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Sample records for dead-box protein ded1

  1. The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus

    PubMed Central

    Senissar, Meriem; Saux, Agnès Le; Belgareh-Touzé, Naïma; Adam, Céline; Banroques, Josette; Tanner, N. Kyle

    2014-01-01

    The DEAD-box helicase Ded1 is an essential yeast protein that is closely related to mammalian DDX3 and to other DEAD-box proteins involved in developmental and cell cycle regulation. Ded1 is considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5′ 7-methylguanosine cap to the AUG start codon. We used IgG pull-down experiments, mass spectrometry analyses, genetic experiments, sucrose gradients, in situ localizations and enzymatic assays to show that Ded1 is a cap-associated protein that actively shuttles between the cytoplasm and the nucleus. NanoLC-MS/MS analyses of purified complexes show that Ded1 is present in both nuclear and cytoplasmic mRNPs. Ded1 physically interacts with purified components of the nuclear CBC and the cytoplasmic eIF4F complexes, and its enzymatic activity is stimulated by these factors. In addition, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes. We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs. PMID:25013175

  2. Coupling between the DEAD-box RNA helicases Ded1p and eIF4A

    PubMed Central

    Gao, Zhaofeng; Putnam, Andrea A; Bowers, Heath A; Guenther, Ulf-Peter; Ye, Xuan; Kindsfather, Audrey; Hilliker, Angela K; Jankowsky, Eckhard

    2016-01-01

    Eukaryotic translation initiation involves two conserved DEAD-box RNA helicases, eIF4A and Ded1p. Here we show that S. cerevisiae eIF4A and Ded1p directly interact with each other and simultaneously with the scaffolding protein eIF4G. We delineate a comprehensive thermodynamic framework for the interactions between Ded1p, eIF4A, eIF4G, RNA and ATP, which indicates that eIF4A, with and without eIF4G, acts as a modulator for activity and substrate preferences of Ded1p, which is the RNA remodeling unit in all complexes. Our results reveal and characterize an unexpected interdependence between the two RNA helicases and eIF4G, and suggest that Ded1p is an integral part of eIF4F, the complex comprising eIF4G, eIF4A, and eIF4E. DOI: http://dx.doi.org/10.7554/eLife.16408.001 PMID:27494274

  3. Duplex unwinding with DEAD-box proteins.

    PubMed

    Jankowsky, Eckhard; Putnam, Andrea

    2010-01-01

    DEAD-box proteins, which comprise the largest helicase family, are involved in virtually all aspects of RNA metabolism. DEAD-box proteins catalyze diverse ATP-driven functions including the unwinding of RNA secondary structures. In contrast to many well-studied DNA and viral RNA helicases, DEAD-box proteins do not rely on translocation on one of the nucleic acid strands for duplex unwinding, but directly load onto helical regions and then locally pry the strands apart in an ATP-dependent fashion. In this chapter, we outline substrate design and unwinding protocols for DEAD-box proteins and focus on the quantitative evaluation of their unwinding activity.

  4. Genome-wide analysis of translational efficiency reveals distinct but overlapping functions of yeast DEAD-box RNA helicases Ded1 and eIF4A

    PubMed Central

    Sen, Neelam Dabas; Zhou, Fujun; Ingolia, Nicholas T.; Hinnebusch, Alan G.

    2015-01-01

    DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5′ end or subsequent scanning of the 5′ UTR, but whether they perform unique or overlapping functions in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in budding yeast Saccharomyces cerevisiae by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A variant encoded by tif1-A79V (in a strain lacking the ortholog TIF2) yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more pervasive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5′ UTR length and propensity for secondary structure, implicating Ded1 in scanning through structured 5′ UTRs. Reporter assays confirmed that cap-distal stem–loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs shows a heightened requirement for eIF4A, dependence on eIF4A is correlated with requirements for Ded1 and 5′ UTR features characteristic of Ded1-dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5′ UTRs, and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs. PMID:26122911

  5. DEAD-Box Helicase Proteins Disrupt RNA Tertiary Structure Through Helix Capture

    PubMed Central

    Pan, Cynthia; Potratz, Jeffrey P.; Cannon, Brian; Simpson, Zachary B.; Ziehr, Jessica L.; Tijerina, Pilar; Russell, Rick

    2014-01-01

    DEAD-box helicase proteins accelerate folding and rearrangements of highly structured RNAs and RNA–protein complexes (RNPs) in many essential cellular processes. Although DEAD-box proteins have been shown to use ATP to unwind short RNA helices, it is not known how they disrupt RNA tertiary structure. Here, we use single molecule fluorescence to show that the DEAD-box protein CYT-19 disrupts tertiary structure in a group I intron using a helix capture mechanism. CYT-19 binds to a helix within the structured RNA only after the helix spontaneously loses its tertiary contacts, and then CYT-19 uses ATP to unwind the helix, liberating the product strands. Ded1, a multifunctional yeast DEAD-box protein, gives analogous results with small but reproducible differences that may reflect its in vivo roles. The requirement for spontaneous dynamics likely targets DEAD-box proteins toward less stable RNA structures, which are likely to experience greater dynamic fluctuations, and provides a satisfying explanation for previous correlations between RNA stability and CYT-19 unfolding efficiency. Biologically, the ability to sense RNA stability probably biases DEAD-box proteins to act preferentially on less stable misfolded structures and thereby to promote native folding while minimizing spurious interactions with stable, natively folded RNAs. In addition, this straightforward mechanism for RNA remodeling does not require any specific structural environment of the helicase core and is likely to be relevant for DEAD-box proteins that promote RNA rearrangements of RNP complexes including the spliceosome and ribosome. PMID:25350280

  6. DEAD-box proteins as RNA helicases and chaperones

    PubMed Central

    Jarmoskaite, Inga; Russell, Rick

    2010-01-01

    DEAD-box proteins are ubiquitous in RNA-mediated processes and function by coupling cycles of ATP binding and hydrolysis to changes in affinity for single-stranded RNA. Many DEAD-box proteins use this basic mechanism as the foundation for a version of RNA helicase activity, efficiently separating the strands of short RNA duplexes in a process that involves little or no translocation. This activity, coupled with mechanisms to direct different DEAD-box proteins to their physiological substrates, allows them to promote RNA folding steps and rearrangements and to accelerate remodeling of RNA-protein complexes. This review will describe the properties of DEAD-box proteins as RNA helicases and the current understanding of how the energy from ATPase activity is used to drive the separation of RNA duplex strands. It will then describe how the basic biochemical properties allow some DEAD-box proteins to function as chaperones by promoting RNA folding reactions, with a focus on the self-splicing group I and group II intron RNAs. PMID:21297876

  7. Unwinding by local strand separation is critical for the function of DEAD-box proteins as RNA chaperones

    PubMed Central

    Campo, Mark Del; Mohr, Sabine; Jiang, Yue; Jia, Huijue; Jankowsky, Eckhard

    2009-01-01

    The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are broadly acting RNA chaperones that function in mitochondria to stimulate group I and group II intron splicing and activate mRNA translation. Previous studies showed that the S. cerevisiae cytosolic/nuclear DEAD-box protein Ded1p could stimulate group II intron splicing in vitro. Here, we show that Ded1p complements the mitochondrial translation and group I and II intron splicing defects in mss116Δ strains, stimulates the in vitro splicing of group I as well as group II introns, and functions indistinguishably from CYT-19 to resolve different non-native secondary and/or tertiary structures in the Tetrahymena thermophila LSU-ΔP5abc group I intron. The Escherichia coli DEAD-box protein SrmB also stimulates group I and II intron splicing in vitro, while the E. coli DEAD-box protein DbpA and vaccinia virus DExH-box protein NPH-II gave little if any group I or II intron splicing stimulation in vitro or in vivo. The four DEAD-box proteins that stimulate group I and II intron splicing unwind RNA duplexes by local strand separation and have little or no specificity, as judged by RNA-binding assays and stimulation of their ATPase activity by diverse RNAs. By contrast, DbpA binds group I and II intron RNAs non-specifically, but its ATPase activity is activated specifically by a helical segment of E. coli 23S rRNA, and NPH-II unwinds RNAs by directional translocation. The ability of DEAD-box proteins to stimulate group I and II intron splicing correlates primarily with their RNA-unwinding activity, which for the protein preparations used here was greatest for Mss116p, followed by Ded1p, CYT-19, and SrmB. Further, this correlation holds for all group I and II intron RNAs tested, implying a fundamentally similar mechanism for both types of introns. Our results support the hypothesis that DEAD-box proteins have an inherent ability to function as RNA chaperones by virtue of their

  8. Dead-box proteins: a family affair—active and passive players in RNP-remodeling

    PubMed Central

    Linder, Patrick

    2006-01-01

    DEAD-box proteins are characterized by nine conserved motifs. According to these criteria, several hundreds of these proteins can be identified in databases. Many different DEAD-box proteins can be found in eukaryotes, whereas prokaryotes have small numbers of different DEAD-box proteins. DEAD-box proteins play important roles in RNA metabolism, and they are very specific and cannot mutually be replaced. In vitro, many DEAD-box proteins have been shown to have RNA-dependent ATPase and ATP-dependent RNA helicase activities. From the genetic and biochemical data obtained mainly in yeast, it has become clear that these proteins play important roles in remodeling RNP complexes in a temporally controlled fashion. Here, I shall give a general overview of the DEAD-box protein family. PMID:16936318

  9. Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3.

    PubMed

    Floor, Stephen N; Condon, Kendall J; Sharma, Deepak; Jankowsky, Eckhard; Doudna, Jennifer A

    2016-01-29

    DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins.

  10. Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3*

    PubMed Central

    Floor, Stephen N.; Condon, Kendall J.; Sharma, Deepak; Jankowsky, Eckhard; Doudna, Jennifer A.

    2016-01-01

    DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins. PMID:26598523

  11. Toward a molecular understanding of RNA remodeling by DEAD-box proteins

    PubMed Central

    Russell, Rick; Jarmoskaite, Inga; Lambowitz, Alan M.

    2013-01-01

    DEAD-box proteins are superfamily 2 helicases that function in all aspects of RNA metabolism. They employ ATP binding and hydrolysis to generate tight, yet regulated RNA binding, which is used to unwind short RNA helices non-processively and promote structural transitions of RNA and RNA-protein substrates. In the last few years, substantial progress has been made toward a detailed, quantitative understanding of the structural and biochemical properties of DEAD-box proteins. Concurrently, progress has been made toward a physical understanding of the RNA rearrangements and folding steps that are accelerated by DEAD-box proteins in model systems. Here, we review the recent progress on both of these fronts, focusing on the mitochondrial DEAD-box proteins Mss116 and CYT-19 and their mechanisms in promoting the splicing of group I and group II introns. PMID:22995827

  12. Analog sensitive chemical inhibition of the DEAD-box protein DDX3.

    PubMed

    Floor, Stephen N; Barkovich, Krister J; Condon, Kendall J; Shokat, Kevan M; Doudna, Jennifer A

    2016-03-01

    Proper maintenance of RNA structure and dynamics is essential to maintain cellular health. Multiple families of RNA chaperones exist in cells to modulate RNA structure, RNA-protein complexes, and RNA granules. The largest of these families is the DEAD-box proteins, named after their catalytic Asp-Glu-Ala-Asp motif. The human DEAD-box protein DDX3 is implicated in diverse biological processes including translation initiation and is mutated in numerous cancers. Like many DEAD-box proteins, DDX3 is essential to cellular health and exhibits dosage sensitivity, such that both decreases and increases in protein levels can be lethal. Therefore, chemical inhibition would be an ideal tool to probe the function of DDX3. However, most DEAD-box protein active sites are extremely similar, complicating the design of specific inhibitors. Here, we show that a chemical genetic approach best characterized in protein kinases, known as analog-sensitive chemical inhibition, is viable for DDX3 and possibly other DEAD-box proteins. We present an expanded active-site mutant that is tolerated in vitro and in vivo, and is sensitive to chemical inhibition by a novel bulky inhibitor. Our results highlight a course towards analog sensitive chemical inhibition of DDX3 and potentially the entire DEAD-box protein family.

  13. DEAD-box Helicases as Integrators of RNA, Nucleotide and Protein Binding

    PubMed Central

    Putnam, Andrea A.

    2013-01-01

    DEAD-box helicases perform diverse cellular functions in virtually all steps of RNA metabolism from Bacteria to Humans. Although DEAD-box helicases share a highly conserved core domain, the enzymes catalyze a wide range of biochemical reactions. In addition to the well established RNA unwinding and corresponding ATPase activities, DEAD-box helicases promote duplex formation and displace proteins from RNA. They can also function as assembly platforms for larger ribonucleoprotein complexes, and as metabolite sensors. This review aims to provide a perspective on the diverse biochemical features of DEAD-box helicases and connections to structural information. We discuss these data in the context of a model that views the enzymes as integrators of RNA, nucleotide, and protein binding. PMID:23416748

  14. The mechanism of ATP-dependent RNA unwinding by DEAD box proteins.

    PubMed

    Hilbert, Manuel; Karow, Anne R; Klostermeier, Dagmar

    2009-12-01

    DEAD box proteins catalyze the ATP-dependent unwinding of double-stranded RNA (dsRNA). In addition, they facilitate protein displacement and remodeling of RNA or RNA/protein complexes. Their hallmark feature is local destabilization of RNA duplexes. Here, we summarize current data on the DEAD box protein mechanism and present a model for RNA unwinding that integrates recent data on the effect of ATP analogs and mutations on DEAD box protein activity. DEAD box proteins share a conserved helicase core with two flexibly linked RecA-like domains that contain all helicase signature motifs. Variable flanking regions contribute to substrate binding and modulate activity. In the presence of ATP and RNA, the helicase core adopts a compact, closed conformation with extensive interdomain contacts and high affinity for RNA. In the closed conformation, the RecA-like domains form a catalytic site for ATP hydrolysis and a continuous RNA binding site. A kink in the backbone of the bound RNA locally destabilizes the duplex. Rearrangement of this initial complex generates a hydrolysis- and unwinding-competent state. From this complex, the first RNA strand can dissociate. After ATP hydrolysis and phosphate release, the DEAD box protein returns to a low-affinity state for RNA. Dissociation of the second RNA strand and reopening of the cleft in the helicase core allow for further catalytic cycles.

  15. Amplification of a DEAD box protein gene in retinoblastoma cell lines.

    PubMed Central

    Godbout, R; Squire, J

    1993-01-01

    DEAD box proteins, characterized by the conserved motif Asp-Glu-Ala-Asp, are putative RNA helicases implicated in a number of cellular processes involving alteration of RNA secondary structure such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. Based on their distribution patterns, some members of this family are believed to be involved in embryogenesis, spermatogenesis, and cellular growth and division. Here, we report that the mRNA encoding a DEAD box protein, designated HuDBP-RB, is present at elevated levels in two of six retinoblastoma (RB) cell lines tested and is preferentially expressed in fetal tissues of neuroectodermal origin. It is not possible to classify HuDBP-RB as a member of any of the DEAD box protein subgroups identified to date since the regions of amino acid similarity between HuDBP-RB and other DEAD box proteins are restricted to the conserved motifs found in all members of this family. The HuDBP-RB gene, which has been mapped to chromosome band 2p24, is amplified in the RB cell lines that overexpress HuDBP-RB RNA. Furthermore, the MYCN gene is also present in multiple copies in these two cell lines, suggesting coamplification of the two genes. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:7689221

  16. DEAD-Box RNA Binding Protein DDX5: Not a Black-Box during Reprogramming.

    PubMed

    Nefzger, Christian M; Polo, Jose M

    2017-04-06

    The role of RNA binding proteins (RBPs) during nuclear reprogramming is poorly characterized. In this issue of Cell Stem Cell,Li et al. (2017) show that DEAD-box RBP DDX5 acts as a reprogramming roadblock and give important mechanistic insights into the establishment of pluripotency by characterizing the intricate downstream events.

  17. p72: a human nuclear DEAD box protein highly related to p68.

    PubMed Central

    Lamm, G M; Nicol, S M; Fuller-Pace, F V; Lamond, A I

    1996-01-01

    P72, a novel human member of the DEAD box family of putative RNA-dependent ATPases and ATP-dependent RNA helicases was isolated from a HeLa cDNA library. The predicted amino acid sequence of p72 is highly homologous to that of the prototypic DEAD box protein p68. In addition to the conserved core domains characteristic of DEAD box proteins, p72 contains several N-terminal RGG RNA-binding domains and a serine/glycine rich C-terminus likely involved in mediating protein-protein interactions. A p72-specific probe detects two mRNAs of approximately 5300 and 9300 bases which, although ubiquitously expressed, show variability in their expression levels in different tissues. Purified recombinant p72 exhibits ATPase activity in the presence of a range of RNA moieties. Immunocytochemical studies of p68 and p72 show that these proteins localise to similar locations in the nucleus of HeLa cells, suggesting their involvement in a nuclear process. PMID:8871553

  18. Structure of the Yeast DEAD Box Protein Mss116p Reveals Two Wedges that Crimp RNA

    SciTech Connect

    Del Campo, Mark; Lambowitz, Alan M.

    2010-01-12

    The yeast DEAD box protein Mss116p is a general RNA chaperone that functions in mitochondrial group I and II intron splicing, translational activation, and RNA end processing. Here we determined high-resolution X-ray crystal structures of Mss116p complexed with an RNA oligonucleotide and ATP analogs AMP-PNP, ADP-BeF{sub 3}, or ADP-AlF{sub 4}{sup -}. The structures show the entire helicase core acting together with a functionally important C-terminal extension. In all structures, the helicase core is in a closed conformation with a wedge {alpha} helix bending RNA 3' of the central bound nucleotides, as in previous DEAD box protein structures. Notably, Mss116p's C-terminal extension also bends RNA 5' of the central nucleotides, resulting in RNA crimping. Despite reported functional differences, we observe few structural changes in ternary complexes with different ATP analogs. The structures constrain models of DEAD box protein function and reveal a strand separation mechanism in which a protein uses two wedges to act as a molecular crimper.

  19. Do DEAD-box proteins promote group II intron splicing without unwinding RNA?

    PubMed

    Del Campo, Mark; Tijerina, Pilar; Bhaskaran, Hari; Mohr, Sabine; Yang, Quansheng; Jankowsky, Eckhard; Russell, Rick; Lambowitz, Alan M

    2007-10-12

    The DEAD-box protein Mss116p promotes group II intron splicing in vivo and in vitro. Here we explore two hypotheses for how Mss116p promotes group II intron splicing: by using its RNA unwinding activity to act as an RNA chaperone or by stabilizing RNA folding intermediates. We show that an Mss116p mutant in helicase motif III (SAT/AAA), which was reported to stimulate splicing without unwinding RNA, retains ATP-dependent unwinding activity and promotes unfolding of a structured RNA. Its unwinding activity increases sharply with decreasing duplex length and correlates with group II intron splicing activity in quantitative assays. Additionally, we show that Mss116p can promote ATP-independent RNA unwinding, presumably via single-strand capture, also potentially contributing to DEAD-box protein RNA chaperone activity. Our findings favor the hypothesis that DEAD-box proteins function in group II intron splicing as in other processes by using their unwinding activity to act as RNA chaperones.

  20. Gene duplication in trypanosomatids - two DED1 paralogs are functionally redundant and differentially expressed during the life cycle.

    PubMed

    Zinoviev, Alexandra; Akum, Yael; Yahav, Tal; Shapira, Michal

    2012-10-01

    DED1/VAS belong to the DEAD-box family of RNA helicases that are associated with translation initiation in higher eukaryotes. Here we report on two DED1/VAS homologs that were identified in the genome of Leishmania. The two paralogs include all the domains that are typical of DEAD-box proteins and a phylogenetic analysis suggests that their duplication predates the branching of DED1 and VAS, which took place along with the appearance of early metazoans. The two Leishmania DED1 paralogs complement a yeast strain that fails to express the endogenous DED1, suggesting that they are responsible for a similar function. This is also supported by RNAi-mediated silencing experiments performed in Trypanosoma brucei. The two proteins are functionally redundant, since defects in protein synthesis and cell growth arrest were observed only when both paralogs were eliminated. A partial stage-specific specialization is observed, as LeishDED1-2 is more abundant in promastigotes, whereas expression of LeishDED1-1 increases in amastigotes. Duplication of an essential gene usually offers a safety net against mutations but in this case it also generated two proteins with stage specific expression. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. The DEAD box protein Mrh4 functions in the assembly of the mitochondrial large ribosomal subunit.

    PubMed

    De Silva, Dasmanthie; Fontanesi, Flavia; Barrientos, Antoni

    2013-11-05

    Proteins in a cell are universally synthesized by ribosomes. Mitochondria contain their own ribosomes, which specialize in the synthesis of a handful of proteins required for oxidative phosphorylation. The pathway of mitoribosomal biogenesis and factors involved are poorly characterized. An example is the DEAD box proteins, widely known to participate in the biogenesis of bacterial and cytoplasmic eukaryotic ribosomes as either RNA helicases or RNA chaperones, whose mitochondrial counterparts remain completely unknown. Here, we have identified the Saccharomyces cerevisiae mitochondrial DEAD box protein Mrh4 as essential for large mitoribosome subunit biogenesis. Mrh4 interacts with the 21S rRNA, mitoribosome subassemblies, and fully assembled mitoribosomes. In the absence of Mrh4, the 21S rRNA is matured and forms part of a large on-pathway assembly intermediate missing proteins Mrpl16 and Mrpl39. We conclude that Mrh4 plays an essential role during the late stages of mitoribosome assembly by promoting remodeling of the 21S rRNA-protein interactions. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Developmental and cell biological functions of the Drosophila DEAD-box protein abstrakt.

    PubMed

    Irion, U; Leptin, M

    1999-12-02

    DEAD-box proteins are a large family of proteins found in bacteria, plants and animals, but only few have been analysed functionally. They are involved in the regulation of various aspects of RNA processing and metabolism, including splicing, transport and translation. The study of their function in multicellular organisms has been restricted to a few special cases, such as the Vasa protein in the fruit fly Drosophila. We show that abstrakt, a gene originally identified genetically by its effect on axon outgrowth and fasciculation of the Bolwig nerve, encodes a new Drosophila DEAD-box protein of which the closest homologue is a human gene of unknown function. Using temperature-sensitive alleles to assay its function, we found that abstrakt is essential for survival at all stages throughout the life cycle of the fly. Mutants show specific defects in many developmental processes, including cell-shape changes, localisation of RNA and apoptosis. Abstrakt is not globally required for RNA splicing, transport, subcellular localisation or translation. Nevertheless, there is a widespread requirement for Abstrakt during post-transcriptional gene expression. Abstrakt must affect processing of specific subsets of RNAs, suggesting that differential post-translational control during development is more common than previously suspected.

  3. DbpA: a DEAD box protein specifically activated by 23s rRNA.

    PubMed Central

    Fuller-Pace, F V; Nicol, S M; Reid, A D; Lane, D P

    1993-01-01

    The Escherichia coli protein DbpA is a member of the 'DEAD box' family of putative RNA-dependent ATPases and RNA helicases, so called because they share the highly conserved motif Asp-Glu-Ala-Asp, together with several other conserved elements. We have investigated DbpA expression under conditions where an endogenous promoter is used. In this context, translation initiation does not occur at the previously identified AUG, but at an upstream, in-frame GUG. Mutation of the GUG initiation codon to AUG virtually abolishes DbpA expression, suggesting an unusual translation initiation mechanism. Using an inducible overexpression plasmid, we have purified milligram quantities of DbpA to homogeneity and shown that the purified protein hydrolyses ATP in an RNA-dependent manner. This ATPase activity is interesting in that, unlike that of other DEAD box proteins investigated to date, it absolutely requires a specific bacterial RNA, which we have identified as 23S rRNA. This observation is particularly significant since DbpA will bind other RNAs and DNA, but will only hydrolyse ATP in the presence of 23S rRNA. Images PMID:8253085

  4. Structure-guided mutational analysis of a yeast DEAD-box protein involved in mitochondrial RNA splicing.

    PubMed

    Bifano, Abby L; Turk, Edward M; Caprara, Mark G

    2010-05-07

    DEAD-box proteins are RNA-dependent ATPase enzymes that have been implicated in nearly all aspects of RNA metabolism. Since many of these enzymes have been shown to possess common biochemical properties in vitro, including the ability to bind and hydrolyze ATP, to bind nucleic acid, and to promote helix unwinding, DEAD-box proteins are generally thought to modulate RNA structure in vivo. However, the extent to which these enzymatic properties are important for the in vivo functions of DEAD-box proteins remains unclear. To evaluate how these properties influence DEAD-box protein native function, we probed the importance of several highly conserved residues in the yeast DEAD-box protein Mss116p, which is required for the splicing of all mitochondrial catalytic introns in Saccharomyces cerevisiae. Using an MSS116 deletion strain, we have expressed plasmid-borne variants of MSS116 containing substitutions in residues predicted to be important for extensive networks of interactions required for ATP hydrolysis and helix unwinding. We have analyzed the importance of these residues to the splicing functions of Mss116p in vivo and compared these results with the biochemical properties of recombinant proteins determined here and in previously published work. We observed that the efficiency by which an Mss116p variant catalyzes ATP hydrolysis correlates with facilitating mitochondrial splicing, while efficient helix unwinding appears to be insufficient for splicing. In addition, we show that each splicing-defective variant affects the splicing of structurally diverse introns to the same degree. Together, these observations suggest that the efficiency by which Mss116p catalyzes the hydrolysis of ATP is critical for all of its splicing functions in vivo. Given that ATP hydrolysis stimulates the recycling of DEAD-box proteins, these observations support a model in which enzyme turnover is a crucial factor in Mss116p splicing function. These results are discussed in the context

  5. Structure-Guided Mutational Analysis of a Yeast DEAD-box Protein Involved in Mitochondrial RNA Splicing

    PubMed Central

    Bifano, Abby L.; Turk, Edward M.; Caprara, Mark G.

    2010-01-01

    Summary DEAD-box proteins are RNA-dependent ATPase enzymes that have been implicated in nearly all aspects of RNA metabolism. Since many of these enzymes have been shown to possess common biochemical properties in vitro, including the ability to bind and hydrolyze ATP, bind nucleic acid, and promote helix unwinding, DEAD-box proteins are generally thought to modulate RNA structure in vivo. However, the extent to which these enzymatic properties are important for the in vivo functions of DEAD-box proteins remains unclear. To evaluate how these properties influence DEAD-box protein native function, we probe the importance of several highly conserved residues in the yeast DEAD-box protein, Mss116p, which is required for the splicing of all mitochondrial catalytic introns in Saccharomyces cerevisiae. Using a MSS116 deletion strain, we have expressed plasmid-borne variants of MSS116 containing substitutions in residues predicted to be important for extensive networks of interactions required for ATP hydrolysis and helix unwinding. We have analyzed the importance of these residues to the splicing functions of Mss116p in vivo and compared these results with the biochemical properties of recombinant proteins determined here and in previously published work. We observe that the efficiency by which an Mss116p variant catalyzes ATP hydrolysis correlates with facilitating mitochondrial splicing, while efficient helix unwinding appears to be insufficient for splicing. In addition, we show that each splicing-defective variant affects the splicing of structurally diverse introns to the same degree. Together, these observations suggest that the efficiency by which Mss116p catalyzes the hydrolysis of ATP is critical for all of its splicing functions in vivo. Given that ATP hydrolysis stimulates the recycling of DEAD-box proteins, these observations support a model in which enzyme turnover is a crucial factor in Mss116p splicing function. These results are discussed in the context

  6. A DEAD-box-family protein is required for nucleocytoplasmic transport of yeast mRNA.

    PubMed Central

    Liang, S; Hitomi, M; Hu, Y H; Liu, Y; Tartakoff, A M

    1996-01-01

    An enormous variety of primary and secondary mRNA structures are compatible with export from the nucleus to the cytoplasm. Therefore, there seems to be a mechanism for RNA export which is independent of sequence recognition. There nevertheless is likely to be some relatively uniform mechanism which allows transcripts to be packaged as ribonucleoprotein particles, to gain access to the periphery of the nucleus and ultimately to translocate across nuclear pores. To study these events, we and others have generated temperature-sensitive recessive mRNA transport (mtr) mutants of Saccharomyces cerevisiae which accumulate poly(A)+ RNA in the nucleus at 37 degrees C. Several of the corresponding genes have been cloned. Upon depletion of one of these proteins, Mtr4p, conspicuous amounts of nuclear poly(A)+ RNA accumulate in association with the nucleolus. Corresponding dense material is also seen by electron microscopy. MTR4 is essential for growth and encodes a novel nuclear protein with a size of approximately 120 kDa. Mtr4p shares characteristic motifs with DEAD-box RNA helicases and associates with RNA. It therefore may well affect RNA conformation. It shows extensive homology to a human predicted gene product and the yeast antiviral protein Ski2p. Critical residues of Mtr4p, including the mtr4-1 point mutation, have been identified. Mtr4p may serve as a chaperone which translocates or normalizes the structure of mRNAs in preparation for export. PMID:8756671

  7. Characterization of a DEAD box ATPase/RNA helicase protein of Arabidopsis thaliana.

    PubMed Central

    Okanami, M; Meshi, T; Iwabuchi, M

    1998-01-01

    We have isolated cDNAs encoding a novel member of the DEAD box RNA helicase family from Arabidopsis. The protein, named AtDRH1, is composed of 619 amino acids and the central portion has high similarity with the helicase core region of a prototypic RNA helicase, the human nuclear protein p68. The N- and C-terminal regions are considerably diverged from the animal and yeast p68 homologs at the amino acid sequence level, but like the p68 subfamily members, an RGG box-like domain is present near the C-terminus. RNA blot analysis showed that the AtDRH1 transcript accumulates at a high level and almost equally in every part of the Arabidopsis plant. The purified, recombinant AtDRH1 was capable of unwinding double-stranded RNA in the presence of ATP or dATP and of hydrolyzing ATP. The ATPase activity was stimulated by some single-stranded RNAs and DNAs, including poly(A) and poly(dT), but not by poly(dA). The ability of the polynucleotides to stimulate the ATPase activity was largely consistent with their affinity for AtDRH1. These results show that AtDRH1 is a novel type of ATP/dATP-dependent RNA helicase and polynucleotide-dependent ATPase. PMID:9592148

  8. Genetic interactions of conserved regions in the DEAD-box protein Prp28p.

    PubMed Central

    Chang, T H; Latus, L J; Liu, Z; Abbott, J M

    1997-01-01

    The yeast PRP28 g ene has been implicated in nuclear precursor messenger RNA (pre-mRNA) splicing, a two-step reaction involved in a multitude of RNA structural alterations. Prp28p, the gene product of PRP28 , is a member of the evolutionarily conserved DEAD-box proteins (DBPs). Members of DBPs are involved in a variety of RNA-related biochemical processes, presumably by their putative RNA helicase activities. Prp28p has been speculated to play a role in melting the duplex between U4 and U6 small nuclear RNAs (snRNAs), leading to the formation of an active spliceosome. To study the function of Prp28p and its interactions with other components of the splicing machinery, we have isolated and characterized a large number of prp28 conditional mutants. Strikingly, many of these prp28 mutations are localized in the highly conserved motifs found in all the DBPs. Intragenic reversion analysis suggests that regions of motifs II, III and V, as well as of motifs I and IV, in Prp28p are likely to be in close proximity to each other. Our results thus provide the first hint of the local structural arrangement for Prp28p, and perhaps for other DBPs as well. PMID:9396812

  9. PRH75, a new nucleus-localized member of the DEAD-box protein family from higher plants.

    PubMed Central

    Lorković, Z J; Herrmann, R G; Oelmüller, R

    1997-01-01

    The putative RNA helicases of the DEAD-box protein family are involved in pre-mRNA splicing, rRNA maturation, ribosome assembly, and translation. Members of this protein family have been identified in organisms from Escherichia coli to humans, but except for the translation initiation factor 4A, there have been no reports on the characterization of other DEAD-box proteins from plants. Here we report on a novel member of the DEAD-box protein family, the plant RNA helicase 75 (PRH75). PRH75 is localized in the nucleus and contains two domains for RNA binding. One is located at the C terminus and is similar to RGG RNA-binding domains of nucleus-localized RNA-binding proteins. The other one is located between amino acids 308 and 622, a region containing the conserved motif VI characteristic of DEAD-box proteins and known as the RNA-binding site of eIF-4A. The N-terminal 81 amino acids are sufficient for nuclear targeting of the protein. Northern and Western blot analyses show that PRH75 is mainly expressed in young and rapidly developing tissues. The purified recombinant PRH75 has a weak ATPase activity which is barely stimulated by RNA ligands. The fractionation of spinach whole-cell extracts by glycerol gradient centrifugation and gel filtration on a Superdex 200 column shows that the protein exists in a complex of about 500 kDa. Possible biological functions of PRH75 as well as structure-function relationships in the context of its modular primary structure are discussed. PMID:9121476

  10. High-Throughput Genetic Identification of Functionally Important Regions of the Yeast DEAD-box Protein Mss116p

    PubMed Central

    Mohr, Georg; Campo, Mark Del; Turner, Kathryn G.; Gilman, Benjamin; Wolf, Rachel Z.; Lambowitz, Alan M.

    2011-01-01

    The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionally important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins, but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends upon ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the C-terminal extension results primarily from a steric block. Finally, we identified two conserved regions, one the previously noted post-II region in the helicase core and the other in the C-terminal extension, which may help displace or sequester the opposite RNA strand during RNA unwinding. PMID:21945532

  11. High-Throughput Genetic Identification of Functionally Important Regions of the Yeast DEAD-Box Protein Mss116p

    SciTech Connect

    Mohr, Georg; Del Campo, Mark; Turner, Kathryn G.; Gilman, Benjamin; Wolf, Rachel Z.; Lambowitz, Alan M.

    2012-03-15

    The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension (CTE) induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionally important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends on ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the CTE results primarily from a steric block. Finally, we identified two conserved regions - one the previously noted post II region in the helicase core and the other in the CTE - that may help displace or sequester the opposite RNA strand during RNA unwinding.

  12. The DEAD-box protein MEL-46 is required in the germ line of the nematode Caenorhabditis elegans.

    PubMed

    Minasaki, Ryuji; Puoti, Alessandro; Streit, Adrian

    2009-06-17

    In the hermaphrodite of the nematode Caenorhabditis elegans, the first germ cells differentiate as sperm. Later the germ line switches to the production of oocytes. This process requires the activity of a genetic regulatory network that includes among others the fem, fog and mog genes. The function of some of these genes is germline specific while others also act in somatic tissues. DEAD box proteins have been shown to be involved in the control of gene expression at different steps such as transcription and pre-mRNA processing. We show that the Caenorhabditis elegans gene mel-46 (maternal effect lethal) encodes a DEAD box protein that is related to the mammalian DDX20/Gemin3/DP103 genes. mel-46 is expressed throughout development and mutations in mel-46 display defects at multiple developmental stages. Here we focus on the role of mel-46 in the hermaphrodite germ line. mel-46(yt5) mutant hermaphrodites are partially penetrant sterile and fully penetrant maternal effect lethal. The germ line of mutants shows variable defects in oogenesis. Further, mel-46(yt5) suppresses the complete feminization caused by mutations in fog-2 and fem-3, two genes that are at the top and the center, respectively, of the genetic germline sex determining cascade, but not fog-1 that is at the bottom of this cascade. The C. elegans gene mel-46 encodes a DEAD box protein that is required maternally for early embryogenesis and zygotically for postembryonic development. In the germ line, it is required for proper oogenesis. Although it interacts genetically with genes of the germline sex determination machinery its primary function appears to be in oocyte differentiation rather than sex determination.

  13. Pisum sativum p68 DEAD-box protein is ATP-dependent RNA helicase and unique bipolar DNA helicase.

    PubMed

    Tuteja, Narendra; Tarique, Mohammed; Banu, Mst Sufara Akhter; Ahmad, Moaz; Tuteja, Renu

    2014-08-01

    DEAD-box helicases play essential role in DNA and RNA metabolism such as replication, repair, recombination, transcription, translation, ribosome biogenesis and splicing which regulate plant growth and development. The presence of helicases in the stress-induced ORFs identified by cDNA microarray indicates that helicases might be playing an important role in stabilizing growth in plants under stress. p68 DEAD-box helicase has been identified and characterized from animal systems but the properties and functions of plant p68 are poorly understood. In this study, the identification, purification and characterization of recombinant p68 from Pisum sativum (Psp68) is presented. Psp68 possesses all the characteristic motifs like DEAD-box ATP-binding and helicase C terminal motifs and is structurally similar to human p68 homologue. Psp68 exhibits ATPase activity in the presence of both DNA and RNA and it binds to DNA as well as RNA. It contains the characteristic RNA helicase activity. Interestingly Psp68 also shows the unique DNA helicase activity, which is bipolar in nature (unwinds DNA in both the 5'-3' and 3'-5' directions). The Km values of Psp68 for ATPase are 0.5126 and 0.9142 mM in the presence of DNA and RNA, respectively. The Km values of Psp68 are 1.6129 and 1.14 nM for DNA helicase and RNA helicase, respectively. The unique properties of Psp68 suggest that it could be a multifunctional protein involved in different aspect of DNA and RNA metabolism. This discovery should make an important contribution to better understanding of nucleic acids metabolism plants.

  14. Yeast DEAD Box Protein Mss116p Is a Transcription Elongation Factor That Modulates the Activity of Mitochondrial RNA Polymerase

    PubMed Central

    Wojtas, Ireneusz D.; Tessitore, Kassandra; Henderson, Simmone; McAllister, William T.

    2014-01-01

    DEAD box proteins have been widely implicated in regulation of gene expression. Here, we show that the yeast Saccharomyces cerevisiae DEAD box protein Mss116p, previously known as a mitochondrial splicing factor, also acts as a transcription factor that modulates the activity of the single-subunit mitochondrial RNA polymerase encoded by RPO41. Binding of Mss116p stabilizes paused mitochondrial RNA polymerase elongation complexes in vitro and favors the posttranslocated state of the enzyme, resulting in a lower concentration of nucleotide substrate required to escape the pause; this mechanism of action is similar to that of elongation factors that enhance the processivity of multisubunit RNA polymerases. In a yeast strain in which the RNA splicing-related functions of Mss116p are dispensable, overexpression of RPO41 or MSS116 increases cell survival from colonies that were exposed to low temperature, suggesting a role for Mss116p in enhancing the efficiency of mitochondrial transcription under stress conditions. PMID:24732805

  15. Characterization of DbpA, an Escherichia coli DEAD box protein with ATP independent RNA unwinding activity.

    PubMed Central

    Böddeker, N; Stade, K; Franceschi, F

    1997-01-01

    DbpA is a putative Escherichia coli ATP dependent RNA helicase belonging to the family of DEAD box proteins. It hydrolyzes ATP in the presence of 23S ribosomal RNA and 93 bases in the peptidyl transferase center of 23S rRNA are sufficient to trigger 100% of the ATPase activity of DbpA. In the present study we characterized the ATPase and RNA unwinding activities of DbpA in more detail. We report that-in contrast to eIF-4A, the prototype of the DEAD box protein family-the ATPase and the helicase activities of DbpA are not coupled. Moreover, the RNA unwinding activity of DbpA is not specific for 23S rRNA, since DbpA is also able to unwind 16S rRNA hybrids. Furthermore, we determined that the ATPase activity of DbpA is triggered to a significant extent not only by the 93 bases of the 23S rRNA previously reported but also by other regions of the 23S rRNA molecule. Since all these regions of 23S rRNA are either part of the 'functional core' of the 50S ribosomal subunit or involved in the 50S assembly, DbpA may play an important role in the ribosomal assembly process. PMID:9016593

  16. A novel protein toxin from the deadly box jellyfish (Sea Wasp, Habu-kurage) Chiropsalmus quadrigatus.

    PubMed

    Nagai, Hiroshi; Takuwa-Kuroda, Kyoko; Nakao, Masahiro; Oshiro, Naomasa; Iwanaga, Setsuko; Nakajima, Terumi

    2002-01-01

    The deadly box jellyfish (Sea Wasp, Habu-kurage in Japanese) Chiropsalmus quadrigatus Haeckel (Cubozoa) is distributed widely in the tropical Pacific region. In Japan, three fatal cases due to stings from this species have been reported officially. We successfully isolated C. quadrigatus toxin-A (CqTX-A, 44 kDa), a major proteinaceous toxin, for the first time, from the nematocysts of C. quadrigatus. CqTX-A showed lethal toxicity to crayfish when administered via intraperitoneal injection (LD50 = 80 microg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED50 = 160 ng/ml). Furthermore, we sequenced the cDNA encoding CqTX-A. The deduced amino acid sequence of CqTX-A (462 amino acids) showed 25.2% and 21.6% sequence similarity to Carybdea rastoni toxins (CrTXs) and Carybdea alata toxin-A (CrTX-A), respectively, which are Cubozoan jellyfish toxins.

  17. ATPase activity of the DEAD-box protein Dhh1 controls processing body formation

    PubMed Central

    Mugler, Christopher Frederick; Hondele, Maria; Heinrich, Stephanie; Sachdev, Ruchika; Vallotton, Pascal; Koek, Adriana Y; Chan, Leon Y; Weis, Karsten

    2016-01-01

    Translational repression and mRNA degradation are critical mechanisms of posttranscriptional gene regulation that help cells respond to internal and external cues. In response to certain stress conditions, many mRNA decay factors are enriched in processing bodies (PBs), cellular structures involved in degradation and/or storage of mRNAs. Yet, how cells regulate assembly and disassembly of PBs remains poorly understood. Here, we show that in budding yeast, mutations in the DEAD-box ATPase Dhh1 that prevent ATP hydrolysis, or that affect the interaction between Dhh1 and Not1, the central scaffold of the CCR4-NOT complex and an activator of the Dhh1 ATPase, prevent PB disassembly in vivo. Intriguingly, this process can be recapitulated in vitro, since recombinant Dhh1 and RNA, in the presence of ATP, phase-separate into liquid droplets that rapidly dissolve upon addition of Not1. Our results identify the ATPase activity of Dhh1 as a critical regulator of PB formation. DOI: http://dx.doi.org/10.7554/eLife.18746.001 PMID:27692063

  18. A novel B cell epitope in cold-shock DEAD-box protein A from Mycobacterium tuberculosis.

    PubMed

    Wang, Huanan; Zhu, Ting; Yu, Shenye; Liu, Huifang; Wang, Xiumei; Chen, Liping; Si, Wei; Pang, Hai; Liu, Siguo

    2013-06-01

    In this study, a hybridoma-based technique and phage display technology were used to obtain mouse monoclonal antibodies (mAb) against cold-shock DEAD-box protein A (CsdA) from Mycobacterium tuberculosis and to determine the location of the relevant epitope. One highly specific mAb, named A3G5, was developed against the recombinant CsdA protein (rCsdA) and could detect rCsdA protein in enzyme-linked immunosorbent assays (ELISA) and Western blot assays. By screening a phage displayed library of random 12-mers (Ph.D.-12), 10 positive phage clones were randomly selected after three rounds of bio-panning and identified by ELISA. Eight of these clones were sequenced, and their amino acid sequences were deduced. One B-cell epitope (-APDPPLSRR-) in the rCsdA protein was identified with mAb A3G5. A synthetic peptide (-MAPDPPLSRR-) (Cpep) matched well with the CsdA sequence at 443-451 aa and was confirmed by affinity ELISA, competitive inhibition assays and the development of an immune response in mice. These results may be of great potential value in the further analysis of the function and structure of the CsdA protein from M. tuberculosis.

  19. Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    PubMed Central

    Iwasaki, Shintaro; Floor, Stephen N.; Ingolia, Nicholas T.

    2016-01-01

    Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumor cells and repress translation of specific mRNAs1-4. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its mRNA selectivity is proposed to reflect highly structured 5′ UTRs that depend strongly on eIF4A-mediated unwinding5. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA1,2,6. Here, we show that secondary structure in 5′ UTRs is only a minor determinant for RocA selectivity and RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA-protein interactions. PMID:27309803

  20. The "DEAD box" protein DbpA interacts specifically with the peptidyltransferase center in 23S rRNA.

    PubMed Central

    Nicol, S M; Fuller-Pace, F V

    1995-01-01

    The Escherichia coli DEAD (Asp-Glu-Ala-Asp) box protein DbpA is a putative RNA helicase and established RNA-dependent ATPase and is the only member of the DEAD box protein family for which a specific RNA substrate, bacterial 23S rRNA, has been identified. We have investigated the nature of this specificity in depth and have localized by deletion mutagenesis and PCR a single region of 93 bases (bases 2496-2588) in 23S rRNA that is both necessary and sufficient for complete activation of ATPase activity of DbpA. This target region forms part of the peptidyltransferase center and includes many bases involved in interaction with the 3' terminal adenosines of both A- and P-site tRNAs. Deletion of stem loops within the 93-base segment abolished ATPase activation. Similarly, point mutations that disrupt base pairing within stem structures ablated stimulation of ATPase activity. These data are consistent with roles for DbpA either in establishing and/or maintaining the correct three-dimensional structure of the peptidyltransferase center in 23S rRNA during ribosome assembly or in the peptidyltransferase reaction. Images Fig. 1 Fig. 2 PMID:8524828

  1. DDX3 DEAD-box RNA helicase plays a central role in mitochondrial protein quality control in Leishmania

    PubMed Central

    Padmanabhan, Prasad Kottayil; Zghidi-Abouzid, Ouafa; Samant, Mukesh; Dumas, Carole; Aguiar, Bruno Guedes; Estaquier, Jerome; Papadopoulou, Barbara

    2016-01-01

    DDX3 is a highly conserved member of ATP-dependent DEAD-box RNA helicases with multiple functions in RNA metabolism and cellular signaling. Here, we describe a novel function for DDX3 in regulating the mitochondrial stress response in the parasitic protozoan Leishmania. We show that genetic inactivation of DDX3 leads to the accumulation of mitochondrial reactive oxygen species (ROS) associated with a defect in hydrogen peroxide detoxification. Upon stress, ROS production is greatly enhanced, causing mitochondrial membrane potential loss, mitochondrial fragmentation, and cell death. Importantly, this phenotype is exacerbated upon oxidative stress in parasites forced to use the mitochondrial oxidative respiratory machinery. Furthermore, we show that in the absence of DDX3, levels of major components of the unfolded protein response as well as of polyubiquitinated proteins increase in the parasite, particularly in the mitochondrion, as an indicator of mitochondrial protein damage. Consistent with these findings, immunoprecipitation and mass-spectrometry studies revealed potential interactions of DDX3 with key components of the cellular stress response, particularly the antioxidant response, the unfolded protein response, and the AAA-ATPase p97/VCP/Cdc48, which is essential in mitochondrial protein quality control by driving proteosomal degradation of polyubiquitinated proteins. Complementation studies using DDX3 deletion mutants lacking conserved motifs within the helicase core support that binding of DDX3 to ATP is essential for DDX3's function in mitochondrial proteostasis. As a result of the inability of DDX3-depleted Leishmania to recover from ROS damage and to survive various stresses in the host macrophage, parasite intracellular development was impaired. Collectively, these observations support a central role for the Leishmania DDX3 homolog in preventing ROS-mediated damage and in maintaining mitochondrial protein quality control. PMID:27735940

  2. Formation of a Trimeric Xpo1-Ran[GTP]-Ded1 Exportin Complex Modulates ATPase and Helicase Activities of Ded1.

    PubMed

    Hauk, Glenn; Bowman, Gregory D

    2015-01-01

    The DEAD-box RNA helicase Ded1, which is essential in yeast and known as DDX3 in humans, shuttles between the nucleus and cytoplasm and takes part in several basic processes including RNA processing and translation. A key interacting partner of Ded1 is the exportin Xpo1, which together with the GTP-bound state of the small GTPase Ran, facilitates unidirectional transport of Ded1 out of the nucleus. Here we demonstrate that Xpo1 and Ran[GTP] together reduce the RNA-stimulated ATPase and helicase activities of Ded1. Binding and inhibition of Ded1 by Xpo1 depend on the affinity of the Ded1 nuclear export sequence (NES) for Xpo1 and the presence of Ran[GTP]. Association with Xpo1/Ran[GTP] reduces RNA-stimulated ATPase activity of Ded1 by increasing the apparent KM for the RNA substrate. Despite the increased KM, the Ded1:Xpo1:Ran[GTP] ternary complex retains the ability to bind single stranded RNA, suggesting that Xpo1/Ran[GTP] may modulate the substrate specificity of Ded1. These results demonstrate that, in addition to transport, exportins such as Xpo1 also have the capability to alter enzymatic activities of their cargo.

  3. Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families.

    PubMed

    de la Cruz, J; Kressler, D; Linder, P

    1999-05-01

    Members of the RNA-helicase family are defined by several evolutionary conserved motifs. They are found in all organisms - from bacteria to humans - and many viruses. The minimum number of RNA helicases present within a eukaryotic cell can be predicted from the complete sequence of the Saccharomyces cerevisiae genome. Recent progress in the functional analysis of various family members has given new insights into, and confirmed the significance of these proteins for, most cellular RNA metabolic processes.

  4. Identification of the human DEAD-box protein p68 as a substrate of Tlk1

    SciTech Connect

    Kodym, Reinhard . E-mail: reinhard.kodym@meduniwien.ac.at; Henoeckl, Christian; Fuerweger, Christoph

    2005-07-29

    The activity of the human protein kinase Tlk1 is down-regulated within minutes after exposure of cells to ionizing radiation. In order to identify signaling pathways which might be relevant in the radiation response of mammalian cells we screened nuclear proteins for substrates of Tlk1. Among several proteins one could be identified as p68 RNA helicase. Furthermore, it could be shown that Tlk1 phosphorylates immunoprecipitated p68. The phosphorylation of the C-terminal fragment of p68 by rTlk1 reduced its affinity to single stranded RNA in a gel shift assay. In addition, it could be demonstrated that increasing the Tlk1 activity in HT1080 cells by forced Tlk1 overexpression leads to an increased phosphorylation of endogenous p68, arguing that p68 might be a physiological substrate of Tlk1. These findings open the possibility that Tlk1 might participate in diverse biologic functions like cell growth and differentiation, pre-mRNA splicing, and transcriptional coactivation.

  5. Interaction of the plant glycine-rich RNA-binding protein MA16 with a novel nucleolar DEAD box RNA helicase protein from Zea mays.

    PubMed

    Gendra, Elisenda; Moreno, Alicia; Albà, M Mar; Pages, Montserrat

    2004-06-01

    The maize RNA-binding MA16 protein is a developmentally and environmentally regulated nucleolar protein that interacts with RNAs through complex association with several proteins. By using yeast two-hybrid screening, we identified a DEAD box RNA helicase protein from Zea mays that interacted with MA16, which we named Z. maysDEAD box RNA helicase 1 (ZmDRH1). The sequence of ZmDRH1 includes the eight RNA helicase motifs and two glycine-rich regions with arginine-glycine-rich (RGG) boxes at the amino (N)- and carboxy (C)-termini of the protein. Both MA16 and ZmDRH1 were located in the nucleus and nucleolus, and analysis of the sequence determinants for their cellular localization revealed that the region containing the RGG motifs in both proteins was necessary for nuclear/nucleolar localization The two domains of MA16, the RNA recognition motif (RRM) and the RGG, were tested for molecular interaction with ZmDRH1. MA16 specifically interacted with ZmDRH1 through the RRM domain. A number of plant proteins and vertebrate p68/p72 RNA helicases showed evolutionary proximity to ZmDRH1. In addition, like p68, ZmDRH1 was able to interact with fibrillarin. Our data suggest that MA16, fibrillarin, and ZmDRH1 may be part of a ribonucleoprotein complex involved in ribosomal RNA (rRNA) metabolism.

  6. Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein.

    PubMed

    Kellner, Julian N; Meinhart, Anton

    2015-09-01

    The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein-protein interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein-protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.

  7. Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein

    SciTech Connect

    Kellner, Julian N.; Meinhart, Anton

    2015-08-25

    The structure of the SPRY domain of the human RNA helicase DDX1 was determined at 2.0 Å resolution. The SPRY domain provides a putative protein–protein interaction platform within DDX1 that differs from other SPRY domains in its structure and conserved regions. The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein–protein interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein–protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.

  8. An unanticipated early function of DEAD-box ATPase Prp28 during commitment to splicing is modulated by U5 snRNP protein Prp8

    PubMed Central

    Price, Argenta M.; Görnemann, Janina; Guthrie, Christine; Brow, David A.

    2014-01-01

    The stepwise assembly of the highly dynamic spliceosome is guided by RNA-dependent ATPases of the DEAD-box family, whose regulation is poorly understood. In the canonical assembly model, the U4/U6.U5 triple snRNP binds only after joining of the U1 and, subsequently, U2 snRNPs to the intron-containing pre-mRNA. Catalytic activation requires the exchange of U6 for U1 snRNA at the 5′ splice site, which is promoted by the DEAD-box protein Prp28. Because Prp8, an integral U5 snRNP protein, is thought to be a central regulator of DEAD-box proteins, we conducted a targeted search in Prp8 for cold-insensitive suppressors of a cold-sensitive Prp28 mutant, prp28-1. We identified a cluster of suppressor mutations in an N-terminal bromodomain-like sequence of Prp8. To identify the precise defect in prp28-1 strains that is suppressed by the Prp8 alleles, we analyzed spliceosome assembly in vivo and in vitro. Surprisingly, in the prp28-1 strain, we observed a block not only to spliceosome activation but also to one of the earliest steps of assembly, formation of the ATP-independent commitment complex 2 (CC2). The Prp8 suppressor partially corrected both the early assembly and later activation defects of prp28-1, supporting a role for this U5 snRNP protein in both the ATP-independent and ATP-dependent functions of Prp28. We conclude that the U5 snRNP has a role in the earliest events of assembly, prior to its stable incorporation into the spliceosome. PMID:24231520

  9. Enhanced expression of tandem multimers of the antimicrobial peptide buforin II in Escherichia coli by the DEAD-box protein and trxB mutant.

    PubMed

    Lee, J H; Kim, M S; Cho, J H; Kim, S C

    2002-05-01

    The tandem multimeric expression of various peptides has been explored by many researchers. However, expression levels have usually not been proportional to the degree of multimerization. To increase the expression level in Escherichia coli of tandem multimers of a cationic antimicrobial peptide, buforin II, fused to an anionic peptide, we studied the effect of the DEAD-box protein and the trxB mutant on the expression of tandem multimers. An expression vector with a tac promoter was more effective in directing multimeric expression than one with a T7 promoter. The expression level of large multimers was substantially increased with the tac promoter, possibly through stabilization of long transcripts by synchronization of transcription and translation. Coexpression of the DEAD-box protein, an RNA-binding protein, with the T7 expression system increased the expression level of multimers, especially large multimers, due to protection of the long RNA transcripts. In addition, the use of the trxB mutant also enhanced the expression level of tandem multimers, which contain two cysteine residues at both ends of the monomeric unit. It seems that disulfide bonds formed in the multimers in the trxB mutant might help efficient charge neutralization for inclusion body formation of the multimers, resulting in enhancement of expression. Our results show that the expression of multimers can be improved through the stabilization of the long transcripts by the DEAD-box protein or the expression, under an oxidizing environment, of the trxB mutant in which covalent cross-links through disulfide bonds facilitate inclusion body formation of the multimeric fusion peptide.

  10. A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

    PubMed

    Garbelli, Anna; Beermann, Sandra; Di Cicco, Giulia; Dietrich, Ursula; Maga, Giovanni

    2011-05-12

    DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

  11. A chicken embryo protein related to the mammalian DEAD box protein p68 is tightly associated with the highly purified protein-RNA complex of 5-MeC-DNA glycosylase.

    PubMed

    Jost, J P; Schwarz, S; Hess, D; Angliker, H; Fuller-Pace, F V; Stahl, H; Thiry, S; Siegmann, M

    1999-08-15

    We have shown previously that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. Amino acid sequences of nine peptides derived from a highly purified 5-MeC-DNA glycosylase complex were identified by Nanoelectrospray ionisation mass spectrometry to be identical to the mammalian nuclear DEAD box protein p68 RNA helicase. Antibodies directed against human p68 helicase cross-reacted with the purified 5-MeC-DNA glycosylase complex and immunoprecipitated the glycosylase activity. A 2690 bp cDNA coding for the chicken homologue of mammalian p68 was isolated and sequenced. Its derived amino acid sequence is almost identical to the human p68 DEAD box protein up to amino acid position 473 (from a total of 595). This sequence contains all the essential conserved motifs from the DEAD box proteins which are the ATPase, RNA unwinding and RNA binding motifs. The rest of the 122 amino acids in the C-terminal region rather diverge from the human p68 RNA helicase sequence. The recombinant chicken DEAD box protein expressed in Escherichia coli cross-reacts with the same p68 antibodies as the purified chicken embryo 5-MeC-DNA glycosylase complex. The recombinant protein has an RNA-dependent ATPase and an ATP-dependent helicase activity. However, in the presence or absence of RNA the recombinant protein had no 5-MeC-DNA glycosylase activity. In situ hybridisation of 5 day-old chicken embryos with antisense probes of the chicken DEAD box protein shows a high abundance of its transcripts in differentiating embryonic tissues.

