Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta.

PubMed

Engelke, Michael; Friedrich, Olaf; Budde, Petra; Schäfer, Christina; Niemann, Ursula; Zitt, Christof; Jüngling, Eberhard; Rocks, Oliver; Lückhoff, Andreas; Frey, Jürgen

2002-07-17

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.

Crystal Structure of the Human, FIC-Domain Containing Protein HYPE and Implications for Its Functions

PubMed Central

Bunney, Tom D.; Cole, Ambrose R.; Broncel, Malgorzata; Esposito, Diego; Tate, Edward W.; Katan, Matilda

2014-01-01

Summary Protein AMPylation, the transfer of AMP from ATP to protein targets, has been recognized as a new mechanism of host-cell disruption by some bacterial effectors that typically contain a FIC-domain. Eukaryotic genomes also encode one FIC-domain protein, HYPE, which has remained poorly characterized. Here we describe the structure of human HYPE, solved by X-ray crystallography, representing the first structure of a eukaryotic FIC-domain protein. We demonstrate that HYPE forms stable dimers with structurally and functionally integrated FIC-domains and with TPR-motifs exposed for protein-protein interactions. As HYPE also uniquely possesses a transmembrane helix, dimerization is likely to affect its positioning and function in the membrane vicinity. The low rate of autoAMPylation of the wild-type HYPE could be due to autoinhibition, consistent with the mechanism proposed for a number of putative FIC AMPylators. Our findings also provide a basis to further consider possible alternative cofactors of HYPE and distinct modes of target-recognition. PMID:25435325

Crystal structure of the human, FIC-domain containing protein HYPE and implications for its functions.

PubMed

Bunney, Tom D; Cole, Ambrose R; Broncel, Malgorzata; Esposito, Diego; Tate, Edward W; Katan, Matilda

2014-12-02

Protein AMPylation, the transfer of AMP from ATP to protein targets, has been recognized as a new mechanism of host-cell disruption by some bacterial effectors that typically contain a FIC-domain. Eukaryotic genomes also encode one FIC-domain protein,HYPE, which has remained poorly characterized.Here we describe the structure of human HYPE, solved by X-ray crystallography, representing the first structure of a eukaryotic FIC-domain protein. We demonstrate that HYPE forms stable dimers with structurally and functionally integrated FIC-domains and with TPR-motifs exposed for protein-protein interactions. As HYPE also uniquely possesses a transmembrane helix, dimerization is likely to affect its positioning and function in the membrane vicinity. The low rate of auto AMPylation of the wild-type HYPE could be due to autoinhibition, consistent with the mechanism proposed for a number of putative FIC AMPylators. Our findings also provide a basis to further consider possible alternative cofactors of HYPE and distinct modes of target-recognition.

Identification and functional analysis of two Golgi-localized UDP-galactofuranose transporters with overlapping functions in Aspergillus niger.

PubMed

Park, Joohae; Tefsen, Boris; Heemskerk, Marc J; Lagendijk, Ellen L; van den Hondel, Cees A M J J; van Die, Irma; Ram, Arthur F J

2015-11-02

Galactofuranose (Galf)-containing glycoconjugates are present in numerous microbes, including filamentous fungi where they are important for morphology, virulence and maintaining cell wall integrity. The incorporation of Galf-residues into galactomannan, galactomannoproteins and glycolipids is carried out by Golgi-localized Galf transferases. The nucleotide sugar donor used by these transferases (UDP-Galf) is produced in the cytoplasm and has to be transported to the lumen of the Golgi by a dedicated nucleotide sugar transporter. Based on homology with recently identified UDP-Galf-transporters in A. fumigatus and A. nidulans, two putative UDP-Galf-transporters in A. niger were found. Their function and localization was determined by gene deletions and GFP-tagging studies, respectively. The two putative UDP-Galf-transporters in A. niger are homologous to each other and are predicted to contain eleven transmembrane domains (UgtA) or ten transmembrane domains (UgtB) due to a reduced length of the C-terminal part of the UgtB protein. The presence of two putative UDP-Galf-transporters in the genome was not unique for A. niger. From the twenty Aspergillus species analysed, nine species contained two additional putative UDP-Galf-transporters. Three of the nine species were outside the Aspergillus section nigri, indication an early duplication of UDP-Galf-transporters and subsequent loss of the UgtB copy in several aspergilli. Deletion analysis of the single and double mutants in A. niger indicated that the two putative UDP-Galf-transporters (named UgtA and UgtB) have a redundant function in UDP-Galf-transport as only the double mutant displayed a Galf-negative phenotype. The Galf-negative phenotype of the double mutant could be complemented by expressing either CFP-UgtA or CFP-UgtB fusion proteins from their endogenous promoters, indicating that both CFP-tagged proteins are functional. Both Ugt proteins co-localize with each other as well as with the GDP-mannose nucleotide transporter, as was demonstrated by fluorescence microscopy, thereby confirming their predicted localization in the Golgi. A. niger contains two genes encoding UDP-Galf-transporters. Deletion and localization studies indicate that UgtA and UgtB have redundant functions in the biosynthesis of Galf-containing glycoconjugates.

Domain fusion analysis by applying relational algebra to protein sequence and domain databases

PubMed Central

Truong, Kevin; Ikura, Mitsuhiko

2003-01-01

Background Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. Results This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at . Conclusion As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time. PMID:12734020

Are plant formins integral membrane proteins?

PubMed

Cvrcková, F

2000-01-01

The formin family of proteins has been implicated in signaling pathways of cellular morphogenesis in both animals and fungi; in the latter case, at least, they participate in communication between the actin cytoskeleton and the cell surface. Nevertheless, they appear to be cytoplasmic or nuclear proteins, and it is not clear whether they communicate with the plasma membrane, and if so, how. Because nothing is known about formin function in plants, I performed a systematic search for putative Arabidopsis thaliana formin homologs. I found eight putative formin-coding genes in the publicly available part of the Arabidopsis genome sequence and analyzed their predicted protein sequences. Surprisingly, some of them lack parts of the conserved formin-homology 2 (FH2) domain and the majority of them seem to have signal sequences and putative transmembrane segments that are not found in yeast or animals formins. Plant formins define a distinct subfamily. The presence in most Arabidopsis formins of sequence motifs typical or transmembrane proteins suggests a mechanism of membrane attachment that may be specific to plant formins, and indicates an unexpected evolutionary flexibility of the conserved formin domain.

Functional Analysis of the Gene Cluster Involved in Production of the Bacteriocin Circularin A by Clostridium beijerinckii ATCC 25752

PubMed Central

Kemperman, Robèr; Jonker, Marnix; Nauta, Arjen; Kuipers, Oscar P.; Kok, Jan

2003-01-01

A region of 12 kb flanking the structural gene of the cyclic antibacterial peptide circularin A of Clostridium beijerinckii ATCC 25752 was sequenced, and the putative proteins involved in the production and secretion of circularin A were identified. The genes are tightly organized in overlapping open reading frames. Heterologous expression of circularin A in Enterococcus faecalis was achieved, and five genes were identified as minimally required for bacteriocin production and secretion. Two of the putative proteins, CirB and CirC, are predicted to contain membrane-spanning domains, while CirD contains a highly conserved ATP-binding domain. Together with CirB and CirC, this ATP-binding protein is involved in the production of circularin A. The fifth gene, cirE, confers immunity towards circularin A when expressed in either Lactococcus lactis or E. faecalis and is needed in order to allow the bacteria to produce bacteriocin. Additional resistance against circularin A is conferred by the activity of the putative transporter consisting of CirB and CirD. PMID:14532033

Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion

PubMed Central

Boswell, Kristin L.; James, Declan J.; Esquibel, Joseph M.; Bruinsma, Stephen; Shirakawa, Ryutaro; Horiuchi, Hisanori

2012-01-01

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca2+, but Ca2+-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca2+ and restored Ca2+-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca2+-binding residues in C2A and C2B. Munc13-4 exhibited Ca2+-stimulated SNARE interactions dependent on C2A and Ca2+-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca2+-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca2+-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca2+ sensor at rate-limiting priming steps in granule exocytosis. PMID:22508512

Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion.

PubMed

Boswell, Kristin L; James, Declan J; Esquibel, Joseph M; Bruinsma, Stephen; Shirakawa, Ryutaro; Horiuchi, Hisanori; Martin, Thomas F J

2012-04-16

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca(2+), but Ca(2+)-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca(2+) and restored Ca(2+)-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca(2+)-binding residues in C2A and C2B. Munc13-4 exhibited Ca(2+)-stimulated SNARE interactions dependent on C2A and Ca(2+)-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca(2+)-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca(2+)-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca(2+) sensor at rate-limiting priming steps in granule exocytosis.

The cysteine-rich domain regulates ADAM protease function in vivo.

PubMed

Smith, Katherine M; Gaultier, Alban; Cousin, Helene; Alfandari, Dominique; White, Judith M; DeSimone, Douglas W

2002-12-09

ADAMs are membrane-anchored proteases that regulate cell behavior by proteolytically modifying the cell surface and ECM. Like other membrane-anchored proteases, ADAMs contain candidate "adhesive" domains downstream of their metalloprotease domains. The mechanism by which membrane-anchored cell surface proteases utilize these putative adhesive domains to regulate protease function in vivo is not well understood. We address this important question by analyzing the relative contributions of downstream extracellular domains (disintegrin, cysteine rich, and EGF-like repeat) of the ADAM13 metalloprotease during Xenopus laevis development. When expressed in embryos, ADAM13 induces hyperplasia of the cement gland, whereas ADAM10 does not. Using chimeric constructs, we find that the metalloprotease domain of ADAM10 can substitute for that of ADAM13, but that specificity for cement gland expansion requires a downstream extracellular domain of ADAM13. Analysis of finer resolution chimeras indicates an essential role for the cysteine-rich domain and a supporting role for the disintegrin domain. These and other results reveal that the cysteine-rich domain of ADAM13 cooperates intramolecularly with the ADAM13 metalloprotease domain to regulate its function in vivo. Our findings thus provide the first evidence that a downstream extracellular adhesive domain plays an active role in regulating ADAM protease function in vivo. These findings are likely relevant to other membrane-anchored cell surface proteases.

Chimeras taking shape: Potential functions of proteins encoded by chimeric RNA transcripts

PubMed Central

Frenkel-Morgenstern, Milana; Lacroix, Vincent; Ezkurdia, Iakes; Levin, Yishai; Gabashvili, Alexandra; Prilusky, Jaime; del Pozo, Angela; Tress, Michael; Johnson, Rory; Guigo, Roderic; Valencia, Alfonso

2012-01-01

Chimeric RNAs comprise exons from two or more different genes and have the potential to encode novel proteins that alter cellular phenotypes. To date, numerous putative chimeric transcripts have been identified among the ESTs isolated from several organisms and using high throughput RNA sequencing. The few corresponding protein products that have been characterized mostly result from chromosomal translocations and are associated with cancer. Here, we systematically establish that some of the putative chimeric transcripts are genuinely expressed in human cells. Using high throughput RNA sequencing, mass spectrometry experimental data, and functional annotation, we studied 7424 putative human chimeric RNAs. We confirmed the expression of 175 chimeric RNAs in 16 human tissues, with an abundance varying from 0.06 to 17 RPKM (Reads Per Kilobase per Million mapped reads). We show that these chimeric RNAs are significantly more tissue-specific than non-chimeric transcripts. Moreover, we present evidence that chimeras tend to incorporate highly expressed genes. Despite the low expression level of most chimeric RNAs, we show that 12 novel chimeras are translated into proteins detectable in multiple shotgun mass spectrometry experiments. Furthermore, we confirm the expression of three novel chimeric proteins using targeted mass spectrometry. Finally, based on our functional annotation of exon organization and preserved domains, we discuss the potential features of chimeric proteins with illustrative examples and suggest that chimeras significantly exploit signal peptides and transmembrane domains, which can alter the cellular localization of cognate proteins. Taken together, these findings establish that some chimeric RNAs are translated into potentially functional proteins in humans. PMID:22588898

Distinct roles for extracellular and intracellular domains in neuroligin function at inhibitory synapses

PubMed Central

Nguyen, Quynh-Anh; Horn, Meryl E; Nicoll, Roger A

2016-01-01

Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that interact trans-synaptically with neurexins to mediate synapse development and function. NLGN2 is only at inhibitory synapses while NLGN3 is at both excitatory and inhibitory synapses. We found that NLGN3 function at inhibitory synapses in rat CA1 depends on the presence of NLGN2 and identified a domain in the extracellular region that accounted for this functional difference between NLGN2 and 3 specifically at inhibitory synapses. We further show that the presence of a cytoplasmic tail (c-tail) is indispensible, and identified two domains in the c-tail that are necessary for NLGN function at inhibitory synapses. These domains point to a gephyrin-dependent mechanism that is disrupted by an autism-associated mutation at R705 and a gephyrin-independent mechanism reliant on a putative phosphorylation site at S714. Our work highlights unique and separate roles for the extracellular and intracellular regions in specifying and carrying out NLGN function respectively. DOI: http://dx.doi.org/10.7554/eLife.19236.001 PMID:27805570

Mapping of the minimal inorganic phosphate transporting unit of human PiT2 suggests a structure universal to PiT-related proteins from all kingdoms of life

PubMed Central

2011-01-01

Background The inorganic (Pi) phosphate transporter (PiT) family comprises known and putative Na+- or H+-dependent Pi-transporting proteins with representatives from all kingdoms. The mammalian members are placed in the outer cell membranes and suggested to supply cells with Pi to maintain house-keeping functions. Alignment of protein sequences representing PiT family members from all kingdoms reveals the presence of conserved amino acids and that bacterial phosphate permeases and putative phosphate permeases from archaea lack substantial parts of the protein sequence when compared to the mammalian PiT family members. Besides being Na+-dependent Pi (NaPi) transporters, the mammalian PiT paralogs, PiT1 and PiT2, also are receptors for gamma-retroviruses. We have here exploited the dual-function of PiT1 and PiT2 to study the structure-function relationship of PiT proteins. Results We show that the human PiT2 histidine, H502, and the human PiT1 glutamate, E70, - both conserved in eukaryotic PiT family members - are critical for Pi transport function. Noticeably, human PiT2 H502 is located in the C-terminal PiT family signature sequence, and human PiT1 E70 is located in ProDom domains characteristic for all PiT family members. A human PiT2 truncation mutant, which consists of the predicted 10 transmembrane (TM) domain backbone without a large intracellular domain (human PiT2ΔR254-V483), was found to be a fully functional Pi transporter. Further truncation of the human PiT2 protein by additional removal of two predicted TM domains together with the large intracellular domain created a mutant that resembles a bacterial phosphate permease and an archaeal putative phosphate permease. This human PiT2 truncation mutant (human PiT2ΔL183-V483) did also support Pi transport albeit at very low levels. Conclusions The results suggest that the overall structure of the Pi-transporting unit of the PiT family proteins has remained unchanged during evolution. Moreover, in combination, our studies of the gene structure of the human PiT1 and PiT2 genes (SLC20A1 and SLC20A2, respectively) and alignment of protein sequences of PiT family members from all kingdoms, along with the studies of the dual functions of the human PiT paralogs show that these proteins are excellent as models for studying the evolution of a protein's structure-function relationship. PMID:21586110

Mapping of the minimal inorganic phosphate transporting unit of human PiT2 suggests a structure universal to PiT-related proteins from all kingdoms of life.

PubMed

Bøttger, Pernille; Pedersen, Lene

2011-05-17

The inorganic (Pi) phosphate transporter (PiT) family comprises known and putative Na(+)- or H(+)-dependent Pi-transporting proteins with representatives from all kingdoms. The mammalian members are placed in the outer cell membranes and suggested to supply cells with Pi to maintain house-keeping functions. Alignment of protein sequences representing PiT family members from all kingdoms reveals the presence of conserved amino acids and that bacterial phosphate permeases and putative phosphate permeases from archaea lack substantial parts of the protein sequence when compared to the mammalian PiT family members. Besides being Na(+)-dependent P(i) (NaP(i)) transporters, the mammalian PiT paralogs, PiT1 and PiT2, also are receptors for gamma-retroviruses. We have here exploited the dual-function of PiT1 and PiT2 to study the structure-function relationship of PiT proteins. We show that the human PiT2 histidine, H(502), and the human PiT1 glutamate, E(70),--both conserved in eukaryotic PiT family members--are critical for P(i) transport function. Noticeably, human PiT2 H(502) is located in the C-terminal PiT family signature sequence, and human PiT1 E(70) is located in ProDom domains characteristic for all PiT family members.A human PiT2 truncation mutant, which consists of the predicted 10 transmembrane (TM) domain backbone without a large intracellular domain (human PiT2ΔR(254)-V(483)), was found to be a fully functional P(i) transporter. Further truncation of the human PiT2 protein by additional removal of two predicted TM domains together with the large intracellular domain created a mutant that resembles a bacterial phosphate permease and an archaeal putative phosphate permease. This human PiT2 truncation mutant (human PiT2ΔL(183)-V(483)) did also support P(i) transport albeit at very low levels. The results suggest that the overall structure of the P(i)-transporting unit of the PiT family proteins has remained unchanged during evolution. Moreover, in combination, our studies of the gene structure of the human PiT1 and PiT2 genes (SLC20A1 and SLC20A2, respectively) and alignment of protein sequences of PiT family members from all kingdoms, along with the studies of the dual functions of the human PiT paralogs show that these proteins are excellent as models for studying the evolution of a protein's structure-function relationship. © 2011 Bøttger and Pedersen; licensee BioMed Central Ltd.

Tamoxifen Dependent Interaction Between the Estrogen Receptor and a Novel P21 Activated Kinase

DTIC Science & Technology

2002-06-01

binding domain at the C Cv1 cells were cotransfected with ARE4-Luc reporter (100 teric G prti Tse bindin dominat the C ng), pRL-SV40 ( Renilla control...48 h and assayed for luciferase and Renilla Three of the four residues defining a putative het- activity. erotrimeric G protein binding motif were...nases. Taken together, these results demonstrated expression of the control Renilla reporter regulated by that the PAK6 CRIB domain was functional with re

Essential role of the HMG domain in the function of yeast mitochondrial histone HM: functional complementation of HM by the nuclear nonhistone protein NHP6A.

PubMed

Kao, L R; Megraw, T L; Chae, C B

1993-06-15

The yeast mitochondrial histone protein HM is required for maintenance of the mitochondrial genome, and disruption of the gene encoding HM (HIM1/ABF2) results in formation of a respiration-deficient petite mutant phenotype. HM contains two homologous regions, which share sequence similarity with the eukaryotic nuclear nonhistone protein, HMG-1. Experiments with various deletion mutants of HM show that a single HMG domain of HM is functional and can restore respiration competency to cells that lack HM protein (him1 mutant cells). The gene encoding the putative yeast nuclear HMG-1 homolog, the NHP6A protein, can functionally complement the him1 mutation. These results suggest that the HMG domain is the basic unit for the function of HM in mitochondria and that the function of HMG-1 proteins in the nucleus and HM in the mitochondrion may be equivalent.

Ras-Association Domain of Sorting Nexin 27 Is Critical for Regulating Expression of GIRK Potassium Channels

PubMed Central

Bodhinathan, Karthik; Taura, Jaume J.; Taylor, Natalie M.; Nettleton, Margaret Y.; Ciruela, Francisco; Slesinger, Paul A.

2013-01-01

G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-ΔRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability. PMID:23536889

Transcriptomics of the Bed Bug (Cimex lectularius)

PubMed Central

Rajarapu, Swapna P.; Jones, Susan C.; Mittapalli, Omprakash

2011-01-01

Background Bed bugs (Cimex lectularius) are blood-feeding insects poised to become one of the major pests in households throughout the United States. Resistance of C. lectularius to insecticides/pesticides is one factor thought to be involved in its sudden resurgence. Despite its high-impact status, scant knowledge exists at the genomic level for C. lectularius. Hence, we subjected the C. lectularius transcriptome to 454 pyrosequencing in order to identify potential genes involved in pesticide resistance. Methodology and Principal Findings Using 454 pyrosequencing, we obtained a total of 216,419 reads with 79,596,412 bp, which were assembled into 35,646 expressed sequence tags (3902 contigs and 31744 singletons). Nearly 85.9% of the C. lectularius sequences showed similarity to insect sequences, but 44.8% of the deduced proteins of C. lectularius did not show similarity with sequences in the GenBank non-redundant database. KEGG analysis revealed putative members of several detoxification pathways involved in pesticide resistance. Lamprin domains, Protein Kinase domains, Protein Tyrosine Kinase domains and cytochrome P450 domains were among the top Pfam domains predicted for the C. lectularius sequences. An initial assessment of putative defense genes, including a cytochrome P450 and a glutathione-S-transferase (GST), revealed high transcript levels for the cytochrome P450 (CYP9) in pesticide-exposed versus pesticide-susceptible C. lectularius populations. A significant number of single nucleotide polymorphisms (296) and microsatellite loci (370) were predicted in the C. lectularius sequences. Furthermore, 59 putative sequences of Wolbachia were retrieved from the database. Conclusions To our knowledge this is the first study to elucidate the genetic makeup of C. lectularius. This pyrosequencing effort provides clues to the identification of potential detoxification genes involved in pesticide resistance of C. lectularius and lays the foundation for future functional genomics studies. PMID:21283830

Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking

PubMed Central

Creelman, Robert A.; Griffith, Cara; Ahrens, Jeffrey E.; Taylor, J. Philip; Murphy, Lesley R.; Manjunath, Siva; Thompson, Rebecca L.; Lingard, Matthew J.; Back, Stephanie L.; Larue, Huachun; Brayton, Bonnie R.; Burek, Amanda J.; Tiwari, Shiv; Adam, Luc; Morrell, James A.; Caldo, Rico A.; Huai, Qing; Kouadio, Jean-Louis K.; Kuehn, Rosemarie; Sant, Anagha M.; Wingbermuehle, William J.; Sala, Rodrigo; Foster, Matt; Kinser, Josh D.; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E.; Huang, Mingya G.; Kuriakose, Saritha V.; Skottke, Kyle; Repetti, Peter P.; Reuber, T. Lynne; Ruff, Thomas G.; Petracek, Marie E.; Loida, Paul J.

2014-01-01

ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize. PMID:24736658

Expression of a truncated ATHB17 protein in maize increases ear weight at silking.

PubMed

Rice, Elena A; Khandelwal, Abha; Creelman, Robert A; Griffith, Cara; Ahrens, Jeffrey E; Taylor, J Philip; Murphy, Lesley R; Manjunath, Siva; Thompson, Rebecca L; Lingard, Matthew J; Back, Stephanie L; Larue, Huachun; Brayton, Bonnie R; Burek, Amanda J; Tiwari, Shiv; Adam, Luc; Morrell, James A; Caldo, Rico A; Huai, Qing; Kouadio, Jean-Louis K; Kuehn, Rosemarie; Sant, Anagha M; Wingbermuehle, William J; Sala, Rodrigo; Foster, Matt; Kinser, Josh D; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E; Huang, Mingya G; Kuriakose, Saritha V; Skottke, Kyle; Repetti, Peter P; Reuber, T Lynne; Ruff, Thomas G; Petracek, Marie E; Loida, Paul J

2014-01-01

ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.

Domain fusion analysis by applying relational algebra to protein sequence and domain databases.

PubMed

Truong, Kevin; Ikura, Mitsuhiko

2003-05-06

Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at http://calcium.uhnres.utoronto.ca/pi. As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time.

PDZ Binding Domains, Structural Disorder and Phosphorylation: A Menage-a-trois Tailing Dcp2 mRNA Decapping Enzymes.

PubMed

Gunawardana, Dilantha

2016-01-01

Diverse cellular activities are mediated through the interaction of protein domains and their binding partners. One such protein domain widely distributed in the higher metazoan world is the PDZ domain, which facilitates abundant protein-protein interactions. The PDZ domain-PDZ binding domain interaction has been implicated in several pathologies including Alzheimer's disease, Parkinson's disease and Down syndrome. PDZ domains bind to C-terminal peptides/proteins which have either of the following combinations: S/T-X-hydrophobic-COOH for type I, hydrophobic-Xhydrophobic- COOH for type II, and D/E-X-hydrophobic-COOH for type III, although hydrophobicity in the termini form the key characteristic of the PDZ-binding domains. We identified and characterized a Dcp2 type mRNA decapping enzyme from Arabidopsis thaliana, a protein containing a putative PDZ-binding domain using mutagenesis and protein biochemistry. Now we are using bioinformatics to study the Cterminal end of mRNA decapping enzymes from complex metazoans with the aim of (1) identifying putative PDZ-binding domains (2) Correlating structural disorder with PDZ binding domains and (3) Demonstrating the presence of phosphorylation sites in C-terminal extremities of Dcp2 type mRNA decapping enzymes. It is proposed here that the trinity of PDZbinding domains, structural disorder and phosphorylation-susceptible sites are a feature of the Dcp2 family of decapping enzymes and perhaps is a wider trick in protein evolution where scaffolding/tethering is a requirement for localization and function. It is critical though laboratory-based supporting evidence is sought to back-up this bioinformatics exploration into tail regions of mRNA decapping enzymes.

Structural Basis and Function of XRN2-Binding by XTB Domains

PubMed Central

Richter, Hannes; Katic, Iskra; Gut, Heinz; Großhans, Helge

2016-01-01

The ribonuclease XRN2 is an essential player in RNA metabolism. In Caenorhabditis elegans, XRN2 functions with PAXT-1, which shares a putative XRN2-binding domain (XTBD) with otherwise unrelated mammalian proteins. Here, we characterize structure and function of an XTBD – XRN2 complex. Although XTBD stably interconnects two XRN2 domains through numerous interacting residues, mutation of a single critical residue suffices to disrupt XTBD – XRN2 complexes in vitro, and recapitulates paxt-1 null mutant phenotypes in vivo. Demonstrating conservation of function, vertebrate XTBD-containing proteins bind XRN2 in vitro, and human CDKN2AIPNL (C2AIL) can substitute for PAXT-1 in vivo. In vertebrates, where three distinct XTBD-containing proteins exist, XRN2 may partition to distinct stable heterodimeric complexes, likely differing in subcellular localization or function. In C. elegans, complex formation with the unique PAXT-1 serves to preserve the stability of XRN2 in the absence of substrate. PMID:26779609

Functional role of STIM1 and Orai1 in silkmoth (Bombyx mori) sex pheromone production

USDA-ARS?s Scientific Manuscript database

Store-operated Ca2+ influx has recently been shown to require the activation of two proteins, stromal interaction molecule 1(STIM1) and Orai1. In mammals the putative channel ion selectivity filter is thought to comprise conserved charged residues in the first and third transmembrane domains of Ora...

Structure of a two-CAP-domain protein from the human hookworm parasite Necator americanus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu

2011-05-01

The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite N. americanus refined to a resolution limit of 2.2 Å is presented. Major proteins secreted by the infective larval stage hookworms upon host entry include Ancylostoma secreted proteins (ASPs), which are characterized by one or two CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) domains. The CAP domain has been reported in diverse phylogenetically unrelated proteins, but has no confirmed function. The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite Necator americanus was refined to a resolution limit of 2.2 Å. The structuremore » was solved by molecular replacement (MR) using Na-ASP-2, a one-CAP-domain ASP, as the search model. The correct MR solution could only be obtained by truncating the polyalanine model of Na-ASP-2 and removing several loops. The structure reveals two CAP domains linked by an extended loop. Overall, the carboxyl-terminal CAP domain is more similar to Na-ASP-2 than to the amino-terminal CAP domain. A large central cavity extends from the amino-terminal CAP domain to the carboxyl-terminal CAP domain, encompassing the putative CAP-binding cavity. The putative CAP-binding cavity is a characteristic cavity in the carboxyl-terminal CAP domain that contains a His and Glu pair. These residues are conserved in all single-CAP-domain proteins, but are absent in the amino-terminal CAP domain. The conserved His residues are oriented such that they appear to be capable of directly coordinating a zinc ion as observed for CAP proteins from reptile venoms. This first structure of a two-CAP-domain ASP can serve as a template for homology modeling of other two-CAP-domain proteins.« less

Comprehensive structural and substrate specificity classification of the Saccharomyces cerevisiae methyltransferome.

PubMed

Wlodarski, Tomasz; Kutner, Jan; Towpik, Joanna; Knizewski, Lukasz; Rychlewski, Leszek; Kudlicki, Andrzej; Rowicka, Maga; Dziembowski, Andrzej; Ginalski, Krzysztof

2011-01-01

Methylation is one of the most common chemical modifications of biologically active molecules and it occurs in all life forms. Its functional role is very diverse and involves many essential cellular processes, such as signal transduction, transcriptional control, biosynthesis, and metabolism. Here, we provide further insight into the enzymatic methylation in S. cerevisiae by conducting a comprehensive structural and functional survey of all the methyltransferases encoded in its genome. Using distant homology detection and fold recognition, we found that the S. cerevisiae methyltransferome comprises 86 MTases (53 well-known and 33 putative with unknown substrate specificity). Structural classification of their catalytic domains shows that these enzymes may adopt nine different folds, the most common being the Rossmann-like. We also analyzed the domain architecture of these proteins and identified several new domain contexts. Interestingly, we found that the majority of MTase genes are periodically expressed during yeast metabolic cycle. This finding, together with calculated isoelectric point, fold assignment and cellular localization, was used to develop a novel approach for predicting substrate specificity. Using this approach, we predicted the general substrates for 24 of 33 putative MTases and confirmed these predictions experimentally in both cases tested. Finally, we show that, in S. cerevisiae, methylation is carried out by 34 RNA MTases, 32 protein MTases, eight small molecule MTases, three lipid MTases, and nine MTases with still unknown substrate specificity.

Functional and structural characterization of a novel putative cysteine protease cell wall-modifying multi-domain enzyme selected from a microbial metagenome.

PubMed

Faheem, Muhammad; Martins-de-Sa, Diogo; Vidal, Julia F D; Álvares, Alice C M; Brandão-Neto, José; Bird, Louise E; Tully, Mark D; von Delft, Frank; Souto, Betulia M; Quirino, Betania F; Freitas, Sonia M; Barbosa, João Alexandre R G

2016-12-09

A current metagenomics focus is to interpret and transform collected genomic data into biological information. By combining structural, functional and genomic data we have assessed a novel bacterial protein selected from a carbohydrate-related activity screen in a microbial metagenomic library from Capra hircus (domestic goat) gut. This uncharacterized protein was predicted as a bacterial cell wall-modifying enzyme (CWME) and shown to contain four domains: an N-terminal, a cysteine protease, a peptidoglycan-binding and an SH3 bacterial domain. We successfully cloned, expressed and purified this putative cysteine protease (PCP), which presented autoproteolytic activity and inhibition by protease inhibitors. We observed cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most bacterial CWME. Fluorimetric binding analysis yielded a K b of 1.8 × 10 5  M -1 for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space R g of 28.35 Å. The elongated molecular envelope corroborates the dynamic light scattering (DLS) estimated size. Furthermore, homology modeling and SAXS allowed the construction of a model that explains the stability and secondary structural changes observed by circular dichroism (CD). In short, we report a novel cell wall-modifying autoproteolytic PCP with insight into its biochemical, biophysical and structural features.

Crystal structures of Hsp104 N-terminal domains from Saccharomyces cerevisiae and Candida albicans suggest the mechanism for the function of Hsp104 in dissolving prions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Peng; Li, Jingzhi; Weaver, Clarissa

Hsp104 is a yeast member of the Hsp100 family which functions as a molecular chaperone to disaggregate misfolded polypeptides. To understand the mechanism by which the Hsp104 N-terminal domain (NTD) interacts with its peptide substrates, crystal structures of the Hsp104 NTDs fromSaccharomyces cerevisiae(ScHsp104NTD) andCandida albicans(CaHsp104NTD) have been determined at high resolution. The structures of ScHsp104NTD and CaHsp104NTD reveal that the yeast Hsp104 NTD may utilize a conserved putative peptide-binding groove to interact with misfolded polypeptides. In the crystal structures ScHsp104NTD forms a homodimer, while CaHsp104NTD exists as a monomer. The consecutive residues Gln105, Gln106 and Lys107, and Lys141 around themore » putative peptide-binding groove mediate the monomer–monomer interactions within the ScHsp104NTD homodimer. Dimer formation by ScHsp104NTD suggests that the Hsp104 NTD may specifically interact with polyQ regions of prion-prone proteins. The data may reveal the mechanism by which Hsp104 NTD functions to suppress and/or dissolve prions.« less

Comparative analyses of putative toxin gene homologs from an Old World viper, Daboia russelii

PubMed Central

Krishnan, Neeraja M.

2017-01-01

Availability of snake genome sequences has opened up exciting areas of research on comparative genomics and gene diversity. One of the challenges in studying snake genomes is the acquisition of biological material from live animals, especially from the venomous ones, making the process cumbersome and time-consuming. Here, we report comparative sequence analyses of putative toxin gene homologs from Russell’s viper (Daboia russelii) using whole-genome sequencing data obtained from shed skin. When compared with the major venom proteins in Russell’s viper studied previously, we found 45–100% sequence similarity between the venom proteins and their putative homologs in the skin. Additionally, comparative analyses of 20 putative toxin gene family homologs provided evidence of unique sequence motifs in nerve growth factor (NGF), platelet derived growth factor (PDGF), Kunitz/Bovine pancreatic trypsin inhibitor (Kunitz BPTI), cysteine-rich secretory proteins, antigen 5, andpathogenesis-related1 proteins (CAP) and cysteine-rich secretory protein (CRISP). In those derived proteins, we identified V11 and T35 in the NGF domain; F23 and A29 in the PDGF domain; N69, K2 and A5 in the CAP domain; and Q17 in the CRISP domain to be responsible for differences in the largest pockets across the protein domain structures in crotalines, viperines and elapids from the in silico structure-based analysis. Similarly, residues F10, Y11 and E20 appear to play an important role in the protein structures across the kunitz protein domain of viperids and elapids. Our study highlights the usefulness of shed skin in obtaining good quality high-molecular weight DNA for comparative genomic studies, and provides evidence towards the unique features and evolution of putative venom gene homologs in vipers. PMID:29230357

Listeria monocytogenes PdeE, a phosphodiesterase that contributes to virulence and has hydrolytic activity against cyclic mononucleotides and cyclic dinucleotides

USDA-ARS?s Scientific Manuscript database

We have identified and partially characterized a putative HD domain hydrolase, LMOf2365_2464, which is highly expressed during listerial intracellular replication. LMOf2365_2464 is annotated as a putative HD domain-containing hydrolase. The ability of an isogenic mutant strain, F2365'2464, to adhere...

Structural Insights into the Protease-like Antigen Plasmodium falciparum SERA5 and Its Noncanonical Active-Site Serine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hodder, Anthony N.; Malby, Robyn L.; Clarke, Oliver B.

The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putativemore » nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S{sub 2} specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.« less

The PYRIN domain: A member of the death domain-fold superfamily

PubMed Central

Fairbrother, Wayne J.; Gordon, Nathaniel C.; Humke, Eric W.; O'Rourke, Karen M.; Starovasnik, Melissa A.; Yin, Jian-Ping; Dixit, Vishva M.

2001-01-01

PYRIN domains were identified recently as putative protein–protein interaction domains at the N-termini of several proteins thought to function in apoptotic and inflammatory signaling pathways. The ∼95 residue PYRIN domains have no statistically significant sequence homology to proteins with known three-dimensional structure. Using secondary structure prediction and potential-based fold recognition methods, however, the PYRIN domain is predicted to be a member of the six-helix bundle death domain-fold superfamily that includes death domains (DDs), death effector domains (DEDs), and caspase recruitment domains (CARDs). Members of the death domain-fold superfamily are well established mediators of protein–protein interactions found in many proteins involved in apoptosis and inflammation, indicating further that the PYRIN domains serve a similar function. An homology model of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1, a member of the Apaf-1/Ced-4 family of proteins, was constructed using the three-dimensional structures of the FADD and p75 neurotrophin receptor DDs, and of the Apaf-1 and caspase-9 CARDs, as templates. Validation of the model using a variety of computational techniques indicates that the fold prediction is consistent with the sequence. Comparison of a circular dichroism spectrum of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1 with spectra of several proteins known to adopt the death domain-fold provides experimental support for the structure prediction. PMID:11514682

The DnaJ Gene Family in Pepper (Capsicum annuum L.): Comprehensive Identification, Characterization and Expression Profiles.

PubMed

Fan, FangFei; Yang, Xian; Cheng, Yuan; Kang, Yunyan; Chai, Xirong

2017-01-01

The DnaJ proteins which function as molecular chaperone played critical roles in plant growth and development and response to heat stress (HS) and also called heat shock protein 40 based on molecular weight. However, little was reported on this gene family in pepper. Recently, the release of the whole pepper genome provided an opportunity for identifying putative DnaJ homologous. In this study, a total of 76 putative pepper DnaJ genes (CaDnaJ01 to CaDnaJ76) were identified using bioinformatics methods and classified into five groups by the presence of the complete three domains (J-domain, zinc finger domain, and C-terminal domain). Chromosome mapping suggested that segmental duplication and tandem duplication were occurred in evolution. The multiple stress-related cis -elements were found in the promoter region of these CaDnaJ genes, which indicated that the CaDnaJs might be involved in the process of responding to complex stress conditions. In addition, expression profiles based on RNA-seq showed that the 47 CaDnaJs were expressed in at least one tissue tested. The result implied that they could be involved in the process of pepper growth and development. qRT-PCR analysis found that 80.60% (54/67) CaDnaJs were induced by HS, indicated that they could participated in pepper response to high temperature treatments. In conclusion, all these results would provide a comprehensive basis for further analyzing the function of CaDnaJ members and be also significant for elucidating the evolutionary relationship in pepper.

The DnaJ Gene Family in Pepper (Capsicum annuum L.): Comprehensive Identification, Characterization and Expression Profiles

PubMed Central

Fan, FangFei; Yang, Xian; Cheng, Yuan; Kang, Yunyan; Chai, Xirong

2017-01-01

The DnaJ proteins which function as molecular chaperone played critical roles in plant growth and development and response to heat stress (HS) and also called heat shock protein 40 based on molecular weight. However, little was reported on this gene family in pepper. Recently, the release of the whole pepper genome provided an opportunity for identifying putative DnaJ homologous. In this study, a total of 76 putative pepper DnaJ genes (CaDnaJ01 to CaDnaJ76) were identified using bioinformatics methods and classified into five groups by the presence of the complete three domains (J-domain, zinc finger domain, and C-terminal domain). Chromosome mapping suggested that segmental duplication and tandem duplication were occurred in evolution. The multiple stress-related cis-elements were found in the promoter region of these CaDnaJ genes, which indicated that the CaDnaJs might be involved in the process of responding to complex stress conditions. In addition, expression profiles based on RNA-seq showed that the 47 CaDnaJs were expressed in at least one tissue tested. The result implied that they could be involved in the process of pepper growth and development. qRT-PCR analysis found that 80.60% (54/67) CaDnaJs were induced by HS, indicated that they could participated in pepper response to high temperature treatments. In conclusion, all these results would provide a comprehensive basis for further analyzing the function of CaDnaJ members and be also significant for elucidating the evolutionary relationship in pepper. PMID:28507559

A bioinformatic survey of distribution, conservation, and probable functions of LuxR solo regulators in bacteria.

PubMed

Subramoni, Sujatha; Florez Salcedo, Diana Vanessa; Suarez-Moreno, Zulma R

2015-01-01

LuxR solo transcriptional regulators contain both an autoinducer binding domain (ABD; N-terminal) and a DNA binding Helix-Turn-Helix domain (HTH; C-terminal), but are not associated with a cognate N-acyl homoserine lactone (AHL) synthase coding gene in the same genome. Although a few LuxR solos have been characterized, their distributions as well as their role in bacterial signal perception and other processes are poorly understood. In this study we have carried out a systematic survey of distribution of all ABD containing LuxR transcriptional regulators (QS domain LuxRs) available in the InterPro database (IPR005143), and identified those lacking a cognate AHL synthase. These LuxR solos were then analyzed regarding their taxonomical distribution, predicted functions of neighboring genes and the presence of complete AHL-QS systems in the genomes that carry them. Our analyses reveal the presence of one or multiple predicted LuxR solos in many proteobacterial genomes carrying QS domain LuxRs, some of them harboring genes for one or more AHL-QS circuits. The presence of LuxR solos in bacteria occupying diverse environments suggests potential ecological functions for these proteins beyond AHL and interkingdom signaling. Based on gene context and the conservation levels of invariant amino acids of ABD, we have classified LuxR solos into functionally meaningful groups or putative orthologs. Surprisingly, putative LuxR solos were also found in a few non-proteobacterial genomes which are not known to carry AHL-QS systems. Multiple predicted LuxR solos in the same genome appeared to have different levels of conservation of invariant amino acid residues of ABD questioning their binding to AHLs. In summary, this study provides a detailed overview of distribution of LuxR solos and their probable roles in bacteria with genome sequence information.

A bioinformatic survey of distribution, conservation, and probable functions of LuxR solo regulators in bacteria

PubMed Central

Subramoni, Sujatha; Florez Salcedo, Diana Vanessa; Suarez-Moreno, Zulma R.

2015-01-01

LuxR solo transcriptional regulators contain both an autoinducer binding domain (ABD; N-terminal) and a DNA binding Helix-Turn-Helix domain (HTH; C-terminal), but are not associated with a cognate N-acyl homoserine lactone (AHL) synthase coding gene in the same genome. Although a few LuxR solos have been characterized, their distributions as well as their role in bacterial signal perception and other processes are poorly understood. In this study we have carried out a systematic survey of distribution of all ABD containing LuxR transcriptional regulators (QS domain LuxRs) available in the InterPro database (IPR005143), and identified those lacking a cognate AHL synthase. These LuxR solos were then analyzed regarding their taxonomical distribution, predicted functions of neighboring genes and the presence of complete AHL-QS systems in the genomes that carry them. Our analyses reveal the presence of one or multiple predicted LuxR solos in many proteobacterial genomes carrying QS domain LuxRs, some of them harboring genes for one or more AHL-QS circuits. The presence of LuxR solos in bacteria occupying diverse environments suggests potential ecological functions for these proteins beyond AHL and interkingdom signaling. Based on gene context and the conservation levels of invariant amino acids of ABD, we have classified LuxR solos into functionally meaningful groups or putative orthologs. Surprisingly, putative LuxR solos were also found in a few non-proteobacterial genomes which are not known to carry AHL-QS systems. Multiple predicted LuxR solos in the same genome appeared to have different levels of conservation of invariant amino acid residues of ABD questioning their binding to AHLs. In summary, this study provides a detailed overview of distribution of LuxR solos and their probable roles in bacteria with genome sequence information. PMID:25759807

Atomic resolution structure of the E. coli YajR transporter YAM domain.

PubMed

Jiang, Daohua; Zhao, Yan; Fan, Junping; Liu, Xuehui; Wu, Yan; Feng, Wei; Zhang, Xuejun C

2014-07-25

YajR is an Escherichia coli transporter that belongs to the major facilitator superfamily. Unlike most MFS transporters, YajR contains a carboxyl terminal, cytosolic domain of 67 amino acid residues termed YAM domain. Although it is speculated that the function of this small soluble domain is to regulate the conformational change of the 12-helix transmembrane domain, its precise regulatory role remains unclear. Here, we report the crystal structure of the YAM domain at 1.07-Å resolution, along with its structure determined using nuclear magnetic resonance. Detailed analysis of the high resolution structure revealed a symmetrical dimer in which a belt of well-ordered poly-pentagonal water molecules is embedded. A mutagenesis experiment and a thermal stability assay were used to analyze the putative role of this dimerization in response to changes in halogen concentration. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification and Characterization of Putative Integron-Like Elements of the Heavy-Metal-Hypertolerant Strains of Pseudomonas spp.

PubMed

Ciok, Anna; Adamczuk, Marcin; Bartosik, Dariusz; Dziewit, Lukasz

2016-11-28

Pseudomonas strains isolated from the heavily contaminated Lubin copper mine and Zelazny Most post-flotation waste reservoir in Poland were screened for the presence of integrons. This analysis revealed that two strains carried homologous DNA regions composed of a gene encoding a DNA_BRE_C domain-containing tyrosine recombinase (with no significant sequence similarity to other integrases of integrons) plus a three-component array of putative integron gene cassettes. The predicted gene cassettes encode three putative polypeptides with homology to (i) transmembrane proteins, (ii) GCN5 family acetyltransferases, and (iii) hypothetical proteins of unknown function (homologous proteins are encoded by the gene cassettes of several class 1 integrons). Comparative sequence analyses identified three structural variants of these novel integron-like elements within the sequenced bacterial genomes. Analysis of their distribution revealed that they are found exclusively in strains of the genus Pseudomonas .

A comprehensive analysis of the Omp85/TpsB protein superfamily structural diversity, taxonomic occurrence, and evolution

PubMed Central

Heinz, Eva; Lithgow, Trevor

2014-01-01

Members of the Omp85/TpsB protein superfamily are ubiquitously distributed in Gram-negative bacteria, and function in protein translocation (e.g., FhaC) or the assembly of outer membrane proteins (e.g., BamA). Several recent findings are suggestive of a further level of variation in the superfamily, including the identification of the novel membrane protein assembly factor TamA and protein translocase PlpD. To investigate the diversity and the causal evolutionary events, we undertook a comprehensive comparative sequence analysis of the Omp85/TpsB proteins. A total of 10 protein subfamilies were apparent, distinguished in their domain structure and sequence signatures. In addition to the proteins FhaC, BamA, and TamA, for which structural and functional information is available, are families of proteins with so far undescribed domain architectures linked to the Omp85 β-barrel domain. This study brings a classification structure to a dynamic protein superfamily of high interest given its essential function for Gram-negative bacteria as well as its diverse domain architecture, and we discuss several scenarios of putative functions of these so far undescribed proteins. PMID:25101071

Interaction of a putative BH3 domain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Dong-Hwa; Ha, Ji-Hyang; Kim, Yul

Highlights: {yields} Identification of a conserved BH3 motif in C-terminal coiled coil region of nCLU. {yields} The nCLU BH3 domain binds to BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. {yields} A conserved binding mechanism of nCLU BH3 and the other pro-apoptotic BH3 peptides with Bcl-X{sub L}. {yields} The absolutely conserved Leu323 and Asp328 of nCLU BH3 domain are critical for binding to Bcl-X{sub L.} {yields} Molecular understanding of the pro-apoptotic function of nCLU as a novel BH3-only protein. -- Abstract: Clusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers. Although CLU is knownmore » to be involved in the regulation of apoptosis and cell survival, the precise molecular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apoptotic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy. The chemical shift perturbation data demonstrated that the nCLU BH3 domain binds to pro-apoptotic BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. A structural model of the Bcl-X{sub L}/nCLU BH3 peptide complex reveals that the binding mode is remarkably similar to those of other Bcl-X{sub L}/BH3 peptide complexes. In addition, mutational analysis confirmed that Leu323 and Asp328 of nCLU BH3 domain, absolutely conserved in the BH3 motifs of BH3-only protein family, are critical for binding to Bcl-X{sub L}. Taken altogether, our results suggest a molecular basis for the pro-apoptotic function of nCLU by elucidating the residue specific interactions of the BH3 motif in nCLU with anti-apoptotic Bcl-2 family proteins.« less

CLAVATA1 Dominant-Negative Alleles Reveal Functional Overlap between Multiple Receptor Kinases That Regulate Meristem and Organ Development

PubMed Central

Diévart, Anne; Dalal, Monica; Tax, Frans E.; Lacey, Alexzandria D.; Huttly, Alison; Li, Jianming; Clark, Steven E.

2003-01-01

The CLAVATA1 (CLV1) receptor kinase controls stem cell number and differentiation at the Arabidopsis shoot and flower meristems. Other components of the CLV1 signaling pathway include the secreted putative ligand CLV3 and the receptor-like protein CLV2. We report evidence indicating that all intermediate and strong clv1 alleles are dominant negative and likely interfere with the activity of unknown receptor kinase(s) that have functional overlap with CLV1. clv1 dominant-negative alleles show major differences from dominant-negative alleles characterized to date in animal receptor kinase signaling systems, including the lack of a dominant-negative effect of kinase domain truncation and the ability of missense mutations in the extracellular domain to act in a dominant-negative manner. We analyzed chimeric receptor kinases by fusing CLV1 and BRASSINOSTEROID INSENSITIVE1 (BRI1) coding sequences and expressing these in clv1 null backgrounds. Constructs containing the CLV1 extracellular domain and the BRI1 kinase domain were strongly dominant negative in the regulation of meristem development. Furthermore, we show that CLV1 expressed within the pedicel can partially replace the function of the ERECTA receptor kinase. We propose the presence of multiple receptors that regulate meristem development in a functionally related manner whose interactions are driven by the extracellular domains and whose activation requires the kinase domain. PMID:12724544

Membrane anchoring of aminoacyl-tRNA synthetases by convergent acquisition of a novel protein domain.

PubMed

Olmedo-Verd, Elvira; Santamaría-Gómez, Javier; Ochoa de Alda, Jesús A G; Ribas de Pouplana, Lluis; Luque, Ignacio

2011-11-25

Four distinct aminoacyl-tRNA synthetases (aaRSs) found in some cyanobacterial species contain a novel protein domain that bears two putative transmembrane helices. This CAAD domain is present in glutamyl-, isoleucyl-, leucyl-, and valyl-tRNA synthetases, the latter of which has probably recruited the domain more than once during evolution. Deleting the CAAD domain from the valyl-tRNA synthetase of Anabaena sp. PCC 7120 did not significantly modify the catalytic properties of this enzyme, suggesting that it does not participate in its canonical tRNA-charging function. Multiple lines of evidence suggest that the function of the CAAD domain is structural, mediating the membrane anchorage of the enzyme, although membrane localization of aaRSs has not previously been described in any living organism. Synthetases containing the CAAD domain were localized in the intracytoplasmic thylakoid membranes of cyanobacteria and were largely absent from the plasma membrane. The CAAD domain was necessary and apparently sufficient for protein targeting to membranes. Moreover, localization of aaRSs in thylakoids was important under nitrogen limiting conditions. In Anabaena, a multicellular filamentous cyanobacterium often used as a model for prokaryotic cell differentiation, valyl-tRNA synthetase underwent subcellular relocation at the cell poles during heterocyst differentiation, a process also dependent on the CAAD domain.

Defining Functional Areas in Individual Human Brains using Resting Functional Connectivity MRI

PubMed Central

Cohen, Alexander L.; Fair, Damien A.; Dosenbach, Nico U.F.; Miezin, Francis M.; Dierker, Donna; Van Essen, David C.; Schlaggar, Bradley L.; Petersen, Steven E.

2009-01-01

The cerebral cortex is anatomically organized at many physical scales starting at the level of single neurons and extending up to functional systems. Current functional magnetic resonance imaging (fMRI) studies often focus at the level of areas, networks, and systems. Except in restricted domains, (e.g. topographically-organized sensory regions), it is difficult to determine area boundaries in the human brain using fMRI. The ability to delineate functional areas non-invasively would enhance the quality of many experimental analyses allowing more accurate across-subject comparisons of independently identified functional areas. Correlations in spontaneous BOLD activity, often referred to as resting state functional connectivity (rs-fcMRI), are especially promising as a way to accurately localize differences in patterns of correlated activity across large expanses of cortex. In the current report, we applied a novel set of image analysis tools to explore the utility of rs-fcMRI for defining wide-ranging functional area boundaries. We find that rs-fcMRI patterns show sharp transitions in correlation patterns and that these putative areal boundaries can be reliably detected in individual subjects as well as in group data. Additionally, combining surface-based analysis techniques with image processing algorithms allows automated mapping of putative areal boundaries across large expanses of cortex without the need for prior information about a region’s function or topography. Our approach reliably produces maps of bounded regions appropriate in size and number for putative functional areas. These findings will hopefully stimulate further methodological refinements and validations. PMID:18367410

Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons.

PubMed

Leung, C L; Sun, D; Zheng, M; Knowles, D R; Liem, R K

1999-12-13

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends-PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH(2) terminus. However, unlike dystonin, mACF7 does not contain a coiled-coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest-specific protein, Gas2. In this paper, we demonstrate that the NH(2)-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

Crystal structure and mutational analysis of aminoacylhistidine dipeptidase from Vibrio alginolyticus reveal a new architecture of M20 metallopeptidases.

PubMed

Chang, Chin-Yuan; Hsieh, Yin-Cheng; Wang, Ting-Yi; Chen, Yi-Chin; Wang, Yu-Kuo; Chiang, Ting-Wei; Chen, Yi-Ju; Chang, Cheng-Hsiang; Chen, Chun-Jung; Wu, Tung-Kung

2010-12-10

Aminoacylhistidine dipeptidases (PepD, EC 3.4.13.3) belong to the family of M20 metallopeptidases from the metallopeptidase H clan that catalyze a broad range of dipeptide and tripeptide substrates, including L-carnosine and L-homocarnosine. Homocarnosine has been suggested as a precursor for the neurotransmitter γ-aminobutyric acid (GABA) and may mediate the antiseizure effects of GABAergic therapies. Here, we report the crystal structure of PepD from Vibrio alginolyticus and the results of mutational analysis of substrate-binding residues in the C-terminal as well as substrate specificity of the PepD catalytic domain-alone truncated protein PepD(CAT). The structure of PepD was found to exist as a homodimer, in which each monomer comprises a catalytic domain containing two zinc ions at the active site center for its hydrolytic function and a lid domain utilizing hydrogen bonds between helices to form the dimer interface. Although the PepD is structurally similar to PepV, which exists as a monomer, putative substrate-binding residues reside in different topological regions of the polypeptide chain. In addition, the lid domain of the PepD contains an "extra" domain not observed in related M20 family metallopeptidases with a dimeric structure. Mutational assays confirmed both the putative di-zinc allocations and the architecture of substrate recognition. In addition, the catalytic domain-alone truncated PepD(CAT) exhibited substrate specificity to l-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepD(CAT) for GABAergic therapies or neuroprotection.

Overlapping and Specific Functions of the Hsp104 N Domain Define Its Role in Protein Disaggregation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jungsoon; Sung, Nuri; Mercado, Jonathan M.

Hsp104 is a ring-forming protein disaggregase that rescues stress-damaged proteins from an aggregated state. To facilitate protein disaggregation, Hsp104 cooperates with Hsp70 and Hsp40 chaperones (Hsp70/40) to form a bi-chaperone system. How Hsp104 recognizes its substrates, particularly the importance of the N domain, remains poorly understood and multiple, seemingly conficting mechanisms have been proposed. Although the N domain is dispensable for protein disaggregation, it is sensitive to point mutations that abolish the function of the bacterial Hsp104 homolog in vitro, and is essential for curing yeast prions by Hsp104 overexpression in vivo. Here, we present the crystal structure of anmore » N-terminal fragment of Saccharomyces cerevisiae Hsp104 with the N domain of one molecule bound to the C-terminal helix of the neighboring D1 domain. Consistent with mimicking substrate interaction, mutating the putative substrate-binding site in a constitutively active Hsp104 variant impairs the recovery of functional protein from aggregates. We fnd that the observed substrate-binding defect can be rescued by Hsp70/40 chaperones, providing a molecular explanation as to why the N domain is dispensable for protein disaggregation when Hsp70/40 is present, yet essential for the dissolution of Hsp104-specifc substrates, such as yeast prions, which likely depends on a direct N domain interaction.« less

Overlapping and Specific Functions of the Hsp104 N Domain Define Its Role in Protein Disaggregation

DOE PAGES

Lee, Jungsoon; Sung, Nuri; Mercado, Jonathan M.; ...

2017-09-11

Hsp104 is a ring-forming protein disaggregase that rescues stress-damaged proteins from an aggregated state. To facilitate protein disaggregation, Hsp104 cooperates with Hsp70 and Hsp40 chaperones (Hsp70/40) to form a bi-chaperone system. How Hsp104 recognizes its substrates, particularly the importance of the N domain, remains poorly understood and multiple, seemingly conficting mechanisms have been proposed. Although the N domain is dispensable for protein disaggregation, it is sensitive to point mutations that abolish the function of the bacterial Hsp104 homolog in vitro, and is essential for curing yeast prions by Hsp104 overexpression in vivo. Here, we present the crystal structure of anmore » N-terminal fragment of Saccharomyces cerevisiae Hsp104 with the N domain of one molecule bound to the C-terminal helix of the neighboring D1 domain. Consistent with mimicking substrate interaction, mutating the putative substrate-binding site in a constitutively active Hsp104 variant impairs the recovery of functional protein from aggregates. We fnd that the observed substrate-binding defect can be rescued by Hsp70/40 chaperones, providing a molecular explanation as to why the N domain is dispensable for protein disaggregation when Hsp70/40 is present, yet essential for the dissolution of Hsp104-specifc substrates, such as yeast prions, which likely depends on a direct N domain interaction.« less

Functional domains of the poliovirus receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koike, Satoshi; Ise, Iku; Nomoto, Akio

1991-05-15

A number of mutant cDNAs of the human poliovirus receptor were constructed to identify essential regions of the molecule as the receptor. All mutant cDNAs carrying the sequence coding for the entire N-terminal immunoglobulin-like domain (domain I) confer permissiveness for poliovirus to mouse L cells, but a mutant cDNA lacking the sequence for domain I does not. The transformants permissive for poliovirus were able to bind the virus and were also recognized by monoclonal antibody D171, which competes with poliovirus for the cellular receptor. These results strongly suggest that the poliovirus binding site resides in domain I of the receptor.more » Mutant cDNAs for the sequence encoding the intracellular peptide were also constructed and expressed in mouse L cells. Susceptibility of these cells to poliovirus revealed that the entire putative cytoplasmic domain is not essential for virus infection. Thus, the cytoplasmic domain of the molecule appears not to play a role in the penetration of poliovirus.« less

Unigenic Evolution: A Novel Genetic Method Localizes a Putative Leucine Zipper That Mediates Dimerization of the Saccharomyces Cerevisiae Regulator Gcr1p

PubMed Central

Deminoff, S. J.; Tornow, J.; Santangelo, G. M.

1995-01-01

The GCR1 gene of Saccharomyces cerevisiae encodes a transcriptional activator that complexes with Rap1p and, through UAS(RPG) elements (Rap1p DNA binding sites), stimulates efficient expression of glycolytic and translational component genes. To map the functionally important domains in Gcr1p, we combined multiple rounds of random mutagenesis in vitro with in vivo selection of functional genes to locate conserved, or hypomutable, regions. We name this method unigenic evolution, a statistical analysis of mutations in evolutionary variants of a single gene in an otherwise isogenic background. Examination of the distribution of 315 mutations in 24 variant alleles allowed the localization of four hypomutable regions in GCR1 (A, B, C, and D). Dispensable N-terminal (intronic) and C-terminal portions of the evolved region of GCR1 were included in the analysis as controls and were, as expected, not hypomutable. The analysis of several insertion, deletion, and point mutations, combined with a comparison of the hypomutability and hydrophobicity plots of Gcr1p, suggested that some of the hypomutable regions may individually or in combination correspond to functionally important surface domains. In particular, we determined that region D contains a putative leucine zipper and is necessary and sufficient for Gcr1p homodimerization. PMID:8601472

Crystal structure of a DEAD box protein from the hyperthermophile Methanococcus jannaschii

PubMed Central

Story, Randall M.; Li, Hong; Abelson, John N.

2001-01-01

We have determined the structure of a DEAD box putative RNA helicase from the hyperthermophile Methanococcus jannaschii. Like other helicases, the protein contains two α/β domains, each with a recA-like topology. Unlike other helicases, the protein exists as a dimer in the crystal. Through an interaction that resembles the dimer interface of insulin, the amino-terminal domain's 7-strand β-sheet is extended to 14 strands across the two molecules. Motifs conserved in the DEAD box family cluster in the cleft between domains, and many of their functions can be deduced by mutational data and by comparison with other helicase structures. Several lines of evidence suggest that motif III Ser-Ala-Thr may be involved in binding RNA. PMID:11171974

The C-terminal domain of the betaine carrier BetP of Corynebacterium glutamicum is directly involved in sensing K+ as an osmotic stimulus.

PubMed

Schiller, Dirk; Rübenhagen, René; Krämer, Reinhard; Morbach, Susanne

2004-05-18

The glycine betaine carrier BetP of Corynebacterium glutamicum was recently shown to function both as an osmosensor and as an osmoregulator in proteoliposomes by sensing changes in the internal K(+) concentration as a measure of hyperosmotic stress. In vivo analysis of mutants carrying deletions at the C-terminal extension of BetP indicated that this domain participates in osmostress-dependent activity regulation. To address the question, whether a putative K(+) sensor is located within the C-terminal domain, several mutants with truncations in this domain were purified and reconstituted in proteoliposomes of Escherichia coli phospholipids, since this in vitro system allowed variation of the K(+) concentration at the lumenal side. Truncation of 12 amino acids led to a partly deregulated BetP in terms of osmoregulation; however, K(+) sensitivity was not impaired in this mutant. The deletion of 25 amino acid residues at the C-terminal end of BetP led to both deregulation of the carrier activity, i.e., high activity independent of external osmolality, and loss of K(+)-dependent transport stimulation, indicating that this region of the C-terminal domain is necessary for K(+) sensing and/or K(+)-dependent carrier activation. Immunological and proteolysis analyses showed that BetP and its recombinant forms were reconstituted in a right-side-out orientation, i.e., the C-terminal domain faces the lumen of the proteoliposomes and is thus able to detect the K(+) signal at the inside. This is the first experimental demonstration of a direct connection between an osmotic stimulus, i.e., the change in internal K(+), and a putative sensor domain.

A domain-based approach for analyzing the function of aluminum-activated malate transporters from wheat (Triticum aestivum) and Arabidopsis thaliana in Xenopus oocytes.

PubMed

Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Ryan, Peter R; Furuichi, Takuya; Yamamoto, Yoko

2014-12-01

Wheat and Arabidopsis plants respond to aluminum (Al) ions by releasing malate from their root apices via Al-activated malate transporter. Malate anions bind with the toxic Al ions and contribute to the Al tolerance of these species. The genes encoding the transporters in wheat and Arabidopsis, TaALMT1 and AtALMT1, respectively, were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the two-electrode voltage clamp system. The Al-activated currents generated by malate efflux were detected for TaALMT1 but not for AtALMT1. Chimeric proteins were generated by swapping the N- and C-terminal halves of TaALMT1 and AtALMT1 (Ta::At and At::Ta). When these chimeras were characterized in oocytes, Al-activated malate efflux was detected for the Ta::At chimera but not for At::Ta, suggesting that the N-terminal half of TaALMT1 is necessary for function in oocytes. An additional chimera, Ta(48)::At, generated by swapping 17 residues from the N-terminus of AtALMT1 with the equivalent 48 residues from TaALMT1, was sufficient to support transport activity. This 48 residue region includes a helical region with a putative transmembrane domain which is absent in AtALMT1. The deletion of this domain from Ta(48)::At led to the complete loss of transport activity. Furthermore, truncations and a deletion at the C-terminal end of TaALMT1 indicated that a putative helical structure in this region was also required for transport function. This study provides insights into the structure-function relationships of Al-activated ALMT proteins by identifying specific domains on the N- and C-termini of TaALMT1 that are critical for basal transport function and Al responsiveness in oocytes. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Structure-Based Phylogenetic Analysis of the Lipocalin Superfamily.

PubMed

Lakshmi, Balasubramanian; Mishra, Madhulika; Srinivasan, Narayanaswamy; Archunan, Govindaraju

2015-01-01

Lipocalins constitute a superfamily of extracellular proteins that are found in all three kingdoms of life. Although very divergent in their sequences and functions, they show remarkable similarity in 3-D structures. Lipocalins bind and transport small hydrophobic molecules. Earlier sequence-based phylogenetic studies of lipocalins highlighted that they have a long evolutionary history. However the molecular and structural basis of their functional diversity is not completely understood. The main objective of the present study is to understand functional diversity of the lipocalins using a structure-based phylogenetic approach. The present study with 39 protein domains from the lipocalin superfamily suggests that the clusters of lipocalins obtained by structure-based phylogeny correspond well with the functional diversity. The detailed analysis on each of the clusters and sub-clusters reveals that the 39 lipocalin domains cluster based on their mode of ligand binding though the clustering was performed on the basis of gross domain structure. The outliers in the phylogenetic tree are often from single member families. Also structure-based phylogenetic approach has provided pointers to assign putative function for the domains of unknown function in lipocalin family. The approach employed in the present study can be used in the future for the functional identification of new lipocalin proteins and may be extended to other protein families where members show poor sequence similarity but high structural similarity.

Regulation of the aceI multidrug efflux pump gene in Acinetobacter baumannii.

PubMed

Liu, Qi; Hassan, Karl A; Ashwood, Heather E; Gamage, Hasinika K A H; Li, Liping; Mabbutt, Bridget C; Paulsen, Ian T

2018-06-01

To investigate the function of AceR, a putative transcriptional regulator of the chlorhexidine efflux pump gene aceI in Acinetobacter baumannii. Chlorhexidine susceptibility and chlorhexidine induction of aceI gene expression were determined by MIC and quantitative real-time PCR, respectively, in A. baumannii WT and ΔaceR mutant strains. Recombinant AceR was prepared as both a full-length protein and as a truncated protein, AceR (86-299), i.e. AceRt, which has the DNA-binding domain deleted. The binding interaction of the purified AceR protein and its putative operator region was investigated by electrophoretic mobility shift assays and DNase I footprinting assays. The binding of AceRt with its putative ligand chlorhexidine was examined using surface plasmon resonance and tryptophan fluorescence quenching assays. MIC determination assays indicated that the ΔaceI and ΔaceR mutant strains both showed lower resistance to chlorhexidine than the parental strain. Chlorhexidine-induced expression of aceI was abolished in a ΔaceR background. Electrophoretic mobility shift assays and DNase I footprinting assays demonstrated chlorhexidine-stimulated binding of AceR with two sites upstream of the putative aceI promoter. Surface plasmon resonance and tryptophan fluorescence quenching assays suggested that the purified ligand-binding domain of the AceR protein was able to bind with chlorhexidine with high affinity. This study provides strong evidence that AceR is an activator of aceI gene expression when challenged with chlorhexidine. This study is the first characterization, to our knowledge, of a regulator controlling expression of a PACE family multidrug efflux pump.

Functional Characterization of the Putative Hepatitis B Virus Core Protein Late Domain Using Retrovirus Chimeras

PubMed Central

Garcia, Mayra L.; Reynolds, Tracy D.; Mothes, Walther; Robek, Michael D.

2013-01-01

The hepatitis B virus (HBV) Core protein encodes a late (L)-domain like motif (129PPAYRPPNAP138) that has been purported to serve as a docking site for recruitment of host factors such as Nedd4 that can mediate viral particle release from infected cells. 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. PMID:24009707

The HhH(2)/NDD Domain of the Drosophila Nod Chromokinesin-like Protein Is Required for Binding to Chromosomes in the Oocyte Nucleus

PubMed Central

Cui, Wei; Hawley, R. Scott

2005-01-01

Nod is a chromokinesin-like protein that plays a critical role in segregating achiasmate chromosomes during female meiosis. The C-terminal half of the Nod protein contains two putative DNA-binding domains. The first of these domains, known as the HMGN domain, consists of three tandemly repeated high-mobility group N motifs. This domain was previously shown to be both necessary and sufficient for binding of the C-terminal half of Nod to mitotic chromosomes in embryos. The second putative DNA-binding domain, denoted HhH(2)/NDD, is a helix-hairpin-helix(2)/Nod-like DNA-binding domain. Although the HhH(2)/NDD domain is not required or sufficient for chromosome binding in embryos, several well-characterized nod mutations have been mapped in this domain. To characterize the role of the HhH(2)/NDD domain in mediating Nod function, we created a series of UAS-driven transgene constructs capable of expressing either a wild-type Nod-GFP fusion protein or proteins in which the HhH(2)/NDD domain had been altered by site-directed mutagenesis. Although wild-type Nod-GFP localizes to the oocyte chromosomes and rescues the segregation defect in nod mutant oocytes, two of three proteins carrying mutants in the HhH(2)/NDD domain fail to either rescue the nod mutant phenotype or bind to oocyte chromosomes. However, these mutant proteins do bind to the polytene chromosomes in nurse-cell nuclei and enter the oocyte nucleus. Thus, even though the HhH(2)/NDD domain is not essential for chromosome binding in other cell types, it is required for chromosome binding in the oocyte. These HhH(2)/NDD mutants also block the localization of Nod to the posterior pole of stage 9–10A oocytes, a process that is thought to facilitate the interaction of Nod with the plus ends of microtubules (Cui et al. 2005). This observation suggests that the Nod HhH2/NDD domain may play other roles in addition to binding Nod to meiotic chromosomes. PMID:16143607

Fragments of Bacterial Endoglycosidase S and Immunoglobulin G Reveal Subdomains of Each That Contribute to Deglycosylation*

PubMed Central

Dixon, Emma V.; Claridge, Jolyon K.; Harvey, David J.; Baruah, Kavitha; Yu, Xiaojie; Vesiljevic, Snezana; Mattick, Susan; Pritchard, Laura K.; Krishna, Benjamin; Scanlan, Christopher N.; Schnell, Jason R.; Higgins, Matthew K.; Zitzmann, Nicole; Crispin, Max

2014-01-01

Endoglycosidase S (EndoS) is a glycoside-hydrolase secreted by the bacterium Streptococcus pyogenes. EndoS preferentially hydrolyzes the N-linked glycans from the Fc region of IgG during infection. This hydrolysis impedes Fc functionality and contributes to the immune evasion strategy of S. pyogenes. Here, we investigate the mechanism of human serum IgG deactivation by EndoS. We expressed fragments of IgG1 and demonstrated that EndoS was catalytically active against all of them including the isolated CH2 domain of the Fc domain. Similarly, we sought to investigate which domains within EndoS could contribute to activity. Bioinformatics analysis of the domain organization of EndoS confirmed the previous predictions of a chitinase domain and leucine-rich repeat but also revealed a putative carbohydrate binding module (CBM) followed by a C-terminal region. Using expressed fragments of EndoS, circular dichroism of the isolated CBM, and a CBM-C-terminal region fusion revealed folded domains dominated by β sheet and α helical structure, respectively. Nuclear magnetic resonance analysis of the CBM with monosaccharides was suggestive of carbohydrate binding functionality. Functional analysis of truncations of EndoS revealed that, whereas the C-terminal of EndoS is dispensable for activity, its deletion impedes the hydrolysis of IgG glycans. PMID:24668806

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jackson, R.N.; Robinson, H.; Klauer, A. A.

The essential RNA helicase, Mtr4, performs a critical role in RNA processing and degradation as an activator of the nuclear exosome. The molecular basis for this vital function is not understood and detailed analysis is significantly limited by the lack of structural data. In this study, we present the crystal structure of Mtr4. The structure reveals a new arch-like domain that is specific to Mtr4 and Ski2 (the cytosolic homologue of Mtr4). In vivo and in vitro analyses demonstrate that the Mtr4 arch domain is required for proper 5.8S rRNA processing, and suggest that the arch functions independently of canonicalmore » helicase activity. In addition, extensive conservation along the face of the putative RNA exit site highlights a potential interface with the exosome. These studies provide a molecular framework for understanding fundamental aspects of helicase function in exosome activation, and more broadly define the molecular architecture of Ski2-like helicases.« less

Protein Composition of Trypanosoma brucei Mitochondrial Membranes

PubMed Central

Acestor, Nathalie; Panigrahi, Aswini K.; Ogata, Yuko; Anupama, Atashi; Stuart, Kenneth D.

2010-01-01

Mitochondria consist of four compartments, outer membrane, intermembrane space, inner membrane and matrix; each harboring specific functions and structures. In this study, we used mass spectrometry (LC-MS/MS) to characterize the protein composition of Trypanosoma brucei mitochondrial membranes, which were enriched by different biochemical fractionation techniques. The analyses identified 202 proteins that contain one or more transmembrane domain(s) and/or positive GRAVY scores. Of these, various criteria were used to assign 72 proteins to mitochondrial membranes with high confidence, and 106 with moderate to low confidence. The sub-cellular localization of a selected subset of 13 membrane assigned proteins was confirmed by tagging and immunofluorescence analysis. While most proteins assigned to mitochondrial membrane have putative roles in metabolic, energy generating, and transport processes, ~50% have no known function. These studies result in a comprehensive profile of the composition and sub-organellar location of proteins in the T. brucei mitochondrion thus, providing useful information on mitochondrial functions. PMID:19834910

Analysis of Tomato spotted wilt virus NSs protein indicates the importance of the N-terminal domain for avirulence and RNA silencing suppression.

PubMed

de Ronde, Dryas; Pasquier, Adrien; Ying, Su; Butterbach, Patrick; Lohuis, Dick; Kormelink, Richard

2014-02-01

Recently, Tomato spotted wilt virus (TSWV) nonstructural protein NSs has been identified unambiguously as an avirulence (Avr) determinant for Tomato spotted wilt (Tsw)-based resistance. The observation that NSs from two natural resistance-breaking isolates had lost RNA silencing suppressor (RSS) activity and Avr suggested a link between the two functions. To test this, a large set of NSs mutants was generated by alanine substitutions in NSs from resistance-inducing wild-type strains (NSs(RI) ), amino acid reversions in NSs from resistance-breaking strains (NSs(RB)), domain deletions and swapping. Testing these mutants for their ability to suppress green fluorescent protein (GFP) silencing and to trigger a Tsw-mediated hypersensitive response (HR) revealed that the two functions can be separated. Changes in the N-terminal domain were found to be detrimental for both activities and indicated the importance of this domain, additionally supported by domain swapping between NSs(RI) and NSs(RB). Swapping domains between the closely related Tospovirus Groundnut ringspot virus (GRSV) NSs and TSWV NSs(RI) showed that Avr functionality could not simply be transferred between species. Although deletion of the C-terminal domain rendered NSs completely dysfunctional, only a few single-amino-acid mutations in the C-terminus affected both functions. Mutation of a GW/WG motif (position 17/18) rendered NSs completely dysfunctional for RSS and Avr activity, and indicated a putative interaction between NSs and Argonaute 1 (AGO1), and its importance in TSWV virulence and viral counter defence against RNA interference. © 2013 BSPP AND JOHN WILEY & SONS LTD.

The cysteine synthase complex from plants. Mitochondrial serine acetyltransferase from Arabidopsis thaliana carries a bifunctional domain for catalysis and protein-protein interaction.

PubMed

Wirtz, M; Berkowitz, O; Droux, M; Hell, R

2001-02-01

Serine acetyltransferase (SAT) catalyzes the rate-limiting step of cysteine biosynthesis in bacteria and plants and functions in association with O-acetylserine (thiol) lyase (OAS-TL) in the cysteine synthase complex. Very little is known about the structure and catalysis of SATs except that they share a characteristic C-terminal hexapeptide-repeat domain with a number of enzymatically unrelated acyltransferases. Computational modeling of this domain was performed for the mitochondrial SAT isoform from Arabidopsis thaliana, based on crystal structures of bacterial acyltransferases. The results indicate a left-handed parallel beta-helix consisting of beta-sheets alternating with turns, resulting in a prism-like structure. This model was challenged by site-directed mutagenesis and tested for a suspected dual function of this domain in catalysis and hetero-oligomerization. The bifunctionality of the SAT C-terminus in transferase activity and interaction with OAS-TL is demonstrated and discussed with respect to the putative role of the cysteine synthase complex in regulation of cysteine biosynthesis.

A systematic analysis of the in vitro and in vivo functions of the HD-GYP domain proteins of Vibrio cholerae.

PubMed

McKee, Robert W; Kariisa, Ankunda; Mudrak, Benjamin; Whitaker, Courtney; Tamayo, Rita

2014-10-25

The second messenger cyclic diguanylate (c-di-GMP) plays a central role in bacterial adaptation to extracellular stimuli, controlling processes such as motility, biofilm development, cell development and, in some pathogens, virulence. The intracellular level of c-di-GMP is controlled by the complementary activities of diguanylate cyclases containing a GGDEF domain and two classes of c-di-GMP phosphodiesterases containing an EAL or HD-GYP hydrolytic domain. Compared to the GGDEF and EAL domains, the functions of HD-GYP domain family proteins are poorly characterized. The human diarrheal pathogen Vibrio cholerae encodes nine putative HD-GYP domain proteins. To determine the contributions of HD-GYP domain proteins to c-di-GMP signaling in V. cholerae, we systematically analyzed the enzymatic functionality of each protein and their involvement in processes known to be regulated by c-di-GMP: motility, biofilm development and virulence. Complementary in vitro and in vivo experiments showed that four HD-GYP domain proteins are active c-di-GMP phosphodiesterases: VC1295, VC1348, VCA0210 and VCA0681. Mutation of individual HD-GYP domain genes, as well as combinatorial mutations of multiple HD-GYP domain genes, had no effect on motility or biofilm formation of V. cholerae under the conditions tested. Furthermore, no single HD-GYP domain gene affected intestinal colonization by V. cholerae in an infant mouse model. However, inactivation of multiple HD-GYP domain genes, including the four encoding functional phosphodiesterases, significantly attenuated colonization. These results indicate that the HD-GYP family of c-di-GMP phosphodiesterases impacts signaling by this second messenger during infection. Altogether, this work greatly furthers the understanding of this important family of c-di-GMP metabolic enzymes and demonstrates a role for HD-GYP domain proteins in the virulence of V. cholerae.

