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Sample records for gras proteins form

  1. Nanocarriers from GRAS Zein Proteins to Encapsulate Hydrophobic Actives.

    PubMed

    Weissmueller, Nikolas T; Lu, Hoang D; Hurley, Amanda; Prud'homme, Robert K

    2016-11-14

    One factor limiting the expansion of nanomedicines has been the high cost of the materials and processes required for their production. We present a continuous, scalable, low cost nanoencapsulation process, Flash Nanoprecipitation (FNP) that enables the production of nanocarriers (NCs) with a narrow size distribution using zein corn proteins. Zein is a low cost, GRAS protein (having the FDA status of "Generally Regarded as Safe") currently used in food applications, which acts as an effective encapsulant for hydrophobic compounds using FNP. The four-stream FNP configuration allows the encapsulation of very hydrophobic compounds in a way that is not possible with previous precipitation processes. We present the encapsulation of several model active compounds with as high as 45 wt % drug loading with respect to zein concentration into ∼100 nm nanocarriers. Three examples are presented: (1) the pro-drug antioxidant, vitamin E-acetate, (2) an anticholera quorum-sensing modulator CAI-1 ((S)-3-hydroxytridecan-4-one; CAI-1 that reduces Vibrio cholerae virulence by modulating cellular communication), and (3) hydrophobic fluorescent dyes with a range of hydrophobicities. The specific interaction between zein and the milk protein, sodium caseinate, provides stabilization of the NCs in PBS, LB medium, and in pH 2 solutions. The stability and size changes in the three media provide information on the mechanism of assembly of the zein/active/casein NC.

  2. Bacterial GRAS domain proteins throw new light on gibberellic acid response mechanisms

    PubMed Central

    Zhang, Dapeng; Iyer, Lakshminarayan M.; Aravind, L.

    2012-01-01

    Summary: Gibberellic acids (GAs) are key plant hormones, regulating various aspects of growth and development, which have been at the center of the ‘green revolution’. GRAS family proteins, the primary players in GA signaling pathways, remain poorly understood. Using sequence-profile searches, structural comparisons and phylogenetic analysis, we establish that the GRAS family first emerged in bacteria and belongs to the Rossmann fold methyltransferase superfamily. All bacterial and a subset of plant GRAS proteins are likely to function as small-molecule methylases. The remaining plant versions have lost one or more AdoMet (SAM)-binding residues while preserving their substrate-binding residues. We predict that GRAS proteins might either modify or bind small molecules such as GAs or their derivatives. Contact: aravind@ncbi.nlm.nih.gov Supplementary Information: Supplementary Material for this article is available at Bioinformatics online. PMID:22829623

  3. Structural and Functional Analysis of the GRAS Gene Family in Grapevine Indicates a Role of GRAS Proteins in the Control of Development and Stress Responses

    PubMed Central

    Grimplet, Jérôme; Agudelo-Romero, Patricia; Teixeira, Rita T.; Martinez-Zapater, Jose M.; Fortes, Ana M.

    2016-01-01

    GRAS transcription factors are involved in many processes of plant growth and development (e.g., axillary shoot meristem formation, root radial patterning, nodule morphogenesis, arbuscular development) as well as in plant disease resistance and abiotic stress responses. However, little information is available concerning this gene family in grapevine (Vitis vinifera L.), an economically important woody crop. We performed a model curation of GRAS genes identified in the latest genome annotation leading to the identification of 52 genes. Gene models were improved and three new genes were identified that could be grapevine- or woody-plant specific. Phylogenetic analysis showed that GRAS genes could be classified into 13 groups that mapped on the 19 V. vinifera chromosomes. Five new subfamilies, previously not characterized in other species, were identified. Multiple sequence alignment showed typical GRAS domain in the proteins and new motifs were also described. As observed in other species, both segmental and tandem duplications contributed significantly to the expansion and evolution of the GRAS gene family in grapevine. Expression patterns across a variety of tissues and upon abiotic and biotic conditions revealed possible divergent functions of GRAS genes in grapevine development and stress responses. By comparing the information available for tomato and grapevine GRAS genes, we identified candidate genes that might constitute conserved transcriptional regulators of both climacteric and non-climacteric fruit ripening. Altogether this study provides valuable information and robust candidate genes for future functional analysis aiming at improving the quality of fleshy fruits. PMID:27065316

  4. Phylogenetic analysis of GRAS proteins from moss, lycophyte and vascular plant lineages reveals that GRAS genes arose and underwent substantial diversification in the ancestral lineage common to bryophytes and vascular plants

    PubMed Central

    2011-01-01

    GRAS genes are a large family of streptophyte specific transcription factors that function in a diverse set of physiological and developmental processes. GRAS proteins of the HAIRY MERISTEM (HAM) sub-family are required for maintenance of shoot and root indeterminacy. The transcriptional targets of HAM proteins and the signaling inputs regulating HAM activity are completely unknown. Understanding the relationship of HAM proteins to other members of the GRAS family may inform hypotheses relating to cellular level HAM functions. Here I report a phylogenetic analysis of GRAS proteins employing the complete set of known and probable GRAS proteins from the sequenced genomes of the flowering plants Arabidopsis and Rice, the lycophyte Selaginella moellendorffii, and the bryophyte Physcomitrella patens. HAM proteins are most closely related to DELLA proteins, key components of gibberellin perception. However, GRAS proteins diversified into a minimum of 12 discreet monophyletic lineages, including the HAM and DELLA subfamilies, prior to divergence of the moss and flowering plant lineages. Substantial diversification of GRAS proteins at so early a point in land plant evolution suggests that relative relatedness among GRAS protein sub-families may not substantially reflect shared protein function. PMID:21543899

  5. Phylogenetic analysis of GRAS proteins from moss, lycophyte and vascular plant lineages reveals that GRAS genes arose and underwent substantial diversification in the ancestral lineage common to bryophytes and vascular plants.

    PubMed

    Engstrom, Eric M

    2011-06-01

    GRAS genes are a large family of streptophyte specific transcription factors that function in a diverse set of physiological and developmental processes. GRAS proteins of the HAIRY MERISTEM (HAM) sub-family are required for maintenance of shoot and root indeterminacy. The transcriptional targets of HAM proteins and the signaling inputs regulating HAM activity are completely unknown. Understanding the relationship of HAM proteins to other members of the GRAS family may inform hypotheses relating to cellular level HAM functions. I here report a phylogenetic analysis of GRAS proteins employing the complete set of known and probable GRAS proteins from the sequenced genomes of the flowering plants Arabidopsis and Rice, the lycophyte Selaginella moellendorffii, and the bryophyte Physcomitrella patens. HAM proteins are most closely related to DELLA proteins, key components of gibberellin perception. However, GRAS proteins diversified into a minimum of twelve discreet monophyletic lineages, including the HAM and DELLA subfamilies, prior to divergence of the moss and flowering plant lineages. Substantial diversification of GRAS proteins at so early a point in land plant evolution suggests that relative relatedness sequence homology among GRAS proteins sub-families may not substantially reflect shared protein function. 

  6. Protein matrix involved in the lipid retention of foie gras during cooking: a multimodal hyperspectral imaging study.

    PubMed

    Théron, Laëtitia; Vénien, Annie; Jamme, Frédéric; Fernandez, Xavier; Peyrin, Frédéric; Molette, Caroline; Dumas, Paul; Réfrégiers, Matthieu; Astruc, Thierry

    2014-06-25

    Denaturation of the protein matrix during heat treatment of duck foie gras was studied in relationship to the amount of fat loss during cooking. A low fat loss group was compared with a high fat loss group by histochemistry, FT-IR, and synchrotron UV microspectroscopy combination to characterize their protein matrix at different scales. After cooking, the high fat loss group showed higher densification of its matrix, higher ultraviolet tyrosine autofluorescence, and an infrared shift of the amide I band. These results revealed a higher level of protein denaturation and aggregation during cooking in high fat loss than in low fat loss foie gras. In addition, the fluorescence and infrared responses of the raw tissue revealed differences according to the level of fat losses after cooking. These findings highlight the importance of the supramolecular state of the protein matrix in determining the fat loss of foie gras.

  7. Amyloidogenic potential of foie gras.

    PubMed

    Solomon, Alan; Richey, Tina; Murphy, Charles L; Weiss, Deborah T; Wall, Jonathan S; Westermark, Gunilla T; Westermark, Per

    2007-06-26

    The human cerebral and systemic amyloidoses and prion-associated spongiform encephalopathies are acquired or inherited protein folding disorders in which normally soluble proteins or peptides are converted into fibrillar aggregates. This is a nucleation-dependent process that can be initiated or accelerated by fibril seeds formed from homologous or heterologous amyloidogenic precursors that serve as an amyloid enhancing factor (AEF) and has pathogenic significance in that disease may be transmitted by oral ingestion or parenteral administration of these conformationally altered components. Except for infected brain tissue, specific dietary sources of AEF have not been identified. Here we report that commercially available duck- or goose-derived foie gras contains birefringent congophilic fibrillar material composed of serum amyloid A-related protein that acted as a potent AEF in a transgenic murine model of secondary (amyloid A protein) amyloidosis. When such mice were injected with or fed amyloid extracted from foie gras, the animals developed extensive systemic pathological deposits. These experimental data provide evidence that an amyloid-containing food product hastened the development of amyloid protein A amyloidosis in a susceptible population. On this basis, we posit that this and perhaps other forms of amyloidosis may be transmissible, akin to the infectious nature of prion-related illnesses.

  8. Mardi Gras Magic.

    ERIC Educational Resources Information Center

    Ladson, Henrietta O.

    2002-01-01

    Describes a kindergarten art activity in which students created masks in celebration of Mardi Gras (New Orleans, Louisiana). Explains that the students learned about Mardi Gras and saw a coloring demonstration to prepare them for the project. Discusses in detail the process of creating the masks. (CMK)

  9. Genome-wide analysis of the GRAS gene family in Chinese cabbage (Brassica rapa ssp. pekinensis).

    PubMed

    Song, Xiao-Ming; Liu, Tong-Kun; Duan, Wei-Ke; Ma, Qing-Hua; Ren, Jun; Wang, Zhen; Li, Ying; Hou, Xi-Lin

    2014-01-01

    The GRAS gene family is one of the most important families of transcriptional regulators. In this study, 48 GRAS genes are identified from Chinese cabbage, and they are classified into eight groups according to the classification of Arabidopsis. The characterization, classification, gene structure and phylogenetic construction of GRAS proteins are performed. Distribution mapping shows that GRAS proteins are nonrandomly localized in 10 chromosomes. Fifty-five orthologous gene pairs are shared by Chinese cabbage and Arabidopsis, and interaction networks of these orthologous genes are constructed. The expansion of GRAS genes in Chinese cabbage results from genome triplication. Among the 17 species examined, 14 higher plants carry the GRAS genes, whereas two lower plants and one fungi species do not. Furthermore, the expression patterns of GRAS genes exhibit differences in three tissues based on RNA-seq data. Taken together, this comprehensive analysis will provide rich resources for studying GRAS protein functions in Chinese cabbage.

  10. More than Mardi Gras

    ERIC Educational Resources Information Center

    Wilson, Kathy

    2012-01-01

    Telling art students to do anything they want can be dangerous. It's not something teachers often do, but this is a project where anything goes. In this article, the author describes how her students created a Mardi Gras type mask, then incorporated it into a mixed-media composition.

  11. Genome-wide identification and characterization of GRAS transcription factors in sacred lotus (Nelumbo nucifera)

    PubMed Central

    Zhou, Ying; Zhou, Yu; Yang, Jie

    2016-01-01

    The GRAS gene family is one of the most important plant-specific gene families, which encodes transcriptional regulators and plays an essential role in plant development and physiological processes. The GRAS gene family has been well characterized in many higher plants such as Arabidopsis, rice, Chinese cabbage, tomato and tobacco. In this study, we identified 38 GRAS genes in sacred lotus (Nelumbo nucifera), analyzed their physical and chemical characteristics and performed phylogenetic analysis using the GRAS genes from eight representative plant species to show the evolution of GRAS genes in Planta. In addition, the gene structures and motifs of the sacred lotus GRAS proteins were characterized in detail. Comparative analysis identified 42 orthologous and 9 co-orthologous gene pairs between sacred lotus and Arabidopsis, and 35 orthologous and 22 co-orthologous gene pairs between sacred lotus and rice. Based on publically available RNA-seq data generated from leaf, petiole, rhizome and root, we found that most of the sacred lotus GRAS genes exhibited a tissue-specific expression pattern. Eight of the ten PAT1-clade GRAS genes, particularly NnuGRAS-05, NnuGRAS-10 and NnuGRAS-25, were preferentially expressed in rhizome and root. In summary, this is the first in silico analysis of the GRAS gene family in sacred lotus, which will provide valuable information for further molecular and biological analyses of this important gene family. PMID:27635351

  12. Evolutionary Analyses of GRAS Transcription Factors in Angiosperms

    PubMed Central

    Cenci, Alberto; Rouard, Mathieu

    2017-01-01

    GRAS transcription factors (TFs) play critical roles in plant growth and development such as gibberellin and mycorrhizal signaling. Proteins belonging to this gene family contain a typical GRAS domain in the C-terminal sequence, whereas the N-terminal region is highly variable. Although, GRAS genes have been characterized in a number of plant species, their classification is still not completely resolved. Based on a panel of eight representative species of angiosperms, we identified 29 orthologous groups or orthogroups (OGs) for the GRAS gene family, suggesting that at least 29 ancestor genes were present in the angiosperm lineage before the “Amborella” evolutionary split. Interestingly, some taxonomic groups were missing members of one or more OGs. The gene number expansion usually observed in transcription factors was not observed in GRAS while the genome triplication ancestral to the eudicots (γ hexaploidization event) was detectable in a limited number of GRAS orthogroups. We also found conserved OG-specific motifs in the variable N-terminal region. Finally, we could regroup OGs in 17 subfamilies for which names were homogenized based on a literature review and described 5 new subfamilies (DLT, RAD1, RAM1, SCLA, and SCLB). This study establishes a consistent framework for the classification of GRAS members in angiosperm species, and thereby a tool to correctly establish the orthologous relationships of GRAS genes in most of the food crops in order to facilitate any subsequent functional analyses in the GRAS gene family. The multi-fasta file containing all the sequences used in our study could be used as database to perform diagnostic BLASTp to classify GRAS genes from other non-model species. PMID:28303145

  13. Evolutionary Analyses of GRAS Transcription Factors in Angiosperms.

    PubMed

    Cenci, Alberto; Rouard, Mathieu

    2017-01-01

    GRAS transcription factors (TFs) play critical roles in plant growth and development such as gibberellin and mycorrhizal signaling. Proteins belonging to this gene family contain a typical GRAS domain in the C-terminal sequence, whereas the N-terminal region is highly variable. Although, GRAS genes have been characterized in a number of plant species, their classification is still not completely resolved. Based on a panel of eight representative species of angiosperms, we identified 29 orthologous groups or orthogroups (OGs) for the GRAS gene family, suggesting that at least 29 ancestor genes were present in the angiosperm lineage before the "Amborella" evolutionary split. Interestingly, some taxonomic groups were missing members of one or more OGs. The gene number expansion usually observed in transcription factors was not observed in GRAS while the genome triplication ancestral to the eudicots (γ hexaploidization event) was detectable in a limited number of GRAS orthogroups. We also found conserved OG-specific motifs in the variable N-terminal region. Finally, we could regroup OGs in 17 subfamilies for which names were homogenized based on a literature review and described 5 new subfamilies (DLT, RAD1, RAM1, SCLA, and SCLB). This study establishes a consistent framework for the classification of GRAS members in angiosperm species, and thereby a tool to correctly establish the orthologous relationships of GRAS genes in most of the food crops in order to facilitate any subsequent functional analyses in the GRAS gene family. The multi-fasta file containing all the sequences used in our study could be used as database to perform diagnostic BLASTp to classify GRAS genes from other non-model species.

  14. Genome-wide analysis of the GRAS gene family in physic nut (Jatropha curcas L.).

    PubMed

    Wu, Z Y; Wu, P Z; Chen, Y P; Li, M R; Wu, G J; Jiang, H W

    2015-12-29

    GRAS proteins play vital roles in plant growth and development. Physic nut (Jatropha curcas L.) was found to have a total of 48 GRAS family members (JcGRAS), 15 more than those found in Arabidopsis. The JcGRAS genes were divided into 12 subfamilies or 15 ancient monophyletic lineages based on the phylogenetic analysis of GRAS proteins from both flowering and lower plants. The functions of GRAS genes in 9 subfamilies have been reported previously for several plants, while the genes in the remaining 3 subfamilies were of unknown function; we named the latter families U1 to U3. No member of U3 subfamily is present in Arabidopsis and Poaceae species according to public genome sequence data. In comparison with the number of GRAS genes in Arabidopsis, more were detected in physic nut, resulting from the retention of many ancient GRAS subfamilies and the formation of tandem repeats during evolution. No evidence of recent duplication among JcGRAS genes was observed in physic nut. Based on digital gene expression data, 21 of the 48 genes exhibited differential expression in four tissues analyzed. Two members of subfamily U3 were expressed only in buds and flowers, implying that they may play specific roles. Our results provide valuable resources for future studies on the functions of GRAS proteins in physic nut.

  15. Studying how protein crystals form

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Watching molecules of the iron-storing protein apoferritin come together to form a nucleus reveals some interesting behavior. In this series of images, researchers observed clusters of four molecules at the corners of a diamond shape (top). As more molecules attach to the cluster, they arrange themselves into rods (second from top), and a raft-like configuration of molecules forms the critical nucleus (third from top), suggesting that crystal growth is much slower than it could be were the molecules arranged in a more compact formation. In the final image, a crystallite consisting of three layers containing approximately 60 to 70 molecules each is formed. Atomic force microscopy made visualizing the process of nucleation possible for the first time. The principal investigator is Peter Vekilov, of the University of Alabama in Huntsville. Vekilov's team at UAH studies protein solutions as they change phases from liquids to crystalline solids. They want to know if the molecules in the solution interact with one another, and if so, how, from the perspectives of thermodynamics and kinetics. They want to understand which forces -- electrical, electrostatic, hydrodynamic, or other kinds of forces -- are responsible for the interactions. They also study nucleation, the begirning stage of crystallization. This process is important to understand because it sets the stage for crystal growth in all kinds of solutions and liquid melts that are important in such diverse fields as agriculture, medicine, and the fabrication of metal components. Nucleation can determine the rate of crystal growth, the number of crystals that will be formed, and the quality and size of the crystals.

  16. Network of GRAS Transcription Factors Involved in the Control of Arbuscule Development in Lotus japonicus1[OPEN

    PubMed Central

    Xue, Li; Cui, Haitao; Buer, Benjamin; Vijayakumar, Vinod; Delaux, Pierre-Marc; Junkermann, Stefanie; Bucher, Marcel

    2015-01-01

    Arbuscular mycorrhizal (AM) fungi, in symbiosis with plants, facilitate acquisition of nutrients from the soil to their host. After penetration, intracellular hyphae form fine-branched structures in cortical cells termed arbuscules, representing the major site where bidirectional nutrient exchange takes place between the host plant and fungus. Transcriptional mechanisms underlying this cellular reprogramming are still poorly understood. GRAS proteins are an important family of transcriptional regulators in plants, named after the first three members: GIBBERELLIC ACID-INSENSITIVE, REPRESSOR of GAI, and SCARECROW. Here, we show that among 45 transcription factors up-regulated in mycorrhizal roots of the legume Lotus japonicus, expression of a unique GRAS protein particularly increases in arbuscule-containing cells under low phosphate conditions and displays a phylogenetic pattern characteristic of symbiotic genes. Allelic rad1 mutants display a strongly reduced number of arbuscules, which undergo accelerated degeneration. In further studies, two RAD1-interacting proteins were identified. One of them is the closest homolog of Medicago truncatula, REDUCED ARBUSCULAR MYCORRHIZATION1 (RAM1), which was reported to regulate a glycerol-3-phosphate acyl transferase that promotes cutin biosynthesis to enhance hyphopodia formation. As in M. truncatula, the L. japonicus ram1 mutant lines show compromised AM colonization and stunted arbuscules. Our findings provide, to our knowledge, new insight into the transcriptional program underlying the host’s response to AM colonization and propose a function of GRAS transcription factors including RAD1 and RAM1 during arbuscule development. PMID:25560877

  17. [Isolation of proteins with complex forming agents].

    PubMed

    Schwenke, K D; Raab, B; Ender, B

    1975-01-01

    Taking vegetable albumins for models, the authors report of the possibilities of isolating proteins (which cannot be precipitated isoelectrically) by using their property of forming complexes with tannin or poly-anions. The precipitation of proteins with dextran sulphate or polyphosphates, which is due to electrostatic interaction, depends on the pH value and the electrolyte content of the solution. Under appropriate experimental conditions, protein yields of 100% are achieved. By means of tannin, the proteins are completely precipitated in a wide range of pH. The protein component of the poly-anion-containing complexes is isolated by precipitation with salt or by thermal coagulation after dissolving of the complexes. The isolation of protein from the tannin complexes is preferably realized by reaction with coffeine.

  18. GRAS NRT Precise Orbit Determination: Operational Experience

    NASA Technical Reports Server (NTRS)

    MartinezFadrique, Francisco M.; Mate, Alberto Agueda; Rodriquez-Portugal, Francisco Sancho

    2007-01-01

    EUMETSAT launched the meteorological satellite MetOp-A in October 2006; it is the first of the three satellites that constitute the EUMETSAT Polar System (EPS) space segment. This satellite carries a challenging and innovative instrument, the GNSS Receiver for Atmospheric Sounding (GRAS). The goal of the GRAS instrument is to support the production of atmospheric profiles of temperature and humidity with high accuracy, in an operational context, based on the bending of the GPS signals traversing the atmosphere during the so-called occultation periods. One of the key aspects associated to the data processing of the GRAS instrument is the necessity to describe the satellite motion and GPS receiver clock behaviour with high accuracy and within very strict timeliness limitations. In addition to these severe requirements, the GRAS Product Processing Facility (PPF) must be integrated in the EPS core ground segment, which introduces additional complexity from the data integration and operational procedure points of view. This paper sets out the rationale for algorithm selection and the conclusions from operational experience. It describes in detail the rationale and conclusions derived from the selection and implementation of the algorithms leading to the final orbit determination requirements (0.1 mm/s in velocity and 1 ns in receiver clock error at 1 Hz). Then it describes the operational approach and extracts the ideas and conclusions derived from the operational experience.

  19. Genome-Wide Identification, Evolutionary Analysis, and Stress Responses of the GRAS Gene Family in Castor Beans.

    PubMed

    Xu, Wei; Chen, Zexi; Ahmed, Naeem; Han, Bing; Cui, Qinghua; Liu, Aizhong

    2016-06-24

    Plant-specific GRAS transcription factors play important roles in regulating growth, development, and stress responses. Castor beans (Ricinus communis) are important non-edible oilseed plants, cultivated worldwide for its seed oils and its adaptability to growth conditions. In this study, we identified and characterized a total of 48 GRAS genes based on the castor bean genome. Combined with phylogenetic analysis, the castor bean GRAS members were divided into 13 distinct groups. Functional divergence analysis revealed the presence of mostly Type-I functional divergence. The gene structures and conserved motifs, both within and outside the GRAS domain, were characterized. Gene expression analysis, performed in various tissues and under a range of abiotic stress conditions, uncovered the potential functions of GRAS members in regulating plant growth development and stress responses. The results obtained from this study provide valuable information toward understanding the potential molecular mechanisms of GRAS proteins in castor beans. These findings also serve as a resource for identifying the genes that allow castor beans to grow in stressful conditions and to enable further breeding and genetic improvements in agriculture.

  20. Genome-Wide Identification, Evolutionary Analysis, and Stress Responses of the GRAS Gene Family in Castor Beans

    PubMed Central

    Xu, Wei; Chen, Zexi; Ahmed, Naeem; Han, Bing; Cui, Qinghua; Liu, Aizhong

    2016-01-01

    Plant-specific GRAS transcription factors play important roles in regulating growth, development, and stress responses. Castor beans (Ricinus communis) are important non-edible oilseed plants, cultivated worldwide for its seed oils and its adaptability to growth conditions. In this study, we identified and characterized a total of 48 GRAS genes based on the castor bean genome. Combined with phylogenetic analysis, the castor bean GRAS members were divided into 13 distinct groups. Functional divergence analysis revealed the presence of mostly Type-I functional divergence. The gene structures and conserved motifs, both within and outside the GRAS domain, were characterized. Gene expression analysis, performed in various tissues and under a range of abiotic stress conditions, uncovered the potential functions of GRAS members in regulating plant growth development and stress responses. The results obtained from this study provide valuable information toward understanding the potential molecular mechanisms of GRAS proteins in castor beans. These findings also serve as a resource for identifying the genes that allow castor beans to grow in stressful conditions and to enable further breeding and genetic improvements in agriculture. PMID:27347937

  1. Mule duck "foie gras" shows different metabolic states according to its quality phenotype by using a proteomic approach.

    PubMed

    François, Yoannah; Marie-Etancelin, Christel; Vignal, Alain; Viala, Didier; Davail, Stéphane; Molette, Caroline

    2014-07-23

    This study aimed at identifying the mechanisms implicated in "foie gras" quality variability through the study of the relationships between liver protein compositions and four liver quality phenotypes: liver weight, melting rate, and protein contents on crude or dry matter. Spots of soluble proteins were separated by bidimensional electrophoresis, and the relative abundance of proteins according to quality traits values was investigated. Twenty-three protein spots (19 unique identified proteins) showed different levels of abundance according to one or more of the traits' values. These abundance differences highlighted two groups of livers with opposite trends of abundance levels. Proteins of the first group, associated with low liver weight and melting rate, are involved in synthesis and anabolism processes, whereas proteins of the second group, associated with high liver weight and melting rate, are proteins involved in stress response. Altogether, these results highlight the variations in metabolic states underlying foie gras quality traits.

  2. GRAS radio occultation on-board of Metop

    NASA Astrophysics Data System (ADS)

    von Engeln, A.; Andres, Y.; Marquardt, C.; Sancho, F.

    2011-01-01

    The GRAS radio occultation instrument is flying on Metop-A and belongs to the EPS (EUMETSAT Polar System). GRAS observes GPS satellites in occultation. Within this work, validation of GRAS closed-loop bending angle data against co-located ECMWF profiles extracted from model fields and occultations from the COSMIC constellation of radio occultation instruments is shown. Results confirm the high data quality and robustness, where GRAS shows lower bending angle noise against ECMWF than COSMIC and in terms of occultations per day, one GRAS ≈ two COSMIC satellites. This is partly due to the operational setup of EPS. For the investigation we focus on two observation periods where updates in the ECMWF (March 2009) and COSMIC processing (October 2009) have improved the statistics further. Bending angles biases agree to within 0.5% against ECMWF and to within 0.1% against COSMIC after the updates for altitudes between 8 and 40 km. In addition, we also analyze the impact of the Metop orbit processing on the derived GRAS bending angle data, where different GPS and Metop orbit solutions are analyzed. Results show that a batch based orbit processing would improve in particular the bending angle bias behavior at higher altitudes. Requirements for the operational processing of GRAS data are briefly outlined, options to ease the use of other positioning system satellites in the near future are discussed. A simplified analysis on the observation of several of these systems, e.g. GPS and Galileo, from one platform shows that about 16% of occultations are found within 300 km, ±3 h, thus providing similar information. A constellation of 2 GRAS like instruments would have only about 10% close-by.

  3. A hard look at FDA's review of GRAS notices.

    PubMed

    Roberts, Ashley; Haighton, Lois A

    2016-08-01

    Generally Recognized as Safe (GRAS) substances are exempt from premarket approval; however, the standard of "reasonable certainty of no harm" is the same. In 1997, the voluntary GRAS affirmation process was replaced with the voluntary U.S. Food and Drug Administration (FDA) GRAS notice process. Under the GRAS notice process, pivotal safety data are required to be in the public domain, and consensus of safety among experts is required. FDA issues responses of "FDA has no questions", "Notice does not provide a basis for a GRAS determination", or, "At Notifier's request, FDA ceased to evaluate the notice." Of 528 notices reviewed, there were 393 "no questions letters", 17 "insufficient basis letters", and 84 "cease to evaluate letters". Of those deemed to be insufficient, most failed to meet the general recognition criteria. Only four raised questions about potential safety, of which three received a no questions letter upon providing more data. Of the 84 withdrawn notices, 22 received a no questions letter upon resubmission. In spite of criticisms, the FDA GRAS notice process is clearly defined, efficient, and cost-effective, and there have been no known public health issues following its implementation.

  4. Were protein internal repeats formed by "bricolage"?

    PubMed

    Lavorgna, G; Patthy, L; Boncinelli, E

    2001-03-01

    Is evolution an engineer, or is it a tinkerer--a "bricoleur"--building up complex molecules in organisms by increasing and adapting the materials at hand? An analysis of completely sequenced genomes suggests the latter, showing that increasing repetition of modules within the proteins encoded by these genomes is correlated with increasing complexity of the organism.

  5. Analytical applications for pore-forming proteins.

    PubMed

    Kasianowicz, John J; Balijepalli, Arvind K; Ettedgui, Jessica; Forstater, Jacob H; Wang, Haiyan; Zhang, Huisheng; Robertson, Joseph W F

    2016-03-01

    Proteinaceous nanometer-scale pores are ubiquitous in biology. The canonical ionic channels (e.g., those that transport Na(+), K(+), Ca(2+), and Cl(-) across cell membranes) play key roles in many cellular processes, including nerve and muscle activity. Another class of channels includes bacterial pore-forming toxins, which disrupt cell function, and can lead to cell death. We describe here the recent development of these toxins for a wide range of biological sensing applications. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

  6. Risk assessment of proteolytic Clostridium botulinum in canned foie gras.

    PubMed

    Membré, Jeanne-Marie; Diao, Moctar; Thorin, Chantal; Cordier, Grégoire; Zuber, François; André, Stéphane

    2015-10-01

    In this study, a risk assessment of proteolytic Clostridium botulinum in canned foie gras was performed, the number of illnesses per year in France due to C. botulinum in foie gras was estimated. Data on initial level in raw materials were collected at manufacturers and analysed using a Negative Binomial distribution. The effect of the usual foie gras heat treatment (equivalent time at 121 °C: F0=0.5 min) was considered at two levels: first, it led to an inactivation (estimated to 2.3 log); second it led to a spore injury and then to a spore inhibition. This latter effect was assessed by analysing data from a challenge test study carried out with Clostridium sporogenes spores in the foie gras product. The probability of spore recovering after thermal inhibition was estimated to 9.5×10(-8) (corresponding to 7.0 log). The data on the consumption pattern were collected on the French market. The Quantitative Microbiological Risk Assessment (QMRA) model and all the assumptions are reported in detail in the study. The initial contamination of raw materials, effect of thermal treatment on microbial inactivation and spore inhibition were handled mathematically using a probabilistic framework, considering only the variability dimension. The model was implemented in Excel and run through Monte Carlo simulation, using @Risk software. In parallel, epidemiological data collected from the French Institute for Public Health Surveillance during the period 2001-2012 were used to estimate an Appropriate Level Of Protection (ALOP) and then a Food Safety Objective (FSO): ALOP equalled to 2.5×10(-3) illnesses per million inhabitant per year, FSO equalled to 1.4×10(-9) foie gras portions containing C. botulinum spore (expressed in decimal logarithm, FSO=-8.9). The QMRA model output values were smaller, but on the same order of magnitude as these two figures: 8.0×10(-4) illnesses per million inhabitants per year, and, 4.5×10(-10) (-9.3 log) foie gras portions containing C

  7. Highly viscous dough-forming properties of marama protein.

    PubMed

    Amonsou, Eric O; Taylor, John R N; Naushad Emmambux, M; Gyebi Duodu, K; Minnaar, Amanda

    2012-10-01

    Marama bean is an indigenous southern African oilseed legume with an unusual protein composition. Hence, its rheological properties were studied. Marama protein formed a highly viscous and extensible dough when compared to soya and gluten. With a dough of 38% moisture, marama protein extensibility was very high (304% of its original length), twice that of gluten and soya, and this increased considerably (>3-fold) when the moisture content was increased to 45%. With added peroxidase, the storage modulus (G') of marama protein dough increased with time, suggesting the formation of new and strong protein networks. Dityrosine crosslinks were detected in the doughs. Marama protein showed a single transition with a denaturation temperature higher than soya glycinin. Marama protein was more hydrophobic and contained more β-sheet structure than did soya. Thus, the highly viscous and extensible rheological behaviour of marama protein is probably related to its high β-sheet conformation, hydrophobic interactions and tyrosine crosslinks.

  8. Synthesis of peptide sequences derived from fibril-forming proteins.

    PubMed

    Scanlon, Denis B; Karas, John A

    2011-01-01

    The pathogenesis of a large number of diseases, including Alzheimer's Disease, Parkinson's Disease, and Creutzfeldt-Jakob Disease (CJD), is associated with protein aggregation and the formation of amyloid, fibrillar deposits. Peptide fragments of amyloid-forming proteins have been found to form fibrils in their own right and have become important tools for unlocking the mechanism of amyloid fibril formation and the pathogenesis of amyloid diseases. The synthesis and purification of peptide sequences derived from amyloid fibril-forming proteins can be extremely challenging. The synthesis may not proceed well, generating a very low quality crude product which can be difficult to purify. Even clean crude peptides can be difficult to purify, as they are often insoluble or form fibrils rapidly in solution. This chapter presents methods to recognise and to overcome the difficulties associated with the synthesis, and purification of fibril-forming peptides, illustrating the points with three synthetic examples.

  9. Acides gras oméga-3 et dyslexie

    PubMed Central

    Zelcer, Michal; Goldman, Ran D.

    2015-01-01

    Résumé Question À la lumière de la hausse du nombre d’enfants d’âge scolaire ayant reçu un diagnostic de dyslexie, quel est le rôle des suppléments d’acides gras oméga-3 dans la prise en charge de cette affection? Réponse La dyslexie est le trouble d’apprentissage le plus répandu et elle est connue pour ses causes multifactorielles. De récentes données probantes pointent vers une corrélation entre le métabolisme défectueux des acides gras polyinsaturés et les troubles de neurodéveloppement, tels que la dyslexie. Bien que l’administration de suppléments d’acides gras oméga-3 aux enfants dyslexiques ait fait l’objet d’études, les données probantes sont limitées. Les critères diagnostiques homogènes de dyslexie, les mesures objectives de carence en acides gras et la surveillance étroite de l’apport alimentaire ne sont que quelques-uns des facteurs pouvant améliorer la qualité de la recherche dans ce domaine.

  10. Neighborhood Walkable Urban Form and C-Reactive Protein

    EPA Science Inventory

    Background: Walkable urban form predicts physical activity and lower body mass index, which lower C-reactive protein (CRP). However, urban form is also related to pollution, noise, social and health behavior, crowding, and other stressors, which may complement or contravene walka...

  11. The cross-sectional GRAS sample: A comprehensive phenotypical data collection of schizophrenic patients

    PubMed Central

    2010-01-01

    Background Schizophrenia is the collective term for an exclusively clinically diagnosed, heterogeneous group of mental disorders with still obscure biological roots. Based on the assumption that valuable information about relevant genetic and environmental disease mechanisms can be obtained by association studies on patient cohorts of ≥ 1000 patients, if performed on detailed clinical datasets and quantifiable biological readouts, we generated a new schizophrenia data base, the GRAS (Göttingen Research Association for Schizophrenia) data collection. GRAS is the necessary ground to study genetic causes of the schizophrenic phenotype in a 'phenotype-based genetic association study' (PGAS). This approach is different from and complementary to the genome-wide association studies (GWAS) on schizophrenia. Methods For this purpose, 1085 patients were recruited between 2005 and 2010 by an invariable team of traveling investigators in a cross-sectional field study that comprised 23 German psychiatric hospitals. Additionally, chart records and discharge letters of all patients were collected. Results The corresponding dataset extracted and presented in form of an overview here, comprises biographic information, disease history, medication including side effects, and results of comprehensive cross-sectional psychopathological, neuropsychological, and neurological examinations. With >3000 data points per schizophrenic subject, this data base of living patients, who are also accessible for follow-up studies, provides a wide-ranging and standardized phenotype characterization of as yet unprecedented detail. Conclusions The GRAS data base will serve as prerequisite for PGAS, a novel approach to better understanding 'the schizophrenias' through exploring the contribution of genetic variation to the schizophrenic phenotypes. PMID:21067598

  12. The SARS coronavirus nucleocapsid protein--forms and functions.

    PubMed

    Chang, Chung-ke; Hou, Ming-Hon; Chang, Chi-Fon; Hsiao, Chwan-Deng; Huang, Tai-huang

    2014-03-01

    The nucleocapsid phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV N protein) packages the viral genome into a helical ribonucleocapsid (RNP) and plays a fundamental role during viral self-assembly. It is a protein with multifarious activities. In this article we will review our current understanding of the N protein structure and its interaction with nucleic acid. Highlights of the progresses include uncovering the modular organization, determining the structures of the structural domains, realizing the roles of protein disorder in protein-protein and protein-nucleic acid interactions, and visualizing the ribonucleoprotein (RNP) structure inside the virions. It was also demonstrated that N-protein binds to nucleic acid at multiple sites with a coupled-allostery manner. We propose a SARS-CoV RNP model that conforms to existing data and bears resemblance to the existing RNP structures of RNA viruses. The model highlights the critical role of modular organization and intrinsic disorder of the N protein in the formation and functions of the dynamic RNP capsid in RNA viruses. This paper forms part of a symposium in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses."

  13. Sampling Protein Form and Function with the Atomic Force Microscope*

    PubMed Central

    Baclayon, Marian; Roos, Wouter H.; Wuite, Gijs J. L.

    2010-01-01

    To study the structure, function, and interactions of proteins, a plethora of techniques is available. Many techniques sample such parameters in non-physiological environments (e.g. in air, ice, or vacuum). Atomic force microscopy (AFM), however, is a powerful biophysical technique that can probe these parameters under physiological buffer conditions. With the atomic force microscope operating under such conditions, it is possible to obtain images of biological structures without requiring labeling and to follow dynamic processes in real time. Furthermore, by operating in force spectroscopy mode, it can probe intramolecular interactions and binding strengths. In structural biology, it has proven its ability to image proteins and protein conformational changes at submolecular resolution, and in proteomics, it is developing as a tool to map surface proteomes and to study protein function by force spectroscopy methods. The power of AFM to combine studies of protein form and protein function enables bridging various research fields to come to a comprehensive, molecular level picture of biological processes. We review the use of AFM imaging and force spectroscopy techniques and discuss the major advances of these experiments in further understanding form and function of proteins at the nanoscale in physiologically relevant environments. PMID:20562411

  14. A GRAS-like gene of sunflower (Helianthus annuus L.) alters the gibberellin content and axillary meristem outgrowth in transgenic Arabidopsis plants.

    PubMed

    Fambrini, M; Mariotti, L; Parlanti, S; Salvini, M; Pugliesi, C

    2015-11-01

    The GRAS proteins belong to a plant transcriptional regulator family that function in the regulation of plant growth and development. Despite their important roles, in sunflower only one GRAS gene (HaDella1) with the DELLA domain has been reported. Here, we provide a functional characterisation of a GRAS-like gene from Helianthus annuus (Ha-GRASL) lacking the DELLA motif. The Ha-GRASL gene contains an intronless open reading frame of 1,743 bp encoding 580 amino acids. Conserved motifs in the GRAS domain are detected, including VHIID, PFYRE, SAW and two LHR motifs. Within the VHII motif, the P-H-N-D-Q-L residues are entirely maintained. Phylogenetic analysis reveals that Ha-GRASL belongs to the SCARECROW LIKE4/7 (SCL4/7) subfamily of the GRAS consensus tree. Accumulation of Ha-GRASL mRNA at the adaxial boundaries from P6/P7 leaf primordia suggests a role of Ha-GRASL in the initiation of median and basal axillary meristems (AMs) of sunflower. When Ha-GRASL is over-expressed in Arabidopsis wild-type plants, the number of lateral bolts increases differently from untransformed plants. However, Ha-GRASL slightly affects the lateral suppressor (las-4-) mutation. Therefore, we hypothesise that Ha-GRASL and LAS are not functionally equivalent. The over-expression of Ha-GRASL reduces metabolic flow of gibberellins (GAs) in Arabidopsis and this modification could be relevant in AM development. Phylogenetic analysis includes LAS and SCL4/7 in the same major clade, suggesting a more recent separation of these genes with respect to other GRAS members. We propose that some features of their ancestor, as well as AM initiation and outgrowth, are partially retained in both LAS and SCL4/7.

  15. Overview of the ISRTP October 2014 workshop on GRAS determinations.

    PubMed

    McColl, Diane B; Janus, Erik R

    2016-08-01

    On October 12-13, 2014 the ISRTP held a very successful Workshop on GRAS Determinations in Washington DC that was not only well-attended by seasoned public and private professionals from a wide swath of food safety disciplines but featured a series of very insightful and informative presentations from current and past officials from the US Food & Drug Administration (FDA). To stay true to our international nature as a Society, we had regulatory and industry representatives from Canada and Europe.

  16. The PIN-FORMED (PIN) protein family of auxin transporters

    PubMed Central

    2009-01-01

    Summary The PIN-FORMED (PIN) proteins are secondary transporters acting in the efflux of the plant signal molecule auxin from cells. They are asymmetrically localized within cells and their polarity determines the directionality of intercellular auxin flow. PIN genes are found exclusively in the genomes of multicellular plants and play an important role in regulating asymmetric auxin distribution in multiple developmental processes, including embryogenesis, organogenesis, tissue differentiation and tropic responses. All PIN proteins have a similar structure with amino- and carboxy-terminal hydrophobic, membrane-spanning domains separated by a central hydrophilic domain. The structure of the hydrophobic domains is well conserved. The hydrophilic domain is more divergent and it determines eight groups within the protein family. The activity of PIN proteins is regulated at multiple levels, including transcription, protein stability, subcellular localization and transport activity. Different endogenous and environmental signals can modulate PIN activity and thus modulate auxin-distribution-dependent development. A large group of PIN proteins, including the most ancient members known from mosses, localize to the endoplasmic reticulum and they regulate the subcellular compartmentalization of auxin and thus auxin metabolism. Further work is needed to establish the physiological importance of this unexpected mode of auxin homeostasis regulation. Furthermore, the evolution of PIN-based transport, PIN protein structure and more detailed biochemical characterization of the transport function are important topics for further studies. PMID:20053306

  17. Oligomeric forms of G protein-coupled receptors (GPCRs)

    PubMed Central

    Palczewski, Krzysztof

    2010-01-01

    Oligomerization is a general characteristic of cell membrane receptors that is shared by G protein-coupled receptors (GPCRs) together with their G protein partners. Recent studies of these complexes, both in vivo and in purified reconstituted forms, unequivocally support this contention for GPCRs, perhaps with only rare exceptions. As evidence has evolved from experimental cell lines to more relevant in vivo studies and from indirect biophysical approaches to well defined isolated complexes of dimeric receptors alone and complexed with G proteins, there is an expectation that the structural basis of oligomerization and the functional consequences for membrane signaling will be elucidated. Oligomerization of cell membrane receptors is fully supported by both thermodynamic calculations and the selectivity and duration of signaling required to reach targets located in various cellular compartments. PMID:20538466

  18. Oligomeric and polymeric aggregates formed by proteins containing expanded polyglutamine

    PubMed Central

    Iuchi, S.; Hoffner, G.; Verbeke, P.; Djian, P.; Green, H.

    2003-01-01

    Neurological diseases resulting from proteins containing expanded polyglutamine (polyQ) are characteristically associated with insoluble neuronal inclusions, usually intranuclear, and neuronal death. We describe here oligomeric and polymeric aggregates formed in cells by expanded polyQ. These aggregates are not dissociated by concentrated formic acid, an extremely effective solvent for otherwise insoluble proteins. Perinuclear inclusions formed in cultured cells by expanded polyQ can be completely dissolved in concentrated formic acid, but a soluble protein oligomer containing the expanded polyQ and released by the formic acid is not dissociated to monomer. In Huntington's disease, a formic acid-resistant oligomer is present in cerebral cortex, but not in cerebellum. Cortical nuclei contain a polymeric aggregate of expanded polyQ that is insoluble in formic acid, does not enter polyacrylamide gels, but is retained on filters. This finding shows that the process of polymerization is more advanced in the cerebral cortex than in cultured cells. The resistance of oligomer and polymer to formic acid suggests the participation of covalent bonds in their stabilization. PMID:12591956

  19. The relevance of international assessments to GRAS determinations.

    PubMed

    Kruger, Claire

    2016-08-01

    A discussion of the risk assessment process as applied to the Generally Recognized As Safe (GRAS) determination of safety for new ingredients can benefit from an international perspective. When we think about how risk assessments are performed around the world it is critical to assess what can be learned. What are the similarities? What are the differences? What are the takeaways? It is important to talk about the similarities in processes, because it validates the approach taken by risk assessors who are charged with protecting the food supply. It is also instructive to evaluate the differences in order to determine where improvements can be made to our process.

  20. Chromatographic resolution of altered forms of protein kinase C

    SciTech Connect

    Ashendel, C.L.; Minor, P.L.; Baudoin, P.A.; Carlos, M.

    1987-05-01

    Rapid chromatographic resolution of protein kinase C (PKC) in extracts of rat brain on DEAE-cellulose yielded two major peaks of activity. These fractions bound phorbol esters with identical affinity and specificity and had similar ratios of PKC to phorbol ester-binding activities. Chicken egg yolk antibodies raised to PKC in the first fraction reacted with 74 to 76 kilodalton peptides in the second fraction. Chromatography of each fraction on hydroxylapatite yielded similar distributions of three PKC isozymes. Rechromatography of the DEAE-cellulose fractions on DEAE-cellulose confirmed that these forms of PKC were not rapidly interconvertible. Results of experiments in which extracts or fractions were incubated with MgATP and phosphatase inhibitors were consistent with elution of dephospho-PKC in the first fraction while the second fraction contained phospho-PKC. If confirmed, this suggests that a substantial fraction of PKC in rat and mouse tissues exists in the phosphorylated form.

  1. Tagatose, the new GRAS sweetener and health product.

    PubMed

    Levin, Gilbert V

    2002-01-01

    Tagatose, a low-calorie, full-bulk natural sugar, has just attained GRAS (Generally Recognized As Safe) status under U.S. Food and Drug Administration (FDA) regulations, thereby permitting its use as a sweetener in foods and beverages. This paper presents all current aspects of tagatose with respect to demonstrated food and beverage applications and the potential health and medical benefits of this unique substance. Summarized studies are referenced to detailed peer-reviewed papers. The safety studies followed the recommendations in the FDA "Red Book." Results were submitted to an Expert Panel for determination of GRAS status under FDA regulation. Small phase 2 clinical trials showed tagatose to be effective in treating type 2 diabetes. The results, buttressed by the references cited, support the efficacy of the various applications disclosed for tagatose. Tagatose has been found to be safe and efficacious for use as a low-calorie, full-bulk sweetener in a wide variety of foods, beverages, health foods, and dietary supplements. It fills broad, heretofore unmet needs for a low-calorie sweetener in products in which the bulk of sugar is important, such as chocolates, chewing gum, cakes, ice cream, and frosted cereals. Its synergism with high-intensity sweeteners also makes it useful in sodas. Various health and medical benefits are indicated, including the treatment of type 2 diabetes, hyperglycemia, anemia, and hemophilia and the improvement of fetal development.

  2. Ordered nanoparticle arrays formed on engineered chaperonin protein templates

    NASA Technical Reports Server (NTRS)

    McMillan, R. Andrew; Paavola, Chad D.; Howard, Jeanie; Chan, Suzanne L.; Zaluzec, Nestor J.; Trent, Jonathan D.

    2002-01-01

    Traditional methods for fabricating nanoscale arrays are usually based on lithographic techniques. Alternative new approaches rely on the use of nanoscale templates made of synthetic or biological materials. Some proteins, for example, have been used to form ordered two-dimensional arrays. Here, we fabricated nanoscale ordered arrays of metal and semiconductor quantum dots by binding preformed nanoparticles onto crystalline protein templates made from genetically engineered hollow double-ring structures called chaperonins. Using structural information as a guide, a thermostable recombinant chaperonin subunit was modified to assemble into chaperonins with either 3 nm or 9 nm apical pores surrounded by chemically reactive thiols. These engineered chaperonins were crystallized into two-dimensional templates up to 20 microm in diameter. The periodic solvent-exposed thiols within these crystalline templates were used to size-selectively bind and organize either gold (1.4, 5 or 10nm) or CdSe-ZnS semiconductor (4.5 nm) quantum dots into arrays. The order within the arrays was defined by the lattice of the underlying protein crystal. By combining the self-assembling properties of chaperonins with mutations guided by structural modelling, we demonstrate that quantum dots can be manipulated using modified chaperonins and organized into arrays for use in next-generation electronic and photonic devices.

  3. Neighborhood Walkable Urban Form and C-Reactive Protein

    PubMed Central

    King, Katherine

    2013-01-01

    Background Walkable urban form predicts physical activity and lower body mass index, which lower C-reactive protein (CRP). However, urban form is also related to pollution, noise, social and health behavior, crowding, and other stressors, which may complement or contravene walkability effects. Purpose This paper assesses within-neighborhood correlation of CRP, and whether three features of walkable urban form (residential density, street connectivity, and land use mix) are associated with CRP levels. Methods CRP measures (n=610) and sociodemographic data come from the 2001–3 Chicago Community Adult Health Study, linked with objective built environment data. Results Within-neighborhood correlations of CRP are greater than those of related health measures. A one standard deviation increase in residential density predicts significantly higher log CRP (e.g. β=0.11, p<.01) in Chicago, while a one standard deviation increase in land use mix predicts significantly lower CRP (e.g. β=−0. 19, p<0.01). Street connectivity is unrelated to CRP in this highly walkable city. Discussion Results suggest residential density may be a risk factor for inflammation, while greater walkability of mixed land use areas may be protective. It may be that negative aspects of density overcome the inflammatory benefits of walking. PMID:24096140

  4. The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

    PubMed

    Satoh, Jun-ichi; Onoue, Hiroyuki; Arima, Kunimasa; Yamamura, Takashi

    2005-10-01

    The 14-3-3 protein family consists of acidic 30-kDa proteins composed of 7 isoforms expressed abundantly in neurons and glial cells of the central nervous system (CNS). The 14-3-3 protein identified in the cerebrospinal fluid provides a surrogate marker for premortem diagnosis of Creutzfeldt-Jakob disease, although an active involvement of 14-3-3 in the pathogenesis of prion diseases remains unknown. By protein overlay and mass spectrometric analysis of protein extract of NTera2-derived differentiated neurons, we identified heat shock protein Hsp60 as a 14-3-3-interacting protein. The 14-3-3zeta and gamma isoforms interacted with Hsp60, suggesting that the interaction is not isoform-specific. Furthermore, the interaction was identified in SK-N-SH neuroblastoma, U-373MG astrocytoma, and HeLa cervical carcinoma cells. The cellular prion protein (PrPC) along with Hsp60 was coimmunoprecipitated with 14-3-3 in the human brain protein extract. By protein overlay, 14-3-3 interacted with both recombinant human Hsp60 and PrPC produced by Escherichia coli, indicating that the molecular interaction is phosphorylation-independent. The 14-3-3-binding domain was located in the N-terminal half (NTF) of Hsp60 spanning amino acid residues 27-287 and the NTF of PrPC spanning amino acid residues 23-137. By immunostaining, the 14-3-3 protein Hsp60 and PrPC were colocalized chiefly in the mitochondria of human neuronal progenitor cells in culture, and were coexpressed most prominently in neurons and reactive astrocytes in the human brain. These observations indicate that the 14-3-3 protein forms a molecular complex with Hsp60 and PrPC in the human CNS under physiological conditions and suggest that this complex might become disintegrated in the pathologic process of prion diseases.

  5. Systematic mining of generally recognized as safe (GRAS) flavor chemicals for bioactive compounds.

    PubMed

    Martinez-Mayorga, Karina; Peppard, Terry L; López-Vallejo, Fabian; Yongye, Austin B; Medina-Franco, José L

    2013-08-07

    Bioactive food compounds can be both therapeutically and nutritionally relevant. Screening strategies are widely employed to identify bioactive compounds from edible plants. Flavor additives contained in the so-called FEMA GRAS (generally recognized as safe) list of approved flavoring ingredients is an additional source of potentially bioactive compounds. This work used the principles of molecular similarity to identify compounds with potential mood-modulating properties. The ability of certain GRAS molecules to inhibit histone deacetylase-1 (HDAC1), proposed as an important player in mood modulation, was assayed. Two GRAS chemicals were identified as HDAC1 inhibitors in the micromolar range, results similar to what was observed for the structurally related mood prescription drug valproic acid. Additional studies on bioavailability, toxicity at higher concentrations, and off-target effects are warranted. The methodology described in this work could be employed to identify potentially bioactive flavor chemicals present in the FEMA GRAS list.

  6. TaSCL14, a novel wheat (Triticum aestivum L.) GRAS gene, regulates plant growth, photosynthesis, tolerance to photooxidative stress, and senescence.

    PubMed

    Chen, Kunmei; Li, Hongwei; Chen, Yaofeng; Zheng, Qi; Li, Bin; Li, Zhensheng

    2015-01-20

    Rates of photosynthesis, tolerance to photooxidative stress, and senescence are all important physiological factors that affect plant development and thus agricultural productivity. GRAS proteins play essential roles in plant growth and development as well as in plant responses to biotic and abiotic stresses. So far few GRAS genes in wheat (Triticum aestivum L.) have been characterized. A previous transcriptome analysis indicated that the expression of a GRAS gene (TaSCL14) was induced by high-light stress in Xiaoyan 54 (XY54), a common wheat cultivar with strong tolerance to high-light stress. In this study, TaSCL14 gene was isolated from XY54 and mapped on chromosome 4A. TaSCL14 was expressed in various wheat organs, with high levels in stems and roots. Our results confirmed that TaSCL14 expression was indeed responsive to high-light stress. Barley stripe mosaic virus (BSMV)-based virus-induced gene silencing (VIGS) of TaSCL14 in wheat was performed to help characterize its potential functions. Silencing of TaSCL14 resulted in inhibited plant growth, decreased photosynthetic capacity, and reduced tolerance to photooxidative stress. In addition, silencing of TaSCL14 in wheat promoted leaf senescence induced by darkness. These results suggest that TaSCL14 may act as a multifunctional regulator involved in plant growth, photosynthesis, tolerance to photooxidative stress, and senescence.

  7. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID.

    PubMed

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês C R; Willige, Björn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-06-19

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the--in many cells--asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  8. Chemoinformatic analysis of GRAS (Generally Recognized as Safe) flavor chemicals and natural products.

    PubMed

    Medina-Franco, José L; Martínez-Mayorga, Karina; Peppard, Terry L; Del Rio, Alberto

    2012-01-01

    Food materials designated as "Generally Recognized as Safe" (GRAS) are attracting the attention of researchers in their attempts to systematically identify compounds with putative health-related benefits. In particular, there is currently a great deal of interest in exploring possible secondary benefits of flavor ingredients, such as those relating to health and wellness. One step in this direction is the comprehensive characterization of the chemical structures contained in databases of flavoring substances. Herein, we report a comprehensive analysis of the recently updated FEMA GRAS list of flavoring substances (discrete chemical entities only). Databases of natural products, approved drugs and a large set of commercial molecules were used as references. Remarkably, natural products continue to be an important source of bioactive compounds for drug discovery and nutraceutical purposes. The comparison of five collections of compounds of interest was performed using molecular properties, rings, atom counts and structural fingerprints. It was found that the molecular size of the GRAS flavoring substances is, in general, smaller cf. members of the other databases analyzed. The lipophilicity profile of the GRAS database, a key property to predict human bioavailability, is similar to approved drugs. Several GRAS chemicals overlap to a broad region of the property space occupied by drugs. The GRAS list analyzed in this work has high structural diversity, comparable to approved drugs, natural products and libraries of screening compounds. This study represents one step towards the use of the distinctive features of the flavoring chemicals contained in the GRAS list and natural products to systematically search for compounds with potential health-related benefits.

  9. Punching Holes in Membranes: How Oligomeric Pore-Forming Proteins and Lipids Cooperate to Form Aqueous Channels in Membranes

    NASA Astrophysics Data System (ADS)

    Fradin, Cécile; Satsoura, Dmitri; Andrews, David W.

    Many important biological processes are carried out by a small number of proteins working together as a team to accomplish a specific task. Cooperation between the different proteins is often accomplished through the formation of a supramolecular complex, comprised of either identical or different subunits. Although the formation of protein assemblies is a favored mechanism throughout the cell, it becomes especially important in lipid membranes, as evidenced by the numerous cellular events that are either triggered by or result in the formation of protein complexes in membranes. However, due to the difficulties associated with the study of membrane proteins, the formation of oligomers in lipid membranes is perhaps one of the least understood cellular processes. In this chapter we focus our attention on a subset of membrane complexes — namely, those formed by proteins that are able to pass from a water-soluble to a transmembrane form in order to create a water-filled channel through the lipid membrane. These pore-forming proteins (PFPs) are found in many organisms throughout different kingdoms of life, from bacteria to human. They are often involved in cell death mechanisms through their capacity to break membrane permeability barriers, which can lead to dissipation of the membrane potential as well as introduction or leakage of enzymatic proteins. In fact, a large subset of the PFPs are toxins, and referred to in the literature as pore-forming toxins (PFTs). The association of several monomers into an oligomer is almost always an important aspect of the modus operandi of these proteins. Oligomerization can be useful in several ways: it results in structures large enough to delineate nanometer-size water-filled channels in lipid bilayers, it ensures the presence of large hydrophobic surfaces that can support insertion in the membrane, and it permits cooperative formation and insertion mechanisms.

  10. Characterisation and toxicological assessment of Neutral Methacrylate Copolymer for GRAS evaluation.

    PubMed

    Eisele, Johanna; Haynes, Geoff; Kreuzer, Knut; Rosamilia, Tiana

    2013-12-01

    Neutral Methacrylate Copolymer is a fully polymerised copolymer used in the pharmaceutical industry to permit pH-independent delayed release of active ingredients from oral dosage forms. This function has potential use with food supplements and this article describes available information on the safety of the substance. Oral administration of radiolabelled copolymer to rats resulted in the detection of chemically unchanged copolymer in the faeces, with negligible absorption. Safety studies revealed no adverse toxicity following repeated administration at doses of up to 2000 mg/kg bw/d in a sub-chronic study in rats or 250 mg/kg bw/d in a sub-chronic study in dogs. No reproductive toxicity occurred at up to 2000 mg/kg bw/d in rats or rabbits. The substance shows no evidence of genotoxicity, has low acute toxicity and no irritation or sensitisation potential. An ADI value of 20 mg/kg bw was concluded from two alternative approaches. Daily exposure from use in dietary supplements is estimated as up to 10.0 mg/kg bw in adults and 13.3 mg/kg bw in children. There would therefore appear to be no safety concerns under the intended conditions of use. The information provided is intended to support an evaluation that the substance may be "generally recognized as safe" (GRAS).

  11. Electrophoretic separation method for membrane pore-forming proteins in multilayer lipid membranes.

    PubMed

    Okamoto, Yukihiro; Tsujimoto, Yusuke; Umakoshi, Hiroshi

    2016-03-01

    In this paper, we report on a novel electrophoretic separation and analysis method for membrane pore-forming proteins in multilayer lipid membranes (MLMs) in order to overcome the problems related to current separation and analysis methods of membrane proteins, and to obtain a high-performance separation method on the basis of specific properties of the lipid membranes. We constructed MLMs, and subsequently characterized membrane pore-forming protein behavior in MLMs. Through the use of these MLMs, we were able to successfully separate and analyze membrane pore-forming proteins in MLMs. To the best of our knowledge, this research is the first example of membrane pore-forming protein separation in lipid membranes. Our method can be expected to be applied for the separation and analysis of other membrane proteins including intrinsic membrane proteins and to result in high-performance by utilizing the specific properties of lipid membranes.

  12. Origins and Evolution of the HET-s Prion-Forming Protein: Searching for Other Amyloid-Forming Solenoids

    PubMed Central

    Gendoo, Deena M. A.; Harrison, Paul M.

    2011-01-01

    The HET-s prion-forming domain from the filamentous fungus Podospora anserina is gaining considerable interest since it yielded the first well-defined atomic structure of a functional amyloid fibril. This structure has been identified as a left-handed beta solenoid with a triangular hydrophobic core. To delineate the origins of the HET-s prion-forming protein and to discover other amyloid-forming proteins, we searched for all homologs of the HET-s protein in a database of protein domains and fungal genomes, using a combined application of HMM, psi-blast and pGenThreader techniques, and performed a comparative evolutionary analysis of the N-terminal alpha-helical domain and the C-terminal prion-forming domain of HET-s. By assessing the tandem evolution of both domains, we observed that the prion-forming domain is restricted to Sordariomycetes, with a marginal additional sequence homolog in Arthroderma otae as a likely case of horizontal transfer. This suggests innovation and rapid evolution of the solenoid fold in the Sordariomycetes clade. In contrast, the N-terminal domain evolves at a slower rate (in Sordariomycetes) and spans many diverse clades of fungi. We performed a full three-dimensional protein threading analysis on all identified HET-s homologs against the HET-s solenoid fold, and present detailed structural annotations for identified structural homologs to the prion-forming domain. An analysis of the physicochemical characteristics in our set of structural models indicates that the HET-s solenoid shape can be readily adopted in these homologs, but that they are all less optimized for fibril formation than the P. anserina HET-s sequence itself, due chiefly to the presence of fewer asparagine ladders and salt bridges. Our combined structural and evolutionary analysis suggests that the HET-s shape has “limited scope” for amyloidosis across the wider protein universe, compared to the ‘generic’ left-handed beta helix. We discuss the implications of

  13. Diarylthiophenes as inhibitors of the pore-forming protein perforin

    PubMed Central

    Miller, Christian K.; Huttunen, Kristiina M.; Denny, William A.; Jaiswal, Jagdish K.; Ciccone, Annette; Browne, Kylie A.; Trapani, Joseph A.; Spicer, Julie A.

    2016-01-01

    Evolution from a furan-containing high-throughput screen (HTS) hit (1) resulted in isobenzofuran-1(3H)-one (2) as a potent inhibitor of the function of both isolated perforin protein and perforin delivered in situ by intact KHYG-1 NK cells. In the current study, structure–activity relationship (SAR) development towards a novel series of diarylthiophene analogues has continued through the use of substituted-benzene and -pyridyl moieties as bioisosteres for 2-thioxoimidazolidin-4-one (A) on a thiophene (B) -isobenzofuranone (C) scaffold. The resulting compounds were tested for their ability to inhibit perforin lytic activity in vitro. Carboxamide (23) shows a 4-fold increase over (2) in lytic activity against isolated perforin and provides good rationale for continued development within this class. PMID:26711151

  14. Integral and differential form of the protein folding problem

    NASA Astrophysics Data System (ADS)

    Tramontano, Anna

    2004-07-01

    The availability of the complete genomic sequences of many species, including human, has raised enormous expectations in medicine, pharmacology, ecology, biotechnology and forensic sciences. However, knowledge is only a first step toward understanding, and we are only at the early stage of a scientific process that might lead us to satisfy all the expectations raised by the genomic projects. In this review I will discuss the present status of computational methods that attempt to infer the unique three-dimensional structure of proteins from their amino acid sequences. Although this problem has been defined as the “holy grail” of biology, it represents only one of the many hurdles in our path towards the understanding of life at a molecular level.

  15. [Antirestriction activity of T7 Ocr protein in monomeric and dimeric forms].

    PubMed

    Zavil'gelskiĭ, G B; Kotova, V Iu

    2014-01-01

    The Ocr protein, encoded by 0.3 (ocr) gene of bacteriophage T7, belongs to the family of antirestriction proteins that specifically inhibit the type I restriction-modification systems. Native Ocr forms homodimer (Ocr)2 both in solution and in the crystalline state. The Ocr protein belongs to the family of mimicry proteins. F53D A57E and E53R V77D mutant proteins were obtained, which form monomers. It was shown that the values of the dissociation constants Kd for Ocr, Ocr F53D A57E and Ocr F53RV77D proteins with EcoKI enzyme differ in 1000 times: Kd (Ocr) = 10(-10) M, Kd (Ocr F53D A57E and Ocr F53R V77D) = 10(-7) M. Antimodification activity of the Ocr monomeric forms is significantly reduced. We have shown, that Ocr dimeric form has fundamental importance for high inhibitory activity.

  16. New Structural Approaches to Understand the Disease Related Forms of the Prion Protein

    DTIC Science & Technology

    2005-07-01

    AD Award Number: DAMD17-03-1-0476 TITLE: New Structural Approaches to Understand the Disease Related Forms of the Prion Protein PRINCIPAL...TITLE AND SUBTITLE 5a. CONTRACT NUMBER New Structural Approaches to Understand the Disease Related Forms of the Prion Protein 5b. GRANT NUMBER DAMD17...Understanding these fundamental steps in the processes of initiation and propagation of the disease related state of the protein may lead to new

  17. Engineering a pH responsive pore forming protein.

    PubMed

    Kisovec, Matic; Rezelj, Saša; Knap, Primož; Cajnko, Miša Mojca; Caserman, Simon; Flašker, Ajda; Žnidaršič, Nada; Repič, Matej; Mavri, Janez; Ruan, Yi; Scheuring, Simon; Podobnik, Marjetka; Anderluh, Gregor

    2017-02-08

    Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH.

  18. Engineering a pH responsive pore forming protein

    PubMed Central

    Kisovec, Matic; Rezelj, Saša; Knap, Primož; Cajnko, Miša Mojca; Caserman, Simon; Flašker, Ajda; Žnidaršič, Nada; Repič, Matej; Mavri, Janez; Ruan, Yi; Scheuring, Simon; Podobnik, Marjetka; Anderluh, Gregor

    2017-01-01

    Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH. PMID:28176876

  19. Engineering a pH responsive pore forming protein

    NASA Astrophysics Data System (ADS)

    Kisovec, Matic; Rezelj, Saša; Knap, Primož; Cajnko, Miša Mojca; Caserman, Simon; Flašker, Ajda; Žnidaršič, Nada; Repič, Matej; Mavri, Janez; Ruan, Yi; Scheuring, Simon; Podobnik, Marjetka; Anderluh, Gregor

    2017-02-01

    Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH.

  20. System and method for forming synthetic protein crystals to determine the conformational structure by crystallography

    DOEpatents

    Craig, George D.; Glass, Robert; Rupp, Bernhard

    1997-01-01

    A method for forming synthetic crystals of proteins in a carrier fluid by use of the dipole moments of protein macromolecules that self-align in the Helmholtz layer adjacent to an electrode. The voltage gradients of such layers easily exceed 10.sup.6 V/m. The synthetic protein crystals are subjected to x-ray crystallography to determine the conformational structure of the protein involved.

  1. System and method for forming synthetic protein crystals to determine the conformational structure by crystallography

    DOEpatents

    Craig, G.D.; Glass, R.; Rupp, B.

    1997-01-28

    A method is disclosed for forming synthetic crystals of proteins in a carrier fluid by use of the dipole moments of protein macromolecules that self-align in the Helmholtz layer adjacent to an electrode. The voltage gradients of such layers easily exceed 10{sup 6}V/m. The synthetic protein crystals are subjected to x-ray crystallography to determine the conformational structure of the protein involved. 2 figs.

  2. Analysis of nanoparticle-protein coronas formed in vitro between nanosized welding particles and nasal lavage proteins.

    PubMed

    Ali, Neserin; Mattsson, Karin; Rissler, Jenny; Karlsson, Helen Marg; Svensson, Christian R; Gudmundsson, Anders; Lindh, Christian H; Jönsson, Bo A G; Cedervall, Tommy; Kåredal, Monica

    2016-01-01

    Welding fumes include agglomerated particles built up of primary nanoparticles. Particles inhaled through the nose will to some extent be deposited in the protein-rich nasal mucosa, and a protein corona will be formed around the particles. The aim was to identify the protein corona formed between nasal lavage proteins and four types of particles with different parameters. Two of the particles were formed and collected during welding and two were manufactured iron oxides. When nasal lavage proteins were added to the particles, differences were observed in the sizes of the aggregates that were formed. Measurements showed that the amount of protein bound to particles correlated with the relative size increase of the aggregates, suggesting that the surface area was associated with the binding capacity. However, differences in aggregate sizes were detected when nasal proteins were added to UFWF and Fe2O3 particles (having similar agglomerated size) suggesting that yet parameters other than size determine the binding. Relative quantitative mass spectrometric and gel-based analyses showed differences in the protein content of the coronas. High-affinity proteins were further assessed for network interactions. Additional experiments showed that the inhibitory function of secretory leukocyte peptidase inhibitor, a highly abundant nasal protein, was influenced by particle binding suggesting that an understanding of protein function following particle binding is necessary to properly evaluate pathophysiological events. Our results underscore the importance of including particles collected from real working environments when studying the toxic effects of particles because these effects might be mediated by the protein corona.

  3. Analysis of nanoparticle–protein coronas formed in vitro between nanosized welding particles and nasal lavage proteins

    PubMed Central

    Ali, Neserin; Mattsson, Karin; Rissler, Jenny; Karlsson, Helen Marg; Svensson, Christian R.; Gudmundsson, Anders; Lindh, Christian H.; Jönsson, Bo A. G.; Cedervall, Tommy; Kåredal, Monica

    2016-01-01

    Abstract Welding fumes include agglomerated particles built up of primary nanoparticles. Particles inhaled through the nose will to some extent be deposited in the protein-rich nasal mucosa, and a protein corona will be formed around the particles. The aim was to identify the protein corona formed between nasal lavage proteins and four types of particles with different parameters. Two of the particles were formed and collected during welding and two were manufactured iron oxides. When nasal lavage proteins were added to the particles, differences were observed in the sizes of the aggregates that were formed. Measurements showed that the amount of protein bound to particles correlated with the relative size increase of the aggregates, suggesting that the surface area was associated with the binding capacity. However, differences in aggregate sizes were detected when nasal proteins were added to UFWF and Fe2O3 particles (having similar agglomerated size) suggesting that yet parameters other than size determine the binding. Relative quantitative mass spectrometric and gel-based analyses showed differences in the protein content of the coronas. High-affinity proteins were further assessed for network interactions. Additional experiments showed that the inhibitory function of secretory leukocyte peptidase inhibitor, a highly abundant nasal protein, was influenced by particle binding suggesting that an understanding of protein function following particle binding is necessary to properly evaluate pathophysiological events. Our results underscore the importance of including particles collected from real working environments when studying the toxic effects of particles because these effects might be mediated by the protein corona. PMID:26186033

  4. The HPr Proteins from the Thermophile Bacillus stearothermophilus Can Form Domain-swapped Dimers

    SciTech Connect

    Sridharan, Sudharsan; Razvi, Abbas; Scholtz, J. Martin; Sacchettini, James C.

    2010-07-20

    The study of proteins from extremophilic organisms continues to generate interest in the field of protein folding because paradigms explaining the enhanced stability of these proteins still elude us and such studies have the potential to further our knowledge of the forces stabilizing proteins. We have undertaken such a study with our model protein HPr from a mesophile, Bacillus subtilis, and a thermophile, Bacillus stearothermophilus. We report here the high-resolution structures of the wild-type HPr protein from the thermophile and a variant, F29W. The variant proved to crystallize in two forms: a monomeric form with a structure very similar to the wild-type protein as well as a domain-swapped dimer. Interestingly, the structure of the domain-swapped dimer for HPr is very different from that observed for a homologous protein, Crh, from B. subtilis. The existence of a domain-swapped dimer has implications for amyloid formation and is consistent with recent results showing that the HPr proteins can form amyloid fibrils. We also characterized the conformational stability of the thermophilic HPr proteins using thermal and solvent denaturation methods and have used the high-resolution structures in an attempt to explain the differences in stability between the different HPr proteins. Finally, we present a detailed analysis of the solution properties of the HPr proteins using a variety of biochemical and biophysical methods.