  12. DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs

    PubMed Central

    Soto-Rifo, Ricardo; Rubilar, Paulina S; Limousin, Taran; de Breyne, Sylvain; Décimo, Didier; Ohlmann, Théophile

    2012-01-01

    Here, we have characterized a step in translation initiation of viral and cellular mRNAs that contain RNA secondary structures immediately at the vicinity of their m7GTP cap. This is mediated by the DEAD-box helicase DDX3 which can directly bind to the 5′ of the target mRNA where it clamps the entry of eIF4F through an eIF4G and Poly A-binding protein cytoplasmic 1 (PABP) double interaction. This could induce limited local strand separation of the secondary structure to allow 43S pre-initiation complex attachment to the 5′ free extremity of the mRNA. We further demonstrate that the requirement for DDX3 is highly specific to some selected transcripts, cannot be replaced or substituted by eIF4A and is only needed in the very early steps of ribosome binding and prior to 43S ribosomal scanning. Altogether, these data define an unprecedented role for a DEAD-box RNA helicase in translation initiation. PMID:22872150

  13. Overexpression of DEAD box protein pMSS116 promotes ATP-dependent splicing of a yeast group II intron in vitro.

    PubMed Central

    Niemer, I; Schmelzer, C; Börner, G V

    1995-01-01

    The group II intron bI1, the first intron of the mitochondrial cytochrome b gene in yeast is self-splicing in vitro. Genetic evidence suggests that trans-acting factors are required for in vivo splicing of this intron. In accordance with these findings, we present in vitro data showing that splicing of bI1 under physiological conditions depends upon the presence of proteins of a mitochondrial lysate. ATP is an essential component is this reaction. Overexpression of the nuclear-encoded DEAD box protein pMSS-116 results in a marked increase in the ATP-dependent splicing activity of the extract, suggesting that pMSS116 may play an important role in splicing of bI1. Images PMID:7659519

  14. The DEAD-box helicase eIF4A

    PubMed Central

    Andreou, Alexandra Z.; Klostermeier, Dagmar

    2013-01-01

    DEAD-box helicases catalyze the ATP-dependent unwinding of RNA duplexes. They share a helicase core formed by two RecA-like domains that carries a set of conserved motifs contributing to ATP binding and hydrolysis, RNA binding and duplex unwinding. The translation initiation factor eIF4A is the founding member of the DEAD-box protein family, and one of the few examples of DEAD-box proteins that consist of a helicase core only. It is an RNA-stimulated ATPase and a non-processive helicase that unwinds short RNA duplexes. In the catalytic cycle, a series of conformational changes couples the nucleotide cycle to RNA unwinding. eIF4A has been considered a paradigm for DEAD-box proteins, and studies of its function have revealed the governing principles underlying the DEAD-box helicase mechanism. However, as an isolated helicase core, eIF4A is rather the exception, not the rule. Most helicase modules in other DEAD-box proteins are modified, some by insertions into the RecA-like domains, and the majority by N- and C-terminal appendages. While the basic catalytic function resides within the helicase core, its modulation by insertions, additional domains or a network of interaction partners generates the diversity of DEAD-box protein functions in the cell. This review summarizes the current knowledge on eIF4A and its regulation, and discusses to what extent eIF4A serves as a model DEAD-box protein. PMID:22995829

  15. ATP-dependent roles of the DEAD-box protein Mss116p in group II intron splicing in vitro and in vivo

    PubMed Central

    Potratz, Jeffrey P.; Campo, Mark Del; Wolf, Rachel Z.; Lambowitz, Alan M.; Russell, Rick

    2011-01-01

    The yeast DEAD-box protein Mss116p functions as a general RNA chaperone in splicing mitochondrial group I and group II introns. For most of its functions, Mss116p is thought to use ATP-dependent RNA unwinding to facilitate RNA structural transitions, but it has been suggested to assist folding of one group II intron (aI5γ) primarily by stabilizing a folding intermediate. Here we compare three aI5γ constructs: one with long exons, one with short exons, and a ribozyme construct lacking exons. The long exons result in slower splicing, suggesting that they misfold and/or stabilize non-native intronic structure. Nevertheless, Mss116p acceleration of all three constructs depends upon ATP and is inhibited by mutations that compromise RNA unwinding, suggesting similar mechanisms. Results of splicing assays and a new two-stage assay that separates ribozyme folding and catalysis indicate that maximal folding of all three constructs by Mss116p requires ATP-dependent RNA unwinding. ATP-independent activation is appreciable for only a subpopulation of the minimal ribozyme construct and not for constructs containing exons. As expected for a general RNA chaperone, Mss116p can also disrupt the native ribozyme, which can refold after Mss116p removal. Finally, using yeast strains with mtDNA containing only the single intron aI5γ, we show that Mss116p mutants promote splicing in vivo to degrees that correlate with their residual ATP-dependent RNA-unwinding activities. Together, our results indicate that, although DEAD-box proteins play multiple roles in RNA folding, the physiological function of Mss116p in aI5γ splicing includes a requirement for ATP-dependent local unfolding, allowing conversion of non-functional to functional RNA structure. PMID:21679717

  16. A Conserved Phenylalanine of Motif IV in Superfamily 2 Helicases Is Required for Cooperative, ATP-Dependent Binding of RNA Substrates in DEAD-Box Proteins▿ †

    PubMed Central

    Banroques, Josette; Cordin, Olivier; Doère, Monique; Linder, Patrick; Tanner, N. Kyle

    2008-01-01

    We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be an anchor that maintains the rigidity of the RecA-like domain. For DEAD-box proteins, the phenylalanine also aligns a highly conserved arginine of motif VI through van der Waals and cation-π interactions, thereby helping to maintain the network of interactions that exist between the different motifs involved in ATP and RNA binding. PMID:18332124

  17. An Arabidopsis ATP-Dependent, DEAD-Box RNA Helicase Loses Activity upon IsoAsp Formation but Is Restored by PROTEIN ISOASPARTYL METHYLTRANSFERASE[C][W

    PubMed Central

    Nayak, Nihar R.; Putnam, Andrea A.; Addepalli, Balasubrahmanyam; Lowenson, Jonathan D.; Chen, Tingsu; Jankowsky, Eckhard; Perry, Sharyn E.; Dinkins, Randy D.; Limbach, Patrick A.; Clarke, Steven G.; Downie, A. Bruce

    2013-01-01

    Orthodox seeds are capable of withstanding severe dehydration. However, in the dehydrated state, Asn and Asp residues in proteins can convert to succinimide residues that can further react to predominantly form isomerized isoAsp residues upon rehydration (imbibition). IsoAsp residues can impair protein function and can render seeds nonviable, but PROTEIN ISOASPARTYL METHYLTRANSFERASE (PIMT) can initiate isoAsp conversion to Asp residues. The proteins necessary for translation upon imbibition in orthodox seeds may be particularly important to maintain in an active state. One such protein is the large, multidomain protein, Arabidopsis thaliana PLANT RNA HELICASE75 (PRH75), a DEAD-box helicase known to be susceptible to isoAsp residue accumulation. However, the consequences of such isomerization on PRH75 catalysis and for the plant are unknown. Here, it is demonstrated that PRH75 is necessary for successful seed development. It acquires isoAsp rapidly during heat stress, which eliminates RNA unwinding (but not rewinding) competence. The repair by PIMT is able to restore PRH75’s complex biochemical activity provided isoAsp formation has not led to subsequent, destabilizing conformational alterations. For PRH75, an important enzymatic activity associated with translation would be eliminated unless rapidly repaired by PIMT prior to additional, deleterious conformational changes that would compromise seed vitality and germination. PMID:23903319

  18. Recruitment, Duplex Unwinding and Protein-Mediated Inhibition of the Dead-Box RNA Helicase Dbp2 at Actively Transcribed Chromatin.

    PubMed

    Ma, Wai Kit; Paudel, Bishnu P; Xing, Zheng; Sabath, Ivan G; Rueda, David; Tran, Elizabeth J

    2016-03-27

    RNA helicases play fundamental roles in modulating RNA structures and facilitating RNA-protein (RNP) complex assembly in vivo. Previously, our laboratory demonstrated that the DEAD-box RNA helicase Dbp2 in Saccharomyces cerevisiae is required to promote efficient assembly of the co-transcriptionally associated mRNA-binding proteins Yra1, Nab2, and Mex67 onto poly(A)(+)RNA. We also found that Yra1 associates directly with Dbp2 and functions as an inhibitor of Dbp2-dependent duplex unwinding, suggestive of a cycle of unwinding and inhibition by Dbp2. To test this, we undertook a series of experiments to shed light on the order of events for Dbp2 in co-transcriptional mRNP assembly. We now show that Dbp2 is recruited to chromatin via RNA and forms a large, RNA-dependent complex with Yra1 and Mex67. Moreover, single-molecule fluorescence resonance energy transfer and bulk biochemical assays show that Yra1 inhibits unwinding in a concentration-dependent manner by preventing the association of Dbp2 with single-stranded RNA. This inhibition prevents over-accumulation of Dbp2 on mRNA and stabilization of a subset of RNA polymerase II transcripts. We propose a model whereby Yra1 terminates a cycle of mRNP assembly by Dbp2.

  19. Crystallization and preliminary X-ray diffraction of the DEAD-box protein Mss116p complexed with an RNA oligonucleotide and AMP-PNP

    SciTech Connect

    Del Campo, Mark; Lambowitz, Alan M.

    2009-09-02

    The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone which functions in mitochondrial group I and group II intron splicing, translation and RNA-end processing. For crystallization trials, full-length Mss116p and a C-terminally truncated protein (Mss116p/{Delta}598-664) were overproduced in Escherichia coli and purified to homogeneity. Mss116p exhibited low solubility in standard solutions ({le}1 mg ml{sup -1}), but its solubility could be increased by adding 50 mM L-arginine plus 50 mM L-glutamate and 50% glycerol to achieve concentrations of {approx}10 mg ml{sup -1}. Initial crystals were obtained by the microbatch method in the presence of a U{sub 10} RNA oligonucleotide and the ATP analog AMP-PNP and were then improved by using seeding and sitting-drop vapor diffusion. A cryocooled crystal of Mss116p/{Delta}598-664 in complex with AMP-PNP and U{sub 10} belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 88.54, b = 126.52, c = 55.52 {angstrom}, and diffracted X-rays to beyond 1.9 {angstrom} resolution using synchrotron radiation from sector 21 at the Advanced Photon Source.

  20. From unwinding to clamping - the DEAD box RNA helicase family.

    PubMed

    Linder, Patrick; Jankowsky, Eckhard

    2011-07-22

    RNA helicases of the DEAD box family are present in all eukaryotic cells and in many bacteria and Archaea. These highly conserved enzymes are required for RNA metabolism from transcription to degradation and are therefore important players in gene expression. DEAD box proteins use ATP to unwind short duplex RNA in an unusual fashion and remodel RNA-protein complexes, but they can also function as ATP-dependent RNA clamps to provide nucleation centres that establish larger RNA-protein complexes. Structural, mechanistic and molecular biological studies have started to reveal how these conserved proteins can perform such diverse functions and how accessory proteins have a central role in their regulation.

  1. Mediation of CTCF transcriptional insulation by DEAD-box RNA-binding protein p68 and steroid receptor RNA activator SRA

    PubMed Central

    Yao, Hongjie; Brick, Kevin; Evrard, Yvonne; Xiao, Tiaojiang; Camerini-Otero, R. Daniel; Felsenfeld, Gary

    2010-01-01

    CCCTC-binding factor (CTCF) is a DNA-binding protein that plays important roles in chromatin organization, although the mechanism by which CTCF carries out these functions is not fully understood. Recent studies show that CTCF recruits the cohesin complex to insulator sites and that cohesin is required for insulator activity. Here we showed that the DEAD-box RNA helicase p68 (DDX5) and its associated noncoding RNA, steroid receptor RNA activator (SRA), form a complex with CTCF that is essential for insulator function. p68 was detected at CTCF sites in the IGF2/H19 imprinted control region (ICR) as well as other genomic CTCF sites. In vivo depletion of SRA or p68 reduced CTCF-mediated insulator activity at the IGF2/H19 ICR, increased levels of IGF2 expression, and increased interactions between the endodermal enhancer and IGF2 promoter. p68/SRA also interacts with members of the cohesin complex. Depletion of either p68 or SRA does not affect CTCF binding to its genomic sites, but does reduce cohesin binding. The results suggest that p68/SRA stabilizes the interaction of cohesin with CTCF by binding to both, and is required for proper insulator function. PMID:20966046

  2. Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae.

    PubMed Central

    Kressler, D; de la Cruz, J; Rojo, M; Linder, P

    1997-01-01

    A previously uncharacterized Saccharomyces cerevisiae gene, FAL1, was found by sequence comparison as a homolog of the eukaryotic translation initiation factor 4A (eIF4A). Fal1p has 55% identity and 73% similarity on the amino acid level to yeast eIF4A, the prototype of ATP-dependent RNA helicases of the DEAD-box protein family. Although clearly grouped in the eIF4A subfamily, the essential Fal1p displays a different subcellular function and localization. An HA epitope-tagged Fal1p is localized predominantly in the nucleolus. Polysome analyses in a temperature-sensitive fal1-1 mutant and a Fal1p-depleted strain reveal a decrease in the number of 40S ribosomal subunits. Furthermore, these strains are hypersensitive to the aminoglycoside antibiotics paromomycin and neomycin. Pulse-chase labeling of pre-rRNA and steady-state-level analysis of pre-rRNAs and mature rRNAs by Northern hybridization and primer extension in the Fal1p-depleted strain show that Fal1p is required for pre-rRNA processing at sites A0, A1, and A2. Consequently, depletion of Fal1p leads to decreased 18S rRNA levels and to an overall deficit in 40S ribosomal subunits. Together, these results implicate Fal1p in the 18S rRNA maturation pathway rather than in translation initiation. PMID:9372960

  3. The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation.

    PubMed

    Khoshnevis, Sohail; Askenasy, Isabel; Johnson, Matthew C; Dattolo, Maria D; Young-Erdos, Crystal L; Stroupe, M Elizabeth; Karbstein, Katrin

    2016-06-01

    DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked.

  4. The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation

    PubMed Central

    Khoshnevis, Sohail; Askenasy, Isabel; Dattolo, Maria D.; Young-Erdos, Crystal L.; Stroupe, M. Elizabeth; Karbstein, Katrin

    2016-01-01

    DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed “helicases,” their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked. PMID:27280440

  5. Crystal structure of the human eIF4AIII–CWC22 complex shows how a DEAD-box protein is inhibited by a MIF4G domain

    PubMed Central

    Buchwald, Gretel; Schüssler, Steffen; Basquin, Claire; Le Hir, Hervé; Conti, Elena

    2013-01-01

    DEAD-box proteins are involved in all aspects of RNA processing. They bind RNA in an ATP-dependent manner and couple ATP hydrolysis to structural and compositional rearrangements of ribonucleoprotein particles. Conformational control is a major point of regulation for DEAD-box proteins to act on appropriate substrates and in a timely manner in vivo. Binding partners containing a middle domain of translation initiation factor 4G (MIF4G) are emerging as important regulators. Well-known examples are eIF4G and Gle1, which bind and activate the DEAD-box proteins eIF4A and Dbp5. Here, we report the mechanism of an inhibiting MIF4G domain. We determined the 2.0-Å resolution structure of the complex of human eIF4AIII and the MIF4G domain of the splicing factor Complexed With Cef1 (CWC22), an essential prerequisite for exon junction complex assembly by the splicing machinery. The CWC22 MIF4G domain binds both RecA domains of eIF4AIII. The mode of RecA2 recognition is similar to that observed in the activating complexes, yet is specific for eIF4AIII. The way the CWC22 MIF4G domain latches on the eIF4AIII RecA1 domain is markedly different from activating complexes. In the CWC22–eIF4AIII complex, the RNA-binding and ATP-binding motifs of the two RecA domains do not face each other, as would be required in the active state, but are in diametrically opposite positions. The binding mode of CWC22 to eIF4AIII reveals a facet of how MIF4G domains use their versatile structural frameworks to activate or inhibit DEAD-box proteins. PMID:24218557

  6. Cryptococcus neoformans growth and protection from innate immunity are dependent on expression of a virulence-associated DEAD-box protein, Vad1.

    PubMed

    Qiu, Jin; Olszewski, Michal A; Williamson, Peter R

    2013-03-01

    The fungus Cryptococcus neoformans has emerged as a major cause of meningoencephalitis worldwide. Host response to the fungus involves both innate and adaptive immunity, but fungal genes that modulate these processes are poorly understood. Previous studies demonstrated attenuated virulence of a mutant of a virulence-associated DEAD-box protein (VAD1) in mice, despite normal growth at host temperatures, suggesting modulation of the immune response. In the present study, the Δvad1 mutant demonstrated progressive clearance from lung and was unable to induce pathological lesions or to cause extrapulmonary disease, despite retaining its ability to grow in mouse serum and a J774.16 macrophage cell line. Pulmonary clearance occurred with a minimal cellular infiltrate, marked by reduced CD4 cells, CD11b(+) Ly6C(high) monocytes, and F4/80(+) macrophages, but the mutant strain retained recruitment of CD8 cells, compared to infections with wild-type fungi. Adaptive cytokine responses were reduced, including Th1, Th2, and Th17 cytokines; however, early gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) responses were retained while nonprotective interleukin 4 (IL-4) and IL-5 were diminished. Furthermore, the Δvad1 mutant was controlled in lungs despite CD4/CD8 cell depletion. These data, along with improved phagocytosis by macrophages and increases in early/innate IL-1α, IFN-γ, and chemokines elicited in the lungs within 3 days of infection with the Δvad1 mutant, indicate that VAD1 expression reduces innate recognition of C. neoformans, rendering the yeast resistant to elimination by the innate mechanisms of host defense. Thus, our studies define a novel role of the cryptococcal Vad1 protein as a central regulator of cryptococcal virulence and illustrate that Vad1 promotes microbe resistance to innate host defenses.

  7. A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1.

    PubMed

    Lin, Min-Hsuan; Sivakumaran, Haran; Jones, Alun; Li, Dongsheng; Harper, Callista; Wei, Ting; Jin, Hongping; Rustanti, Lina; Meunier, Frederic A; Spann, Kirsten; Harrich, David

    2014-12-14

    Previously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex. To seek a possible mechanism that could explain how Nullbasic inhibits Rev activity, we used a proteomics approach to identify host cellular proteins that interact with Nullbasic. Forty-six Nullbasic-binding proteins were identified by mass spectrometry including the DEAD-box RNA helicase, DDX1. To determine the effect of DDX1 on Nullbasic-mediated Rev activity, we performed cell-based immunoprecipitation assays, Rev reporter assays and bio-layer interferometry (BLI) assays. Interaction between DDX1 and Nullbasic was observed by co-immunoprecipitation of Nullbasic with endogenous DDX1 from cell lysates. BLI assays showed a direct interaction between Nullbasic and DDX1. Nullbasic affected DDX1 subcellular distribution in a Rev-independent manner. Interestingly overexpression of DDX1 in cells not only restored Rev-dependent mRNA export and gene expression in a Rev reporter assay but also partly reversed Nullbasic-induced Rev subcellular mislocalization. Moreover, HIV-1 wild type Tat co-immunoprecipitated with DDX1 and overexpression of Tat could rescue the unspliced viral mRNA levels inhibited by Nullbasic in HIV-1 expressing cells. Nullbasic was used to further define the complex mechanisms involved in the Rev-dependent nuclear export of the 9 kb and 4 kb viral RNAs. All together, these data indicate that DDX1 can be sequestered by Nullbasic leading to destabilization of the Rev nucleocytoplasmic transport complex and decreased

  8. The Arabidopsis STRESS RESPONSE SUPPRESSOR DEAD-box RNA helicases are nucleolar- and chromocenter-localized proteins that undergo stress-mediated relocalization and are involved in epigenetic gene silencing.

    PubMed

    Khan, Asif; Garbelli, Anna; Grossi, Serena; Florentin, Assa; Batelli, Giorgia; Acuna, Tania; Zolla, Gaston; Kaye, Yuval; Paul, Laju K; Zhu, Jian-Kang; Maga, Giovanni; Grafi, Gideon; Barak, Simon

    2014-07-01

    DEAD-box RNA helicases are involved in many aspects of RNA metabolism and in diverse biological processes in plants. Arabidopsis thaliana mutants of two DEAD-box RNA helicases, STRESS RESPONSE SUPPRESSOR1 (STRS1) and STRS2 were previously shown to exhibit tolerance to abiotic stresses and up-regulated stress-responsive gene expression. Here, we show that Arabidopsis STRS-overexpressing lines displayed a less tolerant phenotype and reduced expression of stress-induced genes confirming the STRSs as attenuators of Arabidopsis stress responses. GFP-STRS fusion proteins exhibited localization to the nucleolus, nucleoplasm and chromocenters and exhibited relocalization in response to abscisic acid (ABA) treatment and various stresses. This relocalization was reversed when stress treatments were removed. The STRS proteins displayed mis-localization in specific gene-silencing mutants and exhibited RNA-dependent ATPase and RNA-unwinding activities. In particular, STRS2 showed mis-localization in three out of four mutants of the RNA-directed DNA methylation (RdDM) pathway while STRS1 was mis-localized in the hd2c mutant that is defective in histone deacetylase activity. Furthermore, heterochromatic RdDM target loci displayed reduced DNA methylation and increased expression in the strs mutants. Taken together, our findings suggest that the STRS proteins are involved in epigenetic silencing of gene expression to bring about suppression of the Arabidopsis stress response.

  9. Defective chloroplast development inhibits maintenance of normal levels of abscisic acid in a mutant of the Arabidopsis RH3 DEAD-box protein during early post-germination growth.

    PubMed

    Lee, Kwang-Hee; Park, Jiyoung; Williams, Donna S; Xiong, Yuqing; Hwang, Inhwan; Kang, Byung-Ho

    2013-03-01

    The plastid has its own translation system, and its ribosomes are assembled through a complex process in which rRNA precursors are processed and ribosomal proteins are inserted into the rRNA backbone. DEAD-box proteins have been shown to play roles in multiple steps in ribosome biogenesis. To investigate the cellular and physiological roles of an Arabidopsis DEAD-box protein, RH3, we examined its expression and localization and the phenotypes of rh3-4, a T-DNA insertion mutant allele of RH3. The promoter activity of RH3 is strongest in the greening tissues of 3-day and 1-week-old seedlings but reduced afterwards. Cotyledons were pale and seedling growth was retarded in the mutant. The most obvious abnormality in the mutant chloroplasts was their lack of normal ribosomes. Electron tomography analysis indicated that ribosome density in the 3-day-old mutant chloroplasts is only 20% that of wild-type chloroplasts, and the ribosomes in the mutant are smaller. These chloroplast defects in rh3-4 were alleviated in 2-week-old cotyledons and true leaves. Interestingly, rh3-4 seedlings have lower amounts of abscisic acid prior to recovery of their chloroplasts, and were more sensitive to abiotic stresses. Transcriptomic analysis indicated that nuclear genes for chloroplast proteins are down-regulated, and proteins mediating chloroplast-localized steps of abscisic acid biosynthesis are expressed to a lower extent in 1-week-old rh3-4 seedlings. Taken together, these results suggest that conversion of eoplasts into chloroplasts in young seedlings is critical for the seedlings to start carbon fixation as well as for maintenance of abscisic acid levels for responding to environmental challenges. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

  10. RNA CATALYSIS AS A PROBE FOR CHAPERONE ACTIVITY OF DEAD-BOX HELICASES

    PubMed Central

    Potratz, Jeffrey P.; Russell, Rick

    2012-01-01

    DEAD-box proteins are vitally important to cellular processes and make up the largest class of helicases. Many DEAD-box proteins function as RNA chaperones by accelerating structural transitions of RNA, which can result in the resolution of misfolded conformers or conversion between functional structures. While the biological importance of chaperone proteins is clear, their mechanisms are incompletely understood. Here we illustrate how the catalytic activity of certain RNAs can be used to measure RNA chaperone activity. By measuring the amount of substrate converted to product, the fraction of catalytically active molecules is measured over time, providing a quantitative measure of the formation or loss of native RNA. The assays are described with references to group I and group II introns and their ribozyme derivatives, and examples are included that illustrate potential complications and indicate how catalytic activity measurements can be combined with physical approaches to gain insights into the mechanisms of DEAD-box proteins as RNA chaperones. PMID:22713317

  11. The LOTUS domain is a conserved DEAD-box RNA helicase regulator essential for the recruitment of Vasa to the germ plasm and nuage

    PubMed Central

    Jeske, Mandy; Müller, Christoph W.; Ephrussi, Anne

    2017-01-01

    DEAD-box RNA helicases play important roles in a wide range of metabolic processes. Regulatory proteins can stimulate or block the activity of DEAD-box helicases. Here, we show that LOTUS (Limkain, Oskar, and Tudor containing proteins 5 and 7) domains present in the germline proteins Oskar, TDRD5 (Tudor domain-containing 5), and TDRD7 bind and stimulate the germline-specific DEAD-box RNA helicase Vasa. Our crystal structure of the LOTUS domain of Oskar in complex with the C-terminal RecA-like domain of Vasa reveals that the LOTUS domain occupies a surface on a DEAD-box helicase not implicated previously in the regulation of the enzyme's activity. We show that, in vivo, the localization of Drosophila Vasa to the nuage and germ plasm depends on its interaction with LOTUS domain proteins. The binding and stimulation of Vasa DEAD-box helicases by LOTUS domains are widely conserved. PMID:28536148

  12. Two highly similar DEAD box proteins, OsRH2 and OsRH34, homologous to eukaryotic initiation factor 4AIII, play roles of the exon junction complex in regulating growth and development in rice.

    PubMed

    Huang, Chun-Kai; Sie, Yi-Syuan; Chen, Yu-Fu; Huang, Tian-Sheng; Lu, Chung-An

    2016-04-12

    The exon junction complex (EJC), which contains four core components, eukaryotic initiation factor 4AIII (eIF4AIII), MAGO/NASHI (MAGO), Y14/Tsunagi/RNA-binding protein 8A, and Barentsz/Metastatic lymph node 51, is formed in both nucleus and cytoplasm, and plays important roles in gene expression. Genes encoding core EJC components have been found in plants, including rice. Currently, the functional characterizations of MAGO and Y14 homologs have been demonstrated in rice. However, it is still unknown whether eIF4AIII is essential for the functional EJC in rice. This study investigated two DEAD box RNA helicases, OsRH2 and OsRH34, which are homologous to eIF4AIII, in rice. Amino acid sequence analysis indicated that OsRH2 and OsRH34 had 99 % identity and 100 % similarity, and their gene expression patterns were similar in various rice tissues, but the level of OsRH2 mRNA was about 58-fold higher than that of OsRH34 mRNA in seedlings. From bimolecular fluorescence complementation results, OsRH2 and OsRH34 interacted physically with OsMAGO1 and OsY14b, respectively, which indicated that both of OsRH2 and OsRH34 were core components of the EJC in rice. To study the biological roles of OsRH2 and OsRH34 in rice, transgenic rice plants were generated by RNA interference. The phenotypes of three independent OsRH2 and OsRH34 double-knockdown transgenic lines included dwarfism, a short internode distance, reproductive delay, defective embryonic development, and a low seed setting rate. These phenotypes resembled those of mutants with gibberellin-related developmental defects. In addition, the OsRH2 and OsRH34 double-knockdown transgenic lines exhibited the accumulation of unspliced rice UNDEVELOPED TAPETUM 1 mRNA. Rice contains two eIF4AIII paralogous genes, OsRH2 and OsRH34. The abundance of OsRH2 mRNA was about 58-fold higher than that of OsRH34 mRNA in seedlings, suggesting that the OsRH2 is major eIF4AIII in rice. Both OsRH2 and OsRH34 are core components of the EJC

  13. Structural basis for RNA-duplex recognition and unwinding by the DEAD-box helicase Mss116p.

    PubMed

    Mallam, Anna L; Del Campo, Mark; Gilman, Benjamin; Sidote, David J; Lambowitz, Alan M

    2012-10-04

    DEAD-box proteins are the largest family of nucleic acid helicases, and are crucial to RNA metabolism throughout all domains of life. They contain a conserved 'helicase core' of two RecA-like domains (domains (D)1 and D2), which uses ATP to catalyse the unwinding of short RNA duplexes by non-processive, local strand separation. This mode of action differs from that of translocating helicases and allows DEAD-box proteins to remodel large RNAs and RNA-protein complexes without globally disrupting RNA structure. However, the structural basis for this distinctive mode of RNA unwinding remains unclear. Here, structural, biochemical and genetic analyses of the yeast DEAD-box protein Mss116p indicate that the helicase core domains have modular functions that enable a novel mechanism for RNA-duplex recognition and unwinding. By investigating D1 and D2 individually and together, we find that D1 acts as an ATP-binding domain and D2 functions as an RNA-duplex recognition domain. D2 contains a nucleic-acid-binding pocket that is formed by conserved DEAD-box protein sequence motifs and accommodates A-form but not B-form duplexes, providing a basis for RNA substrate specificity. Upon a conformational change in which the two core domains join to form a 'closed state' with an ATPase active site, conserved motifs in D1 promote the unwinding of duplex substrates bound to D2 by excluding one RNA strand and bending the other. Our results provide a comprehensive structural model for how DEAD-box proteins recognize and unwind RNA duplexes. This model explains key features of DEAD-box protein function and affords a new perspective on how the evolutionarily related cores of other RNA and DNA helicases diverged to use different mechanisms.

  14. Factor-dependent processivity in human eIF4A DEAD-box helicase

    PubMed Central

    García-García, Cuauhtémoc; Frieda, Kirsten L.; Feoktistova, Kateryna; Fraser, Christopher S.; Block, Steven M.

    2015-01-01

    During eukaryotic translation initiation, the small ribosomal subunit, assisted by initiation factors, locates the messenger RNA start codon by scanning from the 5′ cap. This process is powered by the eukaryotic initiation factor 4A (eIF4A), a DEAD-box helicase. eIF4A has been thought to unwind structures formed in the untranslated 5′ region via a nonprocessive mechanism. Using a single-molecule assay, we found that eIF4A functions instead as an adenosine triphosphate–dependent processive helicase when complexed with two accessory proteins, eIF4G and eIF4B. Translocation occurred in discrete steps of 11 ± 2 base pairs, irrespective of the accessory factor combination. Our findings support a memory-less stepwise mechanism for translation initiation and suggest that similar factor-dependent processivity may be shared by other members of the DEAD-box helicase family. PMID:26113725

  15. The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote compartmentalized translation initiation of the HIV-1 genomic RNA

    PubMed Central

    Soto-Rifo, Ricardo; Rubilar, Paulina S.; Ohlmann, Théophile

    2013-01-01

    Here, we show a novel molecular mechanism promoted by the DEAD-box RNA helicase DDX3 for translation of the HIV-1 genomic RNA. This occurs through the adenosine triphosphate-dependent formation of a translation initiation complex that is assembled at the 5′ m7GTP cap of the HIV-1 mRNA. This is due to the property of DDX3 to substitute for the initiation factor eIF4E in the binding of the HIV-1 m7GTP 5′ cap structure where it nucleates the formation of a core DDX3/PABP/eIF4G trimeric complex on the HIV-1 genomic RNA. By using RNA fluorescence in situ hybridization coupled to indirect immunofluorescence, we further show that this viral ribonucleoprotein complex is addressed to compartmentalized cytoplasmic foci where the translation initiation complex is assembled. PMID:23630313

  16. Transcriptome-wide analysis of DEAD-box RNA helicase gene family in an Antarctic psychrophilic alga Chlamydomonas sp. ICE-L.

    PubMed

    Liu, Chenlin; Huang, Xiaohang

    2015-09-01

    DEAD-box RNA helicase family proteins have been identified in almost all living organisms. Some of them play a crucial role in adaptation to environmental changes and stress response, especially in the low-temperature acclimation in different kinds of organisms. Compared with the full swing study in plants and bacteria, the characters and functions of DEAD-box family proteins had not been surveyed in algae. To identify genes critical for freezing acclimation in algae, we screened DEAD-box RNA helicase genes from the transcriptome sequences of a psychrophilic microalga Chlamydomonas sp. ICE-L which was isolated from Antarctic sea ice. Totally 39 DEAD-box RNA helicase genes had been identified. Most of the DEAD-box RNA helicase have 1:1 homologous relationships in Chlamydomonas reinhardtii and Chlamydomonas sp. ICE-L with several exceptions. The homologous proteins in ICE-L to the helicases critical for cold or freezing tolerance in Arabidopsis thaliana had been identified based on phylogenetic comparison studies. The response of these helicase genes is not always identical in the Chlamydomonas sp. ICE-L and Arabidopsis under the same low-temperature treatment. The expression of several DEAD-box RNA helicase genes including CiRH5, CiRH25, CiRH28, and CiRH55 were significantly up-regulated under freezing treatment of ICE-L and their function in freezing acclimation of ICE-L deserved further investigation.

  17. A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo.

    PubMed

    Ruminski, Dana J; Watson, Peter Y; Mahen, Elisabeth M; Fedor, Martha J

    2016-03-01

    RNAs must assemble into specific structures in order to carry out their biological functions, but in vitro RNA folding reactions produce multiple misfolded structures that fail to exchange with functional structures on biological time scales. We used carefully designed self-cleaving mRNAs that assemble through well-defined folding pathways to identify factors that differentiate intracellular and in vitro folding reactions. Our previous work showed that simple base-paired RNA helices form and dissociate with the same rate and equilibrium constants in vivo and in vitro. However, exchange between adjacent secondary structures occurs much faster in vivo, enabling RNAs to quickly adopt structures with the lowest free energy. We have now used this approach to probe the effects of an extensively characterized DEAD-box RNA helicase, Mss116p, on a series of well-defined RNA folding steps in yeast. Mss116p overexpression had no detectable effect on helix formation or dissociation kinetics or on the stability of interdomain tertiary interactions, consistent with previous evidence that intracellular factors do not affect these folding parameters. However, Mss116p overexpression did accelerate exchange between adjacent helices. The nonprocessive nature of RNA duplex unwinding by DEAD-box RNA helicases is consistent with a branch migration mechanism in which Mss116p lowers barriers to exchange between otherwise stable helices by the melting and annealing of one or two base pairs at interhelical junctions. These results suggest that the helicase activity of DEAD-box proteins like Mss116p distinguish intracellular RNA folding pathways from nonproductive RNA folding reactions in vitro and allow RNA structures to overcome kinetic barriers to thermodynamic equilibration in vivo.

  18. A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo

    PubMed Central

    Ruminski, Dana J.; Watson, Peter Y.; Mahen, Elisabeth M.; Fedor, Martha J.

    2016-01-01

    RNAs must assemble into specific structures in order to carry out their biological functions, but in vitro RNA folding reactions produce multiple misfolded structures that fail to exchange with functional structures on biological time scales. We used carefully designed self-cleaving mRNAs that assemble through well-defined folding pathways to identify factors that differentiate intracellular and in vitro folding reactions. Our previous work showed that simple base-paired RNA helices form and dissociate with the same rate and equilibrium constants in vivo and in vitro. However, exchange between adjacent secondary structures occurs much faster in vivo, enabling RNAs to quickly adopt structures with the lowest free energy. We have now used this approach to probe the effects of an extensively characterized DEAD-box RNA helicase, Mss116p, on a series of well-defined RNA folding steps in yeast. Mss116p overexpression had no detectable effect on helix formation or dissociation kinetics or on the stability of interdomain tertiary interactions, consistent with previous evidence that intracellular factors do not affect these folding parameters. However, Mss116p overexpression did accelerate exchange between adjacent helices. The nonprocessive nature of RNA duplex unwinding by DEAD-box RNA helicases is consistent with a branch migration mechanism in which Mss116p lowers barriers to exchange between otherwise stable helices by the melting and annealing of one or two base pairs at interhelical junctions. These results suggest that the helicase activity of DEAD-box proteins like Mss116p distinguish intracellular RNA folding pathways from nonproductive RNA folding reactions in vitro and allow RNA structures to overcome kinetic barriers to thermodynamic equilibration in vivo. PMID:26759451

  19. Human DEAD-box ATPase DDX3 shows a relaxed nucleoside substrate specificity.

    PubMed

    Franca, Raffaella; Belfiore, Amalia; Spadari, Silvio; Maga, Giovanni

    2007-06-01

    Human DDX3 (hDDX3) is a DEAD-box protein shown to possess RNA-unwinding and adenosine triphosphatase (ATPase) activities. The hDDX3 protein has been implicated in nuclear mRNA export, cell growth control, and cancer progression. In addition, a role of this protein in the replication of human immunodeficiency virus Type 1 and in the pathogenesis of hepatitis C virus has been recently proposed. Its enzymological properties, however, are largely unknown. In this work, we characterized its ATPase activity. We show that hDDX3 ATPase activity is stimulated by various ribo- and deoxynucleic acids. Comparative analysis with different nucleoside triphosphate analogs showed that the hDDX3 ATPase couples high catalytic efficiency to a rather relaxed substrate specificity, both in terms of base selection and sugar selection. In addition, its ability to recognize the L-stereoisomers of both 3' deoxy- and 2',3' dideoxy-ribose, points to a relaxed stereoselectivity. On the basis of these results, we hypothesize the presence of structural determinants on both the base and the sugar moieties, critical for nucleoside binding to the enzyme. Our results expand the knowledge about the DEAD-box RNA helicases in general and can be used for rational design of selective inhibitors of hDDX3, to be tested as potential antitumor and antiviral agents.

  20. A role for the cytoplasmic DEAD box helicase Dbp21E2 in rhodopsin maturation and photoreceptor viability

    PubMed Central

    Hibbard, Karen L.; O’Tousa, Joseph. E.

    2013-01-01

    The Dbp21E2 (Dead box protein 21E2) is a member of a family of DEAD box helicases active in RNA processing and stability. We used genetic mosaics to identify mutants in Dbp21E2 that affect rhodopsin biogenesis and the maintenance of photoreceptor structure. Analysis of a GFP-tagged Rh1 rhodopsin construct placed under control of a heat shock promoter showed that Dbp21E21 fails to efficiently transport Rh1 from the photoreceptor cell body to the rhabdomere. Retinal degeneration is not dependent on the Rh1 transport defects. We also show that GFP- and RFP-tagged Dbp21E2 proteins are localized to discrete cytoplasmic structures that are not associated with organelles known to be active in rhodopsin transport. The molecular genetic analysis described here reveals an unexpected role for the Dbp21E2 helicase and provides an experimental system to further characterize its function. PMID:22794106

  1. A conserved mechanism of DEAD-box ATPase activation by nucleoporins and IP6 in mRNA export

    PubMed Central

    Montpetit, Ben; Thomsen, Nathan D.; Helmke, Kara J.; Seeliger, Markus A.; Berger, James M.; Weis, Karsten

    2011-01-01

    Superfamily 1 (SF1) and superfamily 2 (SF2) RNA helicases are ubiquitous mRNA-protein (mRNP) remodelling enzymes that play critical roles in all aspects of RNA metabolism1, 2. The SF2 DEAD-box ATPase Dbp5/Ddx19 functions in mRNA export and is thought to remodel mRNPs at the nuclear pore complex (NPC)3–8. Dbp5 is localized to the NPC via an interaction with Nup159/Nup2143–5, 8, 9 and is locally activated there by Gle1 together with the small-molecule inositol hexakisphosphate (IP6) 10, 11. Local activation of Dbp5 at the NPC by Gle1 is essential for mRNA export in vivo11, 12; however, the mechanistic role of Dbp5 in mRNP export is poorly understood and it is not known how Gle1IP6 and Nup159 regulate the activity of Dbp5. Here we report structures of Dbp5 in complex with Gle1IP6, Nup159/Gle1IP6, and RNA. These structures reveal that IP6 functions as a small-molecule tether for the Gle1-Dbp5 interaction. Surprisingly, the Gle1IP6-Dbp5 complex is structurally similar to another DEAD-box ATPase complex essential for translation initiation, eIF4G-eIF4A, and we demonstrate that Gle1IP6 and eIF4G both activate their DEAD-box partner by stimulating RNA release. Furthermore, Gle1IP6 relieves Dbp5 auto- regulation and cooperates with Nup159 in stabilizing an open Dbp5-intermediate that precludes RNA binding. These findings explain how Gle1IP6, Nup159, and Dbp5 collaborate in mRNA export and provide a general mechanism for DEAD-box ATPase regulation by Gle1/eIF4G-like activators. PMID:21441902

  2. Structural Insights into a Unique Dimeric DEAD-Box Helicase CshA that Promotes RNA Decay.

    PubMed

    Huen, Jennifer; Lin, Chia-Liang; Golzarroshan, Bagher; Yi, Wan-Li; Yang, Wei-Zen; Yuan, Hanna S

    2017-03-07

    CshA is a dimeric DEAD-box helicase that cooperates with ribonucleases for mRNA turnover. The molecular mechanism for how a dimeric DEAD-box helicase aids in RNA decay remains unknown. Here, we report the crystal structure and small-angle X-ray scattering solution structure of the CshA from Geobacillus stearothermophilus. In contrast to typical monomeric DEAD-box helicases, CshA is exclusively a dimeric protein with the RecA-like domains of each protomer forming a V-shaped structure. We show that the C-terminal domains protruding outward from the tip of the V-shaped structure is critical for mediating strong RNA binding and is crucial for efficient RNA-dependent ATP hydrolysis. We also show that RNA remains bound with CshA during ATP hydrolysis cycles and thus bulk RNAs could be unwound and degraded in a processive manner through cooperation between exoribonucleases and CshA. A dimeric helicase is hence preserved in RNA-degrading machinery for efficient RNA turnover in prokaryotes and eukaryotes.

  3. Pathway of ATP utilization and duplex rRNA unwinding by the DEAD-box helicase, DbpA.

    PubMed

    Henn, Arnon; Cao, Wenxiang; Licciardello, Nicholas; Heitkamp, Sara E; Hackney, David D; De La Cruz, Enrique M

    2010-03-02

    DEAD-box RNA helicase proteins use the energy of ATP hydrolysis to drive the unwinding of duplex RNA. However, the mechanism that couples ATP utilization to duplex RNA unwinding is unknown. We measured ATP utilization and duplex RNA unwinding by DbpA, a non-processive bacterial DEAD-box RNA helicase specifically activated by the peptidyl transferase center (PTC) of 23S rRNA. Consumption of a single ATP molecule is sufficient to unwind and displace an 8 base pair rRNA strand annealed to a 32 base pair PTC-RNA "mother strand" fragment. Strand displacement occurs after ATP binding and hydrolysis but before P(i) product release. P(i) release weakens binding to rRNA, thereby facilitating the release of the unwound rRNA mother strand and the recycling of DbpA for additional rounds of unwinding. This work explains how ATPase activity of DEAD-box helicases is linked to RNA unwinding.

  4. The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes

    PubMed Central

    Geissler, Rene; Golbik, Ralph P.; Behrens, Sven-Erik

    2012-01-01

    The DEAD-box helicase DDX3 has suggested functions in innate immunity, mRNA translocation and translation, and it participates in the propagation of assorted viruses. Exploring initially the role of DDX3 in the life cycle of hepatitis C virus, we observed the protein to be involved in translation directed by different viral internal ribosomal entry sites. Extension of these studies revealed a general supportive role of DDX3 in translation initiation. DDX3 was found to interact in an RNA-independent manner with defined components of the translational pre-initiation complex and to specifically associate with newly assembling 80S ribosomes. DDX3 knock down and in vitro reconstitution experiments revealed a significant function of the protein in the formation of 80S translation initiation complexes. Our study implies that DDX3 assists the 60S subunit joining process to assemble functional 80S ribosomes. PMID:22323517

  5. Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

    SciTech Connect

    Möhlmann, Sina; Mathew, Rebecca; Neumann, Piotr; Schmitt, Andreas; Lührmann, Reinhard; Ficner, Ralf

    2014-06-01

    The crystal structure of the helicase domain of the human spliceosomal DEAD-box protein Prp28 was solved by SAD. The binding of ADP and ATP by Prp28 was studied biochemically and analysed with regard to the crystal structure. The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP.

  6. Allosteric regulation of helicase core activities of the DEAD-box helicase YxiN by RNA binding to its RNA recognition motif.

    PubMed

    Samatanga, Brighton; Andreou, Alexandra Z; Klostermeier, Dagmar

    2017-01-23

    DEAD-box proteins share a structurally similar core of two RecA-like domains (RecA_N and RecA_C) that contain the conserved motifs for ATP-dependent RNA unwinding. In many DEAD-box proteins the helicase core is flanked by ancillary domains. To understand the regulation of the DEAD-box helicase YxiN by its C-terminal RNA recognition motif (RRM), we investigated the effect of RNA binding to the RRM on its position relative to the core, and on core activities. RRM/RNA complex formation substantially shifts the RRM from a position close to the RecA_C to the proximity of RecA_N, independent of RNA contacts with the core. RNA binding to the RRM is communicated to the core, and stimulates ATP hydrolysis and RNA unwinding. The conformational space of the core depends on the identity of the RRM-bound RNA. Allosteric regulation of core activities by RNA-induced movement of ancillary domains may constitute a general regulatory mechanism of DEAD-box protein activity.

  7. Evolution of the DEAD box helicase family in chicken: chickens have no DHX9 ortholog.

    PubMed

    Sato, Haruko; Oshiumi, Hiroyuki; Takaki, Hiromi; Hikono, Hirokazu; Seya, Tsukasa

    2015-10-01

    Viral RNA represents a pattern molecule that can be recognized by RNA sensors in innate immunity. Humans and mice possess cytoplasmic DNA/RNA sensors for detecting viral replication. There are a number of DEAD (Asp-Glu-Ala-Asp; DExD/H) box-type helicases in mammals, among which retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5 (MDA50) are indispensable for RNA sensing; however, they are functionally supported by a number of sensors that directly bind viral RNA or replicative RNA intermediates to convey signals to RIG-I and MDA5. Some DEAD box helicase members recognize DNA irrespective of the origin. These sensors transmit IFN-inducing signals through adaptors, including mitochondrial antiviral signaling. Viral double-stranded RNAs are reportedly sensed by the helicases DDX1, DDX21, DHX36, DHX9, DDX3, DDX41, LGP2 and DDX60, in addition to RIG-I and MDA5, and induce type I IFNs, thereby blocking viral replication. Humans and mice have all nucleic acid sensors listed here. In the RNA sensing system in chicken, it was found in the present study that most DEAD box helicases are conserved; however, DHX9 is genetically deficient in addition to reported RIG-I. Based on the current genome databases, similar DHX9 deficiency was observed in ducks and several other bird species. Because chicken, but not duck, was found to be deficient in RIG-I, the RNA-sensing system of chicken lacks RIG-I and DHX9 and is thus more fragile than that of duck or mammal. DHX9 may generally compensate for the function of RIG-I and deficiency of DHX9 possibly participates in exacerbations of viral infection such as influenza in chickens.

  8. Chloroplast- or Mitochondria-Targeted DEAD-Box RNA Helicases Play Essential Roles in Organellar RNA Metabolism and Abiotic Stress Responses

    PubMed Central

    Nawaz, Ghazala; Kang, Hunseung

    2017-01-01

    The yields and productivity of crops are greatly diminished by various abiotic stresses, including drought, cold, heat, and high salinity. Chloroplasts and mitochondria are cellular organelles that can sense diverse environmental stimuli and alter gene expression to cope with adverse environmental stresses. Organellar gene expression is mainly regulated at posttranscriptional levels, including RNA processing, intron splicing, RNA editing, RNA turnover, and translational control, during which a variety of nucleus-encoded RNA-binding proteins (RBPs) are targeted to chloroplasts or mitochondria where they play essential roles in organellar RNA metabolism. DEAD-box RNA helicases (RHs) are enzymes that can alter RNA structures and affect RNA metabolism in all living organisms. Although a number of DEAD-box RHs have been found to play important roles in RNA metabolism in the nucleus and cytoplasm, our understanding on the roles of DEAD-box RHs in the regulation of RNA metabolism in chloroplasts and mitochondria is only at the beginning. Considering that organellar RNA metabolism and gene expression are tightly regulated by anterograde signaling from the nucleus, it is imperative to determine the functions of nucleus-encoded organellar RBPs. In this review, we summarize the emerging roles of nucleus-encoded chloroplast- or mitochondria-targeted DEAD-box RHs in organellar RNA metabolism and plant response to diverse abiotic stresses. PMID:28596782

  9. Chloroplast- or Mitochondria-Targeted DEAD-Box RNA Helicases Play Essential Roles in Organellar RNA Metabolism and Abiotic Stress Responses.

    PubMed

    Nawaz, Ghazala; Kang, Hunseung

    2017-01-01

    The yields and productivity of crops are greatly diminished by various abiotic stresses, including drought, cold, heat, and high salinity. Chloroplasts and mitochondria are cellular organelles that can sense diverse environmental stimuli and alter gene expression to cope with adverse environmental stresses. Organellar gene expression is mainly regulated at posttranscriptional levels, including RNA processing, intron splicing, RNA editing, RNA turnover, and translational control, during which a variety of nucleus-encoded RNA-binding proteins (RBPs) are targeted to chloroplasts or mitochondria where they play essential roles in organellar RNA metabolism. DEAD-box RNA helicases (RHs) are enzymes that can alter RNA structures and affect RNA metabolism in all living organisms. Although a number of DEAD-box RHs have been found to play important roles in RNA metabolism in the nucleus and cytoplasm, our understanding on the roles of DEAD-box RHs in the regulation of RNA metabolism in chloroplasts and mitochondria is only at the beginning. Considering that organellar RNA metabolism and gene expression are tightly regulated by anterograde signaling from the nucleus, it is imperative to determine the functions of nucleus-encoded organellar RBPs. In this review, we summarize the emerging roles of nucleus-encoded chloroplast- or mitochondria-targeted DEAD-box RHs in organellar RNA metabolism and plant response to diverse abiotic stresses.

  10. When core competence is not enough: functional interplay of the DEAD-box helicase core with ancillary domains and auxiliary factors in RNA binding and unwinding.

    PubMed

    Rudolph, Markus G; Klostermeier, Dagmar

    2015-08-01

    DEAD-box helicases catalyze RNA duplex unwinding in an ATP-dependent reaction. Members of the DEAD-box helicase family consist of a common helicase core formed by two RecA-like domains. According to the current mechanistic model for DEAD-box mediated RNA unwinding, binding of RNA and ATP triggers a conformational change of the helicase core, and leads to formation of a compact, closed state. In the closed conformation, the two parts of the active site for ATP hydrolysis and of the RNA binding site, residing on the two RecA domains, become aligned. Closing of the helicase core is coupled to a deformation of the RNA backbone and destabilization of the RNA duplex, allowing for dissociation of one of the strands. The second strand remains bound to the helicase core until ATP hydrolysis and product release lead to re-opening of the core. The concomitant disruption of the RNA binding site causes dissociation of the second strand. The activity of the helicase core can be modulated by interaction partners, and by flanking N- and C-terminal domains. A number of C-terminal flanking regions have been implicated in RNA binding: RNA recognition motifs (RRM) typically mediate sequence-specific RNA binding, whereas positively charged, unstructured regions provide binding sites for structured RNA, without sequence-specificity. Interaction partners modulate RNA binding to the core, or bind to RNA regions emanating from the core. The functional interplay of the helicase core and ancillary domains or interaction partners in RNA binding and unwinding is not entirely understood. This review summarizes our current knowledge on RNA binding to the DEAD-box helicase core and the roles of ancillary domains and interaction partners in RNA binding and unwinding by DEAD-box proteins.

  11. The embryonic RNA helicase gene (ERH): a new member of the DEAD box family of RNA helicases.

    PubMed Central

    Sowden, J; Putt, W; Morrison, K; Beddington, R; Edwards, Y

    1995-01-01

    DEAD box proteins share several highly conserved motifs including the characteristic Asp-Glu-Ala-Asp (D-E-A-D in the amino acid single-letter code) motif and have established or putative ATP-dependent RNA helicase activity. These proteins are implicated in a range of cellular processes that involve regulation of RNA function, including translation initiation, RNA splicing and ribosome assembly. Here we describe the isolation and characterization of an embryonic RNA helicase gene, ERH, which maps to mouse chromosome 1 and encodes a new member of the DEAD box family of proteins. The predicted ERH protein shows high sequence similarity to the testes-specific mouse PL10 and to the maternally acting Xenopus An3 helicase proteins. The ERH expression profile is similar, to that of An3, which localizes to the animal hemisphere of oocytes and is abundantly expressed in the embryo. ERH is expressed in oocytes and is a ubiquitous mRNA in the 9 days-post-conception embryo, and at later stages of development shows a more restricted pattern of expression in brain and kidney. The similarities in sequence and in expression profile suggest that ERH is the murine equivalent of the Xenopus An3 gene, and we propose that ERH plays a role in translational activation of mRNA in the oocyte and early embryo. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8948440

  12. An ecdysone-inducible putative "DEAD box" RNA helicase in the spruce budworm (Choristoneura fumiferana).

    PubMed

    Zhang, D-Y; Zheng, S-C; Zheng, Y-P; Ladd, T R; Pang, A S D; Davey, K G; Krell, P J; Arif, B M; Retnakaran, A; Feng, Q-L

    2004-03-01

    RNA helicases are a family of enzymes that unwind nucleic acid duplexes, such as RNA/RNA and RNA/DNA, in a 3' to 5' direction into single-stranded polynucleotides. A putative RNA helicase cDNA (CfrHlc64) was isolated from the spruce budworm, Choristoneura fumiferana. CfrHlc64 was 1998 nucleotides in length, and the deduced protein had 565 amino acids with a predicted molecular mass of 64 kDa. It contained eight functional motifs conserved in the "DEAD box" family of RNA helicases. The deduced amino acid sequence showed 10-50% identities to homologues of other species from bacteria to human. In vitro expression of the cDNA resulted in recombinant proteins of 64 kDa as expected from the deduced amino acid sequence. Northern blotting and RT-PCR analyses revealed the presence of CfrHlc64 mRNA in all developmental stages from embryo to adult. Higher levels of CfrHlc64 mRNA were detected in the fat body and midgut than in the epidermis of sixth instar larvae. The CfrHlc64 protein was distributed mainly in the fat body. Female adults expressed CfrHlc64 mRNA at higher levels than male adults. The nonsteroidal ecdysone agonist, tebufenozide, enhanced the expression of CfrHlc64 in a dose-dependent manner.

  13. Requirement of DDX39 DEAD box RNA helicase for genome integrity and telomere protection.

    PubMed

    Yoo, Hyun Hee; Chung, In Kwon

    2011-08-01

    Human chromosome ends associate with shelterin, a six-protein complex that protects telomeric DNA from being recognized as sites of DNA damage. The shelterin subunit TRF2 has been implicated in the protection of chromosome ends by facilitating their organization into the protective capping structure and by associating with several accessory proteins involved in various DNA transactions. Here we describe the characterization of DDX39 DEAD-box RNA helicase as a novel TRF2-interacting protein. DDX39 directly interacts with the telomeric repeat binding factor homology domain of TRF2 via the FXLXP motif (where X is any amino acid). DDX39 is also found in association with catalytically competent telomerase in cell lysates through an interaction with hTERT but has no effect on telomerase activity. Whereas overexpression of DDX39 in telomerase-positive human cancer cells led to progressive telomere elongation, depletion of endogenous DDX39 by small hairpin RNA (shRNA) resulted in telomere shortening. Furthermore, depletion of DDX39 induced DNA-damage response foci at internal genome as well as telomeres as evidenced by telomere dysfunction-induced foci. Some of the metaphase chromosomes showed no telomeric signal at chromatid ends, suggesting an aberrant telomere structure. Our findings suggest that DDX39, in addition to its role in mRNA splicing and nuclear export, is required for global genome integrity as well as telomere protection and represents a new pathway for telomere maintenance by modulating telomere length homeostasis.

  14. Stress responsive DEAD-box helicases: a new pathway to engineer plant stress tolerance.

    PubMed

    Vashisht, Ajay Amar; Tuteja, Narendra

    2006-08-01

    Abiotic stresses including various environmental factors adversely affect plant growth and limit agricultural production worldwide. Minimizing these losses is a major area of concern for all countries. Therefore, it is desirable to develop multi-stress tolerant varieties. Salinity, drought, and cold are among the major environmental stresses that greatly influence the growth, development, survival, and yield of plants. UV-B radiation of sunlight, which damages the cellular genomes, is another growth-retarding factor. Several genes are induced under the influence of various abiotic stresses. Among these are DNA repair genes, which are induced in response to the DNA damage. Since the stresses affect the cellular gene expression machinery, it is possible that molecules involved in nucleic acid metabolism including helicases are likely to be affected. The light-driven shifts in redox-potential can also initiate the helicase gene expression. Helicases are ubiquitous enzymes that catalyse the unwinding of energetically stable duplex DNA (DNA helicases) or duplex RNA secondary structures (RNA helicases). Most helicases are members of DEAD-box protein superfamily and play essential roles in basic cellular processes such as DNA replication, repair, recombination, transcription, ribosome biogenesis and translation initiation. Therefore, helicases might be playing an important role in regulating plant growth and development under stress conditions by regulating some stress-induced pathways. There are now few reports on the up-regulation of DEAD-box helicases in response to abiotic stresses. Recently, salinity-stress tolerant tobacco plants have already been raised by overexpressing a helicase gene, which suggests a new pathway to engineer plant stress tolerance [N. Sanan-Mishra, X.H. Pham, S.K. Sopory, N. Tuteja, Pea DNA helicase 45 overexpression in tobacco confers high salinity tolerance without affecting yield. Proc. Natl. Acad. Sci. USA 102 (2005) 509-514]. Presently the

  15. A role for Gle1, a regulator of DEAD-box RNA helicases, at centrosomes and basal bodies

    PubMed Central

    Jao, Li-En; Akef, Abdalla; Wente, Susan R.

    2017-01-01

    Control of organellar assembly and function is critical to eukaryotic homeostasis and survival. Gle1 is a highly conserved regulator of RNA-dependent DEAD-box ATPase proteins, with critical roles in both mRNA export and translation. In addition to its well-defined interaction with nuclear pore complexes, here we find that Gle1 is enriched at the centrosome and basal body. Gle1 assembles into the toroid-shaped pericentriolar material around the mother centriole. Reduced Gle1 levels are correlated with decreased pericentrin localization at the centrosome and microtubule organization defects. Of importance, these alterations in centrosome integrity do not result from loss of mRNA export. Examination of the Kupffer’s vesicle in Gle1-depleted zebrafish revealed compromised ciliary beating and developmental defects. We propose that Gle1 assembly into the pericentriolar material positions the DEAD-box protein regulator to function in localized mRNA metabolism required for proper centrosome function. PMID:28035044

  16. DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

    PubMed Central

    Li, Lei; Germain, Devon R.; Poon, Ho-Yin; Hildebrandt, Matthew R.; Monckton, Elizabeth A.; McDonald, Darin; Hendzel, Michael J.

    2016-01-01

    Although RNA and RNA-binding proteins have been linked to double-strand breaks (DSBs), little is known regarding their roles in the cellular response to DSBs and, if any, in the repair process. Here, we provide direct evidence for the presence of RNA-DNA hybrids at DSBs and suggest that binding of RNA to DNA at DSBs may impact repair efficiency. Our data indicate that the RNA-unwinding protein DEAD box 1 (DDX1) is required for efficient DSB repair and cell survival after ionizing radiation (IR), with depletion of DDX1 resulting in reduced DSB repair by homologous recombination (HR). While DDX1 is not essential for end resection, a key step in homology-directed DSB repair, DDX1 is required for maintenance of the single-stranded DNA once generated by end resection. We show that transcription deregulation has a significant effect on DSB repair by HR in DDX1-depleted cells and that RNA-DNA duplexes are elevated at DSBs in DDX1-depleted cells. Based on our combined data, we propose a role for DDX1 in resolving RNA-DNA structures that accumulate at DSBs located at sites of active transcription. Our findings point to a previously uncharacterized requirement for clearing RNA at DSBs for efficient repair by HR. PMID:27550810

  17. Molecular cloning and characterization of a putative nuclear DEAD box RNA helicase in the spruce budworm, Choristoneura fumiferana.

    PubMed

    Zhang, D-Y; Ampasala, D R; Zheng, S-C; Cusson, M; Cheng, X-W; Krell, P J; Feng, Q-L

    2006-04-01

    RNA helicases play important roles in cellular processes such as pre-mRNA splicing, rRNA processing, ribosomal biogenesis, and translation. A full-length DEAD box RNA helicase cDNA (CfrHlc113) was isolated from the spruce budworm, Choristoneura fumiferana. CfrHlc113 contained the eight functional motifs, which are highly conserved in the DEAD box RNA helicase family, and an arginine-serine-aspartate (RSD) domain at its N-terminal end. CfrHlc113 was highly homologous to Rattus norvegicus HEL117 and human prp5 genes, both of which are suggested to be involved in RNA splicing. The results of Northern and Western blotting showed that expression of the CfrHlc113 gene was low or undetectable in eggs, larvae, pupae, and adults. High levels of expression were, however, detected in the three in vitro cultured cell lines, CF-203, CF-124T, and CF-70, which were developed from the midgut, ovaries, and neonate larvae, respectively. Immunocytochemistry revealed that CfrHlc113 protein was present exclusively in the nuclei of these cell lines.

  18. A Cold-Inducible DEAD-Box RNA Helicase from Arabidopsis thaliana Regulates Plant Growth and Development under Low Temperature.

    PubMed

    Liu, Yuelin; Tabata, Daisuke; Imai, Ryozo

    2016-01-01

    DEAD-box RNA helicases comprise a large family and are involved in a range of RNA processing events. Here, we identified one of the Arabidopsis thaliana DEAD-box RNA helicases, AtRH7, as an interactor of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3), which is an RNA chaperone involved in cold adaptation. Promoter:GUS transgenic plants revealed that AtRH7 is expressed ubiquitously and that its levels of the expression are higher in rapidly growing tissues. Knockout mutant lines displayed several morphological alterations such as disturbed vein pattern, pointed first true leaves, and short roots, which resemble ribosome-related mutants of Arabidopsis. In addition, aberrant floral development was also observed in rh7 mutants. When the mutants were germinated at low temperature (12°C), both radicle and first leaf emergence were severely delayed; after exposure of seedlings to a long period of cold, the mutants developed aberrant, fewer, and smaller leaves. RNA blots and circular RT-PCR revealed that 35S and 18S rRNA precursors accumulated to higher levels in the mutants than in WT under both normal and cold conditions, suggesting the mutants are partially impaired in pre-rRNA processing. Taken together, the results suggest that AtRH7 affects rRNA biogenesis and plays an important role in plant growth under cold.