Metallofullerenol Gd@C82(OH)22 distracts the proline-rich-motif from putative binding on the SH3 domain

NASA Astrophysics Data System (ADS)

Kang, Seung-Gu; Huynh, Tien; Zhou, Ruhong

2013-03-01

Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials.Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr33756a

The structure of Tim50(164–361) suggests the mechanism by which Tim50 receives mitochondrial presequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jingzhi; Sha, Bingdong, E-mail: bdsha@uab.edu

2015-08-25

The Tim50 crystal structure indicates that the IMS domain of Tim50 exhibits significant structural plasticity within the putative presequence-binding groove. Mitochondrial preproteins are transported through the translocase of the outer membrane (TOM) complex. Tim50 and Tim23 then transfer preproteins with N-terminal targeting presequences through the intermembrane space (IMS) across the inner membrane. The crystal structure of the IMS domain of Tim50 [Tim50(164–361)] has previously been determined to 1.83 Å resolution. Here, the crystal structure of Tim50(164–361) at 2.67 Å resolution that was crystallized using a different condition is reported. Compared with the previously determined Tim50(164–361) structure, significant conformational changes occurmore » within the protruding β-hairpin of Tim50 and the nearby helix A2. These findings indicate that the IMS domain of Tim50 exhibits significant structural plasticity within the putative presequence-binding groove, which may play important roles in the function of Tim50 as a receptor protein in the TIM complex that interacts with the presequence and multiple other proteins. More interestingly, the crystal packing indicates that helix A1 from the neighboring monomer docks into the putative presequence-binding groove of Tim50(164–361), which may mimic the scenario of Tim50 and the presequence complex. Tim50 may recognize and bind the presequence helix by utilizing the inner side of the protruding β-hairpin through hydrophobic interactions. Therefore, the protruding β-hairpin of Tim50 may play critical roles in receiving the presequence and recruiting Tim23 for subsequent protein translocations.« less

Prediction of CpG-island function: CpG clustering vs. sliding-window methods

PubMed Central

2010-01-01

Background Unmethylated stretches of CpG dinucleotides (CpG islands) are an outstanding property of mammal genomes. Conventionally, these regions are detected by sliding window approaches using %G + C, CpG observed/expected ratio and length thresholds as main parameters. Recently, clustering methods directly detect clusters of CpG dinucleotides as a statistical property of the genome sequence. Results We compare sliding-window to clustering (i.e. CpGcluster) predictions by applying new ways to detect putative functionality of CpG islands. Analyzing the co-localization with several genomic regions as a function of window size vs. statistical significance (p-value), CpGcluster shows a higher overlap with promoter regions and highly conserved elements, at the same time showing less overlap with Alu retrotransposons. The major difference in the prediction was found for short islands (CpG islets), often exclusively predicted by CpGcluster. Many of these islets seem to be functional, as they are unmethylated, highly conserved and/or located within the promoter region. Finally, we show that window-based islands can spuriously overlap several, differentially regulated promoters as well as different methylation domains, which might indicate a wrong merge of several CpG islands into a single, very long island. The shorter CpGcluster islands seem to be much more specific when concerning the overlap with alternative transcription start sites or the detection of homogenous methylation domains. Conclusions The main difference between sliding-window approaches and clustering methods is the length of the predicted islands. Short islands, often differentially methylated, are almost exclusively predicted by CpGcluster. This suggests that CpGcluster may be the algorithm of choice to explore the function of these short, but putatively functional CpG islands. PMID:20500903

In vivo functional mapping of the conserved protein domains within murine Themis1.

PubMed

Zvezdova, Ekaterina; Lee, Jan; El-Khoury, Dalal; Barr, Valarie; Akpan, Itoro; Samelson, Lawrence; Love, Paul E

2014-09-01

Thymocyte development requires the coordinated input of signals that originate from numerous cell surface molecules. Although the majority of thymocyte signal-initiating receptors are lineage-specific, most trigger 'ubiquitous' downstream signaling pathways. T-lineage-specific receptors are coupled to these signaling pathways by lymphocyte-restricted adapter molecules. We and others recently identified a new putative adapter protein, Themis1, whose expression is largely restricted to the T lineage. Mice lacking Themis1 exhibit a severe block in thymocyte development and a striking paucity of mature T cells revealing a critical role for Themis1 in T-cell maturation. Themis1 orthologs contain three conserved domains: a proline-rich region (PRR) that binds to the ubiquitous cytosolic adapter Grb2, a nuclear localization sequence (NLS), and two copies of a novel cysteine-containing globular (CABIT) domain. In the present study, we evaluated the functional importance of each of these motifs by retroviral reconstitution of Themis1(-/-) progenitor cells. The results demonstrate an essential requirement for the PRR and NLS motifs but not the conserved CABIT cysteines for Themis1 function.

Unusual varieties and duplication of Rig-I like receptors encoded in the marine mollusk, Crassostrea gigas

NASA Astrophysics Data System (ADS)

Tian, Z. H.; Jiao, C. Z.

2017-07-01

RIG-I like receptors (RLRs) play key roles in sensing non-self nucleic acids in cytoplasm and trigger antiviral innate immune response in vertebrates and human body. Here we carried out in silico analysis to identify and investigate the putative RLRs encoded in the genome of marine mollusk, Crassostrea gigas (cgRLRs), an invertebrate species. We found the unusual duplication and varieties on domain architecture of putative cgRLRs encoded in the genome of C. gigas. Three putative cgRLRs (accessions numbers are EKC24603, EKC31344.1 and EKC38304.1 on GenBank), have the similar domain architecture with that of human RIG-I or MDA5, and one protein (EKC34573.1) with that of human LGP2; The fifth putative cgRLRs (EKC38303.1) is somewhat similar with human RIG-I/MDA5 except that it has only one caspase activation and recruitment domain (CARD) in its N-terminal. Other nine proteins were identified to be partialy similar with RLRs while with the incomplete sequences, which maybe reflect the events of partial duplication of cgRLRs genes occurred in the oyster genome.

Distinct domains of the AVRPM3A2/F2 avirulence protein from wheat powdery mildew are involved in immune receptor recognition and putative effector function

USDA-ARS?s Scientific Manuscript database

Recognition of the AVRPM3A2/F2 avirulence protein from powdery mildew by the wheat PM3A/F immune receptor induces a hypersensitive response after coexpression in Nicotiana benthamiana. The molecular determinants of this interaction and how they shape natural AvrPm3a2/f2 allelic diversity is unknown....

Systems-based biological concordance and predictive reproducibility of gene set discovery methods in cardiovascular disease.

PubMed

Azuaje, Francisco; Zheng, Huiru; Camargo, Anyela; Wang, Haiying

2011-08-01

The discovery of novel disease biomarkers is a crucial challenge for translational bioinformatics. Demonstration of both their classification power and reproducibility across independent datasets are essential requirements to assess their potential clinical relevance. Small datasets and multiplicity of putative biomarker sets may explain lack of predictive reproducibility. Studies based on pathway-driven discovery approaches have suggested that, despite such discrepancies, the resulting putative biomarkers tend to be implicated in common biological processes. Investigations of this problem have been mainly focused on datasets derived from cancer research. We investigated the predictive and functional concordance of five methods for discovering putative biomarkers in four independently-generated datasets from the cardiovascular disease domain. A diversity of biosignatures was identified by the different methods. However, we found strong biological process concordance between them, especially in the case of methods based on gene set analysis. With a few exceptions, we observed lack of classification reproducibility using independent datasets. Partial overlaps between our putative sets of biomarkers and the primary studies exist. Despite the observed limitations, pathway-driven or gene set analysis can predict potentially novel biomarkers and can jointly point to biomedically-relevant underlying molecular mechanisms. Copyright © 2011 Elsevier Inc. All rights reserved.

Genome-wide identification, classification, and functional analysis of the basic helix-loop-helix transcription factors in the cattle, Bos Taurus.

PubMed

Li, Fengmei; Liu, Wuyi

2017-06-01

The basic helix-loop-helix (bHLH) transcription factors (TFs) form a huge superfamily and play crucial roles in many essential developmental, genetic, and physiological-biochemical processes of eukaryotes. In total, 109 putative bHLH TFs were identified and categorized successfully in the genomic databases of cattle, Bos Taurus, after removing redundant sequences and merging genetic isoforms. Through phylogenetic analyses, 105 proteins among these bHLH TFs were classified into 44 families with 46, 25, 14, 3, 13, and 4 members in the high-order groups A, B, C, D, E, and F, respectively. The remaining 4 bHLH proteins were sorted out as 'orphans.' Next, these 109 putative bHLH proteins identified were further characterized as significantly enriched in 524 significant Gene Ontology (GO) annotations (corrected P value ≤ 0.05) and 21 significantly enriched pathways (corrected P value ≤ 0.05) that had been mapped by the web server KOBAS 2.0. Furthermore, 95 bHLH proteins were further screened and analyzed together with two uncharacterized proteins in the STRING online database to reconstruct the protein-protein interaction network of cattle bHLH TFs. Ultimately, 89 bHLH proteins were fully mapped in a network with 67 biological process, 13 molecular functions, 5 KEGG pathways, 12 PFAM protein domains, and 25 INTERPRO classified protein domains and features. These results provide much useful information and a good reference for further functional investigations and updated researches on cattle bHLH TFs.

An in silico algorithm for identifying stabilizing pockets in proteins: test case, the Y220C mutant of the p53 tumor suppressor protein

PubMed Central

Bromley, Dennis; Bauer, Matthias R.; Fersht, Alan R.; Daggett, Valerie

2016-01-01

The p53 tumor suppressor protein performs a critical role in stimulating apoptosis and cell cycle arrest in response to oncogenic stress. The function of p53 can be compromised by mutation, leading to increased risk of cancer; approximately 50% of cancers are associated with mutations in the p53 gene, the majority of which are in the core DNA-binding domain. The Y220C mutation of p53, for example, destabilizes the core domain by 4 kcal/mol, leading to rapid denaturation and aggregation. The associated loss of tumor suppressor functionality is associated with approximately 75 000 new cancer cases every year. Destabilized p53 mutants can be ‘rescued’ and their function restored; binding of a small molecule into a pocket on the surface of mutant p53 can stabilize its wild-type structure and restore its function. Here, we describe an in silico algorithm for identifying potential rescue pockets, including the algorithm's integration with the Dynameomics molecular dynamics data warehouse and the DIVE visual analytics engine. We discuss the results of the application of the method to the Y220C p53 mutant, entailing finding a putative rescue pocket through MD simulations followed by an in silico search for stabilizing ligands that dock into the putative rescue pocket. The top three compounds from this search were tested experimentally and one of them bound in the pocket, as shown by nuclear magnetic resonance, and weakly stabilized the mutant. PMID:27503952

Structure of Drosophila Oskar reveals a novel RNA binding protein

PubMed Central

Yang, Na; Yu, Zhenyu; Hu, Menglong; Wang, Mingzhu; Lehmann, Ruth; Xu, Rui-Ming

2015-01-01

Oskar (Osk) protein plays critical roles during Drosophila germ cell development, yet its functions in germ-line formation and body patterning remain poorly understood. This situation contrasts sharply with the vast knowledge about the function and mechanism of osk mRNA localization. Osk is predicted to have an N-terminal LOTUS domain (Osk-N), which has been suggested to bind RNA, and a C-terminal hydrolase-like domain (Osk-C) of unknown function. Here, we report the crystal structures of Osk-N and Osk-C. Osk-N shows a homodimer of winged-helix–fold modules, but without detectable RNA-binding activity. Osk-C has a lipase-fold structure but lacks critical catalytic residues at the putative active site. Surprisingly, we found that Osk-C binds the 3′UTRs of osk and nanos mRNA in vitro. Mutational studies identified a region of Osk-C important for mRNA binding. These results suggest possible functions of Osk in the regulation of stability, regulation of translation, and localization of relevant mRNAs through direct interaction with their 3′UTRs, and provide structural insights into a novel protein–RNA interaction motif involving a hydrolase-related domain. PMID:26324911

ELMO Domains, Evolutionary and Functional Characterization of a Novel GTPase-activating Protein (GAP) Domain for Arf Protein Family GTPases*

PubMed Central

East, Michael P.; Bowzard, J. Bradford; Dacks, Joel B.; Kahn, Richard A.

2012-01-01

The human family of ELMO domain-containing proteins (ELMODs) consists of six members and is defined by the presence of the ELMO domain. Within this family are two subclassifications of proteins, based on primary sequence conservation, protein size, and domain architecture, deemed ELMOD and ELMO. In this study, we used homology searching and phylogenetics to identify ELMOD family homologs in genomes from across eukaryotic diversity. This demonstrated not only that the protein family is ancient but also that ELMOs are potentially restricted to the supergroup Opisthokonta (Metazoa and Fungi), whereas proteins with the ELMOD organization are found in diverse eukaryotes and thus were likely the form present in the last eukaryotic common ancestor. The segregation of the ELMO clade from the larger ELMOD group is consistent with their contrasting functions as unconventional Rac1 guanine nucleotide exchange factors and the Arf family GTPase-activating proteins, respectively. We used unbiased, phylogenetic sorting and sequence alignments to identify the most highly conserved residues within the ELMO domain to identify a putative GAP domain within the ELMODs. Three independent but complementary assays were used to provide an initial characterization of this domain. We identified a highly conserved arginine residue critical for both the biochemical and cellular GAP activity of ELMODs. We also provide initial evidence of the function of human ELMOD1 as an Arf family GAP at the Golgi. These findings provide the basis for the future study of the ELMOD family of proteins and a new avenue for the study of Arf family GTPases. PMID:23014990

Membrane topology of Golgi-localized probable S-adenosylmethionine-dependent methyltransferase in tobacco (Nicotiana tabacum) BY-2 cells.

PubMed

Liu, Jianping; Hayashi, Kyoko; Matsuoka, Ken

2015-01-01

S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) transfer methyl groups to substrates. In this study, a novel putative tobacco SAM-MTase termed Golgi-localized methyl transferase 1 (GLMT1) has been characterized. GLMT1 is comprised of 611 amino acids with short N-terminal region, putative transmembrane region, and C-terminal SAM-MTase domain. Expression of monomeric red fluorescence protein (mRFP)-tagged protein in tobacco BY-2 cell indicated that GLMT1 is a Golgi-localized protein. Analysis of the membrane topology by protease digestion suggested that both C-terminal catalytic region and N-terminal region seem to be located to the cytosolic side of the Golgi apparatus. Therefore, GLMT1 might have a different function than the previously studied SAM-MTases in plants.

Solubilization of a membrane protein by combinatorial supercharging.

PubMed

Hajduczki, Agnes; Majumdar, Sudipta; Fricke, Marie; Brown, Isola A M; Weiss, Gregory A

2011-04-15

Hydrophobic and aggregation-prone, membrane proteins often prove too insoluble for conventional in vitro biochemical studies. To engineer soluble variants of human caveolin-1, a phage-displayed library of caveolin variants targeted the hydrophobic intramembrane domain with substitutions to charged residues. Anti-selections for insolubility removed hydrophobic variants, and positive selections for binding to the known caveolin ligand HIV gp41 isolated functional, folded variants. Assays with several caveolin binding partners demonstrated the successful folding and functionality by a solubilized, full-length caveolin variant selected from the library. This caveolin variant allowed assay of the direct interaction between caveolin and cavin. Clustered along one face of a putative helix, the solubilizing mutations suggest a structural model for the intramembrane domain of caveolin. The approach provides a potentially general method for solubilization and engineering of membrane-associated proteins by phage display.

Human tRNA genes function as chromatin insulators

PubMed Central

Raab, Jesse R; Chiu, Jonathan; Zhu, Jingchun; Katzman, Sol; Kurukuti, Sreenivasulu; Wade, Paul A; Haussler, David; Kamakaka, Rohinton T

2012-01-01

Insulators help separate active chromatin domains from silenced ones. In yeast, gene promoters act as insulators to block the spread of Sir and HP1 mediated silencing while in metazoans most insulators are multipartite autonomous entities. tDNAs are repetitive sequences dispersed throughout the human genome and we now show that some of these tDNAs can function as insulators in human cells. Using computational methods, we identified putative human tDNA insulators. Using silencer blocking, transgene protection and repressor blocking assays we show that some of these tDNA-containing fragments can function as barrier insulators in human cells. We find that these elements also have the ability to block enhancers from activating RNA pol II transcribed promoters. Characterization of a putative tDNA insulator in human cells reveals that the site possesses chromatin signatures similar to those observed at other better-characterized eukaryotic insulators. Enhanced 4C analysis demonstrates that the tDNA insulator makes long-range chromatin contacts with other tDNAs and ETC sites but not with intervening or flanking RNA pol II transcribed genes. PMID:22085927

Expression analysis of the Arabidopsis thaliana AtSpen2 gene, and its relationship with other plant genes encoding Spen proteins

PubMed Central

Solís-Guzmán, María Gloria; Argüello-Astorga, Gerardo; López-Bucio, José; Ruiz-Herrera, León Francisco; López-Meza, Joel; Sánchez-Calderón, Lenin; Carreón-Abud, Yazmín; Martínez-Trujillo, Miguel

2017-01-01

Abstract Proteins of the Split ends (Spen) family are characterized by an N-terminal domain, with one or more RNA recognition motifs and a SPOC domain. In Arabidopsis thaliana, the Spen protein FPA is involved in the control of flowering time as a component of an autonomous pathway independent of photoperiod. The A. thaliana genome encodes another gene for a putative Spen protein at the locus At4g12640, herein named AtSpen2. Bioinformatics analysis of the AtSPEN2 SPOC domain revealed low sequence similarity with the FPA SPOC domain, which was markedly lower than that found in other Spen proteins from unrelated plant species. To provide experimental information about the function of AtSpen2, A. thaliana plants were transformed with gene constructs of its promoter region with uidA::gfp reporter genes; the expression was observed in vascular tissues of leaves and roots, as well as in ovules and developing embryos. There was absence of a notable phenotype in knockout and overexpressing lines, suggesting that its function in plants might be specific to certain endogenous or environmental conditions. Our results suggest that the function of Atspen2 diverged from that of fpa due in part to their different transcription expression pattern and divergence of the regulatory SPOC domain. PMID:28850635

Crystal structure of the TRIM25 B30.2 (PRYSPRY) domain: a key component of antiviral signalling.

PubMed

D'Cruz, Akshay A; Kershaw, Nadia J; Chiang, Jessica J; Wang, May K; Nicola, Nicos A; Babon, Jeffrey J; Gack, Michaela U; Nicholson, Sandra E

2013-12-01

TRIM (tripartite motif) proteins primarily function as ubiquitin E3 ligases that regulate the innate immune response to infection. TRIM25 [also known as Efp (oestrogen-responsive finger protein)] has been implicated in the regulation of oestrogen receptor α signalling and in the regulation of innate immune signalling via RIG-I (retinoic acid-inducible gene-I). RIG-I senses cytosolic viral RNA and is subsequently ubiquitinated by TRIM25 at its N-terminal CARDs (caspase recruitment domains), leading to type I interferon production. The interaction with RIG-I is dependent on the TRIM25 B30.2 domain, a protein-interaction domain composed of the PRY and SPRY tandem sequence motifs. In the present study we describe the 1.8 Å crystal structure of the TRIM25 B30.2 domain, which exhibits a typical B30.2/SPRY domain fold comprising two N-terminal α-helices, thirteen β-strands arranged into two β-sheets and loop regions of varying lengths. A comparison with other B30.2/SPRY structures and an analysis of the loop regions identified a putative binding pocket, which is likely to be involved in binding target proteins. This was supported by mutagenesis and functional analyses, which identified two key residues (Asp(488) and Trp(621)) in the TRIM25 B30.2 domain as being critical for binding to the RIG-I CARDs.

Crystal structure of the TRIM25 B30.2 (PRYSPRY) domain: a key component of antiviral signalling

PubMed Central

D'Cruz, Akshay A.; Kershaw, Nadia J.; Chiang, Jessica J.; Wang, May K.; Nicola, Nicos A.; Babon, Jeffrey J.; Gack, Michaela U.; Nicholson, Sandra E.

2014-01-01

TRIM (tripartite motif) proteins primarily function as ubiquitin E3 ligases that regulate the innate immune response to infection. TRIM25 [also known as Efp (oestrogen-responsive finger protein)] has been implicated in the regulation of oestrogen receptor α signalling and in the regulation of innate immune signalling via RIG-I (retinoic acid-inducible gene-I). RIG-I senses cytosolic viral RNA and is subsequently ubiquitinated by TRIM25 at its N-terminal CARDs (caspase recruitment domains), leading to type I interferon production. The interaction with RIG-I is dependent on the TRIM25 B30.2 domain, a protein-interaction domain composed of the PRY and SPRY tandem sequence motifs. In the present study we describe the 1.8 Å crystal structure of the TRIM25 B30.2 domain, which exhibits a typical B30.2/SPRY domain fold comprising two N-terminal α-helices, thirteen β-strands arranged into two β-sheets and loop regions of varying lengths. A comparison with other B30.2/SPRY structures and an analysis of the loop regions identified a putative binding pocket, which is likely to be involved in binding target proteins. This was supported by mutagenesis and functional analyses, which identified two key residues (Asp488 and Trp621) in the TRIM25 B30.2 domain as being critical for binding to the RIG-I CARDs. PMID:24015671

Origin, evolution, and divergence of plant class C GH9 endoglucanases.

PubMed

Kundu, Siddhartha; Sharma, Rita

2018-05-30

Glycoside hydrolases of the GH9 family encode cellulases that predominantly function as endoglucanases and have wide applications in the food, paper, pharmaceutical, and biofuel industries. The partitioning of plant GH9 endoglucanases, into classes A, B, and C, is based on the differential presence of transmembrane, signal peptide, and the carbohydrate binding module (CBM49). There is considerable debate on the distribution and the functions of these enzymes which may vary in different organisms. In light of these findings we examined the origin, emergence, and subsequent divergence of plant GH9 endoglucanases, with an emphasis on elucidating the role of CBM49 in the digestion of crystalline cellulose by class C members. Since, the digestion of crystalline cellulose mandates the presence of a well-defined set of aromatic and polar amino acids and/or an attributable domain that can mediate this conversion, we hypothesize a vertical mode of transfer of genes that could favour the emergence of class C like GH9 endoglucanase activity in land plants from potentially ancestral non plant taxa. We demonstrated the concomitant occurrence of a GH9 domain with CBM49 and other homologous carbohydrate binding modules, in putative endoglucanase sequences from several non-plant taxa. In the absence of comparable full length CBMs, we have characterized several low strength patterns that could approximate the CBM49, thereby, extending support for digestion of crystalline cellulose to other segments of the protein. We also provide data suggestive of the ancestral role of putative class C GH9 endoglucanases in land plants, which includes detailed phylogenetics and the presence and subsequent loss of CBM49, transmembrane, and signal peptide regions in certain populations of early land plants. These findings suggest that classes A and B of modern vascular land plants may have emerged by diverging directly from CBM49 encompassing putative class C enzymes. Our detailed phylogenetic and bioinformatics analysis of putative GH9 endoglucanase sequences across major taxa suggests that plant class C enzymes, despite their recent discovery, could function as the last common ancestor of classes A and B. Additionally, research into their ability to digest or inter-convert crystalline and amorphous forms of cellulose could make them lucrative candidates for engineering biofuel feedstock.

Effects of mutations in the Arabidopsis Cold Shock Domain Protein 3 (AtCSP3) gene on leaf cell expansion.

PubMed

Yang, Yongil; Karlson, Dale

2012-08-01

The cold shock domain is among the most evolutionarily conserved nucleic acid binding domains from prokaryotes to higher eukaryotes, including plants. Although eukaryotic cold shock domain proteins have been extensively studied as transcriptional and post-transcriptional regulators during various developmental processes, their functional roles in plants remains poorly understood. In this study, AtCSP3 (At2g17870), which is one of four Arabidopsis thaliana c old s hock domain proteins (AtCSPs), was functionally characterized. Quantitative RT-PCR analysis confirmed high expression of AtCSP3 in reproductive and meristematic tissues. A homozygous atcsp3 loss-of-function mutant exhibits an overall reduced seedling size, stunted and orbicular rosette leaves, reduced petiole length, and curled leaf blades. Palisade mesophyll cells are smaller and more circular in atcsp3 leaves. Cell size analysis indicated that the reduced size of the circular mesophyll cells appears to be generated by a reduction of cell length along the leaf-length axis, resulting in an orbicular leaf shape. It was also determined that leaf cell expansion is impaired for lateral leaf development in the atcsp3 loss-of-function mutant, but leaf cell proliferation is not affected. AtCSP3 loss-of-function resulted in a dramatic reduction of LNG1 transcript, a gene that is involved in two-dimensional leaf polarity regulation. Transient subcellular localization of AtCSP3 in onion epidermal cells confirmed a nucleocytoplasmic localization pattern. Collectively, these data suggest that AtCSP3 is functionally linked to the regulation of leaf length by affecting LNG1 transcript accumulation during leaf development. A putative function of AtCSP3 as an RNA binding protein is also discussed in relation to leaf development.

Substituted cysteine accessibility method (SCAM) analysis of the transport domain of human concentrative nucleoside transporter 3 (hCNT3) and other family members reveals features of structural and functional importance

PubMed Central

Mulinta, Ras; Yao, Sylvia Y. M.; Ng, Amy M. L.; Cass, Carol E.; Young, James D.

2017-01-01

The human SLC28 family of concentrative nucleoside transporter (CNT) proteins has three members: hCNT1, hCNT2, and hCNT3. Na+-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 transports both pyrimidine and purine nucleosides utilizing Na+ and/or H+ electrochemical gradients. Escherichia coli CNT family member NupC resembles hCNT1 in permeant selectivity but is H+-coupled. Using heterologous expression in Xenopus oocytes and the engineered cysteine-less hCNT3 protein hCNT3(C−), substituted cysteine accessibility method analysis with the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate was performed on the transport domain (interfacial helix 2, hairpin 1, putative transmembrane domain (TM) 7, and TM8), as well as TM9 of the scaffold domain of the protein. This systematic scan of the entire C-terminal half of hCNT3(C−) together with parallel studies of the transport domain of wild-type hCNT1 and the corresponding TMs of cysteine-less NupC(C−) yielded results that validate the newly developed structural homology model of CNT membrane architecture for human CNTs, revealed extended conformationally mobile regions within transport-domain TMs, identified pore-lining residues of functional importance, and provided evidence of an emerging novel elevator-type mechanism of transporter function. PMID:28385889

Structural and temporal requirements of Wnt/PCP protein Vangl2 function for convergence and extension movements and facial branchiomotor neuron migration in zebrafish.

PubMed

Pan, Xiufang; Sittaramane, Vinoth; Gurung, Suman; Chandrasekhar, Anand

2014-02-01

Van gogh-like 2 (Vangl2), a core component of the Wnt/planar cell polarity (PCP) signaling pathway, is a four-pass transmembrane protein with N-terminal and C-terminal domains located in the cytosol, and is structurally conserved from flies to mammals. In vertebrates, Vangl2 plays an essential role in convergence and extension (CE) movements during gastrulation and in facial branchiomotor (FBM) neuron migration in the hindbrain. However, the roles of specific Vangl2 domains, of membrane association, and of specific extracellular and intracellular motifs have not been examined, especially in the context of FBM neuron migration. Through heat shock-inducible expression of various Vangl2 transgenes, we found that membrane associated functions of the N-terminal and C-terminal domains of Vangl2 are involved in regulating FBM neuron migration. Importantly, through temperature shift experiments, we found that the critical period for Vangl2 function coincides with the initial stages of FBM neuron migration out of rhombomere 4. Intriguingly, we have also uncovered a putative nuclear localization motif in the C-terminal domain that may play a role in regulating CE movements. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Gene expression profiles of prohibitin in testes of Octopus tankahkeei (ot-phb) revealing its possible role during spermiogenesis.

PubMed

Mao, Hai-Tao; Wang, Da-Hui; Lan, Zhou; Zhou, Hong; Yang, Wan-Xi

2012-05-01

Prohibitin is essential for intracellular homeostasis and stabilization of mitochondrial respiratory chain complexes. To explore its functions during spermiogenesis of Octopus tankahkeei (O. tankahkeei), we have cloned and sequenced the cDNA of this mammalian PHB homologue (termed ot-PHB) from the testes of O. tankahkeei. The 1165 bp ot-phb cDNA contains a 100 bp 5' UTR, a 882 bp open reading frame and a 183 bp 3' UTR. The putative ot-PHB protein owns a transmembrane domain from 6 to 31 amino acid (aa) and a putative PHB domain from 26 to 178 aa. Protein alignment demonstrated that ot-PHB had 73.3, 73.6, 74.0, 75.1, and 45.4% identity with its homologues in Homo sapiens, Mus muculus, Danio rerio, Xenopus tropicalis and Trypanosoma brucei, respectively. Tissue distribution profile analysis revealed its presence in all the tissues examined. In situ hybridization in spermiogenic cells demonstrated that ot-phb was expressed moderately at the beginning of the spermiogenesis. The abundance of transcripts increased in intermediate spermatids and in drastically remodeling final spermatids. In mature spermatozoa, the residuary transcripts concentrated around the chondriosomal mantle where mitochondria assemble around. In summary, the expression of ot-phb during spermiogenesis implicates a potential function of this protein during mitochondrial ubiquitination. It is the first time to implicate the role of prohibitin in cephalopod spermiogenesis.

Nuclear localization and transactivation by Vitis CBF transcription factors are regulated by combinations of conserved amino acid domains.

PubMed

Carlow, Chevonne E; Faultless, J Trent; Lee, Christine; Siddiqua, Mahbuba; Edge, Alison; Nassuth, Annette

2017-09-01

The highly conserved CBF pathway is crucial in the regulation of plant responses to low temperatures. Extensive analysis of Arabidopsis CBF proteins revealed that their functions rely on several conserved amino acid domains although the exact function of each domain is disputed. The question was what functions similar domains have in CBFs from other, overwintering woody plants such as Vitis, which likely have a more involved regulation than the model plant Arabidopsis. A total of seven CBF genes were cloned and sequenced from V. riparia and the less frost tolerant V. vinifera. The deduced species-specific amino acid sequences differ in only a few amino acids, mostly in non-conserved regions. Amino acid sequence comparison and phylogenetic analysis showed two distinct groups of Vitis CBFs. One group contains CBF1, CBF2, CBF3 and CBF8 and the other group contains CBF4, CBF5 and CBF6. Transient transactivation assays showed that all Vitis CBFs except CBF5 activate via a CRT or DRE promoter element, whereby Vitis CBF3 and 4 prefer a CRT element. The hydrophobic domains in the C-terminal end of VrCBF6 were shown to be important for how well it activates. The putative nuclear localization domain of Vitis CBF1 was shown to be sufficient for nuclear localization, in contrast to previous reports for AtCBF1, and also important for transactivation. The latter highlights the value of careful analysis of domain functions instead of reliance on computer predictions and published data for other related proteins. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Structural Characterization of the Ectodomain of a Disintegrin and Metalloproteinase-22 (ADAM22), a Neural Adhesion Receptor Instead of Metalloproteinase INSIGHTS ON ADAM FUNCTION

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Heli; Shim, Ann H.R.; He, Xiaolin

2009-12-01

ADAMs (adisintegrin and metalloproteinases) are a family of multidomain transmembrane glycoproteins with diverse roles in physiology and diseases, with several members being drug targets for cancer and inflammation therapies. The spatial organization of the ADAM extracellular segment and its influence on the function of ADAMs have been unclear. Although most members of the ADAM family are active zinc metalloproteinases, 8 of 21 ADAMs lack functional metalloproteinase domains and are implicated in protein-protein interactions instead of membrane protein ectodomain shedding. One of such non-proteinase ADAMs, ADAM22, acts as a receptor on the surface of the postsynaptic neuron to regulate synaptic signalmore » transmission. The crystal structure of the full ectodomain of mature human ADAM22 shows that it is a compact four-leaf clover with the metalloproteinase-like domain held in the concave face of a rigid module formed by the disintegrin, cysteine-rich, and epidermal growth factor-like domains. The loss of metalloproteinase activity is ensured by the absence of critical catalytic residues, the filling of the substrate groove, and the steric hindrance by the cysteine-rich domain. The structure, combined with calorimetric experiments, suggests distinct roles of three putative calcium ions bound to ADAM22, with one in the metalloproteinase-like domain being regulatory and two in the disintegrin domain being structural. The metalloproteinase-like domain contacts the rest of ADAM22 with discontinuous, hydrophilic, and poorly complemented interactions, suggesting the possibility of modular movement of ADAM22 and other ADAMs. The ADAM22 structure provides a framework for understanding how different ADAMs exert their adhesive function and shedding activities.« less

New genes and new biological roles for expansins

NASA Technical Reports Server (NTRS)

Cosgrove, D. J.

2000-01-01

Expansins are extracellular proteins that loosen plant cell walls in novel ways. They are thought to function in cell enlargement, pollen tube invasion of the stigma (in grasses), wall disassembly during fruit ripening, abscission and other cell separation events. Expansins are encoded by two multigene families and each gene is often expressed in highly specific locations and cell types. Structural analysis indicates that one expansin region resembles the catalytic domain of family-45 endoglucanases but glucanase activity has not been detected. The genome projects have revealed numerous expansin-related sequences but their putative wall-loosening functions remain to be assessed.

Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

PubMed

Frébortová, Jitka; Greplová, Marta; Seidl, Michael F; Heyl, Alexander; Frébort, Ivo

2015-01-01

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120

PubMed Central

Frébortová, Jitka; Greplová, Marta; Seidl, Michael F.; Heyl, Alexander; Frébort, Ivo

2015-01-01

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants. PMID:26376297

Removal of a putative inhibitory element reduces the calcium-dependent calmodulin activation of neuronal nitric-oxide synthase.

PubMed

Montgomery, H J; Romanov, V; Guillemette, J G

2000-02-18

Neuronal nitric-oxide synthase (NOS) and endothelial NOS are constitutive NOS isoforms that are activated by binding calmodulin in response to elevated intracellular calcium. In contrast, the inducible NOS isoform binds calmodulin at low basal levels of calcium in resting cells. Primary sequence comparisons show that each constitutive NOS isozyme contains a polypeptide segment within its reductase domain, which is absent in the inducible NOS enzyme. To study a possible link between the presence of these additional polypeptide segments in constitutive NOS enzymes and their calcium-dependent calmodulin activation, three deletion mutants were created. The putative inhibitory insert was removed from the FMN binding regions of the neuronal NOS holoenzyme and from two truncated neuronal NOS reductase enzymes in which the calmodulin binding region was either included or deleted. All three mutant enzymes showed reduced incorporation of FMN and required reconstitution with exogenous FMN for activity. The combined removal of both the calmodulin binding domain and the putative inhibitory insert did not result in a calmodulin-independent neuronal NOS reductase. Thus, although the putative inhibitory element has an effect on the calcium-dependent calmodulin activation of neuronal NOS, it does not have the properties of the typical autoinhibitory domain found in calmodulin-activated enzymes.

A Novel Tenebrio molitor Cadherin Is a Functional Receptor for Bacillus thuringiensis Cry3Aa Toxin*

PubMed Central

Fabrick, Jeff; Oppert, Cris; Lorenzen, Marcé D.; Morris, Kaley; Oppert, Brenda; Jurat-Fuentes, Juan Luis

2009-01-01

Cry toxins produced by the bacterium Bacillus thuringiensis are effective biological insecticides. Cadherin-like proteins have been reported as functional Cry1A toxin receptors in Lepidoptera. Here we present data that demonstrate that a coleopteran cadherin is a functional Cry3Aa toxin receptor. The Cry3Aa receptor cadherin was cloned from Tenebrio molitor larval midgut mRNA, and the predicted protein, TmCad1, has domain structure and a putative toxin binding region similar to those in lepidopteran cadherin B. thuringiensis receptors. A peptide containing the putative toxin binding region from TmCad1 bound specifically to Cry3Aa and promoted the formation of Cry3Aa toxin oligomers, proposed to be mediators of toxicity in lepidopterans. Injection of TmCad1-specific double-stranded RNA into T. molitor larvae resulted in knockdown of the TmCad1 transcript and conferred resistance to Cry3Aa toxicity. These data demonstrate the functional role of TmCad1 as a Cry3Aa receptor in T. molitor and reveal similarities between the mode of action of Cry toxins in Lepidoptera and Coleoptera. PMID:19416969

The Arabidopsis SOS5 Locus Encodes a Putative Cell Surface Adhesion Protein and Is Required for Normal Cell Expansion

PubMed Central

Shi, Huazhong; Kim, YongSig; Guo, Yan; Stevenson, Becky; Zhu, Jian-Kang

2003-01-01

Cell surface proteoglycans have been implicated in many aspects of plant growth and development, but genetic evidence supporting their function has been lacking. Here, we report that the Salt Overly Sensitive5 (SOS5) gene encodes a putative cell surface adhesion protein and is required for normal cell expansion. The sos5 mutant was isolated in a screen for Arabidopsis salt-hypersensitive mutants. Under salt stress, the root tips of sos5 mutant plants swell and root growth is arrested. The root-swelling phenotype is caused by abnormal expansion of epidermal, cortical, and endodermal cells. The SOS5 gene was isolated through map-based cloning. The predicted SOS5 protein contains an N-terminal signal sequence for plasma membrane localization, two arabinogalactan protein–like domains, two fasciclin-like domains, and a C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. The presence of fasciclin-like domains, which typically are found in animal cell adhesion proteins, suggests a role for SOS5 in cell-to-cell adhesion in plants. The SOS5 protein was present at the outer surface of the plasma membrane. The cell walls are thinner in the sos5 mutant, and those between neighboring epidermal and cortical cells in sos5 roots appear less organized. SOS5 is expressed ubiquitously in all plant organs and tissues, including guard cells in the leaf. PMID:12509519

Plasmodium berghei MAPK1 Displays Differential and Dynamic Subcellular Localizations during Liver Stage Development

PubMed Central

Wierk, Jannika Katharina; Langbehn, Annette; Kamper, Maria; Richter, Stefanie; Burda, Paul-Christian; Heussler, Volker Theo; Deschermeier, Christina

2013-01-01

Mitogen-activated protein kinases (MAPKs) regulate key signaling events in eukaryotic cells. In the genomes of protozoan Plasmodium parasites, the causative agents of malaria, two genes encoding kinases with significant homology to other eukaryotic MAPKs have been identified (mapk1, mapk2). In this work, we show that both genes are transcribed during Plasmodium berghei liver stage development, and analyze expression and subcellular localization of the PbMAPK1 protein in liver stage parasites. Live cell imaging of transgenic parasites expressing GFP-tagged PbMAPK1 revealed a nuclear localization of PbMAPK1 in the early schizont stage mediated by nuclear localization signals in the C-terminal domain. In contrast, a distinct localization of PbMAPK1 in comma/ring-shaped structures in proximity to the parasite’s nuclei and the invaginating parasite membrane was observed during the cytomere stage of parasite development as well as in immature blood stage schizonts. The PbMAPK1 localization was found to be independent of integrity of a motif putatively involved in ATP binding, integrity of the putative activation motif and the presence of a predicted coiled-coil domain in the C-terminal domain. Although PbMAPK1 knock out parasites showed normal liver stage development, the kinase may still fulfill a dual function in both schizogony and merogony of liver stage parasites regulated by its dynamic and stage-dependent subcellular localization. PMID:23544094

The distal short consensus repeats 1 and 2 of the membrane cofactor protein CD46 and their distance from the cell membrane determine productive entry of species B adenovirus serotype 35.

PubMed

Fleischli, Christoph; Verhaagh, Sandra; Havenga, Menzo; Sirena, Dominique; Schaffner, Walter; Cattaneo, Roberto; Greber, Urs F; Hemmi, Silvio

2005-08-01

The human regulator of complement activation membrane cofactor protein (CD46) has recently been identified as an attachment receptor for most species B adenoviruses (Ads), including Ad type 3 (Ad3), Ad11, and Ad35, as well as species D Ad37. To characterize the interaction between Ad35 and CD46, hybrid receptors composed of different CD46 short consensus repeat (SCR) domains fused to immunoglobulin-like domains of CD4 and a set of 36 CD46 mutants containing semiconservative changes of single amino acids within SCR domains I and II were tested in binding and in Ad35-mediated luciferase transduction assays. In addition, anti-CD46 antibodies and soluble polypeptides constituting various CD46 domains were used in binding inhibition studies. Our data indicate that (i) CD46 SCR I or SCR II alone confers low but significant Ad35 binding; (ii) the presence of SCR I and II is required for optimal binding and transgene expression; (iii) transduction efficiencies equivalent to that of full-length CD46 are obtained if SCR I and II are at an appropriate distance from the cell membrane; (iv) ablation of the N-glycan attached to SCR I has no influence on receptor function, whereas ablation of the SCR II N-glycan results in about a two- to threefold reduction of binding and transgene expression; (v) most putative Ad35 binding residues are located on the same solvent-exposed face of the SCR I or SCR II domain, which are twisted by about 90 degrees ; and (vi) the putative Ad35 binding sites partly overlap with the measles virus binding surface.

The Distal Short Consensus Repeats 1 and 2 of the Membrane Cofactor Protein CD46 and Their Distance from the Cell Membrane Determine Productive Entry of Species B Adenovirus Serotype 35

PubMed Central

Fleischli, Christoph; Verhaagh, Sandra; Havenga, Menzo; Sirena, Dominique; Schaffner, Walter; Cattaneo, Roberto; Greber, Urs F.; Hemmi, Silvio

2005-01-01

The human regulator of complement activation membrane cofactor protein (CD46) has recently been identified as an attachment receptor for most species B adenoviruses (Ads), including Ad type 3 (Ad3), Ad11, and Ad35, as well as species D Ad37. To characterize the interaction between Ad35 and CD46, hybrid receptors composed of different CD46 short consensus repeat (SCR) domains fused to immunoglobulin-like domains of CD4 and a set of 36 CD46 mutants containing semiconservative changes of single amino acids within SCR domains I and II were tested in binding and in Ad35-mediated luciferase transduction assays. In addition, anti-CD46 antibodies and soluble polypeptides constituting various CD46 domains were used in binding inhibition studies. Our data indicate that (i) CD46 SCR I or SCR II alone confers low but significant Ad35 binding; (ii) the presence of SCR I and II is required for optimal binding and transgene expression; (iii) transduction efficiencies equivalent to that of full-length CD46 are obtained if SCR I and II are at an appropriate distance from the cell membrane; (iv) ablation of the N-glycan attached to SCR I has no influence on receptor function, whereas ablation of the SCR II N-glycan results in about a two- to threefold reduction of binding and transgene expression; (v) most putative Ad35 binding residues are located on the same solvent-exposed face of the SCR I or SCR II domain, which are twisted by about 90°; and (vi) the putative Ad35 binding sites partly overlap with the measles virus binding surface. PMID:16014961

Blunt Snout Bream (Megalobrama amblycephala) MyD88 and TRAF6: Characterisation, Comparative Homology Modelling and Expression

PubMed Central

Tran, Ngoc Tuan; Liu, Han; Jakovlić, Ivan; Wang, Wei-Min

2015-01-01

MyD88 and TRAF6 play an essential role in the innate immune response in most animals. This study reports the full-length MaMyD88 and MaTRAF6 genes identified from the blunt snout bream (Megalobrama amblycephala) transcriptome profile. MaMyD88 is 2501 base pairs (bp) long, encoding a putative protein of 284 amino acids (aa), including the N-terminal DEATH domain of 78 aa and the C-terminal TIR domain of 138 aa. MaTRAF6 is 2252 bp long, encoding a putative protein of 542 aa, including the N-terminal low-complexity region, RING domain (40 aa), a coiled-coil region (64 aa) and C-terminal MATH domain (147 aa). Coding regions of MaMyD88 and MaTRAF6 genomic sequences consisted of five and six exons, respectively. Physicochemical and functional characteristics of the proteins were analysed. Alpha helices were dominant in the secondary structure of the proteins. Homology models of the MaMyD88 and MaTRAF6 domains were constructed applying the comparative modelling method. RT-qPCR was used to analyse the expression of MaMyD88 and MaTRAF6 mRNA transcripts in response to Aeromonas hydrophila challenge. Both genes were highly upregulated in the liver, spleen and kidney during the first 24 h after the challenge. While MyD88 and TRAF6 have been reported in various aquatic species, this is the first report and characterisation of these genes in blunt snout bream. This research also provides evidence of the important roles of these two genes in the blunt snout bream innate immune system. PMID:25830478

Identification of YTH Domain-Containing Proteins as the Readers for N1-Methyladenosine in RNA.

PubMed

Dai, Xiaoxia; Wang, Tianlu; Gonzalez, Gwendolyn; Wang, Yinsheng

2018-06-05

N1-methyladenosine (m 1 A) is an important post-transcriptional modification in RNA; however, the exact biological role of m 1 A remains to be determined. By employing a quantitative proteomics method, we identified multiple putative protein readers of m 1 A in RNA, including several YTH domain family proteins. We showed that YTHDF1-3 and YTHDC1, but not YTHDC2, could bind directly to m 1 A in RNA. We also found that Trp 432 in YTHDF2, a conserved residue in the hydrophobic pocket of the YTH domain that is necessary for its binding to N 6 -methyladenosine (m 6 A), is required for its recognition of m 1 A. An analysis of previously published data revealed transcriptome-wide colocalization of YTH domain-containing proteins and m 1 A sites in HeLa cells, suggesting that YTH domain-containing proteins can bind to m 1 A in cells. Together, our results uncovered YTH domain-containing proteins as readers for m 1 A in RNA and provided new insight into the functions of m 1 A in RNA biology.

Mutational analysis of TRAF6 reveals a conserved functional role of the RING dimerization interface and a potentially necessary but insufficient role of RING-dependent TRAF6 polyubiquitination towards NF-κB activation

PubMed Central

Megas, Charilaos; Hatzivassiliou, Eudoxia G.; Yin, Qian; Vignali, Dario A.A.; Mosialos, George

2011-01-01

TRAF6 is an E3 ubiquitin ligase that plays a pivotal role in the activation of NF-κB by innate and adaptive immunity stimuli. TRAF6 consists of a highly conserved carboxyl terminal TRAF-C domain which is preceded by a coiled coil domain and an amino terminal region that contains a RING domain and a series of putative zinc-finger motifs. The TRAF-C domain contributes to TRAF6 oligomerization and mediates the interaction of TRAF6 with upstream signaling molecules whereas the RING domain comprises the core of the ubiquitin ligase catalytic domain. In order to identify structural elements that are important for TRAF6-induced NF-κB activation, mutational analysis of the TRAF-C and RING domains was performed. Alterations of highly conserved residues of the TRAF-C domain of TRAF6 did not affect significantly the ability of the protein to activate NF-κB. On the other hand a number of functionally important residues (L77, Q82, R88, F118, N121 and E126) for the activation of NF-κB were identified within the RING domain of TRAF6. Interestingly, several homologues of these residues in TRAF2 were shown to have a conserved functional role in TRAF2-induced NF-κB activation and lie at the dimerization interface of the RING domain. Finally, whereas alteration of Q82, R88 and F118 compromised both the K63-linked polyubiquitination of TRAF6 and its ability to activate NF-κB, alteration of L77, N121 and E126 diminished the NF-κB activating function of TRAF6 without affecting TRAF6 K63-linked polyubiquitination. Our results support a conserved functional role of the TRAF RING domain dimerization interface and a potentially necessary but insufficient role for RING-dependent TRAF6 K63-linked polyubiquitination towards NF-κB activation in cells. PMID:21185369

Mutational analysis of TRAF6 reveals a conserved functional role of the RING dimerization interface and a potentially necessary but insufficient role of RING-dependent TRAF6 polyubiquitination towards NF-κB activation.

PubMed

Megas, Charilaos; Hatzivassiliou, Eudoxia G; Yin, Qian; Marinopoulou, Elli; Hadweh, Paul; Vignali, Dario A A; Mosialos, George

2011-05-01

TRAF6 is an E3 ubiquitin ligase that plays a pivotal role in the activation of NF-κB by innate and adaptive immunity stimuli. TRAF6 consists of a highly conserved carboxyl terminal TRAF-C domain which is preceded by a coiled coil domain and an amino terminal region that contains a RING domain and a series of putative zinc-finger motifs. The TRAF-C domain contributes to TRAF6 oligomerization and mediates the interaction of TRAF6 with upstream signaling molecules whereas the RING domain comprises the core of the ubiquitin ligase catalytic domain. In order to identify structural elements that are important for TRAF6-induced NF-κB activation, mutational analysis of the TRAF-C and RING domains was performed. Alterations of highly conserved residues of the TRAF-C domain of TRAF6 did not affect significantly the ability of the protein to activate NF-κB. On the other hand a number of functionally important residues (L77, Q82, R88, F118, N121 and E126) for the activation of NF-κB were identified within the RING domain of TRAF6. Interestingly, several homologues of these residues in TRAF2 were shown to have a conserved functional role in TRAF2-induced NF-κB activation and lie at the dimerization interface of the RING domain. Finally, whereas alteration of Q82, R88 and F118 compromised both the K63-linked polyubiquitination of TRAF6 and its ability to activate NF-κB, alteration of L77, N121 and E126 diminished the NF-κB activating function of TRAF6 without affecting TRAF6 K63-linked polyubiquitination. Our results support a conserved functional role of the TRAF RING domain dimerization interface and a potentially necessary but insufficient role for RING-dependent TRAF6 K63-linked polyubiquitination towards NF-κB activation in cells. Copyright © 2010 Elsevier Inc. All rights reserved.

Mapping Interactions between Myosin Relay and Converter Domains That Power Muscle Function*

PubMed Central

Kronert, William A.; Melkani, Girish C.; Melkani, Anju; Bernstein, Sanford I.

2014-01-01

Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile508, Asn509, and Asp511) in communicating with converter domain residue Arg759. We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle. PMID:24627474

A novel class of dual-family immunophilins.

PubMed

Adams, Brian; Musiyenko, Alla; Kumar, Rajinder; Barik, Sailen

2005-07-01

Immunophilins are protein chaperones with peptidylprolyl isomerase activity that belong to one of two large families, the cyclosporin-binding cyclophilins (CyPs) and the FK506-binding proteins (FKBPs). Each family displays characteristic and conserved sequence features that differ between the two families. We report a novel group of dual-family immunophilins that contain both CyP and FKBP domains for which we propose the name FCBP (FK506- and cyclosporin-binding protein). The FCBP of Toxoplasma gondii, a protozoan parasite, contained N-terminal FKBP and C-terminal CyP domains joined by tetratricopeptide repeats. Structure-function analysis revealed that both domains were functional and exhibited family-specific drug sensitivity. The individual domains of FCBP inhibited calcineurin (protein phosphatase 2B) in the presence of the appropriate drugs. In binding studies, FCBP recruited calcineurin in the presence of FK506 and a putative target of rapamycin homolog in the presence of rapamycin. Two additional FCBP sequences in Flavobacterium and one in Treponema (spirochete) were also identified in which the CyP and FKBP domains were in the reverse order. T. gondii growth was inhibited by cyclosporin and FK506 in a moderately synergistic manner. The knockdown of FCBP by RNA interference revealed its essentiality for T. gondii growth. Clearly, the FCBPs are novel chaperones and potential targets of multiple immunosuppressant drugs.

The General Definition of the p97/Valosin-containing Protein (VCP)-interacting Motif (VIM) Delineates a New Family of p97 Cofactors*

PubMed Central

Stapf, Christopher; Cartwright, Edward; Bycroft, Mark; Hofmann, Kay; Buchberger, Alexander

2011-01-01

Cellular functions of the essential, ubiquitin-selective AAA ATPase p97/valosin-containing protein (VCP) are controlled by regulatory cofactors determining substrate specificity and fate. Most cofactors bind p97 through a ubiquitin regulatory X (UBX) or UBX-like domain or linear sequence motifs, including the hitherto ill defined p97/VCP-interacting motif (VIM). Here, we present the new, minimal consensus sequence RX5AAX2R as a general definition of the VIM that unites a novel family of known and putative p97 cofactors, among them UBXD1 and ZNF744/ANKZF1. We demonstrate that this minimal VIM consensus sequence is necessary and sufficient for p97 binding. Using NMR chemical shift mapping, we identified several residues of the p97 N-terminal domain (N domain) that are critical for VIM binding. Importantly, we show that cellular stress resistance conferred by the yeast VIM-containing cofactor Vms1 depends on the physical interaction between its VIM and the critical N domain residues of the yeast p97 homolog, Cdc48. Thus, the VIM-N domain interaction characterized in this study is required for the physiological function of Vms1 and most likely other members of the newly defined VIM family of cofactors. PMID:21896481

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae.

PubMed

Albornos, Lucía; Martín, Ignacio; Iglesias, Rebeca; Jiménez, Teresa; Labrador, Emilia; Dopico, Berta

2012-11-07

Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found.

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

PubMed Central

2012-01-01

Background Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. Results ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. Conclusions We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found. PMID:23134664

Identification and Characterization of the Pyridomycin Biosynthetic Gene Cluster of Streptomyces pyridomyceticus NRRL B-2517*

PubMed Central

Huang, Tingting; Wang, Yemin; Yin, Jun; Du, Yanhua; Tao, Meifeng; Xu, Jing; Chen, Wenqing; Lin, Shuangjun; Deng, Zixin

2011-01-01

Pyridomycin is a structurally unique antimycobacterial cyclodepsipeptide containing rare 3-(3-pyridyl)-l-alanine and 2-hydroxy-3-methylpent-2-enoic acid moieties. The biosynthetic gene cluster for pyridomycin has been cloned and identified from Streptomyces pyridomyceticus NRRL B-2517. Sequence analysis of a 42.5-kb DNA region revealed 26 putative open reading frames, including two nonribosomal peptide synthetase (NRPS) genes and a polyketide synthase gene. A special feature is the presence of a polyketide synthase-type ketoreductase domain embedded in an NRPS. Furthermore, we showed that PyrA functioned as an NRPS adenylation domain that activates 3-hydroxypicolinic acid and transfers it to a discrete peptidyl carrier protein, PyrU, which functions as a loading module that initiates pyridomycin biosynthesis in vivo and in vitro. PyrA could also activate other aromatic acids, generating three pyridomycin analogues in vivo. PMID:21454714

White collar 2, a partner in blue-light signal transduction, controlling expression of light-regulated genes in Neurospora crassa.

PubMed Central

Linden, H; Macino, G

1997-01-01

A saturating genetic dissection of 'blind' mutants in Neurospora crassa has identified a total of two non-redundant loci (wc-1 and wc-2) each of which is required for blue-light perception/signal transduction. Previously, we demonstrated that WC1 is a putative zinc finger transcription factor able to bind specifically to a light-regulated promoter. Here, we present the cloning and characterization of the wc-2 gene. We demonstrate using mutation analysis and in vitro DNA-binding assays that WC2, the second partner of this light signal transduction system, encodes a functional zinc finger DNA-binding protein with putative PAS dimerization and transcription activation domains. This molecular genetic dissection of the second of two components of this light signal transduction system has enabled us to devise a model whereby WC1 and WC2 are proposed to interact via homologous PAS domains, bind to promoters of light-regulated genes and activate transcription. As such, this study provides the first insight into two co-operating partners in blue-light signal transduction in any organism and describes the molecular tools with which to dissect this enigmatic process. PMID:9009271

Analysis and Functional Annotation of an Expressed Sequence Tag Collection for Tropical Crop Sugarcane

PubMed Central

Vettore, André L.; da Silva, Felipe R.; Kemper, Edson L.; Souza, Glaucia M.; da Silva, Aline M.; Ferro, Maria Inês T.; Henrique-Silva, Flavio; Giglioti, Éder A.; Lemos, Manoel V.F.; Coutinho, Luiz L.; Nobrega, Marina P.; Carrer, Helaine; França, Suzelei C.; Bacci, Maurício; Goldman, Maria Helena S.; Gomes, Suely L.; Nunes, Luiz R.; Camargo, Luis E.A.; Siqueira, Walter J.; Van Sluys, Marie-Anne; Thiemann, Otavio H.; Kuramae, Eiko E.; Santelli, Roberto V.; Marino, Celso L.; Targon, Maria L.P.N.; Ferro, Jesus A.; Silveira, Henrique C.S.; Marini, Danyelle C.; Lemos, Eliana G.M.; Monteiro-Vitorello, Claudia B.; Tambor, José H.M.; Carraro, Dirce M.; Roberto, Patrícia G.; Martins, Vanderlei G.; Goldman, Gustavo H.; de Oliveira, Regina C.; Truffi, Daniela; Colombo, Carlos A.; Rossi, Magdalena; de Araujo, Paula G.; Sculaccio, Susana A.; Angella, Aline; Lima, Marleide M.A.; de Rosa, Vicente E.; Siviero, Fábio; Coscrato, Virginia E.; Machado, Marcos A.; Grivet, Laurent; Di Mauro, Sonia M.Z.; Nobrega, Francisco G.; Menck, Carlos F.M.; Braga, Marilia D.V.; Telles, Guilherme P.; Cara, Frank A.A.; Pedrosa, Guilherme; Meidanis, João; Arruda, Paulo

2003-01-01

To contribute to our understanding of the genome complexity of sugarcane, we undertook a large-scale expressed sequence tag (EST) program. More than 260,000 cDNA clones were partially sequenced from 26 standard cDNA libraries generated from different sugarcane tissues. After the processing of the sequences, 237,954 high-quality ESTs were identified. These ESTs were assembled into 43,141 putative transcripts. Of the assembled sequences, 35.6% presented no matches with existing sequences in public databases. A global analysis of the whole SUCEST data set indicated that 14,409 assembled sequences (33% of the total) contained at least one cDNA clone with a full-length insert. Annotation of the 43,141 assembled sequences associated almost 50% of the putative identified sugarcane genes with protein metabolism, cellular communication/signal transduction, bioenergetics, and stress responses. Inspection of the translated assembled sequences for conserved protein domains revealed 40,821 amino acid sequences with 1415 Pfam domains. Reassembling the consensus sequences of the 43,141 transcripts revealed a 22% redundancy in the first assembling. This indicated that possibly 33,620 unique genes had been identified and indicated that >90% of the sugarcane expressed genes were tagged. PMID:14613979

Conserved Domains in the Transformer Protein Act Complementary to Regulate Sex-Specific Splicing of Its Own Pre-mRNA.

PubMed

Tanaka, Arisa; Aoki, Fugaku; Suzuki, Masataka G

2018-05-26

The transformer (tra) gene, which is a female-determining master gene in the housefly Musca domestica, acts as a memory device for sex determination via its auto-regulatory function, i.e., through the contribution of the TRA protein to female-specific splicing of its own pre-mRNA. The TRA protein contains 4 small domains that are specifically conserved among TRA proteins (domains 1-4). Domain 2, also named TRA-CAM domain, is the most conserved, but its function remains unknown. To examine whether these domains are involved in the auto-regulatory function, we performed in vitro splicing assays using a tra minigene containing a partial genomic sequence of the M. domestica tra (Mdtra) gene. Co-transfection of the Mdtra minigene and an MdTRA protein expression vector into cultured insect cells strongly induced female-specific splicing of the minigene. A series of deletion mutation analyses demonstrated that these domains act complementarily to induce female-specific splicing. Domain 1 and the TRA-CAM domain were necessary for the female-specific splicing when the MdTRA protein lacked both domains 3 and 4. In this situation, mutation of the well-conserved 3 amino acids (GEG) in the TRA-CAM domain significantly reduced the female-specific splicing activity of MdTRA. GST-pull down analyses demonstrated that the MdTRA protein specifically enriched on the male-specific exonic region (exon 2b), which contains the putative TRA/TRA-2 binding sites, and that the GEG mutation disrupts this enrichment. Since the MdTRA protein interacts with its own pre-mRNA through TRA-2, our findings suggest that the conserved amino acid residues in the TRA-CAM domain may be crucial for the interaction between MdTRA and TRA-2, enhancing MdTRA recruitment on its pre-mRNA to induce female-specific splicing of tra in the housefly. © 2018 S. Karger AG, Basel.

BioGraph: unsupervised biomedical knowledge discovery via automated hypothesis generation

PubMed Central

2011-01-01

We present BioGraph, a data integration and data mining platform for the exploration and discovery of biomedical information. The platform offers prioritizations of putative disease genes, supported by functional hypotheses. We show that BioGraph can retrospectively confirm recently discovered disease genes and identify potential susceptibility genes, outperforming existing technologies, without requiring prior domain knowledge. Additionally, BioGraph allows for generic biomedical applications beyond gene discovery. BioGraph is accessible at http://www.biograph.be. PMID:21696594

Serine protease-related proteins in the malaria mosquito, Anopheles gambiae.

PubMed

Cao, Xiaolong; Gulati, Mansi; Jiang, Haobo

2017-09-01

Insect serine proteases (SPs) and serine protease homologs (SPHs) participate in digestion, defense, development, and other physiological processes. In mosquitoes, some clip-domain SPs and SPHs (i.e. CLIPs) have been investigated for possible roles in antiparasitic responses. In a recent test aimed at improving quality of gene models in the Anopheles gambiae genome using RNA-seq data, we observed various discrepancies between gene models in AgamP4.5 and corresponding sequences selected from those modeled by Cufflinks, Trinity and Bridger. Here we report a comparative analysis of the 337 SP-related proteins in A. gambiae by examining their domain structures, sequence diversity, chromosomal locations, and expression patterns. One hundred and ten CLIPs contain 1 to 5 clip domains in addition to their protease domains (PDs) or non-catalytic, protease-like domains (PLDs). They are divided into five subgroups: CLIPAs (22) are clip 1-5 -PLD; CLIPBs (29), CLIPCs (12) and CLIPDs (14) are mainly clip-PD; most CLIPEs (33) have a domain structure of PD/PLD-PLD-clip-PLD 0-1 . While expression of the CLIP genes in group-1 is generally low and detected in various tissue- and stage-specific RNA-seq libraries, some putative GPs/GPHs (i.e. single domain gut SPs/SPHs) in group-2 are highly expressed in midgut, whole larva or whole adult libraries. In comparison, 46 SPs, 26 SPHs, and 37 multi-domain SPs/SPHs (i.e. PD/PLD-PLD ≥1 ) in group-3 do not seem to be specifically expressed in digestive tract. There are 16 SPs and 2 SPH containing other types of putative regulatory domains (e.g. LDLa, CUB, Gd). Of the 337 SP and SPH genes, 159 were sorted into 46 groups (2-8 members/group) based on similar phylogenetic tree position, chromosomal location, and expression profile. This information and analysis, including improved gene models and protein sequences, constitute a solid foundation for functional analysis of the SP-related proteins in A. gambiae. Copyright © 2017 Elsevier Ltd. All rights reserved.

Two aspartate residues at the putative p10 subunit of a type II metacaspase from Nicotiana tabacum L. may contribute to the substrate-binding pocket.

PubMed

Acosta-Maspons, Alexis; Sepúlveda-García, Edgar; Sánchez-Baldoquín, Laura; Marrero-Gutiérrez, Junier; Pons, Tirso; Rocha-Sosa, Mario; González, Lien

2014-01-01

Metacaspases are cysteine proteases present in plants, fungi, prokaryotes, and early branching eukaryotes, although a detailed description of their cellular function remains unclear. Currently, three-dimensional (3D) structures are only available for two metacaspases: Trypanosoma brucei (MCA2) and Saccharomyces cerevisiae (Yca1). Furthermore, metacaspases diverged from animal caspases of known structure, which limits straightforward homology-based interpretation of functional data. We report for the first time the identification and initial characterization of a metacaspase of Nicotiana tabacum L., NtMC1. By combining domain search, multiple sequence alignment (MSA), and protein fold-recognition studies, we provide compelling evidences that NtMC1 is a plant metacaspase type II, and predict its 3D structure using the crystal structure of two type I metacaspases (MCA2 and Yca1) and Gsu0716 protein from Geobacter sulfurreducens as template. Analysis of the predicted 3D structure allows us to propose Asp353, at the putative p10 subunit, as a new member of the aspartic acid triad that coordinates the P1 arginine/lysine residue of the substrate. Nevertheless, site-directed mutagenesis and expression analysis in bacteria and Nicotiana benthamiana indicate the functionality of both Asp348 and Asp353. Through the co-expression of mutant and wild-type proteins by transient expression in N. benthamiana leaves we found that polypeptide processing seems to be intramolecular. Our results provide the first evidence in plant metacaspases concerning the functionality of the putative p10 subunit.

Target of Rapamycin Regulates Development and Ribosomal RNA Expression through Kinase Domain in Arabidopsis1[W][OA

PubMed Central

Ren, Maozhi; Qiu, Shuqing; Venglat, Prakash; Xiang, Daoquan; Feng, Li; Selvaraj, Gopalan; Datla, Raju

2011-01-01

Target of rapamycin (TOR) is a central regulator of cell growth, cell death, nutrition, starvation, hormone, and stress responses in diverse eukaryotes. However, very little is known about TOR signaling and the associated functional domains in plants. We have taken a genetic approach to dissect TOR functions in Arabidopsis (Arabidopsis thaliana) and report here that the kinase domain is essential for the role of TOR in embryogenesis and 45S rRNA expression. Twelve new T-DNA insertion mutants, spanning 14.2 kb of TOR-encoding genomic region, have been characterized. Nine of these share expression of defective kinase domain and embryo arrest at 16 to 32 cell stage. However, three T-DNA insertion lines affecting FATC domain displayed normal embryo development, indicating that FATC domain was dispensable in Arabidopsis. Genetic complementation showed that the TOR kinase domain alone in tor-10/tor-10 mutant background can rescue early embryo lethality and restore normal development. Overexpression of full-length TOR or kinase domain in Arabidopsis displayed developmental abnormalities in meristem, leaf, root, stem, flowering time, and senescence. We further show that TOR, especially the kinase domain, plays a role in ribosome biogenesis by activating 45S rRNA production. Of the six putative nuclear localization sequences in the kinase domain, nuclear localization sequence 6 was identified to confer TOR nuclear targeting in transient expression assays. Chromatin immunoprecipitation studies revealed that the HEAT repeat domain binds to 45S rRNA promoter and the 5′ external transcribed spacer elements motif. Together, these results show that TOR controls the embryogenesis, postembryonic development, and 45S rRNA production through its kinase domain in Arabidopsis. PMID:21266656

An ankyrin-like protein with transmembrane domains is specifically lost after oncogenic transformation of human fibroblasts.

PubMed

Jaquemar, D; Schenker, T; Trueb, B

1999-03-12

We have identified a novel transformation-sensitive mRNA, which is present in cultured fibroblasts but is lacking in SV40 transformed cells as well as in many mesenchymal tumor cell lines. The corresponding gene is located on human chromosome 8 in band 8q13. The open reading frame of the mRNA encodes a protein of 1119 amino acids forming two distinct domains. The N-terminal domain consists of 18 repeats that are related to the cytoskeletal protein ankyrin. The C-terminal domain contains six putative transmembrane segments that resemble many ion channels. This overall structure is reminiscent of TRP-like proteins that function as store-operated calcium channels. The novel protein with an Mr of 130 kDa is expressed at a very low level in human fibroblasts and at a moderate level in liposarcoma cells. Overexpression in eukaryotic cells appears to interfere with normal growth, suggesting that it might play a direct or indirect role in signal transduction and growth control.

Quaternary structure of human, Drosophila melanogaster and Caenorhabditis elegans MFE-2 in solution from synchrotron small-angle X-ray scattering.

PubMed

Mehtälä, Maija L; Haataja, Tatu J K; Blanchet, Clément E; Hiltunen, J Kalervo; Svergun, Dmitri I; Glumoff, Tuomo

2013-02-14

Multifunctional enzyme type 2 (MFE-2) forms part of the fatty acid β-oxidation pathway in peroxisomes. MFE-2s from various species reveal proteins with structurally homologous functional domains assembled in different compilations. Crystal structures of all domain types are known. SAXS data from human, fruit fly and Caenorhabditiselegans MFE-2s and their constituent domains were collected, and both ab initio and rigid body models constructed. Location of the putative substrate binding helper domain SCP-2L (sterol carrier protein 2-like), which is not part of MFE-2 protein in every species and not seen as part of any previous MFE-2 structures, was determined. The obtained models of human and C. elegans MFE-2 lend a direct structural support to the idea of the biological role of SCP-2L. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The Clock gene clone and its circadian rhythms in Pelteobagrus vachelli

NASA Astrophysics Data System (ADS)

Qin, Chuanjie; Shao, Ting

2015-05-01

The Clock gene, a key molecule in circadian systems, is widely distributed in the animal kingdom. We isolated a 936-bp partial cDNA sequence of the Clock gene ( Pva-clock) from the darkbarbel catfish Pelteobagrus vachelli that exhibited high identity with Clock genes of other species of fish and animals (65%-88%). The putative domains included a basic helix-loop-helix (bHLH) domain and two period-ARNT-single-minded (PAS) domains, which were also similar to those in other species of fish and animals. Pva-Clock was primarily expressed in the brain, and was detected in all of the peripheral tissues sampled. Additionally, the pattern of Pva-Clock expression over a 24-h period exhibited a circadian rhythm in the brain, liver and intestine, with the acrophase at zeitgeber time 21:35, 23:00, and 23:23, respectively. Our results provide insight into the function of the molecular Clock of P. vachelli.

Structure of a eukaryotic voltage-gated sodium channel at near-atomic resolution.

PubMed

Shen, Huaizong; Zhou, Qiang; Pan, Xiaojing; Li, Zhangqiang; Wu, Jianping; Yan, Nieng

2017-03-03

Voltage-gated sodium (Na v ) channels are responsible for the initiation and propagation of action potentials. They are associated with a variety of channelopathies and are targeted by multiple pharmaceutical drugs and natural toxins. Here, we report the cryogenic electron microscopy structure of a putative Na v channel from American cockroach (designated Na v PaS) at 3.8 angstrom resolution. The voltage-sensing domains (VSDs) of the four repeats exhibit distinct conformations. The entrance to the asymmetric selectivity filter vestibule is guarded by heavily glycosylated and disulfide bond-stabilized extracellular loops. On the cytoplasmic side, a conserved amino-terminal domain is placed below VSD I , and a carboxy-terminal domain binds to the III-IV linker. The structure of Na v PaS establishes an important foundation for understanding function and disease mechanism of Na v and related voltage-gated calcium channels. Copyright © 2017, American Association for the Advancement of Science.

Identification of critical functional residues of receptor-like kinase ERECTA.

PubMed

Kosentka, Pawel Z; Zhang, Liang; Simon, Yonas A; Satpathy, Binita; Maradiaga, Richard; Mitoubsi, Omar; Shpak, Elena D

2017-03-01

In plants, extracellular signals are primarily sensed by plasma membrane-localized receptor-like kinases (RLKs). ERECTA is a leucine-rich repeat RLK that together with its paralogs ERECTA-like 1 (ERL1) and ERL2 regulates multiple aspects of plant development. ERECTA forms complexes with a range of co-receptors and senses secreted cysteine-rich small proteins from the EPF/EPFL family. Currently the mechanism of the cytoplasmic domain activation and transmission of the signal by ERECTA is unclear. To gain a better understanding we performed a structure-function analysis by introducing altered ERECTA genes into erecta and erecta erl1 erl2 mutants. These experiments indicated that ERECTA's ability to phosphorylate is functionally significant, and that while the cytoplasmic juxtamembrane domain is important for ERECTA function, the C-terminal tail is not. An analysis of multiple putative phosphorylation sites identified four amino acids in the activation segment of the kinase domain as functionally important. Homology of those residues to functionally significant amino acids in multiple other plant RLKs emphasizes similarities in RLK function. Specifically, our data predicts Thr812 as a primary site of phosphor-activation and potential inhibitory phosphorylation of Tyr815 and Tyr820. In addition, our experiments suggest that there are differences in the molecular mechanism of ERECTA function during regulation of stomata development and in elongation of above-ground organs. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

BASH, a novel signaling molecule preferentially expressed in B cells of the bursa of Fabricius.

PubMed

Goitsuka, R; Fujimura, Y; Mamada, H; Umeda, A; Morimura, T; Uetsuka, K; Doi, K; Tsuji, S; Kitamura, D

1998-12-01

The bursa of Fabricius is a gut-associated lymphoid organ that is essential for the generation of a diversified B cell repertoire in the chicken. We describe here a novel gene preferentially expressed in bursal B cells. The gene encodes an 85-kDa protein, designated BASH (B cell adaptor containing SH2 domain), that contains N-terminal acidic domains with SH2 domain-binding phosphotyrosine-based motifs, a proline-rich domain, and a C-terminal SH2 domain. BASH shows a substantial sequence similarity to SLP-76, an adaptor protein functioning in TCR-signal transduction. BASH becomes tyrosine-phosphorylated with the B cell Ag receptor (BCR) cross-link or by coexpression with Syk and Lyn and associates with signaling molecules including Syk and a putative chicken Shc homologue. Overexpression of BASH results in suppression of the NF-AT activation induced by BCR-cross-linking. These findings suggest that BASH is involved in BCR-mediated signal transduction and could play a critical role in B cell development in the bursa.

Horizontal functional gene transfer from bacteria to fishes.

PubMed

Sun, Bao-Fa; Li, Tong; Xiao, Jin-Hua; Jia, Ling-Yi; Liu, Li; Zhang, Peng; Murphy, Robert W; He, Shun-Min; Huang, Da-Wei

2015-12-22

Invertebrates can acquire functional genes via horizontal gene transfer (HGT) from bacteria but fishes are not known to do so. We provide the first reliable evidence of one HGT event from marine bacteria to fishes. The HGT appears to have occurred after emergence of the teleosts. The transferred gene is expressed and regulated developmentally. Its successful integration and expression may change the genetic and metabolic repertoire of fishes. In addition, this gene contains conserved domains and similar tertiary structures in fishes and their putative donor bacteria. Thus, it may function similarly in both groups. Evolutionary analyses indicate that it evolved under purifying selection, further indicating its conserved function. We document the first likely case of HGT of functional gene from prokaryote to fishes. This discovery certifies that HGT can influence vertebrate evolution.

The initial step in the archaeal aspartate biosynthetic pathway catalyzed by a monofunctional aspartokinase

PubMed Central

Faehnle, Christopher R.; Liu, Xuying; Pavlovsky, Alexander; Viola, Ronald E.

2006-01-01

The activation of the β-carboxyl group of aspartate catalyzed by aspartokinase is the commitment step to amino-acid biosynthesis in the aspartate pathway. The first structure of a microbial aspartokinase, that from Methanococcus jannaschii, has been determined in the presence of the amino-acid substrate l-­aspartic acid and the nucleotide product MgADP. The enzyme assembles into a dimer of dimers, with the interfaces mediated by both the N- and C-terminal domains. The active-site functional groups responsible for substrate binding and specificity have been identified and roles have been proposed for putative catalytic functional groups. PMID:17012784

d-Omix: a mixer of generic protein domain analysis tools.

PubMed

Wichadakul, Duangdao; Numnark, Somrak; Ingsriswang, Supawadee

2009-07-01

Domain combination provides important clues to the roles of protein domains in protein function, interaction and evolution. We have developed a web server d-Omix (a Mixer of Protein Domain Analysis Tools) aiming as a unified platform to analyze, compare and visualize protein data sets in various aspects of protein domain combinations. With InterProScan files for protein sets of interest provided by users, the server incorporates four services for domain analyses. First, it constructs protein phylogenetic tree based on a distance matrix calculated from protein domain architectures (DAs), allowing the comparison with a sequence-based tree. Second, it calculates and visualizes the versatility, abundance and co-presence of protein domains via a domain graph. Third, it compares the similarity of proteins based on DA alignment. Fourth, it builds a putative protein network derived from domain-domain interactions from DOMINE. Users may select a variety of input data files and flexibly choose domain search tools (e.g. hmmpfam, superfamily) for a specific analysis. Results from the d-Omix could be interactively explored and exported into various formats such as SVG, JPG, BMP and CSV. Users with only protein sequences could prepare an InterProScan file using a service provided by the server as well. The d-Omix web server is freely available at http://www.biotec.or.th/isl/Domix.

Murine c-mpl: a member of the hematopoietic growth factor receptor superfamily that transduces a proliferative signal.

PubMed Central

Skoda, R C; Seldin, D C; Chiang, M K; Peichel, C L; Vogt, T F; Leder, P

1993-01-01

The murine myeloproliferative leukemia virus has previously been shown to contain a fragment of the coding region of the c-mpl gene, a member of the cytokine receptor superfamily. We have isolated cDNA and genomic clones encoding murine c-mpl and localized the c-mpl gene to mouse chromosome 4. Since some members of this superfamily function by transducing a proliferative signal and since the putative ligand of mpl is unknown, we have generated a chimeric receptor to test the functional potential of mpl. The chimera consists of the extracellular domain of the human interleukin-4 receptor and the cytoplasmic domain of mpl. A mouse hematopoietic cell line transfected with this construct proliferates in response to human interleukin-4, thereby demonstrating that the cytoplasmic domain of mpl contains all elements necessary to transmit a growth stimulatory signal. In addition, we show that 25-40% of mpl mRNA found in the spleen corresponds to a novel truncated and potentially soluble isoform of mpl and that both full-length and truncated forms of mpl protein can be immunoprecipitated from lysates of transfected COS cells. Interestingly, however, although the truncated form of the receptor possesses a functional signal sequence and lacks a transmembrane domain, it is not detected in the culture media of transfected cells. Images PMID:8334987

Structural and Functional Analysis of the Pyocyanin Biosynthetic Protein PhzM from Pseudomonas aeruginosa†‡

PubMed Central

Parsons, James F.; Greenhagen, Bryan T.; Shi, Katherine; Calabrese, Kelly; Robinson, Howard; Ladner, Jane E.

2008-01-01

Pyocyanin is a biologically active phenazine produced by the human pathogen Pseudomonas aeruginosa. It is thought to endow P. aeruginosa with a competitive growth advantage in colonized tissue and is also thought to be a virulence factor in diseases such as cystic fibrosis and AIDS where patients are commonly infected by pathogenic Pseudomonads due to their immunocompromised state. Pyocyanin is also a chemically interesting compound due to its unusual oxidation-reduction activity. Phenazine-1-carboxylic acid, the precursor to the bioactive phenazines, is synthesized from chorismic acid by enzymes encoded in a seven-gene cistron in Pseudomonas aeruginosa and in other Pseudomonads. Phenzine-1-carboxylic acid is believed to be converted to pyocyanin by the sequential actions of the putative S-adenosylmethionine dependent N-methyltransferase PhzM and the putative flavin-dependent hydroxylase PhzS. Here we report the 1.8 Å crystal structure of PhzM solved by single anomalous dispersion. Unlike many methyltransferases, PhzM is a dimer in solution. The 36 kDa PhzM polypeptide folds into three domains. The C-terminal domain exhibits the α/β-hydrolase fold typical of small molecule methyltransferases. Two smaller N-terminal domains form much of the dimer interface. Structural alignments with known methyltransferases show that PhzM is most similar to the plant O-methyltransferases that are characterized by an unusual intertwined dimer interface. The structure of PhzM contains no ligands and the active site is open and solvent exposed when compared to structures of similar enzymes. In vitro experiments using purified PhzM alone demonstrate that it has little or no ability to methylate phenzine-1-carboxylic acid. However, when the putative hydroxylase PhzS is included, pyocyanin is readily produced. This observation suggests that a mechanism has evolved in P. aeruginosa that ensures efficient production of pyocyanin by preventing the formation and release of an unstable and potentially deleterious intermediate. PMID:17253782

Double function hydroperoxide lyases/epoxyalcohol synthases (CYP74C) of higher plants: identification and conversion into allene oxide synthases by site-directed mutagenesis.