  5. The surface layer protein of Bacillus thuringiensis CTC forms unique intracellular parasporal inclusion body.

    PubMed

    Zhu, Chenguang; Yu, Ziniu

    2008-08-01

    Bacillus thuringiensis subsp. finitimus strain CTC forms round parasporal inclusion body. The inclusion body protein gene ctc has been cloned and characterized. Sequence homology analysis reveals that the amino acid sequence of CTC protein shows 87% identity with the surface layer (S-layer) protein Sap (GenBank Z36946) in B. anthracis. In this report, transmission electron microscope observation showed that CTC formed intracellular parasporal inclusion body and sheet structure of S-layer-like protein at the spore phase. Furthermore, the ctc gene was transformed into an acrystalliferous B. thuringiensis strain BMB171. The resulting transformant could form parasporal body which had the same shape and molecular weight of protein with that of B. thuringiensis CTC. These results, together with the sequence homology analysis of ctc gene, confirmed that the unique intracellular parasporal inclusion body of B. thuringiensis was comprised of S-layer protein.

  6. Aspartic acid protease from Botrytis cinerea removes haze-forming proteins during white winemaking.

    PubMed

    Van Sluyter, Steven C; Warnock, Nicholas I; Schmidt, Simon; Anderson, Peter; van Kan, Jan A L; Bacic, Antony; Waters, Elizabeth J

    2013-10-09

    White wines suffer from heat-induced protein hazes during transport and storage unless the proteins are removed prior to bottling. Bentonite fining is by far the most commonly used method, but it is inefficient and creates several other process challenges. An alternative to bentonite is the enzymatic removal of haze-forming grape pathogenesis-related proteins using added proteases. The major problem with this approach is that grape pathogenesis-related proteins are highly protease resistant unless they are heat denatured in combination with enzymatic treatment. This paper demonstrates that the protease BcAP8, from the grape fungal pathogen Botrytis cinerea , is capable of degrading chitinase, a major class of haze-forming proteins, without heat denaturation. Because BcAP8 effectively removes haze-forming proteins under normal winemaking conditions, it could potentially benefit winemakers by reducing bentonite requirements.

  7. 76 FR 7701 - Special Local Regulations; Krewe of Charleston Mardi Gras Boat Parade, Charleston Harbor...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-11

    ... consist of a series of buffer zones around vessels participating in the Krewe of Charleston Mardi Gras Boat Parade. These buffer zones are as follows: (1) All waters within 500 yards in front of the lead... from entering, transiting through, anchoring, or remaining within the buffer zones unless...

  8. 21 CFR 570.35 - Affirmation of generally recognized as safe (GRAS) status.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... arsenic and heavy metals. (Recommendation for any change in the Food Chemicals Codex monograph should be... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Affirmation of generally recognized as safe (GRAS) status. 570.35 Section 570.35 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  9. 21 CFR 570.35 - Affirmation of generally recognized as safe (GRAS) status.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... arsenic and heavy metals. (Recommendation for any change in the Food Chemicals Codex monograph should be... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Affirmation of generally recognized as safe (GRAS) status. 570.35 Section 570.35 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  10. 21 CFR 170.35 - Affirmation of generally recognized as safe (GRAS) status.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...) Structural formula. (f) Specifications for food grade material, including arsenic and heavy metals... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Affirmation of generally recognized as safe (GRAS) status. 170.35 Section 170.35 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  11. 21 CFR 570.35 - Affirmation of generally recognized as safe (GRAS) status.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... arsenic and heavy metals. (Recommendation for any change in the Food Chemicals Codex monograph should be... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Affirmation of generally recognized as safe (GRAS) status. 570.35 Section 570.35 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  12. 21 CFR 570.35 - Affirmation of generally recognized as safe (GRAS) status.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... arsenic and heavy metals. (Recommendation for any change in the Food Chemicals Codex monograph should be... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Affirmation of generally recognized as safe (GRAS) status. 570.35 Section 570.35 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  13. 21 CFR 170.35 - Affirmation of generally recognized as safe (GRAS) status.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...) Specifications for food grade material, including arsenic and heavy metals. (Recommendation for any change in the... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Affirmation of generally recognized as safe (GRAS) status. 170.35 Section 170.35 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  14. 21 CFR 570.35 - Affirmation of generally recognized as safe (GRAS) status.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... arsenic and heavy metals. (Recommendation for any change in the Food Chemicals Codex monograph should be... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Affirmation of generally recognized as safe (GRAS) status. 570.35 Section 570.35 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  15. 21 CFR 170.35 - Affirmation of generally recognized as safe (GRAS) status.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...) Structural formula. (f) Specifications for food grade material, including arsenic and heavy metals... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Affirmation of generally recognized as safe (GRAS) status. 170.35 Section 170.35 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  16. 21 CFR 170.35 - Affirmation of generally recognized as safe (GRAS) status.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...) Structural formula. (f) Specifications for food grade material, including arsenic and heavy metals... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Affirmation of generally recognized as safe (GRAS) status. 170.35 Section 170.35 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  17. 21 CFR 170.35 - Affirmation of generally recognized as safe (GRAS) status.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...) Structural formula. (f) Specifications for food grade material, including arsenic and heavy metals... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Affirmation of generally recognized as safe (GRAS) status. 170.35 Section 170.35 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  18. 21 CFR 170.30 - Eligibility for classification as generally recognized as safe (GRAS).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Eligibility for classification as generally recognized as safe (GRAS). 170.30 Section 170.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES Food...

  19. 21 CFR 570.30 - Eligibility for classification as generally recognized as safe (GRAS).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Eligibility for classification as generally recognized as safe (GRAS). 570.30 Section 570.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES...

  20. 21 CFR 170.30 - Eligibility for classification as generally recognized as safe (GRAS).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Eligibility for classification as generally recognized as safe (GRAS). 170.30 Section 170.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES Food...

  1. 21 CFR 170.30 - Eligibility for classification as generally recognized as safe (GRAS).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Eligibility for classification as generally recognized as safe (GRAS). 170.30 Section 170.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD ADDITIVES Food Additive Safety § 170.30 Eligibility...

  2. 21 CFR 570.30 - Eligibility for classification as generally recognized as safe (GRAS).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Eligibility for classification as generally recognized as safe (GRAS). 570.30 Section 570.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES...

  3. 21 CFR 170.30 - Eligibility for classification as generally recognized as safe (GRAS).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Eligibility for classification as generally recognized as safe (GRAS). 170.30 Section 170.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES Food...

  4. 21 CFR 570.30 - Eligibility for classification as generally recognized as safe (GRAS).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Eligibility for classification as generally recognized as safe (GRAS). 570.30 Section 570.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES...

  5. 21 CFR 570.30 - Eligibility for classification as generally recognized as safe (GRAS).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Eligibility for classification as generally recognized as safe (GRAS). 570.30 Section 570.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES...

  6. 21 CFR 570.30 - Eligibility for classification as generally recognized as safe (GRAS).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Eligibility for classification as generally recognized as safe (GRAS). 570.30 Section 570.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES...

  7. 21 CFR 170.30 - Eligibility for classification as generally recognized as safe (GRAS).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Eligibility for classification as generally recognized as safe (GRAS). 170.30 Section 170.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES Food...

  8. Minor coat protein composition and location of the A protein in bacteriophage f1 spheroids and I-forms.

    PubMed Central

    Lopez, J; Webster, R E

    1982-01-01

    The filamentous bacteriophage f1 can be transformed into a spherical particle (spheroid) or an intermediate shortened filament with a flared end (I-forms) by exposure to a chloroform-water interface at 22 or 4 degrees C, respectively. The protein composition of bacteriophage f1 spheroids and I-forms was examined by separating the proteins from the purified. [35S]cysteine-labeled particles by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. Quantitation of the radioactivity on the gels showed that I-forms and spheroids contain the same complement of minor coat proteins as do untreated f1 phage. This composition is unchanged after removal of the DNA, either by digestion with micrococcal nuclease or by centrifugation of the particles through CsCl density gradients, indicating that none of the minor coat proteins is held in the particles solely through an interaction with the DNA. We also examined the location of the A protein in I-forms by decoration with ferritin-conjugated antibodies and examination under the electron microscope and found that the A protein is located specifically at the flared end of the I-form particle, through which the DNA is extruded and at which contraction into spheroids begins. The implications of these results with regard to the orientation of the DNA within the capsid and the process of infection are discussed. Images PMID:7097858

  9. The crystal structure of the thiocyanate-forming protein from Thlaspi arvense, a kelch protein involved in glucosinolate breakdown.

    PubMed

    Gumz, Frauke; Krausze, Joern; Eisenschmidt, Daniela; Backenköhler, Anita; Barleben, Leif; Brandt, Wolfgang; Wittstock, Ute

    2015-09-01

    Kelch repeat-containing proteins are involved in diverse cellular processes, but only a small subset of plant kelch proteins has been functionally characterized. Thiocyanate-forming protein (TFP) from field-penny cress, Thlaspi arvense (Brassicaceae), is a representative of specifier proteins, a group of kelch proteins involved in plant specialized metabolism. As components of the glucosinolate-myrosinase system of the Brassicaceae, specifier proteins determine the profile of bioactive products formed when plant tissue is disrupted and glucosinolates are hydrolyzed by myrosinases. Here, we describe the crystal structure of TaTFP at a resolution of 1.4 Å. TaTFP crystallized as homodimer. Each monomer forms a six-blade β-propeller with a wide "top" and a narrower "bottom" opening with distinct strand-connecting loops protruding far beyond the lower propeller surface. Molecular modeling and mutational analysis identified residues for glucosinolate aglucone and Fe(2+) cofactor binding within these loops. As the first experimentally determined structure of a plant kelch protein, the crystal structure of TaTFP not only enables more detailed mechanistic studies on glucosinolate breakdown product formation, but also provides a new basis for research on the diverse roles and mechanisms of other kelch proteins in plants.

  10. Extracellular matrix-associated proteins form an integral and dynamic system during Pseudomonas aeruginosa biofilm development

    PubMed Central

    Zhang, Weipeng; Sun, Jin; Ding, Wei; Lin, Jinshui; Tian, Renmao; Lu, Liang; Liu, Xiaofen; Shen, Xihui; Qian, Pei-Yuan

    2015-01-01

    Though the essential role of extracellular matrix in biofilm development has been extensively documented, the function of matrix-associated proteins is elusive. Determining the dynamics of matrix-associated proteins would be a useful way to reveal their functions in biofilm development. Therefore, we applied iTRAQ-based quantitative proteomics to evaluate matrix-associated proteins isolated from different phases of Pseudomonas aeruginosa ATCC27853 biofilms. Among the identified 389 proteins, 54 changed their abundance significantly. The increased abundance of stress resistance and nutrient metabolism-related proteins over the period of biofilm development was consistent with the hypothesis that biofilm matrix forms micro-environments in which cells are optimally organized to resist stress and use available nutrients. Secreted proteins, including novel putative effectors of the type III secretion system were identified, suggesting that the dynamics of pathogenesis-related proteins in the matrix are associated with biofilm development. Interestingly, there was a good correlation between the abundance changes of matrix-associated proteins and their expression. Further analysis revealed complex interactions among these modulated proteins, and the mutation of selected proteins attenuated biofilm development. Collectively, this work presents the first dynamic picture of matrix-associated proteins during biofilm development, and provides evidences that the matrix-associated proteins may form an integral and well regulated system that contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of the biofilm. PMID:26029669

  11. Quantitative Protein Profiling of Chlamydia trachomatis Growth Forms Reveals Defense Strategies Against Tryptophan Starvation*

    PubMed Central

    Østergaard, Ole; Follmann, Frank; Olsen, Anja W.; Heegaard, Niels H.; Andersen, Peter; Rosenkrands, Ida

    2016-01-01

    Chlamydia trachomatis is one of the most common sexually transmitted bacterial pathogens in humans. The infection is often asymptomatic and can lead to chronic manifestations. The infectious elementary body and the replicating reticulate body are the two growth forms in the normal developmental cycle. Under the influence of interferon-γ, the normal cycle is disrupted because of tryptophan degradation, leading to a third persistent form, the aberrant reticulate body. For the genital strain C. trachomatis D/UW-3/CX we established a quantitative, label-free proteomic approach, and identified in total 655 out of 903 (73%) predicted proteins, allowing the first quantitative comparison of all three growth forms. Inclusion membrane proteins and proteins involved in translation were more abundant in the reticulate body (RB)1 and aberrant reticulate body (ARB) forms, whereas proteins of the type III Secretion System and the cell envelope were more abundant in the elementary body (EB) form, reflecting the need for these proteins to establish infection and for host interactions. In the interferon-γ induced ARB proteome, the tryptophan synthase subunits were identified as biomarkers with a strong increase from less than 0.05% to 9% of the total protein content, reflecting an inherent defense strategy for the pathogen to escape interferon-γ mediated immune pressure. Furthermore, the total tryptophan content in the ARB form was 1.9-fold lower compared with the EB form, and we demonstrate that modulation of the protein repertoire toward lower abundance of proteins with high tryptophan content, is a mechanism which contributes to establish and maintain chlamydial persistence. Thus, quantitative proteomics provides insights in the Chlamydia defense mechanisms to escape interferon-γ mediated immune pressure. PMID:27784728

  12. New Structural Approaches to Understanding the Disease Related Forms of the Prion Protein

    DTIC Science & Technology

    2006-07-01

    amyloidogenic proteins. Prion diseases are different from other amyloid diseases in that pathogenic (also called scrapie or PrPSc) forms of the prion...that some residues in the 89–145 segment are required for the conformational change to the infectious, scrapie form [16]. A ‘mini-prion’ containing

  13. The Saccharomyces cerevisiae Fin1 protein forms cell cycle-specific filaments between spindle pole bodies.

    PubMed

    van Hemert, Martijn J; Lamers, Gerda E M; Klein, Dionne C G; Oosterkamp, Tjerk H; Steensma, H Yde; van Heusden, G Paul H

    2002-04-16

    The FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion protein is visible as a filament between the two spindle pole bodies. In resting cells the protein is undetectable, and in small-budded cells it is localized in the nucleus. During late mitosis it localizes on the spindle pole bodies. Filaments of cyano fluorescent protein-tagged Fin1 colocalize with filaments of green fluorescent protein-tagged Tub1 only in large-budded cells. By electron and atomic force microscopy we showed that purified recombinant Fin1p self-assembles into filaments with a diameter of approximately 10 nm. Our results indicate that the Fin1 protein forms a cell cycle-specific filament, additional to the microtubules, between the spindle pole bodies of dividing yeast cells.

  14. The Saccharomyces cerevisiae Fin1 protein forms cell cycle-specific filaments between spindle pole bodies

    PubMed Central

    van Hemert, Martijn J.; Lamers, Gerda E. M.; Klein, Dionne C. G.; Oosterkamp, Tjerk H.; Steensma, H. Yde; van Heusden, G. Paul H.

    2002-01-01

    The FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion protein is visible as a filament between the two spindle pole bodies. In resting cells the protein is undetectable, and in small-budded cells it is localized in the nucleus. During late mitosis it localizes on the spindle pole bodies. Filaments of cyano fluorescent protein-tagged Fin1 colocalize with filaments of green fluorescent protein-tagged Tub1 only in large-budded cells. By electron and atomic force microscopy we showed that purified recombinant Fin1p self-assembles into filaments with a diameter of ≈10 nm. Our results indicate that the Fin1 protein forms a cell cycle-specific filament, additional to the microtubules, between the spindle pole bodies of dividing yeast cells. PMID:11929974

  15. SVP-like MADS-box protein from Carya cathayensis forms higher-order complexes.

    PubMed

    Wang, Jingjing; Hou, Chuanming; Huang, Jianqin; Wang, Zhengjia; Xu, Yingwu

    2015-03-01

    To properly regulate plant flowering time and construct floral pattern, MADS-domain containing transcription factors must form multimers including homo- and hetero-dimers. They are also active in forming hetero-higher-order complexes with three to five different molecules. However, it is not well known if a MADS-box protein can also form homo-higher-order complex. In this study a biochemical approach is utilized to provide insight into the complex formation for an SVP-like MADS-box protein cloned from hickory. The results indicated that the protein is a heterogeneous higher-order complex with the peak population containing over 20 monomers. Y2H verified the protein to form homo-complex in yeast cells. Western blot of the hickory floral bud sample revealed that the protein exists in higher-order polymers in native. Deletion assays indicated that the flexible C-terminal residues are mainly responsible for the higher-order polymer formation and the heterogeneity. Current results provide direct biochemical evidences for an active MADS-box protein to be a high order complex, much higher than a quartermeric polymer. Analysis suggests that a MADS-box subset may be able to self-assemble into large complexes, and thereby differentiate one subfamily from the other in a higher-order structural manner. Present result is a valuable supplement to the action of mechanism for MADS-box proteins in plant development.

  16. Pore-forming ability of major outer membrane proteins from Wolinella recta ATCC 33238.

    PubMed Central

    Kennell, W L; Egli, C; Hancock, R E; Holt, S C

    1992-01-01

    Three major outer membrane proteins with apparent molecular masses of 43, 45, and 51 kDa were purified from Wolinella recta ATCC 33238, and their pore-forming abilities were determined by the black lipid bilayer method. The non-heat-modifiable 45-kDa protein (Omp 45) showed no pore-forming activity even at high KCl concentrations. The single-channel conductances in 1 M KCl of the heat-modifiable proteins with apparent molecular masses of 43 kDa (Omp 43) and 51 kDa (Omp 51) were 0.49 and 0.60 nS, respectively. The proteins formed nonselective channels and, as determined by experiments of ion selectivity and zero-current potential, were weakly anion selective. Images PMID:1370429

  17. Development of a polymerase chain reaction assay for species identification of goose and mule duck in foie gras products.

    PubMed

    Rodrı X0301 Guez, Miguel A; Garcı X0301 A, Teresa; González, Isabel; Asensio, Luis; Mayoral, Belén; López-Calleja, Inés; Hernández, Pablo E; Martı X0301 N, Rosario

    2003-12-01

    Polymerase chain reaction amplification of a conserved region of the α-actin gene has been used for the specific identification of goose (Anser anser) and mule duck (Anas platyrhynchos×Cairina moschata) foie gras. Universal primers were used for the amplification of a DNA fragment containing three introns and four exons of the α-actin gene in goose and mule duck. Sequence analysis of the amplified fragments was necessary for the design of forward species-specific primers in the goose and mule duck α-actin genes. The use of species-specific forward primers, together with a reverse universal primer, produced amplicons of different length, allowing clear identification of goose and mule duck foie gras samples. Analysis of experimental mixtures demonstrated that 1% of duck can be easily detected in goose foie gras using the PCR method developed here. This genetic marker can be very useful for the accurate identification of these two species in foie gras products.

  18. The phosphorylated form of the ORF3 protein of hepatitis E virus interacts with its non-glycosylated form of the major capsid protein, ORF2.

    PubMed

    Tyagi, Shweta; Korkaya, Hasan; Zafrullah, Mohammad; Jameel, Shahid; Lal, Sunil K

    2002-06-21

    Hepatitis E virus (HEV) is a human RNA virus containing three open reading frames. Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein, which exists in a glycosylated and non-glycosylated form; and ORF3 codes for a phosphoprotein of undefined function. Using fluorescence-based colocalization, yeast two-hybrid experiments, transiently transfected COS-1 cell co-immunoprecipitation, and cell-free coupled transcription-translation techniques, we have shown that the ORF3 protein interacts with the ORF2 protein. The domains involved in this ORF2-ORF3 association have been identified and mapped. Our deletion analysis showed that a 25-amino acid region (residues 57-81) of the ORF3 protein is required for this interaction. Using a Mexican HEV isolate, site-directed mutagenesis of ORF3, and a phosphatase digestion assay, we showed that the ORF2-ORF3 interaction is dependent upon the phosphorylation at Ser(80) of ORF3. Finally, using COS-1 cell immunoprecipitation experiments, we found that the phosphorylated ORF3 protein preferentially interacts with the non-glycosylated ORF2 protein. These findings were confirmed using tunicamycin inhibition, point mutants, and deletion mutants expressing only non-glycosylated ORF2. ORF3 maps in the structural region of the HEV genome and now interacts with the major capsid protein, ORF2, in a post-translational modification-dependent manner. Such an interaction of ORF2 with ORF3 suggests a possible well regulated role for ORF3 in HEV structural assembly.

  19. Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes

    PubMed Central

    Dygut, Jacek; Kalinowska, Barbara; Banach, Mateusz; Piwowar, Monika; Konieczny, Leszek; Roterman, Irena

    2016-01-01

    The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific) hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains—In this context we can distinguish: (1) Shared hydrophobic cores (spanning the whole dimer); (2) Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above) and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model. PMID:27763556

  20. Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes.

    PubMed

    Dygut, Jacek; Kalinowska, Barbara; Banach, Mateusz; Piwowar, Monika; Konieczny, Leszek; Roterman, Irena

    2016-10-18

    The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific) hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains-In this context we can distinguish: (1) Shared hydrophobic cores (spanning the whole dimer); (2) Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above) and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model.

  1. The Native Form and Maturation Process of Hepatitis C Virus Core Protein

    PubMed Central

    Yasui, Kohichiroh; Wakita, Takaji; Tsukiyama-Kohara, Kyoko; Funahashi, Shin-Ichi; Ichikawa, Masumi; Kajita, Tadahiro; Moradpour, Darius; Wands, Jack R.; Kohara, Michinori

    1998-01-01

    The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles. PMID:9621068

  2. Purification in a functional form of the terminal protein of Bacillus subtilis phage phi 29.

    PubMed Central

    Prieto, I; Lázaro, J M; García, J A; Hermoso, J M; Salas, M

    1984-01-01

    Phage phi 29 terminal protein, p3, essentially pure, was isolated in a denatured form from viral particles, and anti-p3 antiserum was obtained. A radioimmunoassay to detect and quantitate protein p3 was developed. By using this assay, native protein p3 was highly purified from Escherichia coli cells harboring a gene 3-containing recombinant plasmid. After three purification steps, the protein was more than 96% pure; its amino acid composition was very similar to that deduced from the nucleotide sequence of gene 3. The purified protein was active in the formation of the covalent p3-dAMP initiation complex when supplemented with extracts of B. subtilis infected with a sus mutant of phi 29 in gene 3. No DNA polymerase or ATPase activities were present in the final preparation of protein p3. Images PMID:6424120

  3. G4IPDB: A database for G-quadruplex structure forming nucleic acid interacting proteins

    PubMed Central

    Mishra, Subodh Kumar; Tawani, Arpita; Mishra, Amit; Kumar, Amit

    2016-01-01

    Nucleic acid G-quadruplex structure (G4) Interacting Proteins DataBase (G4IPDB) is an important database that contains detailed information about proteins interacting with nucleic acids that forms G-quadruplex structures. G4IPDB is the first database that provides comprehensive information about this interaction at a single platform. This database contains more than 200 entries with details of interaction such as interacting protein name and their synonyms, their UniProt-ID, source organism, target name and its sequences, ∆Tm, binding/dissociation constants, protein gene name, protein FASTA sequence, interacting residue in protein, related PDB entries, interaction ID, graphical view, PMID, author’s name and techniques that were used to detect their interactions. G4IPDB also provides an efficient web-based “G-quadruplex predictor tool” that searches putative G-quadruplex forming sequences simultaneously in both sense and anti-sense strands of the query nucleotide sequence and provides the predicted G score. Studying the interaction between proteins and nucleic acids forming G-quadruplex structures could be of therapeutic significance for various diseases including cancer and neurological disease, therefore, having detail information about their interactions on a single platform would be helpful for the discovery and development of novel therapeutics. G4IPDB can be routinely updated (twice in year) and freely available on http://bsbe.iiti.ac.in/bsbe/ipdb/index.php. PMID:27905517

  4. The fusion of lipid droplets is involved in fat loss during cooking of duck "foie gras".

    PubMed

    Théron, L; Astruc, T; Bouillier-Oudot, M; Molette, C; Vénien, A; Peyrin, F; Vitezica, Z G; Fernandez, X

    2011-12-01

    Fat loss during cooking of duck "foie gras" is the main quality issue in processing plants. To better understand this phenomenon, a histological and ultrastructural study was conducted. The aim was to characterize changes in lipid droplets of duck "foie gras" related to fat loss during cooking. Ten fatty livers were sampled before and after cooking and prepared for optical and transmission electron microscopy. In raw livers, the lipid droplets were nearly spherical while after cooking, they were larger and lost their spherical shape. We also observed a decrease in the number of droplets after cooking, probably due to droplet fusion caused by the heat treatment. Before cooking, there were fewer lipid droplets and a higher osmium tetroxyde staining intensity in the fatty liver, which later gave a lower technological yield. Fat loss during cooking was higher when there was more fusion of lipid droplets before cooking.

  5. Development of GRAS strains for nutraceutical production using systems and synthetic biology approaches: advances and prospects.

    PubMed

    Liu, Long; Guan, Ningzi; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Chen, Jian

    2017-03-01

    Nutraceuticals are food substances with medical and health benefits for humans. Limited by complicated procedures, high cost, low yield, insufficient raw materials, resource waste, and environment pollution, chemical synthesis and extraction are being replaced by microbial synthesis of nutraceuticals. Many microbial strains that are generally regarded as safe (GRAS) have been identified and developed for the synthesis of nutraceuticals, and significant nutraceutical production by these strains has been achieved. In this review, we systematically summarize recent advances in nutraceutical research in terms of physiological effects on health, potential applications, drawbacks of traditional production processes, characteristics of production strains, and progress in microbial fermentation. Recent advances in systems and synthetic biology techniques have enabled comprehensive understanding of GRAS strains and its wider applications. Thus, these microbial strains are promising cell factories for the commercial production of nutraceuticals.

  6. Effect of different GRAS compounds in the control of apples blue mould.

    PubMed

    Venditti, T; Cubaiu, L; Ladu, G; D'Hallewin, G

    2013-01-01

    The most important pathogen for apples is Penicillium expansum that is the causal organism of blue mould. Postharvest losses are controlled with chemical fungicides such as TBZ but a growing concern for human health and a greater awareness for environmental conservation have multiplied the studies on new ecological technologies. In the search of new environment and consumer friendly technologies that can reduce toxic residues, the use of GRAS compounds represent a valid alternative to the use of synthetic postharvest fungicides. The aims of the present work were: (1) To evaluate the effectiveness of different GRAS compounds in the control of P. expansum in artificially inoculated fruit; (2) To assess the capability of injured and treated fruit with GRAS compounds, used alone or combined, to heal the wounds in order to resist to infection. Fruit was injured with a steel rod and after 1 hour was (1) Inoculated with the pathogen and after 24 hours treated or (2) Treated and after 24 hours inoculated. Treatments were performed with the following compounds: sodium bicarbonate (SBC), boric acid (BA) and calcium chloride (CC) by using a 1% solution for all of them. After 9 or 14 days fruit lesion diameters were assessed. In the trial (1) the combined treatment with BA and SBC was the most effective reducing the lesion diameter by 86.5% with respect to untreated fruit, after 9 days from infection. A good pathogen control was also obtained with BA used alone or combined with CC. When the treatment was performed before infection the best results were achieved with the combination of SBC and CC, with 87% of reduction of the lesion diameter. The addition of CC also reduced the lesion if combined with BA (66.8%). These preliminary results showed that GRAS compounds can be effective in reducing blue mold by a direct effect on the pathogen, and by modulating fruit responses enhancing host resistance.

  7. Cell surface expression of glycosylated, nonglycosylated, and truncated forms of a cytoplasmic protein pyruvate kinase.

    PubMed

    Hiebert, S W; Lamb, R A

    1988-09-01

    The soluble cytoplasmic protein pyruvate kinase (PK) has been expressed at the cell surface in a membrane-anchored form (APK). The hybrid protein contains the NH2-terminal signal/anchor domain of a class II integral membrane protein (hemagglutinin/neuraminidase, of the paramyxovirus SV5) fused to the PK NH2 terminus. APK contains a cryptic site that is used for N-linked glycosylation but elimination of this site by site-specific mutagenesis does not prevent cell surface localization. Truncated forms of the APK molecule, with up to 80% of the PK region of APK removed, can also be expressed at the cell surface. These data suggest that neither the complete PK molecule nor its glycosylation are necessary for intracellular transport of PK to the cell surface, and it is possible that specific signals may not be needed in the ectodomain of this hybrid protein to specify cell surface localization.

  8. Structure of Ocr from bacteriophage T7, a protein that mimics B-form DNA.

    PubMed

    Walkinshaw, M D; Taylor, P; Sturrock, S S; Atanasiu, C; Berge, T; Henderson, R M; Edwardson, J M; Dryden, D T F

    2002-01-01

    We have solved, by X-ray crystallography to a resolution of 1.8 A, the structure of a protein capable of mimicking approximately 20 base pairs of B-form DNA. This ocr protein, encoded by gene 0.3 of bacteriophage T7, mimics the size and shape of a bent DNA molecule and the arrangement of negative charges along the phosphate backbone of B-form DNA. We also demonstrate that ocr is an efficient inhibitor in vivo of all known families of the complex type I DNA restriction enzymes. Using atomic force microscopy, we have also observed that type I enzymes induce a bend in DNA of similar magnitude to the bend in the ocr molecule. This first structure of an antirestriction protein demonstrates the construction of structural mimetics of long segments of B-form DNA.

  9. Production in Pichia pastoris of protein-based polymers with small heterodimer-forming blocks.

    PubMed

    Domeradzka, Natalia E; Werten, Marc W T; de Vries, Renko; de Wolf, Frits A

    2016-05-01

    Some combinations of leucine zipper peptides are capable of forming α-helical heterodimeric coiled coils with very high affinity. These can be used as physical cross-linkers in the design of protein-based polymers that form supramolecular structures, for example hydrogels, upon mixing solutions containing the complementary blocks. Such two-component physical networks are of interest for many applications in biomedicine, pharmaceutics, and diagnostics. This article describes the efficient secretory production of A and B type leucine zipper peptides fused to protein-based polymers in Pichia pastoris. By adjusting the fermentation conditions, we were able to significantly reduce undesirable proteolytic degradation. The formation of A-B heterodimers in mixtures of the purified products was confirmed by size exclusion chromatography. Our results demonstrate that protein-based polymers incorporating functional heterodimer-forming blocks can be produced with P. pastoris in sufficient quantities for use in future supramolecular self-assembly studies and in various applications.

  10. Identification of lactic acid bacteria involved in the spoilage of pasteurized "foie gras" products.

    PubMed

    Matamoros, S; André, S; Hue, I; Prévost, H; Pilet, M F

    2010-07-01

    The spoiling microflora of a re-packaged French "foie gras" product was studied. A total of 54 isolates, originating from two different factories, were identified using phenotypical and molecular methods (partial 16S rDNA sequencing). Weissella viridescens was the main species detected in the products from factory 1 (64% of the isolates). These products had a low lactic acid concentration and were considered as non-spoiled. The microflora of factory 2 was dominated mainly by the genus Lactobacillus (95% of the isolates), and the high lactic acid concentration of these products was linked with a strong spoilage. Among the 30 Lactobacillus strains, three species were predominant: Lactobacillus sakei (nine isolates), Lactobacillus coryniformis (eight isolates) and Lactobacillus paraplantarum (five isolates). Challenge tests were performed to confirm the involvement of the Lactobacillus strains in the spoilage of the product. Sterile "foie gras" samples were inoculated with 14 LAB strains from the collection. The most acidifying strains belonged to the species L. sakei, Lactobacillus plantarum and L. paraplantarum. This confirmed the role of the strains from the Lactobacillus genus as the main spoilers of "foie gras" products and will be useful to design new quality protocols and extend the shelf-life of these products.

  11. Statistical analysis of protein structures suggests that buried ionizable residues in proteins are hydrogen bonded or form salt bridges.

    PubMed

    Bush, Jeffrey; Makhatadze, George I

    2011-07-01

    It is well known that nonpolar residues are largely buried in the interior of proteins, whereas polar and ionizable residues tend to be more localized on the protein surface where they are solvent exposed. Such a distribution of residues between surface and interior is well understood from a thermodynamic point: nonpolar side chains are excluded from the contact with the solvent water, whereas polar and ionizable groups have favorable interactions with the water and thus are preferred at the protein surface. However, there is an increasing amount of information suggesting that polar and ionizable residues do occur in the protein core, including at positions that have no known functional importance. This is inconsistent with the observations that dehydration of polar and in particular ionizable groups is very energetically unfavorable. To resolve this, we performed a detailed analysis of the distribution of fractional burial of polar and ionizable residues using a large set of ˜2600 nonhomologous protein structures. We show that when ionizable residues are fully buried, the vast majority of them form hydrogen bonds and/or salt bridges with other polar/ionizable groups. This observation resolves an apparent contradiction: the energetic penalty of dehydration of polar/ionizable groups is paid off by favorable energy of hydrogen bonding and/or salt bridge formation in the protein interior. Our conclusion agrees well with the previous findings based on the continuum models for electrostatic interactions in proteins.

  12. Rpf Proteins Are the Factors of Reactivation of the Dormant Forms of Actinobacteria.

    PubMed

    Nikitushkin, V D; Demina, G R; Kaprelyants, A S

    2016-12-01

    As the response to unfavorable growth conditions, nonsporulating mycobacteria transform into the dormant state with the concomitant formation of the specialized dormant forms characterized by low metabolic activity and resistance to antibiotics. Such dormant cells can be reactivated under the influence of several factors including proteins of Rpf (Resuscitation promoting factor) family, which possess peptidoglycan hydrolase activity and were considered to belong to the group of the autocrine growth factors of the bacteria. Remarkable interest toward Rpf family is determined by its participation in resuscitation of the dormant forms of Mycobacterium tuberculosis, what in turn is the key element in resuscitation of the latent tuberculosis - an infectious disease that affects one third of the World's population. Experiments with Rpf mutant forms and with strains deleted in these proteins revealed a relationship between the enzymatic activity of this protein and its ability to resuscitate mycobacteria both in vitro and in vivo. This review discusses possible mechanisms of Rpf action including those related to possible participation of the products of mycobacterial Rpf-mediated cell wall hydrolysis (muropeptides) as signaling molecules. The unique ability of Rpf proteins to resuscitate the dormant forms of mycobacteria and to stimulate their proliferation would allow these proteins to occupy their niche in medicine - in diagnostics and in creation of antituberculosis subunit vaccines.

  13. Carbohydrate-reactive, pore-forming outer membrane proteins of Aeromonas hydrophila.

    PubMed Central

    Quinn, D M; Atkinson, H M; Bretag, A H; Tester, M; Trust, T J; Wong, C Y; Flower, R L

    1994-01-01

    Two outer membrane proteins of Aeromonas hydrophila A6, isolated in a one-step affinity chromatography process based on carbohydrate reactivity, were found to be pore-forming molecules in artificial planar bilayer membranes. These carbohydrate-reactive outer membrane proteins (CROMPs; M(r)s, 40,000 and 43,000) were subjected to amino acid analysis. The amino acid profiles for these two outer membrane proteins were almost identical. A partial protein sequence of a 14-amino-acid fragment of the 40,000-Da protein revealed homology with outer membrane porins of Escherichia coli and A. hydrophila. CROMPs were compared with carbohydrate-reactive porins also extracted from outer membranes of A. hydrophila A6. These porins were isolated by using standard porin purification techniques (insolubility in 2% sodium dodecyl sulfate, solubility in 0.4 M NaCl, and Sephacryl S-200 gel filtration), and then Synsorb H type 2 affinity chromatography was done. The physical and functional properties of the carbohydrate-reactive porins and CROMPs were found to be identical. On the basis of pore-forming properties in planar lipid bilayers and channel inhibition with maltotriose solutions, a nonspecific, general diffusion porin and a LamB-like maltoporin were identified in both CROMP and carbohydrate-reactive porin preparations. To our knowledge, the use of carbohydrate reactivity to isolate channel-forming proteins from bacterial outer membranes has not been reported previously. Images PMID:7520425

  14. More Than a Pore: The Interplay of Pore-Forming Proteins and Lipid Membranes.

    PubMed

    Ros, Uris; García-Sáez, Ana J

    2015-06-01

    Pore-forming proteins (PFPs) punch holes in their target cell membrane to alter their permeability. Permeabilization of lipid membranes by PFPs has received special attention to study the basic molecular mechanisms of protein insertion into membranes and the development of biotechnological tools. PFPs act through a general multi-step mechanism that involves (i) membrane partitioning, (ii) insertion into the hydrophobic core of the bilayer, (iii) oligomerization, and (iv) pore formation. Interestingly, PFPs and membranes show a dynamic interplay. As PFPs are usually produced as soluble proteins, they require a large conformational change for membrane insertion. Moreover, membrane structure is modified upon PFPs insertion. In this context, the toroidal pore model has been proposed to describe a pore architecture in which not only protein molecules but also lipids are directly involved in the structure. Here, we discuss how PFPs and lipids cooperate and remodel each other to achieve pore formation, and explore new evidences of protein-lipid pore structures.

  15. Electrostatics promotes molecular crowding and selects the aggregation pathway in fibril-forming protein solutions

    NASA Astrophysics Data System (ADS)

    Raccosta, S.; Blanco, M.; J. Roberts, C.; Martorana, V.; Manno, M.

    2016-05-01

    The role of intermolecular interaction in fibril-forming protein solutions and its relation with molecular conformation are crucial aspects for the control and inhibition of amyloid structures. Here, we study the fibril formation and the protein-protein interactions for two proteins at acidic p H, lysozyme and α -chymotrypsinogen. By using light scattering experiments and the Kirkwood-Buff integral approach, we show how concentration fluctuations are damped even at moderate protein concentrations by the dominant long-ranged electrostatic repulsion, which determines an effective crowded environment. In denaturing conditions, electrostatic repulsion keeps the monomeric solution in a thermodynamically metastable state, which is escaped through kinetically populated conformational sub-states. This explains how electrostatics acts as a gatekeeper in selecting a specific aggregation pathway.

  16. Isozymic forms of rat brain CA/sup 2 +/-activated and phospholipid-dependent protein kinase

    SciTech Connect

    Huang, K.P.; Huang, F.L.

    1986-05-01

    Three forms of Ca/sup 2 +/-activated and phospholipid-dependent protein kinase (protein kinase C) were purified from the cytosolic fraction of rat brain. These enzymes, designated as type I, II, and III protein kinase C, all have the similar molecular weight of 80 Kd, bind (/sup 3/H)-phorbol dibutyrate in the presence of Ca/sup 2 +/, and undergo autophosphorylation in the presence of Ca/sup 2 +/, phosphatidylserine, and diolein. Autophosphorylation of these kinases resulted in an incorporation of 1- 1.5 mol /sup 32/P/mol of enzyme. Analysis of the /sup 32/P-labeled tryptic peptides derived from the autophosphorylated protein kinase C by two-dimensional peptide mapping revealed that these kinases had different sites of autophosphorylation. Phosphoamino acid analysis revealed that the type I and type III protein kinase C mainly phosphorylated at Ser residue while the type II kinase phosphorylated at both Ser and Thr residues. In addition, polyclonal antibodies previously prepared against a mixed enzyme fraction preferentially inhibited the type I and type II enzymes but less effectively toward the type III enzyme. Monoclonal antibody specifically against the type II protein kinase C did not inhibit the type I or type III enzymes. These kinases also had different susceptibility to limited proteolysis by trypsin and upon proteolytic degradation they generate distinct fragments. These results demonstrate the presence of isozymic forms of protein kinase C in rat brain.

  17. Thermal effect on Aequorea green fluorescent protein anionic and neutral chromophore forms fluorescence.

    PubMed

    dos Santos, Andrea Martins

    2012-01-01

    The emission behaviour of Aequorea green fluorescent protein (A-GFP) chromophore, in both neutral (N) and anionic (A) form, was studied in the temperature range from 20 °C to 75 °C and at pH = 7. Excitation wavelengths of 399 nm and 476 nm were applied to probe the N and A forms environment, respectively. Both forms exhibit distinct fluorescence patterns at high temperature values. The emission quenching rate, following a temperature increase, is higher for the chromophore N form as a result of the hydrogen bond network weakening. The chromophore anionic form emission maximum is red shifted, upon temperature increase, due to a charge transfer process occurring after A form excitation.

  18. Soluble Proteins Form Film by the Treatment of Low Temperature Plasma

    NASA Astrophysics Data System (ADS)

    Ikehara, Sanae; Sakakita, Hajime; Ishikawa, Kenji; Akimoto, Yoshihiro; Nakanishi, Hayao; Shimizu, Nobuyuki; Hori, Masaru; Ikehara, Yuzuru

    2015-09-01

    It has been pointed out that low temperature plasma in atmosphere was feasible to use for hemostasis without heat injury. Indeed, earlier studies demonstrated that low temperature plasma played an important role to stimulate platelets to aggregate and turned on the proteolytic activities of coagulation factors, resulting in the acceleration of the natural blood coagulation process. On the other hands, our developed equips could immediately form clots upon the contact with plasma flair, while the histological appearance was different from natural coagulation. Based on these findings in formed clots, we sought to determine if plasma flair supplied by our devices was capable of forming film using a series of soluble proteins Following plasma treatment, films were formed from bovine serum albumin, and the other plasma proteins at physiological concentration. Analysis of trans-electron microscope demonstrated that plasma treatment generated small protein particles and made them fuse to be larger aggregations The combined results demonstrated that plasma are capable of aggregating soluble proteins and that platelets and coagulation factors are not necessary for plasma induced blood coagulation. Supported in part by Grants-in-Aid for Scientific Research on Priority Area (21590454, 24590498, and 24108006 to Y. I.).

  19. The Postsynaptic Density Proteins Homer and Shank Form a Polymeric Network Structure

    SciTech Connect

    Hayashi, M.; Tang, C; Verpelli, C; Narayanan, R; Stearns, M; Xu, R; Li, H; Sala, C; Hayashi, Y

    2009-01-01

    The postsynaptic density (PSD) is crucial for synaptic functions, but the molecular architecture retaining its structure and components remains elusive. Homer and Shank are among the most abundant scaffolding proteins in the PSD, working synergistically for maturation of dendritic spines. Here, we demonstrate that Homer and Shank, together, form a mesh-like matrix structure. Crystallographic analysis of this region revealed a pair of parallel dimeric coiled coils intercalated in a tail-to-tail fashion to form a tetramer, giving rise to the unique configuration of a pair of N-terminal EVH1 domains at each end of the coiled coil. In neurons, the tetramerization is required for structural integrity of the dendritic spines and recruitment of proteins to synapses. We propose that the Homer-Shank complex serves as a structural framework and as an assembly platform for other PSD proteins.

  20. Pore-Forming Proteins as Mediators of Novel Epigenetic Mechanism of Epilepsy

    PubMed Central

    Surguchov, Andrei; Surgucheva, Irina; Sharma, Mukut; Sharma, Ram; Singh, Vikas

    2017-01-01

    Epilepsy is a disorder of the brain characterized by an enduring predisposition to generate epileptic seizures. In the last two decades, numerous gene defects underlying different forms of epilepsy have been identified with most of these genes encoding ion channel proteins. Despite these developments, the etiology of majority of non-familial epilepsies has no known associated genetic mutations and cannot be explained by defects in identified ion channels alone. We hypothesize that de novo formation of ion channels by naturally unfolded proteins (NUPs) increases neuronal excitability. Altered ionic homeostasis may initiate/contribute to cellular cascades related to epileptogenesis in susceptible individuals. Here, we consider two small proteins, namely, α-synuclein and stefin B, as prototypical candidates to illustrate the underlying mechanism(s). Previous work points to an association between epilepsy and α-synuclein or stefin B, but the mechanism(s) underlying such association remains elusive. We review the evidence to link the structure–function of these proteins with disease processes. Epigenetic mechanisms unrelated to altered DNA sequence(s) that may affect epileptogenesis include transcriptional or posttranscriptional regulation. Such epigenetic mechanisms or their combination(s) enhance the levels of these proteins and as a result the ability to form annular structures, which upon incorporation into membrane form novel ion channels and disturb intracellular ion homeostasis. Alternative epigenetic mechanisms may change amyloidogenic proteins by posttranslational modifications, thereby increasing their propensity to form channels. Further research elucidating the details about the formation of ion channels through these mechanisms and their role in epileptogenesis may define new molecular targets and guide the development of new drug targets. PMID:28149289

  1. Differential range and activity of various forms of the Hedgehog protein

    PubMed Central

    Dawber, Rebecca J; Hebbes, Stephen; Herpers, Bram; Docquier, France; van den Heuvel, Marcel

    2005-01-01

    Background The Hedgehog (Hh) family of secreted proteins act as extracellular messengers to control and coordinate growth and differentiation. The mechanism by which Hh protein travels across a field of cells, and results in a range of specific effects relating to the distance from the source, has been the subject of much debate. It has been suggested that the range and activity of the pathway can be linked to modifications of the Hh protein, specifically the addition of lipid groups at N- and C-terminal sites. Results Here we have addressed the potency of different forms of Hh protein by expressing these in Drosophila, where we are able to precisely establish pathway activity and range in naïve but responsive tissues. As expected, a construct that can produce all forms of Hh recapitulates endogenous signaling potencies. In comparison, expression of a form that lacks the cholesterol moiety (HhN) leads to an extended range, but the product is less effective at inducing maximal Hh responses. Expression of a point mutant that lacks the N-terminal palmitate binding site shows that the palmitoylation of Hh is absolutely required for activity in this system. Conclusion We conclude that the addition of the cholesterol moiety limits the range of the protein and is required for maximal activity, while addition of palmitate is required for all activity. These findings have implications for understanding how Hedgehog proteins move, and thus their potential at influencing distant sites, and concomitantly, how modifications of the signaling protein can affect the efficacy of the response in exposed cells. PMID:16197551

  2. Assembling the puzzle: Oligomerization of α-pore forming proteins in membranes☆

    PubMed Central

    García-Sáez, Ana J.

    2016-01-01

    Pore forming proteins (PFPs) share the ability of creating pores that allow the passage of ions, proteins or other constituents through a wide variety of target membranes, ranging from bacteria to humans. They often cause cell death, as pore formation disrupts the membrane permeability barrier required for maintaining cell homeostasis. The organization into supramolecular complexes or oligomers that pierce the membrane is a common feature of PFPs. However, the molecular pathway of self-assembly and pore opening remains unclear. Here, we review the most recent discoveries in the mechanism of membrane oligomerization and pore formation of a subset of PFPs, the α-PFPs, whose pore-forming domains are formed by helical segments. Only now we are starting to grasp the molecular details of their function, mainly thanks to the introduction of single molecule microscopy and nanoscopy techniques. PMID:26375417

  3. Complement Protein C1q Forms a Complex with Cytotoxic Prion Protein Oligomers

    PubMed Central

    Erlich, Paul; Dumestre-Pérard, Chantal; Ling, Wai Li; Lemaire-Vieille, Catherine; Schoehn, Guy; Arlaud, Gérard J.; Thielens, Nicole M.; Gagnon, Jean; Cesbron, Jean-Yves

    2010-01-01

    A growing number of studies have investigated the interaction between C1q and PrP, but the oligomeric form of PrP involved in this interaction remains to be determined. Aggregation of recombinant full-length murine PrP in the presence of 100 mm NaCl allowed us to isolate three different types of oligomers by size-exclusion chromatography. In contrast to PrP monomers and fibrils, these oligomers activate the classical complement pathway, the smallest species containing 8–15 PrP protomers being the most efficient. We used Thioflavine T fluorescence to monitor PrP aggregation and showed that, when added to the reaction, C1q has a cooperative effect on PrP aggregation and leads to the formation of C1q-PrP complexes. In these complexes, C1q interacts through its globular domains preferentially with the smallest oligomers, as shown by electron microscopy, and retains the ability to activate the classical complement pathway. Using two cell lines, we also provide evidence that C1q inhibits the cytotoxicity induced by the smallest PrP oligomers. The cooperative interaction between C1q and PrP could represent an early step in the disease, where it prevents elimination of the prion seed, leading to further aggregation. PMID:20410306

  4. Functional analysis of stress protein data in a flor yeast subjected to a biofilm forming condition.

    PubMed

    Moreno-García, Jaime; Mauricio, Juan Carlos; Moreno, Juan; García-Martínez, Teresa

    2016-06-01

    In this data article, an OFFGEL fractionator coupled to LTQ Orbitrap XL MS equipment and a SGD filtering were used to detect in a biofilm-forming flor yeast strain, the maximum possible number of stress proteins under the first stage of a biofilm formation conditions (BFC) and under an initial stage of fermentation used as reference, so-called non-biofilm formation condition (NBFC). Protein functional analysis - based on cellular components and biological process GO terms - was performed for these proteins through the SGD Gene Ontology Slim Mapper tool. A detailed analysis and interpretation of the data can be found in "Stress responsive proteins of a flor yeast strain during the early stages of biofilm formation" [1].

  5. 21 CFR 184.1498 - Microparticulated protein product.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Affirmed as GRAS § 184.1498 Microparticulated protein product. (a) Microparticulated protein product is prepared from egg whites or milk protein or a combination of egg whites and milk protein. These protein... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Microparticulated protein product....

  6. Members of the evolutionarily conserved PMT family of protein O-mannosyltransferases form distinct protein complexes among themselves.

    PubMed

    Girrbach, Verena; Strahl, Sabine

    2003-04-04

    Protein O-mannosyltransferases (PMTs) initiate the assembly of O-mannosyl glycans, an essential protein modification. Since PMTs are evolutionarily conserved in fungi but are absent in green plants, the PMT family is a putative target for new antifungal drugs, particularly in fighting the threat of phytopathogenic fungi. The PMT family is phylogenetically classified into PMT1, PMT2, and PMT4 subfamilies, which differ in protein substrate specificity. In the model organism Saccharomyces cerevisiae as well as in many other fungi the PMT family is highly redundant, and only the simultaneous deletion of PMT1/PMT2 and PMT4 subfamily members is lethal. In this study we analyzed the molecular organization of PMT family members in S. cerevisiae. We show that members of the PMT1 subfamily (Pmt1p and Pmt5p) interact in pairs with members of the PMT2 subfamily (Pmt2p and Pmt3p) and that Pmt1p-Pmt2p and Pmt5p-Pmt3p complexes represent the predominant forms. Under certain physiological conditions, however, Pmt1p interacts also with Pmt3p, and Pmt5p with Pmt2p, suggesting a compensatory cooperation that guarantees the maintenance of O-mannosylation. Unlike the PMT1/PMT2 subfamily members, the single member of the PMT4 subfamily (Pmt4p) acts as a homomeric complex. Using mutational analyses we demonstrate that the same conserved protein domains underlie both heteromeric and homomeric interactions, and we identify an invariant arginine residue of transmembrane domain two as essential for the formation and/or stability of PMT complexes in general. Our data suggest that protein-protein interactions between the PMT family members offer a point of attack to shut down overall protein O-mannosylation in fungi.

  7. Imbalanced Expression of Vcan mRNA Splice Form Proteins Alters Heart Morphology and Cellular Protein Profiles

    PubMed Central

    Burns, Tara A.; Dours-Zimmermann, Maria T.; Zimmermann, Dieter R.; Krug, Edward L.; Comte-Walters, Susana; Reyes, Leticia; Davis, Monica A.; Schey, Kevin L.; Schwacke, John H.; Kern, Christine B.; Mjaatvedt, Corey H.

    2014-01-01

    The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart’s pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican’s expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan(tm1Zim), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan(tm1Zim) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles. PMID:24586547

  8. β-hairpin-forming peptides; models of early stages of protein folding

    PubMed Central

    Lewandowska, Agnieszka; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A.

    2010-01-01

    Formation of β-hairpins is considered the initial step of folding of many proteins and, consequently, peptides constituting the β-hairpin sequence of proteins (the β-hairpin-forming peptides) are considered as models of early stages of protein folding. In this article, we discuss the results of experimental studies (circular-dichroism, infrared and nuclear magnetic resonance spectroscopy, and differential scanning calorimetry) of the structure of β-hairpin-forming peptides excised from the B1 domain of protein G, which are known to fold on their own. We demonstrate that local interactions at the turn sequence and hydrophobic interactions between nonpolar residues are the dominant structure-determining factors, while there is no convincing evidence that stable backbone hydrogen bonds are formed in these peptides in aqueous solution. Consequently, the most plausible mechanism for folding of the β-hairpin sequence appears to be the broken-zipper mechanism consisting of the following three steps: (i) bending the chain at the turn sequence owing to favorable local interactions, (ii) formation of loose hydrophobic contacts between nonpolar residues, which occur close to the contacts in the native structure of the protein but not exactly in the same position and, finally, (iii) formation of backbone hydrogen bonds and locking the hydrophobic contacts in the native positions as a hydrophobic core develops, sufficient to dehydrate the backbone peptide groups. This mechanism provides sufficient uniqueness (contacts form between residues that become close together because the chain is bent at the turn position) and robustness (contacts need not occur at once in the native positions) for folding a β-hairpin sequence. PMID:20494507

  9. Protein synthesis patterns of Paracoccidiodes brasiliensis isolates in stage-specific forms and during cellular differentiation.

    PubMed

    Salem-Izacc, S M; Jesuino, R S; Brito, W A; Pereira, M; Felipe, M S; Soares, C M

    1997-01-01

    In this paper we compared the protein synthesis patterns of Paracoccidioides brasiliensis isolates. The protein profiles were compared for both yeast and mycelial forms and similarity analysis among them was performed by calculating similarity matrices and grouping the isolates in dendrograms. The examined isolates exhibited highly variable cellular morphology at 36 degrees C, when typical yeast cells were expected. On the other hand, at 26 degrees C all the isolates showed mycelial morphology. The analysis of protein synthesis profiles made it possible to cluster the P. brasiliensis isolates into groups that correlated with the morphological data. Interestingly, growth at 36 degrees C strongly decreased the heterogeneity of protein synthesis patterns seen in mycelial isolates. It was possible to cluster the isolates grown at 36 degrees C in three groups based on their two-dimensional protein synthesis analysis. The similarity index observed among the mycelial isolates was lower than that obtained with yeast cells, suggesting a more homogenous gene expression pattern in the host-adapted form than in the saprobic phase.

  10. Apoptotic activity of a nuclear form of mitogaligin, a cell death protein

    SciTech Connect

    Gonzalez, Patrick; Robinet, Pauline; Charpentier, Stephane; Mollet, Lucile; Normand, Thierry; Dubois, Martine; Legrand, Alain

    2009-01-23

    Galig, an internal gene to the galectin-3 gene, encodes two proteins and induces cell death in human cells. Mitogaligin, one of these proteins, contains a mitochondrial targeting sequence and promotes the release of cytochrome c into the cytosol. Here, we show that mitogaligin can also localize to nucleus. The nuclear form of mitogaligin induced cell death through a pathway exhibiting typical properties of apoptosis. These observations indicate for the first time that mitogaligin expresses cytotoxic properties not only when addressed to mitochondria but also when targeted to the nucleus.

  11. Activation of human natural killer cells by the soluble form of cellular prion protein

    SciTech Connect

    Seong, Yeon-Jae; Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon; Park, Bum-Chan; Park, Su-Hyung; Park, Young Woo; Shin, Eui-Cheol

    2015-08-21

    Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.

  12. CotG-Like Modular Proteins Are Common among Spore-Forming Bacilli

    PubMed Central

    Saggese, Anella; Isticato, Rachele; Cangiano, Giuseppina; Ricca, Ezio

    2016-01-01

    ABSTRACT CotG is an abundant protein initially identified as an outer component of the Bacillus subtilis spore coat. It has an unusual structure characterized by several repeats of positively charged amino acids that are probably the outcome of multiple rounds of gene elongation events in an ancestral minigene. CotG is not highly conserved, and its orthologues are present in only two Bacillus and two Geobacillus species. In B. subtilis, CotG is the target of extensive phosphorylation by a still unidentified enzyme and has a role in the assembly of some outer coat proteins. We report now that most spore-forming bacilli contain a protein not homologous to CotG of B. subtilis but sharing a central “modular” region defined by a pronounced positive charge and randomly coiled tandem repeats. Conservation of the structural features in most spore-forming bacilli suggests a relevant role for the CotG-like protein family in the structure and function of the bacterial endospore. To expand our knowledge on the role of CotG, we dissected the B. subtilis protein by constructing deletion mutants that express specific regions of the protein and observed that they have different roles in the assembly of other coat proteins and in spore germination. IMPORTANCE CotG of B. subtilis is not highly conserved in the Bacillus genus; however, a CotG-like protein with a modular structure and chemical features similar to those of CotG is common in spore-forming bacilli, at least when CotH is also present. The conservation of CotG-like features when CotH is present suggests that the two proteins act together and may have a relevant role in the structure and function of the bacterial endospore. Dissection of the modular composition of CotG of B. subtilis by constructing mutants that express only some of the modules has allowed a first characterization of CotG modules and will be the basis for a more detailed functional analysis. PMID:26953338

  13. A new bright green-emitting fluorescent protein: Engineered monomeric and dimeric forms

    PubMed Central

    Ilagan, Robielyn P.; Rhoades, Elizabeth; Gruber, David F.; Kao, Hung-Teh; Pieribone, Vincent A.; Regan, Lynne

    2010-01-01

    Summary Fluorescent proteins (FP) have become essential tools in molecular and biological applications. Here, we present a novel fluorescent protein isolated from warm water coral, Cyphastrea microphthalma. The protein, which we named VFP (vivid Verde FP), matures readily at 37 °C and emits bright green light. Further characterizations revealed that VFP has a tendency to form dimers. By creating a homology model of VFP, based on the structure of red fluorescent protein DsRed, we were able to make mutations that alter the protein’s oligomerization state. We present two proteins, mVFP and mVFP1, that are both exclusively monomeric, and one, dVFP, which is dimeric. We characterized the spectroscopic properties of VFP and its variants in comparison with enhance green fluorescent protein (EGFP), a widely use variant of GFP. All the VFP variants are at least twice as bright as EGFP. Finally, we demonstrate the effectiveness of the VFP variants both in vitro and in vivo detection applications. PMID:20345907

  14. Protein stretching V: two forms of carbonic dehydratase detected by force microscopy

    NASA Astrophysics Data System (ADS)

    Alam, M. T.; Ikai, A.

    Two conformers of the human carbonic anhydrase II (HCA II) counterpart, bovine carbonic anhydrase II (CAB), which has 259 amino acid residues, were detected using the force mode of an atomic force microscope (AFM). Genetically engineered CAB (named CAB253Cys) was prepared by adding a cysteine residue at its N-terminus as well as replacing Glu253 with Cys to sandwich the protein molecule between silanized surfaces of silicon wafer and functionalized tip in the AFM. The genetically inserted Cys253 was located upstream of the protein's previously established knot structure, such that the protein could be stretched without forming the ``knot''. Two distinct types of force-extension curves were observed when the protein was stretched using the force-extension mode of the AFM. One represents a native-like state and the other a rather relaxed conformation relative to the native one. The relaxed CAB conformation was not affected by the addition of enzyme's specific inhibitor p-aminomethylbenzenesulfonamide, indicating that, in this state, the protein lacks its active site. As the AFM was able to identify two newly found conformers of CAB by mechanical means, results of this study indicate that AFM is a useful means to describe multiple competent conformations for a variety of biologically important proteins.

  15. Heterogeneity of normal prion protein in two- dimensional immunoblot: presence of various glycosylated and truncated forms.

    PubMed

    Pan, Tao; Li, Ruliang; Wong, Boon-Seng; Liu, Tong; Gambetti, Pierluigi; Sy, Man-Sun

    2002-06-01

    The common use of one-dimensional (1-D) immunoblot with a single monoclonal antibody (Mab) engenders the notion that the normal or cellular prion protein (PrP(C) ) comprises few and simple forms. In this study we used two-dimensional (2-D) immunoblot with a panel Mabs to various regions of the prion protein to demonstrate the complexity of the PrP(C) present in human brain. We distinguished over 50 immunoblot spots, each representing a distinct PrP(C) species based on combinations of different molecular weights and isoelectric points (pIs). The PrP(C) heterogeneity is due to the presence of a full-length and two major truncated forms as well as to the diversity of the glycans linked to most of these forms. The two major truncated forms result from distinct cleavage sites located at the N-terminus. In addition, enzymatic removal of sialic acid and lectin binding studies indicate that the glycans linked to the full-length and truncated PrP(C) forms differ in their structure and ratios of the glycoforms. The truncation of PrP(C) and the heterogeneity of the linked glycans may play a role in regulating PrP(C) function. Furthermore, the presence of relatively large quantities of different PrP(C) species may provide additional mechanisms by which the diversity of prion strains could be generated.

  16. The cellular prion protein traps Alzheimer's Aβ in an oligomeric form and disassembles amyloid fibers

    PubMed Central

    Younan, Nadine D.; Sarell, Claire J.; Davies, Paul; Brown, David R.; Viles, John H.

    2013-01-01

    There is now strong evidence to show that the presence of the cellular prion protein (PrPC) mediates amyloid-β (Aβ) neurotoxicity in Alzheimer's disease (AD). Here, we probe the molecular details of the interaction between PrPC and Aβ and discover that substoichiometric amounts of PrPC, as little as 1/20, relative to Aβ will strongly inhibit amyloid fibril formation. This effect is specific to the unstructured N-terminal domain of PrPC. Electron microscopy indicates PrPC is able to trap Aβ in an oligomeric form. Unlike fibers, this oligomeric Aβ contains antiparallel β sheet and binds to a oligomer specific conformational antibody. Our NMR studies show that a specific region of PrPC, notably residues 95–113, binds to Aβ oligomers, but only once Aβ misfolds. The ability of PrPC to trap and concentrate Aβ in an oligomeric form and disassemble mature fibers suggests a mechanism by which PrPC might confer Aβ toxicity in AD, as oligomers are thought to be the toxic form of Aβ. Identification of a specific recognition site on PrPC that traps Aβ in an oligomeric form is potentially a therapeutic target for the treatment of Alzheimer's disease.—Younan, N. D., Sarell, C. J., Davies, P., Brown, D. R., Viles, J. H. The cellular prion protein traps Alzheimer's Aβ in an oligomeric form and disassembles amyloid fibers. PMID:23335053

  17. Gadd45a Protein Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase MEKK4*♦

    PubMed Central

    Bullard, Steven A.; Seo, Seongjin; Schilling, Birgit; Dyle, Michael C.; Dierdorff, Jason M.; Ebert, Scott M.; DeLau, Austin D.; Gibson, Bradford W.; Adams, Christopher M.

    2016-01-01

    Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo. We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level. PMID:27358404

  18. LINC Complexes Form by Binding of Three KASH Peptides to Domain Interfaces of Trimeric SUN Proteins

    SciTech Connect

    Sosa, Brian A.; Rothballer, Andrea; Kutay, Ulrike; Schwartz, Thomas U.

    2012-08-31

    Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the nuclear envelope and are composed of KASH and SUN proteins residing in the outer and inner nuclear membrane, respectively. LINC formation relies on direct binding of KASH and SUN in the perinuclear space. Thereby, molecular tethers are formed that can transmit forces for chromosome movements, nuclear migration, and anchorage. We present crystal structures of the human SUN2-KASH1/2 complex, the core of the LINC complex. The SUN2 domain is rigidly attached to a trimeric coiled coil that prepositions it to bind three KASH peptides. The peptides bind in three deep and expansive grooves formed between adjacent SUN domains, effectively acting as molecular glue. In addition, a disulfide between conserved cysteines on SUN and KASH covalently links both proteins. The structure provides the basis of LINC complex formation and suggests a model for how LINC complexes might arrange into higher-order clusters to enhance force-coupling.

  19. Structure of struthiocalcin-1, an intramineral protein from Struthio camelus eggshell, in two crystal forms.

    PubMed

    Ruiz-Arellano, Rayana R; Medrano, Francisco J; Moreno, Abel; Romero, Antonio

    2015-04-01

    Biomineralization is the process by which living organisms produce minerals. One remarkable example is the formation of eggshells in birds. Struthiocalcins present in the ostrich (Struthio camellus) eggshell matrix act as biosensors of calcite growth during eggshell formation. Here, the crystal structure of struthiocalcin-1 (SCA-1) is reported in two different crystal forms. The structure is a compact single domain with an α/β fold characteristic of the C-type lectin family. In contrast to the related avian ovocleidin OC17, the electrostatic potential on the molecular surface is dominated by an acidic patch. Scanning electron microscopy combined with Raman spectroscopy indicates that these intramineral proteins (SCA-1 and SCA-2) induce calcium carbonate precipitation, leading to the formation of a stable form of calcite in the mature eggshell. Finally, the implications of these two intramineral proteins SCA-1 and SCA-2 in the nucleation of calcite during the formation of eggshells in ratite birds are discussed.

  20. Effects of substituting yellow corn for sorghum in geese diets on magret and foie gras quality.

    PubMed

    Arroyo, J; Auvergne, A; Dubois, J P; Lavigne, F; Bijja, M; Bannelier, C; Manse, H; Fortun-Lamothe, L

    2013-09-01

    The aim of this trial was to study the effects of substitution of yellow corn with sorghum during the growing-finishing (G period), overfeeding (O period), or both periods on magret and foie gras quality in geese. In total, 260 ganders were divided into 4 groups (65 birds in each) differing in the cereal (yellow corn or sorghum) included in the diet given during the G and the O periods, using a 2 × 2 factorial arrangement of treatments. The groups differed in the nature of the cereal in the diet offered to birds between 44 and 104 d of age (G period: a diet containing 500 g of sorghum/kg (SS and SC groups) or a diet containing 500 g of yellow corn/kg (CS and CC groups). The groups differed also in the diet offered to birds between 105 and 120 d of age (O period): 967 g of yellow corn/kg (SC and CC groups) or 965 g of sorghum/kg (SS and CS groups). At the end of the O period, the birds were slaughtered after 10 h of fasting to measure foie gras and breast muscle weight, color, and chemical composition. The mortality in the SC group was higher (P < 0.05) than in the other 3 groups (14.29 vs. 3.58%, average of the 3 groups). After overfeeding, birds fed with sorghum had foie gras that were heavier 984 vs. 885 g, in CS+SS vs. CC+SC groups, respectively; P < 0.001) and less yellow (18.03 vs. 23.97 for b*, in CS+SS vs. CC+SC groups, respectively, P < 0.001) than birds fed with corn. The substitution of yellow corn with sorghum during the G and O periods (SS group) increased the weight of the foie gras, but altered its color to a paler yellow. In contrast, a substitution during the G period only (SC group) resulted in increased mortality during the O period.

  1. Apolipoprotein E forms stable complexes with recombinant Alzheimer's disease beta-amyloid precursor protein.

    PubMed Central

    Haas, C; Cazorla, P; Miguel, C D; Valdivieso, F; Vázquez, J

    1997-01-01

    Apolipoprotein E (apoE), a protein genetically linked to the incidence of Alzheimer's disease, forms SDS-stable complexes in vitro with beta-amyloid peptide (Abeta), the primary component of senile plaques. In the present study, we investigated whether apoE was able to bind full-length Abeta precursor protein (APP). Using a maltose-binding-protein-APP fusion protein and human very-low-density lipoprotein (VLDL), we detected an interaction of apoE with APP that was inhibited by Abeta or anti-apoE antibody. Saturation-binding experiments indicated a single binding equilibrium with an apparent 1:1 stoichiometry and a dissociation constant of 15 nM. An interaction was also observed using apoE from cerebrospinal fluid or delipidated VLDL, as well as recombinant apoE. APP.apoE complexes were SDS-stable, and their formation was not inhibited by reducing conditions; however, they were dissociated by SDS under reducing conditions. ApoE.APP complexes formed high-molecular-mass aggregates, and competition experiments suggested that amino acids 14-23 of Abeta are responsible for complex-formation. Finally, no differences were found when studying the interaction of APP with apoE3 or apoE4. Taken together, our results demonstrate that apoE may form stable complexes with the Abeta moiety of APP with characteristics similar to those of complexes formed with isolated Abeta, and suggest the intriguing possibility that apoE-APP interactions may be pathologically relevant in vivo. PMID:9224643

  2. The four-transmembrane protein IP39 of Euglena forms strands by a trimeric unit repeat.

    PubMed

    Suzuki, Hiroshi; Ito, Yasuyuki; Yamazaki, Yuji; Mineta, Katsuhiko; Uji, Masami; Abe, Kazuhiro; Tani, Kazutoshi; Fujiyoshi, Yoshinori; Tsukita, Sachiko

    2013-01-01

    Euglenoid flagellates have striped surface structures comprising pellicles, which allow the cell shape to vary from rigid to flexible during the characteristic movement of the flagellates. In Euglena gracilis, the pellicular strip membranes are covered with paracrystalline arrays of a major integral membrane protein, IP39, a putative four-membrane-spanning protein with the conserved sequence motif of the PMP-22/EMP/MP20/Claudin superfamily. Here we report the three-dimensional structure of Euglena IP39 determined by electron crystallography. Two-dimensional crystals of IP39 appear to form a striated pattern of antiparallel double-rows in which trimeric IP39 units are longitudinally polymerised, resulting in continuously extending zigzag-shaped lines. Structural analysis revealed an asymmetric molecular arrangement in the trimer, and suggested that at least four different interactions between neighbouring protomers are involved. A combination of such multiple interactions would be important for linear strand formation of membrane proteins in a lipid bilayer.

  3. Organ distribution and molecular forms of human xanthine dehydrogenase/xanthine oxidase protein.

    PubMed

    Sarnesto, A; Linder, N; Raivio, K O

    1996-01-01

    Xanthine dehydrogenase/xanthine oxidase (XDH/XO) is a major cytoplasmic source of superoxide radicals and hydrogen peroxide, and it is considered important in the pathogenesis of ischemia-reperfusion damage. Because little is known about the enzyme in human tissues, the aims of this study were to purify human XDH/XO and to produce Ab for detection of the protein in Western blots and for quantification by ELISA. We purified human milk XDH/XO, produced Ab for Western blotting and ELISA of the protein, and evaluated the molecular forms and activity-protein relationships in human tissues. The molecular size of the purified protein under nondenaturing conditions was approximately 300 kd. On SDS-PAGE, it was fragmented into four main bands of 143, 125, 87, and 59 kd. Ab recognized bands of similar size in Western blots of the purified preparation and human milk. In fresh liver homogenates treated with anti-proteases, the three largest bands were observed; in the intestine, only the two largest were observed. Serum, brain, heart, and skeletal muscle were negative, whereas some lung and kidney samples showed one faint band of 143 kd. Trypsin treatment of the enzyme converted the large molecular-weight bands into smaller bands, as did incubation of a liver homogenate without anti-proteases. XDH/XO protein concentrations (ng/mg total protein) were 146 +/- 70 in liver and 556 +/- 320 in intestine and less than 5 ng/ml in serum. The relationship of activity to protein (2.7-3.0 mumol/min/mg XDH/XO protein) was constant in liver and intestine during development. We conclude that 1) human XDH/XO has molecular size and subunit structure similar to other mammalian enzymes; 2) the polypeptide chain is unstable, also in the intact cell, despite retained activity; and 3) the amount of inactive XDH/XO in human liver and intestine is apparently small.