  19. A Cold-Inducible DEAD-Box RNA Helicase from Arabidopsis thaliana Regulates Plant Growth and Development under Low Temperature

    PubMed Central

    Liu, Yuelin; Tabata, Daisuke; Imai, Ryozo

    2016-01-01

    DEAD-box RNA helicases comprise a large family and are involved in a range of RNA processing events. Here, we identified one of the Arabidopsis thaliana DEAD-box RNA helicases, AtRH7, as an interactor of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3), which is an RNA chaperone involved in cold adaptation. Promoter:GUS transgenic plants revealed that AtRH7 is expressed ubiquitously and that its levels of the expression are higher in rapidly growing tissues. Knockout mutant lines displayed several morphological alterations such as disturbed vein pattern, pointed first true leaves, and short roots, which resemble ribosome-related mutants of Arabidopsis. In addition, aberrant floral development was also observed in rh7 mutants. When the mutants were germinated at low temperature (12°C), both radicle and first leaf emergence were severely delayed; after exposure of seedlings to a long period of cold, the mutants developed aberrant, fewer, and smaller leaves. RNA blots and circular RT-PCR revealed that 35S and 18S rRNA precursors accumulated to higher levels in the mutants than in WT under both normal and cold conditions, suggesting the mutants are partially impaired in pre-rRNA processing. Taken together, the results suggest that AtRH7 affects rRNA biogenesis and plays an important role in plant growth under cold. PMID:27116354

  20. Cloning, characterization, and expression analysis of the DEAD-box family genes, Fc-vasa and Fc-PL10a, in Chinese shrimp ( Fenneropenaeus chinensis)

    NASA Astrophysics Data System (ADS)

    Zhou, Qianru; Shao, Mingyu; Qin, Zhenkui; Kyoung, Ho Kang; Zhang, Zhifeng

    2010-01-01

    RNA helicases of the DEAD-box and related families are involved in various cellular processes including DNA replication, DNA repair, and RNA processing. However, the function of DEAD-box proteins in aquaculture species is poorly understood at molecular level. We obtained the full-length cDNA sequences of two genes encoding helicase-related proteins, Fc-vasa and Fc-PL10a, from the testes of Chinese shrimp, Fenneropenaeus chinensis. The two predicted amino acid sequences contain all the conserved motifs characterized by the DEAD-box family and several RGG repeats in the N-terminal regions. Homology and phylogenetic analyses indicate that they belong to the vasa and PL10 subfamilies. The three-dimensional structures of the two proteins were predicted with a homology modeling approach. Both core proteins consist of two tandem RecA-like domains similar to those of the DEAD-box RNA helicase. Using reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR we found that Fc-vasa was expressed specifically in the adult gonads. Transcription decreased in the ovary but increased in the testis during gonadal development. Fc-PL10a expression was widely distributed in the tissues we examined. Using in situ hybridization, we demonstrated that the Fc-vasa transcript is localized to the cytoplasm of the spermatogonia and oocytes. Thus, our results suggest that Fc-vasa plays an important role in germ-line development, and has utility as a germ cell lineage marker which will help to generate new insight into the origin and differentiation of germ cells as well as the regulation of reproduction in F. chinensis.

  1. Molecular cloning and characterization of a salinity stress-induced gene encoding DEAD-box helicase from the halophyte Apocynum venetum.

    PubMed

    Liu, H H; Liu, J; Fan, S L; Song, M Z; Han, X L; Liu, F; Shen, F F

    2008-01-01

    The genes encoding DEAD-box helicases play a key role in various abiotic stresses, including temperature, light, oxygen, and salt stress. A salt-responsive gene, designated AvDH1, was isolated from the halophyte dogbane (Apocynum venetum) by using suppression subtractive hybridization and RACE (rapid amplification of cDNA ends) PCR. The deduced amino acid sequence has nine conserved helicase motifs of the DEAD-box protein family. The AvDH1 gene is present as a single copy in the dogbane genome. This gene is expressed in response to NaCl and not polyethlene glycol (PEG) nor abscisic acid, and its expression increases with time. The transcription of AvDH1 is also induced by low temperature (4 degrees C), but its accumulation first increases then decreases with time. The purified recombinant protein contains ATP-dependent DNA helicase activity, ATP-independent RNA helicase activity, and DNA- or RNA-dependent ATPase activity. The ATPase activity of AvDH1 is stimulated more by single-stranded DNA than by double-stranded DNA or RNA. These results suggested that AvDH1 belonging to the DEAD-box helicase family is induced by salinity, functions as a typical helicase to unwind DNA and RNA, and may play an important role in salinity tolerance.

  2. SlDEAD31, a Putative DEAD-Box RNA Helicase Gene, Regulates Salt and Drought Tolerance and Stress-Related Genes in Tomato.

    PubMed

    Zhu, Mingku; Chen, Guoping; Dong, Tingting; Wang, Lingling; Zhang, Jianling; Zhao, Zhiping; Hu, Zongli

    2015-01-01

    The DEAD-box RNA helicases are involved in almost every aspect of RNA metabolism, associated with diverse cellular functions including plant growth and development, and their importance in response to biotic and abiotic stresses is only beginning to emerge. However, none of DEAD-box genes was well characterized in tomato so far. In this study, we reported on the identification and characterization of two putative DEAD-box RNA helicase genes, SlDEAD30 and SlDEAD31 from tomato, which were classified into stress-related DEAD-box proteins by phylogenetic analysis. Expression analysis indicated that SlDEAD30 was highly expressed in roots and mature leaves, while SlDEAD31 was constantly expressed in various tissues. Furthermore, the expression of both genes was induced mainly in roots under NaCl stress, and SlDEAD31 mRNA was also increased by heat, cold, and dehydration. In stress assays, transgenic tomato plants overexpressing SlDEAD31 exhibited dramatically enhanced salt tolerance and slightly improved drought resistance, which were simultaneously demonstrated by significantly enhanced expression of multiple biotic and abiotic stress-related genes, higher survival rate, relative water content (RWC) and chlorophyll content, and lower water loss rate and malondialdehyde (MDA) production compared to wild-type plants. Collectively, these results provide a preliminary characterization of SlDEAD30 and SlDEAD31 genes in tomato, and suggest that stress-responsive SlDEAD31 is essential for salt and drought tolerance and stress-related gene regulation in plants.

  3. SlDEAD31, a Putative DEAD-Box RNA Helicase Gene, Regulates Salt and Drought Tolerance and Stress-Related Genes in Tomato

    PubMed Central

    Zhu, Mingku; Chen, Guoping; Dong, Tingting; Wang, Lingling; Zhang, Jianling; Zhao, Zhiping; Hu, Zongli

    2015-01-01

    The DEAD-box RNA helicases are involved in almost every aspect of RNA metabolism, associated with diverse cellular functions including plant growth and development, and their importance in response to biotic and abiotic stresses is only beginning to emerge. However, none of DEAD-box genes was well characterized in tomato so far. In this study, we reported on the identification and characterization of two putative DEAD-box RNA helicase genes, SlDEAD30 and SlDEAD31 from tomato, which were classified into stress-related DEAD-box proteins by phylogenetic analysis. Expression analysis indicated that SlDEAD30 was highly expressed in roots and mature leaves, while SlDEAD31 was constantly expressed in various tissues. Furthermore, the expression of both genes was induced mainly in roots under NaCl stress, and SlDEAD31 mRNA was also increased by heat, cold, and dehydration. In stress assays, transgenic tomato plants overexpressing SlDEAD31 exhibited dramatically enhanced salt tolerance and slightly improved drought resistance, which were simultaneously demonstrated by significantly enhanced expression of multiple biotic and abiotic stress-related genes, higher survival rate, relative water content (RWC) and chlorophyll content, and lower water loss rate and malondialdehyde (MDA) production compared to wild-type plants. Collectively, these results provide a preliminary characterization of SlDEAD30 and SlDEAD31 genes in tomato, and suggest that stress-responsive SlDEAD31 is essential for salt and drought tolerance and stress-related gene regulation in plants. PMID:26241658

  4. Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo.

    PubMed Central

    Grandori, C; Mac, J; Siëbelt, F; Ayer, D E; Eisenman, R N

    1996-01-01

    The c-Myc protein is involved in cell proliferation, differentiation and apoptosis though heterodimerization with Max to form a transcriptionally active sequence-specific DNA binding complex. By means of sequential immunoprecipitation of chromatin using anti-Max and anti-Myc antibodies, we have identified a Myc-regulated gene and genomic sites occupied by Myc-Max in vivo. Four of 27 sites recovered by this procedure corresponded to the highest affinity 'canonical' CACGTG sequence. However, the most common in vivo binding sites belonged to the group of 'non-canonical' E box-related binding sites previously identified by in vitro selection. Several of the genomic fragments isolated contained transcribed sequences, including one, MrDb, encoding an evolutionarily conserved RNA helicase of the DEAD box family. The corresponding mRNA was induced following activation of a Myc-estrogen receptor fusion protein (Myc-ER) in the presence of a protein synthesis inhibitor, consistent with this helicase gene being a direct target of Myc-Max. In addition, as for c-Myc, the expression of MrDb is induced upon proliferative stimulation of primary human fibroblasts as well as B cells and down-regulated during terminal differentiation of HL60 leukemia cells. Our results indicate that Myc-Max heterodimers interact in vivo with a specific set of E box-related DNA sequences and that Myc is likely to activate multiple target genes including a highly conserved DEAD box protein. Therefore, Myc may exert its effects on cell behavior through proteins that affect RNA structure and metabolism. Images PMID:8861962

  5. Association of the cold-shock DEAD-box RNA helicase RhlE to the RNA degradosome in Caulobacter crescentus.

    PubMed

    Aguirre, Angel A; Vicente, Alexandre M; Hardwick, Steven W; Alvelos, Daniela M; Mazzon, Ricardo R; Luisi, Ben F; Marques, Marilis V

    2017-04-10

    In diverse bacterial lineages, multi-enzyme assemblies have evolved that are central elements of RNA metabolism and RNA-mediated regulation. The aquatic, Gram-negative bacteria Caulobacter crescentus, which has been a model system for studying the bacterial cell cycle, has an RNA degradosome assembly that is formed by the endoribonuclease RNase E and includes the DEAD-box RNA helicase RhlB. Immunoprecipitations of extracts from cells expressing an epitope-tagged RNase E reveal that RhlE, another member of the DEAD-box helicase family, associates with the degradosome at temperatures below the optimum for growth. Phenotype analyses of mutant strains for rhlE, rhlB and rhlE/rhlB show that RhlE is important for cell fitness at low temperature, and its role may not be substituted by RhlB. Transcriptional and translational fusions of rhlE to the lacZ reporter gene and immunoblot analysis of an epitope-tagged RhlE indicate that its expression is induced upon temperature decrease mainly through post-transcriptional regulation. RNase E pulldown assays show that other proteins, including the transcription termination factor Rho, a second DEAD-box RNA helicase and ribosomal protein S1 also associate with the degradosome at low temperature. The results suggest that the RNA degradosome assembly can be remodeled with environmental change to alter its repertoire of helicases and other accessory proteins.IMPORTANCE DEAD-box RNA helicases are often present in the RNA degradosome complex, helping to unwind secondary structures to facilitate degradation. Caulobacter crescentus is an interesting organism to investigate degradosome remodeling with temperature, because it thrives in freshwater bodies and withstands low temperature. In this study we show that at low temperature the cold-induced DEAD-box RNA helicase RhlE is recruited to the RNA degradosome, along with other helicases and the Rho protein. RhlE is essential for bacterial fitness at low temperature, and its function may not

  6. microRNAs targeting DEAD-box helicases are involved in salinity stress response in rice (Oryza sativa L.)

    PubMed Central

    2012-01-01

    Background Rice (Oryza sativa L.), one of the most important food crop in the world, is considered to be a salt-sensitive crop. Excess levels of salt adversely affect all the major metabolic activities, including cell wall damage, cytoplasmic lysis and genomic stability. In order to cope with salt stress, plants have evolved high degrees of developmental plasticity, including adaptation via cascades of molecular networks and changes in gene expression profiles. Posttranscriptional regulation, through the activity of microRNAs, also plays an important role in the plant response to salinity conditions. MicroRNAs are small endogenous RNAs that modulate gene expression and are involved in the most essential physiological processes, including plant development and adaptation to environmental changes. Results In the present study, we investigated the expression profiles of osa-MIR414, osa-MIR408 and osa-MIR164e along with their targeted genes, under salinity stress conditions in wild type and transgenic rice plants ectopically expressing the PDH45 (Pea DNA Helicase) gene. The present miRNAs were predicted to target the OsABP (ATP-Binding Protein), OsDSHCT (DOB1/SK12/helY-like DEAD-box Helicase) and OsDBH (DEAD-Box Helicase) genes, included in the DEAD-box helicase family. An in silico characterization of the proteins was performed and the miRNAs predicted targets were validated by RLM-5′RACE. The qRT-PCR analysis showed that the OsABP, OsDBH and OsDSHCT genes were up-regulated in response to 100 and 200 mM NaCl treatments. The present study also highlighted an increased accumulation of the gene transcripts in wild type plants, with the exception of the OsABP mRNA which showed the highest level (15.1-fold change compared to control) in the transgenic plants treated with 200 mM NaCl. Salinity treatments also affected the expression of osa-MIR414, osa-MIR164e and osa-MIR408, found to be significantly down-regulated, although the changes in miRNA expression were limited

  7. Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

    NASA Astrophysics Data System (ADS)

    Zhang, Yuan; Palla, Mirkó; Sun, Andrew; Liao, Jung-Chi

    2013-09-01

    DEAD-box RNA helicases are ATP-dependent proteins implicated in nearly all aspects of RNA metabolism. The yeast DEAD-box helicase Mss116 is unique in its functions of splicing group I and group II introns and activating mRNA translation, but the structural understanding of why it performs these unique functions remains unclear. Here we used sequence analysis and molecular dynamics simulation to identify residues in the flexible linker specific for yeast Mss116, potentially associated with its unique functions. We first identified residues that are 100% conserved in Mss116 of different species of the Saccharomycetaceae family. The amino acids of these conserved residues were then compared with the amino acids of the corresponding residue positions of other RNA helicases to identify residues that have distinct amino acids from other DEAD-box proteins. Four residues in the flexible linker, i.e. N334, E335, P336 and H339, are conserved and Mss116-specific. Molecular dynamics simulation was conducted for the wild-type Mss116 structure and mutant models to examine mutational effects of the linker on the conformational equilibrium. Relatively short MD simulation runs (within 20 ns) were enough for us to observe mutational effects, suggesting serious structural perturbations by these mutations. The mutation of E335 depletes the interactions between E335 and K95 in domain 1. The interactions between N334/P336 and N496/I497 of domain 2 are also abolished by mutation. Our results suggest that tight interactions between the Mss116-specific flexible linker and the two RecA-like domains may be mechanically required to crimp RNA for the unique RNA processes of yeast Mss116.

  8. The DEAD-box helicase Mss116 plays distinct roles in mitochondrial ribogenesis and mRNA-specific translation

    PubMed Central

    De Silva, Dasmanthie; Poliquin, Sarah; Zeng, Rui; Zamudio-Ochoa, Angelica; Marrero, Natalie; Perez-Martinez, Xochitl; Fontanesi, Flavia

    2017-01-01

    Abstract Members of the DEAD-box family are often multifunctional proteins involved in several RNA transactions. Among them, yeast Saccharomyces cerevisiae Mss116 participates in mitochondrial intron splicing and, under cold stress, also in mitochondrial transcription elongation. Here, we show that Mss116 interacts with the mitoribosome assembly factor Mrh4, is required for efficient mitoribosome biogenesis, and consequently, maintenance of the overall mitochondrial protein synthesis rate. Additionally, Mss116 is required for efficient COX1 mRNA translation initiation and elongation. Mss116 interacts with a COX1 mRNA-specific translational activator, the pentatricopeptide repeat protein Pet309. In the absence of Mss116, Pet309 is virtually absent, and although mitoribosome loading onto COX1 mRNA can occur, activation of COX1 mRNA translation is impaired. Mutations abolishing the helicase activity of Mss116 do not prevent the interaction of Mss116 with Pet309 but also do not allow COX1 mRNA translation. We propose that Pet309 acts as an adaptor protein for Mss116 action on the COX1 mRNA 5΄-UTR to promote efficient Cox1 synthesis. Overall, we conclude that the different functions of Mss116 in the biogenesis and functioning of the mitochondrial translation machinery depend on Mss116 interplay with its protein cofactors. PMID:28520979

  9. The DEAD Box RNA Helicase VBH-1 Is a New Player in the Stress Response in C. elegans

    PubMed Central

    Paz-Gómez, Daniel; Villanueva-Chimal, Emmanuel; Navarro, Rosa E.

    2014-01-01

    For several years, DEAD box RNA helicase Vasa (DDX4) has been used as a bona fide germline marker in different organisms. C. elegans VBH-1 is a close homolog of the Vasa protein, which plays an important role in gametogenesis, germ cell survival and embryonic development. Here, we show that VBH-1 protects nematodes from heat shock and oxidative stress. Using the germline-defective mutant glp-4(bn2) we found that a potential somatic expression of vbh-1 might be important for stress survival. We also show that the VBH-1 paralog LAF-1 is important for stress survival, although this protein is not redundant with its counterpart. Furthermore, we observed that the mRNAs of the heat shock proteins hsp-1 and sip-1 are downregulated when vbh-1 or laf-1 are silenced. Previously, we reported that in C. elegans, VBH-1 was primarily expressed in P granules of germ cells and in the cytoplasm of all blastomeres. Here we show that during stress, VBH-1 co-localizes with CGH-1 in large aggregates in the gonad core and oocytes; however, VBH-1 aggregates do not overlap with CGH-1 foci in early embryos under the same conditions. These data demonstrate that, in addition to the previously described role for this protein in the germline, VBH-1 plays an important role during the stress response in C. elegans through the potential direct or indirect regulation of stress response mRNAs. PMID:24844228

  10. Structural and functional analysis of the interaction between the nucleoporin Nup214 and the DEAD-box helicase Ddx19

    PubMed Central

    Napetschnig, Johanna; Kassube, Susanne A.; Debler, Erik W.; Wong, Richard W.; Blobel, Günter; Hoelz, André

    2009-01-01

    Key steps in the export of mRNA from the nucleus to the cytoplasm are the transport through the nuclear pore complex (NPC) and the subsequent remodeling of messenger RNA-protein (mRNP) complexes that occurs at the cytoplasmic side of the NPC. Crucial for these events is the recruitment of the DEAD-box helicase Ddx19 to the cytoplasmic filaments of the NPC that is mediated by the nucleoporin Nup214. Here, we present the crystal structure of the Nup214 N-terminal domain in complex with Ddx19 in its ADP-bound state at 2.5 Å resolution. Strikingly, the interaction surfaces are not only evolutionarily conserved but also exhibit strongly opposing surface potentials, with the helicase surface being positively and the Nup214 surface being negatively charged. We speculate that the positively charged surface of the interacting ADP-helicase binds competitively to a segment of mRNA of a linearized mRNP, passing through the NPC on its way to the cytoplasm. As a result, the ADP-helicase would dissociate from Nup214 and replace a single bound protein from the mRNA. One cycle of protein replacement would be accompanied, cooperatively, by nucleotide exchange, ATP hydrolysis, release of the ADP-helicase from mRNA and its rebinding to Nup214. Repeat of these cycles would remove proteins from a mRNP, one at a time, akin to a ratchet mechanism for mRNA export. PMID:19208808

  11. Molecular characterization and expression of buffalo (Bubalus bubalis) DEAD-box family VASA gene and mRNA transcript variants isolated from testis tissue.

    PubMed

    Kaushik, Ramakant; Singh, Karn Pratap; Bahuguna, Vivek; Rameshbabu, K; Singh, Manoj Kumar; Manik, Radhey Shyam; Palta, Prabhat; Singla, Suresh Kumar; Chauhan, Manmohan Singh

    2015-11-01

    VASA is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. In the present study, we isolated, sequenced, and characterized VASA gene in buffalo testis. Here, we demonstrated that VASA mRNA is expressed as multiple isoforms and uses four alternative transcriptional start sites (TSSs) and four different polyadenylation sites. The TSSs identified by 5'-RNA ligase-mediated rapid amplification of cDNA ends (RLM-5'-RACE) were positioned at 48, 53, 85, and 88 nucleotides upstream relative to the translation initiation codon. 3'-RACE experiment revealed the presence of tandem polyadenylation signals, which lead to the expression of at least four different 3'-untranslated regions (209, 233, 239 and 605 nucleotides). The full-length coding region of VASA was 2190 bp, which encodes a 729 amino acid (aa) protein containing nine consensus regions of the DEAD box protein family. VASA variants are highly expressed in testis of adult buffalo. We found five variants, one full length VASA (729 aa) and four splice variants VASA 2, 4, 5, 6 (683, 685, 679, 703 aa). The expression level of VASA 1 was significantly higher than rest of all (P < 0.05) except VASA 6. The relative ratio for VASA 1:2:4:5:6 was 100:1.0:1.6:0.9:48.

  12. Identification of the sites of action of SrmB, a DEAD-box RNA helicase involved in Escherichia coli ribosome assembly.

    PubMed

    Proux, Florence; Dreyfus, Marc; Iost, Isabelle

    2011-10-01

    DEAD-box RNA-dependent ATPases are ubiquitous enzymes that participate in nearly all processes involving RNA, but their detailed molecular functions remain generally unknown. SrmB, one of the five Escherichia coli DEAD-box proteins, participates in the assembly of the large ribosomal subunit notably by facilitating the incorporation of L13, one of the ribosomal proteins that bind 23S rRNA earliest. Previously, we showed that SrmB is tethered to nascent ribosome through interactions with L4, L24 and the region from domain I of 23S rRNA that binds them. To identify the sites of action of SrmB, we have characterized rRNA mutations that bypass SrmB requirement. Five of them affect the same position from two repeated heptanucleotides in domain II of 23S rRNA, whereas two others affect a complementary hexanucleotide in 5S rRNA. Thus the sites of action of SrmB differ from its tethering site. In the mature ribosome, one of the heptanucleotides participates in a highly compact structure that contacts L13, the '1024 G-ribo wrench'. In addition, we have observed that the assembly defect of ΔsrmB cells worsens as rRNA synthesis increases. Based on these results, we propose two non-exclusive scenarios for the role of SrmB in ribosome assembly. © 2011 Blackwell Publishing Ltd.

  13. ATP-competitive, marine derived natural products that target the DEAD box helicase, eIF4A.

    PubMed

    Tillotson, Joseph; Kedzior, Magdalena; Guimarães, Larissa; Ross, Alison B; Peters, Tara L; Ambrose, Andrew J; Schmidlin, Cody J; Zhang, Donna D; Costa-Lotufo, Letícia V; Rodríguez, Abimael D; Schatz, Jonathan H; Chapman, Eli

    2017-09-01

    Activation of translation initiation is a common trait of cancer cells. Formation of the heterotrimeric eukaryotic initiation factor F (eIF4F) complex is the rate-limiting step in 5' m7GpppN cap-dependent translation. This trimeric complex includes the eIF4E cap binding protein, the eIF4G scaffolding protein, and the DEAD box RNA helicase eIF4A. eIF4A is an ATP-dependent helicase and because it is the only enzyme in the eIF4F complex, it has been shown to be a potential therapeutic target for a variety of malignancies. To this end, we have used a simple ATPase biochemical screen to survey several hundred marine and terrestrial derived natural products. Herein, we report the discovery of two natural products from marine sources, elisabatin A (1) and allolaurinterol (2), which show low µM inhibition of eIF4A ATPase activity. Enzymological analyses revealed 1 and 2 to be ATP-competitive, and cellular evaluations showed reasonable cytotoxicity against A549 (lung cancer) and MDA-MA-468 (breast cancer) cell lines. However, only compound 2 showed potent inhibition of helicase activity congruent with its ATPase inhibitory activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Tobacco VDL Gene Encodes a Plastid DEAD Box RNA Helicase and Is Involved in Chloroplast Differentiation and Plant Morphogenesis

    PubMed Central

    Wang, Yingchun; Duby, Geoffrey; Purnelle, Bénédicte; Boutry, Marc

    2000-01-01

    The recessive nuclear vdl (for variegated and distorted leaf) mutant of tobacco was obtained by T-DNA insertion and characterized by variegated leaves and abnormal roots and flowers. Affected leaf tissues were white and distorted, lacked palisadic cells, and contained undifferentiated plastids. The variegation was due to phenotypic, rather than genetic, instability. Genomic and cDNA clones were obtained for both the mutant and wild-type VDL alleles. Three transcripts, resulting from alternate intron splicing or polyadenylation, were found for the wild type. The transcripts potentially encode a set of proteins (53, 19, and 15 kD) sharing the same N-terminal region that contains a chloroplast transit peptide capable of importing the green fluorescent protein into chloroplasts. The predicted 53-kD product belongs to the DEAD box RNA helicase family. In the homozygous vdl mutant, T-DNA insertion resulted in accumulation of the shortest transcript and the absence of the RNA helicase–encoding transcript. Genetic transformation of the homozygous mutant by the 53-kD product–encoding cDNA fully restored the wild-type phenotype. These data suggest that a plastid RNA helicase controls early plastid differentiation and plant morphogenesis. PMID:11090214

  15. DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function.

    PubMed

    Soltanieh, Sahar; Osheim, Yvonne N; Spasov, Krasimir; Trahan, Christian; Beyer, Ann L; Dragon, François

    2015-03-01

    DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA.

  16. Structure of the second domain of the Bacillus subtilis DEAD-box RNA helicase YxiN

    SciTech Connect

    Caruthers, Jonathan M.; Hu, YaoXiong; McKay, David B.

    2006-12-01

    The structure of the second helicase domain of the B. subtilis YxiN protein, a DEAD-box RNA helicase, is presented. The Bacillus subtilis RNA helicase YxiN is a modular three-domain protein. The first two domains form a conserved helicase core that couples an ATPase activity to an RNA duplex-destabilization activity, while the third domain recognizes a stem-loop of 23S ribosomal RNA with high affinity and specificity. The structure of the second domain, amino-acid residues 207–368, has been solved to 1.95 Å resolution, revealing a parallel αβ-fold. The crystallographic asymmetric unit contains two protomers; superposition shows that they differ substantially in two segments of peptide that overlap the conserved helicase sequence motifs V and VI, while the remainder of the domain is isostructural. The conformational variability of these segments suggests that induced fit is intrinsic to the recognition of ligands (ATP and RNA) and the coupling of the ATPase activity to conformational changes.

  17. DEAD-Box RNA Helicase Dbp4 Is Required for Small-Subunit Processome Formation and Function

    PubMed Central

    Soltanieh, Sahar; Osheim, Yvonne N.; Spasov, Krasimir; Trahan, Christian; Beyer, Ann L.

    2014-01-01

    DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5′ end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA. PMID:25535329

  18. Characterization of the kinetics of RNA annealing and strand displacement activities of the E. coli DEAD-box helicase CsdA.

    PubMed

    Stampfl, Sabine; Doetsch, Martina; Beich-Frandsen, Mads; Schroeder, Renée

    2013-01-01

    CsdA is one of five E. coli DEAD-box helicases and as a cold-shock protein assists RNA structural remodeling at low temperatures. The helicase has been shown to catalyze duplex unwinding in an ATP-dependent way and accelerate annealing of complementary RNAs, but detailed kinetic analyses are missing. Therefore, we performed kinetic measurements using a coupled annealing and strand displacement assay with high temporal resolution to analyze how CsdA balances the two converse activities. We furthermore tested the hypothesis that the unwinding activity of DEAD-box helicases is largely determined by the substrate's thermodynamic stability using full-length CsdA and a set of RNAs with constant length, but increasing GC content. The rate constants for strand displacement did indeed decrease with increasing duplex stability, with a calculated free energy between -31.3 and -40 kcal/mol being the limit for helix unwinding. Thus, our data generally support the above hypothesis, showing that for CsdA substrate thermal stability is an important rate limiting factor.

  19. Structural and biochemical analyses of the DEAD-box ATPase Sub2 in association with THO or Yra1

    PubMed Central

    Ren, Yi; Schmiege, Philip; Blobel, Günter

    2017-01-01

    mRNA is cotranscrptionally processed and packaged into messenger ribonucleoprotein particles (mRNPs) in the nucleus. Prior to export through the nuclear pore, mRNPs undergo several obligatory remodeling reactions. In yeast, one of these reactions involves loading of the mRNA-binding protein Yra1 by the DEAD-box ATPase Sub2 as assisted by the hetero-pentameric THO complex. To obtain molecular insights into reaction mechanisms, we determined crystal structures of two relevant complexes: a THO hetero-pentamer bound to Sub2 at 6.0 Å resolution; and Sub2 associated with an ATP analogue, RNA, and a C-terminal fragment of Yra1 (Yra1-C) at 2.6 Å resolution. We found that the 25 nm long THO clamps Sub2 in a half-open configuration; in contrast, when bound to the ATP analogue, RNA and Yra1-C, Sub2 assumes a closed conformation. Both THO and Yra1-C stimulated Sub2’s intrinsic ATPase activity. We propose that THO surveys common landmarks in each nuclear mRNP to localize Sub2 for targeted loading of Yra1. DOI: http://dx.doi.org/10.7554/eLife.20070.001 PMID:28059701

  20. The Expanding Functions of Cellular Helicases: The Tombusvirus RNA Replication Enhancer Co-opts the Plant eIF4AIII-Like AtRH2 and the DDX5-Like AtRH5 DEAD-Box RNA Helicases to Promote Viral Asymmetric RNA Replication

    PubMed Central

    Kovalev, Nikolay; Nagy, Peter D.

    2014-01-01

    Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. Several of the co-opted host factors bind to the viral RNA, which plays multiple roles, including mRNA function, as an assembly platform for the viral replicase (VRC), template for RNA synthesis, and encapsidation during infection. It is likely that remodeling of the viral RNAs and RNA-protein complexes during the switch from one step to another requires RNA helicases. In this paper, we have discovered a second group of cellular RNA helicases, including the eIF4AIII-like yeast Fal1p and the DDX5-like Dbp3p and the orthologous plant AtRH2 and AtRH5 DEAD box helicases, which are co-opted by tombusviruses. Unlike the previously characterized DDX3-like AtRH20/Ded1p helicases that bind to the 3′ terminal promoter region in the viral minus-strand (−)RNA, the other class of eIF4AIII-like RNA helicases bind to a different cis-acting element, namely the 5′ proximal RIII(−) replication enhancer (REN) element in the TBSV (−)RNA. We show that the binding of AtRH2 and AtRH5 helicases to the TBSV (−)RNA could unwind the dsRNA structure within the RIII(−) REN. This unique characteristic allows the eIF4AIII-like helicases to perform novel pro-viral functions involving the RIII(−) REN in stimulation of plus-strand (+)RNA synthesis. We also show that AtRH2 and AtRH5 helicases are components of the tombusvirus VRCs based on co-purification experiments. We propose that eIF4AIII-like helicases destabilize dsRNA replication intermediate within the RIII(−) REN that promotes bringing the 5′ and 3′ terminal (−)RNA sequences in close vicinity via long-range RNA-RNA base pairing. This newly formed RNA structure promoted by eIF4AIII helicase together with AtRH20 helicase might facilitate the recycling of the viral replicases for multiple rounds of (+)-strand synthesis, thus resulting in asymmetrical viral replication. PMID:24743583

  1. Cloning and characterization of a DEAD box RNA helicase from the viable seedlings of aged mung bean.

    PubMed

    Li, S C; Chung, M C; Chen, C S

    2001-12-01

    Seeds stored under adverse conditions will reduce the viability of germination as a result of induced aging. We have established a procedure to induce accelerated aging for studying the process of aging in mung bean (Vigna radiata) seeds at the molecular level. A full-length cDNA was isolated from acceleratedly aged mung bean seedlings. The cDNA, VrRH1 (Vigna radiata RNA helicase 1), contains an open reading frame of 2139 bp encoding a protein of 713 amino acids. VrRHI has seven highly conserved motifs including the DEAD box as in the case of other plant RNA helicases. VrRHI was sub-cloned into an expression vector pET-28b (+), over-expressed in Escherichia coli BL 21 and purified by a Ni2+-agarose column. The expressed protein showed double-stranded RNA unwinding and ATPase activities. Either ATP or dATP is required for the unwinding activity, indicating that VrRHI is an ATP/dATP-dependent RNA helicase. Northern blot analysis showed the presence of mRNAs hybridized with a full-length cDNA fragment of VrRHI (VrRH transcripts) in mung bean seeds that were imbibed for 16 to 32 h after accelerated aging treatment. The amount of these mRNAs reached a maximum in 24 h imbibed seeds after the treatment. The accumulation of VrRH transcripts was shown to lead to the appearance of 25S and 18S rRNAs in the imbibed aging mung bean seeds. The results suggest that VrRHI may play a role in the viability of mung bean seeds.

  2. DDX4 (DEAD box polypeptide 4) colocalizes with cancer stem cell marker CD133 in ovarian cancers

    SciTech Connect

    Kim, Ki Hyung; Kang, Yun-Jeong; Jo, Jin-Ok; Ock, Mee Sun; Moon, Soo Hyun; Suh, Dong Soo; Yoon, Man Soo; Park, Eun-Sil; Jeong, Namkung; Eo, Wan-Kyu; Kim, Heung Yeol; Cha, Hee-Jae

    2014-05-02

    Highlights: • Germ cell marker DDX4 was significantly increased in ovarian cancer. • Ovarian cancer stem cell marker CD133 was significantly increased in ovarian cancer. • DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. • CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4. • Germ cell marker DDX4 has the potential of ovarian cancer stem cell marker. - Abstract: DDX4 (DEAD box polypeptide 4), characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), is an RNA helicase which is implicated in various cellular processes involving the alteration of RNA secondary structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. DDX4 is known to be a germ cell-specific protein and is used as a sorting marker of germline stem cells for the production of oocytes. A recent report about DDX4 in ovarian cancer showed that DDX4 is overexpressed in epithelial ovarian cancer and disrupts a DNA damage-induced G2 checkpoint. We investigated the relationship between DDX4 and ovarian cancer stem cells by analyzing the expression patterns of DDX4 and the cancer stem cell marker CD133 in ovarian cancers via tissue microarray. Both DDX4 and CD133 were significantly increased in ovarian cancer compared to benign tumors, and showed similar patterns of expression. In addition, DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. Furthermore, almost all CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4, suggesting a strong possibility that DDX4 plays an important role in cancer stem cells, and/or can be used as an ovarian cancer stem cell marker.

  3. Pea p68, a DEAD-box helicase, provides salinity stress tolerance in transgenic tobacco by reducing oxidative stress and improving photosynthesis machinery.

    PubMed

    Tuteja, Narendra; Banu, Mst Sufara Akhter; Huda, Kazi Md Kamrul; Gill, Sarvajeet Singh; Jain, Parul; Pham, Xuan Hoi; Tuteja, Renu

    2014-01-01

    The DEAD-box helicases are required mostly in all aspects of RNA and DNA metabolism and they play a significant role in various abiotic stresses, including salinity. The p68 is an important member of the DEAD-box proteins family and, in animal system, it is involved in RNA metabolism including pre-RNA processing and splicing. In plant system, it has not been well characterized. Here we report the cloning and characterization of p68 from pea (Pisum sativum) and its novel function in salinity stress tolerance in plant. The pea p68 protein self-interacts and is localized in the cytosol as well as the surrounding of cell nucleus. The transcript of pea p68 is upregulated in response to high salinity stress in pea. Overexpression of p68 driven by constitutive cauliflower mosaic virus-35S promoter in tobacco transgenic plants confers enhanced tolerances to salinity stress by improving the growth, photosynthesis and antioxidant machinery. Under stress treatment, pea p68 overexpressing tobacco accumulated higher K+ and lower Na+ level than the wild-type plants. Reactive oxygen species (ROS) accumulation was remarkably regulated by the overexpression of pea p68 under salinity stress conditions, as shown from TBARS content, electrolyte leakage, hydrogen peroxide accumulation and 8-OHdG content and antioxidant enzyme activities. To the best of our knowledge this is the first direct report, which provides the novel function of pea p68 helicase in salinity stress tolerance. The results suggest that p68 can also be exploited for engineering abiotic stress tolerance in crop plants of economic importance.

  4. The DEAD box helicase RDE-12 promotes amplification of RNAi in cytoplasmic foci in C. elegans

    PubMed Central

    Yang, Huan; Vallandingham, Jim; Shiu, Philip; Li, Hua; Hunter, Craig P.; Mak, Ho Yi

    2014-01-01

    Summary RNA interference (RNAi) is a potent mechanism for down-regulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi and other eukaryotes [1–3]. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary siRNAs [4–6]. Exogenous double stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis [7, 8]. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear if the sub-cellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively [9–11]. We report that RDE-12, a conserved FG domain containing DEAD-box helicase, localizes in P-granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA targeted mRNA in distinct cytoplasmic compartments. PMID:24684930

  5. DEAD-box helicase DDX27 regulates 3′ end formation of ribosomal 47S RNA and stably associates with the PeBoW-complex

    SciTech Connect

    Kellner, Markus; Rohrmoser, Michaela; Forné, Ignasi; Voss, Kirsten; Burger, Kaspar; Mühl, Bastian; Gruber-Eber, Anita; Kremmer, Elisabeth; Imhof, Axel; Eick, Dirk

    2015-05-15

    PeBoW, a trimeric complex consisting of pescadillo (Pes1), block of proliferation (Bop1), and the WD repeat protein 12 (WDR12), is essential for processing and maturation of mammalian 5.8S and 28S ribosomal RNAs. Applying a mass spectrometric analysis, we identified the DEAD-box helicase DDX27 as stably associated factor of the PeBoW-complex. DDX27 interacts with the PeBoW-complex via an evolutionary conserved F×F motif in the N-terminal domain and is recruited to the nucleolus via its basic C-terminal domain. This recruitment is RNA-dependent and occurs independently of the PeBoW-complex. Interestingly, knockdown of DDX27, but not of Pes1, induces the accumulation of an extended form of the primary 47S rRNA. We conclude that DDX27 can interact specifically with the Pes1 and Bop1 but fulfils critical function(s) for proper 3′ end formation of 47S rRNA independently of the PeBoW-complex. - Highlights: • DEAD-box helicase DDX27 is a new constituent of the PeBoW-complex. • The N-terminal F×F motif of DDX27 interacts with the PeBoW components Pes1 and Bop1. • Nucleolar anchoring of DDX27 via its basic C-terminal domain is RNA dependent. • Knockdown of DDX27 induces a specific defect in 3′ end formation of 47S rRNA.

  6. DP97, a DEAD box DNA/RNA helicase, is a target gene-selective co-regulator of the constitutive androstane receptor

    SciTech Connect

    Kanno, Yuichiro; Serikawa, Takafumi; Inajima, Jun; Inouye, Yoshio

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer DP97 interacts with nuclear receptor CAR. Black-Right-Pointing-Pointer DP97 enhances CAR-mediated transcriptional activation. Black-Right-Pointing-Pointer DP97 synergistically enhances transactivity of CAR by the co-expression of SRC-1 or PGC1{alpha}. Black-Right-Pointing-Pointer DP97 is a gene-selective co-activator for hCAR. -- Abstract: The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that DP97, a member of the DEAD box DNA/RNA helicase protein family, is a novel CAR-interacting protein. Using HepG2 cells expressing human CAR in the presence of tetracycline, we showed that knockdown of DP97 with small interfering RNAs suppressed tetracycline-inducible mRNA expression of CYP2B6 and UGT1A1 but not CYP3A4. Thus, DP97 was found to be a gene (or promoter)-selective co-activator for hCAR. DP97-mediated CAR transactivation was synergistically enhanced by the co-expression of SRC-1 or PGC1{alpha}, therefore it might act as mediator between hCAR and appropriate co-activators.

  7. Isolation and functional characterization of the promoter of a DEAD-box helicase Psp68 using Agrobacterium-mediated transient assay.

    PubMed

    Banu, Sufara Akhter; Huda, Kazi Md Kamrul; Tuteja, Narendra

    2014-04-30

    Helicases are molecular motor proteins that perform a variety of cellular functions including transcription, translation, DNA replication and repair, RNA maturation, ribosome synthesis, nuclear export and splicing processes. The p68 is an evolutionarily conserved protein which plays pivotal roles in all aspect RNA metabolism processes. It is well established that helicases provides abiotic stress adaptation in plants but analysis of cis-regulatory elements present in the upstream regions is still infancy. Here we report isolation and functional characterization of the promoter of a DEAD-box helicase Psp68 in response to abiotic stress and hormonal regulation. The promoter of Psp68 was isolated by gene walking PCR from pea genomic DNA library constructed in BD genome walker kit. In silico analysis revealed that promoter of Psp68 contained a TATA, a CAAT motif and also harbors some important stress and hormone associated cis regulatory elements, including E-box, AGAAA, GATA-box, ACGT, GAAAA and GTCTC. Functional analyses were performed by Agrobacterium-mediated transient assay in tobacco leaves. Very high level of GUS activity was observed in agroinfiltrated tobacco leaves by the construct carrying the Psp68 promoter::GUS, subjected to abiotic stress and exogenous hormonal treatments. Stress-inducible nature of Psp68 promoter opens possibility for the study of the gene regulation under stress condition. Therefore, may be useful in the field of agriculture and biotechnology.

  8. Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells.

    PubMed

    Berneking, Laura; Schnapp, Marie; Rumm, Andreas; Trasak, Claudia; Ruckdeschel, Klaus; Alawi, Malik; Grundhoff, Adam; Kikhney, Alexey G; Koch-Nolte, Friedrich; Buck, Friedrich; Perbandt, Markus; Betzel, Christian; Svergun, Dmitri I; Hentschke, Moritz; Aepfelbacher, Martin

    2016-06-01

    Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines.

  9. Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells

    PubMed Central

    Rumm, Andreas; Trasak, Claudia; Ruckdeschel, Klaus; Alawi, Malik; Grundhoff, Adam; Kikhney, Alexey G.; Koch-Nolte, Friedrich; Buck, Friedrich; Perbandt, Markus; Betzel, Christian; Svergun, Dmitri I.; Hentschke, Moritz; Aepfelbacher, Martin

    2016-01-01

    Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines. PMID:27300509

  10. The DEAD-box RNA helicase 51 controls non-small cell lung cancer proliferation by regulating cell cycle progression via multiple pathways

    PubMed Central

    Wang, Xiaojing; Liu, Hongli; Zhao, Chengling; Li, Wei; Xu, Huanbai; Chen, Yuqing

    2016-01-01

    The genetic regulation of cell cycle progression and cell proliferation plays a role in the growth of non-small cell lung cancer (NSCLC), one of the most common causes of cancer-related mortality. Although DEAD-box RNA helicases are known to play a role in cancer development, including lung cancer, the potential involvement of the novel family member DDX51 has not yet been investigated. In the current study we assessed the role of DDX51 in NSCLC using a siRNA-based approach. DDX51 siRNA-expressing cells exhibited a slower cell proliferation rate and underwent arrest in S-phase of the cell cycle compared with control cells. Microarray analyses revealed that DDX51siRNA expression resulted in the dysregulation of a number of cell signalling pathways. Moreover, injection of DDX51 siRNA into an animal model resulted in the formation of smaller tumours compared with the control group. We also assessed the expression of DDX51 in patients with NSCLC, and the data revealed that the expression was correlated with patient age but no other risk factors. Overall, our data suggest for the first time that DDX51 aids cell cancer proliferation by regulating multiple signalling pathways, and that this protein might be a therapeutic target for NSCLC. PMID:27198888

  11. Cellular DEAD-box RNA helicase DDX6 modulates interaction of miR-122 with the 5' untranslated region of hepatitis C virus RNA.

    PubMed

    Biegel, Jason M; Henderson, Eric; Cox, Erica M; Bonenfant, Gaston; Netzband, Rachel; Kahn, Samantha; Eager, Rachel; Pager, Cara T

    2017-07-01

    Hepatitis C virus (HCV) subverts the cellular DEAD-box RNA helicase DDX6 to promote virus infection. Using polysome gradient analysis and the subgenomic HCV Renilla reporter replicon genome, we determined that DDX6 does not affect HCV translation. Rather expression of the subgenomic HCV Renilla luciferase reporter at late times, as well as labeling of newly synthesized viral RNA with 4-thiouridine showed that DDX6 modulates replication. Because DDX6 is an effector protein of the microRNA pathway, we also investigated its role in miR-122-directed HCV gene expression. Similar to sequestering miR-122, depletion of DDX6 modulated HCV RNA stability. Interestingly, miR-122-HCV RNA interaction assays with mutant HCV genomes sites and compensatory exogenous miR-122 showed that DDX6 affects the function of miR-122 at one particular binding site. We propose that DDX6 facilitates the miR-122 interaction with HCV 5' UTR, which is necessary for stabilizing the viral genome and the switch between translation and replication. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. The eukaryotic initiation factor eIF4H facilitates loop-binding, repetitive RNA unwinding by the eIF4A DEAD-box helicase.

    PubMed

    Sun, Yingjie; Atas, Evrim; Lindqvist, Lisa; Sonenberg, Nahum; Pelletier, Jerry; Meller, Amit

    2012-07-01

    Eukaryotic translation initiation is a highly regulated process in protein synthesis. The principal translation initiation factor eIF4AI displays helicase activity, unwinding secondary structures in the mRNAs 5'-UTR. Single molecule fluorescence resonance energy transfer (sm-FRET) is applied here to directly observe and quantify the helicase activity of eIF4AI in the presence of the ancillary RNA-binding factor eIF4H. Results show that eIF4H can significantly enhance the helicase activity of eIF4AI by strongly binding both to loop structures within the RNA transcript as well as to eIF4AI. In the presence of ATP, the eIF4AI/eIF4H complex exhibits persistent rapid and repetitive cycles of unwinding and re-annealing. ATP titration assays suggest that this process consumes a single ATP molecule per cycle. In contrast, helicase unwinding activity does not occur in the presence of the non-hydrolysable analog ATP-γS. Based on our sm-FRET results, we propose an unwinding mechanism where eIF4AI/eIF4H can bind directly to loop structures to destabilize duplexes. Since eIF4AI is the prototypical example of a DEA(D/H)-box RNA helicase, it is highly likely that this unwinding mechanism is applicable to a myriad of DEAD-box helicases employed in RNA metabolism.

  13. DEAD-box helicase DDX27 regulates 3' end formation of ribosomal 47S RNA and stably associates with the PeBoW-complex.

    PubMed

    Kellner, Markus; Rohrmoser, Michaela; Forné, Ignasi; Voss, Kirsten; Burger, Kaspar; Mühl, Bastian; Gruber-Eber, Anita; Kremmer, Elisabeth; Imhof, Axel; Eick, Dirk

    2015-05-15

    PeBoW, a trimeric complex consisting of pescadillo (Pes1), block of proliferation (Bop1), and the WD repeat protein 12 (WDR12), is essential for processing and maturation of mammalian 5.8S and 28S ribosomal RNAs. Applying a mass spectrometric analysis, we identified the DEAD-box helicase DDX27 as stably associated factor of the PeBoW-complex. DDX27 interacts with the PeBoW-complex via an evolutionary conserved F×F motif in the N-terminal domain and is recruited to the nucleolus via its basic C-terminal domain. This recruitment is RNA-dependent and occurs independently of the PeBoW-complex. Interestingly, knockdown of DDX27, but not of Pes1, induces the accumulation of an extended form of the primary 47S rRNA. We conclude that DDX27 can interact specifically with the Pes1 and Bop1 but fulfils critical function(s) for proper 3' end formation of 47S rRNA independently of the PeBoW-complex.

  14. SACY-1 DEAD-Box Helicase Links the Somatic Control of Oocyte Meiotic Maturation to the Sperm-to-Oocyte Switch and Gamete Maintenance in Caenorhabditis elegans

    PubMed Central

    Kim, Seongseop; Govindan, J. Amaranath; Tu, Zheng Jin; Greenstein, David

    2012-01-01

    In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. In Caenorhabditis elegans, major sperm protein triggers meiotic resumption through a mechanism involving somatic Gαs–adenylate cyclase signaling and soma-to-germline gap-junctional communication. Using genetic mosaic analysis, we show that the major effector of Gαs–adenylate cyclase signaling, protein kinase A (PKA), is required in gonadal sheath cells for oocyte meiotic maturation and dispensable in the germ line. This result rules out a model in which cyclic nucleotides must transit through sheath-oocyte gap junctions to activate PKA in the germ line, as proposed in vertebrate systems. We conducted a genetic screen to identify regulators of oocyte meiotic maturation functioning downstream of Gαs–adenylate cyclase–PKA signaling. We molecularly identified 10 regulatory loci, which include essential and nonessential factors. sacy-1, which encodes a highly conserved DEAD-box helicase, is an essential germline factor that negatively regulates meiotic maturation. SACY-1 is a multifunctional protein that establishes a mechanistic link connecting the somatic control of meiotic maturation to germline sex determination and gamete maintenance. Modulatory factors include multiple subunits of a CoREST-like complex and the TWK-1 two-pore potassium channel. These factors are not absolutely required for meiotic maturation or its negative regulation in the absence of sperm, but function cumulatively to enable somatic control of meiotic maturation. This work provides insights into the genetic control of meiotic maturation signaling in C. elegans, and the conserved factors identified here might inform analysis in other systems through either homology or analogy. PMID:22887816

  15. Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28

    SciTech Connect

    Jacewicz, Agata; Schwer, Beate; Smith, Paul; Shuman, Stewart

    2014-10-10

    Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5' splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1–89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127–588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127–588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to the adenine nucleobase. The triphosphate moiety of AMPPNP•Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg2+•RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. As a result, overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.

  16. Binding of DEAD-box helicase Dhh1 to the 5'-untranslated region of ASH1 mRNA represses localized translation of ASH1 in yeast cells.

    PubMed

    Zhang, Qianjun; Meng, Xiuhua; Li, Delin; Chen, Shaoyin; Luo, Jianmin; Zhu, Linjie; Singer, Robert H; Gu, Wei

    2017-06-09

    Local translation of specific mRNAs is regulated by dynamic changes in their subcellular localization, and these changes are due to complex mechanisms controlling cytoplasmic mRNA transport. The budding yeast Saccharomyces cerevisiae is well suited to studying these mechanisms because many of its transcripts are transported from the mother cell to the budding daughter cell. Here, we investigated the translational control of ASH1 mRNA after transport and localization. We show that although ASH1 transcripts were translated after they reached the bud tip, some mRNAs were bound by the RNA-binding protein Puf6 and were non-polysomal. We also found that the DEAD-box helicase Dhh1 complexed with the untranslated ASH1 mRNA and Puf6. Loss of Dhh1 affected local translation of ASH1 mRNA and resulted in delocalization of ASH1 transcript in the bud. Forcibly shifting the non-polysomal ASH1 mRNA into polysomes was associated with Dhh1 dissociation. We further demonstrated that Dhh1 is not recruited to ASH1 mRNA co-transcriptionally, suggesting that it could bind to ASH1 mRNA within the cytoplasm. Of note, Dhh1 bound to the 5'-UTR of ASH1 mRNA and inhibited its translation in vitro These results suggest that after localization to the bud tip, a portion of the localized ASH1 mRNA becomes translationally inactive because of binding of Dhh1 and Puf6 to the 5'- and 3'-UTRs of ASH1 mRNA. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28

    DOE PAGES

    Jacewicz, Agata; Schwer, Beate; Smith, Paul; ...

    2014-10-10

    Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5' splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1–89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127–588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127–588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to themore » adenine nucleobase. The triphosphate moiety of AMPPNP•Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg2+•RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. As a result, overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.« less

  18. A DEAD Box RNA Helicase Is Critical for Pre-mRNA Splicing, Cold-Responsive Gene Regulation, and Cold Tolerance in Arabidopsis[C][W

    PubMed Central

    Guan, Qingmei; Wu, Jianmin; Zhang, Yanyan; Jiang, Changhua; Liu, Renyi; Chai, Chenglin; Zhu, Jianhua

    2013-01-01

    Cold stress resulting from chilling and freezing temperatures substantially reduces crop production worldwide. To identify genes critical for cold tolerance in plants, we screened Arabidopsis thaliana mutants for deregulated expression of a firefly luciferase reporter gene under the control of the C-REPEAT BINDING FACTOR2 (CBF2) promoter (CBF2:LUC). A regulator of CBF gene expression1 (rcf1-1) mutant that is hypersensitive to cold stress was chosen for in-depth characterization. RCF1 encodes a cold-inducible DEAD (Asp-Glu-Ala-Asp) box RNA helicase. Unlike a previously reported DEAD box RNA helicase (LOW EXPRESSION OF OSMOTICALLY RESPONSIVE GENES4 [LOS4]) that regulates mRNA export, RCF1 does not play a role in mRNA export. Instead, RCF1 functions to maintain proper splicing of pre-mRNAs; many cold-responsive genes are mis-spliced in rcf1-1 mutant plants under cold stress. Functional characterization of four genes (PSEUDO-RESPONSE REGULATOR5 [PRR5], SHAGGY-LIKE SERINE/THREONINE KINASE12 [SK12], MYB FAMILY TRANSCRIPTION FACTOR CIRCADIAN1 [CIR1], and SPFH/PHB DOMAIN-CONTAINING MEMBRANE-ASSOCIATED PROTEIN [SPFH]) that are mis-spliced in rcf1-1 revealed that these genes are cold-inducible positive (CIR1 and SPFH) and negative (PRR5 and SK12) regulators of cold-responsive genes and cold tolerance. Together, our results suggest that the cold-inducible RNA helicase RCF1 is essential for pre-mRNA splicing and is important for cold-responsive gene regulation and cold tolerance in plants. PMID:23371945

  19. The plastidic DEAD-box RNA helicase 22, HS3, is essential for plastid functions both in seed development and in seedling growth.

    PubMed

    Kanai, Masatake; Hayashi, Makoto; Kondo, Maki; Nishimura, Mikio

    2013-09-01

    Plants accumulate large amounts of storage products in seeds to provide an energy reserve and to supply nutrients for germination and post-germinative growth. Arabidopsis thaliana belongs to the Brassica family, and oil is the main storage product in Arabidopsis seeds. To elucidate the regulatory mechanisms of oil biosynthesis in seeds, we screened for high density seeds (heavy seed) that have a low oil content. HS3 (heavy seed 3) encodes the DEAD-box RNA helicase 22 that is localized to plastids. The triacylglycerol (TAG) content of hs3-1 seeds was 10% lower than that of wild-type (WT) seeds, while the protein content was unchanged. The hs3-1 plants displayed a pale-green phenotype in developing seeds and seedlings, but not in adult leaves. The HS3 expression level was high in developing seeds and seedlings, but was low in stems, rosette leaves and flowers. The plastid gene expression profile of WT developing seeds and seedlings differed from that of hs3-1 developing seeds and seedlings. The expression of several genes was reduced in developing hs3-1 seeds, including accD, a gene that encodes the β subunit of carboxyltransferase, which is one component of acetyl-CoA carboxylase in plastids. In contrast, no differences were observed between the expression profiles of WT and hs3-1 rosette leaves. These results show that HS3 is essential for proper mRNA accumulation of plastid genes during seed development and seedling growth, and suggest that HS3 ensures seed oil biosynthesis by maintaining plastid mRNA levels.

  20. Global effects of the DEAD-box RNA helicase DeaD (CsdA) on gene expression over a broad range of temperatures

    PubMed Central

    Vakulskas, Christopher A.; Pannuri, Archana; Cortés-Selva, Diana; Zere, Tesfalem R.; Ahmer, Brian M.; Babitzke, Paul; Romeo, Tony

    2014-01-01

    Summary In Escherichia coli, activity of the global regulatory RNA binding protein CsrA is antagonized by two noncoding sRNAs, CsrB and CsrC, which sequester it away from its lower affinity mRNA targets. Transcription of csrB/C requires the BarA-UvrY two component signal transduction system, which responds to short chain carboxylates. We show that two DEAD-box RNA helicases, DeaD and SrmB, activate csrB/C expression by different pathways. DeaD facilitates uvrY translation by counteracting the inhibitory effect of long distance basepairing between the uvrY mRNA leader and coding region, while SrmB does not affect UvrY or UvrY-phosphate levels. Contrary to the prevailing notion that these helicases act primarily at low temperatures, DeaD and SrmB activated csrB expression over a wide temperature range. High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) revealed in vivo interactions of DeaD with 39 mRNAs, including those of uvrY and 9 other regulatory genes. Studies on the expression of several of the identified genes revealed regulatory effects of DeaD in all cases and diverse temperature response patterns. Our findings uncover an expanded regulatory role for DeaD, which is mediated through novel mRNA targets, important global regulators and under physiological conditions that were considered to be incompatible with its function. PMID:24708042

  1. The DEAD-box RNA helicase Vasa functions in embryonic mitotic progression in the sea urchin.

    PubMed

    Yajima, Mamiko; Wessel, Gary M

    2011-06-01

    Vasa is a broadly conserved ATP-dependent RNA helicase that functions in the germ line of organisms from cnidarians to mammals. Curiously, Vasa is also present in the somatic cells of many animals and functions as a regulator of multipotent cells. Here, we report a mitotic function of Vasa revealed in the sea urchin embryo. We found that Vasa protein is present in all blastomeres of the early embryo and that its abundance oscillates with the cell cycle. Vasa associates with the spindle and the separating sister chromatids at metaphase, and then quickly disappears after telophase. Inhibition of Vasa protein synthesis interferes with proper chromosome segregation, arrests cells at M-phase, and delays overall cell cycle progression. Cdk activity is necessary for the proper localization of Vasa, implying that Vasa is involved in the cyclin-dependent cell cycle network, and Vasa is required for the efficient translation of cyclinB mRNA. Our results suggest an evolutionarily conserved role of Vasa that is independent of its function in germ line determination.

  2. A conformational change in the helicase core is necessary but not sufficient for RNA unwinding by the DEAD box helicase YxiN.

    PubMed

    Karow, Anne R; Klostermeier, Dagmar

    2009-07-01

    Cooperative binding of ATP and RNA to DEAD-box helicases induces the closed conformation of their helicase core, with extensive interactions across the domain interface. The bound RNA is bent, and its distortion may constitute the first step towards RNA unwinding. To dissect the role of the conformational change in the helicase core for RNA unwinding, we characterized the RNA-stimulated ATPase activity, RNA unwinding and the propensity to form the closed conformer for mutants of the DEAD box helicase YxiN. The ATPase-deficient K52Q mutant forms a closed conformer upon binding of ATP and RNA, but is deficient in RNA unwinding. A mutation in motif III slows down the catalytic cycle, but neither affects the propensity for the closed conformer nor its global conformation. Hence, the closure of the cleft in the helicase core is necessary but not sufficient for RNA unwinding. In contrast, the G303A mutation in motif V prevents a complete closure of the inter-domain cleft, affecting ATP binding and hydrolysis and is detrimental to unwinding. Possibly, the K52Q and motif III mutants still introduce a kink into the backbone of bound RNA, whereas G303A fails to kink the RNA substrate.

  3. Duplex unwinding and ATPase activities of the DEAD-box helicase eIF4A are coupled by eIF4G and eIF4B.

    PubMed

    Özeş, Ali R; Feoktistova, Kateryna; Avanzino, Brian C; Fraser, Christopher S

    2011-09-30

    Eukaryotic initiation factor (eIF) 4A is a DEAD-box helicase that stimulates translation initiation by unwinding mRNA secondary structure. The accessory proteins eIF4G, eIF4B, and eIF4H enhance the duplex unwinding activity of eIF4A, but the extent to which they modulate eIF4A activity is poorly understood. Here, we use real-time fluorescence assays to determine the kinetic parameters of duplex unwinding and ATP hydrolysis by these initiation factors. To ensure efficient duplex unwinding, eIF4B and eIF4G cooperatively activate the duplex unwinding activity of eIF4A. Our data reveal that eIF4H is much less efficient at stimulating eIF4A unwinding activity than eIF4B, implying that eIF4H is not able to completely substitute for eIF4B in duplex unwinding. By monitoring unwinding and ATPase assays under identical conditions, we demonstrate that eIF4B couples the ATP hydrolysis cycle of eIF4A with strand separation, thereby minimizing nonproductive unwinding events. Using duplex substrates with altered GC contents but similar predicted thermal stabilities, we further show that the rate of formation of productive unwinding complexes is strongly influenced by the local stability per base pair, in addition to the stability of the entire duplex. This finding explains how a change in the GC content of a hairpin is able to influence translation initiation while maintaining the overall predicted thermal stability.