PubMed

Toporkova, Yana Y; Gorina, Svetlana S; Bessolitsyna, Elena K; Smirnova, Elena O; Fatykhova, Valeria S; Brühlmann, Fredi; Ilyina, Tatiana M; Mukhtarova, Lucia S; Grechkin, Alexander N

2018-04-01

The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT (Medicago truncatula) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9(S)-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13(S)-hydroperoxides of linoleic and α-linolenic acids. To verify whether this dualistic behaviour of CYP74C13_MT is occasional or typical, we also examined five similar putative HPLs (CYP74C). These were CYP74C4_ST (Solanum tuberosum), CYP74C2 (Cucumis melo), CYP74C1_CS and CYP74C31 (both of Cucumis sativus), and CYP74C13_GM (Glycine max). All tested enzymes behaved predominantly as EAS toward 9-hydroperoxide of linoleic acid. Oxiranyl carbinols such as (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acids were the major EAS products. Besides, the CYP74C31 possessed an additional minor 9-AOS activity. The mutant forms of CYP74C13_MT, CYP74C1_CS, and CYP74C31 with substitutions at the catalytically essential domains, namely the "hydroperoxide-binding domain" (I-helix), or the SRS-1 domain near the N-terminus, showed strong AOS activity. These HPLs to AOSs conversions were observed for the first time. Until now a large part of CYP74C enzymes has been considered as 9/13-HPLs. Notwithstanding, these results show that all studied putative CYP74C HPLs are in fact the versatile HPL/EASs that can be effortlessly mutated into specific AOSs. Copyright © 2018 Elsevier B.V. All rights reserved.

Multiple abiotic stress tolerance of the transformants yeast cells and the transgenic Arabidopsis plants expressing a novel durum wheat catalase.

PubMed

Feki, Kaouthar; Kamoun, Yosra; Ben Mahmoud, Rihem; Farhat-Khemakhem, Ameny; Gargouri, Ali; Brini, Faiçal

2015-12-01

Catalases are reactive oxygen species scavenging enzymes involved in response to abiotic and biotic stresses. In this study, we described the isolation and functional characterization of a novel catalase from durum wheat, designed TdCAT1. Molecular Phylogeny analyses showed that wheat TdCAT1 exhibited high amino acids sequence identity to other plant catalases. Sequence homology analysis showed that TdCAT1 protein contained the putative calmodulin binding domain and a putative conserved internal peroxisomal targeting signal PTS1 motif around its C-terminus. Predicted three-dimensional structural model revealed the presence of four putative distinct structural regions which are the N-terminal arm, the β-barrel, the wrapping and the α-helical domains. TdCAT1 protein had the heme pocket that was composed by five essential residues. TdCAT1 gene expression analysis showed that this gene was induced by various abiotic stresses in durum wheat. The expression of TdCAT1 in yeast cells and Arabidopsis plants conferred tolerance to several abiotic stresses. Compared with the non-transformed plants, the transgenic lines maintained their growth and accumulated more proline under stress treatments. Furthermore, the amount of H2O2 was lower in transgenic lines, which was due to the high CAT and POD activities. Taken together, these data provide the evidence for the involvement of durum wheat catalase TdCAT1 in tolerance to multiple abiotic stresses in crop plants. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Molecular cloning of a novel receptor tyrosine kinase, tif, highly expressed in human ovary and testis.

PubMed

Dai, W; Pan, H; Hassanain, H; Gupta, S L; Murphy, M J

1994-03-01

Using a combination of polymerase chain reaction and conventional cDNA library screening approaches, we have cloned and characterized a putative receptor tyrosine kinase termed tif. The extracellular domain of tif has an immunoglobulin-like loop and a fibronectin type III structure. The intracellular domain contains a tyrosine kinase domain. Compared with ryk, a ubiquitously expressed receptor tyrosine kinase, tif expression is tissue-specific with human ovary and testis containing the highest amount of tif mRNA. Many other tested human tissues such as heart, liver, pancreas and thymus do not contain detectable levels of tif mRNA. The molecular cloning and characterization of tif cDNA will facilitate the identification of a potential ligand(s) for the putative receptor and the study of its biological role.

Streptococcus suis serotype 9 strain GZ0565 contains a type VII secretion system putative substrate EsxA that contributes to bacterial virulence and a vanZ-like gene that confers resistance to teicoplanin and dalbavancin in Streptococcus agalactiae.

PubMed

Lai, Liying; Dai, Jiao; Tang, Huanyu; Zhang, Shouming; Wu, Chunyan; Qiu, Wancen; Lu, Chengping; Yao, Huochun; Fan, Hongjie; Wu, Zongfu

2017-06-01

Streptococcus suis (SS), an important pathogen for pigs, is not only considered as a zoonotic agent for humans, but is also recognized as a major reservoir of antimicrobial resistance contributing to the spread of resistance genes to other pathogenic Streptococcus species. In addition to serotype 2 (SS2), serotype 9 (SS9) is another prevalent serotype isolated from diseased pigs. Although many SS strains have been sequenced, the complete genome of a non-SS2 virulent strain has been unavailable to date. Here, we report the complete genome of GZ0565, a virulent strain of SS9, isolated from a pig with meningitis. Comparative genomic analysis revealed five new putative virulence or antimicrobial resistance-associated genes in strain GZ0565 but not in SS2 virulent strains. These five genes encode a putative triacylglycerol lipase, a TipAS antibiotic-recognition domain protein, a putative TetR family transcriptional repressor, a protein containing a LPXTG domain and a G5 domain, and a type VII secretion system (T7SS) putative substrate (EsxA), respectively. Western blot analysis showed that strain GZ0565 can secrete EsxA. We generated an esxA deletion mutant and showed that EsxA contributes to SS virulence in a mouse infection model. Additionally, the antibiotic resistance gene vanZ SS was identified and expression of vanZ SS conferred resistance to teicoplanin and dalbavancin in Streptococcus agalactiae. We believe this is the first experimental demonstration of the existence of the T7SS putative substrate EsxA and its contribution to bacterial virulence in SS. Together, our results contribute to further understanding of the virulence and antimicrobial resistance characteristics of SS. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural Determinants of DNA Binding by a P. falciparum ApiAP2 Transcriptional Regulator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lindner, Scott E.; De Silva, Erandi K.; Keck, James L.

2010-11-05

Putative transcription factors have only recently been identified in the Plasmodium spp., with the major family of regulators comprising the Apicomplexan Apetala2 (AP2) proteins. To better understand the DNA-binding mechanisms of these transcriptional regulators, we characterized the structure and in vitro function of an AP2 DNA-binding domain from a prototypical Apicomplexan AP2 protein, PF14{_}0633 from Plasmodium falciparum. The X-ray crystal structure of the PF14{_}0633 AP2 domain bound to DNA reveals a {beta}-sheet fold that binds the DNA major groove through base-specific and backbone contacts; a prominent {alpha}-helix supports the {beta}-sheet structure. Substitution of predicted DNA-binding residues with alanine weakened ormore » eliminated DNA binding in solution. In contrast to plant AP2 domains, the PF14{_}0633 AP2 domain dimerizes upon binding to DNA through a domain-swapping mechanism in which the {alpha}-helices of the AP2 domains pack against the {beta}-sheets of the dimer mates. DNA-induced dimerization of PF14{_}0633 may be important for tethering two distal DNA loci together in the nucleus and/or for inducing functional rearrangements of its domains to facilitate transcriptional regulation. Consistent with a multisite binding mode, at least two copies of the consensus sequence recognized by PF14{_}0633 are present upstream of a previously identified group of sporozoite-stage genes. Taken together, these findings illustrate how Plasmodium has adapted the AP2 DNA-binding domain for genome-wide transcriptional regulation.« less

PlantTFDB: a comprehensive plant transcription factor database

PubMed Central

Guo, An-Yuan; Chen, Xin; Gao, Ge; Zhang, He; Zhu, Qi-Hui; Liu, Xiao-Chuan; Zhong, Ying-Fu; Gu, Xiaocheng; He, Kun; Luo, Jingchu

2008-01-01

Transcription factors (TFs) play key roles in controlling gene expression. Systematic identification and annotation of TFs, followed by construction of TF databases may serve as useful resources for studying the function and evolution of transcription factors. We developed a comprehensive plant transcription factor database PlantTFDB (http://planttfdb.cbi.pku.edu.cn), which contains 26 402 TFs predicted from 22 species, including five model organisms with available whole genome sequence and 17 plants with available EST sequences. To provide comprehensive information for those putative TFs, we made extensive annotation at both family and gene levels. A brief introduction and key references were presented for each family. Functional domain information and cross-references to various well-known public databases were available for each identified TF. In addition, we predicted putative orthologs of those TFs among the 22 species. PlantTFDB has a simple interface to allow users to search the database by IDs or free texts, to make sequence similarity search against TFs of all or individual species, and to download TF sequences for local analysis. PMID:17933783

Sensing charges of the Ciona intestinalis voltage-sensing phosphatase.

PubMed

Villalba-Galea, Carlos A; Frezza, Ludivine; Sandtner, Walter; Bezanilla, Francisco

2013-11-01

Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing.

[Emerging novel therapeutic strategy for α-dystroglycanopathy by Large].

PubMed

Saito, Fumiaki

2011-11-01

The past decade of researches have revealed mutations of known or putative glycosyltransferases in several types of muscular dystrophy, including Fukuyama-type congenital muscular dystrophy. In these disorders, the function of α-dystroglycan is severely decreased, therefore they are called α-dystroglycanopathy. Recently, putative glycosyltransferase Large was shown to restore the defective function of α-dystroglycan, thus, it is an intriguing idea to apply this effect to the therapy of α-dystroglycanopathy. In the present study, we sought to test this possibility. Using several cultured cell lines, we confirmed that the overexpression of Large results in hyperglycosylation and marked enhancement of the function of α-dystroglycan. For this effect, the whole luminal domain of Large was shown to be necessary using several deletion constructs. We further generated transgenic mice overexpressing Large ubiquitously. In each tissue of the mice, the glycosylation of α-dystroglycan and its laminin binding activity was remarkably increased. Moreover, the morphological analyses on each tissue stained by H-E revealed no significant abnormality in the transgenic mice, suggesting no serious side effects by the overexpression of Large. Taken together, these results indicate that the restoration of the function of α-dystroglycan by Large should be an important molecular target to develop therapeutic strategies for α-dystroglycanopathy.

A Cytosolic Amphiphilic α-Helix Controls the Activity of the Bile Acid-sensitive Ion Channel (BASIC)*

PubMed Central

Schmidt, Axel; Löhrer, Daniel; Alsop, Richard J.; Lenzig, Pia; Oslender-Bujotzek, Adrienne; Wirtz, Monika; Rheinstädter, Maikel C.; Gründer, Stefan; Wiemuth, Dominik

2016-01-01

The bile acid-sensitive ion channel (BASIC) is a member of the degenerin/epithelial Na+ channel (Deg/ENaC) family of ion channels. It is mainly found in bile duct epithelial cells, the intestinal tract, and the cerebellum and is activated by alterations of its membrane environment. Bile acids, one class of putative physiological activators, exert their effect by changing membrane properties, leading to an opening of the channel. The physiological function of BASIC, however, is unknown. Deg/ENaC channels are characterized by a trimeric subunit composition. Each subunit is composed of two transmembrane segments, which are linked by a large extracellular domain. The termini of the channels protrude into the cytosol. Many Deg/ENaC channels contain regulatory domains and sequence motifs within their cytosolic domains. In this study, we show that BASIC contains an amphiphilic α-helical structure within its N-terminal domain. This α-helix binds to the cytosolic face of the plasma membrane and stabilizes a closed state. Truncation of this domain renders the channel hyperactive. Collectively, we identify a cytoplasmic domain, unique to BASIC, that controls channel activity via membrane interaction. PMID:27679529

A Cytosolic Amphiphilic α-Helix Controls the Activity of the Bile Acid-sensitive Ion Channel (BASIC).

PubMed

Schmidt, Axel; Löhrer, Daniel; Alsop, Richard J; Lenzig, Pia; Oslender-Bujotzek, Adrienne; Wirtz, Monika; Rheinstädter, Maikel C; Gründer, Stefan; Wiemuth, Dominik

2016-11-18

The bile acid-sensitive ion channel (BASIC) is a member of the degenerin/epithelial Na + channel (Deg/ENaC) family of ion channels. It is mainly found in bile duct epithelial cells, the intestinal tract, and the cerebellum and is activated by alterations of its membrane environment. Bile acids, one class of putative physiological activators, exert their effect by changing membrane properties, leading to an opening of the channel. The physiological function of BASIC, however, is unknown. Deg/ENaC channels are characterized by a trimeric subunit composition. Each subunit is composed of two transmembrane segments, which are linked by a large extracellular domain. The termini of the channels protrude into the cytosol. Many Deg/ENaC channels contain regulatory domains and sequence motifs within their cytosolic domains. In this study, we show that BASIC contains an amphiphilic α-helical structure within its N-terminal domain. This α-helix binds to the cytosolic face of the plasma membrane and stabilizes a closed state. Truncation of this domain renders the channel hyperactive. Collectively, we identify a cytoplasmic domain, unique to BASIC, that controls channel activity via membrane interaction. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural and Functional Characterization of a Ruminal β-Glycosidase Defines a Novel Subfamily of Glycoside Hydrolase Family 3 with Permuted Domain Topology*

PubMed Central

Ramírez-Escudero, Mercedes; del Pozo, Mercedes V.; Marín-Navarro, Julia; González, Beatriz; Golyshin, Peter N.; Polaina, Julio; Ferrer, Manuel; Sanz-Aparicio, Julia

2016-01-01

Metagenomics has opened up a vast pool of genes for putative, yet uncharacterized, enzymes. It widens our knowledge on the enzyme diversity world and discloses new families for which a clear classification is still needed, as is exemplified by glycoside hydrolase family-3 (GH3) proteins. Herein, we describe a GH3 enzyme (GlyA1) from resident microbial communities in strained ruminal fluid. The enzyme is a β-glucosidase/β-xylosidase that also shows β-galactosidase, β-fucosidase, α-arabinofuranosidase, and α-arabinopyranosidase activities. Short cello- and xylo-oligosaccharides, sophorose and gentibiose, are among the preferred substrates, with the large polysaccharide lichenan also being hydrolyzed by GlyA1. The determination of the crystal structure of the enzyme in combination with deletion and site-directed mutagenesis allowed identification of its unusual domain composition and the active site architecture. Complexes of GlyA1 with glucose, galactose, and xylose allowed picturing the catalytic pocket and illustrated the molecular basis of the substrate specificity. A hydrophobic platform defined by residues Trp-711 and Trp-106, located in a highly mobile loop, appears able to allocate differently β-linked bioses. GlyA1 includes an additional C-terminal domain previously unobserved in GH3 members, but crystallization of the full-length enzyme was unsuccessful. Therefore, small angle x-ray experiments have been performed to investigate the molecular flexibility and overall putative shape. This study provided evidence that GlyA1 defines a new subfamily of GH3 proteins with a novel permuted domain topology. Phylogenetic analysis indicates that this topology is associated with microbes inhabiting the digestive tracts of ruminants and other animals, feeding on chemically diverse plant polymeric materials. PMID:27679487

Scallop DMT functions as a Ca2+ transporter.

PubMed

Toyohara, Haruhiko; Yamamoto, Sayuri; Hosoi, Masatomi; Takagi, Masaya; Hayashi, Isao; Nakao, Kenji; Kaneko, Shuji

2005-05-09

We identified a DMT (divalent metal transporter) homologous protein that functions as a Ca(2+) transporter. Scallop DMT cDNA encodes a 539-amino-acid protein with 12 putative membrane-spanning domains and has a consensus transport motif in the fourth extracellular loop. Since its mRNA is significantly expressed in the gill and intestine, it is assumed that scallop DMT transports Ca(2+) from seawater by the gill and from food by the intestine. Scallop DMT lacks the iron-responsive element commonly found in iron-regulatory proteins, suggesting that it is free of the post-transcriptional regulation from intracellular Fe(2+) concentration. Scallop DMT distinctly functions as a Ca(2+) transporter unlike other DMTs, however, it also transports Fe(2+) and Cd(2+) similar to them.

Ultratight crystal packing of a 10 kDa protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trillo-Muyo, Sergio; Jasilionis, Andrius; Domagalski, Marcin J.

2013-03-01

The crystal structure of the C-terminal domain of a putative U32 peptidase from G. thermoleovorans is reported; it is one of the most tightly packed protein structures reported to date. While small organic molecules generally crystallize forming tightly packed lattices with little solvent content, proteins form air-sensitive high-solvent-content crystals. Here, the crystallization and full structure analysis of a novel recombinant 10 kDa protein corresponding to the C-terminal domain of a putative U32 peptidase are reported. The orthorhombic crystal contained only 24.5% solvent and is therefore among the most tightly packed protein lattices ever reported.

Functional analysis of the putative peroxidase domain of FANCA, the Fanconi anemia complementation group A protein.

PubMed

Ren, J; Youssoufian, H

2001-01-01

Fanconi anemia (FA) is an autosomal recessive disorder manifested by chromosomal breakage, birth defects, and susceptibility to bone marrow failure and cancer. At least seven complementation groups have been identified, and the genes defective in four groups have been cloned. The most common subtype is complementation group A. Although the normal functions of the gene products defective in FA cells are not completely understood, a clue to the function of the FA group A gene product (FANCA) was provided by the detection of limited homology in the amino terminal region to a class of heme peroxidases. We evaluated this hypothesis by mutagenesis and functional complementation studies. We substituted alanine residues for the most conserved FANCA residues in the putative peroxidase domain and tested their effects on known biochemical and cellular functions of FANCA. While the substitution mutants were comparable to wild-type FANCA with regard to their stability, subcellular localization, and interaction with FANCG, only the Trp(183)-to-Ala substitution (W183A) abolished the ability of FANCA to complement the sensitivity of FA group A cells to mitomycin C. By contrast, TUNEL assays for apoptosis after exposure to H2O2 showed no differences between parental FA group A cells, cells complemented with wild-type FANCA, and cells complemented with the W183A of FANCA. Moreover, semiquantitative RT-PCR analysis for the expression of the peroxide-sensitive heme oxygenase gene showed appropriate induction after H2O2 exposure. Thus, W183A appears to be essential for the in vivo activity of FANCA in a manner independent of its interaction with FANCG. Moreover, neither wild-type FANCA nor the W183A mutation appears to alter the peroxide-induced apoptosisor peroxide-sensing ability of FA group A cells. Copyright 2001 Academic Press.

PHTS, a novel putative tumor suppressor, is involved in the transformation reversion of HeLaHF cells independently of the p53 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu Dehua; Fan, Wufang; Liu, Guohong

2006-04-01

HeLaHF is a non-transformed revertant of HeLa cells, likely resulting from the activation of a putative tumor suppressor(s). p53 protein was stabilized in this revertant and reactivated for certain transactivation functions. Although p53 stabilization has not conclusively been linked to the reversion, it is clear that the genes in p53 pathway are involved. The present study confirms the direct role of p53 in HeLaHF reversion by demonstrating that RNAi-mediated p53 silencing partially restores anchorage-independent growth potential of the revertant through the suppression of anoikis. In addition, we identified a novel gene, named PHTS, with putative tumor suppressor properties, and showedmore » that this gene is also involved in HeLaHF reversion independently of the p53 pathway. Expression profiling revealed that PHTS is one of the genes that is up-regulated in HeLaHF but not in HeLa. It encodes a putative protein with CD59-like domains. RNAi-mediated PHTS silencing resulted in the partial restoration of transformation (anchorage-independent growth) in HeLaHF cells, similar to that of p53 gene silencing, implying its tumor suppressor effect. However, the observed increased transformation potential by PHTS silencing appears to be due to an increased anchorage-independent proliferation rate rather than suppression of anoikis, unlike the effect of p53 silencing. p53 silencing did not affect PHTS gene expression, and vice versa, suggesting PHTS may function in a new and p53-independent tumor suppressor pathway. Furthermore, over-expression of PHTS in different cancer cell lines, in addition to HeLa, reduces cell growth likely via induced apoptosis, confirming the broad PHTS tumor suppressor properties.« less

Determination of Surface-Exposed, Functional Domains of Gonococcal Transferrin-Binding Protein A

PubMed Central

Yost-Daljev, Mary Kate; Cornelissen, Cynthia Nau

2004-01-01

The gonococcal transferrin receptor is composed of two distinct proteins, TbpA and TbpB. TbpA is a member of the TonB-dependent family of integral outer membrane transporters, while TbpB is lipid modified and thought to be peripherally surface exposed. We previously proposed a hypothetical topology model for gonococcal TbpA that was based upon computer predictions and similarity with other TonB-dependent transporters for which crystal structures have been determined. In the present study, the hemagglutinin epitope was inserted into TbpA to probe the surface topology of this protein and secondarily to test the functional impacts of site-specific mutagenesis. Twelve epitope insertion mutants were constructed, five of which allowed us to confirm the surface exposure of loops 2, 3, 5, 7, and 10. In contrast to the predictions set forth by the hypothetical model, insertion into the plug region resulted in an epitope that was surface accessible, while epitope insertions into two putative loops (9 and 11) were not surface accessible. Insertions into putative loop 3 and β strand 9 abolished transferrin binding and utilization, and the plug insertion mutant exhibited decreased transferrin-binding affinity concomitant with an inability to utilize it. Insertion into putative β strand 16 generated a mutant that was able to bind transferrin normally but that was unable to mediate utilization. Mutants with insertions into putative loops 2, 9, and 11 maintained wild-type binding affinity but could utilize only transferrin in the presence of TbpB. This is the first demonstration of the ability of TbpB to compensate for a mutation in TbpA. PMID:14977987

The structure of the exopolyphsophatase (PPX) from Escherchia coli O157:H7 suggests a binding mode for long polyphosphate chains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rangarajan,E.; Nadeau, G.; Li, Y.

2006-01-01

Polyphosphate (polyP) is a linear polymer consisting of tens to hundreds of phosphate molecules joined together by high-energy anhydride bonds. These polymers are found in virtually all prokaryotic and eukaryotic cells and perform many functions; prominent among them are the responses to many stresses. Polyphosphate is synthesized by polyP kinase (PPK), using the terminal phosphate of ATP as the substrate, and degraded to inorganic phosphate by both endo- and exopolyphosphatases. Here we report the crystal structure and analysis of the polyphosphate phosphatase PPX from Escherichia coli O157:H7 refined at 2.2 Angstroms resolution. PPX is made of four domains. Domains Imore » and II display structural similarity with one another and share the ribonuclease-H-like fold. Domain III bears structural similarity to the N-terminal, HD domain of SpoT. Domain IV, the smallest domain, has structural counterparts in cold-shock associated RNA-binding proteins but is of unknown function in PPX. The putative PPX active site is located at the interface between domains I and II. In the crystal structure of PPX these two domains are close together and represent the 'closed' state. Comparison with the crystal structure of PPX/GPPA from Aquifex aeolicus reveals close structural similarity between domains I and II of the two enzymes, with the PPX/GPPA representing an 'open' state. A striking feature of the dimer is a deep S-shaped canyon extending along the dimer interface and lined with positively charged residues. The active site region opens to this canyon. We postulate that this is a likely site of polyP binding.« less

Targeted Analysis of KLF6/KLF6-SV1 Regulating Pathways in Prostrate Cancer Development and Metastasis

DTIC Science & Technology

2010-09-01

of KLF6 and KLF6-SV1 it will be important to define the functionality of the putative NLS, the 5BR, as well as the role of nucleo-cytoplasmic...presence of basic residues, Lys and Arg. In many cases 18 these signals are located near or within other important domains that regulate protein...transporter protein Crm1/Xpo1, first discovered in yeast (33-36). Subcellular localization and protein turnover are two related events that are tightly

Cell surface expression of channel catfish leukocyte immune-type receptors (IpLITRs) and recruitment of both Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2.

PubMed

Montgomery, Benjamin C S; Mewes, Jacqueline; Davidson, Chelsea; Burshtyn, Deborah N; Stafford, James L

2009-04-01

Channel catfish leukocyte immune-type receptors (IpLITRs) are immunoglobulin superfamily (IgSF) members believed to play a role in the control and coordination of cellular immune responses in teleost. Putative stimulatory and inhibitory IpLITRs are co-expressed by different types of catfish immune cells (e.g. NK cells, T cells, B cells, and macrophages) but their signaling potential has not been determined. Following cationic polymer-mediated transfections into human cell lines we examined the surface expression, tyrosine phosphorylation, and phosphatase recruitment potential of two types of putative inhibitory IpLITRs using 'chimeric' expression constructs and an epitope-tagged 'native' IpLITR. We also cloned and expressed the teleost Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-1 and SHP-2 and examined their expression in adult tissues and developing zebrafish embryos. Co-immunoprecipitation experiments support the inhibitory signaling potential of distinct IpLITR-types that bound both SHP-1 and SHP-2 following the phosphorylation of tyrosine residues within their cytoplasmic tail (CYT) regions. Phosphatase recruitment by IpLITRs represents an important first step in understanding their influence on immune cell effector functions and suggests that certain inhibitory signaling pathways are conserved among vertebrates.

The MB2 gene family of Plasmodium species has a unique combination of S1 and GTP-binding domains

PubMed Central

Romero, Lisa C; Nguyen, Thanh V; Deville, Benoit; Ogunjumo, Oluwasanmi; James, Anthony A

2004-01-01

Background Identification and characterization of novel Plasmodium gene families is necessary for developing new anti-malarial therapeutics. The products of the Plasmodium falciparum gene, MB2, were shown previously to have a stage-specific pattern of subcellular localization and proteolytic processing. Results Genes homologous to MB2 were identified in five additional parasite species, P. knowlesi, P. gallinaceum, P. berghei, P. yoelii, and P. chabaudi. Sequence comparisons among the MB2 gene products reveal amino acid conservation of structural features, including putative S1 and GTP-binding domains, and putative signal peptides and nuclear localization signals. Conclusions The combination of domains is unique to this gene family and indicates that MB2 genes comprise a novel family and therefore may be a good target for drug development. PMID:15222903

The Oncoprotein Tax Binds the SRC-1-Interacting Domain of CBP/p300 To Mediate Transcriptional Activation

PubMed Central

Scoggin, Kirsten E. S.; Ulloa, Aida; Nyborg, Jennifer K.

2001-01-01

Oncogenesis associated with human T-cell leukemia virus (HTLV) infection is directly linked to the virally encoded transcription factor Tax. To activate HTLV-1 transcription Tax interacts with the cellular protein CREB and the pleiotropic coactivators CBP and p300. While extensively studied, the molecular mechanisms of Tax transcription function and coactivator utilization are not fully understood. Previous studies have focused on Tax binding to the KIX domain of CBP, as this was believed to be the key step in recruiting the coactivator to the HTLV-1 promoter. In this study, we identify a carboxy-terminal region of CBP (and p300) that strongly interacts with Tax and mediates Tax transcription function. Through deletion mutagenesis, we identify amino acids 2003 to 2212 of CBP, which we call carboxy-terminal region 2 (CR2), as the minimal region for Tax interaction. Interestingly, this domain corresponds to the steroid receptor coactivator 1 (SRC-1)-interacting domain of CBP. We show that a double point mutant targeted to one of the putative α-helical motifs in this domain significantly compromises the interaction with Tax. We also characterize the region of Tax responsible for interaction with CR2 and show that the previously identified transactivation domain of Tax (amino acids 312 to 319) participates in CR2 binding. This region of Tax corresponds to a consensus amphipathic helix, and single point mutations targeted to amino acids on the face of this helix abolish interaction with CR2 and dramatically reduce Tax transcription function. Finally, we demonstrate that Tax and SRC-1 bind to CR2 in a mutually exclusive fashion. Together, these studies identify a novel Tax-interacting site on CBP/p300 and extend our understanding of the molecular mechanism of Tax transactivation. PMID:11463834

Gene structure, cDNA characterization and RNAi-based functional analysis of a myeloid differentiation factor 88 homolog in Tenebrio molitor larvae exposed to Staphylococcus aureus infection.

PubMed

Patnaik, Bharat Bhusan; Patnaik, Hongray Howrelia; Seo, Gi Won; Jo, Yong Hun; Lee, Yong Seok; Lee, Bok Luel; Han, Yeon Soo

2014-10-01

Myeloid differentiation factor 88 (MyD88), an intracellular adaptor protein involved in Toll/Toll-like receptor (TLR) signal processing, triggers activation of nuclear factor-kappaB (NF-κB) transcription factors. In the present study, we analyzed the gene structure and biological function of MyD88 in a coleopteran insect, Tenebrio molitor (TmMyD88). The TmMyD88 gene was 1380 bp in length and consisted of five exons and four introns. The 5'-flanking sequence revealed several putative transcription factor binding sites, such as STAT-4, AP-1, cJun, cfos, NF-1 and many heat shock factor binding elements. The cDNA contained a typical death domain, a conservative Toll-like interleukin-1 receptor (TIR) domain, and a C-terminal extension (CTE). The TmMyD88 TIR domain showed three significantly conserved motifs for interacting with the TIR domain of TLRs. TmMyD88 was grouped within the invertebrate cluster of the phylogenetic tree and shared 75% sequence identity with the TIR domain of Tribolium castaneum MyD88. Homology modeling of the TmMyD88 TIR domain revealed five parallel β-strands surrounded by five α-helices that adopted loop conformations to function as an adaptor. TmMyD88 expression was upregulated 7.3- and 4.79-fold after 12 and 6h, respectively, of challenge with Staphylococcus aureus and fungal β-1,3 glucan. Silencing of the TmMyD88 transcript by RNA interference led to reduced resistance of the host to infection by S. aureus. These results indicate that TmMyD88 is required for survival against Staphylococcus infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparative genomic analysis of SET domain family reveals the origin, expansion, and putative function of the arthropod-specific SmydA genes as histone modifiers in insects.

PubMed

Jiang, Feng; Liu, Qing; Wang, Yanli; Zhang, Jie; Wang, Huimin; Song, Tianqi; Yang, Meiling; Wang, Xianhui; Kang, Le

2017-06-01

The SET domain is an evolutionarily conserved motif present in histone lysine methyltransferases, which are important in the regulation of chromatin and gene expression in animals. In this study, we searched for SET domain-containing genes (SET genes) in all of the 147 arthropod genomes sequenced at the time of carrying out this experiment to understand the evolutionary history by which SET domains have evolved in insects. Phylogenetic and ancestral state reconstruction analysis revealed an arthropod-specific SET gene family, named SmydA, that is ancestral to arthropod animals and specifically diversified during insect evolution. Considering that pseudogenization is the most probable fate of the new emerging gene copies, we provided experimental and evolutionary evidence to demonstrate their essential functions. Fluorescence in situ hybridization analysis and in vitro methyltransferase activity assays showed that the SmydA-2 gene was transcriptionally active and retained the original histone methylation activity. Expression knockdown by RNA interference significantly increased mortality, implying that the SmydA genes may be essential for insect survival. We further showed predominantly strong purifying selection on the SmydA gene family and a potential association between the regulation of gene expression and insect phenotypic plasticity by transcriptome analysis. Overall, these data suggest that the SmydA gene family retains essential functions that may possibly define novel regulatory pathways in insects. This work provides insights into the roles of lineage-specific domain duplication in insect evolution. © The Authors 2017. Published by Oxford University Press.

Cadherin Domains in the Polysaccharide-Degrading Marine Bacterium Saccharophagus degradans 2-40 Are Carbohydrate-Binding Modules▿

PubMed Central

Fraiberg, Milana; Borovok, Ilya; Bayer, Edward A.; Weiner, Ronald M.; Lamed, Raphael

2011-01-01

The complex polysaccharide-degrading marine bacterium Saccharophagus degradans strain 2-40 produces putative proteins that contain numerous cadherin and cadherin-like domains involved in intercellular contact interactions. The current study reveals that both domain types exhibit reversible calcium-dependent binding to different complex polysaccharides which serve as growth substrates for the bacterium. PMID:21036994

Structure of the two-domain hexameric APS kinase from Thiobacillus denitrificans: structural basis for the absence of ATP sulfurylase activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gay, Sean C.; Segel, Irwin H.; Fisher, Andrew J., E-mail: fisher@chem.ucdavis.edu

2009-10-01

APS kinase from Thiobacillus denitrificans contains an inactive N-terminal ATP sulfurylase domain. The structure presented unveils the first hexameric assembly for an APS kinase, and reveals that structural changes in the N-terminal domain disrupt the ATP sulfurylase active site thus prohibiting activity. The Tbd-0210 gene of the chemolithotrophic bacterium Thiobacillus denitrificans is annotated to encode a 60.5 kDa bifunctional enzyme with ATP sulfurylase and APS kinase activity. This putative bifunctional enzyme was cloned, expressed and structurally characterized. The 2.95 Å resolution X-ray crystal structure reported here revealed a hexameric assembly with D{sub 3} symmetry. Each subunit contains a large N-terminalmore » sulfurylase-like domain and a C-terminal APS kinase domain reminiscent of the two-domain fungal ATP sulfurylases of Penicillium chrysogenum and Saccharomyces cerevisiae, which also exhibit a hexameric assembly. However, the T. denitrificans enzyme exhibits numerous structural and sequence differences in the N-terminal domain that render it inactive with respect to ATP sulfurylase activity. Surprisingly, the C-terminal domain does indeed display APS kinase activity, indicating that this gene product is a true APS kinase. Therefore, these results provide the first structural insights into a unique hexameric APS kinase that contains a nonfunctional ATP sulfurylase-like domain of unknown function.« less

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

PubMed Central

Guaitoli, Giambattista; Raimondi, Francesco; Gilsbach, Bernd K.; Gómez-Llorente, Yacob; Deyaert, Egon; Renzi, Fabiana; Li, Xianting; Schaffner, Adam; Jagtap, Pravin Kumar Ankush; Boldt, Karsten; von Zweydorf, Felix; Gotthardt, Katja; Lorimer, Donald D.; Yue, Zhenyu; Burgin, Alex; Janjic, Nebojsa; Sattler, Michael; Versées, Wim; Ueffing, Marius; Ubarretxena-Belandia, Iban; Kortholt, Arjan; Gloeckner, Christian Johannes

2016-01-01

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson’s disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity—together with the LRR domain—to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD. PMID:27357661

PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System

PubMed Central

Veith, Paul D.; Butler, Catherine A.; Nor Muhammad, Nor A.; Chen, Yu-Yen; Slakeski, Nada; Peng, Benjamin; Zhang, Lianyi; Dashper, Stuart G.; Cross, Keith J.; Cleal, Steven M.; Moore, Caroline; Reynolds, Eric C.

2016-01-01

Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a β-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS. PMID:27711252

Fiber Diffraction of the Prion-Forming Domain HET-s(218-289) Shows Dehydration-Induced Deformation of a Complex Amyloid Structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wan, William; Stubbs, Gerald

2014-05-01

Amyloids are filamentous protein aggregates that can be formed by many different proteins and are associated with both disease and biological functions. The pathogenicities or biological functions of amyloids are determined by their particular molecular structures, making accurate structural models a requirement for understanding their biological effects. One potential factor that can affect amyloid structures is hydration. Previous studies of simple stacked β-sheet amyloids have suggested that dehydration does not impact structure, but other studies indicated dehydration-related structural changes of a putative water-filled nanotube. Our results show that dehydration significantly affects the molecular structure of the fungal prion-forming domain HET-s(218–289),more » which forms a β-solenoid with no internal solvent-accessible regions. The dehydration-related structural deformation of HET-s(218–289) indicates that water can play a significant role in complex amyloid structures, even when no obvious water-accessible cavities are present.« less

Isolation and characterization of two cDNA clones encoding for glutamate dehydrogenase in Nicotiana plumbaginifolia.

PubMed

Ficarelli, A; Tassi, F; Restivo, F M

1999-03-01

We have isolated two full length cDNA clones encoding Nicotiana plumbaginifolia NADH-glutamate dehydrogenase. Both clones share amino acid boxes of homology corresponding to conserved GDH catalytic domains and putative mitochondrial targeting sequence. One clone shows a putative EF-hand loop. The level of the two transcripts is affected differently by carbon source.

Cloning and analysis of the Glwc-1 and Glwc-2 genes encoding putative blue light photoreceptor from Ganoderma lucidum.

PubMed

Xu, Xinran; Chen, Xiangdong; Yu, Wumengxiao; Liu, Yu; Zhang, Weiwei; Lan, Jin

2017-08-01

Blue light plays an important role during the growth of Ganoderma lucidum, one of the best-known medicinal macrofungi in China. In the present study, we cloned Glwc-1 and Glwc-2, the homologue of the blue light photoreceptors Ncwc-1 and Ncwc-2 of Neurospora crassa, from G. lucidum. The deduced amino acid sequence of Glwc-1 contained the similar function domains as NcWC-1 including LOV, PAS B, PAS C, and PAC domains. The deduced amino acid sequence of Glwc-2 contained PAS domain and GATA-type zinc finger (Znf) domain as well as NcWC-2. Phylogenetic analysis based on fungal WC-1 and WC-2 supported GlWC-1 and GlWC-2 were blue light receptors. The expression of Glwc-1 and Glwc-2 indicated that they might play an important role during the primordium differentiation process of G. lucidum, and the external blue light stimulation increased the expression of Glwc-1 and Glwc-2. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Substrates Control Multimerization and Activation of the Multi-Domain ATPase Motor of Type VII Secretion

DOE PAGES

Rosenberg, Oren S.; Dovala, Dustin; Li, Xueming; ...

2015-04-09

We report that Mycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increasemore » in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically but, rather, by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps to explain interdependence of substrates, and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.« less

Impact of altered phosphorylation on loss of function of juvenile Parkinsonism-associated genetic variants of the E3 ligase parkin.

PubMed

Aguirre, Jacob D; Dunkerley, Karen M; Lam, Rica; Rusal, Michele; Shaw, Gary S

2018-04-27

Autosomal recessive juvenile Parkinsonism (ARJP) is an inherited neurodegenerative disease in which 50% of affected individuals harbor mutations in the gene encoding the E3 ligase parkin. Parkin regulates the mitochondrial recycling pathway, which is induced by oxidative stress. In its native state, parkin is auto-inhibited by its N-terminal ubiquitin-like (Ubl) domain, which blocks the binding site for an incoming E2∼ubiquitin conjugate, needed for parkin's ubiquitination activity. Parkin is activated via phosphorylation of Ser-65 in its Ubl domain by PTEN-induced putative kinase 1 (PINK1) and a ubiquitin molecule phosphorylated at a position equivalent to Ser-65 in parkin. Here we have examined the underlying molecular mechanism of phosphorylation of parkin's Ubl domain carrying ARJP-associated substitutions and how altered phosphorylation modulates parkin activation and ubiquitination. We found that three substitutions in the Ubl domain (G12R, R33Q, and R42P) significantly decrease PINK1's ability to phosphorylate the Ubl domain. We noted that two basic loss-of-function substitutions (R33Q and R42P) are close to acidic patches in the proposed PINK1-parkin interface, indicating that ionic interactions at this site may be important for efficient parkin phosphorylation. Increased auto-ubiquitination with unique ubiquitin chain patterns was observed for two other Ubl domain substitutions (G12R and T55I), suggesting that these substitutions, along with phosphorylation, increase parkin degradation. Moreover, Ubl domain phosphorylation decreased its affinity for the potential effector protein ataxin-3, which edits ubiquitin chain building by parkin. Overall, our work provides a framework for the mechanisms of parkin's loss-of-function, indicating an interplay between ARJP-associated substitutions and phosphorylation of its Ubl domain. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of the Xylella fastidiosa PD1671 Gene Encoding Degenerate c-di-GMP GGDEF/EAL Domains, and Its Role in the Development of Pierce’s Disease

PubMed Central

Cursino, Luciana; Athinuwat, Dusit; Patel, Kelly R.; Galvani, Cheryl D.; Zaini, Paulo A.; Li, Yaxin; De La Fuente, Leonardo; Hoch, Harvey C.; Burr, Thomas J.; Mowery, Patricia

2015-01-01

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce’s disease of grapevines. X. fastidiosa is thought to induce disease by colonizing and clogging xylem vessels through the formation of cell aggregates and bacterial biofilms. Here we examine the role in X. fastidiosa virulence of an uncharacterized gene, PD1671, annotated as a two-component response regulator with potential GGDEF and EAL domains. GGDEF domains are found in c-di-GMP diguanylate cyclases while EAL domains are found in phosphodiesterases, and these domains are for c-di-GMP production and turnover, respectively. Functional analysis of the PD1671 gene revealed that it affected multiple X. fastidiosa virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of gum genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility in vitro but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of X. fastidiosa virulence. PMID:25811864

Characterization of the Xylella fastidiosa PD1671 gene encoding degenerate c-di-GMP GGDEF/EAL domains, and its role in the development of Pierce's disease.

PubMed

Cursino, Luciana; Athinuwat, Dusit; Patel, Kelly R; Galvani, Cheryl D; Zaini, Paulo A; Li, Yaxin; De La Fuente, Leonardo; Hoch, Harvey C; Burr, Thomas J; Mowery, Patricia

2015-01-01

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce's disease of grapevines. X. fastidiosa is thought to induce disease by colonizing and clogging xylem vessels through the formation of cell aggregates and bacterial biofilms. Here we examine the role in X. fastidiosa virulence of an uncharacterized gene, PD1671, annotated as a two-component response regulator with potential GGDEF and EAL domains. GGDEF domains are found in c-di-GMP diguanylate cyclases while EAL domains are found in phosphodiesterases, and these domains are for c-di-GMP production and turnover, respectively. Functional analysis of the PD1671 gene revealed that it affected multiple X. fastidiosa virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of gum genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility in vitro but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of X. fastidiosa virulence.

Genome-Wide Comparative Analysis of the Phospholipase D Gene Families among Allotetraploid Cotton and Its Diploid Progenitors

PubMed Central

Tang, Kai; Dong, Chun-Juan; Liu, Jin-Yuan

2016-01-01

In this study, 40 phospholipase D (PLD) genes were identified from allotetraploid cotton Gossypium hirsutum, and 20 PLD genes were examined in diploid cotton Gossypium raimondii. Combining with 19 previously identified Gossypium arboreum PLD genes, a comparative analysis was performed among the PLD gene families among allotetraploid and two diploid cottons. Based on the orthologous relationships, we found that almost each G. hirsutum PLD had a corresponding homolog in the G. arboreum and G. raimondii genomes, except for GhPLDβ3A, whose homolog GaPLDβ3 may have been lost during the evolution of G. arboreum after the interspecific hybridization. Phylogenetic analysis showed that all of the cotton PLDs were unevenly classified into six numbered subgroups: α, β/γ, δ, ε, ζ and φ. An N-terminal C2 domain was found in the α, β/γ, δ and ε subgroups, while phox homology (PX) and pleckstrin homology (PH) domains were identified in the ζ subgroup. The subgroup φ possessed a single peptide instead of a functional domain. In each phylogenetic subgroup, the PLDs showed high conservation in gene structure and amino acid sequences in functional domains. The expansion of GhPLD and GrPLD gene families were mainly attributed to segmental duplication and partly attributed to tandem duplication. Furthermore, purifying selection played a critical role in the evolution of PLD genes in cotton. Quantitative RT-PCR documented that allotetraploid cotton PLD genes were broadly expressed and each had a unique spatial and developmental expression pattern, indicating their functional diversification in cotton growth and development. Further analysis of cis-regulatory elements elucidated transcriptional regulations and potential functions. Our comparative analysis provided valuable information for understanding the putative functions of the PLD genes in cotton fiber. PMID:27213891

Domain-general signals in the cingulo-opercular network for visuospatial attention and episodic memory

PubMed Central

Sestieri, Carlo; Corbetta, Maurizio; Spadone, Sara; Romani, Gian Luca; Shulman, Gordon L.

2014-01-01

We investigated the functional properties of a previously described cingulo-opercular network (CON) putatively involved in cognitive control. Analyses of common fMRI task-evoked activity during perceptual and episodic memory search tasks that differently recruited the dorsal attention (DAN) and default mode network (DMN) established the generality of this network. Regions within the CON (anterior insula/frontal operculum and anterior cingulate/presupplementary cortex) displayed sustained signals during extended periods in which participants searched for behaviourally relevant information in a dynamically changing environment or from episodic memory in the absence of sensory stimulation. The CON was activated during all phases of both tasks, which involved trial initiation, target detection, decision and response, indicating its consistent involvement in a broad range of cognitive processes. Functional connectivity analyses showed that the CON flexibly linked with the DAN or DMN regions during perceptual or memory search, respectively. Aside from the CON, only a limited number of regions, including the lateral prefrontal cortex, showed evidence of domain-general, sustained activity, although in some cases the common activations may have reflected the functional-anatomical variability of domain-specific regions rather than a true domain-generality. These additional regions also showed task-dependent functional connectivity with the DMN and DAN, suggesting that this feature is not a specific marker of cognitive control. Finally, multivariate clustering analyses separated the CON from other fronto-parietal regions previously associated with cognitive control, indicating a unique fingerprint. We conclude that the CON’s functional properties and interactions with other brain regions support a broad role in cognition, consistent with its characterization as a task-control network. PMID:24144246

Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread.

PubMed

Kroj, Thomas; Chanclud, Emilie; Michel-Romiti, Corinne; Grand, Xavier; Morel, Jean-Benoit

2016-04-01

Plant immune receptors of the class of nucleotide-binding and leucine-rich repeat domain (NLR) proteins can contain additional domains besides canonical NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)) and leucine-rich repeat (LRR) domains. Recent research suggests that these additional domains act as integrated decoys recognizing effectors from pathogens. Proteins homologous to integrated decoys are suspected to be effector targets and involved in disease or resistance. Here, we scrutinized 31 entire plant genomes to identify putative integrated decoy domains in NLR proteins using the Interpro search. The involvement of the Zinc Finger-BED type (ZBED) protein containing a putative decoy domain, called BED, in rice (Oryza sativa) resistance was investigated by evaluating susceptibility to the blast fungus Magnaporthe oryzae in rice over-expression and knock-out mutants. This analysis showed that all plants tested had integrated various atypical protein domains into their NLR proteins (on average 3.5% of all NLR proteins). We also demonstrated that modifying the expression of the ZBED gene modified disease susceptibility. This study suggests that integration of decoy domains in NLR immune receptors is widespread and frequent in plants. The integrated decoy model is therefore a powerful concept to identify new proteins involved in disease resistance. Further in-depth examination of additional domains in NLR proteins promises to unravel many new proteins of the plant immune system. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Structure of the Zinc-Bound Amino-Terminal Domain of the NMDA Receptor NR2B Subunit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karakas, E.; Simorowski, N; Furukawa, H

2009-01-01

N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino-terminal domain (ATD) distinct from the L-glutamate-binding domain. The molecular basis for the ATD-mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc-freemore » and zinc-bound states. The structures reveal the overall clamshell-like architecture distinct from the non-NMDA receptor ATDs and molecular determinants for the zinc-binding site, ion-binding sites, and the architecture of the putative phenylethanolamine-binding site.« less

Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis.

PubMed

Zhang, Hongjie; Abraham, Nessy; Khan, Liakot A; Hall, David H; Fleming, John T; Göbel, Verena

2011-09-18

Metazoan internal organs are assembled from polarized tubular epithelia that must set aside an apical membrane domain as a lumenal surface. In a global Caenorhabditis elegans tubulogenesis screen, interference with several distinct fatty-acid-biosynthetic enzymes transformed a contiguous central intestinal lumen into multiple ectopic lumens. We show that multiple-lumen formation is caused by apicobasal polarity conversion, and demonstrate that in situ modulation of lipid biosynthesis is sufficient to reversibly switch apical domain identities on growing membranes of single post-mitotic cells, shifting lumen positions. Follow-on targeted lipid-biosynthesis pathway screens and functional genetic assays were designed to identify a putative single causative lipid species. They demonstrate that fatty-acid biosynthesis affects polarity through sphingolipid synthesis, and reveal ceramide glucosyltransferases (CGTs) as end-point biosynthetic enzymes in this pathway. Our findings identify glycosphingolipids, CGT products and obligate membrane lipids, as critical determinants of in vivo polarity and indicate that they sort new components to the expanding apical membrane.

Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis

PubMed Central

Zhang, Hongjie; Abraham, Nessy; Khan, Liakot A.; Hall, David H.; Fleming, John T.; Gobel, Verena

2011-01-01

Metazoan internal organs are assembled from polarized tubular epithelia that must set aside an apical membrane domain as a lumenal surface. In a global Caenorhabditis elegans tubulogenesis screen, interference with several distinct fatty-acid-biosynthetic enzymes transformed a contiguous central intestinal lumen into multiple ectopic lumens. We show that multiple-lumen formation is caused by apicobasal polarity conversion, and demonstrate that in situ modulation of lipid biosynthesis is sufficient to reversibly switch apical domain identities on growing membranes of single postmitotic cells, shifting lumen positions. Follow-on targeted lipid-biosynthesis pathway screens and functional genetic assays were designed to identify a putative single causative lipid species. They demonstrate that fatty-acid biosynthesis affects polarity via sphingolipid synthesis, and reveal ceramideglucosyltransferases (CGTs) as endpoint biosynthetic enzymes in this pathway. Our findings identify glycosphingolipids (GSLs), CGT products and obligate membrane lipids, as critical determinants of in vivo polarity and suggest they sort new components to the expanding apical membrane. PMID:21926990

Genome-wide profiling of PRC1 and PRC2 Polycomb chromatin binding in Drosophila melanogaster.

PubMed

Tolhuis, Bas; de Wit, Elzo; Muijrers, Inhua; Teunissen, Hans; Talhout, Wendy; van Steensel, Bas; van Lohuizen, Maarten

2006-06-01

Polycomb group (PcG) proteins maintain transcriptional repression of developmentally important genes and have been implicated in cell proliferation and stem cell self-renewal. We used a genome-wide approach to map binding patterns of PcG proteins (Pc, esc and Sce) in Drosophila melanogaster Kc cells. We found that Pc associates with large genomic regions of up to approximately 150 kb in size, hereafter referred to as 'Pc domains'. Sce and esc accompany Pc in most of these domains. PcG-bound chromatin is trimethylated at histone H3 Lys27 and is generally transcriptionally silent. Furthermore, PcG proteins preferentially bind to developmental genes. Many of these encode transcriptional regulators and key components of signal transduction pathways, including Wingless, Hedgehog, Notch and Delta. We also identify several new putative functions of PcG proteins, such as in steroid hormone biosynthesis. These results highlight the extensive involvement of PcG proteins in the coordination of development through the formation of large repressive chromatin domains.

The structure of cell adhesion molecule uvomorulin. Insights into the molecular mechanism of Ca2+-dependent cell adhesion.

PubMed Central

Ringwald, M; Schuh, R; Vestweber, D; Eistetter, H; Lottspeich, F; Engel, J; Dölz, R; Jähnig, F; Epplen, J; Mayer, S

1987-01-01

We have determined the amino acid sequence of the Ca2+-dependent cell adhesion molecule uvomorulin as it appears on the cell surface. The extracellular part of the molecule exhibits three internally repeated domains of 112 residues which are most likely generated by gene duplication. Each of the repeated domains contains two highly conserved units which could represent putative Ca2+-binding sites. Secondary structure predictions suggest that the putative Ca2+-binding units are located in external loops at the surface of the protein. The protein sequence exhibits a single membrane-spanning region and a cytoplasmic domain. Sequence comparison reveals extensive homology to the chicken L-CAM. Both uvomorulin and L-CAM are identical in 65% of their entire amino acid sequence suggesting a common origin for both CAMs. Images Fig. 1. Fig. 4. Fig. 7. PMID:3501370

Chromosomal localization of the mouse Src-like adapter protein (Slap) gene and its putative human homolog SLA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Angrist, M.; Chakravarti, A.; Wells, D.E.

1995-12-10

Molecules containing Src-homology 2 (SH2) and Src-homology 3 (SH3) domains are critical components of signal transduction pathways that serve to relay signals originating from the cell surface to the interior of the cell. Src-like adapter protein (SLAP) is a recently described adapter protein that binds activated the Eck receptor protein-tyrosine kinase. Although SLAP bears a striking homology to the SH3 and SH2 domains of the Src family of nonreceptor tyrosine kinases, it does not contain a tyrosine kinase catalytic domain. In this report, the Slap gene was mapped by linkage analysis to mouse chromosome 15, while its putative human homologmore » (SLA) was identified and mapped to human 8q22.3-qter using a panel of somatic cell hybrids. 10 refs., 2 figs.« less

Prm3p is a pheromone-induced peripheral nuclear envelope protein required for yeast nuclear fusion.

PubMed

Shen, Shu; Tobery, Cynthia E; Rose, Mark D

2009-05-01

Nuclear membrane fusion is the last step in the mating pathway of the yeast Saccharomyces cerevisiae. We adapted a bioinformatics approach to identify putative pheromone-induced membrane proteins potentially required for nuclear membrane fusion. One protein, Prm3p, was found to be required for nuclear membrane fusion; disruption of PRM3 caused a strong bilateral defect, in which nuclear congression was completed but fusion did not occur. Prm3p was localized to the nuclear envelope in pheromone-responding cells, with significant colocalization with the spindle pole body in zygotes. A previous report, using a truncated protein, claimed that Prm3p is localized to the inner nuclear envelope. Based on biochemistry, immunoelectron microscopy and live cell microscopy, we find that functional Prm3p is a peripheral membrane protein exposed on the cytoplasmic face of the outer nuclear envelope. In support of this, mutations in a putative nuclear localization sequence had no effect on full-length protein function or localization. In contrast, point mutations and deletions in the highly conserved hydrophobic carboxy-terminal domain disrupted both protein function and localization. Genetic analysis, colocalization, and biochemical experiments indicate that Prm3p interacts directly with Kar5p, suggesting that nuclear membrane fusion is mediated by a protein complex.

RICD: a rice indica cDNA database resource for rice functional genomics.

PubMed

Lu, Tingting; Huang, Xuehui; Zhu, Chuanrang; Huang, Tao; Zhao, Qiang; Xie, Kabing; Xiong, Lizhong; Zhang, Qifa; Han, Bin

2008-11-26

The Oryza sativa L. indica subspecies is the most widely cultivated rice. During the last few years, we have collected over 20,000 putative full-length cDNAs and over 40,000 ESTs isolated from various cDNA libraries of two indica varieties Guangluai 4 and Minghui 63. A database of the rice indica cDNAs was therefore built to provide a comprehensive web data source for searching and retrieving the indica cDNA clones. Rice Indica cDNA Database (RICD) is an online MySQL-PHP driven database with a user-friendly web interface. It allows investigators to query the cDNA clones by keyword, genome position, nucleotide or protein sequence, and putative function. It also provides a series of information, including sequences, protein domain annotations, similarity search results, SNPs and InDels information, and hyperlinks to gene annotation in both The Rice Annotation Project Database (RAP-DB) and The TIGR Rice Genome Annotation Resource, expression atlas in RiceGE and variation report in Gramene of each cDNA. The online rice indica cDNA database provides cDNA resource with comprehensive information to researchers for functional analysis of indica subspecies and for comparative genomics. The RICD database is available through our website http://www.ncgr.ac.cn/ricd.

SH3 domain-mediated binding of the Drk protein to Dos is an important step in signaling of Drosophila receptor tyrosine kinases.

PubMed

Feller, Stephan M; Wecklein, Heike; Lewitzky, Marc; Kibler, Eike; Raabe, Thomas

2002-08-01

Activation of the Sevenless (Sev) receptor tyrosine kinase (RTK) in the developing Drosophila eye is required for the specification of the R7 photoreceptor cell fate. Daughter of Sevenless (Dos), a putative multi-site adaptor protein, is a substrate of the Sev kinase and is known to associate with the tyrosine phosphatase Corkscrew (Csw). Binding of Csw to Dos depends on the Csw Src homology 2 (SH2) domains and is an essential step for signaling by the Sev RTK. Dos, however, lacks a recognizable phosphotyrosine interaction domain and it was previously unclear how it is recruited to the Sev receptor. Here it is shown that the SH2/SH3 domain adaptor protein Drk can provide this link. Drk binds with its SH2 domain to the autophosphorylated Sev receptor while the C-terminal SH3 domain is able to associate with Dos. The Drk SH3 domain binding motifs on Dos were mapped to two sites which do not conform the known Drk SH3 domain binding motif (PxxPxR) but instead have the consensus PxxxRxxKP. Mutational analysis in vitro and in vivo provided evidence that both Drk binding sites fulfil an important function in the context of Sev and Drosophila epidermal growth factor receptor mediated signaling processes.

Crystal structure of YHI9, the yeast member of the phenazine biosynthesis PhzF enzyme superfamily.

PubMed

Liger, Dominique; Quevillon-Cheruel, Sophie; Sorel, Isabelle; Bremang, Michael; Blondeau, Karine; Aboulfath, Ilham; Janin, Joël; van Tilbeurgh, Herman; Leulliot, Nicolas

2005-09-01

In the Pseudomonas bacterial genomes, the PhzF proteins are involved in the production of phenazine derivative antibiotic and antifungal compounds. The PhzF superfamily however also encompasses proteins in all genomes from bacteria to eukaryotes, for which no function has been assigned. We have determined the three dimensional crystal structure at 2.05 A resolution of YHI9, the yeast member of the PhzF family. YHI9 has a fold similar to bacterial diaminopimelate epimerase, revealing a bimodular structure with an internal symmetry. Residue conservation identifies a putative active site at the interface between the two domains. Evolution of this protein by gene duplication, gene fusion and domain swapping from an ancestral gene containing the "hot dog" fold, identifies the protein as a "kinked double hot dog" fold. Copyright 2005 Wiley-Liss, Inc.

Envelope-like retrotransposons in the plant kingdom: evidence of their presence in gymnosperms (Pinus pinaster).

PubMed

Miguel, Célia; Simões, Marta; Oliveira, Maria Margarida; Rocheta, Margarida

2008-11-01

Retroviruses differ from retrotransposons due to their infective capacity, which depends critically on the encoded envelope. Some plant retroelements contain domains reminiscent of the env of animal retroviruses but the number of such elements described to date is restricted to angiosperms. We show here the first evidence of the presence of putative env-like gene sequences in a gymnosperm species, Pinus pinaster (maritime pine). Using a degenerate primer approach for conserved domains of RNaseH gene, three clones from putative envelope-like retrotransposons (PpRT2, PpRT3, and PpRT4) were identified. The env-like sequences of P. pinaster clones are predicted to encode proteins with transmembrane domains. These sequences showed identity scores of up to 30% with env-like sequences belonging to different organisms. A phylogenetic analysis based on protein alignment of deduced aminoacid sequences revealed that these clones clustered with env-containing plant retrotransposons, as well as with retrotransposons from invertebrate organisms. The differences found among the sequences of maritime pine clones isolated here suggest the existence of different putative classes of env-like retroelements. The identification for the first time of env-like genes in a gymnosperm species may support the ancestrality of retroviruses among plants shedding light on their role in plant evolution.

Distribution of CpG Motifs in Upstream Gene Domains in a Reef Coral and Sea Anemone: Implications for Epigenetics in Cnidarians.

PubMed

Marsh, Adam G; Hoadley, Kenneth D; Warner, Mark E

2016-01-01

Coral reefs are under assault from stressors including global warming, ocean acidification, and urbanization. Knowing how these factors impact the future fate of reefs requires delineating stress responses across ecological, organismal and cellular scales. Recent advances in coral reef biology have integrated molecular processes with ecological fitness and have identified putative suites of temperature acclimation genes in a Scleractinian coral Acropora hyacinthus. We wondered what unique characteristics of these genes determined their coordinate expression in response to temperature acclimation, and whether or not other corals and cnidarians would likewise possess these features. Here, we focus on cytosine methylation as an epigenetic DNA modification that is responsive to environmental stressors. We identify common conserved patterns of cytosine-guanosine dinucleotide (CpG) motif frequencies in upstream promoter domains of different functional gene groups in two cnidarian genomes: a coral (Acropora digitifera) and an anemone (Nematostella vectensis). Our analyses show that CpG motif frequencies are prominent in the promoter domains of functional genes associated with environmental adaptation, particularly those identified in A. hyacinthus. Densities of CpG sites in upstream promoter domains near the transcriptional start site (TSS) are 1.38x higher than genomic background levels upstream of -2000 bp from the TSS. The increase in CpG usage suggests selection to allow for DNA methylation events to occur more frequently within 1 kb of the TSS. In addition, observed shifts in CpG densities among functional groups of genes suggests a potential role for epigenetic DNA methylation within promoter domains to impact functional gene expression responses in A. digitifera and N. vectensis. Identifying promoter epigenetic sequence motifs among genes within specific functional groups establishes an approach to describe integrated cellular responses to environmental stress in reef corals and potential roles of epigenetics on survival and fitness in the face of global climate change.

Mutation of a putative MAP kinase consensus site regulates NCAM endocytosis and NCAM-dependent neurite outgrowth.

PubMed

Goschzik, Tobias; Cremer, Harold; Gnanapragassam, Vinayaga S; Horstkorte, Rüdiger; Bork, Kaya; Diestel, Simone

2017-07-01

The cytoplasmic domain of the neural cell adhesion molecule NCAM contains several putative serine/threonine phosphorylation sites whose functions are largely unknown. Human NCAM140 (NCAM140) possesses a potential MAP kinase phosphorylation site at threonine (T) 803. The aim of this study was to analyze a possible phosphorylation of NCAM140 by MAP kinases and to identify the functional role of T803. We found that NCAM140 is phosphorylated by the MAP kinase ERK2 in vitro. Exchange of T803 to aspartic acid (D) which mimics constitutive phosphorylation at the respective position resulted in increased endocytosis compared to NCAM140 in neuroblastoma cells and primary neurons. Consistently, NCAM140 endocytosis was inhibited by the MEK inhibitor U0126 in contrast to NCAM140-T803D or NCAM140-T803A endocytosis supporting a role of a potential ERK2 mediated phosphorylation at this site in endocytosis. Furthermore, cells expressing NCAM140-T803D developed significantly shorter neurites than NCAM140 expressing cells indicating that a potential phosphorylation of NCAM by ERK2 also regulates NCAM-dependent neurite outgrowth. Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

Sensing charges of the Ciona intestinalis voltage-sensing phosphatase

PubMed Central

Frezza, Ludivine; Sandtner, Walter

2013-01-01

Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing. PMID:24127524

Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM

PubMed Central

Hymes, Jeffrey P.; Johnson, Brant R.; Barrangou, Rodolphe

2016-01-01

Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in L. acidophilus NCFM, an fbpB-deficient strain was constructed. The L. acidophilus mutant with a deletion of fbpB lost the ability to adhere to mucin and fibronectin in vitro. Homologues of fbpB were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside the L. acidophilus homology group. PMID:26921419

Hybrid proline-rich proteins: novel players in plant cell elongation?

PubMed Central

Dvořáková, Lenka; Srba, Miroslav; Opatrny, Zdenek; Fischer, Lukas

2012-01-01

Background and Aims Hybrid proline-rich proteins (HyPRPs) represent a large family of putative cell-wall proteins characterized by the presence of a variable N-terminal domain and a conserved C-terminal domain that is related to non-specific lipid transfer proteins. The function of HyPRPs remains unclear, but their widespread occurrence and abundant expression patterns indicate that they may be involved in a basic cellular process. Methods To elucidate the cellular function of HyPRPs, we modulated the expression of three HyPRP genes in tobacco (Nicotiana tabacum) BY-2 cell lines and in potato (Solanum tuberosum) plants. Key Results In BY-2 lines, over-expression of the three HyPRP genes with different types of N-terminal domains resulted in similar phenotypic changes, namely increased cell elongation, both in suspension culture and on solid media where the over-expression resulted in enhanced calli size. The over-expressing cells showed increased plasmolysis in a hypertonic mannitol solution and accelerated rate of protoplast release, suggesting loosening of the cell walls. In contrast to BY-2 lines, no phenotypic changes were observed in potato plants over-expressing the same or analogous HyPRP genes, presumably due to more complex compensatory mechanisms in planta. Conclusions Based on the results from BY-2 lines, we propose that HyPRPs, more specifically their C-terminal domains, represent a novel group of proteins involved in cell expansion. PMID:22028464

Characterization of the Drosophila Group Ortholog to the Amino-Terminus of the Alpha-Thalassemia and Mental Retardation X-Linked (ATRX) Vertebrate Protein

PubMed Central

Hernández-Rodríguez, Benjamín; Campos, Adam; Montero, Daniel; Rudiño, Enrique; Vázquez, Martha; Zurita, Mario; Valadez-Graham, Viviana

2014-01-01

The human ATRX gene encodes hATRX, a chromatin-remodeling protein harboring an helicase/ATPase and ADD domains. The ADD domain has two zinc fingers that bind to histone tails and mediate hATRX binding to chromatin. dAtrx, the putative ATRX homolog in Drosophila melanogaster, has a conserved helicase/ATPase domain but lacks the ADD domain. A bioinformatic search of the Drosophila genome using the human ADD sequence allowed us to identify the CG8290 annotated gene, which encodes three ADD harboring- isoforms generated by alternative splicing. This Drosophila ADD domain is highly similar in structure and in the amino acids which mediate the histone tail contacts to the ADD domain of hATRX as shown by 3D modeling. Very recently the CG8290 annotated gene has been named dadd1. We show through pull-down and CoIP assays that the products of the dadd1 gene interact physically with dAtrxL and HP1a and all of them mainly co-localize in the chromocenter, although euchromatic localization can also be observed through the chromosome arms. We confirm through ChIP analyses that these proteins are present in vivo in the same heterochromatic regions. The three isoforms are expressed throughout development. Flies carrying transheterozygous combinations of the dadd1 and atrx alleles are semi-viable and have different phenotypes including the appearance of melanotic masses. Interestingly, the dAdd1-b and c isoforms have extra domains, such as MADF, which suggest newly acquired functions of these proteins. These results strongly support that, in Drosophila, the atrx gene diverged and that the dadd1-encoded proteins participate with dAtrx in some cellular functions such as heterochromatin maintenance. PMID:25437195

A three-domain Kazal-type serine proteinase inhibitor exhibiting domain inhibitory and bacteriostatic activities from freshwater crayfish Procambarus clarkii.

PubMed

Li, Xin-Cang; Wang, Xian-Wei; Wang, Zong-Heng; Zhao, Xiao-Fan; Wang, Jin-Xing

2009-12-01

In crustaceans, Kazal-type serine proteinase inhibitors in hemolymph are believed to function as regulators of the host-defense reactions or inhibitors against proteinases from microorganisms. In this study, we report a Kazal-type serine proteinase inhibitor, named hcPcSPI1, from freshwater crayfish (Procambarus clarkii). We found that hcPcSPI1 is composed of a putative signal peptide, an RGD motif, and three tandem Kazal-type domains with the domain P1 residues L, L and E, respectively. Mainly, hcPcSPI1 was detected in hemocytes as well as in the heart, gills, and intestine at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that hcPcSPI1 in hemocytes was upregulated by the stimulation of Esherichia coli (8099) or became decreased after a white spot syndrome virus (WSSV) challenge. In addition, hcPcSPI1 and its three independent domains were overexpressed and purified to explore their potential functions. All four proteins inhibited subtilisin A and proteinase K, but not alpha-chymotypsin or trypsin. Recombinant hcPcSPI1 could firmly attach to Gram-negative bacteria E. coli and Klebsiella pneumoniae; Gram-positive bacteria Bacillus subtilis, Bacillus thuringiensis and Staphylococcus aureus; fungi Candida albicans and Saccharomyce cerevisiae, and only domain 1 was responsible for the binding to E. coli and S. aureus. In addition, recombinant hcPcSPI1 was also found to possess bacteriostatic activity against the B. subtilis and B. thuringiensis. Domains 2 and 3 contributed mainly to these bacteriostatic activities. All results suggested that hcPcSPI1 might play important roles in the innate immunity of crayfish.

Exploiting Amino Acid Composition for Predicting Protein-Protein Interactions

PubMed Central

Roy, Sushmita; Martinez, Diego; Platero, Harriett; Lane, Terran; Werner-Washburne, Margaret

2009-01-01

Background Computational prediction of protein interactions typically use protein domains as classifier features because they capture conserved information of interaction surfaces. However, approaches relying on domains as features cannot be applied to proteins without any domain information. In this paper, we explore the contribution of pure amino acid composition (AAC) for protein interaction prediction. This simple feature, which is based on normalized counts of single or pairs of amino acids, is applicable to proteins from any sequenced organism and can be used to compensate for the lack of domain information. Results AAC performed at par with protein interaction prediction based on domains on three yeast protein interaction datasets. Similar behavior was obtained using different classifiers, indicating that our results are a function of features and not of classifiers. In addition to yeast datasets, AAC performed comparably on worm and fly datasets. Prediction of interactions for the entire yeast proteome identified a large number of novel interactions, the majority of which co-localized or participated in the same processes. Our high confidence interaction network included both well-studied and uncharacterized proteins. Proteins with known function were involved in actin assembly and cell budding. Uncharacterized proteins interacted with proteins involved in reproduction and cell budding, thus providing putative biological roles for the uncharacterized proteins. Conclusion AAC is a simple, yet powerful feature for predicting protein interactions, and can be used alone or in conjunction with protein domains to predict new and validate existing interactions. More importantly, AAC alone performs at par with existing, but more complex, features indicating the presence of sequence-level information that is predictive of interaction, but which is not necessarily restricted to domains. PMID:19936254

Intracellular Domain Fragment of CD44 Alters CD44 Function in Chondrocytes*

PubMed Central

Mellor, Liliana; Knudson, Cheryl B.; Hida, Daisuke; Askew, Emily B.; Knudson, Warren

2013-01-01

The hyaluronan receptor CD44 undergoes sequential proteolytic cleavage at the cell surface. The initial cleavage of the CD44 extracellular domain is followed by a second intramembranous cleavage of the residual CD44 fragment, liberating the C-terminal cytoplasmic tail of CD44. In this study conditions that promote CD44 cleavage resulted in a diminished capacity to assemble and retain pericellular matrices even though sufficient non-degraded full-length CD44 remained. Using stable and transient overexpression of the cytoplasmic domain of CD44, we determined that the intracellular domain interfered with anchoring of the full-length CD44 to the cytoskeleton and disrupted the ability of the cells to bind hyaluronan and assemble a pericellular matrix. Co-immunoprecipitation assays were used to determine whether the mechanism of this interference was due to competition with actin adaptor proteins. CD44 of control chondrocytes was found to interact and co-immunoprecipitate with both the 65- and 130-kDa isoforms of ankyrin-3. Moreover, this interaction with ankyrin-3 proteins was diminished in cells overexpressing the CD44 intracellular domain. Mutating the putative ankyrin binding site of the transiently transfected CD44 intracellular domain diminished the inhibitory effects of this protein on matrix retention. Although CD44 in other cells types has been shown to interact with members of the ezrin/radixin/moesin (ERM) family of adaptor proteins, only modest interactions between CD44 and moesin could be demonstrated in chondrocytes. The data suggest that release of the CD44 intracellular domain into the cytoplasm of cells such as chondrocytes exerts a competitive or dominant-negative effect on the function of full-length CD44. PMID:23884413

S1-S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation.

PubMed

Groome, James R; Winston, Vern

2013-05-01

The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1-S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1-S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNaV1.4. These show that side chains of putative countercharges in hNaV1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I-III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.

Mining novel effector proteins from the esophageal gland cells of Meloidogyne incognita

PubMed Central

Rutter, William B.; Hewezi, Tarek; Abubucker, Sahar; Maier, Tom R.; Huang, Guozhong; Mitreva, Makedonka; Hussey, Richard S.; Baum, Thomas J.

2014-01-01

Meloidogyne incognita is one of the most economically damaging plant pathogens in agriculture and horticulture. Identifying and characterizing the effector proteins, which M. incognita secretes into its host plants during infection, is an important step towards finding new ways to manage this pest. In this study we have identified the cDNAs for 18 putative effectors, i.e., proteins that have the potential to facilitate M. incognita parasitism of host plants. These putative effectors are secretory proteins that do not contain transmembrane domains and whose genes are specifically expressed in the secretory gland cells of the nematode, indicating that they are likely secreted from the nematode through its stylet. We have determined that in the plant cells, these putative effectors are likely to localize to the cytoplasm. Furthermore, the transcripts of many of these novel effectors are specifically up-regulated during different stages of the nematode’s life cycle, indicating that they function at specific stages during M. incognita parasitism. The predicted proteins showed little to no homology to known proteins from free-living nematode species, suggesting that they evolved recently to support the parasitic lifestyle. On the other hand, several of the effectors are part of gene families within the M. incognita genome as well as that of Meloidogyne hapla, which points to an important role that these putative effectors are playing in both parasites. With the discovery of these putative effectors we have increased our knowledge of the effector repertoire utilized by root-knot nematodes to infect, feed, and reproduce on their host plants. Future studies investigating the roles these proteins play in planta will help mitigate the effects of this damaging pest. PMID:24875667

Mining novel effector proteins from the esophageal gland cells of Meloidogyne incognita.

PubMed

Rutter, William B; Hewezi, Tarek; Abubucker, Sahar; Maier, Tom R; Huang, Guozhong; Mitreva, Makedonka; Hussey, Richard S; Baum, Thomas J

2014-09-01

Meloidogyne incognita is one of the most economically damaging plant pathogens in agriculture and horticulture. Identifying and characterizing the effector proteins which M. incognita secretes into its host plants during infection is an important step toward finding new ways to manage this pest. In this study, we have identified the cDNAs for 18 putative effectors (i.e., proteins that have the potential to facilitate M. incognita parasitism of host plants). These putative effectors are secretory proteins that do not contain transmembrane domains and whose genes are specifically expressed in the secretory gland cells of the nematode, indicating that they are likely secreted from the nematode through its stylet. We have determined that, in the plant cells, these putative effectors are likely to localize to the cytoplasm. Furthermore, the transcripts of many of these novel effectors are specifically upregulated during different stages of the nematode's life cycle, indicating that they function at specific stages during M. incognita parasitism. The predicted proteins showed little to no homology to known proteins from free-living nematode species, suggesting that they evolved recently to support the parasitic lifestyle. On the other hand, several of the effectors are part of gene families within the M. incognita genome as well as that of M. hapla, which points to an important role that these putative effectors are playing in both parasites. With the discovery of these putative effectors, we have increased our knowledge of the effector repertoire utilized by root-knot nematodes to infect, feed on, and reproduce on their host plants. Future studies investigating the roles that these proteins play in planta will help mitigate the effects of this damaging pest.

Putative Porin of Bradyrhizobium sp. (Lupinus) Bacteroids Induced by Glyphosate▿

PubMed Central

de María, Nuria; Guevara, Ángeles; Serra, M. Teresa; García-Luque, Isabel; González-Sama, Alfonso; de Lacoba, Mario García; de Felipe, M. Rosario; Fernández-Pascual, Mercedes

2007-01-01

Application of glyphosate (N-[phosphonomethyl] glycine) to Bradyrhizobium sp. (Lupinus)-nodulated lupin plants caused modifications in the protein pattern of bacteroids. The most significant change was the presence of a 44-kDa polypeptide in bacteroids from plants treated with the higher doses of glyphosate employed (5 and 10 mM). The polypeptide has been characterized by the amino acid sequencing of its N terminus and the isolation and nucleic acid sequencing of its encoding gene. It is putatively encoded by a single gene, and the protein has been identified as a putative porin. Protein modeling revealed the existence of several domains sharing similarity to different porins, such as a transmembrane beta-barrel. The protein has been designated BLpp, for Bradyrhizobium sp. (Lupinus) putative porin, and would be the first porin described in Bradyrhizobium sp. (Lupinus). In addition, a putative conserved domain of porins has been identified which consists of 87 amino acids, located in the BLpp sequence 30 amino acids downstream of the N-terminal region. In bacteroids, mRNA of the BLpp gene shows a basal constitutive expression that increases under glyphosate treatment, and the expression of the gene is seemingly regulated at the transcriptional level. By contrast, in free-living bacteria glyphosate treatment leads to an inhibition of BLpp mRNA accumulation, indicating a different effect of glyphosate on BLpp gene expression in bacteroids and free-living bacteria. The possible role of BLpp in a metabolite interchange between Bradyrhizobium and lupin is discussed. PMID:17557843

Regulation of Cell Diameter, For3p Localization, and Cell Symmetry by Fission Yeast Rho-GAP Rga4p

PubMed Central

Das, Maitreyi; Wiley, David J.; Medina, Saskia; Vincent, Helen A.; Larrea, Michelle; Oriolo, Andrea

2007-01-01

Control of cellular dimensions and cell symmetry are critical for development and differentiation. Here we provide evidence that the putative Rho-GAP Rga4p of Schizosaccharomyces pombe controls cellular dimensions. rga4Δ cells are wider in diameter and shorter in length, whereas Rga4p overexpression leads to reduced diameter of the growing cell tip. Consistent with a negative role in cell growth control, Rga4p protein localizes to the cell sides in a “corset” pattern, and to the nongrowing cell tips. Additionally, rga4Δ cells show an altered growth pattern similar to that observed in mutants of the formin homology protein For3p. Consistent with these observations, Rga4p is required for normal localization of For3p and for normal distribution of the actin cytoskeleton. We show that different domains of the Rga4p protein mediate diverse morphological functions. The C-terminal GAP domain mediates For3p localization to the cell tips and maintains cell diameter. Conversely, overexpression of the N-terminal LIM homology domain of Rga4p promotes actin cable formation in a For3p-dependent manner. Our studies indicate that Rga4p functionally interacts with For3p and has a novel function in the control of cell diameter and cell growth. PMID:17377067

PRRSV strain VR-2332 Nsp2 deletion mutants attenuate clinical symptoms in swine

USDA-ARS?s Scientific Manuscript database

PRRSV nonstructural protein 2 (nsp2) contains a N-terminal cysteine proteinase (PL2) domain, a middle hypervariable region and C-terminal putative transmembrane domain. Prior studies had shown that as much as 403 amino acids could be removed from the hypervariable region without losing virus viabil...

