plasticity but mutations causing overexpression of α-synuclein is neurotoxic, inducing apoptosis [In vitro there is a strong connection between α-synuclein fibrillization and τ fibrillization and more evidence indicate frequent disease overlap between the classical tauopathies  and synucleinopathies  [Linkage analysis has led to the discovery of pathogenic mutations or polymorphisms in mtDNA in nine genes that may account for as many as 5–10% of the cases of familial PD. Out of these genes, two are autosomal dominant (coding for  parkin) , 63. Oth2, and τ . These m2, and τ –68. The poptosis , 70. In n in PD) –73.α-synuclein leading to deposition of Lewy bodies by altering posttranslational modification of α-synuclein [α-synuclein [α-synuclein null mice are resistant to MPTP indicating an important role in mediating toxic effects of MPTP [Postmortem tissues from PD patients have shown evidence that a defect in complex I of the mitochondrial electron-transport chain in substantia nigra, resulting in 30–40% decrease in the activity may be the central cause of sporadic PD . The decynuclein . Impaireynuclein . On the  of MPTP .Ubiquitin, another important protein found in Lewy bodies of sporadic PD is associated with proteolytic stress as ubiquitin-proteasome process is impaired by products of oxidative damage like HNE , 89. Thiα-synuclein aggregation [Besides mutations, overexposure to dopamine or metals like iron (Fe) and manganese (Mn) also plays an important role in the pathology of sporadic PD. In cell culture, dopamine is toxic to PC12 cell via oxidative stress, leading to apoptosis . Dopaminregation .From the above-stated various factors causing PD, it is clear that PD not only results from a complex interplay among genetic and environmental factors, but also aspects of mitochondrial dysfunction and oxidative stress play a very important role. For a better therapeutic strategy for PD, all the causes and their interplay need to be taken into consideration for a drug, targeting PD.As previously mentioned, Huntington's disease (HD) is genetically inherited in an autosomal manner. Clinically HD is characterized by psychiatric disturbances, progressive cognitive impairment, and choreiform movements. Pathologically HD is characterized by loss of long projection neurons, resulting in a progressive atrophy of the caudate nucleus, putamen, and globus pallidus .HTT) gene, a cytoplasmic protein of unknown function [The HD mutation is an expansion of CAG trinucleotide repeat within exon 1 of the huntingtin  has been closely associated with various neurodegenerative diseases like AD, ALS, HD, and PD. Cdk5, a proline-directed serine/threonine kinase, plays multiple roles in neuron development, neuronal survival, phosphorylation of cytoskeletal proteins and synaptic plasticity , 117. Th complex , 121–128 complex , 129–131τ, neurofilament (NF) and other cytoskeletal proteins [β in cortical neurons induces cleavage of p35 to p25 resulting in activation of kinases and inhibition of phosphatases proceeding NFT formation [β or glutamate, primary cortical neurons have shown enhanced Cdk5 activity. After treatment with inhibitors like roscovitine or Cdk5 inhibitory peptide (CIP), cells displayed reduction in hyperactive Cdk5 [β and glutamate toxicity cause Cdk5 to promote mitochondrial damage and induce p38 activation by increasing ROS [2+ release, both of which eventually activate Cdk5 [Cdk5 is activated by oxidative stress in AD, resulting in hyperactive and aberrant Cdk5/p25 activity causing hyperphosphorylation of proteins , 133. Oxproteins . Accumulormation , 123. Upive Cdk5 , 136. Alsing ROS , 138. Insing ROS , 140. Fuate Cdk5 , 127. Thate Cdk5 . More evate Cdk5 –144. Cdkate Cdk5 .Silencing Cdk5 via RNA interference (RNAi) using lentiviral or adenoassociated viral vectors in the brains of transgenic AD mouse models show reduction in phosphorylation of tau and decreased number of NFTs in the hippocampus . BesidesAberrant Cdk5 hyperactivity due to oxidative stress is also linked to motor neuron degeneration and ALS by compromising the NF dynamics . In cultα-synuclein inclusions indicating that phosphorylation of parkin by Cdk5 decreases its E3 ubiquitin-ligase activity and regulates the formation of cytosolic inclusions relevant to PD [Evidence of Cdk5 hyperactivity associated with PD comes from the studies involving gyrus cinguli  of PD patients showing higher p25/p35 ratio due to calpain activation compared to age-matched controls . As prevnt to PD . PhosphoDrosophila and mouse models of HD [Contrary to the role of Cdk5 in above-mentioned neurodegenerative diseases like AD, ALS, and PD, Cdk5 shows a neuroprotective role in HD. As mentioned earlier cause of HD is the expansion in polyQ stretch in the huntingtin (htt) protein. More than 40 polyQ tracts leads to htt protein misfolding, making it toxic and resulting in formation of aggregates and cause disease. Inhibiting polyQ aggregation alleviates the symptoms of HD patients, as reported in ls of HD , 158. Shls of HD , 160. Thls of HD . Variousls of HD . Also, Cls of HD . These rls of HD . Anotherls of HD . This CdRecent evidences have greatly increased our knowledge about the AD, ALS, PD, and HD, major neurodegenerative diseases assessed above. From various studies, it has become evident that all the neurodegenerative diseases are to some extent multifactorial, and oxidative stress is inevitably intertwined with the disease mechanisms. Besides biological factors like inflammation, excitotoxicity, and to a certain extent role of genes involved in sporadic cases, environmental contributions like diet and lifestyle are also important contributing factors for the occurrence of these diseases.β or glutamate toxicity, inflammation, and mitochondrial dysfunction, there is intracellular increase in Ca2+ concentration. This leads to the activation of proteases-like Calpain which cleaves p35 into p25. Association of p25 with Cdk5 leads to the diseased condition as Cdk5/p25 activity is deregulated causing aberrant hyperphosphorylation of various proteins like neurofilament, τ, parkin, α-synuclein, and Huntingtin. Hyperphosphorylation of these proteins leads to misfolding and aggregation causing conformational changes. Misfolded proteins self-aggregate disrupting transport and normal synaptic activity ultimately leading to neuronal degeneration and pathology.These disorders have many common factors. One such factor is the deregulation of Cdk5 as depicted in Since oxidative stress is involved in neurodegeneration, selecting antioxidants, metal chelators, or other compounds boosting endogenous enzymatic and nonenzymatic defense mechanism seems to be an obvious choice as a treatment to these disorders. However, using antioxidants like Vitamin E (tocopherol) and others as a therapeutic target comes with a caveat as most antioxidants have metal-reducing capacity. Therefore, devising a successful regimen of antioxidants to retard the progression of these diseases remains a complicated goal. Besides using anti-oxidants as therapeutic targets, Cdk5 with all its involvement in the above mentioned neurodegenerative diseases seems to be potential and ideal candidate to be used as therapeutic target for neurodegenerative diseases.