  4. Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1

    PubMed Central

    Alessi, Amelia F.; Khivansara, Vishal; Han, Ting; Freeberg, Mallory A.; Moresco, James J.; Tu, Patricia G.; Montoye, Eric; Yates, John R.; Karp, Xantha; Kim, John K.

    2015-01-01

    MicroRNAs (miRNAs) play essential, conserved roles in diverse developmental processes through association with the miRNA-induced silencing complex (miRISC). Whereas fundamental insights into the mechanistic framework of miRNA biogenesis and target gene silencing have been established, posttranslational modifications that affect miRISC function are less well understood. Here we report that the conserved serine/threonine kinase, casein kinase II (CK2), promotes miRISC function in Caenorhabditis elegans. CK2 inactivation results in developmental defects that phenocopy loss of miRISC cofactors and enhances the loss of miRNA function in diverse cellular contexts. Whereas CK2 is dispensable for miRNA biogenesis and the stability of miRISC cofactors, it is required for efficient miRISC target mRNA binding and silencing. Importantly, we identify the conserved DEAD-box RNA helicase, CGH-1/DDX6, as a key CK2 substrate within miRISC and demonstrate phosphorylation of a conserved N-terminal serine is required for CGH-1 function in the miRNA pathway. PMID:26669440

  5. Characterization of a homolog of DEAD-box RNA helicases in Toxoplasma gondii as a marker of cytoplasmic mRNP stress granules.

    PubMed

    Cherry, Ahmed Adnan; Ananvoranich, Sirinart

    2014-06-10

    Toxoplasma gondii is an obligate intracellular protozoan which infects one-third of the human population. Due to its high infection prevalence, Toxoplasma offers an ideal system for the study of host-parasite interaction. Similar to other eukaryotes, Toxoplasma maintains levels and localization of cytoplasmic mRNAs throughout its life cycle as part of a gene regulation network to meet all cellular and biochemical requirements. More recently, it was reported that the presence of cytoplasmic mRNA granules could contribute to the parasite pathogenesis and viability. Here we identified a novel Toxoplasma DEAD-box RNA helicase, referred to as Toxoplasma gondiiHomolog of DOZI (TgHoDI), because of its high homology (81%) to Plasmodium DOZI. TgHoDI is the functional ortholog of yeast DHH1, and its function was authenticated by complementation studies in Δdhh1 yeast strain. We demonstrated that TgHoDI is a marker of cytoplasmic RNA stress granules, which assemble when the parasites experience cellular stresses and translational arrest.

  6. An essential role for the Saccharomyces cerevisiae DEAD-box helicase DHH1 in G1/S DNA-damage checkpoint recovery.

    PubMed Central

    Bergkessel, Megan; Reese, Joseph C

    2004-01-01

    The eukaryotic cell cycle displays a degree of plasticity in its regulation; cell cycle progression can be transiently arrested in response to environmental stresses. While the signaling pathways leading to cell cycle arrest are beginning to be well understood, the regulation of the release from arrest has not been well characterized. Here we show that DHH1, encoding a DEAD-box RNA helicase orthologous to the human putative proto-oncogene p54/RCK, is important in release from DNA-damage-induced cell cycle arrest at the G1/S checkpoint. DHH1 mutants are not defective for DNA repair and recover normally from the G2/M and replication checkpoints, suggesting a specific function for Dhh1p in recovery from G1/S checkpoint arrest. Dhh1p has been suggested to play a role in partitioning mRNAs between translatable and nontranslatable pools, and our results implicate this modulation of mRNA metabolism in the recovery from G1/S cell cycle arrest following DNA damage. Furthermore, the high degree of conservation between DHH1 and its human ortholog suggests that this mechanism is conserved among all eukaryotes and potentially important in human disease. PMID:15166134

  7. Overexpression of an Apocynum venetum DEAD-Box Helicase Gene (AvDH1) in Cotton Confers Salinity Tolerance and Increases Yield in a Saline Field

    PubMed Central

    Chen, Jie; Wan, Sibao; Liu, Huaihua; Fan, Shuli; Zhang, Yujuan; Wang, Wei; Xia, Minxuan; Yuan, Rui; Deng, Fenni; Shen, Fafu

    2016-01-01

    Soil salinity is a major environmental stress limiting plant growth and productivity. We have reported previously the isolation of an Apocynum venetum DEAD-box helicase 1 (AvDH1) that is expressed in response to salt exposure. Here, we report that the overexpression of AvDH1 driven by a constitutive cauliflower mosaic virus-35S promoter in cotton plants confers salinity tolerance. Southern and Northern blotting analyses showed that the AvDH1 gene was integrated into the cotton genome and expressed. In this study, the growth of transgenic cotton expressing AvDH1 was evaluated under saline conditions in a growth chamber and in a saline field trial. Transgenic cotton overexpressing AvDH1 was much more resistant to salt than the wild-type plants when grown in a growth chamber. The lower membrane ion leakage, along with increased activity of superoxide dismutase, in AvDH1 transgenic lines suggested that these characteristics may prevent membrane damage, which increases plant survival rates. In a saline field, the transgenic cotton lines expressing AvDH1 showed increased boll numbers, boll weights and seed cotton yields compared with wild-type plants, especially at high soil salinity levels. This study indicates that transgenic cotton expressing AvDH1 is a promising option for increasing crop productivity in saline fields. PMID:26779246

  8. The DEAD-Box RNA Helicase DDX3 Interacts with NF-κB Subunit p65 and Suppresses p65-Mediated Transcription

    PubMed Central

    Gao, Yu; Song, Feifei; Guo, Liang; Ma, Li; Sun, Guihong; Liu, Dan; Guo, Deyin

    2016-01-01

    RNA helicase family members exhibit diverse cellular functions, including in transcription, pre-mRNA processing, RNA decay, ribosome biogenesis, RNA export and translation. The RNA helicase DEAD-box family member DDX3 has been characterized as a tumour-associated factor and a transcriptional co-activator/regulator. Here, we demonstrate that DDX3 interacts with the nuclear factor (NF)-κB subunit p65 and suppresses NF-κB (p65/p50)-mediated transcriptional activity. The downregulation of DDX3 by RNA interference induces the upregulation of NF-κB (p65/p50)-mediated transcription. The regulation of NF-κB (p65/p50)-mediated transcriptional activity was further confirmed by the expression levels of its downstream cytokines, such as IL-6 and IL-8. Moreover, the binding of the ATP-dependent RNA helicase domain of DDX3 to the N-terminal Rel homology domain (RHD) of p65 is involved in the inhibition of NF-κB-regulated gene transcription. In summary, the results suggest that DDX3 functions to suppress the transcriptional activity of the NF-κB subunit p65. PMID:27736973

  9. Regulation of MicroRNA 183 by Cyclooxygenase 2 in Liver Is DEAD-Box Helicase p68 (DDX5) Dependent: Role in Insulin Signaling

    PubMed Central

    Motiño, Omar; Francés, Daniel E.; Castro-Sánchez, Luis; Fernández-Velasco, María; Boscá, Lisardo; García-Monzón, Carmelo; Brea, Rocío; Casado, Marta

    2015-01-01

    Cyclooxygenase (COX) catalyzes the first step in prostanoid biosynthesis and exists as two isoforms. COX-1 is a constitutive enzyme involved in physiological processes, whereas COX-2 is induced by a variety of stimuli. MicroRNAs (miRNAs) are noncoding RNAs that function as key posttranscriptional regulators of gene expression. Although it is known that COX-2 expression is regulated by miRNAs, there are no data regarding COX-2 involvement in miRNA regulation. Considering our previous results showing that COX-2 expression in hepatocytes protects against insulin resistance, we evaluated the role of COX-2 in the regulation of a specific set of miRNAs implicated in insulin signaling in liver cells. Our results provide evidence of the molecular basis for a novel function of COX-2 in miRNA processing. COX-2 represses miRNA 23b (miR-23b), miR-146b, and miR-183 expression in liver cells by increasing the level of DEAD-box helicase p68 (DDX5) through phosphatidylinositol 3-kinase (PI3K)/p300 signaling and by modulating the enzymatic function of the Drosha (RNase type III) complex through its physical association with DDX5. The decrease of miR-183 expression promotes protection against insulin resistance by increasing insulin receptor substrate 1 (IRS1) levels. These results indicate that the modulation of miRNA processing by COX-2 is a key event in insulin signaling in liver and has potential clinical implications for the management of various hepatic dysfunctions. PMID:25963660

  10. An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon iosAsp formation but is restored by Protein Isoaspartyl Methltransferase

    USDA-ARS?s Scientific Manuscript database

    Arabidopsis thaliana PLANT RNA HELICASE75 (AtPRH75) demonstrated an ATP-dependent, RNA duplex unwinding capacity and an ATP-independent, RNA duplex reforming ability. It is known to accumulate isoAsp, but the consequences of isoAsp formation in AtPRH75 are unknown. Duplex unwinding was abolished by ...

  11. DEAD-box RNA helicase DDX3 connects CRM1-dependent nuclear export and translation of the HIV-1 unspliced mRNA through its N-terminal domain.

    PubMed

    Fröhlich, Alvaro; Rojas-Araya, Bárbara; Pereira-Montecinos, Camila; Dellarossa, Alessandra; Toro-Ascuy, Daniela; Prades-Pérez, Yara; García-de-Gracia, Francisco; Garcés-Alday, Andrea; Rubilar, Paulina S; Valiente-Echeverría, Fernando; Ohlmann, Théophile; Soto-Rifo, Ricardo

    2016-05-01

    DEAD-box RNA helicase DDX3 is a host factor essential for HIV-1 replication and thus, a potential target for novel therapies aimed to overcome viral resistance. Previous studies have shown that DDX3 promotes nuclear export and translation of the HIV-1 unspliced mRNA. Although the function of DDX3 during both processes requires its catalytic activity, it is unknown whether other domains surrounding the helicase core are involved. Here, we show the involvement of the N- and C-terminal domains of DDX3 in the regulation of HIV-1 unspliced mRNA translation. Our results suggest that the intrinsically disordered N-terminal domain of DDX3 regulates its functions in translation by acting prior to the recruitment of the 43S pre-initiation complex onto the viral 5'-UTR. Interestingly, this regulation was conserved in HIV-2 and was dependent on the CRM1-dependent nuclear export pathway suggesting a role of the RNA helicase in interconnecting nuclear export with ribosome recruitment of the viral unspliced mRNA. This specific function of DDX3 during HIV gene expression could be exploited as an alternative target for pharmaceutical intervention.

  12. Systematic Determination of Human Cyclin Dependent Kinase (CDK)-9 Interactome Identifies Novel Functions in RNA Splicing Mediated by the DEAD Box (DDX)-5/17 RNA Helicases.

    PubMed

    Yang, Jun; Zhao, Yingxin; Kalita, Mridul; Li, Xueling; Jamaluddin, Mohammad; Tian, Bing; Edeh, Chukwudi B; Wiktorowicz, John E; Kudlicki, Andrzej; Brasier, Allan R

    2015-10-01

    Inducible transcriptional elongation is a rapid, stereotypic mechanism for activating immediate early immune defense genes by the epithelium in response to viral pathogens. Here, the recruitment of a multifunctional complex containing the cyclin dependent kinase 9 (CDK9) triggers the process of transcriptional elongation activating resting RNA polymerase engaged with innate immune response (IIR) genes. To identify additional functional activity of the CDK9 complex, we conducted immunoprecipitation (IP) enrichment-stable isotope labeling LC-MS/MS of the CDK9 complex in unstimulated cells and from cells activated by a synthetic dsRNA, polyinosinic/polycytidylic acid [poly (I:C)]. 245 CDK9 interacting proteins were identified with high confidence in the basal state and 20 proteins in four functional classes were validated by IP-SRM-MS. These data identified that CDK9 interacts with DDX 5/17, a family of ATP-dependent RNA helicases, important in alternative RNA splicing of NFAT5, and mH2A1 mRNA two proteins controlling redox signaling. A direct comparison of the basal versus activated state was performed using stable isotope labeling and validated by IP-SRM-MS. Recruited into the CDK9 interactome in response to poly(I:C) stimulation are HSPB1, DNA dependent kinases, and cytoskeletal myosin proteins that exchange with 60S ribosomal structural proteins. An integrated human CDK9 interactome map was developed containing all known human CDK9- interacting proteins. These data were used to develop a probabilistic global map of CDK9-dependent target genes that predicted two functional states controlling distinct cellular functions, one important in immune and stress responses. The CDK9-DDX5/17 complex was shown to be functionally important by shRNA-mediated knockdown, where differential accumulation of alternatively spliced NFAT5 and mH2A1 transcripts and alterations in downstream redox signaling were seen. The requirement of CDK9 for DDX5 recruitment to NFAT5 and mH2A1

  13. Use of DEAD-box polypeptide-4 (Ddx4) gene promoter-driven fluorescent reporter mice to identify mitotically active germ cells in post-natal mouse ovaries

    PubMed Central

    Park, Eun-Sil; Tilly, Jonathan L.

    2015-01-01

    Several laboratories have independently isolated mitotically active germ cells, termed female germline stem cells or oogonial stem cells (OSCs), from adult mouse ovaries. However, a recent study using Ddx4-Cre;Rosa26 reporter mice concluded that such germ cells do not exist. Given the disparity in conclusions drawn in this study compared with others, we felt it was important to re-assess the utility of Ddx4-Cre;Rosa26 reporter mice for identification of OSCs in adult mouse ovaries. Transgenic Ddx4-Cre mice were crossed with Rosa26tdTm/tdTm mice to drive restricted tomato red (tdTm) gene expression in cells in which the Ddx4 gene promoter has been activated. Crude dispersion of ovaries from recombined offspring generated cell fractions containing tdTm-positive immature oocytes, which are incapable of proliferation and thus probably represent the uncharacterized reporter-positive ovarian cells identified in the paper Zhang et al. (2012) as being mitotically inactive. Dispersed ovaries further subjected to fluorescence-activated cell sorting yielded a large population of non-germline tdTm-positive cells, indicative of promoter ‘leakiness’ in the Ddx4-Cre mouse line. Nonetheless, a small percentage of these tdTm-positive cells exhibited externalized (extracellular, ec) expression of Ddx4 protein (ecDdx4-positive), expressed markers of primitive germ cells but not of oocytes, and actively proliferated in culture, all of which are characteristic features of OSCs. Thus, crude dispersion of ovaries collected from Ddx4 gene promoter-driven reporter mice is not, by itself, a reliable approach to identify OSCs, whereas the same ovarian dispersates further subjected to cell sorting strategies yield purified OSCs that can be expanded in culture. PMID:25147160

  14. Use of DEAD-box polypeptide-4 (Ddx4) gene promoter-driven fluorescent reporter mice to identify mitotically active germ cells in post-natal mouse ovaries.

    PubMed

    Park, Eun-Sil; Tilly, Jonathan L

    2015-01-01

    Several laboratories have independently isolated mitotically active germ cells, termed female germline stem cells or oogonial stem cells (OSCs), from adult mouse ovaries. However, a recent study using Ddx4-Cre;Rosa26 reporter mice concluded that such germ cells do not exist. Given the disparity in conclusions drawn in this study compared with others, we felt it was important to re-assess the utility of Ddx4-Cre;Rosa26 reporter mice for identification of OSCs in adult mouse ovaries. Transgenic Ddx4-Cre mice were crossed with Rosa26(tdTm/tdTm) mice to drive restricted tomato red (tdTm) gene expression in cells in which the Ddx4 gene promoter has been activated. Crude dispersion of ovaries from recombined offspring generated cell fractions containing tdTm-positive immature oocytes, which are incapable of proliferation and thus probably represent the uncharacterized reporter-positive ovarian cells identified in the paper Zhang et al. (2012) as being mitotically inactive. Dispersed ovaries further subjected to fluorescence-activated cell sorting yielded a large population of non-germline tdTm-positive cells, indicative of promoter 'leakiness' in the Ddx4-Cre mouse line. Nonetheless, a small percentage of these tdTm-positive cells exhibited externalized (extracellular, ec) expression of Ddx4 protein (ecDdx4-positive), expressed markers of primitive germ cells but not of oocytes, and actively proliferated in culture, all of which are characteristic features of OSCs. Thus, crude dispersion of ovaries collected from Ddx4 gene promoter-driven reporter mice is not, by itself, a reliable approach to identify OSCs, whereas the same ovarian dispersates further subjected to cell sorting strategies yield purified OSCs that can be expanded in culture. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. A chloroplast-localized DEAD-box RNA helicaseAtRH3 is essential for intron splicing and plays an important role in the growth and stress response in Arabidopsis thaliana.

    PubMed

    Gu, Lili; Xu, Tao; Lee, Kwanuk; Lee, Kwang Ho; Kang, Hunseung

    2014-09-01

    Although many DEAD-box RNA helicases (RHs) are targeted to chloroplasts, the functional roles of the majority of RHs are still unknown. Recently, the chloroplast-localized Arabidopsis thaliana AtRH3 has been demonstrated to play important roles in intron splicing, ribosome biogenesis, and seedling growth. To further understand the functional role of AtRH3 in intron splicing and growth and the stress response in Arabidopsis, the newly-generated artificial microRNA-mediated knockdown plants as well as the previously characterized T-DNA tagged rh3-4 mutant were analyzed under normal and stress conditions. The rh3 mutants displayed retarded growth and pale-green phenotypes, and the growth of mutant plants was inhibited severely under salt or cold stress but marginally under dehydration stress conditions. Splicing of several intron-containing chloroplast genes was defective in the mutant plants. Importantly, splicing of ndhA and ndhB genes was severely inhibited in the mutant plants compared with the wild-type plants under salt or cold stress but not under dehydration stress conditions. Moreover, AtRH3 complemented the growth-defect phenotype of the RNA chaperone-deficient Escherichia coli mutant and had the ability to disrupt RNA and DNA base pairs, indicating that AtRH3 possesses RNA chaperone activity. Taken together, these results demonstrate that AtRH3 plays a prominent role in the growth and stress response of Arabidopsis, and suggest that proper splicing of introns governed by RNA chaperone activity of AtRH3 is crucial for chloroplast function and the growth and stress response of plants. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  16. RNA helicase proteins as chaperones and remodelers

    PubMed Central

    Jarmoskaite, Inga; Russell, Rick

    2014-01-01

    Superfamily 2 helicase proteins are ubiquitous in RNA biology and have an extraordinarily broad set of functional roles. Central among these roles are to promote rearrangements of structured RNAs and to remodel RNA-protein complexes (RNPs), allowing formation of native RNA structure or progression through a functional cycle of structures. While all superfamily 2 helicases share a conserved helicase core, they are divided evolutionarily into several families, and it is principally proteins from three families, the DEAD-box, DEAH/RHA and Ski2-like families, that function to manipulate structured RNAs and RNPs. Strikingly, there are emerging differences in the mechanisms of these proteins, both between families and within the largest family (DEAD-box), and these differences appear to be tuned to their RNA or RNP substrates and their specific roles. This review outlines basic mechanistic features of the three families and surveys individual proteins and the current understanding of their biological substrates and mechanisms. PMID:24635478

  17. Post-translational regulation by gustavus contributes to selective Vasa protein accumulation in multipotent cells during embryogenesis

    PubMed Central

    Gustafson, Eric A.; Yajima, Mamiko; Juliano, Celina E.; Wessel, Gary M.

    2010-01-01

    Vasa is a broadly conserved DEAD-box RNA helicase associated with germ line development and is expressed in multipotent cells in many animals. During embryonic development of the sea urchin Strongylocentrotus purpuratus, Vasa protein is enriched in the small micromeres despite a uniform distribution of vasa transcript. Here we show that the Vasa coding region is sufficient for its selective enrichment and find that gustavus, the B30.2/SPRY and SOCS box domain gene, contributes to this phenomenon. In vitro binding analyses show that Gustavus binds the N-terminal and DEAD-box portions of Vasa protein independently. A knockdown of Gustavus protein reduces both Vasa protein abundance and its propensity for accumulation in the small micromeres, whereas overexpression of the Vasa-interacting domain of Gustavus (GusΔSOCS) results in Vasa protein accumulation throughout the embryo. We propose that Gustavus has a conserved, positive regulatory role in Vasa protein accumulation during embryonic development. PMID:21035437

  18. The Azoarcus Group I Intron Ribozyme Misfolds and Is Accelerated for Refolding by ATP-dependent RNA Chaperone Proteins*

    PubMed Central

    Sinan, Selma; Yuan, Xiaoyan; Russell, Rick

    2011-01-01

    Structured RNAs traverse complex energy landscapes that include valleys representing misfolded intermediates. In Neurospora crassa and Saccharomyces cerevisiae, efficient splicing of mitochondrial group I and II introns requires the DEAD box proteins CYT-19 and Mss116p, respectively, which promote folding transitions and function as general RNA chaperones. To test the generality of RNA misfolding and the activities of DEAD box proteins in vitro, here we measure native folding of a small group I intron ribozyme from the bacterium Azoarcus by monitoring its catalytic activity. To develop this assay, we first measure cleavage of an oligonucleotide substrate by the prefolded ribozyme. Substrate cleavage is rate-limited by binding and is readily reversible, with an internal equilibrium near unity, such that the amount of product observed is less than the amount of native ribozyme. We use this assay to show that approximately half of the ribozyme folds readily to the native state, whereas the other half forms an intermediate that transitions slowly to the native state. This folding transition is accelerated by urea and increased temperature and slowed by increased Mg2+ concentration, suggesting that the intermediate is misfolded and must undergo transient unfolding during refolding to the native state. CYT-19 and Mss116p accelerate refolding in an ATP-dependent manner, presumably by disrupting structure in the intermediate. These results highlight the tendency of RNAs to misfold, underscore the roles of CYT-19 and Mss116p as general RNA chaperones, and identify a refolding transition for further dissection of the roles of DEAD box proteins in RNA folding. PMID:21878649

  19. P68 RNA Helicase Is A Nucleocytoplasm Shuttling Protein

    PubMed Central

    Wang, Haizhen; Gao, Xueliang; Huang, Yun; Yang, Jenny; Liu, Zhi-Ren

    2009-01-01

    P68 RNA helicase is a prototypical DEAD box RNA helicase. The protein plays a very important role in early organ development and maturation. In consistence with the function of the protein in transcriptional regulation and pre-mRNA splicing, p68 was found to predominately localize in the cell nucleus. However, recent experiments demonstrate a transient cytoplasmic localization of the protein. We report here that p68 shuttles between the nucleus and the cytoplasm. The nucleocytoplasmic shuttling of p68 is mediated by two nuclear localization signal (NLS) and two nuclear exporting signal (NES) sequence elements. Our experiments reveal that p68 shuttles via a classical RanGTPase dependent pathway. PMID:19786986

  20. Proteomic profiling of cellular proteins interacting with the hepatitis C virus core protein.

    PubMed

    Kang, Su-Min; Shin, Min-Jung; Kim, Jung-Hee; Oh, Jong-Won

    2005-05-01

    Hepatitis C virus (HCV) is a causative agent of chronic hepatitis and hepatocellular carcinoma. The core protein of HCV packages the viral RNA genome to form a nucleocapsid. In addition to its function as a structural protein, core protein is involved in regulation of cellular transcription, virus-induced transformation, and pathogenesis. To gain insights into cellular functions of the core protein by identification of cellular proteins interacting with the core protein, we employed a proteomic approach. Hepatocytes soluble cytoplasmic proteins were applied to the core proteins immobilized on Ni-nitrilotriacetic resin and total bound cellular proteins were resolved by 2-DE. Analyses of interacting proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry allowed identification of 14 cellular proteins binding to the core protein. These proteins include DEAD-box polypeptide 5, similar in function to a known protein identified previously by yeast two-hybrid screening and 13 newly identified cellular proteins. Interestingly, nine protein spots were identified as intermediate microfilament proteins, including cytokeratins (five spots for cytokeratin 8, two for cytokeratin 19, and one for cytokeratin 18) and vimentin. Cytokeratin 8 and vimentin, which were previously shown to be involved in the infection processes of other viruses, were further analyzed to confirm their in vivo interactions with the core protein by immunoblotting and immunofluorescence microscopy. We discuss the functional implications of the interactions of the core protein with newly identified cellular proteins in HCV infection and pathogenesis.

  1. Cancer-Associated Mutants of RNA Helicase DDX3X Are Defective in RNA-Stimulated ATP Hydrolysis

    DOE PAGES

    Epling, Leslie B.; Grace, Christy R.; Lowe, Brandon R.; ...

    2015-02-25

    The DEAD-box RNA helicase DDX3X is frequently mutated in pediatric medulloblastoma. We dissect how these mutants affect DDX3X function with structural, biochemical, and genetic experiments. We identify an N-terminal extension (“ATP-binding loop”, ABL) that is critical for the stimulation of ATP hydrolysis by RNA. We present crystal structures suggesting that the ABL interacts dynamically with ATP and confirming that the interaction occurs in solution by NMR chemical shift perturbation and isothermal titration calorimetry. DEAD-box helicases require interaction between two conserved RecA-like helicase domains, D1 and D2 for function. We use NMR chemical shift perturbation to show that DDX3X interacts specificallymore » with double-stranded RNA through its D1 domain, with contact mediated by residues G302 and G325. Mutants of these residues, G302V and G325E, are associated with pediatric medulloblastoma. These mutants are defective in RNA-stimulated ATP hydrolysis. We show that DDX3X complements the growth defect in a ded1 temperature-sensitive strain of Schizosaccharomyces pombe, but the cancer-associated mutants G302V and G325E do not complement and exhibit protein expression defects. In conclusion, taken together, our results suggest that impaired translation of important mRNA targets by mutant DDX3X represents a key step in the development of medulloblastoma.« less

  2. Exploration of Csp genes from temperate and glacier soils of the Indian Himalayas and in silico analysis of encoding proteins.

    PubMed

    Latha, Prema K; Soni, Ravindra; Khan, Mahejibin; Marla, Soma S; Goel, Reeta

    2009-04-01

    The metagenomic Csp library was constructed from the temperate and glacier soils of central Himalaya, India followed by polymerase chain reaction (PCR) amplification. The library was further screened for low-temperature adaptation, and the positive recombinants were sorted out by determining changes in the melting temperature (Tm). A homology search of cloned sequence showed their identity with the Csp genes of Pseudomonas fluorescens, Psychrobacter cryohalolentis K5, and Shewanella spp MR-4. Amino acid sequence analysis annotated the presence of conserved aromatic and basic amino acids as well as RNA binding motifs from the cold shock domain. Furthermore, a PROSITE scan showed a moderate identity of less than 60% with the known cold shock-inducible proteins (ribosomal proteins, rbfA, DEAD-box helicases), cold acclimation protein, and temperature-induced protein (SRP1/TIP1). This study highlighted the prevalence of Csp genes from cold Himalayan environments that can be explored for tailor-made crop constructions in future.

  3. Group II Introns and Their Protein Collaborators

    NASA Astrophysics Data System (ADS)

    Solem, Amanda; Zingler, Nora; Pyle, Anna Marie; Li-Pook-Than, Jennifer

    Group II introns are an abundant class of autocatalytic introns that excise themselves from precursor mRNAs. Although group II introns are catalytic RNAs, they require the assistance of proteins for efficient splicing in vivo. Proteins that facilitate splicing of organellar group II introns fall into two main categories: intron-encoded maturases and host-encoded proteins. This chapter will focus on the host proteins that group II introns recruited to ensure their function. It will discuss the great diversity of these proteins, define common features, and describe different strategies employed to achieve specificity. Special emphasis will be placed on DEAD-box ATPases, currently the best studied example of host-encoded proteins with a role in group II intron splicing. Since the exact mechanisms by which splicing is facilitated is not known for any of the host proteins, general mechanistic strategies for protein-mediated RNA folding are described and assessed for their potential role in group II intron splicing.

  4. A Conserved Metal Binding Motif in the Bacillus subtilis Competence Protein ComFA Enhances Transformation.

    PubMed

    Chilton, Scott S; Falbel, Tanya G; Hromada, Susan; Burton, Briana M

    2017-08-01

    Genetic competence is a process in which cells are able to take up DNA from their environment, resulting in horizontal gene transfer, a major mechanism for generating diversity in bacteria. Many bacteria carry homologs of the central DNA uptake machinery that has been well characterized in Bacillus subtilis It has been postulated that the B. subtilis competence helicase ComFA belongs to the DEAD box family of helicases/translocases. Here, we made a series of mutants to analyze conserved amino acid motifs in several regions of B. subtilis ComFA. First, we confirmed that ComFA activity requires amino acid residues conserved among the DEAD box helicases, and second, we show that a zinc finger-like motif consisting of four cysteines is required for efficient transformation. Each cysteine in the motif is important, and mutation of at least two of the cysteines dramatically reduces transformation efficiency. Further, combining multiple cysteine mutations with the helicase mutations shows an additive phenotype. Our results suggest that the helicase and metal binding functions are two distinct activities important for ComFA function during transformation.IMPORTANCE ComFA is a highly conserved protein that has a role in DNA uptake during natural competence, a mechanism for horizontal gene transfer observed in many bacteria. Investigation of the details of the DNA uptake mechanism is important for understanding the ways in which bacteria gain new traits from their environment, such as drug resistance. To dissect the role of ComFA in the DNA uptake machinery, we introduced point mutations into several motifs in the protein sequence. We demonstrate that several amino acid motifs conserved among ComFA proteins are important for efficient transformation. This report is the first to demonstrate the functional requirement of an amino-terminal cysteine motif in ComFA. Copyright © 2017 American Society for Microbiology.

  5. Physics of protein motility and motor proteins

    NASA Astrophysics Data System (ADS)

    Kolomeisky, Anatoly B.

    2013-09-01

    Motor proteins are enzymatic molecules that transform chemical energy into mechanical motion and work. They are critically important for supporting various cellular activities and functions. In the last 15 years significant progress in understanding the functioning of motor proteins has been achieved due to revolutionary breakthroughs in single-molecule experimental techniques and strong advances in theoretical modelling. However, microscopic mechanisms of protein motility are still not well explained, and the collective efforts of many scientists are needed in order to solve these complex problems. In this special section the reader will find the latest advances on the difficult road to mapping motor proteins dynamics in various systems. Recent experimental developments have allowed researchers to monitor and to influence the activity of single motor proteins with a high spatial and temporal resolution. It has stimulated significant theoretical efforts to understand the non-equilibrium nature of protein motility phenomena. The latest results from all these advances are presented and discussed in this special section. We would like to thank the scientists from all over the world who have reported their latest research results for this special section. We are also grateful to the staff and editors of Journal of Physics: Condensed Matter for their invaluable help in handling all the administrative and refereeing activities. The field of motor proteins and protein motility is fast moving, and we hope that this collection of articles will be a useful source of information in this highly interdisciplinary area. Physics of protein motility and motor proteins contents Physics of protein motility and motor proteinsAnatoly B Kolomeisky Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116 Yuan Zhang, Mirkó Palla, Andrew Sun and Jung-Chi Liao The load dependence of the physical properties of a molecular motor

  6. Protein composition of human mRNPs spliced in vitro and differential requirements for mRNP protein recruitment

    PubMed Central

    Merz, Christian; Urlaub, Henning; Will, Cindy L.; Lührmann, Reinhard

    2007-01-01

    The deposition of proteins onto newly spliced mRNAs has far reaching consequences for their subsequent metabolism. We affinity-purified spliced human mRNPs under physiological conditions from HeLa nuclear extract and present the first comprehensive inventory of their protein composition as determined by mass spectrometry. Several proteins previously not known to be mRNP-associated were detected, including the DEAD-box helicases DDX3, DDX5, and DDX9, and the ELG, hNHN1, BCLAF1, and TRAP150 proteins. The association of some of the newly identified mRNP proteins was shown to be splicing-dependent, but not to require EJC formation. Initial recruitment of EJC proteins to the spliceosome did not require an EJC binding platform at the −20/24 region of the 5′ exon. Finally, while recruitment of EJC proteins and stable EJC formation were not dependent on the cap binding complex, several of the newly identified mRNP proteins required the latter for their association with mRNPs. These results provide novel insights into the composition of spliced mRNPs and the requirements for the association of mRNP proteins with the newly spliced mRNA. PMID:17095540

  7. Inhibition of eukaryotic translation by tetratricopeptide-repeat proteins of Orientia tsutsugamushi.

    PubMed

    Bang, Sunyoung; Min, Chan-Ki; Ha, Na-Young; Choi, Myung-Sik; Kim, Ik-Sang; Kim, Yeon-Sook; Cho, Nam-Hyuk

    2016-02-01

    Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of scrub typhus. The genome of Orientia tsutsugamushi has revealed multiple ORFs encoding tetratricopeptide-repeat (TPR) proteins. The TPR protein family has been shown to be involved in a diverse spectrum of cellular functions such as cell cycle control, transcription, protein transport, and protein folding, especially in eukaryotic cells. However, little is known about the function of the TPR proteins in O. tsutsugamushi. To investigate the potential role of TPR proteins in host-pathogen interaction, two oriential TPR proteins were expressed in E. coli and applied for GSTpull down assay. DDX3, a DEAD-box containing RNA helicase, was identified as a specific eukaryotic target of the TPR proteins. Since the RNA helicase is involved in multiple RNA-modifying processes such as initiation of translation reaction, we performed in vitro translation assay in the presence of GST-TPR fusion proteins by using rabbit reticulocyte lysate system. The TPR proteins inhibited in vitro translation of a reporter luciferase in a dose dependent manner whereas the GST control proteins did not. These results suggested TPR proteins of O. tsutsugamushi might be involved in the modulation of eukaryotic translation through the interaction with DDX3 RNA helicase after secretion into host cytoplasm.

  8. The Proteasomal Rpn11 Metalloprotease Suppresses Tombusvirus RNA Recombination and Promotes Viral Replication via Facilitating Assembly of the Viral Replicase Complex

    PubMed Central

    Prasanth, K. Reddisiva; Barajas, Daniel

    2014-01-01

    ABSTRACT RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a “matchmaker” that brings the viral p92pol replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. IMPORTANCE RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA

  9. Identification of BZR1-interacting Proteins as Potential Components of the Brassinosteroid Signaling Pathway in Arabidopsis Through Tandem Affinity Purification*

    PubMed Central

    Wang, Chunming; Shang, Jian-Xiu; Chen, Qi-Xiu; Oses-Prieto, Juan A.; Bai, Ming-Yi; Yang, Yihong; Yuan, Min; Zhang, Yu-Lan; Mu, Cong-Cong; Deng, Zhiping; Wei, Chuang-Qi; Burlingame, Alma L.; Wang, Zhi-Yong; Sun, Ying

    2013-01-01

    Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14–3-3λ, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipid-binding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response. PMID:24019147

  10. eIF4B stimulates translation of long mRNAs with structured 5′ UTRs and low closed-loop potential but weak dependence on eIF4G

    PubMed Central

    Sen, Neelam Dabas; Zhou, Fujun; Harris, Michael S.; Ingolia, Nicholas T.

    2016-01-01

    DEAD-box RNA helicases eukaryotic translation initiation factor 4A (eIF4A) and Ded1 promote translation by resolving mRNA secondary structures that impede preinitiation complex (PIC) attachment to mRNA or scanning. Eukaryotic translation initiation factor 4B (eIF4B) is a cofactor for eIF4A but also might function independently of eIF4A. Ribosome profiling of mutants lacking eIF4B or with impaired eIF4A or Ded1 activity revealed that eliminating eIF4B reduces the relative translational efficiencies of many more genes than does inactivation of eIF4A, despite comparable reductions in bulk translation, and few genes display unusually strong requirements for both factors. However, either eliminating eIF4B or inactivating eIF4A preferentially impacts mRNAs with longer, more structured 5′ untranslated regions (UTRs). These findings reveal an eIF4A-independent role for eIF4B in addition to its function as eIF4A cofactor in promoting PIC attachment or scanning on structured mRNAs. eIF4B, eIF4A, and Ded1 mutations also preferentially impair translation of longer mRNAs in a fashion mitigated by the ability to form closed-loop messenger ribonucleoprotein particles (mRNPs) via eIF4F–poly(A)-binding protein 1 (Pab1) association, suggesting cooperation between closed-loop assembly and eIF4B/helicase functions. Remarkably, depleting eukaryotic translation initiation factor 4G (eIF4G), the scaffold subunit of eukaryotic translation initiation factor 4F (eIF4F), preferentially impacts short mRNAs with strong closed-loop potential and unstructured 5′ UTRs, exactly the opposite features associated with hyperdependence on the eIF4B/helicases. We propose that short, highly efficient mRNAs preferentially depend on the stimulatory effects of eIF4G-dependent closed-loop assembly. PMID:27601676

  11. Multiple binding of repressed mRNAs by the P-body protein Rck/p54

    PubMed Central

    Ernoult-Lange, Michèle; Baconnais, Sonia; Harper, Maryannick; Minshall, Nicola; Souquere, Sylvie; Boudier, Thomas; Bénard, Marianne; Andrey, Philippe; Pierron, Gérard; Kress, Michel; Standart, Nancy; le Cam, Eric; Weil, Dominique

    2012-01-01

    Translational repression is achieved by protein complexes that typically bind 3′ UTR mRNA motifs and interfere with the formation of the cap-dependent initiation complex, resulting in mRNPs with a closed-loop conformation. We demonstrate here that the human DEAD-box protein Rck/p54, which is a component of such complexes and central to P-body assembly, is in considerable molecular excess with respect to cellular mRNAs and enriched to a concentration of 0.5 mM in P-bodies, where it is organized in clusters. Accordingly, multiple binding of p54 proteins along mRNA molecules was detected in vivo. Consistently, the purified protein bound RNA with no sequence specificity and high nanomolar affinity. Moreover, bound RNA molecules had a relaxed conformation. While RNA binding was ATP independent, relaxing of bound RNA was dependent on ATP, though not on its hydrolysis. We propose that Rck/p54 recruitment by sequence-specific translational repressors leads to further binding of Rck/p54 along mRNA molecules, resulting in their masking, unwinding, and ultimately recruitment to P-bodies. Rck/p54 proteins located at the 5′ extremity of mRNA can then recruit the decapping complex, thus coupling translational repression and mRNA degradation. PMID:22836354

  12. SIRT7 and the DEAD-box helicase DDX21 cooperate to resolve genomic R loops and safeguard genome stability.

    PubMed

    Song, Chenlin; Hotz-Wagenblatt, Agnes; Voit, Renate; Grummt, Ingrid

    2017-08-08

    R loops are three-stranded nucleic acid structures consisting of an RNA:DNA heteroduplex and a "looped-out" nontemplate strand. As aberrant formation and persistence of R loops block transcription elongation and cause DNA damage, mechanisms that resolve R loops are essential for genome stability. Here we show that the DEAD (Asp-Glu-Ala-Asp)-box RNA helicase DDX21 efficiently unwinds R loops and that depletion of DDX21 leads to accumulation of cellular R loops and DNA damage. Significantly, the activity of DDX21 is regulated by acetylation. Acetylation by CBP inhibits DDX21 activity, while deacetylation by SIRT7 augments helicase activity and overcomes R-loop-mediated stalling of RNA polymerases. Knockdown of SIRT7 leads to the same phenotype as depletion of DDX21 (i.e., increased formation of R loops and DNA double-strand breaks), indicating that SIRT7 and DDX21 cooperate to prevent R-loop accumulation, thus safeguarding genome integrity. Moreover, DDX21 resolves estrogen-induced R loops on estrogen-responsive genes in breast cancer cells, which prevents the blocking of transcription elongation on these genes. © 2017 Song et al.; Published by Cold Spring Harbor Laboratory Press.

  13. The emerging roles of the DDX41 protein in immunity and diseases.

    PubMed

    Jiang, Yan; Zhu, Yanping; Liu, Zhi-Jie; Ouyang, Songying

    2017-02-01

    RNA helicases are involved in almost every aspect of RNA, from transcription to RNA decay. DExD/H-box helicases comprise the largest SF2 helicase superfamily, which are characterized by two conserved RecA-like domains. In recent years, an increasing number of unexpected functions of these proteins have been discovered. They play important roles not only in innate immune response but also in diseases like cancers and chronic hepatitis C. In this review, we summarize the recent literatures on one member of the SF2 superfamily, the DEAD-box protein DDX41. After bacterial or viral infection, DNA or cyclic-di-GMP is released to cells. After phosphorylation of Tyr414 by BTK kinase, DDX41 will act as a sensor to recognize the invaders, followed by induction of type I interferons (IFN). After the immune response, DDX41 is degraded by the E3 ligase TRIM21, using Lys9 and Lys115 of DDX41 as the ubiquitination sites. Besides the roles in innate immunity, DDX41 is also related to diseases. An increasing number of both inherited and acquired mutations in DDX41 gene are identified from myelodysplastic syndrome and/or acute myeloid leukemia (MDS/AML) patients. The review focuses on DDX41, as well as its homolog Abstrakt in Drosophila, which is important for survival at all stages throughout the life cycle of the fly.

  14. Vasa protein expression is restricted to the small micromeres of the sea urchin, but is inducible in other lineages early in development

    PubMed Central

    Voronina, Ekaterina; Lopez, Manuel; Juliano, Celina E.; Gustafson, Eric; Song, Jia L.; Extavour, Cassandra; George, Sophie; Oliveri, Paola; McClay, David; Wessel, Gary

    2009-01-01

    Vasa is a DEAD-box RNA helicase that functions in translational regulation of specific mRNAs. In many animals it is essential for germ line development and may have a more general stem cell role. Here we identify vasa in two sea urchin species and analyze the regulation of its expression. We find that vasa protein accumulates in only a subset of cells containing vasa mRNA. In contrast to vasa mRNA, which is present uniformly throughout all cells of the early embryo, vasa protein accumulates selectively in the 16 cell stage micromeres, and then is restricted to the small micromeres through gastrulation to larval development. Manipulating early embryonic fate specification by blastomere separations, exposure to lithium, and dominant-negative cadherin each suggest that, although vasa-protein accumulation in the small micromeres is fixed, accumulation in other cells of the embryo is inducible. Indeed, we find that embryos in which micromeres are removed, respond by significant up-regulation of vasa protein translation, followed by spatial restriction of the protein late in gastrulation. Overall, these results support the contention that sea urchins do not have obligate primordial germ cells determined in early development, that vasa may function in an early stem cell population of the embryo, and that vasa-expression in this embryo is restricted early by translational regulation to the small micromere lineage. PMID:18191830

  15. How to proteins move along DNA? Lessons from type-I and type-III restriction endonucleases.

    PubMed

    Szczelkun, M D

    2000-01-01

    Protein-mediated communications on DNA are universally important. The translocation of DNA driven by a high-energy phosphoryl potential allows long stretches of DNA to be traversed without dissociation. Type-I and type-III enzymes both use a common DNA-tracking mechanism to move along DNA, dependent on the hydrolysis of ATP. Type-I enzymes cleave DNA at distant DNA sites (and in some cases close to the site), due to a stall in enzyme motion. This can be due to collision with another translocating type-I enzyme or, on circular DNA, due to an increased topological load. ATP hydrolysis is considerable, and continues after DNA cleavage. Type-III enzymes only cleave DNA proximal to their sites due to collision between two endonucleases tracking with defined polarity. ATP hydrolysis is less than with the type-I enzymes. Homology to DNA helicases has been found within the HsdR and Res subunits. Mutagenesis of the DEAD-box motifs affects both ATP hydrolysis and DNA cleavage. This demonstrates a tight link between ATPase and endonuclease activities. A strand-separation mechanism akin to the DNA helicases is a possibility. The DNA-based motor proteins are mechanistically ill-defined. Further study using some of the techniques pioneered with classical motor proteins will be needed to reveal more detail.

  16. Mechanistic Insights into the Antilithiatic Proteins from Terminalia arjuna: A Proteomic Approach in Urolithiasis

    PubMed Central

    Mittal, Amisha; Tandon, Simran; Singla, Surender Kumar; Tandon, Chanderdeep

    2016-01-01

    Kidney stone formation during hyperoxaluric condition is inherently dependent on the interaction between renal epithelial cells and calcium oxalate (CaOx) crystals. Although modern medicine has progressed in terms of removal of these stones, recurrence and persistent side effects restricts their use. Strategies involving plant based agents which could be used as adjunct therapy is an area which needs to be explored. Plant proteins having antilithiatic activity is a hitherto unexplored area and therefore, we conducted a detailed identification and characterization of antilithiatic proteins from Terminalia arjuna (T. arjuna). Proteins were isolated from the dried bark of T. arjuna and those having molecular weights > 3 kDa were subjected to anion exchange chromatography followed by gel filtration chromatography. Four proteins were identified exhibiting inhibitory activity against CaOx crystallization and crystal growth kinetics The cytoprotective and anti-apoptotic efficacy of these purified proteins was further investigated on oxalate injured renal epithelial cells (MDCK and NRK-52E) wherein, injury due to oxalate was significantly attenuated and led to a dose dependent increase in viability of these cells. These proteins also prevented the interaction of the CaOx crystals to the cell surface and reduced the number of apoptotic cells. Identification of these 4 anionic proteins from the bark of T. arjuna was carried out by Matrix-assisted laser desorption/ionization-time of flight Mass spectrometry (MALDI-TOF MS). This was followed by database search with the MASCOT server and sequence similarity was found with Nuclear pore anchor, DEAD Box ATP-dependent RNA helicase 45, Lon protease homolog 1 and Heat shock protein 90–3. These novel proteins isolated from T. arjuna have the potential to inhibit CaOx crystallization and promote cell survival and therefore, offer novel avenues which need to be explored further for the medical management of urolithiasis. PMID

  17. Mechanistic Insights into the Antilithiatic Proteins from Terminalia arjuna: A Proteomic Approach in Urolithiasis.

    PubMed

    Mittal, Amisha; Tandon, Simran; Singla, Surender Kumar; Tandon, Chanderdeep

    2016-01-01

    Kidney stone formation during hyperoxaluric condition is inherently dependent on the interaction between renal epithelial cells and calcium oxalate (CaOx) crystals. Although modern medicine has progressed in terms of removal of these stones, recurrence and persistent side effects restricts their use. Strategies involving plant based agents which could be used as adjunct therapy is an area which needs to be explored. Plant proteins having antilithiatic activity is a hitherto unexplored area and therefore, we conducted a detailed identification and characterization of antilithiatic proteins from Terminalia arjuna (T. arjuna). Proteins were isolated from the dried bark of T. arjuna and those having molecular weights > 3 kDa were subjected to anion exchange chromatography followed by gel filtration chromatography. Four proteins were identified exhibiting inhibitory activity against CaOx crystallization and crystal growth kinetics The cytoprotective and anti-apoptotic efficacy of these purified proteins was further investigated on oxalate injured renal epithelial cells (MDCK and NRK-52E) wherein, injury due to oxalate was significantly attenuated and led to a dose dependent increase in viability of these cells. These proteins also prevented the interaction of the CaOx crystals to the cell surface and reduced the number of apoptotic cells. Identification of these 4 anionic proteins from the bark of T. arjuna was carried out by Matrix-assisted laser desorption/ionization-time of flight Mass spectrometry (MALDI-TOF MS). This was followed by database search with the MASCOT server and sequence similarity was found with Nuclear pore anchor, DEAD Box ATP-dependent RNA helicase 45, Lon protease homolog 1 and Heat shock protein 90-3. These novel proteins isolated from T. arjuna have the potential to inhibit CaOx crystallization and promote cell survival and therefore, offer novel avenues which need to be explored further for the medical management of urolithiasis.

  18. GBU4-5 — EDRN Public Portal

    Cancer.gov

    GBU-4 is a recently identified protein which was identified using SEREX technologies. GBU4-5 has a DEAD-box domain (like the CAGE protein), but until 2003 had not been described as eliciting an autoantibody response.

  19. Toward understanding life under subzero conditions: the significance of exploring psychrophilic "cold-shock" proteins.

    PubMed

    Kuhn, Emanuele

    2012-11-01

    Understanding the behavior of proteins under freezing conditions is vital for detecting and locating extraterrestrial life in cold environments, such as those found on Mars and the icy moons of Jupiter and Saturn. This review highlights the importance of studying psychrophilic "cold-shock" proteins, a topic that has yet to be explored. A strategy for analyzing the psychrophilic RNA helicase protein CsdA (Psyc_1082) from Psychrobacter arcticus 273-4 as a key protein for life under freezing temperatures is proposed. The experimental model presented here was developed based on previous data from investigations of Escherichia coli, P. arcticus 273-4, and RNA helicases. P. arcticus 273-4 is considered a model for life in freezing environments. It is capable of growing in temperatures as cold as -10°C by using physiological strategies to survive not only in freezing temperatures but also under low-water-activity and limited-nutrient-availability conditions. The analyses of its genome, transcriptome, and proteome revealed specific adaptations that allow it to inhabit freezing environments by adopting a slow metabolic strategy rather than a cellular dormancy state. During growth at subzero temperatures, P. arcticus 273-4 genes related to energy metabolism and carbon substrate incorporation are downregulated, and genes for maintenance of membranes, cell walls, and nucleic acid motion are upregulated. At -6°C, P. arcticus 273-4 does not upregulate the expression of either RNA or protein chaperones; however, it upregulates the expression of its cold-shock induced DEAD-box RNA helicase protein A (CsdA - Psyc_1082). CsdA - Psyc_1082 was investigated as a key helper protein for sustaining life in subzero conditions. Proving CsdA - Psyc_1082 to be functional as a key protein for life under freezing temperatures may extend the known minimum growth temperature of a mesophilic cell and provide key information about the mechanisms that underlie cold-induced biological systems in

  20. Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification

    PubMed Central

    Markov, Dmitriy A; Savkina, Maria; Anikin, Michael; Del Campo, Mark; Ecker, Karen; Lambowitz, Alan M; De Gnore, Jon P; McAllister, William T

    2009-01-01

    The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP–protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP–TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP–mtRNAP fusion, pulled down associated proteins, and identified them by LC–MS–MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity. Copyright © 2009 John Wiley & Sons, Ltd. PMID:19536766

  1. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  2. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  3. Protein

    USDA-ARS?s Scientific Manuscript database

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  4. Protein

    MedlinePlus

    ... Search for: Harvard T.H. Chan School of Public Health Email People Departments Calendar Careers Give my.harvard ... Nutrition Source Harvard T.H. Chan School of Public Health > The Nutrition Source > What Should I Eat? > Protein ...

  5. Maelstrom, a Drosophila spindle-class gene, encodes a protein that colocalizes with Vasa and RDE1/AGO1 homolog, Aubergine, in nuage.

    PubMed

    Findley, Seth D; Tamanaha, Mio; Clegg, Nigel J; Ruohola-Baker, Hannele

    2003-03-01

    A hallmark of germline cells across the animal kingdom is the presence of perinuclear, electron-dense granules called nuage. In many species examined, Vasa, a DEAD-box RNA helicase, is found in these morphologically distinct particles. Despite its evolutionary conservation, the function of nuage remains obscure. We have characterized a null allele of maelstrom (mael) and shown that Maelstrom protein is localized to nuage in a Vasa-dependent manner. By phenotypic characterization, we have defined maelstrom as a spindle-class gene that affects Vasa modification. In a nuclear transport assay, we have determined that Maelstrom shuttles between the nucleus and cytoplasm, which may indicate a nuclear origin for nuage components. Interestingly, Maelstrom, but not Vasa, depends on two genes involved in RNAi phenomena, aubergine and spindle-E (spn-E), for its nuage localization. Furthermore, maelstrom mutant ovaries show mislocalization of two proteins involved in the microRNA and/or RNAi pathways, Dicer and Argonaute2, suggesting a potential connection between nuage and the microRNA-pathway.

  6. Distribution of cold adaptation proteins in microbial mats in Lake Joyce, Antarctica: Analysis of metagenomic data by using two bioinformatics tools.

    PubMed

    Koo, Hyunmin; Hakim, Joseph A; Fisher, Phillip R E; Grueneberg, Alexander; Andersen, Dale T; Bej, Asim K

    2016-01-01

    In this study, we report the distribution and abundance of cold-adaptation proteins in microbial mat communities in the perennially ice-covered Lake Joyce, located in the McMurdo Dry Valleys, Antarctica. We have used MG-RAST and R code bioinformatics tools on Illumina HiSeq2000 shotgun metagenomic data and compared the filtering efficacy of these two methods on cold-adaptation proteins. Overall, the abundance of cold-shock DEAD-box protein A (CSDA), antifreeze proteins (AFPs), fatty acid desaturase (FAD), trehalose synthase (TS), and cold-shock family of proteins (CSPs) were present in all mat samples at high, moderate, or low levels, whereas the ice nucleation protein (INP) was present only in the ice and bulbous mat samples at insignificant levels. Considering the near homogeneous temperature profile of Lake Joyce (0.08-0.29 °C), the distribution and abundance of these proteins across various mat samples predictively correlated with known functional attributes necessary for microbial communities to thrive in this ecosystem. The comparison of the MG-RAST and the R code methods showed dissimilar occurrences of the cold-adaptation protein sequences, though with insignificant ANOSIM (R = 0.357; p-value = 0.012), ADONIS (R(2) = 0.274; p-value = 0.03) and STAMP (p-values = 0.521-0.984) statistical analyses. Furthermore, filtering targeted sequences using the R code accounted for taxonomic groups by avoiding sequence redundancies, whereas the MG-RAST provided total counts resulting in a higher sequence output. The results from this study revealed for the first time the distribution of cold-adaptation proteins in six different types of microbial mats in Lake Joyce, while suggesting a simpler and more manageable user-defined method of R code, as compared to a web-based MG-RAST pipeline.

  7. Use of biotinylated plasmid DNA as a surrogate for HSV DNA to identify proteins that repress or activate viral gene expression.

    PubMed

    Mallon, Stephen; Wakim, Bassam T; Roizman, Bernard

    2012-12-18

    ICP0, a key herpes simplex virus regulatory protein, functions first in the nucleus and then in the cytoplasm. The duration of its nuclear sojourn in cells transfected with DNA and then infected is related to the quantity of transfected DNA. Furthermore, ICP0 transactivates both viral genes and genes encoded by the transfected DNA. The data support the hypothesis that ICP0 is retained in the nucleus until it completes the replacement of repressive chromatin with effector proteins that enable transcription of both DNA templates.To identify the effector proteins, we transfected cells with biotinylated DNA encoding a nonviral gene and then infected the cells with wild-type virus. Proteins bound to transfected biotinylated plasmid recovered from mock-treated and infected cells were identified using mass spectrometry followed by appropriate database search. The transfected DNA from mock-infected cells yielded proteins associated with repression, whereas DNA recovered from infected cells included proteins known to enable transcription and proteins that have not been previously associated with that role. To test the hypothesis that the proteins hitherto not known to associate with viral gene expression are nevertheless essential, we tested the role of the DEAD-box helicase Ddx17. We report that Ddx17 plays a critical role in the expression of early and late viral genes. Thus, biotinylated DNA recovered from transfected infected cells can function as a surrogate for viral DNA and is a rich source of proteins that play a role in viral gene expression but which have not been previously identified in that role.

  8. Drosophila SMN complex proteins Gemin2, Gemin3, and Gemin5 are components of U bodies

    SciTech Connect

    Cauchi, Ruben J.; Sanchez-Pulido, Luis; Liu, Ji-Long

    2010-08-15

    Uridine-rich small nuclear ribonucleoproteins (U snRNPs) play key roles in pre-mRNA processing in the nucleus. The assembly of most U snRNPs takes place in the cytoplasm and is facilitated by the survival motor neuron (SMN) complex. Discrete cytoplasmic RNA granules called U bodies have been proposed to be specific sites for snRNP assembly because they contain U snRNPs and SMN. U bodies invariably associate with P bodies, which are involved in mRNA decay and translational control. However, it remains unknown whether other SMN complex proteins also localise to U bodies. In Drosophila there are four SMN complex proteins, namely SMN, Gemin2/CG10419, Gemin3 and Gemin5/Rigor mortis. Drosophila Gemin3 was originally identified as the Drosophila orthologue of human and yeast Dhh1, a component of P bodies. Through an in silico analysis of the DEAD-box RNA helicases we confirmed that Gemin3 is the bona fide Drosophila orthologue of vertebrate Gemin3 whereas the Drosophila orthologue of Dhh1 is Me31B. We then made use of the Drosophila egg chamber as a model system to study the subcellular distribution of the Gemin proteins as well as Me31B. Our cytological investigations show that Gemin2, Gemin3 and Gemin5 colocalise with SMN in U bodies. Although they are excluded from P bodies, as components of U bodies, Gemin2, Gemin3 and Gemin5 are consistently found associated with P bodies, wherein Me31B resides. In addition to a role in snRNP biogenesis, SMN complexes residing in U bodies may also be involved in mRNP assembly and/or transport.

  9. Hepatitis C Virus Core Protein Interacts with Cellular Putative RNA Helicase

    PubMed Central

    You, Li-Ru; Chen, Chun-Ming; Yeh, Tien-Shun; Tsai, Tzung-Yuan; Mai, Ru-Tsun; Lin, Chi-Hung; Lee, Yan-Hwa Wu

    1999-01-01

    The nucleocapsid core protein of hepatitis C virus (HCV) has been shown to trans-act on several viral or cellular promoters. To get insight into the trans-action mechanism of HCV core protein, a yeast two-hybrid cloning system was used for identification of core protein-interacting cellular protein. One such cDNA clone encoding the DEAD box family of putative RNA helicase was obtained. This cellular putative RNA helicase, designated CAP-Rf, exhibits more than 95% amino acid sequence identity to other known RNA helicases including human DBX and DBY, mouse mDEAD3, and PL10, a family of proteins generally involved in translation, splicing, development, or cell growth. In vitro binding or in vivo coimmunoprecipitation studies demonstrated the direct interaction of the full-length/matured form and C-terminally truncated variants of HCV core protein with this targeted protein. Additionally, the protein’s interaction domains were delineated at the N-terminal 40-amino-acid segment of the HCV core protein and the C-terminal tail of CAP-Rf, which encompassed its RNA-binding and ATP hydrolysis domains. Immunoblotting or indirect immunofluorescence analysis revealed that the endogenous CAP-Rf was mainly localized in the nucleus and to a lesser extent in the cytoplasm, and when fused with FLAG tag, it colocalized with the HCV core protein either in the cytoplasm or in the nucleus. Similar to other RNA helicases, this cellular RNA helicase has nucleoside triphosphatase-deoxynucleoside triphosphatase activity, but this activity is inhibited by various forms of homopolynucleotides and enhanced by the HCV core protein. Moreover, transient expression of HCV core protein in human hepatoma HuH-7 cells significantly potentiated the trans-activation effect of FLAG-tagged CAP-Rf or untagged CAP-Rf on the luciferase reporter plasmid activity. All together, our results indicate that CAP-Rf is involved in regulation of gene expression and that HCV core protein promotes the trans

  10. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

    SciTech Connect

    Kanno, Yuichiro Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

    2015-03-27

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR.

  11. The proteasomal Rpn11 metalloprotease suppresses tombusvirus RNA recombination and promotes viral replication via facilitating assembly of the viral replicase complex.

    PubMed

    Prasanth, K Reddisiva; Barajas, Daniel; Nagy, Peter D

    2015-03-01

    RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role

  12. AZFa protein DDX3Y is differentially expressed in human male germ cells during development and in testicular tumours: new evidence for phenotypic plasticity of germ cells.

    PubMed

    Gueler, B; Sonne, S B; Zimmer, J; Hilscher, B; Hilscher, W; Græm, N; Rajpert-De Meyts, E; Vogt, P H

    2012-06-01

    DDX3Y (DBY), located within AZoospermia Factor a (AZFa) region of the human Y chromosome (Yq11), encodes a conserved DEAD-box RNA helicase expressed only in germ cells and with a putative function at G1-S phase of the cell cycle. Deletion of AZFa results most often in germ cell aplasia, i.e. Sertoli-cell-only syndrome. To investigate the function of DDX3Y during human spermatogenesis, we examined its expression during development and maturation of the testis and in several types of testicular germ cell tumours (TGCTs), including the pre-invasive carcinoma in situ (CIS) precursor cells which are believed to originate from fetal gonocytes. DDX3Y protein expression was analysed during development in different tissues by western blotting. The localization of DDX3Y in normal fetal and prepubertal testis tissue of different ages as well as in a series of distinct TGCT tissue samples (CIS, classical seminoma, spermatocytic seminoma, teratoma and embryonal carcinoma) was performed by immunohistochemistry. Germ cell-specific expression of DDX3Y protein was revealed in fetal prospermatogonia but not in gonocytes and not before the 17th gestational week. After birth, DDX3Y was expressed at first only in the nuclei of Ap spermatogonia, then also in the cytoplasm similarly to that seen after puberty. In CIS cells, DDX3Y was highly expressed and located predominantly in the nuclei. In invasive TGCT, significant DDX3Y expression was found in seminomas of the classical and spermatocytic type, but not in somatically differentiated non-seminomas, consistent with its germ-cell specific function. The fetal germ cell DDX3Y expression suggests a role in early spermatogonial proliferation and implies that, in men with AZFa deletion, germ cell depletion may begin prenatally. The strong expression of DDX3Y in CIS cells, but not in gonocytes, indicates phenotypic plasticity of CIS cells and suggests partial maturation to spermatogonia, likely due to their postpubertal microenvironment.