Putative SF2 helicases of the early-branching eukaryote Giardia lamblia are involved in antigenic variation and parasite differentiation into cysts

PubMed Central

2012-01-01

Background Regulation of surface antigenic variation in Giardia lamblia is controlled post-transcriptionally by an RNA-interference (RNAi) pathway that includes a Dicer-like bidentate RNase III (gDicer). This enzyme, however, lacks the RNA helicase domain present in Dicer enzymes from higher eukaryotes. The participation of several RNA helicases in practically all organisms in which RNAi was studied suggests that RNA helicases are potentially involved in antigenic variation, as well as during Giardia differentiation into cysts. Results An extensive in silico analysis of the Giardia genome identified 32 putative Super Family 2 RNA helicases that contain almost all the conserved RNA helicase motifs. Phylogenetic studies and sequence analysis separated them into 22 DEAD-box, 6 DEAH-box and 4 Ski2p-box RNA helicases, some of which are homologs of well-characterized helicases from higher organisms. No Giardia putative helicase was found to have significant homology to the RNA helicase domain of Dicer enzymes. Additionally a series of up- and down-regulated putative RNA helicases were found during encystation and antigenic variation by qPCR experiments. Finally, we were able to recognize 14 additional putative helicases from three different families (RecQ family, Swi2/Snf2 and Rad3 family) that could be considered DNA helicases. Conclusions This is the first comprehensive analysis of the Super Family 2 helicases from the human intestinal parasite G. lamblia. The relative and variable expression of particular RNA helicases during both antigenic variation and encystation agrees with the proposed participation of these enzymes during both adaptive processes. The putatives RNA and DNA helicases identified in this early-branching eukaryote provide initial information regarding the biological role of these enzymes in cell adaptation and differentiation. PMID:23190735

Activity of a C-terminal plant homeodomain (PHD) of Msc1 is essential for function.

PubMed

Qiu, Xinxing; Dul, Barbara E; Walworth, Nancy C

2010-11-19

Msc1, a member of the Jarid1 family of putative histone demethylases, is required for chromosome stability in fission yeast. Msc1 associates with the Swr1 complex that facilitates deposition of histone H2A.Z into chromatin. To assess the function of Msc1 in the Swr1 complex, domains of Msc1 necessary for interaction with Swr1 were identified. The C-terminal plant homeodomain (PHD) 2 and PHD3 of Msc1 are sufficient to confer association with Swr1 and allow Msc1 to function in the context of kinetochore mutants. On the other hand, a mutant with a single amino acid substitution in PHD2 within the full-length Msc1 protein retains the ability to bind to Swr1 but eliminates the function of Msc1 in combination with kinetochore mutants. Thus, Swr1 association is critical but not sufficient for Msc1 function. An activity of Msc1 that depends on the cysteine residue within PHD2 of Msc1 is likewise critical for function. On the basis of our observation that the PHDs of Msc1 act as E3 ubiquitin ligases and that mutations of cysteine residues within those domains abolish ligase activity, we speculate that the ability of Msc1 to facilitate ubiquitin transfer is critical for the function it mediates through its association with Swr1.

Activity of a C-terminal Plant Homeodomain (PHD) of Msc1 Is Essential for Function*

PubMed Central

Qiu, Xinxing; Dul, Barbara E.; Walworth, Nancy C.

2010-01-01

Msc1, a member of the Jarid1 family of putative histone demethylases, is required for chromosome stability in fission yeast. Msc1 associates with the Swr1 complex that facilitates deposition of histone H2A.Z into chromatin. To assess the function of Msc1 in the Swr1 complex, domains of Msc1 necessary for interaction with Swr1 were identified. The C-terminal plant homeodomain (PHD) 2 and PHD3 of Msc1 are sufficient to confer association with Swr1 and allow Msc1 to function in the context of kinetochore mutants. On the other hand, a mutant with a single amino acid substitution in PHD2 within the full-length Msc1 protein retains the ability to bind to Swr1 but eliminates the function of Msc1 in combination with kinetochore mutants. Thus, Swr1 association is critical but not sufficient for Msc1 function. An activity of Msc1 that depends on the cysteine residue within PHD2 of Msc1 is likewise critical for function. On the basis of our observation that the PHDs of Msc1 act as E3 ubiquitin ligases and that mutations of cysteine residues within those domains abolish ligase activity, we speculate that the ability of Msc1 to facilitate ubiquitin transfer is critical for the function it mediates through its association with Swr1. PMID:20858896

The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase.

PubMed

Lerner, D R; Raikhel, N V

1992-06-05

Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.

Comparative genomic analysis of SET domain family reveals the origin, expansion, and putative function of the arthropod-specific SmydA genes as histone modifiers in insects

PubMed Central

Jiang, Feng; Liu, Qing; Wang, Yanli; Zhang, Jie; Wang, Huimin; Song, Tianqi; Yang, Meiling

2017-01-01

Abstract The SET domain is an evolutionarily conserved motif present in histone lysine methyltransferases, which are important in the regulation of chromatin and gene expression in animals. In this study, we searched for SET domain–containing genes (SET genes) in all of the 147 arthropod genomes sequenced at the time of carrying out this experiment to understand the evolutionary history by which SET domains have evolved in insects. Phylogenetic and ancestral state reconstruction analysis revealed an arthropod-specific SET gene family, named SmydA, that is ancestral to arthropod animals and specifically diversified during insect evolution. Considering that pseudogenization is the most probable fate of the new emerging gene copies, we provided experimental and evolutionary evidence to demonstrate their essential functions. Fluorescence in situ hybridization analysis and in vitro methyltransferase activity assays showed that the SmydA-2 gene was transcriptionally active and retained the original histone methylation activity. Expression knockdown by RNA interference significantly increased mortality, implying that the SmydA genes may be essential for insect survival. We further showed predominantly strong purifying selection on the SmydA gene family and a potential association between the regulation of gene expression and insect phenotypic plasticity by transcriptome analysis. Overall, these data suggest that the SmydA gene family retains essential functions that may possibly define novel regulatory pathways in insects. This work provides insights into the roles of lineage-specific domain duplication in insect evolution. PMID:28444351

The poly(C)-binding proteins: a multiplicity of functions and a search for mechanisms.

PubMed Central

Makeyev, Aleksandr V; Liebhaber, Stephen A

2002-01-01

The poly(C) binding proteins (PCBPs) are encoded at five dispersed loci in the mouse and human genomes. These proteins, which can be divided into two groups, hnRNPs K/J and the alphaCPs (alphaCP1-4), are linked by a common evolutionary history, a shared triple KH domain configuration, and by their poly(C) binding specificity. Given these conserved characteristics it is remarkable to find a substantial diversity in PCBP functions. The roles of these proteins in mRNA stabilization, translational activation, and translational silencing suggest a complex and diverse set of post-transcriptional control pathways. Their additional putative functions in transcriptional control and as structural components of important DNA-protein complexes further support their remarkable structural and functional versatility. Clearly the identification of additional binding targets and delineation of corresponding control mechanisms and effector pathways will establish highly informative models for further exploration. PMID:12003487

The poly(C)-binding proteins: a multiplicity of functions and a search for mechanisms.

PubMed

Makeyev, Aleksandr V; Liebhaber, Stephen A

2002-03-01

The poly(C) binding proteins (PCBPs) are encoded at five dispersed loci in the mouse and human genomes. These proteins, which can be divided into two groups, hnRNPs K/J and the alphaCPs (alphaCP1-4), are linked by a common evolutionary history, a shared triple KH domain configuration, and by their poly(C) binding specificity. Given these conserved characteristics it is remarkable to find a substantial diversity in PCBP functions. The roles of these proteins in mRNA stabilization, translational activation, and translational silencing suggest a complex and diverse set of post-transcriptional control pathways. Their additional putative functions in transcriptional control and as structural components of important DNA-protein complexes further support their remarkable structural and functional versatility. Clearly the identification of additional binding targets and delineation of corresponding control mechanisms and effector pathways will establish highly informative models for further exploration.

Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

PubMed

Osato, Naoki

2018-01-19

Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional enrichments were related to the cellular functions. The normalized number of functional enrichments of human putative transcriptional target genes changed according to the criteria of enhancer-promoter assignments and correlated with the median expression level of the target genes. These analyses and characters of human putative transcriptional target genes would be useful to examine the criteria of enhancer-promoter assignments and to predict the novel mechanisms and factors such as DNA binding proteins and DNA sequences of enhancer-promoter interactions.

Structural analysis of the receptor binding domain of botulinum neurotoxin serotype D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanfeng; Buchko, Garry W.; Qin, Lin

2010-10-28

Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65 Å resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences aremore » located at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10 Å relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.« less

Structural Analysis of the Receptor Binding Domain of Botulinum Neurotoxin Serotype D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Y Zhang; G Buchko; L Qin

2011-12-31

Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65{angstrom} resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences are locatedmore » at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10{angstrom} relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.« less

Predictive Structure and Topology of Peroxisomal ATP-Binding Cassette (ABC) Transporters

PubMed Central

Andreoletti, Pierre; Raas, Quentin; Gondcaille, Catherine; Cherkaoui-Malki, Mustapha; Trompier, Doriane; Savary, Stéphane

2017-01-01

The peroxisomal ATP-binding Cassette (ABC) transporters, which are called ABCD1, ABCD2 and ABCD3, are transmembrane proteins involved in the transport of various lipids that allow their degradation inside the organelle. Defective ABCD1 leads to the accumulation of very long-chain fatty acids and is associated with a complex and severe neurodegenerative disorder called X-linked adrenoleukodystrophy (X-ALD). Although the nucleotide-binding domain is highly conserved and characterized within the ABC transporters family, solid data are missing for the transmembrane domain (TMD) of ABCD proteins. The lack of a clear consensus on the secondary and tertiary structure of the TMDs weakens any structure-function hypothesis based on the very diverse ABCD1 mutations found in X-ALD patients. Therefore, we first reinvestigated thoroughly the structure-function data available and performed refined alignments of ABCD protein sequences. Based on the 2.85  Å resolution crystal structure of the mitochondrial ABC transporter ABCB10, here we propose a structural model of peroxisomal ABCD proteins that specifies the position of the transmembrane and coupling helices, and highlight functional motifs and putative important amino acid residues. PMID:28737695

Low Temperature Extends the Lifespan of Bursaphelenchus xylophilus through the cGMP Pathway

PubMed Central

Wang, Bowen; Ma, Ling; Wang, Feng; Wang, Buyong; Hao, Xin; Xu, Jiayao; Ma, Yan

2017-01-01

The causal agent of pine wilt disease, pine wood nematode (PWN) (Bursaphelenchus xylophilus), revealed extended lifespan at low temperature. To discover the molecular mechanism of this phenomenon, we attempted to study the molecular characterization, transcript abundance, and functions of three genes of the cyclic guanosine monophosphate (cGMP) pathway from B. xylophilus. Three cGMP pathway genes were identified from B. xylophilus. Bioinformatic software was utilized to analyze the characteristics of the three putative proteins. Function of the three genes in cold tolerance was studied with RNA interference (RNAi). The results showed that the deduced protein of Bx-DAF-11 has an adenylate and guanylate cyclase catalytic domain, indicating an ability to bind to extracellular ligands and synthesizing cGMP. Both Bx-TAX-2 and Bx-TAX-4 have cyclic nucleotide-binding domains and ion transport protein domains, illustrating that they are cGMP-gated ion channels. The transcript level of Bx-daf-11, Bx-tax-2, and Bx-tax-4 increased at low temperature. The survival rates of three gene silenced B. xylophilus revealed a significant decrease at low temperature. This study illustrated that the cGMP pathway plays a key role in low-temperature-induced lifespan extension in B. xylophilus. PMID:29099744

Cell envelope stress in mycobacteria is regulated by the novel signal transduction ATPase IniR in response to trehalose

PubMed Central

van Winden, Vincent J. C.; Sparrius, Marion; van de Weerd, Robert; Speer, Alexander; Ummels, Roy; Sherman, David R.

2017-01-01

The cell envelope of mycobacteria is a highly unique and complex structure that is functionally equivalent to that of Gram-negative bacteria to protect the bacterial cell. Defects in the integrity or assembly of this cell envelope must be sensed to allow the induction of stress response systems. The promoter that is specifically and most strongly induced upon exposure to ethambutol and isoniazid, first line drugs that affect cell envelope biogenesis, is the iniBAC promoter. In this study, we set out to identify the regulator of the iniBAC operon in Mycobacterium marinum using an unbiased transposon mutagenesis screen in a constitutively iniBAC-expressing mutant background. We obtained multiple mutants in the mce1 locus as well as mutants in an uncharacterized putative transcriptional regulator (MMAR_0612). This latter gene was shown to function as the iniBAC regulator, as overexpression resulted in constitutive iniBAC induction, whereas a knockout mutant was unable to respond to the presence of ethambutol and isoniazid. Experiments with the M. tuberculosis homologue (Rv0339c) showed identical results. RNAseq experiments showed that this regulatory gene was exclusively involved in the regulation of the iniBAC operon. We therefore propose to name this dedicated regulator iniBAC Regulator (IniR). IniR belongs to the family of signal transduction ATPases with numerous domains, including a putative sugar-binding domain. Upon testing different sugars, we identified trehalose as an activator and metabolic cue for iniBAC activation, which could also explain the effect of the mce1 mutations. In conclusion, cell envelope stress in mycobacteria is regulated by IniR in a cascade that includes trehalose. PMID:29281637

Sequence characterization of S100A8 gene reveals structural differences of protein and transcriptional factor binding sites in water buffalo and yak.

PubMed

Kathiravan, P; Goyal, S; Kataria, R S; Mishra, B P; Jayakumar, S; Joshi, B K

2011-01-01

The present study was undertaken to characterize the structure of S100A8 gene and its promoter in water buffalo and yak. Sequence data of 2.067 kb, 2.071 kb, and 2.052 kb with respect to complete S100A8 gene including 5' flanking region was generated in river buffalo, swamp buffalo, and yak, respectively. BLAST analysis of coding DNA sequences (CDS) of S100A8 gene revealed 95% homology of buffalo sequence with cattle, 85% with pig and horse, 83% with dog, 72-73% with murines, and around 79% with primates and humans. Phylogenetic analysis of predicted CDS revealed distinct clustering of murines, primates, and domestic animals with bovines and bubalines forming a subcluster among farm animals. In silico translation of predicted CDS revealed a sequence of 89 amino acids with 7 amino acid changes between cattle and buffalo and 2 changes between cattle and yak. The search for Pfam family revealed the N-terminal calcium binding domain and the noncanonical EF hand domain in the carboxy terminus, with more variations being observed in the N-terminal domain among different species. Two amino acid changes observed in carboxy terminal EF hand domain resulted in altered secondary structure of yak S100A8 protein. Analysis of S100A8 gene promoter revealed 14 putative motifs for transcriptional factor binding sites. Two putative motifs viz. C/EBP and v-Myb were found to be absent in swamp buffalo as compared to river buffalo and cattle. Differences in the structure of S100A8 protein and the transcriptional factor binding sites identified in the present study need to be analyzed further for their functional significance in yak and swamp buffalo respectively. Copyright © Taylor & Francis Group, LLC

Mosaic genome of endobacteria in arbuscular mycorrhizal fungi: Transkingdom gene transfer in an ancient mycoplasma-fungus association.

PubMed

Torres-Cortés, Gloria; Ghignone, Stefano; Bonfante, Paola; Schüßler, Arthur

2015-06-23

For more than 450 million years, arbuscular mycorrhizal fungi (AMF) have formed intimate, mutualistic symbioses with the vast majority of land plants and are major drivers in almost all terrestrial ecosystems. The obligate plant-symbiotic AMF host additional symbionts, so-called Mollicutes-related endobacteria (MRE). To uncover putative functional roles of these widespread but yet enigmatic MRE, we sequenced the genome of DhMRE living in the AMF Dentiscutata heterogama. Multilocus phylogenetic analyses showed that MRE form a previously unidentified lineage sister to the hominis group of Mycoplasma species. DhMRE possesses a strongly reduced metabolic capacity with 55% of the proteins having unknown function, which reflects unique adaptations to an intracellular lifestyle. We found evidence for transkingdom gene transfer between MRE and their AMF host. At least 27 annotated DhMRE proteins show similarities to nuclear-encoded proteins of the AMF Rhizophagus irregularis, which itself lacks MRE. Nuclear-encoded homologs could moreover be identified for another AMF, Gigaspora margarita, and surprisingly, also the non-AMF Mortierella verticillata. Our data indicate a possible origin of the MRE-fungus association in ancestors of the Glomeromycota and Mucoromycotina. The DhMRE genome encodes an arsenal of putative regulatory proteins with eukaryotic-like domains, some of them encoded in putative genomic islands. MRE are highly interesting candidates to study the evolution and interactions between an ancient, obligate endosymbiotic prokaryote with its obligate plant-symbiotic fungal host. Our data moreover may be used for further targeted searches for ancient effector-like proteins that may be key components in the regulation of the arbuscular mycorrhiza symbiosis.

Nonagonal cadherins: A new protein family found within the Stramenopiles.

PubMed

Fletcher, Kyle I G; van West, Pieter; Gachon, Claire M M

2016-11-15

Cadherins, a group of molecules typically associated with planar cell polarity and Wnt signalling, have been little reported outside of the animal kingdom. Here, we identify a new family of cadherins in the Stramenopiles, termed Nonagonal after their 9 transmembrane passes, which contrast to the one or seven passes found in other known cadherin families. Manual curation and experimental validation reveal two subclasses of nonagonal cadherins, depending on the number of uninterrupted extracellular cadherin (EC) modules presented. Firstly, shorter mono-exonic, unimodular, protein models, with 3 to 12 EC domains occur as duplicate paralogs in the saprotrophic Labyrinthulomycetes Aurantiochytrium limanicum and Schizochytrium aggregatum, the gastrointestinal Blastocystis hominis (Blastocystae) and as a single copy gene in the autotrophic Pelagophyte Aureococcus anophagefferens. Larger, single copy, multi-exonal, tri-modular protein models, with up to 72 EC domain in total, are found in the Oomycete genera Albugo, Phytophthora, Pythium and Eurychasma. No homolog was found in the closely related autotrophic Phaeophyceae (brown algae) or Bacillariophyceae (diatoms), nor in several genera of plant and animal pathogenic oomycetes (Aphanomyces, Saprolegnia and Hyaloperonospora). This potential absence was further investigated by synteny analysis of the genome regions flanking the cadherin gene models, which are found to be highly variable. Novel to this new cadherin family is the presence of intercalated laminin and putative carbohydrate binding in tri-modular oomycete cadherins and at the N-terminus of thraustochytrid proteins. As we were unable to detect any homologs of proteins involved in signalling pathways where other cadherin families are involved, we present a conceptual hypothesis on the function of nonagonal cadherin based around the presence of putative carbohydrate binding domains. Copyright © 2016. Published by Elsevier B.V.

A Novel N-Acetylglutamate Synthase Architecture Revealed by the Crystal Structure of the Bifunctional Enzyme from Maricaulis maris

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Dashuang; Li, Yongdong; Cabrera-Luque, Juan

2012-05-24

Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 {angstrom} resolution indicates that it is a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. The quaternary structure of crystalline NAGS/K from Xanthomonas campestris (xcNAGS/K) is similar, and cross-linking experiments indicate that both mmNAGS/K and xcNAGS aremore » tetramers in solution. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N-acetylglutamate kinase (NAGK) activity and has a putative arginine binding site, and an N-acetyltransferase (NAT) domain that contains the putative NAGS active site. These structures and sequence comparisons suggest that the linker residue 291 may determine whether arginine acts as an allosteric inhibitor or activator in homologous enzymes in microorganisms and vertebrates. In addition, the angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26{sup o} is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS-deficient patients.« less

One precursor, three apolipoproteins: the relationship between two crustacean lipoproteins, the large discoidal lipoprotein and the high density lipoprotein/β-glucan binding protein.

PubMed

Stieb, Stefanie; Roth, Ziv; Dal Magro, Christina; Fischer, Sabine; Butz, Eric; Sagi, Amir; Khalaila, Isam; Lieb, Bernhard; Schenk, Sven; Hoeger, Ulrich

2014-12-01

The novel discoidal lipoprotein (dLp) recently detected in the crayfish, differs from other crustacean lipoproteins in its large size, apoprotein composition and high lipid binding capacity, We identified the dLp sequence by transcriptome analyses of the hepatopancreas and mass spectrometry. Further de novo assembly of the NGS data followed by BLAST searches using the sequence of the high density lipoprotein/1-glucan binding protein (HDL-BGBP) of Astacus leptodactylus as query revealed a putative precursor molecule with an open reading frame of 14.7 kb and a deduced primary structure of 4889 amino acids. The presence of an N-terminal lipid bind- ing domain and a DUF 1943 domain suggests the relationship with the large lipid transfer proteins. Two-putative dibasic furin cleavage sites were identified bordering the sequence of the HDL-BGBP. When subjected to mass spectroscopic analyses, tryptic peptides of the large apoprotein of dLp matched the N-terminal part of the precursor, while the peptides obtained for its small apoprotein matched the C-terminal part. Repeating the analysis in the prawn Macrobrachium rosenbergii revealed a similar protein with identical domain architecture suggesting that our findings do not represent an isolated instance. Our results indicate that the above three apolipoproteins (i.e HDL-BGBP and both the large and the small subunit of dLp) are translated as a large precursor. Cleavage at the furin type sites releases two subunits forming a heterodimeric dLP particle, while the remaining part forms an HDL-BGBP whose relationship with other lipoproteins as well as specific functions are yet to be elucidated.

Structure of N-acetyl-[beta]-D-glucosaminidase (GcnA) from the Endocarditis Pathogen Streptococcus gordonii and its Complex with the Mechanism-based Inhibitor NAG-thiazoline

DOE Office of Scientific and Technical Information (OSTI.GOV)

Langley, David B.; Harty, Derek W.S.; Jacques, Nicholas A.

2008-09-17

The crystal structure of GcnA, an N-acetyl-{beta}-D-glucosaminidase from Streptococcus gordonii, was solved by multiple wavelength anomalous dispersion phasing using crystals of selenomethionine-substituted protein. GcnA is a homodimer with subunits each comprised of three domains. The structure of the C-terminal {alpha}-helical domain has not been observed previously and forms a large dimerization interface. The fold of the N-terminal domain is observed in all structurally related glycosidases although its function is unknown. The central domain has a canonical ({beta}/{alpha}){sub 8} TIM-barrel fold which harbours the active site. The primary sequence and structure of this central domain identifies the enzyme as a familymore » 20 glycosidase. Key residues implicated in catalysis have different conformations in two different crystal forms, which probably represent active and inactive conformations of the enzyme. The catalytic mechanism for this class of glycoside hydrolase, where the substrate rather than the enzyme provides the cleavage-inducing nucleophile, has been confirmed by the structure of GcnA complexed with a putative reaction intermediate analogue, N-acetyl-{beta}-D-glucosamine-thiazoline. The catalytic mechanism is discussed in light of these and other family 20 structures.« less

The prion-like domain of FUS is multiphosphorylated following DNA damage without altering nuclear localization.

PubMed

Rhoads, Shannon N; Monahan, Zachary T; Yee, Debra S; Leung, Andrew Y; Newcombe, Cameron G; O'Meally, Robert N; Cole, Robert N; Shewmaker, Frank P

2018-06-13

FUS is an abundant, predominantly nuclear protein involved in RNA processing. Under various conditions, FUS functionally associates with RNA and other macromolecules to form distinct, reversible phase-separated liquid structures. Persistence of the phase-separated state and increased cytoplasmic localization are both hypothesized to predispose FUS to irreversible aggregation, which is a pathological hallmark of subtypes of amyotrophic lateral sclerosis and frontotemporal dementia. We previously showed that phosphorylation of FUS's prion-like domain suppressed phase separation and toxic aggregation, proportionally to the number of added phosphates. However, phosphorylation of FUS's prion-like domain was previously reported to promote its cytoplasmic localization, potentially favoring pathological behavior. Here, we used mass spectrometry and human cell models to further identify phosphorylation sites within FUS's prion-like domain, specifically following DNA-damaging stress. In total, 28 putative sites have been identified, about half of which are DNA-dependent protein kinase (DNA-PK) consensus sites. Custom antibodies were developed to confirm the phosphorylation of two of these sites (Ser26 and Ser30). Both sites were usually phosphorylated in a sub-population of cellular FUS following a variety of DNA-damaging stresses, but not necessarily equally or simultaneously. Importantly, we found DNA-PK-dependent multi-phosphorylation of FUS's prion-like domain does not cause cytoplasmic localization.

Structural mechanism of voltage-dependent gating in an isolated voltage-sensing domain.

PubMed

Li, Qufei; Wanderling, Sherry; Paduch, Marcin; Medovoy, David; Singharoy, Abhishek; McGreevy, Ryan; Villalba-Galea, Carlos A; Hulse, Raymond E; Roux, Benoît; Schulten, Klaus; Kossiakoff, Anthony; Perozo, Eduardo

2014-03-01

The transduction of transmembrane electric fields into protein motion has an essential role in the generation and propagation of cellular signals. Voltage-sensing domains (VSDs) carry out these functions through reorientations of positive charges in the S4 helix. Here, we determined crystal structures of the Ciona intestinalis VSD (Ci-VSD) in putatively active and resting conformations. S4 undergoes an ~5-Å displacement along its main axis, accompanied by an ~60° rotation. This movement is stabilized by an exchange in countercharge partners in helices S1 and S3 that generates an estimated net charge transfer of ~1 eo. Gating charges move relative to a ''hydrophobic gasket' that electrically divides intra- and extracellular compartments. EPR spectroscopy confirms the limited nature of S4 movement in a membrane environment. These results provide an explicit mechanism for voltage sensing and set the basis for electromechanical coupling in voltage-dependent enzymes and ion channels.

Cryo-electron microscopy structure of the TRPV2 ion channel.

PubMed

Zubcevic, Lejla; Herzik, Mark A; Chung, Ben C; Liu, Zhirui; Lander, Gabriel C; Lee, Seok-Yong

2016-02-01

Transient receptor potential vanilloid (TRPV) cation channels are polymodal sensors involved in a variety of physiological processes. TRPV2, a member of the TRPV family, is regulated by temperature, by ligands, such as probenecid and cannabinoids, and by lipids. TRPV2 has been implicated in many biological functions, including somatosensation, osmosensation and innate immunity. Here we present the atomic model of rabbit TRPV2 in its putative desensitized state, as determined by cryo-EM at a nominal resolution of ∼4 Å. In the TRPV2 structure, the transmembrane segment 6 (S6), which is involved in gate opening, adopts a conformation different from the one observed in TRPV1. Structural comparisons of TRPV1 and TRPV2 indicate that a rotation of the ankyrin-repeat domain is coupled to pore opening via the TRP domain, and this pore opening can be modulated by rearrangements in the secondary structure of S6.

Cryo-electron microscopy structure of the TRPV2 ion channel

PubMed Central

Chung, Ben C; Liu, Zhirui; Lander, Gabriel C; Lee, Seok-Yong

2016-01-01

Transient receptor potential vanilloid (TRPV) cation channels are polymodal sensors involved in a variety of physiological processes. TRPV2, a member of the TRPV family, is regulated by temperature, by ligands, such as probenecid and cannabinoids, and by lipids. TRPV2 has been implicated in many biological functions, including somatosensation, osmosensation and innate immunity. Here we present the atomic model of rabbit TRPV2 in its putative desensitized state, as determined by cryo-EM at a nominal resolution of ~4 Å. In the TRPV2 structure, the transmembrane segment 6 (S6), which is involved in gate opening, adopts a conformation different from the one observed in TRPV1. Structural comparisons of TRPV1 and TRPV2 indicate that a rotation of the ankyrin-repeat domain is coupled to pore opening via the TRP domain, and this pore opening can be modulated by rearrangements in the secondary structure of S6. PMID:26779611

A genome-wide 20 K citrus microarray for gene expression analysis

PubMed Central

Martinez-Godoy, M Angeles; Mauri, Nuria; Juarez, Jose; Marques, M Carmen; Santiago, Julia; Forment, Javier; Gadea, Jose

2008-01-01

Background Understanding of genetic elements that contribute to key aspects of citrus biology will impact future improvements in this economically important crop. Global gene expression analysis demands microarray platforms with a high genome coverage. In the last years, genome-wide EST collections have been generated in citrus, opening the possibility to create new tools for functional genomics in this crop plant. Results We have designed and constructed a publicly available genome-wide cDNA microarray that include 21,081 putative unigenes of citrus. As a functional companion to the microarray, a web-browsable database [1] was created and populated with information about the unigenes represented in the microarray, including cDNA libraries, isolated clones, raw and processed nucleotide and protein sequences, and results of all the structural and functional annotation of the unigenes, like general description, BLAST hits, putative Arabidopsis orthologs, microsatellites, putative SNPs, GO classification and PFAM domains. We have performed a Gene Ontology comparison with the full set of Arabidopsis proteins to estimate the genome coverage of the microarray. We have also performed microarray hybridizations to check its usability. Conclusion This new cDNA microarray replaces the first 7K microarray generated two years ago and allows gene expression analysis at a more global scale. We have followed a rational design to minimize cross-hybridization while maintaining its utility for different citrus species. Furthermore, we also provide access to a website with full structural and functional annotation of the unigenes represented in the microarray, along with the ability to use this site to directly perform gene expression analysis using standard tools at different publicly available servers. Furthermore, we show how this microarray offers a good representation of the citrus genome and present the usefulness of this genomic tool for global studies in citrus by using it to catalogue genes expressed in citrus globular embryos. PMID:18598343

Predicting the Impact of Alternative Splicing on Plant MADS Domain Protein Function

PubMed Central

Severing, Edouard I.; van Dijk, Aalt D. J.; Morabito, Giuseppa; Busscher-Lange, Jacqueline; Immink, Richard G. H.; van Ham, Roeland C. H. J.

2012-01-01

Several genome-wide studies demonstrated that alternative splicing (AS) significantly increases the transcriptome complexity in plants. However, the impact of AS on the functional diversity of proteins is difficult to assess using genome-wide approaches. The availability of detailed sequence annotations for specific genes and gene families allows for a more detailed assessment of the potential effect of AS on their function. One example is the plant MADS-domain transcription factor family, members of which interact to form protein complexes that function in transcription regulation. Here, we perform an in silico analysis of the potential impact of AS on the protein-protein interaction capabilities of MIKC-type MADS-domain proteins. We first confirmed the expression of transcript isoforms resulting from predicted AS events. Expressed transcript isoforms were considered functional if they were likely to be translated and if their corresponding AS events either had an effect on predicted dimerisation motifs or occurred in regions known to be involved in multimeric complex formation, or otherwise, if their effect was conserved in different species. Nine out of twelve MIKC MADS-box genes predicted to produce multiple protein isoforms harbored putative functional AS events according to those criteria. AS events with conserved effects were only found at the borders of or within the K-box domain. We illustrate how AS can contribute to the evolution of interaction networks through an example of selective inclusion of a recently evolved interaction motif in the MADS AFFECTING FLOWERING1-3 (MAF1–3) subclade. Furthermore, we demonstrate the potential effect of an AS event in SHORT VEGETATIVE PHASE (SVP), resulting in the deletion of a short sequence stretch including a predicted interaction motif, by overexpression of the fully spliced and the alternatively spliced SVP transcripts. For most of the AS events we were able to formulate hypotheses about the potential impact on the interaction capabilities of the encoded MIKC proteins. PMID:22295091

Identification of Plasmodium falciparum DNA Repair Protein Mre11 with an Evolutionarily Conserved Nuclease Function

PubMed Central

Badugu, Sugith Babu; Nabi, Shaik Abdul; Vaidyam, Pratap; Laskar, Shyamasree; Bhattacharyya, Sunanda; Bhattacharyya, Mrinal Kanti

2015-01-01

The eukaryotic Meiotic Recombination protein 11 (Mre11) plays pivotal roles in the DNA damage response (DDR). Specifically, Mre11 senses and signals DNA double strand breaks (DSB) and facilitates their repair through effector proteins belonging to either homologous recombination (HR) or non-homologous end joining (NHEJ) repair mechanisms. In the human malaria parasite Plasmodium falciparum, HR and alternative-NHEJ have been identified; however, little is known about the upstream factors involved in the DDR of this organism. In this report, we identify a putative ortholog of Mre11 in P. falciparum (PfalMre11) that shares 22% sequence similarity to human Mre11. Homology modeling reveals striking structural resemblance of the predicted PfalMre11 nuclease domain to the nuclease domain of Saccharomyces cerevisiae Mre11 (ScMre11). Complementation analyses reveal functional conservation of PfalMre11 nuclease activity as demonstrated by the ability of the PfalMre11 nuclease domain, in conjunction with the C-terminal domain of ScMre11, to functionally complement an mre11 deficient yeast strain. Functional complementation was virtually abrogated by an amino acid substitution in the PfalMre11 nuclease domain (D398N). PfalMre11 is abundant in the mitotically active trophozoite and schizont stages of P. falciparum and is up-regulated in response to DNA damage, suggesting a role in the DDR. PfalMre11 exhibits physical interaction with PfalRad50. In addition, yeast 2-hybrid studies show that PfalMre11 interacts with ScRad50 and ScXrs2, two important components of the well characterized Mre11-Rad50-Xrs2 complex which is involved in DDR signaling and repair in S. cerevisiae, further supporting a role for PfalMre11 in the DDR. Taken together, these findings provide evidence that PfalMre11 is an evolutionarily conserved component of the DDR in Plasmodium. PMID:25938776

Disease-Associated Mutations in CEP120 Destabilize the Protein and Impair Ciliogenesis.

PubMed

Joseph, Nimesh; Al-Jassar, Caezar; Johnson, Christopher M; Andreeva, Antonina; Barnabas, Deepak D; Freund, Stefan M V; Gergely, Fanni; van Breugel, Mark

2018-05-29

Ciliopathies are a group of genetic disorders caused by a failure to form functional cilia. Due to a lack of structural information, it is currently poorly understood how ciliopathic mutations affect protein functionality to give rise to the underlying disease. Using X-ray crystallography, we show that the ciliopathy-associated centriolar protein CEP120 contains three C2 domains. The point mutations V194A and A199P, which cause Joubert syndrome (JS) and Jeune asphyxiating thoracic dystrophy (JATD), respectively, both reduce the thermostability of the second C2 domain by targeting residues that point toward its hydrophobic core. Genome-engineered cells homozygous for these mutations have largely normal centriole numbers but show reduced CEP120 levels, compromised recruitment of distal centriole markers, and deficient cilia formation. Our results provide insight into the disease mechanism of two ciliopathic mutations in CEP120, identify putative binding partners of CEP120 C2B, and suggest a complex genotype-phenotype relation of the CEP120 ciliopathy alleles. Copyright © 2018 MRC Laboratory of Molecular Biology. Published by Elsevier Inc. All rights reserved.

Putative Nonribosomal Peptide Synthetase and Cytochrome P450 Genes Responsible for Tentoxin Biosynthesis in Alternaria alternata ZJ33.

PubMed

Li, You-Hai; Han, Wen-Jin; Gui, Xi-Wu; Wei, Tao; Tang, Shuang-Yan; Jin, Jian-Ming

2016-08-02

Tentoxin, a cyclic tetrapeptide produced by several Alternaria species, inhibits the F₁-ATPase activity of chloroplasts, resulting in chlorosis in sensitive plants. In this study, we report two clustered genes, encoding a putative non-ribosome peptide synthetase (NRPS) TES and a cytochrome P450 protein TES1, that are required for tentoxin biosynthesis in Alternaria alternata strain ZJ33, which was isolated from blighted leaves of Eupatorium adenophorum. Using a pair of primers designed according to the consensus sequences of the adenylation domain of NRPSs, two fragments containing putative adenylation domains were amplified from A. alternata ZJ33, and subsequent PCR analyses demonstrated that these fragments belonged to the same NRPS coding sequence. With no introns, TES consists of a single 15,486 base pair open reading frame encoding a predicted 5161 amino acid protein. Meanwhile, the TES1 gene is predicted to contain five introns and encode a 506 amino acid protein. The TES protein is predicted to be comprised of four peptide synthase modules with two additional N-methylation domains, and the number and arrangement of the modules in TES were consistent with the number and arrangement of the amino acid residues of tentoxin, respectively. Notably, both TES and TES1 null mutants generated via homologous recombination failed to produce tentoxin. This study provides the first evidence concerning the biosynthesis of tentoxin in A. alternata.

Loss of function of Saccharomyces cerevisiae kinesin-related CIN8 and KIP1 is suppressed by KAR3 motor domain mutations.

PubMed

Hoyt, M A; He, L; Totis, L; Saunders, W S

1993-09-01

The kinesin-related products of the CIN8 and KIP1 genes of Saccharomyces cerevisiae redundantly perform an essential function in mitosis. The action of either gene-product is required for an outwardly directed force that acts upon the spindle poles. We have selected mutations that suppress the temperature-sensitivity of a cin8-temperature-sensitive kip1-delta strain. The extragenic suppressors analyzed were all found to be alleles of the KAR3 gene. KAR3 encodes a distinct kinesin-related protein whose action antagonizes Cin8p/Kip1p function. All seven alleles analyzed were altered within the region of KAR3 that encodes the putative force-generating (or "motor") domain. These mutations also suppressed the inviability associated with the cin8-delta kip1-delta genotype, a property not shared by a deletion of KAR3. Other properties of the suppressing alleles revealed that they were not null for function. Six of the seven were unaffected for the essential karyogamy and meiosis properties of KAR3 and the seventh was dominant for the suppressing trait. Our findings suggest that despite an antagonistic relationship between Cin8p/Kip1p and Kar3p, aspects of their mitotic roles may be similar.

Evolution of the SOUL Heme-Binding Protein Superfamily Across Eukarya.

PubMed

Fortunato, Antonio Emidio; Sordino, Paolo; Andreakis, Nikos

2016-06-01

SOUL homologs constitute a heme-binding protein superfamily putatively involved in heme and tetrapyrrole metabolisms associated with a number of physiological processes. Despite their omnipresence across the tree of life and the biochemical characterization of many SOUL members, their functional role and the evolutionary events leading to such remarkable protein repertoire still remain cryptic. To explore SOUL evolution, we apply a computational phylogenetic approach, including a relevant number of SOUL homologs, to identify paralog forms and reconstruct their genealogy across the tree of life and within species. In animal lineages, multiple gene duplication or loss events and paralog functional specializations underlie SOUL evolution from the dawn of ancestral echinoderm and mollusc SOUL forms. In photosynthetic organisms, SOUL evolution is linked to the endosymbiosis events leading to plastid acquisition in eukaryotes. Derivative features, such as the F2L peptide and BH3 domain, evolved in vertebrates and provided innovative functionality to support immune response and apoptosis. The evolution of elements such as the N-terminal protein domain DUF2358, the His42 residue, or the tetrapyrrole heme-binding site is modern, and their functional implications still unresolved. This study represents the first in-depth analysis of SOUL protein evolution and provides novel insights in the understanding of their obscure physiological role.

Transcriptional Profiles of Mating-Responsive Genes from Testes and Male Accessory Glands of the Mediterranean Fruit Fly, Ceratitis capitata

PubMed Central

Scolari, Francesca; Gomulski, Ludvik M.; Ribeiro, José M. C.; Siciliano, Paolo; Meraldi, Alice; Falchetto, Marco; Bonomi, Angelica; Manni, Mosè; Gabrieli, Paolo; Malovini, Alberto; Bellazzi, Riccardo; Aksoy, Serap; Gasperi, Giuliano; Malacrida, Anna R.

2012-01-01

Background Insect seminal fluid is a complex mixture of proteins, carbohydrates and lipids, produced in the male reproductive tract. This seminal fluid is transferred together with the spermatozoa during mating and induces post-mating changes in the female. Molecular characterization of seminal fluid proteins in the Mediterranean fruit fly, Ceratitis capitata, is limited, although studies suggest that some of these proteins are biologically active. Methodology/Principal Findings We report on the functional annotation of 5914 high quality expressed sequence tags (ESTs) from the testes and male accessory glands, to identify transcripts encoding putative secreted peptides that might elicit post-mating responses in females. The ESTs were assembled into 3344 contigs, of which over 33% produced no hits against the nr database, and thus may represent novel or rapidly evolving sequences. Extraction of the coding sequences resulted in a total of 3371 putative peptides. The annotated dataset is available as a hyperlinked spreadsheet. Four hundred peptides were identified with putative secretory activity, including odorant binding proteins, protease inhibitor domain-containing peptides, antigen 5 proteins, mucins, and immunity-related sequences. Quantitative RT-PCR-based analyses of a subset of putative secretory protein-encoding transcripts from accessory glands indicated changes in their abundance after one or more copulations when compared to virgin males of the same age. These changes in abundance, particularly evident after the third mating, may be related to the requirement to replenish proteins to be transferred to the female. Conclusions/Significance We have developed the first large-scale dataset for novel studies on functions and processes associated with the reproductive biology of Ceratitis capitata. The identified genes may help study genome evolution, in light of the high adaptive potential of the medfly. In addition, studies of male recovery dynamics in terms of accessory gland gene expression profiles and correlated remating inhibition mechanisms may permit the improvement of pest management approaches. PMID:23071645

Functional Analysis of Vaccinia Virus B5R Protein: Essential Role in Virus Envelopment Is Independent of a Large Portion of the Extracellular Domain

PubMed Central

Herrera, Elizabeth; del Mar Lorenzo, María; Blasco, Rafael; Isaacs, Stuart N.

1998-01-01

Vaccinia virus has two forms of infectious virions: the intracellular mature virus and the extracellular enveloped virus (EEV). EEV is critical for cell-to-cell and long-range spread of the virus. The B5R open reading frame (ORF) encodes a membrane protein that is essential for EEV formation. Deletion of the B5R ORF results in a dramatic reduction of EEV, and as a consequence, the virus produces small plaques in vitro and is highly attenuated in vivo. The extracellular portion of B5R is composed mainly of four domains that are similar to the short consensus repeats (SCRs) present in complement regulatory proteins. To determine the contribution of these putative SCR domains to EEV formation, we constructed recombinant vaccinia viruses that replaced the wild-type B5R gene with a mutated gene encoding a B5R protein lacking the SCRs. The resulting recombinant viruses produced large plaques, indicating efficient cell-to-cell spread in vitro, and gradient centrifugation of supernatants from infected cells confirmed that EEV was formed. In contrast, phalloidin staining of infected cells showed that the virus lacking the SCR domains was deficient in the induction of thick actin bundles. Thus, the highly conserved SCR domains present in the extracellular portion of the B5R protein are dispensable for EEV formation. This indicates that the B5R protein is a key viral protein with multiple functions in the process of virus envelopment and release. In addition, given the similarity of the extracellular domain to complement control proteins, the B5R protein may be involved in viral evasion from host immune responses. PMID:9420227

Structural Analysis of Peptide-Analogues of Human Zona Pellucida ZP1 Protein with Amyloidogenic Properties: Insights into Mammalian Zona Pellucida Formation

PubMed Central

Louros, Nikolaos N.; Iconomidou, Vassiliki A.; Giannelou, Polina; Hamodrakas, Stavros J.

2013-01-01

Zona pellucida (ZP) is an extracellular matrix surrounding and protecting mammalian and fish oocytes, which is responsible for sperm binding. Mammalian ZP consists of three to four glycoproteins, called ZP1, ZP2, ZP3, ZP4. These proteins polymerize into long interconnected filaments, through a common structural unit, known as the ZP domain, which consists of two domains, ZP-N and ZP-C. ZP is related in function to silkmoth chorion and in an evolutionary fashion to the teleostean fish chorion, also fibrous structures protecting the oocyte and embryo, that both have been proven to be functional amyloids. Two peptides were predicted as ‘aggregation-prone’ by our prediction tool, AMYLPRED, from the sequence of the human ZP1-N domain. Here, we present results from transmission electron microscopy, X-ray diffraction, Congo red staining and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR FT-IR), of two synthetic peptide-analogues of these predicted ‘aggregation-prone’ parts of the human ZP1-N domain, that we consider crucial for ZP protein polymerization, showing that they both self-assemble into amyloid-like fibrils. Based on our experimental data, we propose that human ZP (hZP) might be considered as a novel, putative, natural protective amyloid, in close analogy to silkmoth and teleostean fish chorions. Experiments are in progress to verify this proposal. We also attempt to provide insights into ZP formation, proposing a possible model for hZP1-N domain polymerization. PMID:24069181

Olfactory Ionotropic Receptors in Mosquito Aedes albopictus (Diptera: Culicidae).

PubMed

Chen, Qian; Man, Yahui; Li, Jianyong; Pei, Di; Wu, Wenjian

2017-09-01

Ionotropic glutamate receptors (iGluRs) are a conserved family of ligand-gated ion channels that primarily function to mediate neuronal communication at synapses. A variant subfamily of iGluRs, the ionotropic receptors (IRs), was recently identified in insects and proved with the function in odorant recognition. Ionotropic receptors participate in a distinct olfactory signaling pathway that is independent of olfactory receptors activity. In the present study, we identify 102 putative IR genes, dubbed as AalbIr genes, in mosquito Aedes albopictus (Skuse) by in silico comparative sequence analysis. Among AalbIr genes, 19 show expression in the female antenna by RT-PCR. These putative olfactory AalbIRs share four conservative hydrophobic domains of amino acids, similar to the transmembrane and ion channel pore regions found in conventional iGluRs. To determine the potential function of these olfactory AalbIRs in host-seeking, we compared their transcript expression levels in the antennae of blood-fed females with that of non-blood-fed females by quantitative real-time RT-PCR. Three AalbIr genes showed downregulation when the mosquito finished a bloodmeal. These results may help to improve our understanding of the IR-mediated olfactory signaling in mosquitoes. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Rice Ovate Family Protein 2 (OFP2) alters hormonal homeostasis and vasculature development.

PubMed

Schmitz, Aaron J; Begcy, Kevin; Sarath, Gautam; Walia, Harkamal

2015-12-01

OFP (Ovate Family Protein) is a transcription factor family found only in plants. In dicots, OFPs control fruit shape and secondary cell wall biosynthesis. OFPs are also thought to function through interactions with KNOX and BELL transcription factors. Here, we have functionally characterized OsOFP2, a member of the OFP subgroup associated with regulating fruit shape. OsOFP2 was found to localize to the nucleus and to the cytosol. A putative nuclear export signal was identified within the OVATE domain and was required for the localization of OsOFP2 to distinct cytosolic spots. Rice plants overexpressing OsOFP2 were reduced in height and exhibited altered leaf morphology, seed shape, and positioning of vascular bundles in stems. Transcriptome analysis indicated disruptions of genes associated with vasculature development, lignin biosynthesis, and hormone homeostasis. Reduced expression of the gibberellin biosynthesis gene GA 20-oxidase 7 coincided with lower gibberellin content in OsOFP2 overexpression lines. Also, we found that OsOFP2 was expressed in plant vasculature and determined that putative vascular development KNOX and BELL proteins interact with OsOFP2. KNOX and BELL genes are known to suppress gibberellin biosynthesis through GA20ox gene regulation and can restrict lignin biosynthesis. We propose that OsOFP2 could modulate KNOX-BELL function to control diverse aspects of development including vasculature development. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Antibacterial activity of antileukoprotease.

PubMed Central

Hiemstra, P S; Maassen, R J; Stolk, J; Heinzel-Wieland, R; Steffens, G J; Dijkman, J H

1996-01-01

Antileukoprotease (ALP), or secretory leukocyte proteinase inhibitor, is an endogenous inhibitor of serine proteinases that is present in various external secretions. ALP, one of the major inhibitors of serine proteinases present in the human lung, is a potent reversible inhibitor of elastase and, to a lesser extent, of cathepsin G. In equine neutrophils, an antimicrobial polypeptide that has some of the characteristics of ALP has been identified (M. A. Couto, S. S. L. Harwig, J. S. Cullor, J. P. Hughes, and R. I. Lehrer, Infect. Immun. 60:5042-5047, 1992). This report, together with the cationic nature of ALP, led us to investigate the antimicrobial activity of ALP. ALP was shown to display marked in vitro antibacterial activity against Escherichia coli and Staphylococcus aureus. On a molar basis, the activity of ALP was lower than that of two other cationic antimicrobial polypeptides, lysozyme and defensin. ALP comprises two homologous domains: its proteinase-inhibitory activities are known to be located in the second COOH-terminal domain, and the function of its first NH2-terminal domain is largely unknown. Incubation of intact ALP or its isolated first domain with E. coli or S. aureus resulted in killing of these bacteria, whereas its second domain displayed very little antibacterial activity. Together these data suggest a putative antimicrobial role for the first domain of ALP and indicate that its antimicrobial activity may equip ALP to contribute to host defense against infection. PMID:8890201

A resource for characterizing genome-wide binding and putative target genes of transcription factors expressed during secondary growth and wood formation in Populus.

PubMed

Liu, Lijun; Ramsay, Trevor; Zinkgraf, Matthew; Sundell, David; Street, Nathaniel Robert; Filkov, Vladimir; Groover, Andrew

2015-06-01

Identifying transcription factor target genes is essential for modeling the transcriptional networks underlying developmental processes. Here we report a chromatin immunoprecipitation sequencing (ChIP-seq) resource consisting of genome-wide binding regions and associated putative target genes for four Populus homeodomain transcription factors expressed during secondary growth and wood formation. Software code (programs and scripts) for processing the Populus ChIP-seq data are provided within a publically available iPlant image, including tools for ChIP-seq data quality control and evaluation adapted from the human Encyclopedia of DNA Elements (ENCODE) project. Basic information for each transcription factor (including members of Class I KNOX, Class III HD ZIP, BEL1-like families) binding are summarized, including the number and location of binding regions, distribution of binding regions relative to gene features, associated putative target genes, and enriched functional categories of putative target genes. These ChIP-seq data have been integrated within the Populus Genome Integrative Explorer (PopGenIE) where they can be analyzed using a variety of web-based tools. We present an example analysis that shows preferential binding of transcription factor ARBORKNOX1 to the nearest neighbor genes in a pre-calculated co-expression network module, and enrichment for meristem-related genes within this module including multiple orthologs of Arabidopsis KNOTTED-like Arabidopsis 2/6. © 2015 Society for Experimental Biology and John Wiley & Sons Ltd This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Computational Studies of the Active and Inactive Regulatory Domains of Response Regulator PhoP Using Molecular Dynamics Simulations.

PubMed

Qing, Xiao-Yu; Steenackers, Hans; Venken, Tom; De Maeyer, Marc; Voet, Arnout

2017-11-01

The response regulator PhoP is part of the PhoP/PhoQ two-component system, which is responsible for regulating the expression of multiple genes involved in controlling virulence, biofilm formation, and resistance to antimicrobial peptides. Therefore, modulating the transcriptional function of the PhoP protein is a promising strategy for developing new antimicrobial agents. There is evidence suggesting that phosphorylation-mediated dimerization in the regulatory domain of PhoP is essential for its transcriptional function. Disruption or stabilization of protein-protein interactions at the dimerization interface may inhibit or enhance the expression of PhoP-dependent genes. In this study, we performed molecular dynamics simulations on the active and inactive dimers and monomers of the PhoP regulatory domains, followed by pocket-detecting screenings and a quantitative hot-spot analysis in order to assess the druggability of the protein. Consistent with prior hypothesis, the calculation of the binding free energy shows that phosphorylation enhances dimerization of PhoP. Furthermore, we have identified two different putative binding sites at the dimerization active site (the α4-β5-α5 face) with energetic "hot-spot" areas, which could be used to search for modulators of protein-protein interactions. This study delivers insight into the dynamics and druggability of the dimerization interface of the PhoP regulatory domain, and may serve as a basis for the rational identification of new antimicrobial drugs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The functionally conserved nucleoporins Nup124p from fission yeast and the human Nup153 mediate nuclear import and activity of the Tf1 retrotransposon and HIV-1 Vpr.

PubMed

Varadarajan, Padmapriya; Mahalingam, Sundarasamy; Liu, Peiyun; Ng, Sarah Boon Hsi; Gandotra, Sheetal; Dorairajoo, Desmond Suresh Kumar; Balasundaram, David

2005-04-01

We report that the fission yeast nucleoporin Nup124p is required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr. Failure to import Tf1-Gag into the nucleus in a nup124 null mutant resulted in complete loss of Tf1 transposition. Similarly, nuclear import of HIV-1 Vpr was impaired in nup124 null mutant strains and cells became resistant to Vpr's cell-killing activity. On the basis of protein domain similarity, the human nucleoporin Nup153 was identified as a putative homolog of Nup124p. We demonstrate that in vitro-translated Nup124p and Nup153 coimmunoprecipitate Tf1-Gag or HIV-1 Vpr. Though full-length Nup153 was unable to complement the Tf1 transposition defect in a nup124 null mutant, we provide evidence that both nucleoporins share a unique N-terminal domain, Nup124p(AA264-454) and Nup153(AA448-634) that is absolutely essential for Tf1 transposition. Epigenetic overexpression of this domain in a wild-type (nup124(+)) background blocked Tf1 activity implying that sequences from Nup124p and the human Nup153 challenged the same pathway affecting Tf1 transposition. Our results establish a unique relationship between two analogous nucleoporins Nup124p and Nup153 wherein the function of a common domain in retrotransposition is conserved.

Comparative secretome analysis of Colletotrichum falcatum identifies a cerato-platanin protein (EPL1) as a potential pathogen-associated molecular pattern (PAMP) inducing systemic resistance in sugarcane.

PubMed

Ashwin, N M R; Barnabas, Leonard; Ramesh Sundar, Amalraj; Malathi, Palaniyandi; Viswanathan, Rasappa; Masi, Antonio; Agrawal, Ganesh Kumar; Rakwal, Randeep

2017-10-03

Colletotrichum falcatum, an intriguing hemibiotrophic fungal pathogen causes red rot, a devastating disease of sugarcane. Repeated in vitro subculturing of C. falcatum under dark condition alters morphology and reduces virulence of the culture. Hitherto, no information is available on this phenomenon at molecular level. In this study, the in vitro secretome of C. falcatum cultured under light and dark conditions was analyzed using 2-DE coupled with MALDI TOF/TOF MS. Comparative analysis identified nine differentially abundant proteins. Among them, seven proteins were less abundant in the dark-cultured C. falcatum, wherein only two protein species of a cerato-platanin protein called EPL1 (eliciting plant response-like protein) were found to be highly abundant. Transcriptional expression of candidate high abundant proteins was profiled during host-pathogen interaction using qRT-PCR. Comprehensively, this comparative secretome analysis identified five putative effectors, two pathogenicity-related proteins and one pathogen-associated molecular pattern (PAMP) of C. falcatum. Functional characterization of three distinct domains of the PAMP (EPL1) showed that the major cerato-platanin domain (EPL1∆N1-92) is exclusively essential for inducing defense and hypersensitive response (HR) in sugarcane and tobacco, respectively. Further, priming with EPL1∆N1-92 protein induced systemic resistance and significantly suppressed the red rot severity in sugarcane. Being the first secretomic investigation of C. falcatum, this study has identified five potential effectors, two pathogenicity-related proteins and a PAMP. Although many reports have highlighted the influence of light on pathogenicity, this study has established a direct link between light and expression of effectors, for the first time. This study has presented the influence of a novel N-terminal domain of EPL1 in physical and biological properties and established the functional role of major cerato-platanin domain of EPL1 as a potential elicitor inducing systemic resistance in sugarcane. Comprehensively, the study has identified proteins that putatively contribute to virulence of C. falcatum and for the first time, demonstrated the potential role of EPL1 in inducing PAMP-triggered immunity (PTI) in sugarcane. Copyright © 2017 Elsevier B.V. All rights reserved.

Spatial pattern of receptor expression in the olfactory epithelium.

PubMed Central

Nef, P; Hermans-Borgmeyer, I; Artières-Pin, H; Beasley, L; Dionne, V E; Heinemann, S F

1992-01-01

A PCR-based strategy for amplifying putative receptors involved in murine olfaction was employed to isolate a member (OR3) of the seven-transmembrane-domain receptor superfamily. During development, the first cells that express OR3 appear adjacent to the wall of the telencephalic vesicle at embryonic day 10. The OR3 receptor is uniquely expressed in a subset of olfactory cells that have a characteristic bilateral symmetry in the adult olfactory epithelium. This receptor and its specific pattern of expression may serve a functional role in odor coding or, alternatively, may play a role in the development of the olfactory system. Images PMID:1384038

Targeted Analysis of KLF6/KLF6-SV1 Regulating Pathways in Prostate Cancer Development and Metastasis

DTIC Science & Technology

2011-09-01

and KLF6-SV1 it will be important to define the functionality of the putative NLS, the 5BR, as well as the role of nucleo-cytoplasmic shuttling in...aa EGFP -56 aa -16 aa KLF6 KLF6-SV1 5BR ZF,ZF~F3 ZF, ZF2 ZF3 l29KLF6 57KLF6 17KLF6 located near or within other important domains that...the transporter protein Crm1/Xpo1, first discovered in yeast [33–36]. Subcellular localization and protein turnover are two related events that are

Sequoia, a tramtrack-related zinc finger protein, functions as a pan-neural regulator for dendrite and axon morphogenesis in Drosophila.

PubMed

Brenman, J E; Gao, F B; Jan, L Y; Jan, Y N

2001-11-01

Morphological complexity of neurons contributes to their functional complexity. How neurons generate different dendritic patterns is not known. We identified the sequoia mutant from a previous screen for dendrite mutants. Here we report that Sequoia is a pan-neural nuclear protein containing two putative zinc fingers homologous to the DNA binding domain of Tramtrack. sequoia mutants affect the cell fate decision of a small subset of neurons but have global effects on axon and dendrite morphologies of most and possibly all neurons. In support of sequoia as a specific regulator of neuronal morphogenesis, microarray experiments indicate that sequoia may regulate downstream genes that are important for executing neurite development rather than altering a variety of molecules that specify cell fates.

Biochemistry of Mitochondrial Coenzyme Q Biosynthesis.

PubMed

Stefely, Jonathan A; Pagliarini, David J

2017-10-01

Coenzyme Q (CoQ, ubiquinone) is a redox-active lipid produced across all domains of life that functions in electron transport and oxidative phosphorylation and whose deficiency causes human diseases. Yet, CoQ biosynthesis has not been fully defined in any organism. Several proteins with unclear molecular functions facilitate CoQ biosynthesis through unknown means, and multiple steps in the pathway are catalyzed by currently unidentified enzymes. Here we highlight recent progress toward filling these knowledge gaps through both traditional biochemistry and cutting-edge 'omics' approaches. To help fill the remaining gaps, we present questions framed by the recently discovered CoQ biosynthetic complex and by putative biophysical barriers. Mapping CoQ biosynthesis, metabolism, and transport pathways has great potential to enhance treatment of numerous human diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Recycling domains in plant cell morphogenesis: small GTPase effectors, plasma membrane signalling and the exocyst.

PubMed

Zárský, Viktor; Potocký, Martin

2010-04-01

The Rho/Rop small GTPase regulatory module is central for initiating exocytotically ACDs (active cortical domains) in plant cell cortex, and a growing array of Rop regulators and effectors are being discovered in plants. Structural membrane phospholipids are important constituents of cells as well as signals, and phospholipid-modifying enzymes are well known effectors of small GTPases. We have shown that PLDs (phospholipases D) and their product, PA (phosphatidic acid), belong to the regulators of the secretory pathway in plants. We have also shown that specific NOXs (NADPH oxidases) producing ROS (reactive oxygen species) are involved in cell growth as exemplified by pollen tubes and root hairs. Most plant cells exhibit several distinct plasma membrane domains (ACDs), established and maintained by endocytosis/exocytosis-driven membrane protein recycling. We proposed recently the concept of a 'recycling domain' (RD), uniting the ACD and the connected endosomal recycling compartment (endosome), as a dynamic spatiotemporal entity. We have described a putative GTPase-effector complex exocyst involved in exocytic vesicle tethering in plants. Owing to the multiplicity of its Exo70 subunits, this complex, along with many RabA GTPases (putative recycling endosome organizers), may belong to core regulators of RD organization in plants.

Ketoacylsynthase Domains of a Polyunsaturated Fatty Acid Synthase in Thraustochytrium sp. Strain ATCC 26185 Can Effectively Function as Stand-Alone Enzymes in Escherichia coli.

PubMed

Xie, Xi; Meesapyodsuk, Dauenpen; Qiu, Xiao

2017-05-01

Thraustochytrium sp. strain ATCC 26185 accumulates a high level of docosahexaenoic acid (DHA), a nutritionally important ω-3 very-long-chain polyunsaturated fatty acid (VLCPUFA) synthesized primarily by polyunsaturated fatty acid (PUFA) synthase, a type I polyketide synthase-like megaenzyme. The PUFA synthase in this species comprises three large subunits, each with multiple catalytic domains. It was hypothesized that among these domains, ketoacylsynthase (KS) domains might be critical for catalyzing the condensation of specific unsaturated acyl-acyl carrier proteins (ACPs) with malonyl-ACP, thereby retaining double bonds in an extended acyl chain. To investigate the functions of these putative KS domains, two segment sequences from subunit A (KS-A) and subunit B (KS-B) of the PUFA synthase were dissected and then expressed as stand-alone enzymes in Escherichia coli The results showed that both KS-A and KS-B domains could complement the defective phenotypes of both E. coli fabB and fabF mutants. Overexpression of these domains in wild-type E. coli led to increases in total fatty acid production. KS-B produced a higher ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs), while KS-A could improve the overall production of fatty acids more effectively, particularly for the production of SFAs, implying that KS-A is more comparable to FabF, while KS-B is more similar to FabB in catalytic functions. Successful complementation and functional expression of the embedded KS domains in E. coli are the first step forward in studying the molecular mechanism of the PUFA synthase for the biosynthesis of VLCPUFAs in Thraustochytrium IMPORTANCE Very-long-chain polyunsaturated fatty acids (VLCPUFAs) are important for human health. They can be biosynthesized in either an aerobic pathway or an anaerobic pathway in nature. However, abundant VLCPUFAs in marine microorganisms are primarily synthesized by polyunsaturated fatty acid (PUFA) synthase, a megaenzyme with multiple subunits, each with multiple catalytic domains. Furthermore, the fundamental mechanism for this enzyme to synthesize these fatty acids still remains unknown. This report started with dissecting the embedded KS domains of the PUFA synthase from marine protist Thraustochytrium sp. strain ATCC 26185 and then expressing them in wild-type E. coli and mutants defective in condensation of acyl-ACP with malonyl-ACP. Successful complementation of the mutants and improved fatty acid production in the overexpression experiments indicate that these KS domains can effectively function as stand-alone enzymes in E. coli This result has paved the way for further studying of molecular mechanisms of the PUFA synthase for the biosynthesis of VLCPUFAs. Copyright © 2017 American Society for Microbiology.

Ketoacylsynthase Domains of a Polyunsaturated Fatty Acid Synthase in Thraustochytrium sp. Strain ATCC 26185 Can Effectively Function as Stand-Alone Enzymes in Escherichia coli

PubMed Central

Xie, Xi; Meesapyodsuk, Dauenpen

2017-01-01

ABSTRACT Thraustochytrium sp. strain ATCC 26185 accumulates a high level of docosahexaenoic acid (DHA), a nutritionally important ω-3 very-long-chain polyunsaturated fatty acid (VLCPUFA) synthesized primarily by polyunsaturated fatty acid (PUFA) synthase, a type I polyketide synthase-like megaenzyme. The PUFA synthase in this species comprises three large subunits, each with multiple catalytic domains. It was hypothesized that among these domains, ketoacylsynthase (KS) domains might be critical for catalyzing the condensation of specific unsaturated acyl-acyl carrier proteins (ACPs) with malonyl-ACP, thereby retaining double bonds in an extended acyl chain. To investigate the functions of these putative KS domains, two segment sequences from subunit A (KS-A) and subunit B (KS-B) of the PUFA synthase were dissected and then expressed as stand-alone enzymes in Escherichia coli. The results showed that both KS-A and KS-B domains could complement the defective phenotypes of both E. coli fabB and fabF mutants. Overexpression of these domains in wild-type E. coli led to increases in total fatty acid production. KS-B produced a higher ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs), while KS-A could improve the overall production of fatty acids more effectively, particularly for the production of SFAs, implying that KS-A is more comparable to FabF, while KS-B is more similar to FabB in catalytic functions. Successful complementation and functional expression of the embedded KS domains in E. coli are the first step forward in studying the molecular mechanism of the PUFA synthase for the biosynthesis of VLCPUFAs in Thraustochytrium. IMPORTANCE Very-long-chain polyunsaturated fatty acids (VLCPUFAs) are important for human health. They can be biosynthesized in either an aerobic pathway or an anaerobic pathway in nature. However, abundant VLCPUFAs in marine microorganisms are primarily synthesized by polyunsaturated fatty acid (PUFA) synthase, a megaenzyme with multiple subunits, each with multiple catalytic domains. Furthermore, the fundamental mechanism for this enzyme to synthesize these fatty acids still remains unknown. This report started with dissecting the embedded KS domains of the PUFA synthase from marine protist Thraustochytrium sp. strain ATCC 26185 and then expressing them in wild-type E. coli and mutants defective in condensation of acyl-ACP with malonyl-ACP. Successful complementation of the mutants and improved fatty acid production in the overexpression experiments indicate that these KS domains can effectively function as stand-alone enzymes in E. coli. This result has paved the way for further studying of molecular mechanisms of the PUFA synthase for the biosynthesis of VLCPUFAs. PMID:28213537

Stimulus selectivity and response latency in putative inhibitory and excitatory neurons of the primate inferior temporal cortex

PubMed Central

Mruczek, Ryan E. B.

2012-01-01

The cerebral cortex is composed of many distinct classes of neurons. Numerous studies have demonstrated corresponding differences in neuronal properties across cell types, but these comparisons have largely been limited to conditions outside of awake, behaving animals. Thus the functional role of the various cell types is not well understood. Here, we investigate differences in the functional properties of two widespread and broad classes of cells in inferior temporal cortex of macaque monkeys: inhibitory interneurons and excitatory projection cells. Cells were classified as putative inhibitory or putative excitatory neurons on the basis of their extracellular waveform characteristics (e.g., spike duration). Consistent with previous intracellular recordings in cortical slices, putative inhibitory neurons had higher spontaneous firing rates and higher stimulus-evoked firing rates than putative excitatory neurons. Additionally, putative excitatory neurons were more susceptible to spike waveform adaptation following very short interspike intervals. Finally, we compared two functional properties of each neuron's stimulus-evoked response: stimulus selectivity and response latency. First, putative excitatory neurons showed stronger stimulus selectivity compared with putative inhibitory neurons. Second, putative inhibitory neurons had shorter response latencies compared with putative excitatory neurons. Selectivity differences were maintained and latency differences were enhanced during a visual search task emulating more natural viewing conditions. Our results suggest that short-latency inhibitory responses are likely to sculpt visual processing in excitatory neurons, yielding a sparser visual representation. PMID:22933717

Shark class II invariant chain reveals ancient conserved relationships with cathepsins and MHC class II.

PubMed

Criscitiello, Michael F; Ohta, Yuko; Graham, Matthew D; Eubanks, Jeannine O; Chen, Patricia L; Flajnik, Martin F

2012-03-01

The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates. Copyright © 2011 Elsevier Ltd. All rights reserved.

Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM.

PubMed

Hymes, Jeffrey P; Johnson, Brant R; Barrangou, Rodolphe; Klaenhammer, Todd R

2016-05-01

Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in L. acidophilus NCFM, an fbpB-deficient strain was constructed. The L. acidophilus mutant with a deletion off bpB lost the ability to adhere to mucin and fibronectin in vitro Homologues off bpB were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside theL. acidophilus homology group. Copyright © 2016 Hymes et al.

Disruption of the methyltransferase-like 23 gene METTL23 causes mild autosomal recessive intellectual disability

PubMed Central

Bernkopf, Marie; Webersinke, Gerald; Tongsook, Chanakan; Koyani, Chintan N.; Rafiq, Muhammad A.; Ayaz, Muhammad; Müller, Doris; Enzinger, Christian; Aslam, Muhammad; Naeem, Farooq; Schmidt, Kurt; Gruber, Karl; Speicher, Michael R.; Malle, Ernst; Macheroux, Peter; Ayub, Muhammad; Vincent, John B.; Windpassinger, Christian; Duba, Hans-Christoph

2014-01-01

We describe the characterization of a gene for mild nonsyndromic autosomal recessive intellectual disability (ID) in two unrelated families, one from Austria, the other from Pakistan. Genome-wide single nucleotide polymorphism microarray analysis enabled us to define a region of homozygosity by descent on chromosome 17q25. Whole-exome sequencing and analysis of this region in an affected individual from the Austrian family identified a 5 bp frameshifting deletion in the METTL23 gene. By means of Sanger sequencing of METTL23, a nonsense mutation was detected in a consanguineous ID family from Pakistan for which homozygosity-by-descent mapping had identified a region on 17q25. Both changes lead to truncation of the putative METTL23 protein, which disrupts the predicted catalytic domain and alters the cellular localization. 3D-modelling of the protein indicates that METTL23 is strongly predicted to function as an S-adenosyl-methionine (SAM)-dependent methyltransferase. Expression analysis of METTL23 indicated a strong association with heat shock proteins, which suggests that these may act as a putative substrate for methylation by METTL23. A number of methyltransferases have been described recently in association with ID. Disruption of METTL23 presented here supports the importance of methylation processes for intact neuronal function and brain development. PMID:24626631

Characterization of the genetic elements required for site-specific integration of plasmid pSE211 in Saccharopolyspora erythraea.

PubMed Central

Brown, D P; Idler, K B; Katz, L

1990-01-01

The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site. Restriction analysis of the integrated plasmid, pSE211int, and adjacent chromosomal sequences allowed identification of attP, the plasmid attachment site. Nucleotide sequencing of attP, attB, attL, and attR revealed a 57-base-pair sequence common to all sites with no duplications of adjacent plasmid or chromosomal sequences in the integrated state, indicating that integration takes place through conservative, reciprocal strand exchange. An analysis of the sequences indicated the presence of a putative gene for Phe-tRNA at attB which is preserved at attL after integration has occurred. A comparison of the attB site for a number of actinomycete plasmids is presented. Integration at attB was also observed when a 2.4-kilobase segment of pSE211 containing attP and the adjacent plasmid sequence was used to transform a pSE211- host. Nucleotide sequencing of this segment revealed the presence of two complete open reading frames (ORFs) and a segment of a third ORF. The ORF adjacent to attP encodes a putative polypeptide 437 amino acids in length that shows similarity, at its C-terminal domain, to sequences of site-specific recombinases of the integrase family. The adjacent ORF encodes a putative 98-amino-acid basic polypeptide that contains a helix-turn-helix motif at its N terminus which corresponds to domains in the Xis proteins of a number of bacteriophages. A proposal for the function of this polypeptide is presented. The deduced amino acid sequence of the third ORF did not reveal similarities to polypeptide sequences in the current data banks. Images FIG. 2 FIG. 3 PMID:2180909

RNA circularization strategies in vivo and in vitro

PubMed Central

Petkovic, Sonja; Müller, Sabine

2015-01-01

In the plenitude of naturally occurring RNAs, circular RNAs (circRNAs) and their biological role were underestimated for years. However, circRNAs are ubiquitous in all domains of life, including eukaryotes, archaea, bacteria and viruses, where they can fulfill diverse biological functions. Some of those functions, as for example playing a role in the life cycle of viral and viroid genomes or in the maturation of tRNA genes, have been elucidated; other putative functions still remain elusive. Due to the resistance to exonucleases, circRNAs are promising tools for in vivo application as aptamers, trans-cleaving ribozymes or siRNAs. How are circRNAs generated in vivo and what approaches do exist to produce ring-shaped RNAs in vitro? In this review we illustrate the occurrence and mechanisms of RNA circularization in vivo, survey methods for the generation of circRNA in vitro and provide appropriate protocols. PMID:25662225

Identification and Functional Analysis of ZIC3 Mutations in Heterotaxy and Related Congenital Heart Defects

PubMed Central

Ware, Stephanie M.; Peng, Jianlan; Zhu, Lirong; Fernbach, Susan; Colicos, Suzanne; Casey, Brett; Towbin, Jeffrey; Belmont, John W.

2004-01-01

Mutations in the zinc finger transcription factor ZIC3 cause X-linked heterotaxy and have also been identified in patients with isolated congenital heart disease (CHD). To determine the relative contribution of ZIC3 mutations to both heterotaxy and isolated CHD, we screened the coding region of ZIC3 in 194 unrelated patients, including 61 patients with classic heterotaxy, 93 patients with heart defects characteristic of heterotaxy, and 11 patients with situs inversus totalis. Five novel ZIC3 mutations in three classic heterotaxy kindreds and two sporadic CHD cases were identified. None of these alleles was found in 97 ethnically matched control samples. On the basis of these analyses, we conclude that the phenotypic spectrum of ZIC3 mutations should be expanded to include affected females and CHD not typical for heterotaxy. This screening of a cohort of patients with sporadic heterotaxy indicates that ZIC3 mutations account for ∼1% of affected individuals. Missense and nonsense mutations were found in the highly conserved zinc finger–binding domain and in the N-terminal protein domain. Functional analysis of all currently known ZIC3 point mutations indicates that mutations in the putative zinc finger DNA binding domain and in the N-terminal domain result in loss of reporter gene transactivation. It is surprising that transfection studies demonstrate aberrant cytoplasmic localization resulting from mutations between amino acids 253–323 of the ZIC3 protein, indicating that the pathogenesis of a subset of ZIC3 mutations results at least in part from failure of appropriate nuclear localization. These results further expand the phenotypic and genotypic spectrum of ZIC3 mutations and provide initial mechanistic insight into their functional consequences. PMID:14681828

Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels

PubMed Central

Vocke, Kerstin; Dauner, Kristin; Hahn, Anne; Ulbrich, Anne; Broecker, Jana; Keller, Sandro; Frings, Stephan

2013-01-01

Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca2+/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca2+/calmodulin, one at submicromolar Ca2+ concentrations and one in the micromolar Ca2+ range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca2+/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca2+ signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca2+ regulation in anoctamin Cl− channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types. PMID:24081981

The Putative Exchange Factor Gef3p Interacts with Rho3p GTPase and the Septin Ring during Cytokinesis in Fission Yeast*

PubMed Central

Muñoz, Sofía; Manjón, Elvira; Sánchez, Yolanda

2014-01-01

The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3+ and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation. PMID:24947517

Against All Odds: Trehalose-6-Phosphate Synthase and Trehalase Genes in the Bdelloid Rotifer Adineta vaga Were Acquired by Horizontal Gene Transfer and Are Upregulated during Desiccation

PubMed Central

Hespeels, Boris; Li, Xiang; Flot, Jean-François; Pigneur, Lise-Marie; Malaisse, Jeremy; Da Silva, Corinne; Van Doninck, Karine

2015-01-01

The disaccharide sugar trehalose is essential for desiccation resistance in most metazoans that survive dryness; however, neither trehalose nor the enzymes involved in its metabolism have ever been detected in bdelloid rotifers despite their extreme resistance to desiccation. Here we screened the genome of the bdelloid rotifer Adineta vaga for genes involved in trehalose metabolism. We discovered a total of four putative trehalose-6-phosphate synthase (TPS) and seven putative trehalase (TRE) gene copies in the genome of this ameiotic organism; however, no trehalose-6-phosphate phosphatase (TPP) gene or domain was detected. The four TPS copies of A. vaga appear more closely related to plant and fungi proteins, as well as to some protists, whereas the seven TRE copies fall in bacterial clades. Therefore, A. vaga likely acquired its trehalose biosynthesis and hydrolysis genes by horizontal gene transfers. Nearly all residues important for substrate binding in the predicted TPS domains are highly conserved, supporting the hypothesis that several copies of the genes might be functional. Besides, RNAseq library screening showed that trehalase genes were highly expressed compared to TPS genes, explaining probably why trehalose had not been detected in previous studies of bdelloids. A strong overexpression of their TPS genes was observed when bdelloids enter desiccation, suggesting a possible signaling role of trehalose-6-phosphate or trehalose in this process. PMID:26161530

Discovery: an interactive resource for the rational selection and comparison of putative drug target proteins in malaria

PubMed Central

Joubert, Fourie; Harrison, Claudia M; Koegelenberg, Riaan J; Odendaal, Christiaan J; de Beer, Tjaart AP

2009-01-01

Background Up to half a billion human clinical cases of malaria are reported each year, resulting in about 2.7 million deaths, most of which occur in sub-Saharan Africa. Due to the over-and misuse of anti-malarials, widespread resistance to all the known drugs is increasing at an alarming rate. Rational methods to select new drug target proteins and lead compounds are urgently needed. The Discovery system provides data mining functionality on extensive annotations of five malaria species together with the human and mosquito hosts, enabling the selection of new targets based on multiple protein and ligand properties. Methods A web-based system was developed where researchers are able to mine information on malaria proteins and predicted ligands, as well as perform comparisons to the human and mosquito host characteristics. Protein features used include: domains, motifs, EC numbers, GO terms, orthologs, protein-protein interactions, protein-ligand interactions and host-pathogen interactions among others. Searching by chemical structure is also available. Results An in silico system for the selection of putative drug targets and lead compounds is presented, together with an example study on the bifunctional DHFR-TS from Plasmodium falciparum. Conclusion The Discovery system allows for the identification of putative drug targets and lead compounds in Plasmodium species based on the filtering of protein and chemical properties. PMID:19642978

Putative Monofunctional Type I Polyketide Synthase Units: A Dinoflagellate-Specific Feature?

PubMed Central

Eichholz, Karsten; Beszteri, Bánk; John, Uwe

2012-01-01

Marine dinoflagellates (alveolata) are microalgae of which some cause harmful algal blooms and produce a broad variety of most likely polyketide synthesis derived phycotoxins. Recently, novel polyketide synthesase (PKS) transcripts have been described from the Florida red tide dinoflagellate Karenia brevis (gymnodiniales) which are evolutionarily related to Type I PKS but were apparently expressed as monofunctional proteins, a feature typical of Type II PKS. Here, we investigated expression units of PKS I-like sequences in Alexandrium ostenfeldii (gonyaulacales) and Heterocapsa triquetra (peridiniales) at the transcript and protein level. The five full length transcripts we obtained were all characterized by polyadenylation, a 3′ UTR and the dinoflagellate specific spliced leader sequence at the 5′end. Each of the five transcripts encoded a single ketoacylsynthase (KS) domain showing high similarity to K. brevis KS sequences. The monofunctional structure was also confirmed using dinoflagellate specific KS antibodies in Western Blots. In a maximum likelihood phylogenetic analysis of KS domains from diverse PKSs, dinoflagellate KSs formed a clade placed well within the protist Type I PKS clade between apicomplexa, haptophytes and chlorophytes. These findings indicate that the atypical PKS I structure, i.e., expression as putative monofunctional units, might be a dinoflagellate specific feature. In addition, the sequenced transcripts harbored a previously unknown, apparently dinoflagellate specific conserved N-terminal domain. We discuss the implications of this novel region with regard to the putative monofunctional organization of Type I PKS in dinoflagellates. PMID:23139807

Putative Nonribosomal Peptide Synthetase and Cytochrome P450 Genes Responsible for Tentoxin Biosynthesis in Alternaria alternata ZJ33

PubMed Central

Li, You-Hai; Han, Wen-Jin; Gui, Xi-Wu; Wei, Tao; Tang, Shuang-Yan; Jin, Jian-Ming

2016-01-01

Tentoxin, a cyclic tetrapeptide produced by several Alternaria species, inhibits the F1-ATPase activity of chloroplasts, resulting in chlorosis in sensitive plants. In this study, we report two clustered genes, encoding a putative non-ribosome peptide synthetase (NRPS) TES and a cytochrome P450 protein TES1, that are required for tentoxin biosynthesis in Alternaria alternata strain ZJ33, which was isolated from blighted leaves of Eupatorium adenophorum. Using a pair of primers designed according to the consensus sequences of the adenylation domain of NRPSs, two fragments containing putative adenylation domains were amplified from A. alternata ZJ33, and subsequent PCR analyses demonstrated that these fragments belonged to the same NRPS coding sequence. With no introns, TES consists of a single 15,486 base pair open reading frame encoding a predicted 5161 amino acid protein. Meanwhile, the TES1 gene is predicted to contain five introns and encode a 506 amino acid protein. The TES protein is predicted to be comprised of four peptide synthase modules with two additional N-methylation domains, and the number and arrangement of the modules in TES were consistent with the number and arrangement of the amino acid residues of tentoxin, respectively. Notably, both TES and TES1 null mutants generated via homologous recombination failed to produce tentoxin. This study provides the first evidence concerning the biosynthesis of tentoxin in A. alternata. PMID:27490569

Active nuclear import and passive nuclear export are the primary determinants of TDP-43 localization.