  4. Crystal Structures of Protein Glutaminase and Its Pro Forms Converted into Enzyme-Substrate Complex*

    PubMed Central

    Hashizume, Ryota; Maki, Yukiko; Mizutani, Kimihiko; Takahashi, Nobuyuki; Matsubara, Hiroyuki; Sugita, Akiko; Sato, Kimihiko; Yamaguchi, Shotaro; Mikami, Bunzo

    2011-01-01

    Protein glutaminase, which converts a protein glutamine residue to a glutamate residue, is expected to be useful as a new food-processing enzyme. The crystal structures of the mature and pro forms of the enzyme were refined at 1.15 and 1.73 Å resolution, respectively. The overall structure of the mature enzyme has a weak homology to the core domain of human transglutaminase-2. The catalytic triad (Cys-His-Asp) common to transglutaminases and cysteine proteases is located in the bottom of the active site pocket. The structure of the recombinant pro form shows that a short loop between S2 and S3 in the proregion covers and interacts with the active site of the mature region, mimicking the protein substrate of the enzyme. Ala-47 is located just above the pocket of the active site. Two mutant structures (A47Q-1 and A47Q-2) refined at 1.5 Å resolution were found to correspond to the enzyme-substrate complex and an S-acyl intermediate. Based on these structures, the catalytic mechanism of protein glutaminase is proposed. PMID:21926168

  5. Activation of human natural killer cells by the soluble form of cellular prion protein.

    PubMed

    Seong, Yeon-Jae; Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon; Park, Bum-Chan; Park, Su-Hyung; Park, Young Woo; Shin, Eui-Cheol

    2015-08-21

    Cellular prion protein (PrP(C)) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP(C) in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP(C) protein on human natural killer (NK) cells. Recombinant soluble PrP(C) protein was generated by fusion of human PrP(C) with the Fc portion of human IgG1 (PrP(C)-Fc). PrP(C)-Fc binds to the surface of human NK cells, particularly to CD56(dim) NK cells. PrP(C)-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP(C)-Fc facilitated the IL-15-induced proliferation of NK cells. PrP(C)-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP(C)-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP(C)-Fc protein activates human NK cells via the ERK and JNK signaling pathways.

  6. EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles

    DOE PAGES

    Fan, Yao; Tan, Kemin; Chhor, Gekleng; ...

    2015-07-03

    The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (~10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for twomore » alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.« less

  7. The Promyelocytic Leukemia Gene Product (PML) Forms Stable Complexes with the Retinoblastoma Protein

    PubMed Central

    Alcalay, Myriam; Tomassoni, Lucia; Colombo, Emanuela; Stoldt, Stephan; Grignani, Francesco; Fagioli, Marta; Szekely, Laszlo; Helin, Kristian; Pelicci, Pier Giuseppe

    1998-01-01

    PML is a nuclear protein with growth-suppressive properties originally identified in the context of the PML-retinoic acid receptor α (RARα) fusion protein of acute promyelocytic leukemia. PML localizes within distinct nuclear structures, called nuclear bodies, which are disrupted by the expression of PML-RARα. We report that PML colocalizes with the nonphosphorylated fraction of the retinoblastoma protein (pRB) within nuclear bodies and that pRB is delocalized by PML-RARα expression. Both PML and PML-RARα form complexes with the nonphosphorylated form of pRB in vivo, and they interact with the pocket region of pRB. The regions of PML and PML-RARα involved in pRB binding differ; in fact, the B boxes and the C-terminal region of PML, the latter of which is not present in PML-RARα, are essential for the formation of stable complexes with pRB. Functionally, PML abolishes activation of glucocorticoid receptor-regulated transcription by pRB, whereas PML-RARα further increases it. Our results suggest that PML may be part of transcription-regulatory complexes and that the oncogenic potential of the PML-RARα protein may derive from the alteration of PML-regulated transcription. PMID:9448006

  8. An oxazetidine amino acid for chemical protein synthesis by rapid, serine-forming ligations

    NASA Astrophysics Data System (ADS)

    Pusterla, Ivano; Bode, Jeffrey W.

    2015-08-01

    Amide-forming ligation reactions allow the chemical synthesis of proteins by the union of unprotected peptide segments, and enable the preparation of protein derivatives not accessible by expression or bioengineering approaches. The native chemical ligation (NCL) of thioesters and N-terminal cysteines is unquestionably the most successful approach, but is not ideal for all synthetic targets. Here we describe the synthesis of an Fmoc-protected oxazetidine amino acid for use in the α-ketoacid-hydroxylamine (KAHA) amide ligation. When incorporated at the N-terminus of a peptide segment, this four-membered cyclic hydroxylamine can be used for rapid serine-forming ligations with peptide α-ketoacids. This ligation operates at low concentration (100 μM-5 mM) and mild temperatures (20-25 °C). The utility of the reaction was demonstrated by the synthesis of S100A4, a 12 kDa calcium-binding protein not easily accessible by NCL or other amide-forming reactions due to its primary sequence and properties.

  9. Pxmp2 Is a Channel-Forming Protein in Mammalian Peroxisomal Membrane

    PubMed Central

    Rokka, Aare; Soininen, Raija; Immonen, Hanna L.; Pirilä, Päivi L.; Bergmann, Ulrich; Sormunen, Raija T.; Weckström, Matti; Hiltunen, J. Kalervo

    2009-01-01

    Background Peroxisomal metabolic machinery requires a continuous flow of organic and inorganic solutes across peroxisomal membrane. Concerning small solutes, the molecular nature of their traffic has remained an enigma. Methods/Principal Findings In this study, we show that disruption in mice of the Pxmp2 gene encoding Pxmp2, which belongs to a family of integral membrane proteins with unknown function, leads to partial restriction of peroxisomal membrane permeability to solutes in vitro and in vivo. Multiple-channel recording of liver peroxisomal preparations reveals that the channel-forming components with a conductance of 1.3 nS in 1.0 M KCl were lost in Pxmp2−/− mice. The channel-forming properties of Pxmp2 were confirmed with recombinant protein expressed in insect cells and with native Pxmp2 purified from mouse liver. The Pxmp2 channel, with an estimated diameter of 1.4 nm, shows weak cation selectivity and no voltage dependence. The long-lasting open states of the channel indicate its functional role as a protein forming a general diffusion pore in the membrane. Conclusions/Significance Pxmp2 is the first peroxisomal channel identified, and its existence leads to prediction that the mammalian peroxisomal membrane is permeable to small solutes while transfer of “bulky” metabolites, e.g., cofactors (NAD/H, NADP/H, and CoA) and ATP, requires specific transporters. PMID:19352492

  10. Rapamycin-binding FKBP25 associates with diverse proteins that form large intracellular entities

    SciTech Connect

    Galat, Andrzej Thai, Robert

    2014-08-08

    Highlights: • The hFKBP25 interacts with diverse components of macromolecular entities. • We show that the endogenous human FKBP25 is bound to polyribosomes. • The endogenous hFKBP25 co-immunoprecipitated with nucleosomal proteins. • FKBP25 could induce conformational switch in macromolecular complexes. - Abstract: In this paper, we show some evidence that a member of the FK506-binding proteins, FKBP25 is associated to diverse components that are part of several different intracellular large-molecular mass entities. The FKBP25 is a high-affinity rapamycin-binding immunophilin, which has nuclear translocation signals present in its PPIase domain but it was detected both in the cytoplasm compartment and in the nuclear proteome. Analyses of antiFKBP25-immunoprecipitated proteins have revealed that the endogenous FKBP25 is associated to the core histones of the nucleosome, and with several proteins forming spliceosomal complexes and ribosomal subunits. Using polyclonal antiFKBP25 we have detected FKBP25 associated with polyribosomes. Added RNAs or 0.5 M NaCl release FKBP25 that was associated with the polyribosomes indicating that the immunophilin has an intrinsic capacity to form complexes with polyribonucleotides via its charged surface patches. Rapamycin or FK506 treatments of the polyribosomes isolated from porcine brain, HeLa and K568 cells caused a residual release of the endogenous FKBP25, which suggests that the immunophilin also binds to some proteins via its PPIase cavity. Our proteomics study indicates that the nuclear pool of the FKBP25 targets various nuclear proteins that are crucial for packaging of DNA, chromatin remodeling and pre-mRNA splicing whereas the cytosolic pool of this immunophilin is bound to some components of the ribosome.

  11. Identification of goose, mule duck, chicken, turkey, and swine in foie gras by species-specific polymerase chain reaction.

    PubMed

    Rodríguez, Miguel A; García, Teresa; González, Isabel; Asensio, Luis; Mayoral, Belén; López-Calleja, Inés; Hernández, Pablo E; Martín, Rosario

    2003-03-12

    A specific Polymerase Chain Reaction (PCR) has been developed for the identification of goose (Anser anser), mule duck (Anas platyrhynchos x Cairina moschata), chicken (Gallus gallus), turkey (Meleagris gallopavo), and swine (Sus scrofa domesticus) in foie gras. A forward common primer was designed on a conserved DNA sequence in the mitochondrial 12S ribosomal RNA gene (rRNA), and reverse primers were designed to hybridize on species-specific DNA sequences of each species considered. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose, mule duck, chicken, turkey, and swine in foie gras. Analysis of experimental mixtures demonstrated that the detection limit of the assay was approximately 1% for each species analyzed. This genetic marker can be very useful for the accurate identification of these species, avoiding mislabeling or fraudulent species substitution in foie gras.

  12. Crystal Structure of the Oligomeric Form of Lassa Virus Matrix Protein Z

    PubMed Central

    Hastie, Kathryn M.; Zandonatti, Michelle; Liu, Tong; Li, Sheng; Woods, Virgil L.

    2016-01-01

    ABSTRACT The arenavirus matrix protein Z is highly multifunctional and occurs in both monomeric and oligomeric forms. The crystal structure of a dodecamer of Z from Lassa virus, presented here, illustrates a ring-like structure with a highly basic center. Mutagenesis demonstrates that the dimeric interface within the dodecamer and a Lys-Trp-Lys triad at the center of the ring are important for oligomerization. This structure provides an additional template to explore the many functions of Z. IMPORTANCE The arenavirus Lassa virus causes hundreds of thousands of infections each year, many of which develop into fatal hemorrhagic fever. The arenavirus matrix protein Z is multifunctional, with at least four distinct roles. Z exists in both monomeric and oligomeric forms, each of which likely serves a specific function in the viral life cycle. Here we present the dodecameric form of Lassa virus Z and demonstrate that Z forms a “wreath” with a highly basic center. This structure and that of monomeric Z now provide a pair of critical templates by which the multiple roles of Z in the viral life cycle may be interpreted. PMID:26912609

  13. CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins

    PubMed Central

    Zurawel, Ashley A.; Kabeche, Ruth; DiGregorio, Sonja E.; Deng, Lin; Menon, Kartikeya M.; Opalko, Hannah

    2016-01-01

    ABSTRACT Proteins containing polyglutamine (polyQ) regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt) with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington’s disease (HD). To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt) in Schizosaccharomyces pombe. In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms. PMID:27677791

  14. Amphipathic polymers: tools to fold integral membrane proteins to their active form.

    PubMed

    Pocanschi, Cosmin L; Dahmane, Tassadite; Gohon, Yann; Rappaport, Fabrice; Apell, Hans-Jürgen; Kleinschmidt, Jörg H; Popot, Jean-Luc

    2006-11-28

    Among the major obstacles to pharmacological and structural studies of integral membrane proteins (MPs) are their natural scarcity and the difficulty in overproducing them in their native form. MPs can be overexpressed in the non-native state as inclusion bodies, but inducing them to achieve their functional three-dimensional structure has proven to be a major challenge. We describe here the use of an amphipathic polymer, amphipol A8-35, as a novel environment that allows both beta-barrel and alpha-helical MPs to fold to their native state, in the absence of detergents or lipids. Amphipols, which are extremely mild surfactants, appear to favor the formation of native intramolecular protein-protein interactions over intermolecular or protein-surfactant ones. The feasibility of the approach is demonstrated using as models OmpA and FomA, two outer membrane proteins from the eubacteria Escherichia coli and Fusobacterium nucleatum, respectively, and bacteriorhodopsin, a light-driven proton pump from the plasma membrane of the archaebacterium Halobacterium salinarium.

  15. A family of small coiled-coil-forming proteins functioning at the late endosome in yeast.

    PubMed

    Kranz, A; Kinner, A; Kölling, R

    2001-03-01

    The multispanning membrane protein Ste6, a member of the ABC-transporter family, is transported to the yeast vacuole for degradation. To identify functions involved in the intracellular trafficking of polytopic membrane proteins, we looked for functions that block Ste6 transport to the vacuole upon overproduction. In our screen, we identified several known vacuolar protein sorting (VPS) genes (SNF7/VPS32, VPS4, and VPS35) and a previously uncharacterized open reading frame, which we named MOS10 (more of Ste6). Sequence analysis showed that Mos10 is a member of a small family of coiled-coil-forming proteins, which includes Snf7 and Vps20. Deletion mutants of all three genes stabilize Ste6 and show a "class E vps phenotype." Maturation of the vacuolar hydrolase carboxypeptidase Y was affected in the mutants and the endocytic tracer FM4-64 and Ste6 accumulated in a dot or ring-like structure next to the vacuole. Differential centrifugation experiments demonstrated that about half of the hydrophilic proteins Mos10 and Vps20 was membrane associated. The intracellular distribution was further analyzed for Mos10. On sucrose gradients, membrane-associated Mos10 cofractionated with the endosomal t-SNARE Pep12, pointing to an endosomal localization of Mos10. The growth phenotypes of the mutants suggest that the "Snf7-family" members are involved in a cargo-specific event.

  16. A mass weighted chemical elastic network model elucidates closed form domain motions in proteins

    PubMed Central

    Kim, Min Hyeok; Seo, Sangjae; Jeong, Jay Il; Kim, Bum Joon; Liu, Wing Kam; Lim, Byeong Soo; Choi, Jae Boong; Kim, Moon Ki

    2013-01-01

    An elastic network model (ENM), usually Cα coarse-grained one, has been widely used to study protein dynamics as an alternative to classical molecular dynamics simulation. This simple approach dramatically saves the computational cost, but sometimes fails to describe a feasible conformational change due to unrealistically excessive spring connections. To overcome this limitation, we propose a mass-weighted chemical elastic network model (MWCENM) in which the total mass of each residue is assumed to be concentrated on the representative alpha carbon atom and various stiffness values are precisely assigned according to the types of chemical interactions. We test MWCENM on several well-known proteins of which both closed and open conformations are available as well as three α-helix rich proteins. Their normal mode analysis reveals that MWCENM not only generates more plausible conformational changes, especially for closed forms of proteins, but also preserves protein secondary structures thus distinguishing MWCENM from traditional ENMs. In addition, MWCENM also reduces computational burden by using a more sparse stiffness matrix. PMID:23456820

  17. Blood cell markers in Alzheimer Disease: Amyloid Precursor Protein form ratio in platelets.

    PubMed

    Borroni, Barbara; Agosti, Chiara; Marcello, Elena; Di Luca, Monica; Padovani, Alessandro

    2010-01-01

    A correct clinical diagnosis in the early stage of Alzheimer Disease (AD) is mandatory given the current available treatment with acetylcholine esterase inhibitors. Moreover, a early to preclinical diagnosis would allow to identify patients eligible for future disease-modifying therapies. In the last ten years, we have focused our attention on peripheral markers, evaluating the role of platelet Amyloid Precursor Protein (APP) forms as a reliable tool for AD diagnosis since preclinical stages. APP is the key player in AD pathogenesis, and platelets contain all the enzymatic machinery to its processing, thus being the ideal candidate where to study AD pathogenetic mechanisms. In this review, we summarise the published data regarding the usefulness of platelet APP form ratio in the diagnosis of early AD. Approaches combining APP form ratio along with neuroimaging markers show the promise to accurately identify AD, even in the pre-symptomatic stage.

  18. Bromodomain Proteins Contribute to Maintenance of Bloodstream Form Stage Identity in the African Trypanosome

    PubMed Central

    Schulz, Danae; Mugnier, Monica R.; Paulsen, Eda-Margaret; Kim, Hee-Sook; Chung, Chun-wa W.; Tough, David F.; Rioja, Inmaculada; Prinjha, Rab K.; Papavasiliou, F. Nina; Debler, Erik W.

    2015-01-01

    Trypanosoma brucei, the causative agent of African sleeping sickness, is transmitted to its mammalian host by the tsetse. In the fly, the parasite’s surface is covered with invariant procyclin, while in the mammal it resides extracellularly in its bloodstream form (BF) and is densely covered with highly immunogenic Variant Surface Glycoprotein (VSG). In the BF, the parasite varies this highly immunogenic surface VSG using a repertoire of ~2500 distinct VSG genes. Recent reports in mammalian systems point to a role for histone acetyl-lysine recognizing bromodomain proteins in the maintenance of stem cell fate, leading us to hypothesize that bromodomain proteins may maintain the BF cell fate in trypanosomes. Using small-molecule inhibitors and genetic mutants for individual bromodomain proteins, we performed RNA-seq experiments that revealed changes in the transcriptome similar to those seen in cells differentiating from the BF to the insect stage. This was recapitulated at the protein level by the appearance of insect-stage proteins on the cell surface. Furthermore, bromodomain inhibition disrupts two major BF-specific immune evasion mechanisms that trypanosomes harness to evade mammalian host antibody responses. First, monoallelic expression of the antigenically varied VSG is disrupted. Second, rapid internalization of antibodies bound to VSG on the surface of the trypanosome is blocked. Thus, our studies reveal a role for trypanosome bromodomain proteins in maintaining bloodstream stage identity and immune evasion. Importantly, bromodomain inhibition leads to a decrease in virulence in a mouse model of infection, establishing these proteins as potential therapeutic drug targets for trypanosomiasis. Our 1.25Å resolution crystal structure of a trypanosome bromodomain in complex with I-BET151 reveals a novel binding mode of the inhibitor, which serves as a promising starting point for rational drug design. PMID:26646171

  19. Bacillus thuringiensis Cyt2Aa2 toxin disrupts cell membranes by forming large protein aggregates

    PubMed Central

    Tharad, Sudarat; Toca-Herrera, José L.; Promdonkoy, Boonhiang; Krittanai, Chartchai

    2016-01-01

    Bacillus thuringiensis (Bt) Cyt2Aa2 showed toxicity against Dipteran insect larvae and in vitro lysis activity on several cells. It has potential applications in the biological control of insect larvae. Although pore-forming and/or detergent-like mechanisms were proposed, the mechanism underlying cytolytic activity remains unclear. Analysis of the haemolytic activity of Cyt2Aa2 with osmotic stabilizers revealed partial toxin inhibition, suggesting a distinctive mechanism from the putative pore formation model. Membrane permeability was studied using fluorescent dye entrapped in large unilamellar vesicles (LUVs) at various protein/lipid molar ratios. Binding of Cyt2Aa2 monomer to the lipid membrane did not disturb membrane integrity until the critical protein/lipid molar ratio was reached, when Cyt2Aa2 complexes and cytolytic activity were detected. The complexes are large aggregates that appeared as a ladder when separated by agarose gel electrophoresis. Interaction of Cyt2Aa2 with Aedes albopictus cells was investigated by confocal microscopy and total internal reflection fluorescent microscopy (TIRF). The results showed that Cyt2Aa2 binds on the cell membrane at an early stage without cell membrane disruption. Protein aggregation on the cell membrane was detected later which coincided with cell swelling. Cyt2Aa2 aggregations on supported lipid bilayers (SLBs) were visualized by AFM. The AFM topographic images revealed Cyt2Aa2 aggregates on the lipid bilayer at low protein concentration and subsequently disrupts the lipid bilayer by forming a lesion as the protein concentration increased. These results supported the mechanism whereby Cyt2Aa2 binds and aggregates on the lipid membrane leading to the formation of non-specific hole and disruption of the cell membrane. PMID:27612497

  20. The FEMA GRAS assessment of aliphatic and aromatic terpene hydrocarbons used as flavor ingredients.

    PubMed

    Adams, T B; Gavin, C Lucas; McGowen, M M; Waddell, W J; Cohen, S M; Feron, V J; Marnett, L J; Munro, I C; Portoghese, P S; Rietjens, I M C M; Smith, R L

    2011-10-01

    This publication is the thirteenth in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Since then, the number of flavoring substances has grown to more than 2600 substances. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually and in the context of the available scientific information on the group of structurally related substances. Scientific data relevant to the safety evaluation of the use of aliphatic and aromatic terpene hydrocarbons as flavoring ingredients are evaluated. The group of aliphatic and aromatic terpene hydrocarbons was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; their rapid absorption, metabolic detoxication, and excretion in humans and other animals; their low level of flavor use; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies and the lack of significant genotoxic potential.

  1. Abacavir forms novel cross-linking abacavir protein adducts in patients.

    PubMed

    Meng, Xiaoli; Lawrenson, Alexandre S; Berry, Neil G; Maggs, James L; French, Neil S; Back, David J; Khoo, Saye H; Naisbitt, Dean J; Park, B Kevin

    2014-04-21

    Abacavir (ABC), a nucleoside-analogue reverse transcriptase inhibitor, is associated with severe hypersensitivity reactions that are thought to involve the activation of CD8+ T cells in a HLA-B*57:01-restricted manner. Recent studies have claimed that noncovalent interactions of ABC with HLA-B*57:01 are responsible for the immunological reactions associated with ABC. However, the formation of hemoglobin-ABC aldehyde (ABCA) adducts in patients exposed to ABC suggests that protein conjugation might represent a pathway for antigen formation. To further characterize protein conjugation reactions, we used mass spectrometric methods to define ABCA modifications in patients receiving ABC therapy. ABCA formed a novel intramolecular cross-linking adduct on human serum albumin (HSA) in patients and in vitro via Michael addition, followed by nucleophilic adduction of the aldehyde with a neighboring protein nucleophile. Adducts were detected on Lys159, Lys190, His146, and Cys34 residues in the subdomain IB of HSA. Only a cysteine adduct and a putative cross-linking adduct were detected on glutathione S-transferase Pi (GSTP). These findings reveal that ABC forms novel types of antigens in all patients taking the drug. It is therefore vital that the immunological consequences of such pathways of haptenation are explored in the in vitro models that have been used by various groups to define new mechanisms of drug hypersensitivity exemplified by ABC.

  2. Proteomic analysis of duck fatty liver during post-mortem storage related to the variability of fat loss during cooking of "foie gras".

    PubMed

    Theron, Laetitia; Fernandez, Xavier; Marty-Gasset, Nathalie; Chambon, Christophe; Viala, Didier; Pichereaux, Carole; Rossignol, Michel; Astruc, Thierry; Molette, Caroline

    2013-01-30

    Fat loss during cooking of duck "foie gras" is the main problem for both manufacturers and consumers. Despite the efforts of the processing industry to control fat loss, the variability of fatty liver cooking yields remains high and uncontrolled. To understand the biochemical effects of postslaughter processing on fat loss during cooking, this study characterizes for the first time the protein expression of fatty liver during chilling using a proteomic approach. For this purpose the proteins were separated according to their solubility: the protein fraction soluble in a buffer of low ionic strength (S) and the protein fraction insoluble in the same buffer (IS). Two-dimensional electrophoresis was used to analyze the S fraction and mass spectrometry for the identification of spots of interest. This analysis revealed 36 (21 identified proteins) and 34 (26 identified proteins) spots of interests in the low-fat-loss and high-fat-loss groups, respectively. The expression of proteins was lower after chilling, which revealed a suppressive effect of chilling on biological processes. The shot-gun strategy was used to analyze the IS fraction, with the identification of all the proteins by mass spectrometry. This allowed identification of 554 and 562 proteins in the low-fat-loss and high-fat-loss groups, respectively. Among these proteins, only the proteins that were up-regulated in the high-fat-loss group were significant (p value = 3.17 × 10(-3)) and corresponded to protein from the cytoskeleton and its associated proteins. Taken together, these results suggest that the variability of technological yield observed in processing plants could be explained by different aging states of fatty livers during chilling, most likely associated with different proteolytic patterns.

  3. Proteins in aggregates functionally impact multiple neurodegenerative disease models by forming proteasome-blocking complexes

    PubMed Central

    Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Gao, Yuan; Yu, Li-Rong; Alla, Ramani; Shmookler Reis, Robert

    2015-01-01

    Age-dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP-43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation-prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc-54p/Q40::YFP, a model of polyglutamine array diseases such as Huntington's, accrues aggregates in muscle 2–6 days posthatch. These foci, isolated on antibody-coupled magnetic beads, were characterized by high-resolution mass spectrometry. Three Q40::YFP-associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimer's disease models, nematodes with muscle or pan-neuronal Aβ1–42 expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine-rich, favoring hydrophobic interactions with other random-coil domains. A particularly potent modulator of aggregation, CRAM-1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72–86% (P < 10−6). In worms expressing Aβ1–42, knockdown of cram-1 reduced β-amyloid 60% (P < 0.002) and slowed age-dependent paralysis > 30% (P < 10−6). In wild-type worms, cram-1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM-1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM-1 (or human orthologs) with ubiquitin, and we verified that CRAM-1 reduces degradation of a tagged-ubiquitin reporter. We propose that CRAM-1 exemplifies a class of primitive chaperones that are initially protective and highly

  4. Trapping Open and Closed Forms of FitE-A Group III Periplasmic Binding Protein

    SciTech Connect

    Shi, R.; Proteau, A; Wagner, J; Cui, Q; Purisima, E; Matte, A; Cygler, M

    2009-01-01

    Periplasmic binding proteins (PBPs) are essential components of bacterial transport systems, necessary for bacterial growth and survival. The two-domain structures of PBPs are topologically classified into three groups based on the number of crossovers or hinges between the globular domains: group I PBPs have three connections, group II have two, and group III have only one. Although a large number of structures for group I or II PBPs are known, fewer group III PBPs have been structurally characterized. Group I and II PBPs exhibit significant domain motions during transition from the unbound to ligand-bound form, however, no large conformational changes have been observed to date in group III PBPs. We have solved the crystal structure of a periplasmic binding protein FitE, part of an iron transport system, fit, recently identified in a clinical E. coli isolate. The structure, determined at 1.8 {angstrom} resolution, shows that FitE is a group III PBP containing a single {alpha}-helix bridging the two domains. Among the individual FitE molecules present in two crystal forms we observed three different conformations (open, closed, intermediate). Our crystallographic and molecular dynamics results strongly support the notion that group III PBPs also adopt the same Venus flytrap mechanism as do groups I and II PBPs. Unlike other group III PBPs, FitE forms dimers both in solution and in the crystals. The putative siderophore binding pocket is lined with arginine residues, suggesting an anionic nature of the iron-containing siderophore.

  5. Production of a recombinant full-length prion protein in a soluble form without refolding or detergents.

    PubMed

    Arii, Yasuhiro; Oshiro, Satoshi; Wada, Keita; Fukuoka, Shin-ichi

    2011-01-01

    Recombinant prion protein has been produced in insoluble form and refolded following solubilization with denaturants. It is, however, preferable to use a soluble recombinant protein prepared without artificial solubilization. In this study, a soluble recombinant prion protein was produced in Escherichia coli cells by coexpression of neuregulin I-β1 and purified to high purity.

  6. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.

    PubMed

    Zakeri, Bijan; Fierer, Jacob O; Celik, Emrah; Chittock, Emily C; Schwarz-Linek, Ulrich; Moy, Vincent T; Howarth, Mark

    2012-03-20

    Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.

  7. Frameshift proteins in autosomal dominant forms of Alzheimer disease and other tauopathies.

    PubMed

    van Leeuwen, F W; van Tijn, P; Sonnemans, M A F; Hobo, B; Mann, D M A; Van Broeckhoven, C; Kumar-Singh, S; Cras, P; Leuba, G; Savioz, A; Maat-Schieman, M L C; Yamaguchi, H; Kros, J M; Kamphorst, W; Hol, E M; de Vos, R A I; Fischer, D F

    2006-01-24

    Frameshift (+1) proteins such as APP(+1) and UBB(+1) accumulate in sporadic cases of Alzheimer disease (AD) and in older subjects with Down syndrome (DS). We investigated whether these proteins also accumulate at an early stage of neuropathogenesis in young DS individuals without neuropathology and in early-onset familial forms of AD (FAD), as well as in other tauopathies, such as Pick disease (PiD) or progressive supranuclear palsy (PSP). APP(+1) is present in many neurons and beaded neurites in very young cases of DS, which suggests that it is axonally transported. In older DS patients (>37 years), a mixed pattern of APP(+1) immunoreactivity was observed in healthy looking neurons and neurites, dystrophic neurites, in association with neuritic plaques, as well as neurofibrillary tangles. UBB(+1) immunoreactivity was exclusively present in AD type of neuropathology. A similar pattern of APP(+1) and UBB(+1) immunoreactivity was also observed for FAD and much less explicit in nondemented controls after the age of 51 years. Furthermore, we observed accumulation of +1 proteins in other types of tauopathies, such as PiD, frontotemporal dementia, PSP and argyrophylic grain disease. These data suggest that accumulation of +1 proteins contributes to the early stages of dementia and plays a pathogenic role in a number of diseases that involve the accumulation of tau.

  8. Zinc-dependent mechanical properties of Staphylococcus aureus biofilm-forming surface protein SasG.

    PubMed

    Formosa-Dague, Cécile; Speziale, Pietro; Foster, Timothy J; Geoghegan, Joan A; Dufrêne, Yves F

    2016-01-12

    Staphylococcus aureus surface protein SasG promotes cell-cell adhesion during the accumulation phase of biofilm formation, but the molecular basis of this interaction remains poorly understood. Here, we unravel the mechanical properties of SasG on the surface of living bacteria, that is, in its native cellular environment. Nanoscale multiparametric imaging of living bacteria reveals that Zn(2+) strongly increases cell wall rigidity and activates the adhesive function of SasG. Single-cell force measurements show that SasG mediates cell-cell adhesion via specific Zn(2+)-dependent homophilic bonds between β-sheet-rich G5-E domains on neighboring cells. The force required to unfold individual domains is remarkably strong, up to ∼500 pN, thus explaining how SasG can withstand physiological shear forces. We also observe that SasG forms homophilic bonds with the structurally related accumulation-associated protein of Staphylococcus epidermidis, suggesting the possibility of multispecies biofilms during host colonization and infection. Collectively, our findings support a model in which zinc plays a dual role in activating cell-cell adhesion: adsorption of zinc ions to the bacterial cell surface increases cell wall cohesion and favors the projection of elongated SasG proteins away from the cell surface, thereby enabling zinc-dependent homophilic bonds between opposing cells. This work demonstrates an unexpected relationship between mechanics and adhesion in a staphylococcal surface protein, which may represent a general mechanism among bacterial pathogens for activating cell association.

  9. Native and denatured forms of proteins can be discriminated at edge plane carbon electrodes.

    PubMed

    Ostatná, Veronika; Cernocká, Hana; Kurzątkowska, Katarzyna; Paleček, Emil

    2012-07-20

    In an attempt to develop a label-free electrochemical method for detection of changes in protein structures based on oxidizability of tyrosine and tryptophan residues we tested different types of carbon electrodes. We found that using edge plane pyrolytic graphite electrode (EPGE) we can discriminate between native and denatured forms of human serum albumin (HSA) and of other proteins, such as bovine and chicken serum albumin, aldolase and concanavalin. Treatment of natively unfolded α-synuclein with 8 M urea resulted only in a small change in the tyrosine oxidation peak, in a good agreement with absence of highly ordered structure in this protein. Using square wave voltammetry with EPGE we were able to follow the course of HSA denaturation at different urea concentrations. The electrochemical denaturation curve agreed reasonably well with that based on intrinsic fluorescence of tyrosine and tryptophan. It can be expected that the electrochemical method will be applicable to a large number of proteins and may become useful in biomedicine and proteomics.

  10. Protein kinase A inhibits a consolidated form of memory in Drosophila.

    PubMed

    Horiuchi, Junjiro; Yamazaki, Daisuke; Naganos, Shintaro; Aigaki, Toshiro; Saitoe, Minoru

    2008-12-30

    Increasing activity of the cAMP/protein kinase A (PKA) pathway has often been proposed as an approach to improve memory in various organisms. However, here we demonstrate that single-point mutations, which decrease PKA activity, dramatically improve aversive olfactory memory in Drosophila. These mutations do not affect formation of early memory phases or of protein synthesis-dependent long-term memory but do cause a significant increase in a specific consolidated form of memory, anesthesia-resistant memory. Significantly, heterozygotes of null mutations in PKA are sufficient to cause this memory increase. Expressing a PKA transgene in the mushroom bodies, brain structures critical for memory formation in Drosophila, reduces memory back to wild-type levels. These results indicate that although PKA is critical for formation of several memory phases, it also functions to inhibit at least one memory phase.

  11. Electron crystallography of PhoE porin, an outer membrane, channel- forming protein from E. coli

    SciTech Connect

    Walian, P.J.

    1989-11-01

    One approach to studying the structure of membrane proteins is the use of electron crystallography. Dr. Bing Jap has crystallized PhoE pore-forming protein (porin) from the outer membrane of escherichia coli (E. coli) into monolayer crystals. The findings of this research and those of Jap (1988, 1989) have determined these crystals to be highly ordered, yielding structural information to a resolution of better than 2.8 angstroms. The task of this thesis has been to collect and process the electron diffraction patterns necessary to generate a complete three-dimensional set of high resolution structure factor amplitudes of PhoE porin. Fourier processing of these amplitudes when combined with the corresponding phase data is expected to yield the three-dimensional structure of PhoE porin at better than 3.5 angstroms resolution. 92 refs., 33 figs., 3 tabs. (CBS)

  12. FIE, a nuclear PRC2 protein, forms cytoplasmic complexes in Arabidopsis thaliana.

    PubMed

    Oliva, Moran; Butenko, Yana; Hsieh, Tzung-Fu; Hakim, Ofir; Katz, Aviva; Smorodinsky, Nechama I; Michaeli, Daphna; Fischer, Robert L; Ohad, Nir

    2016-11-01

    Polycomb group (PcG) proteins are evolutionarily conserved chromatin modifiers that regulate developmental pathways in plants. PcGs form nuclear multi-subunit Polycomb Repressive Complexes (PRCs). The PRC2 complex mediates gene repression via methylation of lysine 27 on histone H3, which consequently leads to chromatin condensation. In Arabidopsis thaliana, several PRC2 complexes with different compositions were identified, each controlling a particular developmental program.The core subunit FIE is crucial for PRC2 function throughout the plant life cycle, yet accurate information on its spatial and temporal localization was absent. This study focused on identifying FIE accumulation patterns, using microscopy and biochemical approaches. Analysing endogenous FIE and transgenic gFIE-green fluorescent protein fusion protein (gFIE-GFP) showed that FIE accumulates in the nuclei of every cell type examined. Interestingly, gFIE-GFP, as well as the endogenous FIE, also localized to the cytoplasm in all examined tissues. In both vegetative and reproductive organs, FIE formed cytoplasmic high-molecular-mass complexes, in parallel to the nuclear PRC2 complexes. Moreover, size-exclusion chromatography and bimolecular fluorescence complementation assays indicated that in inflorescences FIE formed a cytoplasmic complex with MEA, a PRC2 histone methyltransferase subunit. In contrast, CLF and SWN histone methyltransferases were strictly nuclear. Presence of PRC2 subunits in cytoplasmic complexes has not been previously described in plants. Our findings are in agreement with accumulating evidence demonstrating cytoplasmic localization and function of PcGs in metazoa. The cytosolic accumulation of PRC2 components in plants supports the model that PcGs have alternative non-nuclear functions that go beyond chromatin methylation.

  13. FIE, a nuclear PRC2 protein, forms cytoplasmic complexes in Arabidopsis thaliana

    PubMed Central

    Oliva, Moran; Butenko, Yana; Hsieh, Tzung-Fu; Hakim, Ofir; Katz, Aviva; Smorodinsky, Nechama I.; Michaeli, Daphna; Fischer, Robert L.; Ohad, Nir

    2016-01-01

    Polycomb group (PcG) proteins are evolutionarily conserved chromatin modifiers that regulate developmental pathways in plants. PcGs form nuclear multi-subunit Polycomb Repressive Complexes (PRCs). The PRC2 complex mediates gene repression via methylation of lysine 27 on histone H3, which consequently leads to chromatin condensation. In Arabidopsis thaliana, several PRC2 complexes with different compositions were identified, each controlling a particular developmental program. The core subunit FIE is crucial for PRC2 function throughout the plant life cycle, yet accurate information on its spatial and temporal localization was absent. This study focused on identifying FIE accumulation patterns, using microscopy and biochemical approaches. Analysing endogenous FIE and transgenic gFIE–green fluorescent protein fusion protein (gFIE-GFP) showed that FIE accumulates in the nuclei of every cell type examined. Interestingly, gFIE-GFP, as well as the endogenous FIE, also localized to the cytoplasm in all examined tissues. In both vegetative and reproductive organs, FIE formed cytoplasmic high-molecular-mass complexes, in parallel to the nuclear PRC2 complexes. Moreover, size-exclusion chromatography and bimolecular fluorescence complementation assays indicated that in inflorescences FIE formed a cytoplasmic complex with MEA, a PRC2 histone methyltransferase subunit. In contrast, CLF and SWN histone methyltransferases were strictly nuclear. Presence of PRC2 subunits in cytoplasmic complexes has not been previously described in plants. Our findings are in agreement with accumulating evidence demonstrating cytoplasmic localization and function of PcGs in metazoa. The cytosolic accumulation of PRC2 components in plants supports the model that PcGs have alternative non-nuclear functions that go beyond chromatin methylation. PMID:27811080

  14. Type III secretion as a generalizable strategy for the production of full-length biopolymer-forming proteins.

    PubMed

    Azam, Anum; Li, Cheng; Metcalf, Kevin J; Tullman-Ercek, Danielle

    2016-11-01

    Biopolymer-forming proteins are integral in the development of customizable biomaterials, but recombinant expression of these proteins is challenging. In particular, biopolymer-forming proteins have repetitive, glycine-rich domains and, like many heterologously expressed proteins, are prone to incomplete translation, aggregation, and proteolytic degradation in the production host. This necessitates tailored purification processes to isolate each full-length protein of interest from the truncated forms as well as other contaminating proteins; owing to the repetitive nature of these proteins, the truncated polypeptides can have very similar chemistry to the full-length form and are difficult to separate from the full-length protein. We hypothesized that bacterial expression and secretion would be a promising alternative option for biomaterials-forming proteins, simplifying isolation of the full-length target protein. By using a selective secretion system, truncated forms of the protein are not secreted and thus are not found in the culture harvest. We show that a synthetically upregulated type III secretion system leads to a general increase in secretion titer for each protein that we tested. Moreover, we observe a substantial enhancement in the homogeneity of full-length forms of pro-resilin, tropo-elastin crosslinking domains, and silk proteins produced in this manner, as compared with proteins purified from the cytosol. Secretion via the type III apparatus limits co-purification of truncated forms of the target protein and increases protein purity without extensive purification steps. Demonstrating the utility of such a system, we introduce several modifications to resilin-based peptides and use an un-optimized, single-column process to purify these proteins. The resulting materials are of sufficiently high quantity and yield for the production of antimicrobial hydrogels with highly reproducible rheological properties. The ease of this process and its

  15. A genetic screen in zebrafish identifies the mutants vps18, nf2 and foie gras as models of liver disease.

    PubMed

    Sadler, Kirsten C; Amsterdam, Adam; Soroka, Carol; Boyer, James; Hopkins, Nancy

    2005-08-01

    Hepatomegaly is a sign of many liver disorders. To identify zebrafish mutants to serve as models for hepatic pathologies, we screened for hepatomegaly at day 5 of embryogenesis in 297 zebrafish lines bearing mutations in genes that are essential for embryonic development. Seven mutants were identified, and three have phenotypes resembling different liver diseases. Mutation of the class C vacuolar protein sorting gene vps18 results in hepatomegaly associated with large, vesicle-filled hepatocytes, which we attribute to the failure of endosomal-lysosomal trafficking. Additionally, these mutants develop defects in the bile canaliculi and have marked biliary paucity, suggesting that vps18 also functions to traffic vesicles to the hepatocyte apical membrane and may play a role in the development of the intrahepatic biliary tree. Similar findings have been reported for individuals with arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome, which is due to mutation of another class C vps gene. A second mutant, resulting from disruption of the tumor suppressor gene nf2, develops extrahepatic choledochal cysts in the common bile duct, suggesting that this gene regulates division of biliary cells during development and that nf2 may play a role in the hyperplastic tendencies observed in biliary cells in individuals with choledochal cysts. The third mutant is in the novel gene foie gras, which develops large, lipid-filled hepatocytes, resembling those in individuals with fatty liver disease. These mutants illustrate the utility of zebrafish as a model for studying liver development and disease, and provide valuable tools for investigating the molecular pathogenesis of congenital biliary disorders and fatty liver disease.

  16. Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming

    PubMed Central

    Ishikawa, Kazuya; Miura, Chihiro; Maejima, Kensaku; Komatsu, Ken; Hashimoto, Masayoshi; Tomomitsu, Tatsuya; Fukuoka, Misato; Yusa, Akira; Yamaji, Yasuyuki

    2014-01-01

    ABSTRACT Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. IMPORTANCE Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly

  17. Early stages of probable Alzheimer disease are associated with changes in platelet amyloid precursor protein forms.

    PubMed

    Borroni, B; Colciaghi, F; Corsini, P; Akkawi, N; Rozzini, L; Del Zotto, E; Talarico, G; Cattabeni, F; Lenzi, G L; Di Luca, M; Padovani, A

    2002-12-01

    Previous findings demonstrated an altered pattern of amyloid precursor protein (APP) forms in platelets of Alzheimer disease (AD) patients, compared both with healthy control subjects or patients with non-Alzheimer-type dementia. The present study aims to evaluate whether platelet APP form ratio (APPr) is altered in patients with early stage AD. We selected 40 patients with early stage AD and 40 age-matched healthy controls. Compared with controls (mean+/-SD=0.91+/-0.3), mean APPr was decreased in AD (mean+/-SD=0.46+/-0.26, p<0.0001). Sixteen very mild AD patients (clinical dementia rating=0.5), identified among the AD group, showed a significant decrease of APPr values (mean+/-SD=0.50+/-0.3, p<0.0001). These findings indicate that alteration of APP processing in platelets is an early event and suggest that this assay might be of diagnostic value in differentiating mild AD from normal ageing.

  18. Toxicity and oxidative stress of different forms of organic selenium and dietary protein in mallard ducklings

    USGS Publications Warehouse

    Hoffman, D.J.; Heinz, G.H.; LeCaptain, L.J.; Eisemann, J.D.; Pendleton, G.W.

    1996-01-01

    Concentrations of over 100 ppm (mg/kg) selenium (Se) have been found in aquatic plants and insects associated with irrigation drainwater and toxicity to fish and wildlife. Composition of diet for wild ducklings may vary in selenium-contaminated environments. Earlier studies have compared toxicities and oxidative stress of Se as selenite to those of seleno-DL-methionine (DL) in mallards (Anas platyrhynchos). This study compares DL, seleno-L-methionine (L), selenized yeast (Y) and selenized wheat (W). Day-old mallard ducklings received an untreated diet (controls) containing 75% wheat (22% protein) or the same diet containing 15 or 30 ppm Se in the above forms except for 30 ppm Se as W. After 2 weeks blood and liver samples were collected for biochemical assays and Se analysis. All forms of selenium caused significant increases in plasma and hepatic glutathione peroxidase activities. Se as L at 30 ppm in the diet was the most toxic form, resulting in high mortality (64%) and impaired growth (>50%) in survivors and the greatest increase in ratio of oxidized to reduced hepatic glutathione (GSH). Se as both L and DL decreased the concentrations of hepatic GSH and total thiols. Se as Y accumulated the least in liver (approximately 50% of other forms) and had less effect on GSH and total thiols. In a second experiment, in which the basal diet was a commercial duck feed (22 % protein), survival was not affected by 30 ppm Se as DL, L, or Y and oxidative effects on GSH metabolism were less pronounced than with the wheat diet.

  19. 21 CFR 186.1 - Substances added indirectly to human food affirmed as generally recognized as safe (GRAS).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Substances added indirectly to human food affirmed... added indirectly to human food affirmed as generally recognized as safe (GRAS). (a) The indirect human... ingredient in this part does not authorize the use of such substance for the purpose of adding the...

  20. 21 CFR 186.1 - Substances added indirectly to human food affirmed as generally recognized as safe (GRAS).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Substances added indirectly to human food affirmed... added indirectly to human food affirmed as generally recognized as safe (GRAS). (a) The indirect human... ingredient in this part does not authorize the use of such substance for the purpose of adding the...

  1. 21 CFR 186.1 - Substances added indirectly to human food affirmed as generally recognized as safe (GRAS).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Substances added indirectly to human food affirmed... added indirectly to human food affirmed as generally recognized as safe (GRAS). (a) The indirect human... ingredient in this part does not authorize the use of such substance for the purpose of adding the...

  2. 21 CFR 186.1 - Substances added indirectly to human food affirmed as generally recognized as safe (GRAS).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... of food-contact surface(s), the functional use(s) of the ingredient, and the level(s) of use. Any use... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Substances added indirectly to human food affirmed as generally recognized as safe (GRAS). 186.1 Section 186.1 Food and Drugs FOOD AND...

  3. Quantitation of mule duck in goose foie gras using TaqMan real-time Polymerase Chain Reaction.

    PubMed

    Rodríguez, Miguel A; García, Teresa; González, Isabel; Asensio, Luis; Hernández, Pablo E; Martín, Rosario

    2004-03-24

    A real-time quantitative Polymerase Chain Reaction (PCR) method has been developed for the quantitation of mule duck (Anas platyrhynchos x Cairina moschata) in binary duck/goose foie gras mixtures. The method combines the use of real-time PCR with duck-specific and endogenous control "duck + goose" primers to measure duck content and total foie gras content, respectively. Both PCR systems (duck-specific and duck + goose) were designed on the mitochondrial 12S ribosomal RNA gene (rRNA). The duck-specific system amplifies a 96 bp fragment from duck DNA, whereas the duck + goose system amplifies a 120 bp fragment from duck and goose DNA. The method measures PCR product accumulation through a FAM-labeled fluorogenic probe (TaqMan). The C(t) (threshold cycle) values obtained from the duck + goose system are used to normalize the ones obtained from the duck-specific system. Analysis of experimental duck/goose foie gras binary mixtures demonstrated the suitability of the assay for the detection and quantitation of duck in the range of 1-25%. This genetic marker can be very useful to avoid mislabeling or fraudulent species substitution of goose by duck in foie gras.

  4. 21 CFR 184.1979c - Whey protein concentrate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ..._locations.html. (3) The whey protein concentrate shall be derived from milk that has been pasteurized, or... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Whey protein concentrate. 184.1979c Section 184... as GRAS § 184.1979c Whey protein concentrate. (a) Whey protein concentrate is the substance...

  5. Preliminary validation efforts of GRAS radio occultation data recorded in raw-sampling mode

    NASA Astrophysics Data System (ADS)

    Zus, Florian; Marquardt, Christian; Bonnedal, Magnus

    2010-05-01

    Atmospheric remote soundings of tropo-/stratospheric temperature profiles by the GNSS (Global Navigation Satellite System) radio occultation (RO) method provide valuable input for numerical weather prediction models and climate change studies. The RO-instrument GRAS (GNSS Receiver for Atmospheric Sounding) on-board of EUMETSAT's (European Organisation for the Exploitation of Meteorological Satellites) MetOp satellite has been designed for observing setting and rising occultations from the GPS (Global Positioning System) satellite constellation. A dedicated ESA (European Space Agency, contract 21995/08/NL/FM) funded study was set up to investigate the potential of RO data recorded in RS (raw-sampling) mode. First results from GRAS RO data recorded in RS mode processed at the GFZ are presented. The experimental processing software POCS-X includes full spectrum inversion in order to cope with multi-path regions and enables (in connection with RS data) to retrieve bending angle/refractivity profiles down to the Earth's surface. The retrievals are validated against co-located ECMWF (European Centre for Medium-Range Weather Forecasts) profiles. The focus is on data from October 2007 provided by EUMETSAT. The intercomparison indicates good quality of the retrieved profiles: The global mean fractional refractivity deviation from 0 to 5 km varies between -1.8% to +0.4%. The meridional distribution of the fractional refractivity deviation at low altitudes shows that the observed negative/positive bias mainly stems from the tropical lower troposhere. This feature can be probably attributed to the presence of superrefraction/subrefraction. From 10 to 35 km we notice an increasing bias with altitude in the global mean fractional refractivity ranging from -0.1% to -0.8%. Sensitivity tests indicate that the climatology (MSIS), which is used in the statistical optimization at high altitudes, contributes to this bias. However, excluding the climatology from the processing, at the

  6. Evolution of the Cytolytic Pore-Forming Proteins (Actinoporins) in Sea Anemones.

    PubMed

    Macrander, Jason; Daly, Marymegan

    2016-12-08

    Sea anemones (Cnidaria, Anthozoa, and Actiniaria) use toxic peptides to incapacitate and immobilize prey and to deter potential predators. Their toxin arsenal is complex, targeting a variety of functionally important protein complexes and macromolecules involved in cellular homeostasis. Among these, actinoporins are one of the better characterized toxins; these venom proteins form a pore in cellular membranes containing sphingomyelin. We used a combined bioinformatic and phylogenetic approach to investigate how actinoporins have evolved across three superfamilies of sea anemones (Actinioidea, Metridioidea, and Actinostoloidea). Our analysis identified 90 candidate actinoporins across 20 species. We also found clusters of six actinoporin-like genes in five species of sea anemone (Nematostella vectensis, Stomphia coccinea, Epiactis japonica, Heteractis crispa, and Diadumene leucolena); these actinoporin-like sequences resembled actinoporins but have a higher sequence similarity with toxins from fungi, cone snails, and Hydra. Comparative analysis of the candidate actinoporins highlighted variable and conserved regions within actinoporins that may pertain to functional variation. Although multiple residues are involved in initiating sphingomyelin recognition and membrane binding, there is a high rate of replacement for a specific tryptophan with leucine (W112L) and other hydrophobic residues. Residues thought to be involved with oligomerization were variable, while those forming the phosphocholine (POC) binding site and the N-terminal region involved with cell membrane penetration were highly conserved.

  7. Evolution of the Cytolytic Pore-Forming Proteins (Actinoporins) in Sea Anemones

    PubMed Central

    Macrander, Jason; Daly, Marymegan

    2016-01-01

    Sea anemones (Cnidaria, Anthozoa, and Actiniaria) use toxic peptides to incapacitate and immobilize prey and to deter potential predators. Their toxin arsenal is complex, targeting a variety of functionally important protein complexes and macromolecules involved in cellular homeostasis. Among these, actinoporins are one of the better characterized toxins; these venom proteins form a pore in cellular membranes containing sphingomyelin. We used a combined bioinformatic and phylogenetic approach to investigate how actinoporins have evolved across three superfamilies of sea anemones (Actinioidea, Metridioidea, and Actinostoloidea). Our analysis identified 90 candidate actinoporins across 20 species. We also found clusters of six actinoporin-like genes in five species of sea anemone (Nematostella vectensis, Stomphia coccinea, Epiactis japonica, Heteractis crispa, and Diadumene leucolena); these actinoporin-like sequences resembled actinoporins but have a higher sequence similarity with toxins from fungi, cone snails, and Hydra. Comparative analysis of the candidate actinoporins highlighted variable and conserved regions within actinoporins that may pertain to functional variation. Although multiple residues are involved in initiating sphingomyelin recognition and membrane binding, there is a high rate of replacement for a specific tryptophan with leucine (W112L) and other hydrophobic residues. Residues thought to be involved with oligomerization were variable, while those forming the phosphocholine (POC) binding site and the N-terminal region involved with cell membrane penetration were highly conserved. PMID:27941639

  8. Chlorophyll ring deformation modulates Qy electronic energy in chlorophyll-protein complexes and generates spectral forms.

    PubMed

    Zucchelli, Giuseppe; Brogioli, Doriano; Casazza, Anna Paola; Garlaschi, Flavio M; Jennings, Robert C

    2007-09-15

    The possibility that the chlorophyll (chl) ring distortions observed in the crystal structures of chl-protein complexes are involved in the transition energy modulation, giving rise to the spectral forms, is investigated. The out-of-plane chl-macrocycle distortions are described using an orthonormal set of deformations, defined by the displacements along the six lowest-frequency, out-of-plane normal coordinates. The total chl-ring deformation is the linear combination of these six deformations. The two higher occupied and the two lower unoccupied chl molecular orbitals, which define the Q(y) electronic transition, have the same symmetry as four of the six out-of-plane lowest frequency modes. We assume that a deformation along the normal-coordinate having the same symmetry as a given molecular orbital will perturb that orbital and modify its energy. The changes in the chl Q(y) transition energies are evaluated in the Peridinin-Chl-Protein complex and in light harvesting complex II (LHCII), using crystallographic data. The macrocycle deformations induce a distribution of the chl Q(y) electronic energy transitions which, for LHCII, is broader for chla than for chlb. This provides the physical mechanism to explain the long-held view that the chla spectral forms in LHCII are both more numerous and cover a wider energy range than those of chlb.

  9. Pore-forming activity of OmpA protein of Escherichia coli.

    PubMed

    Sugawara, E; Nikaido, H

    1992-02-05

    Escherichia coli outer membrane protein OmpA was purified to homogeneity, as a monomer, from a K12 derivative deficient in both OmpF and OmpC porins. When proteoliposomes reconstituted from the purified OmpA, phospholipids, and lithium dodecyl sulfate were tested for permeability to small molecules by osmotic swelling, it was found that OmpA produced apparently nonspecific diffusion channels that allowed the penetration of various solutes. The pore-forming activity was destroyed by the heat denaturation of the OmpA protein, and the use of an OmpA-deficient mutant showed that the activity was not caused by copurifying contaminants. The size of the OmpA channel, estimated by comparison of diffusion rates of solutes of different sizes, was rather similar to that of E. coli OmpF and OmpC porins, i.e. about 1 nm in diameter. The rate of penetration of L-arabinose caused by a given amount of OmpA protein, however, was about a hundredfold lower than the rate produced by the same amount of E. coli OmpF porin. The addition of large amounts of lithium dodecyl sulfate to the reconstitution mixture increased the permeability through the OmpA channel, apparently by facilitating the correct insertion of OmpA into the bilayer.

  10. Identification by proteomic analysis of early post-mortem markers involved in the variability in fat loss during cooking of mule duck "foie gras".

    PubMed

    Theron, Laetitia; Fernandez, Xavier; Marty-Gasset, Nathalie; Pichereaux, Carole; Rossignol, Michel; Chambon, Christophe; Viala, Didier; Astruc, Thierry; Molette, Caroline

    2011-12-14

    Fat loss during cooking of duck "foie gras" is the main quality issue for both processors and consumers. Despite the efforts of the processing industry to control fat loss, the variability of fatty liver cooking yield remains high and uncontrolled. To better understand the biological basis of this phenomenon, a proteomic study was conducted. To analyze the protein fraction soluble at low ionic strength (LIS), we used bidimensional electrophoresis and mass spectrometry for the identification of spots of interest. To analyze the protein fraction not soluble at low ionic strength (NS), we used the shotgun strategy. The analysis of data acquired from both protein fractions suggested that at the time of slaughter, livers with low fat loss during cooking were still in anabolic processes with regard to energy metabolism and protein synthesis, whereas livers with high fat loss during cooking developed cell protection mechanisms. The variability in the technological yield observed in processing plants could be explained by a different physiological stage of liver steatosis.

  11. A Clostridium difficile-Specific, Gel-Forming Protein Required for Optimal Spore Germination

    PubMed Central

    Donnelly, M. Lauren; Li, William; Li, Yong-qing; Hinkel, Lauren; Setlow, Peter

    2017-01-01

    ABSTRACT Clostridium difficile is a Gram-positive spore-forming obligate anaerobe that is a leading cause of antibiotic-associated diarrhea worldwide. In order for C. difficile to initiate infection, its aerotolerant spore form must germinate in the gut of mammalian hosts. While almost all spore-forming organisms use transmembrane germinant receptors to trigger germination, C. difficile uses the pseudoprotease CspC to sense bile salt germinants. CspC activates the related subtilisin-like protease CspB, which then proteolytically activates the cortex hydrolase SleC. Activated SleC degrades the protective spore cortex layer, a step that is essential for germination to proceed. Since CspC incorporation into spores also depends on CspA, a related pseudoprotease domain, Csp family proteins play a critical role in germination. However, how Csps are incorporated into spores remains unknown. In this study, we demonstrate that incorporation of the CspC, CspB, and CspA germination regulators into spores depends on CD0311 (renamed GerG), a previously uncharacterized hypothetical protein. The reduced levels of Csps in gerG spores correlate with reduced responsiveness to bile salt germinants and increased germination heterogeneity in single-spore germination assays. Interestingly, asparagine-rich repeat sequences in GerG’s central region facilitate spontaneous gel formation in vitro even though they are dispensable for GerG-mediated control of germination. Since GerG is found exclusively in C. difficile, our results suggest that exploiting GerG function could represent a promising avenue for developing C. difficile-specific anti-infective therapies. PMID:28096487

  12. Phospholipase A2-activating protein is associated with a novel form of leukoencephalopathy.

    PubMed

    Falik Zaccai, Tzipora C; Savitzki, David; Zivony-Elboum, Yifat; Vilboux, Thierry; Fitts, Eric C; Shoval, Yishay; Kalfon, Limor; Samra, Nadra; Keren, Zohar; Gross, Bella; Chasnyk, Natalia; Straussberg, Rachel; Mullikin, James C; Teer, Jamie K; Geiger, Dan; Kornitzer, Daniel; Bitterman-Deutsch, Ora; Samson, Abraham O; Wakamiya, Maki; Peterson, Johnny W; Kirtley, Michelle L; Pinchuk, Iryna V; Baze, Wallace B; Gahl, William A; Kleta, Robert; Anikster, Yair; Chopra, Ashok K

    2017-02-01

    Leukoencephalopathies are a group of white matter disorders related to abnormal formation, maintenance, and turnover of myelin in the central nervous system. These disorders of the brain are categorized according to neuroradiological and pathophysiological criteria. Herein, we have identified a unique form of leukoencephalopathy in seven patients presenting at ages 2 to 4 months with progressive microcephaly, spastic quadriparesis, and global developmental delay. Clinical, metabolic, and imaging characterization of seven patients followed by homozygosity mapping and linkage analysis were performed. Next generation sequencing, bioinformatics, and segregation analyses followed, to determine a loss of function sequence variation in the phospholipase A2-activating protein encoding gene (PLAA). Expression and functional studies of the encoded protein were performed and included measurement of prostaglandin E2 and cytosolic phospholipase A2 activity in membrane fractions of fibroblasts derived from patients and healthy controls. Plaa-null mice were generated and prostaglandin E2 levels were measured in different tissues. The novel phenotype of our patients segregated with a homozygous loss-of-function sequence variant, causing the substitution of leucine at position 752 to phenylalanine, in PLAA, which causes disruption of the protein's ability to induce prostaglandin E2 and cytosolic phospholipase A2 synthesis in patients' fibroblasts. Plaa-null mice were perinatal lethal with reduced brain levels of prostaglandin E2 The non-functional phospholipase A2-activating protein and the associated neurological phenotype, reported herein for the first time, join other complex phospholipid defects that cause leukoencephalopathies in humans, emphasizing the importance of this axis in white matter development and maintenance.

  13. Scaffold-forming and Adhesive Contributions of Synthetic Laminin-binding Proteins to Basement Membrane Assembly.

    PubMed

    McKee, Karen K; Capizzi, Stephanie; Yurchenco, Peter D

    2009-03-27

    Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although laminins containing the alpha4 and alpha5 subunits are expressed in alpha2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind to deficient laminins that provide such missing activities would promote basement membrane assembly in a Schwann cell model. A chimeric fusion protein (alphaLNNd) that adds a short arm terminus to laminin through the nidogen binding locus was generated and compared with the dystrophy-ameliorating protein miniagrin (mAgrin) that binds to the laminin coiled-coil dystroglycan and sulfatides. alphaLNNd was found to mediate laminin binding to collagen IV, to bind to galactosyl sulfatide, and to selectively convert alpha-short arm deletion-mutant laminins LmDeltaalphaLN and LmDeltaalphaLN-L4b into polymerizing laminins. This protein enabled polymerization-deficient laminin but not an adhesion-deficient laminin lacking LG domains (LmDeltaLG) to assemble an extracellular matrix on Schwann cell surfaces. mAgrin, on the other hand, enabled LmDeltaLG to form an extracellular matrix on cell surfaces without increasing accumulation of non-polymerizing laminins. These gain-of-function studies reveal distinct polymerization and anchorage contributions to basement membrane assembly in which the three different LN domains mediate the former, and the LG domains provide primary anchorage with secondary contributions from the alphaLN domain. These findings may be relevant for an understanding of the pathogenesis and treatment of laminin deficiency states.

  14. Purification and biochemical characterization of a monomeric form of papaya mosaic potexvirus coat protein.

    PubMed

    Lecours, Katia; Tremblay, Marie-Hélène; Gagné, Marie-Eve Laliberté; Gagné, Stéphane M; Leclerc, Denis

    2006-05-01

    Papaya mosaic virus (PapMV) is a flexuous rod shape virus made of 1400 subunits that assemble around a plus sense genomic RNA. The structure determination of PapMV and of flexuous viruses in general is a major challenge for both NMR and X-ray crystallography. In this report, we present the characterization of a truncated version of the PapMV coat protein (CP) that is suitable for NMR study. The deletion of the N-terminal 26 amino acids of the PapMV CP (CP27-215) generates a monomer that can be expressed to high level and easily purified for production of an adequate NMR sample. The RNA gel shift assay showed that CP27-215 lost its ability to bind RNA in vitro, suggesting that the multimerization of the subunit is important for this function. The fusion of a 6x His tag at the C-terminus improved the solubility of the monomer and allowed its concentration to 0.2 mM. The CD spectra of the truncated and the wild-type proteins were similar, suggesting that both proteins are well ordered and have a similar secondary structure. CP27-215 was 15N labeled for NMR studies and a 2D 1H-15N-HSQC spectrum confirmed the presence of a well-ordered structure and the monomeric form of the protein. These results show that CP27-215 is amenable to a complete and exhaustive NMR study that should lead to the first three-dimensional structure determination of a flexuous rod shape virus.

  15. SV40 late protein VP4 forms toroidal pores to disrupt membranes for viral release

    PubMed Central

    Raghava, Smita; Giorda, Kristina M.; Romano, Fabian B.; Heuck, Alejandro P.; Hebert, Daniel N.

    2014-01-01

    Nonenveloped viruses are generally released from the cell by the timely lysis of host cell membranes. SV40 has been used as a model virus for the study of the lytic nonenveloped virus life cycle. The expression of SV40 VP4 at later times during infection is concomitant with cell lysis. To investigate the role of VP4 in viral release and its mechanism of action, VP4 was expressed and purified from bacteria as a fusion protein for use in membrane disruption assays. Purified VP4 perforated membranes as demonstrated by the release of fluorescent markers encapsulated within large unilamellar vesicles or liposomes. Dynamic light scattering results found that VP4 treatment did not cause membrane lysis or change the size of the liposomes. Liposomes encapsulated with bodipy-labeled streptavidin were used to show that VP4 formed stable pores in membranes. These VP4 pores had an inner diameter of between 1 and 5 nm. Asymmetrical liposomes containing pyrene-labeled lipids in the outer monolayer were employed to monitor transbilayer lipid diffusion. Consistent with VP4 forming toroidal pore structures in membranes, VP4 induced transbilayer lipid diffusion or lipid flip-flop. Altogether, these studies support a central role for VP4 acting as a viroporin in the disruption of cellular membranes to trigger SV40 viral release by forming toroidal pores that unite the outer and inner leaflets of membrane bilayers. PMID:23651212

  16. Purification in an active form of the phage phi 29 protein p4 that controls the viral late transcription.

    PubMed Central

    Barthelemy, I; Lázaro, J M; Méndez, E; Mellado, R P; Salas, M

    1987-01-01

    The phage phi 29 protein p4, that controls viral late transcription, was highly purified from Escherichia coli cells harbouring a gene 4-containing plasmid. This protein, representing about 6% of the total cellular protein, was obtained in a highly purified form. The protein was characterized as p4 by amino acid analysis and NH2-terminal sequence determination. The purified protein was active in an in vitro transcription assay, allowing specific initiation of transcription at the phi 29 A3 late promoter in the presence of Bacillus subtilis sigma 43-RNA polymerase holoenzyme. Images PMID:3671066

  17. The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation

    PubMed Central

    Rogala, Kacper B; Dynes, Nicola J; Hatzopoulos, Georgios N; Yan, Jun; Pong, Sheng Kai; Robinson, Carol V; Deane, Charlotte M; Gönczy, Pierre; Vakonakis, Ioannis

    2015-01-01

    Centrioles are microtubule-based organelles crucial for cell division, sensing and motility. In Caenorhabditis elegans, the onset of centriole formation requires notably the proteins SAS-5 and SAS-6, which have functional equivalents across eukaryotic evolution. Whereas the molecular architecture of SAS-6 and its role in initiating centriole formation are well understood, the mechanisms by which SAS-5 and its relatives function is unclear. Here, we combine biophysical and structural analysis to uncover the architecture of SAS-5 and examine its functional implications in vivo. Our work reveals that two distinct self-associating domains are necessary to form higher-order oligomers of SAS-5: a trimeric coiled coil and a novel globular dimeric Implico domain. Disruption of either domain leads to centriole duplication failure in worm embryos, indicating that large SAS-5 assemblies are necessary for function in vivo. DOI: http://dx.doi.org/10.7554/eLife.07410.001 PMID:26023830

  18. Structure of an octameric form of the minichromosome maintenance protein from the archaeon Pyrococcus abyssi

    PubMed Central

    Cannone, Giuseppe; Visentin, Silvia; Palud, Adeline; Henneke, Ghislaine; Spagnolo, Laura

    2017-01-01

    Cell division is a complex process that requires precise duplication of genetic material. Duplication is concerted by replisomes. The Minichromosome Maintenance (MCM) replicative helicase is a crucial component of replisomes. Eukaryotic and archaeal MCM proteins are highly conserved. In fact, archaeal MCMs are powerful tools for elucidating essential features of MCM function. However, while eukaryotic MCM2-7 is a heterocomplex made of different polypeptide chains, the MCM complexes of many Archaea form homohexamers from a single gene product. Moreover, some archaeal MCMs are polymorphic, and both hexameric and heptameric architectures have been reported for the same polypeptide. Here, we present the structure of the archaeal MCM helicase from Pyrococcus abyssi in its single octameric ring assembly. To our knowledge, this is the first report of a full-length octameric MCM helicase. PMID:28176822

  19. The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation.

    PubMed

    Rogala, Kacper B; Dynes, Nicola J; Hatzopoulos, Georgios N; Yan, Jun; Pong, Sheng Kai; Robinson, Carol V; Deane, Charlotte M; Gönczy, Pierre; Vakonakis, Ioannis

    2015-05-29

    Centrioles are microtubule-based organelles crucial for cell division, sensing and motility. In Caenorhabditis elegans, the onset of centriole formation requires notably the proteins SAS-5 and SAS-6, which have functional equivalents across eukaryotic evolution. Whereas the molecular architecture of SAS-6 and its role in initiating centriole formation are well understood, the mechanisms by which SAS-5 and its relatives function is unclear. Here, we combine biophysical and structural analysis to uncover the architecture of SAS-5 and examine its functional implications in vivo. Our work reveals that two distinct self-associating domains are necessary to form higher-order oligomers of SAS-5: a trimeric coiled coil and a novel globular dimeric Implico domain. Disruption of either domain leads to centriole duplication failure in worm embryos, indicating that large SAS-5 assemblies are necessary for function in vivo.

  20. HIV Tat protein and amyloid-β peptide form multifibrillar structures that cause neurotoxicity.

    PubMed

    Hategan, Alina; Bianchet, Mario A; Steiner, Joseph; Karnaukhova, Elena; Masliah, Eliezer; Fields, Adam; Lee, Myoung-Hwa; Dickens, Alex M; Haughey, Norman; Dimitriadis, Emilios K; Nath, Avindra

    2017-02-20

    Deposition of amyloid-β plaques is increased in the brains of HIV-infected individuals, and the HIV transactivator of transcription (Tat) protein affects amyloidogenesis through several indirect mechanisms. Here, we investigated direct interactions between Tat and amyloid-β peptide. Our in vitro studies showed that in the presence of Tat, uniform amyloid fibrils become double twisted fibrils and further form populations of thick unstructured filaments and aggregates. Specifically, Tat binding to the exterior surfaces of the Aβ fibrils increases β-sheet formation and lateral aggregation into thick multifibrillar structures, thus producing fibers with increased rigidity and mechanical resistance. Furthermore, Tat and Aβ aggregates in complex synergistically induced neurotoxicity both in vitro and in animal models. Increased rigidity and mechanical resistance of the amyloid-β-Tat complexes coupled with stronger adhesion due to the presence of Tat in the fibrils may account for increased damage, potentially through pore formation in membranes.

  1. Different forms of MARCKS protein are involved in memory formation in the learning process of imprinting.

    PubMed

    Solomonia, Revaz O; Apkhazava, David; Nozadze, Maia; Jackson, Antony P; McCabe, Brian J; Horn, Gabriel

    2008-06-01

    There is strong evidence that a restricted part of the chick forebrain, the IMM (formerly IMHV), stores information acquired through the learning process of visual imprinting. Twenty-four hours after imprinting training, a learning-specific increase in amount of myristoylated, alanine-rich C-kinase substrate (MARCKS) protein is known to occur in the homogenate fraction of IMM. We investigated the two components of this fraction, membrane-bound and cytoplasmic-phosphorylated MARCKS. In IMM, amount of membrane-bound MARCKS, but not of cytoplasmic-phosphorylated MARCKS, increased as chicks learned. No changes were observed for either form of MARCKS in PPN, a control forebrain region. The results indicate that there is a learning-specific increase in membrane-bound, non-phosphorylated MARCKS 24 h after training. This increase might contribute to stabilization of synaptic morphology.

  2. How Does the VSG Coat of Bloodstream Form African Trypanosomes Interact with External Proteins?

    PubMed

    Schwede, Angela; Macleod, Olivia J S; MacGregor, Paula; Carrington, Mark

    2015-12-01

    Variations on the statement "the variant surface glycoprotein (VSG) coat that covers the external face of the mammalian bloodstream form of Trypanosoma brucei acts a physical barrier" appear regularly in research articles and reviews. The concept of the impenetrable VSG coat is an attractive one, as it provides a clear model for understanding how a trypanosome population persists; each successive VSG protects the plasma membrane and is immunologically distinct from previous VSGs. What is the evidence that the VSG coat is an impenetrable barrier, and how do antibodies and other extracellular proteins interact with it? In this review, the nature of the extracellular surface of the bloodstream form trypanosome is described, and past experiments that investigated binding of antibodies and lectins to trypanosomes are analysed using knowledge of VSG sequence and structure that was unavailable when the experiments were performed. Epitopes for some VSG monoclonal antibodies are mapped as far as possible from previous experimental data, onto models of VSG structures. The binding of lectins to some, but not to other, VSGs is revisited with more recent knowledge of the location and nature of N-linked oligosaccharides. The conclusions are: (i) Much of the variation observed in earlier experiments can be explained by the identity of the individual VSGs. (ii) Much of an individual VSG is accessible to antibodies, and the barrier that prevents access to the cell surface is probably at the base of the VSG N-terminal domain, approximately 5 nm from the plasma membrane. This second conclusion highlights a gap in our understanding of how the VSG coat works, as several plasma membrane proteins with large extracellular domains are very unlikely to be hidden from host antibodies by VSG.

  3. Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms.