  13. DDX3 Interacts with Influenza A Virus NS1 and NP Proteins and Exerts Antiviral Function through Regulation of Stress Granule Formation

    PubMed Central

    Thulasi Raman, Sathya N.; Liu, Guanqun; Pyo, Hyun Mi; Cui, Ya Cheng; Xu, Fang; Ayalew, Lisanework E.; Tikoo, Suresh K.

    2016-01-01

    ABSTRACT DDX3 belongs to the DEAD box RNA helicase family and is a multifunctional protein affecting the life cycle of a variety of viruses. However, its role in influenza virus infection is unknown. In this study, we explored the potential role of DDX3 in influenza virus life cycle and discovered that DDX3 is an antiviral protein. Since many host proteins affect virus life cycle by interacting with certain components of the viral machinery, we first verified whether DDX3 has any viral interaction partners. Immunoprecipitation studies revealed NS1 and NP as direct interaction partners of DDX3. Stress granules (SGs) are known to be antiviral and do form in influenza virus-infected cells expressing defective NS1 protein. Additionally, a recent study showed that DDX3 is an important SG-nucleating factor. We thus explored whether DDX3 plays a role in influenza virus infection through regulation of SGs. Our results showed that SGs were formed in infected cells upon infection with a mutant influenza virus lacking functional NS1 (del NS1) protein, and DDX3 colocalized with NP in SGs. We further determined that the DDX3 helicase domain did not interact with NS1 and NP; however, it was essential for DDX3 localization in virus-induced SGs. Knockdown of DDX3 resulted in impaired SG formation and led to increased virus titers. Taken together, our results identified DDX3 as an antiviral protein with a role in virus-induced SG formation. IMPORTANCE DDX3 is a multifunctional RNA helicase and has been reported to be involved in regulating various virus life cycles. However, its function during influenza A virus infection remains unknown. In this study, we demonstrated that DDX3 is capable of interacting with influenza virus NS1 and NP proteins; DDX3 and NP colocalize in the del NS1 virus-induced SGs. Furthermore, knockdown of DDX3 impaired SG formation and led to a decreased virus titer. Thus, we provided evidence that DDX3 is an antiviral protein during influenza virus infection

  14. Large Scale Comparative Proteomics of a Chloroplast Clp Protease Mutant Reveals Folding Stress, Altered Protein Homeostasis, and Feedback Regulation of Metabolism*

    PubMed Central

    Zybailov, Boris; Friso, Giulia; Kim, Jitae; Rudella, Andrea; Rodríguez, Verenice Ramírez; Asakura, Yukari; Sun, Qi; van Wijk, Klaas J.

    2009-01-01

    The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex. To understand the role of Clp proteases in plastid biogenesis and homeostasis, leaf proteomes of young seedlings of clpr2-1 and wild type were compared using large scale mass spectrometry-based quantification using an LTQ-Orbitrap and spectral counting with significance determined by G-tests. Virtually only chloroplast-localized proteins were significantly affected, indicating that the molecular phenotype was confined to the chloroplast. A comparative chloroplast stromal proteome analysis of fully developed plants was used to complement the data set. Chloroplast unfoldase ClpB3 was strongly up-regulated in both young and mature leaves, suggesting widespread and persistent protein folding stress. The importance of ClpB3 in the clp2-1 mutant was demonstrated by the observation that a CLPR2 and CLPB3 double mutant was seedling-lethal. The observed up-regulation of chloroplast chaperones and protein sorting components further illustrated destabilization of protein homeostasis. Delayed rRNA processing and up-regulation of a chloroplast DEAD box RNA helicase and polynucleotide phosphorylase, but no significant change in accumulation of ribosomal subunits, suggested a bottleneck in ribosome assembly or RNA metabolism. Strong up-regulation of a chloroplast translational regulator TypA/BipA GTPase suggested a specific response in plastid gene expression to the distorted homeostasis. The stromal proteases PreP1,2 were up-regulated, likely constituting compensation for reduced Clp protease activity and possibly shared substrates between the ClpP and PreP protease systems. The thylakoid photosynthetic apparatus was decreased in the seedlings, whereas several structural thylakoid-associated plastoglobular proteins were strongly up-regulated. Two thylakoid-associated reductases involved in isoprenoid and chlorophyll synthesis were up-regulated reflecting feedback from rate

  15. DDX59 promotes DNA replication in lung adenocarcinoma

    PubMed Central

    You, Jin; Wang, Xingshun; Wang, Jiuling; Yuan, Baolei; Zhang, Yandong

    2017-01-01

    DEAD box proteins are multifunctional proteins involved in every aspect in RNA metabolism and have essential roles in many cellular activities. Despite their importance, many DEAD box proteins remain uncharacterized. In this report, we found DDX59 overexpressed in lung adenocarcinoma. DDX59 knockdown reduced cell proliferation, anchorage-independent cell growth, and caused reduction of tumor formation in immunocompromised mice. In multiple lung cancer cells, we found that DDX59 knockdown inhibits DNA synthesis; wild-type DDX59 but not helicase-defective mutant of DDX59 enhances DNA synthesis. DDX59 knockdown caused reduction of MCM protein levels, decreased the loading of MCM ring protein onto chromatin, and therefore inhibited DNA replication. Our study reveals for the first time that DDX59 has an important role in lung cancer development through promoting DNA replication. PMID:28090355

  16. Association of Transcription Factor IIA with TATA Binding Protein Is Required for Transcriptional Activation of a Subset of Promoters and Cell Cycle Progression in Saccharomyces cerevisiae

    PubMed Central

    Ozer, Josef; Lezina, Larissa E.; Ewing, Joshua; Audi, Salma; Lieberman, Paul M.

    1998-01-01

    The general transcription factor IIA (TFIIA) interacts with the TATA binding protein (TBP) and promoter DNA to mediate transcription activation in vitro. To determine if this interaction is generally required for activation of all class II genes in vivo, we have constructed substitution mutations in yeast TFIIA which compromise its ability to bind TBP. Substitution mutations in the small subunit of TFIIA (Toa2) at residue Y69 or W76 significantly impaired the ability of TFIIA to stimulate TBP-promoter binding in vitro. Gene replacement of wild-type TOA2 with a W76E or Y69A/W76A mutant was lethal in Saccharomyces cerevisiae, while the Y69F/W76F mutant exhibited extremely slow growth at 30°C. Both the Y69A and W76A mutants were conditionally lethal at higher temperatures. Light microscopy indicated that viable toa2 mutant strains accumulate as equal-size dumbbells and multibudded clumps. Transcription of the cell cycle-regulatory genes CLB1, CLB2, CLN1, and CTS1 was significantly reduced in the toa2 mutant strains, while the noncycling genes PMA1 and ENO2 were only modestly affected, suggesting that these toa2 mutant alleles disrupt cell cycle progression. The differential effect of these toa2 mutants on gene transcription was examined for a number of other genes. toa2 mutant strains supported high levels of CUP1, PHO5, TRP3, and GAL1 gene activation, but the constitutive expression of DED1 was significantly reduced. Activator-induced start site expression for HIS3, GAL80, URA1, and URA3 promoters was defective in toa2 mutant strains, suggesting that the TFIIA-TBP complex is important for promoters which require an activator-dependent start site selection from constitutive to regulated expression. We present evidence to indicate that transcription defects in toa2 mutants can be both activator and promoter dependent. These results suggest that the association of TFIIA with TBP regulates activator-induced start site selection and cell cycle progression in S

  17. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  18. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2007-09-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  19. NDR proteins

    PubMed Central

    Jones, Alan M

    2010-01-01

    N-myc downregulated (NDR) genes were discovered more than fifteen years ago. Indirect evidence support a role in tumor progression and cellular differentiation, but their biochemical function is still unknown. Our detailed analyses on Arabidopsis NDR proteins (deisgnated NDR-like, NDL) show their involvement in altering auxin transport, local auxin gradients and expression level of auxin transport proteins. Animal NDL proteins may be involved in membrane recycling of E-cadherin and effector for the small GTPase. In light of these findings, we hypothesize that NDL proteins regulate vesicular trafficking of auxin transport facilitator PIN proteins by biochemically alterating the local lipid environment of PIN proteins. PMID:20724844

  20. Proteins (image)

    MedlinePlus

    ... is an important nutrient that builds muscles and bones and provides energy. Protein can help with weight control because it helps you feel full and satisfied from your meals. The healthiest proteins are the leanest. This means ...

  1. Dietary Proteins

    MedlinePlus

    ... and maintain bones, muscles and skin. We get proteins in our diet from meat, dairy products, nuts, and certain grains ... level of physical activity. Most Americans eat enough protein in their diet.

  2. Protein Structure

    ERIC Educational Resources Information Center

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  3. Protein Structure

    ERIC Educational Resources Information Center

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  4. Therapeutic proteins.

    PubMed

    Dimitrov, Dimiter S

    2012-01-01

    Protein-based therapeutics are highly successful in clinic and currently enjoy unprecedented recognition of their potential. More than 100 genuine and similar number of modified therapeutic proteins are approved for clinical use in the European Union and the USA with 2010 sales of US$108 bln; monoclonal antibodies (mAbs) accounted for almost half (48%) of the sales. Based on their pharmacological activity, they can be divided into five groups: (a) replacing a protein that is deficient or abnormal; (b) augmenting an existing pathway; (c) providing a novel function or activity; (d) interfering with a molecule or organism; and (e) delivering other compounds or proteins, such as a radionuclide, cytotoxic drug, or effector proteins. Therapeutic proteins can also be grouped based on their molecular types that include antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. They can also be classified based on their molecular mechanism of activity as (a) binding non-covalently to target, e.g., mAbs; (b) affecting covalent bonds, e.g., enzymes; and (c) exerting activity without specific interactions, e.g., serum albumin. Most protein therapeutics currently on the market are recombinant and hundreds of them are in clinical trials for therapy of cancers, immune disorders, infections, and other diseases. New engineered proteins, including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs, and proteins with optimized pharmacokinetics, are currently under development. However, in the last several decades, there are no conceptually new methodological developments comparable, e.g., to genetic engineering leading to the development of recombinant therapeutic proteins. It appears that a paradigm change in methodologies and understanding of mechanisms is needed to overcome major

  5. Protein tentacles.

    PubMed

    Harrison, Stephen C

    2017-05-27

    Virus structures were among the earliest illustrations of how regulated protein assembly can proceed by folding of polypeptide-chain segments into complementary sites on partner proteins. I draw on Caspar's image of protein "tentacles" and his metaphor of SV40 pentamers as five-legged, aquatic organisms ("pentopuses") to suggest a helpful vocabulary. "Tentacular interactions" among component subunits organize most subcellular molecular machines. Their selective advantages include facile regulation of both assembly and disassembly by modifying enzymes and by folding chaperones. Copyright © 2017. Published by Elsevier Inc.

  6. Total protein

    MedlinePlus

    ... 2016:chap 215. Read More Agammaglobulinemia Albumin - blood (serum) test Amino acids Antibody Burns Chronic Congenital nephrotic syndrome Fibrinogen blood test Glomerulonephritis Hemoglobin Liver disease Malabsorption Multiple myeloma Polycythemia vera Protein in diet ...

  7. Protein Crystallizability.

    PubMed

    Smialowski, Pawel; Wong, Philip

    2016-01-01

    Obtaining diffracting quality crystals remains a major challenge in protein structure research. We summarize and compare methods for selecting the best protein targets for crystallization, construct optimization and crystallization condition design. Target selection methods are divided into algorithms predicting the chance of successful progression through all stages of structural determination (from cloning to solving the structure) and those focusing only on the crystallization step. We tried to highlight pros and cons of different approaches examining the following aspects: data size, redundancy and representativeness, overfitting during model construction, and results evaluation. In summary, although in recent years progress was made and several sequence properties were reported to be relevant for crystallization, the successful prediction of protein crystallization behavior and selection of corresponding crystallization conditions continue to challenge structural researchers.

  8. Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  9. Detecting protein-protein interactions using Renilla luciferase fusion proteins.

    PubMed

    Burbelo, Peter D; Kisailus, Adam E; Peck, Jeremy W

    2002-11-01

    We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.

  10. Interaction entropy for protein-protein binding

    NASA Astrophysics Data System (ADS)

    Sun, Zhaoxi; Yan, Yu N.; Yang, Maoyou; Zhang, John Z. H.

    2017-03-01

    Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interaction entropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interaction entropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

  11. Learning about Proteins

    MedlinePlus

    ... What Happens in the Operating Room? Learning About Proteins KidsHealth > For Kids > Learning About Proteins A A ... the foods you eat. continue Different Kinds of Protein Protein from animal sources, such as meat and ...

  12. Protein Microarray Technology

    PubMed Central

    Hall, David A.; Ptacek, Jason

    2007-01-01

    Protein chips have emerged as a promising approach for a wide variety of applications including the identification of protein-protein interactions, protein-phospholipid interactions, small molecule targets, and substrates of proteins kinases. They can also be used for clinical diagnostics and monitoring disease states. This article reviews current methods in the generation and applications of protein microarrays. PMID:17126887

  13. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

    Tan, Taison; Frenkel, Daan; Gupta, Vishal; Deem, Michael W.

    2005-05-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein-protein interactions has driven the selection of longer proteins, as they are more able to distinguish among a larger number of distinct interactions due to their greater average surface area. Annotated protein sequences available from the SWISS-PROT database were analyzed for 13 eukaryotes, eight bacteria, and two archaea species. The number of subcellular locations to which each protein is associated is used as a measure of the number of interactions to which a protein participates. Two databases of yeast protein-protein interactions were used as another measure of the number of interactions to which each S. cerevisiae protein participates. Protein length is shown to correlate with both number of subcellular locations to which a protein is associated and number of interactions as measured by yeast two-hybrid experiments. Protein length is also shown to correlate with the probability that the protein is encoded by an essential gene. Interestingly, average protein length and number of subcellular locations are not significantly different between all human proteins and protein targets of known, marketed drugs. Increased protein length appears to be a significant mechanism by which the increasing complexity of protein-protein interaction networks is accommodated within the natural evolution of species. Consideration of protein length may be a valuable tool in drug design, one that predicts different strategies for inhibiting interactions in aberrant and normal pathways.

  14. EDITORIAL: Precision proteins Precision proteins

    NASA Astrophysics Data System (ADS)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  15. Multifunctional DDX3: dual roles in various cancer development and its related signaling pathways

    PubMed Central

    Zhao, Luqing; Mao, Yitao; Zhou, Jianhua; Zhao, Yuelong; Cao, Ya; Chen, Xiang

    2016-01-01

    DEAD-box RNA helicase 3 (DDX3) is a highly conserved family member of DEAD-box protein, which is a cluster of ATP-dependent and the largest family of RNA helicase. DEAD-box family is characterized by the regulation of ATPase and helicase activities, the modulation of RNA metabolism, and the actors of RNA binding proteins or molecular chaperones to interact with other proteins or RNA. For DDX3, it exerts its multifaceted roles in viral manipulation, stress response, hypoxia, radiation response and apoptosis, and is closely related to cancer development and progression. DDX3 has dual roles in different cancer types and can act as either an oncogene or tumor suppressor gene during cancer progression. In the present review, we mainly provide an overview of current knowledge on dual roles of DDX3 in various types of cancer, including breast cancer, lung cancer, colorectal cancer, hepatocellular carcinoma, oral squamous cell carcinoma, Ewing sarcoma, glioblastoma multiforme and gallbladder carcinoma, and illustrate the regulatory mechanisms for leading these two controversial biological effects. Furthermore, we summarize the essential signaling pathways that DDX3 participated, especially the Wnt/β-catenin signaling and EMT related signaling (TGF-β, Notch, Hedgehog pathways), which are crucial to DDX3 mediated cancer metastasis process. Thoroughly exploring the dual roles of DDX3 in cancer development and the essential signaling pathways it involved, it will help us open new perspectives to develop novel promising targets to elevate therapeutic effects and facilitate the “Personalized medicine” or “Precision medicine” to come into clinic. PMID:27186411

  16. Shotgun protein sequencing.

    SciTech Connect

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  17. Protein Crystal Based Nanomaterials

    NASA Technical Reports Server (NTRS)

    Bell, Jeffrey A.; VanRoey, Patrick

    2001-01-01

    This is the final report on a NASA Grant. It concerns a description of work done, which includes: (1) Protein crystals cross-linked to form fibers; (2) Engineering of protein to favor crystallization; (3) Better knowledge-based potentials for protein-protein contacts; (4) Simulation of protein crystallization.

  18. Human DDX3 protein is a valuable target to develop broad spectrum antiviral agents.

    PubMed

    Brai, Annalaura; Fazi, Roberta; Tintori, Cristina; Zamperini, Claudio; Bugli, Francesca; Sanguinetti, Maurizio; Stigliano, Egidio; Esté, José; Badia, Roger; Franco, Sandra; Martinez, Miguel A; Martinez, Javier P; Meyerhans, Andreas; Saladini, Francesco; Zazzi, Maurizio; Garbelli, Anna; Maga, Giovanni; Botta, Maurizio

    2016-05-10

    Targeting a host factor essential for the replication of different viruses but not for the cells offers a higher genetic barrier to the development of resistance, may simplify therapy regimens for coinfections, and facilitates management of emerging viral diseases. DEAD-box polypeptide 3 (DDX3) is a human host factor required for the replication of several DNA and RNA viruses, including some of the most challenging human pathogens currently circulating, such as HIV-1, Hepatitis C virus, Dengue virus, and West Nile virus. Herein, we showed for the first time, to our knowledge, that the inhibition of DDX3 by a small molecule could be successfully exploited for the development of a broad spectrum antiviral agent. In addition to the multiple antiviral activities, hit compound 16d retained full activity against drug-resistant HIV-1 strains in the absence of cellular toxicity. Pharmacokinetics and toxicity studies in rats confirmed a good safety profile and bioavailability of 16d. Thus, DDX3 is here validated as a valuable therapeutic target.

  19. Human DDX3 protein is a valuable target to develop broad spectrum antiviral agents

    PubMed Central

    Brai, Annalaura; Fazi, Roberta; Tintori, Cristina; Zamperini, Claudio; Bugli, Francesca; Sanguinetti, Maurizio; Stigliano, Egidio; Esté, José; Badia, Roger; Franco, Sandra; Martinez, Javier P.; Meyerhans, Andreas; Saladini, Francesco; Zazzi, Maurizio; Garbelli, Anna; Botta, Maurizio

    2016-01-01

    Targeting a host factor essential for the replication of different viruses but not for the cells offers a higher genetic barrier to the development of resistance, may simplify therapy regimens for coinfections, and facilitates management of emerging viral diseases. DEAD-box polypeptide 3 (DDX3) is a human host factor required for the replication of several DNA and RNA viruses, including some of the most challenging human pathogens currently circulating, such as HIV-1, Hepatitis C virus, Dengue virus, and West Nile virus. Herein, we showed for the first time, to our knowledge, that the inhibition of DDX3 by a small molecule could be successfully exploited for the development of a broad spectrum antiviral agent. In addition to the multiple antiviral activities, hit compound 16d retained full activity against drug-resistant HIV-1 strains in the absence of cellular toxicity. Pharmacokinetics and toxicity studies in rats confirmed a good safety profile and bioavailability of 16d. Thus, DDX3 is here validated as a valuable therapeutic target. PMID:27118832

  20. Protein-losing enteropathy

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  1. Protein and Heart Health

    MedlinePlus

    ... It Works Healthy Workplace Food and Beverage Toolkit Protein and Heart Health Updated:May 5,2015 Protein ... said. What’s the harm in getting too much protein? The main problem is that often the extra ...

  2. The DbpA catalytic core unwinds double-helix substrates by directly loading on them.

    PubMed

    Childs, Jared J; Gentry, Riley C; Moore, Anthony F T; Koculi, Eda

    2016-03-01

    DbpA is a DEAD-box RNA helicase implicated in the assembly of the large ribosomal subunit. Similar to all the members of the DEAD-box family, the DbpA protein has two N-terminal RecA-like domains, which perform the RNA unwinding. However, unlike other members of this family, the DbpA protein also possesses a structured C-terminal RNA-binding domain that mediates specific tethering of DbpA to hairpin 92 of the Escherichia coli 23S ribosomal RNA. Previous studies using model RNA molecules containing hairpin 92 show that the RNA molecules support the DbpA protein's double-helix unwinding activity, provided that the double helix has a 3' single-stranded region. The 3' single-stranded region was suggested to be the start site of the DbpA protein's catalytic unwinding activity. The data presented here demonstrate that the single-stranded region 3' of the double-helix substrate is not required for the DbpA protein's unwinding activity and the DbpA protein unwinds the double-helix substrates by directly loading on them. © 2016 Childs et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  3. Protein splicing: selfish genes invade cellular proteins.

    PubMed

    Neff, N F

    1993-12-01

    Protein splicing is a series of enzymatic events involving intramolecular protein breakage, rejoining and intron homing, in which introns are able to promote the recombinative transposition of their own coding sequences. Eukaryotic and prokaryotic spliced proteins have conserved similar gene structure, but little amino acid identity. The genes coding for these spliced proteins contain internal in-frame introns that encode polypeptides that apparently self-excise from the resulting host protein sequences. Excision of the 'protein intron' is coupled with joining of the two flanking protein regions encoded by exons of the host gene. Some introns of this type encode DNA endonucleases, related to Group I RNA intron gene products, that stimulate gene conversion and self-transmission.

  4. Nanotechnologies in protein microarrays.

    PubMed

    Krizkova, Sona; Heger, Zbynek; Zalewska, Marta; Moulick, Amitava; Adam, Vojtech; Kizek, Rene

    2015-01-01

    Protein microarray technology became an important research tool for study and detection of proteins, protein-protein interactions and a number of other applications. The utilization of nanoparticle-based materials and nanotechnology-based techniques for immobilization allows us not only to extend the surface for biomolecule immobilization resulting in enhanced substrate binding properties, decreased background signals and enhanced reporter systems for more sensitive assays. Generally in contemporarily developed microarray systems, multiple nanotechnology-based techniques are combined. In this review, applications of nanoparticles and nanotechnologies in creating protein microarrays, proteins immobilization and detection are summarized. We anticipate that advanced nanotechnologies can be exploited to expand promising fields of proteins identification, monitoring of protein-protein or drug-protein interactions, or proteins structures.

  5. PREFACE: Protein protein interactions: principles and predictions

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Tsai, Chung-Jung

    2005-06-01

    Proteins are the `workhorses' of the cell. Their roles span functions as diverse as being molecular machines and signalling. They carry out catalytic reactions, transport, form viral capsids, traverse membranes and form regulated channels, transmit information from DNA to RNA, making possible the synthesis of new proteins, and they are responsible for the degradation of unnecessary proteins and nucleic acids. They are the vehicles of the immune response and are responsible for viral entry into the cell. Given their importance, considerable effort has been centered on the prediction of protein function. A prime way to do this is through identification of binding partners. If the function of at least one of the components with which the protein interacts is known, that should let us assign its function(s) and the pathway(s) in which it plays a role. This holds since the vast majority of their chores in the living cell involve protein-protein interactions. Hence, through the intricate network of these interactions we can map cellular pathways, their interconnectivities and their dynamic regulation. Their identification is at the heart of functional genomics; their prediction is crucial for drug discovery. Knowledge of the pathway, its topology, length, and dynamics may provide useful information for forecasting side effects. The goal of predicting protein-protein interactions is daunting. Some associations are obligatory, others are continuously forming and dissociating. In principle, from the physical standpoint, any two proteins can interact, but under what conditions and at which strength? The principles of protein-protein interactions are general: the non-covalent interactions of two proteins are largely the outcome of the hydrophobic effect, which drives the interactions. In addition, hydrogen bonds and electrostatic interactions play important roles. Thus, many of the interactions observed in vitro are the outcome of experimental overexpression. Protein disorder

  6. Protein Structure Prediction by Protein Threading

    NASA Astrophysics Data System (ADS)

    Xu, Ying; Liu, Zhijie; Cai, Liming; Xu, Dong

    The seminal work of Bowie, Lüthy, and Eisenberg (Bowie et al., 1991) on "the inverse protein folding problem" laid the foundation of protein structure prediction by protein threading. By using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. Their follow-up work (Elofsson et al., 1996; Fischer and Eisenberg, 1996; Fischer et al., 1996a,b) and the work by Jones, Taylor, and Thornton (Jones et al., 1992) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term "protein threading." These computational tools have played a key role in extending the utility of all the experimentally solved structures by X-ray crystallography and nuclear magnetic resonance (NMR), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now.

  7. Protein sequence comparison and protein evolution

    SciTech Connect

    Pearson, W.R.

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  8. Whey protein fractionation

    USDA-ARS?s Scientific Manuscript database

    Concentrated whey protein products from cheese whey, such as whey protein concentrate (WPC) and whey protein isolate (WPI), contain more than seven different types of proteins: alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG), bovine serum albumin (BSA), immunoglobulins (Igs), lactoferrin ...

  9. Mirror Image Proteins

    PubMed Central

    Zhao, Le; Lu, Wuyuan

    2017-01-01

    Proteins composed entirely of unnatural D-amino acids and the achiral amino acid glycine are mirror image forms of their native L-protein counterparts. Recent advances in chemical protein synthesis afford unique and facile synthetic access to domain-sized mirror image D-proteins, enabling protein research to be conducted through “the looking glass” and in a way previously unattainable. D-proteins can facilitate structure determination of their native L-forms that are difficult to crystallize (racemic X-ray crystallography); D-proteins can serve as the bait for library screening to ultimately yield pharmacologically superior D-peptide/D-protein therapeutics (mirror image phage display); D-proteins can also be used as a powerful mechanistic tool for probing molecular events in biology. This review examines recent progress in the application of mirror image proteins to structural biology, drug discovery, and immunology. PMID:25282524

  10. Protein- protein interaction detection system using fluorescent protein microdomains

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  11. Molecular modelling of protein-protein/protein-solvent interactions

    NASA Astrophysics Data System (ADS)

    Luchko, Tyler

    The inner workings of individual cells are based on intricate networks of protein-protein interactions. However, each of these individual protein interactions requires a complex physical interaction between proteins and their aqueous environment at the atomic scale. In this thesis, molecular dynamics simulations are used in three theoretical studies to gain insight at the atomic scale about protein hydration, protein structure and tubulin-tubulin (protein-protein) interactions, as found in microtubules. Also presented, in a fourth project, is a molecular model of solvation coupled with the Amber molecular modelling package, to facilitate further studies without the need of explicitly modelled water. Basic properties of a minimally solvated protein were calculated through an extended study of myoglobin hydration with explicit solvent, directly investigating water and protein polarization. Results indicate a close correlation between polarization of both water and protein and the onset of protein function. The methodology of explicit solvent molecular dynamics was further used to study tubulin and microtubules. Extensive conformational sampling of the carboxy-terminal tails of 8-tubulin was performed via replica exchange molecular dynamics, allowing the characterisation of the flexibility, secondary structure and binding domains of the C-terminal tails through statistical analysis methods. Mechanical properties of tubulin and microtubules were calculated with adaptive biasing force molecular dynamics. The function of the M-loop in microtubule stability was demonstrated in these simulations. The flexibility of this loop allowed constant contacts between the protofilaments to be maintained during simulations while the smooth deformation provided a spring-like restoring force. Additionally, calculating the free energy profile between the straight and bent tubulin configurations was used to test the proposed conformational change in tubulin, thought to cause microtubule

  12. Dbp9p, a putative ATP-dependent RNA helicase involved in 60S-ribosomal-subunit biogenesis, functionally interacts with Dbp6p.

    PubMed Central

    Daugeron, M C; Kressler, D; Linder, P

    2001-01-01

    Ribosome synthesis is a highly complex process and constitutes a major cellular activity. The biogenesis of this ribonucleoprotein assembly requires a multitude of protein trans-acting factors including several putative ATP-dependent RNA helicases of the DEAD-box and related protein families. Here we show that the previously uncharacterized Saccharomyces cerevisiae open reading frame YLR276C, hereafter named DBP9 (DEAD-box protein 9), encodes an essential nucleolar protein involved in 60S-ribosomal-subunit biogenesis. Genetic depletion of Dbp9p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This terminal phenotype is likely due to the instability of early pre-ribosomal particles, as evidenced by the low steady-state levels and the decreased synthesis of the 27S precursors to mature 25S and 5.8S rRNAs. In agreement with a role of Dbp9p in 60S subunit synthesis, we find that increased Dbp9p dosage efficiently suppresses certain dbp6 alleles and that dbp6/dbp9 double mutants show synthetic lethality. Furthermore, Dbp6p and Dbp9p weakly interact in a yeast two-hybrid assay. Altogether, our findings indicate an intimate functional interaction between Dbp6p and Dbp9p during the process of 60S-ribosomal-subunit assembly. PMID:11565753

  13. Combinatorial protein reagents to manipulate protein function.

    PubMed

    Colas, P

    2000-02-01

    The design and use of combinatorial protein libraries has become a fast moving field in molecular biology. Different experimental systems supporting various selection schemes are now available. The latest breakthroughs include evolutionary experiments to improve existing binding surfaces, selections of homodimerizing peptides, the use of peptide aptamers to disrupt protein interactions inside living cells, and functional selections of aptamers to probe regulatory networks.

  14. Surface Mediated Protein Disaggregation

    NASA Astrophysics Data System (ADS)

    Radhakrishna, Mithun; Kumar, Sanat K.

    2014-03-01

    Preventing protein aggregation is of both biological and industrial importance. Biologically these aggregates are known to cause amyloid type diseases like Alzheimer's and Parkinson's disease. Protein aggregation leads to reduced activity of the enzymes in industrial applications. Inter-protein interactions between the hydrophobic residues of the protein are known to be the major driving force for protein aggregation. In the current paper we show how surface chemistry and curvature can be tuned to mitigate these inter-protein interactions. Our results calculated in the framework of the Hydrophobic-Polar (HP) lattice model show that, inter-protein interactions can be drastically reduced by increasing the surface hydrophobicity to a critical value corresponding to the adsorption transition of the protein. At this value of surface hydrophobicity, proteins lose inter-protein contacts to gain surface contacts and thus the surface helps in reducing the inter-protein interactions. Further, we show that the adsorption of the proteins inside hydrophobic pores of optimal sizes are most efficient both in reducing inter-protein contacts and simultaneously retaining most of the native-contacts due to strong protein-surface interactions coupled with stabilization due to the confinement. Department of Energy (Grant No DE-FG02-11ER46811).

  15. Hydrodynamic effects in proteins.

    PubMed

    Szymczak, Piotr; Cieplak, Marek

    2011-01-26

    Experimental and numerical results pertaining to flow-induced effects in proteins are reviewed. Special emphasis is placed on shear-induced unfolding and on the role of solvent mediated hydrodynamic interactions in the conformational transitions in proteins.

  16. Learning about Proteins

    MedlinePlus

    ... body, and protecting you from disease. All About Amino Acids When you eat foods that contain protein, the ... called amino (say: uh-MEE-no) acids. The amino acids then can be reused to make the proteins ...

  17. Protein C blood test

    MedlinePlus

    ... a normal substance in the body that prevents blood clotting. A blood test can be done to see ... history of blood clots. Protein C helps control blood clotting. A lack of this protein or problem with ...

  18. Protein S blood test

    MedlinePlus

    ... a normal substance in your body that prevents blood clotting. A blood test can be done to see ... family history of blood clots. Protein S helps control blood clotting. A lack of this protein or problem with ...

  19. Time course of large ribosomal subunit assembly in E. coli cells overexpressing a helicase inactive DbpA protein.

    PubMed

    Gentry, Riley C; Childs, Jared J; Gevorkyan, Jirair; Gerasimova, Yulia V; Koculi, Eda

    2016-07-01

    DbpA is a DEAD-box RNA helicase implicated in Escherichia coli large ribosomal subunit assembly. Previous studies have shown that when the ATPase and helicase inactive DbpA construct, R331A, is expressed in E. coli cells, a large ribosomal subunit intermediate accumulates. The large subunit intermediate migrates as a 45S particle in a sucrose gradient. Here, using a number of structural and fluorescent assays, we investigate the ribosome profiles of cells lacking wild-type DbpA and overexpressing the R331A DbpA construct. Our data show that in addition to the 45S particle previously described, 27S and 35S particles are also present in the ribosome profiles of cells overexpressing R331A DbpA. The 27S, 35S, and 45S independently convert to the 50S subunit, suggesting that ribosome assembly in the presence of R331A and the absence of wild-type DbpA occurs via multiple pathways. © 2016 Gentry et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  20. Protein and older adults.

    PubMed

    Chernoff, Ronni

    2004-12-01

    Body composition changes as people get older. One of the noteworthy alterations is the reduction in total body protein. A decrease in skeletal muscle is the most noticeable manifestation of this change but there is also a reduction in other physiologic proteins such as organ tissue, blood components, and immune bodies as well as declines in total body potassium and water. This contributes to impaired wound healing, loss of skin elasticity, and an inability to fight infection. The recommended dietary allowance (RDA) for adults for protein is 0.8 grams of protein per kilogram of body weight. Protein tissue accounts for 30% of whole-body protein turnover but that rate declines to 20% or less by age 70. The result of this phenomenon is that older adults require more protein/kilogram body weight than do younger adults. Recently, it has become clear that the requirement for exogenous protein is at least 1.0 gram/kilogram body weight. Adequate dietary intake of protein may be more difficult for older adults to obtain. Dietary animal protein is the primary source of high biological value protein, iron, vitamin B(12), folic acid, biotin and other essential nutrients. In fact, egg protein is the standard against which all other proteins are compared. Compared to other high-quality protein sources like meat, poultry and seafood, eggs are the least expensive. The importance of dietary protein cannot be underestimated in the diets of older adults; inadequate protein intake contributes to a decrease in reserve capacity, increased skin fragility, decreased immune function, poorer healing, and longer recuperation from illness.

  1. Modeling Protein Self Assembly

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton Buck; Hull, Elizabeth

    2004-01-01

    Understanding the structure and function of proteins is an important part of the standards-based science curriculum. Proteins serve vital roles within the cell and malfunctions in protein self assembly are implicated in degenerative diseases. Experience indicates that this topic is a difficult one for many students. We have found that the concept…

  2. Overview of Protein Microarrays

    PubMed Central

    Reymond Sutandy, FX; Qian, Jiang; Chen, Chien-Sheng; Zhu, Heng

    2013-01-01

    Protein microarray is an emerging technology that provides a versatile platform for characterization of hundreds of thousands of proteins in a highly parallel and high-throughput way. Two major classes of protein microarrays are defined to describe their applications: analytical and functional protein microarrays. In addition, tissue or cell lysates can also be fractionated and spotted on a slide to form a reverse-phase protein microarray. While the fabrication technology is maturing, applications of protein microarrays, especially functional protein microarrays, have flourished during the past decade. Here, we will first review recent advances in the protein microarray technologies, and then present a series of examples to illustrate the applications of analytical and functional protein microarrays in both basic and clinical research. The research areas will include detection of various binding properties of proteins, study of protein posttranslational modifications, analysis of host-microbe interactions, profiling antibody specificity, and identification of biomarkers in autoimmune diseases. As a powerful technology platform, it would not be surprising if protein microarrays will become one of the leading technologies in proteomic and diagnostic fields in the next decade. PMID:23546620

  3. CSF total protein

    MedlinePlus

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 milligrams per deciliter (mg/dL) ...

  4. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  5. Destabilized bioluminescent proteins

    DOEpatents

    Allen, Michael S.; Rakesh, Gupta; Gary, Sayler S.

    2007-07-31

    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  6. Texturized dairy proteins

    USDA-ARS?s Scientific Manuscript database

    Dairy proteins are amenable to structural modifications induced by high temperature, shear and moisture; in particular, whey proteins can change conformation to new unfolded states. The change in protein state is a basis for creating new foods. The dairy products, nonfat dried milk (NDM), whey prote...

  7. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  8. SAMPyling Proteins in Archaea

    PubMed Central

    Darwin, K. Heran; Hofmann, Kay

    2010-01-01

    For some time post-translational small protein modifications were found only in eukaryotes; much later, such modifications were identified in some species of bacteria. The recent discovery of ubiquitin-like proteins that form polymeric chains and covalently modify proteins in archaea finally closes the evolutionary gap among the domains of life. PMID:20547064

  9. The E5 Proteins

    PubMed Central

    DiMaio, Daniel; Petti, Lisa

    2013-01-01

    The E5 proteins are short transmembrane proteins encoded by many animal and human papillomaviruses. These proteins display transforming activity in cultured cells and animals, and they presumably also play a role in the productive virus life cycle. The E5 proteins are thought to act by modulating the activity of cellular proteins. Here, we describe the biological activities of the best-studied E5 proteins and discuss the evidence implicating specific protein targets and pathways in mediating these activities. The primary target of the 44-amino acid BPV1 E5 is the PDGF β receptor, whereas the EGF receptor appears to be an important target of the 83-amino acid HPV16 E5 protein. Both E5 proteins also bind to the vacuolar ATPase and affect MHC class I expression and cell-cell communication. Continued studies of the E5 proteins will elucidate important aspects of transmembrane protein-protein interactions, cellular signal transduction, cell biology, virus replication, and tumorigenesis. PMID:23731971

  10. Protein-protein interactions in multienzyme megasynthetases.

    PubMed

    Weissman, Kira J; Müller, Rolf

    2008-04-14

    The multienzyme polyketide synthases (PKSs), nonribosomal polypeptide synthetases (NRPSs), and their hybrids are responsible for the construction in bacteria of numerous natural products of clinical value. These systems generate high structural complexity by using a simple biosynthetic logic--that of the assembly line. Each of the individual steps in building the metabolites is designated to an independently folded domain within gigantic polypeptides. The domains are clustered into functional modules, and the modules are strung out along the proteins in the order in which they act. Every metabolite results, therefore, from the successive action of up to 100 individual catalysts. Despite the conceptual simplicity of this division-of-labor organization, we are only beginning to decipher the molecular details of the numerous protein-protein interactions that support assembly-line biosynthesis, and which are critical to attempts to re-engineer these systems as a tool in drug discovery. This review aims to summarize the state of knowledge about several aspects of protein-protein interactions, including current architectural models for PKS and NRPS systems, the central role of carrier proteins, and the structural basis for intersubunit recognition.

  11. Protopia: a protein-protein interaction tool

    PubMed Central

    Real-Chicharro, Alejandro; Ruiz-Mostazo, Iván; Navas-Delgado, Ismael; Kerzazi, Amine; Chniber, Othmane; Sánchez-Jiménez, Francisca; Medina, Miguel Ángel; Aldana-Montes, José F

    2009-01-01

    Background Protein-protein interactions can be considered the basic skeleton for living organism self-organization and homeostasis. Impressive quantities of experimental data are being obtained and computational tools are essential to integrate and to organize this information. This paper presents Protopia, a biological tool that offers a way of searching for proteins and their interactions in different Protein Interaction Web Databases, as a part of a multidisciplinary initiative of our institution for the integration of biological data . Results The tool accesses the different Databases (at present, the free version of Transfac, DIP, Hprd, Int-Act and iHop), and results are expressed with biological protein names or databases codes and can be depicted as a vector or a matrix. They can be represented and handled interactively as an organic graph. Comparison among databases is carried out using the Uniprot codes annotated for each protein. Conclusion The tool locates and integrates the current information stored in the aforementioned databases, and redundancies among them are detected. Results are compatible with the most important network analysers, so that they can be compared and analysed by other world-wide known tools and platforms. The visualization possibilities help to attain this goal and they are especially interesting for handling multiple-step or complex networks. PMID:19828077

  12. Protein crystallization with paper

    NASA Astrophysics Data System (ADS)

    Matsuoka, Miki; Kakinouchi, Keisuke; Adachi, Hiroaki; Maruyama, Mihoko; Sugiyama, Shigeru; Sano, Satoshi; Yoshikawa, Hiroshi Y.; Takahashi, Yoshinori; Yoshimura, Masashi; Matsumura, Hiroyoshi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Takano, Kazufumi

    2016-05-01

    We developed a new protein crystallization method that incorporates paper. A small piece of paper, such as facial tissue or KimWipes, was added to a drop of protein solution in the traditional sitting drop vapor diffusion technique, and protein crystals grew by incorporating paper. By this method, we achieved the growth of protein crystals with reducing osmotic shock. Because the technique is very simple and the materials are easy to obtain, this method will come into wide use for protein crystallization. In the future, it could be applied to nanoliter-scale crystallization screening on a paper sheet such as in inkjet printing.

  13. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  14. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  15. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2011-11-29

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  16. Forces Stabilizing Proteins

    PubMed Central

    Pace, C. Nick; Scholtz, J. Martin; Grimsley, Gerald R.

    2014-01-01

    The goal of this article is to summarize what has been learned about the major forces stabilizing proteins since the late 1980s when site-directed mutagenesis became possible. The following conclusions are derived from experimental studies of hydrophobic and hydrogen bonding variants. 1. Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a –CH2– group on folding contributes 1.1 ± 0.5 kcal/mol to protein stability. 2. The burial of nonpolar side chains contributes to protein stability in two ways: first, a term that depends on the removal of the side chains from water and, more importantly, the enhanced London dispersion forces that result from the tight packing in the protein interior. 3. Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond on folding contributes 1.1 ± 0.8 kcal/mol to protein stability. 4. The contribution of hydrogen bonds to protein stability is strongly context dependent. 5. Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. 6. Polar group burial can make a favorable contribution to protein stability even if the polar group is not hydrogen bonded. 7. Hydrophobic interactions and hydrogen bonds both make large contributions to protein stability. PMID:24846139

  17. Protein Complexes in Bacteria

    PubMed Central

    Caufield, J. Harry; Abreu, Marco; Wimble, Christopher; Uetz, Peter

    2015-01-01

    Large-scale analyses of protein complexes have recently become available for Escherichia coli and Mycoplasma pneumoniae, yielding 443 and 116 heteromultimeric soluble protein complexes, respectively. We have coupled the results of these mass spectrometry-characterized protein complexes with the 285 “gold standard” protein complexes identified by EcoCyc. A comparison with databases of gene orthology, conservation, and essentiality identified proteins conserved or lost in complexes of other species. For instance, of 285 “gold standard” protein complexes in E. coli, less than 10% are fully conserved among a set of 7 distantly-related bacterial “model” species. Complex conservation follows one of three models: well-conserved complexes, complexes with a conserved core, and complexes with partial conservation but no conserved core. Expanding the comparison to 894 distinct bacterial genomes illustrates fractional conservation and the limits of co-conservation among components of protein complexes: just 14 out of 285 model protein complexes are perfectly conserved across 95% of the genomes used, yet we predict more than 180 may be partially conserved across at least half of the genomes. No clear relationship between gene essentiality and protein complex conservation is observed, as even poorly conserved complexes contain a significant number of essential proteins. Finally, we identify 183 complexes containing well-conserved components and uncharacterized proteins which will be interesting targets for future experimental studies. PMID:25723151

  18. Protein and vegetarian diets.

    PubMed

    Marsh, Kate A; Munn, Elizabeth A; Baines, Surinder K

    2013-08-19

    A vegetarian diet can easily meet human dietary protein requirements as long as energy needs are met and a variety of foods are eaten. Vegetarians should obtain protein from a variety of plant sources, including legumes, soy products, grains, nuts and seeds. Eggs and dairy products also provide protein for those following a lacto-ovo-vegetarian diet. There is no need to consciously combine different plant proteins at each meal as long as a variety of foods are eaten from day to day, because the human body maintains a pool of amino acids which can be used to complement dietary protein. The consumption of plant proteins rather than animal proteins by vegetarians may contribute to their reduced risk of chronic diseases such as diabetes and heart disease.

  19. Pigment-protein complexes

    SciTech Connect

    Siegelman, H W

    1980-01-01

    The photosynthetically-active pigment protein complexes of procaryotes and eucaryotes include chlorophyll proteins, carotenochlorophyll proteins, and biliproteins. They are either integral components or attached to photosynthetic membranes. Detergents are frequently required to solubilize the pigment-protein complexes. The membrane localization and detergent solubilization strongly suggest that the pigment-protein complexes are bound to the membranes by hydrophobic interactions. Hydrophobic interactions of proteins are characterized by an increase in entropy. Their bonding energy is directly related to temperature and ionic strength. Hydrophobic-interaction chromatography, a relatively new separation procedure, can furnish an important method for the purification of pigment-protein complexes. Phycobilisome purification and properties provide an example of the need to maintain hydrophobic interactions to preserve structure and function.

  20. Protein Folding: Detailed Models

    NASA Astrophysics Data System (ADS)

    Pande, Vijay

    Proteins play a fundamental role in biology. With their ability to perform numerous biological roles, including acting as catalysts, antibodies, and molecular signals, proteins today realize many of the goals that modern nanotechnology aspires to. However, before proteins can carry out these remarkable molecular functions, they must perform another amazing feat — they must assemble themselves. This process of protein self-assembly into a particular shape, or "fold" is called protein folding. Due to the importance of the folded state in the biological activity of proteins, recent interest from misfolding related diseases [1], as well as a fascination of just how this process occurs [2-4], there has been much work performed in order to unravel the mechanism of protein folding [5].

  1. [Atypical ubiquitination of proteins].

    PubMed

    Buneeva, O A; Medvedev, A E

    2016-07-01

    Ubiquitination is a type of posttranslational modification of intracellular proteins characterized by covalent attachment of one (monoubiquitination) or several (polyubiquitination) of ubiquitin molecules to target proteins. In the case of polyubiquitination, linear or branched polyubiquitin chains are formed. Their formation involves various lysine residues of monomeric ubiquitin. The best studied is Lys48-polyubiquitination, which targets proteins for proteasomal degradation. In this review we have considered examples of so-called atypical polyubiquitination, which mainly involves other lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys63) and also N-terminal methionine. The considered examples convincingly demonstrate that polyubiquitination of proteins not necessarily targets proteins for their proteolytic degradation in proteasomes. Atypically polyubiquitinated proteins are involved in regulation of various processes and altered polyubiquitination of certain proteins is crucial for development of serious diseases.

  2. The Protein Folding Problem

    PubMed Central

    Dill, Ken A.; Ozkan, S. Banu; Shell, M. Scott; Weikl, Thomas R.

    2008-01-01

    The “protein folding problem” consists of three closely related puzzles: (a) What is the folding code? (b) What is the folding mechanism? (c) Can we predict the native structure of a protein from its amino acid sequence? Once regarded as a grand challenge, protein folding has seen great progress in recent years. Now, foldable proteins and nonbiological polymers are being designed routinely and moving toward successful applications. The structures of small proteins are now often well predicted by computer methods. And, there is now a testable explanation for how a protein can fold so quickly: A protein solves its large global optimization problem as a series of smaller local optimization problems, growing and assembling the native structure from peptide fragments, local structures first. PMID:18573083

  3. Protein solubility modeling

    NASA Technical Reports Server (NTRS)

    Agena, S. M.; Pusey, M. L.; Bogle, I. D.

    1999-01-01

    A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process. Copyright 1999 John Wiley & Sons, Inc.

  4. Packing in protein cores

    NASA Astrophysics Data System (ADS)

    Gaines, J. C.; Clark, A. H.; Regan, L.; O'Hern, C. S.

    2017-07-01

    Proteins are biological polymers that underlie all cellular functions. The first high-resolution protein structures were determined by x-ray crystallography in the 1960s. Since then, there has been continued interest in understanding and predicting protein structure and stability. It is well-established that a large contribution to protein stability originates from the sequestration from solvent of hydrophobic residues in the protein core. How are such hydrophobic residues arranged in the core; how can one best model the packing of these residues, and are residues loosely packed with multiple allowed side chain conformations or densely packed with a single allowed side chain conformation? Here we show that to properly model the packing of residues in protein cores it is essential that amino acids are represented by appropriately calibrated atom sizes, and that hydrogen atoms are explicitly included. We show that protein cores possess a packing fraction of φ ≈ 0.56 , which is significantly less than the typically quoted value of 0.74 obtained using the extended atom representation. We also compare the results for the packing of amino acids in protein cores to results obtained for jammed packings from discrete element simulations of spheres, elongated particles, and composite particles with bumpy surfaces. We show that amino acids in protein cores pack as densely as disordered jammed packings of particles with similar values for the aspect ratio and bumpiness as found for amino acids. Knowing the structural properties of protein cores is of both fundamental and practical importance. Practically, it enables the assessment of changes in the structure and stability of proteins arising from amino acid mutations (such as those identified as a result of the massive human genome sequencing efforts) and the design of new folded, stable proteins and protein-protein interactions with tunable specificity and affinity.

  5. Predictions of Protein-Protein Interfaces within Membrane Protein Complexes

    PubMed Central

    Asadabadi, Ebrahim Barzegari; Abdolmaleki, Parviz

    2013-01-01

    Background Prediction of interaction sites within the membrane protein complexes using the sequence data is of a great importance, because it would find applications in modification of molecules transport through membrane, signaling pathways and drug targets of many diseases. Nevertheless, it has gained little attention from the protein structural bioinformatics community. Methods In this study, a wide variety of prediction and classification tools were applied to distinguish the residues at the interfaces of membrane proteins from those not in the interfaces. Results The tuned SVM model achieved the high accuracy of 86.95% and the AUC of 0.812 which outperforms the results of the only previous similar study. Nevertheless, prediction performances obtained using most employed models cannot be used in applied fields and needs more effort to improve. Conclusion Considering the variety of the applied tools in this study, the present investigation could be a good starting point to develop more efficient tools to predict the membrane protein interaction site residues. PMID:23919118

  6. Toxic proteins in plants.

    PubMed

    Dang, Liuyi; Van Damme, Els J M

    2015-09-01

    Plants have evolved to synthesize a variety of noxious compounds to cope with unfavorable circumstances, among which a large group of toxic proteins that play a critical role in plant defense against predators and microbes. Up to now, a wide range of harmful proteins have been discovered in different plants, including lectins, ribosome-inactivating proteins, protease inhibitors, ureases, arcelins, antimicrobial peptides and pore-forming toxins. To fulfill their role in plant defense, these proteins exhibit various degrees of toxicity towards animals, insects, bacteria or fungi. Numerous studies have been carried out to investigate the toxic effects and mode of action of these plant proteins in order to explore their possible applications. Indeed, because of their biological activities, toxic plant proteins are also considered as potentially useful tools in crop protection and in biomedical applications, such as cancer treatment. Genes encoding toxic plant proteins have been introduced into crop genomes using genetic engineering technology in order to increase the plant's resistance against pathogens and diseases. Despite the availability of ample information on toxic plant proteins, very few publications have attempted to summarize the research progress made during the last decades. This review focuses on the diversity of toxic plant proteins in view of their toxicity as well as their mode of action. Furthermore, an outlook towards the biological role(s) of these proteins and their potential applications is discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Reverse Phase Protein Microarrays.

    PubMed

    Baldelli, Elisa; Calvert, Valerie; Hodge, Alex; VanMeter, Amy; Petricoin, Emanuel F; Pierobon, Mariaelena

    2017-01-01

    While genes and RNA encode information about cellular status, proteins are considered the engine of the cellular machine, as they are the effective elements that drive all cellular functions including proliferation, migration, differentiation, and apoptosis. Consequently, investigations of the cellular protein network are considered a fundamental tool for understanding cellular functions.Alteration of the cellular homeostasis driven by elaborate intra- and extracellular interactions has become one of the most studied fields in the era of personalized medicine and targeted therapy. Increasing interest has been focused on developing and improving proteomic technologies that are suitable for analysis of clinical samples. In this context, reverse-phase protein microarrays (RPPA) is a sensitive, quantitative, high-throughput immunoassay for protein analyses of tissue samples, cells, and body fluids.RPPA is well suited for broad proteomic profiling and is capable of capturing protein activation as well as biochemical reactions such as phosphorylation, glycosylation, ubiquitination, protein cleavage, and conformational alterations across hundreds of samples using a limited amount of biological material. For these reasons, RPPA represents a valid tool for protein analyses and generates data that help elucidate the functional signaling architecture through protein-protein interaction and protein activation mapping for the identification of critical nodes for individualized or combinatorial targeted therapy.

  8. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  9. Protein kinesis: The dynamics of protein trafficking and stability

    SciTech Connect

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  10. A modified Lowry protein test for dilute protein solutions

    Treesearch

    Garold F. Gregory; Keith F. Jensen

    1971-01-01

    A modified Lowry protein test for dilute protein solutions modified Lowry protein test was compared with the standard Lowry protein test. The modified test was found to give estimates of protein concentration that were as good as the standard test and has the advange that proteins can be measured in very dilute solutions.

  11. Protein flexibility as a biosignal.

    PubMed

    Zhao, Qinyi

    2010-01-01

    Dynamic properties of a protein are crucial for all protein functions, and those of signaling proteins are closely related to the biological function of living beings. The protein flexibility signal concept can be used to analyze this relationship. Protein flexibility controls the rate of protein conformational change and influences protein function. The modification of protein flexibility results in a change of protein activity. The logical nature of protein flexibility cannot be explained by applying the principles of protein three-dimensional structure theory or conformation concept. Signaling proteins show high protein flexibility. Many properties of signaling can be traced back to the dynamic natures of signaling protein. The action mechanism of volatile anesthetics and universal cellular reactions are related to flexibility in the change of signaling proteins. We conclude that protein dynamics is an enzyme-enhanced process, called dynamicase.

  12. Antimicrobial proteins: From old proteins, new tricks.

    PubMed

    Smith, Valerie J; Dyrynda, Elisabeth A

    2015-12-01

    This review describes the main types of antimicrobial peptides (AMPs) synthesised by crustaceans, primarily those identified in shrimp, crayfish, crab and lobster. It includes an overview of their range of microbicidal activities and the current landscape of our understanding of their gene expression patterns in different body tissues. It further summarises how their expression might change following various types of immune challenges. The review further considers proteins or protein fragments from crustaceans that have antimicrobial properties but are more usually associated with other biological functions, or are derived from such proteins. It discusses how these unconventional AMPs might be generated at, or delivered to, sites of infection and how they might contribute to crustacean host defence in vivo. It also highlights recent work that is starting to reveal the extent of multi-functionality displayed by some decapod AMPs, particularly their participation in other aspects of host protection. Examples of such activities include proteinase inhibition, phagocytosis, antiviral activity and haematopoiesis.

  13. Protein-protein Interactions using Radiolytic Footprinting

    SciTech Connect

    Takamoto,K.; Chance, M.

    2006-01-01

    Structural proteomics approaches using mass spectrometry are increasingly used in biology to examine the composition and structure of macromolecules. Hydroxyl radical-mediated protein footprinting using mass spectrometry has recently been developed to define structure, assembly, and conformational changes of macromolecules in solution based on measurements of reactivity of amino acid side chain groups with covalent modification reagents. Accurate measurements of side chain reactivity are achieved using quantitative liquid-chromatography-coupled mass spectrometry, whereas the side chain modification sites are identified using tandem mass spectrometry. In addition, the use of footprinting data in conjunction with computational modeling approaches is a powerful new method for testing and refining structural models of macromolecules and their complexes. In this review, we discuss the basic chemistry of hydroxyl radical reactions with peptides and proteins, highlight various approaches to map protein structure using radical oxidation methods, and describe state-of-the-art approaches to combine computational and footprinting data.

  14. Why Do Proteins Look Like Protein?

    NASA Astrophysics Data System (ADS)

    Li, Hao

    1998-03-01

    Protein structures in nature exhibit remarkable regularities (common structural motifs, tertiary symmetries etc.) which are absent from random compact conformations. Comparison of known protein structures in the structure databank reveals that certain popular folds are frequently found in protein families unrelated by sequence homology or biological function. Are protein structures selected merely by historical accident or is there some more fundamental reasons behind their selection? Our studies based on a simple model of protein folding suggest that a designability principle plays an important role in the selection of structures(H Li, R. Helling, C. Tang, N. S. Wingreen,Science) 273, 666 (1996). The designability of a structure is measured by the number of sequences which uniquely design the structure. In an exhaustive study of all possible sequences and structures for chains with different length, we find highly designable structures with number of associated sequences much larger than the average. These highly designable structures possess ``protein like'' structural motifs, and have higher mutational and thermodynamic stability. Thus they are more likely to be selected by nature and survive in the course of evolution. I will discuss how to formulate designability in terms of distribution of structures in a structure space. I will give an example where only hydrophobic interaction is considered. In this case designability of a structure can be obtained by a simple geometric construction, which naturally explains the origin of highly designable structure and the correlation between designability and thermodynamic stability. I will show that highly designable structures need to have an atypical pattern of residue solvent exposure along the backbone, which leads to structural regularity.

  15. Mechanisms Regulating Protein Localization.

    PubMed

    Bauer, Nicholas C; Doetsch, Paul W; Corbett, Anita H

    2015-10-01

    Cellular functions are dictated by protein content and activity. There are numerous strategies to regulate proteins varying from modulating gene expression to post-translational modifications. One commonly used mode of regulation in eukaryotes is targeted localization. By specifically redirecting the localization of a pool of existing protein, cells can achieve rapid changes in local protein function. Eukaryotic cells have evolved elegant targeting pathways to direct proteins to the appropriate cellular location or locations. Here, we provide a general overview of these localization pathways, with a focus on nuclear and mitochondrial transport, and present a survey of the evolutionarily conserved regulatory strategies identified thus far. We end with a description of several specific examples of proteins that exploit localization as an important mode of regulation.

  16. Supramolecular Chemistry Targeting Proteins.

    PubMed

    van Dun, Sam; Ottmann, Christian; Milroy, Lech-Gustav; Brunsveld, Luc

    2017-09-28

    The specific recognition of protein surface elements is a fundamental challenge in the life sciences. New developments in this field will form the basis of advanced therapeutic approaches and lead to applications such as sensors, affinity tags, immobilization techniques, and protein-based materials. Synthetic supramolecular molecules and materials are creating new opportunities for protein recognition that are orthogonal to classical small molecule and protein-based approaches. As outlined here, their unique molecular features enable the recognition of amino acids, peptides, and even whole protein surfaces, which can be applied to the modulation and assembly of proteins. We believe that structural insights into these processes are of great value for the further development of this field and have therefore focused this Perspective on contributions that provide such structural data.

  17. Protein Misfolding Diseases.

    PubMed

    Hartl, F Ulrich

    2017-06-20

    The majority of protein molecules must fold into defined three-dimensional structures to acquire functional activity. However, protein chains can adopt a multitude of conformational states, and their biologically active conformation is often only marginally stable. Metastable proteins tend to populate misfolded species that are prone to forming toxic aggregates, including soluble oligomers and fibrillar amyloid deposits, which are linked with neurodegeneration in Alzheimer and Parkinson disease, and many other pathologies. To prevent or regulate protein aggregation, all cells contain an extensive protein homeostasis (or proteostasis) network comprising molecular chaperones and other factors. These defense systems tend to decline during aging, facilitating the manifestation of aggregate deposition diseases. This volume of the Annual Review of Biochemistry contains a set of three articles addressing our current understanding of the structures of pathological protein aggregates and their associated disease mechanisms. These articles also discuss recent insights into the strategies cells have evolved to neutralize toxic aggregates by sequestering them in specific cellular locations.