PubMed

Pinarbasi, Emile S; Cağatay, Tolga; Fung, Ho Yee Joyce; Li, Ying C; Chook, Yuh Min; Thomas, Philip J

2018-05-04

ALS (Amyotrophic Lateral Sclerosis) is a neurodegenerative disease characterized by the redistribution of the RNA binding protein TDP-43 in affected neurons: from predominantly nuclear to aggregated in the cytosol. However, the determinants of TDP-43 localization and the cellular insults that promote redistribution are incompletely understood. Here, we show that the putative Nuclear Export Signal (NES) is not required for nuclear egress of TDP-43. Moreover, when the TDP-43 domain which contains the putative NES is fused to a reporter protein, YFP, the presence of the NES is not sufficient to mediate nuclear exclusion of the fusion protein. We find that the previously studied "∆NES" mutant, in which conserved hydrophobic residues are mutated to alanines, disrupts both solubility and splicing function. We further show that nuclear export of TDP-43 is independent of the exportin XPO1. Finally, we provide evidence that nuclear egress of TDP-43 is size dependent; nuclear export of dTomato TDP-43 is significantly impaired compared to Flag TDP-43. Together, these results suggest nuclear export of TDP-43 is predominantly driven by passive diffusion.

Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks

PubMed Central

George, Tresa; Wen, Qin; Griffiths, Richard; Ganesh, Anil; Meuth, Mark; Sanders, Cyril M.

2009-01-01

Pif-1 proteins are 5′→3′ superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3′ ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity. PMID:19700773

Comprehensively Surveying Structure and Function of RING Domains from Drosophila melanogaster

PubMed Central

Wu, Yuehao; Wan, Fusheng; Huang, Chunhong; Jie, Kemin

2011-01-01

Using a complete set of RING domains from Drosophila melanogaster, all the solved RING domains and cocrystal structures of RING-containing ubiquitin-ligases (RING-E3) and ubiquitin-conjugating enzyme (E2) pairs, we analyzed RING domains structures from their primary to quarternary structures. The results showed that: i) putative orthologs of RING domains between Drosophila melanogaster and the human largely occur (118/139, 84.9%); ii) of the 118 orthologous pairs from Drosophila melanogaster and the human, 117 pairs (117/118, 99.2%) were found to retain entirely uniform domain architectures, only Iap2/Diap2 experienced evolutionary expansion of domain architecture; iii) 4 evolutionary structurally conserved regions (SCRs) are responsible for homologous folding of RING domains at the superfamily level; iv) besides the conserved Cys/His chelating zinc ions, 6 equivalent residues (4 hydrophobic and 2 polar residues) in the SCRs possess good-consensus and conservation- these 4 SCRs function in the structural positioning of 6 equivalent residues as determinants for RING-E3 catalysis; v) members of these RING proteins located nucleus, multiple subcellular compartments, membrane protein and mitochondrion are respectively 42 (42/139, 30.2%), 71 (71/139, 51.1%), 22 (22/139, 15.8%) and 4 (4/139, 2.9%); vi) CG15104 (Topors) and CG1134 (Mul1) in C3HC4, and CG3929 (Deltex) in C3H2C3 seem to display broader E2s binding profiles than other RING-E3s; vii) analyzing intermolecular interfaces of E2/RING-E3 complexes indicate that residues directly interacting with E2s are all from the SCRs in RING domains. Of the 6 residues, 2 hydrophobic ones contribute to constructing the conserved hydrophobic core, while the 2 hydrophobic and 2 polar residues directly participate in E2/RING-E3 interactions. Based on sequence and structural data, SCRs, conserved equivalent residues and features of intermolecular interfaces were extracted, highlighting the presence of a nucleus for RING domain fold and formation of catalytic core in which related residues and regions exhibit preferential evolutionary conservation. PMID:21912646

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hirasaka, Katsuya, E-mail: hirasaka@nagasaki-u.ac.jp; Department of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima, Tokushima; Mills, Edward M.

Uncoupling protein 3 (UCP3) is known to regulate energy dissipation, proton leakage, fatty acid oxidation, and oxidative stress. To identify the putative protein regulators of UCP3, we performed yeast two-hybrid screens. Here we report that UCP3 interacted with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that was localized in the mitochondria, and is involved in cellular responses to Ca{sup 2+}. The hydrophilic sequences within loop 2, and the matrix-localized hydrophilic domain of mouse UCP3, were necessary for binding to Hax-1 at the C-terminal domain, adjacent to the mitochondrial inner membrane. Interestingly, interaction of these proteins occurred in a calcium-dependentmore » manner. Moreover, the NMR spectrum of the C-terminal domain of Hax-1 was dramatically changed by removal of Ca{sup 2+}, suggesting that the C-terminal domain of Hax-1 underwent a Ca{sup 2+}-induced conformational change. In the Ca{sup 2+}-free state, the C-terminal Hax-1 tended to unfold, suggesting that Ca{sup 2+} binding may induce protein folding of the Hax-1 C-terminus. These results suggested that the UCP3-Hax-1 complex may regulate mitochondrial functional changes caused by mitochondrial Ca{sup 2+}. - Highlights: • UCP3 interacts with Hax-1. • The interaction of UCP3 and Hax-1 occurs in a calcium-dependent manner. • The C-terminal domain of Hax-1 undergoes a calcium-induced conformational change.« less

Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I.

PubMed Central

Takeshita, S; Kikuno, R; Tezuka, K; Amann, E

1993-01-01

A cDNA library prepared from the mouse osteoblastic cell line MC3T3-E1 was screened for the presence of specifically expressed genes by employing a combined subtraction hybridization/differential screening approach. A cDNA was identified and sequenced which encodes a protein designated osteoblast-specific factor 2 (OSF-2) comprising 811 amino acids. OSF-2 has a typical signal sequence, followed by a cysteine-rich domain, a fourfold repeated domain and a C-terminal domain. The protein lacks a typical transmembrane region. The fourfold repeated domain of OSF-2 shows homology with the insect protein fasciclin I. RNA analyses revealed that OSF-2 is expressed in bone and to a lesser extent in lung, but not in other tissues. Mouse OSF-2 cDNA was subsequently used as a probe to clone the human counterpart. Mouse and human OSF-2 show a high amino acid sequence conservation except for the signal sequence and two regions in the C-terminal domain in which 'in-frame' insertions or deletions are observed, implying alternative splicing events. On the basis of the amino acid sequence homology with fasciclin I, we suggest that OSF-2 functions as a homophilic adhesion molecule in bone formation. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8363580

Molecular cloning and characterization of human trabeculin-alpha, a giant protein defining a new family of actin-binding proteins.

PubMed

Sun, Y; Zhang, J; Kraeft, S K; Auclair, D; Chang, M S; Liu, Y; Sutherland, R; Salgia, R; Griffin, J D; Ferland, L H; Chen, L B

1999-11-19

We describe the molecular cloning and characterization of a novel giant human cytoplasmic protein, trabeculin-alpha (M(r) = 614,000). Analysis of the deduced amino acid sequence reveals homologies with several putative functional domains, including a pair of alpha-actinin-like actin binding domains; regions of homology to plakins at either end of the giant polypeptide; 29 copies of a spectrin-like motif in the central region of the protein; two potential Ca(2+)-binding EF-hand motifs; and a Ser-rich region containing a repeated GSRX motif. With similarities to both plakins and spectrins, trabeculin-alpha appears to have evolved as a hybrid of these two families of proteins. The functionality of the actin binding domains located near the N terminus was confirmed with an F-actin binding assay using glutathione S-transferase fusion proteins comprising amino acids 9-486 of the deduced peptide. Northern and Western blotting and immunofluorescence studies suggest that trabeculin is ubiquitously expressed and is distributed throughout the cytoplasm, though the protein was found to be greatly up-regulated upon differentiation of myoblasts into myotubes. Finally, the presence of cDNAs similar to, yet distinct from, trabeculin-alpha in both human and mouse suggests that trabeculins may form a new subfamily of giant actin-binding/cytoskeletal cross-linking proteins.

Identification and characterization of MAVS from black carp Mylopharyngodon piceus.

PubMed

Zhou, Wei; Zhou, Jujun; Lv, Ying; Qu, Yixiao; Chi, Mengdie; Li, Jun; Feng, Hao

2015-04-01

MAVS (mitochondria antiviral signaling protein) plays an important role in the host cellular innate immune response against microbial pathogens. In this study, MAVS has been cloned and characterized from black carp (Mylopharyngodon piceus). The full-length cDNA of black carp MAVS (bcMAVS) consists of 2352 nucleotides and the predicted bcMAVS protein contains 579 amino acids. Structural analysis showed that bcMAVS is composed of functional domains including an N-terminal CARD, a central proline-rich domain, a putative TRAF2-binding motif and a C-terminal TM domain, which is similar to mammalian MAVS. bcMAVS is constitutively transcribed in all the selected tissues including gill, kidney, heart, intestine, liver, muscle, skin and spleen; bcMAVS mRNA level in intestine, liver, muscle increased but decreased in spleen right after GCRV or SVCV infection. Multiple bands of bcMAVS were detected in western blot when it was expressed in tissue culture, which is similar to mammalian MAVS. Immunofluorescence assay determined that bcMAVS is a mitochondria protein and luciferase reporter assay demonstrated that bcMAVS could induce zebrafish IFN and EPC IFN expression in tissue culture. Data generated in this manuscript has built a solid foundation for further elucidating the function of bcMAVS in the innate immune system of black carp. Copyright © 2015 Elsevier Ltd. All rights reserved.

POLE and POLD1 screening in 155 patients with multiple polyps and early-onset colorectal cancer

PubMed Central

Esteban-Jurado, Clara; Giménez-Zaragoza, David; Muñoz, Jenifer; Franch-Expósito, Sebastià; Álvarez-Barona, Miriam; Ocaña, Teresa; Cuatrecasas, Miriam; Carballal, Sabela; López-Cerón, María; Marti-Solano, Maria; Díaz-Gay, Marcos; van Wezel, Tom; Castells, Antoni; Bujanda, Luis; Balmaña, Judith; Gonzalo, Victoria; Llort, Gemma; Ruiz-Ponte, Clara; Cubiella, Joaquín; Balaguer, Francesc; Aligué, Rosa; Castellví-Bel, Sergi

2017-01-01

Germline mutations in POLE and POLD1 have been shown to cause predisposition to colorectal multiple polyposis and a wide range of neoplasms, early-onset colorectal cancer being the most prevalent. In order to find additional mutations affecting the proofreading activity of these polymerases, we sequenced its exonuclease domain in 155 patients with multiple polyps or an early-onset colorectal cancer phenotype without alterations in the known hereditary colorectal cancer genes. Interestingly, none of the previously reported mutations in POLE and POLD1 were found. On the other hand, among the genetic variants detected, only two of them stood out as putative pathogenic in the POLE gene, c.1359 + 46del71 and c.1420G > A (p.Val474Ile). The first variant, detected in two families, was not proven to alter correct RNA splicing. Contrarily, c.1420G > A (p.Val474Ile) was detected in one early-onset colorectal cancer patient and located right next to the exonuclease domain. The pathogenicity of this change was suggested by its rarity and bioinformatics predictions, and it was further indicated by functional assays in Schizosaccharomyces pombe. This is the first study to functionally analyze a POLE genetic variant outside the exonuclease domain and widens the spectrum of genetic changes in this DNA polymerase that could lead to colorectal cancer predisposition. PMID:28423643

Identification of the functional domain in the transcription factor RTEF-1 that mediates alpha 1-adrenergic signaling in hypertrophied cardiac myocytes.

PubMed

Ueyama, T; Zhu, C; Valenzuela, Y M; Suzow, J G; Stewart, A F

2000-06-09

Cardiac myocytes respond to alpha(1)-adrenergic receptor stimulation by a progressive hypertrophy accompanied by the activation of many fetal genes, including skeletal muscle alpha-actin. The skeletal muscle alpha-actin gene is activated by signaling through an MCAT element, the binding site of the transcription enhancer factor-1 (TEF-1) family of transcription factors. Previously, we showed that overexpression of the TEF-1-related factor (RTEF-1) increased the alpha(1)-adrenergic response of the skeletal muscle alpha-actin promoter, whereas TEF-1 overexpression did not. Here, we identified the functional domains and specific sequences in RTEF-1 that mediate the alpha(1)-adrenergic response. Chimeric TEF-1 and RTEF-1 expression constructs localized the region responsible for the alpha(1)-adrenergic response to the carboxyl-terminal domain of RTEF-1. Site-directed mutagenesis was used to inactivate eight serine residues of RTEF-1, not present in TEF-1, that are putative targets of alpha(1)-adrenergic-dependent kinases. Mutation of a single serine residue, Ser-322, reduced the alpha(1)-adrenergic activation of RTEF-1 by 70% without affecting protein stability, suggesting that phosphorylation at this serine residue accounts for most of the alpha(1)-adrenergic response. Thus, these results demonstrate that RTEF-1 is a direct target of alpha(1)-adrenergic signaling in hypertrophied cardiac myocytes.

Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes.

PubMed

Ivarsson, Ylva; Arnold, Roland; McLaughlin, Megan; Nim, Satra; Joshi, Rakesh; Ray, Debashish; Liu, Bernard; Teyra, Joan; Pawson, Tony; Moffat, Jason; Li, Shawn Shun-Cheng; Sidhu, Sachdev S; Kim, Philip M

2014-02-18

The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-μM range. Furthermore, we identified several well-established host-virus protein-protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions.

The GDP-switched GAF domain of DcpA modulates the concerted synthesis/hydrolysis of c-di-GMP in Mycobacterium smegmatis.

PubMed

Chen, Hui-Jie; Li, Na; Luo, Ye; Jiang, Yong-Liang; Zhou, Cong-Zhao; Chen, Yuxing; Li, Qiong

2018-04-09

The second messenger c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate] plays a key role in bacterial growth, survival and pathogenesis, and thus its intracellular homeostasis should be finely maintained. Mycobacterium smegmatis encodes a GAF (mammalian c G MP-regulated phosphodiesterases, Anabaena a denylyl cyclases and Escherichia coli transcription activator F hlA) domain containing bifunctional enzyme DcpA ( d iguanylate c yclase and p hosphodiesterase A ) that catalyzes the synthesis and hydrolysis of c-di-GMP . Here, we found that M. smegmatis DcpA catalyzes the hydrolysis of c-di-GMP at a higher velocity, compared with synthetic activity, resulting in a sum reaction from the ultimate substrate GTP to the final product pGpG [5'-phosphoguanylyl-(3'-5')-guanosine]. Fusion with the N-terminal GAF domain enables the GGDEF (Gly-Gly-Asp-Glu-Phe) domain of DcpA to dimerize and accordingly gain synthetic activity. Screening of putative metabolites revealed that GDP is the ligand of the GAF domain. Binding of GDP to the GAF domain down-regulates synthetic activity, but up-regulates hydrolytic activity, which, in consequence, might enable a timely response to the transient accumulation of c-di-GMP at the stationary phase or under stresses. Combined with the crystal structure of the EAL (Glu-Ala-Leu) domain and the small-angle X-ray scattering data, we propose a putative regulatory model of the GAF domain finely tuned by the intracellular GTP/GDP ratio. These findings help us to better understand the concerted control of the synthesis and hydrolysis of c-di-GMP in M. smegmatis in various microenvironments. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Characterization of a stearoyl-acyl carrier protein desaturase gene family from chocolate tree, Theobroma cacao L.

PubMed

Zhang, Yufan; Maximova, Siela N; Guiltinan, Mark J

2015-01-01

In plants, the conversion of stearoyl-ACP to oleoyol-ACP is catalyzed by a plastid-localized soluble stearoyl-acyl carrier protein (ACP) desaturase (SAD). The activity of SAD significantly impacts the ratio of saturated and unsaturated fatty acids, and is thus a major determinant of fatty acid composition. The cacao genome contains eight putative SAD isoforms with high amino acid sequence similarities and functional domain conservation with SAD genes from other species. Sequence variation in known functional domains between different SAD family members suggested that these eight SAD isoforms might have distinct functions in plant development, a hypothesis supported by their diverse expression patterns in various cacao tissues. Notably, TcSAD1 is universally expressed across all the tissues, and its expression pattern in seeds is highly correlated with the dramatic change in fatty acid composition during seed maturation. Interestingly, TcSAD3 and TcSAD4 appear to be exclusively and highly expressed in flowers, functions of which remain unknown. To test the function of TcSAD1 in vivo, transgenic complementation of the Arabidopsis ssi2 mutant was performed, demonstrating that TcSAD1 successfully rescued all AtSSI2 related phenotypes further supporting the functional orthology between these two genes. The identification of the major SAD gene responsible for cocoa butter biosynthesis provides new strategies for screening for novel genotypes with desirable fatty acid compositions, and for use in breeding programs to help pyramid genes for quality and other traits such as disease resistance.

The Myb-domain protein ULTRAPETALA1 INTERACTING FACTOR 1 controls floral meristem activities in Arabidopsis.

PubMed

Moreau, Fanny; Thévenon, Emmanuel; Blanvillain, Robert; Lopez-Vidriero, Irene; Franco-Zorrilla, Jose Manuel; Dumas, Renaud; Parcy, François; Morel, Patrice; Trehin, Christophe; Carles, Cristel C

2016-04-01

Higher plants continuously and iteratively produce new above-ground organs in the form of leaves, stems and flowers. These organs arise from shoot apical meristems whose homeostasis depends on coordination between self-renewal of stem cells and their differentiation into organ founder cells. This coordination is stringently controlled by the central transcription factor WUSCHEL (WUS), which is both necessary and sufficient for stem cell specification in Arabidopsis thaliana ULTRAPETALA1 (ULT1) was previously identified as a plant-specific, negative regulator of WUS expression. However, molecular mechanisms underlying this regulation remain unknown. ULT1 protein contains a SAND putative DNA-binding domain and a B-box, previously proposed as a protein interaction domain in eukaryotes. Here, we characterise a novel partner of ULT1, named ULT1 INTERACTING FACTOR 1 (UIF1), which contains a Myb domain and an EAR motif. UIF1 and ULT1 function in the same pathway for regulation of organ number in the flower. Moreover, UIF1 displays DNA-binding activity and specifically binds to WUS regulatory elements. We thus provide genetic and molecular evidence that UIF1 and ULT1 work together in floral meristem homeostasis, probably by direct repression of WUS expression. © 2016. Published by The Company of Biologists Ltd.

Open and Lys–His Hexacoordinated Closed Structures of a Globin with Swapped Proximal and Distal Sites

PubMed Central

Teh, Aik-Hong; Saito, Jennifer A.; Najimudin, Nazalan; Alam, Maqsudul

2015-01-01

Globins are haem-binding proteins with a conserved fold made up of α-helices and can possess diverse properties. A putative globin-coupled sensor from Methylacidiphilum infernorum, HGbRL, contains an N-terminal globin domain whose open and closed structures reveal an untypical dimeric architecture. Helices E and F fuse into an elongated helix, resulting in a novel site-swapped globin fold made up of helices A–E, hence the distal site, from one subunit and helices F–H, the proximal site, from another. The open structure possesses a large cavity binding an imidazole molecule, while the closed structure forms a unique Lys–His hexacoordinated species, with the first turn of helix E unravelling to allow Lys52(E10) to bind to the haem. Ligand binding induces reorganization of loop CE, which is stabilized in the closed form, and helix E, triggering a large conformational movement in the open form. These provide a mechanical insight into how a signal may be relayed between the globin domain and the C-terminal domain of HGbRL, a Roadblock/LC7 domain. Comparison with HGbI, a closely related globin, further underlines the high degree of structural versatility that the globin fold is capable of, enabling it to perform a diversity of functions. PMID:26094577

Discovery-2: an interactive resource for the rational selection and comparison of putative drug target proteins in malaria

PubMed Central

2013-01-01

Background Drug resistance to anti-malarial compounds remains a serious problem, with resistance to newer pharmaceuticals developing at an alarming rate. The development of new anti-malarials remains a priority, and the rational selection of putative targets is a key element of this process. Discovery-2 is an update of the original Discovery in silico resource for the rational selection of putative drug target proteins, enabling researchers to obtain information for a protein which may be useful for the selection of putative drug targets, and to perform advanced filtering of proteins encoded by the malaria genome based on a series of molecular properties. Methods An updated in silico resource has been developed where researchers are able to mine information on malaria proteins and predicted ligands, as well as perform comparisons to the human and mosquito host characteristics. Protein properties used include: domains, motifs, EC numbers, GO terms, orthologs, protein-protein interactions, protein-ligand interactions. Newly added features include drugability measures from ChEMBL, automated literature relations and links to clinical trial information. Searching by chemical structure is also available. Results The updated functionality of the Discovery-2 resource is presented, together with a detailed case study of the Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase (PfSAHH) protein. A short example of a chemical search with pyrimethamine is also illustrated. Conclusion The updated Discovery-2 resource allows researchers to obtain detailed properties of proteins from the malaria genome, which may be of interest in the target selection process, and to perform advanced filtering and selection of proteins based on a relevant range of molecular characteristics. PMID:23537208

Structural model of FeoB, the iron transporter from Pseudomonas aeruginosa, predicts a cysteine lined, GTP-gated pore

PubMed Central

Seyedmohammad, Saeed; Fuentealba, Natalia Alveal; Marriott, Robert A.J.; Goetze, Tom A.; Edwardson, J. Michael; Barrera, Nelson P.; Venter, Henrietta

2016-01-01

Iron is essential for the survival and virulence of pathogenic bacteria. The FeoB transporter allows the bacterial cell to acquire ferrous iron from its environment, making it an excellent drug target in intractable pathogens. The protein consists of an N-terminal GTP-binding domain and a C-terminal membrane domain. Despite the availability of X-ray crystal structures of the N-terminal domain, many aspects of the structure and function of FeoB remain unclear, such as the structure of the membrane domain, the oligomeric state of the protein, the molecular mechanism of iron transport, and how this is coupled to GTP hydrolysis at the N-terminal domain. In the present study, we describe the first homology model of FeoB. Due to the lack of sequence homology between FeoB and other transporters, the structures of four different proteins were used as templates to generate the homology model of full-length FeoB, which predicts a trimeric structure. We confirmed this trimeric structure by both blue-native-PAGE (BN-PAGE) and AFM. According to our model, the membrane domain of the trimeric protein forms a central pore lined by highly conserved cysteine residues. This pore aligns with a central pore in the N-terminal GTPase domain (G-domain) lined by aspartate residues. Biochemical analysis of FeoB from Pseudomonas aeruginosa further reveals a putative iron sensor domain that could connect GTP binding/hydrolysis to the opening of the pore. These results indicate that FeoB might not act as a transporter, but rather as a GTP-gated channel. PMID:26934982

Molecular dynamics-based model of VEGF-A and its heparin interactions.

PubMed

Uciechowska-Kaczmarzyk, Urszula; Babik, Sándor; Zsila, Ferenc; Bojarski, Krzysztof Kamil; Beke-Somfai, Tamás; Samsonov, Sergey A

2018-06-01

We present a computational model of the Vascular Endothelial Growth Factor (VEGF), an important regulator of blood vessels formation, which function is affected by its heparin interactions. Although structures of a receptor binding (RBD) and a heparin binding domain (HBD) of VEGF are known, there are structural data neither on the 12 amino acids interdomain linker nor on its complexes with heparin. We apply molecular docking and molecular dynamics techniques combined with circular dichroism spectroscopy to model the full structure of the dimeric VEGF and to propose putative molecular mechanisms underlying the function of VEGF/VEGF receptors/heparin system. We show that both the conformational flexibility of the linker and the formation of HBD-heparin-HBD sandwich-like structures regulate the mutual disposition of HBDs and so affect the VEGF-mediated signalling. Copyright © 2018 Elsevier Inc. All rights reserved.

Structure of the novel monomeric glyoxalase I from Zea mays

PubMed Central

Turra, Gino L.; Agostini, Romina B.; Fauguel, Carolina M.; Presello, Daniel A.; Andreo, Carlos S.; González, Javier M.; Campos-Bermudez, Valeria A.

2015-01-01

The glyoxalase system is ubiquitous among all forms of life owing to its central role in relieving the cell from the accumulation of methylglyoxal, a toxic metabolic byproduct. In higher plants, this system is upregulated under diverse metabolic stress conditions, such as in the defence response to infection by pathogenic microorganisms. Despite their proven fundamental role in metabolic stresses, plant glyoxalases have been poorly studied. In this work, glyoxalase I from Zea mays has been characterized both biochemically and structurally, thus reporting the first atomic model of a glyoxalase I available from plants. The results indicate that this enzyme comprises a single polypeptide with two structurally similar domains, giving rise to two lateral concavities, one of which harbours a functional nickel(II)-binding active site. The putative function of the remaining cryptic active site remains to be determined. PMID:26457425

Structure of the novel monomeric glyoxalase I from Zea mays.

PubMed

Turra, Gino L; Agostini, Romina B; Fauguel, Carolina M; Presello, Daniel A; Andreo, Carlos S; González, Javier M; Campos-Bermudez, Valeria A

2015-10-01

The glyoxalase system is ubiquitous among all forms of life owing to its central role in relieving the cell from the accumulation of methylglyoxal, a toxic metabolic byproduct. In higher plants, this system is upregulated under diverse metabolic stress conditions, such as in the defence response to infection by pathogenic microorganisms. Despite their proven fundamental role in metabolic stresses, plant glyoxalases have been poorly studied. In this work, glyoxalase I from Zea mays has been characterized both biochemically and structurally, thus reporting the first atomic model of a glyoxalase I available from plants. The results indicate that this enzyme comprises a single polypeptide with two structurally similar domains, giving rise to two lateral concavities, one of which harbours a functional nickel(II)-binding active site. The putative function of the remaining cryptic active site remains to be determined.

In Silico Prediction and In Vitro Characterization of Multifunctional Human RNase3

PubMed Central

Kuo, Ping-Hsueh; Chen, Chien-Jung; Chang, Hsiu-Hui; Fang, Shun-lung; Wu, Wei-Shuo; Lai, Yiu-Kay; Pai, Tun-Wen; Chang, Margaret Dah-Tsyr

2013-01-01

Human ribonucleases A (hRNaseA) superfamily consists of thirteen members with high-structure similarities but exhibits divergent physiological functions other than RNase activity. Evolution of hRNaseA superfamily has gained novel functions which may be preserved in a unique region or domain to account for additional molecular interactions. hRNase3 has multiple functions including ribonucleolytic, heparan sulfate (HS) binding, cellular binding, endocytic, lipid destabilization, cytotoxic, and antimicrobial activities. In this study, three putative multifunctional regions, 34RWRCK38 (HBR1), 75RSRFR79 (HBR2), and 101RPGRR105 (HBR3), of hRNase3 have been identified employing in silico sequence analysis and validated employing in vitro activity assays. A heparin binding peptide containing HBR1 is characterized to act as a key element associated with HS binding, cellular binding, and lipid binding activities. In this study, we provide novel insights to identify functional regions of hRNase3 that may have implications for all hRNaseA superfamily members. PMID:23484086

Prokaryotic Caspase Homologs: Phylogenetic Patterns and Functional Characteristics Reveal Considerable Diversity

PubMed Central

Asplund-Samuelsson, Johannes; Bergman, Birgitta; Larsson, John

2012-01-01

Caspases accomplish initiation and execution of apoptosis, a programmed cell death process specific to metazoans. The existence of prokaryotic caspase homologs, termed metacaspases, has been known for slightly more than a decade. Despite their potential connection to the evolution of programmed cell death in eukaryotes, the phylogenetic distribution and functions of these prokaryotic metacaspase sequences are largely uncharted, while a few experiments imply involvement in programmed cell death. Aiming at providing a more detailed picture of prokaryotic caspase homologs, we applied a computational approach based on Hidden Markov Model search profiles to identify and functionally characterize putative metacaspases in bacterial and archaeal genomes. Out of the total of 1463 analyzed genomes, merely 267 (18%) were identified to contain putative metacaspases, but their taxonomic distribution included most prokaryotic phyla and a few archaea (Euryarchaeota). Metacaspases were particularly abundant in Alphaproteobacteria, Deltaproteobacteria and Cyanobacteria, which harbor many morphologically and developmentally complex organisms, and a distinct correlation was found between abundance and phenotypic complexity in Cyanobacteria. Notably, Bacillus subtilis and Escherichia coli, known to undergo genetically regulated autolysis, lacked metacaspases. Pfam domain architecture analysis combined with operon identification revealed rich and varied configurations among the metacaspase sequences. These imply roles in programmed cell death, but also e.g. in signaling, various enzymatic activities and protein modification. Together our data show a wide and scattered distribution of caspase homologs in prokaryotes with structurally and functionally diverse sub-groups, and with a potentially intriguing evolutionary role. These features will help delineate future characterizations of death pathways in prokaryotes. PMID:23185476

Putative DHHC-Cysteine-Rich Domain S-Acyltransferase in Plants

PubMed Central

Sun, Meihong; Liu, Shiyang; Qi, Baoxiu; Li, Xinzheng

2013-01-01

Protein S-acyltransferases (PATs) containing Asp-His-His-Cys within a Cys-rich domain (DHHC-CRD) are polytopic transmembrane proteins that are found in eukaryotic cells and mediate the S-acylation of target proteins. S-acylation is an important secondary and reversible modification that regulates the membrane association, trafficking and function of target proteins. However, little is known about the characteristics of PATs in plants. Here, we identified 804 PATs from 31 species with complete genomes. The analysis of the phylogenetic relationships suggested that all of the PATs fell into 8 groups. In addition, we analysed the phylogeny, genomic organization, chromosome localisation and expression pattern of PATs in Arabidopsis, Oryza sative, Zea mays and Glycine max. The microarray data revealed that PATs genes were expressed in different tissues and during different life stages. The preferential expression of the ZmPATs in specific tissues and the response of Zea mays to treatments with phytohormones and abiotic stress demonstrated that the PATs play roles in plant growth and development as well as in stress responses. Our data provide a useful reference for the identification and functional analysis of the members of this protein family. PMID:24155879

Depressive symptoms in schizophrenia and dopamine and serotonin gene polymorphisms.

PubMed

Peitl, Vjekoslav; Štefanović, Mario; Karlović, Dalibor

2017-07-03

Although depressive symptoms seem to be frequent in schizophrenia they have received significantly less attention than other symptom domains. As impaired serotonergic and dopaminergic neurotransmission is implicated in the pathogenesis of depression and schizophrenia this study sought to investigate the putative association between several functional gene polymorphisms (SERT 5-HTTLPR, MAO-A VNTR, COMT Val158Met and DAT VNTR) and schizophrenia. Other objectives of this study were to closely examine schizophrenia symptom domains by performing factor analysis of the two most used instruments in this setting (Positive and negative syndrome scale - PANSS and Calgary depression rating scale - CDSS) and to examine the influence of investigated gene polymorphisms on the schizophrenia symptom domains, focusing on depressive scores. A total of 591 participants were included in the study (300 schizophrenic patients and 291 healthy volunteers). 192 (64%) of schizophrenic patients had significant depressive symptoms. Genotype distribution revealed no significant differences regarding all investigated polymorphisms except the separate gender analysis for MAO-A gene polymorphism which revealed significantly more allele 3 carriers in schizophrenic males. Factor analysis of the PANSS scale revealed the existence of five separate factors (symptom domains), while the CDSS scale revealed two distinct factors. Several investigated gene polymorphisms (mostly SERT and MAO-A, but also COMT) significantly influenced two factors from the PANSS (aggressive/impulsive and negative symptoms) and one from the CDSS scale (suicidality), respectively. Depressive symptoms in schizophrenic patients may be influenced by functional gene polymorphisms, especially those implicated in serotonergic neurotransmission. Copyright © 2017 Elsevier Inc. All rights reserved.

The Functionally Conserved Nucleoporins Nup124p from Fission Yeast and the Human Nup153 Mediate Nuclear Import and Activity of the Tf1 Retrotransposon and HIV-1 VprV⃞

PubMed Central

Varadarajan, Padmapriya; Mahalingam, Sundarasamy; Liu, Peiyun; Ng, Sarah Boon Hsi; Gandotra, Sheetal; Dorairajoo, Desmond Suresh Kumar; Balasundaram, David

2005-01-01

We report that the fission yeast nucleoporin Nup124p is required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr. Failure to import Tf1-Gag into the nucleus in a nup124 null mutant resulted in complete loss of Tf1 transposition. Similarly, nuclear import of HIV-1 Vpr was impaired in nup124 null mutant strains and cells became resistant to Vpr's cell-killing activity. On the basis of protein domain similarity, the human nucleoporin Nup153 was identified as a putative homolog of Nup124p. We demonstrate that in vitro–translated Nup124p and Nup153 coimmunoprecipitate Tf1-Gag or HIV-1 Vpr. Though full-length Nup153 was unable to complement the Tf1 transposition defect in a nup124 null mutant, we provide evidence that both nucleoporins share a unique N-terminal domain, Nup124pAA264–454 and Nup153AA448–634 that is absolutely essential for Tf1 transposition. Epigenetic overexpression of this domain in a wild-type (nup124+) background blocked Tf1 activity implying that sequences from Nup124p and the human Nup153 challenged the same pathway affecting Tf1 transposition. Our results establish a unique relationship between two analogous nucleoporins Nup124p and Nup153 wherein the function of a common domain in retrotransposition is conserved. PMID:15659641

A putative carbohydrate-binding domain of the lactose-binding Cytisus sessilifolius anti-H(O) lectin has a similar amino acid sequence to that of the L-fucose-binding Ulex europaeus anti-H(O) lectin.

PubMed

Konami, Y; Yamamoto, K; Osawa, T; Irimura, T

1995-04-01

The complete amino acid sequence of a lactose-binding Cytisus sessilifolius anti-H(O) lectin II (CSA-II) was determined using a protein sequencer. After digestion of CSA-II with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSA-II with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid sequence of a putative carbohydrate-binding domain of CSA-II was found to be similar to those of several anti-H(O) leguminous lectins, especially to that of the L-fucose-binding Ulex europaeus lectin I (UEA-I).

Insight on an Arginine Synthesis Metabolon from the Tetrameric Structure of Yeast Acetylglutamate Kinase

PubMed Central

de Cima, Sergio; Gil-Ortiz, Fernando; Crabeel, Marjolaine; Fita, Ignacio; Rubio, Vicente

2012-01-01

N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ∼150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the −110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs. PMID:22529931

Insight on an arginine synthesis metabolon from the tetrameric structure of yeast acetylglutamate kinase.

PubMed

de Cima, Sergio; Gil-Ortiz, Fernando; Crabeel, Marjolaine; Fita, Ignacio; Rubio, Vicente

2012-01-01

N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ~150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the -110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs.

Regulation of EGF receptor signaling by the MARVEL domain-containing protein CKLFSF8.

PubMed

Jin, Caining; Ding, Peiguo; Wang, Ying; Ma, Dalong

2005-11-21

It is known that chemokine-like factor superfamily 8 (CKLFSF8), a member of the CKLF superfamily, has four putative transmembrane regions and a MARVEL domain. Its structure is similar to TM4SF11 (plasmolipin) and widely distributed in normal tissue. However, its function is not yet known. We show here that CKLFSF8 is associated with the epidermal growth factor receptor (EGFR) and that ectopic expression of CKLFSF8 in several cell lines suppresses EGF-induced cell proliferation, whereas knockdown of CKLFSF8 by siRNA promotes cell proliferation. In cells overexpressing CKLFSF8, the initial activation of EGFR was not affected, but subsequent desensitization of EGF-induced signaling occurred rapidly. This attenuation was correlated with an increased rate of receptor endocytosis. In contrast, knockdown of CKLFSF8 by siCKLFSF8 delayed EGFR endocytosis. These results identify CKLFSF8 as a novel regulator of EGF-induced signaling and indicate that the association of EGFR with four transmembrane proteins is critical for EGFR desensitization.

NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Dalei; Su, Xiaoyu; Potluri, Nalini

Here, the neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the widermore » mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.« less

NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors

DOE PAGES

Wu, Dalei; Su, Xiaoyu; Potluri, Nalini; ...

2016-10-26

Here, the neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the widermore » mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.« less

Tryptophan Scanning Reveals Dense Packing of Connexin Transmembrane Domains in Gap Junction Channels Composed of Connexin32.

PubMed

Brennan, Matthew J; Karcz, Jennifer; Vaughn, Nicholas R; Woolwine-Cunningham, Yvonne; DePriest, Adam D; Escalona, Yerko; Perez-Acle, Tomas; Skerrett, I Martha

2015-07-10

Tryptophan was substituted for residues in all four transmembrane domains of connexin32. Function was assayed using dual cell two-electrode voltage clamp after expression in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, with the greatest impact in TM1 and TM4. For instance, in TM1, 15 substitutions were made, six abolished coupling and five others significantly reduced function. Only TM2 and TM3 included a distinct helical face that lacked sensitivity to tryptophan substitution. Results were visualized on a comparative model of Cx32 hemichannel. In this model, a region midway through the membrane appears highly sensitive to tryptophan substitution and includes residues Arg-32, Ile-33, Met-34, and Val-35. In the modeled channel, pore-facing regions of TM1 and TM2 were highly sensitive to tryptophan substitution, whereas the lipid-facing regions of TM3 and TM4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also highly sensitive to tryptophan substitution. Although future studies will be required to separate trafficking-defective mutants from those that alter channel function, a subset of interactions important for voltage gating was identified. Interactions important for voltage gating occurred mainly in the mid-region of the channel and focused on TM1. To determine whether results could be extrapolated to other connexins, TM1 of Cx43 was scanned revealing similar but not identical sensitivity to TM1 of Cx32. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional analysis of recombinant human and Yarrowia lipolytica O-GlcNAc transferases expressed in Saccharomyces cerevisiae.

PubMed

Oh, Hye Ji; Moon, Hye Yun; Cheon, Seon Ah; Hahn, Yoonsoo; Kang, Hyun Ah

2016-10-01

O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.

Integrative analysis workflow for the structural and functional classification of C-type lectins

PubMed Central

2011-01-01

Background It is important to understand the roles of C-type lectins in the immune system due to their ubiquity and diverse range of functions in animal cells. It has been observed that currently confirmed C-type lectins share a highly conserved domain known as the C-type carbohydrate recognition domain (CRD). Using the sequence profile of the CRD, an increasing number of putative C-type lectins have been identified. Hence, it is highly needed to develop a systematic framework that enables us to elucidate their carbohydrate (glycan) recognition function, and discover their physiological and pathological roles. Results Presented herein is an integrated workflow for characterizing the sequence and structural features of novel C-type lectins. Our workflow utilizes web-based queries and available software suites to annotate features that can be found on the C-type lectin, given its amino acid sequence. At the same time, it incorporates modeling and analysis of glycans - a major class of ligands that interact with C-type lectins. Thereafter, the results are analyzed together with context-specific knowledge to filter off unlikely predictions. This allows researchers to design their subsequent experiments to confirm the functions of the C-type lectins in a systematic manner. Conclusions The efficacy and usefulness of our proposed immunoinformatics workflow was demonstrated by applying our integrated workflow to a novel C-type lectin -CLEC17A - and we report some of its possible functions that warrants further validation through wet-lab experiments. PMID:22372988

Structure and functional analysis of LptC, a conserved membrane protein involved in the lipopolysaccharide export pathway in Escherichia coli.

PubMed

Tran, An X; Dong, Changjiang; Whitfield, Chris

2010-10-22

LptC is a conserved bitopic inner membrane protein from Escherichia coli involved in the export of lipopolysaccharide from its site of synthesis in the cytoplasmic membrane to the outer membrane. LptC forms a complex with the ATP-binding cassette transporter, LptBFG, which is thought to facilitate the extraction of lipopolysaccharide from the inner membrane and release it into a translocation pathway that includes the putative periplasmic chaperone LptA. Cysteine modification experiments established that the catalytic domain of LptC is oriented toward the periplasm. The structure of the periplasmic domain is described at a resolution of 2.2-Å from x-ray crystallographic data. The periplasmic domain of LptC consists of a twisted boat structure with two β-sheets in apposition to each other. The β-sheets contain seven and eight antiparallel β-strands, respectively. This structure bears a high degree of resemblance to the crystal structure of LptA. Like LptA, LptC binds lipopolysaccharide in vitro. In vitro, LptA can displace lipopolysaccharide from LptC (but not vice versa), consistent with their locations and their proposed placement in a unidirectional export pathway.

Crystal structure of Escherichia coli L-arabinose isomerase (ECAI), the putative target of biological tagatose production.

PubMed

Manjasetty, Babu A; Chance, Mark R

2006-07-07

Escherichia coli L-arabinose isomerase (ECAI; EC 5.3.1.4) catalyzes the isomerization of L-arabinose to L-ribulose in vivo. This enzyme is also of commercial interest as it catalyzes the conversion of D-galactose to D-tagatose in vitro. The crystal structure of ECAI was solved and refined at 2.6 A resolution. The subunit structure of ECAI is organised into three domains: an N-terminal, a central and a C-terminal domain. It forms a crystallographic trimeric architecture in the asymmetric unit. Packing within the crystal suggests the idea that ECAI can form a hexameric assembly. Previous electron microscopic and biochemical studies supports that ECAI is hexameric in solution. A comparison with other known structures reveals that ECAI adopts a protein fold most similar to E. coli fucose isomerase (ECFI) despite very low sequence identity 9.7%. The structural similarity between ECAI and ECFI with regard to number of domains, overall fold, biological assembly, and active site architecture strongly suggests that the enzymes have functional similarities. Further, the crystal structure of ECAI forms a basis for identifying molecular determinants responsible for isomerization of arabinose to ribulose in vivo and galactose to tagatose in vitro.

TAIL1: an isthmin-like gene, containing type 1 thrombospondin-repeat and AMOP domain, mapped to ARVD1 critical region.

PubMed

Rossi, Valeria; Beffagna, Giorgia; Rampazzo, Alessandra; Bauce, Barbara; Danieli, Gian Antonio

2004-06-23

Isthmins represent a novel family of vertebrate secreted proteins containing one copy of the thrombospondin type 1 repeat (TSR), which in mammals is shared by several proteins with diverse biological functions, including cell adhesion, angiogenesis, and patterning of developing nervous system. We have determined the genomic organization of human TAIL1 (thrombospondin and AMOP containing isthmin-like 1), a novel isthmin-like gene encoding a protein that contains a TSR and a C-terminal AMOP domain (adhesion-associated domain in MUC4 and other proteins), characteristic of extracellular proteins involved in adhesion processes. TAIL1 gene encompasses more than 24.4 kb. Analysis of the DNA sequence surrounding the putative transcriptional start region revealed a TATA-less promoter located in a CpG island. Several consensus binding sites for the transcription factors Sp1 and MZF-1 were identified in this promoter region. In humans, TAIL1 gene is located on chromosome 14q24.3 within ARVD1 (arrhythmogenic right ventricular dysplasia/cardiomyopathy, type 1) critical region; preliminary evidence suggests that it is expressed in several tissues, showing multiple alternative splicing.

Crystal Structure of the FERM-SH2 Module of Human Jak2.

PubMed

McNally, Randall; Toms, Angela V; Eck, Michael J

2016-01-01

Jak-family tyrosine kinases mediate signaling from diverse cytokine receptors. Binding of Jaks to their cognate receptors is mediated by their N-terminal region, which contains FERM and SH2 domains. Here we describe the crystal structure of the FERM-SH2 region of Jak2 at 3.0Å resolution. The structure reveals that these domains and their flanking linker segments interact intimately to form an integrated structural module. The Jak2 FERM-SH2 structure closely resembles that recently described for Tyk2, another member of the Jak family. While the overall architecture and interdomain orientations are preserved between Jak2 and Tyk2, we identify residues in the putative receptor-binding groove that differ between the two and may contribute to the specificity of receptor recognition. Analysis of Jak mutations that are reported to disrupt receptor binding reveals that they lie in the hydrophobic core of the FERM domain, and are thus expected to compromise the structural integrity of the FERM-SH2 unit. Similarly, analysis of mutations in Jak3 that are associated with severe combined immunodeficiency suggests that they compromise Jak3 function by destabilizing the FERM-SH2 structure.

Glucagon-Like Peptide-1 Receptor Ligand Interactions: Structural Cross Talk between Ligands and the Extracellular Domain

PubMed Central

West, Graham M.; Willard, Francis S.; Sloop, Kyle W.; Showalter, Aaron D.; Pascal, Bruce D.; Griffin, Patrick R.

2014-01-01

Activation of the glucagon-like peptide-1 receptor (GLP-1R) in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM). Like other class B G protein-coupled receptors (GPCRs), the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX) to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R) were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R) peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R). In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands. PMID:25180755

Crystal Structure of the C-terminal Region of Streptococcus mutans Antigen I/II and Characterization of Salivary Agglutinin Adherence Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Crowley, Paula J.

2012-05-29

The Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein that adheres to salivary components and extracellular matrix molecules. Here we report the 2.5 {angstrom} resolution crystal structure of the complete C-terminal region of AgI/II. The C-terminal region is comprised of three major domains: C{sub 1}, C{sub 2}, and C{sub 3}. Each domain adopts a DE-variant IgG fold, with two {beta}-sheets whose A and F strands are linked through an intramolecular isopeptide bond. The adherence of the C-terminal AgI/II fragments to the putative tooth surface receptor salivary agglutinin (SAG), as monitored by surface plasmon resonance, indicated that the minimalmore » region of binding was contained within the first and second DE-variant-IgG domains (C{sub 1} and C{sub 2}) of the C terminus. The minimal C-terminal region that could inhibit S. mutans adherence to SAG was also confirmed to be within the C{sub 1} and C{sub 2} domains. Competition experiments demonstrated that the C- and N-terminal regions of AgI/II adhere to distinct sites on SAG. A cleft formed at the intersection between these C{sub 1} and C{sub 2} domains bound glucose molecules from the cryo-protectant solution, revealing a putative binding site for its highly glycosylated receptor SAG. Finally, electron microscopy images confirmed the elongated structure of AgI/II and enabled building a composite tertiary model that encompasses its two distinct binding regions.« less

Hematopoietic Colony Formation from Human Growth Factor-Dependent TF1 Cells and Human Cord Blood Myeloid Progenitor Cells Depends on SHP2 Phosphatase Function

PubMed Central

Etienne-Julan, Maryse; Gotoh, Akihiko; Braun, Stephen E.; Lu, Li; Cooper, Scott; Feng, Gen-Sheng; Li, Xing Jun

2013-01-01

The protein tyrosine phosphatase, SHP2, is widely expressed; however, previous studies demonstrated that hematopoietic cell development more stringently requires Shp2 expression compared to other tissues. Furthermore, somatic gain-of-function SHP2 mutants are commonly found in human myeloid leukemias. Given that pharmacologic inhibitors to SHP2 phosphatase activity are currently in development as putative antileukemic agents, we conducted a series of experiments examining the necessity of SHP2 phosphatase activity for human hematopoiesis. Anti-sense oligonucleotides to human SHP2 coding sequences reduced human cord blood- and human cell line, TF1-derived colony formation. Expression of truncated SHP2 bearing its Src homology 2 (SH2) domains, but lacking the phosphatase domain similarly reduced human cord blood- and TF1-derived colony formation. Mechanistically, expression of truncated SHP2 reduced the interaction between endogenous, full-length SHP2 with the adapter protein, Grb2. To verify the role of SHP2 phosphatase function in human hematopoietic cell development, human cord blood CD34+ cells were transduced with a leukemia-associated phosphatase gain-of-function SHP2 mutant or with a phosphatase dead SHP2 mutant, which indicated that increased phosphatase function enhanced, while decreased SHP2 phosphatase function reduced, human cord blood-derived colonies. Collectively, these findings indicate that SHP2 phosphatase function regulates human hematopoietic cell development and imply that the phosphatase component of SHP2 may serve as a pharmacologic target in human leukemias bearing increased SHP2 phosphatase activity. PMID:23082805

Hematopoietic colony formation from human growth factor-dependent TF1 cells and human cord blood myeloid progenitor cells depends on SHP2 phosphatase function.

PubMed

Broxmeyer, Hal E; Etienne-Julan, Maryse; Gotoh, Akihiko; Braun, Stephen E; Lu, Li; Cooper, Scott; Feng, Gen-Sheng; Li, Xing Jun; Chan, Rebecca J

2013-03-15

The protein tyrosine phosphatase, SHP2, is widely expressed; however, previous studies demonstrated that hematopoietic cell development more stringently requires Shp2 expression compared to other tissues. Furthermore, somatic gain-of-function SHP2 mutants are commonly found in human myeloid leukemias. Given that pharmacologic inhibitors to SHP2 phosphatase activity are currently in development as putative antileukemic agents, we conducted a series of experiments examining the necessity of SHP2 phosphatase activity for human hematopoiesis. Anti-sense oligonucleotides to human SHP2 coding sequences reduced human cord blood- and human cell line, TF1-derived colony formation. Expression of truncated SHP2 bearing its Src homology 2 (SH2) domains, but lacking the phosphatase domain similarly reduced human cord blood- and TF1-derived colony formation. Mechanistically, expression of truncated SHP2 reduced the interaction between endogenous, full-length SHP2 with the adapter protein, Grb2. To verify the role of SHP2 phosphatase function in human hematopoietic cell development, human cord blood CD34+ cells were transduced with a leukemia-associated phosphatase gain-of-function SHP2 mutant or with a phosphatase dead SHP2 mutant, which indicated that increased phosphatase function enhanced, while decreased SHP2 phosphatase function reduced, human cord blood-derived colonies. Collectively, these findings indicate that SHP2 phosphatase function regulates human hematopoietic cell development and imply that the phosphatase component of SHP2 may serve as a pharmacologic target in human leukemias bearing increased SHP2 phosphatase activity.

Topologically Diverse Human Membrane Proteins Partition to Liquid-Disordered Domains in Phase-Separated Lipid Vesicles.

PubMed

Schlebach, Jonathan P; Barrett, Paul J; Day, Charles A; Kim, Ji Hun; Kenworthy, Anne K; Sanders, Charles R

2016-02-23

The integration of membrane proteins into "lipid raft" membrane domains influences many biochemical processes. The intrinsic structural properties of membrane proteins are thought to mediate their partitioning between membrane domains. However, whether membrane topology influences the targeting of proteins to rafts remains unclear. To address this question, we examined the domain preference of three putative raft-associated membrane proteins with widely different topologies: human caveolin-3, C99 (the 99 residue C-terminal domain of the amyloid precursor protein), and peripheral myelin protein 22. We find that each of these proteins are excluded from the ordered domains of giant unilamellar vesicles containing coexisting liquid-ordered and liquid-disordered phases. Thus, the intrinsic structural properties of these three topologically distinct disease-linked proteins are insufficient to confer affinity for synthetic raft-like domains.

The BOS1 gene encodes an essential 27-kD putative membrane protein that is required for vesicular transport from the ER to the Golgi complex in yeast

PubMed Central

1991-01-01

We recently described the identification of BOS1 (Newman, A., J. Shim, and S. Ferro-Novick. 1990. Mol. Cell. Biol. 10:3405-3414.). BOS1 is a gene that in multiple copy suppresses the growth and secretion defect of bet1 and sec22, two mutants that disrupt transport from the ER to the Golgi complex in yeast. The ability of BOS1 to specifically suppress mutants blocked at a particular stage of the secretory pathway suggested that this gene encodes a protein that functions in this process. The experiments presented in this study support this hypothesis. Specifically, the BOS1 gene was found to be essential for cellular growth. Furthermore, cells depleted of the Bos1 protein fail to transport pro-alpha-factor and carboxypeptidase Y (CPY) to the Golgi apparatus. This defect in export leads to the accumulation of an extensive network of ER and small vesicles. DNA sequence analysis predicts that Bos1 is a 27-kD protein containing a putative membrane- spanning domain. This prediction is supported by differential centrifugation experiments. Thus, Bos1 appears to be a membrane protein that functions in conjunction with Bet1 and Sec22 to facilitate the transport of proteins at a step subsequent to translocation into the ER but before entry into the Golgi apparatus. PMID:2007627

Identification and Functional Characterization of a Tonoplast Dicarboxylate Transporter in Tomato (Solanum lycopersicum)

PubMed Central

Liu, Ruiling; Li, Boqiang; Qin, Guozheng; Zhang, Zhanquan; Tian, Shiping

2017-01-01

Acidity plays an important role in flavor and overall organoleptic quality of fruit and is mainly due to the presence of organic acids. Understanding the molecular basis of organic acid metabolism is thus of primary importance for fruit quality improvement. Here, we cloned a putative tonoplast dicarboxylate transporter gene (SlTDT) from tomato, and submitted it to the NCBI database (GenBank accession number: KC733165). SlTDT protein contained 13 putative transmembrane domains in silico analysis. Confocal microscopic study using green fluorescent fusion proteins revealed that SlTDT was localized on tonoplast. The expression patterns of SlTDT in tomato were analyzed by RT-qPCR. The results indicated that SlTDT expressed in leaves, roots, flowers and fruits at different ripening stages, suggesting SlTDT may be associated with the development of different tissues. To further explore the function of SlTDT, we constructed both overexpression and RNAi vectors and obtained transgenic tomato plants by agrobacterium-mediated method. Gas chromatography-mass spectrometer (GC-MS) analysis showed that overexpression of SlTDT significantly increased malate content, and reduced citrate content in tomato fruit. By contrast, repression of SlTDT in tomato reduced malate content of and increased citrate content. These results indicated that SlTDT played an important role in remobilization of malate and citrate in fruit vacuoles. PMID:28261242

Identification and Functional Characterization of a Tonoplast Dicarboxylate Transporter in Tomato (Solanum lycopersicum).

PubMed

Liu, Ruiling; Li, Boqiang; Qin, Guozheng; Zhang, Zhanquan; Tian, Shiping

2017-01-01

Acidity plays an important role in flavor and overall organoleptic quality of fruit and is mainly due to the presence of organic acids. Understanding the molecular basis of organic acid metabolism is thus of primary importance for fruit quality improvement. Here, we cloned a putative tonoplast dicarboxylate transporter gene ( SlTDT ) from tomato, and submitted it to the NCBI database (GenBank accession number: KC733165). SlTDT protein contained 13 putative transmembrane domains in silico analysis. Confocal microscopic study using green fluorescent fusion proteins revealed that SlTDT was localized on tonoplast. The expression patterns of SlTDT in tomato were analyzed by RT-qPCR. The results indicated that SlTDT expressed in leaves, roots, flowers and fruits at different ripening stages, suggesting SlTDT may be associated with the development of different tissues. To further explore the function of SlTDT , we constructed both overexpression and RNAi vectors and obtained transgenic tomato plants by agrobacterium-mediated method. Gas chromatography-mass spectrometer (GC-MS) analysis showed that overexpression of SlTDT significantly increased malate content, and reduced citrate content in tomato fruit. By contrast, repression of SlTDT in tomato reduced malate content of and increased citrate content. These results indicated that SlTDT played an important role in remobilization of malate and citrate in fruit vacuoles.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hiyoshi, Masateru; Hashimoto, Michihiro; Yukihara, Mamiko

Highlights: •Many mutations were identified in Fms as a putative genetic cause of HDLS. •All of the mutations tested severely impair the kinase activity. •Most of the mutations also impair the trafficking to the cell surface. •These defects further suggest that HDLS is caused by a loss of Fms function. -- Abstract: The tyrosine kinase Fms, the cell surface receptor for M-CSF and IL-34, is critical for microglial proliferation and differentiation in the brain. Recently, a number of mutations have been identified in Fms as a putative genetic cause of hereditary diffuse leukoencephalopathy with spheroids (HDLS), implying an important rolemore » of microglial dysfunction in HDLS pathogenesis. In this study, we initially confirmed that 11 mutations, which reside within the ATP-binding or major tyrosine kinase domain, caused a severe impairment of ligand-induced Fms auto-phosphorylation. Intriguingly, we found that 10 of the 11 mutants also showed a weak cell surface expression, which was associated with a concomitant increase in the low molecular weight hypo-N-glycosylated immature gp130Fms-like species. Indeed, the mutant proteins heavily accumulated to the Golgi-like perinuclear regions. These results indicate that all of the Fms mutations tested severely impair the kinase activity and most of the mutations also impair the trafficking to the cell surface, further suggesting that HDLS is caused by the loss of Fms function.« less

Disruption of the methyltransferase-like 23 gene METTL23 causes mild autosomal recessive intellectual disability.

PubMed

Bernkopf, Marie; Webersinke, Gerald; Tongsook, Chanakan; Koyani, Chintan N; Rafiq, Muhammad A; Ayaz, Muhammad; Müller, Doris; Enzinger, Christian; Aslam, Muhammad; Naeem, Farooq; Schmidt, Kurt; Gruber, Karl; Speicher, Michael R; Malle, Ernst; Macheroux, Peter; Ayub, Muhammad; Vincent, John B; Windpassinger, Christian; Duba, Hans-Christoph

2014-08-01

We describe the characterization of a gene for mild nonsyndromic autosomal recessive intellectual disability (ID) in two unrelated families, one from Austria, the other from Pakistan. Genome-wide single nucleotide polymorphism microarray analysis enabled us to define a region of homozygosity by descent on chromosome 17q25. Whole-exome sequencing and analysis of this region in an affected individual from the Austrian family identified a 5 bp frameshifting deletion in the METTL23 gene. By means of Sanger sequencing of METTL23, a nonsense mutation was detected in a consanguineous ID family from Pakistan for which homozygosity-by-descent mapping had identified a region on 17q25. Both changes lead to truncation of the putative METTL23 protein, which disrupts the predicted catalytic domain and alters the cellular localization. 3D-modelling of the protein indicates that METTL23 is strongly predicted to function as an S-adenosyl-methionine (SAM)-dependent methyltransferase. Expression analysis of METTL23 indicated a strong association with heat shock proteins, which suggests that these may act as a putative substrate for methylation by METTL23. A number of methyltransferases have been described recently in association with ID. Disruption of METTL23 presented here supports the importance of methylation processes for intact neuronal function and brain development. © The Author 2014. Published by Oxford University Press.

Subcellular localization and vacuolar targeting of sorbitol dehydrogenase in apple seed.

PubMed

Wang, Xiu-Ling; Hu, Zi-Ying; You, Chun-Xiang; Kong, Xiu-Zhen; Shi, Xiao-Pu

2013-09-01

Sorbitol is the primary photosynthate and translocated carbohydrate in fruit trees of the Rosaceae family. NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), which mainly catalyzes the oxidation of sorbitol to fructose, plays a key role in regulating sink strength in apple. In this study, we found that apple NAD-SDH was ubiquitously distributed in epidermis, parenchyma, and vascular bundle in developing cotyledon. NAD-SDH was localized in the cytosol, the membranes of endoplasmic reticulum and vesicles, and the vacuolar lumen in the cotyledon at the middle stage of seed development. In contrast, NAD-SDH was mainly distributed in the protein storage vacuoles in cotyledon at the late stage of seed development. Sequence analysis revealed there is a putative signal peptide (SP), also being predicated to be a transmembrane domain, in the middle of proteins of apple NAD-SDH isoforms. To investigate whether the putative internal SP functions in the vacuolar targeting of NAD-SDH, we analyzed the localization of the SP-deletion mutants of MdSDH5 and MdSDH6 (two NAD-SDH isoforms in apple) by the transient expression system in Arabidopsis protoplasts. MdSDH5 and MdSDH6 were not localized in the vacuoles after their SPs were deleted, suggesting the internal SP functions in the vacuolar targeting of apple NAD-SDH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Knowledge Representation and Ontologies

NASA Astrophysics Data System (ADS)

Grimm, Stephan

Knowledge representation and reasoning aims at designing computer systems that reason about a machine-interpretable representation of the world. Knowledge-based systems have a computational model of some domain of interest in which symbols serve as surrogates for real world domain artefacts, such as physical objects, events, relationships, etc. [1]. The domain of interest can cover any part of the real world or any hypothetical system about which one desires to represent knowledge for com-putational purposes. A knowledge-based system maintains a knowledge base, which stores the symbols of the computational model in the form of statements about the domain, and it performs reasoning by manipulating these symbols. Applications can base their decisions on answers to domain-relevant questions posed to a knowledge base.

Regulation of activation and processing of the cystic fibrosis transmembrane conductance regulator (CFTR) by a complex electrostatic interaction between the regulatory domain and cytoplasmic loop 3.

PubMed

Wang, Guangyu; Duan, Dayue Darrel

2012-11-23

NEG2 regulates CFTR gating but the mechanism is unknown. A putative NEG2-CL3 electrostatic attraction, possibly weakened by Arg-764/Arg-766 of the R domain, prohibited CFTR activation. A charge exchange between NEG2 and CL3 caused misprocessing. Electrostatic regulation of CFTR activation and processing may be asymmetric at the CL3-R interface. The CL3-R interface is optimally designed for multiple regulations of CFTR functions. NEG2, a short C-terminal segment (817-838) of the unique regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, has been reported to regulate CFTR gating in response to cAMP-dependent R domain phosphorylation. The underlying mechanism, however, is unclear. Here, Lys-946 of cytoplasmic loop 3 (CL3) is proposed as counter-ion of Asp-835, Asp-836, or Glu-838 of NEG2 to prevent the channel activation by PKA. Arg-764 or Arg-766 of the Ser-768 phosphorylation site of the R domain is proposed to promote the channel activation possibly by weakening the putative CL3-NEG2 electrostatic attraction. First, not only D835A, D836A, and E838A but also K946A reduced the PKA-dependent CFTR activation. Second, both K946D and D835R/D836R/E838R mutants were activated by ATP and curcumin to a different extent. Third, R764A and R766A mutants enhanced the PKA-dependent activation. However, it is very exciting that D835R/D836R/E838R and K946D/H950D and H950R exhibited normal channel processing and activity whereas D835R/D836R/E838R/K946D/H950D was fractionally misprocessed and silent in response to forskolin. Further, D836R and E838R played a critical role in the asymmetric electrostatic regulation of CFTR processing, and Ser-768 phosphorylation may not be involved. Thus, a complex interfacial interaction among CL3, NEG2, and the Ser-768 phosphorylation site may be responsible for the asymmetric electrostatic regulation of CFTR activation and processing.

Recurrent nonsense mutations in the growth hormone receptor from patients with Laron dwarfism.

PubMed Central

Amselem, S; Sobrier, M L; Duquesnoy, P; Rappaport, R; Postel-Vinay, M C; Gourmelen, M; Dallapiccola, B; Goossens, M

1991-01-01

In addition to its classical effects on growth, growth hormone (GH) has been shown to have a number of other actions, all of which are initiated by an interaction with specific high affinity receptors present in a variety of tissues. Purification of a rabbit liver protein via its ability to bind GH has allowed the isolation of a cDNA encoding a putative human growth hormone receptor that belongs to a new class of transmembrane receptors. We have previously shown that this putative growth hormone receptor gene is genetically linked to Laron dwarfism, a rare autosomal recessive syndrome caused by target resistance to GH. Nevertheless, the inability to express the corresponding full-length coding sequence and the lack of a test for growth-promoting function have hampered a direct confirmation of its role in growth. We have now identified three nonsense mutations within this growth hormone receptor gene, lying at positions corresponding to the amino terminal extremity and causing a truncation of the molecule, thereby deleting a large portion of both the GH binding domain and the full transmembrane and intracellular domains. Three independent patients with Laron dwarfism born of consanguineous parents were homozygous for these defects. Two defects were identical and consisted of a CG to TG transition. Not only do these results confirm the growth-promoting activity of this receptor but they also suggest that CpG doublets may represent hot spots for mutations in the growth hormone receptor gene that are responsible for hereditary dwarfism. Images PMID:1999489

Computational Identification and Comparative Analysis of Secreted and Transmembrane Proteins in Six Burkholderia Species.

PubMed

Nguyen, Thao Thi; Lee, Hyun-Hee; Park, Jungwook; Park, Inmyoung; Seo, Young-Su

2017-04-01

As a step towards discovering novel pathogenesis-related proteins, we performed a genome scale computational identification and characterization of secreted and transmembrane (TM) proteins, which are mainly responsible for bacteria-host interactions and interactions with other bacteria, in the genomes of six representative Burkholderia species. The species comprised plant pathogens ( B. glumae BGR1, B. gladioli BSR3), human pathogens ( B. pseudomallei K96243, B. cepacia LO6), and plant-growth promoting endophytes ( Burkholderia sp. KJ006, B. phytofirmans PsJN). The proportions of putative classically secreted proteins (CSPs) and TM proteins among the species were relatively high, up to approximately 20%. Lower proportions of putative type 3 non-classically secreted proteins (T3NCSPs) (~10%) and unclassified non-classically secreted proteins (NCSPs) (~5%) were observed. The numbers of TM proteins among the three clusters (plant pathogens, human pathogens, and endophytes) were different, while the distribution of these proteins according to the number of TM domains was conserved in which TM proteins possessing 1, 2, 4, or 12 TM domains were the dominant groups in all species. In addition, we observed conservation in the protein size distribution of the secreted protein groups among the species. There were species-specific differences in the functional characteristics of these proteins in the various groups of CSPs, T3NCSPs, and unclassified NCSPs. Furthermore, we assigned the complete sets of the conserved and unique NCSP candidates of the collected Burkholderia species using sequence similarity searching. This study could provide new insights into the relationship among plant-pathogenic, human-pathogenic, and endophytic bacteria.

Sequencing, bioinformatic characterization and expression pattern of a putative amino acid transporter from the parasitic cestode Echinococcus granulosus.

PubMed

Camicia, Federico; Paredes, Rodolfo; Chalar, Cora; Galanti, Norbel; Kamenetzky, Laura; Gutierrez, Ariana; Rosenzvit, Mara C

2008-03-31

We have sequenced and partially characterized an Echinococcus granulosus cDNA, termed egat1, from a protoscolex signal sequence trap (SST) cDNA library. The isolated 1627 bp long cDNA contains an ORF of 489 amino acids and shows an amino acid identity of 30% with neutral and excitatory amino acid transporters members of the Dicarboxylate/Amino Acid Na+ and/or H+ Cation Symporter family (DAACS) (TC 2.A.23). Additional bioinformatics analysis of EgAT1, confirmed the results obtained by similarity searches and showed the presence of 9 to 10 transmembrane domains, consensus sequences for N-glycosylation between the third and fourth transmembrane domain, a highly similar hydropathy profile with ASCT1 (a known member of DAACS family), high score with SDF (Sodium Dicarboxilate Family) and similar motifs with EDTRANSPORT, a fingerprint of excitatory amino acid transporters. The localization of the putative amino acid transporter was analyzed by in situ hybridization and immunofluorescence in protoscoleces and associated germinal layer. The in situ hybridization labelling indicates the distribution of egat1 mRNA throughout the tegument. EgAT1 protein, which showed in Western blots a molecular mass of approximately 60 kD, is localized in the subtegumental region of the metacestode, particularly around suckers and rostellum of protoscoleces and layers from brood capsules. The sequence and expression analyses of EgAT1 pave the way for functional analysis of amino acids transporters of E. granulosus and its evaluation as new drug targets against cystic echinococcosis.

Structural and Genetic Analyses of the Mycobacterium tuberculosis Protein Kinase B Sensor Domain Identify a Potential Ligand-binding Site.

PubMed

Prigozhin, Daniil M; Papavinasasundaram, Kadamba G; Baer, Christina E; Murphy, Kenan C; Moskaleva, Alisa; Chen, Tony Y; Alber, Tom; Sassetti, Christopher M

2016-10-28

Monitoring the environment with serine/threonine protein kinases is critical for growth and survival of Mycobacterium tuberculosis, a devastating human pathogen. Protein kinase B (PknB) is a transmembrane serine/threonine protein kinase that acts as an essential regulator of mycobacterial growth and division. The PknB extracellular domain (ECD) consists of four repeats homologous to penicillin-binding protein and serine/threonine kinase associated (PASTA) domains, and binds fragments of peptidoglycan. These properties suggest that PknB activity is modulated by ECD binding to peptidoglycan substructures, however, the molecular mechanisms underpinning PknB regulation remain unclear. In this study, we report structural and genetic characterization of the PknB ECD. We determined the crystal structures of overlapping ECD fragments at near atomic resolution, built a model of the full ECD, and discovered a region on the C-terminal PASTA domain that has the properties of a ligand-binding site. Hydrophobic interaction between this surface and a bound molecule of citrate was observed in a crystal structure. Our genetic analyses in M. tuberculosis showed that nonfunctional alleles were produced either by deletion of any of single PASTA domain or by mutation of individual conserved residues lining the putative ligand-binding surface of the C-terminal PASTA repeat. These results define two distinct structural features necessary for PknB signal transduction, a fully extended ECD and a conserved, membrane-distal putative ligand-binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Isolation and Expression Analysis of CYP9A11 and Cytochrome P450 Reductase Gene in the Beet Armyworm (Lepidoptera: Noctuidae)

PubMed Central

Zhao, Chunqing; Feng, Xiaoyun; Tang, Tao; Qiu, Lihong

2015-01-01

Cytochrome P450 monooxygenases (CYPs), as an enzyme superfamily, is widely distributed in organisms and plays a vital function in the metabolism of exogenous and endogenous compounds by interacting with its obligatory redox partner, CYP reductase (CPR). A novel CYP gene (CYP9A11) and CPR gene from the agricultural pest insect Spodoptera exigua were cloned and characterized. The complete cDNA sequences of SeCYP9A11 and SeCPR are 1,931 and 3,919 bp in length, respectively, and contain open reading frames of 1,593 and 2,070 nucleotides, respectively. Analysis of the putative protein sequences indicated that SeCYP9A11 contains a heme-binding domain and the unique characteristic sequence (SRFALCE) of the CYP9 family, in addition to a signal peptide and transmembrane segment at the N-terminal. Alignment analysis revealed that SeCYP9A11 shares the highest sequence similarity with CYP9A13 from Mamestra brassicae, which is 66.54%. The putative protein sequence of SeCPR has all of the classical CPR features, such as an N-terminal membrane anchor; three conserved domain flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) domain; and characteristic binding motifs. Phylogenetic analysis revealed that SeCPR shares the highest identity with HaCPR, which is 95.21%. The SeCYP9A11 and SeCPR genes were detected in the midgut, fat body, and cuticle tissues, and throughout all of the developmental stages of S. exigua. The mRNA levels of SeCYP9A11 and SeCPR decreased remarkably after exposure to plant secondary metabolites quercetin and tannin. The results regarding SeCYP9A11 and SeCPR genes in the current study provide foundation for the further study of S. exigua P450 system. PMID:26320261

Mutations in the voltage-sensing domain affect the alternative ion permeation pathway in the TRPM3 channel.

PubMed

Held, Katharina; Gruss, Fabian; Aloi, Vincenzo Davide; Janssens, Annelies; Ulens, Chris; Voets, Thomas; Vriens, Joris

2018-03-31

Mutagenesis at positively charged amino acids (arginines and lysines) (R1-R4) in the voltage-sensor domain (transmembrane segment (S) 4) of voltage-gated Na + , K + and Ca 2+ channels can lead to an alternative ion permeation pathway distinct from the central pore. Recently, a non-canonical ion permeation pathway was described in TRPM3, a member of the transient receptor potential (TRP) superfamily. The non-canonical pore exists in the native TRPM3 channel and can be activated by co-stimulation of the endogenous agonist pregnenolone sulphate and the antifungal drug clotrimazole or by stimulation of the synthetic agonist CIM0216. Alignment of the voltage sensor of Shaker K + channels with the entire TRPM3 sequence revealed the highest degree of similarity in the putative S4 region of TRPM3, and suggested that only one single gating charge arginine (R2) in the putative S4 region is conserved. Mutagenesis studies in the voltage-sensing domain of TRPM3 revealed several residues in the voltage sensor (S4) as well as in S1 and S3 that are crucial for the occurrence of the non-canonical inward currents. In conclusion, this study provides evidence for the involvement of the voltage-sensing domain of TRPM3 in the formation of an alternative ion permeation pathway. Transient receptor potential (TRP) channels are cationic channels involved in a broad array of functions, including homeostasis, motility and sensory functions. TRP channel subunits consist of six transmembrane segments (S1-S6), and form tetrameric channels with a central pore formed by the region encompassing S5 and S6. Recently, evidence was provided for the existence of an alternative ion permeation pathway in TRPM3, which allows large inward currents upon hyperpolarization independently of the central pore. However, very little knowledge is available concerning the localization of this alternative pathway in the native TRPM3 channel protein. Guided by sequence homology with Shaker K + channels, in which mutations in S4 can create an analogous 'omega' pore, we performed site-directed mutagenesis studies and patch clamp experiments to identify amino acid residues involved in the formation of the non-canonical pore in TRPM3. Based on our results, we pinpoint four residues in S4 (W982, R985, D988 and G991) as crucial determinants of the properties of the alternative ion permeation pathway. © 2018 KU Leuven The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Mycobacterium tuberculosis universal stress protein Rv2623 interacts with the putative ATP binding cassette (ABC) transporter Rv1747 to regulate mycobacterial growth.

PubMed

Glass, Lisa N; Swapna, Ganduri; Chavadi, Sivagami Sundaram; Tufariello, JoAnn M; Mi, Kaixia; Drumm, Joshua E; Lam, TuKiet T; Zhu, Guofeng; Zhan, Chenyang; Vilchéze, Catherine; Arcos, Jesus; Chen, Yong; Bi, Lijun; Mehta, Simren; Porcelli, Steven A; Almo, Steve C; Yeh, Syun-Ru; Jacobs, William R; Torrelles, Jordi B; Chan, John

2017-07-01

We have previously shown that the Mycobacterium tuberculosis universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I) of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT)-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623WT), a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623T237A) exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an M. tuberculosis Rv2623 null mutant (ΔRv2623) displays enhanced expression of phosphatidyl-myo-inositol mannosides (PIMs), while the ΔRv1747 mutant expresses decreased levels of PIMs. Animal studies have previously shown that ΔRv2623 is hypervirulent, while ΔRv1747 is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing in vivo growth phenotypes of ΔRv2623 and ΔRv1747, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747; and that Rv1747 might function as a transporter of PIMs. Because these glycolipids are major mycobacterial cell envelope components that can impact on the immune response, our findings raise the possibility that Rv2623 may regulate bacterial growth, virulence, and entry into persistence, at least in part, by modulating the levels of bacillary PIM expression, perhaps through negatively regulating the Rv1747-dependent export of the immunomodulatory PIMs to alter host-pathogen interaction, thereby influencing the fate of M. tuberculosis in vivo.

Evolution of Saxitoxin Synthesis in Cyanobacteria and Dinoflagellates

PubMed Central

Hackett, Jeremiah D.; Wisecaver, Jennifer H.; Brosnahan, Michael L.; Kulis, David M.; Anderson, Donald M.; Bhattacharya, Debashish; Plumley, F. Gerald; Erdner, Deana L.

2013-01-01

Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. Saxitoxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX synthesis genes, including all of the genes directly involved in toxin synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxitoxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some evolutionarily related proteins. The biological role of STX is not well understood in either cyanobacteria or dinoflagellates. However, STX production in these two ecologically distinct groups of organisms suggests that this toxin confers a benefit to producers that we do not yet fully understand. PMID:22628533

Evolution of saxitoxin synthesis in cyanobacteria and dinoflagellates.

PubMed

Hackett, Jeremiah D; Wisecaver, Jennifer H; Brosnahan, Michael L; Kulis, David M; Anderson, Donald M; Bhattacharya, Debashish; Plumley, F Gerald; Erdner, Deana L

2013-01-01

Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. Saxitoxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX synthesis genes, including all of the genes directly involved in toxin synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxitoxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some evolutionarily related proteins. The biological role of STX is not well understood in either cyanobacteria or dinoflagellates. However, STX production in these two ecologically distinct groups of organisms suggests that this toxin confers a benefit to producers that we do not yet fully understand.

Associations between a neurophysiological marker of central cholinergic activity and cognitive functions in young and older adults

PubMed Central

2012-01-01

Background The deterioration of the central cholinergic system in aging is hypothesized to underlie declines in several cognitive domains, including memory and executive functions. However, there is surprisingly little direct evidence regarding acetylcholine’s specific role(s) in normal human cognitive aging. Methods We used short-latency afferent inhibition (SAI) with transcranial magnetic stimulation (TMS) as a putative marker of cholinergic activity in vivo in young (n = 24) and older adults (n = 31). Results We found a significant age difference in SAI, concordant with other evidence of cholinergic decline in normal aging. We also found clear age differences on several of the memory and one of the executive function measures. Individual differences in SAI levels predicted memory but not executive functions. Conclusion Individual differences in SAI levels were better predictors of memory than executive functions. We discuss cases in which the relations between SAI and cognition might be even stronger, and refer to other age-related biological changes that may interact with cholinergic activity in cognitive aging. PMID:22537877

Characterization of ROS1 cDNA from a human glioblastoma cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Birchmeier, C.; O'Neill, K.; Riggs, M.