    PubMed

    Pletneva, Nadya V; Pletnev, Sergei; Pakhomov, Alexey A; Chertkova, Rita V; Martynov, Vladimir I; Muslinkina, Liya; Dauter, Zbigniew; Pletnev, Vladimir Z

    2016-08-01

    The fluorescent protein from Dendronephthya sp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the C(α)-N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double C(α)=C(β) bond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of native green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.

  4. Development of a long-acting, protein-loaded, redox-active, injectable gel formed by a polyion complex for local protein therapeutics.

    PubMed

    Ishii, Shiro; Kaneko, Junya; Nagasaki, Yukio

    2016-04-01

    Although cancer immunotherapies are attracting much attention, it is difficult to develop bioactive proteins owing to the severe systemic toxicity. To overcome the issue, we designed new local protein delivery system by using a protein-loaded, redox-active, injectable gel (RIG), which is formed by a polyion complex (PIC) comprising three components, viz., cationic polyamine-poly(ethylene glycol)-polyamine triblock copolymer possessing ROS-scavenging moieties as side chains; anionic poly(acrylic acid); and a protein. The mixture formed the protein-loaded PIC flower micelles at room temperature, which immediately converted to a gel with high mechanical strength upon exposure to physiological conditions. Because the protein electrostatically interacts with the PIC gel network, RIG provided a sustained release of the protein without a significant initial burst, regardless of the types of proteins in vitro, and much longer retention of the protein at the local injection site in mice than that of the naked protein. Subcutaneous injections of IL-12@RIG in the vicinity of tumor tissue showed remarkable tumor growth inhibition in tumor-bearing mice, compared to that observed with injection of IL-12 alone, suppressing adverse events caused by IL-12-induced ROS. Our results indicate that RIG has potential as a platform technology for an injectable sustained-release carrier for proteins.

  5. Bacterial collagen-like proteins that form triple-helical structures.

    PubMed

    Yu, Zhuoxin; An, Bo; Ramshaw, John A M; Brodsky, Barbara

    2014-06-01

    A large number of collagen-like proteins have been identified in bacteria during the past 10years, principally from analysis of genome databases. These bacterial collagens share the distinctive Gly-Xaa-Yaa repeating amino acid sequence of animal collagens which underlies their unique triple-helical structure. A number of the bacterial collagens have been expressed in Escherichia coli, and they all adopt a triple-helix conformation. Unlike animal collagens, these bacterial proteins do not contain the post-translationally modified amino acid, hydroxyproline, which is known to stabilize the triple-helix structure and may promote self-assembly. Despite the absence of collagen hydroxylation, the triple-helix structures of the bacterial collagens studied exhibit a high thermal stability of 35-39°C, close to that seen for mammalian collagens. These bacterial collagens are readily produced in large quantities by recombinant methods, either in the original amino acid sequence or in genetically manipulated sequences. This new family of recombinant, easy to modify collagens could provide a novel system for investigating structural and functional motifs in animal collagens and could also form the basis of new biomedical materials with designed structural properties and functions.

  6. Cellular COPII Proteins Are Involved in Production of the Vesicles That Form the Poliovirus Replication Complex

    PubMed Central

    Rust, René C.; Landmann, Lukas; Gosert, Rainer; Tang, Bor Luen; Hong, Wanjin; Hauri, Hans-Peter; Egger, Denise; Bienz, Kurt

    2001-01-01

    Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway. PMID:11559814

  7. Structural Studies of Truncated Forms of the Prion Protein PrP

    PubMed Central

    Wan, William; Wille, Holger; Stöhr, Jan; Kendall, Amy; Bian, Wen; McDonald, Michele; Tiggelaar, Sarah; Watts, Joel C.; Prusiner, Stanley B.; Stubbs, Gerald

    2015-01-01

    Prions are proteins that adopt self-propagating aberrant folds. The self-propagating properties of prions are a direct consequence of their distinct structures, making the understanding of these structures and their biophysical interactions fundamental to understanding prions and their related diseases. The insolubility and inherent disorder of prions have made their structures difficult to study, particularly in the case of the infectious form of the mammalian prion protein PrP. Many investigators have therefore preferred to work with peptide fragments of PrP, suggesting that these peptides might serve as structural and functional models for biologically active prions. We have used x-ray fiber diffraction to compare a series of different-sized fragments of PrP, to determine the structural commonalities among the fragments and the biologically active, self-propagating prions. Although all of the peptides studied adopted amyloid conformations, only the larger fragments demonstrated a degree of structural complexity approaching that of PrP. Even these larger fragments did not adopt the prion structure itself with detailed fidelity, and in some cases their structures were radically different from that of pathogenic PrPSc. PMID:25809267

  8. PROTEIN KINASE Cα MEDIATES A NOVEL FORM OF PLASTICITY IN THE ACCESSORY OLFACTORY BULB

    PubMed Central

    DONG, C.; GODWIN, D. W.; BRENNAN, P. A.; HEGDE, A. N.

    2009-01-01

    Modification of synapses in the accessory olfactory bulb (AOB) is believed to underlie pheromonal memory that enables mate recognition in mice. The memory, which is acquired with single-trial learning forms only with coincident noradrenergic and glutamatergic inputs to the AOB. The mechanisms by which glutamate and norepinephrine (NE) alter the AOB synapses are not well understood. Here we present results that not only reconcile the earlier, seemingly contradictory, observations on the role of glutamate and NE in changing the AOB synapses, but also reveal novel mechanisms of plasticity. Our studies suggest that initially, glutamate acting at Group II metabotropic receptors and NE acting at α2-adrenergic receptors inhibit N-type and R-type Ca2+ channels in mitral cells via a G-Protein. The N-type and R-type Ca2+ channel inhibition is reversed by activation of α1-adrenergic receptors and protein kinase Cα (PKCα). Based on these results, we propose a hypothetical model for a new kind of synaptic plasticity in the AOB that accounts for the previous behavioral data on pheromonal memory. According to this model, initial inhibition of the Ca2+ channels suppresses the GABAergic inhibitory feedback to mitral cells, causing disinhibition and Ca2+ influx. NE also activates phospholipase C (PLC) through α1-adrenergic receptors generating inositol 1,4,5-trisphosphate and diacylglycerol (DAG). Calcium and DAG together activate protein kinase Cα (PKCα) which switches the disinhibition to increased inhibition of mitral cells. Thus, PKCα is likely to be a coincidence detector integrating glutamate and NE input in the AOB and bridging the short-term signaling to long-term structural changes resulting in enhanced inhibition of mitral cells that is thought to underlie memory formation. PMID:19580852

  9. Beta-catenin Forms Protein Aggregation at High Concentrations in HEK293TCells

    PubMed Central

    Jazi, Marie Saghaeian; Najafi, Seyed Mahmoud Arab

    2017-01-01

    Background: The canonical Wnt signal transduction (or the Wnt/β-catenin pathway) plays a crucial role in the development of animals and in carcinogenesis. Beta-catenin is the central component of this signaling pathway. The activation of Wnt/β-catenin signaling results in the cytoplasmic and nuclear accumulation of β-catenin. In the nucleus, β-catenin interacts with the TCF/LEF transcription factors and, therefore, participates in the upregulation or downregulation of some important genes involved in diverse cellular activities. In addition, β-catenin is a critical component of the cadherin-mediated cell adherens junction. We had previously noticed that very high cellular concentrations of β-catenin had a negative effect on the transcriptional activity of this protein and, therefore, the aim of this study was to find a mechanism for this negative interaction. Methods: Cell fractionation, western blotting, and immunofluorescence microscopy experiments were performed to measure β-catenin protein levels and β-catenin cellular localization in HEK293Tcells transfected with various amounts of a β-catenin-encoding plasmid. Also, total RNA was extracted from the cells and used for reverse transcriptase-PCR experiments to measure the expression of the β-catenin target genes. SPSS, version 16, was used to analyze the results statistically. Results: We demonstrated that overexpression of β-catenin led to the formation of rod-shaped protein aggregates. The aggregate structures were mainly formed in the cell nucleus and were heavy enough to be isolated by centrifugation. Beta-catenin aggregate formation was accompanied by a decrease in the expression of the β-catenin target genes used in this study. Conclusion: Since deregulation of β-catenin function occurs in several human diseases, including cancer and neurological disorders, the results of this paper further support the possible biological and clinical significance of β-catenin aggregate formation. PMID

  10. Structures of lysenin reveal a shared evolutionary origin for pore-forming proteins and its mode of sphingomyelin recognition.

    PubMed

    De Colibus, Luigi; Sonnen, Andreas F-P; Morris, Keith J; Siebert, C Alistair; Abrusci, Patrizia; Plitzko, Jürgen; Hodnik, Vesna; Leippe, Matthias; Volpi, Emanuela; Anderluh, Gregor; Gilbert, Robert J C

    2012-09-05

    Pore-forming proteins insert from solution into membranes to create lesions, undergoing a structural rearrangement often accompanied by oligomerization. Lysenin, a pore-forming toxin from the earthworm Eisenia fetida, specifically interacts with sphingomyelin (SM) and may confer innate immunity against parasites by attacking their membranes to form pores. SM has important roles in cell membranes and lysenin is a popular SM-labeling reagent. The structure of lysenin suggests common ancestry with other pore-forming proteins from a diverse set of eukaryotes and prokaryotes. The complex with SM shows the mode of its recognition by a protein in which both the phosphocholine headgroup and one acyl tail are specifically bound. Lipid interaction studies and assays using viable target cells confirm the functional reliance of lysenin on this form of SM recognition.

  11. The transforming proteins of PRCII virus and Rous sarcoma virus form a complex with the same two cellular phosphoproteins.

    PubMed Central

    Adkins, B; Hunter, T; Sefton, B M

    1982-01-01

    P105 and P110, the presumptive transforming proteins of PRCII avian sarcoma virus, have been found to be present in transformed chicken cells in two forms: as monomers and as part of a complex which contains both a 50,000-dalton and a 90,000-dalton cellular phosphoprotein. The 90,000-dalton cellular protein was found to be identical to one of the proteins in chicken cells whose synthesis is induced by stress. The 50,000-dalton protein was found to contain phosphotyrosine when isolated from the complex and therefore may be a substrate for the tyrosine protein kinase activity which is associated with P105 and P110. These same two cellular phosphoproteins have previously been shown to be present in a complex with pp60src, the tyrosine protein kinase which is the transforming protein of Rous sarcoma virus. However, not all avian sarcoma virus transforming proteins with associated tyrosine protein kinase activities form a complex efficiently with these cellular proteins. Little if any of P90, the putative transforming protein of Yamaguchi 73 virus, was found in a complex with the 50,000-dalton and 90,000-dalton cellular phosphoproteins. Images PMID:6180178

  12. An adult form of Alexander disease: a novel mutation in glial fibrillary acidic protein.

    PubMed

    Ohnari, K; Yamano, M; Uozumi, T; Hashimoto, T; Tsuji, S; Nakagawa, M

    2007-10-01

    Glial fibrillary acidic protein (GFAP) mutation has been reported in Alexander disease. We report a patient with the adult form of Alexander disease who shows a novel mutation in GFAP. This case presented with progressive dysarthria, dysphagia and spastic gait on the right side. Brain and spinal cord MRI showed marked atrophy of the medulla oblongata and spinal cord. Abnormal high signal intensities in the ventral medulla oblongata were detected bilaterally. There were no white matter lesions or contrast enhancing lesions. Recently, there have been reports of patients with a juvenile form of Alexander disease presenting with atrophy or signal abnormalities of the medulla or spinal cord. Atrophy of the medulla and spinal cord have specifically been described as suggestive of Alexander disease [1]. Sequence analysis of the GFAP gene of this patient showed a heterozygous c.221T>C mutation, predicting a p.M74T amino acid change. In all patients suspected of Alexander disease on the basis of MRI findings, GFAP analysis is necessary to confirm the diagnosis.

  13. Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD*

    PubMed Central

    Chatterjee, Abhishek; Caballero-Franco, Celia; Bakker, Dannika; Totten, Stephanie; Jardim, Armando

    2015-01-01

    Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits. PMID:26324713

  14. '2A-Like' Signal Sequences Mediating Translational Recoding: A Novel Form of Dual Protein Targeting.

    PubMed

    Roulston, Claire; Luke, Garry A; de Felipe, Pablo; Ruan, Lin; Cope, Jonathan; Nicholson, John; Sukhodub, Andriy; Tilsner, Jens; Ryan, Martin D

    2016-08-01

    We report the initial characterization of an N-terminal oligopeptide '2A-like' sequence that is able to function both as a signal sequence and as a translational recoding element. Owing to this translational recoding activity, two forms of nascent polypeptide are synthesized: (i) when 2A-mediated translational recoding has not occurred: the nascent polypeptide is fused to the 2A-like N-terminal signal sequence and the fusion translation product is targeted to the exocytic pathway, and, (ii) a translation product where 2A-mediated translational recoding has occurred: the 2A-like signal sequence is synthesized as a separate translation product and, therefore, the nascent (downstream) polypeptide lacks the 2A-like signal sequence and is localized to the cytoplasm. This type of dual-functional signal sequence results, therefore, in the partitioning of the translation products between the two sub-cellular sites and represents a newly described form of dual protein targeting.

  15. LRRC8 proteins form volume-regulated anion channels that sense ionic strength

    PubMed Central

    Syeda, Ruhma; Qiu, Zhaozhu; Dubin, Adrienne E.; Murthy, Swetha E.; Florendo, Maria N.; Mason, Daniel E.; Mathur, Jayanti; Cahalan, Stuart M.; Peters, Eric C.; Montal, Mauricio; Patapoutian, Ardem

    2015-01-01

    Summary The volume-regulated anion channel (VRAC) is activated when a cell swells, and plays a central role in maintaining cell volume in response to osmotic challenges. SWELL1 (LRRC8A) was recently identified as an essential component of VRAC. However, the identity of the pore-forming subunits of VRAC, and how the channel is gated by cell swelling are unknown. Here we show that SWELL1 with up to four other LRRC8 subunits assemble into heterogeneous complexes of ~800 kDa. When reconstituted into bilayers, LRRC8 complexes are sufficient to form anion channels activated by osmolality gradients. In bilayers as well as in cells, the single-channel conductance of the complexes depends on the LRRC8 composition. Finally, low ionic strength (Γ), in the absence of an osmotic gradient, activates the complexes in bilayers. These data demonstrate that LRRC8 proteins together constitute the VRAC pore, and that hypotonic stress can activate VRAC through a decrease in cytoplasmic Γ. PMID:26824658

  16. A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle

    PubMed Central

    Mackinder, Luke C. M.; Meyer, Moritz T.; Mettler-Altmann, Tabea; Chen, Vivian K.; Mitchell, Madeline C.; Caspari, Oliver; Freeman Rosenzweig, Elizabeth S.; Pallesen, Leif; Reeves, Gregory; Itakura, Alan; Roth, Robyn; Sommer, Frederik; Geimer, Stefan; Mühlhaus, Timo; Schroda, Michael; Goodenough, Ursula; Stitt, Mark; Griffiths, Howard; Jonikas, Martin C.

    2016-01-01

    Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2. Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2. We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1’s four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency. PMID:27166422

  17. Design of a PROTAC that antagonizes and destroys the cancer-forming X-protein of the hepatitis B virus

    SciTech Connect

    Montrose, Kristopher; Krissansen, Geoffrey W.

    2014-10-31

    Highlights: • A novel proteolysis targeting chimeric molecule (PROTAC) to treat hepatitis B. • The PROTAC antagonizes and destroys the X-protein of the hepatitis B virus. • The PROTAC is a fusion of the X-protein oligomerization and instability domains. • The oligomerization domain is a dominant-negative inhibitor of X-protein function. • X-protein-targeting PROTACs have potential to prevent hepatocellular carcinoma. - Abstract: The X-protein of the hepatitis B virus (HBV) is essential for virus infection and contributes to the development of HBV-induced hepatocellular carcinoma (HCC), a disease which causes more than one million deaths each year. Here we describe the design of a novel PROTAC (proteolysis targeting chimeric molecule) capable of simultaneously inducing the degradation of the X-protein, and antagonizing its function. The PROTAC was constructed by fusing the N-terminal oligomerization and C-terminal instability domains of the X-protein to each other, and rendering them cell-permeable by the inclusion of a polyarginine cell-penetrating peptide (CPP). It was predicted that the oligomerization domain would bind the X-protein, and that the instability domain would cause the X-protein to be targeted for proteasomal degradation. Addition of the PROTAC to HepG2 liver cancer cells, engineered to express full-length and C-terminally truncated forms of the X-protein, resulted in the degradation of both forms of the X-protein. A cell-permeable stand-alone form of the oligomerization domain was taken up by HepG2 cells, and acted as a dominant-negative inhibitor, causing inhibition of X-protein-induced apoptosis. In summary, the PROTAC described here induces the degradation of the X-protein, and antagonizes its function, and warrants investigation in a preclinical study for its ability to prevent or treat HBV infection and/or the development of HCC.

  18. 21 CFR 184.1979c - Whey protein concentrate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... derived from milk that has been pasteurized, or the whey protein concentrate shall be subjected to... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Whey protein concentrate. 184.1979c Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1979c Whey protein concentrate. (a) Whey...

  19. 21 CFR 184.1979c - Whey protein concentrate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... derived from milk that has been pasteurized, or the whey protein concentrate shall be subjected to... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Whey protein concentrate. 184.1979c Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1979c Whey protein concentrate. (a) Whey...

  20. 21 CFR 184.1979c - Whey protein concentrate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... derived from milk that has been pasteurized, or the whey protein concentrate shall be subjected to... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Whey protein concentrate. 184.1979c Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1979c Whey protein concentrate. (a) Whey...

  1. 21 CFR 184.1979c - Whey protein concentrate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... derived from milk that has been pasteurized, or the whey protein concentrate shall be subjected to... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Whey protein concentrate. 184.1979c Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1979c Whey protein concentrate. (a) Whey...

  2. Heterogeneity of high-mobility-group protein 2. Enrichment of a rapidly migrating form in testis.

    PubMed Central

    Bucci, L R; Brock, W A; Meistrich, M L

    1985-01-01

    A determination of the absolute amounts of high-mobility-group proteins 1 and 2 (HMG1 and HMG2) in rat tissues demonstrated that amounts of HMG2 were low in non-proliferating tissues, somewhat higher in proliferating and lymphoid tissues, but were extremely elevated in the testis. This increase was due to a germ-cell-specific form of HMG2 with increased mobility relative to somatic HMG2 on acid/urea/polyacrylamide-gel electrophoresis. To determine if the findings in the rat were a general feature of spermatogenesis, testis (germinal), spleen (lymphoid), and liver (non-proliferating) tissues from various vertebrate species were examined for their relative amounts of HMG1 and HMG2, and for HMG2 heterogeneity. Bull, chimpanzee, cynomologus monkey, dog, gopher, guinea pig, hamster, mouse, opossum, rabbit, rat, rhesus monkey, squirrel and toad (Xenopus) tissues were analysed. Nearly all species showed relatively high contents of HMG2 in testis tissue, whereas HMG1 contents were similar in all species and tissues. Ten of thirteen species showed a rapidly migrating HMG2 subtype in testis tissue, separable by acid/urea/polyacrylamide-gel electrophoresis. Xenopus, which lacks HMG2 in somatic tissues, showed an HMG2-like protein in testis tissue. Although the rapidly migrating HMG2 subtype in species other than rat was not testis-specific, it was always enriched in the testis. This study indicates that increased amounts of HMG2 and the enrichment of a rapidly migrating HMG2 subtype are general features of spermatogenic cells. Images Fig. 1. Fig. 2. Fig. 3. PMID:4038257

  3. DNA Recombinase Proteins, their Function and Structure in the Active Form, a Computational Study

    NASA Technical Reports Server (NTRS)

    Carra, Claudio; Cucinotta, Francis A.

    2007-01-01

    Homologous recombination is a crucial sequence of reactions in all cells for the repair of double strand DNA (dsDNA) breaks. While it was traditionally considered as a means for generating genetic diversity, it is now known to be essential for restart of collapsed replication forks that have met a lesion on the DNA template (Cox et al., 2000). The central stage of this process requires the presence of the DNA recombinase protein, RecA in bacteria, RadA in archaea, or Rad51 in eukaryotes, which leads to an ATP-mediated DNA strand-exchange process. Despite many years of intense study, some aspects of the biochemical mechanism, and structure of the active form of recombinase proteins are not well understood. Our theoretical study is an attempt to shed light on the main structural and mechanistic issues encountered on the RecA of the e-coli, the RecA of the extremely radio resistant Deinococcus Radiodurans (promoting an inverse DNA strand-exchange repair), and the homolog human Rad51. The conformational changes are analyzed for the naked enzymes, and when they are linked to ATP and ADP. The average structures are determined over 2ns time scale of Langevian dynamics using a collision frequency of 1.0 ps(sup -1). The systems are inserted in an octahedron periodic box with a 10 Angstrom buffer of water molecules explicitly described by the TIP3P model. The corresponding binding free energies are calculated in an implicit solvent using the Poisson-Boltzmann solvent accessible surface area, MM-PBSA model. The role of the ATP is not only in stabilizing the interaction RecA-DNA, but its hydrolysis is required to allow the DNA strand-exchange to proceed. Furthermore, we extended our study, using the hybrid QM/MM method, on the mechanism of this chemical process. All the calculations were performed using the commercial code Amber 9.

  4. Polymer-Induced Heteronucleation for Protein Single Crystal Growth: Structural Elucidation of Bovine Liver Catalase and Concanavalin A Forms

    SciTech Connect

    Foroughi, Leila M.; Kang, You-Na; Matzger, Adam J.

    2012-05-09

    Obtaining single crystals for X-ray diffraction remains a major bottleneck in structural biology; when existing crystal growth methods fail to yield suitable crystals, often the target rather than the crystallization approach is reconsidered. Here we demonstrate that polymer-induced heteronucleation, a powerful technique that has been used for small molecule crystallization form discovery, can be applied to protein crystallization by optimizing the heteronucleant composition and crystallization formats for crystallizing a wide range of protein targets. Applying these advances to two benchmark proteins resulted in dramatically increased crystal size, enabling structure determination, for a half century old form of bovine liver catalase (BLC) that had previously only been characterized by electron microscopy, and the discovery of two new forms of concanavalin A (conA) from the Jack bean and accompanying structural elucidation of one of these forms.

  5. A nacre protein, n16.3, self-assembles to form protein oligomers that dimensionally limit and organize mineral deposits.

    PubMed

    Perovic, Iva; Chang, Eric P; Lui, Michael; Rao, Ashit; Cölfen, Helmut; Evans, John Spencer

    2014-04-29

    The mollusk shell is a complex biological material that integrates mineral phases with organic macromolecular components such as proteins. The role of proteins in the formation of the nacre layer (aragonite mineral phase) is poorly understood, particularly with regard to the organization of mineral deposits within the protein extracellular matrix and the identification of which proteins are responsible for this task. We report new experiments that provide insight into the role of the framework nacre protein, n16.3 (Pinctada fucata), as an organizer or assembler of calcium carbonate mineral clusters. Using a combination of biophysical techniques, we find that recombinant n16.3 (r-n16.3) oligomerizes to form amorphous protein films and particles that possess regions of disorder and mobility. These supramolecular assemblies possess an intrinsically disordered C-terminal region (T64-W98) and reorganize in the presence of Ca(2+) ions to form clustered protein oligomers. This Ca(2+)-induced reorganization leads to alterations in the molecular environments of Trp residues, the majority of which reside in putative aggregation-prone cross-β strand regions. Potentiometric Ca(2+) titrations reveal that r-n16.3 does not significantly affect the formation of prenucleation clusters in solution, and this suggests a role for this protein in postnucleation mineralization events. This is verified in subsequent in vitro mineralization assays in which r-n16.3 demonstrates its ability to form gel-like protein phases that organize and cluster nanometer-sized single-crystal calcite relative to protein-deficient controls. We conclude that the n16 nacre framework proteome creates a protein gel matrix that organizes and dimensionally limits mineral deposits. This process is highly relevant to the formation of ordered, nanometer-sized nacre tablets in the mollusk shell.

  6. Gastro-resistant characteristics of GRAS-grade enteric coatings for pharmaceutical and nutraceutical products.

    PubMed

    Czarnocka, Justyna K; Alhnan, Mohamed A

    2015-01-01

    The use of naturally derived excipients to develop enteric coatings offers significant advantages over conventional synthetic polymers. Unlike synthetic polymers, they are biodegradable, relatively abundant, have no daily intake limits or restrictions on use for dietary and nutraceutical products. However, little information is available on their dissolution properties under different gastrointestinal conditions and in comparison to each other. This work investigated the gastric resistance properties of commercially available GRAS-based coating technologies. Three coating systems were evaluated: ethyl cellulose+carboxymethyl cellulose (EC-CMC), ethyl cellulose+sodium alginate (EC-Alg) and shellac+sodium alginate (Sh-Alg) combinations. The minimum coating levels were optimized to meet USP pharmacopoeial criteria for delayed release formulations (<10% release after 2h in pH 1.2 followed by >80% release after 45 min of pH change). Theophylline 150 mg tablets were coated with 6.5%, 7%, and 2.75% coating levels of formulations EC-CMC, EC-Alg and Sh-Alg, respectively. In vitro dissolution test revealed a fast release in pH 6.8 for ethyl cellulose based coatings: t80% value of 65 and 45 min for EC-CMC and EC-Alg respectively, while a prolonged drug release from Sh-Alg coating was observed in both pH 6.8 and 7.4 phosphate buffers. However, when more biologically relevant bicarbonate buffer was used, all coatings showed slower drug release. Disintegration test, carried out in both simulated gastric and intestinal fluid, confirmed good mechanical resistance of EC-CMC and EC-Alg coating, and revealed poor durability of the thinner Sh-Alg. Under elevated gastric pH conditions (pH 2, 3 and 4), EC-CMC and EC-Alg coatings were broken after 70, 30, 55 min and after 30, 15, 15 min, respectively, while Sh-Alg coated tablets demonstrated gastric resistance at all pH values. In conclusion, none of the GRAS-grade coatings fully complied with the different biological demands of delayed

  7. The major form of hepatitis C virus alternate reading frame protein is suppressed by core protein expression

    PubMed Central

    Wolf, Marie; Dimitrova, Maria; Baumert, Thomas F.; Schuster, Catherine

    2008-01-01

    Hepatitis C virus (HCV) is a human RNA virus encoding 10 proteins in a single open reading frame. In the +1 frame, an ‘alternate reading frame’ (ARF) overlaps with the core protein-encoding sequence and encodes the ARF protein (ARFP). Here, we investigated the molecular regulatory mechanisms of ARFP expression in HCV target cells. Chimeric HCV-luciferase reporter constructs derived from the infectious HCV prototype isolate H77 were transfected into hepatocyte-derived cell lines. Translation initiation was most efficient at the internal AUG codon at position 86/88, resulting in the synthesis of a truncated ARFP named 86/88ARFP. Interestingly, 86/88ARFP synthesis was markedly enhanced in constructs containing an inactivated core protein reading frame. This enhancement was reversed by co-expression of core protein in trans, demonstrating suppression of ARFP synthesis by HCV core protein. In conclusion, our results indicate that translation of ARFP occurs mainly by alternative internal initiation at position 86/88 and is regulated by HCV core protein expression. The suppression of ARFP translation by HCV core protein suggests that ARFP expression is inversely linked to the level of viral replication. These findings define key mechanisms regulating ARFP expression and set the stage for further studies addressing the function of ARFP within the viral life cycle. PMID:18400784

  8. Comparison of Replica Exchange Simulations of a Kinetically Trapped Protein Conformational State and its Native Form.

    PubMed

    Olson, Mark A; Legler, Patricia M; Goldman, Ellen R

    2016-03-10

    Recently an X-ray crystallographic structure of a single-domain antibody was reported with the protein chain trapped in a rare homodimeric form. One of the conformers appears to exhibit a misfolded region, and thus presumably the configurational stability is less favorable. To investigate whether simulation methods can detect any difference between the conformers and buttress the notion that one conformation is trapped on a pathway that incurs lower activation energy to unfold, adaptive temperature-based replica exchange simulations were applied to each chain to model conformational transitions. Simulation results found that the observed crystallographic difference between the two chains in the complementarity determining region CDR2 induces a stark distinction in conformational populations on the energy landscape. An appraisal of the energetic difference between the CDR2 conformations at 300 K revealed a localized order-disorder free-energy transition of roughly equivalent to two peptide hydrogen bonds in solution. It was also found that interconversion between the conformers is slower than the rate to unfold and that near an unfolding transition temperature one conformer retained a greater fraction of native-like contacts and energy over a longer time span before fully populating the denatured state, thus verifying the coexistence of a metastable conformation in the crystallographic assembly.

  9. The HhH domain of the human DNA repair protein XPF forms stable homodimers.

    PubMed

    Das, Devashish; Tripsianes, Konstantinos; Jaspers, Nicolaas G J; Hoeijmakers, Jan H J; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E

    2008-03-01

    The human XPF-ERCC1 protein complex plays an essential role in nucleotide excision repair by catalysing positioned nicking of a DNA strand at the 5' side of the damage. We have recently solved the structure of the heterodimeric complex of the C-terminal domains of XPF and ERCC1 (Tripsianes et al., Structure 2005;13:1849-1858). We found that this complex comprises a pseudo twofold symmetry axis and that the helix-hairpin-helix motif of ERCC1 is required for DNA binding, whereas the corresponding domain of XPF is functioning as a scaffold for complex formation with ERCC1. Despite the functional importance of heterodimerization, the C-terminal domain of XPF can also form homodimers in vitro. We here compare the stabilities of homodimeric and heterodimeric complexes of the C-terminal domains of XPF and ERCC1. The higher stability of the XPF HhH complexes under various experimental conditions, determined using CD and NMR spectroscopy and mass spectrometry, is well explained by the structural differences that exist between the HhH domains of the two complexes. The XPF HhH homodimer has a larger interaction interface, aromatic stacking interactions, and additional hydrogen bond contacts as compared to the XPF/ERCC1 HhH complex, which accounts for its higher stability.

  10. Ex vivo mammalian prions are formed of paired double helical prion protein fibrils

    PubMed Central

    Terry, Cassandra; Wenborn, Adam; Gros, Nathalie; Sells, Jessica; Joiner, Susan; Hosszu, Laszlo L. P.; Tattum, M. Howard; Panico, Silvia; Clare, Daniel K.; Collinge, John; Saibil, Helen R.

    2016-01-01

    Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity and the three-dimensional structure of infectious prions has remained obscure. Recently, we developed novel methods to obtain exceptionally pure preparations of prions from mouse brain and showed that pathogenic PrP in these high-titre preparations is assembled into rod-like assemblies. Here, we have used precise cell culture-based prion infectivity assays to define the physical relationship between the PrP rods and prion infectivity and have used electron tomography to define their architecture. We show that infectious PrP rods isolated from multiple prion strains have a common hierarchical assembly comprising twisted pairs of short fibres with repeating substructure. The architecture of the PrP rods provides a new structural basis for understanding prion infectivity and can explain the inability to systematically generate high-titre synthetic prions from recombinant PrP. PMID:27249641

  11. Ex vivo mammalian prions are formed of paired double helical prion protein fibrils.

    PubMed

    Terry, Cassandra; Wenborn, Adam; Gros, Nathalie; Sells, Jessica; Joiner, Susan; Hosszu, Laszlo L P; Tattum, M Howard; Panico, Silvia; Clare, Daniel K; Collinge, John; Saibil, Helen R; Wadsworth, Jonathan D F

    2016-05-01

    Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity and the three-dimensional structure of infectious prions has remained obscure. Recently, we developed novel methods to obtain exceptionally pure preparations of prions from mouse brain and showed that pathogenic PrP in these high-titre preparations is assembled into rod-like assemblies. Here, we have used precise cell culture-based prion infectivity assays to define the physical relationship between the PrP rods and prion infectivity and have used electron tomography to define their architecture. We show that infectious PrP rods isolated from multiple prion strains have a common hierarchical assembly comprising twisted pairs of short fibres with repeating substructure. The architecture of the PrP rods provides a new structural basis for understanding prion infectivity and can explain the inability to systematically generate high-titre synthetic prions from recombinant PrP.

  12. Topographic labelling of pore-forming proteins from the outer membrane of Escherichia coli.

    PubMed Central

    Page, M G; Rosenbusch, J P

    1986-01-01

    The topography of three pore-forming proteins from the outer membrane of Escherichia coli has been explored by using two labelling techniques. Firstly, the distribution of nucleophilic residues has been investigated by selective chemical modification using arylglyoxals (for arginine residues), isothiocyanates (for lysine residues), carbodi-imides (for carboxy residues) and diazonium salts. Secondly, the membrane-embedded domains have been investigated by labelling with photoactivatable phospholipid analogues and a reagent that partitions into the membrane. Few nucleophilic groups are found to be freely accessible to pore-impermeant probes reacting in the aqueous medium. More groups are accessible to small, pore-permeant probes, suggesting that several groups of each sort are contained within the pore. In addition, there appear to be a number of arginine, lysine, carboxyl and many tyrosine residues that are rather inaccessible and that react only with small, hydrophobic probes, if at all. Amongst these more deeply buried residues there are four arginine residues and an as-yet-undetermined number of carboxy residues that appear to be essential to the structural integrity of the oligomeric molecule. Images Fig. 4. PMID:2428354

  13. Functional Analysis of the Hydrophilic Loop in Intracellular Trafficking of Arabidopsis PIN-FORMED Proteins.

    PubMed

    Ganguly, Anindya; Park, Minho; Kesawat, Mahipal Singh; Cho, Hyung-Taeg

    2014-04-01

    Different PIN-FORMED proteins (PINs) contribute to intercellular and intracellular auxin transport, depending on their distinctive subcellular localizations. Arabidopsis thaliana PINs with a long hydrophilic loop (HL) (PIN1 to PIN4 and PIN7; long PINs) localize predominantly to the plasma membrane (PM), whereas short PINs (PIN5 and PIN8) localize predominantly to internal compartments. However, the subcellular localization of the short PINs has been observed mostly for PINs ectopically expressed in different cell types, and the role of the HL in PIN trafficking remains unclear. Here, we tested whether a long PIN-HL can provide its original molecular cues to a short PIN by transplanting the HL. The transplanted long PIN2-HL was sufficient for phosphorylation and PM trafficking of the chimeric PIN5:PIN2-HL but failed to provide the characteristic polarity of PIN2. Unlike previous observations, PIN5 showed clear PM localization in diverse cell types where PIN5 is natively or ectopically expressed and even polar PM localization in one cell type. Furthermore, in the root epidermis, the subcellular localization of PIN5 switched from PM to internal compartments according to the developmental stage. Our results suggest that the long PIN-HL is partially modular for the trafficking behavior of PINs and that the intracellular trafficking of PIN is plastic depending on cell type and developmental stage.

  14. Microscopic relaxations in a protein sustained down to 160 K in a non-glass forming organic solvent

    SciTech Connect

    Mamontov, Eugene; O'Neil, Hugh

    2016-05-03

    In this paper, we have studied microscopic dynamics of a protein in carbon disulfide, a non-glass forming solvent, down to its freezing temperature of ca. 160 K. We have utilized quasielastic neutron scattering. A comparison of lysozyme hydrated with water and dissolved in carbon disulfide reveals a stark difference in the temperature dependence of the protein's microscopic relaxation dynamics induced by the solvent. In the case of hydration water, the common protein glass-forming solvent, the protein relaxation slows down in response to a large increase in the water viscosity on cooling down, exhibiting a well-known protein dynamical transition. The dynamical transition disappears in non-glass forming carbon disulfide, whose viscosity remains a weak function of temperature all the way down to freezing at just below 160 K. The microscopic relaxation dynamics of lysozyme dissolved in carbon disulfide is sustained down to the freezing temperature of its solvent at a rate similar to that measured at ambient temperature. Finally, our results demonstrate that protein dynamical transition is not merely solvent-assisted, but rather solvent-induced, or, more precisely, is a reflection of the temperature dependence of the solvent's glass-forming dynamics.

  15. Microscopic relaxations in a protein sustained down to 160 K in a non-glass forming organic solvent

    DOE PAGES

    Mamontov, Eugene; O'Neil, Hugh

    2016-05-03

    In this paper, we have studied microscopic dynamics of a protein in carbon disulfide, a non-glass forming solvent, down to its freezing temperature of ca. 160 K. We have utilized quasielastic neutron scattering. A comparison of lysozyme hydrated with water and dissolved in carbon disulfide reveals a stark difference in the temperature dependence of the protein's microscopic relaxation dynamics induced by the solvent. In the case of hydration water, the common protein glass-forming solvent, the protein relaxation slows down in response to a large increase in the water viscosity on cooling down, exhibiting a well-known protein dynamical transition. The dynamicalmore » transition disappears in non-glass forming carbon disulfide, whose viscosity remains a weak function of temperature all the way down to freezing at just below 160 K. The microscopic relaxation dynamics of lysozyme dissolved in carbon disulfide is sustained down to the freezing temperature of its solvent at a rate similar to that measured at ambient temperature. Finally, our results demonstrate that protein dynamical transition is not merely solvent-assisted, but rather solvent-induced, or, more precisely, is a reflection of the temperature dependence of the solvent's glass-forming dynamics.« less

  16. Lipids as cofactors in protein folding: Stereo-specific lipid–protein interactions are required to form HAMLET (human α-lactalbumin made lethal to tumor cells)

    PubMed Central

    Svensson, Malin; Mossberg, Ann-Kristin; Pettersson, Jenny; Linse, Sara; Svanborg, Catharina

    2003-01-01

    Proteins can adjust their structure and function in response to shifting environments. Functional diversity is created not only by the sequence but by changes in tertiary structure. Here we present evidence that lipid cofactors may enable otherwise unstable protein folding variants to maintain their conformation and to form novel, biologically active complexes. We have identified unsaturated C18 fatty acids in the cis conformation as the cofactors that bind apo α-lactalbumin and form HAMLET (human α-lactalbumin made lethal to tumor cells). The complexes were formed on an ion exchange column, were stable in a molten globule-like conformation, and had attained the novel biological activity. The protein–fatty acid interaction was specific, as saturated C18 fatty acids, or unsaturated C18:1trans conformers were unable to form complexes with apo α-lactalbumin, as were fatty acids with shorter or longer carbon chains. Unsaturated cis fatty acids other than C18:1:9cis were able to form stable complexes, but these were not active in the apoptosis assay. The results demonstrate that stereo-specific lipid–protein interactions can stabilize partially unfolded conformations and form molecular complexes with novel biological activity. The results offer a new mechanism for the functional diversity of proteins, by exploiting lipids as essential, tissue-specific cofactors in this process. PMID:14627740

  17. Comparison of four different crystal forms of the Mycobacterium tuberculosis ESX-1 secreted protein regulator EspR.

    PubMed

    Gangwar, Shanti P; Meena, Sita R; Saxena, Ajay K

    2014-04-01

    The Mycobacterium tuberculosis ESX-1 secreted protein regulator (EspR, Rv3849) is the key protein that delivers bacterial proteins into the host cell during mycobacterial infection. EspR binds directly to the espACD operon and is involved in transcriptional activation. In the current study, M. tuberculosis EspR has been crystallized and its X-ray structure has been determined at 3.3 Å resolution in a P3221 crystal form. EspR forms a physiological dimer in the crystal. Each EspR monomer contains an N-terminal helix-turn-helix DNA-binding domain and a C-terminal dimerization domain. The EspR structure in the P3221 crystal form was compared with previously determined EspR structures in P32, P21 and P212121 crystal forms. Structural comparison analysis indicated that the N-terminal helix-turn-helix domain of EspR acquires a rigid structure in the four crystal forms. However, significant structural differences were observed in the C-terminal domain of EspR in the P21 crystal form when compared with the P3221 and P32 crystal forms. The interaction, stabilization energy and buried surface area analysis of EspR in the four different crystal forms have provided information about the physiological dimer interface of EspR.

  18. Immunological and Chemical Identification of Intracellular Forms of Adenovirus Type 2 Terminal Protein

    PubMed Central

    Green, Maurice; Symington, Janey; Brackmann, Karl H.; Cartas, Maria A.; Thornton, Helen; Young, Leann

    1981-01-01

    Highly purified adenovirus type 2 terminal protein (TP) with an apparent Mr of 55,000 (55K) was prepared in quantities of 10 to 30 μg from guanidine hydrochloride- or sodium dodecyl sulfate-disrupted virions (60 to 120 mg). Guinea pigs were immunized with 14 to 20 injections of TP in amounts of 1 to 2 μg. Antiserum to TP was used to study the intracellular polypeptides related to adenovirus type 2 TP. By immunoprecipitation with anti-TP serum, we identified 80K and 76K polypeptides in the nucleoplasmic and cytoplasmic S100 fractions of [35S]methionine-labeled cells early and late after infection with Ad2. By immunoautoradiographic analysis which eliminates coprecipitation of unrelated proteins, we identified an 80K polypeptide (probably an 80K-76K doublet) in unlabeled, late infected cells, using anti-TP serum and 125I-labeled staphylococcal protein A. About two- to threefold-higher levels of the 80K and 76K polypeptides were present in the nucleoplasm than in the S100 fraction, and two- to threefold-higher levels were found in late infected cells than in early infected cells (cycloheximide enhanced, arabinofuranosylcytosine treated). We did not detect the 80K or 76K polypeptide in uninfected cells, indicating that these polypeptides are virus coded. Tryptic peptide map analysis showed that the 80K and 76K polypeptides are very closely related and that they share peptides with the DNA-bound 55K TP. Our data provide the first direct demonstration of intracellular 80K and 76K forms of TP. The intracellular 80K and 76K polypeptides are closely related or identical to the 80K polypeptide that Challberg and co-workers (Proc. Natl. Acad. Sci. U.S.A. 77:5105-5109, 1980) detected at the termini of adenovirus DNA synthesized in vitro and to the 87K polypeptide that Stillman and co-workers (Cell 23:497-508, 1981) translated in vitro. We did not detect the 55K TP in early or late infected cells, consistent with the proposal by Challberg and co-workers that the 80K

  19. Reduction of Listeria monocytogenes in queso fresco cheese by a combination of listericidal and listeriostatic GRAS antimicrobials.

    PubMed

    Soni, Kamlesh A; Desai, Monil; Oladunjoye, Ademola; Skrobot, Frederick; Nannapaneni, Ramakrishna

    2012-04-02

    Single and combined effects of three GRAS (generally recognized as safe) antimicrobials including, bacteriophage P100 (phage P100), lauric arginate (LAE), and potassium lactate-sodium diacetate mixture (PL-SD) were evaluated against Listeria monocytogenes cold growth in queso fresco cheese (QFC). The fate of phage P100 when exposed to LAE (200 ppm) or PL-SD (2.8% PL and 0.2% SD) was determined at 4°C and 30°C in a broth model. Phage P100 was found to be stable in the presence of these antimicrobial agents as plaque forming units (PFU) did not vary between control, LAE or PL-SD treatments. When 9 log CFU/ml of stationary phase cells of L. monocytogenes was exposed to these antimicrobials in tryptic soy broth, there was a 3 to 5 log CFU/ml reduction with phage P100 and a complete 9 log CFU/ml reduction with LAE but no measurable reduction with PL-SD after 24h at 4°C or 30°C. In QFC, the L. monocytogenes populations increased from the initial 3.5 log CFU/cm(2) to 7.7 log CFU/cm(2) in 28 days at 4°C. Treatment with 7.8 log PFU/cm(2) of phage P100 or 200 ppm of LAE showed strong listericidal effect initially by reducing L. monocytogenes counts by 2 to 3.5-4 log CFU/cm(2) while there was a subsequent regrowth of L. monocytogenes at 4°C. Treatment with PL-SD showed strong listeriostatic effect without decreasing L. monocytogenes counts but growth was prevented for 28 days at 4°C. Only the combined treatment of listericidal phage P100 or LAE with listeriostatic PL-SD reduced the initial L. monocytogenes counts by 2-4 log CFU/cm(2) and also kept the L. monocytogenes counts at that reduced level in QFC for 28 days at 4°C.

  20. Screening and Characterization of Hydrate Forms of T-3256336, a Novel Inhibitor of Apoptosis (IAP) Protein Antagonist.

    PubMed

    Takeuchi, Shoko; Kojima, Takashi; Hashimoto, Kentaro; Saito, Bunnai; Sumi, Hiroyuki; Ishikawa, Tomoyasu; Ikeda, Yukihiro

    2015-01-01

    Different crystal packing of hydrates from anhydrate crystals leads to different physical properties, such as solubility and stability. Investigation of the potential of varied hydrate formation, and understanding the stability in an anhydrous/hydrate system, are crucial to prevent an undesired transition during the manufacturing process and storage. Only one anhydrous form of T-3256336, a novel inhibitor of apoptosis (IAP) protein antagonist, was discovered during synthesis, and no hydrate form has been identified. In this study, we conducted hydrate screening such as dynamic water vapor sorption/desorption (DVS), and the slurry experiment, and characterized the solid-state properties of anhydrous/hydrate forms to determine the most desirable crystalline form for development. New hydrate forms, both mono-hydrate and hemi-hydrate forms, were discovered as a result of this hydrate screening. The characterization of two new hydrate forms was conducted, and the anhydrous form was determined to be the most desirable development form of T-3256336 in terms of solid-state stability. In addition, the stability of the anhydrous form was investigated using the water content and temperature controlled slurry experiment to obtain the desirable crystal form in the crystallization process. The water content regions of the stable phase of the desired form, the anhydrous form, were identified for the cooling crystallization process.

  1. Sexual risk behavior in men attending Mardi Gras celebrations in New Orleans, Louisiana.

    PubMed

    Benotsch, Eric G; Nettles, Christopher D; Wong, Felicia; Redmann, Jean; Boschini, Jill; Pinkerton, Steven D; Ragsdale, Kathleen; Mikytuck, John J

    2007-10-01

    Previous research with travelers points to higher risk behaviors during vacations. Relative to their day-to-day lives, leisure travelers have more free time to pursue sexual activities and are likely to engage in higher rates of substance use than when at home. Risk behaviors during vacation have not been thoroughly examined in men who have sex with men (MSM), a key group at risk for HIV. The present investigation examined substance use, sexual risk behaviors, and components of the Information-Motivation-Behavioral Skills (IMB) Model in MSM attending Mardi Gras celebrations in New Orleans. Almost half of the sexually active men reported having sex with a partner of unknown HIV status while in New Orleans and a similar number did not disclose their own HIV status to all of their sexual partners. Drug use and excessive alcohol use were associated with unprotected sex (ps < .05). Components of the IMB model also predicted sexual risk behavior: individuals with more accurate HIV transmission information reported fewer unprotected sex acts, and motivation to engage in sexual activity on vacation was associated with more unprotected sex (ps < .05). Findings suggest that some MSM on vacation are placing themselves at risk for HIV. Traditional HIV prevention interventions do not readily lend themselves for use with transient populations. New intervention approaches are needed to reduce sexual risk behaviors in persons traveling for leisure.

  2. Platelet factor 4 binds to glycanated forms of thrombomodulin and to protein C. A potential mechanism for enhancing generation of activated protein C.

    PubMed

    Dudek, A Z; Pennell, C A; Decker, T D; Young, T A; Key, N S; Slungaard, A

    1997-12-12

    Platelet factor 4 (PF4) is an abundant platelet alpha-granule heparin-binding protein. We have previously shown that PF4 accelerates up to 25-fold the proteolytic conversion of protein C to activated protein C by the thrombin.thrombomodulin complex by increasing its affinity for protein C 30-fold. This stimulatory effect requires presence of the gamma-carboxyglutamic acid (Gla) domain in protein C and is enhanced by the presence of a chondroitin sulfate glycosaminoglycan (GAG) domain on thrombomodulin. We hypothesized that cationic PF4 binds to both protein C and thrombomodulin through these anionic domains. Qualitative SDS-polyacrylamide gel electrophoresis analysis of avidin extracts of solutions containing biotinylated PF4 and candidate ligands shows that PF4 binds to GAG+ but not GAG- forms of thrombomodulin and native but not Gla-domainless protein C. Quantitative analysis using the surface plasmon resonance-based BIAcoreTM biosensor system confirms the extremely high affinity of PF4 for heparin (KD = 4 nM) and shows that PF4 binds to GAG+ thrombomodulin with a KD of 31 nM and to protein C with a KD of 0.37 microM. In contrast, PF4 had no measurable interaction with GAG- thrombomodulin or Gla-domainless protein C. Western blot analysis of normal human plasma extracted with biotinylated PF4 demonstrates PF4 binding to protein C in a physiologic context. Thus, PF4 binds with relative specificity and high affinity to the GAG- domain of thrombomodulin and the Gla domain of protein C. These interactions may enhance the affinity of the thrombin.thrombomodulin complex for protein C and thereby promote the generation of activated protein C.

  3. The fungal cerato-platanin protein EPL1 forms highly ordered layers at hydrophobic/hydrophilic interfaces.

    PubMed

    Bonazza, K; Gaderer, R; Neudl, S; Przylucka, A; Allmaier, G; Druzhinina, I S; Grothe, H; Friedbacher, G; Seidl-Seiboth, V

    2015-03-07

    Cerato-platanin proteins (CPPs) and hydrophobins are two classes of small, secreted proteins that are exclusively found in fungi. CPPs are known as chitin-binding proteins, and were recently also shown to form protein layers at air/water interfaces, but the features of these layers were not investigated on the molecular level yet. In this study, by means of atomic force microscopy (AFM), EPL1, a member of the CPP family was shown to form highly ordered monolayers at a hydrophobic surface/liquid-interface. Furthermore, two new hydrophobins were analysed, and the influence of EPL1 on hydrophobin layers was studied in situ. Hydrophobins are amphiphilic proteins that are able to self-assemble at hydrophobic/hydrophilic interfaces, thereby inverting the polarity of the surface. This renders fungal growth structures such as spores water repellent. The combination of AFM data and wettability experiments led to the conclusion that in presence of both, hydrophobins and EPL1, a previously unknown hybrid layer is formed. This mixed protein layer is on one hand not inverting but enhancing the hydrophobicity of HOPG (highly oriented pyrolytic graphite), typical for EPL1, and on the other hand, it is stable and water insoluble, which is reminiscent of hydrophobin layers.

  4. LexA repressor forms stable dimers in solution. The role of specific dna in tightening protein-protein interactions.

    PubMed

    Mohana-Borges, R; Pacheco, A B; Sousa, F J; Foguel, D; Almeida, D F; Silva, J L

    2000-02-18

    Cooperativity in the interactions among proteins subunits and DNA is crucial for DNA recognition. LexA repressor was originally thought to bind DNA as a monomer, with cooperativity leading to tighter binding of the second monomer. The main support for this model was a high value of the dissociation constant for the LexA dimer (micromolar range). Here we show that the protein is a dimer at nanomolar concentrations under different conditions. The reversible dissociation of LexA dimer was investigated by the effects of hydrostatic pressure or urea, using fluorescence emission and polarization to monitor the dissociation process. The dissociation constant lies in the picomolar range (lower than 20 pM). LexA monomers associate with an unusual large volume change (340 ml/mol), indicating the burial of a large surface area upon dimerization. Whereas nonspecific DNA has no stabilizing effect, specific DNA induces tightening of the dimer and a 750-fold decrease in the K(d). In contrast to the previous model, a tight dimer rather than a monomer is the functional repressor. Accordingly, the LexA dimer only loses its ability to recognize a specific DNA sequence by RecA-induced autoproteolysis. Our work provides insights into the linkage between protein-protein interactions, DNA recognition, and DNA repair.

  5. Outer Membrane Proteins form Specific Patterns in Antibiotic-Resistant Edwardsiella tarda

    PubMed Central

    Peng, Bo; Wang, Chao; Li, Hui; Su, Yu-bin; Ye, Jin-zhou; Yang, Man-jun; Jiang, Ming; Peng, Xuan-xian

    2017-01-01

    Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. The comparison of a clinically isolated strain to the laboratory derived kanamycin-, tetracycline-, or chloramphenicol-resistant strains identified their respective outer membrane proteins expression patterns, which are distinct to each other. Similarly, the same approach was utilized to profile the patterns in double antibiotic-resistant bacteria. Surprisingly, one pattern is always dominant over the other as to these three antibiotics; the pattern of chloramphenicol is over tetracycline, which is over kanamycin. This type of pattern was also confirmed in clinically relevant multidrug-resistant bacteria. In addition, the presence of plasmid encoding antibiotic-resistant genes also alters the outer membrane protein profile in a similar manner. Our results demonstrate that bacteria adapt the antibiotic stress through the regulation of outer membrane proteins expression. And more importantly, different outer membrane protein profiles were required to cope with different antibiotics. This type of specific pattern provides the rationale for the development of novel strategy to design outer membrane protein arrays to identify diverse multidrug resistance profiles as biomarkers for clinical medication. PMID:28210241

  6. BHMP39 PROTEINS OF B. HYODYSENTERIAE FORM HIGH MOLECULAR WEIGHT COMPLEXES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brachyspira hyodysenteriae is the aetiological agent of swine dysentery, a severe mucohaemorrhagic diarrhoeal disease of pigs, with economic significance for the global pork industry. The most abundant outer membrane proteins of B. hyodysenteriae are from the Bhmp39 family of proteins. Eight bhmp39 ...

  7. Outer Membrane Proteins form Specific Patterns in Antibiotic-Resistant Edwardsiella tarda.

    PubMed

    Peng, Bo; Wang, Chao; Li, Hui; Su, Yu-Bin; Ye, Jin-Zhou; Yang, Man-Jun; Jiang, Ming; Peng, Xuan-Xian

    2017-01-01

    Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. The comparison of a clinically isolated strain to the laboratory derived kanamycin-, tetracycline-, or chloramphenicol-resistant strains identified their respective outer membrane proteins expression patterns, which are distinct to each other. Similarly, the same approach was utilized to profile the patterns in double antibiotic-resistant bacteria. Surprisingly, one pattern is always dominant over the other as to these three antibiotics; the pattern of chloramphenicol is over tetracycline, which is over kanamycin. This type of pattern was also confirmed in clinically relevant multidrug-resistant bacteria. In addition, the presence of plasmid encoding antibiotic-resistant genes also alters the outer membrane protein profile in a similar manner. Our results demonstrate that bacteria adapt the antibiotic stress through the regulation of outer membrane proteins expression. And more importantly, different outer membrane protein profiles were required to cope with different antibiotics. This type of specific pattern provides the rationale for the development of novel strategy to design outer membrane protein arrays to identify diverse multidrug resistance profiles as biomarkers for clinical medication.

  8. Mitochondria: a kinase anchoring protein 1, a signaling platform for mitochondrial form and function.

    PubMed

    Merrill, Ronald A; Strack, Stefan

    2014-03-01

    Mitochondria are best known for their role as cellular power plants, but they also serve as signaling hubs, regulating cellular proliferation, differentiation, and survival. A kinase anchoring protein 1 (AKAP1) is a scaffold protein that recruits protein kinase A (PKA) and other signaling proteins, as well as RNA, to the outer mitochondrial membrane. AKAP1 thereby integrates several second messenger cascades to modulate mitochondrial function and associated physiological and pathophysiological outcomes. Here, we review what is currently known about AKAP1's macromolecular interactions in health and disease states, including obesity. We also discuss dynamin-related protein 1 (Drp1), the enzyme that catalyzes mitochondrial fission, as one of the key substrates of the PKA/AKAP1 signaling complex in neurons. Recent evidence suggests that AKAP1 has critical roles in neuronal development and survival, which are mediated by inhibitory phosphorylation of Drp1 and maintenance of mitochondrial integrity.

  9. Cytochrome c peroxidase. Interconversion of chemically and enzymatically reactive and unreactive forms of the ferric protein.

    PubMed

    Mathews, R A; Wittenberg, J B

    1979-07-10

    Ferric yeast cytochrome c peroxidase in the presence of different anions may assume a number of forms which differ in optical spectra and chemical properties. In solutions whose only anion is acetate, two spectral forms are present together in an equilibrium. Each of these spectral species is believed to bear bound acetate anion. A form characterized by an intense absorption maximum at 620 nm is unreactive enzymatically and does not react with hydrogen peroxide or with dithionite. A form characterized by a less intense absorption near 645 nm is enzymatically and chemically reactive. Increasing temperature and increasing pH displace the equilibrium toward the 645 nm form. Increasing cytochrome c peroxidase concentration favors the 620 nm form. In kinetic experiments in which the 645 nm form is removed by rapid reaction with H2O2 or dithionite, the 620 nm form is converted in a first order reaction (k = 0.36 s-1, 15 degrees C) to the 645 nm form. In solutions whose sole anion is phosphate a 645 nm form is the only demonstrable spectral species. The enzymatic activity and rates of chemical reaction of 645 nm spectral forms occurring in acetate and in phosphate buffers are the same.

  10. The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin

    PubMed Central

    de Virgilio, Maddalena; Bellucci, Michele; Mainieri, Davide; Rossi, Marika; Benvenuto, Eugenio; Arcioni, Sergio; Vitale, Alessandro

    2008-01-01

    Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein γ-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of γ-zein). Protein blots and pulse–chase indicate that fusions between Nef and the same γ-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin–Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its γ-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants. PMID:18540021

  11. The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin.

    PubMed

    de Virgilio, Maddalena; De Marchis, Francesca; Bellucci, Michele; Mainieri, Davide; Rossi, Marika; Benvenuto, Eugenio; Arcioni, Sergio; Vitale, Alessandro

    2008-01-01

    Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein gamma-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of gamma-zein). Protein blots and pulse-chase indicate that fusions between Nef and the same gamma-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin-Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its gamma-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants.

  12. Iron-regulatory proteins DmdR1 and DmdR2 of Streptomyces coelicolor form two different DNA-protein complexes with iron boxes.

    PubMed Central

    Flores, Francisco J; Martín, Juan F

    2004-01-01

    In high G+C Gram-positive bacteria, the control of expression of genes involved in iron metabolism is exerted by a DmdR [divalent (bivalent) metal-dependent regulatory protein] in the presence of Fe2+ or other bivalent ions. The dmdR1 and dmdR2 genes of Streptomyces coelicolor were overexpressed in Escherichia coli and the DmdR1 and DmdR2 proteins were purified to homogeneity. Electrophoretic mobility-shift assays showed that both DmdR1 and DmdR2 bind to the 19-nt tox and desA iron boxes forming two different complexes in each case. Increasing the concentrations of DmdR1 or DmdR2 protein shifted these complexes from their low-molecular-mass form to the high-molecular-mass complexes. Formation of the DNA-protein complexes was prevented by the bivalent metal chelating agent 2,2'-dipyridyl and by antibodies specific against the DmdR proteins. Cross-linking with glutaraldehyde of pure DmdR1 or DmdR2 proteins showed that DmdR1 forms dimers, whereas DmdR2 is capable of forming dimers and probably tetramers. Ten different iron boxes were found in a search for iron boxes in the genome of S. coelicolor. Most of them correspond to putative genes involved in siderophore biosynthesis. Since the nucleotide sequence of these ten boxes is identical (or slightly different) with the synthetic DNA fragment containing the desA box used in the present study, it is proposed that DmdR1 and DmdR2 bind to the iron boxes upstream of at least ten different genes in S. coelicolor. PMID:14960152

  13. Novel Protein Substrates of the Phospho-Form Modification System in Neisseria gonorrhoeae and Their Connection to O-Linked Protein Glycosylation

    PubMed Central

    Anonsen, Jan Haug; Egge-Jacobsen, Wolfgang; Aas, Finn Erik; Børud, Bente; Koomey, Michael

    2012-01-01

    The zwitterionic phospho-form moieties phosphoethanolamine (PE) and phosphocholine (PC) are important components of bacterial membranes and cell surfaces. The major type IV pilus subunit protein of Neisseria gonorrhoeae, PilE, undergoes posttranslational modifications with these moieties via the activity of the pilin phospho-form transferase PptA. A number of observations relating to colocalization of phospho-form and O-linked glycan attachment sites in PilE suggested that these modifications might be either functionally or mechanistically linked or interact directly or indirectly. Moreover, it was unknown whether the phenomenon of phospho-form modification was solely dedicated to PilE or if other neisserial protein targets might exist. In light of these concerns, we screened for evidence of phospho-form modification on other membrane glycoproteins targeted by the broad-spectrum O-linked glycosylation system. In this way, two periplasmic lipoproteins, NGO1043 and NGO1237, were identified as substrates for PE addition. As seen previously for PilE, sites of PE modifications were clustered with those of glycan attachment. In the case of NGO1043, evidence for at least six serine phospho-form attachment sites was found, and further analyses revealed that at least two of these serines were also attachment sites for glycan. Finally, mutations altering glycosylation status led to the presence of pptA-dependent PC modifications on both proteins. Together, these results reinforce the associations established in PilE and provide evidence for dynamic interplay between phospho-form modification and O-linked glycosylation. The observations also suggest that phospho-form modifications likely contribute biologically at both intracellular and extracellular levels. PMID:22083701

  14. The Proline Rich Homeodomain Protein PRH/Hhex Forms Stable Oligomers That Are Highly Resistant to Denaturation

    PubMed Central

    Shukla, Anshuman; Burton, Nicholas M.; Jayaraman, Padma-Sheela; Gaston, Kevin

    2012-01-01

    Background Many transcription factors control gene expression by binding to specific DNA sequences at or near the genes that they regulate. However, some transcription factors play more global roles in the control of gene expression by altering the architecture of sections of chromatin or even the whole genome. The ability to form oligomeric protein assemblies allows many of these proteins to manipulate extensive segments of DNA or chromatin via the formation of structures such as DNA loops or protein-DNA fibres. Principal Findings Here we show that the proline rich homeodomain protein PRH/Hhex forms predominantly octameric and/or hexadecameric species in solution as well as larger assemblies. We show that these assemblies are highly stable resisting denaturation by temperature and chemical denaturants. Conclusion These data indicate that PRH is functionally and structurally related to the Lrp/AsnC family of proteins, a group of proteins that are known to act globally to control gene expression in bacteria and archaea. PMID:22540015

  15. Comparative protein expression in different strains of the bloom-forming cyanobacterium Microcystis aeruginosa.

    PubMed

    Alexova, Ralitza; Haynes, Paul A; Ferrari, Belinda C; Neilan, Brett A

    2011-09-01

    Toxin production in algal blooms presents a significant problem for the water industry. Of particular concern is microcystin, a potent hepatotoxin produced by the unicellular freshwater species Microcystis aeruginosa. In this study, the proteomes of six toxic and nontoxic strains of M. aeruginosa were analyzed to gain further knowledge in elucidating the role of microcystin production in this microorganism. This represents the first comparative proteomic study in a cyanobacterial species. A large diversity in the protein expression profiles of each strain was observed, with a significant proportion of the identified proteins appearing to be strain-specific. In total, 475 proteins were identified reproducibly and of these, 82 comprised the core proteome of M. aeruginosa. The expression of several hypothetical and unknown proteins, including four possible operons was confirmed. Surprisingly, no proteins were found to be produced only by toxic or nontoxic strains. Quantitative proteome analysis using the label-free normalized spectrum abundance factor approach revealed nine proteins that were differentially expressed between toxic and nontoxic strains. These proteins participate in carbon-nitrogen metabolism and redox balance maintenance and point to an involvement of the global nitrogen regulator NtcA in toxicity. In addition, the switching of a previously inactive toxin-producing strain to microcystin synthesis is reported.

  16. Drosophila Torsin Protein Regulates Motor Control and Stress Sensitivity and Forms a Complex with Fragile-X Mental Retardation Protein

    PubMed Central

    Ahn, Hyo-Min; Koh, Young Ho

    2016-01-01

    We investigated unknown in vivo functions of Torsin by using Drosophila as a model. Downregulation of Drosophila Torsin (DTor) by DTor-specific inhibitory double-stranded RNA (RNAi) induced abnormal locomotor behavior and increased susceptibility to H2O2. In addition, altered expression of DTor significantly increased the numbers of synaptic boutons. One important biochemical consequence of DTor-RNAi expression in fly brains was upregulation of alcohol dehydrogenase (ADH). Altered expression of ADH has also been reported in Drosophila Fragile-X mental retardation protein (DFMRP) mutant flies. Interestingly, expression of DFMRP was altered in DTor mutant flies, and DTor and DFMRP were present in the same protein complexes. In addition, DTor and DFMRP immunoreactivities were partially colocalized in several cellular organelles in larval muscles. Furthermore, there were no significant differences between synaptic morphologies of dfmrp null mutants and dfmrp mutants expressing DTor-RNAi. Taken together, our evidences suggested that DTor and DFMRP might be present in the same signaling pathway regulating synaptic plasticity. In addition, we also found that human Torsin1A and human FMRP were present in the same protein complexes, suggesting that this phenomenon is evolutionarily conserved. PMID:27313903

  17. A novel tubulin-dependent protein kinase forming a paired helical filament epitope on tau.

    PubMed

    Ishiguro, K; Ihara, Y; Uchida, T; Imahori, K

    1988-09-01

    From rat brain microtubule proteins, we purified a protein kinase that phosphorylated tau, one of microtubule-associated proteins. The electrophoretic mobility of the phosphorylated tau on SDS-polyacrylamide gel decreased. The enzyme was not activated by cyclic nucleotides, calmodulin, or phospholipids, and was inhibited by the calcium ions. The kinase bound to tau. The phosphorylation of tau was stimulated by tubulin under the condition of microtubule formation. From these results we propose an idea that the phosphorylation could occur concomitantly with microtubule formation in the brain. Human tau phosphorylated by the kinase carried an epitope of the paired helical filaments that accumulate in the brain in Alzheimer's disease.

  18. A group 6 late embryogenesis abundant protein from common bean is a disordered protein with extended helical structure and oligomer-forming properties.

    PubMed

    Rivera-Najera, Lucero Y; Saab-Rincón, Gloria; Battaglia, Marina; Amero, Carlos; Pulido, Nancy O; García-Hernández, Enrique; Solórzano, Rosa M; Reyes, José L; Covarrubias, Alejandra A

    2014-11-14

    Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association.

  19. A Group 6 Late Embryogenesis Abundant Protein from Common Bean Is a Disordered Protein with Extended Helical Structure and Oligomer-forming Properties*

    PubMed Central

    Rivera-Najera, Lucero Y.; Saab-Rincón, Gloria; Battaglia, Marina; Amero, Carlos; Pulido, Nancy O.; García-Hernández, Enrique; Solórzano, Rosa M.; Reyes, José L.; Covarrubias, Alejandra A.

    2014-01-01

    Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association. PMID:25271167

  20. Folding forms of Escherichia coli DmsD, a twin-arginine leader binding protein.

    PubMed

    Sarfo, Kwabena J; Winstone, Tara L; Papish, Andriyka L; Howell, Jenika M; Kadir, Hakan; Vogel, Hans J; Turner, Raymond J

    2004-03-05

    Escherichia coli DmsD interacts with the twin-arginine leader sequence of the catalytic sub-unit (DmsA) of DMSO reductase. DmsD was purified as a mixture of a number of different folding forms including: dimer (A); monomer (B); a minor thiol oxidized form; a heterogeneously folded or multi-conformational monomer form which displayed a ladder of bands on native-PAGE (D); and proteolytically degraded and aggregated forms. Polyacrylamide gel electrophoresis (PAGE), under denaturing and non-denaturing conditions, was used to examine the folding and stability of DmsD. Additionally, the biophysical methods of dynamic light scattering, circular dichroism, fluorescence, and mass spectroscopy were also used. Form D could be converted to form B by treatment with 4M urea, which is the concentration at which form B begins to denature. Forms A/B could be converted to D by incubation at pH 5.0. Forms A/B and D all had twin-arginine leader binding activity.

  1. The Tobacco etch virus P3 protein forms mobile inclusions via the early secretory pathway and traffics along actin microfilaments.

    PubMed

    Cui, Xiaoyan; Wei, Taiyun; Chowda-Reddy, R V; Sun, Guangyu; Wang, Aiming

    2010-02-05

    Plant potyviruses encode two membrane proteins, 6K and P3. The 6K protein has been shown to induce virus replication vesicles. However, the function of P3 remains unclear. In this study, subcellular localization of the Tobacco etch virus (TEV) P3 protein was investigated in Nicotiana benthamiana leaf cells. The TEV P3 protein localized on the endoplasmic reticulum (ER) membrane and formed punctate inclusions in association with the Golgi apparatus. The trafficking of P3 to the Golgi was mediated by the early secretory pathway. The Golgi-associated punctate structures originated from the ER exit site (ERES). Deletion analyses identified P3 domains required for the retention of P3 at the Golgi. Moreover, the P3 punctate structure was found to traffic along the actin filaments and colocalize with the 6K-containing replication vesicles. Taken together, these data support previous suggestions that P3 may play dual roles in virus movement and replication.

  2. Protein turnover forms one of the highest maintenance costs in Lactococcus lactis.

    PubMed

    Lahtvee, Petri-Jaan; Seiman, Andrus; Arike, Liisa; Adamberg, Kaarel; Vilu, Raivo

    2014-07-01

    Protein turnover plays an important role in cell metabolism by regulating metabolic fluxes. Furthermore, the energy costs for protein turnover have been estimated to account for up to a third of the total energy production during cell replication and hence may represent a major limiting factor in achieving either higher biomass or production yields. This work aimed to measure the specific growth rate (μ)-dependent abundance and turnover rate of individual proteins, estimate the ATP cost for protein production and turnover, and compare this with the total energy balance and other maintenance costs. The lactic acid bacteria model organism Lactococcus lactis was used to measure protein turnover rates at μ = 0.1 and 0.5 h(-1) in chemostat experiments. Individual turnover rates were measured for ~75% of the total proteome. On average, protein turnover increased by sevenfold with a fivefold increase in growth rate, whilst biomass yield increased by 35%. The median turnover rates found were higher than the specific growth rate of the bacterium, which suggests relatively high energy consumption for protein turnover. We found that protein turnover costs alone account for 38 and 47% of the total energy produced at μ = 0.1 and 0.5 h(-1), respectively, and gene ontology groups Energy metabolism and Translation dominated synthesis costs at both growth rates studied. These results reflect the complexity of metabolic changes that occur in response to changes in environmental conditions, and signify the trade-off between biomass yield and the need to produce ATP for maintenance processes.

  3. Characterisation of different forms of the accessory gp3 canine coronavirus type I protein identified in cats.

    PubMed

    d'Orengiani, Anne-Laure Pham-Hung d'Alexandry; Duarte, Lidia; Pavio, Nicole; Le Poder, Sophie

    2015-04-16

    ORF3 is a supplemental open reading frame coding for an accessory glycoprotein gp3 of unknown function, only present in genotype I canine strain (CCoV-I) and some atypical feline FCoV strains. In these latter hosts, the ORF3 gene systematically displays one or two identical deletions leading to the synthesis of truncated proteins gp3-Δ1 and gp3-Δ2. As deletions in CoV accessory proteins have already been involved in tissue or host switch, studies of these different gp3 proteins were conducted in canine and feline cell. All proteins oligomerise through covalent bonds, are N-glycosylated and are maintained in the ER in non-infected but also in CCoV-II infected cells, without any specific retention signal. However, deletions influence their level of expression. In canine cells, all proteins are expressed with similar level whereas in feline cells, the expression of gp3-Δ1 is higher than the two other forms of gp3. None of the gp3 proteins modulate the viral replication cycle of heterologous genotype II CCoV in canine cell line, leading to the conclusion that the gp3 proteins are probably advantageous only for CCoV-I and atypical FCoV strains.