  18. TRIM proteins and diseases.

    PubMed

    Watanabe, Masashi; Hatakeyama, Shigetsugu

    2017-01-07

    Ubiquitination is one of the posttranslational modifications that regulates a number of intracellular events including signal transduction, protein quality control, transcription, cell cycle, apoptosis and development. The ubiquitin system functions as a garbage machine to degrade target proteins and as a regulator for several signalling pathways. Biochemical reaction of ubiquitination requires several enzymes including E1, E2 and E3, and E3 ubiquitin ligases play roles as receptors for recognizing target proteins. Most of the tripartite motif (TRIM) proteins are E3 ubiquitin ligases. Recent studies have shown that some TRIM proteins function as important regulators for a variety of diseases including cancer, inflammatory diseases, infectious diseases, neuropsychiatric disorders, chromosomal abnormalities and developmental diseases. In this review, we summarize the involvement of TRIM proteins in the aetiology of various diseases.

  19. Mayaro virus proteins.

    PubMed

    Mezencio, J M; Rebello, M A

    1993-01-01

    Mayaro virus was grown in BHK-21 cells and purified by centrifugation in a potassium-tartrate gradient (5-50%). The electron microscopy analyses of the purified virus showed an homogeneous population of enveloped particles with 69 +/- 2.3 nm in diameter. Three structural virus proteins were identified and designated p1, p2 and p3. Their average molecular weight were p1, 54 KDa; p2, 50 KDa and p3, 34 KDa. In Mayaro virus infected Aedes albopictus cells and in BHK-21 infected cells we detected six viral proteins, in which three of them are the structural virus proteins and the other three were products from processing of precursors of viral proteins, whose molecular weights are 62 KDa, 64 KDa and 110 KDa. The 34 KDa protein was the first viral protein synthesized at 5 hours post-infection in both cell lines studied.

  20. Protein-protein interactions as drug targets.

    PubMed

    Skwarczynska, Malgorzata; Ottmann, Christian

    2015-01-01

    Modulation of protein-protein interactions (PPIs) is becoming increasingly important in drug discovery and chemical biology. While a few years ago this 'target class' was deemed to be largely undruggable an impressing number of publications and success stories now show that targeting PPIs with small, drug-like molecules indeed is a feasible approach. Here, we summarize the current state of small-molecule inhibition and stabilization of PPIs and review the active molecules from a structural and medicinal chemistry angle, especially focusing on the key examples of iNOS, LFA-1 and 14-3-3.

  1. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell, Post-Doctoral Fellow the National Research Council (NRC) uses a reciprocal space mapping diffractometer for macromolecular crystal quality studies. The diffractometer is used in mapping the structure of macromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystallized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  2. Computer Models of Proteins

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Dr. Marc Pusey (seated) and Dr. Craig Kundrot use computers to analyze x-ray maps and generate three-dimensional models of protein structures. With this information, scientists at Marshall Space Flight Center can learn how proteins are made and how they work. The computer screen depicts a proten structure as a ball-and-stick model. Other models depict the actual volume occupied by the atoms, or the ribbon-like structures that are crucial to a protein's function.

  3. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell, Post-Doctoral Fellow the National Research Council (NRC) uses a reciprocal space mapping diffractometer for macromolecular crystal quality studies. The diffractometer is used in mapping the structure of macromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystallized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  4. Protein oxidation and peroxidation

    PubMed Central

    Davies, Michael J.

    2016-01-01

    Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function. Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides inhibit these pathways and this may contribute to the accumulation of modified proteins in cells. Available evidence supports an association between protein oxidation and multiple human pathologies, but whether this link is causal remains to be established. PMID:27026395

  5. Pressure cryocooling protein crystals

    DOEpatents

    Kim, Chae Un [Ithaca, NY; Gruner, Sol M [Ithaca, NY

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  6. Acanthamoeba castellanii STAT Protein

    PubMed Central

    Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups. PMID:25338074

  7. Protein intakes in India.

    PubMed

    Swaminathan, Sumathi; Vaz, Mario; Kurpad, Anura V

    2012-08-01

    Indian diets derive almost 60 % of their protein from cereals with relatively low digestibility and quality. There have been several surveys of diets and protein intakes in India by the National Nutrition Monitoring Board (NNMB) over the last 25 years, in urban and rural, as well as in slum dwellers and tribal populations. Data of disadvantaged populations from slums, tribals and sedentary rural Indian populations show that the protein intake (mainly from cereals) is about 1 gm/kg/day. However, the protein intake looks less promising in terms of the protein digestibility corrected amino acid score (PDCAAS), using lysine as the first limiting amino acid, where all populations, particularly rural and tribal, appear to have an inadequate quality to their protein intake. The protein: energy (PE) ratio is a measure of dietary quality, and has been used in the 2007 WHO/FAO/UNU report to define reference requirement values with which the adequacy of diets can be evaluated in terms of a protein quality corrected PE ratio. It is likely that about one third of this sedentary rural population is at risk of not meeting their requirements. These levels of risk of deficiency are in a population with relatively low BMI populations, whose diets are also inadequate in fruits and vegetables. Therefore, while the burden of enhancing the quality of protein intake in rural India exists, the quality of the diet, in general, represents a challenge that must be met.

  8. Self assembling proteins

    DOEpatents

    Yeates, Todd O.; Padilla, Jennifer; Colovos, Chris

    2004-06-29

    Novel fusion proteins capable of self-assembling into regular structures, as well as nucleic acids encoding the same, are provided. The subject fusion proteins comprise at least two oligomerization domains rigidly linked together, e.g. through an alpha helical linking group. Also provided are regular structures comprising a plurality of self-assembled fusion proteins of the subject invention, and methods for producing the same. The subject fusion proteins find use in the preparation of a variety of nanostructures, where such structures include: cages, shells, double-layer rings, two-dimensional layers, three-dimensional crystals, filaments, and tubes.

  9. Consensus protein design

    PubMed Central

    Porebski, Benjamin T.; Buckle, Ashley M.

    2016-01-01

    A popular and successful strategy in semi-rational design of protein stability is the use of evolutionary information encapsulated in homologous protein sequences. Consensus design is based on the hypothesis that at a given position, the respective consensus amino acid contributes more than average to the stability of the protein than non-conserved amino acids. Here, we review the consensus design approach, its theoretical underpinnings, successes, limitations and challenges, as well as providing a detailed guide to its application in protein engineering. PMID:27274091

  10. Engineering therapeutic protein disaggregases

    PubMed Central

    Shorter, James

    2016-01-01

    Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD. PMID:27255695

  11. PIC: Protein Interactions Calculator

    PubMed Central

    Tina, K. G.; Bhadra, R.; Srinivasan, N.

    2007-01-01

    Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic–aromatic interactions, aromatic–sulphur interactions and cation–π interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard, published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can also recognize specific kind of interactions, such as apolar–apolar residue interactions or ionic interactions, that are formed between buried or exposed residues or near the surface or deep inside. PMID:17584791

  12. PIC: Protein Interactions Calculator.

    PubMed

    Tina, K G; Bhadra, R; Srinivasan, N

    2007-07-01

    Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic-aromatic interactions, aromatic-sulphur interactions and cation-pi interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard, published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can also recognize specific kind of interactions, such as apolar-apolar residue interactions or ionic interactions, that are formed between buried or exposed residues or near the surface or deep inside.

  13. Chemical Synthesis of Proteins

    PubMed Central

    Nilsson, Bradley L.; Soellner, Matthew B.; Raines, Ronald T.

    2010-01-01

    Proteins have become accessible targets for chemical synthesis. The basic strategy is to use native chemical ligation, Staudinger ligation, or other orthogonal chemical reactions to couple synthetic peptides. The ligation reactions are compatible with a variety of solvents and proceed in solution or on a solid support. Chemical synthesis enables a level of control on protein composition that greatly exceeds that attainable with ribosome-mediated biosynthesis. Accordingly, the chemical synthesis of proteins is providing previously unattainable insight into the structure and function of proteins. PMID:15869385

  14. TRIM proteins in development.

    PubMed

    Petrera, Francesca; Meroni, Germana

    2012-01-01

    TRIM proteins play important roles in several patho-physiological processes. Their common activity within the ubiquitylation pathway makes them amenable to a number of diverse biological roles. Many of the TRIM genes are highly and sometimes specifically expressed during embryogenesis, it is therefore not surprising that several of them might be involved in developmental processes. Here, we primarily discuss the developmental implications of two subgroups of TRIM proteins that conserved domain composition and functions from their invertebrate ancestors. The two groups are: the TRIM-NHL proteins implicated in miRNA processing regulation and the TRIM-FN3 proteins involved in ventral midline development.

  15. Human Mitochondrial Protein Database

    National Institute of Standards and Technology Data Gateway

    SRD 131 Human Mitochondrial Protein Database (Web, free access)   The Human Mitochondrial Protein Database (HMPDb) provides comprehensive data on mitochondrial and human nuclear encoded proteins involved in mitochondrial biogenesis and function. This database consolidates information from SwissProt, LocusLink, Protein Data Bank (PDB), GenBank, Genome Database (GDB), Online Mendelian Inheritance in Man (OMIM), Human Mitochondrial Genome Database (mtDB), MITOMAP, Neuromuscular Disease Center and Human 2-D PAGE Databases. This database is intended as a tool not only to aid in studying the mitochondrion but in studying the associated diseases.

  16. Staining Proteins in Gels

    PubMed Central

    Gallagher, Sean; Chakavarti, Deb

    2008-01-01

    Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here. PMID:19066521

  17. Staining proteins in gels.

    PubMed

    Gallagher, Sean; Chakavarti, Deb

    2008-07-08

    Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

  18. Protein unfolding through nanopores.

    PubMed

    Oukhaled, Abdelghani; Pastoriza-Gallego, Manuela; Bacri, Laurent; Mathé, Jérôme; Auvray, Loïc; Pelta, Juan

    2014-03-01

    In this mini-review we introduce and discuss a new method, at single molecule level, to study the protein folding and protein stability, with a nanopore coupled to an electric detection. Proteins unfolded or partially folded passing through one channel submitted to an electric field, in the presence of salt solution, induce different detectable blockades of ionic current. Their duration depends on protein conformation. For different studies proteins through nanopores, completely unfolded proteins induce only short current blockades. Their frequency increases as the concentration of denaturing agent or temperature increases, following a sigmoidal denaturation curve. The geometry or the net charge of the nanopores does not alter the unfolding transition, sigmoidal unfolding curve and half denaturing concentration or half temperature denaturation. A destabilized protein induces a shift of the unfolding curve towards the lower values of the denaturant agent compared to the wild type protein.Partially folded proteins exhibit very long blockades in nanopores. The blockade duration decreases when the concentration of denaturing agent increases. The variation of these blockades could be associated to a possible glassy behaviour.

  19. Dietary proteins and angiogenesis.

    PubMed

    Medina, Miguel Ángel; Quesada, Ana R

    2014-01-17

    Both defective and persistent angiogenesis are linked to pathological situations in the adult. Compounds able to modulate angiogenesis have a potential value for the treatment of such pathologies. Several small molecules present in the diet have been shown to have modulatory effects on angiogenesis. This review presents the current state of knowledge on the potential modulatory roles of dietary proteins on angiogenesis. There is currently limited available information on the topic. Milk contains at least three proteins for which modulatory effects on angiogenesis have been previously demonstrated. On the other hand, there is some scarce information on the potential of dietary lectins, edible plant proteins and high protein diets to modulate angiogenesis.

  20. Ultrafiltration of pegylated proteins

    NASA Astrophysics Data System (ADS)

    Molek, Jessica R.

    There is considerable clinical interest in the use of "second-generation" therapeutics produced by conjugation of a native protein with various polymers including polyethylene glycol (PEG). PEG--protein conjugates, so-called PEGylated proteins, can exhibit enhanced stability, half-life, and bioavailability. One of the challenges in the commercial production of PEGylated proteins is the purification required to remove unreacted polymer, native protein, and in many cases PEGylated proteins with nonoptimal degrees of conjugation. The overall objective of this thesis was to examine the use of ultrafiltration for the purification of PEGylated proteins. This included: (1) analysis of size-based separation of PEGylated proteins using conventional ultrafiltration membranes, (2) use of electrically-charged membranes to exploit differences in electrostatic interactions, and (3) examination of the effects of PEGylation on protein fouling. The experimental results were analyzed using appropriate theoretical models, with the underlying physical properties of the PEGylated proteins evaluated using size exclusion chromatography, capillary electrophoresis, dynamic light scattering, and reverse phase chromatography. PEGylated proteins were produced by covalent attachment of activated PEG to a protein via primary amines on the lysine residues. A simple model was developed for the reaction kinetics, which was used to explore the effect of reaction conditions and mode of operation on the distribution of PEGylated products. The effective size of the PEGylated proteins was evaluated using size exclusion chromatography, with appropriate correlations developed for the size in terms of the molecular weight of the native protein and attached PEG. The electrophoretic mobility of the PEGylated proteins were evaluated by capillary electrophoresis with the data in good agreement with a simple model accounting for the increase in protein size and the reduction in the number of protonated amine

  1. Hydrophobic folding units at protein-protein interfaces: implications to protein folding and to protein-protein association.

    PubMed Central

    Tsai, C. J.; Nussinov, R.

    1997-01-01

    A hydrophobic folding unit cutting algorithm, originally developed for dissecting single-chain proteins, has been applied to a dataset of dissimilar two-chain protein-protein interfaces. Rather than consider each individual chain separately, the two-chain complex has been treated as a single chain. The two-chain parsing results presented in this work show hydrophobicity to be a critical attribute of two-state versus three-state protein-protein complexes. The hydrophobic folding units at the interfaces of two-state complexes suggest that the cooperative nature of the two-chain protein folding is the outcome of the hydrophobic effect, similar to its being the driving force in a single-chain folding. In analogy to the protein-folding process, the two-chain, two-state model complex may correspond to the formation of compact, hydrophobic nuclei. On the other hand, the three-state model complex involves binding of already folded monomers, similar to the association of the hydrophobic folding units within a single chain. The similarity between folding entities in protein cores and in two-state protein-protein interfaces, despite the absence of some chain connectivities in the latter, indicates that chain linkage does not necessarily affect the native conformation. This further substantiates the notion that tertiary, non-local interactions play a critical role in protein folding. These compact, hydrophobic, two-chain folding units, derived from structurally dissimilar protein-protein interfaces, provide a rich set of data useful in investigations of the role played by chain connectivity and by tertiary interactions in studies of binding and of folding. Since they are composed of non-contiguous pieces of protein backbones, they may also aid in defining folding nuclei. PMID:9232644

  2. Regulation of protein secretion by ... protein secretion?

    PubMed

    Atmakuri, Krishnamohan; Fortune, Sarah M

    2008-09-11

    Mycobacterium tuberculosis (Mtb) requires an alternative protein secretion system, ESX1, for virulence. Recently, Raghavan et al. (2008) reported a new regulatory circuit that may explain how ESX1 activity is controlled during infection. Mtb appears to regulate ESX1 by modulating transcription of associated genes rather than structural components of the secretion system itself.

  3. Human Plasma Protein C

    PubMed Central

    Kisiel, Walter

    1979-01-01

    Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (Mr = 62,000) contains 23% carbohydrate and is composed of a light chain (Mr = 21,000) and a heavy chain (Mr = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human α-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity. Images PMID:468991

  4. Proteins on the move: insights gained from fluorescent protein technologies.

    PubMed

    Miyawaki, Atsushi

    2011-09-23

    Proteins are always on the move, and this may occur through diffusion or active transport. The realization that the regulation of signal transduction is highly dynamic in space and time has stimulated intense interest in the movement of proteins. Over the past decade, numerous new technologies using fluorescent proteins have been developed, allowing us to observe the spatiotemporal dynamics of proteins in living cells. These technologies have greatly advanced our understanding of protein dynamics, including protein movement and protein interactions.

  5. Single-molecule analysis of Mss116-mediated group II intron folding

    PubMed Central

    Karunatilaka, Krishanthi S.; Solem, Amanda; Pyle, Anna Marie; Rueda, David

    2015-01-01

    DEAD-box helicases are conserved enzymes involved in nearly all aspects of RNA metabolism, but their mechanisms of action remain unclear. Here, we investigated the mechanism of the DEAD-box protein Mss116 on its natural substrate, the group II intron ai5γ. Group II introns are structurally complex catalytic RNAs considered evolutionarily related to the eukaryotic spliceosome, and an interesting paradigm for large RNA folding. We used single-molecule fluorescence to monitor the effect of Mss116 on folding dynamics of a minimal active construct, ai5γ–D135. The data show that Mss116 stimulates dynamic sampling between states along the folding pathway, an effect previously observed only with high Mg2+ concentrations. Furthermore, the data indicate that Mss116 promotes folding through discrete ATP-independent and ATP-dependent steps. We propose that Mss116 stimulates group II intron folding through a multi-step process that involves electrostatic stabilization of early intermediates and ATP hydrolysis during the final stages of native state assembly. PMID:20944626

  6. Structural modelling and phylogenetic analyses of PgeIF4A2 (Eukaryotic translation initiation factor) from Pennisetum glaucum reveal signature motifs with a role in stress tolerance and development

    PubMed Central

    Agarwal, Aakrati; Mudgil, Yashwanti; Pandey, Saurabh; Fartyal, Dhirendra; Reddy, Malireddy K

    2016-01-01

    Eukaryotic translation initiation factor 4A (eIF4A) is an indispensable component of the translation machinery and also play a role in developmental processes and stress alleviation in plants and animals. Different eIF4A isoforms are present in the cytosol of the cell, namely, eIF4A1, eIF4A2, and eIF4A3 and their expression is tightly regulated in cap-dependent translation. We revealed the structural model of PgeIF4A2 protein using the crystal structure of Homo sapiens eIF4A3 (PDB ID: 2J0S) as template by Modeller 9.12. The resultant PgeIF4A2 model structure was refined by PROCHECK, ProSA, Verify3D and RMSD that showed the model structure is reliable with 77 % amino acid sequence identity with template. Investigation revealed two conserved signatures for ATP-dependent RNA Helicase DEAD-box conserved site (VLDEADEML) and RNA helicase DEAD-box type, Q-motif in sheet-turn-helix and α-helical region respectively. All these conserved motifs are responsible for response during developmental stages and stress tolerance in plants. PMID:28358146

  7. Screening of genes regulated by cold shock in Shewanella piezotolerans WP3 and time course expression of cold-regulated genes.

    PubMed

    Li, Shengkang; Xiao, Xiang; Sun, Ping; Wang, Fengping

    2008-06-01

    The differential gene transcription of a deep-sea bacterium Shewanella piezotolerans WP3 in response to cold shock was analyzed by RNA arbitrarily primed PCR. Ninety primer sets were used to scan two different RNA pools derived from the culture of cold shock and its control (culture at its optimal grown temperature). Ninety-four putative differentially expressed fragments were identified and cloned. Six out of the 94 fragments were confirmed to be truly differentially transcribed in terms of cold shock by reverse Northern dot blot and then sequenced. Sequence blast analysis showed that the six differentially transcribed genes are putative genes for zonular occludens toxin, chaperon GroEL, efflux transporter, Sua5/YciO/YrdC/YwlC family protein, betaine-aldehyde dehydrogenase, and DEAD box RNA helicase, respectively. The time course expression profiles of these six genes from 0 to 90 min upon cold shock were quantified by real-time PCR. Deletion mutation of the highest induced gene--RNA helicase gene, had no significant impact on the growth of the strain no matter upon cold shock or under permanent low temperature. It is suggested that one or more additional DEAD box RNA helicase genes compensate for the loss of the function of the mutated gene.

  8. A genetic link between epigenetic repressor AS1-AS2 and a putative small subunit processome in leaf polarity establishment of Arabidopsis

    PubMed Central

    Matsumura, Yoko; Ohbayashi, Iwai; Takahashi, Hiro; Kojima, Shoko; Ishibashi, Nanako; Keta, Sumie; Nakagawa, Ayami; Hayashi, Rika; Saéz-Vásquez, Julio; Echeverria, Manuel; Sugiyama, Munetaka; Nakamura, Kenzo; Machida, Chiyoko

    2016-01-01

    ABSTRACT Although the DEAD-box RNA helicase family is ubiquitous in eukaryotes, its developmental role remains unelucidated. Here, we report that cooperative action between the Arabidopsis nucleolar protein RH10, an ortholog of human DEAD-box RNA helicase DDX47, and the epigenetic repressor complex of ASYMMETRIC-LEAVES1 (AS1) and AS2 (AS1-AS2) is critical to repress abaxial (ventral) genes ETT/ARF3 and ARF4, which leads to adaxial (dorsal) development in leaf primordia at shoot apices. Double mutations of rh10-1 and as2 (or as1) synergistically up-regulated the abaxial genes, which generated abaxialized filamentous leaves with loss of the adaxial domain. DDX47 is part of the small subunit processome (SSUP) that mediates rRNA biogenesis. In rh10-1 we found various defects in SSUP-related events, such as: accumulation of 35S/33S rRNA precursors; reduction in the 18S/25S ratio; and nucleolar hypertrophy. Double mutants of as2 with mutations of genes that encode other candidate SSUP-related components such as nucleolin and putative rRNA methyltransferase exhibited similar synergistic defects caused by up-regulation of ETT/ARF3 and ARF4. These results suggest a tight link between putative SSUP and AS1-AS2 in repression of the abaxial-determining genes for cell fate decisions for adaxial development. PMID:27334696

  9. Multidomain proteins under force.

    PubMed

    Valle-Orero, Jessica; Rivas-Pardo, Jaime Andrés; Popa, Ionel

    2017-04-28

    Advancements in single-molecule force spectroscopy techniques such as atomic force microscopy and magnetic tweezers allow investigation of how domain folding under force can play a physiological role. Combining these techniques with protein engineering and HaloTag covalent attachment, we investigate similarities and differences between four model proteins: I10 and I91-two immunoglobulin-like domains from the muscle protein titin, and two α + β fold proteins-ubiquitin and protein L. These proteins show a different mechanical response and have unique extensions under force. Remarkably, when normalized to their contour length, the size of the unfolding and refolding steps as a function of force reduces to a single master curve. This curve can be described using standard models of polymer elasticity, explaining the entropic nature of the measured steps. We further validate our measurements with a simple energy landscape model, which combines protein folding with polymer physics and accounts for the complex nature of tandem domains under force. This model can become a useful tool to help in deciphering the complexity of multidomain proteins operating under force.

  10. Archaeal chromatin proteins.

    PubMed

    Zhang, ZhenFeng; Guo, Li; Huang, Li

    2012-05-01

    Archaea, along with Bacteria and Eukarya, are the three domains of life. In all living cells, chromatin proteins serve a crucial role in maintaining the integrity of the structure and function of the genome. An array of small, abundant and basic DNA-binding proteins, considered candidates for chromatin proteins, has been isolated from the Euryarchaeota and the Crenarchaeota, the two major phyla in Archaea. While most euryarchaea encode proteins resembling eukaryotic histones, crenarchaea appear to synthesize a number of unique DNA-binding proteins likely involved in chromosomal organization. Several of these proteins (e.g., archaeal histones, Sac10b homologs, Sul7d, Cren7, CC1, etc.) have been extensively studied. However, whether they are chromatin proteins and how they function in vivo remain to be fully understood. Future investigation of archaeal chromatin proteins will lead to a better understanding of chromosomal organization and gene expression in Archaea and provide valuable information on the evolution of DNA packaging in cellular life.

  11. Synthesis of Lipidated Proteins.

    PubMed

    Mejuch, Tom; Waldmann, Herbert

    2016-08-17

    Protein lipidation is one of the major post-translational modifications (PTM) of proteins. The attachment of the lipid moiety frequently determines the localization and the function of the lipoproteins. Lipidated proteins participate in many essential biological processes in eukaryotic cells, including vesicular trafficking, signal transduction, and regulation of the immune response. Malfunction of these cellular processes usually leads to various diseases such as cancer. Understanding the mechanism of cellular signaling and identifying the protein-protein and protein-lipid interactions in which the lipoproteins are involved is a crucial task. To achieve these goals, fully functional lipidated proteins are required. However, access to lipoproteins by means of standard expression is often rather limited. Therefore, semisynthetic methods, involving the synthesis of lipidated peptides and their subsequent chemoselective ligation to yield full-length lipoproteins, were developed. In this Review we summarize the commonly used methods for lipoprotein synthesis and the development of the corresponding chemoselective ligation techniques. Several key studies involving full-length semisynthetic lipidated Ras, Rheb, and LC3 proteins are presented.

  12. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  13. Poxviral Ankyrin Proteins

    PubMed Central

    Herbert, Michael H.; Squire, Christopher J.; Mercer, Andrew A

    2015-01-01

    Multiple repeats of the ankyrin motif (ANK) are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range. PMID:25690795

  14. Protein Flexibilty and Folding

    NASA Astrophysics Data System (ADS)

    Thorpe, Michael

    2003-10-01

    In this talk we apply a novel approach to the exploration of energy landscapes of macromolecules and proteins that uses constraint theory. Constraints fix the bond lengths and bond angles and allow the use of theorems from graph theory to perform a rigid region decomposition of the network of atoms, which identifies the rigid regions, the flexible joints between them and also the stressed regions. We will show movies of the diffusive motion of various proteins. The protein unfolding transition is an example of a rigid to floppy transition and is shown to be more first order than second order because of the self-organized nature of the cross-linked polypeptide chain in the native protein. This approach emphasizes the universality in protein unfolding and allows the folding core and the transition state to be identified. Useful reference are: M.F. Thorpe, Ming Lei, A.J. Rader, Donald J. Jacobs and Leslie A. Kuhn Protein Flexibility Predictions using Graph Theory, Proteins 44, 150 - 165, (2001). A. J. Rader, Brandon M. Hespenheide, Leslie A. Kuhn and M. F. Thorpe Protein Unfolding: Rigidity Lost Proceedings of the National Academy of Sciences 99, 3540-3545 (2002). More details of this work can be found via http://physics.asu.edu/mfthorpe

  15. Proteins and Amino Acids

    USDA-ARS?s Scientific Manuscript database

    Proteins are the most abundant substances in living organisms and cells. All proteins are constructed from the same twenty amino acids that are linked together by covalent bonds. Shorter chains of two or more amino acids can be linked by covalent bonds to form polypeptides. There are twenty amino...

  16. Knotting pathways in proteins.

    PubMed

    Sułkowska, Joanna I; Noel, Jeffrey K; Ramírez-Sarmiento, César A; Rawdon, Eric J; Millett, Kenneth C; Onuchic, José N

    2013-04-01

    Most proteins, in order to perform their biological function, have to fold to a compact native state. The increasing number of knotted and slipknotted proteins identified suggests that proteins are able to manoeuvre around topological barriers during folding. In the present article, we review the current progress in elucidating the knotting process in proteins. Although we concentrate on theoretical approaches, where a knotted topology can be unambiguously detected, comparison with experiments is also reviewed. Numerical simulations suggest that the folding process for small knotted proteins is composed of twisted loop formation and then threading by either slipknot geometries or flipping. As the size of the knotted proteins increases, particularly for more deeply threaded termini, the prevalence of traps in the free energy landscape also increases. Thus, in the case of longer knotted and slipknotted proteins, the folding mechanism is probably supported by chaperones. Overall, results imply that knotted proteins can be folded efficiently and survive evolutionary pressure in order to perform their biological functions.

  17. Protein aggregation and prionopathies.

    PubMed

    Renner, M; Melki, R

    2014-06-01

    Prion protein and prion-like proteins share a number of characteristics. From the molecular point of view, they are constitutive proteins that aggregate following conformational changes into insoluble particles. These particles escape the cellular clearance machinery and amplify by recruiting the soluble for of their constituting proteins. The resulting protein aggregates are responsible for a number of neurodegenerative diseases such as Creutzfeldt-Jacob, Alzheimer, Parkinson and Huntington diseases. In addition, there are increasing evidences supporting the inter-cellular trafficking of these aggregates, meaning that they are "transmissible" between cells. There are also evidences that brain homogenates from individuals developing Alzheimer and Parkinson diseases propagate the disease in recipient model animals in a manner similar to brain extracts of patients developing Creutzfeldt-Jacob's disease. Thus, the propagation of protein aggregates from cell to cell may be a generic phenomenon that contributes to the evolution of neurodegenerative diseases, which has important consequences on human health issues. Moreover, although the distribution of protein aggregates is characteristic for each disease, new evidences indicate the possibility of overlaps and crosstalk between the different disorders. Despite the increasing evidences that support prion or prion-like propagation of protein aggregates, there are many unanswered questions regarding the mechanisms of toxicity and this is a field of intensive research nowadays. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  18. Protein Attachment on Nanodiamonds.

    PubMed

    Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

    2015-07-16

    A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery.

  19. Intracellular protein topogenesis

    PubMed Central

    Blobel, Günter

    1980-01-01

    Concurrently with or shortly after their synthesis on ribosomes, numerous specific proteins are unidirectionally translocated across or asymmetrically integrated into distinct cellular membranes. Thereafter, subpopulations of these proteins need to be sorted from each other and routed for export or targeted to other intracellular membranes or compartments. It is hypothesized here that the information for these processes, termed “protein topogenesis,” is encoded in discrete “topogenic” sequences that constitute a permanent or transient part of the polypeptide chain. The repertoire of distinct topogenic sequences is predicted to be relatively small because many different proteins would be topologically equivalent—i.e., targeted to the same intracellular address. The information content of topogenic sequences would be decoded and processed by distinct effectors. Four types of topogenic sequences could be distinguished: signal sequences, stop-transfer sequences, sorting sequences, and insertion sequences. Signal sequences initiate translocation of proteins across specific membranes. They would be decoded and processed by protein translocators that, by virtue of their signal sequence-specific domain and their unique location in distinct cellular membranes, effect unidirectional translocation of proteins across specific cellular membranes. Stop-transfer sequences interrupt the translocation process that was previously initiated by a signal sequence and, by excluding a distinct segment of the polypeptide chain from translocation, yield asymmetric integration of proteins into translocation-competent membranes. Sorting sequences would act as determinants for posttranslocational traffic of subpopulations of proteins, originating in translocation-competent donor membranes (and compartments) and going to translocation-incompetent receiver membranes (and compartments). Finally, insertion sequences initiate unilateral integration of proteins into the lipid bilayer

  20. Structures of membrane proteins

    PubMed Central

    Vinothkumar, Kutti R.; Henderson, Richard

    2010-01-01

    In reviewing the structures of membrane proteins determined up to the end of 2009, we present in words and pictures the most informative examples from each family. We group the structures together according to their function and architecture to provide an overview of the major principles and variations on the most common themes. The first structures, determined 20 years ago, were those of naturally abundant proteins with limited conformational variability, and each membrane protein structure determined was a major landmark. With the advent of complete genome sequences and efficient expression systems, there has been an explosion in the rate of membrane protein structure determination, with many classes represented. New structures are published every month and more than 150 unique membrane protein structures have been determined. This review analyses the reasons for this success, discusses the challenges that still lie ahead, and presents a concise summary of the key achievements with illustrated examples selected from each class. PMID:20667175

  1. Protein disulfide engineering.

    PubMed

    Dombkowski, Alan A; Sultana, Kazi Zakia; Craig, Douglas B

    2014-01-21

    Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnological tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. We review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  2. Artificially Engineered Protein Polymers.

    PubMed

    Yang, Yun Jung; Holmberg, Angela L; Olsen, Bradley D

    2017-06-07

    Modern polymer science increasingly requires precise control over macromolecular structure and properties for engineering advanced materials and biomedical systems. The application of biological processes to design and synthesize artificial protein polymers offers a means for furthering macromolecular tunability, enabling polymers with dispersities of ∼1.0 and monomer-level sequence control. Taking inspiration from materials evolved in nature, scientists have created modular building blocks with simplified monomer sequences that replicate the function of natural systems. The corresponding protein engineering toolbox has enabled the systematic development of complex functional polymeric materials across areas as diverse as adhesives, responsive polymers, and medical materials. This review discusses the natural proteins that have inspired the development of key building blocks for protein polymer engineering and the function of these elements in material design. The prospects and progress for scalable commercialization of protein polymers are reviewed, discussing both technology needs and opportunities.

  3. Protein expression-yeast.

    PubMed

    Nielsen, Klaus H

    2014-01-01

    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline.

  4. Protein crystallization in microgravity.

    PubMed

    Aibara, S; Shibata, K; Morita, Y

    1997-12-01

    A space experiment involving protein crystallization was conducted in a microgravity environment using the space shuttle "Endeavour" of STS-47, on a 9-day mission from September 12th to 20th in 1992. The crystallization was carried out according to a batch method, and 5 proteins were selected as flight samples for crystallization. Two of these proteins: hen egg-white lysozyme and co-amino acid: pyruvate aminotransferase from Pseudomonas sp. F-126, were obtained as single crystals of good diffraction quality. Since 1992 we have carried out several space experiments for protein crystallization aboard space shuttles and the space station MIR. Our experimental results obtained mainly from hen egg-white lysozyme are described below, focusing on the effects of microgravity on protein crystal growth.

  5. Protein Unfolding and Alzheimer's

    NASA Astrophysics Data System (ADS)

    Cheng, Kelvin

    2012-10-01

    Early interaction events of beta-amyloid (Aβ) proteins with neurons have been associated with the pathogenesis of Alzheimer's disease. Knowledge pertaining to the role of lipid molecules, particularly cholesterol, in modulating the single Aβ interactions with neurons at the atomic length and picosecond time resolutions, remains unclear. In our research, we have used atomistic molecular dynamics simulations to explore early molecular events including protein insertion kinetics, protein unfolding, and protein-induced membrane disruption of Aβ in lipid domains that mimic the nanoscopic raft and non-raft regions of the neural membrane. In this talk, I will summarize our current work on investigating the role of cholesterol in regulating the Aβ interaction events with membranes at the molecular level. I will also explain how our results will provide new insights into understanding the pathogenesis of Alzheimer's disease associated with the Aβ proteins.

  6. Junin virus structural proteins.

    PubMed Central

    De Martínez Segovia, Z M; De Mitri, M I

    1977-01-01

    Polyacrylamide gel electrophoresis of purified Junin virus revealed six distinct structural polypeptides, two major and four minor ones. Four of these polypeptides appeared to be covalently linked with carbohydrate. The molecular weights of the six proteins, estimated by coelectrophoresis with marker proteins, ranged from 25,000 to 91,000. One of the two major components (number 3) was identified as a nucleoprotein and had a molecular weight of 64,000. It was the most prominent protein and was nonglycosylated. The other major protein (number 5), with a molecular weight of 38,000, was a glucoprotein and a component of the viral envelope. The location on the virion of three additional glycopeptides with molecular weights of 91,000, 72,000, and 52,000, together with a protein with a molecular weight of 25,000, was not well defined. PMID:189088

  7. Sac phosphatase domain proteins.

    PubMed Central

    Hughes, W E; Cooke, F T; Parker, P J

    2000-01-01

    Advances in our understanding of the roles of phosphatidylinositol phosphates in controlling cellular functions such as endocytosis, exocytosis and the actin cytoskeleton have included new insights into the phosphatases that are responsible for the interconversion of these lipids. One of these is an entirely novel class of phosphatase domain found in a number of well characterized proteins. Proteins containing this Sac phosphatase domain include the yeast Saccharomyces cerevisiae proteins Sac1p and Fig4p. The Sac phosphatase domain is also found within the mammalian phosphoinositide 5-phosphatase synaptojanin and the yeast synaptojanin homologues Inp51p, Inp52p and Inp53p. These proteins therefore contain both Sac phosphatase and 5-phosphatase domains. This review describes the Sac phosphatase domain-containing proteins and their actions, with particular reference to the genetic and biochemical insights provided by study of the yeast Saccharomyces cerevisiae. PMID:10947947

  8. Transdermal delivery of proteins.

    PubMed

    Kalluri, Haripriya; Banga, Ajay K

    2011-03-01

    Transdermal delivery of peptides and proteins avoids the disadvantages associated with the invasive parenteral route of administration and other alternative routes such as the pulmonary and nasal routes. Since proteins have a large size and are hydrophilic in nature, they cannot permeate passively across the skin due to the stratum corneum which allows the transport of only small lipophilic drug molecules. Enhancement techniques such as chemical enhancers, iontophoresis, microneedles, electroporation, sonophoresis, thermal ablation, laser ablation, radiofrequency ablation and noninvasive jet injectors aid in the delivery of proteins by overcoming the skin barrier in different ways. In this review, these enhancement techniques that can enable the transdermal delivery of proteins are discussed, including a discussion of mechanisms, sterility requirements, and commercial development of products. Combination of enhancement techniques may result in a synergistic effect allowing increased protein delivery and these are also discussed.

  9. Manipulating and Visualizing Proteins

    SciTech Connect

    Simon, Horst D.

    2003-12-05

    ProteinShop Gives Researchers a Hands-On Tool for Manipulating, Visualizing Protein Structures. The Human Genome Project and other biological research efforts are creating an avalanche of new data about the chemical makeup and genetic codes of living organisms. But in order to make sense of this raw data, researchers need software tools which let them explore and model data in a more intuitive fashion. With this in mind, researchers at Lawrence Berkeley National Laboratory and the University of California, Davis, have developed ProteinShop, a visualization and modeling program which allows researchers to manipulate protein structures with pinpoint control, guided in large part by their own biological and experimental instincts. Biologists have spent the last half century trying to unravel the ''protein folding problem,'' which refers to the way chains of amino acids physically fold themselves into three-dimensional proteins. This final shape, which resembles a crumpled ribbon or piece of origami, is what determines how the protein functions and translates genetic information. Understanding and modeling this geometrically complex formation is no easy matter. ProteinShop takes a given sequence of amino acids and uses visualization guides to help generate predictions about the secondary structures, identifying alpha helices and flat beta strands, and the coil regions that bind them. Once secondary structures are in place, researchers can twist and turn these pre-configurations until they come up with a number of possible tertiary structure conformations. In turn, these are fed into a computationally intensive optimization procedure that tries to find the final, three-dimensional protein structure. Most importantly, ProteinShop allows users to add human knowledge and intuition to the protein structure prediction process, thus bypassing bad configurations that would otherwise be fruitless for optimization. This saves compute cycles and accelerates the entire process, so

  10. Proteins in unexpected locations.

    PubMed Central

    Smalheiser, N R

    1996-01-01

    Members of all classes of proteins--cytoskeletal components, secreted growth factors, glycolytic enzymes, kinases, transcription factors, chaperones, transmembrane proteins, and extracellular matrix proteins--have been identified in cellular compartments other than their conventional sites of action. Some of these proteins are expressed as distinct compartment-specific isoforms, have novel mechanisms for intercompartmental translocation, have distinct endogenous biological actions within each compartment, and are regulated in a compartment-specific manner as a function of physiologic state. The possibility that many, if not most, proteins have distinct roles in more than one cellular compartment has implications for the evolution of cell organization and may be important for understanding pathological conditions such as Alzheimer's disease and cancer. PMID:8862516

  11. Drugging Membrane Protein Interactions

    PubMed Central

    Yin, Hang; Flynn, Aaron D.

    2016-01-01

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind the cell to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally “undruggable” regions of membrane proteins, enabling modulation of protein–protein, protein–lipid, and protein–nucleic acid interactions. In this review, we survey the state of the art in high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  12. Proteins, fluctuations and complexity

    SciTech Connect

    Frauenfelder, Hans; Chen, Guo; Fenimore, Paul W

    2008-01-01

    Glasses, supercooled liquids, and proteins share common properties, in particular the existence of two different types of fluctuations, {alpha} and {beta}. While the effect of the {alpha} fluctuations on proteins has been known for a few years, the effect of {beta} fluctuations has not been understood. By comparing neutron scattering data on the protein myoglobin with the {beta} fluctuations in the hydration shell measured by dielectric spectroscopy we show that the internal protein motions are slaved to these fluctuations. We also show that there is no 'dynamic transition' in proteins near 200 K. The rapid increase in the mean square displacement with temperature in many neutron scattering experiments is quantitatively predicted by the {beta} fluctuations in the hydration shell.

  13. [Controversies around diet proteins].

    PubMed

    Cichosz, Grazyna; Czeczot, Hanna

    2013-12-01

    Critical theories regarding proteins of anima origin are still and still popularized, though they are ungrounded from scientific point of view. Predominance of soya proteins over the animal ones in relation to their influence on calcium metabolism, bone break risk or risk of osteoporosis morbidity has not been confirmed in any honest, reliable research experiment. Statement, that sulphur amino acids influence disadvantageously on calcium metabolism of human organism and bone status, is completely groundless, the more so as presence of sulphur amino acids in diet (animal proteins are their best source) is the condition of endogenic synthesis of glutathione, the key antioxidant of the organism, and taurine stimulating brain functioning. Deficiency of proteins in the diet produce weakness of intellectual effectiveness and immune response. There is no doubt that limitation of consumption of animal proteins of standard value is not good for health.

  14. Protein crystal growth

    NASA Technical Reports Server (NTRS)

    Bugg, Charles E.

    1993-01-01

    Proteins account for 50% or more of the dry weight of most living systems and play a crucial role in virtually all biological processes. Since the specific functions of essentially all biological molecules are determined by their three-dimensional structures, it is obvious that a detailed understanding of the structural makeup of a protein is essential to any systematic research pertaining to it. At the present time, protein crystallography has no substitute, it is the only technique available for elucidating the atomic arrangements within complicated biological molecules. Most macromolecules are extremely difficult to crystallize, and many otherwise exciting and promising projects have terminated at the crystal growth stage. There is a pressing need to better understand protein crystal growth, and to develop new techniques that can be used to enhance the size and quality of protein crystals. There are several aspects of microgravity that might be exploited to enhance protein crystal growth. The major factor that might be expected to alter crystal growth processes in space is the elimination of density-driven convective flow. Another factor that can be readily controlled in the absence of gravity is the sedimentation of growing crystal in a gravitational field. Another potential advantage of microgravity for protein crystal growth is the option of doing containerless crystal growth. One can readily understand why the microgravity environment established by Earth-orbiting vehicles is perceived to offer unique opportunities for the protein crystallographer. The near term objectives of the Protein Crystal Growth in a Microgravity Environment (PCG/ME) project is to continue to improve the techniques, procedures, and hardware systems used to grow protein crystals in Earth orbit.

  15. Non-equilibrium proteins.

    PubMed

    Klonowski, W

    2001-07-01

    There exist no methodical studies concerning non-equilibrium systems in cellular biology. This paper is an attempt to partially fill this shortcoming. We have undertaken an extensive data-mining operation in the existing scientific literature to find scattered information about non-equilibrium subcellular systems, in particular concerning fast proteins, i.e. those with short turnover half-time. We have advanced the hypothesis that functionality in fast proteins emerges as a consequence of their intrinsic physical instability that arises due to conformational strains resulting from co-translational folding (the interdependence between chain elongation and chain folding during biosynthesis on ribosomes). Such intrinsic physical instability, a kind of conformon (Klonowski-Klonowska conformon, according to Ji, (Molecular Theories of Cell Life and Death, Rutgers University Press, New Brunswick, 1991)) is probably the most important feature determining functionality and timing in these proteins. If our hypothesis is true, the turnover half-time of fast proteins should be positively correlated with their molecular weight, and some experimental results (Ames et al., J. Neurochem. 35 (1980) 131) indeed demonstrated such a correlation. Once the native structure (and function) of a fast protein macromolecule is lost, it may not be recovered--denaturation of such proteins will always be irreversible; therefore, we searched for information on irreversible denaturation. Only simulation and modeling of protein co-translational folding may answer the questions concerning fast proteins (Ruggiero and Sacile, Med. Biol. Eng. Comp. 37 (Suppl. 1) (1999) 363). Non-equilibrium structures may also be built up of protein subunits, even if each one taken by itself is in thermodynamic equilibrium (oligomeric proteins; sub-cellular sol-gel dissipative network structures).

  16. Purifying protein complexes for mass spectrometry: applications to protein translation.

    PubMed

    Link, Andrew J; Fleischer, Tracey C; Weaver, Connie M; Gerbasi, Vincent R; Jennings, Jennifer L

    2005-03-01

    Proteins control and mediate most of the biological activities in the cell. In most cases, proteins either interact with regulatory proteins or function in large molecular assemblies to carryout biological processes. Understanding the functions of individual proteins requires the identification of these interacting proteins. With its speed and sensitivity, mass spectrometry has become the dominant method for identifying components of protein complexes. This article reviews and discusses various approaches to purify protein complexes and analyze the proteins using mass spectrometry. As examples, methods to isolate and analyze protein complexes responsible for the translation of messenger RNAs into polypeptides are described.

  17. New Compound Classes: Protein-Protein Interactions.

    PubMed

    Ottmann, C

    2016-01-01

    "Protein-protein interactions (PPIs) are one of the most promising new targets in drug discovery. With estimates between 300,000 and 650,000 in human physiology, targeted modulation of PPIs would tremendously extend the "druggable" genome. In fact, in every disease a wealth of potentially addressable PPIs can be found making pharmacological intervention based on PPI modulators in principle a generally applicable technology. An impressing number of success stories in small-molecule PPI inhibition and natural-product PPI stabilization increasingly encourage academia and industry to invest in PPI modulation. In this chapter examples of both inhibition as well as stabilization of PPIs are reviewed including some of the technologies which has been used for their identification."

  18. Tear fluid protein biomarkers.

    PubMed

    You, Jingjing; Willcox, Mark D; Madigan, Michele C; Wasinger, Valerie; Schiller, Belinda; Walsh, Bradley J; Graham, Peter H; Kearsley, John H; Li, Yong

    2013-01-01

    The tear film covers and protects the ocular surface. It contains various molecules including a large variety of proteins. The protein composition of the tear fluid can change with respect to various local and systemic diseases. Prior to the advent of the proteomic era, tear protein analysis was limited to a few analytical techniques, the most common of which was immunoelectrophoresis, an approach dependent on antibody availability. Using proteomics, hundreds of tear proteins could potentially be identified and subsequently studied. Although detection of low-abundance proteins in the complex tear proteome remains a challenge, advances in sample fractionation and mass spectrometry have greatly enhanced our ability to detect these proteins. With increasing proteomic applications, tears show great potential as biomarkers in the development of clinical assays for various human diseases. In this chapter, we discuss the structure and functions of the tear film and methods for its collection. We also summarize potential tear protein biomarkers identified using proteomic techniques for both ocular and systemic diseases. Finally, modern proteomic techniques for tear biomarker research and future challenges are explored.

  19. Endometrial proteins: a reappraisal.

    PubMed

    Seppälä, M; Julkunen, M; Riittinen, L; Koistinen, R

    1992-06-01

    Uterine factors influence reproduction at the macro-anatomy level, and the effects of hormonal steroids on endometrial morphology are well recognized in the histopathological diagnosis of dysfunctional bleeding and infertility. During the past decade, attention has been paid to endometrial protein synthesis and secretion with respect to endocrine stimuli and implantation, and to the paracrine/autocrine effects of endometrial peptide growth factors, their binding proteins and other factors. The emphasis of this presentation is on protein secretion of the secretory endometrium, in which progesterone plays a pivotal role. Insulin-like growth factors have receptors on the endometrium, and IGF-binding proteins, stimulated by progesterone, modulate the effects of IGFs locally. Also other protein products of the secretory endometrium have been reviewed in this communication, with special emphasis on studies of a progesterone-associated endometrial protein which has many names in the literature, such as PEP, PP14, alpha 2-PEG and AUP. Extensive studies are ongoing in many laboratories to elucidate the regulation, function, interplay at tissue and cellular levels, and clinical significance of these proteins.

  20. Multidomain proteins under force

    NASA Astrophysics Data System (ADS)

    Valle-Orero, Jessica; Andrés Rivas-Pardo, Jaime; Popa, Ionel

    2017-04-01

    Advancements in single-molecule force spectroscopy techniques such as atomic force microscopy and magnetic tweezers allow investigation of how domain folding under force can play a physiological role. Combining these techniques with protein engineering and HaloTag covalent attachment, we investigate similarities and differences between four model proteins: I10 and I91—two immunoglobulin-like domains from the muscle protein titin, and two α + β fold proteins—ubiquitin and protein L. These proteins show a different mechanical response and have unique extensions under force. Remarkably, when normalized to their contour length, the size of the unfolding and refolding steps as a function of force reduces to a single master curve. This curve can be described using standard models of polymer elasticity, explaining the entropic nature of the measured steps. We further validate our measurements with a simple energy landscape model, which combines protein folding with polymer physics and accounts for the complex nature of tandem domains under force. This model can become a useful tool to help in deciphering the complexity of multidomain proteins operating under force.

  1. TRIM proteins in cancer.

    PubMed

    Cambiaghi, Valeria; Giuliani, Virginia; Lombardi, Sara; Marinelli, Cristiano; Toffalorio, Francesca; Pelicci, Pier Giuseppe

    2012-01-01

    Some members of the tripartite motif (TRIM/RBCC) protein family are thought to be important regulators of carcinogenesis. This is not surprising as the TRIM proteins are involved in several biological processes, such as cell growth, development and cellular differentiation and alteration of these proteins can affect transcriptional regulation, cell proliferation and apoptosis. In particular, four TRIM family genes are frequently translocated to other genes, generating fusion proteins implicated in cancer initiation and progression. Among these the most famous is the promyelocytic leukaemia gene PML, which encodes the protein TRIM19. PML is involved in the t(15;17) translocation that specifically occurs in Acute Promyelocytic Leukaemia (APL), resulting in a PML-retinoic acid receptor-alpha (PML-RARalpha) fusion protein. Other members of the TRIM family are linked to cancer development without being involved in chromosomal re-arrangements, possibly through ubiquitination or loss of tumour suppression functions. This chapter discusses the biological functions of TRIM proteins in cancer.

  2. Direct polymerization of proteins.

    PubMed

    Albayrak, Cem; Swartz, James R

    2014-06-20

    We report the synthesis of active polymers of superfolder green fluorescent protein (sfGFP) in one step using Click chemistry. Up to six copies of the non-natural amino acids (nnAAs) p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) were site-specifically inserted into sfGFP by cell-free protein synthesis (CFPS). sfGFP containing two or three copies of these nnAAs were coupled by copper-catalyzed azide-alkyne cycloaddition to synthesize linear or branched protein polymers, respectively. The protein polymers retained ≥63% of their specific activity (i.e., fluorescence) after coupling. Polymerization of a concentrated solution of triply substituted sfGFP resulted in fluorescent macromolecular particles. Our method can be generalized to synthesize polymers of a protein or copolymers of any two or more proteins, and the conjugation sites can be determined exactly by standard genetic manipulation. Polymers of proteins and small molecules can also be created with this technology to make a new class of scaffolds or biomaterials.

  3. NMCP/LINC proteins

    PubMed Central

    Ciska, Malgorzata; Moreno Díaz de la Espina, Susana

    2013-01-01

    Lamins are the main components of the metazoan lamina, and while the organization of the nuclear lamina of metazoans and plants is similar, there are apparently no genes encoding lamins or most lamin-binding proteins in plants. Thus, the plant lamina is not lamin-based and the proteins that form this structure are still to be characterized. Members of the plant NMCP/LINC/CRWN protein family share the typical tripartite structure of lamins, although the 2 exhibit no sequence similarity. However, given the many similarities between NMCP/LINC/CRWN proteins and lamins (structural organization, position of conserved regions, sub-nuclear distribution, solubility, and pattern of expression), these proteins are good candidates to carry out the functions of lamins in plants. Moreover, functional analysis of NMCP/LINC mutants has revealed their involvement in maintaining nuclear size and shape, another activity fulfilled by lamins. This review summarizes the current understanding of NMCP/LINC proteins and discusses future studies that will be required to demonstrate definitively that these proteins are plant analogs of lamins. PMID:24128696

  4. Fluctuations in Proteins

    NASA Astrophysics Data System (ADS)

    Frauenfelder, Hans

    2007-03-01

    Proteins are the machines of life. In order to perform their functions, they must move continuously. The motions correspond to equilibrium fluctuations and to non-equilibrium relaxations. At least three different fluctuation processes occur: α- and β-fluctuations and processes that occur even below one Kelvin. The α-fluctuations can be approximated by the Vogel-Tammann-Fulcher relation, while the β-fluctuations appear to follow a conventional Arrhenius law (but may in some cases be better characterized by a Ferry law). Both are usually nonexponential in time. These phenomena are similar in proteins and glasses, but there is a fundamental difference between fluctuations in glasses and proteins: In glasses, they are independent of the environment, in proteins the α-fluctuations are slaved to the α-fluctuations in the solvent surrounding the protein; they follow their rate coefficients but they are entropically slowed. The studies of the protein motions are actually still in their infancy, but we can expect that future work will not only help understanding protein functions, but will also feed back to the physics of glasses.

  5. Prediction of protein-protein interactions: unifying evolution and structure at protein interfaces.

    PubMed

    Tuncbag, Nurcan; Gursoy, Attila; Keskin, Ozlem

    2011-06-01

    The vast majority of the chores in the living cell involve protein-protein interactions. Providing details of protein interactions at the residue level and incorporating them into protein interaction networks are crucial toward the elucidation of a dynamic picture of cells. Despite the rapid increase in the number of structurally known protein complexes, we are still far away from a complete network. Given experimental limitations, computational modeling of protein interactions is a prerequisite to proceed on the way to complete structural networks. In this work, we focus on the question 'how do proteins interact?' rather than 'which proteins interact?' and we review structure-based protein-protein interaction prediction approaches. As a sample approach for modeling protein interactions, PRISM is detailed which combines structural similarity and evolutionary conservation in protein interfaces to infer structures of complexes in the protein interaction network. This will ultimately help us to understand the role of protein interfaces in predicting bound conformations.

  6. Prediction of protein-protein interactions: unifying evolution and structure at protein interfaces

    NASA Astrophysics Data System (ADS)

    Tuncbag, Nurcan; Gursoy, Attila; Keskin, Ozlem

    2011-06-01

    The vast majority of the chores in the living cell involve protein-protein interactions. Providing details of protein interactions at the residue level and incorporating them into protein interaction networks are crucial toward the elucidation of a dynamic picture of cells. Despite the rapid increase in the number of structurally known protein complexes, we are still far away from a complete network. Given experimental limitations, computational modeling of protein interactions is a prerequisite to proceed on the way to complete structural networks. In this work, we focus on the question 'how do proteins interact?' rather than 'which proteins interact?' and we review structure-based protein-protein interaction prediction approaches. As a sample approach for modeling protein interactions, PRISM is detailed which combines structural similarity and evolutionary conservation in protein interfaces to infer structures of complexes in the protein interaction network. This will ultimately help us to understand the role of protein interfaces in predicting bound conformations.

  7. Bacterial Ice Crystal Controlling Proteins

    PubMed Central

    Lorv, Janet S. H.; Rose, David R.; Glick, Bernard R.

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  8. Bacterial ice crystal controlling proteins.

    PubMed

    Lorv, Janet S H; Rose, David R; Glick, Bernard R

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions.

  9. Proteins : paradigms of complexity /

    SciTech Connect

    Frauenfelder, Hans,

    2001-01-01

    Proteins are the working machines of living systems. Directed by the DNA, of the order of a few hundred building blocks, selected from twenty different amino acids, are covalently linked into a linear polypeptide chain. In the proper environment, the chain folds into the working protein, often a globule of linear dimensions of a few nanometers. The biologist considers proteins units from which living systems are built. Many physical scientists look at them as systems in which the laws of complexity can be studied better than anywhere else. Some of the results of such studies will be sketched.

  10. Emerging fluorescent protein technologies.

    PubMed

    Enterina, Jhon Ralph; Wu, Lanshi; Campbell, Robert E

    2015-08-01

    Fluorescent proteins (FPs), such as the Aequorea jellyfish green FP (GFP), are firmly established as fundamental tools that enable a wide variety of biological studies. Specifically, FPs can serve as versatile genetically encoded markers for tracking proteins, organelles, or whole cells, and as the basis for construction of biosensors that can be used to visualize a growing array of biochemical events in cells and tissues. In this review we will focus on emerging applications of FPs that represent unprecedented new directions for the field. These emerging applications include new strategies for using FPs in biosensing applications, and innovative ways of using FPs to manipulate protein function or gene expression.

  11. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell (standing), Post-Doctoral Fellow the National Research Council (NRC),and Marc Pusey of Marshall Space Flight Center (MSFC) use a reciprocal space mapping diffractometer for marcromolecular crystal quality studies. The diffractometer is used in mapping the structure of marcromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystalized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  12. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell (standing), Post-Doctoral Fellow the National Research Council (NRC),and Marc Pusey of Marshall Space Flight Center (MSFC) use a reciprocal space mapping diffractometer for marcromolecular crystal quality studies. The diffractometer is used in mapping the structure of marcromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystalized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  13. Piezoelectric allostery of protein

    NASA Astrophysics Data System (ADS)

    Ohnuki, Jun; Sato, Takato; Takano, Mitsunori

    2016-07-01

    Allostery is indispensable for a protein to work, where a locally applied stimulus is transmitted to a distant part of the molecule. While the allostery due to chemical stimuli such as ligand binding has long been studied, the growing interest in mechanobiology prompts the study of the mechanically stimulated allostery, the physical mechanism of which has not been established. By molecular dynamics simulation of a motor protein myosin, we found that a locally applied mechanical stimulus induces electrostatic potential change at distant regions, just like the piezoelectricity. This novel allosteric mechanism, "piezoelectric allostery", should be of particularly high value for mechanosensor/transducer proteins.

  14. Protein Crystal Malic Enzyme

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  15. Protein Crystal Malic Enzyme

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  16. Protein folding and misfolding

    NASA Astrophysics Data System (ADS)

    Dobson, Christopher M.

    2003-12-01

    The manner in which a newly synthesized chain of amino acids transforms itself into a perfectly folded protein depends both on the intrinsic properties of the amino-acid sequence and on multiple contributing influences from the crowded cellular milieu. Folding and unfolding are crucial ways of regulating biological activity and targeting proteins to different cellular locations. Aggregation of misfolded proteins that escape the cellular quality-control mechanisms is a common feature of a wide range of highly debilitating and increasingly prevalent diseases.

  17. Protein based Block Copolymers

    PubMed Central

    Rabotyagova, Olena S.; Cebe, Peggy; Kaplan, David L.

    2011-01-01

    Advances in genetic engineering have led to the synthesis of protein-based block copolymers with control of chemistry and molecular weight, resulting in unique physical and biological properties. The benefits from incorporating peptide blocks into copolymer designs arise from the fundamental properties of proteins to adopt ordered conformations and to undergo self-assembly, providing control over structure formation at various length scales when compared to conventional block copolymers. This review covers the synthesis, structure, assembly, properties, and applications of protein-based block copolymers. PMID:21235251

  18. [Phosphorylation of tau protein].

    PubMed

    Uchida, T; Ishiguro, K

    1990-05-01

    In aged human brain and particularly in Alzheimer's disease brain, paired helical filaments (PHFs) accumulate in the neuronal cell. Recently, it has been found that the highly phosphorylated tau protein, one of the microtubule-associated proteins (MAPs), is a component of PHF. The authors attempted to clarify the mechanism underlying the accumulation of PHF from the following two aspects; 1) What is the mechanism of phosphorylation of tau protein? 2) Is the highly phosphorylated tau protein capable of forming PHFs? From rat or bovine microtubule proteins we partially purified and characterized a novel protein kinase that specifically phosphorylated tau and MAP2 among many proteins in the brain extract, and which formed a PHF epitope on the phosphorylated human tau. This enzyme was one of the protein serine/threonine kinases and was independent of known second messengers. The phosphorylation of tau by this enzyme was stimulated by tubulin under the condition of microtubule formation, suggesting that the phosphorylation of tau could occur concomitantly with microtubule formation in the brain. Since this kinase was usually bound to tau but not directly to tubulin, the enzyme was associated with microtubules through tau. From these properties related to tau, this kinase is designated as tau protein kinase. The tau that been phosphorylated with this kinase using [gamma-32P]ATP as a phosphate donor, was digested by endoprotinase Lys-C to produce three labeled fragments, K1, K2 and K3. These three fragments were sequenced and the phosphorylation sites on tau by this kinase were identified. The K2 fragment overlapped with the tau-1 site known to be one of the phosphorylation site in PHF. This result strengthens the possibility that tau protein phosphorylated by tau protein kinase is incorporated into PHF. Tubulin binding sites on tau were located between K1 and K3 fragments, while K2 fragment was located in the neighboring to N-terminus of K1. No phosphorylated sites were

  19. Teaching resources. Protein phosphatases.

    PubMed

    Salton, Stephen R

    2005-03-01

    This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein phosphatases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the importance of phosphatases in physiology, recognized by the award of a Nobel Prize in 1992, and then proceeds to describe the two types of protein phosphatases: serine/threonine and tyrosine phosphatases. The information covered includes the structure, regulation, and substrate specificity of protein phosphatases, with an emphasis on their importance in disease and clinical settings.