1990-06-01

The authors have isolated and characterized a human ROS1 cDNA from the glioblastoma cell line SW-1088. The cDNA, 8.3 kilobases long, has the potential to encode a transmembrane tyrosine-specific protein kinase with a predicted molecular mass of 259 kDa. The putative extracellular domain of ROS1 is homologous to the extracellular domain of the sevenless gene product from Drosophila. No comparable similarities in the extracellular domains were found between ROS1 and other receptor-type tyrosine kinases. Together, ROS1 and sevenless gene products define a distinct subclass of transmember tyrosine kinases.

Transcriptional repression mediated by the KRAB domain of the human C2H2 zinc finger protein Kox1/ZNF10 does not require histone deacetylation.

PubMed

Lorenz, P; Koczan, D; Thiesen, H J

2001-04-01

The KRAB domain of human Kox1, a member of the KRAB C2H2 zinc finger family, confers strong transcriptional repressor activities even to remote promoter positions. Here, HDAC inhibitors were used to demonstrate that histone deacetylation is not required for mediating transcriptional repression of KRAB zinc finger proteins. Two reporter systems with either stably integrated or transiently transfected templates, both under control of strong viral promoters, were analyzed. Under all circumstances, HDAC inhibition did not alter the repression potential of the KRAB domain. In case of the stably integrated luciferase reporter gene system, neither expression levels of the KRAB fusion protein nor complex formation with its putative co-repressor TIF1beta were significantly changed. Furthermore, the TIF1beta/KRAB complex was devoid of mSin3A and HDAC1. In the transient transfection system, the transcriptional repression induced by TIF1beta and HP1alpha was not diminished by HDAC inhibitors, whereas the repressory activity of TIF1alpha was significantly affected. Thus, KRAB, TIF1beta and HP1alpha are likely to be functionally linked. In conclusion, HDAC activity is not essential for the strong transcriptional repressor activity mediated by the KRAB domain of Kox1 in particular and, presumably, by KRAB domains in general. This feature might be helpful in identifying and characterizing target genes under the control of

The Role of RaxST, a Prokaryotic Sulfotransferase, and RaxABC, a Putative Type I Secretion System, in Activation of the Rice XA21-Mediated Immune Response

PubMed Central

Ronald, Pamela C.

2014-01-01

Tyrosine sulfation is an important posttranslational modification that determines the outcome of serious diseases in plants and animals. We have recently demonstrated that the plant pathogen Xanthomonas oryzae pv. oryzae (Xoo) carries a functional sulfotransferase (RaxST). raxST is required for activation of rice Xa21-mediated immunity indicating the critical, but unknown, function of raxST in mediating the Xoo/rice interaction. The raxST gene resides in the same operon (raxSTAB) as components of a predicted type I secretion and processing system (RaxA and RaxB). These observations suggest a model where RaxST sulfates a molecule that contains a leader peptide, which is cleaved by the peptidase domain of the RaxB protein and secreted outside the bacterial cell by the RaxABC T1SS. PMID:25386383

The Role of RaxST, a Prokaryotic Sulfotransferase, and RaxABC, a Putative Type I Secretion System, in Activation of the Rice XA21-Mediated Immune Response.

PubMed

Ronald, Pamela C

2014-01-01

Tyrosine sulfation is an important posttranslational modification that determines the outcome of serious diseases in plants and animals. We have recently demonstrated that the plant pathogen Xanthomonas oryzae pv. oryzae (Xoo) carries a functional sulfotransferase (RaxST). raxST is required for activation of rice Xa21-mediated immunity indicating the critical, but unknown, function of raxST in mediating the Xoo/rice interaction. The raxST gene resides in the same operon (raxSTAB) as components of a predicted type I secretion and processing system (RaxA and RaxB). These observations suggest a model where RaxST sulfates a molecule that contains a leader peptide, which is cleaved by the peptidase domain of the RaxB protein and secreted outside the bacterial cell by the RaxABC T1SS.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Swenson, Joel M.; Colmenares, Serafin U.; Strom, Amy R.

Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors andmore » regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions.« less

How disordered is my protein and what is its disorder for? A guide through the “dark side” of the protein universe

PubMed Central

Lieutaud, Philippe; Uversky, Alexey V.; Uversky, Vladimir N.; Longhi, Sonia

2016-01-01

ABSTRACT In the last 2 decades it has become increasingly evident that a large number of proteins are either fully or partially disordered. Intrinsically disordered proteins lack a stable 3D structure, are ubiquitous and fulfill essential biological functions. Their conformational heterogeneity is encoded in their amino acid sequences, thereby allowing intrinsically disordered proteins or regions to be recognized based on properties of these sequences. The identification of disordered regions facilitates the functional annotation of proteins and is instrumental for delineating boundaries of protein domains amenable to structural determination with X-ray crystallization. This article discusses a comprehensive selection of databases and methods currently employed to disseminate experimental and putative annotations of disorder, predict disorder and identify regions involved in induced folding. It also provides a set of detailed instructions that should be followed to perform computational analysis of disorder. PMID:28232901

Identification of Delta5-fatty acid desaturase from the cellular slime mold dictyostelium discoideum.

PubMed

Saito, T; Ochiai, H

1999-10-01

cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) information of the Dictyostelium cDNA project. Using this sequence, we have determined the cDNA sequence and genomic sequence of a desaturase. The cloned cDNA is 1489 nucleotides long and the deduced amino acid sequence comprised 464 amino acid residues containing an N-terminal cytochrome b5 domain. The whole sequence was 38.6% identical to the initially identified Delta5-desaturase of Mortierella alpina. We have confirmed its function as Delta5-desaturase by over expression mutation in D. discoideum and also the gain of function mutation in the yeast Saccharomyces cerevisiae. Analysis of the lipids from transformed D. discoideum and yeast demonstrated the accumulation of Delta5-desaturated products. This is the first report concering fatty acid desaturase in cellular slime molds.

Characterization of a stearoyl-acyl carrier protein desaturase gene family from chocolate tree, Theobroma cacao L

PubMed Central

Zhang, Yufan; Maximova, Siela N.; Guiltinan, Mark J.

2015-01-01

In plants, the conversion of stearoyl-ACP to oleoyol-ACP is catalyzed by a plastid-localized soluble stearoyl-acyl carrier protein (ACP) desaturase (SAD). The activity of SAD significantly impacts the ratio of saturated and unsaturated fatty acids, and is thus a major determinant of fatty acid composition. The cacao genome contains eight putative SAD isoforms with high amino acid sequence similarities and functional domain conservation with SAD genes from other species. Sequence variation in known functional domains between different SAD family members suggested that these eight SAD isoforms might have distinct functions in plant development, a hypothesis supported by their diverse expression patterns in various cacao tissues. Notably, TcSAD1 is universally expressed across all the tissues, and its expression pattern in seeds is highly correlated with the dramatic change in fatty acid composition during seed maturation. Interestingly, TcSAD3 and TcSAD4 appear to be exclusively and highly expressed in flowers, functions of which remain unknown. To test the function of TcSAD1 in vivo, transgenic complementation of the Arabidopsis ssi2 mutant was performed, demonstrating that TcSAD1 successfully rescued all AtSSI2 related phenotypes further supporting the functional orthology between these two genes. The identification of the major SAD gene responsible for cocoa butter biosynthesis provides new strategies for screening for novel genotypes with desirable fatty acid compositions, and for use in breeding programs to help pyramid genes for quality and other traits such as disease resistance. PMID:25926841

Molecular evolution of bovine Toll-like receptor 2 suggests substitutions of functional relevance.

PubMed

Jann, Oliver C; Werling, Dirk; Chang, Jung-Su; Haig, David; Glass, Elizabeth J

2008-10-20

There is accumulating evidence that polymorphism in Toll-like receptor (TLR) genes might be associated with disease resistance or susceptibility traits in livestock. Polymorphic sites affecting TLR function should exhibit signatures of positive selection, identified as a high ratio of non-synonymous to synonymous nucleotide substitutions (omega). Phylogeny based models of codon substitution based on estimates of omega for each amino acid position can therefore offer a valuable tool to predict sites of functional relevance. We have used this approach to identify such polymorphic sites within the bovine TLR2 genes from ten Bos indicus and Bos taurus cattle breeds. By analysing TLR2 gene phylogeny in a set of mammalian species and a subset of ruminant species we have estimated the selective pressure on individual sites and domains and identified polymorphisms at sites of putative functional importance. The omega were highest in the mammalian TLR2 domains thought to be responsible for ligand binding and lowest in regions responsible for heterodimerisation with other TLR-related molecules. Several positively-selected sites were detected in or around ligand-binding domains. However a comparison of the ruminant subset of TLR2 sequences with the whole mammalian set of sequences revealed that there has been less selective pressure among ruminants than in mammals as a whole. This suggests that there have been functional changes during ruminant evolution. Twenty newly-discovered non-synonymous polymorphic sites were identified in cattle. Three of them were localised at positions shaped by positive selection in the ruminant dataset (Leu227Phe, His305Pro, His326Gln) and in domains involved in the recognition of ligands. His326Gln is of particular interest as it consists of an exchange of differentially-charged amino acids at a position which has previously been shown to be crucial for ligand binding in human TLR2. Within bovine TLR2, polymorphisms at amino acid positions 227, 305 and 326 map to functionally important sites of TLR2 and should be considered as candidate SNPs for immune related traits in cattle. A final proof of their functional relevance requires further studies to determine their functional effect on the immune response after stimulation with relevant ligands and/or their association with immune related traits in animals.

Role of transmembrane segment 5 of the plant vacuolar H+-pyrophosphatase.

PubMed

Van, Ru C; Pan, Yih J; Hsu, Shen H; Huang, Yun T; Hsiao, Yi Y; Pan, Rong L

2005-08-15

Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase consisting of a single type of polypeptide with a molecular mass of approximately 81 kDa. Topological analysis tentatively predicts that mung bean V-PPase contains 14 transmembrane domains. Alignment analysis of V-PPase demonstrated that the transmembrane domain 5 (TM5) of the enzyme is highly conserved in plants and located at the N-terminal side of the putative substrate-binding loop. The hydropathic analysis of V-PPase showed a relatively lower degree of hydrophobicity in the TM5 region as compared to other domains. Accordingly, it appears that TM5 is probably involved in the proton translocation of V-PPase. In this study, we used site-directed mutagenesis to examine the functional role of amino acid residues in TM5 of V-PPase. A series of mutants singly replaced by alanine residues along TM5 were constructed and over-expressed in Saccharomyces cerevisiae; they were then used to determine their enzymatic activities and proton translocations. Our results indicate that several mutants displayed minor variations in enzymatic properties, while others including those mutated at E225, a GYG motif (residues from 229 to 231), A238, and R242, showed a serious decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase. Moreover, the mutation at Y230 relieved several cation effects on the V-PPase. The GYG motif presumably plays a significant role in maintaining structure and function of V-PPase.

VP3 is crucial for the stability of Nora virus virions.

PubMed

Sadanandan, Sajna Anand; Ekström, Jens-Ola; Jonna, Venkateswara Rao; Hofer, Anders; Hultmark, Dan

2016-09-02

Nora virus is an enteric virus that causes persistent, non-pathological infection in Drosophila melanogaster. It replicates in the fly gut and is transmitted via the fecal-oral route. Nora virus has a single-stranded positive-sense RNA genome, which is translated in four open reading frames. Reading frame three encodes the VP3 protein, the structure and function of which we have investigated in this work. We have shown that VP3 is a trimer that has an α-helical secondary structure, with a functionally important coiled-coil domain. In order to identify the role of VP3 in the Nora virus life cycle, we constructed VP3-mutants using the cDNA clone of the virus. Our results show that VP3 does not have a role in the actual assembly of the virus particles, but virions that lack VP3 or harbor VP3 with a disrupted coiled coil domain are incapable of transmission via the fecal-oral route. Removing the region downstream of the putative coiled coil appears to have an effect on the fitness of the virus but does not hamper its replication or transmission. We also found that the VP3 protein and particularly the coiled coil domain are crucial for the stability of Nora virus virions when exposed to heat or proteases. Hence, we propose that VP3 is imperative to Nora virus virions as it confers stability to the viral capsid. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins.

PubMed

Larsen, Ida Signe Bohse; Narimatsu, Yoshiki; Joshi, Hiren Jitendra; Siukstaite, Lina; Harrison, Oliver J; Brasch, Julia; Goodman, Kerry M; Hansen, Lars; Shapiro, Lawrence; Honig, Barry; Vakhrushev, Sergey Y; Clausen, Henrik; Halim, Adnan

2017-10-17

The cadherin (cdh) superfamily of adhesion molecules carry O-linked mannose (O-Man) glycans at highly conserved sites localized to specific β-strands of their extracellular cdh (EC) domains. These O-Man glycans do not appear to be elongated like O-Man glycans found on α-dystroglycan (α-DG), and we recently demonstrated that initiation of cdh/protocadherin (pcdh) O-Man glycosylation is not dependent on the evolutionary conserved POMT1/POMT2 enzymes that initiate O-Man glycosylation on α-DG. Here, we used a CRISPR/Cas9 genetic dissection strategy combined with sensitive and quantitative O-Man glycoproteomics to identify a homologous family of four putative protein O-mannosyltransferases encoded by the TMTC1-4 genes, which were found to be imperative for cdh and pcdh O-Man glycosylation. KO of all four TMTC genes in HEK293 cells resulted in specific loss of cdh and pcdh O-Man glycosylation, whereas combined KO of TMTC1 and TMTC3 resulted in selective loss of O-Man glycans on specific β-strands of EC domains, suggesting that each isoenzyme serves a different function. In addition, O-Man glycosylation of IPT/TIG domains of plexins and hepatocyte growth factor receptor was not affected in TMTC KO cells, suggesting the existence of yet another O-Man glycosylation machinery. Our study demonstrates that regulation of O-mannosylation in higher eukaryotes is more complex than envisioned, and the discovery of the functions of TMTCs provide insight into cobblestone lissencephaly caused by deficiency in TMTC3.

Definition of a putative pathological region in PARK2 associated with autism spectrum disorder through in silico analysis of its functional structure.

PubMed

Conceição, Inês C; Rama, Maria M; Oliveira, Bárbara; Café, Cátia; Almeida, Joana; Mouga, Susana; Duque, Frederico; Oliveira, Guiomar; Vicente, Astrid M

2017-04-01

The PARK2 gene encodes Parkin, a component of a multiprotein E3 ubiquitin ligase complex that targets substrate proteins for proteasomal degradation. PARK2 mutations are frequently associated with Parkinson's disease, but structural alterations have also been described in patients with neurodevelopmental disorders (NDD), suggesting a pathological effect ubiquitous to neurodevelopmental and neurodegenerative brain processes. The present study aimed to define the critical regions for NDD within PARK2. To clarify PARK2 involvement in NDDs, we examined the frequency and location of copy number variants (CNVs) identified in patients from our sample and reported in the literature and relevant databases, and compared with control populations. Overall, the frequency of PARK2 CNVs was higher in controls than in NDD cases. However, closer inspection of the CNV location in PARK2 showed that the frequency of CNVs targeting the Parkin C-terminal, corresponding to the ring-between-ring (RBR) domain responsible for Parkin activity, is significantly higher in NDD cases than in controls. In contrast, CNVs targeting the N-terminal of Parkin, including domains that regulate ubiquitination activity, are very common both in cases and in controls. Although PARK2 may be a pathological factor for NDDs, likely not all variants are pathogenic, and a conclusive assessment of PARK2 variant pathogenicity requires an accurate analysis of their location within the coding region and encoded functional domains.

Identification of Germ Plasm-Associated Transcripts by Microarray Analysis of Xenopus Vegetal Cortex RNA

PubMed Central

Cuykendall, Tawny N.; Houston, Douglas W.

2011-01-01

RNA localization is a common mechanism for regulating cell structure and function. Localized RNAs in Xenopus oocytes are critical for early development, including germline specification by the germ plasm. Despite the importance of these localized RNAs, only approximately 25 have been identified and fewer are functionally characterized. Using microarrays, we identified a large set of localized RNAs from the vegetal cortex. Overall, our results indicate a minimum of 275 localized RNAs in oocytes, or 2–3% of maternal transcripts, which are in general agreement with previous findings. We further validated vegetal localization for 24 candidates and further characterized three genes expressed in the germ plasm. We identified novel germ plasm expression for reticulon 3.1, exd2 (a novel exonuclease-domain encoding gene), and a putative noncoding RNA. Further analysis of these and other localized RNAs will likely identify new functions of germ plasm and facilitate the identification of cis-acting RNA localization elements. PMID:20503379

Deep transcriptome annotation enables the discovery and functional characterization of cryptic small proteins

PubMed Central

Delcourt, Vivian; Lucier, Jean-François; Gagnon, Jules; Beaudoin, Maxime C; Vanderperre, Benoît; Breton, Marc-André; Motard, Julie; Jacques, Jean-François; Brunelle, Mylène; Gagnon-Arsenault, Isabelle; Fournier, Isabelle; Ouangraoua, Aida; Hunting, Darel J; Cohen, Alan A; Landry, Christian R; Scott, Michelle S

2017-01-01

Recent functional, proteomic and ribosome profiling studies in eukaryotes have concurrently demonstrated the translation of alternative open-reading frames (altORFs) in addition to annotated protein coding sequences (CDSs). We show that a large number of small proteins could in fact be coded by these altORFs. The putative alternative proteins translated from altORFs have orthologs in many species and contain functional domains. Evolutionary analyses indicate that altORFs often show more extreme conservation patterns than their CDSs. Thousands of alternative proteins are detected in proteomic datasets by reanalysis using a database containing predicted alternative proteins. This is illustrated with specific examples, including altMiD51, a 70 amino acid mitochondrial fission-promoting protein encoded in MiD51/Mief1/SMCR7L, a gene encoding an annotated protein promoting mitochondrial fission. Our results suggest that many genes are multicoding genes and code for a large protein and one or several small proteins. PMID:29083303

Voltage-sensing phosphatase: its molecular relationship with PTEN.

PubMed

Okamura, Yasushi; Dixon, Jack E

2011-02-01

Voltage-sensing phosphoinositide phosphatase (VSP) contains voltage sensor and cytoplasmic phosphatase domains. A unique feature of this protein is that depolarization-induced motions of the voltage sensor activate PtdIns(3,4,5)P(3) and PtdIns(4,5)P(2) phosphatase activities. VSP exhibits remarkable structural similarities with PTEN, the phosphatase and tensin homolog deleted on chromosome 10. These similarities include the cytoplasmic phosphatase region, the phosphoinositide binding region, and the putative membrane interacting C2 domain.

Molecular Cloning, Characterization, and Differential Expression of a Glucoamylase Gene from the Basidiomycetous Fungus Lentinula edodes

PubMed Central

Zhao, J.; Chen, Y. H.; Kwan, H. S.

2000-01-01

The complete nucleotide sequence of putative glucoamylase gene gla1 from the basidiomycetous fungus Lentinula edodes strain L54 is reported. The coding region of the genomic glucoamylase sequence, which is preceded by eukaryotic promoter elements CAAT and TATA, spans 2,076 bp. The gla1 gene sequence codes for a putative polypeptide of 571 amino acids and is interrupted by seven introns. The open reading frame sequence of the gla1 gene shows strong homology with those of other fungal glucoamylase genes and encodes a protein with an N-terminal catalytic domain and a C-terminal starch-binding domain. The similarity between the Gla1 protein and other fungal glucoamylases is from 45 to 61%, with the region of highest conservation found in catalytic domains and starch-binding domains. We compared the kinetics of glucoamylase activity and levels of gene expression in L. edodes strain L54 grown on different carbon sources (glucose, starch, cellulose, and potato extract) and in various developmental stages (mycelium growth, primordium appearance, and fruiting body formation). Quantitative reverse transcription PCR utilizing pairs of primers specific for gla1 gene expression shows that expression of gla1 was induced by starch and increased during the process of fruiting body formation, which indicates that glucoamylases may play an important role in the morphogenesis of the basidiomycetous fungus. PMID:10831434

Annotation of differentially expressed genes in the somatic embryogenesis of musa and their location in the banana genome.

PubMed

Maldonado-Borges, Josefina Ines; Ku-Cauich, José Roberto; Escobedo-Graciamedrano, Rosa Maria

2013-01-01

Analysis of cDNA-AFLP was used to study the genes expressed in zygotic and somatic embryogenesis of Musa acuminata Colla ssp. malaccensis, and a comparison was made between their differential transcribed fragments (TDFs) and the sequenced genome of the double haploid- (DH-) Pahang of the malaccensis subspecies that is available in the network. A total of 253 transcript-derived fragments (TDFs) were detected with apparent size of 100-4000 bp using 5 pairs of AFLP primers, of which 21 were differentially expressed during the different stages of banana embryogenesis; 15 of the sequences have matched DH-Pahang chromosomes, with 7 of them being homologous to gene sequences encoding either known or putative protein domains of higher plants. Four TDF sequences were located in all Musa chromosomes, while the rest were located in one or two chromosomes. Their putative individual function is briefly reviewed based on published information, and the potential roles of these genes in embryo development are discussed. Thus the availability of the genome of Musa and the information of TDFs sequences presented here opens new possibilities for an in-depth study of the molecular and biochemical research of zygotic and somatic embryogenesis of Musa.

CMTM5 exhibits tumor suppressor activity through promoter methylation in oral squamous cell carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Heyu; Nan, Xu; Li, Xuefen

Highlights: • Down-regulation of CMTM5 expression in OSCC tissues was found. • The promoter methylation status of CMTM5 was measured. • CMTM5-v1 inhibited cell proliferation and migration and induced apoptosis. • CMTM5 might act as a putative tumor suppressor gene in OSCC. - Abstract: Oral squamous cell carcinoma (OSCC) is one of the most common types of malignancies in the head and neck region. CKLF-like MARVEL transmembrane domain-containing member 5 (CMTM5) has been recently implicated as a tumor suppressor gene in several cancer types. Herein, we examined the expression and function of CMTM5 in oral squamous cell carcinoma. CMTM5 wasmore » down-regulated in oral squamous cell lines and tumor samples from patients with promoter methylation. Treatment with the demethylating agent 5-aza-2′-deoxycytidine restored CMTM5 expression. In the OSCC cell lines CAL27 and GNM, the ectopic expression of CMTM5-v1 strongly inhibited cell proliferation and migration and induced apoptosis. In addition, CMTM5-v1 inhibited tumor formation in vivo. Therefore, CMTM5 might act as a putative tumor suppressor gene through promoter methylation in oral squamous cell carcinoma.« less

Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes.

PubMed

McDowell, T L; Gibbons, R J; Sutherland, H; O'Rourke, D M; Bickmore, W A; Pombo, A; Turley, H; Gatter, K; Picketts, D J; Buckle, V J; Chapman, L; Rhodes, D; Higgs, D R

1999-11-23

ATRX is a member of the SNF2 family of helicase/ATPases that is thought to regulate gene expression via an effect on chromatin structure and/or function. Mutations in the hATRX gene cause severe syndromal mental retardation associated with alpha-thalassemia. Using indirect immunofluorescence and confocal microscopy we have shown that ATRX protein is associated with pericentromeric heterochromatin during interphase and mitosis. By coimmunofluorescence, ATRX localizes with a mouse homologue of the Drosophila heterochromatic protein HP1 in vivo, consistent with a previous two-hybrid screen identifying this interaction. From the analysis of a trap assay for nuclear proteins, we have shown that the localization of ATRX to heterochromatin is encoded by its N-terminal region, which contains a conserved plant homeodomain-like finger and a coiled-coil domain. In addition to its association with heterochromatin, at metaphase ATRX clearly binds to the short arms of human acrocentric chromosomes, where the arrays of ribosomal DNA are located. The unexpected association of a putative transcriptional regulator with highly repetitive DNA provides a potential explanation for the variability in phenotype of patients with identical mutations in the ATRX gene.

BuD, a helix–loop–helix DNA-binding domain for genome modification

PubMed Central

Stella, Stefano; Molina, Rafael; López-Méndez, Blanca; Juillerat, Alexandre; Bertonati, Claudia; Daboussi, Fayza; Campos-Olivas, Ramon; Duchateau, Phillippe; Montoya, Guillermo

2014-01-01

DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing ‘toolkits’ for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing. PMID:25004980

TM6SF2 is a regulator of liver fat metabolism influencing triglyceride secretion and hepatic lipid droplet content

PubMed Central

Mahdessian, Hovsep; Taxiarchis, Apostolos; Popov, Sergej; Silveira, Angela; Franco-Cereceda, Anders; Hamsten, Anders; Eriksson, Per; van't Hooft, Ferdinand

2014-01-01

Genome-wide association studies have identified a locus on chromosome 19 associated with plasma triglyceride (TG) concentration and nonalcoholic fatty liver disease. However, the identity and functional role of the gene(s) responsible for these associations remain unknown. Of 19 expressed genes contained in this locus, none has previously been implicated in lipid metabolism. We performed gene expression studies and expression quantitative trait locus analysis in 206 human liver samples to identify the putative causal gene. Transmembrane 6 superfamily member 2 (TM6SF2), a gene with hitherto unknown function, expressed predominantly in liver and intestine, was identified as the putative causal gene. TM6SF2 encodes a protein of 351 amino acids with 7–10 predicted transmembrane domains. Otherwise, no other protein features were identified which could help to elucidate the function of TM6SF2. Protein subcellular localization studies with confocal microscopy demonstrated that TM6SF2 is localized in the endoplasmic reticulum and the ER-Golgi intermediate compartment of human liver cells. Functional studies for secretion of TG-rich lipoproteins (TRLs) and lipid droplet content were performed in human hepatoma Huh7 and HepG2 cells using confocal microscopy and siRNA inhibition and overexpression techniques. In agreement with the genome-wide association data, it was found that TM6SF2 siRNA inhibition was associated with reduced secretion of TRLs and increased cellular TG concentration and lipid droplet content, whereas TM6SF2 overexpression reduced liver cell steatosis. We conclude that TM6SF2 is a regulator of liver fat metabolism with opposing effects on the secretion of TRLs and hepatic lipid droplet content. PMID:24927523

Distinct spatiotemporal expression of ISM1 during mouse and chick development.

PubMed

Osório, Liliana; Wu, Xuewei; Zhou, Zhongjun

2014-01-01

Isthmin 1 (ISM1) constitutes the founder of a new family of secreted proteins characterized by the presence of 2 functional domains: thrombospondin type 1 repeat (TSR1) and adhesion-associated domain in MUC4 and other proteins (AMOP). ISM1 was identified in the frog embryo as a member of the FGF8 synexpression group due to its expression in the brain midbrain-hindbrain boundary (MHB) or isthmus. In zebrafish, ISM1 was described as a WNT- and NODAL-regulated gene. The function of ISM1 remains largely elusive. So far, ISM1 has been described as an angiogenesis inhibitor that has a dual function in endothelial cell survival and cell death. For a better understanding of ISM1 function, we examined its spatiotemporal distribution in mouse and chick using RT-PCR, ISH, and IHC analyses. In the mouse, ISM1 transcripts are found in tissues such as the anterior mesendoderm, paraxial and lateral plate mesoderm, MHB and trunk neural tube, as well as in the somites and dermomyotome. In the newborn and adult, ISM1 is prominently expressed in the lung and brain. In addition to its putative role during embryonic and postnatal development, ISM1 may also be important for organ homeostasis in the adult. In the chick embryo, ISM1 transcripts are strongly detected in the ear, eye, and spinal cord primordia. Remarkable differences in ISM1 spatiotemporal expression were found during mouse and chick development, despite the high homology of ISM1 orthologs in these species.

Characterization of HKE2: an ancient antigen encoded in the major histocompatibility complex.

PubMed

Ostrov, D A; Barnes, C L; Smith, L E; Binns, S; Brusko, T M; Brown, A C; Quint, P S; Litherland, S A; Roopenian, D C; Iczkowski, K A

2007-02-01

Genes at the centromeric end of the human leukocyte antigen region influence adaptive autoimmune diseases and cancer. In this study, we characterized protein expression of HKE2, a gene located in the centromeric portion of the class II region of the major histocompatibility complex encoding subunit 6 of prefoldin. Immunohistochemical analysis using an anti-HKE2 antibody indicated that HKE2 protein expression is dramatically upregulated as a consequence of activation. In a tissue microarray and in several tumors, HKE2 was overexpressed in certain cancers compared with normal counterparts. The localization of the HKE2 gene to the class II region, its cytoplasmic expression and putative protein-binding domain suggest that HKE2 may function in adaptive immunity and cancer.

Identification and preliminary characterization of a protein motif related to the zinc finger.

PubMed Central

Lovering, R; Hanson, I M; Borden, K L; Martin, S; O'Reilly, N J; Evan, G I; Rahman, D; Pappin, D J; Trowsdale, J; Freemont, P S

1993-01-01

We have identified a protein motif, related to the zinc finger, which defines a newly discovered family of proteins. The motif was found in the sequence of the human RING1 gene, which is proximal to the major histocompatibility complex region on chromosome six. We propose naming this motif the "RING finger" and it is found in 27 proteins, all of which have putative DNA binding functions. We have synthesized a peptide corresponding to the RING1 motif and examined a number of properties, including metal and DNA binding. We provide evidence to support the suggestion that the RING finger motif is the DNA binding domain of this newly defined family of proteins. Images Fig. 1 Fig. 4 PMID:7681583

Novel sequence variants in the TMIE gene in families with autosomal recessive nonsyndromic hearing impairment

PubMed Central

Santos, Regie Lyn P.; El-Shanti, Hatem; Sikandar, Shaheen; Lee, Kwanghyuk; Bhatti, Attya; Yan, Kai; Chahrour, Maria H.; McArthur, Nathan; Pham, Thanh L.; Mahasneh, Amjad Abdullah; Ahmad, Wasim

2010-01-01

To date, 37 genes have been identified for nonsyndromic hearing impairment (NSHI). Identifying the functional sequence variants within these genes and knowing their population-specific frequencies is of public health value, in particular for genetic screening for NSHI. To determine putatively functional sequence variants in the transmembrane inner ear (TMIE) gene in Pakistani and Jordanian families with autosomal recessive (AR) NSHI, four Jordanian and 168 Pakistani families with ARNSHI that is not due to GJB2 (CX26) were submitted to a genome scan. Two-point and multipoint parametric linkage analyses were performed, and families with logarithmic odds (LOD) scores of 1.0 or greater within the TMIE region underwent further DNA sequencing. The evolutionary conservation and location in predicted protein domains of amino acid residues where sequence variants occurred were studied to elucidate the possible effects of these sequence variants on function. Of seven families that were screened for TMIE, putatively functional sequence variants were found to segregate with hearing impairment in four families but were not seen in not less than 110 ethnically matched control chromosomes. The previously reported c.241C>T (p.R81C) variant was observed in two Pakistani families. Two novel variants, c.92A>G (p.E31G) and the splice site mutation c.212–2A>C, were identified in one Pakistani and one Jordanian family, respectively. The c.92A>G (p.E31G) variant occurred at a residue that is conserved in the mouse and is predicted to be extracellular. Conservation and potential functionality of previously published mutations were also examined. The prevalence of functional TMIE variants in Pakistani families is 1.7% [95% confidence interval (CI) 0.3–4.8]. Further studies on the spectrum, prevalence rates, and functional effect of sequence variants in the TMIE gene in other populations should demonstrate the true importance of this gene as a cause of hearing impairment. PMID:16389551

Cytosolic delivery of multi-domain cargos by the N-terminus of Pasteurella multocida toxin.

PubMed

Clemons, Nathan C; Bannai, Yuka; Haywood, Elizabeth E; Xu, Yiting; Buschbach, James D; Ho, Mengfei; Wilson, Brenda A

2018-05-21

The zoonotic pathogen Pasteurella multocida produces a 146-kDa modular toxin (PMT) that enters host cells and manipulates intracellular signaling through action on its Gα-protein targets. The N-terminus of PMT (PMT-N) mediates cellular uptake through receptor-mediated endocytosis, followed by delivery of the C-terminal catalytic domain from acidic endosomes into the cytosol. The putative native cargo of PMT consists of a 710-residue polypeptide of three distinct modular subdomains (C1-C2-C3), where C1 contains a membrane localization domain (MLD), C2 has as-of-yet undefined function, and C3 catalyzes deamidation of a specific active-site glutamine residue in Gα-protein targets. However, whether the three cargo subdomains are delivered intact or undergo further proteolytic processing during or after translocation from the late endosome is unclear. Here, we demonstrate that PMT-N mediates delivery of its native C-terminal cargo as a single polypeptide, corresponding to C1-C2-C3, including the MLD, with no evidence of cleavage between subdomains. We show that PMT-N also delivers into the cytosol non-native GFP cargo, further supporting that the receptor-binding and translocation functions reside within PMT-N. Our findings further show that PMT-N can deliver C1-C2 alone but that the presence of C1-C2 is important for cytosolic delivery of the catalytic C3 subdomain by PMT-N. In addition, we further refine the minimum C3 domain required for intracellular activity as comprising residues 1105-1278. These findings reinforce that PMT-N serves as the cytosolic delivery vehicle for C-terminal cargo and demonstrate that its native cargo is delivered intact as C1-C2-C3. Copyright © 2018 American Society for Microbiology.

Contemplating the plasmalemmal control center model

NASA Technical Reports Server (NTRS)

Pickard, B. G.

1994-01-01

An abundant epidermal mechanosensory calcium-selective ion channel appears able not only to detect mechanical stimuli such as those that initiate gravitropism but also to detect thermal, electrical, and various chemical stimuli. Because it responds to multimodal input with a second messenger output, this channel system seems likely to be an integrator that can engage in feedbacks with many other systems of the cell--and feedback is the hallmark of regulation. In general, the mechanical tension required for channel activation is likely transmitted from the relatively rigid cell wall to the plasma membrane system via linkage or adhesion sites that display antigenicities recognized by antibodies to animal beta-1 integrin, vitronectin, and fibronectin and which have mechanical connections to the cytoskeleton. Thus, functionally, leverage exerted against any given adhesion site will tend to control channels within a surrounding domain. Reactions initiated by passage of calcium ions through the channels could presumably be more effectively regulated if channels within the domains were somewhat clustered and if appropriate receptors, kinases, porters, pumps, and some key cytoskeletal anchoring sites were in turn clustered about them. Accumulating evidence suggests not only that activity of clusters of channels may contribute to control of cytoskeletal architecture and of regulatory protein function within their domain, but also that both a variety of regulatory proteins and components of the cortical cytoskeleton may contribute to control of channel activity. The emerging capabilities of electronic optical microscopy are well suited for resolving the spatial distributions of many of these cytoskeletal and regulatory molecules in living cells, and for following some of their behaviors as channels are stimulated to open and cytosolic calcium builds in their vicinity. Such microscopy, coupled with biochemical and physiological probing, should help to establish the nature of the feedback loops putatively controlled by the linkage sites and their channel domains.

Identification of a probable pore-forming domain in the multimeric vacuolar anion channel AtALMT9.

PubMed

Zhang, Jingbo; Baetz, Ulrike; Krügel, Undine; Martinoia, Enrico; De Angeli, Alexis

2013-10-01

Aluminum-activated malate transporters (ALMTs) form an important family of anion channels involved in fundamental physiological processes in plants. Because of their importance, the role of ALMTs in plant physiology is studied extensively. In contrast, the structural basis of their functional properties is largely unknown. This lack of information limits the understanding of the functional and physiological differences between ALMTs and their impact on anion transport in plants. This study aimed at investigating the structural organization of the transmembrane domain of the Arabidopsis (Arabidopsis thaliana) vacuolar channel AtALMT9. For that purpose, we performed a large-scale mutagenesis analysis and found two residues that form a salt bridge between the first and second putative transmembrane α-helices (TMα1 and TMα2). Furthermore, using a combination of pharmacological and mutagenesis approaches, we identified citrate as an "open channel blocker" of AtALMT9 and used this tool to examine the inhibition sensitivity of different point mutants of highly conserved amino acid residues. By this means, we found a stretch within the cytosolic moiety of the TMα5 that is a probable pore-forming domain. Moreover, using a citrate-insensitive AtALMT9 mutant and biochemical approaches, we could demonstrate that AtALMT9 forms a multimeric complex that is supposedly composed of four subunits. In summary, our data provide, to our knowledge, the first evidence about the structural organization of an ion channel of the ALMT family. We suggest that AtALMT9 is a tetramer and that the TMα5 domains of the subunits contribute to form the pore of this anion channel.

Retrovirus-specific differences in matrix and nucleocapsid protein-nucleic acid interactions: implications for genomic RNA packaging.

PubMed

Sun, Meng; Grigsby, Iwen F; Gorelick, Robert J; Mansky, Louis M; Musier-Forsyth, Karin

2014-01-01

Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.

The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease

PubMed Central

King, Oliver D.; Gitler, Aaron D.; Shorter, James

2012-01-01

Prions are self-templating protein conformers that are naturally transmitted between individuals and promote phenotypic change. In yeast, prion-encoded phenotypes can be beneficial, neutral or deleterious depending upon genetic background and environmental conditions. A distinctive and portable ‘prion domain’ enriched in asparagine, glutamine, tyrosine and glycine residues unifies the majority of yeast prion proteins. Deletion of this domain precludes prionogenesis and appending this domain to reporter proteins can confer prionogenicity. An algorithm designed to detect prion domains has successfully identified 19 domains that can confer prion behavior. Scouring the human genome with this algorithm enriches a select group of RNA-binding proteins harboring a canonical RNA recognition motif (RRM) and a putative prion domain. Indeed, of 210 human RRM-bearing proteins, 29 have a putative prion domain, and 12 of these are in the top 60 prion candidates in the entire genome. Startlingly, these RNA-binding prion candidates are inexorably emerging, one by one, in the pathology and genetics of devastating neurodegenerative disorders, including: amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), Alzheimer’s disease and Huntington’s disease. For example, FUS and TDP-43, which rank 1st and 10th among RRM-bearing prion candidates, form cytoplasmic inclusions in the degenerating motor neurons of ALS patients and mutations in TDP-43 and FUS cause familial ALS. Recently, perturbed RNA-binding proteostasis of TAF15, which is the 2nd ranked RRM-bearing prion candidate, has been connected with ALS and FTLD-U. We strongly suspect that we have now merely reached the tip of the iceberg. We predict that additional RNA-binding prion candidates identified by our algorithm will soon surface as genetic modifiers or causes of diverse neurodegenerative conditions. Indeed, simple prion-like transfer mechanisms involving the prion-like domains of RNA-binding proteins could underlie the classical non-cell-autonomous emanation of neurodegenerative pathology from originating epicenters to neighboring portions of the nervous system. PMID:22445064

Functional analysis of the theobroma cacao NPR1 gene in arabidopsis

PubMed Central

2010-01-01

Background The Arabidopsis thaliana NPR1 gene encodes a transcription coactivator (NPR1) that plays a major role in the mechanisms regulating plant defense response. After pathogen infection and in response to salicylic acid (SA) accumulation, NPR1 translocates from the cytoplasm into the nucleus where it interacts with other transcription factors resulting in increased expression of over 2000 plant defense genes contributing to a pathogen resistance response. Results A putative Theobroma cacao NPR1 cDNA was isolated by RT-PCR using degenerate primers based on homologous sequences from Brassica, Arabidopsis and Carica papaya. The cDNA was used to isolate a genomic clone from Theobroma cacao containing a putative TcNPR1 gene. DNA sequencing revealed the presence of a 4.5 kb coding region containing three introns and encoding a polypeptide of 591 amino acids. The predicted TcNPR1 protein shares 55% identity and 78% similarity to Arabidopsis NPR1, and contains each of the highly conserved functional domains indicative of this class of transcription factors (BTB/POZ and ankyrin repeat protein-protein interaction domains and a nuclear localization sequence (NLS)). To functionally define the TcNPR1 gene, we transferred TcNPR1 into an Arabidopsis npr1 mutant that is highly susceptible to infection by the plant pathogen Pseudomonas syringae pv. tomato DC3000. Driven by the constitutive CaMV35S promoter, the cacao TcNPR1 gene partially complemented the npr1 mutation in transgenic Arabidopsis plants, resulting in 100 fold less bacterial growth in a leaf infection assay. Upon induction with SA, TcNPR1 was shown to translocate into the nucleus of leaf and root cells in a manner identical to Arabidopsis NPR1. Cacao NPR1 was also capable of participating in SA-JA signaling crosstalk, as evidenced by the suppression of JA responsive gene expression in TcNPR1 overexpressing transgenic plants. Conclusion Our data indicate that the TcNPR1 is a functional ortholog of Arabidopsis NPR1, and is likely to play a major role in defense response in cacao. This fundamental knowledge can contribute to breeding of disease resistant cacao varieties through the application of molecular markers or the use of transgenic strategies. PMID:21078185

Functional analysis of the Theobroma cacao NPR1 gene in Arabidopsis.

PubMed

Shi, Zi; Maximova, Siela N; Liu, Yi; Verica, Joseph; Guiltinan, Mark J

2010-11-15

The Arabidopsis thaliana NPR1 gene encodes a transcription coactivator (NPR1) that plays a major role in the mechanisms regulating plant defense response. After pathogen infection and in response to salicylic acid (SA) accumulation, NPR1 translocates from the cytoplasm into the nucleus where it interacts with other transcription factors resulting in increased expression of over 2000 plant defense genes contributing to a pathogen resistance response. A putative Theobroma cacao NPR1 cDNA was isolated by RT-PCR using degenerate primers based on homologous sequences from Brassica, Arabidopsis and Carica papaya. The cDNA was used to isolate a genomic clone from Theobroma cacao containing a putative TcNPR1 gene. DNA sequencing revealed the presence of a 4.5 kb coding region containing three introns and encoding a polypeptide of 591 amino acids. The predicted TcNPR1 protein shares 55% identity and 78% similarity to Arabidopsis NPR1, and contains each of the highly conserved functional domains indicative of this class of transcription factors (BTB/POZ and ankyrin repeat protein-protein interaction domains and a nuclear localization sequence (NLS)). To functionally define the TcNPR1 gene, we transferred TcNPR1 into an Arabidopsis npr1 mutant that is highly susceptible to infection by the plant pathogen Pseudomonas syringae pv. tomato DC3000. Driven by the constitutive CaMV35S promoter, the cacao TcNPR1 gene partially complemented the npr1 mutation in transgenic Arabidopsis plants, resulting in 100 fold less bacterial growth in a leaf infection assay. Upon induction with SA, TcNPR1 was shown to translocate into the nucleus of leaf and root cells in a manner identical to Arabidopsis NPR1. Cacao NPR1 was also capable of participating in SA-JA signaling crosstalk, as evidenced by the suppression of JA responsive gene expression in TcNPR1 overexpressing transgenic plants. Our data indicate that the TcNPR1 is a functional ortholog of Arabidopsis NPR1, and is likely to play a major role in defense response in cacao. This fundamental knowledge can contribute to breeding of disease resistant cacao varieties through the application of molecular markers or the use of transgenic strategies.

A Toxoplasma gondii Class XIV Myosin, Expressed in Sf9 Cells with a Parasite Co-chaperone, Requires Two Light Chains for Fast Motility*

PubMed Central

Bookwalter, Carol S.; Kelsen, Anne; Leung, Jacqueline M.; Ward, Gary E.; Trybus, Kathleen M.

2014-01-01

Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors. PMID:25231988

Structural determinants of Actinomyces sortase SrtC2 required for membrane localization and assembly of type 2 fimbriae for interbacterial coaggregation and oral biofilm formation.

PubMed

Wu, Chenggang; Mishra, Arunima; Reardon, Melissa E; Huang, I-Hsiu; Counts, Sarah C; Das, Asis; Ton-That, Hung

2012-05-01

As a pioneer colonizer of the oral cavity, Actinomyces oris expresses proteinaceous pili (also called fimbriae) to mediate the following two key events in biofilm formation: adherence to saliva deposits on enamel and interbacterial associations. Assembly of type 2 fimbriae that directly facilitate coaggregation with oral streptococci and Actinomyces biofilm development requires the class C sortase SrtC2. Although the general sortase-associated mechanisms have been elucidated, several structural attributes unique to the class C sortases require functional investigation. Mutational studies reported here suggest that the N-terminal transmembrane (TM) region of SrtC2, predicted to contain a signal peptide sequence, is cleaved off the mature protein and that this processing is critical for the proper integration of the enzyme at the cytoplasmic membrane, which is mediated by the extended hydrophobic C terminus containing a TM domain and a cytoplasmic tail. Deletion of this putative TM or the entire cytoplasmic domain abolished the enzyme localization and functionality. Alanine substitution of the conserved catalytic Cys-His dyad abrogated the SrtC2 enzymatic activity. In contrast, mutations designed to alter a "lid" domain that covers the catalytic pocket of a class C sortase showed no effect on enzyme activity. Finally, each of the deleterious mutations that affected SrtC2 activity or membrane localization also eliminated Actinomyces species biofilm development and bacterial coaggregation with streptococci. We conclude that the N terminus of SrtC2, which contains the signal sequence, is required for proper protein translocation and maturation, while the extended C-terminal hydrophobic region serves as a stable membrane anchor for proper enzyme functionality.

Structural Determinants of Actinomyces sortase SrtC2 Required for Membrane Localization and Assembly of Type 2 Fimbriae for Interbacterial Coaggregation and Oral Biofilm Formation

PubMed Central

Wu, Chenggang; Mishra, Arunima; Reardon, Melissa E.; Huang, I-Hsiu; Counts, Sarah C.; Das, Asis

2012-01-01

As a pioneer colonizer of the oral cavity, Actinomyces oris expresses proteinaceous pili (also called fimbriae) to mediate the following two key events in biofilm formation: adherence to saliva deposits on enamel and interbacterial associations. Assembly of type 2 fimbriae that directly facilitate coaggregation with oral streptococci and Actinomyces biofilm development requires the class C sortase SrtC2. Although the general sortase-associated mechanisms have been elucidated, several structural attributes unique to the class C sortases require functional investigation. Mutational studies reported here suggest that the N-terminal transmembrane (TM) region of SrtC2, predicted to contain a signal peptide sequence, is cleaved off the mature protein and that this processing is critical for the proper integration of the enzyme at the cytoplasmic membrane, which is mediated by the extended hydrophobic C terminus containing a TM domain and a cytoplasmic tail. Deletion of this putative TM or the entire cytoplasmic domain abolished the enzyme localization and functionality. Alanine substitution of the conserved catalytic Cys-His dyad abrogated the SrtC2 enzymatic activity. In contrast, mutations designed to alter a “lid” domain that covers the catalytic pocket of a class C sortase showed no effect on enzyme activity. Finally, each of the deleterious mutations that affected SrtC2 activity or membrane localization also eliminated Actinomyces species biofilm development and bacterial coaggregation with streptococci. We conclude that the N terminus of SrtC2, which contains the signal sequence, is required for proper protein translocation and maturation, while the extended C-terminal hydrophobic region serves as a stable membrane anchor for proper enzyme functionality. PMID:22447896

Structural basis for host membrane remodeling induced by protein 2B of hepatitis A virus.

PubMed

Vives-Adrián, Laia; Garriga, Damià; Buxaderas, Mònica; Fraga, Joana; Pereira, Pedro José Barbosa; Macedo-Ribeiro, Sandra; Verdaguer, Núria

2015-04-01

The complexity of viral RNA synthesis and the numerous participating factors require a mechanism to topologically coordinate and concentrate these multiple viral and cellular components, ensuring a concerted function. Similarly to all other positive-strand RNA viruses, picornaviruses induce rearrangements of host intracellular membranes to create structures that act as functional scaffolds for genome replication. The membrane-targeting proteins 2B and 2C, their precursor 2BC, and protein 3A appear to be primarily involved in membrane remodeling. Little is known about the structure of these proteins and the mechanisms by which they induce massive membrane remodeling. Here we report the crystal structure of the soluble region of hepatitis A virus (HAV) protein 2B, consisting of two domains: a C-terminal helical bundle preceded by an N-terminally curved five-stranded antiparallel β-sheet that displays striking structural similarity to the β-barrel domain of enteroviral 2A proteins. Moreover, the helicoidal arrangement of the protein molecules in the crystal provides a model for 2B-induced host membrane remodeling during HAV infection. No structural information is currently available for the 2B protein of any picornavirus despite it being involved in a critical process in viral factory formation: the rearrangement of host intracellular membranes. Here we present the structure of the soluble domain of the 2B protein of hepatitis A virus (HAV). Its arrangement, both in crystals and in solution under physiological conditions, can help to understand its function and sheds some light on the membrane rearrangement process, a putative target of future antiviral drugs. Moreover, this first structure of a picornaviral 2B protein also unveils a closer evolutionary relationship between the hepatovirus and enterovirus genera within the Picornaviridae family. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Structural Basis for Host Membrane Remodeling Induced by Protein 2B of Hepatitis A Virus

PubMed Central

Vives-Adrián, Laia; Garriga, Damià; Buxaderas, Mònica; Fraga, Joana; Pereira, Pedro José Barbosa

2015-01-01

ABSTRACT The complexity of viral RNA synthesis and the numerous participating factors require a mechanism to topologically coordinate and concentrate these multiple viral and cellular components, ensuring a concerted function. Similarly to all other positive-strand RNA viruses, picornaviruses induce rearrangements of host intracellular membranes to create structures that act as functional scaffolds for genome replication. The membrane-targeting proteins 2B and 2C, their precursor 2BC, and protein 3A appear to be primarily involved in membrane remodeling. Little is known about the structure of these proteins and the mechanisms by which they induce massive membrane remodeling. Here we report the crystal structure of the soluble region of hepatitis A virus (HAV) protein 2B, consisting of two domains: a C-terminal helical bundle preceded by an N-terminally curved five-stranded antiparallel β-sheet that displays striking structural similarity to the β-barrel domain of enteroviral 2A proteins. Moreover, the helicoidal arrangement of the protein molecules in the crystal provides a model for 2B-induced host membrane remodeling during HAV infection. IMPORTANCE No structural information is currently available for the 2B protein of any picornavirus despite it being involved in a critical process in viral factory formation: the rearrangement of host intracellular membranes. Here we present the structure of the soluble domain of the 2B protein of hepatitis A virus (HAV). Its arrangement, both in crystals and in solution under physiological conditions, can help to understand its function and sheds some light on the membrane rearrangement process, a putative target of future antiviral drugs. Moreover, this first structure of a picornaviral 2B protein also unveils a closer evolutionary relationship between the hepatovirus and enterovirus genera within the Picornaviridae family. PMID:25589659

Cobra CRISP functions as an inflammatory modulator via a novel Zn2+- and heparan sulfate-dependent transcriptional regulation of endothelial cell adhesion molecules.

PubMed

Wang, Yu-Ling; Kuo, Je-Hung; Lee, Shao-Chen; Liu, Jai-Shin; Hsieh, Yin-Cheng; Shih, Yu-Tsung; Chen, Chun-Jung; Chiu, Jeng-Jiann; Wu, Wen-Guey

2010-11-26

Cysteine-rich secretory proteins (CRISPs) have been identified as a toxin family in most animal venoms with biological functions mainly associated with the ion channel activity of cysteine-rich domain (CRD). CRISPs also bind to Zn(2+) at their N-terminal pathogenesis-related (PR-1) domain, but their function remains unknown. Interestingly, similar the Zn(2+)-binding site exists in all CRISP family, including those identified in a wide range of organisms. Here, we report that the CRISP from Naja atra (natrin) could induce expression of vascular endothelial cell adhesion molecules, i.e. intercellular adhesion molecule-1, vascular adhesion molecule-1, and E-selectin, to promote monocytic cell adhesion in a heparan sulfate (HS)- and Zn(2+)-dependent manner. Using specific inhibitors and small interfering RNAs, the activation mechanisms are shown to involve both mitogen-activated protein kinases and nuclear factor-κB. Biophysical characterization of natrin by using fluorescence, circular dichroism, and x-ray crystallographic methods further reveals the presence of two Zn(2+)-binding sites for natrin. The strong binding site is located near the putative Ser-His-Glu catalytic triad of the N-terminal domain. The weak binding site remains to be characterized, but it may modulate HS binding by enhancing its interaction with long chain HS. Our results strongly suggest that natrin may serve as an inflammatory modulator that could perturb the wound-healing process of the bitten victim by regulating adhesion molecule expression in endothelial cells. Our finding uncovers a new aspect of the biological role of CRISP family in immune response and is expected to facilitate future development of new therapeutic strategy for the envenomed victims.

Binding of the Extracellular Eight-Cysteine Motif of Opy2 to the Putative Osmosensor Msb2 Is Essential for Activation of the Yeast High-Osmolarity Glycerol Pathway

PubMed Central

Yamamoto, Katsuyoshi

2015-01-01

To adapt to environmental high osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase, which regulates diverse osmoadaptive responses. Hog1 is activated through the high-osmolarity glycerol (HOG) pathway, which consists of independent upstream signaling routes termed the SLN1 branch and the SHO1 branch. Here, we report that the extracellular cysteine-rich (CR) domain of the transmembrane-anchor protein Opy2 binds to the Hkr1-Msb2 homology (HMH) domain of the putative osmosensor Msb2 and that formation of the Opy2-Msb2 complex is essential for osmotic activation of Hog1 through the MSB2 subbranch of the SHO1 branch. By analyzing the phenotypes of mutants with Opy2 cysteine-to-alanine mutations, we deduced that the CR domain forms four intramolecular disulfide bonds. To probe for the potential induction of conformational changes in the Opy2-Msb2 complex by osmostress, we constructed mutants with a site-specific Cys-to-Ala mutation of the Opy2 CR domain and mutants with a Cys substitution of the Msb2 HMH domain. Each of these mutants had a reduced cysteine. These mutants were then combinatorially cross-linked using chemical cross-linkers of different lengths. Cross-linking between Opy2 Cys48 and Msb2 Cys1023 was sensitive to osmotic changes, suggesting that osmostress induced a conformational change. We therefore propose that the Opy2-Msb2 complex might serve as an osmosensor. PMID:26598606

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kendall, Amy; Bian, Wen; Maris, Alexander

We have used fiber diffraction, cryo-electron microscopy, and scanning transmission electron microscopy to confirm the symmetry of three potexviruses, potato virus X, papaya mosaic virus, and narcissus mosaic virus, and to determine their low-resolution structures. All three viruses have slightly less than nine subunits per turn of the viral helix. Our data strongly support the view that all potexviruses have approximately the same symmetry. The structures are dominated by a large domain at high radius in the virion, with a smaller domain, which includes the putative RNA-binding site, extending to low radius.

Molecular characterization and origin of novel bipartite cold-regulated ice recrystallization inhibition proteins from cereals.

PubMed

Tremblay, Karine; Ouellet, François; Fournier, Julie; Danyluk, Jean; Sarhan, Fathey

2005-06-01

To understand the molecular basis of freezing tolerance in plants, several low temperature-responsive genes have been identified from wheat. Among these are two genes named TaIRI-1 and TaIRI-2 (Triticum aestivum ice recrystallization inhibition) that are up-regulated during cold acclimation in freezing-tolerant species. Phytohormones involved in pathogen defense pathways (jasmonic acid and ethylene) induce the expression of one of the two genes. The encoded proteins are novel in that they have a bipartite structure that has never been reported for antifreeze proteins. Their N-terminal part shows similarity with the leucine-rich repeat-containing regions present in the receptor domain of receptor-like protein kinases, and their C-terminus is homologous to the ice-binding domain of some antifreeze proteins. The recombinant TaIRI-1 protein inhibits the growth of ice crystals, confirming its function as an ice recrystallization inhibition protein. The TaIRI genes were found only in the species belonging to the Pooideae subfamily of cereals. Comparative genomic analysis suggested that molecular evolutionary events took place in the genome of freezing-tolerant cereals to give rise to these genes with putative novel functions. These apparent adaptive DNA rearrangement events could be part of the molecular mechanisms that ensure the survival of hardy cereals in the harsh freezing environments.

The auto-inhibitory state of Rho guanine nucleotide exchange factor ARHGEF5/TIM can be relieved by targeting its SH3 domain with rationally designed peptide aptamers.

PubMed

He, Ping; Tan, De-Li; Liu, Hong-Xiang; Lv, Feng-Lin; Wu, Wei

2015-04-01

The short isoform of Rho guanine nucleotide exchange factor ARHGEF5 is known as TIM, which plays diverse roles in, for example, tumorigenesis, neuronal development and Src-induced podosome formation through the activation of its substrates, the Rho family of GTPases. The activation is auto-inhibited by a putative helix N-terminal to the DH domain of TIM, which is stabilized by the intramolecular interaction of C-terminal SH3 domain with a poly-proline sequence between the putative helix and the DH domain. In this study, we systematically investigated the structural basis, energetic landscape and biological implication underlying TIM auto-inhibition by using atomistic molecular dynamics simulations and binding free energy analysis. The computational study revealed that the binding of SH3 domain to poly-proline sequence is the prerequisite for the stabilization of TIM auto-inhibition. Thus, it is suggested that targeting SH3 domain with competitors of the poly-proline sequence would be a promising strategy to relieve the auto-inhibitory state of TIM. In this consideration, we rationally designed a number of peptide aptamers for competitively inhibiting the SH3 domain based on modeled TIM structure and computationally generated data. Peptide binding test and guanine nucleotide exchange analysis solidified that these designed peptides can both bind to the SH3 domain potently and activate TIM-catalyzed RhoA exchange reaction effectively. Interestingly, a positive correlation between the peptide affinity and induced exchange activity was observed. In addition, separate mutation of three conserved residues Pro49, Pro52 and Lys54 - they are required for peptide recognition by SH3 domain -- in a designed peptide to Ala would completely abolish the capability of this peptide activating TIM. All these come together to suggest an intrinsic relationship between peptide binding to SH3 domain and the activation of TIM. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Structure of the SANT domain from the Xenopus chromatin remodeling factor ISWI

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horton, John R.; Elgar, Stuart J.; Khan, Seema I.

2008-09-17

The SANT (Swi3, Ada2, N-Cor, and TFIIIB) module was first described as a putative DNA-binding domain with strong similarity to the helix-turn-helix DNA binding domain of Myb-related proteins. The X-ray structure of the C-terminal one third portion of the ATPase ISWI of Drosophila melangoaster, containing both SANT and SLIDE (SANT-Like ISWI Domain), confirmed the overall helix-turn-helix structural architecture of SANT as well as SLIDE. However, the DNA-contacting residues in Myb are not conserved in SANT and the structurally corresponding residues in the ISWI SANT domain are acidic, and therefore incompatible with DNA interaction. Recent studies suggested that SANT domains mightmore » be a histone-tail-binding module, including the DNA binding SANT domain of c-Myb. Here they present the X-ray structure of Xenopus laevis ISWI SANT domain, derived from limited proteolysis of a C-terminal fragment of ISWI protein.« less

Genome-wide identification and expression profiling of serine proteases and homologs in the diamondback moth, Plutella xylostella (L.).

PubMed

Lin, Hailan; Xia, Xiaofeng; Yu, Liying; Vasseur, Liette; Gurr, Geoff M; Yao, Fengluan; Yang, Guang; You, Minsheng

2015-12-10

Serine proteases (SPs) are crucial proteolytic enzymes responsible for digestion and other processes including signal transduction and immune responses in insects. Serine protease homologs (SPHs) lack catalytic activity but are involved in innate immunity. This study presents a genome-wide investigation of SPs and SPHs in the diamondback moth, Plutella xylostella (L.), a globally-distributed destructive pest of cruciferous crops. A total of 120 putative SPs and 101 putative SPHs were identified in the P. xylostella genome by bioinformatics analysis. Based on the features of trypsin, 38 SPs were putatively designated as trypsin genes. The distribution, transcription orientation, exon-intron structure and sequence alignments suggested that the majority of trypsin genes evolved from tandem duplications. Among the 221 SP/SPH genes, ten SP and three SPH genes with one or more clip domains were predicted and designated as PxCLIPs. Phylogenetic analysis of CLIPs in P. xylostella, two other Lepidoptera species (Bombyx mori and Manduca sexta), and two more distantly related insects (Drosophila melanogaster and Apis mellifera) showed that seven of the 13 PxCLIPs were clustered with homologs of the Lepidoptera rather than other species. Expression profiling of the P. xylostella SP and SPH genes in different developmental stages and tissues showed diverse expression patterns, suggesting high functional diversity with roles in digestion and development. This is the first genome-wide investigation on the SP and SPH genes in P. xylostella. The characterized features and profiled expression patterns of the P. xylostella SPs and SPHs suggest their involvement in digestion, development and immunity of this species. Our findings provide a foundation for further research on the functions of this gene family in P. xylostella, and a better understanding of its capacity to rapidly adapt to a wide range of environmental variables including host plants and insecticides.

In Silico Analysis of the Structural and Biochemical Features of the NMD Factor UPF1 in Ustilago maydis.

PubMed

Martínez-Montiel, Nancy; Morales-Lara, Laura; Hernández-Pérez, Julio M; Martínez-Contreras, Rebeca D

2016-01-01

The molecular mechanisms regulating the accuracy of gene expression are still not fully understood. Among these mechanisms, Nonsense-mediated Decay (NMD) is a quality control process that detects post-transcriptionally abnormal transcripts and leads them to degradation. The UPF1 protein lays at the heart of NMD as shown by several structural and functional features reported for this factor mainly for Homo sapiens and Saccharomyces cerevisiae. This process is highly conserved in eukaryotes but functional diversity can be observed in various species. Ustilago maydis is a basidiomycete and the best-known smut, which has become a model to study molecular and cellular eukaryotic mechanisms. In this study, we performed in silico analysis to investigate the structural and biochemical properties of the putative UPF1 homolog in Ustilago maydis. The putative homolog for UPF1 was recognized in the annotated genome for the basidiomycete, exhibiting 66% identity with its human counterpart at the protein level. The known structural and functional domains characteristic of UPF1 homologs were also found. Based on the crystal structures available for UPF1, we constructed different three-dimensional models for umUPF1 in order to analyze the secondary and tertiary structural features of this factor. Using these models, we studied the spatial arrangement of umUPF1 and its capability to interact with UPF2. Moreover, we identified the critical amino acids that mediate the interaction of umUPF1 with UPF2, ATP, RNA and with UPF1 itself. Mutating these amino acids in silico showed an important effect over the native structure. Finally, we performed molecular dynamic simulations for UPF1 proteins from H. sapiens and U. maydis and the results obtained show a similar behavior and physicochemical properties for the protein in both organisms. Overall, our results indicate that the putative UPF1 identified in U. maydis shows a very similar sequence, structural organization, mechanical stability, physicochemical properties and spatial organization in comparison to the NMD factor depicted for Homo sapiens. These observations strongly support the notion that human and fungal UPF1 could perform equivalent biological activities.

The S-layer homology domain-containing protein SlhA from Paenibacillus alvei CCM 2051(T) is important for swarming and biofilm formation.

PubMed

Janesch, Bettina; Koerdt, Andrea; Messner, Paul; Schäffer, Christina

2013-01-01

Swarming and biofilm formation have been studied for a variety of bacteria. While this is well investigated for Gram-negative bacteria, less is known about Gram-positive bacteria, including Paenibacillus alvei, a secondary invader of diseased honeybee colonies infected with Melissococcus pluton, the causative agent of European foulbrood (EFB). Paenibacillus alvei CCM 2051(T) is a Gram-positive bacterium which was recently shown to employ S-layer homology (SLH) domains as cell wall targeting modules to display proteins on its cell surface. This study deals with the newly identified 1335-amino acid protein SlhA from P. alvei which carries at the C‑terminus three consecutive SLH-motifs containing the predicted binding sequences SRGE, VRQD, and LRGD instead of the common TRAE motif. Based on the proof of cell surface location of SlhA by fluorescence microscopy using a SlhA-GFP chimera, the binding mechanism was investigated in an in vitro assay. To unravel a putative function of the SlhA protein, a knockout mutant was constructed. Experimental data indicated that one SLH domain is sufficient for anchoring of SlhA to the cell surface, and the SLH domains of SlhA recognize both the peptidoglycan and the secondary cell wall polymer in vitro. This is in agreement with previous data from the S-layer protein SpaA, pinpointing a wider utilization of that mechanism for cell surface display of proteins in P. alvei. Compared to the wild-type bacterium ΔslhA revealed changed colony morphology, loss of swarming motility and impaired biofilm formation. The phenotype was similar to that of the flagella knockout Δhag, possibly due to reduced EPS production influencing the functionality of the flagella of ΔslhA. This study demonstrates the involvement of the SLH domain-containing protein SlhA in swarming and biofilm formation of P. alvei CCM 2051(T).

Domain cooperativity in the β1a subunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle

PubMed Central

Dayal, Anamika; Bhat, Vinayakumar; Franzini-Armstrong, Clara; Grabner, Manfred

2013-01-01

The dihydropyridine receptor (DHPR) β1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα1S charge movement—the three prerequisites of skeletal muscle excitation–contraction coupling. Despite the ability to fully target α1S into triadic junctions and tetradic arrays, the neuronal isoform β3 was unable to restore considerable charge movement (measure of α1S voltage sensing) upon expression in β1-null zebrafish relaxed myotubes, unlike the other three vertebrate β-isoforms (β1a, β2a, and β4). Thus, we used β3 for chimerization with β1a to investigate whether any of the five distinct molecular regions of β1a is dominantly involved in inducing the voltage-sensing function of α1S. Surprisingly, systematic domain swapping between β1a and β3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of β1a in charge movement restoration. More interestingly, β1a SH3 domain and C terminus, when simultaneously engineered into β3 sequence background, were able to fully restore charge movement together with proper intracellular Ca2+ release, suggesting cooperativity of these two domains in induction of the α1S voltage-sensing function in skeletal muscle excitation–contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of β1a (also of β2a and β4) fully obstructed α1S charge movement. Consequently, we postulate a model according to which β subunits, probably via the SH3–C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage sensing in skeletal muscle. PMID:23589859

Domain cooperativity in the β1a subunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle.

PubMed

Dayal, Anamika; Bhat, Vinayakumar; Franzini-Armstrong, Clara; Grabner, Manfred

2013-04-30

The dihydropyridine receptor (DHPR) β1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα1S charge movement--the three prerequisites of skeletal muscle excitation-contraction coupling. Despite the ability to fully target α1S into triadic junctions and tetradic arrays, the neuronal isoform β3 was unable to restore considerable charge movement (measure of α1S voltage sensing) upon expression in β1-null zebrafish relaxed myotubes, unlike the other three vertebrate β-isoforms (β1a, β2a, and β4). Thus, we used β3 for chimerization with β1a to investigate whether any of the five distinct molecular regions of β1a is dominantly involved in inducing the voltage-sensing function of α1S. Surprisingly, systematic domain swapping between β1a and β3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of β1a in charge movement restoration. More interestingly, β1a SH3 domain and C terminus, when simultaneously engineered into β3 sequence background, were able to fully restore charge movement together with proper intracellular Ca(2+) release, suggesting cooperativity of these two domains in induction of the α1S voltage-sensing function in skeletal muscle excitation-contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of β1a (also of β2a and β4) fully obstructed α1S charge movement. Consequently, we postulate a model according to which β subunits, probably via the SH3-C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage sensing in skeletal muscle.

A novel extracellular metallopeptidase domain shared by animal host-associated mutualistic and pathogenic microbes.

PubMed

Nakjang, Sirintra; Ndeh, Didier A; Wipat, Anil; Bolam, David N; Hirt, Robert P

2012-01-01

The mucosal microbiota is recognised as an important factor for our health, with many disease states linked to imbalances in the normal community structure. Hence, there is considerable interest in identifying the molecular basis of human-microbe interactions. In this work we investigated the capacity of microbes to thrive on mucosal surfaces, either as mutualists, commensals or pathogens, using comparative genomics to identify co-occurring molecular traits. We identified a novel domain we named M60-like/PF13402 (new Pfam entry PF13402), which was detected mainly among proteins from animal host mucosa-associated prokaryotic and eukaryotic microbes ranging from mutualists to pathogens. Lateral gene transfers between distantly related microbes explained their shared M60-like/PF13402 domain. The novel domain is characterised by a zinc-metallopeptidase-like motif and is distantly related to known viral enhancin zinc-metallopeptidases. Signal peptides and/or cell surface anchoring features were detected in most microbial M60-like/PF13402 domain-containing proteins, indicating that these proteins target an extracellular substrate. A significant subset of these putative peptidases was further characterised by the presence of associated domains belonging to carbohydrate-binding module family 5/12, 32 and 51 and other glycan-binding domains, suggesting that these novel proteases are targeted to complex glycoproteins such as mucins. An in vitro mucinase assay demonstrated degradation of mammalian mucins by a recombinant form of an M60-like/PF13402-containing protein from the gut mutualist Bacteroides thetaiotaomicron. This study reveals that M60-like domains are peptidases targeting host glycoproteins. These peptidases likely play an important role in successful colonisation of both vertebrate mucosal surfaces and the invertebrate digestive tract by both mutualistic and pathogenic microbes. Moreover, 141 entries across various peptidase families described in the MEROPS database were also identified with carbohydrate-binding modules defining a new functional context for these glycan-binding domains and providing opportunities to engineer proteases targeting specific glycoproteins for both biomedical and industrial applications.

Differentiation-dependent expression of hypothetical proteins in the neuroblastoma cell line N1E-115.

PubMed

Oh, Ji-eun; Karlmark, Karlin Raja; Shin, Jooho; Hengstschläger, Markus; Lubec, Gert

2006-05-15

Several protein cascades, including signaling, cytoskeletal, chaperones, metabolic, and antioxidant proteins, have been shown to be involved in the process of neuronal differentiation (ND) of neuroblastoma cell lines. No systematic approach to detect hitherto unknown and unnamed proteins or structures that have been predicted upon nucleic acid sequences in ND has been published so far. We therefore decided to screen hypothetical protein (HP) expression by protein profiling. Two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF) identification was used for expression analysis of undifferentiated and dimethylsulfoxide-induced neuronally differentiated N1E-115 cells. We unambiguously identified six HPs: Q8C520, Q99LF4, Q9CXS1, Q9DAF8, Q91WT0, and Q8C5G2. A prefoldin domain in Q91WT0, a t-SNARE domain in Q9CXS1, and a bromodomain were observed in Q8C5G2. For the three remaining proteins, no putative function using Pfam, BLOCKS, PROSITE, PRINTS, InterPro, Superfamily, CoPS, and ExPASy could be assigned. While two proteins were present in both cell lines, Q9CXS1 was switched off (i.e., undetectably low) in differentiated cells only, and Q9DAF8, Q91WT0, and Q8C5G2 were switched on in differentiated cells exclusively. Herein, using a proteomic approach suitable for screening and identification of HP, we present HP structures that have been only predicted so far based upon nucleic acid sequences. The four differentially regulated HPs may play a putative role in the process of ND. (c) 2006 Wiley-Liss, Inc.

A Lys-Trp cation-π interaction mediates the dimerization and function of the chloride intracellular channel protein 1 transmembrane domain.

PubMed

Peter, Bradley; Polyansky, Anton A; Fanucchi, Sylvia; Dirr, Heini W

2014-01-14

Chloride intracellular channel protein 1 (CLIC1) is a dual-state protein that can exist either as a soluble monomer or in an integral membrane form. The oligomerization of the transmembrane domain (TMD) remains speculative despite it being implicated in pore formation. The extent to which electrostatic and van der Waals interactions drive folding and association of the dimorphic TMD is unknown and is complicated by the requirement of interactions favorable in both aqueous and membrane environments. Here we report a putative Lys37-Trp35 cation-π interaction and show that it stabilizes the dimeric form of the CLIC1 TMD in membranes. A synthetic 30-mer peptide comprising a K37M TMD mutant was examined in 2,2,2-trifluoroethanol, sodium dodecyl sulfate micelles, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes using far-ultraviolet (UV) circular dichroism, fluorescence, and UV absorbance spectroscopy. Our data suggest that Lys37 is not implicated in the folding, stability, or membrane insertion of the TMD peptide. However, removal of this residue impairs the formation of dimers and higher-order oligomers. This is accompanied by a 30-fold loss of chloride influx activity, suggesting that dimerization modulates the rate of chloride conductance. We propose that, within membranes, individual TMD helices associate via a Lys37-mediated cation-π interaction to form active dimers. The latter findings are also supported by results of modeling a putative TMD dimer conformation in which Lys37 and Trp35 form cation-π pairs at the dimer interface. Dimeric helix bundles may then associate to form fully active ion channels. Thus, within a membrane-like environment, aromatic interactions involving a polar lysine side chain provide a thermodynamic driving force for helix-helix association.

Sequence analyses and evolutionary relationships among the energy-coupling proteins Enzyme I and HPr of the bacterial phosphoenolpyruvate: sugar phosphotransferase system.

PubMed Central

Reizer, J.; Hoischen, C.; Reizer, A.; Pham, T. N.; Saier, M. H.

1993-01-01

We have previously reported the overexpression, purification, and biochemical properties of the Bacillus subtilis Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) (Reizer, J., et al., 1992, J. Biol. Chem. 267, 9158-9169). We now report the sequencing of the ptsI gene of B. subtilis encoding Enzyme I (570 amino acids and 63,076 Da). Putative transcriptional regulatory signals are identified, and the pts operon is shown to be subject to carbon source-dependent regulation. Multiple alignments of the B. subtilis Enzyme I with (1) six other sequenced Enzymes I of the PTS from various bacterial species, (2) phosphoenolpyruvate synthase of Escherichia coli, and (3) bacterial and plant pyruvate: phosphate dikinases (PPDKs) revealed regions of sequence similarity as well as divergence. Statistical analyses revealed that these three types of proteins comprise a homologous family, and the phylogenetic tree of the 11 sequenced protein members of this family was constructed. This tree was compared with that of the 12 sequence HPr proteins or protein domains. Antibodies raised against the B. subtilis and E. coli Enzymes I exhibited immunological cross-reactivity with each other as well as with PPDK of Bacteroides symbiosus, providing support for the evolutionary relationships of these proteins suggested from the sequence comparisons. Putative flexible linkers tethering the N-terminal and the C-terminal domains of protein members of the Enzyme I family were identified, and their potential significance with regard to Enzyme I function is discussed. The codon choice pattern of the B. subtilis and E. coli ptsI and ptsH genes was found to exhibit a bias toward optimal codons in these organisms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7686067

Identification of Genes Involved in Biofilm Formation and Respiration via Mini-Himar Transposon Mutagenesis of Geobacter sulfurreducens▿ †

PubMed Central

Rollefson, Janet B.; Levar, Caleb E.; Bond, Daniel R.

2009-01-01

Electron transfer from cells to metals and electrodes by the Fe(III)-reducing anaerobe Geobacter sulfurreducens requires proper expression of redox proteins and attachment mechanisms to interface bacteria with surfaces and neighboring cells. We hypothesized that transposon mutagenesis would complement targeted knockout studies in Geobacter spp. and identify novel genes involved in this process. Escherichia coli mating strains and plasmids were used to develop a conjugation protocol and deliver mini-Himar transposons, creating a library of over 8,000 mutants that was anaerobically arrayed and screened for a range of phenotypes, including auxotrophy for amino acids, inability to reduce Fe(III) citrate, and attachment to surfaces. Following protocol validation, mutants with strong phenotypes were further characterized in a three-electrode system to simultaneously quantify attachment, biofilm development, and respiratory parameters, revealing mutants defective in Fe(III) reduction but unaffected in electron transfer to electrodes (such as an insertion in GSU1330, a putative metal export protein) or defective in electrode reduction but demonstrating wild-type biofilm formation (due to an insertion upstream of the NHL domain protein GSU2505). An insertion in a putative ATP-dependent transporter (GSU1501) eliminated electrode colonization but not Fe(III) citrate reduction. A more complex phenotype was demonstrated by a mutant containing an insertion in a transglutaminase domain protein (GSU3361), which suddenly ceased to respire when biofilms reached approximately 50% of the wild-type levels. As most insertions were not in cytochromes but rather in transporters, two-component signaling proteins, and proteins of unknown function, this collection illustrates how biofilm formation and electron transfer are separate but complementary phenotypes, controlled by multiple loci not commonly studied in Geobacter spp. PMID:19395486

Echinoderm phosphorylated matrix proteins UTMP16 and UTMP19 have different functions in sea urchin tooth mineralization.

PubMed

Alvares, Keith; Dixit, Saryu N; Lux, Elizabeth; Veis, Arthur

2009-09-18

Studies of mineralization of embryonic spicules and of the sea urchin genome have identified several putative mineralization-related proteins. These predicted proteins have not been isolated or confirmed in mature mineralized tissues. Mature Lytechinus variegatus teeth were demineralized with 0.6 N HCl after prior removal of non-mineralized constituents with 4.0 M guanidinium HCl. The HCl-extracted proteins were fractionated on ceramic hydroxyapatite and separated into bound and unbound pools. Gel electrophoresis compared the protein distributions. The differentially present bands were purified and digested with trypsin, and the tryptic peptides were separated by high pressure liquid chromatography. NH2-terminal sequences were determined by Edman degradation and compared with the genomic sequence bank data. Two of the putative mineralization-related proteins were found. Their complete amino acid sequences were cloned from our L. variegatus cDNA library. Apatite-binding UTMP16 was found to be present in two isoforms; both isoforms had a signal sequence, a Ser-Asp-rich extracellular matrix domain, and a transmembrane and cytosolic insertion sequence. UTMP19, although rich in Glu and Thr did not bind to apatite. It had neither signal peptide nor transmembrane domain but did have typical nuclear localization and nuclear exit signal sequences. Both proteins were phosphorylated and good substrates for phosphatase. Immunolocalization studies with anti-UTMP16 show it to concentrate at the syncytial membranes in contact with the mineral. On the basis of our TOF-SIMS analyses of magnesium ion and Asp mapping of the mineral phase composition, we speculate that UTMP16 may be important in establishing the high magnesium columns that fuse the calcite plates together to enhance the mechanical strength of the mineralized tooth.

PRMT7 is a member of the protein arginine methyltransferase family with a distinct substrate specificity.