  4. VAT-1 from Torpedo electric organ forms a high-molecular-mass protein complex within the synaptic vesicle membrane.

    PubMed

    Linial, M

    1993-08-15

    VAT-1 is an abundant 41-kDa protein from Torpedo cholinergic synaptic vesicles. Most of VAT-1 immunoreactivity (70%) is localized to the synaptic vesicle membrane while the rest (30%) copurifies with larger membranous fragments. VAT-1 forms a high-molecular-mass complex within the synaptic vesicle membrane. The Stokes radius of the VAT-1 complex is 4.85 nm and the sedimentation coefficient is 8.0 x 10(-13) S. Using these values, the calculated apparent mass of the VAT-1 complex is 176 kDa and the friction coefficient is consistent with that for a globular protein. Electrophoresis of solubilized synaptic vesicle proteins following cross-linking resulted in a 40-kDa ladder which was detected by VAT-1 antibodies. This is in accord with VAT-1 protein complex being composed primarily of VAT-1 subunits. The hydrodynamic characteristics of the VAT-1 protein complex suggest that it is composed of three or four VAT-1 subunits. Synaptophysin, an abundant component of Torpedo synaptic vesicle membranes, which has a similar apparent size as VAT-1, is not part of the VAT-1 protein complex. Interactions between the subunits within the protein complex do not depend on disulfide bonds or on lowering the ionic strength. However, partial dissociation of VAT-1 subunits from the complex occurs by chelating calcium ions.

  5. A protein fold classifier formed by fusing different modes of pseudo amino acid composition via PSSM.

    PubMed

    Kavousi, Kaveh; Moshiri, Behzad; Sadeghi, Mehdi; Araabi, Babak N; Moosavi-Movahedi, Ali Akbar

    2011-02-01

    Protein function is related to its chemical reaction to the surrounding environment including other proteins. On the other hand, this depends on the spatial shape and tertiary structure of protein and folding of its constituent components in space. The correct identification of protein domain fold solely using extracted information from protein sequence is a complicated and controversial task in the current computational biology. In this article a combined classifier based on the information content of extracted features from the primary structure of protein has been introduced to face this challenging problem. In the first stage of our proposed two-tier architecture, there are several classifiers each of which is trained with a different sequence based feature vector. Apart from the application of the predicted secondary structure, hydrophobicity, van der Waals volume, polarity, polarizability, and different dimensions of pseudo-amino acid composition vectors in similar studies, the position specific scoring matrix (PSSM) has also been used to improve the correct classification rate (CCR) in this study. Using K-fold cross validation on training dataset related to 27 famous folds of SCOP, the 28 dimensional probability output vector from each evidence theoretic K-NN classifier is used to determine the information content or expertness of corresponding feature for discrimination in each fold class. In the second stage, the outputs of classifiers for test dataset are fused using Sugeno fuzzy integral operator to make better decision for target fold class. The expertness factor of each classifier in each fold class has been used to calculate the fuzzy integral operator weights. Results make it possible to provide deeper interpretation about the effectiveness of each feature for discrimination in target classes for query proteins.

  6. Trafficking protein particle complex 6A delta (TRAPPC6AΔ) is an extracellular plaque-forming protein in the brain

    PubMed Central

    Chang, Jean-Yun; Lee, Ming-Hui; Lin, Sing-Ru; Yang, Li-Yi; Sun, H. Sunny; Sze, Chun-I; Hong, Qunying; Lin, Yee-Shin; Chou, Ying-Tsen; Hsu, Li-Jin; Jan, Ming-Shiou; Gong, Cheng-Xin; Chang, Nan-Shan

    2015-01-01

    Tumor suppressor WWOX is involved in the progression of cancer and neurodegeneration. Here, we examined whether protein aggregation occurs in the brain of nondemented, middle-aged humans and whether this is associated with WWOX downregulation. We isolated an N-terminal internal deletion isoform, TPC6AΔ, derived from alternative splicing of the TRAPPC6A (TPC6A) gene transcript. TPC6AΔ proteins are present as aggregates or plaques in the extracellular matrix of the brain such as in the cortex. Filter retardation assays revealed that aggregate formation of TPC6AΔ occurs preceding Aβ generation in the hippocampi of middle-aged postmortem normal humans. In a Wwox gene knockout mouse model, we showed the plaques of pT181-Tau and TPC6AΔ in the cortex and hippocampus in 3-week-old mice, suggesting a role of WWOX in limiting TPC6AΔ aggregation. To support this hypothesis, in vitro analysis revealed that TGF-β1 induces dissociation of the ectopic complex of TPC6AΔ and WWOX in cells, and then TPC6AΔ undergoes Ser35 phosphorylation-dependent polymerization and induces caspase 3 activation and Aβ production. Similarly, knockdown of WWOX by siRNA resulted in dramatic aggregation of TPC6AΔ. Together, when WWOX is downregulated, TPC6AΔ is phosphorylated at Ser35 and becomes aggregated for causing caspase activation that leads to Tau aggregation and Aβ formation. PMID:25650666

  7. Properties and Phylogeny of 76 Families of Bacterial and Eukaryotic Organellar Outer Membrane Pore-Forming Proteins

    PubMed Central

    Reddy, Bhaskara L.; Saier, Milton H.

    2016-01-01

    We here report statistical analyses of 76 families of integral outer membrane pore-forming proteins (OMPPs) found in bacteria and eukaryotic organelles. 47 of these families fall into one superfamily (SFI) which segregate into fifteen phylogenetic clusters. Families with members of the same protein size, topology and substrate specificities often cluster together. Virtually all OMPP families include only proteins that form transmembrane pores. Nine such families, all of which cluster together in the SFI phylogenetic tree, contain both α- and β-structures, are multi domain, multi subunit systems, and transport macromolecules. Most other SFI OMPPs transport small molecules. SFII and SFV homologues derive from Actinobacteria while SFIII and SFIV proteins derive from chloroplasts. Three families of actinobacterial OMPPs and two families of eukaryotic OMPPs apparently consist primarily of α-helices (α-TMSs). Of the 71 families of (putative) β-barrel OMPPs, only twenty could not be assigned to a superfamily, and these derived primarily from Actinobacteria (1), chloroplasts (1), spirochaetes (8), and proteobacteria (10). Proteins were identified in which two or three full length OMPPs are fused together. Family characteristic are described and evidence agrees with a previous proposal suggesting that many arose by adjacent β-hairpin structural unit duplications. PMID:27064789

  8. The GraS Sensor in Staphylococcus aureus Mediates Resistance to Host Defense Peptides Differing in Mechanisms of Action

    PubMed Central

    Chaili, Siyang; Cheung, Ambrose L.; Bayer, Arnold S.; Xiong, Yan Q.; Waring, Alan J.; Memmi, Guido; Donegan, Niles; Yang, Soo-Jin

    2015-01-01

    Staphylococcus aureus uses the two-component regulatory system GraRS to sense and respond to host defense peptides (HDPs). However, the mechanistic impact of GraS or its extracellular sensing loop (EL) on HDP resistance is essentially unexplored. Strains with null mutations in the GraS holoprotein (ΔgraS) or its EL (ΔEL) were compared for mechanisms of resistance to HDPs of relevant immune sources: neutrophil α-defensin (human neutrophil peptide 1 [hNP-1]), cutaneous β-defensin (human β-defensin 2 [hBD-2]), or the platelet kinocidin congener RP-1. Actions studied by flow cytometry included energetics (ENR); membrane permeabilization (PRM); annexin V binding (ANX), and cell death protease activation (CDP). Assay conditions simulated bloodstream (pH 7.5) or phagolysosomal (pH 5.5) pH contexts. S. aureus strains were more susceptible to HDPs at pH 7.5 than at pH 5.5, and each HDP exerted a distinct effect signature. The impacts of ΔgraS and ΔΕL on HDP resistance were peptide and pH dependent. Both mutants exhibited defects in ANX response to hNP-1 or hBD-2 at pH 7.5, but only hNP-1 did so at pH 5.5. Both mutants exhibited hyper-PRM, -ANX, and -CDP responses to RP-1 at both pHs and hypo-ENR at pH 5.5. The actions correlated with ΔgraS or ΔΕL hypersusceptibility to hNP-1 or RP-1 (but not hBD-2) at pH 7.5 and to all study HDPs at pH 5.5. An exogenous EL mimic protected mutant strains from hNP-1 and hBD-2 but not RP-1, indicating that GraS and its EL play nonredundant roles in S. aureus survival responses to specific HDPs. These findings suggest that GraS mediates specific resistance countermeasures to HDPs in immune contexts that are highly relevant to S. aureus pathogenesis in humans. PMID:26597988

  9. Comparison of the effect of homocysteine in the reduced form, its thiolactone and protein homocysteinylation on hemostatic properties of plasma.

    PubMed

    Malinowska, Joanna; Nowak, Paweł; Olas, Beata

    2011-03-01

    Mechanisms involved in the relationship between hyperhomocysteinemia and hemostatic process are still unclear. In the literature there are few papers describing studies on the effects of homocysteine (Hcys) on proteins that participate in blood coagulation and fibrinolysis in human. The aim of our study was to establish and compare the influence of a reduced form of Hcys (at final doses of 0.01 - 1 mM) and the most reactive form of Hcys - its cyclic thioester, homocysteine thiolactone (HTL, 0.1 - 1 μM) on the clot formation (using whole human plasma and purified fibrinogen) and the fibrin lysis. Moreover, the aim of our study was to explain the effect of plasma protein modifications (S- and N-homocysteinylation) on selected parameters of hemostasis. We observed that HTL, like its precursor, a reduced form of Hcys stimulated polymerization of fibrinogen, but this process was not dose-dependent. In the presence of HTL (at the lowest tested concentration - 0.1μM) the increase was about 55%. Our present results also demonstrated that Hcys in the reduced form (0.01 - 1 mM) and HTL at lower doses than Hcys (0.1 - 1 μM) reduced the fibrin lysis in whole human plasma. Our results reported that HTL, like the reduced form of Hcys (at concentrations corresponding to concentrations in plasma during hyperhomocysteinemia) induced modifications of hemostatic plasma proteins, and the consequence of these modifications may be alteration in protein structure associated with changes of hemostatic functions.

  10. The Amyloid Precursor Protein Forms Plasmalemmal Clusters via Its Pathogenic Amyloid-β Domain

    PubMed Central

    Schreiber, Arne; Fischer, Sebastian; Lang, Thorsten

    2012-01-01

    The amyloid precursor protein (APP) is a large, ubiquitous integral membrane protein with a small amyloid-β (Aβ) domain. In the human brain, endosomal processing of APP produces neurotoxic Aβ-peptides, which are involved in Alzheimer's disease. Here, we show that the Aβ sequence exerts a physiological function when still present in the unprocessed APP molecule. From the extracellular site, Aβ concentrates APP molecules into plasmalemmal membrane protein clusters. Moreover, Aβ stabilization of clusters is a prerequisite for their targeting to endocytic clathrin structures. Therefore, we conclude that the Aβ domain directly mediates a central step in APP trafficking, driving its own conversion into neurotoxic peptides. PMID:22455924

  11. Entropic potential field formed for a linear-motor protein near a filament: Statistical-mechanical analyses using simple models.

    PubMed

    Amano, Ken-Ichi; Yoshidome, Takashi; Iwaki, Mitsuhiro; Suzuki, Makoto; Kinoshita, Masahiro

    2010-07-28

    We report a new progress in elucidating the mechanism of the unidirectional movement of a linear-motor protein (e.g., myosin) along a filament (e.g., F-actin). The basic concept emphasized here is that a potential field is entropically formed for the protein on the filament immersed in solvent due to the effect of the translational displacement of solvent molecules. The entropic potential field is strongly dependent on geometric features of the protein and the filament, their overall shapes as well as details of the polyatomic structures. The features and the corresponding field are judiciously adjusted by the binding of adenosine triphosphate (ATP) to the protein, hydrolysis of ATP into adenosine diphosphate (ADP)+Pi, and release of Pi and ADP. As the first step, we propose the following physical picture: The potential field formed along the filament for the protein without the binding of ATP or ADP+Pi to it is largely different from that for the protein with the binding, and the directed movement is realized by repeated switches from one of the fields to the other. To illustrate the picture, we analyze the spatial distribution of the entropic potential between a large solute and a large body using the three-dimensional integral equation theory. The solute is modeled as a large hard sphere. Two model filaments are considered as the body: model 1 is a set of one-dimensionally connected large hard spheres and model 2 is a double helical structure formed by two sets of connected large hard spheres. The solute and the filament are immersed in small hard spheres forming the solvent. The major findings are as follows. The solute is strongly confined within a narrow space in contact with the filament. Within the space there are locations with sharply deep local potential minima along the filament, and the distance between two adjacent locations is equal to the diameter of the large spheres constituting the filament. The potential minima form a ringlike domain in model 1

  12. Bovine serum albumin oligomers in the E- and B-forms at low protein concentration and ionic strength

    PubMed Central

    Babcock, Jeremiah J.; Brancaleon, Lorenzo

    2013-01-01

    The manuscript describes the study of the oligomerization process of bovine serum albumin (BSA) in two different structural monomeric forms: the extended-form (E) at pH 2.0 and the basic-form (B) at pH 9.0. The study was conducted at low protein concentration (1 mg/ml) and relatively short incubation time (maximum 56 days) in order to investigate early oligomerization events rather than the formation of mature fibrils. The comparison between the two isoforms show that oligomers form much faster (∼6 days) in the E-form than in the B-form where formation of oligomers requires ∼4 weeks. The oligomers appear to be limited to a maximum of tetramers with size <30nm. Hydrophobic interactions from exposed neutral amino acid residues in the elongated E-form are the likely cause for the quick formation of aggregates at acidic pH. We used an array of biophysical techniques for the study and determined that oligomerization occurs without further large changes in the secondary structure of the monomers. Under the conditions adopted in this study, aggregation does not seem to exceed the formation of tetramers, even though a very small amount of much larger aggregates seem to form. PMID:23148944

  13. Expression of a truncated form of ribosomal protein L3 confers resistance to pokeweed antiviral protein and the Fusarium mycotoxin deoxynivalenol.

    PubMed

    Di, Rong; Tumer, Nilgun E

    2005-08-01

    The contamination of important agricultural products such as wheat, barley, or maize with the trichothecene mycotoxin deoxynivalenol (DON) due to infection with Fusarium species is a worldwide problem. Trichothecenes inhibit protein synthesis by targeting ribosomal protein L3. Pokeweed antiviral protein (PAP), a ribosome-inactivating protein binds to L3 to depurinate the alpha-sarcin/loop of the large rRNA. Plants transformed with the wild-type PAP show lesions and express very low levels of PAP because PAP autoregulates its expression by destabilizing its own mRNA. We show here that transgenic tobacco plants expressing both the wild-type PAP and a truncated form of yeast L3 (L3delta) are phenotypically normal. PAP mRNA and protein levels are very high in these plants, indicating that L3delta suppresses the autoregulation of PAP mRNA expression. Ribosomes are not depurinated in the transgenic plants expressing PAP and L3delta, even though PAP is associated with ribosomes. The expression of the endogenous tobacco ribosomal protein L3 is up-regulated in these plants and they are resistant to the Fusarium mycotoxin DON. These results demonstrate that expression of an N-terminal fragment of yeast L3 leads to trans-dominant resistance to PAP and the trichothecene mycotoxin DON, providing evidence that both toxins target L3 by a common mechanism.

  14. A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal triglyceride transfer protein (MTTP).

    PubMed

    Pons, Véronique; Rolland, Corinne; Nauze, Michel; Danjoux, Marie; Gaibelet, Gérald; Durandy, Anne; Sassolas, Agnès; Lévy, Emile; Tercé, François; Collet, Xavier; Mas, Emmanuel

    2011-07-01

    Abetalipoproteinemia is a rare autosomal recessive disease characterized by low lipid levels and by the absence of apoB-containing lipoproteins. It is the consequence of microsomal triglyceride transfer protein (MTTP) deficiency. We report two patients with new MTTP mutations. We studied their functional consequences on the triglyceride transfer function using duodenal biopsies. We transfected MTTP mutants in HepG2 and HeLa cells to investigate their association with protein disulfide isomerase (PDI) and their localization at the endoplasmic reticulum. These children have a severe abetalipoproteinemia. Both of them had also a mild hypogammaglobulinemia. They are compound heterozygotes with c.619G>T and c.1237-28A>G mutations within the MTTP gene. mRNA analysis revealed abnormal splicing with deletion of exon 6 and 10, respectively. Deletion of exon 6 (Δ6-MTTP) introduced a frame shift in the reading frame and a premature stop codon at position 234. Despite the fact that Δ6-MTTP and Δ10-MTTP mutants were not capable of binding PDI, both MTTP mutant proteins normally localize at the endoplasmic reticulum. However, these two mutations induce a loss of MTTP triglyceride transfer activity. These two mutations lead to abnormal truncated MTTP proteins, incapable of binding PDI and responsible for the loss of function of MTTP, thereby explaining the severe abetalipoproteinemia phenotype of these children.

  15. Primate TRIM5 proteins form hexagonal nets on HIV-1 capsids

    PubMed Central

    Li, Yen-Li; Chandrasekaran, Viswanathan; Carter, Stephen D; Woodward, Cora L; Christensen, Devin E; Dryden, Kelly A; Pornillos, Owen; Yeager, Mark; Ganser-Pornillos, Barbie K; Jensen, Grant J; Sundquist, Wesley I

    2016-01-01

    TRIM5 proteins are restriction factors that block retroviral infections by binding viral capsids and preventing reverse transcription. Capsid recognition is mediated by C-terminal domains on TRIM5α (SPRY) or TRIMCyp (cyclophilin A), which interact weakly with capsids. Efficient capsid recognition also requires the conserved N-terminal tripartite motifs (TRIM), which mediate oligomerization and create avidity effects. To characterize how TRIM5 proteins recognize viral capsids, we developed methods for isolating native recombinant TRIM5 proteins and purifying stable HIV-1 capsids. Biochemical and EM analyses revealed that TRIM5 proteins assembled into hexagonal nets, both alone and on capsid surfaces. These nets comprised open hexameric rings, with the SPRY domains centered on the edges and the B-box and RING domains at the vertices. Thus, the principles of hexagonal TRIM5 assembly and capsid pattern recognition are conserved across primates, allowing TRIM5 assemblies to maintain the conformational plasticity necessary to recognize divergent and pleomorphic retroviral capsids. DOI: http://dx.doi.org/10.7554/eLife.16269.001 PMID:27253068

  16. The propensity of the bacterial rodlin protein RdlB to form amyloid fibrils determines its function in Streptomyces coelicolor

    PubMed Central

    Yang, Wen; Willemse, Joost; Sawyer, Elizabeth B.; Lou, Fei; Gong, Weibin; Zhang, Hong; Gras, Sally L.; Claessen, Dennis; Perrett, Sarah

    2017-01-01

    Streptomyces bacteria form reproductive aerial hyphae that are covered with a pattern of pairwise aligned fibrils called rodlets. The presence of the rodlet layer requires two homologous rodlin proteins, RdlA and RdlB, and the functional amyloid chaplin proteins, ChpA-H. In contrast to the redundancy shared among the eight chaplins, both RdlA and RdlB are indispensable for the establishment of this rodlet structure. By using a comprehensive biophysical approach combined with in vivo characterization we found that RdlB, but not RdlA, readily assembles into amyloid fibrils. The marked difference in amyloid propensity between these highly similar proteins could be largely attributed to a difference in amino acid sequence at just three sites. Further, an engineered RdlA protein in which these three key amino acids were replaced with the corresponding residues from RdlB could compensate for loss of RdlB and restore formation of the surface-exposed amyloid layer in bacteria. Our data reveal that RdlB is a new functional amyloid and provide a biophysical basis for the functional differences between the two rodlin proteins. This study enhances our understanding of how rodlin proteins contribute to formation of an outer fibrillar layer during spore morphogenesis in streptomycetes. PMID:28211492

  17. Quinone-reactive proteins devoid of haem b form widespread membrane-bound electron transport modules in bacterial respiration.

    PubMed

    Simon, Jörg; Kern, Melanie

    2008-10-01

    Many quinone-reactive enzyme complexes that are part of membrane-integral eukaryotic or prokaryotic respiratory electron transport chains contain one or more haem b molecules embedded in the membrane. In recent years, various novel proteins have emerged that are devoid of haem b but are thought to fulfil a similar function in bacterial anaerobic respiratory systems. These proteins are encoded by genes organized in various genomic arrangements and are thought to form widespread membrane-bound quinone-reactive electron transport modules that exchange electrons with redox partner proteins located at the outer side of the cytoplasmic membrane. Prototypic representatives are the multihaem c-type cytochromes NapC, NrfH and TorC (NapC/NrfH family), the putative iron-sulfur protein NapH and representatives of the NrfD/PsrC family. Members of these protein families vary in the number of their predicted transmembrane segments and, consequently, diverse quinone-binding sites are expected. Only a few of these enzymes have been isolated and characterized biochemically and high-resolution structures are limited. This mini-review briefly summarizes predicted and experimentally demonstrated properties of the proteins in question and discusses their role in electron transport and bioenergetics of anaerobic respiration.

  18. Channels Formed by Botulinum, Tetanus, and Diphtheria Toxins in Planar Lipid Bilayers: Relevance to Translocation of Proteins across Membranes

    NASA Astrophysics Data System (ADS)

    Hoch, David H.; Romero-Mira, Miryam; Ehrlich, Barbara E.; Finkelstein, Alan; Dasgupta, Bibhuti R.; Simpson, Lance L.

    1985-03-01

    The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltage-dependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as ``tunnel proteins'' for translocation of active peptide fragments. These findings support the hypothesis that the active fragments of botulinum neurotoxin and tetanus toxin, like that of diphtheria toxin, are translocated across the membranes of acidic vesicles.

  19. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication?

    PubMed Central

    Imhof, Simon; Fragoso, Cristina; Hemphill, Andrew; von Schubert, Conrad; Li, Dong; Legant, Wesley; Betzig, Eric; Roditi, Isabel

    2016-01-01

    Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute) was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature. PMID:27239276

  20. The Outer Membrane Protein OmpW Forms an Eight-Stranded beta-Barrel with a Hydrophobic Channel

    SciTech Connect

    Hong,H.; Patel, D.; Tamm, L.; van den Berg, B.

    2006-01-01

    Escherichia coli OmpW belongs to a family of small outer membrane (OM) proteins that are widespread in Gram-negative bacteria. Their functions are unknown, but recent data suggest that they may be involved in the protection of bacteria against various forms of environmental stress. In order to gain insight into the function of these proteins we have determined the crystal structure of Escherichia coli OmpW to 2.7 Angstroms resolution. The structure shows that OmpW forms an eight-stranded beta-barrel with a long and narrow hydrophobic channel that contains a bound LDAO detergent molecule. Single channel conductance experiments show that OmpW functions as an ion channel in planar lipid bilayers. The channel activity can be blocked by the addition of LDAO. Taken together, the data suggest that members of the OmpW family could be involved in the transport of small hydrophobic molecules across the bacterial OM.

  1. Host-derived, pore-forming toxin-like protein and trefoil factor complex protects the host against microbial infection.

    PubMed

    Xiang, Yang; Yan, Chao; Guo, Xiaolong; Zhou, Kaifeng; Li, Sheng'an; Gao, Qian; Wang, Xuan; Zhao, Feng; Liu, Jie; Lee, Wen-Hui; Zhang, Yun

    2014-05-06

    Aerolysins are virulence factors belonging to the bacterial β-pore-forming toxin superfamily. Surprisingly, numerous aerolysin-like proteins exist in vertebrates, but their biological functions are unknown. βγ-CAT, a complex of an aerolysin-like protein subunit (two βγ-crystallin domains followed by an aerolysin pore-forming domain) and two trefoil factor subunits, has been identified in frogs (Bombina maxima) skin secretions. Here, we report the rich expression of this protein, in the frog blood and immune-related tissues, and the induction of its presence in peritoneal lavage by bacterial challenge. This phenomena raises the possibility of its involvement in antimicrobial infection. When βγ-CAT was administrated in a peritoneal infection model, it greatly accelerated bacterial clearance and increased the survival rate of both frogs and mice. Meanwhile, accelerated Interleukin-1β release and enhanced local leukocyte recruitments were determined, which may partially explain the robust and effective antimicrobial responses observed. The release of interleukin-1β was potently triggered by βγ-CAT from the frog peritoneal cells and murine macrophages in vitro. βγ-CAT was rapidly endocytosed and translocated to lysosomes, where it formed high molecular mass SDS-stable oligomers (>170 kDa). Lysosomal destabilization and cathepsin B release were detected, which may explain the activation of caspase-1 inflammasome and subsequent interleukin-1β maturation and release. To our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter microbial infection by eliciting rapid and effective host innate immune responses. The findings will also largely help to elucidate the possible involvement and action mechanisms of aerolysin-like proteins and/or trefoil factors widely existing in vertebrates in the host defense against pathogens.

  2. Host-derived, pore-forming toxin–like protein and trefoil factor complex protects the host against microbial infection

    PubMed Central

    Xiang, Yang; Yan, Chao; Guo, Xiaolong; Zhou, Kaifeng; Li, Sheng’an; Gao, Qian; Wang, Xuan; Zhao, Feng; Liu, Jie; Lee, Wen-Hui; Zhang, Yun

    2014-01-01

    Aerolysins are virulence factors belonging to the bacterial β-pore–forming toxin superfamily. Surprisingly, numerous aerolysin-like proteins exist in vertebrates, but their biological functions are unknown. βγ-CAT, a complex of an aerolysin-like protein subunit (two βγ-crystallin domains followed by an aerolysin pore-forming domain) and two trefoil factor subunits, has been identified in frogs (Bombina maxima) skin secretions. Here, we report the rich expression of this protein, in the frog blood and immune-related tissues, and the induction of its presence in peritoneal lavage by bacterial challenge. This phenomena raises the possibility of its involvement in antimicrobial infection. When βγ-CAT was administrated in a peritoneal infection model, it greatly accelerated bacterial clearance and increased the survival rate of both frogs and mice. Meanwhile, accelerated Interleukin-1β release and enhanced local leukocyte recruitments were determined, which may partially explain the robust and effective antimicrobial responses observed. The release of interleukin-1β was potently triggered by βγ-CAT from the frog peritoneal cells and murine macrophages in vitro. βγ-CAT was rapidly endocytosed and translocated to lysosomes, where it formed high molecular mass SDS-stable oligomers (>170 kDa). Lysosomal destabilization and cathepsin B release were detected, which may explain the activation of caspase-1 inflammasome and subsequent interleukin-1β maturation and release. To our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter microbial infection by eliciting rapid and effective host innate immune responses. The findings will also largely help to elucidate the possible involvement and action mechanisms of aerolysin-like proteins and/or trefoil factors widely existing in vertebrates in the host defense against pathogens. PMID:24733922

  3. Identification of Novel Potentially Toxic Oligomers Formed in Vitro from Mammalian-derived Expanded huntingtin Exon-1 Protein*

    PubMed Central

    Nucifora, Leslie G.; Burke, Kathleen A.; Feng, Xia; Arbez, Nicolas; Zhu, Shanshan; Miller, Jason; Yang, Guocheng; Ratovitski, Tamara; Delannoy, Michael; Muchowski, Paul J.; Finkbeiner, Steven; Legleiter, Justin; Ross, Christopher A.; Poirier, Michelle A.

    2012-01-01

    Huntington disease is a genetic neurodegenerative disorder that arises from an expanded polyglutamine region in the N terminus of the HD gene product, huntingtin. Protein inclusions comprised of N-terminal fragments of mutant huntingtin are a characteristic feature of disease, though are likely to play a protective role rather than a causative one in neurodegeneration. Soluble oligomeric assemblies of huntingtin formed early in the aggregation process are candidate toxic species in HD. In the present study, we established an in vitro system to generate recombinant huntingtin in mammalian cells. Using both denaturing and native gel analysis, we have identified novel oligomeric forms of mammalian-derived expanded huntingtin exon-1 N-terminal fragment. These species are transient and were not previously detected using bacterially expressed exon-1 protein. Importantly, these species are recognized by 3B5H10, an antibody that recognizes a two-stranded hairpin conformation of expanded polyglutamine believed to be associated with a toxic form of huntingtin. Interestingly, comparable oligomeric species were not observed for expanded huntingtin shortstop, a 117-amino acid fragment of huntingtin shown previously in mammalian cell lines and transgenic mice, and here in primary cortical neurons, to be non-toxic. Further, we demonstrate that expanded huntingtin shortstop has a reduced ability to form amyloid-like fibrils characteristic of the aggregation pathway for toxic expanded polyglutamine proteins. Taken together, these data provide a possible candidate toxic species in HD. In addition, these studies demonstrate the fundamental differences in early aggregation events between mutant huntingtin exon-1 and shortstop proteins that may underlie the differences in toxicity. PMID:22433867

  4. Biochemical characteristics of cytosolic and particulate forms of protein tyrosine kinases from methyl nitrosourea (MNU)-induced rat mammary carcinoma

    SciTech Connect

    Srivastava, A.K.; Chiasson, J.C.; Chiasson, J.L.; Lacroix, A.; Windisch, L. )

    1991-03-11

    Protein tyrosine kinase (PTK) activities in MNU-induced rat mammary carcinoma has been investigated by using poly (glu: tyr; 4:1) as an exogenous substrate. The PTK activity of the mammary carcinoma was about equally distributed between the particulate and cytosolic fractions at 110 000 x g. Both particulate and cytosolic PTKs catalyzed the phosphorylation of several tyrosine containing synthetic substrates to various degrees, however, poly (glu: tyr; 4:1) was the best substrate. Both the forms utilized ATP as the phosphoryl group donor. Among various divalent cations tested, Co{sup 2+}, Mn{sup 2+} and Mg{sup 2+} were able to fulfill the divalent cation requirement. Poly-lysine exerted a stimulatory effect on the particulate, but not on the cytosolic form. On the other hand, though heparin and quercetin inhibited both the forms in a concentration dependent manner, the particulate form was more sensitive to inhibition. These data indicate that MNU-induced rat mammary carcinoma expresses both particulate and cytosolic forms of PTKs and that there are significant differences in the properties of the two forms. Differential differences in the properties of the two forms. Differential effects of some agents on mammary carcinoma PTKs suggest that these enzymes may be acutely regulated in vivo and could play important role in mammary carcinogenesis.

  5. [Isolation and purification of human blood plasma proteins able to form potassium channels in artificial bilayer lipid membrane].

    PubMed

    Venediktova, N I; Kuznetsov, K V; Gritsenko, E N; Gulidova, G P; Mironova, G D

    2012-01-01

    Protein fraction able to induce K(+)-selective transport across bilayer lipid membrane was isolated from human blood plasma with the use of the detergent and proteolytic enzyme-free method developed at our laboratory. After addition of the studied sample to the artificial membrane in the presence of 100 mM KCl, a discrete current change was observed. No channel activity was recorded in the presence of calcium and sodium ions. Channel forming activity of fraction was observed only in the presence of K+. Using a threefold gradient of KCl in the presence of studied proteins the potassium-selective potential balanced by voltage of -29 mV was registered. This value is very close to the theoretical Nernst potential in this case. This means that the examined ion channel is cation-selective. According to data obtained with MS-MALDI-TOF/TOF and database NCBI three protein components were identified in isolated researched sample.

  6. Identification of goose (Anser anser) and mule duck (Anasplatyrhynchos x Cairina moschata) foie gras by multiplex polymerase chain reaction amplification of the 5S RDNA gene.

    PubMed

    Rodríguez, M A; García, T; González, I; Asensio, L; Fernández, A; Lobo, E; Hernández, P E; Martín, R

    2001-06-01

    Polymerase chain reaction (PCR) amplification of the nuclear 5S rDNA gene has been used for the identification of goose and mule duck foie gras. Two species-specific reverse primers were designed and used in a multiplex reaction, together with a forward universal primer, to amplify specific fragments of the 5S rDNA in each species. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose and mule duck foie gras samples. This genetic marker can be useful for detecting fraudulent substitution of the duck liver for the more expensive goose liver.

  7. EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles

    SciTech Connect

    Fan, Yao; Tan, Kemin; Chhor, Gekleng; Butler, Emily K.; Jedrzejczak, Robert P.; Missiakas, Dominique; Joachimiak, Andrzej

    2015-07-03

    The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (~10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for two alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.

  8. Planes formed with four intron-positions in tertiary structures of retinol binding protein and calpain domain VI.

    PubMed

    Nosaka, Michiko; Hirata, Katsuki; Tsuji, Ryotarou; Sunaba, Syunya

    2014-01-07

    Eukaryotic genes have intervening sequences, introns, in their coding regions. Since introns are spliced out from m-RNA before translation, they are considered to have no effect on the protein structure. Here, we report a novel relationship between introns and the tertiary structures of retinol binding protein and calpain domain VI. We identified "intron-positions" as amino acid residues on which or just after which introns are found in their corresponding nucleotide sequences, and then found that four intron-positions form a plane. We also found that the four intron-positions of retinol-binding protein encloses its ligand retinol. The tertiary structure of calpain domain VI changes after Ca(2+) binding, and the four intron-positions form a plane that includes its ligand calpastatin. To evaluate the statistical significance of the planarity, we calculated the mean distance of each intron-position from the plane defined by the other three intron-positions, and showed that it is significantly smaller than the one calculated for randomly generated locations based on exon size distribution. On the basis of this finding, we discuss the evolution of retinol binding protein and the origin of introns.

  9. Study of the Protein Complex, Pore Diameter, and Pore-forming Activity of the Borrelia burgdorferi P13 Porin*

    PubMed Central

    Bárcena-Uribarri, Iván; Thein, Marcus; Barbot, Mariam; Sans-Serramitjana, Eulalia; Bonde, Mari; Mentele, Reinhard; Lottspeich, Friedrich; Bergström, Sven; Benz, Roland

    2014-01-01

    P13 is one of the major outer membrane proteins of Borrelia burgdorferi. Previous studies described P13 as a porin. In the present study some structure and function aspects of P13 were studied. P13 showed according to lipid bilayer studies a channel-forming activity of 0.6 nanosiemens in 1 m KCl. Single channel and selectivity measurements demonstrated that P13 had no preference for either cations or anions and showed no voltage-gating up to ±100 mV. Blue native polyacrylamide gel electrophoresis was used to isolate and characterize the P13 protein complex in its native state. The complex had a high molecular mass of about 300 kDa and was only composed of P13 monomers. The channel size was investigated using non-electrolytes revealing an apparent diameter of about 1.4 nm with a 400-Da molecular mass cut-off. Multichannel titrations with different substrates reinforced the idea that P13 forms a general diffusion channel. The identity of P13 within the complex was confirmed by second dimension SDS-PAGE, Western blotting, mass spectrometry, and the use of a p13 deletion mutant strain. The results suggested that P13 is the protein responsible for the 0.6-nanosiemens pore-forming activity in the outer membrane of B. burgdorferi. PMID:24825899

  10. The Reticulon and Dp1/Yop1p Proteins Form Immobile Oligomers in the Tubular Endoplasmic Reticulum*S⃞

    PubMed Central

    Shibata, Yoko; Voss, Christiane; Rist, Julia M.; Hu, Junjie; Rapoport, Tom A.; Prinz, William A.; Voeltz, Gia K.

    2008-01-01

    We recently identified a class of membrane proteins, the reticulons and DP1/Yop1p, which shape the tubular endoplasmic reticulum (ER) in yeast and mammalian cells. These proteins are highly enriched in the tubular portions of the ER and virtually excluded from other regions. To understand how they promote tubule formation, we characterized their behavior in cellular membranes and addressed how their localization in the ER is determined. Using fluorescence recovery after photobleaching, we found that yeast Rtn1p and Yop1p are less mobile in the membrane than normal ER proteins. Sucrose gradient centrifugation and cross-linking analyses show that they form oligomers. Mutants of yeast Rtn1p, which no longer localize exclusively to the tubular ER or are even totally inactive in inducing ER tubules, are more mobile and oligomerize less extensively. The mammalian reticulons and DP1 are also relatively immobile and can form oligomers. The conserved reticulon homology domain that includes the two membrane-embedded segments is sufficient for the localization of the reticulons to the tubular ER, as well as for their diffusional immobility and oligomerization. Finally, ATP depletion in both yeast and mammalian cells further decreases the mobilities of the reticulons and DP1. We propose that oligomerization of the reticulons and DP1/Yop1p is important for both their localization to the tubular domains of the ER and for their ability to form tubules. PMID:18442980

  11. Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense.

    PubMed

    Nishikawa, C Y; Araújo, L M; Kadowaki, M A S; Monteiro, R A; Steffens, M B R; Pedrosa, F O; Souza, E M; Chubatsu, L S

    2012-02-01

    Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ(54) co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH(4)Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

  12. The Role of a Novel Topological Form of the Prion Protein in Prion Disease

    DTIC Science & Technology

    2006-07-01

    CtmPrP [designated Tg(L9R-3AV)], and these mice develop a spontaneous neurological illness similar to scrapie [Stewart et al. 2005]. We are...that CtmPrP is not produced at appreciable levels in scrapie -infected animals. Task 2: Characterization of the CtmPrP-induced neurotoxic pathway in...Caughey, E. Masliah, and M.Oldstone (2005). Anchorless Prion Protein Results in Infectious Amyloid Disease Without Clinical Scrapie . Science 308:1435

  13. Secretion of sulfated and nonsulfated forms of parathyroid chromogranin A (secretory protein-I)

    SciTech Connect

    Gorr, S.U.; Cohn, D.V. )

    1990-02-25

    Chromogranin A (secretory protein-I) is an acidic, sulfated glycoprotein found in secretory granules of most endocrine cells but not in exocrine or epithelial cells. Parathyroid chromogranin A is sulfated on tyrosine residues, whereas adrenal chromogranin A appears to be sulfated mainly on oligosaccharide residues. Chromogranin B, on the other hand, is tyrosine-sulfated in the bovine adrenal whereas this protein is absent from the parathyroid. The role of this tissue- or species-specific sulfation of chromogranin is not known. Tyrosine sulfation is a common post-translational modification of proteins in the exocytotic pathway and has been suggested to play a role in the sorting or intracellular transport of secretory proteins. To test this, porcine parathyroid tissue slices were metabolically labeled with 35SO4 and (3H)Lys, and the tissue and incubation medium analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoprecipitation with chromogranin A-specific antiserum or by radioimmunoassay for parathormone. Secretion of total and 3H-labeled chromogranin A was about 3- and 7-fold higher, respectively, at 0.5 mM than at 3.0 mM Ca2+, and secretion of 35SO4-labeled chromogranin A was 67-fold higher. This indicates that either sulfated chromogranin A is directed primarily to the Ca2+-regulated pathway or that sulfation occurs following sorting to this pathway. Sodium chlorate (1-10 mM) inhibited sulfation in a dose-dependent manner by up to 95% but it had no effect on the onset or rate of chromogranin A secretion. These data indicate that regulated secretion of parathyroid chromogranin A does not require sulfation of tyrosine residues.

  14. The origins of life -- the 'protein interaction world' hypothesis: protein interactions were the first form of self-reproducing life and nucleic acids evolved later as memory molecules.

    PubMed

    Andras, Peter; Andras, Csaba

    2005-01-01

    The 'protein interaction world' (PIW) hypothesis of the origins of life assumes that life emerged as a self-reproducing and expanding system of protein interactions. In mainstream molecular biology, 'replication' refers to the material copying of molecules such as nucleic acids. However, PIW is conceptualized as an abstract communication system constituted by the interactions between proteins, in which 'replication' happens at the level of self-reproduction of these interactions between proteins. Densely concentrated peptide interaction systems may have reproduced and expanded as 'protocell' vesicles surrounded by lipid bi-layer membranes. Protocells led to the emergence of proto-RNA molecules of greater chemical stability which served as chemically differentiated 'memories' of peptide interaction states, thereby facilitating the reproduction and expansion of protocells. Simplification-driven expansion led to the selection of biotic amino acids and the reduction of the typical RNA alphabet to the four usual bases (A, C, G and U). Dense interactions between RNA molecules led to the emergence of the RNA interaction subsystem of the cell, and to the emergence of 'memories' of RNA interactions in the form of DNA molecules with greater chemical stability. The expansion of DNA molecule interactions led to the dense clustering and encapsulation of DNA molecules within the cell nucleus. RNA molecules therefore serve as memories of protein interactions and DNA molecules are memories of RNA interactions. We believe that the PIW hypothesis is more evolutionarily plausible than the mainstream RNA world hypothesis, and has greater explanatory power.

  15. The evolution of calcite-bearing kimberlites by melt-rock reaction: evidence from polymineralic inclusions within clinopyroxene and garnet megacrysts from Lac de Gras kimberlites, Canada

    NASA Astrophysics Data System (ADS)

    Bussweiler, Y.; Stone, R. S.; Pearson, D. G.; Luth, R. W.; Stachel, T.; Kjarsgaard, B. A.; Menzies, A.

    2016-07-01

    Megacrystic (>1 cm) clinopyroxene (Cr-diopside) and garnet (Cr-pyrope) xenocrysts within kimberlites from Lac de Gras (Northwest Territories, Canada) contain fully crystallized melt inclusions. These `polymineralic inclusions' have previously been interpreted to form by necking down of melts at mantle depths. We present a detailed petrographical and geochemical investigation of polymineralic inclusions and their host crystals to better understand how they form and what they reveal about the evolution of kimberlite melt. Genetically, the megacrysts are mantle xenocrysts with peridotitic chemical signatures indicating an origin within the lithospheric mantle (for the Cr-diopsides studied here ~4.6 GPa, 1015 °C). Textural evidence for disequilibrium between the host crystals and their polymineralic inclusions (spongy rims in Cr-diopside, kelyphite in Cr-pyrope) is consistent with measured Sr isotopic disequilibrium. The preservation of disequilibrium establishes a temporal link to kimberlite eruption. In Cr-diopsides, polymineralic inclusions contain phlogopite, olivine, chromite, serpentine, and calcite. Abundant fluid inclusion trails surround the inclusions. In Cr-pyropes, the inclusions additionally contain Al-spinel, clinopyroxene, and dolomite. The major and trace element compositions of the inclusion phases are generally consistent with the early stages of kimberlite differentiation trends. Extensive chemical exchange between the host phases and the inclusions is indicated by enrichment of the inclusions in major components of the host crystals, such as Cr2O3 and Al2O3. This chemical evidence, along with phase equilibria constraints, supports the proposal that the inclusions within Cr-diopside record the decarbonation reaction: dolomitic melt + diopside → forsterite + calcite + CO2, yielding the observed inclusion mineralogy and producing associated (CO2-rich) fluid inclusions. Our study of polymineralic inclusions in megacrysts provides clear mineralogical

  16. A Long-Distance Translocatable Phloem Protein from Cucumber Forms a Ribonucleoprotein Complex In Vivo with Hop Stunt Viroid RNA†

    PubMed Central

    Gómez, Gustavo; Pallás, Vicente

    2004-01-01

    Viroids are highly structured plant pathogenic RNAs that do not code for any protein, and thus, their long-distance movement within the plant must be mediated by direct interaction with cellular factors, the nature of which is presently unknown. In addition to this type of RNAs, recent evidence indicates that endogenous RNAs move through the phloem acting as macromolecular signals involved in plant defense and development. The form in which these RNA molecules are transported to distal parts of the plant is unclear. Viroids can be a good model system to try to identify translocatable proteins that could assist the vascular movement of RNA molecules. Here, we demonstrate by use of immunoprecipitation experiments, that the phloem protein 2 from cucumber (CsPP2) is able to interact in vivo with a viroid RNA. Intergeneric graft assays revealed that both the CsPP2 and the Hop stunt viroid RNA were translocated to the scion. The translocated viroid is symptomatic in the nonhost scion, indicating that the translocated RNA is functional. The CsPP2 gene was cloned and sequenced. The analysis of its primary structure revealed the existence of a potential double-spaced-RNA-binding motif, previously identified in a set of proteins that bind to highly structured RNAs, which could explain its RNA-binding properties. The possible involvement of this phloem protein in assisting the long-distance movement of the viroid RNA within the plant is discussed. PMID:15331743

  17. Adenovirus DNA-binding protein forms a multimeric protein complex with double-stranded DNA and enhances binding of nuclear factor I.

    PubMed Central

    Stuiver, M H; van der Vliet, P C

    1990-01-01

    The 72-kilodalton adenovirus DNA-binding protein (DBP) binds to single-stranded DNA as well as to RNA and double-stranded DNA and is essential for the replication of viral DNA. We investigated the binding of DBP to double-stranded DNA by gel retardation analysis. By using a 114-base-pair DNA fragment, five or six different complexes were observed by gel retardation. The mobility of these complexes is dependent on the DBP concentration, suggesting that the complexes arise by sequential binding of DBP molecules to the DNA. In contrast to binding to single-stranded DNA, the binding of DBP to double-stranded DNA appears to be noncooperative. DBP binds to linear DNA as well as to circular DNA, while linear DNA containing the adenovirus terminal protein was also recognized. No specificity for adenovirus origin sequences was observed. To study whether the binding of DBP could influence initiation of DNA replication, we analyzed the effect of DBP on the binding of nuclear factor I (NFI) and NFIII, two sequence-specific origin-recognizing proteins that enhance initiation. At subsaturating levels of NFI, DBP increases the rate of binding of NFI considerably, while no effect was seen on NFIII. This stimulation of NFI binding is specific for DBP and was not observed with another protein (NFIV), which forms a similar DNA-multimeric protein complex. In agreement with enhanced NFI binding, DBP stimulates initiation of adenovirus DNA replication in vitro especially strongly at subsaturating NFI concentrations. We explain our results by assuming that DBP forms a complex with origin DNA that promotes formation of an alternative DNA structure, thereby facilitating the binding of NFI as well as the initiation of DNA replication via NFI. Images PMID:2293667

  18. Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate.

    PubMed

    Chen, Wen-Hsiang; Du, Lanying; Chag, Shivali M; Ma, Cuiqing; Tricoche, Nancy; Tao, Xinrong; Seid, Christopher A; Hudspeth, Elissa M; Lustigman, Sara; Tseng, Chien-Te K; Bottazzi, Maria Elena; Hotez, Peter J; Zhan, Bin; Jiang, Shibo

    2014-01-01

    Development of vaccines for preventing a future pandemic of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and for biodefense preparedness is urgently needed. Our previous studies have shown that a candidate SARS vaccine antigen consisting of the receptor-binding domain (RBD) of SARS-CoV spike protein can induce potent neutralizing antibody responses and protection against SARS-CoV challenge in vaccinated animals. To optimize expression conditions for scale-up production of the RBD vaccine candidate, we hypothesized that this could be potentially achieved by removing glycosylation sites in the RBD protein. In this study, we constructed two RBD protein variants: 1) RBD193-WT (193-aa, residues 318-510) and its deglycosylated forms (RBD193-N1, RBD193-N2, RBD193-N3); 2) RBD219-WT (219-aa, residues 318-536) and its deglycosylated forms (RBD219-N1, RBD219-N2, and RBD219-N3). All constructs were expressed as recombinant proteins in yeast. The purified recombinant proteins of these constructs were compared for their antigenicity, functionality and immunogenicity in mice using alum as the adjuvant. We found that RBD219-N1 exhibited high expression yield, and maintained its antigenicity and functionality. More importantly, RBD219-N1 induced significantly stronger RBD-specific antibody responses and a higher level of neutralizing antibodies in immunized mice than RBD193-WT, RBD193-N1, RBD193-N3, or RBD219-WT. These results suggest that RBD219-N1 could be selected as an optimal SARS vaccine candidate for further development.

  19. The Role of a Novel Topological Form of the Prion Protein in Prion Disease

    DTIC Science & Technology

    2005-07-01

    experimental scrapie infection, which suggests that CtmPrP may be the ultimate toxic trigger. We have identified mutations in the prion protein sequence...which express CtmPrP [designated Tg(L9R-3AV)], and these mice develop a spontaneous neurological illness similar to scrapie . We are characterizing the...for the presence of CtmPrP in terminally ill scrapie -infected mice, and did not detect any CtmPrP [Stewart and Harris 2003]. We have proposed

  20. HorC, a hop-resistance related protein, presumably functions in homodimer form.

    PubMed

    Iijima, Kazumaru; Suzuki, Koji; Asano, Shizuka; Ogata, Tomoo; Kitagawa, Yasushi

    2009-08-01

    To determine whether two HorC molecules coordinately form a single unit, the functional properties of covalently linked dimers of HorC encoded by tandemly fused horC genes were studied. Lactobacillus brevis introduced with the fused horC genes and a single horC gene exhibited same degree of resistance to hop compounds and cetyltrimethylammonium bromide. This suggests that HorC functions as a homodimer.

  1. The membrane protein Pannexin1 forms two open channel conformations depending on the mode of activation

    PubMed Central

    Wang, Junjie; Ambrosi, Cinzia; Qiu, Feng; Jackson, David G.; Sosinsky, Gina; Dahl, Gerhard

    2014-01-01

    Pannexin1 (Panx1) participates in several signaling events that involve ATP release, including the innate immune response, ciliary beat in airway epithelia and oxygen supply in the vasculature. The view that Panx1 forms a large ATP-release channel has been challenged by the association of a low conductance, small anion-selective channel with the presence of Panx1. We showed that Panx1 membrane channels can function in two distinct modes with different conductances and permeabilities when heterologously expressed in Xenopus oocytes. When stimulated by potassium ions (K+), Panx1 formed a high conductance channel of ~500 pS that was permeable to ATP. Various physiological stimuli can induce this ATP-permeable conformation of the channel in several cell types. In contrast, the channel had a low conductance (~50 pS) with no detectable ATP permeability when activated by voltage in the absence of K+. The two channel states were associated with different reactivities of the terminal cysteine of Panx1 to thiol reagents, suggesting different conformations. Single particle electron microscopic analysis revealed that K+ stimulated the formation of channels with a larger pore diameter than those formed in the absence of K+. These data suggest that different stimuli lead to distinct channel structures with distinct biophysical properties. PMID:25056878

  2. Deposition of bacteriorhodopsin protein in a purple membrane form on nitrocellulose membranes for enhanced photoelectric response.

    PubMed

    Kim, Young Jun; Neuzil, Pavel; Nam, Chang-Hoon; Engelhard, Martin

    2012-12-27

    Bacteriorhodopsin protein (bR)-based systems are one of the simplest known biological energy converters. The robust chemical, thermal and electrochemical properties of bR have made it an attractive material for photoelectric devices. This study demonstrates the photoelectric response of a dry bR layer deposited on a nitrocellulose membrane with indium tin oxide (ITO) electrodes. Light-induced electrical current as well as potential and impedance changes of dried bR film were recorded as the function of illumination. We have also tested bR in solution and found that the electrical properties are strongly dependent on light intensity changing locally proton concentration and thus pH of the solution. Experimental data support the assumption that bR protein on a positively charged nitrocellulose membrane (PNM) can be used as highly sensitive photo- and pH detector. Here the bR layer facilitates proton translocation and acts as an ultrafast optoelectric signal transducer. It is therefore useful in applications related to bioelectronics, biosensors, bio-optics devices and current carrying junction devices.

  3. Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

    PubMed Central

    Gilbert, Leona; Toivola, Jouni; Välilehto, Outi; Saloniemi, Taija; Cunningham, Claire; White, Daniel; Mäkelä, Anna R; Korhonen, Eila; Vuento, Matti; Oker-Blom, Christian

    2006-01-01

    Fluorescence correlation spectroscopy (FCS) monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids) were fused to the C-terminus of the enhanced green fluorescent protein (EGFP). The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs). The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles. PMID:17156442

  4. Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles.

    PubMed

    Gilbert, Leona; Toivola, Jouni; Välilehto, Outi; Saloniemi, Taija; Cunningham, Claire; White, Daniel; Mäkelä, Anna R; Korhonen, Eila; Vuento, Matti; Oker-Blom, Christian

    2006-12-08

    Fluorescence correlation spectroscopy (FCS) monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids) were fused to the C-terminus of the enhanced green fluorescent protein (EGFP). The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs). The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.

  5. Structural and biochemical characterization of the essential DsbA-like disulfide bond forming protein from Mycobacterium tuberculosis

    PubMed Central

    2013-01-01

    Background Bacterial Disulfide bond forming (Dsb) proteins facilitate proper folding and disulfide bond formation of periplasmic and secreted proteins. Previously, we have shown that Mycobacterium tuberculosis Mt-DsbE and Mt-DsbF aid in vitro oxidative folding of proteins. The M. tuberculosis proteome contains another predicted membrane-tethered Dsb protein, Mt-DsbA, which is encoded by an essential gene. Results Herein, we present structural and biochemical analyses of Mt-DsbA. The X-ray crystal structure of Mt-DsbA reveals a two-domain structure, comprising a canonical thioredoxin domain with the conserved CXXC active site cysteines in their reduced form, and an inserted α-helical domain containing a structural disulfide bond. The overall fold of Mt-DsbA resembles that of other DsbA-like proteins and not Mt-DsbE or Mt-DsbF. Biochemical characterization demonstrates that, unlike Mt-DsbE and Mt-DsbF, Mt-DsbA is unable to oxidatively fold reduced, denatured hirudin. Moreover, on the substrates tested in this study, Mt-DsbA has disulfide bond isomerase activity contrary to Mt-DsbE and Mt-DsbF. Conclusion These results suggest that Mt-DsbA acts upon a distinct subset of substrates as compared to Mt-DsbE and Mt-DsbF. One could speculate that Mt-DsbE and Mt-DsbF are functionally redundant whereas Mt-DsbA is not, offering an explanation for the essentiality of Mt-DsbA in M. tuberculosis. PMID:24134223

  6. The copper transport-associated protein Ctr4 can form prion-like epigenetic determinants in Schizosaccharomyces pombe

    PubMed Central

    Sideri, Theodora; Yashiroda, Yoko; Ellis, David A.; Rodríguez-López, María; Yoshida, Minoru; Tuite, Mick F.; Bähler, Jürg

    2017-01-01

    Prions are protein-based infectious entities associated with fatal brain diseases in animals, but also modify a range of host-cell phenotypes in the budding yeast, Saccharomyces cerevisiae. Many questions remain about the evolution and biology of prions. Although several functionally distinct prion-forming proteins exist in S. cerevisiae, [HET-s] of Podospora anserina is the only other known fungal prion. Here we investigated prion-like, protein-based epigenetic transmission in the fission yeast Schizosaccharomyces pombe. We show that S. pombe cells can support the formation and maintenance of the prion form of the S. cerevisiae Sup35 translation factor [PSI+], and that the formation and propagation of these Sup35 aggregates is inhibited by guanidine hydrochloride, indicating commonalities in prion propagation machineries in these evolutionary diverged yeasts. A proteome-wide screen identified the Ctr4 copper transporter subunit as a putative prion with a predicted prion-like domain. Overexpression of the ctr4 gene resulted in large Ctr4 protein aggregates that were both detergent and proteinase-K resistant. Cells carrying such [CTR+] aggregates showed increased sensitivity to oxidative stress, and this phenotype could be transmitted to aggregate-free [ctr-] cells by transformation with [CTR+] cell extracts. Moreover, this [CTR+] phenotype was inherited in a non-Mendelian manner following mating with naïve [ctr-] cells, but intriguingly the [CTR+] phenotype was not eliminated by guanidine-hydrochloride treatment. Thus, Ctr4 exhibits multiple features diagnostic of other fungal prions and is the first example of a prion in fission yeast. These findings suggest that transmissible protein-based determinants of traits may be more widespread among fungi. PMID:28191457

  7. Truncated Form of the Epstein-Barr Virus Protein EBNA-LP Protects against Caspase-Dependent Apoptosis by Inhibiting Protein Phosphatase 2A▿

    PubMed Central

    Garibal, Julie; Hollville, Émilie; Bell, Andrew I.; Kelly, Gemma L.; Renouf, Benjamin; Kawaguchi, Yasushi; Rickinson, Alan B.; Wiels, Joëlle

    2007-01-01

    The Epstein-Barr virus (EBV)-encoded leader protein, EBNA-LP, strongly activates the EBNA2-mediated transcriptional activation of cellular and viral genes and is therefore important for EBV-induced B-cell transformation. However, a truncated form of EBNA-LP is produced in cells infected with variant EBV strains lacking EBNA2 due to a genetic deletion. The function of this truncated form is unknown. We show here that some Burkitt's lymphoma cells harboring defective EBV strains are specifically resistant to the caspase-dependent apoptosis induced by verotoxin 1 (VT-1) or staurosporine. These cells produced low-molecular-weight Y1Y2-truncated isoforms of EBNA-LP, which were partly localized in the cytoplasm. The transfection of sensitive cells with constructs encoding truncated EBNA-LP isoforms, but not full-length EBNA-LP, induced resistance to caspase-mediated apoptosis. Furthermore, VT-1 induced protein phosphatase 2A (PP2A) activation in sensitive cells but not in resistant cells, in which the truncated EBNA-LP interacted with this protein. Thus, the resistance to apoptosis observed in cells harboring defective EBV strains most probably results from the inactivation of PP2A via interactions with low-molecular-weight Y1Y2-truncated EBNA-LP isoforms. PMID:17494066

  8. Pore-forming proteins with built-in triggers and switches

    NASA Astrophysics Data System (ADS)

    Bayley, Hagan

    1996-02-01

    Genetic engineering and targeted chemical modification are being used to produce polypeptides with pore-forming activity that can be triggered or switched on-and-off by biochemical, chemical or physical stimuli. The principal target of our studies has been the (alpha) -hemolysin ((alpha) HL) from the bacterium Staphylococcus aureus. The remodeled hemolysins include protease-activated pores, metal-regulated pores, pores that are activated by chemical alkylation and pores that are turned on with light. These polypeptides have several potential applications. For example, they might serve as components of sensors or they might be useful for mediating the controlled release of encapsulated drugs.

  9. Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins.

    PubMed

    Malet, Hélène; Liu, Kaiyin; El Bakkouri, Majida; Chan, Sze Wah Samuel; Effantin, Gregory; Bacia, Maria; Houry, Walid A; Gutsche, Irina

    2014-08-05

    A 3.3 MDa macromolecular cage between two Escherichia coli proteins with seemingly incompatible symmetries-the hexameric AAA+ ATPase RavA and the decameric inducible lysine decarboxylase LdcI-is reconstructed by cryo-electron microscopy to 11 Å resolution. Combined with a 7.5 Å resolution reconstruction of the minimal complex between LdcI and the LdcI-binding domain of RavA, and the previously solved crystal structures of the individual components, this work enables to build a reliable pseudoatomic model of this unusual architecture and to identify conformational rearrangements and specific elements essential for complex formation. The design of the cage created via lateral interactions between five RavA rings is unique for the diverse AAA+ ATPase superfamily.

  10. Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB

    PubMed Central

    Coelho, Miguel B; Attig, Jan; Bellora, Nicolás; König, Julian; Hallegger, Martina; Kayikci, Melis; Eyras, Eduardo; Ule, Jernej; Smith, Christopher WJ

    2015-01-01

    Matrin3 is an RNA- and DNA-binding nuclear matrix protein found to be associated with neural and muscular degenerative diseases. A number of possible functions of Matrin3 have been suggested, but no widespread role in RNA metabolism has yet been clearly demonstrated. We identified Matrin3 by its interaction with the second RRM domain of the splicing regulator PTB. Using a combination of RNAi knockdown, transcriptome profiling and iCLIP, we find that Matrin3 is a regulator of hundreds of alternative splicing events, principally acting as a splicing repressor with only a small proportion of targeted events being co-regulated by PTB. In contrast to other splicing regulators, Matrin3 binds to an extended region within repressed exons and flanking introns with no sharply defined peaks. The identification of this clear molecular function of Matrin3 should help to clarify the molecular pathology of ALS and other diseases caused by mutations of Matrin3. PMID:25599992

  11. FRAXE-associated mental retardation protein (FMR2) is an RNA-binding protein with high affinity for G-quartet RNA forming structure

    PubMed Central

    Bensaid, Mounia; Melko, Mireille; Bechara, Elias G.; Davidovic, Laetitia; Berretta, Antonio; Catania, Maria Vincenza; Gecz, Jozef; Lalli, Enzo; Bardoni, Barbara

    2009-01-01

    FRAXE is a form of mild to moderate mental retardation due to the silencing of the FMR2 gene. The cellular function of FMR2 protein is presently unknown. By analogy with its homologue AF4, FMR2 was supposed to have a role in transcriptional regulation, but robust evidences supporting this hypothesis are lacking. We observed that FMR2 co-localizes with the splicing factor SC35 in nuclear speckles, the nuclear regions where splicing factors are concentrated, assembled and modified. Similarly to what was reported for splicing factors, blocking splicing or transcription leads to the accumulation of FMR2 in enlarged, rounded speckles. FMR2 is also localized in the nucleolus when splicing is blocked. We show here that FMR2 is able to specifically bind the G-quartet-forming RNA structure with high affinity. Remarkably, in vivo, in the presence of FMR2, the ESE action of the G-quartet situated in mRNA of an alternatively spliced exon of a minigene or of the putative target FMR1 appears reduced. Interestingly, FMR1 is silenced in the fragile X syndrome, another form of mental retardation. All together, our findings strongly suggest that FMR2 is an RNA-binding protein, which might be involved in alternative splicing regulation through an interaction with G-quartet RNA structure. PMID:19136466

  12. Crystallization and preliminary X-ray diffraction data for the aconitase form of human iron-regulatory protein 1

    SciTech Connect

    Dupuy, J.; Darnault, C.; Moulis, J. M.

    2005-05-01

    Two crystal forms of the aconitase version of recombinant human IRP1 are reported. Iron-regulatory proteins (IRPs) 1 and 2 are closely related molecules involved in animal iron metabolism. Both proteins can bind to specific mRNA regions called iron-responsive elements and thereby control the expression of proteins involved in the uptake, storage and utilization of iron. In iron-replete cells, IRP1, but not IRP2, binds a [4Fe–4S] cluster and functions as a cytoplasmic aconitase, with simultaneous loss of its RNA-binding ability. Whereas IRP2 is known to be involved in Fe homeostasis, the role of IRP1 is less clear; it may provide a link between citrate and iron metabolisms and be involved in oxidative stress response. Here, two crystal forms of the aconitase version of recombinant human IRP1 are reported. An X-ray fluorescence measurement performed on a gold-derivative crystal showed the unexpected presence of zinc, in addition to gold and iron. Both native and MAD X-ray data at the Au, Fe and Zn absorption edges have been collected from these crystals.

  13. Arf1 and Arf6 Promote Ventral Actin Structures formed by acute Activation of Protein Kinase C and Src

    PubMed Central

    Caviston, Juliane P.; Cohen, Lee Ann; Donaldson, Julie G.

    2016-01-01

    Arf proteins regulate membrane traffic and organelle structure. Although Arf6 is known to initiate actin-based changes in cell surface architecture, Arf1 may also function at the plasma membrane. Here we show that acute activation of protein kinase C (PKC) induced by the phorbol ester PMA led to the formation of motile actin structures on the ventral surface of Beas-2b cells, a lung bronchial epithelial cell line. Ventral actin structures also formed in PMA-treated HeLa cells that had elevated levels of Arf activation. For both cell types, formation of the ventral actin structures was enhanced by expression of active forms of either Arf1 or Arf6, and by the expression of guanine nucleotide exchange factors that activate these Arfs. By contrast, formation of these structures was blocked by inhibitors of PKC and Src, and required phosphatidylinositol 4, 5-bisphosphate, Rac, Arf6 and Arf1. Furthermore, expression of ASAP1, an Arf1 GTPase activating protein (GAP) was more effective at inhibiting the ventral actin structures than was ACAP1, an Arf6 GAP. This study adds to the expanding role for Arf1 in the periphery and identifies a requirement for Arf1, a “Golgi Arf”, in the reorganization of the cortical actin cytoskeleton on ventral surfaces, against the substratum. PMID:24916416

  14. A Novel and Conserved Plasmodium Sporozoite Membrane Protein SPELD is Required for Maturation of Exo-erythrocytic Forms

    PubMed Central

    Al-Nihmi, Faisal Mohammed Abdul; Kolli, Surendra Kumar; Reddy, Segireddy Rameswara; Mastan, Babu S.; Togiri, Jyothi; Maruthi, Mulaka; Gupta, Roshni; Sijwali, Puran Singh; Mishra, Satish; Kumar, Kota Arun

    2017-01-01

    Plasmodium sporozoites are the infective forms of malaria parasite to vertebrate host and undergo dramatic changes in their transcriptional repertoire during maturation in mosquito salivary glands. We report here the role of a novel and conserved Plasmodium berghei protein encoded by PBANKA_091090 in maturation of Exo-erythrocytic Forms (EEFs) and designate it as Sporozoite surface Protein Essential for Liver stage Development (PbSPELD). PBANKA_091090 was previously annotated as PB402615.00.0 and its transcript was recovered at maximal frequency in the Serial Analysis of the Gene Expression (SAGE) of Plasmodium berghei salivary gland sporozoites. An orthologue of this transcript was independently identified in Plasmodium vivax sporozoite microarrays and was designated as Sporozoite Conserved Orthologous Transcript-2 (scot-2). Functional characterization through reverse genetics revealed that PbSPELD is essential for Plasmodium liver stage maturation. mCherry transgenic of PbSPELD localized the protein to plasma membrane of sporozoites and early EEFs. Global microarray analysis of pbspeld ko revealed EEF attenuation being associated with down regulation of genes central to general transcription, cell cycle, proteosome and cadherin signaling. pbspeld mutant EEFs induced pre-erythrocytic immunity with 50% protective efficacy. Our studies have implications for attenuating the human Plasmodium liver stages by targeting SPELD locus. PMID:28067322

  15. Maltose binding protein-fusion enhances the bioactivity of truncated forms of pig myostatin propeptide produced in E. coli

    PubMed Central

    Lee, Sang Beum; Park, Sung Kwon

    2017-01-01

    Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth. MSTN propeptide (MSTNpro) inhibits MSTN binding to its receptor through complex formation with MSTN, implying that MSTNpro can be a useful agent to improve skeletal muscle growth in meat-producing animals. Four different truncated forms of pig MSTNpro containing N-terminal maltose binding protein (MBP) as a fusion partner were expressed in E. coli, and purified by the combination of affinity chromatography and gel filtration. The MSTN-inhibitory capacities of these proteins were examined in an in vitro gene reporter assay. A MBP-fused, truncated MSTNpro containing residues 42–175 (MBP-Pro42-175) exhibited the same MSTN-inhibitory potency as the full sequence MSTNpro. Truncated MSTNpro proteins containing either residues 42–115 (MBP-Pro42-115) or 42–98 (MBP-Pro42-98) also exhibited MSTN-inhibitory capacity even though the potencies were significantly lower than that of full sequence MSTNpro. In pull-down assays, MBP-Pro42-175, MBP-Pro42-115, and MBP-Pro42-98 demonstrated their binding to MSTN. MBP was removed from the truncated MSTNpro proteins by incubation with factor Xa to examine the potential role of MBP on MSTN-inhibitory capacity of those proteins. Removal of MBP from MBP-Pro42-175 and MBP-Pro42-98 resulted in 20-fold decrease in MSTN-inhibitory capacity of Pro42-175 and abolition of MSTN-inhibitory capacity of Pro42-98, indicating that MBP as fusion partner enhanced the MSTN-inhibitory capacity of those truncated MSTNpro proteins. In summary, this study shows that MBP is a very useful fusion partner in enhancing MSTN-inhibitory potency of truncated forms of MSTNpro proteins, and MBP-fused pig MSTNpro consisting of amino acid residues 42–175 is sufficient to maintain the full MSTN-inhibitory capacity. PMID:28369115

  16. Visualization by atomic force microscopy of tobacco mosaic virus movement protein-RNA complexes formed in vitro.

    PubMed

    Kiselyova, O I; Yaminsky, I V; Karger, E M; Frolova, O Y; Dorokhov, Y L; Atabekov, J G

    2001-06-01

    The structure of complexes formed in vitro by tobacco mosaic virus (TMV)-coded movement protein (MP) with TMV RNA and short (890 nt) synthetic RNA transcripts was visualized by atomic force microscopy on a mica surface. MP molecules were found to be distributed along the chain of RNA and the structure of MP-RNA complexes depended on the molar MP:RNA ratios at which the complexes were formed. A rise in the molar MP:TMV RNA ratio from 20:1 to 60-100:1 resulted in an increase in the density of the MP packaging on TMV RNA and structural conversion of complexes from RNase-sensitive 'beads-on-a-string' into a 'thick string' form that was partly resistant to RNAse. The 'thick string'-type RNase-resistant complexes were also produced by short synthetic RNA transcripts at different MP:RNA ratios. The 'thick string' complexes are suggested to represent clusters of MP molecules cooperatively bound to discrete regions of TMV RNA and separated by protein-free RNA segments.

  17. Human pancreas-specific protein disulfide-isomerase (PDIp) can function as a chaperone independently of its enzymatic activity by forming stable complexes with denatured substrate proteins.

    PubMed

    Fu, Xin-Miao; Zhu, Bao Ting

    2010-07-01

    Members of the PDI (protein disulfide-isomerase) family are critical for the correct folding of secretory proteins by catalysing disulfide bond formation as well as by serving as molecular chaperones to prevent protein aggregation. In the present paper, we report that the chaperone activity of the human pancreas-specific PDI homologue (PDIp) is independent of its enzymatic activity on the basis of the following lines of evidence. First, alkylation of PDIp by iodoacetamide fully abolishes its enzymatic activity, whereas it still retains most of its chaperone activity in preventing the aggregation of reduced insulin B chain and denatured GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Secondly, mutation of the cysteine residues in PDIp's active sites completely abolishes its enzymatic activity, but does not affect its chaperone activity. Thirdly, the b-b' fragment of PDIp, which does not contain the active sites and is devoid of enzymatic activity, still has chaperone activity. Mechanistically, we found that both the recombinant PDIp expressed in Escherichia coli and the natural PDIp present in human or monkey pancreas can form stable complexes with thermal-denatured substrate proteins independently of their enzymatic activity. The high-molecular-mass soluble complexes between PDIp and GAPDH are formed in a stoichiometric manner (subunit ratio of 1:3.5-4.5), and can dissociate after storage for a certain time. As a proof-of-concept for the biological significance of PDIp in intact cells, we demonstrated that its selective expression in E. coli confers strong protection of these cells against heat shock and oxidative-stress-induced death independently of its enzymatic activity.

  18. Bcl-2 proteins bid and bax form a network to permeabilize the mitochondria at the onset of apoptosis

    PubMed Central

    Gahl, Robert F; Dwivedi, Pallavi; Tjandra, Nico

    2016-01-01

    The most critical step in the initiation of apoptosis is the activation of the Bcl-2 family of proteins to oligomerize and permeabilize the outer-mitochondrial membrane (OMM). As this step results in the irreversible release of factors that enhance cellular degradation, it is the point of no return in programmed cell death and would be an ideal therapeutic target. However, the arrangement of the Bcl-2 proteins in the OMM during permeabilization still remains unknown. It is also unclear whether the Bcl-2 protein, Bid, directly participates in the formation of the oligomers in live cells, even though it is cleaved and translocates to the OMM at the initiation of apoptosis. Therefore, we utilized confocal microscopy to measure Förster resonance energy transfer (FRET) efficiencies in live cells to determine the conformation(s) and intermolecular contacts of Bid within these Bcl-2 oligomers. We found that Bid adopts an extended conformation, which appears to be critical for its association with the mitochondrial membrane. This conformation is also important for intermolecular contacts within the Bid oligomer. More importantly for the first time, direct intermolecular contacts between Bid and Bax were observed, thereby, confirming Bid as a key component of these oligomers. Furthermore, the observed FRET efficiencies allowed us to propose an oligomeric arrangement of Bid, Bax, and possibly other members of the Bcl-2 family of proteins that form a self-propagating network that permeabilizes the OMM. PMID:27763642

  19. Electron microscopy visualization of DNA-protein complexes formed by Ku and DNA ligase IV.

    PubMed

    Grob, Patricia; Zhang, Teri T; Hannah, Ryan; Yang, Hui; Hefferin, Melissa L; Tomkinson, Alan E; Nogales, Eva

    2012-01-02

    The repair of DNA double-stranded breaks (DSBs) is essential for cell viability and genome stability. Aberrant repair of DSBs has been linked with cancer predisposition and aging. During the repair of DSBs by non-homologous end joining (NHEJ), DNA ends are brought together, processed and then joined. In eukaryotes, this repair pathway is initiated by the binding of the ring-shaped Ku heterodimer and completed by DNA ligase IV. The DNA ligase IV complex, DNA ligase IV/XRRC4 in humans and Dnl4/Lif1 in yeast, is recruited to DNA ends in vitro and in vivo by an interaction with Ku and, in yeast, Dnl4/Lif1 stabilizes the binding of yKu to in vivo DSBs. Here we have analyzed the interactions of these functionally conserved eukaryotic NHEJ factors with DNA by electron microscopy. As expected, the ring-shaped Ku complex bound stably and specifically to DNA ends at physiological salt concentrations. At a ratio of 1 Ku molecule per DNA end, the majority of DNA ends were occupied by a single Ku complex with no significant formation of linear DNA multimers or circular loops. Both Dnl4/Lif1 and DNA ligase IV/XRCC4 formed complexes with Ku-bound DNA ends, resulting in intra- and intermolecular DNA end bridging, even with non-ligatable DNA ends. Together, these studies, which provide the first visualization of the conserved complex formed by Ku and DNA ligase IV at juxtaposed DNA ends by electron microscopy, suggest that the DNA ligase IV complex mediates end-bridging by engaging two Ku-bound DNA ends.