  20. Evolution of proteins.

    NASA Technical Reports Server (NTRS)

    Dayhoff, M. O.

    1971-01-01

    The amino acid sequences of proteins from living organisms are dealt with. The structure of proteins is first discussed; the variation in this structure from one biological group to another is illustrated by the first halves of the sequences of cytochrome c, and a phylogenetic tree is derived from the cytochrome c data. The relative geological times associated with the events of this tree are discussed. Errors which occur in the duplication of cells during the evolutionary process are examined. Particular attention is given to evolution of mutant proteins, globins, ferredoxin, and transfer ribonucleic acids (tRNA's). Finally, a general outline of biological evolution is presented.

  1. Protein crystallography prescreen kit

    DOEpatents

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2007-10-02

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  2. Protein crystallography prescreen kit

    DOEpatents

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2005-07-12

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  3. Electrochromatographic separation of proteins

    NASA Technical Reports Server (NTRS)

    Basak, S. K.; Velayudhan, A.; Kohlmann, K.; Ladisch, M. R.; Mitchell, C. A. (Principal Investigator)

    1995-01-01

    We have developed a modified electrochromatography system which minimizes Joule heating at electric field strengths up to 125 V/cm. A non-linear equilibrium model is described which incorporates electrophoretic mobility, hydrodynamic flow velocity, and an electrically induced concentration polarization at the surface of the stationary phase. This model is able to provide useful estimates of protein retention time and velocity in a column packed with Sephadex gel and subjected to an electric field. A correlation of electrophoretic mobility of peptide and proteins with respect to their charge, molecular mass, and asymmetry enables the selection of solute target molecules for electrochromatographic separations. Good separation of protein mixtures have been obtained.

  4. (PCG) Protein Crystal Growth Canavalin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Canavalin. The major storage protein of leguminous plants and a major source of dietary protein for humans and domestic animals. It is studied in efforts to enhance nutritional value of proteins through protein engineerings. It is isolated from Jack Bean because of it's potential as a nutritional substance. Principal Investigator on STS-26 was Alex McPherson.

  5. Protein in diet

    MedlinePlus

    ... MyPlate , can help you make healthy eating choices. Alternative Names ... of Medicine, Food and Nutrition Board. Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein, and ...

  6. Protein Colloidal Aggregation Project

    NASA Technical Reports Server (NTRS)

    Oliva-Buisson, Yvette J. (Compiler)

    2014-01-01

    To investigate the pathways and kinetics of protein aggregation to allow accurate predictive modeling of the process and evaluation of potential inhibitors to prevalent diseases including cataract formation, chronic traumatic encephalopathy, Alzheimer's Disease, Parkinson's Disease and others.

  7. Protein dimerization. Inside job.

    PubMed

    Metzger, H

    1994-04-01

    In a sophisticated combination of genetic engineering and organic synthesis, a general method for dimerizing recombinant intracellular proteins has been devised; the usefulness of the method should now be testable.

  8. Interactive protein manipulation

    SciTech Connect

    SNCrivelli@lbl.gov

    2003-07-01

    We describe an interactive visualization and modeling program for the creation of protein structures ''from scratch''. The input to our program is an amino acid sequence -decoded from a gene- and a sequence of predicted secondary structure types for each amino acid-provided by external structure prediction programs. Our program can be used in the set-up phase of a protein structure prediction process; the structures created with it serve as input for a subsequent global internal energy minimization, or another method of protein structure prediction. Our program supports basic visualization methods for protein structures, interactive manipulation based on inverse kinematics, and visualization guides to aid a user in creating ''good'' initial structures.

  9. Protein Model Database

    SciTech Connect

    Fidelis, K; Adzhubej, A; Kryshtafovych, A; Daniluk, P

    2005-02-23

    The phenomenal success of the genome sequencing projects reveals the power of completeness in revolutionizing biological science. Currently it is possible to sequence entire organisms at a time, allowing for a systemic rather than fractional view of their organization and the various genome-encoded functions. There is an international plan to move towards a similar goal in the area of protein structure. This will not be achieved by experiment alone, but rather by a combination of efforts in crystallography, NMR spectroscopy, and computational modeling. Only a small fraction of structures are expected to be identified experimentally, the remainder to be modeled. Presently there is no organized infrastructure to critically evaluate and present these data to the biological community. The goal of the Protein Model Database project is to create such infrastructure, including (1) public database of theoretically derived protein structures; (2) reliable annotation of protein model quality, (3) novel structure analysis tools, and (4) access to the highest quality modeling techniques available.

  10. Constructing arrays of proteins.

    PubMed

    Sinclair, John C

    2013-12-01

    The construction of crystalline arrays allows proteins to be presented in a dense, oriented and functional way that also facilitates determination of their structure. Rational design of these supramolecular structures is becoming increasingly tractable with recent successes exploiting both innate protein symmetry and advances in protein–protein interface design. Pre-existing symmetry minimizes the number of non-native interfaces that must be produced, and the use of symmetric interfaces facilitates protein alignment. Arrays in which metal coordination or peptide binding are responsible for the inter-particle associations show particular promise due to the malleable and reversible nature of these interactions. Cross-pollination of the principles that underlie successful strategies is likely to produce rapid advances in this field and consequent benefits to both nanotechnology and structural biology.

  11. Protein fabrication automation

    PubMed Central

    Cox, J. Colin; Lape, Janel; Sayed, Mahmood A.; Hellinga, Homme W.

    2007-01-01

    Facile “writing” of DNA fragments that encode entire gene sequences potentially has widespread applications in biological analysis and engineering. Rapid writing of open reading frames (ORFs) for expressed proteins could transform protein engineering and production for protein design, synthetic biology, and structural analysis. Here we present a process, protein fabrication automation (PFA), which facilitates the rapid de novo construction of any desired ORF from oligonucleotides with low effort, high speed, and little human interaction. PFA comprises software for sequence design, data management, and the generation of instruction sets for liquid-handling robotics, a liquid-handling robot, a robust PCR scheme for gene assembly from synthetic oligonucleotides, and a genetic selection system to enrich correctly assembled full-length synthetic ORFs. The process is robust and scalable. PMID:17242375

  12. Fully automated protein purification

    PubMed Central

    Camper, DeMarco V.; Viola, Ronald E.

    2009-01-01

    Obtaining highly purified proteins is essential to begin investigating their functional and structural properties. The steps that are typically involved in purifying proteins can include an initial capture, intermediate purification, and a final polishing step. Completing these steps can take several days and require frequent attention to ensure success. Our goal was to design automated protocols that will allow the purification of proteins with minimal operator intervention. Separate methods have been produced and tested that automate the sample loading, column washing, sample elution and peak collection steps for ion-exchange, metal affinity, hydrophobic interaction and gel filtration chromatography. These individual methods are designed to be coupled and run sequentially in any order to achieve a flexible and fully automated protein purification protocol. PMID:19595984

  13. Microgravity protein crystallization

    PubMed Central

    McPherson, Alexander; DeLucas, Lawrence James

    2015-01-01

    Over the past 20 years a variety of technological advances in X-ray crystallography have shortened the time required to determine the structures of large macromolecules (i.e., proteins and nucleic acids) from several years to several weeks or days. However, one of the remaining challenges is the ability to produce diffraction-quality crystals suitable for a detailed structural analysis. Although the development of automated crystallization systems combined with protein engineering (site-directed mutagenesis to enhance protein solubility and crystallization) have improved crystallization success rates, there remain hundreds of proteins that either cannot be crystallized or yield crystals of insufficient quality to support X-ray structure determination. In an attempt to address this bottleneck, an international group of scientists has explored use of a microgravity environment to crystallize macromolecules. This paper summarizes the history of this international initiative along with a description of some of the flight hardware systems and crystallization results. PMID:28725714

  14. Protein Nitrogen Determination

    NASA Astrophysics Data System (ADS)

    Nielsen, S. Suzanne

    The protein content of foods can be determined by numerous methods. The Kjeldahl method and the nitrogen combustion (Dumas) method for protein analysis are based on nitrogen determination. Both methods are official for the purposes of nutrition labeling of foods. While the Kjeldahl method has been used widely for over a hundred years, the recent availability of automated instrumentation for the Dumas method in many cases is replacing use of the Kjeldahl method.

  15. Plant protein extraction.

    PubMed

    Conlon, Helen E; Salter, Michael G

    2007-01-01

    A method is presented for the extraction of total protein from Arabidopsis thaliana tissue. The protocol was designed for the solubilization of a range of proteins and their efficient and quantitative recovery. It is especially compatible with the small quantities of available tissue often associated with this species and was originally intended for Western blot preparations. Samples extracted using this method can be quantitated directly using a commercially available kit.

  16. Colorimetric protein assay techniques.

    PubMed

    Sapan, C V; Lundblad, R L; Price, N C

    1999-04-01

    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  17. Chirality and protein biosynthesis.

    PubMed

    Banik, Sindrila Dutta; Nandi, Nilashis

    2013-01-01

    Chirality is present at all levels of structural hierarchy of protein and plays a significant role in protein biosynthesis. The macromolecules involved in protein biosynthesis such as aminoacyl tRNA synthetase and ribosome have chiral subunits. Despite the omnipresence of chirality in the biosynthetic pathway, its origin, role in current pathway, and importance is far from understood. In this review we first present an introduction to biochirality and its relevance to protein biosynthesis. Major propositions about the prebiotic origin of biomolecules are presented with particular reference to proteins and nucleic acids. The problem of the origin of homochirality is unresolved at present. The chiral discrimination by enzymes involved in protein synthesis is essential for keeping the life process going. However, questions remained pertaining to the mechanism of chiral discrimination and concomitant retention of biochirality. We discuss the experimental evidence which shows that it is virtually impossible to incorporate D-amino acids in protein structures in present biosynthetic pathways via any of the two major steps of protein synthesis, namely aminoacylation and peptide bond formation reactions. Molecular level explanations of the stringent chiral specificity in each step are extended based on computational analysis. A detailed account of the current state of understanding of the mechanism of chiral discrimination during aminoacylation in the active site of aminoacyl tRNA synthetase and peptide bond formation in ribosomal peptidyl transferase center is presented. Finally, it is pointed out that the understanding of the mechanism of retention of enantiopurity has implications in developing novel enzyme mimetic systems and biocatalysts and might be useful in chiral drug design.

  18. Protein tyrosine nitration

    PubMed Central

    Chaki, Mounira; Leterrier, Marina; Barroso, Juan B

    2009-01-01

    Nitric oxide metabolism in plant cells has a relative short history. Nitration is a chemical process which consists of introducing a nitro group (-NO2) into a chemical compound. in biological systems, this process has been found in different molecules such as proteins, lipids and nucleic acids that can affect its function. This mini-review offers an overview of this process with special emphasis on protein tyrosine nitration in plants and its involvement in the process of nitrosative stress. PMID:19826215

  19. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  20. Protein conducting nanopores

    NASA Astrophysics Data System (ADS)

    Harsman, Anke; Krüger, Vivien; Bartsch, Philipp; Honigmann, Alf; Schmidt, Oliver; Rao, Sanjana; Meisinger, Christof; Wagner, Richard

    2010-11-01

    About 50% of the cellular proteins have to be transported into or across cellular membranes. This transport is an essential step in the protein biosynthesis. In eukaryotic cells secretory proteins are transported into the endoplasmic reticulum before they are transported in vesicles to the plasma membrane. Almost all proteins of the endosymbiotic organelles chloroplasts and mitochondria are synthesized on cytosolic ribosomes and posttranslationally imported. Genetic, biochemical and biophysical approaches led to rather detailed knowledge on the composition of the translocon-complexes which catalyze the membrane transport of the preproteins. Comprehensive concepts on the targeting and membrane transport of polypeptides emerged, however little detail on the molecular nature and mechanisms of the protein translocation channels comprising nanopores has been achieved. In this paper we will highlight recent developments of the diverse protein translocation systems and focus particularly on the common biophysical properties and functions of the protein conducting nanopores. We also provide a first analysis of the interaction between the genuine protein conducting nanopore Tom40SC as well as a mutant Tom40SC (\\mathrm {S}_{54} \\to E ) containing an additional negative charge at the channel vestibule and one of its native substrates, CoxIV, a mitochondrial targeting peptide. The polypeptide induced a voltage-dependent increase in the frequency of channel closure of Tom40SC corresponding to a voltage-dependent association rate, which was even more pronounced for the Tom40SC S54E mutant. The corresponding dwelltime reflecting association/transport of the peptide could be determined with \\bar {t}_{\\mathrm {off}} \\cong 1.1 ms for the wildtype, whereas the mutant Tom40SC S54E displayed a biphasic dwelltime distribution (\\bar {t}_{\\mathrm {off}}^1 \\cong 0.4 ms \\bar {t}_{\\mathrm {off}}^2 \\cong 4.6 ms).

  1. Single-Molecule Study of Protein-Protein and Protein-DNA Interaction Dynamics

    SciTech Connect

    Lu, H. PETER

    2005-03-01

    Protein-protein and protein-DNA interactions play critical roles in biological functions of living cells, such as cell signaling, receptor-ligand activation, cellular metabolism, DNA damage recognition and repair, gene expression, replication, etc. These protein interactions often involve complex mechanisms and inhomogeneous dynamics with significant conformational changes. Protein-protein, protein-ligand, and protein-DNA interactions are often intrinsically single-molecule processes at an induction stage associated with the initiation of crucial early eents in living cells. For example, cell-signaling processes are often initiated through a few copies of protein-interaction complexes, being amplified along the signaling pathway.

  2. Cotton and Protein Interactions

    SciTech Connect

    Goheen, Steven C.; Edwards, J. V.; Rayburn, Alfred R.; Gaither, Kari A.; Castro, Nathan J.

    2006-06-30

    The adsorbent properties of important wound fluid proteins and cotton cellulose are reviewed. This review focuses on the adsorption of albumin to cotton-based wound dressings and some chemically modified derivatives targeted for chronic wounds. Adsorption of elastase in the presence of albumin was examined as a model to understand the interactive properties of these wound fluid components with cotton fibers. In the chronic non-healing wound, elastase appears to be over-expressed, and it digests tissue and growth factors, interfering with the normal healing process. Albumin is the most prevalent protein in wound fluid, and in highly to moderately exudative wounds, it may bind significantly to the fibers of wound dressings. Thus, the relative binding properties of both elastase and albumin to wound dressing fibers are of interest in the design of more effective wound dressings. The present work examines the binding of albumin to two different derivatives of cotton, and quantifies the elastase binding to the same derivatives following exposure of albumin to the fiber surface. An HPLC adsorption technique was employed coupled with a colorimetric enzyme assay to quantify the relative binding properties of albumin and elastase to cotton. The results of wound protein binding are discussed in relation to the porosity and surface chemistry interactions of cotton and wound proteins. Studies are directed to understanding the implications of protein adsorption phenomena in terms of fiber-protein models that have implications for rationally designing dressings for chronic wounds.

  3. Disease specific protein corona

    NASA Astrophysics Data System (ADS)

    Rahman, M.; Mahmoudi, M.

    2015-03-01

    It is now well accepted that upon their entrance into the biological environments, the surface of nanomaterials would be covered by various biomacromolecules (e.g., proteins and lipids). The absorption of these biomolecules, so called `protein corona', onto the surface of (nano)biomaterials confers them a new `biological identity'. Although the formation of protein coronas on the surface of nanoparticles has been widely investigated, there are few reports on the effect of various diseases on the biological identity of nanoparticles. As the type of diseases may tremendously changes the composition of the protein source (e.g., human plasma/serum), one can expect that amount and composition of associated proteins in the corona composition may be varied, in disease type manner. Here, we show that corona coated silica and polystyrene nanoparticles (after interaction with in the plasma of the healthy individuals) could induce unfolding of fibrinogen, which promotes release of the inflammatory cytokines. However, no considerable releases of inflammatory cytokines were observed for corona coated graphene sheets. In contrast, the obtained corona coated silica and polystyrene nanoparticles from the hypofibrinogenemia patients could not induce inflammatory cytokine release where graphene sheets do. Therefore, one can expect that disease-specific protein coronas can provide a novel approach for applying nanomedicine to personalized medicine, improving diagnosis and treatment of different diseases tailored to the specific conditions and circumstances.

  4. Immunoassays of soy proteins.

    PubMed

    Brandon, David L; Friedman, Mendel

    2002-10-23

    Proteins of soybeans (Glycine max) are widely used in animal and human nutrition. In addition to the bulk of the seed storage proteins, which are classified as albumins and globulins, approximately 6% of soybean proteins are classified as inhibitors of trypsin and chymotrypsin and approximately 0.5% are sugar-binding lectins. The two major classes of inhibitors are the Kunitz trypsin inhibitor, which inhibits trypsin, and the Bowman-Birk inhibitor (BBI), which inhibits both trypsin and chymotrypsin. Unless removed or inactivated, these inhibitors and lectins can impair the nutritional quality and safety of soy-based diets. On the other hand, several studies suggest that BBI can also function as an anticarcinogen, possibly through interaction with a cellular serine protease. Good-quality soybean proteins contribute to the nutritional value of many specialty foods including infant soy formulas and milk replacers for calves, and provide texture to many processed foods. However, they may also induce occasional allergic responses in humans. This paper outlines immunoassays developed to analyze for soy proteins in different soybean lines, in processed foods, and in nonsoy foods fortified with soy proteins. An assessment of the current status of immunoassays, especially of enzyme-linked immunosorbent assays for soybean inhibitors of digestive enzymes, soy globulins, and soy lectins, demonstrates the usefulness of these methods in plant and food sciences and in medicine.

  5. Protein phosphorylation and photorespiration.

    PubMed

    Hodges, M; Jossier, M; Boex-Fontvieille, E; Tcherkez, G

    2013-07-01

    Photorespiration allows the recycling of carbon atoms of 2-phosphoglycolate produced by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenase activity, as well as the removal of potentially toxic metabolites. The photorespiratory pathway takes place in the light, encompasses four cellular compartments and interacts with several other metabolic pathways and functions. Therefore, the regulation of this cycle is probably of paramount importance to plant metabolism, however, our current knowledge is poor. To rapidly respond to changing conditions, proteins undergo a number of different post-translational modifications that include acetylation, methylation and ubiquitylation, but protein phosphorylation is probably the most common. The reversible covalent addition of a phosphate group to a specific amino acid residue allows the modulation of protein function, such as activity, subcellular localisation, capacity to interact with other proteins and stability. Recent data indicate that many photorespiratory enzymes can be phosphorylated, and thus it seems that the photorespiratory cycle is, in part, regulated by protein phosphorylation. In this review, the known phosphorylation sites of each Arabidopsis thaliana photorespiratory enzyme and several photorespiratory-associated proteins are described and discussed. A brief account of phosphoproteomic protocols is also given since the published data compiled in this review are the fruit of this approach.

  6. Recombinant human milk proteins.

    PubMed

    Lönnerdal, Bo

    2006-01-01

    Human milk provides proteins that benefit newborn infants. They not only provide amino acids, but also facilitate the absorption of nutrients, stimulate growth and development of the intestine, modulate immune function, and aid in the digestion of other nutrients. Breastfed infants have a lower prevalence of infections than formula-fed infants. Since many women in industrialized countries choose not to breastfeed, and an increasing proportion of women in developing countries are advised not to breastfeed because of the risk of HIV transmission, incorporation of recombinant human milk proteins into infant foods is likely to be beneficial. We are expressing human milk proteins known to have anti-infective activity in rice. Since rice is a normal constituent of the diet of infants and children, limited purification of the proteins is required. Lactoferrin has antimicrobial and iron-binding activities. Lysozyme is an enzyme that is bactericidal and also acts synergistically with lactoferrin. These recombinant proteins have biological activities identical to their native counterparts. They are equally resistant to heat processing, which is necessary for food applications, and to acid and proteolytic enzymes which are needed to maintain their biological activity in the gastrointestinal tract of infants. These recombinant human milk proteins may be incorporated into infant formulas, baby foods and complementary foods, and used with the goal to reduce infectious diseases.

  7. Motor proteins 1: kinesins.

    PubMed

    Bloom, G S; Endow, S A

    1995-01-01

    Progress regarding the kinesins is now being made at a rapid and accelerating rate. The in vivo-functions, and biophysical and enzymatic properties of kinesin itself are being explored at ever increasing levels of detail. The kinesin-related proteins now number several dozen, and although more is known about primary structure than function for most of the proteins, this trend is already reversing. For example, knowledge about the kinesin-related protein, ncd, is expanding rapidly, and more is already known about its three-dimensional structure than is known for kinesin heavy chain. This volume presents a comprehensive review of the major published works on kinesin and kinesin-related proteins. Hopefully, this manuscript will complement other recent review articles [17, 20, 25, 37, 60-62, 67, 69, 75, 85-88, 231, 233, 238, 244, 269-271, 281, 282, 292] or books [49, 227, 293] that have focused on more selective aspects of the kinesin family, or have been aimed more generally at MT motor proteins. In line with the stated purpose of the Protein Profile series, annual updates of the review on the kinesins are planned for at least the next few years.

  8. Fast protein folding kinetics.

    PubMed

    Gelman, Hannah; Gruebele, Martin

    2014-05-01

    Fast-folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast-folding proteins has provided insight into the mechanisms, which allow some proteins to find their native conformation well <1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even 'slow' folding processes: fast folders are small; relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast-folding proteins and provides an overview of the major findings of fast-folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general, as well as some work that is left to do.

  9. Moonlighting Proteins in Yeasts

    PubMed Central

    Gancedo, Carlos; Flores, Carmen-Lisset

    2008-01-01

    Proteins able to participate in unrelated biological processes have been grouped under the generic name of moonlighting proteins. Work with different yeast species has uncovered a great number of moonlighting proteins and shown their importance for adequate functioning of the yeast cell. Moonlighting activities in yeasts include such diverse functions as control of gene expression, organelle assembly, and modification of the activity of metabolic pathways. In this review, we consider several well-studied moonlighting proteins in different yeast species, paying attention to the experimental approaches used to identify them and the evidence that supports their participation in the unexpected function. Usually, moonlighting activities have been uncovered unexpectedly, and up to now, no satisfactory way to predict moonlighting activities has been found. Among the well-characterized moonlighting proteins in yeasts, enzymes from the glycolytic pathway appear to be prominent. For some cases, it is shown that despite close phylogenetic relationships, moonlighting activities are not necessarily conserved among yeast species. Organisms may utilize moonlighting to add a new layer of regulation to conventional regulatory networks. The existence of this type of proteins in yeasts should be taken into account when designing mutant screens or in attempts to model or modify yeast metabolism. PMID:18322039

  10. Food protein sources.

    PubMed

    Pirie, N W

    1976-07-01

    Work on food, planned by the U.M. (Use and Management) Section of the U.K. committe, was limited to sources of protein because we agreed that more problems calling for research were likely to arise in getting adequate supplies of protein than of other types of food. Deer meat can be produced on land too rough and exposed for sheep; parts of the work on their metabolism and food requirements necessitated building a mobile laboratory. The manner in which the nutritive value of maize is affected by changes in the ratios in which the component proteins are present, stimulated similar studies on barley and groundnut. There is good quality protein in coconuts and leaves but its use in human food is restricted by the presence of fibre. Methods for separating protein from fibre and other deleterious components were improved. In cooperation with scientists in India and Nigeria, the potential yield of protein-deficient foods. e.g. cassava, were 'ennobled' by growing micro-organisms on them with the addition of a cheap source of nitrogen.

  11. Fast protein folding kinetics

    PubMed Central

    Gelman, Hannah; Gruebele, Martin

    2014-01-01

    Fast folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast folding proteins has provided insight into the mechanisms which allow some proteins to find their native conformation well less than 1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even “slow” folding processes: fast folders are small, relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast folding proteins and provides an overview of the major findings of fast folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general as well as some work that is left to do. PMID:24641816

  12. Protein-Protein Interfaces in Viral Capsids Are Structurally Unique.

    PubMed

    Cheng, Shanshan; Brooks, Charles L

    2015-11-06

    Viral capsids exhibit elaborate and symmetrical architectures of defined sizes and remarkable mechanical properties not seen with cellular macromolecular complexes. Given the uniqueness of the higher-order organization of viral capsid proteins in the virosphere, we explored the question of whether the patterns of protein-protein interactions within viral capsids are distinct from those in generic protein complexes. Our comparative analysis involving a non-redundant set of 551 inter-subunit interfaces in viral capsids from VIPERdb and 20,014 protein-protein interfaces in non-capsid protein complexes from the Protein Data Bank found 418 generic protein-protein interfaces that share similar physicochemical patterns with some protein-protein interfaces in the capsid set, using the program PCalign we developed for comparing protein-protein interfaces. This overlap in the structural space of protein-protein interfaces is significantly small, with a p-value <0.0001, based on a permutation test on the total set of protein-protein interfaces. Furthermore, the generic protein-protein interfaces that bear similarity in their spatial and chemical arrangement with capsid ones are mostly small in size with fewer than 20 interfacial residues, which results from the relatively limited choices of natural design for small interfaces rather than having significant biological implications in terms of functional relationships. We conclude based on this study that protein-protein interfaces in viral capsids are non-representative of patterns in the smaller, more compact cellular protein complexes. Our finding highlights the design principle of building large biological containers from repeated, self-assembling units and provides insights into specific targets for antiviral drug design for improved efficacy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Protein-Protein Interface and Disease: Perspective from Biomolecular Networks.

    PubMed

    Hu, Guang; Xiao, Fei; Li, Yuqian; Li, Yuan; Vongsangnak, Wanwipa

    Protein-protein interactions are involved in many important biological processes and molecular mechanisms of disease association. Structural studies of interfacial residues in protein complexes provide information on protein-protein interactions. Characterizing protein-protein interfaces, including binding sites and allosteric changes, thus pose an imminent challenge. With special focus on protein complexes, approaches based on network theory are proposed to meet this challenge. In this review we pay attention to protein-protein interfaces from the perspective of biomolecular networks and their roles in disease. We first describe the different roles of protein complexes in disease through several structural aspects of interfaces. We then discuss some recent advances in predicting hot spots and communication pathway analysis in terms of amino acid networks. Finally, we highlight possible future aspects of this area with respect to both methodology development and applications for disease treatment.

  14. Protein crystal growth in space

    NASA Technical Reports Server (NTRS)

    Bugg, C. E.; Clifford, D. W.

    1987-01-01

    The advantages of protein crystallization in space, and the applications of protein crystallography to drug design, protein engineering, and the design of synthetic vaccines are examined. The steps involved in using protein crystallography to determine the three-dimensional structure of a protein are discussed. The growth chamber design and the hand-held apparatus developed for protein crystal growth by vapor diffusion techniques (hanging-drop method) are described; the experimental data from the four Shuttle missions are utilized to develop hardware for protein crystal growth in space and to evaluate the effects of gravity on protein crystal growth.

  15. Protein crystal growth in space

    NASA Technical Reports Server (NTRS)

    Bugg, C. E.; Clifford, D. W.

    1987-01-01

    The advantages of protein crystallization in space, and the applications of protein crystallography to drug design, protein engineering, and the design of synthetic vaccines are examined. The steps involved in using protein crystallography to determine the three-dimensional structure of a protein are discussed. The growth chamber design and the hand-held apparatus developed for protein crystal growth by vapor diffusion techniques (hanging-drop method) are described; the experimental data from the four Shuttle missions are utilized to develop hardware for protein crystal growth in space and to evaluate the effects of gravity on protein crystal growth.

  16. Parallel Computational Protein Design

    PubMed Central

    Zhou, Yichao; Donald, Bruce R.; Zeng, Jianyang

    2016-01-01

    Computational structure-based protein design (CSPD) is an important problem in computational biology, which aims to design or improve a prescribed protein function based on a protein structure template. It provides a practical tool for real-world protein engineering applications. A popular CSPD method that guarantees to find the global minimum energy solution (GMEC) is to combine both dead-end elimination (DEE) and A* tree search algorithms. However, in this framework, the A* search algorithm can run in exponential time in the worst case, which may become the computation bottleneck of large-scale computational protein design process. To address this issue, we extend and add a new module to the OSPREY program that was previously developed in the Donald lab [1] to implement a GPU-based massively parallel A* algorithm for improving protein design pipeline. By exploiting the modern GPU computational framework and optimizing the computation of the heuristic function for A* search, our new program, called gOSPREY, can provide up to four orders of magnitude speedups in large protein design cases with a small memory overhead comparing to the traditional A* search algorithm implementation, while still guaranteeing the optimality. In addition, gOSPREY can be configured to run in a bounded-memory mode to tackle the problems in which the conformation space is too large and the global optimal solution cannot be computed previously. Furthermore, the GPU-based A* algorithm implemented in the gOSPREY program can be combined with the state-of-the-art rotamer pruning algorithms such as iMinDEE [2] and DEEPer [3] to also consider continuous backbone and side-chain flexibility. PMID:27914056

  17. Modeling Mercury in Proteins

    SciTech Connect

    Smith, Jeremy C; Parks, Jerry M

    2016-01-01

    Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively non-toxic, other forms such as Hg2+ and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg2+ can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg2+ to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed picture and circumvent issues associated with toxicity. Here we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intra-protein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confers mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multi-scale model of environmental mercury cycling.

  18. Direct protein-protein conjugation by genetically introducing bioorthogonal functional groups into proteins.

    PubMed

    Kim, Sanggil; Ko, Wooseok; Sung, Bong Hyun; Kim, Sun Chang; Lee, Hyun Soo

    2016-11-15

    Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein-protein conjugates has gained research interest. In this study, site-specific protein-protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein-protein conjugates and protein complexes of biochemical significance. Copyright © 2016. Published by Elsevier Ltd.

  19. Benchtop Detection of Proteins

    NASA Technical Reports Server (NTRS)

    Scardelletti, Maximilian C.; Varaljay, Vanessa

    2007-01-01

    A process, and a benchtop-scale apparatus for implementing the process, have been developed to detect proteins associated with specific microbes in water. The process and apparatus may also be useful for detection of proteins in other, more complex liquids. There may be numerous potential applications, including monitoring lakes and streams for contamination, testing of blood and other bodily fluids in medical laboratories, and testing for microbial contamination of liquids in restaurants and industrial food-processing facilities. A sample can be prepared and analyzed by use of this process and apparatus within minutes, whereas an equivalent analysis performed by use of other processes and equipment can often take hours to days. The process begins with the conjugation of near-infrared-fluorescent dyes to antibodies that are specific to a particular protein. Initially, the research has focused on using near-infrared dyes to detect antigens or associated proteins in solution, which has proven successful vs. microbial cells, and streamlining the technique in use for surface protein detection on microbes would theoretically render similar results. However, it is noted that additional work is needed to transition protein-based techniques to microbial cell detection. Consequently, multiple such dye/antibody pairs could be prepared to enable detection of multiple selected microbial species, using a different dye for each species. When excited by near-infrared light of a suitable wavelength, each dye fluoresces at a unique longer wavelength that differs from those of the other dyes, enabling discrimination among the various species. In initial tests, the dye/antibody pairs are mixed into a solution suspected of containing the selected proteins, causing the binding of the dye/antibody pairs to such suspect proteins that may be present. The solution is then run through a microcentrifuge that includes a membrane that acts as a filter in that it retains the dye/antibody/protein

  20. Protein quality control and cancerogenesis.

    PubMed

    Trcka, F; Vojtesek, B; Muller, P

    2012-01-01

    Both nascent and mature proteins are prone to damaging changes induced by either external or internal stimuli. Dysfunctional or misfolded proteins cause direct physiological risk in crowded cellular environment and must be readily and efficiently eliminated. To ensure protein homeostasis, eukaryotic cells have evolved several protein quality control machineries. Protein quality control plays a special role in cancer cells. Genetic instability causing increased production of damaged and/or deregulated proteins is a hallmark of cancer cells. Therefore, intrinsic genetic instability together with hostile tumour microenvironment represents a demanding task for protein quality control machineries in tumours. Regulation of general protein turnover as well as degradation of tumour-promoting/suppressing proteins by protein quality control machineries thus represent an important processes involved in cancer development and progression. The review focuses on the description of three major protein quality control pathways and their roles in cancer.

  1. Nanotechnologies in protein delivery.

    PubMed

    Salmaso, Stefano; Bersani, Sara; Semenzato, Alessandra; Caliceti, Paolo

    2006-01-01

    The growth rate for biotech drugs, namely proteins, peptides, and oligonucleotides, is dictated by the parallel progresses in biotechnology and nanotechnology. Actually, biotechnology techniques have expanded enormously the arsenal of therapeutically useful peptides and proteins making these products of primary interest for future pharmaceutical market. Nevertheless, the exploitation of protein and peptide drugs is strictly related to the development of innovative delivery systems which should provide for controlled, prolonged, or targeted delivery, improved stability during storage and delivery, reduced adverse effects, increased bioavailability, improved patient compliance and allow for administration through the desired route and cope with cost-containment therapeutic protocols. Colloidal formulations ideally possess the physicochemical and biopharmaceutical requisites for protein delivery. Pharmaceutical nanotechnology is a tool of techniques applied to design, develop and produce these systems. It involves the investigation of innovative materials and production procedures for preparation of a variety of nanosized dosage forms, which range from solid nanoparticles to soluble bioconjugates. The research and development of innovative tailor made protein delivery systems, which must be designed according to the drug candidate pharmacological and physicochemical properties, is one of the primary aim of modern pharmaceutical technology. Therefore, as an unmet need exists for technologies that combine innovative drug delivery solutions, a close un-prejudicial interaction between academic and industrial researchers as well as business thought leaders is required.

  2. The ras superfamily proteins.

    PubMed

    Chardin, P

    1988-07-01

    Several recent discoveries indicate that the ras genes, frequently activated to a transforming potential in some human tumours, belong to a large family that can be divided into three main branches: the first branch represented by the ras, ral and rap genes; the second branch, by the rho genes; and the third branch, by the rab genes. The C-terminal end of the encoded proteins always includes a cystein, which may become fatty-acylated, suggesting a sub-membrane localization. The ras superfamily proteins share four regions of high homology corresponding to the GTP binding site; however, even in these regions, significant differences are found, suggesting that the various proteins may possess slightly different biochemical properties. Recent reports show that some of these proteins play an essential role in the control of physical processes such as cell motility, membrane ruffling, endocytosis and exocytosis. Nevertheless, the characterization of the proteins directly interacting with the ras or ras-related gene-products will be required to precisely understand their function.

  3. Nanophotonics of protein amyloids

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Mily; Mukhopadhyay, Samrat

    2014-04-01

    Technological breakthroughs in the super-resolution optical imaging techniques have enriched our current understanding of a range of biological systems and biomolecular processes at the nanoscopic spatial resolution. Protein amyloids are an important class of ordered protein assemblies consisting of misfolded proteins that are implicated in a wide range of devastating human diseases. In order to decipher the structural basis of the supramolecular protein assembly in amyloids and their detrimental interactions with the cell membranes, it is important to employ high-resolution optical imaging techniques. Additionally, amyloids could serve as novel biological nanomaterials for a variety of potential applications. In this review, we summarize a few examples of the utility of near-field scanning optical imaging methodologies to obtain a wealth of structural information into the nanoscale amyloid assembly. Although the near-field technologies were developed several decades ago, it is only recently that these methodologies are being applied and adapted for amyloid research to yield novel information pertaining to the exciting nanoscopic world of protein aggregates. We believe that the account on the nanophotonics of amyloids described in this review will be useful for the future studies on the biophysics of amyloids.

  4. A Stevedore's Protein Knot

    PubMed Central

    Hsu, Hsiao-Ping; Mirny, Leonid A.; Kardar, Mehran; Onuchic, José N.; Virnau, Peter

    2010-01-01

    Protein knots, mostly regarded as intriguing oddities, are gradually being recognized as significant structural motifs. Seven distinctly knotted folds have already been identified. It is by and large unclear how these exceptional structures actually fold, and only recently, experiments and simulations have begun to shed some light on this issue. In checking the new protein structures submitted to the Protein Data Bank, we encountered the most complex and the smallest knots to date: A recently uncovered α-haloacid dehalogenase structure contains a knot with six crossings, a so-called Stevedore knot, in a projection onto a plane. The smallest protein knot is present in an as yet unclassified protein fragment that consists of only 92 amino acids. The topological complexity of the Stevedore knot presents a puzzle as to how it could possibly fold. To unravel this enigma, we performed folding simulations with a structure-based coarse-grained model and uncovered a possible mechanism by which the knot forms in a single loop flip. PMID:20369018

  5. Heat Capacity in Proteins

    NASA Astrophysics Data System (ADS)

    Prabhu, Ninad V.; Sharp, Kim A.

    2005-05-01

    Heat capacity (Cp) is one of several major thermodynamic quantities commonly measured in proteins. With more than half a dozen definitions, it is the hardest of these quantities to understand in physical terms, but the richest in insight. There are many ramifications of observed Cp changes: The sign distinguishes apolar from polar solvation. It imparts a temperature (T) dependence to entropy and enthalpy that may change their signs and which of them dominate. Protein unfolding usually has a positive ΔCp, producing a maximum in stability and sometimes cold denaturation. There are two heat capacity contributions, from hydration and protein-protein interactions; which dominates in folding and binding is an open question. Theoretical work to date has dealt mostly with the hydration term and can account, at least semiquantitatively, for the major Cp-related features: the positive and negative Cp of hydration for apolar and polar groups, respectively; the convergence of apolar group hydration entropy at T ≈ 112°C; the decrease in apolar hydration Cp with increasing T; and the T-maximum in protein stability and cold denaturation.

  6. Protein Dynamics in Enzymology

    NASA Astrophysics Data System (ADS)

    Brooks, , III

    2001-03-01

    Enzymes carry-out the chemical activity essential for living processes by providing particular structural arrangements of chemically functional moieties through the structure of their constituent proteins. They are suggested to be optimized through evolution to specifically bind the transition state for the chemical processes they participate in, thereby enhancing the rate of these chemical events by 6-12 orders of magnitude. However, proteins are malleable and fluctuating many-body systems and may also utilize coupling between motional processes with catalysis to regulate or promote these processes. Our studies are aimed at exploring the hypothesis that motions of the protein couple distant regions of the molecule to assist catalytic processes. We demonstrate, through the use of molecular simulations, that strongly coupled motions occur in regions of protein molecules distant in sequence and space from each other, and the enzyme’s active site, when the protein is in a reactant state. Further, we find that the presence of this coupling disappears in complexes no longer reactive-competent, i.e., for product configurations and mutant sequences. The implications of these findings and aspects of evolutionary relationships and mutational studies which support the coupling hypothesis will be discussed in the context of our work on dihydrofolate reductase.

  7. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  8. Folding mechanism of proteins and protein-like polymers

    NASA Astrophysics Data System (ADS)

    Pande, Vijay

    2000-03-01

    Proteins are amazing biomaterials: they both perform biological activity as well as assemble themselves. In order to understand how proteins fold and to design synthetic polymers with protein-like properties, we need to understand how these molecules assemble themselves. I will discuss results from recent simulations of proteins and protein-like polymers in order to examine which is common and potentially ``universal'' about the folding (self-assembly) mechanism. These results may shed light on protein and protein-like polymer design, experiments on folding, as well as areas in which misfolding may be important such as many neurodegenerative diseases.

  9. Protein folding and de novo protein design for biotechnological applications

    PubMed Central

    Khoury, George A.; Smadbeck, James; Kieslich, Chris A.; Floudas, Christodoulos A.

    2014-01-01

    In the post-genomic era, the medical/biological fields are advancing faster than ever. However, before the power of full-genome sequencing can be fully realized, the connection between amino acid sequence and protein structure, known as the protein folding problem, needs to be elucidated. The protein folding problem remains elusive, with significant difficulties still arising when modeling amino acid sequences lacking an identifiable template. Understanding protein folding will allow for unforeseen advances in protein design, often referred as the inverse protein folding problem. Despite challenges in protein folding, de novo protein design has recently demonstrated significant success via computational techniques. We review advances and challenges in protein structure prediction and de novo protein design, and highlight their interplay in successful biotechnological applications. PMID:24268901

  10. Solid State NMR and Protein-Protein Interactions in Membranes

    PubMed Central

    Miao, Yimin; Cross, Timothy A.

    2013-01-01

    Solid state NMR spectroscopy has evolved rapidly in recent years into an excellent tool for the characterization of membrane proteins and their complexes. In the past few years it has also become clear that the structure of membrane proteins, especially helical membrane proteins is determined, in part, by the membrane environment. Therefore, the modeling of this environment by a liquid crystalline lipid bilayer for solid state NMR has generated a unique tool for the characterization of native conformational states, local and global dynamics, and high resolution structure for these proteins. Protein-protein interactions can also benefit from this solid state NMR capability to characterize membrane proteins in a native-like environment. These complexes take the form of oligomeric structures and hetero-protein interactions both with water soluble proteins and other membrane proteins. PMID:24034903

  11. Protein crystallization studies

    NASA Technical Reports Server (NTRS)

    Lyne, James Evans

    1996-01-01

    The Structural Biology laboratory at NASA Marshall Spaceflight Center uses x-ray crystallographic techniques to conduct research into the three-dimensional structure of a wide variety of proteins. A major effort in the laboratory involves an ongoing study of human serum albumin (the principal protein in human plasma) and its interaction with various endogenous substances and pharmaceutical agents. Another focus is on antigenic and functional proteins from several pathogenic organisms including the human immunodeficiency virus (HIV) and the widespread parasitic genus, Schistosoma. My efforts this summer have been twofold: first, to identify clinically significant drug interactions involving albumin binding displacement and to initiate studies of the three-dimensional structure of albumin complexed with these agents, and secondly, to establish collaborative efforts to extend the lab's work on human pathogens.

  12. Protein Crystal Serum Albumin

    NASA Technical Reports Server (NTRS)

    1998-01-01

    As the most abundant protein in the circulatory system albumin contributes 80% to colloid osmotic blood pressure. Albumin is also chiefly responsible for the maintenance of blood pH. It is located in every tissue and bodily secretion, with extracellular protein comprising 60% of total albumin. Perhaps the most outstanding property of albumin is its ability to bind reversibly to an incredible variety of ligands. It is widely accepted in the pharmaceutical industry that the overall distribution, metabolism, and efficiency of many drugs are rendered ineffective because of their unusually high affinity for this abundant protein. An understanding of the chemistry of the various classes of pharmaceutical interactions with albumin can suggest new approaches to drug therapy and design. Principal Investigator: Dan Carter/New Century Pharmaceuticals

  13. Thermal hysteresis proteins.

    PubMed

    Barrett, J

    2001-02-01

    Extreme environments present a wealth of biochemical adaptations. Thermal hysteresis proteins (THPs) have been found in vertebrates, invertebrates, plants, bacteria and fungi and are able to depress the freezing point of water (in the presence of ice crystals) in a non-colligative manner by binding to the surface of nascent ice crystals. The THPs comprise a disparate group of proteins with a variety of tertiary structures and often no common sequence similarities or structural motifs. Different THPs bind to different faces of the ice crystal, and no single mechanism has been proposed to account for THP ice binding affinity and specificity. Experimentally THPs have been used in the cryopreservation of tissues and cells and to induce cold tolerance in freeze susceptible organisms. THPs represent a remarkable example of parallel and convergent evolution with different proteins being adapted for an anti-freeze role.

  14. Matricellular proteins and biomaterials.

    PubMed

    Morris, Aaron H; Kyriakides, Themis R

    2014-07-01

    Biomaterials are essential to modern medicine as components of reconstructive implants, implantable sensors, and vehicles for localized drug delivery. Advances in biomaterials have led to progression from simply making implants that are nontoxic to making implants that are specifically designed to elicit particular functions within the host. The interaction of implants and the extracellular matrix during the foreign body response is a growing area of concern for the field of biomaterials, because it can lead to implant failure. Expression of matricellular proteins is modulated during the foreign body response and these proteins interact with biomaterials. The design of biomaterials to specifically alter the levels of matricellular proteins surrounding implants provides a new avenue for the design and fabrication of biomimetic biomaterials.

  15. Thermodynamics of Protein Aggregation

    NASA Astrophysics Data System (ADS)

    Osborne, Kenneth L.; Barz, Bogdan; Bachmann, Michael; Strodel, Birgit

    Amyloid protein aggregation characterizes many neurodegenerative disorders, including Alzheimer's, Parkinson's, and Creutz- feldt-Jakob disease. Evidence suggests that amyloid aggregates may share similar aggregation pathways, implying simulation of full-length amyloid proteins is not necessary for understanding amyloid formation. In this study we simulate GNNQQNY, the N-terminal prion-determining domain of the yeast protein Sup35 to investigate the thermodynamics of structural transitions during aggregation. We use a coarse-grained model with replica-exchange molecular dynamics to investigate the association of 3-, 6-, and 12-chain GNNQQNY systems and we determine the aggregation pathway by studying aggregation states of GN- NQQNY. We find that the aggregation of the hydrophilic GNNQQNY sequence is mainly driven by H-bond formation, leading to the formation of /3-sheets from the very beginning of the assembly process. Condensation (aggregation) and ordering take place simultaneously, which is underpinned by the occurrence of a single heat capacity peak only.

  16. Protein Crystal Serum Albumin

    NASA Technical Reports Server (NTRS)

    1998-01-01

    As the most abundant protein in the circulatory system albumin contributes 80% to colloid osmotic blood pressure. Albumin is also chiefly responsible for the maintenance of blood pH. It is located in every tissue and bodily secretion, with extracellular protein comprising 60% of total albumin. Perhaps the most outstanding property of albumin is its ability to bind reversibly to an incredible variety of ligands. It is widely accepted in the pharmaceutical industry that the overall distribution, metabolism, and efficiency of many drugs are rendered ineffective because of their unusually high affinity for this abundant protein. An understanding of the chemistry of the various classes of pharmaceutical interactions with albumin can suggest new approaches to drug therapy and design. Principal Investigator: Dan Carter/New Century Pharmaceuticals

  17. Polarizable protein packing.

    PubMed

    Ng, Albert H; Snow, Christopher D

    2011-05-01

    To incorporate protein polarization effects within a protein combinatorial optimization framework, we decompose the polarizable force field AMOEBA into low order terms. Including terms up to the third-order provides a fair approximation to the full energy while maintaining tractability. We represent the polarizable packing problem for protein G as a hypergraph and solve for optimal rotamers with the FASTER combinatorial optimization algorithm. These approximate energy models can be improved to high accuracy [root mean square deviation (rmsd) < 1 kJ mol(-1)] via ridge regression. The resulting trained approximations are used to efficiently identify new, low-energy solutions. The approach is general and should allow combinatorial optimization of other many-body problems. Copyright © 2011 Wiley Periodicals, Inc.

  18. Advanced protein formulations

    PubMed Central

    Wang, Wei

    2015-01-01

    It is well recognized that protein product development is far more challenging than that for small-molecule drugs. The major challenges include inherent sensitivity to different types of stresses during the drug product manufacturing process, high rate of physical and chemical degradation during long-term storage, and enhanced aggregation and/or viscosity at high protein concentrations. In the past decade, many novel formulation concepts and technologies have been or are being developed to address these product development challenges for proteins. These concepts and technologies include use of uncommon/combination of formulation stabilizers, conjugation or fusion with potential stabilizers, site-specific mutagenesis, and preparation of nontraditional types of dosage forms—semiaqueous solutions, nonfreeze-dried solid formulations, suspensions, and other emerging concepts. No one technology appears to be mature, ideal, and/or adequate to address all the challenges. These gaps will likely remain in the foreseeable future and need significant efforts for ultimate resolution. PMID:25858529

  19. Tracking protein aggregate interactions

    PubMed Central

    Bartz, Jason C; Nilsson, K Peter R

    2011-01-01

    Amyloid fibrils share a structural motif consisting of highly ordered β-sheets aligned perpendicular to the fibril axis.1, 2 At each fibril end, β-sheets provide a template for recruiting and converting monomers.3 Different amyloid fibrils often co-occur in the same individual, yet whether a protein aggregate aids or inhibits the assembly of a heterologous protein is unclear. In prion disease, diverse prion aggregate structures, known as strains, are thought to be the basis of disparate disease phenotypes in the same species expressing identical prion protein sequences.4–7 Here we explore the interactions reported to occur when two distinct prion strains occur together in the central nervous system. PMID:21597336

  20. Bioinformatics and Moonlighting Proteins

    PubMed Central

    Hernández, Sergio; Franco, Luís; Calvo, Alejandra; Ferragut, Gabriela; Hermoso, Antoni; Amela, Isaac; Gómez, Antonio; Querol, Enrique; Cedano, Juan

    2015-01-01

    Multitasking or moonlighting is the capability of some proteins to execute two or more biochemical functions. Usually, moonlighting proteins are experimentally revealed by serendipity. For this reason, it would be helpful that Bioinformatics could predict this multifunctionality, especially because of the large amounts of sequences from genome projects. In the present work, we analyze and describe several approaches that use sequences, structures, interactomics, and current bioinformatics algorithms and programs to try to overcome this problem. Among these approaches are (a) remote homology searches using Psi-Blast, (b) detection of functional motifs and domains, (c) analysis of data from protein–protein interaction databases (PPIs), (d) match the query protein sequence to 3D databases (i.e., algorithms as PISITE), and (e) mutation correlation analysis between amino acids by algorithms as MISTIC. Programs designed to identify functional motif/domains detect mainly the canonical function but usually fail in the detection of the moonlighting one, Pfam and ProDom being the best methods. Remote homology search by Psi-Blast combined with data from interactomics databases (PPIs) has the best performance. Structural information and mutation correlation analysis can help us to map the functional sites. Mutation correlation analysis can only be used in very specific situations – it requires the existence of multialigned family protein sequences – but can suggest how the evolutionary process of second function acquisition took place. The multitasking protein database MultitaskProtDB (http://wallace.uab.es/multitask/), previously published by our group, has been used as a benchmark for the all of the analyses. PMID:26157797

  1. Antioxidants and protein oxidation.

    PubMed

    Griffiths, H R

    2000-11-01

    Proteins are susceptible to oxidation by reactive oxygen species, where the type of damage induced is characteristic of the denaturing species. The induction of protein carbonyls is a widely applied biomarker, arising from primary oxidative insult. However, when applied to complex biological and pathological conditions it can be subject to interference from lipid, carbohydrate and DNA oxidation products. More recently, interest has focused on the analysis of specific protein bound oxidised amino acids. Of the 22 amino acids, aromatic and sulphydryl containing residues have been regarded as being particularly susceptible to oxidative modification, with L-DOPA from tyrosine, ortho-tyrosine from phenylalanine; sulphoxides and disulphides from methionine and cysteine respectively; and kynurenines from tryptophan. Latterly, the identification of valine and leucine hydroxides, reduced from hydroperoxide intermediates, has been described and applied. In order to examine the nature of oxidative damage and protective efficacy of antioxidants the markers must be thoroughly evaluated for dosimetry in vitro following damage by specific radical species. Antioxidant protection against formation of the biomarker should be demonstrated in vitro. Quantification of biomarkers in proteins from normal subjects should be within the limits of detection of any analytical procedure. Further to this, the techniques for isolation and hydrolysis of specific proteins should demonstrate that in vitro oxidation is minimised. There is a need for the development of standards for quality assurance material to standardise procedures between laboratories. At present, antioxidant effects on protein oxidation in vivo are limited to animal studies, where dietary antioxidants have been reported to reduce dityrosine formation during rat exercise training. Two studies on humans have been reported last year. The further application of these methods to human studies is indicated, where the quality of the

  2. Modeling Mercury in Proteins.

    PubMed

    Parks, J M; Smith, J C

    2016-01-01

    Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively nontoxic, other forms such as Hg(2+) and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg(2+) can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg(2+) to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed molecular picture and circumvent issues associated with toxicity. Here, we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intraprotein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand-binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confer mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multiscale model of environmental mercury cycling. © 2016 Elsevier Inc. All rights reserved.

  3. Amyloidogenesis of Tau protein.

    PubMed

    Nizynski, Bartosz; Dzwolak, Wojciech; Nieznanski, Krzysztof

    2017-08-17

    The role of microtubule-associated protein Tau in neurodegeneration has been extensively investigated since the discovery of Tau amyloid aggregates in the brains of patients with Alzheimer's disease (AD). The process of formation of amyloid fibrils is known as amyloidogenesis and attracts much attention as a potential target in the prevention and treatment of neurodegenerative conditions linked to protein aggregation. Cerebral deposition of amyloid aggregates of Tau is observed not only in AD but also in numerous other tauopathies and prion diseases. Amyloidogenesis of intrinsically unstructured monomers of Tau can be triggered by mutations in the Tau gene, post-translational modifications, or interactions with polyanionic molecules and aggregation-prone proteins/peptides. The self-assembly of amyloid fibrils of Tau shares a number of characteristic features with amyloidogenesis of other proteins involved in neurodegenerative diseases. For example, in vitro experiments have demonstrated that the nucleation phase, which is the rate-limiting stage of Tau amyloidogenesis, is shortened in the presence of fragmented preformed Tau fibrils acting as aggregation templates ("seeds"). Accordingly, Tau aggregates released by tauopathy-affected neurons can spread the neurodegenerative process in the brain through a prion-like mechanism, originally described for the pathogenic form of prion protein. Moreover, Tau has been shown to form amyloid strains-structurally diverse self-propagating aggregates of potentially various pathological effects, resembling in this respect prion strains. Here, we review the current literature on Tau aggregation and discuss mechanisms of propagation of Tau amyloid in the light of the prion-like paradigm. © 2017 The Protein Society.

  4. Epistasis in protein evolution

    PubMed Central

    Starr, Tyler N.

    2016-01-01

    Abstract The structure, function, and evolution of proteins depend on physical and genetic interactions among amino acids. Recent studies have used new strategies to explore the prevalence, biochemical mechanisms, and evolutionary implications of these interactions—called epistasis—within proteins. Here we describe an emerging picture of pervasive epistasis in which the physical and biological effects of mutations change over the course of evolution in a lineage‐specific fashion. Epistasis can restrict the trajectories available to an evolving protein or open new paths to sequences and functions that would otherwise have been inaccessible. We describe two broad classes of epistatic interactions, which arise from different physical mechanisms and have different effects on evolutionary processes. Specific epistasis—in which one mutation influences the phenotypic effect of few other mutations—is caused by direct and indirect physical interactions between mutations, which nonadditively change the protein's physical properties, such as conformation, stability, or affinity for ligands. In contrast, nonspecific epistasis describes mutations that modify the effect of many others; these typically behave additively with respect to the physical properties of a protein but exhibit epistasis because of a nonlinear relationship between the physical properties and their biological effects, such as function or fitness. Both types of interaction are rampant, but specific epistasis has stronger effects on the rate and outcomes of evolution, because it imposes stricter constraints and modulates evolutionary potential more dramatically; it therefore makes evolution more contingent on low‐probability historical events and leaves stronger marks on the sequences, structures, and functions of protein families. PMID:26833806

  5. [Protein-losing enteropathy].

    PubMed

    Parfenov, A I; Krums, L M

    2017-01-01

    Protein-losing enteropathy (PLE) is a rare complication of intestinal diseases. Its main manifestation is hypoproteinemic edema. The diagnosis of PLE is based on the verification of protein loss into the intestinal lumen, by determining fecal α1-antitrypsin concentration and clearance. The localization of the affected colonic segment is clarified using radiologic and endoscopic techniques. The mainstay of treatment for PLE is a fat-free diet enriched with medium-chain triglycerides. Surgical resection of the affected segment of the colon may be the treatment of choice for severe hypoproteinemia resistant to drug therapy.

  6. DELIVERY OF THERAPEUTIC PROTEINS

    PubMed Central

    Pisal, Dipak S.; Kosloski, Matthew P.; Balu-Iyer, Sathy V.

    2009-01-01

    The safety and efficacy of protein therapeutics are limited by three interrelated pharmaceutical issues, in vitro and in vivo instability, immunogenicity and shorter half-lives. Novel drug modifications for overcoming these issues are under investigation and include covalent attachment of poly(ethylene glycol) (PEG), polysialic acid, or glycolic acid, as well as developing new formulations containing nanoparticulate or colloidal systems (e.g. liposomes, polymeric microspheres, polymeric nanoparticles). Such strategies have the potential to develop as next generation protein therapeutics. This review includes a general discussion on these delivery approaches. PMID:20049941

  7. Protein biosynthesis in mitochondria.

    PubMed

    Kuzmenko, A V; Levitskii, S A; Vinogradova, E N; Atkinson, G C; Hauryliuk, V; Zenkin, N; Kamenski, P A

    2013-08-01

    Translation, that is biosynthesis of polypeptides in accordance with information encoded in the genome, is one of the most important processes in the living cell, and it has been in the spotlight of international research for many years. The mechanisms of protein biosynthesis in bacteria and in the eukaryotic cytoplasm are now understood in great detail. However, significantly less is known about translation in eukaryotic mitochondria, which is characterized by a number of unusual features. In this review, we summarize current knowledge about mitochondrial translation in different organisms while paying special attention to the aspects of this process that differ from cytoplasmic protein biosynthesis.

  8. Protein Biosynthesis in Mitochondria

    PubMed Central

    Kuzmenko, A. V.; Levitskii, S. A.; Vinogradova, E. N.; Atkinson, G. C.; Hauryliuk, V.; Zenkin, N.; Kamenski, P. A.

    2013-01-01

    Translation, that is biosynthesis of polypeptides in accordance with information encoded in the genome, is one of the most important processes in the living cell, and it has been in the spotlight of international research for many years. The mechanisms of protein biosynthesis in bacteria and in the eukaryotic cytoplasm are now understood in great detail. However, significantly less is known about translation in eukaryotic mitochondria, which is characterized by a number of unusual features. In this review, we summarize current knowledge about mitochondrial translation in different organisms while paying special attention to the aspects of this process that differ from cytoplasmic protein biosynthesis. PMID:24228873

  9. Congenital protein hypoglycosylation diseases

    PubMed Central

    Sparks, Susan E

    2012-01-01

    Glycosylation is an essential process by which sugars are attached to proteins and lipids. Complete lack of glycosylation is not compatible with life. Because of the widespread function of glycosylation, inherited disorders of glycosylation are multisystemic. Since the identification of the first defect on N-linked glycosylation in the 1980s, there are over 40 different congenital protein hypoglycosylation diseases. This review will include defects of N-linked glycosylation, O-linked glycosylation and disorders of combined N- and O-linked glycosylation. PMID:23776380

  10. SAP family proteins.

    PubMed

    Fujita, A; Kurachi, Y

    2000-03-05

    Thus far, five members including Dlg, SAP97/hDlg, SAP90/PSD-95, SAP102, and PSD-93/chapsyn110 which belong to SAP family have been identified. Recent studies have revealed that these proteins play important roles in the localization and function of glutamate receptors and K(+) channels. Although most of them have been reported to be localized to the synapse, only one member, SAP97, is expressed also in the epithelial cells. In this review, we have summarized structural characters of SAP family proteins and discuss their functions in neurons and epithelial cells.

  11. Protein energy malnutrition.

    PubMed

    Grover, Zubin; Ee, Looi C

    2009-10-01

    Protein energy malnutrition (PEM) is a common problem worldwide and occurs in both developing and industrialized nations. In the developing world, it is frequently a result of socioeconomic, political, or environmental factors. In contrast, protein energy malnutrition in the developed world usually occurs in the context of chronic disease. There remains much variation in the criteria used to define malnutrition, with each method having its own limitations. Early recognition, prompt management, and robust follow up are critical for best outcomes in preventing and treating PEM.

  12. Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

    PubMed

    Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

    2014-10-01

    Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets. © 2014 Wiley Periodicals, Inc.