PubMed

Miranda, Tina Branscombe; Miranda, Mark; Frankel, Adam; Clarke, Steven

2004-05-28

We have identified a mammalian arginine N-methyltransferase, PRMT7, that can catalyze the formation of omega-NG-monomethylarginine in peptides. This protein is encoded by a gene on human chromosome 16q22.1 (human locus AK001502). We expressed a full-length human cDNA construct in Escherichia coli as a glutathione S-transferase (GST) fusion protein. We found that GST-tagged PRMT7 catalyzes the S-adenosyl-[methyl-3H]-l-methionine-dependent methylation of the synthetic peptide GGPGGRGGPGG-NH2 (R1). The radiolabeled peptide was purified by high-pressure liquid chromatography and acid hydrolyzed to free amino acids. When the hydrolyzed products were separated by high-resolution cation-exchange chromatography, we were able to detect one tritiated species which co-migrated with an omega-NG-monomethylarginine standard. Surprisingly, GST-PRMT7 was not able to catalyze the in vitro methylation of a GST-fibrillarin (amino acids 1-148) fusion protein (GST-GAR), a methyl-accepting substrate for the previously characterized PRMT1, PRMT3, PRMT4, PRMT5, and PRMT6 enzymes. Nor was it able to methylate myelin basic protein or histone H2A, in vitro substrates of PRMT5. This specificity distinguishes PRMT7 from all of the other known arginine methyltransferases. An additional unique feature of PRMT7 is that it seems to have arisen from a gene duplication event and contains two putative AdoMet-binding motifs. To see if both motifs were necessary for activity, each putative domain was expressed as a GST-fusion and tested for activity with peptides R1 and R2 (acetyl-GGRGG-NH2). These truncated proteins were enzymatically inactive, suggesting that both domains are required for functionality.

Molecular cloning of the potato Gro1-4 gene conferring resistance to pathotype Ro1 of the root cyst nematode Globodera rostochiensis, based on a candidate gene approach.

PubMed

Paal, Jürgen; Henselewski, Heike; Muth, Jost; Meksem, Khalid; Menéndez, Cristina M; Salamini, Francesco; Ballvora, Agim; Gebhardt, Christiane

2004-04-01

The endoparasitic root cyst nematode Globodera rostochiensis causes considerable damage in potato cultivation. In the past, major genes for nematode resistance have been introgressed from related potato species into cultivars. Elucidating the molecular basis of resistance will contribute to the understanding of nematode-plant interactions and assist in breeding nematode-resistant cultivars. The Gro1 resistance locus to G. rostochiensis on potato chromosome VII co-localized with a resistance-gene-like (RGL) DNA marker. This marker was used to isolate from genomic libraries 15 members of a closely related candidate gene family. Analysis of inheritance, linkage mapping, and sequencing reduced the number of candidate genes to three. Complementation analysis by stable potato transformation showed that the gene Gro1-4 conferred resistance to G. rostochiensis pathotype Ro1. Gro1-4 encodes a protein of 1136 amino acids that contains Toll-interleukin 1 receptor (TIR), nucleotide-binding (NB), leucine-rich repeat (LRR) homology domains and a C-terminal domain with unknown function. The deduced Gro1-4 protein differed by 29 amino acid changes from susceptible members of the Gro1 gene family. Sequence characterization of 13 members of the Gro1 gene family revealed putative regulatory elements and a variable microsatellite in the promoter region, insertion of a retrotransposon-like element in the first intron, and a stop codon in the NB coding region of some genes. Sequence analysis of RT-PCR products showed that Gro1-4 is expressed, among other members of the family including putative pseudogenes, in non-infected roots of nematode-resistant plants. RT-PCR also demonstrated that members of the Gro1 gene family are expressed in most potato tissues.

Peptide Synthetase Gene in Trichoderma virens

PubMed Central

Wilhite, S. E.; Lumsden, R. D.; Straney, D. C.

2001-01-01

Trichoderma virens (synonym, Gliocladium virens), a deuteromycete fungus, suppresses soilborne plant diseases caused by a number of fungi and is used as a biocontrol agent. Several traits that may contribute to the antagonistic interactions of T. virens with disease-causing fungi involve the production of peptide metabolites (e.g., the antibiotic gliotoxin and siderophores used for iron acquisition). We cloned a 5,056-bp partial cDNA encoding a putative peptide synthetase (Psy1) from T. virens using conserved motifs found within the adenylate domain of peptide synthetases. Sequence similarities with conserved motifs of the adenylation domain, acyl transfer, and two condensation domains support identification of the Psy1 gene as a gene that encodes a peptide synthetase. Disruption of the native Psy1 gene through gene replacement was used to identify the function of this gene. Psy1 disruptants produced normal amounts of gliotoxin but grew poorly under low-iron conditions, suggesting that Psy1 plays a role in siderophore production. Psy1 disruptants cannot produce the major T. virens siderophore dimerum acid, a dipetide of acylated Nδ-hydroxyornithine. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the Psy1 disruption, suggesting that iron competition through dimerum acid production does not contribute significantly to disease suppression activity under the conditions used. PMID:11679326

Genome-wide identification and analysis of basic helix-loop-helix domains in dog, Canis lupus familiaris.

PubMed

Wang, Xu-Hua; Wang, Yong; Liu, A-Ke; Liu, Xiao-Ting; Zhou, Yang; Yao, Qin; Chen, Ke-Ping

2015-04-01

The basic helix-loop-helix (bHLH) domain is a highly conserved amino acid motif that defines a group of DNA-binding transcription factors. bHLH proteins play essential regulatory roles in a variety of biological processes in animal, plant, and fungus. The domestic dog, Canis lupus familiaris, is a good model organism for genetic, physiological, and behavioral studies. In this study, we identified 115 putative bHLH genes in the dog genome. Based on a phylogenetic analysis, 51, 26, 14, 4, 12, and 4 dog bHLH genes were assigned to six separate groups (A-F); four bHLH genes were categorized as ''orphans''. Within-group evolutionary relationships inferred from the phylogenetic analysis were consistent with positional conservation, other conserved domains flanking the bHLH motif, and highly conserved intron/exon patterns in other vertebrates. Our analytical results confirmed the GenBank annotations of 89 dog bHLH proteins and provided information that could be used to update the annotations of the remaining 26 dog bHLH proteins. These data will provide good references for further studies on the structures and regulatory functions of bHLH proteins in the growth and development of dogs, which may help in understanding the mechanisms that underlie the physical and behavioral differences between dogs and wolves.

Large-Scale, Lineage-Specific Expansion of a Bric-a-Brac/Tramtrack/Broad Complex Ubiquitin-Ligase Gene Family in Rice[W

PubMed Central

Gingerich, Derek J.; Hanada, Kousuke; Shiu, Shin-Han; Vierstra, Richard D.

2007-01-01

Selective ubiquitination of proteins is directed by diverse families of ubiquitin-protein ligases (or E3s) in plants. One important type uses Cullin-3 as a scaffold to assemble multisubunit E3 complexes containing one of a multitude of bric-a-brac/tramtrack/broad complex (BTB) proteins that function as substrate recognition factors. We previously described the 80-member BTB gene superfamily in Arabidopsis thaliana. Here, we describe the complete BTB superfamily in rice (Oryza sativa spp japonica cv Nipponbare) that contains 149 BTB domain–encoding genes and 43 putative pseudogenes. Amino acid sequence comparisons of the rice and Arabidopsis superfamilies revealed a near equal repertoire of putative substrate recognition module types. However, phylogenetic comparisons detected numerous gene duplication and/or loss events since the rice and Arabidopsis BTB lineages split, suggesting possible functional specialization within individual BTB families. In particular, a major expansion and diversification of a subset of BTB proteins containing Meprin and TRAF homology (MATH) substrate recognition sites was evident in rice and other monocots that likely occurred following the monocot/dicot split. The MATH domain of a subset appears to have evolved significantly faster than those in a smaller core subset that predates flowering plants, suggesting that the substrate recognition module in many monocot MATH-BTB E3s are diversifying to ubiquitinate a set of substrates that are themselves rapidly changing. Intriguing possibilities include pathogen proteins attempting to avoid inactivation by the monocot host. PMID:17720868

FusionHub: A unified web platform for annotation and visualization of gene fusion events in human cancer.

PubMed

Panigrahi, Priyabrata; Jere, Abhay; Anamika, Krishanpal

2018-01-01

Gene fusion is a chromosomal rearrangement event which plays a significant role in cancer due to the oncogenic potential of the chimeric protein generated through fusions. At present many databases are available in public domain which provides detailed information about known gene fusion events and their functional role. Existing gene fusion detection tools, based on analysis of transcriptomics data usually report a large number of fusion genes as potential candidates, which could be either known or novel or false positives. Manual annotation of these putative genes is indeed time-consuming. We have developed a web platform FusionHub, which acts as integrated search engine interfacing various fusion gene databases and simplifies large scale annotation of fusion genes in a seamless way. In addition, FusionHub provides three ways of visualizing fusion events: circular view, domain architecture view and network view. Design of potential siRNA molecules through ensemble method is another utility integrated in FusionHub that could aid in siRNA-based targeted therapy. FusionHub is freely available at https://fusionhub.persistent.co.in.

The rise and fall of long-lived humoral immunity: terminal differentiation of plasma cells in health and disease

PubMed Central

O'Connor, Brian P.; Gleeson, Michael W.; Noelle, Randolph J.; Erickson, Loren D.

2010-01-01

Summary Long-lived humoral immune responses are a hallmark of thymus-dependent immunity. The cellular basis for enduring antibody-mediated immunity is long-lived memory B cells and plasma cells (PCs). Both of these cell populations acquire longevity as a result of antigen-specific, CD40–dependent, cognate interactions with helper T cells within germinal centers (GCs). At the molecular level, defined functional domains of CD40 control the post-GC fate of B cells. PC precursors that emerge from these GC reactions are highly proliferative and terminally differentiate to end-stage cells within the bone marrow (BM). The striking phenotypic similarities between the PC precursors and the putative malignant cell in multiple myeloma (MM) suggests that MM may result from the transformation of PC precursors. Within the domain of autoimmune disease, recent studies have shown that dysregulated migration of PCs to the BM may impact immune homeostasis and the development of lupus. Understanding the processes of normal PC differentiation will provide strategic insights into identifying therapeutic targets for the treatment of differentiated B-cell disorders. PMID:12846808

Predominance of null mutations in ataxia-telangiectasia.

PubMed

Gilad, S; Khosravi, R; Shkedy, D; Uziel, T; Ziv, Y; Savitsky, K; Rotman, G; Smith, S; Chessa, L; Jorgensen, T J; Harnik, R; Frydman, M; Sanal, O; Portnoi, S; Goldwicz, Z; Jaspers, N G; Gatti, R A; Lenoir, G; Lavin, M F; Tatsumi, K; Wegner, R D; Shiloh, Y; Bar-Shira, A

1996-04-01

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity and cancer predisposition. The responsible gene, ATM, was recently identified by positional cloning and found to encode a putative 350 kDa protein with a Pl 3-kinase-like domain, presumably involved in mediating cell cycle arrest in response to radiation-induced DNA damage. The nature and location of A-T mutations should provide insight into the function of the ATM protein and the molecular basis of this pleiotropic disease. Of 44 A-T mutations identified by us to date, 39 (89%) are expected to inactivate the ATM protein by truncating it, by abolishing correct initiation or termination of translation, or by deleting large segments. Additional mutations are four smaller in-frame deletions and insertions, and one substitution of a highly conserved amino acid at the Pl 3-kinase domain. The emerging profile of mutations causing A-T is thus dominated by those expected to completely inactivate the ATM protein. ATM mutations with milder effects may result in phenotypes related, but not identical, to A-T.

Crystal structure of the human astrovirus capsid spike.

PubMed

Dong, Jinhui; Dong, Liping; Méndez, Ernesto; Tao, Yizhi

2011-08-02

Astroviruses are single-stranded, plus-sense RNA viruses that infect both mammals and birds, causing gastroenteritis and other extraintestinal diseases. Clinical studies have established astroviruses as the second leading cause of viral diarrhea in young children. Here we report the crystal structure of the human astrovirus dimeric surface spike determined to 1.8-Å resolution. The overall structure of each spike/projection domain has a unique three-layered β-sandwiches fold, with a core, six-stranded β-barrel structure that is also found in the hepatitis E virus capsid protrusions, suggesting a closer phylogenetic relationship between these two viruses than previously acknowledged. Based on a hepatitis E virus capsid model, we performed homology modeling and produced a complete, T = 3 astrovirus capsid model with features remarkably similar to those observed in a cryoelectron microscopy reconstruction image of a human astrovirus. Mapping conserved residues onto the astrovirus projection domain revealed a putative receptor binding site with amino acid compositions characteristic for polysaccharide recognition. Our results will have an important impact on future characterization of astrovirus structure and function, and will likely have practical applications in the development of vaccines and antivirals.

AfAP2-1, An Age-Dependent Gene of Aechmea fasciata, Responds to Exogenous Ethylene Treatment

PubMed Central

Lei, Ming; Li, Zhi-Ying; Wang, Jia-Bin; Fu, Yun-Liu; Ao, Meng-Fei; Xu, Li

2016-01-01

The Bromeliaceae family is one of the most morphologically diverse families with a pantropical distribution. To schedule an appropriate flowering time for bromeliads, ethylene is commonly used to initiate flower development in adult plants. However, the mechanism by which ethylene induces flowering in adult bromeliads remains unknown. Here, we identified an APETALA2 (AP2)-like gene, AfAP2-1, in Aechmea fasciata. AfAP2-1 contains two AP2 domains and is a nuclear-localized protein. It functions as a transcriptional activator, and the activation domain is located in the C-terminal region. The expression level of AfAP2-1 is higher in juvenile plants than in adult plants, and the AfAP2-1 transcript level was rapidly and transiently reduced in plants treated with exogenous ethylene. Overexpression of AfAP2-1 in Arabidopsis thaliana results in an extremely delayed flowering phenotype. These results suggested that AfAP2-1 responds to ethylene and is a putative age-dependent flowering regulator in A. fasciata. PMID:26927090

Sequence of a cDNA and expression of the gene encoding a putative epidermal chitin synthase of Manduca sexta.

PubMed

Zhu, Yu-Cheng; Specht, Charles A; Dittmer, Neal T; Muthukrishnan, Subbaratnam; Kanost, Michael R; Kramer, Karl J

2002-11-01

Glycosyltransferases are enzymes that synthesize oligosaccharides, polysaccharides and glycoconjugates. One type of glycosyltransferase is chitin synthase, a very important enzyme in biology, which is utilized by insects, fungi, and other invertebrates to produce chitin, a polysaccharide of beta-1,4-linked N-acetylglucosamine. Chitin is an important component of the insect's exoskeletal cuticle and gut lining. To identify and characterize a chitin synthase gene of the tobacco hornworm, Manduca sexta, degenerate primers were designed from two highly conserved regions in fungal and nematode chitin synthase protein sequences and then used to amplify a similar region from Manduca cDNA. A full-length cDNA of 5152 nucleotides was assembled for the putative Manduca chitin synthase gene, MsCHS1, and sequencing of genomic DNA verified the contiguity of the sequence. The MsCHS1 cDNA has an ORF of 4692 nucleotides that encodes a transmembrane protein of 1564 amino acid residues with a mass of approximately 179 kDa (GenBank no. AY062175). It is most similar, over its entire length of protein sequence, to putative chitin synthases from other insects and nematodes, with 68% identity to enzymes from both the blow fly, Lucilia cuprina, and the fruit fly, Drosophila melanogaster. The similarity with fungal chitin synthases is restricted to the putative catalytic domain, and the MsCHS1 protein has, at equivalent positions, several amino acids that are essential for activity as revealed by mutagenesis of the fungal enzymes. A 5.3-kb transcript of MsCHS1 was identified by northern blot hybridization of RNA from larval epidermis, suggesting that the enzyme functions to make chitin deposited in the cuticle. Further examination by RT-PCR showed that MsCHS1 expression is regulated in the epidermis, with the amount of transcript increasing during phases of cuticle deposition.

Solution structure of the Big domain from Streptococcus pneumoniae reveals a novel Ca2+-binding module

PubMed Central

Wang, Tao; Zhang, Jiahai; Zhang, Xuecheng; Xu, Chao; Tu, Xiaoming

2013-01-01

Streptococcus pneumoniae is a pathogen causing acute respiratory infection, otitis media and some other severe diseases in human. In this study, the solution structure of a bacterial immunoglobulin-like (Big) domain from a putative S. pneumoniae surface protein SP0498 was determined by NMR spectroscopy. SP0498 Big domain adopts an eight-β-strand barrel-like fold, which is different in some aspects from the two-sheet sandwich-like fold of the canonical Ig-like domains. Intriguingly, we identified that the SP0498 Big domain was a Ca2+ binding domain. The structure of the Big domain is different from those of the well known Ca2+ binding domains, therefore revealing a novel Ca2+-binding module. Furthermore, we identified the critical residues responsible for the binding to Ca2+. We are the first to report the interactions between the Big domain and Ca2+ in terms of structure, suggesting an important role of the Big domain in many essential calcium-dependent cellular processes such as pathogenesis. PMID:23326635

Expression, purification and preliminary biochemical and structural characterization of the leucine rich repeat namesake domain of leucine rich repeat kinase 2.

PubMed

Vancraenenbroeck, Renée; Lobbestael, Evy; Weeks, Stephen D; Strelkov, Sergei V; Baekelandt, Veerle; Taymans, Jean-Marc; De Maeyer, Marc

2012-03-01

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, Ras of complex proteins) and kinase domains and a connecting COR (C-terminus of ROC) domain. In contrast, the precise functions of the protein-protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR(LRRK2)) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR(LRRK2) in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR(LRRK2) thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% α-helices and 21% β-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR(LRRK2) multimerization was detected via cross-linking studies. Finally, the LRR(LRRK2) clinical mutations did not influence LRR(LRRK2) secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR(LRRK2) inter- and intramolecular interactions. Copyright © 2011 Elsevier B.V. All rights reserved.

C-Terminal β9-Strand of the Cyclic Nucleotide-Binding Homology Domain Stabilizes Activated States of Kv11.1 Channels

PubMed Central

Ng, Chai Ann; Ke, Ying; Perry, Matthew D.; Tan, Peter S.; Hill, Adam P.; Vandenberg, Jamie I.

2013-01-01

Kv11.1 potassium channels are important for regulation of the normal rhythm of the heartbeat. Reduced activity of Kv11.1 channels causes long QT syndrome type 2, a disorder that increases the risk of cardiac arrhythmias and sudden cardiac arrest. Kv11.1 channels are members of the KCNH subfamily of voltage-gated K+ channels. However, they also share many similarities with the cyclic nucleotide gated ion channel family, including having a cyclic nucleotide-binding homology (cNBH) domain. Kv11.1 channels, however, are not directly regulated by cyclic nucleotides. Recently, crystal structures of the cNBH domain from mEAG and zELK channels, both members of the KCNH family of voltage-gated potassium channels, revealed that a C-terminal β9-strand in the cNBH domain occupied the putative cyclic nucleotide-binding site thereby precluding binding of cyclic nucleotides. Here we show that mutations to residues in the β9-strand affect the stability of the open state relative to the closed state of Kv11.1 channels. We also show that disrupting the structure of the β9-strand reduces the stability of the inactivated state relative to the open state. Clinical mutations located in this β9-strand result in reduced trafficking efficiency, which suggests that binding of the C-terminal β9-strand to the putative cyclic nucleotide-binding pocket is also important for assembly and trafficking of Kv11.1 channels. PMID:24204727

The Human Metapneumovirus Small Hydrophobic Protein Has Properties Consistent with Those of a Viroporin and Can Modulate Viral Fusogenic Activity

PubMed Central

Masante, Cyril; El Najjar, Farah; Chang, Andres; Jones, Angela; Moncman, Carole L.

2014-01-01

ABSTRACT Human metapneumovirus (HMPV) encodes three glycoproteins: the glycoprotein, which plays a role in glycosaminoglycan binding, the fusion (F) protein, which is necessary and sufficient for both viral binding to the target cell and fusion between the cellular plasma membrane and the viral membrane, and the small hydrophobic (SH) protein, whose function is unclear. The SH protein of the closely related respiratory syncytial virus has been suggested to function as a viroporin, as it forms oligomeric structures consistent with a pore and alters membrane permeability. Our analysis indicates that both the full-length HMPV SH protein and the isolated SH protein transmembrane domain can associate into higher-order oligomers. In addition, HMPV SH expression resulted in increases in permeability to hygromycin B and alteration of subcellular localization of a fluorescent dye, indicating that SH affects membrane permeability. These results suggest that the HMPV SH protein has several characteristics consistent with a putative viroporin. Interestingly, we also report that expression of the HMPV SH protein can significantly decrease HMPV F protein-promoted membrane fusion activity, with the SH extracellular domain and transmembrane domain playing a key role in this inhibition. These results suggest that the HMPV SH protein could regulate both membrane permeability and fusion protein function during viral infection. IMPORTANCE Human metapneumovirus (HMPV), first identified in 2001, is a causative agent of severe respiratory tract disease worldwide. The small hydrophobic (SH) protein is one of three glycoproteins encoded by all strains of HMPV, but the function of the HMPV SH protein is unknown. We have determined that the HMPV SH protein can alter the permeability of cellular membranes, suggesting that HMPV SH is a member of a class of proteins termed viroporins, which modulate membrane permeability to facilitate critical steps in a viral life cycle. We also demonstrated that HMPV SH can inhibit the membrane fusion function of the HMPV fusion protein. This work suggests that the HMPV SH protein has several functions, though the steps in the HMPV life cycle impacted by these functions remain to be clarified. PMID:24672047

Systematic analysis of human kinase genes: a large number of genes and alternative splicing events result in functional and structural diversity

PubMed Central

Milanesi, Luciano; Petrillo, Mauro; Sepe, Leandra; Boccia, Angelo; D'Agostino, Nunzio; Passamano, Myriam; Di Nardo, Salvatore; Tasco, Gianluca; Casadio, Rita; Paolella, Giovanni

2005-01-01

Background Protein kinases are a well defined family of proteins, characterized by the presence of a common kinase catalytic domain and playing a significant role in many important cellular processes, such as proliferation, maintenance of cell shape, apoptosys. In many members of the family, additional non-kinase domains contribute further specialization, resulting in subcellular localization, protein binding and regulation of activity, among others. About 500 genes encode members of the kinase family in the human genome, and although many of them represent well known genes, a larger number of genes code for proteins of more recent identification, or for unknown proteins identified as kinase only after computational studies. Results A systematic in silico study performed on the human genome, led to the identification of 5 genes, on chromosome 1, 11, 13, 15 and 16 respectively, and 1 pseudogene on chromosome X; some of these genes are reported as kinases from NCBI but are absent in other databases, such as KinBase. Comparative analysis of 483 gene regions and subsequent computational analysis, aimed at identifying unannotated exons, indicates that a large number of kinase may code for alternately spliced forms or be incorrectly annotated. An InterProScan automated analysis was perfomed to study domain distribution and combination in the various families. At the same time, other structural features were also added to the annotation process, including the putative presence of transmembrane alpha helices, and the cystein propensity to participate into a disulfide bridge. Conclusion The predicted human kinome was extended by identifiying both additional genes and potential splice variants, resulting in a varied panorama where functionality may be searched at the gene and protein level. Structural analysis of kinase proteins domains as defined in multiple sources together with transmembrane alpha helices and signal peptide prediction provides hints to function assignment. The results of the human kinome analysis are collected in the KinWeb database, available for browsing and searching over the internet, where all results from the comparative analysis and the gene structure annotation are made available, alongside the domain information. Kinases may be searched by domain combinations and the relative genes may be viewed in a graphic browser at various level of magnification up to gene organization on the full chromosome set. PMID:16351747

Mutagenesis of the C2 domain of protein kinase C-alpha. Differential roles of Ca2+ ligands and membrane binding residues.

PubMed

Medkova, M; Cho, W

1998-07-10

The C2 domains of conventional protein kinase C (PKC) have been implicated in their Ca2+-dependent membrane binding. The C2 domain of PKC-alpha contains several Ca2+ ligands that bind multiple Ca2+ ions and other putative membrane binding residues. To understand the roles of individual Ca2+ ligands and protein-bound Ca2+ ions in the membrane binding and activation of PKC-alpha, we mutated five putative Ca2+ ligands (D187N, D193N, D246N, D248N, and D254N) and measured the effects of mutations on vesicle binding, enzyme activity, and monolayer penetration of PKC-alpha. Altered properties of these mutants indicate that individual Ca2+ ions and their ligands have different roles in the membrane binding and activation of PKC-alpha. The binding of Ca2+ to Asp187, Asp193, and Asp246 of PKC-alpha is important for the initial binding of protein to membrane surfaces. On the other hand, the binding of another Ca2+ to Asp187, Asp246, Asp248, and Asp254 induces the conformational change of PKC-alpha, which in turn triggers its membrane penetration and activation. Among these Ca2+ ligands, Asp246 was shown to be most essential for both membrane binding and activation of PKC-alpha, presumably due to its coordination to multiple Ca2+ ions. Furthermore, to identify the residues in the C2 domain that are involved in membrane binding of PKC-alpha, we mutated four putative membrane binding residues (Trp245, Trp247, Arg249, and Arg252). Membrane binding and enzymatic properties of two double-site mutants (W245A/W247A and R249A/R252A) indicate that Arg249 and Arg252 are involved in electrostatic interactions of PKC-alpha with anionic membranes, whereas Trp245 and Trp247 participate in its penetration into membranes and resulting hydrophobic interactions. Taken together, these studies provide the first experimental evidence for the role of C2 domain of conventional PKC as a membrane docking unit as well as a module that triggers conformational changes to activate the protein.

Computational Approach using Mouse Embryonic Stem Cells to Define a Mechanistic Applicability Domain for Prenatal Developmental Toxicity

EPA Science Inventory

Identification of mechanisms responsible for adverse developmental effects is the first step in creating predictive toxicity models. Identification of putative mechanisms was performed by co-analyzing three datasets for the effects of ToxCast phase Ia and II chemicals: 1.In vitro...

Biotechnology Conference: Protein Engineering Held in Oxford, United Kingdom on 5-8 April 1987.

DTIC Science & Technology

1987-07-27

engineered by protein engineering was reported by J. new variants which are now being checked. Brange (Novo Research Institute, Bags- Studies of a cassette...to Brange . Therefore, multidomain protein consisting of five Brange and his group applied protein en- putative domains: the fribonectin finger

Fine mapping and identification of a candidate gene for the barley Un8 true loose smut resistance gene.

PubMed

Zang, Wen; Eckstein, Peter E; Colin, Mark; Voth, Doug; Himmelbach, Axel; Beier, Sebastian; Stein, Nils; Scoles, Graham J; Beattie, Aaron D

2015-07-01

The candidate gene for the barley Un8 true loose smut resistance gene encodes a deduced protein containing two tandem protein kinase domains. In North America, durable resistance against all known isolates of barley true loose smut, caused by the basidiomycete pathogen Ustilago nuda (Jens.) Rostr. (U. nuda), is under the control of the Un8 resistance gene. Previous genetic studies mapped Un8 to the long arm of chromosome 5 (1HL). Here, a population of 4625 lines segregating for Un8 was used to delimit the Un8 gene to a 0.108 cM interval on chromosome arm 1HL, and assign it to fingerprinted contig 546 of the barley physical map. The minimal tilling path was identified for the Un8 locus using two flanking markers and consisted of two overlapping bacterial artificial chromosomes. One gene located close to a marker co-segregating with Un8 showed high sequence identity to a disease resistance gene containing two kinase domains. Sequence of the candidate gene from the parents of the segregating population, and in an additional 19 barley lines representing a broader spectrum of diversity, showed there was no intron in alleles present in either resistant or susceptible lines, and fifteen amino acid variations unique to the deduced protein sequence in resistant lines differentiated it from the deduced protein sequences in susceptible lines. Some of these variations were present within putative functional domains which may cause a loss of function in the deduced protein sequences within susceptible lines.

Phylogenetic analysis and expression profiling of the pattern recognition receptors: Insights into molecular recognition of invading pathogens in Manduca sexta.

PubMed

Zhang, Xiufeng; He, Yan; Cao, Xiaolong; Gunaratna, Ramesh T; Chen, Yun-ru; Blissard, Gary; Kanost, Michael R; Jiang, Haobo

2015-07-01

Pattern recognition receptors (PRRs) detect microbial pathogens and trigger innate immune responses. Previous biochemical studies have elucidated the physiological functions of eleven PRRs in Manduca sexta but our understanding of the recognition process is still limited, lacking genomic perspectives. While 34 C-type lectin-domain proteins and 16 Toll-like receptors are reported in the companion papers, we present here 120 other putative PRRs identified through the genome annotation. These include 76 leucine-rich repeat (LRR) proteins, 14 peptidoglycan recognition proteins, 6 EGF/Nim-domain proteins, 5 β-1,3-glucanase-related proteins, 4 galectins, 4 fibrinogen-related proteins, 3 thioester proteins, 5 immunoglobulin-domain proteins, 2 hemocytins, and 1 Reeler. Sequence alignment and phylogenetic analysis reveal the evolution history of a diverse repertoire of proteins for pathogen recognition. While functions of insect LRR proteins are mostly unknown, their structure diversification is phenomenal: In addition to the Toll homologs, 22 LRR proteins with a signal peptide are expected to be secreted; 18 LRR proteins lacking signal peptides may be cytoplasmic; 36 LRRs with a signal peptide and a transmembrane segment may be non-Toll receptors on the surface of cells. Expression profiles of the 120 genes in 52 tissue samples reflect complex regulation in various developmental stages and physiological states, including some likely by Rel family transcription factors via κB motifs in the promoter regions. This collection of information is expected to facilitate future biochemical studies detailing their respective roles in this model insect. Copyright © 2015 Elsevier Ltd. All rights reserved.

Phylogenetic analysis and expression profiling of the pattern recognition receptors: insights into molecular recognition of invading pathogens in Manduca sexta

PubMed Central

Zhang, Xiufeng; He, Yan; Cao, Xiaolong; Gunaratna, Ramesh T.; Chen, Yun-ru; Blissard, Gary; Kanost, Michael R.; Jiang, Haobo

2015-01-01

Pattern recognition receptors (PRRs) detect microbial pathogens and trigger innate immune responses. Previous biochemical studies have elucidated the physiological functions of eleven PRRs in Manduca sexta but our understanding of the recognition process is still limited, lacking genomic perspectives. While 34 C-type lectin-domain proteins and 16 Toll-like receptors are reported in the companion papers, we present here 120 other putative PRRs identified through the genome annotation. These include 76 leucine-rich repeat (LRR) proteins, 14 peptidoglycan recognition proteins, 6 EGF/Nim-domain proteins, 5 β-1,3-glucanase-related proteins, 4 galectins, 4 fibrinogen-related proteins, 3 thioester proteins, 5 immunoglobulin-domain proteins, 2 hemocytins, and 1 Reeler. Sequence alignment and phylogenetic analysis reveal the evolution history of a diverse repertoire of proteins for pathogen recognition. While functions of insect LRR proteins are mostly unknown, their structure diversification is phenomenal: In addition to the Toll homologs, 22 LRR proteins with a signal peptide are expected to be secreted; 18 LRR proteins lacking signal peptides may be cytoplasmic; 36 LRRs with a signal peptide and a transmembrane segment may be non-Toll receptors on the surface of cells. Expression profiles of the 120 genes in 52 tissue samples reflect complex regulation in various developmental stages and physiological states, including some likely by Rel family transcription factors via κB motifs in the promoter regions. This collection of information is expected to facilitate future biochemical studies detailing their respective roles in this model insect. PMID:25701384

Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins

PubMed Central

Larsen, Ida Signe Bohse; Narimatsu, Yoshiki; Siukstaite, Lina; Harrison, Oliver J.; Brasch, Julia; Goodman, Kerry M.; Hansen, Lars; Shapiro, Lawrence; Honig, Barry; Vakhrushev, Sergey Y.; Clausen, Henrik

2017-01-01

The cadherin (cdh) superfamily of adhesion molecules carry O-linked mannose (O-Man) glycans at highly conserved sites localized to specific β-strands of their extracellular cdh (EC) domains. These O-Man glycans do not appear to be elongated like O-Man glycans found on α-dystroglycan (α-DG), and we recently demonstrated that initiation of cdh/protocadherin (pcdh) O-Man glycosylation is not dependent on the evolutionary conserved POMT1/POMT2 enzymes that initiate O-Man glycosylation on α-DG. Here, we used a CRISPR/Cas9 genetic dissection strategy combined with sensitive and quantitative O-Man glycoproteomics to identify a homologous family of four putative protein O-mannosyltransferases encoded by the TMTC1–4 genes, which were found to be imperative for cdh and pcdh O-Man glycosylation. KO of all four TMTC genes in HEK293 cells resulted in specific loss of cdh and pcdh O-Man glycosylation, whereas combined KO of TMTC1 and TMTC3 resulted in selective loss of O-Man glycans on specific β-strands of EC domains, suggesting that each isoenzyme serves a different function. In addition, O-Man glycosylation of IPT/TIG domains of plexins and hepatocyte growth factor receptor was not affected in TMTC KO cells, suggesting the existence of yet another O-Man glycosylation machinery. Our study demonstrates that regulation of O-mannosylation in higher eukaryotes is more complex than envisioned, and the discovery of the functions of TMTCs provide insight into cobblestone lissencephaly caused by deficiency in TMTC3. PMID:28973932

Hal Is a Bacillus anthracis Heme Acquisition Protein

PubMed Central

Balderas, Miriam A.; Nobles, Christopher L.; Honsa, Erin S.; Alicki, Embriette R.

2012-01-01

The metal iron is a limiting nutrient for bacteria during infection. Bacillus anthracis, the causative agent of anthrax and a potential weapon of bioterrorism, grows rapidly in mammalian hosts, which suggests that it efficiently attains iron during infection. Recent studies have uncovered both heme (isd) and siderophore-mediated (asb) iron transport pathways in this pathogen. Whereas deletion of the asb genes results in reduced virulence, the loss of three surface components from isd had no effect, thereby leaving open the question of what additional factors in B. anthracis are responsible for iron uptake from the most abundant iron source for mammals, heme. Here, we describe the first functional characterization of bas0520, a gene recently implicated in anthrax disease progression. bas0520 encodes a single near-iron transporter (NEAT) domain and several leucine-rich repeats. The NEAT domain binds heme, despite lacking a stabilizing tyrosine common to the NEAT superfamily of hemoproteins. The NEAT domain also binds hemoglobin and can acquire heme from hemoglobin in solution. Finally, deletion of bas0520 resulted in bacilli unable to grow efficiently on heme or hemoglobin as an iron source and yielded the most significant phenotype relative to that for other putative heme uptake systems, a result that suggests that this protein plays a prominent role in the replication of B. anthracis in hematogenous environments. Thus, we have assigned the name of Hal (heme-acquisition leucine-rich repeat protein) to BAS0520. These studies advance our understanding of heme acquisition by this dangerous pathogen and justify efforts to determine the mechanistic function of this novel protein for vaccine or inhibitor development. PMID:22865843

Quantifying domain-ligand affinities and specificities by high-throughput holdup assay

PubMed Central

Vincentelli, Renaud; Luck, Katja; Poirson, Juline; Polanowska, Jolanta; Abdat, Julie; Blémont, Marilyne; Turchetto, Jeremy; Iv, François; Ricquier, Kevin; Straub, Marie-Laure; Forster, Anne; Cassonnet, Patricia; Borg, Jean-Paul; Jacob, Yves; Masson, Murielle; Nominé, Yves; Reboul, Jérôme; Wolff, Nicolas; Charbonnier, Sebastian; Travé, Gilles

2015-01-01

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this aim, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to a thousand domain-motif equilibrium binding affinities per day. Extracts of overexpressed domains are incubated with peptide-coated resins and subjected to filtration. Binding affinities are deduced from microfluidic capillary electrophoresis of flow-throughs. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from Human Papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human PDZome. We obtained exquisite sequence-dependent binding profiles, describing quantitatively the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has a wide potential for quantifying the specificities of interactomes. PMID:26053890

Dynamic Strength of Titin's Z-Disk End

PubMed Central

Kollár, Veronika; Szatmári, Dávid; Grama, László; Kellermayer, Miklós S. Z.

2010-01-01

Titin is a giant filamentous protein traversing the half sarcomere of striated muscle with putative functions as diverse as providing structural template, generating elastic response, and sensing and relaying mechanical information. The Z-disk region of titin, which corresponds to the N-terminal end of the molecule, has been thought to be a hot spot for mechanosensing while also serving as anchorage for its sarcomeric attachment. Understanding the mechanics of titin's Z-disk region, particularly under the effect of binding proteins, is of great interest. Here we briefly review recent findings on the structure, molecular associations, and mechanics of titin's Z-disk region. In addition, we report experimental results on the dynamic strength of titin's Z1Z2 domains measured by nanomechanical manipulation of the chemical dimer of a recombinant protein fragment. PMID:20414364

Dynamic strength of titin's Z-disk end.

PubMed

Kollár, Veronika; Szatmári, Dávid; Grama, László; Kellermayer, Miklós S Z

2010-01-01

Titin is a giant filamentous protein traversing the half sarcomere of striated muscle with putative functions as diverse as providing structural template, generating elastic response, and sensing and relaying mechanical information. The Z-disk region of titin, which corresponds to the N-terminal end of the molecule, has been thought to be a hot spot for mechanosensing while also serving as anchorage for its sarcomeric attachment. Understanding the mechanics of titin's Z-disk region, particularly under the effect of binding proteins, is of great interest. Here we briefly review recent findings on the structure, molecular associations, and mechanics of titin's Z-disk region. In addition, we report experimental results on the dynamic strength of titin's Z1Z2 domains measured by nanomechanical manipulation of the chemical dimer of a recombinant protein fragment.

The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines.

PubMed

Kristensen, Tatjana P; Maria Cherian, Reeja; Gray, Fiona C; MacNeill, Stuart A

2014-01-01

The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies.

Structural and functional characterization of the LldR from Corynebacterium glutamicum: a transcriptional repressor involved in l-lactate and sugar utilization

PubMed Central

Gao, Yong-Gui; Suzuki, Hiroaki; Itou, Hiroshi; Zhou, Yong; Tanaka, Yoshikazu; Wachi, Masaaki; Watanabe, Nobuhisa; Tanaka, Isao; Yao, Min

2008-01-01

LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism. In the present study, the crystal structure of LldR was determined at 2.05-Å resolution. The structure consists of N- and C-domains similar to those of FadR, but with distinct domain orientations. LldR and FadR dimers achieve similar structures by domain swapping, which was first observed in dimeric assembly of transcription factors. A structural feature of Zn2+ binding in the regulatory domain was also observed, as a difference from the FadR subfamily. DNA microarray and DNase I footprint analyses suggested that LldR acts as a repressor regulating cgl2917-lldD and cgl1934-fruK-ptsF operons, which are indispensable for l-lactate and fructose/sucrose utilization, respectively. Furthermore, the stoichiometries and affinities of LldR and DNAs were determined by isothermal titration calorimetry measurements. The transcriptional start site and repression of LldR on the cgl2917-lldD operon were analysed by primer extension assay. Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding. The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed. PMID:18988622

Killing machines: three pore-forming proteins of the immune system

PubMed Central

McCormack, Ryan; de Armas, Lesley; Shiratsuchi, Motoaki

2014-01-01

The evolution of early multicellular eukaryotes 400–500 million years ago required a defensive strategy against microbial invasion. Pore-forming proteins containing the membrane-attack-complex-perforin (MACPF) domain were selected as the most efficient means to destroy bacteria or virally infected cells. The mechanism of pore formation by the MACPF domain is distinctive in that pore formation is purely physical and unspecific. The MACPF domain polymerizes, refolds, and inserts itself into bilayer membranes or bacterial outer cell walls. The displacement of surface lipid/carbohydrate molecules by the polymerizing MACPF domain creates clusters of large, water-filled holes that destabilize the barrier function and provide access for additional anti-bacterial or anti-viral effectors to sensitive sites that complete the destruction of the invader via enzymatic or chemical attack. The highly efficient mechanism of anti-microbial defense by a combined physical and chemical strategy using pore-forming MACPF-proteins has been retargeted during evolution of vertebrates and mammals for three purposes: (1) to kill extracellular bacteria C9/polyC9 evolved in conjunction with complement, (2) to kill virus infected and cancer cells perforin-1/polyperforin-1 CTL evolved targeted by NK and CTL, and (3) to kill intracellular bacteria transmembrane perforin-2/putative polyperforin-2 evolved targeted by phagocytic and nonphagocytic cells. Our laboratory has been involved in the discovery and description of each of the three pore-formers that will be reviewed here. PMID:24293008

1-Oleoyl-2-acetylglycerol stimulates 5-lipoxygenase activity via a putative (phospho)lipid binding site within the N-terminal C2-like domain.

PubMed

Hörnig, Christina; Albert, Dana; Fischer, Lutz; Hörnig, Michael; Rådmark, Olof; Steinhilber, Dieter; Werz, Oliver

2005-07-22

5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2+ ions and phospholipids that both act via the N-terminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca2+ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 x g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca2+, OAG caused no stimulation of 5-LO. Also, phospholipids or cellular membranes abolished the effects of OAG. As found previously for Ca2+, OAG renders 5-LO activity resistant against inhibition by glutathione peroxidase activity, and this effect of OAG is reversed by phospholipids. Intriguingly, a 5-LO mutant lacking tryptophan residues (Trp-13, -75, and -102) important for the binding of the 5-LO C2-like domain to phospholipids was not stimulated by OAG. We conclude that OAG directly stimulates 5-LO by acting at a phospholipid binding site located within the C2-like domain.

Gain-of-function mutations identify amino acids within transmembrane domains of the yeast vacuolar transporter Zrc1 that determine metal specificity

PubMed Central

Lin, Huilan; Burton, Damali; Li, Liangtao; Warner, David E.; Phillips, John D.; Ward, Diane McVEY; Kaplan, Jerry

2015-01-01

Cation diffusion facilitator transporters are found in all three Kingdoms of life and are involved in transporting transition metals out of the cytosol. The metals they transport include Zn2+, Co2+, Fe2+, Cd2+, Ni2+ and Mn2+; however, no single transporter transports all metals. Previously we showed that a single amino acid mutation in the yeast vacuolar zinc transporter Zrc1 changed its substrate specificity from Zn2+ to Fe2+ and Mn2+ [Lin, Kumanovics, Nelson, Warner, Ward and Kaplan (2008) J. Biol. Chem. 283, 33865–33873]. Mutant Zrc1 that gained iron transport activity could protect cells with a deletion in the vacuolar iron transporter (CCC1) from high iron toxicity. Utilizing suppression of high iron toxicity and PCR mutagenesis of ZRC1, we identified other amino acid substitutions within ZRC1 that changed its metal specificity. All Zrc1 mutants that transported Fe2+ could also transport Mn2+. Some Zrc1 mutants lost the ability to transport Zn2+, but others retained the ability to transport Zn2+. All of the amino acid substitutions that resulted in a gain in Fe2+ transport activity were found in transmembrane domains. In addition to alteration of residues adjacent to the putative metal-binding site in two transmembrane domains, alteration of residues distant from the binding site affected substrate specificity. These results suggest that substrate selection involves co-operativity between transmembrane domains. PMID:19538181

Structural Analyses of the Ankyrin Repeat Domain of TRPV6 and Related TRPV Ion Channels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phelps, C.B.; Huang, R.J.; Lishko, P.V.

2008-06-03

Transient receptor potential (TRP) proteins are cation channels composed of a transmembrane domain flanked by large N- and C-terminal cytoplasmic domains. All members of the vanilloid family of TRP channels (TRPV) possess an N-terminal ankyrin repeat domain (ARD). The ARD of mammalian TRPV6, an important regulator of calcium uptake and homeostasis, is essential for channel assembly and regulation. The 1.7 A crystal structure of the TRPV6-ARD reveals conserved structural elements unique to the ARDs of TRPV proteins. First, a large twist between the fourth and fifth repeats is induced by residues conserved in all TRPV ARDs. Second, the third fingermore » loop is the most variable region in sequence, length and conformation. In TRPV6, a number of putative regulatory phosphorylation sites map to the base of this third finger. Size exclusion chromatography and crystal packing indicate that the TRPV6-ARD does not assemble as a tetramer and is monomeric in solution. Adenosine triphosphate-agarose and calmodulin-agarose pull-down assays show that the TRPV6-ARD does not interact with either ligand, indicating a different functional role for the TRPV6-ARD than in the paralogous thermosensitive TRPV1 channel. Similar biochemical findings are also presented for the highly homologous mammalian TRPV5-ARD. The implications of the structural and biochemical data on the role of the ankyrin repeats in different TRPV channels are discussed.« less

Sphingosine kinase 2 activates autophagy and protects neurons against ischemic injury through interaction with Bcl-2 via its putative BH3 domain.

PubMed

Song, Dan-Dan; Zhang, Tong-Tong; Chen, Jia-Li; Xia, Yun-Fei; Qin, Zheng-Hong; Waeber, Christian; Sheng, Rui

2017-07-06

Our previous findings suggest that sphingosine kinase 2 (SPK2) mediates ischemic tolerance and autophagy in cerebral preconditioning. The aim of this study was to determine by which mechanism SPK2 activates autophagy in neural cells. In both primary murine cortical neurons and HT22 hippocampal neuronal cells, overexpression of SPK2 increased LC3II and enhanced the autophagy flux. SPK2 overexpression protected cortical neurons against oxygen glucose deprivation (OGD) injury, as evidenced by improvement of neuronal morphology, increased cell viability and reduced lactate dehydrogenase release. The inhibition of autophagy effectively suppressed the neuroprotective effect of SPK2. SPK2 overexpression reduced the co-immunoprecipitation of Beclin-1 and Bcl-2, while Beclin-1 knockdown inhibited SPK2-induced autophagy. Both co-immunoprecipitation and GST pull-down analysis suggest that SPK2 directly interacts with Bcl-2. SPK2 might interact to Bcl-2 in the cytoplasm. Notably, an SPK2 mutant with L219A substitution in its putative BH3 domain was not able to activate autophagy. A Tat peptide fused to an 18-amino acid peptide encompassing the native, but not the L219A mutated BH3 domain of SPK2 activated autophagy in neural cells. The Tat-SPK2 peptide also protected neurons against OGD injury through autophagy activation. These results suggest that SPK2 interacts with Bcl-2 via its BH3 domain, thereby dissociating it from Beclin-1 and activating autophagy. The observation that Tat-SPK2 peptide designed from the BH3 domain of SPK2 activates autophagy and protects neural cells against OGD injury suggest that this structure may provide the basis for a novel class of therapeutic agents against ischemic stroke.

Cloning, characterization, and heat stress-induced redistribution of a protein homologous to human hsp27 in the zebrafish Danio rerio

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mao Li; Bryantsev, Anton L.; Chechenova, Maria B.

Hsp27 is a small heat shock protein (shsp) regulating stress tolerance and increasingly thought to play roles in tissue homeostasis and differentiation. The zebrafish Danio rerio is an important model for the study of developmental processes, but little is known regarding shsps in this animal. Here, we report the sequence, expression, regulation, and function of a zebrafish protein (zfHsp27) homologous to human Hsp27. zfHsp27 contains three conserved phosphorylatable serines and a cysteine important for regulation of apoptosis, but it lacks much of a C-terminal tail domain and shows low homology in two putative actin interacting domains that are features ofmore » mammalian Hsp27. zfHsp27 mRNA is most abundant in adult skeletal muscle and heart and is upregulated during early embryogenesis. zfHsp27 expressed in mammalian fibroblasts was phosphorylated in response to heat stress and anisomycin, and this phosphorylation was prevented by treatment with SB202190, an inhibitor of p38 MAPK. Expression of zfHsp27 and human Hsp27 in mammalian fibroblasts promoted a similar degree of tolerance to heat stress. zfHsp27 fusion proteins entered the nucleus and associated with the cytoskeleton of heat stressed cells in vitro and in zebrafish embryos. These results reveal conservation in regulation and function of mammalian and teleost Hsp27 proteins and define zebrafish as a new model for the study of Hsp27 function.« less

Pht2;1 encodes a low-affinity phosphate transporter from Arabidopsis.

PubMed Central

Daram, P; Brunner, S; Rausch, C; Steiner, C; Amrhein, N; Bucher, M

1999-01-01

An Arabidopsis genomic sequence was recently shown to share similarity with bacterial and eukaryotic phosphate (Pi) transporters. We have cloned the corresponding cDNA, which we named Pht2;1, and subsequently performed gene expression studies and functional analysis of the protein product. The cDNA encodes a 61-kD protein with a putative topology of 12 transmembrane (TM) domains interrupted by a large hydrophilic loop between TM8 and TM9. Two boxes of eight and nine amino acids, located in the N- and C-terminal domains, respectively, are highly conserved among species across all kingdoms (eubacteria, archea, fungi, plants, and animals). The Pht2;1 gene is predominantly expressed in green tissue, the amount of transcript staying constant in leaves irrespective of the Pi status of the shoot; in roots, however, there is a marginal increase in mRNA amounts in response to Pi deprivation. Although the protein is highly similar to eukaryotic sodium-dependent Pi transporters, functional analysis of the Pht2;1 protein in mutant yeast cells indicates that it is a proton/Pi symporter dependent on the electrochemical gradient across the plasma membrane. Its fairly high apparent K(m) for Pi (0.4 mM) and high mRNA content in the shoot, especially in leaves, suggest a role for shoot organs in Pi loading. Pht2;1 thus differs from members of the recently described plant Pi transporter family in primary structure, affinity for Pi, and presumed function. PMID:10559441

Distinct spatiotemporal expression of ISM1 during mouse and chick development

PubMed Central

Osório, Liliana; Wu, Xuewei; Zhou, Zhongjun

2014-01-01

Isthmin 1 (ISM1) constitutes the founder of a new family of secreted proteins characterized by the presence of 2 functional domains: thrombospondin type 1 repeat (TSR1) and adhesion-associated domain in MUC4 and other proteins (AMOP). ISM1 was identified in the frog embryo as a member of the FGF8 synexpression group due to its expression in the brain midbrain–hindbrain boundary (MHB) or isthmus. In zebrafish, ISM1 was described as a WNT- and NODAL-regulated gene. The function of ISM1 remains largely elusive. So far, ISM1 has been described as an angiogenesis inhibitor that has a dual function in endothelial cell survival and cell death. For a better understanding of ISM1 function, we examined its spatiotemporal distribution in mouse and chick using RT-PCR, ISH, and IHC analyses. In the mouse, ISM1 transcripts are found in tissues such as the anterior mesendoderm, paraxial and lateral plate mesoderm, MHB and trunk neural tube, as well as in the somites and dermomyotome. In the newborn and adult, ISM1 is prominently expressed in the lung and brain. In addition to its putative role during embryonic and postnatal development, ISM1 may also be important for organ homeostasis in the adult. In the chick embryo, ISM1 transcripts are strongly detected in the ear, eye, and spinal cord primordia. Remarkable differences in ISM1 spatiotemporal expression were found during mouse and chick development, despite the high homology of ISM1 orthologs in these species. PMID:24675886

Putative and unique gene sequence utilization for the design of species specific probes as modeled by Lactobacillus plantarum

USDA-ARS?s Scientific Manuscript database

The concept of utilizing putative and unique gene sequences for the design of species specific probes was tested. The abundance profile of assigned functions within the Lactobacillus plantarum genome was used for the identification of the putative and unique gene sequence, csh. The targeted gene (cs...

Characterization of Phytochrome Interacting Factors from the Moss Physcomitrella patens Illustrates Conservation of Phytochrome Signaling Modules in Land Plants

PubMed Central

Xu, Tengfei; Paik, Inyup; Hanke, Sebastian; Keim, Sarah; Hermann, Helen-Maria; Wolf, Luise; Becker, Claude

2017-01-01

Across the plant kingdom, phytochrome (PHY) photoreceptors play an important role during adaptive and developmental responses to light. In Arabidopsis thaliana, light-activated PHYs accumulate in the nucleus, where they regulate downstream signaling components, such as phytochrome interacting factors (PIFs). PIFs are transcription factors that act as repressors of photomorphogenesis; their inhibition by PHYs leads to substantial changes in gene expression. The nuclear function of PHYs, however, has so far been investigated in only a few non-seed plants. Here, we identified putative target genes of PHY signaling in the moss Physcomitrella patens and found light-regulated genes that are putative orthologs of PIF-controlled genes in Arabidopsis. Phylogenetic analyses revealed that an ancestral PIF-like gene was already present in streptophyte algae, i.e., before the water-to-land transition of plants. The PIF homologs in the genome of P. patens resemble Arabidopsis PIFs in their protein domain structure, molecular properties, and physiological effects, albeit with notable differences in the motif-dependent PHY interaction. Our results suggest that P. patens PIFs are involved in PHY signaling. The PHY-PIF signaling node that relays light signals to target genes has been largely conserved during land plant evolution, with evidence of lineage-specific diversification. PMID:28123107

Identification of mutations in regions corresponding to the two putative nucleotide (ATP)-binding folds of the cystic fibrosis gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kerem, B.; Zielenski, J.; Markiewicz, D.

1990-11-01

Additional mutations in the cystic fibrosis (CF) gene were identified in the regions corresponding to the two putative nucleotide (ATP)-binding folds (NBFs) of the predicted polypeptide. The patient cohort included 46 Canadian CF families with well-characterized DNA marker haplotypes spanning the disease locus and several other families from Israel. Eleven mutations were found in the first NBF, 2 were found in the second NBF, but none was found in the R-domain. Seven of the mutations were of the missense type affecting some of the highly conserved amino acid residues in the first NBF; 3 were nonsense mutations; 2 would probablymore » affect mRNA splicing; 2 corresponded to small deletions, including another 3-base-pair deletion different from the major mutation ({delta}F508), which could account for 70% of the CF chromosomes in the population. Nine of these mutations accounted for 12 of the 31 non-{delta}F508 CF chromosomes in the Canadian families. The highly heterogeneous nature of the remaining CF mutations provides important insights into the structure and function of the protein, but it also suggests that DNA-based genetic screening for CF carrier status will not be straightforward.« less

Annotation of Differentially Expressed Genes in the Somatic Embryogenesis of Musa and Their Location in the Banana Genome

PubMed Central

Maldonado-Borges, Josefina Ines; Ku-Cauich, José Roberto; Escobedo-GraciaMedrano, Rosa Maria

2013-01-01

Analysis of cDNA-AFLP was used to study the genes expressed in zygotic and somatic embryogenesis of Musa acuminata Colla ssp. malaccensis, and a comparison was made between their differential transcribed fragments (TDFs) and the sequenced genome of the double haploid- (DH-) Pahang of the malaccensis subspecies that is available in the network. A total of 253 transcript-derived fragments (TDFs) were detected with apparent size of 100–4000 bp using 5 pairs of AFLP primers, of which 21 were differentially expressed during the different stages of banana embryogenesis; 15 of the sequences have matched DH-Pahang chromosomes, with 7 of them being homologous to gene sequences encoding either known or putative protein domains of higher plants. Four TDF sequences were located in all Musa chromosomes, while the rest were located in one or two chromosomes. Their putative individual function is briefly reviewed based on published information, and the potential roles of these genes in embryo development are discussed. Thus the availability of the genome of Musa and the information of TDFs sequences presented here opens new possibilities for an in-depth study of the molecular and biochemical research of zygotic and somatic embryogenesis of Musa. PMID:24027442

Cloning of cellobiose phosphoenolpyruvate-dependent phosphotransferase genes: Functional expression in recombinant Escherichia coli and identification of a putative binding region for disaccharides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lai, Xiaokuang; Davis, F.C.; Ingram, L.O.

1997-02-01

Genomic libraries from nine cellobiose-metabolizing bacteria were screened for cellobiose utilization. Positive clones were recovered from six libraries, all of which encode phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) proteins. Clones from Bacillus subtilis, Butyrivibrio fibrisolvens, and Klebsiella oxytoca allowed the growth of recombinant Escherichia coli in cellobiose-M9 minimal medium. The K. oxytoca clone, pLOI1906, exhibited an unusually broad substrate range (cellobiose, arbutin, salicin, and methylumbelliferyl derivatives of glucose, cellobiose, mannose, and xylose) and was sequenced. The insert in this plasmid encoded the carboxy-terminal region of a putative regulatory protein, cellobiose permease (single polypeptide), and phospho-{beta}-glucosidase, which appear to form an operon (casRAB).more » Subclones allowed both casA and casB to be expressed independently, as evidenced by in vitro complementation. An analysis of the translated sequences from the EIIC domains of cellobiose, aryl-{beta}-glucoside, and other disaccharide permeases allowed the identification of a 50-amino-acid conserved region. A disaccharide consensus sequence is proposed for the most conserved segment (13 amino acids), which may represent part of the EIIC active site for binding and phosphorylation. 63 refs., 4 figs., 4 tabs.« less

A single type of cadherin is involved in Bacillus thuringiensis toxicity in Plutella xylostella.

PubMed

Park, Y; Herrero, S; Kim, Y

2015-12-01

Cadherins have been described as one the main functional receptors for the toxins of the entomopathogenic bacterium, Bacillus thuringiensis (Bt). With the availability of the whole genome of Plutella xylostella, different types of cadherins have been annotated. In this study we focused on determining those members of the cadherin-related proteins that potentially play a role in the mode of action of Bt toxins. For this, we mined the genome of P. xylostella to identify these putative cadherins. The genome screening revealed 52 genes that were annotated as cadherin or cadherin-like genes. Further analysis revealed that six of these putative cadherins had three motifs common to all Bt-related cadherins: a signal peptide, cadherin repeats and a transmembrane domain. From the six selected cadherins, only P. xylostella cadherin 1 (PxCad1) was expressed in the larval midgut and only the silencing of this gene by RNA interference (double-stranded RNA feeding) reduce toxicity and binding to the midgut of the Cry1Ac type toxin from Bt. These results indicate that from the whole set of cadherin-related genes identified in P. xylostella, only PxCad1 is associated with the Cry1Ac mode of action. © 2015 The Royal Entomological Society.

Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity.

PubMed

Pauly, Thorsten; Ratliff, Miriam; Pietrowski, Eweline; Neugebauer, Rainer; Schlicksupp, Andrea; Kirsch, Joachim; Kuhse, Jochen

2008-07-16

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

Activity-Dependent Shedding of the NMDA Receptor Glycine Binding Site by Matrix Metalloproteinase 3: A PUTATIVE Mechanism of Postsynaptic Plasticity

PubMed Central

Pietrowski, Eweline; Neugebauer, Rainer; Schlicksupp, Andrea; Kirsch, Joachim; Kuhse, Jochen

2008-01-01

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS. PMID:18629001

Protective and compensatory factors mitigating the influence of deviant friends on delinquent behaviours during early adolescence.

PubMed

Fergusson, David M; Vitaro, Frank; Wanner, Brigitte; Brendgen, Mara

2007-02-01

This study examined factors that could moderate or compensate the link between exposure to deviant friends and delinquent behaviours in a sample of 265 early adolescents. The putative moderating or compensatory factors referred to the behavioural domain (i.e. novelty seeking, harm avoidance), the biological domain (i.e. physical maturation), the sociofamily domain (i.e. sociofamily adversity, parental practices), the school domain (i.e. academic performance), and the social domain (i.e. peer acceptance). A series of regression analyses showed that novelty seeking and puberty status moderated the link between friends' self-reported delinquency and participants' self-reported delinquency. In addition, all the factors except peer acceptance also had main effects that, cumulatively, reduced the association between friends' delinquency and self-rated delinquency through compensatory main effects. These results are discussed in light of the differential roles of moderating and of compensatory factors.

Transition from wind pollination to insect pollination in sedges: experimental evidence and functional traits.

PubMed

Wragg, Peter D; Johnson, Steven D

2011-09-01

Transitions from wind pollination to insect pollination were pivotal to the radiation of land plants, yet only a handful are known and the trait shifts required are poorly understood. We tested the hypothesis that a transition to insect pollination took place in the ancestrally wind-pollinated sedges (Cyperaceae) and that floral traits modified during this transition have functional significance. We paired putatively insect-pollinated Cyperus obtusiflorus and Cyperus sphaerocephalus with related, co-flowering, co-occurring wind-pollinated species, and compared pairs in terms of pollination mode and functional roles of floral traits. Experimentally excluding insects reduced seed set by 56-89% in putatively insect-pollinated species but not in intermingled wind-pollinated species. The pollen of putatively insect-pollinated species was less motile in a wind tunnel than that of wind-pollinated species. Bees, beetles and flies preferred inflorescences, and color-matched white or yellow models, of putatively insect-pollinated species over inflorescences, or color-matched brown models, of wind-pollinated species. Floral scents of putatively insect-pollinated species were chemically consistent with those of other insect-pollinated plants, and attracted pollinators; wind-pollinated species were unscented. These results show that a transition from wind pollination to insect pollination occurred in sedges and shed new light on the function of traits involved in this important transition. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

The mitochondrial genome of Pocillopora (Cnidaria: Scleractinia) contains two variable regions: the putative D-loop and a novel ORF of unknown function.

PubMed

Flot, Jean-François; Tillier, Simon

2007-10-15

The complete mitochondrial genomes of two individuals attributed to different morphospecies of the scleractinian coral genus Pocillopora have been sequenced. Both genomes, respectively 17,415 and 17,422 nt long, share the presence of a previously undescribed ORF encoding a putative protein made up of 302 amino acids and of unknown function. Surprisingly, this ORF turns out to be the second most variable region of the mitochondrial genome (1% nucleotide sequence difference between the two individuals) after the putative control region (1.5% sequence difference). Except for the presence of this ORF and for the location of the putative control region, the mitochondrial genome of Pocillopora is organized in a fashion similar to the other scleractinian coral genomes published to date. For the first time in a cnidarian, a putative second origin of replication is described based on its secondary structure similar to the stem-loop structure of O(L), the origin of L-strand replication in vertebrates.

LDlink: a web-based application for exploring population-specific haplotype structure and linking correlated alleles of possible functional variants.

PubMed

Machiela, Mitchell J; Chanock, Stephen J

2015-11-01

Assessing linkage disequilibrium (LD) across ancestral populations is a powerful approach for investigating population-specific genetic structure as well as functionally mapping regions of disease susceptibility. Here, we present LDlink, a web-based collection of bioinformatic modules that query single nucleotide polymorphisms (SNPs) in population groups of interest to generate haplotype tables and interactive plots. Modules are designed with an emphasis on ease of use, query flexibility, and interactive visualization of results. Phase 3 haplotype data from the 1000 Genomes Project are referenced for calculating pairwise metrics of LD, searching for proxies in high LD, and enumerating all observed haplotypes. LDlink is tailored for investigators interested in mapping common and uncommon disease susceptibility loci by focusing on output linking correlated alleles and highlighting putative functional variants. LDlink is a free and publically available web tool which can be accessed at http://analysistools.nci.nih.gov/LDlink/. mitchell.machiela@nih.gov. Published by Oxford University Press 2015. This work is written by US Government employees and is in the public domain in the US.

Isolation and functional characterization of an ammonium transporter gene, PyAMT1, related to nitrogen assimilation in the marine macroalga Pyropia yezoensis (Rhodophyta).

PubMed

Kakinuma, Makoto; Nakamoto, Chika; Kishi, Kazuki; Coury, Daniel A; Amano, Hideomi

2017-07-01

Ammonium and nitrate are the primary nitrogen sources in natural environments, and are essential for growth and development in photosynthetic eukaryotes. In this study, we report on the isolation and characterization of an ammonium transporter gene (PyAMT1) which performs a key function in nitrogen (N) metabolism of Pyropia yezoensis thalli. The predicted length of PyAMT1 was 483 amino acids (AAs). The AA sequence included 11 putative transmembrane domains and showed approximately 33-44% identity to algal and plant AMT1 AA sequences. Functional complementation in an AMT-defective yeast mutant indicated that PyAMT1 mediated ammonium transport across the plasma membrane. Expression analysis showed that the PyAMT1 mRNA level was strongly induced by N-deficiency, and was more highly suppressed by resupply of inorganic-N than organic-N. These results suggest that PyAMT1 plays important roles in the ammonium transport system, and is highly regulated in response to external/internal N-status. Copyright © 2016 Elsevier Ltd. All rights reserved.

Putative bovine topological association domains and CTCF binding motifs can reduce the search space for causative regulatory variants of complex traits.

PubMed

Wang, Min; Hancock, Timothy P; Chamberlain, Amanda J; Vander Jagt, Christy J; Pryce, Jennie E; Cocks, Benjamin G; Goddard, Mike E; Hayes, Benjamin J

2018-05-24

Topological association domains (TADs) are chromosomal domains characterised by frequent internal DNA-DNA interactions. The transcription factor CTCF binds to conserved DNA sequence patterns called CTCF binding motifs to either prohibit or facilitate chromosomal interactions. TADs and CTCF binding motifs control gene expression, but they are not yet well defined in the bovine genome. In this paper, we sought to improve the annotation of bovine TADs and CTCF binding motifs, and assess whether the new annotation can reduce the search space for cis-regulatory variants. We used genomic synteny to map TADs and CTCF binding motifs from humans, mice, dogs and macaques to the bovine genome. We found that our mapped TADs exhibited the same hallmark properties of those sourced from experimental data, such as housekeeping genes, transfer RNA genes, CTCF binding motifs, short interspersed elements, H3K4me3 and H3K27ac. We showed that runs of genes with the same pattern of allele-specific expression (ASE) (either favouring paternal or maternal allele) were often located in the same TAD or between the same conserved CTCF binding motifs. Analyses of variance showed that when averaged across all bovine tissues tested, TADs explained 14% of ASE variation (standard deviation, SD: 0.056), while CTCF explained 27% (SD: 0.078). Furthermore, we showed that the quantitative trait loci (QTLs) associated with gene expression variation (eQTLs) or ASE variation (aseQTLs), which were identified from mRNA transcripts from 141 lactating cows' white blood and milk cells, were highly enriched at putative bovine CTCF binding motifs. The linearly-furthermost, and most-significant aseQTL and eQTL for each genic target were located within the same TAD as the gene more often than expected (Chi-Squared test P-value < 0.001). Our results suggest that genomic synteny can be used to functionally annotate conserved transcriptional components, and provides a tool to reduce the search space for causative regulatory variants in the bovine genome.

Association of an SNP in a novel DREB2-like gene SiDREB2 with stress tolerance in foxtail millet [Setaria italica (L.)].

PubMed

Lata, Charu; Bhutty, Sarita; Bahadur, Ranjit Prasad; Majee, Manoj; Prasad, Manoj

2011-06-01

The DREB genes code for important plant transcription factors involved in the abiotic stress response and signal transduction. Characterization of DREB genes and development of functional markers for effective alleles is important for marker-assisted selection in foxtail millet. Here the characterization of a cDNA (SiDREB2) encoding a putative dehydration-responsive element-binding protein 2 from foxtail millet and the development of an allele-specific marker (ASM) for dehydration tolerance is reported. A cDNA clone (GenBank accession no. GT090998) coding for a putative DREB2 protein was isolated as a differentially expressed gene from a 6 h dehydration stress SSH library. A 5' RACE (rapid amplification of cDNA ends) was carried out to obtain the full-length cDNA, and sequence analysis showed that SiDREB2 encoded a polypeptide of 234 amino acids with a predicted mol. wt of 25.72 kDa and a theoretical pI of 5.14. A theoretical model of the tertiary structure shows that it has a highly conserved GCC-box-binding N-terminal domain, and an acidic C-terminus that acts as an activation domain for transcription. Based on its similarity to AP2 domains, SiDREB2 was classified into the A-2 subgroup of the DREB subfamily. Quantitative real-time PCR analysis showed significant up-regulation of SiDREB2 by dehydration (polyethylene glycol) and salinity (NaCl), while its expression was less affected by other stresses. A synonymous single nucleotide polymorphism (SNP) associated with dehydration tolerance was detected at the 558th base pair (an A/G transition) in the SiDREB2 gene in a core set of 45 foxtail millet accessions used. Based on the identified SNP, three primers were designed to develop an ASM for dehydration tolerance. The ASM produced a 261 bp fragment in all the tolerant accessions and produced no amplification in the sensitive accessions. The use of this ASM might be faster, cheaper, and more reproducible than other SNP genotyping methods, and thus will enable marker-aided breeding of foxtail millet for dehydration tolerance.

crm-1 facilitates BMP signaling to control body size in Caenorhabditis elegans.

PubMed

Fung, Wong Yan; Fat, Ko Frankie Chi; Eng, Cheah Kathryn Song; Lau, Chow King

2007-11-01

We have identified in Caenorhabditis elegans a homologue of the vertebrate Crim1, crm-1, which encodes a putative transmembrane protein with multiple cysteine-rich (CR) domains known to have bone morphogenetic proteins (BMPs) binding activity. Using the body morphology of C. elegans as an indicator, we showed that attenuation of crm-1 activity leads to a small body phenotype reminiscent of that of BMP pathway mutants. We showed that the crm-1 loss-of-function phenotype can be rescued by constitutive supply of sma-4 activity. crm-1 can enhance BMP signaling and this activity is dependent on the presence of the DBL-1 ligand and its receptors. crm-1 is expressed in neurons at the ventral nerve cord, where the DBL-1 ligand is produced. However, ectopic expression experiments reveal that crm-1 gene products act outside the DBL-1 producing cells and function non-autonomously to facilitate dbl/sma pathway signaling to control body size.

Facilitation of Endosomal Recycling by an IRG Protein Homolog Maintains Apical Tubule Structure in Caenorhabditis elegans

PubMed Central

Grussendorf, Kelly A.; Trezza, Christopher J.; Salem, Alexander T.; Al-Hashimi, Hikmat; Mattingly, Brendan C.; Kampmeyer, Drew E.; Khan, Liakot A.; Hall, David H.; Göbel, Verena; Ackley, Brian D.; Buechner, Matthew

2016-01-01

Determination of luminal diameter is critical to the function of small single-celled tubes. A series of EXC proteins, including EXC-1, prevent swelling of the tubular excretory canals in Caenorhabditis elegans. In this study, cloning of exc-1 reveals it to encode a homolog of mammalian IRG proteins, which play roles in immune response and autophagy and are associated with Crohn’s disease. Mutants in exc-1 accumulate early endosomes, lack recycling endosomes, and exhibit abnormal apical cytoskeletal structure in regions of enlarged tubules. EXC-1 interacts genetically with two other EXC proteins that also affect endosomal trafficking. In yeast two-hybrid assays, wild-type and putative constitutively active EXC-1 binds to the LIM-domain protein EXC-9, whose homolog, cysteine-rich intestinal protein, is enriched in mammalian intestine. These results suggest a model for IRG function in forming and maintaining apical tubule structure via regulation of endosomal recycling. PMID:27334269

The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic

DOE Office of Scientific and Technical Information (OSTI.GOV)

Swenson, Joel M.; Colmenares, Serafin U.; Strom, Amy R.

Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors andmore » regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions.« less

The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic

DOE PAGES

Swenson, Joel M.; Colmenares, Serafin U.; Strom, Amy R.; ...

2016-08-11

Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors andmore » regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions.« less

Cloning and functional expression of a plant voltage-dependent chloride channel.

PubMed Central

Lurin, C; Geelen, D; Barbier-Brygoo, H; Guern, J; Maurel, C

1996-01-01

Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants. PMID:8624442

The structure of free L11 and functional dynamics of L11 in free, L11-rRNA(58 nt) binary and L11-rRNA(58 nt)-thiostrepton ternary complexes.

PubMed

Lee, Donghan; Walsh, Joseph D; Yu, Ping; Markus, Michelle A; Choli-Papadopoulou, Theodora; Schwieters, Charles D; Krueger, Susan; Draper, David E; Wang, Yun-Xing

2007-04-06

The L11 binding site is one of the most important functional sites in the ribosome. The N-terminal domain of L11 has been implicated as a "reversible switch" in facilitating the coordinated movements associated with EF-G-driven GTP hydrolysis. The reversible switch mechanism has been hypothesized to require conformational flexibility involving re-orientation and re-positioning of the two L11 domains, and warrants a close examination of the structure and dynamics of L11. Here we report the solution structure of free L11, and relaxation studies of free L11, L11 complexed to its 58 nt RNA recognition site, and L11 in a ternary complex with the RNA and thiostrepton antibiotic. The binding site of thiostrepton on L11 was also defined by analysis of structural and dynamics data and chemical shift mapping. The conclusions of this work are as follows: first, the binding of L11 to RNA leads to sizable conformation changes in the regions flanking the linker and in the hinge area that links a beta-sheet and a 3(10)-helix-turn-helix element in the N terminus. Concurrently, the change in the relative orientation may lead to re-positioning of the N terminus, as implied by a decrease of radius of gyration from 18.5 A to 16.2 A. Second, the regions, which undergo large conformation changes, exhibit motions on milliseconds-microseconds or nanoseconds-picoseconds time scales. Third, binding of thiostrepton results in more rigid conformations near the linker (Thr71) and near its putative binding site (Leu12). Lastly, conformational changes in the putative thiostrepton binding site are implicated by the re-emergence of cross-correlation peaks in the spectrum of the ternary complex, which were missing in that of the binary complex. Our combined analysis of both the chemical shift perturbation and dynamics data clearly indicates that thiostrepton binds to a pocket involving residues in the 3(10)-helix in L11.

The Structure of Free L11 and Functional Dynamics of L11 in Free, L11-rRNA(58nt) Binary and L11-rRNA(58nt)-thiostrepton Ternary Complexes

PubMed Central

Lee, Donghan; Walsh, Joseph D.; Yu, Ping; Markus, Michelle A.; Choli-Papadopoulou, Theodora; Schwieters, Charles D.; Krueger, Susan; Draper, David E.; Wang, Yun-Xing

2007-01-01

Summary The L11 binding site is one of the most important functional sites in the ribosome. The N-terminal domain of L11 has been implicated as a “reversible switch” in facilitating the coordinated movements associated with EF-G–driven GTP hydrolysis. The “reversible switch” mechanism has been hypothesized to require conformational flexibility involving re-orientation and re-positioning of the two L11 domains, and warrants a close examination of the structure and dynamics of L11. Here we report the solution structure of free L11, and relaxation studies of free L11, L11complexed to its 58 nt RNA recognition site, and L11 in a ternary complex with the RNA and thiostrepton antibiotic. The binding site of thiostrepton on L11 was also defined by analysis of structural and dynamics data and chemical shift mapping. The conclusions of this work are as follows: First, the binding of L11 to RNA leads to sizable conformation changes in the regions flanking the linker and in the hinge area that links a β-sheets and a 310-helix-turn-helix element in the N-terminus. Concurrently, the change in the relative orientation may lead to re-positioning of the N-terminus, as implied by a decrease of radius of gyration from 18.5 Å to 16.2 Å. Second, the regions, which undergo large conformation changes, exhibit motions on ms-μs or ns-ps time scales. Third, binding of thiostrepton results in more rigid conformations near the linker (Thr71) and near its putative binding site (Leu12). Lastly, conformational changes in the putative thiostrepton binding site are implicated by the re-emergence of cross-correlation peaks in the spectrum of the ternary complex, which were missing in that of the binary complex. Our combined analysis of both the chemical shift perturbation and dynamics data clearly indicates that thiostrepton binds to a pocket involving residues in the 310-helix in L11. PMID:17292917

Campylobacter fetus subspecies contain conserved type IV secretion systems on multiple genomic islands and plasmids

USDA-ARS?s Scientific Manuscript database

The features contributing to the differences in pathogenicity of the C. fetus subspecies are unknown. Putative factors involved in pathogenesis are located in genomic islands that encode type IV secretion system (T4SS) and fic-domain (filamentation induced by cyclic AMP) proteins. In the genomes of ...

Identification of Cytoplasmic Proteins Interacting with the Mammary Cell Transforming Domain of Ese-1

DTIC Science & Technology

2007-04-01

experiments using antibodies targeting epitope-tagged recombinant forms of these three putative SBCPs and recombinant and endogenous Ese- 1. These...The antagonistic regulation of human MUC4 and ErbB-2 genes by the Ets protein PEA3 in pancreatic cancer cells: implications for the proliferation

Conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of SARS coronavirus envelope protein

PubMed Central

Torres, Jaume; Maheswari, Uma; Parthasarathy, Krupakar; Ng, Lifang; Liu, Ding Xiang; Gong, Xiandi

2007-01-01

The coronavirus responsible for the severe acute respiratory syndrome (SARS-CoV) contains a small envelope protein, E, with putative involvement in host cell apoptosis and virus morphogenesis. It has been suggested that E protein can form a membrane destabilizing transmembrane (TM) hairpin, or homooligomerize to form a regular TM α-helical bundle. We have shown previously that the topology of the α-helical putative TM domain of E protein (ETM), flanked by two lysine residues at C and N termini to improve solubility, is consistent with a regular TM α-helix, with orientational parameters in lipid bilayers that are consistent with a homopentameric model. Herein, we show that this peptide, reconstituted in lipid bilayers, shows sodium conductance. Channel activity is inhibited by the anti-influenza drug amantadine, which was found to bind our preparation with moderate affinity. Results obtained from single or double mutants indicate that the organization of the transmembrane pore is consistent with our previously reported pentameric α-helical bundle model. PMID:17766393

PRGdb: a bioinformatics platform for plant resistance gene analysis

PubMed Central

Sanseverino, Walter; Roma, Guglielmo; De Simone, Marco; Faino, Luigi; Melito, Sara; Stupka, Elia; Frusciante, Luigi; Ercolano, Maria Raffaella

2010-01-01

PRGdb is a web accessible open-source (http://www.prgdb.org) database that represents the first bioinformatic resource providing a comprehensive overview of resistance genes (R-genes) in plants. PRGdb holds more than 16 000 known and putative R-genes belonging to 192 plant species challenged by 115 different pathogens and linked with useful biological information. The complete database includes a set of 73 manually curated reference R-genes, 6308 putative R-genes collected from NCBI and 10463 computationally predicted putative R-genes. Thanks to a user-friendly interface, data can be examined using different query tools. A home-made prediction pipeline called Disease Resistance Analysis and Gene Orthology (DRAGO), based on reference R-gene sequence data, was developed to search for plant resistance genes in public datasets such as Unigene and Genbank. New putative R-gene classes containing unknown domain combinations were discovered and characterized. The development of the PRG platform represents an important starting point to conduct various experimental tasks. The inferred cross-link between genomic and phenotypic information allows access to a large body of information to find answers to several biological questions. The database structure also permits easy integration with other data types and opens up prospects for future implementations. PMID:19906694

Tentacle Transcriptome and Venom Proteome of the Pacific Sea Nettle, Chrysaora fuscescens (Cnidaria: Scyphozoa).

PubMed

Ponce, Dalia; Brinkman, Diane L; Potriquet, Jeremy; Mulvenna, Jason

2016-04-05

Jellyfish venoms are rich sources of toxins designed to capture prey or deter predators, but they can also elicit harmful effects in humans. In this study, an integrated transcriptomic and proteomic approach was used to identify putative toxins and their potential role in the venom of the scyphozoan jellyfish Chrysaora fuscescens. A de novo tentacle transcriptome, containing more than 23,000 contigs, was constructed and used in proteomic analysis of C. fuscescens venom to identify potential toxins. From a total of 163 proteins identified in the venom proteome, 27 were classified as putative toxins and grouped into six protein families: proteinases, venom allergens, C-type lectins, pore-forming toxins, glycoside hydrolases and enzyme inhibitors. Other putative toxins identified in the transcriptome, but not the proteome, included additional proteinases as well as lipases and deoxyribonucleases. Sequence analysis also revealed the presence of ShKT domains in two putative venom proteins from the proteome and an additional 15 from the transcriptome, suggesting potential ion channel blockade or modulatory activities. Comparison of these potential toxins to those from other cnidarians provided insight into their possible roles in C. fuscescens venom and an overview of the diversity of potential toxin families in cnidarian venoms.