  20. The first mutant of the Aequorea victoria green fluorescent protein that forms a red chromophore.

    PubMed

    Mishin, Alexander S; Subach, Fedor V; Yampolsky, Ilia V; King, William; Lukyanov, Konstantin A; Verkhusha, Vladislav V

    2008-04-22

    Green fluorescent protein (GFP) from a jellyfish, Aequorea victoria, and its mutants are widely used in biomedical studies as fluorescent markers. In spite of the enormous efforts of academia and industry toward generating its red fluorescent mutants, no GFP variants with emission maximum at more than 529 nm have been developed during the 15 years since its cloning. Here, we used a new strategy of molecular evolution aimed at generating a red-emitting mutant of GFP. As a result, we have succeeded in producing the first GFP mutant that substantially matures to the red-emitting state with excitation and emission maxima at 555 and 585 nm, respectively. A novel, nonoxidative mechanism for formation of the red chromophore in this mutant that includes a dehydration of the Ser65 side chain has been proposed. Model experiments showed that the novel dual-color GFP mutant with green and red emission is suitable for multicolor flow cytometry as an additional color since it is clearly separable from both green and red fluorescent tags.

  1. A Secreted Effector Protein of Ustilago maydis Guides Maize Leaf Cells to Form Tumors

    PubMed Central

    Redkar, Amey; Hoser, Rafal; Schilling, Lena; Zechmann, Bernd; Krzymowska, Magdalena; Walbot, Virginia; Doehlemann, Gunther

    2015-01-01

    The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize. PMID:25888589

  2. Determination of Free-Form and Peptide Bound Pyrraline in the Commercial Drinks Enriched with Different Protein Hydrolysates

    PubMed Central

    Liang, Zhili; Li, Lin; Qi, Haiping; Zhang, Xia; Xu, Zhenbo; Li, Bing

    2016-01-01

    Pyrraline, a causative factor for the recent epidemics of diabetes and cardiovascular disease, is also employed as an indicator to evaluate heat damage and formation of advanced glycation end-products (AGEs) in foods. Peptide-enriched drinks (PEDs) are broadly consumed worldwide due to rapid rate of absorption and perceived health effects. It can be hypothesized that PED is an important source of pyrraline, especially peptide bound pyrraline (Pep-Pyr). In this study we determined free-form pyrraline (Free-Pyr) and Pep-Pyr in drinks enriched with whey protein hydrolysate (WPH), soy protein hydrolysate (SPH) and collagen protein hydrolysate (CPH). A detection method was developed using ultrahigh-performance liquid chromatography with UV-visible detector coupled with tandem mass spectrometry after solid-phase extraction (SPE). The SPE led to excellent recovery rates ranging between 93.2% and 98.5% and a high reproducibility with relative standard deviations (RSD) of <5%. The limits of detection and quantification obtained were 30.4 and 70.3 ng/mL, respectively. Pep-Pyr was identified as the most abundant form (above 96 percent) of total pyrraline, whereas Free-Pyr was present in a small proportion (less than four percent) of total pyrraline. The results indicate that PED is an important extrinsic source of pyrraline, especially Pep-Pyr. As compared with CPH- and SPH-enriched drinks, WPH-enriched drinks contained high content of Pep-Pyr. The Pep-Pyr content is associated with the distribution of peptide lengths and the amino acid compositions of protein in PEDs. PMID:27384561

  3. Islet amyloid polypeptide forms rigid lipid-protein amyloid fibrils on supported phospholipid bilayers.

    PubMed

    Domanov, Yegor A; Kinnunen, Paavo K J

    2008-02-08

    Islet amyloid polypeptide (IAPP) forms fibrillar amyloid deposits in the pancreatic islets of Langerhans of patients with type 2 diabetes mellitus, and its misfolding and aggregation are thought to contribute to beta-cell death. Increasing evidence suggests that IAPP fibrillization is strongly influenced by lipid membranes and, vice versa, that the membrane architecture and integrity are severely affected by amyloid growth. Here, we report direct fluorescence microscopic observations of the morphological transformations accompanying IAPP fibrillization on the surface of supported lipid membranes. Within minutes of application in submicromolar concentrations, IAPP caused extensive remodeling of the membrane including formation of defects, vesiculation, and tubulation. The effects of IAPP concentration, ionic strength, and the presence of amyloid seeds on the bilayer perturbation and peptide aggregation were examined. Growth of amyloid fibrils was visualized using fluorescently labeled IAPP or thioflavin T staining. Two-color imaging of the peptide and membranes revealed that the fibrils were initially composed of the peptide only, and vesiculation occurred in the points where growing fibers touched the lipid membrane. Interestingly, after 2-5 h of incubation, IAPP fibers became "wrapped" by lipid membranes derived from the supported membrane. Progressive increase in molecular-level association between amyloid and membranes in the maturing fibers was confirmed by Förster resonance energy transfer spectroscopy.

  4. Relative increase in Alzheimer's disease of soluble forms of cerebral Abeta amyloid protein precursor containing the Kunitz protease inhibitory domain.

    PubMed

    Moir, R D; Lynch, T; Bush, A I; Whyte, S; Henry, A; Portbury, S; Multhaup, G; Small, D H; Tanzi, R E; Beyreuther, K; Masters, C L

    1998-02-27

    Although a number of studies have examined amyloid precursor protein (APP) mRNA levels in Alzheimer's disease (AD), no clear consensus has emerged as to whether the levels of transcripts for isoforms containing a Kunitz protease inhibitory (KPI)-encoded region are increased or decreased in AD. Here we compare AD and control brain for the relative amounts of APP protein containing KPI to APP protein lacking this domain. APP protein was purified from the soluble subcellular fraction and Triton X-100 membrane pellet extract of one hemisphere of AD (n = 10), normal (n = 7), and neurological control (n = 5) brains. The amount of KPI-containing APP in the purified protein samples was determined using two independent assay methods. The first assay exploited the inhibitory action of KPI-containing APP on trypsin. The second assay employed reflectance analysis of Western blots. The proportion of KPI-containing forms of APP in the soluble subcellular fraction of AD brains is significantly elevated (p < 0.01) compared with controls. Species containing a KPI domain comprise 32-41 and 76-77% of purified soluble APP from control and AD brains, respectively. For purified membrane-associated APP, 72-77 and 65-82% of control and AD samples, respectively, contain a KPI domain. Since KPI-containing species of APP may be more amyloidogenic (Ho, L., Fukuchi, K., and Yonkin, S. G. (1996) J. Biol. Chem. 271, 30929-30934), our findings support an imbalance of isoforms as one possible mechanism for amyloid deposition in sporadic AD.

  5. High resolution structures of the bone morphogenetic protein type II receptor in two crystal forms: Implications for ligand binding

    SciTech Connect

    Mace, Peter D.; Cutfield, John F.; Cutfield, Sue M. . E-mail: sue.cutfield@otago.ac.nz

    2006-12-29

    BMPRII is a type II TGF-{beta} serine threonine kinase receptor which is integral to the bone morphogenetic protein (BMP) signalling pathway. It is known to bind BMP and growth differentiation factor (GDF) ligands, and has overlapping ligand specificity with the activin type II receptor, ActRII. In contrast to activin and TGF-{beta} type ligands, BMPs bind to type II receptors with lower affinity than type I receptors. Crystals of the BMPRII ectodomain were grown in two different forms, both of which diffracted to high resolution. The tetragonal form exhibited some disorder, whereas the entire polypeptide was seen in the orthorhombic form. The two structures retain the basic three-finger toxin fold of other TGF-{beta} receptor ectodomains, and share the main hydrophobic patch used by ActRII to bind various ligands. However, they present different conformations of the A-loop at the periphery of the proposed ligand-binding interface, in conjunction with rearrangement of a disulfide bridge within the loop. This particular disulfide (Cys94-Cys117) is only present in BMPRII and activin receptors, suggesting that it is important for their likely shared mode of binding. Evidence is presented that the two crystal forms represent ligand-bound and free conformations of BMPRII. Comparison with the solved structure of ActRII bound to BMP2 suggests that His87, unique amongst TGF-{beta} receptors, may play a key role in ligand recognition.

  6. The myth of Brucella L-forms and possible involvement of Brucella penicillin binding proteins (PBPs) in pathogenicity.

    PubMed

    Banai, M; Adams, L G; Frey, M; Pugh, R; Ficht, T A

    2002-12-20

    Brucella spp. L-forms have been proposed to be stationary phase organisms in the evolution of new variants and enduring entities in the host in complicated cases of brucellosis and during latent brucellosis. In vitro formation of Brucella L-forms has been achieved by treating the cells with sub-lethal doses of penicillin. Interestingly, Brucella spp. have classified during the evolution into two groups, penicillin susceptible or penicillin resistant, yet both types grow on 20 microg/ml of methicillin. Strains proven susceptible to penicillin grew in the presence of methicillin as L-forms as demonstrated by light and electron microscopy. In addition, the B. melitensis vaccine strain Rev.1, a penicillin susceptible organism, responded to sheep serum by development of L-form-like structures unlike wild type, strain 16M. The two strains grew normally in sheep macrophages. We propose, for the first time, a model that associates Brucella pathogenicity with the structure and activity of two of their penicillin binding proteins (PBPs). According to the model, PBP1 has evolved as the major cell wall synthesizing enzyme of the genus, capable of responding to host serum growth factor(s) necessary for Brucella survival in the host. This property is associated with high avidity to beta-lactam antibiotics. PBP2 complements the activity of PBP1. New beta-lactam antibiotics and improved vaccines might be developed based on this property.

  7. Membrane damage by an α-helical pore-forming protein, Equinatoxin II, proceeds through a succession of ordered steps.

    PubMed

    Rojko, Nejc; Kristan, Katarina Č; Viero, Gabriella; Žerovnik, Eva; Maček, Peter; Dalla Serra, Mauro; Anderluh, Gregor

    2013-08-16

    Actinoporin equinatoxin II (EqtII) is an archetypal example of α-helical pore-forming toxins that porate cellular membranes by the use of α-helices. Previous studies proposed several steps in the pore formation: binding of monomeric protein onto the membrane, followed by oligomerization and insertion of the N-terminal α-helix into the lipid bilayer. We studied these separate steps with an EqtII triple cysteine mutant. The mutant was engineered to monitor the insertion of the N terminus into the lipid bilayer by labeling Cys-18 with a fluorescence probe and at the same time to control the flexibility of the N-terminal region by the disulfide bond formed between cysteines introduced at positions 8 and 69. The insertion of the N terminus into the membrane proceeded shortly after the toxin binding and was followed by oligomerization. The oxidized, non-lytic, form of the mutant was still able to bind to membranes and oligomerize at the same level as the wild-type or the reduced form. However, the kinetics of the N-terminal helix insertion, the release of calcein from erythrocyte ghosts, and hemolysis of erythrocytes was much slower when membrane-bound oxidized mutant was reduced by the addition of the reductant. Results show that the N-terminal region needs to be inserted in the lipid membrane before the oligomerization into the final pore and imply that there is no need for a stable prepore formation. This is different from β-pore-forming toxins that often form β-barrel pores via a stable prepore complex.

  8. Membrane Damage by an α-Helical Pore-forming Protein, Equinatoxin II, Proceeds through a Succession of Ordered Steps*

    PubMed Central

    Rojko, Nejc; Kristan, Katarina Č.; Viero, Gabriella; Žerovnik, Eva; Maček, Peter; Dalla Serra, Mauro; Anderluh, Gregor

    2013-01-01

    Actinoporin equinatoxin II (EqtII) is an archetypal example of α-helical pore-forming toxins that porate cellular membranes by the use of α-helices. Previous studies proposed several steps in the pore formation: binding of monomeric protein onto the membrane, followed by oligomerization and insertion of the N-terminal α-helix into the lipid bilayer. We studied these separate steps with an EqtII triple cysteine mutant. The mutant was engineered to monitor the insertion of the N terminus into the lipid bilayer by labeling Cys-18 with a fluorescence probe and at the same time to control the flexibility of the N-terminal region by the disulfide bond formed between cysteines introduced at positions 8 and 69. The insertion of the N terminus into the membrane proceeded shortly after the toxin binding and was followed by oligomerization. The oxidized, non-lytic, form of the mutant was still able to bind to membranes and oligomerize at the same level as the wild-type or the reduced form. However, the kinetics of the N-terminal helix insertion, the release of calcein from erythrocyte ghosts, and hemolysis of erythrocytes was much slower when membrane-bound oxidized mutant was reduced by the addition of the reductant. Results show that the N-terminal region needs to be inserted in the lipid membrane before the oligomerization into the final pore and imply that there is no need for a stable prepore formation. This is different from β-pore-forming toxins that often form β-barrel pores via a stable prepore complex. PMID:23803608

  9. Water in the Cratonic Mantle: Insights from FTIR Data on Lac De Gras Xenoliths (Slave Craton, Canada)

    NASA Technical Reports Server (NTRS)

    Peslier, Anne H.; Brandon, Alan D.; Schaffer, Lillian Aurora; O'Reilly, Suzanne Yvette; Griffin, William L.; Morris, Richard V.; Graff, Trevor G.; Agresti, David G.

    2014-01-01

    The mantle lithosphere beneath the cratonic part of continents is the deepest (> 200 km) and oldest (>2-3 Ga) on Earth, remaining a conundrum as to how these cratonic roots could have resisted delamination by asthenospheric convection over time. Water, or trace H incorporated in mineral defects, could be a key player in the evolution of continental lithosphere because it influences melting and rheology of the mantle. Mantle xenoliths from the Lac de Gras kimberlite in the Slave craton were analyzed by FTIR. The cratonic mantle beneath Lac de Gras is stratified with shallow (<145 km) oxidized ultradepleted peridotites and pyroxenites with evidence for carbonatitic metasomatism, underlain by reduced and less depleted peridotites metasomatized by kimberlite melts. Peridotites analyzed so far have H O contents in ppm weight of 7-100 in their olivines, 58 to 255 in their orthopyroxenes (opx), 11 to 84 in their garnet, and 139 in one clinopyroxene. A pyroxenite contains 58 ppm H2O in opx and 5 ppm H2O in its olivine and garnet. Olivine and garnet from the deep peridotites have a range of water contents extending to higher values than those from the shallow ones. The FTIR spectra of olivines from the shallow samples have more prominent Group II OH bands compared to the olivines from the deep samples, consistent with a more oxidized mantle environment. The range of olivine water content is similar to that observed in Kaapvaal craton peridotites at the same depths (129-184 km) but does not extend to as high values as those from Udachnaya (Siberian craton). The Slave, Kaapvaal and Siberian cratons will be compared in terms of water content distribution, controls and role in cratonic root longevity.

  10. The FEMA GRAS assessment of alpha,beta-unsaturated aldehydes and related substances used as flavor ingredients.

    PubMed

    Adams, T B; Gavin, C Lucas; Taylor, S V; Waddell, W J; Cohen, S M; Feron, V J; Goodman, J; Rietjens, I M C M; Marnett, L J; Portoghese, P S; Smith, R L

    2008-09-01

    This publication is the 12th in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Since then, the number of flavoring substances has grown to more than 2200 chemically-defined substances. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, toxicodynamics and toxicology. Scientific data relevant to the safety evaluation for the use of aliphatic, linear alpha,beta-unsaturated aldehydes and structurally related substances as flavoring ingredients are evaluated. The group of substances was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; their low level of flavor use; the rapid absorption and metabolism of low in vivo concentrations by well-recognized biochemical pathways; adequate metabolic detoxication at much higher levels of exposure in humans and animals; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies. While some of the compounds described here have exhibited positive in vitro genotoxicity results, evidence of in vivo genotoxicity and carcinogenicity occurs only under conditions in which animals are repeatedly and directly exposed to high irritating concentrations of the aldehyde. These conditions are not relevant to humans who consume alpha,beta-unsaturated aldehydes as flavor ingredients at low concentrations distributed in a food or beverage matrix.

  11. Danger signaling protein HMGB1 induces a distinct form of cell death accompanied by formation of giant mitochondria.

    PubMed

    Gdynia, Georg; Keith, Martina; Kopitz, Jürgen; Bergmann, Marion; Fassl, Anne; Weber, Alexander N R; George, Julie; Kees, Tim; Zentgraf, Hans-Walter; Wiestler, Otmar D; Schirmacher, Peter; Roth, Wilfried

    2010-11-01

    Cells dying by necrosis release the high-mobility group box 1 (HMGB1) protein, which has immunostimulatory effects. However, little is known about the direct actions of extracellular HMGB1 protein on cancer cells. Here, we show that recombinant human HMGB1 (rhHMGB1) exerts strong cytotoxic effects on malignant tumor cells. The rhHMGB1-induced cytotoxicity depends on the presence of mitochondria and leads to fast depletion of mitochondrial DNA, severe damage of the mitochondrial proteome by toxic malondialdehyde adducts, and formation of giant mitochondria. The formation of giant mitochondria is independent of direct nuclear signaling events, because giant mitochondria are also observed in cytoplasts lacking nuclei. Further, the reactive oxygen species scavenger N-acetylcysteine as well as c-Jun NH(2)-terminal kinase blockade inhibited the cytotoxic effect of rhHMGB1. Importantly, glioblastoma cells, but not normal astrocytes, were highly susceptible to rhHMGB1-induced cell death. Systemic treatment with rhHMGB1 results in significant growth inhibition of xenografted tumors in vivo. In summary, rhHMGB1 induces a distinct form of cell death in cancer cells, which differs from the known forms of apoptosis, autophagy, and senescence, possibly representing an important novel mechanism of specialized necrosis. Further, our findings suggest that rhHMGB1 may offer therapeutic applications in treatment of patients with malignant brain tumors.

  12. Toxicity and oxidative stress of different forms of organic selenium (Se) and dietary protein in mallard (Anas platyrhynchos) ducklings

    USGS Publications Warehouse

    Hoffman, D.; Heinz, G.; Eisemann, J.; Pendleton, G.

    1994-01-01

    High concentrations of Se have been found in aquatic food chains associated with irrigation drainwater and toxicity to fish and wildlife. Earlier studies have compared toxicities of Se as selenite and as seleno-DL-methionine (DL) in mallards. This study compares DL, seleno-L-methionine (L), selenized yeast (Y) and selenized wheat (W). Day-old mallard ducklings received an untreated diet (controls) containing 75% wheat (22% protein) or the same diet containing 15 or 30 ppm Se in the above forms. After 2 weeks blood and liver samples were collected for biochemical assays and Se analysis. All forms of selenium caused significant increases in plasma and hepatic glutathione peroxidase activities. Se as L was the most toxic, resulting in high mortality (64%) and impaired growth (>50%) and the greatest increase in ratio of oxidized to reduced glutathione with 30 ppm in the diet. Se as Y accumulated the least in liver. In a subsequent experiment with 30% dietary protein Se as L was less toxic.

  13. A novel mutation of myelin protein zero associated with an axonal form of Charcot–Marie–Tooth disease

    PubMed Central

    Santoro, L; Manganelli, F; Di, M; Bordo, D; Cassandrini, D; Ajmar, F; Mandich, P; Bellone, E

    2004-01-01

    Objective: To report a new mutation in the MPZ gene which encodes myelin protein zero (P0), associated with an axonal form of Charcot–Marie–Tooth disease (CMT). Methods: Three patients from an Italian family with a mild, late onset axonal peripheral neuropathy are described clinically and electrophysiologically. To detect point mutation in MPZ gene the whole coding sequence was examined. The structure of the mutated protein was investigated using the three dimensional model of P0. Results: All patients showed a relatively mild CMT phenotype characterised by late onset and heterogeneity of the clinical and electrophysiological features. Molecular analysis demonstrated a novel heterozygous T/A transversion in the exon 3 of MPZ gene that predicts an Asp109Glu amino acid substitution in the extracellular domain of the P0. Asp109 is found at the protein surface, on ß strand E, in the interior of the P0 tetramer. Conclusions: The identification of Asp109Glu mutation confirms the pivotal role of P0 in axonal neuropathies and stresses the phenotypic heterogeneity associated with MPZ mutations. This study suggests the value of screening for MPZ mutations in CMT family members with minor clinical and electrophysiological signs of peripheral neuropathy. PMID:14742601

  14. Floating gate memory with charge storage dots array formed by Dps protein modified with site-specific binding peptides

    NASA Astrophysics Data System (ADS)

    Kamitake, Hiroki; Uenuma, Mutsunori; Okamoto, Naofumi; Horita, Masahiro; Ishikawa, Yasuaki; Yamashita, Ichro; Uraoka, Yukiharu

    2015-05-01

    We report a nanodot (ND) floating gate memory (NFGM) with a high-density ND array formed by a biological nano process. We utilized two kinds of cage-shaped proteins displaying SiO2 binding peptide (minTBP-1) on their outer surfaces: ferritin and Dps, which accommodate cobalt oxide NDs in their cavities. The diameters of the cobalt NDs were regulated by the cavity sizes of the proteins. Because minTBP-1 is strongly adsorbed on the SiO2 surface, high-density cobalt oxide ND arrays were obtained by a simple spin coating process. The densities of cobalt oxide ND arrays based on ferritin and Dps were 6.8 × 1011 dots cm-2 and 1.2 × 1012 dots cm-2, respectively. After selective protein elimination and embedding in a metal-oxide-semiconductor (MOS) capacitor, the charge capacities of both ND arrays were evaluated by measuring their C-V characteristics. The MOS capacitor embedded with the Dps ND array showed a wider memory window than the device embedded with the ferritin ND array. Finally, we fabricated an NFGM with a high-density ND array based on Dps, and confirmed its competent writing/erasing characteristics and long retention time.

  15. Hexanucleotide repeats in ALS/FTD form length-dependent RNA foci, sequester RNA binding proteins, and are neurotoxic.

    PubMed

    Lee, Youn-Bok; Chen, Han-Jou; Peres, João N; Gomez-Deza, Jorge; Attig, Jan; Stalekar, Maja; Troakes, Claire; Nishimura, Agnes L; Scotter, Emma L; Vance, Caroline; Adachi, Yoshitsugu; Sardone, Valentina; Miller, Jack W; Smith, Bradley N; Gallo, Jean-Marc; Ule, Jernej; Hirth, Frank; Rogelj, Boris; Houart, Corinne; Shaw, Christopher E

    2013-12-12

    The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration.

  16. AglZ Is a Filament-Forming Coiled-Coil Protein Required for Adventurous Gliding Motility of Myxococcus xanthus

    PubMed Central

    Yang, Ruifeng; Bartle, Sarah; Otto, Rebecca; Stassinopoulos, Angela; Rogers, Matthew; Plamann, Lynda; Hartzell, Patricia

    2004-01-01

    The aglZ gene of Myxococcus xanthus was identified from a yeast two-hybrid assay in which MglA was used as bait. MglA is a 22-kDa cytoplasmic GTPase required for both adventurous and social gliding motility and sporulation. Genetic studies showed that aglZ is part of the A motility system, because disruption or deletion of aglZ abolished movement of isolated cells and aglZ sglK double mutants were nonmotile. The aglZ gene encodes a 153-kDa protein that interacts with purified MglA in vitro. The N terminus of AglZ shows similarity to the receiver domain of two-component response regulator proteins, while the C terminus contains heptad repeats characteristic of coiled-coil proteins, such as myosin. Consistent with this motif, expression of AglZ in Escherichia coli resulted in production of striated lattice structures. Similar to the myosin heavy chain, the purified C-terminal coiled-coil domain of AglZ forms filament structures in vitro. PMID:15342587

  17. The Nitrogenase FeMo-Cofactor Precursor Formed by NifB Protein: A Diamagnetic Cluster Containing Eight Iron Atoms.

    PubMed

    Guo, Yisong; Echavarri-Erasun, Carlos; Demuez, Marie; Jiménez-Vicente, Emilio; Bominaar, Emile L; Rubio, Luis M

    2016-10-04

    The biological activation of N2 occurs at the FeMo-cofactor, a 7Fe-9S-Mo-C-homocitrate cluster. FeMo-cofactor formation involves assembly of a Fe6-8 -SX -C core precursor, NifB-co, which occurs on the NifB protein. Characterization of NifB-co in NifB is complicated by the dynamic nature of the assembly process and the presence of a permanent [4Fe-4S] cluster associated with the radical SAM chemistry for generating the central carbide. We have used the physiological carrier protein, NifX, which has been proposed to bind NifB-co and deliver it to the NifEN protein, upon which FeMo-cofactor assembly is ultimately completed. Preparation of NifX in a fully NifB-co-loaded form provided an opportunity for Mössbauer analysis of NifB-co. The results indicate that NifB-co is a diamagnetic (S=0) 8-Fe cluster, containing two spectroscopically distinct Fe sites that appear in a 3:1 ratio. DFT analysis of the (57) Fe electric hyperfine interactions deduced from the Mössbauer analysis suggests that NifB-co is either a 4Fe(2+) -4Fe(3+) or 6Fe(2+) -2Fe(3+) cluster having valence-delocalized states.

  18. AglZ is a filament-forming coiled-coil protein required for adventurous gliding motility of Myxococcus xanthus.

    PubMed

    Yang, Ruifeng; Bartle, Sarah; Otto, Rebecca; Stassinopoulos, Angela; Rogers, Matthew; Plamann, Lynda; Hartzell, Patricia

    2004-09-01

    The aglZ gene of Myxococcus xanthus was identified from a yeast two-hybrid assay in which MglA was used as bait. MglA is a 22-kDa cytoplasmic GTPase required for both adventurous and social gliding motility and sporulation. Genetic studies showed that aglZ is part of the A motility system, because disruption or deletion of aglZ abolished movement of isolated cells and aglZ sglK double mutants were nonmotile. The aglZ gene encodes a 153-kDa protein that interacts with purified MglA in vitro. The N terminus of AglZ shows similarity to the receiver domain of two-component response regulator proteins, while the C terminus contains heptad repeats characteristic of coiled-coil proteins, such as myosin. Consistent with this motif, expression of AglZ in Escherichia coli resulted in production of striated lattice structures. Similar to the myosin heavy chain, the purified C-terminal coiled-coil domain of AglZ forms filament structures in vitro.

  19. A flagellar A-kinase anchoring protein with two amphipathic helices forms a structural scaffold in the radial spoke complex

    PubMed Central

    Sivadas, Priyanka; Dienes, Jennifer M.; St. Maurice, Martin; Meek, William D.

    2012-01-01

    A-kinase anchoring proteins (AKAPs) contain an amphipathic helix (AH) that binds the dimerization and docking (D/D) domain, RIIa, in cAMP-dependent protein kinase A (PKA). Many AKAPs were discovered solely based on the AH–RIIa interaction in vitro. An RIIa or a similar Dpy-30 domain is also present in numerous diverged molecules that are implicated in critical processes as diverse as flagellar beating, membrane trafficking, histone methylation, and stem cell differentiation, yet these molecules remain poorly characterized. Here we demonstrate that an AKAP, RSP3, forms a dimeric structural scaffold in the flagellar radial spoke complex, anchoring through two distinct AHs, the RIIa and Dpy-30 domains, in four non-PKA spoke proteins involved in the assembly and modulation of the complex. Interestingly, one AH can bind both RIIa and Dpy-30 domains in vitro. Thus, AHs and D/D domains constitute a versatile yet potentially promiscuous system for localizing various effector mechanisms. These results greatly expand the current concept about anchoring mechanisms and AKAPs. PMID:23148234

  20. Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos

    PubMed Central

    1991-01-01

    To gain insight into the mechanisms involved in the formation of maternally stored mRNPs during Xenopus laevis development, we searched for soluble cytoplasmic proteins of the oocyte that are able to selectively bind mRNAs, using as substrate radiolabeled mRNA. In vitro mRNP assembly in solution was followed by UV-cross-linking and RNase digestion, resulting in covalent tagging of polypeptides by nucleotide transfer. Five polypeptides of approximately 54, 56 60, 70, and 100 kD (p54, p56, p60, p70, and p100) have been found to selectively bind mRNA and assemble into mRNPs. These polypeptides, which correspond to previously described native mRNP components, occur in three different particle classes of approximately 4.5S, approximately 6S, and approximately 15S, as also determined by their reactions with antibodies against p54 and p56. Whereas the approximately 4.5S class contains p42, p60, and p70, probably each in the form of individual molecules or small complexes, the approximately 6S particles appears to consist only of p54 and p56, which occur in a near-stoichiometric ratio suggestive of a heterodimer complex. The approximately 15S particles contain, in addition to p54 and p56, p60 and p100 and this is the single occurring form of RNA-binding p100. We have also observed changes in the in vitro mRNA binding properties of these polypeptides during oogenesis and early embryonic development, in relation to their phosphorylation state and to the activity of an approximately 15S particle-associated protein kinase, suggesting that these proteins are involved in the developmental translational regulation of maternal mRNAs. PMID:1670777

  1. Vpr Protein of Human Immunodeficiency Virus Type 1 Forms Cation-Selective Channels in Planar Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Piller, S. C.; Ewart, G. D.; Premkumar, A.; Cox, G. B.; Gage, P. W.

    1996-01-01

    A small (96-aa) protein, virus protein R (Vpr), of human immunodeficiency virus type 1 contains one hydrophobic segment that could form a membrane-spanning helix. Recombinant Vpr, expressed in Escherichia coli and purified by affinity chromatography, formed ion channels in planar lipid bilayers when it was added to the cis chamber and when the trans chamber was held at a negative potential. The channels were more permeable to Na+ than to Cl- ions and were inhibited when the trans potential was made positive. Similar channel activity was caused by Vpr that had a truncated C terminus, but the potential dependence of channel activity was no longer seen. Antibody raised to a peptide mimicking part of the C terminus of Vpr (AbC) inhibited channel activity when added to the trans chamber but had no effect when added to the cis chamber. Antibody to the N terminus of Vpr (AbN) increased channel activity when added to the cis chamber but had no effect when added to the trans chamber. The effects of potential and antibodies on channel activity are consistent with a model in which the positive C-terminal end of dipolar Vpr is induced to traverse the bilayer membrane when the opposite (trans) side of the membrane is at a negative potential. The C terminus of Vpr would then be available for interaction with AbC in the trans chamber, and the N terminus would be available for interaction with AbN in the cis chamber. The ability of Vpr to form ion channels in vitro suggests that channel formation by Vpr in vivo is possible and may be important in the life cycle of human immunodeficiency virus type 1 and/or may cause changes in cells that contribute to AIDS-related pathologies.

  2. Transcribed processed pseudogenes in the human genome: an intermediate form of expressed retrosequence lacking protein-coding ability.

    PubMed

    Harrison, Paul M; Zheng, Deyou; Zhang, Zhaolei; Carriero, Nicholas; Gerstein, Mark

    2005-01-01

    Pseudogenes, in the case of protein-coding genes, are gene copies that have lost the ability to code for a protein; they are typically identified through annotation of disabled, decayed or incomplete protein-coding sequences. Processed pseudogenes (PPsigs) are made through mRNA retrotransposition. There is overwhelming genomic evidence for thousands of human PPsigs and also dozens of human processed genes that comprise complete retrotransposed copies of other genes. Here, we survey for an intermediate entity, the transcribed processed pseudogene (TPPsig), which is disabled but nonetheless transcribed. TPPsigs may affect expression of paralogous genes, as observed in the case of the mouse makorin1-p1 TPPsig. To elucidate their role, we identified human TPPsigs by mapping expressed sequences onto PPsigs and, reciprocally, extracting TPPsigs from known mRNAs. We consider only those PPsigs that are homologous to either non-mammalian eukaryotic proteins or protein domains of known structure, and require detection of identical coding-sequence disablements in both the expressed and genomic sequences. Oligonucleotide microarray data provide further expression verification. Overall, we find 166-233 TPPsigs ( approximately 4-6% of PPsigs). Proteins/transcripts with the highest numbers of homologous TPPsigs generally have many homologous PPsigs and are abundantly expressed. TPPsigs are significantly over-represented near both the 5' and 3' ends of genes; this suggests that TPPsigs can be formed through gene-promoter co-option, or intrusion into untranslated regions. However, roughly half of the TPPsigs are located away from genes in the intergenic DNA and thus may be co-opting cryptic promoters of undesignated origin. Furthermore, TPPsigs are unlike other PPsigs and processed genes in the following ways: (i) they do not show a significant tendency to either deposit on or originate from the X chromosome; (ii) only 5% of human TPPsigs have potential orthologs in mouse. This

  3. Playing RNase P evolution: swapping the RNA catalyst for a protein reveals functional uniformity of highly divergent enzyme forms.

    PubMed

    Weber, Christoph; Hartig, Andreas; Hartmann, Roland K; Rossmanith, Walter

    2014-08-01

    The RNase P family is a diverse group of endonucleases responsible for the removal of 5' extensions from tRNA precursors. The diversity of enzyme forms finds its extremes in the eukaryal nucleus where RNA-based catalysis by complex ribonucleoproteins in some organisms contrasts with single-polypeptide enzymes in others. Such structural contrast suggests associated functional differences, and the complexity of the ribonucleoprotein was indeed proposed to broaden the enzyme's functionality beyond tRNA processing. To explore functional overlap and differences between most divergent forms of RNase P, we replaced the nuclear RNase P of Saccharomyces cerevisiae, a 10-subunit ribonucleoprotein, with Arabidopsis thaliana PRORP3, a single monomeric protein. Surprisingly, the RNase P-swapped yeast strains were viable, displayed essentially unimpaired growth under a wide variety of conditions, and, in a certain genetic background, their fitness even slightly exceeded that of the wild type. The molecular analysis of the RNase P-swapped strains showed a minor disturbance in tRNA metabolism, but did not point to any RNase P substrates or functions beyond that. Altogether, these results indicate the full functional exchangeability of the highly dissimilar enzymes. Our study thereby establishes the RNase P family, with its combination of structural diversity and functional uniformity, as an extreme case of convergent evolution. It moreover suggests that the apparently gratuitous complexity of some RNase P forms is the result of constructive neutral evolution rather than reflecting increased functional versatility.

  4. Uptake and Degradation of Protease-Sensitive and -Resistant Forms of Abnormal Human Prion Protein Aggregates by Human Astrocytes

    PubMed Central

    Choi, Young Pyo; Head, Mark W.; Ironside, James W.; Priola, Suzette A.

    2015-01-01

    Sporadic Creutzfeldt-Jakob disease is the most common of the human prion diseases, a group of rare, transmissible, and fatal neurologic diseases associated with the accumulation of an abnormal form (PrPSc) of the host prion protein. In sporadic Creutzfeldt-Jakob disease, disease-associated PrPSc is present not only as an aggregated, protease-resistant form but also as an aggregated protease-sensitive form (sPrPSc). Although evidence suggests that sPrPSc may play a role in prion pathogenesis, little is known about how it interacts with cells during prion infection. Here, we show that protease-sensitive abnormal PrP aggregates derived from patients with sporadic Creutzfeldt-Jakob disease are taken up and degraded by immortalized human astrocytes similarly to abnormal PrP aggregates that are resistant to proteases. Our data suggest that relative proteinase K resistance does not significantly influence the astrocyte's ability to degrade PrPSc. Furthermore, the cell does not appear to distinguish between sPrPSc and protease-resistant PrPSc, suggesting that sPrPSc could contribute to prion infection. PMID:25280631

  5. Room temperature spectrally resolved single-molecule spectroscopy reveals new spectral forms and photophysical versatility of aequorea green fluorescent protein variants.

    PubMed

    Blum, Christian; Meixner, Alfred J; Subramaniam, Vinod

    2004-12-01

    It is known from ensemble spectroscopy at cryogenic temperatures that variants of the Aequorea green fluorescent protein (GFP) occur in interconvertible spectroscopically distinct forms which are obscured in ensemble room temperature spectroscopy. By analyzing the fluorescence of the GFP variants EYFP and EGFP by spectrally resolved single-molecule spectroscopy we were able to observe spectroscopically different forms of the proteins and to dynamically monitor transitions between these forms at room temperature. In addition to the predominant EYFP B-form we have observed the blue-shifted I-form thus far only seen at cryogenic temperatures and have followed transitions between these forms. Further we have identified for EYFP and for EGFP three more, so far unknown, forms with red-shifted fluorescence. Transitions between the predominant forms and the red-shifted forms show a dark time which indicates the existence of a nonfluorescent intermediate. The spectral position of the newly-identified red-shifted forms and their formation via a nonfluorescent intermediate hint that these states may account for the possible photoactivation observed in bulk experiments. The comparison of the single-protein spectra of the red-shifted EYFP and EGFP forms with single-molecule fluorescence spectra of DsRed suggest that these new forms possibly originate from an extended chromophoric pi-system analogous to the DsRed chromophore.

  6. Salicylate-inducible antibiotic resistance in Pseudomonas cepacia associated with absence of a pore-forming outer membrane protein.

    PubMed Central

    Burns, J L; Clark, D K

    1992-01-01

    The most common mechanism of antibiotic resistance in multiply resistant Pseudomonas cepacia is decreased porin-mediated outer membrane permeability. In some gram-negative organisms this form of antibiotic resistance can be induced by growth in the presence of weak acids, such as salicylates, which suppress porin synthesis. To determine the effects of salicylates on outer membrane permeability of P. cepacia, a susceptible laboratory strain, 249-2, was grown in 10 mM sodium salicylate. Antibiotic susceptibility and uptake, as well as outer membrane protein patterns, were compared between strain 249-2 grown with and without salicylates. The MICs of chloramphenicol, trimethoprim, ciprofloxacin, and ceftazidime were compared between organisms grown in standard and salicylate-containing medium and are as follows: chloramphenicol, 12.5 versus 100 micrograms/ml; trimethoprim, 0.78 versus 3.125 micrograms/ml; ciprofloxacin, 0.4 versus 1.56 micrograms/ml; ceftazidime, 3.125 versus 3.125 micrograms/ml. The permeability of beta-lactam antibiotics was calculated from the rate of hydrolysis of the chromogenic cephalosporin, PADAC. There was no significant difference between strains grown in the presence and absence of salicylate. By using high-pressure liquid chromatography quantitation of loss from culture medium, the effect of 10 mM salicylate on the cellular permeability of chloramphenicol was measured in strain 249-2 by introduction of a plasmid which encodes production of chloramphenicol acetyltransferase. After 1 h of incubation, 18.5% +/- 1.54% versus 70.1% +/- 3.52%, and after 2 h, 4.20% +/- 1.65% versus 41.90% +/- 2.16% remained in supernatants from organisms grown in the absence and presence of 10 mM salicylate, respectively. Outer membrane protein pattern analysis demonstrated the absence of a protein of apparent molecular weight of 40,000 when strain 249-2 was grown in the presence of 10 mM salicylate. To determine whether this protein functioned as a porin

  7. Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive.

    PubMed

    Oka, T; Kujiraoka, T; Ito, M; Egashira, T; Takahashi, S; Nanjee, M N; Miller, N E; Metso, J; Olkkonen, V M; Ehnholm, C; Jauhiainen, M; Hattori, H

    2000-10-01

    Plasma phospholipid transfer protein (PLTP) plays an important role in the maintenance of plasma high-density lipoprotein (HDL) content and remodeling of HDL in the circulation. In the present study we have used different fractionation methods to investigate the distribution of PLTP in human plasma. A novel enzyme-linked immunosorbent assay developed during the study allowed for simultaneous assessment of both PLTP mass and activity in the fractions obtained. Size-exclusion chromatography and plasma fractionation by nondenaturing polyacrylamide gel electrophoresis (PAGE) yielded similar results demonstrating that PLTP associates in native plasma with two distinct particle populations, while ultracentrifugation with high salt leads to detachment of PLTP from lipoprotein particles and loss of a majority of its phospholipid transfer activity. Interestingly, analysis of the size-exclusion chromatography fractions demonstrated that PLTP exists in the circulation as an active population that elutes in the position of HDL corresponding to an average molecular mass of 160+/-40 kDa and an inactive form with an average mass of 520+/-120 kDa. The inactive fraction containing approximately 70% of the total PLTP protein eluted between HDL and low density lipoprotein (LDL). Thus, the two PLTP pools are associated with different types of lipoprotein particles, suggesting that the PLTP activity in circulation is modulated by the plasma lipoprotein profile and lipid composition.

  8. Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry

    PubMed Central

    Hilbert, Manuel; Erat, Michèle C.; Hachet, Virginie; Guichard, Paul; Blank, Iris D.; Flückiger, Isabelle; Slater, Leanne; Lowe, Edward D.; Hatzopoulos, Georgios N.; Steinmetz, Michel O.; Gönczy, Pierre; Vakonakis, Ioannis

    2013-01-01

    Centrioles are evolutionary conserved organelles that give rise to cilia and flagella as well as centrosomes. Centrioles display a characteristic ninefold symmetry imposed by the spindle assembly abnormal protein 6 (SAS-6) family. SAS-6 from Chlamydomonas reinhardtii and Danio rerio was shown to form ninefold symmetric, ring-shaped oligomers in vitro that were similar to the cartwheels observed in vivo during early steps of centriole assembly in most species. Here, we report crystallographic and EM analyses showing that, instead, Caenorhabotis elegans SAS-6 self-assembles into a spiral arrangement. Remarkably, we find that this spiral arrangement is also consistent with ninefold symmetry, suggesting that two distinct SAS-6 oligomerization architectures can direct the same output symmetry. Sequence analysis suggests that SAS-6 spirals are restricted to specific nematodes. This oligomeric arrangement may provide a structural basis for the presence of a central tube instead of a cartwheel during centriole assembly in these species. PMID:23798409

  9. In vivo neuronal synthesis and axonal transport of Kunitz protease inhibitor (KPI)-containing forms of the amyloid precursor protein.

    PubMed

    Moya, K L; Confaloni, A M; Allinquant, B

    1994-11-01

    We have shown previously that the amyloid precursor protein (APP) is synthesized in retinal ganglion cells and is rapidly transported down the axons, and that different molecular weight forms of the precursor have different developmental time courses. Some APP isoforms contain a Kunitz protease inhibitor (KPI) domain, and APP that lacks the KPI domain is considered the predominant isoform in neurons. We now show that, among the various rapidly transported APPs, a 140-kDa isoform contains the KPI domain. This APP isoform is highly expressed in rapidly growing retinal axons, and it is also prominent in adult axon endings. This 140-kDa KPI-containing APP is highly sulfated compared with other axonally transported isoforms. These results show that APP with the KPI domain is a prominent isoform synthesized in neurons in vivo, and they suggest that the regulation of protease activity may be an important factor during the establishment of neuronal connections.

  10. Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry.

    PubMed

    Hilbert, Manuel; Erat, Michèle C; Hachet, Virginie; Guichard, Paul; Blank, Iris D; Flückiger, Isabelle; Slater, Leanne; Lowe, Edward D; Hatzopoulos, Georgios N; Steinmetz, Michel O; Gönczy, Pierre; Vakonakis, Ioannis

    2013-07-09

    Centrioles are evolutionary conserved organelles that give rise to cilia and flagella as well as centrosomes. Centrioles display a characteristic ninefold symmetry imposed by the spindle assembly abnormal protein 6 (SAS-6) family. SAS-6 from Chlamydomonas reinhardtii and Danio rerio was shown to form ninefold symmetric, ring-shaped oligomers in vitro that were similar to the cartwheels observed in vivo during early steps of centriole assembly in most species. Here, we report crystallographic and EM analyses showing that, instead, Caenorhabotis elegans SAS-6 self-assembles into a spiral arrangement. Remarkably, we find that this spiral arrangement is also consistent with ninefold symmetry, suggesting that two distinct SAS-6 oligomerization architectures can direct the same output symmetry. Sequence analysis suggests that SAS-6 spirals are restricted to specific nematodes. This oligomeric arrangement may provide a structural basis for the presence of a central tube instead of a cartwheel during centriole assembly in these species.

  11. Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms

    SciTech Connect

    Pletneva, Nadya V.; Pletnev, Sergei; Pakhomov, Alexey A.; Chertkova, Rita V.; Martynov, Vladimir I.; Muslinkina, Liya; Dauter, Zbigniew; Pletnev, Vladimir Z.

    2016-07-13

    The fluorescent protein fromDendronephthyasp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the Cα—N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double Cα=Cβbond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of native green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.

  12. Protein structure refinement based on paramagnetic NMR shifts: applications to wild-type and mutant forms of cytochrome c.

    PubMed Central

    Gochin, M.; Roder, H.

    1995-01-01

    A new approach to NMR solution structure refinement is introduced that uses paramagnetic effects on nuclear chemical shifts as constraints in energy minimization or molecular dynamics calculations. Chemical shift differences between oxidized and reduced forms of horse cytochrome c for more than 300 protons were used as constraints to refine the structure of the wild-type protein in solution and to define the structural changes induced by a Leu 94 to Val mutation. A single round of constrained minimization, using the crystal structure as the starting point, converged to a low-energy structure with an RMS deviation between calculated and observed pseudo-contact shifts of 0.045 ppm, 7.5-fold lower than the starting structure. At the same time, the procedure provided stereospecific assignments for more than 45 pairs of methylene protons and methyl groups. Structural changes caused by the mutation were determined to a precision of better than 0.3 A. Structure determination based on dipolar paramagnetic (pseudocontact) shifts is applicable to molecules containing anisotropic paramagnetic centers with short electronic relaxation times, including numerous naturally occurring metalloproteins, as well as proteins or nucleic acids to which a paramagnetic metal ion or ligand may be attached. The long range of paramagnetic shift effects (up to 20 A from the iron in the case of cytochrome c) provides global structural constraints, which, in conjunction with conventional NMR distance and dihedral angle constraints, will enhance the precision of NMR solution structure determination. PMID:7757018

  13. The DNA mismatch repair protein MutS forms a one-dimensional Tonks gas on DNA

    NASA Astrophysics Data System (ADS)

    Bundschuh, Ralf; Klajner, Piotr; Hanne, Jeungphill; Britton, Brooke M.; Liu, Jianquan; Park, Jonghyun; Lee, Jong-Bong; Fishel, Richard

    2014-03-01

    MutS is a protein involved in DNA mismatch repair. It recognizes the mismatch, forms a sliding clamp around the DNA, and displaces other proteins bound to the DNA prior to the actual repair process. Here, we present a quantitative model of an ensemble of MutS molecules on a short strand of DNA with one mismatch. We model the ensemble as a Tonks gas of passively diffusing one-dimensional particles of finite extension and include clamp formation at the mismatch and random detachment. The distributions of MutS number bound to the DNA for different mismatch positions and different MutS concentrations in solution fit very well with distributions determined by single molecule experiments, thereby establishing the Tonks gas as an excellent model of MutS action on DNA. This material is based upon work supported by the National Science Foundation under Grant No. 01105458 (RB), the National Institutes of Health under Grant No. CA67007 (RF), and the National Research Foundation of Korea under Grant No. 2011-0013901 (JBL).

  14. Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure.

    PubMed

    Wan, William; Stöhr, Jan; Kendall, Amy; Stubbs, Gerald

    2015-01-01

    Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled the complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.

  15. Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin.

    PubMed

    Chaudhry, Faisal; Breitsprecher, Dennis; Little, Kristin; Sharov, Grigory; Sokolova, Olga; Goode, Bruce L

    2013-01-01

    Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert.

  16. Crystal Structure of a Novel Dimeric Form of NS5A Domain I Protein from Hepatitis C Virus

    SciTech Connect

    Love, Robert A.; Brodsky, Oleg; Hickey, Michael J.; Wells, Peter A.; Cronin, Ciarán N.; Pfizer

    2009-07-10

    A new protein expression vector design utilizing an N-terminal six-histidine tag and tobacco etch virus protease cleavage site upstream of the hepatitis C virus NS5A sequence has resulted in a more straightforward purification method and improved yields of purified NS5A domain I protein. High-resolution diffracting crystals of NS5A domain I (amino acids 33 to 202) [NS5A(33-202)] were obtained by using detergent additive crystallization screens, leading to the structure of a homodimer which is organized differently from that published previously (T. L. Tellinghuisen, J. Marcotrigiano, and C. M. Rice, Nature 435:374-379, 2005) yet is consistent with a membrane association model for NS5A. The monomer-monomer interface of NS5A(33-202) features an extensive buried surface area involving the most-highly conserved face of each monomer. The two alternate structural forms of domain I now available may be indicative of the multiple roles emerging for NS5A in viral RNA replication and viral particle assembly.

  17. D6 PROTEIN KINASE activates auxin transport-dependent growth and PIN-FORMED phosphorylation at the plasma membrane.

    PubMed

    Barbosa, Inês C R; Zourelidou, Melina; Willige, Björn C; Weller, Benjamin; Schwechheimer, Claus

    2014-06-23

    The directed cell-to-cell transport of the phytohormone auxin by efflux and influx transporters is essential for proper plant growth and development. Like auxin efflux facilitators of the PIN-FORMED (PIN) family, D6 PROTEIN KINASE (D6PK) from Arabidopsis thaliana localizes to the basal plasma membrane of many cells, and evidence exists that D6PK may directly phosphorylate PINs. We find that D6PK is a membrane-bound protein that is associated with either the basal domain of the plasma membrane or endomembranes. Inhibition of the trafficking regulator GNOM leads to a rapid internalization of D6PK to endomembranes. Interestingly, the dissociation of D6PK from the plasma membrane is also promoted by auxin. Surprisingly, we find that auxin transport-dependent tropic responses are critically and reversibly controlled by D6PK and D6PK-dependent PIN phosphorylation at the plasma membrane. We conclude that D6PK abundance at the plasma membrane and likely D6PK-dependent PIN phosphorylation are prerequisites for PIN-mediated auxin transport.

  18. Trigger factor: a soluble protein that folds pro-OmpA into a membrane-assembly-competent form

    SciTech Connect

    Crooke, E.; Wickner, W.

    1987-08-01

    Pro-OmpA that is synthesized in vitro can assemble into bacterial inner membrane vesicles in the presence of ATP and NADH. The authors have purified pro-OmpA to determine which additional soluble proteins are necessary for its membrane assembly. (/sup 35/S)Pro-OmpA was bound to Sepharose-linked antibody to OmpA, then eluted with 8 M urea and chromatographed on an anion-exchange resin in 8 M urea. This pro-OmpA is purified 2000-fold and is radiochemically pure. After dialysis, it is soluble but incompetent for membrane assembly. Addition of an Escherichia coli cytoplasmic fraction (S100) to the assembly reaction does not allow translocation. However, when S100 is added to pro-OmpA prior to dialysis, full assembly competence is restored, suggesting that a soluble factor, termed trigger factor, triggers the folding of pro-OmpA into an assembly-competent form as the urea is removed. They noted that, prior to the last purification step, the immunoaffinity-purified pro-OmpA was partially competent for membrane assembly without addition of trigger factor. To test whether trigger factor had bound to the antibody column by means of its association with pro-OmpA, the crude pro-OmpA was acid-denatured prior to immunoadsorption. In this experiment, the trigger factor did not bind to the anti-OmpA column, and S100 was required for renaturation of this (/sup 35/S)pro-OmpA. As suggested by this experiment, the crude (/sup 35/S)pro-OmpA was in complex with other proteins. Sedimentation velocity studies showed that the trigger factor has an apparent molecular weight of approx. = 60,000. They propose that it is required for translocation-competent folding of pro-OmpA and other precursor proteins.

  19. Production, characterization, and immunogenicity of a secreted form of Plasmodium falciparum merozoite surface protein 4 produced in Bacillus subtilis.

    PubMed

    Chittibabu, G; Ma, Charles; Netter, Hans J; Noronha, Santosh B; Coppel, Ross L

    2014-04-01

    Plasmodium falciparum is the causative agent of the most serious form of malaria. Although a combination of control measures has significantly limited malaria morbidity and mortality in the last few years, it is generally agreed that sustained control or even eradication will require additional tools including an effective malaria vaccine. Merozoite surface protein 4, MSP4, which is present during the asexual stage of P. falciparum, is a recognized target that would be useful in a subunit vaccine against blood stages of malaria. Falciparum malaria is most prevalent in developing countries, and this in turn leads to a requirement for safe, low-cost vaccines. We have attempted to utilize the nonpathogenic, gram-positive organism Bacillus subtilis to produce PfMSP4. PfMSP4 was secreted into the culture medium at a yield of 4.5 mg/L. Characterization studies including SDS-PAGE, mass spectrometry, and N-terminal sequencing indicated that the B. subtilis expression system secreted a full length PfMSP4 protein compared to a truncated version in Escherichia coli. Equivalent amounts of purified B. subtilis and E. coli-derived PfMSP4 were used for immunization studies, resulting in statistically significant higher mean titer values for the B. subtilis-derived immunogen. The mouse antibodies raised against B. subtilis produced PfMSP4 that were reactive to parasite proteins as evidenced by immunoblotting on parasite lysate and indirect immunofluorescence assays of fixed parasites. The B. subtilis expression system, in contrast to E. coli, expresses higher amounts of full length PfMSP4 products, decreased levels of aggregates, and allows the development of simplified downstream processing procedures.

  20. A nonstructural protein of feline panleukopenia virus: expression in Escherichia coli and detection of multiple forms in infected cells.

    PubMed Central

    Carlson, J O; Lynde-Maas, M K; Shen, Z D

    1987-01-01

    Sequences coding for the nonstructural protein NS1 of the autonomous parvovirus feline panleukopenia virus were expressed in Escherichia coli as fusion proteins. The fusion proteins were specifically bound by antisera from canine parvovirus-infected dogs. Antisera against one of the fusion proteins bound to several proteins found only in feline panleukopenia virus-infected feline cells. Images PMID:3027392

  1. Increased Understanding of the Biochemistry and Biosynthesis of MUC2 and Other Gel-Forming Mucins Through the Recombinant Expression of Their Protein Domains

    PubMed Central

    Ambort, Daniel; Thomsson, Elisabeth; Johansson, Malin E. V.; Hansson, Gunnar C.

    2016-01-01

    The gel-forming mucins are large and heavily O-glycosylated proteins which build up mucus gels. The recombinant production of full-length gel-forming mucins has not been possible to date. In order to study mucin biosynthesis and biochemistry, we and others have taken the alternative approach of constructing different recombinant proteins consisting of one or several domains of these large proteins and expressing them separately in different cell lines. Using this approach, we have determined that MUC2, the intestinal gel-forming mucin, dimerizes via its C-terminal cysteine-knot domain and also trimerizes via one of the N-terminal von Willebrand D domains. Both of these interactions are disulfide bond mediated. Via this assembly, a molecular network is built by which the mucus gel is formed. Here we discuss not only the functional understanding obtained from studies of the recombinant proteins, but also highlight the difficulties encountered when these proteins were produced recombinantly. We often found an accumulation of the proteins in the ER and consequently no secretion. This was especially apparent when the cysteine-rich domains of the N- and C-terminal parts of the mucins were expressed. Other proteins that we constructed were either not secreted or not expressed at all. Despite these problems, the knowledge of mucin biosynthesis and assembly has advanced considerably through the studies of these recombinant proteins. PMID:23359125

  2. The Generation of Turnip Crinkle Virus-Like Particles in Plants by the Transient Expression of Wild-Type and Modified Forms of Its Coat Protein

    PubMed Central

    Saunders, Keith; Lomonossoff, George P.

    2015-01-01

    Turnip crinkle virus (TCV), a member of the genus carmovirus of the Tombusviridae family, has a genome consisting of a single positive-sense RNA molecule that is encapsidated in an icosahedral particle composed of 180 copies of a single type of coat protein. We have employed the CPMV-HT transient expression system to investigate the formation of TCV-like particles following the expression of the wild-type coat protein or modified forms of it that contain either deletions and/or additions. Transient expression of the coat protein in plants results in the formation of capsid structures that morphologically resemble TCV virions (T = 3 structure) but encapsidate heterogeneous cellular RNAs, rather than the specific TCV coat protein messenger RNA. Expression of an amino-terminal deleted form of the coat protein resulted in the formation of smaller T = 1 structures that are free of RNA. The possibility of utilizing TCV as a carrier for the presentation of foreign proteins on the particle surface was also explored by fusing the sequence of GFP to the C-terminus of the coat protein. The expression of coat protein-GFP hybrids permitted the formation of VLPs but the yield of particles is diminished compared to the yield obtained with unmodified coat protein. Our results confirm the importance of the N-terminus of the coat protein for the encapsidation of RNA and show that the coat protein's exterior P domain plays a key role in particle formation. PMID:26734041

  3. Killing of Microbes and Cancer by the Immune System with Three Mammalian Pore-Forming Killer Proteins

    PubMed Central

    Podack, Eckhard R.; Munson, George P.

    2016-01-01

    Immunology is the science of biological warfare between the defenses of our immune systems and offensive pathogenic microbes and cancers. Over the course of his scientific career, Eckhard R. Podack made several seminal discoveries that elucidated key aspects of this warfare at a molecular level. When Eckhard joined the complement laboratory of Müller-Eberhard in 1974, he was fascinated by two questions: (1) what is the molecular mechanism by which complement kills invasive bacteria? and (2) which one of the complement components is the killer molecule? Eckhard’s quest to answer these questions would lead to the discovery C9 and later, two additional pore-forming killer molecules of the immune system. Here is a brief account of how he discovered poly-C9, the pore-forming protein of complement in blood and interstitial fluids: Perforin-1, expressed by natural killer cells and cytotoxic T lymphocytes; and Perforin-2 (MPEG1), expressed by all cell types examined to date. All the three killing systems are crucial for our survival and health. PMID:27857713

  4. Design of whey protein nanostructures for incorporation and release of nutraceutical compounds in food.

    PubMed

    Ramos, Oscar L; Pereira, Ricardo N; Martins, Artur; Rodrigues, Rui; Fuciños, Clara; Teixeira, José A; Pastrana, Lorenzo; Malcata, F Xavier; Vicente, António A

    2017-05-03

    Whey proteins are widely used as nutritional and functional ingredients in formulated foods because they are relatively inexpensive, generally recognized as safe (GRAS) ingredient, and possess important biological, physical, and chemical functionalities. Denaturation and aggregation behavior of these proteins is of particular relevance toward manufacture of novel nanostructures with a number of potential uses. When these processes are properly engineered and controlled, whey proteins may be formed into nanohydrogels, nanofibrils, or nanotubes and be used as carrier of bioactive compounds. This review intends to discuss the latest understandings of nanoscale phenomena of whey protein denaturation and aggregation that may contribute for the design of protein nanostructures. Whey protein aggregation and gelation pathways under different processing and environmental conditions such as microwave heating, high voltage, and moderate electrical fields, high pressure, temperature, pH, and ionic strength were critically assessed. Moreover, several potential applications of nanohydrogels, nanofibrils, and nanotubes for controlled release of nutraceutical compounds (e.g. probiotics, vitamins, antioxidants, and peptides) were also included. Controlling the size of protein networks at nanoscale through application of different processing and environmental conditions can open perspectives for development of nanostructures with new or improved functionalities for incorporation and release of nutraceuticals in food matrices.

  5. Super-resolution Stimulated Emission Depletion-Fluorescence Correlation Spectroscopy Reveals Nanoscale Membrane Reorganization Induced by Pore-Forming Proteins.

    PubMed

    Sarangi, Nirod Kumar; P, Ilanila I; Ayappa, K G; Visweswariah, Sandhya S; Basu, Jaydeep Kumar

    2016-09-20

    Membrane-protein interactions play a central role in membrane mediated cellular processes ranging from signaling, budding, and fusion, to transport across the cell membrane. Of particular significance is the process of efficient protein olgomerization and transmembrane pore formation on the membrane surface; the primary virulent pathway for the action of antimicrobial peptides and pore forming toxins (PFTs). The suggested nanoscopic length scales and dynamic nature of such membrane lipid-protein interactions makes their detection extremely challenging. Using a combination of super-resolution stimulated emission depletion nanoscopy with fluorescence correlation spectroscopy (STED-FCS) we unravel the emergence of nanoscale lateral heterogeneity in supported bilayer membranes made up of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol upon interaction with the PFT, listeriolysin O (LLO). A distinct length scale-dependent dynamical crossover (<200 nm) from a Brownian diffusive regime is observed at 33 and 50% cholesterol compositions, indicating the partitioning of lipids into domains with variable cholesterol content. At 25% cholesterol content, this dyamical crossover is observed only in bilayers incubated with LLO providing evidence for the existence of sub ∼100 nm dynamical lipid nanodomains bound to LLO pore assemblies. By introducing asymmetry in cholesterol composition across the bilayer leaflets we infer that this domain formation is driven largely due to active cholesterol sequestration and transient trapping of lipids to the membrane bound motifs present in the toxins, en route to LLO oligomerization and subsequent pore formation. Bilayers prepared with labeled lipids present in either the proximal or distal leaflet allow us to track the dynamical perturbation in a leaflet-dependent manner upon LLO incubation. From the differences in the extent and intensity of the dynamical crossover as observed with STED-FCS, these experiments reveal that

  6. Water in the Cratonic Mantle: Insights from FTIR Data on Lac de Gras Xenoliths (Slave Craton, Canada)

    NASA Astrophysics Data System (ADS)

    Peslier, A. H.; Brandon, A. D.; Schaffer, L. A.; O'Reilly, S. Y.; Griffin, W. L.; Morris, R. V.; Graff, T. G.; Agresti, D. G.

    2014-12-01

    The mantle lithosphere beneath the cratonic part of continents is the deepest (> 200 km) and oldest (>2-3 Ga) on Earth, remaining a conundrum as to how these cratonic roots could have resisted delamination by asthenospheric convection over time. Water, or trace H incorporated in mineral defects, could be a key player in the evolution of continental lithosphere because it influences melting and rheology of the mantle [e.g., 1]. Mantle xenoliths from the Lac de Gras kimberlite in the Slave craton [2] were analyzed by FTIR. The cratonic mantle beneath Lac de Gras is stratified with shallow (<145 km) oxidized ultradepleted peridotites and pyroxenites with evidence for carbonatitic metasomatism, underlain by reduced and less depleted peridotites metasomatized by kimberlite melts [3,4]. Peridotites analyzed so far have H2O contents in ppm weight of 7-100 in their olivines, 58 to 255 in their orthopyroxenes (opx), 11 to 84 in their garnet, and 139 in one clinopyroxene. A pyroxenite contains 58 ppm H2O in opx and 5 ppm H2O in its olivine and garnet. Olivine and garnet from the deep peridotites have a range of water contents extending to higher values than those from the shallow ones. The FTIR spectra of olivines from the shallow samples have more prominent Group II OH bands compared to the olivines from the deep samples, consistent with a more oxidized mantle environment [5]. The range of olivine water content is similar to that observed in Kaapvaal craton peridotites at the same depths (129-184 km [1]) but does not extend to as high values as those from Udachnaya (Siberian craton [6]). The Slave, Kaapvaal and Siberian cratons will be compared in terms of water content distribution, controls and role in cratonic root longevity. [1] Peslier et al. 2010 Nature 467, 78-81. [2] Aulbach et al. 2007 CMP 154, 409-427. [3] Creighton et al. 2009 CMP 157, 491-504. [4] Aulbach et al. 2013 CG 352, 153-169. [5] Bai & Kohlstedt 1993 PCM 19, 460-471. [6] Doucet et al. 2014 GCA 137, 159-187.

  7. Geochemistry of hypabyssal kimberlites from Lac de Gras, Canada: Comparisons to a global database and applications to the parent magma problem

    NASA Astrophysics Data System (ADS)

    Kjarsgaard, B. A.; Pearson, D. G.; Tappe, S.; Nowell, G. M.; Dowall, D. P.

    2009-11-01

    We present 104 whole-rock geochemical analyses of hypabyssal kimberlite from the Lac de Gras field. Screens using Yb versus Al 2O 3 and ln Si/Al versus ln Mg/Yb effectively discriminate crustally contaminated samples. The remaining "non-contaminated" kimberlites samples have variable (5 to 50%) entrainment of cratonic peridotite. It is problematic to effectively screen for small amounts (< 5%) of digested crust in samples with higher (> 20%) contents of peridotite contamination. We utilize the Lac de Gras data suite to calculate, by two different methods, parent magma compositions and identify two (and potentially three) geochemically distinct parent magma types. The Lac de Gras parent magma compositions are compared to those calculated from other localities in Canada, Greenland, South Africa and Russia. Together, these calculated parent magmas define a range, albeit limited, of viable, yet distinct, kimberlite parent magma compositions. Geochemically, kimberlite parent magmas have high volatile contents (H 2O and CO 2), high MgO, and low SiO 2, Al 2O 3 and alkalis, with K > Na and Na + K/Al < 1. It is difficult to reconcile differences between various calculated kimberlite parent magma compositions from different cratonic areas as merely due to the effects of craton specific lithospheric mantle contamination, indicating the intra- and inter-cratonic variation of parent magma compositions reflect differing source region characteristics and/or partial melting regimes.

  8. Procoagulant platelets form an α-granule protein-covered "cap" on their surface that promotes their attachment to aggregates.

    PubMed

    Abaeva, Anastasia A; Canault, Matthias; Kotova, Yana N; Obydennyy, Sergey I; Yakimenko, Alena O; Podoplelova, Nadezhda A; Kolyadko, Vladimir N; Chambost, Herve; Mazurov, Aleksei V; Ataullakhanov, Fazoil I; Nurden, Alan T; Alessi, Marie-Christine; Panteleev, Mikhail A

    2013-10-11

    Strongly activated "coated" platelets are characterized by increased phosphatidylserine (PS) surface expression, α-granule protein retention, and lack of active integrin αIIbβ3. To study how they are incorporated into thrombi despite a lack of free activated integrin, we investigated the structure, function, and formation of the α-granule protein "coat." Confocal microscopy revealed that fibrin(ogen) and thrombospondin colocalized as "cap," a single patch on the PS-positive platelet surface. In aggregates, the cap was located at the point of attachment of the PS-positive platelets. Without fibrin(ogen) retention, their ability to be incorporated in aggregates was drastically reduced. The surface fibrin(ogen) was strongly decreased in the presence of a fibrin polymerization inhibitor GPRP and also in platelets from a patient with dysfibrinogenemia and a fibrinogen polymerization defect. In contrast, a fibrinogen-clotting protease ancistron increased the amount of fibrin(ogen) and thrombospondin on the surface of the PS-positive platelets stimulated with collagen-related peptide. Transglutaminases are also involved in fibrin(ogen) retention. However, platelets from patients with factor XIII deficiency had normal retention, and a pan-transglutaminase inhibitor T101 had only a modest inhibitory effect. Fibrin(ogen) retention was normal in Bernard-Soulier syndrome and kindlin-3 deficiency, but not in Glanzmann thrombasthenia lacking the platelet pool of fibrinogen and αIIbβ3. These data show that the fibrin(ogen)-covered cap, predominantly formed as a result of fibrin polymerization, is a critical mechanism that allows coated (or rather "capped") platelets to become incorporated into thrombi despite their lack of active integrins.

  9. Purification, pore-forming ability, and antigenic relatedness of the major outer membrane protein of Shigella dysenteriae type 1.

    PubMed Central

    Roy, S; Das, A B; Ghosh, A N; Biswas, T

    1994-01-01

    The major outer membrane protein (MOMP), the most abundant outer membrane protein, was purified to homogeneity from Shigella dysenteriae type 1. The purification method involved selective extraction of MOMP with sodium dodecyl sulfate in the presence of 0.4 M sodium chloride followed by size exclusion chromatography with Sephacryl S-200 HR. MOMP was found to form hydrophilic diffusion pores by incorporation into artificial liposome vesicles composed of egg yolk phosphatidylcholine and dicetylphosphate, indicating that MOMP of S. dysenteriae type 1 exhibited significant porin activity. However, the liposomes containing heat-denatured MOMP were barely active. The molecular weight of MOMP found by size exclusion chromatography was 130,000, and in sodium dodecyl sulfate-10% polyacrylamide gel it moved as an oligomer of 78,000 molecular weight. Upon boiling, fully dissociated monomers of 38,000 molecular weight were seen for S. dysenteriae type 1. However, among the four Shigella spp., the monomeric MOMP generated upon boiling ranged from 38,000 to 35,000 in molecular weight. Antibody raised in BALB/c mice immunized with MOMP of S. dysenteriae type 1 reacted strongly with purified MOMP of S. dysenteriae type 1 in an enzyme-linked immunosorbent assay (ELISA). The antibody reacted with whole-cell preparations of S. dysenteriae type 1 in an ELISA, suggesting that MOMP possessed surface components. Moreover, MOMP could be visualized on the bacterial surface by immunoelectron microscopy with anti-MOMP antibody. S. dysenteriae type 1 MOMP-specific immunoglobulin eluted from MOMP bound to a nitrocellulose membrane was found to cross-react with MOMP preparations of S. flexneri, S. boydii, and S. sonnei, indicating that MOMPs were antigenically related among Shigella species. The strong immunogenicity, surface exposure, and antigenic relatedness make MOMP of Shigella species an immunologically significant macromolecule for study. Images PMID:7927692

  10. Existence of two forms of rat liver arginyl-tRNA synthetase suggests channeling of aminoacyl-tRNA for protein synthesis.