  13. How Many Protein-Protein Interactions Types Exist in Nature?

    PubMed Central

    Mitra, Pralay; Zhang, Yang

    2012-01-01

    Protein quaternary structure universe” refers to the ensemble of all protein-protein complexes across all organisms in nature. The number of quaternary folds thus corresponds to the number of ways proteins physically interact with other proteins. This study focuses on answering two basic questions: Whether the number of protein-protein interactions is limited and, if yes, how many different quaternary folds exist in nature. By all-to-all sequence and structure comparisons, we grouped the protein complexes in the protein data bank (PDB) into 3,629 families and 1,761 folds. A statistical model was introduced to obtain the quantitative relation between the numbers of quaternary families and quaternary folds in nature. The total number of possible protein-protein interactions was estimated around 4,000, which indicates that the current protein repository contains only 42% of quaternary folds in nature and a full coverage needs approximately a quarter century of experimental effort. The results have important implications to the protein complex structural modeling and the structure genomics of protein-protein interactions. PMID:22719985

  14. Predicting Permanent and Transient Protein-Protein Interfaces

    PubMed Central

    La, David; Kong, Misun; Hoffman, William; Choi, Youn Im; Kihara, Daisuke

    2014-01-01

    Protein-protein interactions are involved in many diverse functions in a cell. To optimize functional roles of interactions, proteins interact with a spectrum of binding affinities. Interactions are conventionally classified into permanent and transient, where the former denotes tight binding between proteins that result in strong complexes, while the latter compose of relatively weak interactions that can dissociate after binding to regulate functional activity at specific time point. Knowing the type of interactions has significant implications for understanding the nature and function of protein-protein interactions. In this study, we constructed amino acid substitution models that capture mutation patterns at permanent and transient type of protein interfaces, which were found to be different with statistical significance. Using the substitution models, we developed a novel computational method that predicts permanent and transient protein binding interfaces in protein surfaces. Without knowledge of the interacting partner, the method employs a single query protein structure and a multiple sequence alignment of the sequence family. Using a large dataset of permanent and transient proteins, we show that our method performs very well in protein interface classification. A very high Area Under the Curve (AUC) value of 0.957 was observed when predicted protein binding sites were classified. Remarkably, near prefect accuracy was achieved with an AUC of 0.991 when actual binding sites were classified. The developed method will be also useful for protein design of permanent and transient protein binding interfaces. PMID:23239312

  15. An introduction to protein moonlighting.

    PubMed

    Jeffery, Constance J

    2014-12-01

    Moonlighting proteins comprise a class of multifunctional proteins in which a single polypeptide chain performs multiple physiologically relevant biochemical or biophysical functions. Almost 300 proteins have been found to moonlight. The known examples of moonlighting proteins include diverse types of proteins, including receptors, enzymes, transcription factors, adhesins and scaffolds, and different combinations of functions are observed. Moonlighting proteins are expressed throughout the evolutionary tree and function in many different biochemical pathways. Some moonlighting proteins can perform both functions simultaneously, but for others, the protein's function changes in response to changes in the environment. The diverse examples of moonlighting proteins already identified, and the potential benefits moonlighting proteins might provide to the organism, such as through coordinating cellular activities, suggest that many more moonlighting proteins are likely to be found. Continuing studies of the structures and functions of moonlighting proteins will aid in predicting the functions of proteins identified through genome sequencing projects, in interpreting results from proteomics experiments, in understanding how different biochemical pathways interact in systems biology, in annotating protein sequence and structure databases, in studies of protein evolution and in the design of proteins with novel functions.

  16. Protein Folding: Then and Now

    PubMed Central

    Chen, Yiwen; Ding, Feng; Nie, Huifen; Serohijos, Adrian W.; Sharma, Shantanu; Wilcox, Kyle C.; Yin, Shuangye; Dokholyan, Nikolay V.

    2007-01-01

    Over the past three decades the protein folding field has undergone monumental changes. Originally a purely academic question, how a protein folds has now become vital in understanding diseases and our abilities to rationally manipulate cellular life by engineering protein folding pathways. We review and contrast past and recent developments in the protein folding field. Specifically, we discuss the progress in our understanding of protein folding thermodynamics and kinetics, the properties of evasive intermediates, and unfolded states. We also discuss how some abnormalities in protein folding lead to protein aggregation and human diseases. PMID:17585870

  17. Protein domain connectivity and essentiality

    NASA Astrophysics Data System (ADS)

    da F. Costa, L.; Rodrigues, F. A.; Travieso, G.

    2006-10-01

    Protein-protein interactions can be properly modeled as scale-free complex networks, while the lethality of proteins has been correlated with the node degrees, therefore defining a lethality-centrality rule. In this work the authors revisit this relevant problem by focusing attention not on proteins as a whole, but on their functional domains, which are ultimately responsible for their binding potential. Four networks are considered: the original protein-protein interaction network, its randomized version, and two domain networks assuming different lethality hypotheses. By using formal statistical analysis, they show that the correlation between connectivity and essentiality is higher for domains than for proteins.

  18. Conformation Distributions in Adsorbed Proteins.

    NASA Astrophysics Data System (ADS)

    Meuse, Curtis W.; Hubbard, Joseph B.; Vrettos, John S.; Smith, Jackson R.; Cicerone, Marcus T.

    2007-03-01

    While the structural basis of protein function is well understood in the biopharmaceutical and biotechnology industries, few methods for the characterization and comparison of protein conformation distributions are available. New methods capable of measuring the stability of protein conformations and the integrity of protein-protein, protein-ligand and protein-surface interactions both in solution and on surfaces are needed to help the development of protein-based products. We are developing infrared spectroscopy methods for the characterization and comparison of molecular conformation distributions in monolayers and in solutions. We have extracted an order parameter describing the orientational and conformational variations of protein functional groups around the average molecular values from a single polarized spectrum. We will discuss the development of these methods and compare them to amide hydrogen/deuterium exchange methods for albumin in solution and on different polymer surfaces to show that our order parameter is related to protein stability.

  19. Protein Catalysts by Computational Design

    NASA Astrophysics Data System (ADS)

    Mayo, Stephen

    2001-03-01

    Understanding the relationships between protein sequence, protein structure, and protein function remains as a central challenge in chemistry and biology. Combined computational and experimental approaches aimed at elucidating these relationships have led to a powerful method for the enhancement of naturally occurring proteins and the creation of new protein function. This presentation will briefly cover the development of our computational protein design methodology and will focus primarily on our recent success in designing an enzyme-like protein catalyst that functions as an esterase. The computational protein design methodology that will be described in this presentation promises not only to revolutionize the conduct of biotechnology in the 21st century, but also to provide a tractable approach for studying the relationships between protein fold and the evolvability of protein function.

  20. Comparative Proteomics of Two Life Cycle Stages of Stable Isotope-labeled Trypanosoma brucei Reveals Novel Components of the Parasite's Host Adaptation Machinery*

    PubMed Central

    Butter, Falk; Bucerius, Ferdinand; Michel, Margaux; Cicova, Zdenka; Mann, Matthias; Janzen, Christian J.

    2013-01-01

    Trypanosoma brucei developed a sophisticated life cycle to adapt to different host environments. Although developmental differentiation of T. brucei has been the topic of intensive research for decades, the mechanisms responsible for adaptation to different host environments are not well understood. We developed stable isotope labeling by amino acids in cell culture in trypanosomes to compare the proteomes of two different life cycle stages. Quantitative comparison of 4364 protein groups identified many proteins previously not known to be stage-specifically expressed. The identification of stage-specific proteins helps to understand how parasites adapt to different hosts and provides new insights into differences in metabolism, gene regulation, and cell architecture. A DEAD-box RNA helicase, which is highly up-regulated in the bloodstream form of this parasite and which is essential for viability and proper cell cycle progression in this stage is described as an example. PMID:23090971

  1. NextGen protein design

    PubMed Central

    Regan, Lynne

    2014-01-01

    Protein engineering is at an exciting stage because designed protein–protein interactions are being used in many applications. For instance, three designed proteins are now in clinical trials. Although there have been many successes over the last decade, protein engineering still faces numerous challenges. Often, designs do not work as anticipated and they still require substantial redesign. The present review focuses on the successes, the challenges and the limitations of rational protein design today. PMID:24059497

  2. Chaos in protein dynamics.

    PubMed

    Braxenthaler, M; Unger, R; Auerbach, D; Given, J A; Moult, J

    1997-12-01

    MD simulations, currently the most detailed description of the dynamic evolution of proteins, are based on the repeated solution of a set of differential equations implementing Newton's second law. Many such systems are known to exhibit chaotic behavior, i.e., very small changes in initial conditions are amplified exponentially and lead to vastly different, inherently unpredictable behavior. We have investigated the response of a protein fragment in an explicit solvent environment to very small perturbations of the atomic positions (10(-3)-10(-9) A). Independent of the starting conformation (native-like, compact, extended), perturbed dynamics trajectories deviated rapidly, leading to conformations that differ by approximately 1 A RMSD within 1-2 ps. Furthermore, introducing the perturbation more than 1-2 ps before a significant conformational transition leads to a loss of the transition in the perturbed trajectories. We present evidence that the observed chaotic behavior reflects physical properties of the system rather than numerical instabilities of the calculation and discuss the implications for models of protein folding and the use of MD as a tool to analyze protein folding pathways.

  3. Protein Crystal Bovine Insulin

    NASA Technical Reports Server (NTRS)

    1991-01-01

    The comparison of protein crystal, Bovine Insulin space-grown (left) and earth-grown (right). Facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  4. Protein thin film machines.

    PubMed

    Federici, Stefania; Oliviero, Giulio; Hamad-Schifferli, Kimberly; Bergese, Paolo

    2010-12-01

    We report the first example of microcantilever beams that are reversibly driven by protein thin film machines fueled by cycling the salt concentration of the surrounding solution. We also show that upon the same salinity stimulus the drive can be completely reversed in its direction by introducing a surface coating ligand. Experimental results are throughout discussed within a general yet simple thermodynamic model.

  5. Protein Stability in Ice

    PubMed Central

    Strambini, Giovanni B.; Gonnelli, Margherita

    2007-01-01

    This study presents an experimental approach, based on the change of Trp fluorescence between native and denatured states of proteins, which permits to monitor unfolding equilibria and the thermodynamic stability (ΔG°) of these macromolecules in frozen aqueous solutions. The results obtained by guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, in the temperature range from −8 to −16°C, demonstrate that the stability of the native fold may be significantly perturbed in ice depending mainly on the size of the liquid water pool (VL) in equilibrium with the solid phase. The data establish a threshold, around VL = 1.5%, below which in ice ΔG° decreases progressively relative to liquid state, up to 3 kcal/mole for VL = 0.285%. The sharp dependence of ΔG° on VL is consistent with a mechanism based on adsorption of the protein to the ice surface. The reduction in ΔG° is accompanied by a corresponding decrease in m-value indicating that protein-ice interactions increase the solvent accessible surface area of the native fold or reduce that of the denatured state, or both. The method opens the possibility for examining in a more quantitative fashion the influence of various experimental conditions on the ice perturbation and in particular to test the effectiveness of numerous additives used in formulations to preserve labile pharmaco proteins. PMID:17189314

  6. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.

    1998-11-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  7. Protein Crystal Bovine Insulin

    NASA Technical Reports Server (NTRS)

    1991-01-01

    The comparison of protein crystal, Bovine Insulin space-grown (left) and earth-grown (right). Facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  8. Cellulose binding domain proteins

    SciTech Connect

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  9. Protein states and proteinquakes.

    PubMed Central

    Ansari, A; Berendzen, J; Bowne, S F; Frauenfelder, H; Iben, I E; Sauke, T B; Shyamsunder, E; Young, R D

    1985-01-01

    After photodissociation of carbon monoxide bound to myoglobin, the protein relaxes to the deoxy equilibrium structure in a quake-like motion. Investigation of the proteinquake and of related intramolecular equilibrium motions shows that states and motions have a hierarchical glass-like structure. PMID:3860839

  10. Tuber Storage Proteins

    PubMed Central

    SHEWRY, PETER R.

    2003-01-01

    A wide range of plants are grown for their edible tubers, but five species together account for almost 90 % of the total world production. These are potato (Solanum tuberosum), cassava (Manihot esculenta), sweet potato (Ipomoea batatus), yams (Dioscorea spp.) and taro (Colocasia, Cyrtosperma and Xanthosoma spp.). All of these, except cassava, contain groups of storage proteins, but these differ in the biological properties and evolutionary relationships. Thus, patatin from potato exhibits activity as an acylhydrolase and esterase, sporamin from sweet potato is an inhibitor of trypsin, and dioscorin from yam is a carbonic anhydrase. Both sporamin and dioscorin also exhibit antioxidant and radical scavenging activity. Taro differs from the other three crops in that it contains two major types of storage protein: a trypsin inhibitor related to sporamin and a mannose‐binding lectin. These characteristics indicate that tuber storage proteins have evolved independently in different species, which contrasts with the highly conserved families of storage proteins present in seeds. Furthermore, all exhibit biological activities which could contribute to resistance to pests, pathogens or abiotic stresses, indicating that they may have dual roles in the tubers. PMID:12730067

  11. Conserved herpesvirus protein kinases

    PubMed Central

    Gershburg, Edward; Pagano, Joseph S.

    2008-01-01

    Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr Virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task. PMID:17881303

  12. Tuber storage proteins.

    PubMed

    Shewry, Peter R

    2003-06-01

    A wide range of plants are grown for their edible tubers, but five species together account for almost 90 % of the total world production. These are potato (Solanum tuberosum), cassava (Manihot esculenta), sweet potato (Ipomoea batatus), yams (Dioscorea spp.) and taro (Colocasia, Cyrtosperma and Xanthosoma spp.). All of these, except cassava, contain groups of storage proteins, but these differ in the biological properties and evolutionary relationships. Thus, patatin from potato exhibits activity as an acylhydrolase and esterase, sporamin from sweet potato is an inhibitor of trypsin, and dioscorin from yam is a carbonic anhydrase. Both sporamin and dioscorin also exhibit antioxidant and radical scavenging activity. Taro differs from the other three crops in that it contains two major types of storage protein: a trypsin inhibitor related to sporamin and a mannose-binding lectin. These characteristics indicate that tuber storage proteins have evolved independently in different species, which contrasts with the highly conserved families of storage proteins present in seeds. Furthermore, all exhibit biological activities which could contribute to resistance to pests, pathogens or abiotic stresses, indicating that they may have dual roles in the tubers.

  13. Protein crystal growth

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Atomic force microscopy uses laser technology to reveal a defect, a double-screw dislocation, on the surface of this crystal of canavalin, a major source of dietary protein for humans and domestic animals. When a crystal grows, attachment kinetics and transport kinetics are competing for control of the molecules. As a molecule gets close to the crystal surface, it has to attach properly for the crystal to be usable. NASA has funded investigators to look at those attachment kinetics from a theoretical standpoint and an experimental standpoint. Dr. Alex McPherson of the University of California, Irvine, is one of those investigators. He uses X-ray diffraction and atomic force microscopy in his laboratory to answer some of the many questions about how protein crystals grow. Atomic force microscopy provides a means of looking at how individual molecules are added to the surface of growing protein crystals. This helps McPherson understand the kinetics of protein crystal growth. McPherson asks, How fast do crystals grow? What are the forces involved? Investigators funded by NASA have clearly shown that such factors as the level of supersaturation and the rate of growth all affect the habit [characteristic arrangement of facets] of the crystal and the defects that occur in the crystal.

  14. Dynamics of protein conformations

    NASA Astrophysics Data System (ADS)

    Stepanova, Maria

    2010-10-01

    A novel theoretical methodology is introduced to identify dynamic structural domains and analyze local flexibility in proteins. The methodology employs a multiscale approach combining identification of essential collective coordinates based on the covariance analysis of molecular dynamics trajectories, construction of the Mori projection operator with these essential coordinates, and analysis of the corresponding generalized Langevin equations [M.Stepanova, Phys.Rev.E 76(2007)051918]. Because the approach employs a rigorous theory, the outcomes are physically transparent: the dynamic domains are associated with regions of relative rigidity in the protein, whereas off-domain regions are relatively soft. This also allows scoring the flexibility in the macromolecule with atomic-level resolution [N.Blinov, M.Berjanskii, D.S.Wishart, and M.Stepanova, Biochemistry, 48(2009)1488]. The applications include the domain coarse-graining and characterization of conformational stability in protein G and prion proteins. The results are compared with published NMR experiments. Potential applications for structural biology, bioinformatics, and drug design are discussed.

  15. Proteins of Excitable Membranes

    PubMed Central

    Nachmansohn, David

    1969-01-01

    Excitable membranes have the special ability of changing rapidly and reversibly their permeability to ions, thereby controlling the ion movements that carry the electric currents propagating nerve impulses. Acetylcholine (ACh) is the specific signal which is released by excitation and is recognized by a specific protein, the ACh-receptor; it induces a conformational change, triggering off a sequence of reactions resulting in increased permeability. The hydrolysis of ACh by ACh-esterase restores the barrier to ions. The enzymes hydrolyzing and forming ACh and the receptor protein are present in the various types of excitable membranes. Properties of the two proteins directly associated with electrical activity, receptor and esterase, will be described in this and subsequent lectures. ACh-esterase has been shown to be located within the excitable membranes. Potent enzyme inhibitors block electrical activity demonstrating the essential role in this function. The enzyme has been recently crystallized and some protein properties will be described. The monocellular electroplax preparation offers a uniquely favorable material for analyzing the properties of the ACh-receptor and its relation to function. The essential role of the receptor in electrical activity has been demonstrated with specific receptor inhibitors. Recent data show the basically similar role of ACh in the axonal and junctional membranes; the differences of electrical events and pharmacological actions are due to variations of shape, structural organization, and environment. PMID:19873642

  16. The Protein Ensemble Database.

    PubMed

    Varadi, Mihaly; Tompa, Peter

    2015-01-01

    The scientific community's major conceptual notion of structural biology has recently shifted in emphasis from the classical structure-function paradigm due to the emergence of intrinsically disordered proteins (IDPs). As opposed to their folded cousins, these proteins are defined by the lack of a stable 3D fold and a high degree of inherent structural heterogeneity that is closely tied to their function. Due to their flexible nature, solution techniques such as small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy and fluorescence resonance energy transfer (FRET) are particularly well-suited for characterizing their biophysical properties. Computationally derived structural ensembles based on such experimental measurements provide models of the conformational sampling displayed by these proteins, and they may offer valuable insights into the functional consequences of inherent flexibility. The Protein Ensemble Database (http://pedb.vib.be) is the first openly accessible, manually curated online resource storing the ensemble models, protocols used during the calculation procedure, and underlying primary experimental data derived from SAXS and/or NMR measurements. By making this previously inaccessible data freely available to researchers, this novel resource is expected to promote the development of more advanced modelling methodologies, facilitate the design of standardized calculation protocols, and consequently lead to a better understanding of how function arises from the disordered state.

  17. Ribosome-inactivating proteins

    PubMed Central

    Walsh, Matthew J; Dodd, Jennifer E; Hautbergue, Guillaume M

    2013-01-01

    Ribosome-inactivating proteins (RIPs) were first isolated over a century ago and have been shown to be catalytic toxins that irreversibly inactivate protein synthesis. Elucidation of atomic structures and molecular mechanism has revealed these proteins to be a diverse group subdivided into two classes. RIPs have been shown to exhibit RNA N-glycosidase activity and depurinate the 28S rRNA of the eukaryotic 60S ribosomal subunit. In this review, we compare archetypal RIP family members with other potent toxins that abolish protein synthesis: the fungal ribotoxins which directly cleave the 28S rRNA and the newly discovered Burkholderia lethal factor 1 (BLF1). BLF1 presents additional challenges to the current classification system since, like the ribotoxins, it does not possess RNA N-glycosidase activity but does irreversibly inactivate ribosomes. We further discuss whether the RIP classification should be broadened to include toxins achieving irreversible ribosome inactivation with similar turnovers to RIPs, but through different enzymatic mechanisms. PMID:24071927

  18. Protein specific polymeric immunomicrospheres

    NASA Technical Reports Server (NTRS)

    Rembaum, Alan (Inventor); Yen, Shiao-Ping S. (Inventor); Dreyer, William J. (Inventor)

    1980-01-01

    Small, round, bio-compatible microspheres capable of covalently bonding proteins and having a uniform diameter below about 3500 A are prepared by substantially instantaneously initiating polymerization of an aqueous emulsion containing no more than 35% total monomer including an acrylic monomer substituted with a covalently bondable group such as hydroxyl, amino or carboxyl and a minor amount of a cross-linking agent.

  19. Protein crystal growth

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Atomic force microscopy uses laser technology to reveal a defect, a double-screw dislocation, on the surface of this crystal of canavalin, a major source of dietary protein for humans and domestic animals. When a crystal grows, attachment kinetics and transport kinetics are competing for control of the molecules. As a molecule gets close to the crystal surface, it has to attach properly for the crystal to be usable. NASA has funded investigators to look at those attachment kinetics from a theoretical standpoint and an experimental standpoint. Dr. Alex McPherson of the University of California, Irvine, is one of those investigators. He uses X-ray diffraction and atomic force microscopy in his laboratory to answer some of the many questions about how protein crystals grow. Atomic force microscopy provides a means of looking at how individual molecules are added to the surface of growing protein crystals. This helps McPherson understand the kinetics of protein crystal growth. McPherson asks, How fast do crystals grow? What are the forces involved? Investigators funded by NASA have clearly shown that such factors as the level of supersaturation and the rate of growth all affect the habit [characteristic arrangement of facets] of the crystal and the defects that occur in the crystal.

  20. Monobodies and other synthetic binding proteins for expanding protein science.

    PubMed

    Sha, Fern; Salzman, Gabriel; Gupta, Ankit; Koide, Shohei

    2017-03-01

    Synthetic binding proteins are constructed using nonantibody molecular scaffolds. Over the last two decades, in-depth structural and functional analyses of synthetic binding proteins have improved combinatorial library designs and selection strategies, which have resulted in potent platforms that consistently generate binding proteins to diverse targets with affinity and specificity that rival those of antibodies. Favorable attributes of synthetic binding proteins, such as small size, freedom from disulfide bond formation and ease of making fusion proteins, have enabled their unique applications in protein science, cell biology and beyond. Here, we review recent studies that illustrate how synthetic binding proteins are powerful probes that can directly link structure and function, often leading to new mechanistic insights. We propose that synthetic proteins will become powerful standard tools in diverse areas of protein science, biotechnology and medicine.

  1. Accessory proteins for heterotrimeric G-proteins in the kidney

    PubMed Central

    Park, Frank

    2015-01-01

    Heterotrimeric G-proteins play a fundamentally important role in regulating signal transduction pathways in the kidney. Accessory proteins are being identified as direct binding partners for heterotrimeric G-protein α or βγ subunits to promote more diverse mechanisms by which G-protein signaling is controlled. In some instances, accessory proteins can modulate the signaling magnitude, localization, and duration following the activation of cell membrane-associated receptors. Alternatively, accessory proteins complexed with their G-protein α or βγ subunits can promote non-canonical models of signaling activity within the cell. In this review, we will highlight the expression profile, localization and functional importance of these newly identified accessory proteins to control the function of select G-protein subunits under normal and various disease conditions observed in the kidney. PMID:26300785

  2. Production of specific antibodies against protein A fusion proteins.

    PubMed Central

    Löwenadler, B; Nilsson, B; Abrahmsén, L; Moks, T; Ljungqvist, L; Holmgren, E; Paleus, S; Josephson, S; Philipson, L; Uhlén, M

    1986-01-01

    The gene for Staphylococcal protein A was fused to the coding sequence of bacterial beta-galactosidase, alkaline phosphatase and human insulin-like growth factor I (IGF-I). The fusion proteins, expressed in bacteria, were purified by affinity chromatography on IgG-Sepharose and antibodies were raised in rabbits. All three fusion proteins elicited specific antibodies against both the inserted protein sequences and the protein A moiety. In the case of IGF-I, the protein A moiety in the fusion protein may act as an adjuvant since native IGF-I alone is a poor immunogen. The results suggest that the protein A fusion system can be used for efficient antibody production against peptides or proteins expressed from cloned or synthetic genes. To facilitate such gene fusions a set of optimized vectors have been constructed. Images Fig. 2. Fig. 3. Fig. 4. Fig. 6. PMID:3096719

  3. Protein-protein interactions: methods for detection and analysis.

    PubMed Central

    Phizicky, E M; Fields, S

    1995-01-01

    The function and activity of a protein are often modulated by other proteins with which it interacts. This review is intended as a practical guide to the analysis of such protein-protein interactions. We discuss biochemical methods such as protein affinity chromatography, affinity blotting, coimmunoprecipitation, and cross-linking; molecular biological methods such as protein probing, the two-hybrid system, and phage display: and genetic methods such as the isolation of extragenic suppressors, synthetic mutants, and unlinked noncomplementing mutants. We next describe how binding affinities can be evaluated by techniques including protein affinity chromatography, sedimentation, gel filtration, fluorescence methods, solid-phase sampling of equilibrium solutions, and surface plasmon resonance. Finally, three examples of well-characterized domains involved in multiple protein-protein interactions are examined. The emphasis of the discussion is on variations in the approaches, concerns in evaluating the results, and advantages and disadvantages of the techniques. PMID:7708014

  4. Protein-protein interactions: scoring schemes and binding affinity.

    PubMed

    Gromiha, M Michael; Yugandhar, K; Jemimah, Sherlyn

    2017-06-01

    Protein-protein interactions mediate several cellular functions, which can be understood from the information obtained using the three-dimensional structures of protein-protein complexes and binding affinity data. This review focuses on computational aspects of predicting the best native-like complex structure and binding affinities. The first part covers the prediction of protein-protein complex structures and the advantages of conformational searching and scoring functions in protein-protein docking. The second part is devoted to various aspects of protein-protein interaction thermodynamics, such as databases for binding affinities and other thermodynamic parameters, computational methods to predict the binding affinity using either the three-dimensional structures of complexes or amino acid sequences, and change in binding affinities of the complexes upon mutations. We provide the latest developments on protein-protein docking and binding affinity studies along with a list of available computational resources for understanding protein-protein interactions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. HIV protein sequence hotspots for crosstalk with host hub proteins.

    PubMed

    Sarmady, Mahdi; Dampier, William; Tozeren, Aydin

    2011-01-01

    HIV proteins target host hub proteins for transient binding interactions. The presence of viral proteins in the infected cell results in out-competition of host proteins in their interaction with hub proteins, drastically affecting cell physiology. Functional genomics and interactome datasets can be used to quantify the sequence hotspots on the HIV proteome mediating interactions with host hub proteins. In this study, we used the HIV and human interactome databases to identify HIV targeted host hub proteins and their host binding partners (H2). We developed a high throughput computational procedure utilizing motif discovery algorithms on sets of protein sequences, including sequences of HIV and H2 proteins. We identified as HIV sequence hotspots those linear motifs that are highly conserved on HIV sequences and at the same time have a statistically enriched presence on the sequences of H2 proteins. The HIV protein motifs discovered in this study are expressed by subsets of H2 host proteins potentially outcompeted by HIV proteins. A large subset of these motifs is involved in cleavage, nuclear localization, phosphorylation, and transcription factor binding events. Many such motifs are clustered on an HIV sequence in the form of hotspots. The sequential positions of these hotspots are consistent with the curated literature on phenotype altering residue mutations, as well as with existing binding site data. The hotspot map produced in this study is the first global portrayal of HIV motifs involved in altering the host protein network at highly connected hub nodes.

  6. Pet food safety: dietary protein.

    PubMed

    Laflamme, D P

    2008-08-01

    The goal of this article was to review the evidence surrounding the risks posed by insufficient or excessive dietary protein. Dietary protein is required to provide essential amino acids and replenish protein reserves. When intake is deficient, protein turnover slows and lean body mass is gradually depleted. These changes lead to increased morbidity and mortality. Dogs can maintain nitrogen balance (typically used to define minimum requirements in adults), yet be in a protein-depleted state due to physiologic adaptations. Preservation of protein turnover and lean body mass requires about threefold more protein than nitrogen balance. The ability of excess dietary protein to induce renal pathology was studied in both dogs with chronic kidney failure and older dogs without kidney failure. Numerous studies have confirmed that protein does not adversely affect the kidneys. However, phosphorus- and protein-restricted diets are clinically beneficial in dogs with existing chronic kidney failure. Protein restriction for healthy older dogs is not only unnecessary, it can be detrimental. Protein requirements actually increase by about 50% in older dogs, while their energy requirements tend to decrease. When insufficient protein is provided, it can aggravate the age-associated loss of lean body mass and may contribute to earlier mortality. Older dogs should receive at least 25% of their calories from protein, typically provided by diets containing at least 7 g protein/100 Kcal ME.

  7. Protein Molecular Structures, Protein SubFractions, and Protein Availability Affected by Heat Processing: A Review

    SciTech Connect

    Yu,P.

    2007-01-01

    The utilization and availability of protein depended on the types of protein and their specific susceptibility to enzymatic hydrolysis (inhibitory activities) in the gastrointestine and was highly associated with protein molecular structures. Studying internal protein structure and protein subfraction profiles leaded to an understanding of the components that make up a whole protein. An understanding of the molecular structure of the whole protein was often vital to understanding its digestive behavior and nutritive value in animals. In this review, recently obtained information on protein molecular structural effects of heat processing was reviewed, in relation to protein characteristics affecting digestive behavior and nutrient utilization and availability. The emphasis of this review was on (1) using the newly advanced synchrotron technology (S-FTIR) as a novel approach to reveal protein molecular chemistry affected by heat processing within intact plant tissues; (2) revealing the effects of heat processing on the profile changes of protein subfractions associated with digestive behaviors and kinetics manipulated by heat processing; (3) prediction of the changes of protein availability and supply after heat processing, using the advanced DVE/OEB and NRC-2001 models, and (4) obtaining information on optimal processing conditions of protein as intestinal protein source to achieve target values for potential high net absorbable protein in the small intestine. The information described in this article may give better insight in the mechanisms involved and the intrinsic protein molecular structural changes occurring upon processing.

  8. Exploring NMR ensembles of calcium binding proteins: Perspectives to design inhibitors of protein-protein interactions

    PubMed Central

    2011-01-01

    Background Disrupting protein-protein interactions by small organic molecules is nowadays a promising strategy employed to block protein targets involved in different pathologies. However, structural changes occurring at the binding interfaces make difficult drug discovery processes using structure-based drug design/virtual screening approaches. Here we focused on two homologous calcium binding proteins, calmodulin and human centrin 2, involved in different cellular functions via protein-protein interactions, and known to undergo important conformational changes upon ligand binding. Results In order to find suitable protein conformations of calmodulin and centrin for further structure-based drug design/virtual screening, we performed in silico structural/energetic analysis and molecular docking of terphenyl (a mimicking alpha-helical molecule known to inhibit protein-protein interactions of calmodulin) into X-ray and NMR ensembles of calmodulin and centrin. We employed several scoring methods in order to find the best protein conformations. Our results show that docking on NMR structures of calmodulin and centrin can be very helpful to take into account conformational changes occurring at protein-protein interfaces. Conclusions NMR structures of protein-protein complexes nowadays available could efficiently be exploited for further structure-based drug design/virtual screening processes employed to design small molecule inhibitors of protein-protein interactions. PMID:21569443

  9. An evaluation of in vitro protein-protein interaction techniques: assessing contaminating background proteins.

    PubMed

    Howell, Jenika M; Winstone, Tara L; Coorssen, Jens R; Turner, Raymond J

    2006-04-01

    Determination of protein-protein interactions is an important component in assigning function and discerning the biological relevance of proteins within a broader cellular context. In vitro protein-protein interaction methodologies, including affinity chromatography, coimmunoprecipitation, and newer approaches such as protein chip arrays, hold much promise in the detection of protein interactions, particularly in well-characterized organisms with sequenced genomes. However, each of these approaches attracts certain background proteins that can thwart detection and identification of true interactors. In addition, recombinant proteins expressed in Escherichia coli are also extensively used to assess protein-protein interactions, and background proteins in these isolates can thus contaminate interaction studies. Rigorous validation of a true interaction thus requires not only that an interaction be found by alternate techniques, but more importantly that researchers be aware of and control for matrix/support dependence. Here, we evaluate these methods for proteins interacting with DmsD (an E. coli redox enzyme maturation protein chaperone), in vitro, using E. coli subcellular fractions as prey sources. We compare and contrast the various in vitro interaction methods to identify some of the background proteins and protein profiles that are inherent to each of the methods in an E. coli system.

  10. Fluorescent Protein Tracers

    PubMed Central

    Chadwick, C. S.; McEntegart, M. G.; Nairn, R. C.

    1958-01-01

    With the object of simplifying the fluorescent protein tracer technique, the following fluorochromes were examined as possible alternatives to fluorescein: aminoeosin, aminorhodamine B, 3-phenyl-7-isocyanatocumarin (Geigy), 5-β-carboxyethylaminoacridine, R 4388 (Geigy), fluolite C (I.C.I.), lissamine flavine FFS (I.C.I.), lissamine rhodamine GS (I.C.I.), and lissamine rhodamine B 200 (I.C.I.) (RB 200). With the exception of RB 200, none was suitable as a protein label largely because of unsatisfactory fluorescence intensity or colour. RB 200 has proved a successful alternative to fluorescein. The conjugation of dye to protein by a sulphonamido linkage is quick and simple and does not materially affect the physico-chemical or biological properties of the protein. The resulting conjugates are stable, have a brilliant orange fluorescence in ultraviolet light and good contrast with tissue autofluorescence. The contrast is sufficient to permit the use in microscopy of ultraviolet plus blue light with a yellow filter above the object to ensure a black background; fluorescence is greatly enhanced in this way. When injected intravenously into rats or rabbits, conjugates are distributed in the tissues and eliminated from the plasma in much the same way as proteins labelled with fluorescein or radio-active isotopes. Serum antibody conjugated with RB 200 retains immunological specificity as demonstrated by the staining of the corresponding antigen. Practical use has been made of RB 200 conjugates as plasma tracers and as specific immunological stains: they have been applied alone and in combination with fluorescein conjugates in double tracing experiments. ImagesFIG. 4FIG. 5FIG. 6FIG. 7FIG. 8FIG. 9 PMID:13610415

  11. Direct Probing of Protein-Protein Interactions

    SciTech Connect

    Noy, A; Sulchek, T A; Friddle, R W

    2005-03-10

    This project aimed to establish feasibility of using experimental techniques based on direct measurements of interaction forces on the single molecule scale to characterize equilibrium interaction potentials between individual biological molecules. Such capability will impact several research areas, ranging from rapid interaction screening capabilities to providing verifiable inputs for computational models. It should be one of the enabling technologies for modern proteomics research. This study used a combination of Monte-Carlo simulations, theoretical considerations, and direct experimental measurements to investigate two model systems that represented typical experimental situations: force-induced melting of DNA rigidly attached to the tip, and force-induced unbinding of a protein-antibody pair connected to flexible tethers. Our results establish that for both systems researchers can use force spectroscopy measurements to extract reliable information about equilibrium interaction potentials. However, the approaches necessary to extract these potentials in each case--Jarzynski reconstruction and Dynamic Force Spectroscopy--are very different. We also show how the thermodynamics and kinetics of unbinding process dictates the choice between in each case.

  12. In-cell protein NMR and protein leakage.

    PubMed

    Barnes, Christopher O; Pielak, Gary J

    2011-02-01

    In-cell nuclear magnetic resonance spectroscopy is a tool for studying proteins under physiologically relevant conditions. In some instances, however, protein signals from leaked protein are observed in the liquid surrounding the cells. Here, we examine the expression of four proteins in Escherichia coli. We describe the controls that should be used for in-cell NMR experiments and show that leakage is likely when the protein being studied exceeds ∼20% of the total cellular protein. © 2010 Wiley-Liss, Inc.

  13. High quality protein microarray using in situ protein purification

    PubMed Central

    Kwon, Keehwan; Grose, Carissa; Pieper, Rembert; Pandya, Gagan A; Fleischmann, Robert D; Peterson, Scott N

    2009-01-01

    Background In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC). This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays. Results Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG) coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST) composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents protein solubility and

  14. INTERACT: an object oriented protein-protein interaction database.

    PubMed

    Eilbeck, K; Brass, A; Paton, N; Hodgman, C

    1999-01-01

    Protein-protein interactions provide vital information concerning the function of proteins, complexes and networks. Currently there is no widely accepted repository of this interaction information. Our aim is to provide a single database with the necessary architecture to fully store, query and analyse interaction data. An object oriented database has been created which provides scientists with a resource for examining existing protein-protein interactions and inferring possible interactions from the data stored. It also provides a basis for examining networks of interacting proteins, via analysis of the data stored. The database contains over a thousand interactions. k.eilbeck@stud.man.ac.uk

  15. Bayesian Estimator of Protein-Protein Association Probabilities

    SciTech Connect

    2008-05-28

    The Bayesian Estimator of Protein-Protein Association Probabilities (BEPro3) is a software tool for estimating probabilities of protein-protein association between bait and prey protein pairs using data from multiple-bait, multiple-replicate, protein LC-MS/MS affinity isolation experiments. BEPro3 is public domain software, has been tested on Windows XP and version 10.4 or newer of the Mac OS 10.4, and is freely available. A user guide, example dataset with analysis and additional documentation are included with the BEPro3 download.

  16. Septins: Regulators of Protein Stability

    PubMed Central

    Vagin, Olga; Beenhouwer, David O.

    2016-01-01

    Septins are small GTPases that play a role in several important cellular processes. In this review, we focus on the roles of septins in protein stabilization. Septins may regulate protein stability by: (1) interacting with proteins involved in degradation pathways, (2) regulating the interaction between transmembrane proteins and cytoskeletal proteins, (3) affecting the mobility of transmembrane proteins in lipid bilayers, and (4) modulating the interaction of proteins with their adaptor or signaling proteins. In this context, we discuss the role of septins in protecting four different proteins from degradation. First we consider botulinum neurotoxin serotype A (BoNT/A) and the contribution of septins to its extraordinarily long intracellular persistence. Next, we discuss the role of septins in stabilizing the receptor tyrosine kinases EGFR and ErbB2. Finally, we consider the contribution of septins in protecting hypoxia-inducible factor 1α (HIF-1α) from degradation. PMID:28066764

  17. The quality of microparticulated protein.

    PubMed

    Erdman, J W

    1990-08-01

    The purpose of this paper is to describe the effects of microparticulation upon the quality of microparticulated protein products and to confirm that microparticulation does not result in changes in protein structure or quality different from those that occur with cooking. Two products were tested: microparticulated egg white and skim milk proteins and microparticulated whey protein concentrate. Three approaches were used to monitor for changes in amino acid and protein value: amino acid analysis, protein efficiency ratio (PER) bioassay, and both one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Evaluation of the results of these tests indicates that no significant differences were found when comparing the premix before and after microparticulation. Significant differences also did not occur when the premix was cooked using conventional methods. Collectively, the data provide strong evidence that the protein microparticulation process used to prepare microparticulated protein products (e.g., Simplesse) does not alter the quality or nutritional value of protein in the final products.

  18. Dissecting protein-protein interactions using directed evolution.

    PubMed

    Bonsor, Daniel A; Sundberg, Eric J

    2011-04-05

    Protein-protein interactions are essential for life. They are responsible for most cellular functions and when they go awry often lead to disease. Proteins are inherently complex. They are flexible macromolecules whose constituent amino acid components act in combinatorial and networked ways when they engage one another in binding interactions. It is just this complexity that allows them to conduct such a broad array of biological functions. Despite decades of intense study of the molecular basis of protein-protein interactions, key gaps in our understanding remain, hindering our ability to accurately predict the specificities and affinities of their interactions. Until recently, most protein-protein investigations have been probed experimentally at the single-amino acid level, making them, by definition, incapable of capturing the combinatorial nature of, and networked communications between, the numerous residues within and outside of the protein-protein interface. This aspect of protein-protein interactions, however, is emerging as a major driving force for protein affinity and specificity. Understanding a combinatorial process necessarily requires a combinatorial experimental tool. Much like the organisms in which they reside, proteins naturally evolve over time, through a combinatorial process of mutagenesis and selection, to functionally associate. Elucidating the process by which proteins have evolved may be one of the keys to deciphering the molecular rules that govern their interactions with one another. Directed evolution is a technique performed in the laboratory that mimics natural evolution on a tractable time scale that has been utilized widely to engineer proteins with novel capabilities, including altered binding properties. In this review, we discuss directed evolution as an emerging tool for dissecting protein-protein interactions.

  19. 14-3-3 proteins: regulators of numerous eukaryotic proteins.

    PubMed

    van Heusden, G Paul H

    2005-09-01

    14-3-3 proteins form a family of highly conserved proteins capable of binding to more than 200 different mostly phosphorylated proteins. They are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. 14-3-3 binding partners are involved in almost every cellular process and 14-3-3 proteins play a key role in these processes. 14-3-3 proteins interact with products encoded by oncogenes, with filament forming proteins involved in Alzheimer'ss disease and many other proteins related to human diseases. Disturbance of the interactions with 14-3-3 proteins may lead to diseases like cancer and the neurological Miller-Dieker disease. The molecular consequences of 14-3-3 binding are diverse and only partly understood. Binding of a protein to a 14-3-3 protein may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization or to the interaction with other proteins. Currently genome- and proteome-wide studies are contributing to a wider knowledge of this important family of proteins.

  20. Protein-protein interaction network of celiac disease.

    PubMed

    Zamanian Azodi, Mona; Peyvandi, Hassan; Rostami-Nejad, Mohammad; Safaei, Akram; Rostami, Kamran; Vafaee, Reza; Heidari, Mohammadhossein; Hosseini, Mostafa; Zali, Mohammad Reza

    2016-01-01

    The aim of this study is to investigate the Protein-Protein Interaction Network of Celiac Disease. Celiac disease (CD) is an autoimmune disease with susceptibility of individuals to gluten of wheat, rye and barley. Understanding the molecular mechanisms and involved pathway may lead to the development of drug target discovery. The protein interaction network is one of the supportive fields to discover the pathogenesis biomarkers for celiac disease. In the present study, we collected the articles that focused on the proteomic data in celiac disease. According to the gene expression investigations of these articles, 31 candidate proteins were selected for this study. The networks of related differentially expressed protein were explored using Cytoscape 3.3 and the PPI analysis methods such as MCODE and ClueGO. According to the network analysis Ubiquitin C, Heat shock protein 90kDa alpha (cytosolic and Grp94); class A, B and 1 member, Heat shock 70kDa protein, and protein 5 (glucose-regulated protein, 78kDa), T-complex, Chaperon in containing TCP1; subunit 7 (beta) and subunit 4 (delta) and subunit 2 (beta), have been introduced as hub-bottlnecks proteins. HSP90AA1, MKKS, EZR, HSPA14, APOB and CAD have been determined as seed proteins. Chaperons have a bold presentation in curtail area in network therefore these key proteins beside the other hub-bottlneck proteins may be a suitable candidates biomarker panel for diagnosis, prognosis and treatment processes in celiac disease.

  1. Regulation of protein turnover by heat shock proteins.

    PubMed

    Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

    2014-12-01

    Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin-proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Quantification of the Influence of Protein-Protein Interactions on Adsorbed Protein Structure and Bioactivity

    PubMed Central

    Wei, Yang; Thyparambil, Aby A.; Latour, Robert A.

    2013-01-01

    While protein-surface interactions have been widely studied, relatively little is understood at this time regarding how protein-surface interaction effects are influenced by protein-protein interactions and how these effects combine with the internal stability of a protein to influence its adsorbed-state structure and bioactivity. The objectives of this study were to develop a method to study these combined effects under widely varying protein-protein interaction conditions using hen egg-white lysozyme (HEWL) adsorbed on silica glass, poly(methyl methacrylate), and polyethylene as our model systems. In order to vary protein-protein interaction effects over a wide range, HEWL was first adsorbed to each surface type under widely varying protein solution concentrations for 2 h to saturate the surface, followed by immersion in pure buffer solution for 15 h to equilibrate the adsorbed protein layers in the absence of additionally adsorbing protein. Periodic measurements were made at selected time points of the areal density of the adsorbed protein layer as an indicator of the level of protein-protein interaction effects within the layer, and these values were then correlated with measurements of the adsorbed protein’s secondary structure and bioactivity. The results from these studies indicate that protein-protein interaction effects help stabilize the structure of HEWL adsorbed on silica glass, have little influence on the structural behavior of HEWL on HDPE, and actually serve to destabilize HEWL’s structure on PMMA. The bioactivity of HEWL on silica glass and HDPE was found to decrease in direct proportion to the degree of adsorption-induce protein unfolding. A direct correlation between bioactivity and the conformational state of adsorbed HEWL was less apparent on PMMA, thus suggesting that other factors influenced HEWL’s bioactivity on this surface, such as the accessibility of HEWL’s bioactive site being blocked by neighboring proteins or the surface

  3. Biological Applications of Protein Splicing

    PubMed Central

    Vila-Perelló, Miquel; Muir, Tom W.

    2010-01-01

    Protein splicing is a naturally-occurring process in which a protein editor, called an intein, performs a molecular disappearing act by cutting itself out of a host protein in a traceless manner. In the two decades since its discovery, protein splicing has been harnessed for the development of several protein-engineering methods. Collectively, these technologies help bridge the fields of chemistry and biology, allowing hitherto impossible manipulations of protein covalent structure. These tools and their application are the subject of this Primer. PMID:20946979

  4. Misfolded Proteins and Retinal Dystrophies

    PubMed Central

    Lin, Jonathan H.; LaVail, Matthew M.

    2010-01-01

    Many mutations associated with retinal degeneration lead to the production of misfolded proteins by cells of the retina. Emerging evidence suggests that these abnormal proteins cause cell death by activating the Unfolded Protein Response, a set of conserved intracellular signaling pathways that detect protein misfolding within the endoplasmic reticulum and control protective and proapoptotic signal transduction pathways. Here, we review the misfolded proteins associated with select types of retinitis pigmentosa, Stargadt-like macular degeneration, and Doyne Honeycomb Retinal Dystrophy and discuss the role that endoplasmic reticulum stress and UPR signaling play in their pathogenesis. Last, we review new therapies for these diseases based on preventing protein misfolding in the retina. PMID:20238009

  5. Protein misfolding disorders and macroautophagy

    PubMed Central

    Menzies, Fiona M; Moreau, Kevin; Rubinsztein, David C

    2011-01-01

    A large group of diseases, termed protein misfolding disorders, share the common feature of the accumulation of misfolded proteins. The possibility of a common mechanism underlying either the pathogenesis or therapy for these diseases is appealing. Thus, there is great interest in the role of protein degradation via autophagy in such conditions where the protein is found in the cytoplasm. Here we review the growing evidence supporting a role for autophagic dysregulation as a contributing factor to protein accumulation and cellular toxicity in certain protein misfolding disorders and discuss the available evidence that upregulation of autophagy may be a valuable therapeutic strategy. PMID:21087849

  6. Redox control of protein degradation

    PubMed Central

    Pajares, Marta; Jiménez-Moreno, Natalia; Dias, Irundika H.K.; Debelec, Bilge; Vucetic, Milica; Fladmark, Kari E.; Basaga, Huveyda; Ribaric, Samo; Milisav, Irina; Cuadrado, Antonio

    2015-01-01

    Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies. PMID:26381917

  7. A magnetic protein biocompass

    NASA Astrophysics Data System (ADS)

    Qin, Siying; Yin, Hang; Yang, Celi; Dou, Yunfeng; Liu, Zhongmin; Zhang, Peng; Yu, He; Huang, Yulong; Feng, Jing; Hao, Junfeng; Hao, Jia; Deng, Lizong; Yan, Xiyun; Dong, Xiaoli; Zhao, Zhongxian; Jiang, Taijiao; Wang, Hong-Wei; Luo, Shu-Jin; Xie, Can

    2016-02-01

    The notion that animals can detect the Earth’s magnetic field was once ridiculed, but is now well established. Yet the biological nature of such magnetosensing phenomenon remains unknown. Here, we report a putative magnetic receptor (Drosophila CG8198, here named MagR) and a multimeric magnetosensing rod-like protein complex, identified by theoretical postulation and genome-wide screening, and validated with cellular, biochemical, structural and biophysical methods. The magnetosensing complex consists of the identified putative magnetoreceptor and known magnetoreception-related photoreceptor cryptochromes (Cry), has the attributes of both Cry- and iron-based systems, and exhibits spontaneous alignment in magnetic fields, including that of the Earth. Such a protein complex may form the basis of magnetoreception in animals, and may lead to applications across multiple fields.

  8. A magnetic protein biocompass.

    PubMed

    Qin, Siying; Yin, Hang; Yang, Celi; Dou, Yunfeng; Liu, Zhongmin; Zhang, Peng; Yu, He; Huang, Yulong; Feng, Jing; Hao, Junfeng; Hao, Jia; Deng, Lizong; Yan, Xiyun; Dong, Xiaoli; Zhao, Zhongxian; Jiang, Taijiao; Wang, Hong-Wei; Luo, Shu-Jin; Xie, Can

    2016-02-01

    The notion that animals can detect the Earth's magnetic field was once ridiculed, but is now well established. Yet the biological nature of such magnetosensing phenomenon remains unknown. Here, we report a putative magnetic receptor (Drosophila CG8198, here named MagR) and a multimeric magnetosensing rod-like protein complex, identified by theoretical postulation and genome-wide screening, and validated with cellular, biochemical, structural and biophysical methods. The magnetosensing complex consists of the identified putative magnetoreceptor and known magnetoreception-related photoreceptor cryptochromes (Cry), has the attributes of both Cry- and iron-based systems, and exhibits spontaneous alignment in magnetic fields, including that of the Earth. Such a protein complex may form the basis of magnetoreception in animals, and may lead to applications across multiple fields.

  9. Collaborative protein filaments.

    PubMed

    Ghosal, Debnath; Löwe, Jan

    2015-09-14

    It is now well established that prokaryotic cells assemble diverse proteins into dynamic cytoskeletal filaments that perform essential cellular functions. Although most of the filaments assemble on their own to form higher order structures, growing evidence suggests that there are a number of prokaryotic proteins that polymerise only in the presence of a matrix such as DNA, lipid membrane or even another filament. Matrix-assisted filament systems are frequently nucleotide dependent and cytomotive but rarely considered as part of the bacterial cytoskeleton. Here, we categorise this family of filament-forming systems as collaborative filaments and introduce a simple nomenclature. Collaborative filaments are frequent in both eukaryotes and prokaryotes and are involved in vital cellular processes including chromosome segregation, DNA repair and maintenance, gene silencing and cytokinesis to mention a few. In this review, we highlight common principles underlying collaborative filaments and correlate these with known functions.

  10. Electron flow through proteins

    NASA Astrophysics Data System (ADS)

    Gray, Harry B.; Winkler, Jay R.

    2009-11-01

    Electron transfers in photosynthesis and respiration commonly occur between metal-containing cofactors that are separated by large molecular distances. Employing laser flash-quench triggering methods, we have shown that 20-Å, coupling-limited Fe II-Ru III and Cu I-Ru III electron tunneling in Ru-modified cytochromes and blue copper proteins can occur on the microsecond timescale both in solutions and crystals. Redox equivalents can be transferred even longer distances by multistep tunneling, often called hopping, through intervening amino acid side chains. Our work has established that 20-Å hole hopping through an intervening tryptophan is two orders of magnitude faster than single-step electron tunneling in a Re-modified blue copper protein.

  11. Electron Flow through Proteins

    PubMed Central

    Gray, Harry B.; Winkler, Jay R.

    2009-01-01

    Electron transfers in photosynthesis and respiration commonly occur between metal-containing cofactors that are separated by large molecular distances. Employing laser flash-quench triggering methods, we have shown that 20-Å, coupling-limited FeII to RuIII and CuI to RuIII electron tunneling in Ru-modified cytochromes and blue copper proteins can occur on the microsecond timescale both in solutions and crystals. Redox equivalents can be transferred even longer distances by multistep tunneling, often called hopping, through intervening amino acid side chains. Our work has established that 20-Å hole hopping through an intervening tryptophan is two orders of magnitude faster than single-step electron tunneling in a Re-modified blue copper protein. PMID:20161522

  12. Protein engineering of subtilisin.

    PubMed

    Bryan, P N

    2000-12-29

    The serine protease subtilisin is an important industrial enzyme as well as a model for understanding the enormous rate enhancements affected by enzymes. For these reasons along with the timely cloning of the gene, ease of expression and purification and availability of atomic resolution structures, subtilisin became a model system for protein engineering studies in the 1980s. Fifteen years later, mutations in well over 50% of the 275 amino acids of subtilisin have been reported in the scientific literature. Most subtilisin engineering has involved catalytic amino acids, substrate binding regions and stabilizing mutations. Stability has been the property of subtilisin which has been most amenable to enhancement, yet perhaps least understood. This review will give a brief overview of the subtilisin engineering field, critically review what has been learned about subtilisin stability from protein engineering experiments and conclude with some speculation about the prospects for future subtilisin engineering.

  13. Statistical analysis and prediction of protein-protein interfaces.

    PubMed

    Bordner, Andrew J; Abagyan, Ruben

    2005-08-15

    Predicting protein-protein interfaces from a three-dimensional structure is a key task of computational structural proteomics. In contrast to geometrically distinct small molecule binding sites, protein-protein interface are notoriously difficult to predict. We generated a large nonredundant data set of 1494 true protein-protein interfaces using biological symmetry annotation where necessary. The data set was carefully analyzed and a Support Vector Machine was trained on a combination of a new robust evolutionary conservation signal with the local surface properties to predict protein-protein interfaces. Fivefold cross validation verifies the high sensitivity and selectivity of the model. As much as 97% of the predicted patches had an overlap with the true interface patch while only 22% of the surface residues were included in an average predicted patch. The model allowed the identification of potential new interfaces and the correction of mislabeled oligomeric states.

  14. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion.

    PubMed

    Dong, Jinlan; Bruening, Merlin L

    2015-01-01

    This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO₂ nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

  15. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

    NASA Astrophysics Data System (ADS)

    Dong, Jinlan; Bruening, Merlin L.

    2015-07-01

    This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

  16. Mx proteins: antiviral proteins by chance or by necessity?

    PubMed

    Arnheiter, H; Meier, E

    1990-10-01

    The interferon-inducible Mx1 protein is responsible for inborn resistance of mice to influenza. It is now recognized that this protein is a member of a family of interferon-inducible, putative GTP-binding proteins found in many organisms. Thus, these proteins, called the Mx proteins, are found in species that are naturally infected with influenza virus, and also in species that are not. Some Mx proteins display a broader antiviral profile than the one observed for Mx1 in mice. Others, however, may not be antiviral. Two recently discovered GTP-binding proteins, Vps1p in yeast and dynamin in rat, are also related to Mx1. These proteins are synthesized constitutively and serve basic cellular functions.

  17. Epigenetic down-regulated DDX10 promotes cell proliferation through Akt/NF-κB pathway in ovarian cancer

    SciTech Connect

    Gai, Muhuizi; Bo, Qifang; Qi, Lixia

    2016-01-22

    Ovarian cancer contributes to the majority of ovarian cancer, while the molecular mechanisms remain elusive. Recently, some DEAD box protein 1 has been reported play a tumor suppressor role in ovarian cancer progression. However, the functions of DEAD box protein (DDX) members in ovarian cancer development remain largely unknown. In current study, we retrieved GEO databases and surprisingly found that DDX10 is significantly down-regulated in ovarian cancer tissues compared with normal ovary. These findings suggest that DDX10 might also play a suppressive role in ovarian cancer. We then validated the down-regulated expression pattern of DDX10 in fresh ovarian cancer tissues. Furthermore, both loss- and gain-functions assays reveal that the down-regulated DDX10 could promote ovarian cancer proliferation in vitro and the xenograft subcutaneous tumor formation assays confirmed these findings in vivo. In addition, we found that DDX10 is epigenetic silenced by miR-155-5p in ovarian cancer. Moreover, we further preliminary illustrated that down-regulated DDX10 promotes ovarian cancer cell proliferation through Akt/NF-κB pathway. Taken together, in current study, we found a novel tumor suppressor, DDX10, is epigenetic silenced by miR-155-5p in ovarian cancer, and the down-regulated expression pattern of DDX10 promotes ovarian cancer proliferation through Akt/NF-κB pathway. Our findings shed the light that DDX families might be a novel for ovarian cancer treatment. - Highlights: • A novel DEAD box protein, DDX10 is significantly down-regulated in ovarian cancer tissues. • Down-regulated DDX10 promotes ovarian cancer cell proliferation and growth both in vitro and in vivo. • miR-155-5p is highly expressed in ovarian cancer tissues and epigenetically targets DDX10. • DDX10 and miR-155-5p regulates Akt/p65 axis in ovarian cancer cells.

  18. Dissecting Amelogenin Protein Nanospheres

    PubMed Central

    Bromley, Keith M.; Kiss, Andrew S.; Lokappa, Sowmya Bekshe; Lakshminarayanan, Rajamani; Fan, Daming; Ndao, Moise; Evans, John Spencer; Moradian-Oldak, Janet

    2011-01-01

    Amelogenin self-assembles to form an extracellular protein matrix, which serves as a template for the continuously growing enamel apatite crystals. To gain further insight into the molecular mechanism of amelogenin nanosphere formation, we manipulated the interactions between amelogenin monomers by altering pH, temperature, and protein concentration to create isolated metastable amelogenin oligomers. Recombinant porcine amelogenins (rP172 and rP148) and three different mutants containing only a single tryptophan (Trp161, Trp45, and Trp25) were used. Dynamic light scattering and fluorescence studies demonstrated that oligomers were metastable and in constant equilibrium with monomers. Stable oligomers with an average hydrodynamic radius (RH) of 7.5 nm were observed at pH 5.5 between 4 and 10 mg·ml−1. We did not find any evidence of a significant increase in folding upon self-association of the monomers into oligomers, indicating that they are disordered. Fluorescence experiments with single tryptophan amelogenins revealed that upon oligomerization the C terminus of amelogenin (around residue Trp161) is exposed at the surface of the oligomers, whereas the N-terminal region around Trp25 and Trp45 is involved in protein-protein interaction. The truncated rP148 formed similar but smaller oligomers, suggesting that the C terminus is not critical for amelogenin oligomerization. We propose a model for nanosphere formation via oligomers, and we predict that nanospheres will break up to form oligomers in mildly acidic environments via histidine protonation. We further suggest that oligomeric structures might be functional components during maturation of enamel apatite. PMID:21840988

  19. Autotransporter protein secretion.

    PubMed

    Tame, Jeremy R H

    2011-12-01

    Autotransporter proteins are a large family of virulence factors secreted from Gram-negative bacteria by a unique mechanism. First described in the 1980s, these proteins have a C-terminal region that folds into a β-barrel in the bacterial outer membrane. The so-called passenger domain attached to this barrel projects away from the cell surface and may be liberated from the cell by self-cleavage or surface proteases. Although the majority of passenger domains have a similar β-helical structure, they carry a variety of sub-domains, allowing them to carry out widely differing functions related to pathogenesis. Considerable biochemical and structural characterisation of the barrel domain has shown that 'autotransporters' in fact require a conserved and essential protein complex in the outer membrane for correct folding. Although the globular domains of this complex projecting into the periplasmic space have also been structurally characterised, the overall secretion pathway of the autotransporters remains highly puzzling. It was presumed for many years that the passenger domain passed through the centre of the barrel domain to reach the cell surface, driven at least in part by folding. This picture is complicated by conflicting data, and there is currently little hard information on the true nature of the secretion intermediates. As well as their medical importance therefore, autotransporters are proving to be an excellent system to study the folding and membrane insertion of outer membrane proteins in general. This review focuses on structural aspects of autotransporters; their many functions in pathogenesis are beyond its scope.

  20. Protein Crystal Isocitrate Lyase

    NASA Technical Reports Server (NTRS)

    1998-01-01

    The comparison of protein crystal, Isocitrate Lyase earth-grown (left) and space-grown (right). This is a target enzyme for fungicides. A better understanding of this enzyme should lead to the discovery of more potent fungicides to treat serious crop diseases such as rice blast; it regulates the flow of metabolic intermediates required for cell growth. Principal Investigator is Larry DeLucas.