Characterization of noncoding regulatory DNA in the human genome.

PubMed

Elkon, Ran; Agami, Reuven

2017-08-08

Genetic variants associated with common diseases are usually located in noncoding parts of the human genome. Delineation of the full repertoire of functional noncoding elements, together with efficient methods for probing their biological roles, is therefore of crucial importance. Over the past decade, DNA accessibility and various epigenetic modifications have been associated with regulatory functions. Mapping these features across the genome has enabled researchers to begin to document the full complement of putative regulatory elements. High-throughput reporter assays to probe the functions of regulatory regions have also been developed but these methods separate putative regulatory elements from the chromosome so that any effects of chromatin context and long-range regulatory interactions are lost. Definitive assignment of function(s) to putative cis-regulatory elements requires perturbation of these elements. Genome-editing technologies are now transforming our ability to perturb regulatory elements across entire genomes. Interpretation of high-throughput genetic screens that incorporate genome editors might enable the construction of an unbiased map of functional noncoding elements in the human genome.

The SH2 domain interaction landscape.

PubMed

Tinti, Michele; Kiemer, Lars; Costa, Stefano; Miller, Martin L; Sacco, Francesca; Olsen, Jesper V; Carducci, Martina; Paoluzi, Serena; Langone, Francesca; Workman, Christopher T; Blom, Nikolaj; Machida, Kazuya; Thompson, Christopher M; Schutkowski, Mike; Brunak, Søren; Mann, Matthias; Mayer, Bruce J; Castagnoli, Luisa; Cesareni, Gianni

2013-04-25

Members of the SH2 domain family modulate signal transduction by binding to short peptides containing phosphorylated tyrosines. Each domain displays a distinct preference for the sequence context of the phosphorylated residue. We have developed a high-density peptide chip technology that allows for probing of the affinity of most SH2 domains for a large fraction of the entire complement of tyrosine phosphopeptides in the human proteome. Using this technique, we have experimentally identified thousands of putative SH2-peptide interactions for more than 70 different SH2 domains. By integrating this rich data set with orthogonal context-specific information, we have assembled an SH2-mediated probabilistic interaction network, which we make available as a community resource in the PepspotDB database. A predicted dynamic interaction between the SH2 domains of the tyrosine phosphatase SHP2 and the phosphorylated tyrosine in the extracellular signal-regulated kinase activation loop was validated by experiments in living cells. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Homologous SV40 RNA trans-splicing

PubMed Central

Eul, Joachim; Patzel, Volker

2013-01-01

Simian Virus 40 (SV40) is a polyomavirus found in both monkeys and humans, which causes cancer in some animal models. In humans, SV40 has been reported to be associated with cancers but causality has not been proven yet. The transforming activity of SV40 is mainly due to its 94-kD large T antigen, which binds to the retinoblastoma (pRb) and p53 tumor suppressor proteins, and thereby perturbs their functions. Here we describe a 100 kD super T antigen harboring a duplication of the pRB binding domain that was associated with unusual high cell transformation activity and that was generated by a novel mechanism involving homologous RNA trans-splicing of SV40 early transcripts in transformed rodent cells. Enhanced trans-splice activity was observed in clones carrying a single point mutation in the large T antigen 5′ donor splice site (ss). This mutation impaired cis-splicing in favor of an alternative trans-splice reaction via a cryptic 5′ss within a second cis-spliced SV40 pre-mRNA molecule and enabled detectable gene expression. Next to the cryptic 5′ss we identified additional trans-splice helper functions, including putative dimerization domains and a splice enhancer sequence. Our findings suggest RNA trans-splicing as a SV40-intrinsic mechanism that supports the diversification of viral RNA and phenotypes. PMID:24178438

The C-terminal coiled-coil of the bacterial voltage-gated sodium channel NaChBac is not essential for tetramer formation, but stabilizes subunit-to-subunit interactions.

PubMed

Mio, Kazuhiro; Mio, Muneyo; Arisaka, Fumio; Sato, Masahiko; Sato, Chikara

2010-09-01

The NaChBac is a prokaryotic homologue of the voltage-gated sodium channel found in the genome of the alkalophilic bacterium Bacillus halodurans C-125. Like a repeating cassette of mammalian sodium channel, the NaChBac possesses hydrophobic domains corresponding to six putative transmembrane segments and a pore loop, and exerts channel function by forming a tetramer although detailed mechanisms of subunit assembly remain unclear. We generated truncated mutants from NaChBac, and investigated their ability to form tetramers in relation to their channel functions. A mutant that deletes almost all of the C-terminal coiled-coil structure lost its voltage-dependent ion permeability, although it was properly translocated to the cell surface. The mutant protein was purified as a tetramer using a reduced concentration of detergent, but the association between the subunits was shown to be much weaker than the wild type. The chemical cross-linking, blue native PAGE, sedimentation velocity experiments, size exclusion chromatography, immunoprecipitation, and electron microscopy all supported its tetrameric assembly. We further purified the C-terminal cytoplasmic domain alone and confirmed its self-oligomerization. These data suggest that the C-terminal coiled-coil structure stabilizes subunit-to-subunit interactions of NaChBac, but is not critical for their tetramer formation. 2010 Elsevier Ltd. All rights reserved.

A STE12 homologue of the homothallic ascomycete Sordaria macrospora interacts with the MADS box protein MCM1 and is required for ascosporogenesis.

PubMed

Nolting, Nicole; Pöggeler, Stefanie

2006-11-01

The MADS box protein MCM1 controls diverse developmental processes and is essential for fruiting body formation in the homothallic ascomycete Sordaria macrospora. MADS box proteins derive their regulatory specificity from a wide range of different protein interactions. We have recently shown that the S. macrospora MCM1 is able to interact with the alpha-domain mating-type protein SMTA-1. To further evaluate the functional roles of MCM1, we used the yeast two-hybrid approach to identify MCM1-interacting proteins. From this screen, we isolated a protein with a putative N-terminal homeodomain and C-terminal C2/H2-Zn2+ finger domains. The protein is a member of the highly conserved fungal STE12 transcription factor family of proteins and was therefore termed STE12. Furthermore, we demonstrate by means of two-hybrid and far western analysis that in addition to MCM1, the S. macrospora STE12 protein is able to interact with the mating-type protein SMTA-1. Unlike the situation in the closely related heterothallic ascomycete Neurospora crassa, deletion (Delta) of the ste12 gene in S. macrospora neither affects vegetative growth nor fruiting body formation. However, ascus and ascospore development are highly impaired by the Deltaste12 mutation. Our data provide another example of the functional divergence within the fungal STE12 transcription factor family.

Functional Domains of the TOL Plasmid Transcription Factor XylS

PubMed Central

Kaldalu, Niilo; Toots, Urve; de Lorenzo, Victor; Ustav, Mart

2000-01-01

The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the Pm promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions. PMID:10648539

Replication domains are self-interacting structural chromatin units of human chromosomes

NASA Astrophysics Data System (ADS)

Arneodo, Alain

2011-03-01

In higher eukaryotes, the absence of specific sequence motifs marking the origins of replication has been a serious hindrance to the understanding of the mechanisms that regulate the initiation and the maintenance of the replication program in different cell types. In silico analysis of nucleotide compositional skew has predicted the existence, in the germline, of replication N-domains bordered by putative replication origins and where the skew decreases rather linearly as the signature of a progressive inversion of the average fork polarity. Here, from the demonstration that the average fork polarity can be directly extracted from the derivative of replication timing profiles, we develop a wavelet-based pattern recognition methodology to delineate replication U-domains where the replication timing profile is shaped as a U and its derivative as a N. Replication U-domains are robustly found in seven cell lines as covering a significant portion (40-50%) of the human genome where the replication timing data actually displays some plasticity between cell lines. The early replication initiation zones at U-domains borders are found to be hypersensitive to DNase I cleavage, to be associated with transcriptional activity and to present a significant enrichment in insular-binding proteins CTCF, the hallmark of an open chromatin structure. A comparative analysis of genome-wide chromatin interaction (HiC) data shows that replication-U domains correspond to self-interacting structural high order chromatin units of megabase characteristic size. Taken together, these findings provide evidence that the epigenetic compartmentalization of the human genome into autonomous replication U-domains comes along with an extensive remodelling of the threedimensional chromosome architecture during development or in specific diseases. The observed cell specific conservation of the replication timing between the human and mouse genomes strongly suggests that this chromosome organization into self-interacting structural and functional units is a general feature of mammalian organisms.

CalA, a Cyanobacterial AbrB Protein, Interacts with the Upstream Region of hypC and Acts as a Repressor of Its Transcription in the Cyanobacterium Nostoc sp. Strain PCC 7120▿ †

PubMed Central

Agervald, Åsa; Zhang, Xiaohui; Stensjö, Karin; Devine, Ellenor; Lindblad, Peter

2010-01-01

The filamentous, heterocystous, nitrogen-fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain, depending on growth conditions, up to two hydrogenases directly involved in hydrogen metabolism. HypC is one out of at least seven auxiliary gene products required for synthesis of a functional hydrogenase, specifically involved in the maturation of the large subunit. In this study we present a protein, CalA (Alr0946 in the genome), belonging to the transcription regulator family AbrB, which in protein-DNA assays was found to interact with the upstream region of hypC. Transcriptional investigations showed that calA is cotranscribed with the downstream gene alr0947, which encodes a putative protease from the abortive infection superfamily, Abi. CalA was shown to interact specifically not only with the upstream region of hypC but also with its own upstream region, acting as a repressor on hypC. The bidirectional hydrogenase activity was significantly downregulated when CalA was overexpressed, demonstrating a correlation with the transcription factor, either direct or indirect. In silico studies showed that homologues to both CalA and Alr0947 are highly conserved proteins within cyanobacteria with very similar physical organizations of the corresponding structural genes. Possible functions of the cotranscribed downstream protein Alr0947 are presented. In addition, we present a three-dimensional (3D) model of the DNA binding domain of CalA and putative DNA binding mechanisms are discussed. PMID:20023111

Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter.

PubMed

Gál, Zita; Hegedüs, Csilla; Szakács, Gergely; Váradi, András; Sarkadi, Balázs; Özvegy-Laczka, Csilla

2015-02-01

Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter. Copyright © 2014. Published by Elsevier B.V.

Involvement of MoVMA11, a Putative Vacuolar ATPase c’ Subunit, in Vacuolar Acidification and Infection-Related Morphogenesis of Magnaporthe oryzae

PubMed Central

Chen, Guoqing; Liu, Xiaohong; Zhang, Lilin; Cao, Huijuan; Lu, Jianping; Lin, Fucheng

2013-01-01

Many functions of vacuole depend on the activity of vacuolar ATPase which is essential to maintain an acidic lumen and create the driving forces for massive fluxes of ions and metabolites through vacuolar membrane. In filamentous fungus Magnaporthe oryzae , subcellular colocalization and quinacrine staining suggested that the V1V0 domains of V-ATPase were fully assembled and the vacuoles were kept acidic during infection-related developments. Targeted gene disruption of MoVMA11 gene, encoding the putative c’ subunit of V-ATPase, impaired vacuolar acidification and mimicked the phenotypes of yeast V-ATPase mutants in the poor colony morphology, abolished asexual and sexual reproductions, selective carbon source utilization, and increased calcium and heavy metals sensitivities, however, not in the typical pH conditional lethality. Strikingly, aerial hyphae of the MoVMA11 null mutant intertwined with each other to form extremely thick filamentous structures. The results also implicated that MoVMA11 was involved in cell wall integrity and appressorium formation. Abundant non-melanized swollen structures and rare, small appressoria without penetration ability were produced at the hyphal tips of the ΔMovma11 mutant on onion epidermal cells. Finally, the MoVMA11 null mutant lost pathogenicity on both intact and wounded host leaves. Overall, our data indicated that MoVMA11, like other fungal VMA genes, is associated with numerous cellular functions and highlighted that V-ATPase is essential for infection-related morphogenesis and pathogenesis in M . oryzae . PMID:23826342

Functional analysis of the Helicobacter pullorum N-linked protein glycosylation system.

PubMed

Jervis, Adrian J; Wood, Alison G; Cain, Joel A; Butler, Jonathan A; Frost, Helen; Lord, Elizabeth; Langdon, Rebecca; Cordwell, Stuart J; Wren, Brendan W; Linton, Dennis

2018-04-01

N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from Campylobacter jejuni is encoded by pgl genes present at a single chromosomal locus. This gene cluster includes the pglB oligosaccharyltransferase responsible for transfer of glycan from lipid carrier to protein. Although all genomes from species of the Campylobacter genus contain a pgl locus, among the related Helicobacter genus only three evolutionarily related species (H. pullorum, H. canadensis and H. winghamensis) potentially encode N-linked protein glycosylation systems. Helicobacter putative pgl genes are scattered in five chromosomal loci and include two putative oligosaccharyltransferase-encoding pglB genes per genome. We have previously demonstrated the in vitro N-linked glycosylation activity of H. pullorum resulting in transfer of a pentasaccharide to a peptide at asparagine within the sequon (D/E)XNXS/T. In this study, we identified the first H. pullorum N-linked glycoprotein, termed HgpA. Production of histidine-tagged HgpA in the background of insertional knockout mutants of H. pullorum pgl/wbp genes followed by analysis of HgpA glycan structures demonstrated the role of individual gene products in the PglB1-dependent N-linked protein glycosylation pathway. Glycopeptide purification by zwitterionic-hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry identified six glycosites from five H. pullorum proteins, which was consistent with proteins reactive with a polyclonal antiserum generated against glycosylated HgpA. This study demonstrates functioning of a H. pullorum N-linked general protein glycosylation system.

The Anisakis Transcriptome Provides a Resource for Fundamental and Applied Studies on Allergy-Causing Parasites.

PubMed

Baird, Fiona J; Su, Xiaopei; Aibinu, Ibukun; Nolan, Matthew J; Sugiyama, Hiromu; Otranto, Domenico; Lopata, Andreas L; Cantacessi, Cinzia

2016-07-01

Food-borne nematodes of the genus Anisakis are responsible for a wide range of illnesses (= anisakiasis), from self-limiting gastrointestinal forms to severe systemic allergic reactions, which are often misdiagnosed and under-reported. In order to enhance and refine current diagnostic tools for anisakiasis, knowledge of the whole spectrum of parasite molecules transcribed and expressed by this parasite, including those acting as potential allergens, is necessary. In this study, we employ high-throughput (Illumina) sequencing and bioinformatics to characterise the transcriptomes of two Anisakis species, A. simplex and A. pegreffii, and utilize this resource to compile lists of potential allergens from these parasites. A total of ~65,000,000 reads were generated from cDNA libraries for each species, and assembled into ~34,000 transcripts (= Unigenes); ~18,000 peptides were predicted from each cDNA library and classified based on homology searches, protein motifs and gene ontology and biological pathway mapping. Using comparative analyses with sequence data available in public databases, 36 (A. simplex) and 29 (A. pegreffii) putative allergens were identified, including sequences encoding 'novel' Anisakis allergenic proteins (i.e. cyclophilins and ABA-1 domain containing proteins). This study represents a first step towards providing the research community with a curated dataset to use as a molecular resource for future investigations of the biology of Anisakis, including molecules putatively acting as allergens, using functional genomics, proteomics and immunological tools. Ultimately, an improved knowledge of the biological functions of these molecules in the parasite, as well as of their immunogenic properties, will assist the development of comprehensive, reliable and robust diagnostic tools.

HUNT: launch of a full-length cDNA database from the Helix Research Institute.

PubMed

Yudate, H T; Suwa, M; Irie, R; Matsui, H; Nishikawa, T; Nakamura, Y; Yamaguchi, D; Peng, Z Z; Yamamoto, T; Nagai, K; Hayashi, K; Otsuki, T; Sugiyama, T; Ota, T; Suzuki, Y; Sugano, S; Isogai, T; Masuho, Y

2001-01-01

The Helix Research Institute (HRI) in Japan is releasing 4356 HUman Novel Transcripts and related information in the newly established HUNT database. The institute is a joint research project principally funded by the Japanese Ministry of International Trade and Industry, and the clones were sequenced in the governmental New Energy and Industrial Technology Development Organization (NEDO) Human cDNA Sequencing Project. The HUNT database contains an extensive amount of annotation from advanced analysis and represents an essential bioinformatics contribution towards understanding of the gene function. The HRI human cDNA clones were obtained from full-length enriched cDNA libraries constructed with the oligo-capping method and have resulted in novel full-length cDNA sequences. A large fraction has little similarity to any proteins of known function and to obtain clues about possible function we have developed original analysis procedures. Any putative function deduced here can be validated or refuted by complementary analysis results. The user can also extract information from specific categories like PROSITE patterns, PFAM domains, PSORT localization, transmembrane helices and clones with GENIUS structure assignments. The HUNT database can be accessed at http://www.hri.co.jp/HUNT.

Expression, purification and preliminary X-ray diffraction studies of VERNALIZATION1208–341 from Arabidopsis thaliana

PubMed Central

King, Gordon; Hill, Justine M.; Martin, Jennifer L.; Mylne, Joshua S.

2009-01-01

VERNALIZATION1 (VRN1) is required in the model plant Arabidopsis thaliana for the epigenetic suppression of the floral repressor FLC by prolonged cold treatment. Stable suppression of FLC accelerates flowering, a physiological process known as vernalization. VRN1 is a 341-residue DNA-binding protein that contains two plant-specific B3 domains (B3a and B3b), a putative nuclear localization sequence (NLS) and two putative PEST domains. VRN1208–341 includes the second B3 domain and a region upstream that is highly conserved in the VRN1 orthologues of other dicotyledonous plants. VRN1208–341 was crystallized by the hanging-drop method in 0.05 M sodium acetate pH 6.0 containing 1.0 M NaCl and 18%(w/v) PEG 3350. Preliminary X-ray diffraction data analysis revealed that the VRN1208–341 crystal diffracted to 2.1 Å and belonged to space group C2, with unit-cell parameters a = 105.2, b = 47.9, c = 61.2 Å, α = 90.0, β = 115.4, γ = 90.0°. Assuming that two molecules occupy the asymmetric unit, a Matthews coefficient of 2.05 Å3 Da−1 and a solvent content of 40.1% were calculated. PMID:19255487

Stereospecific Synthesis of threo- and erythro-β-Hydroxyglutamic Acid During Kutzneride Biosynthesis

PubMed Central

Strieker, Matthias; Nolan, Elizabeth M.; Walsh, Christopher T.; Marahiel, Mohamed A.

2009-01-01

The antifungal and antimicrobial kutznerides, hexadepsipeptides comprised of one α-hydroxy acid and five non-proteinogenic amino acids, are remarkable examples of the structural diversity found in nonribosomally-produced natural products. They contain D-3-hydroxyglutamic acid, which is found in the threo and erythro isomers in mature kutznerides. In this study, two putative non-heme iron oxygenase enzymes, KtzO and KtzP, were recombinantly expressed, characterized biochemically in vitro, and found to stereospecifically hydroxylate the β-position of glutamic acid. KtzO generates threo-L-hydroxyglutamic acid and KtzP catalyzes the formation of the erythro-isomer bound to the peptidyl carrier protein of the third module of the nonribosomal peptide synthetase KtzH. This module has a truncated adenylation domain and is unable to activate and incorporate glutamic acid. The lack of a functional adenylation domain in the third KtzH module is compensated in trans by the stand-alone adenylation domain KtzN, which activates and transfers glutamic acid onto the carrier of KtzH in the presence of the truncated adenylation domain and either KtzO or KtzP. A method that employs non-hydrolyzable coenzyme A analogs was developed and used to determine the kinetic parameters for KtzO- and KtzP-catalyzed hydroxylation of glutamic acid bound to the carrier protein. A detailed mechanism for the in trans compensation of the truncated adenylation domain and the stereospecific hydroxyglutamic acid generation and incorporation is presented. These insights may guide the use of KtzO/KtzP and KtzN or other in trans modification/restoration tools in biocombinatorial engineering approaches. PMID:19722489

The CC2D1A, a member of a new gene family with C2 domains, is involved in autosomal recessive non‐syndromic mental retardation

PubMed Central

Basel‐Vanagaite, L; Attia, R; Yahav, M; Ferland, R J; Anteki, L; Walsh, C A; Olender, T; Straussberg, R; Magal, N; Taub, E; Drasinover, V; Alkelai, A; Bercovich, D; Rechavi, G; Simon, A J; Shohat, M

2006-01-01

Background The molecular basis of autosomal recessive non‐syndromic mental retardation (NSMR) is poorly understood, mostly owing to heterogeneity and absence of clinical criteria for grouping families for linkage analysis. Only two autosomal genes, the PRSS12 gene on chromosome 4q26 and the CRBN on chromosome 3p26, have been shown to cause autosomal recessive NSMR, each gene in only one family. Objective To identify the gene causing autosomal recessive NSMR on chromosome 19p13.12. Results The candidate region established by homozygosity mapping was narrowed down from 2.4 Mb to 0.9 Mb on chromosome 19p13.12. A protein truncating mutation was identified in the gene CC2D1A in nine consanguineous families with severe autosomal recessive NSMR. The absence of the wild type protein in the lymphoblastoid cells of the patients was confirmed. CC2D1A is a member of a previously uncharacterised gene family that carries two conserved motifs, a C2 domain and a DM14 domain. The C2 domain is found in proteins which function in calcium dependent phospholipid binding; the DM14 domain is unique to the CC2D1A protein family and its role is unknown. CC2D1A is a putative signal transducer participating in positive regulation of I‐κB kinase/NFκB cascade. Expression of CC2D1A mRNA was shown in the embryonic ventricular zone and developing cortical plate in staged mouse embryos, persisting into adulthood, with highest expression in the cerebral cortex and hippocampus. Conclusions A previously unknown signal transduction pathway is important in human cognitive development. PMID:16033914

The CC2D1A, a member of a new gene family with C2 domains, is involved in autosomal recessive non-syndromic mental retardation.

PubMed

Basel-Vanagaite, L; Attia, R; Yahav, M; Ferland, R J; Anteki, L; Walsh, C A; Olender, T; Straussberg, R; Magal, N; Taub, E; Drasinover, V; Alkelai, A; Bercovich, D; Rechavi, G; Simon, A J; Shohat, M

2006-03-01

The molecular basis of autosomal recessive non-syndromic mental retardation (NSMR) is poorly understood, mostly owing to heterogeneity and absence of clinical criteria for grouping families for linkage analysis. Only two autosomal genes, the PRSS12 gene on chromosome 4q26 and the CRBN on chromosome 3p26, have been shown to cause autosomal recessive NSMR, each gene in only one family. To identify the gene causing autosomal recessive NSMR on chromosome 19p13.12. The candidate region established by homozygosity mapping was narrowed down from 2.4 Mb to 0.9 Mb on chromosome 19p13.12. A protein truncating mutation was identified in the gene CC2D1A in nine consanguineous families with severe autosomal recessive NSMR. The absence of the wild type protein in the lymphoblastoid cells of the patients was confirmed. CC2D1A is a member of a previously uncharacterised gene family that carries two conserved motifs, a C2 domain and a DM14 domain. The C2 domain is found in proteins which function in calcium dependent phospholipid binding; the DM14 domain is unique to the CC2D1A protein family and its role is unknown. CC2D1A is a putative signal transducer participating in positive regulation of I-kappaB kinase/NFkappaB cascade. Expression of CC2D1A mRNA was shown in the embryonic ventricular zone and developing cortical plate in staged mouse embryos, persisting into adulthood, with highest expression in the cerebral cortex and hippocampus. A previously unknown signal transduction pathway is important in human cognitive development.

Genome-wide identification and phylogenetic analysis of the AP2/ERF gene superfamily in sweet orange (Citrus sinensis).

PubMed

Ito, T M; Polido, P B; Rampim, M C; Kaschuk, G; Souza, S G H

2014-09-26

Sweet orange (Citrus sinensis) plays an important role in the economy of more than 140 countries, but it is grown in areas with intermittent stressful soil and climatic conditions. The stress tolerance could be addressed by manipulating the ethylene response factor (ERF) transcription factors because they orchestrate plant responses to environmental stress. We performed an in silico study on the ERFs in the expressed sequence tag database of C. sinensis to identify potential genes that regulate plant responses to stress. We identified 108 putative genes encoding protein sequences of the AP2/ERF superfamily distributed within 10 groups of amino acid sequences. Ninety-one genes were assembled from the ERF family containing only one AP2/ERF domain, 13 genes were assembled from the AP2 family containing two AP2/ERF domains, and four other genes were assembled from the RAV family containing one AP2/ERF domain and a B3 domain. Some conserved domains of the ERF family genes were disrupted into a few segments by introns. This irregular distribution of genes in the AP2/ERF superfamily in different plant species could be a result of genomic losses or duplication events in a common ancestor. The in silico gene expression revealed that 67% of AP2/ERF genes are expressed in tissues with usual plant development, and 14% were expressed in stressed tissues. Because the AP2/ERF superfamily is expressed in an orchestrated way, it is possible that the manipulation of only one gene may result in changes in the whole plant function, which could result in more tolerant crops.

Regulation of Lactobacillus casei Sorbitol Utilization Genes Requires DNA-Binding Transcriptional Activator GutR and the Conserved Protein GutM▿

PubMed Central

Alcántara, Cristina; Sarmiento-Rubiano, Luz Adriana; Monedero, Vicente; Deutscher, Josef; Pérez-Martínez, Gaspar; Yebra, María J.

2008-01-01

Sequence analysis of the five genes (gutRMCBA) downstream from the previously described sorbitol-6-phosphate dehydrogenase-encoding Lactobacillus casei gutF gene revealed that they constitute a sorbitol (glucitol) utilization operon. The gutRM genes encode putative regulators, while the gutCBA genes encode the EIIC, EIIBC, and EIIA proteins of a phosphoenolpyruvate-dependent sorbitol phosphotransferase system (PTSGut). The gut operon is transcribed as a polycistronic gutFRMCBA messenger, the expression of which is induced by sorbitol and repressed by glucose. gutR encodes a transcriptional regulator with two PTS-regulated domains, a galactitol-specific EIIB-like domain (EIIBGat domain) and a mannitol/fructose-specific EIIA-like domain (EIIAMtl domain). Its inactivation abolished gut operon transcription and sorbitol uptake, indicating that it acts as a transcriptional activator. In contrast, cells carrying a gutB mutation expressed the gut operon constitutively, but they failed to transport sorbitol, indicating that EIIBCGut negatively regulates GutR. A footprint analysis showed that GutR binds to a 35-bp sequence upstream from the gut promoter. A sequence comparison with the presumed promoter region of gut operons from various firmicutes revealed a GutR consensus motif that includes an inverted repeat. The regulation mechanism of the L. casei gut operon is therefore likely to be operative in other firmicutes. Finally, gutM codes for a conserved protein of unknown function present in all sequenced gut operons. A gutM mutant, the first constructed in a firmicute, showed drastically reduced gut operon expression and sorbitol uptake, indicating a regulatory role also for GutM. PMID:18676710

Mechanism of auxiliary β-subunit-mediated membrane targeting of L-type (CaV1.2) channels

PubMed Central

Fang, Kun; Colecraft, Henry M

2011-01-01

Abstract Ca2+ influx via CaV1/CaV2 channels drives processes ranging from neurotransmission to muscle contraction. Association of a pore-forming α1 and cytosolic β is necessary for trafficking CaV1/CaV2 channels to the cell surface through poorly understood mechanisms. A prevalent idea suggests β binds the α1 intracellular I–II loop, masking an endoplasmic reticulum (ER) retention signal as the dominant mechanism for CaV1/CaV2 channel membrane trafficking. There are hints that other α1 subunit cytoplasmic domains may play a significant role, but the nature of their potential contribution is unclear. We assessed the roles of all intracellular domains of CaV1.2-α1C by generating chimeras featuring substitutions of all possible permutations of intracellular loops/termini of α1C into the β-independent CaV3.1-α1G channel. Surprisingly, functional analyses demonstrated α1C I–II loop strongly increases channel surface density while other cytoplasmic domains had a competing opposing effect. Alanine-scanning mutagenesis identified an acidic-residue putative ER export motif responsible for the I–II loop-mediated increase in channel surface density. β-dependent increase in current arose as an emergent property requiring four α1C intracellular domains, with the I–II loop and C-terminus being essential. The results suggest β binding to the α1C I–II loop causes a C-terminus-dependent rearrangement of intracellular domains, shifting a balance of power between export signals on the I–II loop and retention signals elsewhere. PMID:21746784

Molecular cloning and characterization of a membrane associated NAC family gene, SiNAC from foxtail millet [Setaria italica (L.) P. Beauv].

PubMed

Puranik, Swati; Bahadur, Ranjit Prasad; Srivastava, Prem S; Prasad, Manoj

2011-10-01

The plant-specific NAC (NAM, ATAF, and CUC) transcription factors have diverse role in development and stress regulation. A transcript encoding NAC protein, termed SiNAC was identified from a salt stress subtractive cDNA library of S. italica seedling (Puranik et al., J Plant Physiol 168:280-287, 2011). This single/low copy gene containing four exons and four introns within the genomic-sequence encoded a protein of 462 amino acids. Structural analysis revealed that highly divergent C terminus contains a transmembrane domain. The NAC domain consisted of a twisted antiparallel beta-sheet packing against N terminal alpha helix on one side and a shorter helix on the other side. The domain was predicted to homodimerize and control DNA-binding specificity. The physicochemical features of the SiNAC homodimer interface justified the dimeric form of the predicted model. A 1539 bp fragment upstream to the start codon of SiNAC gene was cloned and in silico analysis revealed several putative cis-acting regulatory elements within the promoter sequence. Transactivation analysis indicated that SiNAC activated expression of reporter gene and the activation domain lied at the C terminal. The SiNAC:GFP was detected in the nucleus and cytoplasm while SiNAC ΔC(1-158):GFP was nuclear localized in onion epidermal cells. SiNAC transcripts mostly accumulated in young spikes and were strongly induced by dehydration, salinity, ethephon, and methyl jasmonate. These results suggest that SiNAC encodes a membrane associated NAC-domain protein that may function as a transcriptional activator in response to stress and developmental regulation in plants.

Small molecule correctors of F508del-CFTR discovered by structure-based virtual screening

NASA Astrophysics Data System (ADS)

Kalid, Ori; Mense, Martin; Fischman, Sharon; Shitrit, Alina; Bihler, Hermann; Ben-Zeev, Efrat; Schutz, Nili; Pedemonte, Nicoletta; Thomas, Philip J.; Bridges, Robert J.; Wetmore, Diana R.; Marantz, Yael; Senderowitz, Hanoch

2010-12-01

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.

A novel paired domain DNA recognition motif can mediate Pax2 repression of gene transcription.

PubMed

Håvik, B; Ragnhildstveit, E; Lorens, J B; Saelemyr, K; Fauske, O; Knudsen, L K; Fjose, A

1999-12-20

The paired domain (PD) is an evolutionarily conserved DNA-binding domain encoded by the Pax gene family of developmental regulators. The Pax proteins are transcription factors and are involved in a variety of processes such as brain development, patterning of the central nervous system (CNS), and B-cell development. In this report we demonstrate that the zebrafish Pax2 PD can interact with a novel type of DNA sequences in vitro, the triple-A motif, consisting of a heptameric nucleotide sequence G/CAAACA/TC with an invariant core of three adjacent adenosines. This recognition sequence was found to be conserved in known natural Pax5 repressor elements involved in controlling the expression of the p53 and J-chain genes. By identifying similar high affinity binding sites in potential target genes of the Pax2 protein, including the pax2 gene itself, we obtained further evidence that the triple-A sites are biologically significant. The putative natural target sites also provide a basis for defining an extended consensus recognition sequence. In addition, we observed in transformation assays a direct correlation between Pax2 repressor activity and the presence of triple-A sites. The results suggest that a transcriptional regulatory function of Pax proteins can be modulated by PD binding to different categories of target sequences. Copyright 1999 Academic Press.

Discrimination and divergence among Lactobacillus plantarum-group (LPG) isolates with reference to their probiotic functionalities from vegetable origin.

PubMed

Devi, Sundru Manjulata; Aishwarya, Subramanian; Halami, Prakash M

2016-12-01

The present study was aimed to evaluate the diversity and probiotic properties of Lactobacillus plantarum-group cultures from vegetable origin. First, genotypic diversity of L. plantarum (n=34) was achieved by PCR of Random Amplified Polymorphic DNA and recA gene-specific multiplex PCR. The isolates were segregated into five groups namely, Lactobacillus pentosus, Lactobacillus paraplantarum, Lactobacillus arizonensis, Lactobacillus plantarum subsp. plantarum and argentoratensis. Further discrimination was achieved by restriction fragment length polymorphism of probiotic adhesion genes viz.fbp, mub and msa gene. As determined by nucleotide sequence analysis and bioinformatics Pfam database, the putative Fbp protein had only one FBP domain, whereas Mub protein had 8-10 MUB domain repeats. However, L. pentosus (except CFR MFT9), L. plantarum subsp. argentoratensis (except CFR MFT5) and L. arizonensis (except CFR MFT2) isolates gave no amplicon for the tested marker genes. Selected cultures (n=15) showed tolerance to simulated digestive fluids (20-85%), exhibited auto-aggregation (10-77%), cellular hydrophobicity (12-78%), and broad spectrum of anti-microbial activity. Concurrently, high adherence capacity to mucin was achieved for L. plantarum subsp. plantarum (MCC 2974 and CFR MFT1) and L. paraplantarum (MTCC 9483, MCC 2977, MCC 2978), which had an additional MUB domain repeat. Copyright © 2016 Elsevier GmbH. All rights reserved.

Characterization of a novel single-stranded RNA virus, closely related to fusariviruses, infecting the plant pathogenic fungus Alternaria brassicicola.

PubMed

Zhong, Jie; Shang, Hong Hong; Zhu, Chuan Xia; Zhu, Jun Zi; Zhu, Hong Jian; Hu, Yan; Gao, Bi Da

2016-06-02

The alternaria blackspot of rapeseed is one of the most prominent diseases of rapeseed. It is caused by three species of the genus Alternaria: Alternaria brassicicola, Alternaria brassicae, and Alternaria raphanin. Here we report a novel positive-sense RNA virus from an A. brassicicola strain 817-14. The virus has a 6639 nucleotide (nt) long genome, excluding a poly (A)-tail, and was predicted to contain three putative open reading frames (ORF1, ORF2, and ORF3). The large ORF1 encoded a 174-kDa polyprotein (composed of 1522 amino acid residues) containing a conserved RNA-dependent RNA polymerase (RdRp) domain and a helicase domain. The other two smaller ORFs encoded polypeptides with unknown function. Homology search and phylogenetic analysis, based on the RdRp and helicase domains, suggest that this virus is related to and grouped with Sclerotinia sclerotiorum fusarivirus 1 (SsFV1), Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1), and Penicillium roqueforti RNA mycovirus 1 (PrRV1), all of which belong to a newly proposed family Fusariviridae. For this study, we designed the virus as "Alternaria brassicicola fusarivirus 1" (AbFV1). Virus elimination revealed that AbFV1 has no conspicuous impact on the biological properties of its host. Copyright © 2016. Published by Elsevier B.V.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoon, Bohyun; Kim, Inki; Nam, Ja-Ae

EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengthsmore » of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system. - Highlights: • EFC-1 integrase-mediated recombination was site-specific and unidirectional system. • Serine 21 of EFC-1 integrase plays a major role in the catalytic domain. • The functional minimal sizes of attB and attP was defined 48 and 54 bp.« less

A new putative deltapartitivirus recovered from Dianthus amurensis.

PubMed

An, Hongliu; Tan, Guanlin; Xiong, Guihong; Li, Meirong; Fang, Shouguo; Islam, Saif Ul; Zhang, Songbai; Li, Fan

2017-09-01

Two double stranded RNAs (dsRNA), likely representing the genome of a novel deltapartitivirus, provisionally named carnation cryptic virus 3 (CCV3), were recovered from Dianthus amurensis. The two dsRNAs were 1,573 (dsRNA1) and 1,561 (dsRNA2) bp in size, each containing a single open reading frame (ORF) encoding a 475- and 411-aa protein, respectively. The 475-aa protein contains a conserved RNA dependent RNA polymerase (RdRp) domain which shows significant homology to RdRps of established or putative partitiviruses, particularly those belonging to the genus Deltapartitivirus. However, it shares an amino acid identity of 75% with its closest relative, the RdRp of the deltapartitivirus beet cryptic virus 2 (BCV2), and is <62% identical to the RdRps of other partitiviruses. In a phylogenetic tree constructed with RdRps of selected partitiviruses, CCV3 clustered with BCV2 and formed a well-supported monophyletic clade with known or putative deltapartitiviruses.

Phylogenetics and evolution of Su(var)3-9 SET genes in land plants: rapid diversification in structure and function.

PubMed

Zhu, Xinyu; Ma, Hong; Chen, Zhiduan

2011-03-09

Plants contain numerous Su(var)3-9 homologues (SUVH) and related (SUVR) genes, some of which await functional characterization. Although there have been studies on the evolution of plant Su(var)3-9 SET genes, a systematic evolutionary study including major land plant groups has not been reported. Large-scale phylogenetic and evolutionary analyses can help to elucidate the underlying molecular mechanisms and contribute to improve genome annotation. Putative orthologs of plant Su(var)3-9 SET protein sequences were retrieved from major representatives of land plants. A novel clustering that included most members analyzed, henceforth referred to as core Su(var)3-9 homologues and related (cSUVHR) gene clade, was identified as well as all orthologous groups previously identified. Our analysis showed that plant Su(var)3-9 SET proteins possessed a variety of domain organizations, and can be classified into five types and ten subtypes. Plant Su(var)3-9 SET genes also exhibit a wide range of gene structures among different paralogs within a family, even in the regions encoding conserved PreSET and SET domains. We also found that the majority of SUVH members were intronless and formed three subclades within the SUVH clade. A detailed phylogenetic analysis of the plant Su(var)3-9 SET genes was performed. A novel deep phylogenetic relationship including most plant Su(var)3-9 SET genes was identified. Additional domains such as SAR, ZnF_C2H2 and WIYLD were early integrated into primordial PreSET/SET/PostSET domain organization. At least three classes of gene structures had been formed before the divergence of Physcomitrella patens (moss) from other land plants. One or multiple retroposition events might have occurred among SUVH genes with the donor genes leading to the V-2 orthologous group. The structural differences among evolutionary groups of plant Su(var)3-9 SET genes with different functions were described, contributing to the design of further experimental studies.

Is there an optimal level of open-endedness in prebiotic evolution?

PubMed

Markovitch, Omer; Sorek, Daniel; Lui, Leong Ting; Lancet, Doron; Krasnogor, Natalio

2012-10-01

In this paper we explore the question of whether there is an optimal set up for a putative prebiotic system leading to open-ended evolution (OEE) of the events unfolding within this system. We do so by proposing two key innovations. First, we introduce a new index that measures OEE as a function of the likelihood of events unfolding within a universe given its initial conditions. Next, we apply this index to a variant of the graded autocatalysis replication domain (GARD) model, Segre et al. (P Natl Acad Sci USA 97(8):4112-4117, 2000; Markovitch and Lancet Artif Life 18(3), 2012), and use it to study--under a unified and concise prebiotic evolutionary framework--both a variety of initial conditions of the universe and the OEE of species that evolve from them.

Is There an Optimal Level of Open-Endedness in Prebiotic Evolution?

NASA Astrophysics Data System (ADS)

Markovitch, Omer; Sorek, Daniel; Lui, Leong Ting; Lancet, Doron; Krasnogor, Natalio

2012-10-01

In this paper we explore the question of whether there is an optimal set up for a putative prebiotic system leading to open-ended evolution (OEE) of the events unfolding within this system. We do so by proposing two key innovations. First, we introduce a new index that measures OEE as a function of the likelihood of events unfolding within a universe given its initial conditions. Next, we apply this index to a variant of the graded autocatalysis replication domain (GARD) model, Segre et al. (P Natl Acad Sci USA 97(8):4112-4117, 2000; Markovitch and Lancet Artif Life 18(3), 2012), and use it to study - under a unified and concise prebiotic evolutionary framework - both a variety of initial conditions of the universe and the OEE of species that evolve from them.

First insights into a type II toxin-antitoxin system from the clinical isolate Mycobacterium sp. MHSD3, similar to epsilon/zeta systems.

PubMed

Jaén-Luchoro, Daniel; Aliaga-Lozano, Francisco; Gomila, Rosa Maria; Gomila, Margarita; Salvà-Serra, Francisco; Lalucat, Jorge; Bennasar-Figueras, Antoni

2017-01-01

A putative type II toxin-antitoxin (TA) system was found in the clinical isolate Mycobacterium sp. MHSD3, a strain closely related to Mycobacterium chelonae. Further analyses of the protein sequences of the two genes revealed the presence of domains related to a TA system. BLAST analyses indicated the presence of closely related proteins in the genomes of other recently published M. chelonae strains. The functionality of both elements of the TA system was demonstrated when expressed in Escherichia coli cells, and the predicted structure of the toxin is very similar to those of well-known zeta-toxins, leading to the definition of a type II TA system similar to epsilon/zeta TA systems in strains that are closely related to M. chelonae.

Rab27a regulates epithelial sodium channel (ENaC) activity through synaptotagmin-like protein (SLP-5) and Munc13-4 effector mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saxena, Sunil K.; Horiuchi, Hisanori; Fukuda, Mitsunori

Liddle's syndrome (excessive absorption of sodium ions) and PHA-1 (pseudohypoaldosteronism type 1) with decreased sodium absorption are caused by the mutations in the amiloride-sensitive epithelial sodium channel ENaC. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. Earlier, we reported that Rab27a inhibits ENaC-mediated currents through protein-protein interaction in HT-29 cells. We hereby report that Rab27a-dependent inhibition is associated with the GTP/GDP status as constitutively active or GTPase-deficient mutant Q78L inhibits amiloride-sensitive currents whereas GDP-locked inactive mutant T23N showed no effect. In order to further explore the molecular mechanism of this regulation, we performed competitive assays withmore » two Rab27a-binding proteins: synaptotagmin-like protein (SLP-5) and Munc13-4 (a putative priming factor for exocytosis). Both proteins eliminate negative modulation of Rab27a on ENaC function. The SLP-5 reversal of Rab27a effect was restricted to C-terminal C2A/C2B domains assigned for putative phospholipids-binding function while the Rab27a-binding SHD motif imparted higher inhibition. The ENaC-mediated currents remain unaffected by Rab27a though SLP-5 appears to strongly bind it. The immunoprecipitation experiments suggest that in the presence of excessive Munc13-4 and SLP-5 proteins, Rab27a interaction with ENaC is diminished. Munc13-4 and SLP-5 limit the Rab27a availability to ENaC, thus minimizing its effect on channel function. These observations decisively prove that Rab27a inhibits ENaC function through a complex mechanism that involves GTP/GDP status, and protein-protein interactions involving Munc13-4 and SLP-5 effector proteins.« less

Electron Microscopy Structural Insights into CPAP Oligomeric Behavior: A Plausible Assembly Process of a Supramolecular Scaffold of the Centrosome

PubMed Central

Alvarez-Cabrera, Ana L.; Delgado, Sandra; Gil-Carton, David; Mortuza, Gulnahar B.; Montoya, Guillermo; Sorzano, Carlos O. S.; Tang, Tang K.; Carazo, Jose M.

2017-01-01

Centrosomal P4.1-associated protein (CPAP) is a cell cycle regulated protein fundamental for centrosome assembly and centriole elongation. In humans, the region between residues 897–1338 of CPAP mediates interactions with other proteins and includes a homodimerization domain. CPAP mutations cause primary autosomal recessive microcephaly and Seckel syndrome. Despite of the biological/clinical relevance of CPAP, its mechanistic behavior remains unclear and its C-terminus (the G-box/TCP domain) is the only part whose structure has been solved. This situation is perhaps due in part to the challenges that represent obtaining the protein in a soluble, homogeneous state for structural studies. Our work constitutes a systematic structural analysis on multiple oligomers of HsCPAP897−1338, using single-particle electron microscopy (EM) of negatively stained (NS) samples. Based on image classification into clearly different regular 3D maps (putatively corresponding to dimers and tetramers) and direct observation of individual images representing other complexes of HsCPAP897−1338 (i.e., putative flexible monomers and higher-order multimers), we report a dynamic oligomeric behavior of this protein, where different homo-oligomers coexist in variable proportions. We propose that dimerization of the putative homodimer forms a putative tetramer which could be the structural unit for the scaffold that either tethers the pericentriolar material to centrioles or promotes procentriole elongation. A coarse fitting of atomic models into the NS 3D maps at resolutions around 20 Å is performed only to complement our experimental data, allowing us to hypothesize on the oligomeric composition of the different complexes. In this way, the current EM work represents an initial step toward the structural characterization of different oligomers of CPAP, suggesting further insights to understand how this protein works, contributing to the elucidation of control mechanisms for centriole biogenesis. PMID:28396859

Role of the interfacial binding domain in the oxidative susceptibility of lecithin:cholesterol acyltransferase.

PubMed Central

Wang, Kewei; Subbaiah, Papasani V

2002-01-01

We had previously shown that the cholesterol esterification activity of lecithin:cholesterol acyltransferase (LCAT) is destroyed by oxidation, but still it retains the ability to hydrolyse water-soluble substrates. This suggested that the inactivation of the enzyme is not due to its catalytic function, but due to a loss of its hydrophobic binding. Since recent studies have shown that a tryptophan residue in the putative interfacial domain (Trp(61)) is critical for the activity, we determined the possible role of this residue in the oxidative susceptibility and substrate specificity of LCAT by site-directed mutagenesis. Deletion of Trp(61) resulted in a 56% loss of cholesterol esterification (LCAT) activity, but the phospholipase A(2) (PLA(2)) and the esterase activities of the enzyme were stimulated slightly. Replacing Trp(61) with another aromatic residue [Trp(61)-->Tyr (W61Y)] resulted in an increase in all activities (14-157%), whereas replacing it with an aliphatic residue [Trp(61)-->Gly (W61G)] caused a dramatic loss of LCAT (-90%) and PLA(2) (-82%) activities, but not the esterase activity (-5%). W61Y was the most sensitive to oxidation, whereas W61G was the most resistant, with respect to the LCAT and PLA(2) activities. However, the activities which do not involve interfacial binding, namely the esterase activity and the transesterification of short-chain phospholipids, were more resistant to oxidation in all LCATs, indicating a selective loss of the interfacial binding by oxidation. Furthermore, replacing the two cysteines (Cys(31) and Cys(184)) in the Trp(61) deletion mutant caused additional resistance of the enzyme to oxidizing agents, showing that both domains of the enzyme contribute independently to its oxidative susceptibility. Since the hydrolysis of truncated phospholipids, generated during the oxidation of low-density lipoproteins, does not require the interfacial-binding domain, our results suggest that LCAT may take part in the detoxification of these compounds even after the loss of its cholesterol esterification function. PMID:11966470

Frequency and Distribution of Single-Nucleotide Polymorphisms within mprF in Methicillin-Resistant Staphylococcus aureus Clinical Isolates and Their Role in Cross-Resistance to Daptomycin and Host Defense Antimicrobial Peptides.

PubMed

Bayer, Arnold S; Mishra, Nagendra N; Chen, Liang; Kreiswirth, Barry N; Rubio, Aileen; Yang, Soo-Jin

2015-08-01

MprF is responsible for the lysinylation of phosphatidylglycerol (PG) to synthesize the positively charged phospholipid (PL) species, lysyl-PG (L-PG). It has been proposed that the single-nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) are associated with a gain-in-function phenotype in terms of daptomycin resistance in Staphylococcus aureus. (Note that although the official term is daptomycin nonsusceptibility, we use the term daptomycin resistance in this paper for ease of presentation.) Using 22 daptomycin-susceptible (DAP(s))/daptomycin-resistant (DAP(r)) clinical methicillin-resistant S. aureus (MRSA) strain pairs, we assessed (i) the frequencies and distribution of putative mprF gain-in-function SNPs, (ii) the relationships of the SNPs to both daptomycin resistance and cross-resistance to the prototypical endovascular host defense peptide (HDP) thrombin-induced platelet microbicidal protein (tPMP), and (iii) the impact of mprF SNPs on positive surface charge phenotype and modifications of membrane PL profiles. Most of the mprF SNPs identified in our DAP(r) strains were clustered within the two MprF loci, (i) the central bifunctional domain and (ii) the C-terminal synthase domain. Moreover, we were able to correlate the presence and location of mprF SNPs in DAP(r) strains with HDP cross-resistance, positive surface charge, and L-PG profiles. Although DAP(r) strains with mprF SNPs in the bifunctional domain showed higher resistance to tPMPs than DAP(r) strains with SNPs in the synthase domain, this relationship was not observed in positive surface charge assays. These results demonstrated that both charge-mediated and -unrelated mechanisms are involved in DAP resistance and HDP cross-resistance in S. aureus. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Monoclonal antibodies recognizing the non-tandem repeat regions of the human mucin MUC4 in pancreatic cancer.

PubMed

Jain, Maneesh; Venkatraman, Ganesh; Moniaux, Nicolas; Kaur, Sukhwinder; Kumar, Sushil; Chakraborty, Subhankar; Varshney, Grish C; Batra, Surinder K

2011-01-01

The MUC4 mucin is a high molecular weight, membrane-bound, and highly glycosylated protein. It is a multi-domain protein that is putatively cleaved into a large mucin-like subunit (MUC4α) and a C-terminal growth-factor like subunit (MUC4β). MUC4 plays critical roles in physiological and pathological conditions and is aberrantly overexpressed in several cancers, including those of the pancreas, cervix, breast and lung. It is also a potential biomarker for the diagnosis, prognosis and progression of several malignancies. Further, MUC4 plays diverse functional roles in cancer initiation and progression as evident from its involvement in oncogenic transformation, proliferation, inhibition of apoptosis, motility and invasion, and resistance to chemotherapy in human cancer cells. We have previously generated a monoclonal antibody 8G7, which is directed against the TR region of MUC4, and has been extensively used to study the expression of MUC4 in several malignancies. Here, we describe the generation of anti-MUC4 antibodies directed against the non-TR regions of MUC4. Recombinant glutathione-S-transferase (GST)-fused MUC4α fragments, both upstream (MUC4α-N-Ter) and downstream (MUC4α-C-Ter) of the TR domain, were used as immunogens to immunize BALB/c mice. Following cell fusion, hybridomas were screened using the aforementioned recombinant proteins ad lysates from human pancreatic cell lines. Three anti MUC4α-N-Ter and one anti-MUC4α-C-Ter antibodies were characterized by several inmmunoassays including enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunofluorescene, flow cytometry and immunoprecipitation using MUC4 expressing human pancreatic cancer cell lines. The antibodies also reacted with the MUC4 in human pancreatic tumor sections in immunohistochemical analysis. The new domain-specific anti-MUC4 antibodies will serve as important reagents to study the structure-function relationship of MUC4 domains and for the development of MUC4-based diagnostics and therapeutics.

Monoclonal Antibodies Recognizing the Non-Tandem Repeat Regions of the Human Mucin MUC4 in Pancreatic Cancer

PubMed Central

Jain, Maneesh; Venkatraman, Ganesh; Moniaux, Nicolas; Kaur, Sukhwinder; Kumar, Sushil; Chakraborty, Subhankar; Varshney, Grish C.; Batra, Surinder K.

2011-01-01

The MUC4 mucin is a high molecular weight, membrane-bound, and highly glycosylated protein. It is a multi-domain protein that is putatively cleaved into a large mucin-like subunit (MUC4α) and a C-terminal growth-factor like subunit (MUC4β). MUC4 plays critical roles in physiological and pathological conditions and is aberrantly overexpressed in several cancers, including those of the pancreas, cervix, breast and lung. It is also a potential biomarker for the diagnosis, prognosis and progression of several malignancies. Further, MUC4 plays diverse functional roles in cancer initiation and progression as evident from its involvement in oncogenic transformation, proliferation, inhibition of apoptosis, motility and invasion, and resistance to chemotherapy in human cancer cells. We have previously generated a monoclonal antibody 8G7, which is directed against the TR region of MUC4, and has been extensively used to study the expression of MUC4 in several malignancies. Here, we describe the generation of anti-MUC4 antibodies directed against the non-TR regions of MUC4. Recombinant glutathione-S-transferase (GST)-fused MUC4α fragments, both upstream (MUC4α-N-Ter) and downstream (MUC4α-C-Ter) of the TR domain, were used as immunogens to immunize BALB/c mice. Following cell fusion, hybridomas were screened using the aforementioned recombinant proteins ad lysates from human pancreatic cell lines. Three anti MUC4α-N-Ter and one anti-MUC4α-C-Ter antibodies were characterized by several inmmunoassays including enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunofluorescene, flow cytometry and immunoprecipitation using MUC4 expressing human pancreatic cancer cell lines. The antibodies also reacted with the MUC4 in human pancreatic tumor sections in immunohistochemical analysis. The new domain-specific anti-MUC4 antibodies will serve as important reagents to study the structure-function relationship of MUC4 domains and for the development of MUC4-based diagnostics and therapeutics. PMID:21886786

Complete nucleotide sequence and annotation of the temperate corynephage ϕ16 genome.

PubMed

Lobanova, Juliya S; Gak, Evgueni R; Andreeva, Irina G; Rybak, Konstantin V; Krylov, Alexander A; Mashko, Sergey V

2017-08-01

The complete genome of ϕ16, a temperate corynephage from Corynebacterium glutamicum ATCC 21792, was sequenced and annotated (GenBank: KY250482). The electron microscopy study of ϕ16 virion confirmed that it belongs to the family Siphoviridae. The ϕ16 genome consists of a linear double-stranded DNA molecule of 58,200 bp (G+C = 52.2%) with protruding cohesive 3'-ends of 14 nt. Four major structural proteins were separated by SDS-PAGE and identified by peptide mass fingerprinting technique. Using bioinformatics analysis, 101 putative ORFs and 5 tRNA genes were predicted. Only 27 putative gene products could be assigned to known biological functions. The ϕ16 genome was divided into functional modules. Seven putative promoters and eight putative unidirectional intrinsic terminators were predicted. One site of putative «-1» programmed ribosomal frameshifting was proposed in the phage tail assembly genome region. C. glutamicum genetic tools could be broadened by exploiting the known integrase gene (gp33) and the newly identified excisionase gene (gp47), participating in site-specific recombination between ϕ16-attP/attB.

The Na+-phosphate cotransport system (NaPi-II) with a cleaved protein backbone: implications on function and membrane insertion

PubMed Central

Kohl, Beate; Wagner, Carsten A; Huelseweh, Birgit; Busch, Andreas E; Werner, Andreas

1998-01-01

Renal handling of inorganic phosphate (Pi) involves a Na+-Pi cotransport system which is well conserved between vertebrates. The members of this protein family, denoted NaPi-II, share a topology with, it is thought, eight transmembrane domains. The transporter is proposed to be proteolytically cleaved within a large hydrophilic loop in vivo. The consequences of an interrupted backbone were tested by constructing cDNA clones encoding different N- (1-3 and 1-5) and C-terminal (4-8 and 6-8) complementary fragments of NaPi-II from winter flounder. When the cognate fragments were used in combination (1-3 plus 4-8; 1-5 plus 6-8) they comprised the full complement of the putative transporter domains. None of the four individual fragments or the 1-5 plus 6-8 combination when expressed in Xenopus oocytes increased Pi flux. Coexpression of fragments 1-3 plus 4-8 stimulated transport activity identical to that for expressed wild-type NaPi-II with regard to pH dependency and Km for Na+ and Pi binding; however, the maximal transport rate (vmax) was lower. Immunohistochemistry on cryosections confined the functionally active 1-3 plus 4-8 combination to the oocyte membrane. This was not the case for the 1-5 plus 6-8 combination or any of the individual fragments, all of which failed to induce fluorescence. A second immunohistochemical approach using intact oocytes allowed determination of the extracellular regions of the protein. Epitopes within the loop between transmembrane domains 3 and 4 enhanced fluorescence. Neither N- nor C-terminal tags induced fluorescence. PMID:9508800

Carboxy-terminal cleavage of the human foamy virus Gag precursor molecule is an essential step in the viral life cycle.

PubMed Central

Enssle, J; Fischer, N; Moebes, A; Mauer, B; Smola, U; Rethwilm, A

1997-01-01

Foamy viruses (FVs) express the Gag protein as a precursor with a molecular mass of 74 kDa (pr74) from which a 70-kDa protein (p70) is cleaved by the viral protease. To gain a better understanding of FV Gag protein processing and function, we have generated and analyzed mutants in the C-terminal gag region of an infectious molecular clone. Our results show that p70 is an N-terminal cleavage product of pr74. However, we were unable to identify a p4 molecule. A virus mutant expressing p70 only was found to be replication competent, albeit at very low titers compared to those of wild-type virus. A strong tendency to synthesize and cleave a pr74 molecule was deduced from the occurrence of revertants upon transfection of this mutant. Substitution of the p6gag domain of human immunodeficiency virus type 1 for the p4 domain of FV resulted in a stable chimeric virus which replicated to titers 10 times lower than those of wild-type virus. FV Gag protein was found to be phosphorylated at serine residues. Mutagenesis of serines conserved in the p4 domain had no influence on viral replication in cell culture. The p70/p74 Gag cleavage was found to be required for viral infectivity, since mutagenesis of the putative cleavage site led to replication-incompetent virus. Interestingly, the cleavage site mutants were defective in the intracellular cDNA synthesis of virion DNA, which indicates that correct FV particle formation and the generation of virion DNA are functionally linked. PMID:9311808

Functional analysis of the isoforms of an ABI3-like factor of Pisum sativum generated by alternative splicing.

PubMed

Gagete, Andrés P; Riera, Marta; Franco, Luis; Rodrigo, M Isabel

2009-01-01

At least seven isoforms (PsABI3-1 to PsABI3-7) of a putative, pea ABI3-like factor, originated by alternative splicing, have been identified after cDNA cloning. A similar variability had previously only been described for monocot genes. The full-length isoform, PsABI3-1, contains the typical N-terminal acidic domains and C-terminal basic subdomains, B1 to B3. Reverse transcriptase-PCR analysis revealed that the gene is expressed just in seeds, starting at middle embryogenesis; no gene products are observed in embryo axes after 18 h post-imbibition although they are more persistent in cotyledons. The activity of the isoforms was studied by yeast one-hybrid assays. When yeast was transformed with the isoforms fused to the DNA binding domain of Gal4p, only the polypeptides PsABI3-2 and PsABI3-7 failed to complement the activity of Gal4p. Acidic domains A1 and A2 exhibit transactivating activity, but the former requires a small C-terminal extension to be active. Yeast two-hybrid analysis showed that PsABI3 is able to heterodimerize with Arabidopsis thaliana ABI5, thus proving that PsABI3 is functionally active. The minimum requirement for the interaction PsABI3-AtABI5 is the presence of the subdomain B1 with an extension, 81 amino acids long, at their C-terminal side. Finally, a transient onion transformation assay showed that both the active PsABI3-1 and the inactive PsABI3-2 isoforms are localized to nuclei. Considering that the major isoforms remain approximately constant in developing seeds although their relative proportion varied, the possible role of splicing in the regulatory network of ABA signalling is discussed.

Genome-Wide Analysis of the RAV Family in Soybean and Functional Identification of GmRAV-03 Involvement in Salt and Drought Stresses and Exogenous ABA Treatment

PubMed Central

Zhao, Shu-Ping; Xu, Zhao-Shi; Zheng, Wei-Jun; Zhao, Wan; Wang, Yan-Xia; Yu, Tai-Fei; Chen, Ming; Zhou, Yong-Bin; Min, Dong-Hong; Ma, You-Zhi; Chai, Shou-Cheng; Zhang, Xiao-Hong

2017-01-01

Transcription factors play vital roles in plant growth and in plant responses to abiotic stresses. The RAV transcription factors contain a B3 DNA binding domain and/or an APETALA2 (AP2) DNA binding domain. Although genome-wide analyses of RAV family genes have been performed in several species, little is known about the family in soybean (Glycine max L.). In this study, a total of 13 RAV genes, named as GmRAVs, were identified in the soybean genome. We predicted and analyzed the amino acid compositions, phylogenetic relationships, and folding states of conserved domain sequences of soybean RAV transcription factors. These soybean RAV transcription factors were phylogenetically clustered into three classes based on their amino acid sequences. Subcellular localization analysis revealed that the soybean RAV proteins were located in the nucleus. The expression patterns of 13 RAV genes were analyzed by quantitative real-time PCR. Under drought stresses, the RAV genes expressed diversely, up- or down-regulated. Following NaCl treatments, all RAV genes were down-regulated excepting GmRAV-03 which was up-regulated. Under abscisic acid (ABA) treatment, the expression of all of the soybean RAV genes increased dramatically. These results suggested that the soybean RAV genes may be involved in diverse signaling pathways and may be responsive to abiotic stresses and exogenous ABA. Further analysis indicated that GmRAV-03 could increase the transgenic lines resistance to high salt and drought and result in the transgenic plants insensitive to exogenous ABA. This present study provides valuable information for understanding the classification and putative functions of the RAV transcription factors in soybean. PMID:28634481

A novel L-ficolin/mannose-binding lectin chimeric molecule with enhanced activity against Ebola virus.

PubMed

Michelow, Ian C; Dong, Mingdong; Mungall, Bruce A; Yantosca, L Michael; Lear, Calli; Ji, Xin; Karpel, Marshall; Rootes, Christina L; Brudner, Matthew; Houen, Gunnar; Eisen, Damon P; Kinane, T Bernard; Takahashi, Kazue; Stahl, Gregory L; Olinger, Gene G; Spear, Gregory T; Ezekowitz, R Alan B; Schmidt, Emmett V

2010-08-06

Ebola viruses constitute a newly emerging public threat because they cause rapidly fatal hemorrhagic fevers for which no treatment exists, and they can be manipulated as bioweapons. We targeted conserved N-glycosylated carbohydrate ligands on viral envelope surfaces using novel immune therapies. Mannose-binding lectin (MBL) and L-ficolin (L-FCN) were selected because they function as opsonins and activate complement. Given that MBL has a complex quaternary structure unsuitable for large scale cost-effective production, we sought to develop a less complex chimeric fusion protein with similar ligand recognition and enhanced effector functions. We tested recombinant human MBL and three L-FCN/MBL variants that contained the MBL carbohydrate recognition domain and varying lengths of the L-FCN collagenous domain. Non-reduced chimeric proteins formed predominantly nona- and dodecameric oligomers, whereas recombinant human MBL formed octadecameric and larger oligomers. Surface plasmon resonance revealed that L-FCN/MBL76 had the highest binding affinities for N-acetylglucosamine-bovine serum albumin and mannan. The same chimeric protein displayed superior complement C4 cleavage and binding to calreticulin (cC1qR), a putative receptor for MBL. L-FCN/MBL76 reduced infection by wild type Ebola virus Zaire significantly greater than the other molecules. Tapping mode atomic force microscopy revealed that L-FCN/MBL76 was significantly less tall than the other molecules despite similar polypeptide lengths. We propose that alterations in the quaternary structure of L-FCN/MBL76 resulted in greater flexibility in the collagenous or neck region. Similarly, a more pliable molecule might enhance cooperativity between the carbohydrate recognition domains and their cognate ligands, complement activation, and calreticulin binding dynamics. L-FCN/MBL chimeric proteins should be considered as potential novel therapeutics.

Evolution of the α-Subunit of Na/K-ATPase from Paramecium to Homo sapiens: Invariance of Transmembrane Helix Topology.

PubMed

Morrill, Gene A; Kostellow, Adele B; Liu, Lijun; Gupta, Raj K; Askari, Amir

2016-05-01

Na/K-ATPase is a key plasma membrane enzyme involved in cell signaling, volume regulation, and maintenance of electrochemical gradients. The α-subunit, central to these functions, belongs to a large family of P-type ATPases. Differences in transmembrane (TM) helix topology, sequence homology, helix-helix contacts, cell signaling, and protein domains of Na/K-ATPase α-subunit were compared in fungi (Beauveria), unicellular organisms (Paramecia), primitive multicellular organisms (Hydra), and vertebrates (Xenopus, Homo sapiens), and correlated with evolution of physiological functions in the α-subunit. All α-subunits are of similar length, with groupings of four and six helices in the N- and C-terminal regions, respectively. Minimal homology was seen for protein domain patterns in Paramecium and Hydra, with high correlation between Hydra and vertebrates. Paramecium α-subunits display extensive disorder, with minimal helix contacts. Increases in helix contacts in Hydra approached vertebrates. Protein motifs known to be associated with membrane lipid rafts and cell signaling reveal significant positional shifts between Paramecium and Hydra vulgaris, indicating that regional membrane fluidity changes occur during evolution. Putative steroid binding sites overlapping TM-3 occurred in all species. Sites associated with G-protein-receptor stimulation occur both in vertebrates and amphibia but not in Hydra or Paramecia. The C-terminus moiety "KETYY," necessary for the Na(+) activation of pump phosphorylation, is not present in unicellular species indicating the absence of classical Na(+)/K(+)-pumps. The basic protein topology evolved earliest, followed by increases in protein domains and ordered helical arrays, correlated with appearance of α-subunit regions known to involve cell signaling, membrane recycling, and ion channel formation.

The mitochondrial outer membrane protein hFis1 regulates mitochondrial morphology and fission through self-interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Serasinghe, Madhavika N.; Mitochondrial Research and Innovation Group, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642; Yoon, Yisang

2008-11-15

Mitochondrial fission in mammals is mediated by at least two proteins, DLP1/Drp1 and hFis1. DLP1 mediates the scission of mitochondrial membranes through GTP hydrolysis, and hFis1 is a putative DLP1 receptor anchored at the mitochondrial outer membrane by a C-terminal single transmembrane domain. The cytosolic domain of hFis1 contains six {alpha}-helices ({alpha}1-{alpha}6) out of which {alpha}2-{alpha}5 form two tetratricopeptide repeat (TPR) folds. In this study, by using chimeric constructs, we demonstrated that the cytosolic domain contains the necessary information for hFis1 function during mitochondrial fission. By using transient expression of different mutant forms of the hFis1 protein, we found thatmore » hFis1 self-interaction plays an important role in mitochondrial fission. Our results show that deletion of the {alpha}1 helix greatly increased the formation of dimeric and oligomeric forms of hFis1, indicating that {alpha}1 helix functions as a negative regulator of the hFis1 self-interaction. Further mutational approaches revealed that a tyrosine residue in the {alpha}5 helix and the linker between {alpha}3 and {alpha}4 helices participate in hFis1 oligomerization. Mutations causing oligomerization defect greatly reduced the ability to induce not only mitochondrial fragmentation by full-length hFis1 but also the formation of swollen ball-shaped mitochondria caused by {alpha}1-deleted hFis1. Our data suggest that oligomerization of hFis1 in the mitochondrial outer membrane plays a role in mitochondrial fission, potentially through participating in fission factor recruitment.« less

Parcellation in Left Lateral Parietal Cortex Is Similar in Adults and Children

PubMed Central

Nelson, Steven M.; Cohen, Alexander L.; Power, Jonathan D.; Coalson, Rebecca S.; Miezin, Francis M.; Vogel, Alecia C.; Dubis, Joseph W.; Church, Jessica A.; Petersen, Steven E.; Schlaggar, Bradley L.

2012-01-01

A key question in developmental neuroscience involves understanding how and when the cerebral cortex is partitioned into distinct functional areas. The present study used functional connectivity MRI mapping and graph theory to identify putative cortical areas and generate a parcellation scheme of left lateral parietal cortex (LLPC) in 7 to 10-year-old children and adults. Results indicated that a majority of putative LLPC areas could be matched across groups (mean distance between matched areas across age: 3.15 mm). Furthermore, the boundaries of children's putative LLPC areas respected the boundaries generated from the adults' parcellation scheme for a majority of children's areas (13/15). Consistent with prior research, matched LLPC areas showed age-related differences in functional connectivity strength with other brain regions. These results suggest that LLPC cortical parcellation and functional connectivity mature along different developmental trajectories, with adult-like boundaries between LLPC areas established in school-age children prior to adult-like functional connectivity. PMID:21810781

Working Memory Training in Typically Developing Children: A Meta-Analysis of the Available Evidence

ERIC Educational Resources Information Center

Sala, Giovanni; Gobet, Fernand

2017-01-01

The putative effectiveness of working memory (WM) training at enhancing cognitive and academic skills is still ardently debated. Several researchers have claimed that WM training fosters not only skills such as visuospatial WM and short-term memory (STM), but also abilities outside the domain of WM, such as fluid intelligence and mathematics.…

Therapeutic inhibition of prolyl hydroxylase domain-containing enzymes in surgery: putative applications and challenges

PubMed Central

Harnoss, Jonathan Michael; Strowitzki, Moritz Johannes; Radhakrishnan, Praveen; Platzer, Lisa Katharina; Harnoss, Julian Camill; Hank, Thomas; Cai, Jun; Ulrich, Alexis; Schneider, Martin

2015-01-01

Oxygen is essential for metazoans to generate energy. Upon oxygen deprivation adaptive and protective pathways are induced, mediated by hypoxia-inducible factors (HIFs) and prolyl hydroxylase domain-containing enzymes (PHDs). Both play a pivotal role in various conditions associated with prolonged ischemia and inflammation, and are promising targets for therapeutic intervention. This review focuses on aspects of therapeutic PHD modulation in surgically relevant disease conditions such as hepatic and intestinal disorders, wound healing, innate immune responses, and tumorigenesis, and discusses the therapeutic potential and challenges of PHD inhibition in surgical patients. PMID:27774478

Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

PubMed

Fazio, M J; Da Silva, A C; Rosiere, T K; Bouck, G B

1995-01-01

Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

A split motor domain in a cytoplasmic dynein

PubMed Central

Straube, Anne; Enard, Wolfgang; Berner, Alexandra; Wedlich-Söldner, Roland; Kahmann, Regine; Steinberg, Gero

2001-01-01

The heavy chain of dynein forms a globular motor domain that tightly couples the ATP-cleavage region and the microtubule-binding site to transform chemical energy into motion along the cytoskeleton. Here we show that, in the fungus Ustilago maydis, two genes, dyn1 and dyn2, encode the dynein heavy chain. The putative ATPase region is provided by dyn1, while dyn2 includes the predicted microtubule-binding site. Both genes are located on different chromosomes, are transcribed into independent mRNAs and are translated into separate polypeptides. Both Dyn1 and Dyn2 co-immunoprecipitated and co-localized within growing cells, and Dyn1–Dyn2 fusion proteins partially rescued mutant phenotypes, suggesting that both polypeptides interact to form a complex. In cell extracts the Dyn1–Dyn2 complex dissociated, and microtubule affinity purification indicated that Dyn1 or associated polypeptides bind microtubules independently of Dyn2. Both Dyn1 and Dyn2 were essential for cell survival, and conditional mutants revealed a common role in nuclear migration, cell morphogenesis and microtubule organization, indicating that the Dyn1–Dyn2 complex serves multiple cellular functions. PMID:11566874

The Y-located gonadoblastoma gene TSPY amplifies its own expression through a positive feedback loop in prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kido, Tatsuo; Lau, Yun-Fai Chris, E-mail: Chris.Lau@UCSF.edu

2014-03-28

Highlights: • Y-encoded proto-oncoprotein TSPY amplifies its expression level via a positive feedback loop. • TSPY binds to the chromatin/DNA at exon 1 of TSPY gene. • TSPY enhances the gene expression in a TSPY exon 1 sequence dependent manner. • The conserved SET/NAP-domain is essential for TSPY transactivation. • Insights on probable mechanisms on TSPY exacerbation on cancer development in men. - Abstract: The testis-specific protein Y-encoded (TSPY) is a repetitive gene located on the gonadoblastoma region of the Y chromosome, and has been considered to be the putative gene for this oncogenic locus on the male-only chromosome. Itmore » is expressed in spermatogonial cells and spermatocytes in normal human testis, but abundantly in gonadoblastoma, testicular germ cell tumors and a variety of somatic cancers, including melanoma, hepatocellular carcinoma and prostate cancer. Various studies suggest that TSPY accelerates cell proliferation and growth, and promotes tumorigenesis. In this report, we show that TSPY could bind directly to the chromatin/DNA at exon 1 of its own gene, and greatly enhance the transcriptional activities of the endogenous gene in the LNCaP prostate cancer cells. Domain mapping analyses of TSPY have localized the critical and sufficient domain to the SET/NAP-domain. These results suggest that TSPY could efficiently amplify its expression and oncogenic functions through a positive feedback loop, and contribute to the overall tumorigenic processes when it is expressed in various human cancers.« less

Genome-wide analysis of the Solanum tuberosum (potato) trehalose-6-phosphate synthase (TPS) gene family: evolution and differential expression during development and stress.

PubMed

Xu, Yingchun; Wang, Yanjie; Mattson, Neil; Yang, Liu; Jin, Qijiang

2017-12-01

Trehalose-6-phosphate synthase (TPS) serves important functions in plant desiccation tolerance and response to environmental stimuli. At present, a comprehensive analysis, i.e. functional classification, molecular evolution, and expression patterns of this gene family are still lacking in Solanum tuberosum (potato). In this study, a comprehensive analysis of the TPS gene family was conducted in potato. A total of eight putative potato TPS genes (StTPSs) were identified by searching the latest potato genome sequence. The amino acid identity among eight StTPSs varied from 59.91 to 89.54%. Analysis of d N /d S ratios suggested that regions in the TPP (trehalose-6-phosphate phosphatase) domains evolved faster than the TPS domains. Although the sequence of the eight StTPSs showed high similarity (2571-2796 bp), their gene length is highly differentiated (3189-8406 bp). Many of the regulatory elements possibly related to phytohormones, abiotic stress and development were identified in different TPS genes. Based on the phylogenetic tree constructed using TPS genes of potato, and four other Solanaceae plants, TPS genes could be categorized into 6 distinct groups. Analysis revealed that purifying selection most likely played a major role during the evolution of this family. Amino acid changes detected in specific branches of the phylogenetic tree suggests relaxed constraints might have contributed to functional divergence among groups. Moreover, StTPSs were found to exhibit tissue and treatment specific expression patterns upon analysis of transcriptome data, and performing qRT-PCR. This study provides a reference for genome-wide identification of the potato TPS gene family and sets a framework for further functional studies of this important gene family in development and stress response.

The low density lipoprotein receptor-related protein 1B retains beta-amyloid precursor protein at the cell surface and reduces amyloid-beta peptide production.

PubMed

Cam, Judy A; Zerbinatti, Celina V; Knisely, Jane M; Hecimovic, Silva; Li, Yonghe; Bu, Guojun

2004-07-09

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family that shares high homology with the LDL receptor-related protein (LRP). LRP1B was originally described as a putative tumor suppressor in lung cancer cells; however, its expression profile in several regions of adult human brain suggests it may have additional functions in the central nervous system. Since LRP1B has overlapping ligand binding properties with LRP, we investigated whether LRP1B, like LRP, could interact with the beta-amyloid precursor protein (APP) and modulate its processing to amyloid-beta peptides (Abetas). Using an LRP1B minireceptor (mLRP1B4) generated to study the trafficking of LRP1B, we found that mLRP1B4 and APP form an immunoprecipitable complex. Furthermore mLRP1B4 bound and facilitated the degradation of a soluble isoform of APP containing a Kunitz proteinase inhibitor domain but not soluble APP lacking a Kunitz proteinase inhibitor domain. A functional consequence of mLRP1B4 expression was a significant accumulation of APP at the cell surface, which is likely related to the slow endocytosis rate of LRP1B. More importantly, mLRP1B4-expressing cells that accumulated cell surface APP produced less Abeta and secreted more soluble APP. These findings reveal that LRP1B is a novel binding partner of APP that functions to decrease APP processing to Abeta. Consequently LRP1B expression could function to protect against the pathogenesis of Alzheimer's disease.

EphB4 localises to the nucleus of prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mertens-Walker, Inga, E-mail: inga.mertenswalker@qut.edu.au; Australian Prostate Cancer Research Centre—Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD; Lisle, Jessica E.

2015-04-10

The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of bothmore » NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor. - Highlights: • The EphB4 protein can be found in the nucleus of prostate cancer cell lines. • EphB4 contains two functional nuclear localisation signals. • Chromatin immunoprecipitation has identified potential genome sequences to which EphB4 binds. • Lef1 is a confirmed target for EphB4-mediated gene regulation.« less

The single transmembrane segment drives self-assembly of OutC and the formation of a functional type II secretion system in Erwinia chrysanthemi.

PubMed

Login, Frédéric H; Shevchik, Vladimir E

2006-11-03

Many pathogenic Gram-negative bacteria secrete toxins and lytic enzymes via a multiprotein complex called the type II secretion system. This system, named Out in Erwinia chrysanthemi, consists of 14 proteins integrated or associated with the two bacterial membranes. OutC, a key player in this process, is probably implicated in the recognition of secreted proteins and signal transduction. OutC possesses a short cytoplasmic sequence, a single transmembrane segment (TMS), and a large periplasmic region carrying a putative PDZ domain. A hydrodynamic study revealed that OutC forms stable dimers of an elongated shape, whereas the PDZ domain adopts a globular shape. Bacterial two-hybrid, cross-linking, and pulldown assays revealed that the self-association of OutC is driven by the TMS, whereas the periplasmic region is dispensable for self-association. Site-directed mutagenesis of the TMS revealed that cooperative interactions between three polar residues located at the same helical face provide adequate stability for OutC self-assembly. An interhelical H-bonding mediated by Gln(29) appears to be the main driving force, and two Arg residues located at the TMS boundaries are essential for the stabilization of OutC oligomers. Stepwise mutagenesis of these residues gradually diminished OutC functionality and self-association ability. The triple OutC mutant R15V/Q29L/R36A became monomeric and nonfunctional. Self-association and functionality of the triple mutant were partially restored by the introduction of a polar residue at an alternative position in the interhelical interface. Thus, the OutC TMS is more than just a membrane anchor; it drives the protein self-association that is essential for formation of a functional secretion system.