    PubMed Central

    Sivaram, P; Deutscher, M P

    1990-01-01

    Arginyl-tRNA synthetase (arginine-tRNA ligase, EC 6.1.1.19) is found in extracts of mammalian cells both as a free protein (Mr = 60,000) and as a component (Mr approximately 72,000) of the high molecular weight aminoacyl-tRNA synthetase complex (Mr greater than 10(6). Several pieces of evidence indicate that the low molecular weight free form is not a proteolytic degradation product of the complex-bound enzyme but that it preexists in vivo: (i) the endogenous free form differs in size from the active proteolytic fragment generated in vitro, (ii) conditions expected to increase or decrease the amount of proteolysis do not alter the ratio of the two forms of the enzyme, and (iii) the free form contains an NH2-terminal methionine residue. A model is presented that provides a rationale for the existence of two forms of arginyl-tRNA synthetase in cells. In this model the complexed enzyme supplies arginyl-tRNA for protein synthesis, whereas the free enzyme provides arginyl-tRNA for the NH2-terminal arginine modification of proteins by arginyl-tRNA:protein arginyltransferase. This latter process targets certain proteins for removal by the ubiquitin-dependent protein degradation pathway. The necessity for an additional pool of arginyl-tRNA for the modification reaction leads to the conclusion that the arginyl-tRNA destined for protein synthesis (and/or protein modification) is channeled and unavailable for other processes. Other evidence supporting channeling in protein synthesis is discussed. Images PMID:2187187

  11. Cytotoxic activities of Leptospira interrogans hemolysin SphH as a pore-forming protein on mammalian cells.

    PubMed

    Lee, Seoung Hoon; Kim, Sangduk; Park, Seung Chul; Kim, Min Ja

    2002-01-01

    Leptospirosis is a spirochetal zoonosis that causes an acute febrile systemic illness in humans. Leptospira sp. hemolysins have been shown to be virulence factors for the pathogenesis of leptospirosis. Previously, we cloned a hemolysin SphH of Leptospira interrogans serovar lai, a homologue of L. borgpetersenii sphingomyelinase (SphA), from a genomic library (S. H. Lee, K. A. Kim, Y. K. Kim, I. W. Seong, M. J. Kim, and Y. J. Lee, Gene 254:19-28, 2000). Escherichia coli lysate harboring the sphH showed high hemolytic activities on sheep erythrocytes. However, it neither showed sphingomyelinase nor phospholipase activities, in contrast to SphA which was known to have sphingomyelinase activity. Interestingly, the SphH-mediated hemolysis on erythrocytes was osmotically protected by PEG 5000, suggesting that the SphH might have caused pore formation on the erythrocyte membrane. In the present study, we have prepared the Leptospira hemolysin SphH and investigated its hemolytic and cytotoxic activities on mammalian cells. SphH was shown to be a pore-forming protein on several mammalian cells: When treated with the SphH, the sheep erythrocyte membranes formed pores, which were morphologically confirmed by transmission electron microscopy. Furthermore, the SphH-mediated cytotoxicities on mammalian cells were demonstrated by the release of LDH and by inverted microscopic examinations. Finally, the immune serum against the full-length hemolysin could effectively neutralize the SphH-mediated hemolytic and cytotoxic activities. In conclusion, these results suggest that the virulence of Leptospira SphH was due to the pore formation on mammalian cell membranes.

  12. The Petunia GRAS Transcription Factor ATA/RAM1 Regulates Symbiotic Gene Expression and Fungal Morphogenesis in Arbuscular Mycorrhiza1

    PubMed Central

    Rich, Mélanie K.

    2015-01-01

    Arbuscular mycorrhiza (AM) is a mutual symbiosis that involves a complex symbiotic interface over which nutrients are exchanged between the plant host and the AM fungus. Dozens of genes in the host are required for the establishment and functioning of the interaction, among them nutrient transporters that mediate the uptake of mineral nutrients delivered by the fungal arbuscules. We have isolated in a genetic mutant screen a petunia (Petunia hybrida) GIBBERELLIC ACID INSENSITIVE, REPRESSOR of GIBBERELLIC ACID INSENSITIVE, and SCARECROW (GRAS)-type transcription factor, ATYPICAL ARBUSCULE (ATA), that acts as the central regulator of AM-related genes and is required for the morphogenesis of arbuscules. Forced mycorrhizal inoculations from neighboring wild-type plants revealed an additional role of ATA in restricting mycorrhizal colonization of the root meristem. The lack of ATA, which represents the ortholog of REQUIRED FOR ARBUSCULAR MYCORRHIZA1 in Medicago truncatula, renders the interaction completely ineffective, hence demonstrating the central role of AM-related genes for arbuscule development and function. PMID:25971550

  13. Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions.

    PubMed

    Vanarsdall, Adam L; Howard, Paul W; Wisner, Todd W; Johnson, David C

    2016-04-01

    Human cytomegalovirus (HCMV) is a ubiquitous virus that is a major pathogen in newborns and immunocompromised or immunosuppressed patients. HCMV infects a wide variety of cell types using distinct entry pathways that involve different forms of the gH/gL glycoprotein: gH/gL/gO and gH/gL/UL128-131 as well as the viral fusion glycoprotein, gB. However, the minimal or core fusion machinery (sufficient for cell-cell fusion) is just gH/gL and gB. Here, we demonstrate that HCMV gB and gH/gL form a stable complex early after their synthesis and in the absence of other viral proteins. gH/gL can interact with gB mutants that are unable to mediate cell-cell fusion. gB-gH/gL complexes included as much as 16-50% of the total gH/gL in HCMV virus particles. In contrast, only small amounts of gH/gL/gO and gH/gL/UL128-131 complexes were found associated with gB. All herpesviruses express gB and gH/gL molecules and most models describing herpesvirus entry suggest that gH/gL interacts with gB to mediate membrane fusion, although there is no direct evidence for this. For herpes simplex virus (HSV-1) it has been suggested that after receptor binding gH/gL binds to gB either just before, or coincident with membrane fusion. Therefore, our results have major implications for these models, demonstrating that HCMV gB and gH/gL forms stable gB-gH/gL complexes that are incorporated virions without receptor binding or membrane fusion. Moreover, our data is the best support to date for the proposal that gH/gL interacts with gB.

  14. Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions

    PubMed Central

    Vanarsdall, Adam L.; Howard, Paul W.; Wisner, Todd W.; Johnson, David C.

    2016-01-01

    Human cytomegalovirus (HCMV) is a ubiquitous virus that is a major pathogen in newborns and immunocompromised or immunosuppressed patients. HCMV infects a wide variety of cell types using distinct entry pathways that involve different forms of the gH/gL glycoprotein: gH/gL/gO and gH/gL/UL128-131 as well as the viral fusion glycoprotein, gB. However, the minimal or core fusion machinery (sufficient for cell-cell fusion) is just gH/gL and gB. Here, we demonstrate that HCMV gB and gH/gL form a stable complex early after their synthesis and in the absence of other viral proteins. gH/gL can interact with gB mutants that are unable to mediate cell-cell fusion. gB-gH/gL complexes included as much as 16–50% of the total gH/gL in HCMV virus particles. In contrast, only small amounts of gH/gL/gO and gH/gL/UL128-131 complexes were found associated with gB. All herpesviruses express gB and gH/gL molecules and most models describing herpesvirus entry suggest that gH/gL interacts with gB to mediate membrane fusion, although there is no direct evidence for this. For herpes simplex virus (HSV-1) it has been suggested that after receptor binding gH/gL binds to gB either just before, or coincident with membrane fusion. Therefore, our results have major implications for these models, demonstrating that HCMV gB and gH/gL forms stable gB-gH/gL complexes that are incorporated virions without receptor binding or membrane fusion. Moreover, our data is the best support to date for the proposal that gH/gL interacts with gB. PMID:27082872

  15. Porphyrin-loaded liposomes and graphene oxide used for the membrane pore-forming protein assay and inhibitor screening.

    PubMed

    Liu, Zhongde; Long, Tengfei; Wu, Shuang; Li, Chong

    2015-08-21

    The interaction of planar aromatic molecules with the graphene oxide (GO) sheets is often marked by the fluorescence quenching of the former. Here, the α,β,γ,δ-tetrakis[4-(trimethylammoniumyl)phenyl]porphyrin (TAPP) molecules and the GO, corresponding to the energy donor and the acceptor respectively, are initially separated by encapsulating the TAPP molecules within the liposomes, to obstruct the formation of the self-assembled energy transfer-based quenching system. Upon disruption of the liposome membranes by the PLA2 or the α-toxin, the encapsulated TAPP molecules are released and subsequently result in significant fluorescence changes. Thus, a platform based on the fluorescence signal for monitoring the activity of the membrane pore-forming protein with advantages of high sensitivity and commonality was established. Using this strategy, we can detect the PLA2 and the α-toxin concentrations as low as 200 pM and 9.0 nM, respectively. Furthermore, by taking chlorpromazine and baicalin as the examples, we use the assay to evaluate the prohibition effects on the PLA2 and the α-toxin, and the IC50 values of chlorpromazine toward the PLA2 (9.6 nM) and that of baicalin toward the α-toxin (289.2 nM) were found to be 12.0 ± 0.62 μM and 26.9 ± 2.6 μM, respectively.

  16. Thermodynamics of nucleotide and inhibitor binding to wild-type and ispinesib-resistant forms of human kinesin spindle protein.

    PubMed

    Sheth, Payal R; Basso, Andrea; Duca, José S; Lesburg, Charles A; Ogas, Polina; Gray, Kimberly; Nale, Lissette; Mannarino, Anthony F; Prongay, Andrew J; Le, Hung V

    2009-11-24

    Current antimitotic cancer chemotherapy based on vinca alkaloids and taxanes target tubulin, a protein required not only for mitotic spindle formation but also for the overall structural integrity of terminally differentiated cells. Among many innovations targeting specific mitotic events, inhibition of motor enzymes including KSP (or Eg5) has been validated as a highly productive approach. Many reported KSP inhibitors bind to an induced allosteric site near the site of ATP hydrolysis, and some have been tested in clinical trials with varying degrees of success. This allosteric site was defined in detail by X-ray crystallography of inhibitor complexes, yet complementary information on binding thermodynamics is still lacking. Using two model ATP-uncompetitive inhibitors, monastrol and ispinesib, we report here the results of thermal denaturation and isothermal titration calorimetric studies. These binding studies were conducted with the wild-type KSP motor domain as well as two ispinesib mutants (D130V and A133D) identified to confer resistance to ispinesib treatment. The thermodynamic parameters obtained were placed in the context of the available structural information and corresponding models of the two ispinesib-resistant mutants. The resulting overall information formed a strong basis for future structure-based design of inhibitors of KSP and related motor enzymes.

  17. A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin.

    PubMed Central

    Melia, T J; Cowan, C W; Angleson, J K; Wensel, T G

    1997-01-01

    Activation of the photoreceptor G protein transducin (Gt) by opsin, the ligand-free form of rhodopsin, was measured using rod outer segment membranes with densities of opsin and Gt similar to those found in rod cells. When GTPgammaS was used as the activating nucleotide, opsin catalyzed transducin activation with an exponential time course with a rate constant k(act) on the order of 2 x 10(-3)s(-1). Comparison under these conditions to activation by flash-generated metarhodopsin II (MII) revealed that opsin- and R*-catalyzed activation showed similar kinetics when MII was present at a surface density approximately 10(-6) lower than that of opsin. Thus, in contrast to some previous reports, we find that the catalytic potency of opsin is only approximately 10(-6) that of MII. In the presence of residual retinaldehyde-derived species present in membranes treated with hydroxylamine after bleaching, the apparent k(act) observed was much higher than that for opsin, suggesting a possible explanation for previous reports of more efficient activation by opsin. These results are important for considering the possible role of opsin in the diverse phenomena in which it has been suggested to play a key role, such as bleaching desensitization and retinal degeneration induced by continuous light or vitamin A deprivation. PMID:9414230

  18. Trimeric Structure of (+)-Pinoresinol-forming Dirigent Protein at 1.95 Å Resolution with Three Isolated Active Sites

    SciTech Connect

    Kim, Kye-Won; Smith, Clyde A.; Daily, Michael D.; Cort, John R.; Davin, Laurence B.; Lewis, Norman G.

    2014-11-19

    Control over phenoxy radical-radical coupling reactions in vivo in vascular plants was enigmatic until our discovery of dirigent proteins (DPs, from the Latin dirigere, to guide or align). The first three-dimensional structure of a DP ((+)-pinoresinol-forming DP, 1.95 Å resolution, rhombohedral space group H32)) is reported herein. It has a tightly packed trimeric structure with an eight-stranded β-barrel topology for each DP monomer. Each putative substrate binding and orientation coupling site is located on the trimer surface but too far apart for intermolecular coupling between sites. It is proposed that each site enables stereoselective coupling (using either two coniferyl alcohol radicals or a radical and a monolignol). Interestingly, there are six differentially conserved residues in DPs affording either the (+)- or (₋)-antipodes in the vicinity of the putative binding site and region known to control stereoselectivity. We find DPs are involved in lignan biosynthesis, whereas dirigent domains/sites have been implicated in lignin deposition.

  19. Trimeric Structure of (+)-Pinoresinol-forming Dirigent Protein at 1.95 Å Resolution with Three Isolated Active Sites

    DOE PAGES

    Kim, Kye-Won; Smith, Clyde A.; Daily, Michael D.; ...

    2014-11-19

    Control over phenoxy radical-radical coupling reactions in vivo in vascular plants was enigmatic until our discovery of dirigent proteins (DPs, from the Latin dirigere, to guide or align). The first three-dimensional structure of a DP ((+)-pinoresinol-forming DP, 1.95 Å resolution, rhombohedral space group H32)) is reported herein. It has a tightly packed trimeric structure with an eight-stranded β-barrel topology for each DP monomer. Each putative substrate binding and orientation coupling site is located on the trimer surface but too far apart for intermolecular coupling between sites. It is proposed that each site enables stereoselective coupling (using either two coniferyl alcoholmore » radicals or a radical and a monolignol). Interestingly, there are six differentially conserved residues in DPs affording either the (+)- or (₋)-antipodes in the vicinity of the putative binding site and region known to control stereoselectivity. We find DPs are involved in lignan biosynthesis, whereas dirigent domains/sites have been implicated in lignin deposition.« less

  20. Dimerization, oligomerization, and aggregation of human amyotrophic lateral sclerosis copper/zinc superoxide dismutase 1 protein mutant forms in live cells.

    PubMed

    Kim, Jiho; Lee, Honggun; Lee, Joo Hyun; Kwon, Do-yoon; Genovesio, Auguste; Fenistein, Denis; Ogier, Arnaud; Brondani, Vincent; Grailhe, Regis

    2014-05-23

    More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol. In vitro studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and in vivo studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins.

  1. The tubule-forming NSm protein from Tomato spotted wilt virus complements cell-to-cell and long-distance movement of Tobacco mosaic virus hybrids.

    PubMed

    Lewandowski, Dennis J; Adkins, Scott

    2005-11-10

    A Florida isolate of Tomato spotted wilt virus (TSWV) was able to complement cell-to-cell movement of a movement-defective Tobacco mosaic virus (TMV) vector expressing the jellyfish green fluorescent protein (GFP). To test for complementation of movement in the absence of other TSWV proteins, the open reading frame for the NSm protein was expressed from TMV constructs encoding only the TMV replicase proteins. NSm was expressed from either the coat protein or movement protein subgenomic promoter, creating virus hybrids that moved cell to cell in inoculated leaves of tobacco, providing the first functional demonstration that NSm is the TSWV movement protein. Furthermore, these CP-deficient hybrids moved into upper leaves of Nicotiana benthamiana, demonstrating that NSm can support long-distance movement of viral RNAs. Tubules, characteristic of the NSm protein, were also formed in tobacco protoplasts infected with the TMV-TSWV hybrids. The C-terminus of the NSm protein was shown to be required for movement. TMV-TSWV hybrids expressing NSm and GFP moved within inoculated leaves. Our combination of single-cell and intact plant experiments to examine multiple functions of a heterologous viral protein provides a generalized strategy with wider application to other viruses also lacking a reverse genetic system.

  2. ZipperDB: Predictions of Fibril-forming Segments within Proteins Identified by the 3D Profile Method (from the UCLA-DOE Institute for Genomics and Proteomics)

    DOE Data Explorer

    Goldschmidt, L.; Teng, P. K.; Riek, R.; Eisenberg, D.

    ZipperDB contains predictions of fibril-forming segments within proteins identified by the 3D Profile Method. The UCLA-DOE Institute for Genomics and Proteomics has analyzed over 20,000 putative protein sequences for segments with high fibrillation propensity that could form a "steric zipper"ùtwo self-complementary beta sheets, giving rise to the spine of an amyloid fibril. The approach is unique in that structural information is used to evaluate the likelihood that a particular sequence can form fibrils. [copied with edits from http://www.doe-mbi.ucla.edu/]. In addition to searching the database, academic and non-profit users may also submit their protein sequences to the database.

  3. Three RNA binding proteins form a complex to promote differentiation of germline stem cell lineage in Drosophila.

    PubMed

    Chen, Di; Wu, Chan; Zhao, Shaowei; Geng, Qing; Gao, Yu; Li, Xin; Zhang, Yang; Wang, Zhaohui

    2014-11-01

    In regenerative tissues, one of the strategies to protect stem cells from genetic aberrations, potentially caused by frequent cell division, is to transiently expand the stem cell daughters before further differentiation. However, failure to exit the transit amplification may lead to overgrowth, and the molecular mechanism governing this regulation remains vague. In a Drosophila mutagenesis screen for factors involved in the regulation of germline stem cell (GSC) lineage, we isolated a mutation in the gene CG32364, which encodes a putative RNA-binding protein (RBP) and is designated as tumorous testis (tut). In tut mutant, spermatogonia fail to differentiate and over-amplify, a phenotype similar to that in mei-P26 mutant. Mei-P26 is a TRIM-NHL tumor suppressor homolog required for the differentiation of GSC lineage. We found that Tut binds preferentially a long isoform of mei-P26 3'UTR, and is essential for the translational repression of mei-P26 reporter. Bam and Bgcn are both RBPs that have also been shown to repress mei-P26 expression. Our genetic analyses indicate that tut, bam, or bgcn is required to repress mei-P26 and to promote the differentiation of GSCs. Biochemically, we demonstrate that Tut, Bam, and Bgcn can form a physical complex in which Bam holds Tut on its N-terminus and Bgcn on its C-terminus. Our in vivo and in vitro evidence illustrate that Tut acts with Bam, Bgcn to accurately coordinate proliferation and differentiation in Drosophila germline stem cell lineage.

  4. Three RNA Binding Proteins Form a Complex to Promote Differentiation of Germline Stem Cell Lineage in Drosophila

    PubMed Central

    Zhao, Shaowei; Geng, Qing; Gao, Yu; Li, Xin; Zhang, Yang; Wang, Zhaohui

    2014-01-01

    In regenerative tissues, one of the strategies to protect stem cells from genetic aberrations, potentially caused by frequent cell division, is to transiently expand the stem cell daughters before further differentiation. However, failure to exit the transit amplification may lead to overgrowth, and the molecular mechanism governing this regulation remains vague. In a Drosophila mutagenesis screen for factors involved in the regulation of germline stem cell (GSC) lineage, we isolated a mutation in the gene CG32364, which encodes a putative RNA-binding protein (RBP) and is designated as tumorous testis (tut). In tut mutant, spermatogonia fail to differentiate and over-amplify, a phenotype similar to that in mei-P26 mutant. Mei-P26 is a TRIM-NHL tumor suppressor homolog required for the differentiation of GSC lineage. We found that Tut binds preferentially a long isoform of mei-P26 3′UTR, and is essential for the translational repression of mei-P26 reporter. Bam and Bgcn are both RBPs that have also been shown to repress mei-P26 expression. Our genetic analyses indicate that tut, bam, or bgcn is required to repress mei-P26 and to promote the differentiation of GSCs. Biochemically, we demonstrate that Tut, Bam, and Bgcn can form a physical complex in which Bam holds Tut on its N-terminus and Bgcn on its C-terminus. Our in vivo and in vitro evidence illustrate that Tut acts with Bam, Bgcn to accurately coordinate proliferation and differentiation in Drosophila germline stem cell lineage. PMID:25412508

  5. Expression, purification, crystallization and preliminary X-ray characterization of two crystal forms of stationary-phase survival E protein from Campylobacter jejuni

    SciTech Connect

    Gonçalves, A. M. D.; Rêgo, A. T.; Thomaz, M.; Enguita, F. J.; Carrondo, M. A.

    2008-03-01

    Survival E (SurE) protein from Campylobacter jejuni, a Gram-negative mesophile, has been overexpressed in Escherichia coli as a soluble protein, successfully purified and crystallized in two distinct crystal forms. Survival E (SurE) protein from Campylobacter jejuni, a Gram-negative mesophile, has been overexpressed in Escherichia coli as a soluble protein, successfully purified and crystallized in two distinct crystal forms. The first form belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with a tetramer in the asymmetric unit and unit-cell parameters a = 80.5, b = 119.0, c = 135.3 Å. The second form belongs to space group C2, with unit-cell parameters a = 121.4, b = 47.1, c = 97.8 Å, and contains a dimer in the asymmetric unit. Diffraction data have been collected from these crystal forms to 2.5 and 2.95 Å resolution, respectively.

  6. Coexistence of two forms of disease-associated prion protein in extracerebral tissues of cattle infected with H-type bovine spongiform encephalopathy.

    PubMed

    Okada, Hiroyuki; Miyazawa, Kohtaro; Masujin, Kentaro; Yokoyama, Takashi

    2016-08-01

    H-type bovine spongiform encephalopathy (H-BSE) is an atypical form of BSE in aged cattle. H-BSE is characterized by the presence of two proteinase K-resistant forms of disease-associated prion protein (PrP(Sc)), identified as PrP(Sc) #1 and PrP(Sc) #2, in the brain. To investigate the coexistence of different PrP(Sc) forms in the extracerebral tissues of cattle experimentally infected with H-BSE, immunohistochemical and molecular analyses were performed by using N-terminal-, core-region- and C-terminal-specific anti-prion protein antibodies. Our results demonstrated that two distinct forms of PrP(Sc) coexisted in the various extracerebral tissues.

  7. Helicobacter pylori CheZ(HP) and ChePep form a novel chemotaxis-regulatory complex distinct from the core chemotaxis signaling proteins and the flagellar motor.

    PubMed

    Lertsethtakarn, Paphavee; Howitt, Michael R; Castellon, Juan; Amieva, Manuel R; Ottemann, Karen M

    2015-09-01

    Chemotaxis is important for Helicobacter pylori to colonize the stomach. Like other bacteria, H. pylori uses chemoreceptors and conserved chemotaxis proteins to phosphorylate the flagellar rotational response regulator, CheY, and modulate the flagellar rotational direction. Phosphorylated CheY is returned to its non-phosphorylated state by phosphatases such as CheZ. In previously studied cases, chemotaxis phosphatases localize to the cellular poles by interactions with either the CheA chemotaxis kinase or flagellar motor proteins. We report here that the H. pylori CheZ, CheZ(HP), localizes to the poles independently of the flagellar motor, CheA, and all typical chemotaxis proteins. Instead, CheZ(HP) localization depends on the chemotaxis regulatory protein ChePep, and reciprocally, ChePep requires CheZ(HP) for its polar localization. We furthermore show that these proteins interact directly. Functional domain mapping of CheZ(HP) determined the polar localization motif lies within the central domain of the protein and that the protein has regions outside of the active site that participate in chemotaxis. Our results suggest that CheZ(HP) and ChePep form a distinct complex. These results therefore suggest the intriguing idea that some phosphatases localize independently of the other chemotaxis and motility proteins, possibly to confer unique regulation on these proteins' activities.

  8. Medicago truncatula ERN transcription factors: regulatory interplay with NSP1/NSP2 GRAS factors and expression dynamics throughout rhizobial infection.

    PubMed

    Cerri, Marion R; Frances, Lisa; Laloum, Tom; Auriac, Marie-Christine; Niebel, Andreas; Oldroyd, Giles E D; Barker, David G; Fournier, Joëlle; de Carvalho-Niebel, Fernanda

    2012-12-01

    Rhizobial nodulation factors (NFs) activate a specific signaling pathway in Medicago truncatula root hairs that involves the complex interplay of Nodulation Signaling Pathway1 (NSP1)/NSP2 GRAS and Ethylene Response Factor Required for Nodulation1 (ERN1) transcription factors (TFs) to achieve full ENOD11 transcription. ERN1 acts as a direct transcriptional regulator of ENOD11 through the activation of the NF-responsive "NF box." Here, we show that NSP1, when combined with NSP2, can act as a strong positive regulator of ERN1 and ENOD11 transcription. Although ERN1 and NSP1/NSP2 both activate ENOD11, two separate promoter regions are involved that regulate expression during consecutive symbiotic stages. Our findings indicate that ERN1 is required to activate NF-elicited ENOD11 expression exclusively during early preinfection, while NSP1/NSP2 mediates ENOD11 expression during subsequent rhizobial infection. The relative contributions of ERN1 and the closely related ERN2 to the rhizobial symbiosis were then evaluated by comparing their regulation and in vivo dynamics. ERN1 and ERN2 exhibit expression profiles compatible with roles during NF signaling and subsequent infection. However, differences in expression levels and spatiotemporal profiles suggest specialized functions for these two TFs, ERN1 being involved in stages preceding and accompanying infection thread progression while ERN2 is only involved in certain stages of infection. By cross complementation, we show that ERN2, when expressed under the control of the ERN1 promoter, can restore both NF-elicited ENOD11 expression and nodule formation in an ern1 mutant background. This indicates that ERN1 and ERN2 possess similar biological activities and that functional diversification of these closely related TFs relies primarily on changes in tissue-specific expression patterns.

  9. Site-specific mutations provide new insights into the origin of pH effects and alternative spectral forms in the photoactive yellow protein from Halorhodospira halophila.

    PubMed

    Meyer, T E; Devanathan, S; Woo, T; Getzoff, E D; Tollin, G; Cusanovich, M A

    2003-03-25

    Acid/base titrations of wild-type PYP and mutants, either in buffer or in the presence of chaotropes such as thiocyanate, establish the presence of four spectral forms including the following: a neutral form (446-476 nm), an acidic form (350-355 nm), an alkaline form (430-440 nm), and an intermediate wavelength form (355-400 nm). The acidic species is formed by protonation of the oxyanion of the para-hydroxy-cinnamyl cysteine chromophore as a secondary result of acid denaturation (with pK(a) values of 2.8-5.4) and often results in precipitation of the protein, and in the case of wild-type PYP, eventual hydrolysis of the chromophore thioester bond at pH values below 2. Thus, the large and complex structural changes associated with the acidic species make it a poor model for the long-lived photocycle intermediate, I(2), which undergoes more moderate structural changes. Mutations at E46, which is hydrogen-bonded to the chromophore, have only two spectral forms accessible to them, the neutral and the acidic forms. Thus, an intact E46 carboxyl group is essential for observation of either intermediate or alkaline wavelength forms. The alkaline form is likely to be due to ionization of E46 in the folded protein. We postulate that the intermediate wavelength form is due to a conformational change that allows solvent access to E46 and formation of a hydrogen-bond from a water molecule to the carboxylic acid group, thus weakening its interaction with the chromophore. Increasing solvent access to the intermediate spectral form with denaturant concentration results in a continuously blue-shifted wavelength maximum.

  10. Amyloid Core Formed of Full-Length Recombinant Mouse Prion Protein Involves Sequence 127–143 but Not Sequence 107–126

    PubMed Central

    Chatterjee, Biswanath; Lee, Chung-Yu; Lin, Chen; Chen, Eric H.-L.; Huang, Chao-Li; Yang, Chien-Chih; Chen, Rita P.-Y.

    2013-01-01

    The principal event underlying the development of prion disease is the conversion of soluble cellular prion protein (PrPC) into its disease-causing isoform, PrPSc. This conversion is associated with a marked change in secondary structure from predominantly α-helical to a high β-sheet content, ultimately leading to the formation of aggregates consisting of ordered fibrillar assemblies referred to as amyloid. In vitro, recombinant prion proteins and short prion peptides from various species have been shown to form amyloid under various conditions and it has been proposed that, theoretically, any protein and peptide could form amyloid under appropriate conditions. To identify the peptide segment involved in the amyloid core formed from recombinant full-length mouse prion protein mPrP(23–230), we carried out seed-induced amyloid formation from recombinant prion protein in the presence of seeds generated from the short prion peptides mPrP(107–143), mPrP(107–126), and mPrP(127–143). Our results showed that the amyloid fibrils formed from mPrP(107–143) and mPrP(127–143), but not those formed from mPrP(107–126), were able to seed the amyloidogenesis of mPrP(23–230), showing that the segment residing in sequence 127–143 was used to form the amyloid core in the fibrillization of mPrP(23–230). PMID:23844138

  11. Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells

    PubMed Central

    1993-01-01

    GPI-linked protein molecules become Triton-insoluble during polarized sorting to the apical cell surface of epithelial cells. These insoluble complexes, enriched in cholesterol, glycolipids, and GPI-linked proteins, have been isolated by flotation on sucrose density gradients and are thought to contain the putative GPI-sorting machinery. As the cellular origin and molecular protein components of this complex remain unknown, we have begun to characterize these low-density insoluble complexes isolated from MDCK cells. We find that these complexes, which represent 0.4-0.8% of the plasma membrane, ultrastructurally resemble caveolae and are over 150-fold enriched in a model GPI-anchored protein and caveolin, a caveolar marker protein. However, they exclude many other plasma membrane associated molecules and organelle-specific marker enzymes, suggesting that they represent microdomains of the plasma membrane. In addition to caveolin, these insoluble complexes contain a subset of hydrophobic plasma membrane proteins and cytoplasmically-oriented signaling molecules, including: (a) GTP- binding proteins--both small and heterotrimeric; (b) annex II--an apical calcium-regulated phospholipid binding protein with a demonstrated role in exocytic fusion events; (c) c-Yes--an apically localized member of the Src family of non-receptor type protein- tyrosine kinases; and (d) an unidentified serine-kinase activity. As we demonstrate that caveolin is both a transmembrane molecule and a major phospho-acceptor component of these complexes, we propose that caveolin could function as a transmembrane adaptor molecule that couples luminal GPI-linked proteins with cytoplasmically oriented signaling molecules during GPI-membrane trafficking or GPI-mediated signal transduction events. In addition, our results have implications for understanding v- Src transformation and the actions of cholera and pertussis toxins on hetero-trimeric G proteins. PMID:8349730

  12. In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis

    PubMed Central

    Nadipuram, Santhosh M.; Kim, Elliot W.; Vashisht, Ajay A.; Lin, Andrew H.; Bell, Hannah N.; Coppens, Isabelle; Wohlschlegel, James A.

    2016-01-01

    ABSTRACT Toxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δgra38 and Δgra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo. Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis. PMID:27486190

  13. Structure of the fimbrial protein Mfa4 from Porphyromonas gingivalis in its precursor form: implications for a donor-strand complementation mechanism

    PubMed Central

    Kloppsteck, Patrik; Hall, Michael; Hasegawa, Yoshiaki; Persson, Karina

    2016-01-01

    Gingivitis and periodontitis are chronic inflammatory diseases that can lead to tooth loss. One of the causes of these diseases is the Gram-negative Porphyromonas gingivalis. This periodontal pathogen is dependent on two fimbriae, FimA and Mfa1, for binding to dental biofilm, salivary proteins, and host cells. These fimbriae are composed of five proteins each, but the fimbriae assembly mechanism and ligands are unknown. Here we reveal the crystal structure of the precursor form of Mfa4, one of the accessory proteins of the Mfa1 fimbria. Mfa4 consists of two β-sandwich domains and the first part of the structure forms two well-defined β-strands that run over both domains. This N-terminal region is cleaved by gingipains, a family of proteolytic enzymes that encompass arginine- and lysine-specific proteases. Cleavage of the N-terminal region generates the mature form of the protein. Our structural data allow us to propose that the new N-terminus of the mature protein may function as a donor strand in the polymerization of P. gingivalis fimbriae. PMID:26972441

  14. Developmentally Regulated RNA-binding Protein 1 (Drb1)/RNA-binding Motif Protein 45 (RBM45), a Nuclear-Cytoplasmic Trafficking Protein, Forms TAR DNA-binding Protein 43 (TDP-43)-mediated Cytoplasmic Aggregates.

    PubMed

    Mashiko, Takafumi; Sakashita, Eiji; Kasashima, Katsumi; Tominaga, Kaoru; Kuroiwa, Kenji; Nozaki, Yasuyuki; Matsuura, Tohru; Hamamoto, Toshiro; Endo, Hitoshi

    2016-07-15

    Cytoplasmic protein aggregates are one of the pathological hallmarks of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Several RNA-binding proteins have been identified as components of inclusion bodies. Developmentally regulated RNA-binding protein 1 (Drb1)/RNA-binding motif protein 45 is an RNA-binding protein that was recently described as a component in ALS- and FTLD-related inclusion bodies. However, the molecular mechanism underlying cytoplasmic Drb1 aggregation remains unclear. Here, using an in vitro cellular model, we demonstrated that Drb1 co-localizes with cytoplasmic aggregates mediated by TAR DNA-binding protein 43, a major component of ALS and FTLD-related inclusion bodies. We also defined the domains involved in the subcellular localization of Drb1 to clarify the role of Drb1 in the formation of cytoplasmic aggregates in ALS and FTLD. Drb1 predominantly localized in the nucleus via a classical nuclear localization signal in its carboxyl terminus and is a shuttling protein between the nucleus and cytoplasm. Furthermore, we identify a double leucine motif serving as a nuclear export signal. The Drb1 mutant, presenting mutations in both nuclear localization signal and nuclear export signal, is prone to aggregate in the cytoplasm. The mutant Drb1-induced cytoplasmic aggregates not only recruit TAR DNA-binding protein 43 but also decrease the mitochondrial membrane potential. Taken together, these results indicate that perturbation of Drb1 nuclear-cytoplasmic trafficking induces toxic cytoplasmic aggregates, suggesting that mislocalization of Drb1 is involved in the cause of cytotoxicity in neuronal cells.

  15. The vaccinia virus 14-kilodalton (A27L) fusion protein forms a triple coiled-coil structure and interacts with the 21-kilodalton (A17L) virus membrane protein through a C-terminal alpha-helix.

    PubMed

    Vázquez, M I; Rivas, G; Cregut, D; Serrano, L; Esteban, M

    1998-12-01

    The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third alpha-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and

  16. Steady-state and time-resolved fluorescence studies on wild type and mutant chromatium vinosum high potential iron proteins: holo- and apo-forms.

    PubMed Central

    Sau, A K; Chen, C A; Cowan, J A; Mazumdar, S; Mitra, S

    2001-01-01

    Detailed circular dichroism (CD), steady-state and time-resolved tryptophan fluorescence studies on the holo- and apo- forms of high potential iron protein (HiPIP) from Chromatium vinosum and its mutant protein have been carried out to investigate conformational properties of the protein. CD studies showed that the protein does not have any significant secondary structure elements in the holo- or apo- HiPIP, indicating that the metal cluster does not have any effect on formation of secondary structure in the protein. Steady-state fluorescence quenching studies however, suggested that removal of the iron-sulfur ([Fe(4)S(4)](3+)) cluster from the protein leads to an increase in the solvent accessibility of tryptophans, indicating change in the tertiary structure of the protein. CD studies on the holo- and apo- HiPIP also showed that removal of the metal prosthetic group drastically affects the tertiary structure of the protein. Time-resolved fluorescence decay of the wild type protein was fitted to a four-exponentials model and that of the W80N mutant was fitted to a three-exponentials model. The time-resolved fluorescence decay was also analyzed by maximum entropy method (MEM). The results of the MEM analysis agreed with those obtained from discrete exponentials model analysis. Studies on the wild type and mutants helped to assign the fast picosecond lifetime component to the W80 residue, which exhibits fast fluorescence energy transfer to the [Fe(4)S(4)](3+) cluster of the protein. Decay-associated fluorescence spectra of each tryptophan residues were calculated from the time-resolved fluorescence results at different emission wavelengths. The results suggested that W80 is in the hydrophobic core of the protein, but W60 and W76 are partially or completely exposed to the solvent. PMID:11566801

  17. Nuclear export signal-interacting protein forms complexes with lamin A/C-Nups to mediate the CRM1-independent nuclear export of large hepatitis delta antigen.

    PubMed

    Huang, Cheng; Jiang, Jia-Yin; Chang, Shin C; Tsay, Yeou-Guang; Chen, Mei-Ru; Chang, Ming-Fu

    2013-02-01

    Nuclear export is an important process that not only regulates the functions of cellular factors but also facilitates the assembly of viral nucleoprotein complexes. Chromosome region maintenance 1 (CRM1) that mediates the transport of proteins bearing the classical leucine-rich nuclear export signal (NES) is the best-characterized nuclear export receptor. Recently, several CRM1-independent nuclear export pathways were also identified. The nuclear export of the large form of hepatitis delta antigen (HDAg-L), a nucleocapsid protein of hepatitis delta virus (HDV), which contains a CRM1-independent proline-rich NES, is mediated by the host NES-interacting protein (NESI). The mechanism of the NESI protein in mediating nuclear export is still unknown. In this study, NESI was characterized as a highly glycosylated membrane protein. It interacted and colocalized well in the nuclear envelope with lamin A/C and nucleoporins. Importantly, HDAg-L could be coimmunoprecipitated with lamin A/C and nucleoporins. In addition, binding of the cargo HDAg-L to the C terminus of NESI was detected for the wild-type protein but not for the nuclear export-defective HDAg-L carrying a P205A mutation [HDAg-L(P205A)]. Knockdown of lamin A/C effectively reduced the nuclear export of HDAg-L and the assembly of HDV. These data indicate that by forming complexes with lamin A/C and nucleoporins, NESI facilitates the CRM1-independent nuclear export of HDAg-L.

  18. Structure of the apo form of the catabolite control protein A (CcpA) from Bacillus megaterium with a DNA-binding domain

    SciTech Connect

    Singh, Rajesh Kumar; Palm, Gottfried J.; Panjikar, Santosh

    2007-04-01

    Crystal structure analysis of the apo form of catabolite control protein A reveals the three-helix bundle of the DNA-binding domain. In the crystal packing, this domain interacts with the binding site for the corepressor protein. Crystal structure determination of catabolite control protein A (CcpA) at 2.6 Å resolution reveals for the first time the structure of a full-length apo-form LacI-GalR family repressor protein. In the crystal structures of these transcription regulators, the three-helix bundle of the DNA-binding domain has only been observed in cognate DNA complexes; it has not been observed in other crystal structures owing to its mobility. In the crystal packing of apo-CcpA, the protein–protein contacts between the N-terminal three-helix bundle and the core domain consisted of interactions between the homodimers that were similar to those between the corepressor protein HPr and the CcpA N-subdomain in the ternary DNA complex. In contrast to the DNA complex, the apo-CcpA structure reveals large subdomain movements in the core, resulting in a complete loss of contacts between the N-subdomains of the homodimer.

  19. Expression and properties of wild-type and mutant forms of the Drosophila sex comb on midleg (SCM) repressor protein.

    PubMed

    Bornemann, D; Miller, E; Simon, J

    1998-10-01

    The Sex comb on midleg (Scm) gene encodes a transcriptional repressor of the Polycomb group (PcG). Here we show that SCM protein is nuclear and that its expression is widespread during fly development. SCM protein contains a C-terminal domain, termed the SPM domain, which mediates protein-protein interactions. The biochemical function of another domain consisting of two 100-amino-acid-long repeats, termed "mbt" repeats, is unknown. We have determined the molecular lesions of nine Scm mutant alleles, which identify functional requirements for specific domains. The Scm alleles were tested for genetic interactions with mutations in other PcG genes. Intriguingly, three hypomorphic Scm mutations, which map within an mbt repeat, interact with PcG mutations more strongly than do Scm null alleles. The strongest interactions produce partial synthetic lethality that affects doubly heterozygous females more severely than males. We show that mbt repeat alleles produce stable SCM proteins that associate with normal sites in polytene chromosomes. We also analyzed progeny from Scm mutant germline clones to compare the effects of an mbt repeat mutation during embryonic vs. pupal development. We suggest that the mbt repeat alleles produce altered SCM proteins that incorporate into and impair function of PcG protein complexes.

  20. The Coxsackievirus and adenovirus receptor (CAR) forms a complex with the PDZ domain-containing protein ligand-of-numb protein-X (LNX).

    PubMed

    Sollerbrant, Kerstin; Raschperger, Elisabeth; Mirza, Momina; Engstrom, Ulla; Philipson, Lennart; Ljungdahl, Per O; Pettersson, Ralf F

    2003-02-28

    The Coxsackievirus and adenovirus receptor (CAR) functions as a virus receptor, but its primary biological function is unknown. A yeast two-hybrid screen was used to identify Ligand-of-Numb protein-X (LNX) as a binding partner to the intracellular tail of CAR. LNX harbors several protein-protein interacting domains, including four PDZ domains, and was previously shown to bind to and regulate the expression level of the cell-fate determinant Numb. CAR was able to bind LNX both in vivo and in vitro. Efficient binding to LNX required not only the consensus PDZ domain binding motif in the C terminus of CAR but also upstream sequences. The CAR binding region in LNX was mapped to the second PDZ domain. CAR and LNX were also shown to colocalize in vivo in mammalian cells. We speculate that CAR and LNX are part of a larger protein complex that might have important functions at discrete subcellular localizations in the cell.

  1. Cloning, purification, crystallization and preliminary X-ray analysis of the catalytic domain of human receptor-like protein tyrosine phosphatase [gamma] in three different crystal forms

    SciTech Connect

    Kish, Kevin; McDonnell, Patricia A.; Goldfarb, Valentina; Gao, Mian; Metzler, William J.; Langley, David R.; Bryson, James W.; Kiefer, Susan E.; Carpenter, Brian; Kostich, Walter A.; Westphal, Ryan S.; Sheriff, Steven

    2013-03-07

    Protein tyrosine phosphatase {gamma} is a membrane-bound receptor and is designated RPTP{gamma}. RPTP{gamma} and two mutants, RPTP{gamma}(V948I, S970T) and RPTP{gamma}(C858S, S970T), were recombinantly expressed and purified for X-ray crystallographic studies. The purified enzymes were crystallized using the hanging-drop vapor-diffusion method. Crystallographic data were obtained from several different crystal forms in the absence and the presence of inhibitor. In this paper, a description is given of how three different crystal forms were obtained that were used with various ligands. An orthorhombic crystal form and a trigonal crystal form were obtained both with and without ligand, and a monoclinic crystal form was only obtained in the presence of a particularly elaborated inhibitor.

  2. Prediction of protein subcellular localization by incorporating multiobjective PSO-based feature subset selection into the general form of Chou's PseAAC.

    PubMed

    Mandal, Monalisa; Mukhopadhyay, Anirban; Maulik, Ujjwal

    2015-04-01

    In this article, the possible subcellular location of a protein is predicted using multiobjective particle swarm optimization-based feature selection technique. In general form of pseudo-amino acid composition, the protein sequences are used for constructing protein features. Here, the different amino acids compositions are used to construct the feature sets. Therefore, the data are presented as sample of protein versus amino acid compositions as features. The proposed algorithm tries to maximize the feature relevance and minimize the feature redundancy simultaneously. After proposed algorithm is executed on the multiclass dataset, some features are selected. On this resultant feature subset, tenfold cross-validation is applied and corresponding accuracy, F score, entropy, representation entropy and average correlation are calculated. The performance of the proposed method is compared with that of its single objective versions, sequential forward search, sequential backward search, minimum redundancy maximum relevance with two schemes, CFS, CBFS, [Formula: see text], Fisher discriminant and a Cluster-based technique.

  3. RNA binding by Hfq and ring-forming (L)Sm proteins: a trade-off between optimal sequence readout and RNA backbone conformation.

    PubMed

    Weichenrieder, Oliver

    2014-01-01

    The eukaryotic Sm and the Sm-like (LSm) proteins form a large family that includes LSm proteins in archaea and the Hfq proteins in bacteria. Commonly referred to as the (L)Sm protein family, the various members play important roles in RNA processing, decay, and riboregulation. Particularly interesting from a structural point of view is their ability to assemble into doughnut-shaped rings, which allows them to bind preferentially the uridine-rich 3'-end of RNA oligonucleotides. With an emphasis on Hfq, this review compares the RNA-binding properties of the various (L)Sm rings that were recently co-crystallized with RNA substrates, and it discusses how these properties relate to physiological function.

  4. Protein Interaction between Ameloblastin and Proteasome Subunit α Type 3 Can Facilitate Redistribution of Ameloblastin Domains within Forming Enamel.

    PubMed

    Geng, Shuhui; White, Shane N; Paine, Michael L; Snead, Malcolm L

    2015-08-21

    Enamel is a bioceramic tissue composed of thousands of hydroxyapatite crystallites aligned in parallel within boundaries fabricated by a single ameloblast cell. Enamel is the hardest tissue in the vertebrate body; however, it starts development as a self-organizing assembly of matrix proteins that control crystallite habit. Here, we examine ameloblastin, a protein that is initially distributed uniformly across the cell boundary but redistributes to the lateral margins of the extracellular matrix following secretion thus producing cell-defined boundaries within the matrix and the mineral phase. The yeast two-hybrid assay identified that proteasome subunit α type 3 (Psma3) interacts with ameloblastin. Confocal microscopy confirmed Psma3 co-distribution with ameloblastin at the ameloblast secretory end piece. Co-immunoprecipitation assay of mouse ameloblast cell lysates with either ameloblastin or Psma3 antibody identified each reciprocal protein partner. Protein engineering demonstrated that only the ameloblastin C terminus interacts with Psma3. We show that 20S proteasome digestion of ameloblastin in vitro generates an N-terminal cleavage fragment consistent with the in vivo pattern of ameloblastin distribution. These findings suggest a novel pathway participating in control of protein distribution within the extracellular space that serves to regulate the protein-mineral interactions essential to biomineralization.

  5. Zfrp8 forms a complex with fragile-X mental retardation protein and regulates its localization and function.

    PubMed

    Tan, William; Schauder, Curtis; Naryshkina, Tatyana; Minakhina, Svetlana; Steward, Ruth

    2016-02-15

    Fragile-X syndrome is the most commonly inherited cause of autism and mental disabilities. The Fmr1 (Fragile-X Mental Retardation 1) gene is essential in humans and Drosophila for the maintenance of neural stem cells, and Fmr1 loss results in neurological and reproductive developmental defects in humans and flies. FMRP (Fragile-X Mental Retardation Protein) is a nucleo-cytoplasmic shuttling protein, involved in mRNA silencing and translational repression. Both Zfrp8 and Fmr1 have essential functions in the Drosophila ovary. In this study, we identified FMRP, Nufip (Nuclear Fragile-X Mental Retardation Protein-interacting Protein) and Tral (Trailer Hitch) as components of a Zfrp8 protein complex. We show that Zfrp8 is required in the nucleus, and controls localization of FMRP in the cytoplasm. In addition, we demonstrate that Zfrp8 genetically interacts with Fmr1 and tral in an antagonistic manner. Zfrp8 and FMRP both control heterochromatin packaging, also in opposite ways. We propose that Zfrp8 functions as a chaperone, controlling protein complexes involved in RNA processing in the nucleus.

  6. A ΩXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence

    PubMed Central

    Cyr, Normand; de la Fuente, Cynthia; Lecoq, Lauriane; Guendel, Irene; Chabot, Philippe R.; Kehn-Hall, Kylene; Omichinski, James G.

    2015-01-01

    Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ΩXaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ΩXaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ΩXaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from other Bunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of other Bunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ΩXaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH. PMID:25918396

  7. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

    NASA Astrophysics Data System (ADS)

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy

    2015-07-01

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  8. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules.

    PubMed

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy

    2015-07-28

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  9. A genetically encoded probe for the identification of proteins that form sulfenic acid in response to H2O2 in Saccharomyces cerevisiae.

    PubMed

    Takanishi, Christina L; Wood, Matthew J

    2011-06-03

    It is widely known that reactive oxygen species (ROS), such as hydrogen peroxide, play important roles in cellular signaling and initiation of oxidative stress responses via thiol modifications. Identification of the targets of these modifications will provide a better understanding of the relationship between ROS and human diseases, such as cancer and atherosclerosis. Sulfenic acid is the principle product of a reaction between hydrogen peroxide and a reactive protein cysteine. This reversible post-translational modification plays an important role in enzyme active sites, signaling transduction via disulfide bond formation, as well as an intermediate to overoxidation products during oxidative stress. By re-engineering the C-terminal cysteine rich domain (cCRD) of the Yap1 transcription factor, we were able to create a genetically encoded probe for the general detection and identification of proteins that form sulfenic acid in vivo. The Yap1-cCRD probe has been used previously in the identification of proteins that form sulfenic acid in Escherichia coli. Here we demonstrate the successful use of the Yap1-cCRD probe in the identification of proteins that form sulfenic acid in response to hydrogen peroxide in Saccharomyces cerevisiae.

  10. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

    SciTech Connect

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy

    2015-07-28

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  11. Translational repression of human matrix metalloproteinases-13 by an alternatively spliced form of T-cell-restricted intracellular antigen-related protein (TIAR).

    PubMed

    Yu, Qing; Cok, Steven J; Zeng, Chenbo; Morrison, Aubrey R

    2003-01-17

    Human matrix metalloproteinases-13 (HMMP13) shows a wide substrate specificity, and its expression is limited to pathological situations such as chronic inflammation and cancer. The coding sequence for HMMP13 is 86% identical to rat matrix metalloproteinases-13 (RMMP13); however, the regulation of HMMP13 and RMMP13 protein synthesis in renal mesangial cells is strikingly different. In human cells there is a discordance between HMMP13 mRNA levels and protein expression. Following IL-1 beta or TGF-beta(1) stimulation, HMMP13 mRNA levels increase significantly, whereas the protein expression is absent. This discordance is because of a species-dependent translational repression. In addition to the 3'-untranslated region of the matrix metalloproteinases-13 (MMP13) gene, the differential expression of an alternatively spliced transcript of the RNA-binding protein TIAR in human cell cultures is also critical for this post-transcriptional regulation. Transient expression of the 17-amino acid insert of the alternatively spliced form of TIAR reverses the HMMP13 mRNA silencing observed in human and primate species. In addition, co-transfection of the alternatively spliced form of TIAR and HMMP13 into Rat2 cells suppresses HMMP13 protein expression. Thus, we report for the first time that a species-dependent TIAR isoform plays a major role in the post-transcriptional silencing for HMMP13.

  12. The irre cell recognition module (IRM) protein Kirre is required to form the reciprocal synaptic network of L4 neurons in the Drosophila lamina.

    PubMed

    Lüthy, Kevin; Ahrens, Birgit; Rawal, Shilpa; Lu, Zhiyuan; Tarnogorska, Dorota; Meinertzhagen, Ian A; Fischbach, Karl-Friedrich

    2014-01-01

    Each neuropil module, or cartridge, in the fly's lamina has a fixed complement of cells. Of five types of monopolar cell interneurons, only L4 has collaterals that invade neighboring cartridges. In the proximal lamina, these collaterals form reciprocal synapses with both the L2 of their own cartridge and the L4 collateral branches from two other neighboring cartridges. During synaptogenesis, L4 collaterals strongly express the cell adhesion protein Kirre, a member of the irre cell recognition module (IRM) group of proteins ( Fischbach et al., 2009 , J Neurogenet, 23, 48-67). The authors show by mutant analysis and gene knockdown techniques that L4 neurons develop their lamina collaterals in the absence of this cell adhesion protein. Using electron microscopy (EM), the authors demonstrate, however, that without Kirre protein these L4 collaterals selectively form fewer synapses. The collaterals of L4 neurons of various genotypes reconstructed from serial-section EM revealed that the number of postsynaptic sites was dramatically reduced in the absence of Kirre, almost eliminating any synaptic input to L4 neurons. A significant reduction of presynaptic sites was also detected in kirre(0) mutants and gene knockdown flies using RNA interference. L4 neuron reciprocal synapses are thus almost eliminated. A presynaptic marker, Brp-short(GFP) confirmed these data using confocal microscopy. This study reveals that removing Kirre protein specifically disrupts the functional L4 synaptic network in the Drosophila lamina.

  13. A mutant form of the rho protein can restore stress fibers and adhesion plaques in v-src transformed fibroblasts.

    PubMed

    Mayer, T; Meyer, M; Janning, A; Schiedel, A C; Barnekow, A

    1999-03-25

    The organization of polymerized actin in the mammalian cell is regulated by several members of the rho family. Three rho proteins, cdc42, rac and rho act in a cascade to organize the intracellular actin cytoskeleton. Rho proteins are involved in the formation of actin stress fibers and adhesion plaques in fibroblasts. During transformation of mammalian cells by oncogenes the cytoskeleton is rearranged and stress fibers and adhesion plaques are disintegrated. In this paper we investigate the function of the rho protein in RR1022 rat fibroblasts transformed by the Rous sarcoma virus. Two activated mutants of the rho protein, rho G14V and rho Q63L, and a dominant negative mutant, rho N1171, were stably transfected into RR1022 cells. The resulting cell lines were analysed for the organization of polymerized actin and adhesion plaques. Cells expressing rho Q63L, but not rho wt, rho G14V or rho N1171, showed an altered morphology. These cells displayed a flat, fibroblast like shape when compared with the RR1022 ancestor cells. Immunofluorescence analyses revealed that actin stress fibers and adhesion plaques were rearranged in these cells. We conclude from these data that an active rho protein can restore elements of the actin cytoskeleton in transformed cells by overriding the tyrosine kinase phosphorylation induced by the pp60(v-src).

  14. The Peripheral Neuropathy-Linked Trembler and Trembler-J Mutant Forms of Peripheral Myelin Protein 22 are Folding-Destabilized†

    PubMed Central

    Myers, Jeffrey K.; Mobley, Charles K.; Sanders, Charles R.

    2008-01-01

    Dominant mutations in the tetraspan membrane protein peripheral myelin protein 22 (PMP22) are known to result in peripheral neuropathies such as Charcot-Marie-Tooth Type 1A (CMT1A) disease via mechanisms that appear to be closely linked to misfolding of PMP22 in the membrane of the endoplasmic reticulum (ER). To characterize the molecular defects in PMP22, we examined the structure and folding stability of two human disease mutant forms of PMP22 that are also the basis for mouse models of peripheral neuropathies: G150D (Trembler phenotype), and L16P (Trembler-J phenotype). Circular dichroism and NMR spectroscopic studies indicated that, when folded, the 3-D structures of these disease-linked mutants are similar to the folded wild type protein. However, the folded forms of the mutants were observed to be destabilized relative to the wild type protein, with the L16P mutant being particularly unstable. The rate of refolding from an unfolded state was observed to be very slow for the wild type protein, and no refolding was observed for either mutant. These results lead to the hypothesis that ER quality control recognizes the G150D and L16P mutant forms of PMP22 as defective through mechanisms closely related to their conformational instability and/or slow folding. It was also seen that wild type PMP22 binds Zn(II) and Cu(II) with micromolar affinity, a property that may be important to the stability and function of this protein. Zn(II) was able to rescue the stability defect of the Tr mutant. PMID:18795802

  15. The olivine macrocryst problem: New insights from minor and trace element compositions of olivine from Lac de Gras kimberlites, Canada

    NASA Astrophysics Data System (ADS)

    Bussweiler, Yannick; Foley, Stephen F.; Prelević, Dejan; Jacob, Dorrit E.

    2015-04-01

    This study presents detailed petrographical and geochemical investigations on remarkably fresh olivines in kimberlites from the EKATI Diamond Mine™ located in the Tertiary/Cretaceous Lac de Gras kimberlite field within the Slave craton of Canada. Olivine, constituting about 42 vol.% of the analyzed samples, can be divided into two textural groups: (i) macrocrystic olivines, > 100 μm sub-rounded crystals and (ii) groundmass olivines, < 100 μm subhedral crystals. Olivines from both populations define two distinct chemical trends; a "mantle trend" with angular cores, showing low Ca (< 0.1 wt.% CaO) and high Ni (0.3-0.4 wt.% NiO) at varying Mg# (0.86-0.93), contrasts with a "melt trend" typified by thin (< 100 μm) rims with increasing Ca (up to 1.0 wt.% CaO) and decreasing Ni (down to 0.1 wt.% NiO) contents at constant Mg# (~ 0.915). These findings are in agreement with recent studies suggesting that virtually all olivine is composed of xenocrystic (i.e. mantle-related) cores with phenocrystic (i.e. melt-related) overgrowths, thereby challenging the traditional view that the origin of kimberlitic olivine can be distinguished based on size and morphology. The two main trends can be further resolved into sub-groups refining the crystallization history of olivine; the mantle trend indicates a multi-source origin that samples the layered lithosphere below the Slave craton, whereas the melt trend represents multi-stage crystallization comprising a differentiation trend starting at mantle conditions and a second trend controlled by the crystallization of additional phases (e.g. chromite) and changing magma conditions (e.g. oxidation). These trends are also seen in the concentrations of trace elements not routinely measured in olivine (e.g. Na, P, Ti, Co, Sc, Zr). Trace element mapping with LA-ICP-MS reveals the distribution of these elements within olivine grains. The trace element distribution between the two trends appears to be consistent with phenocrystic olivine

  16. Acidic pH triggers conformational changes at the NH2-terminal propeptide of the precursor of pulmonary surfactant protein B to form a coiled coil structure.

    PubMed

    Bañares-Hidalgo, A; Pérez-Gil, J; Estrada, P

    2014-07-01

    Pulmonary surfactant protein SP-B is synthesized as a larger precursor, proSP-B. We report that a recombinant form of human SP-BN forms a coiled coil structure at acidic pH. The protonation of a residue with pK=4.8±0.06 is the responsible of conformational changes detected by circular dichroism and intrinsic fluorescence emission. Sedimentation velocity analysis showed protein oligomerisation at any pH condition, with an enrichment of the species compatible with a tetramer at acidic pH. Low 2,2,2,-trifluoroethanol concentration promoted β-sheet structures in SP-BN, which bind Thioflavin T, at acidic pH, whereas it promoted coiled coil structures at neutral pH. The amino acid stretch predicted to form β-sheet parallel association in SP-BN overlaps with the sequence predicted by several programs to form coiled coil structure. A synthetic peptide ((60)W-E(85)) designed from the sequence of the amino acid stretch of SP-BN predicted to form coiled coil structure showed random coil conformation at neutral pH but concentration-dependent helical structure at acidic pH. Sedimentation velocity analysis of the peptide indicated monomeric state at neutral pH (s20, w=0.55S; Mr~3kDa) and peptide association (s20, w=1.735S; Mr=~14kDa) at acidic pH, with sedimentation equilibrium fitting to a Monomer-Nmer-Mmer model with N=6 and M=4 (Mr=14692Da). We propose that protein oligomerisation through coiled-coil motifs could then be a general feature in the assembly of functional units in saposin-like proteins in general and in the organization of SP-B in a functional surfactant, in particular.

  17. Identification of novel in vivo phosphorylation sites of the human proapoptotic protein BAD: pore-forming activity of BAD is regulated by phosphorylation.

    PubMed

    Polzien, Lisa; Baljuls, Angela; Rennefahrt, Ulrike E E; Fischer, Andreas; Schmitz, Werner; Zahedi, Rene P; Sickmann, Albert; Metz, Renate; Albert, Stefan; Benz, Roland; Hekman, Mirko; Rapp, Ulf R

    2009-10-09

    BAD is a proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD, little data are available with respect to phosphorylation of human BAD protein. Using mass spectrometry, we identified here besides the established phosphorylation sites at serines 75, 99, and 118 several novel in vivo phosphorylation sites within human BAD (serines 25, 32/34, 97, and 124). Furthermore, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating serine residues 75, 99, and 118. Our results indicate that RAF kinases represent, besides protein kinase A, PAK, and Akt/protein kinase B, in vivo BAD-phosphorylating kinases. RAF-induced phosphorylation of BAD was reduced to control levels using the RAF inhibitor BAY 43-9006. This phosphorylation was not prevented by MEK inhibitors. Consistently, expression of constitutively active RAF suppressed apoptosis induced by BAD and the inhibition of colony formation caused by BAD could be prevented by RAF. In addition, using the surface plasmon resonance technique, we analyzed the direct consequences of BAD phosphorylation by RAF with respect to association with 14-3-3 and Bcl-2/Bcl-X(L) proteins. Phosphorylation of BAD by active RAF promotes 14-3-3 protein association, in which the phosphoserine 99 represented the major binding site. Finally, we show here that BAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation.

  18. The Escherichia coli BolA Protein IbaG Forms a Histidine-Ligated [2Fe-2S]-Bridged Complex with Grx4.

    PubMed

    Dlouhy, Adrienne C; Li, Haoran; Albetel, Angela-Nadia; Zhang, Bo; Mapolelo, Daphne T; Randeniya, Sajini; Holland, Ashley A; Johnson, Michael K; Outten, Caryn E

    2016-12-13

    Two ubiquitous protein families have emerged as key players in iron metabolism, the CGFS-type monothiol glutaredoxins (Grxs) and the BolA proteins. Monothiol Grxs and BolA proteins form heterocomplexes that have been implicated in Fe-S cluster assembly and trafficking. The Escherichia coli genome encodes members of both of these proteins families, namely, the monothiol glutaredoxin Grx4 and two BolA family proteins, BolA and IbaG. Previous work has demonstrated that E. coli Grx4 and BolA interact as both apo and [2Fe-2S]-bridged heterodimers that are spectroscopically distinct from [2Fe-2S]-bridged Grx4 homodimers. However, the physical and functional interactions between Grx4 and IbaG are uncharacterized. Here we show that co-expression of Grx4 with IbaG yields a [2Fe-2S]-bridged Grx4-IbaG heterodimer. In vitro interaction studies indicate that IbaG binds the [2Fe-2S] Grx4 homodimer to form apo Grx4-IbaG heterodimer as well as the [2Fe-2S] Grx4-IbaG heterodimer, altering the cluster stability and coordination environment. Additionally, spectroscopic and mutagenesis studies provide evidence that IbaG ligates the Fe-S cluster via the conserved histidine that is present in all BolA proteins and by a second conserved histidine that is present in the H/C loop of two of the four classes of BolA proteins. These results suggest that IbaG may function in Fe-S cluster assembly and trafficking in E. coli as demonstrated for other BolA homologues that interact with monothiol Grxs.

  19. The Yersinia enterocolitica phage shock proteins B and C can form homodimers and heterodimers in vivo with the possibility of close association between multiple domains.

    PubMed

    Gueguen, Erwan; Flores-Kim, Josué; Darwin, Andrew J

    2011-10-01

    The Yersinia enterocolitica phage shock protein (Psp) stress response is essential for virulence and for survival during the mislocalization of outer membrane secretin proteins. The cytoplasmic membrane proteins PspB and PspC are critical components involved in regulating psp gene expression and in facilitating tolerance to secretin-induced stress. Interactions between PspB and PspC monomers might be important for their functions and for PspC stability. However, little is known about these interactions and there are conflicting reports about the ability of PspC to dimerize. To address this, we have used a combination of independent approaches to systematically analyze the ability of PspB and PspC to form dimers in vivo. Formaldehyde cross-linking of the endogenous chromosomally encoded proteins in Y. enterocolitica revealed discrete complexes corresponding in size to PspB-PspB, PspC-PspC, and PspB-PspC. Bacterial two-hybrid analysis corroborated these protein associations, but an important limitation of the two-hybrid approach was uncovered for PspB. A series of PspB and PspC proteins with unique cysteine substitutions at various positions was constructed. In vivo disulfide cross-linking experiments with these proteins further supported close association between PspB and PspC monomers. Detailed cysteine substitution analysis of predicted leucine zipper-like amphipathic helices in both PspB and PspC suggested that their hydrophobic faces could form homodimerization interfaces.

  20. Protein ordered sequences are formed by random joining of amino acids in protein 0(th)-order structure, followed by evolutionary process.

    PubMed

    Ikehara, Kenji

    2014-12-01

    Only random processes should occur on the primitive Earth. In contrast, many ordered sequences are synthesized according to genetic information on the present Earth. In this communication, I have proposed an idea that protein 0(th)-order structures or specific amino acid compositions would mediate the transfer from random process to formation of ordered sequences, after formation of double-stranded genes.

  1. Inhibition of glucuronidation and oxidative metabolism of buprenorphine using GRAS compounds or dietary constituents/supplements: in vitro proof of concept.

    PubMed

    Maharao, Neha V; Joshi, Anand A; Gerk, Phillip M

    2017-03-01

    The present study investigated the potential of generally recognized as safe (GRAS) compounds or dietary substances to inhibit the presystemic metabolism of buprenorphine and to increase its oral bioavailability. Using IVIVE, buprenorphine extraction ratios in intestine and liver were predicted as 96% and 71%, respectively. In addition, the relative fraction of buprenorphine metabolized by oxidation and glucuronidation in these two organs was estimated using pooled human intestinal and liver microsomes. In both organs, oxidation appeared to be the major metabolic pathway with a 6 and 4 fold higher intrinsic clearance than glucuronidation in intestine and liver, respectively. The oral bioavailability of buprenorphine was predicted to be 1.16%. Inhibition of 75% and 50% of intestinal and hepatic presystemic metabolism would result in an Foral of 49%, which is comparable to the bioavailability of sublingual buprenorphine. In human liver microsomes, chrysin, curcumin, ginger extract, hesperitin, magnolol, quercetin and silybin inhibited ≥50% glucuronidation, whereas chrysin, curcumin, ginger extract, 6-gingerol, pterostilbene, resveratrol and silybin exhibited ≥30% inhibition of oxidation. In human intestinal microsomes, curcumin, ginger extract, α-mangostin, quercetin and silybin inhibited ≥50% glucuronidation while chrysin, ginger extract, α-mangostin, pterostilbene and resveratrol exhibited ≥30% inhibition of oxidation. These results demonstrate the feasibility of our proposed approach of using GRAS or dietary compounds to inhibit the presystemic metabolism of buprenorphine and thus improve its oral bioavailability. An oral buprenorphine formulation containing these inhibitors or their combinations has promising potential to replace sublingual buprenorphine. Copyright © 2016 John Wiley & Sons, Ltd.

  2. An assessment of the use of native and denatured forms of okra seed proteins as coagulants in drinking water treatment.

    PubMed

    Jones, Alfred Ndahi; Bridgeman, John

    2016-10-01

    The effects of temperature, storage time and water pH on the coagulation performance of okra seed protein in water treatment were assessed. In a jar test experiment, okra salt extract achieved a notable improvement in treatment efficiency with storage time and showed good performance in quality after thermal treatment at 60, 97 and 140 °C temperatures for 6, 4 and 2 hours, respectively. The performance improvement of more than 8% is considered to be due to the denaturation and subsequent removal of coagulation-hindering proteins in okra seed. Furthermore, the results of a sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis show two distinctive bands of protein responsible for the coagulation process after denaturation. It was further shown that at optimal coagulant dose, the pH of the treated water remained unaffected as a result of the protein's buffering capability during coagulation. Therefore, denatured okra seed exhibited improved performance compared to the native crude extract and offers clear benefits as a water treatment coagulant.

  3. Human common salivary protein 1 (CSP-1) promotes binding of Streptococcus mutans to experimental salivary pellicle and glucans formed on hydroxyapatite surface.

    PubMed

    Ambatipudi, Kiran S; Hagen, Fred K; Delahunty, Claire M; Han, Xuemei; Shafi, Rubina; Hryhorenko, Jennifer; Gregoire, Stacy; Marquis, Robert E; Melvin, James E; Koo, Hyun; Yates, John R

    2010-12-03

    The saliva proteome includes host defense factors and specific bacterial-binding proteins that modulate microbial growth and colonization of the tooth surface in the oral cavity. A multidimensional mass spectrometry approach identified the major host-derived salivary proteins that interacted with Streptococcus mutans (strain UA159), the primary microorganism associated with the pathogenesis of dental caries. Two abundant host proteins were found to tightly bind to S. mutans cells, common salivary protein-1 (CSP-1) and deleted in malignant brain tumor 1 (DMBT1, also known as salivary agglutinin or gp340). In contrast to gp340, limited functional information is available on CSP-1. The sequence of CSP-1 shares 38.1% similarity with rat CSP-1. Recombinant CSP-1 (rCSP-1) protein did not cause aggregation of S. mutans cells and was devoid of any significant biocidal activity (2.5 to 10 μg/mL). However, S. mutans cells exposed to rCSP-1 (10 μg/mL) in saliva displayed enhanced adherence to experimental salivary pellicle and to glucans in the pellicle formed on hydroxyapatite surfaces. Thus, our data demonstrate that the host salivary protein CSP-1 binds to S. mutans cells and may influence the initial colonization of this pathogenic bacterium onto the tooth surface.

  4. A study of renaturation of reduced hen egg white lysozyme. Enzymically active intermediates formed during oxidation of the reduced protein.

    PubMed

    Acharya, A S; Taniuchi, H

    1976-11-25

    The material obtained from reduced hen egg white lysozyme after complete air oxidation at pH 8.0 and 37 degrees has yielded, by gel filtration on a Bio-Gel P-30 column, enzymically active species and an enzymically inactive form which eluted sooner than the active species but later than expected for a dimer of lysozyme. Reduced lysozyme also elutes at the same position as this inactive material. Examination of the fragments produced on CNBr cleavage of the inactive form indicates that at least 24% of the population contains incorrect disulfide bonds involving half-cystine residues 6, 30, 115, and 127. Tryptophan fluorescence and the intrinsic viscosity of the inactive form show an enlarged molecular domain with a disordered conformation. The yield of the inactive form increases as the oxidation of reduced lysozyme is accelerated using cupric ion. In the presence of 4 X 10(-5) M cupric ion, reduced lysozyme forms almost quantitatively the inactive form, which is almost completely converted to the native form by sulfhydryl-disulfide interchange catalyzed by thiol groups of either reduced lysozyme or beta-mercaptoethanol. The material trapped by alkylation of the free sulfhydryl groups with [1-14C]iodoacetic acid during the early stage of air oxidation of reduced lysozyme was fractionated by gel filtration to permit separation of the active species from the inactive form. Ion exchange chromatography of the active species yielded completely renatured lysozyme and three major enzymically active radioactive derivatives. Two of these derivatives contained approximately 2 mol of S-carboxymethylcysteine. Isolation and characterization of radioactive tryptic peptides from each of the three active forms, permitted the identification of Cys 6 and Cys 127, Cys 76 and 94, and Cys 80 as the sulfhydryl groups alkylated in these three incompletely oxidized, partially active forms. Thus, it appears that the interatomic interactions maintaining the compact three-dimensional structure

  5. Multiple phosphorylated forms of the Saccharomyces cerevisiae Mcm1 protein include an isoform induced in response to high salt concentrations.

    PubMed Central

    Kuo, M H; Nadeau, E T; Grayhack, E J

    1997-01-01

    The Saccharomyces cerevisiae Mcm1 protein is an essential multifunctional transcription factor which is highly homologous to human serum response factor. Mcm1 protein acts on a large number of distinctly regulated genes: haploid cell-type-specific genes, G2-cell-cycle-regulated genes, pheromone-induced genes, arginine metabolic genes, and genes important for cell wall and cell membrane function. We show here that Mcm1 protein is phosphorylated in vivo. Several (more than eight) isoforms of Mcm1 protein, resolved by isoelectric focusing, are present in vivo; two major phosphorylation sites lie in the N-terminal 17 amino acids immediately adjacent to the conserved MADS box DNA-binding domain. The implications of multiple species of Mcm1, particularly the notion that a unique Mcm1 isoform could be required for regulation of a specific set of Mcm1's target genes, are discussed. We also show here that Mcm1 plays an important role in the response to stress caused by NaCl. G. Yu, R. J. Deschenes, and J. S. Fassler (J. Biol. Chem. 270:8739-8743, 1995) showed that Mcm1 function is affected by mutations in the SLN1 gene, a signal transduction component implicated in the response to osmotic stress. We find that mcm1 mutations can confer either reduced or enhanced survival on high-salt medium; deletion of the N terminus or mutation in the primary phosphorylation site results in impaired growth on high-salt medium. Furthermore, Mcm1 protein is a target of a signal transduction system responsive to osmotic stress: a new isoform of Mcm1 is induced by NaCl or KCl; this result establishes that Mcm1 itself is regulated. PMID:9001236

  6. Effect of Fish Protein Hydrolysate on Gel-forming Ability, State of Water, and Denaturation of Wanieso Lizardfish Surimi during Frozen Storage

    NASA Astrophysics Data System (ADS)

    Khan, Mohammed Abu Ali; Hossain, Mohammed Anwar; Hara, Kenji; Osatomi, Kiyoshi; Ishihara, Tadashi; Nozaki, Yukinori

    Fish protein hydrolysate (FPH) was prepared from the underutilized residues of 3-marine fish species disposed of by-products processing by protease treatment. The major component of the FPH was hydrolyzed protein (82-86%). The concentration dependent protective effect of FPH (2.5-10.0%, dry weight/wet weight) on the wanieso lizardfish (Saurida wnieso) frozen surimi at -25°C was evaluated by determining gel-forming ability, unfrozen water, and myofibrillar Ca-ATPase activity. The gel-forming ability and total myofibrillar Ca-ATPase activity of the surimi with FPH showed significantly higher values than those of without FPH (control) during frozen storage. The amount of unfrozen water in the frozen surimi with FPH was significantly increased compared to that in the control. The results indicate that FPH stabilized the hydrated water surrounding myofibrils. These findings suggest that the FPH prevent the freeze-induced denaturation of the surimi during frozen storage.

  7. Conversion of protein kinase C from a Ca/sup 2 +/-dependent to an independent form of phorbol ester-binding protein by digestion with trypsin

    SciTech Connect

    Huang, K.P.; Huang, F.L.

    1986-08-29

    Tryptic fragments of protein kinase C containing the kinase (45 KDa) and phorbol ester-binding activity (38 KDa) were separated by Mono O column chromatography. The purified phorbol ester-binding fragment exhibits a higher affinity for phosphatidylserine than the native enzyme but comparable Kd for (/sup 3/H)phorbol 12,13-dibutyrate as the native enzyme. This proteolytic fragment binds phorbol ester equally efficient either in the presence or absence of Ca/sup 2 +/ and the addition of the kinase fragment did not restore the Ca/sup 2 +/-requirement for the binding. These results indicate that protein kinase C is composed of two functionally distinct units which can be expressed independently after limited proteolysis with trypsin.

  8. GRAS Notice 608

    Center for Food Safety and Applied Nutrition (CFSAN)

    ... non-consecutive batches for each analysis parameter are presented in Tables 4-8 ... 5-~~~!nalysi~- D~ta _!~~~~u te~~~~~_!~~!~lin __~~-ncentrate ...

  9. GRAS Notice 000076: ERYTHRITOL

    Center for Food Safety and Applied Nutrition (CFSAN)

    ... J'::l!:l I:::;mSI'O~:'I'.,e, t~(l fl3rr~~I.~~:~l -,... _ l ! t-u'~ NIf.. NF::.. NR:. NR. (l.\\l~;\\ t(R:. NR. ... <:P'~'~'I\\l.rAM~~ft~'t: <:U~:";:I::c,~,:.6 o ~~. ';1!.2~:11 ...

  10. Mardi Gras Math

    ERIC Educational Resources Information Center

    Eubanks-Turner, Christina; Hajj, Najat

    2015-01-01

    In this article, Christina Eubanks-Turner and Najat Hajj describe a planning process that they used to create a fun-filled eighth-grade math activity that focused on parade planning. The activity was designed to enhance and supplement the eighth-grade algebra curriculum on linear equations and functions, help students use mathematical habits of…

  11. The Fusobacterium nucleatum major outer-membrane protein (FomA) forms trimeric, water-filled channels in lipid bilayer membranes.

    PubMed

    Kleivdal, H; Benz, R; Jensen, H B

    1995-10-01

    The pore-forming activity of the major outer-membrane protein FomA of the anaerobic Fusobacterium nucleatum was studied in artificial lipid bilayer membranes. FomA was isolated from F. nucleatum strains Fev1, ATCC 10953, and ATCC 25586 by extraction with lithium dodecyl sulfate and lithium chloride and had an apparent molecular mass of about 40 kDa. When solubilized at low temperatures, the protein ran with an apparent molecular mass of about 62 kDa on SDS/PAGE. Cross-linking experiments and two-dimensional SDS/PAGE gave evidence that the 62-kDa protein band represented the trimeric form of FomA. The protein trimers were susceptible to SDS and temperature. The stability of the porin trimers varied among the strains. The properties of the FomA channels were studied in reconstitution experiments with black lipid bilayer membranes. The F. nucleatum porins formed channels with single-channel conductances in the range 0.66-1.30 nS in M KCl. The single-channel conductance was a function of the mobilities of the ions present in the aqueous solution bathing the bilayer membrane. This means that FomA forms general diffusion channels since (a) the conductance showed a linear dependence on the salt concentration, (b) the ion selectivity was small and varied for the three strains, and (c) the channels did not exhibit any binding site for maltotriose or triglycine. The water-filled channel was voltage dependent, and conductance decrements were observed at transmembrane potentials of +/- 50 mV. The conductance decrement steps were about one-third of the total conductance of a functional unit in its fully 'open' state. This strongly suggests that the trimer is the functional unit of the porin.

  12. Systematic investigation of crystallization parameters for protein-nucleic acid complexes: application to an active truncated form of Escherichia coli topoisomerase I

    NASA Astrophysics Data System (ADS)

    Weber, Patricia C.; Zhu, Chang-Xi; Tse-Dinh, Yuk-Ching

    1992-08-01

    An experimental strategy to systematically screen possible crystallization parameters for protein:oligonucleotide complexes is described and successfully used in the cocrystallization of a truncated form of Escherichia coli topoisomerase I with an oligo(thymidylic acid) polymer of 8 bases. Crystals are orthorhombic ( a = 65.9A˚, b =74.0A˚, and c = 140.2A˚) and diffract to at least 3A˚resolution.

  13. Discs large 1 (Dlg1) scaffolding protein participates with clathrin and adaptator protein complex 1 (AP-1) in forming Weibel-Palade bodies of endothelial cells.

    PubMed

    Philippe, Monique; Léger, Thibaut; Desvaux, Raphaëlle; Walch, Laurence

    2013-05-03

    Weibel-Palade bodies (WPBs) are specific cigar-shaped granules that store von Willebrand factor (VWF) for its regulated secretion by endothelial cells. The first steps of the formation of these granules at the trans-Golgi network specifically require VWF aggregation and an external scaffolding complex that contains the adaptator protein complex 1 (AP-1) and clathrin. Discs large 1 (Dlg1) is generally considered to be a modular scaffolding protein implicated in the control of cell polarity in a large variety of cells by specific recruiting of receptors, channels, or signaling proteins to specialized zones of the plasma membrane. We propose here that in endothelial cells, Dlg1, in a complex with AP-1 and clathrin, participates in the biogenesis of WPBs. Supporting data show that Dlg1 colocalizes with microtubules, intermediate filaments, and Golgi markers. Tandem mass spectrometry experiments led to the identification of clathrin as an Dlg1-interacting partner. Interaction was confirmed by in situ proximity ligation assays. Furthermore, AP-1 and VWF immunoprecipitate and colocalize with Dlg1 in the juxtanuclear zone. Finally, Dlg1 depletion by siRNA duplexes disrupts trans-Golgi network morphology and WPB formation. Our results provide the first evidence for an unexpected role of Dlg1 in controlling the formation of specific secretory granules involved in VWF exocytosis in endothelial cells.

  14. A sorting nexin-1 homologue, Vps5p, forms a complex with Vps17p and is required for recycling the vacuolar protein-sorting receptor.

    PubMed Central

    Horazdovsky, B F; Davies, B A; Seaman, M N; McLaughlin, S A; Yoon, S; Emr, S D

    1997-01-01

    A number of the Saccharomyces cerevisiae vacuolar protein-sorting (vps) mutants exhibit an altered vacuolar morphology. Unlike wild-type cells that contain 1-3 large vacuolar structures, the class B vps5 and vps17 mutant cells contain 10-20 smaller vacuole-like compartments. To explore the role of these VPS gene products in vacuole biogenesis, we cloned and sequenced VPS5 and characterized its protein products. The VPS5 gene is predicted to encode a very hydrophilic protein of 675 amino acids that shows significant sequence homology with mammalian sorting nexin-1. Polyclonal antiserum directed against the VPS5 gene product detects a single, cytoplasmic protein that is phosphorylated specifically on a serine residue(s). Subcellular fractionation studies indicate that Vps5p is associated peripherally with a dense membrane fraction distinct from Golgi, endosomal, and vacuolar membranes. This association was found to be dependent on the presence of another class B VPS gene product, Vps17p. Biochemical cross-linking studies demonstrated that Vps5p and Vps17p physically interact. Gene disruption experiments show that the VPS5 genes product is not essential for cell viability; however, cells carrying the null allele contain fragmented vacuoles and exhibit defects in vacuolar protein-sorting similar to vps17 null mutants. More than 95% of carboxypeptidase Y is secreted from these cells in its Golgi-modified p2 precursor form. Additionally, the Vps10p vacuolar protein-sorting receptor is mislocalized to the vacuole in vps5 mutant cells. On the basis of these and other observations, we propose that the Vps17p protein complex may participate in the intracellular trafficking of the Vps10p-sorting receptor, as well as other later-Golgi proteins. Images PMID:9285823

  15. Involvement of a non-hormone-binding 90-kilodalton protein in the nontransformed 8S form of the rabbit uterus progesterone receptor

    SciTech Connect

    Renoir, J.M.; Buchou, T.; Baulieu, E.E.

    1986-10-21

    Nontransformed 8S progesterone receptor (8S-PR) was purified by hormone-specific affinity chromatography from rabbit uterine low-salt cytosol containing 20 mM molybdate. In the eluate obtained with radioactive progestin, sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) showed the presence of several bands, including three that corresponded to approx.90, approx.120, and approx.85-kDa proteins. None of these three proteins was found in the eluate of the affinity column when the molybdate-containing cytosol was chromatographed in the presence of nonradioactive progesterone (mock purification). Subsequent purification of the affinity eluate by DEAE-Sephacel chromatography gave a single radioactive receptor peak at 0.15 M KCl with a sedimentation coefficient of 8.5 S. Silver staining after SDS-PAGE revealed that this purified 8S-PR fraction contained mainly the 120-, 90-, and 85-kDa proteins. (/sup 3/H)R5020-labeled 8S-PR purified by DEAE-Sephacel column chromatography was UV irradiated, and after SDS-PAGE the 120- and 85-kDa proteins were revealed, but the 90-kDa protein was not. Further evidence for the presence of the 90-kDa non-hormone-binding protein in the purified molybdate-stabilized nontransformed 8S-PR structure was demonstrated. In the course of this work, it was verified that 0.3 M KCl added in cytosol in the absence of molybdate dissociated the 8S-PR complex, and purified 120- and 85-kDa progestin binding proteins were obtained by hormone-specific affinity chromatography of the salt-treated cytosol. In summer, the results demonstrated that, as for the nontransformed avian 8S-PR the nontransformed 8S form of the rabbit uterus PR includes a non-hormone-binding 90-kDa protein.

  16. Crystal structure of Helicobacter pylori neutrophil-activating protein with a di-nuclear ferroxidase center in a zinc or cadmium-bound form

    SciTech Connect

    Yokoyama, Hideshi; Tsuruta, Osamu; Akao, Naoya; Fujii, Satoshi

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Structures of a metal-bound Helicobacter pylori neutrophil-activating protein were determined. Black-Right-Pointing-Pointer Two zinc ions were tetrahedrally coordinated by ferroxidase center (FOC) residues. Black-Right-Pointing-Pointer Two cadmium ions were coordinated in a trigonal-bipyramidal and octahedral manner. Black-Right-Pointing-Pointer The second metal ion was more weakly coordinated than the first at the FOC. Black-Right-Pointing-Pointer A zinc ion was found in one negatively-charged pore suitable as an ion path. -- Abstract: Helicobacter pylori neutrophil-activating protein (HP-NAP) is a Dps-like iron storage protein forming a dodecameric shell, and promotes adhesion of neutrophils to endothelial cells. The crystal structure of HP-NAP in a Zn{sup 2+}- or Cd{sup 2+}-bound form reveals the binding of two zinc or two cadmium ions and their bridged water molecule at the ferroxidase center (FOC). The two zinc ions are coordinated in a tetrahedral manner to the conserved residues among HP-NAP and Dps proteins. The two cadmium ions are coordinated in a trigonal-bipyramidal and distorted octahedral manner. In both structures, the second ion is more weakly coordinated than the first. Another zinc ion is found inside of the negatively-charged threefold-related pore, which is suitable for metal ions to pass through.

  17. Interleukin-13 receptors on human prostate carcinoma cell lines represent a novel target for a chimeric protein composed of IL-13 and a mutated form of Pseudomonas exotoxin.

    PubMed

    Maini, A; Hillman, G; Haas, G P; Wang, C Y; Montecillo, E; Hamzavi, F; Pontes, J E; Leland, P; Pastan, I; Debinski, W; Puri, R K

    1997-09-01

    We have discovered a new cell surface protein in the form of interleukin-13 receptor on several solid tumor cells, including human renal cell carcinoma cells (Obiri et al., 1995; Debinski et al., 1995). This study reports that human prostate cancer cell lines also express high affinity IL-13 receptors (Kd = 159 pM). These receptors are functional because IL-13 surprisingly increased proliferation of all three prostate cancer cell lines studied as determined by thymidine uptake and clonogenic assays. IL-13 receptors on prostate cancer cell lines were targeted using a chimeric protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (PE38QQR). This molecule, termed IL13-PE38QQR, has been found cytotoxic to all three prostate cancer cell lines as determined by the inhibition of protein synthesis. The IC50 ranged between 1 nmol/l, to 15 nmol/l. These data were confirmed by clonogenic assays in which IL13-PE38QQR almost completely inhibited colony formation at 10 nmol/l. IL13-PE38QQR was not cytotoxic to cells that express little or no IL-13R. Heat inactivated IL13-PE38QQR was not cytotoxic to prostate cancer cells indicating specificity. IL13-PE38QQR was also cytotoxic to colonies when they were allowed to form first for several days before the addition of toxins. Our data suggest that additional studies should be performed to target IL-13 receptor bearing prostate cancer.

  18. Förster Resonance Energy Transfer-Paired Hydrogel Forming Silk-Elastin-Like Recombinamers by Recombinant Conjugation of Fluorescent Proteins.

    PubMed

    Ibáñez-Fonseca, Arturo; Alonso, Matilde; Arias, Francisco Javier; Rodríguez-Cabello, José Carlos

    2017-02-15

    In the last decades, recombinant structural proteins have become very promising in addressing different issues such as the lack of traceability of biomedical devices or the design of more sensitive biosensors. Among them, we find elastin-like recombinamers (ELRs), which can be designed to self-assemble into diverse structures, such as hydrogels. Furthermore, they might be combined with other protein polymers, such as silk, to give silk-elastin-like recombinamers (SELRs), holding the properties of both proteins. In this work, due to their recombinant nature, we have fused two different fluorescent proteins (FPs), i.e., the green Aequorea coerulescens enhanced green fluorescent protein and the near-infrared eqFP650, to a SELR able to form irreversible hydrogels through physical cross-linking. These recombinamers showed an emission of fluorescence similar to the single FPs, and they were capable of forming hydrogels with different stiffness (G' = 60-4000 Pa) by varying the concentration of the SELR-FPs. Moreover, the absorption spectrum of SELR-eqFP650 showed a peak greatly overlapping the emission spectrum of the SELR-Aequorea coerulescens enhanced green fluorescent protein. Hence, this enables Förster resonance energy transfer (FRET) upon the interaction between two SELR molecules, each one containing a different FP, due to the stacking of silk domains at any temperature and to the aggregation of elastin-like blocks above the transition temperature. This effect was studied by different methods, and a FRET efficiency of 0.06-0.2 was observed, depending on the technique used for its calculation. Therefore, innovative biological applications arise from the combination of SELRs with FPs, such as enhancing the traceability of hydrogels based on SELRs intended for tissue engineering, the development of biosensors, and the prediction of FRET efficiencies of novel FRET pairs.

  19. RNA-binding proteins SOP-2 and SOR-1 form a novel PcG-like complex in C. elegans.

    PubMed

    Zhang, Tingting; Sun, Yinyan; Tian, E; Deng, Hansong; Zhang, Yuxia; Luo, Xin; Cai, Qingchun; Wang, Huayi; Chai, Jijie; Zhang, Hong

    2006-03-01

    We describe the identification and characterization of a novel PcG gene in C. elegans, sor-1, which is involved in global repression of Hox genes. sor-1 encodes a novel protein with an RNA-binding activity. We provide evidence that SOR-1 and the previously identified RNA-binding protein SOP-2 may constitute an RNA-binding complex in Hox gene repression. SOR-1 and SOP-2 directly interact with each other and are colocalized in nuclear bodies. The localization of SOR-1 depends on SOP-2. Surprisingly, homologs of SOR-1 and SOP-2 are not found in other organisms, including the congeneric species C. briggsae, suggesting an unexpected lack of evolutionary constraint on an essential global gene regulatory system.

  20. The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export.

    PubMed

    Port, Sarah A; Mendes, Adélia; Valkova, Christina; Spillner, Christiane; Fahrenkrog, Birthe; Kaether, Christoph; Kehlenbach, Ralph H

    2016-10-28

    Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A)(+) RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A)(+) RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors.

  1. Noncanonical FK506-binding Protein BDBT Binds DBT to Enhance its Circadian Function and Forms Foci at Night

    PubMed Central

    Fan, Jin-Yuan; Agyekum, Boadi; Venkatesan, Anandakrishnan; Hall, David R.; Keightley, Andrew; Bjes, Edward S.; Bouyain, Samuel; Price, Jeffrey L.

    2013-01-01

    SUMMARY The kinase DOUBLETIME is a master regulator of the Drosophila circadian clock, yet the mechanisms regulating its activity remain unclear. A proteomic analysis of DOUBLETIME interactors led to the identification of an unstudied protein designated CG17282. RNAi-mediated knock-down of CG17282 produced behavioral arrhythmicity and long periods, high levels of hypophosphorylated nuclear PERIOD and phosphorylated DOUBLETIME. Overexpression of DOUBLETIME in flies suppresses these phenotypes and overexpression of CG17282 in S2 cells enhances DOUBLETIME-dependent PERIOD degradation, indicating that CG17282 stimulates DOUBLETIME’s circadian function. In photoreceptors, CG17282 accumulates rhythmically in PERIOD- and DOUBLETIME-dependent cytosolic foci. Finally, structural analyses demonstrated CG17282 is a noncanonical FK506-binding protein with an inactive peptide prolyl-isomerase domain that binds DOUBLETIME and tetratricopeptide repeats that may promote assembly of larger protein complexes. We have named CG17282 Bride of Doubletime and established it as a mediator of DOUBLETIME’s effects on PERIOD, most likely in cytosolic foci that regulate PERIOD nuclear accumulation. PMID:24210908

  2. Photochemically Generated Elemental Selenium Forms Conjugates with Serum Proteins that Are Preferentially Cytotoxic to Leukemia and Selected Solid Tumor Cells

    PubMed Central

    Daziano, Jean-Pierre; Günther, Wolfgang H.H.; Krieg, Marianne; Tsujino, Ichiro; Miyagi, Kiyoko; Anderson, Gregory S.; Sampson, Reynée W.; Ostrowski, Martin D.; Muir, Sarah A.; Bula, Raymond J.; Sieber, Fritz

    2012-01-01

    The objective of this study was to determine if and how photoproducts contribute to the anti-tumor effect of merocyanine-mediated PDT. A panel of barbituric, thiobarbituric and selenobarbituric acid analogues of Merocyanine 540 was photobleached, and the resulting photoproducts were characterized by absorption, fluorescence emission, mass, energy dispersive X-ray, and X-ray photoelectron spectroscopy, and tested for cytotoxic activity against tumor cell lines and freshly explanted bone marrow cells. While all dyes were readily photobleached, only photoproducts of selone dyes showed cytotoxic activity. One-hour incubations with micromolar concentrations of selone-derived photoproducts were sufficient to reduce leukemia/lymphoma cells ≥10,000 fold while preserving virtually all normal CD34-positive bone marrow cells. Of 6 multi-drug resistant tumor cell lines tested, 5 were as sensitive or more sensitive to photoproducts than the corresponding wild-type lines. Physicochemical characterizations of the cytotoxic activity indicated that it consisted of conjugates of subnano particles of elemental selenium and (lipo)proteins. The discovery of cytotoxic Se-protein conjugates provides a rare example of photoproducts contributing substantially to the anti-tumor effect of PDT and challenges the long-held view that Se in oxidation state zero is biologically inert. Agents modeled after our Se-protein conjugates may prove useful for the treatment of leukemia. PMID:22211823

  3. Enhanced extraction of proteins using cholinium-based ionic liquids as phase-forming components of aqueous biphasic systems.

    PubMed

    Quental, Maria V; Caban, Magda; Pereira, Matheus M; Stepnowski, Piotr; Coutinho, João A P; Freire, Mara G

    2015-09-01

    Aqueous biphasic systems (ABS) composed of ionic liquids (ILs) are promising platforms for the extraction and purification of proteins. In this work, a series of alternative and biocompatible ABS composed of cholinium-based ILs and polypropylene glycol were investigated. The respective ternary phase diagrams, tie-lines, tie-line lengths and critical points were determined at 25°C. The extraction performance of these systems for commercial bovine serum albumin (BSA) was then evaluated. The stability of BSA at the IL-rich phase was ascertained by size exclusion high-performance liquid chromatography and Fourier transform infrared spectroscopy. Appropriate ILs lead to the complete extraction of BSA for the IL-rich phase, in a single step, while maintaining the protein's native conformation. Furthermore, to evaluate the performance of these systems when applied to real matrices, the extraction of BSA from bovine serum was additionally carried out, revealing that the complete extraction of BSA was maintained and achieved in a single step. The remarkable extraction efficiencies obtained are far superior to those observed with typical polymer-based ABS. Therefore, the proposed ABS may be envisaged as a more effective and biocompatible approach for the separation and purification of other value-added proteins.

  4. PTPN14 Forms a Complex with Kibra and LATS1 Proteins and Negatively Regulates the YAP Oncogenic Function*

    PubMed Central

    Wilson, Kayla E.; Li, Ying-Wei; Yang, Nuo; Shen, He; Orillion, Ashley R.; Zhang, Jianmin

    2014-01-01

    The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. Pivotal effectors of this pathway are YAP/TAZ, transcriptional co-activators whose dysfunction contributes to epithelial-to-mesenchymal transition and malignant transformation. Therefore, it is of great importance to decipher the mechanisms underlying the regulations of YAP/TAZ at various levels. Here we report that non-receptor tyrosine phosphatase 14 (PTPN14) interacts with the Kibra protein. The interaction between PTPN14 and Kibra is through the PPXY domain of PTPN14 and WW domain of Kibra. PTPN14 and Kibra can induce the LATS1 activation independently and cooperatively. Interestingly, activation of LATS1 by PTPN14 is dependent on the C terminus of PTPN14 and independent of the upstream mammalian STE20-like kinase (MST) proteins. Furthermore, we demonstrate that PTPN14 increases the LAST1 protein stability. Last, overexpression of Kibra rescues the increased cell migration and aberrant three-dimensional morphogenesis induced by knockdown of PTPN14, and this rescue is mediated through the activation of the upstream LATS1 kinase and subsequent cytoplasmic sequestration of YAP. In summary, our results indicate a potential regulatory role of PTPN14 in the Hippo pathway and demonstrate another layer of regulation in the YAP oncogenic function. PMID:25023289

  5. Combination of collagen and fibronectin to design biomimetic interfaces: Do these proteins form layer-by-layer assemblies?

    PubMed

    Mauquoy, Sara; Dupont-Gillain, Christine

    2016-11-01

    Layer-by-layer (LbL) assembly is a surface modification method which may bring complexity to biointerfaces designed to control cell-material interactions. This work aims at investigating the LbL assembly of two extracellular matrix proteins, collagen (Col) and fibronectin (Fn), on polystyrene substrates. LbL assembly, which is widely applied to polyelectrolytes, is not easily transferred to proteins. Different buffers and conditions are tested, and LbL assembly is compared to the simultaneous adsorption of Fn and Col. Build-up and properties of the films are monitored using quartz crystal microbalance, ellipsometry, water contact angle measurements, and atomic force microscopy. Results show that denatured Col leads to smoother films, and that the addition of a polyethyleneimine anchoring layer increases film thickness. A more regular construction and thicker films are obtained with Hepes (pH 7.4) compared to other buffers. However, the LbL assembly is not sustainable and stops after the deposition of a few layers. Films obtained by simultaneous adsorption have lower water contact angles, different morphologies, lower water content and are as thick or thicker compared to the ones prepared by the LbL method. The present work shows that collagen and fibronectin are not involved in a true LbL assembly process. The obtained biointerfaces however exhibit different properties compared to those obtained by the one-step adsorption of these proteins. These differences could be exploited to control cell fate.

  6. A Fusion between Domains of the Human Bone Morphogenetic Protein-2 and Maize 27 kD γ-Zein Accumulates to High Levels in the Endoplasmic Reticulum without Forming Protein Bodies in Transgenic Tobacco

    PubMed Central

    Ceresoli, Valentina; Mainieri, Davide; Del Fabbro, Massimo; Weinstein, Roberto; Pedrazzini, Emanuela

    2016-01-01

    Human Bone Morphogenetic Protein-2 (hBMP2) is an osteoinductive agent physiologically involved in bone remodeling processes. A commercialized recombinant hBMP2 produced in mammalian cell lines is available in different clinical applications where bone regeneration is needed, but widespread use has been hindered due to an unfavorable cost/effective ratio. Protein bodies are very large insoluble protein polymers that originate within the endoplasmic reticulum by prolamine accumulation during the cereal seed development. The N-terminal domain of the maize prolamin 27 kD γ-zein is able to promote protein body biogenesis when fused to other proteins. To produce high yield of recombinant hBMP2 active domain (ad) in stably transformed tobacco plants we have fused it to the γ-zein domain. We show that this zein-hBMP2ad fusion is retained in the endoplasmic reticulum without forming insoluble protein bodies. The accumulation levels are above 1% of total soluble leaf proteins, indicating that it could be a rapid and suitable strategy to produce hBMP2ad at affordable costs. PMID:27047526

  7. A Protein Synthesis and Nitric Oxide-Dependent Presynaptic Enhancement in Persistent Forms of Long-Term Potentiation

    ERIC Educational Resources Information Center

    Johnstone, Victoria P. A.; Raymond, Clarke R.

    2011-01-01

    Long-term potentiation (LTP) is an important process underlying learning and memory in the brain. At CA3-CA1 synapses in the hippocampus, three discrete forms of LTP (LTP1, 2, and 3) can be differentiated on the basis of maintenance and induction mechanisms. However, the relative roles of pre- and post-synaptic expression mechanisms in LTP1, 2,…

  8. Expression of the constitutive and inducible forms of heat shock protein 70 in human proximal tubule cells exposed to heat, sodium arsenite, and CdCl(2).

    PubMed Central

    Somji, S; Todd, J H; Sens, M A; Garrett, S H; Sens, D A

    1999-01-01

    We determined the expression of the constitutive (hsc 70) and inducible (hsp 70) forms of heat shock protein 70 mRNA and protein in human proximal tubule (HPT) cells exposed to lethal and sublethal concentrations of Cd(+2) under both acute and extended conditions of exposure. The HPT cells exhibited the classic heat shock response when subjected to a physical (heat) or chemical stress (sodium arsenite); hsc 70 mRNA and protein levels were constant or slightly increased, whereas hsp 70 mRNA and protein were greatly elevated. Acute exposure to 53.4 microM CdCl(2) for 4 hr failed to increase either hsc 70 mRNA or protein, a finding similar to that observed under classic conditions of stress. However, under identical conditions of acute exposure to Cd(2+), the expected increase in hsp 70 protein level was suppressed as compared to that found under classic conditions of physical or chemical stress. The decrease in hsp 70 protein level correlated to the reduced expression of mRNA from the hsp 70B gene. The expression of mRNA from the hsp 70A and hsp 70C genes was similar to that found when the cells were treated with heat shock or sodium arsenite. We modeled an extended exposure to Cd(2+) by treating the cells continuously with Cd(2+) at both lethal and sublethal levels over a 16-day time course. Chronic exposure to Cd(2+) failed to increase either hsc 70 mRNA or protein levels in the HPT cells at a nonlethal dosage level and decreased hsc 70 mRNA and protein levels late in the time course of lethal exposure. Under identical conditions, the expression of hsp 70 protein remained at basal levels that were only marginally detectable throughout the time course. Hsp 70A and hsp 70C mRNA levels were unaltered by extended exposure to Cd(2+), and hsp 70B mRNA was not detected during the 16-day time course. Cd(2+) is a poor inducer of hsc 70 and hsp 70 in the proximal tubule under both acute and long-term exposure. These results reinforce the fact that the expression of hsp 70

  9. Sent packing: protein engineering generates a new crystal form of Pseudomonas aeruginosa DsbA1 with increased catalytic surface accessibility

    SciTech Connect

    McMahon, Roisin M. Coinçon, Mathieu; Tay, Stephanie; Heras, Begoña; Morton, Craig J.; Scanlon, Martin J.; Martin, Jennifer L.

    2015-11-26

    The crystal structure of a P. aeruginosa DsbA1 variant is more suitable for fragment-based lead discovery efforts to identify inhibitors of this antimicrobial drug target. In the reported structures the active site of the protein can simultaneously bind multiple ligands introduced in the crystallization solution or via soaking. Pseudomonas aeruginosa is an opportunistic human pathogen for which new antimicrobial drug options are urgently sought. P. aeruginosa disulfide-bond protein A1 (PaDsbA1) plays a pivotal role in catalyzing the oxidative folding of multiple virulence proteins and as such holds great promise as a drug target. As part of a fragment-based lead discovery approach to PaDsbA1 inhibitor development, the identification of a crystal form of PaDsbA1 that was more suitable for fragment-soaking experiments was sought. A previously identified crystallization condition for this protein was unsuitable, as in this crystal form of PaDsbA1 the active-site surface loops are engaged in the crystal packing, occluding access to the target site. A single residue involved in crystal-packing interactions was substituted with an amino acid commonly found at this position in closely related enzymes, and this variant was successfully used to generate a new crystal form of PaDsbA1 in which the active-site surface is more accessible for soaking experiments. The PaDsbA1 variant displays identical redox character and in vitro activity to wild-type PaDsbA1 and is structurally highly similar. Two crystal structures of the PaDsbA1 variant were determined in complex with small molecules bound to the protein active site. These small molecules (MES, glycerol and ethylene glycol) were derived from the crystallization or cryoprotectant solutions and provide a proof of principle that the reported crystal form will be amenable to co-crystallization and soaking with small molecules designed to target the protein active-site surface.

  10. A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice

    SciTech Connect

    Valdes, Iris; Bernardo, Lidice; Pavon, Alekis; Guzman, Maria G.

    2009-11-25

    Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-gamma secretion and protection experiments, mediated by CD4{sup +} and CD8{sup +} cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

  11. TYPE-ONE PROTEIN PHOSPHATASE4 Regulates Pavement Cell Interdigitation by Modulating PIN-FORMED1 Polarity and Trafficking in Arabidopsis1

    PubMed Central

    Guo, Xiaola; Qin, Qianqian; Yan, Jia; Niu, Yali; Huang, Bingyao; Guan, Liping; Li, Yuan; Ren, Dongtao; Li, Jia; Hou, Suiwen

    2015-01-01

    In plants, cell morphogenesis is dependent on intercellular auxin accumulation. The polar subcellular localization of the PIN-FORMED (PIN) protein is crucial for this process. Previous studies have shown that the protein kinase PINOID (PID) and protein phosphatase6-type phosphatase holoenzyme regulate the phosphorylation status of PIN1 in root tips and shoot apices. Here, we show that a type-one protein phosphatase, TOPP4, is essential for the formation of interdigitated pavement cell (PC) pattern in Arabidopsis (Arabidopsis thaliana) leaf. The dominant-negative mutant topp4-1 showed severely inhibited interdigitated PC growth. Expression of topp4-1 gene in wild-type plants recapitulated the PC defects in the mutant. Genetic analyses suggested that TOPP4 and PIN1 likely function in the same pathway to regulate PC morphogenesis. Furthermore, colocalization, in vitro and in vivo protein interaction studies, and dephosphorylation assays revealed that TOPP4 mediated PIN1 polar localization and endocytic trafficking in PCs by acting antagonistically with PID to modulate the phosphorylation status of PIN1. In addition, TOPP4 affects the cytoskeleton pattern through the Rho of Plant GTPase-dependent auxin-signaling pathway. Therefore, we conclude that TOPP4-regulated PIN1 polar targeting through direct dephosphorylation is crucial for PC morphogenesis in the Arabidopsis leaf. PMID:25560878

  12. Aberrant processing forms of lung surfactant proteins SP-B and SP-C revealed by high-resolution mass spectrometry.

    PubMed

    Galetskiy, Dmitry; Woischnik, Markus; Ripper, Jan; Griese, Matthias; Przybylski, Michael

    2008-01-01

    The mutation (g.1286T>C) of the pulmonary surfactant-associated protein C gene (SFTPC) leads to the I73T substitution in the precursor protein (pro-SP-C) and results in interstitial lung disease with the histological pattern of non-specific interstitial pneumonia and pulmonary alveolar proteinosis. Central for the disease is the abnormal processing of the SP-C pro-protein to mature SP-C; however little is known about the nature of intermediates and processing products. We report here the application of high resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry to the characterization of processing intermediates of hydrophobic pulmonary surfactant proteins SP-B and SP-C in intra- alveolar surfactant material of a patient with I73T mutation. SP-C and SP-B processing forms were separated from broncho-alveolar lavage fluid using chloroform/methanol extraction and sodium dodecyl sulfate poly acrylamide gel electrophoreis, detected by Western blot and identified by electrospray- and matrix-assisted laser desorption/ionization-FT-ICR mass spectrometry. The mass spectrometric and immuno-analytical results show the intra-alveolar accumulation of an aberrant C-terminal SP-C processing products in which the mature SP-C protein part is missing and aberrant processing intermediates of SP-B.

  13. A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice.

    PubMed

    Valdés, Iris; Bernardo, Lidice; Gil, Lázaro; Pavón, Alekis; Lazo, Laura; López, Carlos; Romero, Yaremis; Menendez, Ivón; Falcón, Viviana; Betancourt, Lázaro; Martín, Jorge; Chinea, Glay; Silva, Ricardo; Guzmán, María G; Guillén, Gerardo; Hermida, Lisset

    2009-11-25

    Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-gamma secretion and protection experiments, mediated by CD4(+) and CD8(+) cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

  14. DELLA proteins regulate expression of a subset of AM symbiosis-induced genes in Medicago truncatula.

    PubMed

    Floss, Daniela S; Lévesque-Tremblay, Véronique; Park, Hee-Jin; Harrison, Maria J

    2016-01-01

    The majority of the vascular flowering plants form symbiotic associations with fungi from the phylum Glomeromycota through which both partners gain access to nutrients, either mineral nutrients in the case of the plant, or carbon, in the case of the fungus. (1) The association develops in the roots and requires substantial remodeling of the root cortical cells where branched fungal hyphae, called arbuscules, are housed in a new membrane-bound apoplastic compartment. (2) Nutrient exchange between the symbionts occurs over this interface and its development and maintenance is critical for symbiosis. Previously, we showed that DELLA proteins, which are well known as repressors of gibberellic acid signaling, also regulate development of AM symbiosis and are necessary to enable arbuscule development. (3) Furthermore, constitutive overexpression of a dominant DELLA protein (della1-Δ18) is sufficient to induce transcripts of several AM symbiosis-induced genes, even in the absence of the fungal symbiont. (4) Here we further extend this approach and identify AM symbiosis genes that respond transcriptionally to constitutive expression of a dominant DELLA protein and also genes that do respond to this treatment. Additionally, we demonstrate that DELLAs interact with REQUIRED FOR ARBUSCULE DEVELOPMENT 1 (RAD1) which further extends our knowledge of GRAS factor complexes that have the potential to regulate gene expression during AM symbiosis.

  15. Mixed anhydrides (phosphoric-carboxyl) are also formed in the esterification of 5'-amp with n-acetylaminoacyl imidazolides - Implications regarding the origin of protein synthesis

    NASA Technical Reports Server (NTRS)

    Wickramasinghe, Nalinie S. M. D.; Lacey, James C., Jr.

    1992-01-01

    Procedure for the formation of aminoacyl esters of monoribonucleotides with aminoacyl imidazolides were first reported by Gottikh et al. (1970) and summarized in 1970. This reaction has been widely used by us and numbers of other workers as a convenient means of preparing aminoacyl esters of nucleotides. We have previously reported that, under conditions of excess imidazolide, large amounts of bis 2', 3' esters are formed in addition to the monoesters. However, to our knowledge, no one has reported that in addition to the esters, relatively large amounts of the mixed anhydride, with the amino acid carboxyl attached to the phosphate, are also formed at short reaction times. We report here on the relative amounts of anhydride and esters formed in this reaction of racemic mixtures of eleven N-acetyl amino acid imidazolides with 5'-AMP and discuss the relevance of the findings to the origin of protein synthesis.

  16. A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

    NASA Technical Reports Server (NTRS)

    Karpova, E. A.; Kubareva, E. A.; Shabarova, Z. A.

    1999-01-01

    To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

  17. Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA.

    PubMed

    Tullman, Jennifer; Guntas, Gurkan; Dumont, Matthew; Ostermeier, Marc

    2011-11-01

    We demonstrate that S1 nuclease converts supercoiled plasmid DNA to unit-length, linear dsDNA through the creation of a single, double-stranded break in a plasmid molecule. These double-stranded breaks occur not only in the origin of replication near inverted repeats but also at a wide variety of locations throughout the plasmid. S1 nuclease exhibits this activity under conditions typically employed for the nuclease's single-stranded nuclease activity. Thus, S1 nuclease digestion of plasmid DNA, unlike analogous digestion with DNaseI, effectively halts after the first double-stranded break. This property makes easier the construction of large domain insertion libraries in which the goal is to insert linear DNA at a variety of locations throughout a plasmid. We used this property to create a library in which a circularly permuted TEM1 β-lactamase gene was inserted throughout a plasmid containing the gene encoding Escherichia coli ribose binding protein. Gene fusions that encode allosteric switch proteins in which ribose modulates β-lactamase catalytic activity were isolated from this library using a combination of a genetic selection and a screen.

  18. Human cord blood T-cell receptor alpha beta cell responses to protein antigens of Paracoccidioides brasiliensis yeast forms.

    PubMed Central

    Munk, M E; Kaufmann, S H

    1995-01-01

    Paracoccidioides brasiliensis causes a chronic granulomatous mycosis, prevalent in South America, and cell-mediated immunity represents the principal mode of protection against this fungal infection. We investigated the response of naive cord blood T cells to P. brasiliensis lysates. Our results show: (1) P. brasiliensis stimulates T-cell expansion, interleukin-2 (IL-2) production and differentiation into cytotoxic T cells; (2) T-cell stimulation depends on P. brasiliensis processing and major histocompatibility complex (MHC) class II expression; (3) the responsive T-cell population expresses alpha beta T-cell receptors (TCR) with different V beta gene products, CD4 and CD45RO; (4) the P. brasiliensis components involved in T-cell expansion primarily reside in a high molecular weight (100,000 MW) and a low molecular weight (< 1000 MW) protein fraction. These results indicate that protein antigens of P. brasiliensis stimulate cord blood CD4 alpha beta T cells, independent from in vivo presensitization, and thus question direct correlation of positive in vitro responses with protective immunity in vivo. PMID:7890308

  19. Cysteine-rich secretory proteins in snake venoms form high affinity complexes with human and porcine beta-microseminoproteins.

    PubMed

    Hansson, Karin; Kjellberg, Margareta; Fernlund, Per

    2009-08-01

    BETA-microseminoprotein (MSP), a 10 kDa protein in human seminal plasma, binds human cysteine-rich secretory protein-3 (CRISP-3) with high affinity. CRISP-3 is a member of the family of CRISPs, which are widespread among animals. In this work we show that human as well as porcine MSP binds catrin, latisemin, pseudecin, and triflin, which are CRISPs present in the venoms of the snakes Crotalus atrox, Laticauda semifasciata, Pseudechis porphyriacus, and Trimeresurus flavoviridis, respectively. The CRISPs were purified from the venoms by affinity chromatography on a human MSP column and their identities were settled by gel electrophoresis and mass spectrometry. Their interactions with human and porcine MSPs were studied with size exclusion chromatography and surface plasmon resonance measurements. The binding affinities at 25 degrees C were between 10(-10)M and 10(-7)M for most of the interactions, with higher affinities for the interactions with porcine MSP compared to human MSP and with Elapidae CRISPs compared to Viperidae CRISPs. The high affinities of the bindings in spite of the differences in amino acid sequence between the MSPs as well as between the CRISPs indicate that the binding is tolerant to amino acid sequence variation and raise the question how universal this cross-species reaction between MSPs and CRISPs is.

  20. Tl(+) showed negligible interaction with inner membrane sulfhydryl groups of rat liver mitochondria, but formed complexes with matrix proteins.

    PubMed

    Korotkov, Sergey M; Brailovskaya, Irina V; Kormilitsyn, Boris N; Furaev, Viktor V

    2014-04-01

    The effects of Tl(+) on protein sulfhydryl (SH) groups, swelling, and respiration of rat liver mitochondria (RLM) were studied in a medium containing TlNO3 and sucrose, or TlNO3 and KNO3 as well as glutamate plus malate, or succinate plus rotenone. Detected with Ellman's reagent, an increase in the content of the SH groups was found in the inner membrane fraction, and a simultaneous decline was found in the content of the matrix-soluble fraction for RLM, incubated and frozen in 25-75 mM TlNO3 . This increase was greater in the medium containing KNO3 regardless of the presence of Ca(2+) . It was eliminated completely for RLM injected in the medium containing TlNO3 and then washed and frozen in the medium containing KNO3 . Calcium-loaded RLM showed increased swelling and decreased respiration. These results suggest that a ligand interaction of Tl(+) with protein SH groups, regardless of the presence of calcium, may underlie the mechanism of thallium toxicity.

  1. Group 1 metabotropic glutamate receptors 1 and 5 form a protein complex in mouse hippocampus and cortex

    PubMed Central

    Pandya, Nikhil J.; Klaassen, Remco V.; van der Schors, Roel C.; Slotman, Johan A.; Houtsmuller, Adriaan; Smit, August B.

    2016-01-01

    The group 1 metabotropic glutamate receptors 1 and 5 (mGluR1/5) have been implicated in mechanisms of synaptic plasticity and may serve as potential therapeutic targets in autism spectrum disorders. The interactome of group 1 mGluRs has remained largely unresolved. Using a knockout‐controlled interaction proteomics strategy we examined the mGluR5 protein complex in two brain regions, hippocampus and cortex, and identified mGluR1 as its major interactor in addition to the well described Homer proteins. We confirmed the presence of mGluR1/5 complex by (i) reverse immunoprecipitation using an mGluR1 antibody to pulldown mGluR5 from hippocampal tissue, (ii) coexpression in HEK293 cells followed by coimmunoprecipitation to reveal the direct interaction of mGluR1 and 5, and (iii) superresolution microscopy imaging of hippocampal primary neurons to show colocalization of the mGluR1/5 in the synapse. PMID:27392515

  2. Functional convergence of signalling by GPI-anchored and anchorless forms of a salamander protein implicated in limb regeneration.

    PubMed

    Blassberg, Robert A; Garza-Garcia, Acely; Janmohamed, Azara; Gates, Phillip B; Brockes, Jeremy P

    2011-01-01

    The GPI-anchor is an established determinant of molecular localisation and various functional roles have been attributed to it. The newt GPI-anchored three-finger protein (TFP) Prod1 is an important regulator of cell behaviour during limb regeneration, but it is unclear how it signals to the interior of the cell. Prod1 was expressed by transfection in cultured newt limb cells and activated transcription and expression of matrix metalloproteinase 9 (MMP9) by a pathway involving ligand-independent activation of epidermal growth factor receptor (EGFR) signalling and phosphorylation of extracellular regulated kinase 1 and 2 (ERK1/2). This was dependent on the presence of the GPI-anchor and critical residues in the α-helical region of the protein. Interestingly, Prod1 in the axolotl, a salamander species that also regenerates its limbs, was shown to activate ERK1/2 signalling and MMP9 transcription despite being anchorless, and both newt and axolotl Prod1 co-immunoprecipitated with the newt EGFR after transfection. The substitution of the axolotl helical region activated a secreted, anchorless version of the newt molecule. The activity of the newt molecule cannot therefore depend on a unique property conferred by the anchor. Prod1 is a salamander-specific TFP and its interaction with the phylogenetically conserved EGFR has implications for our view of regeneration as an evolutionary variable.

  3. The Arabidopsis acetylated histone-binding protein BRAT1 forms a complex with BRP1 and prevents transcriptional silencing

    PubMed Central

    Zhang, Cui-Jun; Hou, Xiao-Mei; Tan, Lian-Mei; Shao, Chang-Rong; Huang, Huan-Wei; Li, Yong-Qiang; Li, Lin; Cai, Tao; Chen, She; He, Xin-Jian

    2016-01-01

    Transposable elements and other repetitive DNA sequences are usually subject to DNA methylation and transcriptional silencing. However, anti-silencing mechanisms that promote transcription in these regions are not well understood. Here, we describe an anti-silencing factor, Bromodomain and ATPase domain-containing protein 1 (BRAT1), which we identified by a genetic screen in Arabidopsis thaliana. BRAT1 interacts with an ATPase domain-containing protein, BRP1 (BRAT1 Partner 1), and both prevent transcriptional silencing at methylated genomic regions. Although BRAT1 mediates DNA demethylation at a small set of loci targeted by the 5-methylcytosine DNA glycosylase ROS1, the involvement of BRAT1 in anti-silencing is largely independent of DNA demethylation. We also demonstrate that the bromodomain of BRAT1 binds to acetylated histone, which may facilitate the prevention of transcriptional silencing. Thus, BRAT1 represents a potential link between histone acetylation and transcriptional anti-silencing at methylated genomic regions, which may be conserved in eukaryotes. PMID:27273316

  4. The T=1 capsid protein of Penicillium chrysogenum virus is formed by a repeated helix-rich core indicative of gene duplication.

    PubMed

    Luque, Daniel; González, José M; Garriga, Damiá; Ghabrial, Said A; Havens, Wendy M; Trus, Benes; Verdaguer, Nuria; Carrascosa, José L; Castón, José R

    2010-07-01

    Penicillium chrysogenum virus (PcV), a member of the Chrysoviridae family, is a double-stranded RNA (dsRNA) fungal virus with a multipartite genome, with each RNA molecule encapsidated in a separate particle. Chrysoviruses lack an extracellular route and are transmitted during sporogenesis and cell fusion. The PcV capsid, based on a T=1 lattice containing 60 subunits of the 982-amino-acid capsid protein, remains structurally undisturbed throughout the viral cycle, participates in genome metabolism, and isolates the virus genome from host defense mechanisms. Using three-dimensional cryoelectron microscopy, we determined the structure of the PcV virion at 8.0 A resolution. The capsid protein has a high content of rod-like densities characteristic of alpha-helices, forming a repeated alpha-helical core indicative of gene duplication. Whereas the PcV capsid protein has two motifs with the same fold, most dsRNA virus capsid subunits consist of dimers of a single protein with similar folds. The spatial arrangement of the alpha-helical core resembles that found in the capsid protein of the L-A virus, a fungal totivirus with an undivided genome, suggesting a conserved basic fold. The encapsidated genome is organized in concentric shells; whereas the inner dsRNA shells are well defined, the outermost layer is dense due to numerous interactions with the inner capsid surface, specifically, six interacting areas per monomer. The outermost genome layer is arranged in an icosahedral cage, sufficiently well ordered to allow for modeling of an A-form dsRNA. The genome ordering might constitute a framework for dsRNA transcription at the capsid interior and/or have a structural role for capsid stability.

  5. A protective and safe intranasal RSV vaccine based on a recombinant prefusion-like form of the F protein bound to bacterium-like particles.

    PubMed

    Rigter, Alan; Widjaja, Ivy; Versantvoort, Hanneke; Coenjaerts, Frank E J; van Roosmalen, Maarten; Leenhouts, Kees; Rottier, Peter J M; Haijema, Bert Jan; de Haan, Cornelis A M

    2013-01-01

    Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease in infants and the elderly. Currently, no licensed vaccine against RSV is available. Here we describe the development of a safe and effective intranasal subunit vaccine that is based on recombinant fusion (F) protein bound to the surface of immunostimulatory bacterium-like particles (BLPs) derived from the food-grade bacterium Lactococcus lactis. Different variants of F were analyzed with respect to their conformation and reactivity with neutralizing antibodies, assuming that F proteins mimicking the metastable prefusion form of RSV F expose a more extensive and relevant epitope repertoire than F proteins corresponding to the postfusion structure. Our results indicate that the recombinant soluble ectodomain of RSV F readily adopts a postfusion conformation, generation of which cannot be prevented by C-terminal addition of a trimerization motif, but whose formation is prevented by mutation of the two furin cleavage sites in F. While the putative postfusion form of F is recognized well by the monoclonal antibody Palivizumab, this is much less so for the more potently neutralizing, prefusion-specific antibodies D25 and AM22. Both addition of the trimerization motif and mutation of the furin cleavage sites increased the reactivity of F with D25 and AM22, with the highest reactivity being observed for F proteins in which both these features were combined. Intranasal vaccination of mice or cotton rats with BLPs loaded with this latter prefusion-like F protein (BLP-F), resulted in the potent induction of F-specific immunoglobulins and in significantly decreased virus titers in the lungs upon RSV challenge. Moreover, and in contrast to animals vaccinated with formalin-inactivated RSV, animals that received BLP-F exhibited high levels of F-specific secretory IgA in the nose and RSV-neutralizing antibodies in sera, but did not show symptoms of enhanced disease after challenge with RSV.

  6. ABIN-2 Forms a Ternary Complex with TPL-2 and NF-κB1 p105 and Is Essential for TPL-2 Protein Stability†

    PubMed Central

    Lang, V.; Symons, A.; Watton, S. J.; Janzen, J.; Soneji, Y.; Beinke, S.; Howell, S.; Ley, S. C.

    2004-01-01

    NF-κB1 p105 forms a high-affinity, stoichiometric interaction with TPL-2, a MEK kinase essential for TLR4 activation of the ERK mitogen-activated protein kinase cascade in lipopolysaccharide (LPS)-stimulated macrophages. Interaction with p105 is required to maintain TPL-2 metabolic stability and also negatively regulates TPL-2 MEK kinase activity. Here, affinity purification identified A20-binding inhibitor of NF-κB 2 (ABIN-2) as a novel p105-associated protein. Cotransfection experiments demonstrated that ABIN-2 could interact with TPL-2 in addition to p105 but preferentially formed a ternary complex with both proteins. Consistently, in unstimulated bone marrow-derived macrophages (BMDMs), a substantial fraction of endogenous ABIN-2 was associated with both p105 and TPL-2. Although the majority of TPL-2 in these cells was complexed with ABIN-2, the pool of TPL-2 which could activate MEK after LPS stimulation was not, and LPS activation of TPL-2 was found to correlate with its release from ABIN-2. Depletion of ABIN-2 by RNA interference dramatically reduced steady-state levels of TPL-2 protein without affecting levels of TPL-2 mRNA or p105 protein. In addition, ABIN-2 increased the half-life of cotransfected TPL-2. Thus, optimal TPL-2 stability in vivo requires interaction with ABIN-2 as well as p105. Together, these data raise the possibility that ABIN-2 functions in the TLR4 signaling pathway which regulates TPL-2 activation. PMID:15169888

  7. Three wall-associated kinases required for rice basal immunity form protein complexes in the plasma membrane.

    PubMed

    Cayrol, Bastien; Delteil, Amandine; Gobbato, Enrico; Kroj, Thomas; Morel, Jean-Benoit

    2016-01-01

    Receptor-like kinases (RLKs) play key roles in disease resistance, in particular basal immunity. They recognize patterns produced by the pathogen invasion and often work as complexes in the plasma membrane. Among these RLKs, there is increasing evidence in several plant species of the key role of Wall-associated kinases (WAKs) in disease resistance. We recently showed using rice (Oryza sativa) loss-of-function mutants of three transcriptionally co-regulated OsWAK genes that individual OsWAKs are positively required for quantitative resistance to the rice blast fungus, Magnaporthe oryzae. This finding was unexpected since WAK genes belong to large gene families where functional redundancy is expected. Here we provide evidence that this may be due to complex physical interaction between OsWAK proteins.

  8. MARTINI bead form factors for the analysis of time-resolved X-ray scattering of proteins.

    PubMed

    Niebling, Stephan; Björling, Alexander; Westenhoff, Sebastian

    2014-08-01

    Time-resolved small- and wide-angle X-ray scattering (SAXS and WAXS) methods probe the structural dynamics of proteins in solution. Although technologically advanced, these methods are in many cases limited by data interpretation. The calculation of X-ray scattering profiles is computationally demanding and poses a bottleneck for all SAXS/WAXS-assisted structural refinement and, in particular, for the analysis of time-resolved data. A way of speeding up these calculations is to represent biomolecules as collections of coarse-grained scatterers. Here, such coarse-graining schemes are presented and discussed and their accuracies examined. It is demonstrated that scattering factors coincident with the popular MARTINI coarse-graining scheme produce reliable difference scattering in the range 0 < q < 0.75 Å(-1). The findings are promising for future attempts at X-ray scattering data analysis, and may help to bridge the gap between time-resolved experiments and their interpretation.

  9. Two distinct forms of Factor VIII coagulant protein in human plasma. Cleavage by thrombin, and differences in coagulant activity and association with von Willebrand factor.

    PubMed Central

    Weinstein, M J; Chute, L E

    1984-01-01

    We have characterized Factor VIII coagulant protein, present in normal human plasma, that reacts with a specific human 125I-labeled anti-human VIII:C antigen Fab antibody fragment. Two major Factor VIII coagulant antigen populations were present. The first, approximately 85% of the total antigen, was bound to von Willebrand factor and when tested in a standard one-stage assay had Factor VIII coagulant activity. The second antigenic population, eluting near fibrinogen when plasma was gel filtered, was not bound to von Willebrand protein, did not have Factor VIII coagulant activity unless activated, but did block anti-VIII:C Fab neutralization of clotting activity. The two antigenic populations were separable by cryoprecipitation and agarose gel electrophoresis. Although the two antigenic populations differed in their Factor VIII coagulant activity and in their binding to von Willebrand factor, the principal member of both populations is of mol wt 2.4 X 10(5). Both antigens, when proteolyzed by thrombin, were quickly converted to a 1 X 10(5)-mol wt form in association with the appearance of VIII:C activity. The 1 X 10(5)-mol wt antigen was further slowly degraded to an 8 X 10(4)-mol wt form while Factor VIII coagulant activity declined. These results demonstrate the presence of an inactive Factor VIII coagulant protein in plasma, not associated with von Willebrand factor, that can react with thrombin to yield Factor VIII coagulant activity. Images PMID:6421875

  10. Nonionic and zwitterionic forms of glycylglycylarginine as a part of spider silk protein: Spectroscopic and theoretical study

    NASA Astrophysics Data System (ADS)

    Arı, Hatice; Özpozan, Talat

    2016-01-01

    Glycylglycylarginine as a part of GGX motif of spider silk spidroin in nonionic (non-GGR) and zwitterionic (zwt-GGR) forms have been examined from theoretical and spectroscopic aspects. The most stable conformational isomers of non-GGR and zwt-GGR were obtained through relaxed scan using the DFT/B3LYP with 6-31G(d) basis set. Nonionic and zwitterionic forms of 310-helix structures of GGR have also been calculated and compared with the most stable conformers obtained as a result of conformer analysis of isolated three peptide structures. This comparison should give an idea about the stability contribution of intermolecular interactions between the 310-helix structured peptide chains. O3LYP and B3PW91 hybrid functionals beside B3LYP have also been used for further calculations of geometry optimization, vibrational analysis, Natural Bond Orbital (NBO) analysis, HOMO-LUMO analysis and hydrogen bonding analysis. Normal Mode Analysis was carried through Potential Energy Distribution (PED) calculations by means of VEDA4 program package. IR and Raman spectra of GGR have also been used to relate the spectroscopic data obtained to electronic and structural features.

  11. Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor

    PubMed Central

    Amsalem, Ayelet R.; Marom, Barak; Shapira, Keren E.; Hirschhorn, Tal; Preisler, Livia; Paarmann, Pia; Knaus, Petra; Henis, Yoav I.; Ehrlich, Marcelo

    2016-01-01

    The expression and function of transforming growth factor-β superfamily receptors are regulated by multiple molecular mechanisms. The type II BMP receptor (BMPRII) is expressed as two alternatively spliced forms, a long and a short form (BMPRII-LF and –SF, respectively), which differ by an ∼500 amino acid C-terminal extension, unique among TGF-β superfamily receptors. Whereas this extension was proposed to modulate BMPRII signaling output, its contribution to the regulation of receptor expression was not addressed. To map regulatory determinants of BMPRII expression, we compared synthesis, degradation, distribution, and endocytic trafficking of BMPRII isoforms and mutants. We identified translational regulation of BMPRII expression and the contribution of a 3’ terminal coding sequence to this process. BMPRII-LF and -SF differed also in their steady-state levels, kinetics of degradation, intracellular distribution, and internalization rates. A single dileucine signal in the C-terminal extension of BMPRII-LF accounted for its faster clathrin-mediated endocytosis relative to BMPRII-SF, accompanied by mildly faster degradation. Higher expression of BMPRII-SF at the plasma membrane resulted in enhanced activation of Smad signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane. PMID:26739752

  12. NPC1 late endosomes contain elevated levels of non-esterified (‘free’) fatty acids and an abnormally glycosylated form of the NPC2 protein

    PubMed Central

    2005-01-01

    NPC (Niemann–Pick type C) disease is a rare lipidosis characterized by the accumulation of LDL (low-density lipoprotein)-derived non-esterified cholesterol in the E/L (endosomal/lysosomal) system. The gene products that are responsible for the two NPC complementation groups are distinct and dissimilar, yet their cellular and disease phenotypes are virtually indistinguishable. To investigate the relationship between NPC1 and NPC2 and their potential role in NPC disease pathogenesis, we have developed a method for the rapid and efficient isolation of late endocytic vesicles from mouse liver by magnetic chromatography. Late endosomes from Wt (wild-type) and NPC1 mice were found to differ not only in their cholesterol and sphingomyelin content, as expected, but also in their non-esterified (‘free’) fatty acid content, with NPC1 vesicles showing an approx. 7-fold increase in non-esterified fatty acid levels compared with Wt vesicles. Furthermore, we show that the NPC2 protein is in an incompletely deglycosylated form in NPC1 late endosomes by a mechanism that is specific to the NPC2 protein and not a global aberration of protein glycosylation/deglycosylation or trafficking, since NPC2 secreted from NPC1 cells is indistinguishable from that secreted from Wt cells. Also, a greater proportion of the normally soluble cellular NPC2 protein partitions with detergent-insoluble late endosomal internal membrane domains in NPC1 vesicles. In addition, we show that, although a small amount of the NPC2 protein associates with these membranes in Wt vesicles, this localization becomes much more pronounced in NPC1 vesicles. These results suggest that the function of the NPC2 protein may be compromised as well in NPC1 endosomes, which might explain the paradoxical phenotypic similarities of the two NPC disease complementation groups. PMID:15896196

  13. The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.

    PubMed

    Fiorillo, Annarita; di Marino, Daniele; Bertuccini, Lucia; Via, Allegra; Pozio, Edoardo; Camerini, Serena; Ilari, Andrea; Lalle, Marco

    2014-01-01

    The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.

  14. Defective RNA Replication and Late Gene Expression in Temperature-Sensitive Influenza Viruses Expressing Deleted Forms of the NS1 Protein

    PubMed Central

    Falcón, Ana M.; Marión, Rosa M.; Zürcher, Thomas; Gómez, Paulino; Portela, Agustín; Nieto, Amelia; Ortín, Juan

    2004-01-01

    Influenza A virus mutants expressing C-terminally deleted forms of the NS1 protein (NS1-81 and NS1-110) were generated by plasmid rescue. These viruses were temperature sensitive and showed a small plaque size at the permissive temperature. The accumulation of virion RNA in mutant virus-infected cells was reduced at the restrictive temperature, while the accumulation of cRNA or mRNA was not affected, indicating that the NS1 protein is involved in the control of transcription versus replication processes in the infection. The synthesis and accumulation of late virus proteins were reduced in NS1-81 mutant-infected cells at the permissive temperature and were essentially abolished for both viruses at the restrictive temperature, while synthesis and accumulation of nucleoprotein (NP) were unaffected. Probably as a consequence, the nucleocytoplasmic export of virus NP was strongly inhibited at the restrictive temperature. These results indicate that the NS1 protein is essential for nuclear and cytoplasmic steps during the virus cycle. PMID:15047804

  15. Structure of the apo form of the catabolite control protein A (CcpA) from Bacillus megaterium with a DNA-binding domain

    PubMed Central

    Singh, Rajesh Kumar; Palm, Gottfried J.; Panjikar, Santosh; Hinrichs, Winfried

    2007-01-01

    Crystal structure determination of catabolite control protein A (CcpA) at 2.6 Å resolution reveals for the first time the structure of a full-length apo-form LacI-GalR family repressor protein. In the crystal structures of these transcription regulators, the three-helix bundle of the DNA-binding domain has only been observed in cognate DNA complexes; it has not been observed in other crystal structures owing to its mobility. In the crystal packing of apo-CcpA, the protein–protein contacts between the N-terminal three-helix bundle and the core domain consisted of interactions between the homodimers that were similar to those between the corepressor protein HPr and the CcpA N-subdomain in the ternary DNA complex. In contrast to the DNA complex, the apo-CcpA structure reveals large subdomain movements in the core, resulting in a complete loss of contacts between the N-subdomains of the homodimer. PMID:17401189

  16. Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis

    PubMed Central

    Bammert, Lukas; Jonas, Stefanie; Ungricht, Rosemarie; Kutay, Ulrike

    2016-01-01

    Mammalian AATF/Che-1 is essential for embryonic development, however, the underlying molecular mechanism is unclear. By immunoprecipitation of human AATF we discovered that AATF forms a salt-stable protein complex together with neuroguidin (NGDN) and NOL10, and demonstrate that the AATF-NGDN-NOL10 (ANN) complex functions in ribosome biogenesis. All three ANN complex members localize to nucleoli and display a mutual dependence with respect to protein stability. Mapping of protein-protein interaction domains revealed the importance of both the evolutionary conserved WD40 repeats in NOL10 and the UTP3/SAS10 domain in NGDN for complex formation. Functional analysis showed that the ANN complex supports nucleolar steps of 40S ribosomal subunit biosynthesis. All complex members were required for 18S rRNA maturation and their individual depletion affected the same nucleolar cleavage steps in the 5′ETS and ITS1 regions of the ribosomal RNA precursor. Collectively, we identified the ANN complex as a novel functional module supporting the nucleolar maturation of 40S ribosomal subunits. Our data help to explain the described role of AATF in cell proliferation during mouse development as well as its requirement for malignant tumor growth. PMID:27599843

  17. Human Bone-Forming Chondrocytes Cultured in the Hydrodynamic Focusing Bioreactor Retain Matrix Proteins: Similarities to Spaceflight Results

    NASA Technical Reports Server (NTRS)

    Duke, P. J.; Hecht, J.; Montufar-Solis, D.

    2006-01-01

    Fracture healing, crucial to a successful Mars mission, involves formation of a cartilaginous fracture callus which differentiates, mineralizes, ossifies and remodels via the endochondral process. Studies of spaceflown and tailsuspended rats found that, without loading, fracture callus formation and cartilage differentiation within the callus were minimal. We found delayed differentiation of chondrocytes within the rat growth plate on Cosmos 1887, 2044, and Spacelab 3. In the current study, differentiation of human bone-forming chondrocytes cultured in the hydrodynamic focusing bioreactor (HFB) was assessed. Human costochondral chondrocytes in suspension were aggregated overnight, then cultured in the HFB for 25 days. Collagen Type II, aggrecan and unsulfated chondroitin were found extracellularly and chondroitin sulfates 4 and 6 within the cell. Lack of secretion was also found in pancreatic cells of spaceflown rats, and in our SL3 studies. The HFB can be used to study cartilage differentiation in simulated microgravity.

  18. Properties of a Pseudomonas stutzeri outer membrane channel-forming protein (NosA) required for production of copper-containing N sub 2 O reductase

    SciTech Connect

    Lee, H.S.; Ingraham, J.L. ); Hancock, R.E.W. )

    1989-04-01

    A protein (NosA) in the outer membrane of Pseudomonas stutzeri that is required for copper to be inserted into N{sub 2}O reductase has been extracted and purified to homogeneity. The purified protein could form channels in black lipid bilayers. Line N{sub 2}O reductase, NosA contained copper and was only made anaerobically. In contrast to N{sub 2}O reductase, its synthesis was repressed by exogenous copper (but not by Mn, Co, Ni, Zn, or Fe). Also in contrast to N{sub 2}O reductase, NosA homologs were not immunologically detectable in Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, or other strains of P. stutzeri.

  19. Inhibitory protein controls the reversion of protoplasts and L forms of Bacillus subtilis to the walled state.

    PubMed Central

    DeCastro-Costa, M R; Landman, O E

    1977-01-01

    When the cell wall of Bacillus subtilis is removed by lysozyme and the resultant protoplasts are plated on hypertonic soft agar medium, each protoplast forms an L colony. L bodies from such L colonies again plate as L-colony-forming units (CFU). However, if protoplasts or L bodies are "conditioned" by 1 h of incubation in 0.4% casein hydrolysate medium and then incubated in 25% gelatin medium for 1 h, 60 to 100% of the formerly naked cells give rist to bacillary colonies. The present experiments largely explain the mechanism responsible for the "heritable" persistence of the wall-less state in B. subtilis. It is shown that protoplasts produce a reversion inhibitory factor (RIF) which blocks reversion when the cell concentration exceeds 5 x 105 CFU/ml. This inhibitor is nondialyzable and sensitive to trypsin, heat, and detergent. Efficient reversion at 2 x 107 CFU/ml is obtained if the protoplasts are treated with trypsin after conditioning and chloramphenicol is incorporated into the gelatin reversion medium. In the presence of 500 mug of trypsin per ml, the requirement for gelatin is sharply reduced, and reversion occurs rapidly in liquid medium containing only 10% gelatin. Trypsin also stimulates reversion in L colonies growing on soft agar. Latent RIF is activated by beta-mercaptoethanol. This reagent blocks reversion of protoplast suspensions at densities of 5 x 105 CFU/ml. Comparison of the autolytic behavior of B. subtilis and of the RIF revealed that several or the properties of the two activities coincide: both are inhibited by high concentrations of gelatin, both are activated by beta-mercaptoethanol, and both have high affinity for cell wall. Going on the assumption that RIF is autolysin, models for protoplast reversion is suggested by the finding that mutants with altered teichoic acid show altered reversion behavior. PMID:402356

  20. Brain Endothelial Cells Produce Amyloid β from Amyloid Precursor Protein 770 and Preferentially Secrete the O-Glycosylated Form*

    PubMed Central

    Kitazume, Shinobu; Tachida, Yuriko; Kato, Masaki; Yamaguchi, Yoshiki; Honda, Takashi; Hashimoto, Yasuhiro; Wada, Yoshinao; Saito, Takashi; Iwata, Nobuhisa; Saido, Takaomi; Taniguchi, Naoyuki

    2010-01-01

    Deposition of amyloid β (Aβ) in the brain is closely associated with Alzheimer disease (AD). Aβ is generated from amyloid precursor protein (APP) by the actions of β- and γ-secretases. In addition to Aβ deposition in the brain parenchyma, deposition of Aβ in cerebral vessel walls, termed cerebral amyloid angiopathy, is observed in more than 80% of AD individuals. The mechanism for how Aβ accumulates in blood vessels remains largely unknown. In the present study, we show that brain endothelial cells expressed APP770, a differently spliced APP mRNA isoform from neuronal APP695, and produced Aβ40 and Aβ42. Furthermore, we found that the endothelial APP770 had sialylated core 1 type O-glycans. Interestingly, Ο-glycosylated APP770 was preferentially processed by both α- and β-cleavage and secreted into the media, suggesting that O-glycosylation and APP processing involved related pathways. By immunostaining human brain sections with an anti-APP770 antibody, we found that APP770 was expressed in vascular endothelial cells. Because we were able to detect O-glycosylated sAPP770β in human cerebrospinal fluid, this unique soluble APP770β has the potential to serve as a marker for cortical dementias such as AD and vascular dementia. PMID:20952385

  1. Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) forms a Ca2+/H+ antiporter

    PubMed Central

    Shao, Juan; Fu, Zhenglin; Ji, Yanli; Guan, Xiangchen; Guo, Shang; Ding, Zhanyu; Yang, Xue; Cong, Yao; Shen, Yuequan

    2016-01-01

    Leucine zipper-EF-hand-containing transmembrane protein1 (LETM1) is located in the mitochondrial inner membrane and is defective in Wolf-Hirschhorn syndrome. LETM1 contains only one transmembrane helix, but it behaves as a putative transporter. Our data shows that LETM1 knockdown or overexpression robustly increases or decreases mitochondrial Ca2+ level in HeLa cells, respectively. Also the residue Glu221 of mouse LETM1 is identified to be necessary for Ca2+ flux. The mutation of Glu221 to glutamine abolishes the Ca2+-transport activity of LETM1 in cells. Furthermore, the purified LETM1 exhibits Ca2+/H+ anti-transport activity, and the activity is enhanced as the proton gradient is increased. More importantly, electron microscopy studies reveal a hexameric LETM1 with a central cavity, and also, observe two different conformational states under alkaline and acidic conditions, respectively. Our results indicate that LETM1 is a Ca2+/H+ antiporter and most likely responsible for mitochondrial Ca2+ output. PMID:27669901

  2. Detecting the form of selection in the outer membrane protein C of Enterobacter aerogenes strains and Salmonella species.

    PubMed

    Padhi, Abinash; Verghese, Bindhu; Otta, Subhendu K

    2009-01-01

    The types of selective pressure operating on the outer membrane protein C (ompC) of Enterobacter aerogenes strains, the causative agent for nosocomial infections, and Salmonella sp., the hazardous pathogen are investigated using the maximum likelihood-based codon substitution models. Although the rate of amino acid replacement to the silent substitution (omega) across the entire codon sites of ompC of E. aerogenes (omega=0.3194) and Salmonella sp. (omega=0.2047) indicate that the gene is subjected to purifying selection (i.e. omega<1), approximately 3.7% of ompC codon sites in E. aerogenes (omega=21.52) are under the influence of positive Darwinian selection (i.e. omega>1). Such contrast in the intensity of selective pressures in both pathogens could be associated with the differential response to the adverse environmental changes. In E. aerogenes, majority of the positively selected sites are located in the hypervariable cell-surface-exposed domains whereas the trans-membrane domains are functionally highly constrained.

  3. Voltage-dependent gap junction channels are formed by connexin32, the major gap junction protein of rat liver.

    PubMed Central

    Moreno, A P; de Carvalho, A C; Verselis, V; Eghbali, B; Spray, D C

    1991-01-01

    We report here experiments undertaken in pairs of hepatocytes that demonstrate a marked voltage sensivity of junctional conductance and, thus, contradict earlier findings reported by this laboratory (Spray, D.C., R.D.ginzberg, E.A., E. A. Morales, Z. Gatmaitan and I.M. Arias, 1986, J. Cell Biol. 101:135-144; Spray C.D. R.L. White, A.C. Campos de Carvalho, and M.V.L. Bennett. 1984. Biophys. J. 45:219-230) and by others (Dahl, G., T. Moller, D. Paul, R. Voellmy, and R. Werner. 1987. Science [Wash. DC] 236:1290-1293; Riverdin, E.C., and R. Weingart. 1988. Am. J. Physiol. 254:C226-C234). Expression in exogenous systems, lipid bilayers in which fragments of isolated gap junction membranes were incorporated (Young, J.D.-E., Z. Cohn, and N.B. Gilula. 1987. Cell. 48:733-743.) and noncommunicating cells transfected with connexin32 cDNA (Eghbali, B., J.A. Kessler, and D.C. Spray. 1990. Proc. Natl. Acad. Sci. USA. 87:1328-1331), support these findings a